Synthesis of high-mannose 1-thio glycans and their conjugation to protein

Article abstract of DOI:10.1039/c3ob42194e

The oligosaccharides Man₄ and Man₅, substructures of the high-mannose glycans of HIV glycoprotein gp120, were synthesized with a terminal 1-thiomannopyranose residue. The anomeric thiol can be readily converted to an azidomethyl aglycone through reaction with dichloromethane and displacement with sodium azide. The resulting oligomannans were then conjugated to ubiquitin utilizing thiol alkylation or azide/alkyne reactive tethers of minimal length. By combining high efficiency conjugation reactions and a short tether, we sought to establish conjugation conditions that would permit high density clustering of oligomannans in conjugate vaccines that could produce antibodies able to bind gp120 and potentially neutralize virus. LC-UV-MS was used to separate, identify and quantify the ubiquitin glycoconjugates with differing degrees of oligomannan incorporation. Binding of the HIV protective monoclonal antibody 2G12 and concanavalin A to microtitre plates coated with glycoconjugates was measured by ELISA. This journal is

Full text of DOI:10.1039/c3ob42194e

Volume 12 Number 14 14 April 2014 Pages 2153–2324
Organic &
Biomolecular
Chemistry
www.rsc.org/obc
ISSN 1477-0520
PAPER
Justin J. Bailey and David R. Bundle
Synthesis of high-mannose 1-thio glycans and their conjugation to protein

Organic &
Biomolecular Chemistry
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Synthesis of high-mannose 1-thio glycans and
their conjugation to protein†
Cite this: Org. Biomol. Chem., 2014,
12, 2193
Justin J. Bailey* and David R. Bundle
The oligosaccharides Man4 and Man5, substructures of the high-mannose glycans of HIV glycoprotein
gp120, were synthesized with a terminal 1-thiomannopyranose residue. The anomeric thiol can be readily
converted to an azidomethyl aglycone through reaction with dichloromethane and displacement with
sodium azide. The resulting oligomannans were then conjugated to ubiquitin utilizing thiol alkylation or
azide/alkyne reactive tethers of minimal length. By combining high efficiency conjugation reactions and a
short tether, we sought to establish conjugation conditions that would permit high density clustering of
oligomannans in conjugate vaccines that could produce antibodies able to bind gp120 and potentially
neutralize virus. LC-UV-MS was used to separate, identify and quantify the ubiquitin glycoconjugates with
differing degrees of oligomannan incorporation. Binding of the HIV protective monoclonal antibody 2G12
and concanavalin A to microtitre plates coated with glycoconjugates was measured by ELISA.
Received 5th November 2013,
Accepted 3rd February 2014
DOI: 10.1039/c3ob42194e
www.rsc.org/obc
Introduction
The HIV-1 envelope glycoprotein gp120 is the primary focus of
humoral responses against HIV-1 and is an important target
for vaccine development against the virus. The surface of
gp120 is heavily glycosylated, with approximately half its
weight attributed to N-linked glycans.1 These densely clustered
glycans appear to play a protective role, shielding the protein
surface of gp120 from immune recognition.2,3 The glycans
themselves can also be immunological targets, as broadly neu-
tralizing antibodies have been isolated from asymptomatic
HIV-1 infected individuals that recognize carbohydrate epi-
topes, and the existence of these antibodies has been used by
many as a rationale for the construction of glycoconjugate vac-
cines that mimic the glycan shield. Such broadly neutralizing
antibodies include antibody (Ab) 2G12, which recognizes a
unique cluster of high-mannose oligosaccharides on gp120,4–7
and the antibodies PG9 and PG16 and the PGT class of anti-
bodies which recognize both glycan and protein elements of
gp120.8–11
Fig. 1 Structure of the Man9GlcNAc2 oligosaccharide with the Man4
and Man5 substructures outlined in red and green.
Synthetic glycoconjugate vaccines against HIV-1 have
focused on clustering high mannose oligosaccharides that are
recognized by Ab 2G12 onto various carrier scaffolds.12–15 The
oligomannosides utilized in these vaccines were the Man9 and
Man4 substructures of Man9GlcNAc2 oligosaccharide (Fig. 1),
both of which have been implemented with or without the
GlcNAc2 chitobiose core. The conjugate vaccines reported to
date have used a variety of linker chemistries to attach glycans
to immunogenic carriers, either as prearranged clustered
haptens or directly as univalent haptens. In nearly all of the
above cited examples, the length of the linker that attaches
glycan to immunogenic carrier ranged between 9 to greater
than 20 atoms in length. Unfortunately these glycoconjugate
vaccines did not induce protective antibodies against HIV-1.
Alberta Glycomics Centre and the Department of Chemistry, University of Alberta,
Edmonton, Alberta T6G 2G2, Canada. E-mail: jjbailey@ualberta.ca
†Electronic supplementary information (ESI) available: LC-UV-MS chromato-
grams and analysis. NMR spectra data available for compounds 1, 2, 16, 17, 21,
22, 24, 26, 27, 28, 29, 31, 32, 33 and 34. See DOI: 10.1039/c3ob42194e
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Accurate simulation of the crowded oligomannose glycan
surface of gp120 is thought to be crucial to elicit protective
anti-glycan antibodies by vaccination. Thus, appropriate
Results and discussion
Retrosynthetic analysis
glycan presentation, via choice of carrier, conjugation strategy, An anomeric thiol group was chosen to act as the shortest
and glycan selection, may yield a more effective glycoconjugate possible tether to conjugate Man4 and Man5 oligosaccharides
vaccine.
to carrier molecules. Since thiols are highly reactive nucleo-
It seems reasonable to assume that the most relevant philes we anticipated that it would facilitate high efficiency
choice of glycan for use in an HIV-1 vaccine would be those conjugation under a variety of conditions and thereby achieve
found on the native virion. Recent mass spectrometry analysis high density presentation of the target epitopes.
of the gp120 glycans isolated from HIV-1 pseudovirus and
The synthesis of the tetra- and penta-mannopyranosyl thiol
replication competent HIV-1 particles revealed a remarkably analogues 1 and 2 posed two synthetic challenges:
simple and conserved glycan profile compared to recombinant
gp120 expressed in the same 293 T cell line.16–18 While recom-
1. The installation and protection of the β-anomeric thiol.
2. Protection of a glycosyl acceptor for creating 3,6-branched
binant gp120 displays over ∼70% complex-type glycans,16,19–21 mannosides.
the glycans of gp120 derived from virions are predominantly of
Both the tetrasaccharide 1 and pentasaccharide 2 share
the high-mannose Man5–9GlcNAc2 glycan series. Glycans iso- common structural features that can be broken down into the
lated from the envelope of various clades of infectious virus monosaccharide building blocks 4 and 5, and synthon A
particles that were derived from peripheral blood mononuclear (Scheme 1). Well precedented chemistry has been reported for
cells (PBMCs), the natural medium of HIV-1, displayed the the synthesis of the glycosyl imidate 424 and the orthogonally
same predominant Man5–9GlcNAc2 glycan profile. The appar- protected allyl mannoside 5.25,26 These building blocks
ent universal conservation and abundance of the high- provide access to the trisaccharide donor 3 through the 3,6-
mannose type glycans on virion-associated gp120 provides a deprotection of 5 followed by glycosylation with donor 4. In
prospective target for eliciting broadly neutralizing antibodies.
addition, glycosyl donor 4 can be utilized to prepare α1,2-
To achieve a high loading and dense packing of glycan on a linked oligomannosides of the type found in tetrasaccharide 1.
carrier protein scaffold, an efficient and robust conjugation
chemistry is required. Crosslinkers of moderate length are
Synthesis of building blocks
commonly preferred to alleviate steric hindrance at the glycan– For synthon A, we elected to introduce a protected anomeric
carrier interface, but longer linkers add flexibility which may thiol at the beginning of our synthetic route to 1 and 2. An
impede adequate replication of the more rigid structural unsymmetrical tert-butyl disulfide appeared to be the protect-
environment at the gp120 surface. Reducing the length of the ing group most tolerant to protecting group manipulations
tether could restrict the glycan flexibility which may better rep- and glycosylation conditions when evaluated with other
resent the naturally occurring glycans on gp120.
anomeric thiol protecting groups.27 Reaction of tetracetyl-1-S-
We set out to address the issue of maintaining conjugation acetyl-1-thio-β-D-mannopyranose 628,29 with diisopropyl-N-(tert-
efficiency as steric crowding increases during the course of gly- butylsulfanyl) hydrazodicarboxylate 7 gave 8 in 66% yield as a
coconjugate formation, while retaining structural rigidity colourless syrup containing both β and α anomers in the ratio
through the use of a tethering group of the shortest possible 22 : 1 (Scheme 2). Transesterification of 8 produced tetrol 9,
length. To mimic the N-glycans of the crowded surface of which was recrystallized as a white powder of anomers in the
gp120 we elected to synthesize the Man5 epitope which ratio 100 : 64 β/α. It was anticipated that separation of pure
appears with high frequency in clinical isolates,16,18 and the anomers would be most practical following selective
Man4 epitope which is recognized by Ab 2G12.22 To facilitate protection.
the conjugation of these oligosaccharides in high copy
Various protection strategies were explored to selectively
number to a carrier molecule, the Man4 and Man5 structures protect the thiol mannopyranoses 9 for glycosylation at O-3
were synthesized as 1-thiopyranose derivatives. The nucleophi- and O-6. Foremost amongst the approaches was the intro-
lic sulfhydryl group is useful in multiple highly reactive conju- duction of a trityl group at O-6 and a bulky silyl group at
gation reactions and it can also be readily converted to a short O-3 followed by benzoylation, as reported for allyl α-D-
azidomethyl tether23 for use in the copper(I)-catalyzed azide mannopyranoside 5.25 Application of this regioselective meth-
alkyne cycloaddition (CuAAC).
odology to the anomeric mixture 9 afforded the 3,6-protected
Here we describe the synthesis of the mannosyl thiols and product 10 as an inseparable mixture of anomers (Scheme 2).
their azidomethyl analogues and the corresponding thiol alky- Benzoylation of 10 produced the monobenzoate and dibenzo-
lation and CuAAC conjugations of oligosaccharide to ubiquitin. ate 11 and 12. The β-anomer of 10 was extremely resistant to
These methods are a prelude to high loading of the oligosac- 4-O-benzoylation, giving almost exclusively the monobenzoate
charide on phage particles. Since phage particles permit con- product 10. Only after
5 days at reflux with excess
jugation of several thousand copies to a single virion, this benzoyl chloride could appreciable amounts of the β-anomer
may mimic the dense crowding of glycans found on gp120 of 12 be obtained. Since separation of these anomeric and
and enable the use of such conjugates as a candidate HIV-1 incompletely benzoylated mixtures was impractical on
a
vaccine.
preparative scale, an alternate approach was used. Reaction of
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Scheme 1 Retrosynthesis of tetrasaccharide 1 and pentasaccharide 2. R and R’ represent temporary orthogonal protecting groups.
drive the reaction to higher yields resulted in formation of
N-glycosyl trichloroacetamide and limited the yield of 14 in the
range 43–67%. A yield of 83% was eventually achieved through
an inverse glycosylation procedure,30 in which donor 4 was
slowly added to a premixed solution of acceptor 13 and
activator.
Disaccharide 14 is a convenient intermediate to access both
tetrasaccharide 1 and pentasaccharide 2. For use in the syn-
thesis of tetrasaccharide 1, the TBDPS group was first removed
to give triol 15 followed by benzoylation to provide the pro-
tected disaccharide 16 (Scheme 3). The acetyl group was selec-
tively removed using anhydrous HCl in methanol to give 17 in
78%. The reaction was carefully monitored and extended over
170 hours at −10 °C to minimize loss of benzoates. For use in
the synthesis of pentasaccharide 2, disaccharide 14 was first
perbenzoylated to give 18. Removal of the silyl ether gave the
selectively protected disaccharide alcohol 19.
Assembly of tetrasaccharide 22
Glycosylation of acceptor 17 with donor 4 afforded trisacchar-
ide 20 in high yield (Scheme 4). Following acid catalyzed trans-
esterification, trisaccharide acceptor 21 was obtained in
moderate yield together with quantitative recovery of unreacted
trisaccharide 20. The trisaccharide acceptor 21 was reacted
with 4 to give the protected target tetrasaccharide 22 in 91%
yield. The anomeric configuration of each newly introduced
α-D-mannopyranosyl residue for the sequence 14→20→22 was
Scheme 2 (a) 7, DEA, CH2Cl2, 66%; (b) NaOMe, MeOH, 91% 100 : 64
α/β; (c) (i) TrCl, pyridine 81%, (ii) TDSCl, DMF, imidazole, 92%; (d) BzCl,
1-methylimidazole, pyridine, 80% for 11 (α/β mixture); (e) TBDPSCl,
pyridine, 71%.
tert-butylchlorodiphenylsilane with
8 gave an anomeric
mixture of the 6-O-TBDPS ether 13 in 94% yield from which
the pure β-anomer could be obtained in 71% yield by recrystal-
lization (Scheme 2).
1
unambiguously confirmed by heteronuclear JC1,H1 coupling
constants of 171 Hz.31
Conveniently, glycosylation of 13 by the donor 4 was selec-
tive for the formation of the O-3 linked disaccharide 14 with
Assembly of pentasaccharide 26
only trace amounts of O-2 or O-4 linked disaccharides The pentasaccharide was assembled in a 3 + 2 fashion, requir-
(Scheme 3). Use of a 1.5–2 fold molar excess of donor 4 to ing the synthesis of the branched trisaccharide imidate 3 for
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Scheme 3 (a) BF3·OEt2, CH2Cl2, 0 °C, 83%; (b) HF·pyridine (70%), pyridine, 93%; (c) BzCl, pyridine, 87%; (d) CH2Cl2–MeOH 2 : 3, AcCl, −10 °C, 78%;
(e) BzCl, pyridine, 1-methylimidazole, 72%, (f) HF·pyridine (70%), pyridine, 93%.
Scheme 4 (a) 4, TMSOTf, CH2Cl2, rt, 95%; (b) CH2Cl2–MeOH 1 : 1, AcCl, −10 °C, 48%; (c) 4, TMSOTf, CH2Cl2, 0 °C, 91%.
glycosylation to disaccharide 19. The trisaccharide donor was trimethylphosphine (TMP) and then removing benzoates. This
prepared by selectively deprotecting 525,26 followed by glycosy- deprotection order allows for the chromatographic removal of
lation with donor 4 to afford trisaccharide 2426 in 93% yield excess reducing agent which was necessary to avoid the for-
(Scheme 5). The configuration of each newly formed α1,3 and mation of a complex mixture of side products upon disulfide
1
α1,6 glycosidic linkage was confirmed by JC1,H1 heteronuclear reduction.
coupling constants of 171 and 175 Hz,31 and the position of
Tetrasaccharide 22 was cleanly reduced to thiol 27 with
3
3
the linkages by the JH1,C3 and JH1,C6 correlations. Deallyla- TMP (Scheme 6). Subsequent transesterification was per-
tion of 24 with PdCl2 and NaOAc in aqueous acetic acid32 pro- formed in deuterated solvents in order to monitor reaction
duced hemiacetal 25 as an anomeric mixture along with a progress by 1H NMR. Anomerization was observed and the for-
2-oxopropyl side-product. The formation of a Wacker ketone mation of the unwanted α-anomer was found to be time rather
byproduct during anomeric deallylation has been previously than pH dependent, whereas deacylation was more dependent
reported.33–35 The hemiacetal and side-product were difficult on pH than reaction time. Thus, the formation of the
to separate from each other by chromatography and the α-anomer could be kept to a minimum by increasing the
mixture was carried to the next step for separation. Reaction of amount of base. The 2-O-benzoate was extremely resistant to
25 with trichloroacetonitrile furnished the trisaccharide treatment with methoxide solution, even at 1 M concen-
imidate 3 in 52% yield from 24.
trations. This benzoate was removed by saponification of
Pentasaccharide 26 was obtained in 93% yield by glycosyla- mono-benzoylated intermediate with 1 M NaOD to produce
tion of 19 with 3. The configuration of the newly formed α1,6- the fully deprotected tetrasaccharide anomers in 70% yield as
1
linkage was confirmed by the heteronuclear JC1,H1 coupling a 1 : 3 α/β anomeric mixture. The mixture could be cleanly sepa-
constant of 175 Hz.31
rated via HPLC to produce tetrasaccharide 1 as the β-anomer,
as verified by anomeric 1JC1,H1 heteronuclear coupling constant
of 153 Hz.31
Deprotection of tetrasaccharide 22 and pentasaccharide 26
Pentasaccharide 26 was deprotected in analogous fashion
to tetrasaccharide 22 (Scheme 6). Treatment with TMP
Global deprotection of tetrasaccharide 22 and pentasaccharide
26 was achieved by first reducing the disulphide with
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Scheme 5 (a) 90% TFA aq., Et3SiH, 75%; (b) 4, TMSOTf, CH2Cl2, 0 °C, 93%; (c) PdCl2, 90% AcOH aq., NaOAc; (d) Cl3CCN, DBU, CH2Cl2, 52%
(2 steps); (e) 19, TMSOTf, CH2Cl2, 0 °C, 93%.
Scheme 6 (a) TMP (1 M in THF), NaHCO3 aq. (1 M), 99%; (b) (i) NaOCD3 (0.5 M), (ii) NaOD (1 M), 70% 1 : 3 α/β for 1, 63% 1 : 3 α/β for 2.
produced the thiol 28 in quantitative yield. Transesterification Synthesis and deprotection of azidomethyl mannosides
33 and 34
and saponification produced the fully deprotected pentasac-
charide 2 in 63% yield with a 1 : 3 α/β ratio. The β-anomer of
pentasaccharide 2 was obtained after HPLC and its anomeric
configuration verified by
158 Hz.31
Two additional reactive glycans were produced employing a
convenient method to introduce a short azidomethyl tether.23
The tetra- and pentasaccharides were converted into azido-
methyl analogues through the reaction of 27 and 28 with
a
1JC1,H1 coupling constant of
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Scheme 7 (a) DBU, CH2Cl2; (b) NaN3, acetone, H2O, reflux, 77% for 31, 83% for 32 (2 steps); (c) NaOCD3 (0.14 M), 84% for 33, 71% for 34.
dichloromethane in the presence of a hindered base to form 4-pentynoate 36 tethers (Scheme 8). In brief, 5 equivalents of
the corresponding chloromethylthio glycosides 29 and 30 activated tether per amine were reacted with ubiquitin in phos-
(Scheme 7). Displacement with sodium azide yielded the phate buffer, pH 8.3. After 3 hours, buffer and excess crosslin-
azidomethyl products 31 and 32. Saponification gave the ker were removed by size exclusion desalting chromatography.
deprotected tetra- and pentasaccharides 33 and 34.
The degree of tether conjugation was assessed by
LC-UV-MS, where differentially conjugated ubiquitin species
were separated by liquid chromatography (LC) and identified
Glycoconjugation to ubiquitin
Ubiquitin is
a non-glycosylated small molecular weight by mass spectroscopy (MS) (Fig. 2). The identified conjugate
(8.5 kDa) protein with eight exposed amino groups available species were quantified using PeakFit analysis of their UV
for conjugation. Glycoconjugates of ubiquitin were prepared absorbance at 210 nm. Using this method, an average labelling
by first functionalizing the amino groups by reaction of 5.3 iodoacetyl and 6.1 pentynyl functionalities for 37
with either N-succinimidyl iodoacetate 35 or N-succinimidyl and 38 were observed. Approximately 71% of the iodoacetyl
Scheme 8 (a) 35 or 36, sodium borate, pH 8.3; (b) 1 or 2, sodium phosphate, pH 8.0; (c) 33 or 34, sodium phosphate, pH 7.2, CuSO4, THPTA, amino
guanidine, sodium ascorbate.
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Fig. 3 ELISA titration curves of (left) ConA and (right) Ab 2G12 against
39: , 40: , 41: , 42: , and ubiquitin: . ELISAs performed in triplicate.
moieties compared to the one of the tetrasaccharide. There was
no apparent preference in avidity with ConA between the ubi-
quitin conjugates with the iodoacetyl or alkynyl linkers despite
differences in conjugation density. The choice of linker did not
appear to affect the avidity of ConA to the hapten as indicated
by the identical binding curves between the Man4-bearing con-
jugates 39 and 41, and the Man5-bearing conjugates 40 and 42.
Titration of Ab 2G12 against the ubiquitin glycoconjugates
revealed a stark preference for the tetrasaccharide conjugates 39
and 41 over that of the pentasaccharide conjugates (Fig. 3, right).
