Thioester analogues of peptidoglycan fragment MurNAc-L-Ala-γ-D-Glu as substrates for peptidoglycan hydrolase MurNAc-L-Ala amidase

Article abstract of DOI:10.1039/b200921h

MurNAc-L-amidase is one of a family of peptidoglycan hydrolases which catalyses the breakdown of bacterial peptidoglycan. Analogues of the peptidoglycan fragment MurNAc-L-Ala-γ-D-Glu containing S-thiolactic acid in place of L-alanine were synthesised as thioester substrates for this enzyme. Triphenylmethanethiol was used to develop a stereoselective synthesis of S-thiolactic acid, which was elaborated synthetically into MurNAc-dipeptide analogues. MurNAc-S-thioacetyl-N-propylamide 13 and MurNAc-S-thiolactyl-2R-alaninamide 16 were found not to be substrates for recombinant MurNAc-L-Ala amidases CwlA from Bacillus subtilis and Ply21 from bacteriophage TP21, however, turnover of tripeptide thioester S-propionylthiolactyl-γ-D-Glu-L-Lys-OMe 21 was observed using amidase Ply21. Therefore, recognition of the amino acid at position 3 of the pentapeptide sidechain appears to be important for enzymatic turnover.

Full text of DOI:10.1039/b200921h

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Thioester analogues of peptidoglycan fragment MurNAc-L-Ala-ꢀ-
D-Glu as substrates for peptidoglycan hydrolase MurNAc-L-Ala
amidase
1
Ross L. Harding,a Joanne Henshaw,a Joannah Tillingb and Timothy D. H. Bugg*a
a Department of Chemistry, University of Warwick, Coventry, UK CV4 7AL.
E-mail: mssgv@warwick.ac.uk; Fax: 02476-524112; Tel: 02476-573018
b Department of Chemistry, University of Southampton, Highfield, Southampton,
UK SO17 1BJ
Received (in Cambridge, UK) 24th January 2002, Accepted 22nd May 2002
First published as an Advance Article on the web 18th June 2002
MurNAc--amidase is one of a family of peptidoglycan hydrolases which catalyses the breakdown of bacterial
peptidoglycan. Analogues of the peptidoglycan fragment MurNAc--Ala-γ--Glu containing S-thiolactic acid
in place of -alanine were synthesised as thioester substrates for this enzyme. Triphenylmethanethiol was used to
develop a stereoselective synthesis of S-thiolactic acid, which was elaborated synthetically into MurNAc-dipeptide
analogues. MurNAc-S-thioacetyl-N-propylamide 13 and MurNAc-S-thiolactyl-2R-alaninamide 16 were found not
to be substrates for recombinant MurNAc--Ala amidases CwlA from Bacillus subtilis and Ply21 from bacteriophage
TP21, however, turnover of tripeptide thioester S-propionylthiolactyl-γ--Glu--Lys-OMe 21 was observed using
amidase Ply21. Therefore, recognition of the amino acid at position 3 of the pentapeptide sidechain appears to be
important for enzymatic turnover.
Introduction
The peptidoglycan layer of bacterial cell walls, consisting of
N-acetylmuramic acid (MurNAc)-GlcNAc polysaccharide
cross-linked via -Ala-γ--Glu-X--Ala--Ala (where X =
-Lys or m-DAP, meso-diaminopimelic acid) pentapeptide
sidechains, provides much of the strength and rigidity of
bacterial cell envelopes.1,2 The biosynthesis of peptidoglycan is
known to be essential for bacterial growth, and the inhibition
of this pathway is the mechanism of action of a number
of antibacterial agents, including the penicillin family of
β-lactams, and the vancomycin group of glycopeptides.1,2 Dur-
ing cell growth and cell division, a family of peptidoglycan
hydrolase enzymes is required in order to effect the local break-
down of peptidoglycan, concomitant with the synthesis of new
peptidoglycan.3 The peptidoglycan hydrolases can be divided
into two groups: the glycosidases, which hydrolyse the poly-
saccharide backbone; and the peptidases, which hydrolyse the
peptide sidechain.3 The molecular enzymology of the peptido-
glycan hydrolases has been little studied, and is hindered by the
lack of small molecule substrates for their assay. The cellular
consequences of inhibition of these enzymes is also unclear, the
only existing inhibitor being bulgecin A, an inhibitor of lytic
Fig. 1 A. Structure of peptidoglycan repeating unit, showing site of
glycosidase, which is known to augment the antibacterial effects
hydrolytic cleavage by MurNAc--Ala amidase. R = H (-Lys) or CO2H
of penicillin.4
(meso-diaminopimelic acid); B. General structure of MurNAc-S-
A well-proven strategy for the assay of protease enzymes util-
ises synthetic substrates containing a thioester linkage in place
of the scissile amide bond. Cleavage of the thioester functional
group liberates a thiol, which can be reacted in situ with a
chromogenic thiol reagent such as 5,5Ј-dithiobis(2-nitrobenzoic
acid)† (DTNB), giving a colour change at 412 nm.5 This manu-
script describes the synthesis and assay of thioester-containing
substrates for MurNAc--Ala amidase, which catalyses the
hydrolysis of the amide bond between the MurNAc lactyl
sidechain and the -alanine residue at position 1 of the
pentapeptide sidechain (see Fig. 1).
thiolactyl thioester substrates designed for MurNAc--Ala amidase.
MurNAc--amidase enzyme activities have been purified
from Escherichia coli K12,6 Bacillus subtilis,7,8 Bacillus mega-
terium,9 and Staphylococcus aureus.10 The cwlA gene encoding
MurNAc--Ala amidase in B. subtilis 168 has been cloned and
expressed in E. coli.11,12 This enzyme is also found as a bacterio-
lytic enzyme in certain bacteriophages: notably, the bacterio-
phage T7 lysozyme is a bifunctional RNA polymerase/
MurNAc--Ala amidase.13,14 Recently the ply genes encoding
MurNAc--Ala amidases have been identified in bacteriophage
12826 and TP21, which infect Bacillus cereus.15
Little is known about the substrate requirements of MurNAc-
-Ala amidase, beyond the requirement for a divalent metal ion,
† The IUPAC name for 5,5Ј-dithiobis(2-nitrobenzoic acid) is
5-[(3-carboxy-4-nitrophenyl)disulfanyl]-2-nitrobenzoic acid.
1714
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b200921h

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usually Mg2ϩ for the bacterial enzymes,6–12 and Zn2ϩ for the
bacteriophage T7 enzyme.13,14 Using radiolabelled peptido-
glycan fragments, van Heijenoort et al. have previously shown
that MurNAc--Ala-γ--Glu-m-DAP--Ala--Ala and Mur-
NAc--Ala-γ--Glu-m-DAP are substrates for the E. coli
enzyme.6 It was anticipated that, in common with most
peptidase enzymes, the specificity of the enzyme would be
largely determined by the groups immediately adjacent to the
site of cleavage.5 In order to undertake molecular studies of this
enzyme, a reliable continuous enzyme assay was required.
Therefore, the synthesis of thioester analogues of MurNAc--
Ala-γ--Glu was undertaken, in which the -alanine residue at
position one is replaced with S-thiolactic acid (see Fig. 1).
spectroscopy in the presence of chiral shift reagent tris[3-(hepta-
fluoropropylhydroxymethylene)-(ϩ)-camphorato]europium()
gave no splitting of the δ 1.3 ppm CH3 doublet, whereas a
sample of racemic thiolactic acid under the same conditions
gave two doublets. DCC–HOBT coupling of acid 4 with
-alanine diphenylmethyl ester gave a single diastereomer of
a highly crystalline derivative 6, whose X-ray crystal structure
was determined (see Fig. 2). The structure establishes the
Results
Stereoselective synthesis of protected S-thiolactic acid
S-Thiolactic acid has previously been synthesised using thio-
acetic acid, a noxious liquid whose thioester linkage is prone to
base-catalysed cleavage.16 We have investigated the use of
triphenylmethanethiol (1) for the stereoselective introduction
of sulfur, and have found that it is a convenient and versatile
reagent for this purpose (a communication describing this part
of the work has previously been published17). Triphenylmeth-
anethiol (1) can be prepared in 97% yield by passage of
hydrogen sulfide gas through an acidic solution of triphenyl-
methanol,18 and is isolated as an odourless white solid which is
stable to extended storage at room temperature.
