1378
J.Org. Chem., Vol. 40, No. 10, 1975
Kappler and Hampton
Synthesis of 5’- C-Acylaminomethyl Derivatives of Adenosine 5’-Phosphate and Adenosine 5‘-Triphosphate Francis Kappler and Alexander Hampton* The Institute for Cancer Research, Philadelphia, Pennsylvania 19112 Received September 20,1974 Novel nucleotides have been synthesized in which the phosphoester (POCH2) grouping of adenosine 5’-phosphate (AMP) is replaced by POCH(CH2NHR) where R = H, C(O)Me, C(O)Et, C(O)Ph, or CHzCHzCN. Reaction of 2’,3’-0-isopropylideneadenosine-5’-aldehyde(produced in situ from 2’,3’-0-isopropylideneadenosine) with nitromethane in dimethyl sulfoxide in the presence of triethylamine gave 19%overall yield of the all0 epimer 8a and 6%overall yield of the tal0 epimer 8b of 5‘-C-nitromethyl-2’,3’-0-isopropylideneadenosine. Catalytic reduction of these gave the corresponding 5’-C-aminomethyl nucleosides (6a and 6b) in high yield; 6a was also obtained, but in low yield, by reduction with diborane of the previously synthesized all0 epimer of 5/-C-carbamoyl-2’,3’-0-isopropylideneadenosine. The configuration of 6a and 6b was determined by treating them with nitrous acid followed by an acidic hydrolysis which produced D-allose and L-talose, respectively. Treatment of 6a or 6b with acetic, propionic, benzoic, or p- nitrobenzoic acids in the presence of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline gave the respective 5’-acylaminomethyl derivatives of 2’,3’-0-isopropylideneadenosine.Phosphorylation of these with 2-cyanoethyl phosphate-dicyclohexylcarbodiimide and removal of blocking groups gave the all0 and tal0 5’-Cacetamidomethyl and 5’-C-propionamidomethyl derivatives 14a, 14b, 15a, and 15b, respectively, of AMP and the all0 5’-C- benzamidomethyl derivative 16 of AMP. A more efficient route to these nucleotides comprised protection of the aliphatic amino group of 6a and 6b with a phenoxycarbonyl group, phosphorylation of the 5’ hydroxyl, and removal of blocking groups, which gave the pure epimeric 5’-C-aminomethyl derivatives 18a and 18b of AMP in 10%overall yield from 8a and 8b, respectively. Treatment of 18a and 18b in methanol with acetic or propionic acids together with N-ethoxycarbonyl-2-ethoxy-l,2-dihydroquinoline gave high yields of 14a, 14b, and E a , and treatment of 18a with benzoyl chloride in aqueous sodium carbonate gave 16 in high yield. The AMP derivatives 14a and 14b were converted to the corresponding ATP derivatives by a known anion-exchange procedure by which nucleoside 5’”phosphates can be converted to phosphoanhydrides. The all0 and tal0 epimers of 5’-C-cyanoethylaminomethyl AMP were identified as by-products formed in the synthesis of 18a and 18b and were shown to arise from reaction of 18a and 18b with cyanoethylene released during one of the deblocking operations. The synthetic routes developed appear to be general for the introduction of acyl- and aroylaminomethyl substituents a t C-5’ of AMP and ATP.
Adenosine 5’-phosphate (AMP) and more especially adenosine 5’-triphosphate (ATP) are substrates of a large proportion of known enzymes and in addition are utilized frequently in metabolism as allosteric regulators of enzyme activity.l This laboratory has synthesized a number of derivatives of AMP and ATP with mono-C substituents on the sugar moiety and has found them to be valuable agents with which to delineate electronic and steric features of enzymes in and around their binding sites for AMP or ATP.2~3,4 One phase of those studies4 revealed that the complexes of AMP with several AMP-utilizing enzymes have sufficient room to accommodate a methyl group substituted on C-5‘ of AMP. In order to assess the extent of bulk tolerance a t C-5’ of AMP and ATP in their complexes with enzymes it became necessary to extend these studies to AMP and ATP derivatives with larger and more varied substituents at (2-5’. The only 5’-C-substituted adenine nucleotides yet synthesized appear to be 5’X-methyl AMP,4,5 5’-C-carbamyl AMP,4 5’-di-C-methyl AMP,5 and 8,5’~ y c l o - A M P .This ~ , ~ report describes the synthesis of 5’-Cacylaminomethyl derivatives of AMP and ATP which were obtained in the two possible 5’ epimeric configurations, i.e., as derivatives of 0-D-allose (1 and 3) or of a-L-talose (2 and 4). Evidence summarized previously*,6 shows that binding of AMP and ATP to enzymes usually involves interactions of enzymic groups with both the phosphate and the ribose segments of the nucleotides and that rotation about the 4’,5’ bond in the enzyme-substrate complexes is thereby considerably restricted. As a consequence, a group attached to C-5’ will, in such a complex, bear a spatial relationship t o nearby groups of the enzyme which is determined by the stereochemical configuration a t C-5’. The 5’-C-substituted adenine nucleotides made accessible by the present work thus constitute two distinct groups of molecular probes
with which to study the numerous enzymes for which adenine nucleotides are substrates or effectors. An account of the interactions of these analogs with enzymes for which AMP and ATP are substrates will be presented elsewhere. The required 5’-C-acylaminomethyl AMP derivatives 1 and 2, which served as precursors of the ATP derivatives 3 and 4, were prepared by two routes. In the first of these,
OH OH 1, R’ = CH,NHCQR; R2 = H 2, R’ = H; R2 = CH,NHCOR
OH OH
3, R’ = CH2NHCOCH3;R2 = H 4, R’ = H; R2 = CH,NHCOCH,
the hitherto undescribed nucleosides 9-(6-amino-6-deoxy2,3-0-isopropylidene-~-D-allofuranosyl)adenine (6a) and
5’-C-Acylaminomethyl Derivatives of Adenosine 5’-Phosphate
J.Org. Chem., Vol. 40, No. 10, 1975 1379
9-(6-amino-6-deoxy-2,3-0isopropylidene-a-L-talofurano- and 19%yields, respectively. The l H NMR, ir, and uv specsy1)adenine (6b) were prepared and selectively N-acylated tral characteristics of the crystalline epimers Sa and 8b acon the sugar moiety, after which the 5‘ hydroxyl group was phosphorylated; the second approach differed in that phosphorylation of 6a and 6b was performed prior to the N-acylation. We initially attempted to secure the 5’-C-aminomethyl nucleosides 6a and 6b by reduction of the corresponding 5’-C-carbamoyl nucleosides, since we had previously found4 that these epimers can be synthesized readily from 2’,3’-0-isopropylideneadenosine-5’-aldehyde and easily separated from each other. Both epimers proved resistant to reduction by aluminum hydride or lithium aluminum hydride, but when treated with an excess of diborane the all0 epimer ( 5 ) did yield a small amount of the 5’-C-aminomethyl nucleoside 6a whereas the tal0 epimer gave no CONH2
CH2NH2
I
I
corded with their assigned structures, which, as described below, were further supported by subsequent reduction of the nitro group, N-acylation of the resulting amine, and 5’0-phosphorylation of the 5’ hydroxyl group. An unusual feature of the uv spectra of 8a and 8b is a 35% hyperchromic effect observed at pH 11 but not at pH 2-7 which is apparently due to interaction of the acinitro anion with the purine ring. A similar hyperchromic effect is given by the epimeric 5’-C-nitromethyl derivatives of nonblocked adenosine, the synthesis of which was reported by Hogenkamp and coworkersgafter this phase of our work had been completed. These workers isolated the all0 and tal0 epimers in 17 and 5% yields, respectively, following treatment of preisolated adenosine-5’-aldehyde with nitromethane and sodium hydroxide in aqueous dioxane. The absolute configuration at C-5’ of Sa and Sb was established in a sequence of three operations. Firstly, the nitro group was reduced catalytically with platinum to furnish the respective 5’-C-aminomethyl nucleosides 6a and 6b. The yield in this conversion was high provided that rel-