This disparity can be attributed to differences in hapten structure,
as the pentasaccharide does not contain any Manα1→2Man link-
ages. Interestingly, the binding preference of 2G12 is not equivalent
for the Man4-bearing conjugates 39 and 41, unlike with ConA. This
may be due to differences in the linker structures, as the linker
used in 41 is longer and more flexible, and contains a triazole
moiety which could disfavorably orientate the glycan for 2G12
recognition. The degree of glycan loading on ubiquitin may also be
a factor as 2G12 is known to exhibit increased binding to glycans
that are clustered.22,38 Thus, it could be expected that the more
densely conjugated 39 would bind 2G12 with higher avidity. These
observations in differences of avidity highlight the importance of
correctly displaying glycans for immunological recognition.
Fig. 2 Example LC-UV-MS analysis of 37. (Top) extracted-ion chroma-
tograms (EICs) are used to identify the corresponding peaks in the UV
trace (bottom).
functionalized ubiquitin contained 1 or 2 internal crosslinks,
as indicated by the presence of mass + 40 ion species. This
crosslinking is likely due to reaction of the reactive iodoacetyl
functionalized amines with neighbouring amino acid func-
tional groups, such as the ε-amine of lysine, which accounts
for the lower linker loading of 37.
Iodoacetyl functionalized ubiquitin 37 was then reacted with
either the Man4 or Man5 1-thio-mannans 1 and 2 in phosphate
buffer, pH 8.0 for 3 hours. Excess oligosaccharide was removed by
size exclusion chromatography to obtain the desired glycoconju-
gates 39 and 40, with an average glycan loading of 5.3 Man4 or
Man5 oligosaccharides per ubiquitin, as confirmed by LC-UV-MS
(ESI). Similarly, the alkyne functionalized ubiquitin was reacted with
the azidomethyl mannosides 33 and 34 under CuAAC conditions,
to produce the 41 and 42 glycoconjugate products bearing an
average of 4.9 Man4 and 4.7 Man5 oligosaccharides per ubiquitin.
Conclusions
Man4 and Man5 oligosaccharides terminated by a 1-thioman-
nopyranose residue are shown to be effective derivatives for
efficient conjugation of iodoacetyl-functionalized proteins. The
ready conversion of 1-thio derivatives to azidomethyl glyco-
sides provides allows access to the highly effectual CuAAC click
chemistry for conjugation. These conjugation strategies should
facilitate the congested conjugation of oligomannose struc-
tures on phage particles with the objective of simulating the
crowded glycan shield of gp120 for a potential HIV-1 vaccine.
The conjugation to phage, characterization of the conjugates
and their immunogenicity will be reported elsewhere.
ConA and Ab 2G12 binding to ubiquitin glycoconjugates
The ubiquitin glycoconjugates were analyzed by ELISA for
their ability to bind concanavalin A (ConA), a lectin with speci-
ficity for terminal α-D-mannosyl groups,36,37 and Ab 2G12,
which displays specificity for the Manα1→2Man-linked
moiety.7 The pentasaccharide glycoconjugates 40 and 42
exhibited higher relative avidity for ConA than for its tetrasac-
Experimental
General methods
charide counterparts (Fig. 3, left). This is reasonable since the All chemical reagents were of analytical grade and used as
pentasaccharide structure has three terminal α-D-mannose obtained from commercial sources unless otherwise specified.
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Solvents used for water sensitive reactions were distilled under
Thiol alkylation. To a solution of 37 (0.49 mg, 0.057 µmol)
an inert atmosphere. Unless otherwise noted, all reactions in 0.55 mL H2O was added 100 µL of 0.2 M sodium phosphate
were carried out at room temperature and water sensitive reac- pH 8.0 (31 mM final conc.) and 1 mg of 1 (1.4 µmol) or 2 (1.2
tions were performed under a positive pressure of argon. Sol- µmol). The reaction mixture was rotated at room temperature
vents were removed under reduced pressure between 20–40 °C for 3 hours and purified by size exclusion chromatography as
with a rotary evaporator unless otherwise indicated. Molecular previously described. Protein was quantified using a Pierce™
sieves (4 Å) were stored in an oven at 500 °C and were cooled in BCA Protein Assay Kit, yielding ∼80% recovery of glycoconju-
vacuo prior to use. Analytical thin layer chromatography (TLC) gates 39 and 40.
was conducted on silica gel 60-F254 (Merck). TLC plates were
CuAAC. To a solution of 38 (0.27 mg, 0.032 µmol) in 0.3 mL
visualized under UV light and an anisaldehyde stain composed H2O was added 100 µL of 0.2 M sodium phosphate pH 7.2
of 4% H2SO4, 1.2% AcOH and 0.4% p-anisaldehyde in ethanol, (63 mM final conc.) and sparged with argon for 30 minutes.
followed by heating. Amberlite® IR 120 resin (strongly acidic To this degassed solution was added 1 mg of 33 (1.3 µmol) or
H+ form) was used where H+ resin is indicated. Medium 34 (1.1 µmol), 2.5 µL of CuSO4 (2.5 µL, 20 mM, 0.05 nmol),
pressure chromatography was conducted using silica gel tris(3-hydroxypropyltriazolylmethyl)amine (THPTA)39 (5 µL,
(230–400 mesh, Silicycle, Montreal) or FluoroFlash® silica gel 50 mM, in H2O, 0.25 nmol), aminoguanidine hydrochloride
(40 µm, Fluorous Technologies Incorporated) with flow rates (25 µL, 100 mM, in H2O, 2.5 nmol), and sodium ascorbate
between 5–10 mL min−1. Following deprotection, final com- (25 µL, 100 mM, in H2O, 2.5 nmol). The reaction mixture was
pounds were purified by HPLC conducted on a Waters Delta rotated at room temperature for 3 hours and purified by size
600 system using a Waters 2996 Photodiode Array detector. exclusion chromatography as previously described. Protein was
Separations were performed on a TOSOH TSKgel Amide-80 quantified using a Pierce™ BCA Protein Assay Kit, yielding
column with a matched TSKgel Amide-80 column guard using 90% recovery of glycoconjugates 41 and 42.
a gradient of water and acetonitrile. NMR experiments were
recorded on Varian INOVA 500 or 600 MHz spectrometers, or
LC-UV-MS protocol
on Varian VNMR 500 or 700 MHz spectrometers equipped Reverse-phase HPLC-UV-MS was performed using an Agilent
with cryogenic probes. The 600 and 700 MHz spectrometers 1200 SL HPLC System with a ProSwift® RP-4H Analytical
utilize inverse probes (1H{13C/15N}) allowing for 1200 : 1 and column (1 × 250 mm) (Dionex, USA), thermostated at 60 °C,
7000 : 1 1H sensitivity, respectively. 13C NMR was recorded at with a buffer gradient system composed of 0.1% formic acid in
approximately 125 MHz. 1H and 13C NMR chemical shifts, water as mobile phase A and 0.1% formic acid in acetonitrile
reported in δ (ppm), were referenced to internal residual proto- as mobile phase B. Gradients were optimized separately for
nated solvent signals or to external acetone (0.1% ext. acetone the linker labeled proteins and glycoconjugates.
@ 2.225 ppm) in the case of D2O. Electrospray ionization mass
An aliquot of 2 µL equivalent to an amount of 2 µg ubiqui-
spectra were recorded on a Micromass Zabspec TOF mass tin conjugate was loaded onto the column at a flow rate of
spectrometer by the analytical services facility at the University 0.20 mL min−1 and an initial buffer composition of 95%
of Alberta’s Department of Chemistry. Optical rotations were mobile phase A and 5% mobile phase B. After injection, the
determined with a Perkin-Elmer model 241 polarimeter at 22
column was washed using the initial loading conditions for
2 °C using the sodium D-line and are reported in units of 3 minutes to effectively remove salts. Elution of the protein-
degree mL g−1 dm−1. Concentrations (c) are reported in mg linker analytes was done by using a linear gradient from 5% to
mL−1. Combustion analysis was performed by the analytical 15% mobile phase B over a period of 12 minutes, 15% to 50%
services facility at the University of Alberta’s Department of mobile phase B over a period of 25 minutes and 50% to 85%
Chemistry.
mobile phase B over a period of 5 minutes. Elution of the
protein-linker-oligosaccharide analytes was done by using a
linear gradient from 5% to 18% mobile phase B over a period
of 2 minutes, 18% to 38% mobile phase B over a period of
Procedure for ubiquitin conjugation
Linker addition. To a solution of ubiquitin (0.5 mg, 0.058 35 minutes and 38% to 85% mobile phase B over a period of
µmol) in sodium borate (0.5 mL, 50 mM, pH 8.3, 5 mM EDTA) 8 minutes.
was added a solution of tether 35 (commercially available) or
UV absorbance was monitored at 210, 214, 254 and 280 nm.
36 (2.5 µmol in 30 µL DMSO, 85 mM). The reaction mixture Mass spectra were acquired in positive mode of ionization
was rotated at room temperature for 3 hours. The mixture was using an Agilent 6220 Accurate-Mass TOF HPLC/MS system
passed through a Zeba™ size exclusion column (7 K MW (Santa Clara, CA, USA) equipped with a dual sprayer electro-
cutoff) according to the manufacturer’s guidelines. In brief, a spray ionization source with the second sprayer providing a
2.0 mL column was washed three times with water and 0.5 mL reference mass solution. Mass spectrometric conditions were
of protein solution loaded onto the resin. A stacker volume of drying gas 9 L min−1 at 300 °C, nebulizer 30 psi, mass range
20 µL H2O was added and the column centrifuged at 1000×g 100–3000 Da, acquisition rate of ∼1.03 spectra s−1, fragmentor
for 3 minutes. Approximately 98% of the linker addition pro- 175 V, skimmer 63 V, capillary 3200 V, instrument state 4 GHz
ducts 37 and 38 were recovered, as determined by the UV High Resolution. Mass correction was performed for every
absorption at 280 nm.
individual spectrum using peaks at m/z 121.0509 and 922.0098
2200 | Org. Biomol. Chem., 2014, 12, 2193–2213
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from the reference solution. Data acquisition was performed Experimental procedures
using the Mass Hunter software package (ver. B.02.01.) Analy-
α-D-Mannopyranosyl-(1→2)-α-D-mannopyranosyl-(1→2)-α-D-
sis of the HPLC-UV-MS data was done using the Agilent Mass mannopyranosyl-(1→3)-1-thio-β-D-mannopyranose 1. Tetrasac-
Hunter Qualitative Analysis software (ver. B.04.01).
charide 27 (13.2 mg, 6.7 µmol) was dissolved in deuterated
sodium hydroxide (1 mL, 0.5 M) and the reaction monitored
by 1H NMR. After 2 hours, the reaction was neutralized with
Analysis of LV-UV-MS chromatograms
Peak regions within the TIC were extracted and deconvoluted H+ resin, filtered, and concentrated under reduced pressure.
using the Qualitative Analysis with BioConfirm software that is The reaction was dissolved in H2O and lyophilized to remove
part of the Agilent MassHunter Workstation. The resulting methyl benzoate. The crude powder was dissolved in deuter-
peaks were matched with theoretical masses for each analyte. ated sodium hydroxide (1 mL, 1 M) to remove the tenacious
Miscellaneous peaks due to side reactions were also investi- 2-O-Bz. After 2 hours, the reaction was neutralized with H+
gated. In-depth elution profiles for each analyte were then con- resin, filtered and lyophilized. Purification of the crude yellow
structed using extracted-ion chromatograms (EICs) generated product via HPLC to yield 1 (3.2 mg, 4.7 µmol, 70%) as a white
from the most intense isotope of the most intense charge state powder.
of each analyte. Since all other isotope peaks and other charge
1H NMR (700 MHz, D2O): δ 5.34 (br. s., 1H, H-1), 5.30 (s,
states were ignored, peak intensities and peak areas do not 1H, H-1), 5.05 (s, 1H, H-1), 4.99 (s, 1H, H-1a), 4.11 (br. s., 1H,
represent a real relative abundance of analytes in the sample, H-2), 4.08 (br. s., 1H, H-2), 4.07 (br. s., 1H, H-2), 3.26–4.04 (m,
therefore quantitation was performed solely using the corres- 20H), 3.37–3.44 (m, 1H, H-5a); coupled HSQC (700 MHz, D2O):
ponding UV spectra. For illustrative purposes, signals for the δ 103.1/5.05 (JC1/H1 172 Hz), 101.7/5.30 (JC1/H1 173 Hz), 101.5/
least abundant analytes were magnified by 10–30%.
UV chromatograms were exported in excel format by use of C24H42O20S: HR ESIMS [M + Na]+: 705.1882, found: 705.1881.
the Agilent MassHunter Workstation before being imported α-D-Mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→3)]-
5.33 (JC1/H1 174 Hz), 82.5/5.00 (JC1a/H1a 153 Hz); anal. calc. for
into the PeakFit software (Version 4.12; SeaSolve Software, San α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-(1→3)]-1-
Jose, USA) for deconvolution and processing. Peak simulations thio-β-D-mannopyranoside 2. Pentasaccharide 28 (54.6 mg,
were obtained by use of the 4-parameter exponentially modi- 22.6 µmol) was dissolved in deuterated sodium hydroxide
fied Gaussian function of the PeakFit software. Peaks were (1 mL, 0.5 M) and the reaction monitored by 1H NMR. After
then manually adjusted to include additional peaks in areas of 2 hours, the reaction was neutralized with H+ resin, filtered,
significant overlap as identified in the EIC elution profiles. and concentrated under reduced pressure. The reaction was
Fitting was iterated until the correlation reached a stable dissolved in H2O and lyophilized to remove methyl benzoate.
maximum value. The integration results were used to deter- The crude powder was dissolved in deuterated sodium hydrox-
mine the relative abundance of differentially conjugated ide (1 mL, 1 M) to remove the tenacious 2-O-Bz. After 2 hours,
ubiquitin.
the reaction was neutralized with H+ resin, filtered and lyophi-
lized. Purification of the crude yellow product via HPLC to
yield 2 (12.1 mg, 14.2 µmol, 63%) as a white powder.
Procedure for ELISA analysis
Maxisorp 96-well plates (Nunc) were incubated overnight at
1H NMR (700 MHz, D2O): δ 5.15 (s, 1H, H-1), 5.10 (s, 1H,
4 °C with 10 µg per well of ubiquitin or ubiquitin conjugate in H-1), 5.00 (s, 1H, H-1a), 4.92 (d, 1H, J1,2 1.5 Hz, H-1), 4.87 (d,
PBS. The plates were washed (Molecular Devices Skan Washer 1H, J1,2 1.5 Hz, H-1), 4.16 (dd, 1H, J2,3 3.2 Hz, J1,2 1.8 Hz, H-2),
400) 5 times with PBS containing 0.05% Tween-20 (PBST). A 3.64–4.10 (m, 29H); coupled HSQC (700 MHz, D2O): δ 103.3/
1% solution of BSA in PBS was used to block plates by incubat- 5.10 (JC1/H1 172 Hz), 103.0/5.15 (JC1/H1 171 Hz), 100.4/4.87
ing for 30 m at room temperature. The plates were then (JC1/H1 172 Hz), 101.1/4.92 (JC1/H1 172 Hz), 92.1/5.00 (JC1a/H1a
washed 5 times with PBST. 2G12 antibody (produced in CHO 158 Hz); anal. calc. for C30H52O25S: HR ESIMS [M + Na]+:
cells, NIH AIDS Reagent Program Cat No: 1476) or biotinylated 867.2411, found: 867.2411.
ConA were then titrated against ubiquitin or ubiquitin conju-
2-O-Acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-
pffiffiffiffiffi
gates using 10 serial dilutions. After 2 h incubation at room O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-
temperature, the plate was washed 5 times with PBST and 100 µL O-benzoyl-α-D-mannopyranosyl trichloroacetimidate 3. Trisac-
per well of goat anti-human IgG conjugated to horseradish charide 24 (0.73 g, 0.5 mmol) was dissolved in a solution of
peroxidase (HRP) (Kirkegaard & Perry Laboratories, 1 : 5000 NaOAc (0.5 g, 6 mmol) in AcOH (6.65 mL) and H2O (0.35 mL).
dilution in PBST) for 2G12 or streptavidin–HRP conjugate PdCl2 (0.54 g, 3.0 mmol) was added and the reaction mixture
diluted 1 : 20 000 for biotinylated ConA added. After 40 min stirred for 10 hours. Palladium black precipitated from the
incubation, plates were washed 5 times with PBST and develo- solution over the course of the reaction. The reaction mixture
ped for 15 minutes at room temperature with 100 µL per well was poured into saturated NaHCO3 and the aqueous layer was
of a 1 : 1 mixture of 3,3′,5,5′-tetramethylbenzidine (0.4 g L−1
)
extracted with EtOAc three times. The combined organic
and 0.002% H2O2 solution (Kirkegaard & Perry Laboratories). phases were dried over Na2SO4 and concentrated under
Reaction was quenched by addition 100 µL of 1 M phosphoric reduced pressure. Purification of the crude product by chrom-
acid per well. Absorbance was read at 450 nm (Molecular atography (8.5 : 1.5 toluene–EtOAc) yielded trisaccharide 25 as
Devices Spectra Max 190 plate reader).
a white powder that was inseparable from a trisaccharide
This journal is © The Royal Society of Chemistry 2014
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Organic & Biomolecular Chemistry
byproduct. Trisaccharide 25 (∼0.60 g), was dissolved in a solu-
Mecapto-tert-butyl 2,3,4,6-tetra-O-acetyl-1-thio-β-D-manno-
tion of trichloroacetonitrile (0.5 mL, 5.0 mmol) in CH2Cl2 pyranoside 8. Monosaccharide 628,29 (17.9 g, 44.1 mmol) was
(3 mL) and a drop of DBU added. After 24 hours, the reaction added to a solution of 7 (13.6 g, 46.3 mmol) in CH2Cl2
mixture was diluted with toluene and concentrated under (300 mL) and cooled to 0 °C. Diethylamine (4.8 mL, 46 mmol)
reduced pressure. Purification of the crude syrup by chromato- was added to the reaction mixture. After 96 hours, reaction
graphy (8 : 2 toluene–EtOAc) yielded 3 (0.41 g, 0.26 mmol, 52% was diluted with CH2Cl2 and washed with sat. NH4Cl, sat.
from 23) as a white foam.
NaHCO3, H2O, and brine. The organic was dried over Na2SO4
[α]2D5 +6 (c 8.1, CH2Cl2); H NMR (600 MHz, CDCl3): δ 9.00 and concentrated under reduced pressure. Purification of the
(s, 1H, NH), 7.27–8.28 (m, 40H, ArH), 6.56 (d, 1H, J1,2 1.4 Hz, crude product required two rounds of chromatographic purifi-
H-1a), 5.95 (dd, 1H, J3,4 ≈ J4,5 10.1 Hz, H-4a), 5.90 (dd, 1H, cation (6 : 4 hexanes–EtOAc, then 9 : 1 CH2Cl2–EtOAc). The
J3,4 ≈ J4,5 10.1 Hz H-4b), 5.87 (dd, 1H, J2,3 3.3 Hz, J1,2 2.0 Hz, bulk of the diisopropyl hydrazodicarboxylate byproduct can be
H-2a), 5.84 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4c), 5.81 (dd, 1H, J3,4 removed through recrystallization (hexanes–EtOAc), yielding 8
10.0 Hz, J2,3 3.3 Hz, H-3b), 5.59 (dd, 1H, J3,4 9.8 Hz, J2,3 3.3 Hz, (13.1 g, 29.0 mmol, 66%) as a colorless syrup in a 1 : 22 α/β
1
H-3c), 5.46 (dd, 1H, J2,3 3.2 Hz, J1,2 1.8 Hz, H-2b), 5.24 (d, 1H, ratio, containing trace diisopropyl hydrazodicarboxylate.
