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Branched-chain carbohydrates and nucleosides Cliff, Brian Leonard 1978

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BRANCHED-CHAIN CARBOHYDRATES AND NUCLEOSIDES by BRIAN LEONARD CLIFF B.Sc. (Honours)-, U n i v e r s i t y o f B r i t i s h Columbia, 1972 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF • — DOCTOR OF PHILOSOPHY i n THE FACULTY OF.GRADUATE STUDIES In the.Department of Chemistry We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1978 (cp B r i a n Leonard C l i f f , 1978 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Chemistry The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date March 14, 1979. •6 B P 75-51 1 E S u p e r v i s o r : Dr. A. Rosenthal ABSTRACT The syntheses of a number of branched-chain carbo-hydrates and n u c l e o s i d e s are r e p o r t e d . The a p p l i c a t i o n of the K i l i a n i - F i s c h e r cyanohydrin s y n t h e s i s to 1 , 2 : 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - a - g - r i b o -hexofuranos-3-ulose (25) y i e l d e d e i t h e r the 3-C-cyano-1,2 : 5 , 6-di-0_-isopropylidene-a-D-gluco or a l l o f u r a n o s e , (26 and 2_7, r e s p e c t i v e l y ) predominantly, depending upon the c o n d i t i o n s employed. The s t e r e o s e l e c t i v i t y of the r e a c t i o n was examined. Reduction of the cyaho group and a c e t y l a t i o n o f the r e s u l t i n g amine a f f o r d e d 3-C-acetamido-methyl-1, 2 : 5 ,6 - d i - 0 _ - i s o p r o p y l i d e n e - a - g - a l l o and gl u c o -furanose (5_3 and 5_4, r e s p e c t i v e l y ) and pr o v i d e d an unequivocal proof of s t r u c t u r e f o r the o r i g i n a l cyanohydrins. The y l i d generated by the r e a c t i o n of potassium t -butoxide and carbomethoxymethyldimethyl phosphonate was condensed with 1,2:5,6-di-0-isopropylidene-ot-g-ribo-hexofuranos-3-ulose (25_) to a f f o r d , a f t e r r e d u c t i o n of the a,B-unsaturated e s t e r s (135 and 136), 3-C-(carbomethoxy-methyl) -3-deoxy-l ,2 :5 , 6 - d i - 0 _ - i s o p r o p y l i d e n e - a - g - a l l o f uranose (35) . The 5,6-0-isopropylidene group of 35_ was h y d r o l y z e d , - iicv-the r e s u l t i n g d i o l was o x i d a t i v e l y c l e a v e d and reduced, and the r e s u l t i n g a l c o h o l was ben z o y l a t e d t o give 5-0_-benzoyl-3-C- (carbomethoxymethyl) -3-deoxy-l, 2-0_-isopropy lidene-a-D-r i b o f u r a n o s e (172) . The 1,2-0_-isopropylidene group of 172 was hy d r o l y z e d to y i e l d the 5-0-benzoyl-3-C-carboxymethyl-3-deoxy-a,B-D-ribofuranose-2,3-y-lactone (173) which was a c e t y l a t e d to a f f o r d 1-0-acetyl-5-0_-benzoyl-3-C-carboxy-methyl-3-deoxy-p-D-ribofuranose-2,3-y-lactone (169). Compound 169 was fused d i r e c t l y w i t h 2 , 6 - d i c h l o r o p u r i n e to give 2,6-dichloro-9-[3'-C-(carboxymethyl-2',3'-y-lactone)-3 1-deoxy-B and a - D - r i b o f u r a n o s y l ] p u r i n e (174 6 6 and 175, r e s p e c t i v e l y ) and r e a c t e d w i t h N -benzoyl-N , 9 - b i s ( t r i m e t h y l s i l y l ) a d e n i n e , i n the presence of stannous c h l o r i d e , to a f f o r d N -benzoyl-9-(3 1-C-carboxymethyl-2 1,3 1 -y - l a c t o n e - 3 1 - d e o x y - B - D - r i b o f u r a n o s y l ) a d e n i n e (176) and the a-anomer 177. De-benzoylation of the 6-nucleoside 176 y i e l d e d 9-[3'-C-(carboxymethyl-2 1,3 1-y-lactone)-3'-deoxy-B-D-ribofuranosyl]adenine (17 8) while the a-nucleoside 177 a f f o r d e d 9 —[3'-C-carboxymethyl-3'-deoxy-a-D-ribofuranosyl] adenine (181). Condensation of methyl n i t r o a c e t a t e with 5-0_-benzoyl-1,2-0-isopropylidene-a-D-erythro-pentofuranos-3-ulose (182) and 5 - 0 - b e n z y l - l , 2 - 0 - i s o p r o p y l i d e n e - a - D - e r y t h r o - p e n t o f u r a n o s -3-ulose (19 4) i n the presence of ammonium a c e t a t e , f o l l o w e d by immediate treatment with p - t o l u e n e s u l p h o n i c a c i d mono-hydrate i n a c e t i c anhydride, y i e l d e d (E) and (Z^ ) 5-0-- i i i -benzoyl and 5 - 0 - b e n z y l - 3 - d e o x y - l , 2 - 0 - i s o p r o p y l i d e n e - 3 - C -n i t r o (methoxycarbonyl) me thy lene-ct-g-ery thro-pentof uranose , r e s p e c t i v e l y ( 1 8 9 and 1 9 8 ) . In a s i m i l a r s e t of r e a c t i o n s with 1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose (2J5) , the major product, o b t a i n e d i n good y i e l d , was 3 - 0 - ace t y 1 - 1 , 2 : 5 , 6 - d i - 0 _ - i s o p r o p y l i d e n e - 3 - C - [ (R, S) n i t r o -(methoxycarbonyl)methyl]-a-D-allofuranose ( 1 6 5 ) . The 5 , 6 - 0 _ - i s o p r o p y l i d e n e group of compound 1 6 5 was hy d r o l y z e d and the r e s u l t i n g d i o l was t r e a t e d w i t h p-t o l u e n e s u l p h o n i c a c i d monohydrate i n a c e t i c anhydride a t e l e v a t e d temperature to a f f o r d , a f t e r r e d u c t i o n w i t h sodium cyanoborohydride , 5 , 6 - d i - 0 _ - a c e t y l - 3 - d e o x y - l , 2 - 0 _ - i s o p r o p y l i d e n e -3 - C - [ ( R , S ) - n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] - a - D - a l l o f u r a n o s e ( 2 4 1 ) . A c e t o l y s i s o f compound 2 4 1 gave 1 , 2 , 5 , 6 - t e t r a - 0 _ -a c e t y l - 3 - d e o x y - 3 - C - [ ( R , S ) - n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] -a , 3 - D - a l l o f u r a n o s e ( 2 4 3) which was used i n an u n s u c c e s s f u l n u c l e o s i d e s y n t h e s i s with b i s ( t r i m e t h y l s i l y l ) t h y m i n e . Reduction of the n i t r o group of 3 - 0 _ - a c e t y l - l , 2 : 5 , 6 -di - 0_-isopropy lidene- 3-C-[ (R, S ) - n i t r o (methoxycarbonyl) methyl] -a-D-allofuranose by hydrogenation over Raney n i c k e l a f f o r d methyl L- and D - 2 - ( 1 , 2 : 5 , 6-di-O^-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e ( 2 0 5 and 2 0 6 , r e s p e c t i v e l y ) . The major product, methyl L - g l y c i n a t e 2 0 5 , was a c e t y l a t e d and the 0_-acetate and 5 , 6 - 0 _ - i s o p r o p y l i d e n e groups were hy d r o l y z e d to y i e l d a compound which was s u c c e s s i v e l y t r e a t e d with sodium p e r i o d a t e and sodium borohydride to g i v e , a f t e r a c e t y l a t i o n , methyl N - a c e t y l - L - 2 - ( 1 , 2 - 0 -- i v -i s o p r o p y l i d e n e - a - D - r i b o f u r a n o s - 3 - y l ) g l y c i n a t e (232) and i t s 5-0_-acetyl d e r i v a t i v e (233) . T r i f l u o r o a c e t y l a t i o n of methyl L-2-(1, 2 : 5 ,6-di-0_-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (205), f o l l o w e d by s u c c e s s i v e replacement of the 5,6 and 1,2-0-isopropylidene a c e t a l s with 0_-acetate b l o c k i n g groups, a f f o r d e d methyl L-2- (1,2,3,5, 6-penta-0_-acetyl-a , B - D - a l l o f uranos-3-yl) -N-t r i f l u o r o a c e t y l g l y c i n a t e (250) . The attempted f u s i o n of the bromo-sugar d e r i v a t i v e o f compound 250 w i t h N -b e n z o y l - N 6 - 9 - b i s - ( t r i m e t h y l s i l y l ) a d e n i n e y i e l d e d 1,1'-anhydro-2,3,5,6-tetra-0-acetyl-3-C-(R)-methoxycarbonyl-1 ( R ) , 1 ' ( S ) - N - t r i f l u o r o a c e t o e p i m o - a - g - a l l o f u r a n o s e (252) whereas the s i l v e r t r i f l u o r o m e t h y l s u l f o n a t e c a t a l y z e d condensation of the same bromo-sugar with b i s ( t r i m e t h y l -s i l y l ) t h y m i n e a f f o r d e d 1 - [ 2 ' , 3 ' , 5 ' , 6 ' - t e t r a - 0 - a c e t y l - 3 ' -C - ( ( S ) - N - t r i f l u o r o a c e t y l - c a r b o m e t h o x y ( a m i n o ) m e t h y l ) - 8 -D - a l l o f u r a n o s y l ] t h y m i n e (257) . - v -TABLE OF CONTENTS Page ABSTRACT i LIST OF TABLES x i i LIST OF FIGURES x i i i ACKNOWLEDGEMENTS x v i i I OBJECTIVE 1 II INTRODUCTION 4 1. Branched-Chain Sugars 4 1.1 Synthesis of Branched-Chain Sugars 5 1.1.1 Synthesis of Type A Sugars 5 1.1.2 Synthesis.of Type B Sugars 7 2. Methods of Oxidation 9 2.1 Oxidation of Secondary Hydroxyl Groups 9 3. Condensation Reactions Involving Keto-Sugars 12 3.1 Cyanide Condensations 12 3.2 The Witt i g Reaction 13 3.2.1 The C l a s s i c a l Wittig Reaction 14 3.2.2 The Modified Wittig Reaction 16 - v i -Page 3.3 Condensation I n v o l v i n g N i t r o Compounds of the Type R-CH 2-N0 2 18 3.3.1 Condensations of Nitromethane 19 with 3-Keto Sugars 3.3.2 Condensation of A l k y l N i t r o - 23 ace t a t e s with Aldehydo-Sugars 4. Nucleosides 26 4.1 Branched-Chain Nucleosides 26 4.2 Nucleoside S y n t h e s i s 28 4.2.1 Fusion Reactions 29 4.2.2 The H i l b e r t - J o h n s o n and S i l y l 33 Procedures 4.2.3 C a t a l y s e d H i l b e r t - J o h n s o n and 38 S i l y l Procedures 4.2.3.1 Mercury S a l t C a t a l y s t s 39 4.2.3.2 F r i e d e l - C r a f t s 41 C a t a l y s t s 4.2.3.3 P e r f l u o r o a l k a n e S u l f o n a t e 46 and P e r c h l o r a t e C a t a l y s t s 4.3 P e p t i d y l N u c l e o s i d e s 48 4.3.1 The Pol y o x i n s 49 5. Sy n t h e s i s of G l y c o s y l Amino A c i d s 53 II I RESULTS AND DISCUSSION 6 2 1. Synt h e s i s of Branched-chain Amino Sugars. 6 2 Reaction of 1,2:5,6-Di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose with Sodium Cyanide and Methyl N i t r o a c e t a t e 1.1 Synthesis o f 3-C-Cyano-l, 2 :5 ,6-di-^O- 63 isop r o p y l i d e n e - a - D - g l u c o f u r a n o s e (26) and 3-C-Cyano-l, 275 , 6-di-0_-iso-p r o p y l i d e n e - a - D - a l l o f u r a n o s e (27) - v i i -Page 1.1.1 Procedure A to Y i e l d 3-C-Cyano- 6 3 1,2:5,6-di-O-isopropylidene-a-p-glucofuranose (26) 1.1.2 Procedure B to Y i e l d 3-C-Cyano- 66 1,2:5, 6-di-0_-isopropylidene-a-p - a l l o f u r a n o s e (27) 1.1.3 Procedure C to Y i e l d 3-0-Acetyl- 6 7 3-C-cyano-l, 2 : 5 , 6-di-0_-iso-propylidene-a-D-gluco and a l l o -furanose (16 3 and 164), 3-0-Acetyl-1,2:5,6-di-0_-isopropylidene-3-C-[ (R,S)-nitro (methoxycarbonyl) -me t h y l ] - a - D - a l l o f u r a n o s e (165) and 3,5, 6-tri-0^-Acetyl-3-C-cyano-1 , 2 - 0 - i s o p r o p y l i d e n e - a - D - a l l o -furanose (166) ~ 1.2 Syn t h e s i s of 3-C-Acetaminomethyl-l,2:5,6- 68 is o p r o p y l i d e n e - a - D - g l u c o f u r a n o s e (54) and 3-C-Acetaminomethyl-l,2:5,6-di-0-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s e (53) 2. Syn t h e s i s of Branched-Chain N u c l e o s i d e s . 70 Analogues o f the Nucl e o s i d e Moiety of Puromycin 2.1 S y n t h e s i s of l-0-Acetyl-5-0_-benzoyl- 71 3-C-carboxymethyl-3-deoxy-3-D-ribo-furanose-2,3-y-lactone (169)~ 2.2 S y n t h e s i s of 2,6-Dichloro-9-[3 1-C- 74 (carboxymethyl-2',3'-y-lactone)-3 1-deoxy-B and a - D - r i b o f u r a n o s y l ] p u r i n e (174 and 175)~ 2.3 S y n t h e s i s o f 9-[3'-C-(Carboxymethyl- 77 2',3 1-y-lactone)-3'-deoxy-B-g-ribo-f u r a n o s y l ] adenine (178) and~9-[3'-C-Carboxymethyl-3'-deoxy-a-D-ribofuranosyl] adenine (181) ~ 3. Syn t h e s i s of Analogues of the Nucleoside 88 Moiety o f the P o l y o x i n s . 3.1 S y n t h e s i s of 3-Deoxy-3-C-nitro(carbo- 89 methoxy)methy1-D-erythro-pentofuranoses - v i i i -Page 3.1.1 Condensation of Methyl N i t r o - 90 acetate w i t h 5-0-Benzoyl-l,2-O - i s o p r o p y l i d e n e - g - D - e r y t h r o -pentos-3-ulose (182)" 3.1.2 Condensation o f Methyl N i t r o - 95 acetate with 5 - 0 - B e n z y l - l , 2-0-i s o p r o p y l i d e n e - a - D - e r y t h r o -pentos-3-ulose (19"4) 3.2 Condensation of Methyl N i t r o a c e t a t e 9 7 with 1,2:5,6-Di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose (25) 3.2.1 S y n t h e s i s of 1,2:5,6-Di-0- 98 i s o p r o p y l i d e n e - 3 - C - [ ( R , S ) -n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] -a-D-allofuranose (162) 3.2.2 S y n t h e s i s of 3-0-Acetyl-l,2:5,6- 102 di-0_-isopropylidene-3-C- [ (R,S) -n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] -a - g - a l l o f u r a n o s e (165) 3.3 Reduction and Proof o f S t r u c t u r e o f the 104 Methyl N i t r o a c e t a t e Adducts 3.3.1 S y n t h e s i s o f Methyl N - a c e t y l - L - 105 and D-2-(1,2:5,6-di-O-iso-propylidene-a-D-glucof uranos-3 - y l ) g l y c i n a t e " ( 2 0 2 and 203) 3.3.2 S y n t h e s i s o f 3-0-Acetyl-l,2:5,6- 106 di-0_-isopropylidene-3-C-(methoxydicarbony1)-a-D-a l l o f u r a n o s e oxime (204") 3.3.3 S y n t h e s i s of Methyl L- and D- 1 0 7 2- (1, 2 : 5 , 6-di-0_-isopropylidene-a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (20"5 and 206) 3.3.4 S y n t h e s i s of L- and D-2-(1,2: 109 5,6-Di-O^-isopropylidene-a-D - a l l o f u r a n o s - 3 - y l ) g l y c i n e T 2 0 7 and 208)" 3.4 O x i d a t i v e Cleavage of Methyl N - a c e t y l - 117 L-2-(1,2-£-isopropylidene-a-D-allo-f u r a n o s - 3 - y l ) g l y c i n a t e (226) with Sodium P e r i o d a t e - i x -Page 3.4.1 Synthesis of Methyl N-acetyl- 119 L-2-(1,2-0-isopropylidene-a-D-allofuranos-3-yl)glycinate T226) 3.4.2 Attempted Synthesis of 3-C- 125 [(S)-Acetamino(carbomethoxy)-methyl]-1,2-0-isopropylidene-q-D-ribo-pentodialdofuranose-5,N-aminal (228). 3.4.3 Synthesis of Methyl N-acetyl- 126 L-2- (1,2-0_-isopropylidene-a-5-ribofuranos-3-yl)glycinate T232) and Methyl N-acetyl-L-2- (5-0_-acetyl-l, 2-0_-isopropyl-idene-a-D-ribofuranos-3-yl) glycinate (233) 3.5 Synthesis of 3-Deoxy-3-C-nitro(carbo- 129 methoxy)methyl allofuranoses 3.5.1 Attempted Synthesis of 5,6-Di- 129 O-acetyl-3-C-[acetamino-Tmethoxycarbonyl)methylene]-3- deoxy-l,2-0-isopropylidene-g-D-ribo-hexofuranose (234) 3.5.2 Synthesis of 3-Deoxy-l,2:5,6- 131 di-0_-isopropylidene-3-C- [ (R,S) -nitro(methoxycarbonyl)methyl]-a-D-allofuranose (236) 3.5.3 Synthesis of 5,6-Di-0_-acetyl-3- 138 deoxy-1,2-0_-isopropylidene-3--C-[(R,S)-nitro(methoxycarbonyl)-methyl]-a-D-allofuranose (241) 3.5.4 Synthesis of 1,2:5,6-Tetra-O- 145 acetyl-3-deoxy-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a,B-D-allofuranose (24 3) 3.5.5 Attempted Nucleoside Synthesis 148 with Compound 243 3.6 Attempted Synthesis of 1-[3'-C-((S)- 149 Carbomethoxy(amino)methyl)-B-D-a l l o f uranosyl] thymine ~ - x -Page 3.6.1 S y n t h e s i s of Methyl L-2-(3-0- 149 a c e t y l - 1 , 2 : 5 , 6-di-0_-isopro-p y l i d e n e - a - D - a l l o f u r a n o s - 3 -y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (245T 3.6.2 S y n t h e s i s of Methyl L-2-(3,5,6- 151 t r i - 0 _ - a c e t y l - l , 2-0_-isopropylidene-a - D - a l l o f u r a n o s - 3 - y l ) - N - t r i -f l u o r o a c e t y l g l y c i n a t e (246) and Methyl L - 2 - ( 5 , 6 - d i - 0 - a c e t y l -1,2-0_-isopr6pylidene-a-D-allo-f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (247) 3.6.3 S y n t h e s i s of Methyl L-2-(1,2,3, 156 5 , 6-penta-0_-acetyl-a , 6-D-allo-f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (250) 3.6.4 Attempted Nucleoside S y n t h e s i s 159 Using P e r a c y l Amino E s t e r 250. 3.6.4.1 Stannous C h l o r i d e 159 C a t a l y s e d S i l y l H i l b e r t -Johnson Procedure 3.6.4.2 Fusion Procedures. 160 S y n t h e s i s of 1,1'-Anhydro-2,3,5,6-tetra-O-acetyl-3-C-(R)-methoxycarbonylmethyl-1 ( R ) , 1 ' ( S ) - N - t r i f l u o r o -acetoepimo-6-D-allo-furanose (2527 3.6.5 S y n t h e s i s of 1-[2',3',5•,6'- 166 Tetra-0-acetyl-3'-C-((S)-N-t r i f l u o r o a c e t y l - c a r b o m e t h o x y -(amino)methyl)-B-D-allofuranosyl] thymine (257) ~ 3.6.6 Attempted Unblocking of l-[2',3', 170 5',6 ,-Tetra-0-acetyl-3 ,-C-((S) -N-trifluoroacety1-carbomethoxy-Tamino)methyl)-B-D-allofuranosyl] thymine (257)- ~ IV EXPERIMENTAL 1. General Methods 174 174 - x i -1.1 Chromatography 1.1.1 Column Chromatography 1.1.2 T h i n Layer Chromatography 1.1.3 Paper Chromatography 1.2 Nuclear Magnetic Resonance Spectro-scopy 2. S y n t h e s i s o f Branched-Chain Amino Sugars 3. S y n t h e s i s of Branched-Chain Nucleosides 4. S y n t h e s i s of Glycos-3-yl-a-Amino A c i d s an Analogues of the Nucleoside Moiety of P o l y o x i n J REFERENCES - x i i -LIST OF TABLES Table Page 1 Reaction of l-0_-Acetyl-2 , 3 , 5-tri-0_-benzoyl- 44 B-D-ribofuranose (110) with S i l y l a t e d U r a c i l s (109) i n the Presence of S n C l 4 a t 23°C. 2 S t r u c t u r e of the P o l y o x i n s (120). 50 3 A p p l i c a t i o n of the Cyanohydrin S y n t h e s i s 65 to 1,2:5,6-di-0-Isopropylidene-a-D-ribo-hexofuranos-3-ulose (2_5) i n the Presence of Methyl N i t r o a c e t a t e (MNA). 4 Comparison of P h y s i c a l Constants of Various 110 G l y c o s - 3 - y l Amino A c i d s . 5 Low F i e l d Resonances i n the 100 MHz Proton 132 Magnetic Resonance Sp e c t r a of the a, 3 -Unsaturated N i t r o E s t e r s 189, 190 and 235. 6 Chemical S h i f t s and C o u p l i n g Constants of 144 Protons of 5 , 6-di-0_-Acetyl-3-deoxy-l, 2-0_-is o p r o p y l i d e n e - 3 - C - [ ( R , S ) - n i t r o ( m e t h o x y -carbonyl) methyl ] - a - D - a l l o f uranose (241) . - x i i i -LIST OF FIGURES Figure Page 1A 270 MHz P.M.R. Spectrum o f 9-[3'-C- 80 (Carboxymethyl-2 1,3'-y-lactone)-3'-deoxy-6-D-ribofuranosyl]adenine (178) i n DMSO-d^. IB P a r t i a l 270 MHz P.M.R. Spectrum of 9-[3'-C- 81 (Carboxymethyl-2' , 3 '-y-lactone)-3 1-deoxy-B-D - r i b o f u r a n o s y l ] a d e n i n e (178) i n DMSO-d^ . 2A 270 MHz P.M.R. Spectrum of 9-[3'-C-Carboxy- 85 methyl-3 ' -deoxy - c t-D-ribofuranosyl] adenine (181) i n DMSO-d,. 6 2B P a r t i a l 270 MHz P.M.R. Spectrum of 9-[3'- 86 C-Carboxymethyl-3 ' -deoxy-a-p_-ribof uranosyl] -adenine (181) i n DMSO-dg. 3 P a r t i a l 100 MHz P.M.R. Spectrum o f 1,2:5,6- 100 di-0- I s o p r o p y l i d e n e - 3 - C - [ ( R , S ) - n i t r o ( m e t h o x y -carbonyl) methyl] - a - D - a l l o f u r a n o s e (162) i n CDC1 3. - x i v -Fi g u r e 4 60 MHz P.M.R. Spectrum of 3-0_-Acetyl-l, 2 : 5 ,6 di-0_-isopropylidene-3-C- [ (R,S) - n i t r o (methoxy c a r b o n y l ) m e t h y l ] - a - D - a l l o f u r a n o s e (165) i n CDC1 3. 5 100 MHz P.M.R. Spectrum o f Methyl N - a c e t y l -L-2- (1, 2-0_-isopropylidene-a-D-gluco and a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (202 and 209) i n CDC1 3. 6 100 MHz P.M.R. Spectrums of Methyl N - a c e t y l -D-2-(1,2-0-isopropylidene-a-p-gluco and a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (203 and 210) i n CDC1 3. 7 100 MHz P.M.R. Spectrum o f Methyl N - a c e t y l -L-2- (6-0_- a c e t y l - l , 2-0-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (223) i n DMS0-d 6. 8 P a r t i a l 100 MHz P.M.R. Spectrum o f 3-Deoxy-l , 2 : 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - 3 - C - [ ( R , S ) -n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] - a - g - a l l o -furanose (236) i n CDCl^. * - XV -F i g u r e Page 9 P a r t i a l 100 MHz P.M.R. Spectrum o f 5,6- 142 di-0_-Acetyl-3-deoxy-l, 2 - 0 - i o s p r o p y l i d e n e -3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-allofuranose (241) i n CDCl.^. 10 P a r t i a l 100 MHz P.M.R. Spectrum o f Methyl 153 JJ-2- ( 3 ,5 , 6 - t r i - 0 _ - a c e t y l - l , 2-0-i sopropy l i d e n e -a - D - a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (246) i n CDCl^. 11 P a r t i a l 100 MHz P.M.R. Spectrum o f Methyl 154 - (5 , 6 - d i - 0 - a c e t y l - l , 2-0_-i sopropy l i d e n e -a - D - a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (247) i n CDC1 3. 12 P a r t i a l 100 MHz P.M.R. Spectrum o f Methyl 158 L-2 - (1, 2 , 3 ,5 , 6-penta-0_-acetyl-a , 0-D-a l l o f uranos-3-yl). - N - t r i f l u o r o a c e t y l g l y c i n a t e (250) i n CDC1 3. 13 P a r t i a l 100 MHz P.M.R. Spectrum o f 1,1'- 162 Anhydro-2 ,3,5 ,6-tetra-0_-acetyl-3-C- (R) -methoxycarbonylmethyl-1(R),1'(S)-N-tri-fluoroacetoepimo-B-D-allofuranose (252) i n CDC1-,. - x v i -Figure Page 14 P a r t i a l 100 MHz P.M.R. Spectrum of 1- 168 [2 1 , 3 ' , 5 ' , 6 ' - T e t r a - O - a c e t y l - 3 1 - C - ( ( S ) -N - t r i f l u o r o a c e t y l - c a r b o m e t h o x y ( a m i n o ) -m e t h y l ) - 3 - D - a l l o f u r a n o s y l ] t h y m i n e (257) i n CDC1-.. - x v i i -ACKNOWLEDGEMENTS The author wishes t o express h i s g r a t i t u d e to Dr. A l e x Rosenthal f o r h i s encouragement and s k i l l f u l guidance throughout the course of t h i s work. My thanks are extended to Dr. L.D. H a l l , Dr. E. P i e r s , Dr. G.G.S. Dutton and the members of t h e i r r e s e a r c h groups f o r t h e i r many h e l p f u l suggestions d u r i n g my p e r i o d a t U.B.C. I would a l s o l i k e to thank the members of Dr. Rosenthal's r e s e a r c h group and i n p a r t i c u l a r Don Baker, C o l i n Richards, Murray R a t c l i f f e , Kent Dooley, and Robert Dodd, f o r t h e i r i n v a l u a b l e p r a c t i c a l a s s i s t a n c e d u r i n g our a s s o c i a t i o n . To Anne, f o r her encouragement, a s s i s t a n c e and p a t i e n t understanding d u r i n g the p r e p a r a t i o n of t h i s t h e s i s , I wish to express my deepest a p p r e c i a t i o n . F i n a l l y , the f i n a n c i a l support of the U n i v e r s i t y of B r i t i s h Columbia (1973-1978), the H.R. MacMillan Family S c h o l a r s h i p Fund (1973-1975), and the N a t i o n a l Research C o u n c i l of Canada (1972-1973, 1975-1978) i s acknowledged. - 1 -I. OBJECTIVE Since the e a r l y 1950's the i n t e r e s t i n s y n t h e s i z i n g branched-chain carbohydrates and n u c l e o s i d e s has markedly i n c r e a s e d owing to the f i n d i n g t h a t such m o d i f i c a t i o n s of n a t u r a l l y o c c u r r i n g compounds have r e s u l t e d i n i n t e r e s t -i n g changes i n t h e i r b i o l o g i c a l a c t i v i t y . In p a r t i c u l a r , a change of f u n c t i o n a l i t y or branching at C-3 p o s i t i o n i s observed i n many such n a t u r a l l y o c c u r r i n g carbohydrates. The work d e s c r i b e d h e r e i n i n v o l v e s the i n t r o d u c t i o n of s u b s t i t u e n t s at t h i s p o s i t i o n . The K i l i a n i - F i s c h e r cyanohydrin s y n t h e s i s has r e c e n t l y been a p p l i e d to 1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose to y i e l d e i t h e r the 3-C-cyano-l,2:5,6-di-0_-isopropylidene-a-D-gluco or a l l o f u r a n o s e predom-i n a n t l y , depending on the c o n d i t i o n s employed. Reduction o f the cyano group thus pro v i d e s a f a c i l e route to the c o r r e s -ponding 3-C-aminomethyl carbohydrates. The o b j e c t i v e of the f i r s t p a r t of t h i s work was: 1) to examine the i n f l u e n c e o f the pH o f the r e a c t i o n medium on the s t e r e o c h e m i s t r y of the products obtained; - 2 -2) to examine the degree of s t e r e o s e l e c t i v i t y o p e r a t i v e i n the p r o d u c t i o n of the epimeric cyanohydrins and; 3) to provide a p r o o f of s t r u c t u r e f o r the products obtained. In the second p a r t of t h i s work two o b j e c t i v e s were sought. The f i r s t o b j e c t i v e was the e f f i c i e n t s y n t h e s i s of l-0-acetyl-5-0_-benzoyl-3-C- (carboxymethyl) -3-deoxy-g-D-ribofuranose-2,3-y-lactone and the second was to use t h i s l a c t o n e to s y n t h e s i z e 3'-C-carboxymethyl a and 3-adenine n u c l e o s i d e s t h a t would serve as analogues of the a n t i -b i o t i c puromycin. A range o f commercially important g l y c o s y l amino a c i d n u c l e o s i d e s , known as the P o l y o x i n s , has r e c e n t l y been the c e n t r e of much s y n t h e t i c endeavour. These t h i r t e e n p o l y o x i n s a l l c o n t a i n an a-L_-amino a c i d r e s i d u e a t C-4' of a f u r a n o s y l p y r i m i d i n e and i t seemed to us t h a t i n c o r p o r a t i o n of the amino a c i d a t the C-3 1 p o s i t i o n , r a t h e r than the C-4' p o s i t i o n , might r e s u l t i n i n t e r e s t i n g b i o l o g i c a l consequences. The primary o b j e c t i v e o f the f i n a l p o r t i o n of t h i s work was, t h e r e f o r e , t w o - f o l d i n nature. The f i r s t g oal was the s y n t h e s i s of a f u l l y b l o c k e d d e r i v a t i v e of L - 2 - ( D - a l l o f u r a n o s - 3 - y l ) g l y c i n e and the second goal was to use such a compound f o r the s y n t h e s i s of 1-[(S)-3'-C-carboxy(amino)methyl-g-D-a l l o f u r a n o s y l ] t h y m i n e . Such a thymine d e r i v a t i v e would - 3 -serve as an analogue of the nucleoside moiety of the a n t i -b i o t i c p o l y o x i n J . Towards t h i s end, the condensation of methyl n i t r o a c e t a t e w i t h various 3-ketofuranoses was i n v e s t i g a t e d and as a r e s u l t the synthesis of 3-deoxy-3-C-nitro(carbomethoxy)methyl furanoses was a l s o i n v e s t i g a t e d . In order t o provide some background f o r subsequent d i s c u s s i o n s , a b r i e f summary w i l l be made of the synthesis of branched-chain sugars, g l y c o s y l amino acids and nucleosides. - 4 -II . INTRODUCTION 1. Branched-Chain Sugars The enormous i n t e r e s t i n branched-chain carbohydrates which has grown up i n the past twenty years i s l a r g e l y the r e s u l t of numerous r e p o r t s concerning the i s o l a t i o n of such compounds from n a t u r a l sources. Branched-chain 1 2 3 6 sugars ' have been i s o l a t e d from micro-organisms ' , 7 8 — 11 hi g h e r p l a n t s , as components o f c e l l w a l l p o l y s a c c h a r i d e s and i t has been suggested t h a t branched-chain sugars may 12 a l s o occur i n man . P a r t of the reason f o r the i n c r e a s e d i n t e r e s t i n the s y n t h e s i s of these compounds i s undoubtedly due to the f i n d i n g t h a t n u c l e o s i d e s with branched-chain sugars can e x h i b i t c y t o s t a t i c or v i r o s t a t i c a c t i v i t y o f 13-15 p o s s i b l e t h e r a p e u t i c value 16 Branched-chain sugars are d i v i d e d i n t o two types Type A sugars are d e f i n e d as those i n which a hydrogen atom has been s u b s t i t u t e d by a group R while Type B sugars are d e f i n e d as those i n which a hydroxyl group has been s u b s t i t u t e d . Type B compounds are a l s o d e f i n e d as "deoxy" sugars. The f i r s t two r e p o r t e d branched-chain sugars 17 18 19-20 D-apiose ' (1) and g-hamamelose (2) are r e p r e s e n -- 5 -t a t i v e of Type A sugars while L-garosamine (3) e x e m p l i f i e s a Type B sugar. 1.1 S y n t h e s i s o f Branched-Chain Sugars A very b r i e f summary of some of the r e a c t i o n s a p p l i e d to carbohydrates i n order to i n t r o d u c e branching i n t o the sugar s k e l e t o n w i l l be d i s c u s s e d below. There have been 1 2 2 3 s e v e r a l e x t e n s i v e reviews ' ' on t h i s s u b j e c t and t h e r e -fore o n l y the most recent advances w i l l be d i s c u s s e d . Many of the syntheses of branched-chain sugars i n v o l v e condensation r e a c t i o n s with keto-sugars and these w i l l be d i s c u s s e d i n s e c t i o n 3. The condensation r e a c t i o n s which bear d i r e c t l y on the work d e s c r i b e d i n t h i s t h e s i s w i l l a l s o be d i s c u s s e d i n more d e t a i l i n s e c t i o n 3. 1.1.1 S y n t h e s i s of Type A Sugars S e v e r a l methods have been employed to i n t r o d u c e branch-chains onto sugars while r e t a i n i n g a hydroxyl group a t the b r a n c h - p o i n t . - 6 -Diazomethane has been r e a c t e d with keto-sugars to form an epoxide 4_ which may then be opened wi t h a v a r i e t y of reagents. Thus the epoxide 4_ may be t r e a t e d w i t h l i t h i u m aluminum hydride to give a C-methyl sugars (5a); a l k a l i to give a C-hydroxymethyl sugar (5b); or ammonia to give a C-aminomethyl sugar (5_c) . In a s i m i l a r r e a c t i o n the dimethylsulfoxonium m e t h y l i d 26 27 was condensed w i t h the ketose 6_ ' to a f f o r d an epoxide which was subsequently reduced t o give the L-arabino product 7_ (R=CH3) . I n t e r e s t i n g l y , the r e a c t i o n o f d i a z o -methane with ketose 6_ a f f o r d e d , a f t e r l i t h i u m aluminum hydride r e d u c t i o n , the C-methyl sugar 8_ (R=CH3) which i s 31 the C-2 epimer of compound 7_ (R=CH3) Organolithium and organomagnesium compounds have been used e x t e n s i v e l y i n the s y n t h e s i s o f e p i m e r i c Type A sugars. Thus treatment o f the ketose 6_ wit h o r g a n o l i t h i u m reagents a f f o r d s the L - r i b o sugar 8*° whereas organo-magnesium reagents y i e l d predominantly the L-arabino , • .. -,27,29,30 d e r i v a t i v e s 7_ . 1.1.2 S y n t h e s i s o f Type B Sugars The a p p l i c a t i o n o f the W i t t i g r e a c t i o n to keto-sugars has g r e a t l y i n c r e a s e d the a v a i l a b i l i t y o f type B sugars and t h i s r e a c t i o n w i l l be reviewed s e p a r a t e l y i n s e c t i o n 3.2. N u c l e o p h i l i c a d d i t i o n to anhydro-sugars has a l s o found c o n s i d e r a b l e use i n the s y n t h e s i s of branched-chain 32 sugars . Reaction of compound 9_ w i t h a l k y l l i t h i u m reagents proceeded as expected w i t h t r a n s - d i a x i a l cleavage 33 of the epoxide r i n g to give o n l y the a l i o sugars 10_ S i m i l a r l y other carbohydrate o x i r a n e s have been opened with c y a n i d e 3 4 and d i e t h y l malonate carbanions 3 ^ . Improved methods f o r the s y n t h e s i s of unsaturated s u g a r s 3 8 - 4 4 have i n c r e a s e d the use of photochemistry - 8 -i n carbohydrate chemistry. The photoamidation of the unsaturated sugars 1_1, 1_2, and 1_3 to y i e l d carbamoyl branched-chain and ch a i n extended sugars 14-20 i s an example of the use of photochemistry to a f f o r d both Type 4 5 4 6 4 7 B ' and Type A sugars. A c O C H - 9 -2. Methods of Oxidation 2.1 Oxidation of Secondary Hydroxyl Groups The increase i n the number of synthetic branched-chain sugars has p a r a l l e l e d the development of more e f f e c t i v e and e f f i c i e n t methods of o x i d i z i n g secondary alcohols. In addition to the c l a s s i c a l methods of oxidation which employ platinum 4 8 49 oxide and oxygen , chromium trioxide-pyridine , or lead . . 50 tetraacetate-pyridine , two reagents have largely dominated - 10 -the f i e l d o f carbohydrate o x i d a t i o n s . These reagents are dimethyl s u l f o x i d e (DMSO) and ruthenium t e t r o x i d e (RuO^). The methods o f o x i d a t i o n i n carbohydrate chemistry 51 have been thoroughly reviewed by Butterworth and Hanessian and t h e r e f o r e o n l y the methods which were employed i n t h i s work (DMSO and RuO^) w i l l be d i s c u s s e d . 52 Since DMSO was f i r s t used as an o x i d i z i n g agent many v a r i a t i o n s have been s u c c e s s f u l l y employed i n which an a c t i v a t i n g e l e c t r o p h i l e (E) such as N, N - d i c y c l o h e x y l -53 54 55 carbod i i m i d e , a c e t i c anhydride o r phosphorus pentoxide i s used i n combination with DMSO. I t i s prop o s e d 5 ^ t h a t most DMSO o x i d a t i o n s i n v o l v e the formation o f a d i m e t h y l -a l k o x y s u l f o n i u m i n t e r m e d i a t e 21_ which i s then d i s p l a c e d by the a l c o h o l to form the s u l f onium i n t e r m e d i a t e 2_2_. The subsequent l o s s o f a proton c o l l a p s e s the in t e r m e d i a t e 22 to give a ketone 23_ and dimethyl s u l f i d e . The f i r s t a p p l i c a t i o n o f a ruthenium t e t r o x i d e o x i d a -t i o n to a carbohydrate was i n 1964 when Overend and 57 co-workers r e p o r t e d the o x i d a t i o n of a 1,2 :5 ,6-di-0_-i s o p r o p y l i d e n e - a - D - g l u c o f u r a n o s e (24) to give the keto-sugar 2_5, and s i n c e then i t has been proved to be a 58 59 powerful and u s e f u l reagent. Jones and co-workers ' have found t h a t i n s t e a d o f adding a molar e q u i v a l e n t o f the prepared t e t r o x i d e to the r e a c t i o n medium, the t e t r -oxide can be generated i n s i t u from a c a t a l y t i c amount of - 11 -C H 3 S = / C H 3 0 + C H 3 S C H 3 o=c / 1 \ Ri 21 C H 3 + S - 0 - E / RI O H - C H - R 2 C H , V H -R* C H , R 1 2 3 22 ruthenium d i o x i d e and sodium or potassium p e r i o d a t e . As the t e t r o x i d e i s consumed and converted back i n t o the d i o x i d e , more p e r i o d a t e i s added and the c y c l e i s continued - 12 -u n t i l a l l o f the a l c o h o l has been o x i d i z e d . T h i s i s the manner i n which ruthenium t e t r o x i d e o x i d a t i o n s are g e n e r a l l y performed. 3. Condensation Reactions I n v o l v i n g Keto and Aldehydo-Sugars The ready a v a i l a b i l i t y o f keto and aldehydo-sugars has l e d to a p p l i c a t i o n o f a myriad o f condensation r e a c t i o n s to the s y n t h e s i s o f both Type A and Type B sugars. A d i s -c u s s i o n of a l l such r e a c t i o n s i s beyond the scope o f t h i s work and o n l y a b r i e f review o f the p e r t i n e n t r e a c t i o n s w i l l be given. 3.1 Cyanide Condensations The use of cyanide i o n as a n u c l e o p h i l e has been most 61 w i d e l y a p p l i e d to aldoses and i s probably b e s t known as the i n i t i a l r e a c t i o n o f the K i l i a n i - F i s c h e r s y n t h e s i s i n which the carbon chain of an aldose i s lengthened by one carbon atom. The cyanohydrin s y n t h e s i s has not been used very much to prepare branched-chain sugars from k e t o s e s . 6 2 Tronchet and Bourgeois r e p o r t e d the s y n t h e s i s o f the a l i o - c y a n o h y d r i n 27 by the r e a c t i o n o f potassium cyanide w i t h 1 , 2 : 5 , 6 - d i - O - i s o p r o p y l i d i n e - a - D - r i b o -hexofuranos-3-ulose (25). The product was as s i g n e d the a l i o s t r u c t u r e i n the b e l i e f t h a t the cyanide i o n would a t t a c k the c a r b o n y l o f the ketose from the l e s s hindered - 13 -3 face. However, Bourgeois l a t e r r e p o r t e d t h a t e i t h e r the gluco or a l l o - c y a n o h y d r i n (26 and 27_, r e s p e c t i v e l y ) c o u l d be s y n t h e s i z e d e x c l u s i v e l y depending upon the con-d i t i o n s employed. I t was a l s o p o i n t e d out t h a t the cyano-h y d r i n o r i g i n a l l y d e s c r i b e d by Tronchet was the thermo-dynamically more s t a b l e product and t h e r e f o r e the assignment of i t s c o n f i g u r a t i o n on m e c h a n i s t i c grounds was not v a l i d . 2 6 R = OH; R 1 = CN 2 7 R = CNjR^OH The c o n f i g u r a t i o n o f e i t h e r cyanohydrin had not been e s t a b l i s h e d with any c e r t a i n t y u n t i l the completion o f the r e s e a r c h o u t l i n e d h e r e i n . 3.2 The W i t t i g Reaction V a r i o u s compounds have been shown^ to a f f o r d phosphonium y l i d s i f there e x i s t s a hydrogen atom adjacent to a phosphorus atom c a r r y i n g a reasonable degree of p o s i t i v e charge as i n the case o f phosphoranes (28_) , phosphine oxides (29) , phosphinates (30) , and phosphonates (3_1) . Of these, compounds 2 8 and 31 are the most commonly employed - 14 -- C e 0 R - P — C -k 1 0 R - P - C OR 1 0 R O - P — C — OR 1 2 8 2 9 30 31 phosphonium y l i d s . Phosphorones 2_8_ are used i n the c l a s s i c a l W i t t i g r e a c t i o n s while the phosphonates 3_1 are used i n the m o d i f i e d W i t t i g r e a c t i o n . Phosphoranes were used i n condensation r e a c t i o n s as 6 6 e a r l y as 1919 but i t was not u n t i l t h i r t y years l a t e r t h a t t h e i r use developed i n t o a w i d e l y a p p l i c a b l e s y n t h e t i c procedure. 6 7 In 1949 W i t t i g and Rieber t r e a t e d t e t r a m e t h y l -phosphonium i o d i d e 3_2 w i t h methyl l i t h i u m to give the methylenetrimethylphosphorane 33_ which was then r e a c t e d w i t h benzophenone to a f f o r d compound 3_4. In 195 3 W i t t i g 6 8 and G e i s s l e r r e p o r t e d the condensation of methylene-triphenylphosphorane with benzophenone to a f f o r d , i n high y i e l d , d i p h e n y l e t h y l e n e . From t h a t year the number o f a p p l i c a t i o n s o f the W i t t i g r e a c t i o n m u l t i p l i e d g r e a t l y 3.2.1 The C l a s s i c a l W i t t i g Reaction - 15 -and both the mechanism and s t e r e o c h e m i s t r y of the r e a c t i o n i 4 . - 4 . ,.65,69-74 were e x t e n s i v e l y i n v e s t i g a t e d e ( C H 3 ) 3 P - C H 3 I + C H 3 L i 32 -> ( C H 3 ) 3 P = C H 2 + Li I .+ C H 4 33 3 3 ( C 6 H 5 ) 2 C = 0 > ( C H 3 ) 3 P - C H 2 - C - ( C 6 H 5 ) 2 f O H 34 While the W i t t i g r e a c t i o n has been used e x t e n s i v e l y i n o ther areas i t s a p p l i c a t i o n i n the carbohydrate f i e l d has been r a t h e r l i m i t e d . Kochetkov and co-workers r e p o r t e d the r e a c t i o n of carbethoxymethylenetriphenylphosphorane with 7 5 76 a l d o s e s , having e i t h e r f r e e or blocked h y d r o x y l s , to a f f o r d a, B-unsaturated a l d o n i c acids.' The double bond was d i h y d r o x y l a t e d and the e s t e r reduced to y i e l d the expected 77 chain-extended aldoses . In s i m i l a r a p p l i c a t i o n s the W i t t i g r e a c t i o n has a l s o been used to prepare h i g h e r 78 79 8 0 ketoses , a l d o n i c a c i d s , deoxy sugars , and C-• A 81 g l y c o s i d e s . - 16 -C 0 2 E t C H O C H ( C H O A c ) 3 o r 4 + ( C g H 5 ) 3 P C ' H C 0 2 E t > C H C H 2 O A c ( ( f H O A c ) 3 o r 4 C H 2 O A c 3.2.2 The M o d i f i e d W i t t i g Reaction 8 2 In 1961, Wadworth and Emmons showed t h a t s t a b i l i z e d phosphonate carbanions were more r e a c t i v e than t r i a r y l -phosphoranes towards some aldehydes and ketones. In a d d i t i o n they r e q u i r e d m i l d e r c o n d i t i o n s , were l e s s expensive, and c o u l d be r e a d i l y worked up i n water s i n c e the r e s u l t a n t phosphate by-products were water s o l u b l e and d i d not cause the same problems i n product i s o l a t i o n t h a t t r i p h e n y l p h o s p h i n e oxide does i n the c l a s s i c a l W i t t i g r e a c t i o n . 