Synthesis and conformational analysis of His-Phe-Arg-Trp-NH2 and analogues with antifungal properties

Article abstract of DOI:10.1016/j.bmc.2006.07.007

The synthesis, in vitro evaluation, and conformational study of His-Phe-Arg-Trp-NH₂ and related derivatives acting as antifungal agents are reported. Among them, His-Phe-Arg-Trp-NH₂ and His-Tyr-Arg-Trp-NH₂ exhibited antifungal activity against Cryptococcus neoformans. Antifungal activity of these compounds appears to be closely related to the α-MSH effect. A conformational and electronic study allows us to propose a biologically relevant conformation for these tetrapeptides acting as antifungal agents. In addition, these theoretical calculations permit us to determine the minimal structural requirements to produce the antifungal response and may provide a guide for the design of compounds with this biological activity.

Full text of DOI:10.1016/j.bmc.2006.07.007

Bioorganic & Medicinal Chemistry 14 (2006) 7604–7614
Synthesis and conformational analysis of His-Phe-Arg-Trp-NH2
and analogues with antifungal properties
a
a
b
b
´
Marcelo F. Masman, Ana M. Rodrıguez, Laura Svetaz, Susana A. Zacchino,
Csaba Somlai,c Imre G. Csizmadia,c Botond Penkec and Ricardo D. Enriza,*
aFacultad de Qu´ımica, Bioqu´ımica y Farmacia, Universidad Nacional de San Luis, Chacabuco 915, 5700 San Luis, Argentina
bFarmacognosia, Facultad de Ciencias Bioqu´ımicas y Farmace´uticas, Universidad Nacional de Rosario,
Suipacha 531, 2000 Rosario, Argentina
cDepartment of Medical Chemistry, University of Szeged, Do´m ter 8, H-6720 Szeged, Hungary
Received 22 May 2006; accepted 1 July 2006
Available online 22 August 2006
Abstract—The synthesis, in vitro evaluation, and conformational study of His-Phe-Arg-Trp-NH2 and related derivatives acting as
antifungal agents are reported. Among them, His-Phe-Arg-Trp-NH2 and His-Tyr-Arg-Trp-NH2 exhibited antifungal activity
against Cryptococcus neoformans. Antifungal activity of these compounds appears to be closely related to the a-MSH effect. A con-
formational and electronic study allows us to propose a biologically relevant conformation for these tetrapeptides acting as anti-
fungal agents. In addition, these theoretical calculations permit us to determine the minimal structural requirements to produce
the antifungal response and may provide a guide for the design of compounds with this biological activity.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
Most of these peptides are believed to exert their antimi-
crobial activities either forming multimeric pores in the
lipid bilayer of the cell membranes,10 or interacting with
DNA or RNA after penetration into the cell.13–15
Nevertheless, a-melanocyte stimulating hormone
(a-MSH) and its C-terminal tripeptide Lys-Pro-Val,
which showed antimicrobial activity against two repre-
sentative pathogens: Staphylococcus aureus and Candida
albicans,16 appear to act through a mechanism substan-
tially different from that of other natural antimicrobial
peptides. Previous research16–18 suggests that the candi-
dacidal effect of a-MSH is linked to the cAMP produc-
tion in C. albicans and the adenylyl cyclase inhibitor
ddAdo partly reversed the candidacidal effect of the pep-
tide.16 It was suggested by Eberle and Schwyzer19–21 that
a-MSH might have two message sequences, the first
active sequence centered around the central tetrapeptide
His-Phe-Arg-Trp and the second active site centered
around the C-terminal tripeptide Lys-Pro-Val-NH2.
Thus, we focused our attention on both sequences in
order to evaluate their potential antifungal effects.
In the early 1980s, it was believed that virtually any
fungal infection could be successfully treated with the
wide range of available antifungal agents. Nevertheless,
this belief was soon proved to be false.1 The continuing
increase in the incidence of fungal infections together
with the gradual rise in resistance mainly to azoles in
the last two decades highlighted the need to search novel
compounds not tested previously in antifungal assays.2–5
This event resulted in the identification of novel mole-
cules of diverse structures, which could be useful for a
future development. Among them, some natural pep-
tides have been recently reported as antifungal com-
pounds. They showed to inhibit a broad spectrum of
pathogens and microorganisms6–9 and possess a very
important characteristic, that they do not usually induce
bacterial resistance.10 Regarding their structural fea-
tures, they are cationic, but differing considerably in
other characteristics such as size and presence of disul-
fide bonds and structural motifs.11,12
As part of our ongoing program aimed at identifying
novel antifungal agents, we have reported several natu-
ral and synthetic compounds22–31 exhibiting antifungal
activities against different human pathogenic fungi.
Keywords: His-Phe-Arg-Trp-NH2; Small peptides; Antifungal activi-
ties; Conformational and electronic study.
