Synthesis and immunological evaluation of MUC1 glycopeptide conjugates bearing: N -acetyl modified STn derivatives as anticancer vaccines

Article abstract of DOI:10.1039/c6ob01092j

Glycoprotein MUC1 is an attractive target for anti-tumor vaccine development. However, the weak immunogenicity of MUC1 remains a significant problem. To solve this problem, several STn derivatives with N-acetyl modifications were synthesized and incorporated into a 20-amino acid MUC1 tandem repeat sequence. The modified STn-MUC1 glycopeptides were further connected to a carrier protein keyhole limpet hemocyanin (KLH). The immunological effects of these synthetic vaccine conjugates were evaluated using the BALB/c mouse model. The results showed that vaccine V2 elicited higher titers of antibodies which cross-reacted with the native STn-MUC1 antigen. Moreover, the elicited antisera reacted with the STn-MUC1 antigen-positive tumor cells, indicating that the carbohydrate antigen modification strategy may hold potential to overcome the weak immunogenicity of natural MUC1 glycopeptides.

Full text of DOI:10.1039/c6ob01092j

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DOI: 10.1039/C6OB01092J
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Synthesis and immunological evaluation of MUC1 glycopeptide
conjugates bearing N-acetyl modified STn derivatives as
anticancer vaccines†
Received 00th January 20xx,
Accepted 00th January 20xx
An Xiao,† Xiu-Jing Zheng,† Chengcheng Song, Yue Gui, Chang-Xin Huo and Xin-Shan Ye*
DOI: 10.1039/x0xx00000x
Glycoprotein MUC1 is an attractive target for anti-tumor vaccine development. However, the weak immunogenicity of
MUC1 remains a significant problem. To solve this problem, several STn derivatives with N-acetyl modifications were
www.rsc.org/
synthesized and incorporated into
a 20-amino acid MUC1 tandem repeat sequence. The modified STn-MUC1
glycopeptides were further connected to a carrier protein keyhole limpet hemocyanin (KLH). The immunological effects of
these synthetic vaccine conjugates were evaluated using the BALB/c mouse model. The results showed that vaccine V2
elicited higher titers of antibodies which cross-reacted with the native STn-MUC1 antigen. Moreover, the elicited antisera
reacted with the STn-MUC1 antigen-positive tumor cells, indicating that the carbohydrate antigen modification strategy
may hold the potential to break the weak immunogenicity of natural MUC1 glycopeptide.
therapeutic vaccines. Fluorination is one of the most
commonly-used modifications in drug design. The similar atom
radius and lipophilicity make fluorine a good mimic of
Introduction
Immunotherapy is a new and effective cancer therapy strategy,
which requires antitumor vaccines with high immunogenicity
and specificity.1 The transmembrane glycoprotein MUC1,
which is highly over-expressed in most breast, pancreatic and
ovarian malignancies,2 is one of the most optimal targets for
antitumor vaccine development. MUC1 consists of variable
number of tandem repeats (VNTRs), which is a 20-amino acid
sequence HGVTSAPDTRPAPGSTAPPA.3 Compared with MUC1
on normal cells, the MUC1 expressed on tumor cells is
significantly different, which is typically featured by over-
glycosylation with truncated and over-sialylated glycans.4 The
STn antigen, an O-linked disaccharide *Neu5Acα-(2-6)GalNAc],
is one of the most common tumor-associated carbohydrate
antigens (TACAs) expressed on MUC1.5
A main drawback in the development of anticancer
vaccines is the weak immunogenicity of MUC1 and TACAs. To
address this problem, the MUC1 glycopeptides are usually
connected with high immunogenic carrier proteins such as
keyhole limpet hemocyanin (KLH),6 bovine serum albumin
(BSA), and tetanus toxoid (TT),7 since the T-helper epitopes of
the carrier proteins contribute to the presentation of antigens
and the activation of T-helper cells, resulting in a strong IgG
immnune response. The chemical modification of antigens is
another approach to improving the immunogenicity of
hydrogen, which is crucial for the recognition of fluorinated
analogues by the enzymes and immune system. Moreover,
fluorinated analogues can be highly immunogenic since they
are exogenetic to most organisms. Some MUC1 glycopeptides
with deoxyfluoro TACAs, in which a hydroxyl group in the sugar
moiety was substituted by fluorine, were explored as
anticancer vaccines with promising antibody titers.8-13 Recently,
our group identified some N-acetyl modified STn antigens
eliciting stronger immune response than the native STn
antigen.14 The modified STn conjugates elicited a higher titer
of antibodies which could recognize the native STn antigen.
Among these modified antigens, three fluorinated STn
analogues (Figure 1) possessing good activities are worthy of
further studies. Based on our previous work, we herein report
the synthesis of glycopeptides incorporating these N-acetyl
modified STn analogues into a 20-amino acid MUC1 tandem
repeat, in the hope of improving the immunogenicity of
natural MUC1 glycopeptide. These modified STn-containing
glycopeptides as well as the native STn-MUC1 glycopeptide are
linked to the carrier protein KLH. The immunological
evaluation of these vaccines is also reported.
State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical
Sciences, Peking University, Xue Yuan Road No. 38, Beijing 100191, China. E-mail:
xinshan@bjmu.edu.cn; Fax: (+86) 10-82802724; Tel: (+86) 10-82805736
† These authors contributed equally to this work.
†
Electronic Supplementary Information (ESI) available: Immunological results,
NMR and mass spectra for new compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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the sialic acid moiety (Scheme 2). Starting from tVhieewpArrtoictleeOcntleinde
thiosialoside 7 24
the acetyl and methyl eDstOeIr: 1p0r.1o0t3e9c/tCin6OgBg0r1o0u9p2Js
,
were removed under strong basic conditions. After the
resulting amine functionality on the C5 position was modified
with N-fluoroacetyl group, the free carboxylic acid and
hydroxyl groups were protected with benzyl and acetyl groups
again to produce compound
hydrolyzed by N-bromosuccinimide (NBS) in H2O-acetone and
subsequently converted to the sialyl chloride . Finally,
compound was transformed into the N-fluoroacetylsialyl
8. The thiosialoside 8 was
9
9
xanthate donor 10 smoothly.
Figure 1 Three STn analogues with high immunogenicity.
Results and discussion
The PDTRP region is known as the main immune determining
domain,15-19 thus the MUC1 full length tandem repeat
sequence bearing N-acetyl modified STn derivatives on the
threonine of the PDTRP region was designed. Since the high
density of glycosylation might impede the recognition and
processing of MUC1 by antigen-presenting cells (APCs),19, 20
other potential glycosylation sites in the MUC1 sequence were
not glycosylated. To get the MUC1 sequence bearing STn
antigen and its N-acetyl modified derivatives, STn-Thr
[Neu5Acα-(2-6)GalNAcα-threonine] building blocks 21-24 for
solid-phase peptide synthesis were designed and synthesized.
The native STn-Thr building block 21 was prepared
according to the reported procedure.21 For the N-modification
on the GalNAc moiety, the direct glycosylation of threonine
with N-acetyl modified GalNAc would form a β-glycosidic
linkage due to the neighboring group participation effect,
which is not the desired configuration. Thus, the modification
was carried out after the glycosylation of threonine with 2-
Scheme 1 Synthesis of N-acetyl modified monosaccharide
building blocks. Reagents and conditions: a) Zn, AcOH; b)
NaHCO3, CH2FCOCl, CH3CN, 83% for
NaHCO3, (CHF2CO)2O, CH3CN, 95% for
3
4
over two steps; c)
over two steps; d)
azidogalactoside. As shown in Scheme 1, starting from the
-
glycoside 1 22, 23
,
the reduction of azido group and subsequent
NaOMe, MeOH, 69% for 5, 73% for 6.
modification conditions were carefully inspected since amino
acids are easily isomerized. For the reduction of azido group,
weak basic, neutral, or acidic conditions such as 1,3-
propanedithiol with trimethylamine, trimethylphosphine, and
zinc powder with acetic acid were tested. It was found that the
acidic conditions gave the best result with neither the
racemization of threonine nor the migration of the acetyl
groups. Using the optimized reduction conditions, compound
was obtained without further purification. During the
modification of the free amine in using triethylamine and the
2
2
methyl ester of corresponding carboxylic acid in methanol
under reflux,14 the racemization of the threonine moiety was
observed. To solve this problem, much milder conditions using
fluoroacetyl chloride or difluoroacetic anhydride in the
presence of NaHCO3 as base were employed. Under these
conditions, the corresponding products were isolated in good
yields (83% for
3, 95% for 4) without causing the racemization.
After removal of the acetyl protecting group, the resulting N-
acetyl modified monosaccharide building blocks
afforded to be ready for sialylation.
5 and 6 were
An N-fluoroacetylsialyl xanthate donor was prepared for
the purpose of synthesizing 24 with N-acetyl modification on
2 | J. Name., 2012, 00, 1-3
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for 15, 80% for 16; b) Ac2O, Py, 92% for 17, 86% for 18V,ie9w8%ArtifcolerO1n9li,ne
87% for 20; c) TFA, anisole, CH2Cl2, quantitatDivOeI.: 10.1039/C6OB01092J
Scheme 2 Synthesis of the N-fluoroacetyl sialic acid donor. Reagents
and conditions: a) NaOMe, MeOH; b) 2 M NaOH, 70 oC; c) TEA,
CH2FCOOMe, MeOH, 70 oC; d) KF, BnBr, DMF; e) Ac2O, Py, 61% over
5 steps; f) NBS, H2O, acetone; g) Ac2O, Py; h) AcCl, HCl, CH2Cl2,
sealed tube, 90% over three steps; i) potassium ethyl xanthate,
EtOH, 84%.
