Palmitoyl derivatives of GpMBP epitopes: T-cell response and peptidases susceptibility

Article abstract of DOI:10.1021/jm010913j

We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a palmitoyl moiety at the N^α-terminal of Gln⁷⁴ or at

Full text of DOI:10.1021/jm010913j

3
504
J . Med. Chem. 2001, 44, 3504-3510
P a lm itoyl Der iva tives of Gp MBP Ep itop es: T-Cell Resp on se a n d P ep tid a ses
Su scep tibility†
Anna M. Papini,*,⊥ Benedetta Mazzanti, Elena Nardi, Elisabetta Traggiai, Clara Ballerini, Tiziana Biagioli,
‡
⊥
‡
‡
‡
§
§
§
∇
⊥
‡
Hubert Kalbacher, Hermann Beck, Martin Deeg, Mario Chelli, Mauro Ginanneschi, Luca Massacesi, and
Marco Vergelli‡
Dipartimento di Chimica Organica “Ugo Schiff” and Centro di Studio sulla Chimica e la Struttura dei Composti Eterociclici e
loro Applicazioni del C.N.R., Polo Scientifico Universitario, via della Lastruccia 13, I-50019 Sesto Fiorentino, Italy,
Dipartimento di Scienze Neurologiche e Psichiatriche, Universit a` degli Studi di Firenze, viale Pieraccini 6,
I-50134 Firenze, Italy, and Medizinisch-Naturwissenschaftliches Forschungszentrum, Universit a¨ t T u¨ bingen,
Ob dem Himmelreich 7, D-72074 T u¨ bingen, Germany
Received April 30, 2001
We report for the first time the immunoadjuvant effects of lipoconjugation of peptide antigens
in an in vitro system by using CD4+ T-cells. The lipopeptides obtained by conjugating a
R
74
75
palmitoyl moiety at the N -terminal of Gln or at the ꢀ-NH of Lys of GpMBP(74-85) induced
2
increased T-cell responsiveness compared to the native nonlipidated peptide. On the other hand,
lipoderivatives of GpMBP(82-98) did not increase the T-cell response, demonstrating that the
superagonist inducing effect of lipoconjugation is epitope-specific. Digestion of the two native
peptides with cathepsin D and L, both implicated in antigen processing, and with a complete
lysosomal fraction of a EBV-transformed B cell line shows that GpMBP(74-85) is resistant to
cellular proteases, while GpMBP(82-98) is easily digested by these enzymes. These results
suggest that the first prerequisite for increasing the T-cell response by lipoconjugation is the
stability of the native peptide to peptidases, providing an important insight into the
understanding of the immunoadjuvant effect of lipoderivative antigens.
In tr od u ction
tide by the built-in immunoadjuvant lipopeptide Pam3-
Cys-Ser was described as a reproducible way of stimu-
lating cytotoxic T lymphocytes (CTL) response in vivo.
Therefore these new synthetic mitogens are highly
suitable compounds for the study of the early events of
the immune response for structure-activity studies.10
In the last years, synthetic peptides modified also with
simple lipophilic moieties, such as a palmitoyl group
Multiple sclerosis (MS), an inflammatory disease of
the central nervous system (CNS) white matter, is
considered to be immune mediated. Several observa-
tions have strengthened the hypothesis that an autoim-
mune T-cell response to CNS myelin protein play a role
1
in the pathogenesis of MS lesions. Among the candidate
autoantigens, myelin basic protein (MBP) is the most
deeply studied. Immunization of susceptible animals
with MBP or with MBP peptides induces experimental
autoimmune encephalomyelitis (EAE), an experimental
inflammatory demyelinating disease of the CNS, medi-
ated by encephalitogenic CD4+ T-cells. EAE is consid-
ered the best experimental animal model of MS. In
Lewis rats immunized with GpMBP, encephalitogenic
T-cells recognizing the 74-85 amino acid sequence
(Pam), have been shown to be efficient tools in inducing
specialized CTL. Their different bioactivity, compared
to lipid free analogues, has been attributed to their
permanence in the cell membrane and subsequent
facilitated interaction with membrane receptors.1
Examination of the different posttranslational modifica-
tions, which allow membrane association of proteins,
indicates that even a single lipidic chain is able to
anchor large proteins. For example, the N-terminal
addition of an alkyl chain as small as a myristoyl group
on a glycyl residue is sufficient to anchor cytosolic
proteins to membranes. It has been shown that a single
modification of a relatively long peptide by a lipidic
amino acid resulted in the ability to reproducibly induce,
without immunoadjuvant, CD4, CD8, and antibody
1,12
2
1
3
,4
GpMBP(74-85) dominate the immune response. An-
other subdominant encephalitogenic epitope of GpMBP
in Lewis rat is represented by the sequence 86-98.
There are a lot of examples in the literature demon-
strating that the conjugation of a lipidic moiety to
an immunodominant peptide may affect the T-cell
5
-9
response.
Chemically defined modification of a pep-
9
response. Lipopeptides could therefore represent useful
†
tools for the study of immunological responses. On the
other hand, very little is known on the molecular
mechanism that underlies the immunological effect of
lipoconjugation of peptide antigens.
Dedicated to Prof. Luis Moroder on the occasion of his 60th
birthday.
Author for correspondence: Anna Maria Papini, Dipartimento di
*
Chimica Organica “Ugo Schiff”, Polo Scientifico Universitario, via della
Lastruccia 13, I-50019 Sesto Fiorentino, Italy. Phone: +39 055
4
573561. Fax: +39 055 4573531. E-mail: annamaria.papini@unifi.it.
It has been recently described that lipoconjugation of
a HLA-A2.1-restricted HBV 1 polymerase peptide epitope
dominant for human CD8+ T-cell responses increased
the lifespan of functional presentation of the peptide to
CD8+ cytotoxic T-cells. This effect has been linked to a
⊥
Dipartimento di Chimica Organica.
