Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of ω-Asp p-Nitrophenyl Thioesters in Solution

Article abstract of DOI:10.1021/acs.orglett.6b02288

An efficient N-linked glycosylation reaction between glycosylamines and p-nitrophenyl thioester peptides has been developed. The reaction conditions are mild and compatible with the C-terminal free carboxylic acid group and the unprotected N-linked sialyl

Full text of DOI:10.1021/acs.orglett.6b02288

Letter
pubs.acs.org/OrgLett
Convergent Synthesis of N‑Linked Glycopeptides via Aminolysis of
ω‑Asp p‑Nitrophenyl Thioesters in Solution
†
†
Jing-Jing Du, Xiao-Fei Gao, Ling-Ming Xin, Ze Lei, Zheng Liu,* and Jun Guo*
Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, CCNU−uOttawa Joint Research Centre, College of
Chemistry, Central China Normal University, 152 Luoyu Road, Wuhan, Hubei 430079, P. R. China
*
S Supporting Information
ABSTRACT: An efficient N-linked glycosylation reaction between glycosylamines and p-nitrophenyl thioester peptides has been
developed. The reaction conditions are mild and compatible with the C-terminal free carboxylic acid group and the unprotected
N-linked sialyloligosaccharide. By means of this convergent strategy, a versatile N-glycopeptide fragment containing an N-
terminal Thz and a C-terminal thioester was readily prepared, which is available for the synthesis of long glycopeptides and
glycoproteins using the protocol of native chemical ligation.
N-Linked glycosylation is the attachment of a glycan through
N-acetylglucosamine by an amide bond at the side chain of
asparagine within an Asn-X-Ser/Thr consensus sequence
Scheme 1. Strategies for N-Linked Glycosylation Reaction
(where X is any natural amino acid except proline), and it is
regarded as a kind of important co- and post-translational
1
modification for many proteins. This type of linkage influences
various important biological processes, such as protein quality
control and cell recognition, and its faulty structure may result
2
in protein malfunction and diseases. Thus, understanding the
relationship between the structure and biological function of N-
linked glycopeptides is essential in various biomedical projects.
However, N-linked glycosylation in nature usually results in
heterogeneous mixtures of glycoforms, which hinders the study
3
,4
of their diverse biological phenomena. Fortunately, chemical
5
or chemoenzymatic methods provide an ideal platform for
studying N-linked glycopeptides and glycoproteins with
structure-defined glycans.
glycosylation reaction between the glycosylamines and p-
nitrophenyl thioester peptides in solution.
In recent years, various advances to chemically synthesize N-
linked glycoproteins have been reported. Chemical synthetic
In previous studies, activated C-terminal peptides with thiol
6
8
9
esters and o-benzaldehyde esters have been utilized to directly
condense with peptide fragments in the absence of an N-
terminal cysteine residue, thiol auxiliary, or exogenous
activating reagent. Aminolysis of the activated thioesters to
form amide bonds was conducted smoothly, leading to the
ligation products in good yields. We decided to investigate the
strategy of incorporating the glycosylamine moiety into an
active thiol ester of an Asp residue of the peptide.
strategies can be encompassed into sequential or convergent
approaches (Scheme 1). The synthesis on resin via either the
sequential (Scheme 1a) or convergent (Scheme 1b) approach
often suffers from loss of precious material and low reaction
3
yield. In contrast, the convergent method in solution
3
b,c
(
“Lansbury aspartylation” ) can be advantageous, and
pseudoproline dipeptides incorporated into these peptide
substrates significantly suppressed the formation of undesired
aspartimides during the N-linked glycosylation. However, in
Initially, we investigated the influence of different leaving
groups of activated esters during the reaction with glycosyl-
7
10,3j,m
the practical synthesis of glycoproteins via ligation between
glycopeptides and other peptide fragments, some inconven-
amine 1a
(β-anomer; Figure S44) to obtain the glycosyl
product 3 (Table 1 and Figure 1). The reaction processes were
monitored and analyzed by HPLC (Figure S1). In our study,
3m
iences still remained, such as the use of metal catalysts or
3l
protection of C-terminal carboxylic acids. In this regard, we
decided to develop an efficient convergent method for N-linked
glycopeptides. Herein we report an efficient N-linked
Received: August 2, 2016
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.6b02288
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Table 1. N-Linked Glycosylation Reactions of Esters with
Different Functional Groups
Table 2. Optimization of the Reaction Conditions
a
b
entry
1a
2a
solvent
base (equiv)
yield (%)
b
c
entry
ester
X
R1
R2
time (h)
yield (%)
c
1
2
3
4
5
6
7
8
9
2.0
2.0
2.0
2.0
2.0
2.0
2.0
1.0
3.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
DMSO/PB
NMP
DIPEA (2.0)
DIPEA (2.0)
DIPEA (2.0)
DIPEA (2.0)
NMM (2.0)
TEA (2.0)
−
6
41
60
73
61
64
58
53
1
2
3
4
5
6
7
8
9
2a
2b
2c
2d
2e
2f
S
S
S
S
NO2
H
H
4
26
73
56
42
15
2
CHO
H
DMF
CHO
H
42
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
H
>60
>60
60
CH2S
O
H
H
NO2
CHO
H
H
34
3
2g
2h
2i
O
H
>60
>60
>60
DIPEA (2.0)
O
CHO
H
7
DIPEA (2.0)
73
b
a
O
H
<1
Reaction conditions: 1a (0.1 M), 2a (0.05 M), base, rt, 4 h. Yields
were determined by HPLC. PB = phosphate buffer (pH 7.4, 200
a
c
Reaction conditions: 1a (0.1 M), 2 (0.05 M), DIPEA (0.1 M),
DMSO, rt. Times for >95% conversion of the starting materials to the
amide products at room temperature as determined by HPLC. Yields
b
mM).
c
were determined by HPLC at 60 h.
gave the best performance (entries 2−4). When diisopropyle-
thylamine (DIPEA) was replaced with another base (4-
methylmorpholine (NMM) or triethylamine (TEA)), the
yield of product slightly decreased (entries 5 and 6). For the
amount of DIPEA, 2.0 equiv relative to the thioester was
suitable for the N-linked glycosylation reaction (Table S1,
entries 4 and 7−10). For the amount of glycosylamine 1a, the
