The immunogenicity of the tumor-associated antigen Lewisy may be suppressed by a bifunctional cross-linker required for coupling to a carrier protein

Article abstract of DOI:10.1002/chem.200400074

A Lewis^y (Le^y) tetrasaccharide modified by an artificial aminopropyl spacer was synthesized by a highly convergent approach that employed a levulinoyl ester and a 9-fluorenylmethoxycarbonate for temporary protection of the hydroxy gr

Full text of DOI:10.1002/chem.200400074

FULL PAPER
The Immunogenicity of the Tumor-Associated Antigen Lewisy May Be
Suppressed by a Bifunctional Cross-Linker Required for Coupling
to a Carrier Protein
Therese Buskas, Yanhong Li, and Geert-Jan Boons*[a]
Abstract: A Lewisy (Ley) tetrasacchar-
ide modified by an artificial amino-
propyl spacer was synthesized by a
highly convergent approach that em-
ployed a levulinoyl ester and a 9-fluo-
renylmethoxycarbonate for temporary
protection of the hydroxy groups and a
trichloroethyloxycarbonyl as an amino
protecting group. The artificial amino-
propyl moiety was modified by a thioa-
cetyl group,which allowed efficient
conjugation to keyhole limpet hemo-
cyanin (KLH) modified by electrophil-
ic 4-(maleimidomethyl)cyclohexane-1-
carboxylate (MI). Mice were immu-
nized with the KLH MI Ley antigen.
A detailed analysis of sera by ELISA
was elicited against the linker region.
The use of a smaller and more flexible
3-(bromoacetamido)propionate for the
attachment of Ley to KLH not only re-
duced the IgG antibody response
against the linker but also led to a sig-
nificantly improved immune response
against the Ley antigen. This study
shows that highly antigenic linkers sup-
press antibody responses to weak anti-
gens such as self-antigens.
established that
a strong immuno-
globulin G (IgG) antibody response
Keywords: glycoconjugates ¥ immu-
nogenicity
¥
oligosaccharides
¥
tumor-associated antigens ¥ vaccines
Introduction
tigen will raise opsonizing or cytotoxic antibodies,which rec-
ognize and eliminate circulating cancer cells and micrometa-
stases.[4,5] The poor immunogenicity of tumor-associated sac-
charides presents,however,a major obstacle for the devel-
opment of effective carbohydrate-based cancer vaccines.
Tumor-associated oligosaccharides are autoantigens and
consequently tolerated by the immune system. Furthermore,
the inability of saccharides to activate helper T-lymphocytes
also diminishes their usefulness for vaccine develop-
Oncogenesis is often associated with the over-expression of
various oligosaccharides on cell-surface glycoproteins and
glycosphingolipids.[1 This abnormal glycosylation is an im-
3]
portant criterion for the stage,direction,and fate of tumor
progression. Numerous studies have shown that the pres-
ence of tumor-associated oligosaccharides in primary tumors
is strongly correlated with the poor survival rates of patients.
The Lewis antigens sialyl Lewisx (Slex),Sle Lex,Le ,and
Ley are identified as important human-tumor-associated an-
tigens. These oligosaccharides are also ligands for the endo-
thelial cell-surface receptors E- and P-selectin. Binding of
cancer cells to the endothelium is a necessary step for meta-
stasis. Therefore it has been established that over-expression
of these Lewis antigens promotes metastasis.
ment.[6 For most immunogens,including saccharides,anti-
x
a
10]
body production depends on the cooperative interactions of
[8,11,12]
two types of lymphocytes,B-cells and helper T-cells.
Saccharides alone cannot activate helper T-cells and there-
fore have a limited immunogenicity. The formation of low-
affinity immunoglobulin M (IgM) antibodies and the ab-
sence of IgG antibodies manifest this limited immunogenici-
ty. Furthermore,saccharides alone cannot induce immuno-
logical memory and therefore do not give a booster re-
sponse after repeated exposure with the antigen. Fortunate-
ly,conjugation of a saccharide to a foreign carrier protein
(for example,keyhole limpet hemocyanin (KLH),detoxified
tetanus toxoid) enhances their presentation to the immune
system,thereby overcoming the tolerance and the helper T-
cell independent properties. In this case,the carrier protein
provides T-epitopes (peptide fragments of 12 15 amino
acids) that can activate helper T-cells. The immune response
against tumor-associated saccharide antigens can be further
improved by including a potent adjuvant.
Cancer vaccines are based on the elegant concept that the
immunization of cancer patients with a tumor-associated an-
[a] Dr. T. Buskas,+ Y. Li,+ Prof. Dr. G.-J. Boons
Complex Carbohydrate Research Center
University of Georgia
315 Riverbend Road,Athens,GA 30602-4712 (USA)
Fax : (+1)706-542-4412
E-mail: gjboons@ccrc.uga.edu
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2004, 10,3517 3524
DOI: 10.1002/chem.200400074
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
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FULL PAPER
Elegant studies by Danishefsky,Livingston and co-work-
ers at the Sloan Kettering Institute of Cancer Research
have shown that the Ley oligosaccharide (Scheme 1) conju-
gated to the carrier protein KLH in the presence of the im-
munological adjuvant QS-21 can elicit,in mice,IgG and
IgM antibody responses to naturally occurring forms of
Ley.[13] Although the antibody titers were relatively low,a
phase-one clinical trial with twenty-four patients who had
histologically documented ovarian,fallopian tube,or perito-
neal cancer was conducted.[14] The vaccination was well tol-
erated with no adverse effects related to autoimmunity. The
raised antibodies were capable of reacting with naturally oc-
curring Ley and with Ley-expressing tumor cells. Unfortu-
nately,the antibodies produced were mainly of the IgM
class,with only three patients exhibiting detectable levels of
IgG antibodies. Obviously,alternative strategies need to be
developed for presenting these carbohydrate epitopes that
will give a more efficient class switch to IgG antibodies and
higher titers.
spacer at the anomeric center for selective conjugation to a
carrier protein.
An efficient solution-phase synthesis for 9 was developed,
which employs the building blocks 1,[15] 3,[16] and 7.[17] Thus,
coupling of 1 with 3-[(N-benzyloxycarbonyl)amino]propa-
nol[18] in the presence of NIS/TESOTf gave,after purifica-
tion by silica gel column chromatography, 2 with an excel-
lent yield of 83%. This product was immediately used in the
next NIS/TESOTf-mediated glycosylation with galactosyl
donor 3 to give disaccharide 4. The Fmoc protecting group
of 4 was cleaved by treatment with Et3N in CH2Cl2. The re-
Scheme 1. The Ley tetrasaccharide.
We report here that the chemical nature of a bifunctional
linker greatly influences the immune response towards a
synthetic Lewisy tetrasaccharide hapten. It was found that
the widely used 4-(maleimidomethyl)cyclohexane-1-carbox-
ylate linker (Scheme 2) induces a significant immune re-
sponse towards the linker itself. The application of a 3-(bro-
moacetamido)propionate linker not only diminished the re-
sponse towards the linker but led to a considerably im-
proved immune response towards the Ley antigen as well.
