Int. J . PeptideProtein Res. 14,1979,177-185

METHIONINE ENKEPHALIN AND JSOSTERIC ANALOGUES

I. Synthesis on a Phenolic Resin Support?

DEREK HUDSON, GEORGE W. KENNER*, ROBERT SHARPE and MICHAEL SZELKE

Department o f Chemical Pathology, Royal Postgraduate Medical School, London, and *Department of Organic Chemistry, University of Liverpool, Ltverpool, England

Received 1 1 July 1978, accepted for publication 13 February 1979

A n efficient synthesis o f methionine enkephalin using a phenolic resin support is described. Analogues modified at their C-termini, such as peptide acids, amides, methyl esters and compounds formed by their reduction, were prepared con- veniently from common peptide phenyl ester resins. The resin was used in the synthesis o f complex isosterically modified analogues designed to investigate the role the peptide backbone plays in receptor interaction. Free hexapeptide phenyl ester resins underwent intramolecular aminolysis liberating the corre- sponding cyclic peptides.

Key words: cyclic hexapeptide; methionine enkephalin; phenolic resin; receptor interaction; solid phase peptide synthesis.

Frequently the Merrifield method of solid amino acid to the resin. A 1.4% crosslinked phase peptide synthesis (Merrifield, 1964) is 100-200 mesh resin prepared by copolymeris- complicated by low yields and side reactions ation of acetoxystyrene (10 mol %), styrene and occurring during acidolytic cleavage of the com- divinylbenzene (Arshady et al., 1974) has been pleted peptide from the resin. This is especially used, after deacetylation, for the synthesis of true for methionine enkephalin, where an several protected peptide acids which were additional potential hazard is sulphonium salt partial sequences of a modified lysozyme formation during addition of t-butoxycarbonyl- (Kenner & Hudson, unpublished results; Kenner, methionine to chloromethyl resins. Conven- 1977). The greater ease of formation and tional solution synthesis (Bower et al., 1976) lability to nucleophiles of phenyl esters com- has been the method of choice for high yield

Abbreviations: The following non-systematic abbrevi- and purity. ations have been used: Met E, methionine enkephalin;

thesis reside partly in the nature of the benzyl fomamide; DCCI, NJV'dicyclohexylcarbodi~jmide; ester type bond which links the C-terminal DMAP, 4dimethylaminopyridine; HOBt, l-hydroxy-

benzotriazole; CMC, carboxymethyl cellulose; Aeg, N- (2-aminoethy1)glycine; CH,CI, , dichloromethane; t Dedication

This paper is dedicated to the memory of Professor iPrOH, propan3-ol; TFA, trifluoroacetic acid; G.W. Kenner of Liverpool University who died tragi- DMSO, dimethyl sulphoxide; -Met-ol, methioninol: cally just recently. He is missed deeply by his friends and colleagues; but his memory and his inspiration will live on in our work.

The problems with the Merrifield resin 'yn- DMAE, 2dimethylaminoethano1; DMF, "dimethyl-

CH,CH,SCH, I

NHCHCH, OH.

0367-8377/79/080177-09 002.00/0 0 1979 Munksgaard, Copenhagen 177

D. HUDSON ET AL.

pared with the corresponding benzyl esters is reflected in the properties of the phenolic resin compared with the Merrifield resin. Boc-amino acids couple without side reactions to the resin with DCCI; unreacted phenolic groups are blocked by acetylation. Peroxide ion causes a remarkable acceleration in the rate of hydroly- sis of phenyl esters (Kenner & Seely, 1972), and catalysed hydrolysis is also observed for peptide phenyl ester resins. Typically, in 90% (v/v) aqueous dioxan at pH 10.5 in the presence of equivalent peroxide, peptides are liberated within 1-3 h. These conditions were found to be unsuitable for methionine enkephalin, which was oxidised even in the presence of scavengers. In this case the best procedure (Fig. 1) involved transesterification of the completed peptide resin with dimethylaminoethanol in DMF (Barton et al., 1973), followed by hydrolysis at pH 9.7 of the labile peptide ester generated. Sulphoxidation was avoided and over 90% cleavage of the peptide from the resin was found using 40% (v/v) DMAE for 2 days. With

t ( 1 ) 14 Boc-Tyr-( CIGly-Gly-Phe-Met-OH

I

80% TFA; Sephadex G 25 SF

14 ' H -Tyr -( C )Gly-G I y-Phe-Met-OH

FIGURE 1 Phenolic resin synthesis of 2 q 4 C G1y)-5-Metenkephalin

178

50% (v/v) DMAE for the same time cleavage was near quantitative. The methionine enkephalin sequence built up on the Merrifield resin is not removed under these conditions. and catalysis by thallous ethoxide (Savoie & Barton, 1974) is ineffective.

