Total synthesis of polyprenyl N-Glycolyl Lipid II as a mycobacterial transglycosylase substrate

Article abstract of DOI:10.1021/ol2021687

A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like molecule 1 is described. Compound 1 bears pendant undecaprenol and L-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic ac

Full text of DOI:10.1021/ol2021687

ORGANIC
LETTERS
2011
Vol. 13, No. 19
5306–5309
Total Synthesis of Polyprenyl N-Glycolyl
Lipid II as a Mycobacterial
Transglycosylase Substrate
Fan-Chun Meng,† Kuo-Ting Chen,†,‡ Lin-Ya Huang,†, Hao-Wei Shih,† Han-Hui Chang,†
Fu-Yao Nien,§ Pi-Hui Liang,‡ Ting-Jen R. Cheng,† Chi-Huey Wong,† and Wei-Chieh
Cheng*,†
The Genomics Research Center, Academia Sinica, Nankang, Taipei, Taiwan,
Department of Pharmacy, National Taiwan University, Taipei, Taiwan, Department of
Chemistry, National Cheng Kung University, Tainan, Taiwan, and Department of
Molecular and Biological Agricultural Sciences, Taiwan International Graduate
Program, Academia Sinica, Taipei, Taiwan
wcheng@gate.sinica.edu.tw
Received August 10, 2011
ABSTRACT
A feasible synthetic approach toward the Mycobacterium tuberculosis (Mtb) N-glycolyl lipid II-like molecule 1 is described. Compound 1 bears
pendant undecaprenol and L-lysin moieties instead of the naturally occurring decaprenol and meso-diaminopimelic acid, which are not readily
available. Functionalization of 1 with a fluorophore on the peptide side chain gave 14, which was found to be recognized as an Mtb TGase
substrate. This result suggests it has tremendous utility for mechanistic studies, the characterization of mycobacterial enzymes, and
mycobacterial TGase inhibitor evaluation.
Nearly two million people die annually from tubercu-
losis, an infectious disease caused by Mycobacterium
tuberculosis.1 Recently the rise of antibiotic-resistant
infections caused by MDR- and XDR-TB (multidrug
resistant and extensively drug-resistant tuberculosis) has
been recognized as a serious public health threat,2 and new
antibiotics or new strategies to tackle this problem are
urgently required. One class of possible antibiotic targets
are the enzymes responsible for the assembly of mycobac-
terial cell walls. For example, penicillin-binding protein
(PBP) PonA possesses two catalytic domains for transpep-
tidase and transglycosylase activities, and it plays an
important role for mycobacterial cell wall biosynthesis.3,4
† The Genomics Research Center, Academia Sinica.
‡ National Taiwan University.
Department of Molecular and Biological Agricultural Sciences, Acade-
mia Sinica.
§ National Cheng Kung University.
(1) (a) Chopra, P.; Meena, L. S.; Singh, Y. Indian J. Med. Res. 2003,
117, 1–9. (b) Johnson, R.; Streicher, E. M.; Louw, G. E.; Warren, R. M.;
Helden, P. D. v.; Victor, T. C. Curr. Issues Mol. Biol. 2006, 8, 97–112.
(c) Cole, S. T.; Brosch, R.; Parkhill, J.; Garnier, T.; Churcher, C.; Harris,
D.; Gordon, S. V.; Eiglmeier, K.; Gas, S.; Barry, C. E.; Tekaia, F.;
Badcock, K.; Basham, D.; Brown, D.; Chillingworth, T.; Connor, R.;
Davies, R.; Devlin, K.; Feltwell, T.; Gentles, S.; Hamlin, N.; Holroyd,
S.; Hornsby, T.; Jagels, K.; Krogh, A.; McLean, J.; Moule, S.; Murphy,
L.; Oliver, K.; Osborne, J.; Quail, M. A.; Rajandream, M. A.; Rogers, J.;
Rutter, S.; Seeger, K.; Skelton, J.; Squares, R.; Squares, S.; Sulston, J. E.;
Taylor, K.; Whitehead, S.; Barrell, B. G. Nature 1998, 393, 537–544.
(2) Patru, M.-M.; Pavelka, M. S., Jr. J. Bacteriol. 2010, 192, 3043–
3054.
(3) (a) Hett, E. C.; Chao, M. C.; Rubin, E. J. PLoS Pathog. 2010, 6,
e1001020. (b) Bhakta, S.; Basu, J. Biochem. J. 2002, 361, 635–639.
