COMMUNICATIONS TO THE EDITOR
Aug. 5, 1963
2345
It is hoped t h a t 8,2’-cyclonucleoside will be useful group of pyrimidine3 led us to attempt the synthesis in transforming the sugar moiety of purine nucleosides of 8,2’-cyclonucleoside. in a manner similar t o t h a t which has been used so 2’-O-Acetyl-3’-O-tosyl-5/-0-met hoxycarbonyl-D-xylosuccessfully in the pyrimidine series.* Experiments furanosyl chloride4 was condensed with 2,s-dichloroalong this line are now in progress in our Laboratory. adenine chloromercury salt in refluxing xylene to give 2,8-dichloro-9- (2’-0-acetyl-3’-O-tosyl-5’-O-methoxycar- (8) J. J. Fox and I. Wempen, Advan. Carbohydrate Chem., 14, 283 (1959) bonyl) -6-D-xylofuranosyladenine, m .p . 160-162 ( Anul. FACULTY OF PHARMACEUTICAL SCIENCES Calcd. for C21H2109N&12S: C, 42.85; H, 3.60; N , OF MEDICINE MORIOIKEHARA SCHOOL 11.90. Found: C, 42.56; H, 3.79; N, 11.90. XO,:x”C’ HOKKAIDO UNIVERSITY HIROSHITADA SAPPORO, JAPAN 263 mp, 263 mp. Paper chromatography: RECEIVED MAY6, 1963 Rz~0.90 (1-butanol: water = 86:14). This material was converted t o its 8-mercapto derivative by the reaction with one equivalent of thiourea. Although 2Evidence for Chain Transfer in the Autoxidation of chloro-8-mercapto-9- (2’-0-acetyl-3’-O-tosyl-5/-0- methHydrocarbons Retarded by Phenol oxycarbonyl) -6-D-xylofuranosyladenine( I ) was obtained in the form of 3 hard glass (yield 68.2%), evidence for its Sir : assigned structure was obtained by paper chromatogPeroxy radical-antioxidant complexes have been raphy (Rf 0.90 (1-butanol: water = 86:14)) and spectropostulated by Hammond, et al.,l to be intermediates in photometric analysis (Xli;2xNH”’ 302, 310 mp; ”: :A 302, the inhibition of the autoxidation of hydrocarbons. ’ “‘OH 300 mp). 310 mp, A’.K4SX Despite numerous attempts to establish the existence of Refluxing of I in methanol containing sodium such c ~ m p l e x e s , ~no - ~direct evidence has yet been obmethoxide for 12 min. gave a compound having m.p. tained for their existence and, a t the same time, no 228-229O dec. Xt2xNHC‘ 277 mp, Xt,’,NNaoH 277 mp. plausible alternative mechanism has yet been proposed. Anal. Calcd. for C10H1~O3N6C1S.O.5H20 : C, 36.99; The postulation of these complexes was based on the H , 3.38; N,21.56. Found: C, 36.66; H, 3.82; N, 20.80. kinetic evidence that the rate of oxygen absorption durPaper chromatography [Rr 0.42 (water, pH 10.0), ing the oxidation of tetralin in the presence of phenol or &d5]. From spectrophotometric, chromatographic N-methyl aniline followed the kinetic relation and elemental analytical data, the resulting compound was concluded to be 2-chloro-8-mercapto-8,2’-anhydroD-xylofuranosyladenine (11) and not the presumed where Ri is the rate of radical production from azobisintermediate 2’,3’-anhydronucleoside (111). This was propionitrile) (AIBN). This postulation, further supported by the fact t h a t I1 has no v N ~ ~ ~ (2-methyl ’ however, assumed that the rate was first order with a t 863 cm.-’, which has been assigned to the epoxide respect to hydrocarbon concentration. group. In the present work it has now been demonstrated The structure of I1 confirmed by the desulfurization that the dependence of the rate of oxygen absorption of compound I1 with Raney nickel, which afforded with respect to the concentration of the hydrocarbon is 2-chloro-2‘-deoxyadenosine(IV) [Rr 0.52 (water, pH not first order. Data are presented in Fig. 1 for the 10.0), XkixNHcl 265, A”,‘;: NaoH 265 mp]. Hydrogenation initial rate of oxygen absorption by chlorobenzene soluof compound IV over palladized charcoal as a catalyst tions of phenol, AIBN, and tetralin. When phenol was gave 2’-deoxyadenosine [Rf 0.35 (1-butanol-water = absent, the rate was first order with respect to hydro86:14), 0.50 (water, pH 10.0); X ~ . ’ , N H c l 258, X::xNN80H carbon concentration, demonstrating that the rate of 260 mp]. This material was compared directly with formation of radicals from AIBN did not vary with the authentic 2‘-deo~yadenosine~ and 3’-deoxyadenohydrocarbon concentration. However, when phenol sine’ Hydrolysis was added, the rate became order with respect to the Sample product Rf“ Spray I* Spray 11‘ hydrocarbon. Similar results were obtained using 9,lONatural 2’-de- 2-Deoxyribose 0 . 2 1 Pink + + Pink + dihydroanthracene as the substrate. Thus oxy-adenosine Synthetic 2-Deoxyribose ,21 2’-deoxyadenosine 3’-deoxyadeno- 3-Deoxyribose , 4 1 sine a Paper chromatography, 1-butanol-acetic acid-water = 4 :1:5, after equilibration overnight, upper layer was used. * Cysteine-sulfuric acid reagent: J. G. Buchanan, Nature, 168, 1091 (1951). Aniline hydrogen phthalate reagent: S. M . Partridge, Nature, 164, 443( 1949).