R’
I
0 x 0 5
6a
Ad = adenine
6b. The reduction of 5 produced complex mixtures and the yield of 6a was less than 5%. In another approach to the 5’C- aminomethyl nucleosides, the crude 5’-C-cyano nucleosides, which were unisolated intermediates in the synthesis 6a, R1 = CH2NH2;R2 = H of the epimeric 5’-C-carbamoyl derivatives of 2’,3‘-0-isob, R’ = H Rz = CH2NHz pr~pylideneadenosine,~ were treated with lithium aluminum hydride, but the principal product was 2’,3’-O-isoproOH pylideneadenosine, presumably produced by a retro cyaI nohydrin reaction and reduction of the resultant nucleoOH side 5’-aldehyde 7. Synthesis of the 5’-C-nitromethyl nuD-dlOSe cleosides Sa and Sb was next investigated. For this purpose, 2’,3’-0- isopropylideneadenosine was oxidized in dimethyl or H sulfoxide solution by the carbodiimide procedure of L-talose Pfitzner and Moffatts to give a mixture containing 8 0 4 5 % of 2’,3’-0-isopropylideneadenosine-5’-aldehyde(7) and 15-20% starting material. When 2 equiv of nitromethane 0 x 0 and of triethylamine were added directly to this mixture, 7 was converted within several hours at room temperature to 9a, R1 = CH,OH; Rz = H b, R’ = H R2 = CH20H 9-(6-deoxy-6-nitro-2,3-0-isopropylidene-~,~-talofuranosy1)adenine (Sb) and 9-(6-deoxy-6-nitro-2,3-0-isopropyliatively large amounts of platinum were employed; two uvdene-P-D-allofuranosyl)adenine(8a). The two epimers were absorbing by-products were also detected, but in trace amounts. As expected, 6a and 6b migrated as monocations on paper electrophoretograms run at pH 4.5. Treatment of 6a and 6b with nitrous acid yielded the 5’-hydroxymethyl2’,3’-0-isopropylideneinosines9a and 9b, which were directly treated with an aqueous suspension of Dowex-50 0 x 0 (H+) ion-exchange resin under conditions which removed the isopropylidene group and cleaved the glycosidic bond H I to produce hypoxanthine and a single sugar corresponding to allose and talose respectively. These sugars were identified by paper chromatographic comparison with authentic samples in several solvent systems. The chromatograms also established that neither 9a nor 9b had given rise to Lmannose, thus showing that the reaction of 2’,3’-O-isopropylideneadenosine-5’-aldehyde with nitromethane was not accompanied by inversion a t C-4’, a possibility suggested 8a 8b by the tendency of nucleoside 5’-aldehydes to invert at C-4’ readily separated from each other and from 2’,3’-0- isoprounder mild conditions.1° In addition, Walker et aL9 have pylideneadenosine by means of column chromatography on established that reaction of adenosine-5’-aldehyde with nisilica gel in two different solvent systems and isolated in 6 tromethane produced nucleosides derived from allose and
&J+
OXb
1380 J. Org. Chem., Vol. 40, No. 10, 1975
Kappler and Hampton
R’ talose but not from mannose or gulose. The 5’X-aminomethyl nucleosides (6a,6b)could be distinguished from each other on paper chromatograms, thus enabling us to determine the absolute configuration of the 5’4’- carbamoyl nucleoside 5, from which a small amount of 6a was obtained by the action of diborane, as described above. The all0 configuration thus indicated for 5 is in agreement with that previously suggested4 on the basis of its specific rotation. O X 0 In further confirmation of this assignment, the aminometh10%R’ = CHZNBCOCH3; R2 = H yl nucleoside obtained by reduction of 5 with diborane was lob, R’ = H Rz= CH,NHCOCH, treated successively with nitrous acid and with Dowex-50 11% R’ = CH,yHCOC,H6; R2 = H (H+),when it produced a sugar which was chromatographillb, R’ = H; RZ= CH,NHCOC,H, cally identical with allose. 12, R’ = CH,NHCOC&&; ’R2= H Phosphorylation of the 5‘ hydroxyl group of 8a or 8b was 13, R‘ = C H ~ N H C ~ P P O ~ J C ~Rz H ,= ; H briefly investigated with several of the agents known to be R’ useful phosphorylating agents for nucleosides. Phosphorus oxychloride in trimethyl phosphate,ll which phosphorylates the 5’ hydroxyl group of the epimeric 5’Z-methyl2’,3’-0-isopropylideneadenosines,4did not phosphorylate the 5’ hydroxyl of 8a or 8b. Dibenzyl phosphorochloridate in pyridine12 also failed to phosphorylate 8a or 8b. A 2-cyOH OH anoethylphosphoryl group could be attached to the 5’ oxyMa, R’ = CHzNHCOCH3;R2 = H gen of 8a or 8b by means of the 2-cyanoethyl phosphatedicyclohexylcarbodiimide procedure,13 but attempted 14b, R’ = H RZ = CHzNHCOCH3 base-promoted release of cyanoethylene from this interme15a, R’ = CH2NHCOCZH5; R2 = H diate, while furnishing a small yield of material with the 15b, R’ = H; R2 = CH,NHCOCzHS 16, R’ = CHJWCOC6HE; ’R2= H properties of the desired 5’-C-nitromethyl-2’,3’-O-isopropylideneadenosine 5’-phosphate, gave predominantly a phosphorus-free nucleoside which had less polar properties bonyl-2-ethoxy-1,2-dihydroquinoline.15J6By the latter than 8a or 8b as judged from paper partition chromatograprocedure we obtained also 9-(6-benzamidomethyl-6phy and which was tentatively concluded to be the 5’,6’(12) deoxy-2,3-0-isopropylidene-0-D-a1lofuranosyl)adenine dehydro derivative of 8a or 8b resulting from more rapid and its p-nitrobenzamidomethyl analog 13. The foregoing p-elimination of the 2-cyanoethyl phosphate dianion than four 5’-C-acylaminomethyl nucleosides as well as the 5’-Cof the nucleoside 5’-phosphate dianion. benzamidomethyl nucleoside were converted in 25-50% Difficulties were also encountered when direct phosphooverall yields to chromatographically homogeneous 5’-Crylation of the 5’-C-aminomethyl nucleosides 6a and 6b acylaminomethyl derivatives of AMP (14a to 16) by appliwas attempted. These compounds remained largely uncation of the 6-cyanoethyl phosphate-dicyclohexylcarbodichanged after prolonged treatment (72 hr) with excess (4 imide (DCC) method13 for the phosphorylation of nucleoequiv) of 2-cyanoethyl phosphate in the presence of dicysides followed by stepwise removal of the cyanoethyl and clohexylcarbodiimide. The principal product had the same isopropylidene protecting groups by successive basic and ultraviolet absorption properties as 6a or 6b but did nat acidic treatments. undergo the ninhydrin reaction and behaved as a monoanSubsequent studies disclosed a suitable method for synion upon paper electrophoresis at pH 7.6; it was acid labile, thesis of the unprotected epimeric 5’-C-aminomethyl debeing decomposed within 30 min at pH 2 and 25’. This rivatives of AMP (18a and 18b), and these compounds limited evidence suggests that this nucleoside may possess proved to be more valuable than the 5’-C-methylamino nua (2-cyanoethyl)phosphorylaminomethylgroup at C-5’. By phosphorylation of both the aminomethyl and the hydroxyl groups of 6a or 6b with polyfunctional phosphorylating agents it was then hoped to obtain a cyclic phosphoramidate which might undergo P-N bond cleavage under acidic conditions to give the required 5’-phosphate. However, pnitrophenyl pho~phorodichloridate~~ or phosphorus oxychloride in pyridine as well as phosphorus oxychloride in trimethyl phosphate upon reaction with 6a or 6b gave complex mixtures which after mild acidic treatment gave no 17% R’ = CH2NHCOOC,H6;R2 = H 5’-C- aminomethyl nucleoside 5’-phosphates. b, R’ = H; Rz = CH,NHCOOC6HS Selective acetylation of the amino group of the 5’-C-aminomethyl nucleosides 6a and 6b could be achieved by the 8’ action of acetic anhydride in boiling pyridine. Analytically pure 9-(6-acetylaminomethyl-6-deoxy-2,3-0-isopropylidene-P-D-allofuranosy1)adenine(loa) and 9-(6-acetylaminomethyl -6-deoxy-2,3-0-isopropylidene-a!-L -talofuranosy1)adenine (lob)were obtained in this way in 24-45% yields. bH 6H The corresponding 5’-C-propionylaminomethyl nucleo18a, R’ CH2NH2; R2 = H sides lla and llb were similarly obtained. Slightly higher 18b, R’ = H, R2= CH,NH2 yields of these 5’-C-acylaminomethyl nucleosides were ob19% R’ = CH2NHCH2CHzCN;R2= H tained by the action of acetic or propionic acids on 6a or 6b 19b, R’ H; R2 = CHZNHCHZCHZCN in methanolic solution in the presence of N-ethoxycar-
