Divergent and convergent synthesis of polymannosylated dibranched antigenic peptide of the immunodominant epitope MBP(83-99)

Article abstract of DOI:10.1016/j.bmc.2013.08.008

Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity. Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary synthetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope MBP(83-99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradiation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide mannan since mannosylation is a promising technique to achieve modulation in immune response. The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination protocol, prior to EAE induction in Lewis rats.

Full text of DOI:10.1016/j.bmc.2013.08.008

Bioorganic & Medicinal Chemistry 21 (2013) 6718–6725
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Divergent and convergent synthesis of polymannosylated
dibranched antigenic peptide of the immunodominant
epitope MBP(83–99)
f
Irene Friligou a,b,c,e, , Fabio Rizzolo b,c,d,e, , Francesca Nuti b,c,e, , Theodore Tselios a, , Maria Evangelidou ,
⇑
Mary Emmanouil f, Maria Karamita f, John Matsoukas a, Mario Chelli b,c,e, , Paolo Rovero b,c,g,
,
Anna Maria Papini b,c,d,e, ,
⇑
a Department of Chemistry, University of Patras, GR-26504 Patras, Greece
b Laboratory of Peptide & Protein Chemistry & Biology, Italy
c Laboratory of Peptide & Protein Chemistry & Biology, France
d c/o SOSCO – EA4505 Université de Cergy-Pontoise, F-95031 Cergy-Pontoise, France
e c/o Department of Chemistry ‘‘Ugo Schiff’’, University of Florence, I-50019 Sesto Fiorentino, Italy
f Laboratory of Molecular Genetics, Hellenic Pasteur Institute, GR-11521 Athens, Greece
g c/o Department of Neurosciences, Psychology, Drug Research and Child Health, University of Florence, I-50019 Sesto Fiorentino, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Multiple antigenic peptide (MAP) systems are dendrimeric structures bearing multiple copies of identical
or different peptide epitopes, and they have been demonstrated to show enhanced immunogenicity.
Herein, we report the direct (divergent) and indirect (convergent) synthesis, using contemporary syn-
thetic approaches, of a di-branched antigenic peptide (di-BAP) containing the immunodominant epitope
MBP(83–99), which is implicated in experimental autoimmune encephalomyelitis (EAE), an animal
model of multiple sclerosis (MS). The direct synthesis (di-BAP 1) was performed using microwave irradi-
ation. The indirect synthesis (di-BAP 2) was carried out performing an efficient chemoselective coupling
reaction through the formation of a thioether bond. Both di-BAPs were conjugated to polysaccharide
mannan since mannosylation is a promising technique to achieve modulation in immune response.
The conjugation was achieved through free amino groups of both di-BAPs via the formation of Schiff
bases. The mannan-conjugated di-BAPs were further evaluated in vivo in a prophylactic vaccination pro-
tocol, prior to EAE induction in Lewis rats.
Received 8 May 2013
Revised 17 July 2013
Accepted 5 August 2013
Available online 13 August 2013
Keywords:
Di-branched antigenic peptide
Experimental autoimmune
encephalomyelitis
Divergent synthesis
Convergent synthesis
Microwave-assisted synthesis
Mannosylation
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
induced in Lewis rats by immunization with encephalitogenic gui-
nea pig or rat MBP epitopes.3 More specifically, previous studies
Multiple sclerosis (MS) is a chronic inflammatory and demye-
linating disease of the central nervous system (CNS), believed to
be mediated by an autoimmune T cell response directed to pro-
teins of the myelin sheath, such as myelin basic protein (MBP), pro-
teolipid protein (PLP), and myelin oligodendrocyte glycoprotein
(MOG). Experimental autoimmune encephalomyelitis (EAE) is the
best studied animal model for MS. EAE is mediated by CD4+ T lym-
phocytes and shares many of the features seen in MS.1,2 EAE is in-
duced by immunization with myelin antigens emulsified in
complete Freund’s adjuvant (CFA). Depending upon the immuniza-
tion protocol and background of mice used, EAE can take an acute,
chronic progressive or relapsing–remitting course. EAE can be
have shown that the MBP(83–99) epitope is bound to the DR2 mol-
ecule expressed on inflammatory cells.4 EAE induction with MBP
peptides tends to induce EAE with considerable inflammation in
the CNS, but with little or no demyelination which is a hallmark
of human MS.5,6
A variety of therapeutic approaches have been developed in or-
der to modulate immune responses, such as the use of altered pep-
tide ligands (APLs) of immunodominant myelin epitopes in which
the primary TCR contact residues are substituted. APLs may sup-
press the EAE symptoms triggering different immunological
responses.7,8 Another approach in the induction of tolerance is
the administration of myelin or immunodominant myelin epi-
topes,9 such as Dirucotide. Dirucotide is a synthetic peptide with
a sequence corresponding to hMBP(82–98), which is the immuno-
dominant epitope in MS patients with HLA haplotype DR2 and/or
DR4. However, its further clinical use was terminated because it
did not meet the primary endpoint of delaying disease progression
⇑
Corresponding authors. Tel.: +30 2610 997905; fax: +30 2610 997118 (T.T.);
tel.: +39 055 4573561; fax: +39 055 4573584 (A.M.P.).
E-mail addresses: ttselios@upatras.gr (T. Tselios), annamaria.papini@unifi.it
(A.M. Papini).
www.peptlab.eu.