Fig. 1 shows the synthesis of 2-(14C-glycine)- 5-methionine enkephalin; the labelled derivative was used to facilitate detection and quanti- tation. The protected peptide acid (I), obtained in good yield and purity after chromatography on Sephadex LH-20 in DMF, is a potentially useful intermediate for fragment condensation synthesis of other peptides derived from the C-terminus of 0-lipotropin. In this method pro- tection for the tyrosine phenolic hydroxyl group is unnecessary, and deprotection of (I) with aqueous trifluoroacetic acid under nitrogen gave methionine enkephalin. This was shown to be chromatographically and biologically identi- cal to material synthesised by solution methods and was completely digested by aminopeptidase M. In repeated syntheses, and syntheses of 4- D-Phe-5-Metenkephalin and 1 -desarnino-S-Met- enkephalin, overall yields ranged from 50 to 70%.

During the course of Merrifield solid phase peptide synthesis loss of peptide occurs from the resin, principally during the acid depro- tection steps, With a phenolic ester linking the peptide to the resin, loss at this stage is virtually eliminated; however, the relative danger of loss during base wash and coupling steps is en- hanced. This loss was anticipated particularly during elongation of dipeptide to tripeptide and hexapeptide to heptapeptide. In the cases so far studied when brief base treatment and washing procedures were used only trace impurities were generated. These were readily removed by chromatography on Sephadex LH-20 in DMF, which proved to be a system of high resolving power for peptides of this size. However, slightly better results were obtained using different coupling schedules. Exchange of the peptide resin trifluoroacetate salt obtained after deprotection to its hydrochloride salt followed by addition of four equivalents of preactivated Boc-amino acid and then two equivalents of N-methylmorpholine gave excellent results as shown in examples (1) and (4) of the Exper- imental Section. Alternatively,as in example (6 ) ,

PHENOLIC RESIN SYNTHESIS OF ENKEPHALINS

separate and brief base wash followed by 1 -hydroxybenzotriazole catalysed DCCI coupling (Konig & Geiger, 1970; Hruby et al., 1973) were used equally successfully. The com- pleteness of each coupling reaction was moni- tored using the fluorescamine test (Felix & Jimenez, 1973).

This useful procedure combined with the efficiency of the phenolic resin method has facilitated greatly the synthesis of complex isosterically modified analogues. Instead of modifying the configuration or structure of each individual amino acid side chain as in con- ventional analogue synthesis, analogues have been prepared where the individual peptide bonds have been replaced by sterically com- patible chemically and enzymically stable groups. Reported here are cases where the peptide bond -CO-NH- has been replaced by -CH2-CH2- (“hydrocarbon” analogues) and by -CH2-NH- (reduced analogues). Results from a series of such related compounds pro- vide a way of assessing the role each peptide bond plays in receptor binding and activity, and the importance of hydrogen bonding in stabil- ising conformations. During the synthesis suitably protected modified dipeptide units (Fig. 2) must be incorporated. The preparation

R1 R 2 I I

BOC -NH C H C H2-CH2C H C O2 H (a 1

R 2 I

R1

BN-NI-I - 6 ~ -CH~-N-CH - C O ~ H I

X

X = B o c - or Z- FIGURE 2 Protected isosteric units for the synthesis of “hydro- carbon” and reduced enkephalin analogues.

of such isosteric units is discussed elsewhere (Szelke el al., 1977). In brief, simple hydro- carbon units (Fig. 2(a), R2=H) are prepared by repetitive Arndt-Eistert reaction (Sharpe & Szelke, 1976); whereas reduced isosteric units can be prepared by a variety of approaches, including substitution (Szelke et al., 1977; Experimental ( 5 ) ; Atherton et al., 1971), reductive alkylation (Parry et al., 1972), or by reduction of dipeptides with borane (Roeske et al., 1976) or sodium dihydro-bis(2-methoxy- ethoxy)aluminate (Szelke et al., 1977). During synthesis protection of the secondary nitrogen of the reduced linkage is necessary (Fig. 2(b)). For N-terminal units the Boc-group is satis- factory, in other positions benzyloxycarbonyl (Z-) was used. These isosteric units couple less readily than do Bocamino acids because of electronic and possibly steric factors. Overnight coupling in the presence of HOBt to the appro- priately assembled peptide phenyl ester resin with very slight excess of isosteric unit gave good incorporations; unreacted amino groups were blocked by acetylation.

