Synthesis of a Fragment of Bacterial Cell Wall

Article abstract of DOI:10.1021/jo035583k

Cell wall is indispensable for survival of bacteria. This large molecular "mesh" encases the entire cytoplasm of bacteria, and it is comprised of repeating backbone units of N-acetyl-glucosamine (NAG)-N-acetyl-muramic acid (NAM). A pentapeptide is attached to each of the lactyl units of the N-acetyl-muramic acid. The cell wall has both cross-linked and non-cross-linked components. In the present paper, we have devised a synthetic route for the preparation of a fragment of the cell wall comprised of a tetrasaccharide (NAG-NAM-NAG-NAM), along with the two appended peptides. We also report the syntheses of three glycosyl donors (compounds 5, 7, and 9) and three glycosyl acceptors (compounds 4, 6, and 8) based on the D-glucosamine structure as a building unit. The synthetic strategy that is disclosed is generally useful in construction of other natural products containing the D-glucosamine as a building block.

Full text of DOI:10.1021/jo035583k

Syn th esis of a F r a gm en t of Ba cter ia l Cell Wa ll
Dusan Hesek, Mijoon Lee, Ken-ichiro Morio, and Shahriar Mobashery*
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556
mobashery@nd.edu
Received October 28, 2003
Cell wall is indispensable for survival of bacteria. This large molecular “mesh” encases the entire
cytoplasm of bacteria, and it is comprised of repeating backbone units of N-acetyl-glucosamine
(NAG)-N-acetyl-muramic acid (NAM). A pentapeptide is attached to each of the lactyl units of the
N-acetyl-muramic acid. The cell wall has both cross-linked and non-cross-linked components. In
the present paper, we have devised a synthetic route for the preparation of a fragment of the cell
wall comprised of a tetrasaccharide (NAG-NAM-NAG-NAM), along with the two appended peptides.
We also report the syntheses of three glycosyl donors (compounds 5, 7, and 9) and three glycosyl
acceptors (compounds 4, 6, and 8) based on the D-glucosamine structure as a building unit. The
synthetic strategy that is disclosed is generally useful in construction of other natural products
containing the D-glucosamine as a building block.
Bacteria are not capable of maintaining their internal
osmotic pressure. Therefore, they have developed the cell
wall (also known as the cross-linked peptidoglycan or
murein) as a rigid “molecular mesh”, which encloses the
cell and maintains the structural integrity of bacteria.
This single macromolecule that encases the cytoplasm
is also referred to as the sacculus (the “sack”), an entity
that supports and controls the turgor pressure (of an
ever-expanding cytoplasm). The polysaccharide part of
the peptidoglycan is a repeating disaccharide unit of N-
acetyl-glucosamine (NAG)-N-acetyl-muramic acid (NAM).
A uniquely bacterial pentapeptide is attached to the
D-lactyl moiety of NAM (1). The peptides from two
separate strands are cross-linked (2) by DD-transpepti-
dases to give the rigid cell wall.1
peptidoglycan sequestered in its active site, en route to
the cross-linked species.4 We are also interested in
visualizing the solution structure of the non-cross-linked
peptidoglycan. Toward this goal, we have devised a
versatile synthetic route that allows for the assembly of
sizable fragments of the bacterial cell wall. The synthetic
work for the preparation of a fragment of cell wall that
encompasses a tetrasaccharide made up of two repeats
of NAG-NAM along with the two requisite appended
peptides that are found in many Gram-positives (com-
pound 3) is disclosed herein. Insofar as lysine may serve
A variety of techniques have been applied over the past
20-30 years to study the nature of the cell wall, resulting
in four hypotheses for the formation of the sacculus.2,3
Despite these efforts, there is no detailed knowledge of
what the three-dimensional structure of sacculus looks
like at the present. The major difficulty with the earlier
efforts on the studies of the peptidoglycan had been the
nonhomogeneous nature of the samples. These studies
have invariably used samples from bacteria, which were
mixtures of bits and pieces of different sizes. We have
undertaken a series of studies to elucidate the structural
aspects of the bacterial cell wall. In this vein, a glimpse
into the cross-linked peptidoglycan was provided recently
by the publication of a high-resolution X-ray structure
for a DD-transpeptidase that had two mimics of the
as a simplified surrogate for diaminopimelate, the struc-
ture may also represent the situation seen in many
Gram-negative organisms as well.5 We hasten to add that
2-amino-2-deoxyglucose (i.e., glucosamine) is found in
many natural products,6,7 hence the synthetic approaches
that we have developed should be of use for a much
broader purpose.
(4) Lee, W.; McDonough, M. A.; Kotra, L. P.; Li, Z. H.; Silvaggi, N.
R.; Takeda, Y.; Kelly, J . A.; Mobashery, S. A 1.2 Å Snapshot of the
Final Step of Bacterial Cell Wall Biosynthesis. Proc. Natl. Acad. Sci.
U.S.A. 2001, 98, 1427-1431.
(5) Bugg, T. D. H. In Comprehensive Natural Products Chemistry;
Pinto, B. M., Ed.; Elsevier Science B.V.: Amsterdam, The Netherlands,
1999; Vol. III, pp 241-294.
(6) (a) Wittmann, V. In Glycoscience: Chemistry and Chemical
Biology; Fraser-Reid, B., Tatsuta, K., Thiem, J ., Eds.: Springer-
Verlag: Heidelberg, Germany, 2001; Vol. III, pp 2253-2287. (b)
Herzner, H.; Reipen, T.; Schultz, M.; Kunz, H. Chem. Rev. 2000, 100,
4495-4537.
(7) Schmidt, R. R.; Kinzy, W. Adv. Carbohydr. Chem. Biochem. 1994,
50, 21-123.
(1) Goffin, C.; Ghuysen, J . M. Microbiol. Mol. Biol. Rev. 2002, 66,
702-738.
(2) Koch, A. L. Orientation of the Peptidoglycan chains in the
Sacculus of Escherichia coli. Res. Microbiol. 1998, 149, 689-701.
(3) Dmitriev, B. A.; Ehlers, S.; Rietchel, E. T. Layered Murein
Revisited:
a Fundamentally New Concept of Bacterial Cell Wall
Structure, Biogenesis and Function. Med. Microbiol. Immunol. 1999,
187, 173-181.
10.1021/jo035583k CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/13/2004
J . Org. Chem. 2004, 69, 2137-2146
2137

Hesek et al.
SCHEME 1
Resu lts a n d Discu ssion
glycosyl building blocks and develop a synthetic protocol
involving protection-deprotection motifs. The choice of
commonly used protecting groups in sugar chemistry to
mask free hydroxyl groups (e.g., O-benzylidene-, O-
benzyl-, O-acetyl-, and O-tert-butyldimethylsilyl) allowed
us to selectively introduce our desired functionality prior
to coupling. Furthermore, the desired synthetic route
should demonstrate an excellent regioselectivity and/or
stereoselectivity, and be amenable to a single deprotec-
tion step of the final glycopeptide units for practical
reasons. The linkages among the aminosugars are all
â-(1-4). Hence, the terminal sugar should bear a â-meth-
oxy group at the anomeric C-1 position of the terminal
NAM residue (see 3). Considering how the inherent
properties of the individual glycosyl donor/acceptor build-
ing blocks could lead to the formation of several different
derivatives as possible reaction intermediates, we syn-
thesized a focused library of monosaccharide glycosyl
donors and glycosyl acceptors that would serve the
purpose (Figure 1). The syntheses of these building blocks
(the number of synthetic steps and the overall reaction
yields were calculated starting from D-glucosamine),
leading to glycosyl acceptors 4 (7 steps, 16%), 6 (5 steps,
21%), and 8 (7 steps, 18%), and glycosyl donors 5 (3 steps,
32%), 7 (9 steps, 9%) and 9 (9 steps, 10%), are outlined
in Scheme 2.
There is a paucity of precedent for construction of
repeating units of the sugar backbone of peptidoglycan
(PGN),8 although some literature exists for construction
of the NAG-NAM-containing derivatives of lipid II, a
biosynthetic precursor for the bacterial cell wall.9,10 Most
of the effort has been focused on coupling of orthogonally
protected monosaccharide building blocks derived from
NAG and NAM, which already include the lactyl ether
attached at the 3-O-position of D-GlcNAc. The presence
of the (R)-lactate moieties creates difficulties in as-
sembling building blocks into complex structures, such
as intramolecular lactonization at the 4-hydroxy posi-
tion,10 and more importantly racemization of the lactate
residue. Therefore, we decided to introduce the lactate
residue at a later stage in the synthesis and develop
synthetic routes involving orthogonal protective groups
that differentiate the hydroxyl groups in NAM from that
in the NAG unit.
While the choices for N-protection of D-glucosamine are
many,11 we chose the recently developed N-dimethylma-
leoyl protective group that participates in stereoselective
â-(1-4)-glycoside bond formation.12,13 We anticipated that
this protective group would result in high coupling yields
and complete removal of the DMM after coupling and
would allow us to avoid potential solubility complications.
