Model for antibiotic optimization via neoglycosylation: Synthesis of liponeoglycopeptides active against VRE

Article abstract of DOI:10.1021/ja068602r

The neoglycosylation of a methoxyamine-appended vancomycin aglycon with all possible N′-decanoylglucopyranose and N′-biphenoylglucopyranose regioisomers led to the production of a focused set of liponeoglycopeptide variants in good yields and with excellent stereoselectivity. High-throughput antibacterial assays employing a unique set of vancomycin-resistant Enterococci faecalis and Enterococci faecium clinical isolates revealed that the nature and regiochemistry of glycosyl lipidation modulated vancomycin-resistent Enterococci potency. In contrast to prior work with lipoglycopeptides, this study reveals the glucose C3′ or C4′ as the optimal position for neoglycopeptide lipidation. This purely chemical method for the diversification of the glycolipid portion of lipoglycopeptide antibiotics is simple to perform on a large scale, requires minimal synthetic effort in sugar donor preparation, and provides access to highly active antibiotics that are not easily prepared by other state-of-the-art methods.

Full text of DOI:10.1021/ja068602r

Published on Web 06/12/2007
Model for Antibiotic Optimization via Neoglycosylation:
Synthesis of Liponeoglycopeptides Active against VRE
Byron R. Griffith,†,‡ Candace Krepel,§ Xun Fu,†,‡ Sophie Blanchard,†,‡
Aqeel Ahmed,†,‡ Charles E. Edmiston,§ and Jon S. Thorson*,†,‡
Contribution from the DiVision of Pharmaceutical Sciences and the National Drug DiscoVery
Group, UniVersity of WisconsinsMadison, Madison, Wisconsin 53706, and Department of
Surgery, Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Received December 11, 2006; E-mail: jsthorson@pharmacy.wisc.edu.
Abstract: The neoglycosylation of a methoxyamine-appended vancomycin aglycon with all possible N′-
decanoylglucopyranose and N′-biphenoylglucopyranose regioisomers led to the production of a focused
set of liponeoglycopeptide variants in good yields and with excellent stereoselectivity. High-throughput
antibacterial assays employing a unique set of vancomycin-resistant Enterococci faecalis and Enterococci
faecium clinical isolates revealed that the nature and regiochemistry of glycosyl lipidation modulated
vancomycin-resistent Enterococci potency. In contrast to prior work with lipoglycopeptides, this study reveals
the glucose C3′ or C4′ as the optimal position for neoglycopeptide lipidation. This purely chemical method
for the diversification of the glycolipid portion of lipoglycopeptide antibiotics is simple to perform on a large
scale, requires minimal synthetic effort in sugar donor preparation, and provides access to highly active
antibiotics that are not easily prepared by other state-of-the-art methods.
Introduction
moiety, illustrated by teicoplanin (2) and dalbavancin (4), as a
replacement for the natural disaccharide of vancomycin (1). The
The global emergence of vancomycin-resistant Enterococci
(VRE),1 coupled with the recent transfer of vancomycin (1)
resistance to highly pathogenic Staphylococcus aureus (S.
aureus) strains,2,3 has provided a major impetus for developing
new glycopeptide derivatives (Figure 1, 2-11). Although
efficient chemoenzymatic strategies for glycopeptide optimiza-
tion have recently emerged,4 the chemical modification of
glycopeptide sugar substituents remains among the most suc-
cessful strategies to date.5 From these extensive studies, two
major “pharmacophores” have emerged, the first of which was
inspired by the natural lipoglycopeptide teicoplanin (2, Figure
1). These analogues (e.g., 3)6 carry a signature 2′-N-acyl glucosyl
alternative sugar-modified pharmacophore derives from the
chlorobiphenyl-based 3′-alkylation of a glycopeptide-attached
vancosamine as exemplified by oritavancin (5) and chlorobi-
phenyl vancomycin (6).5a,7 In general, compounds containing
the straight-chain 2′-N-acyl glucolipid retain activity against
VanB-type VRE by avoiding the induction of resistance genes
in the targeted bacteria, which allows these antibiotics to bind
to the transglycosylase substrate Lipid II and inhibit bacterial
cell wall biosynthesis. In contrast, compounds containing the
chlorobiphenyl-substituted vancosminylglucosyl disaccharide
retain activity against both VanA- and VanB-type VRE by direct
inhibition of the transglycosylase, even in the absence of the
Lipid II substrate.5b Yet, despite advances in understanding the
mechanisms of vancomycin resistance and how to overcome
them, the systematic study of the lipoglycopeptide structure-
activity relationship (SAR) and its application toward antibiotic
optimization remains restricted by the lack of efficient, diver-
gent, synthetic strategies for lipoglycopeptide sugar modifica-
tion.
