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Protein - carbohydrate interactions in glycogen phosphorylase Street, Ian Philip 1985

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PROTEIN - CARBOHYDRATE INTERACTIONS IN GLYCOGEN PHOSPHORYLASE By IAN PHILIP STREET B.Sc.(Hons), The U n i v e r s i t y of Sussex, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF CHEMISTRY Ve accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA J u l y 1985 P h i l i p S t r e e t , J u l y 1985. In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and study. I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the head o f my department or by h i s or her r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n p e r m i s s i o n . Department o f Chemistry  The U n i v e r s i t y of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date August 1985 i i ABSTRACT I t has long been observed that some o r g a n o - f l u o r i n e compounds e x h i b i t enhanced b i o l o g i c a l a c t i v i t y over t h e i r n o n - f l u o r i n a t e d p r e c u r s o r s , however reasons f o r these unusual p r o p e r t i e s s t i l l remain p o o r l y understood. An e x p l a n a t i o n which has been widely used r e l a t e s to the a b i l i t y of the C-F fragment of the analog to p a r t i c i p a t e i n hydrogen-bonding i n t e r a c t i o n s with i t s p r o t e i n r e c e p t o r . For t h i s reason, f l u o r i n a t e d carbohydrates have been used as hydrogen-bonding probes with a number of p r o t e i n s . Thus there e x i s t s a need f o r a systematic i n v e s t i g a t i o n i n t o the hydrogen-bonding a b i l i t y of the C-F fragment, and the enzyme glycogen phosphorylase provides an e x c e l l e n t s u b j e c t f o r such a study. The glucopyranose b i n d i n g s i t e i n the i n a c t i v e ( T - s t a t e ) conformation of the enzyme has been w e l l c h a r a c t e r i s e d and high r e s o l u t i o n c r y s t a l l o g r a p h i c data i s a v a i l a b l e . Thus by comparison of k i n e t i c and c r y s t a l l o g r a p h i c data f o r the n a t u r a l e f f e c t o r s and the f l u o r i n a t e d s u b s t r a t e analogs c o n s i d e r a b l e i n s i g h t i n t o the hydrogen bonding a b i l i t y of the C-F fragment and the nature of c a r b o h y d r a t e - p r o t e i n i n t e r a c t i o n s should be gained. L i t t l e i s known about the a c t i v e (R-state) conformation of the enzyme and about the T - s t a t e to R-state t r a n s i t i o n . Use of f l u o r i n a t e d analogs of the enzymes n a t u r a l s u b s t r a t e , glucose-l-phosphate, could a l s o shed l i g h t on these q u e s t i o n s . With these aims i n mind, a l l of the isomeric mono-fluorinated d e r i v a t i v e s of glucose and glucose-l-phosphate have been s y n t h e s i s e d . Some deoxy and d i f l u o r i n a t e d analogs of glucose and mannose have a l s o been prepared. K i n e t i c r e s u l t s obtained using i i i the analogs of glucose i n d i c a t e that the 3 and 6 p o s i t i o n s of the sugar p a r t i c i p a t e i n strong hydrogen-bonding i n t e r a c t i o n s with the p r o t e i n while the other p o s i t i o n s are only i n v o l v e d i n r e l a t i v e l y weak i n t e r a c t i o n s . These r e s u l t s agree w e l l with recent X-ray c r y s t a l l o g r a p h i c data. None of the analogs of glucose-l-phosphate e x h i b i t e d any s u b s t r a t e a c t i v i t y . The 2-deoxyfluoro analog had a s i m i l a r a f f i n i t y to glucose-1-phosphate and t h e r e f o r e probably binds i n the same mode. The l a c k of s u b s t r a t e a c t i v i t y i n t h i s case can be explained by the d e s t a b i 1 i s a t i o n of the p u t a t i v e oxo-carbonium io n intermediate at C ( l ) , by the adjacent f l u o r i n e s u b s t i t u e n t . The other analogs of glucose-l-phosphate showed lower a f f i n i t y f o r the enzyme. The s i m i l a r i n h i b i t i o n constants obtained f o r these compounds suggested a b i n d i n g mode i n which the glucopyranose r i n g c o n t r i b u t e s l i t t l e to the o v e r a l l b i n d i n g energy. T h i s has l e d to the proposal of a molecular mechanism f o r the T - s t a t e to R-state t r a n s i t i o n . i v Table of Contents. Page. A b s t r a c t i i L i s t of Tables v i L i s t of F i g u r e s v i i L i s t of A b b r e v i a t i o n s x Acknowledgements x i I n t r o d u c t i o n 1 (A) The l i g a n d ; The e f f e c t s of f l u o r i n e s u b s t i t u t i o n . 1 ( i ) S t e r i c e f f e c t s 2 ( i i ) E l e c t r o n i c e f f e c t s 4 ( i i i ) Conformational e f f e c t s 9 ( i v ) E f f e c t s on hydrogen-bonding 11 (B) The p r o t e i n ; Glycogen phosphorylase 14 (C) Methods of f l u o r i n a t i o n 20 (D) Summary 27 R e s u l t s and D i s c u s s i o n 29 (A) Synthesis of f l u o r i n a t e d analogs of glucose, mannose 29 and glucose-l-phosphate. ( i ) F l u o r i n a t i o n of the 6 - p o s i t i o n 29 ( i i ) F l u o r i n a t i o n of the 4 - p o s i t i o n 32 ( i i i ) F l u o r i n a t i o n of the 3 - p o s i t i o n 35 ( i v ) F l u o r i n a t i o n of the 2 - p o s i t i o n 37 V Table of Contents. Page. (B) Synthesis of deoxy analogs of glucose-l-phosphate. 4 3 and g l u c o s e . ( 1 ) Deoxygenation of the 6 - p o s i t l o n . .- 4 3 ( i l ) Deoxygenation of the 4 - p o s i t i o n 4 4 ( i i i ) Deoxygenation of the 1 - p o s i t i o n 4 5 (C) I n h i b i t i o n s t u d i e s on glycogen phosphorylase b ( T - s t a t e ) 4 6 (D) A comparison with recent c r y s t a 1 l o g r a p h i c data. 5 8 (E) I n h i b i t i o n s t u d i e s on glycogen phosphorylase b (R-state) 7 0 M a t e r i a l s and Methods 8 1 (A) S y n t h e t i c methods 8 1 (B) Enzymological methods 1 0 5 I I* Appendix 1 3r6~8-\& Appendix 2 1 0 9 B i b l i o g r a p h y 1-24-v i L i s t of Tables. Table II A comparison of s i z e f o r some f u n c t i o n a l groups attached to carbon. F l u o r i n a t i o n c o n d i t i o n s f o r the 6 - p o s i t i o n . Page 2 30 I I I F l u o r i n a t i o n c o n d i t i o n s f o r the 4 - p o s i t i o n . 33 IV F l u o r i n a t i o n c o n d i t i o n s f o r the 3 - p o s i t i o n , 36 V Chemical s h i f t data f o r the monofluorinated 39 analogs of glucose-l-phosphate. 1 1 1 19 1 31 VI H - H, H - F, H - P, c o u p l i n g constants f o r 39 the monofluorinated analogs of glucose-l-phosphate VII D i s s o c i a t i o n constants f o r the v a r i o u s T - s t a t e 52 i n h i b i t o r s of glycogen phosphorylase. V I I I D i s s o c i a t i o n constants f o r the v a r i o u s T - s t a t e 53 i n h i b i t o r s of glycogen phosphorylase. IX The hydrogen-bonding r o l e s of the g l u c o s y l 57 hydroxyl groups. X C r y s t a l l o g r a p h i c data f o r the glucose b i n d i n g 60 s i t e of phosphorylase a. XI I n h i b i t o r constants f o r the deoxyfluoro analogs of 74 glucose-l-phosphate XII Concentration data used f o r the determination of 106-107 i n h i b i t o r c o n s t a n t s . v i i L i s t of F i g u r e s . F i g u r e Page 1 The r e a c t i o n c a t a l y s e d by pyruvate k i n a s e . 2 2 The r e a c t i o n c a t a l y s e d by g l y c e r o l k i n a s e . 4 3 The r e a c t i o n c a t a l y s e d by p r e n y l t r a n s f e r a s e . 6 4 A proposed mechanism f o r p r e n y l t r a n s f e r a s e . 6 5 The e f f e c t of f l u o r i n e s u b s t i t u t i o n on the s t a b i l i t y 7 of an a l l y l i c carbonium i o n . 6 The c r y s t a l packing of 2-deoxyfluoro - /J-D-mannosy 1 12 f l u o r i d e . 7 The r e a c t i o n c a t a l y s e d by glycogen phosphorylase. 14 8 The glycogen cascade. 16 9 A schematic representatiom of the phosphorylase 18 monomer showing l i g a n d b i n d i n g s i t e s . 10 A space f i l l i n g model of the phosphorylase dimer 18 and i t s diagrammatic r e p r e s e n t a t i o n . 11 Epoxide cleavage by the f l u o r i d e i o n . 21 12 P r e p a r a t i o n of primary a l k y l f l u o r i d e s . 23 13 F l u o r i d e i o n c a t a l y s e d e l i m i n a t i o n r e a c t i o n s . 23 14 The r e a c t i o n mechanism of DAST. 26 15 F l u o r i n a t i o n of the 6 - p o s i t i o n . 29 16 F l u o r i n a t i o n of the 4 - p o s i t i o n . 32 17 F l u o r i n a t i o n of the 3 - p o s i t i o n . 35 v i i i F l u o r i n a t i o n of the 2 - p o s i t i o n . The s y n t h e t i c route f o r p r e p a r a t i o n of 6-deoxy-a-D-glucose-l-phosphate. The s y n t h e t i c route f o r p r e p a r a t i o n of 4-deoxy-a-D-glucose-l-phosphate. I n h i b i t i o n of glycogen phosphorylase b by 2-deoxyfluoro-a-D-glucosyl f l u o r i d e . I n h i b i t i o n of glycogen phosphorylase b by 2-deoxyfluoro-y3-D-glucosyl f l u o r i d e . I n h i b i t i o n of glycogen phosphorylase b by 2-deoxyfluoro-a-D-mannosy1 f l u o r i d e . I n h i b i t i o n of glycogen phosphorylase b by 4-deoxyfluoro-D-glucose. I n h i b i t i o n of glycogen phosphorylase b by 1, 5-anhydro-D-gluc i t o 1. I n h i b i t i o n of glycogen phosphorylase b by 2-deoxyfluoro-/?-D-mannosyl f l u o r i d e . I n h i b i t i o n of glycogen phosphorylase b by 2- deoxyfluoro-D-mannose. I n h i b i t i o n of glycogen phosphorylase b by a-D-mannosyl f l u o r i d e . I n h i b i t i o n of glycogen phosphorylase b by 3- deoxyfluoro-D-glucose. I n h i b i t i o n of glycogen phosphorylase b by 6-deoxyfluoro-D-glucose. I n h i b i t i o n of glycogen phosphorylase b by 1,2-dideoxy-D-glucose. A schematic r e p r e s e n t a t i o n of p o t e n t i a l donor-acceptor i n t e r a c t i o n s of glucose and phosphorylase a. The a c t i v e s i t e r e g i o n of. the phosphorylase glucose complex. The a c t i v e s i t e r e g i o n of the phosphorylase glucose complex. i x F i g u r e . 35 36 37 38 39 40 41 42 43 44 45 Page The a c t i v e s i t e r e g i o n of the phosphorylase a 69 glucose complex. I n h i b i t i o n of glycogen phosphorylase b by 71 2- deoxyfluoro-a-D-glucose-l-phosphate. I n h i b i t i o n of glycogen phosphorylase' b by 71 3- deoxyfluoro-a-D-glucose-l-phosphate. I n h i b i t i o n of glycogen phosphorylase b by 72 4- deoxyfluoro-a-D-glucose-l-phosphate. I n h i b i t i o n of glycogen phosphorylase b by 72 6-deoxyfluoro-a-D-glucose-l-phosphate. The s l i d i n g domain hyptheses of Madsen and 76 Withers. P r o d u c t i v e and unproductive b i n d i n g modes f o r 77 glucose-l-phosphate i n phosphorylase. Dependence of i n i t i a l r a t e (v) on the i n i t i a l 109 c o n c e n t r a t i o n ( [ S ] ) f o r the uncatalysed and enzyme c a t a l y s e d r e a c t i o n s ^ P-Determination of the i n h i b i t o r constant ( K i ) . 113 S a t u r a t i o n curve f o r Haemoglobin with molecular 114 oxygen. A H i l l p l o t f o r the b i n d i n g of oxygen to 115 Haemoglobin. X L i s t of A b b r e v i a t i o n s . NMR = n u c l e a r magnetic resonance s = s i n g l e t d • doublet t = t r i p l e t m = m u l t i p l e t ATP = adenosine t r i p h o s p h a t e PEP = phosphoenolpyruvate ADP = adenosine diphosphate DAST = d i a l k y l a m i n o s u l p h u r t r i f l u o r i d e DMAP = 4-(dimethyl amino)-pyridine TMP = 2,4,6-trimethyl p y r i d i n e . ABP = arabinose b i n d i n g p r o t e i n DMF = N,N-dime thylformamide AIBN = c s ( , o l ' - a z o b i s - i s o b u t y r o n i t r i l e UDP • u r i d i n e diphosphate PLPP = p y r i d o x a l (5') diphosphate A b b r e v i a t i o n s a s s o c i a t e d with amino a c i d s t r u c t u r e LEU = l e u c i n e , TYR = t y r o s i n e , ASN = asparagine GLU = glutamic a c i d , HIS = h i s t i d i n e , GLY = g l y c i n e x i Acknowledgments. I would l i k e express my g r a t i t u d e to Dr.S.G.Vithers f o r h i s support and guidance over the past three years. I would a l s o l i k e to thank Mr. C.Armstrong and the s t a f f of the NMR l a b o r a t o r y f o r t h e i r e x c e l l e n t t e c h n i c a l a s s i s t a n c e . L a s t l y I would l i k e to thank my wife C h r i s t i n e and my daughter Lucy f o r a p l e n t i f u l supply of c o f f e e and sandwiches during the p r e p a r a t i o n of t h i s manuscript. INTRODUCTION (A) The l i g a n d : E f f e c t s of f l u o r i n e s u b s t i t u t i o n . The s p e c i f i c i t y and c a t a l y t i c e f f i c i e n c y achieved by an enzyme has long been the envy of the s y n t h e t i c organic chemist. Both of these p r o p e r t i e s are e s s e n t i a l f o r the enzyme to f u n c t i o n w i t h i n a b i o l o g i c a l system and are achieved through s p e c i f i c i n t e r a c t i o n s of the enzyme and i t s s u b s t r a t e . Recently p r o t e i n -carbohydrate i n t e r a c t i o n s have become important not only to the (1) enzymologist but a l s o to the understanding of immunochemistry (2) and of the chemistry of blood group determinants .Thus the nature of these p r o t e i n - l i g a n d i n t e r a c t i o n s i s a q u e s t i o n which appears to be of growing importance i n the understanding of a l l b i o l o g i c a l mechanism. Many approaches to the study of t h i s problem have been developed. A r e l a t i v e l y r e c e n t p r o t o c o l has been to modify a s p e c i f i c group on the l i g a n d and observe how the modified l i g a n d a f f e c t s the p r o t e i n ' s a c t i v i t y . A very popular a l t e r a t i o n has been the replacement of hydrogen or a hydroxyl group with (3-5) f l u o r i n e . T h i s approach has many advantages a few of which w i l l be d i s c u s s e d i n the f o l l o w i n g t e x t . -2-( A ) ( i ) S t e r i c e f f e c t s . F l u o r i n e i s q u i t e a small element (see table I) and i t s short bond length when attached to carbon makes i t a s t e r i c a l l y c o n s e r v a t i v e replacement f o r hydrogen. GROUP BOND LENGTH VAN DER WAALS TOTAL (A) RADIUS (A) (A) C-H 1.09 1.20 2.29 C-F 1.39 1 .35 2. 74 C-O(H) 1.A3 1.40 2.83 C-OH 1.43 2 .10 3 . 53 -Table I. A comparison of s i z e f o r some f u n c t i o n a l groups attached to carbon. Data from Walsh ( r e f e r e n c e 10). However the high e l e c t r o n e g a t i v i t y of f l u o r i n e i n t r o d u c e s a p o l a r i t y more a k i n to a C-OH s u b s t i t u e n t and thus, C-F has o f t e n been used as a replacement f o r a hydroxyl group'. In p r a c t i c e an enzyme can view C-F as a C-H or C-OH e q u i v a l e n t . Pyruvate kinase c a t a l y s e s the p r o d u c t i o n of adenosine tr i p h o s p h a t e (ATP) from phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP). A D P 4. F i g u r e 1. The Reaction c a t a l y s e d by pyruvate k i n a s e . -3-The same enzyme w i l l a l s o c a t a l y s e the e n o l i s a t i o n of pyruvate i n (6) the presence of i n o r g a n i c phosphate . I f the f l u o r i n a t e d analog of PEP, 3-fluorophosphoenolpyruvate ( l ) i s presented to pyruvate (7) k i n a s e , both the E and Z isomers are converted to products (1) Likewise both the pro R- and pro S-C(3) hydrogens of 3-f l u o r o p y r u v a t e are removed by the enzyme at the same r a t e i n (8) the presence of i n o r g a n i c phosphate . I f pyruvate kinase were able to d i s t i n g u i s h C-H from C-F, c h i r a l p r o c e s s i n g of the f l u o r i n a t e d analogs would be expected. However the t o t a l l a c k of any c h i r a l r e c o g n i t i o n would suggest that the enzyme cannot d i s t i n g u i s h between C-H and C-F and t h e r e f o r e views C-F as a C-H e q u i v a l e n t . In s t u d i e s of carbohydrate metabolism C-F has o f t e n been used as (9) a s u b s t i t u t e f o r a h y d r o x y l group. E i s e n t h a l et a l . have prepared a l l three i s o m e r i c deoxyfluoro analogs of s n - g l y c e r o l and observed t h e i r e f f e c t upon g l y c e r o l k i n a s e . T h i s enzyme i s s p e c i f i c f o r the p h o s p h o r y l a t i o n of the C(3) hydroxyl group of g l y c e r o l , see f i g u r e 2. -4-F i g u r e 2. The r e a c t i o n c a t a l y s e d by g l y c e r o l k i n a s e . Both 1-deoxyfluoro and 2-deoxyfluoro analogs were phosphorylated at low r a t e s to g i v e the normal product. 3-deoxyfluoro g l y c e r o l could only a c t as an i n h i b i t o r . T h i s l a t t e r r e s u l t i s c o n s i s t e n t with the 3-deoxyfluoro analog b i n d i n g i n the normal p h o s p h o r y l a t i o n s i t e . I f g l y c e r o l kinase were to r e c o g n i s e C-F as C-H, i t would be unable to d i s t i n g u i s h between e i t h e r end of the g l y c e r o l molecule and both the 1- and 3-, 2-deoxyfluoro g l y c e r o l phosphates would be produced. Only the normal 3-phosphate was observed, t h e r e f o r e i n t h i s case the enzyme was able to d i s t i n g u i s h between C-H and C-F. G l y c e r o l kinase thus recognised C-F as an e q u i v a l e n t f o r the C(2) hydroxyl group. ( A ) ( i i ) E l e c t r o n i c e f f e c t s . While s t e r i c a l l y C-F i s a good replacement f o r both C-H and C-OH the chemical s i m i l a r i t i e s are not g r e a t . Since H © i s s t a b l e while F ® i s not, when f l u o r i n e i s used as a replacement f o r hydrogen the p o s s i b i l i t y of proton a b s t r a c t i o n i s suppressed. The hydroxyl group can be o x i d i s e d to a carbonyl while C = F © i s too unstable to e x i s t , t h e r e f o r e a l d o l chemistry could be suppressed. -5-The carbon f l u o r i n e bond i s a l s o very strong (bond energy 107 (10) kcal/mol) and t h i s makes i t very s l u g g i s h towards SN2 type displacement; about one tenthousandth as r e a c t i v e as the (11) corresponding carbon c h l o r i n e bond When f l u o r i n e i s s i t u a t e d adjacent to a r e a c t i o n centre i t s high e l e c t r o n e g a t i v i t y w i l l d e s t a b i l i z e any carbonium i o n which might form. The p o s s i b i l i t y of using t h i s e f f e c t to i n h i b i t g l y c o s y l t r a n s f e r a s e r e a c t i o n s and observe any enzyme induced (12) s t r a i n i n the s u b s t r a t e has been d i s c u s s e d by Dolphin et a l . The l a r g e electron-withdrawing e f f e c t of f l u o r i n e has been used to make a d i s t i n c t i o n between p o s s i b l e SN1 and SN2 mechanisms (13) o p e r a t i n g i n p r e n y l t r a n s f e r a s e . This enzyme i s i n v o l v e d i n the b i o s y n t h e s i s of s t e r o i d s and c a t a l y s e s the -addition of i s o p e n t e n y l pyrophosphate (2) to an a l l y l i c pyrophosphate (14) c o s u b s t r a t e (3) , see f i g u r e 3. P P P P = p y r o p h o s p h a t e (2) (3) The mechanism under s c r u t i n y suggested an i n i t i a l SN1 cleavage of the a l l y l i c pyrophosphate to y i e l d pyrophosphate and an a l l y l i c carbonium i o n ( f i g u r e 4 ) . The carbonium i o n i s then attacked by the TT e l e c t r o n s of the 3,4-double bond of i s o p e n t e n y l pyrophosphate, f o l l o w e d by a b s t r a c t i o n of a proton to gi v e the f i n a l product. -6-F i g u r e 3. The r e a c t i o n c a t a l y s e d by p r e n y l t r a n s f e r a s e . ^ ^ B - E n z F i g u r e 4. A proposed mechanism f o r p r e n y l t r a n s f e r a s e . - 7 -The authors argued that i n t r o d u c t i o n of a s t r o n g l y e l e c t r o n withdrawing group adjacent to the r e a c t i o n centre of the a l l y l i c s u b s t r a t e would d i s f a v o r the formation of the a l l y l i c carbonium i o n , thus g r e a t l y reducing the r a t e of r e a c t i o n . I f however i t proceeded by an SN2 type mechanism the r e a c t i o n r a t e would not be (14) g r e a t l y a f f e c t e d , see f i g u r e 5 (4) R = CF3 (5 ) R = CH3 ( 6 ) R = CH2F F i g u r e 5. The e f f e c t of f l u o r i n e s u b s t i t u t i o n on the s t a b i l i t y of an a l l y l i c carbonium i o n . The r a t e of r e a c t i o n f o r the t r i f l u o r o m e t h y l analog (4) was 1.5 m i l l i o n times slower than f o r d i m e t h y l a l l y l - p y r o p h o s p h a t e (5) while the monofluorinated d e r i v a t i v e (6) was converted with a r a t e i n t e r m e d i a t e between that of (4) and ( 5 ) , thus i m p l i c a t i n g the SN1 mechanism. -8-A f u t h e r e f f e c t a r i s i n g from the la r g e e l e c t r o n e g a t i v i t y of f l u o r i n e i s the i n c r e a s e i n a c i d i t y of v i c i n a l h y droxyl groups. T h i s e f f e c t can be q u i t e l a r g e ( e t h a n o l pKa=16, 1 , 1 , 1 - t r i f l u o r o -(15) ethanol pKa=12.4 ) and the enhanced b i o l o g i c a l a c t i v i t y of (16) 9 - f l u o r o c o r t i c o s t e r o i d s has been a t t r i b u t e d to t h i s In a study on the s u b s t r a t e s p e c i f i c i t y of yeast hexokinase, (17) conducted by Westwood and coworkers . I t was found that f l u o r i n a t i o n of the enzymes s u b s t r a t e , g l ucose, at p o s i t i o n s 3,4 and 6 caused a s u b s t a n t i a l l o s s i n s u b s t r a t e a c t i v i t y . However 2-deoxyfluoro-D-glucose (7) ,2-deoxyfluoro-D-mannose (8) and 2-deoxy-2,2-difluoro-D-arabino-hexose (9) were a l l good subs t r a t e s . (7) (8) (9) In f a c t i t was found that (9) bound to the enzyme more t i g h t l y than gl u c o s e . T h i s o b s e r v a t i o n was a t t r i b u t e d to the a c i d i f i c a t i o n of the C(3) hy d r o x y l group by the gem-difluoro s u b s t i t u e n t s at C(2), making i t a b e t t e r hydrogen-bond donor. I t has a l s o been shown that 3 -fluoropyruvate e x i s t s mainly as (8,19) the hydrated ketone r a t h e r than the normal ca r b o n y l form a f u r t h e r e f f e c t which can be a t t r i b u t e d to the l a r g e e l e c t r o n -withdrawing a b i l i t y of f l u o r i n e . -9-( A ) ( i i i ) Conformational e f f e c t s . For the f l u o r i n a t e d analog to be of any use to the enzymologist i t must be able to bind to the enzyme. This i s u n l i k e l y to happen i f the conformation of the analog i s s i g n i f i c a n t l y d i f f e r e n t from that of the enzymes n a t u r a l subs t r a t e . F l u o r i n a t e d carbohydrates have been e x t e n s i v e l y s t u d i e d by both proton and f l u o r i n e NMR and they provide a good system to study the e f f e c t s of f l u o r i n a t i o n on conformational p o p u l a t i o n s . Most of the conformational p e r t u r b a t i o n s appear to be r e l a t e d to the strong anomeric e f f e c t a s s o c i a t e d with the l a r g e d i p o l e (19) moment of the C-F bond .This e f f e c t means the f l u o r i n e s u b s t i t u e n t at C ( l ) w i l l t r y to adopt an a x i a l o r i e n t a t i o n even (20) at the expense of some unfavorable s t e r i c i n t e r a c t i o n s .This was found to be the case i n a s e r i e s of a c e t y l a t e d and (20) benzoylated pentopyranosy1 f l u o r i d e s examined by H a l l et a l . 