highest yield of 3 was obtained when 2.0 equiv of 1a was added
to thioester 2a (Table 2, entries 4, 8, and 9). Therefore, the
optimal conditions are to employ 2.0 equiv of glycosylamine 1a
and 2.0 equiv of DIPEA in DMSO at room temperature for 4 h.
With the optimized reaction conditions in hand, we prepared
a range of peptides containing ω-Asp p-nitrophenyl thioesters
(
4a−10a) as the substrates for N-linked glycosylation. The
peptides were prepared by solid-phase peptide synthesis and
obtained in good yields after cleavage from the resin (the
detailed procedure is available in the Supporting Information).
Figure 1. Yields of N-linked glycosylation product 3 vs time for
different esters.
As shown in Scheme 2, these substrates were conjugated with
10,12
unprotected glycosylamines (1a−c)
to give N-linked
glycopeptides (4b−10b, 4c, 8c, 9c, 8d, 9d), and the desired
coupling products bearing the amide bond linkage at ω-Asp
were achieved in good yields. Notably, in this protocol just
mixing two shelf-stable components affords the desired
glycopeptide quickly; no other coupling reagents or catalysts
are required, and the workup is simple. Furthermore, this
coupling reaction is compatible with the free carboxylic acid
group at the C-terminus of peptide substrates or the sialic acid
of unprotected sialyloligosaccharides. The C-terminal free
carboxylic group of glycopeptides can be directly converted
into thioesters for further ligation with other peptide fragments.
Using the unprotected complex-type sialyloligosaccharide 1c
as the substrate greatly simplified the procedure for synthesiz-
ing naturally occurring N-linked glycopeptides and glycopro-
teins. The amino acid protecting groups in glycopeptides can be
easily removed using the common trifluoroacetic acid
deprotection protocol. For the orthogonal masking group
Thz (7b), deprotection with methoxyamine hydrochloride
gives free N-terminal Cys, which can be employed in the next
reaction of native chemical ligation.
the reactivities of thioesters and oxoesters were compared; the
influence of different substituents on the aromatic moiety was
11
also investigated, i.e., p-nitro (electron-withdrawing group), o-
aldehyde (electron-withdrawing + neighbor-participating
9
9c
group), and p-aldehyde (electron-withdrawing group) For
thioesters (2a−e), 2a containing a p-nitro group had the best
reactivity and achieved the highest yield of product, which was
followed by 2b containing an o-aldehyde group and 2c
containing a p-aldehyde group; thioesters 2d and 2e, the
common kind of activated esters usually employed to active C-
terminal peptides in peptide and protein synthesis, have a lower
reaction rate. For oxoesters (2f−i), the influence of different
substituents on the reactivity has a similar tendency as for the
thioesters (2a−d), but the reactivities of the oxoesters are too
low (≥60 h for 95% conversion) to accommodate the N-linked
glycosylation processes, even with prolonged time. Therefore,
thioester 2a containing a p-nitro group, which had the best
reactivity and gave the highest yield of product, was the
optimized activated ester used for N-linked glycosylation.
To further probe the efficiency of the N-glycosylation
reaction, different conditions were investigated for thioester 2a
to react with 1a (Table 2). In aqueous solution (entry 1), the
predominant product was the hydrolysis product of 2a. For
different organic solvents (NMP, DMF, and DMSO), DMSO
12
In light of these encouraging results, we continued to
examine the applications of N-linked glycopeptide products by
extending the glycopeptide sequence from either the N-
terminus or the C-terminus via peptide fragment condensation
(Scheme 3; boxed residues are potential ligation sites).
B
DOI: 10.1021/acs.orglett.6b02288
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
a
Scheme 2. Substrate Scope of the N-Glycosylation Reaction between Glycosylamines and p-Nitrophenyl Thioester Peptides
a
b
Reaction conditions: glycosylamine (1a or 1b) (0.1 M), p-nitrophenyl thioester peptide (0.05 M), DIPEA (0.1 M), DMSO, rt, 4 h. Reaction
conditions: sialyloligosaccharide 1c (2.7 mM), p-nitrophenyl thioester peptide (1.8 mM), DIPEA (3.6 mM), 4 Å MS, rt, 10 h. Yields of isolated
products are shown.
Scheme 3. Condensation of N-Linked Glycopeptide
Fragments through Ligation/Desulfurization
developed a protocol of visible-light-induced specific desulfur-
ization of cysteinyl peptides and glycopeptides at room
13
temperature. Under such conditions of visible light with
Ru(bpy) Cl2 and TPPTS, the thiol group of N-linked
3
glycopeptide 11 was selectively removed to give glycopeptide
12 in good yield (Scheme 3 and Figure S22). It should be
noticed that the orthogonal masking group Thz in 11 and 12
can be removed in the next step, endowing these glycopeptides
with the ability to undergo further NCL from their N-terminus.
In our vision, this N-linked glycosylation strategy can be
extended to prepare various long biologically relevant
glycopeptide and glycoprotein fragments.
In conclusion, we have demonstrated a convergent and facile
synthesis of N-linked glycopeptides via combination of
glycosylamines and p-nitrophenyl thioester peptides. This
glycosylation protocol has the merits of simple operation and
good yields. We believe that this method can be particularly
interesting for the preparation of large N-linked glycopeptides
and glycoproteins.
ASSOCIATED CONTENT
■
Glycopeptide 7b was prepared from 7b after conversion of the
*
S
Supporting Information
1
C-terminus into a thioester and deprotection; glycopeptide 6b1
bearing an N-terminal Cys was prepared from 6b after
deprotection. Under standard native chemical ligation con-
ditions, the C-terminus of 7b and N-terminus of 6b were
selectively covalently joined to give the longer glycopeptide 11
in good yield (Scheme 3 and Figure S21). We recently
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b02288.
1
1
Detailed experimental procedures, HPLC traces, and
ESI-MS and characterization data (PDF)
C
DOI: 10.1021/acs.orglett.6b02288
Org. Lett. XXXX, XXX, XXX−XXX

