- Email: [email protected]
Chapter 29. Nucleosides and Nucleotides
30 4 Chapter 29. Nucleosides and Nucleotides Howard J . Schaeffer, State University of New York at Buffalo, Buffalo, New York During 1966, a review of methods in nucleoside syntheses' and a review of the ionization and metal complex formation of adenosine and adenine nucleotides were published. 2 The preparation of nucleosides by the fusion method continues to be employed extensively. Iodine has been found to be a good catalyst for the synthesis of certain nucleosides by the fusion method? Nebularine has been synthesized in high yield by the fusion technique using &-@nitropheny1)hydrogen phosphate as the catalyst .4 The synthesis of 2'-Cmethyladenosine has been accomplished from the protected 2-C-methyl-Dribono-7-lactone (I) which was reduced with &-(3-methyl-2-buty1)borane(disiamylborane). The corresponding tetrabenzoyl derivative was converted into the chloro sugar which after condensation with chloromercuri-6-benzamidopurine followed by deblocking gave 2 '-C-methyladen~sine.~ This compound as well as 3'-C-methyladenosine are both cytotoxic against KB cells in ~ u l t u r e . ~ Nucleosides were prepared by the fusion method from homoribose with 6-chloro-, and 2,6-diI chloropurines. The corresponding 6-mercaptoand 2-chloro-6-aminopurinenucleosides were nontoxic in tissue culture (KB and HEp-2/MP).6 A variety of aldofuranosyl nucleosides have been prepared by the chloromercuri procedure.' The utilization of the 2,4-dinitrophenyl group as a blocking group in the preparation of 2'-amino-2'deoxy-nucleosides has been described in the synthesis of 2'-amino-2'deoxy-adenosine. Urbas and Whistler have prepared several 1- (4-thio-Dribofuranosy1)pyrimidine nucleosides by the Hilbert-Johnson procedure. In all instances a 2:l mixture of anomers was obtained with the f3-2 anomer predominating over the a-2 anomer. The synthesis of 4'-acetamidoadenosine has been accomplished by the condensation of 4-acetamido-1,5-di-O-acetyl2 ,3-di-0- benzoy l-4-deoxy-D -ribofuranose with chloromercuri-6-benzamidopurine using a titanium tetrachloride catalyzed reaction. lo 1-Deaza-6-methylthiopurine ribonucleoside and 3-deaza-6-methylthiopurine ribonucleoside have been prepared by fusion of the corresponding deaza-6-chloropurine with t e t r a - 0 - a c e t y l r i b o f u r a n o s e followed by reaction with sodium methyl mercaptide.11,l2 Both of these deaza nucleosides were much less cytoxic than 6-methylthiopurine ribonucleoside, but l-deaza6-methylthiopurine ribonucleoside was cytotoxic to HEp-2 cells which were resistant to 6-merca topurine. 3-Deazaadenosine has been synthesized by a related procedure.P3 Replacement of the 6-chloro group could not be accomplished with ammonia. However, treatment of 3-deaza-6-chloropurine ribonucleoside with hydrazine followed by reduction with Raney nickel gave 3-deazaadenosine . 13
Montgomery and Thomas have studied the infrared, ultraviolet and NMR
Chap. 2 9
Nucleosides and Nucleotides
Schaeffer
30 5 -
spectra of a number of mercury or chloromercury purines and compared these spectra with the spectra of the corresponding sodium s a l t s and N-7 and N - 9 a1kylp~rines.l~Based on these studies, they concluded that the mercury or chloromercuri are covalent. The chloromercury group is attached to N-7 of 3-benzylhypoxanthine and the mercury group is at N-7 of theophylline. The chloromercuri group is attached to N-9 of l-benzylhypoxanthine, 1-benzylpurine-6(1 H) -thione, and 6-dimethylaminopurine. When these mercury or chloromercury derivatives are allowed to react with acylglycosyl halides the attack is on the nitrogen bearing the mercury or chloromercury group. 14 The utility of a 3-substituent on hypoxanthine to direct substitution to the 7-position has been shown by the preparation of 7a- and 7B-D-arabinofuranosylhypoxanthines. l5 Thus , 3-benzyl- o r 3-benzhydryladenines were treated with nitrosyl chloride to give the corresponding 3-substituted hypoxanthines. The chloromercury derivatives of these compounds were condensed with 2,3,5-tri-O-benzoyl-D-arabinofuranosyl bromide. Catalytic hydrogenolysis of the benzyl or benzhydryl group gave 7a- and 7B-D-arab i n o f u r a n o s y l h y p o x a n t h i n e s . l5 The synthesis of 7-glycosylpurines as the exclusive product of the reactions is exemplified by the preparation of 7B-D-ribofuranosyladenine.16 4 (5) -Bromo-5(4)-nitroimidazole was fused with tetra-0-acetyl-@-D-ribofuranose to give a N-glycosyl derivative which on treatment with potassium cyanide, then Raney nickel and finally with acetic anhydride and triethyl orthoformate gave 7-B-D-ribofuranosyladenine.16 Shimizu and Miyaki have shown that the migration of the ribosyl group in N-benzoyl-3-~-D-ribofuranosyLadenine from N 3 to N 9 occurs by an intermolecular mechanism. 