The sialylation reactions of N-acetyl modified
galactosylthreonine acceptors (11, 5, 6) using sialyl xanthate
donors (12, 10) were carried out in a similar way to the
synthesis of natural STn-Thr (Scheme 3).21 The sialyl xanthate
donors were activated by CH3SBr/AgOTf in the presence of
glycosyl acceptors, producing the sialosides 13
linkage. After sialylation, the free hydroxyl groups were
protected with acetyl groups to give disaccharides 17 20
-16 with α-(2-6)-
-
.
Removal of the tert-butyl ester by trifluoroacetic acid (TFA)
offered the natural and modified STn-Thr disaccharide building
blocks 21-24 ready for the solid-phase peptide synthesis.
With all the four STn-Thr building blocks in hand, solid-
phase peptide synthesis (SPPS) was performed to synthesize
the MUC1 sequence bearing these natural or unnatural STn
antigens (Scheme 4). Starting from the Wang-resin25 preloaded
with alanine, Fmoc-protected amino acids were coupled using
HATU/HOAt26 as the coupling reagent and N,N-
diisopropylethylamine (DIPEA) as the base in N,N-
dimethylformamide (DMF). The glycosylated threonines were
coupled with the stronger conditions using HATU/HOAt as the
coupling reagent and N-methyl morpholine (NMM) as the base
in N-methyl-2-pyrrolidone (NMP).27 DMF/Ac2O/Py = 3:2:1 was
used as the capping condition. 20% piperidine in DMF was
used for the deprotection of Fmoc group. Once the assembly
of the glycopeptides and the subsequent incorporation of
triethylene glycol spacer were finished, the glycopeptides were
detached from the resin under the conditions of TFA/TIS/H2O =
12:1:1, affording the glycopeptides 25-28 in 13-28% yields. The
protecting groups on the carbohydrates were removed under
basic conditions using NaOH aqueous solution (pH = 11),
obtaining the glycopeptides 29
-
32 in the yields of 51-71%.
32 with diethyl squarate28,
under basic conditions (pH = 8) afforded the corresponding
squaric acid monoamides 33 36, which were finally conjugated
with KLH to form the target protein conjugates V1 V4. Since
Treatment of the glycopeptides 29
-
29
-
-
KLH is a huge protein with a mass beyond measure of mass
spectrometer, the epitope ratios of the glycoconjugates were
determined by a resorcinol method.14, 30 This method was used
to determine the content of sialic acid, based on which the
content of STn-MUC1 glycopeptide was calculated. The
epitope ratios were expressed by mass percentages of 36-44%.
Scheme 3 Synthesis of the STn-Thr disaccharide building block and
its N-acetyl modified analogues. Reagents and conditions: a) CH3SBr,
AgOTf, CH3CN, CH2Cl2, 4Å MS, -62 oC, 37% for 13, 68% for 14, 52%
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DOI: 10.1039/C6OB01092J
Scheme 4 Solid-phase synthesis of the STn-MUC1 glycopeptides and their conjugation to the carrier protein KLH to afford vaccine
candidates V1 V4. Fmoc = fluorenyl-9-methoxycarbonyl, HATU = O-(7-azabenzotriazole-1-yl)-N,N,N‘,N‘-tetramethyluronium
hexafluorophosphate, HOAt = N-hydroxy-7-azabenzotriazole, NMM = N-methylmorpholine, TFA = trifluoroacetic acid, TIS =
triisopropylsilane, KLH = keyhole limpet hemocyanin
-
.
To evaluate the immunogenicity of these vaccines, groups elicited by V1 against STn-MUC1-BSA (Figure 2b). However,
of six female BALB/c mice were vaccinated intraperitoneally whether these antibodies induced by the modified vaccines
four times. The antibody titers of pooled sera elicited by V1
V2 V3 and V4 were determined. All of IgG titers against The IgG titers of individual mouse against the natural STn-
corresponding modified STn-MUC1-BSA were higher than that MUC1-BSA showed that all the three modified vaccines elicited
,
could recognize the native antigen was the very concern of us.
,
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comparable antibody titers when compared with V1 (Figure
2a). Especially, the median serum IgG antibody titer induced
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In our previous work, all the three SDTnOId: 1e0r.i1v0a3t9i/vCe6sOsBh0o1w09e2dJ
by V2 against STn-MUC1-BSA was 1.5 folds higher than that good activities. However, after these STn derivatives were
induced by V1 (Table S1 in the ESI ). These results supported incorporated into the MUC1 peptide sequence, V2 showed the
the expected cross-reactivity against the native antigen. better activity when compared with V3 and V4. This might be
†
To further identify the IgG subclasses, the pooled sera explained by the effect of the carbohydrate moiety on the
from different groups were tested with different second- peptide backbone. It was proved that the incorporation of
antibodies (Figure 3a). IgG1 antibody was the predominant carbohydrates could affect the conformation of the peptide
subtype. The modified antigens generated comparable IgG1 backbone significantly.15, 34 Compared with the fluoroacetyl
titers with the native antigen. More interestingly, all other group in V2, the difluoroacetyl group in V3 is much more
subclasses of IgG2a, IgG2b and IgG3 antibodies of serum from different from the native STn antigen in the aspects such as
V2 were obviously higher than those from other groups. atom radius, lipophilicity, and electronegativity, which might
Enlightened by this result, we chose the IgG2a having lead to a greater conformational change of the MUC1 peptide
the biggest difference between groups to detect the titers of backbone. Vaccine V4 has one more fluoroacetyl group
individual mouse against STn-MUC1-BSA. The result showed compared with V2, which could also amplify the difference
that the median serum IgG2a titer induced by V2 against STn- between the STn analogue and the native STn antigen, and
MUC1-BSA was still significantly higher than other groups could subsequently affect the MUC1 peptide moiety. The
(Figure 3b). It was 5.4 folds higher than that induced by V1 structural differences between V3
(Table S3 in the ESI ). The presence of IgG2a is the evidence of MUC1 antigen finally impede the cross-recognition, resulting in
a type 1 T-helper response,31 which is very important for the lower antibody titers compared with V2, which has more
, V4 and the native STn-
†
successful design of antitumor vaccines.32, 33
structural similarity to the native STn-MUC1 antigen.
To test whether the mouse sera after vaccination were
able to recognize the STn-MUC1 antigen presented on cancer
cells, the binding of the sera to sarcoma cells Ag104A
(transfected with MUC1 gene) was examined by flow
cytometry analysis. The results demonstrated that all the
antisera elicited by V1, V2, V3 and V4 could react with the
Ag104A cells very well (Figure 4).
a
b
a
Figure 2. (a) Serum antibody titers against STn-MUC1-BSA of
individual mouse immunized with V1, V2, V3 and V4 after the 4th
vaccination. Each dot represents the average titer of two detections,
and each black line represents the median antibody level of a group
of six mice. (b) Pooled sera IgG titers against STn-MUC1-BSA (hollow
bar) and against the corresponding modified-STn-MUC1-BSA
(dotted bar) after the 4th vaccination with V1, V2, V3 and V4. The
titers were separately detected twice, and three parallel micropores
were arranged. Each titer represents the average of two detections.
Figure 4 Serological IgG analysis results on Ag104A cells after the
4th immunization by flow cytometry.
a
b
Conclusions
To overcome the low immunogenicity of MUC1 glycopeptides
as therapeutic vaccines, STn-Thr derivatives 22, 23, 24 with N-
acetyl modifications were synthesized as the building blocks
for peptide synthesis under mild conditions that prevented the
isomerization of threonine. These modified STn-Thr building
blocks as well as the native STn-Thr unit were incorporated
into a 20-amino acid MUC1 tandem repeat sequence. These
glycopeptides were connected to a carrier protein KLH to
produce the vaccine conjugates V1, V2, V3 and V4, and their
immunological effects were evaluated using the BALB/c mouse
model. The increased antibody titers confirmed an
Figure 3 (a) Reactivity of pooled sera IgG subclasses with STn-
MUC1-BSA by ELISA when diluted 1:50000 for IgG1 and 1:1000
for other subclasses. Triplicate wells were arranged. Data were
shown with mean ± SD of three triplicate optical density. (b)
Serum IgG2a titers against STn-MUC1-BSA of individual mouse
after the 4th vaccination. Each dot represents the average titer
of two detections of an individual mouse, and each black line
represents the median IgG2a level of a group of six mice.
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improvement of immunogenicity, and the antisera after = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.79, 170.50, 170.35,
View Article Online
DOI: 10.1039/C6OB01092J
vaccination could react with the tumor cells expressing the 169.49, 168.12 (d, J = 17.7 Hz), 156.56, 143.93, 141.44, 127.89,
STn-MUC1 antigen. Especially, the serum IgG antibody titer 127.87, 127.24, 125.36, 125.27, 120.14, 120.11, 98.74, 83.40, 80.31
induced by vaccine V2 against STn-MUC1-BSA was much (d, J = 186.5 Hz), 77.36, 76.01, 68.27, 67.57, 67.40, 67.37, 62.13,
higher than that induced by vaccine V1, and the IgG2a titer 58.71, 47.28, 47.13, 28.14, 20.83, 20.78, 18.57; HRMS (ESI) Anal.
induced by V2 against STn-MUC1-BSA was significantly higher Calcd for C37H46N2O13F [M+H]+: 745.2984, Found: 745.2985.
than those induced by the other vaccine conjugates. These N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-(3,4,6-tri-O-acetyl-2-
findings demonstrated that the carbohydrate antigen difluoroacetamido-2-deoxy-α-D-galactopyranosyl)-L-threonine
modification strategy has the potential to break the weak tert-butyl ester (4): To a solution of 1 (841.2 mg, 1.18 mmol) in
immunogenicity of natural MUC1 glycopeptide.
