2 9 4 F. M. VERONESE, E. BOCCU’. C. A. BENASSI, AND E. SCOFFONE Preferential Hydrolysis of Kynurenine Peptides F. M, V e r o n e s e , E. Boccu’, C. A. B e n a s s i, and E. S c o f f o n e * Institute of Pharmaceutical Chemistry (Centro Naz. di Chimica del Farmaco e dei Prodotti Bio- logicamente Attivi del C.N.R.), and * Institute of Organic Chemistry (Centro Naz. di Chimica delle Macromolecole del C.N.R. — Sez. VIII), University of Padova (Italy) (Z. Naturforschg. 21 b, 294—300 [1969] ; eingegangen am 7. August 1968) The preferential cleavage of kynurenine peptides bonds was studied through reduction to y-(o- aminophenyl)-homoserine derivatives followed by mild acidic as well as mild alkaline hydrolysis. The reduction of keto group of kynurenine to hydroxy one was achieved by controlled potential electrolysis at —1.05 Volts. In order to understand the mechanism of hydrolysis some model com­ pounds related to kynurenine peptides were also synthesized and studied. Hydrolysis of y-(o-amino- phenyl)-homoserine peptides in acidic media is related only to the assistance of y-hydroxy function, whereas in alkaline media also aromatic amino group is involved. The conversion of tryptophan to kynurenine was These studies, already accomplished with kynu­ extensively investigated in our laboratories through renine alone, have now been extended to some kynu­ performic acid oxydation 1 and, with better results, renine peptides in 5% and 50% acetic acid. in peptides and proteins by ozonization 2’ 3 and dye As reported in Table I the examined kynurenine sensitized photooxidation 4 w ith the aim to reach a derivatives present similar half-wave potentials of preferential chemical cleavage at the tryptophan polarographic reduction as that done by kynurenine residue. The preparation of kynurenine peptides itself. Some variations among the compounds could was firstly achieved from the corresponding trypto­ depend on the different molecular structure. phan peptides 5 and modified lysozyme w as obtained and studied 6. Compounds Volts vs. SCE In this paper we present results about the pre­ CH3COOH 5% CH3COOH 50% * ferential hydrolysis of kynurenine peptides through Kynurenine - 0.960 - 0.952 reduction by controlled potential electrolysis of the HC1 • H-Kyn-Glv-OEt - 0.992 — — keto group to the hydroxy followed by mild acidic HC1 • H-Ala-Kyn-OMe - 0.996 HC1 • H-Phe-Kyn-OMe - 0.990 — or basic hydrolysis. HC1 • H-Ala-Kyn- In addition some kynetic studies on the cleavage Glv-OEt - 0.992 — Z-Kyn-Gly-OEt — - 1.008 of selectively reduced kynurenine peptides and of Z-Leu-Kyn-OMe — — 1.006 related derivatives are reported *. Z-Ala-Kvn-Gly-OEt — - 1.000 Selective reduction of the keto group of kynure­ Z-Phe-Kyn-OMe — - 1.001 nine (1) to hydroxy group of y-(o-aminophenyl) - Table I. Polarographic Reduction: Half-Wave Potentials of homoserine (2) was previously studied polarogra- Kynurenine and Kynurenine Peptides in 5% and 50% Acetic phically by one of us 8 and was found to occur over Acid a. a For experimental conditins, see text. * 50% acetic acid was employed owing to the insolubility of protected kynu­ a large interval of pH with the consumption of two renine peptides in 5% acetic acid. electrons: 0II NH,I H \ / OH NH,I Controlled potential electrolysis may be better ac­ CH c - c h 2- c h complished at —1.05 Volts vs. SCE, the reduction COOH 2 e " C 00H of keto group being controlled by the lowering of 1 2 current flow and also checked by recording polaro- 1 C. A. Benassi, F . M. Veronese. A. De Antoni, and P. 8 G . G a l i a z z o , G . J o r i , and E. S c o f f o n e , Biochem. bio­ P a je t t a , Gazz. chim. ital. 97, 3 [1967]. physic. Res. Commun. 31. 158 [1968]. 2 E. S c o f f o n e , A. P r e v i e r o , C. A. B e n a s s i , and P . P a j e t t a , * A preliminary account of this wTork was presented at the Peptides, Proc. VIth European Sympos. Athens, 1963 VIII Peptides Symposium, Noordwijk, September 1966 7. (Pergamon Press, Oxford 1966), p. 183 — 188. 7 E. S c o f f o n e , A. F o n t a n a . F . M a r c h i o r i , and C. A. 3 A. P r e v i e r o and E. B o r d i g n o n , Gazz. chim. ital. 94, 630 B e n a s s i , Peptides. Proc. V I I I th European Sympos. Noord­ [1964], wijk (1967) [North Holland Publishing Company, Amster­ 4 C. A. B e n a s s i , E. S c o f f o n e , G . G a l i a z z o , and G . J o r i , dam] , p. 189. Photochem. Photobiol. 