- Email: [email protected]
Studies on polycysteine peptides and proteins. I. Isomeric cystinylcystine peptides
Studies on Polycysteine Peptides and Proteins. I. Isomeric Cystinylcystine Peptides Nobuo Izumiyal and Jesse P. Greenstein Fwnc the Laboratory of Biochemistry, Institutes of Health, Received
NatiorLal (‘ancer Ilutitute, Bethesda, Mar!gland
February
Xatiorlal
10, 1954
INTRODUCTION The oxidative conversion of cysteine to cystine has long been known. What configurations result whan a peptide or a protein containing two or more cysteine residues is oxidized is little known. Some 17 years ago one of the present authors undertook such a study with the synthesis of L-cysteinyl-L-cysteine, which was the first of this type of compounds to be prepared, and which on oxidation yielded the dimeric molecule L-cystinyl-L-cystine (1). The resumption of this investigation has now become possible because of the development of new and more powerful methods of peptide synthesis, and because of the availability of optically pure L- and n-amino acids in quantity, and of new and readily accessible types of physical instruments. The first study in the present series deals with the preparation and properties of optically active cystinylcystine peptides of relatively simple configuration. L-Cystinyl-L-cystine has been synthesized by the following sequence of reactions (1) : dicarbobenzoxy+-cystinyldichloride plus L-cysteinyl ethyl ester + dicarbobenzoxy-L-cystinyl-di(L-cysteinyl ethyl ester) --f L-cysteinyl-L-cysteine ethyl ester ----f anhydro-L-cysteinyl-L-cysteine -+ L-cysteinyl-L-cysteine -+ L-cystinyl-L-cystine. The last-mentioned reaction was accomplished by aerating the ammoniacal solution of cysteinylcysteine at pH 8.5 until the sulfhydryl reaction was negative. The crystalline, oxidized peptide possessed an [cY]~~= -60” in 1 N HCI, and crystallized from water as the dihydrate. Its dihydrochloride salt crystallized as the tetrahydrate. On hydrolysis with HCl, the peptide 1 Fulbright .J:lp:Ul.
and Smith-Mundt
Scholar;
203
on leave
from
Kyushu
University,
204
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
yielded nearly the equivalent amount of L-cystine with [a]E2 = -211’. The structure of the peptide may be tentatively represented by the following isomeric formulations: COOH 1 SCH2CHCONHCHCHk.S
SCHzCHCONHCHCHzS
SCH~CHCONHCHCH~S
SCHZHNHCOCHCHZS
NH2
&Hz
COOH
NH2
COOH
COOH
I
NH2 II
It is difficult to distinguish these two structures experimentally when the residues are both derived from L-cystine. However, if one residue is L-cystine and the other D-cystine, i.e., the dipeptide being either L-cystinyl-D-cystine or its optical enantiomorph, D-cystinyl-L-cystine, hydrolysis of either of these peptides would yield DL-cystine if the structure is I, and meso-cystine if the structure is II. Accordingly, we have prepared L-cysteinyl-D-cysteine and D-cysteinyl-L-cysteine by the mixed anhydride procedure developed by Vaughan and Osato (2, 3), and oxidized these products with air at pH 6.5 and 8.5 to the corresponding cystinylcystines. The same procedure was employed to synthesize L-cystinyl-L-cystine for comparison with the earlier preparation described above. The present method involved essentially the following reactions : carbobenzoxy- S-benzylcysteine plus S-benzylcysteine ethyl ester in presence of isobutylchlorocarbonate and triethylamine -+ carbobenzoxy- S-benzylcysteinyl- S-benzylcysteine ethyl ester -+ carbobenzoxy-S-benzylcysteinyl-S-benzylcysteine --+ S-benzylcysteinyl-S-+ cysteinylcysteine -+ cystinylcystine. That no benzylcysteine apparent racemization occurred in the first step of this procedure was shown by comparison of the optical rotation values of the coupling product with those of the same product obtained by the azide method. All of the cystinylcystine preparations were crystalline. L-CystinylD-cystine whether prepared by oxidation at pH 6.5 or pH 8.5 possessed the same value of specific optical rotation which was equal and opposite to that of the D-cystinyl-L-cystines similarly prepared. All of these isomers gave a single ninhydrin spot on the paper chromatogram with Rf = 0.16 in phenol.2 On acid hydrolysis, they yielded optically inactive cystine. Probable identification of these hydrolytic products as DL-cystine 2 In all cases SOo/, phenol
was employed
with Whatman
No. 1 paper.
POLYCYSTEINE PEPTIDES AND PROTEINS. I
205
was based on the observation that they were oxidized by snake venom n-amino acid oxidase at the same rate as a known specimen of nn-cystine, a rate considerably in excess of that noted with a known specimen of meso-cystine. This observation does not exclude the presence of relatively small amounts of meso-cystine in the optically inactive cystine hydrolytic products, but it does suggest that the greater part at least of these particular cystinylcystine peptides may exist in configuration I. In contrast with the case of the above-mentioned optically enantiomorphic cystinylcystines, the properties of n-cystinyl-n-cystine appeared to be related to the pH at which it was prepared by oxidation of the preceding n-cysteinyl-n-cysteine. Thus, when this pH was maintained at 6.5, the product had an [(Y]:~ = -31” in HCl, it yielded a single ninhydrin spot on the paper chromatogram with R, = 0.49 in phenol, a dihydrochloride was prepared which crystallized as the dihydrate, and finally, the dibenzoyl derivative possessed an [(Y]:’ = -48.0” in NaOH. On the other hand, when the pH at oxidation was maintained at 8.5, the product had an [&? = -58.7” in HCl, it yielded two ninhydrin spots on the paper chromatogram, one of which was relatively intense with Rf = 0.20 whereas the other spot, with Rf = 0.49, was very faint, a dihydrochloride was prepared which crystallized as the tetrahydrate, and finally, the dibenzoyl derivative possessed an [ali = -35.2” in NaOH. Thus, the dibenzoyl derivatives of the two n-cystinyl-n-cystine preparations showed by their differences in specific rotation values their derivation from two different compounds. Both n-cystinyl-n-cystines crystallized from water as the dihydrate, and possessed nearly identical elemental analytical values. Both yielded n-cystine on HCl hydrolysis with [(Y]:~ = -210” to -212” in HCl. It is difficult to avoid the impression that the two n-cystinyl-n-cystines are structural isomers, represented perhaps by configurations I and II above. Both n-cystinyl-n-cystines are nearly equally susceptible to the action of peptidases. Based upon the rotation and other data described, the L-cystinyl-t-cystine prepared in the present instance by oxidation at pH 8.5 is probably identical with that described earlier (l), and which had also been prepared by oxidation at this pH. The reason for the effect of pH on the configuration of the disulfide forms obtained by oxidation of n-cysteinyl-n-cysteine, as contrasted with the absence of such effect at the pH values studied on the configuration of the disulfide forms obtained by oxidation of n-cysteinyl-n-cysteine or n-cysteinyl-L-cysteine, is not apparent at the present time. Formation of a monomeric cyclic
206
N~BUO
IZUMIYA
AND
JESSE
p.
