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Studies on polypeptide antibiotics
ARCHIVES
OF BIOCHEMISTRY
AND
BIOPHYSICS
Studies I. 2,!5Diketopiperazines MILTON From the Life
108,
292-300
(1964)
on Polypeptide as Models WINITZ
Sciences Laboratory,
Antibiotics
of Cyclic
AND NOBUO United
Received
Technology July
Polypeptide
Antibiotics’
IZUMIYA2 Center, Sunnyvale,
California
13, 1964
The D-D, L-L, D-L, and L-D stereomers of valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride were synthesized for purposes of comparison with the cyclic polypeptide antibiotics. Synthesis was achieved via the dicyclohexylcarbodiimide-mediated condensation of formyl-L(or n)-valine or formyl-r. (or n)-phenylalanine with Ne-carbobenzoxy-n(or n)-lysine methyl ester, deformylation with methanolic hydrochloric acid of the !Va-formyl-NC carbobenzoxydipeptide methyl ester so secured, cyclization of the resulting product with methanolic ammonia to the corresponding N’-carbobenzoxylated 2,5-diketopiperazine, and decarbobenzoxylation of the latter to the desired product by palladium catalyzed hydrogenolysis. All four stereomers of each of the synthetic 2,5-diketopiperazines were insusceptible to the hydrolytic action of proteolytic enzymes, a behavior reminiscent of that of the natural cyclic polypeptide antibiotics; however, none exhibited the antibacterial propensities of the latter. The relationship between the chemical structure of polypeptide antibiotics and their antibacterial properties is discussed. INTRODUCTION
Certain of the polypeptides elaborated by bacteria, such as the gramicidins, bacitracins, tyrocidines, and polymixins, in addition to their remarkable antibacterial properties, possess several striking structural and chemical features in common (1). These include (a) a basic character due to the presence of one or more diamino acid residues, (b) a cyclic conformation, and (c) the possession of at least one amino acid residue of the n-configuration. As 2,5diketopiperazines, wherein one or both of the component amino acid residues possess a n-configuration and a basic character, incorporate these same characteristics, it 1 The experimental portion of this work was carried out at the National Cancer Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland, and was previously reported in part [Abstracts, 137th Meeting of the American Chemical Society, 30C (1960)]. 2 Present address : Laboratory of Biochemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
became of interest to ascertain whether these relatively simple molecules would also exhibit the antibact’erial properties of their more complex naturally occurring counterparts. The present study, which was conceived with this in mind, includes the synthesis of each of the four stereomers of valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride as well as experiments designed to test both t’he antibacterial propensity and susceptibility to enzymic hydrolysis of these “antibiotic models.” Also included in the present study is the synthesis of the pertinent stereomers of lysylphenylalanine, phenylalanyllysine, and valyllysine, which were prepared as reference compounds for purposes of chromat’ographic comparison. EXPERIMENTAL
PREPARATION OF THE VALYLLYSINE ANHYDRIDES Formyl-L-valine. This compound was prepared according to the general formylation procedure of Sheehan and Yang (2). Thus, a solution of 35.2 292
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gm of L-valine in 630 ml of 98yo formic acid is chilled in an ice-bath and treated dropwise, over a period of 20 minutes, with 210 ml of acetic anhydride. Stirring at ice-bath temperature is continued for an additional 30 minutes, after which time the reaction mixture is stirred at room temperature for 1 hour. The reaction mixture is then treated with 300 ml of water, with cooling in the ice-bath, and is concentrated to dryness under reduced pressure. The resulting residue is repeatedly treated with a little water and concentrated to dryness in order to remove the final traces of formic acid. A crystalline residue is ultimately obtained which is collected with the aid of a little cold water. Recrystallization is effected from boiling water; yield 28.5 gm. An additional 9.6 gm may be recovered from the aqueous filtrates; total yield 38.1 gm (85%); m.p. 152”153” [lit. values, 156” (3) and 148”149” (2)]. Formyl-D-valine. Preparation of this compound was achieved in the same manner as described for the L-antipode above; yield 84%; m.p. 152”153” [lit. values, 156” (3) and 148”149” (2)]. N’-Carbobenzoxy-z-lysine methyl ester hydrochloride. This compound was prepared according to the procedure of Bergmann et al. (4); m.p. 110”113” [lit. value, 117” (4)]; [a]$ = +17.2” (2$& in water). Ne-Carbobenzoxy-L-lysine methyl ester hydrochloride. Preparation of this compound was achieved in the same manner as the L-antipode above; m.p. 109”-113”. Recrystallization from hot acetone-ether raised the melting point to 114”115’; [CX]~~= -16.9” (2yo in water).$ Anal. Calcd. for C15H2304N2Cl: C, 54.5; H, 7.0; K, 8.5. Found: C, 54.5; H, 7.2; N, 8.4. Formylvalyl-NE-carbobenzoxylysine methyl esters. Directions for the preparation for each of the four stereomers (L-L, D-D, D-L, and L-D) of this compound are given in what follows: To a stirred solution of 7.26 gm of formyl-L (or n)-valine, 16.55 gm of N*-carbobenzoxy-L (or n)-lysine methyl ester hydrochloride, and 7.0 ml of triethylamine in a mixture of 150 ml of tetrahydrofuran and 150 ml of chloroform, at O”, is added 10.3 gm of N, N’-dicyclohexylcarbodiimide. The reaction mixture is stored at 4” overnight, after which time the L-L and D-D stereomers are recovered according to the procedure given under (a) and the D-L and L-D stereomers are recovered as described 3 The n-lysine employed in this preparation was secured by conversion of Ne-benzoyl-n-lysine to the corresponding a-bromo analog by treatment with nitrosyl bromide, followed by aminolysis and subsequent acid hydrolysis of the Nf-benzoyln-lysine so derived (5, 6).
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under (b). (a) The precipitated N, N’-disubstituted urea is removed by filtration and the filtrate is concentrated under a jet of dry air to an oil. After dissolving the latter in 250 ml. of chloroform, the solution is washed successively with 3% hydrochloric acid, water, 5oJo sodium bicarbonate and water, and then concentrated to dryness under a jet of air. The syrupy residue, when treated with 35 ml of ether and 80 ml of petroleum ether, separates as a solid material which is recrystallized from ethyl acetate-ether-petroleum ether (30 ml:60 ml:60 ml); yield 56% for L-L and 59% for D-D stereomer; m.p. 140”143” for both stereomers; [a]Z5 = -11.9” and +12.0” (2% in dimethylformamide), respectively, for L-L and D-D stereomers. (b) The N,N-disubstituted urea, admixed with a small amount of the desired product, precipitates. After taking the crystals of the latter into solution by warming at 50”, the reaction mixture is cooled to room temperature under tap water and the urea derivative separated by filtration. The remainder of the procedure is identical with that described under (a) above. Recrystallization is effected from ethyl acetateether-petroleum ether (90 ml:50 ml:50 ml); yield 55yo for D-L and 58% for L-D stereomers; m.p. 149” for D-L and 148” for L-D stereomer; [a]: = -16.0” and +14.9” (2% in dimethylformamide), respectively, for D-L and L-D stereomers. Anal. Calcd. for CzlH,,06N,: C, 59.8; H, 7.4; C, 60.3; H, 7.4; N, 10.0. Found for L-L stereomer: N, 9.8. Found for D-D stereomer: C, 60.1; H, 7.3; N, 9.8. Found for D-L stereomer: C, 60.2; H, 7.4; N, 9.9. Found for L-D stereomer: C, 60.0; H, 7.5; N, 9.8. Valyl-Ne-carbobenzoxylysine methyl ester hydrochlorides. Each of the four stereomers of this compound was prepared from the corresponding N-formylated dipeptide derivative as follows : A suspension of 8.43 gm of the pertinent formylvalyl-N’-carbobenzoxylysine methyl ester stereomer in 60 ml of 0.5 N methanolic-HCl is stored at room temperature for 48 hours. The resulting clear solution is concentrated under reduced pressure at a temperature not exceeding 40”. Treatment of the residual material with acetone and ether leads to a crystalline product! which is recrystallized from acetone-ether; yield 77, 73, 80, 161”, and 161”, and 75%; m.p. 154”, 153”154”, and [ol]z5 = O”, O”, -43.0” and -l-43.8” (2% in water), for the L-L, D-D, D-L, and L-D stereomers, respectively. 4 Although the crystallization of stereomers occurs more readily the L-L and D-D stereomers, the latter crystallize within the space of an procedure described. D-L
the L-D and than that of nevertheless hour by the
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Anal. Calcd. for C&H3206N3C1: C, 55.8; H, 7.5; N, 9.8; Cl, 8.3. Found for L-L stereomer: C, 55.8; H, 7.5; N, 9.7; Cl, 8.4. Found for D-D stereomer: C, 55.5; H, 7.6; N, 9.7; Cl, 8.2. Found for D-L stereomer: C, 55.6; H, 7.7; N, 9.7; Cl, 8.5. C, 55.8; H, 7.6; N, 9.8; Found for L-D stereomer: Cl, 8.6. Valyl-NC-carbobenzoxylysine anhydrides. Each of the four stereomers of this compound was prepared as follows: To a solution of 4.3 gm of the pertinent valyl-Ne-carbobenzoxylysine methyl ester hydrochloride in 10 ml of methanol is added 100 ml of methanol which has been previously saturated with dry ammonia gas at 0”. After storage for 24 hours at room temperature in a glass-stoppered pressure bottle,6 the reaction mixture is concentrated to dryness under a jet of air, and the crystalline residue is recovered by filtration with the aid of a little water. Recrystallization of the L-L and D-D stereomers is achieved from 85 ml of 3Oyc ethanol, whereas 100 ml of 50% ethanol is used with the L-D and D-L stereomers; yield 88,86,90, and 91%; m.p. 162”164”, 161”-164”; 198”-2OO”, and 199”-201”, and [LY]~~ = -46.1”, t45.00, -4.3”, and +3.2” (2% in dimethylformamide), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for C9Hz704N3: C, 63.1; H, 7.5; N, 11.6. Found for L-L stereomer : C, 63.0; H, 7.5; N, 11.6. Found for D-D stereomer: C, 63.1; H, 7.3; N, 11.4. Found for D-L stereomer; C, 63.2; H, 7.4; N, 11.6. Found for L-D stereomer: C, 63.1, H, 7.4; N, 11.7. Valyllysine anhydride hydrochlorides. Each of the four stereomers of this compound was prepared as follows: A suspension of 1.81 gm of the pertinent stereomer of valyl-Nf-carbobenzoxylysine anhydride in a mixture of 11 ml of 0.5 N methanolic-HCl and 40 ml of methanol is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon completion of the reaction, the catalyst is removed by filtration, with prior warming if necessary to dissolve any product which may have precipitated, and the filtrate is concentrated to dryness under reduced pressure. The crystalline product is isolated by filtration with the aid of a mixture of 12 ml of acetone and 18 ml of ether, and subsequently recrystallized from methanol-acetone; yield 77, 84, 90, and 90%; m.p. 254”256” (dec.), 253”255” (dec.), 282” (dec.), and 282”-283” (dec.), and [ali = -54.1”, +53.6”, -5.4”, and +5.8” (2% in a 5 In the case of the L-D and D-L stereomers, mass of crystals appears after about 1 hour of storage, whereas complete solution is maintained with the L-L and D-D stereomers over the entire 24-hour reaction period.