Table 1 includes the structures, yields and properties of analogues of enkephalin iso- sterically modified at the first two peptide bonds. Most of the analogues are modified additionally at their C-termini, either as the amide or as methioninol (Pless et al., 1977). These derivatives were prepared in good yield using the phenolic resin. Treatment of the assembled isosteric peptide resins with ammonia in 1 : 1 methanol/DMF gave in 1 day quantitative cleavage. Protected peptide methyl esters were formed in good yield after 2-day treatment of the peptide resins with diisopropylethylamine in 1 : 1 methanol/DMF. Overnight reduction of the methyl ester with excess sodium boro- hydride gave the methioninol derivative cleanly (Experimental examples (9) and (8), respec- tively). After purification on Sephadex LH-20 in DMF, deprotection was effected either with aqueous trifluoroacetic acid or with anhydrous hydrogen fluoride at 0” for 30min in the presence of methionine and anisole.

Peptide phenyl ester resins are not only susceptible to intermolecular attack by nucleo- philes but also to intramolecular reaction. Since in the proposed conformations for methionine enkephalin (Bradbury et al., 1976; Momany,

179

TAB

LE 1

En

keph

alin

ana

logu

es - st

ruct

ures

, yields a

nd c

hara

cter

isatio

n

Cod

e N

o.

Com

poun

d A

min

o ac

id a

naly

sis

Ove

rall

t.1.c.

t.1

.e.

yiel

d (%

) R

fA

RfB

Rf

,.,

Rf,.,

G

ly

Tyr

Ph

e M

et

Aeg

Met

E

H 2

11

H 2

12

H 2

15

H 2

16

H 2

18

H 2

19

H 2

20

H 2

13

H 2

14

H-T

yrC

lyG

ly-P

he-M

et-O

H

C,H

,OH

I CH

1 I

NH

, CH

CH

, CH

, CH

,CO

Gly

-Phe

-Met

-NH

,

H-Tyr-NHCH,CH,CH,CH,CO-Phe-Met-NH,

C,H

,OH

I CH

, I C,H

,OH

I CH

, I

NH

, CH

CH

, NH

CH

, CO

Gly

-Phe

-Met

-NH

NH

,CH

CH

, N

HCH

,CO

Gly

-Phe

-Met

-ol

H-T

yr-N

HC

H, C

H, N

HC

H, C

O-P

he-M

etaH

H-T

yr-N

HC

H,C

H,

NH

CH

,CO

-Phe

-Met

4

H-T

yr-N

HC

H,C

H,

NH

CH

,CO

-Phe

-Met

-NH

,

cycl

o(G

1y-T

yrG

lyG

lyP

he-M

et-)

cycl

o [-(

2-am

inoe

thyl

)Tyr

G1y

Cly

-Phe

-Met

-]

55

4Ia

81

36a

4 Oa

32

42

43

40

40

0.68

0.

69

1.0

0.76

0.

73

1.00

0.81

0.

74

1.00

0.73

0.

68

1.30

0.75

0.

68

1.30

0.54

0.

70

1.30

0.67

0.

73

1.33

0.65

0.

69

1.30

-

0.72

-

0.89

0.

67

0.91

1 .o

2.00

1.33

1.

01

1.33

-

1.33

1.

03

1.33

1.

07

1.12

-

1.33

-

1.33

-

-

3.14

1.05

1.

95

1 .oo

-

1.04

-

-

1 .oo

1.01

1.03

0.91

-

0.99

0.

93

0.99

0.

92

1.02

0.

95

0.97

1.

00

0.93

-

1.01

0.

84

0.94

-

0.94

0.

99

0.99

0.

98

0.99

1.

07

P X

C

W

0

2:

m

4 * r v)

0.99

1.05

1.04

-

-

a Y

ield

bas

ed o

n am

ount

of i

sost

eric

uni

t use

d

PHENOLIC RESIN SYNTHESIS OF ENKEPHALINS

1977; Isogai eral., 1977) the amino and carboxy termini can be brought into proximity with little distortion, a cyclic analogue of enkephalin bridged between the termini by a glycine residue (Table 1, H 213) was of interest. Cyclic peptides are difficult to prepare since cyclis- ation must be done at high dilution to avoid dimerisation and polymerisation side reactions which lead to low yields. As originally pointed out by Fridkin and his coworkers (Fridkin ef al., 1965) these side reactions should be minimised on a polymeric support. The peptide Boc-Gly- Tyr(B~l)-('~C)Gly-Gly-Phe-Met phenyl ester resin was assembled using the method of in situ neutralisation during the coupling reaction. After deprotection and neutralisation the resin was stirred gently in DMF. Release of radio- activity into the solution was used to monitor the reaction. Addition of 4dimethylamino- pyridine catalysed the cyclisation and approx- imately doubled the rate of release. HOBt, which catalyses active ester reactions in solution, suppressed the reaction. After 8 days in the presence of DMAP the pure cyclic peptide was obtained after chromatography on Sephadex LH-20 in DMF. Only trace amounts of dimeric and polymeric material were detected. After deprotection the desired cyclic peptide was ob- tained in greater than 40% overall yield. A further cyclic peptide (Table 1, H 214) was prepared by incorporation of a Gly-Tyr reduced isosteric unit in place of glycine and tyrosine (Hudson etal., 1977). In this case the cyclisation was more rapid and was essentially complete after 7 days in DMF without the addition of DMAP. The yield obtained (40%) reflected the poor incorporation of the hindered isosteric dipeptide unit.