AcOH (10 mL), activated zinc dust (1.00 g, 15.4 mmol) was added,
and the mixture was stirred at room temperature for 3 hours. The
mixture was filtered through Celite and concentrated in vacuo. The
residue was dissolved in CH3CN (10 mL), to which NaHCO3 (0.40 g,
4.76 mmol) and (CHF2CO)2 (200 μL, 1.70 mmol) were added. After
10 minutes, the solution was diluted with CH2Cl2 and washed with
water. The organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was purified by column chromatography
Experimental Section
General information for chemical synthesis
All chemicals were purchased as reagent grade and used without
further purification, unless otherwise noted. Dichloromethane and
pyridine were distilled over calcium hydride (CaH2). Ether was dried
and distilled over sodium metal. Acetonitrile was dried and distilled
over P2O5. Methanol was distilled over magnesium. N,N-
Dimethylformamide (DMF) was distilled over P2O5 under reduced
pressure. Analytical TLC was performed on silica gel 60-F254
precoated on aluminium plates (E. Merck), with detection by UV
light and/or by staining with acidic ceric ammonium molybdate.
Solvents were evaporated under reduced pressure below 40 °C.
Column chromatography was performed employing silica gel (200-
300 mesh or 230-400 mesh). Optical rotations were measured on a
Hanon P850 automatic polarimeter and are given in 10-1 deg cm2 g-1.
1H NMR spectra were recorded on a Bruker AVANCE III-400
spectrometer at 25 °C. Chemical shifts (in ppm) were referenced to
tetramethylsilane (δ = 0 ppm) or residual proton solvent. 13C NMR
spectra were obtained by using the same NMR spectrometer and
were calibrated with CDCl3 (δ = 77.00 ppm). High-resolution mass
spectrometry was performed on a Thermo Scientific LTQ Orbitrap
Discovery or Waters Xevo G2 Qtof spectrometers.
(petroleum ether/ ethyl acetate, 3:1) to give 4 (856.1 mg, 95% over
two steps) as a colorless amorphous solid. +50.2º (c 1.31,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.62 (d, J
= 7.1 Hz, 2H), 7.48 – 7.28 (m, 9H), 6.94 (d, J = 8.7 Hz, 1H), 6.06 (d, J =
7.9 Hz, 1H), 5.72 (d, J = 8.8 Hz, 1H), 5.48 – 5.34 (m, 1H), 5.34 – 5.29
(m, 1H), 5.26 (d, J = 12.0 Hz, 1H), 5.21 (d, J = 12.0 Hz, 1H), 5.03 –
4.70 (m, 5H), 4.64 (dd, J = 28.7, 14.4 Hz, 1H), 4.49 – 4.34 (m, 3H),
4.31 – 4.20 (m, 2H), 4.20 – 4.06 (m, 3H), 4.07 – 3.86 (m, 4H), 3.83 (s,
2H), 3.53 (dd, J = 10.5, 3.4 Hz, 1H), 2.83 (s, 2H), 2.67 (dd, J = 12.7,
4.5 Hz, 1H), 2.15 (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.96
(t, J = 12.4 Hz, 1H), 1.44 (s, 9H), 1.26 (d, J = 5.6 Hz, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.82, 170.48, 170.29, 169.66, 163.13 (t, J =
25.6 Hz), 156.48, 143.90, 141.49, 127.93, 127.27, 125.28, 125.21,
120.18, 120.16, 108.40 (t, J = 251.6 Hz), 98.66, 83.68, 77.36, 76.36,
68.30, 67.60, 67.50, 67.35, 62.08, 58.71, 47.83, 47.35, 28.18, 20.81,
20.77, 20.67, 18.36; HRMS (ESI) Anal. Calcd for C37H44N2O13F2Na
[M+Na]+: 785.2709, Found: 785.2729.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-(2-fluoroacetamido-2-
deoxy-α-D-galactopyranosyl)-L-threonine tert-butyl ester (5): To a
stirred solution of 3 (956.2 mg, 1.28 mmol) in MeOH (10 mL) was
added 30% NaOMe solution in MeOH (0.1 mL) dropwise. The
mixture was stirred for 30 minutes (monitered below a pH of 9.0).
Dowex®50WX4 ion-exchange resin (H+) was added and the mixture
was stirred for another 30 minutes. The resin was filtered off and
the solvent was concentrated in vacuo. The crude product was
Synthesis of natural and modified STn-Thr building blocks.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-(3,4,6-tri-O-acetyl-2-
fluoroacetamido-2-deoxy-α-D-galactopyranosyl)-L-threonine tert-
butyl ester (3): To a solution of 1 (1.11 g, 1.56 mmol) in AcOH (15
mL), activated zinc dust (1.00 g, 15.4 mmol) was added, and the
mixture was stirred at room temperature for 3 hours. The mixture
was filtered through Celite and concentrated in vacuo. The residue
was dissolved in CH3CN (20 mL), to which NaHCO3 (1.00g, 11.9
mmol) and CH2FCOCl (162 μL, 2.32 mmol) were added. After 15
minutes, the solution was diluted with CH2Cl2 and washed with
water. The organic layer was dried over Na2SO4 and concentrated in
vacuo. The crude product was purified by column chromatography
purified by column chromatography (CH2Cl2/MeOH, 20:1) to give 5
(546.8 mg, 69%) as a white amorphous solid. +79.3º (c 1.82,
1
MeOH); H NMR (400 MHz, CD3OD) δ 7.80 (d, J = 7.5 Hz, 2H), 7.69
(dd, J = 7.3, 3.5 Hz, 2H), 7.45 – 7.35 (m, 2H), 7.35 – 7.27 (m, 2H),
4.85 (d, J = 47.0 Hz, 2H), 4.83 (d, J = 3.9 Hz, 1H), 4.47 (d, J = 6.6 Hz,
2H), 4.40 – 4.29 (m, 2H), 4.25 (t, J = 6.5 Hz, 1H), 4.13 – 4.07 (m, 1H),
3.94 – 3.86 (m, 2H), 3.80 – 3.67 (m, 3H), 1.44 (s, 9H), 1.26 (d, J = 6.5
Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 171.09, 170.86 (d, J = 18.8
Hz), 159.23, 145.30, 145.15, 142.64, 128.79, 128.18, 126.20, 126.13,
120.95, 120.92, 100.32, 83.45, 80.93 (d, J = 182.0 Hz), 75.92, 73.02,
70.27, 69.33, 67.83, 62.76, 60.85, 50.67, 28.37, 19.78; HRMS (ESI)
Anal. Calcd for C31H39N2O10FNa [M+Na]+: 641.2486, Found:
641.2496.
(petroleum ether/ ethyl acetate, 2:1) to give 3 (956.2 mg, 83% over
two steps) as a colorless amorphous solid. +52.8º (c 3.10,
CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.64 (d, J
= 7.4 Hz, 2H), 7.47 – 7.37 (m, 2H), 7.36 – 7.29 (m, 2H), 6.50 (d, J =
9.8 Hz, 1H), 5.51 (dd, J = 8.6, 3.0 Hz, 1H), 5.43 (d, J = 2.2 Hz, 1H),
5.18 (dd, J = 11.4, 2.9 Hz, 1H), 4.97 (d, J = 3.6 Hz, 1H), 4.90 (dd, J =
37.4, 14.4 Hz, 1H), 4.78 (dd, J = 37.5, 14.2 Hz, 1H), 4.66 (td, J = 11.0,
3.4 Hz, 1H), 4.41 (d, J = 7.1 Hz, 2H), 4.31 – 4.21 (m, 4H), 4.17 – 4.06
(m, 2H), 2.17 (s, 3H), 2.05 (s, 3H), 2.01 (s, 3H), 1.47 (s, 9H), 1.32 (d, J
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-(2-difluoroacetamido-2-
deoxy-α-D-galactopyranosyl)-L-threonine tert-butyl ester (6): The
6 | J. Name., 2012, 00, 1-3
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procedure is the same as described in the synthesis of 5, yield 73%. 1H), 5.56 – 5.46 (m, 2H), 5.37 (d, J = 12.2 Hz, 1H), 5.23V(idew, JAr=tic1le2O.3nlHinez,
1
+1.82º (c 3.10, MeOH); H NMR (400 MHz, CD3OD) δ 7.83 (d, J 1H), 5.21 – 5.17 (m, 1H), 4.76 (dd, J = 26.4, D14O.I4: 1H0z.1,013H9/)C, 64O.6B501(d0d92,JJ
= 7.5 Hz, 2H), 7.71 (dd, J = 7.3, 3.3 Hz, 2H), 7.49 – 7.39 (m, 2H), 7.39 = 27.2, 14.4 Hz, 1H), 4.54 (dd, J = 10.7, 2.4 Hz, 1H), 4.41 (dd, J = 12.5,
– 7.29 (m, 2H), 6.10 (t, J = 54.1 Hz, 1H), 4.87 – 4.84 (m, 1H), 4.58 – 2.6 Hz, 1H), 4.26 (q, J = 10.3 Hz, 1H), 4.10 (dd, J = 12.5, 5.9 Hz, 1H),
4.48 (m, 2H), 4.39 – 4.32 (m, 2H), 4.28 (t, J = 6.3 Hz, 1H), 4.13 (s, 1H), 2.82 (dd, J = 13.9, 4.9 Hz, 1H), 2.27 (dd, J = 13.9, 11.2 Hz, 1H), 2.10 (s,
3.96 – 3.89 (m, 2H), 3.83 – 3.72 (m, 3H), 1.46 (s, 9H), 1.28 (d, J = 6.4 3H), 2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H); 13C NMR (100 MHz, CDCl3)
Hz, 3H); 13C NMR (100 MHz, CD3OD) δ 171.21, 165.39 (t, J = 25.5 Hz), δ 170.95, 170.70, 170.12, 169.79, 168.35 (d, J = 17.9 Hz), 164.86,
159.17, 145.28, 145.16, 142.66, 128.79, 128.16, 126.13, 126.06, 134.70, 128.73, 128.37, 96.60, 80.22 (d, J = 186.1 Hz), 77.36, 73.67,
120.95, 120.92, 109.89 (t, J = 245.1 Hz), 99.97, 83.61, 75.90, 73.09, 70.05, 68.64, 68.49, 66.96, 61.98, 48.09, 40.72, 20.96, 20.88, 20.84,
70.29, 69.17, 67.74, 62.73, 60.73, 51.20, 28.38, 19.75; HRMS (ESI) 20.82; HRMS (ESI) Anal. Calcd for C26H31NO12FNaCl [M+Na]+:
Anal. Calcd for C31H38N2O10F2Na [M+Na]+: 659.2392, Found: 626.1417, Found: 626.1434.
