Cyclolinopeptide derivatives modify methotrexate-induced suppression of the humoral immune response in mice

Article abstract of DOI:10.1016/j.ejmech.2011.07.040

High doses of chemotherapeutics in clinical treatment, leading to cell toxicity, can be lowered by co-administration of other immunoregulatory drugs. The aim of this study was to evaluate effects of several derivatives of cyclolinopeptide A (CLA), derived

Full text of DOI:10.1016/j.ejmech.2011.07.040

European Journal of Medicinal Chemistry 46 (2011) 4608e4617
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Cyclolinopeptide derivatives modify methotrexate-induced suppression
of the humoral immune response in mice
a
a
c
b
b
ꢀ
ꢀ
Joanna Katarzynska , Adam Mazur , Ewa Rudzinska , Jolanta Artym , Micha1 Zimecki ,
Stefan Jankowski a , Janusz Zabrocki
,
a,
**
*
a
_
ꢀ
ꢀ
Institute of Organic Chemistry, Faculty of Chemistry, Technical University of Łódz, Zeromskiego 116, 90-924 Łódz, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wrocław, Poland
c
_
ꢀ
Department of Bioorganic Chemistry, Faculty of Chemistry, Technical University of Wrocław, Wybrzeze Wyspianskiego 27, 50-370 Wrocław, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
High doses of chemotherapeutics in clinical treatment, leading to cell toxicity, can be lowered by co-
administration of other immunoregulatory drugs. The aim of this study was to evaluate effects of
several derivatives of cyclolinopeptide A (CLA), derived from linen seeds, on the suppressive action of
metothrexate (MTX) in a mouse model of humoral immune response in vitro. New CLA analogues 1 and
2, and their linear precursors 3 and 4, containing conformationally restricted dipeptide fragment Phe-
Phe or d-Phe-d-Phe with ethylene bridge (eCH2eCH2e) between phenylalanine nitrogens were
synthesized. NMR studies and theoretical calculations showed that introduction of locally constraining
fragment into CLA molecule increased its overall conformational flexibility.
The bioactivity of new CLA analogues was examined in the mouse model of the in vitro secondary
humoral immune response, suppressed by methotrexate (MTX). The results revealed differential actions
of the peptides such as 1/augmentation of the suppressive activity of MTX or 2/antagonistic effects of the
peptides on MTX-induced suppression. Potential advantages for the application of CLA-derived peptides
in therapy and structureeactivity relationships were discussed.
Received 28 January 2011
Received in revised form
20 July 2011
Accepted 22 July 2011
Available online 28 July 2011
Keywords:
Cyclolinopeptide A
Immune suppression
Methotrexate
Ó 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction
cytotoxicity [10] it remains to be a candidate as a therapeutic agent,
possibly as a modified molecule.
The folic acid analogue e methotrexate (MTX) is widely applied
for suppression of graft-versus-host disease and rheumatoid
arthritis. MTX is effective alone or in combination with other drugs,
such as: cyclosporine A (CsA) [1], or/and prednisone [2], and
Tacrolimus (FK506) [3]. CsA, beside its known immunosuppressive
properties, exhibits at low doses anti-apoptotic activities [4e6].
Application of CsA and Tacrolimus is limited due to their side-
effects, mainly nephrotoxicity.
The immunosuppressive activities of cyclolinopeptide A (CLA)
extracted from the linseeds [7], and its analogues, were investi-
gated in a number of immunological assays [8]. The mechanism of
CLA action is similar to that of CsA since it binds to cyclophilin A
albeit to a much lesser affinity [9e11]. Since the peptide is devoid of
It was suggested that the Pro-Pro-Phe-Phe sequence is
responsible for the interaction of the CLA molecule with
a respective cellular receptor [12e14]. We applied several modi-
fications within this unit to study structureebiological activity
relationship. Both proline residues were replaced by (S)-
proline or (S)-
3-homo-proline [15], 3-phenylalanine [16] and
homophenylalanine [17].
b
2-iso-
b
b
In the present investigation we report results on the biological
activities of new CLA analogues containing the ethylene bridge
between phenylalanine nitrogen atoms. Several studies showed
that such a modification effectively changed biological activities of
peptides playing key roles in physiology. For example, the ethylene
junction of phenylalanines introduced into arginine of vasopressin
(AVP) molecule in positions 2 and 3 abolished its activity [18]. In
turn, the modification of bradykinin in positions 7 and 8 by d-Phe-
d-Phe fragment was found to be advantageous in terms of
increased antagonism in blood pressure tests, but practically
eliminated interaction of the peptide with its uterus receptors [19].
Of interest, the presence of the d-Phe-d-Phe fragment in positions
* Corresponding author. Tel.: þ48 42 631 3222; fax: þ48 42 636 5530.
** Corresponding author. Tel.: þ48 42 631 3153; fax: þ48 42 636 5530.
E-mail addresses: stefan.jankowski@p.lodz.pl (S. Jankowski), janusz.zabrocki@p.
lodz.pl (J. Zabrocki).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.07.040

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4609
cyclo ( Leu1 - Val2 - Pro3 - Pro4 - Phe-Phe - Leu7 - Ile8 - Ile9 )
cyclo ( Leu1 - Val2 - Pro3 - Pro4 - D-Phe-D-Phe - Leu7 - Ile8 - Ile9 )
H - Leu1 - Val2 - Pro3 - Pro4 - Phe-Phe - Leu7 - Ile8 - Ile9 - OH
H - Leu1 - Val2 - Pro3 - Pro4 - D-Phe-D-Phe - Leu7 - Ile8 - Ile9 - OH
H - Leu1 - Val2 - Pro3 - Pro4 - Phe5-Phe6 - Leu7 - Ile8 - Ile9 - OH
1
2
3
4
5
dipeptide fragment Phe-Phe or d-Phe-d-Phe with ethylene bridge
(eCH2eCH2e) between phenylalanine nitrogens (Fig. 1) [23].
The synthesis of the piperazinone rings has been achieved
according to the standard procedure [18,19,24]. The esterification
and cyclization of N,N0-ethylene bridged bis
a-amino acid proceed
simultaneously in boiling ethanol using p-toluenesulfonic acid
monohydrate as an acidic catalyst, followed by alkali hydrolysis.
Desired dipeptide units were used for further experiments as N-9-
fluorenylmethyloxycarbonyl derivatives.
Both linear peptides 3 and 4 were prepared manually using
standard solid-phase procedure on Wang resin (Scheme 1), using
Fmoc group for Na-amino protection and TBTU as a coupling
reagent for naturally occurring amino acids. The acylation of NH-
groups in the piperazinone rings was achieved by using Fmoc-
Pro-OH chloride, in situ generated by BTC (bis(trichloromethyl)
carbonate) [25].
HN
N
O
HN
N
O
OH
OH
O
O
The cyclization of linear precursors 3 and 4 was performed in
dichloromethane solution at dilution 1:104 using EDC/HOBt as
coupling reagent with 21 and 27% yield, respectively.
CLA was obtained according to known procedure [12]. Linear
nonapeptide 5, analogue of peptide 3, was synthesized using
microwave peptide synthesizer.