4 The a - f l u o r i d e s were found to e x i s t i n the normal C 1 conformation (10) while f l u o r i d e s of the opposite anomeric c o n f i g u r a t i o n adopted the 1C conformation (11). (10) (11) -10-However In a s e r i e s of hexopyranosy1 f l u o r i d e s H a l l et a l . (21) found that the unfavourable d i a x i a l i n t e r a c t i o n s of the C(6) (22) hydroxymethy1 group and the r i n g hydroxyl groups were s u f f i c i e n t to prevent any s i g n i f i c a n t p o p u l a t i o n of the conformation, even f o r the f l u o r i d e s . S u b s t i t u t i o n of f l u o r i n e f o r the hydroxyl groups at p o s i t i o n s 3,4 and 6 of glucose d i d not have any s i g n i f i c a n t e f f e c t on t h e i r (23) conformational p o p u l a t i o n s Data from c r y s t a l l o g r a p h i c s t u d i e s tend to support the r e s u l t s obtained by NMR. Compound (8) was found to occupy a s l i g h t l y (24) d i s t o r t e d C 1 conformation , however the d i s t o r t i o n was no g r e a t e r than that observed i n the parent compound, mannose. Methyl 4-deoxy-4-fluoro-a-D-glucopyranoside (12) was again observed to (25) occupy the 4 C 1 conformation . In t h i s case the only d i f f e r e n c e i n conformation between (12) and methyl a-D-glucopyranoside was a s l i g h t l y d i f f e r e n t o r i e n t a t i o n of the C(6) hydroxymethy1 group. 6Me (12) - 1 1 -On c o n s i d e r a t i o n of d i p o l a r r e p u l s i o n s between the e q u a t o r i a l f l u o r i n e s u b s t i t u e n t at C ( l ) , the a x i a l f l u o r i n e s u b s t i t u e n t at C(2) and the r i n g oxygen of 2-deoxy-2-fluoro-/3-D-manno-pyranosyl f l u o r i d e (13), i t might be expected that some d i s t o r t i o n from the normal c h a i r conformation would occur. However both the c r y s t a l l o g r a p h i c data and an NMR study shows that t h i s compound e x i s t s i n an u n d i s t o r t e d Cj conformation ( A ) ( i v ) E f f e c t s on hydrogen-bonding. While C-F i s a good replacement f o r C-OH i n terms of both s i z e and p o l a r i t y , the hydroxyl group can act as both a hydrogen-bond donor and an ac c e p t o r . At best f l u o r i n e can only a ct as an ac c e p t o r . T h i s f a c t o r i s of great importance when c o n s i d e r i n g p r o t e i n - s u b s t r a t e i n t e r a c t i o n s . Most nonenzymic evidence f o r the p a r t i c i p a t i o n of the C-F fragment i n the formation of hydrogen-bonds has come from c r y s t a l l o g r a p h i c data and here i t s r o l e i s s t i l l u n c l e a r . (13) ( 2 6 ) -12-In the cases of compounds p a r t i c i p a t e i n the hydrogen the c r y s t a l packing diagram i n v o l v i n g the f l u o r i n e were (8) and (12) the f l u o r i n e d i d not (24,25) bonding network . However i n of (13) s e v e r a l i n t e r a c t i o n s (26) observed F i g u r e 6.The c r y s t a l packing of 2-deoxy-2-fluoro-^-mannosy1 f l u o r i d e . The f l u o r i n e at C(2) forms an i n t r a m o l e c u l a r hydrogen-bond with the hydroxyl group a t C(3) while the anomeric f l u o r i n e s u b s t i t u e n t i s i n v o l v e d i n two weak i n t e r a c t i o n s with the C-H of C(2) and C ( l ) . Such i n t e r a c t i o n s are unusual but not (27,28) unprecedented A recent survey of c r y s t a 1 l o g r a p h i c data f o r over 260 s t r u c t u r e s c o n t a i n i n g the C-F fragment has been c a r r i e d out by J.P.Glusker (29) and co workers . In only one case was there a c l a s s i c i s o l a t e d (30,31) C-F--H-0 con t a c t . A f u t h e r search f o r contacts i n v o l v i n g N-H as the hydrogen-bond donor re v e a l e d e i g h t such i n t e r a c t i o n s . -13-One p o s s i b l e e x p l a n a t i o n f o r the inc r e a s e d frequency of t h i s type © © of hydrogen-bond i s that protonated groups eg. NH,RNH e t c . are 4 3 good proton donors but cannot a c t as acceptors thus i n c r e a s i n g the p r o b a b i l i t y of C-F--H-N i n t e r a c t i o n . Glusker et a l . concluded that although r e l a t i v e l y few s t r u c t u r e s with p o s s i b l e C-F--donor i n t e r a c t i o n s had been found, there were enough to propose that C-F can a c t as a weak proton a c c e p t o r . Where there i s an excess of proton donors over a c c e p t o r s , or a s u i t a b l e hydrogen-bond donor i s a p p r o p r i a t e l y p o s i t i o n e d i t appears that C-F--HX hydrogen-bonds, a l b e i t weak ones, are formed. The a b i l i t y of f l u o r i n e to a c t only as a hydrogen-bond acceptor has been used to enable a d i s t i n c t i o n to be made between the r o l e s of donor and or acceptor f o r a given s u b s t r a t e hydroxyl group, while i t i s bound to the a c t i v e s i t e of an enzyme. Yeast (32) g a l a c t o k i n a s e has been the focus of such a study . A l l the isom e r i c deoxyfluoro-D-galactopyranoses were s y n t h e s i s e d and a l l were found to be s u b s t r a t e s . By comparison of the k i n e t i c parameters obtained for- the 2-deoxy and the 2-deoxyfluoro analogs, i t was suggested that the C(2) hydroxyl group acted as a hydrogen-bond acceptor when bound at the a c t i v e s i t e . A l o s s i n s u b s t r a t e a c t i v i t y (compared with glucose) was observed f o r the 3-,4- and 6-deoxyfluoro analogs. These r e s u l t s were i n t e r p r e t e d as i n d i c a t i n g a s i m i l a r f u n c t i o n f o r these s u b s t r a t e hydroxyl groups, probably that of a hydrogen-bond donor. -14-(B) The p r o t e i n : Glycogen Phophorylase, a b r i e f i n t r o d u c t i o n . a-glucan phosphorylases are found i n many d i v e r s e sources, from mammalian muscle and l i v e r to b a c t e r i a ( E . c o l i ) and y e a s t . They a l l c a t a l y s e the degradation of glycogen to .a-D-glucose-1-phosphate. F i g u r e 7. The r e a c t i o n c a t a l y s e d by glycogen phosphorylase. (33) The e q u i l i b r i u m constant f o r the r e a c t i o n i s 0.28 at pH 6.8 , the e q u i l i b r i u m l y i n g i n favour of glycogen s y n t h e s i s . However under p h y s i o l o g i c a l c o n d i t i o n s (high phosphate and low glucose-l-phosphate c o n c e n t r a t i o n s ) the r e a c t i o n proceeds i n the (34) d i r e c t i o n of glycogen degradation The s o l e f u n c t i o n of t h i s enzyme i s to provide the t i s s u e with a t i g h t l y r e g u l a t e d supply of phosphorylated glucose from i t s s t o r e of glycogen. The muscle enzyme i s c l o s e l y l i n k e d to the requirements of muscle c o n t r a c t i o n and has to cope with a l a r g e v a r i a t i o n i n demand as the muscle goes from r e s t i n g to an a c t i v e s t a t e . Likewise the l i v e r enzyme must cope with v a r i a t i o n s i n -15-blood sugar l e v e l s . These requirements have led to the development of a very complex c o n t r o l system f o r glycogen phosphorylase which i n v o l v e s both a l l o s t e r i c and c o v a l e n t forms of r e g u l a t i o n . Glycogen phosphorylase e x i s t s i n two forms; f i r s t l y , phosphorylase b, which i s found i n r e s t i n g muscle. This form of the enzyme i s normally i n a c t i v e ( c o n t a i n i n g a high p r o p o r t i o n of the i n a c t i v e T - s t a t e ) and i s s u b j e c t to c o n t r o l by a l l o s t e r i c a c t i v a t o r s such as adenosine monophosphate (AMP). Phosphorylase b can a l s o be i n h i b i t e d by a l l o s t e r i c i n h i b i t o r s such as ATP, ADP, glucose and glucose-6-phosphate. The second form of the enzyme i s known as phosphorylase a, here the enzyme has been c o v a l e n t l y modified by attachment of a (35) phosphate r e s i d u e to s e r i n e 14 , and contains a high p r o p o r t i o n of the a c t i v e R - s t a t e . T h i s form of the the enzyme has l a r g e l y escaped the c o n t r o l of a l l o s t e r i c a c t i v a t o r s and i n h i b i t o r s . However a d d i t i o n of AMP w i l l cause a s l i g h t i n c r e a s e (36) i n a c t i v i t y . L i k e phosphorylase b , t h i s form of the enzyme can be i n h i b i t e d by g l u c o s e . Phosphorylase b i s converted i n t o the more a c t i v e phosphorylase a by a s p e c i f i c p r o t e i n k i n a s e . The kinase i s dependent on calcium ions f o r a c t i v i t y and i s under c o n t r o l of the c e n t r a l nervous system. Augmenting t h i s nervous c o n t r o l i s the hormonally induced (37,38) glycogen cascade , see f i g u r e 8. Glycogen phosphorylase contains one molecule of p y r i d o x a l - 5 * -(39) phosphate per enzyme monomer . T h i s coenzyme i s e s s e n t i a l f o r (40) a c t i v i t y . I t s r o l e i n c a t a l y s i s however, i s unique i n that -16-th e aldehyde group Is not r e q u i r e d f o r phosphorylase a c t i v i t y , whereas the f r e e aldehyde i s e s s e n t i a l i n a l l other enzymes c o n t a i n i n g t h i s c o f a c t o r . hornonsl signal* ++ Ca cAHP protein kinase synthetase b-P eyntbstsse a pbosphorylaaa phospborjlsse (sctlve) klnaaa kinase (ectiT.) phosphatase * • / \ Inhibitor I DDPC / C1P \ \ \ \ \ \ Insulin v tlncocortleolds \ \ (locos. 4 phospborylsse m-t phosphoxrlass » F i g u r e 8. The glycogen cascade; Showing how hormonal s i g n a l s a c t i v a t e phosphorylase through the two kinases and i n a c t i v t e glycogen synthase. The i n a c t i v a t i o n (broken arrows) i s by a c t i o n of phosphatases that are r e g u l a t e d by phosphorylase and i n h i b i t o r p r o t e i n s . Glucose i n a c t i v a t e s phosphorylase a d i r e c t l y . The machanism by which i n s u l i n and other hormones do t h i s i s not known. Th i s was demonstrated by Krebs and h i s coworkers, who found that under m i l d l y a c i d i c c o n d i t i o n s the l i n k a g e between the coenzyme and l y s i n e 679 could be reduced by sodium borohydride with (41) r e t e n t i o n of most of the enzymes a c t i v i t y . Many d i f f e r e n t groups, have s i n c e demonstrated, by means of a v a r i e t y of analog replacement s t u d i e s that the 5'-phosphate moiety of the c o f a c t o r (42) i s e s s e n t i a l f o r the a c t i v i t y of the enzyme However the p r e c i s e r o l e which the 5'-phosphate group plays i n the c a t a l y t i c mechanism i s s t i l l a matter of great c o n j e c t u r e . Both phosphorylase a and b can e x i s t as a tetramer or a dimer (43,44) of i d e n t i c a l subunits i n s o l u t i o n , the dimer-tetramer e q u i l i b r i u m depending on the s t a t e of a c t i v a t i o n of the enzyme -17-(45) . The f u l l sequence of the 841 amino a c i d s (molecular weight (46) 97,412) f o r the phosphorylase monomer, has been determined Extensive c r y s t a l l o g r a p h i c s t u d i e s have been c a r r i e d out on both phosphorylase a and b . In both cases moderately h i g h (47,48,49) r e s o l u t i o n s t r u c t u r e s are a v a i l a b l e , 2.1 A f o r (50,51) phosphorylase a and 2.0 A f o r phosphorylase b . The monomeric subunit i s found to comprise two domains, the N-terminal domain extending from r e s i d u e 1 to 489 and the C-terminal domain from 490 to 841. Each domain c o n s i s t s of approximately 2 57. jg-sheet and 4 57. o t - h e l i x ; the core of the domain c o n t a i n i n g most of the /3-sheet which i s surrounded by a - h e I l e a l segments. Each subunit i n the dimer i s i n t i m a t e l y a s s o c i a t e d with i t s symmetry r e l a t e d p a r t n e r , mainly through contacts w i t h i n t h e i r N-terminal domains (see f i g u r e s 9 and 10). A l l of the primary l i g a n d b i n d i n g s i t e s have been i d e n t i f i e d w i t h i n the c r y s t a l s t r u c t u r e . The phosphate r e s i d u e attached to s e r i n e 14 can be seen near the AMP ( a c t i v a t o r ) b i n d i n g s i t e . I t has a l s o been shown, mainly through s t u d i e s on phosphorylase b (52) that ATP,ADP and glucose-6-phosphate bind at t h i s s i t e The glycogen storage s i t e i s s i t u a t e d on the N-terminal domain. Both glycogen and glycogen fragments such as maltopentaose bind s t r o n g l y to t h i s s i t e while only weak i n t e r a c t i o n s are observed (53) with the a c t i v e s i t e . In v i v o t h i s glycogen b i n d i n g s i t e i s thought to a c t as an anchor p o i n t f i r m l y a f f i x i n g the enzyme to the glycogen molecule. A second i n h i b i t o r s i t e (the purine b i n d i n g s i t e ) i s s i t u a t e d a t the boundaries of the N and C t e r m i n a l domains. -18-F l g u r e 9. A schematic diagram of the phosphorylase a monomer showing l i g a n d b i n d i n g s i t e s . From F l e t t e r i c k and Sprang ( r e f e r e n c e 122). G l u c o s e E f f e c t o r S t l e 2 A d e n o s i n e S i t . 2 A d e n o s i n e S i t e I F i g u r e 10. A space f i l l i n g r e p r e s e n t a t i o n of the phosphorylase dimer and i t s diagrammatic r e p r e s e n t a t i o n . From F l e t t e r i c k and Sprang ( r e f e r e n c e 122). - 1 9 -Th i s s i t e appears to be s p e c i f i c f o r b i n d i n g b i c y c l i c and t r i c y c l i c aromatic molecules, but i t s p h y s i o l o g i c a l r o l e i s not unders tood. Glucose i s found i n a high s p e c i f i c i t y b i n d i n g pocket i n the N-terminal domain with i t s anomeric hydroxyl group p r o j e c t i n g i n t o the c a v i t y between the two domains. The glucopyranosy1 moiety of glucose-l-phosphate binds i n the same pocket with a small t r a n s l a t i o n . The phosphate group of glucose-l-phosphate binds to a separate s u b s i t e d i r e c t l y adjacent to the glucopyranose s i t e . I t s phosphate group i s o r i e n t e d towards the 5'-phosphate of the p y r i d o x a l phosphate c o f a c t o r . D e t a i l e d d e s c r i p t i o n s of the glucose b i n d i n g s i t e s i n both (48) (50) phosphorylase a and phosphorylase b are a v a i a b l e . -20 (C) Methods of f l u o r i n a t i o n . With recent i n c r e a s e d i n t e r e s t i n o r g a n o - f l u o r i n e compounds, many mild and s e l e c t i v e methods f o r the i n t r o d u c t i o n of a C-F (53,54) fragment i n t o a given carbon s k e l e t o n have been developed However f o r the puposes of t h i s i n t r o d u c t o r y chapter, the d i s c u s s i o n w i l l be r e s t r i c t e d to the methods used f o r the replacement of the hy d r o x y l f u n c t i o n a l i t y by f l u o r i n e i n carbohydrate systems. Two g e n e r a l approaches to t h i s problem have been f o l l o w e d ; 1) n u c l e o p h i l i c displacements of an a p p r o p r i a t e l e a v i n g group and 2) e l e c t r o p h i l i c a d d i t i o n s to g l y c a l s . D i s c u s s i o n w i l l be r e s t r i c t e d to the former method. Sugars with f l u o r i n e s i t u a t e d on C ( l ) were among the e a r l i e s t fluorodeoxy carbohydrates to be prepared. The s y n t h e s i s of t h i s type of compound was e f f e c t e d by d i s s o l u t i o n and r e a c t i o n of (55) the p e r - a c e t y l a t e d sugars i n l i q u i d hydrogen f l u o r i d e . T h i s method u s u a l l y gives r i s e to the most thermodynamically s t a b l e f l u o r i d e , although exceptions to t h i s g e n e r a l i s a t i o n have been (56) rep o r t e d . A much more convenient experimental procedure f o r the p r e p a r a t i o n of g l y c o s y l f l u o r i d e s , u t i l i s i n g p o l y hydrogen f l u o r i d e i n p y r i d i n e , i n place of l i q u i d hydrogen f l u o r i d e , has (58) (59) r e c e n t l y been provided by Noyori et a l . and Szarek et a l . R e a c t i o n of a c y l a t e d g l y c o s y l h a l i d e s (bromide or c h l o r i d e ) with s i l v e r f l u o r i d e i n a c e t o n i t r i l e , can be used to prepare the l e s s (57) s t a b l e anomeric f l u o r i d e s F l u o r i n a t i o n at other secondary carbon centres w i t h i n the sugar r i n g , has been accomplished by epoxide cleavage, u s i n g the f l u o r i d e i o n . A previous s y n t h e s i s of 4-deoxy-4-fluoro-D-glucose -21-(60) u t i l i s e d t h i s method f o r i n t r o d u c t i o n of the C-F fragment The p r e c u r s o r , 1, 6 : 3 , 4-dianhydro-/3-D-galactopyranoside (14) was t r e a t e d with potassium hydrogen f l u o r i d e i n b o i l i n g 1,2-ethanedio1 to y i e l d 1,6-anhydro-4-deoxy-4-fluoro-^-D-gluco-pyranose (15) i n 457. y i e l d , see f i g u r e 11. M ( 1 5 ) F i g u r e 11. Epoxide cleavage by the f l u o r i d e i o n . However, the s y n t h e s i s of the d e s i r e d epoxide p r e c u r s o r i s not a t r i v i a l task ( (14) was prepared i n 6 steps from 1,6-anhydro-/?-D-glucopyranose) and f o r t h i s reason alone, t h i s method i s not u s u a l l y amenable to the l a r g e s c a l e p r e p a r a t i o n of deoxyfluoro sugars. Displacement of sulphonate l e a v i n g groups by the f l u o r i d e i o n has provided the most popular method f o r the replacement of a hydroxyl group by f l u o r i n e . R e a c t i o n of 1 ,6-anhydro-4-0-tosy 1-^J-D-galactopyranose (16) with potassium hydrogen f l u o r i d e i n (60) b o i l i n g 1 , 2-ethanedio1 and the r e a c t i o n of methyl 2,3,6-tri-O-benzy1-4-0-mesyl-a-D-galactopyranoside (17) with t e t r a (62) butylammonium f l u o r i d e , have both been u t i l i s e d as the key r e a c t i o n step i n p r e p a r a t i o n of 4-deoxy-4-fluoro-D-glucose. -22-(16) (17) Primary a l k y l f l u o r i d e s have a l s o been prepared i n a s i m i l a r manner. Treatment of 1,2-isopropylidene-3,5-0-benzylidene-6-0-mesyl-a-D-glucofuranose (18) with the d i h y d r a t e of potassium f l u o r i d e i n methanol has provided a method f o r the p r e p a r a t i o n of (61) 6-deoxy-6-fluoro-D-glucose . (18) In a l a t e r m o d i f i c a t i o n of t h i s s y n t h e s i s the p a r t i a l l y p r o t e c t e d d e r i v a t i v e , methyl 6-0-tosy1-a-D-glucopyranoside (19) (62a) was used . In t h i s case a byproduct, the 3,6-anhydro d e r i v a t i v e (20) was a l s o formed, F i g u r e 12. While t h i s type of method has been widely used, i t has a number of disadvantages. F i r s t l y the low n u c l e o p h i l i c i t y of the f l u o r i d e i o n n e c e s s i t a t e s the use of harsh r e a c t i o n c o n d i t i o n s to e f f e c t displacement, and t h i s o f t e n leads to low y i e l d s . -23-(19) C20) F i g u r e 12. P r e p a r a t i o n of primary a l k y l f l u o r i d e s . Secondly, the a b i l i t y of the f l u o r i d e i o n to c a t a l y s e e l i m i n a t i (63) r e a c t i o n s a l s o tends to lower y i e l d s . E l i m i n a t i o n r e a c t i o n s competing with f l u o r i d e i o n displacement have been observed i n the p r e p a r a t i o n of the 3-deoxyfluoro d e r i v a t i v e s of glucose, (64,65) a l l o s e and g a l a c t o s e . While displacement of a t o s y l a t e l e a v i n g group from 1,2;5,6-diisopropylidene-3-0-tosyl-a-D-a l l o f u r a n o s e by f l u o r i d e i o n allowed p r e p a r a t i o n of the 3-deoxyfluoro d e r i v a t i v e of glucose, an attempted displacement (under the same c o n d i t i o n s ) from the gluco-isomer y i e l d e d only the e l i m i n a t i o n product, See f i g u r e 13. F i g u r e 13. F l u o r i d e i o n c a t a l y s e d e l i m i n a t i o n r e a c t i o n s . -24-To a c e r t a i n extent these d i f f i c u l t i e s have been overcome by the use of a more r e a c t i v e l e a v i n g group, the t r i f l u o r o m e t h a n e s u l p h o n y l ( t r i f l a t e ) group. The use of t h i s group has allowed p r e p a r a t i o n of 2-deoxy-2-fluoro-D-glucose, the key step being the r e a c t i o n of methyl 4 , 6 - 0 - b e n z y l i d e n e - 3 - 0 - m e t h y l - 2 - 0 - t r i f l y l -(66) /3-D-mannopyranose (21) with cesium f l u o r i d e . Likewise p r e p a r a t i o n of the 2-deoxyfluoro d e r i v a t i v e of mannose has been achieved by treatment of the corresponding g l u c o s y l d e r i v a t i v e (67,68) with f l u o r i d e i o n (21) Recently a number of reagents designed to promote the d i r e c t displacement of oxygen f u n c t i o n s by f l u o r i n e have gained p o p u l a r i t y with the carbohydrate chemist. Sulphur t e t r a f l u o r i d e has been a p p l i e d to the p r e p a r a t i o n of deoxyfluoro d e r i v a t i v e s (69) of c e l l u l o s e D i a l k y l a m i n o s u l p h u r t r i f l u o r i d e s (DAST) have been found to be e f f e c t i v e reagents f o r p r e p a r a t i o n of a l k y l f l u o r i d e s from (70,71) a l c o h o l s . These reagents w i l l r e a c t with primary or secondary a l c o h o l s , under m i l d r e a c t i o n c o n d i t i o n s (atmospheric p r e s s u r e , low temperature and i n g l a s s apparatus) and can be used (71) i n the presence of a c i d s e n s i t i v e groups . DAST a l s o e x i b i t s a -25-(72) high degree of s t e r e o and r e g i o s e l e c t i v i t y . Card et a l . have reported that methyl or phenyl a - g l u c o s i d e s may be s e l e c t i v e l y f l u o r i n a t e d by DAST at C(4) or C(-6) to a f f o r d the corresponding f l u o r i n a t e d g a l a c t o - or gl u c o p y r a n o s i d e s . In c o n t r a s t to the a-glucopyranosides, the ^-glucopyranosides underwent f l u o r i n a t i o n p r i m a r i l y a t C(3) to give the 3-deoxy-3-fluoro - / J-D-allo d e r i v a t i v e s . High y i e l d s of the primary f l u o r i n a t e d products could be obtained from both a and R (73) glucopyranosides by use of s h o r t r e a c t i o n times DAST has a l s o been used with p a r t i a l l y p r o t e c t e d carbohydrate d e r i v a t i v e s . 1, 2,3,4, - tetra-0-acetyl-6-deoxy-6-fluoro-/?