Synthesis and immunosuppressive activity of cyclolinopeptide A analogues containing homophenylalanine

Article abstract of DOI:10.1016/j.ejmech.2009.03.037

Immune response suppressors are used in the medical praxis to prevent graft rejection after organ transplantation and in the therapy of some autoimmune diseases. Cyclolinopeptide A, naturally existing immunomodulatory peptide, was modified with homophenyl

Full text of DOI:10.1016/j.ejmech.2009.03.037

European Journal of Medicinal Chemistry 44 (2009) 3731–3738
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and immunosuppressive activity of cyclolinopeptide A analogues
containing homophenylalanine
,
Patrycja Dryga1a a, Jadwiga Olejnik a, Adam Mazur a, Krzysztof Kierus a, Stefan Jankowski a
**
*
,
,
Micha1 Zimecki b, Janusz Zabrocki a
a
_
Institute of Organic Chemistry, Department of Chemistry, Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
b Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, R. Weigla 12, 53-114 Wroclaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Immune response suppressors are used in the medical praxis to prevent graft rejection after organ
transplantation and in the therapy of some autoimmune diseases. Cyclolinopeptide A, naturally existing
immunomodulatory peptide, was modified with homophenylalanine in positions 3 (4), 4 (5) or both 3
and 4 (6). The conformational influence of the replacement of Phe by Hphe was analyzed by NMR
spectroscopy. Peptides 4–6 exist as single isomers with all trans peptide bonds except cis Pro–Pro
peptide bond. The peptides were tested for their ability to suppress the proliferative response of mouse
splenocytes to T- and B-cell mitogens and the secondary humoral immune response to sheep eryth-
rocytes in vitro in parallel with a reference drug – cyclosporine A. The substitution of Phe with Hphe in
positions 3 and 4 of CLA led to three different activities in the studied immunological assays. Very
potent inhibition of AFC number of peptide 4 was not associated with cell toxicity. This compound
caused a complete block of T- and B-cell proliferation. Peptides 5 and 6, containing Hphe in position 3 or
3 and 4, respectively, gave similar effects on the proliferative response of splenocytes to mitogens.
Peptide 6 was a moderate suppressor of the humoral immune response, peptide 5 was exceptionally
inhibitory. The presence of Hphe in position 4 of CLA backbone markedly reduced the viability of the
tested cell line, however addition of the second Hphe in position 3 improved cell survival in comparison
with the solvent.
Received 25 April 2008
Received in revised form
17 March 2009
Accepted 26 March 2009
Available online 5 April 2009
Keywords:
Cyclolinopeptide A
Immune suppression
Homophenylalanine
Cytotoxity
Ó 2009 Elsevier Masson SAS. All rights reserved.
1. Introduction
group of naturally existing immunomodulatory peptides and its
analogues, is an important task for medicinal chemistry.
The problem of transplantology, a dynamically expanding
branch of contemporary medicine, is not merely the problem of
exquisite surgical technique, but also, and even more importantly,
the problem of transplanted organ survival. The inhibitors of PPI-
ases, such as cyclosporine A (CsA) [1], Tacrolimus (FK-506) [2] and
rapamycin [1] are effective immune suppressors able to cause
a selective inhibition of lymphocyte T activation during immune
response. All three compounds are potent drugs, preventing graft
rejection after organ transplantation and in the therapy of some
autoimmune diseases. Because of the significant side effects of CsA
and Tacrolimus therapies, a wider use of these drugs meets great
limitation [2]. Thus, the search for new immune suppressors with
a comparable efficacy but with lower toxicity, especially in the
Cyclolinopeptide A (CLA), a natural highly hydrophobic cyclic
nonapeptide: c(-Pro1-Pro2-Phe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9-) [3],
isolated from linseed oil [4], was found to possess a strong immu-
nosuppressive activity comparable at low doses with that of (CsA)
[5,6] with a mechanism that depends on the inhibition of the
interleukin-1 and interleukin-2 action. The determination of the
X-ray structure and the NMR structural analysis of CLA [7–9], gave
the basis to study the biological, structural, and conformational
properties of CLA and related compounds [10–20].
As a part of structure–activity relationships studies a great
number of CLA analogues were synthesized and their biological
activity was evaluated, but none of these analogues showed the
immunosuppressive potency higher than the native peptide
[21,22]. The conformational flexibility of CLA molecule is believed
to be one of the most important factors responsible for its biological
activity. In addition, it was suggested, that the presence of the Pro-
Pro-Phe-Phe tetrapeptide unit sequence, containing the Pro–Pro
peptide bond in cis geometry and the ‘‘edge to face’’ interaction
* Corresponding author. Tel.: þ48 42 631 3152; fax: þ48 42 636 5530.
** Corresponding author. Tel.: þ48 42 631 3153; fax: þ48 42 636 5530.
E-mail addresses: stafan.jankowski@p.lodz.pl (S. Jankowski), janusz.zabrocki@
p.lodz.pl (J. Zabrocki).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.03.037

3732
P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
between the two phenylalanine’s aromatic rings, was important for
the immunosuppressive activity of CLA.
(A) 0.05% trifluoroacetic acid in water and (B) 0.038% trifluoro-
acetic acid in acetonitrile/water 90:10 with a gradient application.
Purification of peptides was performed by the preparative
reversed-phase HPLC on a Vydac C18 column (2.2 ꢀ 25 cm): flow
rate 16 ml/min, UV detection at 220 nm. The identities of the pure
peptides were confirmed by Maldi-TOF mass spectrometry on
In order to evaluate the role and significance of ‘‘edge to face’’
interaction between the two phenylalanine’s aromatic rings for
immunosuppressive activity of the native peptide, we synthesized
three linear precursors and three cyclic analogues of CLA, in which
one or both phenylalanine residues were replaced by homo-
phenylalanine residues (Fig. 1).