8 3 With t h i s i n mind,Rosenthal and Nguyen condensed the y l i d formed by the r e a c t i o n o f potassium t-butoxide on carbomethoxymethyldimethylphosphonate with the keto -sugar 25_ to y i e l d , a f t e r hydrogenation, 3-C- (carbomethoxy-methyl) -3-deoxy-l ,2:5, 6 - d i - 0 _ - i s o p r o p y l i d e n e - a - D - a l l o f uranose (35). S i m i l a r i l y , r e a c t i o n o f the y l i d formed from diethylcyanomethylphosphonate w i t h compound 25_ a f f o r d e d , - 17 -T r O C H 2 T r O C H 2 4 0 a) R s H j R ^ O M e b) R=OMe;R 1 =H T r O C H 2 ° \ OMe C H 2 C N 4 2 - 18 -OA Q C a f t e r hydrogenation, the cyanomethyl sugar 36^  ' . Rosenthal, Catsoutacos, Richards, and C l i f f a p p l i e d the same r e a c t i o n s to the 2-deoxy-hexopyranos-3-ulose 3_7 and the 2-deoxy-pentofuranos-3-ulose 4_0 to a f f o r d the c o r r e s -8 6 ponding 3-C-(carbomethoxymethyl) sugar 38 and 3-C-(cyanomethyl) sugars 3 9 8 7 , 4 1 8 8 , and 4 2 8 8 . 3.3 Condensations I n v o l v i n g N i t r o Compounds of the Type  R-CH 2-N0 2 The condensation o f nitromethane and i t s d e r i v a t i v e s with aldehydo-sugars has long been i n v e s t i g a t e d . As e a r l y as 1894 the nitromethane s y n t h e s i s served as an a l t e r n a t i v e s y n t h e t i c method f o r p r e p a r i n g higher-carbon 8 9 aldoses by use of the Nef r e a c t i o n . A route to the 2-deoxyaldoses was pro v i d e d by a p p l y i n g the Nef r e a c t i o n to unsaturated n i t r o sugars which were a v a i l a b l e from the 90 a c e t y l a t e d n i t r o a l d i t o l s by way of the Schmidt-Rutz r e a c t i o n . Some years l a t e r the "sugar d i a l d e h y d e s " were e l e g a n t l y c y c l i z e d with nitromethane to a f f o r d d e o x y n i t r o , 91 sugar i n t e r m e d i a t e s The wide occurrence of amino sugars as c o n s t i t u e n t s 92 5 93 of a n t i b i o t i c s ' , and the d i s c o v e r y o f a n a t u r a l l y o c c u r r i n g n i t r o sugar ( e v e r n i t r o s e ) l e d to a number of i n v e s t i g a t i o n s of the r e a c t i o n of nitromethane with s u i t a b l y b l o c k e d 3-keto-sugars. - 19 -3.3.1 Condensations Involving Nitromethane and 3-Keto Sugars 94 In the l a t t e r part of 1969 Lourens reported the condensation of nitromethane with two pentofuranos-3-uloses. When ketose 4_3 was treated with a suspension of 1.2 equivalents of sodium hydride i n nitromethane the A c reaction yielded the ribo-nitromethyl sugar 4_4_. Proof of the ribo configuration was provided by reducing compound 44 over palladium-on-charcoal and acetylating the product to afford compound 4_5. The t e r t i a r y hydroxyl of compound 44 was acetylated and the product 4_6 was subjected to standard Schmidt-Rutz reaction conditions to afford the ni t r o o l e f i n 47_. The double bond of compound 47_ was then s e l e c t i v e l y reduced with sodium borohydride to y i e l d the 3-deoxy-3-C-nitromethy1-D-ribo-furanose 4 8. At approximately the same time Rosenthal, Ong and 94 95 Baker reported ' the condensation of nitromethane with - 20 -the hexofuranos-3-ulose 2_5. When a s o l u t i o n of one e q u i v a l e n t of sodium methoxide was added to a s o l u t i o n of the ketose 25 i n nitromethane the r e a c t i o n y i e l d e d 1,2 : 5 , 6-di-0_-i s o p r o p y l i d e n e - 3 - C - n i t r o m e t h y l - a - D - g l u c o f u r a n o s e (49) . The c o n f i g u r a t i o n a t C-3 was determined by s e l e c t i v e l y h y d r o l y z i n g the 5 , 6 - i s o p r o p y l i d e n e group, o x i d i z i n g the d i o l w i t h sodium p e r i o d a t e , r e d u c i n g the aldehyde with sodium borohydride and condensing the r e s u l t i n g product with acetone to g i v e 1,2 : 3 , 5-di-0_-isopropylidene-3-C-n i t r o m e t h y l - a - r j - xylofuranose (50) . In the p r e l i m i n a r y 94 communication the i n i t i a l condensation product was e r r o n e o u s l y a s s i g n e d the a l i o c o n f i g u r a t i o n (51) which was c o n s i s t e n t with the r e p o r t e d s t e r i c c o n t r o l e x e r t e d 9 6 by 1,2-0^-isopropylidene groups . I t was subsequently found t h a t a mixture of the gluco and a l i o compounds (4:1 o f 4_9 and 51) was formed i f the r e a c t i o n was c a r r i e d out i n 1,2-dimethoxyethane and sodium hydride was used as - 21 -the base. L a t e r , M o f f a t t and co-workers^' r e p o r t e d t h a t the a l i o compound 51_ was s e l e c t i v e l y produced by conden-s a t i o n o f nitromethane and ketose 2_5 i n anhydrous N,N-dimethylformamide u s i n g 0.1 e q u i v a l e n t s o f potassium t-b u t o x i d e . Dehydration o f compound 5_1 i n d i m e t h y l s u l f o x i d e and a c e t i c anhydride gave the n i t r o o l e f i n 5_2 which, when su b j e c t e d to base c a t a l y z e d h y d r a t i o n , y i e l d e d e x c l u s i v e l y the gluco isomer 49. - 22 -Yoshimura et a l . 3 0 provided confirmation of the structures of the epimeric nitromethyl sugars by examining whetheror not the corresponding 3-C-acetamidomethyl-l,2-0-isopropylidene-a-D-pentodialdofuranoses could form an aminal-ring between the aldehyde and acetamido group. Hydrogenation and acetylation of compounds 5_1 and 4_9_ gave the corresponding 3-C-acetamidomethyl derivatives 53 and 5_4, respectively. Hydrolysis of the 5,6-0_-isopropylidene groups of compounds 53_ and 5_4 and oxidation - 23 -of the products a f f o r d e d the p e n t o d i a l d o f u r a n o s e s 5_5 and 56, r e s p e c t i v e l y . The i . r . and p.m.r. s p e c t r a o f 5_5 c l e a r l y showed t h a t i t e x i s t e d as the aminal 5_5' while the s p e c t r a o f compound 5_6_ i n d i c a t e d the presence of an aldehyde. The c o n f i g u r a t i o n s of the g l u c o - n i t r o m e t h y l sugar £9_ and the a l i o - n i t r o m e t h y 1 sugar 51_ were t h e r e f o r e u n e q u i v o c a l l y proven. 3.3.2 Condensations I n v o l v i n g A l k y l N i t r o a c e t a t e s With Aldehydo Sugars The range of n i t r o compounds used i n condensations w i t h keto-and aldehydo-sugars has, u n t i l r e c e n t l y , been l i m i t e d to n i t r o a l k y l compounds. The use of a l k y l n i t r o -a c e t a t e s i n such condensations i s very l i m i t e d and has been r e s t r i c t e d to aldehydo-sugars. In 1969, Umezawa e t a l . " * " ^ c y c l i z e d D'-methoxy-d i g l y c o l a l d e h y d e (5_8) , prepared by p e r i o d a t e cleavage o f methyl B-J^-arabinopyranoside (5_7) , with e t h y l n i t r o a c e t a t e to a f f o r d a mixture of c y c l i c a - n i t r o e s t e r stereoisomers (59). - 24 -C a t a l y t i c reduction of the a-nitro esters, followed by chromatographic separation, gave four diastereomeric a-amino esters. Hydrolysis of each ester yielded the corresponding c y c l i c ( + )-a-amino acid (60-63) . The stereo-chemistry at C-2 and C-4 was determined by study of the n.m.r. spectra of the f u l l y acetylated derivatives and by chemical degradation. In an analogous series of reactions a-D-xylopyranoside afforded three enantiomeric c y c l i c (_)-a-amino acids. 6 0 and 61 6 2 6 3 In 1973 Kornilov and co-workers^"" condensed ethyl nitroacetate with isopropylidenated aldehydo-arabinose (64). When condensing agents such as triethylamine, piperidine, diethylamine, or butylamine were used the only product is o l a t e d was compound 6_6_. I t was proposed that such con-densations proceed through the intermediate n i t r o o l e f i n 65 to which another molecule of ethyl nitroacetate adds in a Michael-type reaction. The condensation of 6_4_ with ethyl nitroacetate i n 1:1 proportion proved to be possible - 25 -CHO C O ? E t C N O -E t O X C 0 2 E t 0?NCH CHNO? 2 \ / 1 64 65 .66 o n l y when ammonium a c e t a t e was used as the condensing agent i n an a q u e o u s - a l c o h o l i c medium. Under such c o n d i t i o n s the n i t r o adduct 67 was produced in.50% y i e l d . The a c e t y l a t i o n C 0 2 E t C H N 0 2 CHOH 0 C 0 2 E t A c O N=C C H O A c x: H — — 0 0 — 0 — — H 0 — 11 H 6 9 6 7 6 8 - 26 -of compound 6_7 with acetic anhydride i n pyridine produced the n i t r o l i c ester 6_8_ whereas, i n the presence of c a t a l y t i c amounts of sulphuric acid,the n i t r o group remained unaffected and the acetate 6_9_ was the sole product. 4. Nucleosides 4.1 Branched-Chain Nucleosides Branched-chain nucleoside i s the term commonly used to describe a compound i n which a nitrogen heterocycle, usually a purine or pyrimidine, i s linked through nitrogen to the anomeric position of a carbohydrate which contains branching i n i t s carbon skeleton. 102 In 1954 i t was demonstrated by Baker and co-workers that 6-dimethylamino-9-(3'-amino-3'-deoxy-3-D-ribo-furanosyl)purine (JO), obtained from the hydrolysis of puromycin, had b i o l o g i c a l a c t i v i t y that d i f f e r e d q u a l i t a -t i v e l y and quantitatively from that of the a n t i b i o t i c i t s e l f . This finding stimulated the synthesis of many other nucleosides modified at C-3' and other positions of the carbohydrate moiety and a number of these have been , . - . n • • x . 103,104 investigated for b i o l o g i c a l a c t i v i t y Excellent examples of branched-chain nucleosides possessing b i o l o g i c a l a c t i v i t y are the 2'-C and 31-C 105 methyl nucleosides (71-73) which, have shown i n h i b i t i o n of KB cells"*" 0^ i n culture and are e f f e c t i v e a n t i v a c i n i a 107 agents i n mice . The nitromethyl branched-cham nucleo-- 27 -NMe-HOCH HOCH OH OH 71 R = H , R ^ M e 72 R - M e . R ^ H HOCH-2 y ^ ^  , <^ Q HOCH CH2N02 74 CH \ lo2  N^ 75 - 28 -s i d e s 7_4 and T5_ have r e c e n t l y been s y n t h e s i z e d and shown 108 to be a c t i v e a g a i n s t KB tumor c e l l s Many of the branched-chain n u c l e o s i d e s which possess b i o l o g i c a l a c t i v i t y c o n t a i n peptide l i n k a g e s and/or amino a c i d f u n c t i o n a l i t i e s attached to the carbohydrate moiety. These compounds and t h e i r s y n t h e s i s w i l l be d i s c u s s e d i n more d e t a i l i n s e c t i o n 4.3. 4.2 Nucleoside S y n t h e s i s There have been s e v e r a l reviews of the s y n t h e t i c m e t h o d s 1 ^ ' ^ ® used i n t h i s g r e a t l y expanded f i e l d of carbohydrate chemistry and an e x c e l l e n t review1''"''" of the mechanisms of these r e a c t i o n s has r e c e n t l y appeared. A d i s c u s s i o n o f a l l . t h e v a r i o u s methods i s beyond the scope o f t h i s t h e s i s and t h e r e f o r e o n l y the procedures used i n the s y n t h e s i s of compounds i n the experimental s e c t i o n , and the background to these procedures, w i l l be d e a l t w i t h here. 4.2.1 F u s i o n Reaction 112 The f i r s t a p p l i c a t i o n o f H e l f e r i c h ' s f u s i o n r e a c t i o n to the p r e p a r a t i o n o f purine n u c l e o s i d e s was r e p o r t e d by 113 Sato e t a l . i n 1960. In t h i s r e a c t i o n t e t r a - 0 _ - a c e t y l -B-g-ribofuranose was fused w i t h v a r i o u s p u r i n e s i n the presence of a c a t a l y t i c amount of p_-toluenesulfonic a c i d under vacuum f o r 10-20 minutes. - 29 -Since these e a r l y r e p o r t s a number o f a c i d s or Lewis . c i * . i n c l u d i n , E - t o ^ e n e s u l p h o n i c a c i d " \ T i C l ^ ,and d i c h l o r o a c e t i c a c i d , have been found to be e f f e c t i v e c a t a l y s t s f o r f u s i o n r e a c t i o n s , while I s h i d o 117 118 e t a_l. ' have demonstrated t h a t with c e r t a i n p u rines V no a c i d c a t a l y s t i s r e q u i r e d . In p r e s e n t i n g a u n i f i e d mechanism f o r the f u s i o n of p e r a c e t y l a t e d sugars with p u r i n e bases, with or without le good i 117,118 c a t a l y s t s , Watanabe, H o l l e n b e r g , and F o x 1 1 1 mad use o f the e l e g a n t s t u d i e s of I s h i d o , Hosono e t a l . A f t e r i n t e r p r e t i n g the k i n e t i c data on the e x c l u s i v e formation of 9 - B - D - r i b o f u r a n o s y l - 2 , 6 - d i c h l o r o p u r i n e (78) from the f u s i o n of 2 , 6 - d i c h l o r o p u r i n e (76) with g-D-ribo-furanose t e t r a c e t a t e (77), without c a t a l y s t , Hosono e t 118 a l . proposed the f o l l o w i n g mechanism: - 30 -I t had p r e v i o u s l y been noted"1""1"' t h a t the a c i d i t y o f the purine was a l s o a f a c t o r i n n o n - c a t a l y t i c f u s i o n r e a c t i o n s 118 and i t was suggested t h a t the mechanism of the c a t a l y z e d and non-catalyzed r e a c t i o n s were s i m i l a r except t h a t "usual purines may be a c t i v a t e d by i n t e r a c t i o n w i t h a c i d i c c a t a l y s t s to r e a c t w i t h f u l l y a c e t y l a t e d sugars, and the co r r e s p o n d i n g n u c l e o s i d e s may be produced accompanied by an e l e c t r o n t r a n s f e r such as d e p i c t e d i n the scheme" (see mechanism 77-78) . Watanabe e t a l . 1 " ' " 1 expanded on t h i s and proposed a mechanism s i m i l a r to the t r a n s - g l y c o s y l a t i o n mechanism described1''""'" f o r the heavy-metal procedure w i t h p u r i n e s . - 31 -In the case of a Lewis a c i d c a t a l y s t , such as Z n C ^ r the mechanism proposed i n v o l v e s formation of a molecular complex between the purine and the c a t a l y s t w i t h the Lewis a c i d a ttached to N-3 (7_9) . T h i s complex would be i n e q u i l i b r i u m with an e l e c t r o n i c a l l y imbalanced " a c t i v a t e d " form o f the purine (80) . Intermediate 8_0 then a t t a c k s the a c e t y l a t e d sugar c a t i o n to form the N-9 n u c l e o s i d e (8_2) with accompany-i n g r e g e n e r a t i o n o f the c a t a l y s t . In the case o f a c i d c a t a l y s t s , t h e a c i d protonates the p u r i n e a t N-3 to produce a purine c a t i o n analogous to 7_9. The conjugate base of the a c i d then a b s t r a c t s the N-9 proton to form an " a c t i v a t e d " purine ( l i k e 80) which i s converted to the N-9 n u c l e o s i d e 8_2 with l i b e r a t i o n of the a c i d c a t a l y s t . 113 119 From the e a r l i e s t r e p o r t s ' the formation of anomeric mixtures was observed i n f u s i o n r e a c t i o n s . An i l l u s t r a t i v e example of the s t e r e o s e l e c t i v i t y of the r e a c t i o n , w i t h and without c a t a l y s t , i s , t h e f u s i o n of 2 , 6 - d i c h l o r o -purine (7_6_) w i t h a , 3 - r i b o f u r a n o s y l t e t r a a c e t a t e (8_5 and 118 77). Hosono e t a l . r e p o r t e d the f o l l o w i n g p e r t i n e n t o b s e r v a t i o n s : a) without c a t a l y s t the 3 - r i b o f u r a n o s y l t e t r a a c e t a t e 77 y i e l d e d the N-9 - 3-nucleoside e x c l u s i v e l y , whereas the a - r i b o f u r a n o s y l t e t r a a c e t a t e 8_5 a f f o r d e d an anomeric mixture (a,3=2:3). - 32 -b) with an a c i d c a t a l y s t , f u s i o n o f the B-sugar 77_ with 76_ f o r 1 minute gave o n l y g-nucleoside, whereas f u s i o n f o r 10 minutes y i e l d e d an a,B mixture. c) treatment of the pure $-nucleoside w i t h an a c i d c a t a l y s t a t ^150° f o r 10 minutes produced an anomeric mixture. Watanabe r a t i o n a l i z e d these data by proposing the u n i f i e d mechanism d e p i c t e d below: 0 II 85 8 6 8 7 - 33 -In the auto-catalytic fusion reaction of the B-acetate 77, attack of the C-l acetoxy group on the purine proton would be anchimerically assisted by the 2-acetoxy group to produce a 1,2-acyloxonium ion 83_ which would be attacked by the purine anion to y i e l d the 8-nucleoside 84. In the case of the a-sugar 85,assistance would come from the lone pair of electrons of the r i n g oxygen to produce the carboxonium ion 8_6. Such a carboxonium could rearrange to the c y c l i c acyloxonium ion 83_ or react d i r e c t l y with the purine anion r e s u l t i n g i n a mixture of anomers (84 and 8_7) . The conversion of the B-nucleoside to the a-nucleoside under fusion conditions i n the presence of an acid catalyst i s explained by assuming that the protonated nucleoside 84_ undergoes a reversal of the process to give sugar ions 8_3 and 86_ and an "activated" purine analogous to compound 80. 4.2.2 The Hilbert-Johnson and S i l y l Procedures 120 In 1930, H i l b e r t and Johnson reacted 2,4-dimethoxy-pyrimidine (8_8, R=CH^) with methyl iodide at room temperature to afford, a f t e r treatment with acid, 1-methyluracil. Later the same year, the scope of the reaction was extended by reaction of acetobromoglucose (8_9) with 2,4-diethoxy-pyrimidine (_88, R=C2Hj.) to give the f u l l y blocked nucleoside 9 0 1 2 1 . - 34 -OR N RO^kN 88 + RiO Q Br RiO The mechanism of the H i l b e r t - J o h n s o n r e a c t i o n i s g e n e r a l l y d e p i c t e d as f o l l o w s : RO-^N 91 92 93 The s t e r e o s e l e c t i v i t y o f t h i s r e a c t i o n i s analogous to t h a t o f the aforementioned f u s i o n r e a c t i o n i n t h a t even halogenoses c o n t a i n i n g a 2-acyloxy s u b s t i t u e n t produce anomeric mixtures o f n u c l e o s i d e s . One of the mechanisms - 35 -proposed suggested t h a t s i n c e most o f the r e p o r t e d r e a c t i o n s i n v o l v e d anomeric mixtures of tr i - 0 _ - a c e t y l - D - p e n t o f u r a n o s y l h a l i d e s the products arose from a simple i n t e r m o l e c u l a r S N 2 displacement of the h a l i d e on C - l by the N-1 of the base. This mechanism f a i l s , however, to e x p l a i n the pro-d u c t i o n of anomeric mixtures when o n l y one anomeric h a l i d e , 122,123 i s employed. I t i s now suggested"*"''""'" t h a t a mechanism analogous to t h a t presented f o r the f u s i o n r e a c t i o n i s o p e r a t i v e . In such a case "the r e l a t i v e c o n t r i b u t i o n of carboxonium i o n 98_ to the o v e r a l l r e a c t i o n i n e f f e c t would determine the r e l a t i v e amount of 1',2'-cis n u c l e o s i d e i n the product". When the acyloxonium i o n 9_5 i s excluded from the mechanism, - 36 -e i t h e r by the use o f 2-deoxy halogenoses or by b l o c k i n g of the C-2 hydroxyl with a f u n c t i o n a l i t y i n c a p a b l e of p a r t i c i p a t i o n , then one might expect a and 6 n u c l e o s i d e s to be produced i n approximately equal amounts. Quite o f t e n , however, t h i s i s not the case and one isomer i s formed predominantly. Some o f the t h e o r i e s o f f e r e d to e x p l a i n such r e s u l t s have i n c l u d e d : a) the p a r t i c i p a t i o n of an e x o c y c l i c 5-0_-acyl s u b s t i t u e n t to form a 1 , 5 - c y c l i c acyloxonium i o n 123 i n t e r m e d i a t e (100) b) s t e r i c hinderance by 5-acyloxy s u b s t i t u e n t s . I t 122 123 was concluded ' t h a t t h i s " s t e r i c e f f e c t " i s more pronounced than the aforementioned " p a r t i c i p a t i o n e f f e c t " . and c) formation of a c l o s e i o n p a i r 1 1 1 (eg. 101) r e s u l t i n g i n a t t a c k of the n u c l e o p h i l e from the s i d e o p p o s i t e to where the counter i o n i s l o c a t e d . 100 R = C H 3 , Cl ,N0 2 101 R = ( C 6 H 5 ) C H 2 - 37 -The substitution of a tr i m e t h y l s i l y l o x y function-a l i t y ^ " ^ ^"^ i n place of the alkoxy group on the pyrimidine offered several advantages. F i r s t l y , the lower e l e c t r o -negativity of s i l i c o n makes i t more susceptible to nucleophilic attack and displacement and, secondly, the electron-releasing nature of the t r i m e t h y l s i l y l o x y group increases the n u c l e o p h i l i c i t y of the corresponding pyrimidine. The mechanism of nucleoside formation i n the s i l y l modification i s sim i l a r to that of the Hilbert-Johnson procedure (91->-93) except that the leaving group (R) i n 92 i s a t r i m e t h y l s i l y l group instead of an a l k y l group and the rate determining step i s probably formation of the C-N bond"'""''"'" instead of de-O-alkylation. The stereochemistry of the s i l y l procedure also p a r a l l e l s that of the Hilbert-Johnson reaction i n that mixtures of anomeric nucleosides are obtained even when 12 6 X 3 3 halogenoses bearing a 2-acyloxy function are employed ' (see 94_ and 97 -> 96 and 9_9) . The application of the s i l y l modification to the synthesis of purine nucleosides bears many s i m i l a r i t i e s to the fusion reactions discussed previously. In t h i s case the i n i t i a l product i s an N-3 glycosyl derivative 103 which loses a t r i m e t h y l s i l y l group to form an e l e c t r o n i c a l l y imbalanced speciejs 104 analogous to intermediate 8_0. Compound 104 then undergoes an N-3 -> N-9 transglycosylation to afford the f i n a l nucleoside - 38 -product 105. Mixtures o f anomeric n u c l e o s i d e s are sometimes 129 obtained f o r the same reasons d i s c u s s e d f o r the f u s i o n and H i l b e r t - J o h n s o n r e a c t i o n s . 4.2.3 C a t a l y z e d H i l b e r t - J o h n s o n and S i l y l Procedures A number of c a t a l y s t s have been used w i t h the H i l b e r t -Johnson and s i l y l procedures i n attempts to s y n t h e s i z e n u c l e o s i d e s i n higher y i e l d s u s i n g m i l d e r c o n d i t i o n s . To - 39 -date the catalysts have been of three general types - mercury s a l t s , F riedel-Crafts catalysts, and derivatives of perfluoroalkane sulfonic and perchloric acids - and each w i l l be discussed separately below. 4.2.3.1 Mercury Salt Catalysts 122 The addition of mercury s a l t s to Hilbert-Johnson ' 130,131 , .. , , 127,128 .. . . and s i l y l procedure reactions not only increases the rate of reaction but also influences the stereochemistry of the products. Watanabe, Hollenberg, and Fox p roposed 1 1 1 a two-fo l d role for HgBr 2 i n Hilbert-Johnson reactions. F i r s t l y , that the mercury a s s i s t s i n the formation of the 1,2-acyloxonium ion (e.g. 95) from halogenoses bearing an a-2-acyloxy group thus favouring the formation of B-n u c l e o s i d e s 1 3 ^ and increasing the rate of reaction. In the case of 2-deoxy sugar halides (106), HgBr 2 a s s i s t s in the formation of the carboxonium ion (107), thus increasing the rate of reaction and giving a/B product 122 132 r a t i o s closer to one ' . Secondly, they state, "the intermediate quaternary s a l t formed i n the course of the reaction could complex with HgX2 (structure 108) which would withdraw i r electrons from the pyrimidine r i n g and enhance the cleavage of the 2-alkoxyalkyl group by halide ion". With mercury s a l t s [i.e. HgBr 2, HgCl 2, Hg(OAc) 2 or 127 128 133 HgO] ' ' the mechanism of the s i l y l procedure i s proposed to be very similar to that of the Hilbert-Johnson - 40 -- 41 -4.2.3.2 F r i e d e l - C r a f t s C a t a l y s t s 134 In 1970, N i e d b a l l a and Vorbruggen showed t h a t treatment of 2 , 4 - b i s - ( t r i m e t h y l s i l y l o x y ) p y r i m i d i n e s w i t h p e r - a c e t y l a t e d sugars i n the presence of F r i e d e l - C r a f t s c a t a l y s t s a f f o r d e d N-1 p y r i m i d i n e n u c l e o s i d e s i n good y i e l d s . Comparable r e s u l t s were o b t a i n e d w i t h a v a r i e t y of c a t a l y s t s i n c l u d i n g S n C l 4 , Z n C l 2 , T i C l ^ , BF^-ether, and A l C l ^ i n 1,2-dichloroethane, a c e t o n i t r i l e , benzene, carbon-d i s u l f i d e , or dimethylformamide. For reasons of experimental 135-140 s i m p l i c i t y n e a r l y a l l of the subsequent r e p o r t s i n v o l v e d the use o f 0.25 to 1.5 e q u i v a l e n t s of SnCl^ i n 1,2-dichloromethane and/or a c e t o n i t r i l e . T h i s r e a c t i o n 134-140 o f f e r s the advantage t h a t l-0_-acetyl sugars , and 134 135 i n a few cases l-0_-methyl sugars ' , may be used d i r e c t l y i n s t e a d of c o n v e r t i n g them i n t o t h e i r more r e a c t i v e and u n s t a b l e sugar h a l i d e s . 135 In the case of a c y l a t e d 2-deoxy sugars and sugars 135 without a p a r t i c i p a t i n g group on C-2 a n e a r l y constant product r a t i o of a / B = l was o b t a i n e d which was not i n f l u e n c e d 141 by v a r i a t i o n of the r e a c t i o n c o n d i t i o n s The r e a c t i o n s with sugars b e a r i n g a 2a-acyloxy group show a high degree of s t e r e o s e l e c t i v i t y , w i t h r e s p e c t to the sugar, i n t h a t o n l y 6 - g l y c o s y l . d e r i v a t i v e s are o b t a i n e d . 142 (Some p r o d u c t i o n of o-anomers has been observed when very small amounts o f SnCl^ are used.) With r e s p e c t to - 42 -0 B z O - i OAc = S - O A c S n C l 4 ClCH 2 CH 2 Cl or CH 3 CN OSiMe N R; M e 3 S i O ^ N ^ R 1 109 a) r H = R 2 b) R 1 = H ; R 2 =N0 2 c) R 1 = H ,. R2=OMe d) R 1 = H ; R 2 = l Q > e) R 1 = C H 3 = R 2 f) R i = C H 3 ; R 2 = C H ( C H 3 ) 2 g) R1 = C H 3 ; R 2 = N 0 2 HN' y R 2 s 111 0 ° ^ N ^ R l I 5 113 - 43 -the base, a complex combination of e l e c t r o n i c and s t e r i c factors determine the rate of the reaction and the type of product obtained. Thus, depending on the pyrimidine deriva-t i v e used, one may obtain N-1 nucleosides (111), N-3 nucleosides (112) , or N-1, N-3-bis-nucleosides (113) . 14 0 The study of Niedballa and Vorbruggen on the influence of 5 and 6 substituents i n s i l y l a t e d u r a c i l s (109) i s an i l l u s t r a t i v e example. The results of the reactions are summarized i n Table I. The findings seem to support the contention of the authors that the SnCl^-catalyzed 143 s i l y l Hilbert-Johnson reaction between the hard sugar 14 3 cation and the hard nitrogen of the s i l y l a t e d base i s similar to Priedel-Crafts acylations and that the formation of stable donor-acceptor complexes between the ca t a l y s t and 143 base prevent or impede the acylation by hard acyl cations. Thus, electron-donating substituents on C-5 of the base such as OCH^ (109c) or NR^ (109d), r e s u l t i n formation of stable 1:1 donor-acceptor or TT complexes analogous to complex 108 so that these bases only react with compound 110 i f additional SnCl. i s added to e f f e c t the formation 4 of sugar cations. Even with an excess of c a t a l y s t the reaction rates of 109c and 109d are much slower than that of 5 - n i t r o u r a c i l (109b) where no complex formation seems to occur. Such complex formation also seems to favour the formation of the apparently k i n e t i c a l l y and (in the presence of a 6 substituent) s t e r i c a l l y favoured N-3 product 112 - 44 -TABLE 1. Reaction of 1 - 0 - A c e t y l - 2 ^ 5 - t r i - 0 - b e n z o y l-n - D - r i b o f u r a n o s e (1J0) w i t h Silylated U r a c i l s (109) in the Presence of S n C l , at 2 3 ° C . Uracil (silylated) SnCLj; equiv. a b Time, h a b N a 1°/o b N 3 a % b N i , N 3 % a b Uracil .7 1.4 .7 12 1 12 84 73 89 9 11 4 tr. 7 tr. 6-Azauracil .2 1.4 .2 4 1 12 97 90 93 2 1 3 5-Nitrouracil .2 .2 0.1 0.1 97 98 5-Methoxy uracil 1.4 .9 2.5 12 53 90 27 3 13 tr. 5-Morpholinouracil 1.4 1.4 2 1 39 53 18 32 42 12 5-Isopropyl-6-methyl-uracil 1.2 1.2 72 24 18 53 41 25 5,6-Dimethyluracil 1.1 1.1 6 18 10 66 60 17 6-Methy l-5-nitrouraci I 9 9 09 0 6 84 73 14 19 a In 1,2-dichloroethane. In a c e t o n i t r i l e . Data from r e f e r e n c e s 136 and 140. and the N-1, N-3 product 113. Conversely, when complex formation i s i n h i b i t e d e l e c t r o n i c a l l y (109b and g ) , s t e r i c a l l y (compare 109f and 109e), or by the use of a more p o l a r s o l v e n t ( a c e t o n i t r i l e ) then i n most cases the N-1/ N-3 product r a t i o i s i n c r e a s e d . - 45 -In 1974, L i c h t e n t h a l e r e t a_l. used S n C l 4 to c a t a l y z e the s y n t h e s i s of a number o f N -benzoyl-adenine 6 -n u c l e o s i d e s from p e r - a c e t y l a t e d sugars and N^-benzoyl-N , 9 - b i s ( t r i m e t h y l s i l y l ) a d e n i n e (114). H N B z The r e a c t i o n o f f e r s c e r t a i n advantages over the methods p r e v i o u s l y d i s c u s s e d . F i r s t l y , the method i s a p p l i c a b l e to a wide range of purine d e r i v a t i v e s . In the non-117 113 c a t a l y t i c f u s i o n ' method (see s e c t i o n 4.2.1) onl y " a c i d i c " p u r i n e s , such as 6-chloro, 2 , 6 - d i c h l o r o , 2,6,8-t r i c h l o r o , and 6-cyano-purine, gave n u c l e o s i d e products, whereas 6-methoxypurine (115; R^=Me, R2 = H^ n o t ' T n e use of the SnCl^ method wi t h the corresponding s i l y l d e r i v a t i v e (115; R 1=R2=SiMe 3) and p e r - a c y l sugar 110 146 a f f o r d e d t r i b e n z o y l i n o s i n e (116) i n a 71% y i e l d . Secondly, t h i s method shows a h i g h degree of s t e r e o -s e l e c t i v i t y f a v o u r i n g the formation of 3-nucleosides. 118 In c a t a l y z e d f u s i o n r e a c t i o n s and T i C l . c a t a l y z e d con-- 46 -B z O O B z 14 7-14 9 densations of c h l o r o m e r c u r i - p u r i n e s the product i s o f t e n a mixture o f a,6 anomers. The s t e r e o c h e m i s t r y o f the r e a c t i o n i s probably c o n t r o l l e d by the 2-a-acyloxy group v i a a mechanism analogous to the a u t o - c a t a l y z e d f u s i o n r e a c t i o n ( s e c t i o n 4.2.1) and the mercury s a l t c a t a l y z e d H i l b e r t - J o h n s o n procedure ( s e c t i o n 4 . 2 . 3 . 1 ) . 4.2.3.3 P e r f l u o r o a l k a n e S u l f o n a t e and P e r c h l o r a t e C a t a l y s t s In 1975, Vorbriiggen and K r o l i k i e w i c z r e p o r t e d 1 " ^ the s y n t h e s i s of a number of 6 - n u c l e o s i d e s by r e a c t i o n of 1,2-dichloroethane s o l u t i o n s of' p e r - a c y l r i b o f u r a n o s e 110 with s i l y l a t e d p y r i m i d i n e s and p u r i n e s i n the presence o f t r i m e t h y l s i l y l p e r c h l o r a t e or t r i m e t h y l s i l y l t r i f l u o r o -methane s u l f o n a t e . The most important f e a t u r e o f these r e a c t i o n s , as compared to the analogous SnCl^ c a t a l y z e d r e a c t i o n s , i s the s t r i k i n g absence o f N-3 and N-1, N-3 i . . . , . 136,140,151 _ . .. n u c l e o s i d e by-products . S i m i l a r success has 15 2 15 3 been achieved ' u s i n g 2 , 3 , 5-tri - 0 _ - b e n z o y l - D - r i b o -- 47 -f u r a n o s y l bromide and s i l v e r t r i f l u o r o m e t h y l s u l f o n a t e ( s i l v e r t r i f l a t e ) . 154 Very r e c e n t l y , Vorbruggen and Bennua have r e p o r t e d a procedure f o r the i n s i t u g e n e r a t i o n of both the s i l y l a t e d h e t e r o c y c l e and c a t a l y s t to e f f e c t n u c l e o s i d e formation i n one s t e p . As an example of the procedure, u r a c i l was r e a c t e d w i t h 1.0 e q u i v a l e n t . o f a c y l sugar 110 and 2.4 e q u i v a l e n t s o f potassium n o n a f l a t e i n a b s o l u t e a c e t o n i t r i l e i n the presence of 3.1 e q u i v a l e n t s of t r i m e t h y l c h l o r o s i l a n e (TCS) and 0.7 e q u i v a l e n t s o f h e x a m e t h y l d i s i l a z a n e (HMDS) to a f f o r d N-1-(2',3',5'-tri-O-benzoyl-g-D-ribofuranosyl) u r i d i n e (111a) i n 84% y i e l d . Comparable r e s u l t s were obt a i n e d i n analogous r e a c t i o n s u s i n g f r e e t r i f l u o r o m e t h a n e s u l f o n i c a c i d o r S n C l 4 as c a t a l y s t ; (81% and 83% y i e l d s , - 48 -r e s p e c t i v e l y , were a c h i e v e d ) . T h i s compares very f a v o u r a b l y w i t h the r e s u l t s shown i n Table I. 4.3 P e p t i d y l N u c l e o s i d e s Besides the i n t e r e s t t h a t has been shown i n branched-c h a i n sugars i n g e n e r a l , there has been p a r t i c u l a r i n t e r e s t shown i n amino sugars. Amino sugars occur as the b a s i c 155 c o n s t i t u e n t s o f many a n t i b i o t i c s and many o f these n u c l e o s i d e a n t i b i o t i c s c o n t a i n amino a c i d f u n c t i o n a l i t i e s and/or peptide l i n k a g e s attached to the sugar. B l a s t i c i d i n S (117), gougerotin (118), and puromycin (119) are examples of such p e p t i d y l n u c l e o s i d e s . NH< H 2 NC0 O C - N I H OH HC-CHoOH fslH 0=0 C H 2 N H C H 3 0 HOoC N -a N' C-NH CH2 CHNH2 CH2 NH CH2-^-C-NH2 CH, NMe-r^H OH 0 = 0 :H2N-CH CH OCH: 118 1 1 7 119 - 49 -A new group of p e p t i d y l n u c l e o s i d e s t h a t are a n t i -fungal i n t h e i r a c t i o n i s the p o l y o x i n s . 4.3.1 The P o l y o x i n s The r e s u l t o f a s c r e e n i n g program f o r a n t i b i o t i c s a c t i v e a g a i n s t a photopathogenic fungus, P e l l i c u l a r i a f i l a m e n t o s a f . s a s a k i i , which causes s h e a t h b l i g h t i n r i c e p l a n t s , was the recovery o f a p o l y o x i n complex from the c u l t u r e b roths o f three s t r a i n s o f Streptomyces be l o n g i n g to S. c a c a o i ^ ^ 1 6 0 ^ T q ^a^e t h i r t e e n p o l y o x i n s (designated A to M) have been i s o l a t e d and c h a r a c t e r i z e d . The s t r u c t u r e s o f the p o l y o x i n s , as e l u c i d a t e d by 1 6 1 — 1 6 4 Isono et a_l. , are d e p i c t e d i n Table 2. Each of the p o l y o x i n s c o n t a i n s one of four p o s s i b l e (S)-amino urono n u c l e o s i d e s - each n u c l e o s i d e d i f f e r i n g o n l y i n the nature of i t s p y r i m i d i n e base. Each of the p o l y o x i n s , except p o l y o x i n C, c o n t a i n s one or two of three p o s s i b l e L-amino a c i d s which are desi g n a t e d p o l y o x i m i c a c i d ( 1 2 1 ) , carbamoylpolyoxamic a c i d ( 1 2 2 ) and carbamoyldeoxypoly-oxamic a c i d ( 1 2 3 ) . The p o l y o x i n s show h i g h l y s p e c i f i c a c t i v i t y a g a i n s t v a. • * . 1 5 6 , 1 5 8 , 1 6 5 , 1 6 6 , ., . ., . . . photopathogenic f u n g i while e x h i b i t i n g no t o x i c i t y towards b a c t e r i a , food crops, f i s h , or l a b o r a t o r y , 1 5 6 , 1 5 8 R . , , . . 1 6 7 _ . animals . A r e c e n t study has a l s o shown t h a t p o l y o x i n A i s a more e f f e c t i v e i n h i b i t o r o f tobacco mosaic v i r u s than b l a s t i c i d i n S. - 50 -T A B L E 2. Structure of the Polyoxins ( 1 2 0 ) , 0 HN R 2 O C 0 COHNCH H 7 N C H L I H C R 3 HOCH 0 OH OH C H 2 O C O N H 2 ROC H 2 N C H Polyoxin R CH 2OH A B D E G H J K L M H H JL OH COOH COOH R-C H 2 O H /=<5>N OH C H 2 O H COOH COOH OH OH OH OH OH H COOH COOH OH C H 2 O H OH H C H 3 COOH OH C H 3 OH OH H COOH /=<6N OH OH OH OH H OH OH - 51 -C 0 2 H C 0 2 H H 2 N - J H 3 C - ^ ~ \ / ' " HO NH •R C H 2 O C N H 2 II 121 0 122 R = OH 123 R = H The f i r s t i n d i c a t i o n s of the mode o f a c t i o n of the p o l y o x i n s came wit h the o b s e r v a t i o n t h a t c e l l s grown i n the presence of i n h i b i t o r y c o n c e n t r a t i o n s of the complex n 4r , x . v, 166,168-170 were o s m o t i c a l l y f r a g i l e and prone to b u r s t i n g T h i s was taken to be i n d i c a t i v e of weakened c e l l w a l l s and i n v e s t i g a t i o n s were then focused upon the e f f e c t s of 171 p o l y o x i n s on c e l l w a l l s y n t h e s i s . I t was subsequently determined t h a t the p o l y o x i n s impede c e l l w a l l formation by i n h i b i t i n g the enzyme c h i t i n synthetase which i s r e s p o n s i b l e f o r the p o l y m e r i z a t i o n of N - a c e t y l glucosamine to a f f o r d c h i t i n . A f t e r the p u b l i c a t i o n of the s t r u c t u r a l e l u c i d a t i o n of the p o l y o x i n complex, i t was not u n t i l 19 71 t h a t the f i r s t s y n t h e s i s o f a p o l y o x i n component was r e p o r t e d . 172 Naka and co-workers were the f i r s t w ith t h e i r s y n t h e s i s of methyl (5-benzamido-3-0_-benzoyl-5-deoxy-l, 2 - 0 - i s o p r o p y l -i d e n e - a - D - a l l o f u r a n ) u r o n a t e (124). A few months l a t e r 173 M o f f a t t and co-workers r e p o r t e d the s y n t h e s i s of u r a c i l - 52 -p o l y o x i n C (125a) and i t s g - g - t a l o - i s o m e r (125b) . The f i r s t s y n t h e s i s of an e x i s t i n g p o l y o x i n was c a r r i e d out COoMe B z H N _ H N ?0z  ok OH OH 125 Q) R-, = N H 2 ; R 2 = H b) RT =H ; R 2 =NH 2 0 HN^Sr" T° 2 Ho^ N J CH-by Kuzuhara and Emoto. T h e i r s y n t h e s i s of the most a c t i v e o f the p o l y o x i n s , p o l y o x i n J . (120-J), was composed of three p a r t s : 171 1) s y n t h e s i s of thymine p o l y o x i n C (126) 174 - 53 -175 2) s y n t h e s i s of carbamoylpolyoxamic a c i d (122) 176 and 3) p e p t i d y l c o u p l i n g o f the above two components 5. Synthesis o f G l y c o s y l * TAmino A c i d s Many of the syntheses of g l y c o s y l amino a c i d s r e p o r t e d to date have i n c o r p o r a t e d r e a c t i o n s i n v o l v i n g an S^2 displacement of a secondary methanesulfonyloxy o r t o l u e n e -s u l f o n y l o x y group with sodium a z i d e f o l l o w e d by r e d u c t i o n o f the r e s u l t i n g azide to a f f o r d an amine. 172 174 The methods of Naka and Emoto both employed two s u c c e s s i v e displacement r e a c t i o n s i n order to achieve the c o r r e c t s t e r e o c h e m i s t r y i n the f i n a l product. The r e a d i l y a v a i l a b l e f u l l y b l o c k e d sugar (127) was t r e a t e d w i t h sodium benzoate to a f f o r d the di-O-benzoyl compound 128 w i t h i n v e r s i o n of c o n f i g u r a t i o n a t C-5. De-£-benzoylation of compound 128, f o l l o w e d by s e l e c t i v e t r i t y l a t i o n o f the C-6 hydroxyl and s u l f o n y l a t i o n of the C-5 hydroxyl, a f f o r d e d compound 129. Treatment o f compound 129 with sodium a z i d e gave the a z i d o sugar 130 which was then reduced to g i v e an amino sugar with the c o r r e c t c o n f i g u r a t i o n a t C-5. 