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Glycos-3-Y1 amino acids : synthetic studies of structural analogs of the polyoxin complex Dooley, Kent Cosford 1976

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GLYC0S-3-YL AMINO ACIDS: SYNTHETIC STUDIES OF. STRUCTURAL ANALOGS OF THE POLYOXIN COMPLEX BY KENT COSFORD DOOLEY B.Sc. (Honours), University of British Columbia, 1971 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in the Department of CHEMISTRY We accept this thesis as conforming to the required standard (c) Kent Cosford Dooley, 1976 THE UNIVERSITY OF BRITISH COLUMBIA September, 1976 In presenting this thesis in partia l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library shall make i t 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 B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date November 1, 1976 ABSTRACT The syntheses o f g l y c o s - 3 - y l amino a c i d s , 2-deoxy g l y c o s - 3 - y l amino acids,and deoxy amino ur o n i c a c i d d e r i v a t i v e s (homologous of the sugar moiety of the p o l y o x i n s ) are reported. 5,3-Spiro-pyrrolidone carbohydrate d e r i v a t i v e s , formed by i n t r a m o l e c u l a r c y c l i z a t i o n from doubly branched-chain a, y-diami.no a c i d s , are a l s o d e s c r ibed. The ketose, 1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose (8)'', was condensed w i t h 2-phenyl-5 (4)-oxazolinone (89), i n the presence of lead ( I I ) acetate as c a t a l y s t , to y i e l d (E)-and (Z)-2-phenyl-4- (1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5(4) oxazolone (143) and (144), as a 1:1 m i x t u r e , i n a 75% y i e l d . The s t e r e o -s p e c i f i c s y n t h e s i s o f 143 was achieved by changing the r e a c t i o n s o l v e n t from dimethoxyethane to t e t r a h y d r o f u r a n . Methanolysis of a mixture of 143 and 144 a f f o r d e d a mixture of (E)- and (Z)-methyl-N-benzamido-a-(1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)glycinate (145) and (146) i n a 90% y i e l d . C a t a l y t i c hydrogenation o f the mixture of 145 and 146 a f f o r d e d methyl-D-2 (and L-2) -3-deoxy-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 ) - N - b e n z o y l g l y c i n a t e (147) and (148) which were separated by s i l i c a gel chromatography. Methyl 4,6-0-benzylidene-2-deoxy-q-D-erythro-hexopyranoside-3-ulose (153), was allowed to r e a c t w i t h 2-phenyl-5(4)-oxazolone (89) to y i e l d (E)- and (Z)-2-phenyl~4-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) i n 13% and 27% y i e l d s , r e s p e c t i v e l y . Methanolysis of 154 and 155_ a f f o r d e d ( E l -and (Z)-methyl-N-benzamido-a-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)glycinate (156) and (157) i n q u a n t i t a t i v e y i e l d s . Compound 156 was c a t a l y t i c a l l y " h y d r o g e n a t e d to a f f o r d methyl-D-2-(methyl-4,6-0-cyclohexylmethylidene-2,3-dideoxy - q -g-arabino-hexopyranos-3-yl)-N-cyclohexylcarboxylglycinate (167) i n a 71% y i e l d . S i m i l a r l y , _15_7 afforded methyl-D-2- (methyl-4 ,6-0-cyclohexylmethylidene-2 , 3-dideoxy - q-D-ribo-hexopyranos- 5-yl) -N-c y c l o h e x y l c a r b o x y l g l y c i n a t e (170) i n a 68% y i e l d . 2-Phenyl-5(4)-oxazolone (89) was condensed w i t h 3-0-benzyl-l,2-O-isopropylidene-q-g-xylo-pentodialdo-1,4-furanose (173), prepared by known procedures, to a f f o r d a mixture of (E)- and (Z)-4-(3-0_-benzyl-5-deoxy-1,2-0-isopropylidene - q-D-xylofuranos-S- y l i d e n e ) - 2 - p h e n y l - 5 ( 4 ) -oxazolone (174) and (175) i n a 45% y i e l d . Methanolysis of the mixture o f 174 and 175 afforded (E)- and (Z)-methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene - q-D-xylo-heptofur-5-enuronate (176) and (177) i n an 85% y i e l d . Hydrogenation f o l l o w e d by hydrogenolysis o f the mixture o f 176 and 177 gave a mixture o f methyl-6-N-benzamido-5,6-dideoxy - 1,2-0-isopropy'lidene-a-D-gluco (and g-L-ido) -heptofuranuronat (180) and (181) . T r a n s e s t e r i f i c a t i o n o f 180 and 181 a f f o r d e d the e t h y l e s t e r s which were separated i n t o pure ethyl-6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-a-D-gluco (and g-L-ido)-heptofuranuronate (182) and (183) by f r a c t i o n a l c r y s t a l l i z a t i o n . Compound 183 was h y d r o l y z e d i n hot aqueous e t h a n o l i c barium hydroxide s o l u t i o n to a f f o r d 6-amino-5,6-dideoxy-1,2-0-isopropylidene-g-L-ido-heptofuranuronic a c i d (184) i n a 77% y i e l d . The 1,3-dipolar a d d i t i o n o f diazomethane to (Z)-3-deoxy-1,2:5,6-di-0-isopropylidene-3-C-methoxycarbonylmethylene-a-g-ribo-hexofuranose (13) , prepared from 8_ by a p p l i c a t i o n of the W i t t i g r e a c t i o n , a f f o r d e d a i i i -mixture o f s p i r o - A A - and A^-p y r a z o l i n e s , which were hydrogenated at high pressure w i t h Raney n i c k e l , to a f f o r d spiro-3,4'-S_- (3,3-dideoxy-1,2:5,6-di-O-isopropylidene-q-p-ribo-hexofuranose)-3'-R-amino-2'-p y r r o l i d o n e (192) , spiro-3,4'-S~(3,3-dideoxy-l,2:5,6~di-0-isop ropy lidene-a-D-ribo--hexo furanose)-3'-S_-amino-2'pyrrolidone (195) , s p i r o - 3,4' - R- (3, 3-dideoxy-l ,2 :5 ,6-di-^-isopi'opylidene-a-D-ri_bo_-hexofuranose)-3'-R-amino-2'-pyrrolidone (194) , and spiro-3,4*-R-(3, 3-dideoxy-1,2:5,6-di-0-isopropylidene-a-p-ribo-hexofuranose)- 3'-S-amino-2'-pyrrolidone (195) i n y i e l d s o f 16, 32, 14, and 1 8 % ^ r e s p e c t i v e l y . Compounds 192, 195, 194, and 195 were a c e t y l a t e d to y i e l d the c r y s t a l l i n e d e r i v a t i v e s 196, 197.. 198, and 199, S e l e c t i v e de-0-i sop ropy l . i den at i o n o f compound 196 followed by o x i d a t i v e cleavage w i t h sodium meta-periodate gave an aminal 200 which was a c e t y l a t e d to a f f o r d spiro-3,4' -S_- (3, 3-dideoxy- 1,2-0-isopropylidene-a-D-erythro-pentodia.ldo-1,4-furanose)-3'-R-acetamido-2'-pyrrolidone 3',5 - R-aminal-5,1'-d i a c e t a t e (202). Compound 19 7 w a s ^ s e l e c t i v e l y de-O-isopropylidenated and o x i d i z e d w i t h p e r i o d a t e to y i e l d s p i r o - 3 ,4 '-S-(3,3-dideoxy--1,2-0-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose) -3' -S_- acetamido-2 ' -p y r r o l i d o n e (201) . Complete de-0_-isopropylidenation o f 197 a f f o r d e d s p i r o - 3,4'-S_~ (3,3-dideoxy-g-D-ribo-hexopyranose) -3' -S_-acetamido-2 ' -p y r r o l i d o n e (203) i n a 30% y i e l d . An o p t i c a l l y a c t i v e amino a c i d W i t t i g reagent was prepared as f o l l o w s . L-Cystine d i e t h y l e s t e r d i h y d r o c h l o r i d e (205) was c h l o r i n a t e d to y i e l d 3-chloro-L-alanine e t h y l e s t e r h y d r o c h l o r i d e (206) which was converted to N - a c e t y l - g - c h l o r o - L - a l a n i n e e t h y l e s t e r (207). Compound - i v -207 was reacted with tr:i phenyl nhosplunc and sodium i o d i d e to a f f o r d c t h y l - N - a c e t y l - 3 - (triphenyJphosphoniuiniodo)-a-L-a] an ate (204) i n a 51% y i e l d . TABLE OF CONTENTS OBJECTIVE INTRODUCTION 1. Branched-Chain Sugars 1.1 Synthesis of Branched-Chain Sugars 1.2 Synthesis of Glycosyl Amino Acids 2. The Erlenmeyer Azlactone Synthesis 2.1 The Chemistry of 5(4)-0xazolones 2.2 5(4)-0xazolones in Amino Acid Synthesis 3. Cycloaddition Reactions of Diazoalkanes 3.1 The Diazomethane Cycloaddition in Carbohydrate Synthesis 4. The Wittig Reaction 4.1 The Wittig Reaction in Carbohydrate Chemistry. 4.2 The Phosphonate Modification of the Wittig Reaction RESULTS AND DISCUSSION 1. Glycos-3-yl Amino Acids from the Azlactone Synthesis Structural Analogs of the Sugar Moiety of the Polyoxins 1.1 Synthesis of Derivatives of D-2- and L-2-(3-Deoxy- a-D-allofuranos-3-yl)glycine 1.2 Synthesis of Derivatives of D-2'-[2,3-dideoxy-•arabino-(and ^Ibo)-hexopyranos-3-yl]glycine... 1.3 Synthesis of Derivatives- of 6-Amino-5,6-dideoxy-a-D-gluco(and B-L-ido)heptofuranuronic Acid .... .7 7 - v i -Page 2. Synthesis of Glycos-3-yl Spiro-Pyrrolidones 83 2.1 Diazomethane Addition to Branched-Chain Unsaturated Esters 83 2.2 Synthesis and Chemistry of 3,3'-Spiro-(3-deoxy-q-D-ribo-hexofuranos-3-yl)pyrrolidones 97 3. An Optically Active Amino Acid-Wittig Reagent 105 3.1 Synthesis of Ethyl-N-acetyl-3-(triphenyl-phosphonium iodo)-a-L-alanate 106 3.2 Attempted Wittig Reaction Using Ethyl-N-acetyl-B-(triphenylphosphonium iodo)-q-L-alanate 1 0 8 IV. EXPERIMENTAL 1 1 0 1. General Methods '. 1 1 0 2. Chromatography HO 2.1 Column Chromatography HO 2.2 Thin Layer Chromatography HI 3. Azlactone Condensation: General Considerations ... HI 4. Anhydrous Diazomethane Generation H I 5. Synthesis of: 2-Phenyl-5(4)-oxazolinone (89) HI 1,2 •.5,6-Di-0-isopropylidene-a-D-ribo-hexofuranose-3-ulose (8) . 7 7 1 1 2 (E)- and (Z)-2-Phenyl-4-(1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5(4)-oxazolone (143) an3 1144) 1 1 3 (E)-2-Phenyl-4-(1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5(4)-oxazolone (143)... H4 (E_) -Methyl-N^-benzamido-a- (1,2:5,6-di-0_-isopropylidene-q-D-ribo-hexofuranos-3-ylidene)glycinate (145) 115 - v i i -Page (E)- and (Z)-Methyl-N-benzamido-a-(1,2 :5,6-di-0-isopropylidene-q-g-ribo-hexofuranos-3-ylidene) glycinate (145) and (146) : 1 1 5 Methyl D-2- (3-deoxy-l ,2 :5,6-di-0-isopropylidene-a-D-allofuranos-3-yl)-N-benzoylglycinate (147) 1 1 6 Methyl L-2- (3-deoxy-l,2 :5,6-di-0-isopropylidene-q-D-allofuranos-3-yl)-N-benzoylglycinate (148) 1 1 6 Attempted Base Hydrolysis of the Branched-Chain Amino Acid Sugar N-Benzoates 147 and 148 1 1 8 Attempted Enzyme Hydrolysis of 148 with Hog Kidney Acylase 120 Methyl 4,6-0-benzylidene-2-deoxy-q-D-ribo-hexopyranoside (152) 1 2 1 Methyl 4,6-0-benzylidene-2,3-dideoxy-q-D-erythro-hexopyranos-3-ulose (153) 1 2 1 (E,)- and (Z)-2-Phenyl-4-(methyl-4,6-0-benzylidene-2,3-dideoxy-q-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) 1 2 2 (Z)-Methyl-N-benzamido- -(methyl-4,6-0-benzylidene-2,3-dideoxy-q-p-erythro-hexopyranos-3-ylidene) glycinate (157) 1 2 3 (E)-Methyl-N-benzamido-q-(methyl-4,6-0-benzylidene-2,3-dideoxy-q-D-erythro-hexopyranos-3-ylidene) glycinate (156) 1 2 4 Methyl-D-2-(methyl-4,6-0-benzylidene-2,3-dideoxy-q-D-arabino-hexopyranos-3-yl)-N-benzoylglycinate (166) 7 125 Methyl-D-2-(methyl-4,6-O-cyclohexylmethylidene-2,3-dideoxy-q-D-arabino-hexopyranos-5-yl)-N-cyclohexyl-carboxylglycinate (167) Methyl-D-2- (methyl-4,6-0^-cyclohexylmethylidene-2,3-dideoxy-q-D-ribo-hexopyranos-3-yl)-N-cyclohexyl-carboxylglycinate (170) 7 1 2 6 Attempted Barium Hydroxide Hydrolysis of 167 and 170 127 3-0-Benzyl-l,2-0-isopropylidene-q-D-glucofuranose . (172) 7 7 1 2 7 - v i i i -3-0-Benzyl-1,2-0-isopropylidene-q-D-xylo-pento-dialdo-l,4-furanose (173) 7...-(E)- and (Z)-4-(3-0-benzyl-5-deoxy-l,2-0-isopropyl-idene-q-D-xylofuranose-5-ylidene)-2-phenyl-5(4)-oxazolone (174) and (175) (E)- and (Z)-Methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-1,2-0-isopropyl idene-a - D-jcylo-heptof ur-5-enuronate (176) and (177) 7. Methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-ct-D-gluco- (and 3-L-ido) -heptofur-anuronate (178) and (179) 7 .777 Methyl-6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-a-g-gluco-(and 3-L-ido) heptofuranuronate (180) and (181) 7 Ethyl-6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-q-D-gluco-(and 3-L-ido) heptofuranuronate (182) and (183) 7 6-Amino-5,6-dideoxy-l,2-0-isopropylidene-3-L-ido-heptofuranuronic Acid (184) Attempted Enzyme Hydrolyses of 182 and 183 with Hog Kidney Acylase I (E)- and (Z)-3-Deoxy-3-C_-methoxy-carbonylmethylene-1,2-5,6-di-O-isopropylidene-q-D-ribo-hexofuranose (12) 'and (13) 7 .7777 Treatment of 1_3 with Diazomethane to Afford Spiro-Al-pyrazolines (185 , 186 , 187, and 188) and Spiro-A 2-pyrazolines (189 and 190) Treatment of 12^  with Diazomethane Spiro-3,4'-S-(and R)-(3,3-dideoxy-l,2:5,6-di-0-isopropylidene-q-D-ribo-hexofuranose)-3'-R-(and S)-amino-2'-pyrrolidones (192, 193, 194, and_195) . 7 . . Sprio-3,4 1(3,3-dideoxy-l,2:5,6-di-0-isopropyli-dene-q-D-ribo-hexofuranose)-3'-R-acetamido-21 -pyrrolidone (196) 7 Spiro-3,4'-S-(3,3-dideoxy-l,2:5,6-di-0-isopropyli-dene-q-D-ribo-hexofuranose)-3'-S-acetamido-21 -pyrrolidione (197) 7 - ix -Page Spiro-3,4'-R-(3,3-dideoxy-l,2:5,6-di-0-isopropyli-dene-ot-p-ribo-hexofuranose) -3' -R-ace tImido-2 1 -pyrrolidone (198) 7 140 Spiro-3,4'-R-(3,3-dideoxy-l,2:5,6-di-0-isopropyli-dene-a-p-ribo-hexofuranose)-3'-S-acetamido-21 -pyrrolidone (199) 141 Spiro-3,4' -S- (3,3-dideoxy-l,2-0_-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose)-3'-R-acetamido-2' -pyrrolidone-3*,5-R-aminal-5,1'-diacetate (202) 1 4 1 Spiro-3,4' -S_- (3,3-dideoxy-l,2-0-isopropylidene-a- n-erythro-pentodialdo-1,4-furanose)-3' -S-acetamido-2' -pyrrolidone (201) 1 4 2 Spiro-3,4 1 -S_- (3,3-dideoxy-B-D-ribo-hexopyranose)-3' -S-acetamido-2'-pyrrolidone (203) 1 4 3 (E) -Methyl-4,6-0_-benzylidene-3-£- (carbomethoxy-methylene)-2,3-dideoxy-a-p-erythro-hexopyranoside (163) 7 1 4 3 Attempted Reaction of 163 with Diazomethane 144 B-Chloro-L-alanine Ethyl Ester Hydrochloride (206). 144 Acetyl-B-chloro-L-alanine Ethyl Ester (207) 145 Ethyl-N-acetyl-6-(triphenylphosphoniumiodo)-a-L-alanate (204) 145 Attempted Reaction of 207 with Triphenylphosphine.. 146 Attempted Reaction of 207 with Sodium Iodide 146 Attempted Wittig Reaction with 204 and Benzaldehyde 147 BIBLIOGRAPHY 148 - X -LIST OF FIGURES Figure Page I Branched-Chain Glycos-3-yl Amino Acids 16 II PMR Spectrum of Compounds 145 and 146 51 III Synthetic Scheme for Compounds 147 and 148 55 IV PMR Spectrum of Compound 156 64 V PMR Spectrum of Compound 157 65 VI PMR Spectrum of Compound 167 68 VII PMR Spectrum of Compound 170 72 VIII PMR Spectrum of Compounds 176 and 177_ 77 IX PMR Spectrum of Compound 196 94 X PMR Spectrum of Compound 197 95 XI PMR Spectrum of Compound 198 96 XII PMR Spectrum of Compound 199 97 XIII Alternative Stereochemistry of the Diazomethane Addition 98 XIV PMR Spectrum of Compound 201_ 102 XV PMR Spectrum of Compound 202^  103 - xi -ACKNOWLEDGEMENTS The stimulating direction of Dr. A. Rosenthal and the cheerful support of my colleagues are gratefully acknowledged. The author thanks Dr. L. Weiler for reading the thesis and making helpful comments. - 1 -I. OBJECTIVE Considerable interest in the synthesis of peptidyl nucleosides has resulted from the discovery of the antibiotics puromycin, gougerotin, and blast i c i d i n S. The commercial importance of the polyoxin complex of peptidyl nucleosides as potent inhibitors of rice blast has also prompted synthetic endeavor in the area of amino acid derivatives of carbohydrates and nucleosides. The common features of these compounds are a nucleoside skeleton and a peptide moiety linked, through an amide nitrogen, to the a carbohydrate portion of the nucleoside. The sugar moiety of blast i c i d i n S possesses, as well, a B-amino acid functionality as part of i t s structure/ Similarly the polyoxin sugar moiety has an a-amino acid incorporated in i t s structure. The objective of this thesis was to develop an efficient route for the general synthesis of amino acid derivatives of carbohydrates, linked through a non-hydrolysable carbon-carbon bond. The Erlenmeyer azlactone synthesis with carbohydrate ketoses and aldoses was of interest as a potential route to deoxy branched-chain and extended-chain glycosyl amino acids. A more direct route to branched-chain amino acid sugars, using an optically active amino acid Wittig reagent, was also investigated. - 2 -In the second part of this thesis the synthesis of branched-chain a,Y-diamino acids was undertaken. A method using branched-chain unsaturated sugars as synthetic precursors was chosen since these compounds are readily available using the Wittig reaction. A brief review of the chemistry of branched-chain sugars, oxazolones and 1,3-dipolar additions w i l l be presented in order to provide a background to subsequent discussions. - 3 -II. INTRODUCTION The interest in branched-chain carbohydrates has been stimulated by numerous reports of their presence in nature. At the turn of the century the f i r s t reported branched-chain sugar, Apiose (1 ) , was isolated from parsley.* The structure of Apiose was not elaborated 2 until almost thirty years later. In 1919 hamamelose (2) was isolated from a tannin in Hamamelis virginiana by E. Fischer and C. Freudenberg. In the last two decades several branched-chain sugars have been 4-7 9 isolated from microorganisms and higher plants, from c e l l wall 8—11 12 polysaccharides and as glycosides. The discovery of branched-chain glycosides in antibiotics has increased interest in the synthesis of these compounds. A prominent example is streptose (3J in the structure of the antibiotic streptomycin (4) . HOCH HO OH HO OH - 4 -1. Branched-Chain Sugars 1.1 Synthesis of Branched-Chain Sugars Over the last decade a great many synthetic routes to branched-chain sugars have evolved. These compounds can be divided into two 13 classes. Those which have a group -OR attached to the branching point carbon are designated Type A, while those without this functionality are of Type B. These la t ter , "deoxy", branched-chain sugars shal l be dealt with more completely in the ensuing discussion. The branched-chain sugars with a hydroxyl group at the branch-point have been synthesized by a variety of methods, mainly involving condensation reactions with keto sugars. For example organolithium and organomagnesium compounds react with the ketose (5) to give the 13-15 branched-chain sugars (6) or (7). An interesting stereochemical preference is shown since the former reaction affords the L-ribo 14 sugar (7) whereas L-arabino derivatives (6) are predominantly formed 13 15 with the la t ter reagents. ' -5 -The base catalyzed condensation of nitromethane with keto 16—18 sugars (eg.8) is of particular u t i l i t y since i t can lead to sugars of type A (9) which may be reduced to amines, or dehydrated to c t-nitro olefins (10). The latter compounds can be catalytically reduced to sugars of type B (11). The application of the Wittig reaction to keto sugars, developed 19 by Rosenthal and Nguyen, gave rise to numerous "deoxy" branched-chain sugars which were previously unavailable. There followed considerable activity in the synthesis of branched-chain deoxy sugars by hydrogenation 20-25 of Wittig reaction products (eg. 12-15). H y d r a t i o n o f t h e u n s a t u r a t e d p r o d u c t s o f t h e W i t t i g r e a c t i o n 2 6 a f f o r d s t y p e A s u g a r s . F o r e x a m p l e , W i l l i a m s e t a l . u s e d t h e 27 o x y m e r c u r a t i o n - d e m e r c u r a t i o n p r o c e d u r e o f B r o w n a n d G e o g h e g a n o n a n u n s a t u r a t e d s u g a r t o y i e l d t h e 3 -C m e t h y l s u g a r ( 1 4 ) . 14 - 7 -The olefin Wittig product (15) has been converted into 1_6 by 28 29 Tronchet and coworkers. Recently Brimacombe obtained the azido branched-chain sugar 1_7 by addition of azide, with mercuric acetate as catalyst, to the same Wittig reaction product 15. Anydro sugars are an abundant source of "deoxy" branched compounds, 30a since they can undergo nucleophilic addition. The reaction of 1_8 with alkyllithium reagents proceeded as expected with trans-diaxial * +U 'A  3 0 b opening of the epoxide. - 8 -When 2,3-anhydro sugars were reacted with cyanide,''"1'""' the necessarily strong basic conditions resulted in isomerization of the tertiary carbon. This was overcome by using triethylaluminum-HCN 33 complex in ether. The carbanion of diethyl malonate also cleaved a 34 35 2,3-anhydro sugar ' giving rise, after reduction, to a hydroxyethyl branched-chain sugar. The application of addition reactions to unsaturated sugars also can give "deoxy" branched-chain carbohydrates. Thus hydroformylation of the unsaturated species (20) using high pressures of carbon monoxide and hydrogen with an appropriate catalyst, gave the branched-chain 36 carbohydrate (21) . The oxo reaction with carbohydrate derivatives 37 has been reviewed by Rosenthal. 