A novel linker methodology for the synthesis of tailored conjugate vaccines composed of complex carbohydrate antigens and specific TH-cell peptide epitopes

Article abstract of DOI:10.1002/chem.200800065

Pathogenic organisms or oncogenically transformed cells often express complex carbohydrate structures at their cell surface, which are viable targets for active immunotherapy. We describe here a novel, immunologically neutral, linker methodology for the efficient preparation of highly defined vaccine conjugates that combine complex saccharide antigens with specific T _H-cell peptide epitopes. This novel heterobifunctional approach was employed for the conjugation of a (1→-2)-β-mannan trisaccharide from the pathogenic fungus Candida albicans as well as the carbohydrate portion of tumor-associated ganglioside GM₂ to a T_H-cell peptide epitope derived from the murine 60 kDa self heat-shock protein (hsp60). Moreover, the linkage chemistry has proven well suited for the synthesis of more complex target structures such as a biotinylated glycopeptide, a three component vaccine containing an immunostimulatory peptide epitope from interleukin-1β (IL-1β), and for the conjugation of complex carbohydrates to carrier proteins such as bovine serum albumin.

Full text of DOI:10.1002/chem.200800065

DOI: 10.1002/chem.200800065
A Novel Linker Methodology for the Synthesis of Tailored Conjugate
Vaccines Composed ofComplex Carbohydrate Antigens and
Specific TH-Cell Peptide Epitopes
Sebastian Dziadek, Sandra Jacques, and David R. Bundle*[a]
Abstract: Pathogenic organisms or on-
cogenically transformed cells often ex-
press complex carbohydrate structures
at their cell surface, which are viable
targets for active immunotherapy. We
describe here a novel, immunologically
neutral, linker methodology for the ef-
ficient preparation of highly defined
vaccine conjugates that combine com-
plex saccharide antigens with specific
TH-cell peptide epitopes. This novel
heterobifunctional approach was em-
ployed for the conjugation of a (1!2)-
b-mannan trisaccharide from the
pathogenic fungus Candida albicans as
well as the carbohydrate portion of
tumor-associated ganglioside GM2 to a
TH-cell peptide epitope derived from
the murine 60 kDa self heat-shock pro-
tein (hsp60). Moreover, the linkage
chemistry has proven well suited for
the synthesis of more complex target
structures such as a biotinylated glyco-
peptide, a three component vaccine
containing an immunostimulatory pep-
tide epitope from interleukin-1b (IL-
1b), and for the conjugation of com-
plex carbohydrates to carrier proteins
such as bovine serum albumin.
Keywords: Candida albicans · gly-
coconjugate vaccines · glycopepti-
des · tumor-associated gangliosides
Introduction
poor immunogenicity, which is a key limitation to their suc-
cessful application as vaccines. Extensive research has there-
fore focused on the creation of synthetic vaccines[2–4] based
on complex carbohydrate epitopes conjugated to foreign
carrier proteins such as tetanus toxoid, KLHor BSA, which
after processing liberate peptide fragments (MHC peptides)
for the recruitment of TH cells. The use of large carrier pro-
teins for the construction of conjugate vaccines has, howev-
er, some disadvantages: A strong immune response against
the carrier protein may result in suppression of carbohy-
drate-specific antibody production.[5,6] Alternatively, the
large protein might contain immunosuppressive epitopes,[7–9]
which can reduce the effectiveness of the vaccine. In addi-
tion, conjugation chemistry is often difficult to control lead-
ing to glycoproteins with ambiguities in structure and com-
position, which may affect the reproducibility of an immune
response. In the search for effective vaccines and as a solu-
tion to these obstacles, specific TH-cell peptide epitopes de-
rived from various carrier proteins have been conjugated to
different antigens including monosaccharides,[10] polysaccha-
rides,[11] and tumor-associated glycopeptides.[12] However,
due to the complexity of both the target antigens and the T-
cell peptides, the preparation of vaccine conjugates is a cum-
bersome task often necessitating laborious protecting group
manipulations and accompanied by loss of precious materi-
al.
Vaccine development has been one of the major accomplish-
ments of modern medicine. A variety of different strategies
including the use of killed or attenuated microorganisms, in-
activated bacterial toxins, and protein subunit vaccines have
been successfully employed for the prevention of a variety
of infections.[1] However, there remains a need for effective
vaccines for the treatment of serious diseases such as malar-
ia, AIDS, and cancer as well as for the fight against antibiot-
ic-resistant microbial infections. It has been shown that com-
plex polysaccharides displayed on the surface of pathogenic
organisms as well as tumor-associated carbohydrate antigens
overexpressed on oncogenically transformed cells are viable
targets for active immunotherapy. However, due to their in-
ability to activate TH cells through presentation via MHC
class II molecules, saccharide antigens alone exhibit only
[a] Dr. S. Dziadek, S. Jacques, Prof. Dr. D. R. Bundle
Alberta Ingenuity Centre for Carbohydrate Science
Department of Chemistry, University of Alberta
Edmonton, Alberta, T6G 2G2 (Canada)
Fax : (+1)780-492-7705
E-mail: dave.bundle@ualberta.ca
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
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We describe here the development of a novel linker meth-
odology for the efficient synthesis of highly defined vaccine
conjugates that combine complex carbohydrate antigens
with specific TH-cell peptide epitopes.
succinimide (NHS) and dicyclohexylcarbodiimide (DCC) in
THF to give the heterobifunctional linker 1 in 72% yield
after purification.
Results and Discussion
In order to minimize the loss of valuable synthetic building
blocks, we envisaged a conjugation strategy that would link
the two completely deprotected components—the oligosac-
charide antigen and the specific TH-cell epitope—at the last
step of the synthesis under mild conditions in an aqueous
system and without the need for an organic co-solvent, acti-
vating reagents or inconvenient experimental procedures. To
be able to selectively couple the deprotected building blocks
containing multiple functionalities, we sought a heterobi-
functional linker that could, in the first step, be coupled to
one component to form an intermediate that was sufficiently
stable to permit both purification and long-term storage.
This would furnish a versatile building block that can be
readily utilized for different applications. In recent years a
variety of conjugation methods[13] have been developed for
the preparation of glycoconjugates including the addition of
sulfhydryl groups to maleimides,[14] reaction of thiols with
iodoacetamides,[15] 1,3-dipolar cycloadditions of azides to al-
kynes[16,17] or the use of bissuccinimidyl esters and diethyl
squarate.[18,19] While being very effective for rapid conjugate
formation, these procedures entail major drawbacks. They
either form unstable intermediates, which are too reactive
to allow chromatographic purification and storage, or they
incorporate aromatic or other cyclic structural elements,
which are potentially highly immunogenic themselves, and
may suppress an antibody response towards the target anti-
genic determinant.[20–22] Based on our earlier development of
an adipate p-nitro phenyl diester coupling reagent,[23,24] we
concluded that a suitable linker for vaccine preparation
Scheme 1. Synthesis of novel heterobifunctional linker 1: a) tert-Butyl ac-
rylate, Na, THF, 89%; b) acryloyl chloride, DIPEA, CH2Cl2, 82%; c)
TFA/anisole/CH2Cl2 9:1:10, 82%; d) DCC, NHS, THF, 72%.
