Enantioselective synthesis of α-benzylated lanthionines and related tripeptides for biological incorporation into E. coli peptidoglycan

Article abstract of DOI:10.1039/c4ob01476f

The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantioselective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, followed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal, Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines. These compounds were further converted in a one pot process to the corresponding protected tripeptides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A₂pm. These compounds were evaluated for introduction, by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under different tested conditions probably due to the bulky benzyl group at the C_α carbon of the C-terminal amino acid. This journal is

Full text of DOI:10.1039/c4ob01476f

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Enantioselective synthesis of α-benzylated
lanthionines and related tripeptides for biological
incorporation into E. coli peptidoglycan†
Cite this: Org. Biomol. Chem., 2014,
12, 9853
Thibaut Denoël,a Astrid Zervosen,*a Christian Lemaire,a Bernard Joris,b
Mireille Hervé,c Didier Blanot,c Guillermo Zaragozad and André Luxena
The synthesis of modified tripeptides (S)-Ala-γ-(R)-Glu-X, where X = (R,S) or (R,R) diastereomers of
α-benzyl or α-(4-azidobenzyl)lanthionine, was carried out. The chemical strategy involved the enantio-
selective alkylation of a 4-MeO-phenyloxazoline. The reductive opening of the alkylated oxazolines, fol-
lowed by cyclization and oxidation, led to four PMB-protected sulfamidates. Subsequent PMB removal,
Boc protection and regioselective opening with cysteine methyl ester led to protected lanthionines.
These compounds were further converted in a one pot process to the corresponding protected tripep-
tides. After ester and Boc deprotection, the four tripeptides were evaluated as potential analogues of
the natural tripeptide (S)-Ala-γ-(R)-Glu-meso-A2pm. These compounds were evaluated for introduction,
by means of the biosynthetic recycling pathway, into the peptidoglycan of Escherichia coli. A successful
in vitro biosynthesis of UDP-MurNAc-tripeptides from the tripeptides containing α-benzyl lanthionine was
achieved using purified murein peptide ligase (Mpl). Bioincorporation into E. coli W7 did not occur under
different tested conditions probably due to the bulky benzyl group at the Cα carbon of the C-terminal
amino acid.
Received 14th July 2014,
Accepted 10th October 2014
DOI: 10.1039/c4ob01476f
www.rsc.org/obc
pentapeptide is transferred to membrane-bound bactoprenyl
phosphate by MraY. The next step is the formation of lipid II
Introduction
Peptidoglycan (PG) is unique to bacteria and essential for cell by reaction with UDP-GlcNAc under catalysis by MurG. This
survival. It lies outside the cytoplasmic membrane. It provides molecule is translocated to the periplasmic side of the plasma
the cells with osmotic stability and has also a vital function in membrane and is used there as a substrate for the poly-
bacterial morphogenesis. Enzymes catalysing the biosynthesis merisation reactions, transglycosylation followed by transpepti-
of PG are attractive targets for the development of new anti- dation, catalysed by penicillin-binding proteins (PBPs) (Fig. 1).
biotics.1 The biosynthesis starts in the cytoplasm by the for-
During growth and cell division a high fraction of PG is
mation of UDP-MurNAc from UDP-GlcNAc through the broken down by the action of lytic transglycosylases, amidases
involvement of MurA and MurB. The successive additions of and endopeptidases. Besides de novo biosynthesis of PG,
different amino acids are catalysed by different Mur ligases recycling of PG fragments generated during the growth
((S)-Ala/MurC, (R)-Glu/MurD, meso-A2pm/MurE, (R)-Ala-(R)- and division process is observed. Thus the end-product of the
Ala/MurF). Then the MurNAc-peptide moiety of UDP-MurNAc- degradation process, the tripeptide (S)-Ala-γ-(R)-Glu-meso-
A2pm, is transported by the MppA (murein peptide permease
A) to the cytoplasm where it is directly bound to UDP-MurNAc
under catalysis by Mpl (murein peptide ligase) (Fig. 1).
Herein we report the recycling of PG fragments as a novel
labeling approach of the PG of E. coli through the replacement
aCentre de Recherches du Cyclotron, Université de Liège, Allée du 6 Août 8, Bât B30,
B-4000 Sart-Tilman, Liège, Belgium. E-mail: azervosen@ulg.ac.be;
Fax: +32-4-366-2946; Tel: +32-4-366-3383
bCentre d’Ingénierie des Protéines, Université de Liège, Institut de Chimie, Bât B6,
of natural A2pm 1 (Fig. 2) in the tripeptide (S)-Ala-γ-(R)-Glu-
B-4000 Sart-Tilman, Liège, Belgium
cEnveloppes Bactériennes et Antibiotiques, UMR 8619 CNRS, Univ Paris-Sud, Bât
meso-A2pm by derivatives of lanthionine 2 (Fig. 2). Modified
amino acids (A2pm and Lys derivatives) in the form of tripep-
tide analogs of (S)-Ala-γ-(R)-Glu-meso-A2pm can be introduced
in vivo by the use of the permeases and Mpl activity of E. coli
to allow the biosynthesis of modified UDP-MurNAc-
tripeptides.2–4 The incorporation of the diastereomers of
430, 91405 Orsay, France
dUnidad de Rayos X, RIAIDT Edificio CACTUS, Universidad de Santiago de
Compostela, 15782 Santiago de Compostela, Spain
†Electronic supplementary information (ESI) available. CCDC 958602 and
958603. For ESI and crystallographic data in CIF or other electronic format see
DOI: 10.1039/c4ob01476f
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tripeptides 3 and 4 (Fig. 2), containing α-substituted lanthio-
nines, may open the door to the study of PG synthesis and
turnover in relation to cell growth and division.
Olrichs et al.5,6 have reported a lysine tagged with a fluore-
scent dye in the form of the tripeptide (S)-Ala-γ-(R)-Glu-(S)-Lys-
NBD (5, Fig. 2). Tripeptides containing [35S]lanthionine and a
photoactivatable group situated on an α-carbon could poten-
tially be used to study PG biosynthesis. In vivo, upon UV
irradiation, identification of proteins involved in the bio-
synthesis or biodegradation of the PG or in the cell division
should be feasible.
In this report, we describe a diastereoselective synthesis of
α-substituted tripeptides 3 and 4 (Fig. 2). The benzyl moiety
was used as a model for the benzylazido group, one of
the smallest photoactivatable groups. Moreover, results of bio-
logical incorporation of those molecules into E. coli PG are
presented.
Fig. 1 Metabolism of PG in E. coli. The de novo biosynthesis of PG
starts with the synthesis of UDP-MurNAc-pentapeptide from UDP-Glc-
NAc in the cytoplasm (enzymes: MurA, MurB, MurC, MurD, MurE and
MurF). The second part, the synthesis of lipid II catalyzed by MraY and
MurG, takes place at the level of the cytoplasmic membrane. Finally, the
polymerisation reactions (transglycosylation and transpeptidation) take
place in the periplasm. During the cell growth and cell division the PG is
partly degraded by numerous hydrolases. The degradation products are
re-imported by permeases (AmpG, MppA) and further degraded (AmpD,
NagZ, LdcA). The end-product, the tripeptide (S)-Ala-γ-(R)-Glu-meso-
A2pm, is re-introduced into the biosynthesis of PG by murein peptide
ligase (Mpl).
Results and discussion
Chemical synthesis
Retrosynthetic analysis. The synthesis of tripeptides 3 and 4
(Fig. 2) could be envisioned from a protected lanthionine
through amide coupling and usual deprotection (Scheme 1).
The lanthionine derivative could be obtained via SN2 opening
of a sterically crowded sulfamidate with a cysteine derivative.7,8
The sulfamidate could arise from an α-alkylated serine9 avail-
able in an enantioselective way by alkylation of an oxazoline
precursor.8,10 A phenyloxazoline, substituted with a para-
methoxy group, will provide a sulfamidate carrying a para-
methoxy benzyl (PMB) protective group (removable11 by
oxidative treatment with CAN) (Scheme 1).
Preparation of precursor 12. Phenyloxazoline 12 was pre-
pared from anisaldehyde 6 in six steps (Scheme 2). First, a
reaction of 6 with hydroxylamine in the presence of formic
acid afforded nitrile
7 on a multimolar scale (step i,
Scheme 2).12 Likewise, conversion to the imidate 8 was done at
the molar scale (step ii). The reaction conditions previously
described13 were modified using AcCl to generate HCl in situ.