Dipartimento di Scienze Neurologiche e Psichiatriche. (Dr. M.
‡
Vergelli passed away at age 37 on J uly 7, 2001. His memory will
hearten those pursuing this research.)
§
Universit a¨ t T u¨ bingen.
Centro di Studio.
∇
1
0.1021/jm010913j CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/13/2001

Palmitoyl Derivatives of GpMBP Epitopes
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3505
Ta ble 1. Chemical Data for the Synthesized GpMBP Peptides and Lipopeptides
gradients at
mL min for
semipreparative HPLC
ESI-MS
[M + H] :
found (calcd)
-
1
yielda
mg (%)
+
3
HPLC
(tR, min)
b
no.
peptides
GpMBP(74-85)
Pam-GpMBP(74-85)
[Lys (Pam)]GpMBP(74-85)
Met-Ser-GpMBP(74-85)
Pam3Cys-Ser-GpMBP(74-85)
GpMBP(82-98)
Pam-GpMBP(82-98)
Lys(Pam)-GpMBP(82-98)
1
2
3
4
5
6
7
8
5-45% B/50 min
20-70% B/65 min
25-70% B/65 min
5-45% B/40 min
84 (23)
55 (14)
100 (44)
17 (7)
110 (35)
8 (7)
1415 (1414.7)
1653 (1652.9)
1653 (1652.9)
1632 (1632.8)
2393 (2393.5)
1995 (1995.1)
2233 (2233.3)
2361 (2361.4)
13.20c
d
5.65
9.90
8.00
25.30
11.42
6.64
7
5
e
f
g
h
10-45% B/25 min
50-80% B/30 min
40-85% B/50 min
i
13 (10)
14 (10)
6.51l
a
Calculated as TFA salts. Analytical HPLC gradients at 1 mL min . c 5-25% B in 20 min. d 35-55% B in 20 min. e 20-50% B in 20
b
-1
min. f 5-45% B in 20 min. 30-95% B in 22 min then 95% B isocratic. h 25-50% B in 15 min. 65-85% B in 10 min. 70-100% B in 10
min.
g
i
l
favored internalization and cytoplasm delivery in APC,
resulting in a prolonged expression of conformationally
correct MHC-peptide complexes.13,14
Lys(Pam)-OH during the SPPS. Pam-OPfp was also
used for the synthesis of the building-block Fmoc-Lys-
(Pam)-OH.
The lipopeptide Pam3Cys-Ser-GpMBP(74-85) (5),
containing the built-in immunoadjuvant lipopeptide
Pam3Cys-Ser, was synthesized by coupling Pam-Cys-
(CH2-CHOH-CH2OH)-OH to the resin-linked peptide
Ser-GpMBP(74-85). The two additional palmitoyl resi-
dues were introduced, still in solid phase, on the
hydroxyl functions of the S-(2,3-dihydroxypropyl) present
In the context of developing more stable molecules,
cyclic analogues of GpMBP(74-85), which maintain the
biological function of the original peptide, were previ-
ously designed, synthesized, and evaluated for activity
in the EAE system.15 The authors demonstrated that
the cyclic conformation of the immunodominant peptide
elicits a response in pharmacological quantities thanks
to the increased resistance to degradation in comparison
with the native sequence.16
on the side chain of Cys, by recirculation of palmitoyl
18,20
chloride, as previously described.
As control peptide,
In this study, we designed and synthesized a series
of lipopeptides of the two different immunodominant
peptide epitopes for CD4+ T-cells in Lewis rats, i.e.,
we synthesized Met-Ser-GpMBP(74-85) (4), elongating
the wild-type sequence 1 with Met and Ser at the
N-terminus.
4
GpMBP (74-85) and GpMBP (82-98), to investigate
In the case of GpMBP(82-98) (6), the corresponding
lipopeptides were synthesized as acids on a Fmoc-Pro-
NovaSyn-TGA resin. Lipopeptides 7 and 8 were syn-
thesized introducing, during solid-phase peptide syn-
thesis (SPPS) of 6, a palmitoyl moiety (by Pam-OPfp)
the T-cell response in an in vitro system. Our results
demonstrate, for the first time, that lipoconjugation can
also increase the responsiveness of CD4+ T-cells. In
addition, we show that the biological effect on T-cell
response can be different among different peptide
antigens. The two different effects are correlated to the
specific susceptibility of the peptide antigens to pepti-
dases.
ꢀ
and a N -palmitoyllysine residue [by Fmoc-Lys(Pam)-
OH], respectively, in the N-terminal position. All the
compounds were purified by RP-HPLC and character-
ized by ESI-MS and analytical HPLC (Table 1).
Resu lts a n d Discu ssion
Im m u n ology
Ch em istr y. As synthetic lipopeptides have been
demonstrated to have immunoadjuvant properties both
in vitro and in vivo,9 we synthesized two different
wild-type immunodominant MBP epitopes in Lewis rats,
GpMBP(74-85) [QKSQRSQDENPV, (1)] and GpMBP-
In Vitr o Effects of Lip ocon ju ga tion of Gp MBP -
(74-85) by a P a lm itoyl Moiety on T-Cell Resp on se.