J2,3 1.7 Hz, H-1c), 5.17 (dd, 1H, J2,3 2.9 Hz, J1,2 2.3 Hz, H-2c),
[α]2D5 +31 (c 24.3, CHCl3); H NMR (700 MHz, CDCl3): δ 5.62
1
4.95 (d, 1H, J1,2 1.4 Hz, H-1b), 4.65 (dd, 1H, J3,4 9.7 Hz, J2,3 (br. d, 1H, J2,3 2.6 Hz, H-2), 5.20 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz,
3.4 Hz, H-3a), 4.42–4.50 (m, 5H, H-5a, H-5b, H-5c, H-6b, H-6c), H-4), 5.03 (dd, 1H, J3,4 10.1 Hz, J2,3 3.5 Hz, H-3), 4.65 (d, 1H,
4.38 (dd, 1H, Jgem 12.2 Hz, J5,6 3.2 Hz, H-6b), 4.36 (dd, 1H, J1,2 0.9 Hz, H-1), 4.25 (dd, 1H, Jgem 12.3 Hz, J5,6 6.1 Hz, H-6),
Jgem 12.4 Hz, J5,6 5.2 Hz, H-6c), 4.09 (dd, 1H, Jgem 10.9 Hz, 4.14 (dd, 1H, Jgem 12.3 Hz, J5,6 2.3 Hz, H-6), 3.67 (ddd, 1H, J4,5
J5,6 6.2 Hz, H-6a), 3.78 (dd, 1H, Jgem 10.9 Hz, J5,6 1.9 Hz, H-6a), 9.9 Hz, J5,6 6.2 Hz, J5,6 2.3 Hz, H-5), 2.19 (s, 3H, CH3(CvO)),
2.10 (s, 3H, CH3(CvO)), 1.91 (s, 3H, CH3(CvO)); 13C NMR 2.07 (s, 3H, CH3(CvO)), 2.03 (s, 3H, CH3(CvO)), 1.97 (s, 3H,
(151 MHz, CDCl3): δ 169.7 (CvO), 169.0 (CvO), 166.0 (2 × CH3(CvO)), 1.34 (s, 9H, C(CH3)3); 13C NMR (176 MHz, CDCl3):
CvO), 165.9 (CvO), 165.6 (CvO), 165.4 (CvO), 165.2 (CvO), δ 170.6 (CvO), 170.0 (CvO), 170.0 (CvO), 169.6 (CvO), 92.1
165.0 (CvO), 164.7 (CvO), 159.5 (CvNH), 133.8 (Ar), 133.6 (C-1), 76.9 (C-5), 71.7 (C-3), 70.5 (C-2), 65.6 (C-4), 62.8 (C-6),
(Ar), 133.3 (Ar), 133.3 (Ar), 133.1 (Ar), 133.0 (Ar), 133.0 (Ar), 47.8 (C(CH3)3), 29.9 (C(CH3)3), 20.7 (CH3(CvO)), 20.7
132.9 (Ar), 130.2 (Ar), 130.0 (Ar), 129.9 (Ar), 129.8 (Ar), 129.8 (CH3(CvO)), 20.6 (CH3(CvO)), 20.5 (CH3(CvO)); anal. calc.
(Ar), 129.7 (Ar), 129.6 (Ar), 129.6 (Ar), 129.6 (Ar), 129.4 (Ar), for C18H28O9S2: HR ESIMS [M + Na]+: 475.1067, found:
129.3 (Ar), 129.2 (Ar), 128.9 (Ar), 128.9 (Ar), 128.8 (Ar), 128.7 475.1065; elem. Anal: C, 47.77; H, 6.24; S, 14.17; found: C,
(Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (Ar), 128.3 (Ar), 128.3 (Ar), 47.98; H, 6.21; S, 13.80.
128.3 (Ar), 128.2 (Ar), 99.7 (C-1b), 97.2 (C-1c), 94.3 (C-1a),
Mecapto-tert-butyl 1-thio-D-mannopyranoside 9. Monosac-
90.7 (CCl3), 76.1 (C-3a), 72.2 (C-2a), 70.5, 69.8, 69.8, 69.7, charide 8 (10.68 g, 23.6 mmol) was suspended in methanol
69.6, 69.1, 68.7, 67.8 (C-2b, C-2c, C-3b, C-3c, C-4a, C-5a, C-5b, (110 mL) and 1 M sodium methoxide (0.2 mL) was added.
C-5c), 66.9, 66.6, 66.3 (C-4b, C-4c, C-6a), 63.0, 62.7 (C-6b, C-6c), After 4 hours, reaction was neutralized with H+ resin, filtered,
20.7 (CH3(CvO)), 20.4 (CH3(CvO)); anal. calc. for and concentrated under reduced pressure. Purification of the
C80H68O26NCl3: HR ESIMS [M + Na]+: 1586.2987, found: crude product by recrystallization (hexanes–EtOAc) yielded 9
1586.3012; elem. Anal: C, 61.37; H, 4.38; N, 0.89; found: (6.13 g, 21.5 mmol, 91%) in a 64 : 100 α/β mixture as a white
C, 61.19; H, 4.54; N, 1.05.
powder.
β-Anomer. 1H NMR (600 MHz, CD3OD): δ 4.59 (d, 1H, J1,2 1.1
Diisopropyl-N-(tert-butylsulfanyl)hydrazodicarboxylate 7. To
a solution of 2-methyl-2-propanethiol (5.5 mL, 48.8 mmol) in
CH2Cl2 (150 mL) was added diisopropyl azodicarboxylate
(10.2 mL, 49.2 mmol) dropwise. The reaction orange mixture
gradually turned yellow over the course of the reaction. After 1
week, the reaction mixture was concentrated under reduced
pressure and the yellow syrup purified by chromatography
(8 : 2 hexanes–EtOAc) to yield 15 (13.6 g, 46.3 mmol, 94%) as a
colorless syrup.
Hz, H-1), 4.05 (dd, 1H, J2,3 3.4 Hz, J1,2 1.0 Hz, H-2), 3.85 (dd,
1H, Jgem 11.9 Hz, J5,6 2.4 Hz, H-6), 3.74 (dd, 1H, Jgem 11.7 Hz,
J5,6 5.5 Hz, H-6), 3.61 (dd, 1H, J3,4 ≈ J4,5 9.5 Hz, H-4), 3.45 (dd,
1H, J3,4 9.5, J2,3 3.3 Hz, H-3), 3.22 (ddd, 1H, J4,5 9.7 Hz, J5,6 5.1
Hz, J5,6 2.4 Hz, H-5), 1.36 (s, 9H, C(CH3)3); 13C NMR (126 MHz,
CD3OD): δ 94.4 (C-1), 81.4 (H-5), 74.7 (C-3), 72.4 (C-2), 66.5
(C-4), 61.3 (C-6), 46.6 (SC(CH3)3), 28.9 (SC(CH3)3).
α-Anomer. 1H NMR (600 MHz, CD3OD): δ 5.10 (d, 1H, J1,2 1.8
Hz, H-1), 4.10 (dd, 1H, J2,3 3.1 Hz, J1,2 1.8 Hz, H-2), 3.80–3.83
(m, 1H, H-6), 3.75–3.79 (m, 1H, H-6), 3.73–3.76 (m, 1H, H-5),
3.72 (dd, 1H, J3,4 ≈ J4,5 9.7 Hz, H-4), 3.66 (m, 1H, H-3), 1.36 (s,
9H, C(CH3)3); anal. calc. for C10H20O5S2: HR ESIMS [M + Na]+:
307.0644, found: 307.0641; elem. Anal: C, 42.23; H, 7.09; S,
22.55; found: C, 42.07; H, 7.05; S, 22.54.
[α]2D5 −0.1 (c 16.0 in CHCl3); H NMR (700 MHz, CDCl3): δ
1
7.19–7.35 (br. d., 1H, NH), 4.90 (spt, 1H, J 6.2 Hz, CH(CH3)2),
4.86 (spt, 1H, J 6.1 Hz, CH(CH3)2), 1.27 (br. s., 9H, C(CH3)3),
1.21 (d, 3H, J 6.4 Hz, C(CH3)2), 1.19 (d, 3H, J 6.3 Hz, C(CH3)2);
13C NMR (126 MHz, CDCl3): δ 157.3 (CvO), 155.3 (CvO), 72.2
(OCH(CH3)2), 70.0 (OCH(CH3)2), 49.9 (SC(CH3)3), 28.8 (SC-
(CH3)3), 22.0 (OCH(CH3)2), 21.8 (OCH(CH3)2); anal. calc. for
C12H24N2O4S: HR ESIMS [M + Na]+: 315.1349, found: 315.1349;
elem. Anal: C, 49.29; H, 8.27; N, 9.58; S, 10.97; found: C, 48.69;
H, 7.61; N, 9.21; S, 10.56.
Mecapto-tert-butyl 3-O-thexyldimethylsilyl-1-thio-6-O-triphe-
nylmethyl-β-D-mannopyranoside 10. Tetrol
9
(0.34 g,
1.2 mmol) and TrCl (0.32 g, 1.5 mmol) were dissolved in pyri-
dine (10 mL) and stirred at room temperature. After 68 hours,
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solid NaHCO3 was added and the reaction stirred until bub- 7.40–7.45 (m, 8H, ArH), 7.38 (m, 2H, ArH) 7.14 (m, 6H, ArH),
bling ceased. The reaction mixture was diluted with EtOAc and 7.09 (m, 3H, ArH), 5.84 (d, 1H, J2,3 2.8 Hz, H-2), 5.62 (dd, 1H,
washed with 1 M NaHCO3, H2O, and brine. The organic was J3,4 ≈ J4,5 8.8 Hz, H-4), 4.81 (s, 1H, H-1), 4.03 (dd, 1H, J2,3 3.1
dried over Na2SO4 and concentrated under reduced pressure. Hz, J3,4 9.1 Hz, H-3), 3.73 (m, 1H, H-5), 3.30 (dd, 2H, H-6), 1.32
The crude product was dissolved in a solution of imidazole (spt, 1H, J 7.1 Hz, CH(CH3)2), 0.55 (m, 6H, C(CH3)2), 0.54 (m,
(38 mg, 0.56 mmol) and DMF (3 mL). A solution of TDSCl (64 6H, CH(CH3)2), 0.16 (s, 3H, SiCH3), −0.15 (s, 3H, SiCH3); 13C
µL, 0.33 mmol) in DMF (1 mL) was added dropwise to the reac- NMR (126 MHz, CDCl3): δ 165.8 (CvO), 164.7 (CvO), 143.7
tion mixture over 1 hour. After 24 hours, the reaction was (Ar), 130.1 (Ar), 130.0 (Ar), 128.7 (Ar), 128.4 (Ar), 127.9 (Ar),
diluted with EtOAc and washed with 1 M NaHCO3, H2O, and 127.6 (Ar), 127.3 (Ar), 93.1 (C-1), 86.1 (CPh3) 78.9 (C-5), 73.9
brine. The organic was dried over Na2SO4 and concentrated (C-2), 72.5 (C-3), 69.7 (C-4), 63.1 (C-6), 47.6 (SC(CH3)3), 33.8
under reduced pressure. Purification of the crude yellow (CH(CH3)2), 30.1 (SC(CH3)3), 24.6 (SiCH(CH3)2), 19.9 (CH-
product by chromatography (7 : 3 hexanes–EtOAc) yielded 10 (CH3)2), 19.7 (SiC(CH3)2), 18.3 (SiC(CH3)2), −2.5 (SiCH3), −3.0
(0.17 g, 0.26 mmol, 92%) in a 64 : 100 α/β mixture as a white (SiCH3); anal. calc. for C51H60O7S2Si: HR ESIMS [M + Na]+:
powder.
β-Anomer. Rf 0.53 (8 : 2 hexanes–EtOAc); H NMR (500 MHz,
899.3442, found: 899.3443.
α-Product of 12. Mercapto-tert-butyl 2,4-di-O-benzoyl-3-O-tert-
1
CDCl3): δ 7.43–7.47 (m, 6H, ArH), 7.28–7.33 (m, 6H, ArH), butyldimethylsilyl-1-thio-6-O-triphenylmethyl-α-D-mannopyra-
7.22–7.26 (m, 3H, ArH), 4.52 (dd, 1H, J1,2 ≈ 4J1,OH 1.5 Hz, H-1), noside: Rf 0.68 (toluene); 1H NMR (500 MHz, CDCl3): δ 8.18 (d,
4.03 (ddd, 1H, J2,3 3.6 Hz, J1,2 1.6 Hz, J2,OH 1.5 Hz, H-2), 3.66 2H, Jortho 8.4 Hz, ArH), 7.87 (d, 2H, Jortho 7.1 Hz, ArH), 7.61 (t,
(ddd, 1H, J3,4 ≈ J4,5 9.2 Hz, J4,OH 2.6 Hz, H-4), 3.57 (dd, 1H, J3,4 1H, Jortho 7.5 Hz, ArH), 7.57 (t, 1H, Jortho 7.5 Hz, ArH), 7.49 (t,
8.7 Hz, J2,3 3.6 Hz, H-3), 3.41–3.44 (m, 2H, H-6), 3.37 (m, 1H, 2H, Jortho 7.7 Hz, ArH), 7.38–7.45 (m, 8H, ArH), 7.10–7.15 (m,
4
H-5), 2.60 (dd, 1H, J1,OH ≈ J2,OH 1.6 Hz, 2-OH), 2.30 (d, 1H, J4, 6H, ArH), 7.06 (m, 3H, ArH), 5.81 (dd, 1H, J3,4 ≈ J4,5 9.7 Hz,
2.7, 4-OH), 1.64 (spt., 1H, J 7.1 Hz, CH(CH3)2), 1.35 (s, 9H, H-4), 5.63 (dd, 1H, J2,3 2.9 Hz, J1,2 2.0 Hz, H-2), 5.45 (d, 1H, J1,2
OH
C(CH3)3), 0.90 (d, 3H, J 6.8, CH(CH3)2), 0.89 (d, 3H, J 6.8 Hz, 1.6 Hz, H-1), 4.31 (m, 1H, H-5), 4.29 (m, 1H, H-3), 3.33 (dd,
CH(CH3)3), 0.86 (s, 6H, C(CH3)2), 0.18 (s, 3H, SiCH3), 0.17 (s, 1H, Jgem 10.4 Hz, J5,6 2.2 Hz, H-6), 3.22 (dd, 1H, Jgem 10.4 Hz,
3H, SiCH3); 13C NMR (126 MHz, CDCl3): δ 146.9 (Ar), 143.7 J5,6 4.4 Hz, H-6), 1.38 (spt, 1H, J 7.1 Hz, CH(CH3)2), 0.62 (m,
(Ar), 143.6 (Ar), 128.7 (Ar), 127.9 (Ar), 127.3 (Ar), 127.1 (Ar), 6H, C(CH3)2), 0.59 (m, 6H, CH(CH3)2), 0.11 (s, 3H, SiCH3),
93.5 (C-1), 82.0 (CPh3), 78.6 (C-5), 76.0 (C-2), 72.7 (C-3), 69.6 −0.11 (s, 3H, SiCH).
β-Product of 11. Mercapto-tert-butyl 2-O-benzoyl-3-O-tert-
butyldimethylsilyl-1-thio-6-O-triphenylmethyl-β-D-mannopyra-
noside: Rf 0.32 (toluene); 1H NMR (500 MHz, CDCl3): δ 8.11
(m, 2H, ArH), 7.52–7.60 (m, 7H, ArH), 7.43 (m, 2H, ArH),
7.31–7.37 (m, 6H, ArH), 7.24–7.30 (m, 3H, ArH), 5.75 (d, 1H,
(C-4), 64.9 (C-6), 47.5 (SC(CH3)3), 34.2 (CH(CH3)2), 30.1 (C-
(CH3)3), 25.0 (SiC(CH3)2), 20.5 (CH(CH3)2), 20.1 (CH(CH3)2),
18.7 (SiC(CH3)2), 18.5 (SiC(CH3)2), −2.5 (SiCH3), −2.7 (SiCH3);
anal. calc. for C37H52O5S2Si: HR ESIMS [M + Na]+: 691.2918,
found: 691.2923.
1
α-Anomer. Rf 0.50 (8 : 2 hexanes–EtOAc); H NMR (500 MHz, J1,2 2.9 Hz, H-2), 4.71 (s, 1H, H-1), 3.97 (dd, 1H, J3,4 ≈ J4,5 8.7
CDCl3): δ 7.41–7.52 (m, 6H, ArH), 7.27–7.33 (m, 3H, ArH), Hz, H-4), 3.72 (dd, 1H, J2,3 3.2 Hz, J3,4 9.1 Hz, H-3), 3.56–3.63
7.21–7.28 (m, 3H, ArH), 5.24 (d, 1H, J1,2 1.3 Hz, H-1), 4.03 (m, 1H, H-6), 3.44 (m, 1H, H-5), 3.42 (m, 1H, H–6), 1.96 (br. s,
(ddd, 1H, J2,3 3.3 Hz, J1,2 1.3 Hz, H-2), 3.97 (ddd, 1H, J5,6 9.4 1H, 4-OH), 1.51 (spt, 1H, J 6.8 Hz, CH(CH3)2), 1.37 (s, 9H,
Hz, J4,5 ≈ J5,6 4.6 Hz, H-5), 3.83 (dd, 1H, J3,4 8.8 Hz, J2,3 3.3 Hz, C(CH3)3), 0.75 (s, 3H, C(CH3)2), 0.74 (d, 3H, J 7.0 Hz, CH(CH3)2),
H-3), 3.73 (ddd, 1H, J4,5 9.2 Hz, J3,4 ≈ J4,OH 2.6 Hz, H-4), 3.40 0.74 (d, 3H, J 7.0 Hz, C(CH3)2), 0.72 (m, 6H, CH(CH3)2), 0.21
(br. d, 2H, J 4.6 Hz, H-6), 2.67 (d, 1H, J1,2 1.3 Hz, 2-OH), 2.20 (s, 3H, SiCH3), 0.14 (s, 3H, SiCH3).
α-Product of 11. Mercapto-tert-butyl 2-O-benzoyl-3-O-tert-
butyldimethylsilyl-1-thio-6-O-triphenylmethyl-α-D-mannopyra-
noside: Rf 0.47 (toluene); 1H NMR (500 MHz, CDCl3): δ 8.07
(m, 2H, ArH), 7.58 (m, 1H, ArH), 7.52 (m, 6H, ArH), 7.38–7.48
(m, 2H, ArH), 7.22–7.31 (m, 9H, ArH), 5.53 (dd, 1H, J1,2 3.1 Hz,
J2,3 1.8 Hz, H-2), 5.36 (d, 1H, J1,2 1.5 Hz, H-1), 4.10 (ddd, 1H,
J3,4 ≈ J4,5 9.3 Hz, J4,OH 2.7 Hz, H-4), 4.02 (m, 1H, H-5), 3.99 (m,
1H, H-3), 3.53 (dd, 1H, Jgem 10.3 Hz, J5,6 2.9 Hz, H-6), 3.40 (dd,
1H, Jgem 10.1 Hz, J5,6 3.8 Hz, H-6), 1.95 (d, 1H, J4,OH 2.7, 4-OH),
1.52 (spt, 1H, J 6.8 Hz, CH(CH3)2), 1.38 (s, 9H, C(CH3)3), 0.76
(s, 3H, C(CH3)2), 0.74 (s, 3H, C(CH3)2), 0.74 (d, 6H, J 6.4, CH-
(CH3)2), 0.15 (s, 6H, Si(CH3)2).
(d, 1H, J4,OH 2.9 Hz, 4-OH), 1.65 (spt, 1H, J 7.3 Hz, CH(CH3)2),
1.37 (m, 6H, C(CH3)3), 0.90 (d, 3H, J 6.8 Hz, CH(CH3)2), 0.89 (d,
3H, J 6.8 Hz, CH(CH3)2), 0.87 (s, 6H, C(CH3)2), 0.18 (br. s., 6H,
Si(CH3)2).
Mercapto-tert-butyl 2,4-di-O-benzoyl-3-O-tert-butyldimethyl-
silyl-1-thio-6-O-triphenylmethyl-β-D-mannopyranoside 12. To a
solution of diol 10 (0.32 g, 0.48 mmol) in pyridine (5 mL) was
added benzoyl chloride (0.14 mL, 1.21 mmol) and 1-methyl-
imidazole (1 drop). The reaction was heated to 60 °C for
120 hours, cooled and diluted with EtOAc. The reaction
mixture was washed with sat. NaHCO3, H2O and brine. The
organic was dried over Na2SO4 and concentrated under
reduced pressure. Purification of the crude product by chrom-
atography (toluene) produced products 11 and 12 as an in-
separable mixture (0.37 g, 0.38 mmol, 80%).
Mecapto-tert-butyl
mannopyranoside 13. The anomeric mixture
6-O-tert-butyldiphenylsilyl-1-thio-β-D-
(2.38 g,
9
8.4 mmol) was dissolved in pyridine (40 mL) and TBDPSCl
(2.4 mL, 9.4 mmol) was slowly added. After 48 hours, the reac-
tion was diluted with toluene and extracted with 1 M NaHCO3,
Rf 0.65 (toluene); 1H NMR (500 MHz, CDCl3): δ 8.16 (m, 2H,
ArH), 7.79 (m, 2H, ArH), 7.58 (m, 1H, ArH), 7.54 (m, 1H, ArH),
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H2O, and brine. The organic layer was dried over Na2SO4 and 157 Hz, C-1a); anal. calc. for C55H62O14S2Si: HR ESIMS [M + Na]+:
concentrated under reduced pressure. Purification of the 1061.3246, found: 1061.3242; elem. Anal: C, 63.56; H, 6.01; S,
crude product by chromatography (6 : 3 CH2Cl2–EtOAc) fol- 6.17; found: C, 63.56; H, 6.29; S, 6.17.
lowed by recrystallization (heptane–EtOAc) yielded the
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno-
β-product 13 (1.86 g, 3.57 mmol, 71%) a white crystalline solid. pyranosyl-(1→3)-1-thio-β-D-mannopyranoside 15. Disaccharide
[α]2D5 −35 (c 6.2, CH2Cl2); 1H NMR (498 MHz, CDCl3): δ 14 (0.21 g, 0.17 mmol) was dissolved in a pyridine (0.25 mL)
7.65–7.70 (m, 4H, ArH), 7.37–7.47 (m, 6H, ArH), 4.53 (s, 1H, and hydrogen fluoride in pyridine (25 µL, 70% HF) added. The
H-1), 4.20 (dd, 1H, J2,3 ≈ J2,OH 3.8 Hz, H-2), 3.96 (dd, 1H, Jgem reaction was performed in
a polypropylene flask. After
10.8 Hz, J5,6 4.8 Hz, H-6), 3.92 (dd, 1H, Jgem 10.8 Hz, J5,6 5.8 45 minutes, a suspension of CaCO3 in 1 M NaHCO3 (0.5 mL)
Hz, H-6), 3.87 (ddd, 2H, J3,4 ≈ J4,5 9.2 Hz, J4,OH 1.6 Hz, H-4), was added and the reaction mixture stirred for 30 minutes.
