Synthesis of oligosaccharide fragments of the lipoarabinomannan from Rhodococcus ruber

Article abstract of DOI:10.1081/CAR-200067114

The synthesis of two oligosaccharide fragments (1 and 2) of the lipoarabinomannan from Rhodococcus ruber is reported. Thioglycoside donors were used to assemble these glycans, which were prepared as their 8-(methoxycarbonyl)octyl glycosides for potential

Full text of DOI:10.1081/CAR-200067114

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Synthesis of Oligosaccharide Fragments
of the Lipoarabinomannan from
Rhodococcus ruber
Gladys C. Completo a , Jessica A. Ponto a & Todd L. Lowary a
a Alberta Ingenuity Centre for Carbohydrate Science and Department
of Chemistry , The University of Alberta, Gunning‐Lemieux
Chemistry Centre , Edmonton, AB, Canada
Published online: 16 Aug 2006.
To cite this article: Gladys C. Completo , Jessica A. Ponto & Todd L. Lowary (2005) Synthesis
of Oligosaccharide Fragments of the Lipoarabinomannan from Rhodococcus ruber , Journal of
Carbohydrate Chemistry, 24:4-6, 517-527
To link to this article: http://dx.doi.org/10.1081/CAR-200067114
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Journal of Carbohydrate Chemistry, 24:517–527, 2005
Copyright # Taylor & Francis, Inc.
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1081/CAR-200067114
Synthesis of
Oligosaccharide Fragments
of the Lipoarabinomannan
from Rhodococcus ruber
Gladys C. Completo, Jessica A. Ponto, and Todd L. Lowary
Alberta Ingenuity Centre for Carbohydrate Science and Department of Chemistry, The
University of Alberta, Gunning-Lemieux Chemistry Centre, Edmonton, AB, Canada
The synthesis of two oligosaccharide fragments (1 and 2) of the lipoarabinomannan from
Rhodococcus ruber is reported. Thioglycoside donors were used to assemble these
glycans, which were prepared as their 8-(methoxycarbonyl)octyl glycosides for potential
incorporation into neoglycoconjugates.
Keywords Arabinofuranosides, Glycosylation, Lipoarabinomannan, Thioglycosides
INTRODUCTION
A major immunomodulatory molecule in mycobacteria, including the human
pathogen Mycobacterium tuberculosis, is lipoarabinomannan (LAM), a cell
wall polysaccharide composed of mannopyranose and arabinofuranose
residues bound to a phosphatidylinositol moiety.[1,2] Structural differences in
LAM molecules are observed among different mycobacterial species,[3–6] and
an increasing number of recent papers have reported the structures of LAM
molecules from other actinomycetes.[7–14] Among these[9] is the LAM from
Rhodococcus ruber (RruLAM), a species closely related to the opportunistic
human pathogen Rhodococcus equi.
The structure of RruLAM is similar to that of mycobacterial LAM in that
the mannan domain consists of an a-(1 ! 6)-linked chain of mannopyranose
residues. However, unlike mycobacterial LAM, which has a large arabinan
Received February 7, 2005; accepted March 9, 2005.
Dedicated to the memory of Jacques H. van Boom.
Address correspondence to Todd L. Lowary, Alberta Ingenuity Centre for Carbohydrate
Science and Department of Chemistry, The University of Alberta, Gunning-Lemieux
Chemistry Centre, Edmonton, AB, T6G 2G2 Canada. E-mail: tlowary@ualberta.ca
517

G. C. Completo, J. A. Ponto, and T. L. Lowary
518
motif attached to this mannan backbone, RruLAM has no separate arabinan
domain per se. Instead, the arabinofuranose residues are found as single
“capping” units attached a-(1 ! 2) to the mannan backbone. Approximately
45% of the residues in the mannan core are capped at O-2, and the majority
of these capping motifs are a-arabinofuranose residues, although in a small
percentage of cases the a-arabinofuranose cap is replaced with an a-mannopyr-
anose moiety. The proposed structure of RruLAM is shown in Figure 1.
