Analogues of the mycobacterial arabinogalactan linkage disaccharide as cell wall biosynthesis inhibitors

Article abstract of DOI:10.1016/S0968-0896(03)00366-3

The mycobacterial arabinogalactan linkage disaccharide [α-L-Rha-(1→3)-α-D-GlcNAc] provides a basis for the design of new antitubercular drugs, since it supports a key skeletal structure in the bacterial cell wall. A series of analogues of the linker was s

Full text of DOI:10.1016/S0968-0896(03)00366-3

Bioorganic & Medicinal Chemistry 11 (2003) 3579–3587
Analogues of the Mycobacterial Arabinogalactan Linkage
Disaccharide as Cell Wall Biosynthesis Inhibitors
Xianghui Wen,a Dean C. Crick,b Patrick J. Brennanb and Philip G. Hultina,*
aDepartment of Chemistry, University of Manitoba, Winnipeg, MB, Canada R3T 2N2
bDepartment of Microbiology, Colorado State University, Ft. Collins, CO 80523-1677, USA
Received 14 February 2003; revised 14 May 2003; accepted 2 June 2003
Abstract—The mycobacterial arabinogalactan linkage disaccharide [a-l-Rha-(1!3)-a-d-GlcNAc] provides a basis for the design of
new antitubercular drugs, since it supports a key skeletal structure in the bacterial cell wall. A series of analogues of the linker was
synthesized by coupling appropriate thiorhamnosyl donors modified at their 4-positions, with an N-acetyl glucosamine acceptor. In
a cell-free enzyme inhibition assay, three analogues inhibited the activity of the galactosyltransferase that adds a Galf residue to the
linkage disaccharide. Although the compounds were modest inhibitors, these data confirm the viability of this approach to anti-
mycobacterial agents. It is especially significant that the three effective compounds are modified at the site of the acceptor atom in
the natural substrate.
# 2003 Elsevier Ltd. All rights reserved.
Introduction
disaccharide: a-l-Rha-(1!3)-a-d-GlcNAc (Fig. 1).4 In
the biosynthesis of the mycobacterial cell wall,5 the di-
saccharide linker is formed first, as a diphosphoprenol
ester. The poly(galactofuranose) chain of the AG is then
constructed from the C-40 hydroxyl of the linker, after
which the arabinofuranosyl and mycolate components
are added. It is not clear whether the mycolic acids are
attached to AG before or after ligation to peptidogly-
can. However, recent experiments using whole cell
labeling suggest that mycolylation of the arabinan ter-
mini follows ligation of AG to peptidoglycan.6 The
linkage disaccharide is thus a key structure in the overall
biosynthesis of the cell wall. Although this fact has not
yet been widely exploited in the quest for anti-myco-
bacterial agents,7,8 the McNeil laboratory has identified
the biosynthesis of dTDP-Rha as a potential source of
drug targets.9
Mycobacterium tuberculosis is responsible for over 2
million deaths worldwide annually, largely in the devel-
oping countries. Multidrug resistance and the problem
of dual HIV and M. tuberculosis infections are causes
for growing global concern.1 There is therefore a clear
need for new therapeutic strategies. The search for such
strategies is facilitated by the detailed information
about mycobacterial biochemistry that has become
available in recent years.
The cell wall of Mycobacterium spp. is composed of a
covalently linked complex of mycolic acids, arabinan,
and galactan attached to the underlying peptidoglycan
layer.2 The arabinogalactan (AG) is a central support-
ing structure of the cell wall. Disruption of AG bio-
synthesis is known to be lethal to mycobacteria, and
some front-line drugs interfere with aspects of this key
process. In the quest for new anti-mycobacterial agents,
considerable effort has been directed to the synthesis of
arabinofuranose derivatives and other compounds rela-
ted to the AG.3
We report here our preparation of a set of a-l-Rha-
(1!3)-a-d-GlcNAc analogues in which the C-40 position
In 1990, Brennan et al. reported that the AG is linked to
the underlying peptidoglycan via a unique pyranosidic
*Corresponding author. Tel.:+1-204-474-9814; fax: +1-204-474-
7608; e-mail: hultin@cc.umanitoba.ca
Figure 1. The AG linkage disaccharide.
0968-0896/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0968-0896(03)00366-3

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X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
Scheme 1. Reagents and conditions: (i) NIS, TMSOTf, CH2Cl2, 4 A molecular sieves, rt; (ii) (a) 80% HOAc, 70 ꢀC; (b) NaOMe/MeOH, rt; (iii) (a)
80% HOAc, 70 ꢀC; (2) Ac2O, pyridine, DMAP, rt; (iv) Pd/C, H2, HOAc, rt.
of the disaccharide has been modified to hinder the for-
mation of the glycosidic bond to the AG polymer. We
show that some of these linkage region analogues are
modest inhibitors of the biosynthesis of mycobacterial
cell wall components.
We also prepared some disaccharide analogues con-
taining modified GlcNAc units (Scheme 2). C-Glycosyl
GlcNAc derivatives 18 and 20 were chosen as contain-
ing sterically conservative replacements for the labile
1-phosphate group in the mycobacterial linkage dis-
accharide. They were prepared from known precursors
1714 and 1915 as shown in Scheme 2. Glycosidation
reactions of 18 and 20 with rhamnosyl donors 1 and 2
(Scheme 3) proceeded smoothly, affording heptonic acid
disaccharides 21 (61%) and 22 (83%), and phosphonate
27 (75%). Deprotection of 27 was routine, giving the
free disaccharide analogue 28 in 89% yield. However,
all attempts to remove the benzoyl groups from the
rhamnosyl moieties in 21 and 22 led to some degree of
epimerization of the methyl ester. The desired axial C-
glycosides 23 and 24 predominated, as expected based
on the known anomeric effect of C-linked carboxylic
ester groups,16 but we were unable to suppress this base-
catalyzed process. Nevertheless, the axially- and equa-
torially-substituted C-glycosyl disaccharide analogues
were separable by chromatography, and reasonable
yields of 23–26 were obtained.
Results and Discussion
We elected to use the thioglycoside approach10,11 to
prepare our disaccharides. Thus, we obtained thiogly-
cosyl donors 1, 2 and 3 using literature methods.8,12 The
acceptor, benzyl N-acetyl-2-amino-2-deoxy-glucoside
4,13 was likewise a known compound. Preliminary
experiments showed that NIS/TfOH was a less effective
glycosylation promoter than NIS/TMSOTf. Using the
latter reagent, 5–7 were obtained in 92–95% yields
(Scheme 1). The a anomer was the exclusive product in
each case, which was confirmed by the absence of NOE
among H-10, H-30 and H-50.
Deprotection of 5–7 was achieved in two steps. Initial
treatment with 80% acetic acid cleaved the 4,6-O-ben-
zylidene acetals, after which the benzoyl protecting
groups on the rhamnosyl moieties were removed using
NaOMe. Products 8–10 retaining the benzyl glycoside
group were thus obtained in 90–93% yields. Performing
these deprotection steps in the opposite order rendered
the removal of the benzylidene group extremely difficult.
The effectiveness of our compounds as inhibitors of
mycobacterial AG biosynthesis was assayed in a cell-
free model system. A membrane and cell wall enriched
fraction isolated from Mycobacterium smegmatis17 is
able to catalyze the transfer of N-acetyl glucosamine 1-
phosphate (GlcNAc-1-P) and l-rhamnose (Rha) from
their respective nucleotide donors to endogenous poly-
prenyl phosphate (Pol-P), yielding Pol-P-P-GlcNAc
Pathak et al. had found that octyl d-Galf-a-l-Rha
compounds blocked by acetyl groups on the Galf resi-
due, or by a 2,3-O-isopropylidene group on rhamnose
inhibited the growth of mycobacteria in a whole cell
assay.7 We therefore also prepared derivatives in which
the non-anomeric hydroxyls were blocked by ester
groups, which might be removed by nonspecific ester-
ases present in the cell wall environment. Removal of
the benzylidene protecting group followed by acetyla-
tion gave 11–13 in good yields. Hydrogenolysis of 11–13
afforded the reducing disaccharides 14–16. These reac-
tions were quite sluggish and proceeded only in moder-
ate yields, even when performed in acetic acid solvent.
Scheme 2. Reagents and conditions: (i) H2, Pd/C, HOAc, rt;
(ii) C6H5CH(OMe)2, p-TsOH, DMF, 40 ꢀC.