*
Corresponding author. Tel.: +54 2652 423789; fax: +54 2652
431301; e-mail: denriz@unsl.edu.ar
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.07.007

M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
7605
However, peptide structures had not been tested during
this program until now. In the present study, we report
the design, synthesis and antifungal activity of novel
small-size peptides: 15 tripeptides structurally related
to Lys-Pro-Val (the 11–13 sequence of a-MSH) and 13
tetrapeptides structurally related to His-Phe-Arg-Trp
(the 6–9 sequence of a-MSH). We tested these peptides
against various human pathogenic strains including
yeasts, filamentous as well as dermatophyte fungi. In
addition, a conformational and electronic study on the
most representative peptides reported here was carried
out in order to determine a possible biologically relevant
conformation for these compounds acting as antifungal
agents. Thus, one of the goals of this study was to iden-
tify a topographical and/or substructural template,
which may be the starting structure for the design of
new antifungal compounds.
activation34 and antimicrobial activity.16 However, this
tripeptide did not show any antifungal activity at the
concentrations reported here against any of the strains
of the fungal panel (yeasts: Saccharomyces cerevisiae
and Cryptococcus neoformans; hialohyphomycetes:
Aspergillus flavus, A. niger, and A. fumigatus; dermato-
phytes: Microsporum gypseum, Trichophyton rubrum,
and T. mentagrophytes). It should be noted that com-
pound 1 has been previously reported as antifungal
against a strain of C. albicans.16 However, compound
1 was inactive against all the fungi tested here. Next,
14 tripeptides with different residues (peptides
2–15) were tested against the panel of opportunistic
pathogenic fungi. Results showed that none of these
tripeptides were active against the yeasts, hialohypho-
mycetes and/or dermatophytes neither. It should be not-
ed that peptide 6 was inactive against all the fungi tested
here. This result is in agreement with those previously
reported for peptide 6, by Grieco et al.18 who reported
only 2.7% of inhibition of C. albicans for this tripeptide.
2. Results and discussion
2.1. Chemistry
In a second step of our study, we focused on the tetra-
peptide His-Phe-Arg-Trp-NH2 (16), which is common
to all melanocortin peptides and is important for bind-
ing to the known melanocortin receptors.34 A very inter-
esting result was that this tetrapeptide displayed a
moderate but significant antifungal activity against C.
neoformans (Table 1) as well as against dermatophyte
fungi (MIC values of 125, 250, and 250 lg/ml against
T. mentagrophytes, M. gypseum, and T. rubrum, respec-
tively). Peptide 16 was the only peptide reported here
showing antifungal activity against dermatophytes.
Peptides 1–28 were prepared manually by solid-phase
synthesis on a p-methylbenzhydrylamine resin (1 g
MBHA, 0.39 mmol/g) and Merrifield resin (1 g, 1%,
200–400 mesh) with standard methodology using Boc-
strategy. The peptides were cleaved from the resin with
simultaneous side-chain deprotection by acidolysis with
anhydrous hydrogen fluoride containing 2% anisole, 8%
dimethyl sulfide and indole. Details of the synthesis are
given in Section 4.1 (Synthetic methods).
2.2. Antifungal activity
The next step consisted in testing 12 tetrapeptides struc-
turally related to peptide 16 (peptides 17–28), in which
different replacing in the His-Phe-Arg-Trp-NH2 se-
quence were performed to determine the contribution
of each amino acid to antifungal activity. None of
these tetrapeptides displayed significant effect against
C. albicans, S. cerevisiae, Aspergillus spp., and der-
matophytes. However, six of these tetrapeptides (com-
pounds 17–22) displayed antifungal activity only against
To carry out the antifungal evaluation, concentrations
of peptides up to 250 lg/ml were incorporated to growth
media following the guidelines of the National Commit-
tee for Clinical and Laboratory Standards for yeasts32
and for filamentous fungi.33 Compounds producing no
inhibition of fungal growth at 250 lg/ml level were con-
sidered inactive.
In a first step we focused our attention on the activity of
the sequence Ac-Lys-Pro-Val-NH2 (peptide 1, Scheme 1),
which has been reported as relevant for both receptor
Table 1. Antifungal activity (MIC) of tetrapeptides against Crypto-
coccus neoformans ATCC 32264
Peptide
Structure
MIC (lg/ml)
16
17
His-Phe-Arg-Trp-NH2
His-Tyr-Arg-Trp-NH2
His-Phe-Lys-Trp-NH2
125
125
Ac-Lys-Pro-Val-NH2 (1)
Ac-Lys-Pro-Val-OH (2)
Lys-Pro-Val-NH2 (3)
Lys-Pro-Val-OH (4)
Ac-Lys-Phe-Val-NH2 (5)
Lys-Phe-Val-NH2 (6)
Lys-Phe-Val-OH (7)
Ac-Arg-Pro-Val-NH2 (8)
Arg-Pro-Val-NH2 (9)
Arg-Pro-Val-OH (10)
Ac-His-Pro-Val-NH2 (11)
Ac-Lys-Gly-Ala-NH2 (12)
Ac-Lys-Gly-Gly-NH2 (13)
Ac-Lys-Ala-Ala-NH2 (14)
Ac-Val-Pro-Lys-NH2 (15)
His-Phe-Arg-Trp-NH2 (16)
18
19
250
250
His-Tyr-Arg-Trp-NH2 (17)
His-Phe-Lys-Trp-NH2 (18)
His-Tyr-Lys-Trp-NH2 (19)
His-Phe-D-Arg-Trp-NH2 (20)
His-D-Phe-Arg-Trp-NH2 (21)
His-D-Phe-Lys-Trp-NH2 (22)
Ac-His-Phe-Arg-Trp-NH2 (23)
Ac-His-Tyr-Arg-Trp-NH2 (24)
Ac-His-Phe-Lys-Trp-NH2 (25)
Ac-His-Tyr-Lys-Trp-NH2 (26)
Ac-Trp-Arg-Phe-His-NH2 (27)
For-His-Phe-Arg-Trp-NH2 (28)
His-Tyr-Lys-Trp-NH2
20
21
His-Phe-D-Arg-Trp-NH2
His-D-Phe-Arg-Trp-NH2
His-D-Phe-Lys-Trp-NH2
Ac-His-Phe-Arg-Trp-NH2
Ac-His-Tyr-Arg-Trp-NH2
Ac-His-Phe-Lys-Trp-NH2
Ac-His-Tyr-Lys-Trp-NH2
Ac-Trp-Arg-Phe-His-NH2
For-His-Phe-Arg-Trp-NH2
250
250
22
23
250
>250
>250
>250
>250
>250
>250
24
25
26
27
28
St. drug
Amph B
Ket.