We have a long-standing interest in the synthesis of oligosaccharide
fragments of cell wall polysaccharides from mycobacteria and related organ-
isms.[15–19] As part of this program, we were interested in the synthesis of
RruLAM fragments, functionalized to allow future preparation of neoglycocon-
jugates. Reported here is the preparation of disaccharide 1 and trisaccharide 2
(Fig. 2), as their 8-(methoxycarbonyl)octyl glycosides. Another similarly
functionalized fragment of RruLAM, disaccharide 3, has previously been
synthesized.[20]
RESULTS AND DISCUSSION
The synthesis of 1 and 2 relied on the use of the previously reported protected
8-methoxycarbonyloctyl glycosides 4[20] and 5[21] and the known thioglycosides
6[22] and 7.[23] With these building blocks in hand, the preparation of the two
targets was straightforward. The synthesis of 1 (Sch. 1) involved first the
Figure 1: Proposed structure of the lipoarabinomannan from Rhodococcus ruber (RruLAM).
Approximately 45% of the a-(1 ! 6)-linked D-Manp residues are capped. The values for A,
B, and C are approximately ꢀ1, 15, and 11, respectively.

Fragments of Rhodococcus ruber Lipoarabinomannan
519
Figure 2: Synthetic targets 1 and 2, related structure 3, and monosaccharide building
blocks (4–7) required for synthesis of 1 and 2.
coupling of alcohol 4, with thioglycoside 7 promoted by N-iodosuccinimide and
silver triflate.[24] The expected disaccharide, 8, was obtained in 75% yield. The
anomeric stereochemistry of the arabinofuranosyl residue could be clearly
established by NMR spectroscopy. In the 1H NMR spectrum of 8, the resonance
for the anomeric hydrogen appeared as a singlet, which is consistent with the
a-arabinofuranose stereochemistry.[25] Had the b-isomer been formed, this
signal would have appeared as a doublet with a 4–5 Hz coupling constant.
Similarly, in the 13C NMR spectrum of 8, the chemical shift of the anomeric
carbon was 106.8 ppm as would be expected for an a-arabinofuranoside. This
disaccharide was deprotected in two steps. Treatment of 8 with sodium meth-
oxide removed the benzoyl groups and gave 9 in 95% yield. Subsequent hydro-
genation of the benzyl ethers afforded a 92% yield of the final target, 1.
The preparation of 2 followed a similar route, as illustrated in Scheme 2.
Reaction of alcohol 5, with thioglycoside 6, in the presence of N-iodosuccinimide
Scheme 1: (a) NIS, AgOTf, CH2Cl2, 08C, 75%; (b) NaOCH3, CH3OH, rt, 95%; (c) H2, Pd/C,
CH3OH, rt, 92%.

G. C. Completo, J. A. Ponto, and T. L. Lowary
520
Scheme 2: (a) NIS, AgOTf, CH2Cl2, 08C, 65%; (b) AcCl, CH3OH, 08C ! rt, 60%; (c) 7, NIS,
AgOTf, CH2Cl2, 08C, 68%; (d) NaOCH3, CH3OH, rt, 85%; (e) H2, Pd(OH)2, CH3OH, rt, 82%.
and silver triflate afforded the product disaccharide 10, in 65% yield. The acetyl
group was then selectively cleaved in the presence of the benzoate esters upon
treatment of 10 with methanolic HCl.[26] The product, disaccharide alcohol 11,
was obtained in 60% yield. The arabinofuranose residue was introduced by way
of thioglycoside 7 under conditions identical to those used for the other
glycosylations and the protected trisaccharide 12 was obtained in 68% yield.
The a-stereochemistry of the arabinofuranose moiety could be established
from the chemical shift of the anomeric carbon of this residue, which was
106.6 ppm.[25] Deprotection of the product proceeded without incident.
Compound 12 was first treated with sodium methoxide, which provided, in
85% yield, a product, 13, in which all of the acyl groups had been cleaved.
Trisaccharide 2 was obtained in 82% yield by hydrogenation of the benzyl
ethers in 13.
In summary, we have completed the synthesis of two fragments of LAM
from Rhodoccus ruber. LAM fragments of known structure are expected to be
useful tools in elucidating the biologic role of these glycans. The ester function-
ality present in the aglycone of these oligosaccharides will facilitate the
preparation of neoglycoconjugates for use in, for example, assays requiring
microtiter plates.