X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
3581
Scheme 3. Reagents and conditions: (i) 1 or 2, NIS/TMSOTf, CH2Cl2, 4 A molecular sieves, rt; (ii) (a) 80% HOAc, 70 ꢀC; (b) NaOMe/MeOH, rt;
(iii) 2, NIS/TMSOTf, CH2Cl2, 4 A molecular sieves, rt.
reduce the enzymatic activity, but not significantly
(Fig. 3). In other experiments, only the UDP-Gal was
omitted from the reaction mixture, assaying the forma-
tion of GL-2, with similar results to those of Figure 3
(data not shown). These data strongly suggest that we
are indeed blocking the biosynthetic conversion of GL-2
to GL-3, as planned.
Figure 2.
The value of the disaccharide linker between the myco-
(GL-1) and Pol-P-P-GlcNAc-Rha (GL-2). Galactosyl
transferase-1 (Galtase-1) mediates the transfer of a
galactofuranosyl (Galf) residue from UDP-Galf to GL-
2, giving rise to Pol-P-P-GlcNAc-Rha-Galf (GL-3). Our
compounds were designed to disrupt the conversion of
GL-2 to GL-3.
bacterial peptidoglycan and arabinogalactan as a drug
target has been demonstrated by the observations that
the dTDP-Rha synthase (dTDP-Rha is the Rha donor
in the biosynthesis of the linker),19 the rhamnosyl
transferase that forms the linker20 and a subsequent
Galf transferase (Rv3808c)21 are all essential enzymes in
mycobacteria. The inhibition of the Galtase enzyme in
vitro by 14–16 clearly demonstrates the validity of
designing inhibitors with the purpose of disrupting the
processing of the linker between the mycobacterial pep-
tidoglycan and arabinogalactan.
The inhibition of Galtase-1-mediated Galf transfer was
assayed in mixtures containing radiolabeled UDP-Gal,
UDP-Galp mutase,18 dTDP-Rha, UDP-GlcNAc, and
the cell-wall-enriched fraction (as the source of Galtase-
1 and Pol-P) essentially as previously described.5 The
disaccharides 5–16, 23–26 and 28 prepared in this work,
as well as analogues 29 and 30 (Fig. 2) available from
previous work,8 were assayed. The identities of the
radiolabeled products were confirmed by TLC.
It is surprising that the protected compounds 14, 15 and
16 were the most active inhibitors. This may be due to
their lipophilic nature, which likely gives the inhibitors
physical/solubility properties similar to the native lipid-
linked substrates. Notably, each of these compounds is
modified at the rhamnosyl 4-OH position, which is the
acceptor site for the GL-2 to GL-3 conversion. This is
highly unusual, as very few compounds modified at the
acceptor atom have proven to be effective inhibitors of
Of the 19 analogues tested, three (14, 15 and 16) inhib-
ited the galactosyl transferase activity with IC50 values
shown in Table 1. In order to confirm that formation of
GL-3 was inhibited and not an earlier step, the activity
of N-acetylglucosamine 1-phospate transferase was also
assayed in the presence of 14, 15 and 16 at 300 mg/mL.
Assays were done essentially as described for the galac-
tosyl transferase except the unlabelled UDP-GlcNAc
was replaced with 1 mCi of UDP-[6-3H(N)]GlcNAc (60
Ci/mmol, American Radiolabeled Chemicals Inc., St.
Louis, MO, USA), UDP-Gal and dTDP-Rha were
omitted and the reaction was incubated for 15 min to
measure formation of GL-1. The compounds tended to
Table 1. Galf transferase inhibition by disaccharide analogues
Compd
IC50 (mg/mL)
14
15
16
410
370
240
Figure 3. Effect of disaccharide inhibitors on the formation of GL-1.

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X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
glycosyltransferases,22 let alone analogues bearing
groups as large as a benzoyl.
171.04. Anal. calcd for C17H21NO7: C, 58.11; H, 6.02;
N, 3.99. Found: C, 58.34; H, 6.20;N, 4.11.
Although the synthesized compounds are not ‘drug-like’
in the traditional sense, interest in carbohydrate struc-
tures as leads or drug candidates is growing.23 In the
near term, inhibitors 14–16 are much-needed tools for
the study of the enzymes involved in the early stages of
mycobacterial galactan synthesis. We will use them as
leads for the development of better inhibitors and affi-
nity ligands, which will greatly aid identification of the
Galtase that adds the first Galf residue to the
disaccharide inkage unit.
Diethyl C-(4,6-O-benzylidene-2-acetamido-2-deoxy-ꢀ-D-
glucopyranosyl) methanephosphonate 20. A suspension
of 3,4,6-O-benzyl-protected phosphonate 1915 (449 mg,
0.72 mmol), Pd/C catalyst (10% w/w, 400 mg), and
acetic acid (1.5 mL) was degassed and flushed with H2
gas as described above. The mixture was vigorously
stirred for 3 h at rt, and then filtered through Celite and
concentrated. The residue was dissolved in DMF(1 mL)
.
and treated with p-TsOH H2O (20 mg) and benzalde-
hyde dimethyl acetal (0.25 mL, 1.7 mmol) at 40 ꢀC for 1
h. The reaction was quenched with Et3N and con-
centrated. Chromatography of the residue (EtOAc/
CH2Cl2/MeOH 1/1/0.05–1/1/0.2) afforded 20 as a white
solid (184 mg, 58%).
Experimental
Reactions were performed under a nitrogen atmosphere
in oven-dried glassware except as noted. Reagents were
purchased from the Aldrich Chemical Co. and were
used as received. Solvents were purified or dried
according to standard procedures.24 Flash column
chromatography was performed on silica gel 60 (230–
400 mesh). NMR spectra were recorded at 300 MHz for
1H, 75.5 MHz for 13C and 121.5 MHz for 31P in the
deuterated solvents except as noted. Melting points were
determined on a capillary apparatus and are uncor-
rected. Optical rotations were measured at ambient
temperature using a Rudolph Research Autopol III
polarimeter. Mass spectra were obtained using electro-
spray ionization (ES-MS) on a Micromass Quattro LC
instrument.
Mp 188.5–189.2 ꢀC. [a]D=+3.8ꢀ (c 1.0, CHCl3). 1H
NMR (acetone-d6, 500 MHz): d 1.28 (t, 6H, J=7.1 Hz,
2 ꢁ CH3CH2), 1.92 (s, 3H, CH3C¼O), 2.09–2.13 (m, 1H,
PCH2), 2.43 (ddd, 1H, J=16.1, 16.1 and 11.3 Hz, PCH2),
3.55 (dd, 1H, J=9.1 and 9.1 Hz, H-4), 3.66–3.80 (m, 2H,
H-5, 6), 3.90 (ddd, 1H, J=10.5, 9.1 and 4.6 Hz, H-3),
4.04–4.10 (m, 4H, 2ꢁOCH2CH3), 3.98–4.13 (m, 1H, H-2),
4.16–4.20 (m, 1H, H-6), 4.53–4.61 (m, 1H, H-1), 5.62 (s,
1H, PhCH), 7.30–7.54 (m, 6H, Ph and NHAc). 13C
NMR (acetone-d6, 125.8 MHz): d 16.86 (d, J=3.3 Hz,
CH3CH2), 16.90 (d, J=3.1 Hz, CH3CH2), 23.03
(CH3C¼O), 24.49 (d, J=142.9 Hz, PCH2), 55.56 (d,
J=12.9 Hz, C-2), 61.90 (d, J=5.9 Hz, CH3CH2), 62.37
(d, J=5.9 Hz, CH3CH2), 65.26 (C-5), 68.85 (C-3), 69.76
(C-6), 71.81 (d, J=4.8 Hz, C-1), 84.26 (C-4), 102.37
(PhCH), 170.82. 31P NMR (CDCl3): d 29.20. Anal.
calcd for C20H32NO8P: C, 53.93; H, 7.24; N, 3.15.
Found: C, 54.34; H, 7.31; 3.20.
3-Acetamido-2, 6-anhydro-5,7-benzylidene-3-deoxy-D-gly-
cero-D-ido-heptonic acid methyl ester 18. A vigorously
stirred mixture of 4,5,7-tri-O-benzyl-protected ester 1714
(740 mg, 1.4 mmol), Pd/C catalyst (10% w/w, 500 mg),
and AcOH (10 mL) was degassed under vacuum and
then placed under an atmosphere of H2 gas using a
balloon. The suspension was stirred overnight at rt,
before being filtered through Celite and concentrated.
