Synthesis of Sialidase-Resistant Oligosaccharide and Antibody Glycoform Containing α2,6-Linked 3Fax-Neu5Ac

Article abstract of DOI:10.1021/jacs.9b01991

Fluorinated glycosides are known to resist the glycosidase-catalyzed glycosidic bond cleavage; however, the synthesis of such glycans, especially 3-fluoro-sialic acid (3F-Neu5Ac) containing sialosides, has been a major challenge. Though the enzymatic synthesis of α-2,3-linked 3F-sialosides was reported, until recently there has not been any effective method available for the synthesis of 3F-sialosides in the α-2,6-linkage. In order to understand the biological effect of such modification, we report here a chemical synthesis of 3F^ax-Neu5Ac-α2,6-Gal as a building block for the assembly of 3F^ax-Neu5Ac-containing sialosides and a representative homogeneous antibody glycoform. Our results showed that the sialosides are stable under sialidase catalysis and the rituximab glycoform with a sialylated complex-type biantennary glycan terminated with 3F^ax-Neu5Ac in the α-2,6-linkage (α2,6-F-SCT) has a similar binding avidity as its parent glycoform. These findings open up new opportunities for the development of therapeutic glycoproteins with improved pharmacokinetic parameters.

Full text of DOI:10.1021/jacs.9b01991

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Communication
Synthesis of Sialidase-Resistant Oligosaccharide and
ax
Antibody Glycoform Containing #2,6-Linked 3F-Neu5Ac
Hong-Jay Lo, Larissa Krasnova, Supriya Dey, Ting Cheng, Haitian Liu,
Tsung-I Tsai, Kevin Binchia Wu, Chung-Yi Wu, and Chi-Huey Wong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.9b01991 • Publication Date (Web): 10 Apr 2019
Downloaded from http://pubs.acs.org on April 10, 2019
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Synthesis of Sialidase-Resistant Oligosaccharide and Antibody
Glycoform Containing 2,6-Linked 3Fax-Neu5Ac
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Hong-Jay Lo,†,‡ Larissa Krasnova,‡ Supriya Dey,‡ Ting Cheng,† Haitian Liu,‡ Tsung-I Tsai,‡ Kevin
Binchia Wu,‡ Chung-Yi Wu,† Chi-Huey Wong*,†,‡
† Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nanakang, Taipei 115, Taiwan
‡ The Scripps Research Institute, 10550 N. Torrey Pines Road, La Jolla, CA 92037, USA
Supporting Information Placeholder
monoclonal antibodies (mAbs).5 For example, our group has
ABSTRACT: Fluorinated glycosides are known to resist the
demonstrated that the afucosylated sialylated complex-type
biantennary N-glycan with terminal -2,6-linked sialic acids
(2,6-SCT) is the optimal glycan structure for the
enhancement of antibody-dependent cellular cytotoxicity
(ADCC), complement-dependent cytotoxicity (CDC) and
anti-inflammatory activities.6
glycosidase-catalyzed glycosidic bond cleavage; however the
synthesis of such glycans, especially 3-fluoro-sialic acid (3F-
Neu5Ac) containing sialosides, has been a major challenge.
Though the enzymatic synthesis of -2,3-linked 3F-sialosides
was reported, until recently there has not been any effective
method available for the synthesis of 3F-sialosides in the -
2,6-linkage. In order to understand the biological effect of
such modification, we report here a chemical synthesis of 3Fax-
Neu5Ac-2,6-Gal as building block for the assembly of 3Fax-
Sialic acid derivatives with fluorine at the C-3 position have
been known to inhibit sialyltransferases and sialidases (or
neuraminidases).7 By introducing the fluorine atoms to the
anomeric and C-3 positions, a 2,3-difluorosialic acid (DFSA)
was developed (Scheme 1)8 and used as a biochemical probe,9
and the activity-based protein profiling probe to study
sialidases.10 With the help of DFSA, it was shown that the
fluorine atom at the axial position at C-3 (3Fax) has a greater
effect on slowing down both the deactivation (ki) and
reactivation (khydr) of the enzyme than the 3Feq substituent.9b
Inspired by this observation, we wanted to investigate the
stability of sialosides with 3-fluorosialic acids for their
potential use in glycoprotein therapeutics. Specifically, we
intended to study if incorporation of a 3Fax-Neu5Ac motif at
the terminal end of N-glycan on a mAb could increase its
stability towards sialidases and sustain its effector functions.