GF~EENSTEIN
peptide by combination of each terminal sulfur atom to form a single disulfide link would produce an eight-membered ring, which in the case of n-cystinyl-n-cystine or n-cystinyl-n-cystine would lead on HCl hydrolysis to the formation of pure meso-cystine. The available evidence is not in favor of these considerations. Molecular weight determinations reported earlier for n-cystinyl-n-cystine (1) are compatible with the 16-membered ring represented by the formulas above. This does not exclude the possibility that the other cystinylcystines reported herein may be cyclic peptides of still higher molecular weight. It is not inconceivable that stable rotational isomers may exist among these cyclic disulfide compounds. The crystalline disulfide peptides isolated after oxidation of the various cysteinylcysteines account for only about 2530% of the theoretical yield, and the remainder appears to be ill-defined material probably arising through linear polymerization. Further studies will obviously be necessary to determine the configurations brought aobut by the gentle oxidation under various experimental conditions of polycysteine peptides of biological interest. The importance of such studies has been emphasized by the brilliant synthesis of oxytocin by du Vigneaud and his collaborators (4). In this case the oxidation (at pH 6.5) of two L-cysteinyl residues separated by four amino acid residues along a peptide chain led to the formation of a monomeric cyclic peptide. On a still larger molecular scale, many proteins are known to contain several sulfhydryl groups on their surface, the exposure of which to oxidizing conditions would lead to decided changes in their molecular configurations and sizes. Such studies involving both synthetic polycysteine peptides and proteins are in progress. EXPERIMENTAL
Carbobenzoxy-S-benzylcysteinyl-S-benzykysteink
Ethyl Ester
A solution of 346 g. of carbobenzoxy-S-benzyl-L-cysteine (5, 6) and 139 ml. of triethylamine in 3 1. of toluene was chilled to -5” and treated with 131 ml. of isobutylchlorocarbonate. After 10 min. of standing, a cold solution of 276 g. of S-benzyl-L-cysteine ethyl ester hydrochloride and 139 ml. of triethylamine in 2 1. of chloroform was added, and the mixture allowed to stand overnight at 25”. One additional liter of chloroform was added, and the mixture was washed successively with water, bicarbonate solution, and water, and finally dried over anhydrous sodium sulfate. The filtrate was condensed in pacuo to low bulk, and the residual solution treated with petroleum ether. The carbobenzoxy-S-benzyl-n-
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
207
cysteinyl-S-benzyl-L-cysteine ethyl ester which precipitated was filtered off, and recrystallized from ethanol. The yield was 382 g., or 67 % of the theoretical. M.p. 105” (corr.) ; [a]:’ = -55.3’ (2 % in acetone): Anal. C30H3406N2S2: Calcd. C 63.6, H 6.1, N 4.9; found C 63.5, H 6.0, N 5.0. Carbobenzoxy-S-benzyl-L-cysteinyl-S-benzyl-n-cysteine ethyl ester was prepared in the same manner. The yield was 66 % of the theoretical. M.p. 103” (corr.); [ali = +5.1” (2% in acetone). Found C 63.9, H 6.3, N 5.0. Carbobenzoxy-X-benzyl-n-cysteinyl-S-benzyl-L-cysteine ethyl ester similarly prepared in 68 % yield possessed a m.p. of 103” (corr.) and [a];’ = -4.7” (2% in acetone). Found C 63.4, H 6.1, N 4.9. Two of the compounds were also prepared by the azide procedure as follows. A dry ether solution of carbobenzoxy-S-benzyl+-cysteinylazide from 33.6 g. of carbobenzoxy-S-benzyl-L-cysteinylhydrazide (5, 6) w-as mixed with a dry ether solution of S-benzyl-L-cysteine ethyl ester prcpared from 31.2 g. of the hydrochloride (5, 6), and the reaction mixture was allowed to stand overnight at 25”. The product separated in the form of long needles. After further chilling, it was filtered off, washed I\-ith cold, dry ether, and recrystallized from ethanol. The yield \vas 28.2 g.; m.p. 105” (corr.); [ar]i6 = -55.2” (2% in acetone). Found C 63.7, H 6.2, N 5.0. Carbobenzoxy-S-benzyl-D-cysteinyl-S-benzyl-L-cysteine ethyl ester Tras similarly prepared from carbobenzoxy-S-benzyl-n-cysteinylhydrazide and S-benzyl-L-cysteine ethyl ester hydrochloride. The reaction product however did not precipitate from the ether solut’ion, and consequently the latter was washed successively with dilute HCl, biparbonate solution, and water, and finally dried over anhydrous sodium sulfate. The filtrate was concentrated in raczlo, and, after adding petroleum ether, the product was precipitated, filtered, and recrystallized from ethanol as thin needles. The yield was 58% of the theoretical. M.p. 103” (corr.); [c&’ = -4.9” (2% in acetone). Found C 63.4, H 6.1, N Fj.0. Both mixed anhydride and azide procedures evidently led to the same respective products with nearly identical rotation values. The derivatives of S-benzyl-D-cysteine, not previously reported, were prepared as described for the corresponding L-forms (5, 6). These included S-benzyl-ncysteino et#hyl ester hydrochloride: m.p. 163” ((aon.); [cx]~ = +28. I” (2 % in ethanol); N calcd. 5.1, found 5.1; c~arbobenzoxy-S-benzyl-n-
208
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
cysteine: m.p. 95’ (corr.); [ali6 = +45.1’ (2 % in acetone); N calcd. 4.1, found 4.1; and carbobenzoxy- S-benzyl-n-cysteinylhydrazide : m.p. 135” (corl.); [L-Z]:’= -4.3” (2 per cent in glacial acetic acid); N calcd. 11.7, found 11.5. Carbobenxoxy-S-benzylcysteinyl-S-benzylcy$eine A mixture of 454 g. of carbobenzoxy- S-benzyl-n-cysteinyl- S-benzyln-cysteine ethyl ester in 4.6 1. of dioxane and 480 ml. of 2 N NaOH was shaken for 5 hr. at 5”. To the clear solution 167 ml. of 6 N HCl was added, the mixture condensed in vacua to a low bulk, and treated with water. An oil appeared, which slowly solidified on agitation with a glass rod. The dried solid preparation of carbobenzoxy- S-benzylcysteinyl- Sbenzylcysteine was recrystallized from ethyl acetate solution by careful addition of petroleum ether. The yield was 355 g.; m.p. 152” (corr.); rag5 = -50.6” (2% in acetone). Anal. CIH3006N2S2 : Calcd. C 62.4, H 5.6, N 5.2; found C 62.2, H 5.7, . N 5.3. Carbobenzoxy- S-benzyl-n-cysteinyl- S-benzyl-n-cysteine was similarly prepared in a yield of 85%. M.p. 111’ (corr.); [a]:’ = -4.1” (2% in acetone). Found C 62.2, H 5.8, N 5.3. The yield of carbobenzoxy-Xbenzyl-n-cysteinyl- S-benzyl-L-cysteine was 84 % of the theoretical; m.p. 110” (corr.); [LY]:~ = +4.6 (2% in acetone). Found C 62.1, H 5.7, N 5.4. S-Benzyl-L-cysteinyl-S-benzyh-cyst&e The carbobenzoxy group was removed by the general procedure of Ben-Ishai and Berger (7). A saturated solution of dry HBr in glacial acetic acid in an amount of 1.77 1. was added to 350 g. of carbobenzoxyS-benzyl-n-cysteinyl-S-benzyl-n-cysteine. The mixture was allowed to stand at 25” with occasional shaking for 1 hr., and then treated with a large volume of dry ether. The dipeptide hydrobromide which separated was filtered off, washed with dry ether, dissolved in dilute HBr solution, and precipitated by addition of dilute ammonia solution. The free peptide was filtered off and further purified by suspending in 20 1. of 30 % ethanol and bringing into solution by careful addition of dilute ammonia. From this solution, the X-benzyl-n-cysteinyl-S-benzyl-Lcysteine was crystallized by addition of dilute KC1 with stirring. The recrystallization procedure was repeated. The final yield was 213 g.; m.p. 170-172” (corr.) ; [a]E6 = -14.1’ (2% in 1 N NaOH).
POLYCYSTEINE
PEPTIDES
,4ND
PROTEINS.