† Division of Pharmaceutical Sciences, University of Wisconsins
Madison.
‡ National Drug Discovery Group, University of WisconsinsMadison.
§ Medical College of Wisconsin.
(1) Murray, B. E. N. Engl. J. Med. 2000, 342, 710-721.
(2) Weigel, L. M.; Clewell, D. B.; Gill, S. R.; Clark, N. C.; McDougal, L. K.;
Flannagan, S. E.; Kolonay, J. F.; Shetty, J.; Killgore, G. E.; Tenover, F. C.
Science 2003, 302, 1569-1571.
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Cardo, D.; Fridkin, S. K. N. Engl. J. Med. 2003, 348, 1342-1347.
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Pelczer, I.; Kahne, D.; Walsh, C. T. Biochemistry 2001, 40, 4745-4755.
(b) Losey, H. C.; Jiang, J. Q.; Biggins, J. B.; Oberthur, M.; Ye, X. Y.;
Dong, S. D.; Kahne, D.; Thorson, J. S.; Walsh, C. T. Chem. Biol. 2002, 9,
1305-1314. (c) Fu, X.; Albermann, C.; Jiang, J. Q.; Liao, J. C.; Zhang, C.
S.; Thorson, J. S. Nat. Biotechnol. 2003, 21, 1467-1469. (d) Lin, H. N.;
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425-448.
Neoglycosides are formed by the chemoselective ligation of
an unprotected, unactivated reducing sugar with an alkoxyamine-
containing aglycon.8,9 The stereoselectivity of the neoglycosy-
lation reaction is dictated in part by the sugar donor, and in the
case of glucose and GlcNAc, the â-anomer forms exclusively.8
In the context of natural product glycorandomization, neogly-
(6) Dong, S. D.; Oberthur, M.; Losey, H. C.; Anderson, J. W.; Eggert, U. S.;
Peczuh, M. W.; Walsh, C. T.; Kahne, D. J. Am. Chem. Soc. 2002, 124,
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J. AM. CHEM. SOC. 2007, 129, 8150-8155
10.1021/ja068602r CCC: $37.00 © 2007 American Chemical Society

Antibiotic Optimization via Neoglycosylation
A R T I C L E S
Figure 1. Naturally occurring and synthetically modified (lipo)glycopeptide antibiotics. Vancomycin and teicoplanin are natural products, whereas all the
other compounds have been prepared by semisynthetic methods, including chemoenzymatic techniques. Compounds 5, 6, 9, and 11 are VanA-active, whereas
2-4, 7, 8, and 10 are VanB-active.
cosylation has allowed for the rapid construction of libraries of
biologically active glycosides prohibitively difficult to access
by traditional chemical glycosylation.9 We hypothesized that
the application of this chemistry to lipoglycopeptides would
provide access to a wider range of chemical diversity than has
been achieved thus far in this class of clinically important
compounds. Herein we describe the application of chemose-
lective “neoglycosylation” to expediently evaluate the effect of
both “lipid” regiochemistry and the “lipid” composition of
lipoglycopeptide variants on their biological activity.
Alloc/allyl-protected 1 aglycon 12 was prepared according to a
modified literature procedure.11 To highlight the ease and
scalability of the installation of the reactive methoxyamine,
beginning with 16 g of 1, handle 13 was installed selectively at
the A4 position in five simple steps to provide neoglycoside
aglycon 14 in a 49% overall crude yield. Notably, crude products
were advanced throughout this route and, due to the chemose-
lectivity of neoglycosylation, the crude nature of 14 did not
interfere with the culminating neoglycosylation reaction.