IZUMIYA water), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for CiiH*202N$l: C, 50.0; H, 8.4; N, 15.9: Cl, 13.5. Found for D-D stereomer: C, 49.8; H, 8.5; N, 15.7; Cl, 13.5. Found for D-L stereomer: C, 50.3; H, 8.5; N, 15.7; Cl, 13.6. Found for L-D stereomer: C, 50.0; H, 8.4; N, 15.7; Cl, 13.3.
PREPARATION OF THE PHENYLALANYLLYSINE ANHYDRIDES Formylphenylalanines. The L- and n-antipodes were prepared following the same procedure described above for the formylvalines; yield 75-80%; m.p. 167” for both antipodes [lit. value for L- and n-antipodes was 167” (7)]. Formylphenylalanyl - N’ - carbobenzoxylysine methyl esters. Preparation of each of the four stereomers of this compound is achieved by treatment of a solution of 9.65 gm of formyl-L(or D)phenylalanine, 16.55 gm of N’-carbobenzoxy-n (or n)-lysine methyl ester and 7.0 ml of triethylamine in tetrahydrofuran-chloroform with 10.3 gm of N, N’-dicyclohexylcarbodiimide, following essentially the same procedure employed for the of formylvalyl-NC-carbobenzoxypreparation lysine methyl ester (see above). Recrystallization of the crude products is achieved from ethyl acetate-petroleum ether; yield 75,78,68, and 67%; mp. 125”, 125”, 134”136”, and 134-135”, and [fY]2 = -4.50, +3.9”, -ll.S”, and f11.0” (1% in dimethylformamide), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for C25H3i0eN3: C, 63.9; H, 6.7; C, 64.0; H, 6.8; N, 9.0. Found for L-L stereomer: N, 9.3. Found for D-D stereomer: C, 63.8; H, 6.7; N, 9.0. Found for D-L stereomer: C, 64.2; H, 7.0; N, 9.2. Found for L-D stereomer: C, 64.3; H, 6.7; N, 9.2. Phenylalanyl-Ne-carbobenzoxylysine methyl ester hydrochlorides. The pertinent formyldipeptide ester stereomer was treated in the same manner as described under the preparation of valyl-Necarbobenzoxylysine methyl ester hydrochloride. could be Although the L-L and D-D stereomers recrystallized from methanol-acetone-ether, attempts to solidify the D-L or L-D forms were without success; yield 72% for L-L and 75% for D-D stereomer; m.p. 153”154” and 154”155” for L-L and D-D stereomer respectively; [(Y]:~ = -9.3” (1% in dimethylformamide) for L-L stereomer and +lO.l” for D-D stereomer. Anal. Calcd. for C&H,20sN,Cl: C, 60.3; H, 6.8; N, 8.8. Found for L-L stereomer: C, 60.3; H, 7.0; N, 8.8. Found for D-D stereomer: C, 60.1; H, 7.0; N, 8.6. Phenylalanyl-Nf-carbobenzoxylysine anhydrides. Conversion of each of the precursor methyl ester
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hydrochloride stereomers to the corresponding anhydrides followed the same procedure employed in the preparation of valyl-N%arbobenzoxylysine anhydride. Recrystallization was effected from 7GBOyo ethanol; yield 83,87,85,6 and 75y, m.p. 206”ZOB”, 205”-207”, 171”, and 171”, and [a]? = -26.1”, +25.5”, -lO.O”, and +lO.O” (1% in dimethylformamide), for the L-L, n-n, D-L, and L-D stereomers, respectively. ilnal. Calcd. for C23H2104N3: C, 67.5; H, 6.7; N, 10.3. Found for L-L stereomer: C, 67.5; H, 6.8; N, 10.2. Found for D-D stereomer: C, 67.3; H, 6.8; N, 10.2. Found for D-L stereomer: C, 67.9; H, 7.0; N, C, 67.9; H, 7.1; N, 10.3. Found for L-D stereomer: 10.2. anhydride hydrochlorides. Phenylalanyllysine Each of the stereomera of this compound was prepared as follows: A suspension of 2.05 gm of the pertinent phenylalanyl - N* - carbobenzoxylysine anhydride in 50 ml of glacial acetic acid is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon termination of the reaction, the catalyst is removed by filtration, the filtrate concentrated to dryness under reduced pressure, the residual material dissolved in water, and the aqueous solution treated with 5.3 ml of 1N hydrochloric acid. After concentration of the solution of dryness, the residual product is recovered by filtration with the aid of acetone. Recrystallization is effected from methanolacetone; yield 94,91,88, and 90% for the L-L, D-D, D-L, and L-D stereomers, respectively; [a]? = $2.5” (lo/, in water) for the L-L and -2.0” for the D-D stereomer; [ol]Z5 = -45” (0.3% in water) for the D-L and +48” for the L-D stereomer. Anal. Calcd. for C&H2202N$l: C, 57.8; H, 7.1; S, 13.5. Found for L-L stereomer: C, 57.7; H, 7.3; N, 13.5. Found for D-D stereomer: C, 57.6; H, 7.1; N, 13.5. Found for D-L stereomer: C, 58.1; H, 7.2; N, 13.7. Found for L-D stereomer: C, 57.8; H, 7.3; N, 13.4.
PREPARATION OF REFERENCE COMPOUNDS For purposes of chromatographic comparison, each of the four stereomers of valyllysine was synthesized, as were the L-L and D-L isomers of phenylisomer of lysyland the L-L alanyllysine phenylalanine. Preparative procedures for the intermediates leading to these compounds, as well as for the final products themselves, are given in what follows. Carbobenzoxyvalyl-N’-carbobenzoxylysine methyl 6 Yields of D-L and L-D stereomers based on the formylphenylalanyl-NE-carbobenzoxylyparent sine methyl ester.