The novel linker methodology was subsequently applied
to the development of structurally well-defined glycopeptide
vaccines against cancer and infections with Candida albi-
cans, a pathogenic fungus to which immunocompromised
hospital patients are especially vulnerable.[28] Previous im-
munochemical studies in our group showed that (1!2)-b-
mannan oligomers of the cell wall phosphomannan complex
have potential as key epitopes for the development of con-
jugate vaccines against C. albicans.[24,29] As the antigenic epi-
tope for the incorporation into glycoconjugate vaccines a
(1!2)-b-mannan trisaccharide (I) was prepared according
to a synthetic route that relies on the formation of a b-glu-
copyranosyl linkage with subsequent C-2 epimerization via
an oxidation-reduction sequence as described earlier.[24,30,31]
In our quest to develop an effective anticancer vaccine, our
investigations have focused on tumor-associated ganglioside
antigens.[32,33] For the preparation of model vaccines the tet-
rasaccharide portion of the ganglioside GM2 (II), which is
should incorporate
a non-immunogenic linear aliphatic
chain and exhibit, at the same time, sufficient water-solubili-
ty to facilitate its use. To fulfill these objectives we designed
a coupling reagent 1 based on a water-soluble triethylene
glycol core,[25,26] functionalized at one end with an amine-re-
active N-hydroxysuccinimide activated carboxylate and in-
corporating at the other end an acrylate moiety, which read-
ily reacts with sulfhydryl groups under mildly basic condi-
tions forming non-immunogenic alkyl thioethers.[27]
The synthesis of the novel linker started from triethylene
glycol, which was converted into 12-hydroxy-4,7,10-trioxado-
decanoic acid-tert-butylester 2 in a hetero-Michael reaction
with tert-butyl acrylate in THF. Subsequent treatment with
acryloyl chloride in the presence of ethyldiisopropylamine
as a base gave acrylate 3 in a yield of 82%. Removal of the
tert-butyl ester with trifluoroacetic acid furnished acid 4 in
82% yield. To allow ready conjugation to biomolecules
without the need for coupling reagents, 4 was converted into
the
corresponding
N-hydroxysuccinimidyl
ester
1
(Scheme 1). To this end, acid 4 was treated with N-hydroxy-
Figure 1. bMan3 (I) and GM2 (II) antigens.
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D. R. Bundle et al.
significantly overexpressed in a variety of tumors, was syn-
thesized employing a chemoenzymatic approach developed
in our group.[33] Both target saccharide antigens were
equipped with an amino-functionalized tether that allows
system for purification affording the acrylate modified anti-
gens 5 and 6 in yields of 74 and 79%, respectively
(Scheme 2).
The triethylene glycol acrylate spacer in 5 and 6 provides
a suitable handle to increase the retention on a C18 column
allowing for efficient separation. The derivatives synthesized
in this way are sufficiently stable for long-term storage facil-
itating their convenient application for different aspects of
glycoconjugate preparation.
for the conjugation with heterobifunctional linker
(Figure 1).
1
The oligosaccharide amines I and II were treated with
3 equiv of linker 1 in an aqueous borate buffer (0.02m) at
pH8.1. The reactions were completed in 35 min at which
time the solutions were injected directly into an HPLC-
As specific TH-peptide epitope for the generation of vac-
cine conjugates a peptide fragment from the murine self
60 kDa heat-shock protein (hsp60) identified by Cohen and
co-workers[34] was selected. This heptadecapeptide repre-
senting a strongly immunogenic T-cell epitope of hsp60 be-
tween residues 458 and 472 (p458m) has been shown to
serve as a potent carrier in conjugate vaccines against infec-
tions with S. pneumoniae and N. meningitidis.[11,35,36] The
p458m epitope was assembled in an automated synthesizer
using Fmoc strategy[37] starting from Rapp Tentagel R
resin[38] 7 equipped with Fmoc-isoleucine via the acid-labile
Wang linker[39] and employing a combination of HBTU/
HOBt[40] for activation. After completion of the peptide se-
quence, S-trityl protected mercaptopropionic acid 8, which
is readily available from mercaptopropionic acid and tri-
phenylmethanol, was coupled to install a sulfhydryl moiety
on the peptide to serve as attachment point for the linker
(Scheme 3). The thiol-modified heptadecapeptide 9 was li-
berated from the resin by treatment with TFA which simul-
taneously cleaved all side-chain protecting groups and was
subsequently purified by C18 RP-HPLC.
The conjugation reactions to form vaccines 10 and 11
were performed by simply dissolving the respective oligosac-
charide-acrylate derivatives and the thiol-modified p458m-
peptide 9 in a Tris·HCl buffer system containing 1 mm
EDTA at pH8.9. EDTA coordinates heavy metal ions that
Scheme 2. a) 1 (3 equiv), borate buffer, 0.02m, pH8.1, 35 min, purifica-
tion by C18 RP-HPLC, 74%; b) same as a) 79%.
Scheme 3. a) Peptide synthesis: i) cleavage of Fmoc: 20% piperidine in DMF; ii) amino acid coupling: Fmoc-AA-OH, HBTU, HOBt, DIPEA, DMF; iii)
capping: Ac2O, HOBt, DMF; b) incorporation of thiol moiety: i) Fmoc removal: 20% piperidine in DMF; ii) coupling: 8, HBTU, HOBt, DIPEA; c)
cleavage from resin and removal of acid-labile protecting groups: TFA/TIS/water 90:5:5.
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Glycoconjugate Vaccines
FULL PAPER
Scheme 4. Conjugation reactions to establish vaccine conjugates 10 and 11: a) Tris·HCl buffer, 0.05m, 1 mm EDTA, pH8.9, 4 h, purification by C18 RP-
HPLC, 71% for 10, 75% for 11.
would otherwise catalyze the oxidation of sulfhydryl groups
to from disulfides thereby significantly reducing this side re-
action. The respective reaction mixtures were shaken for 4 h
under argon, and the fully synthetic bMan3-p458m and
GM2-p458m vaccine conjugates 10 and 11 were isolated
after purification by RP-HPLC in 71 and 75% yield, respec-
tively (Scheme 4). The ester moiety contained in the linker
has proven to be sufficiently stable to allow the use of TFA
as ion pairing reagent during the HPLC purification without
complication.
With the aim of generating a useful and versatile tool for
a variety of immunochemical applications, the GM2-p458m
vaccine was re-synthesized incorporating a biotin moiety at
the C-terminal side of the molecule. To this end, an orthogo-
nally protected lysine building block was employed to serve
as a selective attachment point[41,42] for biotin. Starting from
resin 12 modified with ivDde-protected Fmoc-lysine,[43,44]
the p458m peptide was sequentially constructed and
equipped with a thiol-modified tether (Scheme 5). Subse-
quently, the orthogonally stable ivDde group was selectively
removed with 3.5% hydrazine in DMF. In this way, a versa-
tile precursor 13 was generated for the construction of vari-
ous tailor-made immunoconjugates as it allows the coupling
of labels, dyes or peptides on resin and, after cleavage, the
selective conjugation in solution of different antigens acti-
vated with the acrylate linker. After the attachment of
biotin to the C-terminal lysine residue of resin bound pep-
tide 13, the HS-p458m-biotin conjugate 14 was liberated
from the resin with concomitant deprotection by TFA, fol-
lowed by HPLC. After lyophilization, GM2-acrylate building
block 6 was coupled to HS-p458m-bio 14 using the same re-
action conditions as before. The fully synthetic GM2-p458m-
bio immunoconjugate 15 was obtained pure after HPLC in
70% yield (Scheme 5).