The amount of EtOH had to be increased in order to solubilize
7. As indicated by NMR, a small amount of amide that does
Scheme 1 Retrosynthetic approach to α-alkylated lanthionine deriva-
tives. P1 = Me, P2 = Boc, PMB, P3 = tBu.
Fig. 2 Structural formulas of representative molecules.
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Scheme 2 Synthesis of the oxazoline precursor. (i) H2NOH·HCl, pyri-
dine, HCOOH, reflux, 1 h, 85%; (ii) EtOH, AcCl, Et2O, 2 °C, 1 month, 53%;
(iii) Na2CO3, H-(S)-Ser-OMe·HCl, MeOH, reflux, 2 h, 68%; (iv) aq. NaOH,
MeOH, acetone, rt, 1 h, 99%; (v) HCOOH, water, 0 °C, 1 h, 85%; (vi)
Boc2O (2 eq.), DMAP (10 mol%), 1 : 1 CH2Cl2–t-BuOH, reflux, 1 h, 90%.
not disturb the subsequent reaction was formed. To obtain 9,
the free base of 8 was reacted with serine methyl ester hydro-
chloride in MeOH (step iii).8
After saponification with aqueous NaOH, the salt 10 was
precipitated in acetone (step iv).14 To minimize the protona-
tion of the oxazoline ring and subsequent hydrolysis,14 acidifi-
cation of 10 was conducted at 0 °C with formic acid to yield 11
(step v). The tert-butyl ester 12 was then synthesized with
Boc2O–DMAP (step vi).8 Starting from 8, 62 g of 12 were
obtained (four steps, 51%).
Preparation of Maruoka’s catalyst 13. In order to obtain a
high diastereoselectivity in the synthesis of the α-substituted
tripeptides 3 and 4, enantioselective alkylation of oxazoline
precursor 12 is mandatory. Among all the chiral phase transfer
catalysts (PTC) described in the literature,15 Maruoka’s catalyst
13 (Fig. 3) seems the most active while exhibiting a high
enantioselectivity.16 Unfortunately, it is presently commercially
unavailable. The two enantiomers of catalyst 13 were thus pre-
pared according to a slightly modified procedure (see ESI†).17
Scheme 3 Synthesis of sulfamidates 19 and 26. (i) RX, CsOH (5 eq.),
(R)-13, toluene, −20 °C; (ii) NaN3 (2 eq.), sodium ascorbate (15 mol%),
CuI (20 mol%), DMEDA (30 mol%), aq. EtOH, reflux, 5 h, 99%, (iii)
NaBH3CN (6 eq.), AcOH, rt, 16 h; (iv) SOCl2, imidazole, NEt3, CH2Cl2,
−10 °C → rt, 2 h, 98%; (v) NaIO4 (1.2 eq.), RuCl3 (1 mol%), aq. CH3CN, rt,
4 h; (vi) (NH4)2Ce(NO3)6 (CAN) (3 eq.), aq. CH3CN, rt, 20 min, 53–80%;
(vii) Boc2O, DMAP (10 mol%), NEt3 (cat.), CH3CN, rt, 16 h, 90–93%.
Table 1 Enantioselective alkylation of 12
RXa
Entry (eq.)
PTC
(mol%)
Productb
(ee%)
Yieldc
(%)
Solvent
CH2Cl2
T (°C)
1
2
3
4
5
6
7
8
A (3)
rt
rt
rt
rt
(S)-13 (1)
(S)-13 (1)
(S)-13 (2)
(S)-13 (10)
(S)-20 (60) 79
(S)-20 (63) 73
(S)-20 (66)
(S)-20 (64)
(S)-20 (96)
(S)-20 (96) 87
(S)-20 (97)
(S)-20 (90)
A (1.5) CH2Cl2
A (1.5) CH2Cl2
A (1.5) CH2Cl2
—
—
—
A (1.5) Toluene −20 °C (S)-13 (2)
A (1.5) Toluene −20 °C (S)-13 (1)
A (1.5) Toluene −20 °C (S)-13 (0.5)
A (1.5) Toluene −20 °C (S)-13 (0.1)
—
—
—
9
A (1.5) Toluene −20 °C (S)-13 (0.01) (S)-20 (65)
10
11
12
A (1.5) Toluene −20 °C (R)-13 (1)
B (1.5) Toluene −20 °C (S)-13 (1)
(R)-20 (90) 99
(S)-14 (97) 93
(R)-14 (89) 99
B (3)
Toluene −20 °C (R)-13 (0.5)
a A: 4-Br-BnBr, B: BnBr (in molar eq.). b Enantiomeric excesses were
determined by HPLC on a Chiracel OD-H column (Daicel, 150 mm ×
4 mm, 5 µm) with 98 : 2 (n-Hex–i-PrOH) as an eluent at 1 mL min−1
,
37 °C; retention times for isomers S and R were 9.0 min and 15.5 min,
respectively. c Isolated yields.
Enantioselective alkylation of 12. With both antipodes of
the chiral catalyst 13 in hand, we proceeded to perform the
enantioselective alkylation of 12 with benzyl bromide and
para-bromo benzyl bromide (step i, Scheme 3).8,10 The reaction
was investigated in CH2Cl2 or toluene with 5 eq. of CsOH(s) as
a base at various temperatures (Table 1). First, alkylation of 12
was realized with para-bromo benzyl bromide and catalyst
Fig. 3 Phase transfer catalyst (PTC) (R)-13 that was used for the
enantioselective alkylation of 12.
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(S)-13 (Table 1, entries 1–9). When the reaction was carried out
at rt in CH2Cl2, the enantiomeric excesses (ee) of (S)-20 were
poor (Table 1, entries 1–4). By substituting toluene for CH2Cl2
and operating at −20 °C the ee increased from 63 (entries 1–4)
to 96% (entries 5–7) using only 1.5 eq. of electrophile. Decreas-
ing the amount of the catalyst from 0.5 mol% to 0.1 mol%
resulted in a decrease in ee from 97% to 90% (entries 7 and 8).
The use of ten times less equivalents of catalyst (0.01 mol%)
led to a drastic drop in ee (65%, entry 9).
Fig. 4 Capped sticks representation of compounds (R)-18 and (R)-25
solved by single crystal X-ray diffraction.
Thus, alkylation of 12 was performed in multigram quantity
with 1 mol% of (S)-13. After reaction for 72 h at −20 °C, (S)-20
was obtained in 87% yield with an ee of 96% (entry 6).
However, a slightly lower ee (90%) was observed when the
conditions, described in the literature to convert an amide
reaction was conducted with antipode (R)-13 in the same con- into an amido-carbamate,25 Boc-sulfamidates (R)-19 and (R)-26
were obtained in excellent yields.
ditions (entry 10).
Lanthionine 27 was formed after reaction of (R)-19 with the
In the same way, using benzyl bromide as an electrophile,
(S)-14 and (R)-14 were also obtained on a multigram scale methyl ester of (R)-cysteine (step i, Scheme 4) and was used in
the next step without purification. Removal8 of the sulfamate
(entries 11 and 12). Excellent yields (93–99%) and high ee were
group by aqueous NaH2PO4 at 50 °C afforded the protected
The bromine atom carried by (R)-20 was then substituted by lanthionine 28 in good yield (78%, steps i and ii, Scheme 4).
observed (89–97%).
Synthesis of dipeptide 33. To prepare tripeptide 34 from
lanthionine 27 (Scheme 4), dipeptide 33 was necessary
(Scheme 5).
an azido group. The reaction was catalyzed by a Cu(I) salt and
DMEDA (N,N′-dimethylethylenediamine) was used as a ligand
(step ii, Scheme 3).18
The γ-carboxylic acid function of (R)-glutamic acid 29 was
selectively esterified with methanolic HCl for a period of 2 h at
Synthesis of sulfamidates 18 and 25. Sulfamidates 18 and
25 were synthesized in four steps from the 4-alkylated oxazo-
lines (R)-14 and (R)-21 (steps iii–vi, Scheme 3). Steps iii–v were rt (step i, Scheme 5).26 Treatment with pyridine in aqueous
conducted in accordance with our recent publication.8 Briefly,
MeOH gave the zwitterion 30, which was converted to α-tert-
reduction19 of oxazoline (R)-14 with NaBH3CN in acetic acid
butyl ester 31 by transesterification with tert-butyl acetate (step
afforded the α-alkylated serines (R)-15 (step iii). Cyclization ii).26 After basification, diester 31 was reacted with Boc-(S)-
alanine using carbonyl diimidazole (CDI) (step iii). The methyl
ester of 32 was then deprotected with LiOH to give dipeptide
into sulfamidites (R)-16 occurred with SOCl2 in the presence
of imidazole (step iv).20 The crude intermediate was then
oxidized to the sulfamidate (R)-17 with a catalytic amount of 33 (step iv, Scheme 5).