Triphenylmethanethiol (1) was reacted with ethyl (2R)-2-
chloropropionate (2), readily prepared from ethyl S-lactate,16 in
the presence of sodium methoxide–methanol to give the methyl
ester 3 resulting from transesterification. Alkaline hydrolysis of
3 in 0.5 M sodium hydroxide in 1 : 1 water–dioxane, conditions
known to effect the hydrolysis of S-tritylcysteine derivatives
without racemisation,19 gave the acid product 4 in 97% after
column chromatography (see Scheme 1).
Fig. 2 X-Ray crystal structure of S-trityl (S)-thiolactyl-(2R)-alanyl
diphenylmethyl ester 6, used to elucidate the configuration of the
S-thiolactyl chiral centre (C-3 on picture).
S configuration of the thiolactyl C-2 centre, thus establishing
that the reaction of triphenylmethanethiol (1) with ethyl (2R)-
2-chloropropionate occurs with inversion of configuration.
S-Tritylmercaptoacetic acid (7), lacking the methyl sidechain,
was also prepared in 70% yield by reaction of mercaptoacetic
acid and triphenylmethanol in glacial acetic acid.20 Both deriv-
atives were subsequently used for the synthesis of MurNAc
thioesters.
Synthesis of MurNAc-S-thiolactyl-X thioesters
N-Acetylmuramic acid contains an 2S-lactyl ether at the C-3
position of N-acetyl -glucosamine, and has been prepared
previously from N-acetyl -glucosamine either via a furan-
ose oxazoline derivative,21 or via a 4,6-protected pyranose
derivative.22–24 The latter method was found to be amenable for
synthesis of muramic acid derivatives. Thus, the 4,6-benzyl-
idene α-methyl glycoside derivative of N-acetyl--glucosamine
was prepared,22,23 and was alkylated with 2S-chloropropionic
acid using NaH–dioxane to give the protected muramic acid
8.24
Two thioesters were first synthesised in which the S-thiolactic
acid was coupled to n-propylamine, as shown in Scheme 2.
Trityl-protected acids 4 and 7 were coupled to n-propylamine,
using carbonyldiimidazole‡ as coupling agent, to give the
corresponding amides 9 and 10 in 66% and 92% yields
respectively. Amides 9 and 10 were de-tritylated by treatment
with trifluoroacetic acid and triethylsilane, and immediately
coupled with protected muramic acid 8 using DCC–DMAP, to
give the protected thioesters 11 and 12, in 40% and 20% yield
respectively. The presence of the thioester linkage was
confirmed by the appearance of 13C NMR signals at 205.0
and 202.3 ppm respectively, and the observation of parent ions
at m/z 525.5 and 511.3 by electrospray mass spectrometry.
Several methods were attempted for the deprotection of the
benzylidene protecting group present in 11 and 12. Treatment
with 60% acetic acid–water resulted in cleavage of the thioester
linkage. Hydrogenation using Pd/C was unsuccessful, probably
due to the presence of the thioester. Reduction with Na–NH3
led to decomposition. However, the method of Szarek et al.,
involving treatment with 1% iodine in refluxing methanol for
Scheme 1 Synthesis of S-thiolactic acid derivatives from triphenyl-
methanethiol. a, H2S, AcOH–H2SO4, 97%; b, SOCl2, DMF(cat), 77%;
c, NaOMe, MeOH, 100%; d, 0.5 M NaOH, H2O–dioxane (1 : 1), 97%;
e, CF3COOH, Et3SiH, 39%, f, DCC, HOBT, -Ala-OCHPh2, 100%; g,
Ph3COH, BF3ؒOEt2, AcOH, 70%.
In order to determine the stereochemical purity of 4, the
trityl group was deprotected using trifluoroacetic acid–
triethylsilane to give S-thiolactic acid (5). Analysis by 1H NMR
‡ The IUPAC name for carbonyldiimidazole is di(1H-imidazol-1-yl)-
methanone.
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722
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Scheme
2 Synthesis of MurNAc-S-thioacetyl-N-propylamide 13.
a, CH3CH2CH2NH2, carbonyldiimidazole, CH2Cl2, 66% (9), 92% (10);
b, CF3COOH, Et3SiH; c, DCC, DMAP, DMF, 40% (11), 20% (12);
d, 1% I2–MeOH, 75%.
2 hours,25 was successful for the deprotection of thioester 12, to
give the MurNAc thioester 13 in 75% yield. Thioester 13
was characterised by NMR spectroscopy, and showed parent
ion m/z 423 by matrix-assisted laser desorption ionisation
(MALDI) mass spectrometry. Deprotection of thioester 11 was
not successful via this method, for reasons that are not clear,
although the reaction was carried out on a very small scale.
Thioester 13 was used for biological testing.
Scheme 3 Synthesis of MurNAc-S-thiolactyl-2R-alaninamide 16. a,
DCC, HOBT, THF, 33%; b, CF3COOH, Et3SiH; c, DCC, HOBT, THF,
62%; d, 1% I2–MeOH, 40%.
Synthetic studies were continued towards two further
thioesters, which mimicked the carboxylate sidechain of γ--
glutamic acid at position 2 of the pentapeptide sidechain,
by addition of a -alanine residue. Since both γ--Glu and
γ--Gln are found in bacterial peptidoglycan,1,2 a thioester ter-
minating in -alanine amide was also synthesised, as shown in
Scheme 3. -Alanine amide was synthesised by reaction of
-alanine with thionyl chloride in dry methanol, followed by
addition of 25% ammonia solution, to give the amide in 24%
yield. -Alanine amide was coupled to trityl-protected acid 4,
using DCC and HOBT in THF, to give the amide 14 in 33%
yield. Deprotection with trifluoroacetic acid and triethylsilane,
followed by coupling of the thiol to protected muramic acid 8,
gave the protected thioester 15 in 62% yield. The thioester
carbonyl group was observed by 13C NMR spectroscopy at
202.81 ppm, and an (M ϩ Na) peak at m/z 576 was observed
by electrospray mass spectrometry. Deprotection of the benzyl-
idene protecting group was achieved using 1% I2–MeOH to give
MurNAc thioester 16 (m/z 488 (M ϩ Na)) in 38% yield after
chromatography.
It was anticipated that the -alanine-containing thioester
could be synthesised using the acid-labile diphenylmethyl group
to protect the -alanine carboxy. -Alanine diphenylmethyl
ester was synthesised by reaction of Boc--alanine with benzo-
phenone hydrazone and Oxone, in 64% yield, followed by
selective removal of the Boc group using 3 M HCl–MeOH.
As shown in Scheme 1, coupling to trityl-protected acid gave
derivative 6, whose X-ray crystal structure was determined.
Unfortunately, attempts to detritylate 6 and couple to protec-
ted muramic acid 8 were all unsuccessful, perhaps due to
intramolecular cyclisation of the free thiol to form
a
6-membered cyclic thioester (shown in Scheme 3). Attempts to
couple the sodium salt (17) onto 8 gave only a small amount of
coupled product, as observed by NMR spectroscopy, which was
insufficient for deprotection. Therefore, only thioester 16 was
used for biological evaluation.
Synthesis of S-propionyl thiolactyl-ꢀ-D-Glu-L-Lys tripeptide
analogue
For reasons explained below, a thioester analogue mimicking
the -Ala-γ--Glu--Lys tripeptide, but lacking the MurNAc
sugar, was also synthesised, using solution phase peptide
synthesis methods, as shown in Scheme 4.