20 OAc 21 - 9 -Unsaturated sugars may undergo photoaddition reactions. Photoamidation of the unsaturated sugar (22) occurred at both ends of the double bond giving the carbamoyl sugars (25), (24), and (25). These can be converted into carbohydrates with a variety of functionalized branched chains. Branching of sugars may also be 39 reactions, nitroethane addition to 41 . 42 reactions, ring contraction, and achieved using deamination 40 dialdehydes, dimerization reduction of saccharinic acids. 1.2 Synthesis of Glycosyl* Amino Acids Nucleosides have been the subject of intense interest, partly 44 because of their presence in many antibiotics. Recently much attention has focussed upon nucleoside antibiotics which contain peptide linkages and amino acid functionalities attached to the carbohydrate moiety. The great variation in structures among these * Used in the extended sense. - 10 -latter, naturally occurring, compounds is evidenced by the antibiotics bla s t i c i d i n S (26), gougerotin (27), puromycin (28) and the polyoxins (29). Blasticidin S (26) Gougerotin (27) Puromycin (28) 0 - 11 -B l a s t i c i d i n S, gougerotin and puromycin were found to i n h i b i t 45 p r o t e i n synthesis. The former two nucleosides also i n h i b i t e d the rea c t i o n of puromycin with peptidyl-tRNA. These compounds are valuable t o o l s f o r i n v e s t i g a t i n g the mechanisms of pro t e i n synthesis. 46 The polyoxin complex (29), which numbers over twelve v a r i a t i o n s on the poss i b l e combinations presented above, represents a new group of pep t i d y l nucleoside a n t i b i o t i c s that are antifungal i n t h e i r action. The polyoxins are used commercially to cont r o l sheath b l i g h t i n r i c e p l ants. They have been shown to be extremely t o x i c towards phyto-pathogenic fungi, but have no a c t i v i t y towards animals, f i s h , plants 47-49 or b a c t e r i a . Their mode of action involves i n h i b i t i o n of c h i t i n b iosynthesis, presumably by mimicking UDP-N-acetylglucosamine. Almost a l l of the carbohydrate d e r i v a t i v e s of amino acids which have been synthesized are lin k e d through the nitrogen of the amino ac i d moiety. Some of the methods f o r making these N-linked compounds include: (1) condensation of a protected amino a c i d with a halo sugar, (2) d i r e c t condensation of the free sugar aldehyde with the amino 51 52 group of the amino ac i d , ' and (3) a c y l a t i o n of the amino function-53 54 a l i t y of amino sugars with amino a c i d d e r i v a t i v e s . ' A novel approach makes use of the a c t i v a t i n g e f f e c t of the n i t r o group i n an a - n i t r o o l e f i n i c sugar (30). This f a c i l i t a t e s amine addi t i o n of amino 55 a c i d esters. - 12 -The majority of glycosyl amino acids reported to date have been synthesized using displacement of either a methanesulfonyloxy*^ or a toluenesulfonyloxy^ ^ group with azide, followed by reduction to 57 afford the amino functionality. Thus, Moffatt and coworkers reacted the 5 ' aldehydo nucleoside (32) with sodium cyanide in aqueous methanolic potassium carbonate and hydrogen peroxide to form the epimeric hydroxy amides (33) and (34). Reaction of (33) with methane-sulfonyl chloride in pyridine followed by azide displacement of the sulfonate, hydrolysis and reduction afforded the basic polyoxin skeleton (35). Similarly (34) afforded (36). Compounds 34 and 36 are the - 1 3 -corresponding derivatives which are inverted in configuration at C-5'. 62 The Bucherer hydantoin synthesis was used by Umezawa and coworkers^ 3'^ to prepare the novel 3-amino-3-(>carboxy-3-deoxy derivatives (39) and (40). The hydantoin (37) was hydrolyzed to the partially blocked glycosyl amino acid (38) followed by conversion to the nucleosides BzOCH HOCH 38 HOCH NH„ OH 39 NH2 OH 4 Q Recently a one step, highly stereoselective method of synthesizing straight-chain, glycosyl amino acids using copper complexes has been elaborated.^ ^ Thus, N-pyruvilidene-glycinatoaquocopper (II) (41) was reacted with the aldehydes (42) to give adducts (43) which were hydrolyzed to the glycosyl amino acids'(4_4). The threo relation was 66 observed between C-2 and C-3 and between C-3 and C-4. - 14 -T h e B - h y d r o x y - y - a r a i n o a c i d s u g a r ( 4 5 ) , w h i c h w a s v e r y r e c e n t l y s y n t h e s i z e d u s i n g a z i d e d i s p l a c e m e n t o f a s u l p h o n a t e e s t e r , h a s b e e n 69 f o u n d t o i n h i b i t p r o t e a s e s a n d p e p s i n . COOH I C H 2 RNH — OH C H 0 CHMe, 45 - 15 -Rosenthal and Shudo"'"' have stereospecifically synthesized the branched-chain amino acid sugar (49) from the a-hydroxy ester (47). The overall reaction sequence may be seen in Figure I. Rosenthal, Shudo 70 and Richards then applied a similar strategy to synthesize the analogs (48) and (50) (Figure I) with a hydroxy group at C-3 in the gluco configuration. The galacto isomers (53) and (54) (Figure I) were subsequently prepared.^ The amino acid sugar derivatives in Figure I represent a series of structural analogs of the sugar moiety of the polyoxins (55). 71 Jordaan and Brink synthesized the blocked D-amino acid sugar 72 (56), based on the procedure of Schollkopf, by the reaction of the keto-sugar (8) with ethyl isocyanate in the presence of sodium hydride. The unsaturated branched chain product was subsequently reduced to afford compound 56. - 1 6 -) £ 3 H~OH : 0 2 M e 4 6 ' -H,OH (sugar moiety of the polyoxins) H 2 N " H CC^Me 48 ) € 3 H 2 N - H C 0 2 M e 49 M e 0 2 C - ^ 7 6-f-12 52 H - - N 2 C 0 2 M e 50 .0 H N H 2 C 0 2 M e 53 H . - + N H 2 C O o M e L A 54 Figure I. Branched-Chain Glycos-3-yl Amino Acids. - 17 -Very recently, Rosenthal and C l i f f obtained the a-nitroester branched-chain sugar (57) by condensation of methyl nitroacetate with the ketose (8). Acetylation of the product followed by reduction of the nitro group afforded the amino acid branched-chain sugars (58) and (59). 2. The Erlenmeyer Azlactone Synthesis 7. In an attempt to extend Perkin's cinnamic acid synthesis,Plochl heated a mixture of benzaldehyde, hippuric acid and acetic anhydride. He obtained a crystalline compound which was erroneously analyzed. 75 Erlenmeyer developed the reaction with other aromatic aldehydes and 76 some years later proposed the structure (60) which he called the azlactone of a-benzamidocinnamic acid. The names 5(4)-oxazolones are now generally applied to these types of structures. - 18 -PhCJIO + C1L-CO H I 1 NHBz PhCH 0 The reactions are of value.because a carbon-carbon linkage has formed under mild conditions. The products yield a-acylamino acids upon hydrolysis and reduction or a-keto acids under vigorous hydrolysis. The chemistry of oxazolones has grown into a vast area of research 77 78 which has been reviewed extensively by Cornforth and Steglich. Material relevant to this work w i l l be discussed in the following section. 2.1 The Chemistry of 5(4)-Oxazolones The most common method of oxazolone synthesis involves cyclization of a-acylamino acids with acetic anhydride, at times aided with a weak base. Those azlactones with aryl or alkylidene groups at position 2 or 4 are readily obtained due to stabilization of the oxazolone by conjugation. In fact 2-phenyl-4-benzylidene-oxazolone (60) can be formed simply by heating a-benzamidocinnamic acid. - 19 -0 Ph // \ ' \ r . CH \ 2 y . \ PhCH C C0oH » I NHCOPh A H' ' 0 6 0 Ph Acid halides of a-acylamino acids react with diazomethane or mild 77 base to give 5(4)-oxazolones. This anomalous behaviour supports the 78 hypothesis that these acid halides are actually oxazolone hydrohalides. As previously mentioned, 4-alkylidene oxazolones were f i r s t 75 prepared by the Erlenmeyer synthesis. They can also be prepared by the action of acetic anhydride or benzoyl chloride in pyridine upon 79 3-hydroxy-a-acylamino acids. - 20 -C,HcCHOHCHCOOH 6 5 I NHL (CH3CO)20 NH-CHCHOHC,Hc I I 6 5 -> CH3CO COOH CH N C CHC H The action of acetic anhydride on N-chloroacetylphenylalanine 80 (62) was shown by Bergmann and Stern to give the 4-alkylidene oxazolone (64) through a 5(2)-oxazolone (63). This resembles the dehydration of 8-hydroxyl-a-acylamino acids because the anion i s eliminated from the a position of a substituent on C-2 instead of C-4. Bergmann's method is particularly useful for preparing unsaturated oxazolones of low molecular weight since the product can be isolated by d i s t i l l a t i o n at low pressure. - 21 -In their reactions with nucleophiles, the 5(4)-oxazolones behave essentially like acid anhydrides. Water, acids, alcohols, thiols, and amines usually attack the electrophilic center at C-5 giving ring opening and irreversible formation of a stable amide group (66). Although some a-amino acids form 2-methyloxazolones on treatment with excess acetic anhydride, others give polymers arising from reaction of the a-amino acid with the oxazolone. Only a 20% yield of - 22 -oxazolone (68) is obtained from a-aminoisobutyric acid (67) due to 81 polymerization. ( C H 3 ) 2 CjCOOH NH 67 Ac 20 ^ 0 3' 27—t + 67 CH, 68 (CH 3) 2 — C (CH 3) 2 t = 0 NHCOCH, Ac 0 < ( C H 3 ) ^ ~ c c o N H C v C H 3 ) 2 C 0 0 H 69 NHCOCH, When C-5 of an oxazolone is st e r i c a l l y hindered, nucleophilic g attack can occur at the imino ether carbon, C-2. Bergmann and Grafe postulated that the ortho ester anhydride (71) is the i n i t i a l product of the action of alcohols on the substituted oxazolone (70). - 23 The oxazolone (72) undergoes alkyl cleavage when reacted with 83 hydrazoic acid. The tetrazolecarboxylic acid (73) is formed by cyclization of the unstable imidazide. Generally, oxazolones behave as cyclic O-acylimino ethers to azidolysis. 73 CH Saturated azlactones undergo Friedel-Crafts acylation giving acylamino 84 ketones in high yields. Thus, compound (74) reacts with aluminum chloride and aromatic hydrocarbons to give the ketone (75). H R1 74 A1C1. RCH -C I R' O-AICI. ArH 0 II CHC _ A r NHCOR' 75 - 24 -Grignard reagents give a variety of products when reacted with azlactones. Phenylmagnesium bromide attacks C-5 of the saturated 85 oxazolone (76) as expected. However, ethylmagnesium bromide induces 8 6 formation of a dimer which cyclizes to the diketopiperazine (77). , 6 5 C H 3 ^ \ C 2 H 5 M ^ ^ CH3CH<"N I c—0 0 C6 H5 COC,H 76 77 0 — ^ C H C H , I nn w 6 5 The diketopiperazine (77) can also be produced from compound (76) by reaction with hydrogen cyanide in triethylamine or with 87 potassium and methyl iodide. Some unusual reactions of 5(4)-oxazolone anions have been 88 89 pursued over the last decade by W. Steglich. ' With triethylamine as a base, Steglich observed that oxazolones, substituted at C-2 and C-4 with phenyl or fluoro groups (78), would undergo Michael type additions at C-2. - 25 -2.2 5(4)-Oxazolones in Amino Acid Synthesis As described in the previous sections, 5(4)-oxazolones can be elaborated in many ways. Once elaborated they may be readily hydrolyzed to a variety of amino acids. The Erlenmeyer azlactone synthesis can be applied to aromatic aldehydes. For example, phenylalanine (81) can be produced from glycine 90 and benzaldehyde as shown below. 0 0 II PhC-Cl OH Ph PhCHO (Ac20-OAc) 81 CHCO H I NH2 Ph The 4-arylidene oxazolone (57) can also undergo Friedel-Crafts 91 alkylation to afford an arylated oxazolone (82). The product can then be transformed into the B,3-diaryl-a-amino acid.(85). - 26 -It was observed by Homer that 4-alkylidene or 4-arylidene oxazolones (84) reacted with Grignard reagents by conjugate addition to give the precursors to g-disubstituted amino acids (85). - 27 -When applied to aliphatic carbonyl compounds the Erlenmeyer azlactone condensation yielded only their respective enol-acetates due to the reaction of acetic anhydride and a base. Baltazzi and 93 Robinson overcame this problem by using tetrahydrofuran as the solvent and lead II acetate as the catalyst. The synthesis of Y»Y , _ dihydroxyvaline (88) from diacetoxyacetone (86) was accomplished using Robinson's conditions, followed by hydrogenation and hydrolysis of the 94 azlactone (87). CH.OAc i i + fj — 0 ^ CH2OAc CHoC0_H I 2 2 NHCO I Ph Ph((OAc)2 + A c2° "THF * 86 CHo0Ac Ac0H2C / 87 \ Ph 1 ) H2/Pd Dioxane 2) HCI 3) NH OH (CH20II)2CHCHC02H NH 88 A Crawford and L i t t l e were able to increase the yield of the azlactone condensation by using 2-phenyl-5-(4)-oxazolinone (89). This was prepared from the reaction of hippuric acid and acetic 96 anhydride. The reaction was performed in a neat solution of ketone or aldehyde. The yield of 4-alkylidene-5(4)-oxazolones (90) - 28 -was optimum with lead acetate as catalyst. Sodium acetate caused polymerization of the oxazolone reactant whereas catalytic amounts of acetic acid gave incomplete reaction. 89 Ph 90 Ph The access to unsaturated oxazolones by the azlactone condensation is limited when the appropriate carbonyl component is not available. A useful synthesis of unsaturated oxazolones uses the chlorovinyl 97 derivative (91) which undergoes addition elimination reactions with 93 - 29 -carbanions. Thus 5-methoxytryptophan (93) was synthesized from the Grignard adduct of 5-methoxy-indole (92). Another example of the u t i l i t y of oxazolones in the synthesis of elaborated amino acids involves the synthesis of the serine derivative (96) from an oxazolone (94) derived from alanine. The key 98 step in this sequence is the base catalyzed 0- to C-migration of the enol acetate (95). A variation on this synthesis of elaborated serines involves introduction of the aryl group to oxazolone (94) with 99 an organometallic reagent. - 30 -Oxazolones may be used in peptide synthesis since they can be ring-opened at C-5 by the free amine of an amino acid or a peptide. The unsaturated 5(4)-oxazolones may be used in an analogous way, as in the synthesis of the tripeptide (98) from the oxazolone (97).*^ C,HC H O D / \ H2NCH2C02H N \ 0 7 ZZ NOCH. I C 6 H 5 97 C,HcCH=CC0NHCH„C0oH 6 o | 1 1 NHCOCCH„C,Hc , , 2 6 5 NOCH H, Pd/C C,HPCH_CHC0NHCHoC0oH D b Z | Z Z NHC0CH(NH )CH2C H5 98 There are many more examples of the use of oxazolones in synthesis, 102 103 a number of which are found in reviews by Cornforth, F i l l e r , and Meyers. 3. Cycloaddition Reactions of Diazoalkanes The cycloaddition of diazoalkanes with unsaturated compounds has been known for many years. It was not until the recent outstanding r. „ • - - . . 106,107 ^ series of studies by Huisgen and his coworkers that these reactions were represented as 1,3-dipolar cycloadditions. Other molecules which undergo 1,3-dipolar cycloadditions include n i t r i l e oxides, azides, nitrones and n i t r i l e amines. - 31 -NHN+-C"R2 4 > N =N+=CR2 N+=N-C R2 < => N~=N-C+R2 99 The resonance hybrid of diazoalkanes is described by the various 108 canonical structures (99) . There are two schools of thought 109 regarding the mechanism of these particular cycloadditions. Huisgen proposed that the vast majority of these reactions proceed through a concerted mechanism. Firestone'^^ argues that the mechanism involves a diradical species. Evidence exists for a two-step mechanism, of ionic character, with some resonance-stabilized r e a c t a n t s . F o r the reaction between diazomethane (101) and methyl methacrylate (100) three reaction paths can be envisaged as follows: - 32 -The independence of reaction rate relative to solvent polarity, and the large negative entropy of reaction favour the concerted mechanism. However, certain aspects of regioselectivity and stereo-chemistry presented by Firestone"*""^ are best explained by the diradical 112 mechanism. Recently, perturbation theory has been used to reconcile these regioselectivity aspects to the concerted mechanism. - 33 -The cycloaddition of diazoalkanes with double bonds, which are activated by electron withdrawing substituents or ring strain, can yield a variety of interesting heterocyclic compounds. Thus, reaction of diazomethane with the perfluorinated b i c y c l i c compound (102) gives 113 the tetracyclic compound (103). F 103 The dipolarophile (104) when reacted with diphenyl diazomethane 114 gives the trimethylsilyl pyrazoline (105). A weak dipolarophile such as ethylene (107) w i l l undergo cycloaddition with the highly 115 activated trifluoromethyl diazoalkane (106). Diazoalkanes w i l l also cycloadd to alkynes such as the reaction of the unsaturated diazo compound (108) with 109 to give the heterocyclic compound (110). - 34 -Me3Si-CH=CH2 + Ph 2CN 2 104 CF3CHN2 + C H 2 = C H 2 106 107 CH2=CH-CHN2 + R1C~CR2 108 109 Me 3Si -CH ~ / CH \ CPh. N 105 CH. CH i f CF, CH N 3 \ N \ / c C -CH \ CH, :CH-C CH 'N 110 R The 1-pyrazolines resulting from cycloadditions are of great synthetic value. In most cases 1-pyrazolines (111) are thermally and 117 118 photochemically unstable, ' permitting convenient generation of cyclopropanes (112) or alkylated olefins (115). - 35 -Pyrazolines may be converted to pyrazolidines by catalytic 119 reduction at low pressure. High pressure hydrogenation of the carbomethoxy pyrazolines (114), with Raney nickel as the catalyst, was found to yield the esters of a,y-diamino acids (115) . 3.1 The Diazomethane Cycloaddition in Carbohydrate Synthesis The addition of diazomethane to the activated double bonds of carbohydrates has only recently been reported. The scarcity of suitably activated double bonds has undoubtedly delayed research 121 in this area. Tronchet obtained the spiro-pyrazoline (117) by addition of diazomethane to the Wittig reaction product (116). The double bond, in this case, is more reactive than a normal olefin. - 36 -The unsaturated keto-sugar (118) was reacted with diazomethane 122 to give the "pyrano-pyrazoline" (119) which was hydrogenated to the mixture of diamino sugars (120). - 37 -The pyrazoline structure offers interesting p o s s i b i l i t i e s for homologation of nucleoside bases. Thus, the C-nucleoside (122) was 123 synthesized by diazomethane addition to the unsaturated ester (121). C0oCoH_ HN N AcO-l 0 ^ A c 0 _ , . O ^ N ^ ^ C 02 C2 H5 121 4. The Wittig Reaction A portion of this thesis involves the use of amino acid Wittig reagents for the direct synthesis of amino acid branched-chain sugars. It would be appropriate, therefore, to discuss the Wittig reaction with more detail than previously given in Section 1.1. Since this reaction 124 125 has been reviewed several times ' the discussion w i l l be kept brief. 126 In 1949 Wittig and Rieber synthesized the methylenetrimethyl-phosphorane (124) by the action of methyllithium on tetramethyl-phosphonium iodide (123). Upon reaction of the y l i d with benzophenone, compound (125) was formed. - 38 -(CH 3) 3P +-CH 3r + CH 3Li > (CH3)3P=CH2 + Li I + CH^  123 124 HI (CH3)3P=CH2 + (C 6H 5) 2C=0 -^-> [(CH 3) 3PCH 2CCC 6H 5) 2] . 