659.2405.
O-Ethyl S-[benzyl (5-fluoroacetamido-4,7,8,9-tetra-O-acetyl-2,3,5-
Benzyl
[5-fluoroacetamido-4,7,8,9-tetra-O-acetyl-2-(4- trideoxy-D-glycero-D-galacto-2-nonulopyranosyl)onate]
methylphenylthio)-2,3,5-trideoxy-D-glycero-D-galacto-2-
dithiocarbonate (10): To a solution of 9 (786.5 mg, 1.30 mmol) in
nonulopyranosyl]onate (8): To a stirred solution of 7 (1.00 g, 1.67 EtOH (15 mL) was added potassium ethyl xanthate (0.34 g, 2.10
mmol) in MeOH (10 mL) was added 30% NaOMe solution in MeOH mmol) under argon protection. The reaction was left in the dark at
(0.1 mL). After being stirred at room temperature for 1 hour, the room temperature for 12 hours. The solution was diluted with
solution was concentrated to dryness. To the residue was then CH2Cl2 and washed with ice-cold water and brine. The organic layer
added 2 M NaOH solution (20 mL) and allowed to stir under reflux was dried over Na2SO4 and concentrated in vacuo. The crude
for 10 hours. The solution was neutralized with 1 M HCl solution product was purified by column chromatography (petroleum
and concentrated. The residue was redissolved in MeOH (10 mL). ether/ethyl acetate, 1:1) to give 10 (754.0 mg, α:β = 6:1, 84%) as a
Under an atmosphere of argon, triethylamine (1.2 mL, 8.34 mmol) colorless amorphous solid. This anomer mixture was directly used
and methyl fluoroacetate (1.3 mL, 16.6 mL) was added. The mixture for next step without further purification.
was then stirred under reflux for 12 hours and concentrated. The N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-acetamido-2-deoxy-6-
residue was dissolved in DMF (5 mL), KF (0.24 g, 4.1 mmol) and O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-α-
benzyl bromide (0.3 mL, 2.5 mmol) were added and the reaction glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
was stirred for 12 hours. After filtered through Celite, the solution galactopyranosyl}-L-threonine tert-butyl ester (13): To prepare a
was diluted with ethyl acetate and washed with water and brine. 1.6
M solution of methylsulfenylbromide in dichloroethane,
The organic layer was dried over Na2SO4 and concentrated. The bromine (82 µL, 1.60 mmol) was added dropwise to a solution of
residue was dissovled in a mixture of pyridine (5 mL) and Ac2O (5 dimethyldisulfide (142 µL, 1.60 mmol) in dry 1,2-dichloroethane
o
mL) and stirred for 12 hours. The solvent was concentrated in vacuo (1.8 mL) at 0 C under argon atmosphere. The solution was left in
and the crude product was purified by column chromatography the dark for 12 hours and ready as an activator for the glycosylation.
(petroleum ether/ethyl acetate, 2:1) to give 8 (701.3 mg, α:β = 1:6, Compounds 11 (341.0 mg, 0.57 mmol) and 12 (771 mg, 1.15 mmol)
61% over five steps) as a colorless amorphous solid. This anomer were dissolved in a mixture of dry CH3CN (16 mL) and CH2Cl2 (8 mL).
mixture was directly used for next step without further purification. This solution was stirred with powdered 4 Å molecular sieve 4 Å
Benzyl (5-fluoroacetamido-4,7,8,9-tetra-O-acetyl-2-chloro-2,3,5- (2.5 g) for 1 hour under argon atmophere. AgOTf (294.5 mg, 1.15
trideoxy-β-D-glycero-D-galacto-2-nonulopyranosyl)onate (9): To a mmol) was added to the mixture and the temperature was cooled
stirred solution of 8 (1.00 g, 1.45 mmol) in acetone (7.5 mL) was to -62 oC. The methylsulfenylbromide solution (0.79 mL, 1.26 mmol)
added H2O (0.6 mL) and NBS (1.50 g, 8.44 mmol). After being stirred mentioned above cooled to 0 °C were slowly added via an
at room temperature for 30 minutes, the solution was diluted with automatic sampler within 1 hour. The reaction was stirred for 3
o
CH2Cl2, washed with saturated NaHCO3, water and brine, and the hours at -62 C. DIPEA (0.40 mL, 2.30 mmol) was added to quench
organic layer was dried over Na2SO4. The solvent was concentrated the reaction and the mixture was brought to room temperature.
in vacuo and the crude product was purified by column After filtered through Celite, the filtrate was diluted with CH2Cl2 and
chromatography (petroleum ether/ ethyl acetate, 1:1). The product washed with saturated NaHCO3 and brine. The organic layer was
was dissovled in a mixture of pyridine (5 mL) and Ac2O (2.5 mL) and dried over Na2SO4 and concentrated. The crude product was
stirred for 12 hours. The reaction mixture was concentrated in purified by column chromatography (CH2Cl2/acetone, 1:1) to give 13
vacuo and the crude product was purified by column (243.8 mg, 37%) as a colorless foam. 1H NMR (400 MHz, CDCl3) δ
chromatography (petroleum ether/ ethyl acetate, 2:1). The product 7.77 (d, J = 7.4 Hz, 2H), 7.62 (d, J = 6.7 Hz, 2H), 7.47 – 7.28 (m, 9H),
was dissoled in CH2Cl2 in a sealed tube. At 0 oC, AcCl (2 mL) and 6.70 (d, J = 7.3 Hz, 1H), 5.41 (d, J = 9.3 Hz, 1H), 5.39 – 5.34 (m, 1H),
concentrated HCl (0.25 mL) was added dropwise and the reaction 5.33 – 5.29 (m, 1H), 5.26 (d, J = 12.0 Hz, 1H), 5.18 (d, J = 12.0 Hz, 1H),
was sealed for 12 hours. The solution was diluted with CH2Cl2, 5.12 (d, J = 9.1 Hz, 1H), 4.91 – 4.82 (m, 1H), 4.81 (d, J = 3.2 Hz, 1H),
washed with ice-cold water, saturated NaHCO3 and brine. The 4.61 (d, J = 4.1 Hz, 1H), 4.49 (d, J = 6.9 Hz, 2H), 4.37 (dd, J = 12.3, 2.3
organic layer was dried over Na2SO4 and concentrated in vacuo. The Hz, 1H), 4.31 – 4.15 (m, 3H), 4.14 – 4.03 (m, 4H), 3.93 (dd, J = 10.3,
crude product was purified by column chromatography (petroleum 7.2 Hz, 1H), 3.80 – 3.60 (m, 3H), 3.53 (dd, J = 10.3, 4.5 Hz, 1H), 2.88
ether/ethyl acetate, 2:1) to give 9 (786.5 mg, 90% over three steps) (s, 1H), 2.61 (dd, J = 12.8, 4.5 Hz, 1H), 2.13 (s, 3H), 2.12 (s, 3H), 2.10
1
as a colorless amorphous solid. -54.6º (c 2.86, CHCl3); H NMR (s, 3H), 2.02 (s, 6H), 1.96 (t, J = 12.5 Hz, 1H), 1.87 (s, 3H), 1.47 (s, 9H),
(400 MHz, CDCl3) δ 7.45 – 7.31 (m, 5H), 6.68 (dd, J = 10.3, 2.8 Hz,
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1.29 (d, J = 6.1 Hz, 3H). The spectroscopic data coincide with the 7.48 – 7.28 (m, 9H), 6.94 (d, J = 8.7 Hz, 1H), 6.06 (d, JV=iew7.A9rtiHclez,O1nHlin)e,