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esters. The L-L, D-D, D-L, and L-D stereomers of this compound were prepared as follows: A solution of 5.03 gm of carbobenxoxy-L (or n)-valine and 2.8 ml of triethylamine in 40 ml of toluene is chilled to -5” and treated with 2.7 ml of isobutyl chlorocarbonate. Ten minutes later, a chilled solution of 6.61 gm of N’-carbobenzoxy-L (or u)-lysine methyl ester hydrochloride and 2.8 ml of triethylamine in 40 ml of chloroform is added thereto, and the reaction mixture is stored at room temperature overnight. The L-L and D-D stereomers are then recovered according to the procedure given under (a) and the D-L and I,-D stereomers are recovered as described under (b). (a) After being washed successively with water, dilute hydrochloric acid, water, half-saturated sodium bicarbonate solution and water, the organic fraction is dried over anhydrous sodium sulfate and concentrated to dryness. The crystalline residue is recrystallized from ethyl acetatepetroleum ether; yield 76% for L-L and 74% for D-D stereomer; m.p. 124”126” and [ol]$ = 0” (2y0 in dimethylformamide) for both stereomers. (b) The mass of crystals which precipitate are filtered off after the addition of 70 ml. of petroleum ether. Recrystallization is achieved from acetonepetroleum ether; yield 71 and 7770, m.p. 163” and 164”, and [a]:’ = -15.4” and +16.1” (2% in dimethylformamide), for the D-L and L-D stereomers respectively. Anal. Calcd. for GsH,TO,N3: C, 63.7; H, 7.1; N, 8.0. Found for L-L stereomer: C, 63.7; H, 7.1; N, 8.0. Found for D-D stereomer: C, 63.3; H, 7.3; N, 7.9. Found for D-L stereomer: C, 63.6; H, 7.2; N, 7.8. Found for L-D stereomer: C, 63.3; H, 7.1; N, 8.0. Carbobenzoxyvalyl-N’-carbobenzoxylysines. The four stereomers of this compound were prepared via saponification of the corresponding methyl ester derivatives. Conditions employed for saponification of the L-L and D-D antipodes are given under (a), while the D-L and L-D antipodes were saponified as described under (b). (a) A solution of 7.39 gm of the pertinent carbobenzoxyvalyl-Ncarbobenzoxylysine methyl ester in 100 ml of methanol is treated with 16 ml of IN sodium hydroxide. The reaction mixture is stored at room temperature for 25 hours, after which time it is neutralized with 17 ml of IN hydrochloric acid and concentrated to dryness under a jet of air. The residual material is filtered with the aid of a little water, air dried, and recrystallized from ethyl acetate-petroleum ether; yield 90% for L-L and 94% for D-D stereomers; m.p. 139’-140” for both stereomers; [ol]? = +5.0” and -5.9” (2% in respectively, for L-L and dimethylformamide), D-D stereomera. (b) A suspension of 7.39 gm of the
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pertinentc arbobenzoxyvalylN’carbobenzoxylysine methyl ester in a mixture of 220 ml of methanol, 55 ml of dioxane, and 32 ml of 0.5 N sodium hydroxide is stirred at 25” for 7 hours. The resulting clear solution is neutralized with 17 ml of IN hydrochloric acid and is concentrated to dryness under a jet of air; the residual material is recovered and recrystallized as given under (a) above; yield 97% for D-L and 98% for L-D stereomers; m.p. 124”126” for both stereomers; [ol]$ = +13.7” and -15.0” (2% in dimethylformamide), respectively, for D-L and L-D stereomers. Anal. Calcd. for CLH3507N3: C, 63.1; H, 6.9; N, 8.2. Found for L-L stereomer: C, 62.8; H, 6.9; N, 8.1. Found for D-D stereomer: C, 62.7; H, 6.7; N, 8.2. Found for D-L stereomer: C, 62.9; H, 7.0; N, 8.2. Found for L-D stereomer: C, 63.1; H, 6.8; N, 8.1. Valyllysine hydrochlorides. A solution of 5.14 gm of each of the four stereomers of carbobenzoxyvalyl-NC-carbobenzoxylysine in a mixture of 120 ml of methanol and 20.6 ml of 0.5 N hydrochloric acid is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon termination of the reaction, the pertinent solution is filtered and then concentrated to dryness under a jet of air. (a) The concentrates of the L-L and D-D stereomers yield crystalline residual masses, each of which is recovered by filtration with the aid of a little ethanol. Recrystallization is effected from a solution in 5 ml of water by the addition of 30 ml of ethanol; yield 85% for L-L and 84% for D-D stereomers; m.p. 241”243” (dec.) for both stereomers; [ali = f25.4” and -25.3” (2% in water), respec(b) The concentively, for L-L and D-D antipodes. trates of the L-D and D-L stereomers both yield a syrupy residue which can be solidified by treatment with ethanol and acetone. The solids so obtained are slightly hygroscopic and do not yield satisfactory elementary analytical values. However, their paper chromatographic behavior in a number of solvent systems indicates that they give only a single ninhydrin-positive spot and hence are suitable as chromatographic reference compounds for the present purposes; [ol]t5 = +48” and -50” (2% in water) for the L-D and D-L antipodes, respectively. Anal. Calcd. for CiiH2403N&1: C, 46.8; H, 8.6; N, 14.9; Cl, 12.6. Found for L-L stereomer: C, 46.8; H, 8.9; N, 14.9; Cl, 12.3. Found for D-D stereomer: C, 46.6; H, 8.8; N, 15.0; Cl, 12.4. NC-Carbobenzoxy-L-lysine ethyl ester p-toluenesuljonate. A suspension of 56.0 gm of N’-carbobenaoxy-n-lysine and 42 gm of p-toluenesulfonic acid monohydrate in a mixture of 80 ml of ethanol and 400 ml of carbon tetrachloride is refluxed for 24 hours in an apparatus patterned after that
developed by Clarke and Davis (8) for the removal of wrater azeotropically. Upon termination of the reaction, as indicated by a cessation of water liberation, the reaction mixture is concentrated to a small volume under a jet of dry air and the residual material treated with an excess of ether. The precipitate is recovered by filtration and recrystallized from acetone-ether; yield 92 gm (96%); m.p. 87”. Anal. Calcd. for C&H3207N2S: C, 57.5; H, 6.7; N, 5.8. Pound: C, 57.3; H, 6.6; N, 5.9. Carbobenzoxyphenylalanyl - NC carbobenzoxylysine ethyl esters. The L-L and D-L stereomera of this compound are prepared via the mixed carboxylic-carbonic acid anhydride procedure (cf. 9) in a manner analogous to that described above for the D-L and L-D stereomers of carbobenzoxyvalylN’carbobenzoxylysine methyl ester. Recrystallization of the crude products is achieved from ethyl acetate-petroleum ether; yields for L-L and D-L stereomers are 85 and 82%, respectively; m.p. 150” and [a]E5 = -8.7” (1% in dimethylformamide) for L-L isomer; m.p. 148” and [ol]i5 = +4.0° (1% in dimethylformamide) for D-L isomer. Anal. Calcd. for C33H390,N3: C, 67.2; H, 6.7; C, 66.7; H, 6.5; N, 7.1. Found for L-L stereomer: N, 6.8. Found for D-L stereomer: C, 67.2; H, 6.7; IT, 7.0. Carbobenzoxyphenylalanyl - hTf - carbobenzoxylysines. Saponification of the L-L and D-L stereomers of carbobensoxyphenylalanyl-N’-carbobenzoxylysine ethyl ester is achieved as follows: To a solution of 3.54 gm of either of these materials, in a mixture of 100 ml of methanol and 50 ml of dioxane, is added 7.0 ml of 1.0 N sodium hydroxide. After storage at room temperature for 10 hours, the pertinent reaction mixture is neutralized with 7.5 ml of 1.0 N hydrochloric acid and concentrated to dryness under a jet of air. Recrystallization of the residual material is effected from ethyl acetate-petroleum ether; yields 95 and m.p. 93ojc for L-L and D-L stereomers, respectively; 108”-111” and [a]:’ = -3.3” (lo/O in dimethylformamide) for L-L isomer; m.p. 147” and [~l]zr’ = $4.0” (lye in dimethylformamide) for D-L isomer. Anal. Calcd. for GH3507N3: C, 66.3; H, 6.3; N, 7.5. Found for L-L stereomer: C, 66.2; H, 6.3; N, C, 66.3; H, 6.3; 7.5. Found for D-L stereomer: N, 7.4. Phenylalanyllysine hydrochlorides. Palladiumcatalyzed hydrogenolysis of the L-L and D-L stereomers of carbobenzoxyphenylalanyl-N’-carbobenzoxylysine is carried out in the same manner as was described above for the preparation of valyllysine hydrochloride. The free dipeptide hydrochlorides are obtained as hygroscopic crystals which do not give satisfactory elemental
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analytical values. However, as each yielded only :L single ninhydrin-positive spot when subjected to paper chromatographic criteria in several solvent systems, they sufficed as chromatographic standards for the present purposes; yield 79% and [a]$ = +24” (lye in water) for L-L isomer; yield 74y0 and [ol]i5 = -80” (1% in water) for n-r. isomer. Na,Nc - Dicarbobenzoxy-L-lysyl-h-phenylalanine benzyl ester. This compound is prepared by the condensation of Ne, NC-dicarbobenzoxy-r-lysine with L-phenylalanine benzyl ester p-toluenesulfonate, via the mixed carboxylic-carbonic acid anhydride procedure (9), in a manner analogous to that described above for the preparation of carbobenzoxy - D - valyl - N’ - carbobenzoxy L-lysine methyl ester. Recrystallization of the crude product is achieved from ethyl acetatepetroleum ether; yield 67%; m.p. 141’; [a]z5 = - 10.4” (1% in dimethylformamide). Anal. Calcd. for &H4iO,N;3: C, 70.0; HZ 6.3; N, 6.5. Found: C, 70.0; H, 6.3; N, 6.5. L-Lysyl-A-phenylalanine hydrochloride. The palladium-catalyzed hydrogenolysis of N*, N*dicarbobenzoxy-r-lysyl-L-phenylalanine benzyl ester is achieved in the same manner as was described above for the preparation of valyllysine hydrochloride. The dipeptide hydrochloride is recrystallized from water-ethanol; yield 76%; m.p. 143” (dec.); [~l]z’ = $35.0” (l’% in water). Anal. Calcd. for C,H,,O,N,Cl: N, 12.7. Found: N, 12.6. RESULTS
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Although the classification of ant,ibiotics is permissible on the basis of their unique antibacterial behavior, a comparison of the gross chemical composition and structure of different antibiotics, such as gramicidin S on t,he one hand, and penicillin or chloramphenicol on the other, gives little indication t,hat they fall within any particular class of chemical compounds. However, certain of the antibiotics which possess a polypeptide structure, e.g., gramicidins S and J, bacitracin A, tyrocidines A and B, and polymixin B, likewise exhibit several ot,her st,riking structural features in common. Thus, antibiotics in this group all reveal a cyclic conformation by virtue of the fact that the a-amino function and a-carboxyl group of each of their constituent amino acid residues are bound in peptide linkage; that the possession of a cyclic conformation is presumably a necessary prerequisite to