Trichloroacetimidate was used as the leaving group and
trifluoromethanesulfonic acid (TfOH) as the main pro-
moter for the stereoselective â-(1-4)-glycosyl bond for-
mation reaction.7
A systematic analysis of assembly of suitable libraries
of donors and acceptors en route to cell wall fragments
is absent from the literature. Five possible patterns for
the syntheses of the NAG-NAM-NAG-NAM(â-OMe) sugar
sequence were considered (Figure 1).
With these requirements in mind, commercially avail-
able D-glucosamine hydrochloride was used to design
Str a tegy 1: Propagation of the sugar units one-by-one
starting from a terminal glycosyl acceptor 4, which
represents ring A, seemed most logical at first glace. One
advantage is the better freedom to choose the reaction
conditions throughout the synthesis, because a desired
â-OCH3 substituent at C-1 is generally stable (in contrast
to the O-silyl group). The second advantage is the
chemical properties of the substrates (A, A-B, or A-B-
C) being introduced into the next reaction steps that
serve as glycosyl acceptors, without the added difficulty
of employing acid-sensitive silyl-protected groups. How-
ever, this approach is complicated since glycosylation
reactions with donors 7 and 9 (each made in 9 steps) are
required, followed by regioselective reduction of 4,6-O-
benzylidene protective groups, which dramatically would
(8) (a) Inamura, S.; Fukase, K.; Kusumoto, S. Tetrahedron Lett.
2001, 42, 7613-7616. (b) Termin, A.; Schmidt, R. R. Liebigs Ann.
Chem. 1992, 527-533. (c) Termin, A.; Schmidt, R. R. Liebigs Ann.
Chem. 1989, 789-795.
(9) (a) Van Nieuwenhze, M. S.; Mauldin, S. C.; Zia-Ebrahimi, M.;
Winger, B. E.; Hornback, W. J .; Saha, S. L.; Aikins, J . A.; Blaszczak,
L. C. J . Am. Chem. Soc. 2002, 124, 3656-3660. (b) Schwartz, B.;
Markwalder, J . A.; Wang, Y. J . Am. Chem. Soc. 2001, 123, 11638-
11643. (c) Ye, X.-Y.; Lo, M.-C.; Brunner, L.; Walker, D.; Kahne, D.;
Walker, S. J . Am. Chem. Soc. 2001, 123, 3155-3156. (d) Ha, S.; Chang,
E.; Lo, M.-C.; Men, H.; Park, P.; Ge, M.; Walker, S. J . Am. Chem. Soc.
1999, 121, 8415-8426.
(10) Saha, S. L.; Nieuwenhze, S. V.; Hornback, W. J .; Aikins, J . A.;
Blaszczak, L. C. Org. Lett. 2001, 3, 3575-3577.
(11) Debenham, J .; Rodebaugh, R.; Fraser-Reid, B. Liebigs Ann./
Recl. 1997, 791-802.
2138 J . Org. Chem., Vol. 69, No. 6, 2004

Bacterial Cell Wall
F IGURE 1. Synthetic strategy for construction of the tetrasaccharide made of repeating D-glucosamine units. The structures of
the six glycosyl acceptors and donors employed in this study are depicted.
SCHEME 2a
a
Reagents and conditions: (a) (i) 2,3-dimethylmaleic anhydride, (ii) Ac2O, 55%; (b) TiCl4, 76%; (c) HgO, HgCl2, MeOH, 81%; (d) (i)
NaOMe, Amberlite IR-120 (H+), (ii) benzaldehyde dimethylacetal, p-TsOH, (iii) Ac2O, 67%; (e) BH3‚NMe3, BF3‚OEt2, 76%; (f) N2H4‚AcOH,
68%; (g) TBDMS-Cl, imidazole, 78%; (h) Cl3CCN, DBU, 85%; (i) NaOMe, 73%; (j) Bu2SnO, BnBr, 73%; (k) benzaldehyde dimethylacetal,
p-TsOH; 91%; (l) Ac2O, 85%; (m) BH3‚NHMe2, BF3‚OEt2, 90%; (n) n-Bu4NF, 55%; (o) Cl3CCN, DBU, 85%; (p) Cl3CCdNHOBn, TfOH,
67%; (q) n-Bu4NF, 72%; (r) Cl3CCN, DBU, 80%.
decrease the overall reaction yield. Therefore, this strat-
egy would appear to be less economical.
Str a tegy 2: This strategy builds sugar units one-by-
one, starting from glycosyl donor 7 (ring D). After many
J . Org. Chem, Vol. 69, No. 6, 2004 2139

Hesek et al.
SCHEME 3a
attempts to couple the building blocks using strategy 2,
we concluded that this route was the least effective of
the methods proposed herein. During the coupling reac-
tions, the purity of the coupling products was poor and
they had to be resubjected to further purification with
flash-column chromatography due to the similar nonpolar
nature of product and starting materials. As a result, the
major product typically had a maximum yield of 40% for
each consecutive coupling step.
Str a tegy 3: This approach required the formation of
each of the individual monoglycosyl entities on a large
scale, which is a major disadvantage, followed by con-
struction of the two separate disaccharide units to give
the coupled intermediates 7-8 (D-C) and 7-4 (B-A).
Subsequently, the dimers would be converted into their
corresponding donor-acceptor counterparts, followed by
the activation of the donor component, resulting in their
linkage to give the tetrasaccharide moiety.
a
Reagents and conditions: (a) (i) NaOMe, Amberlite IR-120
(H+), (ii) benzaldehyde dimethylacetal, p-TsOH, 60% in two steps;
(b) Ac2O, 85%.
Str a tegies 4 a n d 5: To construct the disaccharide
core C-B from our library first appeared to be the most
favorable when acceptor B was coupled with donor C in
the presence of a catalytic amount of the promoter. The
C-B disaccharide could be prepared by stereoselective
glycosylation of either trichloracetimidate glycosyl donor
5 or 9 with the corresponding acceptor 6, resulting in
disaccharides 5-6 (compound 22) and 9-6 (compound
23), respectively, in good overall yield in both cases.
However, the different nature of disaccharide products
formed would be distinguished. The synthesis of a
disaccharide intermediate similar to 5-6 was recently
described by Schmidt et al.13 However, further transfor-
mations (three additional steps) should be implemented
to obtain 23.
sensitive to the reaction conditions employed (e.g., TBAF/
acetic acid, prolonged exposure 1 h; temperature over 10
°C), resulting in the formation of undesirable side prod-
ucts. However, this strategy readily offers the highly
desirable glycosyl dimer 23, without further manipula-
tion.
The dimer 23 can be converted to either a glycosyl
acceptor, ready for coupling with the donor 4, or to a
glycosyl donor for coupling with the acceptor 7; one or
the other approach makes the difference between strate-
gies 4 and 5. The more extensive synthetic demand for
preparation of acceptor 7 (over donor 4), as well as the
need for purification via strategy 5, makes strategy 4
more favorable for the preparation of compound 3.
After a small modification of disaccharide 23, it was
employed in the construction of the trisaccharide in which
acceptor 4 was used (Strategy 4). This step was optimized
and was scaled up in our synthesis. It is noteworthy that
dimer 23 is a useful intermediate, because it can be
converted to both a glycosyl acceptor and a donor, which
has the potential desired application for construction of
oligosaccharide by a repeat of the coupling procedures.
The route that was ultimately chosen was based on a
combination of the donors and acceptors depicted in
Scheme 3. The donor 9 and acceptor 6 were converted to
disaccharide â-(1f4) 23 in a TfOH-mediated reaction at
low temperature (approximately -10 °C, 71%) in a single
step. The use of scrupulously dry conditions prevented
the formation of any byproducts. However, when TM-
SOTf was used as the promoter, it was noted that on
longer reaction time decomposition occurred to give
undesired products, along with the formation of a bis-
silylated version of 6, with TMS-ether attached at the
4-position. Alternatively, the identical disaccharide 23
was obtained more circuitously when derivative 22 was
converted to 24. This product was obtained after hydroly-
sis of the acetyl groups without affecting dimethylmaleoyl
(DMM) and treatment with benzaldehyde dimethyl acetal
in the presence of p-TsOH as a catalyst. Subsequently,
acetylation of the remaining free 4′-hydroxyl group in the
product 24 afforded 23 in a slightly lower overall reaction
yield in comparison to that described above.
Mainly, one needs to convert 3,4,6-O-triacethyl protec-
tive groups in 22 into the corresponding 3-O-acetyl-4,6-
O-benzylidene derivatives 23 to obtain the suitable
disaccharide building blocks needed for sugar expansion.