0.25
0.25
Scheme 1. Sequence of the peptides studied in this work.
St., Standard; Amph B, Amphotericin B; Ket., Ketoconazole.

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M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
C. neoformans (Table 1). This is an important finding be-
cause cryptococcosis remains an important life-threaten-
ing complication for immunocompromised hosts and
new compounds acting against this fungus are actually
welcome.3
(Tables 1S–8S are available as supplementary materi-
al). Calculations yielded a large set of conformation-
al families for each peptide studied. The total
number of conformations generated for each peptide
varied between 60,983 and 110,063, and the number
of those accepted was 5000 for all the cases. In the
clustering procedure, an RMSD (root meanꢀ1square
From the structure–activity analysis of the active pep-
tides, it appears that the position of His and Trp-NH2
in the amino acid sequence is essential for such activity,
whereas the position of Phe and Arg is not. Replace-
ment of the Phe residue in position 2 by Tyr (16 ! 17)
was possible without any loss of activity. Replacing
Arg in position 3 by Lys was tolerated but with loss of
activity (compare 16 with 18). Peptide 19 showed that
the replacement of the two central residues was tolerated
but with loss of activity. An identical result was
obtained replacing Arg with D-Arg (compare 16 with
20). Our results indicate that replacement of Phe by
Tyr gives a peptide as potent as the starting structure,
whereas replacing Phe by its isomer D-Phe gives an
active tetrapeptide but with loss of activity (compare
16 with 21 and 22). In addition, all the acetylated
(peptides 23–27) and formylated (28) derivatives were
inactive compounds.
˚
deviation) of 0.75 A and a DE of 20 kcal mol were
used. The number of families after clustering varied
between 435 and 789. The total number of families
accepted with
a relative population higher than
0.20% varied between 37 and 66. Their populations
sum up to ca 80% of all conformations in each case
(see Table 2).
All low-energy conformers of tetrapeptides studied here
were then compared to each other. The comparison
involved the spatial arrangements, relative energy, and
populations. It was found that all the active tetrapep-
tides 16–19 possess similar low-energy conformers.
Figure 1 shows a spatial view of most populates and
energetically preferred conformations of compounds
16 and 17. These conformations have been overlapped
with conformation number 19 obtained for compounds
18, and 19 (Tables 1S–8S in supplementary material).
The conformers shown in Figure 1 are very close one
each other considering the values of the dihedral
angles for their backbones as well as comparing the
dihedral angles defining the mutual spatial arrangement
of side chains.
In order to better understand the above experimental re-
sults and to establish some basis to obtain more active
compounds, we performed a conformational and elec-
tronic study on the most representative peptides report-
ed here, using theoretical calculations.
2.3. Conformational and electronic study of His-Phe-Arg-
Trp-NH2 and its analogues
It is worth mentioning that the families 19 are not the
preferred forms for peptides 18, and 19, which possess
1.62 and 2.13 kcal/mol above their respective global
minimum, and also they are not the most populate
forms. In other words, compounds 18, and 19 can
adopt a very similar spatial ordering to those obtained
for peptides 16 and 17 but this is not possible without
some energetic cost. Figure 2 gives a spatial view of
the preferred conformations obtained for compounds
16, 18 and 19. It is clear that there is not a complete
conformational overlapping between these conformers.
In this case, a good fit among the two first amino
acids (His-Phe in peptides 16 and 18 and His-Tyr in
Linear peptides are highly flexible and therefore the
determination of the biologically relevant conforma-
tions is not an easy task. It is necessary to perform an
exhaustive conformational analysis for these structures.
Therefore, eight tetrapeptides (16–19 and 23–26) were
selected for energy calculations.
Theoretical calculations were carried out as described
in Section 4.4 (Computational methods). These re-
sults are summarized in Table 2 and Tables 1S–8S
Table 2. Selected conformational search and clustering results for peptides 16–19 and 23–26 optimized at the EDMC/SRFOPT/ECCEP/3 level of
theory
Generateda
Acceptedb
# F c
# F0.20%
% Pe
d
Peptide
Electrost.
Random
Thermal
Total
Electrost.