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antibiotic activity in molecules of this type is supported by the finding that synthetic open-chain decapeptides, with the same sequence of amino acid residues as are found in gramicidin S, show little if any antibacterial capacity (10-12). Another feature in common lies in the presence, in each of the antibiotics under consideration, of one or more amino acid residues of the n-configuration; although n-phenylalanine residues have hitherto been found most frequently, n-residues of valine, leucine, alloisoleucine and other amino acids also occur. Finally, the aforementioned polypeptide antibiotics exhibit a basic character because they possess free amino groups by virtue of their possession of one or more diamino acid residues; the diamino acid residues that have most frequentfly been encountered include those of ornithine and a, y-diaminobutyric acid. Although dest’ruction of the free amino group by deamination with nitrous acid, or masking its basicity by acylation, results in an appreciable diminution of antibiotic activity, no loss in activity occurs upon the replacement of an ornithine residue with a lysine residue (cf. 13), or upon the conversion of the w-amino group of the diamino acid residue to a guanidino group through treatment with S-methylisothiourea. In any case, it would appear that the possession of a basic character is essential for optimal antibiotic activit.y in molecules of this type. The present studies were undertaken with the dual purpose of ascert’aining, firstly, whether the possession of a cyclic structure, a diamino acid residue, and an amino acid residue of the n-configuration, by relatively small peptides, would suffice to confer antibacterial properties upon these compounds, and secondly, whether such compounds would be susceptible to peptidase action. Among the simplest cyclic peptides which exhibit the desired structural features are 2 : 5-diketopiperazines wherein one or both of the component amino acid residues are of bhe n-configuration and possess a basic character. For the purpose at hand, the L-L, D-D, D-L, and L-D stereomers of both valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride
298
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were selected as model compounds, and these were synthesized according to the procedure described below. Lysine was the diamino acid of choice in each case because of its ready availability, whereas valine was chosen in the former instance and phenylalanine in the latter because of the relatively high frequency of occurrence of these amino acids in cyclic polypeptide antibiotics of natural origin. The reaction sequence employed for the synthesis of the anhydrides of valyllysine and phenylalanyllysine is depicted in Fig. 1. The first step involved the N ,N’-dicyclohexylcarbodiimide-mediated (cf. 14, 15) condensation of formyl-L(or D)-valine [I; R = (CH&CH-] or formyl-L(or D)phenylalanine (I; R = CsHsCH2-) with Nf-carbobenzoxy-r,(or n)-lysine methyl ester (II), in a chloroform-tetrahydrofuran mixture, to yield the corresponding acylated dipeptide methyl ester (III). Treatment of the latter compound with methanolic hydrochloric acid at room temperature resulted in a selective cleavage of the formyl group while leaving the N+-carbobenzoxy group intact. Cyclization of the NC-carbobenzoxydipeptide methyl ester hydrochloride (IV), so derived, to the corresponding N ‘-carbobenzoxylated 2: 5-diketopiperazine (V) was then accomplished via the action of methanolic ammonia; attempts t,o achieve the cyclization reaction in methanol solution cont’aining no added ammonia, or where triethylamine served as the base, were without success. The desired stereomers of valyllysine anhydride hydrochloride [VI;
C02H
R = (CH&CH-] and phenylalanyllysine anhydride hydrochloride (VI; R = CsH,CH2 -) were ultimat,ely secured through catalyt’ic hydrogenolysis of the pertinent, precursor N ‘-carbobenzoxy intermediates (V) in t,he presence of palladium black. All final products were cry&alline materials which showed satisfactory elemental analyses. The L-L and D-D antipodes of the final products exhibited specific rotation values t.hat were equal and opposite within the limits of experimental error, as did the D-L and L-D antipodes. Finally, the products revealed no det,ectable contamination with other ninhydrin-positive mat’erials when subjected to paper chromatography; the Rf values, employing n-butanolacetic acid-pyridinewater (15 : 3: 10: 12, v/v) as t,he solvent system, are given in Table I. With the desired anhydrides of valyllysine and phenylalanyllysine now at hand, an attempt was made to ascertain the susceptibility of these compounds to the hydrolytic action of suitable peptidases. Since L-valyl-L-lysine amide and L-phenylalanyl-L-lysine amide are susceptible to the hydrolybic action of trypsin (16) at’ the lysine carbonyl linkage, it became relevant to determine whether this same enzyme would similarly cleave valyllysine anhydride and phenylalanyllysine anhydride at, the same linkage with t’he formation of valyllysine and phenylalanyllysine, respectively, as t,he hydrolytic product’s. In addition, the known suscept,ibilit,y of phenylalanine-contraining peptides to t’he hydrolytic action of chymot,rypsin at the phenylalanine carbonyl HCO -ljH
?2cH3
HCO-tfH RFH
IZUMIYA
+
CH(CHZ)~NH - CO2CH#k
RtH
-
$OgCH3 tH(CH&NH
I;H2
,,
HCI*tfH2 RFH
tO2Cti3 fHKH&NH
CO - NH IV
- CO2CH$6H5-
-
III
II
I
-COf+C6H5
hO-h
NH-CO / \ RCH CHKH&NH \ CO-NC;
- CO#i&H5+
V
NH-CO / \ RCH CH(CH2)4NH2’HCI \ / CO-NH VI
FIG. 1. Synthetic route to the stereomers of valyllysine anhydride hydrochloride (CH,)&H-) and phenylalanyllysine anhydride hydrochloride (R = C,HsCH2-).
(R =
STUDIES TABLE &
ON POLYPEPTIDE
I
VALUES OF 2,.!-DIKETOPIPERAZINES DIPEPTIDE HYDROCHLORIDES Compound
1-alyllysine anhydride’HC1 1.alyllysine anhydride.HCl Phenylalanyllysine anhydride.HCl Phenylalanyllysine anhydride.HCl J7alyllysine.HCl Valyllysine.HCI Phenylalanyllysine.HCl Phenylalanyllysine.HCl Lysylphenylalanine.HCl 6 Solvent pyridine-water
AND
Configuration
Rf V&lea
L-L Or D-D D-L Or L-D L-L Or D-D
0.50 0.52 0.54
D-L Or L-D
0.60
L-L Or D-D D-L Or L-D L-L D-L L-L
0.25 0.20 0.32 0.27 0.34
system was n-butanol-acetic (15:3:10:12, v/v).
acid-
linkage (cf. 17, 18) indicated that the possible conversion of phenylalanyllysine anhydride to lysylphenylalanine by t’his enzyme should likewise be studied. With these considerations in mind, a 0.01 A4 solution of the pertinent 2,5-diketopiperazine stereomer in 0.1 M phosphate buffer at, pH 7.5 was treated with a saILfree preparation of crystalline trypsin and/or chymotrypsin (0.5 mg of protein N per milliliter of solution), and the mixture incubat,ed at 37” for 24 hours; as the hydrolytic cleavage of these diketopiperazines would lead to the formation of the free dipeptides mentioned above, valyllysine, phenylalanyllysine and lysylphenylalanine were synt,hesized7 for chromatographic con7 Synthesis of each of the four stereomers of valyllysine hydrochloride was achieved by condensation of carbobenzoxy-L (or u)-valine with I\i*-carbobenzoxy-L(or o)-lysine methyl ester via the mixed carboxylic-carbonic acid anhydride method (9), followed by saponification of the dicarbobenzoxylated dipeptide methyl ester, so derived, to the corresponding free acid. Palladiumcatalyzed hydrogenolysis of the pertinent dicarbobenzoxydipeptide then permitted the isolation of L-L, D-D, D-L, and L-D stereomers of ValyllySine as their hydrochloride salts. Essentially the same procedure was employed for the synthesis of the L-L and D-L stereomers of phenylalanyllysine hydrochloride from carbobenzoxy-L (or o)-phenylalanine and NC-carbobenzoxy-L-lysine ethyl ester.
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parison purposes and their Rf values ascertained (see Table I). Paper chromatographic analysis of an aliquot sample of the enzymic digests revealed no ninhydrinpositive material corresponding to any of the dipeptides mentioned above, but rather yielded only a single ninhydrin-positive spot which, in each instance, possessed the same Rf value as the unhydrolyzed 2,5diketopiperazine. Such insusceptibility of t’hese 2,5-diketopiperazines t,o peptidase action is reminiscent of the well-known resistance of the cyclic polypeptide antibiotics to hydrolysis by proteolytic enzymes, a characteristic which is presumably associated with the antibacterial tendencies of these latter compounds. In order to ascertain whether the 2,5diketopiperazines described herein possessed antibacterial act’ivity, the effect of various levels of the L-L, D-D, D-L, and L-D stereomers of both valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride on the growth response of Escherichia coli and penicillin-resistant Staphylococcus aureus was examined. No retardation of growth was noted with either of these microorganisms, even at levels for each compound as high as 100 y per ml. of assay medium. From t’hese findings, it would appear that perhaps other structural characteristics in addition to those alluded to previously, such as a minimum ring size, are necessary before peptide molecules of this type can exhibit) antibacterial properties. Studies presently in progress have as t’heir purpose the determination of just what these additional characteristics may be. REFERENCES 1. GREENSTEIX, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” pp. 1631-1643. Wiley, New York, 1961. 2. SHEEHAN, J. C., AND YANG, D-D.H., J. Am. Chem. Sot. 80, 1154 (1958). In addition, L-phenylalanine hydrochloride was prepared via the condensation of dicarbobenzoxyL-lysine with L-phenylalanine benzyl ester by the mixed anhydride method, followed by catalytic hydrogenolysis of the dicarbobenzoxy-L-lysyl-Lphenylalanine benzyl ester so secured.
300
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3. FISCHER, E., Chem. Ber. 39, 2320 (1906). 4. BERGMANN, M., ZERVAS, L., AND Ross, W. F., J. Biol. Chem. 111, 245 (1935). 5. NEUBERGER, A., AND SANGER, F., Biochem. J. 38, 125 (1944). 6. IZUMIYA, N., AND OTA, S., J. Chem. Sot. Japan 72, 445 (1951). 7. FISCHER, E., AND SCHOELLER, W., Ann. Chem. 367, 1 (1907). 8. CLARKE, H. T., AND DAVIS, A. W., Org. Sgn. 1, 262 (1947). 9. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 978. Wiley. New York, 1961. 10. HARRIS, J. I., AND WORK, T. S., Biochem. J. 46, 582 (1950). 11. BOISSONNAS, P. A., AND SCHUMANN, J., Helv. Chim. Ada 36, 2229, 2237 (1952).
IZUMIYA 12. SCHWYZER, R., AND SIEBER, P., HeEv. Chim. Ada 40, 624 (1957). 13. SCHWYZER, R., AND SIEBER, P., Helv. Chim. Acta 41, 1582 (1958). 14. SHEEHAP;, J. C.,ANDHESS, G. P., J. Am. Chem. Sot. ‘77, 1067 (1955); SHEEHAN, J. C., GOODMAN, M., AXD HESS, G. P., b. Am. Chem. Sot. 78, 1367 (1956). 15. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 1016. Wiley, New York, 1961. 16. IZUMIYA, N., YAMASHITA, T., UCHIO, H., AND KITAGAWA, K., Arch. Biochem. Biophys. 90, 170 (1960). 17. NEURATH, H., APTD SCHWERT, G. W., Chem. Revs. 46, 69 (1950). 18. YAMASHITA, T., AND IZUMIYA, N., J. Biochem. 46, 991 (1959).