Scheme 2. 4-(N-Maleimidomethyl)-cyclohexane-1-carboxylate and 3-(bro-
moacetamido)propionate.
Scheme 3. Synthesis of the Ley tetrasaccharide. a) HO(CH2)3NH-Z,NIS,
TESOTf,CH 2Cl2, 08C,83%; b) NIS,TESOTf,CH
2Cl2, 08C,76%;
c) Et3N/CH2Cl2 (1:1 v/v),84%; d) NH 2NH2¥HOAc,MeOH,CH 2Cl2,
87%; e) 1. Zn,HOAc; 2. Ac 2O,pyridine; 3. MeONa,MeOH,pH 10;
4. Pd/C,H 2,HOAc/EtOH (5:1 v/v),52% over four steps; f) SAMA-
Results and Discussion
Synthesis: As part of a program to develop a fully synthetic
anticancer vaccine,we required substantial quantities of the
tumor-associated antigen Ley. Furthermore,as a reference
antigen,a conjugate of the saccharide to a carrier protein
was required. The target compound,Le
OPfp,Et 3N,DMF; g) 7% NH
(g)/DMF,H 2O; h) sulfo-SMCC,0.1 m
3
sodium phosphate buffer (pH 6.2). Fmoc=9-fluorenylmethoxycarbonyl,
Bn=benzyl,Troc =2,2,2-trichloroethyloxycarbonyl, Z=benzyloxycarbon-
yl,Lev =levulinoyl,NIS =N-iodosuccinimide,TES =triethylsilyl,Tf =tri-
flate=trifluoromethanesulfonyl,SAMA-OPfp =S-acetylthioglycolic acid
pentafluorophenyl ester,DMF =N,N-dimethylformamide,sulfo-SMCC =
sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate.
y
derivative 9
(Scheme 3),was selected as it has an artificial aminopropyl
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Linker Effects on Lewisy Immunogenicity
3517 3524
sulting compound 5 was subjected to hydrazine buffered
with acetic acid to remove the Lev group to give diol 6.
Coupling of 6 with 3.8 equivalents of fucosyl donor 7 result-
ed in clean and stereoselective glycosylation at the C-3 and
C-2’ hydroxy groups and the fully protected tetrasaccharide
8 was isolated with a yield of 61%. The fucosylation pro-
repeated four times and sera were collected seven days after
the last boost. Titers of anti-Ley antibodies (Abs) were de-
termined by ELISA whereby microtiter plates were coated
with BSA MI Ley and serial dilutions of sera added. An
anti-mouse IgG (heavy-chain specific) antibody labeled with
alkaline phosphatase were employed as a secondary anti-
body for detection purposes,for which the measured titers
are listed in Table 1. A surprisingly high IgG antibody titer
was determined (Table 1,entry 1) compared to previously
reported immunizations with Ley KLH conjugates.[13,22] The
two different proteins KLH and BSA had purposely been
used in the immunizations and ELISA experiments,respec-
tively,and consequently,anticarrier antibodies could not ac-
count for the high titers. It was realized that antibodies
raised against the maleimide linker might cause the high
titers. To correct for this possible false positive result,a
3
ceeded with complete a selectivity,as confirmed by JH,H
couplings (J=3.5 Hz). Deprotection of 8 could easily be ac-
complished by a four-step procedure involving removal of
the Troc group by treatment with activated Zn followed by
acetylation of the resulting amine with acetic anhydride.
Next,the acetyl esters were saponified by treatment with
NaOMe in methanol. Finally,the benzyl ethers and the ben-
zyloxycarbonyl moiety were removed by catalytic hydroge-
nation over Pd/C in a mixture of ethanol and acetic acid to
give,after purification by P2 Bio-gel size-exclusion column
chromatography,target compound 9. The amino functionali-
ty of 9 was derivatized with an acetyl thioacetic acid moiety
by treatment with SAMA-OPfp to give 10 and a maleimide
group by reaction with sulfo-SMCC to give 12 in good yield.
Derivatives 10 and 12 may be employed for glycosylation of
proteins that are modified by electrophilic or nucleophilic
moieties,respectively.
y
second conjugate was prepared. In this conjugate,the Le
derivative 11 was conjugated to BSA that had been activat-
ed with electrophilic bromoacetyl groups. This functionality
also reacts with sulfhydryl groups at an appreciable rate at
pH values above 8.0. Thus,BSA was activated by incubation
with succinimidyl 3-(bromoacetamido)propionate (SBAP) in
a sodium phosphate buffer at pH 8.0. The conjugate was pu-
y
rified by centrifugal filter devices. Subsequently,the Le an-
Preparation of carbohydrate protein conjugates and immu-
nizations: In order to obtain an Ley carrier protein conju-
gate,we selected,in the first instance,maleimide thiol liga-
tion methodology involving compound 10 and maleimide-ac-
tivated keyhole limpet hemocyanin (KLH MI). This strat-
egy was thought to be attractive because 1) conjugation of
expensive thiol-modified antigens to KLH in a high-yielding
and reproducible manner is relatively easy,2) KLH MI has
been successfully used as a carrier for various tumor anti-
gens in immunological studies,and 3) maleimide-activated
KLH may conveniently be obtained in lyophilized form
from Pierce Endogen as part of an antibody production
kit.[19] This kit also contains maleimide-activated bovine
serum albumin (BSA MI) for the purpose of selective anti-
body detection by ELISA.
Prior to the conjugation,the S-acetyl group of tetrasac-
charide 10 was cleaved by treatment with ammonia in DMF
under an argon atmosphere to prevent formation of the cor-
responding disulfide. Next,the thiol 11 was added to KLH
MI in a phosphate buffer (pH 7.2) containing sodium azide
and ethylenediaminetetraacetate (EDTA). After a reaction
time of 2 h,the glycoprotein was purified by using a centri-
fugal filter device with a nominal molecular-weight limit of
10 KDa. The number of Ley copies conjugated to KLH was
determined by using Dubois× phenol sulfuric acid assay[20]
and Lowry×s protein-concentration test.[21] The conjugation
efficiency was typically around 80%,based on the number
of available maleimide groups,which correlated to an aver-
age of 620 Ley copies per KLH. For the ELISA assay,a
BSA MI Ley conjugate was prepared by using a similar
procedure but starting from BSA activated with maleimide
groups.
tigen 11 was treated with the bromoacetyl-activated protein
through thioether bond formation in a sodium phosphate
buffer (pH 8.0) containing EDTA; this reaction gave a gly-
coconjugate with an Ley/BSA ratio of 9:1. As expected,
when the BSA BrAc Ley conjugate was used for coating
ELISA plates,much lower titers of IgG antibodies were
measured (Table 1,entry 2). These results clearly demon-
strate that the convenient use of a commercial preactivated
protein kit led to false positive results and mainly antibodies
against the maleimide linker were detected.
Table 1. ELISA antibody titers[a] against Ley after four immunizations
with KLH MI Ley.