To enhance the immunogenicity of our glycopeptide-based
vaccines we designed according to this concept a three-com-
ponent vaccine consisting of the antigenic C. albicans b-
mannan trisaccharide, the p458m TH-cell carrier peptide,
and an immunostimulatory nonapeptide from human inter-
leukin-1b (IL-1b).[45] This IL-1b peptide has been shown to
reproduce the adjuvant effects of the whole IL-1b protein
without any of the IL-1b inflammatory, vasoactive and toxic
properties.[47,48] The synthetic strategy that afforded this vac-
cine relied on the solid-phase assembly of the HS-p458m-
IL-1b conjugate 16, which after cleavage, deprotection and
purification was conjugated to bMan3-acrylate building
block 5. Starting from the selectively amino-deprotected
resin-bound peptide 13 which served as precursor for GM2-
p458m-bio conjugate 15 (Scheme 5) the IL-1b nonapeptide
chain was sequentially assembled on the free amine to form
the HS-p458m-IL-1b conjugate 16, which was subsequently
cleaved from the resin by TFA with concomitant side-chain
deprotection and purified by preparative HPLC. Then, the
b-mannan building block 5 was efficiently linked to this con-
jugate in aqueous solution to yield the fully synthetic three-
component vaccine 17 in 64% after HPLC purification
(Scheme 6).
To investigate the versatility of the new linkage chemistry
the conjugation of ganglioside antigens to carrier proteins
was also studied. A model protein BSA was treated with 25
equivalents of linker 1 in borate buffer at pH8.1 leading to
a substitution of 17 molecules of the acrylate spacer per
molecule of protein as determined by MALDI-TOF mass
spectrometry (Scheme 7a).
For the subsequent conjugation of GM2 analogue II to
BSA–acrylate 18 a sulfhydryl group was incorporated into
the saccharide antigen employing the amine-reactive thiolat-
ing reagent SATA (succinimidyl S-acetylthioacetate)[49] fol-
lowed by deprotection with hydroxylamine (Scheme 7b).
The modified protein 18 was incubated for 8 h with the
GM2-derivative 19 formed in this way (1.5 equiv relative to
Recently, different examples of synthetic vaccines that
combine a B-cell epitope, a promiscuous T-cell peptide and
an immunostimulatory adjuvant have been reported.[10,45,46]
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D. R. Bundle et al.
Scheme 5. a) 3.5% Hydrazine in DMF, 5”5 min; b) biotin, HBTU, HOBt, DIPEA; c) TFA/TIS/water 90:5:5; 6; d) Tris·HCl buffer, 0.05m, 1 mm EDTA,
pH8.9, 4 h, purification by RP-HPLC, 70%; bio =biotinyl.
Scheme 6. Assembly of a three component vaccine: a) Solid-phase synthesis of IL-1b sequence; b) TFA/TIS/water 90:5:5, purification by HPLC, 27%
(for a) and b)); c) 5, Tris·HCl buffer, 0.05m, 1 mm EDTA, pH8.9, 16 h, purification by RP-HPLC, 64%.
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Glycoconjugate Vaccines
FULL PAPER
Scheme 7. Protein conjugation: a) 1 (25 equiv), borate buffer, 0.02m, pH8.1, 1 h; b) SATA (10 equiv), borate buffer, 0.02 m, pH8.1, 30 min, purification
by RP-HPLC; c) H2NOH(0.5 m), sodium phosphate 50 mm, EDTA 25 mm, pH 7.5; d) 18 (0.67 equiv), Tris·HCl buffer, 0.05m, 1 mm EDTA, pH8.9, 8 h,
purification via Sephadex PD-10 column.
the spacer loading in 18) in an EDTA containing borate
buffer at pH8.9 and subsequently freed of salts and unreact-
ed saccharide employing
a Sephadex PD-10 column.
MALDI-TOF analysis showed an average incorporation of
eight molecules of the GM2 saccharide in the BSA conju-
gate 20.
Immunization studies: The synthesized glycopeptide-based
antifungal vaccines Man3-p458m 10 and Man3-p458m-IL-1b
17 were evaluated for their ability to induce a specific anti-
body response against the antigenic b-mannan trisaccharide.
Each conjugate formulated in incomplete Freundꢁs adjuvant
(IFA) was used to immunize groups of five BALB/c mice.
As the p458m carrier peptide was reported[36] to possess ad-
juvant properties, the use of complete Freundꢁs adjuvant
was avoided. A total of five immunizations were adminis-
tered at intervals of three weeks. Between the 3rd and the
4th boost the mice were rested for two months to allow time
for the immunological memory to evolve. Sera from the im-
munized mice were analyzed by ELISA for Man3-specific
antibodies. It was also of particular interest to monitor the
ability of the antibodies to recognize a crude cell wall ex-
tract of native C. albicans. For this purpose, ELISA experi-
ments were carried out using microtitre plates coated with a
conjugate of synthetic b-mannan trisaccharide with BSA
(Man3-BSA)[24] and a cell wall extract from C. albicans.[50]
Figure 2. ELISA Data: Absorbance (OD450) for mouse sera raised against
Man3-p458m vaccine 10 (light grey) and Man3-p458m-IL-1b three-compo-
nent vaccine 17 (dark grey). Synthetic Man3-BSA conjugate was coated
on ELISA plates.
All mice immunized with the conjugate vaccines respond-
ed with markedly increased Man3-specific antibody levels in
comparison with the untreated pre-immune sera. The anti-
bodies raised recognized both the synthetic trisaccharide an-
tigen conjugated to BSA (Figure 2) and, most importantly, a
crude extract of the cell wall of C. albicans (Figure 3). Inter-
estingly, with the exception of only one mouse (mouse 5)
immunizations with the three-component vaccine Man3-
p458m-IL-1b 17 (dark grey) incorporating the peptidic IL-
1b adjuvant epitope reproducibly lead to higher carbohy-
drate specific antibody levels. The same trend was observed
when the ELISA plate was coated with the cell wall extract
from C. albicans (2 ME) (Figure 3). These findings underline
the potential of carefully design three-component vaccines
to induce enhanced immune responses.
Sera were serially diluted starting at a dilution factor of
100. Bound, specific antibodies were visualized with a secon-
1
=
dary goat anti-mouse antibody labeled with horseradish per-
oxidase (HPO). The absorbance (OD) was read at l=
450 nm. To simplify presentation and for better comparison,
1
the OD results are shown at a dilution of = . Figures 2 and
100
3 depict the ELISA data for five mice of each series com-
pared with the pooled pre-immune sera collected from the
same mice prior to immunization.