1 124 125 In 1953, Wittig and Geissler" 1^' reported the formation of a high yield of diphenylmethane from the above reaction. Thereafter, the 128 133 applications of the Wittig reaction increased rapidly ' and the mechanism and stereochemistry of this important reaction were • V • . • 4 j 124,134-137 T extensively investigated. In brief, the Wittig reaction involves the combination of an y l i d (126) (generated by reaction of triphenylphosphine with an alkyl halide followed by dehydrohalogenation using a base) with a carbonyl compound to afford a betaine intermediate (127). Subsequent decomposition of the betaine intermediate to produce an olefin (128) and a tertiary phosphine oxide is thought to occur by the indicated cyclic four-center transition state. - n-C H Li (C 6H 5) 3P • CH3Br (C6H5).3P -CI^Br « > 6 6 (C 6H 5) 3P +-CH 2- > ( W 3 P = C H 2 > 126 - 39 -4.1 The Wittig Reaction in Carbohydrate Chemistry Though the Wittig reaction was used immediately in other f ie lds , i t was not unt i l the last decade that i t was employed extensively in carbohydrate synthesis. In 1963, Kochetkov and coworkers*"^ reported the reaction of carboxymethylene triphenylphosphorane with 138 139 aldoses (129) having either free or blocked hydroxyl groups. The chain-extended aldose (130) was obtained by dihydroxylation of 140 the double bond and reduction of the ester. C0 oEt CHO CHO CH CHOH | II I (CHOAc) 3 + .(.C6H5)3PCHC02Et • CH —> > CHOH CH„0Ac (CHOAc) • (CHOAc)3 *2V CH20Ac CH2OAc 129 - 130 - 40 -The Wittig reaction has also been used to prepare unsaturated u - V. U * 1 4 1 ^A • - A 1 4 2 A 1 4 3 A r higher ketones, aldonic acids, deoxy sugars, and C-144 glycosides. Chain extension has also been obtained at the C-5 terminus of carbohydrates and nucleosides. The synthesis of branched-chain sugars has benefitted particularly from the Wittig reaction. Rosenthal and coworkers have synthesized many branched-chain sugars as mentioned in section 1.1. These are 20 primarily 3-deoxy compounds containing either exocyclic methylenic, 19 23 145 21 22 methoxycarbonylmethylenic, ' ' or cyanomethylenic ' function-a l i t i e s attached to hexoses and pentoses. Recently, Tronchet and coworkers have also synthesized numerous 24 25 28 exocyclic-unsaturated sugars ' ' and investigated their nmr spectral properties (H* and C*^) in relation to conformational analysis. 147 Instead of using a carbonyl-containing sugar, Zhdanov and Polenov converted the carbohydrate moiety into a Wittig reagent (131) and then condensed this reagent with p-nitrobenzaldehyde to afford the a,8-unsaturated ketone (132). 0 C C 6 H 5 ) 3 P ~ C H " ~ C i / ' n p-N0 2(C 6H 5)CH = C H _ C ^ " ' p_-N02(C6H )CH0 \ ^ / ° 132 - 41 4.2 The Phosphonate Modification of the Wittig Reaction 148 Horner and coworkers were the f i r s t to use phosphonates in the so-called modified Wittig reaction, when they condensed diethyl-benzylphosphonate with benzaldehyde using sodium amide to remove the 149 activated benzyl-proton. Later Wadsworth and Emmons showed that stabilized phosphonate carbanions were more reactive then t r i a r y l -phosphoranes towards some aldehydes and ketones, required milder reaction conditions, were less expensive and afforded simpler product isolation. 19 With these facts in mind Rosenthal and Nguyen reacted the blocked ketose (.8) with carbomethoxymethyldimethylphosphonate in the presence of potassium t-butoxide to yield, upon hydrogenation, the branched-chain carbomethoxymethylene sugar (126). Similarly, reaction of the carbanion of diethylcyanomethylphosphonate with (9) afforded the 21 22 cyanomethyl sugar (134) after hydrogenation. ' - 42 -Jones and Moffatt condensed the phosphonate Wittig reagent (135) with adenine and uracil nucleosides (136) and (137) respectively, to afford the 5'-deoxy-51 -(dihydroxyphosphinylmethyl) nucleosides (138) and 139). Ph3P=CHOP(OPh)2 135 136 B 137 B 138 B adenine, R = 0 uracil, R = 0 adenine, R = CHPO(OPh), 139 B = uracil, R = CHPO(OPh). The mechanism of the modified Wittig reaction remains, to some extent, the subject of some speculation. It has been suggested by 124 149 many authors ' that the mechanism is analogous to that of the Wittig reaction, involving the formation of a betaine intermediate. This would undergo cis-elimination to give the olefin product. Recently two groups have investigated the stereochemical course of the reaction with various aldehydes and ketones. ' ^ 2 It was shown"^^ that the ratio of stereoisomers was dependent on steric factors of the ketones (140). Thus, when R2 was increased in size the cis/trans ratio increased as expected for cis-elimination of a betaine intermediate (141). - 43 -Lefebvre and Seyden-Penne''""' have evidence that the erythro and three- betaine intermediates are interconvertible. They suggest that the cis/trans ratio is largely dependent on the relative rates of phosphate elimination of the two possible betaines. - 44 -III. RESULTS AND DISCUSSION The work to be described herein is comprised of three separate synthetic approaches to branched-chain glycosyl amino acids: (1) chemical investigations of the azlactone condensation with suitably blocked ketoses and aldoses; (2) synthesis of glycos-3-yl spiro-pyrrolidones; (3) amino acid Wittig reagent synthesis and reactions. This work w i l l be discussed in the order presented under the following headings: 1. Glycos-3-yl Amino Acids from the Azlactone Synthesis: Structural  Analogues of the Sugar Moiety of the Polyoxins 1.1 Synthesis of Derivatives of Q-2- and L-2-(3-Deoxy-q-g-allo-furanos-3-yl) glycine. 1.2 Synthesis of Derivatives of D-2'-[2,3-dideoxy-arabino-(and ribo)-hexopyranos-3-yl] glycine. 1.3 Synthesis of Derivatives of 6-Amino-5,6-dideoxy-q-D-gluco (and g-L-ido) heptofuranuronic Acid. 2. Synthesis of Glycos-3-yl Spiro-Pyrrolidones 2.1 Diazomethane Addition to Branched-Chain Unsaturated Esters. 2.2 Synthesis and Chemistry of 3,3'-Spiro-(3-deoxy-a-D-ribo-hexofuranos-3-yl) pyrrolidones. - 45 -3. An Optically Active Amino Acid-Wittig Reagent 3.1 Synthesis of Ethyl-N-Acetyl-g-(triphenylphosphonium-iodo)-a-L-alanate. 3.2 Attempted Wittig Reaction Using Ethyl-N-acetyl-g-(triphenyl-phosphonium-iodo)-a-L-alanate. 1. Glycos-3-yl Amino Acids Using the Azlactone Synthesis: Structural Analogs of the Sugar Moiety of the Polyoxins The objective of this work was to realize a facil e route to branched-chain glycosyl amino acids which would be analogous in 46 structure to the sugar moiety of the polyoxins. One may consider the sugar moiety of the polyoxins to contain an L-amino acid residue attached to C-4 of a ribo-furanosyl ring. It was of interest, therefore, to study the properties of similar structures with the amino acid residue at C-3 or C-6 of the sugar moiety. In addition, introduction of branching at C-3 of the sugar portion of naturally occurring nucleosides results in interesting changes in 152 153 their biological activity. Recently, anhydrooctose uronic acid nucleosides, isolated from the culture f i l t r a t e s of Steptomyces cacaoi 154 var. asoensis, were postulated to be biochemical precursors of the polyoxins. With this in mind, the synthesis of polyoxin analogs having a seven carbon sugar moiety was of further interest. The synthesis of branched-chain glycos-3-yl amino acids has been developed in this laboratory"^ , (^'^ as mentioned in the introduction. This stereospecific synthesis has many steps, some of which are relatively - 46 -low yielding. Jordaan and Brink'"""''^ have used a-metallated isocyanates to form the blocked p-amino acid sugar 56_ in two steps. One of our objectives was to develop a simple route to both the D- and L-amino acid sugars. 1.1 Synthesis of Derivatives of D-2- and L-2-(3-Deoxy-cx-D-allofuranos-3-yl) glycine 1.1.1 1,2:5,6-Di-0-isopropylidene-ct-p-ribo-hexofuranos-3-ulose (8) Oxidation of the free hydroxyl group of l,2:5,6-di-0-isopropylidene-a-Q-glucofuranose ( 1 4 1 ) w a s achieved using ruthenium t e t r a o x i d e , ^ generated in situ. The ketose hydrate (142), obtained from the oxidation, was dehydrated to 8_ by azeotropic d i s t i l l a t i o n of toluene and water. - 47 -1.1.2 Azlactone Condensation of l,2:5,6-Di-0-isopropylidene-g-D-ribo-furanos-3-ulose (8) with 2-Phenyl-5(4)-oxazolinone (89) The presence of acid-labile blocking groups on 8^  necessitated 75 76 modification of the original condensation procedure of Erlenmeyer. ' To avoid the presence of acetic acid, the preformed oxazolone 89_ 95 was used. Since' the sugar and oxazolone were solids, tetrahydro-93 furan or dimethoxyethane were used as solvents. As reported by 93 Baltazzi and Robinson, lead acetate was used as the catalyst in order to avoid enolization of the carbohydrate ketone. Compound 8^  was reacted with the oxazolone 89_ in refluxing dimethoxyethane in the presence of ca. 0.3 molar equivalents of lead diacetate. The water produced from the condensation was removed by passing o the refluxing solvent through molecular sieves (3 A). A mixture of the (E)- and (Z)-isomers 143 and 144 was obtained after chromatography with a combined yield of 75%. The yield was optimum with a slight excess of compound 89_ and a reaction time of 24 hours. Extra reaction duration or oxazolone gave rise to decomposition of the product due 81 to polymerization. This effect was also observed by Levene. The mixture of oxazolones 143 and 144 exhibited the presence of a C=N function at 1705 cm * and a C=0 function at 1830 cm * in i t s 94 infrared spectrum. These values were as expected for 5(4)-oxazolones. That 143 and 144 were (E)- and (Z)-2-phenyl-4-(l,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5(4)-oxazolone was shown by pmr and elemental analysis. The assignment of the (E)- and - 48 -(Z)-isomers as 143 and 144 respectively in a ratio of 1:1 required chemical transformations which w i l l be discussed later. H9C C Pb(0Ac)9 _2| | ^ 2, N 89 143 (39%) 144 (33%) 8 + Pb (OAc), 89 THF >^ 143 (69%) It was observed that the above condensation reaction gave exclusively 143 when tetrahydrofuran was used as the reaction solvent instead of dimethoxyethane. The higher reaction temperature in refluxing dimethoxyethane would allow inversion of the intermediate condensation product prior to elimination of water. It has been 158 shown that optically active azlactones are rapidly racemized in 159 acetic anhydride. O'Brien postulated that the geometrical course of the Erlenmeyer reaction can be determined by the interconversion of stereoisomeric forms of the intermediate addition product. Since addition from the 3-face of 8^  is normally expected, there would be two diastereomeric intermediates 143' and 144'. If 143' was the - 49 -kinetically favoured product i t could yield 143 exclusively by trans-elimination. However, interconversion of 143' and 144' at higher temperatures would yield a mixture of products. 143 Ph 144 1.1.3 Methanolysis of (E)- and (Z)-2-Phenyl-4-(1,2;5,6-di-O-isopropyl idene-a-D-rib_o-hexofuranos-3-ylidene)-5(4)-oxazolone (143) and (144 ) The mixture of oxazolones 143 and 144 was reacted with methanol and a catalytic amount of sodium acetate at room temperature. A weake base than u s u a l * ^ was used in order to avoid epimerization at C-5.*^ The unsaturated esters 145 and 146 were obtained in 90% combined yield. - 50 -The presence of two methyl ester peaks in the pmr (Figure II), the i r spectrum and elemental analysis demonstrated that compounds 145 and 146 were (E)- and (Z)-methyl-N-benzamido-a-(1,2:5,6-di-0-isopropylidene-a-g-ribo-hexofuranos-3-ylidene) glycinate. The char-a c t e r i z a t i o n of 145 and 146 as the (E)- and (Z)-isomers was obtained as a result of their subsequent chemical transformations. MeOH Figure II: 60 MHz PMR spectrum of (E)- and m-Methvl-N-benzamido-a- CI .2:5,6-di-0-isopropylidene-a-Q-ribo-hexofuranos-3-ylidene)glycinate (145) and (146). - 52 -When the pure branched-chain oxazolone (145) was reacted with methanol and sodium acetate, the a-benzamido unsaturated ester (145) was obtained in 90% yield. The pmr spectrum had only one methyl ester, indicating the presence of a single isomer. 1.1.4 Hydrogenation of (E)- and (ZJ-Methyl-N-benzamido-a-(1,2;5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene) glycinate (145) and (146) m i -i i . 19,24,162 . There have been numerous examples demonstrating stereo-selectivity in the hydrogenation of exocyclic double bonds of unsaturated sugars. The addition has always occurred from the 3-face of the fused furan and 1,3-dioxolane rings to yield the allo-configuration. Since heterogeneous catalytic hydrogenation gives cis addition of hydrogen to a double bond i t would be possible to determine the configuration of 145 and 146 from the stereochemistry of their reduced products. Subsequently the structures of 143 and 144 could be derived from the structures of their methanolysis products. Thus a mixture of 145 and 146 was hydrogenated with palladium on - 53 -carbon catalyst under one atmosphere of hydrogen. Chromatography of the reaction mixture afforded 147 and 148 in yields of 54% and 44%, respectively. Compound 147 was identified as methyl D-2-(3-deoxy-l,2;5,6-di-0-isopropylidene-a-D-allofuranos-3-yl)-N-benzoylglycinate by direct comparison with the N-benzoyl derivative of 49_. This branched-chain amino acid sugar had been previously stereospecifically synthesized 59 in this laboratory. The structure of 4_9 was verified by comparison with a derivative of 56^ (see Section 1.2) which had undergone X-ray 71 analysis . The pmr, i r , and melting points and mixed melting point a l l verified the identity of 147. Accordingly 148 was found to be methyl L-2-(3-deoxy-l,2;5,6-di-0-isopropylidene-a-D-allofuranos-3-yl)-N-benzoylglycinate by comparison with the N-benzoyl derivative of 51_ which was also synthesized in this laboratory.^ - 54 -Having determined the structures of the branched-chain amino acid sugars (147) and (148) the structures of 145, 146, 145 and 144 became evident, as shown in Figure III. It was found that the D- and L-branched-chain amino acid sugars (147) and (148) could be conveniently synthesized from oxazolones 145 and 144 by simultaneous methanolysis and hydrogenation. Chromatography of the reaction mixture afforded 147 and 148 in a combined yield of 95%. 1.1.5 Attempted Hydrolysis of the N-Benzoyl Blocking Group of Amino Acid Branched-Chain Sugars (147) and (148) Basic hydrolysis of the N-benzoyl blocking group was attempted, since the usual method, refluxing dilute mineral acid, would decompose most carbohydrate derivatives. An amino acid branched nucleoside 163 149 had been obtained from i t s N-benzoyl derivative 150 using aqueous saturated barium hydroxide at 37°C. This prompted an attempt - 56 -at hydrolysing 147 under the same conditions. NHBz OBz NH2 OH 149 150 When 147 was subjected to saturated aqueous barium hydroxide at room temperature, for up to 3 days, there was no observed benzamide hydrolysis, as shown by ninhydrin test on t i c . Refluxing of the barium hydroxide solution afforded no detectable benzamide hydrolysis even after 3 days. The base hydrolysis was also attempted using aqueous sodium hydroxide and sodium hydroxide in dimethyl sulfoxide on compounds 147 and 148. The lack of reactivity of these benzamide groups, under vigorous basic hydrolytic conditions, is most probably due to steric hindrance. Models of compounds 147a and 148a indicate that the solvated carboxylate anion, obtained under basic conditions, would project away from the sugar, leaving the benzamide group close to the endo surface of the furan-dioxolane b i c y c l i c system. Approach of a base would therefore be greatly impeded. - 57 -Enzyme hydrolysis has been used with success in the synthetic resolution of a-amino acids from their N-acyl derivatives. The most widely used enzyme of this type, hog kidney acylase I, has a high spec i f i c i t y for a great variety of N-acyl a-L-amino acids, is relatively stable, and requires uncomplicated conditions. More J67 , recently, i t was discovered that the enzyme hippuricase was identical to hog kidney acylase I and that N-benzoyl groups could be cleaved with equal f a c i l i t y . The optimum pH of 7.0 is usually attained by addition of ammonium hydroxide to a solution of the free acid form of the substrate. Thus, hydrolysis of the N-benzoyl group of the L-amino acid branched sugar (148a) with hog kidney acylase I was attempted. To test the hydrolytic procedure, a control reaction was performed, with hippuric acid (N-benzoylglycine) as the substrate. Charcoal chromatography afforded the product, glycine, and a small amount of unreacted substrate. When the same procedure was applied to the free acid N-benzamide 148a, only substrate was obtained. - 58 -Again steric hindrance must be cited as the prime reason for the lack of reactivity of these compounds to hydrolysis. The amino acid sugar 51_ as i t s free acid v/as found to be unreactive to the enzyme L-amino acid o x i d a s e . T h i s lack of reactivity was probably due to the steric hindrance of the 1,2-0_-isopropylidene functionality. 1.2 Synthesis of Derivatives of D-2-[2,3-dideoxy-arabino-(and ribo)-hexopyranos-3-yl] Glycine 1.2.1 Methyl 4,6-0-benzylidene-2-deoxy-a-D-erythro-hexopyranosid-3-ulose (153) The 3-hydroxy sugar (152), from which the ketose 153 was derived, was i t s e l f obtained using known procedures*^ beginning with methyl 2,3-anhydro-4,6-0_-benzylidene-ct-p-allopyra.noside (151) . Reduct 172 of 151 with lithium aluminum hydride in tetrahydrofuran resulted in axial attack at C-2 by hydride ion to afford 4,6-0-benzylidene-2-deoxy-q-D-ribo-hexopyranoside (152) in quantitative yield. Oxidation of 152 using ruthenium tetraoxide afforded the keto-sugar 153 in 64% yield. Even though the ruthenium tetraoxide oxidation gives lower yields than the Moffatt oxidation using DMSO-174 acetic anhydride (64% as compared to 78%), the former was the method of choice. The latter took longer to complete and afforded a product which always contained dimethylsulfide, even after chromato-graphy and recrystallization. - 59 -0 OH 151 152 153 1.2.2 Azlactone Condensation of Methyl 4,6-0_-benzylidene-2-deoxy-a-D-erythro-hexopyranosid-3-ulose (153) with 2-Phenyl-5(4)-oxazolinone (89) The condensation of 153 with the oxazolinone 89_ was f i r s t attempted using the conditions employed with the furanosulose 8_. The yield, however, was low because the condensation was very slow. Thus, compound 89_ would decompose significantly before being able to condense. It was unexpected that the reactivity of 153 would be less than that of 8_. The C=0 group of 153 should be more reactive to nucleophiles and less st e r i c a l l y hindered than that of compound 8_. A possible explanation i s that the exocyclic double bond of the condensation product of 153 would be of higher energy than that of j5. This extra energy would be due to the higher strain of double bonds which are exocyclic to six-membered rings as opposed to five-membered rings. In the case of the reaction of compound 153, the 8-elimination of water to form the unsaturated product might be the rate determining step. - 60 -0 K o—i 0 0. OMe OMe N Ph 0 Ph 95 Crawford and L i t t l e " " suggested that the lead acetate catalyst aids in the elimination step of the reaction. Thus the condensation was attempted using 5 times as much lead acetate as before. The yield was found to improve significantly in a shorter reaction time. The condensation of 153 with the oxazolinone 89_ in dimethoxy-ethane afforded a mixture of azlactones in a combined yield of 40%. Chromatography afforded (E)- and (Z)-2-phenyl-4-(methyl-4,6-0-benzylidene-2,3-dideoxy-ct-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) in 13% and 27% yield, respectively. The structures of 154 and 155 were pa r t i a l l y determined by their pmr spectra though their assignment as the (_E) - and (Z)-isomers required subsequent chemical transformation. Both recrystallized products had satisfactory elemental analyses. - 61 -155 1.2.3 Methanolysis of (E)- and (ZJ-2-Phenyl-4-(methyl-4,6-0-benzylidene-2,3-dideoxy-ct-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) Compounds 154 and 155 were each subjected to methanolysis using sodium acetate in refluxing methanol. In both cases the products which crystallized from their reaction mixtures gave correct elemental analyses. The assignment of 156 and 157 as respectively (E)- and (Z)-methyl-N-benzamido-a-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-Q-erythro-hexopyranos-3-ylidene) glycinate was based upon their pmr spectra. The - 62 -anisotropy of the carboxymethyl group provides shielding and deshielding influences on adjacent protons. In particular, the y protons of a,B-unsaturated esters are deshielded i f they have a cisoid 175 relationship with the carboxymethyl group. 