For the pilot neoglycosylation, crude aglycon 14 was reacted
with a 10-fold excess of 2′-N-decanoyl-D-glucose in 2.5%
trifluoroacetic acid/dimethyl sulfoxide (TFA/DMSO) at 40 °C,
and the ligation reaction was monitored by HPLC (Figure 2a).
A significant amount of starting material (20 min) was consumed
after 24 h, and a single new peak was observed at 34 min.
Within 48 h, the starting material was consumed, and the newly
generated material was subsequently isolated and fully charac-
terized by NMR spectroscopy and FT-MALDI-MS. An exami-
Results and Discussion
To initiate the application of this unique chemistry toward
lipoglycopeptides, the requisite methoxyamine functionality was
installed at the A4 position of 1sthe natural position of
disaccharide attachmentsthrough an ethylene glycol-type linker
(Scheme 1). This strategy was based on a previous report that
replacing the natural O-glycosidic linkage in chlorobiphenyl
vancomycin with an ethylene glycol linker (see Figure 1, 11)
resulted in only a small decrease in activity.10 Specifically, the
1
nation of the 1D H and 2D TOCSY spectra verified that the
isolated product was homogeneous, representing one compound
and not a mixture of regio- or stereoisomers. The relevant
glucolipid signals were subsequently identified by TOCSY,
HSQC, and HMBC experiments (Figure 2b-d). The attachment
of the glucolipid regioselectively to the methoxyamine was
(8) (a) Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269-12278.
(b) Peri, F.; Deutman, A.; La Ferla, B.; Nicotra, F. Chem. Commun.
(Cambridge) 2002, 1504-1505. (c) Peri, F.; Jimenez-Barbero, J.; Garcia-
Aparicio, V.; Tvaroska, I.; Nicotra, F. Chem.-Eur. J. 2004, 10, 1433-
1444.
(9) (a) Griffith, B. R.; Langenhan, J. M.; Thorson, J. S. Curr. Opin. Biotechnol.
2005, 16, 622-630. (b) Langenhan, J. M.; Peters, N. R.; Guzei, I. A.;
Hoffman, F. M.; Thorson, J. S. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
12305-12310. (c) Ahmed, A.; Peters, N. R.; Fitzgerald, M. K.; Watson, J.
A., Jr.; Hoffman, F. M.; Thorson, J. S. J. Am. Chem. Soc. 2006, 128,
14224-14225.
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1244.
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Griffith et al.
Scheme 1. Synthesis of “Liponeoglycopeptide” Antibioticsa
a (a) (i) 4 equiv of Alloc-OSu, 3.3 equiv of NaHCO3, DMF, rt, 16 h; (ii) 9 equiv of allyl bromide, 4.5 equiv of Cs2CO3, DMF, rt, 16 h; (iii) 1% HBr/
HOAc, 6 equiv of PhSH, rt, 0.5 h. (b) (i) 2 equiv of 13, 1.2 equiv of Cs2CO3, DMF, rt, 48 h; (ii) 0.7 equiv of Cl2Pd(PPh3)2, 120 equiv of Bu3SnH,
DMF/HOAc (3:1), rt, 0.5 h, 49% crude (80% pure) over 5 steps. (c) 10 equiv of N-acyl-D-glucose, 2.5% TFA/DMSO, 40 °C, 24-72 h, 61% conversion
(average).
Table 1. Conversions, Yields, and H1 J Values for the
Neoglycosylation Reactions
appendages without protecting groupssa marked advantage over
classical glycosylation strategies. In contrast, the synthesis of
compound
conversion (%)a
yield (%)b
anomeric 1H J valuec
the 4-substituted glucolipids did require 1,2-protection and
deprotection, before and after lipid attachment, respectively. To
confirm reaction stereoselectivity, each new product was
analyzed by NMR as previously described for 15. TOCSY
experiments established that, as in the case of 15, all of the
isolated materials were single isomers. Further 1D TOCSY
experiments on the relevant glucolipid spin systems provided
anomeric coupling constants of 8.8-10.0 Hz, demonstrating
complete â stereoselectivity, regardless of the sugar donor.12
Table 1 summarizes the synthetic results obtained in this study
and highlights the capacity of our approach to rapidly create
liponeoglycopeptide variants.
