Synthesis of Defined and Functionalized Glycans of Lipoteichoic Acid: A Cell Surface Polysaccharide from Clostridium difficile

Article abstract of DOI:10.1021/acs.orglett.7b01242

Two structurally defined, functionalized glycans of lipoteichoic acid (LTA, also known as PS-III) from C. difficile, which have one or two repeating units of LTA linked to the core trisaccharide, were efficiently synthesized via a convergent [2 + 3] or [2 + 2 + 3] strategy. The α-linkage of both N-acetylglucosamine residues in the repeating unit were constructed with glycosyl imidates of azidosugars as donors, while the phosphodiester bridges between the oligosaccharides were fashioned using H-phosphonate chemistry. Both synthetic targets contained a 3-aminopropyl group at the core trisaccharide reducing end, facilitating their conjugation to other biomolecules to afford conjugates useful for various biological studies and applications.

Full text of DOI:10.1021/acs.orglett.7b01242

Letter
pubs.acs.org/OrgLett
Synthesis of Defined and Functionalized Glycans of Lipoteichoic
Acid: A Cell Surface Polysaccharide from Clostridium difficile
Kang Yu,† Ningning Bi,† Chenghe Xiong,† Shuihong Cai,‡ Zhongzhu Long,‡ Zhongwu Guo,*,†,§
and Guofeng Gu*,†
†National Glycoengineering Research Center and Shandong Provincial Key Laboratory of Carbohydrate Chemistry and Glycobiology,
Shandong University, 27 Shanda Nan Lu, Jinan 250100, China
‡Qidong Dongyue Pharmaceutical Company, 268 Shanghai Road, Qidong, Jiangsu 226200, China
§Department of Chemistry, University of Florida, 214 Leigh Hall, Gainesville, Florida 32611, United States
S
* Supporting Information
ABSTRACT: Two structurally defined, functionalized glycans
of lipoteichoic acid (LTA, also known as PS-III) from C.
difficile, which have one or two repeating units of LTA linked
to the core trisaccharide, were efficiently synthesized via a
convergent [2 + 3] or [2 + 2 + 3] strategy. The α-linkage of
both N-acetylglucosamine residues in the repeating unit were
constructed with glycosyl imidates of azidosugars as donors,
while the phosphodiester bridges between the oligosaccharides
were fashioned using H-phosphonate chemistry. Both synthetic targets contained a 3-aminopropyl group at the core trisaccharide
reducing end, facilitating their conjugation to other biomolecules to afford conjugates useful for various biological studies and
applications.
Clostridium difficile is a Gram-positive, spore-forming anaerobic
bacterium, which has been identified as the main cause of
nosocomial diarrhea and colitis in humans that results in a high
rate of mortality.1−3 In the past two decades, the number of C.
difficile infections (CDIs) has been growing steadily to cause an
enormous medical and social problem. For example, it has been
revealed that the incidence of CDIs in the US has almost doubled
between 1993 and 2003 and the CDI-caused mortality has
quadrupled between 1999 and 2004.4,5 It is estimated that
approximately US$3.2 billion is spent on treating CDIs every
year in the US alone.6 Currently, antibiotic therapy using
vancomycin and fidaxomicin is the most common strategy to
treat CDIs in the clinic.7 Its efficacy, however, has been
challenged by the emergence of hypervirulent C. difficile strains,
such as BI/NAP1/027.8 Therefore, novel strategies for the
prevention and treatment of CDIs are highly desirable.
