Antimalarial activity of ultra-short peptides

Article abstract of DOI:10.3390/molecules14125103

Ultra-short peptides 1-9 were designed and synthesized with phenylalanine, ornithine and proline amino acid residues and their effect on antimalarial activity was analyzed. On the basis of the IC₅₀ data for these compounds, the effects of natur

Full text of DOI:10.3390/molecules14125103

Molecules 2009, 14, 5103-5114; doi:10.3390/molecules14125103
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Antimalarial Activity of Ultra-Short Peptides †
Lemuel Pérez-Picaso 1, Benjamín Velasco-Bejarano 1, A. Berenice Aguilar-Guadarrama 1,
Rocío Argotte-Ramos 2 and María Yolanda Rios 1,*
1
Centro de Investigaciones Químicas, Universidad Autónoma del Estado de Morelos, Avenida
Universidad 1001, Col. Chamilpa, 62209 Cuernavaca, Morelos, México
2
Instituto Nacional de Salud Pública, Centro de Investigación Sobre Enfermedades Infecciosas
(CISEI), Avenida Universidad 655, Col. Santa María Ahuacatitlán, 62100 Cuernavaca, Morelos,
México
†
This paper is taken in part from the Ph.D. thesis of Lemuel Pérez-Picaso.
* Authors to whom correspondence should be addressed; E-Mail: myolanda@uaem.mx;
Tel.: +52-777-329-7000 ext. 6024; Fax: +52-777-329-7997.
Received: 18 November 2009; in revised form: 8 December 2009 / Accepted: 8 December 2009 /
Published: 8 December 2009
Abstract: Ultra-short peptides 1-9 were designed and synthesized with phenylalanine,
ornithine and proline amino acid residues and their effect on antimalarial activity was
analyzed. On the basis of the IC50 data for these compounds, the effects of nature, polarity,
and amino acid sequence on Plasmodium berghei schizont cultures were analyzed too.
Tetrapeptides Phe-Orn-Phe-Orn (4) and Lys-Phe-Phe-Orn (5) showed a very important
activity with IC50 values of 3.31 and 2.57 μM, respectively. These two tetrapeptides are
candidates for subsequent in vivo assays and SARS investigations.
Keywords: tetrapeptides; antimalarial activity; Plasmodium berghei; ultra-short peptides

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1. Introduction
Malaria is one of the most lethal and widespread infectious parasite diseases in the world, affecting
approximately 300 million people annually, mainly from developing countries like Central- and South-
America, Asia, and Sub-Saharan Africa [1,2]. According to the World Health Organization, the
number of clinical cases has reached between 300 and 500 million people per year [3]. About 1.1-2.7
million people in tropical and subtropical regions die of malaria every year [1,4–6]. In humans, it is
caused by protozoan parasites from four Plasmodium species: P. falciparum, P. vivax, P. malariae and
P. ovale. The most serious infections among these species are caused by P. falciparum [7,8]. In recent
years, the social impact of malaria has increased due, to the lower abundance and high cost of
artemisinin and related products (their effective distribution to combat malaria in economically
disadvantaged regions could require an annual global subsidy of $300-500 million) [9], and to the
emergence of resistant strains of P. falciparum and P. vivax to chloroquine, mefloquine,
pyrimethamine, and to immunoprophylactic methods (such as vaccines) [10]. For these reasons, there
is an increasing need for new chemical pharmacophores which may prove effective therapeutic
antimalarial agents.
Several antimalarial peptides have been isolated from natural sources or obtained synthetically
[8,11–21]. Lineal antimalarial peptides with fifteen or more amino acid residues, like leukinostatin A,
efrapeptin, zervamicin and antiamoebin have been isolated [22], however shorter peptides with potent
biological activity have also been described, among them lineal di-, tri-, tetra- and pentapeptides and
their cyclic analogs [23–28]. Several of these peptides showed important activity against P.
