Synthesis of 4-fluorinated UDP-MurNAc pentapeptide as an inhibitor of bacterial growth

Article abstract of DOI:10.1021/ol049598w

Matrix presented. 4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized. In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to be incorporated into the bacterial cell wall through biosynthesis. Compound 2 showed growth-inhibition activity against Gram-positive bacteria when it was added to growth media at 0.01 mg/mL.

Full text of DOI:10.1021/ol049598w

ORGANIC
LETTERS
2004
Vol. 6, No. 11
1753-1756
Synthesis of 4-Fluorinated UDP-MurNAc
Pentapeptide as an Inhibitor of Bacterial
Growth
Taichi Ueda,† Fei Feng,† Reiko Sadamoto,‡ Kenichi Niikura,† Kenji Monde,† and
,†,§
Shin-Ichiro Nishimura*
Laboratory for Bio-Macromolecular Chemistry, DiVision of Biological Sciences,
Graduate School of Science, Frontier Research Center for Post-Genomic Science and
Technology, Hokkaido UniVersity, Kita 21, Nishi 11, Sapporo 001-0021 Japan,
Shionogi Laboratory of Biomolecular Chemistry, DiVision of Biological Sciences,
Graduate School of Science, Hokkaido UniVersity, Kita 21, Nishi 11,
Sapporo 001-0021 Japan, and National Institute of AdVanced Industrial Science and
Technology (AIST), Sapporo 062-8517, Japan
shin@glyco.sci.hokudai.ac.jp
Received March 4, 2004
ABSTRACT
4-Fluorinated UDP-MurNAc pentapeptide, 2, has been synthesized. In our previous study, UDP-MurNAc pentapeptide analogue 1 was found to
be incorporated into the bacterial cell wall through biosynthesis. Compound 2 showed growth-inhibition activity against Gram-positive bacteria
when it was added to growth media at 0.01 mg/mL.
The bacterial cell wall is composed of sugar chains made
up of N-acetyl glucosamine (GlcNAc) and N-acetyl muramic
acid (MurNAc) connected by peptides, and the sugar chain
structure is conserved in all bacteria (Figure 1). In our
previous study,1-3 UDP-MurNAc derivative 1 was found to
be metabolically incorporated into the bacterial cell wall
through its biosynthesis, allowing a fluorescent dye to be
displayed on the surface of the bacteria. This finding has
prompted us to create a novel inhibitor of cell wall synthesis.
In this paper, we synthesized a UDP-MurNAc analogue,
4-fluorinated UDP-MurNAc pentapeptide 2, that cannot be
incorporated into the cell wall, and investigated its inhibition
effect on bacterial growth.
† Laboratory for Bio-Macromolecular Chemistry.
‡ Shionogi Laboratory of Biomolecular Chemistry.
§ National Institute of Advanced Industrial Science and Technology.
(1) Sadamoto, R.; Niikura, K.; Sears, P. S.; Wong, C.-H.; Suksomcheep,
A.; Tomita, F.; Monde, K.; Nishimura, S.-I. J. Am. Chem. Soc. 2002, 124,
9018-9019.
(2) Sadamoto, R.; Niikura, K.; Monde, K.; Nishimura, S.-I. Methods
Enzymol. 2003, 362, 273-286.
(3) Sadamoto, R.; Niikura, K.; Ueda, T.; Monde, K.; Fukuhara, N.;
Nishimura, S.-I. J. Am. Chem. Soc. 2004, 126, 3755-3761.
10.1021/ol049598w CCC: $27.50 © 2004 American Chemical Society
Published on Web 04/30/2004