Following the procedures of Bavetsias et al., orthogonally
protected Cbz--glutamic acid α-tert-butyl ester γ-methyl ester
was synthesised in three steps from -glutamic acid, and the
γ-methyl ester hydrolysed under alkaline conditions to give
Cbz--glutamic acid α-tert-butyl ester (18).26 18 was coupled
onto -Lys(N ε-Boc)-OMe using DCC–HOBT, to give the pro-
tected -Glu-Lys dipeptide (19) in 22% yield. The N-terminal
Cbz group was deprotected by hydrogenation with PdCl2
as catalyst, and the product was coupled onto racemic
S-propionylthiolactic acid using DCC–HOBT, to give pro-
tected tripeptide 20 in 30% yield. The Boc and tert-butyl ester
protecting groups were removed by treatment with trifluoro-
acetic acid, to give the tripeptide thioester 21, which was used
for biological evaluation.
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growing cell walls.15 Using this assay, a clear decrease in A600
could be observed upon addition of 0.5 mg Ply21 or 1 mg egg
white lysozyme (which also functions as a peptidoglycan hydro-
lase), but not for CwlA. The second assay, developed by Leclerc
et al.,27 involves the analysis of the purified protein on an SDS-
polyacrylamide gel which had been pre-treated with intact cell
walls obtained from Bacillus subtilis. Treatment of the gel with
0.1% Triton X-100 (to re-nature the enzyme), followed by
treatment with 0.1% methylene blue gives a blue colouration in
the presence of peptidoglycan, but a clear zone in regions of the
gel where an active peptidoglycan hydrolase is present. Using
this assay, a clear zone was observed at the predicted Mr of
29 kDa for the Ply21 enzyme (Fig. 3), but no clear zone was
Scheme 4 Synthesis of S-propionyl-thiolactyl-γ--Glu--Lys-OMe
21. a, DCC, HOBT, CH2Cl2, 22%; b, H2/PdCl2, 30%; c, S-propionyl-
thiolactic acid, DCC, HOBT, CH2Cl2, 30%; d, CF3COOH, CH2Cl2,
100%.
Expression and assay of MurNAc-L-Ala amidase
Two MurNAc--Ala amidases whose genes had previously been
over-expressed in Escherichia coli were studied: CwlA from
Bacillus subtilis 168 (gift of Dr Simon Foster, University of
Sheffield),12 which was expressed from plasmids pSFP102
(native protein) and Pet24d (N-terminal Hex6 fusion protein);
and the Ply21 amidase from bacteriophage TP21 (gift of Pro-
fessor Martin Loessner, Technische Universität München),
which was expressed from plasmid pBCPL21.15
Fig. 3 Renaturing SDS-PAGE assay of Ply21 amidase from bacterio-
phage PL21. A. SDS-polyacrylamide gel stained with Coomassie blue,
showing Ply21 amidase (29 kDa). B. Duplicate SDS-polyacrylamide
gel, containing B. subtilis cell walls, renatured and stained as described
in Experimental section. Clear zone indicates peptidoglycan hydrolase
activity. Lane 1, sample of Ply21 amidase from JM109/pBCPL21 grown
at 37 ЊC, insoluble fraction, re-suspended in 8 M urea. Lane 2, sample
of Ply21 amidase from JM109/pBCPL21 grown at 25 ЊC, soluble
fraction.
Cell extract from E. coli JM109/pSFP102 (expressing CwlA)
was purified by G-75 Sephadex gel filtration, giving a soluble
fraction in which the desired 30 kDa CwlA protein could be
discerned by sodium dodecyl sulfate (SDS)-polyacrylamide
electrophoresis. Cell extract from E. coli BL21/Pet24d (express-
ing His6-CwlA) showed high levels of the desired 30 kDa CwlA
protein, which was pelleted by centrifugation, indicating that
it is insoluble. Treatment of the pellet with 8 M urea gave a
fraction containing CwlA as the major band by SDS-polyacryl-
amide electrophoresis, which could be dialysed into buffer con-
taining 3 M urea without precipitation. Cell extract from E.coli
JM109/pBCPL21 contained high levels of a 29 kDa protein
corresponding to amidase Ply21. This enzyme was also pelleted
by centrifugation, but growth of the producing strain at 25 ЊC
yielded a soluble fraction after centrifugation containing Ply21
as the major band by SDS-polyacrylamide electrophoresis.
In order to verify the activity of the expressed proteins, two
biological assays were carried out. The first involves the treat-
ment of a growing culture of Bacillus subtilis with a sample of
protein, and observation of a decrease in A600 due to lysis of
observed for CwlA, hence the activity of the CwlA amidase is
undetectable in our hands.
Each of the two MurNAc--Ala enzymes were assayed for
hydrolysis of the thioester substrates, by coupling with chromo-
genic thiol reagent DTNB. Assays were carried out in both
stopped format (adding DTNB at a fixed time-point) and con-
tinuousformat(withDTNBpresentthroughout). Foreachofthe
MurNAc-containing thioesters 13 and 16, no time-dependent
turnover could be detected with either enzyme, relative to con-
trol assays lacking either enzyme or substrate, even at extended
reaction times with high protein concentrations.
In order to test whether recognition of the amino acid at
position 3 is important for enzymatic turnover, the S-propionyl-
thiolactyl-γ--Glu--Lys thioester substrate 21 was sub-
sequently synthesised, and assayed versus amidase Ply21.
Time-dependent release of thiol was observed using a DTNB
stopped assay (see Table 1), indicating that 21 is a substrate
Table 1 Turnover of thioester 21 by amidase Ply21 in DTNB coupled assays, as observed at 412 nm
Rate of turnover of 21
ϩ thioester 21
Ϫ thioester 21
(nmol product (mg protein)Ϫ1 minϪ1
)
Stopped assaya
t = 6 min
t = 10 min
0.100 AU
0.00 AU
39.3
38.4
0.44
0.163 AU
0.00 AU
Continuous assayb
0.0071 AU minϪ1
0.0014 AU minϪ1
a Stopped assay contained 0.15 mg (ml protein)Ϫ1, 200 µl aliquot withdrawn for A412 determination. Mean value from duplicate assays quoted.
b Continuous assay contained 1.15 mg (ml protein)Ϫ1. Mean value from duplicate assays quoted.
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722
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Fig. 4 Summary of biological activity shown by thioesters 13, 16, and 21 as substrates for MurNAc--Ala amidase.
for this enzyme. From the activity over a 10 minute assay,
a specific activity of 39 nmol product minϪ1 (mg protein)Ϫ1 can
be calculated (see Table 1). Much lower activity was measured
in a continuous assay format (0.44 nmol minϪ1 (mg protein)Ϫ1),
implying that the enzyme is inactivated by treatment with
DTNB.
presumably potential inhibitors) for this enzyme will need to
encompass at least a tripeptide in size. Perhaps this unusual
selectivity reflects the fact that the natural substrate for Mur-
NAc--Ala amidase is a physically inaccessible polysaccharide,
rather than a monosaccharide-peptide. It is hoped that the
availability of synthetic substrates for this class of enzymes will
enable enzymologists to study the detailed function of these
enzymes, and the cellular consequences of their inhibition.
Conclusions
Experimental
Although a number of peptidoglycan hydrolase activities have
been identified biologically,3 and a number of corresponding
genes identified, little is known about the molecular details of
their action, or the consequences of inhibition. This is partly
due to the lack of a convenient in vitro enzyme assay. In this
paper we describe the synthesis of three thioester-containing
analogues of the MurNAc-peptide substrate for MurNAc--
Ala amidase.