The purity of all compounds was verified by RP-HPLC and
afterwards the compounds were characterized by NMR spectros-
copy and MS-MALDI spectrometry.
H-Phe-Phe-OH 6a
-D-Phe-D
H -Phe-OH 6b
Fig. 1. CLA analogues 1 and 2, linear peptides 3e5 and dipeptide unit 6a or 6b with
ethylene link bridging nitrogen atoms.
2 and 3 of oxytocin molecule completely suppressed interactions of
new analogues with V1a receptors [20].
We anticipated that introduction of conformationally restricted
dipeptide fragment into CLA molecule should also affect its bioac-
tivity. The bioactivity of new CLA analogues was examined in the
mouse model of the in vitro secondary humoral immune response,
suppressed by methotrexate (MTX), a pro-apoptotic agent [21]. We
recently found that model useful to study an immunorestoring
property of lactoferrin [22]. In the present work we used an
unmodified CLA as reference peptide.
2.2. Biology
CLA and its derivatives were tested for their ability to alter the
secondary immune response to SRBC in vitro suppressed by addi-
tion of MTX. The actions of CLA alone (Fig. 2) were typically
2. Results and discussion
immunoregulatory, i.e. 0.1
stimulated, 1 g/ml concentration was without any effect and
10 g/ml concentration strongly inhibited the AFC numbers in
mg/ml concentration of the compound
m
2.1. Chemistry
m
cultures. The actions of CLA on MTX-induced immune response
suppression were differential. The peptide exhibited suppressive,
synergistic action with MTX (dose combinations: 0.1 mg/ml of CLA
We synthesized new CLA analogues 1, 2 starting from their
linear precursors 3, 4, containing conformationally restricted
Leu
Val
Pro
Pro
Leu
Ile
Ile
Phe - Phe
Fmoc
Fmoc OH
Wang
H
TBTU
Fmoc
20% piperidine
H
Fmoc OH
TBTU
Fmoc
H
20% piperidine
OH
Fmoc
Fmoc
TBTU
20% piperidine
H
OH
Fmoc
Fmoc
BTC
20% piperidine
Fmoc
Fmoc
OH
H
TBTU
20% piperidine
Fmoc
Fmoc
H
OH
TBTU
20% piperidine
Fmoc
Fmoc
OH
H
TBTU
20% piperidine
TFA
H
H
OH
EDC / HOBt
Scheme 1. Synthesis of cyclolinopeptide A analogues 1 and 2 containing conformationally restricted dipeptide fragment 6.

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Fig. 2. Effects of CLA on MTX-induced suppression of the humoral immune response in vitro. Mouse splenocytes from SRBC-primed mice were cultured with antigen (0.05 ml of
0.005% SRBC suspension), MTX was added (concentrations of 0.25, 05 or 1.0 mM) at 24 h following initiation of incubation. CLA (concentrations of 0.1, 1 or 10 g/ml) was added in
the beginning of the incubation period. The majority of differences in AFC numbers between respective groups were significant with p < 0.05 or lower. Non-significant differences
were observed for effects of CLA (1 g/ml) vs. control, CLA (0.1 g/ml) þ MTX (0.5 mM) vs. MTX, CLA (10 g/ml) þ MTX (1 mM) vs. MTX and
g/ml) þ MTX (1.0 mM) vs. MTX, CLA (1
CLA (10
g/ml) þ MTX (0.25 mM) vs. CLA.
m
m
m
m
m
m
and 0.25 mM of MTX). A similar property of CLA was observed at
the 10 g/ml concentration of CLA (MTX at concentrations of
0.25 mM and 0.5 mM). On the other hand, CLA at concentration of
1.0 g/ml counteracted the suppressive action of MTX (1.0 mM)
(Fig. 2).
Next, we wished to test how the modifications of the basic CLA
Interestingly, the compound reversed the inhibitory action of MTX
at higher concentrations of this antimetabolite. The effects of
m
peptide 2 on the immune response were slightly inhibitory (1
mg/
m
ml) or negligible (10 g/ml) (Fig. 4), however, it demonstrated, as
m
compound 1, similar immune response restoring properties in
relation to MTX. Some synergistic, inhibitory action was also
structure would alter the properties of CLA in the studied exper-
observed (1 mg/ml of CLA and 0.25 mM of MTX). The effects of CLA
imental model. The effect of cyclic peptide 1 (Fig. 3) on the
analogue 3 on the immune response were not significant (Fig. 5)
immune response was moderately inhibitory at 10
m
g/ml dose.
but the compound counteracted the immunosuppressive action of
Fig. 3. Effects of peptide 1 on MTX-induced suppression of the humoral immune response in vitro. The culture conditions were described in Fig. 2. Peptide 1 (concentrations of 1 or
10 g/ml) was added in the beginning of the incubation period. The majority of differences in AFC numbers between respective groups were significant with p < 0.02 or lower. Non-
significant differences were observed for effects of peptide 1 (1 g/ml) vs. control, peptide 1 (1 g/ml) þ MTX (0.25 mM) vs. peptide 1,
g/ml) þ MTX (0.5 mM) vs. MTX, peptide 1 (10
peptide 1 (10
g/ml) þ MTX (0.5 mM) vs. peptide 1.
m
m
m
m
m

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4611
Fig. 4. Effects of peptide 2 on MTX-induced suppression of the humoral immune response in vitro. The culture conditions were described in Fig. 2. Peptide 2 (concentrations of 1 or
10 g/ml) was added in the beginning of the incubation period. The differences in AFC numbers between respective groups were significant with p < 0.05 or lower except of the
following comparisons: peptide 2 (10 g/ml) vs. control, MTX (0.25 mM) vs. control, peptide 2 (1
g/ml) þ MTX (0.25 mM) vs. peptide 2 and MTX, peptide 2 (10
m
m
m
g/ml) þ MTX (0.25 mM) vs. peptide 2, peptide 2 (1
m
g/ml) þ MTX (0.25 mM) vs.
g/ml) þ MTX (1 mM) vs.
peptide 2 and MTX, peptide 2 (10
peptide 2.
m
mg/ml) þ MTX (0.5 mM) vs. peptide 2 and peptide 2 (10
m
MTX at 10
immunostimulatory properties (Fig. 6). In addition, its antago-
nistic action with regard to MTX effect at 10 g/ml concentration
seemed to be most potent. Fig. 7 shows that linear peptide 5, CLA
linear analogue without ethylene linker, at the concentrations
used did not change the control AFC response. However, the
peptide 5 like peptide 3, at 10 mg/ml concentration, reversed MTX-
induced suppression (statistically significant action of 5 at all MTX
concentrations).
