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W.F. Altei et al. / Phytochemistry xxx (2014) xxx–xxx
Previous chemical studies of the latex of J. curcas from Indonesia
and Senegal (Auvin et al., 1997) led to characterization and
sequencing of two bioactive cyclic peptides, curcacycline A and B.
Curcacycline A exhibits anti-inflammatory activity (Heller, 1996),
while curcacycline B enhances the rotamase activity of cyclophilin
B (Auvin et al., 1997). In Central and South America, J. curcas L. spe-
cies are cultivated for ornamental, medicinal, environmental and,
recently, biodiesel production (Martinez-Herrera et al., 2006;
Visser et al., 2011). In the present paper, isolation, structural eluci-
dation, conformational studies and biological activities of the cyclic
jatrophidin I (1) isolated from the latex of Brazilian J. curcas L. are
reported.
2. Results and discussion
The main source of cyclic peptides in Jatropha is the latex. Thus,
crude latex (74 mL) of J. curcas L. was partitioned between water
(300 mL) and ethyl acetate. The organic soluble fraction was
subjected to Sephadex G-15 column chromatography to exclude
low-molecular-weight components. A positive reaction in the pres-
ence of the chlorine-o-tolidine reagent suggested the presence of
amide groups, as expected for a peptide, while a negative reaction
with ninhydrin indicated the absence of a free amino group,
indicating a cyclic peptide structure. The peptide-containing frac-
tion was further extracted with SEPACK (Strata C18, Phenomenex,
USA) using octadecylsilane resin (C18) as stationary phase. Final
purification by C18 HPLC afforded a new cyclic octapeptide,
Fig. 1. NOESY correlations of jatrophidin I (1), representing proline in the cis and
trans orientations.
To study the hypothesis about the conformation of the proline
residue, the jatrophidin structure was submitted to conformational
studies using Molecular Dynamics/Simulated Annealing (MD/SA)
simulations, based on NOE correlations. This procedure uses heat-
ing/cooling cycles to sample conformational space and restrains
hydrogen atom distances from jatrophidin I (1) based on NOESY
spectra, thereby predicting specific molecular conformations
expected to exist in solution. The simulations were performed con-
sidering the representations of jatrophidin I (1) with proline in
both cis and trans orientations (Fig. 2), thereby permitting descrip-
tion of the dynamic behavior of this residue. In addition, to inves-
tigate the most stable conformation assumed by the peptide, NMR
spectroscopy was performed at different temperatures. Because in
solution two conformations of the polypeptide chain were simulta-
neously observed, including the cis and the trans forms of an X-Pro
peptide bond, the two species could generally be distinguished on
the basis of the different relative concentrations and different NMR
chemical shifts for some or all carbon and hydrogen atoms. TOCSY
signs were acquired at 25, 35 and 50 °C, and the chemical shifts
corresponding to amino acids residues were assigned. At 25 °C,
obtained as a white powder [a]
25 – 68.0 (c, MeOH). Its ESI spectrum
D
displayed a prominent [M + H]+ at m/z 851.6653. The amino acid
composition indicated 1 mol of tryptophan (Trp), asparagine (Asn)
and proline (Pro), two mol of glycine (Gly), and 3 mol of leucine
(Leu), which, in combination with the ESI data, suggested the
molecular formula C42H61N9O9. Finally, the configuration of each
amino acid residue was assigned as L, which was deduced by acid
hydrolysis and Marfey’s derivatization, followed by HPLC analysis
of the individual amino acids (Bhushan and Bruckner, 2004). The
1H NMR spectroscopic data indicated the presence of seven amide
protons, corresponding to all amino acids present with the exception
of one proline residue. The 1H NMR spectrum of the peptide included
resonances that were characteristic of peptides but displayed
duplicate signals, which were attributed to the presence of the
proline residue in two conformations, cis and trans. This cis–trans
isomerization promotes two conformations in this peptide. The
sequence [cyclo(-Gly-Trp-Leu-Asn-Leu-Leu-Gly-Pro-)] was named
jatrophidin I (1) and its structure elucidated after detailed analysis
of TOCSY (total correlation spectroscopy) and NOESY (nuclear
Overhauser enhancement spectroscopy) spectra. Jatrophidin I (1)
contain a high proportion of hydrophobic amino acids, which is
often observed in cyclic peptides extracted from the Caryophyllaceae
group (Barbosa et al., 2011; Picchi et al., 2009). The asparagine
residue is not, however, common in cyclic peptides from plants.
The influence of these characteristics on peptide function has not
been discussed in the literature; thus, future research is needed
to evaluate the relationship between peptide composition and
biological function.
the ratio between the
a a
H-Asn4 (cis)/ H Asn4 (trans) signals was
1.9. This value decreased to 0.76 at 35° and 0.40 at 50°, suggesting
an interconversion of X-Pro bonds with respect to temperature
enhancement. Other residues exhibited the same behavior, likely
because the trans conformation of proline is favored by increases
in temperature.
Jatrophidin I (1) was inactive in antimalarial, antifungal and
antioxidant assays. However, in a fluorimetric protease inhibition
assay using pepsin as a molecular model for aspartic protease inhi-
bition (Gold et al., 2007) it showed inhibitory activity (Table 2). Its
All the amino acid spin systems were identified using scalar
spin-spin couplings as determined from the 1H–1H TOCSY experi-
ments. Accordingly, the sequence was determined based on the
connectivity between the amide protons of residue i [(NH)i] and
IC50 value was 0.88 lM, as shown in Fig. 5. It did not, however,
inhibit the serine protease subtilisin, showing that protease inhibi-
tion was specific for aspartic proteases. Thus, future experiments
will be done with other aspartic proteases, as renin or HIV prote-
ase. These findings are thus consistent with the results of several
published studies that discuss the c activities of cyclic peptides
(Katsara et al., 2006).
the
Pro8-dH at d 3.50 and Gly7-
(1a) with a trans-Pro8 amide bond. NOEs between Pro8-
4.50 ppm and Gly7- H d 4.04 proved the presence of a cis-Pro8
a
protons of residue i-1[(
a
H)i-1]. NOESY correlations between
H d 3.90 indicated one conformer
H at d
a
a
a
In conclusion, jatrophidin I (1) is a cyclic peptide isolated from
the Brazilian J. curcas plant. Although the description of this class of
amide bond (conformer 1b) (Fig. 1). The chemical shifts for jatro-
phidin I (1) are given in Table 1.
Please cite this article in press as: Altei, W.F., et al. Jatrophidin I, a cyclic peptide from Brazilian Jatropha curcas L.: Isolation, characterization, conforma-
Altei, Wanessa F.