Materials
Triphenylmethanethiol (1) was prepared by the method of
Balfe et al.,18 by passage of hydrogen sulfide through an acidic
solution of triphenylmethanol in acetic acid, in 97% yield,
δH (300 MHz, CDCl3) 7.34–7.24 (15H, m), 3.11 (1H, s) ppm;
δC (75 MHz, CDCl3) 147.3, 129.5, 128.0, 127.0, 63.0 ppm. Ethyl
(2R)-2-chloropropionate (2) was prepared from ethyl S-lactate,
using the method of Hof and Kellogg.16 Boc--alanine di-
phenylmethyl ester was prepared by reaction of Boc--alanine
with benzophenone hydrazone and Oxone,28 in 64% yield.
-Alanine diphenylmethyl ester was prepared by treatment of
Boc--alanine diphenylmethyl ester with 3 M HCl in methanol,
in 67% yield; mp 61–62 ЊC; δH (300 MHz, CDCl3) 7.34 (10H,
m), 6.90 (1H, s), 3.69 (1H, q, J = 6.9 Hz), 2.11 (2H, s), 1.41 (3H,
d, J = 6.9 Hz) ppm; m/z (ESϩ) 256.2 (MHϩ, 100%).
S-Tritylmercaptoacetic acid 7 was prepared by the method of
Alarabi et al.,20 in 70% yield. N-Acetyl--glucosamine was
prepared from -glucosamine by the method of Inouye et al.29
Oxazoline 8 was prepared from N-acetyl--glucosamine by the
method of Mack et al.21 α-Methyl--glucosamine was syn-
thesised by treatment of GlcNAc with Dowex 50W in refluxing
methanol, according to Galemo and Horton,22 in 74% yield,
and was converted to the 4,6-benzylidene derivative by treat-
ment with benzaldehyde and ZnCl2, using the method of
Neuberger.23 Protected muramic acids 8 were prepared by alkyl-
ation with 2S-2-chloropropionic acid, according to Jeanloz et
al.24 Cbz--glutamic acid α-tert-butyl ester (18) was prepared
from -glutamic acid in four steps using the procedures of
Bavetsias et al.26 S-Propionylthiolactic acid was prepared
by acylation of racemic thiolactic acid with propanoyl
chloride in the presence of triethylamine (2 eq.), in 88% yield.
Biological testing of these thioesters was complicated by the
lack of an independent quantitative assay, since a negative
result could be interpreted either as lack of activity as substrate,
or lack of active enzyme present in the assay. Using two
independent biological assays, we have established that the
expressed Ply21 amidase is active as a peptidoglycan hydro-
lase,15 but shows no turnover with thioesters 13 and 16. This
disappointing series of observations suggested that the enzyme
required the recognition of functional groups beyond position 2
3
of the pentapeptide sidechain. Since H-MurNAc--Ala-γ--
Glu-m-DAP has previously been shown to be a substrate for
E.coli MurNAc--Ala amidase,6 it seemed plausible that recog-
nition of the amino acid at position 3 is important for enzym-
atic turnover. Hence we subsequently synthesised the tripeptide
thioester analogue 21, and we have established that 21 is indeed
a substrate for Ply21 amidase, the first synthetic substrate
for this class of enzyme. These observations are summarised in
Fig. 4.
The recognition of a functional group distant from the site of
cleavage is unusual for peptidase enzymes, which normally
show selectivity for the residue preceding (P1 site) or follow-
ing (P1Ј site) the site of cleavage.5 In this case, it appears
that MurNAc--Ala amidase recognises the amino acid three
residues after the cleavage site (P3Ј site). The practical
consequences of this observation are that substrates (and
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δH (300 MHz, CDCl3) 4.25 (1H, q, J = 7.4 Hz), 2.61 (2H, q,
J = 7.5 Hz), 1.53 (3H, d, J = 7.4 Hz), 1.19 (3H, t, 7.5 Hz) ppm.
All other chemicals and biochemicals were purchased from
Sigma-Aldrich.
Plasmids pSFP102 and Pet24d containing the Bacillus
subtilis cwlA gene were gifts of Dr Simon Foster (University of
Sheffield).12 Plasmid pBCPL21 containing the ply gene from
bacteriophage TP21 was a gift of Professor Martin Loessner
(Technische Universität München).15 SDS-polyacrylamide
(2S)-S-Tritylthiolactyl-2R-alanine diphenylmethyl ester 6
To a solution of acid 4 (0.697 g, 2 mmol) in dry THF (20 ml)
under nitrogen was added dicyclohexylcarbodiimide (0.454 g,
2.2 mmol) and hydroxybenzotriazole (0.297 g, 2.2 mmol)
and the solution was stirred at room temperature for 5 min.
-Alanine diphenylmethyl ester (0.500 g, 2 mmol) in THF
(10 ml) was added and the mixture allowed to stir at room
temperature overnight under nitrogen. The solution was filtered
to remove the precipitated urea, washed through with ethyl
acetate and concentrated under reduced pressure to give a white
solid. The crude product was purified by silica column chrom-
atography (50% diethyl ether–petroleum ether, Rf 0.20) to
afford 6 as a white solid (1.190 g, 100%). A sample of 6 was
re-crystallised by vapour diffusion (isopropanol (propan-2-ol)–
water) to give single crystals suitable for X-ray diffraction (see
electrophoresis was carried out using
a Bio-Rad Mini-
Protean apparatus, according to the manufacturer’s instruc-
tions. UV–visible spectrophotometry was carried out using a
Cary 1 spectrophotometer.
Methyl (2R)-S-tritylthiolactate 3
data below; structure shown in Fig. 2). IR (Nujol mull, cmϪ1
)
To methanol (10 ml) at 0 ЊC under nitrogen was added sodium
(12 mg, 0.5 mmol), which was stirred until the sodium
had dissolved (5–10 min). Triphenylmethanethiol 1 (0.138 g,
0.5 mmol) was added and the solution was allowed to warm to
room temperature, and stirring continued for 1 h. Ethyl (2R)-2-
chloropropanoate 2 (0.068 g, 0.5 mmol) in methanol (5 ml) was
added and the mixture was allowed to stir at room temperature
overnight. The solution was concentrated under reduced pres-
sure to give an oil with solid precipitate, which was washed
through a filter with dichloromethane (100 ml), and the filtrate
was concentrated under reduced pressure to give a viscous
yellow oil. The crude product was purified by silica column
chromatography (30% Et2O–petroleum ether Rf 0.37) to give 3
3071 (m), 2992 (s), 1754 (m), 1678 (w), 1586 (m); δH (300 MHz,
CDCl3, 2 rotamers) 7.43–7.12 (25H, m), 6.89 & 6.82 (1H, s,
–CHPh2), 6.70 & 6.50 (1H, d, J = 6.7 Hz, NH), 4.30 & 4.15 (1H,
dq, J = 7.1, 6.7 Hz, –CHNCH3), 2.99 (1H, q, J = 7.6 Hz,
–CHSCH3), 1.42 & 1.39 (3H, d, J = 7.6 Hz, CH3CS), 1.27 &
1.20 (3H, d, J = 7.1 Hz, CH3CN) ppm; δC (75 MHz, CDCl3)
172.1, 171.6, 144.5, 144.3, 139.8, 139.7, 129.9, 129.6, 128.8,
128.8, 128.7, 128.4, 128.2, 128.2, 128.1, 127.3, 127.2, 127.1,
127.0, 78.0, 68.3, 48.6, 48.6, 44.6, 44.4, 20.2, 18.5, 17.9 ppm; m/z
(ESϩ) 342 (60%, M Ϫ Tr).
