hlarch 20, 19ti1
1509
COMMUNICATIONS TRANSFORMATION OF POLYMERIC RUTHENIUM(1V) TO T H E MONOMERIC SPECIES ON ION EXCHANGE RESIN
Sir: -4lthough the existence of polymeric forms of ruthenium(1V) had been suspected, Gortsema and Cobble' were able to prepare both the monomeric and polymeric species and study them extensively in perchloric and nitric acid solutions. Ion exchange provided a simple method for transforming the polymeric form to the monomeric. This was done by equilibrating a quantity of low crosslinked Dowex 50&: resin (2 to 47,) with Ru(1V) solution known to contain extensive amounts of polymer and then eluting the resin with 0.1 :?P ceriuni(II1) perchlorate or 1 M perchloric acid. They make no statement as to whether the ion exchange is a separation of RuO++ (the monomer) from solutions of high polymer concentration, or whether there is a transformation of the polymer to the monomer on the resin. From direct observation of the spectrum of Ru(1V) on the resin it can be concluded t h a t an actual transformation of polymer to monomer occurs as i t passes through the ion exchange resin. This is also significant in that i t shows that the polymeric Ru(1V) structure is not extremely stable and t h a t i t can be broken down into monomer by use of rather mild conditions. The spectra were scanned with a Cary Model 14 recording spectrophotometer. One cm. Beckman fused silica cells were fitted with cell spacers to reduce the optical path to 2 mm. By increasing the dynode voltage on the phototube i t was possible to record data down t o 250 m p or less. The ruthenium(1V) perchlorate solutions were prepared using a procedure described by Yaffe and Voight2 and by Niedrach and T e v e b a ~ g h . Gort~ sema and Cobble' determined the absorbancy index, A (1 cm., moles/liter), to be about 700 for the monomeric Ru(IV) and 800 to 1600 for the polymer a t 480 mp. The 0.0025 M ruthenium perchlorate solutions with A480 ranging from 730 to 1000 were stirred with Dowex 50WX2, 200 to 400 mesh, ion exchange resin. After a short period of time the resin containing Ru(1V) was washed repeatedly with distilled water to remove any ruthenium ion not in the resin phase. A portion of t h e loaded resin then was tapped and settled in one of the 2-mm. cells and the spectrum scanned versus a blank of the unloaded resin in the other 2 mm. cell. After equilibration with the resin, no ruthenium could be found in the residual solution. This enabled calculation of the concentration of the ruthenium in the resin phase. Data were were obtained for several determinations on three different preparations, both of (1) F. P. Gortsema, Doctoral thesis, Purdue University, January, 1980; Dissert. Abstracfs, 21, 48 (1960), L. C . Card No. M i c 60-2206. ( 2 ) R . P. Yaffe a n d A. F. Voight, J . A m . Chem. SOL., 74, 2500 (1952). (3) L. W. Niedrach and A. D . Tevebaugh, i b i d . , 73, 2836 (1951).
To
T H E EDITOR
monomeric Ru(IV), = 733, and polymeric Ru(IV), = 1003. T h e results, given in Table I, indicate that the absorbancy index in the resin phase is a constant, independent of the value for the original solutions and approximates the value for the monomeric Ru(1V). TABLE I ABSORBANCY INDEX OF Ru(1V) ON RESIN Sample no.
A. Monomeric Ru(I1.) Expt.
1
1 2
2
3
Average
800 863 785 806 816 764 B. Polymeric Ru(1Y) 686 895 882 817 842 755 808 782
1 2 3
806
80 1
(4) F r o m tlie P h . D . thesis of D. K. A. entitled "A S t u d y of R u ( I I I ) , and Ru(1V) Species in Aqueous Perchloric Acid Solutions," Purdue Unirersity, August, 1980.
