Tumor-cell-targeted methionine-enkephalin analogues containing unnatural amino acids: Design, synthesis, and in vitro antitumor activity

Article abstract of DOI:10.1021/jm051026+

A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6), was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma (HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues containing Cαα-dialkylated glycine (Aaa1, 1) or Cα-alkylated glycine (Aaa2, 2) amino acid residues showed antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death was confirmed in these tumor cells after exposure to pentapeptide 18.

Full text of DOI:10.1021/jm051026+

3136
J. Med. Chem. 2006, 49, 3136-3142
Tumor-Cell-Targeted Methionine-enkephalin Analogues Containing Unnatural Amino Acids:
Design, Synthesis, and in Vitro Antitumor Activity
Sˇtefica Horvat,*,† Kata Mlinaric´-Majerski,† Ljubica Glavasˇ-Obrovac,‡ Andreja Jakas,† Jelena Veljkovic´,† Sasˇka Marczi,‡
Goran Kragol,†,§ Maja Rosˇcˇic´,† Marija Matkovic´,† and Andrea Milostic´-Srb‡
-
DiVision of Organic Chemistry and Biochemistry, Ruder BosˇkoVic´ Institute, POB 180, 10002 Zagreb, Croatia, Department of Nuclear
Medicine, Radiation and Pathophysiology, Clinical Hospital Osijek, School of Medicine Osijek, 31000 Osijek, Croatia
ReceiVed October 13, 2005
A series of new peptides (8-25) containing different unnatural amino acids of the adamantane type (1-6),
was synthesized. Possible cytotoxic activity on human cervical adenocarcinoma (HeLa), larynx carcinoma
(HEp-2), colon carcinomas (HT-29, Caco-2), poorly differentiated cells from lymph node metastasis of
colon carcinoma (SW-620), mammary gland adenocarcinoma (MCF-7), and melanoma (HBL) cells were
tested by the MTT assay. The results were compared with the effect of methionine-enkephalin (Tyr-Gly-
Gly-Phe-Met, or opioid growth factor, OGF), and its shorter N-terminal fragments. Peptide analogues
containing CRR-dialkylated glycine (Aaa1, 1) or CR-alkylated glycine (Aaa2, 2) amino acid residues showed
antitumor activity against melanoma, larynx carcinoma, colon carcinomas, and colon metastasis cell lines
in vitro. The pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was the most effective analogue especially
against the most antitumor drug-resistant cell lines HEp-2 and SW-620. Apoptosis as a mode of cell death
was confirmed in these tumor cells after exposure to pentapeptide 18.
Introduction
backbone modification to include hydrophobic residue; the sec-
ond would be covalent conjugation with a hydrophobic moiety,
for example, a lipid or a polymeric tail. Increasing hydrophobic-
ity by incorporation of nonnatural amino acids opens up the
possibility for greater chemical diversity, analogous to small-
molecule medicinal chemistry approaches for developing high-
affinity, high-specificity molecular recognition.7
In this paper, we report the synthesis of a series of new
synthetic pentapeptide analogues of methionine-enkephalin, its
structurally related dipeptide, tripeptide, tetrapeptide, and hexapep-
tide derivatives containing a variety of unnatural amino acids
of the adamantane type, and their cytostatic activity toward
malignant diseases.
Chemistry. The adamantane-containing amino acids 1-6
(shown in Figure 1) were prepared by applying modified
literature procedures.8-17 The resultant unnatural amino acid
hydrochlorides were characterized by NMR spectroscopy to
confirm structure and purity. Amino acid 2 [(R,S)-(1-adamantyl)-
glycine, Aaa2] was used in the synthesis of peptides in its
racemic form. Resolution of 2 was carried out by a published
procedure;10 however, this approach resulted in enantiomerically
enriched (R)-Aaa2 and (S)-Aaa2 enantiomers.
Based on the fundamental sequence of methionine-enkephalin
7, 18 new peptide analogues (8-25) containing unnatural amino
acids 1-6 were prepared. Peptides 8-22 were designed by
substituting either glycine (position 2) or glycylglycine (positions
2 and 3) in 7 or in its shorter N-terminal fragments by ada-
mantane-derived amino acids 1-6, while hexapeptides 23-25
were obtained by extending the C-terminal region of the
fundamental structure of methionine-enkephalin. Peptides 8-25
were synthesized by solution-phase methods via the stepwise
or fragment condensation approaches summarized in Figure 2.