94 Galantay and coworkers determined, through observation of the chemical shifts of several methyl benzamido acrylates, that the carboxy-methyl group did deshield any y proton with which i t was cis by up to 0.4 ppm. They also determined that the N-benzoyl group exerts l i t t l e i f any effect on the shift of the y protons. Table I summarizes their relevant data. TABLE I. C H 2 X CCLMe \ / 2 Q Q y \ NHCOPh CH2Y Compound X Y x (CH2X) T (CH^Y) methyl 7.88 8.16 dimethylacrylate 158 H H 7.85 8.15 159 H OAc 7.85 5.35 160 OAc H 4.88 8.10 161 OAc OAc 5.05 5.26 162 OMe OAc 5.83 5.22 - 63 -145 170 177 The chemical shifts of compounds 163, 164, and 165 ' ' are shown in Table II. The cyano functionality is known to have a similar 178 deshielding effect to a carboxymethyl group. Thus the chemical shifts of 164 and 165 demonstrate how H-2e and H-2a of this type of structure are affected by an anisotropic group. TABLE II OMe 163 164 165 Compound T (H-2e) T (H-2a) T (C02CH3) 163 6.22 7.80 6.31 164 6.93 7.43 165 7.40 7.32 156 7.32 7.32 6.62 157 7.09 7.54 6.18 As seen in Table II and Figure V the C-2 equatorial protons of 163, 164 and 157 are deshielded much more than the C-2 axial protons. Models of these compounds indicate that H-2e is in the deshielding M e O O C r ^ \ NHBz 156, 2 I —L I 1 I t 1 I 1 I T I I I I I I I I I I I I I i 4^ J 0 o 15 Hz i i i 1 I i i i I i i i i • i i i Figure IV. Partial 100 MHz PMR Spectrum of (E)-Methyl-N-benzamido-a- (methyl-4,6-0_-benzylidene-2,3-dideoxy-a-g-erythro-hexopyranos-3-ylidene)glycinate (156). Z 2 1 1 L 8 i Figure V: Partial 100 MHz PMR Spectrum of (Z)-Methyl-N-benzamido-a-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)glycinate (157). - 66 -region of the " c i s " carbonyl whereas H-2a is near the shielding cone. The deshielding of H-2e in 157 (0.23 ppm to lower f i e l d than 156) and the shielding of H-2a (by 0.22 ppm) suggests that i t is the Z_ isomer. Further evidence that 156 is the E_ isomer can be found in the chemical shifts of the methyl ester protons of 156 and 157. Usually, the methyl ester protons are found in the region x = 6.1 to T = 6.3. Though this is observed in compound 157, the shift of this group in 156 is 0.38 ppm to higher f i e l d . This significant amount of shielding is caused by the ring current of the phenyl moiety of the 4,6-0-benzylidene acetal. A model of compound 156 clearly places the methyl ester above the center of the plane of the phenyl moiety. The structures of oxazolones 154 and 155 can be deduced from the structures of their methanolysis products 156 and 157. Thus, compound - 67 -1 5 4 is the E_ oxazolone branched sugar and 155 is the isomer. 1 . 2 . 4 Hydrogenation of (E_)-Methyl-N-benzamido-a- (methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene) glycinate (156) Compound 156 was hydrogenated at low pressure with 5% rhodium on alumina as catalyst. Chromatography afforded a small amount of methyl-D-2-(methyl-4,6-0-benzylidene-2,3-dideoxy-q-D-arabino-hexopyranos-3-yl)-N-benzoylglycinate (166) and a larger amount of methyl-D-2-(methyl-4,6-0-cyclohexylmethylidene-2,3-dideoxy-q-D-arabino-hexopyranos-3-yl)-N-cyclohexylcarboxylglycinate (167). The structure of 167 was assigned from i t s pmr spectrum which could be extensively analyzed with the aid of decoupling experiments. The pmr spectrum of 167 (Figure VI) shows an H-3, H-4 coupling constant of 9.8 Hz and an H-3, H-2a coupling of 11.0 Hz. These large couplings 179 suggest that H-3 is in an axial position. Since the pyranose ring 4 is in a C^ conformation the hydrogen must have added to the e x o c y c l i c carbon-carbon double bond, e x c l u s i v e l y from H1 H 4 N l" I '4/5 5 3 Hz T 5 6 ON 00 Figure VI: Partial 100 MHz PMR Spectrum of Methyl-D-2- (methyl-4,6-0-cyclohexylidene-2 f3-dideoxy ra-D-MaMiio-hexopyranose-3-yl)-N-cyclohexylcarboxylglycinate (167). - 69 -the a-face of 156, giving an arabino configuration to the sugar moiety and the D-configuration to the amino acid moiety of 167. The addition of hydrogen from the a-face is unexpected because of a 1,3-diaxial interaction of the incoming catalyst and the methyl glycoside. Indeed, in the case of the unsaturated cyano compounds 164 145 177 and 165 and the unsaturated ester 165 ' hydrogenation took place from the 6-side of the pyranose ring. As w i l l be seen in the next section (1.2.5) hydrogenation of the Z unsaturated amino acid derivative 157 also occurred from the 8-face. OMe C = N OMe 164,165 169 - 70 -Therefore compound 156 clearly exhibits anomalous behaviour upon catalytic reduction. Close examination of the pmr spectrum of 156 may provide an explanation. Unlike i t s Z_ isomer 157 or the unsaturated carboxymethyl or cyano sugars 165, 164 and 165, which a l l have clearly 180 defined signals for H-2 and H-2 , the signals for the H-2 protons a e of 156 (Figure V) behave much more as an ABX system. The couplings of the H-2 protons with H-l are both 3.7 Hz. That they are not well defined axial or equatorial protons suggests that the unsaturated branched sugar 156 is not in a deformed chair but is in a twist boat conformation. 156 The twist boat conformation may be adopted because of steric crowding between the methyl glycoside and the bulky benzamide group on the branched-chain which would be present in the chair conformation. If this conformation was prevalent, then no 1,3-diaxial interaction would occur during hydrogenation from the a-face. In fact, the ot-side would be much less hindered than the B-face. Rhodium on alumina was chosen as the hydrogenation catalyst because 156 was resistant to hydrogenation with palladium on carbon. Prolonged exposure to hydrogen at 5 atm. with palladium on carbon gave mostly - 71 -hydrogenolysis. This phenomenon has been observed previously with benzylidene acetals. 1.2.5 Hydrogenation of (E)-Methyl-N-benzamido-ot-(methyl-4,6-0-benzylidene-2,3-dideoxy-ct-D-erythro-hexopyranos-3-ylidene) glycinate (157) Hydrogenation of unsaturated ester 157 at low pressure with 5% rhodium on alumina afforded crystalline methyl-D-2- (methyl-4,6-0_-cyclohexylidene-2,3-dideoxy-q-D-ribo-hexopyranos-3-yl)-N-cyclohexyl-carboxylglycinate 170. A small amount of material in which only the benzamide was hydrogenated was obtained by chromatography of the mother liquor. Compound 170 was identified by i t s i r spectrum, elemental analysis and pmr spectrum with appropriate decoupling experiments. The negligible coupling of H-3 with H-2 or H-2 (Figure VII) implies that H-3 is in an 179 equatorial orientation. If hydrogenation were from the 3-face of Figure V I I : P a r t i a l 100 MHz PMR Spectrum of Methyl-D-2-(methyl-4,6-0-cyclohexylidene-2,3-dideoxy-ct-D-ribo-h e x o p y r a n o s - 3 - y l ) - N - c y c l o h e x y l c a r b o x y l g l y c i n a t e (170). - 73 -157 then the expected cis-addition of hydrogen would give the D-amino acid branched-chain sugar of ribo configuration. Both blocked amino acid sugars 167 and 170 failed to give a ninhydrin reaction when treated with alcoholic, aqueous barium hydroxide for prolonged intervals. 1.3 Synthesis of Derivatives of 6-Amino-5,6-dideoxy-a-D-gl_uco-(and B-L-ido) heptofuranuronate 1.3.1 Selected Acidic Hydrolysis of 3-0-Benzyl-l,2;5,6-di-O-isopropylidene-a-g-glucofuranose (171) Compound 171 was subjected to mildly acidic hydrolysis with 0.1 N sulfuric acid to give 3-0-benzyl-1,2-0-isopropylidene-a-Q-gluco-181 furanose (172). Chromatographic purification of the product was necessary since a trace amount of material, in which both ketals were hydrolyzed, was produced. Comparison of the optical rotation of 181 172 with the literature, and pmr spectral analysis were used to verify i t s structure. - 74 -1.3.2 Periodate Oxidation 3-0-Benzyl-l,2-0-isopropylidene-a - P - g l u c o f u r a n o s e (172) The 5,6-vicinal diol of compound 172 readily underwent oxidative cleavage by sodium metaperiodate to yield 3-0_-benzyl-l,2-0-isopropylidene-181b q-D-xylo-pentodialdo-1,4-furanose (173). The pmr spectrum of 173 showed the characteristic aldehydic proton resonance at T 0.38. Elemental analysis further supported the structure of the dialdehydo sugar 173. The difference between the optical rotation of 173 and the 181b literature value may be due to the presence of methanol in the chloroform used in the rotation measurement of 175. An alcoholate may 181c be formed since 173 has been shown to form an aldehydrol in a i r . HO—, 1.3.3 Azlactone Condensation of 3-0-Benzyl-l,2-0-isopropylidene-q-D-xylo-pentodialdo-1,4-furanose (173) with 2-Phenyl-5(4)-oxazolinone (89) The free aldehydo sugar 173 was reacted with 89_ in refluxing tetrahydrofuran in the presence of 0.3 molar equivalents of lead diacetate. The water produced from the condensation was removed by o passing the refluxate through molecular seives (3 A). Formation of peroxides must be particularly avoided since they rapidly decompose the starting aldehyde. In this way, an inseparable mixture of the E and Z-isomers 174 and 175 was obtained in a combined yield of - 75 -45% after chromatography on s i l i c a gel. As evidenced by thin layer chromatography there was extensive decomposition of the starting material during the course of the reaction. The highly polar decomposition product defied spectral analysis. The structures of 174 and 175 were established as (E)- and (Z)-4-(3-0_-benzyl-5-deoxy-l, 2-0_-isopropylidene-a-D-xylofuranos-5-ylidene) -2-phenyl-5(4)-oxazolone by their pmr and i r spectra and elemental analyses. The infrared spectrum of the mixture exhibited the presence of a C=N function at 1687 cm"1 and a C=0 function at 1810 cm"1. These 94 values were as expected for 5(4)-oxazolones. 173 174 175 1.3.4 Methanolysis of (E)- and.(Z)-4-(3-0-Benzyl-5-deoxy-1,2-0_-isopropylidene-a-D-xylofuranos-5-ylidene) -2-phenyl-5(4)-oxazolinone [174) and (175) The mixture of oxazolones 174 and 175 was solvolysed in methanol with a catalytic amount of sodium acetate. The reaction proceeded readily at room temperature to give (E)- and (Z)-methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-a-p-xylo-heptofur-5-enuronate (176) and (177) in 85% combined yield. The presence of two - 76 -methyl ester peaks and two amide peaks in the pmr (Figure VIII), in a ratio of ^ 3:1 demonstrated that there was a mixture of unsaturated esters. Though the mixture of isomers could not be separated by chromatography a satisfactory elemental analysis of the mixture was obtained. Figure V I I I : 60 MHz PMR Spectrum of (E)- and (ZJ-Methyl-6-N-benzamido-3-0j-benzyl-5,6-dideoxy-l ,2-0-isopropylidene-a-D-xylo-heptofur-5-enuronate (176) and (177). - 78 -1.3.6 Hydrogenation and Hydrogenolysis of (E)- and (Z)-Methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-a-D-xylo-heptofur-5-enuronate (176) and (177) Careful atmospheric pressure hydrogenation of the unsaturated esters 176 and 177 afforded an analytically pure mixture of methyl 6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-a-D-gluco-heptofuranuronate (178) and methyl 6-t^-benzamido-3-0-benzyl-5,6-dideoxy-1,2-0-isopropylidene-a-L-ido-heptofuranuronate (179) after chromatography. The presence of the saturated esters was verified by the infrared absorption at 1710 cm * and the complex two proton signal for CH^-S at x 7.62 in the pmr. Prolonged atmospheric or low pressure hydrogenation resulted in hydrogenolysis of the 3-0-benzyl ether of 178 and 179. A mixture was s t i l l present as indicated by the pmr spectrum which clearly showed two sets of isopropylidene methyl signals in a ratio of 4:1. The lack of benzyl ether protons in the pmr and the elemental analysis demonstrate that the mixture is methyl 6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-a-D-gluco-heptofuranuronate (180) and i t s g-L-ido-isomer (181). - 79 -C02Me BzNH s\ MeO C • NHBz Nl OCH2Ph OCH2Ph I J _ 0 177 176 H 2 Pd/C ty C02Me CO. Me I 2 H — C NHBz BzNH — C — H I . CH. CH, \1 0 OCH2Ph OCH2Ph 178 BzNH' C02Me C — H I CH. 179 ty 0 CC° Me i ^ "C NHBz I CH0 O-4 o — W o I I 180 181 - 80 -1.3.6 Transesterification and Separation of Methyl 6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-a-D-gluco-heptofuranuronate (180) and Methyl 6-N-benzamido-5,6-dideoxy-1,2-0_-isopropylidene-g-L -ido-heptofuran-uronate (181) The mixture of blocked amino acid sugars readily underwent transesterfication with sodium ethoxide in ethanol to afford ethyl 6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-q-D-gluco-heptofuranuronate (182) and i t s g-L-ido isomer (183) . Fractional crystallization of the mixture gave primarily 183, followed by a small amount of 182. The separation by this technique was confirmed by the distinct melting points, optical rotations and pmr spectra of the two isomers. The identity of 183 as the g-L-ido isomer was established by subsequent chemical transformation. - 81 -1.3.7 Barium Hydroxide Hydrolysis of Ethyl 6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-g-L-ido-heptofuranuronate (183) The blocked amino acid derivative 183 was subjected to hydrolysis by barium hydroxide in a one to one mixture of ethanol and water. Precipitation of barium ions with sulfuric acid followed by ion exchange chromatography on a benzene-sulfonate resin in the pyridinium salt form afforded the free amino acid derivative 6-amino-5,6-dideoxy-1,2-0-isopropylidene-g-L-ido-heptofuranuronic acid (184). This structural assignment was consistent with the pmr and elemental analytical data of the crystalline hemihydrate of 184. The designation of the ch i r a l i t y at C-6 of the product as that of the L-sugar was based on the circular dichroism spectrum of 184. The hydrochloride of 184 had a negative Cotton effect in the wavelength region of the carboxylic acid absorption. This negative Cotton effect is indicative of a D-amino acid function as observed generally . , 182a . . . . . . . , . . 60,61,70 for amino acids. The ammo acid derivatives of carbohydrates have been shown to follow the empirical correlation of positive Cotton effects for L-amino acids and negative Cotton effects for D-amino acids. If C-6 of compound 184 is the a-carbon of a D-amino acid, then compound 184 can be represented as a g-L-ido sugar. - 82 -When compound 182 was subjected t o the same h y d r o l y t i c c o n d i t i o n s as f o r 183 there was no benzamide h y d r o l y s i s observed. Work-up o f the r e a c t i o n under i d e n t i c a l c o n d i t i o n s used to s y n t h e s i z e the g-L-ido d i a s t e r i o m e r a f f o r d e d only the N-benzamido g-D-gluco a c i d . The d i f f e r e n c e i n r e a c t i v i t y w i t h barium hydroxide between 182 and 183 cannot be exp l a i n e d , though i t probably i n v o l v e s an i n t e r a c t i o n w i t h the 3-OH group. 1.3.8 Attempted Enzyme H y d r o l y s i s o f E t h y l 6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-a-D-gluco-hepto-furanuronate (182) and E t h y l 6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-g-L-ido-heptofurano-uronate (183) Since 182 was r e s i s t a n t to base h y d r o l y s i s , an enzymatic method was considered. A l s o an a l t e r n a t i v e proof o f the s t r u c t u r e s o f 182 and 183 was sought, s i n c e c i r c u l a r d i c hroism measurements do not a f f o r d unequivocal proof of the c o n f i g u r a t i o n o f the center a- to - 83 -a carboxyl group in multi-asymetric centered compounds. Hydrolysis of the N-benzoyl group of both 182 and 183 with hog kidney acylase I was attempted. The method used was the same as that employed in Section 1.1.5. Neither 182 nor 183 was hydrolyzed by the enzyme even after prolonged incubation. This lack of reactivity was probably due to steric hindrance to the substrate entering the active site of the enzyme. For further discussion see Section 1.1.5. 2. Synthesis of Carbohydrate Spiro-Pyrrolidones The i n i t i a l aim of this work was to investigate a facile route to carbohydrates possessing an a,y diamino acid moiety as a branched-chain at C-3. The elaboration of amino acid branched-chains of various constitution was attractive because (1) the polyoxins are of considerable biological interest and (2) changes have been observed in biological activity of nucleosides resulting from branching at C-3 152 on the sugar moiety. Recently, in this laboratory, carbohydrates with a 3-amino acid and a,B-diamino acid branched chains at C-3 have 183 been synthesized. Investigation of the a,y-diamino acid branched-chain was an extension of that research. To obtain the a,y-diamino acid moiety, the general synthetic 120 procedure of Carter and coworkers was used (Introduction, Section 3.1). 2.1 Diazomethane Addition to Branched-Chain Unsaturated Esters 2.1.1 Synthesis of (E)- and (Z)-3-Deoxy-1,2;5,6-di-0-isopropylidene-3.rC- (methoxycarbonyl)-methylene-a-D-ribo-hexofuranose (12) and (13) - 84 -The keto sugar 8 was reacted with trimethylphosphonacetate 19 according to the method of Rosenthal and Nguyen to afford a mixture of 12 and L3 after column chromatography. Fractional crystallization gave mostly pure Z_ isomer L3 followed by the E_ isomer 1_2_. These compounds were identified by comparison of their pmr spectra with the literature i 19 values. 