5’-C-Acylaminomethyl Derivatives of Adenosine 5’-Phospharte cleosides 6a and 6b as intermediates for the required 5’acylaminomethyl derivatives of AMP. For the preparation of 18a or 18b,the appropriate 5’-C-nitromethyl-2’,3’-O-ieopropylideneadenosine (sa or 8b) was reduced as previously described and the crude aminomethyl nucleoside was treated in tetrahydrofuran with 1 equiv of phenyl chloroformate. Chromatography on silica gel showed conversion of the aminomethyl nucleoside to a less polar compound, presumed to be the phenylurethane 17a or 17b. The crude mixture was treated with P-cyanoethyl phosphate-DCC, after which the phenoxycarbonyl and 2-cyanoethyl groups were removed with aqueous sodium hydroxide, and finally the isopropylidene group was removed with aqueous acid. The 5‘-C-aminomethyl nucleotides 18a and 18b were then obtained in homogenous form in 10%overall yield (from 8a or 8b) following their elution from a column of Dowex 1 bicarbonate with aqueous triethylammonium bicarbonate. In accord with the presence of the aminomethyl function, 18a and 18b underwent the ninhydrin reaction and migrated as monoanions when subjected to paper electrophoresis a t pH 7.6. The above sequence of reactions provided also an approximately equal amount of a second nucleotide which eluted from a column of Dowex 1 bicarbonate more slowly than the 5’-aminomethyl AMP derivatives 18a and 18b. Its NMR spectrum showed an adenosine moiety and a 5’ substituent which contained six nonexchangeable protons made up of a two-proton triplet at 6 3.58 and a four-proton broad multiplet a t 6 3.85.” The CH2NH grouping in the starting material suggested CH2NHCH2 for the broad resonance a t 6 3.85. The addition to this of a methylene group responsible for the 6 3.58 resonance was indicated by the coupling constant of 6 Hz, and suggested a partial structure in which CH2NHCH2CH2X was substituted at C-5’ of adenosine. The elemental analysis indicated that the compound could be a 5’-C- (2-cyanoethy1)aminomethylderivative of AMP (19a or 19b). This was supported by paper electrophoresis, which showed that the compound, like the aminomethyl nucleotides 18a and 18b, had a net ionic charge of zero at pH 4.5 (AMP had one negative charge), whereas a t pH 7.6 it had 1.6 negative charges compared to 1.0 negative charges in the case of 18a and 18b and 2.0 negative charges for AMP. This suggests that the cyanoethyl group lowers the pK, of the aliphatic amino group of 18a or 18b from a value in excess of 9.0 to a value less than 7.6. An ir absorption peak assignable to the cyano group of 19a or 19b proved difficult to detect, and to confirm the presence of this group, 19a was hydrogenated in 15%aqueous ammonium hydroxide in the presence of rhodium-alumina, when rapid and quantitative reduction occurred to give a single compound which at pH 4.5 migrated on electrophoreto, grams as a monocation and which was concluded to be the 5’-C- (3-aminopropyl)aminomethyl nucleotide 20a. That 19a
+
CHzNH(CH2)3NHZ
CHZNH(CH2)3NH*
OH OH
OH OH
I
20a
I
21a
the ionic charge was due to the net effect of two positive charges on the two aliphatic amino groups and one negative charge on the phosphoryl group was confirmed by treating 20a with alkaline phosphatase to produce a single uv-ab-
J. Org. Chem., Vol. 40, No. 10,1975 1381 sorbing component (presumably the nucleoside 21a) which migrated as a dication at pH 4.5. The 5’-C-aminomethyl nucleotide 18a produced significant amounts of 19a when treated with cyanoethylene under the basic conditions employed to remove the cyanoethyl and phenoxycarbonyl groups in the foregoing synthesis of 18a. The amount of cyanoethylene employed in the reaction was the same as that which would have been generated from the 2-cyanoethyl phosphate employed in the phosphorylation of 17a. This indicates that the nucleotide 19a is produced primarily by intermolecular reaction of the 5’-C-aminomethyl group with cyanoethylene liberated during the alkaline deblocking procedure and that an intramolecular reaction between adjacent 5’-C-aminomethyl and 2-cyanoethylphosphate groups is probably not involved in the formation of 19a. It should be noted that the AMP derivatives 19a and 19b have the same potential value as steric probes of enzyme-AMP complexes as do the 5’42- acylaminomethyl derivatives of AMP. Studies of their interactions with enzymes will be described in a subsequent publication. Selective acetylation or propionylation of the aliphatic amino group of the 5’-C-aminomethyl AMP epimers 18a and 18b was readily accomplished in methanolic solution by acetic or propionic acids which had been converted in situ to mixed carbonic anhydrides by the action of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline.18The only products detected were the starting material, which was quantitatively recovered by paper chromatography, and the required N-acyl derivatives 14a, 14b, and 15a which were isolated in 84-91% yield as their disodium salts. The D-allofuranosyl epimer 18a was employed as its methanolsoluble triethylammonium salt, whereas it was necessary to employ the less soluble L-talofuranosyl epimer 18b as a trioctylammonium salt. When N-benzoylation of 18a was attempted by means of this procedure, the yield of 16 was reduced as a result of the formation of appreciable amounts of an unidentified n~c1eotide.l~ However, treatment of 18a in aqueous sodium carbonate solution with an excess of benzoyl chloride rapidly produced solely the desired 5’-Cbenzamidomethyl AMP derivative 16, which was obtained in high yield as its disodium salt following paper chromatographic purification. That the position of acylation of all these nucleotides was the aliphatic amino group was shown by their inability to undergo the ninhydrin reaction, and by their migration as dianions on electrophoretograms run at pH 7.6. Conversion of the epimeric 5’-C-acetylaminomethyl derivatives of AMP (14aand 14b) to the corresponding derivatives (3 and 4) of ATP was accomplished by the anionexchange reaction of MichelsonFOwhich is of general utility for the conversion of naturally occurring 5‘ nucleotides to a variety of phosphoanhydride derivatives. In the present case, this comprised conversion of 14a and 14b with diphenyl phosphorochloridate to P1,P1-diphenyl-P2-nucleoside 5’-pyrophosphates and reaction of these in situ with tributylammonium pyrophosphate. Paper chromatography satisfactorily separated unchanged 14a and 14b and traces of the corresponding nucleoside 5’-diphosphates from the nucleoside 5’-triphosphates 3 and 4, which were obtained in 35% yield and isolated as their tetrasodium salts. As expected, 3 and 4 possessed the same ultraviolet extinction coefficients as ATP and their paper chromatographic and electrophoretic properties also resembled those of ATP. Further confirmation of the structure of 3 and 4 was provided by the action of alkaline phosphatase, which liberated 3.0 mol of inorganic phosphate per mole of each nucleotide.