-D-glucopyranose has been prepared by the a c t i o n of DAST on the (74) a p p r o p r i a t e t e t r a a c e t a t e . Rea c t i o n of 1,3,4,6-tetra-O-benzyl -jg-D-mannopyranose with DAST a t 40 C gave q u a n t i t a t i v e c o n v e r s i o n to 1,3,4,6,- te t r a - O - b e n z y l - 2-deoxy-2- f luoro - / J-D-g lucopyranose (75) with a r e a c t i o n time of only 5 minutes ! The s t e r e o s p e c i f i c p r e p a r a t i o n of g l y c o s y l f l u o r i d e s by the a c t i o n of DAST on p a r t i a l l y p r o t e c t e d monosaccharides, which have the anomeric hydroxyl group u n d e r i v a t i z e d , has a l s o been r e p o r t e d (76,77) The r e a c t i o n of DAST with an a l c o h o l i s thought to proceed by i n i t i a l n u c l e o p h i l i c a t t a c k of the a l c o h o l at the sulphur atom (70,71) with concomitant l o s s of hydrogen f l u o r i d e ; F i g u r e 14. The i n t e r m e d i a t e i s g e n e r a l l y unstable and forms a very good (78) l e a v i n g group . The r e a c t i o n i s concluded by displacement of the l e a v i n g group by f l u o r i d e a t t a c k i n g a t carbon to y i e l d the d e s i r e d a l k y l f l u o r i d e . -26-R C H 2 O H A T — • RCHiCU/F A i / R R R C H 2 F + R 2 N S F 2 O H F i g u r e 14. The r e a c t i o n mechanism of DAST. When the r e a c t i o n i s performed at a secondary carbon c e n t r e , i t occurs with i n v e r s i o n of c o n f i g u r a t i o n , the displacement probably o c c u r i n g by an SN2 type mechanism. -27-(D) Summary. F l u o r i n a t e d s u b s t r a t e analogs have provided the enzymologist with a powerful t o o l f o r the study of b i o l o g i c a l mechanisms. However there s t i l l e x i s t s some doubt about t h e i r use i n the study of p r o t e i n l i g a n d i n t e r a c t i o n s . A great deal of c r y s t a l l o g r a p h i c i n f o r m a t i o n i s a v a i l a b l e on the glucopyranose b i n d i n g s i t e of both phosphorylase a and phosphorylase b making t h i s enzyme a p a r t i c u l a r l y u s e f u l system f o r the study of p r o t e i n - l i g a n d i n t e r a c t i o n s . Thus a systematic k i n e t i c i n v e s t i g a t i o n u t i l i s i n g a s e r i e s of f l u o r i n a t e d glucose analogs and subsequent r a t i o n a l i s a t i o n of the r e s u l t s with the c r y s t a l l o g r a p h i c data could r e a l i s e c o n s i d e r a b l e i n s i g h t i n t o the nature of c a r b o h y d r a t e - p r o t e i n i n t e r a c t i o n s . T h i s i n f o r m a t i o n could a l s o be u s e f u l i n determining the value of f l u o r i n e as a hydrogen-bond acceptor which would be of much wider i n t e r e s t i n the l i g h t of recent i n t e r e s t i n f l u o r i n e c o n t a i n i n g pharmaceuticals . L i t t l e or no c r y s t a l l o g r a p h i c i n f o r m a t i o n i s a v a i l a b l e f o r the glucose-l-phosphate b i n d i n g s i t e i n the f u l l y a c t i v a t e d form of the enzyme, l a r g e l y because c r y s t a l s of the te r n a r y enzyme-s u b s t r a t e complex are unstable and tend to s h a t t e r . The k i n e t i c r e s u l t s from a s e r i e s of f l u o r i n a t e d analogs of g l u c o s e - l -phosphate could y i e l d v a l u a b l e i n f o r m a t i o n about the a c t i v e s i t e i n the ternary complex and about the nature of the T - s t a t e to R-state t r a n s i t i o n . L a s t l y such a study would a l s o i d e n t i f y p o s i t i o n s on the s u b s t r a t e which could t o l e r a t e a l t e r a t i o n without a d v e r s e l y -28-a f f e c t i n g the b i n d i n g of the s u b s t r a t e . T h i s would be v a l u a b l e i n f o r m a t i o n f o r any ensuing mechanistic i n v e s t i g a t i o n . -29-RESULTS AND DISCUSSION. (A) Synthesis of f l u o r i n a t e d analogs of glucose, mannose and glucose-l-phosphate. A l l of the monofluorinated isomers of glucose have been p r e v i o u s l y s y n t h e s i s e d and consequently many of the int e r m e d i a t e s have a l s o been d e s c r i b e d . The compounds s y n t h e s i s e d were 1 19 c h a r a c t e r i s e d by comparison of the H, F NMR s p e c t r a , melting p o i n t and / or r e s u l t s of elemental a n a l y s i s (see experimental s e c t i o n ) with the r e f e r e n c e d l i t e r a t u r e v a l u e s . However many of the published syntheses have been improved upon by the use of DAST i n place of the c o n v e n t i o n a l method of f l u o r i n a t i o n or by a c t u a l improvements i n the p u b l i s h e d procedures f o r using DAST. ( A ) ( i ) F l u o r i n a t i o n of the 6 - p o s i t i o n . The route used f o r the p r e p a r a t i o n of the 6-deoxyfluoro d e r i v a t i v e s of glucose and glucose-l-phosphate i s gi v e n i n f i g u r e 15. F i g u r e 15. F l u o r i n a t i o n of the 6 - p o s i t i o n . -30-(74) Korytnyk and Sharma have d e s c r i b e d the s y n t h e s i s of l,2,3,4,-tetra-0-acetyl-6-deoxy-6-fluoro-)g-D-glucopyranose (23), by r e a c t i o n of DAST with the t e t r a - a c e t a t e (22) i n dry diglyme. However, the y i e l d s obtained using t h i s method, v a r i e d g r e a t l y . In an attempt to improve the s y n t h e s i s of (23) the r e a c t i o n s o l v e n t was changed to dry dichloromethane, However r e a c t i o n of the t e t r a - a c e t a t e (22) with 2 e q u i v a l e n t s of DAST i n t h i s s o l v e n t gave none of the normal product, see t a b l e I I . EDUCT R 2NSF 3 BASE REACTION TIME(HRS) 7. YIELD FLUORINATED PRODUCT (22) R = Et - 18 - -(22) R=E t DMAP 18 - -(22) R = Et TMP 18 68 (23) (22) R=M e TMP 36 32 (23) Table I I . Reaction c o n d i t i o n s f o r f l u o r i n a t i o n of the 6 - p o s i t i o n . (65) Glaudemans et a l . have noted that i n the r e a c t i o n of 1,2:5,6-di-0-isopropylidene-a-D-gulofuranose with DAST, y i e l d s were g r e a t l y improved by the a d d i t i o n of the base 4-(dimethylamino)-pyridine (DMAP). A d d i t i o n of DMAP to the r e a c t i o n of (14) with DAST, again r e s u l t e d i n none of the d e s i r e d product being produced. However a d d i t i o n of the s t e r i c a l l y hindered base 2 , 4 , 6 - t r i m e t h y l p y r i d i n e (TMP) i n pl a c e of DMAP allowed the i s o l a t i o n of the d e s i r e d 6-deoxyfluoro d e r i v a t i v e (15) i n 687. y i e l d . Presumably the presence of a base f a c i l i t a t e s the r e a c t i o n by -31-a i d i n g the formation of the DAST-alcohol Intermediate and by a c t i n g as an a c i d acceptor f o r hydrogen f l u o r i d e produced during the r e a c t i o n . A p o s s i b l e e x p l a n a t i o n f o r the f a i l u r e of the r e a c t i o n i n the presence of DMAP i s provided by the observ a t i o n s of Ambrose and (79) B i n k l e y . In attempting the s y n t h e s i s of 1,2 , 3 ,4-tetra-0 -ace ty 1-6-0 - [ ( t r i f luorome thy 1) - sulphonyl ] - / J-D-glucopyranose only the 6-N p y r i d i n i u m compound (26) was i s o l a t e d . (26) I n t e r c e p t i o n of the DAST a l c o h o l - i n t e r m e d i a t e by DMAP, i n the same manner could account f o r the f a i l u r e of the f l u o r i n a t i o n of (22) i n the presence of t h i s base. Two types of DAST are commercially a v a i l a b l e , d i e t h y l and dimet h y l . A comparison of r e a c t i o n times and product y i e l d s f o r the two types are a l s o given i n t a b l e I I . G e n e r a l l y the d i e t h y l d e r i v a t i v e g i v e s b e t t e r y i e l d s with s h o r t e r r e a c t i o n times. (80,62a) 6-deoxy-6-fluoro-D-glucose (24) was prepared by c a t a l y t i c d e a c y l a t i o n of the t e t r a - a c e t a t e (23) with 0.1M sodium methoxide i n methanol. Treatment of (23) with anhydrous phosphoric a c i d f o r 2.5 hours at 55°C ac c o r d i n g to the method of (81) MacDonald f o l l o w e d by d e a c y l a t i o n and convers i o n to the -32-cyclohexylammonium s a l t gave 6-deoxy-6-fluoro-a-D-glucose-1-phosphate (25) i n 697. y i e l d . ( A ) ( i i ) F u o r i n a t i o n of the 4 - p o s i t i o n . The s y n t h e t i c route employed i n the p r e p a r a t i o n of the 4-deoxyfluoro d e r i v a t i v e s of glucose and glucose-l-phosphate shown i n f i g u r e 16. F i g u r e 16. F l u o r i n a t i o n of the 4 - p o s i t i o n . f -33 Methyl 2,3,6-tri-O-benzoy1-a-D-galactopyranoside (27) can be (82) prepared i n a s i n g l e step from methyl a-D-galactopyranoside . Reaction of (27) with DAST i n dichloromethane ( t a b l e I I I ) gave methy 1 2,3,6,-tri-0-benzoyl-4-deoxy-4-fluoro-a-D-glucopyranoside (83) (28) i n 457. y i e l d . EDUCT R SNF 2 3 BASE REACTION TIME(HRS) 7. YIELD FLUORINATED PRODUCT (27) R=E t - 18 41 (28) (27) R=E t DMAP 24 46 (28) (27) R=Me DMAP 48 45 (28) Table I I I . F l u o r i n a t i o n c o n d i t i o n s f o r the 4 - p o s i t i o n . U n l i k e the f l u o r i n a t i o n of the 6 - p o s i t i o n the presence of a base had l i t t l e e f f e c t on the y i e l d , although samples of (28) prepared with DMAP were n o t i c e a b l y more e a s i l y c r y s t a l l i s e d . D e a c y l a t i o n of the t r i - b e n z o a t e (28) was accomplished with 0.IM sodium methoxide i n methanol to gi v e methyl 4-deoxy-4-fluoro-a-(62) D-glucopyranoside (29) i n 787. y i e l d . H y d r o l y s i s of (24) with a c i d i c c a t i o n exchange r e s i n gave the f r e e sugar (30) i n 857. y i e l d . Thus 4-deoxy-4-fluoro-D-glucose was prepared In four steps from methyl a-D-galactopyranoside with an o v e r a l l y i e l d of 237., a c o n s i d e r a b l e improvement on any of the p r e v i o u s l y p u b l i s h e d synthesese . The r e a c t i o n of DAST with (27) was independently p u b l i s h e d by (83) P.J.Card while t h i s work was i n progress. Treatment of the f r e e sugar (30) with a c e t i c anhydride and sodium a c e t a t e at 110°C gave an anomeric mixture of -34-l,2,3,6-tetra-0-acetyl-4-deoxy-4-fluoro-D-glucopyranose ( a (31) and 0 ( 3 3 ) ) from which the 0 - t e t r a - a c e t a t e (33) could be p r e f e r e n t i a l l y c r y s t a l l i s e d . 1,2,3,6-tetra-O-acety1 -4-deoxy-4-fluoro-/?-D-glucopyranose was prepared i n 357. y i e l d by t h i s method. The a - t e t r a - a c e t a t e (31) was a l s o prepared d i r e c t l y from the methyl glucopyranoside (29) by treatment i n a mixture c o n s i s t i n g of a c e t i c anhydride and a c e t i c a c i d i n the presence of s u l p h u r i c a c i d . A f t e r i s o l a t i o n by chromatography (31) was obtained i n 45% y i e l d . l ,2,3,6-tetra-0-acetyl-4-deoxy-4-fluoro-a-D-glucopyranose (31) was converted to the /J-acetate (33) by p r e p a r a t i o n of 1,2,3,6-tetra-0-acetyl-4-deoxy-4-fluoro-or-D-glucopyranosy 1 bromide (32) and subsequent r e a c t i o n with mercuric a c e t a t e a c c o r d i n g to the (84) method of Volfrom and Thompson Attempts to convert the methyl glucopyranoside (29) d i r e c t l y to the t e t r a - a c e t y l a t e d a-bromide (32) by using hydrobromic a c i d i n a mixture of a c e t i c a c i d and a c e t i c anhydride gave a complex mixture of products i n which the d e s i r e d compound was only a minor c o n s t i t u e n t . 4-deoxy-4-fluoro-a-D-glucose-l-phosphate (34) was prepared by r e a c t i o n of the 0-tetra-acetate (33) with anhydrous phosphoric a c i d at 55°C f o r 2.5 hours a c c o r d i n g to the method of HacDonald (81) . The 1-phosphate e s t e r (33) was i s o l a t e d as the monohydrate of the dicyclohexylammonium s a l t i n 47% y i e l d . -35-( A ) ( i i i ) F l u o r i n a t i o n of the 3 - p o s i t i o n . The s y n t h e t i c route u t i l i s e d f o r the p r e p a r a t i o n of the 3-deoxyfluoro d e r i v a t i v e s of glucose and glucose-l-phosphate i s given i n f i g u r e 17. D A c F i g u r e 17. F l u o r i n a t i o n of the 3 - p o s i t i o n . -36-l,2:5,6-di-0-isopropylidene-3-deoxy-3-fluoro-a-D-glucofuranose (78) (36) has been prepared by Tewson and Welch by the a c t i o n of DAST on 1,2 : 5 , 6 - d i - 0 - i s o p r o p y l i d e n e - a - D - a l l o f u r a n o s e (35) i n the presence of p y r i d i n e . Again the use of the stronger base DMAP i n pla c e of p y r i d i n e i n c r e a s e d the y i e l d s of the f l u o r i n a t e d product (36), see t a b l e IV. EDUCT R 2NSF 3 BASE REACTION TIME(HRS) 7. YIELD FLUORINATED PRODUCT (35) R=Et PYRIDINE 20 59 (36) (35) R=E t DMAP 18 72 (36) (35) R=Me DMAP 20 58 (36) Table IV. F l u o r i n a t i o n c o n d i t i o n s f o r the 3 - p o s i t i o n . The i s o p r o p y l i d e n e groups were removed by mild a c i d h y d r o l y s i s using Dowex 50 W (H®) c a t i o n exchange r e s i n i n aqueous ethanol at room temperature. A f t e r workup 3-deoxy-3-fluoro-D-glucose (78,64) (37) was i s o l a t e d i n 947. y i e l d as a c o l o u r l e s s gum. A c e t y l a t i o n of the f r e e sugar (37) with sodium a c e t a t e 0 < 6 5 > i n a c e t i c anhydride, at 110 C gave 1,2,4,6-tetra-0-acety1 -3-deoxy-3-fluoro-/3-D-glucopyranose (40), i n 27% y i e l d . An anomeric mixture of the t e t r a - a c e t a t e s (38) and (40) was prepared by r e a c t i o n of the f r e e sugar (37) with a c e t i c anhydride i n c o l d p y r i d i n e . T h i s method of a c e t y l a t i o n has been shown to minimise the amount of a c e t y l a t e d furanose forms which are produced by the (65) sodium a c e t a t e c a t a l y s e d method used above -37-A f t e r removal of the p y r i d i n e the product mixture was converted to 2,4,6-tri-O-acety1-3-deoxy-3-fluoro-a-D-gluco-pyra n o s y l bromide (39) by treatment with 307. hydrobromic a c i d i n a c e t i c a c i d . The a-bromide (39) was then converted to the (84) fi-tetra-acetate (40) by r e a c t i o n with mercuric a c e t a t e . Both the a and fi t e t r a - a c e t a t e s (38) and (40), have p r e v i o u s l y (64,78) been d e s c r i b e d . The a - t e t r a - a c e t a t e (38) was converted to the / J-tetra a c e t a t e (40) with an o v e r a l l y i e l d of 457.. P r e p a r a t i o n of 3-deoxy-3-fluoro-a-D-glucose-1-phosphate (41) was completed by r e a c t i o n of the fi-tetra-acetate (40) (81) with anhydrous phosphoric a c i d and i s o l a t e d as the d i h y d r a t e of the dicyclohexylammonium s a l t i n 3 77. y i e l d . ( A ) ( i v ) F l u o r i n a t i o n of the 2 - p o s i t i o n . The route used f o r the p r e p a r a t i o n of the 2-deoxyfluoro d e r i v a t i v e s of glucose and glucose-l-phosphate i s g i v e n i n f i g u r e 18. F i g u r e 18. F l u o r i n a t i o n of the 2 - p o s i t i o n . -38-1,3,4,6-tetra-O-acety1-2-deoxy-2-fluoro-a-D-glucopyranosy1 (12) bromide (43) was prepared by h y d r o l y s i s of the t r i f l u o r o -(85) methyl g l y c o s i d e (42) ( k i n d l y provided by Dr.D.Dolphin) i n 457. hydrobromic a c i d i n a mixture of a c e t i c a c i d and a c e t i c anhydride. The r e a c t i o n was q u i t e slow t a k i n g s e v e r a l days to go to completion, (43) was i s o l a t e d i n 81% y i e l d . The a-bromide (43) was converted to 1, 3 ,4 , 6-tetra-O-acety 1-2-deoxy-2-f luoro-/?-(85) D-glucopyranose (44) i n 73% y i e l d , by r e a c t i o n of the a-bromide with mercuric a c e t a t e . P r e p a r a t i o n of 2-deoxy-2-fluoro-a -D-glucose-l-phosphate (45) by r e a c t i o n of the / J - t e t r a - a c e t a t e 0 ( 8 1 ) (44) with anhydrous phosphoric a c i d f o r 48 hours at 55 C was completed by d e a c y l a t i o n and c o n v e r s i o n to the eyelohexylammonium s a l t . T h i s allowed i s o l a t i o n of (45) i n 35% y i e l d . Note the long r e a c t i o n times r e q u i r e d . 1 19 (A)(v) H and F NMR assignments f o r the deoxyfluoro analogs of g l u c o s e - l - p h o s p h a t e . 1 19 Chemical s h i f t s and c o u p l i n g constants taken from H and F NMR s p e c t r a of the deoxyfluoro-glucose-l-phosphates are given i n t a b l e s V and VI. Resonances were assigned by comparison of c o u p l i n g p a t t e r n s (based on the expected 4 C ^ conformation) to the experimental data. In a l l cases H ( l ) resonates the f u r t h e s t down f i e l d , appearing as a double doublet, coupled to both H(2) and phosphorus. The magnitude of J i n d i c a t e s an e q u a t o r i a l proton at C ( l ) and hence a l l the compounds possess the a-anomeric c o n f i g u r a t i o n . -39-FLUORINATED POSITION H ( l ) H(2) H(3) H(4) H(5) H(6) H(6') F 6 5.44 3.46 3.78 3.51 4.00 4.74 - 237.48 4 5.52 3 .45 3.97 4.26 4.04 3.80 3.69 199.07 3 5.44 3.72 4.67 3.68 - 3.91 3.84 201.25 2 5.53 4.29 3.96 3.36 3.86 3.81 3.66 200.20 1 H s i g n a l p o s i t i o n s (8) are given with r e f e r e n c e to e x t e r n a l 2,2-dimethyl-2-silapentane-5-sulphonic a c i d (sodium s a l t ) (DSS). 19 F s i g n a l p o s i t i n s (0) are gi v e n with r e f e r e n c e to CFClg. T r i f l u o r o a c e t i c a c i d was used as an e x t e r n a l standard. - resonance p o s i t i o n could not be assigned due to c o i n c i d e n c e with other s i g n a l s Table V. Chemical s h i f t data f o r the monofluorinated analogs of a-D-glucose-l-phosphate. FLUORINATED 1 1 19 1 31 1 POSITION V H \> - H J M J5,6 J5,6' 2 J F - H J J 4 J »/• H 4 J 6 3.4 9.7 10.0 9.4 2.7 10.7 47 .2 31.0 - 7.2 1.7 4 3.5 9.0 9.0 9.0 4.0 12.4 50 .8 15.4 3.5 7.3 -3 3.7 8.0 8.8 8.9 - 12.0 54 .6 13.8 3.7 7.4 -2 3 . 6 9.0 9.6 9.6 4.8 12.0 47 .8 12.4 - 8.0 1.8 A l l v alues are g i v e n i n Hz - indeterminate non-zero c o u p l i n g . 1 1 1 19 1 31 Table VI. H- H, H - F, H - P c o u p l i n g constants f o r the mono f l u o r i n a t e d analogs of glucose-l-phosphate. -40-Where f l u o r i n e i s present a t C(3) and C(4), H ( l ) i s a l s o coupled to f l u o r i n e . The magnitude of these long range c o u p l i n g constants i s s i m i l a r to those observed i n the compounds 1,2,4,6-tetra-0-(86). acetyl-3-deoxy-3-fluoro-a-D-glucopyranose and 1,2,3,6-tetra-0 (85) acetyl-4-deoxy-4-fluoro-a-D-glucopyranose . Again these long range couplings are only observed f o r the a-anomers. For the 2-deoxy-2-fluoro and 6-deoxy-6-fluoro glucose-l-phosphates H(2) i s coupled over 4 bonds to phosphorus, t h i s c o u p l i n g i s a l s o seen (87) 3 4 i n a-glucose-l-phosphate . The magnitude of J and J i s approximately the same as f o r a-glucose-l-phosphate and t h i s suggests that the phosphate moiety e x i s t s trans to C ( 2 ) , as has (87) been suggested p r e v i o u s l y f o r a-glucose-l-phosphate 1 1 The s i z e of the H- H c o u p l i n g constants suggests a trans d i a x i a l c o n f i g u r a t i o n of H(2), H(3), H(4) and H(5) i n d i c a t i n g that the 4 sugar pyranose r i n g occupies the conformation. In g e n e r a l f l u o r i n a t i o n r e s u l t s i n a down f i e l d s h i f t of 0.8-0.9 ppm f o r the proton attached to the carbon bearing the f l u o r i n e . A sma l l e r down f i e l d s h i f t of 0.1-0.26 ppm f o r protons adjacent to the carbon bearing f l u o r i n e i s a l s o observed. 19 The F chemical s h i f t s are i n the normal range f o r f l u o r i n e a ttached to primary and secondary c e n t r e s of a carbohydrate (76,62a) • 19 1 The values observed f o r the geminal and v i c i n a l F- H c o u p l i n g constants are c l o s e to those observed f o r the a p p r o p r i a t e (23) f l u o r i n a t e d glucose . The value of J _ observed i n the 6-deoxyfluoro analog i n d i c a t e s the p r e f e r e d conformation of the f l u o r i n e attached to C(6) i s t r a n s d i a x i a l to C(5), the same as -41-(23,62a) f o r 6-deoxy-6-fluoro-D-glucose S e v e r a l other analogs of glucose and mannose c o n t a i n i n g f l u o r i n e a t C ( l ) and C(2) have been s y n t h e s i s e d . Recently Noyori (58) (59) et a l . and Szarek et a l . have prepared g l y c o s y l f l u o r i d e s by r e a c t i o n of the 1-unprotected or the 1-0-acetylated monosaccharide d e r i v a t i v e with poly hydrogen f l u o r i d e p y r i d i n e . In our hands treatment of 1, 2 ,3 ,4, 6-pen.ta-O-ace ty 1 -D-manno-pyranose with 707«> poly hydrogen f l u o r i d e i n p y r i d i n e f o r 5 hours at room temperature l e d to r e a c t i o n of a l l of the s t a r t i n g m a t e r i a l . P u r i f i c a t i o n of the major product by column chromatography gave 2,3,4,6-tetra-O-acetyl-a-D-mannopyranosy1 f l u o r i d e i n 537. y i e l d . D e a c e t y l a t i o n with 0.01 M sodium methoxide i n methanol at 0°C gave a-D-mannopyranosy1 f l u o r i d e as a c o l o u r l e s s gum which could not be c r y s t a l l i s e d . 3 , 4 , 6 -tri-0-acetyl-2-deoxy -2-fluoro-a-D-mannopyranosy1 f l u o r i d e (46) has been prepared by treatment of the ^ - f l u o r i d e (88) with l i q u i d hydrogen f l u o r i d e Again the use of p o l y hydrogen f l u o r i d e i n p y r i d i n e provided a more a c c e s i b l e route to t h i s compound. Rea c t i o n of the ^ - f l u o r i d e i n 707. poly hydrogen f l u o r i d e i n p y r i d i n e f o r 24 hours at room temperature gave complete c o n v e r s i o n to the thermodynamically favoured i X - f l u o r i d e (46). -42-D e a c e t y l a t i o n with sodium methoxide i n methanol gave 2-deoxy-2-fluoro-a-D-mannopyranosy1 f l u o r i d e . -43-(B) Synthesis of deoxy analogs of glucose and glucose-l-phosphate A l l of the i s o m e r i c deoxy-otrD-glucose-l-phosphates have been (89) (90,91) sy n t h e s i s e d by Zemek et a l . and Shibaev e t a l . . However f o r the purposes of t h i s study only the 6-deoxy and the 4-deoxy-c* -D-glucose-l-phosphates have been prepared. (B)(1) Deoxygenation of the 6 - p o s i t i o n . The route employed f o r the p r e p a r a t i o n of 6-deoxy-<X-glucose-1-phosphate (50) i s g i v e n i n f i g u r e 19. F i g u r e 19.The s y n t h e t i c route f o r 6-deoxy-oVD-glucose-l-phosphate -44-(92) 1,2,3,4-tetra-O-acety1-6-0-tosy1 - 0-D-glucopyranose (47) was t r e a t e d with sodium i o d i d e under r e f l u x i n DMF f o r 1 hour to e f f e c t c o n v e r s i o n to 1,2,3,4-tetra-0-acetyl-6-deoxy-6-iodo-0-D-glucopyranose (48) i n 707. y i e l d . Reduction of (48) with 107. p a l l a d i u m on carbon under an atmosphere of hydrogen, at room temperature gave l,2,3,4-tetra-0-acetyl-6-deoxy - / J-D-glucopyranose (49) . Treatment of (49) with anhydrous phosphoric a c i d by the (81) method of MacDonald gave 6-deoxy-cX-D-glucose-l-phosphate (50) i s o l a t e d i n 337. y i e l d as the dicyclohexylammonium s a l t . ( B ) ( i i ) Deoxygenation of the 4 - p o s i t i o n . The pathway u t i l i s e d f o r the p r e p a r a t i o n of 4-deoxy -oC-D-glucose -1-phosphate (49) i s g i v e n i n f i g u r e 20. H 2 S 0 4 (CH 3CO) 20 CH.COOH WH3PO4 ( 2JLiOH (1) HBr/CH 3 COOH (2) Hg(CH 3 C00) F i g u r e 20.The s y n t h e t i c route f o r 4-deoxy-cxrD-glucose-l-phosphate -45-Methyl 4-deoxy -oC-D-glucopyranoside (51) was prepared by the (62) method of Lopes and T a y l o r and was converted to the oC-tetra-a c e t a t e (52) by treatment with s u l p h u r i c a c i d i n a c e t i c a c i d and a c e t i c anhydride. Conversion to the ^-anomer of the t e t r a - a c e t a t e (53) was achieved by r e a c t i o n of the o<-bromide with mercuric a c e t a t e i n the normal manner. 