Synthesis and immunological characterization of modified hyaluronic acid hexasaccharide conjugates

Article abstract of DOI:10.1021/jo4012442

The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the N-acetyl moiety of the N-acetyl-d-glucosamine residue is replaced with an N-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites.

Full text of DOI:10.1021/jo4012442

Article
pubs.acs.org/joc
Synthesis and Immunological Characterization of Modified
Hyaluronic Acid Hexasaccharide Conjugates
Guofeng Gu,† Pal John Pal Adabala,‡ Monica G. Szczepina,‡ Silvia Borrelli,‡ and B. Mario Pinto*,‡
†National Glycoengineering Research Center, Shandong University, Jinan 250100, PR China
‡Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
S
* Supporting Information
ABSTRACT: The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The
compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A
Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which
the N-acetyl moiety of the N-acetyl-D-glucosamine residue is replaced with an N-propionyl unit in order to enhance
immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction
of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts.
Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide
haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting
that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized
by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with
the amino acid residues of the antibody combining sites.
hydrophilic and hydrodynamic properties by which it retains
water and plays structural and/or lubricant roles, for example, in
joint synovial and eye vitreous fluid, skin, umbilical cord, and
water transport, heart valves, skeletal tissues, supramolecular
assembly of proteoglycans in the extracellular matrix, and control
of tissue hydration.1−3 HA also plays important receptor-
mediated roles (interacting with a variety of binding proteins),
in cell detachment, mitosis, migration, tumor development,
metastasis, and inflammation.1,2 It is now clear that specific
biological roles of HA are related to the length of the
carbohydrate chain and its molecular weight1,2 In addition, HA
is produced in mass during tissue injury, tissue repair, wound
healing, and inflammation,2 and it has also been found in blood
serum in high concentrations.4 Consequently, serum hyaluronan
was described as a disease marker of many pathological
conditions such as liver cirrhosis, rheumatoid arthritis and
other joint diseases, septicemia, uraemia, and renal failure.4 Urine
INTRODUCTION
■
Hyaluronan (or hyaluronic acid, HA) is a linear polysaccharide
composed of repeating disaccharide units of D-glucuronic acid
and N-acetyl-D-glucosamine, which can reach more than 10 000
repeating units of [−β(1,4)-D-glucuronic acid−β(1,3)-N-acetyl-
D-glucosamine]n. The molecular weight of HA is about 4000
kDa, and it has an average extended length of about 10 μm (e.g., n
= 10 000).1
HA has been described in all living organisms, from
prokaryotes to eukaryotes, and is located in the extracellular
and pericellular matrix, but it is also found intracellularly.1 In
mammals, HA can be released by many cell types, although
connective tissue cells are believed to be the predominant source
of HA, which is synthesized by membrane-bound hyaluronan
synthases using the activated nucleotide sugars (UDP-D-
glucuronic acid and UDP-N-acetyl-D-glucosamine) as sub-
strates.1,2 These enzymes are located on the inner surface of
the plasma membrane, and the chains synthesized are secreted
through pore-like structures into the extracellular space.2 The
biological roles of HA in humans are based mainly on its
Received: June 9, 2013
© XXXX American Chemical Society
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Figure 1. Target protein conjugates 1a and 1b.
Scheme 1. Retrosynthetic Analysis of the Hexasaccharides 24a and 24b
HA has been found in patients with bladder cancer angiogenesis
and metastasis; thus, HA appears to be a useful marker in the
diagnosis of these pathologies.5 HA is nonantigenic and
nonimmunogenic because of its highly conserved structure
among species and weak interaction with blood components.1
Streptococcus pyogenes, or Group A Streptococcus (GAS) is the
etiological agent of a number of human diseases ranging from
trivial pharyngitis, to lethal necrotizing fasciitis, and streptococcal
toxic shock syndrome, leading in some cases to delayed sequelae
such as rheumatic fever and rheumatic heart disease.6 GAS causes
∼700 million human infections each year, resulting in over 500
000 deaths.7 Rapid administration of penicillin or amoxicillin
therapy shortens the clinical course, decreases the incidence of
sequelae and the risk of transmission, and prevents acute
rheumatic fever.8 The risk of antibiotic-resistant bacteria9 makes
a vaccine protocol an attractive alternative to the present
antibiotic therapy.10 A safe and effective commercial GAS vaccine
has yet to be developed.11 Synthetic oligosaccharide vaccine
candidates against GAS infections have been reported recently,12
but good protection against heavily encapsulated strains may be
difficult to establish. The surface-anchored GAS M proteins and
its peptides are capable of eliciting protective immunity,11,13 but
cross-reactive antibodies against human tissue antigens have
been elicited, raising vaccine safety concerns.11 GAS is an
B
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a
Scheme 2. Synthesis of the Glucose-Derived Monosaccharide Building Blocks 5 and 6
a
(a) NaOAc, Ac2O, 140 °C. (b) (i) 4-Methoxyphenol, BF3·Et2O, CH2Cl2; (ii) 1 N NaOMe, MeOH; (iii) PhCH(OMe)2, p-TsOH, DMF, 70 °C. (c)
(i) BzCl, pyridine, CH2Cl2, 0 °C to rt, 83 %; (ii) 80% HOAc, CHCl3, reflux, 78%. (d) Levulinic acid, 2-chloro-1-methylpyridinium iodide, 1,4-
diazabicyclo[2,2,2]octane, 1,2-dichloroethane, rt, 90%. (e) (i) Ac2O, pyridine; (ii) (NH4)2Ce(NO3)6, CH3CN/H2O (4:1, v/v); (iii) Cl3CCN, DBU,
CH2Cl2, 0 °C, 70% (3 steps).
a
Scheme 3. Synthesis of the Glucosamine-Derived Monosaccharide Building Block 8
a
(a) (i) Phthalic anhydride, aq NaOH; (ii) Ac2O, pyridine. (b) (i) 4-Methoxyphenol, BF3·Et2O, CH2Cl2, reflux; (ii) 1 N NaOMe, MeOH. (c)
PhCH(OMe)2, p-TsOH, DMF, 70 °C. (d) Chloroacetic anhydride, pyridine/CH2Cl2, 0 °C, 85%. (e) (i) (NH4)2Ce(NO3)6, CH3CN/H2O (4:1, v/
v), 0 °C; (ii) Cl3CCN, DBU, CH2Cl2, 61% (2 steps).
encapsulated bacteria, and its capsule is composed of HA (also
present in Group C Streptococcus), which confers resistance to
phagocytosis. GAS HA is chemically similar to that found in
human connective tissue and is therefore a poor immunogen;
antibodies to GAS HA have been quite difficult to detect in
humans, although they have been elicited in rabbits and mice
immunized with encapsulated GAS.6 Previous work on the
immunogenicities of low molecular weight HA in the prevention
and treatment of Group A and C Streptococcus infections has
shown that these antibodies are directed against the nonreducing
end of the glucuronic acid moiety of low-molecular weight HA.14
Of greater utility would be an antibody that recognizes the
internal epitopes of HA. Ideally, a portion of HA could be used as
part of an antigen to elicit the appropriate antibodies that could
then be used to monitor HA levels as a disease marker. This
oligosaccharide, when conjugated to carrier protein, could also
be used in a vaccine preparation. Alternatively, the antibodies
could be used as therapeutic agents in passive immunization
protocols to treat GAS infections. Various strategies have been
reported for the synthesis of diverse molecular weight HA
oligosaccharides, ranging from enzymatic15,16 to chemical
methods,17−25 with the aim of studying HA-protein interactions
and structure−activity relationships. We report here the
synthesis of a hexasaccharide unit of HA coupled to tetanus
toxoid (TT) for use as an antigen to raise antibodies for
diagnostic purposes. We also report the synthesis of a chemically
modified HA unit and its conjugation to TT to be used as a
vaccine in active immunization protocols. In the latter we replace
the N-acetyl moiety of the N-acetyl-D-glucosamine unit with an
N-propionyl unit, a nonself structural element. This modification
has been shown to work in the case of the polysialic acid antigen
of Neisseria meningitidis B, which shows enhanced immunoge-
nicity.26−29 We also report the immunological studies in mice
with both vaccine candidates, which show very similar antibody
responses for both conjugates, suggesting that the N-acetyl
groups of the glucosaminyl residues of HA-hexasaccharide are
not in close contact with the amino acid residues of the antibody
combining site. Toward this end, we designed the synthesis of
two tetanus toxoid (TT) and two human serum albumin (HSA)
conjugates of the HA-hexasaccharides 1a and 1b (Figure 1).
RESULTS AND DISCUSSION
■
Retrosynthetic analysis indicated that the desired hexasacchar-
ides 24a and 24b could be synthesized through a convergent (3 +
3′) strategy (Scheme 1). Thus, the hexasaccharide derivative 19
could be synthesized by coupling of the trisaccharide donor 16
and trisaccharide acceptor 18, which could be assembled, in turn,
from the following four monosaccharide building blocks:
glucose-derived monosaccharide donor 6, disaccharide acceptor
13, disaccharide donor 14 and glucosamine-derived acceptor 11.
Multiple protecting group manipulations of 19, i.e., removal of
benzylidene, levulinoyl and acyl groups, conversion of the N-
phthalimido group to N-acyl groups, PDC oxidation of 6-OH of
the glucose residues to carboxyl acids and reduction of the azide
to an amine, would then generate the target hexasaccharides
24a/b (Scheme 1). The conjugates of hexasaccharides 24a and
24b with tetanus toxoid (TT) or human serum albumin (HSA)
could then be prepared, using diethyl squarate30−32 as a linker, to
afford the corresponding hexasaccharide neoglycoproteins (TT-
1a/b, HSA-1a/b).
The synthesis of the four key monosaccharide precursors 5, 6,
8, and 11 was examined first. The monosaccharide acceptor 5
was synthesized using a similar procedure described in the
literature (Scheme 2).33 Benzoylation of 4-methoxyphenyl 4,6-
di-O-benzylidene-β-D-glucopyranoside (3) with benzoyl chlor-
ide in pyridine, followed by removal of the benzylidene group
C
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with 80% HOAc in CHCl3, furnished the diol 4 in 61% yield for
two steps. Selective protection of the 6-OH with levulinic acid in
1,2-dichloroethane afforded the glucopyranosyl acceptor 5 in
90% yield. Acetylation of 5, followed by removal of the 4-
methoxyphenyl (MP) group at C-1 with cerium ammonium
nitrate,34 and activation of the anomeric hydroxyl as its
trichloroacetimidate,35 followed by chromatographic purifica-
tion, gave the α-anomer of donor 6 as the major product in a total
yield of 70% (Scheme 2).
trichoroacetimidate 8 in dichloromethane with TMSOTf as
promoter afforded the β-(1 → 4)-linked disaccharide 12 in 81%
yield (Scheme 5). The chemical shifts of H-1′ (δ 5.49 ppm, J1′,2′
8.2 Hz) and C-1′ (δ 98.9 ppm) in the NMR spectra confirmed
the β-linkage. Removal of the chloroacetyl group from 12 with
thiourea gave the disaccharide acceptor 13 (73%). On the other
hand, cerium ammonium nitrate-promoted cleavage of the MP
group on the anomeric carbon of 12, followed by trichlor-
oacetimidate formation with Cl3CCN/DBU in dichlorome-
thane, generated the disaccharide donor 14 with the α-
configuration as the major product in 67% yield for the two
steps (Scheme 5).
The coupling reaction of trichloroacetimidate 6 and the
disaccharide acceptor 13 in dichloromethane was carried out
with promotion of TMSOTf to yield the trisaccharide 15 in 82%
yield (Scheme 6). Then, cleavage of the MP group with cerium
ammonium nitrate followed by activation of the hemiacetal with
trichloroacetonitrile/DBU in CH2Cl2 furnished the α-anomer of
trichloroacetimidate 16 as the major product, after chromatog-
raphy, in 70% yield. Condensation of the acceptor 11 and the
disaccharide donor 14, promoted by TMSOTf at −40 °C, then
furnished the trisaccharide 17 in 83% yield. Thiourea-promoted
dechloroacetylation of 17 generated the trisaccharide acceptor
18 in 78% yield (Scheme 6).
4-Methoxyphenyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-
β-D-glucopyranoside36,37 (7) was prepared from D-glucosamine
hydrochloride as indicated in Scheme 3. Then, protection of the
3-OH with chloroacetic anhydride in pyridine/dichloromethane
at 0 °C, followed by removal of the methoxyphenyl (MP) group
at C-1, and anomeric activation of the hemiacetal as the
trichloroacetimidate furnished the donor 8, after chromatog-
raphy, with the β-configuration as the major product in 61% yield
(Scheme 3). The β-configuration was confirmed by the coupling
1
constant of H-1 (δ 6.72 ppm, J1,2 8.7 Hz) in the H NMR
spectrum.
6-Azido-1-hexanol (9)38−40 was prepared from commercially
available 1,6-hexanediol by (i) selective tosylation with tosyl
chloride in pyridine/DMF (1:2, v/v) at 0 °C and (ii)
displacement of the tosylate with sodium azide in DMF at 70
°C. Glycosylation of 9 with the donor 8 in dichloromethane, with
promotion by trimethylsilyl trifluoromethanesulfonate
(TMSOTf), generated compound 10 in 92% yield. Dechlor-
oacetylation of 10 was carried out with thiourea41 to afford the
acceptor 11 in 83% yield (Scheme 4).
We next turned our attention to the synthesis of the
hexasaccharides. The hexasaccharide 19 was obtained in 70%
yield by condensation of the trisaccharide donor 16 with the
trisaccharide acceptor 18 under standard coupling conditions
(Scheme 7).
Debenzylidenation of 19 with 80% HOAc, followed by
acetylation with acetic anhydride in pyridine, furnished the
hexasaccharide 20 in 78% yield for two steps (Scheme 8).
Delevulinoylation of 20 was carried out smoothly with hydrazine
acetate42 in ethanol/toluene (2:1, v/v) to afford the
hexasaccharide-triol 21 in 89% yield. Oxidation of the triol 21
with pyridinium dichromate43,44 (PDC) in dichloromethane in
the presence of acetic anhydride gave the desired product 22 in
67% yield. The correct structure of 22 was confirmed by spectral
analysis of its methyl-esterified derivative, 22a, whose 1H NMR
spectrum showed three singlets at δ 3.42, 3.44, and 3.71 ppm,
together with the MALDI-TOFMS spectrum, which showed a
molecular-ion peak at 2528 [M + Na]+, indicating the presence of
three methyl-esters. Dephthaloylation of 22 with ethylenedi-
amine45 in n-butanol, followed by acylation with acetic anhydride
or propionic anhydride in pyridine, and then de-O-acetylation of
a
Scheme 4. Synthesis of the Acceptor 11
a
(a) (i) TsCl, pyridine/DMF, 0 °C; (ii) NaN3, DMF, 70 °C. (b)
TMSOTf, CH2Cl2, 4 Å MS, −30 to 0 °C, 92%. (c) Thiourea, 2,6-
lutidine, MeOH/CH2Cl2 (4:1 v/v), 83%.