EXPERIMENTAL
Solvents were distilled from the appropriate drying agents before use. Unless
stated otherwise, all reactions were carried out at rt and under a positive
pressure of argon and were monitored by TLC on silica gel 60 F254 (0.25 mm,
E. Merck). Spots were detected under UV light by charring with 10% H2SO4
in ethanol or by charring with anisaldehyde in ethanol. During the work-up
of reaction mixtures, crude products in organic solvents were washed with

Fragments of Rhodococcus ruber Lipoarabinomannan
521
equal volumes of aqueous solutions. Solvents were evaporated under reduced
pressure and below 408C (bath). Column chromatography was performed on
silica gel 60 (40–60 mM) or Iatrobeads, which refers to a beaded silica gel
6RS–8060 manufactured by Iatron Laboratories (Tokyo). The ratio between
silica gel and crude product ranged from 100 to 50 : 1 (w/w). Optical rotations
1
.
.
were measured at 21 + 28C and are in units of degrees mL/g dm. H NMR
spectra were recorded at 400, 500, or 600 MHz, and chemical shifts are
referenced to TMS (0.0 ppm, CDCl3), CH3OH (4.78 ppm, CD3OD), or HOD
(4.78 ppm, D2O). 13C NMR spectra were recorded at 100 MHz, and 13C
chemical shifts are referenced to CDCl3 (77.00 ppm, CDCl3), CD3OD
(49.15 ppm, CD3OD), or external dioxane (68.11 ppm, D2O). Electrospray
mass spectra were recorded on samples suspended in THF or CH3OH with
added NaCl.
8-(Methoxycarbonyl)octyl 2-O-(2,3,5-tri-O-benzoyl-a-D-arabinofur-
anosyl)-3,4,6-tri-O-benzyl-a-D-mannopyranoside (8). Alcohol 4 (200 mg,
˚
0.322 mmol), thioglycoside 7 (237 mg, 0.418 mmol), and powdered 4A molecular
sieves (0.50 g) were dried overnight under vacuum with P2O5. Freshly distilled
CH2Cl2 (5 mL) was added and the mixture was cooled to 08C before N-iodosuc-
cinimide (108 mg, 0.483 mmol) and AgOTf (17 mg, 0.066) were added. The
reaction mixture was stirred for 30 min and then neutralized with triethyl-
amine, before being filtered through Celite and concentrated. The resulting
residue was purified by column chromatography (6 : 1, hexanes : EtOAc) to
yield 8 (257 mg, 75%) as a clear oil. Rf 0.44 (3 : 1, hexanes : EtOAc);
1
[a]D þ 13.1 (c 0.9, CHCl3); H NMR (500 MHz, CDCl3) : dH, 8.10–8.00 (m, 6 H,
aromatic H), 7.70–7.48 (m, 3 H, aromatic H), 7.44–7.15 (m, 23 H, aromatic
H), 5.73 (d, 1 H, J ¼ 1.2 Hz, H-20), 5.60 (d, 1 H, J ¼ 1.2, 4.5 Hz, H-30), 5.58
(s, 1 H, H-10), 4.99 (d, 1 H, J ¼ 1.8 Hz, H-1), 4.87 (d, 1 H, J ¼ 10.8 Hz,
PhCH2), 4.84 (dd, 1 H, J ¼ 5.1, 11.6 Hz, H-5a0), 4.76 (d, 1 H, J ¼ 12.0 Hz,
PhCH2), 4.73 (d, 1 H, J ¼ 12.0 Hz, PhCH2), 4.70–4.68 (m, 2 H, H-40, H-5b0),
4.58–4.53 (m, 3 H, PhCH2), 4.16–4.12 (s, 1 H, H-20), 3.98 (dd, 1 H, J ¼ 2.9,
9.3 Hz, H-3), 3.95 (dd, 1 H, J ¼ 9.6, 9.6 Hz, H-4), 3.84–3.81 (ddd, 1 H,
J ¼ 1.7, 5.6, 9.6 Hz, H-5), 3.73 (dd, 1 H, J ¼ 1.8, 10.8 Hz, H-6a), 3.71–3.68
(m, 2 H, H-6b, aglycone OCH2), 3.67 (s, 3 H, OCH3), 3.39 (dt, 1 H, J ¼ 6.6,
9.5 Hz, aglycone OCH2), 2.31 (t, 2 H, J ¼ 7.5 Hz, CH2C55O), 1.65–1.55 (m, 4
H, aglycone CH2), 1.35–1.31 (m, 8 H, aglycone CH2); 13C NMR (125 MHz,
CDCl3) : dC, 175.1, 166.2, 165.8, 165.2, 138.5, 138.4, 133.4, 133.0, 130.1, 129.9,
129.8, 129.8, 129.3, 129.3, 128.5, 128.4 (2), 128.3 (3), 128.2 (2), 128.1, 127.6
(2), 127.5 (2), 106.8, 99.1, 82.0, 81.2, 80.0, 77.7, 75.2, 75.1, 74.4, 73.2, 72.2,
71.8, 69.5, 67.8, 63.7, 51.4, 34.1, 29.5, 29.3, 29.2, 29.1, 26.1, 24.9. HRMS
(ESI) calcd. for (M þ Na) C63H68O15 : 1087.4450; found 1087.4448.