The crude products were purified by RP-HPLC. Separation by
RP-HPLC to chiral peptides 9/10 and 15/16 containing indi-
vidual enantiomers of the Aaa2 residue was successful. In
contrast, the diastereoisomeric pentapeptides 18 exhibited no
difference in retention and could thus not be separated under
the RP-HPLC conditions studied. The configurations of the Aaa2
The development of peptide drugs for the treatment of cancer
has undergone dramatic changes in recent years and new types
of peptides and peptidomimetics have been designed to interact
with the tumor cell at specific sites. The spectrum of new targets
include control of cell proliferation, the pathways for transmis-
sion of positive and negative signals from the cell surface to
the nucleus, the control of angiogenesis, and complex processes
that determine apoptosis and DNA repair.1
Opioid peptides act as cell growth factors, in addition to
regulating neurotransmission/neuromodulation in the nervous
system. The native opioid growth factor (OGF), methionine-
enkephalin (Tyr-Gly-Gly-Phe-Met, 7), plays a role in cell
proliferation and tissue organization during development, cellular
renewal, wound healing, and angiogenesis, but also in cancer.2,3
OGF acts as an inhibitor of cell division and does so in a
receptor-mediated fashion.4 The receptor (OGFr) has been
cloned and sequenced in humans, rat, and mouse and its
molecular and protein structure has no resemblance to that of
classical opioid receptors. The action of OGF is constitutive,
stereospecific, reversible, and noncytotoxic. At least in cancer
cells, OGF does not utilize pathways of cell survival (apoptosis,
necrosis).5 OGF, however, suffers from drawbacks which
include rapid degradation in biological fluids wherein the active
compound very rapidly hydrolyzes into inactive form(s).6
To obtain more selective peptides with improved or novel
activity profiles toward malignant diseases, modifications with
lipophilic moieties may be of particular benefit to passive or
active cellular absorption by membrane penetration or attach-
ment. The susceptibility of the modified peptides to enzymatic
degradation may also decrease. Hydrophobization of peptides
may be attempted by two approaches. The first is peptide
* To whom correspondence should be addressed: phone 385-1-4680103;
fax 385-1-4680195; e-mail shorvat@irb.hr.
-
† Ruder Bosˇkovic´ Institute.
‡ School of Medicine Osijek.
§ Present address: PLIVA Research & Development Ltd., Prilaz baruna
Filipovic´a 29, 10000 Zagreb, Croatia.
10.1021/jm051026+ CCC: $33.50 © 2006 American Chemical Society
Published on Web 05/06/2006

Tumor-Cell-Targeted Methionine-enkephalin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3137
behavior of hexapeptides 23-25 confirmed that adamantane-
containing amino acid 1 is the most hydrophobic among the
nonnatural amino acids studied.
Biological Results and Discussion
In Vitro Antitumor Activity. The effect of methionine-
enkephalin (OGF, 7) as well as peptide compounds 8-25,
containing unnatural amino acids, on cell proliferation was tested
on the following malignant tumor cell lines derived from
different tissues: human cervical adenocarcinoma cells (HeLa),
human larynx carcinoma cells (HEp-2), human colon carcinoma
cells (HT-29, Caco-2), poorly differentiated cells from lymph
node metastasis of colon carcinoma (SW-620), human mammary
gland adenocarcinoma cells (MCF-7), human melanoma cells
(HBL), and human normal fibroblasts (WI38). Peptides 8-25
showed great variation in antiproliferative effect on tumor cell
lines, depending on the cell line as well as on the structure and
dose of the compound tested. In general, the novel peptides
displayed moderate (20-50% inhibition) to no or weak (0-
20% inhibition) cytotoxic potency on the tumor cell lines
examined. Only the peptides containing unnatural amino acids
of type Aaa1 (1) and Aaa2 (2) exhibited some selective
cytotoxicities, whereas analogues containing unnatural amino
acids 3-6 as isostere replacements of the Gly-Gly dipeptide
did not show any anticancer activity. As presented in Table 2,
compared to opioid growth factor 7 and untreated cells, peptide
analogues 8-10, 15-18, and 23 showed increased antitumor
activities against HEp-2, HBL, SW-620, and Caco-2 cell lines
in vitro. In the applied concentration of 10-6-10-3 M, peptides
8-10 and 15-18 demonstrated statistically significant stronger
growth inhibition effects on SW-620 and HEp2 cells (see
Supporting Information). The antiproliferative effects of these
compounds on HBL and Caco-2 cell growth were weaker. The
most antitumor drug-resistant cell lines HEp-2 and SW-620 were
especially sensitive to the mixture of diastereoisomers Tyr-(R,S)-
Aaa2-Gly-Phe-Met (18) (Figure 3). In both cell lines, growth
was statistically significantly suppressed by 18 in comparison
to methionine-enkephalin (7) and untreated cells. Diastereomeric
tripeptides 15 and 16, also containing the Aaa2 residue,
displayed moderate inhibitory effects only on the growth of
HEp-2 cells. Among the peptides containing the Aaa1 residue,
dipeptide 8 was cytotoxic against the SW-620 cells and
C-terminally extended hexapeptide 23 significantly inhibited the
growth of HEp-2 and Caco-2 cell lines (Table 2). The cyto-
toxicity of novel peptides on human fibroblasts in the applied
concentrations of 10-6-10-3 M was not observed.