RuO4 (step v).9 The PMB protecting group of (R)-17 was then
removed using CAN in CH3CN (step vi).11 The sulfamidate
(R)-18 was purified by recrystallization.
Using the same approach, sulfamidates (S)-18, (R)-25 and
(S)-25 were obtained in an overall yield of 42–60% (four steps)
Synthesis of tripeptides 3RS, 3RR, 4RS, 4RR. To prepare tri-
peptides 35 and 39, a three step one pot reaction was con-
sidered (steps i, iii and ii; Scheme 4). First, the sulfamidates
(R)-19 and (R)-26 were opened in CH3CN with cysteine methyl
ester and DBU (step i, Scheme 4). After solvent evaporation,
from (S)-14, (R)-21 and (S)-21, respectively. These crystalline coupling of 27 and 37 was realized in CH2Cl2 with activated 33
to afford tripeptides 34 and 38, respectively (step iii). Activation
of dipeptide 33 was previously done with HBTU in CH2Cl2.
Substitution of CH2Cl2 for CH3CN was required for high-yield-
ing amide formation. The sulfamate function of 34 and 38 was
then hydrolysed (step ii) to provide, after chromatography, the
compounds can be stored at
without decomposition. Notably, the azido group was stable
under those reaction conditions.
The solid-state structures of compounds (R)-18 and (R)-25
were determined by X-ray diffraction analysis (XRD) (Fig. 4).
2 °C for several years
Single crystals of both compounds were crystallized in protected tripeptides 35 and 39 in 78–84% yield (three steps).
Removal of the methyl ester of the tripeptides 35 and 39 using
LiOH in aqueous CH3CN (16 h, rt) also gave partial hydrolysis
chiral space groups (R)-18 (CCDC 958602) in monoclinic P21
and (R)-25 (CCDC 958603) in orthorhombic P212121. The abso-
lute configurations of both structures were determined using of the tert-butyl ester on the glutamic acid moiety (step iv). The
the Flack parameter,21 0.03(4) for (R)-18 and 0.10(8) for (R)-25.
target tripeptide 3RS was obtained by treatment of the crude
Synthesis of protected lanthionine 28. Attempts to open7–9
product 36 with a large excess of TFA in CH2Cl2 (10%) (step v).
the sulfamidate (R)-18 by the methyl ester of (R)-cysteine Deprotection of the azido tripeptide 40 with this reagent
resulted in partial tert-butylation of the phenylazido group
in CH3CN with DBU failed to afford the desired product. As
carbamate-protected sulfamidates are more reactive towards
even in the presence of triethylsilane.27 Thus, an alternative
nucleophiles,22,23 the nitrogen of the sulfamidates (R)-18 and deprotection method was selected, involving the use of HCl in
(R)-25 was protected with a tert-butyl carbamate group.24 For
dioxane, followed by a trituration of the dihydrochloride of
4RS in Et2O (step vi, Scheme 4). The synthesis of the epimers
both compounds, the reaction proceeded with Boc2O in the
presence of DMAP in CH3CN (step vii, Scheme 3). Using these 3RR and 4RR (Fig. 2) was done in the same way starting from
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Scheme 4 Synthesis of protected lanthionine 28 and tripeptides 3RS and 4RS. (i) Cysteine Me ester·HCl (1 eq.), DBU (1.1 eq.), CH3CN, rt, 1 h; (ii) 10%
aq. NaH2PO4, EtOAc, 50 °C, 2 h; (iii) 33, HBTU, DiPEA, CH2Cl2, rt, 16 h; (iv) LiOH (1.5 eq.), aq. CH3CN, rt, 16 h, 94–97%; (v) 10% TFA, CH2Cl2, rt, 16 h;
73%; (vi) 10 M HCl –dioxane: 1/3, 50 °C, 8 h; 88%.
Table 2 Specificity of Mpl for tripeptide substrates
Tripeptide
Mpl
(S)-Ala-γ-(R)-Glu-meso-A2pm
(S)-Ala-γ-(R)-Glu-(S)-Lys
(S)-Ala-γ-(R)-Glu-ε-(R)-Lys
kcat/Km = 2900 mM−1 min−1
(ref. 3)
kcat/Km = 3.9 mM−1 min−1
(ref. 3)
kcat/Km = 2400 mM−1 min−1
(ref. 4)
Scheme 5 Synthesis of dipeptide 33. (i) MeOH, AcCl (1.2 eq.), rt, 2 h
and then pyridine (1.3 eq.), aq. MeOH, 0 °C, 16 h, 50%; (ii) t-BuOAc,
H2SO4 (1.5 eq.), rt, 24 h, 49%; (iii) (S)-Boc-Ala, CDI, CH2Cl2, rt, 16 h, 45%;
(iv) LiOH·H2O (1.2 eq.), aq. CH3CN, rt, 4 h and then aq. NaHSO4, 74%.
(S)-Ala-γ-(R)-Glu-p-amino-(S)-
phenylalanine
(S)-Ala-γ-(R)-Glu-p-nitro-(S)-
phenylalanine
(S)-Ala-γ-(R)-Glu-(R,S)-α-benzyl-
lanthionine (3RS)
(S)-Ala-γ-(R)-Glu-(R,R)-α-benzyl-
lanthionine (3RR)
50% conversiona,4
10% conversiona,4
70% conversiona
40% conversiona
the sulfamidates (S)-19 and (S)-26, respectively. The global
yield was 55–69% (five steps). The proposed synthesis
(Scheme 4), featuring a one pot procedure and only one chro-
matographic purification, should be suitable for the future
preparation of [35S]tripeptides from commercially available
[35S]cysteine.
a 1 mM tripeptide, 2 µg Mpl, 0.4 mM UDP-[14C]MurNAc in 50 µL, 16 h
at 37 °C.
Results of biological experiments
In vitro assays with Mpl. Tripeptide 3RS ((S)-Ala-γ-(R)-Glu-
(R,S)-α-benzyl-lanthionine) and its corresponding diastereomer
3RR ((S)-Ala-γ-(R)-Glu-(R,R)-α-benzyl-lanthionine) (Fig. 2) were
incubated with ATP, UDP-[14C]MurNAc and purified murein
peptide ligase (Mpl, Fig. 1). HPLC analysis of the reaction mix-
tures showed the appearance of new radioactive products (70%
conversion with 3RS and 40% with 3RR, with respect to UDP-
MurNAc). However, prolonged incubation (16 h) and a large
amount of enzyme (2 µg per 50 µL) were necessary (Table 2).
To identify these new compounds, the reactions were repeated
with non-labeled UDP-MurNAc and the products were purified
by HPLC. Mass spectrometry analysis confirmed that they were
UDP-MurNAc-tripeptides containing the respective lanthionine
derivatives (Fig. 5).
Fig. 5 MALDI-TOF mass spectrometry (negative mode) of the UDP-
MurNAc-tripeptide obtained from 3RS. Theoretical mass C41H58N7O26
P2S: m/z = 1158.26. (For 3RR, see ESI part.†)
-
In vivo assays of bacterial growth. Tripeptides 3RS and
3RR were evaluated in incorporation experiments. No
growth of E. coli cells W7 was observed in minimal medium28
(supplemented with 50 µg mL−1 (S)-Lys) in the presence of
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Indeed, even if 3RS and 3RR are substrates of Mpl in vitro,
as a high enzyme concentration and a long incubation
time are necessary, these are poor substrates. Therefore, the
tripeptide concentration (50 µg mL−1, 0.1 mM) in our culture
conditions was maybe too low. Thus, the subsequent incorpor-
ation experiment with 3RS was performed in the presence of a
high tripeptide concentration (500 µg mL−1, 1 mM).