N-Propyl-(2S)-S-tritylthiolactamide 9
as a colourless oil (87 mg, 48% yield). IR (liquid film, cmϪ1
)
To acid 4 (0.400 g, 1.1 mmol) in dichloromethane (10 ml) was
added N,N Ј-carbonyldiimidazole (0.185 g, 1.2 mmol) in dry
glassware under nitrogen. The solution was stirred at room
temperature for 3 h. n-Propylamine (90 µl, 1.1 mmol) was added
and stirring continued overnight at room temperature. Water
(10 ml) was added, and the solution was extracted with dichloro-
methane (3 × 10 ml). The combined organic layers were dried
(MgSO4), and concentrated under reduced pressure to give a
yellow solid. The product was purified by silica column chrom-
atography (Et2O, Rf 0.52) to give 9 as a white solid (0.282 g,
66% yield). Mp 122–124 ЊC; IR (Nujol mull, cmϪ1) 3262 (m),
2919 (s), 1644 (s), 1557 (s); δH (300 MHz, CDCl3) 7.41 (6H, d,
J = 7.4 Hz), 7.29–7.17 (9H, m), 5.95 (1H, br s, NH), 3.01 (1H, q,
J = 7.7 Hz, –CHSCH3), 2.90 (1H, ddt, J = 13.2, 7.0 and 6.2 Hz,
–CHHЈN), 2.65 (1H, ddt, J = 12.9, 7.0 and 5.5 Hz, –CHHЈN),
1.43 (3H, d, J = 7.7 Hz, –CHSCH3), 1.27 (2H, m, –CH2–), 0.78
(3H, t, J = 7.4 Hz, –CH2CH3) ppm; δC (75 MHz, CDCl3) 172.2,
144.4, 129.4, 128.1, 127.0, 68.2, 44.7, 41.4, 22.3, 20.3, 11.3 ppm;
m/z (ESϩ) 243.3 (Ph3Cϩ, 100%), 390.6 (MHϩ, 5%).
3057 (s), 2949 (m), 1736 (s), 1595 (m), 1489 (s); δH (300 MHz,
CDCl3) 7.48 (6H, d, J = 7.7 Hz), 7.34–7.21 (9H, m), 3.51 (3H,
s, –COOCH3), 3.02 (1H, q, J = 7.4 Hz, –CHSTr–), 1.25 (3H, d,
J = 7.4 Hz, –CH3) ppm; δC (75 MHz, CDCl3) 174.2, 144.5,
129.8, 128.1, 127.0, 68.3, 52.3, 42.5, 18.9 ppm; m/z (ESϩ) 243
(100%, Ph3Cϩ); [α]2D4 Ϫ3.9 (c 6.0, CH2Cl2).
(2S)-S-Tritylthiolactic acid 4
To methyl ester 3 (0.16 g, 0.44 mmol) was added a solution of
50% 0.5 M sodium hydroxide (10 ml)–50% 1,4-dioxane (10 ml).
The solution was allowed to stir at room temperature for
1 h. When TLC indicated the disappearance of the starting
material, 0.5 M HCl (10 ml) was added until the solution
became acidic. The solution was washed with diethyl ether
(4 × 20 ml). The combined organic layers were dried (MgSO4)
and concentrated under reduced pressure to give a viscous oil.
The product was purified by silica column chromatography
(50% diethyl ether–petroleum ether, Rf 0.29) to afford 4 as a
white solid (0.149 g, 97% yield). Mp 139–140 ЊC; IR (Nujol
mull, cmϪ1) 2968 (s), 2880 (s), 2841 (s), 1710 (m), 1456 (s);
δH (300 MHz, CDCl3) 7.47 (6H, d, J = 7.4 Hz), 7.32–7.19 (9H,
m), 3.04 (1H, q, J = 7.4 Hz, –CHSTr), 1.21 (3H, d, J = 7.4 Hz,
–CH3) ppm; δC (75 MHz, CDCl3) 179.6, 144.3, 129.8, 128.2,
127.1, 68.4, 42.6, 18.7 ppm; m/z (APCIϪ) 347.2 (M Ϫ 1, 11%);
HRMS (FABϪ) 347.1105 (C22H19O2S requires 347.1101); [α]2D4
Ϫ3.9 (c 60, CH2Cl2).
N-Propyl-S-trityl-2-mercaptoacetamide 10
To acid 7 (4.51 g, 13.5 mmol) in dichloromethane (30 ml) at
room temperature under nitrogen was added a solution of
N,N Ј-carbonyldiimidazole (2.40 g, 14.8 mmol) in dichloro-
methane (30 mL). The solution was stirred at room temperature
for 3 h. n-Propylamine (1.11 ml, 13.5 mmol) was added and the
solution was stirred at room temperature for 24 h. The solution
was washed with 0.5 M HCl (20 ml) and saturated ammonium
chloride solution (20 ml), dried (Na2SO4) and concentrated
under reduced pressure to give 10 as a white solid (4.664 g, 92%
yield). Mp 141–142 ЊC; IR (Nujol mull, cmϪ1) 3259 (w), 2921
(s), 1634 (m), 1558 (m); δH (300 MHz, CDCl3) 7.44 (6H, d,
J = 7.4 Hz), 7.34–7.22 (9H, m), 6.07 (1H, br s, NH), 3.15 (2H, s,
–COCH2S), 2.93 (2H, q, J = 6.6 Hz, –CH2N), 1.36 (2H, m), 0.84
(3H, t, J = 7.4 Hz, –CH3) ppm; δC (75 MHz, CDCl3) 168.0,
144.2, 129.6, 128.3, 127.2, 68.0, 41.6, 36.1, 22.6, 11.5 ppm.
(2S)-Thiolactic acid 5
To (2S)-S-tritylthiolactic acid 4 (0.137 g, 0.4 mmol) in tri-
fluoroacetic acid (2 ml) at 0 ЊC was added triethylsilane (80 µl,
0.5 mmol) dropwise. Immediate colour change to a colourless
solution was observed, and a white precipitate was formed. The
ice bath was removed, and hexane (10 ml) and water (10 ml)
were added. The water layer was separated, washed with hexane
(3 × 10 ml), filtered and concentrated under reduced pressure to
give 5 as a colourless liquid (16 mg, 39% yield), which was
identical to authentic material. δH (300 MHz, D2O) 3.42 (1H, q,
J = 7.0 Hz), 1.28 (3H, d, J = 7.0 Hz) ppm; δC (75 MHz, D2O)
178.3, 38.4, 23.4 ppm.
N-Propyl-(2S)-S-{2R-2-O-[O-ꢁ-methyl-4Ј,6Ј-O-benzylidene-2Ј-
acetamido-2Ј-deoxy-D-glucopyranos-3Ј-yl]propionyl}thiolactamide
11
To amide 9 (0.233 g, 0.6 mmol) in trifluoroacetic acid (5 ml) at
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722
1719

View Online
0 ЊC was added triethylsilane (128 µl, 0.8 mmol) dropwise.
Immediate colour change to a colourless solution was observed
and a white precipitate was formed. The ice bath was removed,
and hexane (10 ml) and water (10 m) were added. The water
layer was separated, washed with hexane (3 × 10 ml), filtered
and concentrated under reduced pressure to give the free
thiol as a colourless liquid (98 mg, quant.). δH (300 MHz,
D2O) 3.34 (1H, q, J = 7.0 Hz), 2.92 (2H, t, J = 7.0 Hz), 1.28
(2H, tq, J = 7.4, 7.0 Hz), 1.22 (3H, d, J = 7.0 Hz), 0.66 (3H, t,
J = 7.4 Hz) ppm; δC (75 MHz, D2O) 180.3, 44.4, 40.1, 24.8, 23.9,
13.6 ppm.
(1H, d, J = 3.5 Hz, H-1Ј), 4.59 (1H, q, J = 7.0 Hz, –CHOCH3),
4.29–3.69 (6H, m), 3.54 (2H, AB quartet, J = 14.9 Hz,
–COCH2S), 3.38 (3H, s, –OCH3), 3.21 (2H, q, J = 6.5 Hz,
–CH2N), 2.08 (3H, s, –COCH3), 1.52 (2H, sextet, J = 7.2 Hz,
–CH2–), 1.41 (3H, d, J = 7.0 Hz, –CHOCH3), 0.91 (3H, t,
J = 7.4 Hz, –CH2CH3) ppm; δC (75 MHz, d6-DMSO) 202.3,
169.6, 167.0, 137.6, 128.9, 128.3, 126.0, 100.4, 98.9, 82.2, 81.0,
77.0, 68.0, 62.5, 54.8, 52.7, 40.8, 31.9, 22.9, 22.3, 19.3, 11.4
ppm; m/z (APCIϩ) 511.3 (MHϩ, 100%); HRMS (FABϩ)
511.2114 (C24H35N2O8S requires 511.2111).
