Site-specific protein PEGylation catalyzed by endo-β-N-acetylglucosaminidase

Article abstract of DOI:10.1016/j.tetlet.2019.151475

We demonstrated a novel site-specific method of protein PEGylation that is catalyzed by endo-β-N-acetylglucosaminidase (ENGase) with PEGylated oxazoline (1) as a donor. PEGylation via transglycosylation using 1 proceeded smoothly to GlcNAc compounds such as p-nitrophenyl N-acetylglucosaminide and RNase B derivatives.

Full text of DOI:10.1016/j.tetlet.2019.151475

Journal Pre-proofs
Site-specific protein PEGylation catalyzed by endo-β-N-acetylglucosaminidase
Kohtaro Goto, Masaki Kurogochi, Shou Takashima, Masako Mori, Akio
Matsuda, Mamoru Mizuno
PII:
S0040-4039(19)31274-2
DOI:
https://doi.org/10.1016/j.tetlet.2019.151475
TETL 151475
Reference:
Tetrahedron Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
3 October 2019
28 November 2019
2 December 2019
Please cite this article as: Goto, K., Kurogochi, M., Takashima, S., Mori, M., Matsuda, A., Mizuno, M., Site-specific
protein PEGylation catalyzed by endo-β-N-acetylglucosaminidase, Tetrahedron Letters (2019), doi: https://doi.org/
10.1016/j.tetlet.2019.151475
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Site-specific protein PEGylation catalyzed by
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endo-β-N-acetylglucosaminidase
Kohtaro Goto, Masaki Kurogochi, Shou Takashima, Masako Mori, Akio Matsuda, and Mamoru Mizuno*
: N-Acetylglucosamine
PEG
PEG
: Mannose
oxa
PEG
PEG
ENGase

1
Tetrahedron Letters
journal homepage: www.elsevier.com
Site-specific protein PEGylation catalyzed by endo-β-N-acetylglucosaminidase
Kohtaro Gotoa, Masaki Kurogochia, Shou Takashimaa, Masako Moria, Akio Matsudaa, and Mamoru
Mizunoa
a The Noguchi Institute, 1-9-7, Kaga, Itabashi, Tokyo, Japan
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
We demonstrated a novel site-specific method of protein PEGylation that is catalyzed by
endo-β-N-acetylglucosaminidase (ENGase) with PEGylated oxazoline (1) as a donor.
PEGylation via transglycosylation using 1 proceeded smoothly to GlcNAc compounds such as
p-nitrophenyl N-acetylglucosaminide and RNase B derivatives.
Available online
2009 Elsevier Ltd. All rights reserved.
Keywords:
Site specific
PEGylation
ENGase
Oxazoline
———
 Corresponding author. Tel.: +81-3-5944-3215; fax: +81-3-3964-4071; e-mail: mmizuno@noguchi.or.jp

