June 3 , 1960
COMMUNICATIONS TO THE EDITOR
the mixture a t mass numbers greater than m/e 76. The heptaborane contribution to m/e 78 was only 10% and to mje 77, only 870 of the observed peak heights. From the monoisotopic mass spectrum, the BsHlo+ and B6Hst species of hexaborane-12 are relatively more abundant than those of hexaborane 10. Hexaborane-12 resembles hexaborane-10 in that i t has no B5H11+ or B5HI0+species.
2069 F N
/O\ AMP-I: I, R = - N
PY:
9
LJ
AMP: 11, R=-OH CH20p-R AMP-NH2: 111, R = -NHz OH Q 11
ADP: IV, RE-OPOH I
OH
"1
(8) Xational Engineering Science Co., Pasadena, California. (0) Hughes Tool Company-Aircraft
Division, Culver C i t y , Calif.
OH
YHz
I
OLIN MATHIESON CHEMICAL CORPORATIOSS. G. G I B B I ~ PASADENA, CALIFORNIA I . SHAPIROg RECEIVED FEBRUARY 11, 1960
T H E PREPARATION OF ADENOSINE-5'
IMIDAZOL-1-YLPHOSPHONATE AND ITS REACTIONS
WITH NUCLEOPHILES. A NOVEL SYNTHESIS OF NUCLEOTIDE COENZYMES
Sir: The imidazole ring of a histidine moiety is im plicated in the binding or catalytic activity of esterases, proteases, carbohydrases, etc.' EnzymeATP complexes have been suggested as intermediates in transphosphorylation,2 and an imidazol-ly l p h ~ s p h o n a t ehas ~ been suggested4 as an example of this type of complex. Imidazolylphosphonates, such as imidazol-1-ylphosphonic acid, imidazol1,3-diyldiphosphonic acid and phenyl imidazol-lylphosphonate have been shown to be phosphorylating a g e n t ~ . ~ , ~ , O Adenosine-5' imidazol-1-ylphosphonate(AMP-I, I) was chosen as a model for reaction with nucleophiles to provide information bearing on the nature of transphosphorylation. In addition, these reactions exemplify a novel, facile synthesis of nucleotide coenzymes. Imidazolium AMP-1' is prepared readily in anhydrous dimethylformamide by reaction of the imidazolium salt of adenosine-5' phosphate (,4MP, 11) with 1,l'-carbonyldiimidazole (CDI).8,9 With equimolar quantities of AMP monohydrate and CDI, the products found by paper chromatography are AMP-I in major amount, unchanged AMP, and P1,P2-di-(adenosine-5')pyrophosphate (DAPP, V). With 2-4 moles of CDI to one mole of AMP monohydrate, conversion to AMP-I is nearly quantitative. On Whatman No. 1 paper AMP-I has Rf = 0.43'O in isopropyl alcohol-ammonia(1) For a review a n d leading references see E. A , B a r n a r d and W. D. Stein, Advances i n Enzymol., 20, 51 (1958). (2) M . B. Hoagland, Biochim. et Biophrs. Acta, 16, 288 (1955). (3) "Imidazolylphosphonate," r a t h e r t h a n "phosphoimidazole"~ or "phosphoroimidazole"'~; cf. report of t h e A.C.S. Nomenclature, Spelling and Pronunciation Committee, Chem. Eng. News, 3 0 , 4315 (1952) (4) J. Baddiley, J. G. Buchanan and R . Letters, J . Chem. Soc., 2812 (1956). ( 5 ) T. Rathlev and T. Rosenberg, Avch. Biochem. and B i o p h y s . , 65, 319 (1956). (6) H . A. S t a a b , H. Schaller a n d F. Cramer, Angew. Chem., 71, 738 (1959). (7) A M P , imidazole and dicyclohexylcarbodiimide were reported"] t o give a n unstable solid tentatively identified as a mixture of A M P - I and AMP. (8) H. A. S t a a b , A n n . , 609, 7 5 (1957). (9) After completion of t h e work described here, H . A. S t a a b , el al.,a reported t h e preparation of imidazol-1-ylphosphonic acid and phenyl imidazol-1-ylphosphonateb y t h e reactioo of t h e appropriate pho3phatr with 1,l'-carhonyldiimidazole.