Moreover, the transport of tripeptides from the culture
medium to cells depends on murein peptide permease A
(MppA, Fig. 1).30 If tripeptides are poor ligands of this per-
mease, the tripeptide concentration inside the cells will be
very low. The periplasmic protein MppA binds the tripeptide
(S)-Ala-γ-(R)-Glu-meso-A2pm, which is then transported to the
cytoplasm via membrane components of the oligopeptide per-
mease (Opp) pathway.30 In contrast to most transport systems,
which are tightly regulated, the oligopeptide permease
genes are expressed constitutively.31 It is well known that the
glucose concentration can significantly change the expression
level of genes in E. coli.32 The effect of glucose amount on the
expression of MppA and Mpl is not known but incorporation
experiments with the natural and modified murein tripeptides
described in the literature (Table 2) are mostly performed in
minimal medium supplemented with only 0.2% glucose.4,31
Thus, an incorporation experiment with 3RS was done with
a high tripeptide concentration (500 µg mL−1, 1 mM) and a
lower glucose concentration (0.2% instead of 0.5%) using
minimal medium. However, once more no incorporation was
observed (data not shown). Because of these negative results,
no incorporation experiments were attempted with 4RS
or 4RR.
Fig. 6 Optical density (600 nm) after 18 h of incubation at 37 °C in
minimal medium. In the absence of A2pm no growth was observed.
Fig. 7 Growth of E. coli in presence of A2pm, 3RS and 3RR. Com-
pounds (A2pm, 3RS, 3RR) were added at 50 µg mL−1 after the stationary
phase of bacterial growth was reached. A600 nm: optical density of
different cultures; A0600 nm: optical density of the culture at t = 7 h with
1 µg mL−1 A2pm. Control: no addition at t = 7 h. *p < 0.05: n = 2. aExperi-
ment 1. bExperiment 2 done under the same conditions.
Discussion. In E. coli, the first steps of incorporation into
PG of tripeptides analogous to (S)-Ala-γ-(R)-Glu-meso-A2pm
depend on the MppA and Mpl activities, catalyzing the
tripeptide uptake, and the synthesis of UDP-MurNAc-
tripeptide, respectively.4 The importance of the MppA activity
is nicely shown by the results obtained by Hervé et al. with
the tripeptide (S)-Ala-γ-(R)-Glu-ε-(R)-Lys.3 This tripeptide is a
good substrate of Mpl (Table 2), but probably a very poor
ligand of MppA owing to the lack of the proximal carboxylate
at the C-terminus, and thus was not incorporated in vivo.4
We have shown that the tripeptides (S)-Ala-γ-(R)-Glu-(R,S)-
α-benzyl-lanthionine 3RS and (S)-Ala-γ-(R)-Glu-(R,R)-α-benzyl-
lanthionine 3RR are poor substrates of Mpl in vitro (Table 2).
However, in vivo, no incorporation into the PG was observed
even in the presence of a high tripeptide concentration
(1 mM). In the literature, the tagged (S)-Ala-γ-(R)-Glu-(S)-Lys
analogue, (S)-Ala-γ-(R)-Glu-(S)-Lys-ε-NBD (5, Fig. 2), is incorpor-
ated at a concentration of 1 mM.5 Compared to the natural
(S)-Ala-γ-(R)-Glu-meso-A2pm tripeptide, the NBD-tripeptide 5
lacks one primary amino and one carboxylate. However, the
presence of a bulky substituent like NBD bound to the ε-amino
group is accepted.5 Despite the fact that 3RS and 3RR possess
the same amino and carboxylate functions as the natural
tripeptide, they were not incorporated into PG even at 1 mM. It
has been shown that (R,R) and (R,S)-lanthionine can effectively
replace meso-A2pm in vivo.33–37 Thus, the sulfur group in the
lanthionine
1 ) or tripeptide 3RS or 3RR
(50 µg mL−1
(50 µg mL−1 or 500 µg mL−1). It has been shown that A2pm
auxotrophy in E. coli can be complemented by exogenous
lanthionine in the presence of lysine in minimal medium
after a preliminary growth with small amounts of A2pm.29 It
is shown in Fig. 6 that a minimal concentration of 1 µg mL−1
A2pm is sufficient for the growth of bacterial cells.
Therefore, bacteria were first grown in the presence of 1 µg
mL−1 A2pm. When the stationary phase was reached at t = 7 h,
A2pm, 3RS or 3RR (50 µg mL−1) was added (Fig. 7).
At t = 9 h, with A2pm, an increase of bacterial growth was
observed. In contrast, much smaller variations occurred with
3RS or 3RR. The last results are probably due to a coincidence
arising from random sampling (p > 0.05) and not an
indication of a poor incorporation or a toxic effect of these
tripeptides (Fig. 7).
PG production and analysis with 3RS and 3RR. The in vivo
assays analyzing bacterial growth may not be sensitive enough
for studying the incorporation of 3RS and 3RR. Thus, one-liter
cultures have been performed in minimal medium supple-
mented with 0.5% glucose, 1 µg mL−1 A2pm and 50 µg mL−1
3RS or 3RR. PGs of the different cultures have been purified,
hydrolyzed and subjected to amino acid analysis. However,
under these conditions, no incorporation of compound 3RS or
3RR was observed (data not shown).
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Paper
tripeptides is probably not responsible for the disappointing to give the title compound (62 g, 90%) as pale yellow crystals.
results of our in vivo experiments. However, the bulky benzyl Mp 64–66 °C; 1H NMR (250 MHz, CDCl3) δH 7.89 (d, J = 8.8 Hz,
group at the Cα carbon of the C-terminal amino acid probably 2H, Ar-H), 6.87 (d, J = 8.8 Hz, 2H, Ar-H), 4.77 (dd, J = 10.2,
disturbed the incorporation of 3RS and 3RR.
8.0 Hz, 1H, H-α), 4.63–4.36 (m, 2H, CH2), 3.80 (s, 3H, OCH3),
1.47 (s, 9H, C(CH3)3); 13C NMR (63 MHz, CDCl3) δC 170.70
(CO), 165.85 (CN), 162.39 (Ar-C), 130.39 (Ar-C), 119.71 (Ar-C),
113.67 (Ar-C), 82.01 (C(CH3)3), 69.75 (C-β), 69.45 (C-α), 55.37
(OCH3), 28.06 (C(CH3)3); m/z (ESI) 278 [MH]+; HRMS m/z (ES+)
Conclusion
In conclusion, we have developed a stereoselective synthesis of Calcd for C15H20NO4 278.1387, found 278.1385 [MH]+.
α-alkylated sulfamidates 19 and 26. These efficient electro- (R)-tert-Butyl 4-benzyl-2-(4-methoxyphenyl)-oxazoline-4-carb-
philes were transformed into protected lanthionines 27 and 37 oxylate 14. Phenyloxazoline 12 (6.93 g, 25 mmol) was dis-
which, in turn, were readily converted into tripeptides 3RS, solved in toluene (250 mL). Benzyl bromide (12.8 g, 75 mmol)
3RR, 4RS and 4RR (Scheme 4). The tripeptides (S)-Ala-γ-(R)- and (R)-13 (102 mg, 0.5 mol%) were then added. The flask was
Glu-(R,S)-α-benzyl-lanthionine 3RS and (S)-Ala-γ-(R)-Glu-(R,R)- flushed with nitrogen and cooled to −20 °C. CsOH·H2O (21 g,
α-benzyl-lanthionine 3RR are poor substrates of Mpl in vitro. 125 mmol) was added, the flask was capped and the mixture
No in vivo incorporation of these tripeptides into the PG of vigorously stirred for 72 h at −20 °C. The suspension was
E. coli was observed.
diluted with hexanes (250 mL), filtered and the organic layer
was flash chromatographed on 100 g of silica. Elution was
done first with hexanes to remove excess BnBr, and then with
2 : 3 (EtOAc–hexanes) to recover 14. The solvent was evaporated
in vacuo to give the title compound (9.2 g, 99%) as a yellow oil.
