VOL. 14, 723-731 (197:)

BIOPOLY MII:RS

Studies on Cyclic Peptides. 11. Synthesis of Cyclic Peptides Containing Sarcosine TOSHIHARU SUGIHARA, YUKIO IMANISHI, and TOSHINOBU HIGASHIlIURA, Department of Polymer Chemistry, Kyoto University, Yoshida, Sakyo-ku, Kyoto 606, J a p a n

Synopsis Cyclic peptides containing sarcosine, cyclo-(Pro-Sar-Gly)*, cyclo-(Sar-Sar-Gly)?, cyclo-(Sarc), and cyclo-(Sar6) have been synthesized by the cyclization of the p-nitrophenyl ester of linear peptides. The tert-butoxycarbonyl group was used as the Naprotecting group, which was removed by acid. Benzyl ester was used to protect the Cterminal. tert-butoxycarbonylpeptide was obtained by the stepwise elongation of the peptide bond by the carbodiimide method. 1)ebloeking and cyclization of the linear peptides gave the cyclic peptides.

INTRODUCTION Recent studies on cyclic peptides and related compounds have rcvealed many interesting features of the relation between the conformation and the activity of biomolecule~~-~. The activity of biomolecules arises from thcir interaction with small molecules, and the nature of the interaction is very complex. It is therefore important to investigate the nature of the interaction using a simple model compound for the bimolecules. It mill be advantageous to use peptide as a model compound, because the conformational properties of peptides are closely related t o those of the native biomolecules. This is the primary requirement. Cyclic peptides containing sarcosine were synthesized. They arc soluble in relatively nonpolar solvents such as chloroform. ;\lacrocyclic compounds, called ionophores, participate in the ion transport through a nonpolar lipid layer of biological membranes. The use of a model peptide soluble in nonpolar solvents is desirable. Furthemiore, the imino acid residue as a component of cyclic peptides can assume either tTans or cis peptide linkage5,6so that the conformation of the cyclic peptide should be flexible. The conformational adaptability is essential for the interaction of biomolecules with small molecules. I n the present paper the syntheses of cyclo-(Pro-Sar-Gly)2, cyclo-(SarSar-Gly),, cyclo-(Sar4), and cyclo-(Sar6) are described. Cyclo-(Pro-SarGly), and cyclo-(Sar-Sar-Gly), were first synthesized in this investigation. Though cyclo-(Sar,) and cyclo-(Sars) have been synthesized by Dale and Titlestad,? we have developed a new synthetic route t o the cyclic peptides. 723

@ 1973 by John Wiley & Sons, Inc

SUGIHARA, IMANISHI, AND HIGASHIMUKA

724

SYNTHESIS OF CYCLIC PEPTIDES Sarcosine peptides are in general hydrophobic, so that difficulties arose in synthesizing the peptide fragments by stepwise elongation because oily products were often obtained.8 To avoid the conversion of imino acid peptides into diketopiperazine in the presence of alkali or a~id,~--l-l benzyl ester was used to protect the C-terminal instead of ethyl ester. The benzyl ester group can be removed by hydrogenolysis. The N"-protecting group used was the tert-butoxycarbonyl(Boc) group. The synthetic intermediates and the product cyclic peptides were purified vigorously by recrystallization, partition chromatography, and gel filtration. The purity of the peptides was usually checked by thin-layer chromatography (TLC). The oily product was also analyzed by ir and nmr, especially from the pattern in the N-methyl region of the sarcosine residue. The synthetic route of cyclo-(Pro-Sar-Gly)z is shown in Figure 1. The cyclohexapeptide \$as obtained by the cyclodimerization under a high dilution of Boc-Pro-Sar-Gly-ONp, which was synthesized by stepwise elongation using N,N-dicyclohexylcarbodiimidc (DCC) as a coupling reagent. Cyclo-(Sar-Sar-Gly), was obtained in a similar manner. Cyclo-(Sar6) was synthesized by cyclodimerization of Boc-Sars-ONp. Cyclo-(Sar4) was obtained from Boc-Sar4-OKp. The tripeptide and tetrapeptide esters were synthesized by strpwise elongation from the N-terminal to the C-terminal as shown in Figure 2. The cyclopeptides were identified by a cryoscopic determination of the molecular weight using the lactam of cis-hexahydro-paminobenzoic a ~ i d -asl a~solvent ~ ~ ~ and/or mass spectrometry. In our procedure no dilictopiperazine was formed during the synthesis. Only a t the cyclization of the linear peptides into cyclo-(Pro-Sar-Gly)a and cyclo-(Sar-Sar-Gly)2, were diketopiperazines formed. This would have been caused in part by ion exchangers. By repeating the treatment through ion-exchange resins, the formation of diketopiperazines was checked by TLC and nmr. When sarcosine peptides and strongly acidic or basic ion exchangers were involved, the population of dikctopiperazines *

t

Boc

OH

H

DCC

Boc

OBzl OBzl

4N HCl/dioxane OH

BOC BOC

Boc Boc

T

H

OBzl

DCC

-

OBzl H2/Pd

OH

DCC, p-nitrophenol

.