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.08.008

I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
6719
during the two-year MAESTRO-01 Phase III trial in patients with
secondary progressive multiple sclerosis (SPMS).10
It is evident that there is a compelling need for the development
of improved protocols for the treatment of MS. One current exper-
imental approach is to control this autoimmune disease through
immunization promoting the induction of T regulatory cells (Tregs)
synthesis, since it has been successfully applied as a fast and effi-
cient way to synthesize difficult and large peptide sequences.29–31
The indirect approach includes the separate synthesis of the
core and the peptide epitopes, which are coupled using a chemose-
lective reaction.23,28,32–34 The advantages of this approach are: (i)
the possibility of using partially protected or fully unprotected
fragments which present better solubility, even in aqueous solu-
tion, and (ii) the fragments can be purified in a step before the cou-
pling reaction, thus decreasing the possibilities of any by-products
formation.
or changing the immune response of patients from Th1 (IFN-
c and
TNF- ) to Th2 (IL-4, IL-5, IL-10 and TGF-b) via the secretion of anti-
a
inflammatory cytokines. Mannan derived from Saccharomyces
cerevisae is a polysaccharide (polymannose) which possesses a
backbone of
chains containing
a
(1?6)-linked
D
-mannose units substituted by side
(1?3)-linked -mannose units
In the present study, we report both the microwave-assisted di-
rect synthesis and, through a chemoselective reaction, the indirect
synthesis of a di-BAP, containing MBP(83–99), one of the main
immunodominant epitopes implicated in EAE and MS. The synthe-
sized di-BAPs were further conjugated to polysaccharide mannan
since mannosylation has been considered a promising technique
to achieve modulation of the immune response.35,36
a
(1?2)- and
a
D
(Fig. 1).11 Mannan has successfully been used as a carrier to target
T cell antigens to the mannose receptor (MR) of macrophages and
dendritic cells (DCs) leading to the induction of Th1 or Th2 im-
mune responses in cancer immunotherapy protocols.12–15
The low immune responses of peptides could be overcome
using a carrier protein such as bovine serum albumin (BSA), key-
hole limpet hemocyanin (KLH), tetanus toxoid (TT) and others,16
an approach which can present some disadvantages, such as the
production of irrelevant anti-protein carrier antibodies due to the
immunodominance of the carrier, and the insufficient stability
and sometimes altered antigenic properties of the peptide moiety
because of the conjugation process.17 A successful approach in
the increase of the immunogenicity of single peptides has been
the use of dendrimeric systems,18–20 such as multiple antigenic
peptide (MAP) systems. MAPs were developed by Tam21 and con-
tain an inner core matrix consisting of sequential lysine layers
(known as generations) or other bifunctional units, to which multi-
ple copies of identical or different22 peptide epitopes are attached.
Based on literature data, MAPs exhibit several advantages in com-
parison to the corresponding peptides or peptide polymers, due to
their high molar ratio of antigen to core molecule and their three-
dimensional structure: (i) they do not require further conjugation
to a carrier protein,21 (ii) they are more stable to proteolytic degra-
dation,23 (iii) they present enhanced immunogenicity,21,24–26 and
(iv) they may produce site-specific antibodies.27
2. Materials and methods
2.1. Materials
2-Chlorotrityl chloride polystyrene resin (1% DVB, 200–
400 mesh) was purchased from CBL-Patras (Patras, Greece). Wang
resin was purchased from Novabiochem (Bad Soden, Germany)
Fmoc-protected-amino acids were purchased from CBL-Patras (Pa-
tras, Greece) either from Novabiochem (Bad Soden, Germany). Sol-
vents and other reagents were purchased from Merck (Darmstadt,
Germany), Sigma–Aldrich (Steinheim, Germany), Fluka (Buchs,
Switzerland) and Panreac (Barcelona, Spain).
Mini-cleavages were performed on a Discover™ S Class single-
mode microwave reactor equipped with Explorer-48 autosampler
(CEM Corporation, Matthews, NC) using
a
frequency of
2450 MHz, while peptide synthesis was performed on a Liberty™
Microwave Peptide Synthesizer (CEM Corporation, Matthews, NC).
The determination of resin loading capacity was achieved spec-
trophotometrically using a UV/Visible Spectrophotometer Cary
4000.
Mannan (polymannose from Saccharomyces cerevisiae) was pur-
chased from Sigma–Aldrich Ltd (Steinheim, Germany) and the
PD-10 columns (Sephadex G-25 M) used for the purification of
mannan were purchased from Amersham Biosciences (Uppsala,
Sweden).
MAPs can be synthesized using a direct (divergent) or an indi-
rect (convergent) approach. In the direct approach, multiple copies
of peptide epitopes can be synthesized by standard stepwise solid-
phase peptide synthesis (SPPS) directly on the resin-bound core
using Boc chemistry,21 Fmoc chemistry,28 or
a combination
thereof.24 The introduction of microwave irradiation in peptide
synthesis can be useful even in the case of hindered MAPs
The Complete Freund’s Adjuvant (CFA) was purchased from Sig-
ma–Aldrich Ltd (Steinheim, Germany) and it was supplemented
with H37Ra Mycobacterium tuberculosis by Difco (Detroit, MI).
2.2. Methods
CH2
O
OH
2.2.1. Microwave-assisted solid-phase peptide synthesis (MW-
SPPS) of di-BAP 1
CH2OH
α(1,6)
O
OH
O
O
CH2
O
OH
OH
α(1,2)
Di-BAP 1 was prepared in solid phase on a Wang resin (1 mmol/
g) under microwave irradiation following the Fmoc/tBu strategy
(Scheme 1). The resin was manually functionalized with the first
CH2OH
O
OH
O
α(1,6)
O
α(1,2)
OH
OH
CH2OH
O
CH2OH
O
CH2
O
OH
O
a
α(1,2)
O
N -Fmoc (9-fluorenylmethyloxycarbonyl)-protected amino acid,
OH
OH OH
α(1,3)
OH
Fmoc-Lys(Boc)-OH. Wang resin (830 mg) was pre-swollen in
OH
O
OH
CH2OH
α(1,6)
O
2
OH
CH2OH
O
O
8 mL of DCM for 30 min.