OF BIOCHEMISTRY
AND
BIOPHYSICS
Studies I. 2,!5Diketopiperazines MILTON From the Life
108,
292-300
(1964)
on Polypeptide as Models WINITZ
Sciences Laboratory,
Antibiotics
of Cyclic
AND NOBUO United
Received
Technology July
Polypeptide
Antibiotics’
IZUMIYA2 Center, Sunnyvale,
California
13, 1964
The D-D, L-L, D-L, and L-D stereomers of valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride were synthesized for purposes of comparison with the cyclic polypeptide antibiotics. Synthesis was achieved via the dicyclohexylcarbodiimide-mediated condensation of formyl-L(or n)-valine or formyl-r. (or n)-phenylalanine with Ne-carbobenzoxy-n(or n)-lysine methyl ester, deformylation with methanolic hydrochloric acid of the !Va-formyl-NC carbobenzoxydipeptide methyl ester so secured, cyclization of the resulting product with methanolic ammonia to the corresponding N’-carbobenzoxylated 2,5-diketopiperazine, and decarbobenzoxylation of the latter to the desired product by palladium catalyzed hydrogenolysis. All four stereomers of each of the synthetic 2,5-diketopiperazines were insusceptible to the hydrolytic action of proteolytic enzymes, a behavior reminiscent of that of the natural cyclic polypeptide antibiotics; however, none exhibited the antibacterial propensities of the latter. The relationship between the chemical structure of polypeptide antibiotics and their antibacterial properties is discussed. INTRODUCTION
Certain of the polypeptides elaborated by bacteria, such as the gramicidins, bacitracins, tyrocidines, and polymixins, in addition to their remarkable antibacterial properties, possess several striking structural and chemical features in common (1). These include (a) a basic character due to the presence of one or more diamino acid residues, (b) a cyclic conformation, and (c) the possession of at least one amino acid residue of the n-configuration. As 2,5diketopiperazines, wherein one or both of the component amino acid residues possess a n-configuration and a basic character, incorporate these same characteristics, it 1 The experimental portion of this work was carried out at the National Cancer Institute, National Institutes of Health, U. S. Public Health Service, Bethesda, Maryland, and was previously reported in part [Abstracts, 137th Meeting of the American Chemical Society, 30C (1960)]. 2 Present address : Laboratory of Biochemistry, Faculty of Science, Kyushu University, Fukuoka, Japan.
became of interest to ascertain whether these relatively simple molecules would also exhibit the antibact’erial properties of their more complex naturally occurring counterparts. The present study, which was conceived with this in mind, includes the synthesis of each of the four stereomers of valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride as well as experiments designed to test both t’he antibacterial propensity and susceptibility to enzymic hydrolysis of these “antibiotic models.” Also included in the present study is the synthesis of the pertinent stereomers of lysylphenylalanine, phenylalanyllysine, and valyllysine, which were prepared as reference compounds for purposes of chromat’ographic comparison. EXPERIMENTAL
PREPARATION OF THE VALYLLYSINE ANHYDRIDES Formyl-L-valine. This compound was prepared according to the general formylation procedure of Sheehan and Yang (2). Thus, a solution of 35.2 292
STUDIES
ON POLYPEPTIDE
gm of L-valine in 630 ml of 98yo formic acid is chilled in an ice-bath and treated dropwise, over a period of 20 minutes, with 210 ml of acetic anhydride. Stirring at ice-bath temperature is continued for an additional 30 minutes, after which time the reaction mixture is stirred at room temperature for 1 hour. The reaction mixture is then treated with 300 ml of water, with cooling in the ice-bath, and is concentrated to dryness under reduced pressure. The resulting residue is repeatedly treated with a little water and concentrated to dryness in order to remove the final traces of formic acid. A crystalline residue is ultimately obtained which is collected with the aid of a little cold water. Recrystallization is effected from boiling water; yield 28.5 gm. An additional 9.6 gm may be recovered from the aqueous filtrates; total yield 38.1 gm (85%); m.p. 152”153” [lit. values, 156” (3) and 148”149” (2)]. Formyl-D-valine. Preparation of this compound was achieved in the same manner as described for the L-antipode above; yield 84%; m.p. 152”153” [lit. values, 156” (3) and 148”149” (2)]. N’-Carbobenzoxy-z-lysine methyl ester hydrochloride. This compound was prepared according to the procedure of Bergmann et al. (4); m.p. 110”113” [lit. value, 117” (4)]; [a]$ = +17.2” (2$& in water). Ne-Carbobenzoxy-L-lysine methyl ester hydrochloride. Preparation of this compound was achieved in the same manner as the L-antipode above; m.p. 109”-113”. Recrystallization from hot acetone-ether raised the melting point to 114”115’; [CX]~~= -16.9” (2yo in water).$ Anal. Calcd. for C15H2304N2Cl: C, 54.5; H, 7.0; K, 8.5. Found: C, 54.5; H, 7.2; N, 8.4. Formylvalyl-NE-carbobenzoxylysine methyl esters. Directions for the preparation for each of the four stereomers (L-L, D-D, D-L, and L-D) of this compound are given in what follows: To a stirred solution of 7.26 gm of formyl-L (or n)-valine, 16.55 gm of N*-carbobenzoxy-L (or n)-lysine methyl ester hydrochloride, and 7.0 ml of triethylamine in a mixture of 150 ml of tetrahydrofuran and 150 ml of chloroform, at O”, is added 10.3 gm of N, N’-dicyclohexylcarbodiimide. The reaction mixture is stored at 4” overnight, after which time the L-L and D-D stereomers are recovered according to the procedure given under (a) and the D-L and L-D stereomers are recovered as described 3 The n-lysine employed in this preparation was secured by conversion of Ne-benzoyl-n-lysine to the corresponding a-bromo analog by treatment with nitrosyl bromide, followed by aminolysis and subsequent acid hydrolysis of the Nf-benzoyln-lysine so derived (5, 6).
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under (b). (a) The precipitated N, N’-disubstituted urea is removed by filtration and the filtrate is concentrated under a jet of dry air to an oil. After dissolving the latter in 250 ml. of chloroform, the solution is washed successively with 3% hydrochloric acid, water, 5oJo sodium bicarbonate and water, and then concentrated to dryness under a jet of air. The syrupy residue, when treated with 35 ml of ether and 80 ml of petroleum ether, separates as a solid material which is recrystallized from ethyl acetate-ether-petroleum ether (30 ml:60 ml:60 ml); yield 56% for L-L and 59% for D-D stereomer; m.p. 140”143” for both stereomers; [a]Z5 = -11.9” and +12.0” (2% in dimethylformamide), respectively, for L-L and D-D stereomers. (b) The N,N-disubstituted urea, admixed with a small amount of the desired product, precipitates. After taking the crystals of the latter into solution by warming at 50”, the reaction mixture is cooled to room temperature under tap water and the urea derivative separated by filtration. The remainder of the procedure is identical with that described under (a) above. Recrystallization is effected from ethyl acetateether-petroleum ether (90 ml:50 ml:50 ml); yield 55yo for D-L and 58% for L-D stereomers; m.p. 149” for D-L and 148” for L-D stereomer; [a]: = -16.0” and +14.9” (2% in dimethylformamide), respectively, for D-L and L-D stereomers. Anal. Calcd. for CzlH,,06N,: C, 59.8; H, 7.4; C, 60.3; H, 7.4; N, 10.0. Found for L-L stereomer: N, 9.8. Found for D-D stereomer: C, 60.1; H, 7.3; N, 9.8. Found for D-L stereomer: C, 60.2; H, 7.4; N, 9.9. Found for L-D stereomer: C, 60.0; H, 7.5; N, 9.8. Valyl-Ne-carbobenzoxylysine methyl ester hydrochlorides. Each of the four stereomers of this compound was prepared from the corresponding N-formylated dipeptide derivative as follows : A suspension of 8.43 gm of the pertinent formylvalyl-N’-carbobenzoxylysine methyl ester stereomer in 60 ml of 0.5 N methanolic-HCl is stored at room temperature for 48 hours. The resulting clear solution is concentrated under reduced pressure at a temperature not exceeding 40”. Treatment of the residual material with acetone and ether leads to a crystalline product! which is recrystallized from acetone-ether; yield 77, 73, 80, 161”, and 161”, and 75%; m.p. 154”, 153”154”, and [ol]z5 = O”, O”, -43.0” and -l-43.8” (2% in water), for the L-L, D-D, D-L, and L-D stereomers, respectively. 4 Although the crystallization of stereomers occurs more readily the L-L and D-D stereomers, the latter crystallize within the space of an procedure described. D-L
the L-D and than that of nevertheless hour by the
294
WINITZ
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Anal. Calcd. for C&H3206N3C1: C, 55.8; H, 7.5; N, 9.8; Cl, 8.3. Found for L-L stereomer: C, 55.8; H, 7.5; N, 9.7; Cl, 8.4. Found for D-D stereomer: C, 55.5; H, 7.6; N, 9.7; Cl, 8.2. Found for D-L stereomer: C, 55.6; H, 7.7; N, 9.7; Cl, 8.5. C, 55.8; H, 7.6; N, 9.8; Found for L-D stereomer: Cl, 8.6. Valyl-NC-carbobenzoxylysine anhydrides. Each of the four stereomers of this compound was prepared as follows: To a solution of 4.3 gm of the pertinent valyl-Ne-carbobenzoxylysine methyl ester hydrochloride in 10 ml of methanol is added 100 ml of methanol which has been previously saturated with dry ammonia gas at 0”. After storage for 24 hours at room temperature in a glass-stoppered pressure bottle,6 the reaction mixture is concentrated to dryness under a jet of air, and the crystalline residue is recovered by filtration with the aid of a little water. Recrystallization of the L-L and D-D stereomers is achieved from 85 ml of 3Oyc ethanol, whereas 100 ml of 50% ethanol is used with the L-D and D-L stereomers; yield 88,86,90, and 91%; m.p. 162”164”, 161”-164”; 198”-2OO”, and 199”-201”, and [LY]~~ = -46.1”, t45.00, -4.3”, and +3.2” (2% in dimethylformamide), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for C9Hz704N3: C, 63.1; H, 7.5; N, 11.6. Found for L-L stereomer : C, 63.0; H, 7.5; N, 11.6. Found for D-D stereomer: C, 63.1; H, 7.3; N, 11.4. Found for D-L stereomer; C, 63.2; H, 7.4; N, 11.6. Found for L-D stereomer: C, 63.1, H, 7.4; N, 11.7. Valyllysine anhydride hydrochlorides. Each of the four stereomers of this compound was prepared as follows: A suspension of 1.81 gm of the pertinent stereomer of valyl-Nf-carbobenzoxylysine anhydride in a mixture of 11 ml of 0.5 N methanolic-HCl and 40 ml of methanol is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon completion of the reaction, the catalyst is removed by filtration, with prior warming if necessary to dissolve any product which may have precipitated, and the filtrate is concentrated to dryness under reduced pressure. The crystalline product is isolated by filtration with the aid of a mixture of 12 ml of acetone and 18 ml of ether, and subsequently recrystallized from methanol-acetone; yield 77, 84, 90, and 90%; m.p. 254”256” (dec.), 253”255” (dec.), 282” (dec.), and 282”-283” (dec.), and [ali = -54.1”, +53.6”, -5.4”, and +5.8” (2% in a 5 In the case of the L-D and D-L stereomers, mass of crystals appears after about 1 hour of storage, whereas complete solution is maintained with the L-L and D-D stereomers over the entire 24-hour reaction period.