falciparum: pepstatin A (IC50 = 7.5 μM), a pentapeptide including a statine amino acid that was
isolated from the culture of several Actinomyces species [23]; cyclotetrapeptides HC-toxin I, apicidin
and apicidin A (MIC = 31, 125 and 125 ng/mL, respectively) [24]; linear tetrapeptide hirsutellic A acid
(IC50 = 8.0 μM), isolated from Hirsutella sp. BCC 1528 fermentation broth, possessing an anthranilic
acid residue at the C-terminus [12]; dipeptides 2-(2-tert-butoxycarbonylamino-4-methylpentanoyl-
amino)-cyclooctanecarboxylic acid methyl ester and 2-(2-tert-butoxycarbonylamino-3-phenyl-
propionylamino)-cyclooctanecarboxylic acid methyl ester are very effective in vitro (IC50 = 3.87 and
3.64 μg/mL, respectively); both compounds have maximum activity on maturation schizonts and total
inhibition growth assays probed by total growth inhibition instead of stopping the cell division cycle
[25]. Recent studies have showed that some commercial antiviral dipeptides used for HIV treatment,
among them saquinavir, atazanavir and BR314, inhibit P. falciparum in vitro [26]. Additionally, some
potent plasmepsin II inhibitors (peptidomimetics based on phenylalanine-statine amino acids) and of
cysteine proteins have been studied as potential antimalarial agents [27]; in addition
peptidylfluoromethyl ketones are potent inhibitors of falcipain, and block the degradation of
hemoglobin and the parasite development at nanomolar concentrations [28]; Z-Phe-Arg-CH2F is a very
potent inhibitor of trophozoite cysteine proteinase (TCP), and morpholine urea (Mu)-Phe-HPh-CH2F
has an IC50 = 0.003 μM (after four days of subcutaneous administration) against P. falciparum, and
cured 80% of malarial infected mice [30,31]. Moreover, apicidin (isolated from Fusarium

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pallidoroseum) is a potent in vivo antimalarial agent against P. berghei [29], while 2-(2-tert-
butoxycarbonylamino-3-phenyl-propionylamino)-cyclooctanecarboxylic acid methyl ester was also
active in vivo against this protozoan parasite [25]. In the present study, we report the antimalarial
activity of some ultra-short peptides designed with the purpose of analyzing the effect of
phenylalanine, ornithine and proline amino acid residues present on antimalarial activity.
2. Results and Discussion
Cationic (including arginine, lysine, ornithine and histidine residues, for example) and bulky (like
proline, phenylalanine, valine, isoleucine and tryptophan) side chains are essential for antimicrobial
activity in peptides [32] because they can confer amphiphatic character (simultaneous lipophilicity and
hydrophilicity properties). In this paper, we analyzed the influence of the amphiphatic character of
ultra-short peptides (including four or less amino acids residues) on antimalarial activity.
The amino acids statine, alanine, tyrosine, arginine, leucine, isoleucine, valine, proline,
phenylalanine and lysine are frequently included on antimicrobial and antimalarial peptides [11–21].
On the other hand, it has been established that peptides, including ornithine residue (non-proteinogenic
amino acid), show enzymatic and protein degradation stability [33]. For this reason, we used
isoleucine, valine, proline, phenylalanine, lysine and ornithine amino acids to synthesize the ultra-short
peptides studied in the present work.
Peptides 1-9 were synthesized in solid phase using Fmoc strategy and were designed to investigate
the nature, polarity, and amino acid sequence on Plasmodium berghei schizont cultures. Our main
purpose was to explore their potential antimalarial activity. On this context, peptides 1-4 were prepared
to investigate the effect of N-terminal amino acid, while sequences in compounds 3 and 5 allowed us
to analyze the effect of a change on the second amino acid residue on the antimalarial activity. With
this same purpose, antimalarial activity of compound 2 was compared with its retropeptide sequence at
compound 6. Tetrapeptides 1-5 were synthesized to study the activity of peptides including Xaa-Xbb-
Phe-Orn sequence, while the tetrapeptides 7 and 8, and compound 9 were prepared to analyze the
influence of the presence of proline residue, and tripeptide nature on antimalarial activity, respectively.
Compounds 1-9 were evaluated in vitro for chemosuppression during 16 h with P. berghei schizont
cultures. The results of the biological assay against this parasite blood stage are indicated in Table 1.
Peptide 1 showed moderate antimalarial activity, with an IC50 = 32.35 μM. Substitution of a valine
residue for isoleucine leads to a more active compound (peptide 2), showing an IC50 = 10.00 μM,
suggesting that polarity of the amino acid at the N-terminal residue is important for the antimalarial
activity. In this case, substitution of a hydrogen in valine for a methyl group (isoleucine) confers more
lipophilicity to the peptide and results in diminished antimalarial activity. Probably, the chiral center in
the isoleucine residue is another reason for this decreased activity. The retropeptide of compound 2
(peptide 6) was inactive, indicating that amino acid sequence is very important for the activity.

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Table 1. Chemosuppression of P. berghei schizont cultures of peptides 1-9 after 16 h.
Chemosuppression of P. berghei
Compound (200 μM)
IC50 (μM)
schizont cultures (%)
Ile-Lys-Phe-Orn (1)
Val-Lys-Phe-Orn (2)
Lys-Orn-Phe-Orn (3)
Phe-Orn-Phe-Orn (4)
Lys-Phe-Phe-Orn (5)
Orn-Phe-Lys-Val (6)
Lys-Val-Pro-Orn (7)
Lys-Val-Phe-Pro (8)
Phe-Orn-Val (9)
83.97
85.85
71.60
85.94
75.82
26.58
27.39
72.78
55.95
100
32.35
10.00
13.48
3.31
2.57
>200
>200
51.28
120.22
0.06
Chloroquine
Tetrapeptides 2 and 3 have similar activity (IC50 = 10.00 vs. 13.48 μM) values, however, the IC50
value was about three times lower when the N-terminal lysine residue was substituted by phenyl-
alanine (compound 4, IC50 = 3.31 μM), and almost five times lower when the second amino acid
phenylalanine residue was replaced with ornithine (compound 5, IC50 = 2.57 μM).