17 An interesting stereoselective synthesis of the anomeric 5-mercapto-2'deoxyuridines has recently been reported. l8 When 5-acetylmercapto-2,4-0bis-(trimethylsily1)uracil and the blocked chloro sugar were fused at 100110" for 15-20 minutes, only the B-isomer of the blocked compound could be isolated from the reaction. However, when the reaction was carried out in benzene solution at 37" for 90 hours, only the a-isomer was isolated.18
By modifications of known procedures, the following compounds have been prepared: 1-B-D-arabinofuranosylthymine, l9 2 '-deoxy-5-(trifluoromethyl) uridine and its a-anomer,2o a- and @-5-trifluoromethyl-6-aza-2'-deoxyuridines,21,2 2 l-~-D-arabinofuranosyl-5-fluorouracil, 23 5-fluoro-2'deoxycytidine,24 3'-deoxynucleosides of rimidines,25 pyrLmidine and urine nucleosides of D-glucuronic acid,26,'y 9-D-mannofuranosyladenine, 2f; 3-@-D-arab inofuranosyladenine, 3-B-D- ribofuranosy lor0t ic ac id,30 5- (DLribofuranosyl) -6-azauracil,31 and 5-a-D-arabinitoluracil and 5-a-D-ribitoluracil.32 Chemical transformations of nucleosides have resulted in the syntheses of some 2',3'-unsaturated pyrimidine nucleosides33 as well as a 4',5'unsaturated pyrimidine nucleoside.34 Treatment of 3'-0-tosyl-2'-deoxyadenosine with alkoxide produced the 2' ,3'-unsaturated n u ~ l e o s i d e36 ~ ~ as , well as a smaller amount of the 3',5'-oxetane deri~ative.3~The syntheses of 2 ' ,3'-dideoxy-, 2 ' ,5 ' -dideoxy-, and 2 ' ,3',5 '-trideoxyadenosine have been accomplished by mono- or ditosylation of 2'-deoxyadenosine
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'0 HoxoHp - I2
followed by displacement of the tosylate with sodium ethyl mercaptide and Raney nickel desulfurization.37 F\c L O O H
H
o
~
0 o
I'H~
A novel transformation
in aqueous alkali has been observed with 1-f3-D0 uracil arabinof as uranos outlined.38 yl-5 fluoroThe
-
open cham compound was isolated in 50% yield and by treatment with diazomethane and then with alkali generated 1- f3-D -arabinofuranosyl-3-methyl-5fluorouracil. Moreover, the ribo- or 2'-deoxyribo derivatives did not undergo this reaction.38 The acid catalyzed solvolysis of pyrimidine nucleosides has been studied and the acid stability is markedly influenced by the number o f hydroxyl groups in the sugar. In the 2'-deoxyribonucleosides, the 3'-hydroxyl group exhibits a large effect on the rate of react i ~ n ? A~ convenient synthesis y f 1;p-D-arabinofuranosylcytosine has been reported by tosylation o f ~ 4 , 0 3,O5 -tri-0-acetylcytidine. The proposed 2,2'-anhydrocytidine derivative undergoes rapid hydrolysis to 1-f3-Darabinofuranosylcytosine.40 Hampton and Nichol have described a convenient synthesis of 2,2'-anhydro-l-~-D-arabinofuranosyluracil by treating uridine with diphenyl carbonate.41 In a continuation of some studies on acyl migrations in model compounds related to aminoacyl-S-RNA, a technique has been developed using NMR to determine the rate of acyl migration in some 3'-O-acyl ribonucleosides. It was estimated that the half-time of equilibration into 2' and 3'-isomers of an average aminoacyl-S-RNA derivative in pH 7 buffer at 37' is 2 x sec. It was concluded that equilibration of an average aminoacyl-S-RNA would most likely be much faster than peptide bond synthesis.42 Two studies of ORD on pyrimidine nucleosides have appeared which assist in the assignment o f the anomeric configuration.43,44 The anomeric configuration can be assigned utilizing an NMR technique which compares the pyrimidine nucleoside with its 5,6-dihydroderivative.45 In some 6-substituted purine 3'-deoxyribonucleosides,a direct correlation between the extent of phosphorylation of the nucleosides and inhibition of RNA synthesis in intact Ehrlich ascites cells has been shown.46 The synthesis and properties o f some substituted 6-hydroxylaminopurines has been de~cribed.~7 The structures of f ~ r m y c i n , formycin ~~ B,48 and blasticidin S4' have been described. The syntheses of some nucleosidic peptides related to amicetin have also been reported.50 In an elegant paper, Shealy and Clayton have outlined their synthesis of 9G-DL-2a, 3a-dihydroxy-4P-(hydroxymethyl) cyclopentyl] adenine (11)