To acid 8 (79 mg, 0.2 mmol) in dry DMF (10 ml) at
0 ЊC under nitrogen was added N,N-dimethyl-4-aminopyridine
(30 mg) and dicyclohexylcarbodiimide (41 mg, 0.2 mmol). The
solution was stirred at 0 ЊC for 10 min. The above thiol (26 mg,
0.2 mmol) in DMF (5 ml) was added and stirring continued at
0 ЊC under nitrogen for 5 minutes. The pink mixture was
allowed to warm to room temperature and stirring was con-
tinued overnight. Water (10 ml) and dichloromethane (10 ml)
were added to the yellow solution, and the dichloromethane
layer was washed with water (6 × 10 ml), dried (MgSO4), and
concentrated under reduced pressure to give a yellow solid. The
crude product was purified by silica column chromatography
(EtOAc, Rf 0.20) to afford thioester 11 as a white solid (45.2 mg,
40% yield). Mp 194–195 ЊC; IR (Nujol mull, cmϪ1) 3284 (m),
2971 (s), 2879 (m), 1684 (m), 1649 (s), 1560 (m); δH (300 MHz,
CDCl3, 2 rotamers) 7.41 (5H, m), 6.33 (1H, br, NH), 6.24 &
6.14 (1H, br, NH), 5.58 (1H, s, –CHPh), 4.88 & 4.80 (1H, d,
J = 3.3 Hz, H-1Ј), 4.57 & 4.54 (1H, q, J = 7.0 Hz, –CHOCH3),
4.30–3.68 (7H, m), 3.38 (3H, s, –OCH3), 3.20 (2H, m, –CH2N),
2.10 & 2.08 (3H, s, –COCH3), 1.71 (2H, m, –CH2–), 1.50 (3H,
d, J = 6.6 Hz, –CHSCH3), 1.40 & 1.39 (3H, t, J = 7.4 Hz,
–CHOCH3), 0.91 & 0.90 (3H, t, J = 7.4 Hz, –CH2CH3) ppm;
δC (75 MHz, CDCl3) 205.0, 171.5, 170.4, 137.0, 129.3, 128.5,
126.0, 101.6, 99.2, 99.0, 83.3, 82.4, 76.5, 69.1, 62.5, 55.5, 53.1,
52.8, 40.6, 40.4, 29.8, 22.9, 22.8, 20.2, 16.9, 16.4, 11.4 ppm; m/z
(ESϩ) 525.5 (MHϩ, 100%).
N-Propyl-(2S)-S-[2R-2-O-{O-ꢁ-methyl-2Ј-acetamido-2Ј-deoxy-
D-glucopyranos-3Ј-O-yl}propionyl]thioacetamide 13
Thioester 12 (29 mg, 57 µmol) was dissolved in a 1% iodine–
methanol solution (10 ml), and the mixture was heated to reflux
for 2 h. When TLC indicated the disappearance of the starting
material, sodium thiosulfate (1 g) was added to the deep red
solution. The resulting colourless solution was filtered, and the
solid washed with methanol. The solution was concentrated
under reduced pressure to give a white residue, which was
extracted with isopropanol (5 ml), filtered and concentrated
under reduced pressure. The white residue was dissolved in
water (2 ml) and lyophilised overnight to afford 13 as a white
solid (18 mg, 75% yield). δH (400 MHz, D2O) 4.60 (1H, d,
J = 3.6 Hz, H-1Ј), 4.31 (1H, q, J = 6.9 Hz, –CHOCH3), 3.78–
3.36 (8H, m), 3.22 (3H, s, –OCH3), 3.02 (2H, t, J = 6.8 Hz,
–CH2N), 1.86 (3H, s, –COCH3), 1.37 (2H, tq, J = 7.4, 7.2 Hz,
–CH2–), 1.24 (3H, d, J = 6.9 Hz, –CHOCH3), 0.73 (3H, t,
J = 7.5 Hz, –CH2CH3) ppm; δC (100 MHz, D2O) 177.1, 175.5,
99.4, 81.5, 78.4, 73.0, 71.0, 61.8, 56.6, 54.2, 43.1, 39.2, 23.5,
23.0, 19.7, 12.0 ppm; m/z (MALDI) 423 (MHϩ).
S-Trityl-2S-thiolactyl-2R-alanine amide 14
To acid 4 (0.348 g, 1 mmol) in dry THF (10 ml) under nitrogen
was added dicyclohexylcarbodiimide (0.227 g, 1.1 mmol) and
hydroxybenzotriazole (0.149 g, 1.1 mmol), and the mixture was
stirred at room temperature for 5 min. -Alanine amide (88 mg,
1 mmol) in THF (5 ml) was added, and the mixture stirred at
room temperature overnight under nitrogen. The solution was
filtered to remove the precipitated urea, washed with ethyl acet-
ate, and concentrated under reduced pressure to give a white
solid. The crude product was purified by silica column chrom-
atography (1% Et3N–EtOAc, Rf 0.26) to afford 14 as a white
solid (0.137 g, 33% yield). IR (Nujol mull, cmϪ1) 3172 (m),
1692 (s), 1661 (m), 1586 (m); δH (300 MHz, CDCl3) 7.39 (6H, d,
J = 7.7 Hz), 7.28–7.16 (9H, m), 6.36 (1H, br s, NH), 6.16 (1H, d,
J = 6.7 Hz, NH), 5.46 (1H, br s, NH), 4.08 (1H, dq, J = 7.0, 6.7
Hz, –CHNCH3), 2.96 (1H, q, J = 7.2 Hz, –CHSCH3), 1.37 (3H,
d, J = 7.4 Hz, –CHSCH3), 1.09 (3H, d, J = 7.0 Hz, –CHNCH3),
ppm; δC (75 MHz, CDCl3) 174.1, 173.1, 144.4, 128.3, 128.2,
127.2, 68.4, 48.7, 44.3, 20.1, 17.4 ppm.
N-Propyl-(2S)-S-[2R-2-O-{O-ꢁ-methyl-4Ј,6Ј-O-benzylidene-2Ј-
acetamido-2Ј-deoxy-D-glucopyranos-3Ј-O-yl}propionyl]thio-
acetamide 12
To amide 10 (0.162 g, 0.43 mmol) in trifluoroacetic acid (2 mL)
at 0 ЊC was added triethylsilane (83 µL, 0.52 mmol) dropwise.