6, 857 [1967]. 8 L. G r i g g i o . E. V i a n e l l o , and C. A. B e n a s s i , Gazz. chim. 5 F . M. V e r o n e s e , A. F o n t a n a , E. B o c c u ’, and C. A. ital. 93, 1412 [1965], B e n a s s i , Gazz. chim. ital. 97, 321 [1967], PREFERENTIAL HYDROLYSIS OF KYNURENINE PEPTIDES 2 9 5 grams without changing the experimental condi­ tions 9. Reduction is accompanied by disappearance of the absorption maximum at 360 m/<, whereas a new maximum is noted at 280 m/< (see Fig. 1 ) with a contemporaneous decrease of fluorescence under UV light. J_______ I_______ I_______ L -0.4 -0.6 -0.8 -1.0 -1.2 VOtt-VS SCE Fig. 2. Pattern of polarographic reduction of kynurenine in 5% acetic acid. For these reasons the electrochemical reduction is preferable, especially when small amounts of sub­ stance are available. In order to study the mechanism of cleavage some protected kynurenine peptides were prepared d and the carbobenzoxy group was removed by hydrogeno- Wavelength [m fi] — ► lysis. Their melting points, rotatory power, chro­ Fig. 1. Absorption spectra of kynurenine (1) ( ----------) and matographic behaviour and analytical data are re­ y- (o-aminophenyl) -homoserine (2) ( ---------) in phosphate ported in Table II. buffer 0.05 M, pH 6. All these kynurenine peptides were reduced to the Furthermore, reduced compounds [ 7- (o-amino- corresponding /-(o-aminophenyl)-homoserine deri­ phenyl)-homoserine derivatives] show on paper and vatives and then hydrolized in mild acide (0.2 N TLC chromatograms a bright yellow reaction with HC1) as well as in mild alkaline conditions (0.5 M p-dimethylaminobenzaldehyde reagent. NaHCOg) in a sealed evacuated tube at 100°. Controlled potential electrolysis appears a very The amino acids released from reduced derivati­ selective method of reduction since no other amino ves at various times of hydrolysis were determined acid is affected with the exception of cystine which is by the amino acid analyzer. Fig. 3 a shows the rate quantitatively converted to cysteine 10. Fig. 2 shows of liberation of the N-terminal or C-terminal amino acid from the 7-(o-aminophenyl)-homoserine pepti­ a pattern of polarographic reduction of kynurenine des during acidic hydrolysis. The results obtained in 5% acetic acid. with y- (o-aminophenyl) -homoseryl-glycine ethyl Kynurenine and its peptides may alternatively be ester (3), phenylalanyl-/-(o-aminophenyl)-homo­ reduced to y (o-aminophenyl) -homoserine with serine methyl ester (4), and alanyl- 7- (o-aminophe­ NaBH4 11 but this method is less accurate than the nyl) -homoseryl-glycine ethyl ester (5) are here tabu­ electrochemical one, since over reduction may result, lated. Comparable data have been also obtained with and pure products are obtained only after crystalli­ other model compounds as alanyl-/-(o-aminophe­ zation. nyl)-homoserine methyl ester, carbobenzoxy-y- (o- 9 L. M e i T e s , in: Polarographic Technique (Interscience Pu­ 10 G. M a r k u s , Federat. Proc. 19, 340 [1960]. blishers, Inc., New York, N. Y. 1955). 11 A. P r e v i e r o , M . A. C o l e t t i P r e v i e r o , and P . J o l l e s , Biochim. biophysica Acta [Amsterdam] 124, 400 [1966]. 2 9 6 F. M. VERONESE. E. BOCCU’. C. A. BENASSI, AND E. SCOFFONE X 0 O — aminophenyl)-homoseryl-glycine ethyl ester and car- 1 0 5 0 0 5 0 5 0 " Ö c o -ri bobenzoxy - phenylalanyl - y -(o-aminophenyl) - homo­ 1— 1 ,"H serine methyl ester. CO IO i c IO SO l6 c o CO 0 t - CO 1 0 i q 0 0 X Q 0 5 O 1 0 T* t - i > 0 5 o i Ö CO 1— 1 ,-H ( 0 _ 1—1 0 CO »-H i d 0 O Fig. 3. a) Mild acidic cleavage of the N-terminal and C-ter- 3 5 CO CO 0 5 minal amino acid of y- (o-aminophenyl) -homoserine peptides: 0 5 <N 0 5 0 5 C C H N C H © GO Ö d Gly * ( + — ----h) released from y- (o-aminophenyl) -homo- IO 10 10 seryl-glycine ethyl ester (3). Phe (A ------A released from phenylalanyl-y- (o-aminophenyl) -homoserine methyl ester (4). OiO Ü^ Gly * (G- ■•— G) and Ala (O----O) released from alanyl- o o y- (o-aminophenyl) -homoseryl-glycine ethyl ester (5). b) Mild r f CO acidic cleavage of the N-terminal and C-terminal amino £iO o£ CM acid of 7 -phenyl-homoserine derivatives: Gly (A'----- A) Xl> tcTf released from glycyl- 7-phenyl-}’-hydroxy-propylamide (7).