Oligosaccharides and peptide displayed on an amphiphilic polymer enable solid phase assay of hapten specific antibodies

Article abstract of DOI:10.1021/bc400486w

Copovidone, a copolymer of vinyl acetate and N-vinyl-2-pyrrolidone, was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization, and after deacetylation the polymer was functionalized by introduction of amino, azide, and alkyne pendant groups to allow attachment of glycans and peptide. Candida albicans β-mannan trisaccharides 1 and 2 and M. tuberculosis arabinan hexasaccharide 3 with appropriate tethers were conjugated to the polymers by squarate or click chemistry. C. albicans T-cell peptide 4 bearing a C-terminal I?μ -azidolysine was also conjugated to copovidone by click chemistry. The resulting conjugates provide convenient non-protein-based antigens that are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation or degradation in ELISA readout.

Full text of DOI:10.1021/bc400486w

Article
pubs.acs.org/bc
Oligosaccharides and Peptide Displayed on an Amphiphilic Polymer
Enable Solid Phase Assay of Hapten Specific Antibodies
David R. Bundle,* Pui-Hang Tam, Huu-Anh Tran, Eugenia Paszkiewicz, Jonathan Cartmell,
Joanna M. Sadowska, Susmita Sarkar, Maju Joe, and Pavel I. Kitov
Alberta Glycomics Centre, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
S
* Supporting Information
ABSTRACT: Copovidone, a copolymer of vinyl acetate and
N-vinyl-2-pyrrolidone, was synthesized via reversible addi-
tion−fragmentation chain transfer (RAFT) polymerization,
and after deacetylation the polymer was functionalized by
introduction of amino, azide, and alkyne pendant groups to
allow attachment of glycans and peptide. Candida albicans β-
mannan trisaccharides 1 and 2 and M. tuberculosis arabinan
hexasaccharide 3 with appropriate tethers were conjugated to
the polymers by squarate or click chemistry. C. albicans T-cell
peptide 4 bearing a C-terminal ε-azidolysine was also
conjugated to copovidone by click chemistry. The resulting
conjugates provide convenient non-protein-based antigens that
are readily adsorbed on ELISA plates, and display excellent characteristics for assay of antibody binding to the haptenic group of
interest. Copovidone and BSA glycoconjugates exhibited similar adsorption characteristics when used to coat ELISA plates, and
both conjugates were optimal when used as coating solutions at low nanogram/mL concentrations. Provided that the copovidone
conjugated glycan is stable to acid, assay plates can be easily processed for reuse at least three times without detectable variation
or degradation in ELISA readout.
are functionalized for conjugation to protein by well-defined
chemistry.3−6,9,10 The choice of protein (carrier protein) to
which oligosaccharide is covalently attached is crucial because
carbohydrates are nonimmunogenic, and to provide efficacious
vaccines, they must first be conjugated to strongly immuno-
genic proteins, such as bacterial toxoids (tetanus or diphtheria
toxoids11) or other candidates such outer membrane proteins11
or keyhole limpet hemocyain (KLH).7
Measurement of antibody response following immunization
with conjugate vaccines typically involves conjugation of the
carbohydrate antigen to a protein that is different from that of
the conjugate vaccine. This second glycoconjugate is used to
coat ELISA plates and detect antibodies.9,10 In this way the
antibody response to the glycan component of the vaccine can
be determined without confounding issues of antibodies that
are directed to the carrier protein.
Our work on a Candida albicans glycoconjugate vaccine
typically employed tetanus toxoid as the immunogenic protein,
to which we attached oligosaccharide epitopes.6,9,10 Antibodies
to the oligosaccharide haptenic group are monitored by
conjugating the same hapten to a heterologous protein such
as BSA, and this conjugate is used to coat ELISA plates. The
range of choices for a second protein for synthesis of coating
INTRODUCTION
■
Conjugate vaccines constructed from the capsular polysacchar-
ides of Neisseria meningitidis and Streptococcus pneumoniae and
covalently attached to an immunogenic protein such as tetanus
toxoid or diphtheria toxoid now rank as blockbuster
prophylactic vaccines with annual sales exceeding several billion
dollars.1 The success of these vaccines has stimulated a broad
area of research into other microbial diseases that might benefit
from vaccines composed of cell wall oligosaccharides or
polysaccharides conjugated to immunogenic proteins.2−6
Therapeutic cancer vaccines containing tumor specific
oligosaccharides and glycopeptides conjugated to immunogenic
proteins and peptides are also under investigation in many
laboratories.7,8 Here we describe convenient chemistry that
allows the antibodies produced in response to each component
of a conjugate vaccine to be dissected into antibodies against
the carbohydrate, the linker fragment, and peptide/protein
components.
The design of conjugate vaccines ideally requires either the
isolation and characterization of the cell wall polysaccharide to
be coupled to protein, or identification of the minimum-sized
oligosaccharide fragment of polysaccharide (the protective
epitope), which when conjugated to protein is able to raise
antibodies that kill bacteria,4−6,9 or in the case of cancers, a
tumor specific immune response.7,8 The ability to employ
oligosaccharide epitopes is attractive because it permits the use
of chemical synthesis of relatively small oligosaccharides that
Received: October 21, 2013
Revised: March 5, 2014
Published: March 6, 2014
© 2014 American Chemical Society
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Figure 1. Oligosaccharides 1−3 and peptide 4 haptens for attachment to functionalized copovidone.
antigens is quite limited, because this protein should possess 10
or more sites, usually lysine residues, that permit facile
conjugation of haptenic groups. The second protein should
lack N- or O-glycosylation sites, exhibit good aqueous solubility
before and after conjugation, and ideally the glycoconjugate
should be soluble in water so that it may be freeze-dried to
allow convenient preparation of coating antigens of known
concentration. BSA fulfills these roles well and its conjugates
readily dissolve in aqueous buffer when reconstituted after
lyophilization. Use of BSA may be prohibited if antiserum
contains BSA specific antibodies and especially if BSA or other
serum albumin glycoconjugates are employed as carrier
proteins in conjugate vaccines. These issues highlight the
need for a suitable polymer that contains reactive groups for
attachment of antigenic determinants while being sufficiently
lipophilic to effectively coat plastic microtiter plates.
In search of a nonprotein, biocompatible polymer that would
also be approved for use as a multivalent drug candidate, we
identified copovidone, a widely used excipient in drug
formulations.13 It is a copolymer of vinyl acetate and N-vinyl-
2-pyrrolidone with the attractive feature that it is compatible
with a variety of chemical manipulations performed in both
organic and aqueous media. These properties are retained after
deacetylation and allow the introduction of side chain groups
through which haptens may be readily attached.14
from fructose-bisphosphate aldolase, a protein present in C.
albicans during pathogenesis of human disseminated candidia-
sis.19 A conjugate composed of Fba and a variant of
trisaccharide 2 was shown to be an effective glycopeptide
conjugate vaccine20 and its conjugation to copovidone allows
for convenient detection of peptide specific antibodies. The
third oligosaccharide is a branched hexasaccharide from the cell
wall of Mycobacterium tuberculosis, the crystal structure of which
has been solved as a complex with the monoclonal antibody
(mAb) CS35.21,22
We demonstrate here that copovidone conjugates are
antigens that show excellent coating of microtiter plates at
low nanogram/mL concentrations and that the ELISA data
obtained with them exhibits low nonspecific binding of
antibody to the copovidone coated plates. Moreover, when
the oligosaccharide hapten is stable to acid solution used to
stop color development, ELISA plates can be reused.
RESULTS
■
Reversible addition−fragmentation chain transfer polymer-
ization (RAFT) of vinyl acetate and N-vinyl-2-pyrrolidone
gave polymers of ∼40 kDa molecular weight. Saponification
and subsequent derivatization at hydroxyl groups was used to
create azido P1 and amino P2 substituted polymers as
previously described14 and an alkyne derivative P3 (Figure 2).
Trisaccharide 1 was prepared by using glycosyl donor 5
according to chemistry developed by Crich.23,24 2-Azidoethyl α-
D-mannopyranoside25 6 was glycosylated by 523 to give
disaccharide 7, which was selectively deprotected to give
acceptor 8 for a second glycosylation to afford trisaccharide 9.
Removal of the p-methoxybenzyl group gave 10 and a two-
stage hydrogenation26 gave the acetate salt of 1. Reaction of 1
with dibutyl squarate gave the squarate half ester 11, which may
We report here the synthesis of copovidone with pendant
groups terminated by amine, azide, and alkyne groups, and the
conjugation of three oligosaccharides (1−3) and one peptide
(4) to these polymer derivatives (Figure 1). Trisaccharides 1
and 2 are α and β 2-aminoethyl glycosides of a Candida albicans
trisaccharide that has been shown to be a protective epitope
present in the cell wall phosphomannan of this fungal
pathogen.15−18 The Fba peptide 4 is a T cell peptide derived
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Fba peptide 4 installed with an ε-azido-L-lysine residue at its
C-terminus was conjugated to alkyne substituted copovidone
polymer P3 by a Huisgen 1,3-dipolar cycloaddition reaction to
directly yield conjugate 24 (Scheme 4). The degree of peptide
incorporation was estimated by the NMR method previously
discussed14 and also in this case by HPLC separation and
integration of free and bound peptide (Supporting Information
page 39).
In order to compare the properties of copovidone 22 and
BSA 23 glycoconjugates, plates were coated with these Ara6
antigens in the concentration range 10 μg/mL − 0.1 ηg/mL
and titered against serial dilutions of CS35 antibody. The
results are displayed as three-dimensional plots (Figure 3a and
b). The data used to create the graphs are tabulated as
Supporting Information Tables S1 and S2). The coating
efficiency of the conjugate is most clearly shown by viewing
two-dimensional slices showing absorbance versus antigen
coating at constant antibody concentration (Figure 3c).
Comparison of the coating efficiency for the two conjugates
determined by ELISA response was similar across the
concentration range and clearly demonstrates that each
conjugate has an optimal coating concentration of approx-
imately 100−10 ηg/mL (Figure 3c).
The general utility of povidone conjugates was established
with specific mAbs and polyclonal sera as follows. Conjugates
12 and 20 were used to coat ELISA plates, and a titration assay
was performed with serial dilutions of affinity purified C.
albicans mAb C3.1.16 C3.1 binds to both conjugates with
similar affinity (Figure 4a). In other work,18 we have shown
that either α- or β-mannotriose represented by compound 1 or
2 are equally effective as inhibitors of this mAb, so the small
difference observed in the two titration curves (Figure 4a) that
could be interpreted as a preference for binding to the α-
mannotriose conjugate 12 actually results from the higher
epitope loading of this conjugate (payload 9.8% versus 6.8% for
20). The titration curve obtained with CS35 mAb indicates that
Figure 2. Functionalized copovidone with azide P1, amino P2, and
alkyne P3 pendant groups.
be used directly but here was isolated and characterized.