ONP

I

1

4N HCl/dioxane PYr i d i n e

I ---

1

Fig. 1. Synthetic route of cyclo-(Pro-Sar-Gly)z and cyclo-(Sar-Sar-Gly)*.

CYCLIC PEPTII>ES. I1

BOC Boc

OBzl

DCC H

Boc

H

OH

725

OBzl

2/Pd

OH

H

DCC

Boc

H2/Pd

Boc

DCC

Boc

H

Boc.,

2/Pd

DCC, p-nitrophenol

Boc-

OBzl OBzl

OH

n--

OBzl OBzl

-OH ONP

4N H C l / d i o x a n e

pyridine

I

I

Fig. 2. Synthetic route of cyclo-(Sara).

increased after the treatment. Dale and Titlestad7 isolated cyclo-(SarJ and cyclo-(Sar8) from trisarcosine 2,4,5-trichlorophenyl ester and tetrasarcosine 2,4,5-trichlorophenyl ester, respectively. However, WP did not find any trace of cyclo-(Sar3) and cyclo-(Sar8) in our procedure. The absence of the cyclic peptides in our procedure could have resulted from a mild activation by p-nitrophenyl ester and a mild reaction a t a low temperature (60°C). Dale and Titlestad7 carried out the cyclization a t 115°C or under reflux in pyridine. These vigorous conditions could have raised the population of cyclo-(Sar3) and cyclo-(Sar,) as well as diketopiperazines.

EXPERIMENTAL Boc-Sar-OH A solution of sarcosine (17.3 g, 0.20 mol), sodium bicarbonate (35.4 g, 0.45 mol), and tert-butoxycarbonylazide (34.3 g, 0.24 mol) in dioxane (300 ml) and water (300 ml) was stirred a t 45°C for 24 hr. Adding 100 ml of water, the solution was extracted with ethyl acetate. After being acidified with 1-N hydrochloric acid the solution was extracted with ethyl acetate. The organic layer was dried over sodium sulfate and concentrated t o a small volume. The resulting oil crystallized upon the addition of petroleum ether. It was recrystallized from ethyl acetate; yield 34.5 g (91%); mp 93°C. T L C on Iiieselgel G was performed in the solvent system; I, chloroform :methanol :acetic acid (95 :5 :3) ; 11, chloroform: methanol :pyridine (95 :5 :3) ; 111, n-butanol :acetic acid :water (4:1:1); IV, n-butanol :acetic acid :water: pyridine (30 :6 :24: 20). R f l 0.46, Rf" 0.28, R,"' 0.52, RfIv 0.73. Calcd. for CsH1504N: C, 50.787,; H, 7.99%; N, 7.40%. Found: C, 50.65%; H, 7.90%; N, 7.62%.

A portion of Boc-Sar-OH was converted into dicyclohrxylammonium salt for the ease of the follon-ing coupling reaction. Boc-Sar-OH (lS.9 g, 0.10 mol) was dissolved in 50 ml of ether and dicyclohcxylaminc (18.1 g, 0.10 mol) 1va.s added t o the solution. Itccrystallization from ethyl acetate gave 32.7 g (SSY;) of Boc-Sar-OH. Dcha; mp 13S0-189"C. Calcd. for C ~ O ~ I ~ ~ O C,I 64.87',:{ N ? : ; 13, 10.275;); N, 7.377;. 10.14c/,: N, 7.58%.

Found: C, 64.61%; H,

Boc-Sar-Gly-OBzl T o a methylenc chloride solution (500 nil) of Boc-Sar-0H.Dcha (1S.5 g, 0.05 mol) and Gly-0Bzl.TosOH (16.9 g, 0.05 mol) was added DCC (10.3 g, 0.05 mol) at 0°C. The reaction mixture was allowed to stand at room temperature ovcrnight. It was evapora,ted under reduced pressure and the residue was redissolved in cbhyl acetate (500 ml). After the prccipitatre had been filtered off the filtratc was washed successively with I-N hydrochloric acid, 5% sodium bicarbonatc:, and water. The organic layer mas dried over sodium sulfat,e arid concentrated to a small volume. The residual oil was treated with n-hexane. The crystal formed was recrystallized from et.hyl acetate; yield 13.2 g (7S%); mp SS'C; R,I 0.49, R;I 0.74, Rfrrl0.89, R I V0.90. Calcd. for C17H240jN2: C, (i0.707i.; H, 7.19c/,; N, 8.33'j;. N, S.285;.