Spleen cells were isolated from Lewis rats immunized
with GpMBP(74-85) at day 12 after immunization and
tested in a proliferation assay to evaluate the response
to the wild-type and lipid-bound peptides. In vitro
proliferation of spleen cells from Lewis rats immunized
with 1 was more vigorous and started at lower antigen
concentrations with lipopeptides 2 and 3, corresponding
to the immunodominant epitope GpMBP(74-85) (Fig-
ure 1). Similar results were obtained by using spleen
cells from animals immunized with the lipopeptides,
without significant differences in the ranking of agonist
activity of the peptides (2 > 3 > 1) (data not shown).
Successively, we tested the in vitro activity of lipopep-
tides on the proliferative response of long-term CD4+
T-cell lines (TCL) generated from spleen cells of Lewis
rats immunized with entire MBP and maintained in
culture by periodic restimulation with the whole protein.
The large majority of TCL, obtained from Lewis rats
using entire MBP, proliferated to the immunodominant
peptide 1. When the proliferative response to lipo-
derivatives was compared, it turned out that the lipid
bound analogues exerted an increased stimulatory
,17
(
82-98) [ENPVVHFFKNIVTPRTP, (6)], and several
derived lipid-bound peptides to investigate the T-cell
response (Table 1).
Lipopeptides were synthesized by introducing a palm-
itoyl moiety linked through an amide bond in the
N-terminal position or on a Lys residue in different
positions.
The palmitoyl moiety was introduced in the two
immunodominant MBP epitopes in Lewis rat, GpMBP-
(74-85) (1) and GpMBP(82-98) (6). The syntheses were
performed by SPPS following the Fmoc/tBu strategy, in
the standard synthetic protocol described in the general
procedure. GpMBP(74-85) and its lipoderivatives were
synthesized as peptide amides on a TentaGel S RAM
resin. The lipopeptide 2 was synthesized by introducing
the palmitoyl residue in the N-terminal position of 1,
anchored to the resin, using the pentafluorophenyl ester
of palmitic acid (Pam-OPfp). Lipopeptide 3, bearing a
ꢀ
N -palmitoyllysine residue, was prepared using Fmoc-

3
506 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Papini et al.
F igu r e 3. Proliferative response of a TCL specific for PPD
and tested to different concentrations of PPD itself and to the
lipopeptide Pam-GpMBP(74-85). Data show a representative
experiment out of three, carried out with different TCLs, and
they are expressed as cpm. Background proliferation was 782
cpm.
F igu r e 1. Proliferative response of spleen cells from Lewis
rats immunized with GpMBP(74-85) to different concentra-
tions of the wild-type peptide 1 and its lipoderivatives 2 and
3
. Data are expressed as a percentage of maximal response.
Each point represents the mean value ( standard error of five
different experiments. Maximal response ranged from 4841
to 28095 cpm.
F igu r e 4. Proliferative response of a TCL specific for GpMBP-
(
8
74-85) and tested to different concentrations of GpMBP(74-
5), Met-Ser-GpMBP(74-85), and their lipoderivatives. Data
F igu r e 2. Proliferative response of a TCL generated from
spleen cells of Lewis rats immunized with MBP to different
concentrations of the wild-type peptide 1 and its lipoderivatives
show a representative experiment out of three, carried out with
different TCLs, and they are expressed as cpm. Background
proliferation was 53 cpm.
2
and 3. Data show a representative experiment out of three
carried out with different TCLs, and they are expressed as
cpm. Background proliferation was 315 cpm.
control peptide Met-Ser-GpMBP(74-85) (4). We chose
to introduce a Met residue in order to mimic the
thioether function present in the Pam3Cys moiety.
From the analysis of the proliferative response of a
TCL to increasing concentrations of the tested peptides,
we could observe that also the lipopeptide 5 induced a
higher response compared to the lipid-free analogue 4
activity compared to the lipid free peptide. Proliferation
started at lower antigen concentrations, and the re-
sponse was overall more intense (Figure 2).
To rule out the possibility that this superagonist
activity of the lipopeptides was due to an unspecific
mitogenic activity, the proliferative response of a TCL
specific for a different antigen, i.e., protein purified
derivative (PPD) of Mycobacterium tubercolosis, was
tested to these antigens. No response was observed to
the lipid-conjugated peptides, suggesting that the stim-
ulatory activity of lipid-bound MBP peptides was spe-
cific for MBP-reactive T-cells (Figure 3).
(Figure 4).
This effect was detected only at low peptide concen-
tration. The low solubility of lipopeptide 5 may account
for the lack of immunoadjuvant activity at higher
antigen concentration.
However, considering that 2, the simplest N-terminal
palmitoyl derivative of the immunodominant epitope
GpMBP(74-85), induced the strongest proliferative
response, it can be suggested that the more difficult
synthesis of 5 does not offer significant advantages in
this experimental context.
In Vitr o Effects of Lip ocon ju ga tion of Gp MBP -
(82-98) by a P a lm itoyl Moiety on T-Cell Resp on se.
To assess whether the superagonist activity of the
lipoderivatives was epitope-specific, we investigated the
in vitro effects of lipoconjugation on a different immu-
nodominant epitope. Spleen cells were isolated from
animals immunized with the dominant peptide GpMBP-
(82-98) at day 12 after immunization and tested in a
Finally, we could demonstrate that the increased
responsiveness of T-cells to the lipopeptide required a
stable coupling of the two components. The proliferative
response of TCL to 1 was not increased when an
ꢀ
equimolar amount of N -palmitoyllysine was added
during the proliferation assay (data not shown).