1382
J.Org. Chem., Vol. 40, No. 10, 1975
Kappler and Hampton
1 N NH4OH. The chromatographically homogeneous residue obtained upon evaporation was dissolved in water (1 ml) and acetic 9-(6-Deoxy-6-nitro-2,3- 0-isopropylidene-fl-D-allofuranosacid (0.45 ml). Sodium nitrite (0.25 g) was added and the solution y1)adenine (sa) and 9-(6-Deoxy-6-nitro-2,3-O-isopropyli- was stirred at ca. 22’ for 4 hr. An additional 0.07 g of sodium nidene-a-btalofuranosy1)adenine(8b). To a solution of 2’,3’-0trite and 0.25 ml of acetic acid was added and stirring was continisopropylideneadenosine (8 g, 26 mmol) in dry dimethyl sulfoxide ued for 18 hr. The mixture was then adsorbed onto charcoal. The (50 ml) was added pyridine (2 ml), trifluoroacetic acid (1ml), and charcoal was washed with water, and the nucleoside 9a or 9b [uv dicyclohexylcarbodiimide (16 9). The mixture was stirred at room max (pH 12) 254 and 252 nm, respectively] was then eluted with temperature for 18 hr and dicyclohexylurea was filtered off and 1:l ethanol-0.05 M NH40H and the eluate evaporated. The resiwashed with dimethyl sulfoxide (3 X 25 ml). The combined filtrate due was dissolved in water (5 ml), Dowex-50 (H+) (10 ml wet resin) and washings were extracted with cyclohexane (3 X 25 ml). To the was added, and the mixture was heated at 100’ for 1.5 hr to yield dimethyl sulfoxide solution was added nitromethane (3 g, 50 from 8b hypoxanthine as the only uv-absorbing component and tammol) and triethylamine (6.6 g, 65 mmol), and the red solution lose (Rf0.36 in system G, 0.30 in system H, 0.63 in system J) as the was stirred a t room temperature for 18 hr. Volatile materials were only sugar component. Compound 8a gave allose (Rf 0.27 in G, 0.21 removed a t 35’ (1 mm), during which additional dicyclohexylurea in H, and 0.52 in J) and hypoxanthine. The sugars were detected precipitated. This was filtered off, the dimethyl sulfoxide was diby spraying the papers with 3% p-anisidine in butanol and heating luted with ethyl acetate (100 ml), and the mixture was extracted them for 10 min a t 105’. Mannose had Rj 0.31 in G, 0.23 in H, and with water (3 X 30 ml). The aqueous solutions were extracted with 0.55 in J. ethyl acetate (30 ml). The combined ethyl acetate extracts were General Syntheses of Nucleosides loa-13. Method A. Comdried (MgS04) and then concentrated to small volume and chropound 8a or 8b (1.4 mmol) was added to a suspension of prerematographed over silica gel (2.7 X 120 cm) using a linear gradient duced PtOs (0.5 g) in methanol (100 ml) and hydrogenation was of ethyl acetate to 5% ethanol in ethyl acetate (4 1.). Evaporation of carried out as described above. The catalyst was filtered off and fractions containing 8a and 8b gave a yellow foam (4.5 8). This was the filtrate was evaporated. To the residue was added pyridine (2 dissolved in chloroform and chromatographed over silica gel (2.7 X ml) and the required anhydride (1.9 mmol). This was heated at re120 cm) using a linear gradient of chloroform to 5% methanol in flux for 2 hr. After cooling, the solvents were removed in vacuo and chloroform (5 1.); 15-ml fractions were collected. Compound 8b was the residue was dissolved in chloroform and subjected to preparaeluted in fractions 203-219 and 8a in fractions 228-270. Fractions tive layer chromatography on silica gel with solvent A. 220-227 contained small amounts of both 8a and 8b. Compound Method B. Compound 8a or 8b (1.4 mmol) was reduced as 8b was obtained as a lustrous white solid (0.50 g, 5.6% yield) which above. The catalyst was removed by filtration and the filtrate was crystallized from ethanol as fine needles (0.42 8): mp 216-218’ dec, concentrated to about 50 ml. The required carboxylic acid (2.5 [aIz3D -79.4’ (c 1.1, DMSO); ir 1555, 1375 cm-I (-NOz); uv max mmol) was added followed by N - ethoxycarbonyl-Z-ethoxy-1,Z-dih(pH 2) 257 nm (c 15,500), (pH 11) 254 nm (c 21,700); NMR ydroquinoline (EEDQ, 0.5 g, 2 mmol). The solution was stirred at (DMSO-de) 8.73 (9, 1, H-8), 8.51 (s, 1, H-2), 7.71 (s, 2, -NH2), 6.89 30-35’ for 15 hr. The solvent was evaporated and the residue was (d, 1, J = 4Hz, -OH), 6.50 (d, 1, J = 4 Hz, H-l’), 5.59 (dd, 1, J = 4, dissolved in chloroform (10 ml) and added dropwise to rapidly 6 Hz, H-2’), 5.42 (dd, 1, J = 6, 2 Hz, H-39, 5.20 (m, 1, H-49, 4.77 stirred cold petroleum ether (250 ml). The precipitate was collect(m, 3, H-5’, H-6’, 6’9, 1.92 (s, 3) and 1.68 ppm ( 8 , 3, isopropylied and chromatographed on thick layer silica gel plates with soldene). vent A. This showed that all preparations contained minor Anal. Calcd for C14H1sN606: C, 45.89; H, 4.92; N, 22.95. Found amounts of at least four by-products. [dried at 78’ (0.01 mm)]: C, 45.79; H, 5.09; N, 22.54. 9-(6-Acetamido-6-deoxy-2,3-O-isopropylidene-~-~-alloFrom the foregoing column chromatography, compound 8a was furanosy1)adenine (loa). The crude product obtained from 2 g obtained as a yellow glass (1.8 g, 19% yield) free of uv-absorbing (5.4 mmol) of 8a by method A was chromatographed over silica gel impurities. I t was dissolved in ethyl acetate and chromatographed (46.5 X 2 cm). The column was washed with chloroform (500 ml) on a silica gel column (3.7 X 30 cm) using ethyl acetate as eluent. followed by chloroform-methanol (19:l) (2 1.). Compound 10a was This gave 8a as an off-white solid which separated from ethanol as thereby obtained as a white, chromatographically homogeneous microcrystalline aggregates (0.94 9): mp 179-182’ dec; [ c I ] ~ ~ D glass (0.96 g). This material was sufficiently pure for use in the -60.8’ (c 1.0, DMSO); ir 1560, 1375 cm-‘ (-Nod; uv max (pH 2) phosphorylation described under method C. A solution of the 257 nm (c 15,000), (pH 11) 254 nm (c 20,400); NMR (DMSO-&) product in chloroform (15 ml) was added to petroleum ether (200 8.68 (s, 1,H-8), 8.55 (s,1, H-2), 7.66 (s, 2, -NH2), 6.55 (d, I, J = 2.7 ml). The resulting powder was precipitated twice more from chloHz, H-l’), 6.50 (1,-OH), 5.84 (dd, 1,J = 2.7, 6 Hz, H-2’1, 5.56 (dd, roform using diethyl ether in place of petroleum ether to give 0.75 1,J = 2.6, 6 Hz, H-3’), 4.85 (m, 3, H-4’, H-6’, 6”), 4.58 (m, 1, H-5’1, g (37% yield): mp 123-128’; Rf 0.34 (solvent A); [@Iz2D-61’ (c 1.0, CHC13); ir 1625 cm-l (C=O); uv max (pH 2) 256 nm (c 15,100), 1.93 (s, 31, and 1.69 ppm (s, 3, isopropylidene). (pH 11) 259 nm ( t 15,300); NMR (DMSO-&) 8.75 (s, 1, H-8), 8.55 Anal. Calcd for C14H18N606: C, 45.89; H, 4.92; N, 22.95. Found (9, 1, H-2), 8.22 (9, 1, -CONH), 7.88 (s, 2, -NH2), 6.46 (d, 1,J = 4 [dried at 100’ (0.01 mm)]: C, 45.67; H, 5.05; N, 22.97. Diborane Reduction of 9-(2,3- 0-Isopropylidene-fl-D-allofu- Hz, H-l’), 5.55 (m, 3, H-2’, H-3’, -OH), 4.42 (s, 1, H-49, 4.0 (m, 1, H-5’), 3.37 (m, 2, H-6’, H-6”), 2.15 (s, 3, -CH3), 1.90 and 2.0 ppm ranur0namide)adenine (5). To a stirred solution of 5 (47 mg, 150 fimol) in dry tetrahydrofuran (2 ml) at ca. 22’ was added