Chap. 29
Nucleosides and Nucleotides
HO
OH I1
S cha effe r
307 -
which is the carbocyclic analog of adenosine.51 Norbornadiene was cis-hydroxylated and after protection the olefin was oxidized to yield 2a,3adiacetoxy-l~,4f3-cyclopentanedicarboxylicacid. The cyclic anhydride was converted into the amide acid which on Hofmann rearrangement followed by lithium borohydride reduction on the ester gave a derivative of l~-amino-2a,3a-dihydroxy-4B-hydroxymethylcyclopentane. Condensation of the aminotr iol with 5-amino-4,6-dichloropyrimid ine followed by ring closure with triethyl orthoformate gave the 6-chloropurine derivative which on treatment with ammonia gave II.51
A most unusual rearrangement has been observed by Chheda and Hall with the finding that N6-glycvladenine (111) is converted into N- (6-purinyl) When 111 is kept in aqueous solution for a few minutes glycine (vII),~~ at lOO", it loses the elements of ammonia and forms a cyclic intermediate which has beep assigned structure IV. N6-chloroacetyladenine (V) forms the identical intermediate(1V). Compound IV in neutral solution undergoes ring cleavage, and rearrangement to VII, presumably via VI.52 0
NHCOCH2NH2
H 111
I," /
H
H V
H VI
VII
The initial announcement of a method for the stepwise synthesis of o l i g o d e o x y r i b o n u c l e o t i d e s on aninsoluble polymer support was made in 1965
by Letsinger and M a h a d e ~ a n . ~ n~ 1966 several papers appeared on syntheses with a polymer support.54-5' The procedure used by Letsinger and Mahadevan employs an insoluble copolymer of styrene (88%); 2-vinylbenzoic acid (12%) and p-divinylbenzene (0.2% or 0.02 %). The carboxyl groups on the polymer were converted into acid chlorides with thionyl chloride and allowed to react with 5 '-0-trityl-2'-deoxycytidine (VIII) to yield IX. Condensation of IX with cyanoethylphosphate and mesitylenesulfonyl chloride or DCC gave X. When X was activated with mesitylenesulfonyl chloride and
30 8 -
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condensed with thymidine XI was produced. By repetition of 'the phosphorylation of the free 3'-hydroxyl group, activation and condensation steps several different oligodeoxyribonucle@-co-NH otides were synthesized. The final products can be removed from the polymer with 0.4 M_ sodium hydroxide in a mixture of dioxane, ethanol and 0 water .53354 0
" 4
The second method of deoxyribonucleotide synthesis on a polymer support is different from the procedure of Letsinger and Mahadevan in two respects. The polymer employed is a polystrene polymer which is soluble in the reaction medium and the deoxyribonucleoside is attached to the polymer through its 5I-hydroxyl group by means Ok of a methoxytrityl group on the polymer.55,56 In order to functionalize XI the polymer (XII), it was subjected to a Friedel-Crafts with benzoyl chloride to give XIII. Treatment of XI11 with 2-methoxyphenylmagnesium bromide gave the trityl alcohol (XIV) which was converted to the trit 1 chloride (XV) with acet 1 chloride. Condensation of XV with thymidineT5 or 3I-O-acetylthymidineX6 gave XVI. For internucleotide bond synthesis, XVI was allowed to react with pyridinium 3'-O-acetylthymidine 5 -phosphate and mesitylene chloride in pyridine solution. After treatment with water followed by removal of the 3I-O-acetyl blocking group, XVII was obtained in good yield. By repetitimof the condensation and deblocking steps, several different oligodeoxyribonucleotides were prepared. Removal of the oligodeoxyribonucleotide from the polymer was accomplished by brief treatment with trifluoroacetic acid in chloroform55 or d i ~ x a n e . ~ ~ A review has appeared on the synthesis and biological function of nucle~ t i d e s . A~ ~ paper describing the synthesis of the 64 possible ribotrinucleotides derived from the four major ribomononucleotides has been
Chap. 2 9
Nucleosides and Nucleotides
XI1
XI11
Scha effer
30 9 -
