3252
DAVIDI. MAGRATH AND GEORGE BOSWORTH BROWN
[CONTRIBUTION FROM THE LABORATORIES O F THE SLOAN-E(ET
rERISC
1’01. 7 ! )
DIVISIOSO F CORNELL UXIVERSITY LfEDICAL COLLEGE]
The Synthesis of 9-P-D-Ribofuranosylpurine-5’-phosphate and its Behavior Toward Aqueous Alkali1 BY DAVID I.
? V h G R A T H 2 AND
GEORGI-BOSWORTH BROWS
RECEIVED NOVEMBER l(i,
LOX
The synthesis of 9-4-D-ribofuranosylpurine-5’-phosphate is reported. Two substances formed b y t h e action of dilute aqueous alkali on t h e nucleotide, and also isolated as by-products during its synthesis, have been tentatively identified as 5-amino and 5-formamido derivatives of 4-(5’-phosphoribofuranosylamino)-pyrimidine.
I n studies of potential tumor-inhibiting agents, spectrum in acid, alkali and water was almost 9-P-D-ribofuranosylpurine(I) was synthesized in identical with that of g-P-~-ribofuranosylpurine,~ this Laboratory and shown3v4to be identical with and analysis indicated it to be a monohydrate of nebularine, a toxic, naturally occurring material.4-6 the 5’-monophosphate. It was eluted as a single, The metabolism7 of this compound led to three de- sharp peak from a Dowex-2 (formate) ion-exchange rivatives of p ~ r i n e - C ‘in~ the soluble nucleotide column a t a position on the elution diagram confraction from the liver. From their behavior on the sistent with its being a mononucleotide. ion-exchange resin, these were postulated to be the 9-P-D-Ribofuranosylpurine-5’-phosphate was not 5’-mono-, di- and triphosphates of the administered unstable in 0.1 N acid a t room temperature but in ribosylpurine. Since it was desirable to have an alkali was decomposed readily to give a mixture of authentic specimen for comparison and for investi- two substances similar to the by-products formed gation of potential pharmacodynamic properties, during its synthesis.I2 A mixture of the latter, rethe synthesis of g-P-D-ribofuranosylpurine-5’-phoscovered as a cream-colored powder from the celluphate (11) was undertaken. lose column, yielded, upon acid hydrolysis, a num2’,3‘ - O-Isopropylidene-9-/3-~-ribofuranosylpu-ber of products. The main ultraviolet light-abrine8 was treated with dibenzylphosphorochloridate sorbing component had Rf values and spectral in pyridine solution to give 2 ‘,3‘-O-isopropylidene- characteristics the same as 4,5-diaminopyrimidine. 9 -P-D-ribofuranosylpurine - 5’- dibenzylphosphate. Examination of chromatograms showed a pattern Hydrogenation effected smooth removal of both of phosphorus and aniline phthalate-positive (i.e., benzyl groups, and the isopropylidene group could reducing carbohydrateL3)spots identical with that then be removed from the product by allowing the obtained with a similar hydrolysate of adenosine5‘-phosphate: namely, two aniline phthalate-posifree acid to stand in aqueous s o l ~ t i o n . ~ Examination of this solution by paper chroma- tive spots, one faint and having Rf values similar to tography revealed the main product and two other mribose, the other, present in considerable amount, phosphorus-containing, ultraviolet light-absorbing containing phosphorus and having Rfvalues simimaterials, as well as a small amount of free rihosyl- lar to D-ribose-5-phosphate. Inorganic phosphate purine. The contaminants were separated from was distinguished from the sugar phosphate on the main product by chromatography over a column chromatograms run in acetone-water-acetic acid.I4 The two by-products were separated on a small of powdered cellulose. The main product contained organically-bound phosphbrus and a cis- scale by chromatography on paper. Both conglycol1a system and, in several paper chrornato- tained organically bound phosphate and a cis-glygraphic systems, gave only one ultraviolet light- col system and migrated on paper chromatography absorbing spot with Rf values of the order to be and electrophoresis as typical mononucleotides. expected of a monbnucleotide. Further purifica- The chromatographically faster-moving by-product tion over Dowex-50 resin furnished the free acid could be converted into the slower-moving compoas a pale cream-colored solid. Its ultraviolet nent by further alkaline hydrolysis. The slower-moving component appeared ZL (1) T h i s investigation was supported in p a r t hy funds from t h e fluorescent blue spot under ultraviolet light, gave American Cancer Society upon recommendation of t h e Committee im positive reactions in the alkaline phosphomolybGrowth, KationaI Research Council, G r a n t So. MET-27, a n d t h e Pl?ational Cancer I n s t i t u t e , National Institutes of H e a l t h , Puhlic date testLsand the Bratton-Marshall test16and had H e a l t h Service, G r a n t S o . C-471. a spectrum in acid, alkali and water generally simi(2) D e p a r t m e n t of Biochemistry. Au4tralian Natinnal University. lar to that of 4.5-diaminopyrimidine (Fig. I). Its Canberra, Australia. Research Fellow of t h e Sloan-Kettering I n pk‘,, determined spectrophotometrically, was .7.7 s t i t u t e , 1954-1936. ( 3 ) G . R . Brown a n d 1’. S . R’eliky, J . Azol. Chrnr., 204, 1019 ( 1 9 , S ) . (-l-,.i-diaininopyriniidine, 6.03).17 The chromato(4) hr.Ldfgren, B. 1,iining and 11, ITedstrnm. A < l o CIhFnr. S r o n d . , 8 , graphically faster-moviiig by-prodrict gave a negar i i 0 (19.54). tive reaction i n both the above color tests. T h e ( 5 ) N. Lofgren a n d B. 1,lininx. i h t d . . 7 , 2 2 5 (1953). (6) L. Ehrenherp, H. I-IedctrBm, S. 1,ijfgren and B. l‘akrnan, .U;’t,irsk, K e m . T i d s k r . , 68, 2(iR (1916). ( 7 ) hZ. P. Gordon a n d 0 . B. Brown, .I. B i d C h r m . , 220, 927 (1966). (8) A. H a m p t o n a n d D. I. h f a g r a t h , ’THIS J O U R N A L , 79. 3250 (1957). (9) A t t e m p t s t o remove simultaneously t h e isopropylidene and one benzyl group by acid hydrolysis led t o Considerable hydrolysis of t h e glycosyl linkage. (10) See footnote 26 of ref. 11. JOI-RSAL. (11) M. P. Gordon, V. S. Weliky a n d G . A . Brown. THIS 79, 3246 (1‘257).