Figure 1. Structure of the adamantane-derived unnatural amino acids
1-6.
amino acid residues in pure diastereoisomers 9 and 10 as well
as in 15 and 16 were assigned unambiguously by hydrolysis of
the corresponding peptide, isolation of the pure enantiomer of
amino acid 2, and comparison of the obtained [R]D values with
literature data.9,11 TLC and HPLC analysis on chiral supports
were also used for identification of the individual enantiomers
of Aaa2. According to analytical HPLC with a Chiral-AGP
stationary phase, the diastereoisomeric mixture 18 was slightly
enriched in the (R)-Aaa2-enantiomer (ee[(R)-Aaa2]-18 ) 20%).
Attempts to separate the diastereomers of 18 by fractional
crystallization resulted in diastereomerically enriched pentapep-
tides 18a (ee[(S)-Aaa2]-18 ) 70%) and 18b (ee[(R)-Aaa2]-18 ) 72%).
For screening of cellular uptake, fluorescein-labeled peptides
were prepared from methionine-enkephalin (FITC-7) and pen-
tapeptide 18. FITC-18 diastereoisomers were separated by RP-
HPLC to pure fluorescein-peptides containing individual enan-
tiomers of Aaa2 residue, FITC-[(S)-Aaa2]-18 and FITC-[(R)-
Aaa2]-18. The final compounds were characterized by analytical
RP-HPLC, mass spectrometry, and elemental analyses. The
physicochemical characteristics of the obtained peptides are
summarized in Table 1. A detailed NMR analysis of the target
compounds is given in the Supporting Information.
On the basis of the results of previously published studies,18-21
marked inhibitory activity of pentapeptide 7 against tumor cells
growth was expected. However, under the conditions of this
study, OGF displayed mild or no suppression of cell prolifera-
tion, depending on the tumor cell line tested (Table 2). This
disagreement may be the result of fundamental differences in
the experimental procedure used in previous reports,4,21 in which
all media and compounds were replaced daily. Experiments
employing smaller fragments of methionine-enkephalin (7), such
as Tyr-Gly and Tyr-Gly-Gly, revealed no influence on the tested
tumor cells even at concentrations as great as 10-3 M.
To check whether, due to different chirality of the Aaa2
residue, each diastereomer of pentapeptide 18 will have different
activity compared to racemic 18, the antiproliferative effect of
highly enriched diastereomers (ee ∼ 70%) in either the (S)-
Aaa2- (18a) or the (R)-Aaa2-enantiomer (18b) on HEp-2 and
SW-620 tumor cell lines was studied. It was definitely shown
(Figure 3) that peptides 18a and 18b do not differ substantially
To investigate the possibility of a correlation between peptide
hydrophobicity and biological activity in cancer cells, the
hydrophobic characteristics of peptides 8-25 were investi-
gated by comparing their retention times in RP-HPLC (Table
1). The incorporation of bulky adamantane-containing amino
acid moieties increased the retention times, reflecting the
more hydrophobic character of all analogues in comparison with
Tyr-Gly-Gly-Phe-Met (7) and its shorter N-terminal dipeptide
and tripeptide fragments. The major hydrophobic contribution
of the Aaa1 and Aaa2 residues was evident when the retention
times of pentapeptides 17 or 18 are compared with that of
methionine-enkephalin (7), the differences in the retention times
being about 19 and 15 min, respectively. The retention times
of tetrapeptides 19-22 with unnatural amino acids 3-6 as
isostere replacements of the Gly-Gly dipeptide still indicated a
significant increase in hydrophobicity, but the replacement of
Gly2 in methionine-enkephalin by either Aaa1 or Aaa2 caused
a greater change in hydrophobicity. The differences in retention

3138 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
HorVat et al.
Figure 2. Synthetic pathways to peptides 8-25.
in their ability to inhibit the growth of the examined cell lines.
In fact, they appeared to be less effective than the racemic
compound 18. The obtained data suggest a synergistic effect
between [(S)- and [(R)-Aaa2]-18 when investigated together as
a racemate. Hence, the racemic mixture 18 represents the
optimal formulation for the future investigation of this molecule
in terms of cytotoxic properties. In addition, the diastereomeric
tripeptides 15 and 16, containing either S- or R-Aaa2 showed
similar, cell-selective, cytotoxic effects on tumor cell lines
irrespective of Aaa2 chirality.
