Biomedical Engineering Reference
In-Depth Information
The N-terminal methionine residue of protein can also be employed for selective
PEGylation using aldehyde-terminated PEG via a reductive amination reaction,
because the N-terminal primary amine has a lower p
K
a of 7.8 than other amines
such as lysines, whose p
K
a is 10.1 [
7
]. After reaction with aldehyde-terminated
PEG at low pH, the resultant imine is reduced with sodium cyanoborohydrate to
provide PEGylated protein (Fig.
4
)[
8
,
9
]. This technique was used for the produc-
tion of Neulasta, which was approved for use by the FDA in 2002 [
10
].
2.2 Enzymatic PEGylation
Novel methods have been proposed by various researchers to achieve site-specific
PEGylation using enzymatic PEGylation reactions. Sato et al. utilized transglu-
taminase (TGase; protein-glutamine
g
-glutamyltransferase), an emerging enzyme
[
11
]. This enzyme catalyzes an acyl transfer reaction between the
g
-carboxyamide
group of the glutamine residue (acyl donor) in a protein and a variety of primary
amines (acyl acceptor) (Fig.
5
)[
12
]. TGase is believed to require special sequential
structures of the acyl donor for efficient modification; however, it is interesting to
note that a variety of primary amines are accepted as acyl donors. Thus, TGase is a
very useful reagent for protein modification [
13
]. An amino derivative of PEG, such
as PEG-NH
2
, can be used as an acyl acceptor for PEGylation of proteins. Several
1. PEG aldehyde
amines in lysine
N-terminal amine
CH = O
NH
2
pka = 7.8
CH
2
NH
NH
2
NH
2
NH
2
NH
2
2. NaCNBH
3
NH
2
NH
2
pka = 10.1
Fig. 4 PEGylation at the N-terminal methionine residue. The difference in p
K
a between the
N-terminal amine and other amines in the protein enables site-specific PEGylation. After reaction
with aldehyde-terminated PEG at low pH, reduction of the resultant imine produces PEGylated
protein
protein
PEGylated protein
O
PEG
C
NH(CH
2
)
5
NH
2
CH
CH
2
CH
2
C = O
CH
CH
2
CH
2
C = O
Acyl acceptor
TGase
O
PEG
C
NH(CH
2
)
5
NH
NH
2
Acyl donor
Fig. 5 Enzymatic site-specific PEGylation by transglutaminase (
TGase
). The alkylamine deriva-
tive of PEG can be introduced into proteins in a site-specific manner