4-deoxy-c<-D-glucose-l-phosphate (81) (54) was prepared by the MacDonald procedure and i s o l a t e d as the dicyclohexylammonium s a l t . ( B ) ( i i i ) Deoxygenation of the 1 - p o s i t i o n . (92) The compound 1,5-anhydro-D-glucitol (55) was prepared by r e d u c t i o n of 2 , 3 ,4 , 6-tetra-O-acety 1-CX-D-glucopyranosy 1 bromide (93) with t r i b u t y l t i n h y d r i d e . Q u a n t i t a t i v e c o n v e r s i o n to 2 , 3,4 , 6-tetra-O-acety1-1,5-anhydro-D-glucito 1 a f t e r 1 hour a t 80°C was observed . D e a c e t y l a t i o n of t h i s t e t r a - a c e t a t e with 0.1M sodium methoxide i n methanol gave (55) with an o v e r a l l y i e l d of 7 0%. (55) -46-(C) I n h i b i t i o n s t u d i e s on glycogen phosphorylase b ( T - s t a t e ) . The deoxy and deoxyfluoro analogs of glucose and mannose were t e s t e d as i n h i b i t o r s of glycogen phosphorylase b. Most of the i n h i b i t o r s were tes t e d at s e v e r a l c o n c e n t r a t i o n s f o r each c o n c e n t r a t i o n of s u b s t r a t e , the data p l a c e d on H i l l p l o t s ( f i g u r e s 21-25) and the apparent K i values obtained from a r e p l o t of apparent Km vs i n h i b i t o r c o n c e n t r a t i o n (see appendix 1). K i values f o r compounds which showed no s i g n i f i c a n t i n h i b i t i o n a t c o n c e n t r a t i o n s g r e a t e r than 100 mM were not determined p r e c i s e l y , s i n c e i n h i b i t i o n at such high c o n c e n t r a t i o n s could be (48) the r e s u l t of non s p e c i f i c e f f e c t s . Approximate K i values f o r these compounds were determined by v a r y i n g i n h i b i t o r c o n c e n t r a t i o n at a s i n g l e f i x e d s u b s t r a t e c o n c e n t r a t i o n and p l a c i n g the r e s u l t s on a p l o t a c cording to the method of Dixon (94) . The r e s u l t s obtained i n t h i s f a s h i o n are i n f i g u r e s 26-31. A l l analogs were found to be i n h i b i t o r y , e x h i b i t i n g n o n l i n e a r (48) competitive k i n e t i c s i n the same manner as glucose i t s e l f The r e s u l t s are summarised i n t a b l e s VII and V I I I . E a r l i e r s t u d i e s have suggested t h a t the T - s t a t e enzyme i s s p e c i f i c i n i t s r e c o g n i t i o n of oC-D-glucose as an i n h i b i t o r . (95) C o r i and C o r i have demonstrated that / J-D-glucose i s non i n h i b i t o r y although more r e c e n t r e s e a r c h has c a s t doubt on (96) t h i s f i n d i n g . Withers et a l . have a l s o c a r r i e d out an e x t e n s i v e study on the s p e c i f i c i t y of the glucose b i n d i n g s i t e i n (48) the T - s t a t e enzyme . They found that with the e x c e p t i o n of 3-aminoglucose and D - g l u c a l , analogs s u b s t i t u t e d at any but the 2 - p o s i t i o n had no s i g n i f i c a n t i n h i b i t o r y a c t i v i t y (Ki>>100 mM). 47-8 . *» lmuV' / I N H I B I T O R (mm) F i g u r e 21. I n h i b i t i o n of phosphorylase b by 2-deoxyfluoro-a-D-g l u c o s y l f l u o r i d e . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km (apparent) are given i n parentheses; (+) no i n h i b i t o r (1.8 mM), («) 0.5 mM (7.76 mM), (•) 1.0 mM (13.4 mM), (•) 1.5 mM (18.4 mM), (•) 3.0 mM (38.9). Log ["_!L_1 I N H I B I T O R (mM) F i g u r e 22. I n h i b i t i o n of glycogen phosphorylase b by 2-deoxyfluoro-^-D-glucosyl f l u o r i d e . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km (apparent) are given i n patentheses; ( + ) no i n h i b i t o r (2.34 mM), (*) 2.0 mM (4.76 mM) , (•) 4.0 mm (6.67 mM), (•) 6.0 mM (9.05 mM),(-) 12,0 mM (18.7mM). -48-s e l i e ' INHMUTOR ( M M ) F i g u r e 23. I n h i b i t i o n of phosphorylase b by 2-deoxyf luoro-oC* D-mannopyranosy1 f l u o r i d e . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km (apparent) are given i n parentheses; (•) no i n h i b i t o r (4.49 mM), (•) 62.4 mM (7.00 mM), (•) 83.2 mM ( 7.59mM), (*) 104 mM (9.00 mM), (+) 125 mM (10.5 mM) . -1.0 • 16 20 38 40 I N H t a i T O * ( • » « ) F i g u r e 24. I n h i b i t i o n of phosphorylase b by 4-deoxyfluoro-D-g l u c o s e . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km (apparent) are given i n parentheses;(•) no i n h i b i t o r (4.95) (•) 5.0 mM (4.28 mM),(4) 10.1 mM (4.62 mM), (") 20.1 mM (6.0 mM), (+) 30.2 mM (7.3 mM). -49-.6-. 4 -I N H I B I T O R ( « M ) F i g u r e 25. I n h i b i t i o n of phosphorylase b by 1,5-anhydro - D - g l u c i t o l . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used , values of Km (apparnt) are given i n parentheses; (•) no i n h i b i t o r (4.1 mM), (•) 4.5 mM (5.1 mM), (») 9.04 (6.76 mM), (+) 15.1 mM (8.2 mM), (•) 20.1 mM (10 mM). Vw (ug/min/mg) .03 ^+ -.02 < i r— — i 1 1 1 " 1 ' .01 1 r  1  i  1 1 1 r -800 "600 -400 "200 0 200 INHIBITOR (mM) F i g u r e 26. I n h i b i t i o n of phosphorylase b by 2-deoxyf luoro - J S -D-mannosyl f l u o r i d e . -50-Vv (ug/min/mg) _ 1 .05-.03; Vim, J .01 ' i i i i i 1 i i i i I • l I 1 • • • • 1 . . > I 1 1 • • • I t -50 0 50 100 150 200 INHIBITOR (mM) F i g u r e 27. I n h i b i t i o n of phosphorylase b by 2-deoxyfluoro-D-mannose. Vv (ug/min/mg) 1 .04-A—* . 0 2 Vvm ! . 0 1 ' • i i i 1 1 1 1 1 I I I 1 1 1 -150 -50 50 150 INHIBITOR (mM) F i g u r e 2 8 . I n h i b i t i o n of phosphorylase b by cX-D-mannosyl f l u o r i d e . -51-Vv (ug/min/mg) - . 0 4 - . 0 2 V v m r i - . 0 1 1 - 2 0 0 " 1 0 0 0 1 0 0 INHIBITOR (mM) F i g u r e 29. I n h i b i t i o n of phosphorylase b by 3-deoxyfluoro-D-glucose. Vv ( u g / i n i n / m g ) . 0 7 -. 0 5 -• . 0 1 -i ' i i i —i l l l l -H 1 1 1 1 1 1 1 - 5 0 0 5 0 1 0 0 INHIBITOR (mM) F i g u r e 30. I n h i b i t i o n of phosphorylase b by 6-deoxyfluoro-D-glucose. -52-• * ' l \ 1 1 I 1 1 1 I .01 —• i i i— -469 -366 -206 -106 0 100 200 INHIBITOR (MM] F i g u r e 31. I n h i b i t i o n of phosphorylase b by 1,2-dideoxy-D-glucose INHIBITOR K i (mM) c * * G b i n d (KJ/mol) INHIBITOR K i (mM) c * A G b i n d (kJ/mol) D-glucose 2.0 -b 2 - f l u o r o c<-glucosyl f l u o r i d e 0.2 -3.1 a Ot-glucosyl f l u o r i d e 0.6 -2 - f l u o r o )3-glucosyl f l u o r i d e 1.6 2.1 b J3-glucosyl f l u o r i d e 3.8 4.3 2 - f l u o r o ot-mannosy 1 f l u o r i d e 75 12 1-deoxy glucose 10.7 6.7 2 - f l u o r o jB-mannosy 1 f l u o r i d e K 800 as 18 1,2-dideoxy glucose «600 « 17 OC-mannosy 1 f l u o r i d e « 225 «, 14 a Data from Withers et a 1.(reference 48). b from per s o n a l communication Dr S.G.Withers. c Values c a l c u l a t e d fromAAG « - RT Ln (K 1/ K 2 ) , where R B 8.314 J/K/mol, T - 303 K, K 1- K i of tX-glucosyl f l u o r i d e . Table V I I . D i s s o c i a t i o n constants f o r v a r i o u s T - s t a t e i n h i b i t o r s of glycogen phosphorylase b. -53-INHIBITOR Ki (mM) a 0 A A G b m d (kj/mol) b Anomeric composition (X ot-anomer) D-Glucose 2.0 - 36 c 2-deoxyfluoro -D-glucose 1.9 - 45 c 2-deoxy -D-glucose 27 6.5 47 3-deoxyfluoro -D-glucose «200 <* 12 47 4-deoxyfluoro -D-glucose 25 6.4 41 6-deoxyfluoro -D-glucose «90 « 10 44 c 6-deoxy -D-glucose >>100 >>10 45 2-deoxyfluoro -D-mannose « 90 «1 0 68 C a l c u l a t e d from the equation A A G -= -RT Ln(K 1 / where R - 8.314 J/K/Mol, T - 303 K, K 1 - K i of D-glucose K„ " K i of glucose analog, b Data from Wray and P h i l l i p s ( r e f e r e n c e 23). c Data from Withers et a l . ( r e f e r e n c e 48). Table V I I I . D i s s o c i a t i o n constants f o r v a r i o u s T - s t a t e i n h i b i t o r s of glycogen phosphorylase b. -54-On the other hand, m o d i f i c a t i o n a t the 2 - p o s i t i o n y i e l d e d a range of K i values v a r y i n g over two orders of magnitude ( i n order of i n h i b i t o r y a c t i v i t y glucose = 2-deoxyfluoro glucose > 2-aminoglucose > 2-deoxy glucose >> mannose). These r e s u l t s were r a t i o n a l i s e d i n s t e r e o c h e m i c a l terms, as r e s u l t i n g from s t e r i c c o n f l i c t s with p r o t e i n r e s i d u e s at the b i n d i n g s i t e and v a r i a t i o n s i n hydrogen-bonding a b i l i t y of the analog. With the p o s s i b i l i t y that only one of the anomeric forms of glucose i s i n h i b i t o r y i t i s important to e s t a b l i s h that the K i values obtained with the analogs (presented i n t a b l e V I I I ) are due to the e f f e c t s of f l u o r i n a t i o n and not due to d i f f e r e n c e s i n anomeric composition. A comparison of the anomeric r a t i o s f o r the f l u o r i n a t e d glucose analogs i n d i c a t e s that f l u o r i n a t i o n a t v a r i o u s p o s i t i o n s on the pyranose r i n g has very l i t t l e e f f e c t on anomeric composition. Thus the v a r i a t i o n s of K i seen w i t h i n the s e r i e s are i n t e r n a l l y s e l f - c o n s i s t e n t and can be d i r e c t l y a t t r i b u t e d to the e f f e c t s of f l u o r i n a t i o n . With the p r o v i s o that the analogs must bind to the p r o t e i n i n a p o s i t i o n and o r i e n t a t i o n s i m i l a r to that of glucose i t s e l f i t i s p o s s i b l e to i n t e r p r e t r e d u c t i o n i n i n h i b i t o r y a c t i v i t y i n terms (48) of s t e r i c c o n f l i c t s and or l o s s of hydrogen-bonds 2-fluoro-D-glucose possesses approximately the same K i value as D-glucose and t h e r e f o r e must be i n v o l v e d i n s i m i l a r p r o t e i n l i g a n d i n t e r a c t i o n s . Removal of a l l s u b s t i t u e n t s at C(2) (2-deoxy glucose) r e s u l t s i n an i n c r e a s e i n d i s s o c i a t i o n constant of j u s t over an order of magnitude, i n d i c a t i n g the l o s s of some important i n t e r a c t i o n with the p r o t e i n . -55-Th i s corresponds to a d i f f e r e n c e i n f r e e energy of bi n d i n g of 6.5 kJ/mol between glucose and 2-deoxy-D-glucose. T h i s d i f f e r e n c e could be accounted f o r by l o s s of a weak hydrogen-bond. With the premise that f l u o r i n e can only a ct as a hydrogen-bond a c c e p t o r , the r e s u l t s would i n d i c a t e that the normal f u n c t i o n of the C(2) hydroxyl group i s that of a hydrogen-bond a c c e p t o r . Comparison of the r e s u l t s f o r D-glucose , cA-D-glucosyl f l u o r i d e and 1,5-anhydro-D-glucitol ( t a b l e V I I I ) would i n d i c a t e a s i m i l a r f u n c t i o n f o r the anomeric h y d r o x y l group. T h i s r e s u l t i s (96) i n keeping with the data obtained by A r i k i and Fukui , who found that the value of K i could be r e l a t e d to the e l e c t r o n e g a t i v i t y of the s u b s t i t u e n t at C ( l ) . Thus w i t h i n the s e r i e s that they s t u d i e d , the i n h i b i t o r y p r o p e r t i e s of the analogs were observed to decrease i n the order -F > -OH > -NH > -SH . These r e s u l t s were i n t e r p r e t e d as being c o n s i s t e n t with the C ( l ) h y d r o x y l group a c t i n g as a hydrogen-bond a c c e p t o r . F l u o r i n a t i o n a t p o s i t i o n s 3,4 and 6 lea d s , i n v a r y i n g degrees, to a l o s s of i n h i b i t o r y p r o p e r t i e s . Thus these p o s i t i o n s would appear to have a s i m i l a r f u n c t i o n i n s u b s t r a t e b i n d i n g , probably that of a hydrogen-bond donor. A comparison of the K i values obtained f o r 6-deoxyfluoro-D-glucose (approximately 90 mM) and 6-deoxy-D-glucose (>>100 mM) would i n d i c a t e that the 6 - p o s i t i o n a l s o has the a d d i t i o n a l r o l e of a hydrogen-bond a c c e p t o r . The l o s s of b i n d i n g energy (approximately 12 kj/mol) o c c u r i n g on b i n d i n g 3-fluoro-D-glucose and (approximately 10 kj/mol) on b i n d i n g 6-fluoro-D-glucose i n d i c a t e s that r e l a t i v e l y strong i n t e r a c t i o n s occur with these p o s i t i o n s . - 56-Th e compound 0 - g l u c o s y l f l u o r i d e was found to be i n h i b i t o r y (Ki=3.8 mM) although i t was observed to bind approximately s i x f o l d more weakly than the cX-anomer. Thus i t i s s t i l l a good i n h i b i t o r of glycogen phosphorylase and t h i s suggests that /J-D-glucose might a l s o be an i n h i b i t o r . Presumably the m a j o r i t y of the l o s s i n b i n d i n g f r e e energy between c<-glucosyl f l u o r i d e and /J-glucosyl f l u o r i d e can be accounted f o r by the l o s s of the p r e v i o u s l y observed p o s i t i v e i n t e r a c t i o n with the a x i a l anomeric f l u o r i n e . The r e s u l t s would t h e r e f o r e i n d i c a t e that the presence of the anomeric f l u o r i n e i n an e q u a t o r i a l o r i e n t a t i o n can be accomodated i n the b i n d i n g s i t e without s e r i o u s s t e r i c consequences. R e s u l t s with /J-glucosylamine would tend to support (96) t h i s By comparison of the K i values obtained from the two anomers of 2-deoxyfluoro-D-glucosyl f l u o r i d e and the K i v a l u e s obtained f o r CXand ^ - g l u c o s y l f l u o r i d e , a s i m i l a r trend can be observed. Again i n v e r s i o n of c o n f i g u r a t i o n a t the anomeric carbon ( t o g i v e 2-fluoro-/?-D-glucosy 1 f l u o r i d e ) r e s u l t s i n the l o s s of some p o s i t i v e i n t e r a c t i o n between the p r o t e i n and the a x i a l f l u o r i n e s u b s t i t u e n t a t C ( l ) . However as only a small i n c r e a s e i n K i (8 f o l d ) i s observed i t would appear t h a t a g a i n the e q u a t o r i a l f l u o r i n e of the /J-anoraer i s accomodated wothout s e r i o u s s t e r i c consequences. In complete c o n t r a s t to the 1 - p o s i t i o n , i n v e r s i o n of c o n f i g u r a t i o n at C(2) ( 2-deoxyfluoro-D-mannose and 2-deoxyf luoro-ot-D-mannosy 1 f l u o r i d e ) g i v e s r i s e to a dramatic l o s s i n i n h i b i t o r y e f f e c t . Given the small s i z e of the b i n d i n g energy ( A A G = 6 . 7 kJ/mol) a t t r i b u t e d to the i n t e r a c t i o n s i n v o l v i n g -57-th e e q u a t o r i a l s u b s t i t u e n t at the 2 - p o s i t i o n , i t appears u n l i k e l y that t h i s dramatic i n c r e a s e i n K i i s s o l e l y a consequence of a l o s t hydrogen-bond. Severe s t e r i c i n t e r a c t i o n s between the a x i a l f l u o r i n e s u b s t i t u e n t and a p r o t e i n r e s i d u e provide a more p l a u s i b l e e x p l a n a t i o n . T h i s argument i s r e i n f o r c e d by the value of the d i s s o c i a t i o n constant observed f o r ot-mannosyl f l u o r i d e (Ki=*225 mM) . In t h i s case the unfavorable i n t e r a c t i o n s appear to be much g r e a t e r , the s e v e r i t y of the s t e r i c c l a s h being i n c r e a s e d by the presence of the l a r g e r h ydroxyl group. A second i n v e r s i o n of c o n f i g u r a t i o n ( a t C ( l ) ) to g i v e 2-fluoro -0-D-mannosy1 f l u o r i d e leads to the l o s s of almost a l l i n h i b i t o r y p r o p e r t i e s . L i k e w i s e removal of both s u b s t i t u e n t s at the 1- and 2 - p o s i t i o n s (1,2-dideoxy-D-glucose) a l s o produces a very poor i n h i b i t o r . A summary of the hydrogen-bonding r o l e s f o r each of the g l u c o s y l h ydroxyl groups i s giv e n i n t a b l e IX, below. POSITION ROLE STRENGTH OF INTERACTION 6 D/A medium 4 A weak 3 D 8 trong 2 A weak 1 A weak A = hydrogen-bond a c c e p t o r , D = hydrogen-bond donor. Table IX. The hydrogen-bonding r o l e s of the v a r i o u s g l u c o s y l h y d r o x y l groups. -58-(D) A comparison with recent c r y s t a l l o g r a p h i c data. Throughout the f o l l o w i n g d i s c u s s i o n comparisons w i l l be drawn between the s t r u c t u r e of the phosphorylase glucose s i t e and that of another carbohydrate b i n d i n g p r o t e i n , arabinose b i n d i n g p r o t e i n (ABP). ABP binds both the c* and £ forms of D-arabinose and f u n c t i o n s i n the a c t i v e t r a n s p o r t of the sugar across (97,98) b a c t e r i a l c e l l membranes . Thus the two p r o t e i n s have evolved to f u l f i l l d i f f e r e n t b i o l o g i c a l r o l e s ; ABP has to bind i t s l i g a n d extremely t i g h t l y to t r a n s p o r t i t across the b a c t e r i a l c e l l w a l l (Kd arabinose = l x l 0 ~ 5 M). As no chemical change i s induced i n the l i g a n d , the p r o t e i n has evolved to maximise i t s i n t e r a c t i o n s with the ground s t a t e s t r u c t u r e of the sugar. In comparison phosphorylase has evolved to c a t a l y s e the con v e r s i o n of a s u b s t r a t e to a product and t h e r e f o r e most of the i n t e r a c t i o n s of the p r o t e i n with i t s l i g a n d are maximised f o r a (99,100) t r a n s i t i o n s t a t e and not f o r the ground s t a t e molecule . A t e r t i a r y s t r u c t u r e refinement of the liganded form of ABP has (101) r e c e n t l y been pu b l i s h e d by Quiocho and Vyas . Given the d i f f e r e n c e i n f u n c t i o n of the two p r o t e i n s some i n t e r e s t i n g s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p s can be found. Recently Sprang et a l . have completed a refinement of the c r y s t a 1 l o g r a p h i c data f o r the glucose b i n d i n g s i t e i n (102) phosphorylase a . Data p e r t i n e n t to the i n t e r a c t i o n s of glucose with p r o t e i n r e s i d u e s at the bindin g s i t e are presented l a t e r i n t a b l e X and f i g u r e 32. While i t i s evident that d i f f e r e n c e s i n s t r u c t u r e between the (42,103) c r y s t a l forms of phosphorylase a and b do e x i s t ,a -59-comparison between the k i n e t i c r e s u l t s obtained with phosphorylase b and the s t r u c t u r e of the bindi n g s i t e i n phosphorylase a remains a v a l i d p r o p o s i t i o n f o r the f o l l o w i n g (48) reasons. Withers et a l . have found that a number of i n h i b i t o r s which bound to the T - s t a t e enzyme only, possessed approximately the same d i s s o c i a t i o n constant with both phosphorylase a and b. Most of the s t r u c t u r a l d i f f e r e n c e s between the two enzymic forms t h e r e f o r e appear to manifest themselves i n the R-state enzyme. A comparison of the contacts observed c r y s t a 1 l o g r a p h i c a 1 l y between phosphorylase b and gl u c o s e , glucose-l-phosphate, or glucose cyclic-1,2-phosphate with those between phosphorylase a (103) and glucose show a great many s i m i l a r i t i e s . In a d d i t i o n the amino a c i d s i n v o l v e d i n i n t e r a c t i o n s with the sugar moiety of (103) the potent i n h i b i t o r heptulose-2-phosphate i n phosphorylase b are i d e n t i c a l to those which are seen to i n t e r a c t with glucose i n phosphorylase a. Thus the d i f f e r e n c e s i n the glucose b i n d i n g s i t e between the two enzymic forms i n s o l u t i o n are probably very s m a l l , i f any at a l l . Hydrogen-bonds are recognised from c r y s t a l l o g r a p h i c data by the apparent s h o r t e n i n g of the van der Waals contact d i s t a n c e between a p u t a t i v e donor-acceptor p a i r . The normal van der Waals co n t a c t d i s t a n c e s f o r most b i o l o g i c a l l y s i g n i f i c a n t donor-acceptor p a i r s l i e between 3.5 and 3.75 A (based on summation of van der Waals r a d i i and c o v a l e n t bond l e n g t h s ) . The optimal c o n f i g u r a t i o n f o r a hydrogen-bond i s l i n e a r , but bending causes only small l o s s e s i n energy. Thus donor-acceptor d i s t a n c e s of 2.5 to 3.0 A and 0 C-0--0 angles between 100 and 180 are u s u a l l y i n d i c a t i v e of -60-(48) hydrogen-bond formation, but d i s t a n c e s upto 3.3 A are considered s i g n i f i c a n t . SUGAR ATOM PROTEIN RESIDUE DISTANCE • A ANGLE ABOUT OXYGEN C(1)0H LEU 136 N 3.3 123.7 TYR 572 OH 3.3 151.8 C(2)0H ASN 284 OtU 3.1 115.1 GLU 671 072 3.2 160.9 C(3)0H GLU 671 072 2.7 113.4 C(4)0H ASN 483 OtSl 3.3 162.0 GLY 674 N 3.0 86.9 C(6)0H HIS 376 NtU 2.5 136.9 ASN 483 0<5l 3.0 127.8 AVERAGE 3.1 + /- 0.3 130.9 +/-23 TABLE X. C r y s t a 1 l o g r a p h i c data f o r the glucose b i n d i n g s i t e of phosphorylase a. Data from F l e t t e r i c k and Sprang ( r e f e r e n c e 102). In s p e c t i o n of the data i n t a b l e X r e v e a l s that most of the p o t e n t i a l i n t e r a c t i o n s between the phosphorylase b i n d i n g s i t e and glucose are q u i t e weak. T h i s i s e x e m p l i f i e d by a comparison of the average donor-acceptor d i s t a n c e s i n phosphorylase of 3.1+/-0.3 A and the average d i s t a n c e of 2.8+/-0.1 A observed i n ABP. In phosphorylase only two i n t e r a c t i o n s between the p o t e n t i a l hydrogen-bonding p a i r s C(3)0H-GLU 671 and C(6)0H-HIS 376 possess a donor-aceptor d i s t a n c e of l e s s than 3 A. Out of ten p o t e n t i a l donor-acceptor i n t e r a c t i o n s i n ABP only two are separated by a di s t a n c e g r e a t e r than 3 A ! The thermodynamic data presented i n t a b l e s VII and VII I shows that major c o n t r i b u t i o n s to the bi n d i n g energy of the -61-alpha-D-QluooBe at Phosphorylafle A oatalytlo site F i g u r e 32. A schematic r e p r e s e n t a t i o n of p o t e n t i a l donor acceptor i n t e r a c t i o n s of glucose bound to phosphorylase From F e t t e r i c k and Sprang, ( r e f e r e n c e 102). -62-s u b s t r a t e are provided by the 3- and 6 - p o s i t i o n s of glucose, thus there i s good agreement between c r y s t a 1 l o g r a p h i c and k i n e t i c data. The k i n e t i c data suggests the C(3)0H acts as the hydrogen-bond donor. For t h i s purpose GLU 671, i t s hydrogen-bonding p a r t n e r , could e x i s t i n e i t h e r an i o n i s e d or protonated s t a t e . The r o l e of the C(6)0H as a hydrogen-bond donor could a l s o be f u l f i l l e d p r o v i d i n g Ngl of His 376 i s unprotonated. In t h i s case a p o s s i b l e second, much weaker i n t e r a c t i o n with ASN 483 i s a l s o observed. The i n t e r a c t i o n s of the p r o t e i n with the remaining hydroxyl groups would be p r e d i c t e d (from the c r y s t a l l o g r a p h i c data) to be much weaker. I n s p e c t i o n of the k i n e t i c data shows that again agreement i s good, the hydroxyl groups on carbons 1,2 and 4 p r o v i d i n g an average c o n t r i b u t i o n of 6.5+/-0.15 kj/mol of b i n d i n g energy each. These weak i n t e r a c t i o n s share a number of common f e a t u r e s : 1) The average acceptor d i s t a n c e of 3.2 A i s o u t s i d e the range considered normal f o r hydrogen-bonds, but s t i l l r e p resents a s i g n i f i c a n t s h o r t e n i n g of the van der Waals contact d i s t a n c e . 2) M u l t i p l e i n t e r a c t i o n s between a s i n g l e l i g a n d s i t e and the p r o t e i n are observed. C(2)0H i n t e r a c t s with TYR 572, ASN 284 and shares GLU 671 with the hydroxyl group on C ( 3 ) . C(4)0H i n t e r a c t s with the amide group of GLY 674 and shares ASN 483 with the adjacent hydroxyl group on C(6). 