m
g/ml concentration. Compound 4 exhibited marked
2.3. NMR analysis
m
For conformational studies cyclic analogue of CLA 1 was chosen
and various 1H 1D and 2D NMR techniques, including COSY, TOCSY,
NOESY, ROESY and 1He15N HSQC experiments were applied. 1H
NMR spectra of 1 recorded in DMSO-d6 and CDCl3 solutions at
298 K exhibited broad signals, similar to proton spectra recorded
for unmodified CLA and characteristic for conformational flexibility
of peptide [26]. Due to low solubility of peptide in CDCl3 at low
Fig. 5. Effects of peptide 3 on MTX-induced suppression of the humoral immune response in vitro. The culture conditions were described in Fig. 2. Peptide 3 (concentrations of 1 or
10 g/ml) was added in the beginning of the incubation period. The differences in AFC numbers between respective groups were significant with p < 0.05 or lower except of the
following comparisons: peptide 3 (1 or 10 g/ml) vs. control, peptide 3 (1 g/ml) þ MTX (0.25, 0.5 or 1 mM) vs. peptide 3.
g/ml) þ MTX (0.25, 0.5 or 1 mM) vs. MTX, peptide 3 (10
m
m
m
m

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4612
Fig. 6. Effects of peptide 4 on MTX-induced suppression of the humoral immune response in vitro. The culture conditions were described in Fig. 2. Peptide 4 (concentrations of 1 or
10 g/ml) was added in the beginning of the incubation period. The differences in AFC numbers between respective groups were significant with p < 0.01 or lower except of the
following comparisons: MTX (0.25 mM) vs. control, peptide 4 (1
g/ml) þ MTX (0.25, 0.5 or 1 mM) vs. MTX.
m
m
temperature (max. 0.5 mM) the cryo-probe was applied within the
temperature range 214e298 K to increase the sensitivity of NMR
measurements (Fig. 8). Signals became narrower when tempera-
ture decreased, but even at 214 K 1H NMR spectrum of 1 was poorly
resolved. 1He15N HSQC spectrum (Fig. 9) recorded at 214 K suggests
that CLA analogue 1 is a mixture of at least two isomers, most
probably due to cisetrans isomerization of Pro-Pro amide bond. We
were able to assigned only few resonances for major isomer: Pro2
similar effect of spatial proximity of Pro2 and phenyl ring of Phe3
was observed for the native CLA. Thus, from NMR analysis it can be
concluded that introduction of the ethylene bridge causes the
opposite to expected effect on rigidity of the molecule. Presence of
locally stiffening element causes increase of overall flexibility of
molecule. This effect originates from two factors. First, phenylala-
nine amide nitrogens cannot act as donors of hydrogen bonds
stabilizing CLA ring. Secondly, the presence of piperazinone ring is
a source of additional conformational changes. Theoretical studies
of N, N0-bridged N-acetylated Phe-Phe N00-methylamide (Ac-Phe-
Phe-NHMe) indicated equal preferences of two enantiomeric
(CH
e 3.36, 3.47 ppm) and Phe3 (Ar2,6 protons at 6.94 ppm). The strong
up-field shift of Pro2 CH
protons confirms the assignment, as the
a e 4.48 ppm, CHb e 0.12, 1.04 ppm, CHg e 1.44, 1.55 ppm, CHd
b
Fig. 7. Effects of peptide 5 on MTX-induced suppression of the humoral immune response in vitro. The culture conditions were described in Fig. 2. Peptide 5 (concentrations of 1 or
10 g/ml) was added in the beginning of the incubation period. The differences in AFC numbers between respective groups were significant with p < 0.05 or lower except of the
following comparisons: peptide 5 (10 g/ml) vs. control, peptide 5 (1
g/ml) þ MTX (0.25 or 1 mM) vs. MTX.
m
m
m

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4613
Fig. 8. Temperature effect on 1H NMR spectra of peptide 1 recorded at 214e298 K (600 MHz, CDCl3, cryo-probe).
twisted chair/boat-like pucker types for the piperazinone ring.
Theoretically, it was predicted that N, N0-ethylene bridge
constraints the backbone fragment to a narrow range of confor-
mations strictly related with a conformation of the piperazinone
ring [27]. The presence of three conformers of the piperazinone
rings due to different orientations of the side chains was proposed
on the basis of NMR and X-ray analyses [28,29]. Analysis of 1H NMR
spectra of Fmoc derivatives of dipeptides 7a and 7b in CDCl3
revealed the presence of two conformers in 65:35 ratio.
NH Val9 and Ile7 C]O residues. The results of theoretical calcula-
tions are consistent with observed by NMR spectroscopy the flex-
ibility of new CLA analogues containing the ethylene bridge
between phenylalanine residues.
2.4. Molecular dynamics
Very limited number of NOE restraints made the classical
structure determination and conformational analysis impossible.
Therefore, two different force fields CHARMM27 [30] and GROMOS
53A6 [31] were applied to compare and verify results of unre-
strained molecular dynamics calculations for two isomers of
peptide 1, differentiated on cis or trans geometry of Pro1-Pro2
peptide bond. The calculated structures were similar indepen-
dently on applied force fields and the representative structures of
calculated main clusters of peptide 1 and its all trans isomer are
presented on Fig. 10.
The backbone conformation of CLA in the solid state is
stabilized by the intramolecular hydrogen bonds: NH of Leu5 and
C]O of Phe3, NH of Ile7 and C]O of Phe4, NH of Leu8 and C]O of
Leu5, NH of Phe4 and C]O of Val9, NH of Val9 and C]O of Phe4.
All of these hydrogen bonds were found in the CLA solution
based on temperature coefficients of chemical shifts of NH
protons [26].
The analysis of hydrogen bonds along the trajectories evidenced
that high number of stabilizing structures hydrogen bonds (above
50%) (Table 1) is present only between NH Ile7 and Phe4 C]O and
Fig. 9. 1He15N HSQC spectrum of peptide 1 recorded at 214 K (CDCl3, 600 MHz, cryo-
probe).

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Fig. 10. The representative structures of calculated main clusters of peptide 1 (left) and its all trans isomer (right).
Table 1
MTX. The best antagonistic actions were demonstrated by cyclic
peptide 1 and linear peptide 4.
Populations of hydrogen bonds between different residues in theoretical structure of
CLA analogue 1.
Small peptides exist in the solution as a complex mixture of
conformers and the bioactive conformation can be one of them
[32]. Peptide 1 was more flexible than CLA, as evidenced by NMR
measurements and confirmed by theoretical calculations, but the
conformational flexibility alone appeared to be not sufficient to
change effects of MTX. Linear peptides 3 and 5 exhibited weak
antagonistic activities.
Donor NeH
Acceptor C]O
Population [%]
Leu5
Leu5
Leu5
Ile6
Pro1
Phe3
Val9
Phe4
Phe4
Leu5
Ile6
27
14
18
10
65
6
Ile7
Leu8
Leu8
Val9
The replacement of Phe residues by
fragment of CLA and its linear analogue led to decrease of the
immunosuppressive activity of resulting CLA analogues [14]. or
conformations of phenylalanine residues in cyclic peptides 1 and
D
-Phe residues in Phe3-Phe4
36
87
Ile7
L
D
2 are not very significant for activity towards MTX. More diverse are
activities of linear peptides 3 and 4.
3. Conclusions
In summary, the phenomenon of antagonistic action of CLA
derivatives against pro-apoptotic agents may be of clinical impor-
tance. In addition, synergistic suppressive effects demonstrated by
CLA and MTX may be useful to plan new therapeutic protocols,
allowing to lower doses of chemotherapeutics.