The efficient transformation of the glucoside C ring
having the tri-O-acetyl protected group into the corre-
sponding 4,6-O-benzylidene derivative was performed via
a short route with standard reaction conditions. However,
these transformations are carried out using the disac-
charide segment that would decrease the overall yield
for this strategy. Alternatively, a glycosylation reaction
between the donor 9 and acceptor 6 affords disaccharide
23 in a facile way. To generate the donor 9, it was
necessary to remove the TBDMS protective group at the
anomeric carbon of the corresponding intermediate (18).
This particular desilylation step appears to be unusually
(12) Aly, M. R. E.; Castro-Palomino, J . C.; Ibrahim, E.-S. I.; El Ashry,
E. S. H.; Schmidt, R. R. Eur. J . Org. Chem. 1998, 2305-2316.
(13) Aly, M. R. E.; Ibrahim, E.-S. I.; El-Ashry, E. S. H.; Schmidt, R.
R. Carbohydr. Res. 2001, 331, 129-142.
Elaboration of the substrate 23 into the required
glycosyl donor 26 (Scheme 4) was performed by applying
2140 J . Org. Chem., Vol. 69, No. 6, 2004

Bacterial Cell Wall
SCHEME 4a
a
Reagents and conditions: (a) n-Bu4NF, 75%; (b) Cl3CCN, DBU; (c) 4, TfOH, 55% in two steps; (d) BH3‚NMe3, BF3‚OEt2, 62%; (e)
TfOH, 59-68%.
standard procedures and needs little comment, apart
from the fact that removal of the TBDMS ether at the
C-1 position of disaccharide 23 was accomplished on
treatment with a 1 M solution of tetrabutylammonium
fluoride (TBAF) in ethyl acetate, furnishing 25 in 75%
yield. However, a prolonged reaction time led to a
substantial decrease in yield due to mainly undesired
hydrolysis of the 3-O-acetyl group by the trace of moisture
in the reagent. Stereoselective â-glycosylation between
acceptor 4 and trichloracetimidate 26 in the presence of
TfOH gave the corresponding trisaccharide 27 in good
yield (62%). Regioselective reductive ring opening of the
4,6-O-benzylidene group in 27 was carried out by treat-
ment with the borane-trimethylamine complex (BH3‚
NMe3) and BF3‚OEt2, utilizing a modification of a pub-
lished procedure for similar derivatives.14 This reaction
resulted in the formation of the trisaccharide acceptor
28 with an unmasked 4-hydroxyl functionality typically
in 59-68%. To obtain the desired tetrasaccharide 29, the
reaction sequence was put through an additional O-
glycosyl trichloracetimidate method with 7 to provide
exclusively the tetramer of the â-glycosyl linkage.
Deprotection of the DMM groups in 29 was carried out
by a careful acid/base treatment (NaOH, 6 h; HCl, pH
3.0, 24 h), essentially by the kind of procedure reported
earlier.12 The attendant N-acetylation of 29 gave 30
(Scheme 5). The overall yield for the two steps was 48%.
Whereas the outlined strategy is seemingly laborious,
the assembly of the building blocks into the larger
oligosaccharides without the lactate functionality appears
to be a practical route for the synthesis of derivatives
with repeating NAG-NAM sequence. Due to limited
solubility, the in situ generated compound 31, possessing
free 3-O-hydroxy groups, was committed to the next step
without further purification. The formation of fragment
32 by the reaction of 31 in the presence of an excess of
(S)-2-chloropropionic acid was a relatively simple trans-
formation (Scheme 5). The lactate moieties in 32 were
coupled with the pentapeptide in the next reaction. The
desired pentapeptide (33, L-Ala-D-Glu-R-OBn-γ-N-ꢀ-Cbz-
L-Lys-D-Ala-D-Ala-OBn) was synthesized by a fragment
condensation approach, using standard Boc chemistry
(Scheme 6). The two carboxylic acids and one amino
group in this peptide chain were protected by benzyl and
benzyloxycarbonyl (Cbz) groups, respectively. The p-
nitrophenylester (36) of the known dipeptide (Boc-L-Ala-
D-Glu-R-OBn-γ-OH)15 was prepared from Boc-L-Ala and
Boc-D-Glu-R-OBn by the active ester method. The tripep-
tide (38, N-ꢀ-Cbz-L-Lys-D-Ala-D-Ala-OBn) was readily
made by stepwise condensation using the water-soluble
coupling agent EDCI. The active ester method was
applied again in the final assembly step to obtain the
clean protected pentapeptide 39. The Boc group in 39 was
readily deprotected by trifluoroacetic acid to afford 33 as
a TFA salt.
Subsequently, the lactate moieties in 32 were activated
to the corresponding p-nitrophenol ester derivatives by
treatment with p-nitrophenyl trifluoroacetate. Although
the use of p-nitrophenyl ester is not common for activa-
tion of carboxylates in sugar chemistry, it is noteworthy
that the introduction of the chromophore to the sugar
molecules made it facile to follow the reaction. Further-
more, the p-nitrophenyl ester that was formed was stable
and could be kept at room temperature in a protic solvent
such as methanol without racemization or anomerization.
The active ester was converted to the protected form of
the peptidoglycan tetrasaccharide 34 by coupling to the
requisite pentapeptide 33. The last step of the prepara-
tion was 4,6-O-benzylidene deprotection in 60% acetic
acid, which was allowed to proceed at 70 °C for 1 h. The
global deprotection of the benzyl esters, benzyl ethers,
and benzyloxycarbonyl functionalities was accomplished
by hydrogenolysis over 10% Pd/C in methanol as the final
step in the synthesis to give the crude 3.
The crude compound 3 was purified by preparative
thin-layer chromatography. The sample was pure by
HPLC analysis and produced the desired mass spectro-
metric data. The compound was subsequently studied by
a series of sophisticated high-field NMR analyses for
determination of the solution structure, which will be the
(14) Oikawa, M.; Liu, W.-C.; Nakai, Y.; Koshida, S.; Fukase, K.
Synlett 1996, 1179-1180.
(15) Takeno, H.; Okada, S.; Hemmi, K.; Aratani, M.; Kitaura, Y.;
Hashimoto, M. Chem. Pharm. Bull. 1984, 32, 2925-2931.
J . Org. Chem, Vol. 69, No. 6, 2004 2141

Hesek et al.
SCHEME 5a
a
Reagents and conditions: (a) (i) NaOH, dioxane/water (4:1); HCl, pH 3.0, (ii) Ac2O, 48%; (b) NaOMe, 74%; (c) NaH, (S)-2-chloropropionic
acid, 46%; (d) 4-nitrophenyl trifluoroacetate, pyridine; 33, 41%; (e) 60% AcOH; H2, Pd/C, 76%.
SCHEME 6a
a
Reagents and conditions: (a) (i) L-alanine p-nitrophenyl ester, 70%, (ii) p-nitrophenol, DCC, 75%; (b) (i) D-Ala-D-Ala-OBn, EDCI,
HOBt, 32%, (ii) TFA, 93%; (c) HOBt, 48%; (d) TFA, 96%.
pentapeptide synthesis of Scheme 6 are given in the Support-
ing Information.
subject of a subsequent paper that concerns the struc-
tural biological aspects of our interest in this synthetic
fragment of bacterial cell wall.
ter t-Bu tyld im eth ylsilyl 3-O-Acetyl-4,6-O-ben zylid en e-
2-d eoxy-2-d im eth ylm a leim id o-â-D-glu cop yr a n osyl-(1f4)-
3,6-d i-O-b en zyl-2-d eoxy-2-d im et h ylm a leim id o-â-D-glu -
cop yr a n osid e (23). Compound 23 was obtained by two
methods. One was by coupling of 9 with 6 and the other was
conversion from 22. In the former method, a mixture of
compound 9 (5.6 g, 10 mmol) and compound 6 (6.4 g, 11 mmol)
was dissolved in dry CH2C12 (100 mL, distilled over CaH2 and
then over 4-Å molecular sieves), 4-Å molecular sieves (5 g) was
added, and the suspension was stirred under argon for 2 h.
The mixture was then diluted with cyclohexane (50 mL) and
cooled to -10 °C. The catalyst TfOH (1.5 mL, 1 M solution in
CH2C12) was added in two portions, and the solution was
Meth od s
Compounds 5, 6, 10, 14-17,12 7, 20, 21,16 and 2213 were
prepared by published literature methods. TfOH (trifluoro-
methanesulfonic acid) was used as a 1 M solution in CH2Cl2,
which was kept over 4-Å molecular sieves. Procedures for
syntheses of the monomer compounds in Scheme 2 and the
(16) Hesek, D.; Suvorov, M.; Morio, K.; Lee, M.; Brown, S.; Vaku-
lenko, S. B.; Mobashery, S. J . Org. Chem. 2004, 69, 778-784.
