Automated Solution-Phase Synthesis of S-Glycosides for the Production of Oligomannopyranoside Derivatives

Article abstract of DOI:10.1021/acs.orglett.0c01236

Thioglycosides are more resistant to enzymatic hydrolysis than their O-linked counterparts, thereby becoming attractive targets for carbohydrate-based therapeutic development. We report the first development of methods for the site-selective incorporation

Full text of DOI:10.1021/acs.orglett.0c01236

pubs.acs.org/OrgLett
Letter
Automated Solution-Phase Synthesis of S‑Glycosides for the
Production of Oligomannopyranoside Derivatives
Mallory K. Kern and Nicola L. B. Pohl*
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ABSTRACT: Thioglycosides are more resistant to enzymatic
hydrolysis than their O-linked counterparts, thereby becoming
attractive targets for carbohydrate-based therapeutic development.
We report the first development of methods for the site-selective
incorporation of S-linkages into automated solution-phase
oligosaccharide protocols. The protocols were shown to be
compatible with the formation of S- or O-glycosides for the
synthesis of mannopyranoside trimmers that incorporate both S-
and O-linkages to allow the selective incorporation of an S-
glycoside in various stages in an automated program.
aturally occurring thiosugars are exceedingly rare in
living systems despite sulfur being an essential
mannose linkages are not yet commercially available, thereby
making these particularly important targets for chemical
synthesis. In addition, the syntheses of 1,6- and 1,2-S-linked
disaccharides from thiomannosyl building blocks14 and a 3,6-
O,S-trimannoside17 have been manually made as benchmarks.
Despite these prior approaches, no synthetic procedures
have yet been demonstrated to work in an automated liquid-
handling system.14,17 The use of machine-assisted liquid-
handling systems allows the desired targets to be synthesized
without the variability introduced by multiple manual
operations, thereby increasing batch-to-batch and lab-to-lab
reproducibility.18 Unfortunately, the transfer of manual to
automated processes is not always straightforward.9 Although
solution-phase-based automation platforms can be dosed with
solid reagents when other methods fail, ideally all reaction
components start and remain in solution.19
The automated synthetic method described in this work is
applicable for both highly reactive and less reactive
trichloroacetimidate donors. The addition of a fluorous tag
allows the automated purification of intermediates by fluorous
solid-phase extraction (FSPE).20
We began our investigation into the automated synthesis of
thioglycosides by the design and synthesis of the necessary
building blocks. To this end, we decided to make novel thiol
acceptor 4 and thiol donor 5 along the same pathway (Scheme
1). First, D-(+)-mannose was converted into the known O-allyl,
N
component in other biomolecules. Although naturally rare,
carbohydrates that replace the native ring or glycosidic oxygen
atoms with sulfur constitute an important class of glycomi-
metics.1 Glycosidic linkages that incorporate sulfur are less
prone to hydrolysis than their oxygen-containing analogues2
and can inhibit the activity of some glycosidases.3 These
properties have made thioglycosides attractive therapeutic
targets.4 However, despite great progress in the development
of automated methods to string together carbohydrate
monomers, these efforts have focused primarily on the creation
of the common natural O-linkages.5−9 The development of
protocols to site-selectively incorporate sulfur linkages into
these automated oligosaccharide synthesis protocols could
greatly expand the possibilities for designing glycomimetics.
Herein we report the first automated synthesis protocols that
incorporate S-glycosides while limiting side reactions and
allowing the further incorporation of O-glycosidic linkages.