However, glycosylation with fluorinated sugars as donors is a
major challenge due to the strong electronic effect of the
fluorine group that inactivates glycosylation reaction.
Although there is a sialyltransferase capable of transferring
the 3Fax-sialic acid residue from the corresponding CMP-sialic
acid to form the -2,3-linked sialoside, there is no
corresponding -2,6-sialyltransferase available.11
Neu5Ac-containing sialosides and
a
representative
homogeneous antibody glycoform. Our results showed that
the sialosides are stable under sialidase catalysis and the
rituximab glycoform with
a
sialylated complex-type
biantennary glycan terminated with 3Fax-Neu5Ac in the -2,6-
linkage (2,6-F-SCT) has a similar binding avidity as its parent
glycoform. These findings open up new opportunities for the
development of therapeutic glycoproteins with improved
pharmacokinetic parameters.
Sialic acid is a negatively charged monosaccharide often
displayed at the outmost end of glycans on glycolipids and
glycoproteins, which are involved in many physiological intra-
and intercellular processes, including interactions with other
biomolecules and receptors on cells, viruses, and bacteria.1 In
addition, sialylation plays an important role in regulating the
function and fate of secreted glycoproteins and membrane-
bound receptors. For example, sialylation of the epidermal
growth factor receptor (EGFR) was shown to inhibit EGFR
dimerization, thus interfering with EGF binding and
phosphorylation, which is associated with tumorgenesis.2
Also, sialylation modulates the half-life of glycoproteins in
blood circulation as desialylation of N-glycans exposes the
underlining galactose, which is recognized by the hepatic
asialoglycoprotein receptors leading to a rapid removal of the
glycoprotein from circulation.3 Hence, increasing the degree
of sialylation could improve the half-life and undesirable
effects of glycoprotein therapeutics.
Scheme 1. Steps of hydrolysis with retaining exo-α-
sialidase.
In recent years, the role of glycosylation on protein
structure and function has been intensively studied inspiring
the development of new methods for glycan synthesis4 and
glycoengineering of proteins, particularly therapeutic
Towards our goal, we developed a preparative method for
the synthesis of -2,6-linked 3Fax-Neu5Ac oligosaccharides,
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including the biantennary N-glycan. We also showed that the
The inversion of OHeq to Fax turned out to be a challenging
task. Substitution of OTf and OMs by fluorine using
synthetic 3-Fax-Neu5Ac-α2,6-Gal linkage is stable in the
presence of sialidases and the antibody bearing the
biantennary glycan with 3-Fax-Neu5Ac-α2,6-Gal has the same
binding avidity as a non-fluorinated counterpart.
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tris(dimethylamino)sulfonium
difluorotrimethylsilicate
(TSAF) led to decomposition of the starting material. After
screening a variety of fluorinating reagents,17 we observed that
treatment of 6 with perfluoro-1-butanesulfonyl fluoride (NfF)
in the presence of 1,8-diazabicyclo[5,4,0]-undec-7-ene (DBU)
Several methods were reported for the synthesis of 3F-
Neu5Ac, including (a) fluorination of protected glycals with
XeF2-BF3×OEt2,12 molecular fluorine13 and Selectfluor®;7c (b)
inversion of equatorial hydroxyl group at C3 in a sialic acid
o
in anhydrous toluene for 2 days at 90 C gave 4 in 6 % yield
(Table S4, SI).18 Further optimization of the reaction
conditions, such as decreasing reaction temperature and
increasing reaction times, improved the overall yield of 4.