IC50 data for compounds 4 and 5 corroborate that N-terminal residue nature is very important for
antimalarial activity, and that when a polar residue (like lysine or ornithine) is substituted by a less
polar residue (like phenylalanine) antimalarial activity is increased. In our hands, peptides possessing
the proline residue (compounds 7 and 8) and tripeptide 9 were inactive. These results are in accordance
with the findings of other authors who have suggested that the presence of non polar amino acid
residues (providing lipophilic anchors) dramatically increases the biological activity, because those
residues might be helping their penetration (disruption) through plasma membrane [25,32].
3. Conclusions
Tetrapeptides 1-5 including the sequence Xaa-Xbb-Phe-Orn showed antimalarial activity during in
vitro schizont development assays, which significantly increased when a second phenylalanine amino
acid (aromatic residue) was present, such as in compounds 4 and 5. These compounds showed the
highest antimalarial activity, with IC50 = 3.31 and 2.57 µM, respectively, and will be candidates for
subsequent in vivo assays. These results showed that compounds 4 and 5 include interesting amino
acid sequences for future SAR investigations.
4. Experimental
4.1. General procedures

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Melting points were obtained in a Fisher Johns melting point apparatus and are uncorrected.
Infrared (IR) spectra were obtained in KBr on a Bruker Vector 22 IR spectrometer. Optical rotations
were measured in MeOH with sodium light on a Perkin-Elmer 341 MC polarimeter. 1H- and 13C-NMR
spectra were recorded on a Varian Unity 400 spectrometer operating at 400 MHz for 1H-NMR, 1H-1H
COSY, HSQC, and HMBC, and at 100 MHz for 13C-NMR, using CD3OD as solvent. Chemical shifts
are reported in ppm (δ) relative to the TMS signal. FAB+MS, and HRFAB+MS were recorded on a
JEOL JMStation-JM 700 mass spectrometer at 6 eV in a matrix of glycerol. Flash column
chromatography (FCC) and analytical thin-layer chromatography (TLC) were performed using silica
gel 230-400 mesh and pre-coated silica gel 60 F254 Merck plates, respectively. Fmoc-Lys(Boc)-OH,
Fmoc-Orn(Boc)-OH, Fmoc-Phe-OH, Fmoc-Val-OH, and HBTU were obtained from GENSCRIPT.
Fmoc-Ile-OH, Fmoc-Pro-OH, and HOBt were obtained from ANASPEC, and they were all used
without further purification.
4.2. Cell culture and antimalarial activity measurements
Chloroquine-sensitive cultured Plasmodium berghei schizonts (ANKA 2.34) were used to assess
the antimalarial activity of compounds 1-9. Mature P. berghei schizonts were prepared as described by
Thathy and Menard [34]. Peptides 1-9 were dissolved in PBS containing DMSO 0.1% and 5% ethanol.
Parasites were incubated for 16 h at 37 °C as described. Chloroquine (C6628 Sigma Chloroquine
diphosphate salt solid, ≥98% purity) was used as positive control. Samples of 0.5 mL of each culture
were taken to prepare smears and used to count numbers of schizonts in 2,000 erythrocytes. The IC50
values of each compound were determined using concentrations of 5, 50, 100, and 200 µM, according
to Khalid et al. [35], and by extrapolation from the concentration response curve. The IC50 value
represents the drug concentration producing a 50% reduction in the number of P. berghei schizonts
(compared to drug-free control cultures). For each assay, each drug dilution was analyzed in duplicate,
and the results were averaged in each case. Based on NMR spectroscopic and HPLC chromatographic
analyses, all compounds were at least of 98% purity.
4.3. General synthesis procedure for tri- and tetrapeptides 1-9
Solid phase synthesis of the peptides 1-9 [Ile-Lys-Phe-Orn (1), Val-Lys-Phe-Orn (2), Lys-Orn-Phe-
Orn (3), Phe-Orn-Phe-Orn (4), Lys-Phe-Phe-Orn (5), Orn-Phe-Lys-Val (6), Lys-Val-Pro-Orn (7), Lys-
Val-Phe-Pro (8), Phe-Orn-Val (9)] was carried out manually on a 2-Cl-Trityl-Cl resin (1 g, 1%,
200-400 mesh) using standard Fmoc-strategy methodology. Side-chain protecting groups were
Orn(Boc) and Lys(Boc). All protected amino acids were coupled using a 0.45 M solution of
HBTU/HOBt in DMF (2.1 mL × 0.25 mmol Fmoc aminoacid) and DIEA (2 mmol × 0.25 mmol Fmoc
aminoacid) until completion (30 min to 1 h), judged by the Kaiser ninhydrin test. After coupling of the
appropriate amino acid, Fmoc deprotection was effected by use of 20% piperidine/DMF solution for
2 min first, and then repeated for 30 min, followed by washing with DMF four times (10 mL each).