Voyager Elite, Perseptive Biosystems using
a-cyano-4-hydroxy-
cinnamic acid as a matrix. Cyclic peptides were additionally
analyzed by LSI MS on Finnigan MAT 95 mass spectrometer using
glycerol matrix.
This particular, unnatural amino acid, a homologue of phenyl-
alanine with elongated phenylalkyl side chain, is an agent of
interest to pharmacologists since a long time. It was used as
a precursor for the synthesis of many antihypertensive drugs acting
as angiotensin-converting enzyme inhibitors [23,24] and many
other biologically active compounds including opioid peptides and
proteinase inhibitors [25–29]. Hphe residue, as a non-proteino-
genic amino acid, should enhance the stability of the peptide
toward the enzymatic degradation with preservation of the
aromatic character of Phe. The elongation of the side chain may
change the mutual orientation of aromatic rings and change the
lipophilicity of modified peptide molecule. The effects exerted by
new CLA analogues on the humoral immune response in mice and
the proliferative response of splenocytes to T-cell and B-cell mito-
gens will be compared with those produced by CsA.
The one- and two-dimensional 1H NMR spectra were recorded
on a Bruker Avance II Plus spectrometer at 700.4 MHz in DMSO-d6
or CDCl3, using as an internal standard DMSO signal 2.50 ppm or
TMS (0 ppm), respectively. Spin-lock time in TOCSY experiments
was 80 ms and NOESY experiments were recorded with 300 ms
mixing time. 2D NMR spectra were processed with NMRPipe [30]
and analyzed by CARA [31] software.
All amino acid derivatives and peptide bond forming reagents
were purchased from IRIS Biotech (Germany).
2.2. Peptide synthesis and purification
The linear peptides 1–3 were synthesized by the manual
solid-phase method using chloromethylated Merrifield resin as
a solid support. Attachment of the first Boc-AA-OH (Boc-Phe-OH,
Boc-Leu-OH and Boc-Hphe-OH) to the resin was performed
according to cesium salt procedure [32] and the substitution level
2. Materials and methods
2.1. General remarks
was determined by weight gain measurements (Boc-Phe-
,
0.65 mmol/g; Boc-Leu- , 0.59 mmol/g; Boc-Hphe- , 0.66 mmol/
g). Synthesis of desired peptides was achieved by stepwise
coupling of Boc-amino acids to the growing peptide chain on the
resin. Starting with 0.2 mmol of Boc protected amino acid
attached to the resin, standard single TBTU/HOBt/DIPEA coupling
protocol (but with each deprotonation step omitted) was used for
all amino acids and was repeated if Kaiser test [33] or Chloranil
test (proline residue) [34] was found positive. In all cases where
after second coupling test was slightly positive, remaining free
amino groups were acetylated with the aid of acetic anhydride
All solvents were purified by conventional methods. Evapo-
rations were carried out under reduced pressure. Melting points
were determined on a capillary melting point apparatus and are
uncorrected. 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: (C)
acetonitrile:water (5:1), (D) butan-1-ol:acetic acid:water (4:1:1),
(E) chloroform:methanol:acetic acid (16:4:1), (F) ethyl acetate,
(G) chloroform:methanol (9:1), (H) chloroform:methanol (19:1).
The chromatograms were visualized with chlorine followed by
starch/KI and/or ninhydrin. HPLC was performed on an LDC/
Milton-Roy analytical instrument using a Vydac C18 column
(0.46 ꢀ 25 cm): flow 1.0 ml/min., detection at 220 nm and eluants
(100 ml) in the presence of diisopropylethylamine (100 ml). After
coupling step the Boc protecting group was removed with 50%
TFA in methylene chloride. The peptide resin was treated with
TFMSA (1 ml), TFA (10 ml) and anisole (0.5 ml) at 0 ꢁC and stirred
for 60 min at room temperature. After the resin had been filtered
off and washed with TFA the crude peptide was precipitated
upon concentration of solvents and addition of diethyl ether. The
analytical samples were purified by the preparative HPLC. The
physicochemical properties of the synthesized linear peptide are
summarized in Table 1.
1
2
3
H-Leu5-Ile6-Ile7-Leu8-Val9-Pro1-Pro2-Hphe3-Phe4-OH
H-Hphe4-Leu5-Ile6-Ile7-Leu8-Val9-Pro1-Pro2-Phe3-OH
H-Ile6-Ile7-Leu8-Val9-Pro1-Pro2-Hphe3-Hphe4-Leu5-OH
The crude linear precursors 1–3 (1 eq.) were cyclized (progress
of the cyclization reaction was monitored by HPLC) by means of
EDC (3 eq.) in the presence of HOAt (3 eq.) and diisopropylethyl-
amine (1 eq. to deprotonate the amino group of linear precursor) in
methylene chloride at much lower concentration (40 mg of the
linear peptide in 800 ml of methylene chloride) than usually
described for ‘‘head to tail’’ peptide cyclization reactions, to avoid
the dimer formation [35,36]. Crude cyclic peptides 4–6 were puri-
fied in the same way as their linear precursors. The physicochem-
ical properties of the synthesized cyclic peptide are summarized in
Table 1.
4
5
6
c(Pro1-Pro2-Hphe3-Phe4 -Leu5-Ile6-Ile7-Leu8-Val9)
c(Pro1-Pro2-Phe3-Hphe4-Leu5-Ile6-Ile7-Leu8-Val9)
[Hphe3]CLA
[Hphe4]CLA
c(Pro1-Pro2-Hphe3-Hphe4-Leu5-Ile6-Ile7-Leu8-Val9) [Hphe3,4]CLA
COOH
H2N
2.3. 1H NMR analysis of cyclopeptides 4–6
H
c(Pro1-Pro2-Hphe3-Phe4-Leu5-Ile6-Ile7-Leu8-Val9) (4). (DMSO-d6,
7
25 ꢁC) : 0.69 (3H, Hd, Ile7), 0.71 (3H, Hd, Leu8), 0.75 (3H, Hg, Val9),
d
Fig. 1. The sequences of newly synthesized CLA analogues 1–6 containing (S)-Hphe-
OH 7.