With synthons 5, 6, 8 and 11 in hand, the trisaccharides were
next assembled. First, coupling of the acceptor 5 and
a
Scheme 5. Synthesis of the Disaccharide Acceptor 13 and Donor 14
a
(a) TMSOTf, CH2Cl2, −40 to 0 °C, 81%. (b) Thiourea, 2,6-lutidine, MeOH/CH2Cl2, reflux, 73%. (c) (i) (NH4)2Ce(NO3)6, CH3CN/H2O (4:1),
0 °C; (ii) Cl3CCN, DBU, CH2Cl2, 67% (2 steps).
D
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a
Scheme 6. Synthesis of the Trisaccharide Donor 16 and Acceptor 18
a
(a) TMSOTf, CH2Cl2, −40 to 0 °C, 82% for 15, 83% for 17. (b) (i) (NH4)2Ce(NO3)6, CH3CN/H2O, 0 °C; (ii) Cl3CCN, DBU, CH2Cl2. (c)
Thiourea, MeOH/CH2Cl2, 2,4-lutidine, 78%.
a
Scheme 7. Synthesis of the Hexasaccharide 19
a
(a) TMSOTf, CH2Cl2, −40 to 0 °C, 70%.
a
Scheme 8. Synthesis of the Hexasaccharides 23a and 23b
a
(a) 80% HOAc, 70 ° C, then Ac2O, pyridine, 78%. (b) Hydrazine acetate, 2:1 EtOH−toluene, 89%. (c) PDC/Ac2O, CH2Cl2, 67%. (d) (i)
H2NCH2CH2NH2, 1-butanol; (ii) Ac2O or (CH3CH2CO)2O, pyridine; (iii) 1 N LiOH, THF, 0 °C, 72% for 23a, 78% for 23b.
the resulting products using 1 N LiOH46 in THF at 0 °C,
afforded the free hexasaccharides 23 (72% for 23a; 78% for 23b)
(Scheme 8).
using size-exclusion chromatography. Compounds 25a or 25b
were then directly conjugated to tetanus toxoid (TT) or human
serum albumin (HSA) in 0.1 M carbonate buffer at pH 10.0.47
After dialysis and lyophilization of the crude samples, the
hexasaccharide neoglycoproteins (TT-1a/b, HSA-1a/b) were
obtained as white powders. The average number of hexasac-
charide units incorporated in TT or HSA was assessed by
MALDI-TOF MS analysis (Table 1).
Reduction of the azide function in compounds 23a or 23b with
30
10% Pd−C and NaBH4 in 0.05 M aqueous NaOH gave the
amino compounds 24 (85% for 24a; 81% for 24b; Scheme 9).
Reaction of 24a or 24b with diethyl squarate31,32,47,48 in 1:1
EtOH−phosphate buffer (50 mM, pH 7.3) then generated the
monoethyl squarate derivatives 25a or 25b after purification
E
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a
Scheme 9. Synthesis of the Monoethyl Squarate Adducts 25a and 25b
a
(a) 10% Pd-C, NaBH4, 0.05 M NaOH. (b) Diethyl squarate, pH 7.3
Table 1. TT and HSA-Hexasaccharide Conjugates
hapten
molar ratio of
protein/
residues/
protein
molecule
(n)
squarate
incorporation
neoglycoprotein
R group
Ac
derivative
efficiency (%)
TT-1a
1:60
1:60
1:50
1:50
10.3
7.0
17.2
11.7
15.6
13.8
TT-1b
propionyl
Ac
HSA-1a
HSA-1b
7.8
propionyl
6.9
Immunological Studies. The immunogenicity of the N-
propionylated and N-acetylated (native form) HA-hexasacchar-
ides-TT conjugates (compounds 1b and 1a) in mice was
investigated by ELISA using the homologous hexasaccharide-
human serum albumin (HSA) conjugates as solid-phase antigens.
The absorbances corresponding to antisera on day 38 are shown
(Figures 2 and 3).
The HA hexasaccharideNCOPr-TT 1b-specific IgG response
is shown (Figure 2A) with the HA-hexasaccharideNCOPr-HSA
as a solid-phase antigen on the ELISA plate. The conjugate
induced a strong immune IgG response in virtually all mice
except for one animal. When the same sera were measured for
activity against the native form of the HA-hexasaccharide, the
IgG response was very similar (Figure 2B), indicating that N-
propionylation of the glucosaminyl groups of the oligosacchar-
ides residues did not affect the recognition of the HA-
hexasaccharideNCOPr-specific IgG for the native HA-hexasac-
charide.
Conversely, the native HA-hexasaccharideNAc-TT (com-
pound 1a)-specific IgG response is shown (Figure 3A) with
the HA-hexasaccharideNCOPr-HSA conjugate as an antigen on
the ELISA plate. All sera (except for one) recognized the HA-
hexasaccharideNCOPr immobilized on the plate. When the
same sera were measured against the native form of the HA-
hexasaccharide, the IgG response was very similar (Figure 3B).
These data indicate that N-propionylation of the glucosaminyl
groups of the oligosaccharides residues of HA does not affect the
binding of IgG elicited against either form, NCOPr- or NAc-, of
the hexasaccharides. This surprising result may indicate that the
NAc groups of the glucosaminyl residues of the HA-
hexasaccharide do not form a critical part of the natural epitope
recognized by a polyclonal response, and that the N-acetyl
moiety does not make close contacts with the complementarity
Figure 2. Immunogenicity of the HA-hexasaccharideNCOPr-TT
conjugate (compound TT-1b): antibody titers (IgG) (A) to HA-
hexasaccharideNCOPr-HSA coated plates and (B) to HA-hexasacchar-
ideNAc-HSA coated plates.
determining regions (CDR) residues within the antibody
combining site.
CONCLUSIONS
■
In summary, hyaluronic acid-related hexasaccharide derivative
24a and its N-propionyl analogue 24b were efficiently
synthesized by a highly convergent strategy using glycosyl
trichloroacetimidates as glycosyl donors. These two oligosac-
charide derivatives were linked to tetanus toxoid (TT) or human
serum albumin (HSA) through a squarate linker to provide their
F
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obtained for samples dispersed in a 2,5-dihydroxybenzoic acid matrix.
High resolution mass spectra were obtained by the electrospray
ionization method, using TOF LC−MS high resolution mass
spectrometer. Column chromatography was performed with Silica gel
60 (230−400 mesh), or size-exclusion gel (Sephadex G-10/Bio Gel P-
2). Solvents were evaporated under reduced pressure below 50 °C.
p-Methoxyphenyl 2,3-di-O-benzoyl-β-D-glucopyranoside (4).
To a mixture of 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose (2) (20 g,
51.3 mmol) and p-methoxyphenol (7.0 g, 56.4 mmol) in dichloro-
methane (150 mL) was added borontrifluoride etherate (20 mL) at 0
°C. The reaction mixture was stirred for 0.5 h and then warmed to rt, and
stirring was continued for another 2 h. The mixture was poured into cold
aqueous saturated NaHCO3 (300 mL) and extracted with dichloro-
methane (500 mL). The organic layer was washed with water and brine,
and then dried with anhydrous MgSO4, and filtered. The filtrate was
concentrated to give a syrupy product, which was dissolved in methanol
(200 mL), and 1 M NaOMe in methanol (20 mL) was added. The
reaction mixture was stirred at rt overnight, neutralized with HOAc, and
concentrated to give the crude product as a white solid. This solid was
suspended in EtOAc/hexane (300 mL, v/v 1: 1) and filtered to give the
crude tetrahydroxy compound as a solid (14g, 95%).49 To a solution of
this crude product (14 g) in DMF (100 mL) was added PhH(OMe)2
(12 mL) and a catalytic amount of p-TsOH (500 mg). The mixture was
heated to 70 °C and stirred under reduced pressure for 1 h and then
neutralized with triethylamine, and the solvent was removed under
reduced pressure. This benzylidene product33 was then dissolved in
pyridine (50 mL) and dichloromethane (50 mL), and benzoyl chloride
(13 mL) was added dropwise over 20 min at 0 °C. The reaction mixture
was warmed to rt and stirred overnight. Methanol (2 mL) was then
added to decompose the excess BzCl, and the resulting mixture was
filtered to give part of the desired compound as a white solid (15 g). The
filtrate was concentrated to yield a yellow solid and then suspended in
EtOAc/hexane (50 mL, v/v 1:2). After filtration, p-methoxyphenyl 2,3-
di-O-benzoyl-4,6-di-O-benzylidene-β-D-glucopyranoside was obtained
(21 g). To a solution of this compound in chloroform (100 mL) was
added 80% HOAc (100 mL), and the mixture was heated to 80 °C and
refluxed overnight. The solvent was removed under reduced pressure,
and purification of the resulting residue by flash column (EtOAc/hexane
2:1) gave compound 4 as white crystals (18 g, 78%): [α]2D3 = +96.0 (c 1.0,
CHCl3); mp 88−90 °C; 1H NMR (600 MHz, CDCl3) δ 7.98 (ddd, J =
15.1, 8.2, 1.1 Hz, 4H, Ar-H), 7.55−7.50 (m, 2H, Ar-H), 7.41−7.36 (m,
4H, Ar-H), 6.93−6.89 (m, 2H, Ar-H), 6.80−6.76 (m, 2H, Ar-H), 5.66
(dd, J2,3 = 9.7, J1,2 = 8.0 Hz, 1H, H-2), 5.47 (t, J2,3 = J3,4 = 9.4 Hz, 1H, H-
3), 5.21 (d, J1,2 = 7.9 Hz, 1H, H-1), 4.07−4.03 (m, 2H, H-6a, H-4), 3.94
(dd, J6a,6b = 12.0, J5,6b = 4.8 Hz, 1H, H-6b), 3.75 (s, 3H, −OCH3), 3.71
(ddd, J4,5 = 9.4, J5,6b = 4.7, J5,6a = 3.4 Hz, 1H, H-5), 3.30 (br s, 1H, −OH);
13C NMR (150 MHz, CDCl3) δ 167.7 (−COPh), 165.4 (−COPh),
Figure 3. Immunogenicity of the HA-hexasaccharideNAc-TT conjugate
(compound TT-1a): antibody titers (IgG) (A) to HA-hexasacchar-
ideNCOPr-HSA coated plates and (B) to HA-hexasaccharideNAc-HSA
coated plates.
neoglycoproteins. The TT conjugates were intended for use as
vaccines candidates against Group A Streptococcus infections or
to elicit antibodies directed against HA in order to monitor HA
levels as disease markers in blood serum and urine samples. The
chemically modified N-propionyl hexasaccharide conjugate was
also intended to enhance immunogenicity, but studies in mice
showed similar immunological results for both conjugates,
negating the hypothesis that this modification would differentiate
between self HA antigens and nonself, modified-HA antigens.
The results suggest that the NAc groups of the glucosaminyl
residues of the HA-hexasaccharide are not a critical part of the
epitope recognized by the set of anti-HA polyclonal antibodies.
Furthermore, it would appear that the N-acyl moieties are not in
close contact with the amino acid residues of the antibody
combining sites.
155.8 (Ar-C), 151.1 (Ar-C), 133.8 (Ar-C), 133.4 (Ar-C), 130.1 (Ar-C),
129.8 (Ar-C), 129.3 (Ar-C), 128.8 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C),
118.7 (Ar-C), 114.7 (Ar-C), 100.7 (C-1), 77.3 (C-3), 76.2 (C-5), 71.4
(C-2), 70.0 (C-4), 62.3 (C-6), 55.7 (−OCH3); HRMS m/z calcd for
C27H30NO9 [M + NH4]+ 512.1915, found 512.1924.
p-Methoxyphenyl 2,3-di-O-benzoyl-6-O-levulinoyl-β-D-glu-
copyranoside (5). To a solution of compound 4 (15 g, 31 mmol) in
chloroform (20 mL) and 1,2-dichloroethane (100 mL) was added
levulinic acid (6.4 mL, 62 mmol) and 2-chloro-1-methylpyridinium
iodide (15.8 g, 62 mmol). The reaction mixture was stirred at rt for 20
min, and 1,4-diazabicyclo[2,2,2] octane (10.4 g, 93 mmol) was added.
The resulting mixture was stirred for another 30 min, at which time TLC
(EtOAc/hexane 2:1) showed the complete consumption of diol 4. The
reaction mixture was filtered through Celite, diluted with dichloro-
methane (500 mL), and then washed with 10% NaCl (200 mL). The
organic layer was dried over anhydrous MgSO4, filtered, and
concentrated. Column chromatography (EtOAc/hexane 1:2) of the
residue afforded 5 as a white solid (16.2 g, 88%): [α]2D3 = +66.0 (c 1.5,
CHCl3); mp 147−149 °C; 1H NMR (600 MHz, CDCl3) δ 8.01−7.96
(m, 4H, Ar-H), 7.55−7.49 (m, 2H, Ar-H), 7.41−7.35 (m, 4H, Ar-H),
6.96−6.91 (m, 2H, Ar-H), 6.80−6.75 (m, 2H, Ar-H), 5.66 (dd, J2,3 = 9.7,
EXPERIMENTAL SECTION
■
1
General Methods. Optical rotations were measured at 23 °C. H
and 13C NMR spectra were recorded at 600 and 150 MHz for proton
and carbon respectively. Peak and coupling constant assignments are
based on 1H NMR, 13C NMR, 1H−1H gCOSY, 1H−13C gHSQC, and
1
1H−13C gHMBC experiments. H NMR experiments for the three
hexasaccharides 23a, 23b and 24b were recorded at 800 MHz (D2O; 25
°C). These included gHMBC, gHSQC, TOCSY, ROESY, COSY. The
1
13C NMR spectra were acquired at 600 MHz, as was the H NMR
spectrm of 24a. Severe overlap of resonances prevented accurate
J1,2 = 7.9 Hz, 1H, H-2), 5.50 (t, J2,3 = J3,4 = 9.4 Hz, 1H, H-3), 5.15 (d, J1,2 =
measurement of coupling constants. MALDI-TOF mass spectra were
7.9 Hz, 1H, H-1), 4.58 (dd, J6a,6b = 12.1, J5,6a = 4.8 Hz, 1H, H-6a), 4.44
G
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(dd, J6a,6b = 12.1, J5,6b = 2.1 Hz, 1H, H-6b), 3.95 (td, J3,4 = J4,5 = 9.5, J4,OH
= 2.5 Hz, 1H, H-4), 3.80 (ddd, J4,5 = 9.7, J5,6a = 4.7, J5,6b = 2.2 Hz, 1H, H-
5), 3.74(s, 3H, −OCH3), 3.47 (d, J4,OH = 3.7 Hz, 1H, −OH), 2.80−2.77
(m, 2H, −COCH2CH2), 2.65 (t, J = 6.4 Hz, 2H, −COCH2CH2), 2.19
(s, 3H, −COCH3); 13C NMR (150 MHz, CDCl3) δ 206.9
(−CH2COCH3), 173.3 (−OCOCH2CH2), 167.3 (−COPh), 165.3
(−COPh), 155.8 (Ar-C), 151.2 (Ar-C), 133.6 (Ar-C), 133.4 (Ar-C),
130.1 (Ar-C), 129.8 (Ar-C), 129.3 (Ar-C), 129.0 (Ar-C), 128.5 (Ar-C),
128.5 (Ar-C), 118.9 (Ar-C), 114.6 (Ar-C), 100.9 (C-1), 76.4 (C-3), 74.5
(C-5), 71.4 (C-2), 69.3 (C-4), 63.1 (C-6), 55.7 (−OCH3), 38.1
(−COCH2CH2) 29.9 (−COCH3), 28.0 (−COCH2CH2); HRMS m/z
calcd for C32H36NO11 [M + NH4]+ 610.2283, found 610.2296.