Previous studies2 have demonstrated that ligands selective
for known opioid receptors such as µ, δ, and κ, expressed on
the cell membranes of normal and malignant tissues, did not
affect cell growth either in vivo or in vitro. Only native opioid
peptide methionine-enkephalin (7), also termed opioid growth
factor (OGF), has been identified as a negative growth regulator.

Tumor-Cell-Targeted Methionine-enkephalin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3139
Table 1. Retention Times, Melting Points, and Optical Rotation Data of
Peptides 8-25
Table 2. Growth Inhibitory Effects of Methionine-enkephalin (OGF, 7)
and Selected Novel Peptides on Human Tumor Cells
compd
structure
tRa (min) mp (°C) [R]D,b deg
inhibitiona (%) of cell line
concn
compd (M) HEp-2 HeLa HBL HT-29 SW-620 CaCo-2 MCF-7
8
9
Tyr-Aaa1
12.81 275-280
12.46 180-185
14.05 190-195
8.79 155-158
9.03 192-195
9.76 190-195
10.66 195-200
9.53 200-205
10.79 210-215
26.70 200-205
23.03 194-210
16.51 205-208
18.35 205-208
17.80 202-208
19.93 212-215
31.92 212-215
+40
+8
Tyr-(S)-Aaa2
Tyr-(R)-Aaa2
Tyr-Aaa3
Tyr-Aaa4
Tyr-Aaa5
OGF, 7 10-6
OGF, 7 10-5
OGF, 7 10-4
OGF, 7 10-3
5.2
4.5 11.4
2.4 12.0
2.3
0.4 14.0
1.0 15.9
4.5 16.1
18.9
12.7
15.3
19.1
4.2
6.4
8.4
3.2
3.6
4.1
7.6
10
11
12
13
14
15
16
17
+19
+55
+22
+45
+20
+12
+21
+1
3.3 22.3 10.8 19.3
15.4
8
8
8
8
10-6 14.2
6.5
9.8
1.6
5.4
12.9
16.5
28.1
58.2
3.5
22.7
18.9
26.4
Tyr-Aaa6
10-5 19.7 10.0 14.0
2.4
7.5
Tyr-(S)-Aaa2-Gly
Tyr-(R)-Aaa2-Gly
Tyr-Aaa1-Gly-Phe-Met
10-4 24.9
8.2
7.5
10-3 30.4 36.1 14.0 26.5
18c Tyr-Aaa2-Gly-Phe-Met
+2
9
9
9
9
10-6
10-5
10-4
6.2
6.4
8.2
7.5
6.1
3.1
4.9
8.9
3.4
10.5
21.9
11.8
17.9
3.7
16.5
8.4
19
20
21
22
23
24
25
Tyr-Aaa3-Phe-Met
Tyr-Aaa4-Phe-Met
Tyr-Aaa5-Phe-Met
Tyr-Aaa6-Phe-Met
Tyr-Gly-Gly-Phe-Met-Aaa1
Tyr-Gly-Gly-Phe-DL-Met-Aaa4 23.06 198-204
Tyr-Gly-Gly-Phe-DL-Met-Aaa6 27.34 199-203
+17
+6
3.9
1.1
1.2
17.8
10-3 21.2 14.8
7.3 20.8
14.5
+11
+9
10
10
10
10
10-6
10-5
10-4
8.5
7.2
3.0
4.7
2.7 10.5
7.3 21.8
0.7
8.3
10.7
10.0
9.4
22.2
21.4
17.5
16.0
1.6
2.6
7.7
+4
4.9 17.1
1.9 11.0
+28
+24
10-3 16.2 20.5
18.7
18.7
a Eluent 50.5% MeOH/0.1% TFA. Retention time of methionine-
enkephalin (7) under identical conditions, 8.06 min; Tyr-Gly-Gly and Tyr-
Gly, no retention. b In methanol (c ) 1). c ee[(R)-Aaa2]-18 ) 20%.
15
15
15
15
10-6
10-5
2.7
2.0
9.8
7.3
9.9
1.6
2.9
5.9
5.4
7.4
9.5
13.6
20.4
14.4
23.4
2.2
4.2
6.4
6.2
10-4 18.8
10-3 26.7 28.2 17.9 15.7
21.2
15.6
22.8
OGF, presumably transported actively or passively from the
extracellular milieu, interacts with a receptor (OGFr) residing
on the outer nuclear envelope. Peptide and receptor form a
complex that appears to be associated with karyopherin and is
translocated to the inner nuclear matrix. In view of such
biochemical action and the fact that OGF receptors have been
found in a wide variety of human normal and transformed cells,2
it is reasonable to assume that critical for the magnitude of
change in tumor cell number, invoked in this study by the
addition of either Aaa1- or Aaa2-modified peptides, will be
peptide interaction with membranes and peptide transport
through the cell membrane. This transport has been suggested
to be largely dependent on the hydrophobic properties of the
peptide, its conformation, and molecular size as well as on the
ability of the peptide to change conformation upon lipid-peptide
interaction.22 In this regard, our results showed that the high
hydrophobicity of pentapeptides 17 and 18 and hexapeptide 23
was well correlated with their antiproliferative activity.