Reaction of 11 with the aminated copovidone P2 provided the
conjugate 12 (Scheme 1).
Trisaccharide 2 was prepared in analogous fashion with the
exception that the donor employed was phenyl glycosyl
sulfoxide 13 (Scheme 2).23 It was used to glycosylate 2-
azidoethanol to provide 14 and the starting acceptor 15 after
removal of the p-methoxybenzyl group. Iterative cycles of
glycosylation by 13 or 523 gave first the disaccharide 16 and
subsequently the disaccharide acceptor 17 and finally the
deprotected trisaccharide 2 obtained from 18. Acylation of 2 by
the succinimide ester of glutaric acid propargylamide27 gave 19.
This alkyne derivative was conjugated to azide bearing
copovidone P1 by a Huisgen 1,3-dipolar copper(I)-catalyzed
cycloaddition reaction28−31 to afford the conjugate 20.
Integration of 1H NMR side chain resonances permits
quantification of the degree of substitution. This procedure is
described in detail in a prior publication.14
The branched Ara6 hexasaccharide 8-trifluoroacetamido-octyl
glycoside 321 was treated with base to liberate the amine
terminated tether, which was then acylated by the NHS ester of
glutaric acid monopropargylamide27 and the resulting com-
pound 21 was conjugated to the azido substituted copovidone
P1 to give 22 (Scheme 3). Hexasaccharide 3 was also
conjugated to BSA to give the glycoconjugate Ara6-BSA (23).
a
Scheme 1
a
Reagents and conditions: (a) BSP, TTBP, Tf2O, 4 Å MS, CH2Cl2; (b) Donor 5, BSP, TTBP, Tf2O, 4 Å MS, CH2Cl2; (c) DDQ, CH2Cl2; (d) (i)
Pd/C, pyridine, H2; (ii) Pd/(OH)2, AcOH/H2O, H2; (e) dibutyl squarate, MeOH; (f) P2, Na2CO3 buffer pH 9.
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a
Scheme 2
a
Reagents and conditions: (a) 2-azidoethanol, TTBP, Tf2O, 4 Å MS, CH2Cl2; (b) DDQ, CH2Cl2; (c) 13, TTBP, Tf2O, 4 Å MS, CH2Cl2; (d) (i) 5,
BSP, TTBP, Tf2O, 4 Å MS, CH2Cl2; (ii) DDQ, CH2Cl2; (e) (i) Pd/C, pyridine, H2; (ii) Pd/(OH)2, AcOH/H2O, H2; (f) P1, CuSO4, sodium
ascorbate.
the copovidone conjugate 22 performs as well as the
corresponding Ara6-BSA glyconjugate 23 in ELISA (Figures
3c and 4b). When copovidone without pendant groups was
used to coat plates, nonspecific absorption of mAb can be seen
to be esentially zero across a wide range of antibody
concentrations (Figure 4b).
Sera from a group of mice immunized with a glycopeptide
[Man3-Fba]16-tetanus toxoid conjugate (unpublished results)
were titered on a plate coated with the Fba-copovidone
conjugate 24 (Figure 4c). On the same plate, polyclonal sera
from mice immunized with alum were titered. Fba end point
titers showed that the glycoconjugate raised peptide specific
antibodies that titered to ∼105. The control sera gave negligible
background at dilution of 10−3 or higher.
Under certain circumstances such as iterative on plate
generation and screening protocols used to discover improved
carbohydrate ligands for Shiga like toxins recently described by
us,32 it could be useful to repeat ELISA assays on the same
plate. There have been several reports in the literature
concerning reuse of ELISA plates.33,34 In some of these
methods extensive incubation with detergents and denaturing
agents was necessary. We reasoned that arresting color
development by addition of 1 M phosphoric acid would
cause loss of bound antibody from antigen coated plates. This
encouraged us to attempt reuse of plates because the
developmental stage with horseradish peroxidase-conjugated
second antibody employs 1 M phosphoric acid to stop color
development. This is essentially an elution step for any bound
protein. We observed that plates coated with copovidone
conjugates 12 and 20 could be reused at least 3 times without
any degradation in the reproducibility of end point titers or the
magnitude of the signal at any serum or antibody dilutions
(Figure 5 and Table S3 Supporting Information).
However, carbohydrate epitopes such as the arabinofuranose
residues of conjugate 22 are known to be sensitive to hydrolysis
dilute acid35 and plates coated with antigen 22 show a decrease
in binding capacity with repeated exposure to acid. Peptide
copovidone conjugate 24 was not amenable to a recycling
regime. Having established that plates coated with conjugates
12 and 20 could be recycled, we also observed that that ELISA
plates coated with a mannotriose-BSA conjugate10 prepared
from 2 could also be recycled (Supporting Information, Table
S4).
CONCLUSIONS
■
Originally, we selected copovidone as a biocompatible polymer
to display ligands for potential application as oral therapeutics
to absorb toxins such as the Shiga-like toxin of enter-
ohemorrhagic E. coli.14,32 We have shown that the polymer
exhibits excellent characteristics for repeated cycles of on-plate
chemistry and activity assays in the elaboration of focused
libraries of toxin agonists.32 Here we extend those observations
and describe the application of copovidone as a convenient
nonprotein polymer that is readily synthesized and sub-
sequently modified with a variety of functional groups that
allow for facile conjugation of saccharide and peptide haptens
employing squarate and click chemistry, thereby permitting
determination of epitope-specific antibody response to
synthetic conjugate vaccines. The resulting conjugates display
excellent characteristics in ELISA, and provided the hapten
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a
Scheme 3
EXPERIMENTAL PROCEDURES
General Methods. 1H NMR spectra were recorded at
■
either 400, 500, or 600 MHz, and are referenced to the residual
protonated solvent resonance: δH 7.24 ppm for solutions in
CDCl3, and 0.1% external acetone (δH 2.225) for solutions in
D2O. Mass analysis was performed by positive-mode electro-
spray ionization on a hybrid sector-TOF mass spectrometer
and for protein glycoconjugates by MALDI mass analysis,
employing 2,5-dihydroxybenzoic acid (DHB) as matrix.
Analytical thin-layer chromatography (TLC) was performed
on silica gel 60-F254 (Merck). TLC detection was achieved by
charring with 5% sulfuric acid in ethanol. All commercial
reagents were used as supplied. Column chromatography used
silica gel (SiliCycle, Quebec City, Quebec, 230−400 mesh, 60
Å) and redistilled solvents. HPLC separations were performed
on a Beckmann C18 semipreparative reversed-phase column
with a combination of acetonitrile and water containing 0.1%
acetic acid as eluent. Photoadditions were carried out using a
Spectroline model ENF-260C UV lamp and cylindrical quartz
vessels.
The ligand payload on copovidone polymers, compounds 12,
20, 22, and 24, were estimated by integration of ligand and
polymer resonances and the degree of incorporation was
calculated as reported,13 where payload was calculated as the
percentage of monomers (vinyl pyrrolidone and vinyl alcohol
combined) substituted with the ligand. The ligand payload for
23 was also determined by HPLC of polymer bound versus free
peptide.
a
Reagents and conditions: (a) (i) MeOH, NaOCH3; (ii) H+ ion
Polymers. Copovidone derivatives P1 and P2 were
synthesized as previously described.13 The alkyne substituted
polymer P3 was obtained as follows.
exchange resin; (b) P1, CuSO4, sodium ascorbate.
Poly(prop-2-ynyl 2-(3-(vinyloxy)propylthio)-
ethylcarbamate-co-N-vinyl-2-pyrrolidone) P3. To a solution
of poly(2-(3-(vinyloxy)propylthio)ethanamine-co-N-vinyl-2-
pyrrolidone)13 (100 mg) in 1 M NaHCO3 (3 mL), a solution
of propargyl chloroformate (100 μL, 1 mmol) in methanol (1
groups are stable to strong acid at room temperature, the plates
may be reused at least three times without degradation of
response signals.
a
Scheme 4
a
Reagents and conditions: (a) CuSO4, sodium ascorbate, (Et3NH)HCO3 buffer.
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Figure 4. (a) Titration of C. albicans mAb C3.116 on plates coated
■
■
with conjugates 12 (red ) and 20 (black ); (b) titration of the mAb
CS3522 on plates coated with conjugate 22 (red ), Ara6-BSA 23
●
■
▲
(black ), and unconjugated copovidone (blue ); (c) titration of
polyclonal sera on plates coated with Fba-copovidone conjugate 24
■
●
▲
▼
(red , black , green , red +, and blue ). Five mice were
immunized with Man3-Fba-tetanus toxoid glycoconjugate absorbed on
■
▲
▼
alum and two control mice immunized with alum (gray , purple ).
Two mice had superimposable titration curves (green , red +).
Figure 3. Comparison of ELISA response at different antigen coating
and antibody concentrations. Two antigens were examined,
copovidone conjugate 22 and its BSA counterpart 23. (a) Three
dimensional plot of the variation of ELISA absorbance plotted against
serial dilutions of antigen 22 and antibody CS35. (b) Three-
dimensional plot of the variation of ELISA absorbance plotted against
serial dilutions of antigen 23 and antibody CS35. (c) Comparison of
ELISA response at constant antibody concentration and variable
antigen coating demonstrating the similar antigen coating effciency for
maximum response.