8-(Methoxycarbonyl)octyl
2-O-(D-arabinofuranosyl)-3,4,6-tri-O-
benzyl-a-D-mannopyranoside (9). Disaccharide 8 (219 mg, 0.21 mmol) was

G. C. Completo, J. A. Ponto, and T. L. Lowary
522
dissolved in CH3OH (10 mL) and the solution was stirred for 10 min before solid
NaOCH3 was added in small portions until the pH reached 9. The reaction
mixture was stirred for 2 hr and then neutralized with acetic acid. The crude
product was purified by column chromatography (17 : 1, CH2Cl2 : CH3OH) to
yield 9 as a colorless oil (150 mg, 95%). Rf 0.37 (17 : 1 CH2Cl2 : CH3OH);
1
[a]D þ 63.6 (c 0.6, CHCl3); H NMR (500 MHz, CDCl3) : dH, 7.37–7.25 (m, 15
H, aromatic H), 7.15–7.13 (m, 2 H, aromatic H), 5.14 (s, 1 H, H-10), 4.90 (d, 1
H, J ¼ 2.2 Hz, H-1), 4.80 (d, 1 H, J ¼ 10.7 Hz, PhCH2), 4.72 (d, 1 H, J ¼
11.7 Hz, PhCH2), 4.67 (d, 1 H, J ¼ 11.7 Hz, PhCH2), 4.62 (d, 1 H, J ¼ 12.4 Hz,
PhCH2), 4.48 (d, 1 H, J ¼ 12.4 Hz, PhCH2), 4.39 (d, 1 H, J ¼ 10.7 Hz,
PhCH2), 4.22–4.21 (m, 1 H, H-40), 4.19 (s, 1 H, H-20), 4.01 (s, 1 H, H-30), 3.93
(dd, 1 H, J ¼ 2.8, 9.3 Hz, H-3), 3.89–3.80 (m, 4 H, H-5a0, H-5b0, H-5, H-2),
3.69–3.61 (m, 7 H, aglycone OCH2, H-4, C-6a, C-6b, OCH3), 3.39 (dt, 1 H,
J ¼ 6.6, 9.5 Hz, aglycone OCH2), 2.31 (t, 2 H, J ¼ 7.5 Hz, CH2C55O), 1.65–
1.55 (m, 4 H, aglycone CH2), 1.35–1.31 (m, 8 H, aglycone CH2); 13C NMR
(125 MHz, CDCl3) : dC, 174.4, 138.3, 138.2, 138.1, 128.5, 128.4, 128.3, 128.0
(2), 127.9, 127.6 (2), 109.4, 98. 8, 87.6, 79.2, 78.8, 78.2, 75.2, 74.8, 74.5, 73.3,
72.4, 71.4, 68.5, 67.9, 62.1, 51.5, 34.1, 29.4, 29.3, 29.2, 29.1, 26.0, 24.9. HRMS
(ESI) calcd. for (M þ Na) C42H56O12 : 775.3664; found 775.3664.