Entry
Coating
Titers
1
2
3
4
BSA MI Ley
358928
120
171969
459509
BSA BrAc Ley
BSA MI ME
BSA MI Ley RA
[a] All titers are medians for a group of five mice. Titers were determined
by regression analysis,with log dilution plotted versus absorbance. The
10
titers were calculated to be the highest dilution that gave three times the
absorbance of normal saline mouse sera diluted 1:120. The real antibody
titer against Ley is highlighted in italics.
Intrigued by this remarkable discrepancy,we were com-
pelled to investigate this linker effect further. Quenching
the maleimide groups of BSA MI with 2-mercaptoethanol
(ME) and coating ELISA plates with the obtained linker-
modified protein allowed the detection of only the antibod-
ies against the maleimide cross-linker. As can be seen in
entry 3 of Table 1,significant IgG antibody titers were meas-
ured,a result demonstrating that the cyclohexylmethyl ma-
26]
Groups of five mice were immunized with the KLH MI
Ley conjugate together with the immunoadjuvant QS-21
(Antigenics Inc.,Lexington,MA). The immunizations were
leimide linker is highly immunogenic.[23
It is well known that a maleimide moiety is susceptible to
hydrolysis even at slightly basic conditions. The irreversible
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G.-J. Boons et al.
FULL PAPER
hydrolysis results in the corresponding open form,maleamic
acid,which is unreactive towards sulfhydryl groups
(Scheme 4). It has been shown that the rate of reactivity of
the maleimide moiety with sulfhydryl groups increases with
increasing pH values.[27] However,raising the pH value
above 7.5 also significantly increases the rate of hydrolysis.
To overcome the sensitivity towards hydrolysis,the male-
imide functionality is commonly stabilized by a neighboring
cyclohexane moiety,[28,29] as in the SMCC linker used by
Pierce Endogen in the preactivated protein kits. Despite the
induced stability,it has been established that considerable
hydrolysis of the maleimide groups of the cyclohexylmethyl
maleimide linker occurs even after only 2 h at pH 7.0.[29]
tained by a reverse-activation protocol whereby KLH is
thiolated with Traut×s reagent and subsequently treated with
y
12,Le
modified with a cyclohexylmethyl meleimide
moiety.[30] This conjugate (KLH MI Ley RA) could conven-
iently be prepared as described above for BSA and gave a
remarkably good incorporation of the saccharide epitope
(Ley/KLH 965/1) as determined by Dubois× total-carbohy-
drate assay and the Lowry protein assay.
A more targeted immune response may also be obtained
by employing a linker that is smaller,more flexible,and
hence less immunogenic,such as an aliphatic 3-(bromoacet-
amido)propionate spacer. To investigate the immunological
properties of such a conjugate,Le y 11 was treated with KLH
that was activated with SBAP at pH 8.0 to give KLH
BrAc Ley. In this case,the ratio of Le y/KLH was deter-
mined to be 692:1.
Groups of five mice were immunized with the two conju-
gates four times at weekly intervals and sera were collected
seven days after the last immunization. Gratifyingly,the
KLH MI Ley RA conjugate gave a dramatically improved
titer against Ley,as can be seen from the ELISA assay in
which BSA BrAc Ley was employed as a coating (Table 2,
entry 1). The measured titer is the best reported thus far. A
significant antibody response against the cyclohexylmethyl
maleimide linker was raised (entries 2 and 3) but the re-
sponse was less profound than for immunizations with
KLH MI Ley (Table 1,entry 1). The anti-linker antibodies
recognized both internal and external epitopes since similar
titers were measured with coatings of Ley linked to BSA by
the direct (entry 2) and reverse maleimide activation proce-
dure (entry 3).
Scheme 4. Hydrolysis of the maleimide.
We reasoned that the anti-linker response might be direct-
ed only against terminal hydrolyzed maleimides and not
against maleimides connecting the saccharides to the pro-
tein,that is,internal maleimides (Scheme 5). In this respect,
it is important to realize that only 80% of the maleimides
are functionalized by saccharides and the rest are hydro-
lyzed. To distinguish between the two different modes of in-
teraction,microtiter plates were coated with BSA that was
thiolated by incubation with Traut×s reagent in a triethanola-
mine buffer (pH 8.0) containing EDTA and then treated
with maleimide-activated Ley 12. This reversely activated
conjugate (BSA MI Ley RA) displays only maleimide moi-
eties that are linked to saccharides. As a consequence,anti-
linker antibodies that only recognize terminal hydrolyzed
maleimides should not be detected. As can be seen in
Table 1 (entry 4),this conjugate gave a titer just as high as
the one for BSA MI Ley (entry 1) and,thus,it can be con-
cluded that the anti-linker antibodies recognize both inter-
nal and terminal hydrolyzed maleimide residues.
Table 2. ELISA antibody titers[a] against Ley after four immunizations
with KLH MI Ley RA.
Entry
Coating
Titers
1
2
3
BSA BrAc Ley
BSA MI Ley
10066
116960
103699
BSA MI Ley RA
[a] All titers are medians for a group of five mice. Titers were determined
The high immunogenicity of the linker region may well
suppress the formation of IgG antibodies against the Ley
epitope. Furthermore,although the anti-linker antibodies
recognized both internal and external cyclohexylmethyl ma-
leimide moieties,it was thought that the terminal moieties
are much more immunogenic. Therefore,immunizations
with a KLH Ley conjugate that does not contain any hydro-
lyzed maleimides may give a more targeted immune re-
sponse towards the Ley antigen. Such a conjugate can be ob-
by regression analysis,with log dilution plotted versus absorbance. The
10
titers were calculated to be the highest dilution that gave three times the
absorbance of normal saline mouse sera diluted 1:120. The real antibody
titer against Ley is highlighted in italics.
For the KLH BrAc Ley conjugate,IgG antibody titers
against Ley and the linker region were determined by coat-
y
ing microtiter plates with BSA MI Ley,BSA BrAc Le
and BSA BrAc quenched with 2-mercaptoethanol (BSA
BrAc ME). In this case,a sig-
,
nificantly improved anti-Ley
IgG antibody response was also
obtained (Table 3,entry 1) com-
pared with the KLH MI Ley
conjugate. There was only a
very weak response towards the
linker region,as can be seen
from the experiments with the
BSA BrAc Ley (entry 2) and
BSA BrAc ME (entry 3) coat-
Scheme 5. Internal and terminal maleimides.