an excess (s, 3, CMez). Anal. Calcd for C16Hz~N605:C, 50.79; H, 5.82; N, 22.22. Found of borane in tetrahydrofuran (1ml of a 1 M solution). After stir[dried at 78‘ (0.01 mm)]: C, 50.98; H, 5.91; N, 22.42. ring for 1 hr, water-tetrahydrofuran (1:l) (1 ml) was added fol9-(6-Acetamido-6-deoxy-2,3-0-isopropylidene-a-L-talolowed by 2 N KOH (0.25 ml) and 30% H202 (0.25 ml). The solution furanosy1)adenine (lob). Method B was applied to 0.35 g of 8b, was stirred for an additional 15 min and then evaporated. The resiand 10b was extracted from the silica gel chromatogram with ethyl due was dissolved in water and applied to a column (2 X 20 cm) of acetate. The solvent was volatilized and the residue was dissolved Dowex-50 (pyridinium form). The column was washed with water in chloroform and precipitated from petroleum ether to give 205 and compound 6a was then eluted with 2 N NH4OH. Evaporation mg (54% overall yield) of a white powder: mp 125-132O; Rf 0.35 of this eluate gave a pale yellow powder (2 mg, 4.4%) which was ho-112’ (c 1.0, CHC13); ir 1620 cm-I (C=O); uv (solvent A); [CX]~*.~D mogenous in solvent G ( R j 0.43) and solvent B (Rj 0.76) and indismax (pH 2) 257 nm ( E 15,100), (pH 12) 259 nm ( t 15,300); NMR tinguishable from 6a, but different from 6b which had R f 0.46 and (DMSO-&) 8.70 (s, 1, H-8), 8.45 (s, 1, H-2), 8.15 (d, 1, J = 5 Hz, 0.78 in the respective solvents. The product, like 6a and 6b, react-CONH), 7.60 (s, 2, -NHz), 6.40 (d, 1, J = 4 Hz, H-l’), 5.97 (d, 1, J ed positively to the ninhydrin spray test on chromatograms, mi= 5 Hz, -OH), 5.35 (m, 2, H-2’, H-3’), 4.57 (m, 1, H-4’), 4.05 (m, 1, grated as a monocation on electrophoretograms at pH 4.5, and H-5’), 3.47 (m, 2, H-6’, H-6”), 2.05 (s, 3, -CH3), 1.82 and 1.57 ppm after elution from the electrophoretogram had the same uv absorption maxima a t pH 2 and 7 as adenosine. (s, 3,CMez). Anal. Calcd for C16HzzN605: C, 50.79; H, 5.82; N , 22.22. Found Establishment of the Configurations of 8a and 8b. Com[dried a t 78’ (0.01 mm)]: C, 50.93; H, 5.99; N, 21.99. pound 8a or 8b (50 mg) was added to a suspension of prereduced 9-(6-Deoxy-6-propionamido-2,30-isopropylidene-8-D-alloPtOz (50 mg) in methanol (25 ml). This was shaken at 50’ and 50 furanosy1)adenine ( l l a ) . Method A was applied to 0.5 g (1.4 psi of hydrogen for 18 hr, when TLC (silica gel, EtOAc) showed no mmol) of 8a and l l a was eluted with chloroform from its zone of starting material (Rj 0.44) and a single ninhydrin-positive spot of the thick layer silica gel chromatogram and purified by three prezero Rf. The catalyst was filtered off and the filtrate was evapocipitations from chloroform by addition of diethyl ether. This gave rated. A solution of the residue in water was applied to a Dowex-50 139 mg of material (25% yield): mp 124-129’; Rf 0.40 (solvent A); (pyridinium form) column. The column was washed with water to [aIz3D -62’ (c 1.1, CHCl3); ir 1615 cm-I (C=o); uv max (pH 2) remove nonbasic material and compound 6a or 6b was eluted with
Experimental Sectionz1
J. Org. Chem., Vol. 40, No. 10, 1975 1383
5’-C-AcylaminomethylDerivatives of Adenosine 5’-Phosphate
Table I Paper Chromatography and Electrophoresis Electrophoretic mobility relative to A M P
Rf values system Compd
B
C
E
F
AMP
0.10 0.15 0.17 0.24 0.24 0.26 0.06
0.26 0.31 0.33 0.39 0.41 0.51 0.19
0.56 0.69
0.07
0.18
0.43 0.52 0.54 0.62 0.62 0.64 0.39 0.40
0.17 0.03
0.24 0.09
0.06
0.10
0.05
0.10
14a 14b 15a 15b 16 18a lab 19a ATP
4 3
0.44 0.45 0.44
258 nm ( t 15,000), (pH 12) 259 nm (t 15,200); NMR (DMSO-&) 8.78 (s, 1, H-8), 8.60 (6, 1, H-2), 7.95 (s, 1, -CONH-), 7.60 (s, 2, NHz), 6.54 (d, 1,J = 4 Hz, H-l’), 5.52 (m, 3, H-2’, H-3’, -OH), 4.73 (m, 1, H-47, 4.35 (m, 1, H-5’1, 3.75 (m, 2, H-6’, H-6’9, 2.60 (9, 2, J = 7 Hz, -COCH2-), 1.95 and 1.72 (s, 3, CMez), 1.45 ppm (t,3, J = 7 Hz, -CH3). Anal. Calcd for C17H2dN605: C, 52.04; H, 6.14; N, 21.43. Found [dried at 78’ (0.01 mm)]: C, 51.89; H, 6.33; N, 21.21. 9-( 6-Deoxy-6-propionamido-2,30-isopropylidene-a-L-talofuranosy1)adenine (llb).This was prepared in the same manner as its 5’ epimer lla, except that it was eluted from the silica gel with ethyl acetate and crystallized from the same solvent to give 100 mg of llb (13%overall yield): Rf 0.40 (solvent A); mp 117120’; [o(Iz3D-100’ (c 1.2, CHC13); ir 1620 cm-l (c=o); uv max (pH 2) 258 nm ( t 15,100), (pH 12) 259 nm ( t 15,300); NMR (DMSO-&) 8.80 (s, 1, H-8), 8.55 (s, 1, H-21, 8.15 (s, 1,-CONH-), 7.70 (s, 2, -NH2), 6.48 (d, 1, J = 4 Hz, H-1’), 6.10 (d, 1, J = 5 Hz, -OH), 5.38 (m, 2, H-2’, H-3’), 4.56 (s, 1, H-4’), 4.10 (m, 1, H-59, 3.30 (m, 2, H-6’, H-6”), 2.45 (9, 2, J = 7 Hz, -COCH2-), 1.85 and 1.60 (6, 3, CMeZ), 1.35 ppm (t, 3, J = 7 Hz, -CH3). Anal. Calcd for C17H24N605: C, 52.04; H, 6.14; N, 21.43. Found [dried at 78’ (0.01 mm)]: C, 51.90, H, 6.14; N, 21.52. 9- (6-Benzamido-6-deoxy-2,30-isopropylidene-fl-D-allo furanosy1)adenine (12). Method B was applied to 0.5 g of Sa, and 12 was extracted from the silica gel chromatogram with ethyl acetate. The solvent was volatilized and the residue was dissolved in chloroform and precipitated from petroleum ether to give 206 mg (34% overall yield) of 12 as a white powder: Rj 0.60 (solvent A); mp 105-110’; [ a I z 5 b-120’ (c 1.3, CHCl3); ir 1630 cm-l (c=o);uv max (pH 2) 256 nm (e 15,200), (pH 12) 257 nm ( t 15,600); NMR (DMSO-ds) 8.72 (s, 1,H-8), 8.62 (d, 1, J = 6 Hz, -CONH-), 8.53 (s, I, H-2), 7.77-8.23 (m, 5, aromatic), 7.65 (s, 2, -NH2), 6.51 (d, 1,J = 4 Hz, H-1’), 5.67 (m, l,H-2’), 5.49 (m, 1, H-39, 4.54 (m, 1, H-4’), 4.30 (m, 2, -OH, H-59, 3.77 (m, 2, H-6’, H-6”), 1.91 and 1.69 ppm (s,3 each,CMez). Anal. Calcd for C21H24N&&o CHC13: C, 56.50; H, 5.38; N, 18.83; C1, 1.35. Found [dried at 78’ (0.01 mm)]: C, 56.32; H, 5.72; N, 18.35; C1, 1.23. 9-(6-Deoxy-6-p-nitrobenzamido-2,3O-isopropylidene-flD-allofuranosy1)adenine (13).Method B was applied to 0.25 g of Sa, and 13 was extracted from the silica gel chromatogram with ethyl acetate. The solvent was volatilized and the residue was dissolved in chloroform and precipitated from petroleum ether to give 85 mg (25% overall yield) of 13 as a pale yellow powder (Rf 0.58, solvent A): mp 123-127’ [ o ( ] 2 5 5 D -117’ (c 1.0, CHC13); ir 1635 cm-l (C=O); NMR (DMSO-ds) 8.07-8.75 (m, 7, H-8, H-2, -CONH-, aromatic), 7.60 (s, 2, -NH2), 6.50 (d, 1, J = 4 Hz, H-l’), 5.50 (m, 1, H-39, 4.50 (m, 1, H-49, 4.28 (m, 2, -OH, H-5’), 3.75 (m, 2, H-6’, H-6”), 1.90 and 1.70 ppm (s, 3 each, CMe2). Anal. Calcd for C21H23N707: C, 51.96; H, 4.76; N, 20.21. Found [dried at 78’ (0.01 mm)]: C, 51.40; H, 5.15; N, 20.36. Syntheses of Nucleoside 5‘-Monophosphates. Method C. The 5’-C-substituted 2’,3’-O-isopropylidene nucleoside (0.4 mmol) was dissolved in pyridine (10 ml) and a pyridine solution of 2-cyanoethyl phosphate (1 mmol) was added. The solution was evaporated to dryness and twice again after addition of 10 ml of dry pyridine. The residue was dissolved in anhydrous pyridine (5 ml) and dicyclohexylcarbodiimide (1 g) was added. The mixture was stirred