1H NMR (250 MHz, CDCl3) δH 7.95–7.82 (m, 2H, Ar-H),
Experimental
Reagents
All solvents and chemicals were of analytical grade and used 7.30–7.11 (m, 5H, Ar-H), 6.91–6.79 (m, 2H, Ar-H), 4.63 (d, J =
without further purification. DMF and CH3CN were dried 8.9 Hz, 1H, CH2O), 4.27 (d, J = 8.9 Hz, 1H, CH2O), 3.77 (s, 3H,
on 3 Å molecular sieves. TLC: Macherey-Nagel Polygram SIL OCH3), 3.27 (d, J = 13.8 Hz, 1H, CH2Ph), 3.19 (d, J = 13.8 Hz,
G/UV254 using UV light or an anisaldehyde/sulphuric acid/ 1H, CH2Ph), 1.46 (s, 9H, C(CH3)3); 13C NMR (63 MHz, CDCl3)
AcOH in EtOH stain. CC: silica gel Acros, 0.060–0.200 mm, δC 171.43 (CO), 164.27 (CN), 162.18 (Ar-C), 135.76 (Ar-C),
60 Å. HPLC: Waters system (600 pump, 717 autosampler, 996 130.28 (Ar-C), 130.18 (Ar-C), 128.03 (Ar-C), 126.69 (Ar-C),
PDA detector) with a column XTerra RP18 (4.6 × 150 mm; 119.75 (Ar-C), 113.49 (Ar-C), 81.86 (C(CH3)3), 78.73 (C-α), 72.72
3.5 μm). Mp: Büchi Melting Point B-545 calibrated on three (CH2O), 55.16 (OCH3), 43.21 (CH2Ph), 27.82 (C(CH3)3); m/z
1
points (83, 136 and 237 °C). H and 13C NMR: Bruker Avance (ESI) 368 [MH]+.
DRX 400 (1H at 400 MHz and 13C at 101 MHz) and Bruker AM
(R)-tert-Butyl 2-benzyl-3-hydroxy-2-(4-methoxybenzylamino)
250 (1H at 250 MHz and 13C at 63 MHz); δ in parts per million propanoate 15. A freshly prepared solution of NaBH3CN (10 g,
relative to 13C or the residual proton signal of deuterated 159 mmol) in AcOH (200 mL), cooled to 0 °C, was added to
solvent. MS: Thermoquest Finnigan TSQ 7000 mass spectro- oxazoline 14 (25 mmol) and the mixture was stirred at rt for
meter operating in full scan MS mode with an ESI source. 16 h. The solvent was removed in vacuo and the residue parti-
HRMS: ESI-FT-ICR mass spectrometer (SolariX, Bruker) in the tioned between EtOAc and a saturated Na2CO3 solution. The
positive ion mode. For some samples 1 mM LiI was used for basic aqueous layer was extracted twice with EtOAc and the
adducts. External calibration was done over the range of m/z pooled organic fractions were washed twice with water and
150–700 and the mean residual error obtained was <1 ppm. brine. After drying over MgSO4 and filtration, the solvent was
Elemental analysis: Flash EA 1112 Series (Thermo Electron removed in vacuo to give the title compound (9.2 g, 99%) as a
Corporation).
colourless oil that crystallized on standing at 2 °C. Mp
tert-Butyl 2-(4-methoxyphenyl)-oxazoline-4-carboxylate 12. 49–52 °C; 1H NMR (250 MHz, CDCl3) δH 7.33–7.23 (m, 7H, Ar-H),
The esterification was done as previously described for an ana- 6.94–6.85 (m, 2H, Ar-H), 3.79 (s, 3H, OCH3), 3.79 (d, J =
logous derivative.8 Under nitrogen, in a 500 mL round bottom 11.5 Hz, 1H, CH2O), 3.71 (d, J = 11.8 Hz, 1H, CH2N), 3.66 (d,
flask equipped with an efficient condenser were placed 11 J = 11.5 Hz, 1H, CH2O), 3.65 (d, J = 11.8 Hz, 1H, CH2N), 3.07
(55.2 g, 250 mmol), DMAP (6.1 g, 20 mol%), CH2Cl2 (400 mL) (d, J = 13.4 Hz, 1H, CH2Ph), 2.95 (d, J = 13.4 Hz, 1H, CH2Ph),
and t-BuOH (200 mL). The solution was heated at reflux and 2.46 (br, 2H, NH, OH), 1.46 (s, 9H, C(CH3)3); 13C NMR
Boc2O (108 g, 500 mmol) was added via a syringe in two por- (63 MHz, CDCl3) δC 172.72 (CO), 158.82 (Ar-C), 135.68 (Ar-C),
tions (20 minutes apart). CAUTION: This step produces sub- 131.90 (Ar-C), 130.32 (Ar-C), 129.28 (Ar-C), 128.10 (Ar-C),
stantial gas evolution. The mixture was heated for 1 h and the 126.79 (Ar-C), 113.93 (Ar-C), 81.85 (C(CH3)3), 66.97 (C-α), 60.82
solvents were evaporated. The resulting yellow oil was dis- (CH2O), 55.15 (OCH3), 46.74 (CH2N), 40.13 (CH2Ph), 28.03
solved in CH2Cl2 (400 mL) and MeOH (20 mL). The solution (C(CH3)3); m/z (ESI) 372 [MH]+.
was prepurified by passing through 80 g of silica which was
washed with the same eluent (300 mL). The solvents were oxathiazolidine-4-carboxylate 16. In
removed in vacuo and the oil was crystallized in Et2O–hexanes flask under nitrogen were added compound 15 (25 mmol),
(4R)-tert-Butyl
4-benzyl-3-(4-methoxybenzyl)-2-oxo-1,2,3-
a
dry round bottom
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imidazole20 (6.8 g, 100 mmol), NEt3 (10.4 mL, 75 mmol) and 320.1138 [MLi]+; (R)-18 [α]D20 = +63° (c 2, CH2Cl2) ([α]2D0 = −71°
CH2Cl2 (125 mL). The mixture was cooled to −10 °C and SOCl2 (c 2, CH2Cl2) for (S)-18).
(2.7 mL, 37.5 mmol) was added dropwise via a syringe. The
(4R)-tert-Butyl 4-benzyl-3-(tert-butyloxycarbonyl)-2,2-dioxo-
solution was kept for 30 min at −10 °C and then allowed to 1,2,3-oxathiazolidine-4-carboxylate 19. To a solution of sulfa-
return to rt during 2 h. The mixture was diluted with water midate 18 (207 mg, 0.66 mmol) in CH3CN (4 mL) were added
(50 mL) and thereafter with 10% aqueous NaHSO4 (150 mL). DMAP (8 mg, 10 mol%), Boc2O (174 mg, 0.66 mmol) and one
The organic layer was separated and the aqueous layer was drop of NEt3. After stirring at rt for 16 h, the solvent was evap-
extracted once with CH2Cl2. The pooled organic fractions were orated and the resulting oil was chromatographed on silica
washed twice with water and brine. The solution was dried with 1 : 9 (EtOAc–hexanes). Evaporation of the solvent gave the
1
over MgSO4, filtered and the solvent was removed in vacuo to title compound (253 mg, 93%) as colourless crystals. H NMR
give the title compound (10.2 g, 98%) as a yellow oil, a mixture (250 MHz, CDCl3) δH 7.43–7.24 (m, 3H, Ar-H), 7.24–7.06 (m,
of diastereoisomers.
2H, Ar-H), 4.45 (d, J = 9.7 Hz, 1H, CH2O), 4.41 (d, J = 9.7 Hz,
(4R)-tert-Butyl 4-benzyl-3-(4-methoxybenzyl)-2,2-dioxo-1,2,3- 1H, CH2O), 3.68 (d, J = 14.7 Hz, 1H, CH2Ph), 3.24 (d, J = 14.7
oxathiazolidine-4-carboxylate 17. The crude sulfamidite 16 Hz, 1H, CH2Ph), 1.60 (s, 9H, C(CH3)3), 1.52 (s, 9H, C(CH3)3);
(10.2 g, 24 mmol) was dissolved in CH3CN (500 mL) and the 13C NMR (63 MHz, CDCl3) δC 167.82 (CO), 148.50 (Boc-CO),
solution was cooled to 0 °C. RuCl3·xH2O (52 mg, 1 mol%) was 133.41 (Ar-C), 130.55 (Ar-C), 128.73 (Ar-C), 127.79 (Ar-C), 85.64
added followed by NaIO4 (6 g, 28 mmol) and water (250 mL).9 (C(CH3)3), 84.25 (C(CH3)3), 70.00 (CH2O), 68.55 (C-α), 37.30
The green-brown solution with a white precipitate was stirred (CH2Ph), 27.97 (C(CH3)3), 27.72 (C(CH3)3).
