Synthesis of laminarin fragments and evaluation of a β-(1,3) glucan hexasaccaride-CRM197 conjugate as vaccine candidate against Candida albicans

Article abstract of DOI:10.1080/07328303.2011.604453

Laminarin-CRM₁₉₇ glycoconjugates were previously demonstrated to be immunogenic and confer protection against Candida albicans in mice. Laminarin consists of β-(1,3) glucan repeating units, with sporadic β-(1,6) branches. A set of short glucans was used to study the effect of the β-(1,6) branch on the antigenicity of linear β-(1,3) glucans. A linear β-(1,3) glucan hexasaccharide was selected as the best fragment able to inhibit the binding between laminarin and antilaminarin antibodies. The hexamer was then conjugated to CRM₁₉₇ and induced, in mice, significant titers of specific antilaminarin antibodies comparable with those raised by the Lam-CRM₁₉₇ conjugate. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/07328303.2011.604453

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Synthesis of Laminarin Fragments
and Evaluation of a β-(1,3) Glucan
Hexasaccaride-CRM197 Conjugate as
Vaccine Candidate against Candida
albicans
Roberto Adamo a , Marta Tontini a , Giulia Brogioni a , Maria Rosaria
Romano a , Gabriele Costantini a , Elisa Danieli a , Daniela Proietti a
Francesco Berti a & Paolo Costantino a
,
a Novartis Vaccines & Diagnostics Research Center , Via Fiorentina 1,
53100, Siena, Italy
Published online: 09 Nov 2011.
To cite this article: Roberto Adamo , Marta Tontini , Giulia Brogioni , Maria Rosaria Romano , Gabriele
Costantini , Elisa Danieli , Daniela Proietti , Francesco Berti & Paolo Costantino (2011) Synthesis of
Laminarin Fragments and Evaluation of a β-(1,3) Glucan Hexasaccaride-CRM197 Conjugate as Vaccine
Candidate against Candida albicans , Journal of Carbohydrate Chemistry, 30:4-6, 249-280, DOI:
10.1080/07328303.2011.604453
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Journal of Carbohydrate Chemistry, 30:249–280, 2011
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2011.604453
Synthesis of Laminarin
Fragments and Evaluation
of a β-(1,3) Glucan
Hexasaccaride-CRM197
Conjugate as Vaccine
Candidate against Candida
albicans
Roberto Adamo, Marta Tontini, Giulia Brogioni,
Maria Rosaria Romano, Gabriele Costantini, Elisa Danieli,
Daniela Proietti, Francesco Berti, and Paolo Costantino
Novartis Vaccines & Diagnostics Research Center, Via Fiorentina 1, 53100 Siena,
Italy
Laminarin-CRM197 glycoconjugates were previously demonstrated to be immunogenic
and confer protection against Candida albicans in mice. Laminarin consists of β-(1,3)
glucan repeating units, with sporadic β-(1,6) branches. A set of short glucans was used
to study the effect of the β-(1,6) branch on the antigenicity of linear β-(1,3) glucans. A
linear β-(1,3) glucan hexasaccharide was selected as the best fragment able to inhibit
the binding between laminarin and antilaminarin antibodies. The hexamer was then
conjugated to CRM197 and induced, in mice, significant titers of specific antilaminarin
antibodies comparable with those raised by the Lam-CRM197 conjugate.
Keywords Candida albicans; Glycoconjugate vaccine; Glucans; Delevulinoylation
INTRODUCTION
Deep-seated mycoses represent a serious medical problem, especially in noso-
comial settings where mortality due to Candida and Aspergillus invasive
Received May 9, 2011; accepted July 7, 2011.
Address correspondence to Paolo Costantino, Novartis Vaccines & Diagnostics Research
Center, Via Fiorentina 1, 53100 Siena, Italy. E-mail: paolo.costantino@novartis.com
249

R. Adamo et al.
250
infections is approaching 40%.[1] Antifungal therapy is limited by drug toxi-
city and emergence of resistant strains.[1]
Mucosal infections are also common in nonimmunocompromised subjects;
for example, vulvovaginal candidiasis affects a substantial percentage of all
women (3% to 5%), and often requires chronic antimycotic treatment.[2]
A
prophylactic and/or therapeutic vaccine is increasingly being considered as
one of the most appropriate options to meet the current pressing medical
need.[3]
Fungi are characterized by a cell wall rich in carbohydrates in the form
of polysaccharides or glycoproteins[4]; in particular, glucans containing both
β-(1,3) and β-(1,6) sequences are highly conserved in most if not all pathogenic
fungi, and therefore have been considered as an attractive target for vaccine
development. The first evidence of the possible role of fungal glucans as pro-
tective antigens was reported by Bromuro et al.,[5] who showed that intact C.
albicans cells treated to expose glucans on their surface conferred significant
anti-Candida protection in a mouse model. Subsequent work was then under-
taken in order to further explore this hypothesis. Because of their well-known
suboptimal immunogenicity, polysaccharides are generally conjugated to car-
rier proteins in order to transform them into T-dependent antigens able to elicit
a response characterized by the prevalence of IgG antibodies and immunologi-
cal memory.[6]
Considering possible technical issues associated with the large-scale ex-
traction and purification of β-glucans from pathogenic fungi, we addressed the
question of whether other biological sources of this type of molecules could be
used. In particular, we focused our attention on laminarin (Lam), a β-(1,3) glu-
can with sporadic β-(1,6) branches, extracted from the brown alga Laminaria
digitata. We showed that when Lam is conjugated to CRM197, a nontoxic mu-
tant of diphtheria toxin[7] that has been extensively used as a carrier protein
for a number of commercially available glycoconjugate vaccines, the resulting
Lam-CRM197 conjugate is both immunogenic and protective, in a mouse chal-
lenge model, against infections induced by C. albicans.[8]
Another carbohydrate antigen obtained specifically from C. albicans N-
glycoproteins is represented by mannans, which have been extensively ex-
ploited by other groups for their potential application in the preparation of
a fully synthetic conjugate vaccine against candidiases.[9,10]
Indeed, the advantage of targeting β-glucans is represented by their highly
conserved presence across almost all pathogenic fungi and the stability of their
expression substantially in the entire life cycle of the fungal cells.[4]
In a more recent study we compared CRM197 conjugates of two synthetic
glucans, a linear one composed of 15 β-(1,3) repeating units (15 mer) and an
oligosaccharide with the same backbone but with two β-(1,6) arms spaced by
five repeats (17 mer). Only the linear structure conferred protection against
C. albicans.[11] In the same study we also observed that conjugates of CRM197

Synthesis and Immunogenicity of Laminarin Fragments
251
with oligosaccharides derived from the linear β-(1,3) glucan curdlan polysac-
charide were able to confer a significant level of protection against C. albicans
in mice. Additionally, it has been demonstrated by microarray analysis that
the protective IgG2b mAb named 2G8 specifically recognizes linear β-(1,3) glu-
can epitopes rather than β-(1,4) or β-(1,6).[12] This prompted us to assume that
the protective epitope in the C. albicans glucan cell wall is defined by a linear
β-(1,3) glucan.
The aim of this study was (a) to address more specifically the effect of β-
(1,6) branch on the antigenicity of linear β-(1,3) glucans and (b) to determine
what could be the minimal structure of β-(1,3) glucan oligosaccharides capable
of efficiently binding to antilaminarin antibodies and whether this structure is
immunogenic when conjugated to a carrier protein.
RESULTS AND DISCUSSION
Synthesis of β-glucans
With the scope of studying the role of β-(1,6) linkages in the immunodom-
inant epitope of laminarin, we initially synthesized as methyl glycosides four
laminarin fragments, the three glucans 1–3 reproducing the branching point
of the polysaccharide and the linear trisaccharide 4 (Fig. 1).
Numerous syntheses of β-(1,3) and β-(1,6) glucans have been reported, so
the preparation of the four oligosaccharides was achieved by adaptations of
literature methods[13–18] in a way that we used only the three monosaccharide
building blocks 9,[19] 8, and 12.[20] As a key building block for the introduction
of β-(1,3) and β-(1,6) linkages we synthesized from compound 7b the monosac-
charide 8, possessing two free hydroxyls in the 4 and 6 positions, which allow
regioselective glycosylation of the latter[21] and a levulinoyl ester to protect
position 3.
The levulinoyl group is commonly used in carbohydrate chemistry both
for orthogonal removal in the presence of other esters, such as acetates or ben-
zoates, and to act as a participating group in position 2 during the glycosylation
step.[22] Its cleavage is usually carried out with hydrazine acetate or hydrazine
buffered with acetic acid,[23–25] which are both carcinogenic reagents. Other
reagents, such as hydrogen sulfide at pH 7 or NaH in THF, are also reported,[26]
but they have not found large application in orthogonal deprotection. Our ef-
forts to develop a safer method than hydrazine for its selective removal led us
to test ethylenediamine-acetic acid as reagent. The deprotection of compound
7b proceeded very slowly at rt (longer than 24 h) even with four equivalents
of base and five equivalents of acid, but could be performed in a reasonable
time of 3 to 4 h at 50◦C, without migration of benzoate esters at the 2 position,
giving in nearly quantitative yield the known 3-hydroxy sugar 7a.[27]

R. Adamo et al.
252
Figure 1: Chemical structure of laminarin and synthetic fragments.
This preliminary result on compound 7b convinced us to use this method
during the synthesis of our glucans.
Compounds 2 and 3 were prepared from the common disaccharide building
block 10a, obtained by glycosylation of the diol 8 with thioglycoside 9 in the
presence of NIS-TfOH as promoters (Sch. 1).
The glycosylation reaction occurred with a higher regioselectivity (76%
yield) toward position 6 as previously reported with imidate donor.[21] A dou-
1
blet of doublets at 4.79 ppm in H NMR spectrum, assigned to H-2D with cou-
pling constants J1,2 = 8.1 Hz and J2,3 = 10.2 Hz, allowed to ascertain the
β-configuration of the newly formed glycosidic bond, as expected in the pres-
ence of a participating group at the C-2 position of donor 9.
After acetylation of the hydroxyl group at C-4, a downfield shift of H-4B
1
toward 5.0 ppm in the H NMR spectrum was observed, confirming that the
glycosylation had occurred at the C-6 position. A trisaccharide produced by

Synthesis and Immunogenicity of Laminarin Fragments
253
Scheme 1: Sequence of reactions leading to tetrasaccharide 2 and trisaccharide 3.
Reagents: (a) LevOH, DCC, DMAP, CH2Cl2, 95%; (b) 9:1 AcOH-H2O, 60◦C, 93%; (c) NIS, TfOH,
CH2Cl2, –60◦C, 80%; (d) Ac2O, py, 98%; (e) PdCl2, NaOAc, AcOH, 70%; (f) CCl3CN, DBU,
CH2Cl2, 90%; (g) TMSOTf, CH2Cl2, 75%; (h) H2NCH2CH2NH2.AcOH, CH2Cl2, 50◦C, 90%; (i)
NaOMe; (j) H2, 10% Pd-C, MeOH; (m) 9, NIS, TfOH, CH2Cl2, –40◦C, 75%.
simultaneous glycosylation of 4 and 6 hydroxyls was also isolated as a mi-
nor product (15% yield, as identified by NMR) from the crude mixture. At-
tempts to glycosylate compound 8 with either peracetylated or perbenzoylated
trichloroacetimidate donors and TMSOTf as promoter resulted in a remark-
ably lower yield due to the concomitant formation of the orthoester as a side
product.
The acetylated disaccharide 10b was converted into trichloroacetimidate
11b and glycosylated with acceptor 12 using TMSOTf as Lewis acid. The
1
generated β-anomeric center appeared in H NMR spectrum as a doublet at
4.4 ppm with J1,2 = 7.8 Hz.
The desired compound 3 was smoothly obtained from trisaccharide 13a
by acyl group removal followed by hydrogenation on 10% Pd-C. Delevulinoyla-
tion of trisaccharide 13a with ethylenediamine-acetic acid (90%) delivered the
OH-3B acceptor 13b, which was reacted with 9 to furnish tetrasaccharide 14
(Sch. 1).
The four anomeric centers were detected by 13C NMR analysis at
101.14, 100.81, 100.67, and 100.10 ppm, indicating that they all possessed
β-configuration.[15,24,28]
Deprotection of 14 by methanolysis and subsequent hydrogenation on 10%
Pd-C uneventfully afforded compound 2.
Trisaccharide 1 was assembled by glycosylation of the monosaccharide
acceptor 12 with thioglycoside 9, followed by hydrolysis of the benzylidene-
protecting group and regioselective glycosylation of the primary alcohol of 15b
to yield compound 16 (Sch. 2).
The coupling constants of the three anomeric protons (J1,2 = 8.1 Hz) un-
ambiguously indicated their β-configuration.