H-1), 5.60 (s, 1H, PhCH), 4.71 (dd, J2,3 = 10.5, J3,4 = 8.6 Hz, 1H, H-3),
4.51 (dd, J2,3 10.6, J1,2 = 8.5 Hz, 1H, H-2), 4.41 (dd, J6a,6b = 10.5, J5,6a = 4.6
Hz, 1H, H-6a), 3.93−3.83 (m, 1H, H-6b), 3.76−3.68 (m, 5H, H-5, H-4,
−OCH3); 13C NMR (150 MHz, CDCl3) δ 168.2 (−COPhth), 155.7
(Ar-C), 150.6 (Ar-C), 136.9 (Ar-C), 134.3 (Ar-C), 131.7 (Ar-C), 129.5
(Ar-C), 128.5 (Ar-C), 126.4 (Ar-C), 123.7 (Ar-C), 118.7 (Ar-C), 114.6
(Ar-C), 102.1 (PhCH), 98.2 (C-1), 82.1 (C-4), 68.8 (C-3), 68.7 (C-6),
66.4 (C-5), 56.5 (C-2), 55.7 (−OCH3); HRMS m/z calcd for
C28H26NO8 [M + H]+ 504.1653, found 504.1665; m/z calcd for
C28H29N2O8 [M + NH4]+ 521.1918, found 521.1930.
4,6-Di-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-phthali-
mido-β-D-glucopyranosyl trichloroacetimidate (8). To a mixture
of compound 7 (9 g, 17.3 mmol) in dichloromethane (50 mL) and
pyridine (10 mL) at 0 °C was added chloroacetic anhydride (3.26 g, 19
mmol). The reaction mixture was stirred for 40 min at 0 °C, at which
time TLC (EtOAc/hexane 1:1) indicated the consumption of 7 and the
formation of a new compound. The reaction mixture was then diluted
with dichloromethane (100 mL) and washed with 1 N HCl, water and
brine. The organic layer was dried over MgSO4 and filtered. The filtrate
was concentrated and purified by flash column with 1:1 EtOAc/hexane
as eluent to afford the 3-chloroacetylated compound as a light-yellow
foam (8.8 g, 85%). To a solution of this product in CH3CN/H2O (100
mL, v/v 4:1) at 0 °C was added cerium ammonium nitrate (14.4 g, 26.3
mmol). The reaction mixture was stirred for 40 min at 0 °C, at which
time TLC indicated the disappearance of the starting material. The
resulting mixture was diluted with EtOAc (300 mL), washed with
aqueous saturated NaHCO3 and water. The organic layer was dried over
MgSO4, filtered, and concentrated. The residue was recrystallized from
EtOAc/hexane (1:2) to yield the crude hemiacetal as a light-yellow solid
(5 g). This compound was dissolved in dichloromethane (40 mL), and
trichloroacetonitrile (7 mL) was added. The reaction mixture was stirred
with a catalytic amount of DBU (0.7 mL) at 0 °C for 2 h. After
concentration, the residue was purified on a flash column with EtOAc/
hexane (1:2) as the eluent to afford compound 8 as white crystals (6 g,
56%): [α]2D3 = +31.3 (c 1.5, CHCl3); mp 179−181 °C; 1H NMR (600
MHz, CDCl3) δ 8.67 (s, 1H, −NH), 7.85 (dd, J = 5.2, 2.9 Hz, 2H, Ar-H),
7.76−7.71 (m, 2H, Ar-H), 7.47−7.46 (m, 2H, Ar-H), 7.40−7.36 (m, 3H,
Ar-H), 6.72 (d, J1,2 = 8.7 Hz, 1H, H-1), 6.08 (t, J2,3 = J3,4 = 9.7 Hz,1H, H-
3), 5.57 (s, 1H, −CHPh), 4.66 (dd, J2,3 = 10.3, J1,2 = 8.8 Hz, 1H, H-2),
4.51 (dd, J6a,6b = 10.5, J5,6a = 4.7 Hz, 1H, H-6a), 4.00−3.87 (m, 5H, H-5,
H-4, H-6b, −COCH2Cl); 13C NMR (150 MHz, CDCl3) δ 167.5
(−COPhth), 166.7 (−COCH2Cl), 160.7 (−CNH), 136.6 (Ar-C), 134.6
(Ar-C), 131.2 (Ar-C), 129.4 (Ar-C), 128.4 (Ar-C), 126.3 (Ar-C), 123.9
(Ar-C), 101.9 (−CHPh), 94.0 (C-1), 90.1 (−CCl3), 78.8(C-4), 71.2 (C-
3), 68.5 (C-6), 66.9 (C-5), 54.1 (C-2), 40.4 (−COCH2Cl); HRMS m/z
calcd for C25H21Cl4N2O8 [M + H]+ 617.0047, found 617.0043.
4-O-Acetyl-2,3-di-O-benzoyl-6-O-levulinoyl-α-D-glucopyra-
nosyl trichloroacetimidate (6). To a solution of compound 5 (1.04 g,
1.76 mmol) in pyridine (5 mL) at rt was added acetic anhydride (3 mL).
The reaction mixture was stirred overnight, and the solvent was
coevaporated with toluene under reduced pressure. The resulting
residue was purified by flash column with 1:2 EtOAc/hexane as the
eluent to give the acetylated product as a white solid (1.06 g). To a
solution of this product (1.06 g, 1.67 mmol) in CH3CN/H2O (20 mL,
v/v 4:1) at 0 °C was added cerium ammonium nitrate (2.75 g, 5.01
mmol). The reaction mixture was stirred for 30 min at 0 °C and then
diluted with EtOAc (100 mL). The resulting mixture was washed with
aqueous saturated NaHCO3 and water, and the organic layer was dried
over MgSO4, filtered, and concentrated. The residue was purified by
flash column with 1:1 EtOAc/hexane as the eluent to yield the
hemiacetal as a yellow foam. The product was dissolved in dichloro-
methane (10 mL), trichloroacetonitrile (0.85 mL) and DBU (100 μL)
were added, and the reaction mixture was stirred at 0 °C for 2 h. After
concentration, purification of the residue by flash column (EtOAc/
hexane 1:2) afforded the trichloroacetimidate 6 as a white foam (828
mg, 74%): [α]2D3 = +105.3 (c 1.5, CHCl3); 1H NMR (600 MHz, CDCl3)
δ 8.62 (s, 1H, −NH), 7.96−7.92 (m, 4H, Ar-H), 7.53−7.49 (m, 2H, Ar-
H), 7.40−7.34 (m, 4H, Ar-H), 6.76 (d, J1,2 = 3.5 Hz, 1H, H-1), 6.05 (t,
J2,3 = J3,4 =10.0 Hz, 1H, H-3), 5.50 (dd, J2,3 = 10.2, J1,2 = 3.5 Hz, 1H, H-2),
5.45 (t, J3,4 = J4,5 =9.9 Hz, 1H, H-4), 4.38−4.34 (m, 1H, H-5,), 4.34 (dd,
J6a,6b = 12.0, J5,6a = 4.5, 1H, H-6a), 4.24 (dd, J6a,6b = 12.0, J5,6a = 3.0, 1H,
H-6b), 2.84−2.71 (m, 2H, −COCH2CH2), 2.68−2.64 (m, 2H,
−COCH2CH2), 2.21 (s, 3H, −COCH3), 1.97 (s, 3H, −COCH3); 13
C
NMR (150 MHz, CDCl3) δ 206.4 (−CH2COCH3), 172.4
(−OCOCH2CH2), 169.5 (−OCOCH3), 165.7 (−COPh), 165.5
(−COPh), 160.6 (−CNH), 133.6 (Ar-C), 133.5 (Ar-C), 130.0 (Ar-C),
129.9 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C), 93.2 (C-1), 90.7 (−CCl3),
70.6 (C-2), 70.5 (C-5), 70.3 (C-3), 67.6 (C-4), 61.8 (C-6), 37.9
(−COCH2CH2), 29.9 (−COCH3), 27.9 (−COCH2CH2), 20.6
(−COCH3); HRMS m/z calcd for C29H32Cl3N2O11 [M + NH4]+
689.1066, found 689.1052; m/z calcd for C58H60Cl6N3O22 [2M +
NH4]+ 1360.1794, found 1360.1769.
6-Azidohexyl 4,6-di-O-benzylidene-3-O-chloroacetyl-2-
deoxy-2-phthalimido-β-D-glucopyranoside (10). To a mixture of
compound 8 (360 mg, 0.582 mmol), 6-azido-1-hexanol (9) (84 mg,
0.582 mmol) and molecular sieves 4 Å (500 mg) in dichloromethane (5
mL) at −30 °C was added under N2 trimethylsilyl trifluoromethanesul-
fonate (11 μL, 60 μmol). The reaction mixture was stirred with the
temperature slowly warming to 0 °C for 1 h, at which time TLC
(EtOAc/hexane 1:2) indicated the completion of the reaction. The
reaction mixture was then neutralized with triethylamine and fliltered
through Celite. The fliltrate was evaporated under reduced pressure, and
the residue was purified by column chromatography (EtOAc/hexane
1:2) to give compound 10 as a foam (320 mg, 92%): [α]2D3 = −11.4 (c
p-Methoxyphenyl 4,6-di-O-benzylidene-2-deoxy-2-phthali-
mido-β-D-glucopyranoside (7). A solution of 1,3,4,6-tetra-O-acetyl-
2-deoxy-2-phthalimido-β-D-glucopyranose (15 g, 31.4 mmol), p-
methoxyphenol (4.3 g, 34.54 mmol) and borontrifluoride etherate (15
mL) was refluxed for 1h. The reaction mixture was cooled to rt, poured
into cold aqueous saturated NaHCO3 (50 mL), and then extracted with
dichloromethane (300 mL). The organic layer was washed with water
and brine, dried over MgSO4 and filtered. The filtrate was concentrated
to give light yellow foam, which was used directly in next step. To a
solution of this product in methanol (150 mL) was added 1 M NaOMe/
methanol (10 mL) at rt. The reaction mixture was stirred for 3 h and
neutralized with Amberlyst 15 ion-exchange resin (H+). The resulting
mixture was filtered and concentrated. The generated residue was then
dissolved in DMF (80 mL), and PhH(OMe)2 (10 mL) was added. The
reaction mixture was stirred with a catalytic amount of p-TsOH (300
mg) at 70 °C under diminished pressure for 1.5 h. The reaction was
neutralized with triethylamine and concentrated under reduced
pressure. Column chromatography (EtOAc/hexane 1:1) gave com-
pound 7 as white foam (10.3 g, 63%): [α]2D3 = +12.0 (c 1.0, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 7.90−7.84 (m, 2H, Ar-H), 7.76−7.72 (m,
2H, Ar-H), 7.52−7.49 (m, 2H, Ar-H), 7.41−7.35 (m, 3H, Ar-H), 6.87−
6.83 (m, 2H, Ar-H), 6.76−6.72 (m, 2H, Ar-H), 5.81 (d, J1,2 = 8.5 Hz, 1H,
1
0.35, CHCl3), H NMR (600 MHz, CDCl3) δ 7.88 (br s, 2H, Ar-H),
7.76 (dd, J = 5.3, 2.6 Hz, 2H, Ar-H), 7.47−7.42 (m, 2H, Ar-H), 7.38−
7.34 (m, 3H, Ar-H), 5.96 (t, J2,3 = J3,4 = 9.8 Hz, 1H, H-3), 5.54 (s, 1H,
−CHPh), 5.42 (d, J1,2 = 8.4 Hz, 1H, H-1), 4.43 (dd, J6a,6b = 10.5, J5,6a
=
4.8 Hz, 1H, H-6a), 4.34 (dd, J2,3 = 10.3, J1,2 = 8.5 Hz, 1H, H-2), 3.91 (d, J
= 1.9 Hz, 2H, −COCH2Cl), 3.88−3.80 (m, 3H, H-4, H-6b,
OCHH(CH2)5N3), 3.77−3.73 (m, 1H, H-5), 3.46−3.42 (m, 1H,
OCHH(CH2)5N3), 3.05 (t, J = 7.0 Hz, 2H, −OCH2 (CH2)4CH2N3),
1.50−1.37 (m, 2H, −OCH2CH2(CH2)4N3), 1.32−1.20 (m, 2H,
− O ( C H 2 ) 4 C H 2 C H 2 N 3 ) , 1 . 2 0 − 1 . 0 2 ( m , 4 H , − O -
(CH2)2(CH2)2(CH2)2N3); 13C NMR (150 MHz, CDCl3) δ 166.8
(−COCH2Cl), 136.8 (Ar-C), 134.6 (Ar-C), 134.5 (Ar-C), 131.4 (Ar-C),
H
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129.3 (Ar-C), 128.4 (Ar-C), 126.3 (Ar-C), 123.7 (Ar-C), 101.8
(−CHPh), 98.8 (C-1), 79.3 (C-4), 71.6 (C-3), 70.2
(−OCH2(CH2)5N3), 68.8 (C-6), 66.2 (C-5), 55.2 (C-2), 51.2
(−O(CH2)5CH2N3), 40.5 (−COCH2Cl), 29.2 (−CH2), 28.7 (−CH2),
26.3 (−CH2), 25.4 (−CH2); HRMS m/z calcd for C29H35ClN5O8 [M +
NH4]+ 616.2169, found 616.2171.
p-Methoxyphenyl 4,6-di-O-benzylidene-2-deoxy-2-phthali-
mido-β-D-glucopyranosyl-(1 → 4)-2,3-di-O-benzoyl-6-O-levuli-
noyl-β-D-glucopyranoside (13). To a solution of the disaccharide 12
(966 g, 0.92 mmol) in dichloromethane/methanol (20 mL, v/v 1:4) at rt
was added thiourea (350 mg, 4.6 mmol) and 2,4-lutidine (100 μL). The
reaction mixture was refluxed for 6 h, the solvent was removed under
reduced pressure, and the resulting residue was dissolved in dichloro-
methane (100 mL) and washed with 1 N HCI, water, and brine. The
organic layer was dried over MgSO4, filtered, and concentrated.
Purification of the residue on flash column with 1:1 EtOAc/hexane as
the eluent gave the disaccharide acceptor 13 as a white foam (654 g,
6-Azidohexyl 4,6-di-O-benzylidene-2-deoxy-2-phthalimido-
β-D-glucopyranoside (11). To a solution of compound 10 (381 mg,
0.636 mmol) in dichloromethane/methanol (15 mL, v/v 1:4) at rt was
added thiourea (245 mg, 3.18 mmol) and 2,4-lutidine (50 μL). The
reaction mixture was refluxed for 4 h. The solvent was removed under
reduced pressure, and the resulting residue was dissolved in dichloro-
methane (80 mL) and washed with 1 N HCI, water and brine. The
organic layer was dried over MgSO4, filtered, and concentrated.