In Vitr o Effects of Lip ocon ju ga tion of Gp MBP -
(
74-85) by P a m 3Cys-Ser on T-Cell Resp on se. It was
1
0
previously reported that Pam3Cys-Ser covalently linked
to synthetic peptides in the N-terminal position exerted
potent immunoadjuvant activity. We decided to test
Pam3Cys-Ser-GpMBP(74-85) (5) and the corresponding

Palmitoyl Derivatives of GpMBP Epitopes
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3507
F igu r e 5. Proliferative response of spleen cells from Lewis
rats immunized with GpMBP(82-98) to different concentra-
tions of the wild-type peptide 4 and its lipoderivatives 5 and
6
. Data are expressed as a percentage of maximal response.
Each point represents the mean value ( standard error of
three different experiments. Maximal response ranged from
4
510 to 63022 cpm.
proliferation assay to evaluate the response to the wild-
type and lipid-bound peptides. Differently from the
immunodominant peptide 1, in vitro proliferation of
spleen cells from Lewis rats immunized with 6 was more
vigorous and started at lower antigen concentrations
using the wild-type peptide in comparison to the li-
popeptides 7 and 8. No lipoderivative of the wild-type
peptide 6 increased the T-cell response (Figure 5).
Clea va ge of Gp MBP P ep tid es by Ca th ep sin D,
Ca th ep sin L, a n d Lysosom a l F r a ction s. Very re-
cently, the possible factors that may be critical for CTL
induction by using a dipalmitoylated lipopeptide con-
sisting of an ovalbumin helper T-cell epitope covalently
linked to an influenza virus CTL epitope were described.
Antigen processing of lipopeptide appears to be required
for T-cell induction since there was virtually no in vitro
binding of lipopeptide to purified major histocompat-
ibility complex (MHC) molecules. A major portion of
lipopeptide immunogenicity was due to its particular
nature inasmuch as CTL induction in mice correlated
with insoluble lipopeptide constructs, whereas more
soluble analogues were significantly less immunogenic.
Immunohistological analysis of tissue from immunized
animals revealed that lipopeptide migration from the
subcutaneous injection site to the spleen could be
detected as early as 1 h after immunization, and cell-
associated lipopeptide was observed on macrophages
and dendritic cells, implicating both cell populations in
F igu r e 6. Cathepsin D and L and lysosomal fraction cleavage
sites (V) of GpMBP(82-98) and fragments generated by diges-
tion with lysosomal fraction, as detected by LC-MS analysis
+
(
[M + H] found; calculated values in parentheses).
F igu r e 7. Degradation of the peptides 1-3 and 6-8 after
different incubation times with lysosomal fraction.
carboxypeptidases) could be identified in 6 (Figure 6),
while the epitope 1 remained remarkably stable to
lysosomal proteases (Figure 7).
Degradation studies on the peptides 2, 3, 7, and 8
showed that the lipopeptides were substantially stable
to the lysosomal peptidases (Figure 7).
the processing and presentation of lipopeptide particles
to CTLs.14
Exp er im en ta l Section
On the basis of these findings, in order to try to
explain the effect of lipoconjugation of the two immu-
nodominant MBP peptides, we decided to test their
possible processing pathways, such as their stability to
cellular proteases. In vitro digestion of the two MBP
epitopes for Lewis rats allowed us to determine their
stability. Cathepsin D, an aspartate protease, and
2 5
Dichloromethane (DCM) was freshly distilled from P O .
N,N-Dimethylformamide (DMF), previously stored over 4 Å
molecular sieves, was used for all couplings, 9-fluorenyl-
methoxycarbonyl (Fmoc) cleavage, and washing steps. Piperi-
dine was distilled from KOH. Flash column chromatography
(
FCC) was carried out on SiO
layer chromatography (TLC) was carried out on SiO
2
(ICN; 32-63 µm, 60 Å). Thin-
2
(Merck;
60 Å F254), and spots were located with UV light (254 and 366
nm), Fluram (Fluka; fluorescamine), ninhydrin, or a solution
of p-anisaldehyde (ethanol/sulfuric acid/acetic acid/p-anisal-
cathepsin L, a cysteine protease, both known to be
involved in antigen processing,21 as well as a lysosomal
2
2
3
dehyde, 90:3:1:2). HPLC-grade CH CN was purchased from
fraction of B-lymphoblastoid cell lines (B-LCL), were
used. Digestion mixtures were analyzed by LC-MS.
Several cleavage sites for the different cathepsins and
an asparaginyl endopeptidase,23 all destroying the MHC
binding region, in addition to exopeptidases (amino and
Carlo Erba (Italy). All other chemicals were commercially pure
compounds and were used as received. Melting points were
determined with a B u¨ chi 510 apparatus and are uncorrected.
1
13
H and C NMR spectra were recorded on a Varian Gemini
200 spectrometer. Chemical shifts are reported in ppm relative

3
508 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Papini et al.
1
to CHCl
3
fixed at 7.26 ppm for the H spectra and at 77.0 ppm
deprotected with 25% TFA in DCM (250 mL) for 30 min at 0
°C and 2 h at room temperature. The solution was evaporated
and the residue lyophilized. To a solution of the deprotected
amino acid in DMF (265 mL), brought to pH 8 with N-
methylmorpholine (NMM, 4 mL), were added at 0 °C Pam-
OPfp (4.5 g, 10.7 mmol), 1-hydroxy-7-azabenzotriazole (1.45
g, 10.7 mmol), and NMM (4 mL, 36 mmol). The reaction
mixture was stirred at 0 °C for 30 min and then at room
temperature for 24 h. The solvent was evaporated and the oily
1
3
for the C spectra; coupling constants (J ) are reported in hertz.
Assignments were aided by heteronuclear correlated spectros-
copy (HETCOR) experiments. IR spectra were recorded on a
Perkin-Elmer model 881 spectrometer for KBr pellets. El-
emental analyses were performed on a Perkin-Elmer 240 C
elemental analyzer.