IZUMIYA water), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for CiiH*202N$l: C, 50.0; H, 8.4; N, 15.9: Cl, 13.5. Found for D-D stereomer: C, 49.8; H, 8.5; N, 15.7; Cl, 13.5. Found for D-L stereomer: C, 50.3; H, 8.5; N, 15.7; Cl, 13.6. Found for L-D stereomer: C, 50.0; H, 8.4; N, 15.7; Cl, 13.3.
PREPARATION OF THE PHENYLALANYLLYSINE ANHYDRIDES Formylphenylalanines. The L- and n-antipodes were prepared following the same procedure described above for the formylvalines; yield 75-80%; m.p. 167” for both antipodes [lit. value for L- and n-antipodes was 167” (7)]. Formylphenylalanyl - N’ - carbobenzoxylysine methyl esters. Preparation of each of the four stereomers of this compound is achieved by treatment of a solution of 9.65 gm of formyl-L(or D)phenylalanine, 16.55 gm of N’-carbobenzoxy-n (or n)-lysine methyl ester and 7.0 ml of triethylamine in tetrahydrofuran-chloroform with 10.3 gm of N, N’-dicyclohexylcarbodiimide, following essentially the same procedure employed for the of formylvalyl-NC-carbobenzoxypreparation lysine methyl ester (see above). Recrystallization of the crude products is achieved from ethyl acetate-petroleum ether; yield 75,78,68, and 67%; mp. 125”, 125”, 134”136”, and 134-135”, and [fY]2 = -4.50, +3.9”, -ll.S”, and f11.0” (1% in dimethylformamide), for the L-L, D-D, D-L, and L-D stereomers, respectively. Anal. Calcd. for C25H3i0eN3: C, 63.9; H, 6.7; C, 64.0; H, 6.8; N, 9.0. Found for L-L stereomer: N, 9.3. Found for D-D stereomer: C, 63.8; H, 6.7; N, 9.0. Found for D-L stereomer: C, 64.2; H, 7.0; N, 9.2. Found for L-D stereomer: C, 64.3; H, 6.7; N, 9.2. Phenylalanyl-Ne-carbobenzoxylysine methyl ester hydrochlorides. The pertinent formyldipeptide ester stereomer was treated in the same manner as described under the preparation of valyl-Necarbobenzoxylysine methyl ester hydrochloride. could be Although the L-L and D-D stereomers recrystallized from methanol-acetone-ether, attempts to solidify the D-L or L-D forms were without success; yield 72% for L-L and 75% for D-D stereomer; m.p. 153”154” and 154”155” for L-L and D-D stereomer respectively; [(Y]:~ = -9.3” (1% in dimethylformamide) for L-L stereomer and +lO.l” for D-D stereomer. Anal. Calcd. for C&H,20sN,Cl: C, 60.3; H, 6.8; N, 8.8. Found for L-L stereomer: C, 60.3; H, 7.0; N, 8.8. Found for D-D stereomer: C, 60.1; H, 7.0; N, 8.6. Phenylalanyl-Nf-carbobenzoxylysine anhydrides. Conversion of each of the precursor methyl ester
STUDIES
ON POLYPEPTIDE
hydrochloride stereomers to the corresponding anhydrides followed the same procedure employed in the preparation of valyl-N%arbobenzoxylysine anhydride. Recrystallization was effected from 7GBOyo ethanol; yield 83,87,85,6 and 75y, m.p. 206”ZOB”, 205”-207”, 171”, and 171”, and [a]? = -26.1”, +25.5”, -lO.O”, and +lO.O” (1% in dimethylformamide), for the L-L, n-n, D-L, and L-D stereomers, respectively. ilnal. Calcd. for C23H2104N3: C, 67.5; H, 6.7; N, 10.3. Found for L-L stereomer: C, 67.5; H, 6.8; N, 10.2. Found for D-D stereomer: C, 67.3; H, 6.8; N, 10.2. Found for D-L stereomer: C, 67.9; H, 7.0; N, C, 67.9; H, 7.1; N, 10.3. Found for L-D stereomer: 10.2. anhydride hydrochlorides. Phenylalanyllysine Each of the stereomera of this compound was prepared as follows: A suspension of 2.05 gm of the pertinent phenylalanyl - N* - carbobenzoxylysine anhydride in 50 ml of glacial acetic acid is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon termination of the reaction, the catalyst is removed by filtration, the filtrate concentrated to dryness under reduced pressure, the residual material dissolved in water, and the aqueous solution treated with 5.3 ml of 1N hydrochloric acid. After concentration of the solution of dryness, the residual product is recovered by filtration with the aid of acetone. Recrystallization is effected from methanolacetone; yield 94,91,88, and 90% for the L-L, D-D, D-L, and L-D stereomers, respectively; [a]? = $2.5” (lo/, in water) for the L-L and -2.0” for the D-D stereomer; [ol]Z5 = -45” (0.3% in water) for the D-L and +48” for the L-D stereomer. Anal. Calcd. for C&H2202N$l: C, 57.8; H, 7.1; S, 13.5. Found for L-L stereomer: C, 57.7; H, 7.3; N, 13.5. Found for D-D stereomer: C, 57.6; H, 7.1; N, 13.5. Found for D-L stereomer: C, 58.1; H, 7.2; N, 13.7. Found for L-D stereomer: C, 57.8; H, 7.3; N, 13.4.
PREPARATION OF REFERENCE COMPOUNDS For purposes of chromatographic comparison, each of the four stereomers of valyllysine was synthesized, as were the L-L and D-L isomers of phenylisomer of lysyland the L-L alanyllysine phenylalanine. Preparative procedures for the intermediates leading to these compounds, as well as for the final products themselves, are given in what follows. Carbobenzoxyvalyl-N’-carbobenzoxylysine methyl 6 Yields of D-L and L-D stereomers based on the formylphenylalanyl-NE-carbobenzoxylyparent sine methyl ester.