Purification of the residue on flash column with EtOAc/hexane (2:3) as
73%): [α]2D3 = +20.0 (c 1.0, CHCl3); H NMR (600 MHz, CDCl3) δ
1
8.06−8.05 (m, 2H, Ar-H), 7.93−7.91 (m, 2H, Ar-H), 7.86−7.84 (m, 2H,
Ar-H), 7.76−7.73 (m, 2H, Ar-H), 7.59−7.54 (m, 1H, Ar-H), 7.53−7.48
(m, 1H, Ar-H), 7.45 (t, J = 7.8 Hz, 2H, Ar-H), 7.38−7.34 (m, 4H, Ar-H),
7.33−7.31 (m, 3H, Ar-H), 6.84−6.81 (m, 2H, Ar-H), 6.72−6.69 (m, 2H,
Ar-H), 5.68 (t, J2,3 = J3,4 = 9.0 Hz, 1H, H-3), 5.52 (dd, J2,3 = 9.2, J1,2 = 7.6
Hz, 1H, H-2), 5.35 (d, J1′,2′ = 8.3 Hz, 1H, H-1′), 5.24 (s, 1H, −CHPh),
5.04 (d, J1,2 = 7.6 Hz, 1H, H-1), 4.48 (dd, J2′,3′ = 10.4, J3′,4′ = 8.6 Hz, 1H,
the eluent gave the acceptor 11 as a white foam (277 mg, 83%): [α]D23
=
−36.0 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.88−7.86 (m,
2H, Ar-H), 7.78−7.72 (m, 2H, Ar-H), 7.53−7.47 (m, 2H, Ar-H), 7.41−
7.35 (m, 3H, Ar-H), 5.57 (s, 1H, −CHPh), 5.27 (d, J1,2 = 8.5 Hz, 1H, H-
1), 4.68−4.59 (m, 1H, H-3), 4.40 (dd, J6a,6b = 10.6, J5,6a = 4.5 Hz, 1H, H-
6a), 4.25 (dd, J2,3 = 10.4, J1,2 = 8.6 Hz, 1H, H-2), 3.89−3.79 (m, 2H,
−OCHH(CH2)5N3, H-6b), 3.68−3.58 (m, 2H, H-4, H-5), 3.43 (dt, J =
9.7, 6.5 Hz, 1H, −OCHH(CH2)5N3), 3.05 (t, J = 7.0 Hz, 2H,
−OCH2(CH2)4CH2N3), 2.50 (d, J = 3.5 Hz, 1H, −OH), 1.50−1.36 (m,
2H, −OCH2 CH2 (CH2 )4 N3 ), 1.32−1.21 (m, 2H, −O-
( C H 2 ) 4 C H 2 C H 2 N 3 ) , 1 . 1 7 − 1 . 0 5 ( m , 4 H , − O -
(CH2)2(CH2)2(CH2)2N3); 13C NMR (150 MHz, CDCl3) δ 168.2
(−COPhth), 137.0 (Ar-C), 134.3 (Ar-C), 131.7 (Ar-C), 129.5 (Ar-C),
128.5 (Ar-C), 126.4 (Ar-C), 123.6 (Ar-C), 102.1 (−CHPh), 99.0 (C-1),
82.4 (C-4), 69.9 (−OCH2(CH2)5N3), 68.8 (C-6), 68.7 (C-3), 66.3 (C-
5), 56.7 (C-2), 51.2 (−O(CH2)5CH2N3), 29.2 (−CH2), 28.8 (−CH2),
26.3 (−CH2), 25.4 (−CH2); HRMS m/z calcd for C27H31N4O7 [M +
H]+ 523.2187, found 523.2202; m/z calcd for C27H34N5O7 [M + NH4]+
540.2453, found 540.2465.
H-3′), 4.30 (dd, J6a,6b = 11.9, J5,6a = 1.6 Hz, 1H, H-6a), 4.18 (dd, J2′,3′
=
10.5, J1′,2′ = 8.3 Hz, 1H, H-2′), 4.16−4.13 (m, 1H, H-4), 3.74−3.70 (m,
1H, H-5), 3.71 (s, 3H, −OCH3), 3.68 (dd, J6a,6b = 11.9, J5,6b = 4.2 Hz, 1H,
H-6b), 3.58−3.55 (m, 1H, H-6′a), 3.33−3.29 (m, 2H, H-4′, H-5′),
2.82−2.77 (m, 1H, H-6′b), 2.67 (t, J = 6.9 Hz, 2H, −COCH2CH2),
2.48−2.39 (m, 2H, −COCH2CH2), 2.33 (s, 1H, −OH), 2.20 (s, 3H,
−COCH3); 13C NMR (150 MHz, CDCl3) δ 206.2 (−CH2COCH3),
171.9 (−OCOCH2CH2), 168.1 (−COPhth), 165.2 (−COPh), 165.1
(−COPh), 155.7 (Ar-C), 150.9 (Ar-C), 136.8 (Ar-C), 134.4 (Ar-C),
133.5 (Ar-C), 133.3 (Ar-C), 131.5 (Ar-C), 129.9 (Ar-C), 129.4 (Ar-C),
129.2 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C), 128.4 (Ar-C), 126.3 (Ar-C),
123.9 (Ar-C), 118.9 (Ar-C), 114.5 (Ar-C), 101.7 (−CHPh), 100.3 (C-
1′), 99.2 (C-1), 81.5 (C-4′), 76.4 (C-4), 73.5 (C-3), 72.7 (C-5), 71.8 (C-
2), 68.5 (C-3′), 67.8 (C-6′), 65.9 (C-5′), 61.9 (C-6), 56.7 (C-2′), 55.6
(−OCH3), 38.0 (−COCH2CH2), 29.9 (−COCH3), 27.7
(−COCH2CH2); HRMS m/z calcd for C53H53N2O17 [M + NH4]+
989.3339, found 989.3373.
p-Methoxyphenyl 4,6-di-O-benzylidene-3-O-chloroacetyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl-(1 → 4)-2,4-di-O-
benzoyl-6-O-levulinoyl-β-D-glucopyranoside (12). To a mixture
of compound 5 (1.48 g, 2.49 mmol), 8 (1.85 g, 3.0 mmol) and molecular
4,6-Di-O-benzylidene-3-O-chloroacetyl-2-deoxy-2-phthali-
mido-β-D-glucopyranosyl-(1 → 4)-2,4-di-O-benzoyl-6-O-levuli-
noyl-α-D-glucopyranosyl trichloroacetimidate (14). To a solution
of compound 12 (917 mg, 0.875 mmol) in CH3CN/H2O (20 mL, v/v
4:1) at 0 °C was added cerium ammonium nitrate (1.52 g, 2.62 mmol).
The reaction mixture was stirred for 40 min at 0 °C, diluted with EtOAc
(100 mL), washed with aqueous saturated NaHCO3 and water. The
organic layer was dried over MgSO4 and filtered. The filtrate was
concentrated, and the residue was purified by flash column with EtOAc/
hexane (2:1) as eluent to afford the disaccharide hemiacetal as a yellow
foam. The compound was dissolved in dichloromethane (5 mL),
trichloroacetinitrile (0.4 mL) and DBU (50 μL) were added, and the
reaction mixture was stirred at 0 °C for 3 h. The solvent was removed
under reduced pressure, and the residue was purified by flash column
with EtOAc/hexane (1:1) to yield the disaccharide trichloroacetimidate
14 as a white foam (637 mg, 67%): [α]2D3 = +65.0 (c 1.0, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 8.52 (s, 1H, −NH), 8.06 (d, J = 7.3 Hz, 2H,
Ar-H), 7.90 (d, J = 7.3 Hz, 2H, Ar-H), 7.87−7.82 (m, 2H, Ar-H), 7.77−
7.75 (m, 2H, Ar-H), 7.57 (t, J = 7.4 Hz, 1H, Ar-H), 7.49−7.45 (m, 3H,
Ar-H), 7.34−7.30 (m, 7H, Ar-H), 6.61 (d, J1,2 = 3.6 Hz, 1H, H-1), 6.01
(t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3), 5.77 (t, J2′,3′ = J3′,4′ = 9.8 Hz, 1H, H-3′),
5.58 (d, J1′,2′ = 8.2 Hz, 1H, H-1′), 5.40 (dd, J2,3 = 10.2, J1,2 = 3.7 Hz, 1H,
H-2), 5.23 (s, 1H, −CHPh), 4.32−4.29 (m, 2H, H-2′, H-6a), 4.19 (t, J3,4
= J4,5 = 9.5 Hz, 1H, H-4), 4.13−4.09 (m, 1H, H-5), 3.83 (s, 2H,
−COCH2Cl), 3.61 (dd, J6a,6b = 12.2, J5,6b = 3.2 Hz, 1H, H-6b), 3.58 (dd,
J6′a,6′b = 10.1, J5′,6′a = 4.3 Hz, 1H, H-6′a), 3.55 (t, J3′,4′ = J4′,5′ = 9.4 Hz, 1H,
H-4′), 3.40 (td, J4′,5′ = 9.8, J5′,6′ = 5.2 Hz, 1H, H-5′), 2.91 (t, J6′a,6′b = 10.4
Hz, 1H, H-6′b), 2.75−2.68 (m, 2H, −COCH2CH2), 2.50 (dt, J = 17.0,
6.8 Hz, 1H, −COCH2CHH), 2.39 (dt, J = 17.0, 6.8 Hz, 1H,
−COCH2CHH), 2.23 (s, 3H, −COCH3); 13C NMR (150 MHz,
CDCl3) δ 206.2 (−CH2COCH3), 171.9 (−OCOCH2CH2), 166.8
(−COCH2Cl), 165.5 (−COPh), 165.0 (−COPh), 160.7 (−CNH),
136.6 (Ar-C), 134.6 (Ar-C), 133.6 (2C, Ar-C) 131.2 (br, Ar-C), 130.0
(Ar-C), 129.8 (2C, Ar-C), 129.3 (Ar-C), 128.8 (Ar-C), 128.5 (Ar-C),
́
sieves 4 Å (3 g) in dichloromethane (20 mL) at −30 °C was added
dropwise trimethylsilyl trifluoromethanesulfonate (50 μL) under N2.
The reaction mixture was stirred for 2 h, with the temperature slowly
warming to 0 °C, and neutralized with Et3N. The resulting mixture was
filtered through Celite, and the filtrate was concentrated and purified by
flash column with EtOAc/hexane (2:3) as eluent to yield the
disaccharide 12 as a white foam (2.12 g, 85%): [α]2D3 = +18.0 (c 1.0,
1
CHCl3); H NMR (600 MHz, CDCl3) δ 8.09−8.04 (m, 2H, Ar-H),
7.95−7.90 (m, 2H, Ar-H), 7.88−7.84 (m, 2H, Ar-H), 7.76−7.74 (m, 2H,
Ar-H), 7.58 (t, J = 7.4 Hz, 1H, Ar-H), 7.50 (t, 7.5 Hz, 1H, Ar-H), 7.47 (t,
7.7 Hz, 2H, Ar-H), 7.36 (t, J = 7.8 Hz, 2H), 7.33−7.28 (m, 5H, Ar-H),
6.83 (d, J = 9.1 Hz, 2H, Ar-H), 6.71 (d, J = 9.1 Hz, 2H, Ar-H), 5.79 (t,
J
2′,3′ = J3′,4′ = 9.8 Hz, 1H, H-3′), 5.67 (t, J2,3 = J3,4 = 9.0 Hz, 1H, H-3), 5.53
(dd, J2,3 = 9.1, J1,2 = 7.7 Hz, 1H, H-2), 5.49 (d, J1′,2′ = 8.2 Hz, 1H, H-1′),
5.20 (s, 1H, −CHPh), 5.04 (d, J1,2 = 7.6 Hz, 1H, H-1), 4.31−4.25 (m,
2H, H-2′, H-6a), 4.16 (t, J3,4 = J4,5 = 9.3 Hz, 1H, H-4), 3.83 (s, 2H,
−COCH2Cl), 3.73−3.69 (m, 4H, H-5, −OCH3), 3.62 (m, 2H, H-6′a,
H-6b), 3.52 (t, J3′,4′ = J4′,5′ = 9.4 Hz, 1H, H-4′), 3.44 (m, 1H, H-5′), 2.75
(t, J6′a,6′b = 10.3 Hz, 1H, H-6′b), 2.69 (t, J = 6.8 Hz, 2H, −COCH2CH2),
2.53−2.38 (m, 2H, −COCH2CH2), 2.21 (s, 3H, −OCH3); 13C NMR
(150 MHz, CDCl3) δ 206.2 (−CH2COCH3), 171.9 (−OCOCH2CH2),
166.7 (−COCH2Cl), 165.2 (−COPh), 165.1 (−COPh), 155.8 (Ar-C),
150.9 (Ar-C), 136.6 (Ar-C), 134.6 (Ar-C), 133.6 (Ar-C), 133.3 (Ar-C),
131.2 (Ar-C), 129.9 (Ar-C), 129.3 (Ar-C), 129.2 (Ar-C), 128.7 (Ar-C),
128.5 (Ar-C), 128.3 (Ar-C), 126.3 (Ar-C), 124.0 (Ar-C), 118.9 (Ar-C),
114.5 (Ar-C), 101.5 (−CHPh), 100.3 (C-1), 98.9 (C-1′), 78.4 (C-4′),
76.5 (C-4), 73.5 (C-3), 72.6 (C-5), 71.8 (C-2), 71.5 (C-3′), 67.8 (C-6′),
65.7 (C-5′), 61.9 (C-6), 55.6 (−OCH3), 55.2 (C-2′), 40.3
(−COCH2Cl), 38.0 (−COCH2CH2), 29.9 (−COCH3), 27.7
(−COCH2CH2); HRMS m/z calcd for C55H54ClN2O18 [M + NH4]+
1065.3055, found 1065.3090.
I
dx.doi.org/10.1021/jo4012442 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
128.3 (Ar-C), 126.3 (Ar-C), 124.0 (Ar-C), 101.5 (−CHPh), 99.2 (C-1′),
93.1 (C-1), 90.7 (−CCl3), 78.3 (C-4′), 76.3 (C-4), 71.6 (C-3′), 70.8 (C-
2), 70.7 (C-3, C-5), 67.9 (C-6′), 65.8 (C-5′), 61.5 (C-6), 55.4 (C-2′),
40.3 (−COCH2Cl), 38.1 (−COCH2), 29.9 (−COCH3), 27.7
(−OCOCH2); HRMS m/z calcd for C50H48Cl4N3O17 [M + NH4]+
1102.1732, found 1102.1776.
trisaccharide trichloroacetimidate 16 as a white foam (502 mg, 70%):
[α]2D3 = +110.0 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3) δ 8.48 (s,
1H, −NH), 8.06−8.02 (m, 2H, Ar-H), 7.89−7.87 (m, 2H, Ar-H), 7.70
(dd, J = 8.3, 1.2 Hz, 2H, Ar-H), 7.57−7.54 (m, 1H, Ar-H), 7.48−7.37
(m, 15H, Ar-H), 7.34−7.23 (m, 5H, Ar-H), 7.16 (t, J = 7.8 Hz, 2H, Ar-
H), 6.55 (d, J1,2 = 3.7 Hz, 1H, H-1), 5.94 (t, J2,3 = J3,4 = 9.6 Hz, 1H, H-3),
5.34 (s, 1H, −CHPh), 5.33 (dd, J1,2 = 3.7, J2,3 = 9.6 Hz, 1H, H-2), 5.29−
5.26 (m, 2H, H-1′, H-3″), 5.14 (t, J3″,4″ = J4″,5″ = 9.7, 1H, H-4″), 5.11 (dd,
p-Methoxyphenyl 4-O-acetyl-2,3-di-O-benzoyl-6-O-levulino-
yl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-benzylidene-2-deoxy-
2-phthalimido-β-D-glucopyranosyl-(1 → 4)-2,3-di-O-benzoyl-6-
O-levulinoyl-β-D-glucopyranoside (15). To a mixture of compound
13 (642 mg, 0.661 mmol), 6 (534 mg, 0.793 mmol) and molecular
sieves 4 Å (1.2 g) in dichloromethane (10 mL) at −40 °C was added
dropwise trimethylsilyl trifluoromethanesulfonate (15 μL, 80 μmol)
under N2. The reaction mixture was stirred for 3 h, with the temperature
slowly warming to 0 °C, and neutralized with Et3N. The resulting
mixture was filtered through Celite, and the filtrate was concentrated.