In this investigation we found that modifications of the native
cyclolinopeptide structure may lead to acquisition of new, inter-
esting properties, potentially desirable in medical practice. First, we
found that the typically immunosuppressive compound CLA may
exert opposite actions on the humoral immune response in vitro,
which were strictly dose-dependent. The low dose of that
compound was stimulatory, high one inhibitory, whereas the
moderate dose had no effect on the immune response. However,
when tested together with MTX, an additive, suppressive effect,
even with the lowest concentrations of both agents was observed.
That result may be of clinical importance, since it creates a possi-
bility of obtaining strong immunosuppressive effect by using
suboptimal (less toxic) doses of MTX. The unmodified CLA was,
however, devoid of antagonistic (presumably anti-apoptotic)
activity directed against MTX with one exception (respective
4. Materials and methods
4.1. General remarks
All solvents were purified by conventional methods. Evapora-
tions were carried out under reduced pressure. Melting points were
determined on a capillary melting point apparatus and are uncor-
rected. The optical rotation was measured in a 1 dm cell (1 ml) on
a Horiba high speed automatic polarimeter at 589 nm. For thin
layer chromatography 250 nm silica gel GF precoated uniplates
(Merck) were used with following solvent systems: (A) CHCl3/
MeOH/AcOH 90:10:2, (B) EtOAc/hexane/AcOH 90:10:1 (C) CHCl3/
MeOH 95:5. The chromatograms were visualized with chlorine
followed by starch/KI and/or ninhydrin. Flash chromatography was
performed with Merck silica gel 60 (230e400 mesh). HPLC was
performed for peptides 1e4 on an LDC/Milton-Roy analytical
instrument using a Vydac C18 column or on C-8 Kromasil column
(4.6 ꢀ 250 mm) (peptide 5): flow 1.0 ml/min, detection at 220 nm
and eluants (A) 0.05% trifluoroacetic acid in water and (B) 0.038%
trifluoroacetic acid in acetonitrile/water 90:10 with a gradient
application. Purification of peptides was performed by preparative
doses of MTX and CLA: 1.0 mM and 1 mg/ml).
An analysis of the effects of the modified CLA structures 1 and 2
and the linear peptides 3e5 (Figs. 3e7), showed that the peptides
exhibited negligible or slightly inhibitory effects in the model of the
in vitro humoral immune response in mice. Stimulatory effects
were observed for linear peptide 4 at 10
similarly as for CLA at concentration of 0.1
m
g/ml concentration,
m
g/ml. The stimulatory
action of these peptides could be similar to that of cyclosporine A,
which at small doses could act as an anti-apoptotic agent [4e6]. We
tested CLA derivatives with three concentrations of MTX, which
typically causes cell apoptosis in culture [5]. The results revealed
that the CLA derivatives exhibited antagonistic activities towards

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4615
reversed-phase HPLC on a Vydac C18 column (22 ꢀ 250 mm)
(peptides 1e4) or C-8 Kromasil column (21.2 ꢀ 250 mm) for
peptide 5; flow rate 16 ml/min, UV detection at 220 nm. The
structures of the pure peptides were confirmed by FAB-MS spec-
troscopy on Finningan MAT 95 mass spectrometer and MALDI-MS
spectroscopy on Voyager Elite mass spectrometer.
NMR spectra were recorded at 700 MHz at 298 K and at
600 MHz using cryo-probe at 214e298 K CDCl3 or CD3CN were used
as solvents with tetramethylsilane (TMS) or solvent residual peak
as the internal standards, respectively. The sample of peptide 1 for
temperature measurements was prepared by dissolving of 4 mg of
the peptide in 0.65 ml of CDCl3 or CD3CN. Signals assignments for
7a and 7b were based on analyses of COSY, HSQC and HMBC
spectra.
127.60 (8 ꢀ CH arom Fmoc), 127.00, 128,60, 128.74 (5 ꢀ CH arom
Phe1), 126.40, 128.20, 129.50 (5 ꢀ CH arom Phe2), 136.30, 136.90
(2 ꢀ CIV arom Phe1,2), 141.30, 143.60 (4 ꢀ CIV arom Fmoc), 154.00
(C]O Fmoc), 169.00 (C]O Phe1), 173.20 (C]O Phe2); FAB HRMS:
[M þ H]þ ¼ 561.23884, calcd. for C35H33O5N2 561.23895.
4.2.2. N-(9-Fluorenylmethoxycarbonyl)-[(2R,30R)-3-phenyl-2-(10-
piperazine-30-benzyl-20-on)] propionic acid (7b)
7b was synthesized in the similar manner (starting from the
same quantity of all reactants) yielding the desired product as
a white powder (2.10 g, 3.75 mM, 75%); [
a
]
D
¼ ꢂ11.20ꢁ (c ¼ 1,
MeOH), Rf (A) ¼ 0.61, Rf (B) ¼ 0.52, Rf (C) ¼ 0.38, mp. 76e78 ꢁC; HPLC
purity ¼ 100% (tR ¼ 15.45 min, 50e80% B, 25 min); 1H NMR (CDCl3,
700 MHz) 2 conformers, major (65%): 2.45, 2.50 (brdd, 1H, J ¼ 3.8
and 12.8, NeCH2eCH2), 3.80, 3.82 (brd, 1H, J ¼ 3.8 and 12.8,
NeCH2eCH2), 2.85e2.92 (brm, 1H, NeCH2eCH2), 2.96e3.03 (brm,
