1,6-AnhMurNAc derivatives for assay development of amidase AmiD

Article abstract of DOI:10.1016/j.bmc.2010.09.010

Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives protected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the MurNAc and the α-amino group of l-Ala where the peptide chain was at least a dipeptide (l-Ala-γ-d-Glu) amidated by benzylamine on the γ-carboxyl group of d-Glu. In the presence of a tripeptide chain (l-Ala-γ-d-Glu-l-Lys) or a tetrapeptide chain (l-Ala-γ-d-Glu-m- A₂pm-d-Ala) higher hydrolysis rates were observed. We have also demonstrated that the presence of TNB on the -amino group of l-Lys only has a small influence on the hydrolysis capacity of sAmiD.

Full text of DOI:10.1016/j.bmc.2010.09.010

Bioorganic & Medicinal Chemistry 18 (2010) 7422–7431
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
1,6-AnhMurNAc derivatives for assay development of amidase AmiD
Frédéric Mercier a, Astrid Zervosen a, Nathalie Teller a, Jean-Marie Frère b, Raphaël Herman b,
Anne Pennartz b, Bernard Joris b, André Luxen a,
⇑
a Centre de Recherches du Cyclotron, B30, Université de Liège, Allée du 6 août 8, Sart Tilman, B 4000 Liège, Belgium
b Centre d’ingénierie des Protéines, Institut de Chimie B6a, Université de Liège, Sart Tilman, B 4000 Liège, Belgium
a r t i c l e i n f o
a b s t r a c t
Article history:
Various peptidoglycan fragments were synthesized from two anhydro-muramic acid derivatives pro-
tected with a Bn or a PMB group at the 4th position, in homogenate phase or on a solid support. In order
to facilitate HPLC detection, a chromophoric group was attached to the peptide chain. The periplasmic
amidase sAmiD of Escherichia coli was used to cleave the amide bond between the lactyl group of the
Received 12 March 2010
Revised 10 August 2010
Accepted 2 September 2010
Available online 8 September 2010
MurNAc and the
Glu) amidated by benzylamine on the
-Ala- -Glu- -Lys) or a tetrapeptide chain (
observed. We have also demonstrated that the presence of TNB on the
small influence on the hydrolysis capacity of sAmiD.
a
-amino group of
L
-Ala where the peptide chain was at least a dipeptide (
-carboxyl group of -Glu. In the presence of a tripeptide chain
-Ala- -Glu-m-A2pm- -Ala) higher hydrolysis rates were
-amino group of -Lys only has a
L-Ala-c-D-
c
D
Keywords:
Anhydro-muramic acid derivative
sAmiD
N-Acetyl-L-alanine amidase
Peptidoglycan
(
L
c-
D
L
L
c-
D
D
e
L
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
the production of GlcNAc-anhMurNAc-tri(tetra)peptides. The lib-
erated peptidoglycan fragments (muropeptides) are transported
from the periplasm into the cycloplasm and recycled for further
Peptidoglycan is an essential macromolecule in the bacterial
cell wall, found outside the cytoplasmic membrane of almost all
bacteria. The main functions of peptidoglycan are preserving cell
integrity by withstanding the internal osmotic pressure and main-
taining the cell shape. This macromolecule consists of linear heter-
oglycan chains cross-linked by short peptide chains.1,2 The
heteroglycan chains are composed of alternating b-(1-4)-linked
N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (Mur-
NAc) residues. In Gram-negative bacteria, MurNAc is linked
peptidoglycan biosynthesis. N-Acetylmuramyl-
(MurNAc- -Ala amidases) specifically cleave the amide bond be-
tween the lactyl group of MurNAc and the -amino group of -ala-
L-alanine amidases
L
a
L
nine, the first amino acid of the peptide chain. The cytosolic
amidase AmpD of Escherichia coli specifically catalyses the hydroly-
sis of 1,6-anhydroMurNAc-tri(tetra)peptide after it has been trans-
ported into the cytosol by the permease AmpG. Four periplasmic
amidases have been identified in E. coli: AmiA, AmiB, AmiC and
AmiD. AmiD was first detected in an ampDnagZ double mutant
where a significant amount of the disaccharide GlcNAc-anhMur-
NAc accumulates in addition to the expected GlcNAc-anhMur-
NAc-tripeptide, suggesting that another AmpD-like enzyme must
exist in E. coli.4 The AmpD-like lipoprotein AmiD requires Zn2+
for its activity. It is a member of the amidase_2 family (PF01510).
The AmiD amidase is, in contrast to AmiA, B and C, not involved
in cell separation. AmiD catalyses the hydrolysis of various sub-
through the lactyl amide bond to
minopimelyl- -alanine -Ala-
Gram-positive bacteria, m-A2pm is replaced by
L
-alanyl-
-Glu-m-A2pm-
-Lys.
c
-
D
-glutamyl-meso-dia-
D
(
L
c-
D
D-Ala). In some
L
Both cell growth and cell division require continuous remodel-
ling of peptidoglycan. Up to 60% of the parental peptidoglycan can
be recycled for further peptidoglycan biosynthesis. Up to now, nine
enzymes have been discovered that are specifically involved in this
recycling process.3 Degradation by lytic transglycosidases leads to
strates such as anhMurNAc-
L
-Ala-c-
D-Glu-m-A2pm and GlcNAc
b(1,4)-anhMurNAc- -Ala- -Glu-m-A2pm-D
L
c
-
D
-Ala (TCT) (Fig. 1).
Abbreviations: Ac2O, anhydride acetic; Bn, benzyl; BOC, tert-butoxycarbonyl;
BOP, benzotriazol-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophos-
phate; Cbz, carboxybenzoyl; DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone;
DIEA, N,N-diisopropylethylamine; DMF, dimethylformamide; DMSO, dimethylsulf-
oxide; Fmoc, 9-fluorenylmethoxycarbonyl; HBTU, O-(1H-benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexafluorophosphate; NMM, N-methylmorpholine;
MS, mass spectrometry; PMB, para-methoxybenzyl; PyBOP, benzotriazol-1-yl-oxy-
tris-pyrrolidino-phophonium hexafluorophoshate; TBTU, O-(1H-benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium tetrafluoroborate; TFA, trifluoroacetic acid; TNB,
trinitrobenzene; TNBSA, trinitrobenzensulfonic acid.
Interestingly, AmiD does not have a strict specificity for 1,6-anhy-
dromuropeptides5,6 and is an interesting target to study substrate
specificity on synthetic anhMurNAc and MurNAc derivatives. Up to
now, the lack of authentic fully characterized substrates has lim-
ited the study of this enzyme. AmiD is a periplasmic lipoprotein
anchored in the outer membrane through a modified N-terminal
cysteine. A truncated form of AmiD (sAmiD) has been cloned in
the production vector pBAD, overproduced, purified, character-
ized5 and crystallized.7 In this paper we describe the synthesis of
⇑
Corresponding author. Tel.: +32 4366 2361; fax: +32 4366 2946.
E-mail address: aluxen@ulg.ac.be (A. Luxen).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.010

F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
7423
(compound 27) (Scheme 2). The final step is a complete deprotec-
tion to obtain the molecules, which were subsequently used for
biological experiments. The compound 28 with a trinitrobenzene
(TNB) chromophoric ring on the e-amino group of L-Lys was also
prepared from 27 (Scheme 2).
HO
O
O
CH3
H
N
H
N
H2N
O
N
H
O
O
COOH
H3C
O
CH3
O
O
HN
NHAc
O
NHAc
HO
HO
2.1.2. Solid phase synthesis of anhydro-muramic acid
derivatives containing a peptide chain of three or four amino
acids (compounds 27 and 29)
O
COOH
OH
Syntheses of glycopeptides on solid phase are not very common
in the literature. Synthesis on solid supports (Scheme 3) is simple,
fast and requires small volumes of solvents and compounds. More-
over, the possible side products and excess reagents are removed
by simple washing, which facilitates the purification of the desired
compound.
Figure 1. Structure of TCT.
1,6-anhydro-muramic acid derivatives and their biological evalua-
tion with sAmiD in order to develop a simple biological assay for
this important enzyme.
The synthesis on solid supports of N-acetylglucosamine deriva-
tives containing a peptide chain has been presented by Kumar
et al.11 and the synthesis on solid supports of a pentapeptide by
Chowdhury and Boons.12 According to these literatures, compound
27 was also synthesized by polymeric support synthesis using the
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of anhydro-muramic acid derivatives
Wang-Fmoc-L-Lys(Boc) resin, Fmoc-protected amino acids, and the
containing a (peptidic) chain of variable length (compounds 6,
7, 8, 11, 20, 27, 28) in homogenate phase
properly protected anhydro-muramic acid derivative 2. The peptide
chain was elongated on solid support and 2 was coupled by an acid
carboxylic function to produce the resin-bound glycopeptide. A TFA
treatment of the resin and a complete deprotection of the glycopep-
tide produced the compound 27 with 40% yield for all steps (n = 2).
Compound 27 was previously obtained in four steps in homogenate
phase with a global yield of 6.3%. It was clear that the solid support
synthesis produced a better yield (in a better time).