0.76 (3H, Hd, Ile6), 0.78 (3H, Hg, Ile7), 0.78 (3H, Hd, Leu5), 0.80

P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
3733
Table 1
Physicochemical properties of CLA analogues 4–6 used in the present study and their linear precursors 1–3.
Peptide
Yielda (%)
HPLC
TLC, Rfb
Mol. form.
Formula mass
1071.67
MALDI MS, m/z
LSI MS, m/z
Purity (%)
95.6
tR (min)
1
78.9
10.33 (40–80% B)
0.45 (C)
0.34 (D)
0.47 (E)
C58H89N9O10
[M þ H]þ 1073.3
[M þ Na]þ 1095.1
[M þ K]þ 1125.9
[M þ K]þ 1125.9
[M þ Na]þ 1076.1
[M þ K]þ 1092.1
[M þ Na]þ 1076.1
[M þ K]þ 1092.1
[M þ Na]þ 1090.6
2
3
4
100
100
33.2
100
97.3
99
8.43 (45–65% B)
5.75 (50–90% B)
16.53 (50–90% B)
0.46 (C)
0.54 (C)
0.41 (F)
0.37 (D)
0.50 (D)
0.59 (G)
0.42 (E)
0.56 (E)
0.33 (H)
C58H89N9O10
C59H91N9O10
C58H87N9O9
1071.67
1085.69
1053.66
[M þ H]þ 1054.7
[M ꢂ H]- 1052.6
[M þ H]þ 1054.7
[M ꢂ H]ꢂ 1052.6
[M þ H]þ 1068.6
[M ꢂ H]ꢂ 1066.6
5
6
33.2
42.1
99
99
16.37 (50–90% B)
16.73 (50–90% B)
0.56 (F)
0.39 (F)
0.52 (G)
0.56 (G)
0.30 (H)
0.32 (H)
C58H87N9O9
1053.66
1067.68
C59H89N9O9
a
Yields of crude peptides were calculated on the basis of the loading of the first amino acid.
Solvent systems and conditions are given in the Materials and methods section.
b
(3H, Hd, Leu5), 0.80 (3H, Hd, Leu8), 0.83 (3H, Hg, Ile6), 0.90 (3H, Hg,
(1H, Ha, Pro2), 4.30 (1H, Ha, Pro1), 4.39 (1H, Ha, Ile7), 4.49 (1H, Ha,
Val9), 4.97 (1H, Ha, Phe3), 7.08 (1H, HN, Val9), 7.12 (2H, H2,6, Hphe4),
7.16 (2H, H2,6, Phe3), 7.17 (1H, H4, Phe3), 7.17 (1H, H4, Hphe4), 7.26
(2H, H3,5, Phe3), 7.27 (2H, H3,5, Hphe4), 7.52 (1H, HN, Ile7), 7.68 (1H,
HN, Leu8), 7.75 (1H, HN, Leu5), 7.78 (1H, HN, Ile6), 8.28 (1H, HN,
Hphe4), 8.43 (1H, HN, Phe3).
Val9), 1.00 (1H, Hg, Ile7), 1.07 (1H, Hg, Ile6), 1.35 (1H, Hg, Ile7), 1.38
(1H, Hg, Leu8), 1.39 (1H, Hg, Ile6), 1.39 (1H, Hb, Leu5), 1.47 (1H, Hb,
Leu5), 1.58 (1H, Hg, Leu5), 1.61 (1H, Hb, Leu8), 1.72 (1H, Hg, Pro2), 1.75
(1H, Hb, Pro2), 1.75 (1H, Hb, Pro1), 1.75 (1H, Hb, Leu8), 1.82 (1H, Hg,
Pro1), 1.85 (1H, Hb, Ile7), 1.90 (1H, Hb, Val9), 1.91 (1H, Hg, Pro2), 1.93
(1H, Hg, Pro1), 1.95 (1H, Hg, Hphe3), 2.03 (1H, Hb, Ile6), 2.04 (1H, Hg,
Hphe3); 2.20 (1H, Hb, Pro2), 2.20 (1H, Hb, Pro1), 2.46 (1H, Hb, Hphe3),
2.54 (1H, Hb, Hphe3), 2.88 (1H, Hb, Phe4), 3.05 (1H, Hb, Phe4), 3.39
(1H, Hd, Pro2), 3.55 (1H, Hd, Pro1), 3.56 (1H, Hd, Pro2), 3.59 (1H, Hd,
Pro1), 3.62 (1H, Ha, Ile6), 3.81 (1H, Ha, Leu8), 4.04 (1H, Ha, Leu5), 4.08
(1H, Ha, Pro1), 4.11 (1H, Ha, Phe4), 4.33 (1H, Ha, Ile7), 4.43 (1H, Ha,
Pro2), 4.46 (1H, Ha, Val9), 4.63 (1H, Ha, Hphe3), 6.84 (2H, H2,6, Phe4),
7.14 (1H, H4, Phe4), 7.14 (2H, H2,6, Hphe3), 7.14 (2H, H3,5, Phe4), 7.18
(2H, H3,5, Hphe3), 7.24 (1H, HN, Val9), 7.27 (1H, H4, Hphe3), 7.35 (1H,
HN, Leu5), 7.75 (1H, HN, Ile7), 7.82 (1H, HN, Leu8), 8.03 (1H, HN, Ile6),
8.06 (1H, HN, Phe4), 8.41 (1H, HN, Hphe3).