mL) was slowly added and the solution was stirred for 1 h at
room temperature. The product was dialyzed extensively
against deionized water and freeze-dried to afford the title
1
compound P3 as a white foam (95 mg); H NMR (600 MHz,
D2O) δH 4.8−4.6 (br m under water, CH2 propargyl), 4.3−2.9
(br m, polymer backbone), 4.30−2.90 (br m, polymer
backbone), 2.80−2.55 (br m, CH propargyl), 4.3−2.9 (br m,
polymer backbone), 2.55−1.30 (br m, polymer backbone); IR
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(ESI) calcd. for (M + H) C20H38NO16: 548.2185. Found:
548.2181. FTIR: 3255.6 cm−1.
2-Aminoethyl β-D-mannopyranosyl-(1→2)-β-D-manno-
pyranosyl-(1→2)-β-D-mannopyranoside (2). Pd/C (10 wt
%, 100 mg) was added to trisaccharide 18 (87 mg, 0.078 mmol)
in pyridine (8 mL) and the reaction was stirred under H2
atmosphere overnight.26 The mixture was filtered through
Celite and the solvent was coevaporated with toluene a few
times. The residue was further dried in vacuo for 30 min before
being redissolved in 10:1 AcOH/H2O (10 mL). Pd(OH)2/C
(10 wt %, 140 mg) was added and the reaction was stirred
under H2 atmosphere overnight. The mixture was filtered
through Celite and the solvents were coevaporated several
times with toluene. The residue was further dried in vacuo
overnight to give the crude product as white solid. The crude
was further purified by HPLC using TSK-GEL Amide-80
column to give the pure product 2 (30 mg, 64%) as fluffy white
1
solid: [α]D = −85.1 (c 0.4, H2O); H NMR (500 MHz, D2O)
δH 4.93 (s, 1H, H-1′), 4.91 (s, 1H, H-1″), 4.76 (s, 1H, H-1),
4.33 (d, 1H, J = 3.2 Hz, H-2′), 4.27 (d, 1H, J = 3.0 Hz, H-2),
4.12 (d, 1H, J = 3.0 Hz, H-2″), 4.08 (ddd, 1H, J = 10.0, 4.9, 4.9
Hz, OCH2), 3.88−3.96 (m, 4H, H-6a, H-6a′, H-6a″, OCH2),
3.46−3.79 (m, 9H, H-3, H-4, H-6b, H-3′, H-4′, H-6b′, H-3″,
H-4″, H-6b″), 3.31−3.43 (m, 3H, H-5, H-5′, H-5″), 3.24 (t,
1H, J = 4.9 Hz, CH2NH2), 1.92 (s, 3H, CH3); 13C NMR (125
MHz, D2O) δC 182.4 (CO), 101.5(8), 101.5(5) (C-1′, C-
1″), 101.1 (C-1), 79.1 (C-2), 78.9 (C-2′), 77.4, 77.2(8),
77.2(7) (C-5, C-5′, C-5″), 74.0, 73.2, 72.8 (3C, C-3/C-3′/C-
3″/C-4/C-4′/C-4″), 71.4 (C-2″), 68.3, 67.9, 67.8 (3C, C-3/C-
3′/C-3″/C-4/C-4′/C-4″), 67.3 (OCH2), 62.1, 61.7, 61.6 (3C,
C-6, C-6′, C-6″), 40.2 (CH2NH3), 24.3 (CH3). HRMS (ESI)
calcd. for (M + H) C20H38NO16: 548.2185. Found: 548.2175.
FTIR: 3313.0 cm−1.
Ac-Tyr-Gly-Lys-Asp-Val-Lys-Asp-Leu-Phe-Asp-Tyr-Ala-Gln-
Glu-(ε-N3-Nleu)-OH (4). 2-Chlorotrityl chloride polystyrene
resin (Nova Biochem) was derivatized with Fmoc-ε-N3-Nleu-
OH and immediately used for peptide synthesis according to
standard Fmoc peptide synthesis procedures in an ABI 433A
peptide synthesizer at 0.10 mmol scale. The protocol employed
was the UV FastMoc protocol with single couplings and no
postcoupling capping. Reagents used were N,N,N′,N′-tetra-
methyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate
and N-hydroxybenzotriazole with N,N-diisopropylethylamine
for coupling and 20% piperidine in N-methylpyrrolidin-1-one
(NMP) for removal of Nα-Fmoc groups from the growing
peptides’ N-termini. After removal of the final Nα-Fmoc group
from the fully elongated peptide, the terminal amino group was
capped by treatment with 1 mmol acetic anhydride and 2 mmol
pyridine in 3 mL NMP. The protected peptidyl resin was
washed with 3 × 5 mL NMP, then 3 × 5 mL CH2Cl2. The
peptide was cleaved from the resin and deprotected by gently
shaking the peptidyl resin in a cocktail of H2O (250 μL),
triisopropyl silane (250 μL), and trifluoroacetic acid (TFA, 9.5
mL) for two hours. The resin was filtered from the peptide
solution and washed with two 10 mL portions of TFA. The
filtrates were combined in a round-bottomed flask and
concentrated by rotary evaporation to a volume of approx-
imately 1 mL. Toluene (5 mL) was added and the mixture
concentrated again to give a film of crude peptide. Dry diethyl
ether (15 mL) was added and the peptide film was scraped
from the flask walls and crushed to give a suspension of fine
white powdered peptide in the ether. The peptide precipitate
was allowed to settle for five minutes and the supernatant ether
Figure 5. Reuse of ELISA plates coated with povidone conjugate
conjugate 20. Experiment 1 is the initial use of the ELISA plate
followed by incubation with 1 M phosphoric acid, washing, and
repetition of the assay, experiment 2. After recording absorbance
incubation, washing and reuse gave data for experiment 3. Data are
plotted as a bar graph for plates coated with conjugate 20. The data
recorded in triplicate and used to plot the graph can be found in Table
S3 and for a BSA-Man3 conjugate10 Table S4. Data obtained with
conjugates 22 and 24 (not reported) show poor consistency on reuse.
(cm−1): 3226.79, 3049.4, 2951.04, 2921.97, 2879.45, 2120.27
(alkyne), 1724.52, 1678, 1533.63, 1493.36, 1460. 29, 1423.79,
1371.76, 1316.47, 1287.02, 1231.18, 1168.01, 1130.61, 1094.72,
1036.21, 1000.77, 932.78, 895.84, 844.39, 731.53, 698.48.
2-Aminoethyl β-D-mannopyranosyl-(1→2)-β-D-manno-
pyranosyl-(1→2)-α-D-mannopyranoside (1). Pd/C (10 wt
%, 87 mg) was added to trisaccharide 10 (58 mg, 0.052 mmol)
in pyridine (6 mL) and the reaction was stirred under H2
atmosphere overnight.26 The mixture was filtered through
Celite and the solvent was coevaporated with toluene few
times. The residue was further dried in vacuo for 30 min before
being redissolved in 10:1 AcOH/H2O (6 mL). Pd(OH)2/C
(10 wt %, 110 mg) was added and the reaction was stirred
under H2 atmosphere overnight. The mixture was filtered
through Celite and the solvents were coevaporated with
toluene several times. The residue was further dried in vacuo
overnight to give the crude product as white solid. The crude
was further purified by HPLC using TSK-GEL Amide-80
column to give the pure product 1 (22 mg, 69%) as white fluffy
solid after lyophilization: Rf 0.24 (9:1:0.1 methanol−water−
1
acetic acid); [α]D = −21.1 (c 0.2, H2O); H NMR (600 MHz,
D2O) δH 4.96 (d, 1H, J = 1.4 Hz, H-1), 4.81 (s, 2H, H-1′, H-
1″), 4.24 (d, 1H, J = 3.2 Hz, H-2′), 4.15 (dd, 1H, J = 3.1, 1.9
Hz, H-2), 4.11 (d, 1H, J = 3.1 Hz, H-2″), 3.95 (m, 1H, OCH2),
3.82−3.89 (m, 4H, H-3, H-6a, H-6a′, H-6a″), 3.66−3.74 (m,
4H, H-6b, H-6b′, H-6b″, OCH2), 3.50−3.63 (m, 6H, H-4, H-5,
H-3′, H-4′, H-3″, H-4″), 3.34 (ddd, 1H, J = 9.2, 6.5, 2.5 Hz, H-
5′/H-5″), 3.31 (ddd, 1H, J = 9.3, 6.9, 2.2 Hz, H-5′/H-5″),
3.18−3.26 (m, 2H, CH2NH2), 1.87 (s, 3H, CH3); 13C NMR
(125 MHz, D2O) δC 100.9 (C-1′/C-1″), 99.0 (C-1′/C-1″),
97.9 (C-1), 78.4 (C-2′), 77.6 (C-2), 76.4 (2C, C-5′, C-5″),
73.0, 72.9, 72.1 (C-5, C-3′, C-3″), 70.4 (C-2″), 69.4 (C-3),
67.1, 66.9, 66.8 (C-4, C-4′, C-4″), 63.5 (OCH2), 61.1, 60.7,
60.4 (C-6, C-6′, C-6″), 39.1 (CH2NH3), 23.2 (CH3). HRMS
691
dx.doi.org/10.1021/bc400486w | Bioconjugate Chem. 2014, 25, 685−697

Bioconjugate Chemistry
Article
was removed by decanting. The peptide was washed twice again
with ether and the solid residue was left for two hours to dry in
a fume hood. The dry peptide was dissolved in 3:7
acetonitrile:water (2.5 mL) and purified by reverse phase
HPLC on a 250 mm × 21.2 mm Phenomenex Luna C18(2)
preparative column, eluting with a linear gradient from 7:3 to
67.5:33.5 water:acetonitrile over 25 min, at a flow rate of 10 mL
min−1. The fractions collected from 20 to 23.5 min were
collected, pooled, and lyophilized to give 13.1 mg of the title
compound as a fluffy white solid. Analytical HPLC: tR = 23.177
min, A280 = 98.30% (250 mm × 4.6 mm Phenomenex Luna
C18(2), 1 mL min−1 H2O:CH3CN + 0.1% TFA 75:60 →
50:50, 60 min, A280),. MALDI-TOF-MS (sinapinic acid matrix)
m/z calcd. for (M + H) C85H125N22O27: 1886.91; Found:
(C-6′), 67.8 (C-5′), 66.7 (OCH2), 64.6 (C-5), 55.3 (OCH3),
50.5 (CH2N3). HRMS (ESI) calcd. for (M + Na)
C50H53N3O12: 910.3521. Found: 910.3505. FTIR: 2102.8 cm−1.
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranosyl-(1→2)-3-O-benzyl-4,6-O-benzylidene-α-D-manno-
pyranoside (8). Disaccharide 7 (603 mg, 0.68 mmol) was
dissolved in 4:1 CH2Cl2/H2O (40 mL) and DDQ (192 mg,
0.85 mmol) was added. The reaction was stirred at room
temperature overnight. The mixture was washed with sat.
NaHCO3 (2x) and brine, and dried over anhydrous Na2SO4.