16
16
16
16
10-6
4.6 11.0
4.9 11.7
7.6
3.8
17.0
13.5
13.5
25.3
8.1
6.9
8.1
8.6
10-5
1.2
3.3 12.4
10-4 12.6 11.6
10-3 26.4 28.2 19.4 22.4
17
17
17
17
10-6
1.0
4.9
0.8
1.4 11.0
2.4 7.9
3.3
12.2
15.5
24.7
29.0
3.0
8.4
18.5
10.6
10-5 10.1 10.2
10-4 16.8 13.3
10-3 36.1 28.0 18.3 23.7
18
18
18
18
10-6 13.7
10-5 25.8
8.5
9.5
2.4 16.2
26.4
13.1
14.6
38.4
17.7
10.9
12.1
8.1
4.5
6.6
5.1
10-4 34.7 15.4 11.4
9.6
29.9
10-3 44.5 24.1 23.2 14.5
23
23
23
23
10-6
10-5
10-4
4.1
70.7
2.2
7.1
6.9
3.9
11.08
47.0
10-3 45.0
9.3 34.4
9.2
25.9
a Tumor cell growth inhibition was calculated relative to growth of control
(untreated) cells and shown as a percentage; when no number is shown,
there was no inhibition.
To verify the cell-penetrating properties of peptides 7 and
18 experimentally, fluorescein-labeled methionine-enkephalin
(FITC-7) as well as diastereomers containing individual enan-
tiomers of the Aaa2 residue, FITC-[(S)-Aaa2]-18 and FITC-
[(R)-Aaa2]-18, were prepared. Fluorescence micrographs pre-
sented in Figure 4A (a-d) showed that labeled 7 and FITC-
[(S)-Aaa2]-18 rapidly penetrate to the cellular interior of HEp-2
cells. Microscopy of HEp-2 and SW-620 cells treated with
FITC-[(S)-Aaa2]-18 for 6 h showed both labeled peptides
localized mainly on the outer nuclear membrane of the tumor
cells (see Supporting Information). In contrast, the FITC-(R)-
analogue of compound 18 neither entered the cells nor bound
to their outer membranes, even after prolonged incubation time
[Figure 4A (e,f)].
Induction of active cell death, apoptosis, by antitumor drugs
is vital to the treatment of most tumors. In this study, the number
of annexin V positive cells was increased in SW-620 cells
treated by Aaa2-containing pentapeptide 18 in comparison to
the control, untreated cells. Simultaneous staining with pro-
Figure 3. Growth inhibitory effect of opioid growth factor (OGF, 7) and Aaa2-containing pentapeptides 18, 18a, and 18b on HEp-2 and SW-620
tumor cells.

3140 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
HorVat et al.
HEp-2, HBL, SW-620, and Caco-2 cell lines in vitro. In
particular, the pentapeptide Tyr-(R,S)-Aaa2-Gly-Phe-Met (18)
showed the most potent tumoricidal activity by inducing
apoptosis in HEp-2 and SW-620 cell lines.
The obtained results indicate that in the investigated structural
analogues of methionine-enkephalin (7), such as 18, increased
hydrophobic interaction with membranes and transport through
the cell membrane may play major roles. Therefore, pentapeptide
18 is the most promising candidate for development of new
anticancer agents.
To understand the biological function of peptide 18, further
studies are intended concerning apoptosis/necrosis, conforma-
tion-activity relationships, and other systems related to binding
of 18 to either OGFr or classical opioid receptors.
Experimental Section
General Methods. Melting points were determined on a Tottoli
(Bu¨chi) apparatus and are uncorrected. Optical rotations were
measured at room temperature on an Optical Activity LTD
automatic AA-10 polarimeter. NMR spectra were recorded on a
Bruker AV 600 spectrometer, operating at 150.91 MHz for 13C
and 600.13 MHz for 1H nuclei. The spectra were measured in
DMSO-d6 solutions, unless otherwise specified. Chemical shifts in
parts per million (ppm) were referenced to tetramethylsilane (TMS).
Spectra were assigned on the basis of 2D homonuclear (correlated
spectroscopy, COSY) and heteronuclear multiple quantum coher-
ence (HMQC) and multiple bond correlation (HMBC) experiments.
