Chemical and chemoenzymatic synthesis of a trisaccharide fragment of Tsukamurella paurometabola lipoarabinomannan

Article abstract of DOI:10.1139/V06-049

The synthesis of a trisaccharide fragment (1) of the lipoarabinomannan from Tsukamurella paurometabola is reported. Two approaches were investigated for the synthesis of the target. One was purely chemical, while the other involved the addition of one of

Full text of DOI:10.1139/V06-049

642
Chemical and chemoenzymatic synthesis of a
trisaccharide fragment of Tsukamurella
paurometabola lipoarabinomannan1
Karen M. Ridgway, Wei Shi, Shuang-Jun Lin, Monica M. Palcic, and
Todd L. Lowary
Abstract: The synthesis of a trisaccharide fragment (1) of the lipoarabinomannan from Tsukamurella paurometabola is
reported. Two approaches were investigated for the synthesis of the target. One was purely chemical, while the other
involved the addition of one of the monosaccharide residues via a mannosyltransferase-catalyzed reaction. Both ap-
proaches produced the target in good overall yields. Thus, this chemoenzymatic approach appears to be a useful addi-
tion to the arsenal of methods for the synthesis of lipoarabinomannan-derived oligosaccharides.
Key words: lipoarabinomannan, oligosaccharide, mannosyltransferase, enzymatic synthesis.
Résumé : On a réalisé la synthèse d’un fragment trisaccharide (1) du lipoarabinomannane extrait du Tsukamurella pau-
rometabola. On a étudié deux approches pour la synthèse de ce produit cible. L’une est purement chimique alors que
l’autre implique l’addition des résidus monosaccharides par le biais d’une réaction catalysée par la mannosyltransférase.
Les deux approches ont permis d’arriver à la cible avec de bons rendements globaux. L’approche chimioenzymatique
semble donc être une addition utile à l’arsenal des méthodes de synthèse des oligosaccharides dérivés du lipoarabino-
mannane.
Mots clés : lipoarabinomannane, oligosaccharide, mannosyltransférase, synthèse enzymatique.
[Traduit par la Rédaction] Ridgway et al. 649
Introduction
mycetes and a number of such investigations have been car-
ried out, revealing an impressive range of structural diversity
(7–17).
The human pathogen Mycobacterium tuberculosis pro-
duces an immunomodulatory lipidated polysaccharide, lip-
oarabinomannan (LAM), which plays a critical role in the
progression of tuberculosis (1, 2). This disease, and those
caused by other pathogenic mycobacteria (e.g., M. leprae
and M. avium), are of increasing interest because of their
resurgence in the industrialized world, the emergence of
drug-resistant strains, and the susceptibility of immunocom-
promised individuals to these infections (3, 4). It is now
well-appreciated that the immunomodulatory properties of
mycobacterial LAM depend on structural motifs within the
glycan (1, 2). For example, LAM from M. tuberculosis has
been shown to inhibit the production of the proinflammatory
cytokines including TNF-α (5). In contrast, LAM from the
nonpathogenic M. smegmatis, which differs only slightly in
structure from M. tuberculosis LAM, induces the release of
these same cytokines (6). These observations have sparked
interest in determining the structure of LAM molecules not
only from mycobacterial strains, but also from other actino-
A general model for how LAMs influence the immune re-
sponse has been proposed (10); however, there is relatively
little detailed information on the role of various structural
motifs within these glycans. A systematic structure function
analysis of the activation or suppression of the immune re-
sponse by fragments of LAM should open up new strategies
for the treatment of mycobacterial disease. Among them are
the design of biosynthetic inhibitors of the immuno-
suppressive glycans or the preparation of novel vaccines that
generate antibodies capable of masking these epitopes. The
fundamental biochemical studies that must precede achiev-
ing such a goal require access to discrete oligosaccharide
fragments of a range of LAM motifs, which are most effi-
ciently obtained via chemical synthesis. Previously, we syn-
thesized fragments of LAM from M. tuberculosis (18, 19)
and Rhodococcus ruber (20). Reported here is the synthesis
of a fragment of LAM from Tsukamurella paurometabola
(10) (TpaLAM). Human infections by this actinomycete,
Received 22 September 2005. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 12 May 2006.
Submitted in honor of the 65th birthday of Walter A. Szarek.
K.M. Ridgway, W. Shi, S.-J. Lin, and T.L. Lowary.2 Department of Chemistry and Alberta Ingenuity Centre for Carbohydrate
Science, Gunning–Lemieux Chemistry Centre, University of Alberta, Edmonton, AB T6G 2G2, Canada.
M.M. Palcic. The Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark.
1This article is part of a Special Issue dedicated to Professor Walter A. Szarek.
2Corresponding author (e-mail: tlowary@ualberta.ca).
Can. J. Chem. 84: 642–649 (2006)
doi:10.1139/V06-049
© 2006 NRC Canada

Ridgway et al.
643
Fig. 1. Proposed structure of the LAM from Tsukamurella pauroametabola (TpaLAM) (10). Diacylglycerol (DAG). All monosaccharide
residues are in the D-form.
→5)-α-Araf-(1→5)-α-Araf -(1→?)-α-Manp-(1→6)-α-Manp-(1→6)-α-Manp
16
10
→
(1 2)
→
(1 6)
α
-Manp
Inositol OPO3DAG
→
(1 2)
→
(1 2)
α
-Manp
α-Manp
16
Fig. 2. Target oligosaccharide (1) and the building blocks chosen for its synthesis (2–4).
OH
O
OAc
O
HO
BnO
HO
HO
OH
BnO
BnO
O
SEt
BnO
HO
HO
HO
O
O
3
O(CH2)7CH3
BnO
O
OAc
O
HO
AcO
2
O
AcO
AcO
O(CH2)7CH3
HO
SEt
4
1
although rare, are occasionally found in immunocompromised
patients (21).
Scheme 1. (a) BnBr, NaH, THF, 0 °C → rt, 79%; (b) NaOBn,
BnOH, 100 °C, 88%.
BnO
HO
a
O
O
b
O
O
2
Results and discussion
O(CH2)7CH3
O(CH2)7CH3
The proposed structure of TpaLAM is shown in Fig. 1
(10). Similar to other glycoconjugates of this class, the
glycan has at its core a mannosylated phosphatidylinositol
moiety, to which is attached a chain of α-(1→6)-linked D-
mannopyranose (Manp) residues. This mannan domain is de-
void of branching Manp units, which is a common feature of
all mycobacterial LAM structures reported to date (1, 2).