p~blished.5~A report has appeared describing the use of nucleoside 2’-0benzyl ethers in the synthesis of oligoribonucleotides.59 The use of l-dimethy lamino-1,1-dialkoxymethanes as a blocking group for reactive amino groups of cytidine, adenosine and guanosine in the stepwise synthesis of ribooligonucleotides has been described.60
Detailed kinetic studies on internucleotide bond synthesis using arylXIV, R=OH sulfonyl chlorides are reported, and the XV, R=C1 use of 2,4,6-triisopropylbenzenesulfonyl chloride for internucleotide bond synthesis is described.61 Phosphorylation of nucleosides with organic amine salts of phosphoric acid has been
XVI
XVII
studies on the preparation of nucleotides and ~ t u d i e d . ~ ~Additional >~3 dinucleoside phosphates by the anhydronucleoside method have been reported. 64965 Finally, an interesting phosphorylation of ribonucleosides with phosphorus trichloride has been discovered.66 Reaction of 2 ’ , 3’-O-isopropylidene inosine with phosphorus trichloride in acetone solution followed by an aqueous treatment and removal of the blocking group gave 5’-IMP in a 91% yield. This reaction was applicable to other nucleoside isopropylidene derivatives. The reaction requires oxygen and a solvent which is a ketone or aldehyde (acetone and methyl ethyl ketone are best). It is postulated that phosphorus trichloride reacts with the nucleoside to6give a dichlorophosphite which is then oxidized to the dichlorophosphate.
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References
(1) H. G . Garg, J . S c i . Ind. Research 25, 404 (1966). (2) R. P h i l l i p s , Chem. Rev. 66, 501 (1966). (3) K . I m i a , A. Nohara and M. Honjo, Chem. Pharm. B u l l (Tokyo) E, 1377 (1966). (4) T. Hashizume and H. Iwamura, T e t r a h e d r o n L e t t e r s 643 (1966). (5) E. Walton, S. R. J e n k i n s , R . F. N u t t , M. Zimmerman and F. W . H o l l y , J. Am. Chem. Soc. 88, 4524 (1966). (6) J. A. Montgomery and K. Hewson, J . Med. Chem. 2, 234 (1966). (7) P . Kohn, R. H. S a m a r i t a n 0 and L. M . L e r n e r , J . Org. Chem. 31, 1503 (1966). (8) M. L. Wolfrom and M. W . Winkley, Chem. Comm. 533 (1966). (9) B . Urbas and R. L. W h i s t l e r , J. Org. Chem. 31, 813 (1966). (10) E. J. R e i s t , D. E. Gueffroy, R. W. Blackford and L. Goodman, J . Org. Chem. 3l, 4025 (1966). (11) J. A. Montgomery and K. Hewson, J . Med. Chem. 2, 354 (1966). (12) J. A. Montgomery and K. Hewson, J . Med. Chem. 2, 105 (1966). (13) R. J. Rousseau, L. B. Townsend and R. K. Robins, Biochemistry 5, 756 (1966). (14) J . A. Montgomery and H. J . Thomas, J . Org. Chem. 2,1411 (1966). (15) H. J . Thomas and J . A . Montgomery, J . Org. Chem. 2, 1413 (1966). (16) R. J. Rousseau, L. B. Townsend and R. K. Robins, Chem. Comm. 265 (1966). (17) B. Shimizu and M . Miyaki, Chem. Ind. 664 (1966). '(18) T . J. Bardos, M. P . Kotick and C. Szantay, T e t r a h e d r o n L e t t e r s 1759 (1966). (19) F. K e l l e r and A. R. T y r r i l l , J. Org. Chem. 3l, 1289 (1966). (20) K. J. Ryan, E . M..Acton, and L. Goodman, J . Org. Chem. 3 l , 1181 (1966). (21) M. P . Mertes, S . E . Saheb and D . M i l l e r , J . Med. Chem. 2, 876 (1966). (22) A. D i p p l e and C . H e i d e l b e r g e r , J . Med. Chem. 2, 715 (1966). (23) F. K e l l e r , N. Sugisaka, A. R. T y r r i l l , L. H. Brown, J. E. Bunker and I . J . B o t v i n i c k , J. Org. Chem. 2, 3842 (1966). (24) R. Duschinsky, T. G a b r i e l , M. Hoffner, J . B e r g e r , E . T i t s w o r t h , E . Grunberg, J. H. Burchenal and J. J. Fox, J. Med. Chem. 2, 566 (1966). (25) E . Walton, F. W. H o l l y , G. E. Boxer, R. F. N u t t , J . Org. Chem. 3l, 1163 (1966). (26) T. Kishikawa and H. Yuki, Chem. Pharm. B u l l . (Tokyo) l4, 1360 (1966). (27) T. Kishikawa, T . Yamazaki and H. Yuki, Chem. Pharm. B u l l . (Tokyo) 14, 1354 (1966). (28) L. M. L e r n e r and P . Kohn, J . Org. Chem. 31, 339 (1966). (29) K. R. D a r n a l l and L. B. Townsend, J . H e t e r o c y c l i c Chem. 3, 371 (1966). (30) W. V. Curran and R. B. AngiFr, J . Org. Chem. 3l, 201 (19z6). (31) M . Bobek, J. Farkag and F. Sorm, T e t r a h e d r o n L e t t e r s 3115 (1966). (32) W . Asbun and S . B. B i n k l e y , J. Org. Chem. 3 l , 2215 (1966). (33) J. P. Horwitz, J. Chua, M . A. DaRooge, M. Noel and I. L. Klundt, J . Org. Chem. 3 l , 205 (1966).