8-(Methoxycarbonyl)octyl 2-O-(a-D-arabinofuranosyl)-a-D-manno-
pyranoside (1). Disaccharide 9 (100 mg, 0.13 mmol) was dissolved in
CH3OH (5 mL) and 10% Pd/C (20 mg) was added. The solution was stirred
under a H2 atmosphere for 8 hr and the reaction mixture was then filtered
through Celite and concentrated. The crude product was purified by column
chromatography (10 : 1, CH2Cl2 : CH3OH) to yield 10 as a colorless oil (58 mg,
92%). Rf 0.30 (9 : 1, CH2Cl2 : CH3OH); [a]D þ 56.0 (c 0.4, CH3OH); 1H NMR
(500 MHz, CD3OD) : dH, 5.04 (d, 1 H, J ¼ 1.8 Hz, H-10), 4.91 (s, 1 H, H-1), 4.06
(dd, 1 H, J ¼ 1.8, 5.9 Hz, H-20), 3.98–3.94 (m, 1 H, H-3), 3.82–3.57 (m, 11 H,
H-20, H30, H-40, H-4, H5, H-6a, H-6b, aglycone OCH2, OCH3), 3.51–3.48 (m,
2 H, H-5a0, H-5b0), 3.39 (dt, 1 H, J ¼ 6.6, 9.5 Hz, aglycone OCH2), 2.30 (t, 2
H, J ¼ 7.4 Hz, CH2C55O), 1.61–1.56 (m, 4 H, aglycone CH2), 1.39–1.31 (m, 8
H, aglycone CH2); 13C NMR (125 MHz, CD3OD) : dC, 176.1 (C55O), 111.4
(C-10), 100.7 (C-1), 85.5 (C-40), 83.2 (C-20), 79.4 (C-30), 78.4 (C-2), 74.6 (C-4),
72.3 (C-3), 69.0 (C-5), 68.6 (aglycone OCH2), 63.0 (C-50), 62.9 (C-6), 52.0
(OCH3), 34.8 (aglycone CH2), 30.6 (aglycone CH2), 30.4 (aglycone CH2), 30.3
(aglycone CH2), 30.1 (aglycone CH2), 27.3 (aglycone CH2), 26.0 (aglycone
CH2). HRMS (ESI) calcd. for (M þ Na) C21H38O12 : 505.2256; found 505.2256.
8-(Methoxycarbonyl)octyl 6-O-(2-O-acetyl-3,4,6-tri-O-benzyl-a-D-man
nopyranosyl)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (10). Alcohol 5
(680 mg, 1.03 mmol) was glycosylated with thioglycoside
6
(459 mg,
0.86 mmol) in CH2Cl2 (30 mL) using N-iodosuccinimide (289 mg, 1.28 mmol)
˚
and silver triflate (5 mg, 0.21 mmol) in the presence of powdered 4A molecular

Fragments of Rhodococcus ruber Lipoarabinomannan
523
sieves (1.2 g) as described for the preparation of 8. The product was purified by
column chromatography (4 : 1, hexanes : EtOAc) to yield 10 (636 mg, 65%) as an
oil. Rf 0.33 (3 : 1, hexanes : EtOAc); [a]D –26.9 (c 1.0, CHCl3); 1H NMR
(500 MHz, CDCl3) : dH, 8.11–7.82 (m, 6 H, aromatic H), 7.61–7.14 (m, 24 H,
aromatic H), 5.93 (dd, 1 H, J ¼ 10.0, 10.0 Hz, H-4), 5.87 (dd, 1 H, J ¼ 3.3,
10.2 Hz, H-3), 5.64 (dd, 1 H, J ¼ 1.8, 3.3 Hz, H-2), 5.38 (dd, 1 H, J ¼ 1.8,
3.4 Hz, H-20), 5.04 (d, 1 H, J ¼ 1.7 Hz, H-1), 4.89 (d, 1 H, J ¼ 1.7 Hz, H-10),
4.83 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.57 (d, 1 H, J ¼ 12.1 Hz, PhCH2), 4.51
(d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.44 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.37 (d, 1 H,
J ¼ 12.1 Hz, PhCH2), 4.34 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.26–4.20 (m, 1 H,
H-5), 3.98–3.92 (m, 2 H, H-50, H-30), 3.86 (dd 1 H, J ¼ 9.7, 9.7 Hz, H-40),
3.78–3.72 (m, 2 H, aglycone OCH2), 3.72–3.65 (m, 6 H, OCH3, H-6a, H-6a0,
H-6b), 3.56–3.49 (m, 2 H, H-2, H-6b0), 2.31 (dd, 2 H, J ¼ 7.5, 7.5 Hz,
CH2C55O), 2.12 (s, 3 H C(O)CH3), 1.70–1.60 (m, 4 H, aglycone CH2), 1.45–
1.30 (m, 8 H, aglycone CH2); 13C NMR (125 MHz, CDCl3) : dC, 170.2, 166.2,
165.6, 165.5, 165.4, 138.6, 138.2, 138.0, 133.4, 133.3, 133.1, 129.9, 128.9,
129.7 (2), 129.5, 129.2, 128.4, 128.3 (2), 128.2, 127.8, 127.5, 127.3, 98.0, 97.5,
78.5, 75.0, 74.2, 73.3, 71.8, 71.5, 70.8, 70.2, 69.2, 68.6 (2), 68.5, 67.5, 66.8,
51.4, 34.1, 29.4, 29.3, 29.2, 29.1, 26.1, 25.0, 21.1. HRMS (ESI) calcd. for
(M þ Na) C66H72O17 : 1159.4667; found 1159.4681.