DEPARTMEKT OF CHEMISTRY
PURDCE UNIVERSITY LAFAYETTE, INDIAXA
DONALD K. AT WOOD^ THOS. DE VRIES RECEIVED FEBRUARY 1, 1961
SPECIFIC CLEAVAGE OF METHIONYL PEPTIDES
Sir: Sulfonium derivatives of methionine have been known to undergo easy elimination of the sulfur function with concomitant formation of homoserine l a c t ~ n e . ' , It ~ ~has ~ now been demonstrated that in peptides of methionine this intramolecular displacement occurs with participation and cleavage of the C-peptide bond (I-IV) in high yields. The data presented in Table I show that the extent of TABLE I *ILKYL GROUPO N T H E CLEAVAGE OF Svr.SALTS DERIVEDFROM ETHYL N-.~CETYLMETHIOSYI -
JSFLUESCE FOYIUM
OF
GLY CIpi AT E
Alkylating agent
Molar ..-.
Eauiva-
concn. 01 peptide in reaction mixturea
of alkylating agents
ients
P e r cent. of peptide cleavageb
0,005 3 6 .0 Iodoacetic acid" .01 4 3.6 Methyl iodide"Pd .01 4 43 E t h y l brornoacetatcc,d .O1 3 53 Iodoacetamidr" .01 1 -2 2,4-Dinitrofl~orobenzcne~ ,01 3 5 Diethyl bromomalonatee a Reactions were allowed to proceed at room temperature for 24 hr. * After removal of excess of alkylating agent by ether extraction t h e reaction mixture was heated for one hour at 100'. T h e liberated amino acid was determined b y ninhydrin assay.4 Reaction medium was 0.1 Af f H 3 citrate buffer. d Alkylation was conducted in a sealed tube. e Reaction medium was 1: 1 mixture of E t O H and 0.1 hl pH 3 citrate buffer. (1) G . Toennies a n d J. J . Kolb, THISJOURNAL, 67, 1141 (1945). ( 2 ) R . A. McRorie, G. L. Sutherland, M . I. Lewis, A. D. Barton, M. R . Glazener and W. Shive, rbid., 76, 115 (1954). (3) H. G. Gundlach, W, H. Stein a n d S. Moore, J . Bid. Chem., 234, 1754, 1761 (1959). (4) S . N o o r e and U'. H. Stein, J . B i d . Cheiir., 176, 367 (lQ48).
CONMUNICATIONS TO THE EDITOR
1510 CH3
s CH, 'CH,
0 R-CH-NH-C-CH
I1
1.
COOH
0
I1
AH-C-R' I ICH2CONH2
I
I
COOH
F/ 40') eo ( 95
CHJ I
.-
SH-C-R'
I1
S-CH,CONH,
-
Vol. 83
The procedure was applied to additional dipeptides and the course of the alkylation a t 3540' followed by argentometric t i t r a t i ~ n . ~U'ith over 90% formation of carbamylmethylsulfonium salts only 870 of peptide cleavage occurred a t 40'; however, on brief heating a t 95' the yields of cleaved amino acid increased to 5l-857, (Table 11). Paper chromatography and paper electrophoresis of such reaction mixtures showed that in each case the cleaved amino acid was the only ninhydrin-positive substance present. Since in peptides and proteins alkyl halides a t p H 2.8 react only with the sulfur of methioninej3s7 this procedure permits specific chemical cleavage of methionyl peptide bonds. The application of an improved methionine peptide cleavage to the selective splitting of ribonuclease is described in a following communication. ( 5 ) G. Toennies and J . J. Kolb, THISJ O U R N A L . 67, 849 (1945). (6) D . H. Spackmau, LI'. H. Stein a n d S. Moore, Anal. Chem., 30, 1190 (1958). (7) P. T. Vithayathil and F. M , Richards, J . Bioi. Chem., 238, 2343 (1960). ( S ) Max-Planck Institut flir Biochemie, Munchen, Germany. \\'ILLIAM B. LAWSON* METABOLIC DISEASES ERHARD GROSS CALVIS M. FOLTZ XATIONAL IXSTITUTES O F HEALTH BETHESDA 13, A I D . BERSHARD ~'ITKOP RECEIVEDSEPTEMBER 21, 1060
SATIONAL INSTITUTE OF ARTHRITIS AND
SELECTIVE CLEAVAGE OF T H E METHIONYL PEPTIDE BONDS IN RIBONUCLEASE WITH CYANOGEN BROMIDE'
Sir: cleavage of ethyl N-acetylmethionylglycinate depends on the nature of the alkylating agent. Carbamylmethylsulfonium salts gave the highest yield (-50%). TABLE 11 ALKYLAI10sASD CLEAVAGE OF MEIHIONYL PEPTIDES
Prptide
CarbobeIizoxy-Lmethionyl-Lglutamic acid".b Benzoyl DL-methionylgIycine",* Benz,oyl-DL-methionylglycine e t ~ i yestera.* ~ Carbobenzoxy Lniethionyl-~tyrosine"
Degree of alkylation
Time of alkylation, hr.