Vaccination is one strategy for the effective control of
CDIs.9−11 In the development of vaccines, most present efforts
have been focused on C. difficile toxins, such as toxin A and toxin
B, as target antigens.12,13 Recently, the cell surface poly-
saccharides of C. difficile, including PS-I, PS-II, and lipoteichoic
acid (LTA, also called PS-III),14,15 have been characterized and
identified as promising antigenic epitopes for prophylactic
vaccine development. Accordingly, several oligosaccharides
related to these polysaccharides have been synthesized16−22
and utilized to explore conjugate vaccines that have exhibited
encouraging results.17,19,20,23,24 The structure of the LTA (Figure
1) from C. difficile and Peptostreptococcus anaerobius as well25 is
comprised of a lipophilic diacylglycerol β-1,6-triglucoside core
and many repeating D-glyceric acid α-1,4-diglucosaminoside
Figure 1. Structure of C. difficile LTA. R = lipid chains.
units, which are stitched together via phosphodiester bridges.15
Seeberger and co-workers21 described the first synthesis of the
repeating unit of this LTA and its oligomers and found that they
were recognized by antibodies from C. difficile patients. Further
studies showed that the CRM197 protein conjugates of these
oligosaccharides induced LTA-specific antibodies that could
opsonize C. difficile and inhibit its intestinal colonization.24
Pedersen and co-workers synthesized some LTA analogs that
contained the lipidated core trisaccharide and repeating unit and
used them to explore the noninnate immunity pathway.22
Furthermore, it was shown that the protein conjugates of
deacylated LTA could elicit antibodies to recognize both C.
difficile and other Clostridium strains.26 These results have
highlighted the LTA glycan and related oligosaccharides as
promising antigens for the development of C. difficile vaccines.
To facilitate the study of LTA and the development of LTA-
based C. difficile vaccines, we established a new and efficient
strategy for the synthesis of structurally defined LTA glycans.
The synthetic targets 1 and 2 (Scheme 1) were designed to
Received: April 24, 2017
© XXXX American Chemical Society
A
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in a 68% yield with excellent stereoselectivity (α:β ≈ 10:1). The
newly formed α-glycosidic bond in 12 was confirmed by the
small coupling constant (J1′,2′ = 3.6 Hz) of its H-1′ NMR signal at
δ 4.80 ppm in CDCl3. Deacetylation of 12 with NaOMe in
MeOH and allylation of the resultant free 6′-OH group with allyl
bromide and NaH in DMF gave 13. Regioselective opening of
the 1,6-anhydro ring in 13 with trifluoroacetic acid (TFA) in
acetic anhydride33 gave 14 in an 91% overall yield. Compound 14
was converted into glycosyl donor 15 in two steps, including
selective anomeric deacetylation using hydrazine acetate34 and
trichloroacetimidation of the resultant hemiacetal with trichlor-
oacetonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-
ene (DBU).35 Thereafter, 8 was glycosylated with 15 under the
promotion of TMSOTf to afford 16 as the only anomer. The
small coupling constant (J1,2 = 3.8 Hz) of its H-1 signal at δ 5.18
ppm in CDCl3 verified the α-glycosidic linkage. The 6′-O-allyl
group in 16 was removed on iridium-mediated olefin rearrange-
ment and N-iodosuccinimide (NIS)-promoted hydrolysis,36,37 to
produce 17 in an excellent yield (90%). Subsequently, its 2,2′-
azido groups were converted into acetamido groups by treatment
with thioacetic acid in pyridine21,22,38 to result in 3 used to
construct 2. On the other hand, 6′-O-benzylation of 17 under
acidic conditions with benzyl trichloroacetimidate and triflic acid
(TfOH),39 which had no influence on the base-labile O-acetyl
and glyceric acid groups, was followed by selective 6-O-
deacetylation using 2% acetyl chloride40 in CH2Cl2 and MeOH
(v/v 1:1) and reductive acetylation of the azido group with
thioacetic acid in pyridine to provide 4 (63% overall yield), which
was used for the assembly of both 1 and 2.
