September 1969
7 37
DIPEPTIDES COKTAIKIKG ~ Y C L O P E N T A K E G L Y C I N E. 4 S D P-%THIENYLALBSINE
im-benzylhistidine was detectable in the hydrolysate by electrophoresis a t pH 3.5. This product (100 mg) was purified in the same way as described for the benzyl derivative. The flow rate was 28 ml, hr. The gradient was started after 18.3 hr (320 ml) and the main peak was elrited between 980 and 1040 ml. The combined fractions were lyophilized to give 65 my. After purification via the picrate salt there was 45 mg of white powder. A sample hydrolyzed i n 5.5 S HCl at 110’ for 7% hr had the following amino acid composition: Gly 2.03, T a l 1.03, Tyr 0.95, Ile 0.97, His 0.93, Pro 1.05, Phe 1.00. -4sample hydrolyzed for 40 hr with aminopeptidase-LIZ1 had Gly 2.00, Val 1.10, Tyr 1.00, Ile 0.85, His 0.33, Pro 0.41, Phe 0.94. Electrophore.iis at pH 1.83 showed one spot, EH 0.37: tlc showed one spot, RiII 0.48, R ~ I I0.72; I positive reaction with ninhydrin, Paulg reagent, ch1orinationJ23 and 1iii troso-2-naphthol.24 Ac-Gly-Gly-Val-Tyr-Ile-His-Pro-Phe.-To13 mg of purified
Gly-Gly-Val-Tyr-Ile-His-Pro-Phe in 2 ml of DblF was added 0.05 ml of AcZO and 0.05 ml of Et3N. The solution was stirred for 2 hr a t room temperature. il small amount of insoluble material was removed by centrifugation then 20 ml of Et40 was added to the supernatant giving a voluminous white precipitate. The precipitated peptide was washed with Et20 (two 20-ml portions) then dried in vacuo over KOH pellets at room temperature yielding 10 mg of an off white powder. Elect,rophoresis at p H 1.83 showed one spot, E H 0.30, ninhydrin -, Pauly There was no detectable free peptide a t E H 0.37 under conditions where 1% could have been detected. A 3-mg portion of the peptide was dissolved in 0.2 nil of 0.1 J I S a O K and kept at room temperature for 30 niin to saponify any acetyl groups on tyrosine. The solution was neutralized with 0.2 ml of 0 . 1 JI HC1 giving a gelatinous precipitate. Normal saline containing 0.1% polyvinylpyrrolidone (a0 ml) was added to prevent adsorption on glass. This solution was used directly for biological assay.
+.
Synthesis and Microbiological Properties of Dipeptides Containing Cyclopentaneglycine and ,&Z-Thienylalanine’” JIMT. HILL'^
AND
FLOYD W.
D c x x l c
Department of Bzochemistry, University of Tennessee Medical Cnits, Jlemphis, Y’ennessee Received March 20, 1969 Dipeptides synthesized included ~-cyclopeiitaneglycyl-P-2-thienyl-~-alanine, P-2-thienyl-balanyl-L-cyclopentaneglycine, Lphenylalanyl-L-isoleucine, and glycyl-~cyclopentaneglycine. Cyclopentaneglycine and p-2-thienylalanine may be viewed as structural analogs of isoleucine and phenylalanine, respect,ively. The effect of the dipeptides on the growth of three organisms, Escherichia coli, Leuconostoc mesenteroides, and Lactobacillus arabinoszts, was studied. Under the test conditions, the peptides had greater growth-stimulating or gron-th-inhibiting effects than equivalent amounts of the corresponding free amino acids.
Peptides often display unique growth-stimulating effects in microorganisms. Other peptides, containing amino acid analogs such as P-2-thienylalanine, may be more effective growth inhibitors than the free amino acid analog.J Where an organism was inhibited by a mixture of P-2-thienylalanine and one of its peptides, the inhibition was more effectively nullified by phenylalanine peptides than by free ~ h e n y l a l a n i n e . ~ Iiihara and Snellj described the “double inhibition” of Leuconostoc mesenteroides by high levels of L-alanine and L-leucine. This “double inhibition” was reversed by the addition of a mixture of glycine and L-isoleucine and, more effectively, by a single dipeptide, glycyl-Lisoleucine. This concept of “double inhibition” suggested to us the preparation of a dipeptide containing two amino acid analogs in order to test whether such peptides would be more effective growth inhibitors than the constituent free amino acid analogs. The amino w i d analogs chosen were cyclopentaneglycine6 and
(1) (a) Presented in part at t h e 48th annual meetinn of the Federation of .\merican Societies of Experimental Biology. Chicago, Ill., .ipril 1964. This investigation v a s supported by U. 8 . Puhlic Health Service Grant N o . .\I03710 from the Xational Institute of Allergy and Infectious Diseases. ( b ) Part of this work was taken from the 4I.S. thesis of Jim T. Hill, University of Tennessee, .June 1964. (cj T o whom requests for reprints should lie sent: Department of Cliemistry. .\l>ilene Cliristian College. Al)ilene, Texas 79601. (2) S. Shankman. S. Higa. €1. A. Florslieim, Y. Rcliro, and V. Gold, A r r h . Biorhem. B i o p h y . ~ . 86, , 204 (1960). ( 3 ) F. JV. D u n n . .I. A I . Ravel. and U’.Shiv?. .I. Biol. Chrm.. 219, 809 (195G). ( 4 ) 1 2 . \V. Ihinn, .J. I I i i m ~ l i r e y s .and 11.. Shive. A r c h . Bioehem. Biopkys., 71, 475 (195i). ( 5 ) H.Kiliara and E. E. Hnell. J . Biol. Chem., 236, 415 (1960). (6) (a) TV. 31. Hardine and IV. Shire. ibid., 206, 401 (1954); ( b j J. T. H i l l and 1‘. \V. 1)iinn. . I . Ory. Chem., SO, 1321 (1965).
P-2-thienylalanine,? structural analogs of isoleucine and phenylalanine, respectively. This report describes the synthesis and microbiological properties of ~-cyclopentaneglycyl-P-2-thienyl-~alanine, ~-2-thienyl-~-alanyl-~-cyclopentaneglycine, glycyl-L-cyclopentaneglycine, and ~-phenylalanyl-~-isoleucine.
Experimental Section* Peptide synthesis employed standard coupling and deprotectioii procednres.9 The following compounds iised in the present work were prepared earlier in this laboratory: L-cyclopentaneglycine, carbobenzoxy-p-2-thienyl-~-alanine, carbobenzoxy-L-phenylalanine, carbobeiizoxy-L-i.oleiiciiie, p-2-thienyl-L-alanine methyl ester hydrochloride, phenylalanine methyl ester hydrochloride, L-isolericiiie methyl ester hydrochloride, carbobenzoxyglycine, glycyl-L-i.oleucirie, glj l-~-pheriylala~iiiie, glycyl-p-2-thienyl-~~alanine, and L-isoleiicyl-L-phenylalanine. Carbobenzoxy-L-cyclopentaneglycine (I).-To a solution of 1.45 g of L-cyclopentaneglycine (31 mmoles) in an equivalent amount of 2 N S a O H were slowly added, with stirring a t O”, 6.39 g (36 mmoles) of carbobenzoxy chloride and 18.5 ml (37 mmoles) of 2 S S a O H , maintaining a pH of approximately 8. The prodiict obtained upon acidifying the reaction mixture was piirified by dissolving in ethyl acetate, extracting into 0 . 5 Jf KHCOa, arid reprecipitating with HC1; yield 6.8 g (83c