The mannan is further elaborated by a linear arabinan, con-
sisting of α-(1→5)-linked D-arabinofuranose (Araf) residues.
In contrast to the mycobacterial variant (1, 2), there is no
branching within the arabinan; however, approximately half
of the Araf residues are further glycosylated on O-2 by an α-
(1→2)-linked Manp disaccharide.
With this in mind, we chose to synthesize a trisaccharide
fragment corresponding to the substituted Araf residue pres-
ent in the arabinan domain. The target thus chosen was 1
(Fig. 2), which we envisioned could be synthesized from
three monosaccharide building blocks (2–4). Thioglycosides
3 and 4 were prepared as previously described (22, 23); the
protected octyl glycoside 2 was straightforwardly synthe-
sized as outlined in the following paragraph.
Access to 2 was achieved starting from the known epoxy
glycoside 5 (24), first by benzylation of the hydroxyl group
under standard conditions. The reaction proceeded to give 6
in 79% yield (Scheme 1). Octyl glycoside 2 was then ob-
tained by heating 6 in a solution of benzyl alcohol and so-
dium benzylate at 100 °C overnight. The reaction proceeded
with the expected high degree of regioselectivity (25), af-
fording 2 in 88% yield. Given the relatively sluggish nature
of this reaction, we also evaluated the use of microwave irra-
6
5
diation to effect the conversion of 6 into 2. After optimiza-
tion of the reaction conditions, we were able to carry out this
reaction in only 30 min by microwave irradiation of a solu-
tion of 2 and sodium benzylate in benzyl alcohol at 170 °C.
Under these conditions, an 82% yield of the product was ob-
tained.
Once 2–4 were prepared, the target was synthesized with-
out difficulty as illustrated in Scheme 2. Coupling of thio-
glycoside 3 with alcohol 2 was carried out under the
promotion of N-iodosuccinimide and silver triflate (26),
which afforded disaccharide 7 in 84% yield. The acetate
protecting group in 7 was then cleaved giving a 97% yield of
alcohol 8. The final monosaccharide residue was introduced by
reaction of 8 with 4, again promoted by N-iodosuccinimde
and silver triflate. The expected trisaccharide product (9)
was obtained in 61% yield. Deprotection of 9 was achieved
first by removal of the acetate esters by transesterification
and then hydrogenation of the benzyl ethers, yielding a
quantitative overall yield of 1.
Having in place a route to 1 via a purely chemical ap-
proach, we explored an alternate method, which involved the
introduction of the “nonreducing” mannopyranose unit via
an enzymatic reaction. To test the feasibility of this route,
we synthesized
a fully deprotected disaccharide (12,
Scheme 3), which is a potential substrate for an α-(1→2)-
mannosyltransferase (α-(1→2)-ManT) from Saccharomyces
cerevisiae that has been used in chemoenzymatic syntheses
© 2006 NRC Canada

644
Can. J. Chem. Vol. 84, 2006
Scheme 2. (a) 3, NIS, AgOTf, CH2Cl2, –40 °C → rt, 84%; (b) NaOCH3, CH3OH, rt, 97%; (c) 4, NIS, AgOTf, CH2Cl2, rt, 61%;
(d) K2CO3, CH3OH, rt, quantitative; (e) H2, Pd(OH)2/C, CH3OH, rt, quantitative.
OAc
O
AcO
AcO
OAc
O
OH
O
AcO
BnO
BnO
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
O
b
c
a
d, e
2
1
O
BnO
O
BnO
O
O
BnO
O
O
O(CH2)7CH3
O(CH2)7CH3
BnO
BnO
O(CH2)7CH3
BnO
7
8
9
Scheme 3. (a) 4, NIS, AgOTf, CH2Cl2, –50 °C → rt, 45%; (b) NaOCH3, CH3OH, rt, 83%; (c) H2, Pd(OH)2, CH3OH, rt, 83%;
(d) GDP-Man, ManT, rt, 90%.
OH
O
HO
HO
OH
O
OAc
O
HO
HO
HO
HO
AcO
AcO
AcO
O
O
HO
HO
HO
d
a
b
RO
O
2
BnO
O
O
O
O
HO
O
O(CH2)7CH3
O(CH2)7CH3
RO
BnO
O(CH2)7CH3
HO
11, R = Bn
12, R = H
1
10
c
of oligosaccharides (27–30). Thus, disaccharide 12 was syn-
thesized by first reacting 2 with 4, which gave 10 in 87%
yield. Deprotection of 10 in two steps (transesterification
and hydrogenation) gave 12 via 11, in 69% overall yield.
Fig. 3. Positive and negative substrate controls for the α-(1→2)-
ManT.
OH
O
OH
HO
HO
HO
HO
O
With 12 in hand, we first tested its ability to serve as a
substrate for this α-(1→2)-ManT by incubating it with
radiolabelled guanosine diphosphate mannose ([3H]-GDP-
Man) in the presence of the enzyme and then quantitation by
scintillation counting. Using this assay, the amount of radio-
activity transferred to 12 was approximately 15% of that
transferred to 8-methoxy(carbonyloctyl) α-D-mannopyrano-
side (13, Fig. 3), a substrate for this enzyme. Encouraged by
this result, we then carried out a larger-scale incubation of
12 with GDP-Man and the α-(1→2)-ManT. After shaking
the mixture for 25 h at room temperature (rt), the reaction
was complete and purification of the product gave a 90%
O(CH2)7CH3
O(CH2)8CO2CH3
HO
13
14
thus demonstrating that this simple furanoside is not a sub-
strate for the enzyme, and therefore the synthesis of 12 from
14 via the action of this enzyme is not feasible.
In summary, a trisaccharide fragment of the LAM from T.
paurometabola, has been synthesized using chemical and
chemoenzymatic approaches. Both routes proceeded effi-
ciently to give the target compound. To the best of our
knowledge, this is the first example of a chemoenzymatic
synthesis of an oligosaccharide substructure of LAM. The
success of this approach suggests that similar strategies may
facilitate the synthesis of more complicated fragments of
these structurally diverse glycoconjugates.3
1
yield of 1. The H NMR spectrum for this trisaccharide was
identical to that of 1 obtained from chemical synthesis
(Fig. 4). In addition, the mass spectrum of the enzymatically
formed product also showed a peak at m/z 609.6, which cor-
responds to the sodium adduct of the expected trisaccharide.