(CDCl3, ꢂ60 ꢁC, TMS)
d
: 0.47 (3H, Hd, Ile7), 0.49 (1H, Hg, Pro2),
0.56 (1H, Hg, Ile7), 0.61 (3H, Hd, Leu5), 0.74 (3H, Hg, Ile7), 0.77 (3H,
Hg, Ile6), 0.81 (6H, Hd, Leu8), 0.88 (3H, Hg, Val9), 0.89 (3H, Hd, Leu5),
0.90 (3H, Hd, Ile6), 0.94 (3H, Hg, Val9), 0.95 (1H, Hb, Pro2), 1.07 (1H,
Hg, Ile6), 1.28 (1H, Hg, Ile7), 1.35 (1H, Hg, Pro2), 1.40 (1H, Hg, Leu8),
1.41 (1H, Hg, Ile6), 1.49 (1H, Hg, Leu5), 1.53 (2H, Hb, Leu5), 1.56 (1H,
Hb, Leu8), 1.64 (1H, Hb, Pro1), 1.86 (1H, Hb, Val9), 1.90 (1H, Hb, Ile6),
1.93 (2H, Hg, Pro1), 1.97 (1H, Hb, Pro2), 1.98 (1H, Hb, Pro1), 2.02 (1H,
Hb, Hphe4), 2.28 (1H, Hb, Hphe4), 2.28 (1H, Hb, Ile7), 2.30 (1H, Hb,
Leu8), 2.47 (1H, Hg, Hphe4), 2.68 (1H, Hg, Hphe4), 2.69 (1H, Hb,
Phe3), 3.14 (1H, Hd, Pro2), 3.51 (1H, Ha, Leu5), 3.54 (1H, Ha, Leu8),
3.55 (1H, Hd, Pro2), 3.62 (1H, Hb, Phe3), 3.83 (2H, Hd, Pro1), 4.14 (1H,
Ha, Ile6), 4.15 (1H, Ha, Pro2), 4.26 (1H, Ha, Pro1), 4.66 (1H, Ha, Val9),
4.67 (1H, Ha, Ile7), 4.75 (1H, Ha, Hphe4), 5.07 (1H, Ha, Phe3), 6.30
(1H, HN, Ile6), 6.98 (1H, HN, Ile7), 7.11 (2H, H2,6, Hphe4), 7.18 (2H,
(CDCl3, ꢂ60 ꢁC, TMS)
d
: 0.68 (3H, Hg, Val9), 0.74 (3H, Hd, Ile7),
0.74 (3H, Hd, Leu5), 0.78 (3H, Hg, Ile7), 0.78 (3H, Hg, Val9), 0.84 (3H,
Hd, Leu8), 0.84 (3H, Hd, Leu8), 0.90 (3H, Hd, Leu5), 0.94 (3H, Hd, Ile6),
0.96 (3H, Hg, Ile6), 0.99 (1H, Hg, Ile7), 1.10 (1H, Hg, Ile7), 1.15 (1H, Hg,
Ile6), 1.46 (1H, Hg, Ile6), 1.51 (2H, Hb, Leu5), 1.54 (1H, Hg, Leu5), 1.65
(1H, Hb, Hphe3), 1.66 (1H, Hg, Leu8), 1.66 (1H, Hg, Pro2), 1.69 (1H, Hb,
Hphe3), 1.73 (1H, Hg, Hphe3), 1.79 (1H, Hb, Pro2), 1.84 (1H, Hg,
Hphe3), 1.94 (1H, Hb, Ile7), 1.96 (1H, Hg, Pro1), 2.01 (1H, Hg, Pro1),
2.02 (1H, Hb, Ile6), 2.05 (1H, Hg, Pro2), 2.15 (1H, Hb, Pro2), 2.16 (1H,
Hb, Leu8), 2.16 (1H, Hb, Pro1), 2.26 (1H, Hb, Pro1), 2.34 (1H, Hb, Leu8),
2.38 (1H, Hb, Val9), 2.92 (1H, Hb, Phe4), 3.53 (1H, Hb, Phe4), 3.55 (1H,
Ha, Leu8), 3.58 (1H, Hd, Pro2), 3.66 (1H, Hd, Pro2), 3.69 (1H, Ha, Leu5),
3.88 (2H, Hd, Pro1), 4.16 (1H, Ha, Ile6), 4.30 (1H, Ha, Pro2), 4.37 (1H,
Ha, Pro1), 4.73 (1H, Ha, Val9), 4.73 (1H, Ha, Ile7), 4.78 (1H, Ha, Hphe3),
4.94 (1H, Ha, Phe4), 6.27 (1H, HN, Ile6), 6.46 (1H, HN, Hphe3), 6.86
(1H, HN, Val9), 6.90 (2H, H2,6, Hphe3), 7.10 (1H, H4, Hphe3), 7.10 (2H,
H
2,6, Phe3), 7.18 (2H, H3,5, Hphe4), 7.21 (1H, HN, Val9), 7.26 (2H, H3,5
,
Phe3), 7.26 (1H, H4, Hphe4), 7.34 (1H, H4, Phe3), 7.51 (1H, HN, Leu8),
8.03 (1H, HN, Hphe4).