Random
Thermal
Total
16
17
18
19
23
24
25
26
3808
4592
4602
5403
6158
6517
7514
7104
57,095
65,724
73,490
78,159
94,316
91,519
102,050
95,865
80
125
235
225
340
247
499
382
60,983
70,441
78,327
83,787
100,814
98,283
110,063
103,351
576
651
653
747
536
743
448
518
4359
4240
4168
4074
4199
4078
4190
4206
65
109
179
179
265
179
362
276
5000
5000
5000
5000
5000
5000
5000
5000
442
436
512
449
715
623
786
659
55
51
66
37
45
59
49
51
86.76
87.84
86.12
87.72
79.64
82.22
78.82
81.94
a Number of conformations generated electrostatically, randomly, and thermally during the conformational search.
b Number of conformations accepted from those generated electrostatically, randomly, and thermally during the conformational search.
c # F: Total number of conformational families as result of the clustering run.
d # F0.20%: Number of conformational families with populations above 0.20%.
e % P: Sum of the per cent relative population of # F0.20%
.

M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
7607
Figure 1. Stereoview of overlapping of conformer 1 for peptide 16 (red), conformer 1 for analogue 17 (blue), conformer 19 for analogue 18 (green),
and conformer 19 for analogue 19 (yellow). All hydrogens are omitted for clarity.
Figure 2. Stereoview of overlapping of conformer 1 for peptide 16 (red), conformer 1 for analogue 18 (green), and conformer 2 for analogue 19
(yellow). All hydrogens are omitted for clarity.
peptide 19) was obtained, but there is not a good
overlapping between the two last amino acids (Arg-
Trp for 16 and Lys-Trp in peptides 18 and 19). Inter-
esting enough, compounds 18 and 19 displayed half of
the antifungal activity in comparison with compounds
16 and 17. Thus, our results let us speculate that the
different antifungal effect could be explained by this
conformational difference.
Molecular recognition and the converse concept of spec-
ificity35 are explained in mechanistic and reductionistic
terms by a stereo-electronic ‘complementarity’ between
the ligand and the receptor.36 In this context, it is obvi-
ous that the knowledge of the stereo-electronic attri-
butes and properties of His-Phe-Arg-Trp-NH2 and its
analogues will contribute significantly to the elucidation
of the mechanism of action at the molecular level or, at

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M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
least, to determine a possible pharmacophoric pattern
for these compounds acting as antifungal compounds.
The intermolecular forces that contribute to both affin-
ity and specificity can be schematically classified as
hydrophobic and electrostatic ones. Therefore, molecu-
lar electrostatic potentials (MEPs) are of particular val-
ue because they permit the visualization and assessment
of the capacity of a molecule to interact electrostatically
with a putative binding site.37–39 MEPs can be interpret-
ed in terms of a stereoelectronic pharmacophore con-
densing all available information on the electrostatic
forces underlying affinity and specificity.
for the preferred conformation of peptide 23. This con-
former displays a very different spatial ordering with re-
spect to the preferred conformation obtained for
compound 16 (Fig. 4a). While for active compounds, a
partially extended (b-strand) conformation for the back-
bone is the preferred form, for peptide 23 the preferred
form for the backbone is a folded conformation which is
stabilized through hydrogen bonds. The interaction
shown in Figure 4b is a bifurcated hydrogen bond be-
tween the carbonyl system of the acetyl group and the
guanidine group of Arg residue. This stabilizing interac-
tion might be better appreciated in Figure 5. The differ-
ent electronic distributions obtained for the acetylated
peptides with respect to those obtained for compounds
16–19 might be well appreciated comparing Figure 3
with 5. All the acetylated and formylated derivatives
(peptides 24–28) displayed a conformational behavior
closely related to that obtained for compound 23.
Figures 4c and d give a comparative spatial view be-
tween the preferred conformations of peptide 17 and
its acetylated derivative (compound 24). On the basis
of our results, we can conclude that the lack of anti-
fungal activity of these acetylated peptides could be ex-
plained, at least in part, by their different
conformational and electronic behaviors.
Once the low-energy conformations for the most active
tetrapeptides reported here were obtained and in an
attempt to find the potentially reactive sites for the
ligands, we evaluated the electronic aspects of the mole-
cules using MEPs. Figure 3 shows the MEPs obtained
for compounds 16–19. These results account for the gen-
eral characteristics of the electronic behavior of active
tetrapeptides reported here. The general pattern is simi-
lar for all of the active systems. The MEPs exhibit clear
minimum values (deep red zones) in the vicinity of the
lone pair of nitrogen atom of His and near the carbonyl
groups of the backbone. There are positive regions near
the Arg residue (peptides 16 and 17) or Lys residue
(peptides 18 and 19) and at the amino terminal group.
Also three clear hydrophobic zones might be appreciat-
ed near the aromatic rings of His, Phe (or Tyr), and
Trp.
