Synthesis and antifungal properties of arginine-containing hemin derivatives

Article abstract of DOI:10.1007/s00044-011-9928-2

Due to a steadily increasing number of clinical isolates of microscopic fungi that show resistance to conventionally administered antimycotics, the specialists involved have constantly been searching for novel fungicidal agents among various classes of ch

Full text of DOI:10.1007/s00044-011-9928-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3876–3884
DOI 10.1007/s00044-011-9928-2
ORIGINAL RESEARCH
Synthesis and antifungal properties of arginine-containing
hemin derivatives
•
G. A. Zheltukhina S. A. Okorochenkov
•
•
N. V. Groza V. G. Arzumanyan V. E. Nebolsin
•
Received: 19 May 2011 / Accepted: 1 December 2011 / Published online: 11 December 2011
ꢀ Springer Science+Business Media, LLC 2011
Abstract Due to a steadily increasing number of clinical
isolates of microscopic fungi that show resistance to con-
ventionally administered antimycotics, the specialists
involved have constantly been searching for novel fungicidal
agents among various classes of chemical compounds. The
present study was aimed at synthesizing and assessing the
activity of arginine-containing hemin derivatives against
strains of Candida albicans and Cryptococcus neoformans
insensitive to commonly used antifungal agents. A hemin
conjugate with a branched arginine-containing peptide was
shown to exhibit in vitro fungicidal activity at a concentra-
tion of 25 lM and inhibit the growth of the yeast-like fungus
Candida albicans at concentrations as low as 12.5 lM.
microscopic fungi, which are single- or multi-cellular
hemoorganoheterotrophic eukaryotes. The prevalence of
mycoses is currently growing at a rapid pace due to the
migration of populations and alterations in the way of life in
industrially developed countries. Mycosis-related damage
is particularly perceived in transplantology, oncohematol-
ogy, and neonatology; the most commonly encountered
pathogens of mycoses, such as yeasts of the Candida spp.,
have been steadily ousting nosocomial bacterial infections
from their leading positions in clinical medicine. Mycoses
prevail in the structure of AIDS-associated pathologies and
in individuals with impaired host defenses. Among the well-
known pathogens of mycotic infections, the most dangerous
are the yeasts of the Candida genus, inducing vulvovaginal
candidiasis, bronchomycosis, deep invasive, and dissemi-
nated candidiases. In recent years, frequently encountered
invasive fungal infections have also been associated
with Aspergillus fumigatis and Cryptococcus neoformans
(Clement et al., 2008). Cryptococcosis is typically treated
with combinations of agents known to possess various
mechanisms of action, which is intended to both attain a
synergetic effect and due to increasingly frequent cases
involving drug-resistant strains. Those few currently exist-
ing antifungal agents to which resistance does not develop
at all or develops rarely (e.g., amphotericin B) are typically
known to exhibit considerable human toxicity (Holeman
and Einstein 1963). Taking the abovementioned facts into
consideration, one cannot deny the vital importance of
uninterruptedly searching for novel fungicidal agents
among naturally occurring compounds and structural ana-
logs thereof possessing wide-spectrum activity, especially
against drug-resistant fungal strains. We have therefore
attempted to study hemin conjugates (hereinafter referred to
as HC) with synthetic peptides, including antimicrobial
peptides.
Keywords Hemin Á Arginine-containing peptides Á
Antifungal agents Á Candida albicans Á
Cryptococcus neoformans Á 3-HETE
Introduction
Mycoses appear to have been accompanying the human
civilization from time immemorial. They are induced by
G. A. Zheltukhina Á S. A. Okorochenkov (&) Á N. V. Groza
Moscow State Academy of Fine Chemical Technology Named
After M. V. Lomonosov, Moscow, Russia
e-mail: laboratory211@yandex.ru
V. G. Arzumanyan
Scientific Research Institute for Vaccines and Sera Named After
I. I. Mechnikov Under the Russian Academy of Medical
Sciences, Moscow, Russia
V. E. Nebolsin
Limited Liability Company ‘‘Pharmenterprises’’ Moscow,
Moscow, Russia
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Med Chem Res (2012) 21:3876–3884
3877
The choice of hemin, a naturally occurring metallopor-
phyrin, as the basis for creating new fungicidal agents was
largely predetermined by its known prooxidant properties,
which probably also contribute to the antimicrobial activity
of hemin, especially in the presence of hydrogen peroxide
(Everse and Hsia, 1997; Malik et al., 1988). Production of
reactive oxygen species (ROS) lies at the basis of the
antifungal action of myeloperoxidase present in the spe-
cialized antimicrobial organelles of neutrophils, peroxi-
somes (Rogovin et al., 1977). However, the use of hemin in
clinical practice is complicated due to its toxicity for red
blood cells, water insolubility, and a short-term biocidal
effect (Nitzan et al., 1987). Other promising antifungal
agents are fungicidal peptides possessing high activity
toward pathogenic fungi (Ajesh and Sreejith, 2009).