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D. R. Bundle et al.
enex Luna C18(2) (250”4.6 mm, 5 m) while preparative separations were
conducted on a Phenomenex Luna C18(2) (250”21.20 mm, 10 m) prepa-
rative column, using linear gradients of water and acetonitrile with the
addition of 0.02–0.1% TFA (flow rate 1 mLminꢀ1 for analytical runs and
10 mLminꢀ1 for preparative separations) as eluents. 1Hand 13C NMR
spectra were recorded on Varian INOVA 500 and 600 MHz spectrome-
ters. 1HNMR chemical shifts are reported in d (ppm) units using signals
from residual solvents as a reference. 13C NMR chemical shifts are refer-
enced to internal solvent or to external acetone in the case of D2O. As-
signment of proton and carbon signals was achieved with the assistance
of COSY, HMQC, HMBC, TOCSY and TROESY spectra as necessary.
MALDI TOF mass spectra were recorded on a Voyager Elite spectrome-
ter from Applied Biosystems. Electrospray ionization mass spectra were
recorded on a Micromass Zabspec TOF mass spectrometer. For high res-
olution mass determination, spectra were obtained by voltage scan over a
narrow range at a resolution of approximately 104.
12-Hydroxy-4,7,10-trioxadodecanoic acid-tert-butylester (2): Sodium
(40 mg, 0.9 mmol) was added under argon to a solution of anhydrous tri-
ethylene glycol (25.6 mL, 188.0 mmol) in dry THF (85 mL). After sodium
had completely dissolved, tert-butyl acrylate (9.6 mL, 66.0 mmol) was
added, and the mixture was stirred at RT for 20 h. The solution was neu-
tralized by the addition of 1n HCl (1.6 mL) and subsequently concentrat-
ed in vacuo. The resulting residue was taken up in brine (70 mL), and ex-
tracted with ethyl acetate (3”50 mL). The combined organic phases were
washed with brine, dried over MgSO4, and concentrated in vacuo. The
crude product (16.40 g, 58.9 mmol, 89%), which was obtained as a clear,
colorless solution was used for the subsequent reaction step without fur-
ther purification. Rf =0.30 (ethyl acetate); 1HNMR (600 MHz, CDCl 3):
d = 3.73–3.69 (m, 4H, 3-CH2, 11-CH2), 3.69–3.63 (m, 6H, 3”OCH2),
3.62 (m, 4H, 12-CH2, OCH2), 2.50 (t, 2H, 2-CH2, JCH ,CH =6.6 Hz),
Figure 3. ELISA Data: Same as above. Crude native C. albicans cell wall
extract (2ME) was coated on ELISA plates.
Conclusion
In conclusion, a novel linear non-immunogenic heterobi-
functional linker based on water-soluble triethylene glycol
has been developed and was applied in the efficient synthe-
sis of highly defined glycoconjugate vaccines against cancer
and microbial infections. The new conjugation method is
distinguished by its ease of use, the formation of stable, stor-
able intermediates, and by the mild reaction conditions free
from side reactions. Moreover, the novel linkage chemistry
has proven to be well suited for vaccine preparation by con-
jugation of complex carbohydrate antigens to carrier pro-
teins. The method has been successively applied to a series
of Candida albicans cell wall peptides some of which show
great promise as vaccine candidates in challenge experi-
ments.[51]
2
2
1.44 ppm (s, 9H, CH3-tBu); 13C NMR (125 MHz, CDCl3): d = 170.89 (1-
C=O), 80.52 (Cquart-tBu), 70.63, 70.51, 70.39, 70.35 (4”CH2), 66.91 (3-
CH2), 61.76 (12-CH2), 36.23 (2-CH2), 28.17, 28.16, 28.09 ppm (3”CH3-
tBu); ESI-HRMS: m/z: calcd for C13H26O6Na: 301.1622; found: 301.1621.
14-Oxo-4,7,10,13-tetraoxahexadec-15-enoic
acid-tert-butylester
(3):
Under ice cooling, acryloyl chloride (96%, 3.40 g, 37.60 mmol) was
added dropwise to a solution of 2 (6.00 g, 21.56 mmol) and N,N-diisopro-
pylethylamine (4.68 g, 36.21 mmol) in dry dichloromethane (100 mL).
The solution was allowed to warm up to room temperature over 1 h, and
stirred for 18 h. After diluting with ethyl acetate (100 mL), the reaction
mixture was washed with 10% HCl (100 mL), a sat. solution of NaHCO3
(100 mL) and brine (100 mL). The organic phase was dried over MgSO4
and concentrated in vacuo. The crude product was purified by column
chromatography (hexanes/ethyl acetate 3:1) to give the title compound
(5.85 g, 17.61 mmol, 82%) as a slightly yellow oil. 1HNMR (600 MHz,
CDCl3): d = 6.42 (dd, 1H, H-2tacrylate, J2t/1 =17.4, J2t/2c =1.2 Hz), 6.15 (dd,
1H, H-1acrylate, J1/2t =17.4, J1/2c =10.7 Hz), 5.82 (dd, 1H, H-2cacrylate, J2c/1
=
Experimental Section
10.4, J2c/2t =1.4 Hz), 4.32–4.29 (m, 2H, 12-CH2), 3.75–3.58 (m, 12H, 6”
CH2), 2.49 (t, 2H, 2-CH2, JCH ,CH =6.4 Hz), 1.44 ppm (s, 9H, CH3-tBu);
2
2
Reagents and general experimental procedures: All reagents were pur-
chased from Sigma-Aldrich at the highest available commercial quality
and used without further purification. Fmoc-protected amino acids and
HBTU were obtained from Novabiochem; HOBt was purchased from
Matrix Innovations. As resin for solid-phase peptide synthesis TentaGel
R PHB (Rapp Polymere, Germany) was used. Solvents were dried and
collected using a Pure-Solv solvent purification system manufactured by
Innovative Technologies, Inc. NMP for peptide synthesis was purchased
from Caledon Laboratories Ltd. Unless noted otherwise, all reactions
were carried out at room temperature and non-hydrolytic reactions were
performed under a positive pressure of argon. Solvents were removed at
reduced pressure and a bath temperature of approximately 408C. Reac-
tions were monitored by analytical thin-layer chromatography (TLC)
with pre-coated silica gel 60 F254 glass plates (Merck). Plates were visual-
ized under UV light, and/or by treatment with either acidic cerium am-
monium molybdate or 5% ethanolic sulfuric acid, followed by heating.
Medium pressure chromatography was conducted using silica gel (40–
63 mm, SiliCycle, Quebec) at flow rates of 10–15 mLminꢀ1. RP-HPLC
separation was conducted on a Waters Delta 600 system using a Waters
2996 photodiode array detector. Analyses were performed on a Phenom-
13C NMR (125 MHz, CDCl3): d
= 170.87 (1-C=O), 166.13 (14-C=O),
130.92 (CH2-acrylate), 128.30 (CH-acrylate), 80.49 (Cquart-tBu), 70.64,
70.63, 70.57, 70.38 (4”CH2), 69.12 (11-CH2), 66.91 (3-CH2), 63.69 (12-
CH2), 36.28 (2-CH2), 28.09 ppm (3”CH3-tBu); ESI-HRMS: m/z: calcd
for C16H28O7Na: 355.1727; found: 355.1725.