Various anhydro-muramic acid derivatives containing a pep-
tidic chain of variable length have been synthesized in homogenate
phase using similar synthetic methods, such as Kawasaki et al.8
and Mobashery and co-workers.9,10 The precursor 18–10 or 28–10
is coupled in the presence of an activating agent such as TBTU or
PyBOP with various aromatic amines (Scheme 1: compounds 6,
7, and 8), modified amino acids such as L-Ala-p-nitroanilide 9
Following the same strategy, compound 29 was obtained from
(commercially available) (Scheme 1: compound 11) and peptide
chains containing two (compound 20) or three amino acids
the Wang-Fmoc-D-Ala resin. The meso-diaminopimelic acid
prepared by Teller13 was first coupled, followed by the
CH3
O
HOOC
O
1 : R = Bn
2 : R = PMB
O
OR
NHAc
c
a
CH3
H
N
O
O
O2N
O
O
O
CH3
R'
H
N
O
OR
NHAc
N
H
O
O
(from 1) 3 : R = Bn ; R' = H
(from 2) 4 : R = PMB ; R' = COCH3
(from 2) 5 : R = PMB ; R' = CF3
CH3
10
O
OPMBNHAc
b
b
CH3
H
O2N
O
CH3
N
O
H
N
O
O
O
N
H
O
O
O
R'
CH3
O
OH
NHAc
OH
NHAc
11
(from 3) 6 : R' = H
(from 4) 7 : R' = COCH3
(from 5) 8 : R' = CF3
Scheme 1. Reagents and conditions: (a) 1, CH2Cl2, BOP, NMM, rt, 1 h, N2 and then aniline, rt, N2, 48 h, 50% for 3; 2, DMF, BOP, NMM, rt, 1 h, N2 and then para-
aminoacetophenone or para-trifluoromethylaniline, rt, N2, 48 h, 25% for 4, 40% for 5; (b) 3, MeOH, Pd/C 10%, H2, rt, 6 h, 12% for 6; 4 or 5, CH2Cl2–H2O, DDQ, 0 °C to rt, N2, 24 h,
20% for 7, 31% for 8, 10% for 11; (c) 2, DMF, BOP, NMM, rt, 1 h, N2 and then L-Ala-para-nitroanilide hydrochloride 9, rt, N2, 48 h, 73%.

7424
F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
BnO
O
HOOC
NHBOC
a
a
12
O
OBn
NHR
BnO
O
H
N
NHCbz
O
NHR
O
NH
BnO
O
22 : R = BOC
23 : R = H
b
13 : R = BOC
14 : R = H
a
b
a
BnO
O
BnO
O
O
O
H
N
NHCbz
NHR
CH3
O
b
NHR
CH3
N
H
N
H
NH
O
BnO
O
24 : R = BOC
25 : R = H
16 : R = BOC
17 : R = H
b
c
c
R2O
O
O
CH3
H
N
H
N
O
R3
N
H
O
O
O
CH3
O
OR1 NHAc
18 : R1 = PMB ; R2 = Bn ; R3 = Ph
19 : R1 = PMB ; R2 = H ; R3 = Ph
20 : R1 = R2 = H; R3 = Ph
26 : R1 = PMB ; R2 = Bn ; R3 = CH(COOBn)-(CH2)4-NHCbz
27 : R1 = R2 = H ; R3 = CH(COOH)-(CH2)4-NH2
d
e
d
f
HO
O
NO2
NH
O
CH3
H
N
H
N
O
N
H
O
O
O
CH3
O
O2N
NO2
HO
O
OH
NHAc
28
Scheme 2. Reagents and conditions: (a) CH2Cl2, DIEA, TBTU, rt, 1 h and then desired amine is added (aniline, NHBOC-
L
-Ala 15 or H-Lys(Z)-ObzlꢀHCl 21, rt, 1 h, 75% for 13, 75%
for 16, 98% for 22, 60% for 24 (two steps); (b) CH2Cl2, TFA, rt, 0.5 h, N2, 71% for 14, 74% for 17; (c) 2, CH2Cl2, DIEA, PyBOP or TBTU, rt, 1 h and then 17 or 25, rt, 1 h, 51% for 18
and 18% for 26 (two steps); (d) EtOH, Pd/C 10%, H2, rt, 6 h, 68% for 18, MeOH, Pd/C 10%, H2, 35 °C, 72 h, 60% for 27; (e) CH2Cl2–H2O, DDQ, 0 °C to rt, N2, 24 h, 10%; (f) carbonate
buffer (0.1 N), pH 9.5, TNBSA, 35 °C, 2 h, 97%.
Fmoc-
D
-Glu-OtBu, the Fmoc-
L
-Ala and finally the derivative 2. A TFA
of 1,6-AnhMurNAc glycopeptides could be obtained with this
method.
treatment of the resin and a complete deprotection of the glycopep-
tide produced compound 29 with 51% yield for all steps (n = 1).
Here we show the feasibility of rapidly and automatically cou-
pling the properly protected anhydro-muramic acid derivative 2
to peptides of various lengths which are still bound to the resin.
After cleavage from the resin and final deprotection, a large variety
2.2. Biological experiments
Eight anhydro-muramic derivatives (6, 7, 8, 11, 20, 27, 28 and
29) with various aromatic residues or various peptide chains were

F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
7425
O
BocHN
O
NHFmoc
d, a
NHFmoc
a
O
O
O
O
NHCbz
COOBn
N
H
HN
O
BocHN
O
CH3
O
H
N
O
t-BuOOC
N
H
tBu-O
H
O
O
O
CH3
O
H
N
O
O
N
N
H
OPMB
NHAc
O
O
O
CH3 O
O
O
OPMB
NHAc
b, e
b, c
HO
O
O
HO
O
NH3+ CF3COO-
COOH
O
H
N
H
N
CH3
O
N
H
O
NH2
N
H
HN
O
O
O
CH
3 O
HO
O
OH NHAc
O
CH3
O
H
N
27
O
HOOC
N
H
O
O
29
OH NHAc
Scheme 3. Reagents and conditions: (a) piperidine then Fmoc-D-Glu-OtBu, HOBt; piperidine then Fmoc-L-Ala, HOBt; piperidine then 2, HOBt; (b) TFA, 2 h; (c) MeOH, Pd/C
10%, H2, 35 °C, 72 h, 40% for all steps; (d) piperidine then meso-diaminopimelic acid, HOBt; (e) THF, TFA 0.1%, Pd/C 10%, H2, rt, 2 d, 51% for all steps.
Table 1
The derivatives were incubated with various concentrations of sAmiD in 20 mM sodium phosphate buffer containing 48 nM ZnCl2 at 30 °C for various
incubation times t
Compound
c (lM)
t (min)
sAmiD (
l
M)
(sAmiD) * t (lM min)
Product (%)
TCT (GlcNAc b-(1,4)-anhMurNAc-tetraeptide (with A2pm))
6
7
8
28
1000
250
1000
1000
1000
40
120
120
120
120
120
0.04
3.9
3.9
3.9
3.9
3.9
1.6
74
0
0
0
0
464
464
464
464
464
11
20 (anhMurNAc-dipeptide-Bnz)
9
The solutions were analyzed by HPLC.
synthesized from the anhydro-muramic acid derivatives 1 and 2
previously presented. These derivatives were used for biological
studies with sAmiD.
derivatives 20 and 28 were detected with theoretical masses of
[M+1] = 293 and [M+1] = 558, respectively.
2.2.2. Kinetic studies
2.2.1. Hydrolysis studies
Compounds 20, 27, 28 and 29 were used for kinetic studies. A
linear relationship between the initial rates and substrate concen-
trations up to 1 mM was observed. The hydrolysis of these com-
pounds at a concentration of 0.1 mM was studied and curves
monitoring first order decrease of the substrate concentration over
the incubation time were obtained (Fig. 2).
Hydrolysis of the various compounds catalysed by sAmiD was
studied and the results are summarized in Table 1. sAmiD was
not able to catalyse the cleavage of the amide bond between the
lactyl group of MurNAc and the amino group of substituted aro-
matic rings (6, 7 and 8). No hydrolysis of 11 containing the first
amino acid L-alanine and a para-nitrophenyl residue was observed.
TCT exhibits a burst kinetic (Fig. 3),5 characterized by a rapid
phase preceding a linear phase. Similar kinetics have been found
The anhMurNAc-dipeptide derivative 20, the anhyMurNAc-tripep-
tide derivatives 27 and 28, the anhyMurNAc-tetrapeptide deriva-
tive 29, and GlcNAc b(1,4)-anhMurNAc-tetrapeptide TCT (Fig. 1)
were hydrolysed by sAmiD (Table 2).
The reaction mixtures of hydrolysates of 20, 27, 28 and 29 with
sAmiD were analysed by HPLC and MS. AnhMurNAc with a theo-
retical mass of 275.1 g/mol ((M+1) = 276) was found for 27 and
29 but it was not possible to observe their non-substituted peptide
chains by HPLC-analysis. On the other hand, the peptide
chains (which contain chromophoric groups) of the anhMurNAc
with MurNAc-
L
-Ala-
c-D-Glu-mA2pm and MurNAc-L-Ala-c-D-Glu-
mA2pm- -Ala-
D
D
-Ala.5 The biphasic kinetic curves have been ex-
plained by the rapid and reversible inactivation of sAmiD by the
substrate, which can be described by the kinetic scheme shown
in Figure 4. The sAmiD-dependent product formation in these burst
kinetics obeys Eq. (1):
P ¼ vss ꢁ t ꢂ ðvss
ꢂ
viÞð1 ꢂ eꢂktÞ=k
ð1Þ

7426
F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
Table 2
The derivatives were incubated with various concentrations of sAmiD in 20 mM sodium phosphate buffer containing 48 nM ZnCl2 at 30 °C for various incubation times t
Compound
c (lM)
vi
(l
M minꢂ1
)
sAmiD (E0) (
l
M)
v )
i/E0 (minꢂ1
TCT (GlcNAc b-(1,4)-anhMurNAc-tetraeptide (with A2pm))
20 (anhMurNAc-dipeptide-Bnz)
27 (anhMurNAc-tripeptide (with Lys))
28
100
100
100
100
1.6
0.2
3.4
1.4
0.5
0.04
15.4
0.77
0.34
0.77
40
0.013 (0.004a)
4.4 (1.2a)
28 (anhMurNAc-tripeptide (with
e
-TNBS-Lys))
4.1 (1.1a)
29 (anhMurNAc-tetrapeptide (with A2pm))
0.65 (0.18a)
The solutions were analyzed by HPLC.
a
Estimated
v
i/E0 at a substrate concentration of 28
lM. For these compounds a linear relationship between initial rates and substrate concentrations up to 1 mM was
observed.