Given the success in the synthesis of the trisaccharide by
this chemoenzymatic approach, we also explored the synthe-
sis of disaccharide 12 by the α-(1→2)-ManT catalyzed cou-
pling of GDP-Man with octyl α-D-arabinofuranoside (14,
Fig. 3). To explore this possibility, 14 was incubated with
[3H]-GDP-Man and the α-(1→2)-ManT. However, no trans-
fer of radioactivity to the monosaccharide was observed,
Experimental
General methods
Reactions were carried out in oven-dried glassware. Reac-
tion solvents were distilled from appropriate drying agents
before use. Unless stated otherwise, all reactions were car-
3 Supplementary data for this article are available on the journal Web site (http://canjchem.nrc.ca) or may be purchased from the Depository
of Unpublished Data, Document Delivery, CISTI, National Research Council Canada, Ottawa, ON K1A 0R6, Canada. DUD 5026. For more
information on obtaining material refer to http://cisti-icist.nrc-cnrc.gc.ca/irm/unpub_e.shtml.
© 2006 NRC Canada

Ridgway et al.
645
1
Fig. 4. H NMR spectra for trisaccharide 1 (recorded in D2O). (A) Product obtained via chemical synthesis; (B) product obtained via
chemoenzymatic synthesis. The peak at ~3.35 ppm is a methanol impurity.
(A)
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
(B)
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
ppm
ried out with stirring at rt 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 or by char-
ring with acidified p-anisaldehyde solution in EtOH. In the
processing of reaction mixtures, solutions of organic sol-
vents were washed with equal amounts of aqueous solutions.
Organic solutions were concentrated under vacuum at
<40 °C. All column chromatography was performed on silica
gel (40–60 µmol/L) or Iatrobeads, which refers to a beaded
silica gel 6RS-8060, manufactured by Iatron Laboratories
(Tokyo). In all cases, the ratio between adsorbent and crude
product ranged from 100 to 50:1 (w/w). Optical rotations
were measured at 22 2 °C and in units of degrees mL/g dm.
1H NMR spectra were recorded at 400, 500, or 600 MHz,
and chemical shifts were referenced to internal CHCl3
(7.26 ppm, CDCl3), CHD2OD (3.30 ppm, CD3OD), or exter-
nal acetone (2.23 ppm, D2O). 13C NMR spectra were re-
corded at 100 or 125 MHz, and 13C chemical shifts were
referenced to internal CDCl3 (77.23 ppm, CDCl3) or CD3OD
centration of the filtrate gave 1 (14.5 mg, 100%) as a white
solid. Rf 0.23 (CH2Cl2–CH3OH, 2:1). [α]D +93.9° (c 1.4,
1
CH3OH). H NMR (500 MHz, CD3OD) δH: 5.28 (d, 1H,
J1′ ,2′ = 1.7 Hz, H-1′), 4.98 (d, 1H, J1,2 = 1.6 Hz, H-1), 4.97
(d, 1H, J1′′ ,2′′ = 1.7 Hz, H-1′′), 4.08 (dd, 1H, J1,2 = 1.6 Hz,
J2,3 = 3.8 Hz, H-2), 3.95–3.99 (m, 2H, H-3, H-2′′), 3.88–
3.91 (m, 1H, H-4), 3.78–3.88 (m, 4H, H-2′, H-6a′, H-6a′′,
H-6b′′), 3.64–3.78 (m, 7H, H-5a, H-6b′, H-3′, H-3′′, H-4′,
H-5′′, octyl OCH2), 3.62 (dd, 1H, J4,5b = 5.6 Hz, J5a,5b
12.2 Hz, H-5b), 3.57 (dd, 1H, J3′′ ,4′′ = 9.5 Hz, J4′′ ,5′′
=
=
9.5 Hz, H-4′′), 3.53 (ddd, 1H, J4′ ,5′ = 9.6, J5′ ,6a′ = 2.4 Hz,
J5′ ,6b′ = 5.3 Hz, H-5′), 3.42 (dt, 1H, J = 9.5, 6.6 Hz, octyl
OCH2), 1.53–1.61 (m, 2H, octyl CH2), 1.26–1.38 (m, 10H,
octyl CH2), 0.87 (t, 3H, J = 6.9 Hz, octyl CH3). 13C NMR
(125 MHz, CD3OD) δC: 107.6 (C-1), 104.2 (C-1′′, 1JC-1′′ ,H-1′′
=
1
168.8 Hz), 99.5 (C-1′, JC-1′ ,H-1′ = 168.8 Hz), 88.9 (C-2),
84.5 (C-4), 80.3 (C-2′), 77.2 (C-2′′), 75.1 (C-5), 75.0
(C-5′′), 72.4 (C-3′), 71.9(2) (C-3′′), 71.8(6) (C-3), 68.8(3)
(C-4′), 68.8(2) (octyl OCH2), 68.7(6) (C-4′′), 63.2 (C-6′),
62.8 (C-6′′), 62.7 (C-5), 33.0 (octyl CH2), 30.8 (octyl CH2),
30.5 (octyl CH2), 30.4 (octyl CH2), 27.3 (octyl CH2), 23.7
(octyl CH2), 14.4 (octyl CH3). HRMS (ESI) calcd. for
C25H46O15: 609.2729 (M + Na); found: 609.2729.
1
(49.0 ppm, CD3OD). H NMR data are reported as though
they were first order. Electrospray mass spectra were re-
corded on samples suspended in CH3OH. The recombinant
S. cerevisiae α-(1→2)-ManT used in the enzymatic reactions
was produced as previously reported (31).