for 15 min at 0 °C and allowed to return to rt. After 4 h, the
(S)-tert-Butyl 3-((R)-2-amino-3-methoxy-3-oxopropylthio)-2-benzyl-
mixture was diluted with Et2O (250 mL) and brine (250 mL). 2-(tert-butoxycarbonylamino)propanoate 28. Under nitrogen,
The aqueous layer was extracted with Et2O (3 × 250 mL). The to a degassed solution of sulfamidate 19 (100 mg, 0.24 mmol)
pooled organic fractions were washed twice with saturated and cysteine methyl ester·HCl (42 mg, 0.24 mmol) in CH3CN
NaHCO3 and brine. The organic layer was dried over MgSO4, (4 mL) was added via a syringe a solution of DBU (84 mg,
filtered and the solvents evaporated in vacuo to yield crude 17. 0.55 mmol) in degassed CH3CN (1 mL). After stirring for 1 h at
After flash chromatography on silica with 1 : 4 (EtOAc– rt, EtOAc (20 mL) and 10% aqueous NaH2PO4 (20 mL) were
hexanes), the solvents were removed in vacuo to give the title added. The biphasic mixture containing 27 was heated with
compound (8.5 g, 80%) as a golden oil. 1H NMR (250 MHz, stirring at 50 °C for 2 h. After basification by pouring in an
CDCl3) δH 7.51–7.34 (m, 2H, Ar-H), 7.34–7.16 (m, 3H, Ar-H), excess of saturated Na2CO3 solution, the organic layer was
7.16–7.00 (m, 2H, Ar-H), 7.00–6.77 (m, 2H, Ar-H), 4.63 (d, J = recovered and the aqueous layer was extracted with EtOAc (3 ×
8.8 Hz, 1H, CH2O), 4.62 (d, J = 15.4 Hz, 1H, CH2N), 4.55 (d, J = 20 mL). The pooled organic fractions were washed with brine
15.4 Hz, 1H, CH2N), 4.43 (d, J = 8.8 Hz, 1H, CH2O), 3.77 (s, 3H, and dried over MgSO4. After filtration, evaporation and chrom-
OCH3), 3.38 (d, J = 13.4 Hz, 1H, CH2Ph), 2.83 (d, J = 13.4 Hz, atography on silica with 1 : 1 (EtOAc–hexanes), the title com-
1
1H, CH2Ph), 1.45 (s, 9H, C(CH3)3); 13C NMR (63 MHz, CDCl3) pound (88 mg, 78%) was obtained as a colourless oil. H NMR
δC 167.00 (CO), 159.35 (Ar-C), 132.41 (Ar-C), 129.81 (Ar-C), (250 MHz, CDCl3) δH 7.49–7.14 (m, 3H, Ar-H), 7.14–6.91 (m,
128.72 (Ar-C), 127.82 (Ar-C), 127.64 (Ar-C), 113.89 (Ar-C), 84.38 2H, Ar-H), 5.60 (s, 1H, NH-Boc), 3.80–3.71 (m, 1H, H-α), 3.69
(C(CH3)3), 72.20 (CH2O), 69.02 (C-α), 55.10 (OCH3), 46.49 (s, 3H, OCH3), 3.65 (d, J = 13.5 Hz, 1H, SCH2C), 3.55 (d, J =
(CH2N), 39.85 (CH2Ph), 27.66 (C(CH3)3).
13.5 Hz, 1H, CH2Ph), 3.05 (d, J = 13.5 Hz, 1H, SCH2C), 2.97 (d,
(4R)-tert-Butyl 4-benzyl-2,2-dioxo-1,2,3-oxathiazolidine-4-carb- J = 13.5 Hz, 1H, CH2Ph), 2.95 (dd, J = 13.3, 4.6 Hz, 1H,
oxylate 18. To a stirred solution of sulfamidate 17 (1.0 g, CHCH2S), 2.77 (dd, J = 13.3, 8.0 Hz, 1H, CHCH2S), 1.80 (br, 2H,
2.3 mmol) in CH3CN (23 mL) and water (6 mL) was added NH2), 1.44 (s, 18H, C(CH3)3); 13C NMR (63 MHz, CDCl3) δC
CAN11 (3.8 g, 6.9 mmol). After stirring at rt for 20 min, the 174.30 (CO), 170.27 (CO), 154.22 (CO-Boc), 135.86 (Ar-C),
solution was diluted with CH2Cl2 (100 mL) and decanted. The 129.99 (Ar-C), 128.10 (Ar-C), 127.00 (Ar-C), 83.44 (C(CH3)3),
organic layer was washed with water (100 mL), saturated 79.40 (C(CH3)3), 65.85 (C-α), 54.56 (CH-α), 52.16 (OCH3), 40.82
NaHCO3 (100 mL) and dried over MgSO4. After filtration, the (CH2Ph), 38.40 (SCH2C), 38.05 (CHCH2S), 28.44 (C(CH3)3),
solvents were evaporated in vacuo to yield crude 18. Chromato- 28.01 (C(CH3)3); m/z (ESI) 469 [MH]+; HRMS m/z (ES+) Calcd
graphy on silica with 3 : 7 (EtOAc–hexanes) followed by removal for C23H37N2O6S 469.2367, found 469.2367 [MH]+.
of solvents afforded an oil which was crystallized in Et2O–
(R)-1-tert-Butyl 5-methyl 2-aminopentanedioate 31. In
hexanes. The title compound (380 mg, 53%) was obtained as a reactor equipped with a mechanical stirrer were placed
colourless crystals. Mp 78–80 °C; 1H NMR (250 MHz, CDCl3) (R)-2-amino-5-methoxy-5-oxopentanoic acid 30 (8.05 g) and
δH 7.38 (s, 5H, Ar-H), 5.50 (b, 1H, NH), 4.82 (d, J = 9.2 Hz, 1H, tert-BuOAc (150 mL). The reactor was cooled to 0 °C and con-
CH2O), 4.52 (d, J = 9.2 Hz, 1H, CH2O), 3.38 (d, J = 13.7 Hz, 1H, centrated H2SO4 (4.1 mL, 75 mmol) was added. The reactor
CH2Ph), 3.22 (d, J = 13.7 Hz, 1H, CH2Ph), 1.51 (s, 9H, C(CH3)3); was closed and stirring was continued at rt for 24 h. The
13C NMR (63 MHz, CDCl3) δC 168.65 (CO), 133.82 (Ar-C), mixture was poured on an excess of 10% aqueous Na2CO3.
130.22 (Ar-C), 128.43 (Ar-C), 127.78 (Ar-C), 85.65 (C(CH3)3), After extraction with Et2O (3 × 200 mL), the pooled organic
74.69 (CH2O), 68.57 (C-α), 42.49 (CH2Ph), 27.75 (C(CH3)3); layers were washed with water and brine. Drying over Na2CO3
HRMS m/z (ES+) Calcd for C14H19LiNO5S 320.1139, found followed by filtration and solvent evaporation gave the title
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1
compound (5.3 g, 49%) as a colourless oil. H NMR (250 MHz, added, via a syringe, a degassed solution of DBU (304 mg,
CDCl3) δH 3.52 (s, 3H, OCH3), 3.18 (dd, J = 8.2, 5.3 Hz, 1H), 2 mmol) in CH3CN (10 mL). The resulting solution was stirred
2.31 (t, J = 7.6 Hz, 2H), 1.95–1.79 (m, 1H), 1.74–1.55 (m, 1H), for 1 h at rt under nitrogen and then concentrated to about
1.37 (b, 2H), 1.31 (s, 9H); 13C NMR (63 MHz, CDCl3) δC 174.69 one-fourth of its volume.
(CO), 173.48 (CO), 80.90 (C(CH3)3), 54.16 (CH), 51.37 (OCH3),
30.27 (CH2), 29.70 (CH2), 27.84 (C(CH3)3); m/z (ESI) 218 [MH]+.