1H, NeCH2eCH2), 2.77e2.85 (brm, 1H, CbeH Phe1), 3.05e3.14 (brm,
1H, CbeH Phe1), 3.18, 3.35 (brdd, AB2, 2H, J ¼ 14.4, 2H, CbeH Phe2),
3.97 (brs, 1H, CH Fmoc), 4.24, 4.26 (brdd, 2H, J ¼ 6.1 and 16.4 CH2
Fmoc), 4.50e4.58 (brm, 1H, CaeH Phe1), 4.92e5.01 (brm, 1H, CaeH
Phe2), 6.86 (brs, 1H, CeH arom Phe2), 7.12e7.18 (brm, 4H, CeH arom
Phe2), 7.19e7.30 (brm, 5H, CeH arom Phe1), 7.29e7.33 (brm, 2H,
CeH arom Fmoc), 7.35e7.41 (brm, 2H, CeH arom Fmoc), 7.44 (brd,
2H, J ¼ 7.5, CeH arom Fmoc), 7.72e7.77 (brm, 2H, CeH arom Fmoc);
minor (35%): 2.45, 2.50 (brdd, 1H, J ¼ 3.8 and 12.8, NeCH2eCH2),
3.46e3.54 (brm, 1H, NeCH2eCH2), 2.77e2.85 (brm, 1H,
NeCH2eCH2), 2.96e3.03 (brm, 1H, NeCH2eCH2), 2.69e2.74 (brm,
1H, CbeH Phe1), 3.05e3.14 (brm, 1H, CbeH Phe1), 3.18, 3.35 (brdd,
AB2, 2H, J ¼ 14.4, CbeH Phe2), 4.13 (brs, 1H, CH Fmoc), 4.48e4.55
(brm, 2H, CH2 Fmoc), 4.82e4.91 (brm, 1H, CaeH Phe1) 4.92e5.01
(brm, 1H, CaeH Phe2), 7.01 (brs, 1H, CeH arom Phe2), 7.12e7.18
(brm, 4H, CeH arom Phe2), 7.19e7.30 (brm, 5H, CeH arom Phe1),
7.29e7.33 (brm, 2H, CeH arom Fmoc), 7.35e7.41 (brm, 2H, CeH
arom Fmoc), 7.51 (brd, 2H, J ¼ 7.5, CeH arom Fmoc), 7.72e7.77
(brm, 2H, CeH arom Fmoc); 13C NMR (CDCl3, 62.53 MHz): major
(65%): 34.80 (CbeH Phe2), 38.00 (CbeH Phe1), 39.00 (NeCH2eCH2),
46.60 (NeCH2eCH2), 48.00 (CH Fmoc), 59.05 (CaeH Phe), 61.10
(CaeH Phe), 68.60 (CH2 Fmoc), 121.00, 126.00, 127.50, 128.60
(8 ꢀ CH arom Fmoc), 128.40, 128.60, 129.74 (5 ꢀ CH arom Phe1),
127.40, 129.20, 130.00 (5 ꢀ CH arom Phe2), 137.30, 137.90 (2 ꢀ CIV
arom Phe1,2), 142.30, 145.40 (4 ꢀ CIV arom Fmoc), 155.60 (C]O
Fmoc), 169.50 (C]O Phe1), 174.20 (C]O Phe2); minor (35%): 34.71
(CbeH Phe2), 37.80 (CbeH Phe1), 40.00 (NeCH2eCH2), 47.01
(NeCH2eCH2), 48.10 (CH Fmoc), 59.60 (CaeH Phe), 51.50 (CaeH
Phe), 68.30 (CH2 Fmoc), 121.00, 126.00, 127.50, 128.60 (8 ꢀ CH arom
Fmoc), 128.40, 128,60, 129.74 (5 ꢀ CH arom Phe1), 127.40, 129.20,
123.00 (5 ꢀ CH arom Phe2), 137.30, 137.90 (2 ꢀ CIV arom Phe1,2),
142.30, 145.40 (4 ꢀ CIV arom Fmoc), 155.00 (C]O Fmoc), 169.70
(C]O Phe1), 174.70 (C]O Phe2); FAB HRMS: [M þ H]þ ¼ 561.23801,
calcd. for C35H33O5N2 561.23895.
4.2. Peptide synthesis and purification
4.2.1. N-(9-Fluorenylmethoxycarbonyl)-[(2S,30S)-3-phenyl-2-(10-
piperazine-30-benzyl-20-on)] propionic acid (7a)
To the suspension of [(2S,30S)-3-phenyl-2-(10-piperazine-30-
benzyl-20-on)] propionic acid (1.70 g, 5 mM) and K2CO3 (1.52 g,
11 mM) in water (20 ml) and acetone (25 ml), Fmoc-OSu (1.92 g,
5.7 mM) was added in several portions under vigorous stirring and
pH maintained within the range 8O9. The reaction mixture had
been stirred overnight at room temperature and then acetone was
evaporated. The residue was diluted with water (20 ml), the solu-
tion was extracted with Et2O (3 ꢀ 10 ml) and the ether extracts
were discarded. The water layer was acidified at 0 ꢁC with 6 N HCl
to reach the pH range 2O3 and the white suspension was extracted
with EtOAc (3 ꢀ 20 ml). The combined organic extracts were
washed with water (3 ꢀ 10 ml) and dried over MgSO4. The solvent
was evaporated, the crude product precipitated after addition of
hexane and product was isolated as a white powder (2.30 g,
4.10 mM, 82% yield); mp 77e79 ꢁC; [
a
]
¼ þ12.90ꢁ (c ¼ 0.5, MeOH),
D
Rf (A) ¼ 0.61, Rf (B) ¼ 0.52, Rf (C) ¼ 0.38, HPLC purity 100%
(tR ¼ 15.38 min, 50e80% B, 25 min); 1H NMR (CDCl3, 700 MHz) 2
conformers, major (65%): 2.49e2.58 (brm, 1H, NeCH2eCH2), 3.84
(brd, 1H, J ¼ 12.5, NeCH2eCH2), 2.87e2.96 (brm, 1H, NeCH2eCH2),
2.77e2.84, (brm, 1H, CbeH Phe1), 3.02e3.12 (brm, 2H, NeCH2eCH2
CbeH Phe1) 3.15, 3.37 (brdd, AB2, 2H, J ¼ 14.6, CbeH Phe2), 3.96 (brs,
1H, CH Fmoc), 4.23, 4.25 (brdd, 2H, J ¼ 5.8 and 16.4, CH2 Fmoc), 4.57
(brs, 1H, CaeH Phe1), 5.07 (brs, 1H, CaeH Phe2), 6.88 (brs, 1H, CeH
arom Phe2), 7.13e7.19 (brm, 4H, CeH arom Phe2), 7.17e7.28 (brm,
5H, CeH arom Phe1), 7.28e7.33 (brm, 2H, CeH arom Fmoc),
7.35e7.41 (brm, 2H, CeH arom Fmoc), 7.44 (brd, 2H, J ¼ 7.6, CeH
arom Fmoc), 7.72e7.78 (brm, 2H, CeH arom Fmoc); minor (35%):
2.49e2.58 (brm, 1H, NeCH2eCH2), 3.51, 3.53 (brdd, 1H, J ¼ 6.1 and
12.5, NeCH2eCH2), 2.70e2.75 (brm, 2H, NeCH2eCH2 CbeH Phe1),
3.02e3.12 (brm, 2H, NeCH2eCH2 CbeH Phe1), 3.15, 3.37 (brdd, AB2,
2H, J ¼ 14.6, CbeH Phe2), 4.13 (brs, 1H, CH Fmoc), 4.48e4.54 (brm,
2H, CH2 Fmoc), 4.89 (brs, 1H, CaeH Phe1) 5.07 (brs, 1H, CaeH Phe2),
7.00 (brs, 1H, CeH arom Phe2), 7.13e7.19 (brm, 4H, CeH arom Phe2),
7.17e7.28 (brm, 5H, CeH arom Phe1), 7.28e7.33 (brm, 2H, CeH
arom Fmoc), 7.35e7.41 (brd, 2H, CeH arom Fmoc), 7.51 (brd, 2H,
4.2.3. Solid phase peptide synthesis
The linear peptides were prepared manually using standard
solid-phase procedure on the p-alkoxybenzylalcohol (Wang) resin.