The synthetic cycle was repeated to assemble the resin-bond protected peptide. The peptides were
cleaved from the resin with simultaneous side-chain deprotection by acidolysis with 3% TFA/CH2Cl2

Molecules 2009, 14
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(1:1, 2 × 10 mL) containing 5% anisole at rt for 1 h. Cold diethyl ether (10 mL) was added and the
precipitated peptide was filtered through a fine sintered funnel. The crude peptides were subjected first
to FCC, and then purified to chromatographic homogeneity in the range of 90 to 95% by reverse-phase
high-pressure liquid chromatography, performed on a semipreparative Phenomenex Luna 5C18
column, 250 × 4.6 mm; mobile phase: 80% acetonitrile, 0.1% TFA; flow rate: 1.2 mL/min, 220 nm).
The purified peptides were subjected to amino acid analysis in order to confirm their composition.
Peptides were stored as a lyophilized powder at 220 °C. Prior to experimentation, fresh solutions were
prepared in water, vortexed, sonicated, and centrifuged, and the supernatant was diluted in the
appropriate medium.
Ile-Lys-Phe-Orn (1). White solid; Mp 160-162 °C; Rf 0.30 (BuOH:AcOH:H2O, 2:4:1); [α] – 1.74 (c
0.69, MeOH); IR: 3,434, 2,936, 1,674, 1,534, 1,198, 1,139, 838, 799, 694, 603 cm-1; 1H-NMR: δ 7.31-
7.15 (5H, m, Ar), 4.61 (1H, dd, J = 9.6, 5.2 Hz, Hα-Phe), 4.41 (2H, m, Hα-Lys, Hα-Orn), 3.74 (1H, d,
J = 6.0 Hz, Hα-Ile), 3.18 (1H, dd, J = 14.0, 4.8 Hz, Hβ-Phe), 2.95 (5H, m, Hβ’-Phe, Hδ-Orn, Hε-Lys),
1.97 (1H, m, Hβ-Orn), 1.84 (1H, m, Hβ-Ile), 1.77 (5H, m, Hβ-Orn, Hγ-Orn, Hβ-Lys), 1.67 (2H, m,
Hγ-Lys), 1.51 (1H, m, Hγ-Ile), 1.42 (2H, m, Hγ-Lys), 1.14 (1H, m, Hγ’-Ile), 0.91 (3H, t, J = 7.2 Hz,
Hδ-Ile), 0.89 (3H, d, J = 6.8 Hz, Hβ’-Ile); 13C-NMR: δ 174.37 (s, CO-Phe), 173.59 (s, CO-Orn),
173.37 (s, CO-Lys), 169.39 (s, CO-Ile), 138.16 (s, Ar), 130.30 (d, Ar), 129.48 (d, Ar), 127.82 (d, Ar),
58.82 (d, Cα-Ile), 56.32 (d, Cα-Phe), 54.45 (d, Cα-Lys), 53.06 (d, Cα-Orn), 40.55 (t, Cδ-Orn), 40.29
(t, Cε-Lys), 38.78 (t, Cβ-Phe), 38.11 (d, Cβ-Ile), 32.74 (t, Cβ-Lys), 29.63 (t, Cβ-Orn), 28.16 (t, Cδ-
Lys), 25.58 (t, Cγ-Ile), 25.12 (t, Cγ-Orn), 23.69 (t, Cγ-Lys), 15.28 (q, Cβ’-Ile), 11.78 (q, Cδ-Ile);
FAB+MS m/z (% ra); 521 (49) [M + H]+., 307 (14) [M + H - C11H24N3O]+, 289 (13) [M - C11H24N3O -
NH3]+, 176 (23), 154 (100), 136 (84), 84 (62), 77 (30); HRFAB+MS: observed 521.3468 [M + H]+,
(calcd. for C26H45N6O5, 521.3451).