Resins were purchased from Rapp Polymere (T u¨ bingen,
Germany) or Novabiochem (Bubendorf, Switzerland). Fmoc-
protected amino acids were purchased from Novabiochem and
O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexaflu-
orophosphate (HATU) from PerSeptive Biosystems (Framing-
ham, MA). Semipreparative purifications of peptides were
performed on a Vydac ODS 218TP1010 (250 × 10 mm) column
using a Beckman System Gold gradient apparatus equipped
with a diode array detector. Analytical reversed phase high-
pressure liquid chromatography (RP-HPLC) was used to
determine the purity of the fractions, using a Vydac ODS
18TP54 (250 × 4.6 mm) column. The solvent systems used
were as follows: A [0.1% trifluoroacetic acid (TFA) in H O]
and B (0.1% TFA in CH CN). The flow rates were 1 mL/min
for analytical HPLC and 3 mL/min for semipreparative HPLC,
with the linear gradients indicated in Table 1. Electrospray
ionization-mass spectroscopy ESI-MS was carried out on a
Micromass Model VG Quattro apparatus. Proteolytic products
were characterized by RP-HPLC/ESI-MS using a Vydac ODS
residue dissolved in CHCl
The organic layer was dried over Na
3
/MeOH and washed with 0.1 M HCl.
SO , filtered, and
2
4
evaporated and the residue triturated with diethyl ether to
give the crystalline Fmoc-Lys(Pam)-OH (3.016 g, 47%): mp
1
38-139 °C; TLC R
acid CO), 1692 (urethane CO), 1638 (amide I) cm ; H NMR
CDCl ) δ 0.88 (t, 3 H, CH -(CH 14), 1.0-1.8 (br m, 30 H, CH
CH 12 + â- to δ-H ), 2.03 (pseudo t, 2 H, CH -(CH 13-CH
.05 (m, 2 H, ꢀ-H ), 3.9 (m, 1 H, R-H), 4.26 (m, 3 H, CH
F 2 2
0.69 (CH Cl /MeOH 5:1); IR (KBr) 1738
-
1 1
(
(
(
3
3
2
)
3
-
2
)
2
3
2
)
2
),
-O +
3
2
2
fluorenyl 9-H), 5.78 (br m, 2 H, 2 × NH), 7.36 (m, 4 H, fluorenyl
2
2
7
-H + 3-H + 6-H + 7-H), 7.60 (d, 2 H, fluorenyl 1-H + 8-H),
.75 (d, 2 H, fluorenyl 4-H + 5-H). Anal. C, H, N.
2
3
S-(2,3-Dih ydr oxypr opyl)-N-h exadecan oylcystein e [P am -
Cys(CH
following the previously described pathway. Yield 80%; mp
70-72 °C; TLC R 0.48 (CHCl /MeOH/H O 16:6:1); IR (KBr)
1650 (acid CO), 1640 (amide I), 1520 (amide II) cm ; H NMR
(DMSO-d ) δ 0.88 (t, 3 H, CH -(CH 14), 1.25 (s, 24 H, CH
(CH 12), 1.64 (m, 2 H, CH -(CH 12-CH ), 2.11 (pseudo t, 2 H,
CH ), 2.3-3.0 (m, 4 H, Cys â-H + S-CH CHOH),
.33 (d, 2 H, CH OH), 3.54 (m, 1 H, CHOH), 4.20 (m, 1 H, Cys
2 2
-CHOH-CH OH)-OH]. The synthesis was performed
2
0
F
3
2
-1 1
2
18TP5215 (150 × 2.1 mm) column connected to a TSQ 700
6
3
2
)
3
-
mass spectrometer (Finnigan MAT, San J ose, CA).
2
)
3
2
)
2
3
-(CH
2
)13-CH
2
2
2
2
Gen er a l P r oced u r e for Con tin u ou s-F low Solid -P h a se
P ep tid e Syn th esis. All the peptides (Table 1) were synthe-
sized by the continuous-flow solid-phase method on a semi-
automatic apparatus (NovaSyn Gem Synthesizer) following the
Fmoc/tBu strategy. The resin TentaGel S RAM was used for
the syntheses of peptides 1-5 as amide derivatives, while
Fmoc-Pro-NovaSyn-TGA was used for the syntheses of pep-
tides 6-8 as acids. Fmoc-protected amino acids were used in
a 2.5-fold excess and activated with HATU/NMM in DMF.
Acylation end points were determined by checking that the
absorbance at 597 nm, due to the release of an anionic dye
3
R-H), 7.7 (d, 1 H, NH).
H-Gln -Lys-Ser -Gln -Ar g-Ser -Gln -Asp -Glu -Asn -P r o-Va l-
NH
2
[Gp MBP (74-85)] (1) a n d P a m -Gln -Lys-Ser -Gln -Ar g-
Ser -Gln -Asp -Glu -Asn -P r o-Va l-NH
2
[P a m -Gp MBP (74-85)]
(2). These peptides were synthesized starting from 1 g of resin
(0.21 mmol) and following the General Procedure. For peptide
2, the palmitoyl moiety was introduced at the end of the
synthesis by means of Pam-OPfp, dissolved in DCM in a 2.5-
fold excess (0.222 g, 0.525 mmol) in the presence of 1-hydroxy-
benzotriazole (0.072 g, 0.525 mmol) and NMM (0.087 mL,
(
acid Violet 17) from the protonated resin-bound amino groups,
0
.788 mmol).