R. Adamo et al.
254
Scheme 2: Sequence of reactions leading to trisaccharide 1 and 4. Reagents: (a) 9,
NIS-TfOH, CH2Cl2, –60◦C, 82%; (b) TFA-H2O, CH2Cl2, 90%; (c) 9, NIS, TfOH, CH2Cl2, –60◦C, 93%;
(d) NaOMe, MeOH; (e) H2, 10% Pd-C, MeOH; (f) 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)-Iridium-hexafluorophosphate, THF; I2, H2O, 92%; (g) CCl3CN,
.
DBU, CH2Cl2, 90%; (h) 12, TMSOTf, CH2Cl2, 81%; (i) H2NCH2CH2NH2 AcOH, 50◦C, 95%; (j) 9,
NIS, TfOH, CH2Cl2, –40◦C, 80%; (m) NaOMe, MeOH; (n) H2, 10% Pd-C, MeOH.
For the construction of trisaccharide 4, compound 7b was deal-
lylated by isomerization with hydrogen-activated 1,5-cyclooctadiene-
bis(methyldiphenylphosphine)-Iridium(I)-hexafluorophosphate catalyst and
subsequent oxidative cleavage with I2, which allowed to obtain 7a in 92% yield
instead of the 60% provided by ally removal with PdCl2 catalyst. Following
treatment with trichloroacetonitrile and DBU yielded donor 17b (Sch. 2).
The preparation of 17b was accomplished through a more straight-
forward reaction scheme as compared to the preparation of a similar
trichloroacetimidate donor from ethylthio-2-O-benzoyl-4,6-di-O-benzylidene-
6-O-tertbutyldimethylsylil-β-D-glucopyranoside recently described by Mo et
al.[28] Conversion to trichloroacetimidate in their case was achieved by hydrol-
ysis of the thioethyl group accompanied by migration of the benzoyl ester to
the anomeric position, which needed a further step to rearrange the product
to the desired l-hydroxyl derivative before final conversion into the desired
trichloroacetimidate.
After glycosylation of 12 with donor 17b, the synthesized disaccharide 18a
was selectively deprotected at the OH-3B position by levulinoyl group removal
with ethylenediamine-acetic acid to be glycosylated with 9 and deliver trisac-
charide 19.
While conventional proton-proton coupling constants were found for the
two β-anomeric protons H-1A and H-1D (J1,2 = 7.5 and 8.1 Hz, respectively), a
smaller value (J1,2 = 5.8 Hz) was noticed for H-1B. This observation, however,

Synthesis and Immunogenicity of Laminarin Fragments
255
is consistent with reported coupling constants in similar β-(1,3)-protected oli-
goglucosides.[29] Acyl group removal and following hydrogenation on 10% Pd-C
eventually provided compound 4.
1H NMR analysis of deprotected fragments showed the presence of
anomeric protons belonging to β-(1,3) constructs at around 4.70 ppm with J1,2
ranging from 7.5 to 8.1 ppm, while the ones coming from β-(1,6) linkages fell at
4.50 ppm with J1,2 = 8.0 Hz, similarly to what was observed in the laminarin
spectrum.[30,31]
In addition, 13C NMR signal for the anomeric position C-1D involved in
β-(1,6) linkage was consistently at 103.6 ppm in all the prepared fragments,
while signals attributed to C-1 belonging to β-(1,3) linkages appeared at 103.5
τo 103.6 ppm for C-1C, at 103.5 ppm for C-1B, and 103.2 to 103.4 ppm for C-
1A.[15,32]
Since a preliminary competitive ELISA provided the information that the
linear trisaccharide was a better inhibitor than the branched oligosaccharides,
we synthesized the linear tri- and hexasaccharide 25 and 26, respectively, as
allyl glycosides[33] in view of possible conjugation to carrier protein. For this
purpose we used the approach described by Huang et al.,[32] except that levuli-
noyl ester was introduced for orthogonal removal (Sch. 3).
Glycosylation of monosaccharide 20 with donor 17b promoted by TMSOTf
took place in 75% yield. After delevulinoylation with our method (85%), trisac-
charide 22a was prepared through glycosylation of compound 21b with donor
1
17b (53%). Although the detection of J1,2 was not possible in the H NMR
spectrum, COSY and HMQC experiments allowed to assign the C-1B signal at
98.4 ppm, confirming the formation of a β-glycosidic bond.
Trisaccharide 22a was then subjected to levulinoyl ester removal to
provide acceptor 22b (82%), for which H-1B appeared as a doublet with
J1,2 = 4.4 Hz in the 1H NMR spectrum and as a signal at 97.9 ppm in
Scheme 3: Sequence of reactions leading to protected trisaccharide 25 and
.
hexasaccharide 26. Reagents: (a) 17b, TMSOTf, CH2Cl2, 75%; (b) H2NCH2CH2NH2 AcOH,
.
CH2Cl2, 50◦C, 85%; (c) 17b, TMSOTf, CH2Cl2, 53%; (d) H2NCH2CH2NH2 AcOH, CH2Cl2, 50◦C,
82%; (e) NaOMe, MeOH; (f) 9:1 AcOH-H2O, 50◦C; (g) 1,5-Cyclooctadiene-
bis(methyldiphenylphosphine)-Iridium-hexafluorophosphate catalyst, THF; I2, H2O, 70%; (h)
CCl3CN, DBU, CH2Cl2, 90%; (i) 22b, TMSOTf, CH2Cl2, 54%.

R. Adamo et al.
256
the 13C NMR spectrum as further validation of the assigned stereochem-
istry. Allyl cleavage of 22a with hydrogen activated 1,5-cyclooctadiene-bis
(methyldiphenylphosphine)-Iridium(I)-hexafluorophosphate catalyst followed
by oxidation with I2, which worked also in this case with higher efficiency
in comparison to single-step deallylation with PdCl2 (70% and 63% of yield,
respectively), afforded the donor precursor 23a. The foregoing 1-hydroxy
trisaccharide 23a was then transformed into donor 23b, which was used
for glycosylation of 22b to obtain the hexasaccharide 24 in 54% yield. The
β-configuration of all the anomeric protons could be derived from 13C NMR
analysis where all the anomeric peaks appeared in the range 101.3 to 97.3
ppm.
The utilization of a disaccharide trichloroacetimidate donor closely related
to trisaccharide 23, except for the use of a tert-butyldimethylsylil protection
at the C-3 position instead of levulinoyl ester, to couple with a 2-O-benzoyl-
4,6-O-dibenzylidene-protected disaccharide acceptor for building up a β-(1,3)
glucan tetramer has been reported to give a 4:3 mixture of α-/β-products.[28]
The problem was circumvented by the authors replacing the benzoyl group,
which failed to provide neighboring group participation with a 4-acethoxy-2,2-
dimethylbutanoate ester.
We did not observe any formation of α-glycosylation product in the synthe-
sis of hexasaccharide 24 from our trisaccharide synthons 22b and 23b.
Tri- and hexasaccharide 22a and 24 were deprotected by methanolysis un-
der Zemplen condition and subsequent acid hydrolysis to furnish 25 and 26,
respectively (for J1,2 see Experimental section).
For introduction of an amino group to be used for conjugation to protein,
the allyl functions of 25 and 26 were subjected to thiol-ene reaction with cys-
teamine under UV to afford 5 and 6, respectively (Sch. 4).[34]
Competitive ELISA
Preliminary competitive ELISA experiments on compounds 1–4 showed
that the linear trisaccharide 4 is the best inhibitor of the binding between Lam
and antilaminarin antibodies elicited in the mouse by a Lam-CRM conjugate.
This result supported our hypothesis and previous findings about the lin-
earity of the dominant epitope in C. albicans and suggested to us to focus
only on nonbranched oligosaccharides. In particular, considering that the 17
mer glucan, which failed to induce protection, contains β-(1,6) linkages sepa-
rated by five β-(1,3) glucoside units, the 6 mer seemed a promising target as a
short glucan. We therefore decided to synthesize both the trimer 5 and the hex-
amer 6 carrying an amino linker, suitable for conjugation to the carrier protein
(Sch. 3).
Competitive ELISA of compounds 1–4 and 6 was again performed on plates
coated with laminarin as described in the Experimental section. As shown in

Synthesis and Immunogenicity of Laminarin Fragments
257
Figure 2: Competitive ELISA of synthetic glucans 1–4 and 6. Meningococcal C
polysaccharide was used as negative control (neg). (See Fig. 1 for structures.)
Fig. 2, the hexasaccharide 6 was shown to be the best inhibitor, having an IC50
12-fold lower than trisaccharide 4 and 20-fold lower than tetrasaccharide 2.
Furthermore, compound 6 gave a 95% inhibition at a 4 mMol concentration,
while trisaccharide 4 at the same concentration reached an inhibition level of
85% and the β-(1,6)-branched tetrasaccharide 2 only 73%. The remaining two
trisaccharides 1 and 3 exhibit no inhibition at the assay concentration.
Conjugation to CRM197
The conjugation of hexasaccharide 6 was carried out in two steps: firstly,
the linker-equipped oligosaccharide was reacted with an excess of di-N-
hydroxysuccinimidyl adipate in DMSO (Sch. 4) so that formation of only a half
ester could be obtained (Sch. 4).[11] The half ester was then purified by precip-
itation from the reaction mixture with ethyl acetate and reacted with CRM197
in phosphate buffer (pH 7). Using a 30:1 molar ratio of carbohydrate/protein,
based on spectrophotometric determination of active ester in the oligosac-
charide sample, an average loading of 16.8 sugar chains was achieved as
determined by high-performance anion-exchange chromatography with pulsed
amperometric detection (HPAEC-PAD) and confirmed by MALDI-TOF mass
spectrometry analysis (Fig. 3).
The conjugation efficiency (57%) was higher than previously reported for
the linear 15 mer β-(1,3) glucan (7.5 carbohydrate chains/mol protein, 25% cou-
pling efficiency), although the same reaction stoichiometry was used.[11] This

R. Adamo et al.
258
Scheme 4: Preparation of deprotected trisaccharide 5 and hexasaccharide 6; synthesis of
glycoconjugate 6-CRM197. Reagents: (a) cysteamine, hv, H2O, MeOH; (b) EtN3,
disuccinimidyl adipate, DMSO; (c) CRM197, PBS pH = 7.
observation might indicate a better reactivity of shorter-chain-length oligosac-
charides. A similar trend has been observed for the coupling of Streptococcus
[35]
pneumonia type 3 synthetic oligosaccharides to CRM197
.
Immunogenicity of β-(1,3) Glucan Hexasaccharide-CRM197
Conjugate
Animal experimental guidelines set forth by the Novartis Animal Care De-
partment were followed in the conduct of all animal studies. The β-(1,3) glucan
Figure 3: MALDI-TOF spectra of CRM197 (A) and hexasaccharide(6)-CRM197 conjugate (B).

Synthesis and Immunogenicity of Laminarin Fragments
259
hexasaccharide-CRM197 conjugate was tested in Balb/c mice for its ability to
induce antilaminarin antibodies. For this purpose groups of eight mice were
immunized with a 5-µg carbohydrate-based dose of Lam- and Hexa-CRM197
conjugates, respectively. For Hexa-CRM197 a 1-µg dose was used as well, with
the aim of studying the dose effect. Vaccines were formulated with the ad-
juvant MF59, an oil in water emulsion, which was shown to be effective in
boosting both cellular and humoral immune response and is commonly used
for seasonal flu vaccination.[36] Adjuvant alone in phosphate buffer (PBS) was
used as negative controls.
Sera were analyzed by ELISA for their content of antilaminarin IgG. Lam-
inarin was directly coated onto the plates, determining specifically the binding
to the carbohydrate antigen, thus excluding any effect of the linker.
After three injections, the hexasaccharide conjugate, at both 1- and 5-µg
dosages, induced titers of specific antilaminarin IgG antibody significantly
higher as compared to Lam-CRM197 (geometric mean titers 18,101 vs. 5191;
p < 0.05), which induced a level of antibodies in the same range of those pre-
viously observed although with a different strain of mice.[11] Interestingly, as
shown in Fig. 4, the immunological response of the hexasaccharide conjugate
appeared to be more homogeneous in comparison with Lam-CRM197
.
The effect of saccharide chain length on glycoconjugate vaccine immuno-
genicity has been investigated in other independent studies, indicating that
chain length and degree of saccharide loading onto the carrier protein are
Figure 4: Antibody titers induced in mice by the synthetic β-(1,3) glucan
hexasaccharide-CRM197 conjugate formulated with adjuvant MF59. Horizontal and vertical
bars refer to the geometric means and 95% confidence interval, respectively.