173 In M o f f a t t ' s s y n t h e s i s of u r a c i l p o l y o x i n C (125a) the aldehydo-nucleoside 131 was t r e a t e d with potassium carbonate and sodium cyanide to g i v e epimeric cyanohydrins which were immediately r e a c t e d with hydrogen peroxide to * used i n an extended sense. - 54 -give a mixture of e p i m e r i c hydroxyamides from which the a - T^-taluronamide 132 was separated. The C-5 hydroxy o f compound 132 was then mesylated and the r e s u l t i n g 0^-mesyl group d i s p l a c e d by a z i d e i o n to give the 5-azido-3-p-alluronamide 134. H y d r o l y s i s of the amide and c y c l o h e x y l i d e n e groups and r e d u c t i o n of the a z i d e f u n c t i o n a l i t y o f compound 134 thus a f f o r d e d the p - u r o n i c a c i d 125a. 177-179 Rosenthal, Shudo, and Richards have prepared a number of analogues of the sugar p o r t i o n o f the p o l y o x i n s i n which the a-amino a c i d i s attached to C-3 of the sugar i n s t e a d of C-4. The i n i t i a l compounds i n the r e a c t i o n - 55 -sequences were the E and Z_ unsaturated e s t e r s (135 and 136, r e s p e c t i v e l y ) p r e v i o u s l y prepared by Rosenthal and 83 Nguyen 177 S t e r e o s p e c i f i c h y d r o x y l a t i o n o f compound 136 wi t h potassium permanganate fo l l o w e d by a c e t y l a t i o n o f the secondary hydroxyl group y i e l d e d the (S) - a-O^-acetyl e s t e r 137. S t e r e o s e l e c t i v e d e h y dration o f the t e r t i a r y a l c o h o l - 5 6 -137 with thionyl chloride yielded the enol acetate 138 which i n turn was hydrogenated to afford 3-C-[(R)-acetoxy-(methoxycarbonyl) methyl] -3-deoxy-l ,2:5, 6-di-0_-isopropylidene-a-D-allofuranose (139, R=Ac). The acetyl blocking group was then hydrolyzed and replaced with a to s y l group. When compound 139 (R=Ts) was treated with sodium azide i t under-went the expected displacement reaction, with inversion of configuration, to y i e l d the azide 140 which was immediately reduced to give the L.-amino ester 141. Use of the E-17 unsaturated ester 135 i n an analogous sequence of reactions yielded the corresponding D-amino ester 142 and by omitting - 57 -C02Me Ul R1=H ; R2-NH2 U 2 R1 = NH2;R2 = H C02Me U 3 R1 = H ; R2=NH2 1 U R1=NH2;R2 = H the dehydration r e a c t i o n from the sequence the C-3-hydroxyl 17 8 amino a c i d s 14 3 and 144 were prepared 180-182 Jordaan and co-workers have employed a d i f f e r -ent method f o r the s y n t h e s i s o f g l y c o s y l amino a c i d s . Condensation o f e t h y l i s o c y a n o a c e t a t e and the keto-sugar 25 u s i n g sodium hydride i n t e t r a h y d r o f u r a n a f f o r d e d the unsaturated formylamino e s t e r 145 as the major product. The s a t u r a t e d D-formyl amino e s t e r 146 was obtained by r e d u c t i o n o f compound 145 with Raney n i c k e l . Compound 146 was degraded to a pentofuranose i n the usual manner and was c h a r a c t e r i z e d as i t s c r y s t a l l i n e 183 p_-bromobenzenesulphonate 147. X-ray a n a l y s i s o f compound 147 e s t a b l i s h e d t h a t the branch-chain possessed (R) s t e r e o c h e m i s t r y . An unambiguous proof o f s t r u c t u r e o f 177-179• the a-amino e s t e r s s y n t h e s i z e d by Rosenthal e t a l . - 58 -U 8 147 R=salicyl idene R=brosyl 179 was pr o v i d e d by c o n v e r s i o n of the D-amino e s t e r 142 i n t o the N - s a l i c y l i d e n e compound 148 which was i d e n t i c a l to a compound s y n t h e s i z e d from the D-formylamino e s t e r 1 4 6 1 8 0 . When the condensation of e t h y l i s o c y a n o a c e t a t e and ketose 2_5 was performed i n e t h a n o l , u s i n g sodium cyanide as the c a t a l y s t , the p-formylamino e s t e r a l l o s e d e r i v a t i v e 180 149 was the product . The attempted degradation of compound 14 9 i n t o i t s pentofuranose d e r i v a t i v e l e d to the 181 formation o f the aminal 150 . A c e t o l y s i s o f compound - 59 -150 caused a c e t a l m i g r a t i o n to y i e l d compound 152 which was condensed with b i s ( t r i m e t h y l s i l y l ) u r a c i l to a f f o r d the B - n u c l e o s i d e 153. The 2,3-0-isopropylidene group o f 153 c o u l d not be h y d r o l y z e d without e f f e c t i n g cleavage of the N - g l y c o s y l l i n k a g e . Although the t e t r a - a c e t a t e 154 was subsequently prepared by a c e t o l y s i s of the d i - a c e t a t e 151, a l l attempts to s y n t h e s i z e a n u c l e o s i d e u s i n g 154 f a i l e d . - 6 0 -Jordaan and co-workers have recently synthesized the C-2 glycosyl N-formylamino esters 155 and 156 using a s i m i l a r approach. 155 156 184 Rosenthal and Dooley have applied the azlactone 185 synthesis to the keto-sugar 2_5 to afford the azlactones 157 and 158 which, a f t e r methanolysis and reduction, yielded the benzamido derivatives of compounds 141 and 142 . 157 Ph 158 - 61 -Umezawa and c o - w o r k e r s X C M J used the Bucherer hydantoin 187 s y n t h e s i s to prepare the hydantoin 159 which was h y d r o l y s e d to a f f o r d the p a r t i a l l y b locked c y c l i c a-amino a c i d 160. The amino a c i d 160 was subsequently used to s y n t h e s i z e the g l y c o s y l amino a c i d n u c l e o s i d e 161. I I I . RESULTS AND DISCUSSION 1. S y n t h e s i s of Branched-chain Amino Sugars. Reaction of  1,2:5, 6-Di-p_-isopropylidene-a - B-ribo-hexofuranos-3- ulo s e w i t h Sodium Cyanide and Methyl N i t r o a c e t a t e . Although the K i l i a n i and F i s c h e r cyanohydrin s y n t h e s i s 6 1 has been widely a p p l i e d to aldoses the procedure has been very l i t t l e used to prepare branched-chain sugars from ketoses. T r o n c h e t 6 2 and B o u r g e o i s 6 3 , 6 4 have r e c e n t l y r e p o r t e d a p p l i c a t i o n of the cyanohydrin s y n t h e s i s to 1,2:5,6-di-0-is o p r o p y l i d e n e - a - D -r i b o - h e x o f u r a n o s - 3 - u l o s e (25) to y i e l d an epimeric mixture of cyanohydrins but the s t r u c t u r e s of the cyanohydrins were not proven. In t h i s t h e s i s the proof o f s t r u c t u r e of the cyanohydrins d e r i v e d from ketose 2_5 and the e f f e c t of the pH on c o n t r o l l i n g the p r o p o r t i o n s o f products w i l l be d e s c r i b e d . In a d d i t i o n , some o f the p r e l i m i n a r y work d e a l i n g w i t h the branched-chain methyl n i t r o a c e t a t e adduct of the same ketone, which w i l l be d e a l t with i n d e t a i l i n S e c t i o n s 3.3 and 3.4, w i l l be presented. - 63 -1.1 Synthesis of 3-C-Cyano-l, 2 : 5 , 6-di-0_-isopropylidene-a-g-glucofuranose (26J and 3-C-Cyano-l, 2 : 5 , 6-di-0_-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s e (27_) . The r e a c t i o n of sodium cyanide, methyl n i t r o a c e t a t e , and ketose 2_5 was found to produce e i t h e r the gluco-cyano-h y d r i n 2_6 or the a l l o - c y a n o h y d r i n 27 depending upon the order of a d d i t i o n of the r e a c t a n t s . The r e s u l t s are summarized i n Table 3. 1.1.1 Procedure A to Y i e l d 3-C-Cyano-l,2:5,6-di-0-iso p r o p y l i d e n e - a - D - g l u c o f u r a n o s e (26). The a d d i t i o n of an e q u i v a l e n t of sodium cyanide to a s o l u t i o n of ketose 25_ caused the s o l u t i o n to t u r n b l a c k immediately. A f t e r the r e a c t i o n mixture was s t i r r e d a t room temperature f o r 18 hours, the r e a c t i o n was worked up and the crude product was chromatographed on a column o f s i l i c a g e l , u s i n g 1:1 benzene-ethyl a c e t a t e as the developer, to a f f o r d unreacted ketose 2J5 (31%) and a c l e a r c o l o u r l e s s syrup. The gluco-cyanohydrin 26 c r y s t a l l i z e d spontaneously upon stand i n g . The p.m.r. spectrum of the syrup remaining a f t e r the removal o f the c r y s t a l l i n e gluco-cyanohydrin 26 showed two doublets a t 64.55 and 64 .92 which were a t t r i b u t e d to the C-2 protons of the gluco and a l i o cyanohydrins, r e s p e c t i v e l y . Based on the r e l a t i v e i n t e n s i t i e s of these resonances, the weight of the mother l i q u o r , a n d the weight of c r y s t a l l i n e - 64 -0-OH 0 0 0 0-2 5 ^oJ 0 0 C=N 0- j— 2 6 R= H 1 6 3 R=Ac 0 - , 0- -0 1 fo C H 2 0-\-I HNAc K 3 0 C=N RO 0 0 2 7 R = H 1 6 4 R=Ac X I NH Ac o J 1 . 0 C H 2 OH 0 0 - h On C 0 2 M e " ^ H N 0 2 0 RO 0-1 6 2 R=H 1 6 5 R=Ac - 65 -compound 26_, i t was c a l c u l a t e d t h a t the r e a c t i o n y i e l d e d the gluco-cyanohydrin 26 i n 35% y i e l d and the a l l o -cyanohydrin 2_7 i n 3% y i e l d (based on ketose consumed) . The y i e l d o f compound 26_ was i n c r e a s e d to 57% by adding, a f t e r the a d d i t i o n of sodium cyanide to the ketose 2_5, one molar e q u i v a l e n t of methyl n i t r o a c e t a t e . The y i e l d o f the accompanying compound 21_ was c a l c u l a t e d to be 4% i n t h i s case. TABLE 3 Appl icat ion of the Cyanohydr in Synthesis to Ketose 25 in the Presence of Methy l N i t roaceta te ( M N A ) . REACTION CYANOHYDRIN PROCEDURE NaCN MNA SOLVENT TIME T E M P GLUCO 26 ALLO 27 CMPD 162 (equ iv) (equiv) ( ml ) ( h ) C O (V. ) ( V.) ( V . ) A 1 none EtOH(5) 18 20 35 3 -A 1 1 EtOH(2) 18 20 57 U -B 1 1 EtOH (5) 18 22 6 85 7 B 1 none EtOH(5) 18 22 5 71 -AcOH(Q25) - 66 -1.1.2 Procedure B to Y i e l d 3-C-Cyano-l,2:5,6-di-0-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s e (27). In t h i s procedure i t was found t h a t when the order o f a d d i t i o n o f methyl n i t r o a c e t a t e to ketose 25_ was changed from t h a t of procedure A, then the a l l o - c y a n o h y d r i n 27 was formed predominantly. Thus, a d d i t i o n o f ketose 2_5 to a p r e v i o u s l y formed equimolar mixture o f sodium cyanide and methyl n i t r o a c e t a t e i n ethanol gave, a f t e r chromato-graphy on s i l i c a g e l u s i n g 4:6 benzene-ethyl a c e t a t e as developer, unreacted ketose (22%) and a c l e a r c o l o u r l e s s syrup. The a l l o - c y a n o h y d r i n 27 was c r y s t a l l i z e d from the syrup u s i n g benzene-hexane and an a n a l y s i s of the p.m.r. spectrum of the mother l i q u o r r e v e a l e d the presence o f resonances a t t r i b u t a b l e to H-2 of both cyanohydrin epimers as w e l l as a sharp s i n g l e t a t 6 3.83 a t t r i b u t e d to the methyl e s t e r of 1,2 :5,6-di-0_-isopropylidene-3 - c 4(R,S)-nitro (methoxy carbonyl) m e t h y l l - a - D - a l l o f u r a n o s e (162) . (Compound 162 and i t s proof of s t r u c t u r e w i l l be d e a l t w i t h i n d e t a i l i n S e c t i o n s 3.3 and 3.4.) As was done f o r procedure A, i t was c a l c u l a t e d t h a t procedure B gave the a l l o - c y a n o h y d r i n 27, the gluco-cyanohydrin 26, and compound 162 i n y i e l d s of 85%, 6% and 7%, r e s p e c t i v e l y , based on consumed ketose. Attempts to o b t a i n pure compound 162 by repeated chromato-graphy were u n s u c c e s s f u l because 162. was q u i t e u n s t a b l e . - 67 -1.1.3 Procedure C to Y i e l d 3-0_-Acetyl-3-C-cyano-l, 2 : 5 , 6-di-0_-isopropylidene-a-D-gluco and allofuranose (163) and (164), 3-0-Acetyl-l,2:5,6-di-0-isopropylidene-3-C-[(R,S)-nitro(methoxycarbonyl)-methyl]-a-D-allof uranose (165) and 3,5,6-tri-O-Acetyl-3-C-cyano-l, 2-0_-isopropylidene-a-D-allofuranose (166). Because of the i n s t a b i l i t y of compound 162 towards column chromatography, i t was decided to repeat procedure B and to derivatize the products p r i o r to chromatography in order to determine whether or not compound 162 was produced i n greater than 7% y i e l d . When a molar equivalent of ketose 2_5 was added to a mixture of one equivalent of sodium cyanide and one equivalent of methyl nitroacetate i n ethanol and the r e s u l t i n g mixture allowed to react for 18 hours at room temperature, followed by an immediate acetylation of the p a r t i a l l y p u r i f i e d pro-duct mixture with acetic anhydride i n the presence of p_-toluenesulfonic acid monohydrate, four products were obtained. The mixture of products was chromatographed on s i l i c a gel, using 9:1 benzene-ethyl acetate as the developer,to afford the 3-0_-acetyl derivatives of each product, namely, the gluco-cyanohydrin 163, the allo-cyanohydrin 164, the methyl nitroacetate adduct 165, and the tri-0_-acetyl allo-cyano-hydrin 166 i n a r a t i o of 1:23:4:72, respectively. - 68 -A proof o f s t r u c t u r e o f compounds 163 and 164 was provided by a c i d c a t a l y z e d a c e t y l a t i o n o f the pure cyano-h y d r i n s 2_6 and 2_7 to a f f o r d compounds i d e n t i c a l (m.p., n.m.r., o p t i c a l r o t a t i o n ) to those obtained by procedure C. Proof of s t r u c t u r e of compound 166 was provided by s e l e c t i v e l y h y d r o l y z i n g the 5,6-0-isopropylidene group of the a l l o - c y a n o -h y d r i n 164 and subsequently a c e t y l a t i n g the product to a f f o r d a substance i d e n t i c a l i n a l l r e s p e c t s w i t h the t r i - 0 _ - a c e t y l a l l o - c y a n o h y d r i n 166. T h i s s e l e c t i v e a c e t o l y s i s o f the 5,6-O-i s o p r o p y l i d e n e group of a di-0_-isopropylidene sugar d e r i v a t i v e u s i n g p_-toluenesulfonic a c i d monohydrate i s noteworthy as i t can provide a means of markedly r e d u c i n g the number of steps r e q u i r e d i n some n u c l e o s i d e syntheses. 1.2 Sy n t h e s i s o f 3-C-Acetamidomethyl-l, 2 : 5 , 6-di-0_-isopropyl-idene-a-D-glucof uranose (54) and 3-C-Acetamidomethyl-1, 2 : 5, 6 - d i - 0 - i s o p r o p y l i d e n e - a - p _ - a l l o f uranose (5_3) . The proof of s t r u c t u r e of the epimeric cyanohydrins was pro v i d e d i n the f o l l o w i n g way. L i t h i u m aluminum hydride r e d u c t i o n o f the gluco-cyanohydrin 26 fo l l o w e d by a c e t y l a t i o n of the amino product, a f f o r d e d the corresponding 3-C-acetamido-methyl g_luco-derivative 5_4. Compound 54_ has a p.m.r. spectrum 9 8 i d e n t i c a l to t h a t o f a substance which was obt a i n e d by a p p l i c a t i o n of the nitromethane s y n t h e s i s to ketose 2_5 fo l l o w e d by r e d u c t i o n and a c e t y l a t i o n . Unequivocal proof of s t r u c t u r e o f t h a t compound was provided by Yoshimura e t - 69 -a l . S i m i l a r i l y , r e d u c t i o n and a c e t y l a t i o n of the a l l o -9 8 cyanohydrin 2_7 gave the known 3-C-acetamidomethyl a l l o -sugar 53. - 70 -2. Sy n t h e s i s of Branched-Chain N u c l e o s i d e s . Analogues of the Nucleoside Moiety of Puromycin. 188 In 1973, Rosenthal and Baker r e p o r t e d the s y n t h e s i s o f a number of analogues of the amino sugar n u c l e o s i d e moiety of puromycin (119) . T h e i r s y n t h e t i c sequence u t i l i z e d 84 8 5 the p r e v i o u s l y prepared ' 3-C-cyanomethyl-3-deoxy hexo-fur a n o s i d e 3_6_ which was degraded by the usual s e r i e s o f r e a c t i o n s to a f f o r d 5-0_-benzoyl-3-C-cyanomethyl-3-deoxy-1,2-£-isopropylidene-a-D-ribofuranose (167). When compound 167 was t r e a t e d with 90% t r i f l u o r o a c e t i c a c i d i n order to hydrolyze the a c e t a l b l o c k i n g group, and the r e s u l t i n g pro-duct mixture was a c e t y l a t e d , not o n l y was the expected 1,2-di-0_-acetyl-3-C-cyanomethyl sugar 168 i s o l a t e d but a l s o a small amount (^5%) of the 2,3-y-lactone 169. Obviously, the a c i d h y d r o l y s i s o f the 1,2-0_-isopropylidene group was accompanied by p a r t i a l h y d r o l y s i s of the n i t r i l e group g i v i n g a c a r b o x y l i c a c i d which then underwent i n t r a -m olecular e s t e r i f i c a t i o n to y i e l d a 2,3-y-lactone. In any n u c l e o s i d e s y n t h e s i s r e a c t i o n , compound 169 would be unable to form the 1,2-acyloxonium i o n i n t e r -mediate which leads to predominant formation of 6-nucleosides - 71 -169 168 (see the I n t r o d u c t i o n , s e c t i o n 4.2). I t seemed to us, t h e r e f o r e , t h a t a q u a n t i t a t i v e s y n t h e s i s of l a c t o n e 169 would provide a f a c i l e route t o the l e s s commonly s y n t h e s i z e d a - n u c l e o s i d e s . We thus s e t out to prepare compound 169 f o r use i n the s y n t h e s i s o f a and B-adenine n u c l e o s i d e analogues of the a n t i b i o t i c puromycin. 2.1 S y n t h e s i s of l-0_-Acetyl-5-0-benzoyl-3-C-carboxymethyl-3-deoxy-g-rj-ribofuranose-2 , 3-y-lactone (169) . Rather than t r y i n g to hydrolyze the cyanomethyl group o f compound 167, i t seemed advantageous to i n t r o d u c e the - 72 -c a r b o x y l i c a c i d f u n c t i o n a l i t y a t the beginning of the s y n t h e t i c sequence. 8 3 Using a p r e v i o u s l y r e p o r t e d procedure, the y l i d generated by the r e a c t i o n o f potassium t-butoxide and carbomethoxymethyldimethyl phosphonate was condensed with the keto-sugar 25_ and the r e s u l t i n g mixture of i s o m e r i c a,3-unsaturated e s t e r s (135 and 136) was hydrogenated over 8 3 p a l l a d i u m - o n - c h a r c o a l to a f f o r d the known 3-C-(carbo-1Z2 R=(C6H5)CO - 73 -methoxymethyl) -3-deoxy-l ,2:5, 6-di-0_-isopropylidene-a-D-a l l o f u r a n o s e (35) . The 5,6-0_-isopropylidene b l o c k i n g group of compound 35_ was s e l e c t i v e l y h y drolyzed by d i s s o l v i n g the sugar i n 66% aqueous a c e t i c a c i d . The r e a c t i o n was c a r e f u l l y monitored by t . l . c , u s i n g 4:6 benzene-ethyl acetate as developer, and when the s t a r t i n g m a t e r i a l (R f 0.6) c o u l d no longer be d e t e c t e d , or when compounds having a lower R f than the d e s i r e d product (R^ 0.2) were de t e c t e d , the r e a c t i o n was stopped by e v a p o r a t i n g the a c e t i c a c i d and water under reduced p r e s s u r e . The r e s u l t i n g product mixture c r y s t a l l i z e d spontaneously to a f f o r d 3-C-(carbomethoxymethyl)-1,2-0-isopropylidene-a-D-allofuranose (170) q u a n t i t a t i v e l y and o f such p u r i t y t h a t i t co u l d be used f o r subsequent r e a c t i o n s without f u r t h e r p u r i f i c a t i o n . Compound 170 was degraded t o 3-C-(carbomethoxymethyl)-3-deoxy-1,2-0_-isopropylidene-a - g-ribof uranose (171) by t r e a t -ment with sodium p e r i o d a t e f o l l o w e d by immediate r e d u c t i o n w i t h sodium bor o h y d r i d e . B e n z o y l a t i o n of compound 171 y i e l d e d 5-0_-benzoyl-3-C- (carbomethoxymethyl) -3-deoxy-l, 2-O-is o p r o p y l i d e n e - a - D - r i b o f u r a n o s e (172). The 1,2-0_-isopropylidene group of compound 172 was hydrolyzed u s i n g a 90% (v/v) t r i f l u o r o a c e t i c a c i d s o l u t i o n . A f t e r the r e a c t i o n mixture was s t i r r e d f o r 1 1/2 hours a t room temperature, the v o l a t i l e components were evaporated i n vacuo to y i e l d a white s o l i d which was pre-sumed to be the 2,3-y-lactone 173. T h i s compound was not - 74 -c h a r a c t e r i z e d but was immediately a c e t y l a t e d to a f f o r d a compound which was i d e n t i c a l i n a l l r e s p e c t s (m.p., o p t i c a l r o t a t i o n , n.m.r.) to the y - l a c t o n e 169 obtained by Rosenthal 188 and Baker . The s y n t h e t i c sequence y i e l d e d compound 169 from compound 3_5 i n an o v e r a l l y i e l d of 66%. 2.2 S y n t h e s i s of 2,6-Dichloro-9-[3 1-C-(carboxymethyl-2',3 1-Y-lactone)-3'-deoxy-g and a - D - r i b o f u r a n o s y l ] p u r i n e (174 and 175). The i n i t i a l n u c l e o s i d e s y n t h e s i s attempted w i t h the l a c t o n e sugar 169 was a f u s i o n r e a c t i o n w i t h 2 , 6 - d i c h l o r o -purine . The sugar and one e q u i v a l e n t of the base were i n t i m a t e l y mixed, d r i e d , and then fused at 160° and 20 t o r r . A f t e r approximately 5 minutes, the i n i t i a l v i g o r o u s b u b b l i n g of the melt had subsided and the pressure was reduced to - 75 -0.1 t o r r f o r a f u r t h e r 15 minutes. A f t e r c o o l i n g , the amber g l a s s was chromatographed to a f f o r d unreacted s t a r t i n g m a t e r i a l 169 (38%) , B-nucleoside 174 (23%) , and a-nucleoside 175 (27%) . In an attempt to reduce the amount of unreacted s t a r t -i n g m a t e r i a l and i n c r e a s e the y i e l d s o f the products, a number o f small s c a l e r e a c t i o n s (^1 mg. of m a t e r i a l s ) was c a r r i e d out i n which the lactone/base r a t i o , r e a c t i o n time, and temperature were v a r i e d . The r e s u l t s o f these t e s t s were judged q u a l i t a t i v e l y by t . l . c . and the more promising r e s u l t s repeated on a l a r g e r s c a l e (30-50 mg. of lactone) to a l l o w a q u a n t i t a t i v e a n a l y s i s of the products o b t a i n e d . Some o f the c o n c l u s i o n s a r r i v e d a t i n c l u d e : -a) a base/lactone r a t i o >1 favoured consumption of the s t a r t i n g m a t e r i a l but when the r a t i o approached a v a l u e of 2 or g r e a t e r then p r a c t i c a l d i f f i c u l t i e s were encountered i n s e p a r a t i n g the products from the un-r e a c t e d 2 , 6 - d i c h l o r o p u r i n e . b) a t temperatures much higher than 160° decomposition of the base was observed along w i t h s u b l i m a t i o n of the l a c t o n e 169. c) longer r e a c t i o n times l e d to i n c r e a s e d y i e l d s o f n u c l e o s i d e s . The optimum c o n d i t i o n s found f o r t h i s system i n v o l v e d f u s i n g a 1.5 to 1 r a t i o o f 2 , 6 - d i c h l o r o p u r i n e and l a c t o n e 169 a t 160° and 20 t o r r f o r 5 minutes, then a t 140° and - 76 -0.2 tor r for an additional 25 minutes. Under these conditions the amount of unreacted lactone 169 was reduced to 8% while the yields of the a and B-nucleosides, (17 5, 17 4) were 41% and 48%, respectively. The addition of acid catalysts (dichloroacetic acid or p_-toluenesulphonic acid) to the fusion reaction did not produce any discernable change i n the r e s u l t s . CI The anomeric configurations of the nucleosides were assigned on the basis of t h e i r p.m.r. spectra. The anomeric proton of the g-nucleoside was observed as a sin g l e t at 66.24. However, the H-2' proton appeared as a quartet at 65.64 with coupling constants of 1.0 Hz and 6.8 Hz which suggested that the H-l' resonance was i n fact an unresolved doublet with J., „,= 1.0 Hz. The anomeric proton of the a-nucleoside appeared as a doublet at 66.80 coupled to the - 77 -H-21 proton a t 65.46 with a J value of 4.5 Hz and the H-2' proton was coupled to H-3 1 with , 3 , equal to 7.0 Hz. The r e l a t i v e magnitudes o f the H - l 1 , H-2 1 c o u p l i n g constants and the f a c t t h a t the anomeric s i g n a l o f the ct-D-anomer i s u s u a l l y observed a t lower f i e l d than t h a t o f the B-D-189 anomer c o n f i r m the assignments of the B - g - c o n f i g u r a t i o n to compound 174 and the a - g - c o n f i g u r a t i o n to compound 175. 2.3 Syn t h e s i s of 9-[3'-C-(Carboxymethyl-2',3 1-y-lactone)-3'-deoxy-g-D-ribofuranosyl]adenine (178) and 9-[3'-C-Carboxymethyl-3 1-deoxy-a-D-ribofuranosyl]adenine (181) . The use of the r e a c t i v e 2 , 6 - d i c h l o r o p u r i n e i n nucleo-s i d e syntheses has the disadvantage t h a t subsequent r e -a c t i o n s are necessary to convert the 2 , 6 - d i c h l o r o p u r i n e n u c l e o s i d e i n t o i t s analogous adenine n u c l e o s i d e . I t was t h e r e f o r e deemed advantageous to attempt a condensation o f l a c t o n e 169 d i r e c t l y with an adenine d e r i v a t i v e . Towards t h i s end, a d i c h l o r o e t h a n e s o l u t i o n o f the l a c t o n e 169 and N 6-benzoyl-N 6 , 9-bis ( t r i m e t h y l s i l y l ) a d e n i n e " ^ 1 was heated f o r 18 hours a t 63° i n the presence of stannous c h l o r i d e . A f t e r work-up, the product was chromatographed on a column of s i l i c a g e l to a f f o r d N -benzoyl-9-(3'-C-carboxymethyl-2',3 1-y-lactone-3'-deoxy-B-D-ribofuranosyl)adenine 176 (31%), and the a-anomer 177 (31%). - 78 -169 BzNSiMe-S n C l 4  C 2 H 4 C l 2 The anomeric configuration of the nucleosides was made on the basis of the i r p.m.r. spectrum since the anomeric proton of the B-nucleoside 176 appeared as a si n g l e t at 66 . 20 while the anomeric proton of-the a-nucleoside 177 189 appeared 0.69 p.p.m. further downfield as a doublet coupled to H-21 with 2, = 4.0 Hz. - 79 -The benzoylated n u c l e o s i d e s were not c h a r a c t e r i z e d f u r t h e r but were immediately d r i e d and d i s s o l v e d i n a methanol s o l u t i o n c o n t a i n i n g a c a t a l y t i c amount of sodium methoxide. A f t e r a p e r i o d o f 7 days, Bio-Rex 70 (H +) c a t i o n exchange r e s i n was added to remove the sodium i o n s . The f i l t r a t e remaining a f t e r the removal of the r e s i n was evaporated i n vacuo i n order to remove the methyl benzoate by-product from the n u c l e o s i d e products. In the case of the methanolysis of the 3-nucleoside 176, the l a c t o n e f u n c t i o n a l i t y was immediately regenerated by p a s s i n g a methanol-water s o l u t i o n of the compound through a column of Bio-Rex 70 (H +) r e s i n , u s i n g 8:2 methanol-water as the e l u t i n g s o l v e n t . The l a c t o n e g-nucleoside 178 c r y s t a l l i z e d spontaneously from the e l u e n t and was removed by f i l t r a t i o n i n an o v e r a l l 61% y i e l d w i t h r e s p e c t t o the block e d n u c l e o s i d e 176. The u.v. spectrum of compound 178 e x h i b i t e d maxima a t 205 (el8,700) and 257 nm (el3,200) 190 which confirmed the s i t e o f g l y c o s y l a t i o n at N-9 In the 270 MHz p.m.r. spectrum of compound 178 (see Fi g u r e IA) , the e x i s t e n c e of a l a c t o n e f u n c t i o n a l i t y was i n f e r r e d by the presence of only one exchangeable h y d r o x y l proton (65.21) and no evidence of a low f i e l d a c i d proton resonance. T h i s was confirmed by a c a r b o n y l a b s o r p t i o n a t 1780 cm i n the i . r . spectrum of compound 178. The 3 - c o n f i g u r a t i o n of the n u c l e o s i d e , suggested by the presence of a s i n g l e t anomeric proton resonance i n the p.m.r. spectrum 9 8 7 6 PPM(S) 5 4 3 2 F i g u r e 1A 270 MHz P.M.R. Spectrum of 9 - [ 3 1 - C - ( C a r b o x y m e t h y l - 2 3 ' - y - l a c t o n e ) - 3 ' - d e o x y - g -D - r i b o f u r a n o s y l ] a d e n i n e (178) i n DMSO-d,. F i g u r e IB P a r t i a l 270 MHz P.M.R. Spectrum of 9-[3'-C-(Carboxymethyl-2',3'-y-lactone)-3'-deoxy-B-rj-ribofuranosyl] adenine (178) i n DMSO-d . - 82 -(65.91) , was confirmed by the c d . spectrum which e x h i b i t e d 220 a n e g a t i v e Cotton e f f e c t a t 260 nm. The high r e s o l u t i o n mass spectrum of compound 178 showed mol e c u l a r i o n and (M + + 1) peaks at m/e values of 291.0953 and 292.1032, r e s p e c t i v e l y , i n e x c e l l e n t agree-ment wi t h the corresponding t h e o r e t i c a l values of 291.0967 and 292.1045. The elemental a n a l y s i s o f 178 a l s o agreed w i t h i t s proposed s t r u c t u r e . In the case of the methanolysis of the b e n z o y l a t e d l a c t o n e a - n u c l e o s i d e 177, a white c r y s t a l l i n e product p r e c i p i t a t e d from s o l u t i o n a f t e r the n e u t r a l i z i n g r e s i n was removed by f i l t r a t i o n . The elemental a n a l y s i s o f t h i s pro-duct suggests t h a t i t was 9-(3'-C-carbomethoxymethyl-3 1-deoxy-a-D-ribofuranosyl)adenine (179) . - 83 -In order to regenerate the l a c t o n e , a methanol-water s o l u t i o n of compound 179 was passed through a column of Bio-Rex 70 (H +) r e s i n u s i n g 8:2 methanol-water as the e l u t i n g s o l v e n t . Upon s t a n d i n g f o r 18 hours, a white c r y s t a l l i n e product c r y s t a l l i z e d from the e l u e n t and was removed by f i l t r a t i o n i n a 51% y i e l d , based on the b l o c k e d l a c t o n e c t-nucleoside 177. - 84 -The u.v. spectrum of the product e x h i b i t e d maxima at 203 nm (el7,900) and 252 nm (el2,700) and e s t a b l i s h e d the 190 product as an N-9 n u c l e o s i d e . However, the 270 MHz p.m.r. spectrum of the product (see F i g u r e s 2A and 2B) showed i t to be the 3'-C-carboxymethyl-a-nucleoside 181 r a t h e r than the expected l a c t o n e c t-nucleoside 180. The spectrum showed three exchangeable proton resonances i n a d d i t i o n to the amine protons of the adenine base - a low f i e l d a c i d proton resonance at 612.13 and two h y d r o x y l protons at 6 5.57 and 64.83. The presence of the a c i d f u n c t i o n a l i t y was confirmed by a broad a b s o r p t i o n between 2400 and 3800 cm 1 (OH) i n the i n f r a - r e d spectrum of 181. A l s o present i n the p.m.r. spectrum was an anomeric proton resonance at 66.27 with J , , 9 , = 3.0 Hz. The lower f i e l d . . 189 p o s i t i o n and l a r g e r c o u p l i n g constant of t h i s resonance, compared to the analogous s i g n a l f o r the l a c t o n e 8-nucleoside 178 (65 .91, J, , „ , = 0 Hz), confirmed the c t-nucleoside l ,2 c o n f i r m a t i o n of compound 181. A d d i t i o n a l proof was pro-v i d e d by the c d . spectrum of 181, which e x h i b i t e d a p o s i t i v e Cotton e f f e c t at 256 nm. The elemental a n a l y s i s o f the product was i n agreement with i t s proposed s t r u c t u r e . The e x i s t e n c e of the a-nucleoside 181 i n the f r e e a c i d form r a t h e r than the l a c t o n e form 180 i s most probably due to p a r t i c i p a t i o n o f the h e t e r o c y c l i c base i n the h y d r o l y -s i s o f the l a c t o n e or by a f a v o u r a b l e hydrogen-bonding HO H-2 H - 8 NH-C 0 2 H H-r C H 2 OH \ - A s - ^ N C 0 2 H OH N H , 181 OH i H-2' H-5'a \ H-5(D H-4 '| / L J H-1 a DMSO i i I i I i i | | • • 1 • 12 8 1 1 1 ; 1 1 1 1 * 1 1 1 1 1 1 1 • ' • i i i 1 1 1 PPM(S) 5 1 1 ' 1 ' • ' 1 1 ' • ' 3 2 F i g u r e 2A 270 MHz P.M.R. Spectrum o f 9-[3 1-C-Carboxymethyl-3 1-deoxy-a-D-ribofuranosyl]-adenine (181) i n DMSO-d,. 6 F i g u r e 2B P a r t i a l 270 MHz P.M.R. Spectrum of 9-[3'-C-Carboxymethyl-3'-deoxy-a-Q-ribo-f u r a n o s y l ] a d e n i n e (181) i n DMSO-d . - 87 -i n t e r a c t i o n between the base and the f r e e a c i d which would favour the a c i d s i d e of the a c i d - l a c t o n e e q u i l i b r i u m . A s i m i l a r i n f l u e n c e was shown by the h e t e r o c y c l i c base of 9-[3'-acetamido-3 1-C-(carboxymethyl-2',3'-y-lactone)-3'-d e o x y - a - D - x y l o f u r a n o s y l ] a d e n i n e p r e v i o u s l y s y n t h e s i z e d by 2 22 Rosenthal and R a t c l i f f e The mass spectrum of the carboxy a-nucleoside 181 i s very s i m i l a r to t h a t o f the l a c t o n e 8-nucleoside 178 i n t h a t molecular i o n and (M + + 1) peaks appear at m/e values of 291.0984 and 292.1041, r e s p e c t i v e l y . Obviously, under the o p e r a t i n g c o n d i t i o n s necessary t o o b t a i n a mass spectrum, compound 181 l o s t a molecule of water to y i e l d the lactone a - n u c l e o s i d e 180 and t h i s was the compound whose mass spectrum was ob t a i n e d . - 88 -3. Synt h e s i s o f Analogues o f the Nucleoside Moiety o f the  P o l y o x i n s . The procedures employed, up u n t i l now, f o r the s y n t h e s i s of g l y c o s y l 3-C-amino a c i d s have s u f f e r e d from s e v e r a l 18 5 disadvantages. The a z l a c t o n e s y n t h e s i s employed by 184 Rosenthal and Dooley a f f o r d e d 3-deoxy-N-benzoyl ammo ac i d s which c o u l d not be unblocked to y i e l d the f r e e amino a c i d s . The s t e r e o s p e c i f i c s y n t h e s i s employed by Rosenthal 177-179 e t a l . i n v o l v e s many steps and the o v e r a l l y i e l d i s low. F i n a l l y , the condensation of e t h y l i s o c y a n o a c e t a t e 180—182 wit h keto-sugars used by Jordaan and co-workers i n t r o d u c e d a b l o c k e d amino a c i d p o s s e s s i n g R-(D) s t e r e o -chemistry whereas the n a t u r a l l y o c c u r r i n g p o l y o x i n s c o n t a i n an S-(L)-amino a c i d . Furthermore, degradation o f the r e s u l t i n g hexofuranose to a f f o r d a pentofuranose r e s u l t e d 181 i n i r r e v e r s i b l e formation o f an aminal which e i t h e r would not undergo condensation with a p y r i m i d i n e base or c o u l d not be unblocked when condensation d i d occu r . In o r d e r to overcome the disadvantages encountered by these previous procedures, i t was decided to attempt the condensation of methyl n i t r o a c e t a t e w i t h v a r i o u s keto-sugars . The primary o b j e c t i v e was the s y n t h e s i s o f a p e p t i d y l n u c l e o s i d e composed o f the f o l l o w i n g elements: 1) L - ( S ) - g l y c i n e l i n k e d through the a-carbon to - 89 -2) C-3 of a D - a l l o f u r a n o s e and 3) a 3 - l i n k e d N-1 thymine base. The s y n t h e t i c s t r a t e g y e n v i s i o n e d to accomplish t h i s goal was as f o l l o w s : 1) condensation of methyl n i t r o a c e t a t e with 1,2:5,6-d i - O - i s o p r o p y l i d e n e - g - r j - r i b o - h e x o f u r a n o s - 3 - u l o s e (25) . 2) r e d u c t i o n of the n i t r o group and i s o l a t i o n of the d e s i r e d a l l o f u r a n o s - 3 - y l L-amino a c i d . and 3) condensation of the branch-chain sugar w i t h a thymine base. The work c a r r i e d out towards t h i s end i s d e s c r i b e d i n s e c t i o n s 3.2, 3.3, and 3.6. At the same time i n v e s t i g a t i o n s were made i n t o : 1) s y n t h e s i s of 3-deoxy a l l o f u r a n o s - 3 - y l branch-c h a i n sugars ( d e s c r i b e d i n s e c t i o n 3.5) and 2) s y n t h e s i s of p e n t o f u r a n o s - 3 - y l branch-chain sugars ( d e s c r i b e d i n s e c t i o n 3.1). 3.1 S y n t h e s i s of 3-Deoxy-3-C-nitro(carbomethoxy)methylene-D-erythro-pentofuranoses. In order to overcome the d i f f i c u l t i e s encountered 180—182 by Jordaan and co-workers i n t h e i r attempts to s y n t h e s i z e a p e n t o f u r a n o s - 3 - y l g l y c i n a t e by degradation of a hexofuranos-3-yl g l y c i n a t e , i t was decided to attempt - 90 -the condensation of methyl n i t r o a c e t a t e d i r e c t l y w i t h pentos-3-uloses. 3.1.1 Condensation of Methyl N i t r o a c e t a t e with 5-0_-Benzoyl-1, 2-0-isopropylidene-ct-D-ery thro-pentos-3-ulose (182) . The ketose 182 was o b t a i n e d by a procedure which began with D-xylose. A c c o r d i n g to the procedure d e s c r i b e d 192 by Baker and Schaub , the di-0_-isopropylidene compound 18 3, prepared by a c i d c a t a l y s e d condensation of acetone with D-xylose, was t r e a t e d w i t h d i l u t e a c i d to y i e l d the monoacetone xylose 184. Compound 184 was then r e a c t e d with D - X Y L O S E 1 8 3 185 1 8 4 - 91 -1.1 e q u i v a l e n t s o f benzoyl c h l o r i d e to a f f o r d a product mixture from which 5-0_-benzoy 1-1, 2 - 0 - i s o p r o p y l i d e n e - a -D-xylofuranose (185) was separated by column chromatography i n an 84% y i e l d . Two methods of o x i d a t i o n of compound 185 were employed. In the f i r s t method, ruthenium t e t r o x i d e was used as an oxidant, a c c o r d i n g to a procedure r e p o r t e d by 19 3 R.F. Nutt and co-workers . In our hands, however, the y i e l d s of the ketose 182 were too low to be u s e f u l due to concomitant formation of the product of oxygen i n s e r t i o n , namely, 6-0_-benzoyl-3-deoxy-l, 2-0_-isopropylidene-3-oxa-19 3 g-D-erythro-hexos-4-ulose (186) B z 0 B z O - i In the second method,the o x i d a t i o n of compound 185 was accomplished u s i n g phosphorus pentoxide as the o x i d a n t and a c e t i c anhydride as the " a c t i v a t i n g " agent. Ketose 182 was thus o b t a i n e d i n >80% y i e l d . To a s o l u t i o n of the ketose 182 and ammonium acet a t e (1.1 equiv.) i n anhydrous N,N-dimethylformamide - 92 -(DMF) was added methyl n i t r o a c e t a t e (2.1 e q u i v . ) . A f t e r the r e a c t i o n was s t i r r e d f o r 2 hours, i t was worked-up to y i e l d a dark amber syrup. The t . l . c . o f the crude product, u s i n g 8:2 benzene-ethyl a c e t a t e as the developer, showed 182 NH^OAc MNA 1 DMF B z O 0 2 N C H 0-4— C 0 2 M e 1 8 7 R=H 1 8 8 R=Ac the presence of two components wi t h values of 0.35 and 0.45. I t was assumed t h a t one o f these products was the methyl n i t r o a c e t a t e adduct 187 and, t h e r e f o r e , the crude product mixture was d i s s o l v e d i n a c e t i c anhydride and heated to ^90° f o r 5 hours i n the presence of a c a t a l y t i c amount of £-toluenesulphonic a c i d monohydrate (p-TSA) i n order to convert compound 18 7 i n t o i t s more s t a b l e 3—0—acetyl d e r i v a t i v e 188. S u r p r i s i n g l y , however, the r e a c t i o n e f f e c t e d the t o t a l c o n v e r s i o n of the lower R^ m a t e r i a l i n t o the hi g h e r R^ m a t e r i a l together w i t h s u b s t a n t i a l decomposition of the r e a c t a n t s . Column chromatography of the crude r e a c t i o n mixture a f f o r d e d (E) and (Z_) -5-0_-benzoyl-3-deoxy-1,2-0_-isopropylidene-3-C-nitro (methoxycarbonyl) methylene-- 93 -g-D-erythro-pentofuranose (189) i n an o v e r a l l y i e l d of 23% from compound 185. The f i r s t i n d i c a t i o n of the structure of compound 189 appeared when the t . l . c . plates of the condensation and acetylation reactions were sprayed with a d i l u t e solution of potassium permanganate. A positive r e s u l t for unsatura-tion was exhibited by the higher R^ component. I t i s therefore suggested that the condensation reaction i n i t i a l l y produced the adduct 187 which i n turn underwent spontaneous dehydration to afford a mixture of compound 187 (R^ 0.35) and the unsaturated n i t r o ester 189 (R^ 0.45). The sub-sequent acid catalyzed acetylation conditions then drove the dehydration to completion to give compound 189 as the sole product. The location of the carbon-carbon double bond i n the branch-chain was evidenced by the s h i f t to lower 194 frequencies of the in f r a - r e d absorptions of the ester and n i t r o f u n c t i o n a l i t i e s . The i . r . spectrum of compound 189 exhibits an ester absorption at 1730 cm 1 and a n i t r o absorption at 15 45 cm \ whereas the analogous absorptions for methyl nitroacetate occur at 1755 and 1570 cm 1 . The p.m.r. spectrum of compound 189 revealed a mixture of geometrical isomers. From the r e l a t i v e heights of methyl ester signals at 6 3.90 and 6 3.94, the r a t i o of isomers was calculated to be 1.9:1. The C-2 proton of the major isomer, designated as H-2A, occurred at 65.52 as a - 94 -quartet with coupling constants of ^ = 4.8 Hz and J2 4 = 1.8 Hz while the C-2 proton of the minor isomer, designated as H-2B, occurred s l i g h t l y downfield at 65.67 with i d e n t i c a l coupling constants. The magnitude of the 3^ 4 values i s consistent with values obtained for other systems involving a 195 ^-contribution to long-range couplings . In the un-saturated hexofuranoses 190-19 3 the magnitude of the H-2, 190 R=0Ac R 1 = C 0 2 M e 192 R = OMs R, =C0 2 Me 191 R=C0 2 Me R 1 = 0Ac 193 R= H R, =C0 2 Me H-4 coupling was also i n d i c a t i v e of the configuration at 179 177 C-4 with the ribo sugars 190 and 191 possessing J values of 1.5 Hz and 2.0 Hz, respectively, while the xylo 19 6 196 sugars 192 and 193 had J values equal to 0.0. The elemental analysis of compound 189 was i n agreement with i t s designated structure. Because of the low y i e l d of the product i t was decided to attempt the condensation of methyl nitroacetate with the 5-0_-benzyl ketose 194. - 95 -3.1.2 Condensation of Methyl N i t r o a c e t a t e with 5-0_-B e n z y l - 1 , 2 - 0 - i s o p r o p y l i d e n e - c t - D - e r y t h r o - p e n t o s -3-ulose (194). T o s y l a t i o n of monoacetone xylose 184 and sub-sequent treatment of the r e s u l t i n g 5-0_-tosyl d e r i v a t i v e 195 with an e q u i v a l e n t of base gave 1, 2-0_-isopropylidene-3 , 5-19 7 anhydro-a-D-xylofuranose (196) . Compound 196 was then r e a c t e d with sodium benzylate to a f f o r d the 5-0_-benzyl 198 . 