It is worthwhile to compare the biologically active con-
formation for the backbone of the His-Phe-Arg-Trp-
NH2 proposed in this study to that previously reported
for the same sequence from proton NMR studies in
aqueous solution.40 Sugg et al. reported that the
observed topology was consistent with nonhydrogen-
bonded b-like structure (/ = 139ꢁ and w = 135ꢁ for
All the acetylated derivatives tested here were inactive
compounds. Figure 4b shows the spatial view obtained
Figure 3. Electrostatic potential-encoded electron density surfaces of peptides 16 (a), 17 (b), 18 (c) and 19 (d). The surfaces were generated with
Gaussian 03 after RHF/6-31G(d) single-point calculations. The coloring represents electrostatic potential with red indicating the strongest attraction
to a positive point charge and blue indicating the strongest repulsion. The electrostatic potential is the energy of interaction of the positive point
charge with the nuclei and electrons of a molecule. It provides a representative measure of overall molecular charge distribution.

M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
7609
Figure 4. Spatial view of the preferred conformations obtained for peptides 16 (a), 23 (b), 17 (c), and 24 (d) using EDMC calculations. The hydrogen
bonds are denoted by dotted lines.
Figure 5. Electrostatic potential-encoded electron density surface of peptide 23. The surface was generated with Gaussian 03 after RHF/6-31G(d)
single-point calculation. The color-coded is shown at the left.
L-amino acids) as the predominant solution conforma-
tion. This conformation is closely related to those ob-
tained for the different conformers of the active
peptides reported here. In Figure 6 we overlapped the
conformation reported by Sugg et al. (in white) with
three representative conformations obtained for peptide
16. In the case of conformer 11 (Fig. 6a) a complete
overlap, not only with the backbone but also with the
vicinal side chains, was obtained. For conformers 5
and 1 (Figs. 6b and c) although there is not a complete
fit, the resemblance is still apparent.
At this stage of our study, some general trends might be
established:

7610
M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
Figure 6. Overlapped stereoviews of the backbone conformation from Ref. 40 in white and various conformations for compound 16 in red: (a)
conformer 11, (b) conformer 5, and (c) conformer 1. All hydrogens are omitted for clarity.
• The smallest fragments with measurable antifungal
activity were His-Phe-Arg-Trp-NH2 and its analo-
gues 17–22. It has been shown that the minimal
sequence possessing a-MSH-like activity is the central
tetrapeptide His6-Phe7-Arg8-Trp9.34 This ‘core’
sequence was suggested to be the ‘message’ fragment
for a-MSH, the rest of the molecule being regarded as
the ‘address’ sequence.41
3. Conclusions
We synthesized small-size peptides (tri- and tetrapep-
tides) with different residues and tested their antifungal
activity. Among the compounds tested, His-Phe-Arg-
Trp-NH2 and analogues displayed antifungal activities
mainly against C. neoformans. The antifungal activity
of these compounds appears to be closely related to
the a-MSH effect. These results are in a complete agree-
ment with those previously reported by Grieco et al.
against C. albicans.16–18
• For the a-MSH activity, the importance of His6
was demonstrated by the fact that Ac-a-MSH7–10
-
NH2 lacked measurable activity. Trp9 was also crit-
ical for biological activity as demonstrated by the
lack of biological activity of Ac-a-MSH6–8. Our
results suggest that the presence of His and Trp-
NH2 sequence appears to be a structural require-
ment for the antifungal activity as well. In contrast,
our results indicate that Phe and Arg could be
replaced.
A detailed conformational and electronic study sup-
ported by theoretical calculations helped us to iden-
tify a possible ‘biologically relevant conformation’
and understand the minimal structural requirements
for the antifungal actions of tetrapetides reported
here. Our results are very encouraging in that they
• The group of A. Catania16–18 suggested that the
antimicrobial effects of a-MSH are exerted
through a unique mechanism different from that
of other natural antimicrobial peptides. They sug-
gested that the candidacidal effect of a-MSH is
linked to the cAMP-inducing activity. Our results
may be regarded as an additional support for that
hypothesis.
• It is interesting to note that the tetrapeptides 16–22,
being the smallest fragment with antifungal activity,
contains three aromatic residues. In this regard, aro-
matic amino acids are common in the active site of
many hormones and are often considered critical
for biological activity.42
show a great potential of His-Phe-Arg-Trp-NH2
and analogues as a truly novel class of antifungal
compounds particularly against the yeast C.
neoformans.
Although it is desirable to develop compounds having
a broad spectrum of activity, it is also important to
bear in mind that long treatments of fungal infections
with the same broad spectrum antifungal agent may
lead to a high resistance to the available antifungal
agents.43 Thus, one of the strategies for overcoming
this problem is the treatment of fungal infections with
the appropriate narrow spectrum agent when the etio-
logical agent is known.44 The selective antifungal
activity reported in this paper for peptides 16–22
(and among them for compounds 16 and 17 in partic-
ular) opens an interesting field of research, which
deals with the discovery and further development of
new antifungal peptides useful for treating cryptococ-
• Our conformational and electronic study indicates
that a predominant b-conformation without inter-
nal stabilizing hydrogen bond could be the
‘biologically relevant conformation’ for these
compounds.

M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
7611
cosis, an important life-threatening infection for
immunocompromised hosts.