Fmoc-Ile-OH attached to Wang resin was prepared through ester-
ification reaction using Mitsunobu methods [33,34]. Unreacted
hydroxyl groups were acetylated with the aid of acetic anhydride,
pyridine, dichloromethane (1:2:3). Standard single TBTU protocol
(3 eq) was used for all single amino acid derivatives (3 eq) and was
repeated if Kaiser or chloranil (for proline residue) tests were
slightly positive [35,36]. Deprotection of Fmoc groups were
accomplished by treating of resin with 20% piperidine solution in
dimethylformamide (twice 10 ml: 20 min and 10 min). Standard
washing of resin was accomplished using DMF (3 ꢀ 10 ml), DCM
(3 ꢀ 10 ml), MeOH (3 ꢀ 10 ml), DCM (3 ꢀ 10 ml). The peptide resin,
J ¼ 7.6, CeH arom Fmoc), 7.72e7.78 (brm, 2H, CeH arom Fmoc); 13
C
NMR (CDCl3, 62.53 MHz): major (65%): 33.80 (CbeH Phe2), 37.20
(CbeH Phe1), 38.10 (NeCH2eCH2), 45.00 (NeCH2eCH2), 47.10 (CH
Fmoc), 58.05 (CaeH Phe), 60.10 (CaeH Phe), 67.30 (CH2 Fmoc),
120.00, 124.80, 126.50, 127.60 (8 ꢀ CH arom Fmoc), 127.70, 128.60,
128.74 (5 ꢀ CH arom Phe1), 126.40, 128.20, 129.50 (5 ꢀ CH arom
Phe2), 136.30, 136.90 (2 ꢀ CIV arom Phe1,2), 141.40, 143.47 (4 ꢀ CIV
arom Fmoc), 154.67 (C]O Fmoc), 168.53 (C]O Phe1), 173.70 (C]O
Phe2); minor (35%): 34.00 (CbeH Phe2), 36.80 (CbeH Phe1), 39.00
(NeCH2eCH2), 45.01 (NeCH2eCH2), 47.10 (CH Fmoc), 58.50 (CaeH
Phe), 59.50 (CaeH Phe), 67.50 (CH2 Fmoc), 120.00, 124.80, 126.50,

ꢀ
J. Katarzynska et al. / European Journal of Medicinal Chemistry 46 (2011) 4608e4617
4616
after being dried under reduced pressure over KOH and P2O, was
cleaved with the mixture of TFA/H2O/anisol (95:5:3) for 3 h at room
temperature. TFA was removed by evaporation and the crude
peptide was washed several times with diethyl ether and dried.
Purification of linear precursors has been achieved by preparative
HPLC with the proper linear gradient. After purification peptides
were lyophilized from tert-butanol/water and were cyclized by
means of EDC (3 eq) in the presence of HOBt (3 eq) and DIPEA (6 eq)
in DCM at much lower concentration (40 mg of peptide in 800 ml of
DCM) than was usually described for “head to tail” peptide cycli-
zation reactions. Crude cyclic peptides were purified by preparative
purity ¼ 100%, tR ¼ 15.59 min, 50e90% B in 25 min) MALDI MS:
1088.9 [M þ Na]þ, 1104.8 [M þ K]þ calcd. for C59H87O9N9 1066.28.
4.3. Biological tests
4.3.1. Mice
12-week-old BALB/c female mice were used for the study. The
mice were fed a granulated, commercial food and filtered tap water
ad libitum.
4.3.2. Reagents
HPLC with linear gradient 50e95% B in A in 40 min,
l
¼ 220 nm (B:
Methotrexate was the product of LACHEMA (Czech Republik),
Sheep red blood cells (SRBC) were provided by the Wroclaw Agri-
culture Academy. RPMI-1640 medium (Vibi/Life Technologies, UK),
FCS-foetal calf serum (Gibco).
0.038 trifluoroacetic acid in acetonitrile/water 90:10, A: 0.05% tri-
fluoroacetic acid in water) The homogeneity of all peptides was
checked by analytical HPLC and structures were confirmed by
MALDI MS and NMR spectra.
4.3.3. Preparation of the compounds for the in vitro culture
4.2.3.1. Linear peptides 3e5
The peptides (4 mg) were initially dissolved in a mixture
4.2.3.1.1. H-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile-Ile-OH (3). 300 mg
(0.53 mM/g) of Wang resin has been used for the preparation of the
crude linear peptide 3 (215 mg, HPLC purity ¼ 73%, tR ¼ 11.23 min,
40e80% B in 25 min). The crude peptide (30 mg) was purified using
preparative HPLC (tR ¼ 21.09 min, 40e80% B in 40 min), giving pure
nonapeptide 3 (13.3 mg, 44%, HPLC purity ¼ 100%, tR ¼ 11.41 min,
40e80% B in 25 min). MALDI MS: 1106.3 [M þ Na]þ,1122.3 [M þ K]þ
calcd. for C59H89O10 N9 1084.28.
(100 mL) of Cremophor (Sigma) and ethanol (34:64 ratio). Then the
peptides were diluted with RPMI 1640 medium.
4.3.4. The secondary humoral immune response in vitro
Mice were primed with 0.2 ml 1% sheep red blood cell (SRBC)
suspension intraperitoneally (i.p.). After 4 days the splenocytes
were isolated and a single cell suspension was prepared in a culture
medium consisting of RPMI 1640, supplemented with 10% foetal
calf serum, glutamine, sodium pyruvate, 2-mercaptoethanol and
antibiotics. The cells were incubated in 24-well culture plates
4.2.3.1.2. H-Leu-Val-Pro-Pro- -Phe-d-Phe-Leu-Ile-Ile-OH (4).
D
Starting from the same quantity of reactants, the crude peptide 4
has been synthesized in the similar manner as peptide 3 (210 mg,
HPLC purity ¼ 68%, tR ¼ 13.54 min, 40e80% B in 25 min). After
purification of 30 mg using preparative HPLC: tR ¼ 23.29 min,
40e80% B in 40 min, the pure nonapeptide has been obtained
(12.4 mg, 41%, HPLC purity ¼ 100%, tR ¼ 13.79 min, 40e80% B in
25 min) MALDI MS: 1106.3 [M þ Na]þ, 1122.3 [M þ K]þ calcd. for
C59H89O10 N9 1084.28.
(5 ꢀ 106/well/ml) with addition of 50
mL 0.005% SRBC. MTX was
added to the cell cultures at 24 h following initiation of incubation.
The compounds were added to the cultures in the beginning of the
incubation period. After 4 days of incubation in a cell culture
incubator the number of antibody-forming- cells (AFC) was deter-
mined using an assay of local haemolysis in agar gel [37].
4.2.3.1.3. H-Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile-Ile-OH (5). Peptide
was synthesized using CEM Liberty microwave peptide synthesizer
on 2-chlorotrityl resin loaded with isoleucine 1.5 mM/g. The
synthesis was conducted with temperature limitations 50 ꢁC.
Cleavage was accomplished with 5% TFA in DCM for 5, when
30 min. After purification of 30 mg using preparative HPLC:
tR ¼ 27.1 min, 20e60% B in 32 min, the pure nonapeptide has been
obtained (10 mg, 30%, HPLC purity ¼ 100%, tR ¼ 15.5 min, 20e60% B
in 16 min). FAB MS: 1056.7 [M ꢂ H]ꢂ, 1058.7 [M þ H]þ calcd. for
C57H87O10 N9 1057.66.