Val-Lys-Phe-Orn (2). Colorless solid; Mp 125-127 °C; Rf 0.35 (BuOH:AcOH:H2O, 2:4:1); [α] – 2.07
(c 0.66, MeOH); IR: 3,433, 2,938, 1,675, 1,535, 1,440, 1,200, 1,141, 840, 800, 718, 603 cm-1;
1H- NMR: δ 7.30-7.16 (5H, m, Ar), 4.62 (1H, dd, J = 9.2, 4.8 Hz, Hα-Phe), 4.40 (2H, m, Hα-Lys, Hα-
Orn), 3.70 (1H, d, J = 6.0 Hz, Hα-Val), 3.18 (1H, dd, J = 14.4, 5.2 Hz, Hβ-Phe), 2.94 (5H, m, Hβ’-
Phe, Hδ-Orn, Hε-Lys), 2.11 (1H, dh, J = 6.8, 5.6 Hz, Hβ-Val), 1.97 (1H, m, Hβ-Orn), 1.77 (3H, m,
Hβ’-Orn, Hβ-Lys), 1.66 (4H, m, Hγ-Orn, Hδ-Lys), 1.42 (2H, m, Hγ-Lys), 0.95 (6H, d, J = 6.8 Hz, Hγ-
13
Val, Hγ’-Val); C-NMR: δ 174.35 (s, CO-Phe), 173.56 (s, CO-Orn), 173.43 (s, CO-Lys), 169.46 (s,
CO-Val), 138.13 (s, Ar), 130.32 (d, Ar), 129.50 (d, Ar), 127.81 (d, Ar), 59.59 (d, Cα-Val), 56.27 (d,
Cα-Phe), 54.54 (d, Cα-Orn), 53.04 (d, Cα-Lys), 40.53 (t, Cδ-Orn), 40.30 (t, Cε-Lys), 38.75 (t, Cβ-
Phe), 32.73 (t, Cβ-Lys), 31.70 (t, Cβ-Val), 29.63 (t, Cβ-Orn), 28.16 (t, Cδ-Lys), 25.12 (t, Cγ-Orn),
23.71 (t, Cγ-Lys), 19.16 (q, Cγ-Val), 18.14 (q, Cγ’-Val); FAB+MS m/z (% ra): 507 (39) [M + H]+, 307
(14), 289 (10), 176 (14), 154 (100), 136 (65), 120 (38), 107 (29), 89 (34), 84 (65), 77 (31), 72 (86);
HRFAB+MS: observed 507.3288 [M + H]+, (calcd. for C25H43N6O5, 507.3295).

Molecules 2009, 14
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o
Lys-Orn-Phe-Orn (3). White solid; Mp 123-125 C; Rf 0.20 (BuOH:AcOH:H2O, 2:4:1); [α] + 0.92 (c
2.01, MeOH); IR: 3,434, 3,072, 2,937, 1,675, 1,535, 1,439, 1,198, 1,139, 839, 800, 718, 603 cm-1;
1H- NMR: δ 7.32-7.16 (5H, m, Ar), 5.65 (1H, dd, J = 9.2, 5.2 Hz, Hα-Phe), 4.40 (2H, m, Hα of both
Orn), 3.92 (1H, t, J = 6.4 Hz, Hα-Lys), 3.17 (1H, dd, J = 14.4, 5.2 Hz, Hβ-Phe), 2.93 (5H, m, Hβ’-Phe,
Hδ-Orn, Hε-Lys), 1.96 (2H, m, Hβ of both Orn), 1.83 (2H, m, Hβ-Lys), 1.72 (8H, m, Hβ’ of both Orn,
13
Hδ of both Orn, Hε-Lys), 1.42 (2H, m, Hγ-Lys); C-NMR: δ 174.44 (s, CO-Phe), 173.53, 173.29 (s,
CO of both Orn), 170.09 (s, CO-Lys), 138.21 (s, Ar), 130.47 (d, Ar), 129.51 (d, Ar), 127.84 (d, Ar),
56.25 (d, Cα-Phe), 54.36 (d, Cα-Orn), 54.08 (d, Cα-Lys), 53.20 (d, Cα-Orn), 40.33 (t, Cδ of both
Orn), 40.16 (t, Cε-Lys), 38.93 (t, Cβ-Phe), 32.06 (t, Cβ-Lys), 30.04, 29.70 (t, Cβ of both Orn), 29.00
(t, Cδ-Lys), 25.20, 24.92 (t, Cγ of both Orn), 22.78 (t, Cγ-Lys); FAB+MS m/z (% ra): 522 (24) [M +
H]+, 307 (27), 289 (14), 154 (100), 136 (68), 107 (21), 89 (23), 77 (22); HRFAB+MS: observed
522.3398 [M + H]+, (calcd. for C25H44N7O5, 522.3404).