c(Pro1-Pro2-Hphe3-Hphe4-Leu5-Ile6-Ile7-Leu8-Val9) (6). (DMSO-
d6, 25 ꢁC) : 0.61 (3H, Hd, Ile7), 0.72 (3H, Hd, Ile6), 0.75 (3H, Hg, Ile7),
d
0.78 (3H, Hd, Leu5), 0.78 (3H, Hg, Ile6), 0.79 (3H, Hg, Val9), 0.79 (3H,
Hd, Leu8), 0.81 (3H, Hd, Leu8), 0.88 (3H, Hd, Leu5), 0.92 (3H, Hg, Val9),
0.98 (1H, Hg, Ile7), 1.09 (1H, Hg, Ile6), 1.30 (1H, Hg, Ile7), 1.35 (1H, Hg,
Ile6), 1.39 (1H, Hg, Leu8), 1.52 (1H, Hb, Leu5), 1.58 (1H, Hg, Pro1), 1.60
(1H, Hb, Leu5), 1.61 (1H, Hg, Leu5), 1.66 (1H, Hg, Pro2), 1.68 (1H, Hb,
Leu8), 1.73 (1H, Hb, Ile6), 1.74 (1H, Hb, Pro1), 1.79 (1H, Hg, Hphe4),
1.87 (1H, Hb, Val9),1.90 (1H, Hg, Hphe4),1.91 (1H, Hg, Pro2); 1.91 (1H,
Hg, Pro1); 1.92 (1H, Hg, Hphe3), 2.01 (1H, Hb, Leu8); 2.05 (1H, Hb,
Ile7), 2.10 (1H, Hb, Pro2), 2.14 (1H, Hg, Hphe3), 2.16 (1H, Hb, Pro1),
2.34 (1H, Hb, Hphe4), 2.39 (1H, Hb, Pro2), 2.52 (2H, Hb, Hphe3), 2.52
(1H, Hb, Hphe4), 3.43 (2H, Hd, Pro2), 3.49 (1H, Hd, Pro1), 3.58 (1H, Hd,
Pro1), 3.91 (1H, Ha, Hphe4), 3.95 (1H, Ha, Ile6), 4.00 (1H, Ha, Leu5),
4.05 (1H, Ha, Leu8), 4.40 (1H, Ha, Ile7), 4.47 (1H, Ha, Pro1), 4.51 (1H,
Ha, Val9), 4.52 (1H, Ha, Pro2), 4.80 (1H, Ha, Hphe3), 6.95 (1H, HN,
Val9), 7.10 (2H, H2,6, Hphe4), 7.14 (2H, H2,6, Hphe3), 7.18 (1H, H4,
H
2,6, Phe4), 7.19 (3H, H3,4,5, Phe4), 7.25 (2H, H3,5, Hphe3), 7.27 (1H,
HN, Ile7), 7.33 (1H, HN, Leu5), 7.56 (1H, HN, Leu8), 8.10 (1H, HN, Phe4).
c(Pro1-Pro2-Phe3-Hphe4-Leu5-Ile6-Ile7-Leu8-Val9) (5). (DMSO-d6,
25 ꢁC) : 0.73 (3H, Hd, Ile7); 0.77 (1H, Hg, Pro2), 0.81 (3H, Hd, Leu8),
d
0.82 (3H, Hd, Leu5), 0.82 (3H, Hg, Ile7), 0.83 (3H, Hd, Leu8), 0.83 (3H,
Hd, Ile6), 0.84 (3H, Hg, Val9), 0.87 (3H, Hg, Ile6), 0.90 (3H, Hd, Leu5),
0.94 (3H, Hg, Val9), 1.04 (1H, Hg, Ile7), 1.15 (1H, Hg, Ile6), 1.38 (1H, Hg,
Ile7), 1.43 (1H, Hg, Leu8), 1.48 (1H, Hg, Pro2), 1.49 (1H, Hg, Ile6), 1.56
(1H, Hb, Leu5), 1.60 (1H, Hg, Pro1), 1.62 (1H, Hg, Leu5), 1.64 (1H, Hb,
Pro1), 1.65 (1H, Hb, Leu5), 1.69 (1H, Hb, Leu8), 1.78 (1H, Hb, Pro2), 1.87
(1H, Hb, Ile6), 1.88 (1H, Hg, Hphe4), 1.88 (1H, Hg, Pro1), 1.89 (1H, Hb,
Leu8), 1.90 (1H, Hb, Val9), 1.95 (1H, Hg, Hphe4), 2.04 (1H, Hb, Ile7),
2.05 (1H, Hb, Pro1), 2.40 (1H, Hb, Hphe4); 2.55 (1H, Hb, Hphe4), 2.94
(1H, Hb, Phe3), 3.14 (1H, Hd, Pro2), 3.24 (1H, Hd, Pro2), 3.24 (1H, Hb,
Phe3), 3.51 (1H, Hd, Pro1), 3.58 (1H, Hd, Pro1), 3.95 (1H, Ha, Ile6), 4.05
(1H, Ha, Leu8), 4.06 (1H, Ha, Leu5), 4.13 (1H, Ha, Hphe4), 4.30
Hphe4), 7.18 (1H, H4, Hphe3), 7.27 (2H, H3,5, Hphe3), 7.27 (2H, H3,5
,
Hphe4), 7.38 (1H, HN, Leu8), 7.49 (1H, HN, Ile7), 7.55 (1H, HN, Ile6),
7.81 (1H, HN, Leu5), 8.47 (1H, HN, Hphe4), 8.50 (d, J ¼ 8.5 Hz, 1H, HN,
Hphe3).