3) The assignment of a r o l e as donor or acceptor w i t h i n a given hydrogen-bonding p a i r becomes more d i f f i c u l t . The k i n e t i c r e s u l t s c l e a r l y placed the C(2)0H i n the r o l e of a c c e p t o r . T h i s -63-assignment i s a c c e p t a b l e f o r TYR 572 and GLU 671 (provided i t i s protonated),both of which can a c t as strong hydrogen-bond donors. However the c r y s t a l l o g r a p h i c data suggests that p o t e n t i a l l y the s t r o n g e s t i n t e r a c t i o n i s with the c a r b o n y l oxygen of ASN 284 and i n t h i s case the amino a c i d i s unable to a c t as a hydrogen-bond donor. For the anomeric hydroxyl group the k i n e t i c r e s u l t s suggest a r o l e as a hydrogen-bond acceptor and t h i s r o l e i s e a s i l y r e c o n c i l e d with the backbone amido group of LEU 136 a c t i n g as the donor of t h i s hydrogen-bonding p a i r . However t h i s proposed f u n c t i o n i s somewhat unusual s i n c e i t was observed i n ABP that the anomeric hydroxyls serve s o l e l y as hydrogen-bond donors, whereas the r e s t of the sugar hydroxyls serve as both donors and a c c e p t o r s . T h i s p a t t e r n i s a l s o observed i n the c r y s t a l packing (104) of carbohydrates 4) F l u o r i n e appears to be a very e f f i c i e n t replacement f o r the hydroxyl group i n conserving these weak i n t e r a c t i o n s . D espite the d i s c r e p a n c i e s noted i n p o i n t 3 ) , i n g e n e r a l the k i n e t i c r e s u l t s agree w e l l with the c r y s t a l l o g r a p h i c data. A comparison of these r e s u l t s with those obtained from ABP suggest that the glucose b i n d i n g s i t e i n phosphorylase has evolved to give a network of weak i n t e r a c t i o n s aimed at p r o v i d i n g a high s p e c i f i c i t y pocket f o r the glucopyranose moiety ( r a t h e r than a high s p e c i f i c i t y , high a f f i n i t y s i t e as In ABP ). T h i s f i t s w e l l with the r o l e of glucose as an a l l o s t e r i c i n h i b i t o r of glycogen phosphorylase. While i t i s u n l i k e l y that glucose performs any p h y s i o l o g i c a l l y important r e g u l a t o r y r o l e f o r muscle glycogen phosphorylase (no f r e e glucose i s present i n muscle t i s s u e ) , -64-the form of the enzyme which i s found i n the l i v e r and which i s (105) c l o s e l y r e l a t e d , may play a r o l e i n blood sugar homeostasis T y p i c a l l y blood glucose l e v e l s are kept f a i r l y constant at approximately 5 mM, however t h i s l e v e l w i l l vary between i n d i v i d u a l s and a c c o r d i n g to the time of day. Thus a high a f f i n i t y s i t e with a low d i s s o c i a t i o n constant would be of l i t t l e use i n performing any r e g u l a t o r y f u n c t i o n i n glycogen phosphorylase, as a l a r g e changes i n glucose c o n c e n t r a t i o n would be r e q u i r e d before the enzyme could be switched on or o f f . I t t h e r e f o r e appears that the phosphorylase glucose b i n d i n g s i t e i s p r i m a r i l y designed to provide a high s p e c i f i c i t y pocket which can d i s c r i m i n a t e between glucose and other n a t u r a l l y o c c u r r i n g sugars. Indeed sugars such as mannose (Ki>>1OOmM) , g a l a c t o s e (Ki>>100 mM) and x y l o s e (Ki>>100 mM) were a l l found to (48) be v i r t u a l l y non i n h i b i t o r y . Based on the r e s u l t s obtained from ot and J g-glucosyl f l u o r i d e i t appears that the glucose b i n d i n g s i t e i n phosphorylase i s able to d i s c r i m i n a t e between the two anomeric forms of glucose, b i n d i n g the c*. f o r i with higher a f f i n i t y than thej^anomer. In comparison ABP has evolved a complex system of i n t e r a c t i o n s which enable the p r o t e i n to bind both anomeric forms of the sugar with equal a f f i n i t y . The use of f l u o r i n e as a hydrogen-bonding probe s t i l l remains u n c l e a r . While f o r the 1 and 2 - p o s i t i o n s the replacement of a s u b s t r a t e hydroxyl group by f l u o r i n e was c l e a r l y an e f f i c i e n t s u b s t i t u t i o n , examination of the c r y s t a l l o g r a p h i c data r e v e a l e d that only p o t e n t i a l l y weak i n t e r a c t i o n s with p r o t e i n r e s i d u e s were p o s s i b l e f o r these two positions.. Roles of donor or acceptor - 6 5 -w i t h i n p o t e n t i a l hydrogen-bonding p a i r s , a l s o could not be assigned unambiguously f o r these two p o s i t i o n s . For the remaining s u b s t r a t e hydroxyl groups the r o l e of hydrogen-bond donor was assigned, based upon the o b s e r v a t i o n that the f l u o r i n a t e d analogs e x i b i t e d lower a f f i n i t y than d i d glucose f o r the p r o t e i n . These r e s u l t s could be r e c o n c i l e d with the c r y s t a l l o g r a p h i c data p r o v i d i n g c e r t a i n assumptions about i o n i s a t i o n s t a t e s of the p r o t e i n r e s i d u e s were made. These same r e s u l t s could have been obtained i f f l u o r i n e were to a c t only as a poor hydrogen-bond a c c e p t o r , able to r e p l a c e the hydroxyl group i n some c a p a c i t y but unable to maintain the f u l l i n t e g r i t y of the i n t e r a c t i o n with the p r o t e i n . Therefore care must be taken i n i n t e r p r e t a t i o n of these r e s u l t s . A c t u a l l y the unusual b i o l o g i c a l a c t i v i t y of f l u o r i n a t e d analogs probably does not s o l e l y r e l y on the a b i l i t y of the C-F fragment to conserve hydrogen-bonds, but a l s o on i t s a b i l i t y to promote other i n t e r a c t i o n s with the p r o t e i n . T h i s i s seen most c l e a r l y upon examination of the k i n e t i c data obtained with the analogs rX-glucosyl f l u o r i d e and 2-deoxyfluoro-cx-glucosy 1 f l u o r i d e . Both of these compounds bound to phosphorylase t i g h t e r than d i d glucose i t s e l f , a s i t u a t i o n which i s hard to understand i f f l u o r i n e i s considered as j u s t a hydrogen-bonding replacement f o r the hydroxyl group. The h i g h a f f i n i t y shown by phosphorylase f o r the d i f l u o r i n a t e d compound ( 2-deoxyf luoro-o<r glucosy 1 f l u o r i d e ) , i s somewhat r e m i n i s c e n t of the r e l a t i v e l y t i g h t b i n d i n g of 2,2-difluoro-D-arabinohexose (another d i f l u o r i n a t e d compound) to yeast hexokinase. The a c i d i f i c a t i o n of the C(3) hydroxyl group and consequent 66-strengthening of the hydrogen-bond i n which i t i s i n v o l v e d , could a l s o e x p l a i n the e x t r a a f f i n i t y shown by phosphorylase f o r t h i s compound. T h i s a b i l i t y of f l u o r i n e to i n c r e a s e the e f f e c t i v e n e s s of a neighbouring group's i n t e r a c t i o n s appears to i n c r e a s e as the number of f l u o r i n e s u b s t i t u e n t s i n the analog i n c r e a s e s . T h i s can be seen i n a number of i n s t a n c e s : (1) G l u c o s y l f l u o r i d e e x h i b i t s higher a f f i n i t y than D-glucose. (2) 2-deoxyfluoro-tX-D-glucosyl f l u o r i d e e x h i b i t s higher a f f i n i t y than c X-glucosyl f l u o r i d e . (3) 2-deoxyfluoro-^-D-glucosyl f l u o r i d e a l s o e x h i b i t s higher a f f i n i t y than / J-glucosyl f l u o r i d e . (4) On i n v e r s i o n of c o n f i g u r a t i o n (from &{toft) i n g l u c o s y l f l u o r i d e , 4.6 kj/mol of b i n d i n g energy i s l o s t . On performing the same i n v e r s i o n with 2-deoxyfluoro-ot-D-glucosyl f l u o r i d e 5.2 kj/mol of b i n d i n g energy i s l o s t . T h i s suggests that the i n t e r a c t i o n of the p r o t e i n with the a x i a l f l u o r i n e s u b s t i t u e n t i s some 0.6 kJ/mol stronger f o r the d i f l u o r i n a t e d compounds. Examination of the c r y s t a l packing s t r u c t u r e 2-deoxyf luoro-/?-D-mannosyl f l u o r i d e (see i n t r o d u c t o r y s e c t i o n on hydrogen-bonding) r e v e a l s the presence of weak i n t e r a c t i o n s between f l u o r i n e and protons which are attached to carbon atoms bearing an e l e c t o n e g a t i v e s u b s t i t u e n t . I t t h e r e f o r e could be p o s t u l a t e d that the presence of f l u o r i n e , p a r t i c u l a r l y i n the d i f l u o r i n a t e d compounds, could i n c r e a s e the d i s p o s i t i o n of C-H groups on the analog to p a r t i c i p a t e i n i n t e r a c t i o n s with p o l a r groups on the p r o t e i n . T h i s would r e s u l t i n a d d i t i o n a l " p o s i t i v e " -67-i n t e r a c t i o n s which would i n c r e a s e the a f f i n i t y of the enzyme (51 ) f o r the analog. Sansom et a l . have shown that over 707. of the contact area between the i n h i b i t o r , g l u c o s e - 1 , 2 - c y c l i e phosphate and phosphorylase b came from contacts with the non-polar core of the glucopyranose r i n g . These contacts must t h e r e f o r e provide a s u b s t a n t i a l p o r t i o n of the o v e r a l l b i n d i n g energy. Any f a c t o r which can i n c r e a s e the i n t e r a c t i o n s between the p r o t e i n and the c o n - p o l a r core of the glucopyranose r i n g , e i t h e r i n q u a n t i t y c r t e n a c i t y , w i l l a l s o p l a y a r o l e i n i n c r e a s i n g the a f f i n i t y of •;he p r o t e i n f o r i t s l i g a n d . A diagramatic summary of the p r o t e i n - 1 i g a n d i n t e r a c t i o n s (obtained from the c r y s t a l l o g r a p h i c data) of glucose i n the b i n d i n g s i t e of phosphorylase a are given i n f i g u r e s 33, 34 and 35. These are photographs taken from the g r a p h i c s screen on which was p r o j e c t e d the a c t i v e s i t e r e g i o n of the phosphorylase a glucose complex. These f i g u r e s were k i n d l y provided by Dr S.Sprang. The molecule of glucose i s marked i n l i g h t blue i n the c e n t r e of the diagram (the hydroxyl groups i n r e d ) . Spheres r e p r e s e n t i n g van der Waals r a d i i of the glucose atoms are represented by blue d o t s . Contacts between the van der Waals r a d i i of the p r o t e i n r e s i d u e s with those of the glucose molecule are represented as red d o t s . F i g u r e 33 shows a s i d e view of the CX-D-glucose molecule; note the stong contact between the h y d r o x y l group on C(6) and HIS 376. F i g u r e 34, represents a view from the lower s i d e of the glucopyranose r i n g , again note the strong contact between the C(3) hydroxyl group and ASP 671. In F i g u r e 35, the weaker con t a c t between C(4)0H and the p r o t e i n , -68-as w e l l as the c o n t a c t of the anomeric hydroxyl group with the backbone amido group of LEU 136 can a l s o be seen. * .gure 33. The a c t i v e s i t e r e g i o n of the phosphorylase a -glucose complex-. Figure 34. The a c t i v e s i t e r e g i o n of the phosphorylase a -glucose complex. Figure 35. The a c t i v e s i t e r e g i o n of the phosphorylase a -glucose complex. -70-(D) I n h i b i t i o n s t u d i e s on glycogen phosphorylase b ( R - s t a t e ) . The deoxy and deoxyfluoro analogs of glucose-l-phosphate were t e s t e d f o r s u b s t r a t e a c t i v i t y with glycogen phosphorylase b. None of the analogs were found to a c t as s u b s t r a t e s even a t h i g h enzyme c o n c e n t r a t i o n s (120 u g / r e a c t i o n mix) and prolonged r e a c t i o n times (18 h o u r s ) . The analogs were a l s o analysed as i n h i b i t o r s of glycogen phosphorylase b. As with the glucose analogs a value f o r K i was obtained by determining r e a c t i o n r a t e s at s e v e r a l d i f f e r e n t i n h i b i t o r c o n c e n t r a t i o n s f o r each s u b s t r a t e c o n c e n t r a t i o n . The data were then p l o t t e d on a double r e c i p r o c a l p l o t (1/v vs 1/s) to f a c i l i t a t e the r e c o g n i t i o n of i n h i b i t i o n p a t t e r n s , but values f o r Km(apparent) and e r r o r s a s s o c i a t e d with t h i s v a l u e , were determined by use of the computer program l i s t e d i n appendix 2. (106) The program i s based on the s t a t i s t i c a l method of W i l k i n s o n A p l o t of Km(apparent) vs i n h i b i t o r c o n c e n t r a t i o n gave a value f o r K i . The r e s u l t s are given i n f i g u r e s 36 to 39. A l l compounds e x i b i t e d l i n e a r i n h i b i t i o n k i n e t i c s , showing p a t t e r n s t y p i c a l of c o mpetitive i n h i b i t i o n with glucose-l-phosphate. S i m i l a r r e s u l t s have been obtained f o r other R-state (107) i n h i b i t o r s such as g l u c o s e - 1 , 2 - c y c l i c phosphate , p y r i d o x a l (108) (109) (5 ' )diphospho(l)-o4-D-glucose , heptulose-2-phosphate , (49) UDP-glucose , mannose-l-phosphate, l-phospho glucuronate (48) and phenyl phosphate . The values of K i ( a p p a r e n t ) obtained f o r the deoxyfluoro analogs of glucose-l-phosphate are given i n t a b l e XI. -71-F i g u r e 36. I n h i b i t i o n of phosphorylase b by 2-deoxyfluoro-0< -glucose-l-phosphate. The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km apparent are given i n parentheses. (•) no i n h i b i t o r (3.0 mM) , (•) 1.0 mM (4.5 mM) , (•>) 2.0 mM (6.5 mM), (+) 3.8 mM (9.0 mM) (*) 7.5 mM (15.1 mM). _ 1/V lug/min/mgJ F i g ure 37. I n h i b i t i o n of phosphorylase b by 3deoxyfluoro-0<-glucose-l-phosphate. The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values of Km (apparent) are given i n parentheses. (•) no i n h i b i t o r (2.4 mM), (•) 15.0 mM ( 3.1 mM), (*) 25.0 mM (3.7 mM), (+) 40 mM (4.8 mM). - 7 2 -— • 3 — • 1 • 1 • 3 «5 • 7 1/8 (mM)"' F i g u r e 38. I n h i b i t i o n of phosphorylase b by 4-deoxyfluoro-a-glucose-l-phosphate. The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were used, values f o r Km (apparent) are given i n parentheses. (+) no i n h i b i t o r (2.8 mM), (") 10.0 mM (3.6 mM), (•) 20.0 mM (4.8 mM), (•) 29.5 mM (5.4 mM), 39.5 mM <4.7 mM). . V, (mM)"1 F i g u r e 39. I n h i b i t i o n of phosphorylase b by 6-deoxyfluoro-a-glucose-l-p h o s p h a t e . The f o l l o w i n g i n h i b i t o r c o n c e n t r a t i o n s were as f o l l o w s , values of Km (apparent) are given i n parentheses (••) no i n h i b i t o r (4.4 mM), (•) 13.1 mM (3.5 mM), (•) 21.8 mM (4.1 mM), (*) 34.8 (5.4 mM), (+) 43.5 mM (6.2 mM). -73-FLUORINATED K i (mM) POSITION 6 2 5.1 4 30.2 3 39.0 2 2.0 Table XI. K i values f o r the b i n d i n g of fluorodeoxy analogs of glucose-l-phosphate to phosphorylase b. Determination of the K i valu e s f o r 4-deoxy and 6-deoxy-OC-D-glucose-l-phosphates were hampered by h y d r o l y s i s of the compounds oc c u r i n g during the assay procedure. To some extent t h i s problem was overcome by the use of c o n t r o l s during the assay, but e r r o r s a s s o c i a t e d with t h i s data made i t impossible to determine a value f o r K i with any degree of c e r t a i n t y , t h e r e f o r e r e s u l t s are not presented f o r these compounds. Apart from the value obtained f o r 2-deoxyfluoro-o(-glucose-l-phosphate the d i s s o c i a t i o n constants f o r a l l the other analogs of glucose-l-phosphate f a l l w i t h i n the range 25 to 40 mM, these r e s u l t s are c o n s i s t e n t with those obtained i n an e a r l i e r study by (48) Withers et a l . . They found that the compounds ot>D-mannose-1-phosphate ( K i = 35 mM) and phenyl phosphate ( K i = 19 mM) both possessed s i m i l a r d i s s o c i a t i o n constants and were unable to demonstrate, c r y s t a 1 l o g r a p h i c a 1 l y , the b i n d i n g of e i t h e r x y lose-l-phosphate or phenyl phosphate to c r y s t a l l i n e -74-phosphorylase a. In c o n t r a s t to the T - s t a t e i n h i b i t o r s , i t was found that these analogs bound with decreased d i s s o c i a t i o n constant ( g r e a t e r a f f i n i t y ) to phosphorylase a. They concluded that the o b s e r v a t i o n of such s i m i l a r K i v a l u e s , obtained f o r such d i v e r s e compounds, i n d i c a t e d that they bound at a s i t e which recognised the phosphate moiety o n l y , and that the r e s u l t s were i n c o n s i s t e n t with the compounds b i n d i n g at both the glucose and the phosphate s u b s i t e s . The s m a l l v a r i a t i o n i n K i values observed f o r the deoxyfluoro analogs of glucose-l-phosphate i s hard to r e c o n c i l e with a d i f f e r e n t i a l a b i l i t y to form hydrogen-bonds and does indeed i n d i c a t e that i n t e r a c t i o n s with the glucopyranose r i n g only make a minimal c o n t r i b u t i o n to the o v e r a l l b i n d i n g energy. The e x c e p t i o n to t h i s trend i s the 2-deoxyf luoro-<X>D-glucose-l-phosphate. Ve have a l r e a d y seen i n the T - s t a t e enzyme that an e q u a t o r i a l f l u o r i n e s u b s t i t u e n t a t C(2) d i d not a f f e c t the a b i l i t y of the analog to bind to the enzyme. T h i s r e s u l t c ould r e f l e c t a s i m i l a r i t y i n the hydrogen-bonding network between the R and T-state«» of the enzyme. A l t e r n a t i v e l y the C(2) hydroxyl group may not be i n v o l v e d i n any s u b s t r a t e - p r o t e i n i n t e r a c t i o n s at a l l i n the R-state enzyme. I t appears from the r e s u l t s obtained with these R-state i n h i b i t o r s , that even small changes i n s t r u c t u r e (with the exception of C(2)0H ) render the analog unable to bind a t both the glucose and the phosphate s u b s i t e s . In c o n s i d e r a t i o n of the NMR data presented e a r l i e r , which showed that a l l of the deoxyfluoro glucose-l-phosphate analogs adopted the "normal" 4C -75-c h a i r , t h i s i s u n l i k e l y to be caused by any gross conformational d i s t o r t i o n s present i n the analogs. The small s i z e of the C-F group a l s o renders s t e r i c i n c o m p a t i b i l i t y as an u n l i k e l y e x p l a n a t i o n . I t would appear that a small r e d u c t i o n i n p o t e n t i a l b i n d i n g energy, which i s a s s o c i a t e d with the replacement of the hydroxyl group by f l u o r i n e , renders the analog unable to bind simultaneously at both the glucose and phosphate s u b s i t e s . A mechanism by which the enzyme could accomplish t h i s can be proposed as f o l l o w s . The a c t i v e s i t e (which i s composed of both the glucose and phosphate s u b s i t e s ) i s s i t u a t e d on the boundary of the N and C t e r m i n a l domains. The glucose b i n d i n g s i t e i s s i t u a t e d on the N-terminal domain while the phosphate b i n d i n g s i t e and the p y r i d o x a l phosphate c o f a c t o r r e s i d e on the C-terminal domain. Thus the a c t i v e s i t e i s s i t u a t e d at a l o c a t i o n on the p r o t e i n where movement of the two s u b s i t e s r e l a t i v e to each other i s p o s s i b l e . T h i s o b s e r v a t i o n has a l r e a d y been u t i l i s e d i n the (109) " s l i d i n g domain" hypothesis of Madsen and Withers . They proposed that upon formation of the t e r n a r y enzyme-substrate complex, the N-terminal domain, with the glucose-l-phosphate locked i n t o i t s glucose b i n d i n g pocket, s l i d e s past the C-terminal domain, to which the coenzyme i s a t t a c h e d , b r i n g i n g the two phosphates i n t o c o n t a c t and a l l o w i n g c a t a l y s i s to occur. See f i g u r e 40. By making two assumptions i t i s p o s s i b l e to r e f i n e the " s l i d i n g domain" hypothesis to account f o r the b i n d i n g c h a r a c t e r i s t i c s e x h i b i t e d by the analogs of glucose-l-phosphate. -76-Figure 40. The s l i d i n g domain hypothesis of Madsen and Withers. These assumptions are; 1) The s u b s t r a t e must be bound at both the glucose and the phosphate s u b s i t e f o r c a t a l y s i s to proceed. 