(5 ml) using DCC (2.5 equiv) and HOBt (2.5 equiv) until
completion (3 h) judged by Kaiser ninhydrin test. After
coupling of the appropriate amino acid, Boc deprotec-
tion was effected by use of TFA/CH2Cl2 (1:1, 5 ml) for
5 min first then repeated for 25 min. Following neutral-
ization with 10% TEA/CH2Cl2 three times (5–5 ml of
each), the synthetic cycle was repeated to assemble the
resin-bond protected peptide. The peptides were cleaved
from the resin with simultaneous side-chain deprotec-
tion by acidolysis with anhydrous hydrogen fluoride
(5 ml) containing 2% anisole, 8% dimethyl sulfide and
indole at 5 ꢁC for 45 min. The crude peptides were dis-
solved in aqueous acetic acid and lyophilized. Prepara-
tive and analytical HPLC of the crude and the purified
peptides were performed on an LKB Bromma apparatus
(for preparative HPLC, column: Lichrosorb RP C18,
7 lm, 250 · 16 mm; gradient elution: 30–100%, 70 min;
mobile phase: 80% acetonitrile, 0.1% TFA; flow rate:
4 ml/min, 220 nm, for analytical HPLC, column: Phe-
nomenex Luna 5C18, 250 · 4.6 mm; mobile phase:
80% acetonitrile, 0.1% TFA; flow rate: 1.2 ml/min,
220 nm). ESI-MS: Finnigan TSQ 7000.
4. Experimental section
4.1. Synthetic methods
Solid phase synthesis of the peptides (His-Phe-Arg-Trp-
NH2, His-D-Phe-Arg-Trp-NH2, His-Tyr-Arg-Trp-NH2,
His-Phe-Lys-Trp-NH2, His-D-Phe-Lys-Trp-NH2, His-
Tyr-Lys-Trp-NH2, His-Phe-D-Arg-Trp-NH2, His-D-
Phe-D-Arg-Trp-NH2, His-Phe-D-Lys-Trp-NH2, Arg-
Pro-Val-NH2, Lys-Phe-Val-NH2, Lys-Pro-Val-NH2,
Ac-Arg-Pro-Val-NH2, Ac-Lys-Phe-Val-NH2, Ac-Val-
Pro-Lys-NH2, Ac-His-Pro-Val-NH2, Ac-Lys-Pro-Val-
NH2, Ac-Lys-Gly-Gly-NH2, Ac-Lys-Gly-Ala-NH2, Ac-
Lys-Ala-Gly-NH2, Ac-His-Phe-Arg-Trp-NH2, For-His-
Phe-Arg-Trp-NH2, Ac-Trp-Arg-Phe-His-NH2, Ac-His-
Tyr-Arg-Trp-NH2, Ac-His-Phe-Lys-Trp-NH2, Ac-His-
Tyr-Lys-Trp-NH2, Lys-Pro-Val-OH, Arg-Pro-Val-OH,
Lys-Phe-Val-OH, and Ac-Lys-Pro-Val-OH) was carried
HPLC data of the synthesized peptides
Retention factor (min)
Gradient elution (%)
1.
2.
His-Phe-Arg-Trp-NH2
His-D-Phe-Arg-Trp-NH2
Lys-Pro-Val-NH2
12.912
6.510
9.962
8.048
6.523
5.552
10.570
8.343
9.598
9.079
9.004
6.068
7.434
12.750
11.878
5.674
8.311
8.153
8.919
12.703
6.533
8.364
11.770
7.297
8.785
8.781
6.235
8.945
9.254
11.109
5–60 (20 min)
20–50 (15 min)
0–25 (15 min)
20–50 (15 min)
20–50 (20 min)
5–50 (20 min)
5–50 (20 min)
10–70 (20 min)
10–70 (20 min)
10–70 (20 min)
10–70 (20 min)
20–50 (15 min)
5–80 (20 min)
10–45 (15 min)
5–80 (25 min)
0–30 (10 min)
10–45 (15 min)
5–25 (10 min)
15–45 (15 min)
5–80 (25 min)
10–35 (10 min)
5–35 (10 min)
5–80 (20 min)
20–50 (10 min)
20–50 (10 min)
5–80 (25 min)
0–40 (20 min)
0–30 (15 min)
5–20 (15 min)
5–50 (20 min)
3.
4.
His-Phe-Lys-Trp-NH2
His-Tyr-Arg-Trp-NH2
Arg-Pro-Val-NH2
5.
6.
7.
Lys-Phe-Val-NH2
8.