Scheme 1. Synthetic Targets 1 and 2 and the Retrosynthesis
contain both the core trisaccharide and the repeating unit of
LTA, as well as a 3-aminopropyl group at the trisaccharide
reducing end, to enable their expedient and efficient coupling
with other molecules. Retrosynthetic disconnection of 1 and 2
(Scheme 1) led to specially protected disaccharides 3 and 4 and
trisaccharide 5 as key intermediates. According to this design, 1
and 2 could be assembled by a convergent [2 + 3] or [2 + 2 + 3]
strategy upon bridging 6,6′-O-phosphorylation. In turn, 3 and 4
could be prepared from glycosyl donor 6,27,28 acceptor 7,27,29 and
D-glyceric acid 8.21 In 6 and 7, the azido group was used as a
latent amino group to prevent neighboring group participation
and thereby promote glucosamine 1,2-cis α-glycosylation.30,31
On the other hand, 5 could be prepared from 9−11 via
preactivation-based iterative one-pot glycosylation,32 and the 2-
O-benzoyl group in 9−11 would favor 1,2-trans β-glycosylation
as a result of the neighboring group participation effect.
Monosaccharides 9−11 were prepared from p-tolyl 1-thio-β-
D-glucopyranoside 1941 via a series of established trans-
formations (Scheme 3). First, regioselective silylation of the 6-
Scheme 3. Synthesis of Monosaccharides 9−11
Our syntheses commenced with preparing 6,27,28 7,27,29 and
821 according to reported procedures, which were utilized to
build disaccharides 3 and 4 as outlined in Scheme 2.
Glycosylation of 7 with 6 using trimethylsilyl triflate (TMSOTf)
as a promoter smoothly afforded the α-1,3-linked disaccharide 12
Scheme 2. Synthesis of Intermediates 3 and 4
OH group in 19 with tert-butyldimethylchlorosilane
(TBDMSCl) and benzoylation of the remaining hydroxyl groups
with benzoyl chloride afforded 9 (95% overall yield). Treating 9
with tetrabutylammonium fluoride (TBAF) to remove the
TBDMS group gave 10 (89%). Alternatively, the reaction of 9
with 3-azidopropanol in the presence of silver triflate (AgOTf)
and NIS, followed by desilylation with TBAF, provided 11.
Scheme 4 outlines the assembly of 5 from 9, 10, and 11 by
preactivation-based iterative one-pot glycosylation.32 For each
glycosylation, the glycosyl donor was preactivated at −78 °C for
15 min with p-TolSOTf, generated in situ from p-toluenesulfenyl
chloride (p-TolSCl) and AgOTf, using 2,4,6-tritert-butyl-
pyrimidine (TTBP) as a scavenger of TfOH formed from the
reaction. Then, the glycosyl acceptor was added at −78 °C, and
the reaction was kept at rt for 15 min and monitored with TLC to
ensure completion. Moreover, 1 equiv of p-TolSCl and 0.9 equiv
of 10 or 11 (relative to 9) were utilized to guarantee complete
consumption of glycosyl acceptors, so as to minimize the impact
of unreacted substrate on subsequent reaction. Eventually, 21
was isolated in a 62% yield after two rounds of glycosylation, and
all glycosidic bonds in 21 were β, verified by the coupling
B
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resultant phosphite, which gave H-phosphonate monoester 23
(87%) purified by silica gel column chromatography. Next, 23
was coupled with 4 under the influence of pivaloyl chloride
(PivCl),42 which was followed by oxidation of the resultant H-
phosphonate diester with I2 to afford fully protected phosphate
24 (78%) as the single isomer. Debenzylation of 24 by
hydrogenolysis with concomitant azide reduction afforded
synthetic target 1 in a 75% yield, after purification by size-
exclusion chromatography on an LH-20 column.
Scheme 4. Preactivation-Based Iterative One-Pot Synthesis of
5
Synthetic target 2 was assembled by a procedure of coupling
two disaccharide repeating units first and then coupling with
trisaccharide H-phosphonic monoester 23 (Scheme 6). Com-
pound 3 was converted into H-phosphonate monoester 25
(89%) by the protocol used to synthesize 23, which was then
coupled with 4 under the above-mentioned conditions to
produce tetrasaccharide phosphate 26 in a 76% yield. Removal of
the 6-O-acetyl group in 26 under acidic conditions with 0.5%
AcCl in CH2Cl2 and MeOH (v/v 1:1) did not affect other
functional groups, but this reaction was incomplete even after an
elongated time, and the deacetylated product was inseparable
from 26 (5% remained). Nonetheless, 26 would not interfere
with the next synthetic step, and so the mixture was directly used
for conjugating with 4 by the above-mentioned two-step one-pot
protocol to afford fully protected 27, which was readily separated
from 26 by size-exclusion column chromatography in a 69%
overall yield. Finally, 27 was deprotected and purified as
described above to produce 2. Both synthetic targets 1 and 2,
and the intermediates involved in their syntheses, were fully
characterized with MS and NMR data.