2
Tetrahedron
by using transglycosylation of N-glycan with ENGases.27,28 In
1. Introduction
these ENGase-catalysed transglycosylation reactions, it has been
revealed that sugar oxazoline derivatives can be used as powerful
donors.29 We expected that site-specific PEGylation could be
achieved by using the glycosyl transfer activity of ENGases
together with sugar donors comprising sugar oxazoline
derivatives to which a PEG chain had been connected in advance.
Furthermore, the use of glycans as the linker has the possibility
that it does not significantly affect the structure of the protein and
the activity of protein is not reduced by PEGylation. Herein, we
Proteins and peptides have been used as therapeutic agents for
a long time, and now account for a large share of all newly
approved drugs and are used in almost every field of medicine.1
Most protein therapeutics are highly active owing to their
specific structure, and almost all are characterized by being less
immunogenic than other common drugs as they are based on
endogenously expressed proteins. Due to this versatility, they are
used as therapeutic agents in a wide variety of diseases.
report
a novel site-specific chemo-enzymatic method of
However, protein therapeutics have the disadvantage of
having a short half-life in the body compared to other
therapeutics due to their degradation by proteases in the body and
excretion via the kidneys, and in many cases frequent
administration of the drug is required. This disadvantage can
cause complications such as eliciting an immune response,2,3
which is a huge issue that needs to be resolved. Many methods of
protein modification aiming to extend this short half-life in the
body have been reported.4-6 The PEGylation method, in which a
polyethylene glycol (PEG) chain is introduced to the protein, is
one of the most widely used methods at present.7
PEGylation catalyzed by an ENGase, such as endo--N-
acetylglucosaminidase from Mucor hiemalis (Endo-M) or endo-
-N-acetylglucosaminidase from Arthrobacter protophormiae
(Endo-A), with a PEGylated donor.
2. Results and discussion
In 2001, Fujita et al. reported Endo-M-catalyzed
transglycosylation
with
disaccharide
(Manβ1-4GlcNAc)
oxazoline as a donor.30 Therefore, we designed and synthesized
3′,6′-BisPEG5 oxazoline (1) with N-glycan as a motif, which had
pentaethylene glycol chains connected to the 3′ and 6′ positions
of the mannose residue as a PEGylated disaccharide oxazoline
donor (Fig. 1). Usually, high molecular weight PEG reagents for
protein PEGylation are sold in a state with a wide molecular
weight distribution rather than a single structure. This is indicated
the identification of PEGylated compounds by using NMR are so
difficult. Therefore, we selected pentaethylene glycol derivative
which can be easy to identify the structures as first step of
PEGylati
PEG chains have several desirable properties such as
flexibility, hydrophilicity, low toxicity, and a size that can be
freely changed. Introducing a PEG chain having such properties
into a protein confers many advantages, such as increasing its
half-life in the blood and significantly improving its solubility. In
the 1970s, Davis et al. reported that random PEGylation of
proteins increased their half-life in the body.8,9 Since those
reports, a huge number of studies on protein PEGylation have
been conducted, and over 10 PEGylated products are currently
registered for clinical use and more are in clinical trials. Many of
those PEGylated proteins are prepared by introducing a PEG
chain via an amide bond with the N-terminal amino group of the
protein or as a side chain of a lysine residue.10 Although
PEGylation through an amide bond is excellent because of its
high yields and simple operation, various regioisomers which are
PEGylated at different positions and numbers are also formed.
Mixtures with a plurality of PEGylated regioisomers show a wide
range of activities, and a uniform medicinal effect cannot be
expected.11
A)
OH
OH
OBn
H(OCH2CH2)5O
OBn
O
OBn
OBn
O
O
HO
HO
O
O
Ph
O
O
ii)
iii)
iv)
H(OCH CH ) O
BnO2
O
2
O
HO
O
O
RO
HO5
OBn
OBn
BnO
BnO
O
1
N
NPhth
NPhth
2 : R = PMB
3 : R = H
4
OH
i)
Endo-M(WT)
or
OBn
O
O
HO
HO
OPNP
N175Q
OBn
AcHN
OBn
O
OBn
HO
BnO
HO
AcO
BnO
AcO
v)
9
O
O
O
O
OBn
OBn
BnO
BnO
OH
OH
AcHN
H(OCH2CH2)5O
AcHN
OH
O
O
HO
O
5
6
O
H(OCH2CH2)5O
O
HO
HO
OPNP
To solve this issue, various methods of site-specific
PEGylation that are expected to improve the stability of
proteinaceous pharmaceuticals in the body while minimizing
reduction in their activity have been reported.10,12 These methods
include, for example, a thiol selective method using maleimide,13
an N-terminal amino group selective method using the difference
of pKa with a lysine side chain,14 a C-terminal selective method
using sortase,15 and oligosaccharide on protein selective methods
using glycosyltransferase.16,17
AcHN
OR
AcHN
viii)
OR
O
10
R(OCH2CH2)5O
RO
vi)
O
B)
O
R(OCH2CH2)5O
OR
RO
100
AcHN
7 : R = Bn
8 : R = H
90
vii)
80
[%]
70
OH
60
50
OH
H(OCH2CH2)5O
wild type
yield
O
PEG5
HO
N175Q
O
O
: GlcNAc
: Man
H(OCH2CH2)5O
40
oxa
rsion
HO
30
20
10
0
PEG5
O
N
1
Conve
We have designed a novel method of site-specific enzymatic
PEGylation that makes use of glycosidases, represented by
ENGases. ENGases are used in various situations as useful tools
for the chemo-enzymatic synthesis. ENGases of the glycoside
hydrolase family 85 (GH85) are endoglycosidases that release N-
glycans from glycoproteins by hydrolyzing the glycosidic bond
in the chitobiose (GlcNAcβ1-4GlcNAc) core of asparagine-
linked oligosaccharides (N-glycan) of glycoproteins. Some
ENGases have been used for glycopeptide and glycoprotein
synthesis because of their transglycosylation activity, and more
efficient and higher-yielding glycoprotein synthesis has been
achieved in recent years by using mutant ENGases with both
suppressed hydrolytic activity and enhanced transglycosylation
activities.18-26 For example, Wang and co-workers have achieved
chemo-enzymatic synthesis of homogeneous RNase derivatives
Fig. 1. Synthesis of 3',60'-BisPEG5 oxazoline 1. Reagents and
30
60
90
120
150
180
210
240
Reaction time [min]
conditions: i) DDQ, CH2Cl2-H2O, r.t., 3 h, 83%; ii) 1.0 M
Fig. 2. ENGase-catalyzed PEGylation by Endo-M (WT) and
BH3/THF, 1.0 M n-Bu BOTf/CH Cl2, 0 oC, 1 h, 94%; iii)
N175Q. A) 2Scheme o2f the PEGylation. Assay conditions: 2
mM GlcNAc acceptor 9, 12 mM oxazoline donor 1, Endo-M
(CH2NH2)2, MeOH, reflux, 17 h; iv) Ac2O, Pyridine, MeOH,
r.t., 17 h, 2 steps 84%; v) NaOMe, MeOH, r.t., 19 h, 96%; vi)
(WT) or N175Q (10 mU), 50 mM phosphate buffer (pH 7.0, 40
o
Bn(OCH2CH2)5OTs, NaH, DMF, 0 C 1 h then r.t. 17 h, 87%;
o
L), 30 C. B) Time course of the PEGylation. Yields were
vii) H2 gas, Pd/C, 1,4-dioxane-H2O, r.t., 5 h, 93%; viii) DMC,
determined by integration of the acceptor and product peaks in
Et3N, H2O, 0 oC, 17 h, 99%.
the HPLC profile.
on in this paper.
The p-methoxy benzyl (PMB) group of compound 231 was
treated with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ)
to afford compound 3 in a yield of 83%. This was followed by