His-Phe-D-Lys-Trp-NH2
His-D-Phe-D-Arg-Trp-NH2
His-Phe-D-Arg-Trp-NH2
His-Tyr-Lys-Trp-NH2
His-D-Phe-Lys-Trp-NH2
Ac-Lys-Gly-Gly-NH2
For-His-Phe-Arg-Trp-NH2
Ac-Lys-Phe-Val-NH2
Ac-Lys-Gly-Ala-NH2
Ac-Lys-Pro-Val-OH
Ac-Lys-Pro-Val-NH2
Ac-His-Tyr-Lys-Trp-NH2
Ac-His-Phe-Lys-Trp-NH2
Ac-His-Pro-Val-NH2
Ac-Val-Pro-Lys-NH2
Ac-Trp-Arg-Phe-His-NH2
Ac-His-Tyr-Arg-Trp-NH2
Ac-His-Phe-Arg-Trp-NH2
Ac-Arg-Pro-Val-NH2
Ac-Lys-Ala-Ala-NH2
Lys-Pro-Val-OH
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
Arg-Pro-Val-OH
Lys-Phe-Val-OH
out manually on a p-methylbenzhydrylamine resin (1 g
MBHA, 0.39 mmol/g) and Merrifield resin (1 g, 1%,
200–400 mesh), respectively, with standard methodology
using Boc-strategy. Side-chain protecting groups were as
follows: Arg(Tos), His(Tos), Lys(2Cl-Z), and Tyr(2Br-
Z). All protected amino acids were coupled in CH2Cl2
4.2. Microorganisms and media
For the antifungal evaluation, strains from the Ameri-
can Type Culture Collection (ATCC), Rockville, MD,
USA, and CEREMIC (C), Centro de Referencia
´
´
Micologica, Facultad de Ciencias Bioquımicas
y

7612
M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
´
Farmaceuticas, Suipacha 531-(2000)-Rosario, Argentina
were used: Candida albicans ATCC 10231, Saccharomy-
ces cerevisiae ATCC 9763, Cryptococcus neoformans
ATCC 32264, Aspergillus flavus ATCC 9170, Aspergillus
fumigatus ATCC 26934, Aspergillus niger ATCC 9029,
and Trichophyton mentagrophytes ATCC 9972, and
Trichophyton rubrum C 113, Microsporum gypseum C
115.
applied at this stage, greatly improves the acceptance
coefficient. In this algorithm / and w angles are changed
in a manner which allows the corresponding peptide
group to find the most proper orientation in the electro-
static field of the rest of the peptide chain. The energy of
the new conformation is minimized, compared to the
previous one and may be accepted or discarded on the
basis of energy and/or geometry. If the new energy-min-
imized conformation is similar in shape and in energy to
the starting conformation, it is discarded. Otherwise, the
energy of the new conformation is compared to the ener-
gy of the parent conformation. If the new energy is low-
er, the new conformation is accepted unconditionally,
otherwise the Metropolis criterion54 is applied in order
to accept or reject the new conformation. If the new con-
formation is accepted, it replaces the starting one; other-
wise another perturbation of the parent conformation is
tried. A temperature jump may be included if the pertur-
bation is not successful for an arbitrarily chosen number
of iterations. The process is iterated, until a sufficient
number of conformations have been accepted. The
detailed procedure is described in Ref. 55.
Strains were grown on Sabouraud-chloramphenicol
agar slants for 48 h at 30 ꢁC, maintained on slopes of
Sabouraud-dextrose agar (SDA, Oxoid), and subcul-
tured every 15 days to prevent pleomorphic transforma-
tions. Inocula of cell or spore suspensions were obtained
according to reported procedures and adjusted to 105
cells/spores with colony forming units (CFU)/mL.45
4.3. Antifungal susceptibility testing
Minimal inhibitory concentration (MIC) of each com-
pound was determined by using broth microdilution
techniques according to the guidelines of the National
Committee for Clinical Laboratory Standards for yeasts
(M27-A2)32 and for filamentous fungi (M 38 A).33 MIC
values were determined in RPMI-1640 (Sigma, St. Louis,
MO, USA) buffered to pH 7.0 with MOPS. The starting
inocula were 1 · 103 to 5 · 103 CFU/ml. Microtiter trays
were incubated at 35 ꢁC for yeasts and hialohyphomyce-
tes, and at 28–30 ꢁC for dermatophyte strains in a moist,
dark chamber, and MICs were visually recorded at 48 h
for yeasts, and at a time according to the control fungus
growth, for the rest of fungi.
In order to explore the conformational space extensively,
we carried out 10 different runs, each of them with a dif-
ferent random number, for each peptide studied. Since
the EDMC procedure uses random numbers, there is a
need to initialize the random number generator by pro-
viding an integer. Therefore, we collected a total of
5000 accepted conformations for each peptide studied.
Each EDMC run was terminated after 500 energy-mini-
mized conformations had been accepted. The parameters
controlling the runs were the following: a temperature of
298.15 K was used for the simulations. A temperature
jump of 50,000 K was used; the maximum number of
allowed repetitions of the same minimum was 50; the
maximum number of electrostatically predicted confor-
mations per iteration was 400; the maximum number
of random-generated conformations per iteration was
100; the fraction of random/electrostically predicted con-
formations was 0.30; the maximum number of steps at
one increased temperature was 20; and the maximum
number of rejected conformations until a temperature
jump is executed was 100. Only trans peptide bonds
(x ffi 180ꢁ) were considered.
For the assay, stock solutions of peptides were twofold
diluted with RPMI 250-1 lg/ml (final volume = 100 ll)
and a final DMSO concentration 61%. A volume of
100 ll of inoculum suspension was added to each well
with the exception of the sterility control where sterile
water was added to the well instead. MIC was defined
as the minimum inhibitory concentration of pure com-
pound which resulted in total inhibition of the fungal
growth. Ketoconazole and Amphotericin B were used
as positive controls.
