A programmable one-pot oligosaccharide synthesis for diversifying the sugar domains of natural products: A case study of vancomycin

Article abstract of DOI:10.1002/anie.200351534

The notoriously difficult glycosylation of vancomycin was tackled in a one-pot procedure. A number of oligosaccharides were synthesized and attached to the vancomycin aglycon to create a library of derivatives (see formula) to probe the effect of glycosylation on the antibiotic activity.

Full text of DOI:10.1002/anie.200351534

Angewandte
Chemie
Vancomycin–Oligosaccharide Library
rial cell wall by binding to the terminal peptide fragments of
immature cell walls. In VRE a change in the peptide substrate
weakens its affinity for the vancomycin binding pocket.
Although the hydrogen-bonding interactions between the
vancomycin aglycon and the peptidoglycan precursor are well
understood, reengineering of the binding pocket by replacing
internal amino acids would be a very complex synthetic
endeavor. However, dramatic increases in activity against
resistant strains were achieved by attaching hydrophobic
A Programmable One-Pot Oligosaccharide
Synthesis for Diversifying the Sugar Domains of
Natural Products: A Case Study of Vancomycin
Thomas K. Ritter, Kwok-Kong T. Mong, Haitian Liu,
Takuji Nakatani, and Chi-Huey Wong*
Drug discovery relies heavily on the pool of
natural products for the identification of
lead compounds. Numerous natural prod-
ucts are glycosylated, and their peripheral
carbohydrate moieties are the key recog-
nition elements and often define their
biological activities.[1] Yet, efficient meth-
ods to systematically alter these essential
carbohydrate ligands are still lacking.
There are several strategies for the one-
pot synthesis of oligosaccharides.[2] In par-
ticular, programmed one-pot oligosacchar-
ide synthesis facilitates the convenient
assembly of glycan structures[3] and has
been used in the synthesis of such complex
molecules as Globo H,[4] Lewis Y,[5] and
fucosyl GM1.[6] Desired glycan structures
three to six residues in length can be
constructed by choosing building blocks
with suitable reactivities from the Optimer
database.[7] Furthermore, the one-pot gly-
cosylation methodology may provide a
unique opportunity for convenient assem-
bly of glycosylation libraries of natural
products (Figure 1). Libraries of this type
Figure 1. Strategies for the synthesis of libraries of glycosylated natural products by the direct one-pot
glycosylation (left) or the coupling of preformed oligosaccharides (right).
have been shown to facilitate elucidation of
glycan function as well as the discovery of
new compounds with interesting activi-
ties.[8]
The notoriously difficult glycosylation of vancomycin as
well as the clinical importance of this antibiotic prompted us
to choose vancomycin as a test case for the strategy outlined
in Figure 1. Vancomycin is the most important member of the
glycopeptide class of antibiotics and is considered the drug of
last resort for the treatment of methicillin-resistant Staph-
ylococcus aureus infections. Due to the emergence of
vancomycin-intermediate-resistant S. aureus (VISA) and
vancomycin-resistant enterococci (VRE), there is currently
great interest in improving the efficacy of vancomycin.
Vancomycin inhibits peptidoglycan biosynthesis in the bacte-
groups to vancomycin's terminal sugar residue, vancos-
amine.[9] This increased activity was originally rationalized
by the enhancing effects the substituents might have on
dimerization and membrane anchoring.[10] More detailed
studies suggest that the new vancomycin derivatives inhibit
peptidoglycan biosynthesis by directly interfering with the
enzymes catalyzing transglycosylation.[11–13]
In order to further investigate the role of the glycan
portion, an efficient strategy for the glycosylation of the
vancomycin aglycon is required. Enzymatic glycosylation
procedures are restricted by the substrate tolerance of the
glycosyltransferases and the availability of the nucleotide-
sugar donors.[14–16] Previous chemical protocols suffer from
poor yields in the glycosylation step[17] or lengthy protection/
deprotection procedures.[18] A solid-support approach was
reported, greatly simplifying workup and purification proce-
dures.[19] However, only monoglycosylated derivatives
resulted from this work. In a different approach, a short
ethylene glycol linker was placed between the glycan and the
aglycon, simplifying the coupling step significantly.[20] Yet, no
[*] Prof. Dr. C.-H. Wong, Dr. T. K. Ritter, Dr. K.-K. T. Mong, H. Liu,
Dr. T. Nakatani
Department of Chemistry and
The Skaggs Institute of Chemical Biology
The Scripps Research Institute
10550 N. Torrey Pines Road, La Jolla, CA 92037 (USA)
Fax : (+1)858-784-2409
E-mail: wong@scripps.edu
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DOI: 10.1002/anie.200351534
ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
examples of direct chemical glycosylation of the aglycon to
values (RRV) of these compounds were determined as
described previously.[7] As shown in Scheme 2, the reactivity
differences of the new donors and building blocks available
from our database are in a suitable range for successful one-
pot syntheses. The oligosaccharides were assembled accord-
ing to the standard coupling protocol of the programmable
one-pot synthesis (N-iodosuccinimide, triflic acid). Following