3.58 (ddd, 1H, J3,4 9.3 Hz, J3,OH 6.2 Hz, J2,3 3.3 Hz, H-3), 3.36 The slurry was filtered through a pad of Celite and the eluted
(dt, 2H, J4,5 9.7 Hz, J5,6 5.0 Hz, H-5), 3.09 (d, 2H, J4,OH 1.8 Hz, solution concentrated under reduced pressure. Purification of
4-OH), 2.58 (d, 2H, J3,OH 6.4 Hz, 3-OH), 2.38 (d, 2H, J2,OH 4.8 the crude product by chromatography (9 : 1 toluene–EtOAc)
Hz, 2-OH), 1.30 (s, 9H, SC(CH3)3), 1.06 (s, 9H, SiC(CH3)3); 13C yielded 15 (0.16 g, 0.16 mmol, 93%) as a white solid.
NMR (126 MHz, CDCl3): δ 135.6 (Ar), 135.6 (Ar), 132.6 (Ar),
[α]2D5 +42 (c 11.8, CHCl3); 1H NMR (500 MHz, CDCl3): δ
132.5 (Ar), 130.0 (Ar), 127.9 (Ar), 92.3 (C-1), 78.7 (C-5), 74.9 8.01–8.05 (m, 2H, ArH), 7.89–7.93 (m, 2H, ArH), 7.76–7.81 (m,
(C-3), 71.4 (C-2), 70.3 (C-4), 65.1 (C-6), 47.7 (SC(CH3)3), 29.9 2H, ArH), 7.51–7.56 (m, 1H, ArH), 7.38–7.48 (m, 3H, ArH),
(SC(CH3)3), 26.8 (SiC(CH3)3), 19.2 (SiC(CH3)3); coupled HSQC 7.27–7.35 (m, 3H, ArH), 7.09–7.15 (m, 2H, ArH), 5.95 (dd, 1H,
(500 MHz, CDCl3): δ 92.3/4.53 (JC1/H1 158 Hz, C-1); anal. calc. J3,4 10.0 Hz, J2,3 3.3 Hz, H-3b), 5.92 (dd, 1H, J3,4 ≈ J4,5 9.5 Hz,
for C26H38O5S2Si: HR ESIMS [M + Na]+: 545.1822, found: H-4b), 5.64 (dd, 1H, J2,3 2.4 Hz, J1,2 1.8 Hz, H-2b), 5.45 (d, 1H,
545.1818; elem. Anal: C, 59.73; H, 7.33; S, 12.27; found: C, J1,2 1.6 Hz, H-1b), 4.76 (ddd, 1H, J4,5 8.9 Hz, J5,6 5.5 Hz, J5,6 3.1
59.12; H, 7.24; S, 12.34.
Hz, H-5b), 4.65 (br. s, 1H, 4-OHa), 4.56 (dd, 1H, Jgem 12.1 Hz,
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno- J5,6 3.1 Hz, H-6b), 4.52 (s, 1H, H-1a), 4.52 (dd, 1H, Jgem 12.3 Hz,
pyranosyl-(1→3)-6-O-tert-butyldiphenylsilyl-1-thio-β-D-manno- J5,6 5.5 Hz, H-6b), 4.39 (dd, 1H, J2,3 5.9 Hz, J2,OH 3.3 Hz, H-2a),
pyranoside 14. Acceptor 13 (0.32 g, 0.61 mmol) was dissolved 4.30 (ddd, 1H, J3,4 ≈ J4,5 4.8 Hz, J4,OH 9.5 Hz, H-4a), 4.16 (m,
in a solution of CH2Cl2 (8 mL) containing molecular sieves, 1H, 3-OHa), 3.98 (m, 2H, H-6a), 3.74 (dd, 1H, J3,4 9.3 Hz, J2,3 3.3
cooled to 0 °C, and BF3·OEt2 (12 µL, 96 µmol) added. A solu- Hz, H-3a), 3.61 (br. s, 1H, 6-OHa), 3.31 (ddd, 1H, J4,5 ≈ J5,6 9.7,
tion of donor 4 (0.42 g, 0.61 mmol) in CH2Cl2 (5 mL) was J5,6 2.6 Hz, H-5a), 2.11 (s, 3H, CH3(CvO)), 1.30 (s, 9H,
added dropwise over 30 minutes to the acceptor solution. After C(CH3)3); 13C NMR (126 MHz, CDCl3): δ 170.3 (CvO), 166.5
1 hour, the reaction was quenched with 3 drops of TEA and (CvO), 166.3 (CvO), 165.6 (CvO), 133.6 (Ar), 133.3 (Ar), 133.2
concentrated under reduced pressure. Purification of the (Ar), 129.8 (Ar), 129.7 (Ar), 129.7 (Ar), 129.6 (Ar), 128.8 (Ar),
crude product by chromatography (9 : 1 toluene–EtOAc) yielded 128.7 (Ar), 128.6 (Ar), 128.5 (Ar), 128.3 (Ar), 99.2 (C-1b), 93.2
14 (0.53 g, 0.51 mmol, 83%) as a white powder.
(C-1a), 82.7 (C-3a), 80.5 (C-5a), 72.4 (C-2a), 70.3, 70.1 (C-2b,
[α]2D5 +25 (c 2.0, CHCl3); 1H NMR (500 MHz, CDCl3): δ C-3b), 69.4 (C-5b), 66.9 (C-4b), 65.4 (C-4a), 63.7 (C-6b), 61.2
8.01–8.05 (m, 2H, ArH), 7.95–7.99 (m, 2H, ArH), 7.87–7.91 (m, (C-6a), 47.5 (C(CH3)3), 30.0 (C(CH3)3), 20.8 (CH3(CvO)); anal.
2H, ArH), 7.70 (m, 4H, ArH), 7.33–7.56 (m, 15H, ArH), 5.90 calc. for C39H44O14S2: HR ESIMS [M + Na]+: 823.2065, found:
(dd, 1H, J3,4 ≈ J4,5 10.1 Hz, H–4b), 5.82 (dd, 1H, J3,4 10.7 Hz, J2,3 823.2053; elem. Anal: C, 58.49 H, 5.54; S, 8.01; found: C, 57.97
3.7 Hz, H-3b), 5.62 (dd, 1H, J2,3 3.2 Hz, J1,2 1.9 Hz, H-2b), 5.34 H, 5.87; S, 7.75.
(d, 1H, J1,2 1.6 Hz, H-1b), 4.63 (ddd, 1H, J4,5 9.8 Hz, J5,6 5.6 Hz,
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno-
J5,6 3.1 Hz, H–5b), 4.59 (dd, 1H, Jgem 11.9 Hz, J5,6 2.9 Hz, H-6b), pyranosyl-(1→3)-2,4,6-tri-O-benzoyl-1-thio-β-D-mannopyrano-
4.53 (dd, 1H, Jgem 11.7 Hz, J5,6 5.7 Hz, H-6b), 4.47 (s, 1H, H-1a), side 16. Triol 15 (0.48 g, 0.60 mmol) was dissolved in pyridine
4.36 (dd, 1H, J2,OH 5.4 Hz, J2,3 3.4 Hz, H-2a), 4.12 (ddd, 1H, J3,4 (2.5 mL) and cooled to 0 °C (ice-water bath). Benzoyl chloride
≈ J4,5 9.2 Hz, J4,OH 2.6 Hz, H-4a), 3.94 (d, 2H, Jgem ≈ J5,6 4.8 Hz, (2.5 mL, 21.6 mmol) was added in portions and after 1 hour
H-6a), 3.67 (dd, 1H, J3,4 9.2 Hz, J2,3 3.2 Hz, H-3a), 3.34 (ddd, 1H, the reaction was removed from the ice bath. After 24 hours
J4,5 9.3 Hz, 2 × J5,6 4.6 Hz, H-5a), 2.90 (d, 1H, J4,OH 2.6 Hz, 4- total reaction time, the reaction mixture was diluted with
OH), 2.50 (d, 1H, J2,OH 5.5 Hz, 2-OH), 2.14 (s, 3H, CH3(CvO)), toluene and washed with 1 M NaOH, H2O, and brine. The
1.29 (s, 9H, SC(CH3)3), 1.08 (s, 9H, SiC(CH3)3); 13C NMR organic layer was dried over Na2SO4 and concentrated under
(126 MHz, CDCl3): δ 169.8 (CvO), 166.3 (CvO), 165.6 (CvO), reduced pressure. Purification of the crude product by chrom-
165.6 (CvO), 135.7 (Ar), 135.6 (Ar), 133.5 (Ar), 133.3 (Ar), 133.2 atography (9 : 1 toluene–EtOAc) yielded 16 (0.53 g, 0.47 mmol,
(Ar), 132.9 (Ar), 132.7 (Ar), 129.9 (Ar), 129.8 (Ar), 129.7 (Ar), 87%) as a white powder.
129.1 (Ar), 128.9 (Ar), 128.5 (Ar), 128.4 (Ar), 127.9 (Ar), 99.3
(C-1b), 92.7 (C-1a), 82.9(C-3a), 79.5 (C-5a), 71.7 (C-2a), 69.9, 69.8 7.25–8.26 (m, 30H, Ar), 6.04 (dd, 1H, J2,3 3.5 Hz, J1,2 1.0 Hz,
[α]2D5 −39 (c 3.9, CHCl3); 1H NMR (600 MHz, CDCl3): δ
(C-2b, C-3b), 69.4 (C-5b), 68.2 (C-4a), 67.2 (C-4b), 64.7 (C-6a), H-2a), 5.82 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4a), 5.79 (dd, 1H, J3,4
≈
63.7 (C-6b), 47.6 (SC(CH3)3), 30.0 (SC(CH3)3), 26.9 (SiC(CH3)3), J4,5 9.8 Hz, H-4b), 5.53 (dd, 1H, J3,4 9.7 Hz, J2,3 3.4 Hz, H-3b),
20.8 (CH3(CvO)), 19.3 (SiC(CH3)3); coupled HSQC (500 MHz, 5.09 (dd, 1H, J2,3 3.4 Hz, J1,2 2.0 Hz, H-2b), 5.07 (d, 1H, J1,2
CDCl3): δ 99.3/5.34 (JC1/H1 177 Hz, C-1b), 92.7/4.47 (JC1/H1 1.9 Hz, H-1b), 4.80 (d, 1H, J1,2 1.2 Hz, H-1a), 4.80 (ddd, 1H, J4,5
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9.8 Hz, J5,6 4.7 Hz, J5,6 2.8 Hz, H-5b), 4.68 (dd, 1H, Jgem 12.3 Hz, 0.72 mmol) was dissolved in a solution of pyridine (0.75 mL),
J5,6 2.5 Hz, H-6b), 4.68 (dd, 1H, Jgem 12.1 Hz, J5,6 2.5 Hz, H-6a), benzoyl chloride (0.25 mL, 2.16 mmol) and 1-methylimidazole
4.53 (dd, 1H, Jgem 12.2 Hz, J5,6 4.6 Hz, H-6b), 4.47 (dd, 1H, Jgem (1 drop). After 48 hours, the reaction was diluted with CH2Cl2
12.2 Hz, J5,6 5.3 Hz, H-6a), 4.33 (dd, 1H, J3,4 9.8 Hz, J2,3 3.5 Hz, and washed with 1 M NaOH, H2O and brine. The organic layer
H-3a), 3.99 (ddd, 1H, J4,5 10.0 Hz, J5,6 5.3 Hz, J5,6 2.9 Hz, H-5a), was dried over Na2SO4 and concentrated under reduced
1.78 (s, 3H, CH3(CvO)), 1.32 (s, 9H, C(CH3)3); 13C NMR pressure. Purification of the crude product by chromatography
(126 MHz, CDCl3): δ 166.6 (CvO), 166.5 (CvO), 166.4 (CvO), (9.7 : 0.3 toluene–EtOAc) yielded 18 (0.69 g, 0.52 mmol, 72%)
165.5 (CvO), 164.9 (CvO), 133.79 (Ar), 133.77 (Ar), 133.70 as a white solid.
(Ar), 133.66 (Ar), 133.60 (Ar), 133.50 (Ar), 133.34 (Ar), 133.32
[α]2D5 −40 (c 8.1, CHCl3); 1H NMR (600 MHz, CDCl3): δ
(Ar), 133.26 (Ar), 133.22 (Ar), 133.21 (Ar), 130.63 (Ar), 133.61 7.16–8.30 (m, 35H, ArH), 6.05 (d, 1H, J2,3 3.0 Hz, H-2a), 5.90
(Ar), 130.5 (Ar), 130.18 (Ar), 130.10 (Ar), 130.08 (Ar), 130.06 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4a), 5.80 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz,
(Ar), 129.99 (Ar), 129.95 (Ar), 129.2 (Ar) 128.8 (Ar) 128.61 (Ar), H-4b), 5.55 (dd, 1H, J3,4 9.7 Hz, J2,3 3.3 Hz, H-3b), 5.12 (dd, 1H,
127.8 (Ar), 99.4 (H-1b), 92.6 (H-1a), 77.3 (H-3a), 76.9 (H-5a), 72.9 J2,3 3.0 Hz, J1,2 2.1 Hz, H-2b), 5.01 (d, 1H, J1,2 1.4 Hz, H-1b),
(H-2a), 69.9 (H-5b), 69.5 (H-2b), 69.27 (H-3b), 69.21 (H-4a), 67.2 4.81 (m, 1H, H-5b), 4.79 (s, 1H, H-1a), 4.69 (dd, 1H, Jgem 12.2
(H-4b), 63.52 (H-6a), 63.4 (H-6b), 48.0 (C(CH3)3), 30.1 (C(CH3)3), Hz, J5,6 2.4 Hz, H-6b), 4.52 (dd, 1H, Jgem 12.2 Hz, J5,6 4.4 Hz,
20.4 (CH3(CvO)); anal. calc. for C60H56O17S2: HR ESIMS [M + H-6b), 4.28 (dd, 1H, J3,4 9.9 Hz, J2,3 3.4 Hz, H-3a), 3.86 (m, 2H,
Na]+: 1135.2851, found: 1135.2847; elem. Anal: C, 64.74 H, H-6a), 3.68 (ddd, 1H, J4,5 9.9 Hz, 2 × J5,6 2.9 Hz, H-5a), 1.79 (s,
5.07; S, 5.76; found: C, 62.62 H, 5.02; S, 5.49.
3H, CH3(CvO)), 1.36 (s, 9H, SC(CH3)3), 1.08 (s, 9H, SiC(CH3)3);
Mecapto-tert-butyl 3,4,6-tri-O-benzoyl-α-D-mannopyranosyl- 13C NMR (151 MHz, CDCl3): δ 168.7 (CvO), 166.2 (CvO),
(1→3)-2,4,6-tri-O-benzoyl-1-thio-β-D-mannopyranoside 17. Tri- 166.2 (CvO), 165.4 (CvO), 164.7 (CvO), 164.5 (CvO), 135.8
saccharide 16 (0.36 g, 0.32 mmol) was dissolved in CH2Cl2 (Ar), 135.6 (Ar), 133.4 (Ar), 133.3 (Ar), 133.1 (Ar), 133.1 (Ar),
(1.5 mL) and MeOH (2.5 mL) and cooled to −10 °C (HAAKE 133.0 (Ar), 132.8 (Ar), 130.4 (Ar), 130.0 (Ar), 129.9 (Ar), 129.7
Fisons chiller).
A solution of acetyl chloride (0.25 mL, (Ar), 129.5 (Ar), 129.5 (Ar), 129.4 (Ar), 129.3 (Ar), 129.2 (Ar),
2.9 mmol) in MeOH (1 mL) was added dropwise. The reaction 129.1 (Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2
was stirred at −10 °C for 170 hours, and quenched with 1 M (Ar), 127.6 (Ar), 127.5 (Ar), 99.1 (C-1b), 92.4 (C-1a), 79.7 (C-5a),
NaHCO3. The reaction mixture was extracted three times with 77.5 (C-3a), 72.7 (C-2a), 69.6, 69.5, 68.9, 68.4 (C-4a, C-2b, C-3b,
EtOAc and the combined organic layer concentrated under C-5b), 67.0 (C-4b), 63.1 (C-6b), 62.6 (C-6a), 47.7 (SC(CH3)3), 30.0
reduced pressure. Purification of crude product by chromato- (SC(CH3)3), 26.7 (SiC(CH3)3), 20.3 (CH3(CvO)), 19.2 (SiC-
graphy (9.4 : 0.6 toluene–EtOAc) yielded 17 (0.27 g, 0.25 mmol, (CH3)3); anal. calc. for C69H70O16S2Si: HR ESIMS [M + Na]+:
78%) as a white powder.
[α]2D5 −27 (c 3.0, CHCl3); 1H NMR (500 MHz, CDCl3): 5.14; found: C, 66.10; H, 5.61; S, 5.09.
δ 7.28–8.27 (m, 30H, Ar), 6.05 (dd, 1H, J2,OH 3.4, J1,2 1.0 Hz, Mercapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-man-
H-2a), 5.85 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4b), 5.80 (dd, 1H, J3,4
nopyranosyl-(1→3)-2,4-di-O-benzoyl-1-thio-β-D-mannopyranose
1269.3767, found: 1269.3765; elem. Anal: C, 66.43 H, 5.66; S,
≈
J4,5 9.8 Hz, H-4a), 5.43 (dd, 1H, J3,4 9.5 Hz, J2,3 3.1 Hz, H-3b), 19. Disaccharide 18 (0.21 g, 0.17 mmol) was dissolved in a pyri-
5.11 (d, 1H, J1,2 1.8 Hz, H-1b), 4.87 (ddd, 1H, J4,5 9.9 Hz, J5,6 dine (0.25 mL) and hydrogen fluoride in pyridine (25 µL, 70%
4.6 Hz, J5,6 2.8 Hz, H-5b), 4.82 (d, 1H, J1,2 1.1 Hz, H-1a), 4.66 HF) added. The reaction was performed in a polypropylene
(dd, 1H, Jgem 12.2 Hz, J5,6 2.7 Hz, H-6b), 4.66 (dd, 2H, Jgem flask. After 45 minutes, a suspension of CaCO3 in sat. NaHCO3
12.2 Hz, J5,6 2.8 Hz, H-6a), 4.56 (dd, 1H, Jgem 12.3 Hz, J5,6 (0.5 mL) was added and the reaction mixture stirred for
4.8 Hz, H-6b), 4.47 (dd, 4H, Jgem 12.3 Hz, J5,6 5.3 Hz, H-6a), 4.38 30 minutes. The slurry was filtered through a pad of Celite and
(dd, 1H, J3,4 9.8 Hz, J2,3 3.4 Hz, H-3a), 4.00 (ddd, 1H, J4,5 the eluted solution concentrated under reduced pressure. Puri-
9.9 Hz, J5,6 5.3 Hz, J5,6 2.9 Hz, H-5a), 3.95 (ddd, 1H, J2,OH fication of the crude product by chromatography (9 : 1
3.9 Hz, J2,3 3.2 Hz, J1,2 1.9 Hz, H-2b), 1.92 (d, 1H, J2,OH 4.2 Hz, toluene–EtOAc) yielded 19 (0.16 g, 0.16 mmol, 93%) as a white
2-OH), 1.32 (s, 9H, C(CH3)3); 13C NMR (126 MHz, CDCl3): solid.