The residue was dissolved in DMF(5 mL) and p-
General procedure for glycoside synthesis
The thiorhamnosyl donor 1, 2, or 3 (0.50 mmol),
N-acetyl glucosamine acceptor 4, 18 or 20 (0.60 mmol)
and freshly activated, powdered 4 A molecular sieves
(500 mg) were dissolved in dry CH2Cl2 (4 mL). The
mixture was vigorously stirred for 1 h, after which
N-iodosuccinimide (0.65 mmol) and TMSOTf (10 mL)
were added. The reaction was allowed to proceed at rt
until TLC indicated the complete consumption of the
donor. The reaction mixture was diluted with CH2Cl2
and filtered through Celite. The filtrate was washed
successively with saturated aqueous solutions of
Na2S2O3 and NaHCO3, water and brine, then dried and
concentrated. The residue was purified by flash chro-
matography, eluting with a mixture of hexane and
EtOAc, to obtain 5–7, 21, 22 or 27.
.
TsOH H2O (20 mg) and benzaldehyde dimethyl acetal
(0.25 mL, 1.7 mmol) were added. After stirring at
40 ꢀC for 2 h, the reaction was quenched with Et3N,
and the mixture was concentrated. Crystallization of
the residue in MeOH gave 18 as white crystals (310 mg,
64%).
ꢀ
ꢀ
1
Mp 260.5–261.0 C. [a]D=+74.9 (c 0.35, DMSO). H
NMR (CDCl3): d 2.04 (s, 3H, CH3C¼O), 2.73 (d, 1H,
J=3.0 Hz, OH), 3.53 (ddd, 1H, J=9.7, 9.3 and 4.2 Hz,
H-6), 3.59 (dd, 1H, J=9.3 and 9.3 Hz, H-5), 3.74 (dd,
1H, J=9.7 and 9.7 Hz, H-7), 3.83 (s, 3H, OCH3), 4.05
(ddd, 1H, J=9.3, 9.3 and 3.0 Hz, H-4), 4.33 (dd, 1H,
J=9.7 and 4.2 Hz, H-7), 4.48 (ddd, 1H, J=9.3, 8.7 and
5.7 Hz, H-3), 4.60 (d, 1H, J=5.7 Hz, H-2), 5.55 (s, 1H,
PhCH), 6.62 (d, 1H, J=8.7 Hz, NHAc), 7.36–7.49 (m,
5H, Ph). 13C NMR (CDCl3): d 23.36 (CH3C¼O), 51.60
(C-3), 52.71 (OCH3), 67.98 (C-6), 68.66 (C-7), 70.08 (C-
4), 74.49 (C-2), 82.25 (C-5), 101.96 (PhCH), 170.87,
Benzyl 20,30-di-O-benzoyl-40,60-dideoxy-ꢀ-L-lyxo-hexo-
pyranosyl - (1!3) - 2- acetamido - 4,6 - O - benzylidene - 2-
deoxy-ꢀ-D-glucopyranosidꢀe 5. White solid, 95%. Mp
ꢀ
1
232–233 C. [a]D=+68.5 (c 1.03, CH2Cl2). H NMR
(CDCl3): d 0.77 (d, 3H, J=6.2 Hz, H-60), 1.75–1.90 (m,
2H, H-40), 2.10 (s, 3H, CH3C¼O), 3.70 (dd, 1H, J=9.5
and 9.5 Hz, H-4), 3.79 (dd, 1H, J=10.2 and 10.2 Hz, H-
6), 3.93 (ddd, 1H, J=10.2, 9.5 and 4.7 Hz, H-5), 3.98

X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
3583
(dd, 1H, J=9.5 and 9.5 Hz, H-3), 4.25 (dd, 1H, J=10.2
and 4.7 Hz, H-6), 4.27 (m, 1H, H-50), 4.45 (ddd, 1H,
J=9.9, 9.5 and 3.7 Hz, H-2), 4.49 (d, 1H, J=11.8 Hz,
PhCH2), 4.74 (d, 1H, J=11.8 Hz, PhCH2), 4.87 (d, 1H,
J=3.7 Hz, H-1), 5.17 (m, 2H, H-20, 10), 5.51–5.54 (m,
1H, H-30), 5.57 (s, 1H, PhCH), 5.69 (d, 1H, J=9.9 Hz,
NHAc), 7.30–8.10 (m, 20H, 4ꢁPh). 13C NMR (CDCl3):
d 20.35 (C-60), 23.42 (CH3C¼O), 23.72 (C-40), 53.12 (C-
2), 63.50 (C-5), 64.28 (C-50), 67.75 (C-30), 68.95 (C-6),
69.47 (C-20), 70.03 (PhCH2), 76.58 (C-3), 80.36 (C-4),
97.81 (C-1), 99.09 (C-10), 102.19 (PhCH), 165.45
(PhC¼O), 165.70 (PhC¼O), 170.61 (CH3C¼O). Anal.
calcd for C42H43NO11: C, 68.37; H, 5.87; N, 1.90.
Found: C, 68.54; H, 6.05; N, 1.94.
20,30-Di-O-benzoyl-40,60-dideoxy-ꢀ-L-lyxo-hexopyranosyl-
(1!4) - 3 - acetamido - 2,6 - anhydro - 5,7 - benzylidene - 3 -
deoxy-D-glycero-D-ido-heptonic acid methyl ester 21.
Crystals, 61%, mp 242–243 ꢀC. [a]D=+22.9ꢀ (c 1.25,
1
CH2Cl2). H NMR (CDCl3): d 0.78 (d, 3H, J=6.2 Hz,
H-60), 1.84–2.04 (m, 2H, H-40), 2.16 (s, 3H, CH3C¼O),
3.51 (ddd, 1H, J=9.7, 9.7 and 4.8 Hz, H-6), 3.71 (dd,
1H, J=9.7 and 9.7 Hz, H-5), 3.76 (dd, 1H, J=10.0 and
9.7 Hz, H-7), 3.85 (s, 3H, OCH3), 4.10 (dd, 1H, J=10.0
and 9.7 Hz, H-4), 4.25 (m, 1H, H-50), 4.34 (dd, 1H,
J=10.0 and 4.8 Hz, H-7), 4.51 (d, 1H, J=5.6 Hz, H-2),
4.69 (ddd, 1H, J=10.0, 9.9 and 5.6 Hz, H-3), 5.21–5.24
(m, 2H, H-20, 10), 5.50–5.58 (m, 1H, H-30), 5.57 (s, 1H,
PhCH), 6.39 (d, 1H, J=9.9 Hz, NHAc), 7.33–8.10 (m,
15H, 3ꢁPh). 13C NMR (CDCl3): d 20.35 (C-60), 23.45
(CH3C¼O), 33.70 (C-40), 51.07 (C-3), 52.78 (OCH3),
64.39 (C-50), 67.72 (C-30), 68.72 (C-20), 68.78 (C-6),
69.29 (C-7), 75.03 (C-2), 75.93 (C-4), 80.51 (C-5), 99.13
(C-10), 102.19 (PhCH), 165.47 (PhC¼O), 165.75
(PhC¼O), 170.79 (RC¼O), 170.96 (RC¼O). Anal. calcd
for C37H39NO12: C, 64.43; H, 5.70; N, 2.03. Found: C,
64.15; H, 5.67; N, 2.15.
Benzyl 20,30- di-O-benzoyl-40-O-methyl-ꢀ-L-rhamnopyra-
nosyl-(1!3)-2-acetamido-4,6-O-benzylidene-2-deoxy-ꢀ-
D-glucopyranoside 6. White solid, 93%. Mp 216–217 ꢀC.