To this last solution of 27 was added, via a syringe, the pre-
viously synthesized activated ester solution. Stirring was con-
(R)-1-tert-Butyl 5-methyl 2-((S)-2-(tert-butoxycarbonylamino) tinued for 16 h at rt to afford 34. After solvent evaporation, the
propanamido)pentanedioate 32. (S)-Boc-alanine (4.73 g, solution was diluted with EtOAc (20 mL). Aqueous NaH2PO4
25 mmol) and CDI (4.05 g, 25 mmol) were stirred for 1 h in (10%, 20 mL) was added and the biphasic mixture was heated
CH2Cl2 (75 mL). A solution of 31 (5.2 g, 24 mmol) in CH2Cl2 with stirring at 50 °C for 2 h. The organic layer was recovered
was then added. After stirring at rt for 16 h, the organic layer and the aqueous layer was extracted with EtOAc (3 × 20 mL).
was washed successively with 10% aqueous NaHSO4, water, The pooled organic fractions were washed with brine, dried
saturated NaHCO3 and brine. The organic layer was dried over over MgSO4 and filtered. After solvent evaporation, the crude
MgSO4, filtered and the solvent was removed to give the crude product 35 was chromatographed on silica with 7 : 3 (EtOAc–
product 32. After recrystallisation from Et2O–hexanes the title hexanes). Solvent evaporation afforded the title compound
1
compound (4.2 g, 45%) was obtained as off-white crystals. Mp (580 mg, 78%) as a colourless oil. H NMR (250 MHz, MeOD)
109–110 °C; 1H NMR (250 MHz, CDCl3) δH 6.87 (br d, J = δH 7.46–7.25 (m, 3H, Ar-H), 7.25–7.07 (m, 2H, Ar-H), 6.26 (br,
6.7 Hz, 1H, NH), 5.23 (br d, J = 6.9 Hz, 1H, NH-Boc), 4.50–4.33 1H, NH-Boc), 4.73–4.56 (m, 1H, H-α-Lan), 4.37 (dd, J = 9.0,
(m, 1H, H-α-Glu), 4.14 (b, 1H, H-α-Ala), 3.59 (s, 3H, OCH3), 4.6 Hz, 1H, H-α-Glu), 4.16 (q, J = 7.1 Hz, 1H, H-α-Ala), 3.80
2.45–2.21 (m, 2H, CH2CO), 2.21–2.01 (m, 1H, CH2), 2.01–1.75 (s, 3H, OCH3), 3.44 (d, J = 13.5 Hz, 1H, SCH2C), 3.41 (d, J =
(m, 1H, CH2), 1.38 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3), 1.29 14.1 Hz, 1H, CH2Ph), 3.17 (br d, J = 13.5 Hz, 2H, SCH2C,
(d, J = 7.1 Hz, 3H, CH3); 13C NMR (63 MHz, CDCl3) δC 173.15 CH2Ph), 3.14–3.05 (m, 1H, CHCH2S), 2.98 (dd, J = 13.5, 8.1 Hz,
(CO-γ-Glu), 172.70 (CO-Ala), 170.75 (CO-Glu), 155.41 (CO-Boc), 1H, CHCH2S), 2.40 (t, J = 7.1 Hz, 2H, CH2CO), 2.34–2.14
82.29 (C-(CH3)3), 79.94 (C(CH3)3), 51.95 (C-α-Glu), 51.68 (m, 1H, CH2CH2CO), 2.14–1.88 (m, 1H, CH2CH2CO), 1.66–1.44
(OCH3), 50.16 (C-α-Ala), 29.89 (CH2CO), 28.27 (C(CH3)3), 27.93 (m, 36H, C(CH3)3), 1.40 (d, J = 7.1 Hz, 3H, CH3); 13C NMR
(C(CH3)3), 27.56 (CH2), 18.39 (CH3); m/z (ESI) 389 [MH]+.
(63 MHz, MeOD) δC 175.79 (CO), 174.55 (CO), 172.29 (CO),
(R)-5-tert-Butoxy-4-((S)-2-(tert-butoxycarbonylamino) propan- 172.00 (CO), 171.83 (CO), 157.49 (Boc-CO), 155.81 (Boc-CO),
amido)-5-oxopentanoic acid 33. Compound 32 (4.66 g, 136.86 (Ar-C), 131.19 (Ar-C), 129.09 (Ar-C), 128.03 (Ar-C), 84.00
12 mmol) was dissolved in CH3CN (36 mL). A solution of (C(CH3)3), 82.93 (C(CH3)3), 80.63 (C(CH3)3), 80.49 (C(CH3)3),
LiOH·H2O (605 mg, 14.4 mmol) in water (36 mL) was added. 65.95 (C-α), 54.13 (CH-α-Lan), 53.76 (CH-α-Glu), 52.93 (OCH3),
After stirring at rt for 4 h, the CH3CN was evaporated. The 51.73 (CH-α-Ala), 41.09 (CH2Ph), 38.58 (SCH2C), 35.59
aqueous layer was washed twice with Et2O and acidified with (CHCH2S), 32.73 (CH2CO), 28.85 (C(CH3)3), 28.78 (C(CH3)3),
10% aqueous NaHSO4. The precipitated solid was filtered and 28.30 (C(CH3)3), 28.28 (CH2CH2CO), 28.25 (C(CH3)3), 18.38
washed with cold water. After drying in vacuo to constant (CH3); m/z (ESI) 825 [MH]+.
weight, the title compound (3.3 g, 74%) was obtained as a
(S)-tert-Butyl 3-((R)-2-((R)-5-tert-butoxy-4-((S)-2-(tert-butoxy
colourless solid. Mp 168–170 °C; 1H NMR (250 MHz, MeOD) carbonylamino)propanamido)-5-oxopentanamido)-3-hydroxy-3-
δH 8.01 (b, 1H, NH), 4.41–4.22 (m, 1H, H-α-Glu), 4.07 (q, J = oxopropylthio)-2-benzyl-2-(tert-butoxycarbonylamino) propano-
7.1 Hz, 1H, H-α-Ala), 2.37 (t, J = 7.5 Hz, 2H, CH2CO), 2.24–2.03 ate 36. To tripeptide 35 (540 mg, 0.65 mmol) dissolved in
(m, 1H, CH2), 2.03–1.80 (m, 1H, CH2), 1.47 (s, 9H, C(CH3)3), CH3CN (10 mL) was added LiOH·H2O (41 mg, 0.98 mmol) in
1.45 (s, 9H, C(CH3)3), 1.31 (d, J = 7.1 Hz, 3H, CH3); 13C NMR water (10 mL). After stirring for 16 h at rt under nitrogen
(63 MHz, MeOD) δC 176.05 (CO-γ-Glu), 175.85 (CO-Ala), 172.06 and acidification with 10% aqueous NaHSO4 (2 mL), 36 was
(CO-Glu), 157.37 (CO-Boc), 82.95 (C(CH3)3), 80.57 (C(CH3)3), extracted three times with Et2O. The pooled organic layers
53.64 (C-α-Glu), 51.63 (C-α-Ala), 30.94 (CH2CO), 28.69 were washed with brine and dried over MgSO4. Filtration and
(C(CH3)3), 28.22 (C(CH3)3), 27.73 (CH2), 18.43 (CH3); m/z (ESI) solvent evaporation yielded the title compound (515 mg, 97%)
1
373 [M]−; HRMS m/z (ES+) Calcd for C17H31N2O7 375.2126, as a colourless foam. H NMR (250 MHz, MeOD) δH 7.09–6.91
found 375.2125 [MH]+.
(m, 3H, Ar-H), 6.91–6.74 (m, 2H, Ar-H), 4.32 (dd, J = 7.5,
(S)-tert-Butyl 3-((R)-2-((R)-5-tert-butoxy-4-((S)-2-(tert-butoxy 4.4 Hz, 1H, H-α-Lan), 4.12–3.95 (m, 1H, H-α-Glu), 3.86 (q, J =
carbonylamino)propanamido)-5-oxopentanamido)-3-methoxy- 7.1 Hz, 1H, H-α-Ala), 3.25–2.99 (m, 2H, SCH2C, CH2Ph),
3-oxopropylthio)-2-benzyl-2-(tert-butoxycarbonylamino)
pro- 2.94–2.75 (m, 3H, SCH2C, CH2Ph, CHCH2S), 2.68 (dd, J = 12.7,
panoate 35. Under nitrogen, dipeptide 33 (374 mg, 1 mmol), 8.2 Hz, 1H, CHCH2S), 2.18–2.01 (m, 2H, CH2CO), 2.01–1.84
HBTU (379 mg, 1 mmol) and diisopropylethylamine (DiPEA, (m, 1H, CH2CH2CO), 1.84–1.63 (m, 1H, CH2CH2CO), 1.32–1.10
190 μL, 1.1 mmol) were suspended in dry CH2Cl2 (20 mL) and (m, 36H, C(CH3)3), 1.08 (d, J = 7.1 Hz, 3H, CH3); 13C NMR
stirred for 1 h at rt (i.e. activated ester).