++
+
+
+++
These results clearly show t h a t the resulting deoxynucleoside is 2’-deoxyadenosine and not the 3’-isomer. Thus it must be concluded t h a t the 8,2‘-cyclic bond was formed by the nucleophilic attack of the 8-mercapto group on the 2‘-carbon atom of the epoxide linkage in 111. (3) M. Wilkins. “Nucleoproteins,” R . Stoops, Ed., Solvay Inst. Chem., 11th Chemical Conference, Interscience, New York, N.Y . , 1959, p. 48. (4) C . D.Anderson, L. Goodman and B. R . Baker, J . A m . Chem. Soc., 81, 386’1 (1959). (5) Rad stands for a value obtained from Rr divided by Rf of adenosine. (6) Purchased from Sigma Chemical Co. St. Louis, Mo. (7) Unpublished experiments by A. Yamaraki and M. Ikehara.
dOz -dt
oc
[Ri]’/z [RH]’/s [phenol]’/;-
The kinetic chain lengths in these experiments ranged from 4 to 30. A mechanism which can account for the observed kinetics follows R’
ki
- N = N - R ’ +2aR’.
+ NZ Ri
=
2akl[AIBN] (I)
kn
+ 0%+R‘Oz, ka R’OZ,(or R o t . ) + RH R’OZH + R. R’.
--f
R.
+ Oa +ROz.
(3)
kd
(4)
ka
+ AOH +ROzH + AO. ks AO. + RH +AOH + R . kr ROz, + AO. +inert products
Rot.
(2)
(5) (6)
(7)
where AOH is phenol, AO. is the phenoxy radical and RH is tetralin. If one makes the usual steady state (1) G.S. Hammond, C. S. Boozer, C. E. Hamilton, and J . N . Sen, J Am, Chem. Soc., 17, 3238 (1955). (2) K . U. Ingold, Chem. Rev., 61, 563 (1961). (3) J. R.Thomas and C . A. Tolman, J . Am. Chem. Soc., 84, 2930 (1962). (4) J. R.Thomas, i b i d . , 86, 591, 693 (1963).