R. Adamo et al.
260
probably two interconnected variables. In particular, glycoconjugates contain-
ing short oligosaccharides might work better at higher loading, while increas-
ing the oligosaccharide chain length the optimal glycosylation degree might
be lower.[37, 38] Noteworthy, the β-(1,3) glucan hexasaccharide-CRM197 conju-
gate tested in the present study has a loading degree of approximately 17 as
compared to a value of 7.5 for the Lam-CRM197 conjugate.
CONCLUSIONS
In a previous report we demonstrated that a linear 15 mer synthetic β-(1,3) glu-
can conjugated to CRM197 could be a vaccine candidate against C. albicans.[11]
Interestingly, the introduction of β-(1,6) branching in each of the five units re-
sulted in loss of protection against the fungus infection. On the other hand,
laminarin, whose conjugates with CRM197 resulted in conferring protection,
contains β-(1,6) branches that are statistically spaced by about 10 residues.[11]
Torosantucci et al.[12] showed, through glycoarray analysis, that an IgG murine
monoclonal antibody previously reported to control C. albicans and C. neofor-
mans infections in different animal models[39] specifically recognizes β-(1,3)
glucans oligomers. The authors observed the highest binding strength starting
from chain lengths of seven sugar residues, which correspond to six repeat-
ing units if we consider that the immobilization method used by the authors
results in more than 85% of the reducing sugar being in open form.[40]
To further investigate these findings by well-defined synthetic glucans,
we reported here an alternative synthesis of laminarin fragments based on
regioselective glycosylation at position 6 of 4,6-sugar diols by thioglycoside
donors for the construction of the β-(1,6) and novel orthogonal deprotection
of 3-O-levulinoylated building blocks with ethylenediamine-acetic acid for the
introduction of β-(1,3) linkages. Competitive ELISA assay on prepared glucans
showed that branching is likely not involved in C. albicans epitope recognition
as linear fragments possess higher capacity of inhibiting the interaction of lam-
inarin with anti-Lam-CRM197 antibodies with respect to the β-(1,6)-branched
ones.
In fact, the trisaccharide 4 proved to be a better inhibitor as com-
pared to the β-(1,6)-branched tetrasaccharide 2. In addition, the linear hex-
asaccharide 6 resulted in a better inhibitor than trisaccharide 4. With this
information in our hands, which is in line with findings of other authors,[12]
the synthetic linear hexamer was conjugated to CRM197 for further immuno-
genicity studies. The β-(1,3) glucan hexasaccharide-CRM197 conjugate raised
in mice a more homogeneous antibody response with IgG titers significantly
higher than the Lam-CRM197 vaccine. This outcome suggests that a linear hex-
asaccharide might be long enough to cover the C. albicans epitope.
Following the promising immunogenicity results here described, further
studies are planned to evaluate the protection evoked by the β-(1,3) glucan

Synthesis and Immunogenicity of Laminarin Fragments
261
hexasaccharide-CRM197 conjugate, in the context of a larger panel of glycocon-
jugate vaccine candidates.
EXPERIMENTAL SECTION
General Methods
All chemicals were of reagent grade and were used without further
purification. Reactions were monitored by thin-layer chromatography (TLC)
on Silica Gel 60 F254 (Merck); after exam under UV light, compounds were
visualized by heating with 10% (v/v) ethanolic H2SO4. In the workup pro-
cedures, organic solutions were washed with the amounts of the indicated
aqueous solutions, then dried with anhydrous Na2SO4 and concentrated under
reduced pressure at 30 to 50◦C on a water bath. Column chromatography
was performed on Silica Gel 60 (Sigma Aldrich, 0.040–0.063 nm). Unless
otherwise specified, a gradient 0 → 100% hexane-EtOAc was applied in a
Combiflash Rf (Teledyne-Isco) or Flash Master Plus (Biotage) instrument.
Solvent mixtures less polar than those used for TLC were used at the onset
1
of separation. H NMR spectra were measured at 300 MHz with a Varian
Unity Inova 300 or 400 MHz with a Bruker AvanceIII 400 spectrometer; δH
values are reported in ppm, relative to internal Me4Si (δH = 0.00, CDCl3
and CD3OD) or internal acetone (δH = 2.22, D2O); when not suppressed the
solvent peak for D2O was calibrated at 4.79 ppm. 13C NMR spectra were
measured at 75 MHz with a Varian Unity Inova 300 or 100 MHz with a
Bruker AvanceIII 400 spectrometer; δC values are reported in ppm relative
to the signal of CDCl3 (δC = 77.0, CDCl3), CD3OD (δC = 49.0, CD3OD), or
internal acetone (δC = 30.9, D2O). Assignments of NMR signals were made
by homonuclear and heteronuclear two-dimensional correlation spectroscopy,
run with the software supplied with the spectrometers. Sugar moieties in
the final compounds 1–4 and related building blocks are named using the
letters A, B, C, and D based on the tetrasaccharide structure, as depicted in
Fig. 1. For hexasaccharide 6 letters A–F are used starting from the aglycon.
Nuclei associated with the 3-(2-aminoethylthio)propyl linker are denoted
with a prime. Exact masses were measured by electron spray ionization
mass spectroscopy, using a Q-Tof micro Macromass (Waters) instrument.
Palladium chloride and 1,5-Cyclooctadiene-bis(methyldiphenylphosphine)-
Iridium-hexafluorophosphate catalyst were purchased from Sigma-Aldrich.
Hydrogenation reactions were performed by flow chemistry on an H-Cube
(Thalesnano) instrument.
General procedure for glycosylation with trichloroacetimidate donors
To a stirred solution of acceptor (1 mmol) and donor (1.2 mmol) in CH2Cl2
˚
(15 mL) containing activated 4 A MS (0.75 g), TMSOTf (0.1 mmol) was added

R. Adamo et al.
262
at 0◦C, under nitrogen atmosphere. The mixture was stirred for 30 min when
TLC (2:1 hexane-EtOAc) showed the reaction was complete. Then the mixture
was neutralized with triethylamine (0.1 µL) and filtered through a celite pad,
and the filtrate concentrated. Chromatography of the residue (hexane-EtOAc)
gave the desired product.
General procedure for delevulinoylation
To a solution of the 3-O-Lev oligosaccharide (1 mmol) in CH2Cl2 (25 mL),
ethylenediamine (0.26 mL, 4 mmol) and AcOH (0.29 mL, 5 mmol) were added
at 0◦C. A white solid was formed and the suspension was stirred for 5 to 6 h
at 50◦C, when the deprotection was complete (TLC, hexane-EtOAc 2:1). The
mixture was concentrated and chromatography of the residue (hexane-EtOAc)
yielded the delevulinoylated product.
General procedure for deallylation
A mixture of the 1-O-allyl compound (1 mmol) and 1,5-cyclooctadiene-
bis(methyldiphenylphosphine)-Iridium(I)-hexafluorophosphate catalyst (2 mg)
in dry THF (7 mL) was carefully degassed. The catalyst was activated under
hydrogen atmosphere for 2 min, until the catalyst turned from red to pale
yellow, and the reaction mixture was stirred at rt for 3 h. When NMR anal-
ysis of a small portion showed complete isomerization, water was added in
order to get a 4:1 THF:H2O mixture, followed by iodine (0.5 g, 2 mmol). Af-
ter 15 min (TLC, 1:1 hexane-EtOAc) the solution was diluted with water and
extracted with CH2Cl2 (3 × 10 mL). The organic layers were washed with a
freshly prepared 5% solution of Na2S2O3 until decoloration, dried (Na2SO4),
filtered, and concentrated. Chromatography of the residue (hexane-EtOAc) af-
forded the deallylated compound as a mixture of α/β anomers, which was used
for the next step.
Allyl 2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-α-D-glucopyranoside
7b. To a solution of 3-OH sugar 7a[26] (2.4 g, 5.8 mmol) in dichloromethane
(100 mL), levulinic acid (810 mg, 7 mmol) was added followed by DMAP
(850 mg, 7 mmol) and DCC (1.44 g, 7 mmol). The mixture was stirred for 3 h
when TLC (2:1 hexane-EtOAc) showed the reaction was complete. The mixture
was filtered, and the filtrate was partitioned with aq NaHCO3. Combined
organic layers were dried on MgSO4 and concentrated. Chromatography of
the residue with 5:1 hexane-EtOAc gave the 3-O-Lev product 7b (2.8 g, 94%).
1
[α]D = +77◦ (c = 0.6; CHCl3). H NMR (300 MHz, CDCl3) δ: 8.07–7.20 (m,
10 H, PhCH, PhCO), 5.88–5.75 (m, 2 H, CH2CH CH2, incl. t, 5.84, J = 9.9 Hz,
H-3), 5.56 (s, 1 H, PhCH), 5.31–5.09 (m, 4 H, H-2, CH2CH CH2, incl. s, 5.28,
J1,2 = 3.9 Hz, H-1), 4.34 (dd, J5,6a = 5.1, J6a,6b = 10.1 Hz, H-6a), 4.25–3.97
(m, 2 H, CH2CH CH2), 3.85–3.73 (m, 2 H, H-4,6b), 3.65–3.57 (m, 1 H, H-5),
2.65–2.50 (m, 4 H, CH2CH2), 2.00 (s, 3 H, CH3). 13C NMR (CDCl3, 75 MHz)
δ: 205.75 (CH2CO), 171.77 (2 C, CH2COO, CH3CO), 165.78 (PhCO), 136.86,

Synthesis and Immunogenicity of Laminarin Fragments
263
134.40, 133.13, 132.53, 131.01, 129.86, 129.78, 129.50, 127.29, 125.24, 124.99
(PhCO, PhCH, CH), 117.65 ( CH2), 101.44 (PhCH), 95.77 (C-1), 79.08 (C-4),
72.17 (C-2), 69.07 (C-3), 68.71 (CH2CH CH2), 68.65 (C-6), 62.56 (C-5), 37.87
(CH2CO), 29.53 (CH3), 27.3 (CH2COO). ESI HR-MS (C28H30O9): m/z = found
([M+H]+ 511.1924; calc 511.1968).
Allyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-2-O-benzoyl-
3-O-levulinoyl-α-D-glucopyranoside 10a. A solution of the 3-O-Lev monosac-
charide 7b (1.5 g, 2.9 mmol) in 90% AcOH (100 mL) was stirred overnight at
60◦C, when TLC (2:1 hexane-EtOAc, 1:4 CH2Cl2-EtOAc) showed the reaction
was complete. The mixture was coevaporated with toluene and the residue pu-
rified on silica gel with CH2Cl2-EtOAc 1:4, to afford 8 (1.15 g, 93%).
Allyl 2-O-benzoyl-3-O-levulinoyl-α-D-glucopyranoside 8. [α]D = +118◦ (c
= 0.1; CHCl3). 1H NMR (300 MHz, CDCl3) δ: 8.03–7.41 (m, 5 H, PhCO),
5.87–5.74 (m, 1 H, CH2CH CH2), 5.84 (m, 1 H, H-3), 5.29–5.04 (m, 4
H, H-2, CH2CH=CH2, incl. d, 5.21, J1,2 = 3.9 Hz, H-1), 4.34–3.96 (m, 2
H, CH2CH CH2), 3.91–3.70 (m, 5 H, H-4,5,6a,b, OH), 2.85–2.40 (m, 4 H,
CH2CH2), 2.11 (s, 3 H, CH3), 2.04 (br. s, 1 H, OH). ESI HR-MS (C21H26O9):
m/z = found ([M+H]+ 423.1667; calc 423.1655).
To a stirred solution of acceptor 8 (1.12 g, 2.64 mmol) and donor 9[19]
˚
(1.75 g, 3.17 mmol) in CH2Cl2 (100 mL) containing activated 4 A MS, NIS
(0.89 g, 3.96 mmol) was added at –60◦C, under nitrogen atmosphere, followed
by TfOH (60 µL). The mixture was stirred for 6 h at –60◦C, when TLC (1:1
hexane-EtOAc) showed the formation of the disaccharide. The mixture was
filtered through a celite pad, and partitioned with aq NaHCO3. Chromatogra-
phy of the concentrated organic layers with 5% CH2Cl2-EtOAc gave 1.76 g of
1
product 10a (76%). [α]D = +45◦ (c = 1.2; CHCl3). H NMR (300 MHz, CDCl3)
δ: 8.03–7.14 (m, 20 H, 3 × PhCH2, PhCO), 5.85–5.72 (m, 1 H, CH2CH=CH2),
5.60 (dd, 1 H, J2,3 = 9.0, J3,4 = 10.2 Hz, H-3B), 5.29–5.12 (m, 6 H, PhCH2,
CH2CH=CH2, incl. d, 5.19, J1,2 = 3.6 Hz, H-1B, dd, J2,3 = 10.2 Hz, H-2B),
4.85–4.45 (m, 6 H, 2 × PhCH2, incl. dd, 4.79, J1,2 = 8.1, J2,3 = 10.2 Hz, H-2D;
d, 4.52, H-1D), 4.20–3.54 (m, 12 H, CH2CH=CH2, H-3D,4B,D,5B,D,6aB,D,6bB,D
,
C
OH-4B), 2.75–2.40 (m, 4 H, CH2CH2), 2.07, 1.98 (2 s, 6 H, 2 × CH3CO). 13
NMR (CDCl3, 75 MHz) δ: 207.25 (CH2CO), 172.75 (2 C, CH2COO, CH3CO),
169.64 (CH3CO), 165.81 (PhCO), 138.12–125.14 (PhCO, PhCH, 3 × PhCH2,
=CH), 117.54 (=CH2), 100.60 (C-1D), 95.89 (C-1B), 82.79, 77.65, 75.07, 73.54,
72.43, 71.51, 70.63 (9 C, 3 × PhCH2, C-2B,D,3B,D,4B,D), 69.20, 68.33, 68.21 (5 C,
CH2CH=CH2, C-6B,D,5B,D), 38.26 (CH2CO), 29.56 (CH3CO), 28.32 (CH2COO),
20.88 (CH3CO). ESI HR-MS (C50H56O15): m/z = found ([M+K]+ 935.3203; calc
935.3256); found [M+Na]+ 919.3473; calc 919.3513).
Allyl 2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-4-O-acetyl-2-
O-benzoyl-3-O-levulinoyl-α-D-glucopyranoside 10b. A solution of the disac-
charide 10a (1.75 g, 1.95 mmol) in 9:1 Ac2O-pyridine (50 mL) was stirred