K1
kp
E +P
[ES]
K2
[E'S]
E + S
Figure 4. Kinetic scheme for the substrate-induced inactivation mechanism of
sAmiD (Pennartz et al.). E, native enzyme; E0, inactivated or poorly active form of
the enzyme induced by the substrate; S, substrate; P, product; [ES] and [E0S]
substrate-enzyme complexes. K1 and K2 represent dissociation constants. kp is the
rate constant characterizing the hydrolytic decay of [ES]. This kinetic scheme can be
used because the conversion of E0 to E is fast and the EME0 equilibrium completely
displaced toward E.
difficult to distinguish a burst from a slow down due to substrate
depletion.14 For example, 28 exhibited burst kinetics in the pres-
ence of a high sAmiD concentration (1.9
presence of a lower sAmiD concentration (0.3
behaviour was found (Fig. 2). Thus as postulated by Waley14
l
M) (Fig. 3), while in the
Figure 2. Hydrolysis time courses of 20, 27, 28 and 29, 20, 27, 28 and 29 (100
were hydrolysed with various enzyme concentrations (20: 7.7 M, 27: 0.7 M, 28:
0.3 M and 29: 0.7 M) in 20 mM sodium phosphate buffer containing 48 nM ZnCl2
lM)
lM) no burst kinetic
l
l
a
l
l
branched-pathway mechanism as described in Figure 4 may prevail
even when no burst is observed. This is probably due to the fact that
one of the above conditions is not fulfilled (sufficient substrate and
enzyme concentration).
at 30 °C. The reactions were stopped after various incubation times and analysed as
described in Section 4.29.
In order to compare data, initial rates for all substrates were cal-
culated from various curves (20, 27, 28 and 29) (Fig. 2) and TCT
(Fig. 3). The vi/E0 values (Table 2) show that:
– TCT is about 33 times more active than the anhyMurNAc-tripep-
tide 27 and about 222 times more active than anhyMurNAc-tet-
rapeptide 29, which contains the same peptide chain as TCT,
showing that sAmiD prefers substrates containing GlcNAc-anh-
MurNAc to substrates containing only anhMurNAc, as already
described.5 Thus, GlcNAc-anhMurNAc-tripeptide was described
as 100-fold more active than anhMurNAc-tripeptide.6
– The presence of TNB on the
e-amino group of L-Lys (compound
28) appears to have little influence on the hydrolysis capacity
of sAmiD, such that nearly identical vi/E0 values were observed
for 28 and 27 (Table 2).
– The anhMurNAc derivative 20 containing a dipeptide chain was
10,000 times less active than TCT and the i/E0 value of 20 was +/
ꢂ 300 times smaller than those of 27 and 28. Previously, other
MurNAc-dipeptides (MurNAc- -Ala- -Glu, MurNAc- -Ala- -Glu
and MurNAc- -Ala-
-Glu) studied by Pennartz et al.5 were found
v
Figure 3. Hydrolysis time courses of TCT (28
lM) and 28 (100 lM) in the presence
L
D
D
D
of sAmiD (0.04 M or 1.93 M). The biphasic behaviour is characterized by a rapid
l
l
L
L
phase preceding a linear phase, a typical burst reaction. The curve fitting was
obtained using Eq. (1). TCT:
v
ss = 0.12
l
M minꢂ1
,
v
ii = 1.6
l
M minꢂ1, k = 0.14 minꢂ1
.
not to be substrates of sAmiD. Here we show that the elongation
of the peptide chain by addition of a benzylamine group bound
28: ss = 0.73 ii = 31.4
v
l
M minꢂ1
,
v
l
M minꢂ1, k = 0.5 minꢂ1
.
to the
c-carboxyl group of D-Glu allowed the recognition and
hydrolysis of 20 by sAmiD.
where the concentration (p) of product at time t is given a function
of vi vss and k. vi and vss represent the initial and the stationary
,
In conclusion, a good substrate of sAmiD should contain anh-
MurNAc as a carbohydrate moiety with a tripeptide extension. It
is possible to replace m-A2pm by Lys, which facilitates synthesis.
In agreement with crystallographic data showing that the
rates. k is the burst constant. The burst constant k increases with
initial substrate concentration S. If the initial concentration of sub-
strate is too low, the vi/vss ratio is not far from 1 and no burst is ob-
served. Conversely, if the amount of enzyme is too low, it becomes

F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
7427
side-chain amino group of lysine projects into a solvent,7 the addi-
tion of a chromophoric residue like TNB has no influence on the
substrate specificity and allows easy monitoring of the hydrolysis
reaction in complex samples by HPLC using a PDA-detector.
pressure of 50 bars on HPLC NOVASEP LC50.500.VE150 with
250 g of silica gel Merck (0.015–0.040 mm). Column chromatogra-
phy was performed with silica gel 0.060–0.200 mm from Acros. 1H
NMR and 13C NMR spectra were recorded at room temperature on
Bruker 250 or 400 MHz apparatus. Chemical shifts are given in d
relative to tetramethylsilane as an internal standard, the coupling
constants J are given in hertz. Assignment of peaks was based upon
HMBC, HSQC and COSY experiments. The abbreviations are as fol-
lows: s = singulet, d = doublet, t = triplet, q = quadruplet, m = mul-
2.2.3. Inhibition studies of sAmiD
Amidases play an essential role in the bacterial life cycle and are
considered as appropriate drug targets in the development of anti-
biotics. Thus, amidase inhibitors have been proposed as antibacte-
rial agents.15 Inhibition studies were performed with several
derivatives that were not substrates of sAmiD (6, 7, 8 and 11).
These compounds were incubated for 20 min at 30 °C with sAmiD
(39 nM). The residual activity of the amidase was determined by
tiplet. MS analysis was made on
Finnigan device equipped with an electrospray source (ES). The
co-solvent (injected in 200 l/min) is a mixture 50:50: H2O/MeOH
a TSQ 7000 Thermoquest
l
containing 0.1% of HCOOH or HAc. The automated solid phase pep-
tide synthesizer was a PS3 from Protein Technologies used in Fmoc
strategy employing HBTU as the coupling agent.
measuring the initial rate of the hydrolysis of 29
lM TCT. No inhi-
bition was observed with 100 M of the anhMurNAc derivatives 6,
l
7, 8, 11 and 20. In the presence of 20 at concentrations of 0.5 mM
and 1 mM residual activities of 83% and 64% (respectively) were
detected, demonstrating the competition between the natural sub-
strate TCT and 20. Furthermore, after a pre-incubation of 30 min at
30 °C of 1 mM 1, 6, 7, 8 and 11 with sAmiD, the residual activity of
sAmiD was determined by measuring the initial rate of the hydro-
lysis of 1 mM 20, which is 10,000 less active than TCT (Table 2). De-
spite the fact that these conditions could facilitate the detection of
an inhibition, no such phenomenon was observed.
4.1. General procedure for peptide coupling with BOP and NMM
(Procedure A)
Under
a nitrogen atmosphere, NMM (2.2 equiv) and BOP
(1 equiv) were added at 0 °C to a stirred solution of the free acid
(1 equiv) in dry DMF (approximately 0.08 M per component). After
1 h, the amine (1 equiv) was added. The reaction was stirred under
a nitrogen atmosphere at rt for 48 h. The solvent was concentrated
in vacuum and the residue was coevaporated several times with
toluene in order to remove traces of DMF. The residue is diluted
with CH2Cl2 and the organic phase was washed with three portions
of saturated NH4Cl, dried (MgSO4), filtered and concentrated in a
vacuum to produce an oil which was purified by preparative HPLC.
Our results illustrate that anhMurNAc 1 and anhMurNAc deriv-
atives containing an amide bond between the lactyl group of Mur-
NAc and the amino group of substituted aromatic rings (6, 7 and 8)
or the
a-amino group of L-alanine of the L-alanine-para-nitro-
phenyl residue 11 (Table 1) were neither substrates nor inhibitors
of sAmiD. For binding, the enzyme requires an anhMurNAc deriv-
ative containing at least a dipeptide chain amidated by benzyl-
4.2. General procedure for peptide coupling with TBTU and DIEA
(Procedure B)
amine on the c-carboxylgroup of D-Glu (Section 2.2.1).
DIEA (2.2 equiv) was added at room temperature to a stirred
solution of the free acid (1 equiv) in dry CH2Cl2 (approximately
0.08 M per component). After 1 h, the amine (1 equiv) was added
and the reaction was stirred for another 1 h. The organic phase
was washed with three portions of saturated NH4Cl, dried (MgSO4),
filtered and concentrated in a vacuum to produce a solid or oil.
Sometimes, purification by preparative HPLC was necessary.