However, the transformation of 7 to 4 seemed to be a rate-
limiting step, probably due to steric hindrance. Thus, we were
able to isolate 7 in the presence of NfF and DBU at room
temperature within 1 day, however conversion of 7 to 4 (49%,
or 77% brsm yield) required long reaction times (15 days). The
attempts to improve the conversion by elevating reaction
temperature resulted in decomposition of 7. Finally, we found
that addition of TASF has helped improve the reaction
efficiency reducing the reaction time to only 2 days. The
stereochemistry of fully protected 3Fax-Neu5Ac-α2,6-Gal-STol
disaccharide (4) was confirmed by the X-ray diffraction
analysis.
derivative;7f
and
(c)
aldolase-catalyzed
enzymatic
9
transformation of ManNAc and 3-fluoro-pyruvate into 3Feq-
Neu5Ac and 3Fax-Neu5Ac.7a,11a,14 However, to the best of our
knowledge, there is no method describing the synthesis of N-
glycans terminated with 3Fax-Neu5Ac so far. The only account
disclosing the preparation of oligosaccharides with 3Fax-
Neu5Ac was limited to the enzymatic synthesis of 3-Fax-
Neu5Ac-α2,3-Lac-βOMe, and not the α2,6-linkage.11a We,
therefore, focused our effort on the chemical synthesis of this
linkage.15
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Scheme 2. Synthesis of 3-Fax-Neu5Ac-α2,6-Gal-STol.
Figure 1. Synthetic routes towards 3Fax-Neu5Ac-α2,6-Gal.
After screening a variety of glycosylation conditions using
3Fax-Neu5Ac-based donors without any success (Figure 1a and
Table S1, SI), we investigated alternative strategies, which
encompassed the SN2 reaction of the OHeq to Fax in 3OHeq-
Neu5Ac-α2,6-Gal-STol (Figure 1b).
Starting with the
sialylation conditions reported by Goto et al. (Table S2, SI),16
we were able to optimize the sialylation reaction to give 6 in
35% (99% brsm) yield with excellent α-selectivity (α:β = 13:1)
(Scheme 2).
The 3Fax-Neu5Ac-disaccharide donor 4 was coupled with
the acceptor (8) using NIS/TMSOTf (Scheme 3) to give 9.
Next, the O-allyl group at the anomeric position was removed
by isomerization with PdCl2 in AcOH/NaOAc, and
Scheme 3. Synthesis of 3Fax-Neu5Ac-terminated biantennary N-glycan (17).
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Scheme 4. Programmable one-pot synthesis of
hexasaccharide (14).
analysis6 together with the parent non-fluorinated glycoform
(G2S2) and a commercial sample of rituximab (major
glycoforms: G1F1, G0F1, G2F1). When compared to the
commercial sample of rituximab, the homogeneous
glycoforms of IgG1 bearing 2,6-SCT without core fucose
demonstrated 39.9-fold (2,6-SCT-Rit) and 37.4-fold (2,6-F-
SCT-Rit) improvement in binding avidity (Table 1). The fact
that the avidity of the 3Fax-Neu5Ac-modified glycoform was
similar to that of the parent glycan provides a premise for the
in vivo studies of 3Fax-Neu5Ac-glycosylated mAb. These
results will be reported in a due course.
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Scheme 5. Synthesis of 2,6-F-SCT glycoform of
rituximab.
Table 1. Binding avidity of glycoengineered retuximab
the anomeric hydroxyl group (10) was further transformed
into fluoride (11) and imidate (12). The glycosylation of the
core disaccharide (13) at O-3 with 3Fax-Neu5Ac-terminated
fluoride donor (11) using Cp2HfCl2/AgOTf conditions gave
hexasaccharide 14 in 85% yield. After removal of the
benzylidene group, 15 was glycosylated at the O-6 position to
give the desired decasaccharide (16) in 70% yield with
excellent regio- and α-stereoselectivity. We also tested the
IgG1 to FcγRIIIa.a
Sample
IgG1
ka
(1/Ms)
kd (1/s)
KD (M) Rmax
(RU)
Fold
Rituximabb 2.31E5
0. 07054
3.06E-7
32.33
71.28
1
2.44E5
0.001996 8.18E-9
37.4
2,6-F-
SCT-Rit
TfOH-promoted
glycosylation
with
N-phenyl
2.68E5 0.002059 7.67E-9
60.64
39.9
2,6-SCT-
trifluoroacetimidate donor (12), which, however, gave the
product in a poor yield. Next, the fully deprotected glycan (17)
was obtained in 40% overall yield following a sequence of
steps: (a) saponification with LiOH to remove the esters and
the NHTroc group; (b) acetylation of free amines and alcohols;
(c) removal of the OAc groups with sodium methoxide; and
(d) hydrogenolysis of the O-benzyl groups with Pd/C in a
mixture of MeOH/water/HCO2H.4b
Rit
aAnalyzed antibodies were captured by the human Fab
capture kit and detected with the single cycle kinetic method.