However, these compounds appear to frequently render a
considerable hemolytic effect. Besides, they are unstable in
physiological media as a result of degradation when acted
upon by proteinases. At the same time, hemin has been
shown to inhibit proteinases, including chymotrypsin and
HIV proteinase (Patent RU No. 2238959, 2004). Therefore,
it might be expected that hemin conjugates with peptides,
including antifungal peptides, could have increased resis-
tance to proteinases, while at the same time remain active
toward pathogenic fungi. Since the majority of antifungal
peptides do have sequences enriched with residues of
amino acids with positively charged lateral functions (Lys,
Arg, or His), we have synthesized new hemin conjugates
with the following arginine-containing peptides: HC with
an analog of a combi-1-N-a-acetylated synthetic peptide
possessing high fungicidal activity against Candida albi-
cans (Blondlle and Houghten, 1996), HC with an RGD
peptide from the sequence of a cell adhesion factor
encountered in the sequences of antimicrobial peptides, and
HC with the simplest branched arginine-containing peptide
Glu(ArgOMe)ArgOMe. We investigated the antifungal
activity of these novel HCs, the peptide Ac-RRWWRF-
OH, and a previously synthesized hemin conjugate with an
RGD peptide (Okorochenkov et al., 2010).
Oxylipin, 3-hydroxy-(5Z,8Z,11Z,14Z)-eicosatetraenoic
acid (3-HETE), was obtained by means of complete
chemical synthesis according to a technique we had
worked out previously, with purity of 98–99% (Groza
et al., 2002).
As a polymeric carrier, we used a styrene copolymer
with 1% divinylbenzene and a 2-chlorotrityl chloride
anchor group with the chlorine content amounting to
1.43 mmol/g (Bachem, Switzerland). Solid-phase synthesis
of peptides was carried out according to the Fmoc strategy
(Barlos et al., 1991). The degree of substitution of the
carrier with the starting amino acid was determined spec-
trophotometrically (Dryland and Sheppard, 1986).
The identity of the obtained compounds was checked by
means of TLC on Kieselgel 60 F254 plates (Merck, Ger-
many) in the following systems: chloroform–methanol–
25% ammonia (5:3:0.5) (1), chloroform–methanol 9:1 (2),
chloroform–methanol–25% ammonia 6:1:0.2 (3). The
chromatograms were developed with either the chlorine-
benzidine reagent or UV light.
Analytical HPLC was performed on a Gilson 305
chromatograph (Gilson, France) under the following con-
ditions: column (2 9 150 mm), reverse-phase C18, sorbent
mean pore diameter 5 lm (Phenomenex, USA) in a gra-
dient of 80% aqueous acetonitrile in a 0.05% TFA solution
from 0 to 100%, and detection at 220 nm. Preparative
HPLC was carried out under the following conditions:
column (19 9 250 mm), reverse-phase C18, sorbent pores
mean diameter 5 lm (Phenomenex, USA) at a gradient of
80% aqueous acetonitrile in a 0.15% TFA solution from 0
to 60%, and detection at 280 nm.
Column chromatography was performed on Sephadex
LH-20 (Pharmacia, Sweden) or on Kieselgel 60 F 254
plates (Merck, Germany).
High-performance mass spectra were obtained on an
Ultraflex time-of-flight spectrometer (Bruker, Germany) by
means of matrix-assisted laser desorption ionization
(MALDI-TOF), with 2,5-dihydroxybenzoic acid used as
the matrix.
Electron spectra were registered on a UV–Visible
spectrophotometer (Helios, UK).