3. Conclusion
Using the solid support strategy, we have shown that it was
possible to attach compound 2 with peptides of various lengths
still bound to the resin. After a TFA treatment, the glycopeptide ob-
tained is completely deprotected to produce the desired 1,6-Anh-
MurNAc derivatives containing peptide side chains of various
lengths. With this strategy, we have prepared compounds 27 and
29 containing three and four amino acids (respectively) in the pep-
tide side chains. We have also shown that the solid support synthe-
sis of 27 produces a better yield (40%) than in homogenate phase
(6.3%).
4.3. General procedure for PMB-cleavage by DDQ (Procedure C)
Under a nitrogen atmosphere, DDQ (1.1 equiv) was added at
0 °C to a stirred solution of starting material in a mixture of
CH2Cl2/H2O (20:1 v/v) (approximately 0.05 M for starting mate-
rial). After 24 h, CH2Cl2 (10 v) was added and the organic phase
was washed with saturated NaHCO3 and brine, dried (MgSO4), fil-
tered and concentrated in a vacuum to produce an oil which was
purified with a semi-preparative in reverse phase HPLC.
Biological studies with sAmiD showed that anhMurNAc-
-Glu- -Lys 28 with TNB as chromophoric group on the -amino
group of -Lys is a good candidate for the development of a novel
L-Ala-
c-
D
L
e
L
sensitive enzyme assay of sAmiD.
4. Experimental
4.4. General procedure for BOC-cleavage (Procedure D)
All chemicals and reagents were either purchased puriss. p.a.
from commercial suppliers. Melting points (Mp) were determined
with Büchi Melting Point-B545 and are uncorrected. For analytical
thin layer chromatography (TLC), plastic silica gel plates (Mache-
rey-Nagel Polygram SIL G/UV254) were used, and compounds were
viewed by irradiation with UV light and/or by pulverizing of a solu-
tion of ethanol/anisaldehyde/sulfuric acid (v/v/v: 90:5:5), each fol-
lowed by heating. The HPLC chain consists of a pump (Waters 600)
and an UV detector (PDA Waters 996), (200 and 400 nm). The ana-
lytical HPLC analyses were performed on a XTerra RP18 column
Under a nitrogen atmosphere, the starting material was stirred
for 30 min in a mixture of TFA/CH2Cl2 (1:1 v/v) (approximately
0.1 M for starting material). The solvents were concentrated in a
vacuum and the residue was diluted with saturated Na2CO3 (5 v).
The desired product was extracted with CH2Cl2 and the organic
phase was dried (MgSO4), filtered and concentrated in a vacuum
to produce an oil which was used without further purification
4.5. General procedure for glycopeptide solid phase synthesis
(Procedure E)
(4.6 ꢃ 150 mm, 3.5
tions were performed with a semi-preparative XTerra RP18 col-
umn (7.8 ꢃ 300 mm, 7 m). Preparative HPLC was performed at a
lm) whereas the semi-preparative purifica-
The glycopeptides were prepared by the solid phase peptide
l
synthesis strategy on
a PS3 automated peptide synthesizer

7428
F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
(Protein Technologies, Inc., Tucson, AZ) using N-
oxycarbonyl (Fmoc)-based chemistry on Wang-Fmoc-
resin or Wang-Fmoc- -Ala resin. These resins and N-
a
-fluorenylmeth-
-Lys(Boc)
-Fmoc
ArH-amide, J = 8.9), 7.25 (2H, d, ArH PMB, J = 8.6), 6.91 (2H, d,
ArH PMB, J = 8.6), 6.09 (1H, d, NH NHAc, J = 8.0), 5.51 (1H, s,
H-1), 4.67–4.62 (2H, m, –CH2-phenyl and H-5), 4.54 (1H, d,
–CH2-phenyl, J = 11.6), 4.29–4.24 (2H, m, H-lactyl and H-6endo),
4.12 (1H, d, H-2, J = 8.0), 3.89 (1H, dd, H-6exo, J = 5.0 and 7.0),
3.82 (3H, s, –OCH3), 3.55 (1H, s, H-3), 3.48 (1H, s, H-4), 1.95
(3H, s, CH3 NHAc), 1.43 (3H, d, lactyl-CH3, J = 6.6). 13C NMR
L
D
a
amino acids (0.4 mmol) were purchased from Iris Biotech GmbH,
Marktredwitz, Germany. The side chain of Glu was protected
with the t-butyl derivative (tBu). Compound 2 (0.4 mmol) was
used as an amino acid. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetram-
ethyluronium hexafluorophosphate (Iris Biotech GmbH)
(0.4 mmol) and N-methylmorpholine 0.4 M in N,N-dimethylform-
amide (DMF) (3 ml, 0.4 mmol) were used as coupling and acti-
vating reagents, respectively. Fmoc deprotection at each step
was carried out using 20% piperidine/DMF (12 ml). The glycopep-
tides attached to the resin beads were washed subsequently
with DMF, EtOH, and CH2Cl2. The side-chain protecting groups
were removed, and the glycopeptides were cleaved from the re-
sin using TFA/anisole/water (6 ml; 10:1:1) for 3 h at room tem-
perature. The uncharged resin was separated from the solution
by filtration. The crude glycopeptides were precipitated with
Et2O and used directly in the next step.
(CDCl3, 100 MHz)
d ppm 170.9, 169.7, 159.8, 141.3, 129.5,
126.5 (7), 119.2 (2), 114.2 (2), 100.8, 76.1, 75.5, 74.4 (2), 71.5,
65.7, 55.4, 47.3, 23.2, 17.2. HPLC (CH3CN/H2O: 60:40, 0.7
ml/min): tR = 7.5 min. MS (ES+): m/z = 539 (M+1). MS (ESꢂ):
m/z = 597 (adduct HAc).
4.9. 2-Acetamido-1,6-anhydro-2-deoxy-3-O-{[(2R)-N-phenyl-
propionamide]-2-yl}-b-D-glucopyranose (6)
Catalytic Pd/C (10%) (45 mg) was added to a solution of 3
(35.8 mg, 0.08 mmol) in MeOH (20 ml). A hydrogen atmosphere
(two bars) was maintained for 6 h. The mixture was filtered
through Celite, and the catalyst was washed with hot MeOH. After
concentration of the filtrate, the residual colourless oil was crystal-
lized under cooling to afford 6 as a white solid (12% yield). 1H NMR
(CDCl3, 400 MHz) d ppm 9.34 (1H, s, NH-amide), 7.67 (2H, d, ArH-
amide, J = 7.6), 7.29 (2H, t, ArH-amide, J = 7.6), 7.08 (1H, t, ArH-
amide, J = 7.6), 6.43 (1H, d, NH NHAc, J = 8.0), 5.51 (1H, s, H-1),
4.58 (1H, d, H-5, J = 5.0), 4.30 (1H, d, H-6endo, J = 7.0), 4.28–4.23
(1H, q, H-lactyl, J = 6.6), 4.10 (1H, d, H-2, J = 8.0), 3.86 (1H, dd, H-
4.6. 2-Acetamido-1,6-anhydro-4-O-benzyl-2-deoxy-3-O-{[(2R)-
N-phenylpropionamide]-2-yl}-b-D-glucopyranose (3)
Compound 12 and aniline were coupled according to Proce-
dure A (DMF was replaced by CH2Cl2). Purification by prepara-
tive HPLC (petroleum/AcOEt: 34:66–0:100) afforded
3 as a
white solid (50% yield). TLC (AcOEt/petroleum: 2:1): Rf = 0.4. 1H
NMR (CDCl3, 400 MHz) d ppm 9.32 (1H, s, NH-amide), 7.67–
7.65 (2H, m, ArH-amide), 7.39–7.29 (7H, m, ArH-amide and
ArH PMB), 7.11–7.08 (1H, m, ArH PMB), 6.05 (1H, d, NH NHAc,
J = 8.0), 5.51 (1H, s, H-1), 4.73–4.70 (2H, m, H-5 and –CH2-phe-
6exo, J = 5.0 and 7.0), 3.82–3.73 (2H, m, H-4 and –OH), 3.51 (1H, s,
H-3), 1.98 (3H, s, CH3 NHAc), 1.45 (3H, d, lactyl-CH3, J = 6.6). HPLC
(CH3CN/H2O: 40:60, 0.7 ml/min): tR = 3 min. MS (ES+): m/z = 373
(adduct Na+), 414 (adduct CH3CN + Na+).
nyl), 4.63 (1H, d, –CH2-phenyl, J = 11.6), 4.31 (1H, d, H-6endo
,
J = 7.0), 4.23 (1H, q, H-lactyl, J = 6.6), 4.12 (1H, d, H-2, J = 8.0),
3.89 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.49 (1H, s, H-3), 3.27
(1H, s, H-4), 1.94 (3H, s, CH3 NHAc), 1.43 (3H, d, lactyl-CH3,
J = 6.6). HPLC (CH3CN/H2O: 40:60, 0.7 ml/min): tR = 20 min. MS
(ES+): m/z = 441 (M+1), 463 (adduct Na+).