199 xylose 197 which was o x i d i z e d u s i n g the ruthenium dioxide-sodium p e r i o d a t e method to a f f o r d the 5-0_-benzyl ketose 194. 194 197 - 96 -The condensation o f methyl n i t r o a c e t a t e and ketose 19 4 was c a r r i e d out as p r e v i o u s l y d e s c r i b e d f o r the 5-0_-benzoyl ketose 182 and the product o f the r e a c t i o n was s u b j e c t e d to the same a c i d c a t a l y z e d a c e t y l a t i o n c o n d i t i o n s . Chromatography of the r e s u l t i n g crude product mixture on a column of s i l i c a g e l , which was packed and e l u t e d with 9:1 benzene-ethyl a c e t a t e , a f f o r d e d two com-ponents w i t h R f values o f 0.50 and 0.28. The p.m.r. spectrum o f the lower m a t e r i a l (58% by weight, based on ketose) showed t h a t i t was a mixture of compounds c o n t a i n i n g approximately 80% 5-benzyl ketose 194. The other f r a c t i o n s of t h i s component were u n i d e n t i f i e d although the p.m.r. spectrum d i d not i n d i c a t e the presence of a methyl e s t e r nor d i d the i . r . spectrum show any a b s o r p t i o n s a t t r i b u t a b l e to a n i t r o group. The h i g h e r R^ m a t e r i a l (5% y i e l d ) possessed c h a r a c t e r i s t i c s of an a, 8-unsaturated c t - n i t r o e s t e r i n t h a t when a t . l . c . o f t h i s component was sprayed w i t h potassium permanganate a p o s i t i v e t e s t f o r u n s a t u r a t i o n was o b t a i n e d . The i . r . spectrum a l s o e x h i b i t e d e s t e r and n i t r o a b s o r p t i o n s at 1735 and 1545 cm - 1, r e s p e c t i v e l y . I t i s t h e r e f o r e pro-posed t h a t t h i s component i s 5-0_-benzyl-3-deoxy-l, 2-0_-is o p r o p y l i d e n e - 3 - C - n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l e n e - a - g -e r y t h r o - p e n t o f u r a n o s e (198). In support o f t h i s p r o p o s a l , the p.m.r. spectrum o f compound 198 e x h i b i t e d resonances f o r a t o t a l o f 21 proton s . U n l i k e the unsaturated a - n i t r o e s t e r 189, however, the - 97 -BnO- 1) MNA , NH/OAc » 2) Ac20, p-TSA-H20 BnO n.m.r. spectrum o f compound 198 appeared to be of almost e x c l u s i v e l y one g e o m e t r i c a l isomer. The resonance of H-2 appeared a t 65.48 as a q u a r t e t w i t h a ^ value of 1.5 Hz which, as p r e v i o u s l y d i s c u s s e d , i s t y p i c a l f o r compounds of such s t e r e o c h e m i s t r y . U n f o r t u n a t e l y , a s a t i s f a c t o r y a n a l y s i s o f compound 198 c o u l d not be ob t a i n e d to h e l p s u b s t a n t i a t e i t s proposed s t r u c t u r e , and because of the extremely low y i e l d o f the product i t was decided t o abandon t h i s approach i n favour o f condensations w i t h the more r e a d i l y a v a i l a b l e hexofuran-3-ulose 25. 3.2 Condensation of Methyl N i t r o a c e t a t e with 1, 2 : 5 , 6-Di-0_-is o p r o p y l i d e n e - a - D - r i b o - h e x o f u r a n o s - 3 - u l o s e (25). - 98 -3.2.1 S y n t h e s i s o f 1,2 :5 , 6-Di-0_-isopropylidene-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-a l l o f u r a n o s e (162) . K o r n i l o v , Paidak and Zhdanov e f f e c t e d the con-densation o f e t h y l n i t r o a c e t a t e w i t h d i i s o p r o p y l i d e n e -aldehydo-L-arabinose i n an aqueous a l c o h o l i c medium us i n g ammonium acetate as the condensing agent. However, the use of the ketose 2_5_ i n an aqueous medium r e s u l t s i n the formation o f an u n r e a c t i v e hydrate 199. In f a c t , the pr o d u c t i o n o f the ketose 25_ i n v o l v e s ruthenium t e t r o x i d e o x i d a t i o n 5 8 , 6 6 of the commercially a v a i l a b l e d i i s o p r o p y l i d e n e glucose 200 to a f f o r d q u a n t i t a t i v e l y the ketose hydrate 199 which must then be dehydrated i n r e f l u x i n g benzene or toluene. T h e r e f o r e , the f i r s t attempted condensations of ketose 25_ and methyl n i t r o a c e t a t e i n the presence o f ammonium a c e t a t e employed anhydrous methanol, d i m e t h y l -s u l f o x i d e , o r N,N-dimethylformamide as s o l v e n t s . - 99 -The most complete consumption of ketose, as evidenced by t . l . c , and the h i g h e s t y i e l d s o f products were obtained when two e q u i v a l e n t s o f methyl n i t r o a c e t a t e were added to a s o l u t i o n of ketose 2_5 and one e q u i v a l e n t of ammonium acetate i n anhydrous N,N-dimethylformamide. A f t e r 20 minutes, the r e a c t i o n was worked-up and the crude product was chromatographed on a column of s i l i c a g e l , which was packed and e l u t e d w i t h 1:1 benzene-ethyl a c e t a t e , to a f f o r d the ketose 2_5 (R^ 0.44) and a pale yellow syrup (R^ 0.58) which was shown, by i . r . and n.m.r. spectroscopy, to be composed of methyl n i t r o a c e t a t e and the 3-hydroxy-a-n i t r o e s t e r 16 2. In the p.m.r. spectrum o f the syrup (Figure 3) the presence o f methyl n i t r o a c e t a t e was evidenced by a methyl e s t e r resonance a t 6 3.88 and a methylene proton s i g n a l a t 6 5.18. The e x i s t e n c e o f compound 162 was a t t e s t e d to by the presence o f ; CO ? Me C H N 0 2 C 0 2 M e 0 2 N C H 2 C 0 2 M e HO 162 MNA H-2 H-1' MNA i i I i i i i l i i i i I i i i i I i i i i I I I I I I I I I I i I ' l l I i i i i I I I I 6 5 P P M (6) 4 F i g u r e 3 P a r t i a l 100 MHz P.M.R. Spectrum of 1, 2 : 5 ,6-di-0_-Isopro-pylidene-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-a l l o f u r a n o s e (162) i n CDC1.,. o o - 101 -1) a one proton s i n g l e t at 65.83 a t t r i b u t e d to the s i n g l e branch-chain proton 2) a three proton s i n g l e t a t 63.83 a t t r i b u t e d to the methyl e s t e r , and 3) a broad one proton s i g n a l a t 63.47 - which disappeared upon a d d i t i o n of D 20 - a t t r i b u t e d to the t e r t i a r y h y d r o x y l proton a t C-3. I t was c a l c u l a t e d , from the r e l a t i v e i n t e n s i t i e s of the resonances, t h a t the product was a 5:1 mixture of compound 162 and methyl n i t r o a c e t a t e . The i . r . spectrum of the syrup e x h i b i t e d absorp-t i o n s of h y d r o x y l (3450 cm ^ ) , e s t e r (1755 cm ^ ) , and n i t r o groups (1570 cm "*") i n support of the n.m.r. evidence. In an attempt to i s o l a t e compound 162, the product mixture was rechromatographed on a column of s i l i c a g e l u s i n g 8:2 benzene-ethyl acetate as the developer. Sur-p r i s i n g l y , the o n l y carbohydrate m a t e r i a l recovered was the d i i s o p r o p y l i d e n e ketose 2_5. A l l other s o l v e n t mixtures employed i n an attempt to i s o l a t e compound 162 by column chromatography or p r e p a r a t i v e t . l . c . r e s u l t e d i n e i t h e r no s e p a r a t i o n of the product mixture or decomposition o f the n i t r o e s t e r 162 i n t o the s t a r t i n g m a t e r i a l s . - 10 2 -3.2.2 S y n t h e s i s of 3-0_-Acetyl-l, 2 :5 ,6-di-0_-isopropylidene-3-C-[(R,S)nitro(methoxycarbonyl)methyl]-a-D-a l l o f u r a n o s e (165) . I t was decided t h a t the b e s t way to i s o l a t e the condensation product was to " t r a p " i t by d e r i v a t i z i n g the h y d r o x y l group and thereby prevent the decomposition back i n t o s t a r t i n g m a t e r i a l s . A c i d c a t a l y s e d a c e t y l a t i o n was the method of d e r i v a t i z a t i o n chosen due to the t e r t i a r y nature o f the hydroxy group i n v o l v e d . Thus, the r e a c t i o n of ketose 2_5 and methyl n i t r o -a c e t a t e was c a r r i e d out i n the u s u a l way up to the chromato-graphy. The p a r t i a l l y p u r i f i e d and d r i e d product mixture was then d i s s o l v e d i n a c e t i c anhydride and a c a t a l y t i c amount (10-15% w/w) of p_-toluenesulfonic a c i d monohydrate was added to the s o l u t i o n . A f t e r h e a t i n g at 80-85°C f o r 5 hours, the r e a c t i o n mixture was worked-up and the b l a c k product mixture was chromatographed to y i e l d a p a l e y e l l o w syrup which c r y s t a l l i z e d spontaneously upon s t a n d i n g . R e c r y s t a l l i z a t i o n of the crude product a f f o r d e d the 3-0_-a c e t y l n i t r o e s t e r 165 i n 82% y i e l d . The p.m.r. spectrum o f compound 165 (see F i g u r e 4) c l e a r l y shows the s i n g l e t s of the 0_-acetate (62 .08), the methyl e s t e r (63.80) and the branched-chain proton (65.90) and the presence of the n i t r o group was confirmed by an a b s o r p t i o n i n the i . r . spectrum a t 1570 cm 1 . The CO,Me C H N 0 2 - 0 . AcO i 0 1 6 5 i i I > I i i i l l i i l i i I I I I 4h - L-U - L J I I I I I L J I I I U_J L J I I I I I I I i _ 7.0 60 5.0 ' 4 0 30 2 0 10 F i g u r e 4 60 MHz P.M.R. Spectrum of 3 - 0 - A c e t y l - l , 2 : 5 ,6-di-0_-iso-propylidene-3-C- [ (R,S)-nitro(methoxycarbonyl)methyl]-a-D - a l l o f u r a n o s e (165) i n CDCl^. - 104 -elemental a n a l y s i s of compound 165 was a l s o c o n s i s t e n t w i t h i t s proposed s t r u c t u r e . A c 2 0 p - T S A - H 2 0 8 0 - 8 5 ° / 5 h 3.3 Reduction and Proof o f S t r u c t u r e of the Methyl N i t r o - acetate Adducts. The r e d u c t i o n of the g l y c o s y l a - n i t r o e s t e r s 162 and 165 would not only p l a c e the s y n t h e s i s of p o l y o x i n analogues one step nearer, i t would a l s o provide a means of determining the absolute c o n f i g u r a t i o n at C-3 of the r e s u l t i n g g l y c o s y l a-amino e s t e r s . T h i s i s p o s s i b l e 178 87 because Rosenthal e t a l . ' have p r e v i o u s l y s y n t h e s i z e d g l y c o s - 3 - y l amino a c i d s having the gluco c o n f i g u r a t i o n f o r which an unequivocal proof of s t r u c t u r e has been pr o v i d e d and t h e r e f o r e the c o n f i g u r a t i o n of the amino e s t e r s d e r i v e d from the n i t r o e s t e r s can be determined by d i r e c t comparison with a u t h e n t i c samples. The compounds which are b e s t s u i t e d - 105 -f o r such a comparison are L-2 and D-2-(1, 2 : 5 , 6-di-0_-i s o p r o p y l i d e n e - a - D - g l u c o f u r a n o s - 3 - y l ) g l y c i n e (200 and 201, r e s p e c t i v e l y ) and t h e i r c orresponding N - a c e t y l methyl e s t e r s (202 and 20 3, r e s p e c t i v e l y ) . C 0 2 R 1 200 R = H ; R 1 = H 202 R = Ac ; RT =Me 201 R = H 203 R = Ac ; R-k1 3.3.1 Sy n t h e s i s o f Methyl N - a c e t y l - ^ and g-2-(1, 2:5 ,6-di-0_-isopropylidene-a-D-glucof uranos-3-yl) g l y c i n a t e (20.2 and 203) . 200 97 95 Pa l l a d i u m on c h a r c o a l ' , platinum oxide ' 201-203 , „ . , ,186-204 , , , and Raney n i c k e l have been r o u t i n e l y used to reduce n i t r o f u n c t i o n a l i t i e s on sugars. When the p a l l a d i u m o r platinum c a t a l y s t s were used i n attempts to reduce the mixture of the B-hydroxy a - n i t r o e s t e r 162 and methyl n i t r o a c e t a t e , i n a methanolic medium, a very - 106 -complex mixture composed of a great number of components was produced. Even a f t e r repeated chromatography no one pure compound c o u l d be separated o r i d e n t i f i e d . However, when the r e d u c t i o n was repeated u s i n g p a l l a d i u m on char-c o a l i n the presence of a c e t i c anhydride, and the r e s u l t -i n g crude product mixture was chromatographed on a column of s i l i c a g e l u s i n g 10:5:1 benzene-ether-ethanol as the developer, the N - a c e t y l L-amino e s t e r 20 2 (12%) and the N - a c e t y l D-amino e s t e r 20 3 (9%) were i s o l a t e d along w i t h ketose 25_ and a complex mixture of a t l e a s t nine u n i d e n t i f i e d compounds. Compounds 202 and 203 were i d e n t i c a l (p.m.r., 178 o p t i c a l r o t a t i o n ) to a u t h e n t i c samples p r e v i o u s l y prepared However, due to the complex nature o f the product mixture, and because of the low y i e l d s of the i d e n t i f i a b l e compon-ents, no safe i n f e r e n c e c o u l d be made about the c o n f i g u r a t i o n at C-3 o f the n i t r o e s t e r 162. 3.3.2 S y n t h e s i s of 3 - 0 - A c e t y l - l , 2 : 5 , 6 - d i - 0 -i s o p r o p y l i d e n e - 3 - C - (methoxydicarbonyl) -a-p_-a l l o f u r a n o s e oxime (204) . Hydrogenation of a methanolic s o l u t i o n o f the 3-0-acetyl n i t r o e s t e r 165 over p a l l a d i u m on c h a r c o a l and under an atmosphere of hydrogen f o r 48 hours a f f o r d e d one c h r o m a t o g r a p h i c a l l y pure compound *in 9 4% y i e l d . The c h a r a c t e r i s t i c a b s o r p t i o n s of an amine were absent from both the i . r . and p.m.r. s p e c t r a of the product. Instead, - 107 -the p.m.r. spectrum showed on l y one exchangeable proton as a broad s i g n a l between 65.5 - 4.9. From the elemental a n a l y s i s of the product the m o l e c u l a r formula o f the product was c a l c u l a t e d t o be C]_7 H25 N O10 * T ^ e i n t e 9 r a t i ° n of the p.m.r. spectrum agrees e x a c t l y w i t h the presence of t w e n t y - f i v e protons and i t was thus concluded t h a t the r e d u c t i o n of compound 165 had proceeded only as f a r as the oxime 204. 3.3.3 S y n t h e s i s of Methyl L_- and D-2-(1,2:5,6-di-0-i s o p r o p y l i d e n e - a - Q - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (205 and 206). Hydrogenation o f the 3-0_-acetyl n i t r o e s t e r 165 20 7 over f r e s h l y prepared Raney n x c k e l W-4 c a t a l y s t a f f o r d e d , a f t e r column chromatography, a major component 20 5 (67%) and a minor component 206 (8%), both of which were f a i n t l y n i n h y d r i n p o s i t i v e . The decreased i n t e n s i t y o f the c o l o u r r e a c t i o n compared to t h a t e x h i b i t e d by amino a c i d s i s x. • • 4 . 87,178,179 ,196 _ , c h a r a c t e r i s t i c o f a-ammo e s t e r s . Compounds 20 5 and 206 a l s o have p.m.r. s p e c t r a t y p i c a l of a-amino e s t e r s ; i . e . 1) the broad, h i g h f i e l d resonance (6 1.83 and 6 2.08 r e s p e c t i v e l y ) of the two amine protons which was e a s i l y i d e n t i f i e d s i n c e i t r a p i d l y disappeared upon a d d i t i o n of deuterium oxide, and - 108 -2) the broad, lower f i e l d resonance (64.11 and 6 4.02, r e s p e c t i v e l y ) of the branch-chain proton which, a f t e r a d d i t i o n of deuterium oxide, immediately c o l l a p s e d to a sharp s i n g l e t . - 109 -When the p r e v i o u s l y o b t a i n e d oxime 204 was hydrogenated i n the presence of Raney n i c k e l c a t a l y s t , i t a f f o r d e d a product mixture i d e n t i c a l i n composition to t h a t o b t a i n e d by r e d u c t i o n of compound 165. In f a c t , when r e d u c t i o n of compound 165 was i n t e r r u p t e d by removing the c a t a l y s t before a l l of the s t a r t i n g m a t e r i a l was consumed, the chromatography of the r e s u l t i n g mixture of compounds on a column of s i l i c a g e l , u s i n g 9:1 e t h y l a c e t a t e - e t h e r as the developer, a f f o r d e d the s t a r t i n g m a t e r i a l 16 5, the oxime 204 and the a-amino e s t e r s 205 and 206. 3.3.4 S y n t h e s i s of L- and p_-2-(1, 2 : 5 , 6-Di-0_-isopro-p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n e (207 and 208) . The a l k a l i n e h y d r o l y s i s of compounds 205 and 206 provides a ready proof of s t r u c t u r e by e n a b l i n g one to determine 1) the absolute c o n f i g u r a t i o n of the a-carbon of the amino a c i d by c i r c u l a r d i c h r o i s m and 2) the absolute c o n f i g u r a t i o n at C-3 by d i r e c t comparison w i t h the pre-v i o u s l y d e s c r i b e d gluco-a-amino a c i d s 200 and 201. Thus, compound 205 was t r e a t e d with methanolic sodium hydroxide and a f t e r 4 hours a t room temperature the s o l u t i o n was a p p l i e d to a s h o r t column of Rexyn RG-51 (H +) r e s i n i n order to remove the sodium i o n s . The column was e l u t e d w i t h water and those f r a c t i o n s which gave a s t r o n g - 110 -p o s i t i v e n i n h y d r i n r e a c t i o n were c o l l e c t e d and evaporated to. y i e l d the f r e e a-amino a c i d 207 i n an 82% y i e l d . The c i r c u l a r d i c h r o i s m spectrum of compound 207 i n 0.5 N methanolic h y d r o c h l o r i c a c i d e x h i b i t e d a p o s i t i v e Cotton e f f e c t (Ae + 0.97) a t 209 nm. Compound 207 was t h e r e f o r e a s s i g n e d the L-amino a c i d c o n f i g u r a t i o n i n agreement w i t h the p o s i t i v e Cotton e f f e c t e x h i b i t e d by ot h e r L-amino . , 178,179 ,208 a c i d s TABLE 4. Comparison of Phys ica l Constants of Var ious G l y c o s-3 - y l Amino A c i d s . COMPOUND m.p.rO [oc] D c d . 207 189-191 +89.2° A E 2 0 9 + 0.97 200 185.5-186.5 +51.1° A E 2 1 2 + 1.55 208 157-159 + 25.0° A £209 -0.93 201 193.5-195.0 + 35.0° A £ 2 1 2 -1.41 211 U1-U2 +27.4° 202 52-62 ( g l a s s ) + 71.0° 212 s y r u p + 45.9° 203 54-56 ( g l a s s ) + 50.6° - I l l -An examination of the physical c h a r a c t e r i s t i c s of the L-amino acids 200 and 207, (as shown i n Table 4), reveals that s i g n i f i c a n t differences e x i s t between them. A s i g n i f i c a n t melting point depression was also observed for a mixture of the two compounds. I t i s therefore pro-posed that compounds 207 and 200 are diastereomers d i f f e r i n g i n configuration at C-3 of the sugar and that compound 20 7 i s L-2-(1,2:5, 6-di-0_-isopropylidene-a-D-allof uranos-3-yl) glycine. Compound 205 i s thus designated methyl L-2-(3-0-acetyl-1,2:5, 6-di-0_-isopropylidene-a-D-allof uranos-3-yl) glycinate. The hydrolysis of the amino ester 206 afforded the a-amino acid 208 i n a 85% y i e l d . The c i r c u l a r dichroism spectrum of a methanolic 0.5 N hydrochloric acid solution of compound 208 shows a negative Cotton e f f e c t (Ae -.93) at 209 nm and compound 208 i s therefore assumed to possess a D-amino acid configuration. Table 4 shows that s i g n i f i c a n t differences e x i s t i n the physical charac-t e r i s t i c s of D-amino acids 208 and 201 and on th i s basis i t i s proposed that compound 208 i s D-2-(1, 2 : 5 , 6-di-0_-isopropylidene-a-D-allofuranos-3-yl)glycine. I t therefore follows that the a-amino ester 206 must be methyl D-2-( 3-0_-acetyl-l ,2:5 ,6-di-£-isopropylidene-a-D-allof uranos-3-yl)glycinate. In order to provide another basis for the comparison of the gluco and a l i o amino acids, the syntheses - 112 2 0 0 R=H ; RT = H 2 0 J 2 0 2 R = A c ; R 1 = M e — 2 0 3 - 113 -of methyl N - a c e t y l - L and D-2-(1, 2 : 5 ,6-di-0_-isopropylidene-a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (211 and 212) were under-taken. The L-amino e s t e r 20 5 was a c e t y l a t e d u s i n g a c e t i c anhydride i n methanol to a f f o r d , i n q u a n t i t a t i v e y i e l d , a compound whose p.m.r. spectrum and elemental a n a l y s i s are c o n s i s t e n t with i t s d e s i g n a t i o n as methyl N - a c e t y l - L - 2 -( 3-0_-acetyl-l, 2 :5 , 6-di-0_-isopropy l i d e n e - a - D - a l l o f uranos-3-yl) g l y c i n a t e (209) . To h y d r o l y z e the 0_-acetate group, a c a t a l y t i c amount of sodium methoxide was added t o a s o l u t i o n of compound 209 i n methanol. A f t e r 25 minutes the s o l u t i o n was d e - c a t i o n i z e d w i t h Amberlite IRC-50 (H +) r e s i n and evaporated to a f f o r d a g l a s s . The crude product was chromatographed on s i l i c a g e l to a f f o r d pure compound 211 i n 91% y i e l d . The p.m.r. spectrum of the product shows an acetate methyl s i g n a l a t 62.05 and a broad NH resonance at 66.83 t y p i c a l of amides. The NH resonance e x i s t s as a doublet and i s coupled to the H - l 1 doublet a t 65.2 2 w i t h a c o u p l i n g constant of 9.5 Hz. With the a d d i t i o n o f deuterium oxide t o the sample, the amide resonance d i s -appeared, the H - l ' s i g n a l c o l l a p s e d to a s i n g l e t , and a broad s i n g l e t at 63.09, a t t r i b u t e d to the C-3 h y d r o x y l , disappeared. In a d d i t i o n to the p.m.r. spectrum, the elemental a n a l y s i s i s a l s o i n agreement with the proposed s t r u c t u r e o f compound 211. - 114 -The a c e t y l a t i o n o f compound 206 and the subsequent h y d r o l y s i s o f the 0-acetate of the i n t e r m e d i a t e methyl N-acetyl-D-2- ( 3-0_-acetyl-l ,2:5, 6 - d i - 0 - i s o p r o p y l i d e n e - a - D -a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (210) y i e l d e d methyl N - a c e t y l -D-2- (1, 2: 5 , 6 - d i - 0 _ - i s o p r o p y l i dene-a-D-allof uranos-3-yl) -g l y c i n a t e (212) i n an o v e r a l l 64% y i e l d . In the p.m.r. spectrum of compound 212 the NHAc proton appears as a broad doublet a t 6 7.08 coupled to the H - l ' proton resonance at 65.25 with a J value of 10.1 Hz, and the f r e e C-3 h y d r o x y l i s shown as a broad s i n g l e t a t 62.54. Table 4 shows t h a t the N - a c e t y l amino e s t e r s 211 and 212 and the gluco N - a c e t y l amino e s t e r s 20 2 and 20 3. have d i s t i n c t l y d i f f e r e n t p h y s i c a l c h a r a c t e r i s t i c s , m e l t i n g p o i n t s , and o p t i c a l r o t a t i o n s . T h i s , t o g e t h e r with the d i s s i m i l a r p.m.r. s p e c t r a (shown i n F i g u r e s 5 and 6), leaves no doubt t h a t the fo u r compounds are diastereomers and i s f u r t h e r support of the a l i o c o n f i g u r a t i o n a s s i g n e d to compounds 211 and 212. In s e c t i o n 3.3.1 i t was r e p o r t e d t h a t the r e d u c t i o n o f the 3-hydroxy a - n i t r o e s t e r 162 over p a l l a d i u m on c h a r c o a l , i n the presence o f a c e t i c anhydride, a f f o r d e d a myriad of products and t h a t the only two products i d e n t i -f i e d were of the gluco c o n f i g u r a t i o n . T h i s apparent c o n t r a d i c t i o n can be e x p l a i n e d by p o s t u l a t i n g t h a t under the c o n d i t i o n s employed f o r the r e d u c t i o n , an e q u i l i b r i u m 7 6 5 / . 3 ? 11 ^ • , , , I , . . . , • . • , I i i • • , i , • , I , , i , , , , , i | i i i i i i i i—i—I i i i i i i i l l P P M ( g l F i g u r e 5 100 MHz P.M.R. Spectrums of Methyl N-acetyl-L-2-(1,2-0_-i s o p r o p y l i d e n e - a - D - g l u c o and a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (202 and 211) i n CDCl-,. P P M ( 6) F i g u r e 6 100 MHz P.M.R. Spectrums of Methyl N - a c e t y l - D - 2 - ( 1 , 2 - 0 - i s o p r o p y l i d e n e -a-D-gluco and a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (203 and 212) i n CDC1.,. - 117 -e x i s t s between the a l i o and gluco condensation p r o d u c t s . I t has been p r e v i o u s l y demonstrated t h a t the condensation product w i l l r e v e r t back i n t o ketose and methyl n i t r o a c e t a t e under c e r t a i n chromatographic con-d i t i o n s so the condensation must be regarded as a r e v e r s i b l e r e a c t i o n u n t i l a c i d c a t a l y z e d a c e t y l a t i o n of the condensation product " t r a p s " i t i n the a l i o c o n f i g u r a -t i o n as the proof of s t r u c t u r e has shown. 3.4 O x i d a t i v e Cleavage of Methyl N-acetyl-L-2 - (1, 2-0_-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (226) with Sodium P e r i o d a t e . The p e r i o d a t e cleavage of the 5 , 6 - v i c i n a l d i o l of a 3-C-aminomethyl-allofuranose branched-chain sugar o f t e n r e s u l t s i n spontaneous condensation of the amine f u n c t i o n -a l i t y w i t h the r e s u l t i n g 5-aldehyde to y i e l d a 5-membered - 118 -c y c l i c aminal. T h i s r e a c t i o n was used to prove the c i s r e l a t i o n s h i p between the 5,6 " t a i l " and the 3-C-acetamino-9 8 methyl branch c h a i n o f compound 213 . Jordaan and co-214 R = C 0 2 E t ; R] = C H 0 21_5 150 workers encountered a s i m i l a r r e a c t i o n when they attempted to cle a v e and reduce the 5,6 d i o l 214. S i m i l a r i l y 205 Rosenthal and R a t c l i f f e proved the a l i o s t e r e o c h e m i s t r y of t h e i r 3-C-carbamoyl sugar 216 by formation o f the aminal 217. - 119 -In order to provide a d d i t i o n a l c o n f i r m a t i o n o f the a l i o c o n f i g u r a t i o n of the a-amino e s t e r 205, i t was decided to attempt such an aminal s y n t h e s i s . 3.4.1 Sy n t h e s i s of Methyl N-acetyl-L-2-(1,2-0-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (226) . The s y n t h e t i c s t r a t e g y o r i g i n a l l y e n v i s i o n e d f o r the s y n t h e s i s of an aminal i n v o l v e d : 1) h y d r o l y s i s o f the 5,6-0_-isopropylidene group of the f u l l y b l o c k e d g l y c o s y l amino a c i d 209 to a f f o r d the 5,6 d i o l 218, 2) o x i d a t i v e cleavage o f the d i o l 218 to a f f o r d the 5-aldehydo sugar 219, and 3) spontaneous c y c l i z a t i o n of compound 219 to a f f o r d the aminal 220. - 120 -Towards t h i s end, compound 209 was d i s s o l v e d i n 66% a c e t i c a c i d and a f t e r 24 hours at room temperature the s o l v e n t was removed to a f f o r d a product which c r y s t a l -l i z e d spontaneously. The main f e a t u r e s of the p.m.r. spectrum o f the product i n chloroform-d^ were: 1> two acetate methyl resonances a t 6 2.08 and 62.06, 2) a methyl e s t e r a b s o r p t i o n a t 6 3.75 3) a t y p i c a l broad doublet of the amide proton at 6 7.23 coupled to the branch-chain proton a t 65.23 with J equal to 8.0 Hz, and 4) two broad s i n g l e proton s i g n a l s a t 64.69 and 64 which exchanged i n D 20 and were assig n e d to the C-5 and 6 h y d r o x y l protons. The elemental a n a l y s i s of the product suggested a molecular formula of C,,H„ cNO i r. and on the b a s i s of t h i s lb ±u and the p.m.r. spectrum the product was designated methyl N - a c e t y l - L - 2 - ( 3-0_-acetyl-l, 2-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (218). S u r p r i s i n g l y , treatment of the c r y s t a l l i n e product, o b t a i n e d from the h y d r o l y s i s of compound 209, with sodium metaperiodate under standard o x i d a t i v e - c l e a v a g e c o n d i t i o n s y i e l d e d the s t a r t i n g m a t e r i a l unchanged. I t was a l s o observed t h a t when the h y d r o l y s i s product was a c e t y l a t e d , u s i n g a c e t i c anhydride and p y r i d i n e , the s o l e product i s o l a t e d (compound 222) had an elemental a n a l y s i s - 121 -and p.m.r. spectrum c o n s i s t e n t with a compound p o s s e s s i n g a methyl N - a c e t y l g l y c i n a t e branch-chain, two 0_-acetate groups and one f r e e h y d r o x y l . The remaining f r e e h y d r o x y l group of compound 222 was f i n a l l y a c e t y l a t e d u s i n g a c e t i c anhydride and p - t o l u e n e s u l p h o n i c a c i d monohydrate at e l e v a t e d temperature to y i e l d the f u l l y b l o c k e d methyl N-a c e t y l - L - 2 - (3,5 , 6 - t r i - 0 _ - a c e t y l - l , 2-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (223). From these r e a c t i o n s i t seemed obvious t h a t the h y d r o l y s i s of compound 209 d i d not r e s u l t i n formation of the d i o l 218 but t h a t i n s t e a d the h y d r o l y s i s was f o l l o w e d by m i g r a t i o n of the 3-0_-acetyl group to e i t h e r the C-5 or C-6 hy d r o x y l to y i e l d compound 221. A c . r - K C0 2Me A c y l m i g r a t i o n s i n carbohydrate chemistry have 206—208 been w e l l documented . Although the a l i o s t e r e o -chemistry a t f i r s t seemed p r o h i b i t i v e , the c o n s t r u c t i o n of - 122 -a molecular model showed t h a t the o r t h o - e s t e r i n t e r m e d i a t e 224 c o u l d be e a s i l y a t t a i n e d from compound 218. Since the movement o f the a c y l group i s , i n the m a j o r i t y o f cases, 20 8 towards a primary h y d r o x y l group , i t was f e l t t h a t compound 221 was probably the product of an i n i t i a l 3-0_ to - 123 -5-0_ m i g r a t i o n f o l l o w e d by a 5-0_ to 6-0 m i g r a t i o n . In order to c o n f i r m t h i s s u s p i c i o n the p.m.r. spectrum of compound 221 i n dimethyl s u l f o x i d e - d ^ was o b t a i n e d (see Fi g u r e 7). The t e r t i a r y C-3 hy d r o x y l proton was e v i d e n t as a s i n g l e t a t 65.46. A t 65.28 was a doublet w i t h a c o u p l i n g constant o f 5.2 Hz which was a t t r i b u t e d to the C-5 hydroxyl p r o t o n . Both of these resonances disappeared upon a d d i t i o n of D2O to the sample. On the b a s i s of t h i s evidence compound 221 was assig n e d as methyl N-acetyl-J^-2- (6- 0 _ - a c e t y l - l , 2-0_-isopropylidene-a-D-allof uranos-3-yl) g l y c i n a t e . I t was obvious t h a t p r i o r to o x i d a t i v e cleavage of the 5,6 " t a i l " the 6-£-acetyl group of compound 221 would have to be removed. T h i s was accomplished by t r e a t -i n g a methanolic s o l u t i o n o f 221 with a c a t a l y t i c amount of sodium methoxide, i n a r e a c t i o n i d e n t i c a l to t h a t employed i n the d e - a c e t y l a t i o n of compound 209, to y i e l d methyl N - a c e t y l - L - 2 - (1, 2-0_-isopropylidene-a-D-allof uranos-3- y D g l y c i n a t e (226) i n a 95% y i e l d . A l t e r n a t e l y compound 226 was prepared by a c i d h y d r o l y s i s of the 5, 6 - i s o p r o p y l i d e n e group of compound 211. The t r i o l 226 was c h a r a c t e r i z e d as i t s d i - a c e t a t e 2 22 i n t h a t a c e t y l a t i o n o f 226 u s i n g a c e t i c anhydride and p y r i d i n e produced a compound t h a t -was i d e n t i c a l ( [ a ] D , p.m.r., i . r . ) to the product o f the a c e t y l a t i o n o f compound 221. 9 8 6 5 PPM ( E ) A 3 2 1 F i g u r e 7 100 MHz P.M.R. Spectrum of Methyl N-acetyl-L.-2 - (6-0_-acetyl-l, 2-0-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (221 ) i n DMSO-d, . 3.4.2 Attempted S y n t h e s i s of 3-C-[(S)-Acetamino-(carbomethoxy) methyl] -1, 2-0_-isopropylidene-a-D-ribo-pentodialdofuranose-5,N-aminal (228) . Compound 226 was d i s s o l v e d i n a 2:1 methanol-water s o l u t i o n and to t h i s was slo w l y added a conc e n t r a t e d water s o l u t i o n o f sodium metaperiodate. A f t e r the a d d i t i o n of 1.1 e q u i v a l e n t s o f p e r i o d a t e the t . l . c . o f the r e a c t i o n showed t h a t a l l of the s t a r t i n g m a t e r i a l (R 0.21, 8:2 benzene-ethanol) had been consumed and only one oth e r spot (R^ 0.40) was e v i d e n t . Work-up of the r e a c t i o n y i e l d e d a c o l o u r l e s s g l a s s . 227 228 R ^ R ^ H 229 R i = A c , R 2 = H 230 RT =R 2 = AC 231 RT = R 2 = C 6 H 5 C O I t was assumed t h a t , s i m i l a r to previous 98 180 205 examples ' ' , the product o f the p e r i o d a t e r e a c t i o n - 126 -was the aldehydo-sugar 227 which was i n e q u i l i b r i u m with the aminal 228. In an attempt to i s o l a t e the aminal, as i t s 5-0_-acetyl d e r i v a t i v e 229 , the crude product of the r e a c t i o n was d i s s o l v e d i n a s o l u t i o n of a c e t i c anhydride-p y r i d i n e f o r 24 hours. Chromatography of the product o f the a c e t y l a t i o n , however, a f f o r d e d only a mixture of a t l e a s t f i v e impure components - none of which c o u l d be reasonable a s s i g n e d a s t r u c t u r e or f u r t h e r p u r i f i e d e a s i l y . Attempts to i s o l a t e the d i - a c e t a t e aminal 230 or the d i -benzoyl aminal 231 by a c i d c a t a l y s e d a c e t y l a t i o n or b e n z o y l a t i o n , r e s p e c t i v e l y , o f the crude product of the p e r i o d a t e r e a c t i o n produced s i m i l a r complex mixtures of products. The attempt to s y n t h e s i z e an aminal from com-pound 226 was t h e r e f o r e abandoned. 3.4.3 S y n t h e s i s of Methyl N-acetyl-L-2-(1,2-0-i s o p r o p y l i d e n e - a - D - r i b o f u r a n o s - 3 - y l ) g l y c i n a t e (232) and Methyl N-acetyl-L-2-(5-0-acety1-1, 2-0_-i sopropy l i d e n e - a - D - r i b o f uranos-3-yl) g l y c i n a t e (233) . In o r d e r to ensure t h a t the o x i d a t i v e cleavage of compound 226 was producing the expected product, namely the 5-aldehydo sugar 227, the r e a c t i o n was repeated and a f t e r t . l . c . had shown a l l the s t a r t i n g m a t e r i a l to be consumed an excess of sodium borohydride was added. A f t e r 15 minutes the r e a c t i o n was worked-up to y i e l d a c o l o u r l e s s - 127 -g l a s s . T h i s product was not c h a r a c t e r i z e d but was immediately d i s s o l v e d i n a c e t i c anhydride and p y r i d i n e f o r 24 hours i n an attempt to a c e t y l a t e the primary h y d r o x y l group produced by the r e d u c t i o n . Chromatography of the crude m a t e r i a l from the a c e t y l a t i o n on a column of s i l i c a g e l , u s i n g 8:2 benzene-ethanol as developer, a f f o r d e d two products 232 and 233. HOHoC A c H N COoMe 226 D I O A 2) BH4 232 Ac 20 pyr. A c O H 2 C ' C 0 2 M e A c H N - f - H -0 HO 0 - j — 233 The p.m.r. spectrum o f the lower component (26%) showed the presence of only one a c e t a t e methyl resonance and t h a t was a t t r i b u t e d to the N - a c e t y l group. A l s o e v i d e n t were the resonances of three exchangeable protons. The low f i e l d d oublet at 66 .95 was, of course, a t t r i b u t e d to the amide p r o t o n . The second exchangeable proton appeared as a broad s i n g l e t a t 65.57 w h i l e the t h i r d was hidden under the o v e r l a p p i n g s i g n a l s (6 4.60 -63.95) o f f o u r o t h e r protons. The presence of t h i s l a s t p roton was assumed from the r e d u c t i o n of the i n t e g r a t e d - 128 -area of the s i g n a l s between 6 4.60 and 63.95 from an e q u i v a l e n t of f i v e protons to four protons upon a d d i t i o n of D 20 to the sample. The second and t h i r d exchangeable protons were assumed to be h y d r o x y l p r o t o n s . In a l l , the p.m.r. spectrum i n d i c a t e d the presence of 21 protons c o n s i s t e n t with the s t r u c t u r e o f the p e n t o f u r a n o s y l d i o l 232. The hi g h r e s o l u t i o n mass spectrum of the lower component was a l s o c o n s i s t e n t w i t h i t b e i n g the d i o l 232. An i n t e n s e peak i n the spectrum o c c u r r e d at a mass-to-charge r a t i o o f 30 4.1013 which corresponded to the mass fragment a r i s i n g from l o s s of a methyl group from the parent i o n C-^H^NOg. T * i e t h e o r e t i c a l value f o r a mass of c i 2 H l 8 N 0 8 w a s 304.1032. The elemental a n a l y s i s of the component a l s o agreed w i t h the assig n e d s t r u c t u r e . The h i g h e r R^ m a t e r i a l gave p h y s i c a l and s p e c t r a l evidence c o n s i s t e n t with i t s s t r u c t u r a l assignment as the 5-0 - a c e t y l sugar 233. The p.m.r. spectrum i n d i c a t e d the presence of one 0_-acetate and one f r e e h y d r o x y l group i n a d d i t i o n to the N - a c e t y l amino e s t e r branch-chain. The high r e s o l u t i o n mass spectrum showed a parent peak a t an m/e value of 261.1349 and a much more i n t e n s e peak, c o r r e s -ponding to l o s s o f CH^/ a t 346.1136. The t h e o r e t i c a l v alues f o r masses of c i 5 H 2 3 N O g a n d C 1 4 H 2 0 N O 9 w e r e 361.1372 and 346.1138, r e s p e c t i v e l y . F i n a l l y , the elemental a n a l y s i s was c o n s i s t e n t with a mo l e c u l a r composition of C^t-H^NOg. Q u a n t i t a t i v e c o n v e r s i o n of the d i o l 232 i n t o i t s 5-0_-acetyl d e r i v a t i v e 233 was accomplished by c o n t i n u i n g the a c e t y l a t i o n r e a c t i o n f o r an a d d i t i o n a l 4 8 hours. - 129 3. 