Immediate colour change to a colourless solution was observed
and a white precipitate was formed. The ice bath was removed,
and hexane (20 ml) and water (20 ml) were added. The water
layer was separated and washed with hexane (3 × 20 ml), the
aqueous layer was passed through Celite and concentrated
under reduced pressure to give the free thiol as a yellow oil
(0.071 g, quant.). δH (300 MHz, D2O) 2.82 (2H, s), 2.74
(2H, t, J = 7.0 Hz), 1.10 (2H, m), 0.47 (3H, t, J = 7.4 Hz) ppm;
δC (75 MHz, D2O) 176.1, 43.9, 29.5, 24.0, 12.8 ppm; m/z (ESϩ)
265.1 (MHϩ, 100%). To acid 8 (100 mg, 0.3 mmol) in dry DMF
(10 ml) at 0 ЊC under nitrogen was added N,N-dimethyl-4-
aminopyridine (30 mg) and dicyclohexylcarbodiimide (62 mg,
0.3 mmol). The solution was stirred at 0 ЊC for 10 min. The
above thiol (40 mg, 0.3 mmol) in dichloromethane (5 ml)
was added, and stirring continued at 0 ЊC under nitrogen for
5 minutes. The pink mixture was allowed to warm to
room temperature and stirring was continued overnight. Water
(10 ml) and dichloromethane (10 ml) were added to the yellow
solution. The organic layer was washed with water (6 × 10 ml),
dried (MgSO4) and concentrated under reduced pressure to give
a yellow solid. The crude product was purified twice by silica
column chromatography (EtOAc, Rf 0.12) to give thioester
12 as a white solid (30.3 mg, 20% yield). IR (Nujol mull,
cmϪ1) 3282 (m), 2896 (s), 2852 (s), 1681 (w), 1644 (m), 1556
(w); δH (300 MHz, CDCl3) 7.47–7.38 (5H, m), 6.30 (1H, d,
J = 7.4 Hz, NH), 6.16 (1H, br, NH), 5.58 (1H, s, –CHPh), 4.83
(2S)-S-[2R-2-O-{ꢁ-Methyl-4Ј,6Ј-O-benzylidene-2Ј-acetamido-
2Ј-deoxy-D-glucopyranos-3Ј-O-yl}propionyl]thiolactyl-2R-
alaninamide 15
To a solution of 14 (50 mg, 0.12 mmol) in trifluoroacetic acid
(2 ml) at 0 ЊC was added triethylsilane (44 µl, 0.28 mmol), to
give a clear solution with a white precipitate. Hexane (10 ml)
and water (10 ml) were added; the water layer was separated,
washed with hexane (3 × 10 ml), filtered, and freeze-dried
to afford the free thiol as a white solid (25 mg, quant.).
δH (300 MHz, D2O) 4.14 (1H, q, J = 7.0 Hz), 3.50 (1H, q,
J = 7.0 Hz), 1.34 (3H, d, J = 7.0 Hz), 1.28 (3H, q, J = 7.4 Hz)
ppm; δC (75 MHz, D2O) 180.4, 179.1, 52.3, 38.9, 23.2, 19.2
ppm; m/z (ESϩ) 177.0 (MHϩ, 30%).
To a solution of acid 8 (47 mg, 0.12 mmol) in THF (10 ml)
in dry glassware was added dicyclohexylcarbodiimide (27 mg,
0.13 mmol) and hydroxybenzotriazole (18 mg, 0.13 mmol). The
1720
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722

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S-(Propionyl)thiolactyl-D-Glu(ꢁ-tBu)-ꢀ-L-Lys(N ꢂ-Boc)-OMe 20
solution was stirred at room temperature under nitrogen for
5 min, then a solution of the above thiol (21 mg, 0.12 mmol)
in THF (5 ml) was added, and stirring continued overnight at
room temperature. The precipitated urea was filtered off, and
the solvent was removed under reduced pressure to give the
solid product, which was purified by silica column chromato-
graphy to give thioester 15 as a white solid (41 mg, 62% yield).
Mp 194–196 ЊC; IR (Nujol mull, cmϪ1) 3284 (m), 2900 (s), 2854
(s), 1748 (m), 1689 (w), 1651 (m); δH (300 MHz, d6-DMSO) 8.18
(1H, d, J = 7.7 Hz), 8.00 (1H, d, J = 8.8 Hz), 7.42–7.37 (5H, m),
7.23 (2H, s, NH), 7.02 (1H, s, NH), 5.72 (1H, s, –CHPh), 4.61
(1H, d, J = 3.7 Hz, H-1Ј), 4.32 (1H, q, J = 6.6 Hz, –CHOCH3),
4.24–3.63 (9H, m), 3.32 (3H, s, –OCH3), 1.83 (3H, s, –COCH3),
1.31 (3H, d, J = 7.0 Hz, –CHOCH3), 1.31 (3H, d, J = 7.0 Hz, –
CHSCH3), 1.17 (3H, d, J = 7.0 Hz, –CHNCH3) ppm; δC (75
MHz, d6-DMSO) 202.8, 173.8, 170.2, 137.5, 128.8, 128.1, 125.9,
100.3, 98.8, 82.1, 80.8, 77.0, 67.9, 62.4, 54.7, 52.7, 48.1, 40.9,
25.3, 22.9, 19.3, 18.2 ppm; m/z (FABϩ) 554 (MHϩ, 8%), 576
(M ϩ Na, 100%); HRMS (FABϩ) 554.2172 (C25H36N3O9S
requires 554.2173).
Dipeptide 19 (93 mg, 0.16 mmol) was dissolved in 1 : 1 dry
THF–methanol, to which palladium() chloride catalyst
(15 mg) was added. The reaction mixture was purged with
hydrogen gas, and stirred under an atmosphere of hydrogen for
16 h at room temperature. Solvent was evaporated under
reduced pressure, and the product was purified by silica column
chromatography (EtOAc, Rf 0.51) to give the deprotected
dipeptide (21 mg, 30%). δH (300 MHz, CDCl3) 4.38 (1H, m,
CαH), 4.28 (1H, m, CαH), 3.62 (3H, s, –OCH3), 3.04 (2H, m,
t
t
–CH2N), 2.35–1.6 (8H, m), 1.41 (9H, s, Bu), 1.36 (9H, s, Bu),
1.20 (2H, m) ppm.
S-(Propionyl)thiolactic acid (6 mg, 0.035 mmol) was dis-
solved in dry dichloromethane (5 ml), to which was added 5 µl
triethylamine (0.035 mmol) and dicyclohexylcarbodiimide
(8 mg, 0.039 mmol). Hydroxybenzotriazole (5 mg, 0.035 mmol)
was added, then the deprotected -Glu--Lys dipeptide (15 mg,
0.034 mmol), and triethylamine (5 µl, 0.035 mmol). The reac-
tion was stirred for 16 h at room temperature. The precipitated
urea was filtered off, and the solvent evaporated under reduced
pressure. The residue was dissolved in ethyl acetate (10 ml), and
the solution was washed with water (5 ml), 5% potassium
hydrogen sulfate solution (5 ml), 5% sodium bicarbonate solu-
tion (5 ml), and sat. sodium chloride solution (5 ml). The
organic layer was dried (Na2SO4) and evaporated at reduced
pressure. The product was purified by silica column chrom-
atography (EtOAc, Rf 0.56) to give tripeptide 20 as a white
powder (6 mg, 30% yield). δH (300 MHz, CDCl3, 2 diastereo-
isomers) 7.05 (1H, br s, NH), 6.96 (1H, br s, NH), 4.57 (1H,
m, CαH), 4.10 (1H, m, CαH), 3.76 & 3.75 (3H, 2 × s, –OCH3),
3.52 (1H, m, –CHSCH3), 3.12 (2H, m, –CH2N), 2.64 (2H, q,
J = 7.0 Hz, –COCH2CH3), 2.25 (2H, m), 2.0–1.6 (6H, m), 1.48
& 1.47 (9H, 2 × s, tBu), 1.42 & 1.41 (9H, 2 × s, tBu), 1.5–1.3 (3H,
2 × d, –CHSCH3), 1.21 (3H, t, J = 7.0 Hz, –COCH2CH3),
1.2–1.0 (2H, m) ppm.
(2S)-S-[2R-2-O-{ꢁ-Methyl-2Ј-acetamido-2Ј-deoxy-D-gluco-
pyranos-3Ј-O-yl}propionyl]thiolactyl-2R-alaninamide 16
To thioester 15 (26.3 mg, 48 µmol) was added 1% iodine in
methanol (2 ml), and the solution heated to reflux for 2 h. After
cooling, sodium thiosulfate was added until the solution
became clear. The mixture was filtered, washed through with
methanol and concentrated under reduced pressure to give a
white residue. The residue was extracted with isopropanol, fil-
tered, and concentrated under reduced pressure. The residue
was then extracted with water, filtered and freeze-dried over-
night. The white residue was purified by silica column chrom-
atography (70% iPrOH–20% H2O–10% MeOH, Rf 0.57)
to afford thioester 16 as a white solid (8.6 mg, 38% yield).