Full details are given in the Experimental procedures section of the synthesis of Met- enkephalin and isosteric analogues H 218, H 21 9 and H 220,as well as of the cyclic peptide H 213. The syntheses of H 211, H 212, H215 and H 216 parallel closely those of the given examples; all isosteric units were incorporated using the DCCI/HOBt method (Experimental (6)). Details of the methods of synthesis of isosteric units will appear elsewhere. Cyclic peptide H 214 was prepared by a method analogous to that used for H 213, which has been reported previously (Hudson et al., 1977).

The biological activities of the analogues appear in Part I1 along with deductions about the manner in which they, and methionine enkephalin, bind to the opiate receptor.

EXPERIMENTAL PROCEDURES

Peptide synthesis was performed manually in a vessel stirred by nitrogen bubbling (Corley el al., 1972). Boc-phenylalanine and Boc-methionine dicyclohexylammonium salt were obtained from Fluka AG, Switzerland; Boc-tyrosine from Sigma Chemical Co., U.S.A. Boc-(14C) glycine, Boc-glycine and Boc-5-aminopentanoic acid (7% yield, m.p. 4 7 5 4 8 . 5 " ) were prepared using tetramethylguanidine and Boc-azide (Ali ef al., 1972). Thin-layer chromatography (t.1.c.) was run on silica gel (Merck Kieselgel60 FZs4) in the following systems (ratios by volume); A: ethyl acetate-pyridine-acetic acid-water (50:20: 6: 11) - in systems A' and A" the ethyl acetate ratios were 60 and 80 respectively; B: ethyl acetate-1-butanol-acetic acid-water (1 : 1 : 1 : 1);C: benzenedioxan-acetic acid (95:25:4); D: chloro- form-methanol (9: 1). E: chloroform-trifluoro- ethanol (7:3); F: 1-butanol-acetic acid-water (3: 1: 1); G: 1-propanol-water (7:3); H: l-butanol- concentrated ammonia (1 : 1). Electrophoresis (t .l.e.) was performed on cellulose plates (Merck) at 1000 V, 20 mA, for 30 min. Rf values are re- ported relative to the mobility of methionine enkephalin at pH 2.1 (formic acid-acetic acid- water, 25:87:888 by vol.) and at pH 6.5 (pyr- idine-acetic acid-water, 100:4:896 by vol.). Chromatography on Sephadex LH-20 in DMF was performed in a 2.5 x 80cm column eluted with distilled DMF at a flow rate of 15 ml/h collecting 190 drop (6-ml) fractions. Chromato- graphy on Sephadex LH-20 in methanol was performed in a 2.5 x 93 cm column eluted with Analar methanol at a flow rate of 12ml/h collecting 9-ml fractions. Chromatography on Sephadex G-25 SF was performed in a 1.6 x 93 cm column eluted with deaerated 1 : 1 distilled water-acetic acid (containing 0.01% v/v 2- mercaptoethanol) at a flow rate of 10ml/h collecting 130 drop (4-ml) fractions. CMC chromatography was performed with Whatman CM52 in a 2.5 x 34 cm column eluted at 20 ml/h collecting 150 drop (9.5-ml) fractions. A Gilson Mixograd gradient former was used to generate

181

D. HUDSON ET AL.

a linear gradient over 2 days, starting either from 0.01 M NH40Ac pH 7 and running to 0.2 M NH,OAc pH 7 or from 0.05 M NH40Ac pH 7 to 0 . 5 ~ NH40Ac pH 7. Amino acid analysis was performed with a Jeol JLC 6 AH analyser. Resin samples were hydrolysed at 130" for 2 h in 1 : 1 (v/v) propionic acid - 12 M HCl. Peptide samples were hydrolysed with 6 M HCl containing phenol at 110" for 18 h. Amino- peptidase M (Sigma) was used in 0.1 M phos- phate buffer pH 7.5 for 1 day at 37" at an enzyme to substrate ratio of 1 :250.