15
16
17
18
19
20
21
22
70
53
54
44
26
81
80
78
25
14
26
14
15
15
26
30
9.6
8.8
8.8
9.0
10.0
8.8
9.0
8.8
a Determined by HPLC. b Isolated yield of pure compound. c Determined
by 1D TOCSY.
verified by a strong HMBC correlation between the anomeric
proton and the methylene protons adjacent to the glycosidic
nitrogen. A subsequent 1D TOCSY experiment on the gluco-
lipid spin system demonstrated an anomeric coupling constant
of 9.6 Hz, consistent with a â-configured glucosidic linkage
in 15.
Following the pilot reaction conditions, seven additional lipid-
variant analogues were prepared in parallel (Figure 3a) using
65 mg of crude aglycon 14 per reaction. The 2-, 3-, and
6-substituted glucolipid donors for these reactions were syn-
thesized directly from the corresponding aminoglucosides, which
were either commercially available (2-amino-D-glucopyranose)
or prepared according to literature procedures (3- and 6-amino-
D-glucopyranose).12 The 3- and 6-amino-D-glucopyranoses were
efficiently prepared on large scale and derivatized with lipid
In the final neoglycosylation reaction, all eight sugar donors
provided the desired products in a single step with conversions
ranging from 26 to 81% and isolated yields from 14 to 30%
(Table 1). While the yields of this culminating glycosylation
reaction are lower than we desired, the general ease and
efficiency of the overall synthetic strategy is advantageous over
existing alternatives. Although the syntheses for the correspond-
ing O-glycosides of compounds 15-22 have not been reported,
the syntheses of similar analogues (Figure 1, compounds 3 and
6-11) may provide some insight into the prior state-of-the-art
in lipoglycopeptide sugar modification. Perhaps the most striking
difference between the current strategy and preexisting ones is
the overall synthetic design. Previous syntheses of lipoglyco-
peptides have been largely target-oriented linear routes,6,10,13,14
leading to the synthesis of desired analogues on a small scale
(12) See the Supporting Information.
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Antibiotic Optimization via Neoglycosylation
A R T I C L E S
Figure 2. Pilot neoglycosylation: Formation and characterization of compound 15. (a) HPLC analysis is shown for the pilot glycosylation reaction: (i) 48
h reaction aliquot; (ii) 24 h reaction aliquot; (iii) 0 h reaction aliquot. After 24 h, the starting material (14) was partially consumed, and a new peak at 34
min was observed. The starting material was mostly consumed after 48 h, giving a 70% conversion to the product 15. (b) An HSQC experiment on 15 at
500 MHz and 77 °C shows the methylene R to the glycosidic nitrogen as a negative signal at 51.1 ppm in the inset. (c) An HMBC experiment under the
same conditions shows a strong correlation between the anomeric proton (H1) at 4.23 ppm and the methylene carbon identified in (b). (d) A 1D TOCSY
experiment at 800 MHz and 45 °C on the glucolipid spin system shows an anomeric coupling constant (J1,2) of 9.6 Hz.
and encompassing a relatively small amount of chemical
diversity by modifying a single sugar position. In contrast, the
use of neoglycosylation is highly divergent from a single
intermediate, which can be prepared on a multigram scale in
49% overall yield (80% pure) without any chromatography.
Once the neoglycosylation aglycon is in hand, a single step can
encompass diversification of the lipid and/or sugar attachment.
Furthermore, while chemoenymatic advances in glycosylation
and acylation hold promise for exploring the SAR of sugar
diversity, applications reported to date remain restricted by
reaction scale and enzyme specificity.4c,4e,15
(13) Kerns, R.; Dong, S. D.; Fukuzawa, S.; Carbeck, J.; Kohler, J.; Silver, L.;
Kahne, D. J. Am. Chem. Soc. 2000, 122, 12608-12609.
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Griffith et al.