previous report.21
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-2-
deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
5.72 (d, J = 8.8 Hz, 1H), 5.48 – 5.34 (m, 1H), 5.34 – 5.29 (m, 1H), 5.26
(d, J = 12.0 Hz, 1H), 5.21 (d, J = 12.0 Hz, 1H), 5.03 – 4.70 (m, 5H),
4.64 (dd, J = 28.7, 14.4 Hz, 1H), 4.49 – 4.34 (m, 3H), 4.31 – 4.20 (m,
2H), 4.20 – 4.06 (m, 3H), 4.07 – 3.86 (m, 4H), 3.83 (s, 2H), 3.53 (dd, J
DOI: 10.1039/C6OB01092J
galactopyranosyl}-L-threonine tert-butyl ester (14): The procedure = 10.5, 3.4 Hz, 1H), 2.83 (s, 2H), 2.67 (dd, J = 12.7, 4.5 Hz, 1H), 2.15
is the same as described in the synthesis of 13. This compound was (s, 3H), 2.11 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.96 (t, 1H), 1.44 (s,
prepared from 5 (236.0 mg, 0.38 mmol) and 12 (0.51 g, 0.76 mmol), 9H), 1.26 (d, J = 5.6 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 171.43,
1
affording 14 (303.2 mg, 68%). +24.9º (c 1.74, CHCl3); H NMR 171.01, 170.53, 170.28, 169.86, 168.20 (d, J = 17.8 Hz), 167.56,
(400 MHz, CDCl3) δ 7.76 (d, J = 7.6 Hz, 2H), 7.62 (d, J = 6.9 Hz, 2H), 156.68, 144.00, 143.90, 141.44, 134.84, 129.09, 128.95, 128.79,
7.46 – 7.28 (m, 9H), 6.81 (d, J = 8.6 Hz, 1H), 5.58 (d, J = 8.8 Hz, 1H), 127.88, 127.24, 125.26, 120.13, 99.23, 98.88, 83.14, 80.22 (d, J =
5.36 (td, J = 6.9, 2.4 Hz, 1H), 5.31 (d, J = 7.5 Hz, 1H), 5.26 (d, J = 12.0 186.0 Hz), 80.06 (d, J = 186.0 Hz), 77.36, 76.55, 72.57, 69.81, 69.46,
Hz, 1H), 5.22 – 5.14 (m, 2H), 5.07 – 4.71 (m, 4H), 4.50 – 4.34 (m, 4H), 69.37, 68.82, 68.21, 67.36, 64.83, 62.48, 59.05, 49.90, 48.60, 47.31,
4.28 – 4.14 (m, 3H), 4.14 – 4.05 (m, 2H), 4.01 (dd, J = 12.4, 6.4 Hz, 37.52, 28.12, 21.26, 20.88, 18.52; HRMS (ESI) Anal. Calcd for
1H), 3.91 (dd, J = 10.2, 6.8 Hz, 1H), 3.84 – 3.75 (m, 2H), 3.71 (d, J = C57H69N3O22F2Na [M+Na]+: 1208.4238, Found: 1208.4327.
8.2 Hz, 1H), 3.59 (s, 1H), 3.53 (dd, J = 10.3, 4.3 Hz, 1H), 3.30 (s, 1H), N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-acetamido-3,4-di-O-
2.62 (dd, J = 12.8, 4.5 Hz, 1H), 2.13 (s, 6H), 2.03 (s, 3H), 2.02 (s, 3H), acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-
1.97 (t, J = 12.8 Hz, 1H), 1.87 (s, 3H), 1.45 (s, 9H), 1.28 (d, J = 6.2 Hz, 3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
3H); 13C NMR (100 MHz, CDCl3) δ 171.27, 170.94, 170.71, 170.34, galactopyranosyl}-L-threonine tert-butyl ester (17): Compound 13
170.25, 169.91, 169.73, 167.67, 156.62, 144.00, 143.87, 141.45, (243.8 mg, 0.21 mmol) was dissovled in a mixture of pyridine (5 mL)
134.87, 129.06, 128.94, 128.77, 127.89, 127.24, 125.26, 120.15, and Ac2O (2.5 mL) and stirred for 12 hours. The solvent was
120.12, 99.27, 98.93, 83.15, 80.14 (d, J = 186.1 Hz), 77.36, 76.41, concentrated in vacuo and the crude product was purified by
73.09, 70.04, 69.57, 69.39, 69.07, 69.01, 68.09, 67.65, 67.40, 64.59, column chromatography (petroleum ether/ethyl acetate, 1:5) to
62.77, 59.04, 50.05, 49.40, 47.33, 37.45, 28.13, 23.31, 21.25, 20.95, give 17 (240.1 mg, 92%) as a colorless foam. 1H NMR (400 MHz,
20.91, 18.69; HRMS (ESI) Anal. Calcd for C57H71N3O22F [M+H]+: CDCl3) δ 7.85 – 7.74 (m, 2H), 7.70 – 7.60 (m, 2H), 7.48 – 7.29 (m, 9H),
1168.4570, Found: 1168.4570.
5.93 (d, J = 9.7 Hz, 1H), 5.58 (d, J = 9.3 Hz, 1H), 5.37 – 5.28 (m, 2H),
5.26 (d, J = 11.9 Hz, 1H), 5.19 – 5.11 (m, 2H), 5.09 – 5.02 (m, 1H),
4.98 (dd, J = 11.3, 2.5 Hz, 1H), 4.90 – 4.78 (m, 2H), 4.56 (td, J = 11.3,
3.6 Hz, 1H), 4.48 (d, J = 7.1 Hz, 2H), 4.32 – 4.16 (m, 4H), 4.09 – 4.02
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-difluoroacetamido-2-
deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
galactopyranosyl}-L-threonine tert-butyl ester (15): The procedure (m, 3H), 4.00 – 3.92 (m, 1H), 3.87 – 3.71 (m, 1H), 3.14 (dd, J = 10.4,
is the same as described in the synthesis of 13. This compound was 4.3 Hz, 1H), 2.56 (dd, J = 12.7, 4.5 Hz, 1H), 2.13 (s, 3H), 2.10 (s, 6H),
prepared from 6 (519.8 mg, 0.816 mmol) and 12 (1.07 g, 1.59 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 6H), 1.91 (t, J = 12.7 Hz, 1H), 1.87 (s,
mmol), affording 15 (503.0 mg, 52%). +26.7º (c 1.67, CHCl3); 3H), 1.46 (s, 9H), 1.30 (d, J = 6.0 Hz, 3H). The spectroscopic data
1H NMR (400 MHz, CDCl3) δ 7.76 (d, J = 7.2 Hz, 2H), 7.62 (d, J = 7.3 coincide with the previous report.21
Hz, 2H), 7.46 – 7.28 (m, 9H), 7.05 (d, J = 8.8 Hz, 1H), 6.00 (t, J = 54.1 N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-3,4-
Hz, 1H), 5.56 (d, J = 9.3 Hz, 1H), 5.35 (td, J = 6.9, 2.5 Hz, 1H), 5.32 – di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
5.29 (m, 1H), 5.26 (d, J = 11.9 Hz, 2H), 5.18 (d, J = 12.0 Hz, 1H), 4.93 acetyl-3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-
– 4.81 (m, 2H), 4.54 – 4.30 (m, 4H), 4.28 – 3.96 (m, 6H), 3.91 (dd, J = α-D-galactopyranosyl}-L-threonine tert-butyl ester (18): The
10.2, 6.7 Hz, 1H), 3.83 – 3.74 (m, 2H), 3.70 (d, J = 9.0 Hz, 1H), 3.54 procedure is the same as described in the synthesis of 17. This
(dd, J = 10.1, 4.5 Hz, 1H), 3.26 (s, 1H), 3.18 (s, 1H), 2.62 (dd, J = 12.8, compound was prepared from 14, yield 86%. +27.3º (c 2.89,
4.4 Hz, 1H), 2.13 (s, 3H), 2.12 (s, 3H), 2.02 (s, 6H), 1.98 (t, J = 12.4 Hz, CHCl3); 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 7.5 Hz, 2H), 7.65 (d, J
1H), 1.87 (s, 3H), 1.45 (s, 9H), 1.27 (d, J = 7.1 Hz, 3H); 13C NMR (100 = 7.4 Hz, 2H), 7.47 – 7.29 (m, 9H), 6.46 (d, J = 9.6 Hz, 1H), 5.51 (d, J =
MHz, CDCl3) δ 171.28, 170.99, 170.72, 170.43, 170.29, 167.66, 9.0 Hz, 1H), 5.40 – 5.29 (m, 2H), 5.26 (d, J = 11.9 Hz, 1H), 5.18 (d, J =
164.31 (t, J = 25.4 Hz), 156.60, 143.92, 143.84, 141.45, 134.85, 2.3 Hz, 1H), 5.15 (d, J = 11.9 Hz, 1H), 5.08 (d, J = 8.6 Hz, 2H), 4.93 (dd,
129.06, 128.94, 128.79, 127.91, 127.24, 125.19, 120.15, 108.43 (t, J J = 8.7, 5.1 Hz, 1H), 4.89 – 4.68 (m, 3H), 4.60 (td, J = 10.8, 3.4 Hz, 1H),
= 250.6 Hz), 99.16, 98.94, 83.39, 77.36, 76.49, 73.11, 69.73, 69.49, 4.51 – 4.36 (m, 2H), 4.32 – 4.18 (m, 4H), 4.10 – 4.02 (m, 3H), 4.01 –