by titration,
68" 13je 136e 13Se
92 91 91 91
68' 135' 13je 13je 68 ' 65"
99 93 93 93 92 84
%
Per cent. of peptide cleavage
8. I d
80f 850 81h
Concentration in ethanol-water (1: 1) was 2 X 10' M. b Three equivalents of alkylating agent were used. Temperature of alkylation was 40'. Mixture was fractionated on columns of Amberlite I R 120 and t h e cleaved amino acid determined with automatic recoiding eqi.'p - 1 menLB e Temperature of alkylatioii was 35-40 . f Reaction mixture was analyzed directly by ninhydrin r n e t h ~ d . ~ Reaction mixture was heated for 1 hr. at 95" before a n a l y ~ i s . ~ Reaction mixture was extracted mith ether, heated for 1 hr. a t 95' aiid a n a l y ~ e d . ~ (1
Cyanogen bromide reacts with ethyl N-benzoylDL-methionylglycinate in alcohol-water at room temperature to yield, via the unstable cyanosulfonium salt, (i) 70% N-benzoyl-DL-homoserinelactone (m.p. 143", rep. 142°),2a (ii) methyl thiocyanate, assayed by gas chromatography and infrared absorption, and (iii) 75-907, of ethyl glycinate. By contrast, the von Braun cyanogen bromide cleavage of dialkyl thioethers in which there is no intramolecular assistance by functional groups requires elevated temperatures for the formation of alkylthiocyanate and alkyl bromide.2b The usefulness and selectivity of this improved method for the cleavage of methionyl peptide bonds3 was demonstrated with ribonuclease, which in a chain of 12-1: amino acids contains four methi0nines.l Bovine pancreatic ribonucleases in 0.10.3 N HC1 solution reacted with up to 30 equivalents of cyanogen bromide a t 20" for 2-1: hours. After removal of solvent, excess reagent and methyl thiocyanate by lyophilization the residue in aliquots of 1.5-2.0 mg. was subjected to paper electrophoresis for 4 hours a t 1100 v., GO m A. and pH 6.5 in a pyridine-acetate buffer system. In order (1) Presented in p a r t a t t h e Annual s l e e t i n g of t h e Chemical Society of Japan (iTIHOh- KAGAKU K.41 N E N K A I ) , April 1-4, 1961, i n Tokyo. (2) (a) E . Fischer a n d H. Blumenthal, Be?., 40, 106 (1910); (b) J. von Braun el al., Ber., 6 6 , 1573 (1923); 69, 1202 (1926); A n n . , 490, 189 (1931). (3) Cf.W. B. Lawson, E. Gross, C. M. Foltz a n d B. Witkop, J . A m . Chem. Soc., 83, 1500 (1961). (4) C . H . W. IIirs, S. Moore a n d W. H. Stein, J . Bioi. Chem., 236, 633 (1960); C. H. W, Hirs, A n n . N . Y . Acad. Sci., 88, 611 (1960). ( 5 ) Sigma Chemical Company, St. Louis, Lot R60B-069.