Organic Letters
Letter
(
5) For some examples of chemoenzymatic synthesis of glycopep-
AUTHOR INFORMATION
■
*
*
tides and glycoproteins, see: (a) Yamamoto, K.; Kadowaki, S.; Fujisaki,
M.; Kumagai, H.; Tochikura, T. Biosci., Biotechnol., Biochem. 1994, 58,
72−77. (b) Mizuno, M.; Haneda, K.; Iguchi, R.; Muramoto, I.;
Kawakami, T.; Aimoto, S.; Yamamoto, K.; Inazu, T. J. Am. Chem. Soc.
Corresponding Authors
E-mail: jguo@mail.ccnu.edu.cn.
E-mail: liuz1118@mail.ccnu.edu.cn.
1
999, 121, 284−290. (c) Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang,
Author Contributions
†J.-J.D. and X.-F.G. contributed equally.
L.-X. J. Am. Chem. Soc. 2005, 127, 9692−9693. (d) Goodfellow, J. J.;
Baruah, K.; Yamamoto, K.; Bonomelli, C.; Krishna, B.; Harvey, D. J.;
Crispin, M.; Scanlan, C. N.; Davis, B. G. J. Am. Chem. Soc. 2012, 134,
Notes
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030−8033. (e) Asahina, Y.; Kamitori, S.; Takao, T.; Nishi, N.; Hojo,
The authors declare no competing financial interest.
H. Angew. Chem., Int. Ed. 2013, 52, 9733−9737. (f) Amin, M. N.;
McLellan, J. S.; Huang, W.; Orwenyo, J.; Burton, D. R.; Koff, W. C.;
Kwong, P. D.; Wang, L.-X. Nat. Chem. Biol. 2013, 9, 521−526.
ACKNOWLEDGMENTS
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6) For selected reviews, see: (a) Herzner, H.; Reipen, T.; Schultz,
We are grateful to the National Natural Science Foundation of
China (21272084 and 21402058) and the Fundamental
Research Funds for the Central Universities
CCNU16A02001 and CCNU15A05017) for support of this
research.
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DOI: 10.1021/acs.orglett.6b02288
Org. Lett. XXXX, XXX, XXX−XXX