Chap. 29
N u c l e o side s and N u c l e o t i d e s
Scha ef f e r
31 1 -
(34) J. P . H. Verheyden and J . H. M o f f a t t , J . Am. Chem. SOC. 88,5684 (1966). (35) J . R . McCarthy, J r . , M . J . Robins, L. R. Townsend and R . K . Robins, J . Am. Chem. S O C . 88, 1549 (1966). (36) J . P . Horwitz, J . Chua and M . Noel, T e t r a h e d r o n L e t t e r s 1343 (1966). (37) M . J . Robins, J . R . McCarthy, J r . and R. K . Robins, B i o c h e m i s t r y 5, 224 (1966). (38) J . J . Fox, N . C . Miller and R. J . Cushley, T e t r a h e d r o n L e t t e r s 4927 (1966)(39) E . R . G a r r e t t , J . K. S e y d e l and A . J . Sharpen, J . Org. Chem. 2, 2219 (1966). (40) H. P . M . Fromageot and C . B . Reese, T e t r a h e d r o n L e t t e r s 3499 (1966). (41) A . Hampton and A . W. N i c h o l , B i o c h e m i s t r y 5, 2076 (1966). (42) B . E . G r i f f i n , M . Jarman, C . B . Reese, J . E . S u l s t o n and D . R . Trentham, B i o c h e m i s t r y 5, 3638 (1966). (43) 1. F r i c , J . Smejkal and J . F a r k a s , T e t r a h e d r o n L e t t e r s 75 (1966). (44) T. L. V. U l b r i c h t , T . R . Emerson and R. J . Swan, T e t r a h e d r o n L e t t e r s 1561 (1966). (45) R . J . Cushley, K . A . Watnabe and J . J . Fox, Chem. Corn. 598 (1966). (46) H . T. S h i g e u r a , G. E . Boxer, M . L. Meloni and S . D . Sampson, B i o c h e m i s t r y 2, 994 (1966). (47) A . G i n e r - S o r o l l a , S . O'Bryant, J . H. Burchenal and A . Bendick, B i o c h e m i s t r y 5, 3057 (1966). (48) R. K . Robins, L. B. Townsend, F . C a s s i d y , J . F . Gerster, A. F . L e w i s and R . L . M i l l e r , J . H e t e r o c y c l i c Chem. 3, 110 (1966). (49) J . J . Fox and K . A . Watanabe, T e t r a h e d r o n L e t t e r s 897 (1966). (50) C. L . S t e v e n s , T . S . Sulkowski and M. E . Munk, J . Org. Chem. 3l, 4014 (1966). (51) Y . F. S h e a l y and J . D . C l a y t o n , J . Am. Chem. SOC. 88, 3885 (1966). (52) G. B . Chheda and R. H. H a l l , B i o c h e m i s t r y 5 , 2 0 8 2 (1966). (53) R. T . L e t s i n g e r and V. Mahadevan, J . Am. Chem. SOC. 87, 3526 (1965). (54) R . T . L e t s i n g e r and V. Mahadevan, J . Am. Chem. SOC. 88, 5319 (1966). (55) H. Hayatsu and H. J . Khorana, 3. Am. Chem. SOC. 88, 3182 (1966). (56) F . C r a m e r , R. H e l b i g , H. H e t t l e r , K. H. S c h e i t and H . S e l i g e r , Angew. Chern.Intern. Ed. E n g l . 5, 601 (1966). (57) F. C r a m e r , Angew. Chern.Intern. Ed. E n g l . 5, 173 (1966). (58) R . Lohrmann, D . S811, H . Hayatsu, E . Ohtsuka and H. G. Khorana, J . Am. Chem. SOC. 8 8 , 8 1 9 (1966). (59) B . E . G r i f f i n , C. B . R e e s e , G . F . S t e p h e n s o n and D . R . Trentham, T e t r a h e d r o n L e t t e r s , 4349 (1966). (60) A , Hal? and J . Smrt, C o l l e c t i o n Czech. Chem. Comun. 31, 3800 (1966). (61) R. Lohrmann and H . G. Khorana, J . Am. Chem. SOC. 88, 829 (1966). 1061 (62) M . Honjo, Y . Furukawa and K . Kobayashi, Chem. Pharm. B u l l . (1966). (63) M . Honjo, Y . Furukawa and K . Kobayashi, Chem. Pharm. B u l l . (Tokyo) 14, 1061 (1966). (64) T M i z u n y and T . S a s a k i , J . Am. Chem. SOC. 88, 863 (1966). (65) J . Nagyvary, B i o c h e m i s t r y 5, 1316 (1966). (66) M . Honjo, R . Marumoto, K. Kobayashi and Y . Yoshioka, T e t r a h e d r o n L e t t e r s 3851 (1966).