2.1.2 The Addition of Diazomethane to (Z)-3-Deoxy-1,2;5,6-di-0_-isopropylidene-3-C_-methoxycarbonylmethylene-a-D-ribo-hexofuranose (13) When a mixture of L3 and diazomethane in anhydrous ether was allowed to react for 8 h at 5° two unstable product mixtures were obtained upon column chromatography. The lower R^  products were shown to be a mixture of the diastereomeric A*-pyrazolines 185, 186, 187 and 2 188. The higher R^  products were designated as the A -pyrazolines 189 and 190. - 85 -189 and 190 The s t r u c t u r e s of 185, 186, 187, and 188 were p a r t i a l l y deduced by a n a l y s i s of t h e i r i r , pmr, and mass s p e c t r a . The i r spectrum o f the mixture e x h i b i t e d a saturat e d carbonyl a b s o r p t i o n at 1723 cm *. The pmr spectrum could not be f u l l y r e s o l v e d ; however, the i n t e g r a t i o n r a t i o between the r e s o l v a b l e anomeric doublet and the complex r e g i o n from x 4.41-6.80 accounted f o r H-2, H-4, H-5, 2H-6 and H-a plus an a d d i t i o n a l 2 protons on the methylenic carbon o f the p y r a z o l i n e r i n g . A d d i t i o n of diazomethane was a l s o confirmed by the mass spectrum o f the mixture which gave a parent peak at m/e = 369. Compounds 189 and 190 were correspondingly deduced by t h e i r i r , - 86 -pmr and mass s p e c t r a . The i r spectrum of the mixture e x h i b i t e d absorptions at 3460 cm *, 1705 cm * and 1548 cm * which i n d i c a t e N-H, C=0 and O N groups, r e s p e c t i v e l y . The i n f r a r e d s p e c t r a l r e s u l t s f o r 1 2 both A - and A - p y r a z o l i n e s agreed with those which have been A^-pyrazolines contained a broad s i n g l e t at x 2.38 which exchanged upon a d d i t i o n of D^ O. This s i g n a l was assigned as the N-H proton resonance 2 of the A - p y r a z o l i n e . The a d d i t i o n of diazomethane was again confirmed by the mass spectrum of the mixture which gave a parent peak at m/e = 369 A "^-Pyrazolines are known to be the primary products o f a d d i t i o n o f diazoalkanes to double bonds. However, these compounds may tautomerize, p a r t i c u l a r l y i f the product i s s t a b i l i z e d by c o n j u g a t i o n . * The A*-pyrazolines 185, 186, 187, and 188 r e a d i l y tautomerized i n t o conjugation w i t h e s t e r carbonyl, as evidenced by the r a t i o (1:3) o f A*-2 and A - p y r a z o l i n e s which were i s o l a t e d . p r e v i o u s l y documented. 184,185 The pmr spectrum of the mixture of 185, 186, 187, and 1_88 189 and 190 - 87 -The presence of the four diastereomeric A - p y r a z o l i n e s 185, 186, 187 and 188 was demonstrated by the stereochemistry o f t h e i r hydrogena-t i o n products which i s to be elaborated (Section 2.1.3). I t i s i n t e r e s t -in g t hat two of these diastereomers must r e s u l t from the a d d i t i o n o f the methylene carbon of diazomethane from the ce-surface of the unsaturated e s t e r 13_. This i s the f i r s t i n s t a n c e , i n our l a b o r a t o r y , of both a- and g-attack upon 13, and may i n d i c a t e t h a t a concerted c i s 109 112 a d d i t i o n i s not o p e r a t i v e . ' P o s s i b l y , as suggested by other w o r k e r s , ' t h e t e r m i n a l n i t r o g e n of the diazomethane f i r s t adds to the methyne carbon of the e x o c y c l i c carbon-carbon double bond fo l l o w e d by r i n g c l o s u r e to y i e l d the mixture o f p y r a z o l i n e s . The (C_)-branched unsaturated e s t e r L2 was completely u n r e a c t i v e to diazomethane. The same u n r e a c t i v i t y was observed f o r (E)-methyl-4,6-0-benzylidene-3-C_- (carbomethoxymethylene)- 2,3-dideoxy-a-D-erythro-hexopyranoside (163). The unsaturated e s t e r 163 was d e r i v e d from the ketose 153 by W i t t i g r e a c t i o n w i t h trimethylphosphonacetate according to 145 the method of Rosenthal and Catsoulacos. A c o r r e l a t i o n between the r e a c t i v i t y o f 1_2, 1_3 and 163 with diazomethane and the 1,3-dipolar 183 a d d i t i o n of azide i o n has been observed. I n t e r e s t i n g l y , 13 was r e a c t i v e to both azide and diazomethane, whereas 12 and 163 were u n r e a c t i v e to both o f these reagents. This suggests t h a t some a d d i t i o n a l r e a c t i v i t y has been imparted to the Z unsaturated e s t e r 13. A space f i l l i n g model o f L3_ showed d e f i n i t e s t e r i c crowding o f the methyl e s t e r by the C-2 oxygen o f the adjacent i s o p r o p y l i d e n e group. S t e r i c s t r a i n , thus imposed on the double bond, could make 13 more r e a c t i v e . I t i s - 89 -well-known that such s t r a i n increases the d i p o l a r o p h i l i c r e a c t i v i t y _ - 187,188 of o l e f i n s . 2.1.3 High Pressure Hydrogenolysis of P y r a z o l i n e s 185, 186, 187, 188, 189, and 190 To optimize the y i e l d i n the o v e r a l l s y n t h e t i c sequence, the crude product mixture of p y r a z o l i n e s 185-190 was hydrogenolyzed according to 120 the general method of C a r t e r and coworkers. Thus, the mixture was subjected to hydrogenation at 2200 p . s . i . i n the presence o f Raney's n i c k e l c a t a l y s t . A f t e r 8 h at 75-80° the c a t a l y s t and s o l v e n t were removed t o a f f o r d a syrupy mixture. The mixture was separated i n t o four components by column chromatography using stepwise g r a d i e n t e l u t i o n w i t h e t h y l acetate-ethanol mixtures o f i n c r e a s i n g s o l v e n t p o l a r i t y . - 90 -In this way, spiro-3,4»-S_- (3,3-dideoxy-l, 2;5,6-di-O-isopropylidene-g-D-ribo-hexofuranose)-31-R-amino-2'-pyrrolidone (192), spiro-3,4'-S-(3,3-dideoxy-1,2;5,6-di-O-isopropylidene-g-g-Tibo-hexofuranose)- 31 -S-amino-2*-pyrrolidone (193), spiro-3,4'-R- (3,3-dideoxy-1,2;5,6-di-O-isopropylidene-ct-D-ribo-hexofuranose)-3'-R-amino-2'-pyrrolidone (194), and spiro-3,4'- R-(3,3-dideoxy-1,2;5,6-di-0-isopropylidene-a-p-hexofur-anose)-3'-S^-amino-21 -pyrrolidone (195) were obtained in overall yields of 16%, 32%, 14%, and 18%, respectively from 1_3. A l l four substances exhibited strong infrared absorption at about 1700 cm \ thus indicating 91 the presence of a 5-mcmbered lactam r i n g . The lactams ( p y r r o l i d o n e s ) were presumed to be formed v i a an i n t r a m o l e c u l a r c y c l i z a t i o n of the amine f u n c t i o n w i t h the e s t e r group. The presence of the amino 2 1 - p y r r o l i d o n e was confirmed by the pmr s p e c t r a o f the f o u r compounds which showed the absence of methyl e s t e r peaks and the presence of two amino protons, i n the r e g i o n x 7.4 to x 8.2, which exchanged r a p i d l y w i t h D^O, and i n a d d i t i o n , one amide proton i n the r e g i o n x 2.4-2.9 which exchanged s l o w l y w i t h D 20. Although the amino 2'-pyrrolidones gave mass s p e c t r a (m/e = 329) which agreed w i t h t h e i r p o s t u l a t e d s t r u c t u r e s , t h e i r chemical analyses were u n s a t i s f a c t o r y . Trace amounts of s i l i c i c a c i d from the chromatography could not be removed from the a n a l y t i c a l samples. 185 H 192 - 93 -2.1.4 Acetylation of Spiro-3,4'-S-(3,3-dideoxy-l,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranose)-3'-R-amino-2'-pyrrolidone (192) and i t s 3,4'-S-3'-S (193), 3,4'-R-3'-R (194), and 3,4'-R-3'-S (195) Diastereomers In order to obtain analytically pure derivatives the spiro-pyrrolidones 192, 193, 194, and 195 were N-acetylated with acetic anhydride in methanol. The crystalline 3'-acetamido-2'-pyrrolidones 196, 197, 198, and 199 gave elemental analyses which were in f u l l agreement with the proposed structures. The presence of the acetamido-2'-pyrrolidone structure was further evidenced by the pmr spectra of the four derivatives (Figures IX, X, XI, and XII). These spectra showed amide protons in the region T 2.36- x 3.78, which were strongly coupled to the 3'-protons in the region x 4.67- x 5.45. The 3'-proton doublets collapsed to singlets upon the addition of D2O. The y-lactam (pyrrolidone) structure can only be formed from 190 the products arising from the "normal" mode of addition of diazo-alkanes to conjugated double bonds, where the carbon of the diazoalkane attacks the 8-carbon of the unsaturated system. The infrequently observed "reverse" addition would ultimately lead to diamino esters which could only form g-lactams. This would be an unlikely result under the reaction conditions which were present. Furthermore, the g-lactam structures were ruled out on the basis of the above pmr data which showed the 3'-protons of a l l four diastereomers coupled to the N-acetyl amide protons. The g-lactams would have acetamido protons which would either be coupled to the methylene AB system in case A or to no protons in case B (Figure XIII). In this way the "reverse" mode of addition was ruled out. Figure IX: P a r t i a l 60 MHz PMR Spectrum of Spiro-3,4'-S-(3,3-dideoxy-l-,2 :5,6-di-0-isopropylidene-a-D-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone (196). Figure X: Partial 60 MHz PMR Spectrum of Spiro-3,4'-S-3,3-dideoxy-l,2:5,6-isopropylidene-a-D-ribo-hexofuranose)-3'-S-acetamido-2'-pyrrolidone (197). Figure XI: Partial 60 MHz PMR Spectrum of Spiro-3,4'-R-3,3-dideoxy-l,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone (198). Figure XII: Partial 60 MHz PMR Spectrum of Spiro-3,41-R-3,3-dideoxy-1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranose)-3'-S-acetamido-2'-pyrrolidone (199) . - 98 -FIGURE XIII. Alternative Stereochemistry of Addition of Diazomethane. - 99 -The assignment of the four structures by pmr analysis was uncertain because C-3 of the sugar ring is tetrasubstituted. Fortunately, the crystals of 198 and 199 were amenable to X-ray 191 analysis. Compound 198 was shown to be spiro-3,4'-R-(3,3-dideoxy-1,2:5,6-di-0-isopropylidene-a-p-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone and compound 199 was the diastereomeric spiro 3,4'-R-(3,3-dideoxy-l,2:5,6-di-0-isopropylidene-q-g-ribo-hexofuranose)-3'-S-acetamido-2' -pyrrolidone. The remaining two pyrrolidones 196 and 197 must therefore be the diastereomers formed by addition of diazomethane to the opposite face of the carbon-carbon double bond followed by reduction. That i s , the chi r a l i t y of the spiro junction (C-3) of 196 and 197 should be S_ instead of R as in 198 and 199. The assignment of the ch i r a l i t y of C-3' of compounds 196 and 197 is described in the next section. 2.2 Structural Assignment of the 3,4'-S-Spiro Pyrrolidones 196 and 197 A sequence of reactions, described by Yoshimura and coworkers,* was used to assign the ch i r a l i t y at C-3' of compounds 196 and 197. The structural proof involves attempted aminal formation between the C-3' amide and an aldehyde group devised at C-5 of the sugar moiety. 2.2.1 Formation of an Aminal Derivative of Spiro-3,4'-S-(3,3-dideoxy-1,2:5,6-di-Q-isopropylidene-a-D-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone (196) The glycosyl pyrrolidones 196 and 197 were de-0_-isopropylidenated at - 100 -199 Compounds 198 and 199 determined by X-ray analysis - 101 -C-5 and C-6 by hydrolysis in 66% aqueous acetic acid at room temperature. The 5,6-diols formed were not characterized. Instead they were immediately cleaved oxidatively by sodium meta-periodate. The pmr spectrum of the aldehydo pyrrolidone (Figure XIV) derived from 197 exhibited an aldehyde hydrogen resonance at x 0.07, whereas the aldehydo pyrrolidone obtained from 196 showed the absence of an aldehyde proton and the presence of an aminal (Figure XV). Acetylation of the aminal product resulting from 196 afforded a triacetate 202. The pmr spectrum of the latter compound showed no coupling between H-4 and H-5 thus indicating that C-5 has the Re-configuration. Obviously, the C-5 aldehyde functionality of the periodate cleaved product from 196 must have undergone intramolecular cyclization with the primary amino acetyl group to yield spiro-3,41-S-(3,3-dideoxy-1,2-0-isopropylidene-a -D-erythro-pentodialdo-1,4-furanose)-3 ' -R-acetamido-2'-pyrrolidone-3',5-R-aminal-5,1'-diacetate (202). The 3'-S-pyrrolidone 201 could not form such an aminal as i t would necessitate a trans ring fusion. Thus, compound 196 must be spiro-3,4 1-S-(3,3-dideoxy-1,2:5,6-di-0- isopropylidene- a-D-ribo-hexofuranose)-3 ' -R-acetamido-2'-pyrrolidone. The diastereomeric pyrrolidone 197, which did not cyclize, must be spiro-3,4 1 -S- (3, 3-dideoxy-1, 2: 5, 6-di-0_-isopropylidene-q-D-ribo-hexofuranose)-3'-S-acetamido-2'-pyrrolidone. offset 200 Hz I Figure XIV: 60 MHz PMR Spectrum of Spiro-3,4'-S-(3,3-dideoxy-l, 2-0-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose)-3'-S-acetamido-2'-pyrrolidone (201). AcCK 1 1 [ • > ' ' 1 ' • I, I I I I I i I l . : i i ' ' 1 ' ' ' ' ' ' 1 1 1 1 1 1 ' •• • ' 1 ' 1 ' 1 • • • • I • • • • ' • • • • I • • • • I , 3 4 5 6 7 : P a r t i a l 100 MHz PMR Spectrum of Spiro-3,4'-S-(3,3-dideoxy-1 ,2-0-isopropylidene-a-D-erythro-p e n t o d i a l d o - l , 4 - f u r a n o s e ) - 3 ' - R - a c e t a m i d o - 2 ' - p y r r o l i d o n e - 3 S 5 - R - a m i n a l - 5 3 l , - d i a c e t a t e (202). - 104 -The unexpected a c e t y l a t i o n o f the lactam n i t r o g e n was confirmed by pmr and elemental a n a l y s i s . I t i s of i n t e r e s t to note t h a t the r a t e o f a c i d i c h y d r o l y s i s of the 5,6-0_-isopropylidene group o f compound 197 was about twenty times g r e a t e r than that o f compound 196. This d i f f e r e n c e i n chemical r e a c t i v i t y might be due to the f a c t that the b a s i c acetamido group i s nearer to the 5,6-0_-isopropylidene moiety i n compound 196 than i n compound 197 and t h i s impeded the p r o t o n a t i o n of the k e t a l . 2.3 Unblocking of Spiro-3,4'-S-(3,3-dideoxy-l,2:5,6-di-0-isopropylidene-q-D-ribo-hexofuranose) -3' -S-acetamido-2' -p y r r o l i d o n e (197) The i s o p r o p y l i d e n e b l o c k i n g groups of the s p i r o p y r r o l i d o n e 197 were removed by h y d r o l y s i s i n 80% aqueous t r i f l u o r o a c e t i c a c i d at 25° f o r 48 h. This very slow r a t e of h y d r o l y s i s was undoubtedly due to the presence of r e l a t i v e l y b a s i c amide f u n c t i o n a l i t i e s i n the v i c i n i t y o f - 105 -the 1,2-O-isopropylidene group. The product of the h y d r o l y s i s was p u r i f i e d by charcoal chromatography. D i s s o l u t i o n i n p y r i d i n e , f o l l o w e d by f i l t r a t i o n , removed any i n s o l u b l e i m p u r i t i e s . The product was f i n a l l y p r e c i p i t a t e d from ethanol wit h hexanes to a f f o r d s p i r o - 3 , 4 ' -S- (3,3-dideoxy-3-D-ribo-hexopyranose)- 3' -S_-acetamido-2 ' - p y r r o l i d o n e (203) . The elemental a n a l y s i s and the lack o f i s o p r o p y l i d e n e methyl s i g n a l s i n the pmr confirmed that h y d r o l y s i s was complete. I n t e r e s t i n g l y , the f r e e sugar 203 was observed to be e x c l u s i v e l y i n the 3-pyranose form, as evidenced by i t s pmr spectrum which showed the anomeric proton at T 5.24 w i t h a coupling constant J 8.0 Hz. This l a r g e c o u p l i n g was i n d i c a t i v e o f a t r a n s - d i a x i a l o r i e n t a t i o n between H-l and H-2 on a 193 pyranose r i n g . A l s o , the r e l a t i v e l y high f i e l d resonance of the anomeric proton supports the 3-pyranose s t r u c t u r e . 3. An O p t i c a l l y A c t i v e Amino A c i d - W i t t i g Reagent The i n v e s t i g a t i o n of the s y n t h e t i c u t i l i t y o f an amino a c i d W i t t i g reagent was undertaken i n the i n t e r e s t of e l a b o r a t i n g branched-chain amino a c i d carbohydrates by the a d d i t i o n of an i n t a c t amino a c i d moiety to r e a d i l y a v a i l a b l e keto-sugars and k e t o - n u c l e o s i d e s . An o p t i c a l l y a c t i v e reagent was chosen to f a c i l i t a t e the s t r u c t u r a l assignment of any products and to lea d d i r e c t l y to the more b i o l o g i c a l l y i n t e r e s t i n g L-amino a c i d d e r i v a t i v e s . 106 -3.1 Synthesis of Ethyl-N-Acetyl- -(triphenylphosphoniumiodo)--^-alanate (204) 3.1.1 Chlorination of L-Cystine Diethyl Ester Dihydro-chloride (205) L-Cystine diethyl ester dihydrochloride (205), which was prepared 194 by known methods, underwent chlorination, according to the method 195 of Baganz and Dranch. This resulted in cleavage of the disulfide with replacement of the sulfur atoms by chlorine atoms. In this way 8-chloro-L-alanine ethyl ester hydrochloride (206) was formed, as 195 evidenced by i t s melting point. The ethyl ester hydrochloride 206 was converted into the free amino ester which was immediately acetylated using acetic anhydride in ethanol. This procedure was somewhat simpler than the method of 196 Benoiton, using thioacetic acid, and afforded N-acetyl-g-chloro-L-alanine ethyl ester (207) which had a slightly improved melting point and optical rotation 196 >H2irCrEt 2ClCH2-CH-C02Et S N H 3 C 1 — C H C l ^ fe^r 206 S CH2-CH-C02Et N»3 C 1" 1 i) NaHC03(EtOH) — I 2) Ac20/EtOH 2ClCH2-CH-C02Et NHAc 207 - 107 -3.1.2 Reaction of N-Acetyl-8-chloro-L-alanine Ethyl Ester (207) with Triphenylphosphine The 8-chloro alanine derivative 207 was found to be completely unreactive toward triphenylphosphine. Thus a synthesis of the 8-iodo derivative was attempted by reacting 207 with sodium iodide in 197 198 acetone. ' The 3-iodo compound was too unstable, probably decompos-ing by elimination of hydrogen iodide. It was thought that generation of the iodo compound with triphenyl-phosphine present, to immediately form the triphenylphosphonium salt, 199 could be a viable method. Thus the 8-chloro alanine derivative 207 was reacted with triphenylphosphine and an equimolar amount of sodium iodide in refluxing ethyl acetate. After 4 h the reaction was cooled and a mixture of ethyl-N-acetyl-8-(triphenylphosphonium-iodo)-a-L-alanate (204) and sodium chloride crystallized. Routine purification afforded 204 in 51% yield. An analytically pure sample of the monohydrate was found to be s t i l l optically active. The postulated structure for 204 was supported by pmr spectral evidence which showed an amide N-H as a doublet at x 1.59, a fifteen proton multiplet of the triphenylphosphonium moiety at x 2.1-2.4, a one proton multiplet at x 5.5 corresponding to H-cx, and a two proton multiplet at x 5.8-6.2 for the two 8 hydrogens. C1CH_-CH-C0-Et 2 | 2 207 NHAc Nal, Ph3P \, EtOAc [ICH2-CH-C02Et] NHAc Ph„P —it*-—> Ph,P -CH_-CH-CC\,Et 204 NHAc - 108 -3.2 Attempted Wittig Reaction Using Ethyl-N-acetyl-8-(triphenyl-phosphonium-iodo)-a-L-alanate (204) To test i t s potential reactivity, the optically active Wittig reagent 204 was reacted with benzaldehyde. n_-Butyllithium was used as the base for generating the y l i d . It is not usually feasible to prepare stable ylids from phosphonium salts which have a 8-carboalkoxy function, due to a competing elimination of triphenylphosphine. Therefore the y l i d was formed as a transient intermediate which hopefully would be trapped by the carbonyl compound. This method has , . j .. * . . . , , . 200 ,201,202 been applied with success in several laboratories. When the potential Wittig reagent (204) was reacted in this way with benzaldehyde there was no Wittig reaction detected. However, a large amount of the product formed by elimination of triphenylphosphine was found as evidenced by pmr. + H H C0 2 E t Ph-P C C — CCLEt " „ > CH =C 3 I I 2 -Ph,P 2 H NHAc NHAc 204 1) B- ; 2) PhCHD The reaction with the corresponding phosphonate derivative was 203 not attempted. It is known that these compounds (eq. 208) undergo a reaction analogous to the Stobbe condensation under the strongly basic conditions of the Horner modification of the Wittig reaction. - 109 -Ph-C= I H 0 0 t (Et-0)2P- CH 2-CH 2-C0 2Et H I Ph-j: o 208 C0 oEt I 2 -CH I CH. -V I 2 0— P-OEt A VOEt Ph-C0 2Et Base P. ^ O E t / C 0 2 E t Ph-C=C I 2 HO-P(OEt) !