(12) I t nil- a l i o foiinrl t h a t extensive dogradation occurrcd d i l r t n x an a t t e m p t t o prepare t h e dimethyl ester b y reaction with diaromethane. (1:3) S. %.I. Partridge, S u t r d ? ’ ~164, , 413 (1949). (14) S . Burrows, F. S. SI. Grylls a n d J. S. Harrison, ibid., 170, 800 (1952). (15) A. Bendich a n d G. C. Clements, Biochim. el Biophrs. Acln, 12, 462 (1953). (16) A. C. Bratton and E. IC. Marshall, Jr., J . Binl. Chem., 128, 637 (1939). (17) S. F. Mason. J . Chrnr. Soc.. ‘2071 (1’254).
RIBOFURANOSYLPURINE-5 ’-PHOSPHATE
June 20, 1957
32.53
OH
OH H
/
2 20
240
260
280
/
300
l
I
.
l
l
l
320
d>mP.
Fig. dine; In 0.1 (-
1.-Ultraviolet spectra of: ( a ) 4,5-diaminopyrimi( b ) chromatographically slower-moving by-product. and HzO N HCl ( . . . , . .), 0.1 N NaOH , pH 6.48 and 4.51, respectively). (--.-a)
similarity of its ultraviolet spectrum with that of 4-amino-5-formamidopyrimidine‘s is apparent (Fig. 2 ) . The latter was found to possess a pK, value of 11.40, which may be attributed to the &formamido group, and a value of 11.43 was found for the by-product. It appears that the two substances produced by the alkaline hydrolysis of 9-P-D-ribofuranosylpurine-5’-phosphate, and also found as by-products during its synthesis, are 5’-phosphoribofuranosyl derivatives of 4,5-diaminopyrimidine (I11 and IV), one (111) still carrying the 8-carbon atom of the original molecule as a formyl group attached to the primarylg amino group of IV. The presence of the 5’-phosphate group precludes furanose-pyranose isomerization, although mutarotation a t the glycosyl linkage is still possible for either compound. The initial step in the alkaline degradation of the purine nucleosidell and of this nucleotide probably involves a nucleophilic attack on the 8-carbon atom by hydroxyl ion; the subsequent ring opening is perhaps facilitated by conjugation of the resulting ionized formamido group with the pyrimidine riny. (18) Authentic samples of this and related compounds were kindly sent us by Professor Adrien Albert and Dr. D. J. Brown, Department of Medical Chemistry, the Australian National University. For proof of the position of the formyl group see D. J. Brown, J. A p p l i e d Chem., 6, 358 (1955). (19) A formyl group attached to the ribosylamino group of I V should not give rise to the p X . value of 11.4 observed for the fastermoving by-product.
I
OH
AH
(IV)
The alkali degradation products of 9-P-D-ribofuranosylpurine are thought“ to be ribofuranosyl and -pyranosyl derivatives of 4,5-diaminopyrimidine, which had resulted from the loss of the 8-carbon atom. The degradation was postulated as proceeding by way of the intermediate formation of the 5-formamido compound, although there was
il,m p . Fig. 2.-Ultraviolet spectra of: (a) 4-amino-5-formamidopyrimidine; ( b ) chromatographically faster-moving by-product, in 0.1 N HC1 ( . . . . . . ) , 0.1 A’ NaOH (--.--.) and H20 (, pH 5.88 and 5.23, respectively).