The inclusion of sulfur into the glycosidic linkage poses
additional synthetic challenges relative to oxygen. Thioglyco-
side donors and common glycosylation promoters (N-
iodosuccinimide, Ph3Bi(OTf)2, and AuCl3) are incompatible
with thioglycoside acceptors for the formation of S-glycosidic
linkages.9−11 Also, thiol acceptors can dimerize prior to
coupling and are susceptible to oxidation reactions.11,12
Despite these challenges, a number of manual thioglycoside
syntheses have been reported.5,6,11,13,14
Received: April 7, 2020
In choosing targets for automated synthesis, we focused our
efforts on the common mannose linkage found in the high-
mannose capping structures of N-glycans and that is of
particular interest in the design of compounds that modulate
viral and parasitic infections.15 D-Mannose has also been useful
in glycan-based drug development.16 Stable enzymes to create
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© XXXX American Chemical Society
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Letter
workup, the disaccharide was obtained in unexpectedly low
yields (<20%), with an appreciable amount of the 1,1-linked
homodimer (observed: m/z 1021.2 [M + Na+]) and orthoester
(observed: m/z 989.1 [M + Na+], 13C peak at 120 ppm) side
products having been formed, as seen by LRMS and NMR
analyses (10 and 11, Scheme 3). These types of side products
Scheme 1. Trisaccharide Targets Discussed in This Work
Scheme 3. Initial Glycosylation Strategy Using the
“Traditional” Glycosylation Method
2,3,4-tribenzyl, and 6-mesyl mannoside in 29% over five steps
(S.6 in the Supporting Information (SI)).21 The allyl group of
the mannopyranoside was then removed using PdCl2, followed
by the introduction of a thioacetate group at the six-position to
give mannopyranoside 7 in 42% yield over two steps (Scheme
2). 7 was then converted into the trichloroacetimidate (TCA)
a
Scheme 2. Synthesis of Mannopyranoside Analog 8
are known in the literature with O-glycosides and fairly
common in glycosylation reactions with reactive TCA
donors.24 The three benzyl protecting groups and an acetate
moiety appended to mannosyl donor 3 make this building
block especially reactive, making it a superarmed donor.25,26
Likewise, thiol donor 5 was shown to be reactive as well,
suggesting armed reactivity. (See the SI for further discussion.)
Automated oligosaccharide synthesis protocols rely on
normal glycosylation reactions in which the donor and
activator solutions are added to a tagged or solid-phase-linked
acceptor. However, in light of the reactivities of TCA donors 3
and 5, an inverse glycosylation procedure was investigated for
the formation of the thioglycoside targets. The inverse
glycosylation procedure was developed by Schmidt and
coworkers and involves the incubation of the acceptor moiety
with the promoter prior to donor addition.27 A batch-mode
solution-phase automated synthesis platform should be
amenable to programming of the inverse glycosylation more
readily than solid-phase-based machines. Indeed, the use of the
inverse procedure significantly improved our glycosylation
yields (73 and 78% of trisaccharides 1 and 2) with minimal
orthoester and homodimer formation.
a
(a) PdCl2, MeOH, 18 h; (b) KSAc, DMF, 80 °C, 3 h; (c) TCA,
DBU, CH2Cl2, 12 h; and (d) BF3OEt2, CH2Cl2/TFT, 2 h.
mannopyranoside donor 5. Fluorinated mannopyranoside
analog 8 was then realized by coupling thiol donor 5 with
the partially fluorinated undecanol in 78% yield. The fluorous
tag addition allows for the facile isolation of target compounds
by FSPE.20 (See the SI.)
Initially the deprotection of the thioacetate moiety of
mannoside analog 8 with LiAlH4, was attempted; however,
yields of thiol acceptor 4 were poor (<20%, see the SI), which
prompted us to explore other deprotection pathways. The
thiomannopyranoside was instead deprotected with hydrazine
monohydrate and acetic acid, yielding the free thiol mannoside
acceptor 4 in 72% yield. (See the SI.) Finally, mannosyl donor
3 was synthesized over six steps with an overall yield of 8%
following previously published procedures20,22 to incorporate a
standard O-linked sugar into the automated synthesis
protocols.