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Chem. Eur. J. 2004, 10,3517 3524

Linker Effects on Lewisy Immunogenicity
3517 3524
ever,more importantly,a considerable improvement of
y
ings. In this respect,the BSA BrAc Le conjugate measures
a combined titer for Ley and linker whereas a coating with
BSA BrAc ME only determines a response for the linker
region.
immune response towards the Ley antigen was observed. A
reverse-conjugation protocol in which the Ley epitope was
modified by a maleimide moiety and then conjugated to
thiolated KLH also gave suppression of anti-linker antibo-
dies,a result indicating that terminal hydrolyzed linker resi-
dues are more immunogenic than internal ones. The anti-
linker IgG antibody titers were,however,significantly
higher than for the KLH BrAc Ley conjugate. The conju-
gate obtained by the reverse-activation method also elicited
higher IgG antibody titers against Ley. This high titer is a
result of the reduced response against the linker region and
a higher incorporation of Ley epitopes into KLH. The stud-
ies reported here call for an awareness of possible false posi-
tives stemming from the use of commercial kits in which the
same linker is employed for conjugation of a hapten to dif-
ferent proteins for antibody production and detection.
Table 3. ELISA antibody titers[a] against Ley after four immunizations
with KLH BrAc Ley.
Entry
Coating
Titers
1
2
3
BSA MI Ley
3294
3957
824[b]
BSA BrAc Ley
BSA BrAc ME
[a] All titers are medians for a group of four mice (one mouse died).
Titers were determined by regression analysis,with log dilution plotted
10
versus absorbance. The titers were calculated to be the highest dilution
that gave three times the absorbance of normal saline mouse sera diluted
1:120. The real antibody titer against Ley is highlighted in italics. [b] One
mouse gave a strong response that was far out of the range of values for
the rest of the group and was consequently omitted.
A remarkable finding is that although KLH MI Ley RA
(obtained by the reverse-activation protocol) gives a signifi-
cantly higher anti-linker antibody response than the KLH
Experimental Section
General: Succinimidyl 3-(bromoacetamido)propionate (SBAP),sulfosuc-
cinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate
y
BrAc Ley conjugate,it also elicited a stronger anti-Le anti-
(sulfo-
SMCC),2-iminothiolane (Traut×s reagent),keyhole limpet hemocyanin
(KLH),maleimide-activated mariculture KLH (mcKLH MI),and
bovine serum albumin (BSA MI) were purchased from Pierce Endogen,
Rockford,IL. BSA was purchased from Sigma. N-Iodosuccinimide (NIS)
was purchased from Fluka and recrystallized from dioxane/CCl4. All
other chemicals were purchased from Aldrich,Acros,or Fluka and used
without further purification. Molecular sieves were activated at 1458C
for 10 h. All solvents employed were of reagent grade and dried by re-
fluxing over appropriate drying agents. All the reactions were performed
under anhydrous conditions and monitored by TLC on Kieselgel 60 F254
(Merck) plates,with detection by UV light (254 nm) and/or by charring
with 8% sulfuric acid in ethanol. Column chromatography was per-
formed on silica gel (Merck,mesh 70 230). Size-exclusion column chro-
matography was performed on Sephadex LH-20 or Sephadex G10 gel
(Pharmacia Biotech AB,Uppsala,Sweden). Extracts were concentrated
under reduced pressure at ꢀ408C (water bath). 1H NMR and 13C NMR
spectra were recorded on a Varian Inova300 spectrometer and a Varian
Inova500 spectrometer equipped with Sun workstations. 1H spectra re-
corded in CDCl3 were referenced to residual CHCl3 at d=7.26 ppm or to
tetramethylsilane at d=0 ppm; 13C spectra were referenced to the central
peak of CDCl3 at d=77.0 ppm. Assignments were made by using stan-
dard 1D and gCOSY,gHSQC,and TOCSY 2D experiments. Positive-ion
matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)
mass spectra were recorded by using an HP-MALDI instrument with
gentisic acid as a matrix. Centrifugal filter devices were purchased from
Millipore Inc. The immunoadjuvant QS-21 was a gift from Antigenics
Inc.,Lexington,MA. Immulon II Hb ELISA plates were purchased from
Fisher Scientific Inc.
body response. An important difference between the two
constructs is that the reverse-activation protocol gave a
much higher incorporation of the Ley epitopes,which may
be relevant for the improved immunogenicity.
Our studies have shown that the selection of appropriate
conjugation technology is of critical importance in eliciting
IgG antibodies for weakly immunogenic epitopes such as
for tumor-associated carbohydrate antigens. In particular,
reducing the immunogenicity of the linker region and opti-
mizing epitope loading are critical factors. In this respect,
Danishefsky and co-workers have shown that conjugation of
a Ley to KLH by reductive amination gave a significantly
better immune response than the use of a construct that was
obtained by coupling of a thiolated saccharide derivative
with KLH modified with maleimides. Probably,this differ-
ence in immunogenicity arises from a linker effect.
Conclusion
Conjugation of a carbohydrate antigen to a carrier protein is
commonly used to overcome its T-cell independence. A
wide variety of coupling methods utilizing various heterobi-
functional cross-linking reagents have been described for
this purpose. Here,we demonstrate that the choice of linker
is of critical importance for the induction of a strong
immune response against a synthetic Lewisy tumor-associat-
ed antigen. The widely used 4-(maleimidomethyl)cyclohex-
ane-1-carboxylate linker proved to be highly immunogenic
and suppressed an IgG antibody response against the Ley
epitope. Detailed studies established that the anti-linker an-
tibodies recognize terminal hydrolyzed maleimides as well
as internal maleimides connecting the saccharides to the
protein. The use of the smaller and more flexible 3-(bro-
moacetamido)propionate cross linker resulted in a signifi-
cant reduction of antibodies against the linker region. How-
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2-{[(2,2,2-tri-
chloroethoxy)carbonyl]amino}-3-O-(9-fluorenylmethoxycarbonyl)-b-d-
glucopyranoside (2): A mixture of thioglycoside 1 (100 mg,0.14 mmol)
and 3-[(N-benzyloxycarbonyl)amino]propanol (33 mg,0.17 mmol) in dry
dichloromethane (10 mL) was dried azeotropically with toluene and then
subjected to high vacuum for 2 h. The mixture was dissolved in dry di-
chloromethane (10 mL) and stirred at room temperature under argon in
the presence of activated molecular sieves for 30 min,whereafter the
mixture was cooled to 08C and treated with NIS (35 mg,0.16 mmol) and
TESOTf (3 mL,0.01 mmol). After the reaction mixture was stirred for
10 min at 08C,TLC showed full conversion of the donor. The solution
was diluted with dichloromethane (60 mL) and the molecular sieves were
removed by filtration through a plug of celite. The filtrate was washed
with aqueous sodium thiosulfate (15%,4 mL) and brine,then dried over
MgSO4 and concentrated. The residue was purified by column chroma-
tography (silica gel,hexane/EtOAc 2:1) to give the product 2 as a white
Chem. Eur. J. 2004, 10,3517 3524
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G.-J. Boons et al.
FULL PAPER
powder (100 mg,83%).