[a]D=+114ꢀ (c 1.07, CH2Cl2). 1H NMR (CDCl3): d
0.95 (d, 3H, J=6.2 Hz, H-60), 2.09 (s, 3H, CH3C¼O),
3.39 (s, 3H, OCH3), 3.40 (dd, 1H, J=9.8 and 9.8 Hz, H-
40), 3.77 (dd, 1H, J=9.8 and 9.6 Hz, H-4), 3.83 (d, 1H,
J=10.1 Hz, H-6), 3.94 (dd, 1H, J=9.8 and 4.6 Hz, H-
5), 4.01 (dd, 1H, J=9.6 and 9.6 Hz, H-3), 4.14 (dq, 1H,
J=9.8 and 6.2 Hz, H-50), 4.28 (dd, 1H, J=10.1 and 4.6
Hz, H-6), 4.51 (ddd, 1H, J=9.8, 9.6 and 3.7 Hz, H-2),
4.52 (d, 1H, J=11.8 Hz, PhCH2), 4.76 (d, 1H, J=11.8
Hz, PhCH2), 4.90 (d, 1H, J=3.7 Hz, H-1), 5.14 (d, 1H,
J=1.7 Hz, H-10), 5.37 (dd, 1H, J=3.4 and 1.7 Hz, H-
20), 5.60 (s, 1H, PhCH), 5.67 (dd, 1H, J=9.8 and 3.4
Hz, H-30), 5.74 (d, 1H, J=9.8 Hz, NHAc), 7.28–8.06
(m, 20H, 4ꢁPh). 13C NMR (CDCl3): d 17.18 (C-60),
23.28 (CH3C¼O), 53.02 (C-2), 59.89 (OCH3), 63.39
(C-5), 67.56 (C-50), 68.88 (C-6), 69.99 (PhCH2), 71.05
(C-30), 71.97 (C-20), 75.71 (C-3), 80.33 (C-4), 80.77
(C-40), 97.73 (C-1), 97.87 (C-10), 101.91 (PhCH),
20,30 -Di-O-benzoyl-40 -O-methyl-ꢀ-L-rhamnopyranosyl-
(1!4)-3-acetamido-2,6-anhydro-5,7-benzylidene-3-deoxy-
D-glycero-D-ido-heptonic acid methyl ester 22. Amor-
phous glassy solid, 83%, [a]D=+91.3ꢀ (c 1.05, EtOAc).
1H NMR (CDCl3): d 0.92 (d, 3H, J=6.2 Hz, H-60), 2.12
(s, 3H, CH3C¼O), 3.39 (s, 3H, OCH3), 3.42 (dd, 1H,
J=9.8 and 9.8 Hz, H-40), 3.51 (ddd, 1H, J=9.8, 9.8 and
4.8 Hz, H-6), 3.77 (dd, 1H, J=9.8 and 9.8 Hz, H-5),
3.77 (ddd, 1H, J=10.6, 9.8 and 4.8 Hz, H-7), 3.84 (s,
3H, OCH3), 4.10 (dd, 1H, J=10.0 and 9.8 Hz, H-4),
4.10 (dq, 1H, J=9.8 and 6.2 Hz, H-50), 4.34 (dd, 1H,
J=10.6 and 4.8 Hz, H-7), 4.53 (d, 1H, J=5.6 Hz, H-2),
4.70 (ddd, 1H, J=10.0, 9.7 and 5.6 Hz, H-3), 5.17 (d,
1H, J=1.6 Hz, H-10), 5.37 (dd, 1H, J=3.4 and 1.6 Hz,
H-20), 5.57 (s, 1H, PhCH), 5.64 (dd, 1H, J=9.8 and 3.4
Hz, H-30), 6.36 (d, 1H, J=9.7 Hz, NHAc), 7.32–8.04
(m, 15H, 3ꢁPh). 13C NMR (CDCl3): d 17.20 (C-60),
23.35 (CH3C¼O), 51.05 (C-3), 52.74 (OCH3), 60.12
(OCH3), 67.76 (C-50), 68.57 (C-6), 68.75 (C-7), 71.22 (C-
30), 71.81 (C-20), 74.93 (C-2), 75.65 (C-4), 80.49 (C-5),
80.79 (C-40), 97.95 (C-10), 101.96 (PhCH), 165.23
(PhC¼O), 165.53 (PhC¼O), 170.70 (RC¼O), 170.87
(RC¼O). Anal. calcd for C38H41NO13: C, 63.41; H,
5.74; N, 1.95. Found: C, 63.01; H, 5.78; N, 1.95.
165.10
(PhC¼O),
165.42
(PhC¼O),
170.38
(CH3C¼O). Anal. calcd for C43H45NO12: C, 67.26;
H, 5.91; N, 1.82. Found: C, 66.91; H, 6.04; N, 1.89.
Benzyl 20,30,40-tri-O-benzoyl-ꢀ-L-rhamnopyranosyl-(1!3)-
2-acetamido-4, 6-O-benzylidene-2-deoxy-ꢀ-D-glucopyra-
noside 7. Glassy solid, 92%. [a]D=+100ꢀ (c 1.03,
1
CHCl3). H NMR (CDCl3): d 0.86 (d, 3H, J=6.2 Hz,
H-60), 2.12 (s, 3H, CH3C¼O), 3.83 (dd, 1H, J=10.0 and
10.0 Hz, H-6), 3.84 (dd, 1H, J=10.0 and 10.0 Hz, H-4),
3.99 (ddd, 1H, J=10.0, 10.0 and 4.6 Hz, H-5), 4.09 (dd,
1H, J=10.0 and 10.0 Hz, H-3), 4.32 (dd, 1H, J=10.0
and 4.6 Hz, H-6), 4.48 (dq, 1H, J=10.0 and 6.2 Hz, H-
50), 4.53 (d, 1H, J=11.8 Hz, PhCH2), 4.60 (ddd, 1H,
J=10.4, 10.0 and 3.7 Hz, H-2), 4.78 (d, 1H, J=11.8 Hz,
PhCH2), 4.92 (d, 1H, J=3.7 Hz, H-1), 5.27 (d, 1H,
J=1.6 Hz, H-10), 5.45 (dd, 1H, J=3.4 and 1.6 Hz, H-
20), 5.60 (dd, 1H, J=10.0 and 10.0 Hz, H-40), 5.67 (s,
1H, PhCH), 5.85 (d, 1H, J=10.4 Hz, NHAc), 5.90 (dd,
1H, J=10.0 and 3.4 Hz, H-30), 7.20–8.15 (m, 20H,
4ꢁPh). 13C NMR (CDCl3): d 16.74 (C-60), 23.14
(CH3C¼O), 53.20 (C-2), 63.58 (C-5), 66.82 (C-50), 69.12
(C-6), 69.70 (C-30), 70.39 (PhCH2), 71.89 (C-20), 72.12
(C-40), 75.70 (C-3), 80.55 (C-4), 98.20 (C-10), 98.43 (C-
1), 102.70 (PhCH). Anal. calcd for C49H47NO13: C,
68.60; H, 5.52; N, 1.63. Found: C, 68.24; H, 5.29; N,
2.07.
Diethyl 20,30-di-O-benzoyl-40-O-methyl-ꢀ-L-rhamnopyra-
nosyl-(1!3)-C-(4,6-O-benzylidene-2-acetamido-2-deoxy-
ꢀ-D-glucopyranosyl) methanephosphonate 27. Amorphous
white solid, 75%, [a]D=+25.0ꢀ (c 0.76, CHCl3). 1H
NMR (CDCl3): d 0.93 (d, 3H, J=6.1 Hz, H-60), 1.23 (t,
3H, J=7.0 Hz, CH3CH2), 1.25 (t, 3H, J=7.0 Hz,
CH3CH2), 2.10 (s, 3H, CH3C¼O), 2.10–2.30 (m, 2H,
PCH2), 3.37 (s, 3H, OCH3), 3.40 (dd, 1H, J=9.7 and
9.7 Hz, H-40), 3.71–3.74 (m, 3H, H-5, 4, 6), 3.94–4.12
(m, 6H, 2ꢁOCH2CH3, H-3, 50), 4.23 (m, 1H, H-6),
4.40–4.50 (m, 2H, H-2, 1), 5.24 (d, 1H, J=1.4 Hz, H-10),
5.42 (dd, 1H, J=3.4 and 1.4 Hz, H-20), 5.57 (s, 1H,
PhCH), 5.62 (dd, 1H, J=9.7 and 3.4 Hz, H-30), 7.32–
8.02 (m, 16H, 3ꢁPh, NHAc). 13C NMR (CDCl3): d
16.29 (CH3CH2), 16.37 (CH3CH2), 17.31 (C-60), 23.03

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X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
(CH3C¼O), 25.68 (d, J=143.4 Hz, PCH2), 53.38 (d,
J=13.5 Hz, C-2), 60.03 (OCH3), 62.14 (d, J=6.5 Hz,
CH3CH2), 62.32 (d, J=6.5 Hz, CH3CH2), 65.10 (C-5),
67.60 (C-50), 69.29 (C-6), 70.75 (d, J=4.5 Hz, C-1),
71.30 (C-20), 71.55 (C-30), 74.68 (C-3), 80.85 (C-4), 81.00
(C-40), 97.55 (C-10), 101.92 (PhCH), 165.21 (PhC¼O),
165.29 (PhC¼O), 171.17 (CH3C¼O). 31P NMR
(CDCl3): d 28.50.
