The Chemical Synthesis of Amino Acyl Adenylates* `I'hc present papcr deals with a description of the mcthod of preparation and partial purification of several amino arid adenyl- ates. Two proccclurcs for the synthesis of these com~~ou~ids have previously been ticscrihd, but these haw certiiiii disarlv:int.ages for gcncriil aplilication. The first of tlicse iiic.t.liodi-: (I) wliicli lends to tl1e s~~ritllc of the lciicyl, aiaiiyl, cind plicnylnlanyl atlenylates in J.ieltls :tLout 10 per cent, involveti tlic rc1:rction of t,hc xiniiio acid :iryI chloride \\.it11 the silvcr salt of ndcnosinc 5'-phosphate (A5P).' In another method, I\-ieland et til. (2) used Ix-valine tliiopiirnol hytlroclil~ride as the :Lcti\-attLcl viiiino acid and they ivere abk to cffcct LL transfer of the i-aline moiety to A5P in yields or 10 to 20 ~JW cwt. lleccntl y the usefulness of S ~ ~'-clicyclolics~-lcarbocliiniitle for the synthesis of nuclcosidc pyrophosphate derivatives was ele- gant,ly demonstrated b Khoraiia et ul. (3-5). Earlicr, Zetzclic aiitl Fredrich (tij had u d thc carbodiimirlcs for tlic synthesis of ca.rbosylic acid anhydr cs. It seemed, therefore, t,ht the car- hodiimides might offer :t ~isrfnl rc.ageiit> for coupling tlic amino acids to A5P by an acyl p1iosph:tte 1iiik:rgc. Soon after our studies wcrc unrlcr way (5) TnlIwrt, and HuennPkeiis (S) Icported t,hc synthesis of butyryl itdenylatc with DCC. The incthod to lie dcscribed here involves the use of DCC to effect a condensn- tion of thc cnrhosyl group of a free amino acid with the phosphate of adenylic acid in aqueous pyridine. Vsing this procedure the nrlcnjlate derivatives of n- and L-methionine, L-iilieii)-lslaiiiiie, L-t,ryptoplian, i~nd L-scrine have been prepared and purified. MATERIALS ANU METHOUS Crystalline A5P (free acid) was obtained from t,lie Sigma Chcinical Co. and the L-amino acids were proditcts of the Cali- fornia Foundation for Biocliernicnl Research or of Nutritional Biocheniical Corp. A5P deaminase wa.s prepared by the procedure for Prepar:ttion A of 1ialck:tr (9) and dialyzed against 0.05 nr potassium succinate buffer, pII G.0, to remove animuniuin sulfate. Hydroxylamine was freshly prepared by neutralizing a stock solutiun ol 4 M liydroxylariiine lydrocliloride witli 3.5 N NaOH to :t pH of 6.5. l`lic corieeritration of thc: arnino acid adenylates \\-as incasuretl spcctropliotoirietricallv by conversion to the amino acid hydros- amatcs. To 0.5 nil. of neutralized hydrosy1:uiiinc (2 ni) were adclctl tlic nniino acyl adeiiy1:ite aiid water to a totul volurnc 01 1 rnl. After threr minutes I ml. of a solution of n,cidic ferric chloride (10) was added and the rnist'urc mas shakcn rapidly to * This work !vas supported by a research grant from the U. S. 1 `I'he abbreviations used are: A5P, adeiiosiiie S'-phosghate: I'ublic Health Service. A4TP, :ideiiosine triphosphate; DCC, dicyc1ohex);lcnrbotliimicle. reinove g:ts hibbles, filtcrcd, and the optical tlcnsitj- at 540 nip was iiicasurd :[gainst a blank cont:tining no aniino acid adeiiyl- ate. Thc concentration \\-as calculated with extinction cocffi- cieiits obtained n-ith synthetic aniino acid hydroxarnnt,es. Total ASP \vas cleteriiiiiicd lvitli 15P dcaininase ($1) after pr~- s of :in :iliquot of tlie amino :wid ntlt~iiylste :it pH 10 for 5 iiiiuutcs nt room tempernturc. Free :15P (in the prescncc of aniino t~cyl adrny1:itc~) was dctcriiiinctl nit11 :I large :mount of A5P c1c:tniiiiasc to complete the rcaction in 1 to 2 minutes, :md thus iiiinirnize the ~IOW libcration of ASP due to destruction of tlie :imino acid :tdrnylate. Ribosc `II-RS cleterniincd bj- tlic Mejbauni mc:thod (1 1) with X5P as the st:tndnrd. Ykthioiiiue was dctcrniined by n nioclifi- cation of the method of RlcC:trtli!