Preparation of a new PEGylation reagent for sulfhydryl-containing polypeptide

Tetrahedron

Letters,

Vol. 35, No. 35. pp. 65 15-6516. 1994 Elsevier Scvmce Ltd Printed in Great Britain 0040-4039/94 $7.Oct+o.00

0040-4039(94)01357-8

Preparation of a New PEGylation Reagent for Sulfhydryl-containing Polypeptide

Wei tang, Yuan Chnng, and Xin-Yuan Shanghai Institute of Biochemistry, Academia

Liu

Sinica, China

320 Yue-Yang Road, Shanghai, 200031

Abstract: N-maleimido-6-amino-caproyl

ester of

of DCC etc., as a new sulthydryl-PEGylation

Recently,

chemical

modification,

widely applied for altering their potency.

especially

PEG was

preparated in the presence

reagent substituded for m&sac-PEG.

PEGylation

The bitictional

of native

crosslinking

or recombinant

proteins

has been

reagent, N-maleimido-6-amide-caproyl

ester of I-hydroxy-2-nitro-4~benzene sulfonic acid (mal-sac-HNSA, (a)) can be used for conjugation of sulfhydryl-containing compound(protein, peptide etc. ) to PEG-NH2(b)l-2. However the synthesis of (a) is rather complicated

and that of (b) is very difficu@-7.

and (b) to make a new activated proteins

PEG ester directly

Here we describe

a procedure

without

preparation

as below, which can be used for sulfhydryl-PEGylation

of (a) of

or peptides.

HCA II HCA’

/? ‘0 + NH*-(CH2)+OOH

------+

B ‘NH-(CH,),-COOH “i HC----COOH

“0

HC--C, -

II H&---C

P

mPEG

N-(CH2)5-COOH-

HC---C 11

S

$0

(d)

(c) At first, maleic anhydride was added and refluxed

t-(CH2)5-COO-mPEG

HC--C’

reacted with 6-aminocaproic

under a Dean-Stark

water separator

6515

acid for 4 hours at 25°C , then more acetic acid over night. The acetic acid was removed

in vacua

6516

and the oily residue containing column in chloroform:acetic sac-PEG)‘can

(c) was dissloved

be made by the reaction of(c)

4-dimethylaminopyridine(DMAP) precipitation

in chloform,

filtered and chmomatogmphed

acid (95:5). Then the pure product (c) was obtained. with mPEG (MWSOOO) in methylene

and N,N-dicylohexylcarbodiimide

with ether, recrystallization

Finally,

from ethanol,

(DCCI),

and lyophization

over a silica gel

the product (d) (mal-

chloride, followed

in the presence by purification

to give a powder.

of by

The total yield was

50-60%. The data of UV-spectra

show that : the yield for the attachment

the PEG is >90%. The activated

PEG ester is readily soluble

several months when stored at -20°C In a first example&ovine with dithiothrcitol(DTT)

buffer (pH6.0), then mal-sac-PEG

crosslinked bioactivity

example,

to the activated

is reported. 7-steps

the

of recombinant

y-interforon

method for the preparation for the preparation

and the difftcult

Because the yield of the reaction of mal-sac-HNSA

for several hours or over night. Results (rIFN-y),

i.c.,

with the previous

be directly used more efficiently

steps

for making

HNSA

of medically

is much higher and therfore can

important

proteins

1990,8,343

3. Manfred, Mutter, Tetrahedron Letters 1978.3 1.2839

6. Dereu, Norbert

Cr., Makromolekukw

Edith, Mabolekular

than

of the products

Robert J.; Katre, Nandini V., Bio/Technologv

Dellaeherie,

reagent

to the mPEG is not high, and the separation

reagent , the purity of the new reagent

for sulfhydryl-PEGylation

A. F.; Morr, M.; Johansson

reagent rather

were averted.

1. Aldwin, Lois; Nitecki, Danute E., Analytical Biochem. 1987,164,494

5. Leonard, Michele;

were had full

and PEG-NH2

Reference

4. Buckmann,

ZSCys-rIFN-y.

of a new sulfhydryl-PEGylation

of sullhydryl--PEGylation

or more specific sites.

2. Coodson,

BSA was treated

in O.OSM phosphate

one. The result will be shown in another paper.

It needs only 3-step reactions

is not easy, compared

PEG(d).The

G-25 column

PEG (d) as above. The yield was about 50-80%. The PEG-ZSCys-rIFN-y

in literature,

acid to

It is stable for

could been bound to one BSA molecule.

mutants

a simple high yielding

that reported

with activated

by a Sephadex

was added in excess, stirred at 2S°C

relative to the unmodified

In conclusion,

was modified

and DTT was removed

indicated that one or more PEG molecules In another

chloride.

.

serum albumin(BSA)

previously

of the N-maleimido-6-amidocapoic

in water or methylene

Chemie 1981,182(4-6),1379

Chemie 1988,189,1809

, Synthetic Cornmunitions 1991,21( 1),85

7. King, Ruth .J: Chem. Sot. 1921,119,2105

(Received in China 19 April 1994, accepted 4 July 1994)

or peptides

at one