14-Oxo-4,7,10,13-tetraoxahexadec-15-enoic acid (4): A solution of pro-
tected linker 3 (2.50 g, 7.52 mmol) in a mixture of dichloromethane
(5 mL), TFA (4.5 mL) and anisole (0.5 mL) was stirred at room tempera-
ture for 2 h. The reaction mixture was diluted with toluene (25 mL), and
the solvent was removed in vacuo. The resulting residue was co-evaporat-
ed with toluene (3”25 mL) and purified by column chromatography
(hexanes/ethyl acetate 3:1!1:1!ethyl acetate) to yield compound 4
(1.70 g, 6.15 mmol, 82%) as a colorless oil. Rf =0.15 (hexanes/ethyl ace-
tate 1:1); 1HNMR (600 MHz, CD 3OD): d = 6.39 (dd, 1H, H-2tacrylate
2t/1 =17.4, 2t/2c =1.2 Hz), 6.17 (dd, 1H, H-1acrylate 1/2t =17.4, J1/2c =
,
J
J
,
J
10.2 Hz), 5.88 (dd, 1H, H-2cacrylate, J2c/1 =10.2, J2c/2t =1.8 Hz), 4.30–4.26 (m,
2H, 12-CH2), 3.73–3.69 (m, 4H, 3-CH2, 11-CH2), 3.66–3.56 (m, 8H, 3”
OCH2, 12-CH2), 2.53 ppm (t, 2H, 2-CH2, JCH ,CH =6.0 Hz); 13C NMR
2
2
(125 MHz, CD3OD): d
=
175.28 (1-C=O), 167.64 (14-C=O), 131.69,
5914
www.chemeurj.org
ꢀ 2008 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim Chem. Eur. J. 2008, 14, 5908 – 5917

Glycoconjugate Vaccines
FULL PAPER
129.48, 71.60, 71.54, 71.42, 67.88, 35.82 ppm; ESI HRMS: m/z: calcd for
1.9 Hz), 3.48–3.38 (m, 2H, 3.36 (dd, 1H, H2Gal, J2,1 =8.1, J2,3 =9.7 Hz),
C12H20O7Na: 299.1101; found: 299.1098.
3.31 (t, 1H, H-2Glc, J2,1 =J2,3 =8.4 Hz), 2.66 (dd, 1H, H-3eqNeuNAc; J3e/3a =
12.6, J3/4 =4.7 Hz), 2.53 (t, 2H, 2-CH2 linker, J2,3 =6.2 Hz), 2.02, 2.00 (2”
s, 2”3H, N(O)CH3), 1.92 ppm (t, 1H, H-3axNeuNAc, J3a/3e =J3/4 =12.0 Hz);
13C NMR (150 MHz, D2O, chemical shifts taken from HSQC, HMBC): d
14-Oxo-4,7,10,13-tetraoxahexadec-15-enoic
acid-N-hydroxysuccinimide
ester (1): N,N’-Dicyclohexylcarbodiimide (248 mg, 1.20 mmol) and N-hy-
droxysuccinimide (97%, 142 mg, 1.20 mmol) were added under argon to
a solution of 4 (300 mg, 1.09 mmol) in dry THF (9 mL). The reaction
mixture was stirred at room temperature for 15 h. After concentration
under reduced pressure, the resulting residue was purified by column
chromatography to afford 1 as a colorless oil (292 mg, 0.78 mmol, 72%).
Rf =0.40 (hexanes/ethyl acetate 1:1); 1HNMR (600 MHz, CDCl 3): d =
=
133.6 (C2acrylate), 128.4 (C1acrylate), 103.7 (2C, C1GalNAc, C1Gal), 103.2
(C1Glc), 38.2 (C3NeuNAc); MALDI-TOF-MS (positive, DHB): m/z: calcd
for C45H75N3O30Na: 1161.1; found: 1160.9 [M+Na]+.
HS-(CH2)2-Asn-Glu-Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-
Leu-Lys-Ile-OH (HS-p458m) (9): Starting from Fmoc-Ile-OHpreloaded
TentaGel R resin 7 (588 mg, 0.1 mmol, substitution: 0.17 mmolgꢀ1, Rapp
Polymere, Germany) the solid-phase peptide synthesis was carried out on
an automated Perkin Elmer ABI 433 A peptide synthesizer using Fmoc
chemistry (protocol, see Supporting Information). Upon completion of
the amino acid sequence and removal of the N-terminal Fmoc group
with piperidine (20% in NMP), S-trityl-b-mercaptopropionic acid (8) (2”
348.4 mg, 1 mmol) was attached in a standard double coupling. For the
cleavage procedure under simultaneous removal of the acid-labile side-
chain protecting groups, the resin was placed into a Merrifield glass reac-
tor, washed with dichloromethane (3”15 mL) and treated with a mixture
of TFA (7.5 mL), water (0.45 mL) and triisopropylsilane (0.45 mL) for
2.5 h. After filtration, the resin was washed with TFA (3”3 mL), and the
combined filtrates were concentrated in vacuo and co-evaporated with
toluene (3”15 mL). The peptide was precipitated by addition of cold
(08C) diethyl ether (15 mL) to furnish a colorless solid, which was re-
peatedly washed with diethyl ether. The crude product was purified by
preparative RP-HPLC (Phenomenex Jupiter C12, acetonitrile/water +
0.1% TFA 5:95!50:50, 60 min, l=212 nm, tR =36 min) to give the thiol
modified peptide 9 (87 mg, 0.042 mmol, 42%) as a colorless solid after
lyophilization. MALDI-TOF-MS (positive, DHB): m/z: calcd for
C91H162N25O27S: 2069.18; found: 2069.94 [M+H]+.
6.43 (dd, 1H, H-2tacrylate, J2t/1 =17.4, J2t/2c =1.2 Hz), 6.16 (dd, 1H, H-1acrylate
1/2t =17.4, 1/2c =10.8 Hz), 5.84 (dd, 1H, H-2cacrylate 2c/1 =10.5, J2c/2t
1.2 Hz), 4.34–4.29 (m, 2H, 12-CH2), 3.85 (t, 2H, CH2, JCH ,CH =6.6 Hz),
,
J
J
,
J
=
2
2
3.75–3.72 (m, 2H, CH2), 3.69–3.61 (m, 8H, 4”CH2), 2.90 (t, 2H, 2-CH2,
J2-CH ,3-CH =JCH gem =6.0 Hz), 2.83 ppm (brs, 4H, CH2-NHS); 13C NMR
2
2
2
(125 MHz, CDCl3): d = 168.92 (C=O-NHS), 166.69 (1-C=O), 166.14 (11-
C=O), 130.92, 128.31, 70.73, 70.64, 70.60, 70.55, 69.09, 32.17, 25.61,
25.57 ppm; ESI HRMS: m/z: calcd for C16H23NO9Na: 396.1265; found:
396.1262.