5 S y n t h e s i s of 3-Deoxy-3-C.-ni.tro (methoxycarbonyl) methyl  a l l o f u r a n o s e s . Although, i n our s y n t h e s i s of an analogue of the p o l y o x i n s , our prime o b j e c t i v e was the s y n t h e s i s of a g l y c o s - 3 - y l amino a c i d i n which the 3-C-hydroxyl group was r e t a i n e d , we were a l s o i n t e r e s t e d to see i f the methyl n i t r o a c e t a t e condensation c o u l d provide a route to the , _ , , . , 177,181,182 3-deoxy-3-C-glycosyl amino acxds 3.5.1 Attempted S y n t h e s i s of 5 , 6-Di-0_-acetyl-3-C-facetamino(methoxycarbonyl)methylene]-3-deoxy-1,2-O-isopropylidene - g-D-ribo-hexofuranose (234) . The i n i t i a l attempt to s y n t h e s i z e a 3-deoxy sugar i n v o l v e d the dehydration of the 13r-hydroxy-a-acetamido e s t e r 222. F r e s h l y d i s t i l l e d t h i o n y l c h l o r i d e was added to a p y r i d i n e s o l u t i o n o f compound 222 (kept at -25°) and the mixture was maintained a t 5° o v e r n i g h t . Work-up of the r e a c t i o n mixture y i e l d e d a dark amber syrup which was prone t o decomposition a t room temperature e i t h e r i n s o l u t i o n o r neat. On the assumption t h a t the product was the unsaturated acetamido e s t e r 2 34 the pro-duct was immediately d i s s o l v e d i n methanol and hydrogenated over Raney n i c k e l f o r 24 hours. The s o l u t i o n was then f i l t e r e d and evaporated and the r e s u l t i n g syrup was chromatographed on a column of s i l i c a g e l to a f f o r d a - 130 -s i n g l e product (29%). Quite unexpectedly, the product was shown (p.m.r. [a] , i . r . ) to be the s t a r t i n g compound 222. One p o s s i b l e e x p l a n a t i o n f o r the observed r e s u l t s , i n the absence of p h y s i c a l or s p e c t r o s c o p i c evidence, i s t h a t the compound i s o l a t e d a f t e r the i n i t i a l "dehydration" r e a c t i o n was an i n t e r m e d i a t e a l k y l c h l o r o s u l f i t e (R0S0C1). 221 Such compounds have been i s o l a t e d . Decomposition o f the unstable c h l o r o s u l f i t e would undoubtedly cause f u r t h e r d e gradation of the a c i d s e n s i t i v e carbohydrate and would account f o r the observed i n s t a b i l i t y o f the i s o l a t e d com-pound at room temperature. In the presence of Raney n i c k e l , however, the a l k y l c h l o r o s u l f i t e was e v i d e n t l y h y d r o l y z e d to y i e l d the o r i g i n a l a l c o h o l . - 131 -3.5.2 Sy n t h e s i s o f 3-Deoxy-l, 2 : 5 , 6-di-0_-isopropylidene-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-a l l o f u r a n o s e (236) . As p r e v i o u s l y d e s c r i b e d i n s e c t i o n 3.1, the condensation o f methyl n i t r o a c e t a t e w i t h the 5-0_-benzoyl-pentos-3-ulose 182 gave a mixture of the simple a d d i t i o n product 187 and the unsaturated n i t r o e s t e r 189. The a c i d c a t a l y z e d a c e t y l a t i o n c o n d i t i o n s used i n an attempt t o i s o l a t e compound 187 as i t s 3-0_-acetyl d e r i v a t i v e r e s u l t e d , i n s t e a d , i n dehydration o f compound 187 to a f f o r d the unsaturated compound 189 as the s o l e p r o d u c t . I t was t h e r e f o r e q u i t e s u r p r i s i n g to f i n d t h a t an i d e n t i c a l s e r i e s of r e a c t i o n s u s i n g the ketose 25_ a f f o r d e d o n l y the 3-0_-acetyl d e r i v a t i v e 165 with no t r a c e o f the 3-deoxy-1,2:5, 6-di-0_-isopropylidene-3-C- [ n i t r o (methoxycarbonyl) -methylene]-a-D-ribo-hexofuranose (235). However, when the a c e t y l a t i o n was performed f o r one hour at 120°C, the chromatography of the bl a c k syrup obtained upon work-up of the r e a c t i o n a f f o r d e d the B-acetoxy n i t r o e s t e r 165 (R^ 0.38 i n 8:2 benzene-ethyl a c e t a t e , 63%) and the a,B-. unsaturated n i t r o e s t e r 235 (R^ 0.42, 4%) . The p.m.r. spectrum of compound 235 was complex and showed a mixture o f two components i n an approximate r a t i o o f 1.5:1 as c a l c u l a t e d from the r e l a t i v e h e i g h t s of the methyl e s t e r resonances a t 63.94 and 3.98, r e s p e c t i v e l y . The H-2 protons appeared as superimposed q u a r t e t s a t - 132 -65.50. In the major isomer H-2 was coupled to the anomeric proton (65.89) and H-4 (65.69) with c o u p l i n g constants of 5.0 Hz and 2.0 Hz, r e s p e c t i v e l y , w h i l e i n the minor isomer H-2 was coupled to H - l (65.87) wi t h 2 = 4.8 Hz and H-4 (65.62) with ^ = 2.0 Hz. As can be seen i n Table 5, the low f i e l d r e g i o n of the p.m.r. spectrum o f compound 235 i s very s i m i l a r to t h a t of the p r e v i o u s l y d e s c r i b e d a,B-unsaturated n i t r o e s t e r s 189 and 190. Compound 235 was not c h a r a c t e r i z e d but was immediately reduced to a f f o r d the 3-deoxy-3-C-nitro-(methoxycarbonyl)methyl sugar 236. TABLE 5 Low Field Resonances in the 100 MHz Proton Magne t i c Resonance S p e c t r a of the a , ft-Unsatu ra ted Nitro E s t e r s 189 ,190, and 2 3 5 . COMPOUND H-1 J 1 , 2 H-2 H-4 . (S) (Hz) ( S ) (Hz) ( S ) 1 8 9 ° 6.02 4.8 5.52 1.8 6.00 1 8 9 b 6.05 4.5 5.67 1.8 5.86 190 5.98 4.8 5.48 1.5 5.73 2 3 5 ° 5.89 5.0 5.50 2.0 5.69 2 3 5 b 5.87 4.8 5.50 2.0 5.62 a - denotes major isomer, b - denotes minor isomer - 133 -Sodium borohydride has been used to reduce double bonds of a,^-unsaturated esters and nitroalkenes For the l a t t e r class of compounds, however, the reaction required that the pH of the medium be maintained between the l i m i t s 3-6 i n order to i n h i b i t the formation of the a-carbanion i n the nitroalkane and retard Michael addition to the nitroalkene which would r e s u l t i n dimeric pro-210-211 ducts . For this reason the more acid stable 212 sodium cyanoborohydride was used i n reduction of compound 235 and the pH of the methanolic medium was main-tained between 3-4 by the periodic addition of 1% hydro-- 134 -c h l o r i c a c i d i n methanol. Compound 236 was thus o b t a i n e d i n a 90% y i e l d from 235. The s t e r e o c h e m i s t r y a t C-3 was deduced from the p.m.r. spectrum (see F i g u r e 8). As i n *the case o f the spectrum of the unsaturated e s t e r 235, the spectrum of compound 236 showed the presence of two components. S t e r e o c h e m i c a l l y , the most important resonances were those of the H-2 protons and t h a t of the major product, designated A, appeared as a t r i p l e t a t 65.09 while t h a t of the minor product, d e s i g n a t e d B, appeared as a t r i p l e t a t 64.87. A component r a t i o of A:B equal to 2.2:1 was c a l c u l a t e d from the i n t e g r a t e d areas of the H-2 resonances. Protons H-1A and H-1B e x i s t e d as a s i n g l e doublet a t 65.84 wit h J.. _ = 3.8 Hz. I r r a d i a t i o n of H - l caused the H-2A and H-2B s i g n a l s to c o l l a p s e to doublets w i t h 3 = 4.9 Hz. I t has been amply demonstrated 2"'" 3' ' t h a t H-2, H-3 c o u p l i n g constants of t h i s magnitude i n 3-deoxy-l, 2-0_-i s o p r o p y l i d e n e - h e x o f u r a n o s e systems are i n d i c a t i v e of a l l o -s t e r e o c h e m i s t r y and t h e r e f o r e , compound 236 must be 3-deoxy-1, 2 : 5 ,6-di-0_-isopropylidene-3-C- [ (R,S) - n i t r o (methoxy-carbonyl) methyl] - a - D - a l l o f u r a n o s e . As a f u r t h e r proof of s t r u c t u r e , compound 236 was converted i n t o a number o f p r e v i o u s l y s y n t h e s i z e d compounds of known s t e r e o c h e m i s t r y . Reduction o f the n i t r o group o f 236 by hydro-genation over Raney n i c k e l c a t a l y s t a f f o r d e d the a-amino I l l 1 I I I I I I -I I I I I I I I I I I I I I I 1 I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I 6 5 P P M ( S ) 4 3 F i g u r e 8 P a r t i a l 100 MHz P.M.R. Spectrum of 3-Deoxy-l, 2 : 5 ,6-di-0_-isopropylidene-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-allofuranose (236) i n CDCl,. - 136 -e s t e r s 141 (46%) and 142 (38%). Compound 141 e x h i b i t e d a p.m.r. spectrum superimposable with the spectrum o f the 177 p r e v i o u s l y d e s c r i b e d methyl L.-2- ( 3-deoxy-l, 2 : 5 , 6-di-0_-i s o p r o p y l i d e n e - c t - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e w h ile compound 142 had a p.m.r. spectrum i d e n t i c a l with the corresponding D - g l y c i n a t e . Furthermore, when the T^-amino e s t e r 141 was h y d r o l y z e d w i t h a methanol-water s o l u t i o n o f sodium hydroxide (0.6%) and the product of the r e a c t i o n was passed through a column of RG-51 (H +) c a t i o n exchange r e s i n , the white c r y s t a l l i n e m a t e r i a l i s o l a t e d (62%) possessed a m e l t i n g p o i n t and o p t i c a l r o t a t i o n which i n d i c a t e d i t was L-2 - ( 3-deoxy-l, 2-0_-isopropylidene-a-D-allof uranos-3-yl) -177 g l y c i n e (237) . B e n z o y l a t i o n of the D-amino e s t e r 142 y i e l d e d a compound i d e n t i c a l (m.p., [ d p ) wit h methyl N-benzoyl-D-2- ( 3-deoxy-l ,2:5 ,6-di-0_-i sopropy lidene- o t-D-184 a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (238) 236 141 R = H ; R-| = N H 2 237 142 R = N H 2 ; R I = H 238 R = N H C O C 6 H 5 ; R 1 = H - 137 -As p r e v i o u s l y d e s c r i b e d (see the I n t r o d u c t i o n , s e c t i o n 5) an unambiguous c o n f i r m a t i o n o f the c o n f i g u r a -t i o n a l assignments of these g l y c o s y l a-amino a c i d d e r i v a -177—179 184 t i v e s , s y n t h e s i z e d by Rosenthal and co-workers ' , 179 was p r o v i d e d by c o n v e r t i n g the D-amino e s t e r 142 i n t o 3-deoxy-3-C-[(R)-hydroxymethyl-(N-salicylideneamino)-methyl] -1,2:5, 6- d i - 0 _ - i s o p r o p y l i d e n e - a - p - a l l o f u r a n o s e 180 (148), a p r e v i o u s l y r e p o r t e d compound whose s t r u c t u r e was c o r r e l a t e d with 5-0_- (p-bromophenylsulphonyl) -3-deoxy-3-C-(R)-(ethoxycarbonylformamido)methyl-1,2-0-180 i s o p r o p y l i d e n e - a - D - r i b o f u r a n o s e (147) . The s t r u c t u r e 180 18 3 of compound 147 has been determined by X-ray a n a l y s i s ' 142 °1 C H R=N-0 RO-ll 0 H o 4 -C H 2 0 H 1 0 148 R= salicylidene 0HCHN-|-H 6 4 -C 0 2 E t 147 R = B r ( C 6 H 5 ) S 0 2 - 138 -3.5.3 S y n t h e s i s of 5 , 6 -Di-0_-acetyl-3-deoxy-l, 2-0_-i s o p r o p y l i d e n e - 3 - C - [ ( R , S ) - n i t r o ( m e t h o x y c a r b o n y l ) -m e t h y l ] - a - D - a l l o f u r a n o s e (241) . I t seemed obvious t h a t a necessary requirement f o r the a c i d c a t a l y z e d g e n e r a t i o n of the unsaturated n i t r o e s t e r 235, from compound 162, was the presence of a f r e e t e r t i a r y h y d r o x y l group a t £-3. T h i s was confirmed by an experiment i n which the g - a c e t o x y - a - n i t r o e s t e r 165 was recovered unchanged a f t e r b e i n g heated f o r f o u r hours a t - 139 -120°C i n a c e t i c anhydride c o n t a i n i n g p - t o l u e n e s u l p h o n i c a c i d monohydrate (15% w/w wit h r e s p e c t t o 165). In s e c t i o n 3.4.1 we r e p o r t e d the 0_-3 to 0_-6 m i g r a t i o n o f an a c y l group d u r i n g the a c i d h y d r o l y s i s o f a 5 ,6-0_-isopropylidene b l o c k i n g group. I t was f e l t t h a t i f such a m i g r a t i o n took p l a c e d u r i n g the h y d r o l y s i s o f compound 165 then the r e s u l t i n g B-hydroxy-a-nitro e s t e r 239, would be s u s c e p t i b l e to a c i d c a t a l y z e d dehydration to a f f o r d the di-0_-acetyl unsaturated n i t r o e s t e r 240 . 165 Ac ro n 9°2 M e Compound 165 was d i s s o l v e d i n 66% a c e t i c a c i d and heated f o r f i v e hours a t 50°. The a c e t i c a c i d was then evaporated and the crude product d e c o l o u r i z e d w i t h a c t i v a t e d c h a r c o a l to a f f o r d a c l e a r c o l o u r l e s s syrup which c r y s t a l l i z e d spontaneously upon s t a n d i n g . The elemental a n a l y s i s o f a r e c r y s t a l l i z e d sample was con-s i s t e n t w i t h the d e s i r e d molecular formula of c i 4 H 2 1 N 0 l l - 140 -The p.m.r. spectrum of the sample was o b t a i n e d i n dimethyl s u l f o x i d e - d g i n an attempt to determine the l o c a t i o n o f the a c e t a t e group by the degree of s p l i t t i n g e x h i b i t e d by the h y d r o x y l protons; however, no d e f i n i t e c o n c l u s i o n s c o u l d be drawn. There were f i v e resonances i n the low f i e l d r e g i o n between 65 and 67 which i n t e g r a t e d to a t o t a l of f o u r protons. The branch-chain proton appeared as a s i n g l e t at 66.22 and the anomeric proton s i g n a l was a doublet a t 65.84 with J 1 2 = 4.0 Hz. The three remaining resonances - a sharp s i n g l e t a t 66.44 e q u i v a l e n t to one proton, a. sharp s i n g l e t a t 65.70 e q u i v a l e n t to ^1/2 proton, and a broad s i n g l e t a t 65.51 e q u i v a l e n t t o % l / 2 proton - a l l disappeared a f t e r a d d i t i o n of deuterium oxide to the sample and were a t t r i b u t e d to the two h y d r o x y l protons of the d i a s t e r e o m e r i c mixture. That the compound was a mixture o f two components was f u r t h e r evidenced by the e x i s t e n c e of two methyl e s t e r peaks a t 6 3.79 and 6 3.74. Compound 239 was t h e r e f o r e d e s i g n a t e d 3-, 5-, or 6-0_-acetyl-l, 2-0_-isopropylidene-3-C- [ (R,S) - n i t r o -(methoxycarbonyl)methyl]-a-D-allofuranose. Compound 239 and p - t o l u e n e s u l p h o n i c a c i d mono-hydrate (13% w/w) were d i s s o l v e d i n a c e t i c anhydride and the s o l u t i o n was maintained at 85° f o r 24 hours or u n t i l a l l of the s t a r t i n g m a t e r i a l had been consumed as evidenced. by t . l . c . The r e a c t i o n was worked-up i n the u s u a l manner and the r e s u l t i n g crude product was p u r i f i e d by column - 141 -chromatography t o a f f o r d E and Z-5,6-di-0_-acetyl-3-deoxy-1, 2-0_-isopropylidene-3-C- [ n i t r o (methoxycarbonyl) methylene] -a-D-allofuranose (240) as an i n s e p a r a b l e mixture i n an 88% y i e l d . The n.m.r. of compound 240 e x h i b i t e d the f a m i l i a r p a t t e r n o f low f i e l d s i g n a l s f o r both E and Z_ isomers. The anomeric protons appeared as o v e r - l a p p i n g doublets a t 65.94 and 65.91 with i d e n t i c a l J 1 2 values of 5.0 Hz. The H-2 protons appeared as q u a r t e t s a t 65.59 and 65.48 wi t h J 2 4 = 2.0 Hz and the H-4 protons e x h i b i t e d q u a r t e t s a t 65.84 and 65.73. The elemental a n a l y s i s o f compound 240 a l s o agrees w i t h i t s proposed s t r u c t u r e . The r e d u c t i o n of the carbon-carbon double bond of compound 240 was accomplished w i t h sodium cyanoborohydride at pH 3-4 to a f f o r d the 3-deoxy-3-C-nitro(methoxycarbonyl)-methyl sugar 241 i n 9 7% y i e l d . A f i r s t - o r d e r a n a l y s i s o f the p.m.r. spectrum of 241 (see F i g u r e 9) r e v e a l e d the f o l l o w i n g : 1) the m u l t i p l i c i t y o f resonances showed t h a t , as expected, the compound was a mixture of two components, 2) the anomeric protons appeared as o v e r - l a p p i n g doublets at 65.87 and 65.77, 3) the branch-chain protons showed as doublets a t 65.41 and 65.36, 4) the H-3 protons appeared as o v e r - l a p p i n g F i g u r e 9 P a r t i a l 100 MHz P.M.R. Spectrum of 5,6-di-0-Acetyl-3-deoxy-l, 2-0-iso-propylidene-3-C-[(R,S)-nitro(methoxycarbonyl)methyl]-a-D-allofuranose (241) i n CDC1 - 143 -m u l t i p l e t s between 6 2.9 and 6 3.3, and 5) H-2 and H-4 were shown as o v e r - l a p p i n g s i g n a l s between 6 4.6 and 6 5.1. The d e t a i l s of the l o c a t i o n and c o u p l i n g constants of the resonances were r e v e a l e d by a s e r i e s o f i r r a d i a t i o n experiments, the r e s u l t s of which are summarized i n Table 6. The i r r a d i a t i o n s were performed as f o l l o w s (as u s u a l , the resonances of the predominant component are l a b e l l e d "A" and those the minor component "B")" 1) i r r a d i a t i o n of the H - l resonances r e v e a l e d H-2 resonances a t 64.97 and 64.79, 2) i r r a d i a t i o n of H-l'A a t 65.36 r e v e a l e d H-3A at 63.01 and i r r a d i a t i o n of the doublet a t 64.97 showed t h a t i t was proton H-2A with a c o u p l i n g constants of J~2 3 = 5.0 Hz and 2 = 3.5 Hz. The value of J ^ , ^ = 9.0 Hz was read d i r e c t l y from the s p l i t t i n g of the H-l'A doublet and t h e r e f o r e the 4 value was determined to be 9.0 Hz. 3) a f i r s t o r d e r " a n a l y s i s thus r e v e a l e d t h a t proton H-2B a t 64.79 was coupled to H-1B a t 5.87 w i t h J 1 2 = 4 Hz and to H-3B (^63.1) with J 2 ^ = 5.5 Hz. The 0"2 -j values o f 5.0 and 5.5 Hz f o r the major 213 177 179 and minor isomers, r e s p e c t i v e l y , i n d i c a t e d ' ' an a l i o c o n f i g u r a t i o n f o r compound 241. A d d i t i o n a l p roof of - 144 -T A B L E 6 Chemica l Shi f ts and Coupl ing Cons tan ts of P r o t o n s of 5 , 6 - D i - 0 - a c e t y l - 3 - d e o x y - 1 , 2 - 0 - i sopropy l idene- . 3 - C - [ ( R , S ) - n i t r o (methoxycarbony l ) methy l ] - c c - Q -al lof u r a n o s e ( 241 ). PROTON $ COUPLING CONSTANTS (Hz ) major isomer H-1 5.77 Jl,2= 3.5 H-2 4.97 Jl,2= 3.5 J 2 ) 3 = 5.0 H-3 3.01 J 2 i 3 = 5.0 J 34 =9.0 J 3 > 1 ' = 9.0 H-f 5.36 minor isomer H-1 5.87 J i 2 = 4.0 H-2 4.79 J 1 | 2 = 4 . 0 J 2 ) 3 = 5 . 5 H-3 3.1 J 2,3 =5.5 J 3,1' = 10.0 H-1' 5.41 J 3 j l - = 10.0 i t s s t r u c t u r e was p r o v i d e d by h y d r o l y z i n g the 5,6-0_-i s o p r o p y l i d e n e b l o c k i n g group of the 3-deoxynitro e s t e r 236 and immediately a c e t y l a t i n g the r e s u l t i n g d i o l 242 to y i e l d a compound i d e n t i c a l to the p r e v i o u s l y d e s c r i b e d d i -a c e t y l n i t r o e s t e r 241. An unequivocal proof of s t r u c t u r e of compound 236 was p r o v i d e d i n s e c t i o n 3.5.2. - 145 -2 4 0 241 R=Ac 2 3 6 242 R = H 3.5.4 Syn t h e s i s of 1, 2, 5 , 6-Tetra-0_-acetyl-3-deoxy-3-C - [ ( R , S ) - n i t r o ( m e t h o x y c a r b o n y l ) m e t h y l ] - a , 6 -D - a l l o f u r a n o s e (243). Before a n u c l e o s i d e s y n t h e s i s c o u l d be attempted, the 1,2-0_-isopropylidene b l o c k i n g group of compound 241 had t o be r e p l a c e d by a c e t a t e s . A c i d c a t a l y z e d h y d r o l y s i s of the a c e t a l , f o l l o w e d by a c e t y l a t i o n was not the method of c h o i c e because of the p o s s i b i l i t y o f l a c t o n e formation between the branch-chain e s t e r and the C-2 h y d r o x y l d u r i n g the h y d r o l y s i s . T h i s would present two problems: 1) s i n c e compound 141 i s a mixture of d i a s t e r e o -meric isomers, d i f f e r i n g i n c o n f i g u r a t i o n a t the branch-chain carbon, one isomer might favour l a c t o n i z a t i o n whereas the other might not due to - 146 -s t e r i c c o n s i d e r a t i o n s . The probable formations of a and 8 anomeric products from each of the isomers would thus r e s u l t i n a complex product mixture which c o u l d prove d i f f i c u l t t o separate and/or p u r i f y , and 2) the formation of a l a c t o n e would prevent a c e t y l a t i o n of the C-2 hydroxy. As p r e v i o u s l y d i s c u s s e d i n the I n t r o d u c t i o n ( s e c t i o n 4.2), the absence of a " p a r t i c i p a t i n g " group a t C-2 favours the p r o d u c t i o n of a and B n u c l e o s i d e s whereas the presence of a 2-0_-acetoxy group favours predominant formation of the d e s i r e d B - n u c l e o s i d e . For these reasons the i s o p r o p y l i d e n e group of compound 241 was removed by a c e t o l y s i s to a f f o r d the t e t r a -0_-acetyl n i t r o e s t e r 243 d i r e c t l y . Thus, compound 241 was d i s s o l v e d i n an a c e t i c a c i d s o l u t i o n c o n t a i n i n g 10 e q u i v a l e n t s of a c e t i c anhydride and 0.2 e q u i v a l e n t s of p - t o l u e n e s u l p h o n i c a c i d . A f t e r h e a t i n g a t 80-85° f o r two hours, the r e a c t i o n mixture was worked-up to a f f o r d compound 243 i n 9 8% y i e l d . The p.m.r. spectrum of compound 243 was under-standably complex. The main f e a t u r e s were: 1) the anomeric protons appeared as a doublet a t 66.44 with J, ^ = 4.0 Hz and a doublet a t - 147 -A c O - i 241 O2N(J:H OAC C 0 2 M e 243 66.19 with J, „ = 2.8 Hz and on the b a s i s of the 1 r 213 c o u p l i n g constants were desi g n a t e d as the H-l protons of a and 3 isomers, r e s p e c t i v e l y . The i n t e g r a t e d areas of the resonances gave an a/3 r a t i o o f 1:4. 2) two e s t e r resonances a t 63.92 and 63.84 e q u i v a l e n t t o 3 protons, and 3) f i v e a c e t a t e s i g n a l s between 62.22 and 62.06 e q u i v a l e n t t o 12 prot o n s . Although the p.m.r. spectrum showed t h a t compound 243 was a mixture of a and 3 anomers, no s o l v e n t system c o u l d be found to a f f o r d s e p a r a t i o n of the isomers. N e v e r t h e l e s s , the elemental a n a l y s i s of the mixture was w e l l w i t h i n a c c e p t a b l e l i m i t s . The high r e s o l u t i o n mass spectrum of the mixture showed an i n t e n s e peak corres p o n d i n g to a mass of 390.1048 and a molecular formula of C,cH~nNO,, - 148 -The c a l c u l a t e d mass of the parent i o n -OAc was 390.1036. 3.5.5 Attempted Nuc l e o s i d e S y n t h e s i s w i t h Compound 243. The stannous c h l o r i d e c a t a l y z e d s i l y l H i l b e r t -134-140,146 , _ . , . . Johnson procedure (see I n t r o d u c t i o n , s e c t i o n 4.2.3.2) was the method chosen f o r the attempted condensa-t i o n of the p e r - a c e t y l n i t r o e s t e r 243 with b i s ( t r i m e t h y l -s i l y l ) thymine ( 2 4 4 ) 1 9 1 . Compounds 243 and 244 (2 equiv.) were d i s s o l v e d i n dichloromethane and 1.4 e q u i v a l e n t s o f stannous c h l o r i d e were added. A f t e r s t i r r i n g f o r 24 hours a t room temperature, the s o l u t i o n was d i l u t e d w i t h a s a t u r a t e d aqueous s o l u t i o n of sodium b i c a r b o n a t e and e x t r a c t e d with e t h y l a c e t a t e . The combined e x t r a c t s were then d r i e d (Na2S0 4) and evaporated to y i e l d an amorphous s o l i d . However, i f the s o l i d was c a r e f u l l y washed w i t h chloroform/ then e v a p o r a t i o n of the f i l t r a t e y i e l d e d a yellow syrup which proved to be (p.m.r. spectroscopy) the s t a r t i n g sugar 243. The remaining s o l i d r e v e a l e d i t s e l f to be predominantly thymine. Even when the r e a c t i o n was repeated u s i n g 1,2-d i c h l o r o e t h a n e as the s o l v e n t and the r e a c t i o n temperature was i n c r e a s e d to 63° f o r p e r i o d s up to 24 hours, no evidence of n u c l e o s i d e formation was found. At t h i s time i t was decided to proceed towards the prime o b j e c t i v e of our r e s e a r c h - the s y n t h e s i s of a - 149 -hex o f u r a n o s y l amino a c i d n u c l e o s i d e analogue o f p o l y o x i n J - r a t h e r than pursue the 3-deoxy analogue sequence any f u r t h e r . 3 - 6 Attempted Sy n t h e s i s of 1-[3'-C-((S)-carbomethoxy(amino)— m e t h y l ) - B - D - a l l o f u r a n o s y l ] t h y m i n e . The s y n t h e t i c s t r a t e g y e n v i s i o n e d f o r the s y n t h e s i s o f an analogue of the n u c l e o s i d e moiety of p o l y o x i n J from the L-amino e s t e r 205 i n v o l v e d : 1) b l o c k i n g of the amino f u n c t i o n a l i t y w i t h a t r i -f l u o r o a c e t y l group 2) h y d r o l y s i s of the 5,6-0_-isopropylidene group with accompanying a c e t y l m i g r a t i o n 3) a c e t y l a t i o n of f r e e hydroxy groups 4) a c e t o l y s i s of the 1,2-0_-isopropylidene group 5) condensation o f the p e r - a c e t y l sugar with b i s -( t r i m e t h y l s i l y l ) t h y m i n e , and 6) removal of the a c e t a t e s . 3.6.1 S y n t h e s i s of Methyl L - 2 - ( 3 - 0 - a c e t y l - l , 2 : 5 , 6 - d i -0 _ - i s o p r o p y l i d e n e - c t - D - a l l o f u r a n o s - 3 - y l ) - N - t r i -f l u o r o a c e t y l g l y c i n a t e (245) . T r i f l u o r o a c e t y l a t i o n of methyl L-2-( 3-0_-acetyl-1,2:5,6-di-0_-isopropylidene-a-p_-allofuranos-3-yl) g l y c i n a t e (205) was accomplished by adding t r i f l u o r o a c e t i c anhydride to a c o o l e d (-10°) s o l u t i o n of the sugar i n ch l o r o f o r m -- 150 -p y r i d i n e . A f t e r 1/2 hour the r e a c t i o n was worked-up and the crude product was chromatographed on a column of s i l i c a g e l to a f f o r d compound 245 i n an 85% y i e l d . evidence f o r the e x i s t e n c e of the N - t r i f l u o r o a c e t a t e . Only one resonance disappeared upon a d d i t i o n of deuterium oxide to the sample and t h a t was a broad one-proton doublet at 67.83, t y p i c a l of N - a c e t y l amide protons which are g e n e r a l l y found between 66.6 and 67.0. The c o r r e s p o n d i n g N - a c e t y l compound 209 e x h i b i t e d an amide resonance a t 66.68 and the d o w n - f i e l d s h i f t t o 67.83 may be a t t r i b u t e d to the i n d u c t i v e e f f e c t of the t r i f l u o r o m e t h y 1 group. The branch-chain proton H - l ' appears as a doublet a t 65.88 with J , , =9.0 Hz. In a l l the spectrum showed resonances of 26 protons c o n s i s t e n t with the proposed s t r u c t u r e of compound 245. C o n f i r m a t i o n of the s t r u c t u r e was p r o v i d e d by the elemental a n a l y s i s . The p.m.r. spectrum of compound 245 gave i n d i r e c t - 151 -3.6.2 S y n t h e s i s of Methyl L - 2 - ( 3 , 5 , 6 - t r i - 0 - a c e t y l - l , 2 - 0 -i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y 1 ) - N - t r i -f l u o r o a c e t y l g l y c i n a t e (246) and Methyl L-2-( 5 , 6 - d i - 0 - a c e t y l - l , 2 - 0 - i s o p r o p y l i d e n e - a - D -a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (247) . In s e c t i o n 3.4.1 we d e s c r i b e d the h y d r o l y s i s of the 5,6-0_-isopropylidine group of compound 209 and the accompanying m i g r a t i o n of the 3-0_-acetyl group to the C-6 h y d r o x y l . I t was expected t h a t a s i m i l a r a c y l m i g r a t i o n would occur d u r i n g the h y d r o l y s i s of compound 245. With t h i s i n mind, the f u l l y b l o c k e d amino e s t e r 245 was d i s s o l v e d i n 66% a c e t i c a c i d and the s o l u t i o n was s t i r r e d f o r 5 hours at 22° and a f u r t h e r 4 h a t 40° a t which time a t . l . c . o f the s o l u t i o n , u s i n g 1:1 benzene-e t h y l acetate as the developer, showed t h a t the s t a r t i n g m a t e r i a l (R^ 0.57) had been consumed to y i e l d a product w i t h an R f value of 0.30. The product i s o l a t e d was not c h a r a c t e r i z e d but was immediately d r i e d and then a c e t y l a t e d with a c e t i c a n h y d r i d e - p y r i d i n e , f o r 13 hours, to a c e t y l a t e primary and secondary h y d r o x y l groups. T . l . c . of the r e a c t i o n mixture showed the presence of two products w i t h R^ values of 0.29 and 0.41. The r e a c t i o n mixture was worked-up and the crude product chromatographed on a column of s i l i c a g e l to a f f o r d a t r a c e amount of the s t a r t -i n g m a t e r i a l 245 (1%) and the two lower R_ components. - 152 -The 0.41 component (29%) possessed a p.m.r. spectrum w i t h the f o l l o w i n g f e a t u r e s : (see F i g u r e 10) 1) the usual resonances o f the branch-chain; methyl e s t e r (63.78) and H - l ' (65.83) coupled to the broad doublet of the amide proton (67.64) wi t h J = 10.0 Hz. 2) three a c e t a t e s i g n a l s , a t 62.10, 62.06 and 62.03, e q u i v a l e n t to a t o t a l o f 9 protons 3) methyl resonances a t t r i b u t e d to an i s o p r o p y -l i d e n e group, and 4) s i x resonances c o n s i s t e n t with H - l , 2, 4, 5 and 6 of an a - a l l o f u r a n o s e system. The l a c k of evidence of any f r e e h y d r o x y l groups, the presence of three a c e t a t e s i g n a l s and an elemental a n a l y s i s and h i g h r e s o l u t i o n mass spectrum c o r r e s p o n d i n g to a molecular formula of Cs~H„,N0,-F-, i n d i c a t e d t h a t the 20 26 12 3 R^ 0.41,component was the f u l l y b l o c k e d t r i - 0 - a c e t y l sugar 246 . The lower R^ component was i s o l a t e d as a c r y s t a l -l i n e s o l i d (65%). I t s p.m.r. spectrum (see F i g u r e 11) was s i m i l a r to t h a t of compound 246 with the f o l l o w i n g important d i f f e r e n c e s : 1) the presence of only two acetate resonances at 62.13 and 62.06, and. 2) the presence o f a sharp s i n g l e t a t 6 3.93 e q u i v a l e n t to one proton and which exchanged i n deuterium oxide. AcO-i AcCM C02Me GFXON-M-H 3 H i I « J H-1 H-1' V 7 5 J5 . 6 b = 5 5 J 5,6a 2.5 C 0 2 M e H-6a H-6b J 6 a , 6 b = 1 2 5 P P M ( S) Figure 10 P a r t i a l 100 MHz P.M.R. Spectrum of Methyl L-2-(3,5,6-tri-0-acetyl-l 0-isopropylidene-a-D-allofuranos-3-yl)-N-trifluoroacetyl glycinate i n CDC1-,. F i g u r e 11 - 155 -The elemental a n a l y s i s a l s o agreed w i t h a compound b e a r i n g two a c e t y l groups and one f r e e hydroxy. Reasoning t h a t the a c e t y l a t i o n c o n d i t i o n s were s u f f i c i e n t to a c e t y l a t e a l l primary and secondary h y d r o x y l s , the lower R^ component must t h e r e f o r e be the 3-hydroxy-5,6-di-0_-acety 1 sugar 24 7 . 246 247 - 156 -I t i s p o s t u l a t e d t h a t the a c i d h y d r o l y s i s of compound 245 y i e l d e d the d i o l 24 8 which underwent an a c y l m i g r a t i o n from the C-3 h y d r o x y l to give the d i o l 249 to the extent of ^70%. The a c e t y l a t i o n of the mixture of compounds 248 and 249 i n a c e t i c a n h y d r i d e - p y r i d i n e thus a f f o r d e d a mixture of the t r i - 0 - a c e t a t e 246 and the d i - 0 -acetate 24 7 i n the same p r o p o r t i o n s as the parent d i o l s . T h i s hypothesis was supported by an experiment i n which compound 247 was recovered unchanged from a s o l u t i o n of a c e t i c a n h y d r i d e - p y r i d i n e a f t e r a p e r i o d of 48 hours. The c o n v e r s i o n of 247 i n t o 246 was, however, accomplished u s i n g a c i d c a t a l y z e d c o n d i t i o n s a t e l e v a t e d temperatures. S i m i l a r l y , compound 246 was s y n t h e s i z e d d i r e c t l y from the h y d r o l y s i s mixture by a c i d c a t a l y z e d a c e t y l a t i o n a t 110° i n a 79% y i e l d based on the s t a r t i n g compound 245. 3.6.3 S y n t h e s i s of Methyl L-2-(1, 2 , 3 ,5 ,6-penta-0_-a c e t y l - a , B - D - a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o -a c e t y l g l y c i n a t e ( 250) . Compound 246 was d i s s o l v e d i n a mixture of 1:1 a c e t i c a n h y d r i d e - a c e t i c a c i d and p - t o l u e n e s u l p h o n i c a c i d monohydrate and the r e s u l t i n g s o l u t i o n was heated at 110° f o r two hours u n t i l the t . l . c . (6:4 benzene-ethyl acetate) showed t h a t the s t a r t i n g m a t e r i a l had been consumed. The - 157 -crude product i s o l a t e d from the r e a c t i o n mixture was chromatographed to a f f o r d the p e r - a c e t y l a t e d compound 250 i n a 47% y i e l d . From i t s p.m.r. spectrum (see F i g u r e 12) i t was e v i d e n t t h a t compound 250 was a mixture of a and B anomers. The anomeric proton of the minor isomer appeared as a doublet a t 66.42 coupled to the H-2 doublet a t 65.64 wi t h J, = 5.1 Hz. H-l and H-2 of the major isomer appeared i , z as superimposed s i g n a l s a t ^66.02. When the spectrum of compound 250 was o b t a i n e d a t 270 MHz, the s i g n a l s were separated i n t o two s i n g l e t s at 66.01 and 66.04 and on 189 t h i s b a s i s the major isomer i s p o s t u l a t e d to be the 3 isomer while the minor isomer i s p o s t u l a t e d to be the a isomer. 2 4 6 Ac 2 0 AcOH p-TSA-H 20 1107 2h C F o C O N 6 H OAc AcO OAc 2 5 0 H-5a 6 P P M ( 6 ) 5 4 F i g u r e 12 P a r t i a l 100 MHz P.M.R. Spectrum of Methyl L-2-(1, 2 , 3 ,5 , 6-penta-0_-acetyl-a , B - D - a l l o f uranos-3-yl) - N - t r i f l u o r o a c e t y l g l y c i n a t e (250) i n CDC1-.. - 159 -3.6.4 Attempted Nuc l e o s i d e Syntheses Using P e r - a c y l Amino E s t e r 250. A number of u n s u c c e s s f u l attempts were made to s y n t h e s i z e n u c l e o s i d e s from compound 250 u s i n g the stannous c h l o r i d e c a t a l y z e d s i l y l H i l b e r t - J o h n s o n and f u s i o n methods (see I n t r o d u c t i o n , s e c t i o n s 4.2.1 and 4.2.3). Although the primary o b j e c t i v e was the s y n t h e s i s o f a thymine n u c l e o s i d e to serve as an analogue of p o l y o x i n J , some attempts were a l s o made to s y n t h e s i z e purine n u c l e o s i d e s of compound 250. 3.6.4.1 Stannous C h l o r i d e C a t a l y z e d S i l y l H i l b e r t - Johnson Procedure. The r e a c t i o n o f compound 250 with N - b e n z o y l -N , 9 - b i s - ( t r i m e t h y l s i l y l ) adenine (114) i n 1 , 2 - d i c h l o r o -ethane a t 70° and i n the presence of 1.4 e q u i v a l e n t s of stannous c h l o r i d e produced no d i s c e r n a b l e n u c l e o s i d e pro-d u c t s . The t . l . c . of the product i s o l a t e d a f t e r work-up of the r e a c t i o n showed the presence of onl y the s t a r t i n g m a t e r i a l 250 and N-benzoyl adenine and f o l l o w i n g removal of the p u r i n e , compound 250 was recovered i n an 86% y i e l d . S i m i l a r r e s u l t s were ob t a i n e d when b i s ( t r i m e t h y l -s i l y l ) thymine 244 was used as the base and even the use of more p o l a r a c e t o n i t r i l e as the s o l v e n t produced no d i s c e r n a b l e r e a c t i o n . - 160 -3.6.4.2 Fusion Procedures. S y n t h e s i s of 1,1'-Anhydro-2,3,5,6-tetra-0-acetyl-3-C-(R)-methoxycarbonyl-1 ( R ) , 1 ' ( S ) - N - t r i f l u o r o a c e t o e p i m o - a - D - a l l o -furanose (252) . The f u s i o n of compound 250 w i t h N-benzoyl adenine, 2 , 6 - d i c h l o r o p u r i n e o r s i l y l a t e d N-benzoyl adenine 114, with o r without a c i d c a t a l y s t , produced no n u c l e o s i d e products. I t was t h e r e f o r e decided to convert compound 250 i n t o i t s more r e a c t i v e bromo sugar d e r i v a t i v e 251 p r i o r to the f u s i o n r e a c t i o n . Compound 250 was d i s s o l v e d i n dichloromethane, the s o l u t i o n was c o o l e d to 0°, and a slow stream o f anhydrous hydrogen bromide was bubbled through the s o l u t i o n f o r 2 1/2 hours. The r e s i d u a l a c i d i c components were then removed by d i s t i l l a t i o n under reduced pressure and the r e s u l t i n g bromo-sugar 251 was i n t i m a t e l y mixed with 1.8 e q u i v a l e n t s of s i l y l a t e d N-benzoyl adenine 114. Th i s mixture was fused f o r 20 minutes at 160°/ 15 t o r r to y i e l d , a f t e r work-up, a crude product t h a t was shown by t . l . c . (6:4 benzene-ethyl acetate) to be composed o f two components. Column chromatography o f the crude product a f f o r d e d a h i g h R^ component (37%) which was shown to be the s t a r t i n g m a t e r i a l 250 and a low R f component 252 (52%, based on s t a r t i n g m a t e r i a l consumed). T . l . c . had a l r e a d y shown t h a t compound 252 was a c h a r r i n g , non-U.V. a c t i v e compound and t h e r e f o r e probably - 161 -a carbohydrate d e r i v a t i v e r a t h e r than a n u c l e o s i d e . The p.m.r. spectrum (see F i g u r e 13) of 252 confirmed t h i s assumption as there were none of the low f i e l d resonances present t h a t would be expected f o r an N-benzoyl adenine n u c l e o s i d e . S e v e r a l o f the e a s i l y r e c o g n i z a b l e s i g n a l s i n c l u d e d : 1) f o u r a c e t a t e methyl peaks a t 62 .10, 2 .06 ,..2..01 and 2.0 0 2) a methyl e s t e r s i g n a l at 63.76, and 3) resonances a t t r i b u t a b l e to H-4, 5 and 6 of a hexofuranose system. The H-4 doublet a t 65.01 was coupled to the H-5 s e x t e t a t 65.30 which was i n t u r n coupled to two H-6 q u a r t e t s a t 64.44 and 4.01. T h i s l e f t t h r e e unaccounted-for resonances, each e q u i v a l e n t to one proton. The f i r s t appeared as a broadened t r i p l e t a t 65.59, the second as a broad doublet or over-l a p p i n g doublets a t 6 5 .39 and the t h i r d as a broad s i n g l e t at 64.76. N o t i c e a b l y absent was a resonance a t t r i b u t a b l e to an amide proton or any other exchangeable proton, s i n c e the a d d i t i o n o f deuterium oxide to the n.m.r. sample d i d not change i t s p.m.r. spectrum. I t was t h e r e f o r e assumed t h a t the proton of the branch-chain n i t r o g e n had been s u b s t i t u t e d by an a c y l or a l k y l group to a f f o r d a t e r t i a r y amine. F i g u r e 13 P a r t i a l 100 MHz P.M.R. Spectrum of 1,1'-Anhydro-2,3,5,6-tetra-0_-acetyl-3-C- (R) -methoxycarbonylmethyl-1 (R) ,1' (S) -N - t r i f l u o r o a c e t o e p i m o - B - D - a l l o f u r a n o s e (252) i n CDCl-.. - 163 -The f i r s t c l u e to the i d e n t i t y of compound 252 was the l i n e broadening and apparent m u l t i p l i c i t y o f the three unassigned resonances. In 1963, Hanessian and 215 H a s k e l l r e p o r t e d the s y n t h e s i s and p.m.r. spectrum of 215 216 5-acetamido-5-deoxy-D-xylofuranose 253 ' . They e x p l a i n e d the e x i s t e n c e of two a c e t y l methyl peaks and two anomeric doublets by p o s t u l a t i n g t h a t 25 3 was a mixture of a and 8 anomers. A s i m i l a r m u l t i p l i c i t y was shown to e x i s t i n the p.m.r. spectrum o f methyl 4-acetamido-4-deoxy-217 218 J^-erythrofuranoside 254 ' w i t h the appearance of two anomeric doublets (64.95 and 65.04), two methoxyl s i n g l e t s (63.36 and 63.41) and two a c e t y l methyl s i n g l e t s (62.08 218 and 62.12). In both cases i t was l a t e r shown t h a t such m u l t i p l i c i t y was not due to a mixture of anomeric com-pounds, but i n s t e a d to the e x i s t e n c e of two r o t a t i o n a l isomers r e s u l t i n g from r e s t r i c t e d r o t a t i o n about the CO-N bond a r i s i n g from resonance c o n j u g a t i o n between the p-o r b i t a l of the n i t r o g e n and the p - o r b i t a l of the T i - e l e c t r o n system. In the case of compound 254 such a c o n j u g a t i o n r e s u l t s i n the d i p o l a r s t r u c t u r e s 254a and 254b. With t h i s i n mind the p.m.r. spectrum of compound 252 was r e a d i l y i n t e r p r e t e d as t h a t o f the two r o t a t i o n a l isomers of 1,1* -anhydro-2 ,3,5 , 6-tetra-0_-acetyl-3-C- (R) -methoxycarbonylmethyl-1(R),1'(S)-N-trifluoroacetoepimino-B-D-allofuranose. The low f i e l d resonance at 65.59 was thus seen as the two o v e r l a p p i n g doublets of the anomeric - 164 -OH A H » 0 H 254 II OH H O / H 254a ,OMe H 3 C / 0 -C II \ O H HO 254b H.OMe proton which are coupled to the o v e r l a p p i n g doublets o f H-2 at 65.39 with 2 % 1.5 Hz. The branch-chain protons thus appeared as a broadened s i n g l e t a t 6 4.76. The high r e s o l u t i o n mass spectrum of compound 252 supported the proposed s t r u c t u r e with the appearance of a peak corre s p o n d i n g to a mass of 514.1194. The c a l c u l a t e d mass o f a c 1 9 H 2 3 N O i 2 F 3 ( M + + ^ i o n w a s 5 1 4 - 1 1 7 3 - T h e elemental a n a l y s i s o f compound 252 a l s o agreed w i t h i t s proposed s t r u c t u r e . The mechanism of the r e a c t i o n i s probably very s i m i l a r to those proposed f o r the formation o f n u c l e o s i d e s (see I n t r o d u c t i o n , s e c t i o n 4.2) wit h the i o n i c i n t e r m e d i a t e s - 165 -A c O -A c O -C F 3 C O N H 251 A c O - i CFoCON H AcO OAc 251 - 166 -255 and 256 b e i n g formed p r i o r t o the i n t r a m o l e c u l a r N-g l y c o s i d a t i o n r e a c t i o n which a f f o r d e d compound 252. 