68.97, 68.09, 67.69, 67.27, 64.48, 62.82, 58.98, 50.68, 49.35, 47.33, 3.96 (m, 1H), 3.84 (dd, J = 10.2, 7.5 Hz, 1H), 3.12 (dd, J = 10.4, 4.3 Hz,
37.41, 28.16, 23.28, 21.25, 20.96, 20.91, 18.77; HRMS (ESI) Anal. 1H), 2.57 (dd, J = 12.8, 4.6 Hz, 1H), 2.13 (s, 3H), 2.11 (s, 3H), 2.10 (s,
Calcd for C57H69N3O22F2Na [M+Na]+: 1208.4238, Found: 1208.4296.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-2-
deoxy-6-O-[benzyl-(5-fluoroacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
3H), 2.02 (s, 3H), 2.02 (s, 3H), 2.00 (s, 3H), 1.93 (t, J = 12.6 Hz, 1H),
1.87 (s, 3H), 1.46 (s, 9H), 1.30 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 170.90, 170.76, 170.66, 170.44, 170.27, 170.24, 169.73,
169.64, 168.04 (d, J = 17.9 Hz), 167.50, 156.62, 143.98, 143.93,
galactopyranosyl}-L-threonine tert-butyl ester (16): The procedure 141.43, 134.84, 129.19, 128.99, 128.79, 127.86, 127.24, 125.35,
is the same as described in the synthesis of 13. This compound was 125.31, 120.13, 120.10, 98.98, 98.69, 83.20, 80.28 (d, J = 186.3 Hz),
prepared from 5 (476.7 mg, 0.77 mmol) and 10 (0.91 g, 1.32 mmol), 77.36, 76.16, 72.75, 69.04, 68.54, 68.48, 68.16, 68.01, 67.62, 67.54,
1
affording 16 (734.4 mg, 80%). +26.8º (c 2.43, CHCl3); H NMR 67.26, 63.95, 62.52, 58.92, 49.31, 47.30, 47.08, 37.78, 28.15, 23.32,
(400 MHz, CDCl3) δ 7.75 (d, J = 7.5 Hz, 2H), 7.62 (d, J = 7.1 Hz, 2H),
8 | J. Name., 2012, 00, 1-3
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21.14, 20.95, 20.94, 20.87, 20.83, 20.73, 18.65; HRMS (ESI) Anal. was purified by column chromatography (ethyl acetate/methonal,
View Article Online
1
Calcd for C61H78N4O24F [M+NH4]+: 1269.4990, Found: 1269.5092.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-difluoroacetamido-
3,4-di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
10:1) to give 21 (227.4 mg, quant.) as a colorless foam. H NMR (400
MHz, DMSO-d6) δ 12.88 (s, 1H), 7.89 (dd, J = 7.4, 2.5 Hz, 2H), 7.73 (d,
J = 8.1 Hz, 3H), 7.62 (d, J = 9.3 Hz, 1H), 7.55 (d, J = 9.7 Hz, 1H), 7.47 –
DOI: 10.1039/C6OB01092J
acetyl-3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]- 7.20 (m, 9H), 5.31 – 5.08 (m, 5H), 4.98 (dd, J = 11.7, 3.0 Hz, 1H), 4.77
α-D-galactopyranosyl}-L-threonine tert-butyl ester (19): The (d, J = 3.8 Hz, 1H), 4.72 (dd, J = 11.1, 3.9 Hz, 1H), 4.48 (dd, J = 10.7,
procedure is the same as described in the synthesis of 17. This 6.9 Hz, 1H), 4.42 (dd, J = 10.7, 6.7 Hz, 1H), 4.35 – 4.23 (m, 2H), 4.21
compound was prepared from 15, yield 98%. +24.0º (c 1.66, – 4.03 (m, 4H), 4.03 – 3.85 (m, 3H), 3.72 (dd, J = 9.7, 7.9 Hz, 1H),
1
CHCl3); H NMR (400 MHz, CDCl3) δ 7.78 (dd, J = 7.4, 2.8 Hz, 2H), 3.08 (dd, J = 10.0, 4.3 Hz, 1H), 2.55 (dd, J = 12.8, 4.4 Hz, 1H), 2.04 (s,
7.64 (d, J = 7.3 Hz, 2H), 7.44 – 7.31 (m, 9H), 6.78 (d, J = 9.6 Hz, 1H), 3H), 2.03 (s, 3H), 2.02 (s, 3H), 1.95 (s, 3H), 1.94 (s, 3H), 1.89 (s, 3H),
5.94 (t, J = 54.1 Hz, 1H), 5.68 (d, J = 9.1 Hz, 1H), 5.38 – 5.28 (m, 2H), 1.83 (s, 3H), 1.71 – 1.69 (m, 1H), 1.68 (s, 3H), 1.12 (d, J = 6.4 Hz, 3H).
5.26 (d, J = 11.9 Hz, 1H), 5.19 – 5.11 (m, 3H), 5.08 (dd, J = 11.4, 2.8 The spectroscopic data coincide with the previous report.21
Hz, 1H), 4.92 (d, J = 3.3 Hz, 1H), 4.88 – 4.77 (m, 1H), 4.61 – 4.40 (m, N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-3,4-
3H), 4.33 – 4.16 (m, 4H), 4.10 – 4.01 (m, 3H), 4.00 – 3.94 (m, 1H), di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
3.82 (dd, J = 10.3, 7.5 Hz, 1H), 3.14 (dd, J = 10.4, 4.2 Hz, 1H), 2.57 acetyl-3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-
(dd, J = 12.8, 4.6 Hz, 1H), 2.13 (s, 3H), 2.11 (s, 3H), 2.10 (s, 3H), 2.02 α-D-galactopyranosyl}-L-threonine (22): The procedure is the same
(s, 6H), 1.99 (s, 3H), 1.92 (t, J = 12.6 Hz, 3H), 1.87 (s, 3H), 1.45 (s, 9H), as described in the synthesis of 21. This compound was prepared
1.28 (d, J = 6.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.91, 170.85, from 18, quantitative yield. +27.9º (c 1.50, CHCl3); 1H NMR
170.70, 170.37, 170.32, 170.25, 169.92, 169.81, 167.46, 163.08 (t, J (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 8.06 (d, J = 9.1 Hz, 1H), 7.94 –
= 25.3 Hz), 156.62, 143.90, 141.46, 134.83, 129.19, 128.99, 128.82, 7.84 (m, 2H), 7.82 – 7.69 (m, 3H), 7.54 (d, J = 9.9 Hz, 1H), 7.46 – 7.26
127.89, 127.26, 125.20, 120.15, 108.33 (t, J = 251.1 Hz), 98.88, (m, 9H), 5.30 – 5.09 (m, 6H), 4.93 – 4.67 (m, 4H), 4.50 (dd, J = 10.7,
98.70, 83.36, 77.36, 76.52, 72.77, 69.02, 68.71, 68.38, 68.22, 68.02, 6.8 Hz, 1H), 4.43 (dd, J = 10.8, 6.6 Hz, 1H), 4.36 – 4.03 (m, 6H), 4.03
67.56, 67.29, 63.92, 62.54, 58.94, 49.28, 47.77, 47.35, 37.75, 28.16, – 3.82 (m, 3H), 3.79 – 3.67 (m, 1H), 3.08 (dd, J = 9.9, 4.0 Hz, 1H),
23.29, 21.13, 20.93, 20.82, 20.70, 18.57; HRMS (ESI) Anal. Calcd for 2.55 (dd, J = 12.9, 4.8 Hz, 1H), 2.04 (s, 6H), 2.02 (s, 3H), 1.95 (s, 3H),
C61H77N4O24F2 [M+NH4]+: 1287.4896, Found: 1287.5007.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[benzyl-(5-fluoroacetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-α-glycero-D-galacto-2-
1.94 (s, 3H), 1.90 (s, 3H), 1.72 – 1.69 (m, 1H), 1.68 (s, 3H), 1.12 (d, J
= 6.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 171.69, 169.96,
169.84, 169.77, 169.65, 169.36, 169.16, 167.45 (d, J = 19.0 Hz),
166.77, 156.84, 143.77, 143.71, 140.83, 140.75, 135.08, 128.51,
nonulopyranosyl)onate]-α-D-galactopyranosyl}-L-threonine tert- 128.47, 127.99, 127.66, 127.63, 127.11, 127.06, 125.19, 125.08,
butyl ester (20): The procedure is the same as described in the 120.19, 120.13, 98.65, 98.08, 79.71(d, J = 179.1 Hz), 75.50, 71.81,
synthesis of 17. This compound was prepared from 16, yield 87%. 69.03, 67.47, 67.40, 67.29, 67.22, 66.93, 65.52, 63.10, 62.02, 58.45,
1
+26.3º (c 1.72, CHCl3); H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 47.67, 46.78, 46.51, 37.46, 22.58, 20.80, 20.65, 20.57, 20.55, 20.48,
7.5 Hz, 2H), 7.65 (d, J = 7.3 Hz, 2H), 7.47 – 7.28 (m, 9H), 6.47 (d, J = 20.25, 18.40; HRMS (ESI) Anal. Calcd for C57H65N3O24F [M-H]-:
9.7 Hz, 1H), 5.95 (d, J = 5.7 Hz, 1H), 5.53 (d, J = 8.9 Hz, 1H), 5.40 – 1194.3942, Found: 1194.3971.
5.33 (m, 1H), 5.33 – 5.24 (m, 2H), 5.22 – 5.12 (m, 2H), 5.08 (dd, J = N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-difluoroacetamido-
11.4, 2.6 Hz, 1H), 4.97 – 4.54 (m, 7H), 4.50 – 4.35 (m, 2H), 4.32 – 3,4-di-O-acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-
4.19 (m, 4H), 4.17 – 4.09 (m, 2H), 4.05 (dd, J = 12.5, 5.0 Hz, 1H), 4.02 acetyl-3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-
– 3.96 (m, 1H), 3.84 (dd, J = 10.1, 7.7 Hz, 1H), 3.12 (dd, J = 10.4, 4.3 α-D-galactopyranosyl}-L-threonine (23): The procedure is the same
Hz, 1H), 2.63 (dd, J = 12.8, 4.6 Hz, 1H), 2.12 (s, 3H), 2.11 (s, 6H), 2.02 as described in the synthesis of 21. This compound was prepared
(s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.91 (t, J = 12.5 Hz, 1H), 1.46 (s, 9H), from 19, quantitative yield. +26.8º (c 2.84, CHCl3); 1H NMR
1.29 (d, J = 6.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 170.80, 170.70, (400 MHz, DMSO-d6) δ 12.86 (s, 1H), 8.61 (d, J = 9.1 Hz, 1H), 7.89
170.50, 170.46, 170.26, 169.71, 169.65, 168.16 (d, J = 17.8 Hz), (dd, J = 7.4, 2.9 Hz, 2H), 7.81 – 7.66 (m, 3H), 7.55 – 7.24 (m, 10H),
168.08 (d, J = 17.4 Hz), 167.39, 156.66, 144.01, 143.94, 141.45, 6.13 (t, J = 54.0 Hz, 1H), 5.33 – 5.06 (m, 6H), 4.84 (d, J = 3.7 Hz, 1H),
134.79, 129.26, 129.01, 128.86, 127.88, 127.26, 125.31, 120.15, 4.80 – 4.67 (m, 1H), 4.52 (dd, J = 10.7, 6.6 Hz, 1H), 4.44 (dd, J = 10.8,
120.11, 99.03, 98.67, 83.19, 80.29 (d, J = 186.6 Hz), 80.23 (d, J = 6.5 Hz, 1H), 4.35 – 4.21 (m, 2H), 4.21 – 4.03 (m, 4H), 4.03 – 3.84 (m,
186.0 Hz), 77.36, 76.21, 72.44, 68.88, 68.53, 68.46, 68.15, 67.84, 3H), 3.73 (dd, J = 9.6, 8.0 Hz, 1H), 3.09 (dd, J = 9.9, 3.9 Hz, 1H), 2.55
67.61, 67.51, 67.10, 64.00, 62.36, 58.95, 48.53, 47.33, 47.12, 37.82, (dd, J = 12.7, 4.5 Hz, 1H), 2.04 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 1.95
28.17, 21.14, 20.95, 20.89, 20.83, 20.73, 18.68; HRMS (ESI) Anal. (s, 3H), 1.93 (s, 3H), 1.89 (s, 3H), 1.70 (t, J = 5.6 Hz, 1H), 1.68 (s, 3H),
Calcd for C61H77N4O24F2 [M+NH4]+: 1287.4896, Found: 1287.5005.
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-acetamido-3,4-di-O-
acetyl-2-deoxy-6-O-[benzyl-(5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-α-glycero-D-galacto-2-nonulopyranosyl)onate]-α-D-
1.11 (d, J = 6.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 171.83,
169.97, 169.84, 169.71, 169.67, 169.37, 169.18, 166.76, 162.31 (t, J
= 25.0 Hz), 156.78, 143.78, 143.70, 140.85, 140.78, 135.09, 128.51,
128.47, 128.08, 128.01, 127.67, 127.64, 127.08, 127.06, 125.14,
galactopyranosyl}-L-threonine (21): Under argon atmosphere, 125.03, 120.21, 120.15, 108.19 (t, J = 245.7 Hz), 98.16, 98.09, 75.47,
trifluoroacetic acid (2 mL) and anisole (0.4 mL) were added to a 71.82, 69.04, 67.48, 67.36, 67.31, 67.08, 66.93, 65.47, 63.07, 62.03,
solution of 17 (240.1 mg, 0.19 mmol) in CH2Cl2 (2 mL). The mixture 58.31, 47.68, 47.04, 46.80, 37.46, 22.58, 20.82, 20.65, 20.58, 20.49,
was stirred for 6 hours and then diluted with toluene (10 mL). The 20.40, 20.24, 18.35; HRMS (ESI) Anal. Calcd for C57H64N3O24F2 [M-H]-:
reaction mixture was concentrated in vacuo and the crude product 1212.3848, Found: 1212.3862.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
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ARTICLE
Journal Name
N-(9H-Fluorene-9-yl)-methoxycarbonyl-O-{2-fluoroacetamido-3,4-
di-O-acetyl-2-deoxy-6-O-[benzyl-(5-fluoroacetamido-4,7,8,9-tetra-
O-acetyl-3,5-dideoxy-α-glycero-D-galacto-2-
Glycopeptide 26: 75.0 mg, 26%, Rt = 9.7 min (A/B: (20:80)→(40:60),
View Article Online
10 min). MALDI-TOF MS: 2944.3171 [M+H]+,DcOalI:d1.:02.1904394/.C326O75B.01092J
Glycopeptide 27: 88.3 mg, 28%, Rt = 10.0 min (A/B: (20:80)→(40:60),
nonulopyranosyl)onate]-α-D-galactopyranosyl}-L-threonine (24): 10 min). MALDI-TOF MS: 2962.2974 [M+H]+, cald.: 2962.3181.
The procedure is the same as described in the synthesis of 21. This Glycopeptide 28: 52.0 mg, 18%, Rt = 10.2 min (A/B: (20:80)→(40:60),
compound was prepared from 20, quantitative yield. +25.4º 10 min). MALDI-TOF MS: 2962.2202 [M+H]+, cald.: 2962.3181.
(c 3.09, CHCl3); 1H NMR (400 MHz, DMSO-d6) δ 12.78 (s, 1H), 8.17 (d,
J = 9.7 Hz, 1H), 8.04 (d, J = 9.2 Hz, 1H), 7.88 (d, J = 7.3 Hz, 2H), 7.72 Deprotection of the glycosyl moiety to get 29-32: Glycopeptides
(d, J = 7.4 Hz, 2H), 7.51 (d, J = 9.4 Hz, 1H), 7.44 – 7.30 (m, 9H), 5.24 25–28 were dissolved in water and the pH of the solution was
(s, 3H), 5.21 – 5.15 (m, 2H), 5.15 – 5.09 (m, 1H), 4.95 – 4.60 (m, 6H), adjusted to 11 by treating with 1M NaOH. The reaction was
4.56 – 4.39 (m, 2H), 4.32 – 4.15 (m, 5H), 4.10 (d, J = 9.9 Hz, 1H), 4.05 monitored by HPLC until it was finished, typically 12-24 hours. Then
– 3.93 (m, 3H), 3.78 – 3.69 (m, 1H), 3.12 (dd, J = 9.9, 4.1 Hz, 1H), the reaction was neutralized by 1M acetic acid. The crude products
2.60 (dd, J = 12.5, 4.4 Hz, 1H), 2.05 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), were purified by HPLC, and the products were lyophilized.
1.95 (s, 3H), 1.94 (s, 3H), 1.90 (s, 3H), 1.67 (t, J = 12.3 Hz, 1H), 1.12 Glycopeptide 29: 11.6 mg, 51%, Rt = 10.0 min (A/B: (5:95)→(30:70),
(d, J = 6.2 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ 171.66, 169.90, 15 min). MALDI-TOF MS: 2293.0049 [M-Sial+H] +, cald.: 2293.1312.
169.79, 169.72, 169.57, 169.43, 169.17, 167.40 (d, J =18.8 Hz), Glycopeptide 30: 44.5 mg, 57%, Rt = 10.1 min (A/B: (5:95)→(30:70),
166.43, 156.78, 143.75, 143.68, 140.79, 140.72, 135.01, 128.45, 15 min). MALDI-TOF MS: 2310.9719 [M-Sial+H] +, cald.: 2310.1139.
128.40, 127.97, 127.61, 127.58, 127.06, 127.02, 125.15, 125.05, Glycopeptide 31: 44.7 mg, 67%, Rt = 10.6 min (A/B: (5:95)→(30:70),
120.14, 120.08, 98.60, 98.03, 79.86 (d, J =182.5 Hz), 79.67 (d, J 15 min). MALDI-TOF MS: 2329.0969 [M-Sial+H] +, cald.: 2329.1124.
=178.7 Hz), 75.47, 71.32, 68.75, 67.58, 67.41, 67.25, 67.20, 66.86, Glycopeptide 32: 32.7 mg, 71%, Rt = 10.1 min (A/B: (5:95)→(30:70),
65.49, 62.99, 61.87, 58.45, 47.24, 46.76, 46.49, 37.41, 20.79, 20.61, 15 min). MALDI-TOF MS: 2310.9895 [M-Sial+H] +, cald.: 2310.1139.
20.54, 20.50, 20.43, 20.22, 18.38; HRMS (ESI) Anal. Calcd for
C57H64N3O24F2 [M-H]-: 1212.3848, Found: 1212.3832.
Synthesis of glycopeptide squaric acid monoamides 33–36: To a
solution of glycopeptides 29–32 dissolved in EtOH/H2O (1:1) was
added 3,4-diethoxy-3-cyclobutene-1,2-di-one (1.2 equiv.). The pH of
the solution was adjusted to 8 by treating with saturated NaHCO3.