Column chromatography of the residue with EtOAc/hexane (2:1) as the
eluent yielded the trisaccharide 15 as a white foam (804 mg, 82%): [α]D23
= +78.0 (c 1.0, CHCl3); 1H NMR (600 MHz, CDCl3) δ 8.04−8.03 (m,
2H, Ar-H), 7.91−7.90 (m, 2H, Ar-H), 7.71−7.69 (m, 2H, Ar-H), 7.59−
7.56 (m, 1H, Ar-H), 7.50−7.44 (m, 5H, Ar-H), 7.42−7.33 (m, 11H, Ar-
H), 7.30−7.29 (m, 2H, Ar-H), 7.25−7.23 (m, 2H, Ar-H), 7.16−7.14 (m,
2H, Ar-H), 6.80−6.77 (m, 2H, Ar-H), 6.69−6.66 (m, 2H, Ar-H), 5.61 (t,
J2,3 = J3,4 = 9.0 Hz, 1H, H-3), 5.48 (dd, J2,3 = 9.2, J1,2 = 7.6 Hz, 1H, H-2),
5.31 (s, 1H, −CHPh), 5.28 (t, J2″,3″ = J3″,4″ = 9.6 Hz, 1H, H-3″), 5.19 (d,
J
1″,2″ = 8.0, J2″,3″ = 9.6, 1H, H-2″), 4.74 (d, J1″,2″ = 8.0 Hz, 1H, H-1″), 4.59
(dd, J = J2′,3′ = 10.2, J3′,4′ = 8.8 Hz, 1H, H-3′), 4.27 (dd, J2′,3′ = 10.3, J1′,2′
8.4 Hz, 1H, H-2′), 4.17 (dd, J6a,6b = 12.1, J5,6a = 1.5 Hz, 1H, H-6a), 4.08
(t, J3,4 = J4,5 = 9.7, 1H, H-4), 4.01−3.98 (m, 1H, H-5), 3.92 (dd, J6″a,6″b
12.1, J5″,6″a = 3.1 Hz, 1H, H-6″a), 3.87 (dd, J6″a,6″b = 12.1, J5″,6″b = 2.2 Hz,
=
=
1H, H-6″b), 3.68 (t, J3′,4′ = J4′,5′ = 9.1 Hz, 1H, H-4′), 3.59 (dd, J6′a,6′b
=
10.6, J5′,6′a = 4.9 Hz, 1H, H-6′a), 3.48 (dd, J6a,6b = 12.2, J5,6b = 3.3 Hz, 1H,
H-6b), 3.32 (td, J4′,5′ = 9.7, J5′,6′ = 4.8 Hz, 1H, H-5′), 3.22 (dt, J4″,5″ = 9.8,
J
5″,6″ =2.9 Hz, 1H, H-5″), 3.00−2.95 (m, 1H, H-6′b), 2.74 (td, J = 6.5, 3.5
Hz, 2H, −COCH2CH2), 2.65 (q, J = 6.8 Hz, 2H, −COCH2CH2), 2.59−
2.55 (m, 2H, −COCH2CH2), 2.42−2.39 (m, 1H, −COCH2CHH),
2.33−2.31 (m, 1H, −COCH2CHH), 2.21 (s, 3H, −COCH3), 2.20 (s,
3H, −COCH3), 1.79 (s, 3H, −COCH3); 13C NMR (150 MHz, CDCl3)
δ
206.6 (−CH2COCH3), 206.2 (−CH2COCH3), 172.5
(−OCOCH2CH2), 171.7 (−OCOCH2CH2), 169.2 (−COCH3),
165.7 (−COPh), 165.5 (−COPh), 164.9 (−COPh), 164.4 (−COPh),
160.7 (−CNH), 136.9 (Ar-C), 134.0 (Ar-C), 133.5 (Ar-C), 133.3 (Ar-
C), 132.9 (Ar-C), 130.8 (Ar-C), 129.9−129.5 (m, Ar-C), 128.7−128.3
(m, Ar-C), 128.1 (Ar-C), 126.2 (Ar-C), 123.5 (Ar-C), 101.6 (−CHPh),
99.9 (C-1′), 98.9 (C-1″), 92.9 (C-1), 90.4 (−CCl3), 80.4 (C-4′), 76.0
(C-4), 75.9 (C-3′), 73.4 (C-3″), 72.1 (C-2″), 71.4 (C-5″), 70.8 (C-5),
70.7 (C-2), 70.5 (C-3), 68.0 (C-6′), 67.7 (C-4″), 65.9 (C-5′), 61.4 (C-6,
C-6″), 55.3 (C-2′), 38.0 (−COCH2CH2), 37.9 (−COCH2CH2), 29.9
(−COCH3), 29.9 (−COCH3), 28.0 (−COCH2CH2), 27.6
(−COCH2CH2), 20.5 (−COCH3); HRMS m/z calcd for
C75H73Cl3N3O26 [M + NH4]+1536.3542, found1536.3604.
6-Azidohexyl 4,6-di-O-benzylidene-3-O-chloroacetyl-2-
deoxy-2-phthalimido-β-D-glucopyranosyl-(1 → 4)-2,4-di-O-
benzoyl-6-O-levulinoyl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-
benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside
(17). To a mixture of compound 14 (543 mg, 0.50 mmol) and 11 (236
mg, 0.452 mmol), containing molecular sieves 4 Å (1 g), in
dichloromethane (8 mL) at −40 °C was added dropwise trimethylsilyl
trifluoromethanesulfonate (9 μL, 50 μmol) under N2. The reaction
mixture was stirred for 2 h, with the temperature slowly warming to 0 °C,
and neutralized with Et3N. The resulting mixture was filtered through
Celite, and the filtrate was concentrated. Column chromatography of
the residue with EtOAc/hexane (3:2) as eluent yielded the trisaccharide
17 as a white foam (535 mg, 82%): [α]2D3 = +42.0 (c 1.0, CHCl3); 1H
NMR (600 MHz, CDCl3) δ 7.88−7.81 (m, 6H, Ar-H), 7.51−7.45 (m,
2H, Ar-H), 7.40−7.29 (m, 7H, Ar-H), 7.29−7.26 (m, 8H, Ar-H), 7.14−
7.11 (m, 5H, Ar-H), 5.68 (dd, J2″,3″ = 10.2, J3″,4″ = 9.4 Hz, 1H, H-3″), 5.51
J
1′,2′ = 8.4 Hz, 1H, H-1′), 5.14 (t, J3″,4″ = J4″,5″ = 9.7 Hz, 1H, H-4″), 5.12
(dd, J1″,2″ = 8.0, J2″,3″ = 9.7 Hz, 1H, H-2″), 4.98 (d, J1,2 = = 7.6 Hz, 1H, H-
1), 4.74 (d, J1″,2″ = 8.0 Hz, 1H, H-1″), 4.62 (dd, J2′,3′ = 10.3, J3′,4′ = 8.8 Hz,
1H, H-3′), 4.25 (dd, J2′,3′ = 10.3, J1′,2′ = 8.4 Hz, 1H, H-2′), 4.13 (dd, J6a,6b
= 11.9, J5,6a = 1.6 Hz, 1H, H-6a), 4.05 (t, J3,4 = J4,5 = 9.0 Hz 1H, H-4), 3.92
(dd, J6″a,6″b = 12.1, J5″,6″a = 3.2 Hz, 1H, H-6″a), 3.86 (dd, J6″a,6″b = 12.1,
J5″,6″a = 2.2 Hz, 1H, H-6″b), 3.69 (s, 3H, −OCH3), 3.63 (t, J3′,4′ = J4′,5′
=
9.1 Hz, 1H, H-4′), 3.61−3.59 (m, 1H, H-5), 3.57 (dd, J6′a,6′b = 10.6, J5′,6′a
= 4.8 Hz, 1H, H-6′a) 3.48 (dd, J6a,6b = 12.0, J5,6b = 4.1 Hz, 1H, H-6b),
3.34 (td, J4′,5′ = 9.7, J5′,6′ = 4.8 Hz 1H, H-5′), 3.23 (dt, J4″,5″ = 10.0, J5″,6″
=
2.8 Hz, 1H, H-5″), 2.82 (t, J6′a,6′b = 10.4 Hz, 1H, H-6′b), 2.74 (td, J = 6.6,
3.2 Hz, 2H, −COCH2CH2), 2.64−2.54 (m, 4H, −COCH2CH2,
−COCH2CH2), 2.42−2.30 (m, 2H, −COCH2CH2), 2.21 (s, 3H,
−COCH3), 2.18 (s, 3H, −COCH3), 1.79 (s, 3H, −COCH3); 13C NMR
(150 MHz, CDCl3) δ 206.6 (−CH2COCH3), 206.2 (−CH2COCH3),
172.5 (−OCOCH2CH2), 171.7 (−OCOCH2CH2), 169.2 (−COCH3),
165.7 (−COPh), 165.2(−COPh), 165.0 (−COPh), 164.4 (−COPh),
155.7 (Ar-C), 150.9 (Ar-C), 136.9 (Ar-C), 133.9 (Ar-C), 133.5 (Ar-C),
133.3 (Ar-C), 133.3 (Ar-C), 132.8 (Ar-C), 130.8 (Ar-C), 129.9−129.5
(m, Ar-C), 129.2 (Ar-C), 128.7−128.3 (m, Ar-C), 128.1 (Ar-C), 126.2
(Ar-C), 123.5 (Ar-C), 118.9 (Ar-C), 114.5(Ar-C), 101.6 (−CHPh),
100.2 (C-1), 100.0 (C-1″), 98.8 (C-1′), 80.5 (C-4′), 76.3 (C-4), 75.9
(C-3′), 73.4 (C-3), 73.3 (C-3″), 72.5 (C-5), 72.1 (C-2″), 71.7 (C-2),
71.4 (C-5″), 67.9 (C-6′), 67.7 (C-4″), 65.9 (C-5′), 61.8 (C-6), 61.4 (C-
6″), 55.6 (−OCH3), 55.2 (C-2′), 37.9 (−COCH2CH2 X 2), 29.9
(−COCH3 X 2), 27.9 (−COCH2CH2), 27.6 (−COCH2CH2), 20.5
(−COCH3); HRMS m/z calcd for C80H79N2O27 [M + NH4]+
1499.4865, found 1499.4895; m/z calcd for C80H83N3O27 [M +
2NH4]2+ 758.7601, found 758.7623.
(s, 1H, −CHPh), 5.33 (t, J2′,3′ = J3′,4′ = 9.3 Hz, 1H, H-3′), 5.32 (d, J1″,2″
=
8.2 Hz, 1H, H-1″), 5.12 (s, 1H, −CHPh), 5.08 (d, J1,2 = 8.5 Hz, 1H, H-
1), 5.05 (dd, J2′,3′ = 9.7, J1′,2′ = 8.2 Hz, 1H, H-2′), 4.68 (d, J1′,2′ = 8.1 Hz,
1H, H-1′), 4.63 (dd, J2,3 = 10.4, J3,4 = 8.7 Hz, 1H, H-3), 4.30 (dd, J6a,6b
=
10.6, J5,6b = 4.8 Hz, 1H, H-6a), 4.21 (dd, J2,3 = 10.4, J1,2 = 8.6 Hz, 1H, H-
2), 4.11 (dd, J2″,3″ = 10.2, J1″,2″ = 8.1 Hz, 1H, H-2″), 3.96 (dd, J6′a,6′b
12.1, J5′,6′a = 1.5 Hz, 1H, H-6′a), 3.88 (t, J3′,4′ = J4′,5′ = 9.3 Hz, 1H, H-4′),
4-O-Acetyl-2,3-di-O-benzoyl-6-O-levulinoyl-β-D-glucopyra-
nosyl-(1 → 3)-4,6-di-O-benzylidene-2-deoxy-2-phthalimido-β-
D-glucopyranosyl-(1 → 4)-2,3-di-O-benzoyl-6-O-levulinoyl-α-D-
glucopyranosyl trichloroacetimidate (16). To a solution of
compound 15 (720 mg, 0.486 mmol) in CH3CN/H2O (10 mL, v/v
4:1) at 0 °C was added cerium ammonium nitrate (845 mg, 1.46 mmol).
The reaction mixture was stirred for 1 h at 0 °C, at which time TLC
(EtOAc/hexane 3:1) indicated completion of the reaction. The reaction
mixture was diluted with EtOAc (100 mL), and washed with aqueous
saturated NaHCO3 and water. The organic layer was dried over MgSO4,
filtered, and concentrated. The residue was purified by flash column with
EtOAc/hexane (5:1) as the eluent to give the hemiacetal as a yellow
foam. This compound was then dissolved in dichloromethane (5 mL),
trichloroacetonitrile (0.3 mL) and DBU (50 μL) were added at 0 °C,
and the reaction mixture was stirred for 2 h, and concentrated. Column
chromatography of the residue (EtOAc/hexane 1:1) gave the
=
3.85 (t, J6a,6b = 10.3 Hz, 1H, H-6b), 3.79 (s, 2H, −CH2Cl), 3.77 (t, J3,4
=
J4,5 = 9.1 Hz, 1H, H-4), 3.77−3.71 (m, 1H, −OCHH(CH2)5N3), 3.59
(td, J4,5 = 9.8, J5,6 = 4.9 Hz, 1H, H-5), 3.48 (dd, J6″a,6″b = 10.8, J5″,6″a = 4.9
Hz, 1H, H-6″a), 3.41 (t, J3″,4″ = J4″,5″ = 9.4 Hz, 1H, H-4″), 3.32 (td, J4″,5″
=
9.7, J5″,6″ = 4.8, 1H, H-5″), 3.31−3.27 (m, 1H, −OCHH(CH2)5N3), 3.00
(dd, J6′a,6′b = 12.1, J5′,6′b = 2.5 Hz, 1H, H-6′b), 2.96 (t, J = 7.0 Hz, 2H,
−OCH2(CH2)4CH2N3), 2.89 (dt, J4′,5′ = 9.7, J5′,6′ = 2.4, 1H, H-5′),
2.83−2.71 (m, 2H, −COCH2CH2), 2.59 (t, J6″a,6″b = 10.4 Hz, 1H, H-
6″b), 2.52−2.39 (m, 2H, −COCH2CH2), 2.27 (s, 3H, −COCH3),
1.41−1.30 (m, 1H, −OCH2CHH(CH2)4N3), 1.30−1.22 (m, 1H,
−OCH2CHH(CH2)4N3), 1.22−1.08 (m, 2H, −O(CH2)4CH2CH2N3),
1.07−0.86 (m, 4H, −O(CH2)2(CH2)2(CH2)2N3); 13C NMR (150
MHz, CDCl3) δ 206.4 (−CH2COCH3), 172.0 (−OCOCH2CH2), 166.7
J
dx.doi.org/10.1021/jo4012442 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
(−COCH2Cl), 164.9 (−COPh), 164.5 (−COPh), 136.9 (Ar-C), 136.6
(Ar-C), 134.6 (br, Ar-C), 133.8 (Ar-C), 133.3 (Ar-C), 132.8 (Ar-C),
131.1 (br, Ar-C), 129.8 (Ar-C), 129.6 (Ar-C), 129.5 (Ar-C), 129.3 (Ar-
C), 129.2 (Ar-C), 128.6 (Ar-C), 128.5 (Ar-C), 128.3 (Ar-C), 128.2 (Ar-
C), 128.1 (Ar-C), 126.2 (Ar-C), 126.1 (Ar-C), 101.9 (−CHPh), 101.4
(−CHPh), 100.2 (C-1′), 98.6 (C-1″, C-1), 81.4 (C-4), 78.3 (C-4″), 76.3
(C-3), 75.8 (C-4′), 73.7 (C-3′), 72.3 (C-2′), 71.9 (C-5′), 71.4 (C-3″),
69.7 (−OCH2(CH2)5N3), 68.9 (C-6), 67.6 (C-6″), 66.3 (C-5), 65.5 (C-
5″), 60.9 (C-6′), 55.1 (C-2), 55.0 (C-2″), 51.1 (−O(CH2)5CH2N3),
40.2 (−COCH2Cl), 38.2 (−COCH2), 30.1 (−COCH3), 29.0
(−OCH2CH2(CH2)4N3), 28.6 (−O(CH2)4CH2CH2N3), 27.8
(−OCOCH2), 26.2 (−O(CH2)2CH2(CH2)3N3), 25.3 (−O-
(CH2)3CH2(CH2)2N3); HRMS m/z calcd for C75H76ClN6O23 [M +
NH4]+ 1463.4645, found 1463.4689.