δ 166.3 (CvO), 166.2 (CvO), 166.0 (CvO), 165.5 (CvO), 165.1
[α]2D5 −63 (c 7.2, CHCl3); 1H NMR (600 MHz, CDCl3): δ
(CvO), 164.8 (CvO), 133.7 (Ar), 133.5 (Ar), 133.3 (Ar), 133.1 7.25–8.28 (m, 25H, ArH), 6.05 (d, 1H, J2,3 3.2 Hz, H-2a), 5.82
(Ar), 133.1 (Ar), 133.0 (Ar), 130.3 (Ar), 129.9 (Ar), 129.8 (Ar), (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4b), 5.58 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz,
129.8 (Ar), 129.7 (Ar), 129.4 (Ar), 129.3 (Ar), 129.2 (Ar), 128.7 H-4a), 5.50 (dd, 1H, J3,4 9.6 Hz, J2,3 3.3 Hz, H-3b), 5.13 (d, 1H,
(Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3 (Ar), J1,2 1.5 Hz, H-1b), 5.09 (dd, 1H, J2,3 2.9 Hz, J1,2 2.1 Hz, H-2b),
101.3 (C-1b), 92.3 (C-1a), 77.2 (C-5a), 76.6 (C-3a), 72.8 (C-2a), 4.80 (s, 1H, H-1a), 4.77 (ddd, 1H, J4,5 9.9 Hz, J5,6 4.1 Hz, J5,6 3.0
71.8 (C-3b), 69.6 (C-5b), 69.3 (C-4a), 69.1 (C-2b), 66.8 (C-4b), 63.4 Hz, H-5b), 4.70 (dd, 1H, Jgem 12.2 Hz, J5,6 2.5 Hz, H-6b), 4.53
(C-6b), 63.3 (C-6a), 47.8 (C(CH3)3), 30.0 (C(CH3)3); anal. calc. (dd, 1H, Jgem 12.2 Hz, J5,6 4.4 Hz, H-6b), 4.36 (dd, 1H, J3,4 9.7
for C56H54O16S2: HR ESIMS [M + Na]+: 1093.2745, found: Hz, J2,3 3.5 Hz, H-3a), 3.84 (ddd, 1H, Jgem 12.7 Hz, J5,6 9.5 Hz,
1093.2742.
J5,6 2.1 Hz, H-6a), 3.78 (dd, 1H, J5,6 12.8 Hz, J5,6 4.9 Hz, H-6a),
Mercapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-man- 3.64 (ddd, 1H, J4,5 9.9 Hz, J5,6 4.7 Hz, J5,6 2.3 Hz, H-5a), 2.62
nopyranosyl-(1→3)-2,4-di-O-benzoyl-6-O-tert-butyldiphenylsi- (dd, 1H, Jgem 9.4 Hz, J5,6 5.0 Hz, 6-OH), 1.80 (s, 3H,
lyl-1-thio-β-D-mannopyranose 18. Disaccharide 14 (0.75 g, CH3(CvO)), 1.38 (s, 9H, SC(CH3)3); 13C NMR (151 MHz,
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CDCl3): δ 168.8 (CvO), 166.2 (CvO), 166.1 (CvO), 166.0
Mecapto-tert-butyl 3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-
(CvO), 165.4 (CvO), 164.6 (CvO), 133.7 (Ar), 133.6 (Ar), 133.4 (1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-tri-
(Ar), 133.1 (Ar), 132.9 (Ar), 130.3 (Ar), 129.9 (Ar), 129.7 (Ar), O-benzoyl-1-thio-β-D-mannopyranoside 21. Trisaccharide 20
129.5 (Ar), 129.3 (Ar), 129.1 (Ar), 129.0 (Ar), 128.7 (Ar), 128.5 (0.24 g, 0.15 mmol) was dissolved in CH2Cl2 (1 mL) and MeOH
(Ar), 128.5 (Ar), 128.4 (Ar), 128.2 (Ar), 99.2 (C-1b), 92.0 (C-1a), (1 mL) and cooled to −10 °C (HAAKE Fisons chiller). A solu-
79.5 (C-5a), 76.9 (C-3a), 72.5 (C-2a), 69.7, 69.4, 69.1, 68.9 (C-4a, tion of acetyl chloride (0.15 mL, 2.1 mmol) in MeOH (0.85 mL)
C-2b, C-3b, C-5b), 66.8 (C-4b), 63.1 (H-6b), 61.7 (H-6a), 47.8 (SC- was added dropwise. The reaction was stirred at −10 °C
(CH3)3), 29.9 (SC(CH3)3), 20.2 (CH3(CvO)O); anal. calc. for for
C53H52O16S2: HR ESIMS [M
Na]+: 1031.2589, found: reaction mixture was extracted three times with EtOAc and the
1031.2591; elem. Anal: C, 63.08; H, 5.19; S, 6.36; found: C, combined organic layer concentrated under reduced pressure.
63.07; H, 5.25; S, 6.35. Purification of crude product by chromatography (9.5 : 0.5
2 weeks, and quenched with 1 M NaHCO3. The
+
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno- toluene–EtOAc) yielded 21 (0.11 g, 71 mmol, 48%) as a white
pyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl- powder.
(1→3)-2,4,6-tri-O-benzoyl-1-thio-β-D-mannopyranoside
[α]2D5 −63 (c 10.4, CHCl3); 1H NMR (700 MHz, CDCl3): δ
20. Donor 4 (0.22 g, 0.33 mmol) and disaccharide acceptor 17 7.30–8.26 (m, 45H, Ar), 5.94 (dd, 1H, J2,3 3.4, J1,2 0.9 Hz, H-2a),
(0.27 g, 0.25 mol) were dissolved in dry CH2Cl2 (8 mL), acti- 5.88 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4b), 5.67 (dd, 1H, J3,4 ≈ J4,5
vated molecular sieves added, and TMSOTf (4.6 µL, 25 µmol) 9.9 Hz, H-4a), 5.65 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4c), 5.58 (dd,
was added at room temperature. After 1 hour the reaction was 1H, J3,4 9.9 Hz, J2,3 3.1 Hz, H-3c), 5.46 (dd, 1H, J3,4 9.9 Hz, J2,3
quenched with a drop of TEA and the reaction mixture concen- 3.4 Hz, H-3b), 5.36 (d, 1H, J1,2 1.5 Hz, H-1b), 4.86 (ddd, 1H, Jgem
trated under reduced pressure. The resulting slurry was puri- 10.0 Hz, J5,6 4.8 Hz, J5,6 2.8 Hz, H-5b), 4.69 (dd, 1H, Jgem 12.4
fied by chromatography (6 : 4 heptane–EtOAc) to yield 20 Hz, J5,6 4.9 Hz, H-6b), 4.62 (dd, 1H, Jgem 12.3 Hz, J5,6 2.5 Hz,
(0.38 g, 0.24 mmol, 95%) as a white powder.
H-6b), 4.59 (d, 1H, J1,2 1.0 Hz, H-1a), 4.57 (dd, 1H, Jgem 12.2 Hz,
[α]D25 −53 (c 6.0, CHCl3); 1H NMR (498 MHz, CDCl3): J5,6 3.0 Hz, H-6a), 4.39 (dd, 1H, Jgem 12.2 Hz, J5,6 5.2 Hz, H-6a),
δ 7.29–8.25 (m, 45H, Ar), 5.93 (dd, 1H, J2,3 3.5 Hz, J1,2 0.7 Hz, 4.36 (m, 1H, H-5c), 4.34 (m, 1H, H-6c), 4.18 (d, 1H, J1,2 1.4 Hz,
H-2a), 5.88 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4b), 5.71 (dd, 1H, J3,4 H-1c), 4.12 (m, 1H, H-6c), 4.09 (m, 1H, H-2c), 3.90 (dd, 1H, J2,3
9.9 Hz, J2,3 3.3 Hz, H-3c), 5.64 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4a), 2.8 Hz, J1,2 1.4 Hz, H-2b), 3.85 (dd, 1H, J3,4 9.7 Hz, J2,3 3.3 Hz,
5.61 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4c), 5.51 (dd, 1H, J3,4 9.9 Hz, H-3a), 3.56 (ddd, 1H, J4,5 9.8 Hz, J5,6 5.2 Hz, J5,6 3.1 Hz, H-5a),
J2,3 3.3 Hz, H-3b), 5.33 (d, 1H, J1,2 1.5 Hz, H-1b), 5.28 (dd, 1H, 1.65 (d, 1H, J2,OH 4.8 Hz, 2-OH), 1.35 (s, 9H, C(CH3)3); 13C NMR
J2,3 3.1 Hz, J1,2 2.2 Hz, H-2c), 4.87 (ddd, 1H, J4,5 9.8 Hz, J5,6 5.0 (126 MHz, CDCl3): δ 166.5 (CvO), 166.1 (CvO), 166.0 (CvO),
Hz, J5,6 2.7 Hz, H-5b), 4.71 (dd, 1H, Jgem 12.4 Hz, J5,6 5.2 Hz, 165.9 (CvO), 165.8 (CvO), 165.3 (CvO), 165.1 (CvO), 165.0
H-6b), 4.61 (dd, 1H, Jgem 12.3 Hz, J5,6 2.9 Hz, H-6b), 4.57 (d, 1H, (CvO), 164.9 (CvO), 133.8 (Ar), 133.5 (Ar), 133.4 (Ar), 133.4
J1,2 0.9 Hz, H-1a), 4.55 (dd, 1H, Jgem 12.1 Hz, J5,6 2.9 Hz, H-6a), (Ar), 133.3 (Ar), 133.2 (Ar), 133.1 (Ar), 133.0 (Ar), 133.0 (Ar),
4.37 (m, 1H, H-5c), 4.35 (m, 1H, H–6a), 4.34 (m, 1H, H-6c), 4.21 130.3 (Ar), 130.2 (Ar), 130.1 (Ar), 130.0 (Ar), 130.0 (Ar), 130.0
(d, 1H, J1,2 1.8 Hz, H-1c), 4.12 (m, 1H, H-6c), 3.87 (dd, 1H, J2,3 (Ar), 129.9 (Ar), 129.9 (Ar), 129.8 (Ar), 129.7 (Ar), 129.7 (Ar),
3.4 Hz, J1,2 1.9 Hz, H-2b), 3.83 (dd, 1H, J3,4 9.6 Hz, J2,3 3.4 Hz, 129.3 (Ar), 129.3 (Ar), 129.3 (Ar), 129.1 (Ar), 128.9 (Ar), 128.8
H-3a), 3.51 (ddd, 1H, J4,5 10.0 Hz, J5,6 5.3 Hz, J5,6 3.2 Hz, H-5a), (Ar), 128.7 (Ar), 128.7 (Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar),
1.95 (s, 3H, CH3(CvO)), 1.34 (s, 9H, C(CH3)3); 13C NMR 128.4 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar), 101.7 (C-1c), 100.1
(126 MHz, CDCl3): δ 169.1 (CvO), 166.30 (CvO), 166.29 (C-1b), 92.2 (C-1a), 78.5 (C-2b), 77.4 (C-3a), 76.4 (C-5a), 72.9
(CvO), 166.25 (CvO), 165.9 (CvO), 166.30 (CvO), 166.28 (C-2a), 72.1 (C-3c), 70.0 (C-3b), 69.7 (C-5c), 69.4 (C-5b), 69.1
(CvO), 166.26 (CvO), 166.24 (CvO), 165.15 (CvO), 133.8 (C-2c), 68.9 (C-4a), 67.2 (C-4b), 66.6 (C-4c), 64.1 (C-6b), 63.7
(Ar), 133.79 (Ar), 133.78 (Ar), 133.27 (Ar), 133.26 (Ar), 133.24 (C-6a), 63.3 (C-6c), 47.8 (C(CH3)3), 30.1 (C(CH3)3); anal. calc. for
(Ar), 133.0 (Ar), 132.78 (Ar), 132.73 (Ar), 130.63 (Ar), 130.61 C85H76O24S2: HR ESIMS [M
(Ar), 130.56 (Ar), 130.26 (Ar), 130.23 (Ar), 130.14 (Ar), 130.11 1567.4042.
+
Na]+: 1567.4060, found:
(Ar), 130.10 (Ar), 130.09 (Ar), 130.08 (Ar), 130.01 (Ar), 129.61
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno-
(Ar), 129.60 (Ar), 129.58 (Ar), 129.57 (Ar), 129.4 (Ar), 129.3 (Ar), pyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-
128.85 (Ar), 128.83 (Ar), 128.56 (Ar), 128.55 (Ar), 128.54 (Ar), (1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-tri-
128.3 (Ar), 102.8 (C-1b), 99.7 (C-1c), 92.5 (C-1a), 78.8 (C-2b), 77.4 O-benzoyl-1-thio-β-D-mannopyranoside 22. Donor 4 (45 mg,
(C-3a), 76.6 (C-5a), 73.0 (C-2a), 70.0 (C-5c), 69.8 (C-3b), 69.78 66 µmol) and trisaccharide acceptor 21 (79 mg, 51 µmol) were
(C-3c), 69.74 (C-2c), 69.6 (C-5b), 67.5 (C-4b), 5.65 (C-4a), 5.62 dissolved in dry CH2Cl2 (0.6 mL), activated molecular sieves
(C-4c), 64.53 (C-6b), 63.80 (C-6c), 63.5 (C-6a), 48.0 (C(CH3)3), added, and the reaction mixture cooled to 0 °C (ice water
30.3 (C(CH3)3), 20.7 (CH3(CvO)); coupled HSQC (700 MHz, bath). TMSOTf (1 µL, 5.5 µmol) was added and after 1.5 hours
D2O): δ 102.8/5.33 (JC1/H1 174 Hz, C-1b), 99.7/4.21 (JC1/H1 the reaction was quenched with a drop of TEA. The reaction
171 Hz, C-1c), 92.5/4.57 (JC1/H1 158 Hz, C-1a); anal. calc. for mixture was concentrated under reduced pressure and the
C87H78O25S2: HR ESIMS [M
1609.4160; elem. Anal: C, 65.82 H, 4.95; S, 4.04; found: C, EtOAc) to yield 22 (95 mg, 46 µmol, 91%) as a white crystalline
+
Na]+: 1609.4160, found: resulting slurry purified by chromatography (7 : 3 heptane–
65.51; H, 5.13; S, 3.92.
solid.
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[α]2D5 −32 (c 43.2, Benzene); 1H NMR (600 MHz, CDCl3): δ Hz, 2 × J 6.0 Hz, Hb), 5.91 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4c), 5.85
7.09–8.32 (m, 60H, Ar), 5.99 (d, 1H, J1,2 3.3 Hz, H-2a), 5.89 (dd, (dd, 1H, J3,4 10.3 Hz, J2,3 3.0 Hz, H-3c), 5.83 (dd, 1H, J3,4 ≈ J4,5
1H, J3,4 ≈ J4,5 9.9 Hz, H-4b), 5.80 (dd, 1H, J3,4 10.1 Hz, J2,3 2.9 10.1 Hz, H-4a), 5.79 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4b), 5.68 (dd,
Hz, H-3c), 5.76 (dd, 1H, J3,4 10.0 Hz, J2,3 3.4 Hz, H-3d), 5.74 (dd, 1H, J2,3 3.4 Hz, J1,2 1.6 Hz, H-2a), 5.57 (dd, 1H, J3,4 9.7 Hz, J2,3
1H, J3,4 ≈ J4,5 7.5 Hz, H-4c), 5.71 (dd, 1H, J3,4 ≈ J4,5 9.7 Hz, 3.3 Hz, H-3b), 5.50 (dd, 1H, J2,3 3.1 Hz, J1,2 1.8 Hz, H-2c), 5.43
4
H-4a), 5.68 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4d), 5.63 (dd, 1H, J2,3 (dq, 1H, Jtrans 17.2 Hz, Jgem ≈ 2 × J 1.3 Hz, Ha), 5.30 (dq, 1H,
4
3.2 Hz, J1,2 1.9 Hz, H-2d), 5.47 (dd, 1H, J3,4 9.9 Hz, J2,3 3.4 Hz, Jcis 10.4 Hz, Jgem ≈ 2 × J 1.2 Hz, Hc), 5.18 (d, 1H, J1,2 1.7 Hz,
H-3b), 5.37 (d, 1H, J1,2 1.2 Hz, H-1b), 4.89 (ddd, 1H, J4,5 10.0 H-1b), 5.13 (s, 1H, H-1a), 5.12 (dd, 1H, J2,3 2.8 Hz, J1,2 2.1 Hz,
Hz, J5,6 4.7 Hz, J5,6 2.6 Hz, H-5b), 4.86 (d, 1H, J1,2 1.3 Hz, H-1d), H-2b), 4.97 (d, 1H, J1,2 1.3 Hz, H-1c), 4.60 (dd, 1H, J3,4 9.7 Hz,
4.71 (dd, 1H, Jgem 12.1 Hz, J5,6 5.0 Hz, H-6b), 4.62 (s, 1H, H-1a), J2,3 3.4 Hz, H-3a), 4.52 (dd, 1H, Jgem 12.1 Hz, J5,6 2.5 Hz, H-6b),
4.62 (br. s., 1H, H-1c), 4.63 (dd, 1H, Jgem 12.3 Hz, J5,6 2.6 Hz, 4.49 (dd, 1H, Jgem 11.9 Hz, J5,6 2.5 Hz, H-6c), 4.45 (ddd, 1H,
H-6b), 4.58 (dd, 1H, Jgem 12.2 Hz, J5,6 2.9 Hz, H-6a), 4.40 (dd, Jgem 9.9 Hz, J5,6 5.2 Hz, J5,6 2.6 Hz, H-5c), 4.42 (ddd, 1H, Jgem
1H, Jgem 12.2 Hz, J5,6 5.2 Hz, H-6a), 4.33 (ddd, 1H, J4,5 9.9 Hz, 9.8 Hz, J5,6 4.3 Hz, J5,6 2.5 Hz, H-5b), 4.36 (dd, 1H, Jgem 12.0 Hz,
J5,6 6.0 Hz, J5,6 2.0 Hz, H-5c), 4.26 (d, 1H, J2,3 1.8 Hz, H-2c), 4.24 J5,6 4.1 Hz, H-6b), 4.36 (dd, 1H, Jgem 11.9 Hz, J5,6 5.2 Hz, H-6c),
3
4
(dd, 1H, Jgem 12.1 Hz, J5,6 6.0 Hz, H-6c), 4.13 (br. d, 1H, Jgem 4.34 (ddt, 1H, Jgem 12.8 Hz, J 5.3 Hz, 2 × J 1.2 Hz, Hd), 4.27
12.2 Hz, H-6c), 4.02 (m, 1H, H-5d), 4.00 (dd, 1H, J3,4 10.4 Hz, (ddd, 1H, J4,5 9.9 Hz, J5,6 6.4 Hz, J5,6 1.8 Hz, H-5a), 4.16 (ddt,
J2,3 3.3 Hz, H-3a), 3.98 (dd, 1H, Jgem 12.6 Hz, J5,6 3.8 Hz, H-6d), 1H, Jgem 12.8 Hz, 3J 6.2 Hz, 2 × 4J 1.1 Hz, Hd), 4.10 (dd, 1H,
3.93 (dd, 1H, Jgem 12.3 Hz, J5,6 3.5 Hz, H-6d), 3.90 (br. s., 1H, Jgem 10.6 Hz, J5,6 6.4 Hz, H-6a), 3.72 (dd, 1H, Jgem 10.6 Hz, J5,6
H-2b), 3.53 (ddd, 1H, J4,5 9.6 Hz, J5,6 5.0 Hz, J5,6 3.3 Hz, H-5a), 1.9 Hz, H-6a), 2.10 (s, 3H, CH3(CvO)), 1.88 (s, 3H, CH3(CvO));
2.00 (s, 3H, CH3(CvO)), 1.36 (s, 9H, C(CH3)3); 13C NMR 13C NMR (151 MHz, CDCl3): δ 169.7 (CvO), 169.0 (CvO),
(126 MHz, CDCl3): δ 169.0 (CvO), 166.5 (CvO), 166.1 (CvO), 166.1 (CvO), 166.1 (CvO), 166.0 (CvO), 165.5 (CvO), 165.5
166.1 (CvO), 165.9 (CvO), 165.7 (CvO), 165.7 (CvO), 165.4 (CvO), 165.2 (CvO), 165.1 (CvO), 164.6 (CvO), 133.5 (Ar),
(CvO), 165.2 (CvO), 165.2 (CvO), 165.1 (CvO), 165.0 (CvO), 133.4 (Ar), 133.4 (Ar), 133.2 (Ar), 133.1 (Ar), 133.1 (CHvCH2),
164.6 (CvO), 134.1 (Ar), 133.6 (Ar), 133.4 (Ar), 133.3 (Ar), 133.2 133.0 (Ar), 132.9 (Ar), 132.9 (Ar), 130.1 (Ar), 129.9 (Ar), 129.9
(Ar), 133.1 (Ar), 133.1 (Ar), 133.0 (Ar), 132.8 (Ar), 130.4 (Ar), (Ar), 129.9 (Ar), 129.8 (Ar), 129.8 (Ar), 129.6 (Ar), 129.6 (Ar),
130.3 (Ar), 130.0 (Ar), 130.0 (Ar), 130.0 (Ar), 129.8 (Ar), 129.8 129.6 (Ar), 129.3 (Ar), 129.3 (Ar), 129.2 (Ar), 129.0 (Ar), 128.9
(Ar), 129.7 (Ar), 129.7 (Ar), 129.7 (Ar), 129.4 (Ar), 129.4 (Ar), (Ar), 128.9 (Ar), 128.8 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3 (Ar),
129.3 (Ar), 129.3 (Ar), 128.9 (Ar), 128.9 (Ar), 128.7 (Ar), 128.7 128.2 (Ar), 118.7 (CHvCH2), 99.5 (C-1b), 97.3 (C-1c), 96.5
(Ar), 128.6 (Ar), 128.5 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), (C-1a), 76.3 (C-3a), 72.0 (C-2a), 69.83, 69.78, 69.71, 69.7, 69.6,
128.3 (Ar), 128.2 (Ar), 100.8 (C-1c), 100.0 (C-1b), 99.3 (C-1d), 69.1, 68.8, 68.7, 68.6 (C-2b, C-2c, C-3b, C-3c, C-4a, C-5a, C-5b,
92.3 (C-1a), 78.2 (C-2b), 76.6 (C-3a), 76.5 (C-5a), 75.1 (C-2c), 72.8 C-5c, CH2CHvCH2), 66.9, 66.8, 66.8 (C-4b, C-4c, C-6a), 63.1,
(C-2a), 71.0 (C-3c), 69.9 (C-5c), 69.8 (C-3b), 69.5 (C-2d), 69.4 63.0 (C-6b, C-6c), 20.7 (CH3(CvO)), 20.4 (CH3(CvO)); coupled
(C-5b), 69.3 (C-3d), 69.3 (C-5d), 68.9 (C-4a), 67.6 (C-4d), 67.4 HSQC (500 MHz, CDCl3): δ 99.7/5.18 (JC1/H1 171 Hz, C-1b),
(C-4b), 66.8 (C-4c), 64.2 (C-6a), 64.0 (C-6c), 63.3 (C-6a), 63.1 97.6/4.97 (JC1/H1 175 Hz, C-1c), 96.8/5.12 (JC1/H1 175 Hz, C-1a);
(C-6d), 47.8 (C(CH3)3), 30.1 (C(CH3)3), 20.6 (CH3(CvO)); anal. calc. for C87H72O26: HR ESIMS [M + Na]+: 1483.4204,
coupled HSQC (600 MHz, CDCl3): δ 100.8/4.62 (JC1/H1 171 Hz, found: 1483.4169.