the synthesis of the oligosaccharides, the protecting groups
were exchanged for acetates. This operation minimizes the
number of deprotection steps required after coupling of the
glycans to the vancomycin aglycon. The peracetylated oligo-
saccharides were activated as trichloroacetimidates 2c–f by a
two-step procedure: deprotection of the anomeric hydroxy
groups (hydrazine/acetate in the case of anomeric acetate, N-
bromosuccinimide in the case of anomeric thioglycosides)
followed by conversion to their corresponding trichloroace-
timidates with trichloroacetonitrile and cesium carbonate.
Compounds 2c–f were then coupled to the vancomycin
aglycon by using the conditions developed for the couplings of
2a and b, and the protecting groups were removed. Deal-
lylation of 3e occurred with concomitant reduction of the C6
azide on the terminal sugar residue by means of a radical-
provide vancomycin derivatives bearing nonnatural di- or
oligosaccharide substituents have been reported. By imple-
menting a programmable one-pot oligosaccharide strategy,
we hoped to construct a set of such derivatives.
In order to investigate the feasibility of our approach, it
was necessary to establish an efficient glycosylation proce-
dure for attaching oligosaccharides to a protected vancomycin
aglycon. Di-O-acetyl-N-alloc-tri-O-allyl vancomycin aglycon
allyl ester 1 was obtained in seven steps from commercially
available vancomycin.[17] Glycosylation of 1 with thioglyco-
side donors could not be achieved under a variety of
conditions, including different promoters such as N-iodosuc-
cinimide and dimethyl(methylthio)sulfonium trifluorometh-
anesulfonate (DMTST). Therefore, glycosylation with pera-
cetylated imidate donors 2a and 2b was performed and
proceeded in good yields after optimization (Scheme 1). The
acetate and allyl protecting groups were subsequently
removed by reaction with hydrazine and palladium-catalyzed
reduction, respectively,[17] providing the vancomycin deriva-
tives 4a and 4b.[21]
The successful synthesis of oligosaccharides by the one-
pot strategy relies on the generation of significant differences
in reactivity between the individual thioglycoside donors.
New nitrogen-containing building blocks were designed by
fine-tuning reactivities through various electron-donating and
-withdrawing protecting groups (Figure 2). Relative reactivity
À
mediated reduction mechanism through a Sn N bond-form-
ing process.
The biological activities of the vancomycin derivatives
were investigated by determination of their minimum inhib-
itory concentrations (MIC) against a vancomycin-sensitive
Scheme 1. Coupling of various oligosaccharides to vancomycin aglycon and deprotection of the resulting vancomycin derivatives. n/a=not
available. Alloc=allyloxycarbonyl.
4658
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Figure 2. New nitrogen-containing building blocks for the Optimer database. Relative reactivity values are in parentheses. Bn=Benzyl,
PMB=para-methoxybenzyl, TBDMS=tert-butyldimethylsilyl, TBDPS=tert-butyldiphenylsilyl, Tol=toluene.
(E. faecalis 29212, MIC(vancomycin) = 2 mgmLÀ1) and a
Van A vancomycin-resistant strain (E. faecalis BM4166,
MIC(vancomycin) = 2000 mgmLÀ1). Compounds 4e–f were
shown to inhibit growth of vancomycin-sensitive bacteria at a
concentration of 5 mgmLÀ1, whereas compounds 4a–d
showed no growth inhibition at concentrations up to
40 mgmLÀ1. This result and other data (not shown) indicate
the importance of the amino functionality on the terminal
sugar for antibacterial activity. Growth inhibition of the
Van A strain by 4a–f was not observed at concentrations up to
100 mgmLÀ1.
In conclusion, by combining an efficient oligosaccharide
segment glycosylation with a straightforward deprotection
scheme, we have developed a method for the facile synthesis
of vancomycin derivatives bearing different sugar substitu-
ents. Compounds 4a–f are the first products of the one-step
chemical glycosylation of the vancomycin aglycon with non-
natural oligosaccharides. Their biological evaluation demon-
strates the importance of the glycan portion for activity, in
particular the requirement for a terminal amino sugar residue.
The chemistry developed sets the stage for a more detailed,
systematic exploration of glycan function. Furthermore, this
study constitutes the first implementation of the program-
mable one-pot glycosylation towards the synthesis of a set of
natural product derivatives. This new strategy, that is, rapid
creation of oligosaccharide sets by one-pot glycosylation
followed by conversion to peracetylated trichloroacetimidate
donors and coupling to an aglycon, should be generally
applicable towards the construction of glycosylation libraries
of natural products and the exploration of the varied bio-
logical functions of their carbohydrate substituents.
Received: March 31, 2003 [Z51534]
Keywords: antibiotics · carbohydrates · glycosylation ·
.
oligosaccharides · vancomycin
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ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Communications
Scheme 2. Programmable one-pot oligosaccharide synthesis, followed by switching of protecting groups and activation as trichloroacetimidates.
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4660 ꢀ 2003 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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