3.6.5 Syn t h e s i s of 1-[2',3',5',6 1-Tetra-O-acetyl-3'-C - ( ( S ) - N - t r i f l u o r o a c e t y l - c a r b o m e t h o x y ( a m i n o ) -m e t h y l ) - B - D - a l l o f u r a n o s y l ] thymine (257). A s u c c e s s f u l n u c l e o s i d e s y n t h e s i s was f i n a l l y 150 accomplished by a p p l i c a t i o n of the r e c e n t l y developed ' 152 153 ' v a r i a t i o n o f the s i l y l H i l b e r t - J o h n s o n procedure i n which s i l v e r t r i f l u o r o m e t h y l s u l f o n a t e ( s i l v e r t r i f l a t e ) i s used as the c a t a l y s t . Compound 250 was d i s s o l v e d i n anhydrous d i c h l o r o -methane and the s o l u t i o n was c o o l e d to 0° p r i o r to i n t r o -d u c t i o n of a slow stream of anhydrous hydrogen bromide. A f t e r 2 hours the s o l v e n t s and r e s i d u a l a c i d were removed by vacuum d i s t i l l a t i o n . The product was r e d i s s o l v e d i n dichloromethane and then slowly added to a mixture of s i l v e r t r i f l a t e and dichloromethane which was maintained at -70°. F i v e minutes a f t e r the a d d i t i o n was complete, a s o l u t i o n o f b i s ( t r i m e t h y l s i l y l ) t h y m i n e (244) was s l o w l y added and the r e s u l t i n g r e a c t i o n mixture was maintained at -70° f o r an a d d i t i o n a l 2 hours b e f o r e a l l o w i n g i t to spontaneously reach room temperature over the next 18 hours. The crude product obtained from the work-up of the r e a c t i o n was chromatographed on a column o f s i l i c a g e l , u s i n g 10:5:1 benzene-ether-ethanol as the developer, - 167 -to a f f o r d the s t a r t i n g m a t e r i a l 250 (54%) and the thymine n u c l e o s i d e 257 (93% based on s t a r t i n g m a t e r i a l consumed). The high r e s o l u t i o n mass spectrum of n u c l e o s i d e 257 showed a parent peak at 6 39.15 30 mass u n i t s ( t h e o r e t i c a l value 639.1515) and a fragment a t 514.1140 corresp o n d i n g to l o s s o f the base from the parent i o n ( t h e o r e t i c a l value 514.1173). The p.m.r. spectrum of compound 257 (see F i g u r e 14) possessed a l l the c h a r a c t e r i s t i c s expected. The presence of a thymine base was shown by a methyl s i n g l e t a t 61.93, a v i n y l proton s i n g l e t a t 67.24 and an amide s i n g l e t a t 69.55. The elements of the branch-chain were e x h i b i t e d i n the methyl e s t e r peak a t 63.84 and the mutually coupled amide and branch-chain proton doublets at 67.63 and 65.61, r e s p e c t i v e l y . The resonances of the s i x remaining protons of the sugar s k e l e t o n and the fo u r a c e t a t e s were a l s o r e a d i l y i d e n t i f i a b l e . One unexpected f e a t u r e was the l a r g e c o u p l i n g constant o f 7.2 Hz between H - l 1 and H-2'. I t i s w e l l e s t a b l i s h e d t h a t the anomeric c o n f i g u r a t i o n o f a l d o f u r a n o s y l d e r i v a t i v e s cannot be 219 determined from the , 2> c o u p l i n g constants o f a s i n g l e isomer and t h e r e f o r e the d e s i g n a t i o n of compound 257 as a 3-nucleoside was based on mechanistic c o n s i d e r a -t i o n s . 134-Compared with the stannous c h l o r i d e method 140 146 ' , the p e r f l u o r o a l k a n e sulphonate or p e r c h l o r a t e I I I I I I I I I ' 1 J ' ' 1 1 1 1 1 I I I I I I I I I I I I I I I I , I I 1 I I I ' l l ' 9 7 6 PPM (S) 5 U 2 Figure 14 P a r t i a l 100 MHz P.M.R. Spectrum of 1- [ 2 1 , 3 1 , 5 1 , 6 ' -Tetra-0_-acetyl-3 ' -C-( (S) -N - t r i f l u o r o a c e t y l - c a r b o m e t h o x y ( a m i n o ) m e t h y l ) - B - D - a l l o f u r a n o s y l ] t h y m i n e (257) i n CDC1 0. - 169 -150-154 catalyzed reaction of s i l y l a t e d pyrimidines, with sugars possessing 2-a-acyloxy groups, shows the same high degree of s t e r e o s e l e c t i v i t y favouring the exclusive forma-tion of 8-N-l-nucleosides from 2-alkyl-l,2-dioxolenium cation intermediates (see Introduction, section 4.2). Indirect spectroscopic evidence for the existence of such an intermediate as the predominant a l k y l a t i n g agent 251 C F 3 S 0 3 A g OSiMe-A c O ' A c O H CFoCON H C 0 2 M e A c O - i A c 0 - 1 C F 3 C 0 N C 0 2 M e C F 3 S 0 3 A c O 0 — 255 A c O - i CF3SO3 AcO 0 258 - 170 -has been pr o v i d e d by p.m.r. o b s e r v a t i o n s 1 3 ^ o f 2 , 3 , 5 - t r i -0_-acetyl-D-ribofuranosyl t r i f l a t e 258 . The formation of the thymine 3-nucleoside 257 i s t h e r e f o r e c o n s i s t e n t with the c a t i o n 255 a c t i n g as a s t e r e o s p e c i f i c a l k y l a t i n g agent. F u r t h e r support f o r the as s i g n e d g - c o n f i g u r a t i o n was pr o v i d e d by the c d . spectrum of compound 257 which 2 26 e x h i b i t e d a p o s i t i v e Cotton e f f e c t a t 268 nm. 3.6.6 Attempted Unblocking of 1- [ 2 1 , 3 1 , 5 ' , 6 ' - T e t r a - 0 -a c e t y l - 3 1 - C - ( S ) - N - t r i f l u o r o a c e t y l - c a r b o m e t h o x y -(amino)methyl-g-D-allofuranosyl]thymine (257) . S e v e r a l methods were employed i n an attempt t o hyd r o l y s e the acetate and e s t e r b l o c k i n g groups of the f u l l y b l o c k e d n u c l e o s i d e 257. D e a c e t y l a t i o n of the 0_-acetyl b l o c k i n g groups 223 usi n g sodium methoxide i n methanol was the i n i t i a l un-b l o c k i n g procedure employed. The n u c l e o s i d e 25 7 was d i s s o l v e d i n anhydrous methanol c o n t a i n i n g a c a t a l y t i c amount of sodium methoxide and a f t e r s e v e r a l hours t . l . c . showed a l a r g e p r o p o r t i o n o f the s t a r t i n g m a t e r i a l s t i l l remained. A f t e r 24 hours, t . l . c . showed t h a t a l l o f the n u c l e o s i d e 25 7 had been consumed and the r e a c t i o n mixture was d e i o n i z e d i n the us u a l manner u s i n g IRC-50 (H +) c a t i o n exchange r e s i n . The amorphous s o l i d i s o l a t e d from the work-up of the r e a c t i o n was then d i s s o l v e d i n deuterium oxide and a p.m.r. spectrum of the s o l u t i o n was o b t a i n e d . I t was immediately obvious t h a t the d e a c e t y l a t i o n r e a c t i o n had not proceeded to completion as the r e g i o n o f the p.m.r. - 171 -spectrum around 6 2 c o n t a i n e d a g r e a t many ac e t a t e methyl resonances of widely v a r y i n g i n t e n s i t i e s . The low f i e l d r e g i o n o f the spectrum, where the anomeric proton resonances are u s u a l l y observed, was s i m i l a r i l y complex i n d i c a t i n g t h a t the crude product was i n f a c t a mixture of a g r e a t many compounds. When the procedure was repeated and the r e a c t i o n mixture was l e f t a t room temperature f o r 7 days, the p.m.r. spectrum of the product obtained from the r e a c t i o n was as complex as t h a t o b t a i n e d p r e v i o u s l y . The complex composition of the product mixture was confirmed by paper chromatography u s i n g 10:4:3 e t h y l a c e t a t e - p y r i d i n e - w a t e r as the s o l v e n t . S i x components were d e t e c t e d on the paper chromatogram with R^ values o f 0.11, 0.15, 0.34, 0.65, 0.73, and 0.81. A l l o f the zones were v i s i b l e under U.V. l i g h t w i t h the e x c e p t i o n of the R f 0.81 component, while the R 0.11, 0.15, 0.34 and 0.81 components gave a p o s i t i v e r e a c t i o n with n i n h y d r i n . The R^ 0.34 zone was by f a r the predominant component o f the product mixture and was t h e r e f o r e i s o l a t e d from a pre-p a r a t i v e s c a l e chromatogram by e l u t i n g the zone with water. U n f o r t u n a t e l y , the p.m.r. spectrum of t h i s component was as complex as t h a t of the o r i g i n a l product mixture. Column chromatography of r e a c t i o n mixture on a column of Bio-Rex 70 (H +) r e s i n a l s o f a i l e d to e f f e c t the s e p a r a t i o n o f an i n d i v i d u a l i d e n t i f i a b l e compound. - 172 -The second method used f o r the attempted de-223 a c e t y l a t i o n of compound 257 employed methanolic ammonia a c c o r d i n g to the procedures o u t l i n e d by Wolfrom and co-workers f o r the unblocking of 0 _ - a c e t y l / N - t r i f l u o r o a c e t y l 214 n u c l e o s i d e s . A c c o r d i n g to t h i s procedure the f u l l y b l o c k e d n u c l e o s i d e was d i s s o l v e d i n anhydrous methanol, the s o l u t i o n was s a t u r a t e d with ammonia at 0 0 and then kept a t room temperature f o r 5 days. E v a p o r a t i o n of the s o l v e n t y i e l d e d a product whose p.m.r. spectrum i n d i c a t e d t h a t incomplete d e a c e t y l a t i o n had once again been achieved. A s i m i l a r i l y complex product mixture was o b t a i n e d a f t e r a prolonged ammonolysis attempt of 14 days d u r a t i o n . Once again, p r e p a r a t i v e paper chromatography f a i l e d to achieve the i s o l a t i o n o f any s i n g l e i d e n t i f i a b l e component. The t h i r d method used f o r the d e a c e t y l a t i o n of the n u c l e o s i d e 257 i n v o l v e d s a t u r a t i n g a c o o l e d (0°C) methanolic s o l u t i o n of 257 w i t h hydrogen c h l o r i d e and keeping the r e s u l t i n g s o l u t i o n a t room temperature f o r 18 hours a c c o r d i n g to the procedure o u t l i n e d by Wolfrom and 214 co-workers . Once again , a complex mixture of p a r t i a l l y d e - a c e t y l a t e d compounds was i s o l a t e d . 224 F i n a l l y , the t r i e t h y l a m i n e - m e t h a n o l and t r i -225 ethylamine-methanol-water methods p r e v i o u s l y employed f o r the d e a c e t y l a t i o n of p_-acetyl n u c l e o s i d e s and p o l y -s a c c h a r i d e s f a i l e d , i n our hands, to completely remove a l l o f the O-acetates o f compound 257. - 173 -Although the f a i l u r e to unblock the p e r - a c e t y l n u c l e o s i d e 257 r e p r e s e n t s a s y n t h e t i c impasse, i n s e v e r a l r e s p e c t s the condensation of methyl n i t r o a c e t a t e with 1,2;5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose (25) as a method f o r producing analogues of the p o l y o x i n complex must s t i l l be c o n s i d e r e d a success. F i r s t l y , i t has been shown (see S e c t i o n 3.3.3) t h a t the r e a c t i o n a f f o r d e d a g l y c o s - 3 - y l a-amino a c i d , i n good y i e l d , t h a t possessed the same absolute c o n f i g u r a t i o n as the n a t u r a l l y occurring compounds and secondly, the r e a c t i o n p r o v i d e d a route to the analogous p e n t o f u r a n o s y l d e r i v a t i v e s (see S e c t i o n 3.4.3). F i n a l l y , the a p p l i c a t i o n o f the t r i f l a t e c a t a l y z e d n u c l e o s i d e s y n t h e s i s proved s u c c e s s f u l d e s p i t e the s t e r i c hinderance o f f e r e d by the branched-chain a l l o f u r a n o s e (see S e c t i o n 3.6.5) . H o p e f u l l y , with the wide v a r i e t y o f a l t e r n a t i v e e s t e r and e t h e r b l o c k i n g groups a v a i l a b l e f o r the p r o t e c t i o n of a l c o h o l s , the s y n t h e s i s of a f r e e p e p t i d y l n u c l e o s i d e w i l l be a t t a i n e d u s i n g t h i s procedure. - 174 -IV. EXPERIMENTAL 1. General Methods Mass s p e c t r a were recorded on an A.E.I. MS9 spectrometer. O p t i c a l r o t a t i o n s were measured with a P e r k i n Elmer model 137 spectrometer and c i r c u l a r d i c h r o i s m ( c d . ) measurements were performed on a JASCO ORD/UV-5 s p e c t r o p o l a r i m e t e r or a JASCO J-20 Automatic Recording S p e c t r o p o l a r i m e t e r . U l t r a -v i o l e t s p e c t r a (u.v.) were recorded on a Carey 15 spectrometer. M e l t i n g p o i n t s were determined on a L e i t z Microscope h e a t i n g stage model 350 and are c o r r e c t e d . I n f r a r e d s p e c t r a ( i . r . ) were recorded on a P e r k i n Elmer model 727B spectrometer. Elemental analyses were performed by Mr. P. Borda of the Department of Chemistry, U n i v e r s i t y o f B r i t i s h Columbia. 1.1 Chromatography 1. 1.1 Column Chromatography Column chromatography was performed u s i n g s i l i c a g e l i n d i c a t e d as " s i l i c a g e l H f o r t . l . c . acc. to S t a h l (Type 60)" (E.M. Reagents). Columns were p r e s s u r i z e d above the s o l v e n t r e s e r v o i r to a pressure of 7-12 p s i . - 175 -1.1.2 T h i n Layer Chromatography A l l t h i n l a y e r chromatography was performed u s i n g s i l i c a g e l f o r t . l . c . (Camag), c o n t a i n i n g 5% CaSO^ and u.v. i n d i c a t o r . Compounds were d e t e c t e d by u l t r a v i o l e t a b s o r p t i o n , by s p r a y i n g w i t h 50% s u l p h u r i c a c i d f o l l o w e d by h e a t i n g on a hot p l a t e , by s p r a y i n g w i t h a 0.3% s o l u t i o n of n i n h y d r i n i n n-butanol f o l l o w e d by warming at 110° i n an oven and/or by s p r a y i n g with a d i l u t e s o l u t i o n of potassium permanganate. 1.1.3 Paper Chromatography Paper chromatograms were performed u s i n g Whatman No. 1 paper. Nucleosides were d e t e c t e d with u l t r a v i o l e t l i g h t . 1.2 Nuclear Magnetic Resonance Spectroscopy Proton magnetic resonance s p e c t r a (p.m.r.) were reco r d e d on V a r i a n T-60, HA-100, XL-100 or Bruker 270 MHz.spectro-meters. Absorptions are given i n 6 u n i t s w i t h t e t r a m e t h y l -s i l a n e (unless otherwise stated) as the i n t e r n a l standard (set a t 6 = 0 ) . The f o l l o w i n g a b b r e v i a t i o n s are used i n d e s c r i b i n g p.m.r. s p e c t r a : s = s i n g l e t , d = doublet, t = t r i p l e t , q = q u a r t e t , qn = q u i n t e t , sex = s e x t e t , sept = s e p t e t , o c t = o c t e t , and m = m u l t i p l e t . Chemical s h i f t s (<5 values) were measured at the mid-point of the a b s o r p t i o n s and are not c o r r e c t e d . P.m.r. s p e c t r a were i n i t i a l l y s u b j e c t e d to a f i r s t o r d e r a n a l y s i s i n order to a r r i v e a t - 176 -an i n t e r n a l l y c o n s i s t e n t set of c o u p l i n g constants (J values) and, wherever p o s s i b l e , the assignments thus a r r i v e d at were confirmed by i r r a d i a t i o n experiments. - 177 -2. S y n t h e s i s of Branched-Chain Amino Sugars. Treatment o f 1,2:5,6-Di-O-isopropylidene - a-D-ribo-hexo-furan-3-ulose (25) w i t h Sodium Cyanide and Methyl N i t r o a c e t a t e . Procedure A to Y i e l d Predominantly 3-C-Cyano-l,2:5,6-d i - 0 _ - i s o p r o p y l i d e n e - a - D - g l u c o f uranose (26) . To a s o l u t i o n of ketose 2_5 (1.43 g) i n anhydrous ethanol (2 ml) was added sodium cyanide (0.290 g ) . A s o l u t i o n of methyl n i t r o a c e t a t e (0.706 g) i n anhydrous ethanol (2 ml) was added dropwise and the r e a c t i o n mixture was s t i r r e d f o r 18 h a t room temperature. The ethanol was removed by e v a p o r a t i o n under reduced p r e s s u r e . A f t e r the a d d i t i o n of 10 ml of chloroform, a p r e c i p i t a t e of non-carbohydrate m a t e r i a l was removed by f i l t r a t i o n . Water (10 ml) was added and the mixture e x t r a c t e d w i t h c h l o r o f o r m (5 x 20 m l ) . The combined c h l o r o f o r m e x t r a c t s were d r i e d over magnesium sulph a t e , f i l t e r e d , and evaporated to y i e l d a syrup (1.13 g ) . The crude product was chromatographed on a column o f s i l i c a g e l (50 g, 24 x 2.8 cm) u s i n g 1:1 benzene-ethyl a c e t a t e as developer to y i e l d ketose 2_5 (0.44 g) and a syrup (0 .739 g,. 61%) which c r y s t a l l i z e d upon s t a n d i n g . Compound 26_ was 24 r e c r y s t a l l i z e d from benzene-hexane; m.p. 99-100°, [ a ] D + 51.6° (c 1.4, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 5.92 (d, 1, J „ 4 Hz, H - l ) , 4.55 (d, 1, H-2), 4.4-4.0 (m, 5, one proton x, Z exchanges i n D 2 0 ) , 1.55, 1.51, 1.36 and 1.34 (4s, 12, CH 3). - 178 -A n a l . C a l c . f o r C 1 3 H 1 9 N 0 6 : c> 54.73; H, 6.71; N, 4.91. Found: C, 54.87; H, 6.77; N, 4.92. The n.m.r. spectrum of the syrup showed the presence of both gluco-cyanohydrin 26 and a l l o - c y a n o h y d r i n 27 i n a r a t i o of.13:1. Procedure B to Y i e l d Predominantly 3-C-Cyano-l,2:5,6-d i - 0 _ - i s o p r o p y l i d e n e - a - D - a l l o f uranose (21) . To a mixture of sodium cyanide (0.219 g) i n anhydrous ethanol (2 ml) was added methyl n i t r o a c e t a t e (0.525 g) , f o l l o w e d immediately by a s o l u t i o n of ketose 2_5 (1.14 g) i n e t hanol (5 ml). A f t e r the r e a c t i o n mixture was s t i r r e d f o r 18 h a t room temperature, the p r e c i p i t a t e o f non-carbohydrate m a t e r i a l was removed by f i l t r a t i o n . E v a p o r a t i o n o f the f i l t r a t e a f f o r d e d a syrup which was t r i t u r a t e d with water (20 ml) and e x t r a c t e d w i t h c h l o r o f o r m (5 x 2 0 m l ) . The combined c h l o r o f o r m e x t r a c t s were d r i e d (MgSO^), f i l t e r e d , and evaporated to y i e l d a c l e a r syrup. T h i s syrup was chromatographed on a column of s i l i c a g e l (130 g, 5 x 16 cm) u s i n g 4:6 benzene-ethyl a c e t a t e as developer to y i e l d ketose 25 (0.247 g) and a syrup (0.986 g ) . The syrup was c r y s t a l l i z e d from benzene-hexane t o a f f o r d the t i t l e com-- 179 -pound 27_ (0.75 g, 85% based on r e a c t e d k e t o s e ) ; m.p. 61-64°, [ a ] 2 4 + 8.6° (c 1.4, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 5.94 (d, 1, J 4 Hz, H - l ) , 4.92 (d, 1, H-2), 4.72 - 3.83 (over-x, z. l a p p i n g s i g n a l s , 4, H-4,5 and 6), 3.77 (s, 1, exchanges i n D 20, OH), 1.61, 1.48 and 1.40 (3s, 12, CH 3). A n a l . C a l c . f o r C^H^NOg: C, 54.73; H, 6.71; N, 4.91. Found: C, 54.89; H, 6.81; N, 4.98. The n.m.r. spectrum of the syrup o b t a i n e d from the mother l i q u o r a f t e r the c r y s t a l l i z a t i o n o f 2_7 showed the presence of 2J7, the gluco-cyanohydrin 26, ' and the methyl n i t r o a c e t a t e a d d i t i o n compound 162. The r a t i o of products 27, 26, and 162 was 87:6:7, r e s p e c t i v e l y . Attempts to o b t a i n pure 162 by repeated column chromatography l e d to i t s decomposition. Procedure C The r e a c t i o n was c a r r i e d out i n the same way as des-c r i b e d i n Procedure B up to the chromatographic s e p a r a t i o n . The product mixture (1.266 g) was a c e t y l a t e d with a c e t i c anhydride (10 ml) and p_-toluenesulfonic a c i d monohydrate (0.17 g) f o r 5 h at 80-85°. The v o l a t i l e components of the mixture were removed by c o - d i s t i l l a t i o n with toluene under reduced pressure to y i e l d a dark syrup which was d i s -s o l v e d i n c h l o r o f o r m (10 0 ml) and then washed wi t h water - 180 -(3 x 15 ml). The ch l o r o f o r m s o l u t i o n was d r i e d over sodium sulphate and then evaporated to a f f o r d a syrup which was chromatographed on a column of s i l i c a g e l (110 g, 3 x 40 cm) u s i n g 9:1 benzene-ethyl a c e t a t e as developer to a f f o r d the ac e t a t e s o f the gluco-cyanohydrin 163 (9 mg, 0.8%) , the a l l o - c y a n o h y d r i n 164 (188 mg, 15.6%), the methyl n i t r o a c e t a t e a d d i t i o n compound 165 (40 mg, 2.6%), and the t r i - 0 _ - a c e t y l a l l o - c y a n o h y d r i n 166 (651 mg, 47.6%) . The r a t i o o f y i e l d s of compounds 163, 164, 165 and 166 was 23:1:4:72, r e s p e c t i v e l y . 3-0-Acetyl-3-C-cyano-1,2:5, 6 - d i - 0 - i s o p r o p y l i d e n e - c t - p -glucofuranose (163) . The gluco-cyanohydrin 26 (400 mg) was a c e t y l a t e d w i t h a c e t i c anhydride (4 ml) and p_-toluenesulfonic monohydrate (20 mg) f o r 0.5 h a t 80°. The product was worked-up i n the u s u a l way to a f f o r d 490 mg of a syrup which was chromato-graphed on s i l i c a g e l (40 g, 2 x 33 cm) u s i n g 8:2 benzene-e t h y l a c e t a t e as the developer. The g l u c o - a c e t a t e 163 (381 mg, 83%) was c r y s t a l l i z e d from hexane; m.p. 79-80°, 2 3 [ot]p + 45° (c 1.3, c h l o r o f o r m ) ; n.m.r. (CDC1 3) : 6 6.00 (d, 1, J, 9 4 Hz, H - l ) , 4.48-4.00 (o v e r l a p p i n g s i g n a l s , 4, H-4, 5, and 6), 2.13 (s, 3, OAc), 1.63, 1.57, 1.43, and 1.38 (4s, 12, CH 3). A n a l . C a l c . f o r C 1 5 H 2 1 N 0 7 : C, 55.04; H, 6.47; N, 4.28. Found: C, 55.22; N, 6.56; N, 4.29. - 181 -3-0_-Acetyl-3-C-cyano-l ,2:5, 6 - d i - O - i s o p r o p y l i d e n e - a - g - a l l o -furanose (164) . The a l l o - c y a n o h y d r i n 27 was a c e t y l a t e d and p u r i f i e d u s i n g the same procedure u t i l i z e d f o r the p r e p a r a t i o n o f compound 16 3. Compound 164 was c r y s t a l l i z e d from hexane; 21 m.p. 115-115.5°, [a] + 38° (c 1.1, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 5.95 (d, 1, J± 2 4 Hz, H - l ) , 5.21 (d, 1, H-2), 4.52-4.32 (oct, 1), 4.25-4.00 ( o v e r l a p p i n g s i g n a l s , 3), 2.14 (s, 3, OAc), 1.50, 1.45, 1.36, and 1.32 (4s, 12, CH 3). A n a l . C a l c . f o r c 1 5 H 2 i N 0 7 : c ' 5 5 - 0 4 ' " H ' 6.47; N, 4.28. Found: C, 55.13; H, 6.45; N, 4.40 3,5, 6 - T r i - 0 - a c e t y l - 3 - C - c y a n o - l , 2 - 0 _ - i s o p r o p y l i d e n e - a - g -a l l o f u r a n o s e (166) . The a l l o - c y a n o a c e t a t e 164 (200 mg) was t r e a t e d with 66% a c e t i c a c i d f o r 24 h at room temperature. The a c e t i c a c i d was then removed by c o - d i s t i l l a t i o n with toluene under reduced p r e s s u r e . The r e s i d u a l syrup was then a c e t y l a t e d i n the usual way u s i n g a c e t i c anhydride and p y r i d i n e to a f f o r d the t r i - 0 _ - a c e t a t e 166 which was p u r i f i e d by column chromatography on s i l i c a g e l (30 gm, 2.3 x 19.5 cm) u s i n g 8:2 benzene-ethyl a c e t a t e as developer. Compound 166 (227 mg, 92.5%) was c r y s t a l l i z e d from benzene-hexane; m.p. 131.5-132.5°, [ a ] p 6 + 73° (c 1.0, c h l o r o f o r m ) ; n.m.r. (CDC13> : 6 5.99 (d, 1, J± 2 4 Hz, H - l ) , 5.44 (oct, 1, 5 7.5 Hz, Jr 5 Hz, J c . 2.8 Hz, H-5), 5.26 (d, 1, H-2), 4.62 (q, b , bo D,ba - 182 -1/ Jc ci. 12.5 Hz, H-6a) , 4.36 (d, 1, H-4), 4.23 (q, 1, ba, D D H-6b) , 2.18, 2.10, and 2.08 (3s, 9, OAc), 1.55 and 1.36 (2s, 6, CH3).. A n a l . C a l c . f o r C 1 6H 2 1NO g: C, 51.75; H, 5.70; N, 3.77. Rmnd: C, 51.61; H, 5.60; N, 3.55. 3-C-Acetami dome thy 1-1,2:5, 6-di-0_-isopropylidene - c t-D-gluco-f uranose (5_4) and 3-C-Acetamidomethyl-l, 2 : 5 , 6-di-0_-i s o p r o p y l i d e n e - c t - D - a l l o f u r a n o s e (53) . The gluco-cyanohydrin 26 (100 mg) was d i s s o l v e d i n t e t r a h y d r o f u r a n (THF) (1 ml) and added dropwise to a mixture o f l i t h i u m aluminum hy d r i d e (LAH) (50 mg) and THF (4 m l ) . A f t e r s t i r r i n g f o r 1 1/2 h, the excess LAH was decomposed by the a d d i t i o n of e t h y l a c e t a t e (3 m l ) . The s o l u t i o n was then f i l t e r e d , evaporated, and d i s s o l v e d i n c h l o r o f o r m (20 m l ) . The ch l o r o f o r m was washed with water (2 x 5 ml), d r i e d over sodium sul p h a t e , and evaporated t o gi v e a c r y s t a l l i n e white s o l i d i n q u a n t i t a t i v e y i e l d ; m.p. 112-113.5°. T h i s s o l i d (100 mg), without f u r t h e r p u r i f i c a t i o n , was d i s s o l v e d i n anhydrous methanol (5 ml) and a c e t i c anhydride (1 m l ) . A f t e r 24 h, the mixture was poured i n t o i c e water (30 ml) and the s o l u t i o n was e x t r a c t e d with c h l o r o f o r m (5 x 20 m l ) . The combined e x t r a c t s were d r i e d over sodium sulphate and evaporated t o y i e l d 90 mg (79%) of a c l e a r - 183 -c o l o u r l e s s syrup. C r y s t a l l i z a t i o n of compound 54_ from benzene-ether gave white c r y s t a l l i n e needles (76 mg, 66%); m.p. 126-127°, [ a j ^ 4 + 67.3° (c 1.46, e t h a n o l ) ; { L i t . v a l u e s ; m.p. 120-121°, [ a ] ^ +63.8 (c 1.44, e t h a n o l ) } ; n.m.r. (CDC1 3): <5 6.56 (br t , 1, J x • a N H 5 H z / J i ' b , N H 7 Hz, exchanges i n D 20, NHAc), 5.81 (d, 1, J 1 2 4.5 Hz, H - l ) , 4.90 (s, 1, exchanges i n D 20, OH), 5.67 (sex, 1, 5 8.0 Hz, J c _ 6.0 Hz, J_ 5.5 Hz, H-5) , 4.31 (d, 1, H-2), J , ba D , D D 4.09 (g, 1, Jc c. 8.5 Hz, H-6a), 3.95 (q, 1, H-6b), 3.75 ba, D D (d, 1, H-4), 3.74 (q, 1, J n , 14.5 Hz, H - l ' a ) , 3.46 X 3. r X 0 (q, 1, H - l ' b ) , 2.00 (s, 3, NAc), 1.47, 1.39, 1.33, and 1.28 (4s, 12, CH 3). Compound 27_ was t r e a t e d i d e n t i c a l l y to y i e l d compound 53 (86 mg, 74%) w h i c h , a f t e r c r y s t a l l i z a t i o n from benzene-ether-hexane,gave white c r y s t a l l i n e needles; m.p. 158-158.5°, 24 98 [ a ] D + 64.3 (c_ 1.43, e t h a n o l ) ; { L i t value ; m.p. 156-156.5°, [ c t ] p 3 + 56.3° ( c l . 0 , e t h a n o l ) } ; n.m.r. (CDC1 3) : 6 6.27 (br q, 1, exchanges i n D 20, NHAc), 5.77 (d, 1, 2 4.0, H - l ) , 4.23 (d, 1, H-2), 4.17-3.69 ( o v e r l a p p i n g s i g n a l s , 4, H-4, 5, and 6), 3.68 (d, 1, J l t g l 1 M 3 14 Hz, J]_, a N H 8.0 Hz, H - l ' a ) , 3.29 (q, 1, J . 3.5 Hz, H - l ' b ) , 3.09 (s, 1, X D , N i l exchanges i n D 20, OH), 2.01 (s, 3, NAc), 1.56, 1.43, and 1.33 (3s, 12, CH 3). - 184 -3. S y n t h e s i s o f Branched-Chain Sugar N u c l e o s i d e s . 3-C- (Carbomethoxymethyl) -3-deoxy-l ,2-0_-isopropylidene-a-D - a l l o f u r a n o s e (170). 3-C- (Carbomethoxymethyl) -3-deoxy-l ,2:5, 6-di-0_-isopro-p y l i d e n e - a - D - a l l o f u r a n o s e (35_) was d i s s o l v e d i n 66% a c e t i c a c i d (370 ml) and l e f t f o r 9 1/2 h at room temperature. The s o l u t i o n was evaporated i n vacuo and the remaining t r a c e s of a c e t i c a c i d were removed by repeated c o - d i s t i l l a t i o n with toluene at 19 t o r r to y i e l d a white c r y s t a l l i n e s o l i d (13.5 g, 100%). An a n a l y t i c a l sample of compound 170 was prepared by r e c r y s t a l l i z a t i o n from e t h a n o l - e t h e r ; m.p. 90-91°, [ a ] 2 5 + 70.0° (c 0.6, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 5.82 (d, 1, J1 2 4Hz, H - l ) , 4.82 ( t , 1, J 2 3 4Hz, H-2), 3.42 and 3.40 (2s, 7, H-4, 5, 6 and C0 2CH 3) , 2.9 - 2.2 (o v e r l a p p i n g s i g n a l s , 3, H-3 and 1'), 2.57 (s, 2, exchanges i n D 20, OH), 1.82 and 1.65 (2s, 6, CH 3). An a l . C a l c . f o r C 1 2 H 2 0 ° 7 : C ' 5 2 * 1 7 ? H ' 7 - 3 0 - Found: C, 51.87; H, 7.23. 3-C- (Carbomethoxymethyl) -3-deoxy-l,2-0_-isopropylide'ne-a-D-ribofuranose (171). To a s o l u t i o n of the d i o l 170 (13.0 g) i n ethanol (200 ml) was added a s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n (15 ml) and a s o l u t i o n of sodium p e r i o d a t e (10.7 g, 1 equiv.) i n 200 ml water. A f t e r s t i r r i n g f o r 1 h, a few drops of - 185 -e t h y l e n e g l y c o l were added to consume any unreacted p e r i o d a t e , Sodium borohydride (0.95 g) was added and the s o l u t i o n was s t i r r e d f o r an a d d i t i o n a l 1 h. A f t e r the a d d i t i o n o f acetone (1 ml) to consume any unreacted sodium borohydride, the s o l u t i o n was f i l t e r e d and e x t r a c t e d with methylene c h l o r i d e (8 x 100 m l ) . The combined e x t r a c t s were d r i e d over sodium sulphate and evaporated to y i e l d a p a l e yellow syrup. The crude product was d i s t i l l e d at 140° and 0.2 t o r r to y i e l d compound 171 as an a n a l y t i c a l l y pure syrup (10.3 g, 84%); 22 [ c t ] D + 64.2° (c 2.5, c h l o r o f o r m ) ; n.m.r. (CDC1 3) : 6 5.85 (d, 1, J± 2 4 Hz, H - l ) , 4.80 ( t , 1, J ~ 2 3 4 Hz, H-2), 4.2 -3.6 ( o v e r l a p p i n g s i g n a l s , 3, H-4, 5), 3.73 (s, 3, C0 2CH 3), 2.7 - 2.3 ( o v e r l a p p i n g s i g n a l s , 3, H-3, 1'), 2.1 (br s, 1, exchanges i n D 20, OH), 1.85 and 1.68 (2s, 6, CH 3). A n a l . C a l c . f o r C, ,H l o0,: C, 5 3.65; H, 7.37. Found: 11 l o b C, 53 .37; H, 7.49. 5-0_-Benzoyl-3-C- (carbomethoxymethyl) -3-deoxy-l ,2-0_-i s o p r o p y l i d e n e - a - p - r i b o f u r a n o s e (172). To a s o l u t i o n of compound 171 (10.3 g) i n anhydrous benzene (200 ml) was added a s o l u t i o n of benzoyl c h l o r i d e (5.35 ml) and p y r i d i n e (6.8 m l ) . A f t e r 18 h a t room temp-e r a t u r e , the r e a c t i o n mixture was passed through a column o f grade I I alumina and e l u t e d w i t h benzene (100 m l ) . The combined e l u e n t s were evaporated to y i e l d a c l e a r y ellow syrup (13.9 g, 95%). Compound 172 was r e c r y s t a l l i z e d from - 186 -25 e t h e r - p e t . ether (30-60); m.p. 85-86°, [a] + 54.3° (c 2.2, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 8.2 - 7.2 (m, 5, COC,H c), 5.88 (d, 1, J . „ 4 Hz, H - l ) , 4.83 ( t , 1, J„ _ 4 Hz, H-2), 4.59 (q, J c 12 Hz, J_ . 3 Hz, H-5a), 4.37 D 3 , D D Da , 4 (q, 1, J 5 b 4 4.5 Hz, H-5b), 4.16 (m, 1, H-4), 3.68 (s, 3, C 0 2CH 3), 2.9 - 2.2 (m, 3, H-3, 1'), 1.50 and 1.31 (2s, 6, CH 3). A n a l . C a l c . f o r C ; L 8 H 2 2 0 7 : C , 61.70; H, 6.33. Found: C, 61.70; H, 6.22. l-0_-Acetyl-5-0_-benzoyl-3-C-carboxymethyl-3-deoxy-B-D-r i b o f uranose-2 , 3-y-lactone (169). Compound 172 (13.9 g) was d i s s o l v e d i n 90% (v/v) t r i -f l u o r o a c e t i c a c i d (130 ml) f o r 1 1/2 h a t room temperature. The s o l u t i o n was evaporated to dryness and the r e s i d u a l t r a c e s of a c i d were removed by repeated c o - d i s t i l l a t i o n w i t h toluene under reduced p r e s s u r e to y i e l d 5-£-benzoyl-3-C-carboxymethyl-3-deoxy-B-D-ribofuranose-2,3-y-lactone (173) as a white s o l i d (10.6 g, 96.5%) . Compound 173 was d i s s o l v e d i n a c e t i c anhydride (45 ml) and p y r i d i n e (45 ml) and s t i r r e d f o r 18 h a t room temperature. The r e a c t i o n was poured i n t o i ce-water (100 ml) to p r e c i p i t a t e the t i t l e compound as a white s o l i d (10.1 g, 83%). Compound 169 was r e c r y s t a l -l i z e d from i s o - p r o p a n o l - e t h y l a c e t a t e ; m.p. 136-137°, ( l i t . value 137°); n.m.r. (CDC1 3) : 6 8.1 - 7.3 (m, 5, COCgH^ , - 187 -6.34 (s, 1, H - l ) , 4.95 (d, 1, J 2 3 6.2 Hz, H-2), 4.40 and 4.39 (2s, 3, H-4, 5), 3.19 (m, 1, J 3 ^ 8.5 Hz, J 3 ^ 2.3 Hz, H-3), 2.89 (q, 1, J, , , 17.5 Hz, H - l ' a ) , 2.54 (q, 1, H - l ' b ) , 1.98 (s, 3, OAc). 2,6-Dichloro-9-[3 1-C-(carboxymethyl-2',3'-y-lactone)-3'-deoxy-3-D-ribofuranosyl] purine (174) and 2,6-Dichloro-9-[3'-C-(carboxymethyl-2',3 *-y-lactone)-3'-deoxy-a-D-r i b o f u r a n o s y l ] p u r i n e (175). Compound 169 (1.04 g) and 2 , 6 - d i c h l o r o p u r i n e (0.92 g, 1.5 equiv.) were i n t i m a t e l y mixed, d r i e d , and then q u i c k l y heated to 160° at a pressure of 20 t o r r f o r 5 min. The temperature was allowed to drop to 14 0° over 2 min a t which time the pre s s u r e was reduced to 0.2 t o r r . A f t e r 25 min the r e a c t i o n was c o o l e d to 100°, the vacuum was r e l i e v e d and e t h y l a c e t a t e (3 ml) was added. When the melt was completely d i s s o l v e d , the s o l u t i o n was co o l e d t o 0° and the r e s u l t i n g tan p r e c i p i t a t e was removed by f i l t r a t i o n . The f i l t r a t e was evaporated to y i e l d a foamy white s o l i d . Column chroma-tography of the crude product on s i l i c a g e l (150 g, 5 x 17.5 cm) u s i n g 1:1 benzene-ethyl a c e t a t e as the developer a f f o r d e d compound 169 (79 mg), 3-nucleoside 174 (547 mg, 41%) and a- n u c l e o s i d e 175 (648 mg, 48%). Compound 175 was r e c r y s t a l l i z e d from c h l o r o f o r m to 25 y i e l d a white c r y s t a l l i n e s o l i d ; m.p. 211-214°, [°0 D M o O H - 102.5 (c 1.0, c h l o r o f o r m ) ; 215 nm ( e 21,800), — Illa. X - 188 ~ and 273 nm (shoulder a t ^230 nm) (E 9850) ; n.m.r. (CDC1 3) : 6 8.60 (s, 1, H-8), 8.12 - 7.40 (m, 5, B z) , 6.80 (d, 1, J±, f 2 , 4.7 Hz, H - l ' ) , 5.46 (q, 1, J 2 , 3 , 7.0 Hz, H-2'), 4.89 (q, 1, J 3 , ^ 4 , 6.0 Hz, J 4 , 5 , a 5.0 Hz, J 4 , 5 , b 4.0 Hz, H-4'), 4.60 (d, 1, H-5'a), 4.58 (d, 1, H-5'b), 3.45 (m, 1, J l " a , 3 ' 8 , 0 H z ' J l " b , 3 ' 3 , 8 H Z ' H - 3 , ) ' 3 * 1 0 ( c3' 1 ' J l " a , l " b 18.2 Hz, H-l"a) , 2.86 (q, 1, H-l"b) . A n a l . C a l c . f o r c i 9 H i 4 N 4 ° 5 C 1 2 : C ' 50.79; H, 3.14; N, 12.47. Found: C, 50.58; H, 3.25; N, 12.25. The 2 , 6 - d i c h l o r o p u r i n e g-nucleoside 174 was i s o l a t e d 22 as an amorphous s o l i d ; m.p. 50-60°, [ c ] D + 36.0 (c 1.0, dichloromethane) ; 215 (e 16,900), 229 (e 11,600), and 274 nm (e 7,110); n.m.r. (CDClg): 8.28 (s, 1, H-8), 8.07 -7.25 (m, 5, CgH 5) , 6.24 (d, 2,1.0 Hz, H - l ' ) , 5.64 (q, 1, J 2 3 6.5 Hz, H-2'), 4.76 - 4.25 ( o v e r l a p p i n g s i g n a l s , 3, H-4' and 5'), 3.71 (q, 1, J 3 , 4 , 8.5 Hz, J ^ , ^ 3 , 8.0 Hz, H-3'), 3.01 (q, 1, J „ , ,,, 18.0 Hz, H-l"a) , 2.62 (d, 1, H-l"b) . M o l e c u l a r weight by mass spectrometry 44 8.0 336. C 1 9 H 1 4 ° 5 N 4 C 1 2 r e c 3 u i r e s 448.0341. 