dichloromethane (8 mL) at rt was stirred with molecular sieves 4 Å (1 g)
for 1 h under N2. Trimethylsilyl trifluoromethanesulfonate (5 μL, 27
μmol) was then added at −40 °C. The reaction mixture was stirred for 2
h, with temperature slowly warming to 0 °C, at which time TLC
(EtOAc/hexane 2:1) indicated the complete consumption of the
trisaccharide trichloroacetimidate 16. The reaction mixture was
neutralized with Et3N, filtered through Celite, the solvent was
evaporated under reduced pressure, and the resulting residue was
purified by flash column with EtOAc/hexane (2:1) as the eluent to yield
the hexasaccharide 19 as a white foam (435 mg, 70%): [α]2D3 = +91.0 (c
1.0, CHCl3); 1H NMR (600 MHz, CDCl3) δ 7.79−7.76 (m, 4H, Ar-H),
7.68−7.66 (m, 3H, Ar-H), 7.48−7.28 (m, 24H, Ar-H), 7.26−7.22 (m,
7H, Ar-H), 7.19−7.03 (m, 16H, Ar-H), 7.00−6.99 (m, 3H, Ar-H), 5.45
(s, 1H, −CHPh), 5.23 (t, J2,3 = J3,4 = 9.3 Hz, 1H, H-3B), 5.22 (t, J2,3 = J3,4
= 9.6 Hz, 1H, H-3F), 5.21 (s, 1H, −CHPh), 5.15 (t, J2,3 = J3,4 = 9.3 Hz,
1H, H-3D), 5.10 (t, J3,4 = J4,5 = 9.7 Hz, 1H, H-4F), 5.05 (dd, 1H, J1,2 = 8.0,
J2,3 = 9.5 Hz, H-2F), 5.05 (d, 1H, J1,2 = 8.5 Hz, H-1A), 5.04 (s, 1H,
−CHPh), 5.00 (d, J1,2 = 8.4 Hz, 1H, H-1E), 4.99 (d, J1,2 = 8.6 Hz, 1H. H-
6-Azidohexyl 4,6-di-O-benzylidene-2-deoxy-2-phthalimido-
β-D-glucopyranosyl-(1 → 4)-2,4-di-O-benzoyl-6-O-levulinoyl-β-
D-glucopyranosyl-(1 → 3)-4,6-di-O-benzylidene-2-deoxy-2-
phthalimido-β-D-glucopyranoside (18). To a solution of com-
pound 17 (491 mg, 0.34 mmol) in dichloromethane/methanol (20 mL,
1:4 v/v) at rt was added thiourea (130 mg, 1.7 mmol) and 2,4-lutidine
(50 μL). The reaction mixture was refluxed for 6 h, the solvent was
removed under reduced pressure, and the resulting residue was
dissolved in dichloromethane (100 mL) and washed with 1 N HCl,
water, and brine. The organic layer was dried over MgSO4, filtered and
concentrated. Purification of the residue on flash column with EtOAc/
hexane (3:2) as eluent gave the trisaccharide acceptor 18 as a white foam
1C), 4.97 (dd, J1,2 = 8.3, J2,3 = 9.5 Hz, 1H, H-2B), 4.88 (dd, J1,2 = 8.3, J2,3
=
9.5 Hz, 1H, H-2D), 4.68 (d, J1,2 = 8.0 Hz, 1H, H-1F), 4.60 (d, J1,2 = 8.3 Hz,
1H, H-1B), 4.57 (dd, J2,3 = 10.2, J3,4 = 8.8 Hz, 1H, H-3A), 4.47 (t, J2,3 = J3,4
= 10.0 Hz, 1H, H-3E), 4.46 (d, J1,2 = 8.3 Hz, 1H, H-1D), 4.35 (dd, J2,3
=
10.0, J3,4 = 9.0 Hz, 1H, H-3C), 4.27 (dd, J6a,6b = 10.6, J5,6a = 4.8 Hz, 1H, H-
6aA), 4.16 (dd, J2,3 = 10.3, J1,2 = 8.6 Hz, 1H, H-2A), 4.04 (dd, J2,3 = 10.2,
J1,2 = 8.5 Hz, 1H, H-2E), 3.98 (dd, J2,3 = 10.2, J1,2 = 8.5 Hz, 1H, H-2C),
3.88−3.78 (m, 5H, H-6aF, 6bA, 6aB, 6aD, 6bF), 3.74−3.69 (m, 1H, 4H, H-
(363 mg, 78%): [α]2D3 = +54.0 (c 1.0, CHCl3); H NMR (600 MHz,
1
4
A,B,D, −OCHH(CH2)4CH2N3), 3.54 (td, J4,5 = 9.7, J5,6 = 4.9 Hz, 1H, H-
CDCl3) δ 7.87−7.83 (m, 4H, Ar-H), 7.81 (dd, J = 5.5, 3.0 Hz, 2H, Ar-H),
7.48−7.45 (m, 2H, Ar-H), 7.40−7.27 (m, 15H, Ar-H), 7.17−7.11 (m,
5H, Ar-H), 5.53 (s, 1H, −CHPh), 5.34 (t, J2′,3′ = J3′,4′ = 9.3 Hz, 1H, H-
3′), 5.20 (d, J1″,2″ = 8.3 Hz, 1H, H-1″), 5.16 (s, 1H, −CHPh), 5.08 (d, J1,2
= 8.5 Hz, 1H, H-1), 5.05 (dd, J2′,3′ = 9.6, J1′,2′ = 8.2 Hz, 1H, H-2′), 4.68
(d, J1′,2′ = 8.1 Hz, 1H, H-1′), 4.63 (dd, J2,3 = 10.4, J3,4 = 8.7 Hz, 1H, H-3),
4.36 (dd, J3″,4″ = 8.8, J2″,3″ = 10.5 Hz, 1H, H-3″), 4.30 (dd, J6a,6b = 10.6,
J5,6b = 4.8 Hz, 1H, H-6a), 4.21 (dd, J2,3 = 10.4, J1,2 = 8.6 Hz, 1H, H-2),
4.02 (dd, J1″,2″ = 8.3, J2″,3″ = 10.4 Hz, 1H, H-2″), 4.00−3.98 (m, 1H, H-
6′a), 3.87 (t, J3′,4′ = J4′,5′ = 9.4, Hz, 1H, H-4′), 3.86−3.83 (m, 1H, H-6b),
5A), 3.50 (t, J3,4 = J4,5 = 9.1 Hz, 1H, H-4E), 3.43 (dd, J6a,6b = 10.7, J5,6a = 4.7
Hz, 1H, H-6aE), 3.36−3.33 (m, 2H, H-4C, H-6aC), 3.29−3.25 (m, 1H,
OCHH(CH2)4CH2N3), 3.21 (td, J = 9.7, 4.9 Hz, 1H, H-5E), 3.17−3.11
(m, 2H, H-5C,F), 2.95 (t, J = 7.0 Hz, 2H, −OCH2(CH2)4CH2N3), 2.86−
2.80 (m, 3H, H-5B, H-6bB,D), 2.74−2.64 (m, 7H, H-5D, 3 ×
−COCH2CH2), 2.61 (t, J6a,6b = 10.6 Hz, 1H, H-6bE), 2.57−2.51 (m,
3H, H-6bC, −COCH2CH2), 2.42−2.28 (m, 4H, 2 × −COCH2CH2),
2.24 (s, 3H, −COCH3), 2.23 (s, 3H, −COCH3), 2.18 (s, 3H,
−COCH3), 1.77 (s, 3H, −COCH3), 1.35−1.29 (m, 1H, −OCH2CHH-
(CH2)4N3) 1.26−1.22 (m, 1H, −OCH2CHH(CH2)4N3), 1.19−1.08
(m, 2H, −O(CH2)4CH2CH2N3), 1.05−0.87 (m, 4H, −O-
(CH2)2(CH2)2(CH2)2N3); 13C NMR (150 MHz, CDCl3) δ 206.6
(−CH2COCH3), 206.4 (−CH2COCH3), 206.3 (−CH2COCH3), 172.4
(−OCOCH2CH2), 171.9 (−OCOCH2CH2), 171.8 (−OCOCH2CH2),
169.2 (−COCH3), 167.9 (br, −COPhth), 166.5 (br, −COPhth), 165.7
(−COPh), 164.8 (−COPh), 164.7 (−COPh), 164.5 (−COPh), 164.4
(−COPh), 164.3 (−COPh), 136.9 (2C, Ar-C), 136.6 (Ar-C), 133.9−
133.8 (m, Ar-C), 133.3 (Ar-C), 133.2 (Ar-C), 132.8 (2C, Ar-C), 132.7
(Ar-C), 130.9 (br, Ar-C), 130.4 (br, Ar-C), 129.8−129.3 (m, Ar-C),
128.7−128.1 (m, Ar-C), 126.1 (Ar-C), 126.0 (Ar-C), 125.9 (Ar-C),
123.4 (br, Ar-C), 101.9 (−CHPh), 101.6 (−CHPh), 101.5 (−CHPh),
100.1 (C-1B), 99.9 (C-1F), 99.6 (C-1D), 98.6 (C-1A), 98.5 (2C, C-1C,E),
81.3 (C-4A), 80.4 (2 C, C-4C,E), 76.2 (C-3A), 75.9 (C-3E), 75.7 (C-3C),
75.6 (C-4B), 75.5 (C-4D), 73.5 (2 C, C-3B,D), 73.3 (C-3F), 72.3 (C-2B),
72.2 (C-2D), 72.1 (C-2F), 71.9 (C-5B), 71.7 (C-5D), 71.3 (C-5F), 69.7
(−OCH2(CH2)5N3), 68.8 (C-6A), 67.8 (C-6E), 67.7 (2 C, C-4F and C-
6C), 66.3 (C-5A), 65.7 (C-5E), 65.6 (C-5C), 61.3 (C-6F), 60.8 (C-6B),
60.8 (C-6D), 55.1 (C-2A), 54.9 (C-2E), 54.9 (C-2C), 51.1 (−O-
(CH2)5CH2N3), 38.1, 38.1, 37.9, 30.1 (−COCH3), 30.0 (−COCH3),
29.9 (−COCH3), 29.1 (−OCH2CH2(CH2)4N3), 28.5 (−O-
(CH2)4CH2CH2N3), 27.9, 27.8, 27.7, 26.1 (−O(CH2)2CH2(CH2)3N3),
25.3 (−O(CH2)3CH2(CH2)2N3), 20.5 (−COCH3); HRMS m/z calcd
for C146H142N7O47 [M + NH4]+ 2744.8931, found 2744.8934; m/z calcd
for C146H146N8O47 [M + 2NH4]2+ 1381.9651, found 1381.9659.
6-Azidohexyl 4-O-acetyl-2,3-di-O-benzoyl-6-O-levulinoyl-β-
D-glucopyranosyl-(1 → 3)-4,6-di-O-acetyl-2-deoxy-2-phthalimi-
do-β-D-glucopyranosyl-(1 → 4)-2,3-di-O-benzoyl-6-O-levulino-
yl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-acetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl-(1 → 4)-2,4-di-O-benzoyl-6-
O-levulinoyl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-acetyl-2-
deoxy-2-phthalimido-β-D-glucopyranoside (20). A solution of
the hexasaccharide 19 (400 mg, 0.147 mmol) in 80% HOAc (20 mL)
was stirred at 80 °C for 5 h, at which time TLC indicated the
3.78−3.71 (m, 2H, H-4, −OCHH(CH2)5N3), 3.59 (td, J4,5 = 9.7, J5,6
4.9 Hz, 1H, H-5), 3.44 (dd, J6″a,6″b = 10.8, J5″,6″a = 4.7 Hz, 1H, H-6″a),
3.29 (ddd, J = 9.9, 7.4, 5.6 Hz, 1H, −OCHH(CH2)5N3), 3.22 (t, J3″,4″
4″,5″ = 9.0 Hz, 1H, H-4″), 3.18 (td, J4″,5″ = 9.4, J5″,6″ = 4.7 Hz, 1H, H-5″),
=
=
J
3.08 (dd, J6′a,6′b = 12.1, J5′,6′a = 2.6 Hz, 1H, H-6′b), 2.96 (t, J = 7.0 Hz, 2H,
−OCH2(CH2)4CH2N3), 2.93 (dt, J4′,5′ = 9.9, J5′,6′ = 2.2 Hz, 1H, H-5′),
2.80−2.69 (m, 2H, −COCH2CH2), 2.65−2.62 (m, 1H, H-6″b), 2.48−
2.37 (m, 2H, −COCH2CH2), 2.27 (s, 3H, −COCH3), 1.34−1.31 (m,
1 H , − O C H 2 C H H ( C H 2 ) 4 N 3 ) , 1 . 2 9 − 1 . 2 3 ( m , 1 H ,
−OCH2CHH(CH2)4N3), 1.20−1.09 (m, 2H, −O(CH2)4CH2CH2N3),
1.07−0.88 (m, 4H, −O(CH2)2(CH2)2(CH2)2N3); 13C NMR (150
MHz, CDCl3) δ 206.4 (−CH2COCH3), 172.0 (−OCOCH2CH2), 164.9
(−COPh), 164.5 (−COPh), 136.9 (Ar-C), 136.8 (Ar-C), 134.4 (Ar-C),
133.8 (Ar-C), 133.2 (Ar-C), 132.7 (Ar-C), 131.5 (Ar-C), 129.9−129.4
(m, Ar-C), 128.6 (Ar-C), 128.4−128.3 (m, Ar-C), 128.1 (Ar-C), 126.3
(Ar-C), 126.1 (Ar-C), 123.8 (Ar-C), 101.9 (−CHPh), 101.7 (−CHPh),
100.1 (C-1′), 98.8 (C-1″), 98.6 (C-1), 81.4 (C-4″), 81.4 (C-4), 76.2 (C-
3), 75.7 (C-4′), 73.7 (C-3′), 72.4 (C-2′), 72.1 (C-5′), 69.7
(−OCH2(CH2)5N3), 68.9 (C-6), 68.4 (C-3″), 67.7 (C-6″), 66.3 (C-
5), 65.7 (C-5″), 60.9 (C-6′), 56.6 (C-2″), 55.1 (C-2), 51.1
(−O(CH2)5CH2N3), 38.2 (−COCH2), 30.1 (−COCH3), 29.1
(−OCH2CH2(CH2)4N3), 28.6 (−O(CH2)4CH2CH2N3), 27.8
(−OCOCH2), 26.2 (−O(CH2)2CH2(CH2)3N3), 25.3 (−O-
(CH2)3CH2(CH2)2N3); HRMS m/z calcd for C73H75N6O22 [M +
NH4]+ 1387.4929, found 1387.4973.