(CDCl3, ꢂ60 ꢁC, TMS)
d
: 0.61 (3H, Hd, Ile7), 0.66 (1H, Hg, Ile7), 0.78
(3H, Hd, Leu5), 0.85 (3H, Hg, Ile7), 0.90 (3H, Hg, Ile6), 0.93 (6H, Hd,
Leu8), 0.99 (3H, Hg, Val9), 0.99 (3H, Hd, Ile6), 1.01 (3H, Hd, Leu5), 1.06
(3H, Hg, Val9), 1.15 (1H, Hg, Ile6), 1.38 (1H, Hg, Ile7), 1.50 (1H, Hg, Ile6),

3734
P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
1.51 (1H, Hg, Leu8), 1.60 (1H, Hg, Leu5), 1.62 (1H, Hb, Leu5), 1.67 (1H,
Hb, Leu8), 1.88 (1H, Hb, Pro2), 1.96 (1H, Hb, Val9), 1.98 (1H, Hb,
Hphe3), 1.98 (1H, Hb, Ile6), 2.00 (1H, Hg, Pro2), 2.07 (1H, Hg, Pro1),
2.11 (1H, Hb, Hphe4), 2.12 (1H, Hg, Pro1), 2.19 (1H, Hg, Pro2), 2.21 (1H,
Hb, Pro2), 2.36 (1H, Hb, Pro1), 2.41 (1H, Hb, Ile7), 2.42 (1H, Hb,
Hphe4), 2.42 (1H, Hb, Leu8), 2.42 (1H, Hb, Hphe3), 2.47 (1H, Hb, Pro1),
2.52 (1H, Hd, Hphe4), 2.68 (1H, Hd, Hphe3), 2.70 (1H, Hd, Hphe3),
2.75 (1H, Hd, Hphe4), 3.64 (1H, Ha, Leu5), 3.65 (1H, Ha, Leu8), 3.74
(1H, Hd, Pro2), 3.82 (1H, Hd, Pro2), 3.94 (1H, Hd, Pro1), 3.98 (1H, Hd,
Pro1), 4.23 (1H, Ha, Ile6), 4.42 (1H, Ha, Pro2), 4.54 (1H, Ha, Pro1), 4.77
(1H, Ha, Ile7), 4.79 (1H, Ha, Hphe4), 4.79 (1H, Ha, Val9), 4.91 (1H, Ha,
Hphe3), 6.32 (1H, HN, Ile6), 6.96 (1H, HN, Ile7), 7.13 (1H, HN, Leu5),
7.17 (2H, H2,6, Hphe4), 7.24 (2H, H2,6, Hphe3), 7.27 (1H, H4, Hphe3),
7.34 (3H, H3,4,5, Hphe4), 7.34 (1H, HN, Val9), 7.41 (2H, H3,5, Hphe3),
7.61 (1H, HN, Leu8), 8.05 (1H, HN, Hphe4).
well) in flat-bottom, 96-well plates were incubated with DMSO
control solutions or the compounds for 24 h in a cell culture
incubator. After the incubation the cell viability was determined by
MTT colorimetric method [36]. The results are presented as the
percentage of OD values in relation to appropriate solvent dilutions,
where the cell viability was regarded as 100%.
Statistics. The data were statistically evaluated using Student’s
t-test. The results were presented as mean values ꢃ SE and regar-
ded significant when p < 0.05.
3. Results and discussion
3.1. Chemistry
The three new analogues of CLA (4–6), modified with homo-
phenyalanine residue in positions 3, 4 and both 3 and 4, were
synthesized by stepwise coupling of Boc-amino acids to the
growing peptide chain on Merrifield resin. The couplings were
mediated by the TBTU/HOBt in the presence of diisopropylethyl-
amine. On completion of the synthesis, the unprotected peptides
were cleaved with the TFMA/TFA reagent and the crude linear
precursors (1–3) were cyclized by means of EDC in the presence of
HOAt and DIPEA. The cyclization reactions were carried out in very
diluted solutions to avoid the dimer formation. The homogeneity of
the purified cyclic peptides was checked by analytical HPLC and
their structures were confirmed by Maldi-TOF MS, LSI MS and NMR
spectroscopy. 1H NMR measurements indicated absence of epi-
merization during the synthesis and confirmed that pure peptides
4–6 contained only one isomer as in the native peptide.
2.4. Biological methods
Mice. 12-week-old CBA mice of both sexes were used for the
study. Mice were fed with a commercial, pelleted food and filtered
tap water ad libitum.
Reagents and media. Concanavalin A (ConA), pokeweed mitogen
(PWM),
93-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium
bromide (MTT) and DMSO were from Sigma, RPMI-1640 and Hanks’
medium from Cibi/Life Technologies, UK, and fetal calf serum from
Gibco. Sheep red blood cells (SRBC) were delivered by Wroclaw
University of Environmental and Life Sciences.
Preparations of the peptides for the tests. The compounds were
initially dissolved in DMSO and subsequently in the culture
medium. Then, the solutions (1 mg/ml) were sterilized by filtration,
aliquoted and stored at ꢂ20 ꢁC until use.
3.2. NMR measurements
Proliferative response of mouse splenocytes to mitogens. Spleens
were homogenized by pressing the organs through a plastic screen
into Hanks’ medium. After centrifugation the cells were treated for
5 min with 0.83% ammonium chloride to lyze erythrocytes. Then,
the cells were washed twice by centrifugation in Hanks’ medium
and filtered through a sterile cotton wool to remove dead cells and
debris. Eventually, the cells were re-suspended in a medium con-
The structure of CLA molecule was studied in the solid state and
in solution [8]. The geometry of peptide bonds is trans except the cis
Pro1–Pro2 bond (
u
¼ 10ꢁ). The backbone conformation of CLA is
stabilized in the solid state by five hydrogen bonds NH/O]C:
HN5 / CO3, HN7 / CO4, HN8 / CO5, HN4 / CO9 and NH9 / CO4.
