Complex Formation in Oligonucleotides and ... - ACS Publications

Malonic acid and di-£-dimethylaminophenyl- carbodiimide havebeen reported to react inpyri- dine medium to give a diacylurea but the fate of malonic a...
0 downloads 0 Views 265KB Size
COMMUKICATIONS TO THE EDITOR

April 5, 1962

R’\ ,C(CO2Hi2 R“ R’,

+

R”’N=C=NR”’

4

R”‘ I

,CO-N C )cO Ret' ‘C0-N I

+

R”’NHC0h”R”’

R“’

I, R’= H, R” = H, R”’= CeHii 11, R’ = H, R ” = CsHsCH2, R”’= CsHii

111, R’ = Et, R ” Iv,R’ = H,R ” V,R’ = Et, R ”

= Et, R”’ = CeHii = H, R“’ = i-pr = Et, R”’ = $-tolyl

1311

acids do not. The role of steric and electronic factors in these reactions is under investigation. The usefulness of this novel reaction of carbodiimides was demonstrated by the synthesis of 6lactam-substituted spirobarbiturates [for example, VIII, m.p. 234-235’1 which could not be prepared by the usual methods of barbituric acid synthesis. We are thankful to J. Zuclich3 and J. Pelosi3 for valuable technical assistance and to Dr. E. R. Mahowski and R . S. Magee3 for the n.m.r. spectra and their interpretation. This investigation was supported in part by a research grant (MU-3930) from the Xational Institute of Mental Health, U. S. Public Health Service, for which grateful acknowledgement is made.

Substituted malonic acids led to substituted barbiturates on reaction with N,N’-dicyclohexyl(3) Kational Science Foundation undergraduate research parcarbodiimide and N, N’-diisopropylcarbodiimide. ticipant. Some of the compounds prepared by this method DEPARTMEST OF CHEMISTRY ASD CHEMICAL ESGIYEERISG are: 11, m.p. 120-121’; 111, m.p. 160-162’; IV, STEVEXS INSTITUTE OF TECHNOLOGY AJAY K. BOSE m.p. 128-129’. SHEILAGARRATT HOBOKEN, SEW JERSEY Malonic acid and di-p-dimethylaminophenylRECEIVED FEBRUARY 23, 1962 carbodiimide have been reported to react in pyridine medium to give a diacylurea but the fate of COMPLEX FORMATION IN OLIGONUCLEOTIDES malonic acid on reaction with di-p-tolylcarbodi- AND ITS APPLICATION T O THE SEPARATION OF imide has not been estab1ished.l We found that POLYNUCLEOTIDES the reaction of malonic acid with N,N’-di-p-tolyl- Sir : carbodiimide in tetrahydrofuran solution afforded Recent studies‘ on oligonucleotides have shown N,N’-di-p-tolylurea and a compound that melted that they arc: capable of complexing with themselves a t 141-142°, solidified a t about 150’ and remelted in buffered salt solutions. The complex formaAnal. Calcd. for C33H3zN404:C, tion appears to be of the “Watson-Crick” type and a t 225-230’. 72.24; H,5.88; N, 10.21;mol. wt., 548.6. Found: depends on the chain length, temperature and C, 72.63; H , 5.68; N > 9.93; mol. wt., 370; A?:’ nature of the nucleotide bases present. For ex3.05-3.20~ (weak), 5.7611 (strong), 5 . 9 5 ~(weak), ample, a mixture of thymidine dodecanucleotide,2 6.00~ (weak); n.m.r. peaks,2 7.68 (6H), 7.55 (BH), (PT)’~and 2 mole equivalents of deoxyadenosine 6 . i i (2H), 2.78 (center of multiple peak, 16H), hexanucleotide, d-(p-k)~.a t 0” in 1 118 NaC1, pH 7 -0.Si (2H). displays an llyGdecrease in optical density a t On the basis of infrared and n.m.r. spectra, we 259 and 267 mp. The complex melts over the prefer structure VI to the acylurea structure VI1 for range 4-30’ with a melting point (Tm)a t ca. 16”. this compound. On heating above the melting ,4 mixture of (pT)6 and d-(pA1)6, however, shows no point, VI was converted into the di-p-toluidide of significant hypochromicity under these conditions. malonic acid. Analogous results were obtained on These results suggested the possibility of devisusing ethylmalonic acid. When, however, di- ing a polynucleotide-cellulose column which would ethylmalonic acid was allowed to react with N,N’- separate polynucleotides not only on the basis of di-p-tolylcarbodiimide the barbiturate V, m.p. 16s- their nucleotide composition, but also by virtue 16S.5’, was formed readily. of their nucleotide sequence. A columii o f ccllu0 -1rNH 0 Ar 0 lose on which polynucleotide chains had hceii I I/ I il 1 incorporated by bonding a t one end of the cliaiiis, 112C(C--0 C N.lr). 1ItC(C--N-c-hlIAl)? was expected to be the most efficient, as this type TI VI1 of coluiiin should allow iliaxiinurn interaction wi tli ’The difference between aroiiiatic and aliphatic the polynucleotides to be separated. carbodiimides in the reaction with malonic acid is Thymidine 5’-phosphate (2 mmole) in dry pyristriking. I t is also remarkable that diethylmalonic dine (3 ml.) was mixed with dicyclohexylcarbodiacid yields a barbiturate on reaction with di-p- imide (4 mmole) and shaken with glass balls for tolylcarbodiimide but ethylmalonic and malonic 5 days. The mixture was added to dry cellulose (5 g.) dicyclohexylcarbodiimide (2 g.) in pyridine Ph C~HII (50 ml.) and shaken for 5 days. The filtered celI I lulose was washed with pyridine and allowed to (IC,/N\C/CO -N, co stand in aqueous pyridine overiiight and thc~t washed with ethanol arid water. Analysis of the CCl/ ‘(’0-N’ I combined washings showed an incorporation of tlic ( -dlI L nucleotide of more than 50%. The cellulose VI11 prepared in this way was expected to contain chains of thymidine polynucleotides (of up to ca. (1) F. Zetzsche and H Lindlar, Bel,., 71, 2095 (1938). (2) Determined in dilute deuterated chloroform solution using tetramethylsilane as a n internal standard. T h e first number indicates the 7 value and the figure in parentheses denotes the number of equivalent protons under the peak.