R. Adamo et al.
264
overnight. After TLC (1:1 hexane-EtOAc) showed the reaction was com-
plete, the mixture was co-concentrated with toluene and the residue purified
with hexane-EtOAc to give compound 10b in theoretically quantitative yield
(1.92 g). [α]D = +65◦ (c = 0.3; CHCl3). 1H NMR (400 MHz, CDCl3) δ: 8.02–7.16
(m, 20 H, 3 × PhCH2, PhCO), 5.82–5.65 (m, 2 H, CH2CH=CH2, incl. t, 5.71,
J = 9.8 Hz, H-3B), 5.24–5.10 (m, 3 H, CH2CH=CH2, incl. d, 5.20, J1,2 = 3.6
Hz, H-1B), 5.02–4.97 (m, 3 H, H-2B, 2D, 4B), 5.79–4.52 (m, 6 H, 3 × PhCH2),
4.40 (d, 1 H, J1,2 = 7.7 Hz, H-1D), 4.20–4.15 (m, 1 H, CH2aCH=CH2), 4.05–4.01
(m, 1 H, H-5B), 3.99–3.97 (m, 2 H, CH2bCH=CH2, H-6aB), 3.76–3.60 (m, 4 H,
H-3D,4D,6bB,6aD), 3.53–3.49 (m, 2 H, H-5D,6bD), 2.69–2.32 (m, 4 H, CH2CH2),
2.07, 1.98 (2 s, 9 H, 3 × CH3CO). 13C NMR (CDCl3, 100 MHz) δ: 205.81
(CH2CO), 171.69, 170.00 (2 C, CH2COO, CH3CO), 169.45, (2 × CH3CO), 165.64
(PhCO), 138.09–127.60 (PhCO, PhCH, 3 × PhCH2, =CH), 117.65 (=CH2),
101.10 (C-1D), 94.47 (C-1B), 82.85, 77.77 (C-3D,4D), 75.10 (5D), 75.00, 74.97,
73.40 (3 × PhCH2), 72.86, 71.57 (3 C, C-2B,2D,4B), 70.15 (C-3B), 68.70 (2 C,
C-3D,4D), 68.53 (CH2CH=CH2), 68.49 (C-5B), 68.07 (C-6D), 67.86 (C-6B), 37.66
(CH2CO), 29.64 (CH3CO), 27.90 (CH2COO), 20.88, 20.69 (2 × CH3CO). ESI
HR-MS (C50H61O21): m/z = found ([M+K]+ 977.3340; calc 977.3362), found
([M+Na]+ 961.3647; calc 961.3627).
2-O-Acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-4-O-acetyl-2-O-
benzoyl-3-O-levulinoyl-α-D-glucopyranosyl trichloroacetimidate 11b. The allyl
sugar 10b (1.8 g, 1.91 mmol) was dissolved in 20:1 AcOH/H2O (25 mL); then
NaOAc (4.2 g, 30.5 mmol) and PdCl2 (288 mg, 1.91 mmol) were added. The
reaction was monitored by TLC (1:1 hexane/EtOAc). The mixture was stirred
for 24 h, when TLC showed the reaction was complete. The mixture was
diluted with CH2Cl2 and filtered over celite, washed with satd NaHCO3 until
neutralization, dried (MgSO4), filtered, and concentrated. Purification by flash
chromatography (hexane-EtOAc) gave 1.35 g of product 11a (80%), which was
used for the next step.
The foregoing 1-OH disaccharide 11a (1.3 g, 1.52 mmol) was dissolved in
CH2Cl2 (20 mL) and CCl3CN (2 mL), to which DBU (60 µL) was added. Af-
ter 30 min the reaction was complete (1:1 hexane-EtOAc). The mixture was
concentrated and after purification with hexane-EtOAc, 1.4 g of product 11b
(92%) was obtained. 1H NMR (300 MHz, CDCl3) δ: 8.54 (s, 1 H, NH), 7.98–7.16
(m, 20 H, 3 × PhCH2, PhCO), 6.68 (d, 1 H, J1,2 = 3.6 Hz, H-1B), 5.79 (t, 1
H, J = 9.6 Hz, H-3B), 5.29 (dd, 1 H, H-2B), 5.18 (t, 1 H, J = 10.2 Hz, H-2D),
4.99 (t, 1 H, J = 9.0 Hz, H-4B), 4.80–4.52 (m, 6 H, 3 × PhCH2), 4.41 (d, 1
H, J1,2 = 8.1 Hz H-1D), 4.24–3.49 (m, 8 H, H-3D,4D,5B,D,6aB,D,6bB,D), 2.70–2.35
(m, 4 H, CH2CH2), 2.07, 2.04, 1.98 (3 s, 9 H, 3 × CH3CO). 13C NMR (CDCl3,
75 MHz) δ: 205.78 (CH2CO), 171.73 (2 C, CH2COO, CH3CO), 169.82, 169.56
(2 × CH3CO), 165.28 (PhCO), 160.43 (OCONH), 138.14–126.91 (PhCO, 3 ×
PhCH2), 100.84 (2 C, C-1B,D), 92.85 (q, CCl3), 82.85, 77.78, 75.15, 75.00, 73.446,

Synthesis and Immunogenicity of Laminarin Fragments
265
72.74, 71.55, 70.43 (9 C, 3 × PhCH2, C-2B,D,3B,D,4B,D), 69.74, 68.54, 67.89,
67.23 (5 C, CH2CH=CH2, C-5B,D,6B,D), 37.62 (CH2CO), 29.46 (CH3CO), 27.64
(CH2COO), 20.89, 20.66 (2 × CH3CO). ESI HR-MS (C50H54Cl3NO15): m/z =
found ([M+Na+H]2+ 1065.2462; calc 1065.2482).
Methyl
[2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-4-O-
acetyl-2-O-benzoyl-β-D-glucopyranoyl]-(1→3)-2-O-benzoyl-4,6-O-benzylidene-
β-D-glucopyranoside 13b. Reaction of the methyl glycoside 12[20] and
1
trichloroacetimidate donor 11b gave the trisaccharide 13a (73%). H NMR
(300 MHz, CDCl3) δ: 8.02–7.18 (m, 30 H, 3 × PhCH2, 2 ×PhCO, PhCH), 5.64
(s, 1H, PhCH), 5.21 (t, 1 H, J = 8.4 Hz, H-3B), 5.16 (d, 1 H, J1,2 = 8.7 Hz,
H-1B), 4.94 (t, 1 H, J = 9.0 Hz, H-2A), 4.84–4.73 (m, 3 H, H-2B,D,4B), 4.69–4.32
(m, 9 H, 3 × PhCH2, 6aA, incl. d, 4.44, J1,2 = 8.1 Hz, H-1D; d, 4.44, J1,2
=
7.8 Hz, H-1A), 4.24–3.49 (m, 12 H, H-3A,D,4A,D,5A,B,D,6aB,D,6bA,B,D), 3.67 (s, 3 H,
OCH3), 2.04, 1.99 (2 s, 6 H, 2 × CH3).
The delevulinoylation procedure was applied to 3-O-Lev disaccharide 13a,
yielding product 13b (91%). [α]D = +55◦ (c = 0.3; CHCl3). 1H NMR (300 MHz,
CDCl3) δ: 7.59–7.14 (m, 30 H, 3 × PhCH2, 2 × PhCO, PhCH), 5.62 (s, 1H,
PhCH), 5.31–4.75 (m, 5 H, H-1B,2A,B,D,4B), 4.65–4.22 (m, 4 H, 3 × PhCH2,
H-1A,D), 4.37 (dd, 1 H, J5,6a = 4.8, J6a,6b = 10.2 Hz, H-6aA), 4.30 (t, 1 H,
J = 9.0 Hz, H-3A), 4.04–3.35 (m, 15 H, H-3B,D,4A,D,5A,B,D,6aB,D,6bA,B,D, incl.
s, 3.41, OCH3), 2.07, 2.00 (2 s, 6 H, 3 × CH3). 13C NMR (CDCl3, 75 MHz)
δ: 170.51 169.57 (2 × CH3CO), 166.20 164.43 (2 × PhCO), 138.45–125.07
(2 × PhCO, PhCH, 3 × PhCH2), 102.11 (PhCH), 100.89, 100.58, 99.53 (C-
1A,B,D), 82.53, 78.93, 77.76, 76.36, 74.83, 74.82, 74.40, 73.96, 73.59, 73.29,
72.79, 71.49 (3 × PhCH2, C-2A,B,D,3A,B,D,4A,B,D), 71.03, 68.47, 68.13, 67.59, 66.43
(6 C, C-5A,B,D,6A,B,D), 56.80 (OCH3), 21.31, 20.75 (2 × CH3CO). ESI HR-MS
(C65H68O20): m/z = found ([M+Na]+ 1191.4202; calc 1191.4243).
Methyl
{[2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl]-(1→3)-[2-O-
acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl]-(1→6)-2-O-benzoyl-4-O-acetyl-β-
D-glucopyranoyl}-(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside
14. To a stirred solution of acceptor 13b (150 mg, 0.13 mmol) and donor 9 (96
˚
mg, 0.17 mmol) in CH2Cl2 (5 mL) containing activated 4 A MS, NIS (47 mg,
0.21 mmol) was added at –40◦C, under nitrogen atmosphere, followed by TfOH
(5 µL). The mixture was stirred for 30 min at –40◦C; then TLC (hexane-EtOAc
2:1) showed the reaction was complete. Then the mixture was filtered through
a celite pad and partitioned with aq NaHCO3. Chromatography of the concen-
trated organic layers (hexane-EtOAc) gave 150 mg of product 14 (77%). [α]D
=
1
+45◦ (c = 0.3; CHCl3). H NMR (300 MHz, CDCl3) δ: 7.72–7.09 (m, 45 H, 6 ×
PhCH2, 2 × PhCO, PhCH), 5.63 (s, 1 H, PhCH), 5.24–4.91 (m, 4 H, H-2A,B,C,D),
4.85–4.21 (m, 16 H, 6 × PhCH2, H-1A,B,C,D), 4.37 (dd, 1 H, J5,6a = 4.8, J6a,6b
=
10.2 Hz, H-6aA), 4.31–3.35 (m, 22 H, H-3A,B,C,D,4A,B,C,D,5A,B,C,D,6aB,C,D,6bA,B,C,D
,