8-(Methoxycarbonyl)octyl 6-O-(3,4,6-tri-O-benzyl-a-D-mannopyra-
nosyl)-2,3,4-tri-O-benzoyl-a-D-mannopyranoside (11).
A solution of
methanolic HCl was prepared by dissolving acetyl chloride (1.5 mL) in
CH3OH (48.5 mL) at 08C. The entirety of this solution was used to dissolve
disaccharide 10 (614 mg, 0.54 mmol) and the reaction mixture was stirred for
6 hr. The solution was partially concentrated, diluted with CH2Cl2, and
washed successively with a saturated aqueous solution of NaHCO3, water,
and brine. The organic phase was dried with MgSO4, filtered through cotton,
and concentrated, and the resulting residue was purified by column chromato-
graphy (4 : 1, hexanes : EtOAc) to afford 11 (281 mg, 60%) as an oil. Rf 0.25 (3 : 1,
1
hexanes : EtOAc); [a]D–15.1 (c 1.0, CHCl3); H NMR (500 MHz, CDCl3) : dH,
8.10–7.83 (m, 6 H, aromatic H), 7.56–7.14 (m, 24 H, aromatic H), 5.97 (dd, 1
H, J ¼ 10.1, 10.1 Hz, H-4), 5.88 (dd, 1 H, J ¼ 3.4, 10.1 Hz, H-3), 5.65 (dd, 1 H,
J ¼ 1.8, 3.3 Hz, H-2), 5.05 (d, 1 H, J ¼ 1.7 Hz, H-1), 5.01 (d, 1 H, J ¼ 1.5 Hz,
H-10), 4.80 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.54 (d, 1 H, J ¼ 12.2 Hz, PhCH2),
4.52 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.49 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.54
(d, 1 H, J ¼ 12.2 Hz PhCH2), 4.39 (d, 1 H, J ¼ 10.9 Hz, PhCH2), 4.23–4.20
(m, 2 H, H-5, H-50), 4.02 (dd, 1 H, J ¼ 1.2, 2.6 Hz, H-20), 3.94 (dd, 1 H,
J ¼ 4.6, 11.2 Hz, H-6a), 3.85–3.70 (m, 5 H, aglycone OCH2, H-30, H-40, H-6a0),
3.66 (s, 3 H, OCH3), 3.62 (dd, 1 H, J ¼ 4.4, 10.8 Hz, H-6b0), 3.50–3.53 (m, 2
H, H-6b, H-6a), 2.32 (dd, 2 H, J ¼ 7.5, 7.5 Hz, CH2C55O), 1.59–1.72 (m, 4 H,
aglycone CH2), 1.30–1.42 (m, 8 H, aglycone CH2); 13C NMR (125 MHz,

G. C. Completo, J. A. Ponto, and T. L. Lowary
524
CDCl3) : dC, 170.3, 165.6, 165.5, 165.4, 138.5, 138.2, 137.9, 133.4, 133.3, 133.1,
129.9, 129.8, 129.8, 129.5, 129.3, 129.2, 128.6, 128.5, 128.4, 128.3, 127.9,
127.8, 127.7, 127.6, 127.5, 99.4, 97.6, 80.3, 75.0, 74.1, 73.3, 71.8, 71.2, 70.8,
70.3, 69.4, 68.7, 68.6, 68.1, 67.6, 66.4, 51.4, 34.1, 29.4, 29.3, 29.2, 29.1, 26.1,