did not change by more than 0.002 absorbance units over 10
min after a recirculation time of 30 min. Cleavage of the Fmoc
group was accomplished with 20% piperidine in DMF. Depro-
tection reactions were followed by monitoring at 365 nm the
resulting dibenzofulvene-piperidine adduct. On completion of
the synthesis, the resin was washed with DCM and ether and
dried in vacuo. Peptide cleavage from the resins and depro-
tection of the amino acid side chains was carried out with TFA/
thioanisole (95:5) for 30 min at 0 °C and 2 h at room
temperature. The resin was filtered off and washed with TFA,
and the filtrate was partially evaporated. The crude products
were precipitated with diethyl ether, collected by centrifuga-
H-Gln -Lys(P am )-Ser -Gln -Ar g-Ser -Gln -Asp-Glu -Asn -P r o-
Va l-NH
2
{[Lys75(P a m )]Gp MBP (74-85)} (3). The peptide
was synthesized starting from 0.5 g of resin (0.12 mmol)
following the general procedure. The introduction of the
lipophilic moiety was performed by using a 2.5-fold excess of
Fmoc-Lys(Pam)-OH in DMF (HATU/NMM activation) as for
the other protected amino acids.
H-Met-Ser -Gln -Lys-Ser -Gln -Ar g-Ser -Gln -Asp -Glu -Asn -
P r o-Va l-NH2 [Met-Ser -Gp MBP (74-85)] (4). The peptide
was synthesized starting from 0.5 g of resin (0.12 mmol)
following the general procedure.
2
tion, suspended or dissolved in H O, and lyophilized. The
P a m -Cys[CH
Ser -Gln -Ar g-Ser -Gln -Asp-Glu -Asn -P r o-Val-NH
2
-CH(O-P a m )-CH
2
O-P a m ]-Ser -Gln -Lys-
[P am Cys-
peptides were purified by semipreparative HPLC. Fractions
containing homogeneous material as monitored by HPLC were
combined and lyophilized. Characterization of the products
was performed using analytical HPLC and ESI-MS spectrom-
etry. The analytical data are reported in Table 1.
2
3
Ser -Gp MBP (74-85)] (5). The peptide Ser-GpMBP(74-85)
was synthesized starting from 0.5 g of resin (0.12 mmol)
following the General Procedure. The lipophilic built-in im-
2 2
munoadjuvant Pam-Cys[CH -CHOH-CH OH]-OH was intro-
Hexa d eca n oic Acid P en ta flu or op h en yl Ester [P a m -
OP fp ]. Pam-OPfp was synthesized, following the procedure
duced at the end of the synthesis, in the N-terminal position
of the resin-linked peptide in a 2.5-fold excess by endo-N-
hydroxy-5-norbornene-2,3-dicarboximide/N,N′-diisopropylcar-
bodiimide activation in DMF/DCM (1:1). Two additional
palmitoyl residues were introduced, in solid phase, on the
hydroxyl functions of 2,3-dihydroxypropyl group present on the
side chain of the Cys residue. Palmitoyl chloride (1.2 mmol)
in pyridine/DCM (1:1) was recirculated on the resin for 14 h.
After treatment with DMF and 20% piperidine in DMF, the
resin was washed with DMF and DCM and the peptide cleaved
from the resin as described in the general procedure. The
peptide was used with no further purification.
2
4
of Kisfaludy et al. for the synthesis of the pentafluorophenyl
ester of amino acids, by adding, at 0 °C, dicyclohexylcarbodi-
imide (DCC, 3.1 g, 15 mmol) in dioxane (20 mL) to a stirred
solution of pentafluorophenol (2.76 g, 15 mmol) and hexade-
canoic acid (3.85 g, 15 mmol) in DMF (8 mL) and dioxane (20
mL). The mixture was stirred overnight at room temperature,
then it was filtered, and the solution was evaporated to
dryness. The oily residue, triturated with hexane, gave a white,
crystalline product (5.85 g, 92%): mp 43-44 °C; IR (KBr) 1775
-
1 1
(
CO), 1516 (aromatic C-C) cm ; H NMR (CDCl
3
) δ 0.88 (t, 3
12), 1.77 (m, 2 H,
) C, H.
H, CH
3
-(CH
2
)
14), 1.26 (br m, 24 H, CH
3
-(CH
2
)
H-Glu -Asn -P r o-Va l-Va l-His-P h e-P h e-Lys-Asn -Ile-Va l-
Th r -P r o-Ar g-Th r -P r o-OH [Gp MBP (82-98)] (6). The pep-
tide was synthesized starting from 0.5 g of resin (0.05 mmol)
â-H ), 2.65 (t, 2 H, R-H
2
2
). Anal. (C22
H
31
O
2
F
5
r
E
N -Fm oc-N -h exadecan oyllysin e [Fm oc-Lys(P am )-OH].
The ꢀ-amino group of Fmoc-Lys(Boc)-OH (5 g, 10.7 mmol) was
9
7
following the general procedure. Deprotection of Thr was

Palmitoyl Derivatives of GpMBP Epitopes
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21 3509
Here we show that the synthetic lipopeptides 2 and
performed with a 5 mL/min piperidine flow for a period shorter
than usual to avoid the formation of the corresponding
diketopiperazine.
P a m -Glu -Asn -P r o-Va l-Va l-H is-P h e-P h e-Lys-Asn -Ile-
Va l-Th r -P r o-Ar g-Th r -P r o-OH [P a m -Gp MBP (82-98)] (7).
