Synthesis of Mannosidase-Stable Man3and Man4Glycans Containing S-linked Manα1→2Man Termini

Article abstract of DOI:10.1021/acs.orglett.1c00726

Oligomannose glycans are of interest as HIV vaccine components, but they are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the nonreducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford trimannose and tetramannose fragments. Saturation transfer difference nuclear magnetic resonance (STD NMR) studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.

Full text of DOI:10.1021/acs.orglett.1c00726

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Letter
Synthesis of Mannosidase-Stable Man3 and Man4 Glycans
Containing S‑linked Manα1→2Man Termini
Mahesh Neralkar,† Leiming Tian,† Richard L. Redman, and Isaac J. Krauss*
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ABSTRACT: Oligomannose glycans are of interest as HIV vaccine
components, but they are subject to mannosidase degradation in vivo.
Herein, we report the synthesis of oligosaccharides containing a thio
linkage at the nonreducing end. A thio-linked dimannose donor
participates in highly stereoselective glycosylations to afford
trimannose and tetramannose fragments. Saturation transfer differ-
ence nuclear magnetic resonance (STD NMR) studies show that
these glycans are recognized by HIV antibody 2G12, and we confirm
that the reducing terminal S-linkage confers complete stability
against x. manihotis mannosidase.
arbohydrate or glycoconjugate vaccines1 are in use or
been reported, with a gluco- configuration.34 Glycosylation
Cdevelopment for the prevention of bacterial infec- with dimannose derivative 1 would offer an efficient route to
tions,2,3 cancer,4 and HIV.5−7 In HIV vaccine development,
serum-stabilized fragments of Man9GlcNAc2, or potentially
the whole oligosaccharide.
there is significant interest in elicitation of antibodies that can
bind to the Manα1→2Man moieties of high mannose
(Man9GlcNAc2) glycans;8−11 however, we and others have
shown that, for glycoconjugate vaccines, mannosidase
trimming degrades this motif, so that the antibody response
is directed against the glycan core or other structures in the
glycoconjugate.12,13 A possible solution to this problem is
chemical stabilization of the Manα1→2Man linkage against
enzymatic hydrolysis, in particular using sulfur14−20 in the
glycosidic linkage. Indeed, antibodies raised against some S-
linked glycan analogues exhibit cross-reactivity with the
natural oxygen-linked sugars,21−25 but such analogues have
not been tested in the case of oligomannose vaccines.
To prepare the requisite thio-disaccharide donor, we began
from known trityl thioglycoside 4a (Scheme 2).35,18 Exchange
Scheme 2. Synthesis of S-Linked Disaccharide Donor
Inspired by a report of anomeric alkylation to produce a
sulfur-linked Manα1→2Man disaccharide,19 we wondered
whether a disaccharide donor containing this S-linkage (see 1,
Scheme 1) would participate in stereospecific glycosylation
with anchimeric assistance from the thioether linkage.
Glycosyl donors containing simple 2-thio substituents are
known,26−33 but only one thio-linked disaccharide donor has
to benzyl protecting groups proceeded in 68% overall yield to
afford building block 4b. We wondered whether 5-derived
thiolate could displace a 2-triflate derivative with a relatively
inert leaving group such as a fluoride already present at C1.
Thus, we prepared 1-fluoro glucose derivative 6 by a
known protocol including epoxidation of tribenzyl glucal,36
followed by TBAF treatment.37 Following triflation of 6 and
Scheme 1. Thioether-Linkage-Assisted Stereospecific
Glycosylation
Received: March 2, 2021
Published: April 1, 2021
https://doi.org/10.1021/acs.orglett.1c00726
© 2021 American Chemical Society
Org. Lett. 2021, 23, 3053−3057
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Figure 1. Binding analysis of S-Man4 to HIV broadly neutralizing antibody 2G12: (a) STD-NMR spectrum of S-Man4 (21) with 2G12 IgG.
Bottom spectrum (blue) shows the reference 800 MHz 1H NMR whereas the top (red) shows corresponding STD spectrum. See the
Supporting Information for details. Numbers indicate selected assignments by carbon number and ring letter. (b) Crystal structure for all O-
linked Man4 (22) bound to 2G12 (PDB ID 6MSY).
triethylsilane/trifluoroacetic acid deprotection of 4b, 5 and 7
were combined and allowed to react in the presence of
sodium tert-butoxide to afford the desired disaccharide 8 in
63% yield.
Scheme 4. Synthesis of S-Linked Man4
With dimannose donor 8 in hand, we prepared a suitable
monomannose acceptor to produce Man3. Starting from
mannose building block 9,19 installation of an azidoethyl
linker and deprotection at the 2-position efficiently afforded