4.3.5. Statistics
The data were statistically evaluated using Student’s t-test. The
results are presented as mean values of AFC from quadruplicate
determinations ꢃstandard error. Results are considered significant
when p value is equal or lower than 0.05. Not significant results
were indicated in description of figures.
4.4. Molecular dynamics
MD simulations were performed using NAMD package and
CHARMM27 force field [30] or GROMACS package with GROMOS
53A6 force field [31]. The peptide and chloroform molecules were
placed in a cubic box with the edge length of 50 Å. In the first 300 ps
period temperature of 300 K and pressure of 1 bar was reached,
with positions of peptide backbone and ethylene bridge atoms
frozen. Then molecular dynamics (20 ns) was performed at
constant temperature and pressure. Integration time was 1 fs and
all bonds were constrained employing the SHAKE algorithm. Elec-
trostatic interactions for distances shorter than 10 Å were evaluated
using Coulomb equation and other interactions were evaluated by
means of Particle-Mesh Ewald algorithm with switching distance of
1 Å. Van der Waals interactions were calculated using Lennard-
Jones potential for distances shorter than 10 Å. Cartesian coordi-
nates for peptide 1 molecule were saved every 10 ps.
4.2.3.2. Cyclic peptides 1 and 2
4.2.3.2.1. c(Leu-Val-Pro-Pro-Phe-Phe-Leu-Ile-Ile) (1). 40 mg of
the linear precursor 3, 17 mg HOBt (0.111 mM) and 19.3 mL DIPEA
(0.111 mM) were dissolved in 800 ml DCM. After 3 min, 21.3 mg EDC
(0.111 mM) was added. The solution was stirred for 10 days at
ambient temperature and then was washed with water
(3 ꢀ 200 ml) and evaporated. The crude residue (42 mg, HPLC
purity ¼ 49%, tR ¼ 14.75 min, 50e90% B in 25 min) was dried under
reduced pressure over KOH and P2O5 and purified using preparative
HPLC (tR ¼ 25.42 min, 50e95% B in 40 min), yielding desired pure
cyclic CLA analogue
1
(10 mg, 24%, HPLC purity
¼
100%,
tR ¼ 14.93 min, 50e90% B in 25 min). MALDI MS: 1088.9 [M þ Na]þ,
1104.8 [M þ K]þ calcd. for C59H87O9N9 1066.28.
4.2.3.2.2. c(Leu-Val-Pro-Pro-d-Phe-d-Phe-Leu-Ile-Ile) (2). Starting
from the same quantity of reactants, the crude peptide 2 has been
synthesized in the similar manner as compound 1 (43 mg, HPLC
purity ¼ 46%, tR ¼ 15.75 min, 50e90% B in 25 min). After purification
(preparative HPLC: tR ¼ 26.37 min, 50e95% B in 40 min), the desired
pure cyclic analogue has been obtained (9 mg, 21%, HPLC
Acknowledgements
We are thankful to Dr. Krzysztof Kierus for synthesis of linear
peptide 5. This work was supported by the Ministry of Science and
Information Technology, Grant No 2 505F 035 28.

ꢀ
J. Katarzynska et al. / European Journal of Medicinal Chemistry 46 (2011) 4608e4617
4617
[14] M. Cebrat, M. Lisowski, I.Z. Siemion, Z. Wieczorek, The cyclolinopeptide A
Abbreviations
analogs with
1401e1412.
ꢀ
D-Phe, D-Tyr, and L and D-Trp residues, Pol. J. Chem. 71 (1997)
[15] J. Katarzynska, A. Mazur, M. Bilska, E. Adamek, M. Zimecki, S. Jankowski,
J. Zabrocki, Synthesis and immunosuppressive activity of new cyclo-
AcOH
BTC
CsA
acetic acid
bis(trichloromethyl)carbonate
cyclosporin A
linopeptide
A analogs modified with b-prolines, J. Pept. Sci. 14 (2008)
1283e1294.
[16] K. Kaczmarek, K.B. Farina, P. Zubrzak, S. Jankowski, M. Zimecki, P. Suder,
E. Benedetti, R. Fattorruss, M. Saviano, J. Zabrocki, Synthesis, conformational
DCM
DIPEA
dichloromethane
diisopropylethylamine
analysis and immunological activity of b3Phe-substituted cyclolinopeptide A
analogues, J. Pept. Sci. 15 (2009) 166e174.
DMSO dimethylsulphooxide
DIPEA diisopropylethylamine
DMAP N,N-dimethylaminopyridine
[17] P. Dryga1a, J. Olejnik, A. Mazur, K. Kierus, S. Jankowski, M. Zimecki, J. Zabrocki,
Synthesis and immunosuppressive activity of cyclolinopeptide A analogues
containing homophenylalanine, Eur. J. Med. Chem. 44 (2009) 3731e3738.
[18] B. Lammek, M. Czaja, I. Derdowska, E. qempicka, P. Sikora, W. Szkróbka,
H.L. Trzeciak, Biologically active analogs of arginine vasopresin containing
conformationally restricted dipeptide fragments, J. Pept. Res. 51 (1998)
149e154.
DMF
EDC
dimethylformamide
N-(3-dimethylaminopropyl)N0-ethylcarbodiimide
ethyl acetate
EtOAc
HOBt
MTX
PPCA
TBTU
[19] I. Trzeciak, W. Kozik, S. Melhem, A. Kania, D. Dobrowolski, A. Prahl,
I. Derdowska, B. Lampek, New bradykinin analogs in contraction of rat uterus,
Peptides (New York) 21 (2000) 829e834.
hydroxybenzotriazol
methotrexate
1-propanephosphonic acid cyclic anhydride
O-benzotriazol-1-yl-N,N,N0,N0-tetramethyl-uronium
tetrafluoroborate
ꢁ
[20] E. qempicka, I. Derdowska, B. Jastrze˛bska, P. Kuncarova, J. Slaninowa,
B. Lammek, Analogs of oxytocin conformationally restricted in the N-terminal
part of the molecule, J. Pept. Res. 57 (2001) 162e167.
[21] C. Toniolo, Conformationally restricted peptides through short-range cycli-
zations, Int. J. Pept. Protein Res. 35 (1990) 287e300.
[22] J. Artym, M. Zimecki, M. Kruzel, Effect of lactoferrin on the methotrexate-
induced suppression of the cellular and humoral immune response in mice,
Anticancer Res. 24 (2004) 3831e3836.
TEA
TFA
THF
triethylamine
trifluoroacetic acid
tetrahydrofuran
[23] L.N. Schoenberg, D.W. Cooke, C.F. Liu, Nuclear magnetic resonance determi-
nation of the absolute configuration of complexes of cobalt (III) with asym-
metric tetradente ligands, Inorg. Chem. 7 (1968) 2386e2393.
Appendix. Supplementary data
[24] T. Yamashita, H. Takenaka, Y. Kojima, Preparations of N, N0-ethylene-bridged
dipeptides (eXX) constructed from (S)-methionine, -tryptophan, -tyrosine and
eN(ε)-benzyloxycarbonyllysine, Amino Acids 4 (1993) 187e192.