The peptide was synthesized starting from 0.5 g of resin (0.05
mmol) following the general procedure. The palmitoyl moiety
was introduced at the end of the synthesis by means of Pam-
OPfp, dissolved in DCM/DMF in a 5-fold excess (0.106 g, 0.25
mmol) in the presence of HATU (0.095 g, 0.25 mmol) and NMM
3
of the immunodominant epitope in Lewis rat, GpMBP-
(
74-85), obtained by SPPS through the conjugation of
R
a lipophilic moiety (palmitoyl) at the terminal N of
7
4
75
Gln and at the ꢀ-NH2 of Lys , respectively, increased
the T-cell responsiveness compared to the native non-
lipidated analogue. On the other hand, lipoconjugation
of the different MBP immunodominant peptide GpMBP-
(82-98) does not result in increased T-cell response. In
(0.042 mL, 0.38 mmol).
addition, the data obtained by the cathepsin cleavage
of the two immunodominant peptides suggest that the
first prerequisite for increasing the T-cell response by
a lipopeptide is the stability of the immunodominant
wild-type peptide to peptidases.
H-Lys(P am )-Glu -Asn -P r o-Val-Val-His-P h e-P h e-Lys-Asn -
Ile-Va l-Th r -P r o-Ar g-Th r -P r o-OH [Lys(P a m )-Gp MBP (82-
9
8)] (8). The peptide was synthesized starting from 0.5 g of
resin (0.05 mmol) following the general procedure. The intro-
duction of the lipophilic moiety was performed by using a 2.5-
fold excess of Fmoc-Lys(Pam)-OH in DMF (HATU/NMM
activation), as for the other protected amino acids.
Im m u n iza tion . Female Lewis rats were immunized intra-
dermally in the hind foot pads with 200 µg of MBP(74-85) or
MBP(82-98) peptide emulsified in incomplete Freund’s adju-
vant (IFA, Sigma, Germany) supplemented with 300 µg of
Mycobacterium tubercolosis H37 RA (DIFCO, Detroit, MI).
In Vitr o Lym p h ocyte P r olifer a tion . Spleens from im-
munized Lewis rats were removed aseptically from rats at day
This is the first demonstration of immunoadjuvant
effects of lipoconjugation in an in vitro system, by using
CD4+ T-cells.
Three different hypotheses can be formulated to
explain the increased T-cell responsiveness to the lipid-
bound peptides:
(
i) A costimulatory activity of the lipophilic moiety can
8
be envisioned. A recent study showing that lipoproteins
can provide costimulatory signals to CD4+ and CD8+
T lymphocytes to produce pro-inflammatory cytokines
would support this hypothesis. Due to the different
effect of lipoconjugation of the two GpMBP epitopes, this
hypothesis is, however, unlikely.
1
2 after immunization. Single cell suspensions of spleen were
prepared by passage of spleens through a stainless steel mesh
and tested in a proliferation assay to evaluate the response to
the immunizing peptides and to its lipoderivatives. Prolifera-
3
tive response of spleen cells was evaluated by H-thymidine
incorporation in the last 8 h of culture. Incorporated radioac-
tivity was measured in a scintillation counter (Microbeta Plus,
Wallac, Finland). Results are expressed as the mean counts
per min (cpm).
(ii) An increased affinity to the MHC molecule and/
or T-cell receptor of responding T-cells can be respon-
sible for the lipopeptides’ biological activity. In this case,
the lipoderivatives of the two immunodominant peptides
should bind the surface MHC molecule, and the differ-
ent effect of lipidation could be explained either by a
different susceptibility of the TCR of the T-cell clones
specific for the two different epitopes or by a different
binding kinetic of the lipopeptides to Ia and Ir, the MHC
class II molecules involved in the recognition of GpMBP-
Digestion of th e P ep tid es w ith Ca th ep sin D. Digestion
mixtures were prepared with 10 µg of each peptide [10 µL of
1
mg/mL solution in water, 1% dimethyl sulfoxide, (DMSO)]
and 0.1 µg of bovine cathepsin D (Sigma) in 1 N citrate buffer
pH 5.0 (10 µL). Each mixture was incubated at 37 °C for 40
min and then frozen. The digestion was followed by HPLC
using a Vydac C18 column (5 µm, 150 × 2.1 mm, 0.2 mL/min
flow rate) with a gradient of 0-90% B in 30 min for GpMBP-
(
82-98) and for lipoderivatives of GpMBP(74-85) and a
gradient of 0-80% B in 30 min for GpMBP(74-85). For
lipoderivatives of GpMBP(82-98) a Vydac C4 column (5 µm,
1
1
(
74-85) and GpMBP(82-98), respectively. This hypoth-
esis is not consistent with previous results showing an
50 × 2.1 mm, 0.2 mL/min flow rate) with a gradient of 20-
impaired binding of lipoderivatives to MHC molecule.14
00% B in 30 min was used.
Digestion of th e P ep tid es w ith Ca th ep sin L. Digestion
mixtures were prepared with 10 µg of each peptide (10 µL of
mg/mL solution in water, 1% DMSO) and 0.1 µg of human
cathepsin L (kindly provided by Dr. E. Weber, Institute of
Biochemistry, University of Halle, Germany) in 0.2 M citrate
buffer pH 5.5 containing 1.2 mg/mL dithiothreitol (DTT,
Cleland’s reagent) and 0.2 mM pepstatin (10 µL). Each mixture
was incubated at 37 °C for 2 h, then frozen, and followed by
HPLC with the methods reported for cathepsin D.
(iii) A more favorable antigen presentation could
explain the lipopeptide superagonist activity. Several
pieces of evidences indicate that lipoconjugation may
favor the interaction of the peptide antigen with cell
membrane. In this context, fluorescence resonance
energy transfer (FRET) experiments of the fluorescence-
labeled lipopeptide AMCA-ωAud-GpMBP(74-85) with
covesicles made by DPPC/AMCA-ωAud-GpMBP(74-85)/
phospholipids bearing a quencher on the alkyl chain
1
Digestion of th e P ep tid es w ith a Lysosom a l F r a ction .