acceptor 12. Glycosylation of 12 with 1.5 equiv of 8, in the
presence of hafnium trifluoromethanesulfonate38 afforded the
desired trisaccharide 13 in 64% yield as a single stereoisomer.
After global deprotection with sodium in liquid ammonia, the
desired S-Man3 14 was isolated in 62% yield (Scheme 3).
Scheme 3. Synthesis of S-Linked Man3
174 Hz for the α linkages, and, as expected, a value of 158
Hz for β linkage (see the Supporting Information).
With these S-Man3 and S-Man4 derivatives in hand, we
proceeded to study their recognition by HIV broadly
neutralizing antibody 2G12, which binds primarily to the
linear trimannose (D1) arm of Man9GlcNAc2. STD-NMR
(Saturation Transfer Difference nuclear magnetic resonance
(NMR)) spectroscopy with 25 μM 2G12 IgG and a 200:1
ratio of sugar:antibody showed that, as expected, the greatest
saturation transfer is seen for the nonreducing mannose unit
in either Man3 or Man4 (Figure S1 in the Supporting
Information and Figure 1a). In the case of the Man4
derivative, negligible STD is observed for the reducing-
terminal mannose unit. These data are closely analogous to
STD NMR data previously acquired for oxygen-linked
oligomannose fragments,43,44 and are consistent with crystal
structure data for Man4 bound to 2G12, in which little if any
interaction is evident between the antibody and residue D
(Figure 1b).
Lastly, we tested natural and sulfur-substituted Man4
derivatives against the action of Xanthomonas manihotis
mannosidase, which cleaves oligomannose Manα1→2Man
and Manα1→3Man linkages. S-Man4 derivative 21 and its
oxygen analogue 22 were labeled by strain-promoted azide/
alkyne cycloaddition45 with DBCO46 amine linker 23, in
order to facilitate separation and detection of degradation
products by LC/MS. After incubation with mannosidase, LC/
MS analysis showed no degradation of sulfur-substituted
The stability of the thio linkage under dissolving metal
conditions has been observed previously,19,20 but is never-
theless noteworthy. The α configuration of all mannose units
was confirmed by carbon-coupled HSQC, which showed all
1JCH to be in the range of 171−178 Hz (see the Supporting
Information).39,40
Similarly, we set about preparation of an S-Man4
containing a reducing-terminal β-mannose analogous to the
core mannose in the natural Man9GlcNAc2. We prepared
dimannose acceptor 19 by coupling our previously described
β-mannose core 1741 to known building block 16,42 followed
by Lev deprotection. 19 coupled smoothly to Man2 fluoride
donor 8 (see Scheme 4) in 77% yield, again as single
stereoisomer. This tetrasaccharide was globally deprotected
and converted to azide 21 in three steps with an overall yield
1
of 40%. 21 exhibited three anomeric JCH values from 169 to
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Letter
Scheme 5. Stability of S-Linked Man4 Against Mannosidase Cleavage
derivative 24 after 48 h, but nearly complete digestion of
natural Man4 derivative 25 to Man1 27 (Scheme 5).
Richard L. Redman − Department of Chemistry, Brandeis
University, Waltham, Massachusetts 02454, United States
In conclusion, we have demonstrated a facile synthetic
route to Man3 and Man4 derivatives with a nonreducing-
terminal sulfur linkage that is highly resistant to enzymatic
degradation. These derivatives are recognized by an HIV
antibody, 2G12, through contacts that are similar to those it
makes with the natural oligomannose structure. This
synthetic strategy should be readily amenable to preparation
of higher branched stabilized oligomannose analogues,
suitable for immunogenicity studies in the near future.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.1c00726
Author Contributions
†These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.1c00726.
■
■
sı
This project was supported by NIH Award Nos.
R01AI090745 and R01AI113737 and Brandeis’ SPROUT
program. The 800 MHz NMR in the Landsman Research
Facility was purchased with a grant from the NCRR High-
End Instrumentation program (No. S10RR017269) and
updated with funds from HHMI and Brandeis University.
Dr. Susan Pochapsky of the Brandeis NMR Facility is
gratefully acknowledged for assistance with STD NMR.
Procedures, spectra for compounds 4b, 5−8, 10−21,
and additional experiments (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Isaac J. Krauss − Department of Chemistry, Brandeis
University, Waltham, Massachusetts 02454, United States;
orcid.org/0000-0003-0984-4085; Email: kraussi@
brandeis.edu
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Authors
Mahesh Neralkar − Department of Chemistry, Brandeis
University, Waltham, Massachusetts 02454, United States
Leiming Tian − Department of Chemistry, Brandeis
University, Waltham, Massachusetts 02454, United States
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