R. Adamo et al.
266
incl. s, 3.36, OCH3), 2.07, 2.00, 1.98 (3 s, 9 H, 3 × CH3). 13C NMR (CDCl3,
75 MHz) δ: 169.67, 169.61, 169.44 (3 × CH3CO), 164.35 164.13 (2 × PhCO),
138.42–126.66 (2 ×PhCO, PhCH, 6 × PhCH2), 102.06 (PhCH), 101.14, 100.88,
100.67, 100.10 (C-1A,B,C,D), 82.86, 82.49, 78.75, 78.74, 77.77, 74.98, 74.67,
74.48, 74.46, 74.22, 73.56, 73.35, 73.32, 73.30, 73.29, 73.27, 72.18, 71.93
(20 C, 6 × PhCH2, C-2A,B,C,D3A,B,C,D,4A,B,C,D), 68.75, 68.41, 68.05, 66.45 (8 C,
C-5A,B,C,D,6A,B,C,D), 56.70 (OCH3), 21.37, 20.38, 20.37 (3 × CH3CO). ESI HR-MS
(C94H98O26): m/z = found ([M+Na]+ 1665.6268; calc 1665.6244).
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)-2-O-
benzoyl-4,6-O-benzylidene-β-D-glucopyranoside 15a. To a stirred solution of
acceptor 12 (100 mg, 0.26 mmol) and donor (151 mg, 0.26 mmol) in CH2Cl2
(5 mL) containing activated 4 A MS, NIS (88 mg, 0.4 mmol) was added under
˚
nitrogen atmosphere at –60◦C followed by TfOH (3 µL). The mixture was
stirred for 30 min at –60◦C, when it turned red. After 30 min the reaction was
finished and one major product was obtained (TLC hexane-EtOAc 2:1). Et3N (5
µL) was added, the mixture was filtered through a celite pad, and the filtrate
concentrated. Chromatography of the residue (98:2 → 95:5 toluene-acetone)
gave the disaccharide 15a (180 mg, 82%). [α]D = –6◦ (c = 0.7; CHCl3). 1H NMR
(300 MHz, CDCl3) δ: 8.04–7.10 (m, 25 H, 3 × PhCH2, PhCO, PhCH), 5.50 (s,
1 H, PhCH), 5.27 (t, 1 H, J = 7.8 Hz, H-2B), 4.93 (dd, 1 H, J1,2 = 7.8 Hz,
J2,3 = 9.6 Hz, H-2C), 4.68–4.34 (m, 9 H, 3 × PhCH2, H-6aB, incl. d, 5.54, H-1C;
d, 4.52, J1,2 = 8.4 Hz, H-1B), 4.15 (t, 1 H, J = 8.7 Hz, H-3B), 3.84–3.76 (m, 2
H, H-4B,6bB), 3.66–3.47 (m, 7 H, H-4C,5B,C,6aC, incl. s, 3.47, OCH3), 3.36–3.30
(m, 2 H, H-6bA, incl. t, 3.33, J = 9.3 Hz, H-3C), 2.04 (s, 3 H, CH3CO). 13C NMR
(CDCl3, 75 MHz) δ: 169.66 (CH3CO), 164.65 (PhCO), 138.35–125.11 (PhCO,
PhCH, 3 × PhCH2), 102.29 (PhCH), 101.91 (C-1B), 100.63 (C-1C), 82.93, 78.94,
77.98, 75.41, 74.98, 73.93, 73.58, 72.53 (C-2B,C,3B,C,4B,C, 3 × PhCH2), 68.92,
68.64 (C-6B,C), 66.70 (2 C, C-5B,C), 56.95 (OCH3), 20.34 (CH3CO). ESI HR-MS
(C50H52O13): m/z = found ([M+Na]+ 883.3266; calc 883.3306).
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→6)-[2-
O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)]-2-O-benzoyl-β-D-
glucopyranoside 16. To a solution of disaccharide 15a (120 mg, 0.14 mmol)
in CH2Cl2 (5 mL), TFA (100 µL) and water (20 µL) were added. The mixture
was stirred overnight, when TLC (9:1 CH2Cl2-acetone, 1:1 hexane-EtOAc)
showed the reaction was complete. The mixture was concentrated and
chromatographed with 95:5 CH2Cl2-acetone to yield the debenzylinated
disaccharide (95 mg, 88%).
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)-2-O-
1
benzoyl-β-D-glucopyranoside 15b. [α]D = +8◦ (c = 0.3; CHCl3). H NMR (300
MHz, CDCl3) δ: 8.05–7.13 (m, 20 H, 3 × PhCH2, PhCO), 5.18 (t, 1 H, J = 8.1

Synthesis and Immunogenicity of Laminarin Fragments
267
Hz, H-2B), 4.95 (t, 1 H, J = 8.1 Hz, H-2C), 4.75–4.42 (m, 8 H, 3 × PhCH2, incl.
4.46, d, J1,2 = 8.4 Hz, H-1C; 4.43, d, J1,2 = 8.1 Hz, H-1D), 3.95 (dd, 1 H, J5,6a
=
3.3, J6a,6b = 11.7 Hz, H-6aB), 4.82–4.45 (m, 12 H, H-3B,C,4B,C,5B,C,6aC,6bB,C
,
incl. s, 3.47, OCH3), 1.61 (br. s, 2H, 2 × OH), 1.48 (s, 3 H, CH3CO). ESI HR-MS
(C43H48O13): m/z = found ([M+Na]+ 790.3366; calc 790.3412).
To a stirred solution of acceptor 15b (80 mg, 0.10 mmol) and donor 7
˚
(73 mg, 0.12 mmol) in CH2Cl2 (5 mL) containing activated 4 A MS, NIS (34
mg, 0.15 mmol) was added at –60◦C, under nitrogen atmosphere, followed by
TfOH (1.5 µL). The mixture was stirred for 30 min at –60◦C, when it turned
red. After 30 min the reaction was complete (TLC, 9.5:0.5 CH2Cl2-acetone,
1:1 hexane-EtOAc). Triethylamine (3 µL) was added, the mixture was filtered
through a celite pad, and the filtrate concentrated. Chromatography of the
residue (9.5:0.5 toluene-acetone) gave the desired compound 16 (88 mg, 68%).
[α]D = +5◦ (c = 0.6 CHCl3).1H NMR (300 MHz, CDCl3) δ: 8.04–7.14 (m, 35 H,
6 × PhCH2, PhCO), 5.15 (t, 1 H, J = 8.7 Hz, H-2D), 5.04 (t, 1 H, J = 8.4 Hz, H-
2B), 4.94 (t, 1 H, J = 8.1 Hz, H-2C), 4.82–4.28 (m, 16 H, 6 × PhCH2, H-6aB, incl.
d, 4.70, J1,2 = 8.1 Hz, H-1C; d, 4.46, J1,2 = 8.1 Hz, H-1B; d, 4.34, J1,2 = 8.1 Hz,
H-1D), 3.76–3.41 (m, 17 H, H-3B,C,D,4 B,C,D,5 B,C,D,6aC,D,6bB,C,D, incl. s, 3.41,
OCH3), 1.96 (s, 3 H, CH3CO), 1.55 (s, 3 H, CH3CO). 13C NMR (CDCl3, 75 MHz)
δ: 169.44 (2 C, 2 × CH3CO), 164.67 (PhCO), 138.13–126.88 (PhCO, 6 × PhCH2),
101.65, 101.41, 101.24 (C-1B,C,D), 84.87, 82.77, 77.93, 77.65, 75.69, 75.01, 74.67,
73.57, 73.42, 73.38, 72.20, 71.38 (15 C, C-2B,C,D,3B,C,D,4B,C,D, 6 × PhCH2),
69.15, 68.99, 68.70, 68.55 (6 C, C-5B,C,D,6B,C,D), 56.59 (OCH3), 20.87, 20.88 (2
× CH3CO). ESI HR-MS (C72H77O19): m/z = found ([M+Na]+ 1269.5045; calc
1269.5035).
4,6-O-Benzylidene-2-O-benzoyl-3-O-levulinoyl-α-D-glucopyranosyl
trichloroacetimidate 17b. Allyl group was removed from sugar 7b to give
17a (92%). The foregoing 1-OH sugar 17a (2.09 g, 4.46 mmol) was dissolved
in CH2Cl2 (50 mL), to which trichloroacetonitrile (1.45 mL, 13.3 mmol) was
added followed by DBU (74 µL, 0.89 mmol). The mixture was stirred for
2 h and monitored by TLC (2:1 hexane-EtOAc). The mixture was concentrated
and chromatographed (hexane-EtOAc) to afford 2.1 g of trichloroacetimidate
17b (84%). 1H NMR (300 MHz, CDCl3) δ: 8.59 (s, 1 H, NH), 8.05–7.26 (m, 10 H,
PhCO, PhCH), 6.69 (d, 1 H, J1,2 = 3.9 Hz, H-1), 5.89 (t, 1 H, J = 9.6 Hz, H-3),
5.89 (s, 1 H, PhCH), 5.37 (dd, 1 H, J2,3 =9.9 Hz, H-2), 4.40 (dd, 1 H, J5,6a = 4.8,
J6a,6b = 10.2 Hz, H-6a), 4.23–4.08 (m, 1 H, H-5), 3.88 (t, partially overlapped,
1 H, J = 9.9 Hz, H-4), 3.83 (t, partially overlapped, 1 H, J = 10.5 Hz, H-6b),
2.64–2.55 (m, 4 H, CH2CH2), 1.98 (s, 3 H, CH3). 13C NMR (CDCl3, 75 MHz)
δ: 205.64 (CH2CO), 171.74 (2 C, CH2COO, CH3CO), 165.46 (PhCO), 160.70
(OCONH), 136.56, 134.44, 132.49, 131.06, 129.93, 129.06, 128.86, 127.51,
127.32, 125.01 (PhCO, PhCH), 101.51 (PhCH), 100.94 (C-1), 93.34 (q, CCl3),

R. Adamo et al.
268
78.40 (C-4), 70.93 (2 C, C-2,3), 68.81 (C-6), 65.17 (C-5), 37.85 (CH2CO), 29.53
(CH3), 27.86 (CH2COO). ESI HR-MS (C27H26Cl3NO9): m/z = found ([M+Na]+
636.0529; calc 636.0571).
Methyl 2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-
(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside 18a. Acceptor 12
and trichloroacetimidate donor 17b afforded disaccharide 18a (81%). [α]D
=
+2◦ (c = 0.8; CHCl3).1H NMR (300 MHz, CDCl3) δ: 7.78–7.24 (m, 20 H, 2 ×
PhCO, 2 × PhCH), 5.58, (s, 1 H, PhCH), 5.34 (s, partially overlapped, 1 H,
PhCH), 5.32 (t, partially overlapped, 1 H, J = 9.0 Hz, H-3B), 5.26–5.19 (m,
2 H, H-2A,2B), 4.95 (d, 1 H, J1,2 = 6.9 Hz, H-1B), 4.49 (d, 1 H, J1,2 = 7.2 Hz,
H-1A), 4.38 (dd, 1 H, J5,6a = 4.8, J6a,6b = 10.8 Hz, H-6aA), 4.24–4.09 (m, 3 H, H-
6aB,6bA,3A), 3.88–3.78 (m, 3 H, H-4A,B,6bB), 3.60–3.45 (m, 2 H, H-5A,B), 3.36 (s, 3
H, OCH3), 3.53–3.29 (m, 4 H, CH2CH2), 1.93 (s, 3 H, CH3CO). 13C NMR (CDCl3,
75 MHz) δ: 205.69 (CH2CO), 171.68 (2 C, CH2COO, CH3CO), 164.81, 164.50
(2 × PhCO), 137.06–124.99 (2 × PhCO, 2 × PhCH), 102.21 (PhCH), 101.33
(PhCH), 101.16 (C-1B), 100.53 (C-1A), 79.16, 78.41, 77.89, 73.11, 72.72, 71.93
(C-2A,B,3A,B,4A,B), 68.54, 68.50, (C-6A,B), 66.29, 66.14 (C-5A,B), 56.81 (OCH3),
37.77 (CH2CO), 29.54 (CH3), 27.88 (CH2COO). ESI HR-MS (C46H46O15):
m/z = found ([M+Na]+ 861.2739; calc 861.2734).
Methyl
2-O-acetyl-3,4,6-tri-O-benzyl-β-D-glucopyranosyl-(1→3)-2-O-
benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranoside 19. From 3-O-Lev disaccharide 18a com-
pound 18b was obtained (85%). [α]D = –4◦ (c = 0.1; CHCl3).1H NMR
(300 MHz, CDCl3) δ: 7.85–7.26 (m, 20 H, 2 × PhCO, 2 × PhCH), 5.58, 5.37 (2 s,
1 H each, 2 × PhCH), 5.25 (t, 1 H, J = 7.5 Hz, H-2B), 5.12 (t, 1 H, J = 7.2 Hz,
H-2A), 4.97 (d, 1 H, J1,2 = 7.2 Hz, H-1B), 4.52 (d, 1 H, J1,2 = 7.2 Hz, H-1A), 4.39
(dd, 1 H, J5,6a = 4.8, J6a,6b = 10.8 Hz, H-6aA), 4.24–4.18 (m, 2 H, H-6aB,3A),
3.89–3.36 (m, 10 H, H-3B,4A,B,5A,B,6bA,B, incl. s, 3.36, 3 H, OCH3).
To a stirred solution of the foregoing disaccharide acceptor 18b (140 mg,
0.19 mmol) and donor 7 (123 mg, 0.23 mmol) in CH2Cl2 (5 mL) containing
˚
activated 4 A MS, NIS (64 mg, 0.29 mmol) was added, under nitrogen atmo-
sphere, at –40◦C followed by TfOH (5 µL). The mixture was stirred for 30 min
at –40◦C; then TLC (hexane-EtOAc 2:1) showed the reaction was complete.
Then the mixture was filtered through a celite pad and partitioned with aq
NaHCO3. Chromatography of the concentrated organic layers (hexane-EtOAc)
gave 185 mg of product 19 (80%). [α]D = +5◦ (c = 0.3; CHCl3). 1H NMR
(300 MHz, CDCl3) δ: 7.78–7.05 (m, 35 H, 2 × PhCO, 2 × PhCH, 3 × PhCH2),
5.54 (s, 1 H, PhCH), 5.13 (t, partially overlapped, 1 H, J = 8.1 Hz, H-2A), 5.08
(t, partially overlapped, 1 H, J = 6.0 Hz, H-2B), 4.94 (s, 1 H, PhCH), 4.84 (t, 1 H,
J = 8.1 Hz, H-2C), 4.82 (d, 1 H, J1,2 = 5.8 Hz, H-1B), 4.31 (m, 9 H, 3 × PhCH2,
H-6aA, incl. 4.43, d, J1,2 = 7.5 Hz, H-1C; 4.32, d, J1,2 = 8.1 Hz, H-1A), 4.16–4.08