14,
Montgomery and Thomas have studied the infrared, ultraviolet and NMR
Chap. 2 9
Nucleosides and Nucleotides
Schaeffer
30 5 -
spectra of a number of mercury or chloromercury purines and compared these spectra with the spectra of the corresponding sodium s a l t s and N-7 and N - 9 a1kylp~rines.l~Based on these studies, they concluded that the mercury or chloromercuri are covalent. The chloromercury group is attached to N-7 of 3-benzylhypoxanthine and the mercury group is at N-7 of theophylline. The chloromercuri group is attached to N-9 of l-benzylhypoxanthine, 1-benzylpurine-6(1 H) -thione, and 6-dimethylaminopurine. When these mercury or chloromercury derivatives are allowed to react with acylglycosyl halides the attack is on the nitrogen bearing the mercury or chloromercury group. 14 The utility of a 3-substituent on hypoxanthine to direct substitution to the 7-position has been shown by the preparation of 7a- and 7B-D-arabinofuranosylhypoxanthines. l5 Thus , 3-benzyl- o r 3-benzhydryladenines were treated with nitrosyl chloride to give the corresponding 3-substituted hypoxanthines. The chloromercury derivatives of these compounds were condensed with 2,3,5-tri-O-benzoyl-D-arabinofuranosyl bromide. Catalytic hydrogenolysis of the benzyl or benzhydryl group gave 7a- and 7B-D-arab i n o f u r a n o s y l h y p o x a n t h i n e s . l5 The synthesis of 7-glycosylpurines as the exclusive product of the reactions is exemplified by the preparation of 7B-D-ribofuranosyladenine.16 4 (5) -Bromo-5(4)-nitroimidazole was fused with tetra-0-acetyl-@-D-ribofuranose to give a N-glycosyl derivative which on treatment with potassium cyanide, then Raney nickel and finally with acetic anhydride and triethyl orthoformate gave 7-B-D-ribofuranosyladenine.16 Shimizu and Miyaki have shown that the migration of the ribosyl group in N-benzoyl-3-~-D-ribofuranosyLadenine from N 3 to N 9 occurs by an intermolecular mechanism. 17 An interesting stereoselective synthesis of the anomeric 5-mercapto-2'deoxyuridines has recently been reported. l8 When 5-acetylmercapto-2,4-0bis-(trimethylsily1)uracil and the blocked chloro sugar were fused at 100110" for 15-20 minutes, only the B-isomer of the blocked compound could be isolated from the reaction. However, when the reaction was carried out in benzene solution at 37" for 90 hours, only the a-isomer was isolated.18
By modifications of known procedures, the following compounds have been prepared: 1-B-D-arabinofuranosylthymine, l9 2 '-deoxy-5-(trifluoromethyl) uridine and its a-anomer,2o a- and @-5-trifluoromethyl-6-aza-2'-deoxyuridines,21,2 2 l-~-D-arabinofuranosyl-5-fluorouracil, 23 5-fluoro-2'deoxycytidine,24 3'-deoxynucleosides of rimidines,25 pyrLmidine and urine nucleosides of D-glucuronic acid,26,'y 9-D-mannofuranosyladenine, 2f; 3-@-D-arab inofuranosyladenine, 3-B-D- ribofuranosy lor0t ic ac id,30 5- (DLribofuranosyl) -6-azauracil,31 and 5-a-D-arabinitoluracil and 5-a-D-ribitoluracil.32 Chemical transformations of nucleosides have resulted in the syntheses of some 2',3'-unsaturated pyrimidine nucleosides33 as well as a 4',5'unsaturated pyrimidine nucleoside.34 Treatment of 3'-0-tosyl-2'-deoxyadenosine with alkoxide produced the 2' ,3'-unsaturated n u ~ l e o s i d e36 ~ ~ as , well as a smaller amount of the 3',5'-oxetane deri~ative.3~The syntheses of 2 ' ,3'-dideoxy-, 2 ' ,5 ' -dideoxy-, and 2 ' ,3',5 '-trideoxyadenosine have been accomplished by mono- or ditosylation of 2'-deoxyadenosine
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'0 HoxoHp - I2
followed by displacement of the tosylate with sodium ethyl mercaptide and Raney nickel desulfurization.37 F\c L O O H
H
o
~
0 o
I'H~
A novel transformation
in aqueous alkali has been observed with 1-f3-D0 uracil arabinof as uranos outlined.38 yl-5 fluoroThe
-