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esters. The L-L, D-D, D-L, and L-D stereomers of this compound were prepared as follows: A solution of 5.03 gm of carbobenxoxy-L (or n)-valine and 2.8 ml of triethylamine in 40 ml of toluene is chilled to -5” and treated with 2.7 ml of isobutyl chlorocarbonate. Ten minutes later, a chilled solution of 6.61 gm of N’-carbobenzoxy-L (or u)-lysine methyl ester hydrochloride and 2.8 ml of triethylamine in 40 ml of chloroform is added thereto, and the reaction mixture is stored at room temperature overnight. The L-L and D-D stereomers are then recovered according to the procedure given under (a) and the D-L and I,-D stereomers are recovered as described under (b). (a) After being washed successively with water, dilute hydrochloric acid, water, half-saturated sodium bicarbonate solution and water, the organic fraction is dried over anhydrous sodium sulfate and concentrated to dryness. The crystalline residue is recrystallized from ethyl acetatepetroleum ether; yield 76% for L-L and 74% for D-D stereomer; m.p. 124”126” and [ol]$ = 0” (2y0 in dimethylformamide) for both stereomers. (b) The mass of crystals which precipitate are filtered off after the addition of 70 ml. of petroleum ether. Recrystallization is achieved from acetonepetroleum ether; yield 71 and 7770, m.p. 163” and 164”, and [a]:’ = -15.4” and +16.1” (2% in dimethylformamide), for the D-L and L-D stereomers respectively. Anal. Calcd. for GsH,TO,N3: C, 63.7; H, 7.1; N, 8.0. Found for L-L stereomer: C, 63.7; H, 7.1; N, 8.0. Found for D-D stereomer: C, 63.3; H, 7.3; N, 7.9. Found for D-L stereomer: C, 63.6; H, 7.2; N, 7.8. Found for L-D stereomer: C, 63.3; H, 7.1; N, 8.0. Carbobenzoxyvalyl-N’-carbobenzoxylysines. The four stereomers of this compound were prepared via saponification of the corresponding methyl ester derivatives. Conditions employed for saponification of the L-L and D-D antipodes are given under (a), while the D-L and L-D antipodes were saponified as described under (b). (a) A solution of 7.39 gm of the pertinent carbobenzoxyvalyl-Ncarbobenzoxylysine methyl ester in 100 ml of methanol is treated with 16 ml of IN sodium hydroxide. The reaction mixture is stored at room temperature for 25 hours, after which time it is neutralized with 17 ml of IN hydrochloric acid and concentrated to dryness under a jet of air. The residual material is filtered with the aid of a little water, air dried, and recrystallized from ethyl acetate-petroleum ether; yield 90% for L-L and 94% for D-D stereomers; m.p. 139’-140” for both stereomers; [ol]? = +5.0” and -5.9” (2% in respectively, for L-L and dimethylformamide), D-D stereomera. (b) A suspension of 7.39 gm of the
296
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AN 1) IZUMIYA
pertinentc arbobenzoxyvalylN’carbobenzoxylysine methyl ester in a mixture of 220 ml of methanol, 55 ml of dioxane, and 32 ml of 0.5 N sodium hydroxide is stirred at 25” for 7 hours. The resulting clear solution is neutralized with 17 ml of IN hydrochloric acid and is concentrated to dryness under a jet of air; the residual material is recovered and recrystallized as given under (a) above; yield 97% for D-L and 98% for L-D stereomers; m.p. 124”126” for both stereomers; [ol]$ = +13.7” and -15.0” (2% in dimethylformamide), respectively, for D-L and L-D stereomers. Anal. Calcd. for CLH3507N3: C, 63.1; H, 6.9; N, 8.2. Found for L-L stereomer: C, 62.8; H, 6.9; N, 8.1. Found for D-D stereomer: C, 62.7; H, 6.7; N, 8.2. Found for D-L stereomer: C, 62.9; H, 7.0; N, 8.2. Found for L-D stereomer: C, 63.1; H, 6.8; N, 8.1. Valyllysine hydrochlorides. A solution of 5.14 gm of each of the four stereomers of carbobenzoxyvalyl-NC-carbobenzoxylysine in a mixture of 120 ml of methanol and 20.6 ml of 0.5 N hydrochloric acid is subjected to hydrogenolysis in the presence of palladium black catalyst. Upon termination of the reaction, the pertinent solution is filtered and then concentrated to dryness under a jet of air. (a) The concentrates of the L-L and D-D stereomers yield crystalline residual masses, each of which is recovered by filtration with the aid of a little ethanol. Recrystallization is effected from a solution in 5 ml of water by the addition of 30 ml of ethanol; yield 85% for L-L and 84% for D-D stereomers; m.p. 241”243” (dec.) for both stereomers; [ali = f25.4” and -25.3” (2% in water), respec(b) The concentively, for L-L and D-D antipodes. trates of the L-D and D-L stereomers both yield a syrupy residue which can be solidified by treatment with ethanol and acetone. The solids so obtained are slightly hygroscopic and do not yield satisfactory elementary analytical values. However, their paper chromatographic behavior in a number of solvent systems indicates that they give only a single ninhydrin-positive spot and hence are suitable as chromatographic reference compounds for the present purposes; [ol]t5 = +48” and -50” (2% in water) for the L-D and D-L antipodes, respectively. Anal. Calcd. for CiiH2403N&1: C, 46.8; H, 8.6; N, 14.9; Cl, 12.6. Found for L-L stereomer: C, 46.8; H, 8.9; N, 14.9; Cl, 12.3. Found for D-D stereomer: C, 46.6; H, 8.8; N, 15.0; Cl, 12.4. NC-Carbobenzoxy-L-lysine ethyl ester p-toluenesuljonate. A suspension of 56.0 gm of N’-carbobenaoxy-n-lysine and 42 gm of p-toluenesulfonic acid monohydrate in a mixture of 80 ml of ethanol and 400 ml of carbon tetrachloride is refluxed for 24 hours in an apparatus patterned after that
developed by Clarke and Davis (8) for the removal of wrater azeotropically. Upon termination of the reaction, as indicated by a cessation of water liberation, the reaction mixture is concentrated to a small volume under a jet of dry air and the residual material treated with an excess of ether. The precipitate is recovered by filtration and recrystallized from acetone-ether; yield 92 gm (96%); m.p. 87”. Anal. Calcd. for C&H3207N2S: C, 57.5; H, 6.7; N, 5.8. Pound: C, 57.3; H, 6.6; N, 5.9. Carbobenzoxyphenylalanyl - NC carbobenzoxylysine ethyl esters. The L-L and D-L stereomera of this compound are prepared via the mixed carboxylic-carbonic acid anhydride procedure (cf. 9) in a manner analogous to that described above for the D-L and L-D stereomers of carbobenzoxyvalylN’carbobenzoxylysine methyl ester. Recrystallization of the crude products is achieved from ethyl acetate-petroleum ether; yields for L-L and D-L stereomers are 85 and 82%, respectively; m.p. 150” and [a]E5 = -8.7” (1% in dimethylformamide) for L-L isomer; m.p. 148” and [ol]i5 = +4.0° (1% in dimethylformamide) for D-L isomer. Anal. Calcd. for C33H390,N3: C, 67.2; H, 6.7; C, 66.7; H, 6.5; N, 7.1. Found for L-L stereomer: N, 6.8. Found for D-L stereomer: C, 67.2; H, 6.7; IT, 7.0. Carbobenzoxyphenylalanyl - hTf - carbobenzoxylysines. Saponification of the L-L and D-L stereomers of carbobensoxyphenylalanyl-N’-carbobenzoxylysine ethyl ester is achieved as follows: To a solution of 3.54 gm of either of these materials, in a mixture of 100 ml of methanol and 50 ml of dioxane, is added 7.0 ml of 1.0 N sodium hydroxide. After storage at room temperature for 10 hours, the pertinent reaction mixture is neutralized with 7.5 ml of 1.0 N hydrochloric acid and concentrated to dryness under a jet of air. Recrystallization of the residual material is effected from ethyl acetate-petroleum ether; yields 95 and m.p. 93ojc for L-L and D-L stereomers, respectively; 108”-111” and [a]:’ = -3.3” (lo/O in dimethylformamide) for L-L isomer; m.p. 147” and [~l]zr’ = $4.0” (lye in dimethylformamide) for D-L isomer. Anal. Calcd. for GH3507N3: C, 66.3; H, 6.3; N, 7.5. Found for L-L stereomer: C, 66.2; H, 6.3; N, C, 66.3; H, 6.3; 7.5. Found for D-L stereomer: N, 7.4. Phenylalanyllysine hydrochlorides. Palladiumcatalyzed hydrogenolysis of the L-L and D-L stereomers of carbobenzoxyphenylalanyl-N’-carbobenzoxylysine is carried out in the same manner as was described above for the preparation of valyllysine hydrochloride. The free dipeptide hydrochlorides are obtained as hygroscopic crystals which do not give satisfactory elemental
STUDIES
ON POLYPEPTIDE
analytical values. However, as each yielded only :L single ninhydrin-positive spot when subjected to paper chromatographic criteria in several solvent systems, they sufficed as chromatographic standards for the present purposes; yield 79% and [a]$ = +24” (lye in water) for L-L isomer; yield 74y0 and [ol]i5 = -80” (1% in water) for n-r. isomer. Na,Nc - Dicarbobenzoxy-L-lysyl-h-phenylalanine benzyl ester. This compound is prepared by the condensation of Ne, NC-dicarbobenzoxy-r-lysine with L-phenylalanine benzyl ester p-toluenesulfonate, via the mixed carboxylic-carbonic acid anhydride procedure (9), in a manner analogous to that described above for the preparation of carbobenzoxy - D - valyl - N’ - carbobenzoxy L-lysine methyl ester. Recrystallization of the crude product is achieved from ethyl acetatepetroleum ether; yield 67%; m.p. 141’; [a]z5 = - 10.4” (1% in dimethylformamide). Anal. Calcd. for &H4iO,N;3: C, 70.0; HZ 6.3; N, 6.5. Found: C, 70.0; H, 6.3; N, 6.5. L-Lysyl-A-phenylalanine hydrochloride. The palladium-catalyzed hydrogenolysis of N*, N*dicarbobenzoxy-r-lysyl-L-phenylalanine benzyl ester is achieved in the same manner as was described above for the preparation of valyllysine hydrochloride. The dipeptide hydrochloride is recrystallized from water-ethanol; yield 76%; m.p. 143” (dec.); [~l]z’ = $35.0” (l’% in water). Anal. Calcd. for C,H,,O,N,Cl: N, 12.7. Found: N, 12.6. RESULTS
AND
DISCUSSIOK
Although the classification of ant,ibiotics is permissible on the basis of their unique antibacterial behavior, a comparison of the gross chemical composition and structure of different antibiotics, such as gramicidin S on t,he one hand, and penicillin or chloramphenicol on the other, gives little indication t,hat they fall within any particular class of chemical compounds. However, certain of the antibiotics which possess a polypeptide structure, e.g., gramicidins S and J, bacitracin A, tyrocidines A and B, and polymixin B, likewise exhibit several ot,her st,riking structural features in common. Thus, antibiotics in this group all reveal a cyclic conformation by virtue of the fact that the a-amino function and a-carboxyl group of each of their constituent amino acid residues are bound in peptide linkage; that the possession of a cyclic conformation is presumably a necessary prerequisite to