9-[3'-C-(Carboxymethyl-2',3'-y-lactone)-3'-deoxy-g-D-r i b o f u r a n o s y l ] a d e n i n e (178) and 9-[3'-C-Carboxymethyl-3'-deoxy-a-D-ribofuranosyl]adenine (181) . To a s o l u t i o n o f the l a c t o n e 169 (2.00 g) and N -b e n z o y l - N 6 , 9 - b i s ( t r i m e t h y l s i l y l ) a d e n i n e (2.50, 1.04 equiv) i n d i c h l o r o e t h a n e (60 ml) was added a s o l u t i o n of t i n - 189 -t e t r a c h l o r i d e (1.46 ml, 2.0 equiv) i n d i c h l o r o e t h a n e (10 ml). The s o l u t i o n was s t i r r e d f o r 18 h a t 63° and a f t e r c o o l i n g to 0°, a s a t u r a t e d s o l u t i o n o f sodium b i c a r b o n a t e (10 ml) was added with vigorous s t i r r i n g . The s o l u t i o n was f i l t e r e d , the p r e c i p i t a t e washed wit h c h l o r o f o r m (2 x 20 ml) and the o r g a n i c and aqueous phases were separat e d . The o r g a n i c phase was d r i e d over sodium sulphate p r i o r to e v a p o r a t i o n to y i e l d a white s o l i d (2.2 g ) . Column chromatography o f the product on s i l i c a g e l (30 g, 2.2 x 19 cm), u s i n g 9:1 e t h y l a c e t a t e - e t h a n o l as developer, a f f o r d e d N 6-benzoyl-9-[3'-C-(carboxymethyl-2',3'-y-lactone)-3 1-deoxy-B-D-ribo-f u r a n o s y l ] a d e n i n e (176) (966 mg, 31%) and N 6-benzoyl-9-[3 1-C-(carboxymethyl-2 1,3'-y-lactone)-3'-deoxy-a-D-ribo-f u r a n o s y l ] a d e n i n e (177) 954 mg, 31%). Both n u c l e o s i d e s were used f o r subsequent r e a c t i o n without f u r t h e r p u r i f i c a t i o n . The B-nucleoside 176: n.m.r. (CDC1 3): 6 9.52 (s, 1, NH), 8.56 and 8.10 (2s, 2, H-2 and 8), 7.95 - 7.31 (over-l a p p i n g s i g n a l s , 10, C^H^), 6.20 (s, 1, H - l ' ) , 5.65 (d, 1, J2,f3, 6.8 Hz, H-2'), 4.70 (q, 1, J 4 . / 5 . a 5.8 Hz, J 5 , a ^ 5 . b 12.0 Hz), 4.48 (q, 1, J 4 , 5 , b 5.4 Hz, H-5'b), 4.27 (qn, 1, J 3 , ^ 4 , 8.4 Hz, H - 4 ' ) , 3.80 (q, 1 , J r . a 3 , 9 - 0 Hz, H-3'), 2.96 (q, 1, J,„ ,,,, 18 Hz, H-l"a) , 2.60 (d, 1, H-l"b) . The a-nuc l e o s i d e 177; n.m.r. (CDC1 3): 6 9.43 (s, 1, NH), 8.70 and 8.25 (2s, 2, H-2 and 8), 8.30 - 7.40 (over-l a p p i n g s i g n a l s , 10, C^E^), 6.89 (d, 1, J 1 , 2 , 4.0 Hz, H - l ' ) , 5.32 (q, 1, J 2 , / 3 I 7.0 Hz, H-2'), 4.77 (q, 1, J 3 , / 4 , 6.0 Hz, J 4 , 5 , a 5.0 Hz, J 4 , 5 , b 4.0 Hz, H-4'), 4.57 (q, 1, - 190 -a,5'b 12.0 Hz, H-5'a), 4.48 (q, 1, H-5'b), 3.32 (q, 1, 9.0 Hz, J l'b,3 ' 3.5 Hz, H-31), 2.98 (q, J l ' a , l ' b 18.0 Hz, H - l ' a ) , 2.81 (q, 1, H - l ' b ) . To a s o l u t i o n o f the B-nucleoside 176 (210 mg) i n anhydrous methanol (16 ml) was added a s o l u t i o n of sodium methoxide (0.66 ml o f a 5 mg sodium/ml methanol s o l u t i o n ; 0.33 equiv) and the mixture was maintained at room temperature f o r 7 days. Bio-Rex 70 (H +) c a t i o n exchange r e s i n (48 mg, 1.1 equiv.) was added to the s o l u t i o n and the mixture was s t i r r e d f o r 15 min. be f o r e the r e s i n was removed by f i l t r a -t i o n . The f i l t r a t e was then evaporated under reduced p r e s s u r e to a f f o r d an amorphous s o l i d . The product was then chromato-graphed on a column of Bio-Rex 70 (H +) r e s i n (1 x 11 cm), usin g 8:2 methanol-water as developer, to a f f o r d the l a c t o n e 3-nucleoside 178 (76 mg, 61%) which c r y s t a l l i z e d spontaneously from s o l u t i o n . An a n a l y t i c a l sample of compound 178 was 24 r e c r y s t a l l i z e d from water; m.p. 196-198°, [ a ] D -29° (c 0.09, methanol); v K B r 1780 cm - 1 (C=0); A M e 0 H 205 (e 18,700), max max 257 nm (e 13,200); c d . Ae -1.82 (A 260 nm, c 0 .0003, ' max — methanol); n.m.r. (DMSO-dg): 8.45 and 8.15 (2s, 2, H-2 and H-8), 7.31 (s, 2, exchanges i n D 20, NH 2), 5.91 (s, 1, H - l ' ) , 5.21 (br s, 1, exchanges i n D20, OH), 4.45 (d, 1, J 2 , f 3 , 4.8 Hz, H-2'), 3.93 (s, 1, J 3 , f i , 9-6 Hz, J 4 , f 5 . b 3.3 Hz, J . , c , 2.7 Hz, H-4 1), 3.78 (q, 1, J c , 13.0 Hz, 4 , D a o a, _> D H-5'a), 3.59 (q, 1, H-5'b), 2.66 (sept, 1, J-. , , 9.6 Hz, J 3 ' , l " b 4 , 8 H z ' H ~ 3 , ) ' 2 * 5 3 1 ' J l " a , l " b 1 7 , 2 H z ' H ~ 1 , , a ) ' - 191 -2.50 (m, DMSO), 2.33 (q, 1, H - l " b ) . A n a l . C a l c . f o r C 1 2 H 1 3 N 5 0 4 • 1 1/2 H 20: C, 45.28; H, 5.07; N, 22.00. Found: C, 45.60; H, 5.07; N, 21.97. M o l e c u l a r weight by mass spectrometry 291.0953. c i 2 H 1 3 N 5 ° 4 r e c 3 u i r e s 291.0967. M + + 1 292.1032 r e q u i r e d 292.1045. To a s o l u t i o n of the a-nucleoside 177 (349 mg) i n anhydrous methanol (22 ml) was added a s o l u t i o n o f sodium methoxide (0.90 ml of a 5 mg sodium/ml methanol s o l u t i o n ; 0.37 equiv) and the mixture was maintained at room temperature f o r 7 days. Bio-Rex 70 (H +) c a t i o n exchange r e s i n (64 mg, 0.9 equiv) was added to the s o l u t i o n and the mixture was s t i r r e d f o r 15 min a f t e r which the r e s i n was removed by f i l t r a t i o n . The f i l t r a t e was evaporated i n vacuo t o a f f o r d an amorphous white s o l i d which was r e c r y s t a l l i z e d from methanol t o a f f o r d 9-(3'-C-carbomethoxymethyl-3'-deoxy-a-g - r i b o f uranosyl) adenine (179.); m.p. 183.0 - 183.5°, U ] 2 2 = +20° (c 0.5, 1:1 methanol-water). A n a l . C a l c . f o r C 1 3 H 1 7 N 5 0 5 : C, 48.29; H, 5.30; N, 21.66. Found: C, 48.29; H, 5.48; N, 21.36. Compound 180 was d i s s o l v e d i n a minimum amount of 8:1 methanol-water and the s o l u t i o n was a p p l i e d t o a column o f Bio-Rex 70 (H +) r e s i n (1 x 12 cm) which was packed and e l u t e d with 8:2 methanol-water. The a c i d i c a - n u c l e o s i d e 181 c r y s t a l l i z e d spontaneously from the e l u a n t s o l u t i o n and was removed by f i l t r a t i o n t o a f f o r d a white c r y s t a l l i n e s o l i d (106 mg, 51%); m.p. 233-236°, [ a ] 2 4 0° (c 0.1, 1:1 - 192 -H«0 methanol-water) ; X 203 (e 17,900), 252 nm (e 12,700); max ' v K B r 2400 - 3600 cm - 1 (OH); c d . Ae +1.53 (X 256 nm, c max max ' — 0. 0003, water); n.m.r. (DMSO-dg); 12.13 (br s, 1, exchanges i n D 20, C0 2H), 8.13 and 8.09 (2s, 2, H-2 and 8), 7.25 (s, 2, exchanges i n D 20, NH 2), 6.27 (d, 1, J±, 2 , 3.0 Hz, H - l ' ) , 5.57 (br s, 1, exchanges i n D 20, OH), 4.83 (br s, 1, exchanges i n D 20, OH), 4.26 ( t , 1, J 2 , 3 , 3.8 Hz, H-2'), 4.03 (sex, 1, J 3 , ^ 4 , 9.5 Hz, J4, f 5 , h 4.0 Hz, J 4 , f 5 , a 3.0 Hz, H-4'), 3.63 (q, 1, J _ , C I K 1 2 ' ° H z ' H-5'a), 3.47 (q, 1, H-5'b), 2.68 (oct, 1, J 1 H b 3 3.2 Hz, H-3'), 2.61 (d, 1, J x i a / 1 i b 16.0 Hz, H - l ' a ) , 2.50 (m, DMSO) , 2.39 (q, 1, H-l"b) . An a l . C a l c f o r c 1 2 H i 5 N 5 ° 5 : C ' 46.60; H, 4.85; N, 22.65. Found: C, 47.65; H, 4.99; N, 22.66. M o l e c u l a r weight by mass spectroscopy 291.0984. c i 2 H 1 3 N 5 ° 4 ~ H 20) r e q u i r e s 291.0968. (M + + 1) - H 20 292.1041 r e q u i r e d 292 .1046 . - 193 -4. Sy n t h e s i s of G l y c o s - 3 - y l a-Amino A c i d s and Analogues  o f the Nucle o s i d e Moiety o f P o l y o x i n J . (E) and (Z) -5-0_-Benzoyl-3-deoxy-l, 2-0_-isopropylidene-3-C-[nitro(methoxycarbonyl)methylene]-a-B-erythro-pento-furanose (189). To a s o l u t i o n o f 5-0_-benzoyl-l, 2-0_-isopropylidene - c t -g-erythro-pentos-3-ulose (182, 554 mg) and ammonium acetate (162 mg, 1.1 equiv.) i n anhydrous N,N-dimethylformamide (3 ml) was added methyl n i t r o a c e t a t e (468 mg, 2.1 e q u i v . ) . A f t e r the r e a c t i o n mixture was s t i r r e d a t room temperature f o r 2 h, i t was f i l t e r e d , the f i l t r a t e was d i l u t e d with c h l o r o f o r m (25 ml) and the s o l u t i o n washed wi t h water (3 x 10 ml), d r i e d over sodium sul p h a t e , and evaporated t o y i e l d an amber syrup. T . l . c . o f the product showed the presence o f two components w i t h v a l u e s o f 0.35 and 0.4 5 when 8:2 benzene-ethyl acetate was used as the developer. The crude product mixture was d i s s o l v e d i n a c e t i c anhydride (10 ml) and t o the r e s u l t i n g s o l u t i o n was added p - t o l u e n e s u l p h o n i c a c i d monohydrate (90 mg). The r e a c t i o n mixture was heated to ^90° f o r 5 h a t which time t . l . c . of the product showed the presence of o n l y the high e r R f component. The s o l u t i o n was evaporated i n vacuo and the r e s u l t i n g crude product was d i s s o l v e d i n c h l o r o f o r m (25 ml). The ch l o r o f o r m s o l u t i o n was then washed w i t h water (3 x 10 ml), d r i e d over sodium su l p h a t e , and evaporated to y i e l d a dark gummy s o l i d (54 7 mg). - 194 -Column chromatography of the crude product on s i l i c a g e l (30 g ) , u s i n g 9:1 benzene-ethyl a c e t a t e as the developer, a f f o r d e d the t i t l e compound as a p a l e yellow syrup (216 mg, 29%). An a n a l y t i c a l sample of compound 189 was o b t a i n e d by d i s t i l l a t i o n at 140-160° and 0.06 t o r r ; U ] 2 6 + 323° (c 1.6, c h l o r o f o r m ) ; v 3 1730 (C=0) and 1545 cm — max (N0 2); n.m.r.* (CDC1 3): 6 8.1 - 7.2 ( o v e r l a p p i n g s i g n a l s , 5, C 6 H 5 ) , 6.05 (d, 1, J l B f 2 B 4 , 5 H z ' H _ 1 B ) ' 6 , 0 2 (d' 1' J1A 2A 4 , 8 H z ' H - 1 A ) ' ^6.00 ( o v e r l a p p i n g s i g n a l s , 1, J2A,4A 1 - 8 H Z ' J4A,5A 1 ' 2 H z ' H " 4 A ) ' 5 ' 8 6 ( m ' 1 ' J2B,4B 1.8 Hz, J 4 B 5 B 3 Hz, H-4B), 5.67 (q, 1, H-2B), 5.52 (q, 1, H-2A), 4.60 and 4.57 (2d, 2, H-5A), 4.52 and 4.46 (2d, 2, H-5B), 3.94 (s, 3, C0 2CH 3"B), 3.90 (s, 3, C0 2CH 3 - A), 1.44 and 1.40 (2s, 6, CH 3). A n a l . C a l c . f o r C 1 8 H 1 9 N 0 9 : C ' 5 4 - 9 6 ; H ' 4- 8 7'" N ' 3 - 5 6 Found: C, 55.00; H, 5.00; N, 3.36 * The n.m.r. spectrum was of a mixture of g e o m e t r i c a l isomers. The major to minor product r a t i o , c a l c u l a t e d by the r e l a t i v e h e i g h t s of the methyl e s t e r resonances a t 5 3.90 and 3.94, r e s p e c t i v e l y , was 1.9:1. Those resonances a t t r i b u t e d to the major isomer are l a b e l l e d A and to the minor isomer B. - 195 -1,2:5 , 6-Di-0_-isopropylidene-3-C- [ (R,S) - n i t r o (methoxy-c a r b o n y l ) m e t h y l ] - a - D - a l l o f u r a n o s e (162) . To a mixture of 1, 2 :5 ,6-di-0_-isopropylidene-a-D-ribo-hexofuranos-3-ulose (25) (1.00 g, 1.0 e q u i v . ) , ammonium ac e t a t e (0.30 g, 1.0 equiv.) and anhydrous N,N-dimethyl-formamide (4 ml) was added dropwise, w i t h s t i r r i n g , methyl n i t r o a c e t a t e (0.92 g, 2.0 e q u i v . ) . A f t e r the r e a c t i o n mixture was s t i r r e d f o r 20 min at room temperature, the s o l u t i o n was f i l t e r e d , water was added (4 ml) and the r e a c t i o n mixture was e x t r a c t e d w i t h c h l o r o f o r m (3 x 10 m l ) . The combined o r g a n i c e x t r a c t s were d r i e d (magnesium s u l p h a t e ) , and evaporated t o y i e l d an orange syrup (1.40 g ) . The crude product was chromatographed on s i l i c a g e l (150 g, 5 x 17 cm, 1:1 benzene-ethyl a c e t a t e as developer) to a f f o r d s t a r t i n g m a t e r i a l 25_ (157 mg, 0.44), and a 1:5 mixture (1 g, R f 0.58) o f methyl n i t r o a c e t a t e and product 16 2; ^ f i l m 3 4 5 Q ( 0 H ) 1 7 5 5 c m - l ( C=o); n.m.r. (CDC1,): 6 5.90 max J (d, 1, J ± 2 4 Hz, H - l ) , 5.83 (s, 1, H - l ' ) , 5.18. (s, CH 2 of methyl n i t r o a c e t a t e ) , 3.88 (s, OCH 3 of methyl n i t r o a c e t a t e ) , 3.83 (s, 3, C0 2CH 3), 3.47 (s, 1, OH, exchanges w i t h D 2 0 ) , 1.46, 1.37 and 1.33 (3s, 12, CH 3). 3 - 0 - A c e t y l - l , 2 : 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - 3 - C - [ ( R , S ) - n i t r o -methoxycarbonyl) methyl] - a - D - a l l o f u r a n o s e (165) . Methyl n i t r o a c e t a t e (4.9 g, 1.9 e q u i v . ) , ketose 2_5 (5.7 g, 1.0 e q u i v . ) , and ammonium a c e t a t e (1.7 g, 1.0 equiv.) - 196 -were r e a c t e d i n N,N-dimethylformamide (20 ml) as p r e v i o u s l y d e s c r i b e d . The crude product o b t a i n e d and p-toluene-s u l p h o n i c a c i d monohydrate (0.8 g) were d i s s o l v e d i n a c e t i c anhydride (40 ml) and s t i r r e d f o r 5 h a t 80-85°C. The a c e t i c anhydride was removed by c o - d i s t i l l a t i o n with toluene (3 x 30 ml) under reduced p r e s s u r e to y i e l d a dark brown syrup (9.0 g ) . The crude product was chromatographed on s i l i c a g e l (200 gm, 5 x 24 cm, 8:2 benzene-ethyl acetate as developer) to y i e l d a c l e a r , p a l e y e l l o w syrup (R f 0.37). Compound 165 was c r y s t a l l i z e d from ether-hexane to a f f o r d a white c r y s t a l l i n e s o l i d (7.5 g, 82% based on ketose 25); m.p. 136-137°, [a]J 4 + 71.2° (c 5.3, c h l o r o f o r m ) ; v C H C l 3 ^ . D — max 1740 (C=0), and 1570 cm - 1 (N0 2); n.m.r. (CDC1 3): 6 5.93 (d, 1, J1 2 3.5 Hz, H - l ) , 5.90 (s, 1, H - l 1 ) , 4.90 (d, 1, H-2), 4.87 (s, 1), 4.34 (s, 3), 3.80 (s, 3, C 0 2CH 3), 2.08 (s, 3, OAc), 1.50, 1.43, 1.37, and 1.33 (4s, 12, CH 3). A n a l . C a l c . f o r c i 7 H 2 5 N O n : C ' 4 8 - 6 9 ; H / 6.01; N, 3.34. Found: C, 4 8.86; H, 6.08; N, 3.10. Methyl N-acety1-^-2-(1,2:5,6-di-O-isopropylidene-a-D-g l u c o f u r a n o s - 3 - y l ) g l y c i n a t e (202) and Methyl N-acetyl^D-2- (1, 2 : 5 , 6-di-0_-isopropylidene-ct-D-glucof uranos-3-yl) g l y c i n a t e (203) . To a pre-hydrogenated mixture o f p a l l a d i u m - o n - c h a r c o a l (0.95 g) i n methanol (100 ml) was added a s o l u t i o n of the n i t r o e s t e r 162 (2.3 g) d i s s o l v e d i n a minimum of methanol. - 19 7 -A c e t i c anhydride (6 ml) was added and the r e a c t i o n mixture hydrogenated a t atmospheric pressure f o r 18 h. The s o l u t i o n was f i l t e r e d , n e u t r a l i z e d with aqueous sodium b i c a r b o n a t e s o l u t i o n , and e x t r a c t e d w i t h c h l o r o f o r m (5 x 100 ml) to y i e l d a pale yellow syrup (1.7 g ) . Column chromatography of the crude product on s i l i c a g e l (300 g) with 10:5:1 benzene-e t h e r - e t h a n o l as developer a f f o r d e d 202 (0.27 g, 12%) and 203 (0.20 g, 9%) as pale y e l l o w syrups, as w e l l as ketose. 25 and two u n i d e n t i f i e d compounds (0.22 g and 0.24 g ) . Compound 202 was rechromatographed on s i l i c a g e l (32 g, 1.8 x 30 cm) w i t h 8:2 e t h y l a c e t a t e - e t h a n o l as developer to y i e l d a c l e a r c o l o u r l e s s syrup (0.17 g) which was d i s t i l l e d a t 130° and 0.2 t o r r to y i e l d a c l e a r hard g l a s s ; m.p. 52-25 62°, [ a ] D + 71° (c 1.5, methylene c h l o r i d e ) ; n.m.r. (CDC1 3) : 6 6 .92 (br d, 1, J„ „ n , 6.5 Hz, NHAc) , 5.83 (d, 1, J1 „ 3.5 Hz, H - l ) , 4.97 (d, 1, H - l ' ) , 4.35 (d, 1, H-2), 4.30 (br s, 1, OH, exchanges i n D 2 0 ) , 3.78 (s, 3, C 0 2CH 3), 2.04 (s, 3, NAc). A n a l . C a l c . f o r C 1 7 H 2 7 N 0 g : C, 52.43; H, 6.99; N, 3.60. Found: C, 52.17; H, 7.10; N, 3.58. M o l e c u l a r weight by mass spectrometry 374.1430. C i 6 H 2 4 N 0 9 ( M + - C H 3 ^ r e q u i r e s 374.1449. Compound 203 (0.20 g) was rechromatographed on s i l i c a g e l (40 g, 1.8 x 38 cm) with 8:2 e t h y l a c e t a t e - e t h a n o l as developer to y i e l d a c l e a r , c o l o u r l e s s syrup (0.12 g); 27 [ a ] D + 50.6° (£ l . l , methylene c h l o r i d e ) ; n.m.r. (CDC1 3): - 198 -6 6.90 (br d,> Is, J r T U „ ., 10 Hz, NHAc), 5.89 (d, 1, J n „ JNri , r l — X X , z. 4 Hz, H - l ) , 5.18 (d, 1, H - l ' ) , 4.48 (d, 1, H-2), 4.28 (s, 1, OH, exchanges i n D 20), 3.80 (s, 3, C0 2CH 3), 2.00 (s, 3, NAc) . Mo l e c u l a r weight by mass spectroscopy 374.1469. C 1 6 H 2 4 N 0 9 ( M + _ C H 3 ) r e q u i r e d 374.1449. Chromatographic i n v e s t i g a t i o n s of the two u n i d e n t i f i e d f r a c t i o n s o f the i n i t i a l chromatography showed t h a t the f i r s t (0.22 g) was a mixture of at l e a s t s i x components wh i l e the second (0.24 g) was a mixture o f th r e e components, a l l o f which remain u n i d e n t i f i e d . 3-0_-Acetyl-l ,2:5 ,6-di-0_-isopropylidene-3-C- (methoxydi-carbonyl)-a-p_-allofuranose oxime (204) . The n i t r o e s t e r 165 (165 mg) i n methanol ( 20 ml) was hydrogenated a t atmospheric p r e s s u r e , w i t h palladium-on-c h a r c o a l as c a t a l y s t (5%, 150 mg), f o r 48 h. The c a t a l y s t was removed by f i l t r a t i o n and the f i l t r a t e evaporated to y i e l d the oxime 204 (148 mg, 94%) as a c l e a r c o l o u r l e s s syrup. An a n a l y t i c a l sample was obt a i n e d by d i s t i l l a t i o n a t 110-120°/0.08 t o r r ; [ a ] ^ 3 + 10.7° (c 1.2, chloroform) n.m.r. (CDC1 3): 6 5.89 (d, 1, 2 3.5 Hz, H - l ) , 5.5 - 4.9 (br s, 1, exchanges i n D 20, NOH), 4.6 - 4.1 ( o v e r l a p p i n g s i g n a l s , 4, H-4, 5, and 6), 4.42 (d, 1, H-2), 3.82 (s, 3, C0 2CH 3), 2.09 (s, 3, OAc), 1.46 and 1.34 (2s, 12, CH 3). A n a l . C a l c . f o r c x 7 H 2 5 N O 1 0 : C ' 5 0 - 6 2 ' H ' 6 - 2 5 ; N ' 3.47. Found: C, 50.49; H, 6.58; N, 3.23. - 199 -Methyl L-2 - ( 3 - 0 - a c e t y l - l ,2:5 , 6-di-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (205) and Methyl g-2-(3-0-a c e t y l - l ,2:5,6-di-0_-isopropylidene-a-D-allof uranose-3-y l ) g l y c i n a t e (206). The 3-0_-acetyl n i t r o e s t e r 165 (200 mg) i n methanol (12 ml) was hydrogenated over f r e s h l y a c t i v a t e d Raney n i c k e l (0.4 ml) c a t a l y s t f o r 4 h a t atmospheric pressure and room temperature. The c a t a l y s t was removed by f i l t r a -t i o n and the f i l t r a t e evaporated to y i e l d a c l e a r c o l o u r l e s s syrup (150 mg, 81%). Column chromatography o f the product on s i l i c a g e l (30 g, 2.3 x 22 cm), wit h 9:1 e t h y l a c e t a t e -e t h e r as developer, y i e l d e d two pure, n i n h y d r i n p o s i t i v e , compounds. Compound 205 (R f 0.35, 124 mg, 6 7%) was d i s t i l l e d a t 100-110° and 0.05 t o r r ; [ a ] 2 4 + 38.4° (c 0.9, methylene c h l o r i d e ) ; n.m.r. ( C D C l 3 ) : 6 5.81 (d, 1, J1 2 3.5 Hz, H - l ) , 4.70 - 4.14 ( o v e r l a p p i n g s i g n a l s , 5), 4.41 (d, H-2), 4.11 (br s, 1, H - l ' , c o l l a p s e s to sharp s i n g l e t a f t e r a d d i t i o n o f D 20), 3.66 (s, 3, C0 2CH 3), 2.00 (s, 3, OAc), 1.83 (br s, 2, NH 2, exchanges i n D 2 0 ) , 1.48 (s, 9, CH 3) , 1.34 (s, 3, C H 3 ) . A n a l . C a l c . f o r C^H^NOg: C, 52.44; H, 6.99; N, 3.60. Found: C, 52 .73; H, 6.87; N, 3.46. Compound 206 (R f 0.42, 15 mg, 8%) was d i s t i l l e d at 2 3 120° and 0.2 t o r r ; + 16.6° (c 1.7, methylene c h l o r i d e ) ; n.m.r. (CDC1 3): 6 5.88 (d, 1, J1 2 4 Hz, H - l ) , 4.68 (d, 1, - 200 -J 4 5 2 Hz, H-4), 4.58 (d, 1, H-2), 4.28 (s, 3), 4.02 ( b r s , 1, H - l ' , sharp s i n g l e a f t e r a d d i t i o n of D 20), 3.76 (s, 3, C0 2CH 3) , 2.08 (s, 5, OAc and NH 2, c o l l a p s e s to 3 proton s i g n a l upon a d d i t i o n o f D 20), 1.53 (s, 3, CH^), 1.44 (s, 6, CH 3), 1.34 (s, 3, CH 3). An a l . C a l c . f o r C 1 7 H 2 7 N 0 9 : C, 52.44; H, 6.99; N, 3.60. Found: C, 52.39; H, 7.10; N, 3.49. L-2- (1,2:5, 6-Di-0_-isopropylidene - c t-D-allof uranos-3-yl) -g l y c i n e (207) and D-2-(1, 2 : 5 , 6 - D i - O - i s o p r o p y l i d e n e - a - D -a l l o f u r a n o s - 3 - y l ) g l y c i n e (20 8) . To a s o l u t i o n of the ^ - g l y c i n a t e 205 (50 mg) i n methanol (1 ml) was added a c a t a l y t i c amount of sodium hydroxide (1 ml of a 2.5% solution) and the s o l u t i o n was kept at room temperature f o r 4 h. The s o l u t i o n was a p p l i e d t o a column of Rexyn RG-51 (H +) r e s i n (15 ml) and the column was e l u t e d w i t h water. The n i n h y d r i n p o s i t i v e f r a c t i o n s were combined and evaporated t o y i e l d the amino a c i d 207 (35 mg, 82%) as a p a l e , y e l l o w g l a s s . Compound 207 was c r y s t a l l i z e d from e t h a n o l - e t h y l a c e t a t e ; m.p. 189-191°, [ a ] ? 4 + 89.2° (c 0.6, water); c d . Ae + 0.97 (A 209, D — max 0.5 N HC1 i n methanol); n.m.r. (D 20): 6 6.08 (d, 1, J 1 2 3.5 Hz, H - l ) , 5.07 (d, 1, 5 3 Hz, H-4), 4.57 (d, 1, H-2), 4.37 - 3.77 (o v e r l a p p i n g s i g n a l s , 4, H - l 1 , 5, and 6), 1.48 and 1.30 (2s, 12, CH 3). An a l . C a l c . f o r C 1 4H 2 3NO g•%H 20: C, 49.77; H, 7.01; N, 4.15. Found: C, 49.74; H, 7.10; N, 4.04. - 201 -I d e n t i c a l base h y d r o l y s i s of the D - g l y c i n a t e 206 (55 mg) y i e l d e d the D-amino a c i d 208 (40 mg, 85%). R e c r y s t a l -l i z a t i o n of 208 from e t h a n o l - e t h e r a f f o r d e d a pure white 25 c r y s t a l l i n e s o l i d ; m.p. 157-159°, [ a ] D + 25° (c 0.7, water); c d . ; A e - 0.93 ( X . 209, 0.5 N HC1 i n methanol); n.m.r. ' mxn ( D 20): 6 6.07 (d, 1, J± 2 3.5 Hz, H - l ) , 4.37 - 3.60 (over-l a p p i n g s i g n a l s , 5), 1.57, 1.53, and 1.38 (3s, 12, CH^). A n a l . C a l c . f o r C 1 4 H 2 3 N 0 8 '%E20: C ' 4 9 - 1 2 ' " H / 7.07; N, 4.09. Found: C, 49.03; H, 7.15; N, 4.10. Methyl N - a c e t y l - ^ - 2 - (3-0_-acetyl-l ,2:5 , 6-di-0_-isopropylidene-a - p - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (209) and methyl N - a c e t y l -D-2- ( 3-0_-acetyl-l ,2:5 , 6 - d i - 0 _ - i s o p r o p y l i d e n e - a - D - a l l o f uranos-3 - y l ) g l y c i n a t e (210). A c e t i c anhydride (3 ml) was added to a s o l u t i o n of the L - g l y c i n a t e 205 (510 mg) i n methanol (10 ml) and the s o l u t i o n was s t i r r e d f o r 21 h a t room temperature. The s o l u t i o n was evaporated under reduced pressure and the r e s i d u a l a c e t i c anhydride was removed by c o - d i s t i l l a t i o n w i t h t o l u e n e , i n  vacuo, to y i e l d 209 as an amorphous s o l i d (579 mg, 100%) . An a n a l y t i c a l sample of the N - a c e t y l L - g l y c i n a t e 209 was o b t ained by d i s t i l l a t i o n a t 140° and 0.2 t o r r ; m.p. 56-59°, 25 [ a ] D + 18.1° (c 3.3, methylene c h l o r x d e ) ; n.m.r. (CDCl^): 6 6 .68 (d, 1, J„„ „ -,,9.5 Hz, exchanges i n D-0, NHAc), 5.93 (d, 1, J, 0 4 Hz, H - l ) , 5.45 (d, 1, c o l l a p s e s to s i n g l e t a f t e r a d d i t i o n of D 20, H - l ' ) , 4.51 (d, 1, H-2), 4.46 - 4.00 - 202 -(ov e r l a p p i n g s i g n a l s , 4, H-4, 5 and 6), 3.76 (s, 3, C0 2 C H 3 ) , 2.14 and 2.10 (2s, 6, A c ) , 1.50, 1.48, 1.42 and 1.37 (4s, 12, CH 3) . An a l . C a l c . f o r c i 9 H 2 9 N O i o : C ' 5 2 - 8 9 ' H ' 6-78; N, 3.25. Found: C, 52.68; H, 6.90; N, 3.27. I d e n t i c a l treatment o f g - g l y c i n a t e 206 (50 mg) y i e l d e d N - a c e t y l g - g l y c i n a t e 210 (56 mg, 100%) as a c l e a r c o l o u r l e s s 25 syrup, which was d i s t i l l e d at 120° and 0.2 t o r r ; [ a ] D + 30.8° (c 3.6, c h l o r o f o r m ) ; n.m.r. • (CDC1 3) : 6 7.00 (d, 1, J M t , „ , , 1 0 Hz, exchanges i n D„0, NHAc) , 5.91 (d, 1, J.. „ JN r l , r r X Z X , Z 3.8 Hz, H - l ) , 5.27 (d, 1, c o l l a p s e s to s i n g l e t a f t e r a d d i t i o n o f D 20, H - l ' ) , 4.59 (d, 1, H-2), 4.42 - 4.12 (over-l a p p i n g s i g n a l s , 4, H-5 and H-6), 3.78 (s, 3, C0 2 C H 3 ) , 2.06 and 2.01 (2s, 6, A c ) , 1.58, 1.50, 1.47 and 1.38 (4s, 12, CH 3) . A n a l . C a l c . f o r c i 9 H 2 9 N O i o : C ' 5 2 - 8 9 ' H ' 6.78; N, 3.25. Found: C, 52.65; H, 6.81; N, 3.15. Methyl N-acetyl-L-2 - (1,2:5 , 6-di-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (211). To a s o l u t i o n o f the N - a c e t y l ^ - g l y c i n a t e 209 (500 mg) i n anhydrous methanol (5 ml) was added a c a t a l y t i c amount o f sodium methoxide (1.5 ml o f a 0.5% s o l u t i o n ) and the s o l u t i o n was kept f o r 25 min. The s o l u t i o n was d e c a t i o n i z e d w i t h Amberlite IRC-50 (H +) r e s i n and evaporated to y i e l d a pale yellow g l a s s (499 mg). The product was chromatographed - 203 -on s i l i c a g e l (50 g, 2 x 39 cm), w i t h 9:1 benzene-ethyl acetate as the developer, to a f f o r d the s t a r t i n g m a t e r i a l 209 (R f 0.31, 16 mg) and the t i t l e compound 211 (R f 0.30, 39 6 mg, 91% based on s t a r t i n g m a t e r i a l consumed). Compound 211 was r e c r y s t a l l i z e d from benzene-hexane; m.p. 141-142°, [ a ] ^ 4 + 27.4° (c 1.7, c h l o r o f o r m ) ; n.m.r. (CDClg): 6 6.83 (d, 1, J, , 9.5 Hz, NHAc, exchanges wi t h D o0) , 5.86 (d, 1 — N H £ 1, J, „ 4.0 Hz, H - l ) , 5.22 (d, 1, H - l 1 , c o l l a p s e s to a 1 , 2. s i n g l e t upon a d d i t i o n of D 20), 4.44 (d, 1, H-2), 4.35 (d, 1, J . c 3.0 Hz, H-4), 4.09 (s, 1, J c c 6.0 Hz, J c 6.5 Hz, H-5), 3.83 (q, 1, J , c. 14 Hz, H-6a), 3.72 (q, b a , D D 1, H-6b), 3.69 (s, 3, C 0 2CH 3), 3.09 (br s, 1, OH, exchanges wi t h D 2 0 ) , 2.05 (s, 3, NAc), 1.46 (s, 6, CH 3), 1.39 and 1.32 (2s, 6, CH 3). A n a l . C a l c . f o r C 1 7 H 2 7 N 0 9 : C, 52.43; H, 6.99; N, 3.60. Found: C, 52.08; H, 6.96; N, 3.42. Methyl N-acetyl-D-2- (1,2:5, 6-di-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (212) . To a s o l u t i o n of the 3-0_-acetyl-N-acetyl D - g l y c i n a t e 210 (417 mg) i n methanol (10 ml) was added 0.5% sodium methoxide i n methanol (0.5 m l ) . IRC-50 (H +) r e s i n was added to the s o l u t i o n a f t e r 1 h and the mixture was s t i r r e d f o r an a d d i t i o n a l 15 min. The r e s i n was removed by f i l t r a -t i o n and the f i l t r a t e evaporated, under d i m i n i s h e d p r e s s u r e , to g ive an amber syrup. Column chromatography o f the crude - 204 -product on s i l i c a g e l (15 cj, 1.2 x 2.15 cm), u s i n a 9:1 benzene-ethyl a c e t a t e as developer, gave a c l e a r c o l o u r l e s s syrup (242 mg, 64%). D i s t i l l a t i o n o f the syrup at 160°/ 0.2 t o r r . y i e l d e d the t i t l e compound 212 as a chromatographi-22 c a l l y pure syrup; [ a l D + 45.9° (c 1.2, c h l o r o f o r m ) ; n.m.r. (CDCl-,) : <5 7.08 (d, 1, J.T„ ,, 10 Hz, NHAc, exchange wi t h D 20), 5.85 (d, 1, J± 2 3.8 Hz, H - l ) , 5.25 (d, 1, c o l l a p s e s t o s upon a d d i t i o n o f D 20, H - l ' ) , 4.58 (d, 1, H-2), 4.30 (d, 1, J . _ 2.5 Hz, H-4), 4.09 (sex, 1, J 5.5 Hz, H-5), 3.72 (s, 5, C0 2CH 3, H-6), 2.54 (br s, 1, exchanges i n D 20, OH), 1.96 (s, 3, Ac), 1.56, 1.47, 1.46 and 1.36 (4s, 12, CH 3) . An a l . C a l c . f o r C 1 7 H 2 7 N 0 9 : C, 52.44; H, 6.99; N, 3.60. Found: C, 51.94; H, 6.86; N, 3.87. Mo l e c u l a r weight by mass spectrometry 389.170. C^H^NO^ r e q u i r e s 389.169. (M +-CH 3) 374.146. C l g H 2 4 N O g r e q u i r e s 374.145. Methyl N - a c e t y l - L - 2 - (6-0_- a c e t y l - l , 2 - 0 - i s o p r o p y l i d e n e - c t -g - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (221). The N - a c e t y l - L - g l y c i n a t e 209 (4 80 mg) was d i s s o l v e d i n 66% aqueous a c e t i c a c i d (10 ml). A f t e r the r e a c t i o n mixture was kept f o r 24 h a t room temperature, the s o l u t i o n was evaporated to y i e l d a c l e a r syrup (431 mg, 99%) which c r y s t a l l i z e d spontaneously upon s t a n d i n g . R e c r y s t a l l i z a -t i o n o f compound 221 from e t h a n o l - e t h e r produced f i n e , white c r y s t a l s ; m.p. 124.5-126.0°, [a]^° + 47.7°(c 1.2, methanol); - 205 -n.m.r. (CDC1 0) : <S 7.23 (d, 1, J„„ , , 8.0 Hz, NHAc), 5.83 (d, 1, J X 2 3.5 Hz, H - l ) , 5.23 (d, 1, H - l ' ) , 4.69 (br s, 1, OH, exchanges i n D 20), 4.36 (d, 1, H-2), 4.50 -3.90 (m, 5, 1 proton exchanges i n D 20, OH, H-4, 5, and 6), 3.75 (s, 3, C0 2CH 3), 2.08 and 2.06 (2s, 6, A c ) , 1.43 and 1.29 (2s, 6, CH 3); n.m.r. (DMSO-d g): 6 8.36 (d, 1, J » T L T i • 9.0 H z ' NHAc, exchanges i n D„0), 5.80 (d, 1, J N _ NH,1 Z 1,Z 3.5 Hz, H - l ) , 5.46 (s, 1, C-3-OH, exchanges i n D 2 0 ) , 5.28 (d, 1, J C 5.2 Hz, exchanges i n D„0, C-5-OH) , 5.09 (d, 1, D , Uri Z — c o l l a p s e s to s upon a d d i t i o n of D2O, H - l " ) , 4.47 (d, 1, H-2), 4.40 - 3.80 (m, 4, H-4, 5, and 6), 3.63 (s, 3, C0 2CH 3), 3.31 (DOH), 2.03 and 1.92 (2s, 6, A c ) , 1.39 and 1.28 (2s, 6, CH 3). A n a l . C a l c . f o r c X 6 H 2 5 N O i o : C ' 4 9 - 1 0 ; H, 6.44; N, 3.58. Found: C, 49.35; H, 6.36; N, 3.63. Methyl N - a c e t y l - L - 2 - (5 , 6 - d i - 0 - a c e t y l - l , 2-0_-isopropylidene-a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (222) . The 6-0-acetyl L - g l y c i n a t e 221 (110 mg) was d i s s o l v e d i n p y r i d i n e (2 ml) and a c e t i c anhydride (1 ml) and was s t i r r e d f o r 24 h at 22°. Work-up of the r e a c t i o n mixture i n the usual manner y i e l d e d the t i t l e compound 222 (117 mg, 96%) as a c l e a r syrup. An a n a l y t i c a l sample of compound 222 was o b t a i n e d by d i s t i l l a t i o n a t 160-170° and 0.3 t o r r ; ta]J3 + 72.5°(c 0.8, c h l o r o f o r m ) ; v C H C l 3 3450 (OH, NH), D — max 1740 (C=0), and 1690 cm" 1 (amide I ) ; n.m.r. (CDC1 3): 6 6.82 (d, 1, J N H ± i 8.5 Hz, NHAc, exchanges i n D 2 0 ) , - 206 -5.90 (d, 1, J1 2 3.8 Hz, H - l ) , 5.49 (oct, 1, J 4 5 3.5 Hz, J r c 2.5 Hz, J c r, 7.8 Hz, H-5), 5.08 (d, 1, H - l ' , c o l l a p s e d b,ba D , bb to s i n g l e t upon a d d i t i o n of D~0), 4.74 (q, 1, Jc 12.5 Hz, H-6a), 4.46 (d, 1, H-4), 4.45 (s, 1, OH, exchanges i n D 20), 4.44 (d, 1, H-2), 4.13 (q, 1, H-6b), 3.79 (s, 3, C0 2CH 3), 2.09 and 2.03 (2s, 9, A c ) , 1.48 and 1.31 (2s, 6, CH 3) . A n a l . C a l c . f o r c 1 8 H 2 7 N O i i : c ' 49.88; H, 6.28; N, 3.23. Found: C, 49.66; H, 6.33; N, 3.20. Methyl N - a c e t y l - L - 2 - (3,5 , 6 - t r i - 0 - a c e t y l - l , 2-0_-isopropylidene-a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (223). To a s o l u t i o n o f the 5,6-di-0_-acetyl-L.-glycinate 222 (38 mg) i n a c e t i c anhydride (1 ml) was added p - t o l u e n e -s u l p h o n i c a c i d monohydrate (3 mg). A f t e r the r e a c t i o n was s t i r r e d f o r 6 h a t 60°, t . l . c . o f the product showed the r e a c t i o n to be complete. The a c e t i c anhydride was removed by repeated c o - d i s t i l l a t i o n w i t h toluene (3 x 5 ml) under reduced p r e s s u r e . The r e s u l t i n g syrup was d i s s o l v e d i n c h l o r o f o r m (5 ml) and washed wi t h water (3 x 1 ml). The c h l o r o f o r m s o l u t i o n was d r i e d over sodium sulphate and evaporated t o y i e l d the t e t r a - a c e t a t e g l y c i n a t e 223 (37 mg, 93%) as a c l e a r , pale yellow syrup. An a n a l y t i c a l sample of compound 223 was obtained by d i s t i l l a t i o n a t 160-165° 20 and 0.3 t o r r ; [ a ] D + 81.1° (c 0.6, c h l o r o f o r m ) ; n.m.r. - 207 -(CDC1,) : 6 6.71 (d, 1, 0\T„ ,, 10.5 Hz, NHAc, exchanges wit h D 20), 6.02 (d, 1, 0^ 2 4.0 Hz, H - l ) , 5.85 (d, 1, H - l ' , c o l l a p s e s t o s i n g l e t upon a d d i t i o n of D 20), 5.51 (sex, 1, J . c 2.5 Hz, J c c 2.5 Hz, Jc 8.0 Hz, H-5) , 5.12 (d, 1, H-2), 4.54 (q, 1, J , 12.5 Hz, H-6a) , 4.46 (d, 1, H-4), 4.03 (q, 1, H-6b), 3.74 (s, 3, C0 2CH 3), 2.12, 2.10, 2.04 and 2.01 (4s, 12, Ac), 1.58 and 1.38 (2s, 6, CH 3) . A n a l . C a l c . f o r C 2 0 H 2 9 N O 1 2 : C, 50.52; H, 6.15; N, 2.95. Found: C, 50.31; H, 6.22; N, 2.80. I d e n t i c a l a c e t y l a t i o n o f the t r i o l g l y c i n a t e 221 with a c e t i c anhydride and p - t o l u e n e s u l p h o n i c a c i d mono-hydrate y i e l d e d the f u l l y a c e t y l a t e d compound 223 i n an 85% y i e l d . Methyl N - a c e t y l - ^ - 2 - (1,2-0_-isopropylidene-ct-D-allofuranos-3 - y l ) g l y c i n a t e (226) . Method A: The d i - O - i s o p r o p y l i d e n e - L - g l y c i n a t e 221 (200 mg) was d i s s o l v e d i n 66% a c e t i c a c i d (10 ml) and l e f t at room temperature f o r 28 h. Ev a p o r a t i o n of the s o l u t i o n gave a pale y e l l o w g l a s s , which was then d i s s o l v e d i n methanol (10 m l ) . T h i s s o l u t i o n was d e c o l o r i z e d u s i n g a c t i v a t e d c h a r c o a l , f i l t e r e d , and evaporated t o give a c l e a r c o l o u r -l e s s g l a s s i n q u a n t i t a t i v e y i e l d ; n.m.r. (DMSO-dg): 6 8.52 (d, 1, J.7„ ,, 8.5 Hz, NHAc, exchanges i n . D~0) , 5.85 NH ,1 Z (d, 1, J , „ 3.5 Hz, H - l ) , 5.70 (br s, 1, OH, exchanges i n . D o 0 ) , 5.13 (d, 1, H - l 1 , c o l l a p s e s t o s upon a d d i t i o n o f - 208 -D 2O),5.09 (br s, 1, OH, exchanges with D 20), 4.92, (br s, 1, OH, exchanges i n D 20), 4.52 (d, 1, H-2), 4.30 (d, 1, J 4 5 6 Hz, H-4), 4.00 - 3.10 (m, 3, H-5, 6), 3.69 (s, 3, C 0 2CH 3), 2.00 (s, 3, A c ) , 1.47 and 1.36 (2s, 6, C H 3 ) . The t r i o l L - g l y c i n a t e 226 was a c e t y l a t e d with a c e t i c a n h y d r i d e - p y r i d i n e to y i e l d the p r e v i o u s l y prepared methyl N-acetyl-L-2- (5 , 6 - d i - 0 _ - a c e t y l - l , 2-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (222, 214 mg, 96%). Method B: The d i o l ^ - g l y c i n a t e 221 (50 mg) was de-0_-a c e t y l a t e d i n a manner s i m i l a r to t h a t employed f o r the d e - a c e t y l a t i o n of the 0-acetate g l y c i n a t e 209 to y i e l d the t r i o l L - g l y c i n a t e 226 as a p a l e y ellow g l a s s i n q u a n t i -t a t i v e y i e l d . D e c o l o r i z a t i o n and a c e t y l a t i o n of the t r i o l 226 with a c e t i c anhydride and p y r i d i n e y i e l d e d the 5,6-di-0_-acetyl L - g l y c i n a t e 222 (53 mg, 95%) as a c l e a r c o l o u r l e s s syrup. Attempted S y n t h e s i s of 3-C-[(S)-Acetamino(carbomethoxy)-m e t h y l ] - 1 , 2 - 0 - i s o p r o p y l i d e n e - a - g - r i b o - p e n t o d i a l d o f u r a n o s e -5,N-aminal (228). The 3 , 5 , 6 - t r i o l L - g l y c i n a t e 226 (50 mg) was d i s s o l v e d i n an aqueous s o l u t i o n (2.5 ml) of sodium p e r i o d a t e (32 mg, 1.0 equiv.) and sodium b i c a r b o n a t e (13 mg, 1.1 e q u i v . ) . A f t e r the r e a c t i o n mixture was allowed to stand f o r 1 1/2 h a t room temperature, e t h a n o l (3 ml) was added and the - 209 -s o l u t i o n was cooled to 0°. The s o l u t i o n was f i l t e r e d and the f i l t r a t e evaporated under di m i n i s h e d pressure to y i e l d a c l e a r c o l o u r l e s s syrup (53 mg). The crude product was used d i r e c t l y i n subsequent r e a c t i o n s . Attempted A c e t y l a t i o n to g i v e 5-0-Acetyl-3-C-[ ( s ) -acetamino (carbomethoxy)methyl] -1,2-0-isopropylidene-oc-D-ribo-pentodialdofuranose-5,N-aminal (229) . The above product 228 was d i s s o l v e d i n a c e t i c anhydride (1 ml) and p y r i d i n e (2 ml) and s t i r r e d f o r 24 h at room temperature. Work up i n the u s u a l manner gave a l i g h t brown syrup (9 0 mg). Column chromatography of the crude product on s i l i c a g e l (10 g, 14 x 1.5 cm), w i t h .8:2 benzene-eth a n o l as developer, y i e l d e d a mixture of g r e a t e r than f i v e components i n low y i e l d . Attempted A c e t y l a t i o n t o g i v e 3-C-[(S)-Acetamino(carbo-methoxy) methyl] -3, 5 - d i - O ^ - a c e t y l - l , 2-0_-isopropylidene-a-D-ribo-pentodialdofuranose-5,N-aminal (230). The crude product 227 obtained from the o x i d a t i o n of the t r i o l 226 was d i s s o l v e d i n a c e t i c anhydride (1 ml) and to t h i s was added p - t o l u e n e s u l p h o n i c a c i d monohydrate (7 mg). The r e s u l t i n g s o l u t i o n was heated f o r 5 h a t 83°. The r e a c t i o n was worked-up i n the u s u a l manner and the r e s u l t i n g dark brown syrup (105 mg) was chromatographed on a column of s i l i c a g e l (10 gm, 14.0 x 1.5 cm), w i t h 8:2 - 210 -benzene-ethanol as developer, t o a f f o r d s i x inhomogeneous components. Attempted B e n z o y l a t i o n to g i v e 3-C-[(S)-Acetamino(carbo-methoxy ) methyl] -3 , 5 - d i - 0 - b e n z o y l - l , 2 - 0 _ - i s o p r o p y l i d e n e - a -D-ribo-pentodialdofuranose-5,N-aminal (231). The crude product 227 obtained from the p e r i o d a t e o x i d a t i o n of the t r i o l 226 was d i s s o l v e d i n a mixture of anhydrous benzene (1 ml), p y r i d i n e (0.046 ml), and benzoyl c h l o r i d e (0.038 ml). A f t e r the r e a c t i o n mixture was allowed t o stand f o r 24 h, the s o l u t i o n was passed through a s h o r t column o f grade I I alumina (0.5 g) and the column was subsequently r i n s e d with an a d d i t i o n a l 5 ml of anhydrous benzene. E v a p o r a t i o n of the e l u a n t and column chromatography of the r e s u l t i n g pale y e l l o w syrup (60 mg) on s i l i c a g e l (10 gm, 14 x 1.5 cm), with 9:1 benzene-e t h y l a c e t a t e as the developer, gave an i n s e p a r a b l e mixture of compounds. Methyl N - a c e t y l - L - 2 - (5 - 0 _ - a c e t y l - l , 2-0_-isopropylidene-a-D-r i b o f u r a n o s - 3 - y l ) g l y c i n a t e (233) and Methyl N - a c e t y l - ^ - 2 -(1, 2-0_-isopropylidene-a-D-ribofuranos-3-yl) g l y c i n a t e (232) . The d i - 0 - i s o p r o p y l i d e n e L - g l y c i n a t e 211 was h y d r o l y z e d as p r e v i o u s l y d e s c r i b e d to a f f o r d the t r i o l L - g l y c i n a t e 226. Without f u r t h e r p u r i f i c a t i o n the crude t r i o l 226 was d i s s o l v e d i n water (10 ml) and methanol (5 ml) and a - 211 - -s o l u t i o n of sodium p e r i o d a t e (109 mg) and water (5 ml) was s l o w l y added over a p e r i o d of 15 min. E thylene g l y c o l (2 drops) was added to consume the unreacted sodium p e r i o d a t e and a f t e r 5 min sodium borohydride (10 mg) was added to the s o l u t i o n . The r e s u l t i n g s o l u t i o n was s t i r r e d f o r 15 min a t room temperature before adding acetone (1 ml). A f t e r a f u r t h e r 15 min the s o l u t i o n was evaporated, i n vacuo,and t o the r e s u l t i n g white s o l i d was added eth a n o l ( 5 m l ) . The r e s u l t i n g mixture was c o o l e d to 0°, f i l t e r e d through s i n t e r e d g l a s s , and the f i l t r a t e evaporated to y i e l d a c o l o u r l e s s g l a s s . The crude product was d i s s o l v e d i n a c e t i c anhydride (1 ml) and p y r i d i n e (2 ml) and s t i r r e d f o r 24 h a t room temperature. The r e a c t i o n was worked-up i n the u s u a l manner to y i e l d a t a n - c o l o u r e d s o l i d which was chromatographed on a column of s i l i c a g e l (20 mg), u s i n g 8:2 benzene-ethanol as the developer, to a f f o r d the 5-fJ-acetyl g l y c i n a t e 233 (21 mg, 13%) and the 3 , 5 - d i o l g l y c i n a t e 232 (38 mg, 26%). Q u a n t i t a t i v e conver-s i o n of the d i o l 232 i n t o i t s 5-0_-acetyl d e r i v a t i v e 233 was accomplished by c o n t i n u i n g the a c e t y l a t i o n r e a c t i o n f o r an a d d i t i o n a l 48 hours. An a n a l y t i c a l sample of compound 233 was o b tained by d i s t i l l a t i o n a t 150° and 0.20 t o r r ; [ a ] ^ 4 + 57° (c 0.4, c h l o r o f o r m ) ; n.m.r. (CDC1.J : <$ 6.79 (br d, 1, J„„ -J NH , 1 8.5 Hz, exchanges i n D 20, NH), 5.95 (d, 1, 2 3.8 Hz, H - l ) , 5.07 (d, 1, H - l ' ) , 4.47 (d, 1, H-2), 4.6 - 4.2 (over-l a p p i n g s i g n a l s , 4, a d d i t i o n of D 20 reduces i n t e g r a t i o n to 3 p r otons, H-4, H-5, and OH), 3.80 (s, 3, C 0 2CH 3), 2.12 and 2.08 (2s, 6, A c ) , 1.50 and 1.32 (2s, 6, CH,,) . - 212 -Ana l . C a l c . f o r C 1 5 H 2 3 N 0 9 : C ' 4 9 « 8 6 ; H, 6.41; N, 3.88, Found: C, 49.47; H, 6.40; N, 3.83. Molecular weight by mass spectrometry 361.1349. c i 5 H 2 3 N 0 9 r e c 3 u i r e s 361.1372. (M +-CH 3) 346.1136 r e q u i r e d 346.1138. An a n a l y t i c a l sample of the 3 , 5 - d i o l L - g l y c i n a t e 232 24 was obt a i n e d by d i s t i l l a t i o n a t 150° and 0.02 t o r r ; [ a ] ^ + 44° (c 1.0, ch l o r o f o r m ) ; n.m.r. (CDC1 3): 6 6.95 (br d, 1, J. T t J ,, 9.0 Hz, exchanges i n D o0, NH) , 5.95 (d, 1, N n ,1 Z 2 3.7 Hz, H - l ) , 5.57 (br s, 1, exchanges i n D 20, OH), 5.20 (d, 1, c o l l a p s e s t o s i n g l e t upon a d d i t i o n of D 20, H - l 1 ) , 4.60 - 3.95 (ov e r l a p p i n g s i g n a l s , 5, a d d i t i o n of D 20 reduces i n t e g r a t i o n to 4 protons, H-2, 4, 5 and OH), 3.80 (s, 3, C0 2CH 3), 2.10 (s, 3, NAc), 1.52 and 1.34 (2s, 6, CH 3). Ana l . C a l c . f o r C 1 3H 2 1NO g: C, 48.89; H, 6.63; N, 4.39 Found: C, 48.51; H, 6.46; N, 4.35. Mo l e c u l a r weight by mass spectrometry 304.1013. c i 2 H 1 8 N 0 8 ( M + - C H 3 ) r e q u i r e s 304.1032. Attempted Dehydration of Methyl N-acetyl-L-2-(5,6-di-0_-a c e t y l - 1 , 2 - 0 - i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e  (222) . To a s o l u t i o n of compound 222 (386 mg) i n p y r i d i n e (12 ml) (kept a t -25°) was added t h i o n y l c h l o r i d e (4 ml). The s o l u t i o n was maintained at +5° o v e r n i g h t a t which time the excess t h i o n y l c h l o r i d e was decomposed by the a d d i t i o n - 213 -of p y r i d i n e - w a t e r (5:1, 2 ml). Water (10 ml) was added and the aqueous s o l u t i o n was then e x t r a c t e d with c h l o r o f o r m (5 x 10 ml). The combined o r g a n i c e x t r a c t s were d r i e d (Na2SO^) and evaporated to give a dark amber syrup. The unstable crude product was immediately hydrogenated i n methanol over Raney n i c k e l f o r 24 h. The c a t a l y s t was removed by f i l t r a t i o n and the f i l t r a t e evaporated to y i e l d a syrup which was chromatographed on a column of s i l i c a g e l (15 g, 1.5 x 25 cm), u s i n g 8:2 benzene-ethanol as the developer, to y i e l d the s t a r t i n g compound 222 (112 mg, 29%) . 