(1) P. T. Gilham, unpublished. ( 2 ) For preparation of oligonucleotides see 11. C . Khorana and J . I’. Vizsolyi, J . Am. Chem. SOC.,83, 073 (1961); R. R. Ralph a n d H G. Rhorana, i h . , 83, 2920 (19fil).

1312

COMMUNICATIONS TO THE EDITOR Temperature, "C. 4.

I

24'

considered responsible for these observations

I

CCl4 -k 2(CaHs)aP + ( CBHL)SPCIZ(I) $- (CeHs)sP=CC1>(II)

35'

I n order to test this hypothesis, an equimolar amount of benzaldehyde was added to a mixture of triphenylphosphine in excess carbon tetrachloride which had been heated for 3 hours a t 65". Gas chromatographic analysis of the resulting solution indicated that most of the benzaldehyde was consumed and two new products, identified as p,pdichlorostyrene and benzal chloride appeared. These lend support to the proposed intermediacy of triphenylphosphine dichloromethylene and triphenylphosphine dichloride. (ceH~,)J'=cCh f CeHsCHO --3 (CeHs)aPO f CeHsC?d=CC12

2 2.0 ( 0 0

U

d-(p A)

..x u

B

w 1.0

.-:: 0" 3

.r,

+A

(C6Hs)sPCL $- CeHsCHO +(CeHs)3PO f C ~ H E . C H C ~ ~ ~

50 100 Volume, ml, Fig. 1.