I
209
Anal. CZ0HU103N2S2 : Calcd. C 59.3, H 6.0, N 6.9; found C 59.7, H 5.9, N 7.1. When ether was added as above to the HBr-glacial acetic acid solution of S-benzyl-n-cysteinyl-S-benzyl-n-cysteine no precipitation of the product occurred. The solution was therefore treated with a little ether followed by addition of an excess of petroleum ether. On chilling to 110” an oil separated which was washed several times by decantation with a 1:3 ether-petroleum ether mixture. The originally oily dipeptide hydrobromide was converted to the crystalline, free dipeptide, and was recrystallized twice as described above. The yield of S-benzyl-n-cysteinyl-S-benzyl-n-cysteine was 78 % of the theoretical; m.p. 184-186” (corr.); [c& = -1.8” (0.5% in 1 N NaOH). Found C 59.3, H 6.2, N 7.0. The yield of the enantiomorphic S-benzyl-n-cysteinyl-S-benzyl-Lcysteine similarly prepared was 76 % of the theoretical. M.p. 183-186” (corr.); [c&’ = +1.5” (0.5 7o in 1 N NaOH). Found C 59.5, H 6.2, N 7.0. L-Cystinyl-L-cystine Removal of the S-benzyl groups was accomplished by the use of sodium in liquid ammonia solution (8). Both carbobenzoxy and S-benzyl groups are simultaneously removable by this single reductive step, but when employed with carbobenzoxy-S-benzyl-L-cysteinyl-S-benzylL-cysteine, the final product was amorphous or gummy in character. The removal of the two groups was therefore accomplished in two successive stages. To a solution of 15 g. of S-benzyl-L-cysteine-S-benzyl-L-cysteine in 500 ml. of liquid ammonia, 4.5 g. of sodium was added in small pieces. The blue color was discharged by addition of 13 g. of ammonium sulfate, and the ammonia was allowed to evaporate. The solid residue was dissolved in 450 ml. of cold 0.7 N HzS04 , and a small amount of insoluble material removed by filtration. Ninety milliliters of mercuric sulfate solution (9) was added with stirring, the resulting precipitate removed by centrifugation, and washed with oxygen-free water. Mercury ion was removed with HZS, and the gas removed by distillation in vacua. The pH of the solution was ajdusted to 6.5 by addition of baryta, the BaS04 removed by filtration, and the filtrate treated with a stream of air until the nitroprusside reaction disappeared. The clear solution was condensed in vucuo to a small volume, and the crystalline precipitate of L-cystinyl-n-cystine filtered off and washed with cold water. It was
210
NOBUO
IZUMIYA
AND
JESSE
P. GREENSTEIN
dissolved in hot water, filtered from a small amount of insoluble material, and the filtrate condensed in vacua. The peptide appeared as long prisms in a yield of 2.2 g., or about 25% of the theoretical; [& = -31.0” (1% in 1 N HC1).3 On drying for 2 hr. at 100” and 1 mm. Hg pressure, the peptide lost 8.0 % in weight; calculated for two molecules of water of crystallization, 7.5 %. Anal. CEHX,O~N& : Calcd. C 32.4, H 4.5, N 12.6, S 28.8; found C 32.2, H 4.6, N 12.5, S 28.6. The dihydrochloride was obtained by successive addition of ethanol, acetone, and ether to the solution of the peptide in a slight excess of aqueous HCl; [aIt = -24.6’ (1% in 1 N HCl). On drying at 100” in vacua, the salt lost 7.1% in weight ; calculated for two molecules of water of crystallization, 6.5 %. And. CLH~O~N& + 2HCl: Calcd. N 10.8, Cl 13.7; found N 10.9, Cl 14.0. A second preparation of the n-cystinyl-n-cystine gave [a]: = -32.1” (1% in 1 N HCl). Both preparations gave a single ninhydrin spot on the paper chromatogram (phenol solvent) with R, = 0.49. Hydrolysis of the peptide in 2 N HCI for 2 hr. yielded n-cystine in about 70% yield with [cw]:’ = -210” (1% in 1 N HCl). Calcd. N 11.7, found 11.7. The Na-liquid NH3 procedure described above was repeated with 15 g. of S-benzyl-n-cysteinyl-S-benzyl-L-cysteine except that for the oxidation step the solution was first brought to pH 6.5 with baryta (the BaSOd filtered off), and then to pH 8.5 with ammonia, and finally aerated until disappearance of the nitroprusside reaction occurred. The resulting n-cystinyl-n-cystine was crystallized from water as above, and yielded 2.7 g. in the form of long prisms. This preparation of L-cystinyl-ncystine also contained two molecules of water of crystallization, for on drying at 100” in vucue, it lost 7.7% in weight. Found C 32.4, H 4.5, N 12.6, S 29.0. The air-dried preparation gave an [c$,’ = -58.7” (1% in 1 N HCl), and yielded a dihydrochloride salt which crystallized with four molecules of water of crystallization; [&’ = -45.6” (1% in 1 N HCl). On drying at 100’ in vacua it lost 11.3 % in weight; calculated for four molecules of water of crystallization, 12.2 %. Found N 11.1, Cl 13.9. *The mother liquors from the crystallization of the various cystinylcystine preparations yielded on evaporation a yellowish gum, which on treatment with ethanol was converted either into an amorphous solid or a rubbery, elastic mass. Paper chromatography in 80% phenol of samples of these ill-defined materials showed the presence of a large number of ninhydrin spots. It is probable that these mixtures represent various polymeric products.
POLYCYSTEINE
PEPTIDES
.4ND
PROTEINS.
I
211
Three other preparations of n-cystinyl-n-cystine obtained as above (by oxidation at pH 8.5) gave [cy]z6values of -56.2”, -52.7’, and -46.0” (1% in 1 N HCl). Each of the four preparations showed two ninhydrin spots on the paper chromatogram (phenol solvent), one a major spot with Rf = 0.20, the other a relatively faint spot with Rf = 0.49. The intensity of the latter spot appeared to vary inversely with the absolute values of [a], for the preparations, that for the preparation with [cy]:’ = -58.7” being nearly indistinct, whereas that for the preparation with [cz]~~= -46.0” was clearly visible. Attempts to remove the component with R/ = 0.49 by repeated cyrstallizations from water were unsuccessful. That the lower values of [a], were not due to appreciable racemization was indicated by the fact that the preparations with [a], = -58.7” and -52.7” both yielded n-cystine on acid hydrolysis with [cr]:’ = -212” (1% in 1 N HCI). Found N 11.5. The RI value for cystine in phenol was 0.15. It is not improbable that the contaminant with R, = 0.49 is that n-cystinyl-n-cystine ordinarily obtained by oxidation at pH 6.5. Dibenzoyl-L-cystinyh-cystines The dibenzoyl derivatives of the L-cystinyl-n-cystine preparation with [c& = -31.0” (by oxidation at pH 6.5), and of the n-cystinyl-ncystine preparation with [aID = -58.7” (by oxidation at pH 8.5), were prepared as described (1). For the former compound, [a]:’ = -48.0” (0.5% in 0.05 N NaOH); m.p. 220” (corr.); found N 8.7, S 19.5. For the latter compound, [cy]i6 = -35.2” (0.5 % in 1 IV NaOH) ; m.p. 218” (corr.); found N 8.5, S 19.5. Calculated N 8.5, S 19.7. L-Cystinyl-mcystine
and D-Cystinyh-cystine
The compounds were prepared by oxidation at pH 6.5 and at pH 8.5 as above. However, they were much more insoluble than the n-cystinyln-cystine preparations, and during the aeration procedure at pH 6.5, they crystallized in the form of fine needles. In all cases they were crystallized twice from dilute HCl solutions by careful addition of ammonia water. The air-dried preparations possessed no water of crystallization. The yields ranged from 1.4 to 2.6 g. of peptide from 10 g. of starting material. For n-cystinyl-n-cystine obtained by oxidation at pH 6.5: [(Y]:~ = - 187”; found C 32.1, H 4.8, N 12.4, S 28.5; for that obtained by oxidation at pH 8.5: [a]:’ = -191”; found C 32.4, H 4.8, N 12.4, S 28.4. For n-cystinyl-n-cystine obtained by oxidation at pH 6.5: [a]iB = +190°;
212
NOBTJO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
found C 32.1, H 4.8, N 12.4, S 28.5; for that obtained by oxidation at pH 8.5: [cy]t6= +195”; found C 32.0, H 4.8, N 12.6, S 28.5. The rotations in all caseswere measured at 1% in 1 N HCl solution. All of the peptides yielded cystine on HCl hydrolysis with no measurable optical rotation in HCl solution. Found, respectively, N values of 11.6, 11.5, 11.7, and 11.7. The four peptide preparations all yielded a single ninhydrin spot on the paper chromatogram with R, = 0.16 for each (phenol solvent). Susceptibility to L-Amino Acid Oxidase of the Cystines Obtained by Hydrolysis The optically inactive cystine preparations obtained by hydrolysis of the four isomeric peptides of cystinylcystine described above were treated with Crotolus adamunteus snake venom L-amino acid oxidase [cf. (lo)], and compared with the results of similar treatment of n-cystine, r-m-cystine, and meso-cystine. The meso-cystine was a generous gift by Dr. Vincent du Vigneaud to whom we are indebted for this courtesy. The results of this study, given in terms of initial rates of oxidation of the substrates, are described in Table I. The oxidative rates for n-cystine, nn-cystine, and the optically inactive cystines obtained by hydrolysis of the peptides appeared to be very nearly the same for all of these compounds. In contrast, the susceptibility of meso-cystine to the action Susceptibility
of Cystine
TABLE I Preparations to Crotalus
adamenteus Venoms
Preparation
0, consumed/hr./mg.
N
pmolcs
n-Cystine nn-Cystine Optically inactive cystine cystine* Optically inactive cystine cystine” Optically inactive cystine cystine* Optically inactive cystine cystine” meso-Cyatine
from hydrolyeate
of n-cystinyl-n-
60 65 69
from hydrolyzate
of n-cystinyl-n-
57
from hydrolyzate
of n-cystinyl-n-
53
from hydrolyzate
of n-cystinyl-n-
66 5
0 Mixtures composed of 1.2-1.4 mg. of n-cystine or 2.3-2.7 mg. of DL- or mesocystine in 2.0 ml. of 0.2 M tris buffer at pH 7.2 plus 0.1 ml. catalase solution. The side arm contained 0.3 ml. (0.7 g.) snake venom solution in water. * n-Cysteinyl-n-cysteine or n-cysteinyl-n-cysteine oxidized at pH 8.5. c n-Cysteinyl-n-cysteine or n-cysteinyl-n-cysteine oxidized at pH 6.5.