(319 mg, 0.68 mmol), HOBt (126 mg, 0.82 mmol), and DIC
(105 L, 0.68 mmol) was dissolved in minimum volume of DMF
A mixture of Fmoc-Lys(Boc)-OH
α(1,3)
CH2
O
OH OH
α(1,2)
O
OH
OH
1
OH
OH
l
OH
CH2OH
O
α(1,6)
O
O
(approximately 1.5 mL) and then added to Wang resin. Subse-
quently, DMAP (8.3 mg, 0.068 mmol) was dissolved in minimum
volume of DMF, it was added to the resin, and the resin was stirred
for 3 h at rt.37 The resin was successively washed with DMF, DCM,
and MeOH (5 times each), and dried overnight under vacuum. The
loading of the resin (0.12 mmol/g resin) was determined spectro-
α(1,2)
CH2
O
OH OH
CH2OH
O
O
α(1,2)
OH
4
OH OH
OH OH
OH
O
OH
5
Figure 1. Structure of mannan from Saccharomyces cerevisae.11

6720
I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
OH
OCH2
Fmoc-Lys(Boc)-OH
HOBt/DIC/DMAP
Fmoc-Lys(Boc)-
MW-SPPS
1. Fmoc-AA-OH, TBTU/ DIPEA, Tmax 75 °C, 30W, 300 sec
2. 20% piperidine/ DMF, 1st cycle: Tmax 75 °C, 35W, 30 sec
2nd cycle: Tmax 75 °C, 60W, 180 sec
H-[Gly-Lys(Boc)]2-
MW-SPPS
1. Fmoc-AA-OH, TBTU/ DIPEA, Tmax 65 °C, 30W, 300sec a
2. 20% piperidine/ DMF, 1st cycle: Tmax 65 °C, 35W, 30 sec
2nd cycle: Tmax 65 °C, 62W, 180 sec
MBP(83-99)-βAla-Lys(Mtt)-[Gly-Lys(Boc)]2-
1. Boc2O/ DIPEA
2. HFIP/ TIS/ DCM
3
: 0.1 : 6.9
Boc-MBP(83-99)-βAla-Lys-[Gly-Lys(Boc)]2-
MW-SPPS
1. Fmoc-AA-OH, TBTU/ DIPEA, Tmax 75 °C, 30W, 300 sec a
2. 20% piperidine/ DMF, 1st cycle: Tmax 65 °C, 35W, 30 sec
nd cycle: Tmax 65 °C, 62W, 180 sec
2
Boc-MBP(83-99)-βAla-Lys-[Gly-Lys(Boc)]2-
(
)
4
NH
MBP(83-99)-βAla
TFA/ TIS/ EDT/ H2O
1 : 2.5 : 2.5
9
:
O
MBP(83-99)
MBP(83-99)
Lys-Gly-Lys-Gly-Lys-OH
(
)
4
NH
di-BAP 1
O
Scheme 1. Microwave-Assisted Solid Phase Peptide Synthesis (MW-SPPS) of di-BAP 1. Arg and His residues were coupled using different conditions as reported in the paper.
photometrically (301 nm,
e
: 7800 MÀ1 cmÀ1) from the amount of
Arg, during coupling, particular coupling cycles were used. The
coupling cycle of His consisted of two steps; the first one was for
120 s at 25 °C and 0 W, and the second one for 240 s at 50 °C and
30 W.38 Arg residues were coupled twice. The first coupling was
a two-step cycle. The first step of the first cycle was for 1500 s at
25 °C and 0 W, while the second step was for 300 s at 65 °C and
25 W. The second coupling cycle was for 300 s at 65 °C and 30 W.39
When the synthesis of the first MBP(83–99) epitope was accom-
the adduct dibenzofulvene-piperidine formed after treatment of a
small amount of Fmoc-Lys(Boc)-resin with piperidine/DCM/MeOH.
The remaining active sites of the resin were acetylated by stirring
with a mixture of Ac2O/pyridine/DCM (2:3:5 v/v/v) at rt for 30 min,
and subsequently it was thoroughly washed with DCM, DMF, and
MeOH (5 times each).
The resin-bound polylysine core was synthesized using micro-
wave irradiation on Fmoc-Lys(Boc)-Wang resin at 0.1 mmol scale.
Branching was achieved by the incorporation of the Fmoc-
Lys(Mtt)-OH derivative. After Fmoc deprotection, followed the
addition of a b-Ala spacer and then the first epitope MBP(83–99)
was synthesized.
The Fmoc protecting group was removed by treatment with a
solution of 20% piperidine in DMF. All coupling reactions of the
appropriate amino acid residue (0.2 M in DMF, 5 equiv) were per-
formed with TBTU (0.5 M in DMF, 5 equiv) and DIPEA (2 M in NMP,
10 equiv). Since Mtt group can be partially removed at high tem-
peratures, after the introduction of Fmoc-Lys(Mtt)-OH in the se-
quence, the coupling and deprotection steps were carried out at
65 °C instead of 75 °C. Each Fmoc-deprotection microwave cycle
was characterized by two steps; the first one was for 30 sec at
65 °C and 35 W, and the second one for 180 sec at 65 °C and
62 W. All coupling reactions were for 300 s at 65 °C and 30 W. In
order to avoid racemization of His and formation of d-lactam of
a
plished, the N -terminus was protected with Boc2O (11 equiv) and
DIPEA (11 equiv) at rt for 2 h. Subsequently, the Mtt orthogonally-
protected lysine residue was deprotected using HFIP/TIS/DCM
solution (3:0.1:6.9 v/v/v) at rt for 8 h and, after the addition of a
b-Ala spacer, the second epitope MBP(83–99) was synthesized
stepwise on the core using microwave irradiation at 75 °C as men-
tioned above, apart from His (50 °C). In order to monitor the syn-
thesis, microwave-assisted mini-cleavages were performed after
e
coupling of the Fmoc-b-Ala and Fmoc-Pro residues on the N -group
of Lys and the intermediate products were characterized by UPLC-
MS. For the microwave-assisted mini-cleavage, the Discover™ S
Class single-mode microwave reactor (CEM) was used, and the
microwave cycle was performed for 15 min at 45 °C, 15 W, with
cooling, using a TFA/TIS/EDT/H2O solution (94:1:2.5:2.5 v/v/v/v).