3-Deoxy-l ,2:5 , 6-di-0_-isopropylidene-3-C- [ (R,S) - n i t r o -( methoxycarbonyl ) m e t h y l ] - a - D - a l l o f u r a n o s e (236) . The ketose 2_5 (4.67 g) , methyl n i t r o a c e t a t e (4.30 g, 2.0 equiv.) and ammonium ac e t a t e (1.39 g, 1.0 equiv.) were r e a c t e d together i n N,N-dimethylformamide (20 ml) as p r e v i o u s l y d e s c r i b e d t o y i e l d the methyl n i t r o a c e t a t e adduct 162, as an amber syrup. The crude product and p-t o l u e n e s u l p h o n i c a c i d monohydrate (0.40 gm) were d i s s o l v e d i n a c e t i c anhydride (40 ml) and the temperature of the s o l u t i o n was q u i c k l y r a i s e d t o 120° where i t was main-t a i n e d f o r 1 h. The r e a c t i o n was worked-up i n the u s u a l manner to y i e l d a t h i c k b l a c k syrup. Column chromato-graphy of the crude product on s i l i c a g e l (500 g, 6 x 45 cm), developed with 8:2 benzene-ethyl acetate,gave the n i t r o - 214 -e s t e r 165 (R f 0.38, 4.78 gm, 63%) and (E) and (Z)-1,2:5,6-di-0_-isopropylidene-3-C- [ n i t r o (methoxycarbonyl) methylene] -ct-D-ribo-hexofuranose (235) (R^ 0.42, 262 mg, 4%); n.m.r.* (CDC1 3): 6 5.89 (d, 1, J 1 A 2 A 5.0 Hz, H-1A), 5.87 (d, 1, J1B,2B 4 ' 8 H Z ' H " 1 B ) ' 5 ' 6 9 1> J2A,4A 2 H Z ' J4A,5A 4 H Z ' H-4A), 5.62 (q, 1, J 2 B ^ 4 B 2 Hz, J 4 B ^ 5 B 5 Hz, H-4B), 5.50 (q, 1, H-2A and H-2B), 4.25 - 3.40 (ove r l a p p i n g s i g n a l s , 3, H-5 and 6), 3.98 (s, 3, C0 2CH 3~B), 3.94 (s, 3, C0 2CH 3~A), 1.39, 1.37, 1.30 and 1.26 (4s, 12,CH 3). Compound 235, without f u r t h e r p u r i f i c a t i o n , was d i s -s o l v e d i n a s o l u t i o n of methanol (20 ml) and bromocresol green and the pH of the s o l u t i o n was adjus t e d t o ^ 4.0 by a d d i t i o n of 1% h y d r o c h l o r i c a c i d i n methanol. Sodium cyanoborohydride (46 mg, 1.0 equiv.) was added to the s o l u t i o n and the pH was maintained a t ^ 4.0 by the p e r i o d i c a d d i t i o n o f a c i d . A f t e r 1 h at room temperature, acetone (1/2 ml) was added to the s o l u t i o n and 10 min l a t e r the s o l u t i o n was d i l u t e d with s a t u r a t e d sodium c h l o r i d e s o l u t i o n (50 m l ) . The aqueous s o l u t i o n was e x t r a c t e d with c h l o r o f o r m (3 x 20 ml) and the combined e x t r a c t s were d r i e d ( N a 2 S 0 4 ) , f i l t e r e d , and evaporated to y i e l d a p a l e y e l l o w syrup (244 mg, 89.5%). An a n a l y t i c a l sample of compound 236 was prepared by d i s t i l l a t i o n at 125°/0.2 t o r r , [ a ] 2 5 + 80.1° (c 0.4, c h l o r o f o r m ) ; n.m.r.* (CDC1 3): 6 5.84 (d, 1, J 2 3.8 Hz, H - l ) , 5.68 (d, 1, J 1 < A / 3 A 6.0 Hz, H-l'A), 5.36 (d, 1, J 1 I B 3 B 9.8 Hz, H-l'B), 5.09 ( t , 1, - 215 -J2A,3A 4 , 9 H Z ' H - 2 A ) ' 4 , 8 7 ( t ' X ' J2B,3B 4 , 9 H z ' H - 2 B ) ' 4.32 - 3.87 (overlapping s i g n a l s , 4, H-4, 5 and 6), 3.82 (s, 3, C0 2CH 3), 2.83 (oct, 1, J 3 A 4 A 9.5 Hz, H-3A), 1.51, 1.48, 1.42, 1.38 and 1.30 (5s, 12, CH 3). Ana l . C a l c . f o r C ± 5 H 2 3 N 0 9 : C, 49.86; H, 6.14; N, 3.88. Found: C, 49.74; H, 6.61; N, 3.77. * Compounds 235 and 236 were a mixture o f g e o m e t r i c a l and d i a s t e r e o m e r i c isomers, r e s p e c t i v e l y . Those resonances, i n the p.m.r. spectrum, a t t r i b u t e d to the major isomer were desi g n a t e d A while those a t t r i b u t e d to the minor isomer were d e s i g n a t e d B. Those resonances common t o both isomers were t o bear no d e s i g n a t i o n . In the p.m.r. spectrum of compound 2 35 the r a t i o of isomers was c a l c u l a t e d , on the b a s i s of the h e i g h t s of the methyl e s t e r resonances, as A:B equals 1.5:1. In the p.m.r. spectrum o f compound 2 36 the r a t i o of isomers was c a l c u l a t e d , on the b a s i s of the i n t e g r a t e d areas of the H-2 protons, as A:B equals 2.2:1. Methyl L-2-(3-deoxy-l,2:5,6-di-O-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (141) and Methyl D-2-(3-deoxy-1,2:5, 6- d i - 0 _ - i s o p r o p y l i d e n e - a - D - a l l o f uranos-3-yl) g l y c i n a t e (142) . To a s o l u t i o n of the n i t r o e s t e r 236 (209 mg) i n - 216 -methanol (25 ml) was added a c t i v a t e d Raney n i c k e l c a t a l y s t ('vl ml) and the r e s u l t i n g mixture was v i g o r o u s l y s t i r r e d under an atmosphere of hydrogen f o r 48 h. The c a t a l y s t was removed by f i l t r a t i o n and the f i l t r a t e was evaporated under reduced pressure t o y i e l d a c l e a r c o l o u r l e s s syrup. Chromatography of t h i s syrup on s i l i c a g e l (30 gm, 2 x 31 cm), u s i n g 9:1 benzene-ethanol as developer, gave compound 141 (R f 0.29, 88 mg, 46%) and compound 142 (R f 0.35, 74 mg, 38%) both as c l e a r c o l o u r l e s s syrups. Compound 141; n.m.r. (CDC1 3): •& 5.78 (d, 1, 2 3.8 Hz, H - l ) , 4.78 ( t , 1, J 2 3 4.8 Hz, H-2), 4.5 - 3.5 (ov e r l a p p i n g s i g n a l s , 5, H-6, 5, 4, and 1'), 3.74 (s, 3, C0 2CH 3), 2.36 (qn, 1, J 3 4 7 Hz, J 3 ± , 7 Hz, H-3), 2.06 (br s, 2, NH2, exchanges i n D 2 0 ) , 1.48, 1.42, 1.38 177 and 1.29 (4s, 12, CH 3); { L i t value; n.m.r. (CDC1 3): 6 5.78 (d, 1, J x 2 4 Hz, H - l ) , 4.77 ( t , 1, J 2 3 4 Hz, H-2), 4.4 - 3.8 (ove r l a p p i n g peaks), 3.74 (s, 3, C0 2CH 3), 2.48 -2.24 (m, H - l 1 , c l e a r l y v i s i b l e a f t e r a d d i t i o n of D 2 0 ) , 2.3 -1.9 (NH 2, disapp e a r s on a d d i t i o n of D,,0), 1.38 - 1.22 (4s, 12, CH 3)};n.m.r. (CD 3OD): 6 5.77 (d, 1, J± 2 3.8 Hz, H - l ) , 4.82 ( t , 1, J 2 3 4.5 Hz, H-2), 4.32 (q, 1, J 3 4 9.2 Hz, J . _ 4.5 Hz, H-4), 4.2 - 3.8 (ov e r l a p p i n g s i g n a l s , 4 , D 3, H-5 and 6), 3.94 (d, 1, J 3 ± , 5.8 Hz, H - l ' ) , 3.73 (s, 3, C 0 2 C H 3 ) , 2.31 (qn, 1, H-3), 1.45, 1.40, 1.32 and 1.28 (4s, 12, CH 3). - 217 -Compound 142; n.m.r. (CDC1 3): 6 5.78 (d, 1, ^ 2 4.0 Hz, H - l ) , 4.77 ( t , 1, J 2 3 4.5 Hz, H-2), 4.5 - 3.6 (ove r l a p p i n g s i g n a l s , 5, H-4, 5, 6 and 1'), 3.79 (s, 3, C0 2CH 3), 2.5 (br s, 3, H-3 and NH 2, c o l l a p s e s t o broad s i g n a l r e p r e s e n t i n g 1 proton upon a d d i t i o n of D 2 0 ) , 1.54, 1.45, 1.34 and 1.30 (4s, 12, C H 3 ) . { L i t . value 1 7 9 ; n.m.r. (CDC1 3): 6 5.79 (d, 1, J± 2 3.8 Hz, H - l ) , 4.78 ( t , 1, J ^ 4.0 Hz, H-2), 4.5 - 6.1 (m, 5, H - l 1 , 4, 5 and 6), 3.80 (s, 3, C0 2CH 3), 2.45 (m, 1, H-3), 1.85 (br s, 2, NH2)}"; n.m.r. (CD3OD) : 6 4 .24 (d, 1, J± 2 3.8 Hz, H - l ) , 4.79 ( t , 1, J 2 3 4.8 Hz, H-2), 6.4 - 3.6 (o v e r l a p p i n g s i g n a l s , 5, H - l ' , 4, 5 and 6), 3.76 (s, 3, C0 2 C H 3 ) , 2.37 (oct, 1, J 9.5 Hz, J_ 6.5 Hz, H-3), 1.50, 1.41 j , H 5 , X and 1.31 (3s, 12, CH 3). L-2 - (3-Deoxy-1, 2-0_-isopropylidene-a-D-allof uranos-3-yl) -g l y c i n e (237). The amino e s t e r 141 (65 mg) was d i s s o l v e d i n methanol (1 ml) and to t h i s was added 1 ml of sodium hydroxide s o l u t i o n (1.25% i n methanol-water 1:1). The s o l u t i o n was s t i r r e d f o r 2 h at room temperature. The s o l u t i o n was then a p p l i e d t o a column of RG 51 (H +) c a t i o n exchange r e s i n (5 ml) and the column was e l u t e d w i t h water. The n i n h y d r i n p o s i t i v e f r a c t i o n s were c o l l e c t e d and evaporated to y i e l d a pa l e y e l l o w c r y s t a l l i n e s o l i d (34 mg, 62%). R e c r y s t a l l i z a t i o n of compound 237 from methanol-water - 218 -y i e l d e d a white c r y s t a l l i n e s o l i d ; m.p. 212-214°, L " ! ^ + 57.5° (c 0.3 water). { L i t . v a l u e 1 7 7 m.p. 213-215°, [ a ] 2 2 + 60° (c 0.5, water) } . Methyl N-benzoyl-D-2- (3-deoxy-l ,2:5, 6-di-0^-isopropylidene - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e (238). To compound 142 (32 mg), d i s s o l v e d i n methanol (3 ml), was added benzoic anhydride (32 mg), and the r e s u l t i n g s o l u t i o n was s t i r r e d o v e r n i g h t at room temperature. The s o l u t i o n was then passed through a s h o r t column o f alumina (grade I I , 2 g) which was packed and e l u t e d w i t h anhydrous benzene. The c h a r r i n g f r a c t i o n s were c o l l e c t e d and evaporated to y i e l d a p a l e y e l l o w g l a s s . The crude product was chromatographed on a column of s i l i c a g e l (5 g, 1 x 15.5 cm), u s i n g 6:4 benzene-ethyl a c e t a t e as the developer, to g i v e the t i t l e compound 238 as a c l e a r c o l o u r l e s s g l a s s . Compound 238 was c r y s t a l l i z e d from hexane-ethanol 22 to g i v e f i n e white needles; m.p. 145-146.5°, [ c ] D + 28° 184 (c 1.8, c h l o r o f o r m ) . { L i t . values ; m.p. 138-140°, 22 [ a ] D + 26° (c 0.5, ch l o r o f o r m ) } . The p.m.r. spectrum of 18 compound 238 was superimposable w i t h t h a t o f an a u t h e n t i c sample o f methyl N-benzoyl-D-2-(3-deoxy-l,2:5,6-di-0-i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s - 3 - y l ) g l y c i n a t e . - 219 -3,5 or 6 - 0 - A c e t y l - l , 2 - 0 - i s o p r o p y l i d e n e - 3 - C - [ ( R , S ) - n i t r o -( methoxycarbonyl ) m e t h y l ] - a - g - a l l o f u r a n o s e (239) . The n i t r o e s t e r 165 (1 gm) was d i s s o l v e d i n 66% a c e t i c a c i d (50 ml) and the s o l u t i o n was maintained f o r 5 h a t 50°. The s o l u t i o n was then evaporated under reduced pressure and the remaining t r a c e s o f a c e t i c a c i d were removed by c o - d i s t i l l a t i o n w i t h t o l u e n e , i n vacuo, t o y i e l d a pink syrup. Treatment o f the syrup w i t h a c t i v a t e d c h a r c o a l and r e f l u x i n g methanol y i e l d e d , a f t e r e v a p o r a t i o n , a c l e a r c o l o u r l e s s syrup which c r y s t a l l i z e d spontaneously upon st a n d i n g (877 mg, 97%). R e c r y s t a l l i z a t i o n of the t i t l e compound 239 from benzene-hexane gave white c r y s t a l -l i n e needles; m.p. 115-120°, [ a ] 2 4 + 43.5° (c 1.5, c h l o r o -form); n.m.r. (DMSO-dg): 6 6.44 (s, 1, exchanges i n D 20, OH), 6.22 (s, 1, H - l ' ) , 5.84 (d, 1, J± 2 4.0 Hz, H - l ) , 5.70 (s, 1/2, exchanges i n D 20, OH), 5.51 (br s, 1/2 exchanges i n D 20, OH), 4.79 (d, 1, H-2), 4.60 - 3.86 (o v e r l a p p i n g s i g n a l s , 4, H-4, 5, and 6), 3.79 and 3.74 (2s, 3, C0 2CH 3), 2.03 (s, 3, OAc), 1.36 and 1.26 (2s, 6, C H 3 ) . Ana l . C a l c . f o r C 1 4H 2 ]NO i ; L: C, 44.33; H, 5.58; N, 3.69. Found: C, 44.25; H, 5.57; N, 3.87. (E) and ( Z ) - 5 , 6 - D i - 0 - a c e t y l - 3 - d e o x y - l , 2 - 0 - i s o p r o p y l i d e n e -3-C- [ n i t r o ( methoxycarbonyl ) methylene] - c t-D-ribo-hexo-furanose (240). Compound 239 (800 mg) was d i s s o l v e d i n a c e t i c anhydride (10 ml) and t o t h i s was added p - t o l u e n e s u l p h o n i c a c i d - 220 -monohydrate (100 mg). The temperature of the s o l u t i o n was e l e v a t e d to 85° where i t was maintained f o r 24 h. The v o l a t i l e components of the s o l u t i o n were removed by e v a p o r a t i o n and repeated c o - d i s t i l l a t i o n w i t h toluene, i n vacuo, and the r e s u l t i n g syrup was then d i s s o l v e d i n c h l o r o f o r m (30 ml). The c h l o r o f o r m s o l u t i o n was washed with s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n (7 ml), washed wit h water ( 2 x 5 ml), d r i e d over sodium s u l p h a t e , and evaporated t o g i v e a y e l l o w syrup. Column chromatography of the crude product on s i l i c a g e l (40 gm, 2 x 38 cm), u s i n g 8:2 benzene-ethyl a c e t a t e as the developer, y i e l d e d the t i t l e compound 240 as a c l e a r c o l o u r l e s s syrup (749 mg, 88%). An a n a l y t i c a l sample of 240 was prepared by d i s t i l -22 l a t i o n at 140°/0.2 t o r r ; [a] + 263° (c 0.7, c h l o r o f o r m ) ; n.m.r. (CDCl^): 6 5.96 - 5.45 ( o v e r l a p p i n g s i g n a l s , 3, H - l , 2, 4), 5.15 - 4.74 ( o v e r l a p p i n g s i g n a l s , 1, H-5), 4.47 - 3.99 ( o v e r l a p p i n g s i g n a l s , 2, H-6), 3.88 (s, 3, C 0 2CH 3), 2.06, 2.04 and 2.00 (3s, 6, OAc), 1.39 and 1.36 (2s, 6, CH 3). A n a l . C a l c . f o r c x 6 H 2 i N O i i : C ' 47.65; H, 5.25; N, 3.47. Found: C, 47.43; H, 5.39; N, 3.32. 5 ,6-Di-0_-acetyl-3-deoxy-l, 2-0_-isopropylidene-3-C-[ ( R , S ) - n i t r o ( methoxycarbonyl ) m e t h y l ] - a - D - a l l o f u r a n o s e (241) . Method A: Compound 240 (25 7 mg) i n methanol (2 ml) was - 221 -added to a s o l u t i o n of sodium cyanoborohydride (40 mg) and bromocresol green i n methanol (3 m l ) . The s o l u t i o n was maintained a t pH 4 by the a d d i t i o n of 0.1% h y d r o c h l o r i c a c i d i n methanol f o r 20 min a f t e r which the s o l u t i o n was d i l u t e d with water (10 ml) and e x t r a c t e d w i t h c h l o r o f o r m (4 x 10 m l ) . The combined o r g a n i c e x t r a c t s were d r i e d over sodium s u l p h a t e , f i l t e r e d , and evaporated t o y i e l d a p a l e y e l l o w syrup (251 mg, 97%). An a n a l y t i c a l sample of compound 241 was o b t a i n e d by d i s t i l l a t i o n a t 125-140° 22 and 0.2 t o r r ; t a l D + 41.9° (c 1.1, c h l o r o f o r m ) ; n.m.r.* (CDC1 3): 6 5.87 (d, 1, J± 2 4.0 Hz, H-1B), 5.77 (d, 1, J . 0 3.5 Hz, H-1A), 5.41 (d, 1, J , , , 10.0 Hz, H- l ' B ) , X , Z X , J 5.36 (d, 1, J l t 3 9.0 Hz, H-l'A), 4.97 (q, 1, J 2 3 5.0 Hz, H-2A), 4.79 (q, 1, J 2 3 5.5 Hz, H-2B), 4.95 - 4.65 (over-l a p p i n g s i g n a l s , 1, H-4), 4.5 - 3.8 ( o v e r l a p p i n g s i g n a l s , 3, H-5 and 6), 3.87 (s, 3, C0 2CH 3~B), 3.82 (s, 3, C0 2CH 3~A), 3.01 (sex, 1, J 3 4 9.0 Hz, H-3A), 3.1 (m, 1, H-3B), 2.06 and 2.02 (2s, 6, OAc), 1.49 and 1.29 (2s, 6, CH 3). A n a l . C a l c . f o r c i 6 H 2 3 N O n : C ' 47.41; H, 5.72; N, 3.46. Found: C, 47.50; H, 5.60; N, 3.19. Method B: Compound 236 (50 mg) was d i s s o l v e d i n 66% a c e t i c a c i d and s t i r r e d f o r 6 1/2 h at room temperature. The s o l v e n t was removed by e v a p o r a t i o n i n vacuo and the r e s u l t i n g crude product, t o g e t h e r w i t h p - t o l u e n e s u l p h o n i c a c i d monohydrate (8 mg), was d i s s o l v e d i n a c e t i c anhydride. - 222 -The s o l u t i o n was s t i r r e d f o r 24 h a t room temperature and then worked-up i n the u s u a l manner to y i e l d an amber syrup (42 mg, 7 5% based on compound 236). The crude product was d i s t i l l e d at 140°/0.2 t o r r t o a f f o r d a compound i d e n t i c a l i n a l l r e s p e c t s t o compound 241. * The n.m.r. spectrum r e v e a l e d a mixture o f two isomers. The major isomer was desi g n a t e d "A" and the minor isomer was desi g n a t e d "B". A r a t i o of A:B = 1.35:1 was c a l c u l a t e d from the r e l a t i v e i n t e n s i t i e s of the methyl e s t e r r e s o n -ances . 1,2,5, 6-Tetra-0_-acetyl-3-deoxy-3-C- [ (R, S) - n i t r o (methoxy-carbonyl) m e t h y l ] - a , B - D - a l l o f u r a n o s e (243). The n i t r o e s t e r 241 (656 mg, i.62 mmoles) was d i s s o l v e d i n a c e t i c anhydride (.153 ml, 16.2 mmoles) and a c e t i c a c i d (10 ml) and t o t h i s s o l u t i o n was added p - t o l u e n e s u l p h o n i c a c i d monohydrate (62 mg, 0.32 mmoles). A f t e r s t i r r i n g f o r 2 h at 80-85°, the s o l u t i o n was co o l e d to room temperature and poured i n t o ice-water (100 m l ) . The water was e x t r a c t e d with e t h e r (4 x 50 ml) and the combined o r g a n i c e x t r a c t s were d r i e d (sodium sulphate) and evaporated, i n vacuo, t o y i e l d a pale yellow syrup (719 mg, 98%) . An a n a l y t i c a l sample of compound 243 was prepared by d i s t i l l a -t i o n a t 150°/0.01 t o r r ; U ] 2 2 - 15.0° (c 0.6, c h l o r o f o r m ) ; - 223 -n.m.r. (CDC1,): 6 6.44 (d, 0.24, J . _ 4.0 Hz, H - l a ) , j l a , za 6.19 (d, 0.76, J . 0 O D 2.8 Hz, H - l g ) , 4.7 - 3.3 ( o v e r l a p p i n g i p , /. p s i g n a l s , 7, H-2, 3, 4, 5, 6, 1'), 3.92 and 3.84 (2s, 3, C0 2CH 3), 2.22, 2.14, 2.10, 2.08 and 2.06 (5s, 12, OAc). A n a l . C a l c . f o r c 1 7 H 2 3 N O i 3 : C ' 45.44; H, 5.16; N, 3.12. Found: C, 45.58; H, 5.24; N, 2.97. M o l e c u l a r weight by mass spectroscopy 390.1048. c i 5 H 2 0 N O l l (M+-OAc) r e q u i r e s 390.1036. Attempted N u c l e o s i d e S y n t h e s i s with 1,2,5,6-Tetra-O-acetyl-3-deoxy-3-C-[(R,S)-nitro( methoxycarbonyl ) m e t h y l ] - a , B - g -a l l o f u r a n o s e (243) and B i s ( t r i m e t h y l s i l y l ) t h y m i n e (244). To a s o l u t i o n o f compound 243 (183 mg) and compound 244 (22 0 mg, 2.0 equiv.) i n anhydrous methylene c h l o r i d e (6 ml) was added a s o l u t i o n of stannous c h l o r i d e (153 mg, 6 8 y l , 1.4 equiv.) i n methylene c h l o r i d e (1 ml) and the r e a c t i o n was s t i r r e d f o r 24 h a t room temperature. The s o l u t i o n was then d i l u t e d with a s a t u r a t e d s o l u t i o n of sodium b i c a r b o n a t e (217 mg) and f i l t e r e d . The f i l t r a t e was e x t r a c t e d with e t h y l a c e t a t e (4 x 10 ml), d r i e d over sodium s u l p h a t e , f i l t e r e d and evaporated to y i e l d an amorphous s o l i d . The product was then t r i t u r a t e d with c h l o r o f o r m (10 ml), the mixture f i l t e r e d , and the f i l t r a t e evaporated to y i e l d the s t a r t i n g m a t e r i a l 243 (105 mg). The f i l t e r e d s o l i d was shown by p.m.r. spectroscopy t o be predominately thymine e o n t a i n i n g minor amounts o f 24 3. - 224 -The above r e a c t i o n was repeated u s i n g 1 , 2 - d i c h l o r o -ethane as the s o l v e n t and a f t e r 5 or 24 h a t 63° the r e a c t i o n was worked-up with s i m i l a r r e s u l t s . No evidence of n u c l e o s i d e m a t e r i a l c o u l d be found. Methyl L- 2- ( 3 - 0 - a c e t y l - l ,2:5 , 6-di-0_-isopropylidene-a-D-a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (245) . To a s o l u t i o n of compound 205 (7.94 g) i n d i c h l o r o -methane (120 ml) and p y r i d i n e (10 ml) at -10° was added t r i f l u o r o a c e t i c anhydride (10 m l ) . A f t e r s t i r r i n g a t -10° f o r 0.5 h, water (50 ml) was added and vigorous s t i r -r i n g maintained u n t i l the s o l u t i o n spontaneously a t t a i n e d room temperature. The aqueous l a y e r was separated and e x t r a c t e d with c h l o r o f o r m (2 x 40 ml) and the combined e x t r a c t s were d r i e d over sodium sulphate and evaporated, i n  vacuo,to a f f o r d a p a l e amber syrup. Column chromatography of the crude product on s i l i c a g e l (450 g, 6 x 40 cm), u s i n g 6:4 benzene-ethyl a c e t a t e as the developer, a f f o r d e d the t i t l e compound 245 as a c l e a r c o l o u r l e s s syrup (8.41 g, 85%). An a n a l y t i c a l sample of 245 was o b t a i n e d by d i s -t i l l a t i o n a t 120-140° and 0.005 t o r r ; [ a ] 2 2 + 5.7° (£ 4.6, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 8.24 (br d, 1, J±, N H 9 Hz, NH), 6.16 (d, 1, J1 2 3.8 Hz, H - l ) , 5.88 (d, 1, H - l ' ) , 4.75 (d, 1, H-2), 5.0 - 4.5 ( o v e r l a p p i n g s i g n a l s , 4, H-4, 5 and 6), 3.70 (s, 3, CC^CH-j) , 2.07 (s, 3, OAc), 1.77, 1.73, 1.66 and 1.51 (4s, 12, CH-j) . - 225 -A n a l . C a l c . f o r c 1 9 H 2 6 N O l O F 3 : C ' 4 7 - 0 1 ' H ' 5.40; N, 2.89. Found: C, 47.21; H, 5.43; N, 3.10. Methyl L - 2 - ( 3 , 5 , 6 - t r i - 0 - a c e t y l - l , 2 - 0 - i s o p r o p y l i d e n e - a -D - a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (246). Compound 245 (716 mg) was d i s s o l v e d i n 66% a c e t i c a c i d (20 ml) and s t i r r e d f o r 48 h a t room temperature. The r e a c t i o n mixture was worked-up i n the u s u a l manner to y i e l d a pale yellow syrup. Without f u r t h e r p u r i f i c a -t i o n , the crude product was d i s s o l v e d i n a s o l u t i o n of a c e t i c anhydride (20 ml) and p - t o l u e n e s u l p h o n i c a c i d mono-hydrate (100 mg) and s t i r r e d f o r 3 h a t 85° and f o r an a d d i t i o n a l 5 1/2 h a t 110°. The r e a c t i o n was worked-up i n the us u a l manner to give an amber syrup which was chromatographed on a column of s i l i c a g e l (40 g, 2 x 32 cm), u s i n g 1:1 benzene-ethyl a c e t a t e as developer, t o a f f o r d the t i t l e compound as a c l e a r syrup (616 mg, 79%) . An a n a l y t i c a l sample of compound 246 was obtained by d i s t i l -l a t i o n at 130° and 0.01 t o r r ; [ a ] 2 2 + 77.3° (C 2.0, c h l o r o -form) ; n.m.r. (CDCl-j) : 6 7 .64 (br d, 1, J ± i N H 10.0 Hz, exchanges i n D 20, NH), 6.05 (d, 1, 2 3.8 Hz, H - l ) , 5.83 (d, 1, c o l l a p s e s t o s i n g l e t upon a d d i t i o n o f D 20, H - l 1 ) , 5.40 (oct, 1, J 4 5 7.5 Hz, J 5 f 6 h 5.5 Hz, J ^ 6 a 2.5 Hz, H-5), 5.12 (d, 1, H-2), 4.61 (d, 1, H-4), 4.58 (q, 1, 3C 12.5 Hz, H-6a) , 4.08 (q, 1, H-6b) , 3.78 (s, 3, 6a, 6b - 226 -C 0 2CH 3), 2.10, 2.06 and 2.03 (3s, 9, OAc), 1.56, 1.38 and 1.27 (3s, 6, CH 3). A n a l . C a l c . f o r C 2 ( ) H 2 6 N 0 1 2 F 3 : C, 45.37; H, 4.95; N, 2.65. Found: C, 45.17; H, 4.82; N, 2.50. M o l e c u l a r weight by mass spectrometry 529.1387. C 2 0 H 2 6 N O 1 2 F 3 r e q u i r e s 529.1406. Methyl L-2- (5 , 6 - d i - 0 _ - a c e t y l - l , 2-0_-isopropylidene-a-p_-a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (247). Compound 24 5 (157 mg) was d i s s o l v e d i n 66% a c e t i c a c i d (12 ml) and a f t e r 5 h a t 22° the temperature was r a i s e d t o 40° f o r an a d d i t i o n a l 4 h. The r e a c t i o n was worked-up i n the u s u a l manner. The crude product was d i s s o l v e d i n a c e t i c anhydride (1 ml) and p y r i d i n e (4 ml) and l e f t at room temperature f o r 13 h. The a c e t i c anhydride and p y r i d i n e were evaporated i n vacuo t o a f f o r d a p a l e y e l l o w syrup which was immediately chromatographed on a column of s i l i c a g e l (15 g, 1.5 x 22 cm), u s i n g 6:4 benzene-ethyl a c e t a t e as developer. The chromatography y i e l d e d the s t a r t i n g m a t e r i a l (2 mg), the t r i - 0 - a c e t a t e 246 (49 mg, 29%), and the d i - 0 -acetate 247 (101 mg, 65%). Compound 247 was r e c r y s t a l -22 l i z e d from ether-hexane; m.p. 141-142°, [ a ] D + 54.4° (c 1.1, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 7.54 (br d, 1, - 227 -J l ' NH 9 , 0 H z ' e x c h a n 9 e s i n D 2 0 ' N R ) ' 5 - 9 4 ( d' !' J 1 2 3.8 Hz, H - l ) , 5.33 (sept, 1, J 5 g b 7.0 Hz, J 4 5 4.5 Hz, J 5 6 a 2.2 Hz, H-5), 5.09 (d, 1, c o l l a p s e s t o s upon a d d i t i o n of D 20, H - l ' ) , 4.69 (q, 1, J g a g b 12.5 Hz, H-6a), 4.51 (d, 2, H-2 and 4), 4.20 (q, 1, H-6b), 3.93 (s, 1, exchanges i n D 20, OH), 3.83 (s, 3, C0 2CH 3), 2.13 and 2.06 (2s, 6, OAc), 1.48 and 1.32 (2s, 6, C H 3 ) . A n a l . C a l c . f o r c 1 8 H 2 4 N O i i F 3 •" c ' 44.36; H, 4.96; N, 2.87. Found: C, 44.57; H, 4.86; N, 2.70. Methyl L-2- (1,2,3,5 , 6-penta-0_-acetyl-a , B - D - a l l o f uranos-3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (250). To a s o l u t i o n o f compound 246 (3.9 g) i n a c e t i c a c i d (78 ml) and a c e t i c anhydride (78 ml) was added p-toluene-s u l p h o n i c a c i d monohydrate (1.9 g), and the r e s u l t i n g s o l u t i o n was s t i r r e d f o r 2 h a t 110°. A f t e r the a d d i t i o n of c h l o r o f o r m (300 ml), the s o l u t i o n was washed with water (2 x 100 ml) and s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n (2 x 125 ml), d r i e d over sodium sulphate and evaporated i n vacuo to y i e l d a dark amber syrup. The crude product was chromatographed on s i l i c a g e l (400 g), u s i n g 6:4 benzene-ethyl a c e t a t e as the developer, to y i e l d compound 250 (2.0 g, 47%) as a b r i g h t y e l l o w foam. An a n a l y t i c a l sample of compound 250 was o b t a i n e d by d i s t i l l a t i o n a t 150° and 0.01 t o r r ; [ a ] 2 2 + 59.7° (c 1.9, c h l o r o f o r m ) ; n.m.r. (CDC1 3): 6 7.56 (br d, 1, ^, 9.6 Hz, exchanges - 228 -i n D 20, NH), 6.42 (d, 1/3, J± 2 5.1 Hz, H - l a ) , 6.04 (s, 2/3, H-1B) , 6.01 (s, 2/3, H-2B) , 5.64 (d, 1/3, H-2a) , 5.56 (1, H - l ' ) , 5.34 (oct, 2/3, J . ',. 7.2 Hz, J c _ 2.6 Hz, J c 6.1 Hz, H-5B) , 5.28 (oct, 1/3, J r , 2.4 Hz, J . c 5.6 Hz, J_ 6.4 Hz, H-5ct) , 4.68 (d, 2/3, H-4B), 4.63 D , D D (q, 2/3, J 6 a / 6 b 12.1 Hz, H-6aB), 4.61 (q, 1/3, J 6 a ^ 6 b 12.5 Hz, H-6aa), 4.51 (d, 1/3, H-4a), 4.02 (q, 1/3, H-6ba), 3.96 (q, 2/3, H-6bB), 3.78 and 3.76 (2s, 3, C 0 2CH 3), 2.14, 2.12, 2.10, 2.08, 2.07, 2.06, 2.03 and 2.01 (8s, 15, OAc) . A n a l . C a l c . f o r c 2 i H 2 6 N 0 1 4 F 3 : C / 4 3 - 9 9 ; H ' 4.57; N, 2.44. Found: C, 43.74; H, 4.31; N, 2.33. A p p l i c a t i o n of the Stannous C h l o r i d e C a t a l y s e d S i l y l H i l b e r t - J o h n s o n Procedure to the Condensation of Methyl L-2- (1,2,3,5 , 6-penta-0_-acetyl-a , B - D - a l l o f uranos-3-yl) -6 6 N - t r i f l u o r o a c e t y l g l y c i n a t e (250) with N -Benzoyl-N ,9-b i s ( t r i m e t h y l s i l y l ) adenine (114) or B i s ( t r i m e t h y l s i l y l ) -thymine (244) . Attempt 1: To a s o l u t i o n of compound 250 (50 mg) and s i l y l a t e d N-benzoyl adenine 114 (40 mg, 1.2 equiv.) i n anhydrous 1,2-dichloroethane (5 ml) was added a s o l u t i o n of stannous c h l o r i d e ( S n C l 4 , 14 y l , 1.4 equiv.) i n 1,2-d i c h l o r o e t h a n e (1 ml) and the mixture was heated to 70°. A f l o c c u l e n t p r e c i p i t a t e was observed a f t e r 20-30 min. - 229 -A f t e r s t i r r i n g f o r 13 h the r e a c t i o n was c o o l e d to 0° and sodium b i c a r b o n a t e (13 mg) i n et h a n o l (3 ml) was added f o l l o w e d by water (10 ml). The r e a c t i o n was f i l t e r e d , the f i l t r a t e was e x t r a c t e d w i t h e t h y l a c etate ( 4 x 5 ml) and the combined e x t r a c t s were d r i e d over sodium sulphate and evaporated t o y i e l d an amber syrup. Chloroform (10 ml) was added t o the crude product and the s o l u t i o n was r e f l u x e d f o r 5 min a f t e r which i t was co o l e d , f i l t e r e d and evaporated to y i e l d the s t a r t i n g sugar (43 mg, 86%) as an amber syrup. Attempt 2: The r e a c t i o n was c a r r i e d out as d e s c r i b e d above except t h a t b i s ( t r i m e t h y l s i l y l ) t h y m i n e (244 , 1.2 equiv.) was used as the base. The s t a r t i n g m a t e r i a l 250 was recovered i n 80% y i e l d . Attempt 3: Compound 250 (200 mg), b i s ( t r i m e t h y l s i l y l ) -thymine (118 mg) and stannous c h l o r i d e (70 y l ) were r e a c t e d together i n a c e t o n i t r i l e (5 ml) f o r 12 1/2 h at •60°. The r e a c t i o n was worked-up as d e s c r i b e d above t o y i e l d o n l y s t a r t i n g m a t e r i a l (190 mg, 95%). - .230 -Reaction of Methyl £-2-(1, 2 , 3 , 5 , 6-penta-0_-acetyl-a , g-D-a l l o f u r a n o s - 3 - y l ) - N - t r i f l u o r o a c e t y l g l y c i n a t e (250) w i t h V a r i o u s Purine Bases by the Fusion Method. Attempt 1: A c a r e f u l l y d r i e d mixture of compound 250 (52 mg), N-benzoyl adenine (40 mg, 2 e q u i v . ) , and p-toluene-s u l p h o n i c a c i d ( 2 mg) were fused f o r 10 min a t 180°/6 t o r r a f t e r which the temperature was lowered to 160° f o r a f u r t h e r 5 min. A f t e r c o o l i n g the crude product was e x t r a c t e d with b o i l i n g e t h a n o l (4 ml) and the r e s u l t i n g s o l u t i o n was f i l t e r e d and evaporated to y i e l d a brown syrup. Column chromatography of the crude product on s i l i c a g e l (5 g, 1.2 x 10.5 cm), u s i n g 9:1 benzene-ethanol as the developer, y i e l d e d o n l y the s t a r t i n g m a t e r i a l 250 (43 mg, 83%). Attempt 2: Compound 250 (35 mg) and 2 , 6 - d i c h l o r o p u r i n e (23 mg) were fused f o r 20 min at 160°/6 t o r r and f o r an a d d i t i o n a l 10 min at 160°/0.1 t o r r . The r e a c t i o n was worked-up as d e s c r i b e d above to y i e l d the s t a r t i n g m a t e r i a l 250 q u a n t i t a t i v e l y . Attempt 3: Compound 250 (40 mg), 2 , 6 - d i c h l o r o p u r i n e (27 mg) and p - t o l u e n e s u l p h o n i c a c i d (1 mg) were fused as d e s c r i b e d i n Attempt 2 with no a p p r e c i a b l e change i n the r e s u l t s . - 2 3 1 -Attempt 4: Compound 250 (50 mg) and s i l y l a t e d N-benzoyl adenine 114 (67 mg) were fused f o r 10 min a t 160°/13 t o r r and f o r an a d d i t i o n a l 10 min at 160°/6 t o r r . T . l . c . of the crude product obtained a f t e r work-up of the r e a c t i o n showed the presence o f predominantly s t a r t i n g m a t e r i a l with no evidence of any U.V. a c t i v e / c h a r r i n g components. 1,1'-Anhydro-2,3,5,6-tetra-0-acetyl-3-C-(R)-methoxy-c a r b o n y l - 1 ( R ) , 1 ' ( S ) - N - t r i f l u o r o a c e t o e p i m o - g - D - a l l o f u r a n o s e (252) . Compound 250 (52 mg) was d i s s o l v e d i n dichloromethane (6 ml), the s o l u t i o n was c o o l e d to 0° and a slow stream of hydrogen bromide was passed through the s o l u t i o n f o r 2 1/2 h. The s o l v e n t and the r e s i d u a l a c i d i c components were removed 'by repeated c o - d i s t i l l a t i o n w i t h toluene ( 3 x 3 ml) a t <35° and 15 t o r r . To the r e s u l t i n g syrup was added a 6 6 s o l u t i o n of N -benzoyl-N , 9 - b i s ( t r i m e t h y l s i l y l ) a d e n i n e (114, 62 mg) i n dichloromethane (3 ml) and a f t e r removal of the s o l v e n t the r e s u l t i n g syrup was fused f o r 20 min at 160° and 15 t o r r . The r e a c t i o n mixture was c o o l e d t o g i v e a dark g l a s s t o which was added e t h a n o l (10 ml) s a t u r a t e d with sodium b i c a r b o n a t e . The s o l u t i o n was f i l t e r e d and the s o l v e n t evaporated, i n vacuo, t o y i e l d 65 mg of a dark brown amorphous s o l i d . The crude product was chromatographed on a column of s i l i c a g e l (17 g, - 232 -1.5 x 25 cm), u s i n g 6:4 benzene-ethyl a c e t a t e as the dev e l o p e r , t o y i e l d the s t a r t i n g m a t e r i a l 250 (19 mg) and the t i t l e compound 252 (15 mg, 52% based on s t a r t i n g m a t e r i a l consumed). An a n a l y t i c a l sample of compound 252 was obtained by d i s t i l l a t i o n at ^150° and 0.02 t o r r ; [ a ] 2 2 + 117° (c 1.8, ch l o r o f o r m ) ; n.m.r. (CDCl,): 6 5.59 (2d, 1, J 1.5 Hz, H - l ) , 5.39 (2d, 1, H-2), 5.30 (sex, 11 <-1, J . c 6.5 Hz, J c , 3.9 Hz, J c 6.2 Hz, H-5) , 5.01 (d, 4,r> D,ba O, DD 1, H-4), 4.76 (s, 1, H - l ' ) , 4.44 (q, 1, J c c , 12.0 Hz, ba, D U H-6a), 4.01 (q, 1, H-6b), 3.76 (s, 3, C0 2CH 3), 2.10, 2.06, 2.01 and 2.00 (4s, 12, OAc) . An a l . C a l c . f o r C 1 9 H 2 2 N 0 1 2 F 3 : C ' 4 4 - 4 5 ' H ' 4.32; N, 2.73. Found: C, 44.40; H, 4.25; N, 2.79. Mass spectrometry shows (M ++l) peak a t 514.1194. c i 9 H 2 3 N O l 2 F 3 r e q u i r e s 514.1173. (M+-OCH3) 482.0897 r e q u i r e s 482.0910. l-[2' , 3 ' , 5 ' , 6 ' - T e t r a - 0 - a c e t y l - 3 ' - C - ( ( S ) - N - t r i f l u o r o a c e t y l -carbomethoxy(amino)methyl)-B-D-allofuranosyl]thymine (25 7). An a,Bmixture of compound 250 (500 mg) was d i s s o l v e d i n anhydrous dichloromethane (30 ml) and the s o l u t i o n was cool e d to 0°. A slow stream of anhydrous hydrogen bromide was passed through the co o l e d s o l u t i o n f o r 2 h. The s o l u t i o n was then evaporated and any remaining a c e t i c a c i d removed by s u c c e s s i v e a z e o t r o p i n g with toluene (2 x 5 ml) under d i m i n i s h e d p r e s s u r e . The r e s u l t i n g syrup was - 233 -d i s s o l v e d i n anhydrous dichloromethane (2.6 ml) and added to a c o o l e d (-70°) mixture of s i l v e r t r i f l a t e (112 mg, 0. 5 equiv.) i n anhydrous dichloromethane (0.7 ml) and to the r e s u l t i n g mixture was added a s o l u t i o n of b i s ( t r i m e t h y l -s i l y l ) thymine (259 mg, 1.0 equiv.) i n anhydrous d i c h l o r o -methane (1.4 ml). The r e a c t i o n was maintained at -70° f o r 2 h and then allowed to spontaneously a t t a i n room temperature. A f t e r 18 h the s o l u t i o n was cooled to 0° and a s a t u r a t e d s o l u t i o n of sodium b i c a r b o n a t e (2 ml) added with v i g o r o u s s t i r r i n g . The mixture was f i l t e r e d and the p r e c i p i t a t e washed with water (5 ml) and dichloromethane (10 ml). The dichloromethane phase was then separated, d r i e d over sodium sulphate, and evaporated, i n vacuo, to a f f o r d a yellow syrup. Column chromatography of the crude product on s i l i c a g e l (60 g, 2.8 x 26 cm), u s i n g 10:5:1 benzene-ether-ethanol as developer, y i e l d e d the s t a r t i n g m a t e r i a l 250 (267 mg, 54%) and the t i t l e compound (242 mg, 93% based on s t a r t i n g m a t e r i a l consumed) as a g l a s s ; m.p. 91-96°, t e t ] 2 2 + 51.8° (c 1.5, dichloromethane); c d . Ae + 1.08 (X 268, c 0.0005, methanol); n.m.r. max — (CDC1 3) : 6 9.55 (s, 1, NH) , 7.63 (d, 1, J N H / 1 , . 10-0 Hz, NHC0CF 3), 7.24 (s, 1, H-6), 6.05 (d, 1, J±, 2 , 7.2 Hz, H - l ' ) , 5.78 (d, 1, H-2'), 5.61 (d, 1, H - l " ) , 5.42 (sept, 1, J 4 , / 5 . 7.5 Hz, J 5 , ^ 6 , a 2 . 2 Hz, J 5 , f 6 , b 6.0 Hz, H-5•), 4.88 (d, 1, H-4'), 4.60 (q, 1, J 6 - a / 6 . b I 2 - 2 H z / H-6'a), 4.03 (q, 1, H-6'b), 3.84 (s, 3, C 0 2CH 3), 2.10, 2.07, and 2.04 (3s, 12, OAc), 1.93 (s, 3, CH 3). - 234 -A n a l . C a l c . f o r C 2 4 H 2 8 N 3 ° 1 4 F 3 : C ' 4 5 - 0 8 ' " H ' 4 « 4 1 ; N, 6.57. Found: C, 45.77; H, 4.57; N, 6.56. M o l e c u l a r weight by mass spectrometry 639.1530. C 2 4 H 2 8 N 3 ° 1 4 F 3 r e q u i r e s 639.1515. M +-thyminyl 514.1140. c i 9 H 2 3 N O i 2 F 3 r e q u i r e s 514.1173. Attempted Unblocking o f 1 - [ 2 ' , 3 1 , 5 1 , 6 1 - T e t r a - O - a c e t y l - 3 ' - C -((S)-N-trifluoroacetyl-carbomethoxy(amino)methyl)-B-D-a l l o f u r a n osyl] thymine (257). Method A: Attempted D e a c e t y l a t i o n with Sodium Methoxide i n Methanol. To a s o l u t i o n of compound 257 (44 mg, 0.07 mmoles) i n anhydrous methanol (5 ml) was added a methanolic s o l u t i o n of sodium methoxide (0.1 ml, 0.1 e q u i v ) . A f t e r 2 hours a t room temperature, t . l . c . (10:5:1 benzene-ether-ethanol) showed the m a j o r i t y of the s t a r t i n g m a t e r i a l was s t i l l p r e s e n t . A f t e r 24 h, t . l . c . (water s a t u r a t e d n-butanol or ethanol) showed t h a t a l l of the s t a r t i n g m a t e r i a l had been consumed and o n l y b a s e l i n e m a t e r i a l was e v i d e n t . The r e a c t i o n mixture was d e i o n i z e d w i t h IRC-50 (H +) c a t i o n exchange r e s i n and e v a p o r a t i o n of the s o l u t i o n , a f t e r removal of the r e s i n by f i l t r a t i o n , y i e l d e d an amorphous t a n -c o l o u r e d s o l i d (30 mg). Paper chromatography of the pro-duct (^10 mg), u s i n g 10:4:3 e t h y l a c e t a t e - p y r i d i n e - w a t e r as developer, r e v e a l e d the presence of s i x components. The - 235 -components w i t h R f v a l u e s of 0.11, 0.15, 0.34, 0.65, and 0.73 were v i s i b l e under U.V. l i g h t and the R f 0.11, 0.15, 0.34, and 0.81 components r e a c t e d p o s i t i v e l y w i t h n i n h y d r i n . The predominant R^ 0.34 zone was e l u t e d with water (25 ml) and the aqueous s o l u t i o n was f r e e z e - d r i e d to y i e l d an amorphous s o l i d (^ 3 mg). The p.m.r. spectrum of t h i s product (E^O, 270 MHz F.T.) showed i t to be a complex mixture of compounds. S i m i l a r r e a c t i o n s employing 2 or 6 e q u i v a l e n t s of sodium methoxide a f f o r d e d s i m i l a r r e s u l t s . A s i m i l a r r e a c t i o n employing 0.1 e q u i v a l e n t s of sodium methoxide f o r 7 days a f f o r d e d s i m i l a r r e s u l t s . Column chromatography of the crude product (^ 3 0 mg) on a column of Bio-Rex 70 (H +) r e s i n (1 x 11 cm), u s i n g water or 8:2 methanol-water as developer, a f f o r d e d two components which were shown by paper chromatography to be a mixture of the R^ 0.11, 0.15, and 0.34 components and the h i g h e r R^ components. Method B: Attempted Unblocking w i t h Methanolic Ammonia. The n u c l e o s i d e 257 (30 mg) was d i s s o l v e d i n methanol (10 ml) and the s o l u t i o n was n e a r l y s a t u r a t e d a t 0° with ammonia. A f t e r keeping at room temperature f o r 5 days, i t was evaporated to dryness to a f f o r d an amorphous s o l i d . Although the paper chromatogram of the product (10:4:3 e t h y l a c e t a t e - p y r i d i n e - w a t e r ) r e v e a l e d only one component - 236 -(R f 0.5) under U.V. l i g h t , the p.m.r. spectrum r e v e a l e d a mixture of components. No a p p r e c i a b l e change was observed a f t e r prolonged (14 days) ammonolysis. Method C: Attempted Unblocking w i t h Methanolic Hydrogen C h l o r i d e . The n u c l e o s i d e (41 mg) was d i s s o l v e d i n methanol (10 ml) and the s o l u t i o n was n e a r l y s a t u r a t e d a t 0° with hydrogen c h l o r i d e . A f t e r the s o l u t i o n had stood f o r 18 hours a t room temperature, the s o l v e n t was evaporated, i n vacuo, to y i e l d an amber c o l o u r e d g l a s s . The above pro-duct was d i s s o l v e d i n water (1/2 ml) and the s o l u t i o n was t r e a t e d w i t h sodium b i c a r b o n a t e (6 mg) i n water (1 ml). A p o r t i o n of the product was chromatographed on paper (10:4:3 e t h y l a c e t a t e - p y r i d e n e - w a t e r ) and the s i n g l e com-ponent d e t e c t e d under U.V. l i g h t was e l u t e d w i t h water. The p.m.r. spectrum of the product (D 20, 270 MHz F.T.) r e v e a l e d a mixture of p a r t i a l l y a c e t y l a t e d compounds. Method D: Attempted D e a c e t y l a t i o n w i t h T r i e t h y l a m i n e . A s o l u t i o n of 257 (50 mg) i n methanol (5 ml) was t r e a t e d w i t h t r i e t h y l a m i n e (0.5 ml) f o r 24 h a t room temperature. 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