2142 J . Org. Chem., Vol. 69, No. 6, 2004

Bacterial Cell Wall
stirred at -10 °C for 1 h, and then at room temperature for
an additional 1 h, after which the reaction was quenched by
adding triethylamine (0.21 mL) and filtered through a layer
of Celite, and the organic solvents were removed in vacuo. The
residue was purified by flash chromatography (n-hexane/
EtOAc, 3.2:1) to give 23 (7.0 g) in 71% yield. 1H NMR (400
MHz, CDCl3) δ -0.10, 0.11, 0.72 (3s, 15H), 1.79 (br s, 6H),
mL, 1.0 M in THF) at -20 °C. The mixture was stirred at the
same temperature until the starting material was consumed
(1 h). After the addition of a few drops of sat. NaHCO3, the
solution was quickly filtered through a small layer of silica
gel and the silica pad was washed with cold ethyl acetate. The
collected filtrate was concentrated in vacuo and the residue
was chromatographed (n-hexane/EtOAc, 2:1 to 1.2:1) to give
I
1.93, 1.96 (2s, 9H), 3.37-3.51 (m, 3H, H-5I, H-6I, H-6′ ), 3.53
1
the title compound (4.0 g, 75%) as a single isomer. H NMR
(dd, 1H, J ) 6.9, 10.1 Hz, H-6II), 3.61 (dd, 1H, J ) 8.9, 9.7 Hz,
H-4II), 3.85 (dd, 1H, J ) 8.1, 10.5 Hz, H-2I), 4.03 (dd, 1H, J )
8.5, 10.1 Hz, H-2II), 4.09-4.13 (m, 2H, H-3I, H-4I), 4.18 (dd,
1H, J ) 4.5, 10.1 Hz, H-6′ II), 4.61 (2d, 2H, J ) 12.2 Hz, CH2-
Ph), 4.46, 4.81 (2d, 2H, J ) 12.2 Hz, CH2Ph), 5.08 (d, 1H, J )
8.1 Hz, H-1I), 5.41 (s, 1H, CHPh), 5.46 (d, 1H, J ) 8.1 Hz,
(400 MHz, CDCl3) δ 1.76, 1.93, 1.97 (3s, 15H), 3.35 (m, 1H,
I
H-5II), 3.41-3.45 (m, 3H, H-5I, H-6I, H-6′ ), 3.46 (t, 1H, J )
9.7 Hz, H-6II), 3.58 (dd, 1H, J ) 9.7, 11.4 Hz, H-4II), 3.80 (dd,
1H, J ) 8.1, 10.5 Hz, H-2I), 4.00 (dd, 1H, J ) 8.1, 10.5 Hz,
H-2II), 4.12-4.16 (m, 2H, H-3I, H-4I), 4.19 (dd, 1H, J ) 5.3,
10.9 Hz, H-6′ II), 4.43, 4.82 (2d, 2H, J ) 12.2 Hz, CH2Ph), 4.57
(s, 2H, CH2Ph), 5.07 (d, 1H, J ) 8.1 Hz, H-1I), 5.39 (s, 1H,
CHPh), 5.40 (d, 1H, J ) 8.1 Hz, H-1II), 5.65 (dd, 1H, J ) 8.9,
10.5 Hz, H-3II), 7.18-7.41 (m, 15H); 13C NMR (100 MHz,
CDCl3) δ 8.9, 20.9, 56.1 (C-2II), 57.4 (C-2I), 66.0 (C-5II), 68.4
(C-6II), 68.8 (C-6I), 70.0 (C-3II), 73.2, 74.5 (2CH2Ph), 74.7 (C-
5I), 76.1 (C-3I), 77.3 (C-4I), 79.4 (C-4II), 93.1 (C-1I), 97.6 (C-
1II), 101.8 (CHPh), 126.5, 127.4, 127.7, 127.8, 128.0, 128.1,
128.4, 128.5, 128.6, 129.4, 137.2, 138.2, 139.1, 170.4, 172.0;
MS (ESI) m/z 889.30 [M + Na]+.
H-1II), 5.69 (t, 1H, J ) 9.7 Hz, H-3II), 7.18-7.41 (m, 15H); 13
C
NMR (100 MHz, CDCl3) δ -5.3, -4.0, 8.8, 9.1, 17.8, 20.9, 25.6,
56.2 (C-2II), 57.8 (C-2I), 66.0 (C-5II), 68.2 (C-6I), 68.9 (C-6II),
70.1 (C-3II), 73.2, 74.3 (2CH2Ph), 74.8 (C-5I), 76.7 (C-3I), 77.5
(C-4I), 79.5 (C-4II), 93.6 (C-1I), 97.9 (C-1II), 101.8 (CHPh), 126.5,
127.3, 127.6, 127.8, 128.1, 128.4, 128.5, 128.6, 129.4, 137.0,
137.2, 138.6, 139.3, 170.4, 171.8; MS (ESI) m/z 1003.41 [M +
Na]+.
Com p ou n d 23 (obta in ed via con ver sion of 22). A
solution of compound 22 (1.8 g, 1.9 mmol) and NaOMe (0.05
g) in anhydrous MeOH (15 mL) was stirred at room temper-
ature. The mixture was stirred at room temperature until
starting material was consumed (1 h). The reaction was
quenched by the addition of an excess amount of Amberlite
IR-120 (H+). After an additional 20 min of stirring, the resin
was filtered and the resulting solution was concentrated to
dryness. The residue was dissolved in acetonitrile and re-
evaporated to dryness, and the residue was kept under vacuum
for 1 h. The residue was dissolved in anhydrous acetonitrile
(15 mL) and the resulting solution was treated with benzal-
dehyde dimethylacetal (0.8 mL, 5.2 mmol) and p-toluene-
sulfonic acid monohydrate (0.06 g). After being stirred at room
temperature for 3 h, the reaction was neutralized by the
addition of a few drops of saturated solution of NaHCO3 and
stirring was continued for an additional 0.5 h. The reaction
mixture was concentrated to dryness and the residue was
chromatographed on silica gel (n-hexane to n-hexane/EtOAc,
3-O-Acet yl-4,6-O-b en zylid en e-2-d eoxy-2-d im et h ylm a -
leim ido-â-D-glu copyr an osyl-(1f4)-3,6-di-O-ben zyl-2-deoxy-
2-d im eth ylm a leim id o-â-D-glu cop yr a n osyl Tr ich lor oa ce-
tim id a te (26). Compound 25 (2.9 g, 3.3 mmol) in anhydrous
CH2Cl2 (15 mL) was treated with 2,2,2-trichloroacetonitrile (1.8
mL, 18.0 mmol) and a catalytic amount of DBU and the
resultant solution was stirred at ice-water temperature. When
the starting material was consumed completely (∼1 h), the
reaction mixture was filtered through a small layer of silica
gel. The filtrate was concentrated under scrupulously dry
condition and the residue was kept under high vacuum for
0.5 h. This material was used without further purification
because of adequate purity for the glycosidation step. 1H NMR
(400 MHz, CDCl3) δ 1.77, 1.94, 1.98 (3s, 15H), 3.35 (m, 1H,
H-5II), 3.47 (dd, 1H, J ) 3.2, 11.4 Hz, H-6I), 3.52 (t, 1H, J )
10.1 Hz, H-6II), 3.55-3.62 (m, 2H, H-5I, H-4II), 3.68 (d, 1H,
I
J ) 11.3 Hz, H-6′ ), 4.02 (dd, 1H, J ) 8.3, 10.3 Hz, H-2II),
4.15-4.20 (m, 3H, H-6′ II, H-4I, H-2I), 4.28 (m, 1H, H-3I), 4.47,
4.84 (2d, 2H, J ) 12.2 Hz, CH2Ph), 4.59, 4.63 (2d, 2H, J )
12.2 Hz, CH2Ph), 5.40 (s, 1H, CHPh), 5.44 (d, 1H, J ) 8.1 Hz,
H-1II), 5.68 (t, 1H, J ) 9.7 Hz, H-3II), 6.15 (d, 1H, J ) 8.9 Hz,
H-1I), 7.17-7.42 (m, 15H), 8.51 (s, 1H, NH); 13C NMR (100
MHz, CDCl3) δ 8.9, 20.9, 54.5 (C-2I), 56.1 (C-2II), 66.0 (C-5II),
67.9 (C-6I), 68.8 (C-6II), 70.0 (C-3II), 73.1, 74.6 (2CH2Ph), 75.7
(C-5I, C-3I), 77.2 (C-4I), 79.5 (C-4II), 94.2 (C-1I), 97.4 (C-1II),
101.8 (CHPh), 126.5, 127.5, 127.8, 127.9, 128.2, 128.4, 128.5,
128.6, 129.4, 137.2, 138.3, 139.0, 161.1, 170.4, 171.4.