(hexane/EtOAc 2:1); H NMR (300 MHz,CDCl ): d=7.80 (d, J=7.5 Hz,
2H,Ar-H),7.72 7.20 (m,16H,Ar-H),5.74 (d,
(s,1H,CH 2NHCOOCH2Ph),5.07 (s,2H,CH 2NHCOOCH2Ph),4.86 (t,
J=9.4 Hz,1H,H-3),4.65 4.51 (m,4H,ArC
(m,3H,H-1,Fmoc-CH 2),4.29 (t,
[
a]D =À32.2 (c=1.0 in CH2Cl2); Rf =0.48
CH2CH2CH2bNHCOOCH2Ph),2.78 2.19 (m,4H,OCOC
H2CH2,
1
Lev),2.12
(s,3H,CH
2COCH3,Lev),1.75
(m,2H,
3
J=8.3 Hz,1H,NH),5.20
CH2CH2CH2NHCOOCH2Ph) ppm; 13C NMR (300 MHz,CDCl 3): d=
171.6 (OCOCH2CH2,Lev),156.8 (NH COOCH2Ph),154.8 (NH CO),
138.6 127.1 (30C,Ar-C),101.6 (C-1 ’),101.3 (C-1),95.9 (CCl 3),81.2 (C-
H2,Troc),4.48 4.37
J=6.3 Hz,1H,Fmoc-CH),
4),80.5 (C-3 ’),74.8,74.7,73.9,73.7,72.4 (5C,4îO
CH2Ph,O CH2CCl3),
3.90 3.71 (m,5H,H-2,H-4,H-6a,C
3.41 (m,3H,H-5,H-6b,CH
H2CH2CH2NHCOOCH2Ph),3.52
2CH2CH2 NHCOOCH2Ph),3.26
73.4 (C-5),74.0 (C-5 ’),72.6 (C-3),72.4 (C-4 ’),71.6 (C-2 ’),68.4,67.3,67.4,
66.8 (4C,C-6,C-6 ’, OCH2CH2CH2,COO CH2Ph),57.7 (C-2),38.1
a
b
3.18 (m,1H,CH
2CH2CH2 NHCOOCH2Ph),1.72 (m,2H,
(OCOCH2CH2,Lev),37.9 (OCH 2CH2CH2),30.1 (CH COCH3,Lev),29.8
2
CH2CH2CH2NHCOOCH2Ph) ppm; 13C NMR (300 MHz,CDCl 3): d=
156.9 (NHCOOCH2Ph),155.9 (CHCH 2COO),154.8 (NH CO),142.5
120.3 (24C,Ar-C),101.4 (C-1),95.7 (CCl 3),79.4 (C-3),74.6,74.1 (2C,
(OCH2CH2CH2),29.0 (OCOCH 2CH2,Lev) ppm; HR MALDI-TOF MS:
m/z: calcd for C59H67Cl3N2O16: 1164.3556; found: 1187.3429 [M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2-[[(2,2,2-tri-
chloroethoxy)carbonyl]amino]-4-O-(3,4,6-tri-O-benzyl-b-d-galatopyrano-
syl)-b-d-glucopyranoside (6): A solution of hydrazine acetate (3 mL,
0.5m in methanol) was added dropwise to a stirred mixture of compound
5 (50 mg,0.043 mmol) in dichloromethane (10 mL). The reaction was
kept at room temperature for 2 h,quenched by addition of acetonylace-
tone (0.2 mL),and diluted by dichloromethane (40 mL). The organic
phase was washed with brine,dried over MgSO 4,and concentrated. The
residue was purified by column chromatography (silica gel,hexane/
EtOAc 2:1) to give the product 6 as a white powder (40 mg,87%).
[a]D =À40.7 (c=1.0 in CH2Cl2); Rf =0.31 (hexane/EtOAc 2:1); 1H NMR
OCH2Ph,O CH2CCl3),73.9 (C-5),70.9 (O
(CHCH2CO,Fmoc),70.2 (C-4),67.5 (C-6),66.9 (COO
(C-2),46.8 CHCH2CO,Fmoc),38.0 (OCH
CH2CH2CH2),70.7
CH2Ph),56.2
2CH2CH2),29.9
(
(OCH2CH2CH2) ppm; HR MALDI-TOF MS: m/z: calcd for
C42H43Cl3N2O11: 856.1932; found: 879.1941 [M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2-{[(2,2,2-tri-
chloroethoxy)carbonyl]amino}-3-O-(9-fluorenylmethoxycarbonyl)-4-O-
(3,4,6-tri-O-benzyl-2-O-levulinoyl-b-d-galactopyranosyl)-b-d-glucopyra-
noside (4): A mixture of acceptor 2 (95 mg,0.11 mmol) and thiogalacto-
side 3 (80 mg,0.13 mmol) in dry dichloromethane (10 mL) was dried
azeotropically with toluene and then subjected to high vacuum for 2 h.
The mixture was dissolved in dry dichloromethane (10 mL) and stirred at
room temperature under argon in the presence of activated molecular
sieves for 30 min. The mixture was cooled to 08C and treated with NIS
(33 mg,0.15 mmol) and TESOTf (3 mL,0.01 mmol). After 30 min,TLC
indicated that the reaction was complete and the mixture was diluted
with dichloromethane (60 mL) and filtered through celite. The filtrate
was washed with aqueous sodium thiosulfate (15%,4 mL) and brine
(20 mL),dried over MgSO 4,and concentrated. The residue was purified
by column chromatography (silica gel,hexane/EtOAc 2:1) to give the
(300 MHz,CDCl ): d=7.39 7.28 (m,25H,Ar-H),5.45 (d, J=8.3 Hz,1H,
3
NH),5.22
(s,1H,CH
2NHCOOCH2Ph),5.08
(s,2H,
CH2NHCOOCH2Ph),4.90 4.31 (m,10H,4îArC H2,Troc),4.61 (m,1H,
H-1),4.26 (d, J=6.3 Hz,1H,H-1 ’),3.99 3.83 (m,3H,H-2 ’,H-4 ’,H-6 ’a),
3.86 3.65 (m,6H,H-3,H-6,H-5
3.35 (m,6H,H-2,H-4,H-5,H-3
3.26 3.18 (m,1H,CH
’, CH2CH2CH2NHCOOCH2Ph),3.59
a
’,H-6 ’a,CH 2CH2CH2 NHCOOCH2Ph),
2CH2CH2bNHCOOCH2Ph),1.75 (m,2H,
CH2CH2CH2NHCOOCH2Ph) ppm; 13C NMR (300 MHz,CDCl 3): d=
156.7 (NHCOOCH2Ph),154.8 (NH CO),138.4 127.9 (30C,Ar-C),104.5
(C-1’),101.3 (C-1),95.9 (CCl 3),83.3 (C-4),82.1 (C-3 ’),74.8,74.7,73.7,
73.6,72.8 (5C,4îO CH2Ph,O CH2CCl3),74.2 (C-5),73.9 (C-5 ’),72.8 (C-
product 4 as a white powder (110 mg,76%). [ a]D =À48.9 (c=1.0 in
1
CH2Cl2); Rf =0.41 (hexane/EtOAc 2:1); H NMR (300 MHz,CDCl ): d=
3),72.7 (C-4 ’),71.3 (C-2 ’),69.6,68.6,67.4,66.8 (4C,C-6,C-6
’,
3
7.76 (d, J=3.0 Hz,2H,Ar-H),7.74 7.08 (m,31H,Ar-H),5.62 (d,
J=
OCH2CH2CH2,COO CH2Ph),57.6 (C-2),38.1 (OCH 2CH2CH2),29.8
8.3 Hz,1H,NH),5.25 (t,
CH2NHCOOCH2Ph),5.07 (s,2H,CH
9.5 Hz,1H,H-3),4.85 4.61 (m,6H,2îArC
H-1,H-1 ’,Fmoc-CH 2,2îArC H2),4.20 (t, J=6.3 Hz,1H,Fmoc-CH),