Di-BAP cleavage from the resin and removal of the amino acids
side chain protecting groups were carried out with the solution
used in mini-cleavages for 5 h at rt. The solvent was partially

I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
6721
evaporated, and the crude di-BAP 1 was precipitated with cold
diethyl ether and collected after filtration (0.24 g).
The crude di-BAP 1 was subjected to a UPLC analysis (Waters
ACQUITY Ultra Performance LC system equipped with UV detector)
achieved by the incorporation of the Fmoc-Lys(Fmoc)-OH
derivative.
a
e
After N ,N -di-Fmoc deprotection of the N-terminal Lys residue,
bromoacetic acid was coupled to the protected polylysine core on
the solid support using symmetric anhydride chemistry. Initially
the core-resin was swollen in DMF for 30 min. Preparation of the
bromoacetic anhydride was carried out by dissolving bromoacetic
acid (278 mg, 2 mmol) in 5 mL dry DCM. The solution was cooled
using an Acquity C18 BEH column (1.7
l
m, 2.1 Â 100 mm). Separa-
tion was achieved by gradient elution of 20% to 90% solvent B (sol-
vent A = 0.1% TFA in H2O; solvent B = 0.1% TFA in ACN) over 3 min
at a flow rate of 0.45 mL/min and a column temperature at 40 °C
(conditions I) (see Supplementary, Fig. S1 A).
to 0 °C, and subsequently DIC (156 lL, 1 mmol) was added. The
The purification of di-BAP 1 was carried out on a semi-prepara-
tive RP-HPLC (Waters 600 solvent delivery system, combined with
a UV 2487 Dual Wavelength Absorbance Detector) using a Jupiter
mixture was stirred for 20 min. The precipitated diisopropylurea
(DIU) was removed by filtration.42 The bromoacetic anhydride
was added to the resin and after 15 min DIPEA (340 lL, 2 mmol)
Phenomenex C18 column (10
l
m, 250 Â 10 mm) at 230 and
was also added to the reaction mixture and it was stirred for 2 h
at rt. The (BrCH2CO)2-Lys-[Gly-Lys(Boc)]2-CLTR 3b was subse-
quently filtered and washed with DCM (1 Â 5 mL), DMF
(3 Â 5 mL), i-PrOH (2 Â 5 mL) and n-hexane (1 Â 5 mL), and dried
under vacuum. The completion of the reaction was verified by Kai-
ser test.
The bromoacetylated polylysine core 3 was then cleaved by
treating 3b with a TFA/anisole solution (97.5:2.5 v/v) for 2 h at
rt. The resin was filtered, the solvent was partially evaporated,
and the crude peptide 3 was precipitated from cold diethyl ether
and collected by filtration (0.27 g).
254 nm. Separation was achieved by gradient elution of 20% to
70% solvent B (solvent A = 0.1% TFA in H2O; solvent B = 0.1% TFA
in ACN) over 40 min at a flow rate of 4 mL/min (conditions II).
After lyophilization, the pure di-BAP 1 was obtained in 20%
yield (480 mg) and analyzed by analytical RP-HPLC (Waters system
equipped with a 600E system controller, an operating system Mil-
lennium 2.1 and a Waters 996 photodiode array detector) using a
C-18 Nucleosil column (5
l
m, 250 Â 5 mm) at 214 and 254 nm.
Separation was achieved by gradient elution of 5% to 100% solvent
B (solvent A = 0.1% TFA in H2O; solvent B = 0.1% TFA in ACN) over
30 min at a flow rate of 1 mL/min (conditions III) (see Supplemen-
tary Information, Fig. S1 B).
The purification of the crude peptide 3 was carried out on a
semi-preparative RP-HPLC (Waters 600 solvent delivery system,
combined with a Waters 996 photodiode array detector) using a
The final di-BAP 1 was identified by MicromassÒ Q-Tof MICRO™
mass spectrometer (Waters) equipped with an ESI source (see Sup-
plementary Information, Fig. S1 C).
Nucleosil C-18 column (7
lm, 250 Â 10 mm) at 230 nm and
254 nm. Separation was achieved by gradient elution of 5% to
50% solvent B (solvent A = 0.1% TFA in H2O; solvent B = 0.1% TFA
in ACN) over 45 min at a flow rate of 3 mL/min (conditions IV).
The lyophilized pure peptide 3 was obtained in 60% yield
(0.162 g) and analyzed by analytical RP-HPLC (conditions III). The
final peptide 3 was identified by ESI-MS (Waters Micromass ZQ
4000 mass detector) (see Supplementary Information, Fig. S2 A).
2.2.2. Synthesis of di-BAP 2 via thioether chemoselective
ligation
2.2.2.1. Synthesis of bromoacetylated polylysine core 3.
protected polylysine core H-Lys-[Gly-Lys(Boc)]2-CLTR 3a
(Scheme 2) was prepared on 862 mg of a manually functionalized
0.58 mmol/g Fmoc-Lys(Boc)-CLTR40,41 under microwave irradia-
tion at 0.5 mmol scale, following the MW-SPPS protocol for 2-chlo-
rotrityl resin (CLTR) we previously reported.29 Branching was
The
2.2.2.2. Synthesis of the peptide epitope Cys-MBP(83–99)
4.