Phe-Orn-Phe-Orn (4). White solid; Mp 148-150 °C; Rf 0.26 (BuOH:AcOH:H2O, 2:4:1); [α] – 3.7 (c
0.62, MeOH); IR: 3,423, 3,073, 2,935, 1,676, 1,528, 1,199, 1,139, 839, 800, 715, 603 cm-1. 1H NMR: δ
7.37-7.12 (10H, m, Ar), 4.62 (1H, dd, J = 9.6, 5.2 Hz, Hα-Phe), 4.43 (2H, m, Hα of both Orn), 4.13
(1H, dd, J = 8.4, 6.0 Hz, Hα-Phe), 3.20, (1H, dd, J = 14.0, 8.4 Hz, Hβ-Phe), 3.13 (1H, dd, J = 14.0, 6.0
Hz, Hβ-Phe), 2.95 (6H, m, Hβ’ of both Phe, Hδ of both Orn), 1.99 (2H, m, Hβ of both Orn), 1.75 (6H,
m, Hβ’ of both Orn, Hγ of both Orn); 13C-NMR: δ 174.61 (s, CO-Orn), 173.90 (s, CO-Phe), 173.07 (s,
CO-Orn), 169.78 (s, CO-Phe), 138.35 and 135.60 (s, Ar), 130.62, 130.53, 130.19 and 129.71 (d each,
C2,6 and C3,5 of both Phe), 128.88, 128.04 (d each, C4 of both Phe), 56.53, 55.69 (d each, Cα of both
Phe), 53.76, 53.08 (d, Cα of both Orn), 40.27, 40.22 (t, Cδ of both Orn), 38.79, 38.72 (t, Cβ of both
Phe), 30.24, 29.58 (t, Cβ of both Orn), 25.10, 24.72 (t, Cγ of both Orn); FAB+MS m/z (% ra): 522 (24)
[M + H]+, 307 (27), 289 (14), 154 (100), 136 (68), 107 (21), 89 (23), 77 (22); HRFAB+MS: observed
522.3398 [M + H]+, (calcd. for C29H43N6O5, 522.3404).
Lys-Phe-Phe-Orn (5). White solid; Mp 114-116 °C; Rf 0.26 (BuOH:AcOH:H2O, 2:4:1); [α] + 6.6 (c
1.27, MeOH); IR: 3,422, 3,278, 3,074, 2,943, 1,677, 1,534, 1,197, 1,140, 839, 801, 713 cm-1; 1H-NMR:
δ 7.36-7.16 (10H, m, Ar), 4.62 (1H, dd, J = 8.8, 5.2 Hz, Hα-Phe), 4.59 (1H, dd, J = 9.6, 5.2 Hz,
Hα-Phe), 3.82 (1H, t, J = 6.8 Hz, Hα-Lys), 3.17 (1H, dd, J = 14.0, 5.2 Hz, Hβ-Phe), 3.07 (1H, dd, J =
14.0, 5.6 Hz, Hβ-Phe), 2.96 (6H, m, Hβ’ of both Phe, Hδ-Orn, Hε-Lys), 1.98 (1H, m, Hβ-Orn), 1.82
13
(1H, m, Hβ-Lys), 1.75 (3H, m, Hβ’-Orn, Hγ-Orn), 1.66 (2H, m, Hδ-Lys), 1.40 (2H,m, Hγ-Lys); C-
NMR: δ 174.32 (s, CO-Orn), 173.44, 173.32 (s, CO of both Phe), 170.07 (s, CO-Lys), 138.30, 138.13
(2s, C1 of both Phe), 130.54, 130.18 (d, C2,6 of both Phe), 129.54 (d, C3,5 of both Phe), 127.87 (d, C4 of
both Phe), 57.12, 56.38 (d, Cα of both Phe), 54.04 (d, Cα-Lys), 53.04 (d, Cα-Orn), 40.33 (t, Cδ-Orn,
Cε-Lys), 38.86, 38.75 (t, Cβ of both Phe), 32.15 (t, Cβ-Lys), 29.74 (t, Cβ-Orn), 28.16 (t, Cδ-Lys),
25.20 (t, Cγ-Orn), 22.72 (t, Cγ-Lys); FAB+MS m/z: 555 (88) [M + H]+, 440 (13), 248 (10), 154 (76),
136 (68), 120 (95), 84 (100), 77 (32) [C6H5]+; HRFAB+MS: observed 555.3320 [M + H]+, (calcd. for
C29H43N6O5, 555.3295).