0.71 0.79
o .a0 0.87 0.76 0.74 0.52 0.42 0.43 0.42
pH 7.5
pH 4.5
pH 3.5
1 .oo
1 .oo 0.98
1 .oo
0.94 0.94 0.93 0.94
0.87 0.51 0.53
0.81
0.98
1.08
1 .oa 0.91 0.00 0.00 0 .oo 2.34
2 -13 2.17
at room temperature for 15 hr.Water (5 ml) was added and stirring was continued for 30 min. Concentrated NHdOH (IO ml) was added and the mixture was heated at 70’ for 60 min and then evaporated to dryness. Water (20 ml) was added and the filtered solution was adjusted to pH 1.5 with concentrated HCl and heated at 70° (bath temperature) for 2 hr. The pH was adjusted to 3.5, Darco G-60 charcoal (1 g) and Celite filter aid (1 g) were added, and the mixture was stirred for 2 hr. The charcoal was collected by filtration and washed with water (500 ml). The nucleotide was desorbed from the charcoal with 200 ml of aqueous 50% ethanol containing 0.6 ml of concentrated NH40H. The extract was concentrated and subjected to downward chromatography for 3 days in solvent D on four sheets (width 20 cm) of Whatman 3MM paper. The aqueous eluate of the nucleotide was lyophilized, giving the ammonium salt of the nucleotide as a white powder which was homogeneous in all the solvent systems listed in Table I. This was dissolved in methanol (5 ml) containing Et3N (2 drops). Volatiles were removed, and a solution of the residue in MeOH (5 ml) was clarified by filtration and treated with 1 M NaI (0.6 ml) followed by acetone (25 ml). The precipitated disodium salt of the nucleotide was collected by centrifugation, washed with acetone (3 X 10 ml), and dried at 0.01 mm (18 hr, P2O5). Method D. Compound Sa or 8b (2.0 g, 5.3 mmol) was reduced in the normal manner. The residue of 6a or 6b obtained on work-up was suspended in THF (100 ml) and EON (1 ml, 6 mmol) was added. Phenyl chloroformate (1 g, 6 mmol) in T H F (30 ml) was added dropwise to the rapidly stirring mixture. After 1 hr addition was complete and Et3N.HCl had separated. TLC in solvent A indicated one major component, R,t 0.5, and several minor components. The mixture was filtered and the filtrate was evaporated. The residue was dissolved in dry pyridine (10 ml) and treated with 2-cyanoethyl phosphate (from 15 mmol of the barium salt) in dry pyridine (10 ml). DCC (10 g) was added and the mixture was stirred for 72 hr. Water (10 ml) was added and after 0.5 hr 2 N NaOH (30 ml) was added. The mixture was stirred at 22’ for 0.5 hr, filtered, and evaporated. The residue was dissolved in H2O (30 ml) and the pH was adjusted to 1.5 with concentrated HCl. The solution was heated at 70’ for 1.5 hr. After cooling, the pH was adjusted to 3.5 with 1 N NaOH and the product was purified by adsorption onto charcoal (10 g) in the normal manner. After elution the nucleotide fraction was concentrated (ca 10 ml) and applied to Dowex-1 (HC03-1 (4.5 X 32 cm). This was eluted stepwise with 0-1.0 M Et3NHC03 (0.1 M increments/l.). With 0-0.2 M salt ca. 40,000 O D Zunits ~ ~ were eluted; with 0.5 M salt compound 18s or 18b was eluted (8,000 OD260 units, 10% yield) and was obtained as a white, chromatographically homogenous triethylammonium salt after desalting it by coevaporating the partially evaporated eluate with ethanol. An analytical sample was prepared by dissolving a portion of the powdery salt in MeOH and adding 1M NaI in acetone. Precipitation with acetone gave 18a or 18b as the monosodium salt, presumably because as the free acid, 18a or 18b exists as an inner salt: uv max (pH 2) 257 nm ( t 15,100, (pH 12) 259 nm ( t 15,300). Anal. Calcd for C ~ I H ~ ~ N ~ O ~ P N C, ~.H 31.65; @ : H, 4.56; N, 20.14; P, 7.43. Found [dried at 25’ (0.01 mm)]: C, 32.08; H, 4.88; N, 20.33; P, 6.88. A solution of the triethylammonium salt of 18a was treated with EEDQ and the required carboxylic acid under conditions employed in method B and heated at 35’ for 3 hr. The methanol was
1384 J . Org. Chem., Vol. 40, No. 10, 1975
Kappler and Hampton
259 nm ( t 15,100); NMR (DzO) 8.83 (s, 1, H-8), 8.51 (s, 1, H-2), evaporated and the residue was triturated several times with pe7.80-8.20 (m, 5, aromatic), 6.48 (d, 1, J = 6 Hz, H-l’), 5.41 (t, 1, troleum ether. The residue was dissolved in water and purified on H-29, 5.10 (H-3’, partially obscured by HDO peak), 4.61 (m, 1, HWhatman 3MM paper in solvent D. The products obtained were 4’), 4.45 (m, 1,H-5’), 3.90 (m, 2, H-6’, H-6”). identical with those from method C. Anal. Calcd for Cl8Hl&@,PNay2H20: P, 5.54. Found [dried at 9-( 6-Cyanoethylaminomethyl-6-deoxy-50-phosphoryl-@25’ (0.01 mm)]: P, 5.42, 5.46. D-allofuranosy1)adenine (19a). Elution of the above ion-exB. By Benzoylation of 18a. Compound 18a (2600 OD260 units; change column with 0.6 M Et3NHC03 gave a mixture of 18a with 190 pmol) and Na2C03 (14.8 mg, 140 pmol) were dissolved in HzO 19a (2000 ODZ60units) and 0.7 M gave homogeneous 19a (12,000 (700 pl) and stirred at 0’. Benzoyl chloride (23.3 pl, 200 pmol) in OD260 units, 15%yield). The triethylammonium salt of 19a was obether (300 p1) was added slowly. Stirring was then continued at 0” tained as described for the same salt of 18a and was converted to for 10 min, then at ca. 22O for 3 hr. Chromatography on Whatman the sodium salt with sodium iodide in acetone: uv max (pH 2) 257 No. 3MM paper (two sheets, 20 X 46 cm) in solvent D gave, after nm ( t 15,100), (pH $2), 259 nm ( E 15,500); NMR (DzO) 8.85 (s, 1, elution and lyophilization, compound 18a (500 OD260 units, 19.3% H-8), 8.50 (s, 1, H-2), 6.45 (d, 1, J = 5 Hz, H-l’), 4.63 (m, 1,H-4’or recovered) and compound 16 (1700 OD260 units, 81% yield and 65% H-5’), 3.85 (m, 4, -CHzNHCHz-), 3.58 ppm (t, 2, J = 6 Hz, conversion) identical with that prepared by method C in the sol-CH2CN). The HDO peak at 5.20 obscured the remaining sugar vent systems listed in Table I. protons. C. From Method D. Paper chromatographic analysis (solvent Anal. Calcd for C14H18N707PNay2.5 HzO: C, 32.43; H, 4.12; N, 18.92; P, 5.98. Found [dried at 25’ (0.01 mm)]: C, 32.38; H, 4.18; N, C) of the mixture obtained upon application of method D gave 16 (57%, spectrophotometrically determined), unchanged 18a (17%), 18.61; P, 5.90. 