Figure 1. Biosynthetic route of the bacterial cell wall.
Fluorine-substituted analogues of biologically active or-
ganic compounds have been widely studied.4,5 Fluorinated
carbohydrate derivatives have widespread medical applica-
tions as substrate mimics for the inhibition of enzymatic
processes because of the inability of enzymes to differentiate
between the fluorinated and original compounds. Some
fluorinated glycosyl donors are recognized as artificial
substrates of glycosyltransferases.6-8 Recently, Walker et al.
reported that fluorinated lipid I analogues can be a potent
inhibitor of MurG, which catalyzes the transfer of N-acetyl
glucosamine from UDP to the C-4 hydroxyl residue of lipid
I.9 As muramic acid is a specific component in all bacteria,
a fluorinated muramic acid derivative would make a promis-
ing candidate for a new type of antibiotic.
As illustrated in Scheme 1, we have established a synthetic
route to the key intermediate 4-F-MurNAc-1-phosphate 13
from the less expensive N-acetyl galactosamine (GalNAc)
rather than MurNAc. The anomeric position of GalNAc was
protected by the allyl group. Using Lewis acid BF3‚Et2O as
a catalyst, heating the commercially available D-GalNAc at
70 °C for 2 h gave the desired R-anomer 3 in 80% yield.
Subsequent regioselective benzylidenation of the C-4 and
C-6 positions of the obtained allyl glycoside was performed
using benzaldehyde dimethyl acetal and a catalytic amount
of camphor-10-sulfonic acid (CSA) in dimethylformamide
(DMF) to produce the partially unprotected saccharide 4 with
a free 3-hydroxyl group. The compound 4 was converted
into the fully protected glycoside 5 by reaction with
4-methoxybenzyl chloride in the presence of sodium hydrate
and tetrabutylammonium iodide in tetrahydrofuran (THF)10
at 40 °C. This reaction could not proceed at all at room
temperature. The benzylidene acetal was reductively opened
with sodium cyanotrihydroborate (NaBH3CN) and hydrogen
chloride in THF to provide the required 6-O-benzyl ether 6
(4) Williams, S. J.; Withhers, S. G. Carbohydr. Res. 2000, 327, 27-46.
(5) Dax, K.; Albert, M.; Ortner, J.; Paul, B. J. Carbohydr. Res. 2000,
327, 47-86.
(6) Burkart, M. D.; Vincent, S. P.; Duffels, A.; Murray, B. W.; Ley, S.
V.; Wong, C.-H. Bioorg. Med. Chem. 2000, 8, 1937-1946.
(7) Kajihara, Y.; Endo, T.; Ogasawara, H.; Kodama, H.; Hashimoto, H.
Carbohydr. Res. 1995, 269, 273-294.
(8) Hartman, M. C.; Coward, J. K. J. Am. Chem. Soc. 2002, 124, 10036-
10053.
(9) Chen, L.; Men, H.; Ha, S.; Ye, X.-Y.; Brunner, L.; Hu, Y.; Walker,
S. Biochemistry 2002, 41, 6824-6833.
(10) Czernecki, S.; Georgoulis, C.; Provelenghiou, C. Tetrahedron Lett.
1976, 17, 3535-3536.
1754
Org. Lett., Vol. 6, No. 11, 2004

Scheme 1
without the formation of the isomeric 4-O-benzyl ether.11
of 30% after purification by silica gel column chromatog-
raphy.16 This oxidation reaction was unsuccessful when
performed with hydrogen peroxide because of a break in the
P-O bond.
We then focused our attention on the fluorination reaction
to obtain the key intermediate 7 by treating 6 with (diethyl-
amino)sulfur trifluoride (DAST)12 under nucleophilic dis-
placement conditions as shown in the scheme to give the
desired 4-fluoroglucosaminide 7 in 60%yield. The p-meth-
oxyphenylmethyl (MPM) group of 7 was successfully
removed using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone
(DDQ) in dichloromethane (DCM) to afford the intermediate
8, which was reacted with (S)-(-)-2-chloropropionic acid
to give the compound 9 in 80% yield. The protection of the
carbonyl group of 9 with 2-phenylsulfonyl ethanol also led
to the formation of the compound 10. The anomeric position
protected by the allyl moiety could be deprotected using (1,5-
cyclooctadiene)bis(methyldiphenylphosphine)iridium(I) hex-
anefluorophosphate reagent13,14 to generate the intermediate
11 as an R-â mixture. This deallylation was also attempted
using palladium chloride in acetic acid, but the yield was
much lower. Phosphorylation of 11 was carried out with
dibenzyl diethylphosphoramidite (DDP)15 and triazole in
DCM to give dibenzyl phosphite 12 as a mixture of R and
â. The stereo mixture of 12 was oxidized with tert-
butylperoxide (TBHP) in THF at -10 °C to generate the
single R-isomer 13 (the coupling constant between the
anomeric and C-2 proton was 3.05 Hz) in an isolated yield
Prior to the coupling reaction with a peptide moiety,
deprotection of the carboxyl group with 1,8-diazabicyclo-
[5.4.0]undec-7-ene (DBU) in CH2Cl2 was carried out.17
Pentapeptide 15 was synthesized from Boc-D-Ala-D-Ala-
OMe by using Boc general protocol. Coupling pentapeptide
15 with the muramyl carboxyl group of 14 gave the
glycopeptide 16 in 66% yield for the two steps. The benzyl-
protecting groups of the phosphate moiety in 16 were
removed in the presence of trioctylamine. The hydrophilic
cationic counterion was used to increase solubility in the
organic solvent in the next step. Subsequent reaction of the
trioctylammonium phosphate salt with uridine 5′-monophos-
phomorpholidate and 1H-tetrazole in pyridine for 3 days
produced the protected UDP-4-F-MurNAc pentapeptide.18
Removal of the protecting groups was accomplished by rapid
treatment with aqueous sodium hydroxide. The mixture was
purified by column chromatography, and the final compound
was confirmed by NMR and ESI mass spectroscopy (see
details in Supporting Information).
Gram-positive bacteria (L. plantarum JCM1149) were
incubated at 37 °C in lactobacilli MRS broth (Difco
Laboratories) in the presence of 2 at a concentration of 0.01
mg/mL. Compound 2 was dissolved in a buffered solution
and sterilized before addition to the bacteria. After 3, 5, 7,
and 10 h, the number of bacteria in the culture was counted
(11) Oikawa, M.; Wada, A.; Yoshizaki, H.; Fukase, K.; Kusumoto, S.
Bull. Chem. Soc. Jpn. 1997, 70, 1435-1440.
(12) Berkin, A.; Szarek, W. A.; Kisilevsky, R. Carbohydr. Res. 2000,
326, 250-263.
(13) Tamura, J.-I.; Nishihara, J. J. Org. Chem. 2001, 66, 3074-3083.
(14) Mochizuki, T.; Iwano, Y.; Shiozaki, M.; Kurakata, S.-I.; Kanai, S.;
Nishijima. M. Carbohydr. Res. 2000, 324, 225-230.
(16) Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260-2267.
(15) Yu, K.-L.; Fraser-Reid, B. Tetrahedron Lett. 1988, 29, 979-982.
Org. Lett., Vol. 6, No. 11, 2004
1755

