8lOCHEMlCAL

Vol. 91, No. 2, 1979 November

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 693-698

28. 1979

ENZYMATIC SYNTHESIS OF LEU- AND MET-ENKEPHALIN Willi Max-Planck-Institut D-3400 Giittingen, Received

September

Kullmann fiir biophysikalische Chemie, Am FaBberg, West Germany

20,1979

Summary: The protease-catalyzed synthesis of Leu- and Met-enkephalin is reported. Each peptide bond of the endogeneous opiate-pentapeptides was formed either by papain or cl-chymotrypsin catalysis. N-acyl amino acids and peptides or their ester derivatives served as substrates whereas amino acid and peptide phenylhydrazides were used as nucleophiles. The free pentapeptides exhibited naloxone-reversible opiate-like activity in guinea-pig ileum and mouse vas deferens assays. The present study suggests the usefulness of enzymic peptide synthesis which allows rapid preparation of homogeneous compounds with high optical purity. Peptide

bond

formation

reported

by Bergmann

in 1938,

respectively

order

to learn

review

enzymes zymic

the

synthesis organic

peptide

bond

Since

A renewed

involved

field

were

in protein

interest the

thus

last

that

shifting

the

in (for

of in vivo

protein

a

of proteolytic

years are

intensified

biosynthesis

in application

during

and Fruton

(4,5,6,7,8).

insoluble

chemical

The en-

in biphasic

equilibrium

towards

formation.

several

in solution,

in this

of products

media

the elucidation

1975 (9),

arose

was first

1937 and by Bergmann

waned when the mechanism

advantage

reaction

in

were

interest

synthesis takes

and ol-chymotrypsin

Investigations

proteases

was elucidated.

aqueous

in

(1,2).

whether

to peptide

by papain

and Fraenkel-Conrat

see (3)).But

synthesis

catalyzed

of the syntheses

structure of these

or by papain-catalyzed

of the peptides

fragment

enkephalins by the

by Hughes

solid

condensation

phase

(10)

have

et al.

method, been

reported. In this

paper

biologically protease-catalyzed

active

I want

to describe

enkephalins reactions

with

for all

the

first

time

the peptide

the preparation bonds

established

of by

( Scheme 1).

Abbreviations used: Boc, t-butyloxycarbonyl; OEt, ethyl ester; OTMB, trimethylbenzyl liquid chromatography.

ester;

Bzl, benzyl; HPLC, high

Ph, phenyl; pressure

0006-291X/79/220693-06$01.00/0 693

Copyright All rights

@ I979 by Academic Press, Inc. in any form reserved

of reproduction

Vol. 91, No. 2, 1979

BIOCHEMICAL

AND BIOPHYSICAL

Papain

CF3CCOH

Nli2NHFll

OH

Boc

RESEARCH COMMUNICATIONS

- N,H,Ph

OH

Boc

H

CF,COOli

N2Y”h

Boc

Y”2h

Boc

Papain

I Boc

OEt

Ii

N,H,Ph

-

1

Boc

L-Chymotrypsin

- N,H,Ph

BOC

I I

Boc

OEt

FeClj

H --

I

- OH

Bzl

I

I

Boc

. OH

WV”

=-l---i

H. Papam

,

N2H2Ph

CL - Chymotrypsin

BOC

,

Nz”,m

CFJ COOH

2 C&OH

I

Boc

--

l. COCli2CH2CoN%

Bzl

NzYr

I

Boc

‘VW’ 1 FcCl, 2CF,CCC+l/tlBr/Cti,OC,n,

IOH

Scheme

Enzymic

as well

ly removable

aqueous

as chemical

protecting

the enzymatic

Synthesis

should

in order

render

to give

be partially

soluble

place

at all.

Boc groups

meet these

quite

soluble

in

by mild

the pa-range

as to allow

On the other

during

hand,

this

work.

acids

were

for in

the reactants

reaction

No-protection

and Boc-amino

chosen

insoluble

the coupling for

and selective-

groups

products

requirements

694

easily

the blocking

yields.

acidolysis applied

require

the reaction

high

at least

removable

synthesis

Furthermore,

must

are readily

of Leu-enkephalin

peptide

groups.

synthesis

medium

1:

to take as they found

to be

BIOCHEMICAL

Vol. 91, No. 2, 1979

Carboxyl easily

groups

available

and were high trypsin

cleaved

amides

nucleophiles

over

formation

nents

Although

all

ty in aqueous

The synthetic

to enable

an initial

Tyr(Bzl)-Gly-Gly-OH product 1.00; peptide tion

acid

1.03;

Leu,

composition 1.00.

of which

Boc-Tyr(Bzl)-Gly-Phe-Leu-OTMB mixture

indicating

ted coupling

split

finding(l4)

the residue

are very

catalysis decided

pH 6.1, tapeptide

which

susceptible

may also to couple

Leu-N2H2Ph.

that

apply

in the presence which

readily

peptides

to papain

containing

action.

condensation of Z-mercaptoethanol

695

specificity

Boc-Tyr(Bzl)-Gly-OH McIlvain

gave

(yield,82%).