The crude was concentrated and purified by chromatography
(3:2 hexane−EtOAc) to give 8 (406 mg, 78%) as colorless oil:
Rf 0.16 (3:2 hexane−EtOAc); [α]D = −44.4 (c 2.6, CH2Cl2);
1H NMR (600 MHz, CDCl3) δH 7.49−7.52 (m, 4H, ArH),
7.27−7.43 (m, 16H, ArH), 5.58 (s, 1H, PhCH), 5.51 (s, 1H,
PhCH), 4.90 (d, 1H, J = 1.5 Hz, H-1), 4.87 (d, 1H, J = 12.2 Hz,
PhCH2), 4.82 (d, 1H, J = 11.8 Hz, PhCH2), 4.79 (d, 1H, J =
11.7 Hz, PhCH2), 4.78 (d, 1H, J = 11.5 Hz, PhCH2), 4.77 (d,
1H, J = 1.2 Hz, H-1′), 4.45 (dd, 1H, J = 3.3, 1.4 Hz, H-2),
4.24−4.32 (m, 3H, H-6a, H-4′, H-6a′), 4.19 (d, 1H, J = 3.8 Hz,
H-2′), 4.16 (dd, 1H, J = 9.8, 9.8 Hz, H-4), 4.03 (dd, 1H, J =
10.0, 3.5 Hz, H-3), 3.90 (ddd, 1H, J = 10.7, 5.9, 3.4 Hz, OCH2),
3.78−3.86 (m, 3H, H-5, H-6b, H-6b′), 3.70 (dd, 1H, J = 9.0,
3.8 Hz, H-3′), 3.63 (ddd, 1H, J = 10.6, 7.1, 3.4 Hz, OCH2),
3.37−3.47 (m, 3H, H-5′, CH2N3), 3.20 (br s, 1H, OH); 13C
NMR (125 MHz, CDCl3) δC 138.2 (Ar), 138.1(9) (Ar), 137.5
(Ar), 137.4(8) (Ar), 128.9(3) (Ar), 128.9(2) (Ar), 128.8(4)
(2C, Ar), 128.3 (2C, Ar), 128.2(4) (2C, Ar), 128.2(2) (2C,
Ar), 127.8(7) (2C, Ar), 127.8(1) (2C, Ar), 127.8 (Ar), 127.7
(Ar), 126.0(9) (2C, Ar), 126.0(6) (2C, Ar), 101.5 (PhCH),
101.4 (PhCH), 98.9 (C-1), 98.4 (C-1′), 78.6 (2C, C-4, C-4′),
76.4 (C-3′), 74.4 (C-3), 72.9 (C-2), 72.6 (PhCH2), 72.5
(PhCH2), 69.6 (C-2′), 68.6(9) (C-6/C-6′), 68.6(8) (C-6/C-
6′), 67.0 (C-5′), 66.7 (OCH2), 64.4 (C-5), 50.4 (CH2N3).
HRMS (ESI) calcd. for (M + Na) C42H45N3O11: 790.2932.
Found: 790.2946. FTIR: 3491.0, 2102.9 cm−1.
1
1887.53. H NMR (500 MHz, 5:2 D2O:CD3CN) δ 7.43−7.61
(m, 5 H, Phe), 7.38 (dd, J = 7.89, 1.85 Hz, 4 H, Hδ-Tyr), 7.06
(dd, J = 7.98, 4.68 Hz, 4 H, Hε-Tyr), 4.84 (t, J = 6.5 Hz, 1 H),
4.43−4.57 (m, 5 H), 4.33−4.43 (m, 2 H), 4.27 (m, 1 H), 4.17
(d, J = 16.87 Hz, 1 H), 4.06 (d, J = 16.87 Hz, 1 H), 3.91 (s, 2
H), 3.53 (t, J = 6.88 Hz, 2 H), 3.25−3.40 (m, 3 H), 3.06−3.24
(m, 7 H), 2.65−3.01 (m, 6 H), 2.60 (t, J = 7.5 Hz, 2 H), 2.41−
2.50 (m, 2 H), 2.31−2.40 (m, 2 H), 2.19−2.26 (m, 1 H), 2.17
(s, 3 H, HAc), 1.52−2.17 (m, 27 H), 1.17 (d, J = 4.65 Hz, 3 H,
Hχ-Val), 1.19 (d, J=4.65 Hz, 3 H, Hχ-Val), 1.10 (d, J = 6.05 Hz,
3 H, Hδ-Leu), 1.03 (d, J = 6.05 Hz, 3 H, Hδ-Leu).
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-2-O-p-me-
thoxybenzyl-β-D-mannopyranosyl-(1→2)-3-O-benzyl-4,6-O-
benzylidene-α-D-mannopyranoside (7). Mannose donor 523
(670 mg, 1.2 mmol) and 4 Å molecular sieve (700 mg) in
CH2Cl2 (15 mL) were stirred at room temperature under argon
for 30 min. BSP19 (304 mg, 1.4 mmol) TTBP (562 mg, 2.2
mmol) were added at −60 °C followed by the addition of Tf2O
(250 μL, 1.5 mmol) and the mixture was stirred at the same
temperature for 30 min. Mannoside acceptor 620 (370 mg, 0.87
mmol) in CH2Cl2 (3 mL) was added at −78 °C and the
reaction was further stirred for 4 h. The reaction was then
quenched with Et3N and filtered through Celite. The filtrate
was washed with sat. NaHCO3, water and brine, and dried over
anhydrous Na2SO4. The crude was concentrated and purified
by chromatography (2:1 hexane−EtOAc) to give 7 (603 mg,
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-2-O-p-me-
thoxybenzyl-β-D-mannopyranosyl-(1→2)-3-O-benzyl-4,6-O-
benzylidene-β-D-mannopyranosyl-(1→2)-3-O-benzyl-4,6-O-
benzylidene-a-D-mannopyranoside (9). Mannose donor 523
(653 mg, 1.1 mmol) and 4 Å molecular sieve (600 mg) in
CH2Cl2 (14 mL) were stirred at room temperature under argon
for 30 min. BSP (283 mg, 1.4 mmol) TTBP (420 mg, 1.7
mmol) were added at −60 °C followed by the addition of Tf2O
(240 μL, 1.4 mmol) and the mixture was stirred at the same
temperature for 30 min.23 Disaccharide acceptor 8 (386 mg,
0.50 mmol) in CH2Cl2 (3 mL) was added at −78 °C and the
reaction was further stirred for 4 h. The reaction was then
quenched with Et3N and filtered through Celite. The filtrate
was washed with sat. NaHCO3, water and brine, and dried over
anhydrous Na2SO4. The crude was concentrated and purified
by chromatography (3:2 hexane−EtOAc) to give 9 (296 mg,
78%) as colorless oil: Rf 0.34 (2:1 hexane−EtOAc); [α]D
=
−72.1 (c 2.5, CH2Cl2); 1H NMR (500 MHz, CDCl3) δH 7.26−
7.55 (m, 22H, ArH), 6.85−6.88 (m, 2H, ArH), 5.63 (s, 1H,
PhCH), 5.55 (s, 1H, PhCH), 5.01 (d, 1H, J = 11.8 Hz,
PhCH2), 4.94 (d, 1H, J = 11.8 Hz, PhCH2), 4.89 (d, 1H, J = 1.5
Hz, H-1), 4.81 (d, 1H, J = 12.2 Hz, PhCH2), 4.76 (d, 1H, J =
12.2 Hz, PhCH2), 4.74 (d, 1H, J = 12.6 Hz, PhCH2), 4.66 (s,
1H, H-1′), 4.64 (d, 1H, J = 12.6 Hz, PhCH2), 4.26−4.32 (m,
4H, H-2, H-6a, H-4′, H-6a′), 4.16 (dd, 1H, J = 9.8, 9.8 Hz, H-
4), 4.02 (dd, 1H, J = 9.8, 3.3 Hz, H-3), 4.01 (d, 1H, J = 3.2 Hz,
H-2′), 3.79−3.93 (m, 4H, H-5, H-6b, H-6b′, OCH2), 3.79 (s,
3H, OCH3), 3.60−3.65 (m, 2H, H-3′, OCH2), 3.47 (ddd, 1H, J
= 13.3, 7.4, 3.3 Hz, CH2N3), 3.33−3.41 (m, 2H, H-5′, CH2N3);
13C NMR (125 MHz, CDCl3) δC 159.2 (Ar), 138.8 (Ar), 138.5
(Ar), 137.6 (Ar), 137.5 (Ar), 130.6 (Ar), 130.3 (2C, Ar), 128.9
(Ar), 128.8(7) (Ar), 128.3 (2C, Ar), 128.2(1) (2C, Ar),
128.2(0) (2C, Ar), 128.1 (2C, Ar), 127.5(8) (2C, Ar),,
127.5(5) (2C, Ar), 127.5(3) (Ar), 127.3(Ar), 126.1 (2C, Ar),
126.0(7) (2C, Ar), 113.6 (2C, Ar), 101.7 (PhCH), 101.4
48%) as colorless oil: Rf 0.32 (3:2 hexane−EtOAc); [α]D
=
−93.9 (c 2.8, CH2Cl2); 1H NMR (600 MHz, CDCl3) δH 7.26−
7.54 (m, 32H, ArH), 6.76−6.78 (m, 2H, ArH), 5.64 (s, 1H,
PhCH), 5.55 (s, 1H, PhCH), 5.44 (s, 1H, PhCH), 5.14 (s, 1H,
H-1″), 4.92 (d, 1H, J = 1.3 Hz, H-1), 4.91 (d, 1H, J = 12.0 Hz,
PhCH2), 4.88 (d, 1H, J = 12.5 Hz, PhCH2), 4.82 (d, 1H, J =
12.5 Hz, PhCH2), 4.71 (d, 1H, J = 12.3 Hz, PhCH2), 4.70 (d,
1H, J = 12.0 Hz, PhCH2), 4.70 (d, 1H, J = 2.1 Hz, H-1′), 4.66
(d, 1H, J = 12.3 Hz, PhCH2), 4.59 (d, 1H, J = 11.7 Hz,
PhCH2), 4.46 (d, 1H, J = 3.3 Hz, H-2″), 4.42 (d, 1H, J = 11.7
Hz, PhCH2), 4.35−4.41 (m, 4H, H-2, H-2′, H-6a′, H-6a″), 4.40
(PhCH), 101.0 (C-1′, JC1−H1 = 155.9 Hz), 98.8 (C-1, JC1−H1
=
171.1 Hz), 78.5(4) (C-4/C-4′), 78.5(2) (C-4/C-4′), 77.7 (C-
3′), 76.0 (C-3), 75.4 (C-2′), 75.0 (C-2), 74.1(4) (PhCH2),
74.1(0) (C-3), 72.3 (PhCH2), 71.6 (PhCH2), 68.9 (C-6), 68.6
692
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Article
(dd, 1H, J = 10.2, 4.7 Hz, H-6a), 4.25 (dd, 1H, J = 9.5, 9.5 Hz,
H-4″), 4.12 (dd, 1H, J = 9.7, 9.7 Hz, H-4′), 3.98−4.04 (m, 2H,
H-3, H-6b″), 3.95 (dd, 1H, J = 9.4, 9.4 Hz, H-4), 3.83−3.94 (m,
2H, H-5,), 3.72−3.82 (m, 2H, H-6b, H-6b′), 3.70 (dd, 1H, J =
9.9, 3.1 Hz, H-3′), 3.65 (s, 3H, OCH3), 3.61−3.66 (m, 1H,
OCH2), 3.57 (dd, 1H, J = 10.0, 3.3 Hz, H-3″), 3.36−3.50 (m,
4H, H-5′, H-5″, CH2N3); 13C NMR (125 MHz, CDCl3) δC
158.8 (Ar), 138.6 (Ar), 138.4 (Ar), 138.3(5) (Ar), 137.6 (Ar),
137.4 (Ar), 137.1 (Ar), 131.5 (Ar), 129.7 (2C, Ar), 129.0 (2C,
Ar), 128.8 (Ar), 128.1 (2C, Ar), 128.0(6) (3C, Ar), 128.0(2)
(3C, Ar), 127.7 (2C, Ar), 127.5(6) (2C, Ar), 127.5(3) (2C,
Ar), 127.5 (Ar), 127.4(6) (2C, Ar), 127.3(6) (2C, Ar), 127.2
(2C, Ar), 127.1(6) (Ar), 126.1(4) (2C, Ar), 126.1(1) (2C, Ar),
126.0(6) (2C, Ar), 113.4 (2C, Ar), 103.2 (C-1″, JC1−H1 = 160.5
Hz), 102.0 (PhCH), 101.7 (PhCH), 101.3 (PhCH), 99.8 (C-1′,
(7.4 mg, 12 μmol) in MeOH (1.0 mL), dibutyl squarate (7.5
μL, 24 μmol) was added and the reaction was stirred for 1 h.