OH
I
OH
OH
OH
water (7:1:2), in which solvent it is partly solvolyzed to adenosine-5' phosphoramidate (AMPNH2, 111))Rf= 0.20. When imidazolium A N P - I (from one mole of AMP monohydrate and 2 moles of CDI) is allowed to react with AMP monohydrate and the reaction mixture is chromatographed on Dowex-1 (formate), 57% of colorless crystalline DAPP (V) sesquihydrate," rn.p. 184-189", is obtained (Calcd. for C20H26N10013P2.1.5Hz0: C, 34.2; H , 4.16; N , 19.9; P, 8.81. Found: C, 34.3; H , 4.20; N, 20.2; P, 8.49, 8.37)) homogeneous by the criteria of paper chromatography in two solvent systems and by paper electrophoresis. Cridine-5' phosphate and AMP-I give P1-(adenosine-5') P2(uridine-5') pyrophosphate (VI) 0.84 as electrophoretically mobile as P1,P2-di-(uridine-5') pyrophosphate on Whatman 3hIM paper in pH 4.S acetate buffer.I2 Imidazolium AMP-I (from one mole of ill\.fP monohydrate and 3 moles of CDI), aqueous ammonia, dimethylformamide and tert-butyl alcohol, (111), kept a t 92" for 11 hours, give AMP-"? isolated in 86% yield as the colorless crystalline 1,3-dicyclohexylguanidinium salt solvated with water and dimethylformamide, m.p. 207-210" dec. (Calcd. for CloH15NGOeP.C13HzsN3~H~O~CaH~NO: C, 47.3; H , 7.47; N,21.2; P, 4.69. Found: C, 47.4; H, 7.25; N , 21.6, 21.3; P, 4.93), which, when recrystallized from aqueous acetone, gives the unsolvated salt, m.p. 236-238" dec.IG With excess S5Y0 phosphoric acid a t -10 to -20°, imidazolium AMP-I (from equal moles of hRlP monohydrate and CDI) is converted to adenosine-5' pyrophosphate (ADP, IV) , isolated in 25% yield as the yellow crystalline acridinium salt,I3 m.p. 218-217" dec. (Calcd. for CloHlsNsOioPz.CiaH&: C, 45.6; H, 3.99; N, 13.9; P, (10) R . W. Chambers a n d 1.G. Moffatt, THISJ O U R N A L , 80, 3752 (1958). (11) An amorphous t e t r a h y d r a t e is described b y S. M . H. Christie, D . T. Elmore, G. W. Kenner, A. R . Todd and F. J . Weymouth, J. Chem. Soc., 2947 (1953), (12) G. W. Kenner, C. B Reese and A. R . T o d d , ibid , ,540 (1958). (13) T. Wagner-Jauregg, Hoppe-Seylrv's Z . phrszol C h c ~ t n .239, , 188
(1936).
2970
COMMUNICATIONS TO THE EDITOR
Vol. 82
i0.2; adenine:pentose: total ??:labile P = 1: 1: 2 : 1. Found: C, 46.0, 45.7;H, 4.32, 1.20; N, 13.7; P, 9.30, 9.56; adenine:pentose:total P:labile P = 1: 0.97 : 1.98 : 0.98), which is indistinguishable from an authentic sample by mixture m.p., comparison (Jf infrared and ultraviolet spectra, and mobility in paper chromatographic and electrophoretic systems, and which is enzymatically active (pyru\-ate kinase coupled with lactic dehydrogenase).l 4 Solutions of AMP-I, prepared from A X P momhydrate and excess CDI, react with phosphoric ncid to produce a mixture of compounds, the nature o f which will be described in a future coiiimunication.
sample: Rf 0.381 (BElY 4l5), 0.267 (AA)] and L-serine DNP, [ " ] Z ~ D + 6 S o ir 0.25, 4yo aq. &aHCOs), lif0.365 (BEW 415), 0.340 (-U)[authentic sample: Rf 0.365 (BEW 413), 0.240 (Ah)]. Identical oxidation of N-2,4-dinitrophenyl-~-ghicosaminol, m.p. 163-164' [A-nal. Found: C, 41.46; H , 4.94; N, 11.74.1, also gave L-serine D N P , [cY]'~D f66" (c 0.325, 4% XaHC03), I