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
213
of the oxidase was very much lower. The results suggest that the optically inactive cystines obtained from the corresponding cystinylcystine peptides by acid hydrolysis are at least largely DL. The data by themselves however would not exclude the presence of small amounts of mesocystine. Attempts to carry the enzymatic oxidation of the cystines to completion failed, for the products of the reaction appeared to be inhibitory to the oxidase. In view of the considerable difference in the susceptibility of DLcystine and of meso-cystine to the oxidase, it is interesting to note that the action of crude rat liver homogenates containing an active cystine desulfurase was equally effective on both substrates. After 2 hr. of incubation at 37” with a 1:3 aqueous liver homogenate (II), 25 pmoles of each substrate (based on the x,-component) yielded 11 pmoles of ammonia N. Enzymatic Susceptibility
of the Cystinylcystines
The cystinylcystine peptides were subjected to the action of renal acylase I (12) and of a partially purified renal aminopeptidase preparation (13) under conditions previously described for the study of these enzymes [cf. (12-15)].4 There appeared to be no difference between the isomeric L-cystinyl-L-cystine peptides in respect to their susceptibility to each of the enzymes employed (Table II). They were quite slowly attacked by acylase I, and although readily susceptible to the action of aminopeptidase, their hydrolytic rates with the latter enzyme were considerably lower than that of glycyl-D-alanine. Still lower were the hydrolytic rates of L-cystinyl-n-cystine and n-cystinyl-L-cystine, which were nearly the same for the two substrates. These findings areof interest in regard to the specificity of the aminopeptidase. This enzyme has been shown to hydrolyze corresponding glycyl-L- and glycyl-n-amino acids equally well; however, it effects the hydrolysis of L-alanyl+alanine at twice the rate of L-alanyl-n-alanine (13). In the case of the cystinylcystine peptides, it catalyzes the hydrolysis of L-cystinyl-L-cystine at nearly ten times the rate of L-cystinyl-n-cystine (Table II). The nearly equal susceptibility of the optically enantiomorphic L-cystinyl-n-cystine and n-cystinyl-L-cystine is perhaps surprising, and suggestive of the 4 The aminopeptidase preparation was not subjected t,o the customary final fractionation with ammonium sulfate [cf. (13)j. On the basis of its action on glycylo-alanine it is nearly 500 times as active as the original hog kidney homogenat,e. We are indebted to Dr. E. Ohmurn for this preparation.
214
NOBUO IZUMIyA
AND JESSE P. GREENSTEIN TABLE
Susceptibility
of the Cystinylcystine
II Peptides
to Enzymatic Hydrolytic
Hydrolysis” rates by action of b
Substrate Acylase I
n-Cyatinyl-n-cystine n-Cystinyl-n-cystine L-Cystinyl-n-cystine n-Cystinyl-L-cystine Clycyl-n-alanine
(by (by (by (by
oxidation oxidation oxidation oxidation
at at at at
pH pH pH pH
6.5) 8.5) 6.5) 6.5)
1.0 1.3 0 0 0
Aminopeptidase
11,800 10,200 1,460 2,080 82,800
4 Digests composed for acylase I studies of 1 ml. enzyme in water, 1 ml. of 0.1 25 pmoles of substrate. Same digests for aminopeptidase studies except that buffer solution was 0.117 M borate at pH 8.1. Substrate and enzyme blanks were negligible. * Hydrolytic rates in terms of micromoles peptide bonds hydrolyzed/hr./mg. of protein N.
M phosphate buffer at pH 7.0, and 1 ml. containing
need for further investigations on aminopeptidase with analogous types of optically isomeric substrates.6 The resistance of the latter two cystinylcystines to the action of acylase I is not unexpected [cf. (15)] in view of the high order of resistance of peptides containing a n-amino acid residue to the action of this enzyme. ACKNOWLEDGEMENTS We are indebted to Mr. Clyde M. Otey for the enzymatic Robert J. Koegel and his staff for the elemental analyses.
data, and to Mr.
SUMMARY
L-Cystinyl-n-cystine, L-cystinyl-n-cystine, and n-cystinyl-n-cystine were synthesized by the mixed anhydride procedure involving the reaction of carbobenzoxy-S-benzylcysteine and S-benzylcysteine ester in the presence of isobutylchlorocarbonate and triethylamine, followed by the successive removal of ester, carbobenzoxy, and X-benzyl groups, and by the oxidation of the respective cysteinylcysteines at pH 6.5 and pH 8.5 to the corresponding cystinylcystines. The peptides were all crystalline. 5 In the presence of excess aminopeptidase, and over a 24-hr. period of incubation, all of the cystinylcystine peptides studied were hydrolyzed to lOO’% as revealed by manometric ninhydrin measurements. This is an additional check on the purity of these compounds.
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
215
The L-cystinyl-n-cystine and D-cystinyl-L-cystine, except for their optical enantiomorphism, appeared to be identical and independent of the pH at which they were formed by oxidation. They yielded optically inactive cystine on HCl hydrolysis, the greater part of which appeared to be nL-cystine. m-Cystine could be distinguished from meso-cystine by the considerably greater susceptibility of the former to the action of snake venom L-amino acid oxidase, although both forms were nearly equal in their susceptibility to cystine desulfurase. The L-cystinyl-L-cystine formed by oxidation at pH 6.5 appeared by several criteria to be different from that formed by oxidation at pH 8.5, although their elemental analyses were practically identical, and it is possible that, the conditions of oxidation led to configurational isomers. Both forms on HCl hydrolysis yielded pure L-cystine. The dihydrochloride salts and the dibenzoyl derivatives prepared from the two forms of L-cystinyl-L-cystine showed in some characteristics their origin from different parent compounds, although in these cases too, the elemental analyses were practically identical. All of the cystinylcystine peptides were completely and quantitatively hydrolyzed by renal aminopeptidase. The hydrolytic rates for the two r,-cystillyl-L-cystine forms mere practically the same, and considerably higher than the nearly equal values for L-cystinyl-n-cystine and D-cyst’inyl-L-cystine. Renal acylase I acted very slowly on the former, and not, at all on the latter peptides. REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
GREEXSTEIN, J. l’., J. Biol. Chem. 118, 321; 121, 9 (1937). VAUC:BAN, T. R., JR., AND OSATO, R. L., J. Am. Chem. Sot. 73, 5553 (1951). VAIX:IIAX, T. It., JR., AND OSATO, R. L., J. Am. Chem. Sot. 74, 676 (1952). Vl(:NI;.4vr), V. I)U,RESSLER,C., SWAN,J. M.,ROBERTS, C.W.,KATROYANNIR, 1'. G., AND GORDOh', S., J. Am. Chem. Sot. 75, 4879 (1953). ~IAZ~INGTOX,(:. R., AND R/IEAD, ‘r. H., Blochem. J. 30, 1598 (1936). HARINCITON, C. R., AND PITT-RIVERS, R. V., Biochem. J. 38, 417 (1944). HEN-ISHAI, D., AP;U BERGER, A., J. Org. Chem. 17, 1564 (1952). LORINC:, IT. S., AXI) VIGNEAUD, V. DIJ, J. Biol. Uwm. 111, 385 (1935). Kk:sr)AI I,, 1,:. C‘., MCKENZIE, B. F., ASD hksoN, H. L., J. Bid. Chem. 84, 657 (lY29). GREEBSTEIN, J. P., BIRNBAUM, S. &I., AND OTEY, RI. C., J. Bid. Chem. 204, 307 (1953). GREENSTEIN, J. P., AND LEUTHARDT, F.M., J.h’atZ. Cancer Inst. 6,197 (196). BIRNRAUM, S. hf., LEVINTOW, L., KINGSLEY, R. B., AND GREENSTEIN, J. l’., J. Niol. Chem. 194, -155 (1952).
216
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
13. ROBINSON, D. S., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 202, 1 (1953). 14. RAO, K. R., BIRNBAUM, S. M., KINGSLEY, R. B., AND GREENSTEIN, J. P., J.
Biol. Chem. 198, 507 (1952). 15. RAO, K. R., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 203, 1 (1953).