6-Azidohexyl 4-O-acetyl-2,3-di-O-benzoyl-6-O-levulinoyl-β-
D-glucopyranosyl-(1 → 3)-4,6-di-O-benzylidene-2-deoxy-2-
phthalimido-β-D-glucopyranosyl-(1 → 4)-2,3-di-O-benzoyl-6-
O-levulinoyl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-benzyli-
dene-2-deoxy-2-phthalimido-β-D-glucopyranosyl-(1 → 4)-2,4-
di-O-benzoyl-6-O-levulioyl-β-D-glucopyranosyl-(1 → 3)-4,6-di-
O-benzylidene-2-deoxy-2-phthalimido-β-D-glucopyranoside
(19). A mixture of the trisaccharide trichloroacetimidate 16 (415 mg,
0.273 mmol), the trisaccharide acceptor 18 (312 mg, 0.228 mmol) in
K
dx.doi.org/10.1021/jo4012442 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
a
1
Table 2. H and 13C NMR Data for Compounds 23a, 23b, 24a, and 24b
resonance
23a δ (ppm)
23b δ (ppm)
24a δ (ppm)
24b δ (ppm)
CH3
22.0, 22.3
CH3 (3C)
CH2ε/CH2ζ
CH2δ/CH2γ
CH2β/CH2ε
CH2-carbonyl
CH2-carbonyl (3C)
CH2β
22.3, 22.5
8.9, 9.3
9.0, 9.4
24.8, 25.7
28.1, 28.5
24.6, 25.4
27.8, 28.3
24.4, 25.1
26.5, 28.1
24.6, 25.1
26.5, 28.3
29.1, 29.3
51.0
29.2, 29.3
51.2
CH2ζ
39.2
39.3
C-2 CE
54.1, 54.1
54.4
54.0, 54.1
54.4
C-2 A
C-2 ACE
C-6 ACE
C-6 CE
54.3, 54.4, 54.6
60.6, 60.8
54.0, 54.1, 54.3
60.3, 60.5
60.2, 60.3
60.5
C-6 A
C-6 ACE (3C)
C-4 A
60.4, 60.6
68.1
C-4 CE
68.3, 68.3
C-4 ACE
CH2α
68.3, 68.4, 68.6
68.1, 68.2, 68.4
68.3, 68.4, 68.5
70.3
70.5
71.4
70.3
71.3
70.2
C-4 F
71.5
71.6
C-2BD
72.2, 72.3
72.5
72.3, 72.3
72.6
C-2F
C-2 BDF
C-3 BD
72.3, 72.3, 72.6
73.7
72.1, 72.4
73.3
73.3, 73.4
73.4
C-5 BDF
C-5 ACE/C-3F
C-5F
74.5
74.9
75.6, 76.0, 76.2
75.1, 75.2, 75.3
75.2, 75.5, 75.5
75.2
75.1, 75.1, 75.2, 75.2
75.6
C-5 BD
76.1, 76.2
C-4 BD
80.1
79.3
79.7, 79.8
79.4, 79.5
C-3 CE
81.8, 82.2
C-3ACE
C-5 ACE
C-3 A
82.4, 82.7,82.8
81.8, 82.1, 82.2
81.6, 82.0, 82.6
82.8
C-1 ACE
C-1 CE
101.0, 101.1
100.5
100.4, 100.5
100.3, 100.3
100.9
C-1 A
100.8
100.8
C-1 BDF
CO (6C)
CH3
102.8, 103.0, 103.1
174.6,174.9, 174.9
102.5,102.7,102.9
178.2, 178.4
0.99
102.8, 102.9, 103.0
174.0,174.0,174.3, 174.8, 175.3
102.7, 102.8, 102.9
173.8, 173.9,175.1, 178.2, 178.6
0.99
H-ζ/H-ε/H-δ/H-γ (8H)
CH2 (6H)
CH3 (9H)
H-ζ (2H)
H-β (2H)
H-2 F
1.37, 1.57, 1.61
2.03
1.47, 1.42, 1.22
2.16
1.27, 1.47, 1.56
1.22, 1.43, 1.47
2.16
1.93
2.89
3.19
3.33
3.36
3.38
3.47
3.50
3.52
3.19
3.22
3.25
3.33
3.36
3.38
3.23
3.26
3.36
3.39
3.41
3.41
3.45
3.27
3.30
3.40
3.42
3.42
3.43
H-2 BD
H-5 A
H-5 CE
H-4 A
H-4 F/H-3 F
H-4 C/H-4 E
H-3 F
3.53
3.56
3.57
3.62
3.64
3.74
3.78
3.80
3.38
3.45
3.44
3.53
3.48
3.64
3.70
3.69
H-4 CE
3.47
H-4 F
H-α
3.50
3.49
H-3 BD
3.50
3.70
H-3 ACE
H-6 ACE
H-4 BD
H-5 BD
3.61
L
dx.doi.org/10.1021/jo4012442 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
Table 2. continued
resonance
H-3 CE
23a δ (ppm)
23b δ (ppm)
24a δ (ppm)
24b δ (ppm)
3.62
3.64
H-3 A
H-5 BDF
H-α
3.53
3.66
3.70
3.70
3.70
H-5 F
3.64
H-5 BD
H-6 ACE
H-4 BD
H-6 A
3.66
3.66
3.68
3.72
3.75
H-6 CE
H-2 A
3.82
3.86
3.90
3.91
3.92
3.69
3.75
3.81
H-2 CE
H-5 BDF
H-α
3.74
3.81
3.84
3.85
3.81
3.84
3.85
H-6 ACE
H-6 A
3.82
3.82
H-6 CE
H-1 BD
H-1 F
4.40
4.42
4.46
4.37
4.42
4.39
H-1 A
4.53
4.47
H-1 BD
H-1 BDF
H-1 CE
4.54
4.59
4.45
4.53
4.47
4.52
a
1H NMR spectra for 23a, 23b, and 24b were recorded at 800 MHz. All 13C NMR spectra and the 1H NMR spectrum for the hexasaccharide 24a
were recorded at 150 and 600 MHz, respectively.
disappearance of the starting material. The solvent was coevaporated
with toluene/methanol (3 × 20 mL, v/v 1:1). The resulting residue was
then dissolved in pyridine (5 mL), and acetic anhydride (3 mL) was
added. The reaction mixture was stirred at rt overnight and then
concentrated with toluene. Purification of the resulting residue by flash
column using EtOAc/hexane (5:1) as eluent gave compound 20 as a
colorless foam (293 mg, 78%): [α]2D3 = +17.0 (c 1.0, CHCl3); 1H NMR
(600 MHz, CDCl3) δ 7.75−7.60 (m, 13H, Ar-H), 7.50−7.35 (m, 13H,
Ar-H), 7.26−7.14 (m, 16H, Ar-H), 5.33 (t, J = 9.6 Hz, 1H), 5.23 (t, J =
9.0 Hz, 1H), 5.18 (t, J = 9.0 Hz, 1H), 5.13 (t, J = 9.8 Hz, 1H), 5.11 (dd, J
= 7.9, 9.7 Hz, 1H), 5.03−4.99 (m, 2H), 4.96 (dd, J = 9.2, 7.8 Hz, 1H),
4.87 (d, J = 8.3 Hz, 1H), 4.83 (t, J = 8.8 Hz, 2H), 4.77 (t, J = 9.4 Hz, 1H),
4.69 (t, J = 9.1 Hz, 1H), 4.64 (dd, J = 9.1, 10.6 Hz, 1H), 4.54 (dd, J = 9.1,
10.7 Hz, 1H), 4.44 (dd, J = 9.2, 10.5 Hz, 1H), 4.42−4.39 (m, 2H), 4.27
(d, J = 7.6 Hz, 1H), 4.19 (dd, J = 12.0, 4.5 Hz, 1H), 4.14−4.00 (m, 5H),
3.98 (dd, J = 10.8, 8.4 Hz, 1H), 3.90−3.80 (m, 4H), 3.70−3.58 (m, 4H),
3.56−3.46 (m, 7H), 3.25−3.15 (m, 3H,), 2.95 (t, J = 7.1 Hz, 2H,
−OCH2(CH2)4CH2N3), 2.80−2.65 (m, 6H), 2.61−2.42 (m, 6H), 2.21
(s, 3H, −COCH3), 2.20 (s, 3H, −COCH3), 2.17 (s, 3H, −COCH3),
2.06 (s, 3H, −COCH3), 1.98 (s, 3H, −COCH3), 1.88 (s, 3H,
−COCH3), 1.85 (s, 3H, −COCH3), 1.83 (s, 6H, −COCH3 x 2), 1.78
(s, 3H, −COCH3), 1.30−1.24 (m, 1H, −OCH2CHH(CH2)4N3), 1.21−
1.16 (m, 1H, −OCH2CHH(CH2)4N3), 1.15−1.06 (m, 2H, m, 2H,
− O ( C H 2 ) 4 C H 2 C H 2 N 3 ) , 0 . 9 9 − 0 . 8 0 ( m , 4 H , − O -
(CH2)2(CH2)2(CH2)2N3); 13C NMR (150 MHz, CDCl3) δ 206.3
(−CH2COCH3), 206.1 (−CH2COCH3), 206.0 (−CH2COCH3), 172.4
(−OCOCH2CH2), 171.8 (−OCOCH2CH2), 171.8 (−OCOCH2CH2),
170.9 (−COCH3), 170.6 (2C, −COCH3), 169.3 (−COCH3), 169.3
(−COCH3), 169.2 (−COCH3), 169.0 (−COCH3), 165.8 (−COPh),
165.0 (2C, −COPh), 164.9 (−COPh), 164.9 (2C, −COPh), 134.4 (Ar-
C), 134.2 (Ar-C), 133.5 (Ar-C), 133.1 (2C, Ar-C), 132.8 (Ar-C), 132.6
(2C, Ar-C), 130.9 (Ar-C), 130.7 (Ar-C), 130.6 (Ar-C), 129.9−129.3 (m,
Ar-C), 128.5 (Ar-C), 128.4 (Ar-C), 128.2−128.1 (m, Ar-C), 124.0 (Ar-
C), 123.6 (br, Ar-C), 101.0, 100.9, 100.7, 98.2, 97.5 (2C), 76.5, 75.9,
75.6, 74.7, 74.5, 73.0, 72.6 (2C), 72.5, 72.4, 72.2, 72.1 (2C), 72.0 (2C),
71.9 (2C), 69.4 (−OCH2(CH2)5N3), 69.3, 68.9, 68.8, 67.8, 62.4 (2C),
62.3, 62.0, 61.8, 61.7, 55.4 (2C), 55.3, 51.1 (−O(CH2)5CH2N3), 37.8
(3C), 30.0 (2C, −COCH3 ), 29.9 (−COCH3 ), 29.0
(−OCH2CH2(CH2)4N3), 28.5 (−O(CH2)4CH2CH2N3), 27.8, 27.7,
2 7. 6 , 26 . 1 ( − O ( C H 2 ) 2 C H2 ( C H 2 ) 3 N 3 ) , 2 5 . 3 ( −O -
(CH2)3CH2(CH2)2N3), 20.9 (2C, −COCH3), 20.8 (−COCH3), 20.7
(−COCH3), 20.6 (−COCH3), 20.5 (2C, −COCH3); HRMS m/z calcd
for C137H142N7O53 [M + NH4]+ 2732.8626, found 2732.8661; m/z calcd
for C137H146N8O53 [M + 2NH4]2+ 1375.9499, found 1375.9515.
6-Azidohexyl 4-O-acetyl-2,3-di-O-benzoyl-β-D-glucopyrano-
syl-(1 → 3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-gluco-
pyranosyl-(1 → 4)-2,3-di-O-benzoyl-β-D-glucopyranosyl-(1 →
3)-4,6-di-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl-
(1 → 4)-2,4-di-O-benzoyl-β-D-glucopyranosyl-(1 → 3)-4,6-di-O-
acetyl-2-deoxy-2-phthalimido-β-D-glucopyranoside (21). To a
solution of hexasaccharide 20 (248 mg, 91.3 μmol) in ethanol/toluene
(21 mL, v/v 2:1) at rt was added hydrazine acetate (120 mg, 1.38
mmol). The reaction mixture was stirred at rt for 3 h, and the solvent was
removed under reduced pressure. The residue was then dissolved in
dichloromethane (50 mL) and washed with 1 N HCl, water, and brine.