Materials and methods
Experimental
Study of the antifungal action of hemin peptides and
their combined effect with oxylipin 3-HETE. The objects
acted upon were cells of museum strains of Candida
albicans No. 927 and Cryptococcus neoformans No. 3465
grown on yeast–peptone–glucose (YPG) medium at 32ꢁC
for 24 h. The antimycotics pymafucin, amphotericin, and
fluconazole were used as comparison compounds.
All experiments were carried out in duplicate using
round-bottom 96-well culture plates (Lenmedpolimer,
Russia). The first well was filled with 10 ll of the stock
solution of water-soluble agents at a concentration of
10-2–10-3 mol/l, the second one with 10 ll plus 10 ll of
General procedures and materials
The amino acids and L-series derivatives used in this study
were manufactured by the Bachem Company (Germany).
DIC, HOBt, DCC, 9-FmocONSu, Et3N, and hemin (from a
porcine source) were supplied by Sigma (USA). All sol-
vents were anhydrous, except those used for extraction
from aqueous solutions.
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Med Chem Res (2012) 21:3876–3884
Solid-Phase Synthesis of Ac-RRWWRF-OH (2)
on the Polymer with a 2-Chlorotrityl Chloride Anchor
Group
the solvent corresponding to the given compound, followed
by suspending and transferring 10 ll into the next well and
so on for all eight wells of the row. The stock solutions of
water-insoluble compounds were prepared in DMSO and
diluted with DMSO and the medium. The DMSO con-
centration in the reaction well was B5% and did not
influence the yeast growth (separate control).
The peptide RRWWRF was synthesized on 2-chlorotrityl
chloride resin using a modified standard Fmoc-SPPS protocol
(Zheltukhina et al., 2006). Since NG-unprotected Fmoc-
Arg-OH was used, after each Fmoc cleavage cycle, the resin
was treated with HOBt (10 eq.) in 1 ml of DMF for arginine
guanidine side chain protonation. Once synthesis and
deblocking of the a-amino group was completed, 0.143 g of
the peptidylpolymer was washed with DMF (1 ml 9 eight
times, each for 2 min), washed with HOBt (3.6 eq.) in 1 ml of
DMF, and acetylated with p-nitrophenyl acetate (3 eq.) in the
presence of triethylamine (1 eq.) for 2 h. Completeness of
amino-group replacement was controlled by means of the
ninhydrin test. The acetylated peptidylpolymer was washed
withDMF(1 ml, eighttimes,eachfor2 min)andDCM(1 ml,
twice, each for 2 min), dried in vacuo, and supplemented with
4 ml of a TFA:TFE:DCM mixture (1:1:8), stirring for 3 h
under a nitrogen atmosphere. The polymer was separated and
washed with 1:1:8 TFA:TFE:DCM (1 min 9 2 ml); the sol-
vents from the filter were removed in vacuo, the residue was
triturated using a 10- to 12-fold excess volume of cooled
anhydrous ether, and the deposit which formed was filtered,
washed twice with ether, and vacuum-dried. The peptide was
isolated with preparative HPLC. The yield amounted to
0.013 g (43%). Rf 0.8 (6). IR (KBr, cm-1): 1724 (COOH),
1656 (C=O amide I), 1543 (C=O amide II); MALDI-MS
(m/z): 1049.2 [M]? (calculated 1049.4); HPLC: individual
peak, retention time 13.76 min.
The experiment involving combined incubation of the
culture with a derivative of fatty acid (3-HETE) and HC
was performed using the stock solution of oxylipin at a
concentration of 550 lM in ethanol with serial dilutions to
obtain the concentrations of 7 and 14 lM per well, with the
ethanol concentration in the reactive well not exceeding
0.2%.
Each well was then supplemented with 190 ll of sus-
pended cellular culture of Candida albicans or Crypto-
coccus neoformans in synthetic nutrient medium (a final
concentration of approximately 103 CFU/ml), containing
the pH indicator bromine-cresol blue (at a pH of 5.5 in the
medium, the indicator turns blue). Once the pH value
decreases to 4.5, the color of the indicator in the medium
changes to yellow, as a sign of fungal growth. The syn-
thetic medium consisted of the following components: salt,
amino acids, trace elements, vitamins, an antibiotic, and
glucose (with a total of 30 components) (Yarrow, 1998).