Octyl 3,5-di-O-benzyl-␣-D-arabinofuranoside (2)
Octyl 2-O-[2-O-(␣-D-mannopyranosyl)-␣-D-
mannopyranosyl]-␣-D-arabinofuranoside (1)
Thermal method
Compound 6 (199 mg, 0.60 mmol) was dissolved in
benzyl alcohol (10 mL). Sodium benzylate (1 mol/L, 7 mL,
7 mmol) was added and the reaction was stirred at 100 °C
overnight. After cooling, a few drops of acetic acid were
added to neutralize the reaction mixture. The solution was
then concentrated to remove all but traces of the benzyl al-
cohol and the resulting residue was purified by column chro-
matography (hexanes–EtOAc, 6:1) to give 2 (231 mg, 88%)
as a yellow syrup. Rf 0.19 (hexanes–EtOAc 6:1). [α]D +81.1°
Trisaccharide 9 (30.0 mg, 0.025 mmol) and K2CO3
(2.7 mg, 0.020 mmol) were stirred in CH3OH (2.0 mL) at rt
overnight. The solution was concentrated and the residue
was purified by column chromatography (CH2Cl2–CH3OH,
20:1) to give a partially deprotected intermediate (25.7 mg,
100%). To a solution of this intermediate (25.7 mg,
0.025 mmol) in methanol (5 mL) was added Pd(OH)2/C
(30 mg). The mixture was stirred overnight under an H2 at-
mosphere and the catalyst was separated by filtration
through Celite and then washed with CH3OH (10 mL). Con-
1
(c 1.3, CH2Cl2). H NMR (400 MHz, CDCl3) δH: 7.27–7.38
(m, 10H, Ph), 5.01 (s, 1H, H-1), 4.68 (d, 1H, J = 12.1 Hz,
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646
Can. J. Chem. Vol. 84, 2006
PhCH2), 4.62 (d, 1H, J = 11.9 Hz, PhCH2), 4.51 (d, 1H, J =
12.1 Hz, PhCH2), 4.49 (d, 1H, J = 11.9 Hz, PhCH2), 4.27
(ddd, 1H, J4,5a = 2.3 Hz, J4,5b = 2.4 Hz, J3,4 = 3.1 Hz, H-4),
4.14 (s, 1H, H-2), 3.87 (d, 1H, J3,4 = 3.1, H-3), 3.70 (dt, 1H,
J = 9.7, 6.9 Hz, octyl OCH2), 3.67 (dd, 1H, J4,5a = 2.3 Hz,
Octyl 2-O-(2-O-acetyl-3,4,6-tri-O-benzyl-␣-D-manno-
pyranosyl)-3,5-di-O-benzyl-␣-D-arabinofuranoside (7)
Alcohol 2 (42.9 mg, 0.097 mmol) and thioglycoside 3
(22) (106.5 mg, 0.18 mmol) were dissolved in CH2Cl2
(7.0 mL). To this solution were added 4 Å molecular sieves
(500 mg). After stirring at rt for 15 min, the reaction mixture
was cooled to –40 °C and stirred for another 15 min. NIS
(43.6 mg, 0.19 mmol) was added, followed by AgOTf
(7.5 mg, 0.03 mmol). After 3.5 h, a few drops of Et3N were
added until the pH was slightly basic. CH2Cl2 (20 mL) was
added and the reaction mixture was filtered through Celite.
The filtrate was concentrated and the resulting product was
purified by column chromatography (hexanes–EtOAc, 8:1)
to give 7 (75.0 mg, 84%) as a yellow syrup. Rf 0.18 (hex-
J5a,5b = 10.4 Hz, H-5a), 3.50 (dd, 1H, J4,5b = 2.4 Hz, J5a,5b
=
10.4 Hz, H-5b), 3.44 (dt, 1H, J = 9.7, 6.6 Hz, octyl OCH2),
1.56–1.65 (m, 2H, octyl CH2), 1.22–1.40 (m, 10H, octyl
CH2), 0.88 (t, 3H, J = 6.8 Hz, octyl CH3). 13C NMR
(125 MHz, CDCl3) δC: 138.0 (Ph), 137.1 (Ph), 128.6 (Ph,
2C), 128.4 (Ph, 2C), 128.0 (Ph), 127.9 (2C, Ph), 127.7(4)
(2C, Ph), 127.7(0) (Ph), 109.1 (C-1), 85.4 (C-3), 83.2 (C-4),
78.1 (C-2), 73.8 (PhCH2), 71.9 (PhCH2), 69.9 (C-5), 67.7
(octyl OCH2), 31.9 (octyl CH2), 29.6 (octyl CH2), 29.4
(octyl CH2), 29.3 (octyl CH2), 26.1 (octyl CH2), 22.7 (octyl
CH2), 14.1 (octyl CH3). HRMS (ESI) calcd. for C27H38O5:
465.2617 (M + Na); found: 465.2612. Anal. calcd. for
C27H38O5: C 73.27, H 8.65; found: C 73.42, H 8.76.
1
anes–EtOAc, 8:1). [α]D +54.4° (c 6.2, CH2Cl2). H NMR
(400 MHz, CDCl3) δH: 7.27–7.38 (m, 23H, Ph), 7.16–7.19
(m, 2H, Ph), 5.36 (dd, 1H, J1′ 2′ = 1.8 Hz, J2′ ,3′ = 2.6 Hz, H-
2′), 5.12 (s, 1H, H-1), 5.01 (d, 1H, J1′ ,2′ = 1.8 Hz, H-1′),
4.90 (d, 1H, J = 10.6 Hz, PhCH2), 4.50–4.78 (m, 9H,
PhCH2), 4.22–4.28 (m, 2H, H-4, H-2), 3.96–4.01 (m, 2H,
H-3′, H-4′), 3.84–3.91 (m, 3H, H-3, H-6a′, H-5′), 3.66–
Microwave method
Sodium benzylate (1 mol/L, 0.126 mL, 0.126 mmol) was
added to 6 (21.2 mg, 0.063 mmol) in benzyl alcohol
(0.37 mL). The mixture was heated, with stirring, in a CEM
Discover/Explorer microwave system at 300 W and 170 °C
for 0.5 h. After cooling, acetic acid was added dropwise un-
til the mixture was neutralized. The solution was filtered
through cotton, concentrated, and the resulting residue was
purified by chromatography (hexanes–EtOAc, 6:1) to give 2
(22.9 mg, 82%) as a yellow syrup.
3.76 (m, 3H, H-5a, H-6b′, octyl OCH2), 3.62 (dd, 1H, J4,5a
=
5.3, J5a,5b = 10.8 Hz, H-5b), 3.37 (dt, 1H, J = 6.6, 9.7 Hz,
octyl OCH2), 2.22 (s, 3H, C(O)CH3), 1.51–1.59 (m, 2H,
octyl CH2), 1.26–1.40 (m, 10H, octyl CH2), 0.93 (t, 3H, J =
6.8 Hz, octyl CH3). 13C NMR (125 MHz, CDCl3) δC: 170.4
(C=O), 138.3 (Ph), 138.1(3) (Ph), 138.1(0) (Ph), 137.9 (Ph),
137.8 (Ph), 128.4 (2C, Ph), 128.3(8) (3C, Ph), 128.3(5) (Ph,
2C), 128.3(2) (2C, Ph), 128.0 (2C, Ph), 127.9 (2C, Ph),
127.8(6) (2C, Ph), 127.7(8) (2C, Ph), 127.7(5) (2C, Ph),
127.6(8) (2C, Ph), 127.6(3) (2C, Ph), 127.6(2) (2C, Ph),
106.1 (C-1), 97.2 (C-1′), 86.4 (C-4), 83.6 (C-3), 80.8 (C-2),
78.0 (C-3′), 75.3 (PhCH2), 74.2 (C-4′), 73.5 (PhCH2), 73.4
(PhCH2), 72.2 (PhCH2), 72.0 (C-5′), 71.9 (PhCH2), 69.7 (C-
5), 68.9 (C-2′), 68.7 (C-6′), 67.7 (octyl OCH2), 31.9 (octyl
CH2), 29.6 (octyl CH2), 29.4 (octyl CH2), 29.3 (octyl CH2),
26.1 (octyl CH2), 22.7 (octyl CH2), 21.1 (C(O)CH3), 14.1
(octyl CH3). HRMS (ESI) calcd. for C56H68O11: 939.4654
(M + Na); found: 939.4655. Anal. calcd. for C56H68O11: C
73.34, H 7.47; found: C 73.21, H 7.48.