20 units long) connected a t their 5' phosphoryl ends to the cellulose by phosphodiester linkages. The cellulose was packed into a column (20 cni. X 1 cm. diam.) and washed extensively with 1 M NaC1-0.01 M NaH2P04 (pH 7) solution. Ten optical density units each of (pT)6 and d-(pA)e were passed slowly through the column in this salt solution a t 4". The elution diagram shows that the d-(pA)B was selectively bound to the column and could not be eluted until the temperature of the column had been raised to 35 O. The separation was complete and the recovery quantitative. Preliminary experiments with E. coli amino acid transfer RNA on this column have resulted in a selective adsorption of 6y0 of the RNA. This fraction is now being examined for the presence of a consecutive adenosine sequence and for its specificity in accepting amino acids. This work was supported by research grants (No. RGSS17, C5178) from thc National Institutes of Health. INSTIIUTE FOR ENZYME RESEARCII UNIVERSITY OF WISCONSIN P. ~VISCONSIN MADISON, RECEIVED FEBRUARY 20, 1962

'r. GILIIAM

For synthetic utility i t is convenient to conduct the entire reaction in one step by adding the carbonyl component to the initial mixture. For example, when a solution of 72.0 g. (0.274 mole) of triphenylphosphine and 29.0 g. (0.274 mole) of benzaldehyde in 150 ml. of carbon tetrachloride was heated a t 60" for 2 hours, gas chromatographic analysis revealed that the benzaldehyde disappeared rapidly while benzal chloride and P,p-diclilorostyrene formed in equal quantities. Addition of 200 ml. of low boiling petroleum ether to the reaction mixture precipitated 69 g. (go%), m.p. 153-156', of triphenylphosphine oxide. Fractional distillation of the filtrate gave less than 1 g. of unreacted benzaldehyde, 15.1 g. (72%) of benzal chloride, b.p. 121-3' (63 mm.) and 16.0 g. (72%) of p,p-dichlorostyrene,* b.p. 135-7' (63 mm.). A careful study of the reaction of 32.7 g. (0.1'25 mole) of triphenylphosphine and 70 ml. of carbon tetrachloride in the presence of 22.8 g. (0.125 mole) of benzophenone a t 60" was conducted and followed by quantitative gas chromatography. After 4 hours, the ratio of the peak area of the product, 1,l-diphenyldichloroethylene,to that of the benzophenone became constant. The solution contained 9.65 g. (0.052 mole) of benzophenone and 12.2 g. (0.049 mole) of 1,l-diphenyldichloroethylene(7h% based on the stoichiometry) :5 2(ceHs)sP f ~(CF,HF,)~CO f CClr --+ 2( C&)sPO f (C,&s)2C=CC12

YLID

Vola s4

INTERMEDIATE IN THE REACTION TRIPHENYLPHOSPHINE WITH CARBON TETRACHLORIDE

OF

Sir : We wish to report an extremely simple, high yield route to prepare the ylid, triphenylphosphine dichloromethylenel [by the direct reaction of carbon tetrachloride with triphenylphosphine] and a convenient one-step synthesis for p,p-dihaloolefins. When a concentrated solution of triphenylphosphine in carbon tetrachloride is allowed to stand a t room temperature for 48 hours or is heated at 60' for 2-3 hours and then hydrolyzed, no triphenylphosphine is recovered.2 This reaction was (1) This ylid was first described by A. J. SpeziaIe, G. L. Marco and K. W. Ratts, J. A m . Chcm. Soc., 82, 1260 (1960). (2) I n the 60' case, 76% of triphenylphosphine oxide was obtained. An additional 12% of the triphenylphosphine was combined in a water soluble unidentified crystalline solid, m.p. 239-241O with an empirical formula of CioHisClZP.

f (ce&):Cc12

However, no diphenyldichloromethane was observed, demonstrating that the triphenylphosphirie dichloride produced in this reaction does not exchange with benzophenone. Addition of benzaldehyde to an aliquot of the reaction misture produced benzal chloride almost immediately but, as anticipated, no p,p-dichlorostyrene was formed. This shows that under special conditions and depending on the carbonyl component, considerable control of the product may be obtained. This type of reaction thus provides a potentially convenient route to otherwise difficultly accessible 1,l-dihaloolefins. (3) I,. Horner, H. Oediger and H. Hoffmann, Ann., 616, 26 (1959). (4) Anal. Calculated for CsHsCli: C, 55.5; H, 3.46; C1, 41 0. Found: C, 5 5 . 9 ; H, 3.65; C1, 41.23. (5) The reaction mixture was conveniently worked up taking advantage of the insolubility of triphenylphosphine oxide in ethyl ether and the much greater solubility of benzophenone in methanol than that of 1,l-diphenyldichloroethylene. The latter, m.p. 78.078.5' was identified by comparison with an authentic sample.