Mass spectra were recorded on a ThermoFinnigan Deca ion trap
mass spectrometer operating in electrospray ionization (ESI) mode.
High-performance liquid chromatography (HPLC) was performed
on a Varian Pro Star 230 or HP 1090 HPLC systems with a
Eurospher 100 reversed-phase C-18 semipreparative (250 × 8 mm
i.d., 5 µm) column (flow rate 1.0 mL/min) or analytical (250 × 4
mm i.d., 5 µm) column (flow rate 0.5 mL/min) eluted isocratically
with different concentrations of MeOH in 0.1% aqueous trifluo-
roacetic acid (TFA). Chiral HPLC separations were performed on
a chiral CrownPak CR(+) (150 × 4 mm i.d., 5 µm) column eluted
with 15% MeOH in 0.1 M HClO4 (flow rate 0.8 mL/min) or on a
Chiral-AGP (100 × 4 mm i.d., 5 µm) column eluted with 15%
MeOH in 0.05 M phosphate buffer (pH 7.0) (flow rate 0.7 mL/
min). UV detection was performed at 280 and 215 nm on a HP
1090 diode-array detector. Enantiomeric resolution of amino acids
by TLC was performed on Chiralplate.
Chemistry. Methionine-enkephalin (7) and Tyr-Gly-Gly were
purchased from Sigma Chemical Co. Tyr-Gly was from Bachem
AG.
Synthesis of Unnatural Amino Acids 1-6. Modified literature
procedures were used for the synthesis of 2-aminoadamantane-2-
carboxylic acid8 (Aaa1, 1), (R,S)-(1-adamantyl)glycine9 (Aaa2, 2),
3-aminoadamantane-1-carboxylic acid12,13 (Aaa3, 3), 3-(aminom-
ethyl)adamantane-1-carboxylic acid15,16 (Aaa4, 4), 3-aminoada-
mantane-1-methylcarboxylic acid14 (Aaa5, 5) and 3-(aminomethyl)-
adamantane-1-methylcarboxylic acid15-17 (Aaa6, 6).
Partial resolution of an analytical sample of (R,S)-(1-adamantyl)-
glycine (2) was achieved through the esterification of amino acid
Aaa2 to the corresponding methyl ester followed by preparation
of diastereomeric salts with (+)-dibenzoyltartaric acid, fractional
crystallization, and hydrolysis to enantiomerically enriched (R)- and
(S)-Aaa2 as described by Krasutskii et al.10 HPLC retention times
on chiral CrownPak CR(+) column: for (R)-2, 35.58 min; for
(S)-2, 41.37 min.
Details of the syntheses and NMR analyses for amino acids 1-6
and their intermediates are available in the Supporting Information.
Synthesis of Peptides 8-25. The peptides were synthesized in
solution by either a stepwise or a fragment condensation approach
(Figure 2). The N-terminal amino and hydroxyl groups of the
tyrosine moiety were tert-butoxycarbonyl- (Boc-) protected. The
coupling reactions were carried out by BOP/HOBt, EDC/HOBt,
HATU/HOBt, or mixed anhydride activation protocols. All the
Figure 4. (A) Fluorescence micrographs of HEp-2 cells stained with
10 µM FITC-7 (a, b), 10 µM FITC-[(S)-Aaa2]-18 (c, d), and FITC-
[(R)-Aaa2]-18 (e, f). The cells were incubated with the test compounds
for 30 min and washed with PBS prior to observation (×400). The
slides were analyzed by use of filters for FITC detection (b, d, f). (B)
Fluorescence microscopic analysis of apoptosis in HEp-2 cells. DNA
strand breaks were detected by use of the In Situ Cell Death Detection
Kit, Fluorescein, after incubation with pentapeptide 18 (10 µM) for 1
h (37 °C, 5% CO2). Slides were analyzed under an Axioskop2 MOT
microscope with 450-490 nm excitation filter and 520 nm barrier filter.
pidium iodide allowed discrimination of the necrotic cells (red)
from the apoptotic cells (green) (see Supporting Information).
At the same time, SW-620 cells exposed to methionine-
enkephalin (7) did not express apoptotic features. Similar
observation has been also previously reported for methionine-
enkephalin.5 By use of the TUNEL method as a relatively late
marker of the apoptotic process, apoptosis as a mode of cell
death was confirmed in HEp-2 (Figure 4B) as well as in
SW-620 cells exposed to racemic pentapeptide 18 (see Sup-
porting Information).