0.81;

Gly,

of the tetraof the reac-

may be explained residue

to the bond

of a phenylalanine

using

Tyr,

in a

by papain-media-

This

group

Boc-

resulted

a phenylalanine

As this

instead

fragments

precipitated

bond followed

the carboxyl

to a tyrosine

to be:

H-Phe-Leu-OTMB.

contributes

the protected

Papain-catalyzed

with

as catalyst

by chromatograms

of the Gly-Gly

of Boc-Tyr(Bzl)-Gly-OH

by the earlier preceding

cleavage

cleavage.

prepared

the formation

was given

solubili-

to be a practicable

enzymatically

for

the reactants,

to proteolytic

was found

evidence

elonga-

had enhanced

1 was found

papain

could

phenylhydrazides,

more susceptible

using

compo-

in the pentapeptides acid

to couple

Further

amino

(13).

bond formation,

and H-Phe-Leu-OTMB

the amino Phe,

attempt

o-chymo-

peptide

in many cases because

in scheme

(11)

as acceptor

as suitable

amides

Boc-amino

were

outlined

with

were

ensured

since

or peptides

described

contained

peptide

and thus

They also

reactions

acids

latter

to the papain-catalyzed

as compared

was impossible

pathway

whereas

respect

as their

chain

solutions

of amino

sequences

enzymatically

deprotected

coupling

the

condensation

FeC13 (12).

have been

dipeptide

of the peptide

procedure

using

With

low basicity the

be synthetized

partially

(6).

since

by papain-catalyzed

or hydrazides

, phenylhydrazides

due to their

tion

acids

by oxidation

esters

RESEARCH COMMUNICATIONS

as phenylhydrazides

of the a-chymotrypsin-mediated

prefers

bond

protected

from Boc-amino

rapidly

yields

were

AND BIOPHYSICAL

to be

of papain residue,

I

and H-Gly-Phebuffer-ethanol

the desired

protected

(6:4), pen-

Vol. 91, No. 2, 1979

BIOCHEMICAL

The protected cubation

fragment

Boc-Tyr(Bzl)-Gly-OH

of Boc-Tyr-OEt

formamide

(2:l),pH

Gly-N2H2Ph

(yield,

chain.

could

in the

72%) which

sence of a-chymotrypsin. sable

because

Gly-OH

as substrate,

All

attempts

ester

probably

or phenylhydrazide

could

be synthetized buffer,pH

(yield,

80%),

4.8,

Boc-Gly-Phe-Leu

subsequent

H-Leu-N2H2Ph (yield, sis

of the

of these

Met-enkephalin

leucine

analogue.

pentapeptides

The enzymatically were

further

will

derived purified

be given

comparison

with

the

corresponding

latter.

failed

because

tripeptide

(Scheme in a 3 M

and 2lnercaptoethanol by an ethyl

of the protected

similiar of the

ester

dipeptide

(2:l),pH

description

with

9.95, to the

synthe-

enzymatic

syntheses

elsewhere. fragments

were

worked

HPLC on prepacked

obtained

Boc-Tyr-

or trimethylbenzyl

this

in a manner

A detailed

by preparative

of the products

of the

buffer-dimethylformamide

protected

Assignment

using

of the phenylhydrazide

was prepared

in the pre-

and H-Phe-N2H2Ph

of papain

condensation

in 0.2 M carbonate

70%).

However,

Boc-Tyr

was indispen-

catalysis

of Boc-Gly-OH

and a-chymotrypsin-catalyzed

(15)

solubility

side

because

ether

as the t-butyl

bond.

replacement

chosen

not be obtained

or thermolysine

in the presence

the tyrosine

the benzyl

could

Boc-Tyr-

FeC15 to cleave

when incubated

due to increased

by incubation

with

above were

hand,

of the Phe-Leu

to give

to benzylate

H-Gly-N2H2Ph

by papain

cleavage

acetate

with

pentapeptide

to prepare

of hydrolytic

bromide

On the other

the desired

treated

by in-

buffer-dimethyl-

of a-chymotrypsin

described

be coupled

1) was prepared

in 0.2 M carbonate

presence

benzyl

procedures

not

(Scheme

was subsequently

and with

The synthetic

(Bzl)-OEt

and H-Gly-N2H2Ph

10.1,

the phenylhydrazide

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

by catalytic peptides

silica

synthesis

prepared

up as usual gel

and

60 columns.

was achieved

by conventional

by

methods

in solution. The fully

protected

pentapeptides

CF3COOH/HBr

in the presence

Boc and Bzl

groups,respectively.

geneous

form

after

were

of anisole

chromatography

treated

to cleave

The free on Bio-Gel

peptides P-2

with

FeC13 followed

the phenylhydrazide were (0.05

obtained M NH4HC03),

by and the

in homosilica

gel

1)

BIOCHEMICAL

Vol. 91, No. 2, 1979

60 (n-butanol-acetic

acid-water,

using

(95:5)

methanol-water

The chromatographic of Leu-

acid

0.89;

Phe, 0.95;

behaviour

analyses

Elementary

of acid

analyses:

2.00;

C, 60.52;

H, 6.71;

S, 5.88;

C27H35N507S

(573.7)

for

filtration

on sephadex

LH-20

Met,

0.98;

N, 12.60

to that

Gly,

Tyr,

2.00;

0.91;

Leu,

Phe,

0.98;

N, 12.35;

C28H37N507

; found:

C, 56.25;

H, 6.37;

C, 56.55; for

H, 6.15;

Leu-enkephalin;

Tyr,

0.96.