3
site-specific ring opening of the benzylidene acetal which yielded
compound 4 in 94%. The phthaloyl group of compound 4 was
removed with ethylenediamine, and subsequent acetylation
afforded compound 5 in a yield of 84%. The acetyl groups of O-
3′ and O-6′ of compound 5 were removed with NaOMe in 96%.
Endo-M(WT) on the basis of the results of PEGylation with
GlcNAc-OPNP 9. However, although various reaction conditions
were investigated, a new PEGylated protein band was only
observed with about 10% yield. Next, we tried Endo-A(WT)34
from Arthrobacter protophormiae as another ENGase for
enzymatic PEGylation based on the finding28 that Endo-A can
transfer various compounds because of its lower substrate
recognition than Endo-M, therefore, we selected Endo-A
accordingly. A mixture of the acceptor 11 (53.5 M), PEGylated
oxazoline 1 (10 mM) as donor, and Endo-A(WT) (3.3 µg/µL) in
Tris-HCl buffer (pH 7.0, 50 mM) was incubated at 30°C. After 3
hours, a new protein band corresponding to PEGylated RNase B
12 (61% HPLC yield, see supporting information Fig. S14),
which was about 1 kDa larger than the GlcNAc-RNase B 11, was
clearly formed (Fig. 3, lane 2). The PEGylated RNase B (12) was
isolated by HPLC (Fig. 3, lane 3), and its identity was
characterized by a combination of analytic means (see supporting
information 2.4).
Compound
6
and Bn(OCH2CH2)5OTs (see supporting
information Fig. S1) as PEGylation reagents were coupled with
NaH in an 87% yield. Hydrogenation of 7 in the presence of
Pd/C was then conducted to remove all benzyl ethers with a yield
of 93%. Finally, conversion of compound 8 into oxazoline
derivative
1
was achieved by using 2-chloro-1,3-
dimethylimidazolinium chloride (DMC)32 and triethylamine at
0°C in an excellent yield (99%).
ENGase-catalyzed PEGylations were then carried out by using
Endo-M wild type (WT) and its mutant N175Q. First, we
examined the transglycosylation abilities of PEGylated donor 1
toward p-nitrophenyl N-acetylglucosaminide 9 (GlcNAc-OPNP)
(Fig 2). Both Endo-M(WT) and N175Q were able to catalyze the
PEGylation via transglycosylation of the Man-GlcNAc moiety to
9, and the corresponding trisaccharide derivative 10 was formed.
However, the transfer efficiency was very different, and Endo-
M(WT) was far superior to N175Q in the reaction yield and time.
Furthermore, the product yield did not decrease with time at all in
Endo-M(WT)-catalyzed transglycosylation under these reaction
conditions. This result indicated that the PEGylated products
were highly resistant to hydrolysis by Endo-M(WT). We
speculated the hydrolysis rate of ENGase is important for the
transfer reaction of PEGylated oxazoline. N175Q is suppressed
its hydrolytic activities, therefore we guessed the
tranglycosylation yield using N175Q is much lower than Endo-
M(WT). In addition, NMR analysis after deuterated acetylation
of compound 10 found that even though the donor was
PEGylated, the resulting glycosidic bond was a β1-4 linkage (see
supporting information Fig. S17).
3. Conclusions
In conclusion, we developed a novel method of site-specific
protein PEGylation which was catalyzed by an ENGase such as
Endo-M or Endo-A with PEGylated disaccharide oxazoline 1 as
a donor. The PEGylated reaction via transglycosylation using 1
proceeded smoothly to form GlcNAc compounds such as
GlcNAc-OPNP and GlcNAc-RNase B. In this method, wild type