C-1c), 100.0/5.37 (JC1/H1 175 Hz, C-1b), 99.2/4.86 (JC1/H1 175 Hz,
C-1d), 92.2/4.62 (JC1/H1 156 Hz, C-1a); anal. calc. for C114H100O33S2: pyranosyl-(1→6)-[2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyra-
HR ESIMS [M + Na]+: 2083.5480, found: 2083.5457.
nosyl-(1→3)]-2,4-di-O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-O-
Mecapto-tert-butyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-manno-
Allyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl- acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-
(1→6)-[2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl- benzoyl-1-thio-β-D-mannopyranoside 26. Trisaccharide donor
(1→3)]-2,4-di-O-benzoyl-α-D-mannopyranoside 24. Donor 4 3 (84.6 mg, 54 µmol) and disaccharide acceptor 19 (53 mg,
(1.55 g, 2.28 mmol) and acceptor 23 (0.47 g, 1.09 mol) were 53 µmol) were dissolved in dry CH2Cl2 (0.7 mL), activated mole-
dissolved in dry CH2Cl2 (18 mL), activated molecular sieves cular sieves added, and the reaction mixture cooled to 0 °C
added, and the reaction mixture cooled to 0 °C (ice water (ice water bath). TMSOTf (1 µL, 5.5 µmol) was added and after
bath). TMSOTf (14.5 µL, 73 µmol) was added and after 30 minutes the reaction was quenched with a drop of TEA.
30 minutes the reaction was removed from the ice bath. Reaction mixture was concentrated under reduced pressure
Additional TMSOTf (8 µL, 27 µmol) was added after 2 hours and the resulting slurry purified by chromatography (9 : 1
total reaction time and the reaction was quenched 30 minutes toluene–EtOAc) to yield 26 (0.119 g, 49 µmol, 93%) as a white
later with the addition of 3 drops of TEA. The reaction mixture glassy solid.
was concentrated under reduced pressure and the resulting
[α]2D5 −10 (c 9.1, CHCl3); 1H NMR (700 MHz, CDCl3): δ
slurry purified by chromatography (8 : 2 hexanes–EtOAc) to 7.24–8.27 (m, 65H, ArH), 6.08 (dd, 1H, J2,3 3.5 Hz, J1,2 1.1 Hz,
yield 24 (1.44 g, 0.99 mol, 93%) as a white glassy solid; Spectral H-2a), 5.95 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4c), 5.89 (dd, 1H, J3,4
data matches literature values.26
≈ J4,5 10.0 Hz, H-4e), 5.84 (dd, 1H, J3,4 10.4 Hz, J2,3 3.1 Hz,
[α]D25 −5 (c 4.3, CHCl3); 1H NMR (600 MHz, CDCl3): δ H-3e), 5.83 (dd, 1H, J3,4 ≈ J4,5 9.6 Hz, H-4d), 5.82 (dd, 1H, J3,4
7.26–8.25 (m, 40H, ArH), 5.95 (ddt, 1H, Jtrans 16.8 Hz, Jcis 10.7 J4,5 10.0 Hz, H-4b), 5.79 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4a), 5.76
≈
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(dd, 1H, J2,3 3.4 Hz, J1,2 1.6 Hz, H-2c), 5.59 (dd, 1H, J3,4 9.8 Hz,
[α]2D5 −45 (c 7.0, CHCl3); 1H NMR (700 MHz, CDCl3): δ
J2,3 3.3 Hz, H-3d), 5.54 (dd, 1H, J3,4 9.6 Hz, J2,3 2.9 Hz, H-3b), 7.06–8.34 (m, 60H, Ar), 5.84 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4b),
5.46 (dd, 1H, J2,3 3.1 Hz, J1,2 1.9 Hz, H-2e), 5.23 (d, 1H, J1,2 1.9 5.81 (dd, 1H, J2,3 3.4 Hz, J1,2 0.9 Hz, H-2a), 5.79 (dd, 1H, J3,4
Hz, H-1d), 5.18 (dd, 1H, J2,3 3.1 Hz, J1,2 2.1 Hz, H-2d), 5.12 (d, 10.2 Hz, J2,3 3.0 Hz, H-3c), 5.78 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz,
1H, J1,2 1.2 Hz, H-1c), 5.10 (br. s, 1H, H-1b), 5.10 (dd, 1H, J2,3 H-4a), 5.75 (dd, 1H, J3,4 9.9 Hz, J2,3 3.4 Hz, H-3d), 5.75 (dd,
3.3 Hz, J1,2 1.9 Hz, H-2b), 4.83 (d, 1H, J1,2 1.2 Hz, H-1a), 4.80 (d, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4c), 5.67 (dd, 1H, J3,4 ≈ J4,5 10.1 Hz,
1H, J1,2 1.8 Hz, H-1e), 4.80 (ddd, 1H, J4,5 9.6 Hz, J5,6 4.0 Hz, J5,6 H-4d), 5.62 (dd, 1H, J2,3 3.3, J1,2 1.8 Hz, H-2d), 5.48 (dd, 1H,
3.0 Hz, H-5b), 4.72 (dd, 1H, Jgem 12.2 Hz, J5,6 2.6 Hz, H-6b), 4.64 J3,4 9.8 Hz, J2,3 3.3 Hz, H-3b), 5.35 (d, 1H, J1,2 1.5 Hz, H-1b),
(dd, 1H, J3,4 9.7 Hz, J2,3 3.5 Hz, H-3c), 4.52 (dd, 1H, Jgem 12.2 4.92 (ddd, 1H, J4,5 10.1 Hz, J5,6 5.8 Hz, J5,6 2.6 Hz, H-5b), 4.86
Hz, J5,6 4.4 Hz, H-6b), 4.51 (dd, 1H, Jgem 13.3 Hz, J5,6 4.0 Hz, (d, 1H, J1,2 1.5 Hz, H-1d), 4.73 (dd, 1H, Jgem 12.2 Hz, J5,6 5.8
H-6d), 4.38 (dd, 1H, Jgem 11.9 Hz, J5,6 2.4 Hz, H-6e), 4.36 (dd, Hz, H-6b), 4.67 (dd, 1H, J1,SH 10.1 Hz, J1,2 1.0 Hz, H-1a), 4.63
1H, J3,4 10.0 Hz, J2,3 3.7 Hz, H-3a), 4.34–4.37 (m, 2H, H-5d, (s, 1H, H-1c), 4.61 (dd, 1H, Jgem 9.2 Hz, J5,6 2.6 Hz, H-6b), 4.59
H-6a), 4.35 (ddd, 1H, J4,5 9.5 Hz, J5,6 4.8 Hz, J5,6 2.9 Hz, H-5e), (dd, 1H, Jgem 12.2 Hz, J5,6 3.1 Hz, H-6a), 4.34 (dd, 1H, Jgem
4.28 (dd, 1H, Jgem 11.8 Hz, J5,6 5.1 Hz, H-6e), 4.24 (ddd, 1H, 12.3 Hz, J5,6 4.4 Hz, H-6a), 4.29 (ddd, 1H, J4,5 9.8, J5,6 5.9 Hz,
J4,5 10.2 Hz, J5,6 5.1 Hz, J5,6 2.0 Hz, H-5c), 4.18 (dd, 1H, Jgem J5,6 2.2 Hz, H-5c), 4.25 (dd, 1H, J2,3 2.7, J1,2 2.1 Hz, H-2c), 4.20
11.1 Hz, J5,6 6.1 Hz, H-6a), 4.01 (dd, 1H, Jgem 10.8 Hz, J5,6 5.2 (dd, 1H, Jgem 12.1 Hz, J5,6 6.1 Hz, H-6c), 4.11 (br. d, 1H, Jgem
Hz, H-6c), 3.92 (ddd, 1H, J4,5 10.0 Hz, J5,6 6.2 Hz, J5,6 1.8 Hz, 11.9 Hz, H-6c), 4.08 (dd, 1H, J3,4 9.9 Hz, J2,3 3.3 Hz, H-3a), 4.02
H-5a), 3.75 (dd, 1H, Jgem 11.1 Hz, J5,6 1.6 Hz, H-6a), 3.49 (dd, (dt, 1H, J4,5 9.9 Hz, J5,6 4.0 Hz, H-5d), 3.96 (d, 2H, J5,6 3.2 Hz,
1H, Jgem 10.6 Hz, J5,6 1.9 Hz, H-6c), 2.10 (s, 3H, CH3(CvO)), H-6d), 3.88 (dd, 1H, J2,3 2.7 Hz, J1,2 1.5 Hz, H-2b), 3.57 (dt, 1H,
1.92 (s, 3H, CH3(CvO)), 1.77 (s, 3H, CH3(CvO)), 1.38 (s, 9H, J4,5 10.0 Hz, 2 × J5,6 3.7 Hz, H-5a), 2.59 (d, 1H, J1,SH 10.1 Hz,
C(CH3)3); 13C NMR (176 MHz, CDCl3): δ 169.5 (CvO), 168.9 SH), 1.99 (s, 3H, CH3(CvO)O); 13C NMR (126 MHz, CDCl3): δ
(CvO), 168.7 (CvO), 166.2 (CvO), 166.2 (CvO), 166.1 168.9 (CvO), 166.4 (CvO), 166.1 (CvO), 166.0 (CvO), 165.9
(CvO), 166.0 (CvO), 165.9 (CvO), 165.6 (CvO), 165.6 (CvO), 165.7 (CvO), 165.7 (CvO), 165.4 (CvO), 165.3
(CvO), 165.4 (CvO), 165.2 (2 × CvO), 165.1 (CvO), 164.6 (CvO), 165.1 (CvO), 165.1 (CvO), 165.0 (CvO), 164.7
(CvO), 164.6 (CvO), 133.5 (Ar), 133.4 (Ar), 133.3 (Ar), 133.1 (CvO), 134.1 (Ar), 133.8 (Ar), 133.4 (Ar), 133.3 (Ar), 133.3 (Ar),
(Ar), 133.1 (Ar), 133.0 (Ar), 133.0 (Ar), 132.9 (Ar), 132.9 (Ar), 133.2 (Ar), 133.2 (Ar), 133.1 (Ar), 133.1 (Ar), 133.0 (Ar), 133.0
130.2 (Ar), 130.2 (Ar), 130.0 (Ar), 130.0 (Ar), 130.0 (Ar), 129.9 (Ar), 132.9 (Ar), 132.9 (Ar), 132.8 (Ar), 130.3 (Ar), 130.3 (Ar),
(Ar), 129.8 (Ar), 129.8 (Ar), 129.7 (Ar), 129.7 (Ar), 129.7 (Ar), 130.0 (Ar), 130.0 (Ar), 130.0 (Ar), 130.0 (Ar), 129.9 (Ar), 129.9
129.6 (Ar), 129.5 (Ar), 129.5 (Ar), 129.4 (Ar), 129.3 (Ar), 129.2 (Ar), 129.9 (Ar), 129.8 (Ar), 129.8 (Ar), 129.7 (Ar), 129.7 (Ar),
(Ar), 129.2 (Ar), 129.1 (Ar), 129.1 (Ar), 128.9 (Ar), 128.9 (Ar), 129.7 (Ar), 129.4 (Ar), 129.4 (Ar), 129.3 (Ar), 129.3 (Ar), 129.3
128.7 (Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3 (Ar), 129.1 (Ar), 128.9 (Ar), 128.9 (Ar), 128.9 (Ar), 128.8 (Ar),
(Ar), 128.3 (Ar), 128.2 (Ar), 128.2 (Ar), 100.0 (C-1d), 99.3 (C-1b), 128.6 (Ar), 128.6 (Ar), 128.5 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4
97.6 (C-1c), 97.3 (C-1e), 92.0 (C-1a), 77.7, 77.4 (C-3c, C-5a), 77.4 (Ar), 128.4 (Ar), 128.3 (Ar), 128.3 (Ar), 128.2 (Ar), 100.7 (C-1c),
(C-3a), 72.6 (C-2a), 71.9 (C-2c), 70.1, 69.67, 2 × 69.67, 69.5, 2 × 100.0 (C-1b), 99.3 (C-1d), 78.1 (C-2b), 76.9 (C-1a), 76.7 (C-3a),
69.4, 69.3, 69.0, 68.7, 68.6 (C-4a, C-2b, C-3b, C-5b, C-5c, C-2d, 76.6 (C-5a), 75.1 (C-2c), 74.1 (C-2a), 71.0 (C-3c), 69.9 (C-5c),
C-3d, C-5d, C-2e, C-3e, C-5e), 67.9 (C-4c), 66.9 (C-4b), 66.74 69.78 (C-3b), 69.74 (C-5b), 69.4 (C-2d), 69.3 (C-3d), 69.3 (C-5d),
(C-6a), 66.66 (C-4d, C-4e), 66.0 (C-6c), 63.1, 63.0, 62.8 (C-6b, 68.5 (C-4a), 67.6 (C-4d), 67.4 (C-4b), 66.7 (C-4c), 64.4 (C-6b),
C-6d, C-6e), 47.9 (C(CH3)3), 29.9 (C(CH3)3), 20.7 (CH3(CvO)), 63.9 (C-6c), 63.07 (C-6a), 63.03 (C-6d), 20.6 (CH3CvO);
20.4 (CH3(CvO)), 20.2 (CH3(CvO)); coupled HSQC coupled HSQC (700 MHz, CDCl3): δ 100.7/4.63 (JC1/H1 175 Hz,
(700 MHz, CDCl3): δ 100.0/5.23 (JC1/H1 174 Hz, C-1d), 99.3/ C-1c), 99.98/5.35 (JC1/H1 178 Hz, C-1b), 99.3/4.85 (JC1/H1
5.10 (JC1/H1 177 Hz, C-1b), 97.6/5.12 (JC1/H1 175 Hz, C-1c), 176 Hz, C-1d), 76.94/4.67 (JC1/H1 152 Hz, C-1a); anal. calc. for
97.3/4.80 (JC1/H1 175 Hz, C-1e), 92.0/4.83 (JC1/H1 157 Hz, C-1a);
anal. calc. for C131H118O41S: HR ESIMS [M + Na]+: 2433.6482, 1995.5105.
found: 2433.6434. 2-O-Acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-
C110H92O33S: HR ESIMS [M +
Na]+: 1995.5134, found:
2-O-Acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)- O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-
3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)-3,4,6-tri-O- O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-O-acetyl-3,4,6-tri-O-
benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-tri-O-benzoyl-1-thio- benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-benzoyl-β-D-man-
β-D-mannopyranose 27. Tetrasaccharide 22 (21.5 mg, 10 µmol) nopyranosyl thiol 28. Pentasaccharide 26 (20.5 mg, 8.5 µmol)
was dissolved in a 1 M solution of trimethylphosphine in THF was dissolved in a 1 M solution of trimethylphosphine in THF
(1 mL, 1 mmol) and a 1 M solution of NaHCO3 (1 mL, pH 9) (1 mL, 1 mmol) and a 1 M solution of NaHCO3 (1 mL, pH 9)
added and stirred vigorously. After 1.5 hours, the reaction added and stirred vigorously. After 30 minutes, the reaction
mixture was neutralized with dilute AcOH and extracted with mixture was neutralized with dilute AcOH and extracted with
EtOAc three times and the organic layer concentrated under EtOAc three times and the organic layer concentrated under
reduced pressure. Purification of the crude product by chrom- reduced pressure. Purification of the crude product by chrom-
atography (1 : 1 heptane–EtOAc) yielded 27 (20.4 mg, 10 µmol, atography (1 : 1 heptane–EtOAc) yielded 28 (19.7 mg, 8.5 µmol,
99%) as a white powder.
99%) as a white powder.
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1H NMR (700 MHz, CDCl3): δ 7.26 (m, 65H, ArH), 5.87 (d, product 29 was sensitive to oxidation and unstable on silica
1H, J2,3 2.4 Hz, H-2a), 5.86 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4e), gel and was used immediately for the next step.