Synthesis and Immunogenicity of Laminarin Fragments
269
(m, 2 H, H-6aB,3A), 3.92–3.33 (m, 15 H, H-3B,C, H-4A,B,C,5A,B,C,6aC,6bA,B,C, incl.
s, 3.36, 3 H, OCH3), 1.94 (s, 3 H, CH3CO). 13C NMR (CDCl3, 75 MHz) δ:
171.68 (CH3CO), 169.71, 164.63 (2 × PhCO), 138.35–125.29 (2 × PhCO, 2
× PhCH, 3 × PhCH2), 102.21 (PhCH), 101.61 (PhCH), 100.86 (C-1B), 99.93,
99.47 (C-1A,C), 83.01, 79.01, 78.00, 77.78, 75.41, 74.96, 74.13, 73.83, 72.48,
70.35, (C-2A,B,C,3A,B,C,4A,B,C, 3 × PhCH2), 68.71, 68.69, 68.67 (C-6A,B,C), 66.49,
66.30, 66.01 (C-5A,B,C), 56.85 (OCH3), 21.94 (CH3CO). ESI HR-MS (C70H70O19):
m/z = found ([M+Na]+ 1237.4437; calc 1237.4409).
General procedure for deprotection of 13b, 14, 16, and 19
To a solution of oligosaccharide (0.1 mmol) in MeOH, 1 M NaOMe was
added until pH was alkaline. The mixture was stirred overnight, then neutral-
ized with Dowex H+. After filtration, the filtrate was concentrated and purified
on silica gel (hexane-EtOAc) to afford the deacetylated product (nearly quanti-
tative yield).
The foregoing oligosaccharide (0.1 mmol) was dissolved in MeOH (10 mL)
and flown in H-Cube apparatus with 10% Pd-C cartridge (flow = 0.5 mL/min,
t = 20◦C, time = 4–8 h), monitored by TLC (1:1 EtOAc-MeOH). The mixture
was concentrated and purified in silica gel (1:1 EtOAc-MeOH) to afford the
deprotected material, which was dissolved in MilliQ water, filtered through a
0.2-µm Nalgene filter, and freeze-dried (75% to 96%).
1
Compound 1: H NMR (400 MHz, D2O) δ: 4.72 (d, 1 H, J1,2 = 8.0 Hz,
H-1C), 4.50 (d, 1 H, J1,2 = 8.0 Hz, H-1D), 4.41 (d, 1 H, J1,2 = 8.0 Hz, H-
1B), 4.23–4.20 (m, 1 H, H-6D), 3.92–3.85 (m, 3 H, H-6bD, 6aB,C), 3.76–3.68 (m,
3 H, H-3B,6bB,C), 3.66–3.61 (m, 1 H, H-5D), 3.60–3.55 (m, 6 H, H-4B,C,D, incl.
s, 3.56, OCH3), 3.53–3.40 (m, 6 H, H-2B,3C,D,5B,C,D), 3.38 (t, 1 H, J = 9.2 Hz,
H-2C), 3.34 (t, 1 H, J = 9.1 Hz, H-2D). 13C NMR (CDCl3, 100 MHz) δ: 103.61
(C-1D), 103.45 (C-1C), 103.35 (C-1B), 84.93 (C-3B), 76.56, 76.44, 76.17, 76.08
(5 C, C-3C,D, C-4A,B,C), 75.15 (C-5D), 73.98 (C-2C), 73.63 (C-2D), 73.29 (C-2B),
70.11 (C-5B/C), 69.12 (C-6D), 68.49 (C-5B/C), 61.93 (2 C, C-6B,C), 57.98 (OCH3).
ESI HR-MS (C25H44O21): m/z = ([M+K]+ 557.1439; calc 557.1484); found
([M+Na]+ 541.1741; calc 541.1745). This compound was previously synthe-
sized,[41] but NMR data were not reported.
Compound 2: [α]D = –5◦ (c = 0.5; H2O). 1H NMR (400 MHz, D2O)
δ: 4.74 (d, 1 H, J1,2 = 7.7 Hz, H-1C), 4.72 (d, 1 H, J1,2 = 7.5 Hz, H-1B),
4.50 (d, 1 H, J1,2 = 8.0 Hz, H-1D), 4.40 (d, 1 H, J1,2 = 7.9 Hz, H-1A),
4.22–4.18 (m, 1 H, H-6D), 3.90–3.85 (m, 4 H, H-6bD,6aA,B,C), 3.80–3.63 (m, 6 H,
H-3A,C,6bA,B,C,5D), 3.63–3.54 (m, 5 H, H-2B,3B, incl. s, 3.56, OCH3), 3.52–3.37
(m, 9 H, H-2A,3D,4A,B,C,D,5B,C,D), 3.34 (t, 1 H, J = 9.2 Hz, H-2C), 3.29 (t, 1 H, J =
9.1 Hz, H-2D). 13C NMR (CDCl3, 100 MHz) δ: 103.62 (C-1D), 103.47 (C-1B,C),
103.35 (C-1A), 85.99 (C-3A), 85.85 (C-3B), 76.68, 76.55 (C-3C,D), 76.26, 76.22,
75.15, 75.18, (C-4A,B,C,D), 74.12 (C-5D), 73.82 (C-2C,D), 73.55 (C-2B), 73.26 (C-2A),
(C-2B), 69.91 (C-5A/B/C), 69.39 (C-6D), 68.95, 68.73 (C-5A/B/C), 61.35 (3 C,

R. Adamo et al.
270
C-6A,B,C), 57.88 (OCH3). ESI HR-MS (C25H44O21): m/z = found ([M+K]+
719.2016; calc 719.2012); found ([M+Na]+ 703.2325; calc 703.2273).
1
Compound 3: [α]D = –15◦ (c = 0.5; H2O). H NMR (400 MHz, D2O) δ:
4.68 (d, 1 H, J1,2 = 8.1 Hz, H-1B), 4.50 (d, 1 H, J1,2 = 8.1 Hz, H-1D), 4.40 (d,
1 H, J1,2 = 8.1 Hz, H-1A), 4.23–4.20 (m, 1 H, H-6aD), 3.94–3.82 (m, 3 H, H-
6bD,6aA,B), 3.75–3.62 (m, 3 H, H-3B,6bA,B), 3.69–3.63 (m, 1 H, H-5D), 3.56 (s,
3 H, OCH3), 3.54–3.38 (m, 9 H, H-2B,3A,D,4A,B,D,5A,B,D), 3.35 (t, 1 H, J = 9.1
Hz, H-2A), 3.29 (dd, 1 H, J3,4 = 9.0 Hz, H-2D). 13C NMR (CDCl3, 100 MHz) δ:
103.64 (C-1D), 103.59 (C-1B), 103.37 (C-1A), 86.03 (C-3B), 76.53, 76.25, 76.12,
76.06, (5 C, C-3A,B, C-4A,B,D), 75.46 (C-5D), 73.96 (C-2B), 73.80 (C-2D), 73.18
(C-2A), 70.18, 70.23 (C-5A/B), 69.39 (C-6D), 68.98 (C-5A/B), 61.34 (2 C, C-
6A,B), 57.88 (OCH3). ESI HR-MS (C19H34O16): found ([M+K]+ 557.1472; calc
557.1484); found ([M+Na]+ 541.1721; calc 541.1745).
1
Compound 4: H NMR (400 MHz, D2O) δ: 4.76 (d, 1 H, J1,2 = 8.4 Hz,
H-1C), 4.73 (d, 1 H, J1,2 = 7.7 Hz, H-1B), 4.40 (d, 1 H, J1,2 = 8.3 Hz, H-1A),
3.94–3.88 (m, 3 H, H-6aA,B,C), 3.78–3.69 (m, 5 H, H-3A,C,6bA,B,C), 3.56 (s, 3
H, OCH3), 3.53–3.34 (m, 9 H, H-2A,C,3B,4A,B,C,5A,B,C), 3.38 (dd, 1 H, J3,4 = 9.3
Hz, H-2B). 13C NMR (CDCl3, 100 MHz) δ: 103.64 (C-1C), 103.47 (C-1B), 103.19
(C-1A), 85.08, 84.85 (C-3A,C), 76.66 (C-3B), 76.29, 76.21, 76.05 (C-4A,B,C), 74.11,
73.90, 73.49 (C-2A,B,C), 70.23, 68.82, 68.77 (C-5A,B,C), 61.35 (3 C, C-6A,B,C), 57.90
(OCH3). ESI HR-MS (C19H34O16): m/z = ([M+K]+ 557.1513; calc 557.1484);
found ([M+Na]+ 541.1711; calc 541.1745). This compound was previously syn-
thesized,[13] but incomplete NMR data were reported.
Allyl
2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-
(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranoside 20. The general
procedure for glycosylation was applied to compounds 20 and 17b. Yield: 63%.
[α]D = –6◦ (c = 0.1; CHCl3). 1H NMR (400 MHz, CDCl3) δ: 7.76–7.25
(m, 20 H, 2 × PhCO, 2 × PhCH), 5.65–5.54 (m, 2 H, CH2CH=CH2,
incl. s, 5.58, PhCH), 5.34, (s, 1 H, PhCH), 5.29 (t, partially overlapped,
1 H, J = 9.6 Hz, H-3B), 5.27 (t, partially overlapped, 1 H, J = 8.2 Hz,
H-2A), 5.22 (t, partially overlapped, 1 H, J = 8.5 Hz, H-2B), 5.13–5.00
(m, 2 H, CH2CH=CH2), 4.94 (d, 1 H, J1,2 = 7.2 Hz, H-1B), 4.61 (d, 1
H, J1,2 = 7.8 Hz, H-1A), 4.37 (dd, 1 H, J5,6a = 4.9, J6a,6b = 10.5 Hz,
H-6aB), 4.24–4.15 (m, 3 H, H-6aA,3B,CH2aCH=CH2), 3.99–3.95 (m, 1 H,
CH2bCH=CH2), 3.87–3.81 (m, 2 H, H-4B,6bB), 3.77 (t, partially overlapped,
1 H, J = 9.6 Hz, H-4A), 3.71 (t, partially overlapped, 1 H, J = 10.2 Hz,
H-6bA), 3.56–3.43 (m, 2 H, H-5A,B), 2.49–2.33 (m, 4 H, CH2CH2), 1.94 (s, 3
H, CH3CO). 13C NMR (CDCl3, 100 MHz) δ: 205.69 (CH2CO), 171.69 (2 C,
CH2COO, CH3CO), 164.78, 164.47 (2 × PhCO), 137.06–125.99 (2 × PhCO,
2 × PhCH, CH=), 117.54 (=CH2), 101.34, 101.20 (2 × PhCH), 101.34 (C-1B),
100.05 (C-1A), 79.19 (C-4B), 78.45 (C-3A), 77.91 (C-4A), 73.21 (C-2A), 72.73
(C-2B), 71.95 (C-3B), 69.80 (CH2CH=CH2), 68.68 (C-6B), 68.52 (C-6A), 66.35