The conformational flexibility of CLA molecule in solution is
comparable to the flexibility of linear peptides.
sisting of RPMI 1640, supplemented with FCS, L-glutamine, sodium
pyruvate, 2-mercaptoethanol and antibiotics (referred later to as
the culture medium). The cells were distributed to flat-bottom
CLA in chloroform solution at 214 K exists as a single conformer
with well-defined structure. In 1H NMR spectrum NH resonances
96-well culture plates at density of 105 cells/100
m
l/well. ConA or
g/ml and the studied peptides at
g/ml concentrations. The cells were incubated for 3 days in
were recorded within 2 ppm region and the NH–CHa coupling
PWM were used at a dose of 2.5
1–100
m
constants were determined for all residues except Leu5. The
temperature dependence of NH chemical shifts indicated that NHs
of Phe4, Ile7, Leu8 and Val9 are hydrogen bonded and Ile6 is exposed
to the solvent. The close proximity of Pro2 side chain and aromatic
Phe3 ring in the CLA molecule causes a strong signal shift of one of
m
a cell culture incubator and the rate of cell proliferation was
determined using colorimetric MTT method [37]. The results were
presented as mean optical density (OD) values at 550/630 nm
wavelength from quadruplicate wells (determinations) ꢃ standard
error (SE). The studied effects of the compounds were compared
with appropriate DMSO controls.
Pro2
g proton to 0.33 ppm [8].
Peptides 4–6 were examined by 1H NMR in DMSO or CDCl3
solutions. In both solvents peptides 4–6 exist as one isomer with all
trans peptide bonds except cis Pro–Pro peptide bond. The diag-
Determination of the humoral immune response in vitro. Mice
were sensitized with 0.2 ml of 1% SRBC suspension. Four days later
the animals were sacrificed and the splenocyte suspension was
prepared as described above. The cells were distributed to flat-
bottom, 24-well culture plates (5 ꢀ106/ml/well), followed by
addition of SRBC (0.1 ml of 0.005% suspension). After four-day
incubation the number of antibody-forming cells (AFC) was
determined [38]. The results were presented as mean AFC numbers
from quadruplicate wells ꢃ SE (standard error).
nostic NOE correlations between C
found for each peptide.
a
H atoms of Pro1 and Pro2 were
The replacement of one or both Phe residues by Hphe in
peptides 4–6 changed their 1H NMR spectra recorded in CDCl3 at
213 K in comparison to that recorded for the unmodified CLA [8].
The most noticeable changes in chemical shifts are observed for
residues 1–4. The presence of Hphe in position 3 as in 4 and 6
causes the increasing of Pro1
C
a
H chemical shift of 0.15 and
0.21 ppm, respectively. The difference in chemical shifts between
the
protons of Pro1 is about 0.1 ppm for 4 and 6, smaller than
Determination of toxicity of the compounds for WEHI 164.13 cells.
The toxicity of the compounds was determined with respect to
WEHI 164.13 cell line [39]. The compounds were dissolved in
DMSO, then in the culture medium at the concentration range of
b
observed for these protons in 5 and in CLA (0.34 and 0.37 ppm,
respectively). The aliphatic protons of Pro2 in peptides 4 and 6 are
strongly shifted down-field in comparison to CLA. Particularly, the
g0 protons of Pro2 in 4 and 6 are observed at 1.71 and 1.94 ppm,
0.1–100 mg/ml. Appropriate concentrations of DMSO served as
respective controls. The monolayers of WEHI 164.13 cells (2 ꢀ 105/

P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
3735
Fig. 2. The 700 MHz 1H NMR spectra of peptides 4–6 in CDCl3 recorded at 213 K.
respectively. It means much smaller shielding effect of Hphe phenyl
rings in comparison to Phe. The mutual orientation of Pro2 and Phe3
in 5 is similar to that in CLA. All resonances of Pro2 are placed in
similar positions, however g0 proton (0.53 ppm) do not experience
unusually large high-field shift observed for CLA (0.33 ppm). The
majority of Leu5, Ile6, Ile7, Leu8 and Val9 protons in 4–6 have reso-
nances placed very close or slightly down-field shifted (<0.25 ppm)
relatively to chemical shifts observed for these residues in CLA.
Only few exceptions are noticeable. The presence of Hphe in posi-
tion 4 as in 5 and 6 causes the down-field shift of Ile7 NH signal of
0.32 and 0.44 ppm, respectively (Fig. 2).
The replacement of CDCl3 by DMSO and increase of temperature
to 333 K simplified proton spectra in a way characteristic for the
chemical exchange among conformers of similar energy (Fig. 4).
However, the rate of exchange for peptide 4 at elevated tempera-
ture remained slow and only Leu5 NH signal became sharp. For
peptide 5 and 6 the exchange was faster and more NH signals
became well resolved, except for NH signals of Hphe4, Leu5 and Ile6
for peptide 5 and Hphe4 for peptide 6.
NH of Leu5 (1), Leu8 and Val9 (5), which moved linearly upfield with
temperatures. The low temperature coefficients (<3 ppb/K) are
characteristic for the presence of intramolecular hydrogen bonds
[40]. Peptides 5 and 6 are stabilized by six intramolecular hydrogen
bonds involving all NH atoms except of NH Hphe4 atom. For peptide
4 only three NH (Phe4, Leu5 and Val9) atoms formed hydrogen
bonds with carbonyl oxygen atoms.
Signals of Phe and HPhe aromatic rings in CDCl3 and DMSO are
placed in similar range of about 0.2 ppm. Only ortho atoms of Hphe3
in peptide 4 in are shifted up-field to 6.84 ppm (DMSO, 298 K) and
6.95 ppm (CDCl3, 213 K). This may suggest, for this CLA analogue,
the ‘‘face to edge’’ interaction of phenyl rings, similarly as in the
native CLA [8,41]. The close proximity of Pro2 side chain and
aromatic Phe3 ring in peptide 5 is consistent with observed strong
up-field shifts of Pro2 g0 signal to 0.77 ppm (DMSO, 298 K) and
0.53 ppm (CDCl3, 213 K).