25.0. HRMS (ESI) calcd. for (M þ Na) C64H70O16 : 1117.4561; found 1117.4576.
8-(Methoxycarbonyl)octyl 6-O-[2-O-(2,3,5-tri-O-benzoyl-a-D-arabi-
nofuranosyl)-3,4,6-tri-O-benzyl-a-D-mannopyranosyl]-2,3,4-tri-O-benz-
oyl-a-D-mannopyranoside (12). Alcohol 11 (261 mg, 0.24 mmol) was
glycosylated with thioglycoside 7 (163 mg, 0.29 mmol) in CH2Cl2 (20 mL)
using N-iodosuccinimide (80 mg, 0.36 mmol) and silver triflate (2 mg,
˚
0.06 mmol) in the presence of powdered 4A molecular sieves (500 mg) as
described for the preparation of 8. The product was purified by column chrom-
atography (4 : 1, hexanes : EtOAc) to yield 12 (250 mg, 68%) as an oil. Rf 0.46
(2 : 1, hexanes : EtOAc); [a]D–19.8 (c 0.6, CHCl3); 1H NMR (500 MHz,
CDCl3) : dH, 8.12–7.81 (m, 12 H, aromatic H), 7.62–7.11 (m, 33 H, aromatic
H), 5.98 (dd, 1 H, J ¼ 10.1, 10.1 Hz, H-4), 5.87 (dd, 1 H, J ¼ 3.3, 10.1 Hz,
H-3), 5.66–5.62 (m, 2 H, H-200, H-2), 5.56 (dd, 1 H, J ¼ 1.0, 3.4 Hz, H-300), 5.43
(s, 1 H, H-1), 5.05 (s, 2 H, H-10, H-100), 4.80 (d, 1 H, J ¼ 11.0 Hz, PhCH2),
4.80–4.75 (m, 1 H, PhCH2), 4.64–4.60 (m, 2 H, PhCH2), 4.52 (d, 1 H, J ¼
11.5 Hz, PhCH2), 4.50 (d, 1 H, J ¼ 11.5 Hz, PhCH2), 4.47 (d, 1 H, J ¼ 11.0 Hz,
PhCH2), 4.43 (d, 1 H, J ¼ 12.0 Hz, PhCH2), 4.22 (d, 1 H, J ¼ 12.0 Hz,
PhCH2), 4.24–4.20 (m, 1 H, H-5), 4.10–4.13 (m, 1 H, H-50), 4.00–3.88 (m, 5
H, H-400, H-30, H-40, H-20, H-6b0), 3.80–3.66 (m, 3 H, aglycone OCH2, H-5a00,
H05b00), 3.64 (s, 3 H, OCH3), 3.58–3.48 (m, 4 H, H-6a, H-6a0, H-6b, aglycone
OCH2), 2.31 (dd, 2 H, J ¼ 7.5, 7.5 Hz, CH2C55O), 1.60–1.70 (m, 4 H, aglycone
CH2), 1.20–1.44 (m, 8 H aglycone CH2); 13C NMR (125 MHz, CDCl3) : dC,
170.2, 166.2, 165.7, 165.6, 165.5, 165.3, 165.1, 138.6, 138.4, 138.3, 133.4,
133.3, 133.2, 133.1, 132.9, 130.0, 130.0, 129.9 (2), 129.8 (2), 129.8, 129.7,
129.5, 129.3, 129.2, 128.6, 128.5, 128.4 (2), 128.3 (2), 128.2 (2), 128.0, 127.6,
127.4 (3), 106.6, 99.3, 97.5, 82.0, 81.2, 80.1, 77.6, 75.0, 74.8, 73.7, 73.0, 72.0,
71.9, 70.9, 69.4, 69.2, 68.6, 67.6, 66.5, 66.5, 63.6, 51.1, 34.1, 29.4, 29.3, 29.2,
29.1, 26.1, 25.0. HRMS (ESI) calcd. for (M þ Na) C90H90O23; 1561.5770,
found 1561.5774.