The pH of the reaction mixture was carefully adjusted with
Et3N to slightly above pH 7. The reaction mixture was then
concentrated and purified on an Iatrobead-packed column.
Excessive squarate reagent was washed out with DCM−MeOH
(9:1) and the desired product was eluted with MeOH−H2O
(9:1) to yield the trimannoside squarate half-ester 11 (6.9 mg,
81%) as white fluffy solid after lyophilization: Rf 0.53 (9:1
1
MeOH−H2O); H NMR (500 MHz, D2O) δH 4.94, 4.93 (s,
1H, H-1), 4.82 (s, 1H, H-1″), 4.79, 4.77 (s, 1H, H-1′), 4.72 (t,
1H, J = 6.3 Hz, OCH2‑squarate), 4.67 (t, 1H, J = 6.1 Hz,
OCH2‑squarate), 4.23 (br s, 1H, H-2′), 4.13 (d, 1H, J = 2.9 Hz, H-
2″), 4.04, 4.02 (br s, 1H, H-2), 3.46−3.92 (m, 17H), 3.28−3.38
(m, 2H), 1.70−1.82 (m, 2H, CH2‑squarate), 1.34−1.48 (m, 2H,
CH2‑squarate), 0.87−0.95 (m, 3H, CH3‑squarate); 13C NMR (125
MHz, D2O) δC 101.0 (C-1″), 99.0 (C-1′), 97.6, 97.3 (C-1),
78.5 (C-2′), 77.8 (C-2), 76.4 (2C, C-5′, C-5″), 74.5
(OCH2‑squarate), 73.0 (C-3″), 72.9, 72.2 (C-3′), 70.4 (C-2″),
69.6 (C-3), 67.1, 66.9, 66.8, 66.6 (1C, OCH2), 61.2, 60.6, 60.4
(C-6, C-6′, C-6″), 44.2, 43.8 (1C, CH2NH), 31.4 (CH2), 18.2
(CH2), 12.9 (CH3). HRMS (ESI) calcd. for (M + Na)
C28H45NO19: 722.2478. Found: 722.2468.
J
C1−H1 = 171.0 Hz), 98.2 (C-1, JC1−H1 = 157.8 Hz), 79.2 (C-3″),
78.8 (C-4), 78.2(9) (C-4′/C-4″), 78.2(0) (C-4′/C-4″), 76.3
(C-2′), 75.9 (C-3′), 75.6 (C-2″), 74.5 (PhCH2), 74.3 (C-3),
73.8 (C-2), 72.1 (PhCH2), 71.8 (PhCH2), 71.1 (PhCH2), 68.9
(C-6/C-6′/C-6″), 68.8 (C-6/C-6′/C-6″), 64.3 (C-5), 55.1
(OCH3), 50.4 (CH2N3). HRMS (ESI) calcd. for (M + Na)
C70H73N3O17: 1250.4832. Found: 1250.4822. FTIR: 2102.2
cm−1.
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranosyl-(1→2)-3-O-benzyl-4,6-O-benzylidene-(1→2)-3-O-
benzyl-4,6-O-benzylidene-α-D-mannopyranoside (10). Tri-
saccharide 9 (240 mg, 0.20 mmol) was dissolved in 4:1
CH2Cl2/H2O (25 mL) and DDQ (90 mg, 0.40 mmol) was
added. The reaction was stirred at room temperature overnight.
The mixture was washed with sat. NaHCO3 (2x) and brine, and
dried over anhydrous Na2SO4. The crude was concentrated and
purified by chromatography (3:2 hexane−EtOAc) to give 10
(153 mg, 71%) as white solid: Rf 0.22 (3:2 hexane−EtOAc);
[α]D = −91.8 (c 0.3, CH2Cl2); 1H NMR (500 MHz, CDCl3) δH
7.16−7.56 (m, 30H, ArH), 5.63 (s, 1H, PhCH), 5.61 (s, 1H,
PhCH), 5.55 (s, 1H, PhCH), 5.13 (s, 1H, H-1″), 4.91 (s, 1H,
H-1), 4.82 (s, 2H, PhCH2), 4.75 (d, 1H, J = 11.9 Hz, PhCH2),
4.70 (s, 1H, H-1′), 4.67 (d, 1H, J = 11.8 Hz, PhCH2), 4.65 (d,
1H, J = 11.4 Hz, PhCH2), 4.62 (d, 1H, J = 12.0 Hz, PhCH2),
4.43 (d, 1H, J = 3.2 Hz, H-2′), 4.20−4.42 (m, 7H, H-2, H-6a,
H-4′, H-6a′, H-2″, H-4″, H-6a″), 3.81−4.01 (m, 6H, H-3, H-4,
H-5, H-6b′, H-6b″, OCH2), 3.74 (dd, 1H, J = 10.2, 10.2 Hz, H-
6b), 3.70 (dd, 1H, J = 9.9, 3.3 Hz, H-3′), 3.63 (ddd, 1H, J =
10.8, 7.6, 3.2 Hz, OCH2), 3.57 (dd, 1H, J = 10.2, 3.2 Hz, H-3″),
3.35−3.51 (m, 4H, H-5′, H-5″, CH2N3), 2.92 (br s, 1H, OH);
13C NMR (125 MHz, CDCl3) δC 138.4 (Ar), 138.3 (Ar), 138.2
(Ar), 137.5 (Ar), 137.4 (Ar), 137.2 (Ar), 129.0 (Ar), 128.8(9)
(Ar), 128.8(6) (Ar), 128.4 (2C, Ar), 128.3(3) (3C, Ar),
128.3(1) (3C, Ar), 128.2(5) (3C, Ar), 128.2 (2C, Ar), 128.1(6)
(2C, Ar), 127.7 (2C, Ar), 127.6(6) (Ar), 127.6(0) (2C, Ar),
127.5 (1C, Ar), 126.1(9) (2C, Ar), 126.1(5) (2C, Ar), 126.1(2)
(2C, Ar), 101.8 (PhCH), 101.5 (PhCH), 101.4(6) (PhCH),
100.8 (C-1″), 99.4 (C-1′), 98.1 (C-1), 78.5 (C-4/C-4″), 78.3
(C-4/C-4″), 78.1 (C-4′), 77.5 (C-3″), 76.3 (C-3′), 74.0 (C-2′),
73.8 (C-3), 73.7 (C-2), 72.2 (PhCH2), 71.8 (PhCH2), 71.6
(PhCH2), 68.9 (C-2″), 68.8 (2C, C-6/C-6′/C-6″), 68.4 (C-6/
C-6′/C-6″), 67.8 (C-5′), 67.3 (C-5″), 66.8 (OCH2), 64.3 (C-
5), 50.4 (CH2N3). HRMS (ESI) calcd. for (M + Na)
C62H65N3O16: 1130.4257. Found: 1130.4255. FTIR: 3504.6,
2102.9 cm−1.
β-D-Mannopyranosyl-(1→2)-β-D-mannopyranosyl(1→2)-
α-D-mannopyranoside−copovidone Glycoconjugate (12).
Poly(prop-2-ynyl 2-(3-(vinyloxy)propylthio)ethylcarbamate-
co-N-vinyl-2-pyrrolidone) (9.6 mg) P214 and 2-(2-butoxy-3,4-
dioxocyclobut-1-enylamino)ethyl 2-O-[(2-O-β-D-mannopyra-
nosyl)-β-D-mannopyranosyl)]-α-D-mannopyranoside (9.2 mg)
11 were dissolved in water (100 μL) and pH was adjusted to 9
with 1 M Na2CO3. The mixture was stirred overnight at room
temperature, then dialized against deionized water and freeze-
1
dried to give 12 as a white powder (14.1 mg); from H NMR
the estimated payload8 is 9.8% corresponding to ∼66% m/m of
the ligand/polymer conjugate composition. 1H NMR (500
MHz, D2O) δH 4.96 (br s, H-1), 4.82 (br s, H-1″), 4.78 (br s,
H-1′), 4.23 (d, J = 2.5, H-2′), 4.12 (d, J = 2.6 Hz, H-2″), 4.03
(br s, 1H, H-2), 4.39−2.9 (br m, H-3−6, H-3′-6′, H-3″-6″,
polymer backbone), 2.80−2.55 (br m, CH2S linker), 2.55−1.30
(br m, polymer backbone).
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-2-O-p-me-
thoxybenzyl-β-D-mannopyranoside (14). A mixture of man-
nosyl sulfoxide 1323 (3.5 g, 5.9 mmol), TTBP (3.0 g, 12 mmol),
and 4 Å molecular sieve (3.5 g) was dissolved in CH2Cl2 (180
mL) and stirred for 30 min at −78 °C. Tf2O (1.1 mL, 6.6
mmol) was added to the mixture and stirred at the same
temperature. After 10 min, 2-azidoethanol (1.0 g, 12 mmol)
was added and the reaction was further stirred for 20 min. The
reaction was slowly warmed to −30 °C, quenched with MeOH,
and filtered through Celite. The filtrate was washed with sat.