Benzyl ꢀ - L - rhamnopyranosyl - (1!3) - 2- acetamido - 2-
deoxy-ꢀ-D-glucopyranoside 10. Crystals, 93%, mp 211–
212 ꢀC. [a]D=+69.0ꢀ (c 0.50, CH3OH). 1H NMR
(D2O): d 1.21 (d, 3H, J=6.3 Hz, H-60), 1.99 (s, 3H,
CH3C¼O), 3.41 (dd, 1H, J=9.6 and 9.6 Hz, H-40), 3.55
(dd, 1H, J=9.1 and 9.1 Hz, H-4), 3.75 (dd, 1H, J=9.6
and 3.3 Hz, H-30), 3.75 (dd, 1H, J=3.3 and 1.2 Hz, H-
20), 3.75 (dd, 1H, J=9.1 and 9.1 Hz, H-3), 3.77–3.84 (m,
3H, H-6, 5), 3.96 (dq, 1H, J=9.6 and 6.3 Hz, H-50), 4.04
(dd, 1H, J=9.1 and 3.6 Hz, H-2), 4.56 (d, 1H, J=11.8
Hz, PhCH2), 4.77 (d, 1H, J=11.8 Hz, PhCH2), 4.83 (d,
1H, J=1.2 Hz, H-10), 4.89 (d, 1H, J=3.6 Hz, H-1),
7.30–7.50 (m, 5H, Ph). 13C NMR (D2O): d 16.83 (C-60),
22.24 (CH3C¼O), 53.54 (C-2), 60.86 (C-6), 68.72 (C-4),
69.24 (C-50), 69.94 (PhCH2), 70.58 (C-30), 71.12 (C-20),
72.27 (C-40), 72.72 (C-3), 79.72 (C-5), 96.54 (C-1),
101.56 (C-10), 174.47 (CH3C¼O). Anal. calcd for
C21H31NO10: C, 55.14; H, 6.83; Found: C, 54.76; H,
6.95.
Preparation of 8–10, 23–26 and 28
The disaccharide 5, 6 7 or 27 (0.2 mmol) was treated
with 80% aqueous acetic acid at 70 ꢀC for 2 h. The
reaction mixture was then evaporated to dryness and
0.1 M NaOMe in MeOH (5 mL) was added. After a
further 1 h at rt, Amberlite IR-120(H+) was added. The
resin was removed by filtration, and the filtrate was
concentrated. Column chromatography using mixtures
of EtOAc, CH2Cl2 and MeOH as the eluant afforded
the pure products 8–10 or 28. The same procedure
applied to 21 or 22 led to mixtures of stereoisomers 23
and 25, or 24 and 26 respectively, which were separated
chromatographically.
40,60-Dideoxy-ꢀ-L-lyxo-hexopyranosyl-(1!4)-3-acetam-
ido-2, 6-anhydro-3-deoxy-D-glycero-D-ido-heptonic acid
methyl ester 23. Amorphous white solid, 44%.
[a]D=+8.2ꢀ (c 0.69, H2O). H NMR (D2O): d 1.17 (d,
1
Benzyl 40,60-dideoxy-ꢀ-L-lyxo-hexopyranosyl-(1!3)-2-
acetamido-2-deoxy-ꢀ-D-glucopyranoside 8. Crystals,
93%. Mp 193–194 ꢀC. [a]D=+81.1ꢀ (c 0.35, CH2Cl2).
1H NMR (D2O): d 1.15 (d, 3H, J=6.3 Hz, H-60), 1.55
(ddd, 1H, J=11.9, 11.9 and 11.9 Hz, H-40), 1.70 (ddd,
1H, J=11.9, 2.3 and 0.7 Hz, H-40), 2.00 (s, 3H,
CH3C¼O), 3.53 (dd, 1H, J=9.0 and 9.0 Hz, H-4), 3.59
(dd, 1H, J=1.7 and 1.7 Hz, H-20), 3.72–3.86 (m, 4H, H-
6, 5, 3), 3.96 (ddd, 1H, J=11.9, 1.7 and 0.7 Hz, H-30),
4.02 (dd, 1H, J=10.4 and 3.6 Hz, H-2), 4.20 (ddd, 1H,
J=11.9, 6.3 and 2.3 Hz, H-50), 4.57 (d, 1H, J=11.8 Hz,
PhCH2), 4.78 (d, 1H, J=11.8 Hz, PhCH2), 4.88 (d, 1H,
J=1.7 Hz, H-10), 4.90 (d, 1H, J=3.6 Hz, H-1), 7.40–
7.46 (m, 5H, Ph). 13C NMR (D2O): d 20.25 (C-60), 22.23
(CH3C¼O), 34.76 (C-40), 53.51 (C-2), 60.85 (C-6), 65.48
(C-30), 66.01 (C-50), 68.79 (C-4), 68.82 (C-20), 69.95
(PhCH2), 72.64 (C-5), 79.60 (C-3), 96.57 (C-1), 102.29
(C-10), 174.49 (CH3C¼O). ESI-MS: C21H31NO9,
[M+H+], 442.48; Found 442.59.
3H, J=6.3 Hz, H-60), 1.58 (q, 1H, J=11.9 Hz, H-40),
1.73 (ddd, 1H, J=11.9, 4.5 and 2.2 Hz, H-40), 2.04 (s,
3H, CH3C¼O), 3.59 (t, 1H, J=7.8 Hz, H-5), 3.69 (dd,
1H, J=3.2 and 1.6 Hz, H-20), 3.75 (m, 1H, H-6), 3.80
(m, 2H, H-7), 3.81 (s, 3H, OCH3), 3.85 (dd, 1H, J=11.0
and 7.8 Hz, H-4), 4.00 (ddd, 1H, J=11.9, 4.5 and 3.2
Hz, H-30), 4.16 (ddq, 1H, J=11.9, 6.3 and 2.2 Hz, H-50),
4.30 (dd, 1H, J=9.1 and 5.2 Hz, H-3), 4.65 (d, 1H,
J=5.2 Hz, H-2), 5.00 (d, 1H, J=1.6 Hz, H-10). 13C
NMR (D2O): d 20.29, 22.32, 34.80, 50.47, 53.27, 60.47,
65.48, 66.19, 68.29, 68.68, 77.94, 78.00, 101.56, 171.53,
174.64.
40,60-Dideoxy-ꢀ-L-lyxo-hexopyranosyl-(1!4)-3-acetam-
ido-2, 6-anhydro-3-deoxy-D-glycero-D-gulo-heptonic acid
methyl ester 25. Amorphous white solid, 12%.
[a]D=ꢂ43.8ꢀ (c 0.37, H2O). H NMR (D2O): d 1.17 (d,
1
3H, J=6.3 Hz, H-60), 1.56 (q, 1H, J=11.9 Hz, H-40),
1.74 (ddd, 1H, J=11.9, 2.3 and 0.7 Hz, H-40), 2.03 (s,
3H, CH3C¼O), 3.52 (ddd, 1H, J=9.8, 5.3 and 1.9 Hz,
H-6), 3.54 (t, 1H, J=9.8 Hz, H-5), 3.65 (dd, 1H, J=5.3
and 1.5 Hz, H-20), 3.68 (dd, 1H, J=10.6 and 9.8 Hz, H-
4), 3.75 (s, 3H, OCH3), 3.76 (dd, 1H, J=12.7 and 5.3
Hz, H-7), 3.91 (dd, 1H, J=12.7 and 1.9 Hz, H-7), 4.00
(ddd, 1H, J=11.9, 5.3 and 0.7 Hz, H-30), 4.01 (t, 1H,
J=10.6 Hz, H-3), 4.09 (d, 1H, J=10.6 Hz, H-2), 4.22
(ddq, 1H, J=11.9, 6.3 and 2.3 Hz, H-50), 4.940(d, 1H,
J=1.5 Hz, H-10). 13C NMR (D2O): d 20.26 (C-6 ), 22.22
(CH3C¼O), 34.77 (C-40), 53.35 (C-3), 53.64 (OCH3),
61.14 (C-7), 65.46 (C-30), 66.13 (C-50), 68.66 (C-5), 68.74
(C-20), 76.45 (C-2), 80.10 (C-6), 81.92 (C-4), 102.41 (C-
10), 171.25 (C¼O), 174.77 (C¼O).