4.10. 2-Acetamido-3-O-{[(2R)-N-4-acetylphenylpropionamide]-
2-yl}-1,6-anhydro-2-deoxy-b-D-glucopyranose (7)
Compound 4 was deprotected according to Procedure C. Purifi-
cation by semi-preparative reverse phase HPLC (CH3CN/H2O:
40:60) produced 7 as a white solid (20% yield). 1H NMR (CDCl3,
400 MHz) d ppm 9.52 (1H, s, NH-amide), 7.91 (2H, d, ArH-amide,
J = 8.9), 7.78 (2H, d, ArH-amide, J = 8.9), 6.33 (1H, d, NH NHAc,
J = 8.0), 5.54 (1H, s, H-1), 4.62 (1H, d, H-5, J = 5.0), 4.35–4.29 (2H,
m, H-lactyl and H-6endo), 4.10 (1H, d, H-2, J = 8.0), 3.89 (1H, dd,
H-6exo, J = 5.0 and 7.0), 3.82–3.73 (2H, m, H-4 and –OH), 3.53
(1H, s, H-3), 2.57 (3H, s, CH3–CO-Ar), 1.99 (3H, s, CH3 NHAc),
1.48 (3H, d, lactyl-CH3, J = 6.6). HPLC (CH3CN/H2O: 40:60, 05 ml/
min): tR = 4.5 min. MS (ES+): m/z = 393 (M+1). MS (ESꢂ): m/
z = 451 (adduct HAc).
4.7. 2-Acetamido-3-O-{[(2R)-N-4-acetylphenylpropionamide]-
2-yl}-1,6-anhydro-2-deoxy-4-O-p-methoxybenzyl-b-D-
glucopyranose (4)
Compound 2 and p-aminoacetophenone were coupled accord-
ing to Procedure A. Purification by preparative HPLC (AcOEt)
afforded 4 as a white solid (25% yield). TLC (AcOEt): Rf = 0.6.
Mp: 145 °C. 1H NMR (CDCl3, 400 MHz) d ppm 9.58 (1H, s, NH-
amide), 7.91 (2H, d, ArH-amide, J = 8.9), 7.78 (2H, d, ArH-amide,
J = 8.9), 7.25 (2H, d, ArH PMB, J = 8.6), 6.91 (2H, d, ArH PMB,
J = 8.6), 6.09 (1H, d, NH NHAc, J = 8.0), 5.52 (1H, s, H-1), 4.69–
4.62 (2H, m, –CH2-phenyl and H-5), 4.57 (1H, d, –CH2-phenyl,
J = 11.6), 4.30 (2H, m, H-6endo and H-lactyl), 4.10 (1H, d, H-2,
J = 8.0), 3.92 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.55 (1H, s, H-
3), 3.48 (1H, s, H-4), 2.57 (3H, s, CH3–CO-Ar), 1.94 (3H, s, CH3
NHAc), 1.43 (3H, d, lactyl-CH3, J = 6.6). HPLC (CH3CN/H2O:
40:60, 0.5 ml/min): tR = 18.0 min. MS (ES+): m/z = 513 (M+1).
MS (ESꢂ): m/z = 511 (Mꢂ1).
4.11. 2-Acetamido-1,6-anhydro-2-deoxy-3-O-{[(2R)-N-4-(2,2,2-
trifluoroacetyl)phenylpropionamide]-2-yl}-b-D-glucopyranose
(8)
Compound 5 was deprotected according to Procedure C. Purifi-
cation by semi-preparative reverse phase HPLC (CH3CN/H2O:
30:70) produced 8 as a white solid (31% yield). 1H NMR (CDCl3,
400 MHz) d ppm 9.55 (1H, s, NH-amide), 7.78 (2H, d, ArH-amide,
J = 8.9), 7.54 (2H, d, ArH-amide, J = 8.9), 6.24 (1H, d, NH NHAc,
J = 8.0), 5.54 (1H, s, H-1), 4.62 (1H, d, H-5, J = 5.0), 4.35–4.29 (2H,
m, H-lactyl and H-6endo), 4.11 (1H, d, H-2, J = 8.0), 3.89 (1H, dd,
H-6exo, J = 5.0 and 7.0), 3.82–3.73 (2H, m, H-4 and –OH), 3.54
(1H, s, H-3), 2.00 (3H, s, CH3 NHAc), 1.48 (3H, d, lactyl-CH3,
J = 6.6). 13C NMR (CDCl3, 100 MHz) d ppm 171.5, 170.4, 141.3,
126.2 (4), 119.3 (2), 100.1, 79.0, 76.5, 76.2, 69.8, 65.1, 47.7, 23.1,
17.5. HPLC (CH3CN/H2O: 60:40, 0.7 ml/min): tR = 3.6 min. MS
(ES+): m/z = 419 (M+1). MS (ESꢂ): m/z = 415 (Mꢂ1), 477 (adduct
HAc).
4.8. 2-Acetamido-1,6-anhydro-4-O-benzyl-2-deoxy-3-O-{[(2R)-
N-4-(2,2,2-trifluoroacetyl)phenylpropionamide]-2-yl}-b-D-
glucopyranose (5)
Compound
2 and p-trifluoromethylaniline were coupled
according to Procedure A. Purification by preparative HPLC
(AcOEt) afforded 5 as a white solid (40% yield). TLC (AcOEt):
Rf = 0.77. Mp: 154 °C. 1H NMR (CDCl3, 400 MHz) d ppm 9.56
(1H, s, NH-amide), 7.78 (2H, d, ArH-amide, J = 8.9), 7.54 (2H, d,

F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
7429
4.12. 2-Acetamido-1,6-anhydro-2-deoxy-4-O-p-methoxybenzyl-
4.17. a-Benzyl-N-a-L-alanyl-N-d-phenyl-D-glutaminate (17)
3-O-{(2R)-propionyl-[L-alanine-p-nitroanilide]-2-yl}-b-D-gluco-
pyranose (10)
Compound 16 was deprotected according to Procedure D to
produce 17 (74% yield). TLC (CH2Cl2/AcOEt: 1:1) Rf = 0.05. 1H
NMR (CDCl3, 400 MHz) d ppm 8.66 (1H, s, NH–C6H5), 8.15 (1H, d,
NH-Glu, J = 7.0), 7.60–7.07 (10H, m, ArH), 5.22–5.12 (2H, d, CH2
Bn, J = 11.6), 4.66 (1H, d, H-Glu, J = 7.0), 3.53 (1H, m, H-Ala), 2.36
(2H, m, CH–CH2-Glu), 2.02 (2H, m, CH–CH2–CH2-Glu), 1.37 (3H,
d, –CH3 Ala, J = 7.0). MS (ES+): m/z = 384 (M+1). MS (ESꢂ):
m/z = 382 (Mꢂ1), 442 (adduct HAc).
Compound 2 and L-Ala-p-nitroanilide hydrochloride 9 were
coupled according to Procedure A. Purification by preparative HPLC
(AcOEt/MeOH: 9:1) produced 10 as a white solid (73% yield). TLC
(AcOEt/MeOH: 9:1): Rf = 0.7. Mp: 61.6 °C. 1H NMR (CDCl3,
400 MHz) d ppm 9.65 (1H, s, NH-Ar), 8.16 (2H, d, ArH, J = 9.2),
8.04 (1H, d, NH-Ala, J = 6.3), 7.68 (2H, d, ArH, J = 9.2), 7.26 (2H, d,
ArH, J = 8.6), 6.90 (2H, d, ArH, J = 8.6), 6.09 (1H, d, NH NHAc,
J = 8.0), 5.43 (1H, s, H-1), 4.62–4.59 (3H, m, 1H CH2-phenyl, H-Ala
and H-5), 4.53 (1H, d, 1H CH2-phenyl, J = 11.6), 4.17–4.15 (2H, m,
H-lactyl and H-6endo), 4.03 (1H, d, H-2, J = 8.0), 3.81 (3H, s,
–OCH3), 3.79 (1H, m, H-6exo), 3.48 (1H, s, H-3), 3.40 (1H, s, H-4),
1.96 (3H, s, CH3 NHAc), 1.48 (3H, d, –CH3 Ala, J = 7.0), 1.37 (3H, d,
lactyl-CH3, J = 6.6). 13C NMR (CDCl3, 100 MHz) d ppm 174.0,
171.1, 169.9, 159.9, 144.4, 143.4, 129.5 (2), 129.1, 124.9 (2),
119.2 (2), 114.3 (2), 100.6, 76.4, 75.7, 75.6, 74.3, 71.5, 65.3, 55.4,
50.0, 47.4, 23.2, 17.7, 16.7. HPLC (CH3CN/H2O: 60:40, 0.7 ml/
min): tR = 4.3 min. MS (ESꢂ): m/z = 585 (Mꢂ1), 645 (adduct HAc).