9-(6-Acetamido-6-deoxy-5-~-phosphoryl-~-D-allofura~ and an additional nucleotide (2396, Rf 0.42 in solvent C). The unnosy1)adenine (14a). Application of method C to compound 10a known compound had R f 0.27 in solvent B and an electrophoretic (100 mg, 0.27 mmol) gave the disodium salt of 14a (32 mg) as a mobility (AMP = 1) at pH 7.5 of 0.89. Compound 16 and the unknown showed a large difference in the 260/235 nm ratio of uv abwhite powder: ir 1610 (C=O), 1240 (P=O), 1100 cm-l (P-0-C); sorption (1.05 and 2.92, respectively). uv max (pH 2) 257 nm (c 14,800), (pH 12) 259 nm ( t 14,900); NMR 9-(6-Acetamido-6-deoxy-5-0-triphosphoryl-P-D-allofura(D20) 8.89 (9, 1, H-8), 8.60 (s, 1, H-2), 6.29 (d, 1, J = 6 Hz, H-1’), 5.30 (t, 1, H-2‘), 5.13 (H-3’, partially obscured by HDO peak), 4.80 nosy1)adenine (3). The homogeneous ammonium salt of com(m, 1, H-4’), 4.56 (m, 1, H-5’), 3.92 (m, 2, H-6’, H-6’9, 2.41 ppm (s, pound 14a (1200 OD260 units, 80 pmol) (purified by chromatogra3, -CH3). phy in solvent D; see method C) was suspended in MeOH (5 ml) Anal. Calcd for C13H17N608PNa2-2H20:C, 31.33; H, 4.22; N, and tri-n-butylamine (300 pl) was added. The mixture was 16.87; P, 6.22. Found [dried at 25’ (0.01 mm)]: C, 31.42; H, 4.32; N, warmed until homogeneous; then the MeOH was evaporated. The 16.62; P, 6.41. residue was rendered anhydrous by three evaporations in vacuo Compound 14a was obtained in 87% yield from 18a (81% converwith pyridine (2 ml). Dioxane (0.5 ml) and N,N-dimethylformamsion) by method D. ide (0.3 ml) were added followed by diphenyl phosphorochloridate 9-( 6-Acetamido-6-deoxy-5-0-phosphoryl-c~-~-talofurano-(30 pl) and tri-n-butylamine (40 pl). The mixture was stirred at sy1)adenine (14b). Phosphorylation of 10b (0.36 mmol) by methroom temperature for 3 hr. Solvents were removed in vacuo at 25” od C gave 0.19 mmol of chromagraphically homogeneous ammoand ether (50 ml) was added to the residue with shaking. The mixnium salt. From this the disodium salt of the nucleotide was obture was kept at 0’ for 1 hr. The ether was decanted and dioxane (1 ml) was added, after which the mixture was evaporated to a tained as a white powder in the usual manner: ir 1620 (C=O), 1230 syrup. A solution of di(tri-n-butylammonium) pyrophosphate (0.1 (P=O), 1100 cm-l (P-0-C); uv max (pH 2) 257 nm ( 6 14,900), (pH mmol) in pyridine (200 pl) was added and the solution was kept at 12) 259 nm (e 15,100); NMR (DzO) 8.97 (s, 1, H-8), 8.40 (s, 1, H-2), room temperature for 1 hr. The pyridine was removed in vacuo 6.43 (d, 1,J = 6 Hz, H-l’), 5.33 (H-2’, partially obscured by HDO and ether (50 ml) was added to precipitate the nucleotide and then peak), 5.17 (m, 1, H-3‘), 4.94 (m, 1, H-49, 4.77 (s, 1,H-5’), 3.98 (d, decanted off. The precipitate was dissolved in water and streaked 2 , J = 6 Hz, H-6’, 6”), 2.43 ppm (s, 3, CH3). on two sheets (20 X 46 cm) of Whatman 3MM paper. This was deAnal. Calcd for C&1?N,jO8PNa2.2Hzo: P, 6.22. Found [dried at veloped for 24 hr in solvent E. The zone corresponding to 3 was 25O (0.01 mm)]: P, 6.38, 6.35. eluted into water and the solution was lyophilized. The product Compound 14b was obtained in 91% yield from 18b (85%conver(405 OD260 units) was chromatographed in solvent F to remove the sion) by method D. 9-( 6-Deoxy-5- O-phosphoryl-6-propionamido-@-D-allofura- corresponding nucleoside diphosphate (30 OD260 units). The developed chromatogram was soaked in l-propanol to remove ammonosy1)adenine (15a). This was obtained as a chromatographically nium butyrate, after which the product was eluted into water and and electrophoretically homogeneous ammonium salt (0.14 mmol) obtained as a solid by lyophilization. It was converted to the tetraby application of method C to l l a (0.57 mmol). This was convertsodium salt (320 OD260 units, 27% yield) by the procedure used to ed in high yield to the disodium salt (a white powder) with sodium prepare disodium salts of the monophosphates, uv max (pH 2) 257 iodide in acetone: ir 1615 (C=O), 1230 (P=O), 1100 cm-l (P-Onm (c 14,900), (pH 12) 259 nm ( e 15,600). C ) ; uv max (pH 2) 257 nm (e 14,9001, (pH 12) 259 nm ( t 15,100); Anal. Calcd for C ~ ~ H ~ ~ N ~ O ~ ~ P ~P,N13.57. ~ C Found: L ~ H ZP,O : NMR (D20) 8.88 (s, 1, H-8), 8.56 (s, 1, H-2), 6.50 (d, 1,J = 6 Hz, 13.72. H-l’), 5.30 (t, 1, H-29, 5.14 (H-3’, partially obscured by HDO The product (12.36 OD260 units) was kept for 60 min a t 2 2 O in 1 peak), 4.86 (m, 1, H-4’), 4.64 (m, 1,H-5’), 3.98 (m, 2, H-6’, 6”), 2.70 ml of Tris buffer, pH 10.4, containing 0.02 mg of alkaline phospha(9, 2, J = 7 Hz, -COCH2-), 1.52 (t, 3, J = 7 Hz, -CH3). tase of calf intestinal mucosa (Type VII, from Sigma Chemical Anal. Calcd for C14Hl~N~O~PNa2-2Hz0: P, 6.05. Found [dried at Co.). The ratio of inorganic phosphatez3 to 5’-C-acetylaminometh25’ (0.01 mm)]: P, 6.10, 6.12. yladenosine was 2.92:l. Compound 15a was obtained in 84% yield from 18a (82% conver9-(6-Acetamido-6-deoxy-5-0-triphosphoryl-c~-~-talofurasion) by method D. 9-(6-Deoxy-5- 0-phosphoryl-6-propionamido-a-L-talofura- nosy1)adenine (4). Compound 14b (1200 OD260 units) was converted to 4 by the foregoing procedure. Purification in solvent E nosy1)adenine (15b). This was obtained as a chromatographically gave 435 OD units of 4, which showed no trace of the nucleoside diand electrophoretically homogeneous ammonium salt (0.13 mmol) phosphate or other impurity in solvent F. Compound 4 was isolatby application of method C to l l b (0.4 mmol). From this the disoed as its tetrasodium salt (350 OD260 units, 29% yield) as described dium salt was prepared as above: ir 1610 (C=O), 1235 (P=O), for its 5’ epimer, uv max (pH 2) 257 nm ( t 14,800), (pH 12) 259 nm 1100 cm-’ (P-0-C); uv max (pH 2) 257 nm (c 15,0001, (pH 12) 259 ( t 15,200). nm ( 6 15,300); NMR (D20) 9.00 (s, 1,H-81, 8.53 (s, 1, H-2), 6.51 (d, Anal. Calcd for C13H17N6014P3Na4‘1.0H~O:P, 13.74. Found: P, 1, J = 6 Hz, H-1’), 5.20 (H-2’, partially obscured by HDO peak), 13.85. 5.08 (ni, 1, H-3’), 4.91 (m, 1, H-4’), 4.80 (s, 1,H-59, 3.99 (d, 2, J = 6 The ratio of the inorganic phosphate released by alkaline phosHz, H-6’, 6”), 2.63 (9, 2, J = 7 Hz, -COCH2-), 1.42 (t, 3, J = 7 Hz, phatase to 5’-C-acetylaminomethyladenosine(determined as for 3) -CH3). was 3.14:l. Anal. Calcd for
[email protected]~0:P, 6.05. Found [dried at 25’ (0.01 mm)]: P, 5.98,6.02. 9-( 6-Benzamido-6-deoxy-5- 0-phosphoryl-j3-D-allofuraAcknowledgments. This work was supported by nosy1)adenine (16). A. From Method C. Compound 16 was obUSPHS Research G r a n t CA-11196 from t h e National Cantained as its ammonium salt (0.08 mmol) by application of method cer Institute, an award from the Pennsylvania Science and C to 12 (0.25 mmol): ir 1620 (C=O), 1230 (P=o), 1100 cm-l (PEngineering Fund, a n d b y grants t o the Institute for CanO-C); uv (disodium salt) max (pH 2) 257 nm ( 6 14,900), (pH 12)
S y n t h e s e s of Diribonucleoside Monophosphates
J . Org. Chem., Vol. 40, No. 10,1975 1386
cer Research (USPHS G r a n t s CA-06927 and RR-05539 and an appropriation from the Commonwealth Of Pennsylvania)
.
Registry No.-3 tetrasodium salt, 54677-85-9; 4 tetrasodium salt, 54677-86-0; 5,50304-49-9; 8a, 54677-87-1; ab, 54677-88-2; loa, 54677-89-3; lob, 54677-90-6; lla, 54677-91-7; l l b , 54724-59-3; 12, 54677-92-8;13,54677-93-9; 14a disodium salt, 54677-94-0; 14a ammonium salt, 54677-95-1; 14b disodium salt, 54677-96-2; 15a disodium salt, 546’77-97-3;15b disodium salt, 54677-98-4; 16 disodium salt, 54677-99-5; 18a sodium salt, 54678-00-1; 18b sodium salt, 54678-01-2; 19a disodium salt, 54678-02-3; 2’,3’-O-isopropylideneadenosine, 362-75-4.