5.85 (dd, 1H, J3,4 ≈ J4,5 9.4 Hz, H-4a), 5.84 (dd, 1H, J3,4 ≈ J4,5 9.8
[α]2D5 −70 (c 16.9, CHCl3); 1H NMR (700 MHz, CDCl3): δ
Hz, H-4d), 5.83 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4c), 5.78 (dd, 1H, 7.10–8.28 (m, 60H, Ar), 5.86 (d, 1H, J2,3 3.5 Hz, H-2a), 5.85 (dd,
J3,4 10.1 Hz, J2,3 3.3 Hz, H-3e), 5.73 (dd, 1H, J2,3 3.5 Hz, J1,2 1.7 1H, J3,4 ≈ J4,5 9.5 Hz, H-4b), 5.81 (dd, 1H, J3,4 10.1 Hz, J2,3 3.1
Hz, H-2c), 5.74 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4b), 5.57 (dd, 1H, Hz, H-3c), 5.76 (dd, 1H, J3,4 10.1 Hz, J2,3 3.6 Hz, H-3d), 5.76 (dd,
J3,4 9.8 Hz, J2,3 3.2 Hz, H-3d), 5.51 (dd, 1H, J3,4 9.6 Hz, J2,3 3.4 1H, J3,4 ≈ J4,5 9.5 Hz, H-4a), 5.72 (dd, 1H, J3,4 ≈ J4,5 10.1 Hz,
Hz, H-3b), 5.35 (dd, 1H, J2,3 3.3 Hz, J1,2 1.8 Hz, H-2e), 5.23 (d, H-4c), 5.68 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4d), 5.63 (dd, 1H, J2,3
1H, J1,2 2.0 Hz, H-1d), 5.18 (dd, 1H, J2,3 3.3 Hz, J1,2 2.0 Hz, 3.2 Hz, J1,2 1.9 Hz, H-2d), 5.44 (dd, 1H, J3,4 9.9 Hz, J2,3 3.3 Hz,
H-2d), 5.11 (d, 1H, J1,2 1.4 Hz, H-1c), 5.10 (dd, 1H, J2,3 3.4 Hz, H-3b), 5.39 (s, 1H, H-1b), 5.05 (s, 1H, H-1a), 5.04 (d, 1H, Jgem
J1,2 1.9 Hz, H-2b), 5.05 (d, 1H, J1,2 2.0 Hz, H-1b), 4.88 (dd, 1H, 11.8 Hz, SCH2Cl), 4.86 (s, 1H, H-1d), 4.77 (ddd, 1H, J4,5 9.8 Hz,
J1,SH 10.4 Hz, J1,2 1.2 Hz, H-1a), 4.85 (ddd, 1H, J4,5 10.0 Hz, J5,6 J5,6 5.7 Hz, J5,6 2.6 Hz, H-5b), 4.72 (dd, 1H, Jgem 12.2 Hz, J5,6 5.5
5.3 Hz, J5,6 2.5 Hz, H-5b), 4.71 (d, 1H, J1,2 1.8 Hz, H-1e), 4.65 Hz, H-6b), 4.70 (d, 1H, Jgem 11.6 Hz, SCH2Cl), 4.59 (dd, 1H,
(dd, 1H, Jgem 11.4 Hz, J5,6 2.5 Hz, H-6b), 4.63 (dd, 1H, J3,4 9.9 Jgem 12.0 Hz, J5,6 2.8 Hz, H-6a), 4.58 (dd, 1H, Jgem 12.0 Hz, J5,6
Hz, J2,3 3.5 Hz, H-3c), 4.58 (dd, 1H, Jgem 12.2 Hz, J5,6 5.3 Hz, 2.2 Hz, H-6b), 4.56 (s, 1H, H-1c), 4.41 (m, 1H, H-5c), 4.40 (dd,
H-6b), 4.53 (dd, 1H, Jgem 13.0 Hz, J5,6 3.5 Hz, H-6d), 4.40 (dd, 1H, Jgem 12.5 Hz, J5,6 5.7 Hz, H-6a), 4.37 (dd, 1H, Jgem 11.7 Hz,
1H, Jgem 11.2 Hz, J5,6 2.5 Hz, H-6e), 4.38 (dd, 1H, J3,4 9.9 Hz, J2,3 J5,6 6.8 Hz, H-6c), 4.26 (br. s., 1H, H-2c), 4.20 (br. d, 1H, Jgem
3.2 Hz, H-3a), 4.36 (ddd, 1H, J4,5 9.8 Hz, J5,6 5.3 Hz, J5,6 2.4 Hz, 11.6 Hz, H-6c), 3.99 (dd, 1H, J3,4 9.3 Hz, J2,3 3.73 Hz, H-3a), 3.98
H-5e), 4.29–4.33 (m, 2H, H-5d, H-6d), 4.28 (dd, 1H, Jgem 11.8 (m, 1H, H-5d), 3.95 (m, 1H, H-6d), 3.92 (s, 1H, H-2b), 3.89 (m,
Hz, J5,6 5.2 Hz, H-6e), 4.20 (ddd, 1H, J4,5 10.2 Hz, J5,6 6.0 Hz, 1H, H-6d), 3.50 (ddd, 1H, J4,5 9.6 Hz, J5,6 5.5 Hz, J5,6 3.0 Hz,
J5,6 2.0 Hz, H-5c), 4.15 (dd, 1H, Jgem 11.2 Hz, J5,6 5.3 Hz, H-6a), H-5a), 1.99 (s, 3H, CH3(CvO)); 13C NMR (176 MHz, CDCl3): δ
3.94 (dd, 1H, Jgem 10.4 Hz, J5,6 5.8 Hz, H-6c), 3.94 (ddd, 1H, J4,5 168.9 (CvO), 166.4 (CvO), 166.0 (CvO), 165.9 (CvO), 165.6
9.9 Hz, J5,6 5.1 Hz, J5,6 1.7 Hz, H-5a), 3.79 (dd, 1H, Jgem 11.2 Hz, (CvO), 165.6 (CvO), 165.4 (CvO), 165.1 (CvO), 165.1 (CvO),
J5,6 1.9 Hz, H-6a), 3.37 (dd, 1H, Jgem 10.5 Hz, J5,6 2.0 Hz, H-6c), 164.9 (CvO), 164.6 (CvO), 134.2 (Ar), 133.6 (Ar), 133.4 (Ar),
2.70 (d, 1H, J1,SH 10.4 Hz, SH), 2.07 (s, 3H, CH3(CvO)), 1.95 (s, 133.3 (Ar), 133.2 (Ar), 133.2 (Ar), 133.1 (Ar), 133.1 (Ar), 132.9
3H, CH3(CvO)), 1.75 (s, 3H, CH3(CvO)); 13C NMR (126 MHz, (Ar), 132.8 (Ar), 130.2 (Ar), 130.0 (Ar), 130.0 (Ar), 129.9 (Ar),
CDCl3): δ 169.6 (CvO), 169.0 (CvO), 168.6 (CvO), 166.3 129.9 (Ar), 129.8 (Ar), 129.7 (Ar), 129.7 (Ar), 129.6 (Ar), 129.6
(CvO), 166.2 (CvO), 166.1 (CvO), 166.0 (CvO), 166.0 (CvO), (Ar), 129.4 (Ar), 129.4 (Ar), 129.2 (Ar), 129.2 (Ar), 129.2 (Ar),
165.6 (CvO), 165.6 (CvO), 165.5 (CvO), 165.2 (CvO), 165.2 129.1 (Ar), 128.9 (Ar), 128.8 (Ar), 128.8 (Ar), 128.7 (Ar), 128.7
(CvO), 165.1 (CvO), 164.6 (CvO), 164.6 (CvO), 133.7 (Ar), (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3 (Ar), 128.2 (Ar),
133.5 (Ar), 133.5 (Ar), 133.4 (Ar), 133.2 (Ar), 133.1 (Ar), 133.0 128.2 (Ar), 128.1 (Ar), 101.0 (C-1c), 100.1 (C-1b), 99.2 (C-1d),
(Ar), 132.9 (Ar), 132.9 (Ar), 130.2 (Ar), 130.2 (Ar), 130.0 (Ar), 79.2 (C-1a), 78.5 (C-2b), 76.60 (C-3a), 76.55 (C-5a), 74.8 (C-2c),
130.0 (Ar), 130.0 (Ar), 129.9 (Ar), 129.8 (Ar), 129.8 (Ar), 129.7 71.7 (C-2a), 71.0 (C-3c), 69.9 (C-5c), 69.7 (C-3b), 69.5 (C-5b), 69.4
(Ar), 129.7 (Ar), 129.7 (Ar), 129.6 (Ar), 129.6 (Ar), 129.5 (Ar), (C-2d), 69.1 (C-3d), 69.1 (C-4a), 68.8 (C-5d), 67.7 (C-4d), 67.3
129.4 (Ar), 129.4 (Ar), 129.4 (Ar), 129.2 (Ar), 129.1 (Ar), 129.1 (C-4b), 66.8 (C-4c), 64.3 (C-6b), 64.1 (C-6c), 63.0 (C-6a), 63.0
(Ar), 129.0 (Ar), 128.9 (Ar), 128.9 (Ar), 128.8 (Ar), 128.7 (Ar), (C-6d), 46.0 (SCH2Cl), 20.5 (CH3(CvO)); anal. calc. for
128.5 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), 128.4 (Ar), 128.3
C
111H93ClO33S: HR ESIMS [M + Na]+: 2043.4901, found:
(Ar), 128.2 (Ar), 128.2 (Ar), 100.1 (C-1d), 99.3 (C-1b), 97.6 (C-1c), 2043.4887.
97.1 (C-1e), 77.9 (C-5a), 77.8 (C-3c), 77.7 (C-3a), 77.5 (C-1a), 74.2
Chloromethyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyra-
(C-2a), 71.9 (C-2c), 70.0, 69.8, 69.7, 69.68, 69.62, 69.4, 69.35, nosyl-(1→6)-[2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyrano-
69.31, 68.9, 68.8, 68.3, 68.1, 67.1, 66.7, 66.6, 66.5, 66.2 (C-4a, syl-(1→3)]-2,4-di-O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-O-
C-6a, C-2b, C-3b, C-4b, C-5b, C-4c, C-5c, C-6c, C-2d, C-3d, C-4d, acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-
C-5d, C-2e, C-3e, C-4e, C-5e), 63.3 (C-6b), 63.0 (C-6e), 62.8 (C-6d), benzoyl-1-thio-β-D-mannopyranoside 30. To
a solution of
20.7 (CH3(CvO)), 20.5 (CH3(CvO)), 20.2 (CH3(CvO)); coupled pentasaccharide 28 (20 mg, 8.6 µmol) in CH2Cl2 (5 mL) was added
HSQC (700 MHz, CDCl3): δ 100.1/5.23 (JC1/H1 175 Hz, C-1d), DBU (3 µL, 20 µmol). After 2.5 hours, reaction was diluted with
99.3/5.05 (JC1/H1 176 Hz, C-1b), 97.6/5.11 (JC1/H1 176 Hz, C-1c), toluene and concentrated under reduced pressure. Crude
97.1/4.71 (JC1/H1 176 Hz, C-1e), 77.9/4.88 (JC1/H1 163 Hz, C-1a); product 30 was sensitive to oxidation and unstable on silica
anal. calc. for C127H110O41S: HR ESIMS [M + Na]+: 2345.6135, gel and was used immediately for the next step.
found: 2345.6132.
1H NMR (600 MHz, CDCl3): δ 7.26 (s, 65H, Ar), 6.00 (dd, 1H,
Chloromethyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyra- J2,3 3.5 Hz, J1,2 0.9 Hz, H-2a), 5.85 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz,
nosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)- H-4e), 5.85 (dd, 1H, J3,4 ≈ J4,5 9.6 Hz, H-4c), 5.83 (dd, 1H, J3,4
≈
3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-tri-O- J4,5 9.8 Hz, H-4a), 5.81 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4b), 5.75
benzoyl-1-thio-β-D-mannopyranoside 29. To a solution of tetra- (dd, 1H, J3,4 9.9 Hz, J2,3 3.2 Hz, H-3e), 5.76 (dd, 1H, J3,4 ≈ J4,5
saccharide 27 (20.4 mg, 10.3 µmol) in CH2Cl2 (5 mL) was 9.9 Hz, H-4d), 5.71 (dd, 1H, J2,3 3.5 Hz, J1,2 1.7 Hz, H-2c), 5.57
added DBU (6 µL, 20 µmol). After 2 hours, reaction was diluted (dd, 1H, J3,4 9.7 Hz, J2,3 3.3 Hz, H-3b), 5.49 (dd, 1H, J3,4 9.6 Hz,
with toluene and concentrated under reduced pressure. Crude J2,3 3.4 Hz, H-3d), 5.36 (dd, 1H, J2,3 3.4 Hz, J1,2 1.7 Hz, H-2e),
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5.32 (d, 1H, J1,2 1.0 Hz, H-1a), 5.21 (d, 1H, J1,2 2.1 Hz, H-1b), (dd, 1H, J3,4 10.1 Hz, J2,3 3.5 Hz, H-3d), 5.74 (dd, 1H, J3,4 ≈ J4,5
5.16 (dd, 1H, J2,3 3.3 Hz, J1,2 2.1 Hz, H-2b), 5.14 (d, 1H, J1,2 1.8 10.1 Hz, H-4c), 5.68 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz, H-4d), 5.62 (dd,
Hz, H-1c), 5.14 (d, 1H, Jgem 11.8 Hz, SCH2Cl), 5.10 (d, 1H, J1,2 1H, J2,3 3.2 Hz, J1,2 1.8 Hz, H-2d), 5.46 (dd, 1H, J3,4 9.8 Hz, J2,3
2.1 Hz, H-1d), 5.08 (dd, 1H, J2,3 3.4 Hz, J1,2 2.0 Hz, H-2d), 4.77 3.4 Hz, H-3b), 5.39 (d, 1H, J1,2 1.5 Hz, H-1b), 4.91 (d, 1H, J1,2 0.5
(d, 1H, J1,2 1.7 Hz, H-1e), 4.77 (d, 1H, Jgem 11.8 Hz, SCH2Cl), Hz, H-1a), 4.86 (d, 1H, J1,2 1.4 Hz, H-1d), 4.76 (ddd, 1H, J4,5 9.9
4.66 (ddd, 1H, J4,5 9.9 Hz, J5,6 4.8 Hz, J5,6 2.8 Hz, H-5d), 4.63 Hz, J5,6 4.9 Hz, J5,6 2.8 Hz, H-5b), 4.69 (dd, 1H, Jgem 12.1 Hz, J5,6
(dd, 1H, Jgem 11.8 Hz, J5,6 2.9 Hz, H-6d), 4.62 (dd, 1H, J3,4 9.9 4.9 Hz, H-6b), 4.62 (br. s, 1H, H-1c), 4.62 (dd, 1H, Jgem 12.2 Hz,
Hz, J2,3 3.2 Hz, H-3c), 4.50 (dd, 1H, Jgem 12.3 Hz, J5,6 3.1 Hz, J5,6 3.0 Hz, H-6b), 4.61 (dd, 1H, Jgem 12.5 Hz, J5,6 2.8 Hz, H-6a),
H-6b), 4.49 (dd, 1H, Jgem 12.1 Hz, J5,6 4.2 Hz, H-6d), 4.45 (dd, 4.50 (d, 1H, Jgem 13.3 Hz, SCH2N3), 4.39 (dd, 1H, Jgem 12.2 Hz,
1H, J3,4 9.7 Hz, J2,3 3.4 Hz, H-3a), 4.38 (dd, 1H, Jgem 11.7 Hz, J5,6 J5,6 5.2 Hz, H-6a), 4.38 (d, 1H, Jgem 13.1 Hz, SCH2N3), 4.35
2.5 Hz, H-6e), 4.33–4.36 (m, 1H, H-5e), 4.32–4.35 (m, 1H, H-6b), (ddd, 1H, J4,5 10.0 Hz, J5,6 6.3 Hz, J5,6 2.2 Hz, H-5c), 4.29 (dd,
4.30–4.34 (m, 1H, H-5b), 4.28 (dd, 1H, J4,5 11.7 Hz, J5,6 5.2 Hz, 1H, Jgem 12.0 Hz, J5,6 6.2 Hz, H-6c), 4.26 (dd, 1H, J2,3 2.8 Hz, J1,2
H-6e), 4.22 (ddd, 1H, J4,5 10.2 Hz, J5,6 5.7 Hz, J5,6 2.9 Hz, H-5c), 1.9 Hz, H-2c), 4.17 (br. d, 1H, Jgem 11.8 Hz, H-6c), 4.04 (dd, 1H,
4.17 (dd, 1H, Jgem 11.1 Hz, J5,6 6.1 Hz, H-6a), 4.03 (ddd, 1H, J4,5 J3,4 9.8 Hz, J2,3 3.2 Hz, H-3a), 4.00 (dt, 1H, J4,5 9.9 Hz, 2 × J5,6
10.0 Hz, J5,6 6.3 Hz, J5,6 2.2 Hz, H-5a), 3.98 (dd, 1H, Jgem 10.7 4.1 Hz, H-5d), 3.95 (dd, 1H, Jgem 12.1 Hz, J5,6 4.3 Hz, H-6d), 3.92
Hz, J5,6 5.4 Hz, H-6c), 3.79 (dd, 1H, Jgem 11.4 Hz, J5,6 2.2 Hz, (br. s., 1H, H-2b), 3.91 (dd, 1H, Jgem 13.7 Hz, J5,6 3.2 Hz, H-6d),
H-6a), 3.50 (dd, 1H, Jgem 10.6 Hz, J5,6 2.8 Hz, H-6c), 2.09 (s, 3H, 3.57 (ddd, 1H, J4,5 9.8 Hz, J5,6 5.0 Hz, J5,6 3.2 Hz, H-5a), 2.00 (s,
CH3(CvO)), 1.92 (s, 3H, CH3(CvO)), 1.76 (s, 3H, CH3(CvO)); 3H, CH3(CvO)); 13C NMR (125 MHz, CDCl3): δ 169.0 (CvO),
13C NMR (176 MHz, CDCl3): δ 169.5 (CvO), 168.9 (CvO), 166.5 (CvO), 166.1 (CvO), 166.0 (CvO), 166.0 (CvO), 165.7
168.7 (CvO), 166.1 (CvO), 166.1 (CvO), 166.1 (CvO), 165.9 (CvO), 165.4 (CvO), 165.1 (CvO), 165.0 (CvO), 164.7 (CvO),
(CvO), 165.9 (CvO), 165.6 (CvO), 165.5 (CvO), 165.3 (CvO), 134.1 (Ar), 133.6 (Ar), 133.4 (Ar), 133.3 (Ar), 133.3 (Ar), 133.2
165.2 (CvO), 165.2 (CvO), 165.1 (CvO), 164.6 (CvO), 164.5 (Ar), 133.1 (Ar), 133.0 (Ar), 132.8 (Ar), 130.3 (Ar), 130.0 (Ar),
(CvO), 133.6 (Ar), 133.5 (Ar), 133.5 (Ar), 133.3 (Ar), 133.3 (Ar), 130.0 (Ar), 130.0 (Ar), 129.9 (Ar), 129.7 (Ar), 129.7 (Ar), 129.7
133.3 (Ar), 133.1 (Ar), 133.0 (Ar), 133.0 (2 × Ar), 132.9 (Ar), (Ar), 129.6 (Ar), 129.5 (Ar), 128.9 (Ar), 128.7 (Ar), 128.7 (Ar),
132.9 (2 × Ar), 130.1 (Ar), 130.1 (Ar), 130.0 (Ar), 130.0 (Ar), 128.6 (Ar), 128.5 (Ar), 128.5 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4
129.9 (Ar), 129.9 (Ar), 129.7 (Ar), 129.7 (Ar), 129.7 (Ar), 129.6 (Ar), 128.3 (Ar), 128.3 (Ar), 128.2 (Ar), 100.9 (C-1c), 100.2 (C-1b),
(Ar), 129.5 (Ar), 129.5 (Ar), 129.3 (Ar), 129.3 (Ar), 129.3 (Ar), 99.2 (C-1d), 80.0 (C-1a), 78.2 (C-2b), 77.2 (C-3a), 76.6 (C-5a), 75.0
129.1 (Ar), 129.0 (Ar), 129.0 (Ar), 128.9 (Ar), 128.9 (Ar), 128.9 (C-2c), 72.0 (C-2a), 71.0 (C-3c), 69.9 (C-5c), 69.8 (C-3b), 69.5
(Ar), 128.6 (Ar), 128.6 (Ar), 128.5 (Ar), 128.4 (Ar), 128.4 (Ar), (C-5b), 69.4 (C-2d), 69.2 (C-3d), 69.2 (C-4a), 68.8 (C-5d), 67.7
128.3 (Ar), 128.3 (Ar), 128.2 (Ar), 128.2 (Ar), 128.1 (Ar), 100.0 (C-4d), 67.3 (C-4b), 66.9 (C-4c), 64.2 (C-6b), 64.0 (C-6c), 63.1
(C-1d), 99.4 (C-1b), 97.4 (C-1c), 97.3 (C-1e), 80.5 (C-1a), 77.9 (C-6a, C-6d), 51.8 (SCH2N3), 20.6 (CH3(CvO)); coupled HSQC
(C-5a), 77.7 (C-3a), 77.1 (C-3b), 71.9, 71.9 (C-2a, C-2c), 69.9, 69.7, (700 MHz, CDCl3): δ 100.9/4.61 (JC1/H1 174 Hz, C-1c), 100.1/5.39
69.59, 69.57, 69.51, 2 × 69.3, 69.2, 68.9, 68.8, 68.6, 68.5 (C-4a, (JC1/H1 177 Hz, C-1b), 99.2/4.86 (JC1/H1 176 Hz, C-1d), 80.0/4.91
C-2b, C-3b, C-5b, C-4c. C-5c, C-2d, C-3d, C-5d, C-2e, C-3e, C-5e), (JC1/H1 156 Hz, C-1a); anal. calc. for C111H93N3O33S: HR ESIMS
66.9 (C-6a), 66.8, 66.7, 66.6 (C-4b, C-4d, C-4e), 66.5 (C-6c), 63.2, [M + Na]+: 2050.5304, found: 2050.5232.