Found: C, 60.76Yc; H,

7.177,;

Sar-Gly-OBzl .HCl T o a solution of Boc-Sar-Gly-OBzl (20 g, 60 mmol) \\-as added 4-N hydrogen chloride in dioxane (180 ml). Thc reaction mixture was allowed to stand a t room temperature for 30 min. After the solvent had been evaporated t o dryness under reduced pressure, the crystalline residue was recrystallized from ethanol-ethyl acetate; yield 14.6 g (89%) ; mp 14S"C; RfrO.OO,R,"0.09, Rf"'O.5S, RtV0.75. Calcd. for C1~Hl~03N2C1: C, 52.847;; H, 6.28c/,; N, 10.26c/.

H, (i.285G; N, 10.27'';.

Found: C, 52.62%;

Boc-Pro-Sar-Gly-OBzl Boc-Pro-OH (5.3s g, 2.5 mmol), HC1.Sar-Gly-OBzl (fiS2 g, 25 mmol), and triethylamirie (3.5 ml, 25 mmol) were dissolved in dichloromcthane (150 ml) at 0°C. DCC (5.15 g, 2.5 mmol) was added to the solut,ion. It mas kept a t 0°C for 2 hr and then ovcrnight at room temperature. Thc prccipit.ated dicyclohexylurea was removed by filtration and the filtrate was washed with 5% aqueous sodium bicarbonate, 1-N hydrochloric acid. and water. After being dried over sodium sulfate thc organic layer was evaporated. The residual oil crystallized on addition of 72-hexanc. It was recrystallized from carbontetrachloride-ether-whexane; yield 8.74 g (81%); mp S2"-SSoC; [ a ] D 2 j -23.1" (c = 1.0, EtOH); Ryr 0.34, R,Ir 0.43, Rf"' O.SG, R," 0,s;. Calcd. for C22H3,0sN3:C, 60.95r/,; H, 7.217;; 7.307,; N, 9.925~6.

N, 9.69vi. Found: C , 60.9,5c/;,; H'

CYCLIC P1:PTII)I'S.

11

727

Boc-Pro-Sar-GIy-OH Boc-Pro-Sar-Gly-OBzl (6.5 g, 15 mmol) was hydrogenated in ethyl acetate (100 ml) using palladium as a catalyst. The catalyst was filtered off and the filt,rate was evaporated to dryness undcr reduced pressure. The oily product was treated with n-hexanc. Crystallizat,ion occurred upon the addition of ethyl acetate. The white prccipit.atc was rccrystallized from ethyl acct,ate-ether-n-hcxan(~; yicld 4.3 g (S3%); mp 111°C; [a]n2j -32.6" (C = 1.0, EtOH); R,' 0.06, Rj" 0.05, Rj"' 0.73, R,'\' 0.72. Calcd. for C~:H~;OGN~: C, 52.477;,; TI, 7.347,; N, 12.24r/l. Found: C, 52.387; ; H, 7.2.57,; N, 12.195.;.

Boc-Pro-Sar-Gly-ONp T o an ethyl acetate solution (60 ml) of Boc-Pro-Sar-Gly-OH (4.31 g, 12 mmol) and p-nitrophenol (1.67 g, 12 mmol) was added DCC (2.47 g, 12 mmolc) at 0°C. The reaction mixture was 1q)t at 0°C for 2 hr and thcn a t room temperat,urc overnight. The prccipit'ate was filtered off and thc filtrate was evaporated. Thc residual oil was washed with &her and uhexane. It gave a single spot on T L C ; yield 4.73 g (91yo); R,' 0.54, R," 0.53, RfrI10.73; Rj"' 0.83.