• 0 - 110 -IV. EXPERIMENTAL 1. General Methods Proton magnetic resonance spectra were determined on a Varian T-60, HA-100 or XL-100 spectrometer. Absorbtions are given in T units -with tetramethylsilane as internal standard (set at 10). The following abbreviations are used in describing nmr spectra: (d) = doublet, (s) = singlet, (t) = t r i p l e t , (q) = quartet, (m) = multiplet. Mass spectra were recorded on an A . E . I . MS9 spectrometer. Optical rotations were measured with a Perkin Elmer model 141 automatic polarimeter. Infrared spectra (ir) were recorded on a Perkin Elmer model 137 spectrometer. Ultraviolet spectra (uv) were recorded on a Unicam SP 800 spectrometer. Melting points were determined on a Lietz Microscope heating stage model 350, and are corrected. Elemental analyses were performed by Mr. P. Borda, Department of Chemistry, University of Br i t i sh Columbia. 2. Chromatography 2.1 Column Chromatography S i l i c a gel column chromatography was performed using s i l i c a gel for t i c (Merck, s i l i c a gel H). If not stated the ratio of material to - 111 -absorbent was approximately 1:100 and the ratio of column length to diameter was approximately 10:1. Columns were pressurized above the solvent reservior to a pressure of 5-10 psi. 2.2 Thin Layer Chromatography A l l thin layer chromatography was performed using s i l i c a gel for t i c (D-0, Mondray Ltd.), containing 1% electronic phosphor. Compounds were detected by ultraviolet absorption, by spraying with 50% sulfuric acid followed by heating on a hot plate, or by spraying with a 0.3% solution of ninhydrin in n-butanol followed by warming at 110° in an oven. 3. Azlactone Condensation: General Considerations The solvents; either tetrahydrofuran or dimethoxyethane, were freshly d i s t i l l e d from lithium aluminum hydride to ensure dryness and freedom from peroxides. During the condensation reaction the o refluxing solvent was d i s t i l l e d into a bed of molecular sieves 3 A then returned to the reaction flask. This was achieved using an addition funnel, with a side arm for vapour transport, f i t t e d with a reflux condenser on top. The funnel was f i l l e d with molecular sieves to a volume equalling half of the reaction volume and the whole apparatus was flushed with nitrogen. 4. Anhydrous Diazomethane Generation 204 The diazomethane was made according to Vogel; but with the following exceptions: 1) a d i s t i l l i n g column O 10 cm x 1 cm), packed with sodium hydroxide pellets, was placed between the reaction - 112 -flask and the condenser ( i i ) . The collecting flasks contained anhydrous ether. 2-Phenyl-5(4)oxazolinone (89) Compound 89_ was prepared using a modification of the method of 95 Crawford and L i t t l e . Hippuric acid (20 g) and acetic anhydride (200 ml) were heated at 70° with s t i r r i n g until the solution cleared. Heating was continued for 60 minutes. The solution was then concentrated under reduced pressure at less than 50° to a dark red syrup. Then xylene (100 ml) was added and the solution reconcentrated to remove traces of acetic anhydride. The syrup was dissolved in benzene (200 ml) and the benzene solution washed with 1% aqueous sodium bicarbonate solution (2 x 100 ml) and water (2 x 100 ml). The benzene layer was dried over anhydrous sodium sulfate and concentrated to a .volume of 40 ml whereupon 2-phenyl-5(4)-oxazolinone (89) crystallized (11 g, 62%). A further 4 g of product could be obtained from the mother liquor to 95 give a total yield of 15 g, 84%; m.p. 85-86° ( l i t . m.p. 86°). 1,2:5,6-di-0-isopropylidene-q-D-ribo-hexofuranos-3-ulose (8) To a solution of 1,2:5, 6-di-0-isopropyl idene-a-rj-gluco-furanose*^ (141, 20 g) in carbon tetrachloride (100 ml) and water (100 ml) containing sodium hydrogen carbonate (1 g) and ruthenium dioxide (hydrate, 0.2 g), was added a 5% solution of sodium metaperiodate with vigorous s t i r r i n g . The metaperiodate was added very slowly (1 ml every 10 minutes) unt i l the characteristic yellow-green colouration - 113 -of ruthenium tetroxide was observed. When the solution had completely-reverted to the black, dioxide stage the addition was repeated as above. After one hour the rate of addition was increased to 1 ml per minute and after 4 h the solution could be stirred with an excess of periodate present. The pH must be maintained at greater than 6 by the addition of sodium hydrogen carbonate. After 10-20 h the reaction was complete as evidenced by t i c on s i l i c a gel eluted with 95:5 dichloro-methane-ethyl acetate. Excess ruthenium tetroxide was decomposed by the addition of isopropanol (1 ml). The reaction mixture was fi l t e r e d and the carbon tetrachloride layer separated from the aqueous solution which was extracted with chloroform (10 x 100 ml). The combined organic solutions were washed with 5% sodium thiosulfate solution (50 ml), dried over anhydrous sodium sulfate, f i l t e r e d and evaporated to afford crystalline 8^  (19.5 g, 97%). The ketose hydrate was recrystallized from hexanes; m.p. 109-110°; ( l i t . 2 ( ^ 109-111°C). Immediately before use, the anhydrous ketose was prepared by azeo-tropically removing water with toluene. (E)- and (Z)-2-Phenyl-4-(1,2:5,6-di-O-isopropylidene-a-D-ribo-hexo-furanos-3-ylidene)-5(4)-oxazolone (143) and (144) The keto-sugar (8) (4 g) and 2-phenyl-5(4)-oxazolone (89) (2.1 g) were refluxed in dry dimethoxyethane (100 ml) under a nitrogen atmosphere in the presence of lead diacetate (0.5 g). The apparatus was as described previously under general considerations. After 24 h, the starting material was completely consumed as evidenced by t i c (benzene-ethyl acetate (80:20)). The solvent was then removed by - 114 -d i s t i l l a t i o n under reduced pressure and the residue dissolved in benzene (100 ml) and extracted with water (2 x 50 ml) to remove the lead acetate. The benzene layer was then dried over anhydrous sodium sulfate and condensed to a thick, red syrup which was chromatographed on a 400 g s i l i c a gel column with benzene-ethyl acetate (80:20) as the eluting solvent. The f i r s t band which absorbed uv but did not char was decomposed oxazolone. The second band which absorbed uv and charred proved to be a mixture of the adducts (145 and 144) obtained as a clear syrup 4.5 g (75%); R f 0.35 [benzene-ethyl acetate (80:20)]; CDC1 T 3 1.8-2.6 (complex, 5, aromatic r ing) , 3.91 (d, 1, ^ 4.1 Hz, H- l ) , 4.23-4.60 (complex, 2, H-2, and H-4), 5.1-5.4 (complex, 1, H-5), 5.96-6.15 (complex, 2, J 5 & 6 Hz, H-6), 8.51-8.92 (3s, 12, CH 3 ) ; i r (nujol) 1830 cm"1 (C=0 5(4)-oxazolone), 1705 cnf 1 (C=N 5(4)-oxazolone). (E)-2-Phenyl-4-(1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5-oxazolone (143) The keto-sugar (8) (4 g) and 2-phenyl-5(4)-oxazolone (89) (2.2 g) were refluxed in dry tetrahydrofuran (100 ml) under a nitrogen atmosphere in the presence of lead diacetate (0.5 g). After 96 h, the reaction was complete according to t i c [benzene-ethyl acetate (80:20)]. The reaction was worked up exactly as for the reaction in dimethoxy-ethane. The product was shown, by subsequent chemical transformation, 22 to be the pure _E isomer (143), a white syrup (4.1 g, 69%); [a]Q +114.7 CDC1 ( £ 0 . 1 5 chloroform); x 3 1.9-2.6 (complex, 5, aromatic r ing) , 3.91 (d, 1, J 4.1 Hz, H- l ) , 4.25-4.60 (complex, 2, H-2 and H-4), l , z 5.1-5.4 (complex, 1, H-5), 5.76-6.1 (complex, 2, d, J 6 Hz, H-6), 8.51-8.92 (3s, 12, CHj). Anal. Calc. for C 2 H N0?: C, 62.81; H, 5.78; N, 3.49. Found: C, 62.42; H, 5.69; N, 3.20. (E)-Methyl-N-benzamido-a-(1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)glycinate (145) The azlactone (143) (2 g) was dissolved in methanol (50 ml) and sodium acetate (500 mg) was added. After 24 h at room temperature, the reaction mixture was concentrated to a yellow solid which was dissolved in benzene (100ml). The benzene solution was washed with water (3 x 50 ml), dried over anhydrous sodium sulfate and concentrated 22 to a clear syrup (1.8 g, 90%); [a]^ +85.1° (c 0.15, chloroform); CDC1 T 3 2.01-2.69 (complex, 5, benzamide), 0.35 (s, 1, exchanges with D20, N-H), 4.12 (d, 1, J1 2 3.6 Hz, H-l), 4.58 (d, 1, J 2 1 3.6 Hz, H-2), 5.22-6.02 (complex, 3, H-4, H-5, H-6), 6.14 (s, 3, ester), 8.32-8.95 (4s, 12, CH3); i r (nujol) 1730 cm"1 (C=0 ester), 1650 cm"1 (C=0 benzamide). Anal. Calc. for C__H__N0o: C, 60.96; H, 6.28; N, 3.23. Found: ZZ ZI o C, 60.83; H, 6.32; N, 3.12. (E)- and (Z)-Methyl-N-benzamido-a-(1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)glycinate (145) and (146) The mixture of azlactones (143) and (144) underwent methanolysis under the same conditions as pure 143. Upon workup an inseparable mi xture of unsaturated esters (145) and (146) was obtained as a clear rnn syrup; x 3 2.01-2.71 (complex 5, benzamide), 0.4 (broad s, 1, N-H), - 116 -4.12 (d, 1, J 1 2 3.6 Hz, H-l), 4.60 (d, 1, J 2 1 3.6 Hz, H-2), 5.1 (complex, H-4), 5.61-6.02 (complex, 2, H-5, H-6), 6.14, 6.18 (2s, 3, ester), 8.32-8.95 (complex, 12 CH 3); i r (nujol) 1730 cm"1 (OO ester), 1650 cm 1 (OO benzamide) . Methyl p-2-(3-deoxy-l,2:5,6-di-O-isopropylidene-a-g-allofuranos-3-yl)-benzoylglycinate (147) To a suspension of 5% palladium on carbon (0.10 g) in methanol (100 ml) was added 145 (1.1 g) and the reaction stirred under 1 atmosphere of hydrogen at room temperature until hydrogen uptake ceased (26 h). F i l t r a t i o n and evaporation of the mixture afforded a colourless syrup which was crystallized from ethanol-hexanes to yield compound 147 (0.86 g). Recrystallization of the mother liquor yielded an additional 0.20 g of material to give a total yield of 97% of 22 cnn compound 147; m.p. 139-141°; [a] +26.2° (£0.5, chloroform); x 3 2.02-2.65 (complex, 5, benzamide), 4.20 (d, 1, J 3.9 Hz, H-l), 4.50 (d, d, 1, J l f N H 4.0 Hz, H-l'), 5.24 (d.d, 1, J 2 3 4.3 Hz, 2 3.9 Hz, H-2), 5.68-5.0 (m, 3, H-4, H-5, H-6), 6.22 (s, 3, ester), 7.2-7.6 (m, 1, H-3), 8.41 and 8.68 ( 2 s , 12 CH 3). Anal. Calc. for C22H29N08: C, 60.68; H, 6.71; N, 3.22. Found: C, 60.73; H, 6.65; N, 3.00. Methyl L-2-(3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-allofuranose-3-yl)-N-benzoylglycinate (148) To a suspension of 5% palladium on carbon (0.20 g) in methanol (150 ml) was added a mixture of unsaturated esters (145) and (146) - 117 -(2.0 g) and the reaction stirred under 1 atmosphere of hydrogen at room temperature until hydrogen uptake ceased (44 h). Fi l t r a t i o n and evaporation of the mixture afforded a colourless syrup which was chromatographed on s i l i c a gel (100 g) with benzene-ethyl acetate (80:20) as eluting solvent. The f i r s t band afforded the D-amino acid sugar (147) (1.1 g, 54%) which was recrystallized from ethanol-hexanes. The second band yielded the L-amino acid sugar (148) (0.90 g, 44%), 22 which also crystallized from ethanol-hexanes; m.p. 120-122°; CDC1 +40° (c 0.5, chloroform); x 3 2.05-2.69 (complex, 5, benzamide), 4.24 (d, 1, J1 2 3.7 Hz, H-l), 4.81 (d.d, 1, J^, N H = 3 5.8 Hz, H- l 1 ) , 5.28 (d.d, 1, J 4.0 Hz, J 3.7 Hz, H-2), 5.58-6.10 (m, 3, H-4, H-5, H-6), 6.21 (s, 3, CH3 of ester), 7.18-7.55 (m, 1, H-3), 8.54 and 8.70 (2s, 12, CH 3). Anal. Calc. for C ^ H ^ N C y C, 60.68; H, 6.71; N, 3.22. Found: C, 60.75; H, 6.71; N, 3.16. The amino-acid sugars (147) and (148) could also be obtained in a "one-pot" reaction from the branched-chain oxazolones (143) and (144) . Thus, a mixture of L43 and 144 (0.50 g) in methanol (100 ml) along with sodium acetate (0.10 g) were hydrogenated in the presence of 5% palladium on carbon (50 mg) for a period of 48 h. Fi l t r a t i o n and evaporation followed by s i l i c a gel column chromatography of the product [benzene-ethyl acetate (80:20)] afforded the two branched-chain amino acid sugar derivatives (147) and (148) in a combined yield of - 118 -Conversion of Methyl g-2-(3-deoxy-1,2:5,6-di-O-isopropylidene-a-Q-59 allofuranos-3-yl)glycinate (49) to Methyl D-2-(3-deoxy-1,2:5,6-di-0_-isopropylidene-a-g-allofuranos-3-yl)-N-benzoylglycinate (147) 59 To compound 49_ (6 mg) in methanol (0.5 ml) was added benzoic anhydride (6 mg). The reaction was s t i rred at room temperature for 3 h then evaporated to dryness to afford a clear syrup which was crysta l l ized (ethanol-hexanes) (4 mg, 48%); m.p. 141-143° (m.p. of 147, 1 3 9 - 1 4 1 ° ) ; R f 0.15, benzene-ethyl acetate (80:20) [R f 147 0.15, CDC1 benzene-ethyl acetate (80:20)]; x 3 same as compound 147; i r (nujol) same as compound 147. Conversion of Methyl L-2-(3-deoxy-l,2:5,6-di-O-isopropylidene-a-D-allofuranos-3-yl)glycinate (51)^* to Methyl L-2-(3-deoxy-l,2:5,6-di-0_-isopropylidene-a-0-allofuranos-3-yl) -N-benzoylglycinate (148) To compound 51^* (5 mg) in methanol (0.5 ml) was added benzoic anhydride (5 mg). The reaction was s t i rred at room temperature for 5 h then evaporated to dryness to afford a clear syrup which was crysta l l ized (ethanol-hexanes) (3 mg, 45%), m.p. 122-125° (m.p. 148 1 2 0 - 1 2 2 ° ) ; R f 0.13, benzene-ethyl acetate (80:20) [R f 148 0.13, CDC1 benzene-ethyl acetate (80:20)]; x 3 same as compound 148). Attempted Base Hydrolysis of the Branched-Chain Amino Acid Sugar N-Benzoates 147 and 148 Sodium Hydroxide Hydrolysis in Water The D-amino acid sugar (147) (40 mg) was dissolved in water (2 ml) - 119 -and sodium hydroxide (200 mg) was added. The reaction was stirred and heated at 80° for 2 days. Neutralization and t i c of an aliquot of the product [ethyl acetate-ethanol (5:1)] indicated that no amide hydrolysis had occurred as there was no ninhydrin positive spot. The L-amino acid sugar (148) (15 mg) also was resistant to hydrolysis under the same conditions. Sodium Hydroxide Hydrolysis in Dimethylsulfoxide The n-amino acid sugar (147) (20 mg) was dissolved in a suspension of finely divided sodium hydroxide (100 mg) in dimethylsulfoxide (1 ml). The solution was heated to 80° with s t i r r i n g for 2 days. Fi l t r a t i o n of the sodium hydroxide and neutralization with 1% ^2^4 f°ll° w ed by evaporation gave a brown solid. Thin layer chromatography with ethyl acetate-ethanol (5:1) gave a negative ninhydrin reaction. Barium Hydroxide Hydrolysis A solution of 147 (53 mg) in methanol (1 ml) and 0.36 N aqueous barium hydroxide (2.4 ml) was stirred at 39° for 3 days. The solution was neutralized with 1 N sulfuric acid and kept at pH 4.5-5.5 for 24 h. The barium sulfate was removed by f i l t r a t i o n and the solution concentra-ted under reduced pressure to a solid (59 mg). Thin layer chromatography [ethyl acetate-isopropanol (3:1)] showed a spot which chars (R^ . 0.48) but gave no ninhydrin reaction. The solid material was then dissolved in 0.36 N barium hydroxide (10 ml) and the solution was refluxed for 3 days. Workup followed by t i c (ninhydrin test) indicated that no amide hydrolysis had occurred. - 120 -Attempted Enzyme Hydrolysis of Methyl-L-2-(3-deoxy-l,2:5,6-di-0-isopropylidene-a-D-allofuranos-3-yl)-N-benzoylglycinate (148) with Hog Kidney Acylase As a control hog kidney acylase (I.C.N. Pharmaceuticals) was used to hydrolyze N-benzoylglycine. Thus hog kidney acylase (2.0 mg) was added to a solution of N-benzoylglycine (10 mg) in water (2 ml) which was ad-justed to pH 7.5 by addition of 1% ammonium hydroxide. The reaction was l e f t at 37° for 16 h whereupon t i c [ethyl acetate-ethanol-water (2:1:1)] indicated a ninhydrin positive product and a large amount of starting material (iodine absorption). A further 2.0 mg of hog kidney acylase was added and the mixture was allowed to react for 24 h. The mixture was then evaporated to a solid which was chromatographed on a charcoal-Celite (3:1 wt/wt) column (0.5 g) under pressure (9 psi) with 5% aqueous methanol as the eluting solvent. The free amino acid, glycine, (2.2 mg) was eluted shortly after the solvent front. After changing the solvent to ethanol (95%) the unreacted N-benzoylglycine (3.0 mg) was eluted from the column. To a solution of 148 (4.0 mg) in methanol (0.5 ml) was added 2.5% aqueous methanolic sodium hydroxide (0.5 ml) and the solution stirred for 20 minutes. The solution was then stirred with Rexyn RG 51 (H ) (Polystyrene Carboxylic Acid Type Resin) (1 ml). Fi l t r a t i o n and evaporation of the product afforded a hard glass (3.5 mg). This deesterified L-amino acid sugar was then treated with hog kidney acylase under the conditions described above. There were no ninhydrin positive fractions obtained from the charcoal-celite column though - 121 -1.8 mg of material which charred on t i c and had the same [0.5, pyridine-ethanol-water (1:2:1)] as the free acid, starting material was recovered. Methyl 4,6-0-benzylidene-2-deoxy-a-D-ribo-hexopyranoside (152) To a solution of methyl 2,5-anhydro-4,6-0-benzylidene-q-D-allo-172 pyranoside (151, 10 g) in anhydrous tetrahydrofuran (280 ml) was added lithium aluminum hydride (6 g) slowly. After the reaction mixture was refluxed for 20 h, the excess lithium aluminum hydride was decomposed by the careful addition of sodium sulfate -decahydrate (10 g) with the temperature maintained below 30°. After s t i r r i n g for 6 h, the solids were removed by f i l t r a t i o n and washed with chloroform (2 x 200 ml). The combined f i l t r a t e was washed twice with water (200 ml), dried with sodium sulfate, and evaporated under reduced 17° pressure to give crystalline 152 (10 g, 100%); m.p. 125-126° ( l i t . m.p. 125-127°). Methyl 4,6-0-benzylidene-2-deoxy-a-D-erythro-hexopyranosid-3-ulose (153) To a vigorously stirred solution of 152 (10 g), sodium hydrogen carbonate (2 g), and ruthenium dioxide (0.17 g) in carbon tetrachloride (50 ml), chloroform (50 ml), and water (50 ml) was added, portionwise, a solution of 5% sodium metaperiodate. After the f i r s t portion (5 ml) was added the solution was stirred for 1 h. Each portion thereafter was added until the characteristic yellow-green colour of the ruthenium tetraoxide had formed. The solution was stirred vigorously until • the colour was discharged to give rise to black ruthenium dioxide. - 122 -The next aliquot of 5% metaperiodate solution was added and the procedure continued until a l l 152 had been converted to 153 as evidenced by t i c [benzene-ethyl acetate (97:3); 152 0.30 and 153 R^  0.40]. Filt r a t i o n and extraction of the solution with chloroform (4 x 200 ml) followed by washing of the chloroform extracts with 5% sodium thiosulfate (1 x 100 ml) afforded 153 (6.3 g, 64%) after drying with anhydrous sodium sulfate and evaporation of solvent under reduced pressure; m.p. 176-177° ( l i t . 1 7 3 m.p. 176-177°). (E)- and (Z)-2-Phenyl-4-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) The keto-sugar (153) (2.6 g) , which had been dried by co-d i s t i l l a t i o n of toluene, and 2-phenyl-5(4)-oxazolinone (89) (2.5 g) were dissolved in anhydrous dimethoxyethane (500 ml). Lead diacetate (2.0 g) was added and the solution refluxed under an atmosphere of nitrogen. The apparatus was as described previously under general considerations. After 2 h, the starting material was consumed as evidenced by t i c [benzene-ethyl acetate (95:5); R^  153, 0.40] and two new compounds appeared (R^ 0.61 and 0.53). The reaction was stopped and xylene (100 ml) was added. The mixture was concentrated under reduced pressure to a volume of 100 ml. Benzene (200 ml) was added and the solution washed with water (100 ml) followed by 10% aqueous sodium hydrogen carbonate (2 x 100 ml) and water (100 ml). After dryi over anhydrous sodium sulfate and evaporation to a solid, the crude mixture was subjected to column chromatography on s i l i c a gel (200 g) with benzene-ethyl acetate (97:3). The f i r s t component to be eluted, - 123 -which charred, was 154 (0.60 g, 13%). The second c h a r r i n g component was 155 (1.3 g, 27%). Compound 155 was r e c r y s t a l l i z e d from ether; m.p. 227-228°; 25 [ a ] Q +88.2 (c 1.6, chloroform); R f 0.61 [benzene-ethyl acetate CDC1 (95:5)]; T 3 1.8-2.5 (complex, 10, ar o m a t i c ) , 4.12 (s, 1, be n z y l i d e n e ) , 4.96 (d.d, 1, J , - = J , ~ 4.0 Hz, H - l ) , 4.2-5.0 (complex, 4, H-4, H-5, H-6), 5.50 (s, 3, CHj) , 6.28, 6.44 (2d, 2, 2 & = J J 2 g 4.0 Hz, H-2 , H-2 ). Anal. Calc. f o r C__H_.N0..: C, 67.81; H, 5.20; N, 3.44. Found: 16 z i 6 C, 67.50; H, 5.40; N, 3.27. 