NaHCO3 and brine, and dried over anhydrous Na2SO4. The
crude was concentrated and purified by chromatography (2:1
hexane−EtOAc) to give 14 (2.2 g, 68%) as colorless syrup: Rf
0.43 (2:1 hexane−EtOAc); [α]D = −89.37 (c 3.9, CH2Cl2); 1H
NMR (500 MHz, CDCl3) δH 7.50−7.52 (m, 2H, ArH), 7.27−
7.41 (m, 10H, ArH), 6.85−6.87 (m, 2H, ArH), 5.63 (s, 1H,
PhCH), 4.92 (d, 1H, J = 11.8 Hz, PhCH2), 4.81 (d, 1H, J =
11.9 Hz, PhCH2), 4.67 (d, 1H, J = 12.5 Hz, PhCH2), 4.56 (d,
1H, J = 12.5 Hz, PhCH2), 4.53 (d, 1H, J = 0.8 Hz, H-1), 4.30
(dd, 1H, J = 10.4, 4.9 Hz, H-6a), 4.21 (app t, 1H, J = 9.6 Hz, H-
4), 4.09−4.13 (m, 1H, OCH2), 3.99 (app d, 1H, J = 3.1 Hz, H-
2), 3.94 (app t, 1H, J = 10.3 Hz, H-6b), 3.80 (s, 3H, OCH3),
3.65 (ddd, 1H, J = 10.5, 8.7, 3.3 Hz, OCH2), 3.59 (dd, 1H, J =
2-(2-Butoxycyclobutene-3,4-dione-1-ylamino)ethyl-β-D-
mannopyranosyl-(1→2)-β-D-mannopyranosyl-(1→2)-α-D-
mannopyranoside (11). To the deprotected trimannoside 1
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9.9, 3.1 Hz, H-3), 3.53−3.59 (m, 1H, CH2N3), 3.32−3.37 (m,
2H, H-5, CH2N3); 13C NMR (125 MHz, CDCl3) δC 159.3
(Ar), 138.3 (Ar), 137.6 (Ar), 130.5 (Ar), 130.3 (2C, Ar), 128.9
(Ar), 128.3 (2C, Ar), 128.2 (2C, Ar), 127.5(9), 127.5(6) (3C,
Ar), 126.1 (2C, Ar), 113.6 (2C, Ar), 102.3 (C-1, JC1−H1 = 156.6
Hz), 101.5 (PhCH), 78.5 (C-4), 77.8 (C-3), 75.4 (C-2), 74.7
(PhCH2), 72.3 (PhCH2), 68.7 (OCH2), 68.6 (C-6), 67.7 (C-
5), 55.3 (OCH3), 50.9 (CH2N3). HRMS (ESI) calcd. for (M +
Na) C30H33N3O7: 570.2211. Found: 570.2197. FTIR: 2105.2
cm−1.
MHz, CDCl3) δC 159.0 (Ar), 138.6(2) (Ar), 138.6(0) (Ar),
137.7 (Ar), 137.4 (Ar), 131.1 (Ar), 130.2 (2C, Ar), 128.1(9),
128.1(4), 128.1(3) (5C, Ar), 127.5(9), 127.5(6), 127.5(3),
127.5(1), 127.4(5), 127.3(8), 127.3(6) (7C, Ar), 126.0(4),
126.0(3), 125.9(8), 125.9(6) (8C, Ar), 113.4 (2C, Ar), 103.8
(C-1′, JC1−H1 = 160.4 Hz), 101.5(9) (PhCH), 101.5(6)
(PhCH), 101.3 (C-1, JC1−H1 = 157.5 Hz), 78.4 (C-4′), 78.1
(C-4), 76.7 (2C, C-2, C-3′), 76.0 (C-3), 75.2 (C-2′), 74.1
(PhCH2), 72.1 (PhCH2), 71.1 (PhCH2), 68.9, 68.6(8), 68.6(5)
(3C, C-6, C-6′, OCH2), 67.7, 67.5 (2C, C-5, C-5′), 55.2
(OCH3), 50.9 (CH2N3). HRMS (ESI) calcd. for (M + Na)
C50H53N3O12: 910.3521. Found: 910.3502. FTIR: 2105.0 cm−1.
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranosyl-(1→2)-3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranoside (17). Disaccharide 16 (140 mg, 0.16 mmol) was
dissolved in 17:1 CH2Cl2/H2O (9 mL) and DDQ (77 mg, 0.34
mmol) was added at 0 °C. The reaction was slowly warmed
back to room temperature and stirred overnight. The reaction
was then washed with sat. NaHCO3 (2x) and brine, and dried
over anhydrous Na2SO4. The crude was concentrated and
purified by chromatography (3:2 hexane−EtOAc) to give 17
(82 mg, 68%) as colorless syrup: Rf 0.33 (1:1 hexane−EtOAc);
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranoside (15). Monosaccharide 14 (2.2 g, 3.9 mmol) was
dissolved in 17:1 CH2Cl2/H2O (120 mL) and DDQ (2.0 g, 9.0
mmol) was added at 0 °C. The reaction was slowly warmed
back to room temperature and stirred overnight. The reaction
was then washed with sat. NaHCO3 (2x) and brine, and dried
over anhydrous Na2SO4. The crude was concentrated and
purified by chromatography (3:2 hexane−EtOAc) to give 15
(1.3 g, 79%) as white solid: Rf 0.22 (3:2 hexane−EtOAc); [α]D
1
= −57.8 (c 0.9, CH2Cl2); H NMR (500 MHz, CDCl3) δH
7.49−7.51 (m, 2H, ArH), 7.27−7.41 (m, 8H, ArH), 5.61 (s,
1H, PhCH), 4.86 (d, 1H, J = 12.2 Hz, PhCH2), 4.78 (d, 1H, J =
12.2 Hz, PhCH2), 4.58 (s, 1H, H-1), 4.32 (dd, 1H, J = 10.4, 5.0
Hz, H-6a), 4.14−4.18 (m, 2H, H-2, H-4), 4.09 (ddd, 1H, J =
10.7, 4.9, 3.9 Hz, OCH2), 3.89 (app t, 1H, J = 10.4 Hz, H-6b),
3.74 (ddd, 1H, J = 10.7, 8.2, 3.7 Hz, OCH2), 3.66 (dd, 1H, J =
9.5, 3.3 Hz, H-3), 3.54 (ddd, 1H, J = 12.1, 8.2, 3.7 Hz, CH2N3),
3.34−3.40 (m, 2H, H-5, CH2N3), 2.53 (d, 1H, J = 1.5 Hz,
OH); 13C NMR (125 MHz, CDCl3) δC 137.8 (Ar), 137.4 (Ar),
129.0 (Ar), 128.5 (2C, Ar), 128.2 (2C, Ar), 127.8(9), 127.8(7)
(3C, Ar), 126.0 (2C, Ar), 101.6 (PhCH), 100.6 (C-1), 78.3 (C-
4), 76.6 (C-3), 72.6 (PhCH2), 69.8 (C-2), 68.6 (OCH2), 68.5
(C-6), 67.0 (C-5), 50.6 (CH2N3). HRMS (ESI) calcd. for (M +
Na) C22H25N3O6: 450.1636. Found: 450.1629. FTIR: 3500.8,
2104.1 cm−1.
1
[α]D = −116.0 (c 0.2, CH2Cl2); H NMR (500 MHz, CDCl3)
δH 7.49−7.52 (m, 4H, ArH), 7.26−7.42 (m, 16H, ArH), 5.59
(s, 1H, PhCH), 5.58 (s, 1H, PhCH), 4.94 (s, 1H, H-1′), 4.85
(d, 1H, J = 12.5 Hz, PhCH2), 4.81 (d, 1H, J = 12.5 Hz,
PhCH2), 4.78 (d, 1H, J = 12.5 Hz, PhCH2), 4.74 (d, 1H, J =
12.5 Hz, PhCH2), 4.54 (s, 1H, H-1), 4.44 (d, 1H, J = 3.3 Hz, H-
2), 4.27−4.34 (m, 3H, H-6a, H-2′, H-6a′), 4.25 (app t, 1H, J =
9.4 Hz, H-4′), 4.07−4.10 (m, 2H, H-4, OCH2), 3.88 (app t,
1H, J = 10.4 Hz, H-6b′), 3.84 (app t, 1H, J = 10.2 Hz, H-6b),
3.64−3.70 (m, 3H, H-3, H-3′, OCH2), 3.32−3.47 (m, 3H, H-5,
H-5′, CH2N3), 3.26 (ddd, 1H, J = 13.5, 4.9, 3.0 Hz, CH2N3),
2.93 (s, 1H, OH); 13C NMR (125 MHz, CDCl3) δC 138.4 (Ar),
138.2 (Ar), 137.6 (Ar), 137.4 (Ar), 128.9 (Ar), 128.8(7) (Ar),
128.3 (3C, Ar), 128.2(5) (3C, Ar), 128.2 (2C, Ar), 127.9 (2C,
Ar), 127.6 (Ar), 127.6(0) (Ar), 127.5 (2C, Ar), 126.1 (2C, Ar),
126.0(5) (2C, Ar), 101.5 (3C, C-1, PhCH), 100.6 (C-1′), 78.4
(C-4′), 77.9 (C-4), 76.6 (C-3′), 76.2 (C-3), 73.2 (C-2), 72.3
(PhCH2), 71.6 (PhCH2), 69.5 (C-2′), 69.1 (OCH2), 68.7 (C-
6), 68.4 (C-6′), 67.6 (C-5′), 67.0 (C-5), 50.8 (CH2N3). HRMS
(ESI) calcd. for (M + Na) C42H45N3O11: 790.2946. Found:
790.2936. FTIR: 3496.2, 2105.2 cm−1.
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranosyl-(1→2)-3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranosyl-(1→2)-3-O-benzyl-4,6-O-benzylidene-β-D-manno-
pyranoside (18). Mannose donor 523 (817 mg, 1.4 mmol) and
4 Å molecular sieves (1 g) in CH2Cl2 (14 mL) were stirred at
room temperature under argon for 30 min. BSP (380 mg, 1.8
mmol) TTBP (570 mg, 2.3 mmol) were added at −60 °C
followed by the addition of Tf2O (314 μL, 1.9 mmol) and the
mixture was stirred at the same temperature for 30 min.23
Disaccharide 17 (710 mg, 0.92 mmol) in CH2Cl2 (3 mL) was
added at −78 °C and the reaction was further stirred for 4 h.