Conclusion
In the present study, we designed and synthesized 18 new
analogues of the endogenous peptide methionine-enkephalin (7)
by substituting either Gly2 or Gly2-Gly3 in 7 and/or in its shorter
N-terminal fragments. Instead of Gly2 or Gly2-Gly3, adamantane-
derived amino acids 1-6 were incorporated while the hexapep-
tide derivatives were obtained by extending the C-terminal
region of the fundamental structure of methionine-enkephalin
by unnatural amino acids 1, 4, or 6. Only the peptide analogues
containing CRR-dialkylated glycine (Aaa1, 1) or CR-alkylated
glycine (Aaa2, 2) residues showed antitumor activity against

Tumor-Cell-Targeted Methionine-enkephalin Analogues
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11 3141
intermediates were used in the next step after chromatographic
purification. After removal of the N-terminal Boc groups with
TFA/H2O (9:1), the crude peptides were purified by semipreparative
RP-HPLC. Diastereomeric mixtures of dipeptides 9/10 and tripep-
tides 15/16 were successfully separated by RP-HPLC due to the
stronger retention of one isomer on the reversed-phase column.
Pentapeptide 18, containing a racemic Aaa2 residue, could not be
separated into individual isomers by RP-HPLC. Determination of
diastereomeric purity was achieved by HPLC analysis on a Chiral-
AGP column. Retention time for [(S)-Aaa2]-18 was 5.6 min; for
[(R)-Aaa2]-18, 8.4 min. Fractional crystallization of 18 (ee[(R)-Aaa2]-18
) 20%) resulted in diastereomerically enriched pentapeptides 18a
(ee[(S)-Aaa2]-18 ) 70%) and 18b (ee[(R)-Aaa2]-18 ) 72%). Assignment
of the S/R configurational features of the Aaa2 residue in 18a and
18b was based on differences in the chemical shifts of the H-δδ′
protons in the tyrosine aromatic ring and the S-CH3 protons of the
cell viability. Cytotoxic effects on tumor cell growth were
determined by the MTT assay.24 Compounds were dissolved in
high-purity water with 10% DMSO and diluted into working
concentration. The concentration of DMSO was too small to affect
the growth as confirmed in the control experiments. All working
dilutions (10-3-10-6 M) were prepared immediately before each
experiment in phosphate-buffered saline (PBS). At day 0 of the
experiment, tumor cells, 2 × 104 cells/mL, were plated onto 96-
microwell plates. Twenty-four hours later, the medium was replaced
with fresh medium containing defined concentrations of the
compounds investigated and the cells were treated for an additional
72 h. Control cells (without any compound) were growing under
the same conditions. After 72 h of incubation, the cell growth rate
was evaluated by performing the MTT assay. Data are expressed
as means of three individual experiments conducted in triplicate.
The percentage of growth of the cell lines was calculated from the
following equation:
1
methionine residue in the H NMR spectra of the two diastereo-
isomers, and on comparison with the corresponding protons in the
NMR spectra of the enantiomerically pure compounds 9, 10, 15,
and 16. Additional confirmation of the stereochemical assignment
of the Aaa2 residue in 18a and 18b was obtained by analysis of
the corresponding hydrolysates on Chiralplate (for details see
Supporting Information).
Peptides 8-25 were at least 95% pure as assessed by analytical
RP-HPLC. Molecular structures were confirmed by NMR, MS, and
elemental analyses. For bioactivity assays, the organic salts present
after preparative HPLC were removed by use of an octadecylsilica
solid-phase extraction (SPE) cartridge. The cartridge was eluted
with water to remove the salt. The peptide compounds were then
recovered with methanol. The effluent was evaporated, dissolved
in water, and lyophilized.
The structures, RP-HPLC retention times, melting points, and
optical rotation data of the novel peptide compounds 8-25 are listed
in Table 1.
Stereochemical Assignment of the Aaa2-residue Configura-
tion in Peptides 9, 10, 15, and 16. Hydrolysis of dipeptide 9 in 6
N HCl and separation of the obtained amino acids on an SPE
cartridge afforded enantiomerically pure (S)-(1-adamantyl)glycine
[(S)-2], [R]D +15° (c 0.53, MeOH) [lit.:11 (S)-2‚HCl [R]D +16°
(c 0.50, MeOH)]. Chiral TLC (eluent CH3CN-MeOH-H2O,
4:1:1): (S)-2 Rf 0.58.
Following the same procedure, hydrolysis of dipeptide 10
afforded enantiomerically pure (R)-(1-adamantyl)glycine hydro-
chloride [(R)-2‚HCl], [R]D -14° (c 0.52, MeOH) [lit.:9 [R]D -18°
(c 0.67, MeOH)]. Chiral TLC (eluent CH3CN-MeOH-H2O,
4:1:1): (R)-2 Rf 0.34.
The same procedure was applied for the stereochemical assign-
ment of the configuration of the Aaa2 residue in tripeptides 15
[Tyr-(S)-Aaa2-Gly] and 16 [Tyr-(R)-Aaa2-Gly].