bMan3-acrylate 5: In a 2 mL-Eppendorf tube bMan3-amine I[24] (30 mg,
0.0482 mmol) and linker 1 (34.5 mg, 0.0925 mmol, 1.9 equiv) were com-
bined in aqueous borate buffer (1 mL; Na2B407·12H2O/H3BO3; 0.02m,
pH8.1), and the solution was tumbled at ambient temperature for
35 min. Subsequently, the mixture was filtered over a syringe filter (Milli-
pore Millex; LCR PTFF 0.45 mM), and the title compound (31.3 mg,
0.0357 mmol, 74%) was isolated by preparative RP-HPLC (Phenomenex
Luna C18(2), acetonitrile/water 5:95!50:50, 65 min, tR =31.7 min);
1HNMR (600 MHz, D 2O): d = 6.47–6.34 (m, 1H, H-2cacrylate), 6.25–6.19
(m, 1H, H-1acrylate), 6.02–5.98 (m, 1H, H-2tacrylate), 4.93 (s, 1H, H-1’’), 4.89
(s, 1H, H-1’), 4.73 (s, 1H, H-1), 4.36–4.31 (m, 3H, 12-CH2 linker {4.34}, H-2’
{4.33}), 4.23- 4.20 (m, 1H, H-2), 4.15–4.12 (m, 1H, H-2’’), 4.00–3.94 (m,
1H, OCH2aCH2CH2-S-), 3.93–3.88 (m, 3H, H-6a, H-6a’, H-6a’’), 3.84–
3.63 (m, 19H, 3-CH2 linker {3.77}, H-6b’, H-6b’’ {3.74}, H-6b {3.73},
OCH2bCH2CH2-S- {3.70}, H-3 {3.67}, 6”CH2 linker), 3.63–3.53 (m, 4H, H-
bMan3-(CH2)3S
A
(CH2CH2O)4CO
G
N
Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-Leu-Lys-Ile-OH
(bMan3-p458m) (10): To Man3-acrylate 5 (1.75 mg, 1.98 mmol) was added
a solution of HS-p458m peptide 9 (4.10 mg, 1.98 mmol) in Tris·HCl buffer
(1.5 mL; 0.05m Tris, 1 mm EDTA; pH8.9) under an argon atmosphere.
The mixture was sonicated for 5 min to afford complete dissolution and
tumbled for 4 h. Subsequently, the mixture was filtered over a syringe
filter (Millipore Millex; LCR PTFF 0.45 mM), and injected directly into
an HPLC system. The target conjugate 10 (4.12 mg, 1.41 mmol, 71%) was
isolated employing an acetonitrile/water gradient (Phenomenex Luna
C18(2), acetonitrile/water + 0.02% TFA 15:85!50:50, 65 min) and ob-
tained as a colorless amorphous solid after lyophilization. Analytical
3’ {3.61}, H-3’’ {3.61}, H-4’ {3.59}, H-4’’ {3.58}), 3.48 (dd, 1H, H-4, J3,4
ꢁ
J4,5 ꢁ10.2 Hz), 3.44–3.38 (m, 2H, S-CH2CH2-NH), 3.38–3.31 (m, 3H, H-5
{3.36}, H-5’’ {3.35}, H-5’ {3.34}), 2.73–2.61 (m, 4H, S-CH2CH2-NH{2.70},
OCH2CH2CH2-S- {2.66}), 2.52 (t, 2H, 2-CH2 linker
, J=6.0 Hz), 1.95–
1.86 ppm (m, 2H, OCH2CH2CH2-S-); 13C NMR (150 MHz, D2O, chemical
shifts taken from HSQC): d = 133.7 (C-2acrylate), 128.5 (C-1acrylate), 102.2
(C-1’), 101.7 (C-1’’), 101.1 (C-1), 79.7 (C-2), 79.1 (C-2’), 77.3 (C-5, C-5’,
C-5’’), 73.8, 73.3 (C-3’, C-3’’), 72.9 (C-3), 71.3 (C-2’’), 70.6 (6”OCH2 linker),
69.5 (C-6’, O-CH2CH2CH2-S), 68.4 (C-4), 67.8 (C-4’, C-4’’), 67.7 (3-
CH2 linker), 64.9 (12-CH2 linker), 61.9 (2C, C-6, C-6’’), 39.7 (S-CH2CH2-NH),
37.1 (2-CH2 linker), 31.6 (S-CH2CH2-NH), 29.9 (OCH2CH2CH2-S),
29.0 ppm (OCH2CH2CH2-S); MALDI-TOF-MS (positive, DHB): m/z:
calcd for C35H61NO22SNa: 902.9; found: 903.0 [M+Na]+.
HPLC: tR =17.4 min (Phenomenx Luna C18(2), acetonitrile/water
+
0.02% TFA 15:85!50:50, 25 min); MALDI-TOF-MS (positive, HCCA):
m/z: calcd for C126H223N26O49S2: 2950.4; found: 2950.1 [M+H]+.
GM2-(CH2)3S
Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-Leu-Lys-Ile-OH
p458m) (11): In 2 mL-Eppendorf tube GM2-acrylate
A
(CH2CH2O)4CO
U
G
(GM2-
(1.5 mg,
GM2-acrylate 6: A solution of GM2-amine II (5.1 mg, 0.0058 mmol) in
aqueous borate buffer (1 mL; 0.02m, pH8.1) was added to heterobifunc-
tional linker 1 (4.6 mg, 0.0123 mmol, 2.1 equiv). The mixture was vor-
texed for 5 min and subsequently the solution was tumbled for 35 min at
room temperature, filtered over a syringe filter (Millipore Millex; LCR
PTFF 0.45 mM), and injected into a preparative HPLC system. The de-
sired GM2 analogue 6 (5.2 mg, 0.00457 mmol, 79%) was isolated using an
acetonitrile/water gradient and obtained as colorless lyophilisate after
freeze-drying. tR =27.0 min (Phenomenex Jupiter C12, acetonitrile/water
6
1.318 mmol) and HS-p458m 9 (2.72 mg, 1.318 mmol) were combined in a
Tris·HCl buffer system (1.5 mL; 0.05m Tris, 1 mm EDTA; pH8.9). The
mixture was sonicated for 10 min to facilitate complete dissolution and
subsequently tumbled for 4 h under argon. The solution was filtered over
a syringe filter, and glycopeptide 11 (3.2 mg, 0.099 mmol, 75%) was iso-
lated by preparative RP-HPLC (Phenomenex Luna C18(2), acetonitrile/
water + 0.02% TFA 15:85!50:50, 65 min) and obtained as a colorless
solid after lyophilization. Analytical HPLC: tR =17.8 min (Phenomenex
+
0.1% TFA 5:95!50:50, 65 min); 1HNMR (600 MHz, D 2O): d
=
=
=
Luna C18(2), acetonitrile/water
MALDI-TOF-MS (positive, HCCA): m/z: calcd for C136H237N28O57S:
3206.6; found: 3206.5 [M+H]+.