The reaction was then quenched with Et3N and filtered through
Celite. The filtrate was washed with sat. NaHCO3 and brine,
and dried over anhydrous Na2SO4. The crude was concentrated
and partially purified by chromatography (3:2 hexane−EtOAc;
Rf 0.32) to give trisaccharide as pale yellow syrup. The resulting
intermediate was then dissolved in 4:1 CH2Cl2/H2O (50 mL)
and DDQ (160 mg, 0.71 mmol) was added. The reaction was
stirred overnight before being washed with sat. NaHCO3 (2x)
and brine, and dried over anhydrous Na2SO4. The crude was
2-Azidoethyl 3-O-benzyl-4,6-O-benzylidene-2-O-p-me-
thoxybenzyl-β-D-mannopyranosyl-(1→2)-3-O-benzyl-4,6-O-
benzylidene-β-D-mannopyranoside (16). A mixture of man-
nosyl sulfoxide 1323 (280 mg, 0.48 mmol), TTBP (240 mg,
0.97 mmol), and 4 Å molecular sieve (300 mg) were dissolved
in CH2Cl2 (18 mL) and stirred for 30 min at −78 °C. Tf2O (88
μL, 0.52 mmol) was added to the mixture and stirred at the
same temperature. After 10 min, glycosyl acceptor 15 (97 mg,
0.23 mmol) was added and the reaction was further stirred for 4
h. The reaction was then quenched with MeOH and filtered
through Celite. The filtrate was washed with sat. NaHCO3 and
brine, and dried over anhydrous Na2SO4. The crude was
concentrated and purified by chromatography (3:2 hexane−
EtOAc) to give 16 (138 mg, 69%) as a colorless syrup: Rf 0.42
1
(3:2 hexane−EtOAc); [α]D = −105.7 (c 1.5, CH2Cl2); H
NMR (500 MHz, CDCl3) δH 7.22−7.52 (m, 22H, ArH), 6.80−
6.84 (m, 2H, ArH), 5.59 (s, 1H, PhCH), 5.44 (s, 1H, PhCH),
5.00 (d, 1H, J = 11.9 Hz, PhCH2), 4.92 (d, 1H, J = 11.9 Hz,
PhCH2), 4.79 (d, 1H, J = 12.6 Hz, PhCH2), 4.78 (s, 1H, H-1′),
4.75 (d, 1H, J = 12.5 Hz, PhCH2), 4.63 (d, 1H, J = 12.6 Hz,
PhCH2), 4.54 (d, 1H, J = 12.5 Hz, PhCH2), 4.52 (s, 1H, H-1),
4.31 (dd, 1H, J = 10.3, 4.8 Hz, H-6a′), 4.25−4.30 (m, 2H, H-2,
H-6a), 4.20 (app t, 1H, J = 9.6 Hz, H-4), 4.12 (d, 1H, J = 3.4
Hz, H-2′), 4.04−4.10 (m, 2H, H-4, OCH2), 3.89 (app t, 1H, J =
10.3 Hz, H-6b′), 3.76 (app t, 1H, J = 10.4 Hz, H-6b), 3.74 (s,
3H, OCH3), 3.61−3.69 (m, 2H, H-3, OCH2), 3.56 (dd, 1H, J =
10.0, 3.1 Hz, H-3′), 3.26−3.37 (m, 3H, H-5, H-5′, CH2N3),
3.20 (ddd, 1H, J = 13.6, 5.0, 3.1 Hz, CH2N3); 13C NMR (125
694
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Article
concentrated and purified by chromatography (3:2 hexane−
EtOAc) to give 18 (370 mg, 45% over two steps) as foamy
white solid: Rf 0.26 (3:2 hexane−EtOAc); [α]D = −134.5 (c
EDTA (30 μL). The reaction mixture was diluted 2-fold with
water and dialyzed against 1 mM EDTA in water (2x) and
deionized water (2x). The final product 20 was lyophilized as
pale yellow solid (24 mg); from 1H NMR the estimated
payload is 6.8% corresponding to ∼49% m/m of the ligand/
polymer conjugate composition.14 1H NMR (500 MHz, D2O)
δH 4.90 (s, 1H, H-1), 4.84 (s, 1H, H-1′), 4.70 (s, 1H, H-1″),
4.42 (br s, 1H), 4.29 (d, 1H, J = 2.9 Hz, H-2′), 4.18 (d, 1H, J =
2.8 Hz, H-2″), 4.11 (d, 1H, J = 2.7 Hz, H-2), 2.80−3.97 (m,
49H), 1.45−2.50 (m, 136H).
1
0.5, CH2Cl2); H NMR (500 MHz, CDCl3) δH 7.48−7.52 (m,
4H, ArH), 7.26−7.44 (m, 23H, ArH), 7.18−7.21 (m, 2H,
ArH), 7.11−7.12 (m, 1H, ArH), 5.60 (s, 2H, PhCH), 5.42 (s,
1H, PhCH), 5.11 (s, 1H, H-1″), 4.87 (s, 1H, H-1′), 4.69−4.78
(m, 6H, PhCH2), 4.53 (d, 1H, J = 3.2 Hz, H-2′), 4.51 (s, 1H,
H-1), 4.42 (d, 1H, J = 3.2 Hz, H-2″), 4.28−4.35 (m, 4H, H-2,
H-4, H-6a, H-6a′), 4.25 (dd, 1H, J = 10.4, 4.8 Hz, H-6a″), 4.18
(app t, 1H, J = 9.6 Hz, H-4′), 4.07 (ddd, 1H, J = 10.7, 5.3, 3.0
Hz, OCH2), 3.89−3.95 (m, 3H, H-6b, H-6b′, H-4″), 3.60−3.71
(m, 3H, H-3, H-3′, H-3″, H-6b″, OCH2), 3.26−3.43 (m, 5H,
H-5, H-5′, H-5″, CH2N3); 13C NMR (125 MHz, CDCl3) δC
138.5 (Ar), 138.2 (2C, Ar), 137.5(5) (Ar), 137.5(0) (Ar), 137.2
(Ar), 128.8(8) (Ar), 128.8(7) (Ar), 128.8(4) (Ar), 128.2(9),
128.2(5), 128.2(1), 128.1(8), 128.1(6) (13C, Ar), 127.7 (2C,
Ar), 127.6(7) (2C, Ar), 127.5(8) (2C, Ar), 127.5(4) (Ar),
127.5(0) (Ar), 126.0(7) (2C, Ar), 126.0(4) (2C, Ar), 126.0(0)
Ara6-Copovidone Conjugate (22). Hexasaccharide 3 (13
mg) was dissolved in MeOH (2.25 mL) and 1 M NaOCH3
(0.25 mL) was added. The reaction was stirred at room
temperature until TLC indicated completion (checked via TLC
using ninhydrin reagent). The solution was then neutralized
with Amberlite 120 (H+), filtered, and concentrated. Without
further purification, the crude product was redissolved in of
NaHCO3 buffer (1 mL 0.1 M, pH 8.3), and succinimidyl N-
propargyl glutariamidate25 (13.5 mg) was added and the
reaction stirred overnight. The mixture was then purified by
HPLC using a TSK gel Amide-80 column (21.5 mm ID × 30
cm, 10 μm; from TOSOH BioScience LLC, Japan) with a
nonlinear gradient (Waters gradient 8) acetonitrile:water
gradient from 80:20 to 65:35 over 45 min at a flow rate of
10 mL/min. Fractions collected were lyophilized and analyzed
by high resolution MS (ESI). The final product, propargylated
Ara6 21, (7.0 mg, 54% theoretical yield) was identified at an m/
z of 1111.5 for M+Na+ [calculated mass for M = C46H76N2O27:
1088.46]. Alkyne 21 (0.97 mg, 0.89 μmol) was used without
further characterization by dissolution in water with 2-
poly(azidoethene-co-N-vinyl-2-pyrrolidone (2.97 mg) and the
mixture was lyophilized. The resulting solid was dissolved in 0.2
M NH4HCO3 buffer (pH: 8; 0.4 mL) and 1 M sodium L-
ascorbate (10 μL) and 0.05 M copper sulfate (20 μL) were
added.25,27 The reaction mixture was left on a tumbler. After 3
days the solution was diluted with water (2 mL) and
concentrated by centrifugation (5000 rpm) using a Millipore
membrane (10,000 MWCO). The concentrate was diluted with
water (2 mL) and concentrated again. Then the concentrate
was diluted with water and filtered through a Millipore syringe
filter (0.02 μm) and lyophilized to provide the conjugate 22 as
(2C, Ar), 102.4 (C-1′, JC1−H1 = 162.3 Hz), 101.7 (C-1, JC1−H1
=
157.6 Hz), 101.5 (PhCH), 101.4(6) (PhCH), 101.3(8)
(PhCH), 101.2 (C-1″, JC1−H1 = 155.1 Hz), 78.5 (C-4), 78.2
(C-4″), 78.0 (C-4′), 77.2 (C-3″), 76.4 (C-3′), 75.8 (C-3), 75.7
(C-2), 74.1 (C-2′), 72.2 (PhCH2), 71.6 (PhCH2), 71.5
(PhCH2), 69.2 (OCH2), 69.0 (C-2″), 68.8, 68.5(1), 68.4(9)
(3C, C-6, C-6′, C-6″), 67.7, 67.5, 67.2 (3C, C-5, C-5′, C-5″),
50.9 (CH2N3). HRMS (ESI) calcd. for (M + Na)
C62H65N3O16: 1130.4257. Found: 1130.4250. FTIR: 3512.8,
2105.3 cm−1.
2-(2-(5-Oxo-5-(prop-2-ynylamino)pentanamido)ethyl) 2-
(N-propargyl glutariamidyl)ethyl β-D-mannopyranosyl-(1→
2)-β-D-mannopyranosyl-(1→2)-β-D-mannopyranoside (19).