Molecules 2009, 14
5110
Orn-Phe-Lys-Val (6). White solid; Mp 136-138 °C; Rf 0.30 (BuOH:AcOH:H2O, 2:4:1); [α] + 2.71 (c
1.7, MeOH); IR: 3,431, 3,293, 2,966, 1,675, 1,539, 1,436, 1,200, 1,135, 840, 803, 722, 449 cm-1;
1H- NMR: δ 7.32-7.17 (5H, m, Ar), 4.70 (1H, dd, J = 10.0, 4.8 Hz, Hα-Phe), 4.46 (1H, dd, J = 8.0,
6.0 Hz, Hα-Orn), 4.27 (1H, d, J = 6.0 Hz, Hα-Val), 3.92 (1H, t, J = 6.0 Hz, Hα-Lys), 3.20 (1H, dd,
J = 14.4, 4.8 Hz, Hβ-Phe), 2.95 (5H, m, Hβ’-Phe, Hδ-Orn, Hε-Lys), 2.19 (1H, dh, J = 6.8, 5.6 Hz,
Hβ-Val), 1.95 (3H, m, Hβ-Orn, Hβ-Lys), 1.91-1.65 (5H, m, Hβ’-Orn, Hδ-Orn, Hδ-Lys), 1.48 (2H, m,
Hγ-Lys), 0.99 (3H, d, J = 6.8 Hz, Hγ-Val), 0.98 (3H, d, J = 6.8 Hz, Hγ’-Val);13C-NMR: δ 174.58 (s,
CO-Val), 173.91 (s, CO-Phe), 173.40 (s, CO-Orn), 169.85 (s, CO-Lys), 138.09 (s, Ar), 130.20 (d, Ar),
129.60 (d, Ar), 127.94 (d, Ar), 59.43 (d, Cα-Val), 56.95 (d, Cα-Phe), 54.42 (d, Cα-Orn), 53.55 (d,
Cα-Lys), 40.60 (t, Cδ-Orn), 40.22 (t, Cε-Lys), 38.86 (t, Cβ-Phe), 32.83 (t, Cβ-Lys), 31.70 (t, Cβ-Val),
29.64 (t, Cβ-Orn), 28.27 (t, Cδ-Lys), 23.85 (t, Cγ-Orn), 22.72 (t, Cγ-Lys), 19.79 (q, Cγ-Val), 18.65 (q,
Cγ’-Val); FAB+MS m/z (% ra): 507 (100) [M + H]+, 307 (12), 246 (11), 154 (64), 136 (53), 120 (25),
107 (22), 89 (30), 77 (29), 70 (42), 63 (18); HRFAB+MS: observed 507.3288 [M + H]+, (calcd. for
C25H43N6O5, 507.3295).
Lys-Val-Pro-Orn (7). Colorless syrup; Rf 0.13 (BuOH:AcOH:H2O, 2:4:1); [α] – 21.2 (c 0.43, MeOH);
IR: 3,426, 3,080, 2,974, 1,680, 1,637, 1,541, 1,448, 1,202, 1,139, 841, 801, 722, 603 cm-1; 1H-NMR: δ
4.48-4.43 (3H, m, Hα-Pro, Hδ-Pro), 4.45 (1H, d, J = 8.0 Hz, Hα-Val), 4.42-4.35 (1H, m, Hα-Orn),
4.00 (1H, t, J = 6.8 Hz, Hα-Lys), 3.00-2.90 (4H, m, Hε-Orn, Hε-Lys), 2.28-2.18 (1H, m, Hβ-Pro),
2.15-2.91 (4H, m, Hβ-Val, Hβ-Pro, Hδ-Pro), 1.90-1.75 (6H, m, Hβ-Lys, Hβ-Orn, Hγ-Orn), 1.73-1.62
(2H, m, Hδ-Lys), 1.60-1.40 (2H, m, Hγ-Lys), 1.07 (3H, d, J = 6.8 Hz, Hγ-Val), 1.03 (3H, d, J = 6.8
13
Hz, Hγ’-Val); C-NMR: δ 174.03 (s, CO-Orn), 172.32 (s, CO-Pro), 170.16 (s, CO-Lys), 169.99 (s,
CO-Val), 61.76 (t, Cδ-Pro), 61.40 (d, Cα-Pro), 58.74 (d, Cα-Val), 53.87 (d, Cα- Lys), 53.10 (d,
Cα-Orn), 40.38 (t, Cδ-Orn), 40.21 (t, Cε-Lys), 32.24 (t, Cβ-Lys), 31.54 (t, Cβ-Val), 30.71 (t, Cβ-Pro),
29.73 (t, Cβ-Orn), 28.10 (t, Cδ-Lys), 26.20 (t, Cγ-Pro), 25.01 (t, Cγ-Orn), 22.21 (t, Cγ-Lys), 19.78 (q,
Cγ-Val), 19.06 (q, Cγ-Val); FAB+MS m/z (% ra): 501 (37) [M + 2Na – H]+, 479 (100) [M + Na]+, 457
(43) [M + H]+, 364 (32), 347 (63), 319 (21), 230 (53); HRFAB+MS: observed 457.3215 [M + H]+,
(calcd. for C21H41N6O5, 457.3138).