open cham compound was isolated in 50% yield and by treatment with diazomethane and then with alkali generated 1- f3-D -arabinofuranosyl-3-methyl-5fluorouracil. Moreover, the ribo- or 2'-deoxyribo derivatives did not undergo this reaction.38 The acid catalyzed solvolysis of pyrimidine nucleosides has been studied and the acid stability is markedly influenced by the number o f hydroxyl groups in the sugar. In the 2'-deoxyribonucleosides, the 3'-hydroxyl group exhibits a large effect on the rate of react i ~ n ? A~ convenient synthesis y f 1;p-D-arabinofuranosylcytosine has been reported by tosylation o f ~ 4 , 0 3,O5 -tri-0-acetylcytidine. The proposed 2,2'-anhydrocytidine derivative undergoes rapid hydrolysis to 1-f3-Darabinofuranosylcytosine.40 Hampton and Nichol have described a convenient synthesis of 2,2'-anhydro-l-~-D-arabinofuranosyluracil by treating uridine with diphenyl carbonate.41 In a continuation of some studies on acyl migrations in model compounds related to aminoacyl-S-RNA, a technique has been developed using NMR to determine the rate of acyl migration in some 3'-O-acyl ribonucleosides. It was estimated that the half-time of equilibration into 2' and 3'-isomers of an average aminoacyl-S-RNA derivative in pH 7 buffer at 37' is 2 x sec. It was concluded that equilibration of an average aminoacyl-S-RNA would most likely be much faster than peptide bond synthesis.42 Two studies of ORD on pyrimidine nucleosides have appeared which assist in the assignment o f the anomeric configuration.43,44 The anomeric configuration can be assigned utilizing an NMR technique which compares the pyrimidine nucleoside with its 5,6-dihydroderivative.45 In some 6-substituted purine 3'-deoxyribonucleosides,a direct correlation between the extent of phosphorylation of the nucleosides and inhibition of RNA synthesis in intact Ehrlich ascites cells has been shown.46 The synthesis and properties o f some substituted 6-hydroxylaminopurines has been de~cribed.~7 The structures of f ~ r m y c i n , formycin ~~ B,48 and blasticidin S4' have been described. The syntheses of some nucleosidic peptides related to amicetin have also been reported.50 In an elegant paper, Shealy and Clayton have outlined their synthesis of 9G-DL-2a, 3a-dihydroxy-4P-(hydroxymethyl) cyclopentyl] adenine (11)
Chap. 29
Nucleosides and Nucleotides
HO
OH I1
S cha effe r
307 -
which is the carbocyclic analog of adenosine.51 Norbornadiene was cis-hydroxylated and after protection the olefin was oxidized to yield 2a,3adiacetoxy-l~,4f3-cyclopentanedicarboxylicacid. The cyclic anhydride was converted into the amide acid which on Hofmann rearrangement followed by lithium borohydride reduction on the ester gave a derivative of l~-amino-2a,3a-dihydroxy-4B-hydroxymethylcyclopentane. Condensation of the aminotr iol with 5-amino-4,6-dichloropyrimid ine followed by ring closure with triethyl orthoformate gave the 6-chloropurine derivative which on treatment with ammonia gave II.51
A most unusual rearrangement has been observed by Chheda and Hall with the finding that N6-glycvladenine (111) is converted into N- (6-purinyl) When 111 is kept in aqueous solution for a few minutes glycine (vII),~~ at lOO", it loses the elements of ammonia and forms a cyclic intermediate which has beep assigned structure IV. N6-chloroacetyladenine (V) forms the identical intermediate(1V). Compound IV in neutral solution undergoes ring cleavage, and rearrangement to VII, presumably via VI.52 0
NHCOCH2NH2
H 111
I," /
H
H V
H VI
VII
The initial announcement of a method for the stepwise synthesis of o l i g o d e o x y r i b o n u c l e o t i d e s on aninsoluble polymer support was made in 1965
by Letsinger and M a h a d e ~ a n . ~ n~ 1966 several papers appeared on syntheses with a polymer support.54-5' The procedure used by Letsinger and Mahadevan employs an insoluble copolymer of styrene (88%); 2-vinylbenzoic acid (12%) and p-divinylbenzene (0.2% or 0.02 %). The carboxyl groups on the polymer were converted into acid chlorides with thionyl chloride and allowed to react with 5 '-0-trityl-2'-deoxycytidine (VIII) to yield IX. Condensation of IX with cyanoethylphosphate and mesitylenesulfonyl chloride or DCC gave X. When X was activated with mesitylenesulfonyl chloride and
30 8 -
Sect. VI
- Topics
in Chemistry
Smi s sman, Ed.