Figure 3. Structure and activity of neolipoglycopeptides. (a) Representation of the 1 substitutions at position A4. (b) Relative potency of liponeoglycopeptides
against a VRE panel, as defined by the MIC of 1 (MIC1) divided by the MIC of the compound of interest (MICcomp.) for each strain. MIC values were
obtained using a standard microdilution assay. The MIC is defined as the lowest concentration at which no growth was visible after incubation at 35 °C for
22 h. VRE strains showed MIC1 from 1 to 1024 µg/mL and included primary clinical isolates and standard ATCC strains. An asterisk (/) indicates no
bacteriostatic activity was observed at or below the compound’s solubility limit (100 µg/mL) under the assay conditions.
Liponeoglycopeptides 15-22 and aglycon 14, in purified
form, were subsequently tested against a panel of 15 different
1-resistant clinical isolates of Enterococci, representing low-
and high-level VRE. Figure 3b illustrates the corresponding
VRE activity of these liponeoglycopeptides, with the best
compound displaying a >40-fold enhancement in potency over
1. This broad analysis revealed the N′-biphenoyl analogues 19-
22 to display specificity toward the low-level VRE strains and
the N′-decanoyl derivatives 15-18 to display specificity toward
the highly resistant strains. Interestingly, while the N′-decanoyl
series performed better overall, both series of compounds
showed a marked preference for substitution at the sugar 3′- or
4′-position. Previous studies with 2′-N-decanoyl-1 and 6′-N-
decanoyl-1 (Figure 1, compouunds 3 and 8, respectively)
revealed these modifications to favor activity against low-level
VRE.6,13 Additionally, the insertion of an ethylene glycol linker
between the aglycon and sugar (Figure 1, compound 11)10 in a
previous report did not alter the low-/high-level VRE bias of
the parent compound. Conversely, N′-decanoyl substitution
within liponeoglycopeptides 16 and 17 favors activity against
high-level VRE. These perceived differences in VRE specifici-
ties among related lipoglycopeptides and liponeoglycopeptides,
although preliminary, may implicate mechanism of action
distinctions. In addition, although structural differences may not
(15) Kruger, R. G.; Lu, W.; Oberthur, M.; Tao, J.; Kahne, D.; Walsh, C. T.
Chem. Biol. 2005, 12, 131-140.
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Antibiotic Optimization via Neoglycosylation
A R T I C L E S
allow for a direct comparison between lipoglycopeptides and
liponeoglycopeptides, the current study implicates the glucosyl
3′- or 4′-position as a target to consider for future glycopeptide
derivatization.
inhibition mechanisms,16 as well as new strategies to delineate
the molecular details of bacterial cell wall biosynthesis.17
Acknowledgment. We thank Prof. J. M. Langenhan for
helpful discussions and Dr. S. Singh, Dr. T. Stringfellow, the
School of Pharmacy Analytical Instrumentation Center, the UW
NMR-FAM, and the UW Chemistry Department Mass Spec-
trometry facility for analytical support. This work was supported
in part by the NIH (Grants AI52218 and GM70637 to J.S.T.).
B.R.G. is an American Cancer Society Postdoctoral Fellow
(Grant PF-05-016-01-CDD).
Conclusions
In summary, we have developed a purely chemical method
for the diversification of the glycolipid portion of lipoglyco-
peptide antibiotics that is simple to perform on a large scale,
requires minimal synthetic effort in sugar donor preparation,
and provides access to highly active antibiotics that are not easily
prepared by other state-of-the-art methods. Our results demon-
strate that the natural glycopeptide O-glycosidic linkage can be
replaced with the neoglycoside N-glycosidic linkage while
enhancing biological activity. We also find that, with liponeo-
glycopeptides, VRE activity is favored by the attachment of
the lipid at the “unnatural” 3- or 4-position, versus the “natural”
2-position, of the glucosyl moiety. In addition, this new class
of “liponeoglycopeptides” may add to the repertoire of novel
reagents to aid in the study of transpeptidase/transglycosylase
Supporting Information Available: Synthetic procedures,
spectroscopic data, procedures for antibiotic testing, strain
identification, and raw biological data. This material is available
free of charge via the Internet at http://pubs.acs.org.
JA068602R
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