+
0.02% TFA 5:95!50:50, 25 min);
6.46–6.42 (m, 1H, H-2cacrylate), 6.21 (dd, 1H, H-1acrylate, J1/2t =17.4, J1/2c
10.8 Hz), 6.01–5.98 (m, 1H, H-2tacrylate), 4.71 (d, 1H, H-1GalNAc, J1/2
8.6 Hz), 4.51 (d, 1H, H-1Gal, J1/2 =7.8), 4.48 (d, 1H, H-1Glc, J1/2 =7.8 Hz),
4.36–4.33 (m, 2H, CH2-OC(O)-), 4.14 (dd, 1H, H-3Gal, J3,2 =9.8, J3,4
3.4 Hz), 4.09 (d, 1H, H-4Gal 4,3 =2.9 Hz), 3.98–3.93 (m, 2H,
=
TrtS-(CH2)2-Asn
Ile-Glu(OtBu)-Ile-Ile-Lys
TentaGel R (13): The p458m amino acid sequence was assembled on
A
G
N
U
E
,
J
A
G
A
ACHTREUNG
-OCH2aCH2N-{3.97}, H-6aGlc {3.95}), 3.92–3.87 (m, 1H, H-2GalNAc {3.90}),
3.86 (dd, 1H, H-9aNeuNAc, J9a,9b =12.0, J9a,8 =2.2 Hz), 3.83–3.55 (m, 29H,
11-CH2 linker {3.81}, H-5NeuNAc {3.80}, -OCH2bCH2N-{3.79}, H-4NeuNAc
{3.78}, H-4GalNac {3.77}, H-6bGlc {3.76}, H-3GalNac {3.68}, H-4Glc, H -3 {3.65},
5”CH2 linker, H-9bNeuNAc {3.61}, H-5Glc {3.59}, H-7NeuNAc, H -N8euNAc, H -
solid phase starting from Fmoc-Lys(ivDde)-OHfunctionalized TentaGel
G
R resin 12 (0.1 mmol, substitution: 0.17 mmolgꢀ1) in an ABI 433 A pep-
tide synthesizer as described above. Upon completion of the peptide se-
quence and removal of the N-terminal Fmoc group, S-trityl-b-mercapto-
Glc
5
GalNac, H -6GalNac, H -5 , H -6 ), 3.49 (dd, 1H, H-6NeuNAc, J6,5 =10.1, J6,7
=
propionic acid
8
(2”348.4 mg, 1 mmol) was attached in
a
standard
Gal
Gal
Chem. Eur. J. 2008, 14, 5908 – 5917
ꢀ 2008 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.chemeurj.org
5915

D. R. Bundle et al.
double coupling, and the resin was extensively washed with NMP and di-
chloromethane. The resin-bound peptide was transferred into a Merri-
field glass reactor, washed with DMF (3”5 mL) and treated with a 3.5%
(relative to anhydrous hydrazine) solution of hydrazine monohydrate in
DMF (3 mL) for 5 min. The resin was filtered and the hydrazine treat-
ment was repeated four more times. The progress of the reaction was fol-
lowed by monitoring the UV absorbance of an aliquot of the deprotec-
tion solutions at l=290 nm. Subsequently, the resin was washed with
DMF (5”5 mL) and dichloromethane (3”5 mL), and dried in vacuo to
afford 768 mg of the selectively deprotected resin-bound peptide 13.
65 min) to furnish title compound 16 (32.2 mg, 0.0095 mmol, 29%) as a
colorless solid after lyophilization; MALDI-TOF-MS (positive, sinapinic
acid): m/z: calcd for C145H251N40O50S: 3386.85; found: 3386.34 [M+H]+.
bMan3-(CH2)3S
A
G
N
Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-Leu-Lys-Ile-Lys(Ac-
Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-CONH-(CH2CH2O)2CH2CH2-
CO-)-OH (Man3-p458m-IL-1b) (17): In a 2 mL Eppendorf tube Man3-ac-
rylate 5 (1.5 mg, 1.70 mmol, 1.8 equiv) and HS-p458m-IL-1b building
block 16 (3.2 mg, 0.945 mmol) were combined in aqueous Tris·HCl buffer
(600 mL; 0.05m Tris, 1 mm EDTA; pH8.9). Acetonitrile (200 mL) was
added and the mixture was vortexed for 10 min. The solution obtained
was tumbled gently for 16 h under argon, filtered over a syringe filter
(Millipore Millex; LCR PTFF 0.45 mM), and the three-component-vac-
cine 17 (2.6 mg, 0.61 mmol, 64%) was isolated by preparative RP-HPLC
(Phenomenex Luna C18(2), acetonitrile/water + 0.02% TFA, 25:75!
50:50, 65 min) and obtained as a colorless solid after lyophilization.
Excess Man3-acrylate 5 (0.9 mg, 1.02 mmol, 90% conversion) was re-iso-
lated during HPLC purification. Analytical HPLC: tR =11.6 min (Phe-
nomenex Luna C18(2), acetonitrile/water + 0.02% TFA 25:75!50:50,
25 min). MALDI-TOF-MS (positive, sinapinic acid): m/z: calcd for
C180H312N41O72S2: 4266.7; found: 4266.1 [M+H]+.
HS-(CH2)2-Asn-Glu-Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-
Leu-Lys-Ile-Lys(biotin)-OH (HS-p458m-bio) (14): A portion of resin 13
A
(268 mg, max. 0.033 mmol) was placed into the ABI A433 peptide syn-
thesizer, and washed with dichloromethane and NMP. The biotinylation
at the selectively deprotected lysine residue was carried out in a double
coupling with extended coupling times of 1 h each employing biotin (2”
244 mg, 2”1 mmol) and the standard activating conditions of the synthe-
sizer (1 mmol HBTU/HOBt, 2 mmol DIPEA in DMF). After extensive
washing with NMP and dichloromethane, the resin was treated in a Mer-
rifield reactor with a cleavage cocktail consisting of TFA (7.5 mL), water
(0.45 mL) and TIS (0.45 mL) for 2.5 h. Subsequently, the resin was fil-
tered and washed with TFA (3”3 mL). The combined filtrates were con-
centrated in vacuo and co-evaporated with toluene (3”15 mL). The pep-
tide was precipitated by addition of cold (08C) diethyl ether (10 mL) to
furnish a colorless solid, which was repeatedly washed with diethyl ether.
The crude product was purified by preparative RP-HPLC (Phenomenex
Protein conjugation I (BSA–acrylate 18): Bovine serum albumin (Sigma)
(ꢁ10.5 mg, 0.158 mmol) in aqueous borate buffer (1.5 mL;
Na2B407·12H2O/H3BO3; 0.02m, pH8.1) was added to heterobifunctional
linker 1 (1.5 mg, 4.02 mmol, ꢁ25 mol equiv). The mixture was gently tum-
bled for 1.5 h, diluted with milliQ water to a volume of 2.5 mL, applied
to a PD-10 desalting column (GE Healthcare, Sephadex G-25 Medium),
and eluted with milliQ water. A volume of 3 mL was collected to afford
a solution of activated BSA conjugate 18 (c ꢁ3.5 mgmLꢀ1 milliQ water).
The protein conjugate solution was stored at ꢀ208C prior to use.
MALDI-TOF-MS (positive, sinapinic acid): m/z: 70807.5; BSA, m/z:
66341.
Luna C18(2), acetonitrile/water + 0.1% TFA, 25:75!35:65
+ 0.1%
TFA, 65 min, l=212 nm, 32 min) to afford biotinylated building block 14
(30.3 mg, 0.0125 mmol, 38%) as a colorless solid after lyophilization. An-
alytical HPLC: tR =17.1 min (Phenomenex Luna C18(2), acetonitrile/
water + 0.1% TFA 25:75!50:50, 25 min); MALDI-TOF-MS (positive,
sinapinic acid): m/z: calcd for C107H188N29O30S2: 2424.94; found: 2424.99
[M+H]+.