Table 2
3
Vicinal coupling constants JNHC [Hz] of peptides 4–6 in DMSO at 333 K and in
The NH–CaH coupling constants determined at 213 K in chlo-
aH
chloroform at 213 K.
roform and at 333 K in DMSO are summarized in Table 2. Most of
3JNHC H values of about 7 Hz were measured. Low number of vicinal
4
5
6
CLAa
a
couplings determined at low temperature in CDCl3 indicate lack of
preferred conformation in the solution, contrary to that observed
for CLA. At elevated temperature in DMSO the fast exchange
between quasi-energetic conformers led to the coalesced peaks
(Fig. 3).
DMSO
8.31
nd
4.96
nd
nd
nd
nd
CDCl3
DMSO
CDCl3
DMSO
CDCl3
CDCl3
Phe3/Hphe3
Phe4/Hphe4
Leu5
nd
7.86
nd
nd
nd
8.57
9.10
8.40
9.10
8.40
6.93
9.12
nd
7.84
nd
8.62
nd
nd
6.63
nd
nd
8.49
nd
8.82
10.01
<2
7.96
11.02
7.81
5.62
6.61
10.08
6.68
8.96
Ile6
nd
Ile7
9.09
3.73
nd
The solvent exposure of NH protons was probed by determining
Leu8
nd
9.31
the temperature coefficients (Dd/D
T) in DMSO from 1H NMR spectra
Val9
nd
11.23
or in the case of the overlapping signals, COSY DQF spectra recorded
in the range of 298–333 K (Table 3). All NH resonances, except of
a
Data from Ref. [10].

3736
P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
Fig. 3. The 700 MHz 1H NMR spectra of peptides 4–6 in DMSO recorded at 333 K.
From parameters obtained from NMR it may be concluded that
peptides 4–6 exist in solutions as mixtures of conformers. Peptide 4
was the most flexible CLA analogue in this study.
100
This compound caused a complete block of T- and B-cell prolifera-
tion at 100 g/ml (Figs. 4 and 5). The presented data clearly showed
mg/ml) of peptide 4 was not associated with cell toxicity (6.03%).
m
that modification of the tetrapeptide sequence (Pro1-Pro2-Phe3-
Phe4) in CLA, by substitution of Phe with Hphe in positions 3 and 4,
led to appearance of three different activities in the studied
immunological assays. Moreover, Hphe included in position 4
markedly reduced the viability of the tested cell line, however
3.3. Biology
The immunomodulatory activities of three cyclic nanopeptides
modified with homophenylalanine residue (4–6) were examined
and compared to that of cyclosporine A. The peptides exhibited
interesting, differential properties in the response of splenocytes to
T- and B-cell mitogens and in the model of secondary humoral
immune response to sheep erythrocytes in vitro. The results
showed that the peptides 5 and 6, containing Hphe in position 3 or 3
and 4, respectively, exhibited similar effects on the proliferative
response of splenocytes to mitogens (Figs. 4 and 5). Both peptides
stimulated T-cell proliferation, the B-cell proliferation was,
however, inhibited. Whereas 6 was a moderate suppressor of the
humoral immune response (Fig. 6), peptide 5 was exceptionally
inhibitory (92% inhibition at 100
mg/ml). That effect was correlated
with relatively high cell toxicity (47.19% at 100
m
g/ml concentration)
– Fig. 7. Interestingly, very potent inhibition of AFC number (94.5% at
Table 3
Temperature dependence of the NH chemical shits (ꢂDd
/DT, ppb/K) of major isomers
of peptides 4ꢂ6 in DMSO, in the range 298–338 K.
Peptide
Phe3 or Hphe3
Phe4 or Hphe4
Leu5
Ile6
Ile7
Leu8
Val9
2.5
0.05
ꢂ0.9
Fig. 4. Effects of peptides 4–6 on concanavalin A-induced splenocyte proliferation.
4
5
6
3.6
2.9
2.7
2.7
6.2
6.4
ꢂ2.7
1.4
2.0
7.3
2.5
1.2
5.4
1.4
2.0
4.4
1.7
ꢂ0.06
Mouse splenocytes were induced by addition of 2.5
mg/ml of ConA and peptides 4–6
were applied at concentrations of 0.1–100
mg/ml. * Indicates p < 0.05, ** indicates
p < 0.02 and *** indicates p < 0.001.

P. Drygała et al. / European Journal of Medicinal Chemistry 44 (2009) 3731–3738
3737
addition of the second Hphe in position 3 improved, in fact, cell
survival in comparison with the solvent.
4. Conclusions
The replacement of one or both Phe residues by Hphe led to
three different biological activities. This observation supports the
view that the balance of the phenyl rings and their relative angle
may control the biological activity of CLA molecule, not only the
immunosuppressive activity, but also its toxicity. Elongation of the
side chain in position 3, as in peptide 4, resulted in a very potent
inhibition of AFC number, not associated with cell toxicity, as well
as a complete block of T- and B-cell proliferation. Peptides 5 and 6
with Hphe in position 3 or 3 and 4, respectively, gave similar effects
on the proliferative response of splenocytes to mitogens. The
viability of cell lines is lower when Hphe is present in both posi-
tions 3 and 4 and the lowest when Hphe is present in position 4.
Only peptide 6 was a moderate suppressor of the humoral immune
response, peptide 5, was, in turn, exceptionally inhibitory. All
peptides (4–6) are attractive for more detailed investigations in
other immunological models; peptide 4 deserves more attention
because it appears to be a universal suppressor of the immune
response and is devoid of toxicity.
Fig. 5. Effects of peptides 4–6 on pokeweed mitogen-induced splenocyte proliferation.
Mouse splenocytes were induced by addition of 2.5 g/ml of PWM and peptides 4–6
g/ml. * Indicates p < 0.05, ** indicates
m
were applied at concentrations of 0.1–100
m
p < 0.02 and *** indicates p < 0.001.
Acknowledgements
This work was supported by the Ministry of Science and Higher
Education, Grant No. 2 505F 035 28.
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