(c) Lepage, S.; Dubois, P.; Ghosh, T.; Joris, B.; Mahapatra, S.; Kundu,
M.; Basu, J.; Chakrabarti, P.; Cole, S.; Nguyen-Disteche, M.; Ghuysen,
J. J. Bacteriol. 1997, 179, 4627–4630.
(4) Goffin, C.; Ghuysen, J.-M. Microbiol. Mol. Biol. Rev. 2002, 66,
702–738.
r
10.1021/ol2021687
Published on Web 09/13/2011
2011 American Chemical Society

Scheme 1. Retrosynthetic Analysis of Mycobacterial N-Glyco-
lyl Lipid II-like Molecule (1)
Figure 1. Mycobacterial Lipid II as the substrate in transglyco-
sylation for mycobacterial peptidoglycan formation.
The transpeptidase responsible for the cross-linking
of peptidoglycans is a target for existing antibiotics.4,5
Although the transglycosylase (TGase) is also a poten-
tial target, no antibiotics have yet been developed.6,7
Mycobacterial TGase catalyzes the polymerization of
Lipid II to form peptidoglycan (Figure 1).8,9 During
transglycosylation, the sugar moiety from the activated
polymeric peptidoglycan (a glycosyl donor) is linked
to the specific hydroxyl group (4-OH) of Lipid II
(a glycosyl acceptor) with associated release of a decapre-
nyl pyrophosphate (Figure 1). Since TGase is located on
the external surface of bacterial membranes, it allows easy
access to inhibitors and, because of the lack of a eukaryotic
counterpart, thus eliminates the risk of serious side effects.
As such, it is thought an attractive target for antibiotic
discovery and development.8 Unfortunately, the study of
mycobacterial transglycosylation for drug discovery has
been hampered by the difficulty in acquiring N-glycolyl
Lipid II from natural sources.10
mycobacterial cell walls, and considered a potential bio-
marker. The N-glycolyl groups in peptidoglycan chains
may play an impotant role in the resistance to lysozyme
and in the innate immune response during a mycobacterial
infection.12
Currently, only Mtb Park’s nucleotide, a peptidoglycan
precursor, has been synthesized by Kurosu and co-
workers.13 To the best of our knowledge, chemical synth-
eses of N-glycolyl Lipid II and its analogues have not been
explored. Herein, we describe the first synthesis of a N-
glycolyl Lipid II-like molecule, GlcNAcMurNglyc-(L-Ala-
D-Glu-L-Lys-D-Ala-D-Ala)-undecaprenyl phosphate (1).
The decaprenyl phosphate and meso-diaminopimelic acid
(meso-DAP) moieties in the original Mtb N-glycolyl Lipid
Structually, Mtb N-glycolyl Lipid II comprises the dis-
accharide of N-acetylglucosamine (GlcNAc) and N-glyco-
lylmuramic acid (MurNGlyc), pyrophosphate, decaprenol
lipid tail, and the pentapeptide moiety (L-Ala-D-Glu-meso-
DAP-D-Ala-D-Ala (Figure 1).9À11 Notably, the N-glycolyl
muramic acid is a special component and only observed in
(12) (a) Raymond, J. B.; Mahapatra, S.; Crick, D. C.; Pavelka, M. S.
J. Biol. Chem. 2005, 280, 326–333. (b) Daryaee, F.; Kobarfard, F.;
Khalaj, A.; Farnia, P. Eur. J. Med. Chem. 2009, 44, 289–295.
(13) Li, K.; Kurosu, M. Heterocycles. 2008, 76, 455–469.
(14) (a) Khidyrova, N. K.; Shakhidoyatov, K. M. Chem. Nat.
Compd. 2002, 38, 107–121. (b) Ruiz-Rodriguez, A.; Bronze, M.-R.;
Ponte, M. N. d. J. Supercrit. Fluids 2008, 45, 171–176.
(5) (a) Chambers, H.; Moreau, D.; Yajko, D.; Miick, C.; Wagner, C.;
Hackbarth, C.; Kocagoz, S.; Rosenberg, E.; Hadley, W.; Nikaido, H.
Antimicrob. Agents Chemother. 1995, 39, 2620–2624. (b) Anishetty, S.;
Pulimi, M.; Pennathur, G. Comput. Biol. Chem. 2005, 29, 368–378.
(6) Barry, C. E.; Crick, D. C.; McNeil, M. R. Infect. Disord.: Drug
Targets 2007, 7, 182–202.