62.9, 62.8 (C-6b, C-6d, C-6e), 46.4 (SCH2Cl), 20.7 (CH3(CvO)),
20.4 (CH3(CvO)), 20.2 (CH3(CvO)); anal. calc. for nosyl-(1→6)-[2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyrano-
128H111ClO41S: HR ESIMS [M + Na]+: 2393.5902, found: syl-(1→3)]-2,4-di-O-benzoyl-α-D-mannopyranosyl-(1→6)-[2-O-
2393.5850. acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)]-2,4-di-O-
Azidomethyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyra-
C
Azidomethyl 2-O-acetyl-3,4,6-tri-O-benzoyl-α-D-mannopyra- benzoyl-1-thio-β-D-mannopyranoside 32. Crude pentasacchar-
nosyl-(1→2)-3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→2)- ide 30 (∼8.6 µmol) was dissolved in acetone (2 mL) and 3 mg
3,4,6-tri-O-benzoyl-α-D-mannopyranosyl-(1→3)-2,4,6-tri-O- (46 µmol) of NaN3 added. Reaction was heated to 60 °C and
benzoyl-1-thio-β-D-mannopyranoside 31. Crude tetrasacchar- H2O was added dropwise until the solution became homo-
ide 29 (∼10.3 µmol) was dissolved in acetone (3 mL) and genous. After 20 hours at 60 °C, the reaction mixture was
15 mg (0.23 mmol) of NaN3 added. Reaction was heated to cooled and diluted with EtOAc and washed with H2O, then
60 °C and H2O was added dropwise until the solution became brine, and dried over Na2SO4. The organic layer was concen-
homogenous. After 24 hours at 60 °C, the reaction mixture was trated under reduced pressure and the crude yellow powder
cooled and diluted with EtOAc, washed with H2O, then brine, purified by chromatography (9.6 : 0.4 toluene–acetonitrile) to
and dried over Na2SO4. The organic layer was concentrated yield 32 (12 mg, 7.15 µmol, 83% from 28) as a white solid.
under reduced pressure and the crude yellow powder purified
by chromatography (9.7 : 0.3 toluene–acetonitrile) to yield 31 7.21–8.21 (m, 65H, Ar), 5.97 (dd, 1H, J2,3 3.5 Hz, J1,2 0.9 Hz,
(16 mg, 7.89 µmol, 77% from 27) as a white solid.
[α]2D5 −49 (c 12.0 in CHCl3); 1H NMR (700 MHz, CDCl3): δ
H-2a), 5.84 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4c), 5.83 (dd, 1H, J3,4
[α]D25 −70 (c 8.7, CHCl3); 1H NMR (700 MHz, CDCl3): δ ≈ J4,5 10.2 Hz, H-4e), 5.80 (dd, 1H, J3,4 ≈ J4,5 9.6 Hz, H-4b), 5.79
7.09–8.29 (m, 60H, ArH), 5.88 (dd, 1H, J3,4 ≈ J4,5 10.0 Hz, H-4b), (dd, 1H, J3,4 ≈ J4,5 9.4 Hz, H-4a), 5.76 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz,
5.86 (dd, 1H, J2,3 3.5, J1,2 0.5 Hz, H-2a), 5.80 (dd, 1H, J3,4 10.2 H-4d), 5.73 (dd, 1H, J3,4 10.1 Hz, J2,3 3.2 Hz, H-3e), 5.68 (dd, 1H,
Hz, J2,3 3.1 Hz, H-3c), 5.79 (dd, 1H, J3,4 ≈ J4,5 9.8 Hz, H-4a), 5.76 J2,3 3.4 Hz, J1,2 1.7 Hz, H-2c), 5.55 (dd, 1H, J3,4 9.7 Hz, J2,3
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3.3 Hz, H-3b), 5.47 (dd, 1H, J3,4 9.5 Hz, J2,3 3.4 Hz, H-3d), 5.34 (dd, 1H, J2,3 3.2 Hz, J1,2 1.7 Hz, H-2b), 4.07 (dd, 1H, J2,3 3.3 Hz,
(dd, 1H, J2,3 3.2 Hz, J1,2 1.7 Hz, H-2e), 5.19 (d, 1H, J1,2 1.9 Hz, J1,2 1.9 Hz, H-2d), 3.99 (dd, 1H, J3,4 9.6 Hz, J2,3 3.3 Hz, H-3b),
H-1b), 5.14 (d, 1H, J1,2 0.8 Hz, H-1a), 5.14 (dd, 1H, J2,3 3.3 Hz, 3.96 (dd, 1H, J3,4 9.3 Hz, J2,3 3.3 Hz, H-3c), 3.93 (dd, 1H, Jgem
J1,2 1.9 Hz, H-2b), 5.11 (d, 1H, J1,2 1.4 Hz, H-1c), 5.08 (d, 1H, J2,3 12.5 Hz, J5,6 2.2 Hz, H-6a), 3.90 (m, 1H, H-6b), 3.87 (m, 1H,
1.9 Hz, H-1d), 5.06 (dd, 1H, J2,3 3.4 Hz, J1,2 2.1 Hz, H-2d), 4.76 H-6d), 3.86 (dd, 1H, Jgem 12.5 Hz, J5,6 1.7 Hz, H-6c), 3.84 (dd,
(d, 1H, J1,2 1.5 Hz, H-1e), 4.66 (s, 1H, H-5d), 4.63–4.66 (m, 1H, 1H, J3,4 9.5 Hz, J2,3 3.4 Hz, H-3d), 3.78 (dd, 1H, J3,4 9.7 Hz, J2,3
H-6d), 4.58–4.61 (dd, 1H, J3,4 9.7 Hz, J2,3 3.4 Hz, H-3c), 4.56 (d, 3.2 Hz, H-3a), 3.77 (dd, 1H, Jgem 11.7 Hz, J5,6 6.0 Hz, H-6c),
1H, Jgem 13.4 Hz, SCH2N3), 4.47–4.50 (m, 1H, H-6b), 4.47 (dd, 3.74–3.78 (m, 2H, H-5b, H-6b), 3.75 (m, 1H, H-6a), 3.75 (dd, 1H,
1H, Jgem 12.2 Hz, J5,6 4.5 Hz, H-6d), 4.43 (d, 1H, Jgem 13.4 Hz, J3,4 ≈ J4,5 9.4 Hz, H-4a), 3.73–3.79 (m, 2H, H-5d, H-6d), 3.73 (m,
SCH2N3), 4.38–4.41 (dd, 1H, J3,4 9.7 Hz, J2,3 3.5 Hz, H-3a), 1H, H-5c), 3.70 (dd, 1H, J3,4 ≈ J4,5 9.4 Hz, H-4b), 3.70 (dd, 1H,
4.33–4.36 (m, 1H, H-6e), 4.32–4.34 (m, 1H, H-5e), 4.31–4.34 (m, J3,4 ≈ J4,5 9.4 Hz, H-4c), 3.64 (dd, 1H, J3,4 ≈ J4,5 9.7 Hz, H-4d),
1H, H-6b), 4.29–4.32 (m, 1H, H-5b), 4.24–4.27 (m, 1H, H-6e), 3.49 (ddd, 1H, J4,5 9.1 Hz, J5,6 6.0 Hz, J5,6 2.1 Hz, H-5a); 13C
4.20 (ddd, 1H, J4,5 10.1 Hz, J5,6 5.4 Hz, J5,6 2.9 Hz, H-5c), 4.14 NMR (126 MHz, D2O): δ 102.2 (C-1d), 100.7 (C-1c), 100.7 (C-1b),
(dd, 1H, Jgem 11.4 Hz, J5,6 6.3 Hz, H-6a), 3.97 (dd, 1H, Jgem 10.6 83.2 (C-1a), 81.3 (C-3a), 80.4 (C-5a), 78.6 (C-2b), 78.5 (C-2c), 73.4
Hz, J5,6 5.4 Hz, H-6c), 3.95 (ddd, 1H, J4,5 10.1 Hz, J5,6 6.3 Hz, J5,6 (C-5b), 73.2 (C-5c), 73.2 (C-5d), 71.5 (C-2a), 70.3 (C-3d), 70.1
2.1 Hz, H-5a), 3.75 (dd, 1H, Jgem 11.3 Hz, J5,6 1.8 Hz, H-6a), 3.49 (C-3b), 70.0 (C-3c), 70.0 (C-2d), 67.0 (C-4c), 66.9 (C-4b), 66.8
(dd, 1H, Jgem 10.6 Hz, J5,6 2.8 Hz, H-6c), 2.07 (s, 3H, (C-4d), 65.9 (C-4a), 61.1 (C-6d), 61.0 (C-6a, C-6b, C-6c), 52.2
CH3(CvO)), 1.91 (s, 3H, CH3(CvO)), 1.74 (s, 3H, CH3(CvO)); (SCH2N3); coupled HSQC (700 MHz, D2O): δ 102.3/5.05 (JC1/H1
13C NMR (176 MHz, CDCl3): δ 169.5 (CvO), 169.0 (CvO), 172 Hz, C-1d), 100.70/5.30 (JC1/H1 173 Hz, C-1c), 100.66/5.36
168.7 (CvO), 166.2 (CvO), 166.1 (CvO), 166.1 (CvO), 166.0 (JC1/H1 174 Hz, C-1b), 83.2/5.06 (JC1/H1 156 Hz, C-1a); anal. calc.
(CvO), 165.9 (CvO), 165.57 (CvO), 165.56 (CvO), 165.3 for C25H43N3O20S: HR ESIMS [M + Na]+: 760.2053, found:
(CvO), 165.2 (CvO), 165.17 (CvO), 165.15 (CvO), 164.61 760.2045.
(CvO), 164.55 (CvO), 133.6 (Ar), 133.5 (Ar), 133.5 (Ar), 133.3
Azidomethyl α-D-mannopyranosyl-(1→6)-[α-D-mannopyrano-
(Ar), 133.3 (Ar), 133.1 (Ar), 133.1 (Ar), 133.0 (Ar), 132.9 (Ar), syl-(1→3)]-α-D-mannopyranosyl-(1→6)-[α-D-mannopyranosyl-
130.2 (Ar), 130.1 (Ar), 130.0 (Ar), 129.93 (Ar), 129.90 (Ar), 129.8 (1→3)]-1-thio-β-D-mannopyranoside 34. Pentasaccharide 32
(Ar), 129.71 (Ar), 129.67 (Ar), 129.6 (Ar), 129.5 (Ar), 129.5 (Ar), (15 mg, 6.3 µmol) was suspended in deuterated methanol
129.4 (Ar), 129.33 (Ar), 129.29 (Ar), 129.18 (Ar), 129.17 (Ar), (1 mL) and deuterated sodium methoxide (0.14 mL, 1 M)
129.03 (Ar), 128.99 (Ar), 128.98 (Ar), 128.92 (Ar), 128.89 (Ar), added. Reaction was monitored by 1H NMR. After 20 hours,
128.67 (Ar), 128.65 (Ar), 128.5 (Ar), 128.46 (Ar), 128.45 (Ar), the reaction was neutralized with H+ resin and the solvent
128.42 (Ar), 128.35 (Ar), 128.26 (Ar), 128.21 (Ar), 128.18 (Ar), removed under reduced pressure. The yellow syrup was puri-
100.0 (C-1d), 99.4 (C-1b), 97.6 (C-1c), 97.3 (C-1e), 80.9 (C-1a), fied via HPLC to yield 34 (4.0 mg, 4.45 µmol, 71%) as a white
77.9 (C-5a), 77.6 (C-3a), 77.0 (C-3c), 72.2, 71.9 (C-2a, C-2c), 70.0, powder.
69.7, 2 × 69.6, 69.6, 69.3, 69.34, 69.33, 69.0, 68.9, 68.6, 68.5
1H NMR (700 MHz, D2O): δ 5.15 (d, 1H, J1,2 1.4 Hz, H-1d),
(C-4a, C-2b, C-3b, C-5b, C-4c C-5c, C-2d, C-3d, C-5d, C-2e, C-3e, 5.12 (d, 1H, J1,2 1.2 Hz, H-1b), 5.07 (s, 1H, H-1a), 4.91 (d, 1H,
C-5e), 67.1 (C-6a), 66.9, 66.7, 66.6 (C-4b, C-4d, C-4e), 66.5 (C-6c), J1,2 1.3 Hz, H-1e), 4.87 (d, 1H, J1,2 1.5 Hz, H-1c), 4.60 (d, 1H,
63.1, 63.0, 62.9 (C-6b, C-6d, C-6e), 51.9 (SCH2N3), 20.7 Jgem 13.4 Hz, SCH2N3), 4.49 (d, 1H, Jgem 13.4 Hz, SCH2N3), 4.27
(CH3(CvO)), 20.5 (CH3(CvO)), 20.2 (CH3(CvO)); coupled (d, 1H, J2,3 3.5 Hz, H-2a), 4.16 (dd, 1H, J2,3 3.2 Hz, J1,2 1.9 Hz,
HSQC (700 MHz, D2O): δ 100.0/5.08 (JC1/H1 175 Hz, C-1d), 99.4/ H-2c), 4.09 (dd, 1H, J2,3 3.4 Hz, J1,2 1.7 Hz, H-2b), 4.07 (dd, 1H,
5.19 (JC1/H1 176 Hz, C-1b), 97.6/5.11 (JC1/H1 175 Hz, C-1c), 97.3/ J2,3 3.4 Hz, J1,2 1.7 Hz, H-2d), 3.99 (dd, 1H, J2,3 3.4 Hz, J1,2 1.7
4.76 (JC1/H1 176 Hz, C-1e), 80.9/5.14 (JC1/H1 155 Hz, C-1a); anal. Hz, H-2e), 3.97–4.00 (m, 1H, H-6c), 3.96 (dd, 1H, Jgem 11.3 Hz,
calc. for C128H111N3O41S: HR ESIMS [M + Na]+: 2400.6306, J5,6 4.8 Hz, H-6a), 3.93 (dd, 1H, J3,4 9.5 Hz, J2,3 3.5 Hz, H-3c),
found: 2400.6308.
3.90 (s, 1H, H-6b), 3.89 (dd, 1H, J3,4 9.5 Hz, J2,3 3.4 Hz, H-3b),
Azidomethyl α-D-mannopyranosyl-(1→2)-α-D-mannopyrano- 3.89 (m, 1H, H-3d), 3.91 (m, 1H, H-6c), 3.88–3.91 (m, 1H, H-6e),
syl-(1→2)-α-D-mannopyranosyl-(1→3)-1-thio-β-D-mannopyrano- 3.89 (m, 1H, H-5c), 3.87–3.89 (m, 1H, H-4c), 3.87 (dd, 1H, J3,4
≈
side 33. Tetrasaccharide 31 (14 mg, 6.9 µmol) was suspended J4,5 9.8 Hz, H-4a), 3.84 (dd, 1H, J3,4 9.2 Hz, J2,3 3.4 Hz, H-3e),
in deuterated methanol (1 mL) and deuterated sodium meth- 3.81 (dd, 1H, J3,4 9.9 Hz, J2,3 3.5 Hz, H-3a), 3.79 (br. s., 1H,
oxide (0.14 mL, 1 M) added. Reaction was monitored by 1H H-6a), 3.76 (dd, 1H, Jgem 12.4 Hz, J5,6 5.9 Hz, H-6e), 3.75–3.77
NMR. After 20 hours, the reaction was neutralized with H+ (m, 1H, H-6c), 3.75–3.80 (m, 2H, H-6d, H-6d), 3.68–3.71 (m, 1H,
resin and the solvent removed under reduced pressure. H-5e), 3.66–3.70 (m, 1H, H-5d), 3.68 (dd, 1H, J3,4 ≈ J4,5 9.9 Hz,
The orange syrup was purified via HPLC to yield 33 (4.3 mg, H-4d), 3.64–3.68 (m, 1H, H-4e), 3.65 (ddd, 1H, J4,5 9.9 Hz, J5,6
5.83 µmol, 84%) as a white powder.
5.3 Hz, J5,6 1.9 Hz, H-5a); 13C NMR (176 MHz, D2O): δ 103.3
[α]2D5 −10 (c 5.1, H2O); H NMR (700 MHz, D2O): δ 5.37 (d, (C-1b), 103.1 (C-1d), 100.4 (C-1c), 100.1 (C-1e), 84.7 (C-1a), 82.4
1H, J1,2 1.5 Hz, H-1b), 5.31 (d, 1H, J1,2 1.5 Hz, H-1c), 5.06 (s, 1H, (C-3a), 79.4 (C-3c), 79.2 (C-5a), 74.2, 74.1, 73.5, 72.3, 71.7, 71.4,
H-1a), 5.05 (d, 1H, J1,2 1.5 Hz, H-1d), 4.59 (d, 1H, Jgem 13.3 Hz, 2 × 71.2, 70.9, 70.85, 70.8, 70.3, 67.61, 2 × 67.56 (C-2a, C-2b,
SCH2N3), 4.52 (d, 1H, Jgem 13.4 Hz, SCH2N3), 4.23 (d, 1H, J2,3 C-3b, C-4b, C-5b, C-2c, C-5c, C-2d, C-3d, C-4d, C-5d, C-2e, C-3e,
3.1 Hz, H-2a), 4.11 (dd, 1H, J2,3 3.1 Hz, J1,2 1.9 Hz, H-2c), 4.09 C-4e, C-5e), 66.6 (C-4c), 66.5 (C-6a), 66.4 (C-4a), 66.1 (C-6c), 61.8,
1
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2 × 61.8 (C-6b. C-6d, C-6e), 53.3 (SCH2N3); coupled HSQC
(700 MHz, D2O): δ 103.3/5.12 (JC1/H1 176 Hz, C-1b), 103.1/5.15
(JC1/H1 175 Hz, C-1d), 100.4/4.87 (JC1/H1 175 Hz, C-1c), 100.0/4.91
(JC1/H1 174 Hz, C-1e), 84.7/5.07 (JC1/H1 157 Hz, C-1a); anal. calc.
for C31H53N3O25S: HR ESIMS [M + Na]+: 922.2581, found:
922.2573.
6 C. N. Scanlan, R. Pantophlet, M. R. Wormald, E. O. Saphire,
R. Stanfield, I. A. Wilson, H. Katinger, R. A. Dwek,
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N-Succinimidyl 4-pentynoate 36. To a 0 °C (ice-water bath)
solution of 4-pentynoic acid (0.5 g, 4.84 mmol) and N-hydroxy-
succinimide (0.57 g, 4.84 mmol) in 1 : 1 EtOAc–1,2-dioxane
(60 mL) was added DCC (1.01 g, 4.90 mmol) in a single
portion. The reaction mixture was allowed to warm to room
temperature overnight. After 20 hours, the mixture was filtered
through Celite and the collected solution diluted with EtOAc
and washed with H2O and brine. The organic layer was con-
centrated under reduced pressure and recrystallized with
hexanes–EtOAc to yield 54 (0.68 g, 3.49 mmol, 72%) as a white
crystalline solid.
9 K. J. Doores and D. R. Burton, J. Virol., 2010, 84, 10510–
10521.
10 L. M. Walker, M. Huber, K. J. Doores, E. Falkowska,
R. Pejchal, J.-P. Julien, S.-K. Wang, A. Ramos, P.-Y. Chan-
Hui, M. Moyle, J. L. Mitcham, P. W. Hammond,
O. A. Olsen, P. Phung, S. Fling, C.-H. Wong, S. Phogat,
T. Wrin, M. D. Simek, W. C. Koff, I. A. Wilson,
D. R. Burton, P. Poignard and P. G. Principal Investigators,
Nature, 2011, 477, 466–470.
1
m.p.: 74.20 °C; H NMR (498 MHz, CDCl3): δ 2.88 (t, 2H, J
7.9 Hz, CH2(CvO)O), 2.84 (br. s., 4H, CH2(CvO)N), 2.62 (td,
4H, J 7.5, J 2.7 Hz, CH2CuC), 2.04 (t, 1H, J 2.7 Hz, CuCH); 13
C
NMR (126 MHz, CDCl3): δ 168.9 (CvO), 167.0 (CvO), 80.8
(CuC), 70.0 (CuC), 30.3 (CH2), 25.6 (CH2), 14.1 (CH2); anal.
calc. for C9H11NO4: HR ESIMS [M + Na]+: 218.0424, found:
218.0424; elem. Anal: 55.39; H, 4.65; N, 7.18; found: C, C, 11 R. Pejchal, K. J. Doores, L. M. Walker, R. Khayat,
55.13; H, 4.65; N, 7.35.
P.-S. Huang, S.-K. Wang, R. L. Stanfield, J.-P. Julien,
A. Ramos, M. Crispin, R. Depetris, U. Katpally,
A. Marozsan, A. Cupo, S. Maloveste, Y. Liu, R. McBride,
Y. Ito, R. W. Sanders, C. Ogohara, J. C. Paulson, T. Feizi,
C. N. Scanlan, C.-H. Wong, J. P. Moore, W. C. Olson,
A. B. Ward, P. Poignard, W. R. Schief, D. R. Burton and
I. A. Wilson, Science, 2011, 334, 1097–1103.
Acknowledgements
This work was supported by a Centres Program grant from
Alberta Innovates, Technology Futures and by a Natural
Science and Engineering Research Council of Canada Discov-
ery grant to DRB.
The following reagent was obtained through the NIH AIDS
Reagent Program, Division of AIDS, NIAID, NIH: HIV-1 gp120
Monoclonal Antibody (2G12) from Dr Hermann Katinger.
We thank Dr Randy Whittal and Mr Béla Reiz in the Mass
Spectrometry Laboratory at the University of Alberta for their
assistance with LC-UV-MS.
12 J. Ni, H. Song, Y. Wang, N. M. Stamatos and L.-X. Wang,
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