bCommercial sample of rituximab contains several glycoforms
(Figure S3, SI)
In conclusion, we have developed a chemical synthesis of
3Fax-Neu5Ac-α2,6-Gal-STol building block for the synthesis of
sialidase-resistant oligosaccharides and α2,6-F-SCT, which
was used for modification of a representative mAb. When
compared with the commercial rituximab sample, the
homogeneous glycoform modified with α2,6-F-SCT showed a
37.4-fold improvement in binding to the FcγRIIIa. The parent
Having established a protocol for the stepwise synthesis, we
streamlined the glycan assembly by developing
a
programmable [2+2+2] one-pot synthesis of hexasaccharide
(14), which is a precursor of 17 (Scheme 4). The one-pot
protocol was initiated by coupling of the 3Fax-Neu5Ac-α2,6-
Gal-STol donor (4) (RRV = 2053) with a less reactive acceptor
(18) (RRV = 537) at -40 oC, followed by injection of the
reducing-end acceptor 13 at – 20 oC. After 1 h at -10 oC and a
standard purification protocol, the hexasaccharide 14 was
isolated in 26% yield.
non-fluorinated
and
3Fax-Neu5Ac-modified
antibody
glycoforms demonstrated similar binding avidity to the
FcγRIIIa receptor. Overall, our results have revealed a new
general strategy for the improvement of half-lives of
therapeutic glycoproteins.
In order to gather preliminary data about the stability of the
3Fax-Neu5Ac-α2,6-Gal motif in the presence of sialidases, we
prepared Neu5Ac-α2,6-Gal-pNP (1) and the 3Fax-Neu5Ac
analog (2) as substrates (Scheme 1) for the in vitro assay19 with
the commercially available sialidases from C. perfingens and
V. cholera. Both enzymes showed the expected hydrolytic
activity for the native substrate 1 but were inactive toward the
3Fax-analog 2 (Figure S1, SI). We also observed that 2 did not
significantly inhibit the hydrolysis of 1 as DANA did.
ASSOCIATED CONTENT
Supporting Information
Synthetic procedures, characterization of compounds and
crystallographic data for 4, as well as protocols for biological
assays are available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
To prepare a homogeneous glycoform of mAb, compound
17 was converted into the oxazoline donor and ligated to the
GlcNAc-primed IgG (without core fucose) in the presence of
Endo S2 (D184Q) following a standard protocol (Scheme 5).6
The binding avidity of the mAb 3Fax-Neu5Ac-glycoform to
FcγRIIIa was measured by the surface plasma resonance
Corresponding Author
*wong@scripps.edu, chwong@gate.sinica.edu.tw
Notes
The authors declare no competing financial interest.
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glycosyltransferases. Bioorg. Med. Chem. 2000, 8, 1937. (f) Sun, X.-L.;
ACKNOWLEDGMENT
Kanie, Y.; Guo, C.-T.; Kanie, O.; Suzuki, Y.; Wong, C.-H. Syntheses of
C-3-Modified Sialylglycosides as Selective Inhibitors of Influenza
Hemagglutinin and Neuraminidase. Eur. J. Org. Chem. 2000, 2643. (g)
Rillahan, C. D.; Antonopoulos, A.; Lefort, C. T.; Sonon, R.; Azadi, P.;
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Chem. Biol. 2012, 8, 661.
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Itoh, M. Tumor uptake study of 18F-labeled N-acetylneuraminic acids.
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sialidase Operates through a Covalent Sialyl−Enzyme Intermediate:ꢀ
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S.; Chavas, L. M.; Withers, S. G. Tuning Mechanism-Based Inactivators
of Neuraminidases: Mechanistic and Structural Insights. Angew.
Chem. Int. Ed. Engl. 2014, 53, 3382. (c) Kim, J. H.; Resende, R.;
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This work was supported by the National Institutes of Health
(AI072155), the National Science Foundation (CHE-1664283),
Academia Sinica and the Kwang Hua Foundation. We thank
Dr. Gembicky (UCSD) for the X-ray diffraction analysis of 4.
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