Synthesis and Immunogenicity of Laminarin Fragments
271
66.35, (C-5B), 66.13, (C-5A), 37.80 (CH2CO), 29.44 (CH3), 27.90 (CH2COO).
ESI HR-MS (C48H48O15): m/z = found ([M+Na]+ 887.2902; calc 887.2891).
Allyl
2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-
benzoyl-4,6-O-benzylidene-β-D-glucopyranoside 21b. Levulinic group was
removed from 21a as described above. Yield: 85%. White crystals from EtOAc,
m.p. 156–157◦C. [α]D = –15◦ (c = 1.0; CHCl3).1H NMR (400 MHz, CDCl3) δ:
7.83–7.26 (m, 20 H, 2 × PhCO, 2 × PhCH), 5.66–5.54 (m, 2 H, CH2CH=CH2,
incl. s, 5.55, PhCH), 5.35, (s, 1 H, PhCH), 5.28 (t, 1 H, J = 7.5 Hz, H-2A),
5.15–5.00 (m, 3 H, CH2CH=CH2, incl. t, 5.10, J = 7.5 Hz, H-2B), 4.95 (d,
1 H, J1,2 = 7.1 Hz, H-1B), 4.63 (d, 1 H, J1,2 = 7.5 Hz, H-1A), 4.37 (dd, 1 H, J5,6a
=
4.9, J6a,6b = 10.5 Hz, H-6aB), 4.24–4.18 (m, 3 H, H-3A,6bB, CH2aCH=CH2),
3.99–3.95 (m, 1 H, CH2bCH=CH2), 3.87–3.79 (m, 3 H, H-3B,4B,6bB), 3.70 (t,
partially overlapped, 1 H, J = 10.1 Hz, H-6bA), 3.66 (t, partially overlapped,
1 H, J = 9.2 Hz, H-4A), 3.58–3.52 (m 1 H H-5B), 3.42–3.35 (m, 1 H, H-5A), 2.35
(br. s, 1 H, OH-3B). 13C NMR (CDCl3, 100 MHz) δ: 165.56, 164.59 (2 × PhCO),
137.09–125.92 (2 × PhCO, 2 × PhCH, CH=), 117.57 (=CH2), 101.63, 101.40
(2 × PhCH), 100.23 (C-1B), 99.95 (C-1A), 80.46 (C-4A), 79.23 (C-4B), 79.91
(C-3A), 75.22 (C-2A), 73.45 (C-2B), 72.57 (C-3B), 69.77 (CH2CH=CH2), 68.73 (C-
6B), 68.58 (C-6A), 66.32 66.35, (C-5B), 66.00, (C-5A). ESI HR-MS (C43H42O13):
m/z = found ([M+H]+ 767.2756; calc 767.2702); found ([M+Na]+ 789.2518;
calc 789.2523); found ([M+K]+ 805.2308; calc 805.2262).
Allyl
2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-
(1→3)-2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-
4,6-O-benzylidene β-D-glucopyranoside 22a. The reaction was carried out
with 21b and 17b according to the general procedure for glycosylation. Yield:
1
53%. [α]D = –2◦ (c = 0.1; CHCl3). H NMR (400 MHz, CDCl3) δ: 7.86–7.21
(m, 30 H, 3 × PhCO, 3 × PhCH), 5.66–5.53 (m, 1 H, CH2CH=CH2), 5.49 (s,
1 H, PhCH), 5.40–5.35 (m, 2 H, PhCH, H-3C), 5.27 (t, 1 H, J = 8.6 Hz, H-2C),
5.14–5.01 (m, 4 H, CH2CH=CH2, H-2B, incl. d, 5.08, J1,2 = 7.4 Hz, H-1C),
4.92–4.88 (m, 2 H, H-2A, H-1B), 4.83 (s, 1 H, PhCH), 4.52 (d, 1 H, J1,2
=
7.5 Hz, H-1A), 4.32 (dd, 1 H, J5,6a = 4.7, J6a,6b = 10.4 Hz, H-6aA), 4.24–3.99
(m, 6 H, H-6aB,C, 6bA,3A,B,CH2aCH=CH2), 3.96–3.91 (m, 1 H, CH2bCH=CH2),
3.77 (t, 1 H, J = 9.5 Hz, H-4C), 3.73–3.51 (m, 5 H, H-4B,5B,C,6bB,C), 3.46–3.40
(m, 1 H H-5A), 3.35 (t, 1 H, J = 9.2 Hz, H-4A), 2.49–2.46 (m, 4 H, CH2CH2),
1.96 (s, 3 H, CH3CO). 13C NMR (CDCl3, 100 MHz) δ: 205.75 (CH2CO), 171.63
(2 C, CH2COO, CH3CO), 164.97, 164.52, 164.31 (3 × PhCO), 137.20–125.99
(3 × PhCO, 3 × PhCH, CH=), 117.44 (=CH2), 101.66, 101.20, 100.59 (3 ×
PhCH), 100.59 (C-1A), 99.87 (C-1C), 98.42 (C-1B), 78.71 (C-4A), 78.20 (C-4B),
77.80 (C-4C), 77.20 (C-3B), 75.00 (C-3A), 74.00 (C-2A), 72.72 (C-2B), 72.37
(C-2C), 71.95 (C-3C), 69.69, 68.63, 68.51 (4 C, CH2CH=CH2, C-6A,B,C), 66.30
(C-5A), 66.13, 65.48 (C-5B,C), 37.86 (CH2CO), 29.46 (CH3), 27.93 (CH2COO).

R. Adamo et al.
272
ESI HR-MS (C68H66O21): m/z = found ([M+Na]+ 1218.4049; calc 1218.4097);
found ([M+K]+ 1257.3718; calc 1257.3734).
2-O-Benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-(1→3)-
2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-O-
benzylidene-β-D-glucopyranosyl trichloroacetimidate 23b. The removal of allyl
group in trisaccharide 22a (1 g, 0.82 mmol) gave compound 23a (70%).
The 1-OH trisaccharide 23a (120 mg, 0.1 mmol) was dissolved in CH2Cl2
(5 mL), to which trichloroacetonitrile (30 µL, 0.3 mmol) was added followed by
DBU (3 µL, 0.02 mmol). The mixture was stirred for 1 h when TLC (2:1 hexane-
EtOAc) showed the reaction was finished. The mixture was concentrated and
chromatographed (hexane-EtOAc) to furnish 100 mg of trichloroacedimidate
23b (76%).
1H NMR (400 MHz, CDCl3) δ: 8.47 (s, 1 H, NH), 7.92–7.22 (m, 30 H, 3 ×
PhCO, 3 × PhCH), 6.43 (d, 1 H, J1,2 = 3.7 Hz, H-1A), 5.57 (s, 1 H, PhCH),
5.46–5.41 (m, 2 H, PhCH, incl. t, 5.43, J = 9.1 Hz, H-3C), 5.31 (t, 1 H, J =
8.7 Hz, H-2C), 5.16–5.11 (m, 2 H, H-2B, incl. d, 5.15, J1,2 = 7.2 Hz, H-1C),
5.07 (d, 1 H, J1,2 = 4.4 Hz, H-1B), 4.70 (s, 1 H, PhCH), 4.62 (dd, 1 H, J2,3
=
9.5 Hz, H-2A), 4.40 (t, 1 H, J = 9.7 Hz, H-3A), 4.33–3.94 (m, 5 H, H-3B,C,6aA,B,C),
4.08–3.88 (m, 2 H, H-5A,B), 3.79 (t, 1 H, J = 9.6 Hz, H-4C), 3.81–3.56 (m, 5 H,
H-4B,5C,6bA,B,C), 3.33 (t, 1 H, J = 9.3 Hz, H-4A), 2.52–2.51 (m, 4 H, CH2CH2),
1.96 (s, 3 H, CH3CO). ESI HR-MS (C67H62Cl3NO21): m/z = found ([M+Na]+
1344.2778; calc 1344.2778).
Allyl bis[2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)]-2-O-
benzoyl-4,6-O-benzylidene-β-D-glucopyranoside 22b. The general procedure
for delevulinoylation was followed with 22a. Yield: 82%. [α]D = +76◦ (c =
0.2; CHCl3).1H NMR (400 MHz, CDCl3) δ: 7.99–7.15 (m, 30 H, 3 × PhCO,
3 × PhCH), 5.67–5.57 (m, 1 H, CH2CH=CH2), 5.46 (s, 1 H, PhCH), 5.39 (s,
1 H, PhCH), 5.18 (t, 1 H, J = 7.9, H-3C), 5.15–4.99 (m, 5 H, CH2CH=CH2,
H-2B,C, incl. d 5.07, J1,2 = 7.4 Hz, H-1C), 4.92 (d, 1 H, J1,2 = 4.5 Hz, H-1B),
4.89 (d, 1 H, J = 8.4 Hz, H-2A), 4.64 (s, 1 H, PhCH), 4.50 (d, 1 H, J1,2
=
7.7 Hz, H-1A), 4.28 (dd, 1 H, J5,6a = 4.7, J6a,6b = 10.4 Hz, H-6aA), 4.22–3.99
(m, 5 H, H-6aB,C,3A,B,CH2aCH=CH2), 3.96–3.89 (m, 2 H, CH2bCH=CH2, incl.
t, 3.91, J = 9.0 Hz, H-4C), 3.69–3.42 (m, 6 H, H-4B,5B,C,6bA,B,C), 3.41–3.36 (m,
1 H, H-5A), 3.21 (t, 1 H, J = 9.3 Hz, H-4A). 13C NMR (CDCl3, 100 MHz) δ:
171.12 (CH3CO), 165.68, 164.65, 164.42 (3 × PhCO), 137.20–126.00 (3 ×
PhCO, 3 × PhCH, CH=), 117.38 (=CH2), 101.79, 101.64, 100.44 (3 × PhCH),
99.87 (C-1A), 98.14 (C-1C), 97.89 (C-1B), 80.68 (C-4B), 78.59 (C-4A), 77.53 (C-4C),
76.25 (C-3B), 74.61 (C-3C), 74.36 (C-3A), 74.16 (C-2A), 72.25, 72.32 (C-2B,C),
69.68 (CH2CH=CH2), 68.64, 68.51 (3 C, C-6A,B,C), 66.29 (C-5A), 65.96 (C-5B).
ESI HR-MS (C63H60O19): m/z = found ([M+Na]+ 1143.3581; calc 1143.3567).

Synthesis and Immunogenicity of Laminarin Fragments
273
Allyl
2-O-benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-
(1→3)-tetrakis[2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)]-
2-O-benzoyl-4,6-O-benzylidene β-D-glucopyranoside 24. Trisaccharide 22a
(1 g, 0.82 mmol) was subjected to allyl group removal according to the general
procedure, yielding compound 23a (70%).
The 1-OH trisaccharide 23a (120 mg, 0.1 mmol) was dissolved in CH2Cl2
(5 mL), to which trichloroacetonitrile (30 µL, 0.3 mmol) was added followed by
DBU (3 µL, 0.02 mmol). The mixture was stirred for 1 h when TLC (2:1 hexane-
EtOAc) showed the reaction was finished. The mixture was concentrated and
chromatographed (hexane-EtOAc) to furnish 100 mg of trichloroacedimidate
23b (76%).
2-O-Benzoyl-4,6-O-benzylidene-3-O-levulinoyl-β-D-glucopyranosyl-(1→3)-
2-O-benzoyl-4,6-O-benzylidene-β-D-glucopyranosyl-(1→3)-2-O-benzoyl-4,6-
O-benzylidene-α-D-glucopyranosyl trichloroacetimidate 23b. 1H NMR (400
MHz, CDCl3) δ: 8.47 (s, 1 H, NH), 7.92–7.22 (m, 30 H, 3 × PhCO, 3 ×
PhCH), 6.43 (d, 1 H, J1,2 = 3.7 Hz, H-1A), 5.57 (s, 1 H, PhCH), 5.46–5.41
(m, 2 H, PhCH, incl. t, 5.43, J = 9.1 Hz, H-3C), 5.31 (t, 1 H, J = 8.7 Hz,
H-2C), 5.16–5.11 (m, 2 H, H-2B, incl. d, 5.15, J1,2 = 7.2 Hz, H-1C), 5.07
(d, 1 H, J1,2 = 4.4 Hz, H-1B), 4.70 (s, 1 H, PhCH), 4.62 (dd, 1 H, J2,3
=
9.5 Hz, H-2A), 4.40 (t, 1 H, J = 9.7 Hz, H-3A), 4.33–3.94 (m, 5 H, H-3B,C,6aA,B,C),
4.08–3.88 (m, 2 H, H-5A,B), 3.79 (t, 1 H, J = 9.6 Hz, H-4C), 3.81–3.56 (m, 5 H,
H-4B,5C,6bA,B,C), 3.33 (t, 1 H, J = 9.3 Hz, H-4A), 2.52–2.51 (m, 4 H, CH2CH2),
1.96 (s, 3 H, CH3CO). ESI HR-MS (C67H62Cl3NO21): m/z = found ([M+Na]+
1344.2778; calc 1344.2778).
Glycosylation of acceptor 22b with compound 23b was performed accord-
ing to the general procedure, except that 0.5 mmol of TMSOTf was used, to
afford 24. Yield: 54%. [α]D = –3◦ (c = 0.1, CHCl3) 1H NMR (400 MHz, CDCl3) δ:
7.85–7.14 (m, 60 H, 6 × PhCO, 6 × PhCH), 5.66–5.57 (m, 1 H, CH2CH=CH2),
5.53 (s, 1 H, PhCH), 5.39 (t, 1 H, J = 9.0, H-3F), 5.38 (s, 1 H, PhCH), 5.28 (dd,
1 H, J1,2 = 8.3, J1,2 = 9.3 Hz, H-2C), 5.19 (t, J = 6.3 Hz, H-2B), 5.15–5.02 (m,
3 H, CH2CH=CH2, incl. d 5.06, J1,2 = 7.7 Hz, H-1F), 4.99 (d, 1 H, J1,2 = 6.0 Hz,
H-1B), 4.97–4.71 (m, 13 H, 4 × PhCH, H-1C-F,2A,C-F), 4.53 (d, 1 H, J1,2 = 7.8 Hz,
H-1A), 4.23 (dd, 1 H, J5,6a = 4.7, J6a,6b = 10.4 Hz, H-6aA), 4.23–4.06 (m, 9 H,
H-3A,B,6aB-F, CH2aCH=CH2), 3.98–3.89 (m, 5 H, CH2bCH=CH2, H-3C-F),
3.77 (t, 1 H, J = 9.3 Hz, H-4F), 3.73–3.40 (m, 16 H, H-4C-F,5A-F,6bA-F),
3.33 (t, 1 H, J = 8.5 Hz, H-4B), 3.25 (t, 1 H, J = 9.0 Hz, H-
4A), 2.52–2.35 (m, 4 H, CH2CH2), 1.96 (s, 3 H, CH3CO). 13C NMR
(CDCl3, 100 MHz) δ: 205.83 (CH2CO), 171.69 (2 C, CH2COO, CH3CO),
165.06, 164.67, 164.55, 164.49 (6 × PhCO), 137.22–126.06 (6 × PhCO,
6 × PhCH, CH=), 117.50 (=CH2), 101.96 (PhCH), 101.25 (C-1), 101.13
(PhCH), 100.96 (2 C, C-1), 100.77 (PhCH), 99.94 (C-1A), 99.16 (C-1F), 98.26
(PhCH), 97.28 (C-1B), 96.94 (PhCH), 96.79 (PhCH), 78.79 (C-4A), 78.36