NatiorLal (‘ancer Ilutitute, Bethesda, Mar!gland
February
Xatiorlal
10, 1954
INTRODUCTION The oxidative conversion of cysteine to cystine has long been known. What configurations result whan a peptide or a protein containing two or more cysteine residues is oxidized is little known. Some 17 years ago one of the present authors undertook such a study with the synthesis of L-cysteinyl-L-cysteine, which was the first of this type of compounds to be prepared, and which on oxidation yielded the dimeric molecule L-cystinyl-L-cystine (1). The resumption of this investigation has now become possible because of the development of new and more powerful methods of peptide synthesis, and because of the availability of optically pure L- and n-amino acids in quantity, and of new and readily accessible types of physical instruments. The first study in the present series deals with the preparation and properties of optically active cystinylcystine peptides of relatively simple configuration. L-Cystinyl-L-cystine has been synthesized by the following sequence of reactions (1) : dicarbobenzoxy+-cystinyldichloride plus L-cysteinyl ethyl ester + dicarbobenzoxy-L-cystinyl-di(L-cysteinyl ethyl ester) --f L-cysteinyl-L-cysteine ethyl ester ----f anhydro-L-cysteinyl-L-cysteine -+ L-cysteinyl-L-cysteine -+ L-cystinyl-L-cystine. The last-mentioned reaction was accomplished by aerating the ammoniacal solution of cysteinylcysteine at pH 8.5 until the sulfhydryl reaction was negative. The crystalline, oxidized peptide possessed an [cY]~~= -60” in 1 N HCI, and crystallized from water as the dihydrate. Its dihydrochloride salt crystallized as the tetrahydrate. On hydrolysis with HCl, the peptide 1 Fulbright .J:lp:Ul.
and Smith-Mundt
Scholar;
203
on leave
from
Kyushu
University,
204
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
yielded nearly the equivalent amount of L-cystine with [a]E2 = -211’. The structure of the peptide may be tentatively represented by the following isomeric formulations: COOH 1 SCH2CHCONHCHCHk.S
SCHzCHCONHCHCHzS
SCH~CHCONHCHCH~S
SCHZHNHCOCHCHZS
NH2
&Hz
COOH
NH2
COOH
COOH
I
NH2 II
It is difficult to distinguish these two structures experimentally when the residues are both derived from L-cystine. However, if one residue is L-cystine and the other D-cystine, i.e., the dipeptide being either L-cystinyl-D-cystine or its optical enantiomorph, D-cystinyl-L-cystine, hydrolysis of either of these peptides would yield DL-cystine if the structure is I, and meso-cystine if the structure is II. Accordingly, we have prepared L-cysteinyl-D-cysteine and D-cysteinyl-L-cysteine by the mixed anhydride procedure developed by Vaughan and Osato (2, 3), and oxidized these products with air at pH 6.5 and 8.5 to the corresponding cystinylcystines. The same procedure was employed to synthesize L-cystinyl-L-cystine for comparison with the earlier preparation described above. The present method involved essentially the following reactions : carbobenzoxy- S-benzylcysteine plus S-benzylcysteine ethyl ester in presence of isobutylchlorocarbonate and triethylamine -+ carbobenzoxy- S-benzylcysteinyl- S-benzylcysteine ethyl ester -+ carbobenzoxy-S-benzylcysteinyl-S-benzylcysteine --+ S-benzylcysteinyl-S-+ cysteinylcysteine -+ cystinylcystine. That no benzylcysteine apparent racemization occurred in the first step of this procedure was shown by comparison of the optical rotation values of the coupling product with those of the same product obtained by the azide method. All of the cystinylcystine preparations were crystalline. L-CystinylD-cystine whether prepared by oxidation at pH 6.5 or pH 8.5 possessed the same value of specific optical rotation which was equal and opposite to that of the D-cystinyl-L-cystines similarly prepared. All of these isomers gave a single ninhydrin spot on the paper chromatogram with Rf = 0.16 in phenol.2 On acid hydrolysis, they yielded optically inactive cystine. Probable identification of these hydrolytic products as DL-cystine 2 In all cases SOo/, phenol
was employed
with Whatman
No. 1 paper.
POLYCYSTEINE PEPTIDES AND PROTEINS. I
205
was based on the observation that they were oxidized by snake venom n-amino acid oxidase at the same rate as a known specimen of nn-cystine, a rate considerably in excess of that noted with a known specimen of meso-cystine. This observation does not exclude the presence of relatively small amounts of meso-cystine in the optically inactive cystine hydrolytic products, but it does suggest that the greater part at least of these particular cystinylcystine peptides may exist in configuration I. In contrast with the case of the above-mentioned optically enantiomorphic cystinylcystines, the properties of n-cystinyl-n-cystine appeared to be related to the pH at which it was prepared by oxidation of the preceding n-cysteinyl-n-cysteine. Thus, when this pH was maintained at 6.5, the product had an [(Y]:~ = -31” in HCl, it yielded a single ninhydrin spot on the paper chromatogram with R, = 0.49 in phenol, a dihydrochloride was prepared which crystallized as the dihydrate, and finally, the dibenzoyl derivative possessed an [(Y]:’ = -48.0” in NaOH. On the other hand, when the pH at oxidation was maintained at 8.5, the product had an [&? = -58.7” in HCl, it yielded two ninhydrin spots on the paper chromatogram, one of which was relatively intense with Rf = 0.20 whereas the other spot, with Rf = 0.49, was very faint, a dihydrochloride was prepared which crystallized as the tetrahydrate, and finally, the dibenzoyl derivative possessed an [ali = -35.2” in NaOH. Thus, the dibenzoyl derivatives of the two n-cystinyl-n-cystine preparations showed by their differences in specific rotation values their derivation from two different compounds. Both n-cystinyl-n-cystines crystallized from water as the dihydrate, and possessed nearly identical elemental analytical values. Both yielded n-cystine on HCl hydrolysis with [(Y]:~ = -210” to -212” in HCl. It is difficult to avoid the impression that the two n-cystinyl-n-cystines are structural isomers, represented perhaps by configurations I and II above. Both n-cystinyl-n-cystines are nearly equally susceptible to the action of peptidases. Based upon the rotation and other data described, the L-cystinyl-t-cystine prepared in the present instance by oxidation at pH 8.5 is probably identical with that described earlier (l), and which had also been prepared by oxidation at this pH. The reason for the effect of pH on the configuration of the disulfide forms obtained by oxidation of n-cysteinyl-n-cysteine, as contrasted with the absence of such effect at the pH values studied on the configuration of the disulfide forms obtained by oxidation of n-cysteinyl-n-cysteine or n-cysteinyl-L-cysteine, is not apparent at the present time. Formation of a monomeric cyclic
206
N~BUO
IZUMIYA
AND
JESSE
p.