After administering the suspension of Candida albicans
cells, the plates were incubated for 1 h at 32ꢁC with agi-
tation, then transferred to the stationary thermostat and
incubated for a further 24 h and 4 days at 27ꢁC. After the
addition of the suspension of Cryptococcus neoformans,
the plates were incubated for 1 h at 32ꢁC with agitation,
then transferred to the stationary thermostat and incubated
for 2 and 5 days at 27ꢁC. In order to detect live cells, we
performed inoculation from the wells containing HC after
4 days.
N-a-[6(7)-(Protohemin IX)-yl]-RRWWRF-OH Á 3
CF3COOH (3)
A total amount of 0.29 g of the peptidylpolymer synthesized
as mentioned above after Fmoc group cleavage was sup-
plemented with 3 eq. of the 6(7)-mono-N-oxy-5-norboren-
2,3-decarboxyimide ester of protohemin IX in 4.5 ml of
DMF, agitated for 5 h, and allowed to stand for 24 h at room
temperature. The hemin peptidylpolymer was separated and
washed with DMF (3 ml, seven times, each for 3 min). The
ninhydrin test was negative. The hemin peptidylpolymer
was washed with DCM (2 ml, twice, each for 2 min),
dried in vacuo, then supplemented with 4.5 ml of a
TFA:TFE:DCM mixture (1:1:8), and stirred in a nitrogen-
containing atmosphere for 3 h. The polymer was separated
and washed with a 1:1:8 mixture of TFA:TFE:DCM (1 ml,
four times, each for 1 min). The residue was triturated with
cooled anhydrous diethyl ether (using a 10- to 12-fold
excess in volume); the precipitate was filtered, washed twice
with ether, and vacuum-dried. The product was purified on a
Sephadex LH-20 column (150 9 20 mm), eluting the target
In the experiment aimed at clarifying the mechanism of
action of 3, a 24-h culture of. Candida albicans strain No.
927 was suspended in a potassium phosphate buffer of pH
4.6 to a cell density of approximately 106 CFU/ml. In a test
tube, 100 ll of the given suspension and 100 ll of 3 at a
concentration of 2 9 10-3 M were combined. In the con-
trol tube, 100 ll of the given suspension and 100 ll of the
corresponding buffer were combined. Two series of
experiments were performed: incubation of tubes for 2 h at
32ꢁC or for 21 h at the same temperature. The samples
were then centrifuged for 7 min at 16,000 rpm, discarding
the supernatant, and the sediment was supplemented with
200 ll of bromocresol purple in the same buffer and
incubated for 45 min at 32ꢁC. The samples were then again
centrifuged in the same way, and the obtained precipitate
was examined by means of an MBI-6 light microscope
(total magnification 17509) and Sony Cybershot 7.2
camera.
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Med Chem Res (2012) 21:3876–3884
3879
substance with a 20:1 ethanol–water mixture. The yield
amounted to 0.027 g (25%). Rf 0.5 (1). Electron spectrum
(methanol), kmax, nm (e 9 10-3, M-1 cm-1): 393.4. (65.8),
474.8 (8.7), 578.0 (3.61), 607.2 (3.19); IR (KBr, cm-1):
1728 (COOH), 1659 (C=O amide I), 1546 (C=O amide II);
MALDI-MS (m/z): 1605 [M]? (calculated 1603.5).
and Arg), 4.37 (t, 1H, a-CH of Glu), 4.31–4.28 (m, 2H,
a-H of Arg), 3.65 (s, 6H 2 9 OCH3), 3.17 (t, 4H, d-CH2 of
Arg), 2.42–2.28 (m, 4H, –CH2–CH2– of Glu), 1.98–1.91
(m, 4H, b-CH2 of Arg), 1.78–1.66 (m, 4H, c-CH2 of Arg),
1.36 (s, 9H, (CH3)3C of Boc group); MALDI-MS (m/z):
[M]? 588.3 (calculated 588.6).