R. Adamo et al.
274
(C-3), 78.27, 77.83, 77.40 (C-4), 74.94 (C-3A), 74.57, 74.27, 74.15, 74.00
(C-3), 73.01 (C-2A,C-F), 72.48 (C-2B,F), 72.02 (C-3F), 69.79 (CH2CH=CH2), 68.67,
68.58 (6 C, C-6A-F), 66.33, 66.13, 65.64, 65.47 (C-5A-F), 37.92 (CH2CO), 29.51
(CH3), 27.98 (CH2COO). ESI HR-MS (C128H120NO39): m/z = found ([M+K]+
2319.6982; calc 2319.7044); found ([M+Na]+ 2303.7092; calc 2303.7304)
General procedure for deprotection of 22a and 24
The allyl oligosaccharide (0.1 mmol) was dissolved in 90% AcOH (5 mL).
After stirring for 6 h at 50◦C the solvent was evaporated, and the material
was dissolved in MeOH (5 mL), to which a solution of 1 M NaOMe was added
until pH was strongly alkaline (TLC 9:1 CH2Cl2-MeOH). The mixture was
stirred overnight and then concentrated. The residue was purified on a C18
Isolute SPE column with H2O. Fractions containing the pure desired com-
pound (NMR) were combined and freeze-dried.
Allyl bis[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside 25. Yield: 92%.
[α]D = –28.2◦ (c = 1.9, CHCl3). 1H NMR (400 MHz, D2O) δ: 6.00–5.91 (m, 1 H,
CH2CH=CH2), 5.39–5.26 (m, 2 H, CH2CH=CH2), 4.76 (d, 1 H, J1,2 = 8.3 Hz,
H-1C), 4.73 (d, 1 H, J1,2 = 8.3 Hz, H-1B), 4.52 (d, 1 H, J1,2 = 8.3 Hz, H-1A),
4.39–4.35 (m, 1 H, CH2aCH=CH2), 4.23–4.18 (m, 1 H, CH2bCH=CH2), 3.90
(d, 3 H, J5,6 = 12.0 Hz, H-6aA,B,C), 3.78–3.66 (m, 5 H, H-3A,C,6bA,B,C), 3.53 (t,
J = 9.3 Hz, H-2C), 3.52–3.35 (m, 7 H, H-2A,4A,B,C,5A,B,C), 3.41–3.31 (m, 3 H,
H-2B,3A,C). 13C NMR (D2O, 100 MHz) δ: 133.82 (CH=), 119.40 (=CH2), 103.36
(C-1C), 103.12 (C-1B), 101.51 (C-1A), 84.96, 84.77, 76.58, 76.20, 76.13, 74.03,
73.82, 73.47, 71.28 (CH2CH=CH2), 70.15, 68.75, 68.70, 61.28 (3 C, C-6A-C). ESI
HR-MS (C21H36NO16): m/z = found ([M+Na]+ 567.1930; calc 567.1901).
Allyl pentakis[β-D-glucopyranosyl-(1→3)]-β-D-glucopyranoside 26. Yield:
1
93%. [α]D = –22.2◦ (c = 0.1, CHCl3). H NMR (400 MHz, D2O) δ: 6.00–5.91
(m, 1 H, CH2CH=CH2), 5.39–5.26 (m, 2 H, CH2CH=CH2), 4.76 (d, 4 H, J1,2
=
8.0 Hz, H-1B-E), 4.73 (d, 1 H, J1,2 = 7.8 Hz, H-1F), 4.52 (d, 1 H, J1,2 = 8.3 Hz,
H-1A), 4.40–4.39 (m,
H, CH2bCH=CH2), 3.90 (d,
1
H, CH2aCH=CH2), 4.24–4.19 (m,
1
6
H, J5,6
=
12.0 Hz, H-6aA-F),
3.79–3.68 (m, 11 H), 3.57–3.30 (m, 18 H). 13C NMR (D2O, 100
MHz) δ: 133.82 (CH=), 119.40 (=CH2), 103.40 (C-1F), 103.12
(4 C, C-1B-E), 101.51 (C-1A), 84.94, 84.77, 76.59, 76.20, 76.13, 74.03, 73.89,
73.84, 73.48, 71.26 (CH2CH=CH2), 70.16, 68.75, 68.66, 61.25 (6 C, C-6A-F). ESI
HR-MS (C39H67NO31): m/z = found ([M+H]+ 1054.3602; calc 1054.3564).
General procedure for thiol-ene reaction with cysteamine
To a solution of allyl oligosaccharide 25 or 26 (0.1 mmol) in CH2Cl2
(0.2 mL), cysteamine (0.3 mmol) dissolved in MeOH (0.2 mL) was added, and
the mixture was irradiated in a quartz vial under UV for 12 h. The residue was

Synthesis and Immunogenicity of Laminarin Fragments
275
purified on a G10 Sephadex column run in an Akta GE Instrument, using wa-
ter for elution. Fractions containing the pure desired compound (NMR) were
combined and freeze-dried.
3-(2-Aminoethylthio)propyl
bis[β-D-glucopyranosyl-(1→3)]-β-D-
glucopyranoside 5. Yield: 61%. 1H NMR (400 MHz, D2O) δ: 4.76 (d, 1 H,
J1,2 = 8.3 Hz, H-1C), 4.74 (d, 1 H, J1,2 = 8.1 Hz, H-1B), 4.49 (d, 1 H, J1,2
=
8.0 Hz, H-1A), 4.04–3.99 (m, 1 H, H-1ꢁa), 3.92 (d, 3 H, J5,6 = 12.0 Hz, H-6aA,B,C),
3.82–3.70 (m, 6 H, H-3A,C,6bA,B,C,1ꢁb), 3.55 (t, 1 H, J = 9.3 Hz, H-2C), 3.54–3.40
(m, 8 H, H-2A,3B,4A,B,C,5A,B,C), 3.36 (t, 1 H, J = 9.1 Hz, H-2B), 3.21 (t, 2 H,
J = 6.8 Hz, H-5ꢁ), 2.86 (t, 2 H, J = 6.5 Hz, H-4ꢁ), 2.70 (t, 2 H, J = 7.1 Hz, H-3ꢁ),
1.96–1.90 (m, 2 H, H-2ꢁ). ESI HR-MS (C23H43NO16S): m/z = found ([M+Na]+
622.2115; calc 622.2143).
3-(2-Aminoethylthio)propyl
pentakis[β-D-glucopyranosyl-(1→3)]-β-D-
glucopyranoside 6. Yield: 73%. 1H NMR (400 MHz, D2O) δ: 4.80–4.74 (m, 5 H,
H-1B-F), 4.48 (d, 1 H, J1,2 = 7.8 Hz, H-1A), 4.02–3.96 (m, 1 H, H-1ꢁa), 3.92 (d,
6 H, J5,6 = 12.0 Hz, H-6aA-F), 3.81–3.68 (m, 12 H, H-3A,C-F,6bA-F,1ꢁb), 3.56–3.38
(m, 18 H, H-2A,C-F,3B,4A-F,5A-F), 3.34 (t, 1 H, J = 8.8 Hz, H-2B), 3.19 (t, 2 H,
J = 7.1 Hz, H-5ꢁ), 2.84 (t, 2 H, J = 6.4 Hz, H-4ꢁ), 2.68 (t, 2 H, J = 7.1 Hz,
H-3ꢁ), 1.95–1.88 (m, 2 H, H-2ꢁ). H NMR (400 MHz, D2O) at 50◦C showed
1
the following anomeric signals, δ: 5.05 (d, 3 H, J1,2 = 8.0 Hz, 3 × H-1), 5.04
(d, 1 H, J1,2 = 5.3 Hz, H-1), 4.74 (d, 1 H, J1,2 = 8.1 Hz, H-1A). ESI HR-MS
(C41H73NO31S): m/z = found ([M+H]+ 1108.3982; calc 1108.3966).
Conjugation of 6 to CRM197
The amino-oligosaccharide 6 was dissolved in DMSO at a 4 mM amino
group concentration, and then triethylamine (10 equiv) and adipic acid N-
hydroxysuccinimide-diester (10 equiv) were added. The reaction was kept
under gentle stirring at rt for 2 h. The activated oligosaccharides were then
separated from the reagents by precipitation with 9 volumes of EtOAc, fol-
lowed by washing of the precipitate 10 times with EtOAc and drying under
vacuum.
Compound 27: ESI HR-MS (C51H85N2O36S): m/z = found ([M+H]+
1333.4535; calc 1333.4603).
Conjugation was carried out by combining the activated oligosaccharides
27 with CRM197 at a molar ratio of 30:1 (moles of active ester per moles of pro-
tein) in 70 mM phosphate buffer at pH 7.0. After reaction overnight at rt, the
conjugate was purified by ammonium sulfate precipitation (500 mg/mL). Pellet
was dissolved in 10 mM phosphate buffer at pH 7.0 and again precipitated with
ammonium sulfate (500 mg/mL). After repeating this operation twice, the solid
was washed twice with a solution of ammonium sulfate (500 mg/mL) in 10 mM
phosphate buffer at pH 7.0, and finally reconstituted with 10 mM phosphate
buffer at pH 7.0.

R. Adamo et al.
276
MALDI-TOF mass spectra of CRM197 and glycoconjugate 6-CRM197 were
recorded by an UltraFlex III MALDI-TOF/TOF instrument (Bruker Daltonics)
in linear mode and with positive ion detection. The samples for analysis were
prepared by mixing 2.5 µL of product and 2.5 µL of Super DHB matrix; 2.5 µL
of each mixture was deposited on a samples plate, dried at rt for 10 min, and
subjected to the spectrometer.
Determination of carbohydrate content in glycoconjugates
Total saccharide content in glycoconjugates was determined by ionic chro-
matography using a CarboPac PA1 column (50 mm × 250 mm) coupled to a
CarboPac PA1 guard column and connected to a Dionex ICS3000 system. Di-
alyzed samples, where free oligosaccharide has been already removed, were
diluted at 5 µg/mL of saccharide and treated with trifluoroacetic acid (TFA) at
a final concentration of 2 M, heated at 100◦C for 2 h, dried, dissolved in 0.5 mL
of distilled water, and filtered 0.45 µm before the analysis.
The separation was performed with a flow rate of 1 mL/min using isocratic
elution of 40 mM NaOH for 12 min, followed by a washing step with 500 mM
NaOH for 5 min. The chromatography was monitored using the pulsed ampero-
metric mode with a gold working electrode and an Ag/AgCl reference electrode;
a quadruple-potential waveform for carbohydrates was applied. The chromato-
graphic data were processed using Dionex Chromeleon software. Calibration
curve, treated as samples, was set up with glucose (Fluka) in the range 0.5 to
10.0 µg/mL.
Immunochemical Evaluation
ELISA analysis
Ninety-six-well Maxisorp plates (Nunc, Thermo Fisher Scientific) were
coated overnight at +4◦C with laminarin 5 µg per well in 0.05 M bicar-
bonate buffer at pH 9.6. After coating, the plates were washed three times
with 300 µL per well of TPBS (PBS with 0.05% Tween 20, pH 7.4) and
blocked with 100 µL/well of 3% BSA (Sigma-Aldrich) for 1 h at 37◦C.
Subsequently, each incubation step was followed by a triple TPBS wash.
Sera, prediluted 1:25–1:100 in TPBS, were transferred into coated-plates
(200 µL) and then serially two-fold diluted followed by 2 h incubation
at 37◦C. Then 100 µL/well of 1:10,000 in TPBS antimouse IgG alkaline
phosphatase conjugated (Sigma–Aldrich) were added and plates were incu-
bated for 1 h at 37◦C. Plates were then developed for 30 min at RT with
100 µL per well of
1
mg/mL p-nitrophenyl phosphate disodium
(Sigma–Aldrich) in 1 M diethanolamine (pH 9.8) and read at 405 nm
with a microplate spectrophotometer (Biorad).