GF~EENSTEIN
peptide by combination of each terminal sulfur atom to form a single disulfide link would produce an eight-membered ring, which in the case of n-cystinyl-n-cystine or n-cystinyl-n-cystine would lead on HCl hydrolysis to the formation of pure meso-cystine. The available evidence is not in favor of these considerations. Molecular weight determinations reported earlier for n-cystinyl-n-cystine (1) are compatible with the 16-membered ring represented by the formulas above. This does not exclude the possibility that the other cystinylcystines reported herein may be cyclic peptides of still higher molecular weight. It is not inconceivable that stable rotational isomers may exist among these cyclic disulfide compounds. The crystalline disulfide peptides isolated after oxidation of the various cysteinylcysteines account for only about 2530% of the theoretical yield, and the remainder appears to be ill-defined material probably arising through linear polymerization. Further studies will obviously be necessary to determine the configurations brought aobut by the gentle oxidation under various experimental conditions of polycysteine peptides of biological interest. The importance of such studies has been emphasized by the brilliant synthesis of oxytocin by du Vigneaud and his collaborators (4). In this case the oxidation (at pH 6.5) of two L-cysteinyl residues separated by four amino acid residues along a peptide chain led to the formation of a monomeric cyclic peptide. On a still larger molecular scale, many proteins are known to contain several sulfhydryl groups on their surface, the exposure of which to oxidizing conditions would lead to decided changes in their molecular configurations and sizes. Such studies involving both synthetic polycysteine peptides and proteins are in progress. EXPERIMENTAL
Carbobenzoxy-S-benzylcysteinyl-S-benzykysteink
Ethyl Ester
A solution of 346 g. of carbobenzoxy-S-benzyl-L-cysteine (5, 6) and 139 ml. of triethylamine in 3 1. of toluene was chilled to -5” and treated with 131 ml. of isobutylchlorocarbonate. After 10 min. of standing, a cold solution of 276 g. of S-benzyl-L-cysteine ethyl ester hydrochloride and 139 ml. of triethylamine in 2 1. of chloroform was added, and the mixture allowed to stand overnight at 25”. One additional liter of chloroform was added, and the mixture was washed successively with water, bicarbonate solution, and water, and finally dried over anhydrous sodium sulfate. The filtrate was condensed in pacuo to low bulk, and the residual solution treated with petroleum ether. The carbobenzoxy-S-benzyl-n-
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
207
cysteinyl-S-benzyl-L-cysteine ethyl ester which precipitated was filtered off, and recrystallized from ethanol. The yield was 382 g., or 67 % of the theoretical. M.p. 105” (corr.) ; [a]:’ = -55.3’ (2 % in acetone): Anal. C30H3406N2S2: Calcd. C 63.6, H 6.1, N 4.9; found C 63.5, H 6.0, N 5.0. Carbobenzoxy-S-benzyl-L-cysteinyl-S-benzyl-n-cysteine ethyl ester was prepared in the same manner. The yield was 66 % of the theoretical. M.p. 103” (corr.); [ali = +5.1” (2% in acetone). Found C 63.9, H 6.3, N 5.0. Carbobenzoxy-X-benzyl-n-cysteinyl-S-benzyl-L-cysteine ethyl ester similarly prepared in 68 % yield possessed a m.p. of 103” (corr.) and [a];’ = -4.7” (2% in acetone). Found C 63.4, H 6.1, N 4.9. Two of the compounds were also prepared by the azide procedure as follows. A dry ether solution of carbobenzoxy-S-benzyl+-cysteinylazide from 33.6 g. of carbobenzoxy-S-benzyl-L-cysteinylhydrazide (5, 6) w-as mixed with a dry ether solution of S-benzyl-L-cysteine ethyl ester prcpared from 31.2 g. of the hydrochloride (5, 6), and the reaction mixture was allowed to stand overnight at 25”. The product separated in the form of long needles. After further chilling, it was filtered off, washed I\-ith cold, dry ether, and recrystallized from ethanol. The yield \vas 28.2 g.; m.p. 105” (corr.); [ar]i6 = -55.2” (2% in acetone). Found C 63.7, H 6.2, N 5.0. Carbobenzoxy-S-benzyl-D-cysteinyl-S-benzyl-L-cysteine ethyl ester Tras similarly prepared from carbobenzoxy-S-benzyl-n-cysteinylhydrazide and S-benzyl-L-cysteine ethyl ester hydrochloride. The reaction product however did not precipitate from the ether solut’ion, and consequently the latter was washed successively with dilute HCl, biparbonate solution, and water, and finally dried over anhydrous sodium sulfate. The filtrate was concentrated in raczlo, and, after adding petroleum ether, the product was precipitated, filtered, and recrystallized from ethanol as thin needles. The yield was 58% of the theoretical. M.p. 103” (corr.); [c&’ = -4.9” (2% in acetone). Found C 63.4, H 6.1, N Fj.0. Both mixed anhydride and azide procedures evidently led to the same respective products with nearly identical rotation values. The derivatives of S-benzyl-D-cysteine, not previously reported, were prepared as described for the corresponding L-forms (5, 6). These included S-benzyl-ncysteino et#hyl ester hydrochloride: m.p. 163” ((aon.); [cx]~ = +28. I” (2 % in ethanol); N calcd. 5.1, found 5.1; c~arbobenzoxy-S-benzyl-n-
208
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
cysteine: m.p. 95’ (corr.); [ali6 = +45.1’ (2 % in acetone); N calcd. 4.1, found 4.1; and carbobenzoxy- S-benzyl-n-cysteinylhydrazide : m.p. 135” (corl.); [L-Z]:’= -4.3” (2 per cent in glacial acetic acid); N calcd. 11.7, found 11.5. Carbobenxoxy-S-benzylcysteinyl-S-benzylcy$eine A mixture of 454 g. of carbobenzoxy- S-benzyl-n-cysteinyl- S-benzyln-cysteine ethyl ester in 4.6 1. of dioxane and 480 ml. of 2 N NaOH was shaken for 5 hr. at 5”. To the clear solution 167 ml. of 6 N HCl was added, the mixture condensed in vacua to a low bulk, and treated with water. An oil appeared, which slowly solidified on agitation with a glass rod. The dried solid preparation of carbobenzoxy- S-benzylcysteinyl- Sbenzylcysteine was recrystallized from ethyl acetate solution by careful addition of petroleum ether. The yield was 355 g.; m.p. 152” (corr.); rag5 = -50.6” (2% in acetone). Anal. CIH3006N2S2 : Calcd. C 62.4, H 5.6, N 5.2; found C 62.2, H 5.7, . N 5.3. Carbobenzoxy- S-benzyl-n-cysteinyl- S-benzyl-n-cysteine was similarly prepared in a yield of 85%. M.p. 111’ (corr.); [a]:’ = -4.1” (2% in acetone). Found C 62.2, H 5.8, N 5.3. The yield of carbobenzoxy-Xbenzyl-n-cysteinyl- S-benzyl-L-cysteine was 84 % of the theoretical; m.p. 110” (corr.); [LY]:~ = +4.6 (2% in acetone). Found C 62.1, H 5.7, N 5.4. S-Benzyl-L-cysteinyl-S-benzyh-cyst&e The carbobenzoxy group was removed by the general procedure of Ben-Ishai and Berger (7). A saturated solution of dry HBr in glacial acetic acid in an amount of 1.77 1. was added to 350 g. of carbobenzoxyS-benzyl-n-cysteinyl-S-benzyl-n-cysteine. The mixture was allowed to stand at 25” with occasional shaking for 1 hr., and then treated with a large volume of dry ether. The dipeptide hydrobromide which separated was filtered off, washed with dry ether, dissolved in dilute HBr solution, and precipitated by addition of dilute ammonia solution. The free peptide was filtered off and further purified by suspending in 20 1. of 30 % ethanol and bringing into solution by careful addition of dilute ammonia. From this solution, the X-benzyl-n-cysteinyl-S-benzyl-Lcysteine was crystallized by addition of dilute KC1 with stirring. The recrystallization procedure was repeated. The final yield was 213 g.; m.p. 170-172” (corr.) ; [a]E6 = -14.1’ (2% in 1 N NaOH).
POLYCYSTEINE
PEPTIDES
,4ND
PROTEINS.