( 1 ) Boc-(I4C) Gly-Gly-Phe-Met-phenyl ester resin Acetoxy resin (1.4% crosslinked, 10 mol% acetoxystyrene, 1 g) was placed in the synthesis vessel and treated overnight with hydrazine hydrate (1 ml) in DMF (10ml) and dioxan (5ml). The resin was washed repeatedly with each of the following: DMF, DMF-H20 (3:1), DMF, CH2C12, iPrOH and CH2C12. Boc- methionine (0.50 g, 2 mmol) in CH, Cl, (7.5 ml) was treated with DCCI (0.5 1 g, 2.47 mmol) and the mixture added to the resin followed by pyridine (1 ml). After 3 h the resin was thoroughly washed: CH, Clz (3 x ), iPrOH (3 x ), CH2 C12 (3 x ) and DMF (3 x ). Acetic anhydride (1 g, 10mmol) and triethylamine (1.4m1, 10mmol) in DMF (10ml) were added. After 90 min, the resin was washed thoroughly as pre- viously, and the acetylation repeated. It was washed repeatedly, dried (1.07 g, amino acid analysis 0.4 mmol/g), then rewashed and treated with 50% TFA in CH2C12 (containing 2% ethanedithiol and 2% diethyl phosphite - all %v/v) for 1 min and repeated for 15 min. This double treatment was repeated after CH2 Cl, (3 x), iF'rOH (3 x ) and CH2C12 (3 x ) washes. After further thorough washing the resin (fluorescamine test positive) was treated twice with 0.075M HCI in DMF (10ml for 2min each time). The wash, exchange and wash steps were repeated. A solution of Boc-phenylalanine (0.43g, 1.6mmol) in CH2Clz (7ml) was treated with DCCI (0.36g, 1.75mmol) and added to the resin followed by N-methylmor- pholine ( 8 0 ~ 1 , 0.73mmol). After 1 h the resin was washed thoroughly (fluorescamine test negative), dried and removed from the apparatus (1.13 9).

182

One half of the resin (0.2 mmol) was washed thoroughly and subjected to the double pre- wash and deprotection, and the 0.075 M HC1 in DMF exchange steps. After washing (fluoresc- amine test positive) a solution of Boc-glycine (0.175 g, 1 mmol) in CHz Clz (5 ml) was treated with a solution of DCCI in CH2 Clz (1.1 mmol) and added to the resin followed by N-methyl- morpholine (50~1) . After 1 h the resin was washed (fluorescamine test negative). The tri- peptide resin was deprotected, washed, ex- changed to its hydrochloride and rewashed. Boc-glycine (70 mg, 0.4 mmol) containing 8.8 x lo5 d.p.m. 14C labelled derivative in CHzC1, (3 ml) was treated with DCCI solution (0.45mmol), and added to the resin followed with N-methylmorpholine (50~1) . After 2 h the thoroughly washed resin (negative fluorescamine test) was removed and dried (0.59 8).

(2) Bo c - T ~ r - 1 ' ~ C ) Gly -Cly -Ph e-Me t-OH ( I ) A sample of the tetrapeptide resin (0.189 g, 0.064 mmol) was subjected to the repeated pre- wash and deprotection steps. After thorough washing the resin was treated with 10% tri- ethylamine in CH2 Cl, (2 x 2 min; fluorescamine test positive). The resin was washed and a ready prepared solution of Boc-tyrosine (0.10 g, 0.35 mmol) and HOBt (0.12 g, 0.78 mmol) in CH2C12 (2ml) - DMF (2ml) treated with DCCI (0.10 g, 0.50 mmol) was added. After 4 h the resin was washed with CH, Clz (3 x ), iPrOH

10% triethylamine in CH2Clz (3 x ) , CH2Clz (3 x ) and DMF (3 x); the fluorescamine test was negative. The resin was treated with 50% DMAE - DMF (v/v) for 2 days, washed with DMF (4x) , and the combined filtrate and washings were evaporated in vacuo. The residue was dissolved in 1 : 1 DMF - H2 0 (1 2 ml) and the pH maintained at 9.7 overnight by the addition of 0.1 M sodium hydroxide. Water (6ml) was added and the pH adjusted to 3.0 by careful addition of saturated potassium bi- sulphate solution. The solution was evaporated in vacuo at room temperature. The residue was suspended in DMF (Sml), filtered and ex- tracted several times with DMF. The combined filtrates were concentrated in vacuo (to 2 ml) and chromatographed on Sephadex LH-20 in DMF. Fractions 42 -46 were combined and

(3 x ), CH2 Clz (3 x ), DMF (3 x ), CH2 Cl2 (3 x ),

PHENOLIC RESIN SYNTHESIS OF ENKEPHALINS

evaporated to give (I) as a colourless glass (54 mg;yield by quantitative amino acid analysis and scintillation counting 72%). T.1.c.: Rf A', 0.73; Rf F 0.70;Rf G 0.63; amino acid analysis: Gly, 1.96; Met, 0.76; Tyr, 1.04; Phe, 1 .OO.