3.90 3.83 (m,5H,H-4,H-4 ’,H-6,H-6 ’a),3.80 3.62 (m,4H,H-2,H-5 ’,H-
J=6.5 Hz,1H,H-2
’),5.20 (s,1H,
(OCH2CH2CH2) ppm; HR MALDI-TOF MS: m/z: calcd for
2NHCOOCH2Ph),4.93 (t, J=
H2,Troc),4.58 4.17 (m,8H,
C54H61Cl3N2O14: 1066.3188; found: 1089.3102 [M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2-[[(2,2,2-tri-
chloroethoxy)carbonyl]amino]-3-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-fuco-
pyranosyl)-4-O-(3,4,6-tri-O-benzyl-2-O-(3,4-di-O-acetyl-2-O-benzyl-a-l-
6’b,C
CH2CH2CH2 NHCOOCH2Ph),3.39 3.15 (m,1H,H-3
1H,CH 2CH2CH2bNHCOOCH2Ph),2.81 2.15 (m,4H,OCOC
Lev),2.12 (s,3H,CH 2COCH3,Lev),1.72 (m,2H,
CH2CH2CH2NHCOOCH2Ph) ppm; 13C NMR (300 MHz,CDCl 3): d=
171.5 (OCOCH2CH2,Lev),156.8 (NH COOCH2Ph),154.9
H2CH2CH2NHCOOCH2Ph),3.60 3.41
(m,2H,H-5,
’),3.26 3.18 (m,
H2CH2,
fucopyranosyl)-b-d-galactopyranosyl)-b-d-glucopyranoside (8):
A
solu-
a
tion of compound (50 mg,0.05 mmol) and compound
6
7 (72 mg,
0.19 mmol) was dried azeotropically with toluene and then subjected to
high vacuum for 2 h. The mixture was dissolved in dry dichloromethane
(10 mL) and stirred at room temperature under argon in the presence of
activated molecular sieves for 30 min. The mixture was cooled to 08C
and NIS (46 mg,0.20 mmol) and TESOTf (4 mL,0.01 mmol) were added.
The reaction mixture was stirred at 08C for 30 min,diluted by dichloro-
methane (60 mL),and filtered through celite. The filtrate was washed
with aqueous sodium thiosulfate (15%,4 mL) and brine,dried over
MgSO4,and concentrated. The residue was purified by column chroma-
tography (silica gel,hexane/EtOAc 2:1) to give the product 8 as a white
(CHCH2COO),154.7 (NH CO),143.7 120.1 (42C,Ar-C),101.4 (C-1
’),
101.2 (C-1),95.7 (CCl ),80.5 (C-3 ’),77.3 (C-3),75.5 (C-4),74.9 (2C,C-5,
3
C-5’),74.6,73.8,73.6,72.0 (5C,4îO
(C-4),72.1 (C-2),70.1 (CH CH2CO,Fmoc),68.1,67.9,67.4,66.8 (4C,C-6,
C-6’, OCH2CH2CH2,COO CH2Ph),56.4 (C-2),46.8 ( CHCH2CO,Fmoc),
38.0 (2C,OCO CH2CH2,(Lev),OCH 2CH2CH2),30.1 (CH 2COCH3,Lev),
29.9 (OCH2CH2CH2),28.1 (OCOCH 2CH2,Lev) ppm; HR MALDI-TOF
CH2Ph,O CH2CCl3),73.4 (C-5),72.5
powder (45 mg,61%).
[
a]D =À87.0 (c=1.0 in CH2Cl2); Rf =0.31
(hexane/EtOAc 2:1); 1H NMR (300 MHz,CDCl 3): d=7.39 6.82 (m,
35H,Ar-H),5.54 (d, J=3.5 Hz,1H,H-1 ’’’),5.22 (s,1H,NH),5.39 4.13
(m,5H,H-1 ’’ (d, J=3.5 Hz),H-3 ’’,H-3 ’’’,H-4 ’’,H-4 ’’’),5.11 4.86 (m,2H,
MS: m/z: calcd for C74H77Cl3N2O18
: 1386.4237; found: 1409.4187
[M+Na]+.
3-[(N-Benzyloxycarbonyl)amino]propyl 6-O-benzyl-2-deoxy-2-[[(2,2,2-tri-
chloroethoxy)carbonyl]amino]-4-O-(3,4,6-tri-O-benzyl-2-O-levulinoyl-b-
H-1,H-5 ’’’),4.82 4.65 (m,3H,H-3,H-4,H-5
’’),4.49 (d,1H,H-1 ’),4.28
d-galactopyranosyl)-b-d-glucopyranoside (5): Compound
4
(100 mg,
3.86 (m,8H,H-2 ’,H-2 ’’,H-2 ’’’,H-4 ’,H-6,H-6 ’),3.39 3.05 (m,5H,H-5 ’,
H-5,H-3 ’,CH 2CH2CH2NHCOOCH2Ph),3.02 2.86 (m,1H,H-2),2.07,
2.06,1.97,1.96 (s,4H,4îCH 3CO),1.69 (s,1H,OCH 2CH2CH2),1.15 (d,
J=6.7 Hz,3H,H-6 ’’’),0.93 (d, J=6.5 Hz,3H,H-6 ’’) ppm; 13C NMR
0.072 mmol) was dissolved in a solution of triethylamine in dichlorome-
thane (5 mL,1:1 v/v). The reaction mixture was stirred at ambient tem-
perature under argon for 18 h and then concentrated to dryness under re-
duced pressure. The residue was purified by column chromatography
(silica gel,hexane/EtOAc 2:1) to give the product 5 as a white powder
(60 mg,84%). [ a]D =À71.2 (c=1.0 in CH2Cl2); Rf =0.21 (hexane/EtOAc
(300 MHz,CDCl 3): d=170.8,170.6,170.5,170.3 (4C,4îCH
3CO),156.7
(NHCOOCH2Ph),153.8 (NH CO),143.9 120.1 (42C,Ar-C),99.7 (C-1 ’),
99.4 (C-1),98.4 (C-1 ’’),97.6 (C-1 ’’’),95.7 (CCl 3),83.7 (C-3 ’),75.9 (C-5),
1
2:1); H NMR (300 MHz,CDCl 3): d=7.39 7.26 (m,25H,Ar-H),5.40 (d,
75.3 (C-3),74.7,74.0,73.3,73.0,72.8,71.9,71.6,71.2,(8C,6îO
OCH2CCl3, COOCH2Ph),74.6,74.1,73.7,72.6,72.4,72.1 (7C,C-2
CH2Ph,
’,C-2 ’’,
J=8.3 Hz,1H,NH),5.28 (t,
CH2NHCOOCH2Ph),5.08 (s,2H,CH
10H,4îArC H2,Troc),4.41 (m,1H,H-1
J=6.5 Hz,1H,H-2
’),5.12 (s,1H,
2NHCOOCH2Ph),4.92 4.30 (m,
’),4.36 (m,1H,H-1),
C-2’’’,C-4,C-4 ’,C-4 ’’,C-4 ’’’),73.7 (C-5 ’),70.9 (C-3 ’’’),70.0 (C-3 ’’),68.0,
65.0,64.8 (C-6,C-6 ’, OCH2CH2CH2),67.3 (C-5 ’’’),66.7 (C-5 ’’),60.0 (C-2),
3.90 3.83 (m,2H,H-4,H-6
’a),3.60 3.50 (m,7H,H-3,H-4,H-6,
35.2 (OCH2CH2CH2),25.0 (OCH CH2CH2),21.2,21.1,20.9,20.8 (4C,4î
2
H-5’, CH2CH2CH2NHCOOCH2Ph,), 3.49 3.35 (m, 5H, H-2, H-5,
H-3’,H-6 ’a,CH 2CH2CH2 NHCOOCH2Ph),3.26 3.18 (m,1H,
CH3CO),15.7 (C-6 ’’),15.5 (C-6 ’’’) ppm; HR MALDI-TOF MS: m/z:
a
calcd for C80H95Cl3N2O24: 1572.5340; found: 1595.5398 [M+Na]+.