To the deprotected trimannoside 2 (14 mg, 23 μmol) in 0.1
M sodium bicarbonate, pH 8.3 (1.0 mL), succinimidyl N-
propargyl glutariamidate26 (25 mg, 92 μmol) was added and
the reaction was stirred for 2 h. The desired product was
purified by HPLC (Luna C18 from Phenomenex) to give the
propargylated trimannoside 19 (8.7 mg, 54%) as fluffy white
solid after lyophilization: [α]D = −53.2 (c 0.7, H2O); 1H NMR
(500 MHz, D2O) δH 4.91 (s, 1H, H-1), 4.85 (s, 1H, H-1′), 4.75
(s, 1H, H-1″), 4.30 (d, 1H, J = 3.0 Hz, H-2′), 4.19 (d, 1H, J =
3.1 Hz, H-2″), 4.13 (d, 1H, J = 2.8 Hz, H-2), 3.87−3.98 (m,
6H, H-6a, H-6a′, H-6a″, CH2‑alkyne, OCH2), 3.52−3.77 (m, 9H,
H-3, H-4, H-6b, H-3′, H-4′, H-6b′, H-3″, H-6b″, OCH2),
3.30−3.50 (m, 6H, H-5, H-5′, H-4″, H-5″, CH2NH2), 2.60 (t,
1H, J = 2.5 Hz, alkyne), 2.29 (t, 4H, J = 7.6 Hz, CH2), 1.88
(quin, 2H, J = 7.8 Hz, CH2); 13C NMR (125 MHz, D2O) δC
176.8 (CO), 176.5 (CO), 101.8 (2C, C-1, C-1′), 101.2
(C-1″), 79.4 (C-2′), 79.2 (C-2″), 77.3 (2C), 77.2 (C-5, C-5′,
C-5″), 73.9 (C-3), 73.2 (C-3′), 72.9 (C-3″), 71.4 (CH2‑alkyne),
69.2 (C-2), 68.3 (OCH2), 68.0 (C-4″), 67.7 (2C, C-4′, C-4),
62.1, 61.7 (2C) (C-6, C-6′, C-6″), 40.2 (CH2NH), 35.9
(CH2CO), 35.6 (CH2CO), 29.7 (C−alkyne), 22.7 (C−alkyne).
HRMS (ESI) calcd. for (M + Na) C28H46N2O18: 721.2638.
Found: 721.2636. FTIR: 3290.4 cm−1.
8
1
white foam (3.08 mg). From H NMR the estimated payload
is 2.65% corresponding to ∼28% m/m of the ligand/polymer
1
conjugate composition. H NMR (600 MHz, D2O): δ 8.00−
7.60 (m, 1 H, CH); 5.23, 5.16, 5. 13, 5.12, 5.09, and 4.99 (6 br
s, H-1a, H-1b, H-1c, H-1d, H-1e, H-1f), 4.44 (br. s.), 4.32−4.25
(m), 4.22−4.16 (m), 4.16−2.9 (br m, remaining carbohydrate
signals, polymer backbone, linker), 2.55−1.25 (br m, polymer
backbone, linker).
Ara6-BSA Conjugate (23). To a solution of Ara6-amine21
related to trifluoracetamide 3 (5.1 mg) and 3,4-diethoxy-3-
cyclobutene-1,2-dione (2 μL) in ethanol−water (1:1, 1.5 mL)
was added sat aq sodium bicarbonate solution (30 μL). The
solution was then stirred at room temperature for 1 h, when
TLC (CH3OH:CH2Cl2, 1:2) showed that almost all of the
Ara6-amine was consumed. The mixture was directly con-
centrated on a rotary evaporator and the residue was purified by
column chromatography (CH3OH:CH2Cl2, 1:3) to give the
squaric acid monoester (5.3 mg, 92%). To this solution was
added BSA (16.6 mg) followed by borate buffer (pH 9.0, 120
μL) and the reaction was left for 36 h at room temperature.
The reaction solution was then diluted with deionized water
(2−3 mL) and transferred to a membrane bag for dialysis
β-D-Mannopyranosyl-(1→2)-β-D-mannopyranosyl(1→2)-
β-D-mannopyranoside−copovidone Glycoconjugate (20).
Propargylated trimannoside (8.7 mg, 12 μmol) 19 and polymer
P114 (18 mg, 9.1 μmol) were dissolved in degassed water (1
mL). The mixture was adjusted to ∼ pH 8 with NaHCO3
followed by the addition of 0.05 M CuSO4 (50 μL) and 1.0 M
sodium ascorbate (25 μL, freshly made).30,31 The reaction was
left at room temperature for two days and quenched with 0.5 M
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against changes of deionized water (3 times over 36 h). The
solution from the membrane bag was then transferred to a glass
vial, and lyophilized to afford the Ara6-BSA conjugate 23 as a
white fluffy solid (20 mg). Analysis by MALDI mass
spectrometry indicated a loading of 14.7 mol of Ara6 per mol
of BSA.
h, then washed 5 times with PBST (0.05% Tween-20 in
phosphate buffer saline, PBS). Plates were blocked using 1%
BSA in PBS buffer and incubated at RT for 45 min, then
washed with PBST prior to use. Serial √10 dilutions of
immune sera or monoclonal antibodies were made in PBST
containing 0.1% BSA (rabbit and mouse sera were used at a
starting dilution of 1:100 and mAb was used at a starting
concentration of 10 μg/mL). The solutions were distributed in
triplicate on coated microtiter plates and incubated at room
temperature for 2 h. Plates were washed with PBST (5 times)
and goat anti-mouse IgG or goat anti-rabbit secondary antibody
conjugated to horseradish peroxidase (Kirkegaard & Perry
Laboratories; 1:5000 dilution in 0.1% BSA/PBST; 100 μL/
well) was added. The mixture was then incubated for 30 min at
room temperature, and then washed (5 times) with PBST.
Peroxidase substrate, 3,3′,5,5′-tetramethylbenzidine (TMB)
with H2O2 (1:1 mixture of 3,3′,5,5′-tetramethylbenzidine (0.4
g/L) and 0.02% H2O2 solution, Kirkegaard & Perry
Laboratories) was added (100 μL/well). After 15 min, the
reaction was quenched by addition of 1 M phosphoric acid
(100 μL/well). Absorbance was read at 450 nm.
Ac-Fba-Copovidone Conjugate (24). To propargyl copovi-
done polymer P3 (10.0 mg) dissolved in water (250 μL) and
methanol (250 μL) in a 1.5 mL microcentrifuge tube was added
1 M sodium ascorbate (25 μmol, 25 μL) 0.05 M CuSO4 (2.5
μmol, 50 μL), peptide 4 (3.0 μmol, 5.6 mg), and 1 M
triethylammonium bicarbonate (100 μmol, 100 μL pH 8.5).
The mixture was vortexed for 21 h on a benchtop shaker. The
reaction mixture was neutralized by addition of trifluoroacetic
acid (104 μmol, 8 μL). The solution was filtered through a 13
mm PVDF filter with 0.45 μm pore size and divided into three
portions for separation. The polymer and peptide were
efficiently separated from each other and other reaction
mixture components by gel filtration on a Superdex Peptide
300/10 GL column eluted with 70:30 H2O:CH3CN containing
0.1% TFA at a flow rate of 0.8 mL min−1. Peptide-containing
eluent was selectively detected by absorbance at 280 nm and
consisted of two fractions; the first, from 6.4 to 9.6 mL,
End point titers are recorded as the dilution giving an
absorbance 0.2 above background.
1
Repeated Use of Coated ELISA Plates. Assays were
performed in an identical manner to that described above.
After plates were read at 450 nm, they were stored with
quenching solution (i.e., TMB + acid) overnight at RT. The
following day, plates were washed with PBST, blocked, and
ELISA was performed as before. After the plates had been read,
they were again stored overnight and reused in the same
manner. In total, 3 sequential assays were performed on plates
coated with each antigen.
contained polymer-bound peptide as determined by H NMR.
The second, from 11.0 to 13.0 mL, contained free peptide as
determined by MALDI-MS. The gel chromatography peak
areas were further compared with the peak area obtained upon
gel filtration of a known amount (0.46 mg) free peptide, and
corresponded to 3.6 mg and 0.34 mg polymer-linked peptide
and free peptide, or 91% conversion, and a combined recovery
of 70%. The fractions from 6.4 to 9.6 mL were combined and
lyophilized, rendering 10.5 mg of peptide-bearing copovidone
polymer, or 70% of the quantity expected from a 91%
conversion. The estimated loading is 34% m/m (0.18 mmol
ASSOCIATED CONTENT
■
1
peptide per gram of polymer); by H NMR the estimated
S
* Supporting Information
payload14 is 1.6% corresponding to ∼33% m/m of the peptide/
Detailed ELISA data, NMR spectra for all new compounds, and
where appropriate mass spectra and HPLC profiles for peptide
4 and its conjugate 23. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
polymer conjugate composition. H NMR (500 MHz, D2O) δ
7.96−7.90 (m, CH), 7.80 (s), 7.23−7.12 (m), 7.07−6.98 (m),
6.74−6.65 (m), 5.14−5.05 (m), 4.90−4.44 (m under water),
4.40−0.7 (br m, peptide signals, polymer backbone, linker).
Antigen Coating Efficiency. Optimal antigen coating for
ELISA plates was established by applying increasing dilutions of
antigens 22 or 24 across the rows of an ELISA plate. Antigen
solution was prepared at 10 μg/mL and successive wells across
the plate were incubated with √10 dilutions of the antigen.
This resulted in 12 wells with antigen coat beginning at 10 μg/
mL and ending at 0.1 ηg/mL. Coated plates were then
incubated with √10 dilutions of antibody CS35 such that each
row received the same dilution of antibody. Bound antibody
was detected as described below and the resulting three-
dimensional matrix is recorded in Tables S1 and S2 and plotted
as a three-dimensional graph (Figure 3).
ELISA. Antisera and mouse monoclonal antibodies were as
follows: rabbit polyclonal antisera were raised to a synthetic β-
mannose trisaccharide tetanus toxoid conjugate;10 mouse
immune sera were raised to a mannose trisaccharide
glycopeptide conjugated to tetanus toxoid (unpublished
data); and two mAbs were used: C3.1 specific for the Candida
albicans β-mannan16 and mAb CS35 specific for the M.
tuberculosis cell wall antigen.21,22 Antigen stock solutions were
prepared by dissolving copovidone or BSA glycoconjugates in
PBS (1 mg/mL). Microtiter plates were coated with antigen
solution (10 μg/mL, 100 μL/well) by incubation at 4 °C for 18
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: dave.bundle@ualberta.ca; Tel: (+1)780-492-8808;
Fax: (+1)780-492-7705.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The research was made possible by grants awarded to D. R.
Bundle; a Discovery grant from the Natural Science and
Engineering Research Council of Canada; and support from the
Alberta Innovates Centres Program. We thank Professor T. L.
Lowary and Mr. Yu Bai for the synthesis of hexasaccharide 3.
ABBREVIATIONS
■
Ara6, arabinose hexasaccharide; BSA, bovine serum albumin;
BSP, 1-benzenesulfinyl piperidine; DDQ, 2,3-dichloro-5,6-
dicyano-1,4-benzoquinone; mAb, monoclonal antibody; Man3,
1,2-β-mannotriose; MS, molecular sieve; PMB, p-methoxyben-
zyl; TTBP, 2,4,6-tritert-butylpyrimidine; Tf2O, trifluorometha-
nesulfonic anhydride
696
dx.doi.org/10.1021/bc400486w | Bioconjugate Chem. 2014, 25, 685−697

Bioconjugate Chemistry
Article
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