4.4. Computational methods
4.4.1. EDMC calculations. The conformational space of
each peptide was explored using the method previously
employed by Liwo et al.46 that included the Electrostat-
ically Driven Monte Carlo (EDMC) method.47,48
Conformational energy was evaluated using the
ECEPP/3 force field.49 This force field employs rigid va-
lence geometry. Hydration energy was evaluated using a
hydration-shell model with a solvent sphere radius of
The ensemble of obtained conformations was then clus-
tered into families using the program ANALYZE,56–58
which applies the minimal-tree clustering algorithm for
separation, using all heavy atoms, energy threshold of
20 kcal molꢀ1, and RMSD of 0.75 A as separation crite-
˚
ria. This procedure allows for substantial reduction of
the number of conformations and eliminates repetitions.
˚
1.4 A and atomic hydration parameters that have been
4.4.2. Molecular electrostatic potentials. The electronic
study of the peptides was carried out using Molecular
Electrostatic Potentials (MEPs). MEPs have shown to
provide reliable information, both on the interaction
sites of molecules with point charges and on the compar-
ative reactivities of these sites.37–39 These MEPs were
calculated using RHF/6-31G(d) wave function from
the Gaussian 03 program.59 EDMC coordinates were
imported to generate the wave functions; thus, RHF/6-
optimized using nonpeptide data (SRFOPT).50,51 In this
model, in addition to a sum of electrostatic, non-bond-
ing, hydrogen-bond and torsional energy terms, the
total conformational energy includes terms accounting
for loop closing and peptide solvation. The conforma-
tion with minimized energy was subsequently perturbed
by changing its torsional / and w angles using the Mon-
te Carlo method.52 Piela’s algorithm,53 which was also

M. F. Masman et al. / Bioorg. Med. Chem. 14 (2006) 7604–7614
7613
18. Grieco, P.; Rossi, C.; Gatti, S.; Colombo, G.; Carlin, A.;
Novellino, E.; Lama, T.; Lipton, J. M.; Catania, A.
J. Med. Chem. 2005, 48, 1384.
19. Eberle, A.; Schwyzer, R. Helv. Chim. Acta 1975, 58, 1528.
20. Eberle, A.; Fauchere, J.-L.; Tesser, G. I.; Schwyzer, R.
Helv. Chim. Acta 1975, 58, 2106.
31G(d) single-point calculations were performed from
Gaussian 03 program. All MEPs’ graphical presenta-
tions were created using Molekel.60
Acknowledgments
21. Eberle, A.; Schwyzer, R. In Surface Membrane Receptors;
Bradshaw, R. A., Frazier, W. A., Merrell, R. C., Gotteib,
I., Hogue-Anselletti, R. A., Eds.; Plenum: New York,
1976; pp 291–304.
22. Zacchino, S.; Rodriguez, G.; Pezzenati, G.; Orellana, G.;
Enriz, R. D.; Gonzalez Sierra, M. J. Nat. Prod. 1997, 60,
661.
This work is part of the Hungarian-Argentine Intergov-
ernmental S & T Cooperation Programme. This re-
search was supported by grants from Universidad
Nacional de San Luis and grants to S.A.Z. and
´
´
´
R.D.E. (Agencia de Promocion Cientıfica y Tecnologica
de la Argentina PICT R 260) and it is part of the Ibero-
american Project X.7 PIBEAFUN (Search and develop-
ment of new antifungal part of the Iberoamerican
Program of Science and Technology for the Develop-
ment (CYTED)). R. D. Enriz is a member of the
´
´
23. Zacchino, S.; Lopez, S. A.; Pezzenati, G.; Furlan, R.;
Santecchia, C.; Mun˜oz, L.; Giannini, F.; Rodrıguez, A.
´
M.; Enriz, R. D. J. Nat. Prod. 1999, 63, 1353.
24. Kouznetsov, V. V.; Urbina, J.; Palma, A.; Lopez, S.;
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Ribas, J.; Enriz, R. D.; Zacchino, S. Bioorg. Med. Chem.
2000, 8, 691.
25. Lopez, S. N.; Castelli, M. V.; Zacchino, S.; Dominguez, J.;
Lobo, G.; Charris-Charris, J.; Cortes, D.; Ribas, J.; Devia,
C.; Rodriguez, A. M.; Enriz, R. D. Bioorg. Med. Chem.
2001, 9, 1999.
CONICET (Argentina) staff. The authors thank
´
´
´
´
E. Menyhart and I. Nogradi for the technical assistance
of peptide synthesis, as well as for the support of OTKA
TS049817 and RET 08/2004 Grants.
26. Vargas, L. Y.; Castelli, M.; Kouznetsov, V.; Urbina, J.
M.; Lopez, S. N.; Sortino, M.; Enriz, R. D.; Ribas, J. C.;
Zacchino, S. Bioorg. Med. Chem. 2003, 11, 1531.
27. Karolyhazy, L.; Freile, M. L.; Anwair, M.; Beke, G.;
Giannini, F.; Sortino, M.; Ribas, J. C.; Zacchino, S.;
Matyus, P.; Enriz, R. D. Arzneimittel-Forsch 2003, 5, 738.
28. Freile, M. L.; Giannini, F.; Pucci, G.; Sturniollo, A.;
Dodero, L. R.; Pucci, O.; Balzareti, V.; Enriz, R. D.
Fitoterapia 2003, 74, 702.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmc.
2006.07.007.
29. Giannini, F.; Aimar, L. M.; Sortino, M. V.; Gomez, R.;
Sturniollo, A.; Juarez, A.; Zacchino, S.; De Rossi, R. H.;
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