(7) Vollmer, W.; Holtje, J.-V. Antimicrob. Agents Chemother. 2000,
44, 1181–1185.
(15) Schwartz, B.; Markwalder, J. A.; Wang, Y. J. Am. Chem. Soc.
2001, 123, 11638–11643.
(8) (a) Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.;
Walker, S. J. Am. Chem. Soc. 2001, 123, 3155–3156. (b) Halliday, J.;
McKeveney, D.; Muldoon, C.; Rajaratnam, P.; Meutermans, W. Bio-
chem. Pharmacol. 2006, 71, 957–967.
(9) Crick, D. C.; Mahapatra, S.; Brennan, P. J. Glycobiology. 2001,
11, 107R–118R.
(16) (a) Liu, C.-Y.; Guo, C.-W.; Chang, Y.-F.; Wang, J.-T.; Shih, H.-
W.; Hsu, Y.-F.; Chen, C.-W.; Chen, S.-K.; Wang, Y.-C.; Cheng, T.-J.
R.; Ma, C.; Wong, C.-H.; Fang, J.-M.; Cheng, W.-C. Org. Lett. 2010, 12,
1608–1611. (b) Stachyra, T.; Dini, C.; Ferrari, P.; Bouhss, A.; van
Heijenoort, J.; Mengin-Lecreulx, D.; Blanot, D.; Biton, J.; Le Beller,
D. Antimicrob. Agents Chemother. 2004, 48, 897–902.
(10) Mahapatra, S.; Yagi, T.; Belisle, J. T.; Espinosa, B. J.; Hill, P. J.;
McNeil, M. R.; Brennan, P. J.; Crick, D. C. J. Bacteriol. 2005, 187, 2747–
2757.
(17) VanNieuwenhze, M. S.; Mauldin, S. C.; Zia-Ebrahimi, M.;
Winger, B. E.; Hornback, W. J.; Saha, S. L.; Aikins, J. A.; Blaszczak,
L. C. J. Am. Chem. Soc. 2002, 124, 3656–3660.
(11) (a) Kaur, D.; Brennan, P. J.; Crick, D. C. J. Bacteriol. 2004, 186,
7564–7570. (b) Vollmer, W. FEMS Microbiol. Rev. 2008, 32, 287–306.
(18) Benakli, K.; Zha, C.; Kerns, R. J. J. Am. Chem. Soc. 2001, 123,
9461–9462.
Org. Lett., Vol. 13, No. 19, 2011
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II were substituted with two much more readily avail-
able alternatives, undecaprenyl phosphate and L-lysine,
respectively.14 The N-glycolyl Lipid II-like molecule (1)
was subsequently converted to a fluorescent probe in order
to study whether or not this structurally modified probe
could be recognized as an Mtb TGase substrate.15,16
Our synthetic strategy toward 1 is illustrated in Scheme
1. We planned to assemble three proposed fragments,
including an undecaprenol phosphate, disaccharide 2,
and tetrapeptide 3, to obtain the target molecule. To
circumvent the problems associated with handling an acid
labile R-glycosyl diphosphate group, construction of the
diphosphate moiety was proposed last, following conjuga-
tion of an undecaprenol phosphate and a phosphoryl
disaccharide, in turn, prepared from tetrapeptide 3 and
disaccharide 2. Notably, the key intermediate 2 could be
prepared from glycosyl donor 4 and acceptor 5.17
acetoxyacetyl chloride at the C2-amino group, and selec-
tive O-acetylation at the C6-hydroxy group, gave the key
intermediate 5 in 42% overall yield over five steps
(Scheme 2).
Scheme 3. Synthesis of Disaccharide 2
We started with preparation of the intermediate, N-
glycolyl muramic acid 5. N-Boc protection of 6, prepared
from D-glucosamine,18 followed by the removal of the N-
acetyl and O-acetyl moieties under Zemplen conditions19
(cat. One M NaOMe solution inMeOH) and regioselective
benzylidene acetalization resulted in 7 in a yield of 67%
over three steps (Scheme 2).18 Attempts to undergo O-
alkylation at the C3-hydroxy position in 7 with S-(À)-2-
chloropropionic acid and NaH at 60 °C were unsuccessful
because the N-Boc moiety was unexpectedly deprotected
and many unidentified side products were observed (see
Supporting Information, Table S1). After examination of
various reaction conditions, saccharide 8 was finally ob-
tained in a yield of 87% by using methyl (S)-lactate
triflate13 as the electrophile. Hydrolysis of 8, followed by
Glycosylation between donor 4 and acceptor 5 mediated
by TMSOTf afforded the corresponding disaccharide 10 in
50% yield (Scheme 3). The β-linkage of the newly formed
glycosidic bond was confirmed by 1H NMR spectroscopy.