Synthesis of Fluorescein-Labeled Peptides. Methionine-en-
kephalin (7) or Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) was reacted with
fluorescein-5-isothiocyanate (FITC isomer I) by using modified
procedures for labeling of small peptides.23 The FITC-peptides were
purified by gel chromatography and/or RP-HPLC, affording pure
FITC-7 as well as the labeled diastereomers of 18 containing the
individual enantiomers of Aaa2, that is, FITC-[(S)-Aaa2]-18 and
FITC-[(R)-Aaa2]-18.
percentage of growth ) (Apeptide - Ablanck)/(Acontrol - Ablanck) × 100
where blanck denotes medium without cells, containing the peptide
and MTT.
Cellular Uptake. To detect cellular uptake and intracellular
distribution of pentapeptide 18 and methionine-enkephalin (7), as
a control substance, SW-620 and HEp-2 cells (1 × 105 cells/
chamber slide) were incubated with 10 µM fluorescein-labeled
methionine-enkephalin (FITC-7) or with 10 µM FITC-[(S)-Aaa2]-
18 or FITC-[(R)-Aaa2]-18, for 30 min and 6 h. After incubation,
cells were rinsed with PBS and analyzed under an Axioskop2 MOT
microscope with excitation filter 450-490 nm and barrier filter
520 nm for FITC detection.
Detection of Apoptosis. Detection of apoptotic cells was
performed by use of an Annexin-V-FLUOS Staining Kit according
to manufacturer’s recommendations. SW-620 cells (1 × 105 cells/
chamber slide) were incubated with either Tyr-Gly-Gly-Phe-Met
(7) or Tyr-(R,S)-Aaa2-Gly-Phe-Met (18) (10 µM) for 1 h and
analyzed under a fluorescent microscope.
DNA strand breaks in treated SW-620 and HEp-2 cells (1 ×
105 cells/chamber slide) were detected by use of the In Situ Cell
Death Detection Kit, Fluorescein, after incubation with pentapeptide
18 (10 µM) for 1 h (37 °C, 5% CO2). Positive controls were
incubated with DNase I (10 µg/mL) for 10 min. Slides were
prepared for fluorescence microscopy according to the manufac-
turer’s instructions (air-dried cell samples are fixed for 1 h at room
temperature; slides were rinsed with PBS and incubated in
permeabilization solution for 2 min on ice; cells were washed twice
with PBS and incubated with TUNEL reaction mixture for 1 h at
37 °C in a humidified atmosphere in the dark; slides were rinsed
3× with PBS) and then analyzed under an Axioskop2 MOT
microscope with a 450-490 nm excitation filter and a 520 nm
barrier filter.
Statistics. The original results of MTT tests were subjected to
statistical analysis. The Kolmogorov-Smirnov test, a normality
distribution test was applied. Differences between groups were
assessed by a nonparametric Kruskal-Wallis test (p < 0.05).
Statistical analyses were performed with Statistica 6 package for
Windows.
Details of the synthesis for all peptide compounds and their
intermediates, NMR and MS data, as well as elemental analyses
are included in the Supporting Information.
Acknowledgment. We thank Milica Perc, Renato Margeta,
and Kristina Kalmar for their excellent technical assistance. The
work described in this study was supported financially by the
Ministry of Science, Education and Sport, Republic of Croatia
(Grant TP-01/0098/32).
Antitumor Activity Assays. Human cervical adenocarcinoma
cells (HeLa), human larynx carcinoma cells (HEp-2), human colon
carcinoma cells (HT-29, Caco-2), poorly differentiated cells from
lymph node metastasis of colon carcinoma (SW-620), human
mammary gland adenocarcinoma cells (MCF-7), human melanoma
cells (HBL), and human fibroblasts (WI38) were obtained from
ATCC. The cells were grown as monolayers in Dulbecco’s modified
Eagle medium (DMEM) with 10% fetal bovine serum (FBS)
supplemented with 2 mM glutamine, 100 units of penicillin, and
0.1 mg of streptomycin in a humidified atmosphere with 5% CO2
at 37 °C. The trypan blue dye exclusion method was used to assess
Supporting Information Available: Details of amino acid and
1
peptide syntheses with H and 13C NMR spectroscopic data and
tables listing HPLC retention times, purities, and MS and elemental
analysis data; in vitro antitumor activities of selected novel peptides;
and cellular uptake and intracellular distribution and detection of

3142 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 11
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one-pot synthesis of nitriles from carboxylic acids. Synlett 2005,
2089-2091.
apoptosis for peptide 18. This material is available free of charge
via the Internet at http://pubs.acs.org.
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