Scheme 2
Figure 2. Changes in bacterial proliferation in the presence of
compound 2, fosfomycin, and compound 2 with fosfomycin: (9)
without adding any antibiotics (negative control); (b) compound
2; (O) fosfomycin; (1) compound 2 with fosfomycin.
by the conventional plate counting method. Figure 2 shows
the time course of the growth of the bacteria in the presence
of 2. Bacterial growth was significantly inhibited as com-
pared to the negative control, which was cultured without
the addition of any antibiotics.
GalNAc as a starting material. Fluorine was introduced
through an interconversion reaction from a galacto- to a
gluco-configuration at the C-4 position. The UDP-4-F-
MurNAc pentapeptide showed growth inhibition activity
against Gram-positive bacteria. We demonstrated that UDP-
MurNAc derivatives are useful not only as precursors to
modify the bacterial cell wall but also as inhibitors of
bacterial growth. The kinetic details of inhibitor 2 will be
reported soon using recombinant MraY and MurG.
In our previous study,3 enhanced incorporation of UDP-
MurNAc derivative 1 in the bacterial cell wall was observed
by the addition of fosfomycin with 1 to the medium.
Fosfomycin is an antibiotic that inhibits the early stage of
cell wall biosynthesis; that is, the transformation from UDP-
GlcNAc to UDP-MurNAc. We consider that the added UDP-
MurNAc derivative 1 was used preferentially as a substrate
for cell wall biosynthesis because the production of natural
UDP-MurNAc in bacteria was depressed by fosfomycin. The
inhibition of bacterial growth was greater in the presence of
2 with fosfomycin than in the presence of 2 or fosfomycin
alone. Compound 2 could be utilized as a substrate mimic
by MraY and then block peptidoglycan synthesis at the MurG
step, or it could inhibit the MraY step directly.
Acknowledgment. We thank Ms. S. Oka (Hokkaido
University) for ESI mass spectrometry measurements. This
work was partly supported by a grant for the “Glycocluster
Project” from NEDO and a Grant-in-Aid for Exploratory
Research from the Ministry of Education, Culture, Sports,
Science and Technology (MEXT).
Supporting Information Available: Experimental pro-
cedures, spectroscopic data for all new molecules, and
procedures used for the inhibition studies of bacterial growth.
This material is available free of charge via the Internet at
http://pubs.acs.org.
In conclusion, we synthesized a UDP-4-F-MurNAc pen-
tapeptide toward a novel inhibitor of bacterial growth from
(17) Hitchcock, S.; Eid, C. N.; Aikins, J. A.; Zia-Ebrahimi, M.; Blaszczak,
L. C. J. Am. Chem. Soc. 1998, 120, 1916-1917.
(18) Wittmann, V.; Wong. C.-H. J. Org. Chem. 1997, 62, 2144-2147.
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