Cys-MBP(83–99) 4 (Scheme 3) was prepared on 794 mg
Fmoc-Lys(Boc)-OH/ DIPEA/ DCM
C
Cl
Fmoc-Lys(Boc)
Cl
MW-SPPS
1. Fmoc-AA-OH, DIC/ HOBt, Tmax 60 °C, 34W, 300 sec
2. 20% piperidine/ DMF, 1st cycle: Tmax 60 °C, 52W, 30 sec
2
nd cycle: Tmax 60 °C, 70W, 180 sec
H-Lys-[Gly-Lys(Boc)]2
(3a)
DIC
(BrCH2CO)2O/ DIPEA
BrCH2CO2H
DCM
2 h
0 °C, 20 min
BrAc-Lys-[Gly-Lys(Boc)]2
(3b)
(
)
4
NH
BrAc
TFA / anisole, 2 h
97.5 : 2.5
(3)
BrAc-Lys-(Gly-Lys)2-OH
(
)
4
NH
BrAc
Scheme 2. Synthesis of the bromoacetylated polylysine core 3.

6722
I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
Fmoc-Pro-OH/ DIPEA/ DCM
Fmoc-Pro-
C
Cl
Cl
MW-SPPS
1. Fmoc-AA-OH, DIC/ HOBt, Tmax 60 °C, 34W, 300 sec a
2. 20% piperidine/ DMF, 1st cycle: Tmax 60 °C, 52W, 30 sec
2nd cycle: Tmax 60 °C, 70W, 180 sec
H-Cys(Trt)-Glu(OtBu)-Asn(Trt)-Pro-Val-Val-His(Trt)-Phe-Phe-
Lys(Boc)-Asn(Trt)-Ile-Val-Thr(OtBu)-Pro-Arg(Pbf)-Thr(OtBu)-Pro-
DCM/ TFE/ AcOH
7
:
2 :
1
H-Cys(Trt)-Glu(OtBu)-Asn(Trt)-Pro-Val-Val-His(Trt)-Phe-Phe-
Lys(Boc)-Asn(Trt)-Ile-Val-Thr(OtBu)-Pro-Arg(Pbf)-Thr(OtBu)-Pro-OH
TFA/ TIS/ EDT/ H2O
94 : 1 : 2.5 : 2.5
H-Cys-Glu-Asn-Pro-Val-Val-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro-OH (4)
Scheme 3. Microwave-assisted solid phase peptide synthesis (MW-SPPS) of peptide 4. Arg and His residues were coupled using different conditions as reported in the paper.
of a manually functionalized Fmoc-Pro-CLTR (0.63 mmol/g)40,41
Br
under microwave irradiation29 on a 0.5 mmol scale.
H
+ 2 H-Cys-MBP(83-99)-OH
N Lys-Gly-Lys-Gly-Lys-OH
The synthesized protected peptide on the resin was cleaved
with the cleavage solution DCM/TFE/AcOH (7:2:1) for 1 h at rt, fol-
lowed by deprotection using TFA/TIS/EDT/H2O (94:1:2.5:2.5 v/v/v/
v) for 5 h at rt. The solvent was partially evaporated and the crude
product 4 was precipitated with cold diethyl ether and collected
after filtration (0.81 g).
(
)
3
4
4
HS
O
NH
O
Br
H-Cys-MBP(83-99)-OH
The purification was carried out on a semi-preparative RP-HPLC
(Waters 600 solvent delivery system, combined with a Waters 996
photodiode array detector) using a Nucleosil C-18 column (7 lm,
250 Â 10 mm) at 230 nm and 254 nm. Separation was achieved
by gradient elution of 10 to 60% solvent B (solvent A = 0.1% TFA
in H2O; solvent B = 0.1% TFA in ACN) over 45 min at a flow rate
of 3 mL/min (conditions V).
S
H
N Lys-Gly-Lys-Gly-Lys-OH + 2 HBr
(
)
4
O
NH
O
di-BAP 2
S
H-Cys-MBP(83-99)-OH
After lyophilization of the pure peptide 4, a yield of 67.3%
(0.545 g) was obtained. The final peptide 4 was further analyzed
by analytical RP-HPLC (conditions III) and identified by ESI-MS
(Waters Micromass ZQ 4000 mass detector) (see Supplementary
Information, Fig. S2 B).
H-Cys-MBP(83-99)-OH
S
H
N Lys-Gly-Lys-Gly-Lys-OH
2.2.2.3. Ligation of peptides 3 and 4.
tion reaction (Scheme 4), (BrCH2CO)2-Lys-(Gly-Lys)2-OH
(0.8 mg, 1 mol) and H-Cys-MBP(83–99)-OH 4 (4.2 mg, 2 mol)
For the thioether liga-
(
)
4
3
O
NH
l
l
O
were dissolved in 2 mL of a degassed solution of ACN/0.1 M borate
buffer (pH 8.72) (1:3 v/v) and the solution was held under N2 for
2 h at rt.22 The completeness of the reaction was verified by analyt-
ical RP-HPLC (conditions III) (see Supplementary Information,
Fig. S2 C). The solvent was partially evaporated and subjected to
semi-preparative RP-HPLC purification (conditions V) to yield, after
by-product 5
OH
Scheme 4. Chemoselective ligation of 3 and 4 to give di-BAP 2.
1 or 2 was added and allowed to react overnight at rt in the dark.