References and Notes (1) (a) D. E. Atkinson, Annu. Rev. Biochem., 35, 85 (1966); (b) E. R . Stadtman, Adv. Enzymol., 28, 41 (1966); (c) H. M. Kaickar et ai., Ed.,.”The Role of Nucleotides for the Function and Conformation of Enzymes”, Academic Press, New York. N.Y., 1969. (2) A. Hampton, T. Sasaki, and B. Paul, J. Am. Chem. SOC., 95, 4404 (1973). (3) A. Hampton, F. Perini, and P.J. Harper, Biochemistry, 12, 1730 (1973). (4)A. Hampton, P. Howgate, P. J. Harper, F. Perini, F. Kappler, and R. K. Preston, Biochemistry, 12, 3328 (1973). (5) R. S. Ranganathan, G. H. Jones, and J. G. Moffatt, J. Org. Chem., 39, 290 (1974). (6) A. Hampton, P. J. Harper, and T. Sasaki, Biochemistry, 11, 4965 (1972). (7) K. Keck, Z.Naturforsch., 238, 1034 (1968). (8) K. E. Pfitzner and J. G. Moffatt, J. Am. Chem. Soc., 85, 3027 (1963); J. G. Moffatt in “Techniques and Applications in Organic Synthesis. Oxidation”, Vol. II, Marcel Dekker. New York, N.Y., 1971, p 1. (9) T. E. Walker, H. Folimann, and H. P. C. Hogenkamp, Carbohydr. Res., 27, 225 (1973). (IO) G. H. Jones and J. G. Moffatt, Abstracts, 158th National Meeting of the American Chemical Society, New York, N.Y., Sept 7-12, 1969, No. CARB-16. (11) M. Yoshikawa, T. Kato, and T. Takenishi, Bull. Chem. Soc. Jpn., 42, 3505 (1969). (12) J. Baddiiey and A. R. Todd, J. Chem. SOC., 648 (1947). (13) G. M. Tener, J. Am. Chem. Soc., 83, 159 (1961). (14) A. F. Turner and H. G. Khorana, J. Am. Chem. Soc., 81, 4651 (1959). (15) J. F. B. Mercer and R. H. Symons, Biochim. Biophys. Acta, 238, 27 (1971).
(16) This reagent had previously proved useful for selective N-acylation of other nucleotides in this laboratory (Slotin and Hampton, in preparation). (17) An external concentric capillary of Me4Si was used for determination of ail ’H NMR spectra and caused a downfieid shift of 0.4-0.5 ppm for ail protons. (18) B. Belleau and G. Maiek, J. Am. Chem. Soc., 90, 1651 (1966). (19) This nucleotide (see Experimental Section) had similar paper eiectrophoretic properties as 16, was more polar than 18 on paper chromatograms, and appeared from its uv spectrum to lack the benzamido group of IS; since it arose from a reaction which involved the mixed anhydride of benzoic acid and ethyicarbonic acid,“ the compound may differ from I 6 by possessing an ethoxycarbonyl group in place of the benzoyl group. (20) A. M. Micheison, Biochim. Biophys. Acta, 91, l(1964). (21) Darco G-60 charcoal was used for adsorbing nucleotides and was activated prior to use by the method of Lipkin et ai.22 Petroleum ether employed in purifications boiled at 30-60’. Thin layer chromatograms were obtained with Merck F-254 silica gel plates in chloroform-methano1 (9:l) (system A). Preparative layer chromatography was conducted with 2-mm layers of silica gel on glass. Paper chromatography (descending) employed Whatman No. l , 3MM or 17 papers in (B) 2-propanoi-concentrated NH40H-water (7: 1:2); (C) I-butanol-acetic acidwater (5:2:3): (D) I-propanol-concentrated NH4OH-water (7:1:2); (E) 1propanol-concentrated NH40H-water (55:10:35); (F) isobutyric acid-I M NH40H (10:6); (G) I-butanol-acetic acid-water (4:1:5, upper layer); (H) I-butanol-pyridine-water (3: 1:1); (J) I-butanol-pyridine (2: 1). Eiectrophoresis was performed on Whatman No. 1 paper at 40-80 V/cm for 30-60 min at pH 7.5 [0.05 M(Et)SNHCOs, pH 4.5 (0.05 Macetate), or pH 3.5 (0.015 M citrate)]. Mobility values ( M A M are ~ ) relative to those of adenosine 5’-phosphate (AMP). Spots on chromatograms were detected by their ultraviolet absorption and (in the case of silica gel chromatograms) by spraying with the Molisch reagent. Melting points (uncorrected) were determined by the capillary method. Ultraviolet spectra were determined with a Cary Model 15 spectrophotometer. infrared spectra were determined in KBr disks with a Perkin-Elmer spectrophotometer Model 137, and ’H NMR spectra were obtained with a Varian XL-100-15 spectrometer and are recorded as parts per million downfield from an external standard (concentric capillary) of SiMe4. Infrared arid NMR spectra of nucleotides were usually obtained on ammonium salts from which the analytically pure sodium salts were obtained. Specific rotations were determined with a Bendix automatic polarimeter 1169. Elemental analyses were performed by Atlantic Microlabs, Atlanta, Ga., and Midwest Microiab, Ltd., Indianapolis, Ind. (22) D. Lipkin, P. T. Talbert, and M. Cohn, J. Am. Chem. Soc., 76, 2871 (1954). (23) Determined by the method of Lowry and Lopez, J. Bioi. Chem., 162, 421 (1946).
Nucleotides. V. Syntheses of 2‘- 0-and 3’- 0-(3-Methyl-2-picolyl 1-oxide) Ribonucleosides and Diribonucleoside Monophosphates by Application of 3-Methyl-2-picolyl 1-Oxide Protection1 Yoshihisa Mizuno,* Takeshi E n d o , and Kazuyoshi I k e d a Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan Received October 25,1974 The alkylation of ribonucleosides (uridine series, 3 and 6; adenosine series, 16 and 19) with 1-oxido-3-methyl2-pyridyldiazomethane (2) afforded an isomeric mixture of 2’-0- and 3’-0- (3-methyl-2-picolyl 1-oxide) ribonucleosides in 63-91% yields. 2/-0-(3-Methyl-2-picolyl1-0xide)uridine (4) and 2’-0- (17), and 3’-0- (3-methyl-2-picolyl 1-0xide)adenosine (18) could be isolated by fractional crystallization of the respective isomeric mixture, whereas 2’-0-(3-methyl-2-picolyl l-oxide)-5’-0-benzoyluridine(7) and 2’-0-(3-methyl-2-picolyl l-oxide)-O5’,W-dibenzoyladenosine (20) were isolated by column chromatography on silica gel. The stability of 3-methyl-2-picolyl 1oxide group toward tritylation, benzoylation, and especially phosphorylation by the general method (phosphate in the presence of TPS) and its removability were found to be compatible with the oligoribonucleotide synthesis. Thus, the synthesis of 2 / - 0 - (3-methyl-2-picolyl l-oxide)uridylyl(3’-5/)-02~,03’,N6triacetyladenosine (26) and uridylyl(3’-5’)adenosine jUpA, 27) was achieved by the application of 3-methyl-2-picolyl protection. Requirements for protecting groups in t h e oligoribonucleotide synthesis have been recently discussed b y Christen a n d Broom.2 T h e development of t h e synthesis b y a “phosphotriester a p p r o a ~ h ” ,i~n particular, has d e p e n d e d to a significant e x t e n t o n t h e design of a new protecting group for 2’-hydroxyl function which meets t h e requirement^.^ Since 2’-0- (2-picolyl 1-oxide) ribonucleoside m i g h t b e useful key intermediates for t h e oligonucleotide synthesis, our interest is selective and direct introduction of a removable
blocking group of this t y p e into t h e cis-glycol system of ribonucleosides. W e have therefore prepared a series of nitrogenous heterocyclic N - oxides bearing a diazomethylene g r o u p and i t was concluded that o u t of these diazoalkanes, l-oxido-3-methyl-2-pyridyldiazomethane (2), m i g h t be a reagent of choice for t h e monoalkylation of t h e cis-glycol system of t h e ribonucleosides because of its easy accessibility and comparatively small 6 value of t h e signal d u e t o t h e diazomethylene proton i n its NMR ~ p e c t r u m . ~