δH (300 MHz, D2O) 4.69 (1H, d, J = 2.6 Hz, H-1Ј), 4.23 (1H, q,
J = 5.3 Hz, –CHOCH3), 4.02–3.40 (8H, m), 3.21 (3H, s,
–OCH3), 1.85 (3H, s, –COCH3), 1.32 (3H, d, J = 5.6 Hz,
–CHOCH3), 1.25 (2 × 3H, d, J = 5.3 Hz) ppm; δC (75 MHz,
D2O) 177.1, 176.2, 172.3, 95.9, 77.6, 75.6, 69.8, 67.8, 58.5, 53.2,
51.3, 47.9, 39.9, 20.1, 16.6, 14.6, 14.1 ppm; m/z (MALDI) 488
(M ϩ Na, 10%).
S-(Propionyl)thiolactyl-D-Glu-ꢀ-L-Lys-OMe 21
Tripeptide 20 (6 mg) was dissolved in a mixture of trifluoro-
acetic acid (1 ml) and dichloromethane (1 ml). The reaction was
stirred for 2 h at room temperature, then the solvent was evap-
orated at reduced pressure. The residue was washed with diethyl
ether, then evaporated at reduced pressure to give tripeptide 21
as a pale green solid (6 mg, 100%). δH (300 MHz, CDCl3,
2 diastereoisomers) 7.3–7.1 (4H, m, NH), 4.45 (1H, m, CαH),
4.28 (1H, m, CαH), 3.65 (3H, s, –OCH3), 3.44 (1H, m,
–CHSCH3), 3.0–2.8 (2H, m), 2.56 (2H, q, J = 7.0 Hz,
–COCH2CH3), 2.25 (2H, m), 2.0–1.5 (6H, m), 1.37 & 1.32 (3H,
2 × d, J = 7.5 Hz, –CHSCH3), 1.15 (3H, t, J = 7.0 Hz,
–COCH2CH3), 1.0–0.9 (2H, m) ppm; m/z (ESϩ) 434.2 (MHϩ,
45%); HRMS (FABϩ) 434.1973 (C18H32N3O7S requires
434.1961).
Cbz-D-Glu(ꢁ-tBu)-ꢀ-L-Lys(N ꢂ-Boc)-OMe 19
To a solution of Cbz--glutamic acid α-tert-butyl ester
(0.615 g, 1.85 mmol) and triethylamine (0.28 ml, 2.0 mmol) in
dry dichloromethane (20 ml) at 0 ЊC was added dicyclohexyl-
carbodiimide (0.47 mg, 2.28 mmol). After stirring for 5 min,
hydroxybenzotriazole (30 mg) was added, followed by
-lysine(Nε-Boc) methyl ester (0.60 g, 2.2 mmol) and triethyl-
amine (0.31 ml, 2.2 mmol). The reaction was stirred for 24 h at
room temperature. The precipitated urea was filtered off,
and the filtrate was evaporated at reduced pressure. The residue
was dissolved in ethyl acetate (50 ml), and the solution was
washed with water (2 × 10 ml), 5% potassium hydrogen
sulfate solution (2 × 10 ml) and 5% sodium bicarbonate
solution (2 × 10 ml), sat. sodium chloride solution (2 × 10 ml).
The organic layer was dried (Na2SO4) and evaporated at
reduced pressure. The product was purified by silica column
chromatography (EtOAc, Rf 0.48) to give dipeptide 19 as a
colourless oil (0.235 g, 22% yield). δH (300 MHz, CDCl3)
7.26 (5H, s, Ar), 6.72 (1H, d, J = 7.7 Hz, NH), 6.47 (1H, d,
J = 7.4 Hz, NH), 5.61 (1H, d, J = 8.3 Hz, NH), 5.03 (2H, s,
–CH2Ph), 4.48 (1H, m, CαH), 4.26 (1H, m, CαH), 3.64 (3H,
s, –OCH3), 3.11 (2H, m, –CH2N), 2.35–1.6 (8H, m), 1.38 (9H, s,
tBu), 1.36 (9H, s, tBu), 1.25 (2H, m) ppm; δC (75 MHz, CDCl3)
173.3, 172.5, 171.4, 156.8, 156.4, 136.6, 128.9, 128.6, 128.5,
82.9, 79.3, 67.5, 54.2, 52.8, 52.5, 40.4, 32.8, 32.1, 29.9, 29.7,
28.8, 28.4, 22.9 ppm.
Expression of MurNAc-L-Ala amidases
Escherichia coli strain JM109/pSFP102 (expressing CwlA) was
grown with aeration at 37 ЊC in Luria broth containing 100 µg
mlϪ1 ampicillin; BL21/Pet24d (expressing CwlA) was grown
with aeration at 25 ЊC in Luria broth containing 0.5% glucose
and 30 µg mlϪ1 kanamycin; and JM109/pBCPL21 (expressing
Ply21) was grown at 25 ЊC with aeration in Luria broth contain-
ing 100 µg mlϪ1 ampicillin. Induction of each culture was
carried out by addition of isopropylthio--galactoside (IPTG,
0.5 mM) at A595 0.6, then growth was continued for a further
5 h. Cells were harvested by centrifugation (6000g, 10 min),
and were re-suspended in 50 mM Tris buffer pH 7.5 containing
20 mM MgCl2, and cells lysed by sonication. Cell debris was
removed by centrifugation (10 000g, 20 min), to give the crude
cell extracts, which were examined by SDS polyacrylamide
electrophoresis. The soluble protein fraction was obtained by
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722
1721

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further centrifugation (100000g, 30 min), which in the case
of Ply21 yielded soluble enzyme. In the case of CwlA, the
pellet was re-suspended by treatment with 8 M urea, followed
by dialysis vs. 3 M urea (1 l).
and the University of Southampton for an MRes studentship
(J. T.), and EPSRC and the University of Warwick for an MSc
studentship (J. H.). We thank Dr Simon Foster (University
of Sheffield) for the gift of plasmids pSFP102 and pET24d
containing the cwlA gene, and Professor Martin Loessner
(Technische Universität München) for the gift of plasmid
pBCPL21 containing the ply gene. We thank Dr Howard Clase
for solving the X-ray crystal structure of compound 6. We also
thank Dr Adrian Lloyd (University of Warwick) for assistance
with biochemical work.
Renaturing SDS-PAGE assay for peptidoglycan hydrolase
activity
The procedure of Leclerc et al. was employed,27 using bacterial
cell walls from Bacillus cereus W23 (prepared using the method
of Mintz et al. 30). The SDS-PAGE gel was prepared in the
presence of 0.1% (w/v) cell walls. After electrophoresis, the gel
was incubated in 1% (v/v) Triton X-100 for 3 × 30 min at 20 ЊC,
then in 0.1% Triton X-100 for 16 h at 37 ЊC. The gel was then
stained with 0.1% (w/v) methylene blue in 0.01% KOH. Bands
containing peptidoglycan hydrolase activity appeared as a clear
zone, against a dark blue background (see Fig. 3).
References
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Crystal data for 617
C38H35NO3S, M = 585.73, triclinic, a = 8.6803(6), b = 9.1105(6),
c = 10.6271(8) Å, U = 773.11(9) Å3, T = 180(2) K, space group
P1, Z = 1, µ = 0.143 mmϪ1, 5048 reflections measured, 4151
unique reflections (Rint 0.0482), R indices (all data) R1 = 0.0618,
wR2 = 0.1178. The configuration of the C-3 chiral centre was
determined by comparison with the known chirality of C-8,
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Acknowledgements
We thank EPSRC for a PhD studentship (R. L. H.), EPSRC
1722
J. Chem. Soc., Perkin Trans. 1, 2002, 1714–1722