25 Compound 154 was r e c r y s t a l l i z e d from ether; m.p. 200-201°; [ a ] ^ -193° (£0.6 chloroform); R f 0.53 [benzene-ethyl acetate (95:5)]; CDC1 x 3 1.9-2.7 (complex, 10, aromatic), 4.19 (s, 1, b e n z y l i d e n e ) , 5.10 ( t , 1, J l j 2 a = J 4.8 Hz, H - l ) , 5.30 (d.d, 1, J 1.0 Hz, 8.5 Hz, H-4), 5.48-6.15 (complex, 3, H-5, H-6), 6.59 (s, 3, CH,,) , 6.72, 6.90 (2d.d, 2, ^  2 a = J . 2 g 4.8 Hz, J 2 4 1.0, H-2^ H-2 g). A n a l . Calc. f o r C--H--N0,: C, 67.81; H, 5.20; N, 3.44. Found: ZJ> z 1 o C, 67.68; H, 5.16; N, 3.25. Z-Methyl-N-benzamido-a-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-Q-eryt h r o - h e x o p y r a n o s - 3 - y l i d e n e ) g l y c i n a t e (157) Compound 155 (0.96 g) and sodium acetate (50 mg) were d i s s o l v e d i n r e f l u x i n g methanol (150 ml) and the s o l u t i o n was kept at 50° f o r 3 h. The r e a c t i o n mixture was cooled to a f f o r d c r y s t a l l i n e 157 (0.85 g). Concentration o f the mother l i q u o r t o a volume of 50 ml gave a f u r t h e r 0.14 g of product f o r a y i e l d of 100% of 157; m.p. - 124 -25 rnn 183-184°; [ a ] D +38.1° (c 0.5, chloroform); x 3 0.50 (s, 1, exchanges in D20, N-H), 2.5-3.3 (complex, 10, aromatic), 4.48 (s, 1, benzylidene), 5.32 (m, 1, J 3.8 Hz, J 0.8 Hz, H-l), 5.5-6.1 1 y «- 3- JL y ^6 (complex, 4, H-4, H-5,2H-6), 6.18 (s, 3, CH3 ester), 6.72 (s, 3, CH3 ether), 7.09 (m, 1, J. _ 0.8 Hz, J_ „ 15 Hz, H-2 J , 7.54 (m, 1, J l , 2 a 3 - 8 H z ' J 2 e , 2 a l 5 H z > H"V ' Anal. Calc. for C^H^NOy C, 65.59; H, 5.73; N, 3.19. Found: C, 65.38; H, 5.50; N, 3.21. E-Methyl-N-benzamido-a- (methyl-4,6-0_-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)glycinate (156) Compound 154 (0.32 g) and sodium acetate (20 mg) were dissolved in refluxing methanol (50 ml) and the solution was kept at 60° for 3 h. When the mixture was cooled,crystalline 156 precipitated (0.33 g, 100%); 25 rnn m.p. 220-221°; [ a ] D +185° (c 0.3, chloroform); x 3 2.1-2.7 (complex, 10, aromatic), 4.40 (s, 1, benzylidene), 5.16 (t, 1, J 0 = JL y Z3-J1 2 e 3.7 Hz, H-l), 5.4-6.4 (complex, 4, H-4, H-5, 2H-6), 6.62 (2s, 6, CH3 ester and ether), 7.32 (m, 2, ABX system, J x 2 3.7 Hz, J 2 & 2e 15 Hz, H-2 , H-2 ) ' e' a J Anal. Calc. for C ^H^NCy C, 65.59; H, 5.73; N, 3.19. Found: C, 65.44; H, 5.74; N, 3.14. Methyl-D-2-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-arabino-hexopyranos-3-yl)-N-benzoylglycinate (166) Compound 156 (150 mg) was hydrogenated in ethanol (100 ml) with 5% rhodium on powdered alumina (80 mg) at 5 atm and 35° for 12 h. - 125 -Filt r a t i o n and evaporation of the reaction mixture afforded a white solid which was chromatographed on s i l i c a gel (15 g) with hexanes-acetone (4:1). A minor amount of high material (15 mg, 9%) 25 corresponding to compound 166 was obtained; m.p. 186-188°; [ a]p +62.4° mn (£0.2, chloroform); x 3 2.19 (broads, 1, NH-amide), 2.5-3.1 (complex, 10, aromatic), 4.51 (s, 1, benzylidene), 4.89 (d.d, 1, J„ 4 10 Hz, J 4 5 4.8 Hz, H-4), 5.36 (d.d., 1, J. _ & 3.8 Hz, J. 2 g 2.4 Hz, H-l), 5.6-6.6 (complex, 4, H-2', H-5, 2H-6), 6.31 (s, 3, CR. ester), 6.71 (s, 3, CH3 ether), 6.3 (m, 1, H-3), 7.02 (m, 1, J. _ a 3.8 Hz, J _ a j 3 5.7 Hz, H-2a), 7.28 (m, 1, H-2 g). Anal. Calc. for C24H_ NO : C, 65.29; H, 6.17; N, 3.17. Found: C, 65.14; H, 6.53; N, 3.23. Methyl-D-2-(methyl-4,6-0-cyclohexylmethylidene-2,3-dideoxy-q-Q-arabino-hexopyranos-3-yl)-N-cyclohexylcarboxylglycinate (167) The major component from the reduction of 156 was compound 167 (115 mg, 71%) which was recrystallized from acetone-hexanes; m.p. 141-25 rnn 142°; [a] +63.2° ( c _ l , chloroform); x 3 2.85 (broad s, 1, NH-amide), 5.12 (q, 1, J 4 3 9.8 Hz, J 4 _ 5.3 Hz, H-4), 5.36 (t, 1, J. 2_ = J - 3.8 Hz, H-l), 5.76 (d, 1, J 4.2 Hz, methylidene), 5.8-6.7 (complex, 4), 6.28 (s, 3, CH_ ester), 6.77 (s, 3, CH3 ether), 7.38 (m, 1, J 3.8 Hz, J_ 11.0 Hz, J_ 9.8 Hz, H-3), 8.0-9.0 (complex, 24, H-2 , H-2 , cyclohexyl). Irradiation at T 7.38 produced a doublet at a e x 5.12. Irradiation at x 8.1 produced a singlet at x 5.36 and a singlet at x 5.76. - 126 -Anal. Calc. for C_ H gN0 : C, 63.55; H, 8.67; N, 3.09. Found: C, 63.28; H, 8.79; N, 3.06. Methyl-D-2-(methyl-4,6-0-eyelohexylmethy1idene-2,3-dideoxy-a-D-ribo-hexopyranos-3-yl)-N-cyclohexylcarboxylglycinate (170) Compound 157 (150 mg) was hydrogenated in ethanol (100 ml) with 5% rhodium on powdered alumina (80 mg) at 5 atm. and 40° for 36 h. Fil t r a t i o n and evaporation of. the reaction mixture afforded 170 as a solid which was recrystallized from acetone-hexanes (110 mg, 68%); 25 m.p. 127-130°; [ a ] n +57.7° (c 0.7, chloroform); T 5.35 (d.d, 1, J 4.5 Hz, J 1 _ e 1.8 Hz, H-l), 5.63 (d, 1, J 4.6 Hz, methylidene), 5.69 (d, 1, J 4.1 Hz), 5.8-6.65 (complex, 4, H-4, H-5, 2H-6), 6.22 z , J (s, 3, CH„ ester), 6.71 (s, 3, CH_ ether), 7.23 (d.d, 1, J 2 q 1.8 Hz, J2e,2a 1 4 " 5 H z ' H" 2 e )> 7 " 6 7 ( 4 d ' J l f 2 a 4 " 5 "Z> J2e,2a 1 4 " 5 H z> J2a,3 0.9 Hz, H-2a), 8.0-9.0 (complex, 23, H-3 cyclohexyl); i r (CHC1.-) 1739 cm - 1 (C=0 ester), 1680 cm"1 (C=0 benzamide). Anal. Calc. for C_.H_oN0_: C, 63.55; H, 8.67; N, 3.09. Found: C, 63.54; H, 8.50; N, 3.02. Column chromatography of the mother liquor with acetone-hexanes (1:4) gave additional f u l l y reduced material and a small amount of r compound in which only the phenyl moiety of the benzamide was reduced. CDC1 This was evidenced by i t s pmr spectrum; x 3 1.18 (broad s, 1, N-H), 2.4-2.6 (complex, 5, benzylidene), 4.30 (s, 1, benzylidene), 5.18 (d.d, 1, J. 2 a 4.2 Hz, 3l 2_.1.5 Hz, H-l), 5.4-6.2 (complex,4, H-4, H-5, 2H-6), 6.12 (s, 3, CH- ester), 6.57 (s, 3, CH- ether), 7.07 (d-d, 1, - 127- -J, _ 1.5 Hz, J . _ 15.8 Hz, H-2e), 7.42 (d.d, 1, _ 4.2 Hz, L,Ze ze,za l,za J 0 „ 15.8 Hz, H-2a), 8.1-9.1 (complex, 11, cyclohexylcarbonyl). Attempted Barium Hydroxide Hydrolysis of 167 and 170 The blocked amino acids 167 and 170 (20 mg) were each subjected to hydrolysis with barium hydroxide in aqueous ethanol using the procedure applied to 184. Thin layer chromatography of the products [ethyl acetate-ethanol-acetic acid (50:10:1)] showed the free acid forms of the starting compounds as charring spots which failed to give a positive ninhydrin reaction. 3-0-Benzyl-l,2-0-isopropylidene-a-g-glucofuranose (172) To 3-0-benzyl-l,2:5,6-di-O-isopropylidene-a-D-glucofuranose (171) (4 g) in methanol (250 ml) was added 0.1 N H2SC>4 (80 ml). The reaction was stirred at 25° for 26 h. Then the solution was neutralized with sodium hydrogen carbonate and concentrated under reduced pressure to a volume of 80 ml. This solution was extracted with methylene chloride (5 x 50 ml). The combined extracts were dried over sodium sulfate and evaporated to a syrup which contained a trace of baseline material as shown by t i c [benzene-ethyl acetate (4:1)]. Therefore the product was purified by column chromatography on s i l i c a gel (100 g) with benzene-ethyl acetate (4:1) as solvent to afford compound 172 (2.6 25 g, 75%) as a clear syrup; R^  0.55 [benzene-ethyl acetate (4:1)]; [ a ] n -44.2° (c 1, chloroform); [ l i t . * 8 ' ' ' [a]iP -46° chloroform)]; - 128 -T 3 2.66 (s, 5, aromatic), 4.12 (d, 1, J 3.9 Hz, H-l), 5.3-5.5 (complex, 6, H-2, H-3, H-4, H-5, 2H-6), 5.92 (s, 2, CH2 benzyl ether), 6.28 (broad s, 2, OH-5, OH-6), 8.50, 8.68 (2s, 6, CH 3). 3-0_- Benzyl -1,2-0-isopropylidene-a-D-xylo-pentodialdo-1,4- f uranos e (173) Compound 172 (6.2 g) was dissolved in ethanol (150 ml) and saturated aqueous sodium hydrogen carbonate (15 ml). To this solution was added sodium metaperiodate (8.6 g) and the reaction stirred with the exclusion of light for 20 h at 25°. The reaction appeared to be complete by t i c [benzene-ethyl acetate (4:1) 172 = 0.55, R^  173, 0.68] so the excess periodate was consumed by the addition of ethylene glycol (2 ml). The mixture was f i l t e r e d and the inorganic salts washed with chloroform (4 x 100 ml). The f i l t r a t e was washed with water (2 x 100 ml) dried over anhydrous sodium sulfate and evaporated to 22 c o n yield syrup 173 (5.5 g, 98%); [ a ] n -36.0° (c 1, chloroform); T 3 0.38 (s, 1, -CHO), 2.72 (s, 5, benzyl ether), 3.94 (d, 1, J 2 4.0 Hz, 18 lb* 2 2 H-l), 5.3-6.0 (complex, 8), 8.58, 8.76 (2s, 6, CH 3). [Lit [ a ] Q -86.5° (c 2.7, chloroform)]. Anal. Calc. for C l cH l o0_: C, 64.74; H, 6.52. Found: C, 64.43; H, 6. lb lo b (E)- and (Z)-4- (3-0-benzy 1-5-deoxy-1, 2-0_-isopropylidene-ct-D-xylofuranos-5-ylidene)-2-phenyl-5(4)-oxazolone (174) and (175) Aldehydo sugar (173) (2.5 g) and 2-phenyl-5(4)-oxazolone (89) (2.5 g) were dissolved in peroxide-free tetrahydrofuran (250 ml) with lead diacetate (500 mg) as catalyst. The reaction mixture was refluxed - 129 -under a nitrogen atmosphere for 6 h. The same apparatus was used as described under general considerations. The reaction appeared to be ^ 75% complete as evidenced by t i c [hexanes-ether (3:1)]; however, as a great deal of baseline material was forming the reaction was stopped. Xylene (50 ml) was added and the reaction mixture was concentrated under reduced pressure to a volume of 50 ml. Benzene (200 ml) was added and the solution washed with water (2 x 100 ml), 10% aqueous sodium hydrogen carbonate (2 x 100 ml), and with water (1 x 100 ml). After drying over sodium sulfate and evaporation to a red syrup the product was purified by column chromatography on s i l i c a gel (200 g) with hexanes-ether (5:2) to yield a mixture of 174 and 175 (1.7 g, 45%) as a clear white syrup (slowly colours at room temperature). Tic using: (1) benzene-ethyl acetate 6:4 and (2) benzene-ether (6:4) CDC1 failed to resolve the components of the mixture, x 3 1.86-2.85 (complex, 10, aromatic), 3.28 (d, 1, J 7.7 Hz, H-5), 5.97 (d, 1, J1 2 3.7 Hz, H-l), 4.59 (d.d, 1, J 3 4 3.9 Hz, g 7.7 Hz, H-4), 5.38 (d, 1, J 3.7 Hz, H-2), 5.47 (d, 2, benzyl ether), 5.80 (d, 1, J 1, Z 3 , 4 3.9 Hz, H-3), 8.46, 8.68 (2s, 6, CH 3); i r (nujol) 1810 cm"1 (OO, oxazolinone), 1687 cm 1 (C=N, oxazolinone). Anal. Calc. for C_.H„„N0,: C, 68.40; H, 5.50; N, 3.32. Found: C, 68.10; H, 5.70; N, 3.00. - 1 3 0 -(E)- and (Z)-Methyl 6-N-benzamido-3-£-benzyl-5,6-dideoxy-l,2-0-isopropylidene-a-D-xylo-heptofur-5-enuronate (176) and (177) The mixture of oxazolone adducts (174) and (175) (2.0 g) was dissolved in methanol (500 ml) containing sodium acetate (1 g). After the reaction was stored for 3 days at 25°, toluene (500 ml) was added and the solution concentrated to a volume of 300 ml which was f i l t e r e d . The f i l t r a t e was washed with water (3 x 100 ml), dried over anhydrous sodium sulfate and evaporated to yield 176 and 177 as a yellow syrup (1.8 g, 85%). Tic using: (1) benzene-ethyl acetate (1:1), (2) benzene-ethyl acetate-ether (6:4:1), or (3) hexane-ether (1:2) failed to resolve CDC1 the components of the mixture, x 3 1.91 (s, 1, exchanges in D^ O, N-H), 2.2-2.9 (complex, 10, aromatic), 3.47 (d, 1, J 6.3 Hz, H-5), 4.16 (d, 1, J± 2 3.5 Hz, H-l), 5.08 (d.d, 1, 5 6.3 Hz, J 3 ^ 2.8 Hz, H-4), 5.39 (s, 2, benzyl ether), 5.43 (d, 1, J 3.5 Hz, H-2), 5.99 (d, 1, J 3 ^ 2.8 Hz, H-3), 6.22 (s, 3, CH3 ester), 8.69 (2s, 6, CH„. isopropylidene); i r (nujol) 1745 cm 1 (C=0, ester), 1690 cm 1 (C=0, benzamide). Anal. Calc. for C^H^NO^ C, 66.21; H, 6.00; N, 3.09. Found: C, 65.83; H, 6.21; N, 2.75. Methyl 6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-a-D-gluco-heptofuranuronate (178) and Methyl 6-_N-benzamido-3-0-benzyl-5,6-dideoxy-1,2-0-isopropylidene-g-^-ido-heptofuranuronate (179) The mixture of unsaturated esters (176) and (177) (1.0 g) was added to a suspension of 5% palladium on carbon (0.20 g) in methanol (100 ml). The reaction was stirred under 1 atmosphere of hydrogen at - 131 -room temperature until the reaction was complete as evidenced by t i c [benzene-ethyl acetate (1:1)]. F i l t r a t i o n and evaporation of the product afforded a white syrup which was purified by column chromatography on s i l i c a gel (100 g) using benzene-ethyl acetate (4:6) as developer. A small amount (0.16 g) of starting material was present as evidenced by t i c and pmr. The major band contained a mixture of 1_78 and 179 (0.82 g, 82%); [ a ] 2 2 mixture +21.6° (c 1.5, CDC1 chloroform); x 3 2.02-2.69 (complex, 10, aromatic), 4.08 (complex, 1, H-l), 5.10-6.18 (complex, 6, H-2, H-3, H-4, H-6, benzyl ether), 6.24 (2s, 3, CH3 ester), 7.62 (m, 2, H-5), 8.7-8.9 (complex, CH3 isopropylidene); i r (nujol) 3280 cm 1 (N-H amide), 1710 cm 1 (C=0 ester), 1630 cm 1 (benzamide). Anal. Calc. for C H2gN0 : C, 65.92; H, 6.42; N, 3.19. Found: C, 65.32; H, 6.42; N, 3.10. Methyl 6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-q-D-gluco-heptofuranuronate (180) and Methyl 6-N-benzamido-5,6-dideoxy-l,2-0_-isopropylidene-8-L-ido-heptofuranuronate (181) The mixture of 178 and 179 (0.50 g) with 5% palladium on carbon (150 mg) catalyst in methanol (50 ml) was hydrogenated at 40° with a hydrogen pressure of 5 atm. After 24 h t i c indicated that hydrogenolysis had occurred [R^ 180, 181 0.15; benzene ethyl acetate (1:1)]. The reaction mixture was f i l t e r e d and the f i l t r a t e evaporated under reduced pressure to a white syrup which was purified by column chromatography on s i l i c a gel (120 g) using benzene-ethyl acetate (1:1) as elution solvent. Starting material (100 mg) was obtained from the - 132 -60th to the 80th ml of eluent followed by a mixture of 3-hydroxy compounds 180 and 181 (0.30 g, 75%). Tic using: (1) ether and, (2) hexanes-acetone (4:1) also f a i l e d to resolve the components of the mixture. 180 and 181 were recrystallized from hexanes-acetone m n m.p. 168-171°; x 3 2.01-2.67 (complex, 5, benzamide), 4.08 (d, 1, J 2 _ 3.9 Hz, H-l), 5.12-6.17 (complex, 3), 5.42 (d, 1, 2 3.9 Hz, H-2), 6.22 (s, 3, CH„ ester), 6.70 (broad s, 1, exchanges with D20, OH), 7.60 (complex, 2, H-5a, H-5b), [8.60 (s, 2.4), 8.62 (s, 0.6), 8.72 (s, 2.4), 8.80 (s, 0.6) isopropylidene]. Anal. Calcd. for C1oHo,N0_: C, 59.14; H, 6.34; N, 3.83. Found: C, 58.S3; H, 6.38; N, 3.54. Ethyl 6-N-benzamido-5,6-dideoxy-l,2-0-isopropylidene-ct-g-gluco-heptofuranuronate (182) and Ethyl 6-N-benzamido-5,6-dideoxy-l,2-0-isopropy1idene-B-L-ido-heptofuranuronate (183) The mixture of methyl esters 180 and 181 (250 mg) was dissolved in a 0.1 N ethanolic (10 ml) solution of sodium ethoxide. After 10 min at 25° the solution was poured into water (100 ml) and the aqueous mixture extracted with chloroform (3 x 50 ml). The chloroform extracts were dried and concentrated under reduced pressure to a clear syrup which slowly crystallized. The diasteriomers 182 and 183 were separated by fractional crystallization from acetone-hexanes. Since 183 was the predominant isomer,it crystallized f i r s t (128 mg). Careful recrystallization of the mother liquor afforded compound 182 (41 mg). Thereafter both compounds were recrystallized two times. - 133 -Compound 182; m.p. 149-150°; [ a ] ^ -17.6° (c 0.4, chloroform); cnn T 3 2.1-2.9 (complex, 5, benzamide), 4.09 (d, 1, ^  - 4.1 Hz, H-l), 5.47 (d, 1, J1 2 4.1 Hz, H-2), 5.6-6.3 (complex, 6, H-3, H-4, OH-3, CH2 ester), 7.79 (2d, 2, 2H-5), 8.58, 8.72 (2s, 6, isopropylidene), 8.72 (t, 3, J 6.1 Hz, CH~ ester). Anal. Calc. for C H--N0 : C, 60.15; H, 6.64; N, 3.69. Found: C, 60.26; H, 6.59; N, 3.57. Compound 183; m.p. 184-186°; [a]^4 -10.7° (c 1, chloroform); CDC1 T 3 2.2-2.8 (complex, 5, benzamide), 4.19 (d, 1, 2 3.8 Hz, H-l), 5.2-6.0 (complex, 6, H-3, H-4, H-6, CH2 ester), 5.60 (d, 1, 2 3.8 Hz, H-2), 7.64-7.93 (complex, 2, 2H-5), 8.64, 8.79 (2s, 6, isopropylidene), 8.79 (t, 3, J 6.2 Hz, CH- ester). Anal. Calc. for C^H ^ N C y C, 60.15; H, 6.64; N, 3.69. Found: C, 59.92; H, 6.51; N, 3.73. 6-Amino-5,6-dideoxy-l,2-0-isopropylidene-g-L-ido-heptofuranuronic acid (184) The blocked amino acid sugar 185 (40 mg) was hydrolyzed in 10 ml ethanol and 10 ml 0.36 N Ba(0H)2 at 65° for 120 h. At that time t i c [ethyl acetate-ethanol-acetic acid (50:10:1)] indicated that the reaction was over 50% completed as evidenced by the formation of low material which gave a positive ninhydrin colour reaction. The solution was neutralized by careful addition of 1 N H 2S0 4 and maintained at pH 5.0-5.5 for 24 h by addition of 1 N H 2S0 4. The barium sulfate was removed by f i l t r a t i o n and the solution concentrated under reduced pressure to a volume of 2 ml. Ion exchange chromatography on Amberlite - 134 -CG 120 (100-200 mesh) in the pyridinium salt form using a 10 ml column eluted with water-methanol-pyridine (80:19:1) afforded a ninhydrin positive fraction (20 mg). Tic using: (1) ethyl acetate-ethanol-acetic acid (50:10:1) and (2) ethanol-acetic acid (99:1) gave single spots for 184 [R^ (0.08) and (0.75) respectively]. Concentration followed by crystallization from ethanol-water afforded the free amino acid 184; m.p. 106-109° (-^0), 215° (dec); [a] 2 5-22° (£0.7, ethanol); x D M S 0 _ D 6 4.29 (d, 1, J : 2 3.5 Hz, H-l), 4.64 (d, 1, 2 3.5 Hz, H-2), 5.8-6.9 (complex, 7), 6.22 (broad s, 1, 1/2 H 20), 7.88-8.38 (complex, 2, 2H-6), 8.64, 8.83 (2s, 6, CH, isopropylidene), c d . Ae- 0.60 (A o max 30 213 nm, c 0.0025, 0.5 N methanolic HCl), [ Q ] 2 U -1980°. Anal. Calc. for C 1 0H i yN0 6•(hemihydrate): C, 46.87; H, 7.08; N, 5.47. Found: C, 46.98; H, 6.93; N, 5.18. The ct-D-gluco blocked amino acid 182 was subjected to the same hydrolytic conditions as 183. However, only a"very faint ninhydrin positive baseline spot was observed on t i c [ethyl acetate-ethanol-acetic acid (50:10:1)]. Workup of the reaction with the same method as for 184 gave only the N-benzoylated acid as evidenced by pmr, x ^ 3 ^ 2.9-3.2 (complex, 5, benzamide), 4.88 (d, 1, J 3.1 Hz, H-l), 5.5-7.1 (complex),8.21, 8.30 (2s, 6, isopropylidene). Attempted Enzyme Hydrolyses of 182 and 183 with Hog Kidney Acylase I The N-benzoyl amino esters 182 and 183 were each (20 mg) treated with ammonium hydroxide (0.5 ml of 6 N NH^  aq.) in methanol (2 ml). Saponification was evidenced by a significant drop in R^  [tic (ether)]. Compound 182 was completely hydrolysed in 30 minutes. Compound 183 - 135 -required an additional 60 minutes. The reaction mixtures were condensed under reduced pressure to amorphous solids which were dissolved in water (2 ml) and incubated with hog kidney acylase I (5 mg) at pH 7.5 and 30° for 24 h. The activity of the enzyme was verified with hippuric acid as substrate. After 24 h t i c [methanol-ether-acetic acid (6:1:1)] showed a trace amount of ninhydrin positive, charring material in the reaction mixture containing 182. Fi l t r a t i o n of the mixtures through charcoal to remove the enzyme and concentration under reduced pressure afforded two solid mixtures which were each passed through columns of Bio-rad AG-1X2 (200-400 mesh) quaternary ammonium ion exchange resin. Elution with 5% acetic acid gave ninhydrin positive fractions for both 182 and 183. The pmr spectra of both ninhydrin positive components (1.0 mg and 0.8 mg respectively) failed to show