2) When the enzyme i s i n the i n a c t i v e conformation, the two s i t e s are e i t h e r too f a r a p a r t or o r i e n t e d such that p r o d u c t i v e i n t e r a c t i o n cannot occur with both s i t e s , (see f i g u r e 41). The i n a c t i v e form of the enzyme would thus represent a p o t e n t i a l energy minima of the p r o t e i n and would normally be adopted i n the absence of a s u b s t r a t e . Under normal c o n d i t i o n s the s u b s t r a t e could bind i n two s t e p s ; F i r s t l y , by the formation of the phosphate b i n d i n g pocket on the C-terminal domain and consequent b i n d i n g of the s u b s t r a t e ' s phosphate moiety. This process causes some conformational change s t a r t i n g the r e l a t i v e movement of the two domains and b r i n g i n g the two s u b s i t e s c l o s e r together. -77-Active Mode (R State) Inact ive Mode (T State) F i g u r e 41. Pro d u c t i v e and unproductive b i n d i n g modes f o r glucose-l-phosphate i n glycogen phosphorylase. Secondly, the energy r e a l i s e d from the i n t e r a c t i o n of the glucopyranose r i n g with i t s b i n d i n g s i t e completes the conformational change b r i n g i n g the two s u b s i t e s c l o s e enough so that p r o d u c t i v e i n t e r a c t i o n with both s i t e s can occur. In r e a l i t y the process probably does not occur as the two stage process d e p i c t e d above, but as a more concerted f i t t i n g of the two p a r t s of the s u b s t r a t e i n t o t h e i r r e s p e c t i v e s u b s i t e s , with the c o n f o r m a t i o n a l changes i n the p r o t e i n a l l o w i n g p r o g r e s s i v e l y more i n t e r a c t i o n with each s i t e . T h i s mechanism r e l i e s on three c h a r a c t e r i s t i c s of the s u b s t r a t e , the presence of a phosphate moiety and a glucopyranose r i n g (which i s capable of p a r t i c i p a t i n g i n a l l of the normal p r o t e i n - l i g a n d I n t e r a c t i o n s ) which are separated by a g i v e n d i s t a n c e . Any of these c r i t e r i a which are not f u l f i l l e d r e s u l t i n i n s u f f i c i e n t b i n d i n g energy -78-being d e r i v e d to a t t a i n the f i n a l a c t i v e conformation and the p r o t e i n c o l l a p s e s back to the i n a c t i v e conformation where i n t e r a c t i o n with only one of the s u b - s i t e s (probably the phosphate s u b - s i t e ) o c c u r s . T h i s mechanism would provide glycogen phosphorylase with a h i g h l y s p e c i f i c method f o r " e d i t i n g " p o t e n t i a l s u b s t r a t e s a l l o w i n g any sugar phosphates other than i t s n a t u r a l s u b s t r a t e to a c t only as weak i n h i b i t o r s . T h i s model p r e d i c t s a number of e f f e c t s that have been observed i n previous experiments. I t p r e d i c t e d s that compounds which could simultaneously occupy both the phosphate and glucose s u b s i t e s i n the same manner as glucose-l-phosphate should produce the a c t i v e conformation of the enzyme. T h i s has been found to occur with a number of compounds; g l u c o s e - 1 , 2 - c y c l i c phosphate (107) (108) (49) , p y r i d o x a l ( 5 1 ) d i p h o s p h o ( l )<5C-D-glucose , UDP-glucose (109) and heptulose-2-phosphate have a l l been shown to promote R-state conformational changes i n phosphorylase. The model a l s o p r e d i c t s t h at s u b s t r a t e present i n the phosphate b i n d i n g pocket w i l l promote a conformational change. T h i s i s seen most c o n v i n c i n g l y i n phosphorylase which c o n t a i n s an analog of the p y r i d o x a l c o f a c t o r pyridoxal-5*-diphosphate (PLPP). When enzyme i s r e c o n s t i t u t e d with t h i s analog, the pyrophosphate group occupies both the normal phosphate s i t e of the c o i a c t o i as w e l l as the phosphate b i n d i n g pocket. T h i s enzyme has been shown to e x i s t i n a more a c t i v a t e d (R) conformation than does (110) n a t i v e phosphorylase and a d d i t i o n of a n u c l e o t i d e a c t i v a t o r t o t a l l y locked the enzyme i n t h i s a c t i v e conformation. - 7 9 -A d d i t i o n of glucose and c a f f e i n e (both of which are T - s t a t e e f f e c t o r s ) f a i l e d to promote the T - s t a t e conformation i n t h i s form of the enzyme; the n a t i v e enzyme would have been converted completely to the i n a c t i v e conformation a t much lower c o n c e n t r a t i o n s of these e f f e c t o r s . T h i s i s c o n s i s t e n t with the previous o b s e r v a t i o n that glucose cannot bind i n the presence of (111) phosphate s i n c e i n t h i s case the phosphate being c o v a l e n t l y bound i s non d i f f u s i b l e . T h i s i s a l s o compatible with the model as i n the a c t i v e conformation of the enzyme a bad s t e r i c i n t e r a c t i o n between the anomeric hydroxyl group of c<-D-glucose and the phosphate i n the phosphate s u b s i t e would a l s o be expected. Thus the simultaneous b i n d i n g of glucose and phosphate could not be accomplished i n the R-state enzyme. In the absence of t h i s unfavorable s t e r i c i n t e r a c t i o n , the presence of both a hexopyranose moiety and i n o r g a n i c phosphate should promote the a c t i v e conformation of the enzyme. A number of compounds which e i t h e r possess a small s u b s t i t u e n t , or none at a l l a t the anomeric carbon, have been shown to a c t as s u b s t r a t e s only i n the presence of phosphate or the phosphate analog, a r s e n a t e . G l u c o s y l f l u o r i d e i s a potent T - s t a t e i n h i b i t o r of glycogen phosphorylase, however i n the presence of i n o r g a n i c phosphate and a g l y c o s y l a cceptor such as maltopentaose the g l u c o s y l moiety i s (112) t r a n s f e r r e d to the a c c e p t o r , although a t a very slow r a t e A more e f f i c i e n t g l y c o s y l donor, D - g l u c a l , i s a l s o t r a n s f e r r e d (113) to glycogen i n the presence of phosphate or arsenate to y i e l d -80-2-deoxy g l u c o s y l d e r i v a t i v e s of the a c c e p t o r . L a s t l y h e p t e n i t o l i s phosphorylated by glycogen phosphorylase i n the presence of i n o r g a n i c phosphate, however i n t h i s case the phosphorylated molecule i s a "dead end product" and i s not t r a n s f e r e d to an (114) acceptor The comparatively high a f f i n i t y of the 2-deoxyfluoro analog f o r phosphorylase suggests that t h i s compound binds i n the p r o d u c t i v e mode, i n t e r a c t i n g with both s u b s i t e s , and t h e r e f o r e might be expected to a c t as a s u b s t r a t e . A p o s s i b l e e x p l a n a t i o n f o r i t s l a c k of a c t i v i t y i s a mechanistic one. I t has been observed that the R-state enzyme can accomodate the h a l f c h a i r (117) (114) conformation adopted by 1,5-gluconolactone and D - g l u c a l (115,116,117) T h i s and other evidence has lead a number of authors to propose the involvement of a carbonium i o n i n t e r m e d i a t e i n the r e a c t i o n mechanism of phosphorylase. The f l u o r i n e s u b s t i t u e n t at C(2) of 2-deoxyfluoro-otrO-glucose-l-phosphate would be expected to d e s t a b i l i s e the proposed oxo-carbonium i o n intermediate and t h e r e f o r e d r a m a t i c a l l y slow or even stop the enzymic r e a c t i o n . Thus the i n a c t i v i t y of the 2-deoxyfluoro analog can be taken as evidence i n support of the proposed carbonium i o n i n t e r m e d i a t e . -81-MATERIALS AND METHODS. General s y n t h e t i c methods. Unless otherwise s t a t e d , the f o l l o w i n g are i m p l i e d . M e l t i n g p o i n t s (m.p.) were determined on a K o f l e r micro-heating stage and are un c o r r e c t e d . Nuclear magnetic resonance (NMR) spectroscopy was performed on the f o l l o w i n g instuments; 1 H: 80 MHz (FT) s p e c t r a on a Bruker model WP-80. 270 MHz (FT) s p e c t r a on a 270 MHz instument c o n s t r u c t e d from a Bruker console, a N i c o l e t computer model 239 A with a Diablo d i s k d r i v e and a super-conducting magnet s u p p l i e d by Oxford instruments. 400 MHz (FT) s p e c t r a on a Bruker model UH-400. 19 F: 254 MHz (FT) s p e c t r a on a Bruker model HXS-270, equipped 19 with a N i c o l e t 1180 computer, a Di a b l o d i s k d r i v e and a 5 mm F probe. 1 For H s p e c t r a s i g n a l p o s i t i o n s are given i n the d e l t a s c a l e (5) with i n t e r n a l t e t r a m e t h y l s i l a n e ( 5 = 0.00) ( a l l s o l v e n t s except DgO) used as r e f e r e n c e . Samples which were d i s s o l v e d i n DgO are ref e r e n c e d to e x t e r n a l 2,2-dimethyl-2-silapentane-5-sulphonate ( 5 = 0.015). 19 For F s p e c t r a s i g n a l p o s i t i o n s are given on the d e l t a s c a l e (0) with r e f e r e n c e to CClgF ( 0 = 0.00). S i g n a l s o c c u r i n g u p - f i e l d of t h i s resonance are giv e n a p o s i t i v e v a l u e . E x t e r n a l t r i f l u o r o a c e t i c a c i d ( 0 - 76.53) was used as r e f e r e n c e . S i g n a l m u l t i p l i c i t y , c o u p l i n g constants (where n e c e s s a r y ) , i n t e g r a t e d area and resonance assignments are i n d i c a t e d i n parentheses. Micro-analyses were performed by Mr P.Borda, M i c r o - a n a l y t i c a l -82-l a b o r a t o r y , U n i v e r s i t y of B r i t i s h Columbia, Vancouver. Solvents and reagents used were e i t h e r reagent grade, c e r t i f i e d or s p e c t r a l grade and were d i s t i l l e d before use. The term "pet ether 35-60 " r e f e r s to the low b o i l i n g f r a c t i o n of reagent grade petroleum d i s t i l l a t e (b.p. ca 35-60°C). Dry s o l v e n t s or reagents where i n d i c a t e d were prepared as f o l l o w s ; Methylene c h l o r i d e , was washed s e v e r a l times with concentrated s u l p h u r i c a c i d , followed by s e v e r a l washings with water and s a t u r a t e d sodium bicarbonate s o l u t i o n . The s o l v e n t was then p r e - d r i e d with sodium sulphate and d i s t i l l e d from calcium h y d r i d e . P y r i d i n e and 2,4,6-trimethy1 p y r i d i n e were p r e - d r i e d f o r s e v e r a l days over p e l l e t s of potassium hydroxide followed by d i s t i l l a t i o n from barium oxide. Toluene, N,N-dimethylformamide (DMF) and a c e t o n i t r i l e were d i s t i l l e d from calcium h y d r i d e . Anhydrous d i e t h y l ether was used as s u p p l i e d . Methanol was d i s t i l l e d from magnesium methoxide prepared i n s i t u by r e a c t i o n of methanol with magnesium tu r n i n g s i n the presence of i o d i n e . C r y s t a l l i n e phosphoric a c i d was obtained from BDH chemicals and was d r i e d i n vacuo over magnesium p e r c h l o r a t e f o r s e v e r a l days p r i o r to use. DAST ( d i e t h y l ) and 70 7. poly h y d r o g e n f l u o r i d e i n p y r i d i n e were obtained from A l d r i c h Chemicals and were used without f u r t h e r p u r i f i c a t i o n . DAST ( d i m e t h y l ) , t r i b u t y l t i n h y d r i d e and 1, 2 , 3 , 4-tetra-0-acety 1 -/?-D-g lucopyranose were obtained from Sigma Chemical Co. and were a l s o used without f u r t h e r p u r i f i c a t i o n . T r i f l u o r o m e t h y 1 3,4,6-tri-0-acety1-2-deoxy-2-fluoro-t* -D-glucopyranoside, t r i f l u o r o m e t h y 1 3 , 4 , 6 - t r i - 0 - a c e t y 1 --83-2-deoxy-2-f luoro-CA-D-mannopyranos id e and 3 ,4 , 6 - t r i - O - a c e t y l -2-deoxy-2-fluoro^S-D-momnopyranosy1 f l u o r i d e were k i n d g i f t s from Dr.D.Dolphin. T h i n l a y e r chromatography ( t i c ) was conducted on aluminium backed p l a t e s of (0.2 mm t h i c k n e s s ) K i e s e l g e l 60 F 4 2 5 • A f t e r development i n the a p p r o p r i a t e s o l v e n t , compounds were v i s u a l i s e d by f l u o r e s c e n c e quenching under U.V. l i g h t and or by c h a r r i n g with 107. s u l p h u r i c a c i d i n methanol. The f o l l o w i n g s o l v e n t systems were commonly employed; (A) n-pentane : e t h y l a c e t a t e : ethanol 20:9:1. (B) e t h y l a c e t a t e : ethanol : water, 7:2:1. Column chromatography was c a r r i e d out according to the method (118) of Khan et a l . ( " f l a s h chromatography"), using a s i l i c a g e l column of K i e s e l g e l 60 (180-230 mesh). The so l v e n t system and Rf of the product are given i n parentheses. General procedures; (1) F l u o r i n a t i o n To a 3-neck round bottom f l a s k equipped with a pressure e q u a l i s e d a d d i t i o n f u n n e l and a calcium c h l o r i d e guard tube, i s added the carbohydrate d e r i v a t i v e and the a p p r o p r i a t e base d i s s o l v e d i n dry methylene c h l o r i d e . DAST i s then added to the a d d i t i o n f u n n e l and the apparatus f l u s h e d with dry n i t r o g e n and cooled to ca. - 20°C. The DAST i s then slowly added to the r e a c t i o n over a p e r i o d of 15 minutes. A f t e r a d d i t i o n i s complete the r e a c t i o n i s allowed to warm slowly to ambient temperature where the r e a c t i o n i s fol l o w e d by t i c . Upon completion the r e a c t i o n mixture i s cooled (0°C) and 5 mis of methanol added c a u t i o u s l y to quench the r e a c t i o n . The s o l v e n t i s removed i n -84-vacuo. and the r e s u l t i n g yellow o i l p u r i f i e d by column chromatography. (2) A c e t y l a t i o n ; (65) T h i s procedure was reported by Kovac and Glaudemans A c e t i c anhydride c o n t a i n i n g 20 mg / ml of anhydrous sodium a c e t a t e i s heated to between 90°and 110°C. The carbohydrate i s then added and the r e a c t i o n maintained a t t h i s temperature f o r 45 minutes. A f t e r c o o l i n g to room temperature, the r e a c t i o n mixture i s added over a p e r i o d of 2 hours to a s a t u r a t e d s o l u t i o n of sodium bicar b o n a t e ( a d d i t i o n of s o l i d sodium bicarbonate to maintain the b a s i c nature of the mixture i s a l s o n e c e s s a r y ) . A f t e r a d d i t i o n i s complete the r e a c t i o n i s l e f t to s t i r f o r a f u r t h e r 1 hour. The r e s u l t i n g suspension i s e x t r a c t e d s e v e r a l times with ch l o r o f o r m . The combined organic phases are then washed with water, d r i e d with sodium sulphate and the s o l v e n t removed i n vacuo. (3) Conversion of r x t o Q anomeric c o n f i g u r a t i o n . (84) T h i s method was o r i g i n a l l y reported by Volfrom and Thompson The c<-tetra-acetate i s d i s s o l v e d i n 45% HBr i n a c e t i c a c i d c o n t a i n i n g a c e t i c anhydride at a c o n c e n t r a t i o n of 0.1 ml / ml. A f t e r 1 hour methylene c h l o r i d e i s added and the mixture washed with 5 p o r t i o n s of i c e c o l d water. The organic phase i s then d r i e d with sodium sulphate and the s o l v e n t removed i n vacuo. The r e s u l t i n g gum i s r e d i s s o l v e d i n g l a c i a l a c e t i c a c i d and mercuric a c e t a t e added. The r e a c t i o n i s p r o t e c t e d from l i g h t and l e f t to s t i r f o r 1 hour a t room temperature. Again methylene c h l o r i d e i s -85-added and the mixture washed with 5 p o r t i o n s of d e i o n i s e d water. A f t e r d r y i n g with sodium sulphate and removal of the s o l v e n t i n vacuo the product i s c r y s t a l l i s e d . (4) D e a c y l a t i o n . (119) This i s a m o d i f i c a t i o n of the procedure of Zemplen To a suspension of the a c y l a t e d carbohydrate i n anhydrous methanol a s o l u t i o n of sodium methoxide i n methanol i s added ( f i n a l c o n c e n t r a t i o n 0.1 M sodium methoxide). The r e a c t i o n i s s t i r r e d a t room temperature u n t i l d e a c y l a t i o n i s complete ( u s u a l l y w i t h i n 2 or 3 minutes). The r e a c t i o n i s then n e u t r a l i s e d by a d d i t i o n of Dowex 50 W (H®) c a t i o n exchange r e s i n , f i l t e r e d and the s o l v e n t removed i n vacuo. (5) P h o s p h o r y l a t i o n . T h i s procedure f o l l o w e d i s that used by HacDonald f o r the p r e p a r a t i o n of © C r D - S l u c o s e - l - p h o s p h a t e from 1, 2 , 3 , 4 , 6-penta-(81) 0 - a c e ty 1 -J3~D - g 1 u c o py rano s e Anhydrous c r y s t a l l i n e phosphoric a c i d i s placed i n a round bottom f l a s k and heated to 55°C under a good vacuum. A f t e r a l l of the phosphoric a c i d has become molten the vacuum i s broken and the carbohydrate i s added. The r e a c t i o n i s then maintained under vacuum a t 55°C f o r a time s p e c i f i e d f o r each sugar. The r e a c t i o n i s quenched by a d d i t i o n of i c e c o l d 2M l i t h i u m hydroxide and l e f t to stand o v e r n i g h t a t room temperature. The i n s o l u b l e l i t h i u m phosphate i s removed by f i l t r a t i o n through a cake of c e l i t e and the remaining s o l u t i o n c ooled i n an i c e bath. The product i s then converted to the dicyclohexylammonium s a l t by passage down a -86-precooled column of Dowex 50 W (H +) c a t i o n exchange r e s i n i n t o a s o l u t i o n of eyelohexylamine i n d e i o n i s e d water. The volume of the e l u e n t i s then reduced by r o t a r y e v aporation (30°C) and c r y s t a l l i s a t i o n induced by a d d i t i o n of acetone to the p o i n t of i n c i p i e n t opalescence. 1,2,3,4-tetra-0-acety1-6-deoxy-6-fluoro-fe-D-glucopyranose (23). 0.103 g (0.3 mmoles) of 1,2,3,4-tetra-0-acety1-^-D-glucopyranose and 0.07 ml (0.6 mmoles) of 2,4,6-trimethyl p y r i d i n e d i s s o l v e d i n 2 ml of dry methylene c h l o r i d e , were reacted with 0.078 ml (0.6 mmoles) of DAST a c c o r d i n g to the general f l u o r i n a t i o n procedure. In t h i s case a f t e r a r e a c t i o n time of 24 hours the r e a c t i o n was quenched and the r e a c t i o n mixture washed with two 5 ml p o r t i o n s of 1 M h y d r o c h l o r i c a c i d . T h i s removed a l l traces of 2 ,4 , 6 - t r i m e t h y l p y r i d i n e . Column chromatography (90 7. methylene c h l o r i d e , 10% e t h y l a c e t a t e , Rf = 0.67) gave a c o l o u r l e s s gum which c r y s t a l l i s e d from methanol (0.071 g, 0.203 mmoles, 687.), m.p. = 125-126°C. 1 H NMR 8 (400 MHz, CDClj) 5.73 (d, J=8.2 Hz, IH, H ( l ) ), 5.27 ( t , J=9.4 Hz, IH), 5.14 ( t , J=8.0 Hz, IH), 5.11 ( t , J=8.0 Hz , IH) H(4), H(3), H(2), 4.49 (dd, J=2.4, 47.2 Hz, IH, H(6) ) 4.45 (dd, J=4.12, 47.0 Hz, IH, H(6') ), 4.10 (m, IH, H(5) ), 2.11, 2.06, 2.04, 2.01 (4 X s, 3H each, 4 X 0C0CH 3). 19 F NMR: 0 (254 MHz, CDC1 ), 233.93 ( d t , J=47.10, 24.83 Hz). -87-6-deoxy-6-fluoro-D-glucopyranose (24), 0.151 g (0.43 mmoles.) of the t e t r a - a c e t a t e (23) was deacylated using the general d e a c y l a t i o n procedure. The r e s u l t i n g gum was d i s s o l v e d i n a minimal volume of methanol and d i e t h y l ether added to induce c r y s t a l l i s a t i o n . R e c r y s t a l l i s a t i o n from the same so l v e n t system gave c o l o u r l e s s c r y s t a l s of (24) (0.075 g, 0.41 mmoles, 95 % ) , m.p. = 144-145°C. 19 F NMR: <p (254 MHz, DgO) £-anomer 235.95 ( d t , J=26.04, 47.38 Hz) ot-anomer 236.49 ( d t , J=28.79 , 47.5 Hz). 6-deoxy-6-f luoro-cX-D-glucose-1-phosphate ( 25) . 0.5 g (1.42 mmoles) of the t e t r a - a c e t a t e (23) and 1.0 g (10 mmoles) of anhydrous phosphoric a c i d were t r e a t e d as i n the general p h o s p h o r y l a t i o n procedure, with a r e a c t i o n time of 2.5 hours. (25) was o b t a i n e d , a f t e r r e c r y s t a l l i s a t i o n from water / acetone as a white s o l i d (0.454 g, 0.98 mmoles, 70 % ) , m.p. = 179-185°C. 1 H NMR: 8 (400 MHz, D 20) 5.44 (dd, J=3.4,7.2 Hz, IH, H ( l ) ), 4.74 (dd, J=47.4, 2.7 Hz, H(6) ), 4.00 (dd, J=31.0, 10 Hz, IH, H(5) ), 3.78 ( t , J=10 Hz, IH, H(3) ), 3.51 ( t , J=10 Hz, IH, H(4) ), 3.46 © (ddd, J=3.4, 9.7, 1.8 Hz, IH, H(2) ), 3.13 (m, 2H, H.N-CH.-), 1.1-2.1 ( 22H, c y c l o h e x y l r i n g ) . 19 F NMR: 0 (254 MHz, D 20) 237.48 ( d t , J=47.0, 31.0 Hz) Elemental a n a l y s i s : Required C, 46.95; H, 8.32; N, 6.08. Found C, 46.96; H, 8.29; N, 6.06. Methyl 2,3, 6- tri-0-benzoyl-4-deoxy-4-f luoro-0^-glucopyranoside . 10 g (20 mmoles) of methyl 2 ,3 , 6-tri-O-benzoyl-CX-D-galacto--88-(82) pyranoside and 4.9 g of DMAP were d i s s o l v e d i n 30 ml of dry methylene c h l o r i d e and t r e a t e d a c c o r d i n g to the general f l u o r i n a t i o n procedure. A f t e r 24 hours the r e a c t i o n was quenched and the product p u r i f i e d by column chromatography (957. methylene c h l o r i d e , 57. e t h y l a c e t a t e , Rf = 0.75). A f t e r r ecrys t a l l i s a t i o n from ethanol c o l o u r l e s s c r y s t a l s of (28) were obtained (4.7 g, 9.2 mmoles, 4 67.), m.p. = 138-139°C. 1 H NMR: 5(400 MHz, CDClj) 8.13-7.96 (m, 6H, a r o m a t i c ) , 7.63-7.35 (m, 9H, a r o m a t i c ) , 6.11 ( d t , J=15.0,9.0 Hz, IH, H(3) ), 5.22 (dd, J=4.0, 10 Hz, IH, H(2) ), 5.18 ( t , J=4.0 Hz, IH, H ( l ) ), 4.78 (dt J-10.0, 51.7 Hz IH, H(4) ), 4.66 (m, 2H, H(6)+H(6') ), 4.33 (m, IH, H(5) ) 3.48 ( s , 3H, -OCH,). 19 3 F NMR: 0 (254 MHz, CDClj) 197.79 (dd, J=50.98, 13.65 Hz). Methyl 4-deoxy-4-fluoro - c<-D-glucopyranoside (29). 5.60 g (11.1 mmoles) of the t r i - b e n z o a t e (28) was suspended i n 24 ml of a b s o l u t e methanol and dea c y l a t e d using the general d e a c y l a t i o n procedure. In t h i s case the r e a c t i o n was l e f t f o r 18 hours and the product p u r i f i e d by column chromatography, (907. e t h y l a c e t a t e , 107. methanol, Rf = 0.31). The r e s u l t i n g c o l o u r l e s s gum, c r y s t a l l i s e d on removal of the s o l v e n t and was r e c r y s t a l l i s e d from e t h y l a c e t a t e : acetone, 1 : 1 (1.71 g, 8.7 mmoles, 787.), m.p. = 129-130°C. 1 H NMR: 5 (80 MHz, D 20) 4.76 ( t , J=3.3 Hz, IH H ( l ) ), 4.12-3.2 (m, 5H, H(2), H(3), H(4), H(6), H(6') ), 3.48 ( s , 3H, OCH, ). 19 3 F NMR: 0 (254 MHz, D 20) 199.23 (dd, J-15.9, 50.91 Hz ). -89-4-deoxy-4-fluoro-D-glucose (30). 0.454 g (2.3 mmoles) of the methyl g l u c o s l d e (29) was d i s s o l v e d i n 40 ml of d e i o n i s e d water and 20 mis of Dowex 50 W (H®) c a t i o n exchange r e s i n added. The mixture was then g e n t l y s t i r r e d and brought to r e f l u x temperature. The r e a c t i o n was monitored by t i c ( s o l v e n t ( b ) , Rf s t a r t i n g m a t e r i a l = 0.62, Rf product= 0.51). A f t e r 25 hours the r e a c t i o n was c o o l e d , the i o n exchange r e s i n removed by f i l t r a t i o n and water removed i n vacuo. The r e s u l t i n g gum was c r y s t a l l i s e d by d i s s o l u t i o n i n a minimal volume of methanol followed by a d d i t i o n of d i e t h y l e t h e r . Compound (30) was i s o l a t e d as a c o l o u r l e s s c r y s t a l l i n e s o l i d (0.355 g, 1.95 mmoles, 85 7.), m.p. = 184-186°C. 19 F NMR: (254 MHz, DgO) Ok-anomer 199.21 (dd, J=15.11, 50.87 Hz) £-anomer 201.24 (dd, J=16.05,50.95 Hz). 1,2,3, 6- tetra-O-acetyl-4-deoxy-4-f luoro-OC-D-glucopyranose (31). 1.89 g (9.6 mmoles) of the methyl g l u c o s i d e (29) was d i s s o l v e d i n 25 ml of a 507. mixture of a c e t i c anhydride i n a c e t i c a c i d . T h i s mixture was cooled to 0°C and 3.75 ml of concentrated s u l p h u r i c a c i d were added dropwise. The mixture was l e f t to stand a t room temperature f o r 4 days. A f t e r c o o l i n g to 0°C, the s u l p h u r i c a c i d was n e u t r a l i s e d by a d d i t i o n of 7.0 g of sodium a c e t a t e . The r e a c t i o n mix was then added to 200 ml of water and e x t r a c t e d with methylene c h l o r i d e . The combined organic phases were washed with water, s a t u r a t e d sodium bicarbonate s o l u t i o n , d r i e d with magnesium sulphate and the s o l v e n t removed i n vacuo. P u r i f i c a t i o n by column chromatography (907. chloroform, 10% e t h y l a c e t a t e , Rf = 0.39) gave a c o l o u r l e s s gum (1.75 g, 4.7 mmoles, 50 7. ). -90-1 H NMR: 6 (270 MHz, CDClj) 6.32 ( t , J=3.5 Hz, IH, H ( l ) ), 5.64 ( d t , J=10, 14 Hz, IH, H(3) ), 5.06 (dd, J=3.5, 10 Hz, IH, H(2) ), 4.45 ( d t , J=10, 51.6 Hz, IH, H(4) ), 4.41-4.28 (m, 2H, H(6)+H(6*) 4.18 (m, IH, H(5) ), 2.04, 2.20 (2 X 8, 3H each, 2 X 0C0CH 3), 2.12 ( s , 6H, 2 X 0C0CH 3). 1,2,3,6-tetra-O-acetyl-4-deoxy-4-fluoro-$-D-glucopyranose ( 3 3 ) . ( i ) From the f r e e sugar (30). 0.10 g (0.55 mmoles) of the f r e e sugar (30) was a c e t y l a t e d a c c o r d i n g to the general a c y l a t i o n procedure. A f t e r 45 minutes t i c ( s o l v e n t (A) ), i n d i c a t e d a s i n g l e compound Rf = 0.51. A f t e r work up the gum was r e d i s s o l v e d i n a minimal volume of d i e t h y l ether and c r y s t a l l i s a t i o n induced by the a d d i t i o n of n-pentane. (0.068 g, 0.19 mmoles, 387.) m.p.= 124-126°C. A f t e r removal of the c r y s t a l s of (33) by f i l t r a t i o n , s o l v e n t from the f i l t r a t e was removed i n vacuo to give a f u r t h e r 0.062 g of c o l o u r l e s s gum. The gum was i d e n t i c a l by t i c to compound (33) ( s o l v e n t (A), Rf = 0.51), however examination of the NMR spectrum of t h i s compound r e v e a l e d that the gum was composed mainly of the &rtetra-acetate (31). ( i i ) From the Q^ - t e t r a - a c e t a t e (31) . 1.75 g (5.0 mmoles) of the o<-tetra-acetate (31) was t r e a t e d i n 5 ml of 457. HBr i n g l a c i a l a c e t i c a c i d a c c ording to the g e n e r a l procedure ( 3 ) . The tetra-acetyl-OQ-bromide (32) thus formed was subsequently r e a c t e d with 3.5 g of mercuric a c e t a t e . A f t e r work up, the product was c r y s t a l l i s e d from methanol to give c o l o u r l e s s c r y s t a l s of (33) (1.62 g, 4.62, 92 7. ), m.p. = 126-128°C. The NMR -91-s p e c t r a of t h i s compound were i d e n t i c a l to the compound prepared by method ( i ) . 1 H NMR: g (270 MHz, CDC1.) 5.78 (d, J=8.0 Hz, IH, H ( l ) ), 5.44 ( d t , J=10,15.7 Hz, IH, H(3) ), 5.13 ( t , J=8.0 Hz, IH, H(2) ), 4.57 ( d t , J=10, 52 Hz, IH, H(4) ), 4.45-4.30 (m, 2H, H(6)+H(6') ) 3.93 (m, IH, H(5) ), 2.06, 2.12 ( 2 X s, 3H each, 3 X OCOCHj), 2.14 ( s , 6H, 2 X OCOCH,). 