Di-pentafluorophenyl ester of Boc-L-Glutamic Acid (4)
Di-(methyl ester N-a-arginyl)-L-Glutamic Acid (6)
A solution of 1.500 g (6.73 mmol) of Boc-L-glutamic acid
in 9 ml of acetonitrile was supplemented with 2.235 g
(12.145 mmol) of pentafluorophenol. The reaction mixture
was cooled to -10ꢁC and supplemented dropwise for
10 min with 2.505 g (12.145 mmol) of DCC in 4 ml of
acetonitrile. The reaction mixture was agitated for 20 min
at 0ꢁC and kept for 24 h at 4–5ꢁC. The reaction was
monitored by means of TLC under conditions (10). The
DCU sediment was filtered, and the solvent was removed
by vacuum. The oil-like residue was twice crystallized
from ethyl acetate with hexane. The solid residue was
vacuum-dried over anhydrous CaCl2. The yield was
3.410 g (84%), Rf 0.70 (10). m. p. 127–132ꢁC. IR (KBr,
cm-1): 1737 (CO ester), 1697 (OCONH), 991, 1113 (C–F),
To 0.073 g (0.0946 mmol) of compound 5, 6 ml of
approximately 3 N HCl/MeOH was poured. The obtained
suspension was agitated for 2 h at room temperature. The
process was monitored by TLC under the conditions
(6).Once complete cleavage of the Boc protection of the
compound was attained, the solvent was removed in vacuo.
The oil-like residue was crystallized over anhydrous die-
thyl ether, with the solvent removed by decanting, and the
residue was dried. The yield was 0.056 g (85 %), Rf 0.2
(2). IR (KBr, cm-1): 1734 (CO ester), 1656 (amide I), 1532
1
(amide II); H NMR (400 MHz, DMSO-d6): d 7.23–7.08
(m, 4H, a-NH2 of Glu, a-NH of Arg), 4.35 (t, 1H, a-CH of
Glu), 4.28–4.20 (m, 2H, a-H of Arg), 3.65 (s, 6H
2 9 OCH3), 3.17 (t, 4H, d-CH2 of Arg), 2.38–2.22 (m, 4H,
–CH2–CH2– of Glu), 1.96–1.89 (m, 4H, b-CH2 of Arg),
1.76–1.66 (m, 4H, c-CH2 of Arg); MALDI-MS (m/z): [M]?
488.9 (calculated 488.3).
1
1521 (aromatic). H NMR (400 MHz, DMSO-d6): d 7.18
(s, 1H, –NH), 4.37 (t, 1H, a-CH of Glu), 1.97–2.33 (m, 4H,
–CH2–CH2–), 1.43 (s, 9H, (CH3)3C of Boc group); Anal.
calcd. for C22H15NO6F10 C, 45.60; H, 2.59; N, 2.42 Found:
C, 45.24; H, 2.60; N, 2.47. Ref. (Khrushchev et al., 2007):
m. p. 128–132.
6,7-Bis-[di-methyl ether of N-a-L-arginyl)-L-glutamyl]-
Protohemin IX (7)
Di-(methyl ester N-a-arginyl)-L-Glutamic Acid (5)
To 0.082 g (0.167 mmol) of compound 6 in 1.5 ml of
DMF, 0.047 ml (0.335 mmol) of Et3N was added. Reac-
tion mixture was stirred at room temperature for 2 min.
The obtained suspension was supplemented with a solution
of 0.047 g (0.0558 mmol) of 6,7,-bis-N-oxy succinimide
ether of protohemin IX (I) in 2 ml of DMF and agitated for
22 h at room temperature. The reaction was monitored by
means of TLC under the conditions (6). The solution was
concentrated to 1 ml and supplemented with 10 ml of
diethyl ether. In order to insert a counterion of Cl-, the
sediment was dissolved in 4 ml of MeOH and supple-
mented with 0.150 ml of *3 N HCl/MeOH to adjust the
pH to 4.0. The solvent was removed under vacuum at 30ꢁC.
The substance was purified by column chromatography on
a Sephadex LH-20 column (13 9 1 cm), eluting with
MeOH. The fractions containing the substance with Rf 0.1
(3) were combined. The solvent was removed under vac-
uum. The yield was 0.034 g (40 %), Rf 0.2 (2). Electron
spectrum, kmax, nm, chloroform:MeOH (8:2), (e 9 10-3):
400 (92.2), 508 (6.31), 636 (2.39); IR (KBr, cm-1): 1737
(CO ester), 1649 (amide I), 1528 (amide II); MALDI-MS
(m/z): [M]? 1554.9 (calculated 1555.2).