4.18. 2-Acetamido-1,6-anhydro-2-deoxy-4-O-p-methoxybenzyl-
3-O-{(2R)-propionyl-[
L-alanyl-(a-benzyl-N-d-phenyl-glutamin-
ate)]-2-yl}-b- -glucopyranose (18)
D
Compounds 17 and 2 were coupled according to Procedure B to
produce 18 as a white solid. However, PyBOP was used instead of
TBTU (51% yield). TLC (AcOEt/MeOH: 10:1): Rf = 0.55. 1H NMR
(CDCl3, 400 MHz) d ppm 8.66 (1H, s, NH-C6H5), 7.96 (1H, d, NH-
Ala, J = 6.3), 7.57–7.55 (2H, m, ArH), 7.31–7.22 (10H, m, ArH), 6.95
(1H, d, NH-Glu, J = 7.0), 6.90 (2H, d, ArH PMB, J = 8.6), 6.06 (1H, d,
NH NHAc, J = 8.0), 5.39 (1H, s, H-1), 5.22–5.12 (2H, d, CH2 Bn,
J = 11.6), 4.61 (1H, d, H-Glu, J = 7.0), 4.60 (1H, d, 1H CH2 PMB,
J = 11.6), 4.57 (1H, d, H-5, J = 5.0), 4.51 (1H, d, 1H CH2 PMB,
J = 11.6), 4.39 (1H, m, H-Ala), 4.17 (1H, d, H-6endo, J = 7.0), 4.11 (1H,
q, H-lactyl, J = 6.6), 4.01 (1H, d, H-2, J = 8.0), 3.79 (3H, s, CH3 PMB),
3.72 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.46 (1H, s, H-3), 3.39 (1H, s,
H-4), 2.35–2.30 (4H, m, 2 ꢃ CH2 Glu), 1.92 (3H, s, CH3 NHAc), 1.37
(3H, d, –CH3 Ala, J = 7.0), 1.31 (3H, d, lactyl-CH3, J = 6.6). 13C NMR
(CDCl3, 100 MHz) d ppm 174.4, 173.4, 172.5, 171.2, 170.6, 159.0,
139.1, 136.0, 130.2 (2), 129.9–129.0 (7), 124.8, 120.7 (2), 115.0 (2),
101.4, 77.2, 76.8, 76.3, 75.1, 72.3, 68.4, 66.0, 56.3, 52.4, 50.6, 48.2,
34.2, 29.9, 24.1, 18.7, 18.0. HPLC (CH3CN/H2O: 60:40, 0.7 ml/min):
tR = 6.7 min. MS (ES+): m/z = 761 (M+1), 783 (adduct Na+). MS
(ESꢂ): m/z = 759 (Mꢂ1), 819 (adduct HAc).
4.13. 2-Acetamido-1,6-anhydro-2-deoxy-3-O-{(2R)-propionyl-
[
L-alanine-p-nitroanilide]-2-yl}-b-
D-glucopyranose (11)
Compound 10 was deprotected according to Procedure C. How-
ever, the reaction was stirred for 48 h. Purification by semi-pre-
parative reverse phase HPLC (CH3CN/H2O: 40:60) produced 11 as
a white solid (10% yield). 1H NMR (CDCl3, 400 MHz) d ppm 9.66
(1H, s, NH-Ar), 8.16 (2H, d, ArH, J = 8.6), 8.06 (1H, d, NH-Ala,
J = 6.3), 7.69 (2H, d, ArH, J = 8.6), 6.25 (1H, d, NH NHAc, J = 8.0),
5.46 (1H, s, H-1), 4.60–4.58 (2H, m, H-Ala and H-5), 4.25–4.23
(2H, m, H-lactyl and H-6endo), 4.02 (1H, d, H-2, J = 8.0), 3.79–3,49
(3H, m, H-6exo and H-4 and OH), 3.07 (1H, s, H-3), 2.01 (3H, s,
CH3 NHAc), 1.48 (3H, d, –CH3 Ala, J = 7.0), 1.40 (3H, d, lactyl-CH3,
J = 6.9). HPLC (CH3CN/H2O: 40:60, 0.5 ml/min): tR = 5.4 min. MS
(ES+): m/z = 467 (M+1). MS (ESꢂ): m/z = 465 (Mꢂ1).
4.19. 2-Acetamido-1,6-anhydro-2-deoxy-4-O-p-methoxybenzyl-
3-O-{(2R)-propionyl-[
L-alanyl-(N-d-phenyl-glutaminate)]-2-yl}-
4.14.
a
-Benzyl-N-(tert-butyloxycarbonyl)-N-d-phenyl-
D-
b-D-glucopyranose (19)
glutamine (13)
Catalytic Pd/C (5%, wet) (120 mg) was added to a stirred solution
Compound 12 and aniline were coupled according to Procedure
B. Purification by preparative HPLC (CH2Cl2/AcOEt: 1:1) produced
13 as a white solid (75% yield). The spectroscopic data were iden-
tical to these reported previously.16
of 18 (496 mg, 0.65 mmol) in EtOH (25 ml). A hydrogen atmosphere
(2.5 bars) was maintained for 6 h. The mixture was filtered through
Celite, and the catalyst was washed with hot EtOH. The filtrate was
concentrated in a vacuum to yield 19 as a colourless oil (68% yield).
TLC (AcOEt/MeOH + HCOOH: 9:1): Rf = 0.30. 1H NMR (CDCl3,
400 MHz) d ppm 8.84 (1H, s, NH-C6H5), 7.96 (1H, d, NH-Ala,
J = 6.3), 7.57–7.55 (2H, m, ArH), 7.31–7.22 (5H, m, ArH), 7.03 (1H,
d, NH-Glu, J = 7.0), 6.86 (2H, d, ArH, J = 8.6), 6.10 (1H, d, NH NHAc,
J = 8.0), 5.36 (1H, s, H-1), 4.55–4.46 (4H, m, H-Glu, H-5 and 2H
CH2 PMB), 4.36 (1H, m, H-Ala), 4.16 (1H, d, H-6endo, J = 7.0), 4.07
(1H, q, H-lactyl, J = 6.6), 3.96 (1H, d, H-2, J = 8,0), 3.78 (3H, s, CH3
PMB), 3.69 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.43 (1H, s, H-3), 3.38
(1H, s, H-4), 2.41–2.29 (4H, m, 2 ꢃ CH2 Glu), 1.92 (3H, s, CH3 NHAc),
1.37 (3H, d, –CH3 Ala, J = 7,0), 1.30 (3H, d, lactyl-CH3, J = 6.6). HPLC
(CH3CN/H2O: 60:40, 0.7 ml/min): tR = 2.1 min. MS (ES+): m/z = 671
(M+1). MS (ESꢂ): m/z = 669 (Mꢂ1).
4.15. a-Benzyl-N-d-phenyl-D-glutamine (14)
Compound 13 was deprotected according to Procedure D (71%
yield). The spectroscopic data were identical to these reported
previously.17
4.16.
a-Benzyl-N-a-(N-tert-butyloxycarbonyl-L-alanyl)-N-d-
phenyl-
D
-glutaminate (16)
Compound 14 and NHBOC-L-Ala 15 (commercially available
from IRIS) were coupled according to Procedure B, and yielded
16 as a white solid (75% yield). Mp: 129 °C. TLC (CH2Cl2/AcOEt:
1:1): Rf = 0.56. 1H NMR (CDCl3, 400 MHz) d ppm 8.42 (1H, s, NH-
C6H5), 7.58–7.29 (10H, m, ArH), 7.07 (1H, d, NH BOC, J = 4.3), 6.99
(1H, d, NH-Glu, J = 7.0), 5.15 (2H, d, CH2 Bn, J = 11.6), 4.66 (1H, d,
H-Glu, J = 7.0), 4.17 (1H, m, H-Ala), 2.36 (2H, m, CH–CH2-Glu),
1.95 (2H, m, CH–CH2–CH2-Glu), 1.45 (9H, s, –C(CH3)3), 1.37 (3H,
d, –CH3 Ala, J = 7.0). HPLC (CH3CN/H2O: 60:40, 0.7 ml/min):
tR = 6.7 min. MS (ES+): m/z = 484 (M+1). MS (ESꢂ): m/z = 482
(Mꢂ1), 542 (adduct HAc).
4.20. 2-Acetamido-1,6-anhydro-2-deoxy-3-O-{(2R)-propionyl-
[L
-alanyl-(N-d-phenyl-glutaminate)]-2-yl}-b-D-glucopyranose
(20)
Compound 19 was deprotected according to Procedure C. Puri-
fication by preparative HPLC (AcOEt/MeOH + HCOOH 0.1%: 82:18)
followed by a semi-preparative reverse phase HPLC (CH3CN/H2O:
20:80) yielded 20 as a white solid (10% yield). TLC (AcOEt/MeOH:

7430
F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
9:2): Rf = 0.45. 1H NMR (CDCl3, 400 MHz) d ppm 9.21 (1H, s, NH-
C6H5), 7.96 (1H, d, NH-Ala, J = 6.3), 7.47–7.20 (5H, m, ArH), 6.98
(1H, d, NH-Glu, J = 7.0), 6.06 (1H, d, NH NHAc, J = 8.0), 5.32 (1H,
s, H-1), 4.46 (2H, m, H-Glu and H-5), 4.30 (1H, m, H-Ala), 4.15
(1H, d, H-6endo, J = 7.0), 4.06 (1H, d, H-lactyl, J = 6.6), 3.89 (1H, d,
H-2, J = 8,0), 3.70 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.61–3.49 (2H,
m, H-4 and –OH), 3.32 (1H, s, H-3), 2.27 (4H, m, 2 ꢃ CH2 Glu),
1.90 (3H, s, CH3 NHAc), 1.34 (3H, d, lactyl-CH3, J = 6.6), 1.28 (3H,
d, –CH3 Ala, J = 7,0). HPLC (CH3CN/H2O: 20:80, 0.7 ml/min):
tR = 5.5 min. MS (ES+): m/z = 551 (M+1), 573 (adduct Na+). MS
(ESꢂ): m/z = 549 (Mꢂ1).
3.47 (1H, s, H-3), 3.42 (1H, s, H-4), 3.14 (2H, m, –CH2-NHZ Lys),
2.30–2.17 (4H, m, 2 ꢃ –CH2-Glu), 1.93 (3H, s, CH3 NHAc), 1.83–
1.75 (2H, m, –CH–CH2-Lys), 1.49 (2H, m, –CH2–CH2-NHZ Lys),
1.38–1.33 (8H, m, –CH3 Ala, CH2–CH2–CH2–NHZ Lys and CH3-lac-
tyl). 13C NMR (CDCl3, 100 MHz) d ppm 173.1–169.8 (6), 159.6,
156.5, 136.6, 135.4 (3), 129.9–127.8 (17), 114.2 (2), 100.4, 76.1,
75.7, 75.5, 74.1, 71.3, 67.1, 67.0, 66.4, 65.1, 55.8, 52.4, 51.6, 49.1,
47.3, 40.5, 31.7, 31.0, 29.3, 26.9, 23.1, 22.6, 17.7, 17.1.