3522
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10,3517 3524

Linker Effects on Lewisy Immunogenicity
3517 3524
Aminopropyl 2-deoxy-2-acetamido-3-O-a-l-fucopyranosyl-4-O-(2-O-a-l-
that had been restored with ddH2O (200 mL) to give the protein in the
conjugation buffer (sodium phosphate buffer (pH 7.2) containing EDTA
and sodium azide). The mixture was incubated for 2 h at room tempera-
ture and then purified by use of a Millipore centrifugal filter device with
a 10000 Da molecular cut-off. All centrifugations were made at 158C for
20 min,with spinning at 13 g. The reaction mixture was centrifuged off
and the filter was washed with 10 mm 2-[4-(2-hydroxyethyl)-1-piperazinyl]-
ethanesulfonic acid (HEPES) buffer (pH 6.5,3î200 mL). The filtrates
were checked for the presence of carbohydrate by TLC. The conjugate
was retrieved and taken up in 10mm HEPES buffer (pH 6.5,1 mL). The
average number of copies of Ley attached to mcKLH and BSA was deter-
mined to be 620 and 9,respectively,according to Dubois× phenol sulfuric
acid total-carbohydrate assay and Lowry×s protein-concentration test.
Conjugation of Ley derivative 11 to BSA BrAc: A solution of SBAP
(5 mg) in DMSO (40 mL) was added to a solution of BSA (2 mg) in
0.1 mm sodium phosphate buffer (pH 8.0) containing 0.1 mm EDTA
(200 mL). The mixture was slowly stirred for 1 h at room temperature and
then purified by using centrifugal filters with a molecular cut-off of
10000 Da. All centrifugations were performed at 158C for 20 min,with
spinning at 13 g. The reaction mixture was centrifuged off and the filter
was washed with conjugation buffer (2î200 mL). The activated protein
was retrieved by spinning at 13 g for 20 min at 158C and taken up in
0.1 mm sodium phosphate buffer (pH 8.0) containing 0.1mm EDTA
(200 mL). A solution of thiol derivative 11 (1.2 mg) in the conjugation
buffer (100 mL) was added to the activated protein and the mixture was
incubated at room temperature overnight. Purification was achieved by
using the centrifugal filters as described above for the KLH/BSA MI
Ley conjugates. This gave a glycoconjugate with nine Ley residues per
BSA as determined by the phenol sulfuric acid total-carbohydrate assay
and Lowry×s protein-concentration test.
fucopyranosyl)-b-d-galactopyranosyl-b-d-glucopyranoside
(9):
Zinc
(10 mg,0.15 mmol,nanosize powder) was added to a stirred solution of
tetrasaccharide 8 (40 mg,0.02 mmol) in acetic acid (2 mL). After 20 min,
the zinc was removed by filtering through celite and the filtrate was con-
centrated to dryness. The residue was dissolved in pyridine (2 mL) and
acetic anhydride (1 mL) and the mixture was stirred at room temperature
overnight,whereafter it was quenched by addition of methanol (2 mL).
The solution was diluted by dichloromethane (60 mL) and was washed
successively with 1m HCl solution,aqueous sodium hydrogencarbonate
(15%),and brine. The organic layer was dried over MgSO and concen-
4
trated. The obtained residue was dissolved in methanol (5 mL),then
sodium methoxide (1m in methanol) was added until the pH value
reached 10. The solution was stirred at room temperature for 24 h,neu-
tralized with Dowex 50 H+ resin,diluted with methanol (50 mL),filtered,
and concentrated. The residue was purified by column chromatography
(silica gel,EtOAc/methanol 10:1). The obtained compound was dissolved
in acetic acid (5 mL) and ethanol (1 mL). The mixture was hydrogenated
over Pd/C (10%,20 mg) at ambient temperature. After 24 h,the mixture
was filtered through celite to remove the catalyst and concentrated to
dryness under reduced pressure. The residue was purified by size-exclu-
sion column chromatography (Biogel P2 column,eluted with H 2O con-
taining 1% nBuOH) to give the product 9 as a white powder (9 mg,
52%). [a]D =À62.9 (c=1.0 in MeOH); 1H NMR (500 MHz,D 2O,30 8C):
d=5.20 (d, J=3.0 Hz,1H),5.02 (d, J=3.9 Hz,1H),4.79 (q, J=6.4 Hz,
1H),4.42 (d, J=8.0 Hz,2H),4.19 (q,
31H),3.36 (brs,1H),3.24 (m,1H),3.08 3.02 (m,2H),1.98 1.79 (m,
5H),1.16 (d, J=6.4 Hz,3H),1.12 (d,
J=6.4 Hz,3H) ppm; 13C NMR
J=6.4 Hz,1H),3.95 3.58 (brs,
(500 MHz,D 2O,30 8C): data of anomeric carbons: d=101.3,100.4,99.6,
98.8 ppm; HR MALDI-TOF MS: m/z: calcd for C29H52N2O19: 732.3164;
found: 755.2945 [M+Na]+.
BSA MI ME and BSA BrAc MI: A solution of 2-mercaptoethanol in
DMF (37 mL,1:100 v/v) was added to 3-(bromoacetamido)propionate-ac-
tivated BSA (2 mg) prepared as described above or (maleimidomethyl)-
cyclohexane-1-carboxylate-activated BSA (2 mg) from Pierce Endogen.