- and Sullivan (12). and phos- phate was ineasured by thr metliocl of Fish and S~ibhRo~ (1 3). RESULTS DCC in nrlueoris pyridiue hrings about, tlic fornutioil of tlic substitutctl acj-1 phosphate derivative from an amino acid and As€'. With iiictliionine, for esaniplr, the rc;iction proceeded to completion (Tdde I). The filial \due attained depended upon the aniouiit of methionine or A5P cmploycd aid reniained ~OII- st:tnt for at least 90 minutes. Klicther this is due to the stability of the rnethionyl adenylatc under these conditions or to the :it- taininetit of :L steady state in n-hich the rate of 11reakdon.n wis equal to the rate of syntlicsis is not known. h detailed descrip- tion of the preparation and isolation of L-methionyl ndenylate follows. Synlhesis of L-methionyl ..I de?aylate-L-llletl~ioiiiiie (2.0 mnioles) aiicl A5P (1.92 inmoles) mere inised with 3.2 ml. of cold water and 10.4 nil. of pyridinc in :L 250 nil. glass-stoppored fl:tsli. 8 N HCl (U.25 nil.) W:LS ridclcd and the niisture w:ts stirred in :in ice bath with the aid of a magnetic stirrer. DCC (50 mnioles), dissolved in 12 nil. of pyridine, was added and the inisture was stirred vigorously. The formation of L-niethionyl :idcriyl:tte was deterniiiietl on aliquots removed at various time intervals (see "hIcthods"). After 3 to 3.5 llours Illere W~S nu furthcr incrwsc in iiiethioiiyl adenylate formation. The value attained was usu- ally between 90 and 95 per cent of the theoretical niaxinium based on the amount of A5P used. Tlie reactioii was terininated and thc crude nietliionyl :idcnyl- ate w:ts prccipiated by thc addition of about 150 ml. of acetone cliilled to -15". Sfter 45 seconds the precipitAte \\'as filtcrcd ra.pidly with the aid of suction, \vaslicd with sinall portions of a inisture of acetone-alcohol (BO:.lOj at, O", then with cthcr (O"), and sucked almost dry on the filter. The niaterial \vas then dried further a,t 3" overiiiglit, in z'ucuo over P205 and paraffin. The precipit:\tion, washing, and air drying were complcted in ap- proximately 8 minutes. The material obtained :it this shge 0.22 0.23 0 .:m (r 21 610 Synthesis of Amino Acyl Adenylates VOl. 233, No. 3 for methionyl adenylatc, compared to 0.22 and 0.84 for free ,\5P .-lnalyses for the various ronstituents of the purified methionyl adenylate (Table I1 j show reasonably good a,greeineiit bet\vecn the ASP, total methionine, and bound niethioninc. Occasionally, certain preparations \v\-ere contaminated with more free methi- onine than shown in Table 11. This, however, rarely exceeded a wlue of 20 per cent free methionine. Paper electrophoresis studies with niethionyl adenylate showed it to be slightly cationic at, pI-I 3.1 and easily separable from A5P which migrates as an anion under t.hcse conditions (Fig. 1). Exposure of methionyl adenylate to neutral hydroxylamine or 0.01 N KOH for 5 minutes at room temperature resulted in the disappearance of the niethionyl adenylate and formation of A5P. &inthesis of Othe? A.inino Acyl .4clenylates-The amino acyl adeiiylates of L-serine, L-plienylalaninc, I,-tryptophan, and D- methionine have been prepared with the use of the same pro- cedure already described for methionyl adenylate. The data foi recoveries, purky, and similar properties are summarized in Ta- ble 111. (14). DISCUSSION `rhe alkyl carbodiimides have proved to be extremely usefu reagents for the synthesis of a number of conipounds of biological interest. In addition to thc nucleotide pyrophosphate dcriva- tives (3-5), the unsymmetrical nucleot,ide pyrophosphate coen- zymes, such as cytidine diphosphocholine (16), diphosphopyridine nucleotide (17), flaviiiadenine dinucleotide (18), and, through the nucleoside 5'-pliosphoraiiiidate, uridine diphosphoglucose (19), have been prepared with DCC. More recently this reagent has been utilized for the synthesis of a deoxydinucleosidc monophos- phatc (20) and didcosynucleotides (21). In the present case DCC has bccn employed to link an amino acid to A5P. The studies with L-methionyl adenylate indicatc that the linkage is an anhydride between the amino acid carboxyl group and the phosphate of ASP (Fig. 2). The evidence for this conclusion is based on the following properties. The purified compound contains -45P and methionine in a 1:l ratio. The absorption specLruni is identical to that of free A5P, indicating that the amino acid is not linked to the adenine group. At pII 3.1 thc cornpound moves slon-ly as a cation on paper electro- phoresis and it is not rctaincd by the strongly cationic adsorbent