the presence of the hydrolyzed sugars as evidenced by the lack of isopropylidene methyl resonances. The ninhydrin positive material was probably a portion of decomposed enzyme. (E)- and (Z)-3-Deoxy-3-C-methoxycarbonylmethylene-l,2:5,6-di-0-isopropylidene-q-D-ribo-hexofuranose (12) and (15) Anhydrous ketose (8) (20 g) was reacted with phosphonacetic acid trimethyl ester (17.5 g) and potassium t-butoxide (9.5 g) in N_,N-dimethylformamide (60 ml) at -20° for 16 h. The mixture was concentrated to a volume of 20 ml and dissolved in ether (200 ml). The ether layer was washed with water (2 x 200 ml), dried over sodium sulfate, and evaporated to afford a red syrup. The product was chromatographed . - 136 -on s i l i c a gel (800 g) using benzene-ethyl acetate (4:1) as elution solvent. The more mobile zone consisted of 1_2 and 13_ (11.0 g, 45%) 19 and has been previously characterized. The syrup was found to fractionally crystallize from hexane, yielding f i r s t the Z-isomer 1_3 (m.p. 61-63°) and then the E-isomer (12) (m.p. 67-70°). The pmr 19 spectrum of 1_3 was found to agree with the literature spectrum : rn n T 3 3.76 (d.d, 1, 2 1.25 Hz, J p 4 2.0 Hz, H-l'), 4.21 (d, 1, „ 4.0 Hz, H01), 4.29 (d.d, 1, _ 4.0 Hz, J 2 J t 1.25 Hz, H-2), 5.36 (d, 1, J 4 _ 6.25 Hz, H-4), 5.80 (m, 2, H-5, H-6), 6.27 (s, 3, CH- ester) . The pmr spectrum of 12_ w a s found to agree with the 19 CDC1 literature : x 3 3.82 (2d, 1, J^, 2 1.75 Hz, and J j , 4 1.5 Hz, H-l»), 4.10 (d, 1, J x 2 4.75 Hz, H-l), 4.94 (d.d, 1, 1 4.75 Hz, J 1.75 Hz, H-2). Treatment of (Z) 3-deoxy-3-C- (methoxycarbony 1) -methylene-1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranose (13) with Diazomethane to _ - . . _ Afford Spiro A -pyrazolines (185, 186, 187 and 188) and Spiro A -pyrazolines (189 and 190) To a solution of 13^  (2.0 g) in purified diethyl ether (4 ml) was added an anhydrous solution of diazomethane (0.42 g) in ether (12 ml). After the reaction was continued for 8 h at 5°, the ether was removed under reduced pressure. The mixture of pyrazolines must be kept under nitrogen at 0° as i t was found to.be unstable. An aliquot (450 mg) of this syrup was chromatographed on s i l i c a gel (50 g) with benzene-ethyl acetate (4:1) as elution solvent, to afford the A^-pyrazolines 2 (185, 186, 187 and 188) (85 mg, 19%) and the A -pyrazolines (189 and 190) (255 mg, 56%). - 137 -Products 185, 186, 187, and 188 were a syrup; Rf 0.24 [benzene-r n n ethyl acetate (4:1)]; T 3 4.41 (d, 1, J 3.0 Hz, H-l), 4.41-4.60 (m, 5), 5.32 (d,. k, J 3.0 Hz, H-2), 5.62-6.80 (m, 2), 6.38 (s, 3, CH3 ester), 8.38-8.80 (12, CH3 isopropylidene); i r (CDC13) 1723 cm - 1 (carbonyl); \ 298 mu ( e 798 in ether); m/e = 369. Products 189 and 190 s o l i d i f i e d from ether hexane; m.p. 78-81°; [ a ] 2 5 +149.9° (c 1, chloroform); T b e n z e n e " D 6 2.38 (s, 1, exchanges in D 20), 3.72 (d, 1, 2 3.5 Hz, H-l), 5.32 (d, 1, J 2 1 3.5 Hz, H-2), 5.60-6.20 (m), 6.30 (s, 3, CH3 ester), 8.48-8.80 (12, CH3 isopropylidene); i r (CDC13) 3460 cm"1 (NH), 1705 cm"1 (OO), and 1548 cm"1 (ON); \ 291 my (e 1050 in ether); m/e 369. max Treatment of (E)-3-deoxy-3-C- (methoxycarbonyl) -methylene-1,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranose (12) with Diazomethane The unsaturated ester 1_2 (1 g) was reacted with diazomethane (0.2 g up to 1.0 g) at -5°, 0°, 5° and 10°C for up to 18 h. There appeared to be no reaction as evidenced by t i c [benzene-ethyl acetate (4:1)]. The characteristic blue fluorescent spot on t i c (as seen with the reaction of 1_3 and diazomethane) did not appear and the starting material was isolated from the reaction (0.95 g) . Spiro-3,4 1-S-(and R)-(3,3-dideoxy-l,2:5,6-di-0-isopropylidene-a-D-ribo-hexofuranose)-3'-R-(and S)-amino-2'-pyrrolidones (192, 193, 194, and 195) The crude product mixture of pyrazolines (185-190) (1.6 g) in anhydrous methanol (40 ml) was hydrogenated with Raney nickel (50 mg) as catalyst at 2200 psi and 75-80° for 8 h. The catalyst was f i l t e r e d - 138 -off and the f i l t r a t e evaporated to afford a mixture of 192-195 as a yellow syrup. Hie mixture was column chromatographed on s i l i c a gel (42 g) using gradient elution as follows: (1) 200 ml of ethyl acetate-ethanol (8:1), (2) 200 ml of (7:1) mixture, (3) 200 ml of (5:1) mixture, (4) sufficient (3:1) mixture to elute off a l l remaining components. Compound 192 (spiro-5,4 1-S-ribo-3'-R-aminopyrrolidone) (220 mg, 16%) was crystallized from ethyl acetate-ethanol; m.p. 123-124°; 25 [a] Q +50° (£0.9, chloroform); Rf 0.30 [ethyl acetate-ethanol (5:1)]; CDC1 x 3 2.79 (s, 1, exchanges with D20, lactam N-H), 3.85 (d, 1, J 3.6 Hz, H-l), 4.82-5.18 (m), 5.25 (d, 1, J 3.6 Hz, H-2), 5.40-i , z z, 1 5.91 (m), 5.98-6.48 (2d, AB system, 2, J , 12 Hz, H-5* -H-5* ), O 3 5 b D 3. D 7.9 (s, 2, exchanges with D20, NH2), 8.17-8.60 (12, CH 3); i r (CDC13) 1720 cm"1 (lactam); m/e (m+l) = 329. Compound 193 (spiro-3,4'-S-ribo-3'-S-aminopyrrolidone ) (450 mg, 25 32%) was a syrup, [a] D +36.3° (c 1.0, chloroform); Rf 0.75 [ethyl CDC1 acetate-ethanol (5:1)]; x 3 2.71 (s, 1, exchanges with D20, lactam NH), 4.38 (d, 1, J 3.7 Hz, H-l), 5.27 (d, 1, J 3.7 Hz, H-2), i , Z Z , 1 5.74-6.41 (m), 6.43-6.95 (2d, AB system, 2, J c , , 11.5 Hz, H-5'a and b 3. j J D H-5'b), 7.71 (s, 2, exchanges with D20, NH2), 8.37-8.80 (12, CH3); i r (CDC13) 1730 cm"1 (lactam); m/e (m+l) = 329. Compound 194 (spiro-3,4'-R-ribo-3'-R-aminopyrrolidone) (185 mg, 25 14%) was a syrup, [a] Q +88.5° (£ 1.6, chloroform); Rf 0.06 [ethyl acetate-CDC1 ethanol (5:1)]; x 3 2.71 (s, 1, exchanges with D20, lactam NH), 4.38 (d, 1, J 3.7 Hz, H-l), 5.27 (d, 1, J 3.7 Hz, H-2), 5.74-6.41 (m), i , z z, i 6.43-6.95 (2d, AB system, J 11.5 Hz, H-5'a, H-5'b), 7.71 (s, 2, b 3 ) b D - 139 -exchanges with D20, NH-), 8.37-8.80 (12, CH-); i r (CDC1-) 1710 cm (lactam); m/a (m+1) = 329, (m+2) = 330. Compound 195 (spiro-3,4 1-R-ribo-3'-S-aminopyrrolidone) (250 mg, 25 rnn 18%) was a syrup; [ a ] D +55.5° (c 1.0, chloroform); x 3 2.48 (s, 1, exchanges with D\-,0, lactam NH), 4.16 (d, 1, J 3.8 Hz, H-l), 5.35 ' Z 1 y Z (d, 1, J„ 1 3.8 Hz, H-2), 5.40-6.23 (m), 6.34-7.03 (2d, AB system, J5'a 5'b 1 1 , 3 H z ' H~ 5' a> H-5'b), 7.4-8.0 (s, 2, exchanges with D20, NH2), 8.28-8.62 (12, CH_); i r (CDC1-) 1720 cm"1 (lactam); m/e (m+1) = 329, (m+2) = 330. Attempted d i s t i l l a t i o n of 192-195 gave decomposition products. Spiro-3,4'-S-(3,3-dideoxy-l,2:5,6-di-O-isopropylidene-q-p-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone (196) To 192 (100 mg) in anhydrous methanol (1 ml) was added acetic anhydride (400 mg) and the mixture was allowed to stand for 48 h at room temperature. After addition of xylene ( 2 x 1 ml), the solution was twice evaporated to dryness under vacuum, and the residue crystallized from ethyl acetate-hexane, (96 mg), m.p. 213-215°, 25 CDC1 [a] Q -6.5° (£3.6, chloroform); x 3 3.48 (d, 1, exchanges with D20, H-3' 6.0 Hz, acetamido NH), 3.97 (s, 1, exchanges with D20, lactam NH), 4.18 (d, 1, J j _ 4.4 Hz, H-l), 5.01 (d, 1, J 2 x 4.4 Hz, H-2), 5.45 (d, 1, becomes s in D„0, J M I I , „ ,, 6.0 Hz, H-3'), 5.52-2 NH ,H-3' 6.22 (m), 6.29, 6.76 (2d, AB system, J 12.1 Hz, H-5'a, H-5'b), D ct j D D 7.98 (s, 3, CH- of acetamido group), 8.42-8.92 (12, CH-) '. Anal. Calc. for C H_6N 0 : C, 55.13; H, 7.08; N, 7.56. Found: C, 54.50; H, 6.90; N, 7.22. - 140 -Spiro-3,4' -S_- (3,3-dideoxy-1,2: 5,6-di-O-isopropylidene-a -D-ribo-hexofuranose)-3'-S-acetamido-2'-pyrrolidone (197) Compound 193 was acetylated to afford the acetamidopyrrolidone 25 (197) by the same procedure described above, m.p. 217-219°, [ a ] n rnn +28.8° (c 1.0,chloroform); x 3 2.36 (d, 1, exchanges with D20, N^H H-3' 8 - 8 acetamido NH), 3.92 (s, 1, exchanges with n 2 0 , lactam NH), 4.34 (d, 2 3.1 Hz, H-l), 4.79 (d, 1, becomes s with D20, J N H H_ 3, 8.8 Hz, H-3'), 5.18 (m, 1, J.4 g 8.5 Hz, J 5 ^ 5.6 Hz, J_ 5.6 Hz, J, ,, 8.6 Hz, H-6a), 6.13 (m, 1, J r 4.5 Hz, J\ 5,6b 6a,6b 5,6b 6a,6b 8.6 Hz, H-6b), 6.24 (d, 1, J 8.5 Hz, H-4), 6.32-6.50 (m), 7.97 4,5 (s, 3, CH3 acetamido group), 8.40-8.79 (12, CH 3). Anal. Calc. for C ^ H ^ ^ G y C, 55.13; H, 7.08; N, 7.56. Found: C, 55.03; H, 7.20; N, 7.26. Spiro-3,4'-R- (3,3-dideoxy-l,2:5,6-di-O-isopropylidene-q-D-ribo-hexofuranose)-3'-R-acetamido-2'-pyrrolidone (198) Compound 194 was acetylated to yield 198 by the same procedure 25 described above, m.p. 261-263°; [ a ] n +108.5° (c_ 0.6, chloroform); CDC1 T 3 2.66 (s, 1, exchanges with D20, lactam NH), 3.09 (d, 1, exchanges with D20, \\ y 8.9 Hz, acetamido NH), 4.21 (d, 1, 2 3.0 Hz, H-l), 4.76 (d, 1, becomes s in D20, J N _ H H _ 3 , 8.9 Hz, H-3'), 5.31 (d, 1, J 3.0 Hz, H-2), 5.55-6.17 (m), 6.34, 7.07 (2d, AB system, J c , C I, 10.2 Hz, H-5'a, H-5'b), 7.99 (s, 3, CH acetamido b ct, b D o group), 8.34-8.73 (12, CH 3). - 141 -Anal. Calc. for C._Ho,N_0_: C, 55.13; H, 7.08; N, 7.56. Found: II ZD Z / C, 55.14; H, 7.16; N, 7.43. Spiro-3,4' -R- (3,3-dideoxy-l, 2 : 5, 6-di-O-isopropylidene-ct-D-ribo-hexofuranose)-3'-S-acetamido-2'-pyrrolidone (199) Compound 195 was acetylated as described above to afford 199, 25 rnn m.p. 284-286°; [a] +98.3° (c 1.4, chloroform); T 3 3.27 (s, 1, exchanges with D2O, lactam NH), 3.78 (d, 1, exchanges with D^ O, JNH H 3' 1 0 , 0 H z ' a c e t a m i d o N H)> 4 - 3 4 (d> 1> J i 2 2 , 7 H z ' H_1)' 4*67 (d, 1, becomes s in D20, J N H __, 10.0 Hz, H-3*), 5.45 (d, J 2 1 2.9 Hz, H-2), 5.68-6.37 (m), 6.61, 6.96 (2d, AB system, J , 10.9 Hz, Z> Si y O D H-5*a, H-5'b), 7.98 (s, 3, CH- acetamido group), 8.50-8.72 (12, CH ). Anal. Calc. for C^H.-N-O-: C, 55.13; H, 7.08; N, 7.56. Found: LI ZD Z / C, 55.13; H, 7.15; N, 7.44. Spiro-3,4 1-S- (3,3-dideoxy-l,2-0-isopropylidene-a-P-erythro-pentodialdo-1,4-furanose)-3'-R-acetamido-2'-pyrrolidone-3',5-R-aminal-5,11 -diacetate (202) A solution of 196 (100 mg) in 66% aqueous acetic acid (2 ml) was stirred at room temperature for 72 h, at which time t i c [ethyl acetate-ethanol (5:1)] indicated completion. Then the mixture was evaporated to dryness to yield a syrup (95 mg). After the syrup was oxidized with aqueous sodium metaperiodate (66 mg) at 10°, the solvent was removed by freeze-drying. The residue was extracted with ethyl acetate and the extract was evaporated under reduced pressure to yield a syrup - 142 -(49 mg, 53%). This product was a mixture of C-5 anomers (200). Acetylation of the mixture (50 mg) with acetic anhydride (300 mg) and pyridine (0.5 ml) required 4 days as evidenced by t i c (ethyl acetate). After removal of the solvents under reduced pressure, p-xylene ( 5 x 3 ml) was added to the residue and removed by evaporation to afford a syrup (53 mg). Column chromatography of this syrup on s i l i c a gel [30 mg, ethyl acetate-ethanol (8:1)] afforded f u l l y acetylated aminal (202) (27 mg, 42%) which was crystallized from ether-hexane, m.p. 156-157°, 25 rnn [a] +164° (c 0.5, chloroform); T 3 3.61 (s, 1, H-5), 4.18 (d, 1, J 3.6 Hz, H-l), 4.75 (s, 1, H-3'), 5.27 (d, 1, J 3.6 Hz, H-2), i , Z Z , i 5.47 (s, 1, H-4), 5.61-6.25 (2d, 2, AB system, J 12.0 Hz, H-5'a, o 3., D D H-5'b), 7.54, 7.70, 8.06 (s, 9, CH- acetate and aceto groups), 8.47, 8.68 (s, 6, CH3); m/e 382.4. Anal. Calc. for C H _ „ N „ 0 o : C, 53.40; H, 5.80; N, 7.33. Found: 1 / ZZ Z o C, 53.53; H, 5.83; N, 7.09. Spiro-3,4 1 -S_- (3,3-dideoxy-1, 2-0-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose)-3'-S-acetamido-2'-pyrrolidone (201) Pyrrolidone (197) was hydrolyzed with 66% aqueous acetic acid for 5.5 h when t i c [ethyl acetate-ethanol (5:1)] indicated that the reaction was complete. The hydrolyzed product (54 mg) was oxidized with sodium metaperiodate as described above to afford a product which CDC1 crystallized from ethanol-hexane, m.p. 200-205°; T 3 0.14 ( s , l , H-5, aldehyde), 1.81 (d, 1, exchanges with D2O, J M _ ^ 3! 9.1 Hz, acetamido NH), 2.73 (s, 1, exchanges with D 20, lactam NH), 4.21 (d, 1, ^ 2 3.1 Hz, H-l), 4.25-6.58 (m), 5.29 (d, 1, J ? 1 3.1 Hz), 2.00 (s, 3, CH-- 143 -acetamido group), 8.40-8.80 (6, CH.); m/e = 297 (m-1). Spiro-3,4 1 -S- (3,3-dideoxy-g-D-ribo-hexopyranose) -3' -S_-acetamido-2' -pyrrolidone (203) The spiro pyrrolidone derivative 197 (30 mg) was hydrolyzed in 80% aqueous trifluoroacetic acid (1 ml) at 25° for 48 h. At this time the reaction was essentially complete as evidenced by t i c [ethyl acetate-ethanol (5:1)]. The solution was concentrated under reduced pressure to a syrup. Toluene was added and the solution reconcentrated to a glass. To remove decomposed material the product was passed through a column of charcoal (0.5 cm x 4 cm) with 10% aqueous methanol. The 10th to 30th ml of eluent contained the free sugar (10 mg). This sample was further purified by dissolving in pyridine (10 ml) and f i l t r a t i o n to remove inorganic impurities. The pyridine solution was concentrated to a glass which was dissolved in ethanol (1.0 ml) and precipitated with hexanes. The product was collected by centrifugation to yield 203, (7 mg, 30%); m.p. 141-145°; [ a ] 2 5 +19.8° (£0.4, ethanol); T D M S 0 _ D 6 2.11 (s, 1, lactam NH), 2.48 (d, 1, Jm „, 9.0 Hz, acetamido NH), 4.84 (d, 1, J N H 9.0 Hz, H-3'), 5.24 (d, 1, ^ _ 8.0 Hz, H-l), 5.32 (broad, s, 4, OH), 5.6-6.8 (complex, 7), 7.76 (s, 3, CH- acetate). Anal. Calc. for C. ..H.. oN_0_: C, 45.52; H, 6.25; N, 9.65. Found: 1 1 1 o Z I C, 45.40; H, 6.35; N, 9.85. (E)-Methy1-4,6-0-benzylidene-3-C-(carbomethoxymethylene)-2,3-dideoxy-a-D-erythro-hexopyranoside (163) A solution of phosphonacetic acid trimethyl ester (6 ml) and - 144 -potassium t>butoxide (1.5 g) in anhydrous N_,N-dimethylformamide (10 ml) was added to a stirred solution cooled to -20°, of methyl-4,6-0-benzylidene-2-deoxy-q-D-erythro-hexopyranos-3-ulose (153) (2.5 g) in 30 ml of anhydrous N,N-dimethylformamide. The reaction was stirred at room temperature for 20 h and was then concentrated under reduced pressure to a volume of 10 ml. The solution was diluted to 50 ml with ether and washed with water (2 x 20 ml). Concentration of the solvent afforded crude 163 which was recrystallized from ethyl acetate-hexane (2.2 g, 88%), m.p. 127-129° ( l i t . 1 4 5 m.p. 128-129°); [ a ] 2 2 +215° (c 1.0, chloroform) [ l i t . E ~ 5 [ a ] 2 2 +213° (c 2, chloroform)]. Attempted Reaction of 163 with Diazomethane Compound 163 was reacted with diazomethane using the same procedure as was applied to compound 1_3. Evaporation of the solvent afforded pure starting material. The reaction time was increased from 8 h to 24 h and temperatures of 0°, 10° and 22° were used with no indication of any reaction product forthcoming as evidenced by the lack of the characteristic blue fluorescing material on t i c using uv light detection. 8-Chloro-L-alanine Ethyl Ester Hydrochloride (206) To a suspension of L-cystine (12 g) in ethanol (300 ml) was added thionyl chloride (26 ml). The reaction was stirred and refluxed until a l l the amino acid had dissolved (8 h). Ether (350 ml) was then added and after cooling the product (16 g) was f i l t e r e d off. This material had m.p. 186-187°; [ a ] 2 2 -47° (c 4, water), [ l i t . 1 9 4 m.p. - 145 -188°, [a] -48° (c 21, water)]. A stream of chlorine was passed into a suspension of L-cystine diethylester dihydrochloride (10 g) in purified chloroform for 1 h at 0°. The reaction was stoppered and kept at 4° for 40 h. Ether (100 ml) was added and air was bubbled through the mixture for 1 h to remove excess chlorine. The precipitate was f i l t e r e d and washed 195 with ether; yield of 206 (7 g, 61% overall); m.p. 140-141° ( l i t . m.p. 141°). Acetyl-8-chloro-L-alanine Ethyl Ester (207) To a solution of 206 (0.77 g) in ethanol (10 ml) was added 0.2 g sodium hydrogen carbonate and the mixture stirred for 15 m. Acetic anhydride (4 g) was added and the solution stirred for 20 h at room temperature. The reaction mixture was added to xylene (50 ml) and evaporated to a solid which was dissolved in methylene-chloride (50 ml). The solution was then f i l t e r e d and concentrated to a white solid (720 mg, 92%) which was recrystallized from ether-hexanes. Compound 22 207 was a white crystalline solid, m.p. 96-97°; [ct]p -15.2° (c 1, dimethylformamide); [ l i t . 1 ^ m.p. 95°; [a]j^ -14.3° (c_ 1, dimethyl-formamide) . Ethyl-N-acetyl-8-(triphenylphosphoniumiodo)-a-L-alanate (204) Compound 207 (1.9 g) and triphenyl phosphine (2.6 g) were dissolved in ethyl acetate (25 ml). The solution was refluxed so that o the condensed ethyl acetate passed through molecular sieves 4A. After 2 h sodium iodide (0.8 ml) was added and the refluxing continued for - 146 -4 h. The reaction mixture was cooled to 0° and f i l t e r e d . The solids were dissolved in ethanol (20 ml) and f i l t e r e d . Compound 204 (2.8 g, 51%) was crystallized by the addition of ether (10 ml) to the ethanol solution: m.p. 187-189°; [ a ] 2 5 -3.42° (c 2, chloroform); R f 0.48 [ methylenechloride-ether-isopropanol (10:5:1)]; ^6 1.59 (s, 1, N-H), 2.1-2.4 (m, 15, phenyl), 5.5 (m, 1, H-a), 5.8-6.2 (m, 2, H-3), 6.05 (q, 2, J 6.6 Hz, CH2 ester), 6.60 (s, 1, H 20), 8.60 (s, 3, acetate), 8.88 (t, 3, J 6.6 Hz, CH3 ester). Anal. Calc. for (monohydrate) C 2 5H 2 gIN0 4P: C, 53.11; H, 5.17; N, 2.56; I, 22.44. Found: C, 53.21; H, 5.00; N, 2.50; I, 22.15. Attempted Reaction of 207 with Triphenylphosphine Compound 207 (50 mg) in xylene (1 ml) and triphenylphosphine (75 mg) were refluxed together for 48 h. No reaction had occurred as evidenced by t i c [benzene-ethyl acetate (4:1)]. Evaporation of the solvent and triphenylphosphine afforded 207 m.p. 90-95° (recrystallized from ether-hexanes). Attempted Reaction of 207 with Sodium Iodide Compound 207 (180 mg) and sodium iodide (450 mg) were dissolved in acetone (2 ml) and the solution was refluxed for 4 h. The white precipitate (sodium chloride) was f i l t e r e d off and the solution was evaporated under reduced pressure to give a yellow solid. Recrystalliz-ation from ether-hexane gave a yellow solid (150 mg). The mother liquor (50 mg) appeared to be material which underwent elimination. The solid material appeared to be a mixture of 3-chloro- and 8-iodo - 147 -compounds (7:3) according to elemental analysis and pmr. Attempted Wittig Reaction with 204 and Benzaldehyde To 204 (0.55 g, dried by azeotfopic benzene d i s t i l l a t i o n ) in dimethoxyethane (50 ml) was added benzaldehyde ( 0 . 1 1 g) followed by n-butyl lithium in hexanes (4.4 ml of a 2.3 M solution) dropwise with s t i r r i n g . The solution was refluxed for 4 h. Evaporation of the solvent and benzaldehyde under reduced pressure gave a solid which was dissolved in benzene ( 2 0 ml). The benzene solution was washed with water ( 2 x 2 0 ml) dried over sodium sulfate and concentrated to a syrup (0.3 g). The pmr spectrum of the syrup had no aromatic C D C 1 proton signals, x 3 2 . 1 (broad s, 1, exchanges in D 2 O , NH), 4.15 (d, 2, vinyl protons), 5.76 (q, 3, methylene), 7.95 (s, 3, acetate), 8.71 (t, 4, methyl). - 148 -BIBLIOGRAPHY E. Vongerichten, Liebigs Ann. Chem., 318, 121 (1901). 0. Th. Schmidt, Liebigs Ann. Chem., 438, 115 (1930). E. Fischer and K. Freudenberg, Ber. Dtsch. Chem. Ges., 4_5, 2709 (1912). H. Grisebach and R. Schmid, Angew. Chem. Int. Ed., U, 159 (1972). J.S. Brimacombe, Angew. Chem. Int. Ed., 8_, 401. (1969). H. Umezawa: Recent Advances in Chemistry and Biochemistry of Antibiotics. Microbial Chemistry Research Foundation, Tokyo, 1964, p. 67. S. Hanessian and T.H. Haskell, in W. Pigman and D. 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