(3) H-Tyr-Gly-Gly-Phe-Met-OH (Met E ) Protected peptide I (75% of product obtained above) was dissolved in 80% aqueous TFA 20ml) under an atmosphere of nitrogen. After 30 min the solution was evaporated in vacuo, the residue dried in vacuo over NaOH pellets and chromatographed on Sephadex G-25 SF. Fractions 27-30 were pooled and evaporated to give 25.9mg. This was chromatographed on CMC (gradient 0 .O 1 M -0.2 M NH4 OAc). Fractions 12-1 5 were pooled and lyophilised. The residue was relyophilised from water and then from 0.01 M HCl. Yield of Met E hydro- chloride 18.1 mg (62% overall; by a quantitative amino acid analysis 55% overall); character- isation: see Table 1. Amino acid analysis after digestion with aminopeptidase M: Gly, 2.02; Met, 1.07; Tyr, 0.94; Phe, 0.96.

(4) cyclo (-Gly-Tyr-Gly-Gly-Phe-Met-) (H 21 3) The tetrapeptide resin described in (1) (0.195 g, 0.064 mmol) was deprotected and exchanged to its hydrochloride salt as usual. Bod-benzyl - tyrosine (0.14g, 0.4mmol) in CH2Clz (2.5 ml) was treated with DCCI (0.10 g, 0.5 mmol) and the solution added to the resin followed by N- methylmorpholine (20 pl). After 1 h the resin was washed thoroughly (fluorescamine test negative). In the next cycle Boc-glycine (70 mg, 0.4mmol) was coupled for 1 h (fluorescamine test negative). The resin was deprotected and exchanged to its hydrochloride salt as usual. After washing, HOBt (34mg, 0.21 mmol) and N-methylmorpholine (40 pl) were added, Build up of radioactivity in the solution showed slow cleavage (<4% after 19h). The resin was washed with 5% (v/v) N-methylmorpholine in DMF and then gently stirred in DMF. The rate of release of radioactivity was doubled, and further increased by the addition of DMAP (1 2.2 mg). After 8 days 50% loss of peptide had occurred from the resin. The suspension was filtered and the resin beads thoroughly washed in DMF, then resuspended in DMF. The com- bined filtrate and washings were evaporated in

vucuo and the residue chromatographed on Sephadex LH-20 in DMF as usual. Fractions 45 -47 gave 21 mg (47% overall) of highly in- soluble homogeneous cyclic peptide; t .l.c.: Rf A", 0.86 (streaking to origin); Rf B, 0.78 (streaking to origin); Rf E, 0.24. The re- suspended resin beads were left in DMF alone for 8 weeks and gave an additional 10 mg (22% overall) of identical material.

The protected cyclic peptide (20.5 mg) was treated at 0' for 30 min with anhydrous hydro- gen fluoride (10 ml) in the presence of methion- ine (50mg) and anisole (1 ml). The solvents were evaporated in vacuo and the residue chromatographed on Sephadex G-25 SF. Fractions 28-30 gave 14.8mg of water in- soluble cyclic peptide; characterisation: see Table 1, also Rf H, 0.52.

(5) N-Benzyloxycarbonyl-N-(2-t-butyloxy- carbonylaminoethy1)glycine 2-Bromo-N-t - butyloxycarbonylaminoethane (prepared by treatment of 2-bromoethylamine hydrochloride with Boc-azide and triethylamine in DMF; 0.9g, 4mmol) was stirred in dry DMSO (10ml) with glycine ethyl ester hydro- chloride (1.4 g, 10 mmol) and triethylamine (1.95m1, 14mmol) for 2 days at 37". The mixture was partitioned between 1 M sodium bicarbonate and ethyl acetate and the organic extract dried and evaporated. Purification on Sephadex LH-20 in methanol (fractions 25-26) gave N-(2-t-butoxycarbonylaminoethyl) glycine ethyl ester (0.31 g, 32% yield); t.1.c.: Rf F0.54. A sample (0.25 g, 1 .O mmol) was stirred with benzyl chloroformate (0.1 7 mi, 1.5 mmol) in dioxan (5ml) and 1 M potassium bicarbonate solution (5 ml) at room temperature overnight. Excess reagent was destroyed by reaction with unsyrn.dimethylethy1enediamine (0.1 1 ml, 1 .O mmol) for 1 h, and the ethyl ester isolated by ethyl acetate extraction of the acidified reaction mixture. Hydrolysis in methanol (15 ml) with 0.2 M sodium hydroxide solution (5.0 ml) gave, after recrystallisation from ethyl acetate - 60-80" petroleum ether, 0.23g of the reduced dipeptide derivative (61% for last 2 step~);m.p.91.5-94~;t.l.c.: RfC,0.22;RfD, 0.05. From ethylenediamine (Atherton et al., 1971) reported m.p. 90-91'.