Prior to developing our strategy for the automated synthesis
of S-linked glycosides, potential glycosylation conditions were
screened. The Lewis acid promoter trimethylsilyl triflate
(TMSOTf) was employed because it has been shown to
work well in the formation of O-glycosidic linkages7,8 as well as
S-linkages17 and works well as a solution for delivery by
automated liquid-handling platforms.6−9,23 Mannosyl acceptor
4 was coupled to TCA donor 3 in the presence of TMSOTf to
form the expected disaccharide (S.9 in the SI). However, upon
With a viable path toward S-linked di- and trisaccharides
using only soluble reactants and reagents, we began to develop
automation protocols for their synthesis using an automated
liquid-handling platform. The platform consists of a robotic
arm coupled to two syringe pumps, which transfer reagents in
solution to an array of double-jacketed reactors vessels under
argon. These vessels can be vortexed and either heated or
cooled.7,9 (See the Supporting Information.) To enable a
successful reaction completion on a solution-phase liquid-
handling platform, all reagents must be stable in solution at
room temperature for the length of the automation cycle.7,9
A
promoter solution of TMSOTf was employed for the
B
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glycosylation steps, followed by hydrazine monohydrate and
acetic acid to deprotect the thioacetate moiety. Hydrazine
monohydrate is a relatively mild liquid reagent, and acetic acid
is not corrosive to the robotic needle, even in the concentrated
form.
O-linked glycosides and avoid the formation of side products
such as orthoesters and homodimers. With the demonstration
of such an automated protocol, glycomimetic production with
the site-selective incorporation of S-glycosidic linkages can
become routine.
The automated synthesis of 1 was carried out by successive
cycles of coupling, deprotection, and purification (Scheme 4).
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.orglett.0c01236.
■
sı
Scheme 4. Automated Solution-Phase Synthesis of
Trisaccharide 1
1
Experimental procedures, characterization data, and H
and 13C spectra for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Nicola L. B. Pohl − Department of Chemistry, Indiana
University, Bloomington, Indiana 47405, United States;
orcid.org/0000-0001-7747-8983; Email: npohl@
indiana.edu
Author
Mallory K. Kern − Department of Chemistry, Indiana
University, Bloomington, Indiana 47405, United States;
orcid.org/0000-0003-4878-4636
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.orglett.0c01236
Author Contributions
Both authors contributed to the design and reporting of this
project. M.K.K. carried out all experimental work.
Notes
First, the disaccharide unit (S.9; see the SI) was synthesized by
the addition of solutions of TMSOTf and 3 to a cooled (−40
°C) solution of acceptor 4. The solution of 4 was vortexed for
10 min at −40 °C before the introduction of TMSOTf and the
dropwise addition of 3. Following the deprotection and FSPE
purification steps to generate and isolate 12 on the platform,
the acceptor was once more cooled to −40 °C, and TMSOTf
was added, followed by donor 3 after 10 min. Upon
purification of the crude mixture by FSPE, trisaccharide 1
was achieved in 73% isolated yield with evidence of only the α-
linked product. These conditions for the formation of the O-
linked glycoside did not lead to any noticeable epimerization
or cleavage of the S-linked glycosides.
The synthesis of trisaccharide 2, which contains two
thioglycoside linkages, was carried out in the same fashion as
trisaccharide 1 from mannosyl acceptor 4 and mannosyl donor
5. Upon the completion of two automation cycles and FSPE
purification, the desired target 2 was synthesized in 78% yield.
Again, only the α-linked product was seen. The use of TCA
donor 3 allowed for the selective formation of the α-anomer
due to anchimeric assistance from the acetate group at the two-
position. TCA donor 5 additionally allowed for the selective
formation of the α-anomer, possibly due to the influence of the
thioacetate moiety at the six-position.25,28(See the Supporting
Information.)
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank former group members Dr. R. Saliba (Pharma-
Cytics), Dr. K. Marion (Vanderbilt University), and Dr. V.
Kohout (University of Utah) as well as J. Dobscha (Indiana
University) for their support in editing the manuscript/
Supporting Information. J. Dobscha provided computational
data assistance. This work was supported in part by Indiana
University, the Joan and Marvin Carmack Fund, the Graduate
Training program in Quantitative and Chemical Biology under
the award number T32 GM109825, and the National Institutes
of Health (5U01GM116248).
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