8-(Methoxycarbonyl)octyl 6-O-[2-O-(a-D-arabinofuranosyl)-3,4,6-
tri-O-benzyl-a-D-mannopyranosyl]-a-D-mannopyranoside (13). Trisac-
charide 12 (249 mg, 0.16 mmol) was deacylated in CH3OH (30 mL) with
sodium methoxide as described for the preparation of 9. The product was
purified by column chromatography (2 : 1, EtOAc : hexanes) to yield 13
(125 mg, 85%) as an oil. Rf 0.20 (2 : 1, EtOAc : hexanes). The product was
1
characterized by H NMR spectroscopy and mass spectrometry and used in
1
the subsequent reaction. H NMR (400 MHz, CD3OD) : dH, 7.36–7.04 (m, 15
H, aromatic H), 5.19 (s, 1 H, H-1), 5.09 (s, 1 H, H-10), 4.72–4.58 (m, 5 H,

Fragments of Rhodococcus ruber Lipoarabinomannan
525
H-100, 4 ꢁ PhCH2), 4.44 (d, 1 H, J ¼ 12.2 Hz, PhCH2), 4.32 (d, 1 H, J ¼ 10.9 Hz,
PhCH2), 4.18–4.10 (m, 3 H, H-30, H-50, H-400), 3.98–3.50 (m, 18 H, H-2, H-3,
H-4, H-6a, H-6b, H-20, H-40, H-6a0, H-6b, aglycone OCH2, OCH3, H-200, H-300,
H-5a00, H-5b00), 3.28 (1 H, ddd, J ¼ 7.5, 7.5, 9.6 Hz, H-5), 2.28 (dd, 2 H,
J ¼ 7.5, 7.5 Hz, CH2C55O), 1.62–1.58 (m, 4 H, aglycone CH2), 1.32–1.20
(m, 8 H, aglycone CH2). HRMS (ESI) calcd. for (M þ Na) C48H66O17
:
937.4192; found 937.4198.
8-(Methoxycarbonyl)octyl
6-O-[2-O-(a-D-arabinofuranosyl)-a-D-
mannopyranosyl]-a-D-mannopyranoside (2). Trisaccharide 13 (96 mg,
0.11 mmol) was dissolved in CH3OH (5 mL) and Pd(OH)2 (60 mg) was added.
The solution was stirred under H2 for 4 hr and then filtered through Celite
and concentrated. The product was purified on Iatrobeads (7 : 3,
CH2Cl2 : CH3OH) to yield
2 (55 mg, 82%) as an oil. Rf 0.19 (4 : 1,
CH2Cl2 : CH3OH); [a]Dþ29.5 (c 1.0, CH3OH); 1H NMR (600 MHz, D2O) : dH,
5.23 (d, 1 H, J ¼ 1.8 Hz, H-100), 5.10 (d, 1 H, J ¼ 1.8 Hz, H-10), 4.92 (d, 1 H,
J ¼ 1.8 Hz, H-1), 4.26 (dd, 1 H, J ¼ 1.8, 3.6 Hz, H-200), 4.17 (ddd, 1 H, J ¼ 3.6,
5.4, 9.0 Hz, H-400), 4.05–3.76 (m, 19, H-2, H-3, H-4, H-5, H-6a, H-6b, H-20,
H-30, H-40, H-6a0, H-6b0, H-300, H-5a00, H-5b00, OCH2 aglycone, OCH3), 3.58
(ddd, 1 H, J ¼ 7.5, 7.5, 9.6 Hz, H-50), 2.39 (dd, 2 H, J ¼ 7.5, 7.5 Hz,
CH2C55O), 1.67–1.54 (m, 4 H, aglycone CH2), 1.39–1.25 (m, 8 H, aglycone
13
CH2); C NMR (125 MHz, D2O) : dC, 178.8 (C55O), 110.3 (C–100), 100.4 (C-10),
99.6 (C-1), 84.5 (C-200), 82.1 (C-400), 78.4, 77.4, 73.5, 71.9, 71.7, 71.2, 71.0,
68.9, 67.8, 67.5, 66.9 (C-1), 62.0 (C-500), 61.6 (C-10), 52.9 (OCH3), 34.6
(aglycone CH2), 29.3 (aglycone CH2), 29.2 (aglycone CH2), 29.1 (aglycone
CH2), 29.0 (aglycone CH2), 26.2 (aglycone CH2), 25.2 (aglycone CH2). HRMS
(ESI) calcd. for (M þ Na) C27H48O17 : 667.2784, found 667.2788.
ACKNOWLEDGMENTS
This work was supported by the Natural Sciences and Engineering Research
Council of Canada, The Alberta Ingenuity Centre for Carbohydrate Science
and The University of Alberta.
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