1
2:1) to give a white solid (24, 1.1 g, 60%). H NMR (400 MHz,
CDCl3) δ -0.10, 0.02, 0.73, (3s, 15H), 1.79 (br s, 6H), 1.96 (s,
6H), 3.33 (dd, 1H, J ) 4.9, 9.7 Hz, 1H, H-5II), 3.37-3.47 (m,
I
2H, H-6I, H-6′ ), 3.39 (m, 1H, H-5I), 3.43 (t, 1H, J ) 8.9 Hz,
H-4II), 3.53 (dd, 1H, J ) 9.7, 12.2 Hz, H-6II), 3.84 (dd, 1H, J )
8.5, 10.1 Hz, H-2I), 3.98 (dd, 1H, J ) 8.1, 10.5 Hz, H-2II), 4.09-
4.11 (m, 2H, H-3I, H-4I), 4.18 (dd, 1H, J ) 4.9, 9.7 Hz, H-6′ II),
4.65 (t, 1H, J ) 8.9 Hz, H-3II), 4.56, 4.62 (2d, 2H, J ) 12.2 Hz,
CH2Ph), 4.48, 4.81 (2d, 2H, J ) 12.2 Hz, CH2Ph), 5.08 (d, 1H,
J ) 8.1 Hz, H-1I), 5.26 (d, 1H, J ) 8.1 Hz, H-1II), 5.44 (s, 1H,
CHPh), 7.17-7.46 (m, 15H); 13C NMR (100 MHz, CDCl3) δ
-5.3, -4.0, 8.8, 9.1, 17.8, 20.9, 25.6, 57.2 (C-2II), 57.8 (C-2I),
66.0 (C-5II), 68.3 (C-6II), 68.6 (C-3II), 68.9 (C-6I), 73.2, 74.3
(2CH2Ph), 74.9 (C-5I), 76.8 (C-3I), 77.3 (C-4I), 82.4 (C-4II), 93.5
(C-1I), 98.2 (C-1II), 102.1 (CHPh), 126.6, 127.3, 127.6, 127.8,
128.0, 128.1, 128.4, 128.6, 129.6, 137.0, 137.3, 137.6, 138.6,
139.3, 171.9; MS (ESI) m/z 961.35 [M + Na]+.
Met h yl
3-O-Acet yl-4,6-O-b en zylid en e-2-d eoxy-2-d i-
m eth ylm aleim ido-â-D-glu copyr an osyl-(1f4)-3,6-di-O-ben -
zyl-2-d eoxy-2-d im et h ylm a leim id o-â-D-glu cop yr a n osyl-
(1f4)-3-O-a cetyl-6-O-ben zyl-2-d eoxy-2-d im eth ylm a leim -
id o-â-D-glu cop yr a n osid e (27). Compound 26 (3.0 g, 3.0
mmol) was dissolved in anhydrous CH2Cl2 (15 mL) and was
treated with compound 4 (1.6 g, 3.7 mmol) and 4-Å molecular
sieves (3 g). After being stirred for 0.5 h at room temperature,
the mixture was cooled to -5 °C and was treated with a
catalytic amount of TfOH (0.56 mL, 1 M solution in CH2C12).
The reaction was monitored by TLC (Rf 0.71 for 26, Rf 0.36
for 4, Rf 0.50 for 27 in n-hexane/EtOAc, 1:2), as well as HPLC.
Reverse-phase HPLC analysis was performed on a Waters
Delta 600 liquid chromatography system, vydac protein/
peptide C-18 column (5 µm, 4.6 × 250 mm), and monitored
with a Waters 2996 photodiode array spectrometer equipped
with the Millenium software. Isocratic condition (80% MeOH)
with a flow rate of 0.8 mL/min was used. The unreacted
glycosyl donor (4, tR ) 3.8 min) and the product (27, tR ) 7.7
min) were readily detected by this procedure. When the
starting material was consumed completely (∼1-2 h), a few
Compound 24 obtained above (1.1 g, 1.2 mmol) was mixed
with Ac2O (3.1 mL) and pyridine (2.7 mL) and the resultant
solution was stirred at room temperature for 3 h. Evaporation
of the solvent gave the crude product, which was taken up in
a mixture of CH2Cl2 and water. Layers were separated and
the organic layer was washed with water (3×). The organic
layer was dried over anhydrous MgSO4 and concentrated in
vacuo to afford compound 23, which was used for the next step
without further purification (1.5 g, 85%).
3-O-Acet yl-4,6-O-b en zylid en e-2-d eoxy-2-d im et h ylm a -
leim ido-â-D-glu copyr an osyl-(1f4)-3,6-di-O-ben zyl-2-deoxy-
2-d im eth ylm a leim id o-â-D-glu cop yr a n ose (25). Compound
23 (6.0 g, 6.1 mmol) in ethyl acetate (30 mL) was treated by
the dropwise addition of tetrabutylammonium fluoride (8.0
J . Org. Chem, Vol. 69, No. 6, 2004 2143

Hesek et al.
drops of Et3N was added and the solution was stirred at room
temperature for 0.5 h. The reaction mixture was filtered
through a small layer of silica gel, and the silica layer was
washed with CH2Cl2. The filtrate was concentrated to dryness
and the resultant residue was chromatographed (n-hexane/
â-D-glu cop yr a n osyl-(1f4)-3-O-a cetyl-6-O-ben zyl-2-d eoxy-
2-dim eth ylm aleim ido-â-D-glu copyr an oside (29). Compound
28 (0.45 g, 0.35 mmol) and compound 7 (0.35 g, 0.57 mmol)
were added to a suspension of activated 4-Å molecular sieves
(0.5 g) in CH2Cl2 (10 mL). The mixture was stirred at room
temperature for 1 h under an argon atmosphere. The reaction
mixture was cooled in an ice-water bath and n-hexane (3 mL)
was added with vigorous stirring, followed by the addition of
TfOH (52 µL, 1 M solution in CH2C12) dropwise over a period
of 15 min. Stirring was continued at the same temperature
for 2 h, the reaction was quenched by the addition of a few
drops of Et3N, and the solution was filtered through a pad of
Celite. The volatiles were removed in vacuo and the residue
was purified by flash chromatography (n-hexane/EtOAc, 1.2:
1
EtOAc, 1:2) to give compound 27 (2.3 g, 55%). H NMR (400
MHz, CDCl3) δ 1.83, 1.91, 1.92, 2.01 (4s, 18H), 1.76 (br s, 6H),
3.37 (s, 3H, OCH3), 3.11 (2m, 1H, H-5II), 3.20 (m, 1H, H-5III),
I
3.32 (m, 1H, H-6II), 3.36-3.43 (m, 2H, H-6I, H-6′ ), 3.41 (m,
1H, H-6III), 3.47 (m, 1H, H-5I), 3.52 (dd, 1H, J ) 8.9, 9.7 Hz,
H-4III), 3.59 (dd, 1H, J ) 6.5, 10.6 Hz, H-6′ II), 3.78 (dd, 1H,
J ) 8.5, 10.9 Hz, H-2II), 3.91-3.94 (m, 2H, H-3II, H-4I), 3.91
(t, 1H, J ) 7.3 Hz, H-2I), 3.97 (t, 1H, J ) 8.1 Hz, H-2III), 4.13
(dd, 1H, J ) 5.3, 10.1 Hz, H-6′ III), 4.20 (dd, 1H, J ) 8.9, 9.7
Hz, H-4II), 4.38, 4.80 (2d, 2H, J ) 12.2 Hz, CH2Ph), 4.53 (s,
2H, CH2Ph), 4.49, 4.60 (2d, 2H, J ) 11.4 Hz, CH2Ph), 4.94 (d,
1H, J ) 8.1 Hz, H-1II), 5.01 (d, 1H, J ) 8.1 Hz, H-1I), 5.35 (s,
1H, CHPh), 5.36 (d, 1H, J ) 8.1 Hz, H-1III), 5.43 (dd, 1H, J )
9.7 10.5 Hz, H-3I), 5.61 (t, 1H, J ) 9.7 Hz, H-3III), 7.12-7.39
(m, 20H); 13C NMR (100 MHz, CDCl3) δ 9.0, 20.8, 20.9, 54.9
(C-2I), 55.0 (C-2III), 56.1 (C-2II), 56.9 (OCH3), 65.9 (C-5III), 68.1
(C-6I, C-6II), 68.8 (C-6III), 70.1 (C-3III), 71.1 (C-3I), 72.7, 73.1
(2CH2Ph), 73.8 (C-3II), 74.7 (C-5II), 74.5 (CH2Ph), 74.7 (C-5I),
75.3 (C-4II), 77.4 (C-4I), 79.4 (C-4III), 97.1 (C-1II, C-1III), 99.1
(C-1I), 101.7 (CHPh), 126.5, 127.4, 127.5, 127.7, 127.9, 128.0,
128.1, 128.4, 128.6, 129.4, 137.2, 138.3, 138.7, 139.2, 170.4,
170.5; MS (ESI) m/z 1304.41 [M + Na]+.