Octyl 2,3-anhydro-5-O-benzyl-␣-D-lyxofuranoside (6)
To a solution of 5 (24) (2.71 g, 11.1 mmol) in THF
(20 mL) at 0 °C, a mixture of NaH (60% in mineral oil,
0.89 g, 22.2 mmol) in THF (20 mL) was added dropwise.
After 10 min, BnBr (1.7 mL, 14.3 mmol) was added
dropwise. The reaction mixture was stirred for 2 h while
warming to rt before water was added. The aqueous layer
was extracted with CH2Cl2 and the organic extract was
washed with brine, dried (Na2SO4), filtered, and concen-
trated. The crude product was purified by chromatography
(hexanes–EtOAc (20:1) → hexanes–EtOAc (8:1)) to give 6
(2.94 g, 79%) as a pale yellow syrup. Rf 0.53 (hexanes–
EtOAc, 8:1). [α]D +13.5° (c 1.2, CH2Cl2). 1H NMR
(400 MHz, CDCl3) δH: 7.27–7.38 (m, 5H, Ph), 5.05 (s, 1H,
H-1), 4.63 (d, 1H, J = 12.0 Hz, PhCH2), 4.58 (d, 1H, J =
12.0 Hz, PhCH2), 4.21 (dd, 1H, J4,5a = J4,5b = 6.4 Hz, H-4),
3.77 (d, 1H, J2,3 = 2.9 Hz, H-3), 3.75 (dt, 1H, J = 9.5,
6.7 Hz, octyl OCH2), 3.66 (d, 2H, J4,5 = 6.4 Hz, H-5), 3.65
(d, 1H, J2,3 = 2.9 Hz, H-2), 3.46 (dt, 1H, J = 9.5, 6.7 Hz,
octyl OCH2), 1.53–1.62 (m, 2H, octyl CH2), 1.23–1.38 (m,
10H, octyl CH2), 0.89 (t, 3H, J = 6.8 Hz, octyl CH3). 13C
NMR (125 MHz, CDCl3) δC: 138.0 (Ph), 128.4 (Ph), 128.4
(Ph), 127.8 (Ph), 127.7 (Ph), 127.7 (Ph), 101.2 (C-1), 75.0
(C-4), 73.6 (PhCH2), 68.6 (C-5), 68.6 (octyl OCH2), 56.3
(C-2), 54.5 (C-3), 31.8 (octyl CH2), 29.6 (octyl CH2), 29.3
(octyl CH2), 29.2 (octyl CH2), 26.1 (octyl CH2), 22.6 (octyl
CH2), 14.1 (octyl CH3). HRMS (ESI) calcd. for C20H30O4:
357.2042 (M + Na); found: 357.2036. Anal. calcd. for
C20H30O4: C 71.82, H 9.04; found: C 71.58, H 8.92.
Octyl 2-O-(3,4,6,-tri-O-benzyl-␣-D-mannopyranosyl)-3,5-
di-O-benzyl-␣-D-arabinofuranoside (8)
Disaccharide 7 (64.4 mg, 0.070 mmol) and K2CO3
(6.9 mg, 0.050 mmol) were stirred together in CH3OH
(2.6 mL) at rt overnight. The solution was then concentrated
and the residue was purified by chromatography (hexanes–
EtOAc, 4:1) to give 8 (54.6 mg, 97%). Rf 0.30 (hexanes–
EtOAc, 4:1). [α]D +65.4° (c 3.9, CH2Cl2). 1H NMR
(500 MHz, CDCl3) δH: 7.24–7.38 (m, 23H, Ph), 7.17–7.21
(m, 2H, Ph), 5.06 (s, 1H, H-1), 4.98 (d, 1H, J1′ ,2′ = 1.7 Hz,
H-1′), 4.83 (d, 1H, J = 10.8 Hz, PhCH2), 4.50–4.78 (m, 9H,
PhCH2), 4.20–4.23 (m, 2H, H-2, H-4), 3.87–3.95 (m, 2H,
H-2′, H-4′), 3.77–3.86 (m, 4H, H-3, H-3′, H-5′, H-6a′),
3.57–3.71 (m, 4H, H-5a, H-5b, H-6b′, octyl OCH2), 3.37
(dt, 1H, J = 6.6, 9.6 Hz, octyl OCH2), 2.04 (br s, 1H, OH),
1.52–1.60 (m, 2H, octyl CH2), 1.22–1.35 (m, 10H, octyl
CH2), 0.86–0.92 (m, 3H, octyl CH3). 13C NMR (125 MHz,
CDCl3) δC: 138.2 (Ph), 138.1(7) (Ph), 138.1(4) (Ph),
137.8(8) (Ph), 137.8(7) (Ph), 128.5 (2C, Ph), 128.4 (2C, Ph),
© 2006 NRC Canada

Ridgway et al.
647
128.3(6) (2C, Ph), 128.3(2) (2C, Ph), 128.0 (2C, Ph),
127.9(6) (Ph), 127.8(7) (2C, Ph), 127.8(0) (2C, Ph), 127.7(7)
(2C, Ph), 127.7(4) (2C, Ph), 127.6(8) (2C, Ph), 127.6(3)
(2C, Ph), 127.5(7) (2C, Ph), 106.5 (C-1), 98.1 (C-1′), 85.2
(C-4), 83.1 (C-3), 81.1 (C-2), 80.0 (C-3′), 75.2 (PhCH2),
74.1 (C-4′), 73.5 (PhCH2), 73.4 (PhCH2), 72.1 (PhCH2),
72.0 (PhCH2), 71.7 (C-5′), 69.7 (C-5), 68.7 (C-6′), 68.5 (C-
2′), 67.6 (octyl OCH2), 31.9 (octyl CH2), 29.6 (octyl CH2),
29.4 (octyl CH2), 29.3 (octyl CH2), 26.1 (octyl CH2), 22.7
(octyl CH2), 14.1 (octyl CH3). HRMS (ESI) calcd. for
C54H66O10: 897.4548 (M + Na); found: 897.4547.