Benzyl 40-O-methyl-ꢀ-L-rhamnopyranosyl-(1!3)-2-acet-
amido-2-deoxy-ꢀ-D-glucopyranoside 9. Crystals, 90%,
mp 161–163 ꢀC. [a]D=+61.9ꢀ (c 0.75, CH3OH). 1H
NMR (D2O): d 1.25 (d, 3H, J=6.3 Hz, H-60), 1.99 (s,
3H, CH3C¼O), 3.15 (dd, 1H, J=9.6 and 9.6 Hz, H-40),
3.52 (s, 3H, OCH3), 3.55 (dd 1H, J=10.0 and 10.0 Hz,
H-4), 3.75–3.85 (m, 3H, H-6, 5), 3.76 (dd, 1H, J=2.9 and
1.4 Hz, H-20), 3.77 (dd, 1H, J=10.0 and 10.0 Hz, H-3),
3.78 (dd, 1H, J=9.6 and 2.9 Hz, H-30), 3.97 (dq, 1H,
J=9.6 and 6.3 Hz, H-50), 4.03 (dd, 1H, J=10.0 and 3.6
Hz, H-2), 4.56 (d, 1H, J=11.8 Hz, PhCH2), 4.76 (d, 1H,
J=11.8 Hz, PhCH2), 4.80 (d, 1H, J=1.4 Hz, H-10), 4.89
(d, 1H, J=3.6 Hz, H-1), 7.30–7.50 (m, 5H, Ph). 13C
NMR (D2O): d 17.02 (C-60), 22.22 (CH3C¼O), 53.53
(C-2), 60.36 (OCH3), 60.84 (C-6), 68.21 (C-50), 68.69
(C-4), 69.94 (PhCH2), 70.30 (C-30), 71.23 (C-20), 72.71
(C-5), 79.58 (C-3), 82.60 (C-40), 96.53 (C-1), 101.40
(C-10), 174.47 (CH3C¼O). Anal. calcd for C22H33NO10:
C, 56.04; H, 7.05; Found: C, 55.51; H, 6.58.
40-O-Methyl-ꢀ-L-rhamnopyranosyl-(1!4)-3-acetamido-2,
6-anhydro-3-deoxy-D-glycero-D-ido-heptonic acid methyl
ester 24. Amorphous white solid, 28%. [a]D=ꢂ1.0ꢀ (c
1
1.23, H2O). H NMR (D2O): d 1.27 (d, 3H, J=6.3 Hz,
H-60), 2.04 (s, 3H, CH3C¼O), 3.18 (dd, 1H, J=9.6 and
9.6 Hz, H-40), 3.54 (s, 3H, OCH3), 3.59 (dd, 1H, J=8.0

X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
3585
and 8.0 Hz, H-5), 3.67–3.73 (m, 1H, H-6), 3.81 (s, 3H,
OCH3), 3.77–3.95 (m, 6H, H-7, 4, 50, 30, 20), 4.29 (dd, 1H,
J=9.3 and 5.3 Hz, H-3), 4.64 (d, 1H, J=5.3 Hz, H-2),
4.94 (d, 1H, J=1.6 Hz, H-10). 13C NMR (D2O): d 17.09,
22.32, 50.60, 53.28, 60.44, 60.55, 68.26, 68.40, 70.33,
71.13, 72.71, 77.98, 78.20, 82.64, 100.79, 171.49, 174.63.
ꢀ-D-glucopyranoside 11. Amorphous white solid, 82%.
[a]D=+99.2ꢀ (c 0.65, CH2Cl2). H NMR (CDCl3): d
1
1.24 (d, 3H, J=6.1 Hz, H-60), 1.92–2.00 (m, 2H, H-40),
2.09 (s, 6H, 2ꢁCH3C¼O), 2.10 (s, 3H, CH3C¼O), 3.89–
3.95 (m, 2H, H-3, 5), 4.00–4.14 (m, 2H, H-6, 50), 4.20
(dd, 1H, J=12.3 and 4.6 Hz, H-6), 4.47 (ddd, 1H,
J=10.0, 9.7 and 3.6 Hz, H-2), 4.51 (d, 1H, J=11.8 Hz,
PhCH2), 4.70 (d, 1H, J=11.8 Hz, PhCH2), 4.94 (d, 1H,
J=3.6 Hz, H-1), 5.10 (d, 1H, J=1.6 Hz, H-10), 5.15 (dd,
1H, J=9.8 and 9.8 Hz, H-4), 5.28 (dd, 1H, J=2.1 and 1.6
Hz, H-20), 5.43–5.50 (m, 1H, H-30), 5.91 (d, 1H, J=9.7
Hz, NHAc), 7.27–8.08 (m, 15H, 3ꢁPh). 13C NMR
(CDCl3): d 20.73 (C-60), 21.10 (CH3C¼O), 23.26
(CH3C¼O), 33.67 (C-40), 52.32 (C-2), 62.15 (C-6), 65.26
(C-50), 67.38 (C-30), 68.31 (C-5), 69.07 (C-20), 69.93 (C-4),
70.01 (PhCH2), 79.98 (C-3), 96.92 (C-1), 100.73 (C-10),
165.01 (PhC¼O), 165.94 (PhC¼O), 169.50 (CH3C¼O),
170.55 (CH3C¼O), 170.73 (CH3C¼O). ESI-MS:
C39H43NO13, [M+H+], 734.77; Found 734.69.
40-O-Methyl-ꢀ-L-rhamnopyranosyl-(1!4)-3-acetamido-2,
6-anhydro-3-deoxy-D-glycero-D-gulo-heptonic acid methyl
ester 26. Amorphous white solid, 15%. [a]D=ꢂ40.0ꢀ (c
1
0.35, CH2Cl2). H NMR (D2O): d 1.26 (d, 3H, J=6.3
Hz, H-60), 2.02 (s, 3H, CH3C¼O), 3.17 (dd, 1H, J=7.8
and 7.8 Hz, H-40), 3.53 (s, 3H, OCH3), 3.48–3.57 (m,
2H, H-5, 30), 3.67 (dd, 1H, J=10.6 and 10.0 Hz, H-4),
3.71–3.81 (m, 3H, H-20 6, 7,), 3.93 (dd, 1H, J=12.5 and
1.9 Hz, H-7), 3.99 (dd, 1H, J=10.0 and 6.3 Hz, H-50),
4.00 (dd, 1H, J=10.6 and 10.6 Hz, H-3), 4.09 (d, 1H,
J=10.6 Hz, H-2), 4.87 (d, 1H, J=1.4 Hz, H-10). 13C
NMR (D2O): d 17.04, 22.22, 53.37, 53.64, 60.37, 61.12,
68.33, 68.54, 70.27, 71.18, 76.41, 80.17, 81.89, 82.60,
101.52, 171.21, 174.76.
Benzyl 20, 30-di-O-benzoyl-40-O-methyl-ꢀ-L-rhamnopyra-
nosyl-(1!3)-2-acetamido-4, 6-di-O-acetyl-2-deoxy-ꢀ-D-
glucopyranoside 12. Amorphous white solid, 90%.