Reduced mannan-di-BAP 1 or reduced mannan-di-BAP 2 com-
plexes were prepared by treating the oxidized ones with sodium
borohydride (1 mg/mL oxidized conjugate) for 3 h at rt.12,43–45
In the current work, the conjugation of the di-BAPs to mannan
was verified by analytical RP-HPLC (conditions III). In particular,
firstly a sample of di-BAP 1 or 2 which contained the same concen-
tration of the di-BAP in the final conjugate (0.5 mg/mL) was sub-
jected to RP-HPLC analysis, and then, at the same conditions, was
injected the related conjugate. The absence of the peak corre-
sponding to the di-BAP alone, without mannan, revealed the com-
plete conjugation of the di-BAPs to mannan (data not shown).
lyophilization, the final di-BAP 2 in 50% yield (2.4 mg, 0.5 lmol).
The final di-BAP 2 was identified by a MicromassÒ Q-Tof MICRO™
mass spectrometer (Waters) equipped with an ESI source (see Sup-
plementary Information, Fig. S3 A–B).
2.2.3. Conjugation of di-BAPs to mannan
Briefly, mannan (14 mg) was dissolved in 0.1 M sodium phos-
phate buffer (1 mL, pH 6.0), followed by the addition of 0.1 M so-
dium metaperiodate (100
incubated for 1 h at 4 °C. Ethylene glycol (10
l
L) in phosphate buffer (pH 6.0) and
L) was added to
l
the mixture and incubated for 30 min at 4 °C. The mixture (oxi-
dized mannan) was passed through a PD-10 column (Sephadex
G-25 M column, Amersham Biosciences, Sweden) pre-equilibrated
with a 0.1 M bicarbonate buffer (pH 9.0) followed by addition of
bicarbonate buffer (1.5 mL, pH 9.0). Oxidized mannan was isolated
by collecting the following 2 mL of eluate, to which 1 mg of di-BAP
2.3. EAE induction and evaluation
Experiments were performed in female Lewis rats of three
months of age that were bred and maintained under specific

I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
6723
pathogen-free conditions in the Experimental Animal Facility of
the Hellenic Pasteur Institute. di-BAP 1 conjugated with oxidized
DIPEA were used during the coupling as activating and base re-
agents respectively, while Fmoc removal was performed by treat-
ment with 20% piperidine in DMF.
In order to reach the desired branching level, Fmoc-Lys(Mtt)-
OH was used in the appropriate position, as Mtt group can be selec-
tively removed under mild acidic conditions. Since Mtt group can
be partially removed at high temperatures, after the introduction
of Fmoc-Lys(Mtt)-OH in the sequence, the rest coupling and depro-
tection steps were carried out at a lower temperature (65 °C in-
stead of 75 °C).
mannan (equivalent to 700
injection) or di-BAP 2 conjugated with reduced mannan (equiva-
lent to 700 g mannan and 30 g di-BAP 2 per injection) or oxi-
dized mannan (equivalent to 700 g mannan and 30 g di-BAP 2
per injection) or as controls, unconjugated reduced mannan
(700 g per injection), unconjugated oxidized mannan (700
lg mannan and 30 lg di-BAP 1 per
l
l
l
l
l
lg
per injection),or PBS vehicle were administered to groups of rats
while they were under isoflurane sedation, in a prophylactic proto-
col by s.c. tail base injection two days prior to induction of EAE. EAE
After the completeness of the MW-SPPS of the first MBP(83–99)
a
was induced by s.c. injection of an emulsion containing 40
lg of
epitope on the N amino group of the resin-bound lysyl core ma-
a
MBP(74–85) dissolved in 50 l of saline and 50 l of CFA (Sigma–
l
l
trix, the N -terminus of MBP(83–99)-core was capped with Boc2O.
Aldrich) supplemented with H37Ra Mycobacterium tuberculosis
(Difco) at a final concentration of 4 mg/ml. Rats were evaluated
daily for weight loss and clinical score. Scoring was based on clin-
ical signs according to the following parameters; (1) floppy tail; (2)
mild paraparesis; (3) severe paraparesis; (4) tetraparesis or mori-
bund condition.46 Rats were allowed free access to food and water
throughout the experiment and all animal procedures were ap-
proved by institutional review boards and national authorities
and conformed to European Union guidelines.
The Mtt orthogonal protecting group was then removed and the
second copy of the MBP(83–99) epitope was synthesized stepwise
on the N amine group of the lysine, under microwave irradiation.
In order to prevent steric hindrance, b-Ala was used as a spacer
between the core and the two epitope copies. The process of the di-
BAP 1 synthesis was monitored by microwave-assisted mini cleav-
ages and subsequent UPLC-MS analysis.
e
The crude product was analyzed by UPLC (conditions I) (see
Supplementary Information, Fig. S1 A), and purified by semi-pre-
parative RP-HPLC (conditions II) to give, after lyophilization, the fi-
nal di-BAP 1 in 10.5% total yield (Table 1). The pure di-BAP 1 was
analyzed by analytical RP-HPLC (conditions III) and identified by
3. Results and discussion
a
MicromassÒ Q-Tof MICRO™ mass spectrometer (Waters)
In this study, the divergent and convergent synthetic proce-
dures of di-BAPs using the immunodominant epitope MBP
(83–99) and their effect upon EAE development were studied.
Thus, we synthesized a MAP with two branches, containing the
immunodominant epitope MBP(83–99), which is implicated in
EAE, following both a direct (MW-SPPS) and an indirect (chemose-
lective ligation) synthetic approach. The C-terminus of both di-BAP
1 and 2 consists of an alternate Gly-Lys motif which acts as a linker
between the MAP and mannan, through the formation of Schiff
bases on the available free amino functions.