constants (>7.2 Hz) of its H-1 NMR signals. The results
indicated the excellent stereoselectivity of the glycosylations due
to the neighboring group participation effect. At this stage, the
benzoyl groups in 21 were swapped for benzyl groups to exploit
the stability of benzyl ethers to base and acid and, in the
meantime, to allow for one-step global deprotection later.
Finally, the 6″-O-TBDMS group in 22 was removed with TBAF
to complete 5 in a 98% yield.
For the synthesis of 1 depicted in Scheme 5, the key step was
the coupling of 4 with 5 via a phosphoryl bridge, which was
Scheme 5. Synthesis of 1
In conclusion, a highly convergent and efficient strategy was
developed for synthesizing the glycans of LTA from C. difficile.
This synthesis was highlighted by separate assembly of the α-
linked disaccharide repeating unit and β-linked core trisaccharide
first and then joining them together by phosphate bridges. The
repeating unit was prepared in excellent overall yields and α-
selectivity with glucosamine derivatives 6 and 15 as glycosyl
donors. Using a reactive bicyclic derivative of glucosamine 727 as
a glycosyl acceptor was also helpful for the outcome.
Trisaccharide 5 was prepared by iterative one-pot glycosylation,
which gave a good yield and stereoselectivity as a result of the
neighboring group participation. The 6,6′-O-phosphodiester
bridge connecting oligosaccharide moieties was fashioned using
H-phosphonate chemistry that turned out to be very efficient.
Ultimately, LTA glycans 1 and 2 were synthesized in a
fashioned by H-phosphonate chemistry. First, the reaction of 5
with 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one42 in the
presence of pyridine was attempted followed by hydrolysis of the
Scheme 6. Synthesis of 2
C
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ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.orglett.7b01242.
Synthetic procedures; NMR and MS spectra and other
analytical data for all new compounds (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
(25) Stortz, C. A.; Cherniak, R.; Jones, R. G.; Treber, T. D.; Reinhardt,
D. J. Carbohydr. Res. 1990, 207, 101−120.
*E-mail: zguo@chem.ufl.edu.
*E-mail: guofenggu@sdu.edu.cn.
ORCID
(26) Cox, A. D.; St. Michael, F.; Aubry, A.; Cairns, C. M.; Strong, P. C.;
Hayes, A. C.; Logan, S. M. Glycoconjugate J. 2013, 30, 843−855.
(27) Xue, J.; Guo, Z. W. Tetrahedron Lett. 2001, 42, 6487−6489.
(28) Li, T.; Ye, H.; Cao, X.; Wang, J.; Liu, Y.; Zhou, L.; Liu, Q.; Wang,
W.; Shen, J.; Zhao, W.; Wang, P. ChemMedChem 2014, 9, 1071−1080.
(29) Paulsen, H.; Stenzel, W. Chem. Ber. 1978, 111, 2334−2347.
(30) Orgueira, H. A.; Bartolozzi, A.; Schell, P.; Seeberger, P. H. Angew.
Chem., Int. Ed. 2002, 41, 2128−2131.
Zhongwu Guo: 0000-0001-5302-6456
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
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Palmacci, E. R.; Seeberger, P. H. Chem. - Eur. J. 2003, 9, 140−169.
(32) Huang, X. F.; Huang, L. J.; Wang, H. S.; Ye, X. S. Angew. Chem.,
Int. Ed. 2004, 43, 5221−5224.
This research was supported by grants from the National Natural
Science Foundation of China (Projects 21672129 and
21472114).
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