(63 MHz, MeOD) δC 175.54 (CO), 174.34 (CO), 173.40 (CO),
Meanwhile, in another flask, containing a degassed suspen- 171.78 (CO), 171.59 (CO), 157.21 (Boc-CO), 155.52 (Boc-CO),
sion of sulfamidate 19 (373 mg, 0.9 mmol) and cysteine 136.71 (Ar-C), 131.01 (Ar-C), 128.95 (Ar-C), 127.89 (Ar-C), 83.87
methyl ester·HCl (154 mg, 0.9 mmol) in CH3CN (10 mL), was (C(CH3)3), 82.76 (C(CH3)3), 80.47 (C(CH3)3), 80.34 (C(CH3)3),
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65.87 (C-α), 54.00 (CH-α-Lan), 53.69 (CH-α-Glu), 51.46 (CH- with water in order to remove SDS. PG was further treated with
α-Ala), 40.98 (CH2Ph), 38.78 (SCH2C), 35.84 (CHCH2S), 32.67 α-amylase (200 µg mL−1, 20 mM sodium phosphate, 7 mM
(CH2CO), 28.81 (C(CH3)3), 28.75 (C(CH3)3), 28.27 (C(CH3)3), NaCl, pH 6.9, 3 h at 37 °C) to remove oligosaccharides, with
28.22 (C(CH3)3), 28.06 (CH2CH2CO), 18.37 (CH3); m/z (ESI) 811 trypsin (200 µg mL−1, 20 mM sodium phosphate, 7 mM NaCl,
[MH]+.
pH 8, 3 h at 37 °C) and pronase (500 µg mL−1, 100 mM Tris,
(R)-5-((R)-2-((S)-2-amino-2-carboxy-3-phenylpropylthio)-1-carb- 10 mM CaCl2, pH 8, 3 h at 40 °C) to remove proteins. After all
oxyethylamino)-2-((S)-2-aminopropanamido)-5-oxo pentanoic treatments, PG was recovered by ultracentrifugation (60 min,
acid bis(trifluoroacetate) salt 3RS. Compound 36 (489 mg, 50 000 rpm). Finally, it was washed with water, with 8 M LiCl
0.6 mmol) was dissolved in CH2Cl2 (50 mL) and the solution to remove any polypeptidic contamination, again with water,
was purged with nitrogen for 15 min. TFA (5 mL) was added. and lyophilized.
The flask was closed with a glass stopper and the mixture was
Analysis of purified PG. Aliquots were hydrolyzed in 6 M
stirred for 16 h at rt. After solvent evaporation, the residue was HCl containing 0.05% (v/v) 2-mercaptoethanol at 95 °C for
repetitively taken up in CH3CN and evaporated. The product 16 h. The reducing agent prevented the oxidation of lanthio-
was transferred into a centrifugation vial with the help of nine to sulfoxide. After evaporation, the pellet was dissolved
CH3CN (15 mL). Then, Et2O (15 mL) was added to precipitate with 67 mM sodium citrate–HCl buffer (pH 2.2) and injected
it. After centrifugation, the pellet was washed with Et2O. The into a Hitachi L-8800 amino acid analyzer equipped with a
product 3RS was taken up in water and lyophilised to yield the 2620MSC-PS column (Science-Tec). Amino acids and hexos-
1
title compound (318 mg, 73%) as a colourless solid. H NMR amines were detected after post-column reaction with nin-
(250 MHz, D2O + MeOD) δH 7.37–7.14 (m, 3H, Ar-H, Ar-H), hydrin. Lanthionine derivatives did not co-elute with any
7.14–6.90 (m, 2H, Ar-H), 4.36 (dd, J = 7.1, 5.2 Hz, 1H, H-α-Lan), other amino acid or hexosamine; retention times were: (R,R)-
4.22 (dd, J = 8.6, 4.8 Hz, 1H, H-α-Glu), 3.96 (q, J = 7.1 Hz, 1H, α-benzyl-lanthionine, 30.7 min, (R,S)-α-benzyl-lanthionine,
H-α-Ala), 3.23 (d, J = 14.4 Hz, 1H, SCH2C), 3.17 (d, J = 14.4 Hz, 31.3 min.
1H, CH2Ph), 3.08–2.83 (m, 3H, CHCH2S, CH2Ph), 2.78 (d, J =
In vitro assay with Mpl
14.4 Hz, 1H, SCH2C), 2.28 (t, J = 7.3 Hz, 2H, CH2CO), 2.18–1.98
(m, 1H, CH2CH2CO), 1.98–1.73 (m, 1H, CH2CH2CO), 1.39 (d,
J = 7.1 Hz, 3H, CH3); 13C NMR (63 MHz, D2O) δC 176.81 (CO),
175.82 (CO), 175.73 (CO), 174.26 (CO), 171.65 (CO), 134.31
(Ar-C), 131.08 (Ar-C), 130.05 (Ar-C), 129.06 (Ar-C), 66.51 (C-α),
54.61 (CH-α-Lan), 54.10 (CH-α-Glu), 50.10 (CH-α-Ala), 42.39
(CH2Ph), 39.25 (SCH2C), 35.86 (CHCH2S), 32.72 (CH2CO),
27.75 (CH2CH2CO), 17.60 (CH3); m/z (ESI) 499 [MH]+; HRMS
m/z (ES+) Calcd for C21H31N4O8S 499.1857, found 499.1855
[MH]+.
The assay mixture (50 µL) contained 100 mM Tris-HCl buffer
(pH 8.4), 5 mM ATP, 15 mM, MgCl2, 0.4 mM UDP-[14C]-
MurNAc (500 Bq), 1 mM tripeptide, and purified Mpl enzyme3
(2 µg of protein). Mixtures were incubated at 37 °C for 16 h
and reactions were stopped by the addition of 10 µL of acetic
acid, followed by lyophilization. The residue was dissolved in
50 mM ammonium formate, pH 3.8 (buffer A), and injected
onto a Nucleosil 100 C18 5 µm column (4.6 × 150 mm; Grace
Davison Discovery Sciences). Elution was performed at 0.6 mL
min−1 with buffer A (isocratic, 10 min) followed by a gradient
of methanol in buffer A (from 0 to 40% over 35 min).
Detection was performed with a radioactive flow detector
(model LB506-C1, Berthold) using the Quicksafe Flow 2 scintil-
lator (Zinsser Analytic) at 0.6 mL min−1. Quantification was
carried out with the Winflow software (Berthold).
Biological experiments
Strains and media. W7 (dap lysA)38 was obtained from
A. Derouaux (Newcastle University, Newcastle, United
Kingdom). The cells were grown at 37 °C in a minimal salts
medium28 containing (per liter) 6.33 g of K2HPO4·3H2O, 2.95 g
of KH2PO4, 1.05 g of (NH4)2SO4, 1.0 g of MgSO4.7H2O, 0.1 mg
of FeSO4·7H2O, 2.8 mg of Ca(NO3)2·4H2O, 4 mg of thiamine,
5 g (0.5%) or 2 g (0.2%) of glucose, and 50 mg of (S)-lysine.
Incorporation experiments were done in the presence of
50 µg mL−1 or 500 µg mL−1 3RS and 3RR, respectively.
Purification of PG. The preparation of highly purified PG
has been done from one-liter culture medium by modifying
a protocol described by Girardin.39 Briefly, bacteria were
harvested in the exponential growth phase at an optical
density (600 nm) of 0.4–0.6. After centrifugation (10 min,
6000g) the pellet was suspended in 20 mL ice-cold water, and
the suspension was added dropwise to 8% boiling SDS
(20 mL). The sample was boiled for 45 min. SDS treatment
removes contaminating proteins, non-covalently bound lipo-
proteins and LPS. Polymeric PG, which remained insoluble,
was recovered by ultracentrifugation (45 min, Beckmann
L2-65B, rotor Type 60Ti, 50 000 rpm) and washed five times
Acknowledgements
This work was financed by an ARC (grant 03/08-297) of
the Belgium French community and by the FP6 European
Integrated Project EurIntafar (LSHM-CT-2004-512138). We
thank Professor Albert Demonceau for insightful discussions
and correction of the draft. We thank Dr Gabriel Mazzucchelli
and co-workers at the GIGA MSL for the HRMS determinations.
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