HPLC (CH3CN/H2O: 75:25, 0.7 ml/min): tR = 5.1 min. MS (ES+):
m/z = 1039 (M+1), 1061 (adduct Na+).
4.24. N-Acetyl-1,6-anhydromuramyl-L-ala-c-D-Glu-L-Lys (27)
4.21. Benzyl N-(N-tert-butyloxycarbonyl-O-benzyl-
c-
D
-
(homogenate phase)
glutamyl)-N- -benzyloxycarbonyl- -lysine (22)
e
L
Catalytic Pd/C (10%) (30 mg) was added to a stirred solution of
26 (118 mg, 0.11 mmol) in MeOH (30 ml). A hydrogen atmosphere
(six bars) was maintained for 72 h at 35 °C. The mixture was fil-
tered through Celite, and the catalyst was washed with hot MeOH.
The filtrate was concentrated in a vacuum and the residue was
purified by semi-preparative reverse phase HPLC (CH3CN/H2O:
50:50) to produce 27 as a white solid (60% yield). 1H NMR (D2O,
400 MHz) d ppm (NH2, 2 ꢃ COOH, 4 ꢃ NH–, OH-4 not detected)
5.40 (1H, s, H-1), 4.60 (1H, d, H-5, J = 5.0), 4.29 (1H, m, H-Ala),
4.22–4.08 (4H, m, H-lactyl, H-6endo, H-Lys and H-Glu), 3.84 (1H,
s, H-2), 3.79 (1H, s, H-4), 3.72 (1H, dd, H-6exo, J = 5.0 and 7.0),
3.34 (1H, s, H-3), 2.90 (2H, m, –CH2-NH2 Lys), 2.40–2.07 (4H, m,
2 ꢃ –CH2-Glu), 1.94 (3H, s, CH3 NHAc), 1.78–1.58 (4H, m, –CH–
CH2-Lys and –CH2–CH2-NHZ Lys), 1.35–1.29 (8H, m, –CH3 Ala,
CH2–CH2–CH2-NHZ Lys and CH3-lactyl). HPLC (CH3CN/H2O:
50:50, 0.7 ml/min): tR = 2.5 min. MS (ES+): m/z = 604 (M+1).
Compound 21 H-Lys(Z)-OBzlꢀHCl (commercially available from
IRIS) and 12 were coupled according to Procedure B, and produced
22 as a white solid after crystallization in CHCl3–petroleum (98%
yield). TLC (petroleum/AcOEt: 1:1): Rf = 0.4. Mp: 109 °C. 1H NMR
(CDCl3, 400 MHz) d ppm 7.36–7.24 (15H, s, ArH), 6.73 (1H, d,
NH-Lys, J = 7.0), 5.33 (1H, d, NHBOC, J = 4.3), 5.20 (6H, m, 3 ꢃ
–CH2-Bn), 4.88 (1H, s, –NHZ), 4.61 (1H, d, H-Lys, J = 7.0), 4.43
(1H, d, H-Glu, J = 7.0), 3.14 (2H, d, –CH2-NHZ, J = 5.0), 2.31–1.95
(4H, m, 2 ꢃ –CH2-Glu), 1.86–1.67 (2H, m, –CH–CH2-Lys), 1.55–1.25
(13H, m, –CH–CH2–CH2–CH2–CH2-NHZ and –C(CH3)3). 13C NMR
(CDCl3, 100 MHz) d ppm 172.1 (2), 156.6, 155.9, 136.6, 135.4,
135.2, 128–110 (15), 80.1, 67.2, 67.0, 66.5, 52.9, 52.2, 40.5, 32.2,
29.3, 28.9, 28.3 (3), 27.7, 22.3. HPLC (CH3CN/H2O: 60:40, 0.7
ml/min): tR = 16 min. MS (ES+): m/z = 690 (M+1), 712 (adduct Na+).
4.22. Benzyl N-((N-tert-butyloxycarbonyl-L-alanyl)-O-benzyl-c-
D
-glutamyl)-N- -benzyloxycarbonyl- -lysine (24)
e
L
4.25. N-Acetyl-1,6-anhydromuramyl-L-ala-c-D-Glu-L-Lys (27)
(on solid support)
Compound 22 was deprotected according to Procedure D and
coupled with NHBOC- -Ala 15 according to Procedure B to produce
L
The glycopeptide was produced according to General Procedure
E and the final deprotection was performed according to Sec-
tion 4.24 (above) with a global yield of 40%. For spectroscopy data
see Section 4.24.
24 as a white solid after crystallization in CHCl3–petroleum (60%
yield). Mp: 115 °C. 1H NMR (CDCl3, 400 MHz) d ppm 7.35 (17H, s,
ArH, NH-Lys and NH-Glu), 5.21–5.09 (7H, m, 3 ꢃ –CH2-Bn and
NH-CBZ), 5.01 (1H, d, NH-Ala, J = 6.3), 4,56 (2H, d, H-Lys and H-
Glu, J = 7.0), 4.11 (1H, m, H-Ala), 3.20–3.08 (2H, m, –CH2-NHZ
Lys), 2.25–2.06 (4H, m, 2 ꢃ –CH2-Glu), 1.83–1.75 (2H, m,
–CH–CH2-Lys), 1.53 (2H, m, –CH2–CH2-NHZ Lys), 1.46 (9H, s,
–C(CH3)3), 1.34 (3H, d, –CH3 Ala, J = 7.0), 1.29 (2H, m, –CH2–CH2–
CH2-NHZ Lys). 13C NMR (CDCl3, 100 MHz) d ppm 173.5, 172.6,
171.8, 171.3, 156.5, 155.6, 136.5, 135.3, 135.2, 128.5, 128.1 (15),
80.2, 67.2, 67.1, 66.6, 53.8, 52.4, 51.6, 40.4, 31.8, 30.9, 29.2, 28.2
(3), 27.7, 22.5, 17.9. HPLC (CH3CN/H2O: 75:25, 0.7 ml/min):
tR = 6.0 min. MS (ES+): m/z = 761 (M+1), 783 (adduct Na+).
4.26. N-Acetyl-1,6-anhydromuramyl-L-ala-c-D-Glu-[N-e-(2,4,6-
trinitrophenyl)- -lysine] (28)
L
TNBSA (1 M in MeOH, Aldrich) (200 ll, 0.2 mmol) was added to
a stirred solution of 27 (49 mg, 0.081 mmol) at 37 °C in a carbonate
buffer (0.1 N) to pH 9.5. After 1.5 h, a novel addition of TNBSA was
performed (200 ll, 0.2 mmol) and the mixture was stirred for 1.5 h
again. The solvent was concentrated in a vacuum and the residue
was purified by semi-preparative reverse phase HPLC (CH3CN/
H2O: 50:50) to produce 28 as a red solid (97% yield). 1H NMR
(MeOD, 400 MHz) d ppm (2 ꢃ COOH, 5 ꢃ NH-, OH-4 not detected)
9.14 (2H, s, ArH), 5.49 (1H, s, H-1), 4.69 (1H, d, H-5, J = 5.0), 4.39–
4.12 (5H, m, H-lactyl, H-6endo, H-Lys, H-Glu and H-Ala), 3.93 (1H, s,
H-2), 3.89 (1H, s, H-4), 3.82 (1H, dd, H-6exo, J = 5.0 and 7.0), 3.44
(1H, s, H-3), 3.12 (2H, s, –CH2-NH2 Lys), 2.40–2.11 (4H, m, 2 ꢃ
–CH2-Glu), 2.02 (3H, s, CH3 NHAc), 1.98–1.58 (4H, m, –CH–CH2-
Lys and –CH2–CH2-NHZ Lys), 1.43–1.29 (8H, m, –CH3 Ala,
CH2–CH2–CH2-NHZ Lys and CH3-lactyl). HPLC (CH3CN/H2O:
50:50, 0.7 ml/min): tR = 2.5 min. MS (ES+): m/z = 815 (M+1). MS
(ESꢂ): m/z = 813 (Mꢂ1).