The mixture was incubated at room temperature overnight. The mercap-
toethanol conjugates were purified by using spin filters as described
above.
Conjugation of Ley derivative 11 to KLH BrAc: A solution of KLH
(2.3 mg) in 0.1m sodium phosphate buffer (pH 7.2) containing 0.15m
NaCl (200 mL) was added to a solution of SBAP (1 mg) in DMSO
(40 mL). The mixture was slowly stirred for 1 h at room temperature and
then purified by using centrifugal filters with a molecular cut-off of
10000 Da. All centrifugations were performed at 158C for 20 min,with
spinning at 13 g. The reaction mixture was centrifuged off and the filter
was washed with conjugation buffer (2î200 mL) The activated protein
was retrieved by spinning at 13 g for 20 min at 158C and taken up in
0.1 mm sodium phosphate buffer (pH 8.0) containing 0.1 mm EDTA
(200 mL). A solution of thiol derivative 11 (0.6 mg) in the conjugation
buffer (100 mL) was added to the activated protein and the mixture was
incubated at room temperature overnight. Purification was achieved by
using the centrifugal filters as described above for the KLH/BSA MI
Ley conjugates. This gave a glycoconjugate with 692 Ley residues per
KLH molecule as determined by the phenol sulfuric acid total-carbohy-
drate assay and Lowry×s protein-concentration test.
3-(S-Acetylthioglycolylamido)-propyl 2-deoxy-2-acetamido-3-O-a-l-fuco-
pyranosyl-4-O-(2-O-a-l-fucopyranosyl)-b-d-galactopyranosyl-b-d-gluco-
pyranoside (10): Compound
9 (6 mg,0.01 mmol) was dried under
vacuum overnight. The sugar was slurried in dry DMF. SAMA-OPfp
(5.3 mg,0.02 mmol) was added. TEA (2.3 mL,0.02 mmol) was added
dropwise into the mixture. After stirring at room temperature for 2 h,the
mixture was evaporated and the residue was purified by size-exclusion
chromatography (Biogel P2 column,eluted with H 2O containing 1%
nBuOH) to give thioacetate 10 as a white powder (4 mg,58%). 1H NMR
(300 MHz,D 2O,30 8C): d=5.21 (d, J=3.0 Hz,1H),4.95 (d, J=3.9 Hz,
1H),4.78 (q, J=6.4 Hz,1H),4.40 (d, J=8.0 Hz,2H),4.11 (q, J=6.4 Hz,
1H),3.85 3.51 (brs,33H),3.36 (brs,1H),3.24 (m,1H),3.08 3.02 (m,
2H),1.98 1.79 (m,8H),1.17 (d,
J=6.4 Hz,3H),1.12 (d,
J=6.4 Hz,
3H) ppm; HR MALDI-TOF MS: m/z: calcd for C33H56N2O21S: 848.3096;
found: 871.2983 [M+Na]+.
3-(Mercaptoacetamido)-propyl 2-deoxy-2-acetamido-3-O-a-l-fucopyrano-
syl-4-O-(2-O-a-l-fucopyranosyl)-b-d-galactopyranosyl-b-d-glucopyrano-
side (11): 7% NH3 (g) in DMF solution (50 mL) was added to a solution
of thioacetate 10 (1 mg) in H2O (15 mL) and the mixture was stirred
under argon atmosphere. The reaction was monitored by MALDI-TOF
MS,which showed the product peak of [ M+Na]+. After 45 min,the mix-
ture was evaporated under reduced pressure and coevaporated twice
with toluene. The thiol was dried under high vacuum for 30 min and then
used immediately for conjugation without further purification.
Thiolation of BSA: BSA (1.2 mg,0.015 mmol) in thiolation buffer con-
taining 50 mm triethanolamine,0.15 m NaCl,and 5 m m EDTA (pH 8.0,
200 mL) was incubated for 1.5 h with Traut×s reagent (1.08 mg,7.83 mmol).
The activated protein was purified on a d-salt dextran column (Pierce
Endogen) preequilibrated with 0.1m sodium phosphate buffer containing
0.15m NaCl and 0.1m EDTA (pH 7.2). The first fractions positive to Ell-
man×s reagent (total of approximately 0.560 mmol of thiol) were pooled
and used directly.
BSA MI Ley (RA): The pooled fractions of Ley derivative 12 and thiol-
activated BSA were mixed and incubated overnight under an argon at-
mosphere. The conjugate was purified by using centrifugal filters with a
molecular cut-off of 10 KDa. All centrifugations were performed at 13 g
for 30 min. The reaction mixture was centrifuged and the filter was
washed with 10 mm HEPES buffer (pH 6.5,2î200 mL). The glycoconju-
gate was retrieved (15 min at 13 g) and taken up in 10 mm HEPES buffer
(pH 6.5). The glycoconjugate had 9 copies of Ley per BSA as determined
3-[4-(N-Maleimidomethyl)cyclohexane-1-carbonylamino]-propyl
2-
deoxy-2-acetamido-3-O-a-l-fucopyranosyl-4-O-(2-O-a-l-fucopyranosyl)-
b-d-galactopyranosyl-b-d-glucopyranoside (12): A mixture of tetrasac-
charide 9 (1.2 mg,1.64 mmol) and sulfo-SMCC (1.07 mg,2.46 mmol) in
0.1m sodium phosphate buffer containing 0.15m NaCl (pH 7.2,600 mL)
was stirred at ambient temperature for 2 h. The compound was purified
by size-exclusion chromatography on a Sephadex G10 column equilibrat-
ed in 0.1m sodium phosphate buffer containing 5 mm EDTA (pH 6.2).
Fractions containing compound 12,as determined by TLC and MALDI-
TOF MS ([M+Na]+ 976.5),were pooled and used immediately without
further characterization for conjugation to thiolated BSA or KLH.
Conjugation of Ley derivative 11 to mcKLH MI and BSA MI: The con-
jugations were performed as suggested by Pierce Endogen. In short,thiol
11 (2.5 equiv excess to available MI groups on the protein),deprotected
just prior to conjugation as described above,was dissolved in ddH 2O
(100 mL) and added to a solution of maleimide-activated protein (2 mg)
Chem. Eur. J. 2004, 10,3517 3524
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¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
3523

G.-J. Boons et al.
FULL PAPER
by using Dubois× carbohydrate assay and Lowry×s protein-concentration
test.
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KLH MI Ley RA: Pooled fractions of Ley derivative 12 (4.92 mmol),pre-
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Acknowledgement
The authors are grateful for financial support for this work by the Na-
tional Cancer Institute of the National Institutes of Health (Grant
no. RO1 CA88986). We thank Dr. Liliana Jaso-Friedmann at University
of Georgia for helpful discussions regarding the immunization protocol
and the immunoassays.
Received: January 21,2004
Published online: May 26,2004
3524
¹ 2004 Wiley-VCH Verlag GmbH & Co. KGaA,Weinheim
www.chemeurj.org
Chem. Eur. J. 2004, 10,3517 3524