A suspension containing 0.069 g (0.266 mmol) of H-Arg-
OMe•2HCl in 2 ml of DMF was supplemented with
0.074 ml (0.531 mmol) of Et3N and agitated at room
temperature for 5 min. The obtained solution was supple-
mented with 0.077 g (0.133 mmol) of di-pentafluorophenyl
ester of Boc-glutamic acid obtained previously and stirred
for 4 h at ambient temperature. The reaction was moni-
tored by means of TLC under the conditions (5). The
solution was concentrated in vacuum to 1 ml and supple-
mented with 10 ml of diethyl ether. The oil-like sediment
was separated from the solvent by decanting, and the
residual solvent was removed under vacuum. The sub-
stance was purified by column chromatography on Kie-
selgel 60 F254 silica gel (Merck, Germany) (23 9 1 cm)
and eluted with an ethanol–acetic acid–water mixture
(4:0.5:0.5). The fractions containing the substance with Rf
0.67 (5) were collected, and then the solvent was removed
under vacuum and the residue was dried. The yield was
0.073 g (77%), Rf 0.67 (3). IR (KBr, cm-1): 1735 (CO
ester), 1653 (amide I), 1542 (amide II). 1H NMR
(400 MHz, DMSO-d6): d 7.23–7.08 (m, 3H, a-NH of Glu
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Med Chem Res (2012) 21:3876–3884
Results and discussion
Synthesis of hemin peptides
and dichloromethane (1:1:8) for 3 h (Scheme 1). The
obtained Ac-RRWWRF-OH was purified by means of
preparative HPLC.
The conjugate of hemin and peptide 3 was obtained on
the solid phase by acylating the appropriate deblocked
peptidylpolymer with a threefold excess of the ONb-ether
of protohemin IX obtained according to (Okorochenkov
et al., 2010) (Scheme 1). Compound 2 was detached from
the respective hemin peptidylpolymer and purified by
column chromatography on Sephadex LH-20 with metha-
nol-mediated elution. The procedure resulted in obtaining
compounds 2 and 3 with satisfactory yields (43 and 25%,
respectively).
Most fungicidal and antimicrobial peptides (AMP) appear
to be amidated at the C-terminus; however, effective AMPs
also include peptides with a C-terminal carboxylic group,
for instance magainine-1 and magainine-2 (Kokryakov,
1999). Therefore, we synthesized a 2-chlorotrityl chloride
polymer-based peptide Ac-RRWWRF-OH 2 amidated at
the C-terminus whose analog (combi-1) exhibits high
antifungal activity (Blondlle and Houghten, 1996), as well
as the corresponding conjugate with hemin 2. Synthesis of
the latter is of special independent interest and associated
with a series of certain difficulties, since the use of con-
ventional polymers for solid-phase synthesis and standard
acid-labile protective groupings is limited by the presence
of the metalloporphyrin residue in the molecule. Analog 2
of the antifungal peptide combi-1 and the corresponding
hemin peptide 3 were synthesized by a solid-phase method
with previously developed methodology on a polymer with
a 2-chlorotrityl chloride anchor group (Blondlle and
Houghten, 1996). While doing so, the guanidine group of
arginine was protected by protonation, leaving the indole
nucleus of tryptophan unprotected, since detachment of the
peptide from the chlorotrityl chloride polymer occurs under
sufficiently mild conditions under which side reactions
along the lateral functional group of tryptophan are
excluded. It is known (Rubina et al., 2000) that at the stage
of deblocking the a-alpha-amino group resulting from
treatment with piperidine having a pKa 11.12, partial de-
protonation of the guanyl group takes place which further
on under the effect of an excess amount of an acylating
agent may lead to the formation of by-products. In fact, the
literature data strongly suggest the ability of the guanine
group of arginine to participate in acetylating reactions and
to engage in other side reactions (Rink et al., 1984). The
introduction of an additional stage of protonation of the
guanidine group after the amino group and prior to the
subsequent stage of condensation makes it possible to
entirely prevent the course of this side reaction (Rubina
et al., 2000). Therefore, in order to avoid the formation of
by-products, we additionally treated the peptidylpolymer
with HOBt after each stage of deblocking with piperidine.
Since exposure to acetic anhydride conventionally used
for the acetylation of amino groups of peptides on a solid
phase might be associated with modifications of the pro-
tonation-protected guanidine group of arginine residues,
acetylation of the N-terminus of peptide 5 was performed
by the action of a mild acylating agent, p-nitrophenyl
acetate, in the presence of triethylamine. Cleavage of pep-
tide 2 and hemin peptide 3 from the polymer was carried
out using a mixture of trifluoroacetic acid, trifluoroethanol,
Synthesized peptide 2 was characterized by means of
thin-layer chromatography (TLC) and high-performance
liquid chromatography (HPLC) (Fig. 1), as well as spec-
trometry, while hemin peptide 3 was assessed by means
of TLC, IR and UV–Vis spectroscopy, and mass
spectrometry.