H, 6.88;

requires:

in methanol)

was identical

methods.

gave:

C, 60.23;

requires:

in methanol)

by solution

hydrolysates

Found:

(c=O.9

and gel

of the pentapeptides

prepared

and Gly,

[a], 25 = + 32.5"

4:1:1),

RESEARCH COMMUNlCATlONS

as eluant.

and Met-enkephalin

Amino

AND BIOPHYSICAL

(555.6) N, 11,92;

N, 12.21;

[u]i5=

S, 5.59.

+ 31.6'

(~1.0

Met-enkephalin.

The biological

activities

of the

enzymatically

peptides

by the naloxone-reversible

tions

of the

guinea-pig

found

to be:

EDSO

0.86

x 10m7 (GPI)

and 1.4 x 10m8 (MVD) (Met-enkephalin);

EDso

4.45

x 10 -7 (GPI)

and 0.85

were

in close

agreement

results

(16).

Enzymatically

nearly

equipotent

D-enantiomer

respect

of ca.

mes as catalysts mixing

study

in peptide

provides

or peptides

Acknowledgment. during enkephalin

this

demonstrate

during

pure

in solution,

to Dr. R. Schulz,

B. Gutte

697

whereas

an all-

effective

of proteolytic

are

available

his

action

of Nu-acyl

result

by

or 37°C.

stereospecific

groups

may often for

readily

enzy-

at room temperature

MPI Psychiatry

samples.

shown to be

was less

due to the

carboxyl

synthesis

were

et al,

assay.

buffers

products

(MVD) were

by Waterfield

assays,

The products

of the

chemical

contrac-

(Leu-enkephalin).

Leu-enkephalins

in aqueous

activation

and to Dr.

reported

the usefulness

synthesis.

I am indebted work

prepared

synthetized

optically

of the enzymes whereas acids

those

to the abovementioned

of the reactants

method

with

x 10m8(MVD)

200 in the mouse vas deferens

of this

induced

and of the mouse vas deferens

and chemically with

The results

This

(GPI)

of Leu-enkephalin,

by a factor

simple

ileum

of electrically

as deter-

mined

These

inhibition

prepared

amino

in partial

support

and interest

Munich,

for

racemization.

assaying

the

BIOCHEMICAL

Vol. 91, No. 2, 1979

AND BlOPHYSlCAL

RESEARCH COMMUNICATIONS

References 1. Bergmann,M., and Fraenkel-Conrat,H.(l937) J.Biol.Chem. 119, 707-720. 2. Bergmann,M., and Fruton,J.S. (1938) J.Biol.Chem. 124, 321-329. 3. Borspok,H.(1953) Peptide Bond Formation. In: Advances in Protein Chemistry. Anson,M.L., Bailey,K., and Edsall,J.T.,eds.,Vol.8,pp.127-174, Academic Press,New York. 4. Isowa,J., Ohmori,M.,Ichikawa,T., Kurita,H., Sato,M., and Mori,K. (1977) Bull.Chem.Soc.Japan 50, 2762-2765. 5. Isowa,J., Ohmori,M., Sato,M., and Mori,K. (1977) Bull.Chem.Soc.Japan 50,

2766-2772

Morihara,K., and Oka,T. (1977) Bi0chem.J. 163, 531-542 Saltman,R., Vlach,D., and Luisi,P.L. (1977) Biopolymers 8. Homandberg,G.A., Mattis,J.A., and Laskowski,M.Jr. (1978) 6. 7.

17,

631-638

Biochemistry

5220-5227

9. Hughes,J., Morris,H.R. 10. Wong,C-H., 576,

16,

Smith,T.W.,

Kosterlitz,H.W., Fothergill,L.A., Nature 258, 577-579 Chen,S-T., and Wang,K-S. (1979) Biochim.

Morgan,B.A.,

and

(1975)

Biophys.

Acta

247-249

11. Milne,B.H., and Carpenter,F.H. (1968) J.Org.Chem. 33, 4476-4479 12. Milne,B.H., Halver,J.E., Ho,D.S., and Mason,M.S. (1957) J.Amer.Chem.Soc. 79, 637-639 13. Waldschmitz-Leitz,E., and Kiihn,K. (1951) Chem.Ber. 84, 381-384 14. Schechter,I., and Berger,A. (1968) Biochem.Biophys.Res.Comm. 32, 898-902 15. Milne,B.H., and Kilday,W. (1965) J.Org.Chem. 30, 64-66 16. Waterfield,A.A., Smokcum,R.W.J., Hughes,J., Kosterlitz,H.W., and Henderson,G. (1977) Eur.J.Pharmacol. 43, 107-116

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