19 3 F NMR: 0 (254 MHz, CDClg) 200.88 (dd, J=14.7,50.65 Hz). 4-deoxy-4-fluoro -ofr-D-glucose-l-phosphate (34). 1.0 g (2.8 mmoles) of t h e t e t r a - a c e t a t e (33) was t r e a t e d with 2.0 g (20 mmoles) of anhydrous phosphoric a c i d a c c o r d i n g to the gene r a l p h o s p h o r y l a t i o n method. A f t e r 2.0 hours the r e a c t i o n was n e u t r a l i s e d with 60 ml of 2M l i t h i u m hydroxide and converted to the dicyclohexylammonium s a l t (0.62 g, 1.3 mmoles, 46 7.), m.p.= 148-151°C. 1 H NMR: 5 (400 MHz, D 20) 5.52 ( d t , J=3.5, 7.3 Hz, IH, H ( l ) ), 4.26 ( d t , J=9.0, 51.0 Hz, IH, H(4) ), 4.05 (m, IH, H(5) ), 3.97 ( d t , J=9.0, 16.0 Hz, IH, H(3) ), 4.04-3.80 (m, 2H, H(6) © +H(6') ), 3.08 (m, 2H, H.N-CH-), 2.0-1.1 (m, 22H, c y c l o h e x y l ) . 19 3 F NMR: <p (254 MHz, D 20) 199.07 (dd, J=50.8, 15.4 Hz) Elemental a n a l y s i s : Required ( f o r the mono hydrate) C, 45.18; H, 8.43; N, 5.85. Found C, 45.89; H, 7.95; N, 6.19. I, 2:5 , 6-di-0 - J 8 o p r o y lidene-3-deoxy - 3-f luoro-o<-glue of uranose ( 35) . 0.5 g (1.9 mmoles) of 1, 2 ; 5 , 6-di-0-isopropylidene-0<rallof uranose (120) and 0.5 g of DMAP (4.3 mmoles) were t r e a t e d with 0.5 ml of DAST acc o r d i n g to the general f l u o r i n a t i o n procedure. A f t e r -92-20 hours the r e a c t i o n was quenched and the product p u r i f i e d by column chromatography (967. methylene c h l o r i d e , 47. e t h y l a c e t a t e ) , to y i e l d a pale yellow o i l (0.32 g, 1.2 mmoles, 647.). 1 H NMR: 5 (400 MHz, CDC1 3) 5.96 (d, J=4 Hz, IH, H ( l ) ), 5.01 (dd, J=1.8, 50.0 Hz, IH, H(3) ), 4.71 (dd, J - l l , 3 Hz, IH, H(2) ) 4.29 ( d t , J=6, 8 Hz, IH, H(5) ), 4.16-4.02 (m, 3H, H(4), H(6), H(6') ), 1.41, 1.36, 1.27, 1.23 (4 X s, 3H each, 4 X -CH,). 19 3 F NMR: 0 (254 MHz, CDClg) 200.49 (ddd, J=10.7, 29.0, 49.8 Hz). 3-deoxy-3-fluoro-D-glucose (37). 1.20 g (4.6 mmoles) of (36) was d i s s o l v e d i n a mixture composed of 60 ml of d e i o n i s e d water and 12 mis of e t h a n o l . The mixture was l e f t to s t i r at ambient temperature f o r 4.5 days i n the presence o f Dowex 50 W (H®) c a t i o n exchange r e s i n . The mixture was then f i l t e r e d and the product passed down a short f l a s h column ( s o l v e n t (B), Rf = 0.51). The product was d r i e d f o r s e v e r a l days i n vacuo to y i e l d a c o l o u r l e s s gum (0.796 g, 4.3 mmo les , 9 37. ) . 19 F NMR: 0(254 MHz, D 20) <*.-anomer 197.78 (dtd, J=4.6, 13.3, 54.2) j3-anomer 202.71 ( d t , J=13.8, 52.9 Hz). Elemental a n a l y s i s : Required, C, 39.56; H, 6.09. Found, C, 39.90; H, 6.23. I, 2,4,6-tetra-O-ace ty1-3-deoxy-3-fluoro-fi-D-glucopyranose (40). ( i ) 0.23 g (1.3 mmoles) of the f r e e sugar (37) was t r e a t e d with 6.1 ml of the a c e t i c anhydride / sodium a c e t a t e mixture as d e t a i l e d i n the general a c e t y l a t i o n procedure. The product was c r y s t a l l i s e d by d i s s o l v i n g the product mixture i n a minimal -93-volume of chloroform followed by a d d i t i o n of pet ether 35-60 u n t i l the p o i n t of i n c i p i e n t opalesence was a t t a i n e d (0.12g, 0.34 mmoles, 267.), m.p. = 118-120°C. ( i i ) 0.593 g (3.2 mmoles) of the f r e e sugar (37) was d i s s o l v e d i n 5 ml of dry p y r i d i n e and cooled to 0°C. 3 ml of a c e t i c anhydride was then added sl o w l y and the r e a c t i o n allowed to warm to room temperature, where i t was l e f t f o r 48 hours. The r e a c t i o n was a g a i n cooled to 0°C and 3ml of methanol added. A f t e r a f u r t h e r 1 hour the s o l v e n t s were removed i n vacuo and any remaining p y r i d i n e removed by coevaporation with toluene. A f t e r d r y i n g f o r 18 hours i n vacuo, there remained 1.02g of a p a l e y e l l o w , chromatographica1ly pure ( t i c , s o l v e n t (A), Rf-0.54) gum. T h i s gum was then t r e a t e d with 5 mis of 45 7. HBr i n g l a c i a l a c e t i c a c i d as d e t a i l e d i n g e n e r a l procedure ( 3 ) . Subsequent r e a c t i o n of the g l u c o s y l bromide with 1.84 g of mercuric a c e t a t e i n 20 ml of g l a c i a l a c e t i c a c i d , gave the product (40) which was c r y s t a l l i s e d by d i s s o l u t i o n i n a minimal volume of methylene c h l o r i d e f o l l o w e d by a d d i t i o n of n-pentane (0.508 g, 1.45 mmoles, 457.) , m.p.= 116-118°C. 1 H NMR: 5(400 MHz, CDC1 3) 5.65 (d, J=8.5 Hz, IH, H ( l ) ), 5.25 ( d t , J=9.0, 12.6 Hz, 2H, H(2), H(3) ), 4.60 ( d t , J=9.1, 52.0 Hz, IH, H(3) ), 4.26-4.12 (m, 2H, H(6), H(6') ), 3.75 (m, IH, H(5) ), 2.11, 2.10, 2.09, 2.08 (4 X s, 3H each, 4 X OCOCH,). 19 F NMR: 0 (254 MHz, CDClg) 196.73 ( d t , J=12.7,51.9 Hz ). 3-deoxy-3-f luoro-ot-D-glucose-l-phosphate (41) . 0.3 g (0.86 mmoles) of the J R , -tetra-acetate (40) was t r e a t e d with 0.6 g (6.0 mmoles) of anhydrous phosphoric a c i d a c c o r d i n g to the -94-g e n e r a l p h o s p h o r y l a t i o n procedure. A f t e r a r e a c t i o n time of 2.5 hours the r e a c t i o n was quenched with 11 ml of 2 M l i t h i u m h y droxide. A f t e r c o n v e r s i o n to the dicyclohexylamraonium s a l t and r e c r y s t a l l i s a t i o n from acetone / water, (41) was i s o l a t e d as a c o l o u r l e s s c r y s t a l l i n e s o l i d (0.157 g, 0.32 mmoles, 377.), m.p. = 140-143°C. 1 H NMR: 8 (400 MHz, D 20) 5.44 (dd, J=3.4, 7.2 Hz, IH, H ( l ) ), 4.67 ( d t , J=10.0, 47.2 Hz, IH, H(3) ), 3.91-3.84 (m, 2H, H(6), H(6') ), 3.72 (m, J=3.5, 9.0, 13.8 Hz, IH, H(2) ), 3.68 (m, 2H, H(4), H(5) ), 3.10 (m, 2H, H®N-CH_ ), 2.04-1.10 (m, 21H, cyclohex) 19 F NMR: <p (254 MHz, D 20) 201.25 ( d t d , J=1.62, 14.4, 54.7). Elemental a n a l y s i s : Required ( f o r the d i h y d r a t e ) , C, 43.54; H, 8.53; N, 5.64. Found, C, 43.05; H, 8.12; N, 5.77. 3,4,6- t r i - O - a c e t y 1-2-deoxy -2-f luoro-QC-glucopyranosy 1 bromide (43). 3 g (8.0 mmoles) of t r i f l u o r o m e t h y l 3,4,6-tri-0-acety1-2-deoxy-2-fluoro-o(-D-glucopyranose (42) was d i s s o l v e d i n 30 ml of 45 7. HBr i n g l a c i a l a c e t i c a c i d c o n t a i n i n g 3 ml of a c e t i c anhydride and l e f t a t room temperature f o r 6 days. 50 ml of methylene c h l o r i d e was then added and the mixture washed with water (0°C) to remove a c e t i c a c i d . The o r g a n i c phase was d r i e d with magnesium sulphate and the s o l v e n t removed i n vacuo. The remaining gum was c r y s t a l l i s e d by d i s s o l u t i o n i n a minimal volume of dry d i e t h y l ether f o l l o w e d by a d d i t i o n of n-pentane. R e c r y s t a l 1 i s a t i o n i n the same manner gave (43) as a white c r y s t a l i n e s o l i d (2.42 g, 6.5 mmoles, 817. ), m.p. = 83°C. 1 H NMR:5(270 MHz, CDClj) 6.30 (d, J=4.0 Hz, IH, H ( l ) ), 5.64 ( d t , J-10.0, 12.0 Hz, IH, H(3) ), 5.14 ( t , J=10.0 Hz, IH, H(4) ), -95-4.56 (ddd, J=4.0, 9.0, 50.0 Hz, IH, H(2) ), 4.38-4.10 (m, 3H, H(5), H(6), H(6') ), 2.05, 2.07, 2.09 (3 X s, 3H each, 3X0C0CH 3). 1,3,4,6- te tra-O-acety 1-2-deoxy - 2-f luoro-J&-D-glucopyranose (44) . 3.0 g (8.1 mmoles) of the g l u c o s y l bromide (43) was d i s s o l v e d i n 60 ml of g l a c i a l a c e t i c a c i d and 5.1 g of mercuric a c e t a t e and l e f t f o r 20 hours at room temperature. The a c e t i c a c i d was then removed i n vacuo (any remaining a c e t i c a c i d was coevaporated with toluene) and the white s o l i d resuspended i n anhydrous d i e t h y l e t h e r . The e t h e r e a l suspension was f i l t e r e d and c r y s t a l l i s t a t i o n of the product induced by a d d i t i o n of n-pentane. A f t e r r e c r y s t a l l i s a t i o n i n the same manner (44) was obtained as a c o l o u r l e s s s o l i d (2.1 g, 5.9 mmoles, 737.), m.p. = 91-92°C. 1 H NMR: 5 (400 MHz, CDC lg) 5.79 (dd, J=3.0, 8.0 Hz, IH, H ( l ) ), 5.38 ( d t , J=9.0, 13.7 Hz, IH, H(3) ), 5.07 ( t , J=9 Hz, IH, H(4) ) 4.45 ( d t , J=8.6, 51.0 Hz, IH, H(2) ), 4.30-4.11 (m, 2H, H(6), H(6') ), 3.86 (m, IH, H(5) ), 2.19, 2.10, 2.09, 2.05 (4 X s, 3H each, 4 X OCOCH,). 19 3 F NMR: <f> (254 MHz, CDClj) 201.71 (ddd, J<=3.1, 14.3, 50.8 Hz). 2-deoxy-2-fluoro -oC-D-glucose-1 -phosphate (45) . 2.0 g (5.7 mmoles) of the t e t r a - a c e t a t e (44) was t r e a t e d with 4.0 g (40.0 mmoles) of anhydrous phosphoric a c i d a c c o r d i n g to the general p h o s p h o r y l a t i o n procedure. In t h i s case a r e a c t i o n time of 48 hours was r e q u i r e d f o r the complete c o n v e r s i o n of a l l the s t a r t i n g m a t e r i a l to sugar-phosphate. The r e a c t i o n was quenched with 50 ml. of 2 M l i t h i u m hydroxide and the product converted to the dicyclohexylammonium s a l t (0.927 g, 2.0 mmoles, 357.). -96-A f t e r repeated c y c l e s of r e c r y s t a l l i s a t i o n and d r y i n g i n vacuo 1 a H NMR spectrum suggested that the product was no longer the dicyclohexylammonium s a l t , but a combination of the s a l t and the f r e e a c i d . The i n t e g r a l r a t i o of the resonances between 8 2.0-1.1 (due to the c y c l o h e x y l r i n g ) to the resonance at 55.60 (due to H ( l ) ) i n d i c a t e d 0.4 moles of c y c l o h e x y l ammonium s a l t to 1 mole of sugar-phosphate. 1 H NMR: 8 (400 MHz, DjO) 5.53 (dd, J=3.6, 8.0 Hz, IH, H ( l ) ), 4.29 (m, J=1.5, 4.0, 9.0, 47.8 Hz, IH, H(2) ), 3.96 ( d t , J=9.6, 12.4 Hz, IH, H(3) ), 3.86 (m, IH, H(5) ), 3.81-3.66 (m, 2H, H(6), H(6') ), 3.36 ( t , J=9.6 Hz, IH, H(4) ). 19 F NMR:0(254 MHz, D ^ ) 200.23 (dd, J = 13.1, 52.3 Hz). Elemental a n a l y s i s : Required ( f o r 0.4 moles of c y c l o h e x y l ammonium s a l t per mole of sugar-phosphate), C, 33.38; H, 5.87; N, 1.85. Found, C, 33.43; H, 6.26; N, 2.46. 2,3,4, 6 - tetra-O-acety 1-oC-D-mannopyranosy 1 f l u o r i d e . 3.0 g (7.7 mmoles) of 1, 2 , 3 ,4, 6-penta-0-ace ty 1 -c<-D-mannopyranose was d i s s o l v e d i n 5 ml of methylene c h l o r i d e and cooled to 0°C. While t h i s s o l u t i o n was s t i r r i n g r a p i d l y 6 mis of 707. poly hydrogen f l u o r i d e i n p y r i d i n e was added and the r e a c t i o n allowed to warm to room temperature. A f t e r 5 hours none of the s t a r t i n g m a t e r i a l remained, ( t i c s o l v e n t (A), Rf product = 0.56, s t a r t i n g m a t e r i a l = 0.33) and the r e a c t i o n was quenched by pouring i n t o a mixture of i c e / water and methylene c h l o r i d e . The o r g a n i c phase was then separated and washed twice with water, d r i e d with sodium sulphate and the s o l v e n t removed i n vacuo. The major product was p u r i f i e d by column chromatography (907. methylene -97-c h l o r i d e , 107. e t h y l a c e t a t e , Rf 0.63) and c r y s t a l l i s e d on seeding from d i e t h y l ether / n-pentane mixtures (1.44 g, 4.1 mmoles, (121) 537.), m.p. = 68-70°C, l i t . m.p. - 68-69°C 1 H NMR: 5 (80 MHz, CDC1 ) 5.58 (dd, J=0.5, 47.0 Hz, IH, H ( l ) ), 5.36 (m, 3H, H(2), H(3), H(4) ), 4.22 (m, 3H, H(5), H(6), H(6') ) 2.20, 2.13, 2.07, 2.02 (4 X s, 3H each, 4 X OCOCHj). <X-D-mannopyranosy1 f l u o r i d e . 1.44 g (4.1 mmoles) of 2 , 3 ,4 , 6-tetra-0-ace ty l-o<-D-mannopyranosy 1 f l u o r i d e was d i s s o l v e d i n a b s o l u t e methanol and cooled to 0°C. Sodium methoxide i n methanol was added to a f i n a l c o n c e n t r a t i o n of 0.01 M and the r e a c t i o n l e f t a t 0°C f o r 55 minutes. A f t e r t h i s time, most of the s t a r t i n g m a t e r i a l had been converted to a product which appeared as a s i n g l e spot on t i c ( s o l v e n t ( B), Rf = 0.61). The r e a c t i o n was n e u t r a l i s e d by a c i d i c i o n exchange r e s i n i n the normal manner and the s o l v e n t removed i n vacuo. The r e s u l t i n g c o l o u r l e s s gum could not be c r y s t a l l i s e d . 19 F NMR: <p (254 MHz, D ^ 0) 138.97 (d, J=46.3 Hz). 3,4,6- t r i-O-ace ty 1- 2-deoxy - 2-f luoro -Q(-D-mannopyranosyl  f l u o r i d e (46) 0.664 g (2.1 mmoles) of 3,4,6-tri-0-acety1-2-deoxy-2-fluoro-J^-D-mannopyranosy 1 f l u o r i d e was d i s s o l v e d i n 5 ml of dry methylene c h l o r i d e and cooled to -20°C. 1.3 mis of 70 7. poly hydrogen f l u o r i d e i n p y r i d i n e was added and the r e a c t i o n allowed to warm to room temperature where i t was l e f t while s t i r r i n g f o r 24 hours. The r e a c t i o n was quenched by pouring the mixture i n t o i c e / water and e x t r a c t e d i n t o methylene c h l o r i d e . A f t e r the organic phase had been washed with water, i t was d r i e d -98-with sodium sulphate and the s o l v e n t removed i n vacuo. The c o l o u r l e s s gum c r y s t a l l i s e d on standing over n i g h t and was r e c r y s t a 1 U s e d by d i s s o l u t i o n i n a minimal volume of d i e t h y l ether f o l l l o w e d by a d d i t i o n of n-pentane (0.429 g, 1.4 mmoles, 657.) , m.p. = 90-92°C. 1 H NMR: 8 (80 MHz, CDClg) 5.80 (ddd, J=2.0, 3.0, 48.5 Hz, IH, H ( l ) ), 5.45-5.00 (m, 2H, H(3), H(4) ), 4.92 ( d t , J=2.0, 48.6 Hz IH, H(2) ), 4.31-4.05 (m, 3H, H(5), H(6), H(6') ), 2.19 ( s , 6H, 2 X 0C0CH 3), 2.12 ( s , 3H, OCOCHg). 2-deoxy-2-f luoro-Oft-D-mannosyl f l u o r i d e . D e a c y l a t i o n of 0.476 g (1.5 mmoles) of the t r i - a c e t a t e (46) ac c o r d i n g to the ge n e r a l d e a c y l a t i o n procedure, followed by p u r i f i c a t i o n by column chromatography ( e t h y l a c e t a t e , Rf 0.36) gave a c o l o u r l e s s gum (0.174 g, 0.94 mmoles, 637.). Elemental a n a l y s i s : Required, C, 39.14; H, 5.47. Found, C, 39.30 H, 5.50. 2,3,6-tri-O-acety1-2-deoxy-2-fluoro-ft-D-glucopyranosy1 f l u o r i d e . 0.5 g (1.4 mmoles) of 3 ,4 , 6-tr i-0-ace ty 1-2-deoxy-2-f luoro-04-D-g l u c o p y r a n o s y l bromide (43) was converted to the J3>- f l u o r i d e by the a c t i o n of s i l v e r f l u o r i d e i n dry a c e t o n i t r i l e a c c o r d i n g to (88) the method of H a l l et a l . . The product was c r y s t a l l i s e d by t r i t u r a t i o n of the r e s u l t i n g gum with d i e t h y l ether f o l l o w e d by a d d i t i o n of n-pentane to complete c r y s t a l l i s a t i o n (0.327g, I. 1 mmoles, 78%), m.p. = 104-105°C. 1 H NMR: 8 (400 MHz, CDC1,) 5.45 (ddd, J=4.0, 6.0, 52.0 Hz, IH, H ( l ) ), 5.34 ( d t , J-15.3, 7.2 Hz, IH, H(3) ), 5.13 ( t , J=9.5 Hz, -99-1H, H(4) ), 4.51 (m, J=6.2, 8.0, 50.1, 14.1 Hz, IH, H(2) ), 4.31-4.17 (m, 2H, H(6), H(6') ), 3.91 (m, IH, H(5) ), 2.10 ( s , 6H, 2 X OCOCHj), 2.05 ( s , 3H, O C O C H 3 ) . 2-deoxy-2-fluoro -r j-D-glucopyranosyl f l u o r i d e . T h i s compound was prepared by d e a c y l a t i o n of 0.16g (0.5 mmoles) of the t r i - a c e t a t e (46) i n 0.2 ml of absolute methanol ac c o r d i n g to the general d e a c y l a t i o n procedure. The product was c r y s t a l l i s e d by d i s s o l v i n g the gum i n a minimal amount of methanol followed by a d d i t i o n of d i e t h y l ether and n-pentane (0.058 g 0.3 mmoles 607.). 1 H NMR: 8 (400 MHz, D 20) 5.40 (ddd, J=3.2, 7.0, 53.0 Hz, IH, H ( l ) ), 4.34 (m, J=7.0, 9.0, 13.5 Hz), 3.92-3.74 (m, 3H, H(3), H(4), H(5) ), 3.64-3.51 (m, 2H, H(6), H(6') ). 2-deoxy-2-fluoro-D-mannose. 2.0 g (5.3 mmoles) of t r i f l u o r o m e t h y l 3,4,6-tri-O-acety1-2-deoxy-2-fluoro-ctf-D-mannopyranoside was dea c y l a t e d a c c o r d i n g to the ge n e r a l d e a c y l a t i o n procedure. The r e s u l t i n g t r i f l u o r o m e t h y l mannoside was then r e d i s s o l v e d i n 15 ml of water and Dowex 50 V (H®) c a t i o n exchange r e s i n added .The r e a c t i o n mixture was then brought to a g e n t l e r e f l u x f o r 1 hour. F i l t r a t i o n f o l l o wed by removal of the s o l v e n t i n vacuo l e f t a c o l o u r l e s s gum which was p u r i f i e d on a small chromatography column (907. e t h y l a c e t a t e , 107. methanol Rf = 0.28). The r e s u l t i n g gum e v e n t u a l l y c r y s t a l l i s e d on standing and was r e c r y s t a l l i s e d from methanol / d i e t h y l ether (0.718 g, 3.9 mmoles, 747. ) m.p. « 113-115°C. M a t e r i a l prepared by t h i s method was ch r o m a t o g r a p h i c a l l y i d e n t i c a l ( t i c sovent (B), -100-(122) Rf = 0.85) to m a t e r i a l prepared by the method of H a l l et a l . 19 F NMR: 0 (254 MHz, D 20) o-anomer 204.46 (dd,J=34.5, 49.0), j^-anomer 223. 11 (m, J=53.0, 18.78, 32.3 Hz). 1,2,3,4-tetra-O-acety1-6-deoxy-6-iodo-fe-D-glucopyranose (48). 5.0 g (10 mmoles) of 1,2,3,4-tetra-0-acety1-6-0-tosy1 - R-D-gluco-(92) pyranose was d i s s o l v e d i n 30 ml of dry DMF and 1.65 g of sodium i o d i d e (11 mmoles) added. The mixture was heated a t r e f l u x f o r 45 minutes. A f t e r c o o l i n g the mixture was tipped i n t o i c e water and the product e x t r a c t e d i n t o d i e t h y l e t h e r . The e t h e r e a l phase was then washed with water, s a t u r a t e d sodium b i c a r b o n a t e s o l u t i o n , sodium t h i o s u l p h a t e s o l u t i o n and d r i e d with sodium s u l p h a t e . The s o l v e n t was removed i n vacuo and the product d i s s o l v e d i n a minimal volume of d i e t h y l e t h e r . C r y s t a l l i s a t i o n was induced by the a d d i t i o n of pet ether 35-60 (3.4 g, 7.6 mmoles, 7 67.). 1 H NMR: 0 (80 MHz, CDClg) 5.74 (d, J=8.0 Hz, IH, H ( l ) ), 5.37-4.85 (m, 3H, H(2), H(3), H(4) ), 3.50 (m, IH, H(5) ), 3.25-3.00 (m, 2H, H(6), H(6') ), 2.07, 2.00 (2 X s, 3H, each, 2 X OCOCHg), 1.96 ( s , 6H, 2 X OCOCHg). 1,2,3,4- tetra-O-ace ty 1- 6- deoxy-ft-D-g lucopyranose ( 49 ) . 3.4 g (7.6 mmoles) of the 6 - i o d o - t e t r a - a c e t a t e (48) was d i s s o l v e d i n a mixture of 60 ml of e t h y l a c e t a t e and 30 mis of 957. ethanol c o n t a i n i n g 3.0 g of sodium a c e t a t e . To t h i s mixture 2.0 g of 107. p a l l a d i u m on carbon was added and the r e a c t i o n placed under an atmosphere of hydrogen. A f t e r 3 hours, hydrogen uptake had ceased; the c a t a l y s t was removed by f i l t r a t i o n -101-washed with small amounts of d i e t h y l ether and the s o l v e n t removed i n vacuo. The remaining gum was r e d i s s o l v e d i n methylene c h l o r i d e and washed with water, s a t u r a t e d sodium bicarbonate s o l u t i o n , sodium t h i o s u l p h a t e s o l u t i o n and d r i e d with sodium s u l p h a t e . The s o l v e n t was removed i n vacuo and the gum r e d i s s o l v e d i n a minimal volume of d i e t h y l e t h e r . C r y s t a l l i s a t i o n was induced by a d d i t i o n of n-pentane (1.6 g, 4.8 mmoles, 637.), m.p. =114-116°C. 1 H NMR: 5 (80 MHz, CDClg) 5.71 (d, J=8.0 Hz, IH, H ( l ) ), 5.35-4.67 (m, 3H, H(2), H(3), H(4) ), 3.72 (m, IH, H(5) ), 2.12, 2.07, 2.00, 1.97 (4 X s, 3H each, 4 X O C O C H 3 ) , 1.22 (d, J=6 Hz, 3H, -CH 3). 6-deoxy-o<-D-glucose-l-phosphate (50) . 1.5 g (4.5 mmoles) of the t e t r a - a c e t a t e (49) were t r e a t e d with 3.5 g (35 mmoles) of anhydrous c r y s t a l l i n e phosphoric a c i d a c c o r d i n g to the general p h o s p h o r y l a t i o n procedure. A f t e r 2 hours the r e a c t i o n was quenched by a d d i t i o n of 73 ml of 2 M l i t h i u m hydroxide. The product was converted to the dicyclohexylammonium s a l t i n the normal manner. A f t e r the volume of the e l u e n t had been reduced, the product was c r y s t a l l i s e d by a d d i t i o n of 2-3 volumes of ethanol and a l l o w i n g the s o l u t i o n to stand a t 4°C f o r 48 hours. R e c r y s t a l l i s a t i o n from the same s o l v e n t gave the product as a c o l o u r l e s s c r y s t a l l i n e s o l i d (0.67g, 1.5 mmoles, 337.), m.p. = 156-160°C. 1 H NMR: 5 (270 MHz, D 20) 5.38 (dd, J=3.5, 7.0 Hz, IH, H ( l ) ), 3.95 ( t , J=8.6 Hz, IH, H(3) ), 3.70 ( t , J=9.2 Hz, IH, H(4) ), © 3.45 (dd, J-3.1, 9.0 Hz, IH, H(2) ), 3.11 (m, 2H, H 3 N-CH ), 1.95-1.24 (m, 20H, c y c l o h e x y l ), 1.23 (d, J=5.5 Hz, 3H, -CH,). -102-Elemental a n a l y s i s : Required, C,48.89; H, 8.59; N, 6.33. Found, C, 48.63; H, 8.50; N, 6.46. l,2,3,6-tetra-0-acetyl-4-deoxy - c*-D-glucopyranose (52) . (62) 1.97 g (11 mmoles) of the methyl g l u c o s i d e (51) was d i s s o l v e d i n an i c e c o l d , 50 7. s o l u t i o n of a c e t i c anhydride i n g l a c i a l a c e t i c a c i d . 3.7 ml of concentrated s u l p h u r i c a c i d was added s l o w l y , and the r e a c t i o n allowed to warm to ambient temperature. A f t e r 48 hours the r e a c t i o n was quenched by a d d i t i o n of 7.0 g of sodium a c e t a t e (mixture was cooled during the a d d i t i o n ) and poured i n t o i c e water. The aqueous suspension was then e x t r a c t e d with methylene c h l o r i d e , the o r g a n i c phase washed with water, s a t u r a t e d sodium bicar b o n a t e s o l u t i o n and d r i e d with sodium s u l p h a t e . A f t e r the s o l v e n t had been removed i n vacuo the major product was p u r i f i e d by column chromatography (907. chloroform, 107. e t h y l a c e t a t e , Rf = 0.32) to y i e l d a pale yellow o i l (1.38 g, 4.1 mmoles, 377.). 1 H NMR: $ (80 MHz, CDC1 3) 6.24 (d, J=3.5 Hz, IH, H ( l ) ), 5.26-4.85 (m, 2H, H(2),H(3) ), 4.31-3.97 (m, H(5), H(6), H(6') ), 2.25-1.87 (m, 14H, 4 X OCOCHj, H(4), H(4') ). 1,2,3,6-tetra-O-acetyl-4-deoxy-fi-D-glucopyranose (53). 1.38 g (4.1 mmoles) of the c x - t e t r a - a c e t a t e (52) was t r e a t e d with 5 ml of 45 7. HBr i n g l a c i a l a c e t i c a c i d and 3.5 g of mercuric a c e t a t e as d e t a i l e d i n g e n e r a l procedure ( 3 ) . The product was d i s s o l v e d i n a minimal volume of d i e t h y l ether and c r y s t a l l i s a t i o n induced by a d d i t i o n of pet ether 35-60 to y i e l d (53), (1.03 g, 2.8 mmoles, 687.), m.p. - 99-103°C. -103-1 H NMR: 5 (270 MHz, CDC1 3) 5.61 (d, J=8.0, IH, H ( l ) ), 5.07 (m, 2H, H(2), H(3) ), 4.18 (m, 2H, H(6), H(6") ), 3.78 (m, IH, H(5) ), 2.20-1.94 (m, 14H, 4 X OCOCH3, H(4), H(4') ). 4-deoxy-ofr-D-glucose-l-phosphate (54). 