Branched positively charged peptides have been shown
(Khrushchev et al., 2007; Bruschi et al., 2010) to possess
high activity toward various pathogens; however, the
available literature seems to contain no reports regarding
the antifungal activity of conjugates of porphyrins and
branched peptides. In order to investigate the promising
nature of such compounds as antifungal agents, we have
obtained a compound, i.e. a hemin conjugate with the
simplest branched positively charged peptide 7.
A suitable syntone for obtaining dendrimers based on
glutamic acid is a di-pentafluorophenyl-activated ether of
its Boc derivative (Khrushchev et al., 2007). Glutamic acid
dipentafluorphenyl ester was obtained by the action of
DCC on Boc-protected glutamic acid in the presence of
pentafluorophenol in acetonitrile. Compound 6 was syn-
thesized by the coupling of HArgOMeÁ2HCl with dipen-
tafluorphenyl ester 4 in the presence of Et3N in DMF with
further cleavage of Boc-protecting group from compound
5. (Scheme 2).
After deblocking the amino group of the obtained
N-Boc-protected peptide in acidic medium, branched
peptide 6 was added to the reaction with 6,7-bis-N-oxy-
succinidic ester of hemin (Hem(ONSu)2), obtained
according to (Okorochenkov et al., 2010), with the result-
ing formation of target HC 7.
Antifungal activity of HCs
We then investigated the activity of the synthesized HCs
toward human opportunistic yeast-like fungi of the. Candida
albicans and Cryptococcus neoformans species. Compound 7
was found to inhibit the growth of. Candida albicans, despite
exhibiting no fungicidal action even at high concentrations,
withthedynamicsofcolonygrowthinthecontrolinoculations
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Med Chem Res (2012) 21:3876–3884
3881
Scheme 1 Synthesis of the peptide on the 2-chlorotrityl chloride polymer
the growth of both Candida albicans and Cryptococcus neo-
formans at concentrations as low as 12.5 lM. Using control
inoculations, it was shown that compound 7 rendered a fun-
gicidal effect on Candida albicans at concentrations down to
25 lM inclusive (the colonies in the control inoculations did
not grow at all) and showed a pronounced activity against
Cryptococcus neoformans (the colonies in the control inocu-
lations did grow; however, their numbers were significantly
lower compared with the control).
The obtained findings are of special value, given the
known resistance of Candida albicans strain No. 927 to
exoderil and low sensitivity to amphotericin, pymafucin,
miramistin, nitrofungin, as well as the resistance of Cryp-
tococcus neoformans strain No. 3465 to amphotericin and
low sensitivity to pimafucin and nitrofungin.
Fig. 1 Chromatogram of Ac-RRWWRF-OH (2). HPLC was per-
formed on a Gilson 305 unit (Gilson, France) using a 2 9 150 mm
column, reverse-phase C18, sorbent pores mean diameter 5 lm
(Phenomenex, USA) with a gradient of acetonitrile in a 0.05%
aqueous solution of TFA from 0 to 100%. Detection at 220 nm
Moreover, Cryptococcus neoformans is known for being
insensitive to azole-series drugs (nizoral, clotrimazole,
fluconazole, and itraconazole), which is typical of basidi-
omycetes class the given strain belongs to.
In order to investigate the mechanisms of the antifungal
activity of compound 7, a microscopic study was carried
out on Candida albicans cells treated with compound 7 for
2 h (Fig. 2a) and for 21 h (Fig. 2b). Figure 2 shows that
the control sample (Fig. 2c) contains intact cells separated
following exposure to compound 3 not differing from that of
the intact culture. The hemin conjugate with the RGD peptide
(HemRGD) exhibited no activity against either of the fungi
examined. As can be seen from Tables 1 and 2, the most
efficient peptide proved to be compound 3, which suppressed
123

3882
Med Chem Res (2012) 21:3876–3884
Scheme 2 Synthesis of 6,7-bis-[di-methyl ether N-a-L-arginyl)-L-glutamyl]-protohemin IX
Table 1 Growth of the Candida albicans culture in the presence of
HC
Table 2 Growth of the Cryptococcus neoformans culture in the
presence of HC
Compounds Growth of the test culture at various concentrations of
the compounds (lM)
Compounds Growth of the test culture at various concentrations of
the compounds (lM)
100 50 25 12.5 6.25 3.13 1.56 0.78
50 25 12.5 6.25 3.13 1.56 0.78 0.39
2
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2
?