[25] E. Falb, T. Yechezkel, Y. Salitra, C. Gilon, In situ generation of Fmoc-amino acid
chlorides using bis-(trichloromethyl)carbonate and its utilization for difficult
couplings in solid-phase peptide synthesis, J. Pept. Res. 53 (1999) 507e517.
[26] B. Di Biasio, F. Rossi, E. Benedetti, V. Pavone, C. Pedone, P.A. Temussi,
G. Zanotti, T. Tancredi, Bioactive peptides: solid-state and solution confor-
mation of cyclolinopeptide A, J. Am. Chem. Soc. 111 (1989) 9089e9098.
[27] C. Czaplewski, B. Lammek, J. Ciarkowski, Conformational constraints emerging
in the N, N0-ethylene- and N, N0-propylene-bridged dipeptide units, Pol. J.
Chem. 68 (1994) 2589e2598.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.ejmech.2011.07.040.
References
[1] R. Storb, H.J. Deeg, J. Whitehead, F. Appelbaum, P. Beatty, W. Bensinger,
D. Buckner, R. Clift, K. Doney, V. Farewell, Methotrexate and cyclosporine
compared with cyclosporine alone for prophylaxis of acute graft-versus-host
disease after marrow transplantation for leukemia, N. Engl. J. Med. 314
(1986) 729e735.
[2] N.J. Chao, G.M. Schmidt, J.C. Niland, M.D. Mylon, A.C. Dagis, G.D. Long,
A.P. Nademanee, R.S. Negrin, M.R. O’Donnell, P.M. Parker, Cyclosporine,
methotrexate, and prednisone compared with cyclosporine and prednisone
for prophylaxis of acute-graft-versus host disease, N. Engl. J. Med. 329 (1993)
1225e1230.
[3] R.A. Nash, L.A. Pineiro, R. Storb, J.H. Deeg, W.E. Fitzsimmons, T. Furlong,
I.A. Hansen, T. Gooley, R.M. Maher, P. Martin, FK506 combination with
methotrexate for the prevention of graft-versus-host disease after marrow
transplantation from matched unrelated donors, Blood 88 (1996) 3634e3641.
[4] L. Genestier, M.eT. Dearden-Badet, N. Bonnefoy-Berard, G. Lizard, J.-
P. Revillard, Cell Immunol. 155 (1994) 283e291.
[5] M.K. Jenkins, R.H. Schwartz, D.M. Pardoll, Science 241 (1988) 1655e1658.
[6] A. Gafter-Gvilli, Y. Kalechman, B. Sredni, R. Gal, U. Gafter, Arch. Dermatol. Res.
296 (2004) 265e269.
[28] A. Michel, G. Evrard, B. Norberg, Etude conformationnelle de cetopiperazines:
(I) 3R-methyl-6-carbethoxy-2 oxopiperazine, (II) 3R-[p-hydroxybenzyl]-6-
carbethoxy-2 oxopiperazine: I, II en solution et structure crystalline de II,
Can. J. Chem. 65 (1987) 1308e1312.
[29] Y. Kojima, Y. Ikeda, E. Kumata, J. Maruo, A. Okamoto, K. Hitotsu, K. Shibata,
A. Ohsuka, Preparations, solution conformations and molecular structures of
N, N0-ethylene-bridged dipeptides and their derivatives, Int. J. Pept. Protein
Res. 37 (1991) 468e475.
[30] A.D. MacKarell, D. Bashford, M. Bellot, R.L. Dunbrack, J.D. Evanseck, M.J. Field,
S. Fischer, J. Gao, H. Guo, S. Ha, D. Joseph-McCarthy, L. Kuchnir, K. Kuczera,
F.T.K. Lau, C. Mattos, S. Michnick, T. Ngo, D.T. Nguyen, B. Prodhom,
W.E. Reiher, B. Roux, M. Schlenkrich, J.C. Smith, R. Stote, J. Straub,
M. Watanabe, J. Wiórkiewicz-Kuczera, D. Yin, M. Karplus, All-atom empirical
potential for molecular modeling and dynamics studies of proteins, J. Phys.
Chem. B. 102 (1998) 3586e3616.
[31] C. Oestenbrink, A. Villa, A.E. Mark, W.F. van Gunsteren, A biomolecular force
field based on the free enthalpy of hydration and solvation: the GROMOS
force-field parameter sets 53A5 and 53A6, J. Comput. Chem. 25 (2004)
1656e1676.
[7] H.P. Kaufmann, A. Tobschirbel, Ube rein Oligopeptid aus Leinsamen, Chem.
Ber. 92 (1959) 2805e2809.
[8] I.Z. Siemion, M. Cebrat, Z. Wieczorek, Cyclolinopeptides and their analogs e
[32] P.A. Temussi, D. Picone, M.A. Castiglione-Morelli, A. Motta, T. Tancredi,
Bioactive conformation of linear peptide in solution: an elusive goal?
Biopolymers 28 (1989) 91e107.
[33] T.A. Rano, K.T. Chapman, Solid phase synthesis of aryl ethers via Mitsunobu
reaction, Tetrahedron Lett. (1995) 3789e3792.
a
new family of peptide immunosuppressants affecting the calcineurin
system, Arch. Immunol. Ther. Exp. 47 (1999) 143e153.
[9] T.J. Gaymes, M. Cebrat, I.Z. Siemion, J.E. Kay, Cyclolinopeptide (CLA) mediates
its immunosuppressive activity through cyclophilin-dependent calcineurin
inactivation, FEBS Lett. 418 (1997) 224e227.
[34] L.S. Richter, T.R. Gadek, A surprising observation about Mitsunobu reactions in
solid phase synthesis, Tetrahedron Lett. 35 (1994) 4705e4706.
[35] E. Kaiser, R.L. Colescot, C.D. Bossinger, P.I. Cook, Color test for detection of free
terminal amino groups in the solid-phase synthesis of peptides, Anal. Bio-
chem. 34 (1970) 595e598.
[36] T. Vojkovsky, Detection of secondary amines on solid phase, Pept. Res. 8
(1995) 236e237.
[37] R.I. Mishell, R.W. Dutton, Immunization of dissociated spleen cell cultures
from normal mice, J. Exp. Med. 126 (1967) 423e442.
[10] E. Benedetti, C. Pedone, Cyclolinopeptide A: inhibitor, immunosuppressor or
other? J. Pept. Sci. 11 (2005) 268e272.
[11] B. Picur, M. Cebrat, J. Zabrocki, I.Z. Siemion, Cyclopeptides of Linum usita-
tissimum, J. Pept. Sci. 12 (2006) 569e574.
[12] Z. Wieczorek, B. Bengtsson, J. Trojmar, I.Z. Siemion, Immunosuppresive
activity of cyclolinopeptide A, Pept. Res. 4 (1991) 275e283.
[13] Z. Wieczorek, M. Zimecki, A. Pe˛dyczak, M. Lisowski, I.Z. Siemion, Immuno-
suppressive activity of alanine analogs of cyclolinopeptide A, Arch. Immunol.
Ther. Exp. 41 (1993) 291e296.