3254
DAVIDI. MAGRATII AND GEORGE BOSWORTH BROWN
VOl. 79
no conclusive evidence for its presence in that hydrolysis mixture. The reason for the increased stability of the 5-formamido group toward alkali with the presence of the Sl-phosphate is not obvious. A comparison has been madezoof the synthetic purine mononucleotide with the relevant metabolite of 9-P-D-ribofuranosylpurine isolated from rats.
tion of this solution by paper chromatography (solvents A, B and C ) indicated a major ultraviolet light-absorbing component (Rr values 0.14, 0.60 and 0.61, respectively) which contained phosphorus but no cis-glycol system. There were also traces of isopropylideneribosylpurine and a substance with Rr values of 0.02, 0.40 and 0.36, respectively, and the latter contained phosphorus and a cis-glycol system. T h e above solution was diluted t o 30 cc. with water and allowed t o stand a t 24" for 48 hr. Paper chromatography (solvents B and C) showed a major ultraviolet light-absorbirig component (I?, values 0.40 and 0.36, respectively) which Experimental contained both phosphorus and a cis-glycol system, and t h e Paper Chromatography.-The follorving solverlt systcrr~s spectra of which in water, acid and alkali closely resembled those of g-P-D-ribofuranosylpurine. I n addition t o a trace were uscd: A, n-butyl alcohol saturated with water; B, 12of t h e latter, two other ultraviolet light-absorbing spots butyl alcohol-ethyl alcohol-water (45: 30: 40); C , n-butyl ':; aqueous also were observed on chromatograms run in solvent B (Rt alcohol-acetic acid-water (50:20:35); 11, 1 values 0.34 and 0.26, respectively); they ran a s one spot in ammoniuni sulfate-isopropyl alcohol ( 1 :2)2l; E, acetone0.26). Both contained phosphorus and a cis30';; acetic acid (1 : l)I4; F, isopropyl alcohol-lCG aqueous solvent C ( R f glycol system. T h e solution was adjusted t o cn. PH 7 with boric acid-lOyo ammonia (50:25: G, pyridine-ethyl acetate-water (l:2:').23 Svlrent D was used with papers ammonium hydroxide and then evaporated t o dryness under reduced pressure. T h e thick, brown, oily residue was previously soaked in 1' C aqueous ammonium sulfate and dried. R f values quoted are for ascending chromatograms evaporated three times with small portions of ethyl alcohol for 2 days. It run on IYhatman S o . 1 paper in freshly made up solvent. and left in a vacuum desiccator (P205) Ultraviolet light-absorbing materials were located on t h e weighed 1.126 g. A column of powdered cellulose (350 g., 6.5 cm. diarn.) chromatograms by visual examination and photography of t h e dried papers under ultra\-iolet light (Corning KO. 9863 was prepared by firmly pressing down successive additions filter), rcducitig carbohydrates by t h e aniline h>-drogen (ca. 50 g.) of t h e dry powder, and then washed with solvent C until t h e eluate was colorless ( c n . 1300 cc.). T h e crude phthalate spray reageiit,'a cis-glycol systems by the periodate spray t e c l i ~ i i q u eand ~ ~ ~phosphates ~~ by Hanes and Ishcr- product above was dissolved in 10 cc. of solvent C, t h e solution carefully applied t o the top of t h e column, t h e container Ivood reagent.z5 T h e spectra were determined with a Bcckrinsed out with a little more solvent a n d t h e column deman spectrophotometer, model D U , x i t h 1-cm. matched veloped with t h e same solvent. T h e first 230 cc. of eluate cells. was discarded and t h e remainder collected in fractions of cn. 9-P-~-Ribofuranosylpurine-5 '-phosphate .-2,3-O-Isopi-o18 cc. at 7-10 minute intervals until ultraviolet light-absorbpylidene-9-P-n-ribofuranosylpurine (1 g., dricd in 'i'acz~oover iiig material ceased t o be eluted (ca. fraction 60). The small phosphorus pentoxide for 3 days) was dissolved in dry pyridine ( 5 cc.) contained in a 25-cc. flask, the solution cooled amount of ribosylpurine present appeared in fractions 19-81 , t o near freezing (f.p. pyridine -42') in a DrJ- Ice-acetonc- t h e bulk of t h e major ultraviolet light-absorbing component bath and diberizylphosphorochloridatc (from 2.17 g. of di- in fractions 34-41 and a mixture of t h e two by-products in benzyl phosphite and 1.44 g. of S-chlorosuccinimide)*~ fractions 49-58. Fractions 34-41 were combined and evaporated t o dryncss added and t h e residual reagent washed from t h e container with cn. 3 cc. of cold pyridine. T h e flask was stoppered in t~acuo; small white crystals separated during the latter and t h e reaction mixture kept in a scini-frozen condition for stages of the evaporation. T h e pale brown residual sirup 5 hr., then left in t h e cooling bath overnight. -4fter re- was evaporated down once with methyl alcohol and then moval from t h e cooling bath, a solution of sodium acetate left in a vacuum desiccator (PIOa) overnight, when it became (4 g. anhydrous\ iii water (10 cc.) was added and t h e mixture converted t o a hygroscopic frothy solid, weight O.61 g. Paper chromatograms of this material, run in solvent C , shaken vigorously for 1.5 hr., then evaporated t o dryness uiidcr reduced pressure (bath temperature