1
1) to give 29 in yields ranging from 59 to 68%. H NMR (500
MHz, acetone-d6) δ 1.78, 1.87, 1.94, 2.00, 2.01 (overlapping s,
30H, CH3 and CH3CO), 3.06 (dd, 1H, J ) 1.5, 9.6 Hz, H-5III),
3.14 (dd, 1H, J ) 1.5, 9.6 Hz, C-5II), 3.21 (dd, 1H, J ) 3.0,
11.1 Hz, C-6III), 3.32 (s, OCH3), 3.43 (m, 1H, H-5IV), 3.42 (dd,
1H, J ) 4.2, 11.0 Hz, H-6I), 3.47 (dd, 1H, J ) 2.0, 9.1 Hz,
H-6′ III), 3.48 (dd, 1H, J ) 4.3, 10.1 Hz, H-5I), 3.49 (dd, 1H,
I
J ) 3.9, 11.0 Hz, H-6II), 3.58 (d, 1H, J ) 9.9 Hz, H-6′ ), 3.66
(d, 1H, J ) 10.6 Hz, H-6′ II), 3.71 (dd, 1H, J ) 8.6, 10.6 Hz,
H-2II), 3.76 (t, 1H, J ) 9.1 Hz, H-6IV), 3.80 (dd, 1H, J ) 8.7,
10.5 Hz, H-2I), 3.81 (dd, 1H, J ) 8.9, 9.9 Hz, H-2IV), 3.81 (dd,
1H, J ) 9.1, 10.2 Hz, H-4IV), 3.94 (dd, 1H, J ) 8.7, 10.5 Hz,
H-2III), 3.94 (t, 1H, J ) 9.5 Hz, H-4I), 3.95 (t, 1H, J ) 9.4 Hz,
H-4III), 3.97 (dd, 1H, J ) 9.1, 10.4 Hz, H-3II), 4.19 (dd, 1H,
J ) 8.8, 10.0 Hz, H-4II), 4.24 (dd, 1H, J ) 8.6, 10.6 Hz, H-3IV),
4.28 (dd, 1H, J ) 4.8, 10.4 Hz, H-6′ IV), 4.42-4.55 (overlapping
ds, 7H, CH2Ph), 4.62 (d, 1H, J ) 11.6 Hz, CH2Ph), 4.75 (d,
1H, J ) 12.2 Hz, CH2Ph), 4.84 (d, 1H, J ) 12.2 Hz, CH2Ph),
4.98 (d, 1H, J ) 8.6 Hz, H-1II), 4.99 (d, 1H, J ) 8.4 Hz, H-1I),
5.14 (d, 1H, J ) 8.6 Hz, H-1IV), 5.29 (d, 1H, J ) 8.6 Hz, H-1III),
5.41 (dd, 1H, J ) 9.1, 10.6 Hz, H-3I), 5.50 (dd, 1H, J ) 8.9,
10.4 Hz, H-3III), 5.72 (s, 1H, CHPh), 7.03-7.54 (m, 30H, 6Ph);
13C NMR (125 MHz, acetone-d6) δ 8.1, 8.2 (CH3), 20.1, 20.3
(2CH3CO), 54.9 (C-2IV), 55.8 (C-2III), 56.0 (C-2II, C-2I), 56.1
(OCH3), 66.2 (C-5IV), 67.3 (C-6III), 68.2 (C-6I, C-6II), 68.5 (C-
6IV), 71.0 (C-3I), 71.5 (C-3III), 72.1 (CH2Ph), 72.5 (CH2Ph), 72.7
(CH2Ph), 73.9 (CH2Ph), 74.2 (C-5II), 74.3 (C-3II), 74.5 (CH2Ph),
74.6 (C-5I), 74.7 (C-5III), 75.0 (C-4II), 75.1 (C-3IV), 75.5 (C-4I),
77.2 (C-4III), 82.7 (C-4IV), 96.3 (C-1III), 97.6 (C-1II), 98.9 (C-1IV),
99.1 (C-1I), 101.2 (CHPh), 126.4, 127.4, 127.5, 127.6, 127.7,
128.1, 128.3, 128.4, 128.5, 128.8, 129.0, 137.1, 137.3, 138.0,
138.2, 138.7, 138.8, 138.9, 139.1, 139.2, 169.4, 169.7, 171.2,
172.1; MS (ESI) m/z 1304.41 [M + Na]+.
Meth yl 3-O-Acetyl-6-O-ben zyl-2-d eoxy-2-d im eth ylm a -
leim ido-â-D-glu copyr an osyl-(1f4)-3,6-di-O-ben zyl-2-deoxy-
2-d im eth ylm a leim id o-â-D-glu cop yr a n osyl-(1f4)-3-O-a c-
e t y l-6-O -b e n zy l-2-d e o x y -2-d im e t h y lm a le im id o -â-D -
glu cop yr a n osid e (28). Compound 27 (1.3 g, 1.0 mmol) was
dissolved in CH3CN (10 mL) and the solution was cooled in
an ice-water bath. Borane-trimethylamine complex (BH3‚
NMe3, 0.15 g, 2.0 mmol) and BF3‚OEt2 (0.25 mL, 2.0 mmol)
were added dropwise to the reaction mixture in the respective
order. The flask was removed from the bath after 1 h and the
solution was stirred at room temperature for 0.5 h, at which
time TLC (n-hexane/EtOAc, 2:3) indicated complete conversion
of a faster moving material. NaHCO3 (0.14 g) was added to
this solution and the solution was evaporated to dryness. The
crude product was dissolved in ethyl acetate and washed with
aq NaCl. The organic layer was dried over MgSO4, filtered,
and concentrated to dryness to afford the crude product.
Purification by chromatography on silica gel (n-hexane/EtOAc,
2:1 to 1.5:1) gave the desired compound, followed by crystal-
1
lization from diethyl ether (0.80 g, 62%). H NMR (500 MHz,
CDCl3) δ 1.75 (br s, 2CH3), 1.80, 1.95 (2s, 2CH3CO), 1.90, 2.00
(2s, 4CH3), 3.11 (dd, 1H, J ) 1.5, 9.6 Hz, H-5II), 3.15 (dd, 1H,
J ) 1.5, 9.6 Hz, H-5III), 3.36 (s, OCH3), 3.37, 3.55 (m, 2H, H-6I,
H-6′ I), 3.39, 3.61 (m, 2H, H-6II, H-6′ II), 3.47 (m, 1H, H-5I), 3.47,
3.58 (m, 2H, H-6III, H-6′ III), 3.68 (t, 1H, J ) 9.1 Hz, H-4III),
3.77 (dd, 1H, J ) 8.6, 10.6 Hz, H-2II), 3.88-3.95 (overlapping
dd, 4H, H-2I, H-2III, H-3II, H-4I), 4.19 (dd, 1H, J ) 8.6, 10.1
Hz, H-4II), 4.35, 4.78 (2d, 2H, J ) 12.2 Hz, CH2Ph), 4.45 (s,
2H, CH2Ph), 4.49, 4.61 (2d, 2H, J ) 11.6 Hz, CH2Ph), 4.52 (d,
2H, J ) 2.0 Hz, CH2Ph), 4.92 (d, 1H, J ) 8.1 Hz, H-1II), 5.00
(d, 1H, J ) 8.6 Hz, H-1I), 5.25 (d, 1H, J ) 8.6 Hz, H-1III), 5.39
(dd, 1H, J ) 8.6, 10.6 Hz, H-3III), 5.42 (dd, 1H, J ) 9.6, 10.6
Hz, H-3I), 7.05-7.35 (m, 20H, 4Ph); 13C NMR (100 MHz,
CDCl3) δ 9.0 (CH3), 20.8, 20.9 (2CH3CO), 54.9 (C-2I), 55.4 (C-
2III), 55.9 (C-2II), 56.9 (OCH3), 68.0 (C-6I), 68.2 (C-6II), 70.3 (C-
6III), 71.1 (C-3III), 72.0 (C-4III), 72.5 (CH2Ph), 73.1 (CH2Ph), 73.1
(C-5III), 73.6 (C-3I), 73.9 (CH2Ph), 73.9 (C-3II), 74.3 (C-5II), 74.5
(CH2Ph), 74.7 (C-5I), 74.9 (C-4II), 77.3 (C-4I), 96.3 (C-1III), 97.2
(C-1II), 99.1 (C-1I), 127.2, 127.3, 127.5, 127.6, 127.9, 128.0,
128.2, 128.3, 128.4, 128.5, 128.7, 137.1, 137.8, 138.4, 138.7,
139.2, 170.5, 171.1, 171.7; MS (ESI) m/z 1306.49 [M + Na]+.