The organic layer was dried over anhydrous Na2SO4, filtered, and
concentrated. Column chromatography of the residue using EtOAc/
hexane (10:1) as the eluent afforded the hexasaccharide triol 21 as a
white foam (198 mg, 89%): [α]2D3 = +36.0 (c 1.0, CHCl3); 1H NMR (600
MHz, CDCl3) δ 7.78−7.71 (m, 6H, Ar-H), 7.66−7.60 (m, 7H, Ar-H),
7.54 (dd, J = 8.3, 1.2 Hz, 2H, Ar-H), 7.51−7.40 (m, 12H, Ar-H), 7.30−
7.17 (m, 15H, Ar-H), 5.43 (t, J = 9.6 Hz, 1H), 5.31 (t, J = 8.9 Hz, 1H),
5.28 (t, J = 8.8 Hz, 1H), 5.10 (dd, J = 9.7, 7.7 Hz, 1H), 5.06−5.02 (m,
3H), 5.00 (d, J = 8.4 Hz, 1H), 4.94 (dd, J = 8.9, 7.4 Hz, 1H), 4.89−4.84
(m, 3H), 4.77 (t, J = 9.4 Hz, 1H), 4.69 (dd, J = 9.2, 10.6 Hz, 1H), 4.65
(dd, J = 9.0, 10.8 Hz, 1H), 4.57 (d, J = 7.7 Hz, 1H), 4.51 (dd, J = 9.1, 10.8
Hz, 1H), 4.49 (d, J = 7.3 Hz, 1H), 4.39 (d, J = 7.2 Hz, 1H), 4.22−4.06
(m, 5H), 3.89 (td, J = 9.2, 3.5 Hz, 2H), 3.74−3.69 (m, 2H), 3.65−3.62
(m, 1H), 3.58−3.50 (m, 5H), 3.47−3.43 (m, 1H), 3.40 (d, J = 11.4 Hz,
1H), 3.37−3.35 (m, 1H), 3.27−3.21 (m, 2H), 3.20−3.17 (m, 1H),
3.15−3.10 (m, 3H), 2.96 (t, J = 7.0 Hz, 2H, −OCH2(CH2)4CH2N3),
2.06 (s, 3H, −COCH3), 1.94 (s, 3H, −COCH3), 1.90 (s, 3H,
−COCH3), 1.89 (s, 6H, −COCH3 x 2), 1.87 (s, 3H, −COCH3), 1.78
(s, 3H, −COCH3), 1.34−1.28 (m, 1H, −OCH2CHH(CH2)4N3), 1.25−
1.19 (m, 1H, −OCH2CHH(CH2)4N3), 1.17−1.08 (m, 2H, −O-
( C H 2 ) 4 C H 2 C H 2 N 3 ) , 1 . 0 4 − 0 . 8 4 ( m , 4 H , − O -
(CH2)2(CH2)2(CH2)2N3); 13C NMR (151 MHz, CDCl3) δ 170.9
M
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The Journal of Organic Chemistry
Article
(−COCH3), 170.6 (−COCH3), 170.6 (−COCH3), 170.2 (−COCH3),
169.8 (−COCH3), 169.7 (−COCH3), 169.5 (−COCH3), 165.7
(−COPh), 164.9 (4C, −COPh) 164.8 (−COPh), 134.5 (Ar-C), 134.3
(Ar-C), 134.2 (Ar-C), 133.5 (Ar-C), 133.2 (2C, Ar-C), 132.9 (Ar-C),
132.8 (Ar-C), 131.1 (Ar-C), 130.9 (Ar-C), 129.9−129.6 (m, Ar-C),
129.3−129.2 (m, Ar-C), 128.6 (Ar-C), 128.4−128.2 (m, Ar-C), 123.5
(br, Ar-C), 99.8 (2C), 99.6, 98.4, 97.9 (2C), 76.2, 75.9, 75.2, 74.8, 74.7,
74.6, 74.4, 74.3, 73.4, 72.9, 72.6, 72.3, 72.0, 71.9, 69.6
(−OCH2(CH2)5N3), 69.4, 68.9, 68.7 (2C), 62.3, 61.6 (2C), 61.2, 60.5
(2C), 55.7, 55.5 (2C), 51.2 (−O(CH2)5CH2N3), 29.0
(−OCH2CH2(CH2)4N3), 28.6 (−O(CH2)4CH2CH2N3), 26.1 (−O-
(CH2)2CH2(CH2)3N3), 25.3 (−O(CH2)3CH2(CH2)2N3), 20.9 (2C,
−COCH3), 20.8 (2C, −COCH3), 20.7 (2C, −COCH3), 20.6
(−COCH3); HRMS m/z calcd for C122H124N7O47 [M + NH4]+
2438.7523, found 2438.7610; m/z calcd for C122H128N8O47 [M +
2NH4]2+ 1228.8947, found 1228.8937.
C51H83N6O34 [M + H]+ 1323.4945, found 1323.4999; m/z calcd for
C51H84N6O34 [M + 2H]2+ 662.2509, found 662.2540.
6-Aminohexyl (β-D-glucopyranosyluronic acid)-(1 → 3)-(2-
deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 4)-(β-D-gluco-
pyranosyluronic acid)-(1 → 3)-(2-deoxy-2-acetamido-β-D-glu-
copyranosyl)-(1 → 4)-(β-D-glucopyranosyluronic acid)-(1 → 3)-
2-deoxy-2-acetamido-β-D-glucopyranoside (24a). To a solution
of 10% Pd−C (2 mg) and NaBH4 (6 mg) in water (1 mL) was added a
solution of compound 23a (12 mg, 9.4 μmol) in 50 mM aqueous NaOH
(1.5 mL). The reaction mixture was stirred at rt for 5 h and then filtered
through Celite. The filtrate was loaded on a size-exclusion column (Bio-
Gel P-2, 50 mM NH4HCO3) and chromatographed to afford the free-
amine 24a, after lyophilization, as a white foam (10 mg, 85%): [α]D23
=
−45 (c 0.1, H2O); The 1H and 13C NMR resonances are listed in Table
2; HRMS m/z calcd for C48H79N4O34 [M + H]+ 1255.4570, found
1255.4525; m/z calcd for C48H80N4O34 [M + 2H]2+ 628.2321, found
628.2293.
6-Azidohexyl (4-O-acetyl-2,3-di-O-benzoyl-β-D-glucopyrano-
syluronic acid)-(1 → 3)-(4,6-di-O-acetyl-2-deoxy-2-phthalimi-
do-β-D-glucopyranosyl)-(1 → 4)-(2,3-di-O-benzoyl-β-D-gluco-
pyranosyluronic acid)-(1 → 3)-(4,6-di-O-acetyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl)-(1 → 4)-(2,4-di-O-benzoyl-β-
D-glucopyranosyluronic acid)-(1 → 3)-4,6-di-O-acetyl-2-deoxy-
2-phthalimido-β-D-glucopyranoside (22). To a solution of the triol
21 (170 mg, 70.2 μmol) in dichloromethane (10 mL) was added
pyridinium dichromate (200 mg, 532 μmol) and Ac2O (150 μL). The
mixture was stirred at rt for 8 h, at which time TLC (EtOAc/HOAc
10:1) indicated the completion of reaction. The reaction mixture was
diluted with EtOAc (15 mL), and the suspended mixture was transferred
to column chromatography (EtOAc/HOAc 10:1 → 5:1) to yield the
triacid 22 as a brown solid (116 mg, 67%): [α]2D3 = +120.0 (c 0.5,
CHCl3); HRMS m/z calcd for C122H118N7O50 [M + NH4]+ 2480.6901,
found 2480.6939; m/z calcd for C122H122N8O50 [M + 2NH4]2+
1249.8636, found 1249.8661.
6-Aminohexyl (β-D-glucopyranosyluronic acid)-(1 → 3)-(2-
deoxy-2-propionamido-β-D-glucopyranosyl)-(1 → 4)-(β-D-glu-
copyranosyluronic acid)-(1 → 3)-(2-deoxy-2-propionamido-β-
D-glucopyranosyl)-(1 → 4)-(β-D-glucopyranosyluronic acid)-(1
→ 3)-2-deoxy-2-propionamido-β-D-glucopyranoside (24b).
Compound 24b (6.5 mg, 81%) was prepared as a white foam using a
similar protocol as 24a from compound 23b (8.2 mg, 6.2 μmol): [α]D23
=
−40.0 (c 0.1, H2O); The 1H and 13C NMR resonances are listed in Table
2; HRMS m/z calcd for C51H85N4O34 [M + H]+ 1297.5040, found
1297.5067; m/z calcd for C51H86N4O34 [M + 2H]2+ 649.2556, found
649.2569.
Preparation of the Hexasaccharide−Tetanus Toxoid (TT)
Conjugates (1a, 1b) and Hexasaccharide−Human Serum
Albumin (HSA) Conjugates. To a solution of 24a/b (5 mg) in 50
mM phosphate buffer (500 μL, pH 7.3) was added a solution of diethyl
squarate (2 equiv) in ethanol (500 μL). The mixture was stirred at rt for
20 h, at which time TLC (1-butanol:EtOH:H2O:HOAc 4:2:2:0.5)
indicated the disappearance of starting material and formation of one
major product. The solvent was removed under reduced pressure, and
size-exclusion chromatography (Bio-Gel P-2, 50 mM NH4HCO3) of the
residue yielded the monoethyl squarate compounds 25a/b (∼5.2 mg),
which were used directly for preparation of the protein conjugates. A
portion of the hexasaccharide (2.5 mg, 40−60 equiv of protein) was
added to a solution of protein (4.5 mg for TT; 3.0 mg for HSA) in 0.1 M
carbonate buffer (pH 10, 100 μL). After incubation for 7 days at rt,
analysis by MALDI-TOF mass spectrometry (sinapinic acid matrix)
indicated no further increase in mass had occurred. The reaction mixture
was dialyzed against distilled water (3 × 5 mL) using an Amicon
ulfrafiltration cell equipped with a Diaflo membrane. The residue was
taken up in water and lyophilized to give the corresponding protein-
hexasaccharide conjugates (4.4 mg for TT-1a; 4.2 mg for TT-1b; 2.9 mg
for HSA-1a and 2.8 mg for HSA-1b) as white powders.
MALDI-TOF MS data (sinapinic acid matrix, 0.1% TFA in 1:1
CH3CN/H2O): TT-hexasaccharide 1a conjugate (163 907 Da); TT-
hexasaccharide 1b conjugate (159 816 Da); HSA-hexasaccharide 1a
conjugate (76 482 Da); HSA-hexasaccharide 1b conjugate (75 548 Da).
The number of haptens per protein for each conjugate is listed in Table
1.
ELISA Solid-Phase Antigens. HSA-hexasaccharideNAc 1a, HSA-
hexasaccharideNCOPr 1b conjugates were used as solid-phase antigens
for the enzyme-linked immunosorbent assays (ELISA).
Esterification of a small amount of 22 with diazomethane in ether
1
gave the methyl ester 22a, which was used for analysis: Selected H
NMR data (500 MHz, CDCl3) δ 4.85, 4.83, 4.79 (3 d, 3 × 1H, J1,2 = 8.5
Hz, H-1A,C,E), 4.45, 4.32, 4.20 (3 d, 3 × 1H, J1,2 = 7.8 Hz, H-1B,D,F), 3.71,
3.44, 3.42 (3 s, 3 × 3H, 3 CH3O−), 2.95 (t, 2H, J = 7.0 Hz,
−CH2CH2N3); MALDI-TOF MS m/z calcd for C125H120N6NaO50 [M
+ Na]+ 2527.69, found 2528.08.
6-Azidohexyl (β-D-glucopyranosyluronic acid)-(1 → 3)-(2-
deoxy-2-acetamido-β-D-glucopyranosyl)-(1 → 4)-(β-D-gluco-
pyranosyluronic acid)-(1 → 3)-(2-deoxy-2-acetamido-β-D-glu-
copyranosyl)-(1 → 4)-(β-D-glucopyranosyluronic acid)-(1 → 3)-
2-deoxy-2-acetamido-β-D-glucopyranoside (23a). To a solution
of compound 22 (50 mg, 34.9 μmol) in n-butanol (10 mL) was added
ethylenediamine (8 mL). The reaction mixture was stirred at 90 °C for
20 h under N2. The solvent was coevaporated with toluene (3 × 10 mL)
under reduced pressure, and the residue was then dissolved in pyridine
(10 mL). Acetic anhydride (10 mL) was added to the above solution.
The reaction mixture was then stirred at rt overnight, concentrated, and
coconcentrated with toluene and methanol (3 × 10 mL). The yellow
residue was then dissolved in THF (15 mL), and aqueous 1 N LiOH (5
mL) was added at 0 °C. The reaction mixture was stirred for 20 h with
temperature slowly warming to rt, and then neutralized with aqueous 1
N HCl (5 mL), and the resulting mixture was concentrated. Purification
of the residue by gel filtration on Sephadex G-10 (water) gave 23a,
isolated after lyophilization as a white foam (18.7 mg, 72%): [α]2D3 = −42
(c 0.1, H2O); The 1H and 13C NMR resonances are listed in Table 2;
HRMS m/z calcd for C48H80N7O34 [M + NH4]+ 1298.4741, found
1298.4724; m/z calcd for C48H78N6O34 [M + 2H]2+ 641.2274, found
641.2253.
Experimental Groups of Mice and Immunization Protocol.
Groups of 10 female CD1 outbred mice (4−6 weeks old) were
immunized subcutaneously with 50 μg (based on the protein amount in
the conjugate) of the synthetic hexasaccharideNAc-TT conjugates 1a
and 1b. They received two doses of the conjugate vaccine at day 0 and
day 28. The first dose was formulated with Freund’s complete adjuvant
(FCA, Sigma Chemical Co., St. Louis, MO) and the second dose with
Incomplete Freund’s adjuvant (IFA, Sigma). Sera from mice were
collected on days 0, 28, and 38 and stored frozen until they were
analyzed for HA-specific IgG titer.
6-Azidohexyl (β-D-glucopyranosyluronic acid)-(1 → 3)-(2-
deoxy-2-propionamido-β-D-glucopyranosyl)-(1 → 4)-(β-D-glu-
copyranosyluronic acid)-(1 → 3)-(2-deoxy-2-propionamido-β-
D-glucopyranosyl)-(1 → 4)-(β-D-glucopyranosyluronic acid)-(1
→ 3)-2-deoxy-2-propionamido-β-D-glucopyranoside (23b).
Compound 23b (20.9 mg, 78%) was prepared by the same process as
for 23a by using propionic anhydride; it was obtained, after
ELISA. Antibody titers to hexasaccharideNAc-HSA and hexasacchar-
ideNCOPr-HSA in sera from mice were determined before vaccination
(day 0) and on days 28 and 38 by enzyme-linked immunosorbent assay
1
lyopholization, as a white foam: [α]2D3 = −60 (c 0.1, H2O); The H
and 13C NMR resonances are listed in Table 2; HRMS m/z calcd for
N
dx.doi.org/10.1021/jo4012442 | J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
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(ELISA). Serum samples were titered against the HSA conjugates in 96-
well plates (NUNC-Polysorp, Rochester, NY). Wells were coated with
100 μL of HSA conjugates (1 μg/mL) in PBS (pH 7.4, Quality
Biologicals, Gaithersburg, MD) per well for 1 h at 37 °C; the plates were
previously covered with adhesive film. Plates were aspirated and washed
three times with a washing buffer of 0.05% Tween 20 (Sigma Chemical
Co., St. Louis, MO) in PBS (PBS-T). The wells were blocked by the
addition of 150 μL of 0.5% BSA (Sigma) in PBS for 1 h at rt. This was
followed by aspiration of blocking buffer solution and washing the plates
(×3) as above. Plates were stored at 4 °C until further use. Antisera
serially diluted in PBS-T to a final 1:500 dilution (starting at 1:20) were
added (100 μL well) and incubated for 1 h at rt. This was followed by
washing three times the plates as above, followed by the addition of 100
μL of peroxidase-labeled goat antimouse IgG (H+L) conjugate
(Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD) (1:2500)
in PBS-T; plates were incubated for 1 h at rt. Plates were then washed
five times as above, and this was followed by addition of 100 μL per well
of substrate solution (SureBlue Reserve TMB, Kirkegaard & Perry
Laboratories), and plates were incubated for 5−10 min at rt. This was
followed by the addition of 100 μL of stop solution (1 N HCl). Plates
were scanned at 450 nm in a microplate reader. Absorbances
corresponding to antisera from day 38 were plotted versus log of
serum dilutions.
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ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of 1H and 13C NMR spectra. This material is available free
of charge via the Internet at http://pubs.acs.org.
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C. Org. Lett. 2012, 14, 3776−3779.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +1 778 782 4152. Fax: +1 778 782 4860. E-mail: bpinto@
sfu.ca.
■
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to the Natural Sciences and Engineering
Research Council of Canada for financial support. We thank F.
Michon for helpful discussions.
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