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-
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-
?
?
-
?
-
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?
-
?
-
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?
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?
-
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?
?
?
?
?
3
3
7
-
?
-
-
?
-
?
?
-
?
-
?
?
7
HemRGD
Pimaricin
?
-
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
?
HemRGD
Pimaricin
Clotrimazole ?
Control
Clotrimazole ?
Control
?
?
(-) No growth, blue indicator
) Weak growth, yellow indicator but the precipitate of the grown
cells is in the form of a fuzzy spot
(-) No growth, blue indicator
(
(?) Intensive growth, with yellow indicator and the cellular precip-
itate in the form of a compact spot
(?) Intensive growth, with yellow indicator and the cellular precip-
itate in the form of a compact spot
debris, since the cellular organelles remained after cell
lysis. When assessing these findings, it should be borne in
mind that under the conditions of the experiment, the ratio
of HC to fungal cells was 25 times lower compared with
that observed under the conditions of the standard experi-
ment (Table 1). This factor may have led to incomplete
destruction of fungal cells (Fig. 2).
from each other, whereas the experimental variants
(Fig. 2a, b) are represented in the form of agglomerates
consisting of intact whole cells and cellular debris.
Apparently, the mechanism of action of compound 7 on
fungal cells consists of complete destruction (lysis) of the
major part of the cells. The interaction between compound
7 and the cells of Candida albicans probably occurs very
rapidly; 2 h and 21 h after adding compound 7 to the
culture, similar results were observed with a small portion
of non-destroyed fungal cells and a large amount cellular
Therefore, compound 7 has fungicidal action upon cells
of Candida albicans, which is probably mediated by
complete lysis of both the cellular wall and membrane.
We then attempted to investigate a possibility of
increasing the efficacy of the most promising HC 7 due to a
123

Med Chem Res (2012) 21:3876–3884
3883
Fig. 2 Cells of Candida albicans, strain No. 927, 5 9 105 CFU/ml after exposure to compound 7 (2 9 10-3 M). a after 2 h; b after 21 h;
c control
potential synergetic effect with another naturally occurring
agent. As a novel potential cellular target of fungi, special
attention has been drawn to their lipid metabolism. It has
been shown that polyunsaturated fatty acids are capable of
inhibiting C. albicans culture growth in vitro at concen-
trations varying from 4 to 20 lM (Clement et al., 2008).
Moreover, they are capable of considerably altering the
proprieties of cellular membranes, including fluidity, per-
meability, and the activity of membrane-bound proteins,
thereby increasing the absorption and accumulation of
therapeutic substances in the cell (Gleissman et al., 2010).
Such compounds may at the same time induce oxidative
stress in the cell, as they are good substrates for oxidation
and generators of reactive oxygen species (Gleissman
et al., 2010). Hydroxy derivatives of unsaturated fatty acids
(oxylipins), in particular 3-HETE, play an important role in
the processes of adhesion, growth, and reproduction of
fungal cells, including Candida albicans (Groza et al.,
2010).
of the antifungal effect, which may have been the result of
their opposite action on the same targets in the fungal cell.
Determining the actual causes of this phenomenon requires
additional study.
Conclusion
In this study, new hemin conjugates with arginine-con-
taining peptides were synthesized, showing the antifungal
activity of a hemin conjugate with a branched arginine-
containing peptide against human opportunistic fungi
resistant or poorly sensitive to known antifungal agents.
Based on the obtained findings, a conclusion may be drawn
that such structural peculiarities of HCs, such as the pres-
ence of several Arg residues, the modification of both
propionic acid groups of hemin, the lack of unprotected
carboxylic groups in the HC molecule, and the total posi-
tive charge of the substituent in the HC, may have con-
tributed to the antifungal activity of this compound.
In regard to the abovementioned results, the 3-HETE-
oxidized derivative of arachidonic acid (C20n-6) was
chosen to investigate its combined effect with HC 7 on
the commonly occurring species of opportunistic fungi
Candida albicans and Cryptococcus neoformans.
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