ENGases were much more suitable than the corresponding
mutants owing to the high resistance of PEGylated products to
hydrolysis by ENGases. This methodology can be expected to be
useful not only in protein PEGylation but also as a method of
site-specific modification of proteins by using a modified short
PEG chain. We are currently improving the method of
PEGylation of site-specific proteins using longer or modified
short PEG chains.
We then attempted ENGase-catalyzed chemo-enzymatic
protein PEGylation by using the PEGylated donor 1 and bovine
pancreatic ribonuclease B (RNase B) as a model system. RNase
B is a natural glycoprotein consisting of 124 amino acids and
carries heterogeneous high-mannose-type N-glycan (Man5-
9GlcNAc2) at the Asn34 residue.33 To test the chemo-enzymatic
approach, first GlcNAc-RNase B (11) which could be used as an
acceptor of PEGylation was prepared by deglycosylation of
natural RNase B with Endo-H based on Wang’s report (Fig 3,
lane 1).27 Protein PEGylation reactions were then conducted with
4. Acknowledgments
We thank Tomohiro Hirose (Instrumental Analysis Division,
Equipment Management Center, Creative Research Institution,
Hokkaido University), for high resolution mass spectra analysis.
5. References and notes
1.
Leader, B.; Baca, Q. J.; Golan, D. E. Nat. Rev. Drug Discovery,
2008, 7, 21.
2.
3.
4.
Groot, A. S. D.; Scott, D. W. Trends Immunol., 2007, 28, 482.
Nagata, S.; Pastan, I. Adv. Drug Deliv. Rev., 2009, 61, 977.
Hutt, M.; Schwarz, A. F.; Unverdorben, F.; Richter, F.;
Kontermann, R. E. J. Biol. Chem., 2012, 287, 4462.
Trüssel, S.; Dumelin, C.; Frey, K.; Villa, A.; Buller, F.; Neri, D.
Bioconjug. Chem., 2009, 20, 2286.
Szlachcic, A.; Zakrzewska, M.; Otlewski, J. Biotechnol. Adv.,
2011, 29, 436.
Kodera, Y.; Matsushima, A.; Hiroto, M.; Nishimura, H.; Ishii, A.;
Ueno, T.; Inada, Y. Prog. Polym. Sci., 1998, 23, 1233.
Abuchowski, A.; van Es, T.; Palczuk, N. C.; Davis, F. F. J. Biol.
Chem., 1977, 252, 3578.
PEG5
A)
oxa
5.
6.
7.
8.
9.
PEG5
PEG5
1
Endo-M(WT)
or
PEG5
12
11
Endo-A(WT)
Abuchowski, A.; McCoy, J. R.; Palczuk, N. C.; van Es, T.; Davis,
F. F. J. Biol. Chem., 1977, 252, 3582.
B)
10. Dozier, J. K.; Distefano, M. D. Int. J. Mol. Sci., 2015, 16, 25831.
11. Foser, S.; Schacher, A.; Weyer, K. A.; Brugger, D.; Dietel, E.;
Marti, S.; Schreitmüller, T. Protein Expr. Purif., 2003, 30, 78.
12. Pasut, G.; Veronese, F. M. J. Controlled Release, 2012, 161, 461.
13. Unterweger, B.; Stoisser, T.; Leitgeb, S.; B.-Grünberger, R.;
Nidetzky, B. Bioconjug. Chem., 2012, 23, 1406.
1
2
3
M
lane
lane
lane
(kDa)
25
20
14. Kinstler, O. B.; Brems, D. N.; Lauren, L. S.; Paige, A. G;
Hamburger, J. B.; Treuheit, M. J. Pharm. Res., 1995, 13, 996.
15. Popp, M. W.; Antos, J. M.; Grotenbreg, G. M.; Spooner, E.;
Ploegh, H. L. Nat. Chem. Biol., 2007, 3, 707.
17
11
16. DeFrees, S.; Wang, Z. G.; Xing, R.; Scott, A. E.; Wang, J.; Zopf,
D.; Gouty, D. L.; Sjoberg, E. R.; Panneerselvam, K.; B-.van der
Fig. 3. ENGase-catalyzed protein PEGylation by Endo-M (WT)
and Endo-A (WT). A) Scheme of the PEGylation. Assay
conditions: 53.5 M GlcNAc-RNase B 11, 10 mM oxazoline
donor 1, Endo-M (1.0 mU/ L) or Endo-A (3.3 g/ L), 50 mM
Tris-HCl buffer (pH 7.0, 30 L), 30 oC. B) SDS-PAGE: lane 1,
GlcNAc-RNase 11; lane 2, Reaction mixture of PEGylation
reaction by Endo-A(WT); lane 3, Pure PEGylated RNase B 12.