Dowes 1. The remaining uncertainty in the proof of structure is in the position of the amino acyl group. It could reside in an ester linkage on the 2'- or 3'-hydrosyl group of the ribose or as shown in Fig. 2 in an anhydride linkage with the 5'-phosphate group. De Moss et a/. (1) have used as evidence for a linkage with the 5'-phosphate group the inability of adenylic deaminase 00 CH,-S-( CH 2) *-CH-C-O-P--O-CHP I1 T /`\:denins I I I NHa+ 0- H `y+ OH OH FIG. 2 to deaminate the amino acid adenylate derivative. However, it is not clear whether substitution in the 2` or 3' position on thc ribose would likewise prevent thc action of adenylic dcarninasc. The rapid and quantitative formation of thc amino acid hy- droxarnate in the presence of hydrosylamine at pH 6.5 would appear to argue in favor of the anhydride linkage. It has been pointed out recently (22), however, that amino acid esters also react with neutral hydrosylamine to form the hydrosainate. It should be emphasized however that Raacke (22) has pointed out that at pH 7 and bclow t,he rate of amino acid hydrosamate formation is very slow and usually incomplete. With the amino acyl adenylates the reaction is complete within a few minutes, a be- havior which is more characteristic of the anhydride. Moreover the enzymat.ic formation of ATP from the amino acyl dcrivatives and inorganic pyrophosphate is more easily reconciled with the formulation shown in Fig. 2. Similar evidence for the structure of L-leucyl adenylate has been presented by De Moss et al. (1). Although the detailed description for the preparation of only a few of the amino acid adenylates is presented here, preliminary experiments with leucine, valine, isoleucine, alanine, glycine, tlireonirie, tyrosine, and arginine have demonstrated that these too are converted to the adenylate derivatives under conditions similar to those described above. With some of these amino acids the reaction proceeded more slowly and the final value reached was only 30 to 60 per cent of the theoretical masirnuin. Attempts to prepare the histidyl, glutamyl, and aspartyl deriva- tives of adenylic acid have been unsuccessful to date. The rea- sons for this are riot clear but in the case of the dicarbosylic acids there is the possibility of internal cyclization to form 5 and 6 membered cyclic anhydrides which might be unstable in aqueous pyridine. With re,gard to histidine, it has been shown (23) that imidazole catalyzes a rapid breakdown of the acyl adenylate de- rivatives and it is conceivable that the imidazole group of histi- dine might promote the breakdown of a carboxyl activated hist,idine in the aqueous pyridine syst,em. It does seem possible however, that with further work including the use of suitable I protected derivatives of the amino acids, all of the nat,urally oc- curring amino acids could be converted to the adenyl derivatives with DCC. SUMMARY The present paper describes the chemical synthesis of the methionyl, seryl, phenylalanyl, and tryptophanyl adenylates from the free amino acids and adenosine 5'-phosphate in the presence of dicyclohesylcarbodiiniide. These compounds havc been obtained in relatively pure form in over-all yields ranging from 20 to 30 per cent by a combination of alcohol precipitation and treatment with Dowex 1 C1- 10 per cent cross-linked resin. Thc properties and analyses of cmethionyl adenylate indicate that the amino acid is linked to the phosphate group of adeno- sine 5'-phosphate in an acyl phosphate linkage. Acknowledgment-I am deeply grateful to Dr. H. G. Khorana for many valuable suggestions in the use of dicyclohexyl-carbo- diimide and for estending to me the hospitality of his labora- tory at the British Research Council Laboratory, Vancouver, British Columbia. Scptember 1958 P. Berg 61 1 REFERENCES 1. DEMOSS, J. A., GENUTH, S. M., AND NOVELLI, G. D., Proc. 2. WIELAND, T., NIEMANN, E., AND PFLEIDERER, G., ilngew. 3. KHORANA, H. G., J. Am. Chem. Soc., 76, 3517 (1954). 13. FISKE, C. H., AND SUBBAROW, Y., J. Bid. Chem., 66, 375 (1925). 14. BEAVEN, G. H., HOLIDAY, E. R., AND JOHNSON, E. A., In E. 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