183

D. HUDSON ET AL.

(6) Boc-Tyr-NHCH2 CH2 N(Z)CHz CO-Phe-Met- phenyl ester resin Boc-methionine phenyl ester resin (0.555 g, 0.22 mmol) was deprotected (as in l), and after thorough washing (fluorescamine test positive), treated with 10% triethylamine in CHzCl2 (4 x 20 s). After rapid washing, immediately was added a solution prepared 2min previously at 4" of Boc-phenylalanine (0.265 g, 1 mmol) and HOBt (0.34 g, 2.2 mmol) in 1 : 1 DMF-CH2 C12 (7 ml) treated with DCCI (0.22 g, 1.1 mmol). After 90min the resin was washed with DMF ( 3 x), CH2C12 (3 x), iPrOH (3 x ) and CH2C12 (fluorescamine test negative). The resin was again treated with 10% triethylamine in CH2C12 (4 x ~ O S ) , thoroughly washed and reacted with acetyl imidazole (0.3 g, 3 mmol) in DMF (7ml). After 30min the resin was washed as after the coupling step. In the next cycle the resin was deprotected, washed (fluorescamine test positive) and repeatedly treated with 10% triethylamine in CH2C12 (4 x 20 s). After rapid washing, immediately was added a solution of protected isostere (5, 100 mg, 0.28 mmol) and HOBt (96 mg, 0.62mmol) in 1:l DMF-CH2C12 (5ml) at 0" treated 2 min previously with DCCI (83 mg, 0.4 mmol). The reaction was left overnight. After thorough washing the fluorescamine test was faintly positive. The resin was treated with 10% triethylamine in CH2C12 (4 x 20 s), thoroughly washed and reacted for 1 h with acetyl imidazole (0.3g, 3mmol) in DMF (7ml). The resin was washed as previously (fluorescarnine test negative), and deprotected with 25% TFA in CH2 C12 containing 2% ethanedithiol and 2% diethyl phosphite (for 1 min, and then for 30 min). After thorough washing (fluorescamine test positive) the resin was treated with 10% triethylamine in CH2C12 (4 x ~ O S ) , rewashed, and then Boc-tyrosine (0.29 g, 1 mmol) was coupled as described previously for Boc- phenylalanine. The resin was washed thoroughly (fluorescamine test negative) and dried to give 0.707 g.

( 7 ) H-Tyr-NHCH2 CH2 NHCH2 CO-Phe-Met-OH (H218) The protected peptide resin (0.235 g, 0.073 mmol) was stirred for 2 days in 40% DMAE- DMF (20 ml). The resin suspension was filtered

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and the beads thoroughly washed with DMF (resin recovered 0.160 g). The combined filtrates were evaporated in vucuo and the residue hydrolysed overnight at pH9.7 in 1 : l DMF- water (12ml). Water (6ml) was added and the pH adjusted to 3.5 with saturated potassium bisulphate. The solution was evaporated in vucuo and the residue extracted several times into DMF. The solution was concentrated (to cu. 2ml) and chromatographed on Sephadex LH-20 in DMF (for this case only, the column dimensions were 2.5 x 88 cm; elution positions are not directly comparable). Fractions 48-5 1 were combined and evaporated to give the protected peptide 35.8 mg (55%); amino acid analysis: Aeg, 0.93; Met, 0.90; Tyr, 1.05; Phe, 1.03. The total product was treated with anhydrous hydrogen fluoride (10 mi) in the presence of anisole (1 ml) and methionine (100mg). After 30min at 0" the solvents were evaporated in vacuo. The residue was chromato- graphed on Sephadex G-25 SF. Fractions 25- 29 were pooled and evaporated, and the residue chromatographed on CMC (gradient 0.01 M- 0.2 M NH4 OAc). Fractions 2 1-33 were pooled and lyophilised repeatedly from water and finally from 0.01 M HCl. Yield of H218 14.8 mg; for characterisation: see Table 1.