I
209
Anal. CZ0HU103N2S2 : Calcd. C 59.3, H 6.0, N 6.9; found C 59.7, H 5.9, N 7.1. When ether was added as above to the HBr-glacial acetic acid solution of S-benzyl-n-cysteinyl-S-benzyl-n-cysteine no precipitation of the product occurred. The solution was therefore treated with a little ether followed by addition of an excess of petroleum ether. On chilling to 110” an oil separated which was washed several times by decantation with a 1:3 ether-petroleum ether mixture. The originally oily dipeptide hydrobromide was converted to the crystalline, free dipeptide, and was recrystallized twice as described above. The yield of S-benzyl-n-cysteinyl-S-benzyl-n-cysteine was 78 % of the theoretical; m.p. 184-186” (corr.); [c& = -1.8” (0.5% in 1 N NaOH). Found C 59.3, H 6.2, N 7.0. The yield of the enantiomorphic S-benzyl-n-cysteinyl-S-benzyl-Lcysteine similarly prepared was 76 % of the theoretical. M.p. 183-186” (corr.); [c&’ = +1.5” (0.5 7o in 1 N NaOH). Found C 59.5, H 6.2, N 7.0. L-Cystinyl-L-cystine Removal of the S-benzyl groups was accomplished by the use of sodium in liquid ammonia solution (8). Both carbobenzoxy and S-benzyl groups are simultaneously removable by this single reductive step, but when employed with carbobenzoxy-S-benzyl-L-cysteinyl-S-benzylL-cysteine, the final product was amorphous or gummy in character. The removal of the two groups was therefore accomplished in two successive stages. To a solution of 15 g. of S-benzyl-L-cysteine-S-benzyl-L-cysteine in 500 ml. of liquid ammonia, 4.5 g. of sodium was added in small pieces. The blue color was discharged by addition of 13 g. of ammonium sulfate, and the ammonia was allowed to evaporate. The solid residue was dissolved in 450 ml. of cold 0.7 N HzS04 , and a small amount of insoluble material removed by filtration. Ninety milliliters of mercuric sulfate solution (9) was added with stirring, the resulting precipitate removed by centrifugation, and washed with oxygen-free water. Mercury ion was removed with HZS, and the gas removed by distillation in vacua. The pH of the solution was ajdusted to 6.5 by addition of baryta, the BaS04 removed by filtration, and the filtrate treated with a stream of air until the nitroprusside reaction disappeared. The clear solution was condensed in vucuo to a small volume, and the crystalline precipitate of L-cystinyl-n-cystine filtered off and washed with cold water. It was
210
NOBUO
IZUMIYA
AND
JESSE
P. GREENSTEIN
dissolved in hot water, filtered from a small amount of insoluble material, and the filtrate condensed in vacua. The peptide appeared as long prisms in a yield of 2.2 g., or about 25% of the theoretical; [& = -31.0” (1% in 1 N HC1).3 On drying for 2 hr. at 100” and 1 mm. Hg pressure, the peptide lost 8.0 % in weight; calculated for two molecules of water of crystallization, 7.5 %. Anal. CEHX,O~N& : Calcd. C 32.4, H 4.5, N 12.6, S 28.8; found C 32.2, H 4.6, N 12.5, S 28.6. The dihydrochloride was obtained by successive addition of ethanol, acetone, and ether to the solution of the peptide in a slight excess of aqueous HCl; [aIt = -24.6’ (1% in 1 N HCl). On drying at 100” in vacua, the salt lost 7.1% in weight ; calculated for two molecules of water of crystallization, 6.5 %. And. CLH~O~N& + 2HCl: Calcd. N 10.8, Cl 13.7; found N 10.9, Cl 14.0. A second preparation of the n-cystinyl-n-cystine gave [a]: = -32.1” (1% in 1 N HCl). Both preparations gave a single ninhydrin spot on the paper chromatogram (phenol solvent) with R, = 0.49. Hydrolysis of the peptide in 2 N HCI for 2 hr. yielded n-cystine in about 70% yield with [cw]:’ = -210” (1% in 1 N HCl). Calcd. N 11.7, found 11.7. The Na-liquid NH3 procedure described above was repeated with 15 g. of S-benzyl-n-cysteinyl-S-benzyl-L-cysteine except that for the oxidation step the solution was first brought to pH 6.5 with baryta (the BaSOd filtered off), and then to pH 8.5 with ammonia, and finally aerated until disappearance of the nitroprusside reaction occurred. The resulting n-cystinyl-n-cystine was crystallized from water as above, and yielded 2.7 g. in the form of long prisms. This preparation of L-cystinyl-ncystine also contained two molecules of water of crystallization, for on drying at 100” in vucue, it lost 7.7% in weight. Found C 32.4, H 4.5, N 12.6, S 29.0. The air-dried preparation gave an [c$,’ = -58.7” (1% in 1 N HCl), and yielded a dihydrochloride salt which crystallized with four molecules of water of crystallization; [&’ = -45.6” (1% in 1 N HCl). On drying at 100’ in vacua it lost 11.3 % in weight; calculated for four molecules of water of crystallization, 12.2 %. Found N 11.1, Cl 13.9. *The mother liquors from the crystallization of the various cystinylcystine preparations yielded on evaporation a yellowish gum, which on treatment with ethanol was converted either into an amorphous solid or a rubbery, elastic mass. Paper chromatography in 80% phenol of samples of these ill-defined materials showed the presence of a large number of ninhydrin spots. It is probable that these mixtures represent various polymeric products.
POLYCYSTEINE
PEPTIDES
.4ND
PROTEINS.
I
211
Three other preparations of n-cystinyl-n-cystine obtained as above (by oxidation at pH 8.5) gave [cy]z6values of -56.2”, -52.7’, and -46.0” (1% in 1 N HCl). Each of the four preparations showed two ninhydrin spots on the paper chromatogram (phenol solvent), one a major spot with Rf = 0.20, the other a relatively faint spot with Rf = 0.49. The intensity of the latter spot appeared to vary inversely with the absolute values of [a], for the preparations, that for the preparation with [cy]:’ = -58.7” being nearly indistinct, whereas that for the preparation with [cz]~~= -46.0” was clearly visible. Attempts to remove the component with R/ = 0.49 by repeated cyrstallizations from water were unsuccessful. That the lower values of [a], were not due to appreciable racemization was indicated by the fact that the preparations with [a], = -58.7” and -52.7” both yielded n-cystine on acid hydrolysis with [cr]:’ = -212” (1% in 1 N HCI). Found N 11.5. The RI value for cystine in phenol was 0.15. It is not improbable that the contaminant with R, = 0.49 is that n-cystinyl-n-cystine ordinarily obtained by oxidation at pH 6.5. Dibenzoyl-L-cystinyh-cystines The dibenzoyl derivatives of the L-cystinyl-n-cystine preparation with [c& = -31.0” (by oxidation at pH 6.5), and of the n-cystinyl-ncystine preparation with [aID = -58.7” (by oxidation at pH 8.5), were prepared as described (1). For the former compound, [a]:’ = -48.0” (0.5% in 0.05 N NaOH); m.p. 220” (corr.); found N 8.7, S 19.5. For the latter compound, [cy]i6 = -35.2” (0.5 % in 1 IV NaOH) ; m.p. 218” (corr.); found N 8.5, S 19.5. Calculated N 8.5, S 19.7. L-Cystinyl-mcystine
and D-Cystinyh-cystine
The compounds were prepared by oxidation at pH 6.5 and at pH 8.5 as above. However, they were much more insoluble than the n-cystinyln-cystine preparations, and during the aeration procedure at pH 6.5, they crystallized in the form of fine needles. In all cases they were crystallized twice from dilute HCl solutions by careful addition of ammonia water. The air-dried preparations possessed no water of crystallization. The yields ranged from 1.4 to 2.6 g. of peptide from 10 g. of starting material. For n-cystinyl-n-cystine obtained by oxidation at pH 6.5: [(Y]:~ = - 187”; found C 32.1, H 4.8, N 12.4, S 28.5; for that obtained by oxidation at pH 8.5: [a]:’ = -191”; found C 32.4, H 4.8, N 12.4, S 28.4. For n-cystinyl-n-cystine obtained by oxidation at pH 6.5: [a]iB = +190°;
212
NOBTJO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
found C 32.1, H 4.8, N 12.4, S 28.5; for that obtained by oxidation at pH 8.5: [cy]t6= +195”; found C 32.0, H 4.8, N 12.6, S 28.5. The rotations in all caseswere measured at 1% in 1 N HCl solution. All of the peptides yielded cystine on HCl hydrolysis with no measurable optical rotation in HCl solution. Found, respectively, N values of 11.6, 11.5, 11.7, and 11.7. The four peptide preparations all yielded a single ninhydrin spot on the paper chromatogram with R, = 0.16 for each (phenol solvent). Susceptibility to L-Amino Acid Oxidase of the Cystines Obtained by Hydrolysis The optically inactive cystine preparations obtained by hydrolysis of the four isomeric peptides of cystinylcystine described above were treated with Crotolus adamunteus snake venom L-amino acid oxidase [cf. (lo)], and compared with the results of similar treatment of n-cystine, r-m-cystine, and meso-cystine. The meso-cystine was a generous gift by Dr. Vincent du Vigneaud to whom we are indebted for this courtesy. The results of this study, given in terms of initial rates of oxidation of the substrates, are described in Table I. The oxidative rates for n-cystine, nn-cystine, and the optically inactive cystines obtained by hydrolysis of the peptides appeared to be very nearly the same for all of these compounds. In contrast, the susceptibility of meso-cystine to the action Susceptibility
of Cystine
TABLE I Preparations to Crotalus
adamenteus Venoms
Preparation
0, consumed/hr./mg.
N
pmolcs
n-Cystine nn-Cystine Optically inactive cystine cystine* Optically inactive cystine cystine” Optically inactive cystine cystine* Optically inactive cystine cystine” meso-Cyatine
from hydrolyeate
of n-cystinyl-n-
60 65 69
from hydrolyzate
of n-cystinyl-n-
57
from hydrolyzate
of n-cystinyl-n-
53
from hydrolyzate
of n-cystinyl-n-
66 5
0 Mixtures composed of 1.2-1.4 mg. of n-cystine or 2.3-2.7 mg. of DL- or mesocystine in 2.0 ml. of 0.2 M tris buffer at pH 7.2 plus 0.1 ml. catalase solution. The side arm contained 0.3 ml. (0.7 g.) snake venom solution in water. * n-Cysteinyl-n-cysteine or n-cysteinyl-n-cysteine oxidized at pH 8.5. c n-Cysteinyl-n-cysteine or n-cysteinyl-n-cysteine oxidized at pH 6.5.