0.8 g (2.4 mmoles) of the j}> - t e t r a - a c e t a t e (53) was t r e a t e d with 1.6 g (16.2 mmoles) of anhydrous c r y s t a l l i n e phosphoric a c i d a c c o r d i n g to the gen e r a l p h o s p h o r y l a t i o n procedure. A f t e r 2 hours the r e a c t i o n was quenched by the a d d i t i o n of 60 ml of 2 M l i t h i u m hydroxide. The product was converted to the d i c y c l o h e x y l ammonium s a l t i n the normal manner, and slow c r y s t a l l i s a t i o n from n-butanol and acetone gave (54) as a c o l o u r l e s s s o l i d (0.372 g, 0.84 mmoles, 357.), m.p. = 138-145°C. 1 H NMR: 6 (400 MHz, D 20) 5.48 (dd, J=3.3, 7.2 Hz, IH, H ( l ) ), 4.18 (m, IH, H(5) ), 3.85 ( d t , J=5.0, 9.3 Hz, IH, H(3) ), 3.66-3.52 (m, 2H, H(6), H(6') ), 3.38 ( d t , J-1.7, 9.2 Hz, IH, H(2) ), 3.13 (m, 2H, H®N-CH ), 1.96-1.17 (m, 22H, c y c l o h e x y l , H(4), H(4«) ). Elemental a n a l y s i s : Required ( f o r the mono h y d r a t e ) , C, 46.74; H, 8.93; N, 6.06. Found, C, 46.11; H, 8.59; N, 6.22. I, 5-anhydro-D-glucitol (55). (123) 1.42 g (3.4 mmoles) of acetobromoglucose and 12.7 mg of AIBN were d i s s o l v e d i n 10 ml of dry toluene contained i n a 2-neck round bottom f l a s k equiped with a condenser and a d r y i n g tube. The apparatus was f l u s h e d with n i t r o g e n and 1.6 ml of t r i b u t y l t i n h y d r i d e added. The r e a c t i o n was heated to 80°C and maintained at t h i s temperature f o r 2 hours. The r e a c t i o n was cooled to room -104-temperature and the toluene removed i n vacuo. The remaining gum was d i s s o l v e d i n a c e t o n i t r i l e and e x t r a c t e d 4 times with equal volumes of pet ether 35-60 . The a c e t o n i t r i l e phase was then separated and the s o l v e n t removed i n vacuo to y i e l d 1.05 g of a c r y s t a l l i n e s o l i d . The s o l i d was d i s s o l v e d i n 10 ml of a b s o l u t e methanol and t r e a t e d a c c o r d i n g to the g e n e r a l d e a c y l a t i o n procedure. The f i n a l product was r e c r y s t a l l i s e d from ethanol (0.372 g, 2.3 mmoles, 687.), m.p. = 140-141°C. 1,2-dideoxy-D-glucose. 3.0 g (13.4 mmoles) of 3 , 4 , 6 - t r i - 0 - a c e t y 1 - D - g l u c a l and 0.25 g of platinum oxide were added to 4 ml of g l a c i a l a c e t i c a c i d (124) and t r e a t e d a c c o r d i n g to the method of F i s c h e r . 1.96 g (8.7 mmoles) of the r e s u l t i n g t r i - a c e t a t e was d i s s o l v e d i n 16 ml of a b s o l u t e methanol and d e a c y l a t e d a c c o r d i n g to the g e n e r a l d e a c y l a t i o n procedure. The product mixture was p u r i f i e d by column chromatography ( s o l v e n t (B), Rf = 0.42) g i v i n g 1,2-dideoxy-D-glucose which c r y s t a l l i s e d a f t e r 72 hours under vacuum (0.7 g, 4.7 mmoles, 547.), m.p. = 82-83°C. Elemental a n a l y s i s : Required, C, 48.64; H, 8.16. Found, C, 48.36; H, 8.11. -105-(B) Enzymology. Absorbance measurments were c a r r i e d out on a Pye Unicam PU-8800 UV / v i s i b l e spectrophotometer. Measurements of pH were c a r r i e d out on a Radiometer PHM 62 pH meter. A l l b u f f e r chemicals were purchased from Sigma Chemical Co. and were of the h i g h e s t q u a l i t y a v a i l a b l e . Rabbit muscle phosphorylase b (EC.2.3.1.1) was prepared from r a b b i t s k e l e t a l muscle obtained from P e l - Freez B i o l o g i c a l s , (125) a c c o r d i n g to the method of F i s c h e r and Krebs , except that DTT was used i n s t e a d of c y s t e i n e and the enzyme was c r y s t a l l i s e d a t l e a s t three times before use. P r o t e i n c o n c e n t r a t i o n s were determined from absorbance measurments at 280 nm by using an (126) absorbance index 1.32 mg /ml /cm . Rabbit l i v e r glycogen (type I I I ) purchased from Sigma Chemical Co., was p u r i f i e d on a Dowex 1-C1 (127) column and assayed by the method of Dische . The c o n c e n t r a t i o n of glycogen i s expressed as the molar e q u i v a l e n t of i t s glucose r e s i d u e s . A l l of the k i n e t i c experiments were conducted with the t e c h n i c a l a s s i s t a n c e of Mr C.Armstong.Initia1 r e a c t i o n r a t e s were (128) determined by the Fiske-SubbaRow phosphate a n a l y s i s i n the d i r e c t i o n of s a c c h a r i d e s y n t h e s i s as de s c r i b e d by Engers et a l . (129) . Reaction volumes were 0.2 or 0.5 ml and r e a t i o n s were performed a t 30°C and pH 6.8 i n a b u f f e r c o n t a i n i n g 100 mM potassium c h l o r i d e , 50 mM t r i e t h a n o l a m i n e h y d r o c h l o r i d e , 1 mM EDTA, and 1 mM DTT. Reactions were i n i t i a t e d by a d d i t i o n of enzyme with AMP and Glycogen (to a f i n a l c o n c e n t r a t i o n of 1 mM and 17. r e s p e c t i v e l y ) to s u b s t r a t e and p o s s i b l e i n h i b i t o r a t -106-a p p r o p r l a t e c o n c e n t r a t i o n s and allowed to proceed f o r 5 minutes. The r e a c t i o n was stopped by the a d d i t i o n of 7 ml of 0.07 N s u l p h u r i c a c i d . An i n i t i a l estimate of the K i value f o r each compound was obtained by measuring the r a t e s at a f i x e d c o n c e n t r a t i o n of glucose-l-phosphate ( u s u a l l y 4.0 mM) and v a r i o u s c o n c e n t r a t i o n s of the i n h i b i t o r . Compounds which showed l i t t l e or no i n h i b i t i o n at c o n c e n t r a t i o n s approaching 100 mM were not s t u d i e d f u r t h e r and an approximate value f o r K i c a l c u l a t e d from t h i s data. Analogs showing an apparent K i value of l e s s than 100 mM were su b j e c t e d to a f u l l k i n e t i c a n a l y s i s to determine the K i v a l u e . The c o n c e n t r a t i o n s of glucose-l-phosphate, phosphorylase b and i n h i b i t o r were as f o l l o w s ; INHIBITOR GLUCOSE-1-PHOSPHATE CONCENTRATION (mM) PHOSPHORYLASE b CONCENTRATION (ug / rexn) INHIBITOR CONCENTRATION (mM) 4-deoxyfluoro D-glucose 1.5 - 10.0 2.5 5.0 - 30.2 1,5-anhydro D - g l u c i t o l 1.5 - 8.0 2.5 4.5 - 15.1 2-deoxyfluoro ot-D-glucosyl f l u o r i d e 1.6 - 16.0 4.6 0.5 - 3.0 2-deoxyfluoro OC-D-mannosy 1 f l u o r i d e 1.5 - 15.2 2.5 62.4 - 125.0 3-deoxyfluoro D-glucose 4.0 2.6 1.0 - 100.0 6-deoxyfluoro D-glucose 4.0 2.5 69.4 - 138.8 2-deoxyfluoro D-mannos e 4.0 2.5 25.3 - 202.0 -107-INHIBITOR GLUCOSE-1-PHOSPHATE CONCENTRATION (mM) PHOSPHORYLASE b CONCENTRATION (jig 1 rexn.) INHIBITOR CONCENTRATION (mM) 1,2-dideoxy D-glucose 4.0 2.6 27.5 - 220.0 2-deoxyfluoro jB-D-mannosyl f l u o r i d e 6.4 3.0 12.6 - 59.8 ot- mannosyl f l u o r i d e 4.0 2.5 24.0 - 192.0 6-deoxyfluoro o k -glucose-l-phosphate 1.5 - 10.0 2.5 13.1 - 43.5 4-deoxyfluoro C<- g l u c o s e - l -phosphate 1.5 - 20.2 2.0 10.0 - 39.9 3-deoxyfluoro C<- g l u c o s e - l -phosphate 1.5 - 20.2 2.5 15.0 - 40.0 2-deoxyfluoro 0<- g l u c o s e - l -phosphate 1.5 - 20.2 2.6 2.0 - 7.5 Table X I I . C o n c e n t r a t i o n data f o r the K i determinations of the glucose-l-phosphate analogs. The analogs of glucose-l-phosphate were tested f o r s u b s t r a t e a c t i v i t y with 1 mM AMP, 17. glycogen, 3 - 120 ug / r e a c t i o n mix of phosphorylase b and r e a c t i o n times of up to 18 hours. -108-BIBLIOGRAPHY (1) Y . l t t a h & CP.J.Glaudemans. Carbohydr.Res , 95, 189 (1981). (2) R.U.Lemieux. Chem.Soc.Revs. 7, 423 (1978). (3) P.Goldman. Sc i e n c e . 164, 1123 (1969). (4) R . 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For the simple e q u i l i b r i u m process; k« S ^ P I t can be shown, that the i n i t i a l r a t e of r e a c t i o n ( i . e [P] C 0) i s d e s c r i b e d by the simple r a t e equation ( 1 ) . v = k r S ] ( D I f the c o n c e n t r a t i o n of S i s kept very low, where the uncatalysed r a t e i s exceedingly slow, the a d d i t i o n of an enzyme w i l l g i v e r i s e to measurable r a t e s . An i n v e s t i g a t i o n of the dependence of r a t e upon c o n c e n t r a t i o n of S i n the presence of a f i x e d c o n c e n t r a t i o n of enzyme now r e v e a l s that the simple equation (1) f o r the i n i t i a l r a t e i n the absence of product no longer h o l d s . A graph of v a g a i n s t [S] now d e s c r i b e s p a r t of a r e c t a n g u l a r hyperbola ( f i g u r e 42) and i s governed by the equation Vm [S] v = (2) Km + [S] Where Vm = the maximum r a t e of r e a c t i o n and Km = constant. I t can be shown that a t high s u b s t r a t e c o n c e n t r a t i o n s ([S] >> Km) equation (2) w i l l s i m p l i f y to v = Vm and a t low c o n c e n t r a t i o n of S ([S] << Km) equation (2) can be s i m p l i f i e d to the f o l l o w i n g f i r s t order r a t e e x p r e s s i o n v - (Vm / Km) [S] - 1 1 6 -s F i g u r e 42. Dependence of i n i t i a l r a t e v on the i n i t i a l c o n c e n t r a t i o n S f o r the uncatalysed and enzyme c a t a l y s e d r e a c t i o n S;==iP. These observations and others led s e v e r a l of the e a r l y enzymologists to make the suggestion that the enzyme (E) a c t u a l l y combines r e v e r s i b l y with the s u b s t r a t e (S) to form a complex (ES). T h i s then breaks down to l i b e r a t e the product (P) together with the unchanged enzyme. This proposal e x p l a i n e d why the i n i t i a l r a t e reached a l i m i t i n g value when the s u b s t r a t e c o n c e n t r a t i o n was i n c r e a s e d to high v a l u e s , because the enzyme would become " s a t u r a t e d " with S. In mathematical terms , the h y p e r b o l i c shape of the curve could a l s o be explained by t h i s p r o p o s a l . Assuming the r e a c t i o n takes place according to the f o l l o w i n g model and that b i n d i n g i s not the r a t e determining step k. k. E + S The i n i t i a l r a t e of appearance of product i s given by d[P] - k 2 [ES] (3) dt Where [ES] = the steady s t a t e c o n c e n t r a t i o n of the enzyme s u b s t r a t e complex, which i s d e s c r i b e d by; -11 7 -d[ES] « = k,(e - [ E S ] ) ( s - [ES]) - [ ES ] (k_«, + k 2 ) = 0 (4) dt e = t o t a l enzyme c o n c e n t r a t i o n and s = i n i t i a l s u b s t r a t e c o n c e n t r a t i o n . Assuming (s - [ES] ) a; t h i s assumption i s g e n e r a l l y v a l i d because e << s thus when a l l the enzyme i s converted to ES i t would not make an a p p r e c i a b l e d i f f e r e n c e to the c o n c e n t r a t i o n of f r e e S. From equation ( 4 ) ; k. (e - [ES] ) s = [ES] (k-, + k 2 ) k, e s - k, [ES] s - [ES] (k_« + k 2 ) [ES] (k-. + k 2 + k t s ) = k. e s k« e s [ES] = (k_, + k 2 + k« s) d i v i d i n g by k« e s [ES] = [ ( k-, + k 2 ) / k< ] + s s u b s t i t u t i n g t h i s value f o r [ES] i n t o equation (3) k, e s v = - (5) [ ( k_ 4 + k 2 ) / k 4 ] + s I f Vm = k 2 e , and Km = (k_ 1 + k 2 ) / k 1 equation (5) becomes e x a c t l y the same as the e x p e r i m e n t a l l y derived equation ( 2 ) . The d e r i v a t i o n of t h i s equation ( u s i n g the steady s t a t e assumption) was o r i g i n a l l y due to Briggs and Haldane, but s i m i l a r r e s u l t s can be obtained i f i t i s assumed that the s u b s t r a t e i s i n r a p i d e q u i l i b r i u m with the enzyme and that the r a t e l i m i t i n g step i s the breakdown of ES to P i e . k 2 << k_1 . In t h i s case Km = Kd (Kd = d i s s o c i a t i o n constant f o r ES), these assumptions were used -118-by M i c h a l i s and Menten i n the o r i g i n a l d e r i v a t i o n of equation ( 2 ) . These assumptions are not true f o r a l l enzyme mechanisms, but the r a p i d e q u i l i b r i u m can be regarded as a s p e c i a l case of the more general steady s t a t e treatment. The p h y s i o l o g i c a l s i g n i f i c a n c e of Km, as d e r i v e d by the steady s t a t e method, i s not so obvious, as c l e a r l y i t i s not a simple d i s s o c i a t i o n c o n s t a n t . However Km can s t i l l be regarded as a measure of the a f f i n i t y that an enzyme shows f o r a s u b s t r a t e . Determination of Km and Vm. Without r e s o r t i n g to computer aided methods, the best way to o b t a i n an accurate estimate of Vm and Km from experimental data i s to r e p l o t them i n a d i f f e r e n t manner. A very common procedure i s due to Lineweaver and Burk i n which 1/v i s p l o t t e d a g a i n s t 1/s. From equation (2) by simple i n v e r s i o n ; 1 Km + s Km 1 1 _ + _ ( 6) v Vm s Vm s Vm Thus t h i s double r e c i p r o c a l p l o t w i l l be a s t a i g h t l i n e of slope Km/Vm and i n t e r c e p t on the X a x i s of 1/Vm. A l s o , i t can be shown that when 1/v = 0, 1/s = -1/Km. R e v e r s i b l e enzyme i n h i b i t i o n . When a l i g a n d binds to an enzyme and prevents b i n d i n g of the s u b s t r a t e , the r a t e of co n v e r s i o n of S to P w i l l be reduced simply because l e s s of the enzyme i s a v a i l a b l e f o r c a t a l y s i s and the enzyme i s s a i d to be " i n h i b i t e d " by the l i g a n d . The simplest type of r e v e r s i b l e i n h i b i t i o n i s known as competitive i n h i b i t i o n . Here the i n h i b i t o r can only bind to the f r e e enzyme - 1 1 9 -at the same s i t e that would normally be occupied by the s u b s t r a t e . T h i s type of i n h i b i t i o n i s o f t e n seen when the i n h i b i t o r i s s t r u c t u r a l l y s i m i l a r to the s u b s t r a t e . + P The c o n c e n t r a t i o n of f r e e X i s assumed to be equal to i t s t o t a l c o n c e n t r a t i o n x. A f t e r a s i m i l a r d e r i v a t i o n to that shown f o r equation (5) the f o l l o w i n g r e s u l t i s obtained; Vm s v - (7) Km ( 1 + x / K i ) s Where K i = kg / k_g, the d i s s o c i a t i o n constant f o r the enzyme i n h i b i t o r complex. I t may be noted that the term Vm remains unchanged, while the Km i s i n c r e a s e d by the f a c t o r x / K i , thus a competitive i n h i b i t o r has the e f f e c t of i n c r e a s i n g the apparent value of Km while Vm remains co n s t a n t . I f r e c i p r o c a l s of (7) are taken, the f o l l o w i n g r e s u l t i s obtained; 1 1 Km / x \ 1 = + 1 + ] (8) v Vm Vm V k i / s A p l o t of 1/v a g a i n s t 1/s a t a constant value of x i s t h e r e f o r e a s t r a i g h t l i n e of slope Km(l + x / K i ) / Vm . The i n t e r c e p t on the 1 / v a x i s ( i e . when 1 / s = 0) i s 1 / Vm whatever f i n i t e value i s g iven to x. T h i s s i t u a t i o n i s shown i n f i g u r e 43. The i n t e r c e p t on the x a x i s (1 / v = 0) = -1 / Km (apparent) t h e r e f o r e a value f o r Km (apparent) can be r e a d i l y o btained. As Km (apparent) = Km(l + x / K i ) a p l o t of Km (apparent) a g a i n s t -120-Partial pressure of oxygen F i g u r e 44. S a t u r a t i o n curve f o r haemoglobin with molecular oxygen L a t e r he suggested that haemoglobin was an o l i g o m e r i c p r o t e i n , whose i n d i v i d u a l protomers could each bind one oxygen molecule i n such a way that the b i n d i n g of s u c c e s s i v e oxygen molecules i n c r e a s e d the a f f i n i t y of the remaining protomers. Hb + 0. Hb0 2 + 0 2 H b ( 0 2 ) 2 + 0 2 Hb0 2 j K1 H b ( 0 2 ) 2 ^ H b ( 0 2 ) 3 H b < ° 2 > n - 1 + °2 ^ H b<°2>n Where the e q u i l b r i u m constants KI - K4 were p r o g r e s s i v e l y b i g g e r , K-j << Kg << Kg << K n . In that case he argued, that oxygen molecules would e f f e c t i v e l y bind n at a time (where n was the number of protomers i n the haemoglobin o l i g o m e r ) , because the co n c e n t r a t i o n s of H b ( 0 2 ) , H b ( 0 2 ) 3 e t c . would be v a n i s h i n g l y s m a l l . The b i n d i n g process could then be summarised as; Hb + n 0 2 [ Hb(0,) ] K ' = n Hb(0 2 ) n (10) [Hb] [ 0 2 ] Where K' = an o v e r a l l constant f o r the h y p o t h e t i c a l process of n oxygen molecules b i n d i n g s i m u l t a n e o u s l y . This i s a h y p o t h e t i c a l -121-x w i l l y i e l d a s t r a i g h t l i n e of slope of Km / Ki and a Y i n t e r c e p t (Km (apparent) = 0) of - K i . Thus a r e p l o t of the values obtained f o r Km (apparent) (form the double r e c i p r o c a l ) a g a i n s t x w i l l y i e l d a value f o r K i . 1 1 1 1 F i g u r e 43. Determination of the i n h i b i t o r constant ( K i ) . ( l ) and (2) represent the p l o t obtained i n the absence and presence of an i n h i b i t o r . A l l o s t e r i c enzymes; Not a l l enzymes e x h i b i t the simple r e l a t i o n s h i p between v and s d e s c r i b e d by equation ( 2 ) ; many show a more complex sigmoid r e l a t i o n s h i p as d e p i c t e d i n f i g u r e 44. Haemoglobin i s t y p i c a l of t h i s type of p r o t e i n and was the s u b j e c t of one of the e a r l i e s t attempts to e x p l a i n t h i s phenomenon. H i l l pointed out that the e m p i r i c a l equation of the form; n [ O i ] Y = n ( 9 ) K + [0^] Where Y = f r a c t i o n a l s a t u r a t i o n with oxygen, K = constant and n = a small p o s i t i v e number would d e s c r i b e the behviour of t h i s type of enzyme. -12 2-process of course because the the l i k e l i h o o d of s e v e r a l molecules of oxygen b i n d i n g at once to a s i n g l e molecule of haemoglobin i s exceedingly s m a l l . However equation (10) can be rearranged to g i v e the e m p i r i c a l equation (9) and i t i s p o s s i b l e to rearrange equation (9) to give a more u s e f u l form (11). Y log n log [ 0 2 ] - log K (11) 1 - Y In t h i s way a p l o t of log (Y / 1 - Y) a g a i n s t log [ 0 A ] should g i v e a s t r a i g h t l i n e of slope n and i n t e r c e p t - l o g K i f H i l l s e x p l a n a t i o n i s c o r r e c t . In f a c t the experimental data f o r haemoglobin, when p l o t t e d i n t h i s manner gives the curve shown i n f i g u r e 45, with a maximum value f o r the slope of 2.5 - 3.0. As we now know haemoglobin i s a t e t r a m e r i c p r o t e i n and n should have had a value of 4 i f H i l l ' s i d e a had been a b s o l u t e l y c o r r e c t and the b i n d i n g t o t a l l y c o o p e r a t i v e . Slope • 1 Log[Oj F i g u r e 45. A H i l l p l o t f o r the b i n d i n g of oxygen to haemoglobin, Although H i l l ' s e x p l a n a t i o n turned out to be i n c o r r e c t , h i s method of p l o t t i n g data i s s t i l l widely used and i s p a r t i c u l a r l y u s e f u l f o r the determination of i n h i b i t o r c o n s t a n t s . I f the r a t e v i s assumed to be p r o p o r t i o n a l to the f r a c t i o n a l s a t u r a t i o n Y, -12 3-a p l o t of log (v / Vm - v) a g a i n s t log s w i l l give a curve of maximum slope n and an i n t e r c e p t of - log Km (appare n t ) . The value of n i s known as the H i l l c o e f f i c i e n t and although i t has no p h y s i o l o g i c a l s i g n i f i c a n c e i s taken as an index of the c o o p e r a t i v i t y of the system. For a s e r i e s of experiments run a t v a r i o u s values of x ( i n the same manner as f o r the double r e c i p r o c a l p l o t ) a r e p l o t of the values obtained f o r Km (apparent) a g a i n s t x w i l l again y i e l d a value f o r K i . -124-APPENDIX 2 10 HOME 20 PRINT "THIS PROGRAM PROVIDES ESTIMATES" 30 PRINT "OF KM AND VMAX FROM A WEIGHTED" 40 PRINT "LINEAR REGRESSION ANALYSIS. 50 DIM S(10 ) , V(10 ) ,S$(10),V|(10) 60 D$ = CHR$ (4) + CHR* (13) 70 KI = 0 80 PRINT : PRINT 90 PRINT "1) KI DETERMINATION" 100 PRINT : PRINT "2) KM DETERMINATION" 110 PRINT 120 GET Q$ 130 IF ASC (Q$) - 49 THEN KI = 1: GOTO 160 140 IF ASC (Q$) = 50 THEN 160 150 GOTO 120 160 HOME 170 PRINT "SAMPLE #"; TAB( 11 ) "SUBSTRATE"; TAB( 23)"RATE"; TAB( 30)"CALC TJLATED" 180 PRINT TAB ( 13)"CONC"; TAB ( 32)"RATE" 190 POKE 34,3 200 HOME 210 N - 0 220 IF KI - 1 THEN IT$ - "INHIBITOR CONC: ":L - 7:IL - 3:FL = 3: GOSU1 1010:IN$ = B$ 230 PRINT 240 GOSDB 910 250 IF N > 10 THEN 280 260 IF VAL (B$) < > 0 THEN 240 270 IF N < 3 THEN 240 280 GOSUB 1390 290 IT$ - "ADD A POINT ? (Y/N) " 300 GOSUB 1560 310 IF Q$ - "Y" THEN GOSUB 1460: GOSUB 910 320 A - 0:B - 0:C = 0:D = 0:E = 0 3S''> FOR Z - 1 TO N 34 > « V(Z) k 2 351 - X / S(Z) 360 ;r- = A + (V(Z) * X) 370 B = B + X k 2 380 C - C + (Y * V(Z)) 390 D - D + (X * Y) 400 E • E + Y k 2 410 NEXT Z 420 DE - ((A * E) - (D * C)) 430 KM - ((B * C) - (A * D)) / DE 440 VM - ((B * E) - (D * D)) / DE 450 A • 0:B - 0:C - 0:D - 0:E - 0:F - 0:G - 0:F1 - 0 460 FOR IC - 1 TO N 470 F - VM * S(IC) / (S(IC) + KM) 480 FI - - VM * S(IC) / ((S(IC) + KM) k 2) 490 A - A + F k 2 500 B - B + FI k 2 - 1 2 5 -510 C - C + (F * FI) 520 D - D + (V(IC) * F) 530 E = E + (V(IC) * FI) 540 G = G + V(IC) k 2 550 NEXT IC 560 DE - (A * B) - (C * C) 570 BI - ((B * D) - (C * E)) / DE 580 B2 = ((A * E) - (C * D)) / DE 590 VR - BI * VM 600 KR • KM + (B2 / BI) 610 S2 - SQR ((G - BI * D - B2 * E) / (U - 2)) 620 EK - S2 / BI * SQR (A / DE) 630 EV - VM * S2 * SQR (B / DE) 640 IL - 3:FL » 3:NO « KR: GOSOB 1270:KM$ = B$ 650 IL « 2:FL - 3:N0 «• EK: GOSUB 1270:EK$ - B$ 660 IL - 3:FL « 3:NO - VR: GOSUB 1270:VM$ = B$ 670 IL - 2:FL - 3:N0 = EV: GOSUB 1270:EV$ - B$ 680 GOSUB 1520 690 FOR Z - 1 TO N 700 VC - VR * S(Z) / (KR + S(Z)) 710 VTAB (Z + 3) 720 HTAB (32) 730 IL - 3:FL » 3:N0 = VC: GOSUB 1270: PRINT ;B$ 740 NEXT Z 750 GOSUB 1390 760 IF VAL (B$) < > 0 THEN 320 770 IT$ - "PRINT RESULTS (Y/N) ": GOSUB 1560 780 IF Q$ = "N" THEN 870 790 IT$ - "PRINTER SLOT # ":IL • 1:FL • 0:L = 1: GOSUB 1010:SL » VAL (B$) 800 PR# SL 810 HOME 820 PRINT ;"SAMPLE 3"; TAB( 11)"SUBSTRATE"; TAB( 22){"RATE" 830 PRINT TAB( 13)"C0NC" 840 GOSUB 1470 850 GOSUB 1520 860 PR# 0 870 IT* = "ANOTHER SET OF DATA ": GOSUB 1560 880 IF Q$ - "Y" THEN 200 890 END 900 IL = 1:FL - 0:L - 1:IT$ - "PRINTER SLOT # ": GOSUB 1010:SL • VAL (B$) 910 N - N + 1 920 VTAB (N + 3): PRINT TAB( 3);N 930 IT$ - "ENTER RATE : ":L - 7:FL - 3:IL - 3: GOSUB 1010 940 IF VAL (B$) - 0 THEN VTAB (N + 3): HTAB (3): PRINT ;" ":N - N - 1: RETURN 950 VTAB (N + 3): HTAB (22): PRINT ;B$ 960 V(N) - VAL (B$):V$(N) - B$ 970 IT$ - "SUBSTRATE CONC: ":L - 7:IL - 3:FL - 3: GOSUB 1010 980 S(N) - VAL (B*):S$(N) - B$ 990 VTAB (N + 3): HTAB (11): PRINT ;B$ 1000 RETURN -126-1010 1020 1030 1040 1050 1060 1070 1080 1090 1100 1110 1120 1130 1140 1150 1160 1170 1180 1190 1200 1210 1220 1230 1240 1250 1260 1270 1280 1290 1300 1310 1320 1330 1340 1350 1360 1370 1380 1390 1400 1410 1420 1430 1440 1450 1460 1470 1480 1490 1500 THEN 1070 < 46 OR ASC (X*) M* - " " S* - " " B* - MID* (M*,1,L) POKE 35,24: VTAB (23): HTAB (1) PRINT ;IT*; I - 1 VTAB (23): HTAB (20): PRINT S* HTAB (20): VTAB (23) PRINT MID* (B*, LEN (B*) VTAB (23) HTAB (20 + L) GET X* IF X* - CHR* (13) THEN 1240 IF X* < > CHR* (8) THEN 1190 IF I - 1 THEN 1070 B* - MID* ( B * , l , LEN (B*) - 1) I - I - 1 GOTO 1070 IF I - L + 1 IF ASC (X*) B* - B* + X* 1 = 1 + 1 GOTO 1070 B* - MID* (B*, LEN (B*) B* - MID* (S*,1,L NO •= VAL (B*) S - SGN (NO) N2 «» 10 k FL Nl - ABS (NO) IP « INT (Nl) FP « INT (N2 * (Nl B* - STR* (IP) :L «= IF IL « 0 AND IP -IF S < 0 THEN L -- "-":L - 1 IF IL > L THEN B* IF FL > 0 THEN B* POKE 35,22 RETURN IT* « "DELETE POINT # IF P - 0 THEN RETURN N •* N - 1 L + 1, LEN (B*)) > 57 THEN 1070 1 + 2 , LEN (B*)) LEN (B*)) + B* 5 / N2 IP)) + N2 LEN (B*):B* 0 THEN B* - ""; RIGHT* (B*,L) L - 0 L + 1:B* - "-" + B*: IF IP - 0 AND IL - 1 THEN B* B* + + B*:L - L + 1: GOTO 1350 ' + RIGHT* ( STR* (FP),FL) ":L - 2: GOSUB 1010:P - VAL (B*) FOR Z - P V(Z) - V(Z S(Z) - S(Z NEXT HOME FOR Z - 1 VTAB (Z + TO N + 1):V*(Z) + 1):S*(Z) TO N 3): PRINT V*(Z S*(Z 1) 1) TAB( 3);Z; PRINT NEXT TAB( 11);S*(Z); TAB( 22);V*(Z) -127-1510 RETURN 1520 VTAB (N + 5): HTAB (2): PRINT "KM - ";KM$;" +/- ";EK$ 1530 VTAB (N + 7): HTAB (2): PRINT "VM - ";VM|;" +/- ";EV$ 1540 IF KI - 1 THEN VTAB (N + 9): HTAB (2): PRINT "[INHIBITOR] - ";IN$ 1550 RETURN 1560 POKE 35,24 1570 VTAB (23): HTAB (1) 1580 PRINT IT$; 1590 VTAB (23): HTAB (20): PRINT S* 1600 VTAB (23): HTAB (21): GET Q$ 1610 IF Q| •= "Y" OR Q$ = "N" THEN POKE 35,22: RETURN 1620 GOTO 1570 

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