[a]D=+137ꢀ (c 1.07, CH2Cl2). 1H NMR (CDCl3): d
1.33 (d, 3H, J=6.1 Hz, H-60), 2.07 (s, 3H, CH3C¼O),
2.09 (s, 6H, 2ꢁCH3C¼O), 3.44 (dd, 1H, J=9.3 and 9.3
Hz, H-40), 3.48 (s, 3H, OCH3), 3.90 (dq, 1H, J=9.3 and
6.1 Hz, H-50), 3.90 (ddd, 1H, J=9.6, 4.6 and 2.2 Hz, H-
5), 3.94 (dd, 1H, J=9.6 and 9.6 Hz, H-3), 4.02 (dd, 1H,
J=12.3 and 2.2 Hz, H-6), 4.15 (dd, 1H, J=12.3 and 4.6
Hz, H-6), 4.45 (dd, 1H, J=9.8 and 3.6 Hz, H-2), 4.50
(d, 1H, J=11.8 Hz, PhCH2), 4.69 (d, 1H, J=11.8 Hz,
PhCH2), 4.94 (d, 1H, J=3.6 Hz, H-1), 5.02 (ws, 1H, H-
10), 5.15 (dd, 1H J=9.6 and 9.6 Hz, H-4), 5.43–5.47 (m,
2H, H-20, 30), 5.82 (d, 1H, J=9.6 Hz, NHAc), 7.34–8.03
(m, 15H, 3ꢁPh). 13C NMR (CDCl3): d 17.67 (C-60),
20.62 (CH3C¼O), 20.94 (CH3C¼O), 23.09 (CH3C¼O),
52.08 (C-2), 60.27 (OCH3), 62.02 (C-6), 68.20 (C-5),
68.44 (C-50), 69.85 (C-4), 69.92 (PhCH2), 71.22 (C-30),
71.67 (C-20), 79.00 (C-3), 80.29 (C-40), 96.82 (C-1), 99.18
(C-10), 164.78 (PhC¼O), 165.60 (PhC¼O), 169.29
(CH3C¼O), 170.39 (CH3C¼O), 170.65 (CH3C¼O).
Anal. calcd for C40H45NO14: C, 62.90; H, 5.94; N, 1.83.
Found: C, 63.29; H, 6.18; N, 1.84.
Diethyl 40-O-methyl-ꢀ-L-rhamnopyranosyl-(1!3)-C-(2-
acetamido-2-deoxy-ꢀ-D-glucopyranosyl)
methanepho-
sphonate 28. Colorless thick oil, 89%. [a]D=+11.0ꢀ (c
1
0.20, CH2Cl2). H NMR (D2O): d 1.27 (d, 3H, J=6.3
Hz, H-60), 1.20–1.40 (m, 6H, 2ꢁCH3CH2), 2.04 (s, 3H,
CH3C¼O), 2.11 (ddd, 1H, J=19.4, 16.0 and 3.5 Hz,
PCH2), 2.45 (ddd, 1H, J=16.0, 16.0 and 11.0 Hz, H-1),
3.18 (dd, 1H, J=9.5 and 9.5 Hz, H-40), 3.54 (s, 3H,
OCH3), 3.64 (dd, 1H, J=7.9 and 7.9 Hz, H-4), 3.66 (dd,
1H, J=9.4 and 7.9 Hz, H-3), 3.71 (ddd, 1H, J=7.9, 4.0
and 2.5 Hz, H-5), 3.77 (dd, 1H, J=11.9 and 2.5 Hz, H-
6), 3.81 (dd, 1H, J=9.5 and 3.0 Hz, H-30), 3.84 (dd, 1H,
J=3.0 and 1.5 Hz, H-20), 3.88 (dd, 1H, J=11.9 and 4.0
Hz, H-6), 3.92 (dq, 1H, J=9.5 and 6.3 Hz, H-50), 4.10–
4.20 (m, 4H, CH3CH2), 4.33–4.43 (m, 1H, H-1), 4.89 (d,
1H, J=1.5 Hz, H-10). 13C NMR (D2O): d 15.95
(CH3CH2), 16.03 (CH3CH2), 17.14 (C-60), 22.33
(CH3C¼O), 23.51 (d, J=142.6 Hz, PCH2), 52.25 (d,
J¼13.5 Hz, C-2), 60.32 (C-6), 60.43 (OCH3), 63.90 (d,
J¼6.7 Hz, CH3CH2), 64.05 (d, J=6.7 Hz, CH3CH2),
68.22 (C-4), 68.47 (C-50), 68.60 (d, J=5.2 Hz, C-1),
70.32 (C-30), 71.15 (C-20), 74.87 (C-3), 77.97 (C-5), 82.64
(C-40), 100.75 (C-10), 174.55 (CH3C¼O). 31P NMR
(D2O): d 32.33. Anal. calcd for C20H38NO12P: C, 46.60;
H, 7.43; N, 2.72. Found: C, 46.26; H, 7.52; N, 2.80.
Benzyl 20, 30, 40-tri-O-benzoyl-ꢀ-L-rhamnopyranosyl-
(1!3)-2-acetamido-4, 6-di-O-acetyl-2-deoxy-ꢀ-D-gluco-
pyranoside ꢀ 13. Amorphous white solid, 88%.
[a]D=+184 (c 0.90, CHCl3). 1H NMR (CDCl3): d 1.29
(d, 3H, J=6.2 Hz, H-60), 2.10 (s, 3H, CH3C¼O), 2.13 (s,
3H, CH3C¼O), 2.17 (s, 3H, CH3C¼O), 3.91–3.98 (m,
2H, H-3, 5), 4.01–4.15 (m, 1H, H-6), 4.21–4.26 (m, 2H,
H-6, 50), 4.54 (d, 1H, J=11.8 Hz, PhCH2), 4.55–4.63
(m, 1H, H-2), 4.73 (d, 1H, J=11.8 Hz, PhCH2), 4.99 (d,
1H, J=3.6 Hz, H-1), 5.11 (d, 1H, J=1.6 Hz, H-10), 5.23
(dd, 1H, J=9.6 and 9.6 Hz, H-4), 5.55 (m, 1H, H-20),
5.62–5.71 (m, 2H, H-30, 40), 5.91 (d, 1H, J=9.7 Hz,
NHAc), 7.21–8.09 (m, 20H, 4ꢁPh). 13C NMR (CDCl3):
d 17.26 (C-60), 20.63 (CH3C¼O), 21.04 (CH3C¼O),
23.14 (CH3C¼O), 51.84 (C-2), 61.92 (C-6), 67.64 (C-50),
68.20 (C-5), 69.72 (C-40), 69.98 (C-4), 70.01 (PhCH2),
70.97 (C-30), 71.18 (C-20), 80.64 (C-3), 96.87 (C-1), 99.84
(C-10), 164.85 (PhC¼O), 165.71 (PhC¼O), 165.82
Preparation of 11–13
Disaccharide 5, 6 or 7 (0.2 mmol) was treated with 80%
HOAc at 70 ꢀC for 2 h. The solvent was then evapo-
rated, and pyridine (2 mL), Ac2O (1 mL) and DMAP
(20 mg) were added. The reaction was stirred at rt until
judged complete by TLC analysis. The mixture was
diluted with CH2Cl2 and washed with 1 M HCl, satd
NaHCO3, water and brine. The organic layer was dried
and concentrated. The residue was purified by chroma-
tography, eluting with a mixture of hexane and EtOAc,
to afford 11, 12 or 13.
Benzyl 20,30 -di-O-benzoyl-40,60 -dideoxy-ꢀ-L-lyxo-hexo-
pyranosyl-(1!3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-

3586
X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
(PhC¼O), 169.43 (CH3C¼O), 170.55 (CH3C¼O),
170.62 (CH3C¼O). Anal. calcd for C46H47NO15: C,
64.71; H, 5.55; N, 1.64. Found: C, 64.36; H, 5.76; N,
1.63.
20, 30, 40-Tri-O-benzoyl-ꢀ-L-rhamnopyranosyl-(1!3)-2-
acetamido-4, 6-di-O-acetyl-2-deoxy-ꢀ-D-glucopyranoside
16. Amorphous white solid, 80%. [a]D=+146ꢀ (c 0.50,
1
CH2Cl2). H NMR (CDCl3): d 1.28 (d, 3H, J=6.1 Hz,
H-60), 2.09 (s, 3H, CH3C¼O), 2.10 (s, 3H, CH3C¼O),
2.19 (s, 3H, CH3C¼O), 4.06–4.22 (m, 2H, H-6), 4.12–
4.28 (m, 2H, H-5,50), 4.11 (dd, 1H, J=10.1 Hz, H-3),
4.41 (ddd, 1H, J=10.1 and 10.1, 9.4 and 3.3 Hz, H-2),
4.60 (d, 1H, J=2.8 Hz, H-OH), 5.09 (d, 1H, J=1.6 Hz,
H-10), 5.22 (dd, 1H, J=9.2 and 9.2 Hz, H-4), 5.38 (dd,
1H, J=3.3 and 2.8 Hz, H-1), 5.52–5.53 (m, 1H, H-20),
5.63–5.68 (m, 2H, H-30, 40), 6.33 (d, 1H, J=9.4 Hz,
NHAc), 7.20–8.10 (m, 15H, 3ꢁPh). 13C NMR (CDCl3):
d 17.41 (C-60), 20.80 (CH3C¼O), 21.25 (CH3C¼O),
23.26 (CH3C¼O), 53.21 (C-2), 62.19 (C-6), 67.93 (C-5,
C-50), 69.92 (C-4), 70.26 (C-40), 71.21 (C-20), 71.24 (C-
30), 80.46 (C-3), 92.34 (C-1), 100.23 (C-10), 165.26
(C¼O), 165.87 (C¼O), 166.26 (C¼O), 169.68 (C¼O),
170.95 (C¼O), 171.62 (C¼O). Anal. calcd for
C39H41NO15: C, 61.33; H, 5.41; N, 1.83. Found: C,
60.91; H, 5.56; N, 1.79.