(8) H-Tyr-NHCH2 CH2 NHCH2 CO-Phe-Met-ol (11219) The protected peptide resin from Exp. (6) (0.235 g, 0.073 mmol) was stirred for 2 days in 1 : 1 methanol - DMF (20 ml) in the presence of diisopropylethylamine (1 ml). The suspension was filtered and the resin beads thoroughly washed with DMF (resin recovered 0.152g). Evaporation of the combined filtrates gave the protected peptide methyl ester which was dissolved in'methanol (5 ml), and water (4 ml) and sodium borohydride (0.1 5 g) added to the stirred solution. After 18h the solvents were evaporated in vacuo and the residue extracted into DMF. The solution was concentrated (to ca. 2ml) and chromatographed on Sephadex LH-20 in DMF. Fractions 34-37 gave 53.9 mg of the protected peptide methioninol derivative (amino acid analysis - methionine absent). The total product was treated at 0" with anhydrous hydrogen fluoride (10 ml) in the presence of anisole (1 ml) and methionine (1 00 mg). After

PHENOLIC RESIN SYNTHESIS OF ENKEPHALINS

30min the solvents were evaporated and the dried residue chromatographed on Sephadex G-25 SF. Fractions 25-29 were pooled, evaporated in vucuo and the residue chromato- graphed on CMC (gradient 0.05 M -0.5 M NH,OAc). Fractions 53-60 were pooled and lyophilised repeatedly from water and finally from 0.01 M HCl to give H219, 18.5mg (42% overall); for characterisation: see Table 1. ,

( 9 ) H-Tyr-NHCH2 CH2 NHCHl CO-Phe-Met-NH2 ( H 220) The protected peptide resin from Exp. (6) , (0.235g. 0.073mmol) was suspended in 1:l methanol-DMF, cooled to O", and saturated with anhydrous ammonia. The flask was tightly stoppered and stirred for 18 h at room tempera- ture. The suspension was filtered and the resin beads washed with DMF (resin recovered 0.145 9). The combined filtrateswere evaporated in vucuo and the residue chromatographed on Sephadex LH-20 in DMF. Fractions 36-40 were combined and evaporated in vacuo to give 56.9 mg (90% overall) of the protected peptide amide (amino acid analysis: Aeg, 0.98; Met, 0.90; Tyr, 1.02; Phe, 1.01). The total protected peptide amide was deprotected and chromato- graphed as described in (7) above. From CMC chromatography fractions 105-1 14 gave 20.0mg of the amide (43% overall); for characterisation: see Table 1.

ACKNOWLEDGEMENTS

We thank R. Arshady and A. Ledwith, Department of Physical and Inorganic Chemistry, Liverpool University for preparation and supply of the phenolic resin; Pearlie Tien for amino acid analyses; and B.A. Morgan, A. Wilson and C.F.C. Smith, Reckitt and Colman, Hull, England for assay and comparison of methionine enkephali samples.

REFERENCES

Ali, A., Fahrenholz, F. & Weinstein, B. (1972) Angew. Chem. Int. Ed. 11,289-290

Arshady, R., Kenner, G.W. & Ledwith, A. (1974)J. Polymer Sci. Polymer Chem. Ed. 12,2017-2025

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Hruby, V.J., Muscio, F., Groginsky, C.M., Gitu, P.M., Saba, D. & Chan, W.Y. (1973) J. Med. Chem. 16,

Hudson, D., MacIntyre, I., Sharpe, R., Szelke, M., Fink, G. & Kenner, G.W. (1977) in Molecular Endocrinology (MacIntyre, 1. & Szelke, M., eds.), pp. 269-277, Elsevier/North Holland Biomedical Press, Amsterdam

Isogai, Y., Nemethy, G. & Scheraga, H.A. (1977) Proc. Natl. Acad. Sci. US 14,414-418

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Kenner, G.W. & Seely, J.H. (1972) J. Am. Chem. SOC.

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Chemistry and Biology of Peptides (Meienhofer, J., ed.), pp. 541-544, Ann Arbor Science Publishers, Ann Arbor

Pless, J., Bauer, W., Cardinaux, F., Closse, A., Hauser, D., Huguenin, R., Romer, D., Buscher, H. & Hill, R.C. (1977) in Molecular Endocrinology (Mac- Intyre, I. & Szelke, M., eds.), pp. 279-285, Elsevier/North Holland Biomedical Press, Amsterdam

Roeske, R.W., Weitl, F.L., Prasad, K.U. & Thompson, R.M. (1976)J. Org. Chem. 41,1260-1261

Savoie, J.Y. & Barton, M.A. (1974) Canad. J. Chem. 52,2832-2839

Sharpe, R. & Szelke, M. (1976) U.K. Pat. Appl. 13 192/76.

Szelke, M., Hudson, D., Sharpe, R., MacIntyre, I., Fink, G. & Picketing, A.M.C. (1977) in Molecular Endocrinology (MacIntyre, I. & Szelke, M., eds.), pp. 57-70, ElsevierlNorth Holland Biomedical Press, Amsterdam

52,377-381

624-629

44 1-45 7

94,3259

798

Address:

Dr. Derek Hudson Dept. of Chemical Pathology Royal Postgraduate Medical School London W12 England

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