O-(5-Acetamido-3,5-dideoxy-d-glycero-a-d-galacto-non-2-ulopyranosyl-
onic acid)-(2!3)-[2-acetamido-2-deoxy-O-b-d-galactopyranosyl-(1!4)]-
O-(b-d-galactopyranosyl)-(1!4)-O-(b-d-glucopyranosyl)-(1!1)-2-(thio-
GM2-(CH2)3S
A
(CH2CH2O)4CO
A
ACHTREUNG
Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-Leu-Lys-Ile-Lys
AHCTREUNG
(GM2-p458m-bio) (15): In a Falcon tube GM2-acrylate building block 6
(2.2 mg, 1.93 mmol) and HS-p458m-bio 14 (4.7 mg, 1.94 mmol) were com-
bined in aqueous Tris·HCl buffer (3 mL; 0.05m Tris, 1 mm EDTA;
pH8.9). The mixture was sonicated for 10 min and subsequently tumbled
for 6 h under argon. The solution was filtered over a syringe filter (Milli-
pore Millex; LCR PTFF 0.45 mM), and the biotinylated glycoconjugate
15 (4.8 mg, 1.35 mmol, 70%) was isolated by preparative RP-HPLC (Phe-
nomenex Luna C18(2), acetonitrile/water + 0.1% TFA, 25:75!35:65 +
0.02% TFA, 65 min, l=212 nm, tR =20.8 min) and obtained as a color-
less solid after lyophilization. Analytical HPLC: tR =13.8 min (Phenom-
acetamido)ethanol (GM2-SH) (19): Succinimidyl S-acetylthioacetate
(6.15 mg, 26.6 mmol, 5 equiv, Calbiochem) in dimethylsulfoxide (30.8 mL,
c = 10 mg per 50 mL) was added to a solution of GM2-amine II (5 mg,
5.32 mmol) in aqueous borate buffer (1 mL; 0.02m, pH8.1). The solution
was tumbled for 30 min, filtered over a syringe filter and injected into an
HPLC system. Acetylated intermediate GM2-SAc (2 mg, 2.0 mmol, 38%)
was isolated employing an acetonitrile/water gradient (Phenomenex
Luna C18(2), acetonitrile/water + 0.02% TFA 5:95!20:80, 65 min) and
obtained as a colorless amorphous solid after lyophilization. MALDI-
TOF-MS (positive, DHB): m/z: calcd for C37H61N3O26SNa: 1018.32;
found: 1018.21 [M+Na]+; calcd for C37H60N3O26SNa2: 1040.31; found:
1040.19 [M+2NaꢀH]+. For the subsequent de-acetylation, GM2-SAc
(1.2 mg, 1.21 mmol) was dissolved in a cleavage mixture (75 mL) consist-
ing of H2NOH(0.5 m), sodium phosphate (50 mm), and EDTA (25 mm),
pH7.5. The solution was tumbled for 2 h and the formed GM 2-SHana-
logue 19 was immediately employed for the protein conjugation without
further purification.
enex Luna C18(2), acetonitrile/water
25 min). MALDI-TOF-MS (positive, sinapinic acid): m/z: calcd for
152H263N32O60S2: 3560.80; found: 3561.61 [M+H]+.
+
0.02% TFA 25:55!35:65,
C
HS-(CH2)2-Asn-Glu-Asp-Gln-Lys-Ile-Gly-Ile-Glu-Ile-Ile-Lys-Arg-Ala-
Leu-Lys-Ile-Lys(Ac-Val-Gln-Gly-Glu-Glu-Ser-Asn-Asp-Lys-CONH-
(CH2CH2O)2CH2CH2-CO-)-OH (HS-p458m-IL-1b) (16): A portion of
resin 13 (268 mg, max. 0.033 mmol) was placed into the ABI A433 pep-
tide synthesizer, and washed with dichloromethane and NMP. Subse-
quently, spacer amino acid Fmoc-NH-(CH2CH2O)2CH2CH2-COOH(2”
399 mg, 2”1 mmol) was attached to the selectively deprotected lysine
residue in an extended double coupling (90 min per coupling), and the
IL-1b sequence was assembled according to the standard protocol of the
synthesizer (Suppl. Information). The N-terminal Fmoc group was re-
moved with piperidine (20% in NMP), and the amino terminus acetylat-
ed with capping reagent. After washing with NMP and dichloromethane,
the resin was treated in a Merrifield reactor with cleavage cocktail (TFA/
water/TIS 7.5 mL:0.45 mL:0.45 mL) for 2.5 h. Subsequently, the resin was
filtered and washed with TFA (3”3 mL). The combined filtrates were
concentrated in vacuo and co-evaporated with toluene (3”15 mL). The
peptide was precipitated by addition of cold (08C) diethyl ether (10 mL)
to furnish a colorless solid, which was repeatedly washed with diethyl
ether. The crude product was purified by preparative RP-HPLC (Phe-
nomenex Luna C18(2), acetonitrile/water + 0.1% TFA, 25:75!35:65,
Protein conjugation II (BSA–GM2 20): A portion of BSA–acrylate 18
dissolved in milliQ water (950 mL, c = 3.5 mgmLꢀ1, 0.047 mmol, 0.67 mo-
l equiv based on acrylate loading of 18) was diluted with Tris·HCl buffer
(1550 mL; 0.05m Tris, 1 mm EDTA; pH8.9), and applied to a PD-10 de-
salting column, which was pre-conditioned with the same buffer. The pro-
tein was eluted with Tris·HCl buffer, and 3 mL were collected. This
BSA–acrylate solution was added to GM2-SHbuilding block
19
(1.21 mmol) in DMSO (75 mL) and tumbled for 8 h. Subsequently, salts
and unbound saccharide were removed with a PD-10 desalting column
and the solution was lyophilized to furnish BSA–GM2 conjugate 20
(3.4 mg) as a colorless solid. MALDI-TOF-MS (positive, sinapinic acid):
m/z: 78216.
Immunization protocol: Two groups of five BALB/c mice were immu-
nized by interperitoneal and subcutaneous injections of vaccine conju-
gates 10 and 17, respectively (10 mg in 100 mL PBS/Freundꢁs incomplete
adjuvant, 1:1 (homogenized) per injection site). Subsequent injections
5916
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ꢀ 2008 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
Chem. Eur. J. 2008, 14, 5908 – 5917

Glycoconjugate Vaccines
FULL PAPER
were made interperitoneally and subcutaneously on days 21, 42, 63, 133,
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Acknowledgements
This research was funded by the Alberta Ingenuity Centre for Carbohy-
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Austauschdient (DAAD) and the Alberta Heritage Foundation for Medi-
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Received: January 13, 2008
Published online: May 14, 2008
Chem. Eur. J. 2008, 14, 5908 – 5917
ꢀ 2008 Wiley-VCHVerlag GmbH& Co. KGaA, Weinheim
www.chemeurj.org
5917