Meth yl 2-Aceta m id o-3-O-ben zyl-4,6-O-ben zylid en e-2-
d eoxy-â-D-glu cop yr a n osyl-(1f4)-2-a ceta m id o-O-ben zyl-
3-O-((R)-1′-ca r b oxyet h yl)-2-d eoxy-â-D-glu cop yr a n osyl-
(1 f4 )-2 -a c e t a m i d o -3 ,6 -d i -O -b e n z y l -2 -d e o x y -â-D -
glu cop yr a n osyl-(1f4)-2-a ceta m id o-6-O-ben zyl-3-O-((R)-
1′-car boxyeth yl)-2-deoxy-â-D-glu copyr an oside (30). NaOH
(1.0 g of solid pellets, 25 mmol) was added to a stirred solution
of 29 (1.7 g, 1 mmol) in a 1,4-dioxane-water mixture (4:1, 50
mL) at 10 °C under a nitrogen atmosphere; the reaction
mixture was sonicated. After 15 min, the mixture was allowed
to warm to room temperature and was stirred for an additional
6 h. The pH was adjusted to 3 by the addition of 0.5 N HCl,
and the pH of the solution was kept at 3.0 for 24 h. Evapora-
tion to dryness of the reaction mixture gave a yellow oily
residue, which was treated with MeOH (60 mL). Precipitated
inorganic material was filtered off and washed well with cold
MeOH, and the filtrate was evaporated to dryness. The residue
in pyridine (5 mL) was treated with an excess of acetic
anhydride (7 mL) and the resultant solution was stirred at
room temperature overnight, followed by evaporation to dry-
ness. The product was purified with flash chromatography
using EtOAc/CH3CN (3:2) to give 30 (0.7 g, 48%) as a white
powder. 1H NMR (500 MHz, 5% CD3OD in CDCl3) δ 1.71, 1.72,
1.76, 1.91, 1.97, 1.98 (6s, 18H), 3.19 (2m, 1H, H-5III), 3.25 (m,
1H, H-5IV), 3.29 (m, 1H, H-5II), 3.44 (t, 1H, J ) 9.2 Hz, H-2IV),
Met h yl 3-O-Ben zyl-4,6-O-b en zylid en e-2-d eoxy-2-d i-
m eth ylm a leim id o-â-D-glu cop yr a n osyl-(1f4)-3-O-a cetyl-
6-O-ben zyl-2-d eoxy-2-d im eth ylm a leim id o-â-D-glu cop yr a -
n osyl-(1f4)-3,6-di-O-ben zyl-2-deoxy-2-dim eth ylm aleim ido-
2144 J . Org. Chem., Vol. 69, No. 6, 2004

Bacterial Cell Wall
3.40-3.50 (m, 4H, H-3II, H-6III, H-5I, H-6I), 3.52-3.64 (m, 6H,
5% CD3OD in CDCl3) δ 1.29, 1.32 (2d, 6H, J ) 6.4 Hz, O-CH-
CH3), 1.76, 1.78, 1.94, 1.95 (4s, 12H, NHCOCH3), 2.85 (m, 1H),
3.12 (m, 1H), 3.20 (m, 1H), 3.30 (m, 1H), 3.38 (s, 3H, OCH3),
3.38-3.51 (m, 5H), 3.54-3.74 (m, 10H), 3.74-3.95 (m, 5H),
4.00-4.62 (m, 9H, 4CH2Ph and H-1), 4.03 (d, 1H, J ) 8.1 Hz,
H-1), 4.27 (d, 1H, J ) 8.1 Hz, H-1), 4.41 (d, 1H, J ) 8.1 Hz,
H-1), 4.54-4.66 (overlapping 2q, 2H, O-CH-CH3), 4.79 (t, 2H,
J ) 12.2 Hz, CH2Ph), 5.58 (s, 1H, CHPh), 7.00-7.45 (m, 30H,
6Ph); 13C NMR (100 MHz, 5% CD3OD in CDCl3) δ 18.4, 18.9,
21.8, 23.2, 23.3, 53.8, 54.3, 55.2, 55.6, 56.6, 65.9, 67.7, 68.6,
68.9, 73.2, 73.5, 73.6, 74.2, 74.5, 74.8, 75.2, 75.3, 75.8, 76.7,
77.1, 77.4, 77.5, 77.7, 78.0, 80.6, 82.6, 100.5, 100.7, 101.3, 101.4,
103.1, 126.1, 127.3, 127.6, 127.8, 128.1, 128.3, 128.6, 128.8,
129.0, 129.3, 137.2, 137.9, 138.0, 138.1, 138.4, 139.2, 171.1,
171.6, 172.8, 173.1, 177.1, 177.6; MS (ESI) m/z 1550.91
[M + Na]+.
I
H-6′ , H-6II, H-6′ II, H-6IV, H-3IV, H-4IV), 3.65-3.73 (m, 2H,
H-3IV, H-6′ III), 3.71 (t, 1H, J ) 7.9 Hz,H-2II), 3.80 (t, 1H, J )
8.4 Hz, H-4I), 3.81 (t, 1H, J ) 9.0 Hz, H-4III), 3.92 (t, 1H, J )
9.6 Hz, H-2III), 3.92 (t, 1H, J ) 7.3 Hz, H-4II), 4.02 (t, 1H, J )
8.6 Hz, H-2I), 4.25 (d, 1H, J ) 7.6 Hz, H-1I), 4.25 (d, 1H, J )
8.4 Hz, H-1III), 4.28 (dd, 1H, J ) 4.9, 10.5 Hz, H-6′ IV), 4.33 (d,
1H, J ) 8.1 Hz, H-1II), 4.49 (d, 1H, J ) 7.1 Hz, H-1IV), 4.75 (t,
1H, J ) 9.6 Hz, H-3III), 4.95 (t, 1H, J ) 8.9 Hz, 1H, H-3I), 5.52
1
(s, 1H, CHPh), 7.15-7.47 (m, 30H, 6Ph); H NMR (500 MHz,
5% CDCl3 in CD3CN) δ 1.90, 1.94, 1.96, 2.01, 2.11 (5s, 18H),
3.29 (2m, 1H, H-5III), 3.35 (m, 1H, H-5II), 3.39 (m, 1H, H-5IV),
3.56 (dd, 1H, J ) 4.9, 11.4 Hz, H-6III), 3.58 (m, 1H, H-3II), 3.62
(m, 2H, H-5I, H-6I), 3.64 (dd, 1H, J ) 8.5, 9.7 Hz, H-2II), 3.69-
3.74 (m, 3H, H-2IV, H-3IV, H-4IV), 3.74-3.72 (m, 2H, H-6II,
I
H-6′ II), 3.78 (dd, 1H, J ) 10.9 Hz, H-6′ ), 3.84 (m, 1H, H-6III),
3.85 (m, 1H, H-6IV), 3.86 (t, 1H, J ) 9.5 Hz, H-2III), 3.86 (m,
1H, dd, 1H, J ) 8.5, 9.3 Hz, H-4III), 3.87 (t, 1H, J ) 9.3 Hz,
H-2I), 3.87 (m, 1H, H-4I), 4.07 (t, 1H, J ) 8.9 Hz, H-4II), 4.36
(dd, 1H, J ) 4.9, 9.7 Hz, H-6′ IV), 4.46 (d, 1H, J ) 8.1 Hz, H-1I),
4.49 (d, 1H, J ) 8.5 Hz, H-1II), 4.57 (d, 1H, J ) 7.3 Hz, H-1IV),
4.68 (d, 1H, J ) 8.1 Hz, H-1III), 5.05 (t, 1H, J ) 9.7 Hz, 1H,
H-3I), 5.06 (t, 1H, J ) 9.7 Hz, H-3III), 5.52 (s, 1H, CHPh), 7.06-
7.50 (m, 30H, 6Ph); 13C NMR (125 MHz, 5% CDCl3 in CD3-
CN) δ 20.5, 20.8 (2CH3CO), 22.4, 22.6, 22.8 (4CH3CONH), 53.8
(C-2I), 54.9 (C-2III), 55.2 (C-2II), 55.5 (C-2IV), 56.5 (OCH3), 66.1
(C-5IV), 68.3 (C-6III), 68.6 (C-6I), 68.7 (C-6IV), 68.9 (C-6II), 72.6
(CH2Ph), 73.0 (CH2Ph), 73.1 (CH2Ph), 73.3 (C-3I), 73.5 (C-3III),
73.9 (CH2Ph), 74.2 (CH2Ph), 74.7 (C-5II), 74.8 (C-5I), 74.7 (C-
5III), 75.1 (C-4II), 75.2 (C-4I), 76.2 (C-4III), 78.7 (C-3IV), 80.7 (C-
3II), 81.9 (C-4IV), 99.6 (C-1III), 101.1 (C-1II), 101.2 (CHPh), 101.8
(C-1IV), 102.0 (C-1I), 126.4, 127.5, 127.8, 128.0, 128.1, 128.4,
128.6, 128.7, 128.8, 129.2, 138.2, 138.7, 139.0, 139.7, 170.4,
170.5, 170.7, 170.8; MS (ESI) m/z 1467.77 [M + H]+, 1489.79
[M + Na]+.