(C-6′′), 31.8 (octyl CH2), 29.6 (octyl CH2), 29.4 (octyl
CH2), 29.3 (octyl CH2), 26.1 (octyl CH2), 22.6 (octyl CH2),
20.9 (C(O)CH3), 20.7(3) (C(O)CH3), 20.7(0) (C, C(O)CH3),
20.6(5) (C, C(O)CH3), 14.1 (octyl CH3). HRMS (ESI) calcd.
for C68H84O19: 1227.5499 (M + Na); found: 1227.5495.
Octyl 2-O-(2,3,4,6-tetra-O-acetyl-␣-D-mannopyranosyl)-
3,5-di-O-benzyl-␣-D-arabinofuranoside (10)
Alcohol 2 (22.7 mg, 0.051 mmol) and thioglycoside 4
(23) (46.4 mg, 0.102 mmol) were dissolved in CH2Cl2
(5 mL). To this solution were added 4 Å molecular sieves
(50 mg). After stirring at rt for 15 min, the reaction mixture
was cooled to 0 °C and stirred for another 15 min. NIS
(25.3 mg, 0.112 mmol) was added, followed by AgOTf
(4.0 mg, 0.016 mmol). The reaction was monitored by TLC
and after the disappearance of 4, 3 drops of Et3N were
added. The reaction mixture was filtered through Celite. The
filtrate was concentrated and the resulting crude product was
purified by column chromatography (hexanes–EtOAc, 3:1)
to give 11 (33.8 mg, 87%). Rf 0.27 (hexanes–EtOAc, 3:1).
Octyl 2-O-[2-O-(2,3,4,6-tetra-O-acetyl-␣-D-mannopyrano-
syl)-3,4,6-tri-O-benzyl-␣-D-mannopyranosyl]-3,5-di-O-
benzyl-␣-D-arabinofuranoside (9)
Disaccharide alcohol 8 (52.4 mg, 0.060 mmol) and
thioglycoside 4 (23) (44.2 mg, 0.092 mmol) were dissolved
in CH2Cl2 (8.0 mL). To this solution were added 4 Å molec-
ular sieves (100 mg). After stirring at rt for 15 min, the reac-
tion mixture was cooled to 0 °C and stirred for another
15 min. NIS (31.2 mg, 0.14 mmol) was added and the reac-
tion was stirred for 20 min before the addition of AgOTf
(4.6 mg, 0.02 mmol). After 4 h, a few drops of Et3N were
added until the pH was slightly basic and then CH2Cl2
(20 mL) was added and the reaction mixture was filtered
through Celite. The filtrate was concentrated and the result-
ing product was purified by column chromatography (hex-
anes–EtOAc, 3:1) to give 9 (44.1 mg, 61%) as a yellow
syrup. Rf 0.44 (hexanes–EtOAc 2:1). [α]D +53.6° (c 4.5,
1
[α]D +66.0° (c 3.4, CH2Cl2). H NMR (400 MHz, CDCl3)
δH: 7.23–7.33 (m, 10H, Ph), 5.22–5.31 (m, 2H, H-4′, H-3′),
5.16 (dd, 1H, J1,2 = 1.9 Hz, J2,3 = 2.9 Hz, H-2′), 5.06 (s, 1H,
H-1), 4.85 (d, 1H, J1,2 = 1.8 Hz, H-1′), 4.46–4.60 (m, 4H,
PhCH2), 4.24 (dd, 1H, J5,6a′ = 5.4 Hz, J6a′ ,6b′ = 12.2 Hz, H-
6a′), 4.12–4.18 (m, 2H, H-2, H-4), 4.07 (dd, 1H, J5,6b′ = 2.3,
J6a′ ,6b′ = 12.2 Hz, H-6b′), 3.97–4.03 (m, 1H, H-5′), 3.88
(dd, 1H, J = 3.0 Hz, J = 6.7 Hz, H-3), 3.68 (dt, 1H, J = 9.5,
7.0 Hz, octyl OCH2), 3.61 (dd, 1H, J4,5a = 3.7 Hz, J5a,5b
10.8 Hz, H-5a), 3.56 (dd, 1H, J4,5b = 5.1 Hz, J5a,5b
=
=
1
CH2Cl2). H NMR (400 MHz, CDCl3) δH: 7.16–7.38 (m,
10.8 Hz, H-5b), 3.39 (dt, 1H, J = 9.5, 7.0 Hz, octyl OCH2),
2.14 (s, 3H, C(O)CH3), 2.06 (s, 3H, C(O)CH3), 2.01 (s, 3H,
C(O)CH3), 1.97 (s, 3H, C(O)CH3), 1.51–1.59 (m, 2H, octyl
CH2), 1.20–1.33 (m, 10H, octyl CH2), 0.85 (t, 3H, J =
6.9 Hz, octyl CH3). 13C NMR (100 MHz, CDCl3) δC: 170.5
(C=O), 169.9 (C=O), 169.8 (C=O), 169.7 (C=O), 138.0
(Ph), 137.6 (Ph), 128.3(9) (2C, Ph), 128.3(6) (2C, Ph), 127.8
(4C, Ph), 127.6 (2C, Ph), 105.6 (C-1), 97.2 (C-1′), 87.4 (C-
4), 83.4 (C-3), 80.5 (C-2), 73.4 (PhCH2), 72.4 (PhCH2), 69.6
(C-3′), 69.5 (C-5), 69.1 (C-2′), 68.8 (C-5′), 67.8 (octyl
OCH2), 66.0 (C-4′), 62.3 (C-6′), 31.8 (octyl CH2), 29.6
(octyl CH2), 29.4 (octyl CH2), 29.2 (octyl CH2), 26.1 (octyl
CH2), 22.6 (octyl CH2), 20.9 (C(O)CH3), 20.6(9)
(C(O)CH3), 20.6(6) (2C, C(O)CH3), 14.1 (octyl CH3).
HRMS (ESI) calcd. for C41H54O14: 795.3568 (M + Na);
found: 795.3562. Anal. calcd. for C41H54O14: C 63.72, H
7.30; found: C 63.73, H 7.35.