4.23. 2-Acetamido-1,6-anhydro-2-deoxy-4-O-p-methoxybenzyl-
3-O-{(2R)-propionyl-[
-benzyloxycarbonyl-
glucopyranose (26)
L
-alanyl-(
c
-
D
-glutamyl
a-benzyl ester)-(N-
e
L
-lysine
a
-benzyl ester)]-2-yl}-b-D-
Compound 24 was deprotected according to Procedure D and
coupled with 2 according to Procedure B to produce 26 as a white
solid after purification by manually chromatography (AcOEt/
MeOH: 9:1) (18% yield). TLC (AcOEt/MeOH: 9:1): Rf = 0.64. 1H
NMR (CDCl3, 400 MHz) d ppm 7.90 (1H, d, NH-Glu, J = 7.0), 7.51
(1H, d, NH-Lys, J = 7.0), 7.35–7.29 (15H, s, ArH); 7,28 (1H, d, NH-
Ala, J = 6.3), 7.28 (2H, d, ArH PMB, J = 8.6), 6.93 (2H, d, ArH PMB,
J = 8.6), 6.10 (1H, d, NH NHAc, J = 8.0), 5.40 (1H, s, H-1), 5.21–
5.09 (7H, m, 3 ꢃ –CH2-Bn and NH-CBZ), 4.63 (3H, m, H-5 and
CH2-PMB), 4.53 (1H, d, H-Lys, J = 7.0), 4.32 (2H, m, H-Ala and H-
Glu), 4.16–4.09 (2H, m, H-6endo and H-lactyl), 4.02 (1H, d, H-2,
J = 8.0), 3.83 (3H, s, –OMe), 3.76 (1H, dd, H-6exo, J = 5.0 and 7.0),
4.27. N-Acetyl-1,6-anhydromuramyl-L-ala-c-D-Glu-meso-DAP-D-
Ala trifluoroacetate salt (29)
The glycopeptide was produced according to General Procedure
E and the final deprotection was performed as follows. Catalytic
Pd/C (10%) (10 mg) was added to a stirred solution of previously

F. Mercier et al. / Bioorg. Med. Chem. 18 (2010) 7422–7431
7431
synthesized glycopeptide (63 mg, 6.69 10ꢂ2 mmol) in THF (15 ml)
and 0.1% of TFA. A hydrogen atmosphere (six bars) was maintained
for 48 h at rt. The mixture was filtered through Celite, and the cat-
alyst was washed with hot THF. The filtrate was concentrated in a
vacuum and the residue was precipitated in a mixture of TFA/Et2O
and collected by centrifugation (51% yield). 1H NMR (D2O,
400 MHz) d ppm: 5.46 (1H, s, H-1), 4.65 (1H, d, J = 6.0, H-5), 4.35
(3H, m, 2 ꢃ H-Ala, H-DAP), 4.25 (2H, m, H-6endo, H-DAP), 4.15
(1H, m, H-lactyl), 3.97 (1H, t, J = 6.0, H-Glu), 3.90 (1H, s, H-2),
3.84 (1H, s, H-4), 3.78 (1H, m, H-6exo), 3.39 (1H, s, H-3), 2.36 (2H,
m, COCH2-Glu), 2.23 (2H, m, COCH2CH2-Glu), 1.72–1.98 (7H, m,
CH3 NHAc, CH2CH2CH2 DAP), 1.47 (2H, m, CH2CH2CH2 DAP), 1.40
activity of sAmiD. The compounds (100
l
M) were incubated in
the presence of 39 nM sAmiD in 20 mM sodium phosphate buffer
containing 48 nM ZnCl2 at 30 °C for 20 min (total volume: 94 l).
The residual activity of sAmiD was determined by adding 6
TCT (0.5 mM). The hydrolysis was stopped after 20 min by addition
of 10 l H3PO4 (1:100 dilution with water). One hundred microli-
l
ll
l
tres of the solutions were injected onto the HPLC column and ana-
lysed as described previously.
Furthermore, compounds 1, 6, 7, 8, 11 (1 mM) were incubated
in the presence of 3.9
fer containing 48 nM ZnCl2 at 30 °C for 30 min (volume total:
50 l). The residual activity of sAmiD was determined by adding
1 mM 20. The hydrolysis of 20 was stopped after 2 h and the solu-
tion (50 l) was analysed by HPLC as described previously.
lM sAmiD in 20 mM sodium phosphate buf-
l
(m, 9H, CH3-lactyl, CH3-L-Ala, CH3-D
-Ala). RMN 13C (D2O,
100 MHz) d: 173.6–176.8 (8), 100.5, 79.1, 76.5, 76.4, 68.6, 65.7,
54.2, 53.6, 52.4, 50.1, 50.0, 49.2, 31.9, 31.1, 30.8, 27.2, 22.3, 21.3,
18.6, 17.4, 16.65. MS (ES+): m/z = 719 (M+1).
l
Acknowledgments
4.28. HPLC analysis for biological experiments
We thank Dr. D. Mengin-Lecreulx (University of Paris-Sud,
France) for the preparation of TCT. This research was supported
in part by the Belgian Program on Interuniversity Poles of Attrac-
tion initiated by the Belgian state (grants P5/33 and P6/19), the Ac-
tions de Recherche Concertées (grant 03/08-297), the Fonds de la
Recherche Fondamentale Collective (grants 2.4511.06 and
2.4524.03), the Fonds de la Recherche en Sciences Médicales (grant
3.4586.05), Eur-Intafar (grant LSHM-CT-2004-512138), and the
Centre National de la Recherche Scientifique (France). A.P. is a re-
cipient of a fellowship from the Fonds pour la Recherche dans
l’Industrie et l’Agriculture.
The following protocol was used: solvent A: water (Millipore
quality) containing 0.05% or 0.1% TFA and solvent B: CH3CN con-
taining 0.05% or 0.1% TFA. Flow: 0.7 ml/min, gradient: 0–2 min:
100% A, 2–4 min: 0–10% B, 4–34 min: 10–70% B, 34–39 min: 70%
B, 39–40 min: 30–100% A, 40–60 min: 100% A. The injection vol-
umes were 10–100 ll.
4.29. Hydrolysis of anhMurNAc derivatives
The anhMurNAc derivatives were dissolved in water (concen-
tration: 6, 20, 27, 28 and 29: 10 mM, 7: 250 lM, 8: 2.5 mM, 11:
Supplementary data
1 mM). The compounds were incubated for various incubation
times with various enzyme concentrations (Table 1) in 20 mM so-
dium phosphate buffer containing 48 nM ZnCl2 at 30 °C. The reac-
tion was stopped by addition of 1:10 of the total assay volume of
H3PO4 (1:100 dilution with water). The solution was analysed by
HPLC as described previously.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.09.010.
References and notes
1. Coyette, J.; van der Ende, A. FEMS Microbiol. Rev. 2008, 32, 147.
2. Vollmer, W.; Blanot, D.; de Pedro, M. A. FEMS Microbiol. Rev. 2008, 32,
149.
4.30. Kinetic studies
3. Park, J. T.; Uehara, T. Microbiol. Mol. Biol. Rev. 2008, 72, 211.
4. Cheng, Q.; Li, H.; Merdek, K.; Park, J. T. J. Bacteriol. 2000, 182, 4836.
5. Pennartz, A.; Genereux, C.; Parquet, C.; Mengin-Lecreulx, D.; Joris, B. Antimicrob.
Agents Chemother. 2009, 53, 2991.
6. Uehara, T.; Park, J. T. J. Bacteriol. 2007, 189, 5634.
7. Kerff, F.; Petrella, S.; Mercier, F.; Sauvage, E.; Herman, R.; Pennartz, A.;
Zervosen, A.; Luxen, A.; Frere, J.-M.; Joris, B.; Charlier, P. J. Mol. Biol. 2010, 397,
249.
8. Kawasaki, A.; Karasudani, Y.; Otsuka, Y.; Hasegawa, M.; Inohara, N.; Fujimoto,
Y.; Fukase, K. Chem. Eur. J. 2008, 14, 10318.
9. Hesek, D.; Lee, M.; Zhang, W.; Noll, B. C.; Mobashery, S. J. Am. Chem. Soc. 2009,
131, 5187.
Kinetic studies were performed with 20, 27, 28 and 29. Various
concentrations of the compounds were incubated with the follow-
ing enzyme concentrations (20: 3.9
lM, 27: 0.7 lM, 28: 0.3 lM
and 29: 0.7 M) in 20 mM sodium phosphate buffer containing
l
48 nM ZnCl2 at 30 °C. The reactions were stopped after various
incubation times (20: 2 h, 27 and 28: 10 min; 29: 30 min) and ana-
lysed as described previously.
100
zyme concentrations (20: 7.7
0.7 M) in 20 mM sodium phosphate buffer containing 48 nM
l
M of 20, 27, 28 and 29 were hydrolysed with various en-
l
M, 27: 0.7 M 28: 0.3 M and 29:
l
l
10. Lee, M.; Zhang, W.; Hesek, D.; Noll, B. C.; Boggess, B.; Mobashery, S. J. Am. Chem.
Soc. 2009, 131, 8742.
11. Kumar, S.; Roychowdhury, A.; Ember, B.; Wang, Q.; Guan, R.; Mariuzza, R. A.;
Boons, G.-J. J. Biol. Chem. 2005, 280, 37005.
l
ZnCl2 at 30 °C. The reaction was stopped after various incubation
times (Fig. 2) and analysed as described previously.
12. Chowdhury, A. R.; Boons, G.-J. Tetrahedron Lett. 2005, 46, 1675.
13. Teller, N. Centre de Recherches du Cyclotron, Faculté des Sciences. Thèse de
doctorat: Université de Liège, 2009.
4.31. Inhibition studies
14. Waley, S. G. Biochem. J. 1991, 279, 87.
15. Lutzner, N.; Patzold, B.; Zoll, S.; Stehle, T.; Kalbacher, H. Biochem. Biophys. Res.
Commun. 2009, 380, 554.
16. Rosowsky, A.; Bader, H.; Radike-Smith, M.; Cucchi, C. A.; Wick, M. M.;
Freisheim, J. H. J. Med. Chem. 1986, 29, 1703.
Inhibition studies were performed with compounds 1, 6, 7, 8, 11
and 20. The anhMurNAc derivatives were dissolved in water (1, 6
and 11: 10 mM, 20: 1 mM, 8: 2.5 mM) or in DMF (7: 100 mM).
The presence of 1% DMF in the assay had no influence on the
17. Ryan, J. W.; Chung, A. U.S. Patent No. 641,395, 1989.