condensed with thymidine XI was produced. By repetition of 'the phosphorylation of the free 3'-hydroxyl group, activation and condensation steps several different oligodeoxyribonucle@-co-NH otides were synthesized. The final products can be removed from the polymer with 0.4 M_ sodium hydroxide in a mixture of dioxane, ethanol and 0 water .53354 0
" 4
The second method of deoxyribonucleotide synthesis on a polymer support is different from the procedure of Letsinger and Mahadevan in two respects. The polymer employed is a polystrene polymer which is soluble in the reaction medium and the deoxyribonucleoside is attached to the polymer through its 5I-hydroxyl group by means Ok of a methoxytrityl group on the polymer.55,56 In order to functionalize XI the polymer (XII), it was subjected to a Friedel-Crafts with benzoyl chloride to give XIII. Treatment of XI11 with 2-methoxyphenylmagnesium bromide gave the trityl alcohol (XIV) which was converted to the trit 1 chloride (XV) with acet 1 chloride. Condensation of XV with thymidineT5 or 3I-O-acetylthymidineX6 gave XVI. For internucleotide bond synthesis, XVI was allowed to react with pyridinium 3'-O-acetylthymidine 5 -phosphate and mesitylene chloride in pyridine solution. After treatment with water followed by removal of the 3I-O-acetyl blocking group, XVII was obtained in good yield. By repetitimof the condensation and deblocking steps, several different oligodeoxyribonucleotides were prepared. Removal of the oligodeoxyribonucleotide from the polymer was accomplished by brief treatment with trifluoroacetic acid in chloroform55 or d i ~ x a n e . ~ ~ A review has appeared on the synthesis and biological function of nucle~ t i d e s . A~ ~ paper describing the synthesis of the 64 possible ribotrinucleotides derived from the four major ribomononucleotides has been
Chap. 2 9
Nucleosides and Nucleotides
XI1
XI11
Scha effer
30 9 -
p~blished.5~A report has appeared describing the use of nucleoside 2’-0benzyl ethers in the synthesis of oligoribonucleotides.59 The use of l-dimethy lamino-1,1-dialkoxymethanes as a blocking group for reactive amino groups of cytidine, adenosine and guanosine in the stepwise synthesis of ribooligonucleotides has been described.60
Detailed kinetic studies on internucleotide bond synthesis using arylXIV, R=OH sulfonyl chlorides are reported, and the XV, R=C1 use of 2,4,6-triisopropylbenzenesulfonyl chloride for internucleotide bond synthesis is described.61 Phosphorylation of nucleosides with organic amine salts of phosphoric acid has been
XVI
XVII
studies on the preparation of nucleotides and ~ t u d i e d . ~ ~Additional >~3 dinucleoside phosphates by the anhydronucleoside method have been reported. 64965 Finally, an interesting phosphorylation of ribonucleosides with phosphorus trichloride has been discovered.66 Reaction of 2 ’ , 3’-O-isopropylidene inosine with phosphorus trichloride in acetone solution followed by an aqueous treatment and removal of the blocking group gave 5’-IMP in a 91% yield. This reaction was applicable to other nucleoside isopropylidene derivatives. The reaction requires oxygen and a solvent which is a ketone or aldehyde (acetone and methyl ethyl ketone are best). It is postulated that phosphorus trichloride reacts with the nucleoside to6give a dichlorophosphite which is then oxidized to the dichlorophosphate.
310 -
Sect. VI
-
T o p i c s in C h e m i s t r y
Smi s sman, Ed.
References
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Chap. 29
N u c l e o side s and N u c l e o t i d e s
Scha ef f e r
31 1 -
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14,