Lys-Val-Phe-Pro (8). White solid; Mp 161-163 °C; Rf 0.43 (BuOH:AcOH:H2O, 2:4:1); [α] – 16.98 (c
1.02, MeOH); IR: 3,421, 3,076, 2,969, 1,679, 1,637, 1,542, 1,452, 1,200, 1,140, 841, 801, 717,
1
604 cm-1; H-NMR: δ 7.35-7.18 (5H, m, Ar), 4.83 (1H, dd, J = 8.0, 5.6 Hz, Hα-Phe), 4.38 (1H, dd,
J = 8.8, 4.4 Hz, Hα-Pro), 4.20 (1H, d, J = 7.6 Hz, Hα-Val), 3.96 (1H, t, J = 6.4 Hz, Hα-Lys), 3.85-
3.65 and 3.52-3.41 (4H, m, Hδ-Pro and Hε-Lys), 3.11 (1H, dd, J = 13.6, 5.2 Hz, Hβ-Phe), 3.03-2.94
(3H, m, Hβ’-Phe), 2.93-2.87 (2H, m, Hε-Lys), 2.30-1.35 (2H, m, Hβ-Pro, Hβ-Val, Hβ-Lys, Hγ-Lys,
13
Hδ-Lys), 0.92 (3H, d, J = 6.4 Hz, Hγ-Val), 0.87 (3H, d, J = 6.4 Hz, Hγ’-Val); C-NMR: δ 175.25 (s,
CO-Val), 173.00 (s, CO-Pro), 171.82 (s, CO-Phe), 170.03 (s, CO-Lys), 138.19 (s, Ar), 130.60 (d, Ar),
129.56 (d, Ar), 127.86 (d, Ar), 73.07 (d, Cα-Pro), 64.92 (t, Cδ-Pro), 60.79 (d, Cα-Pro, Cα-Val), 54.20
(d, Cα-Phe), 54.00 (d, Cα-Lys), 40.38 (t, Cε-Lys), 38.49 (t, Cβ-Phe), 32.28 (t, Cβ-Lys), 31.97 (t,
Cβ-Val), 30.30 (t, Cβ-Pro), 28.13 (t, Cδ-Lys), 26.03 (t, Cγ-Pro), 22.80 (t, Cγ-Lys), 19.85 (q, Cγ-Val),

Molecules 2009, 14
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19.00 (q, Cγ-Val); FAB+MS m/z (% ra): 490 (87) [M + H]+, 375 (11), 307 (10), 263 (15), 200 (16), 175
(49), 154 (83), 136 (76), 120 (50), 84 (100); HRFAB+MS: observed 490.3071 [M + H]+, (calcd. for
C25H40N5O5, 490.3029).
Phe-Orn-Val (9). White solid; Mp 205-207 °C; Rf 0.46 (BuOH:AcOH:H2O, 2:4:1); [α] + 1.57 (c 0.69,
MeOH); IR: 3,433, 3,077, 2,968, 1,681, 1,648, 1,537, 1,400, 1,201, 1,134, 838, 801, 592 cm-1;
1H- NMR: δ 7.36-7.25 (5H, m, Ar), 4.53 (1H, dd, J = 7.2, 6.0 Hz, Hα-Orn), 4.34 (1H, d, J = 5.2 Hz,
Hα-Val), 4.20 (1H, dd, J = 8.0, 5.6 Hz, Hα-Phe), 3.27 (1H, dd, J = 14.0, 5.2 Hz, Hβ-Phe), 3.04 (1H,
m, dd, J = 14.0, 8.0 Hz, Hβ’-Phe), 2.97 (1H, t, J = 7.2 Hz, Hδ-Orn), 2.23 (1H, dh, J = 6.8, 5.6 Hz,
Hβ-Val), 1.90 (1H, m, Hβ-Orn), 1.77 (3H, m, Hβ’-Orn, Hγ-Orn), 1.01 (3H, d, J = 6.8 Hz, Hγ-Val),
13
0.99 (3H, d, J = 6.8 Hz, Hγ’-Val); C-NMR: δ 174.85 (s, CO-Val), 173.35 (s, CO-Orn), 169.77 (s,
CO-Phe), 135.58 (s, Ar), 130.70 (d, Ar), 130.21 (d, Ar), 128.95 (d, Ar), 59.17 (d, Cα-Val), 55.57 (d,
Cα-Phe), 53.92 (d, Cα-Orn), 40.30 (t, Cδ-Orn), 38.61 (t, Cβ-Phe), 31.74 (d, Cβ-Val), 30.36 (t,
Cβ-Orn), 24.79 (t, Cγ-Orn), 19.79 (q, Cγ-Val), 18.41 (q, Cγ’-Val); FAB+MS m/z (% ra): 379 (80) [M +
H]+, 262 (8) [M + H - C5H10NO2]+, 232 (8) [M + H - C9H9NO]+, 213 (8) [C10H17N2O3]+, 120 (100)
[C8H10N]+, 115 (48) [C5H9NO2]+, 91 (13) [C7H7]+, 70 (42); HRFAB+MS: observed 379.2315 [M +
H]+, (calcd. for C19H31N4O4, 379.2345).
Acknowledgements
This work was financially supported by CONACyT (Grants number 79584-Q and 2006-C01-LN-
56431). LPP thanks CONACyT for a predoctoral fellowship (number 181754). We are grateful to
Blanca Domínguez Mendoza, Diana Gabriela Vargas Pineda, Victoria Labastida, and María Medina
Pastor for technical assistance.
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Sample Availability: Samples of the compounds 1-9 are available from the authors.
© 2009 by the authors licensee Molecular Diversity Preservation International, Basel, Switzerland.
This article is an open-access article distributed under the terms and conditions of the Creative
Commons Attribution license (http://creativecommons.org/licenses/by/3.0/).