Debenzylation of 11 by catalytic hydrogenation (Pd(OH)2/C,
H2, 1 atm) was not successful, and only 11 was observed (see
Supporting Information, Table S2). Fortunately, use of high
pressure hydrogen (4.5 bar) in the presence of a catalytic
amount of acetic acid gave lactol 12 in a reasonable yield
(70%). Phosphorylation of 12 was carried out in a phosphi-
tylation/oxidation sequence to deliver phosphoryldiester 2 as
a single diastereomer. The R-configuration of 2 was con-
firmed by 1H NMR spectroscopy (Scheme 3).
Scheme 2. Synthesis of Monosaccharide 5
Selective deprotection of the OTMSE group in 2 by
treatment with TBAF in THF, followed by coupling with
the tetrapeptide 3 (D-Glu-L-Lys-D-Ala-D-Ala), gave disac-
charyl pentapeptide 13 in 77% overall yield in two
steps (Scheme 4). Finally, debenzylation of 13, followed
by conjugation with an undecprenyl phosphate, and glo-
bal deprotection under basic conditions gave the desired
target 1 in a yield of 21% over two steps. A novel fluo-
rescent probe 14 was prepared from 1 by attaching a
conjugation with H-Ala-OTMSE, deprotection of both
benzylidene acetal and N-Boc moieties, N-acetylation with
(19) Chen, Q.; Cheng, Q. Y.; Zhao, Y. C.; Han, B.-H. Macromol.
Rapid Commun. 2009, 30, 1651–1655.
(20) de Kruijff, B.; van Dam, V.; Breukink, E. Prostaglandins,
Leukotrienes Essent. Fatty Acids 2008, 79, 117–21.
5308
Org. Lett., Vol. 13, No. 19, 2011

(98%) in 4 h during the transglycosylation reaction cata-
lyzed by Mtb PonA (10 nM) (Figure 2). This observation
indicates that the undecaprenol and lysine moieties do not
dramatically attenuate the substrate efficiency toward
enzymes. Next, moenomycin A, a known bacterial TGase
inhibitor,20 was used to test the inhibitory activity of Mtb
Pon A in this mycobacterial assay system. Around 87% of
this enzyme reaction progress was blocked in the presence
of 1 μM of moenomycin A (see Supporting Information,
Figure S2).7,21
Scheme 4. Synthesis of Mtb N-Glycolyl Lipid II-like Moelcule
(1) and Its Fluorescent Probe (14)
Figure 2. Measurement of transglycosylation progress by
HPLC. The reactions (aÀe) are at t = 0, 0.5, 1, 2, and 4 h.
Peaks A and B represent 14 and an internal standard,
respectively.
In summary, synthetic protocols for the preparation of
an Mtb N-glycolyl lipid II-based analogue and its fluor-
escent probe have been successfully developed and several
transformations, such as 7 to 8 and 11 to 12, have been
optimized.22 This convergent methodology can be poten-
tially scaledup, andthestructureoftargetmoleculescan be
modified easily. Preliminary studies show this probe to be
an Mtb TGase substrate and, therefore, suggest it has
tremendous utility for mechanistic studies, the character-
ization of mycobacterial enzymes, and mycobacterial
TGase inhibitor evaluation.
nitrobenzoxadiazole (NBD) fluorophore at the terminal
ε-NH2 site of lysine on the peptide side chain.
The feasibility of 14 as a TGase substrate was examined
in our HPLC-based functional enzyme assay system.16a To
our delight, 14 (2.5 μM) was almost completely consumed
Acknowledgment. We thank theNational Science Council
and Academia Sinica for financial support.
Supporting Information Available. Experimental pro-
cedures, characterization of the synthetic compounds,
and HPLC analysis of transglycosylation. This material
is available free of charge via the Internet at http://pubs.
acs.org.
(21) Shih, H.-W.; Chen, K.-T.; Chen, S.-K.; Huang, C.-Y.; Cheng,
T.-J. R.; Ma, C.; Wong, C.-H.; Cheng, W.-C. Org. Biomol. Chem. 2010,
8, 2586–2593.
(22) The studies of transformations are in the Supporting
Information.
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