Hydrogenolysis of benzyl glycosides 11, 12 and 13
The glycoside 11, 12 or 13 (0.2 mmol) was suspended in
glacial AcOH (2 mL) with Pd/C catalyst (10% w/w; 50
mg). The mixture was vigorously stirred under a H2
atmosphere until TLC showed the completion of the
reaction. The catalyst was removed by filtration through
Celite, and the filtrate was then concentrated. The resi-
due was purified by flash column chromatography using
a hexane/EtOAc eluant, to afford the reducing dis-
accharide 14, 15, or 16.
20, 30-Di-O-benzoyl-40,60-dideoxy-ꢀ-L-lyxo-hexopyranosyl-
(1!3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-ꢀ-D-gluco-
pyranoside 14. Amorphous white solid, 63%.
[a]D=+78.7ꢀ (c 1.00, CH2Cl2). H NMR (CDCl3): d
1
1.24 (d, 3H, J=6.0 Hz, H-60), 1.85–2.15 (m, 2H, H-40),
2.05 (s, 3H, CH3C¼O), 2.09 (s, 3H, CH3C¼O), 2.10 (s,
3H, CH3C¼O), 4.01 (dd, 1H, J=9.4 and 9.4 Hz, H-3),
4.01–4.23 (m, 4H, H-6, 5, 50), 4.33 (ddd, 1H, J=9.4, 9.4
and 3.2 Hz, H-2), 5.07 (bs, 1H, H-10), 5.11 (dd, 1H,
J=9.4 and 9.4 Hz, H-4), 5.26 (bs, 1H, H-20), 5.29 (d,
1H, J=3.2 Hz, H-1), 5.50 (ddd, 1H, J=10.7, 4.0 and
4.0 Hz, H-30), 6.62 (d, 1H, J=9.4 Hz, NHAc), 7.26–8.06
Galactosyl transfer assays
Following the procedure described by Mikusova et al.,5
solvent was removed from 1 mCi of UDP-[6-3H]Galp
(American Radiolabeled Chemicals, Inc.; 60 Ci/mmol)
under a stream of N2. The radiolabeled compound was
dissolved in 50 mM pH 8.0 MOPS buffer (22 mL) con-
taining 5 mM 2-mercaptoethanol, and 10 mM MgCl2
(buffer A). The mixture was incubated with 30 mL of
UDP-Galp mutase (100 mg of protein) at 37 ꢀC for 30
min. Subsequently, reagents and buffer A were added to
make a total volume of 320 mL, resulting in final con-
centrations of 20 mM UDP-GlcNAc, 20 mM dTDP-Rha,
60 mM ATP, 5.13 mg/mL protein, 1% DMSO and con-
centrations of analogues 5–6, 23–26 or 28 up to 600 mg/
mL. The reaction mixture was incubated for another 30
min at 37 ꢀC, after which CHCl3/CH3OH (2/1) was
added. Protein was removed by centrifugation and the
supernatant was transferred to a second tube. Deionized
water was added to achieve a biphasic mixture, which was
separated by centrifugation. The aqueous phase was
removed and discarded; the organic phase was washed
with CHCl3/CH3OH/H2O (3/47/48) after which the sol-
vent was evaporated under a stream of N2. The residue
was dissolved in CHCl3/CH3OH (2/1). The amounts of
radiolabeled products formed were measured by liquid
scintillation spectrometry. Their identities were con-
firmed by TLC analysis on silica gel G60 plates developed
in CHCl3/CH3OH/NH4OH/H2O (65/25/0.5/3.6, v/v).
(m, 10H, 2ꢁPh). 13C NMR (CDCl3):
d 20.77
(CH3C¼O), 21.13 (C-60, CH3C¼O), 21.21 (CH3C¼O),
23.19 (C-40), 53.23 (C-2), 62.37 (C-6), 65.33 (C-50), 67.55
(C-30), 67.69 (C-5), 69.18 (C-20), 70.11 (C-4), 80.20 (C-
3), 91.90 (C-1), 101.09 (C-10), 165.32 (PhC¼O), 166.11
(PhC¼O), 169.76 (CH3C¼O), 171.09 (CH3C¼O),
171.69 (CH3C¼O). ESI-MS: C32H37NO13, [M+H+],
644.65; Found 644.69. Anal. calcd for C32H37NO13: C,
59.71; H, 5.79; N, 2.18. Found: C, 59.39; H, 5.72; N,
2.17.
20,30 -Di-O-benzoyl-40 -O-methyl-ꢀ-L-rhamnopyranosyl-
(1!3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-ꢀ-D-gluco-
pyranoside 15. Amorphous white solid, 77%.
[a]D=+136ꢀ (c 0.75, CH2Cl2). 1H NMR (CDCl3): d
1.35 (d, 3H, J=6.1 Hz, H-60), 2.08 (s, 3H, CH3C¼O),
2.12 (s, 3H, CH3C¼O), 2.13 (s, 3H, CH3C¼O), 3.48 (s,
3H, OCH3), 3.44 (dd, 1H, J=9.6 and 9.6 Hz, H-40), 3.96
(dq, 1H, J=9.6 and 6.1 Hz, H-50), 4.08 (dd, 1H, J=10.0
and 9.4 Hz, H-3), 4.08–4.23 (m, 3H, H-6, 5), 4.33 (ddd,
1H, J=10.0, 9.2 and 2.8 Hz, H-2), 5.02 (d, 1H, J=1.3
Hz, H-10), 5.14 (dd, 1H, J=9.4 and 9.4 Hz, H-4), 5.30
(d, 1H, J=2.8 Hz, H-1), 5.44 (dd, 1H, J=3.2 and 1.3
Hz, H-20), 5.51 (dd, 1H, J=9.6 and 3.2 Hz, H-30), 6.54
(d, 1H, J=9.2 Hz, NHAc), 7.30–8.10 (m, 10H, 2ꢁPh).
13C NMR (CDCl3): d 18.15 (C-60), 21.18 (CH3C¼O),
21.56 (CH3C¼O), 23.52 (CH3C¼O), 53.69 (C-2), 60.98
(OCH3), 62.78 (C-6), 68.00 (C-5), 69.00 (C-50), 70.64
(C-4), 71.84 (C-20), 72.28 (C-30), 79.78 (C-3), 81.10 (C-
40), 92.40 (C-1), 100.07 (C-10), 165.52 (PhC¼O), 166.14
(PhC¼O), 170.20 (CH3C¼O), 171.47 (CH3C¼O), 172.15
(CH3C¼O). ESI-MS: C33H39NO14, [M+H+], 674.67;
Found 674.73. Anal. calcd for C33H39NO14: C, 58.84; H,
5.84; N, 2.08. Found: C, 59.21; H, 6.21; N, 2.12.
Acknowledgements
This work was supported by a research grant from the
Natural Sciences and Engineering Research Council of
Canada (to P.G.H.) and grants AI049151 (to D.C.C.)
and AI046393 (to P.J.B.) from the National Institute of
Allergy and Infectious Diseases, NIH. We also thank Dr.
Kirk Marat and Dr. Helene Perreault for their help in
obtaining NMR and mass spectra, and Hataichanok
Scherman for technical assistance in the biological assays.

X. Wen et al. / Bioorg. Med. Chem. 11 (2003) 3579–3587
3587
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