Meth yl 2-Aceta m id o-3-O-ben zyl-4,6-O-ben zylid en e-2-
deoxy-â-D-glu copyr an osyl-(1f4)-2-acetam ido-6-O-ben zyl-
3-O-((R)-1′-ca r b oxyet h yl)-2-d eoxy-â-D-glu cop yr a n osyl-
(1f4)-2-a ceta m id o-3,6-d i-O-ben zyl-2-d eoxy-â-D-glu cop y-
r a n o s y l-(1f4)-2-a c e t a m i d o -6-O -b e n z y l-3-O -((R )-1′-
car boxyeth yl)-2-deoxy-â-D-glu copyr an oside (32). A solution
of NaOMe (0.05 g) in dry MeOH (1 mL) was added dropwise
to a suspension of 30 (1 g, 0.68 mmol) in dry MeOH (15 mL).
The reaction mixture was stirred at room temperature for 1 h
under an atmosphere of nitrogen and then after neutralization
with acetic acid the solution was evaporated to obtain a
residue, which was washed with cold water, then filtered and
dried under vacuum. The white solid material was dispersed
in a minimal amount of dry CH3CN, stirred under an atmo-
sphere of nitrogen for 1 h, and then filtered to give a pure
dihydroxy derivative 31 (0.7 g, 74%), which was used in the
next reaction without further purification. MS (ES) m/z
1383.75 [M + H]+, 1405.70 [M + Na]+.
Com p ou n d 34. p-Nitrophenyl trifluoroacetate (780 mg, 3.3
mmol) was added to a solution of 32 (510 mg, 0.33 mmol) in
pyridine (20 mL) and the mixture was stirred at room
temperature for 18 h. Water and dichloromethane were added
into the reaction mixture and layers were separated. The
organic layer was washed with water and brine, dried over
Na2SO4, and concentrated under reduced pressure to give the
crude material. Subsequently, diethyl ether was added and
the precipitate was filtered, washed with diethyl ether, and
dried over P2O5 to give the p-nitrophenyl ester of 32 (420 mg,
72%) as a white solid.
Peptide 33 (510 mg, 0.56 mmol) in dry DMF (30 mL) was
neutralized with Et3N (78 µL, 0.56 mmol) and was stirred at
room temperature for 15 min. The p-nitrophenyl ester of 32
(300 mg, 0.17 mmol) was added into this amine solution and
the resultant mixture was stirred at room temperature for 18
h. The solvent was removed in vacuo and the residue was
suspended in water and filtered. The residue was purified by
short-path column chromatography (CHCl3/MeOH, 5:1) to give
the fully protected 34 (300 mg, 57%). 1H NMR (400 MHz,
acetone-d6) δ 1.29, 1.32 (2d, 6H, J ) 6.4 Hz, O-CH-CH3), 1.76,
1.78, 1.94, 1.95 (4s, 12H, NHCOCH3), 2.85 (m, 1H), 3.12 (m,
1H), 3.20 (m, 1H), 3.30 (m, 1H), 3.38 (s, 3H, OCH3), 3.38-
3.51 (m, 5H), 3.54-3.74 (m, 10H), 3.74-3.95 (m, 5H), 4.00-
4.62 (m, 9H, 4CH2Ph and H-1), 4.03 (d, 1H, J ) 8.1 Hz, H-1),
4.27 (d, 1H, J ) 8.1 Hz, H-1), 4.41 (d, 1H, J ) 8.1 Hz, H-1),
4.54-4.66 (overlapping 2q, 2H, O-CH-CH3), 4.79 (t, 2H, J )
12.2 Hz, CH2Ph), 5.58 (s, 1H, CHPh), 7.00-7.45 (m, 30H, 6Ph);
13C NMR (100 MHz, acetone-d6) δ 16.9, 17.1, 17.4, 17.6, 17.7,
18.1, 18.5, 19.0, 22.3, 22.8, 23.0, 23.1, 23.2, 27.0, 29.7, 29.8,
29.9, 30.1, 31.4, 40.7, 48.3, 48.5, 48.6, 48.8, 49.6, 51.5, 51.7,
52.6, 54.6, 54.9, 55.1, 55.9, 56.4, 65.8, 66.2, 66.4, 66.5, 66.6,
68.7, 69.8, 72.7, 72.9, 73.5, 74.0, 74.2, 74.6, 75.1, 75.4, 75.8,
76.6, 77.0, 78.4, 79.0, 80.7, 82.6, 99.8, 100.1, 100.9, 102.5, 126.5,
127.5, 127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.5, 128.6,
128.7, 136.3, 136.4, 136.5, 137.8, 138.4, 139.0, 139.1, 139.3,
139.9, 156.7, 169.5, 170.5, 170.9, 171.0, 171.6, 172.4, 172.5,
172.6, 172.9, 173.2, 173.8.
The residue 31 (0.7 g, 0.51 mmol) was dissolved in anhy-
drous DMF (15 mL) and the solution was stirred in the
presence of activated 4-Å molecular sieves (3 g), followed by
treatment with NaH (0.5 g, 12.5 mmol, 60% oil dispersion),
and (S)-2-chloropropionic acid (1.6 g, 14.7 mmol), and the
mixture was allowed to stir at 10 °C for 0.5 h. Additional NaH
(0.8 g, 20 mmol) in two portions was added to the reaction
mixture and the resultant solution was stirred at room
temperature overnight. The pH of the reaction mixture was
brought to pH 4-5 by addition of dilute HCl and the suspen-
sion was filtered through a layer of Celite, which was subse-
quently washed well with MeOH. The volatiles were evapo-
rated to dryness and the residue was taken up in a mixture of
water and chloroform. The layers were separated and the
aqueous portion was washed with chloroform. The combined
organic layer was concentrated in vacuo to obtain a solid
residue, which was purified by chromatography on a small
silica gel column, eluting with CH2Cl2/MeOH (5:1) to afford
Com p ou n d 3. Compound 34 (110 mg, 36 µmol) was
dissolved in 60% acetic acid (10 mL), and the solution was
stirred at 70 °C for 1h. The solvent was removed under reduced
pressure. The residue was dissolved in MeOH (20 mL), and
the stirred mixture was cautiously treated with Pd/C (10%,
0.2 g) and then kept under an atmosphere of hydrogen with
continued stirring at room temperature overnight. The mixture
was diluted with MeOH (20 mL) and filtered through a layer
of Celite, and the Celite pad was washed with MeOH. The
volatiles were evaporated in vacuo. Purification by preparative
thin-layer chromatography (n
BuOH/AcOH/H2O, 3:2:1) afforded
3 (56 mg, 76%). The purity of the isolated product was analyzed
by HPLC. Reverse-phase HPLC analysis was performed on a
Waters Delta 600 liquid chromatography system, delta-pak
C-18 column (5 µm, 3.9 × 150 mm), and monitored with a
Waters 2414 refractive index detector equipped with the
Millenium software. Isocratic condition (90% acetonitrile) with
a flow rate of 0.75 mL/min was used. The retention time of
1
the title compound (0.49 g) in 46% yield. H NMR (400 MHz,
J . Org. Chem, Vol. 69, No. 6, 2004 2145

Hesek et al.
compound 3 was 4.97 min and the purity was 97%. The
chromatogram of 3 is given in the Supporting Information. 1H
NMR (500 MHz, D2O) δ 1.20-1.29 (overlapping d and m, 28H),
1.54 (t, 4H), 1.65 (m, 4H), 1.77-1.92 (overlapping s and m,
15H), 1.95 (m, 2H), 2.19 (m, 4H), 2.85 (t, 4H), 3.32 (s, 3H),
3.25-3.35 (m, 4H), 3.38-3.64 (m, 15H), 3.68-3.77 (m, 7H),
3.97-4.30 (m, 13H), 4.35-4.39 (overlapping d, 3H); 13C NMR
(125 MHz, D2O) δ16.7, 17.1, 17.2, 17.5, 18.0, 18.2, 22.2, 22.3,
22.4, 26.4, 28.5, 30.6, 32.0, 39.3, 49.7, 50.0, 54.2, 54.3, 54.8,
55.5, 56.1, 57.2, 59.9, 60.0, 60.4, 61.2, 70.3, 72.3, 73.7, 74.9,
75.1, 75.2, 75.8, 76.2, 78.1, 78.4, 79.5, 79.6, 100.6, 101.9, 102.2,
Ack n ow led gm en t. This work was supported by the
National Institutes of Health.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for syntheses of the monomer compounds in Scheme
2 and the pentapeptide synthesis of Scheme 6 and 1H, 13C
NMR, H-H COSY, and HMQC spectra for selected com-
pounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
174.2; HRMS (ESI) [M + 2H]2+ calcd for C79H134N16O39
2+
965.4492, found 965.4446.
J O035583K
2146 J . Org. Chem., Vol. 69, No. 6, 2004