25H, Ph), 5.46 (dd, 1H, J1′′ ,2′′ = 2.0 Hz, J2′′ ,3′′ = 3.4 Hz,
H-2′′), 5.40 (dd, 1H, J2′′ ,3′′ = 3.4 Hz, J3′′ ,4′′ = 10.0 Hz, H-3′′),
5.26 (dd, 1H, J3′′ ,4′′ = J4′′ ,5′′ = 10.0 Hz, H-4′′), 5.08 (d, 1H,
J1,2 = 0.9 Hz, H-1), 5.01 (d, 1H, J1′′ ,2′′ = 2.0 Hz, H-1′′),
4.98 (d, 1H, J1′,2′ = 1.8 Hz, H-1′), 4.82 (d, 1H, J = 10.8 Hz,
PhCH2), 4.68 (d, 1H, J = 11.8 Hz, PhCH2), 4.64 (d, 1H, J =
12.3 Hz, PhCH2), 4.48–4.61 (m, 7H, PhCH2), 4.21 (dd, 1H,
J5′′ ,6a′′ = 5.1 Hz, J6a′′ ,6b′′ = 12.1 Hz, H-6a′′), 4.16–4.19 (m,
2H, H-4, H-5′′), 4.15 (dd, 1H, J1,2 = 0.9 Hz, J2,3 = 2.6 Hz,
H-2), 4.03 (dd, 1H, J5′′ ,6b′′ = 2.2 Hz, J6a′′ ,6b′′ = 12.1 Hz, H-
6b′′), 3.91 (dd, 1H, J3′ ,4′ = J4′ ,5′ = 9.5 Hz, H-4′), 3.84 (dd,
1H, J2′ ,3′ = 2.9 Hz, J3′ ,4′ = 9.5 Hz, H-3′), 3.78–3.82 (m, 3H,
H-3, H-2′, H-5′), 3.73 (dd, 1H, J5′ ,6a′
J6a′ ,6b′
Hz,
=
=
=
10.7 Hz, H-6a′), 3.73 (dd, 1H, J5′ ,6b′ = 2.51.1 Hz, J6a′ ,6b′
10.7 Hz, H-6b′), 3.56–3.64 (m, 3H, H-5a, H-5b, octyl
OCH2), 3.32 (dt, 1H, J = 9.5, 6.5 Hz, octyl OCH2), 2.12 (s,
3H, C(O)CH3), 2.03 (s, 3H, C(O)CH3), 2.00 (s, 3H,
C(O)CH3), 1.98 (s, 3H, C(O)CH3), 1.48–1.54 (m, 2H, octyl
CH2), 1.22–1.30 (m, 10H, octyl CH2), 0.87 (t, 3H, J =
7.0 Hz, octyl CH3). 13C NMR (100 MHz, CDCl3) δC: 170.6
(C=O), 169.8 (C=O), 169.7 (C=O), 169.6 (C=O), 138.3
(Ph), 138.2 (2C, Ph), 138.1 (Ph), 137.9 (Ph), 128.4 (2C, Ph),
128.3(7) (4C, Ph), 128.3(3) (2C, Ph), 128.2(9) (2C, Ph),
128.1 (Ph) 127.7 (3C, Ph), 127.7 (2C, Ph), 127.6(3) (Ph),
127.6(2) (Ph), 127.6(0) (2C, Ph), 127.5 (2C, Ph), 127.4 (Ph),
105.9 (C-1), 99.3 (C-1′), 98.5 (C-1′′), 87.2 (C-2), 83.8 (C-5′),
80.3 (C-4), 79.2 (C-3′), 77.8 (C-2′), 75.3 (PhCH2), 74.7 (C-
4′), 73.4 (PhCH2), 73.2 (PhCH2), 72.5 (PhCH2), 72.4 (C-3),
72.1 (PhCH2), 69.8 (C-2′′), 69.5 (C-5), 69.1 (C-3′′), 69.0
(C-5′′), 68.9 (C-6′), 67.6 (octyl OCH2), 66.1 (C-4′′), 62.5
Octyl 2-O-(␣-D-mannopyranosyl)-␣-D-arabinofuranoside
(12)
Disaccharide 10 (41.1 mg, 0.054 mmol) and K2CO3
(4.1 mg, 0.030 mmol) were stirred together in CH3OH
(2 mL) at rt overnight. The solution was then concentrated
and the residue purified by chromatography (CH2Cl2–
CH3OH, 10:1) to give 11 (26.8 mg, 83%). Rf 0.44 (CH2Cl2–
1
CH3OH, 10:1). H NMR (500 MHz, CD3OD) δH: 7.24–7.34
(m, 10H, Ph), 5.08 (s, 1H, H-1), 4.84 (d, 1H, J = 1.6 Hz, H-
1′), 4.61 (d, 1H, J = 12.0 Hz, PhCH2), 4.53 (s, 2H, PhCH2),
4.51 (d, 1H, J = 12.0 Hz, PhCH2), 4.17 (d, 1H, J2,3 = 2.2 Hz,
H-2), 4.14 (ddd 1H, J3,4 = J4,5a = J4,5b = 5.4 Hz, H-4), 3.83
© 2006 NRC Canada

648
Can. J. Chem. Vol. 84, 2006
(dd, 1H, J2,3 = 2.2 Hz, J3,4 = 5.4 Hz, H-3), 3.81 (dd, 1H,
J5′ ,6a′ = 2.2 Hz, J6a′ ,6b′ = 11.9 Hz, H-6a′), 3.54–3.73 (m, 7H,
H-5a, H-5b, H-6b′, H-2′, H-3′, H-4′, octyl OCH2), 3.50–
3.54 (m, 1H, H-5′), 3.42 (dt, 1H, J = 9.5, 6.3 Hz, octyl
OCH2), 1.53–1.60 (m, 2H, octyl CH2), 1.22–1.40 (m, 10H,
octyl CH2), 0.87 (t, 3H, J = 6.4 Hz, octyl CH3). 13C NMR
(125 MHz, CD3OD) δC: 139.5 (Ph), 139.3 (Ph), 129.4 (4C,
Ph), 128.9(9) (2C, Ph), 128.9(5) (2C, Ph), 128.8(5) (Ph),
128.8 (Ph), 107.8 (C-1), 101.3 (C-1′), 87.1 (C-4), 85.0 (C-
2), 82.5 (C-3), 75.3 (C-4′), 74.4 (PhCH2), 73.1 (PhCH2),
72.4 (C-2′), 72.3 (C-3′), 71.0 (C-5), 68.5 (octyl OCH2), 68.4
(C-5′), 62.7 (C-6′), 33.0 (octyl CH2), 30.7 (octyl CH2),
30.5(2) (octyl CH2), 30.4(6) (octyl CH2), 27.3 (octyl CH2),
23.7 (octyl CH2), 14.5 (octyl CH3).
cartridges (Waters) (32) and quantified by liquid scintillation
counting on a Beckman LS6500 scintillation counter. Under
these conditions, [3H]-Man transfer to 12 was 15% of that of
13, whereas the radioactivity transferred to 14 was at back-
ground levels. Modification of the assay with the use of
2.0 mmol/L GDP-Man, 4.0 mmol/L 14, and increased
α-(1→2)-ManT did not lead to greater levels of radioactivity
transfer, thus demonstrating that this monosaccharide is not
a substrate for the enzyme.