Cyclo- (Pro-Sar-Gly), T o Boc-Pro-Sar-Gly-OSp (4.73 g, 10.9 mmol) 4-N hydrogen chloride iu dioxane (36 ml) was added. The solution was kcpt for 1 hr at room tcmperaturc and then evaporated t o drynws undrr reduced prcssure. The syrup was washed with (%herand whcxanc. It was dissolved in dimcthylformamidc (50 ml) containing glacial acetic acid (0.5 ml). Thc solution was added drop by drop into pyridine (1500 ml) at 60°C over a 5-hr period. The reaction mixture was kept at room tempcraturc overnight. After the solvent had been evaporated the residual oil was dissolved in mt:t,hanolwater (3: 1). The solution was clutcd through the columns of Dowex 50 and Don-ex 1, and the effluent was concentrated t o drynoss undcr rcduced pressure. The solid was composed of two ninhydrin-negative components RJ 0.07 and R> 0.33. Each component was separated through a column of Sephadex LH-20 in methanol. Thosc. fractions containing a slower running material n-ere collected (componmt I). component I (R,I 0.07) was recrystallized from ethanol; yield: 121 nig (4.9%); mp 293°C; m/e 450; molecular weight 461 + 19 (theor. 450.49); R,I1 0.11, R/"' 0.05, R I r V 0 . 5 i ;[ a ] D 2 " f l ~ 1 . l(c o = 1.0, EtOH). Calcd. for C,,H3,06N,: 7.017,; N, 18.407,.

C, 53.32Cj,; H, 6.81%; N, 18.66(,&. Found: C, 33.38%; H'

The other component (R/T 0.33) was supposed t o be cyclo-(Pro-Sar) as judged from nmr spectra.

Boc-Sar-Sar-Gly-OBzl This compound was obtained from Boc-Sar-OH.Dcha (9.3 g, 25 mmol), HC1.Sar-Gly-OBzl ( 6 3 g, 25 mmol), and DCC (5.15 g, 25 mmol) as dc-

728

SUGIHARA, IMANISHT, AN11 HIGASHIAIUItA

scribed for the prcparation of Boc-Pro-Sar-Gly-OBzl. Thc product was recrystallized from ethyl acetate-ether-u-hcxane; yield S.4 g (82%) ; mp90'C; Rl0.36, R,110.60, R,"I0.74, R,'V0.S6. Calcd. for CziH&N3: H, 7.17c/,; N, 10.51c/;.

C, .5S.95%o; H, 7.17%; N, 10.30%;. Found: C, 58.7770;

Boc-Sar-Sar-Gly-OH Boc-Sar-Sar-Cly-OBzl (6.1 g, 15 mmol) was hydrogenated for 8 hr in ethyl acetate (50 ml). After the filtrate had been evaporated the crude product was recrystallized from ethyl acetate; yidd 3.6 g (73y0); mp S4"-S;S°C; RI' 0.06, R," 0.00, R,"' 0.44, R,Iv 0.70. Calcd. for Cl3HZ3o6N3:C, 49.200/;.; H, 7.31%; N, 13.249;. H, 7.26Oj,; N, 13.43yi.

Found: C, 49.43%;

Boc-Sar-Sar-Gly-ONp This was prepared from Boc-Sar-Sar-Gly-OH (3.13 g, 10 mmol), p-nitrophenol (1.39 g, 10 mmol), and DCC (2.06 g, 10 mmol) in the same manner as that described for the p r t p r a t i o n of Roc-Pro-Sar-Gly-ONp. A pale yellow solid was obtained and was uscd for t.he follon-ing reaction without further purification; yicld 4.01 g (91%); mp 97"-l0O0C; R,' 0.33, R,Ir 0.41, R/"' 0.73, R,'vO.S:i. Calcd. for C1&&N4: 6.20%; N, 12.66%.

C, 52.05%; H, 5.98%,; N, 12.78YG. Found: C, a2.307,; H,

Cyclo- (Sar-Sar-Gly), T o Boc-Sar-Sar-Gly-OKp (4.0 g, 9.1 mmol) 4-N hydrogen chloride in dioxanc (30 ml) was addcd. The solution was kept a t room temperature for 30 min, and t,hcn the solvent was evaporated. The residual syrup was washed with ether and ?I-hcxane. It was dissolved in dimcthylformamide (30 ml) containing glacial acetic acid (0.3 ml). The solution was slowly added to pyridirie (1500 ml) a t 60°C. After evaporating solvents, the residuc was dissolved in a mixture of methanol-water (3 : 1). The solution was passed through t.he columns of DOWFX 50 and Dowex 1, and the effluent was concentrated under reduced pressure. The residual solid contained cyclo-(Sar-Sar-G13.)~(RlII 0.10) together with cyclo-(Sar-Sar). Cyclo-(Sar-Sar-Cily)2 \\-as obtained by recrystallization from water-ethanol-ether; yield 229 mg (11%); mp 303"-304"C; molecular weight 418 f 25 (t,hoor.407.42); I