The reaction was monitored by HPLC until it was finished, typically
2-3 hours. Then the reaction was neutralized by 1M acetic acid. The
crude products were purified by HPLC and the products were
lyophilized.
Glycopeptide 33: 9.2 mg, 68%, Rt = 12.5 min (A/B: (5:95)→(30:70),
15 min). MALDI-TOF MS: 2417.0164 [M-Sial+H] +, cald.: 2417.1472.
Glycopeptide 34: 39.5 mg, 84%, Rt = 12.6 min (A/B: (5:95)→(30:70),
15 min). MALDI-TOF MS: 2435.0339 [M-Sial+H] +, cald.: 2435.1378.
Glycopeptide 35: 36.6 mg, 79%, Rt = 13.0 min (A/B: (5:95)→(30:70),
15 min). MALDI-TOF MS: 2453.0793 [M-Sial+H] +, cald.: 2453.1284.
Glycopeptide 36: 22.4 mg, 65%, Rt = 12.7 min (A/B: (5:95)→(30:70),
15 min). MALDI-TOF MS: 2434.9424 [M-Sial+H] +, cald.: 2435.1378.
Synthesis of glycopeptides and protein conjugates
General methods for the synthesis of glycopeptides and protein
conjugates: The solid-phase peptide synthesis was carried out
manually using the 25 mL-vessel. Reverse-phase HPLC was done
with Waters 2535 equipment. SunFireTM Prep C18 OBDTM 5 μm
19×150 mm column was used with a flow rate of 15 mL/min.
Elution was performed with solution A (acetonitrile with 0.6%
trifluoroacetic acid) and solution B (water with 0.6 % trifluoroacetic
acid). Signals were detected with a UV detector at a wavelength of
214 nm. MALDI-TOF MS spectra were recorded on AB Sciex
TOF/TOF™ Series Explorer™ 72115 by using α-cyano-4-
hydroxycinnamic acid (CHCA).
Synthesis of MUC1 Glycopeptides 25–28: NH2-Ala-Wang-resin with
a loading rate of 0.18 mmol/g was used for the solid-phase peptide
synthesis in a scale of 0.1 mmol. The Fmoc protecting group was
deprotected under a condition of 20% piperidine in DMF for 5
minutes and 20 minutes. For the coupling reactions of amino acids
and the triethylene glycol linker, typically, the amino acids (4 equiv.),
HATU (4 equiv.), and HOAt (4 equiv.), DIPEA (8 equiv.) were
dissolved in DMF and the reaction was left for 50 minutes. The
glycosylated threonines (STn-Thr) were coupled with a stronger
condition using STn-Thr (2.0 equiv.), HATU (2.5 equiv.), HOAt (2.5
equiv.), and NMM (5.0 equiv.) in NMP for 12 hours. After each
coupling, the unreacted free amino groups were capped by treating
with DMF/Ac2O/Py= 3:2:1 for 40 minutes. After all the coupling
reactions were done, the resin was transferred from the peptide
synthesizer into a flask. The glycopeptides were detached from the
resin using a condition of TFA/TIS/H2O = 12:1:1 for 2 hours. The
crude products were purified by HPLC.
Conjugation of glycopeptide squaric acid monoamides 33–36 with
KLH to get vaccines V1-V4: Glycopeptide squaric acid monoamides
33–36 (3.0 mg) and KLH of equal mass were dissolved in a buffer of
0.07M Na2B4O7/0.035M KHCO3 (1 mL). The reaction was left for 24
hours and dialyzed with deionized water using a membrane of 12
kDa for 48 hours. The water was removed by lyophilization to afford
the glycopeptide–KLH conjugates. Yield: 5.3 mg for V1, 5.1 mg for
V2, 5.0 mg for V3, 5.0 mg for V4.
Epitope ratios of the glycopeptide–KLH conjugates as vaccine
candidates V1-V4: The resorcinol method was used to determine
the content of sialic acid, based on which the content of STn-MUC1
glycopeptide was calculated. The glycoconjugate (100 μL) was
mixed with the resorcinol reagent (100 μL) and heated by boiling
water bath for 30 minutes, then cooled on ice for 10 minutes. An
extraction solution (1-butanol acetate and 1-butanol, 85:15 v/v, 250
μL) was added to the mixture. The mixture was kept standing still
for 15 minutes after it was shaken vigorously to allow the organic
Glycopeptide 25: 38.2 mg, 13%, Rt = 9.5 min (A/B: (20:80)→(40:60),
10 min). MALDI-TOF MS: 2926.91 [M+H]+, cald.: 2926.34.
10 | J. Name., 2012, 00, 1-3
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Page 11 of 13
Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
layer to separate well from the inorganic layer. The absorbance of was added, and the mixture was incubated for 30 minutes on ice.
View Article Online
organic layer at 580 nm was determined by an UV-vis spectrometer, Washed twice and assayed using a FACScan D(BOeIc:t1o0n.10D3i9c/kCin6sOoBn0)1.092J
using a blank organic solution as the control. A measurement of the
STn disaccharide standard was performed under the same
conditions to create a calibration curve. The sialic acid content of
Acknowledgements
the glycoconjugate was determined by this calibration curve and
the content of STn-MUC1 glycopeptide was calculated as V1 (1.9 mg,
36%), V2 (2.0 mg, 39%), V3 (2.0 mg, 40%), V4 (2.2 mg, 44%).
We thank Dr. Yunsen LI (Institutes of Biology and Medical
Sciences, the First Affiliated Hospital, Soochow University) for
kindly providing the AG104A. This work was financially
supported by the grants (2012CB822100, 2013CB910700) from
the Ministry of Science and Technology of China, and the
National Natural Science Foundation of China (21232002,
81172916).
Immunological studies
Immunization of mice. The immunological efficacy of these
glycopeptide–KLH conjugates was examined with BALB/c mice. Six
to eight weeks old female BALB/c mice (Number: SCXKjing2012-
0001, SPF/VAF) were obtained from Department of Laboratory
Animal Science, Peking University Health Science Center. Groups of
six mice were immunized by intraperitoneal injection for four times 1.
at 2-week intervals with 10 μg of glycopeptide–KLH conjugates.
Notes and references
R. O. Dillman, Cancer Biother. Radiopharm., 2011, 26, 1-
64.
2.
B. Acres and J. M. Limacher, Expert Rev. Vaccines, 2005, 4,
493-502.
Mice were bled 14 days after the 4th immunization. Serum was
o
isolated from blood. After complement was inactivated at 37 C for
30 minutes serum was stored at -80 oC.
3.
4.
5.
T. Becker, S. Dziadek, S. Wittrock and H. Kunz, Curr.
Cancer Drug Targets, 2006, 6, 491-517.
R. E. Beatson, J. Taylor-Papadimitriou and J. M. Burchell,
Immunotherapy, 2010, 2, 305-327.
G. E. Kim, H. I. Bae, H. U. Park, S. F. Kuan, S. C. Crawley, J. J.
L. Ho and Y. S. Kim, Gastroenterology, 2002, 123, 1052-
1060.
Serological assay. The antigen-specific antibody titers of the pooled
sera and individual mouse were assessed by ELISA. BSA-conjugates
with glycopeptide dissolved in 0.05 M bicarbonate buffer (pH = 9.6)
and coated 100 μL/well (including 0.125 μg of glycopeptide-BSA) at
ELISA plates and incubated at 4 °C overnight. After three washings
with 200 μL of PBST (0.05% Tween-20 in PBS), 3% BSA in PBS was
6.
7.
8.
E. Kagan, G. Ragupathi, S. S. Yi, C. A. Reis, J. Gildersleeve,
D. Kahne, H. Clausen, S. J. Danishefsky and P. O. Livingston,
Cancer Immunol. Immunother., 2005, 54, 424-430.
A. Kaiser, N. Gaidzik, U. Westerlind, D. Kowalczyk, A.
Hobel, E. Schmitt and H. Kunz, Angew. Chem. Int. Ed.,
2009, 48, 7551-7555.
A. Hoffmann-Roder, A. Kaiser, S. Wagner, N. Gaidzik, D.
Kowalczyk, U. Westerlind, B. Gerlitzki, E. Schmitt and H.
Kunz, Angew. Chem. Int. Ed., 2010, 49, 8498-8503.
S. Wagner, C. Mersch and A. Hoffmann-Roder, Chem. Eur.
J., 2010, 16, 7319-7330.
o
added to block each well at 37 C for 1 hour. Washed three times,
serial 2-fold diluted sera were added in wells and incubated at 37 oC
for 1 hour. The plate was washed and incubated with a 1:5000
dilution of horseradish peroxidase conjugated goat anti-mouse IgG
(γ-chain specific) (Southern Biotechnology Associates, Inc.,
Buckingham, AL) at 37 oC for 1 hour. Washed and developed with O-
phenylenediamine (OPD) substrate in the dark for 15 minutes,
terminated with 2 M H2SO4, and then read at 490 nm. The antibody
titer was defined as the highest dilution showing an absorbance of
0.1, after subtracting background. Every concentration was
triplicate wells. The assays were conducted separately twice. As to
9.
10.
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antibody goat anti-mouse IgG (γ-chain specific) Alex-488 (Jacson)
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DOI: 10.1039/C6OB01092J
Table of Contents:
Unnatural STn disaccharides with N-acetyl modifications were incorporated into a 20-amino acid
MUC1 tandem repeat sequence. The modified STn-MUC1 glycopeptide-protein conjugates
showed high immunogenicity.