4
Tetrahedron
Linden, E. C. M.; Bayer, R. J.; Tarp, M. A.; Clausen, H.
Glycobiology, 2006, 16, 9. 833.
J.; Matsuda, A.; Tomita, M.; Takayanagi, A.; Shoda, S.; Shirai, T.
PLoS One., 2015, 10:e0132848.
17. Stennicke, H. R.; Østergaard, H.; Bayer, R. J.; Kalo, M. S.;
25. Tomabechi, Y.; Katoh, T.; Kunishima, M.; Inazu, T.; Yamamoto,
K. Glycoconj J., 2017, 34, 481.
26. Ishii, N.; Ogiwara, K.; Sano, K.; Kumada, J.; Yamamoto, K.;
Matsuzaki, Y.; Matsuo, I. ChemBioChem, 2018, 19,136.
27. Amin, M. N.; Huang, W.; Mizanur, R. M.; Wang, L. X.; J. Am.
Chem. Soc., 2011, 133, 36, 14404.
Kinealy, K.; Holm, P. K.; Sørensen, B. B.; Zopf, D.; Bjørn, S. E.
Thromb Haemost, 2008, 100, 920.
18. Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.; Kawakami, T.;
Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc., 1999,
121, 2, 284.
19. Yamamoto, K. J. Biosci. Bioeng., 2001, 92, 493.
20. Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L. X. J. Am. Chem.
Soc., 2005, 127, 9692.
21. Rising, T. W.; Claridge, T. D.; Moir, J. W.; Fairbanks, A. J.
ChemBioChem, 2006, 7, 1177.0
22. Murakami, D.; Shimada, Y.; Kamiya, S.; Yamazaki, K.;
Makimura, Y.; Ito, K.; Minamiura, N.; Yamamoto, K. Arch.
Biochem. Biophys., 2008, 477, 299.
28. Huang, W.; Yang, Q.; Umekawa, M.; Yamamoto, K.; Wang, L. X.
ChemBioChem, 2010, 11, 10, 1350.
29. Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto, K.; Zhang,
X.; Wang, L. X. J. Am. Chem. Soc., 2009, 131, 2214.
30. Fujita, M.; Shoda, S.; Haneda, K.; Inazu, T.; Takegawa, K.;
Yamamoto, K. Biochimica et Biophysica Acta, 2001, 1528, 9.
31. Zou, G.; Ochiai, H.; Huang, W.; Yang, Q.; Li, C.; Wang, L. X. J.
Am. Chem. Soc., 2011, 133, 18975.
23. Umekawa, M.; Li, C.; Higashiyama, T.; Huang, W.; Ashida, H.;
Yamamoto, K.; Wang, L. X. J. Biol. Chem., 2010, 285, 511.
24. Kurogochi, M.; Mori, M.; Osumi, K.; Tojino, M.; Sugawara, S.;
Takashima, S.; Hirose, Y.; Tsukimura, W.; Mizuno, M.; Amano,
32. Noguchi, M.; Tanaka, T.; Gyakushi, H.; Kobayashi, A.; Shoda, S.
J. Org. Chem., 2009, 74, 2210.
33. Baynes, J. W.; Wold, F. J. Biol. Chem., 1976, 251, 6016.
34. Fujita, K.; Tanaka, N.; Sano, M.; Kato, I.; Asada, Y.; Takegawa,
K. Biochem. Biophys. Res. Commun., 2000, 267, 134.

5
Site-specific protein PEGylation catalyzed by ENGase
Kohtaro Goto, Masaki Kurogochi, Shou Takashima, Masako Mori, Akio Matsuda, and Mamoru Mizuno*
Highlights
We demonstrated a novel site-specific method of protein PEGylation.
The PEGylation was catalyzed by endo-β-N-acetylglucosaminidase (ENGase).
Wild type ENGases were much more suitable than the corresponding mutants.