COMMUNICATIONS TO THE EDITOR
2346
RESULTSO F
Vol. 85 TABLE1 3'-HYDROXYL GROUPSIS A W D OLIGONUCLEOTIDES
hCETYLATION O F
Compound'
PT d-pd d-pG d-pC
Concentration AcnO (pmole/ml.) (mmoles) Yield
0.72 70 .70 .70
PTPTPTPT
18
d-PAPAp ApA
18
d-pCpCpCpC
.18
d- p -4pAp Ap Ap A p Ap A
,035
1 1 1 1 2 3 1 3 1 3 1 3 1
63 85 88 71 81 96 55 70
75 80 73 87 62
MONO-
Assay
1 1 1 1 1 1 1,2 1,2 1,2 1 2 2 2
phosphomonoester groups in polynucleotides have been reported from this Laboratory. 2 , 3 The present communication describes a method for the selective acetylation of the free hydroxyl groups, on the terminal nucleosides in deoxyribopolynucleotides. The approach thus promises to be complementary to those developed prev i o ~ s l for y ~ the ~ ~ labeling of end groups in nucleic acids. X solution of the mononucleotide or oligonucleotide (Table I ) (0.07-1.4 pmoles of the sodium or ammonium I I I I I 1 1 1 salt) in water (2.0 ml.) was treated with acetic anhyI 2 3 4 5 6 7 8 9 dride (0.1-0.3 ml.) a t room temperature, the mixture log [ t o i r o l i n ] . being stirred by a magnetic stirrer. Acetic anhydride was added in portions of 0.01 ml., the p H of 7 mainFig. 1,---Dependence of rate of oxygen absorption on tetralin tained by the continuous addition of 4 N sodium hyM AIBS: A, no phenol, concentration a t 60" and 37 x droxide from a microsyringe. After the completion of slope = 0.90; B, 7 . 6 X M phenol, slope = 1.42; C, 23 X the addition (about 15 min.), the nucleotidic material M phenol, slope = 1.53; D, 38 x lW4 M phenol, slope = was freed from sodium acetate either by extensive di1.48. alysis against water (for oligonucleotides) or by passage assumptions and the further approximations of the total solution through a column of pyridinium Dowex-50 ion-exchange resin and lyophilization of the resulting solution. Two types of assays were used for and following the extent of the acetylation reaction. In then assay 1, the mixture was chromatographed in the solvent n-butyl alcohol-acetic acid-water (5-2-3) or ethyl alcohoI-(J.5 M ammonium acetate (pK 3.8) Thus, the kinetic expression obtained for the phenol(7-3, v./v.). I n the case of mononucleotides and the tetralin system is consistent with a mechanism in tetranucleotides pTpTpTpT4 and d-pXpApApA,4 the which chain transfer,6,6reaction 6, is of considerable 3 ' O a c e t y l derivatives were clearly resolved from the importance, with no necessity of invoking a peroxy starting materials. The assay 2 involved incubation of radical-inhibitor complex. Other inhibitors whose the mixture with the Escherichia cola phosphodiesterase5 kinetic behavior has been interpreted in terms of in order to degrade the unchanged oligonucleotide to "complex formation" (viz., N-methylaniline, diphenylmono- and dinucleotides and to determine the acetyamine, trialkylamine~~)are substances which, like lated product by paper chromatography. I t has been phenol, would give relatively unreactive monofunctional demonstrated recently that acetylation of the 3'radicals and which therefore could restart chains by a hydroxyl group confers complete resistance on the olisimilar chain transfer mechanism. gonucleotide towards this enzyme.6 The results are ( 5 ) W A Waters and C. Wickham-Jones, J Chem. S O L ,812 (1950) given in Table I. In general, the yields were high and (6) A F Bickel and E C Kooyman, ibid., 221.5 (19.56) probably can be increased further. I t is worth noting CHEMISTRY DEPARTMENT LEE R . MAHONEY that the yields in the case of the tetranucleotides and SCIENTIFIC LABORATORY FREDC. FERRIS the one heptanucleotide studied showed no decrease. MOTORCOMPASY FORD Previous experience' of acylation of nucleotides in DEARBORS, MICHIGA?; RECEIVED J U N E 13, 1963
The Selective Acetylation of Terminal Hydroxyl Groups in Deoxyribo-oligonucleotides Sir : As a part of work on the sequential analysis of nucleic acids, recently, methods for the labeling of terminal (1) This work has been supported by grants from the iYational Cancer Institute of the Kational Institutes of Health, T h e National Science Foundation, Washington and T h e Life Insurance Medical Research F u n d , New Yvrk. K. Y
(2) R . K . Ralph, R . J , Young, and 1%.G . Khorana, J , A m Chem S a c . , 84, 1490 (1962). (3) R . J. Young and H . G . Khorana, ibid , 86, 244 (1963); U L . RajBhandary, R . J . Young, and H . G. Khorana, Federalion P r o c , PI, 350 (1963). (4) T h e system of abbreviations is as in current use in J . Bioi. Chem. and previously described H. G Khorana, "Some Recent Developments in t h e Chemistry of Phosphate Esters of Biological Interest," John Wiley and Sons, I n c , New York, N . Y., 1961. ( 5 ) I R . Lehman, J . Bioi C h e m . , 236, 1479 (1960). (6) A. Falaschi, J . Adler, and H G Khorana, ibid , in press (7) (a) H. G. Khorana, A F Turner, and J . P . Vizsolyi, J A m . Chem. S o c , 83, 686 (1961); (b) R . K Ralph and H G . Khorana, ibid , 8 8 , 2926 (1961).