The MW-assisted solid-phase synthetic strategy described
could be used for the synthesis of large peptides or MAPs with dif-
ferent peptides in a poly-lysine core. We combined the benefits of
microwave irradiation leading to the rapid and efficient synthesis
of difficult peptide sequences and MAPs. Mannosylation is a prom-
ising technique to achieve modulation of immune response. The
lack of immunogenicity of the tested analogues could be overcome
using two or three immunodominant epitopes of myelin proteins
on a poly-lysine core. It is known that many different epitopes of
myelin proteins have been recognized by the encephalitogenic T
cells obtained in MS patients. There is not only one antigen respon-
sible for the appearance of the disease. The protection of mice
using mannan-peptide conjugation is antigen specific and it in-
duces durable peptide-specific T cell tolerance in mice. Moreover,
the mannan conjugated with mucin 1, a tumour cell surface pro-
tein, has been previously successfully used (CVacTM product) as
a therapeutic vaccine for the treatment of ovarian cancer.
equipped with an ESI source (theoretical average mass:
4613.3272; observed mass: 4613.1524) (see Supplementary Infor-
mation, Fig. S1 B–C).
3.2. Synthesis of bromoacetylated polylysine core 3
The protected peptide 3a was synthesized on CLTR-Cl40,41 using
the standard Fmoc/tBu methodology under microwave irradiation.
In order to avoid premature cleavage of the peptide from the CLTR
due to its high sensitivity at elevated temperatures and after per-
forming repetitive cycles of microwave, we adopted the modified
MW-SPPS conditions previously reported by us.29 DIC/HOBt was
used as coupling reagent, while the Fmoc removal was performed
by treatment with 20% piperidine in DMF. Branching was achieved
by the incorporation of Fmoc-Lys(Fmoc)-OH derivative on the re-
sin-bound lysyl core matrix. After deprotection of Fmoc-Lys(F-
moc)-OH followed bromoacetylation of the polylysine core by
using bromoacetic acid, DIC and DIPEA. The completeness of the
reaction was verified by Kaiser test. The cleavage of the bromoacet-
ylated polylysine core 3 from the resin and the removal of the ami-
no acids side chain protecting groups were carried out by
treatment with a 97.5% TFA solution in the presence of anisole as
a scavenger. Thiol or silyl type scavengers were not used in order
to avoid their reaction with the bromine moieties. Also, in order
to avoid the hydrolysis of the bromine, the cleavage reaction was
reduced to 2-2.5 h.
The crude product was subsequently purified by semi-prepara-
tive RP-HPLC (conditions IV) to give after lyophilization the final
bromoacetylated polylysine core 3 in 43% total yield (Table 1).
The pure core 3 was analyzed by analytical RP-HPLC (conditions
III) and identified by ESI-MS (see Supplementary Information,
3.1. MW-assisted SPPS of di-BAP 1
The step by step synthesis of di-BAP 1 using conventional RT-
SPPS protocols presented difficulties in terms of slow and incom-
plete couplings and Fmoc-removal reactions, probably due to chain
aggregation. Thus, we turned to MW-SPPS, which may allow the
fast stepwise synthesis of large and difficult peptide sequences29–
Table 1
List of peptides and di-BAPs synthesized by direct and indirect approach
a
Peptide
Purification yield (%)
Total yield (%)
tR (min)
Mcalc (Da)
31
di-BAP 1
Peptide 3
Peptide 4
di-BAP 2
20.0
60.0
67.3
50.0
10.5
42.7
52.0
50.0
16.74
10.60
16.92
16.53
4613.35
758.50
2098.43
4793.53
Di-BAP 1 was synthesized on a Wang resin, following the stan-
dard Fmoc/tBu strategy, using a Liberty™ microwave peptide syn-
thesizer (CEM). A low resin substitution (0.12 mmol/g resin) was
chosen in order to prevent steric hindrance and chain aggregation,
which increase proportionally to the peptide chain. TBTU and
a
Conditions III.

6724
I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
Fig. S2 A). The existence of both bromine moieties in the final prod-
uct 3 was verified by the presence of three peaks; one correspond-
ing to the M of the peptide, a second one of a double height of the
previous peak corresponding to the M+2, and a third one of the
same height of the first peak corresponding to the M+4.
Thioether ligation relies on the highly chemoselective reaction
between thiols and haloacetyl groups at neutral to mildly alkaline
pH. Thus, it is critical to perform the reaction in a pH range of 8.5,
in order to ensure that no other functional groups of the peptide
sequence, such as lysine, react with the haloacetyl groups. Another
critical step is to keep the reaction under anaerobic conditions in
order to prevent the thiol peptide from forming disulfide dimers.
Ligation of peptides 3 and 4 proceeded almost quantitively to
give di-BAP 2 (see Supplementary Information, Fig. S2 C). After
2 h, the starting peptide 3 could not be detected by analytical
RP-HPLC (conditions III), while in the mixture were present small
quantities of the peptide 4 and of the by-product 5, i.e. the peptide
3 ligated with one of the two bromine moieties of the core 4, while
the other bromine was hydrolyzed (see Supplementary Informa-
tion, Fig. S2 C, Scheme 4).
3.3. MW-SPPS of the peptide epitope Cys-MBP(83–99) 4
a
MBP(83–99) was synthesized bearing a cysteine in its N -termi-
nus in order to react further with the bromine moieties of the core
3. Cys-MBP(83–99)4 was prepared on CLTR-Cl40,41 using the stan-
dard Fmoc/tBu methodology under microwave irradiation and
according to the modified MW-SPPS protocol for CLTR-Cl29 DIC/
HOBt was used as coupling reagent, while the Fmoc removal was
performed by treatment with 20% piperidine in DMF.