Digestion mixtures were prepared with 10 µg of each peptide
(
100:5:1) demonstrated a high efficiency (76%) and a
(
10 µl of 1 mg/mL solution in water, 1% DMSO), 10 µL of 0.15
small AMCA-BODIPY distance (41 Å). The high FRET
efficiency is in agreement with AMCA localized near the
quencher BODIPY, and it can be explained only if the
AMCA-labeled lipopeptide penetrates the phospholipid
bilayer. These data support the hypothesis that the
presence of the lipophilic moiety facilitates the interac-
M phosphate buffer pH 5.4, 1.2 mg/mL DTT, and 1 µL of a
lysosomal fraction of the EBV-transformed B cell line BSM.22
Each system was incubated at 37 °C for 3 h, then frozen, and
followed by HPLC with the methods reported for cathepsin
D.
Con clu sion s
25
tion with the cell membrane. On the other hand, our
It has been recently demonstrated that a single
modification of a relatively long peptide by a lipophilic
amino acid with a long hydrocarbon chain, results in
the ability to reproducibly induce, without immunoad-
juvant, a relevant virus-specific CTL response in vivo,
in the absence of an immunoadjuvant.11 However, the
mechanism is not well understood.
data demonstrate that lipidation induces the two im-
munodominant peptides a comparable stability to pep-
tidases, with respect to their wild-type sequences.
Therefore, we must hypothesize that the lipophilic
moiety, inducing both the lipopeptides to cross the cell
membrane bypassing the binding to the cell surface
MHC, is then removed before the antigens enter the

3
510 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 21
Papini et al.
lysosomal compartment. Here the cathepsins will ex-
plicate their function, before the peptides bind to the
nascent MHC molecules for presentation on the cell
surface. In this context, there are several evidences that
the intracellular processing is fundamental for peptide
antigen recognition.26 It is evident that such a mecha-
nism would be advantageous for an epitope stable to
peptidases such as GpMBP(74-85), but not for GpMBP-
(11) Deprez, B.; Sauzet, J . P.; Boutillon, C.; Martinon, F.; Tartar, A.;
Sergheraert, C.; Guillet, J . G.; Gomard, E.; Gras-Masse, H.
Comparative Efficiency of Simple Lipopeptide Constructs for in
Vivo Induction of Virus-Specific CTL. Vaccine 1996, 14, 375-
3
82.
(
12) Thiam, K.; Loing, E.; Verwaerde, C.; Auriault, C.; Gras-Masse,
H. IFN-γ-Derived Lipopeptides: Influence of Lipid Modification
on the Conformation and the Ability to Induce MHC Class II
Expression on Murine and Human Cells. J . Med. Chem. 1999,
4
2, 3732-3736.
(
13) Livingston, B. D.; Crimi, C.; Grey, H.; Ishioka, G.; Chisari, F.
V.; Fikes, J .; Grey, H.; Chesnut, R. W.; Sette, A. The Hepatitis
B Virus-Specific CTL Responses Induced in Humans by Li-
popeptide Vaccination are Comparable to Those Elicited by
Acute Viral Infection. J . Immunol. 1997, 159, 1383-1392.
(82-98), which contains several cleavage sites for
cathepsins and other peptidases.
In conclusion, we formulate a hypothesis on the
mechanism of immunoadjuvanticity of lipopeptides, by
which the lipidic moiety bound to the examined peptides
would affect the T-cell response. This includes intrac-
ellular processing of the lipopeptide antigens and ac-
counts for the epitope specificity of the effects of
lipoconjugation.
These findings can provide a first insight into the
understanding of the immunoadjuvant effect of lipo-
derivative antigens.
(14) Tsunoda, I.; Sette, A.; Fujinami, R. S.; Oseroff, C.; Ruppert, J .;
Dahlberg, C.; Southwood, S.; Arrhenius, T.; Kuang, L. Q.; Kubo,
R. T.; Chesnut, R. W.; Ishioka, G. Y. Lipopeptide Particles as
the Immunologically Active Component of CTL Inducing Vac-
cines. Vaccine 1999, 17, 675-685.
(
15) Tselios, T.; Probert, L.; Daliani, I.; Matsoukas, E.; Troganis, A.;
Gerothanassis, I. P.; Mavromoustakos, T.; Moore, G. J .; Mat-
soukas, J . M. Design and Synthesis of a Potent Cyclic Analogue
of the Myelin Basic Protein Epitope MBP72-85: Importance of
8
1
the Ala Carboxyl Group and of a Cyclic Conformation for the
Induction of Experimental Aallergic Encephalomyelitis. J . Med.
Chem. 1999, 42, 1170-1177.
(
16) Tselios, T.; Probert, L.; Kollias, G.; Matsoukas, E.; Roumelioti,
P.; Alexopoulos, K.; Moore, G. J .; Matsoukas, J . Design and
Synthesis of Small Semi-Mimetic Peptides with Immunomodu-
latory Activity Based on Myelin Basic Protein (MBP). Amino
Acids 1998, 14, 333-341.
17) Schild, H.; Deres, K.; Wiesm u¨ ller, K.-H.; J ung, G.; Rammensee,
H. G. Efficiency of Peptides and Lipopeptides for in Vivo Priming
of Virus-Specific Cytotoxic T Cells. Eur. J . Immunol. 1991, 21,
Ack n ow led gm en t. This research was supported by
Ministero Sanit a` , ISS, MS Project 1997-1999, MURST-
Cofin98, and Vigoni-DAAD Exchange Program.
(
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