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antibiotic activity in molecules of this type is supported by the finding that synthetic open-chain decapeptides, with the same sequence of amino acid residues as are found in gramicidin S, show little if any antibacterial capacity (10-12). Another feature in common lies in the presence, in each of the antibiotics under consideration, of one or more amino acid residues of the n-configuration; although n-phenylalanine residues have hitherto been found most frequently, n-residues of valine, leucine, alloisoleucine and other amino acids also occur. Finally, the aforementioned polypeptide antibiotics exhibit a basic character because they possess free amino groups by virtue of their possession of one or more diamino acid residues; the diamino acid residues that have most frequentfly been encountered include those of ornithine and a, y-diaminobutyric acid. Although dest’ruction of the free amino group by deamination with nitrous acid, or masking its basicity by acylation, results in an appreciable diminution of antibiotic activity, no loss in activity occurs upon the replacement of an ornithine residue with a lysine residue (cf. 13), or upon the conversion of the w-amino group of the diamino acid residue to a guanidino group through treatment with S-methylisothiourea. In any case, it would appear that the possession of a basic character is essential for optimal antibiotic activit.y in molecules of this type. The present studies were undertaken with the dual purpose of ascert’aining, firstly, whether the possession of a cyclic structure, a diamino acid residue, and an amino acid residue of the n-configuration, by relatively small peptides, would suffice to confer antibacterial properties upon these compounds, and secondly, whether such compounds would be susceptible to peptidase action. Among the simplest cyclic peptides which exhibit the desired structural features are 2 : 5-diketopiperazines wherein one or both of the component amino acid residues are of bhe n-configuration and possess a basic character. For the purpose at hand, the L-L, D-D, D-L, and L-D stereomers of both valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride
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were selected as model compounds, and these were synthesized according to the procedure described below. Lysine was the diamino acid of choice in each case because of its ready availability, whereas valine was chosen in the former instance and phenylalanine in the latter because of the relatively high frequency of occurrence of these amino acids in cyclic polypeptide antibiotics of natural origin. The reaction sequence employed for the synthesis of the anhydrides of valyllysine and phenylalanyllysine is depicted in Fig. 1. The first step involved the N ,N’-dicyclohexylcarbodiimide-mediated (cf. 14, 15) condensation of formyl-L(or D)-valine [I; R = (CH&CH-] or formyl-L(or D)phenylalanine (I; R = CsHsCH2-) with Nf-carbobenzoxy-r,(or n)-lysine methyl ester (II), in a chloroform-tetrahydrofuran mixture, to yield the corresponding acylated dipeptide methyl ester (III). Treatment of the latter compound with methanolic hydrochloric acid at room temperature resulted in a selective cleavage of the formyl group while leaving the N+-carbobenzoxy group intact. Cyclization of the NC-carbobenzoxydipeptide methyl ester hydrochloride (IV), so derived, to the corresponding N ‘-carbobenzoxylated 2: 5-diketopiperazine (V) was then accomplished via the action of methanolic ammonia; attempts t,o achieve the cyclization reaction in methanol solution cont’aining no added ammonia, or where triethylamine served as the base, were without success. The desired stereomers of valyllysine anhydride hydrochloride [VI;
C02H
R = (CH&CH-] and phenylalanyllysine anhydride hydrochloride (VI; R = CsH,CH2 -) were ultimat,ely secured through catalyt’ic hydrogenolysis of the pertinent, precursor N ‘-carbobenzoxy intermediates (V) in t,he presence of palladium black. All final products were cry&alline materials which showed satisfactory elemental analyses. The L-L and D-D antipodes of the final products exhibited specific rotation values t.hat were equal and opposite within the limits of experimental error, as did the D-L and L-D antipodes. Finally, the products revealed no det,ectable contamination with other ninhydrin-positive mat’erials when subjected to paper chromatography; the Rf values, employing n-butanolacetic acid-pyridinewater (15 : 3: 10: 12, v/v) as t,he solvent system, are given in Table I. With the desired anhydrides of valyllysine and phenylalanyllysine now at hand, an attempt was made to ascertain the susceptibility of these compounds to the hydrolytic action of suitable peptidases. Since L-valyl-L-lysine amide and L-phenylalanyl-L-lysine amide are susceptible to the hydrolybic action of trypsin (16) at’ the lysine carbonyl linkage, it became relevant to determine whether this same enzyme would similarly cleave valyllysine anhydride and phenylalanyllysine anhydride at, the same linkage with t’he formation of valyllysine and phenylalanyllysine, respectively, as t,he hydrolytic product’s. In addition, the known suscept,ibilit,y of phenylalanine-contraining peptides to t’he hydrolytic action of chymot,rypsin at the phenylalanine carbonyl HCO -ljH
?2cH3
HCO-tfH RFH
IZUMIYA
+
CH(CHZ)~NH - CO2CH#k
RtH
-
$OgCH3 tH(CH&NH
I;H2
,,
HCI*tfH2 RFH
tO2Cti3 fHKH&NH
CO - NH IV
- CO2CH$6H5-
-
III
II
I
-COf+C6H5
hO-h
NH-CO / \ RCH CHKH&NH \ CO-NC;
- CO#i&H5+
V
NH-CO / \ RCH CH(CH2)4NH2’HCI \ / CO-NH VI
FIG. 1. Synthetic route to the stereomers of valyllysine anhydride hydrochloride (CH,)&H-) and phenylalanyllysine anhydride hydrochloride (R = C,HsCH2-).
(R =
STUDIES TABLE &
ON POLYPEPTIDE
I
VALUES OF 2,.!-DIKETOPIPERAZINES DIPEPTIDE HYDROCHLORIDES Compound
1-alyllysine anhydride’HC1 1.alyllysine anhydride.HCl Phenylalanyllysine anhydride.HCl Phenylalanyllysine anhydride.HCl J7alyllysine.HCl Valyllysine.HCI Phenylalanyllysine.HCl Phenylalanyllysine.HCl Lysylphenylalanine.HCl 6 Solvent pyridine-water
AND
Configuration
Rf V&lea
L-L Or D-D D-L Or L-D L-L Or D-D
0.50 0.52 0.54
D-L Or L-D
0.60
L-L Or D-D D-L Or L-D L-L D-L L-L
0.25 0.20 0.32 0.27 0.34
system was n-butanol-acetic (15:3:10:12, v/v).
acid-
linkage (cf. 17, 18) indicated that the possible conversion of phenylalanyllysine anhydride to lysylphenylalanine by t’his enzyme should likewise be studied. With these considerations in mind, a 0.01 A4 solution of the pertinent 2,5-diketopiperazine stereomer in 0.1 M phosphate buffer at, pH 7.5 was treated with a saILfree preparation of crystalline trypsin and/or chymotrypsin (0.5 mg of protein N per milliliter of solution), and the mixture incubat,ed at 37” for 24 hours; as the hydrolytic cleavage of these diketopiperazines would lead to the formation of the free dipeptides mentioned above, valyllysine, phenylalanyllysine and lysylphenylalanine were synt,hesized7 for chromatographic con7 Synthesis of each of the four stereomers of valyllysine hydrochloride was achieved by condensation of carbobenzoxy-L (or u)-valine with I\i*-carbobenzoxy-L(or o)-lysine methyl ester via the mixed carboxylic-carbonic acid anhydride method (9), followed by saponification of the dicarbobenzoxylated dipeptide methyl ester, so derived, to the corresponding free acid. Palladiumcatalyzed hydrogenolysis of the pertinent dicarbobenzoxydipeptide then permitted the isolation of L-L, D-D, D-L, and L-D stereomers of ValyllySine as their hydrochloride salts. Essentially the same procedure was employed for the synthesis of the L-L and D-L stereomers of phenylalanyllysine hydrochloride from carbobenzoxy-L (or o)-phenylalanine and NC-carbobenzoxy-L-lysine ethyl ester.
ANTIBIOTICS.
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parison purposes and their Rf values ascertained (see Table I). Paper chromatographic analysis of an aliquot sample of the enzymic digests revealed no ninhydrinpositive material corresponding to any of the dipeptides mentioned above, but rather yielded only a single ninhydrin-positive spot which, in each instance, possessed the same Rf value as the unhydrolyzed 2,5diketopiperazine. Such insusceptibility of t’hese 2,5-diketopiperazines t,o peptidase action is reminiscent of the well-known resistance of the cyclic polypeptide antibiotics to hydrolysis by proteolytic enzymes, a characteristic which is presumably associated with the antibacterial tendencies of these latter compounds. In order to ascertain whether the 2,5diketopiperazines described herein possessed antibacterial act’ivity, the effect of various levels of the L-L, D-D, D-L, and L-D stereomers of both valyllysine anhydride hydrochloride and phenylalanyllysine anhydride hydrochloride on the growth response of Escherichia coli and penicillin-resistant Staphylococcus aureus was examined. No retardation of growth was noted with either of these microorganisms, even at levels for each compound as high as 100 y per ml. of assay medium. From t’hese findings, it would appear that perhaps other structural characteristics in addition to those alluded to previously, such as a minimum ring size, are necessary before peptide molecules of this type can exhibit) antibacterial properties. Studies presently in progress have as t’heir purpose the determination of just what these additional characteristics may be. REFERENCES 1. GREENSTEIX, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” pp. 1631-1643. Wiley, New York, 1961. 2. SHEEHAN, J. C., AND YANG, D-D.H., J. Am. Chem. Sot. 80, 1154 (1958). In addition, L-phenylalanine hydrochloride was prepared via the condensation of dicarbobenzoxyL-lysine with L-phenylalanine benzyl ester by the mixed anhydride method, followed by catalytic hydrogenolysis of the dicarbobenzoxy-L-lysyl-Lphenylalanine benzyl ester so secured.
300
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3. FISCHER, E., Chem. Ber. 39, 2320 (1906). 4. BERGMANN, M., ZERVAS, L., AND Ross, W. F., J. Biol. Chem. 111, 245 (1935). 5. NEUBERGER, A., AND SANGER, F., Biochem. J. 38, 125 (1944). 6. IZUMIYA, N., AND OTA, S., J. Chem. Sot. Japan 72, 445 (1951). 7. FISCHER, E., AND SCHOELLER, W., Ann. Chem. 367, 1 (1907). 8. CLARKE, H. T., AND DAVIS, A. W., Org. Sgn. 1, 262 (1947). 9. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 978. Wiley. New York, 1961. 10. HARRIS, J. I., AND WORK, T. S., Biochem. J. 46, 582 (1950). 11. BOISSONNAS, P. A., AND SCHUMANN, J., Helv. Chim. Ada 36, 2229, 2237 (1952).
IZUMIYA 12. SCHWYZER, R., AND SIEBER, P., HeEv. Chim. Ada 40, 624 (1957). 13. SCHWYZER, R., AND SIEBER, P., Helv. Chim. Acta 41, 1582 (1958). 14. SHEEHAP;, J. C.,ANDHESS, G. P., J. Am. Chem. Sot. ‘77, 1067 (1955); SHEEHAN, J. C., GOODMAN, M., AXD HESS, G. P., b. Am. Chem. Sot. 78, 1367 (1956). 15. GREENSTEIN, J. P., AND WINITZ, M., “Chemistry of the Amino Acids,” p. 1016. Wiley, New York, 1961. 16. IZUMIYA, N., YAMASHITA, T., UCHIO, H., AND KITAGAWA, K., Arch. Biochem. Biophys. 90, 170 (1960). 17. NEURATH, H., APTD SCHWERT, G. W., Chem. Revs. 46, 69 (1950). 18. YAMASHITA, T., AND IZUMIYA, N., J. Biochem. 46, 991 (1959).