Enzymatic synthesis of 1 from 12
Disaccharide 12 (2.1 mg, 4.95 µmol) and GDP-mannose
disodium salt (4.32 mg, µmol) were reacted with the α-
(1→2)-ManT (200 mU) in 50 mmol/L MOPS buffer at
pH 7.5, containing 0.1% Triton X-100, 10 mmol/L MnCl2,
1 mg/mL BSA, 2 mmol/L DTT, and alkaline phosphatase
(3 U). The total reaction volume was 600 µL. The reaction
mixture was incubated on an end-to-end shaker at rt for 25 h
and reaction progress was monitored by TLC (CHCl3–
CH3OH–water, 70:35:2, Rf 0.5). When TLC showed the
complete disappearance of 12, cold water (~600 µL) was
added and the incubation mixture was centrifuged to remove
precipitates. The supernatent was loaded onto a C18 Sep-
Pak cartridge, which had been preequilibrated with CH3OH
(50 mL) and then water (50 mL). The cartridge was then
washed with water (50 mL) and the product was eluted with
50% CH3OH–H2O (2 × 20 mL). The eluants were concen-
trated to remove the CH3OH and the resulting aqueous solu-
tion was filtered through a 0.22 µm Millex-GV filter. The
filtrate was lyophilized to yield 1 as a white powder (2.6 mg,
Disaccharide 11 (26.8 mg, 0.045 mmol) was dissolved in
CH3OH (3 mL) and Pd(OH)2/C (10.6 mg) was added. A bal-
loon containing hydrogen was placed over the reaction flask.
The solution stirred overnight at rt until the reaction was
complete, as shown by TLC. The catalyst was removed by
filtration through Celite. After washing the filtered catalyst
with CH3OH, the combined filtrates were concentrated, the
residue dissolved in H2O, and the resulting solution was then
loaded onto a C18 Sep-Pak cartridge, which had been
preequilibrated with CH3OH (10 mL) and then water
(10 mL). The cartridge was then washed with water (10 mL)
and the product was eluted with CH3OH (2 × 5 mL). The
eluants were concentrated, the residue redissolved in water,
and the solution filtered through a 0.22 µm Millex-GV filter.
Freeze-drying of the filtered solution gave 12 (15.6 mg,
83%) as a white solid. Rf 0.55 (CH2Cl2–CH3OH, 3:1. [α]D
1
1
90%). The H NMR spectrum (500 MHz, D2O) obtained on
+52.2° (c 1.1, CH3OH). H NMR (600 MHz, CD3OD) δH:
this product was identical to that obtained from 1 produced
by chemical synthesis. Analysis of the product by ESI-MS
showed a signal and m/z 609.6 corresponding to the [M +
Na]+ peak of the trisaccharide.
4.99 (d, 1H, J1′ ,2′ = 1.2 Hz, H-1′), 4.98 (d, 1H, J1,2
=
1.8 Hz, H-1), 4.08 (dd, 1H, J1,2 = 1.8 Hz, J2,3 = 4.2 Hz, H-
2), 3.94 (dd, 1H, J2,3 = 4.2 Hz, J3,4 = 7.2 Hz, H-3), 3.88
(ddd, 1H, J = 3.0 Hz, J = 5.4 Hz, J3,4 = 7.2 Hz, H-4), 3.77–
3.81 (m, 2H, H-2′, H-6a′), 3.63–3.75 (m, 5H, H-5a, H-3′,
H-4′, H-6b′, octyl OCH2), 3.60 (dd, 1H, J = 4.8, 12.0 Hz,
H-5b), 3.51–3.55 (m, 1H, H-5′), 3.40 (dt, 1H, J = 9.6,
6.6 Hz, octyl OCH2), 1.53–1.59 (m, 2H, octyl CH2), 1.26–
1.37 (m, 10H, octyl CH2), 0.87–0.92 (m, 3H, octyl CH3).
13C NMR (100 MHz, CDCl3) δC: 107.4 (C-1), 101.2 (C-1′),
89.0 (C-2), 84.4 (C-4), 77.1 (C-3), 75.0 (C-5′), 72.4 (C-3′),
72.2 (C-2′), 68.4 (C-4′), 68.8 (octyl OCH2), 62.7 (C-5), 62.6
(C-6′), 33.0 (octyl CH2), 30.7 (octyl CH2), 30.5 (octyl CH2),
30.4 (octyl CH2), 27.3 (octyl CH2), 23.7 (octyl CH2), 14.4
(octyl CH3). HRMS (ESI) calcd. for C19H36O10: 447.2206
(M + Na); found: 447.2201.
Acknowledgments
This work was supported by the University of Alberta, the
Natural Sciences and Engineering Research Council of Can-
ada (NSERC), and the Alberta Ingenuity Centre for Carbo-
hydrate Science. KMR is the recipient of an Undergraduate
Student Research Award from NSERC and a Summer
Studentship from the Alberta Heritage Foundation for Medi-
cal Research. WS is the recipient of a Studentship from the
Alberta Ingenuity Fund. We thank Professor Annette
Herscovics (McGill University, Montreal, Quebec) for the
clone of the recombinant mannosyltransferase used in the
chemoenzymatic synthesis of 1.
␣-(1→2)-ManT assay for determining relative substrate
activity of 12–14
Reaction mixtures for assessing the incorporation of [3H]-
Man into 12–14 consisted of 50 mmol/L MOPS (pH 7.5),
10 mmol/L MgCl2, 0.5 mmol/L GDP-Man, 1.0 mmol/L po-
tential acceptor substrate, the α-(1→2)-ManT (0.0134 mU
for 12, 0.0027 mU for 13, 0.134 mU for 14) (32), 0.1% Tri-
ton X-100, 1 mg/mL of BSA, guanosine diphosphate mannose
(American Radiolabeled Chemicals, Inc., 8.9 Ci/mmol,
0.6 µL) in a total reaction volume of 30 µL. The mixture
was incubated at 37 °C for 30 min. Control assays without
acceptor were also performed. Radiolabeled products were
isolated by reverse-phase chromatography on SepPak C18
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