As with histidine and arginine, also cysteine requires special
attention during MW-SPPS, since it is prone to racemization. Cys-
teine racemization has been attributed to base-catalyzed a-carbon
proton abstraction during coupling. The level of racemization can
be decreased by using weaker bases and avoiding preactivation
in phosphonium or aminium salt mediated coupling protocols.47
Additionally, in order to overcome increases in racemization from
excess microwave energy, coupling temperature was reduced to
50 °C.38
The purification of the di-BAP 2 was performed by semi-pre-
parative RP-HPLC purification (conditions V) after partial evapora-
tion. The pure di-BAP 2 was obtained in 50% total yield after
lyophilization (Table 1), and it was analyzed by analytical RP-HPLC
(conditions III) and identified by a MicromassÒ Q-Tof MICRO™
mass spectrometer (Waters) equipped with an ESI source (theoret-
ical average mass: 4793.5294; observed mass: 4793.5560) (see
Supplementary Information, Fig. S3 A–B).
The crude product was subsequently purified by semi-prepara-
tive RP-HPLC (conditions V) to give, after lyophilization, the final
peptide 4 in 52% total yield (Table 1). The pure peptide 4 was ana-
lyzed by analytical RP-HPLC (conditions III) and identified by ESI-
MS (see Supplementary Information, Fig. S2 B).
3.5. Conjugation of di-BAPs to mannan and monitoring of the
reaction
Di-BAPs 1 and 2, after lyophilization, were conjugated to poly-
saccharide mannan aiming in the diversion of the immune re-
sponse from Th1 to Th2 in EAE. Conjugation of di-BAPs to
mannan occurs via Schiff base formation between the free amino
groups of the di-BAPs and the aldehyde of oxidized mannan.
Reduction of oxidized mannan-di-BAPs complexes by treatment
with NABH4 results in the reduction of aldehydes to alcohols and
of Schiff bases to alkylamines.
In the current work, the conjugation of the di-BAPs to mannan
was verified by analytical RP-HPLC by the absence of the peak cor-
responding to the di-BAP alone (without mannan) in the final con-
jugates (data not shown).
3.4. Synthesis of di-BAP 2 via Thioether Chemoselective ligation
Since the MW-SPPS resulted in a 10.5% total yield of di-BAP 1,
we turned our attention to chemoselective ligation techniques
which are considered to be an efficient route for obtaining dendri-
meric constructs in high yields and purity.
A number of chemoselective ligation methods have been used
to synthesize large and complex peptide vaccines. In a previous
comparative study, Zeng et al.22 evaluated the yields and ease to
carry out three different chemoselective techniques, such as oxime
bond, thioether bond and disulfide bond formation and they found
that thioether bond formation was the simplest method, giving the
highest yield of immunogen. Taking this in consideration as well as
the fact that thioether bonds are stable in the physiological pH
range, we decided to follow this method for the synthesis of di-
BAP 2.
3.6. Assessment of the prophylactic role of mannan-di-BAP 1
and mannan-di-BAP 2 conjugates on the development of
MBP(74-85) induced EAE
To evaluate whether oxidized and reduced mannan- di-BAP 2
and oxidized mannan-di-BAP 1 conjugates can induce tolerance
Figure 2. Immunization of Lewis rats pretreated with oxidized, reduced mannan-di-BAP 2 (n = 4 per group), oxidized mannan-di-BAP 1 (n = 4) or reduced (n = 3) mannan, and
PBS vehicle control (n = 5) two days prior to the induction of EAE (arrow) (⁄, p < 0.05 for comparison between reduced mannan and PBS-treated groups).

I. Friligou et al. / Bioorg. Med. Chem. 21 (2013) 6718–6725
6725
in an MS-like disease, we used an MBP-EAE model inducible in
References and notes
Lewis rats by s.c. immunization with MBP(74–85) emulsified in
CFA. Groups of rats were injected intradermally with dilute solu-
ble oxidized or reduced mannan- di-BAP 2 or oxidized mannan
di-BAP 1 conjugates (Fig. 2), unconjugated oxidized or reduced
mannan or PBS 2 days prior to EAE induction. All rats developed
an acute monophasic disease that was not significantly different
to that developed in PBS-treated controls. Thus, mannan-conju-
gated di-BAP 1 and di-BAP 2 and unconjugated mannan-treated
rats showed similar disease onset and progression of clinical
symptoms as PBS-treated rats, except at one time point (day
16) where reduced mannan-vaccinated rats showed significantly
reduced clinical signs. The finding that mannan MBP(83–99) con-
jugates did not protect against MBP(74–85)-induced EAE is con-
sistent with previous data that mannosylated or mannan-
conjugated myelin antigens (based on the MOG protein) induce
T cell tolerance and protect against the development of EAE in
a peptide-specific manner (Tseveleki V.; Tselios T.; Friligou I.; Kout-
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peptides to target dendritic cells: A pre-clinical study in mice.
Unpublished results). The tolerogenic potential of di-BAP 2 will
need to be further tested upon the induction of MBP(83–99)-in-
duced specific T cell responses.
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Acknowledgments
We thank the Lifelong Learning Programme (LLP) Erasmus for
the financial support of Dr. I. Friligou’s mobility to PeptLab of the
University of Florence. We also thank the Hellenic Republic, Minis-
try of Development, for the financial support of this study through
the ‘‘Cooperation’’, 09SYN 21 609 program. Moreover we thank the
Ente Cassa di Risparmio di Firenze for financial support to PeptLab
of the University of Florence and the Agence Nationale de la
Recherche for the Chaire d’Excellence Programme to A.M. Papini
for the Project: PepKit at PeptLab@UCP.
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Apostolopoulos, V. J. Med. Chem. 2009, 52, 214.
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Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.08.008.
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