Protease-mediated Peptide Bond Formation | Summaries Biochemistry

THE

JOURNAL

BIOLOGICAL

CHEMISTRY

Printed

U.S.A.

Val.

256,

No.

Issue

February

10,

pp.

1301-1304,

1981

Protease-mediated Peptide Bond Formation

SOME UNEXPECTED OUTCOMES DURING ENZYMATIC SYNTHESIS OF LEU-ENKEPHALIN*

(Received for publication, August 6, 1980)

Willi Kullmann

From the Max-Planck-Znstitut

fur

Biophysikalische Chemie,

0.3400

Gottingen, Am Fussberg. Federal Republic

Germany

In the course of a study in protease-controlled peptide

synthesis, several promising pathways to synthetic en-

kephalins had to be discarded or modified because they

failed to give the required products. Partial deprotec-

tion of peptide fragments prior to chain elongation

resulted in an enhanced susceptibility of scissile bonds

to proteolysis. The use of proteases, the specificity

which was not confined solely to the bond to be synthe-

sized, thus led to

priori

cleavage

pre-existing pep-

tide bonds. Subsequently, enzyme-mediated synthesis

new peptide bonds between initial reactants and

nascent degradation products furnished undesired,

of-

ten truncated peptides.

detailed characterization of the undesired products

gave information as to whetber or not the peptide

bonds were susceptible to proteolytic cleavage or ac-

cessible via enzymatic synthesis. In this way, the un-

expected outcome

protease catalysis led to working

predictions regarding enzyme-mediated peptide bond

formation, and thus, finally contributed to the success-

ful synthesis of the target peptides.

Peptide bond formation on a preparative scale by the

agency of proteases has become a rapidly developing field

(1-

10).

In this approach, the enzymatic specificity suppresses the

formation of undesired side-products often found in the course

of conventional synthesis. In particular, it provides for an

outstanding way of maintaining chiral integrity because of the

stereoselectivity of the proteases. Furthermore, since the

products are often largely insoluble in the reaction media, this

method facilitates the purification procedure.

Despite these promising features, the enzymatic procedure

has not yet reached general applicability. Much of this draw-

back is due to the specificity, which is often not sharp enough

to permit predictions whether and to what extent the desired

compounds are going to be formed. On the other hand, it

cannot be reliably determined whether the possible products

formed will be sufficiently insoluble to escape from

poster-

iori

proteolysis of the newly synthesized peptide bonds.

Notwithstanding the above-mentioned objections, the fact

that the lack

unique specificity of some proteolytic enzymes

allows for the formation of various kinds of peptide bonds

should not be ignored. However, it implies a further risk. The

inherent capacity of these proteases to act at

variety of

peptidic substrates jeopardizes pre-existing, scissile bonds

when dealing with fragment condensation.

hydrolysis oc-

curs, the resultant cleavage products may nevertheless act as

substrates and give rise to undesired compounds. Conse-

The costs of publication of this article were defrayed in part by

the payment of page charges. This article must therefore be hereby

marked “adc~ertisement” in accordance with

U.S.C.

Section 1734

solely to indicate this fact.

quently, these imponderables keep the outcome

protease-

catalyzed reactions in suspense, thus making methodical de-

sign of a synthesis more difficult or even rendering it a trial

and error procedure.

Recently, two alternative routes to sequential synthesis of

Leu- and Met-enkephalin were reported

(10).

The peptide

bonds were catalytically formed either by papain or n-chy-

motrypsin. In the progress of this work, several pathways had

to be discontinued because they proved to be dead ends. In

this paper, some unsuccessful strategies for the preparation of

opioid peptides are described. They are depicted in Fig.

together with the synthetic routes that do lead to the desired

products.

EXPERIMENTAL PROCEDURES

All chemicals and solvents were reagent grade. Bocl-amino acids

synthesis of Boc-Tyr-OEt

(I)

and Boc-Phe-OEt

(XXI)

followed the

were purchased from Fluka and were of the L-configuration. The

method reported by Yonemitsu et

al.

(11). Boc-Gly-NLH2Ph (II), Boc-

Phe-NZHsPh

(V),

and Boc-Leu-NLHLPh (VIII) were prepared by

papain-catalyzed condensation in the presence of 2-mercaptoethanol,

referring to methods described by Milne and Carpenter (12). Prior to

use, their purity was confirmed by thin laver chromatography and

elementary analysis.

Deacylation of Boc-amino acid or peptide phenylhydrazides was

achieved as reported previously (10).

Boc-Gly-Phe-OH was obtained from Boc-Gly-Phe-NLHLPh (VI)

after removal of the phenylhydrazide group by FeCIl treatment, as

described for the preparation of Boc-Tyr(Bz1)-Gly-OH (IV) (10).

Papain (EC 3.4.22.2) and thermolysin

(EC

3.4.24.4) were obtained

from Sigma and Bohringer, respectively.

Prepacked Silica Gel 60 columns were purchased from Merck

(Darmstadt). Thin layer chromatograms were developed on precoated

silica gel plates (Merck) using the following solvent systems

(v/v):

System A, chloroform/methanol (3:l); System B. chloroform/meth-

anol/acetic acid (45:4:1).

Enzymatic Synthesis

(Fig.

Boc-Gly-Gly-N2H2Ph (XVZZZ)-Boc-Gly-OH (2.28 g, 13 mniol) and

H-Gly-N,HLPh.CF.lCOOH (3.63

13 mmol) were dissolved in 150 ml

of 3

sodium acetate buffer (pH 6.25) which was 2.7

in potassium

chloride. 2-Mercaptoethanol (1.4 ml) was added following which the

solution was kept at 40°C in the presence of papain

g) for 40 h.

The reaction mixture was extensively extracted with ethyl acetate.

The organic layer was washed in succession with 0.1

HCI, water,

59;

aqueous ammonia, water, and dried over NaZSOt. On evaporation, an

oil was obtained which was further purified bv means of HI’LC on a

prepacked Silica Gel 60 column (44

3.7 cm). The pure dipeptide

(XVIII)

was eluted from the column using chloroform/methanol (50:

(v/v).

Cr~ystallization from methanol with ether yielded 2.58 g (8.85

mmol, 68%). Chromatographically homogeneous in Systems A and

m.p. 175-177°C.

~~~~~ ~~ ~~~~ ~ ~~~~~~~~~~ ~

.~~

The abbreviations used are: Boc, t-butyloxycarbonyl; Bpoc.

2-(4-

biphenyl)-2-propyloxycarbon,~l;

Bz. benzoyl;

Rzl,

benzyl; I’h, phenyl;

OEt, ethyl ester; OTMB,

2,4,fi,-trimethylbenzylester;

HPLC. high

performance liquid chromatography.

1301

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Protease-mediated PeptideBond Formation

ON SOME UNEXPECTED OUTCOMES DURING ENZYMATIC SYNTHESIS OF LEU-ENKEPHALIN*

to proteolysis. The use of proteases, the specificity of

sized, thus led to a priori cleavage of pre-existing pep-

of new peptide bonds between initial reactants and

A detailed characterization of the undesired products

expected outcome of protease catalysis led to working

formed will be sufficiently insoluble to escape from a poster-