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
213
of the oxidase was very much lower. The results suggest that the optically inactive cystines obtained from the corresponding cystinylcystine peptides by acid hydrolysis are at least largely DL. The data by themselves however would not exclude the presence of small amounts of mesocystine. Attempts to carry the enzymatic oxidation of the cystines to completion failed, for the products of the reaction appeared to be inhibitory to the oxidase. In view of the considerable difference in the susceptibility of DLcystine and of meso-cystine to the oxidase, it is interesting to note that the action of crude rat liver homogenates containing an active cystine desulfurase was equally effective on both substrates. After 2 hr. of incubation at 37” with a 1:3 aqueous liver homogenate (II), 25 pmoles of each substrate (based on the x,-component) yielded 11 pmoles of ammonia N. Enzymatic Susceptibility
of the Cystinylcystines
The cystinylcystine peptides were subjected to the action of renal acylase I (12) and of a partially purified renal aminopeptidase preparation (13) under conditions previously described for the study of these enzymes [cf. (12-15)].4 There appeared to be no difference between the isomeric L-cystinyl-L-cystine peptides in respect to their susceptibility to each of the enzymes employed (Table II). They were quite slowly attacked by acylase I, and although readily susceptible to the action of aminopeptidase, their hydrolytic rates with the latter enzyme were considerably lower than that of glycyl-D-alanine. Still lower were the hydrolytic rates of L-cystinyl-n-cystine and n-cystinyl-L-cystine, which were nearly the same for the two substrates. These findings areof interest in regard to the specificity of the aminopeptidase. This enzyme has been shown to hydrolyze corresponding glycyl-L- and glycyl-n-amino acids equally well; however, it effects the hydrolysis of L-alanyl+alanine at twice the rate of L-alanyl-n-alanine (13). In the case of the cystinylcystine peptides, it catalyzes the hydrolysis of L-cystinyl-L-cystine at nearly ten times the rate of L-cystinyl-n-cystine (Table II). The nearly equal susceptibility of the optically enantiomorphic L-cystinyl-n-cystine and n-cystinyl-L-cystine is perhaps surprising, and suggestive of the 4 The aminopeptidase preparation was not subjected t,o the customary final fractionation with ammonium sulfate [cf. (13)j. On the basis of its action on glycylo-alanine it is nearly 500 times as active as the original hog kidney homogenat,e. We are indebted to Dr. E. Ohmurn for this preparation.
214
NOBUO IZUMIyA
AND JESSE P. GREENSTEIN TABLE
Susceptibility
of the Cystinylcystine
II Peptides
to Enzymatic Hydrolytic
Hydrolysis” rates by action of b
Substrate Acylase I
n-Cyatinyl-n-cystine n-Cystinyl-n-cystine L-Cystinyl-n-cystine n-Cystinyl-L-cystine Clycyl-n-alanine
(by (by (by (by
oxidation oxidation oxidation oxidation
at at at at
pH pH pH pH
6.5) 8.5) 6.5) 6.5)
1.0 1.3 0 0 0
Aminopeptidase
11,800 10,200 1,460 2,080 82,800
4 Digests composed for acylase I studies of 1 ml. enzyme in water, 1 ml. of 0.1 25 pmoles of substrate. Same digests for aminopeptidase studies except that buffer solution was 0.117 M borate at pH 8.1. Substrate and enzyme blanks were negligible. * Hydrolytic rates in terms of micromoles peptide bonds hydrolyzed/hr./mg. of protein N.
M phosphate buffer at pH 7.0, and 1 ml. containing
need for further investigations on aminopeptidase with analogous types of optically isomeric substrates.6 The resistance of the latter two cystinylcystines to the action of acylase I is not unexpected [cf. (15)] in view of the high order of resistance of peptides containing a n-amino acid residue to the action of this enzyme. ACKNOWLEDGEMENTS We are indebted to Mr. Clyde M. Otey for the enzymatic Robert J. Koegel and his staff for the elemental analyses.
data, and to Mr.
SUMMARY
L-Cystinyl-n-cystine, L-cystinyl-n-cystine, and n-cystinyl-n-cystine were synthesized by the mixed anhydride procedure involving the reaction of carbobenzoxy-S-benzylcysteine and S-benzylcysteine ester in the presence of isobutylchlorocarbonate and triethylamine, followed by the successive removal of ester, carbobenzoxy, and X-benzyl groups, and by the oxidation of the respective cysteinylcysteines at pH 6.5 and pH 8.5 to the corresponding cystinylcystines. The peptides were all crystalline. 5 In the presence of excess aminopeptidase, and over a 24-hr. period of incubation, all of the cystinylcystine peptides studied were hydrolyzed to lOO’% as revealed by manometric ninhydrin measurements. This is an additional check on the purity of these compounds.
POLYCYSTEINE
PEPTIDES
AND
PROTEINS.
I
215
The L-cystinyl-n-cystine and D-cystinyl-L-cystine, except for their optical enantiomorphism, appeared to be identical and independent of the pH at which they were formed by oxidation. They yielded optically inactive cystine on HCl hydrolysis, the greater part of which appeared to be nL-cystine. m-Cystine could be distinguished from meso-cystine by the considerably greater susceptibility of the former to the action of snake venom L-amino acid oxidase, although both forms were nearly equal in their susceptibility to cystine desulfurase. The L-cystinyl-L-cystine formed by oxidation at pH 6.5 appeared by several criteria to be different from that formed by oxidation at pH 8.5, although their elemental analyses were practically identical, and it is possible that, the conditions of oxidation led to configurational isomers. Both forms on HCl hydrolysis yielded pure L-cystine. The dihydrochloride salts and the dibenzoyl derivatives prepared from the two forms of L-cystinyl-L-cystine showed in some characteristics their origin from different parent compounds, although in these cases too, the elemental analyses were practically identical. All of the cystinylcystine peptides were completely and quantitatively hydrolyzed by renal aminopeptidase. The hydrolytic rates for the two r,-cystillyl-L-cystine forms mere practically the same, and considerably higher than the nearly equal values for L-cystinyl-n-cystine and D-cyst’inyl-L-cystine. Renal acylase I acted very slowly on the former, and not, at all on the latter peptides. REFERENCES 1. 2. 3. 4.
5. 6. 7. 8. 9. 10. 11. 12.
GREEXSTEIN, J. l’., J. Biol. Chem. 118, 321; 121, 9 (1937). VAUC:BAN, T. R., JR., AND OSATO, R. L., J. Am. Chem. Sot. 73, 5553 (1951). VAIX:IIAX, T. It., JR., AND OSATO, R. L., J. Am. Chem. Sot. 74, 676 (1952). Vl(:NI;.4vr), V. I)U,RESSLER,C., SWAN,J. M.,ROBERTS, C.W.,KATROYANNIR, 1'. G., AND GORDOh', S., J. Am. Chem. Sot. 75, 4879 (1953). ~IAZ~INGTOX,(:. R., AND R/IEAD, ‘r. H., Blochem. J. 30, 1598 (1936). HARINCITON, C. R., AND PITT-RIVERS, R. V., Biochem. J. 38, 417 (1944). HEN-ISHAI, D., AP;U BERGER, A., J. Org. Chem. 17, 1564 (1952). LORINC:, IT. S., AXI) VIGNEAUD, V. DIJ, J. Biol. Uwm. 111, 385 (1935). Kk:sr)AI I,, 1,:. C‘., MCKENZIE, B. F., ASD hksoN, H. L., J. Bid. Chem. 84, 657 (lY29). GREEBSTEIN, J. P., BIRNBAUM, S. &I., AND OTEY, RI. C., J. Bid. Chem. 204, 307 (1953). GREENSTEIN, J. P., AND LEUTHARDT, F.M., J.h’atZ. Cancer Inst. 6,197 (196). BIRNRAUM, S. hf., LEVINTOW, L., KINGSLEY, R. B., AND GREENSTEIN, J. l’., J. Niol. Chem. 194, -155 (1952).
216
NOBUO
IZUMIYA
AND
JESSE
P.
GREENSTEIN
13. ROBINSON, D. S., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 202, 1 (1953). 14. RAO, K. R., BIRNBAUM, S. M., KINGSLEY, R. B., AND GREENSTEIN, J. P., J.
Biol. Chem. 198, 507 (1952). 15. RAO, K. R., BIRNBAUM, S. M., AND GREENSTEIN, J. P., J. Biol. Chem. 203, 1 (1953).