Synthetically modified l-histidine-rich peptidomimetics exhibit potent activity against Cryptococcus neoformans

Article abstract of DOI:10.1016/j.bmcl.2014.04.120

We describe the synthesis and antimicrobial evaluation of structurally new peptidomimetics, rich in synthetically modified l-histidine. Two series of tripeptidomimetics were synthesized by varying lipophilicity at the C-2 position of l-histidine and at the N- and C-terminus. The data indicates that peptides (5f, 6f, 9f and 10f) possessing highly lipophilic adamantan-1-yl group displayed strong inhibition of Cryptococcus neoformans. Peptide 6f is the most potent of all with IC₅₀ and MFC values of 0.60 and 0.63 μg/mL, respectively, compared to the commercial drug amphotericin B (IC₅₀ = 0.69 and MFC = 1.25 μg/mL). The selectivity of these peptides to microbial pathogen was examined by a tryptophan fluorescence quenching study and transmission electron microscopy. These studies indicate that the peptides plausibly interact with the mimic membrane of pathogen by direct insertion, and results in disruption of membrane of pathogen.

Full text of DOI:10.1016/j.bmcl.2014.04.120

Accepted Manuscript
Synthetically Modified L-Histidine-Rich Peptidomimetics Exhibit Potent Ac-
tivity Against Cryptococcus neoformans
Amit Mahindra, Nitin Bagra, Nishima Wangoo, Rohan Jain, Shabana I. Khan,
Melissa R. Jacob, Rahul Jain
PII:
S0960-894X(14)00487-9
DOI:
http://dx.doi.org/10.1016/j.bmcl.2014.04.120
BMCL 21613
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
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28 January 2014
1 April 2014
30 April 2014
Please cite this article as: Mahindra, A., Bagra, N., Wangoo, N., Jain, R., Khan, S.I., Jacob, M.R., Jain, R.,
Synthetically Modified L-Histidine-Rich Peptidomimetics Exhibit Potent Activity Against Cryptococcus
neoformans, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.04.120
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Amit Mahindra, Nitin Bagra, Nishima Wangoo, Rohan Jain, Shabana. I. Khan, Melissa R. Jacob and Rahul Jain*

Bioorganic & Medicinal Chemistry Letters
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Synthetically Modified L-Histidine-Rich Peptidomimetics Exhibit Potent Activity
Against Cryptococcus neoformans
Amit Mahindra,a Nitin Bagra,b Nishima Wangoo,b Rohan Jain,a Shabana. I. Khan,c Melissa R. Jacobc and
Rahul Jaina*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, India
bCenter for Nanoscience and Nanotechnology, Panjab University, Sector 14, Chandigarh 160 014, India
cNational Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
We describe the synthesis and antimicrobial evaluation of structurally new peptidomimetics, rich
in synthetically modified L-histidine. Two series of tripeptidomimetics were synthesized by
varying lipophilicity at the C-2 position of L-histidine and at the N- and C-terminus. The data
indicates that peptides (5f, 6f, 9f and 10f) possessing highly lipophilic adamantan-1-yl group
displayed strong inhibition of C. neoformans. Peptide 6f is the most potent of all with IC50 and
MFC values of 0.60 µg/mL and 0.63 µg/mL, respectively, compared to the commercial drug
amphotericin B (IC50= 0.69 and MFC = 1.25 µg/mL). The selectivity of these peptides to
Revised
Accepted
Available online
Keywords:
microbial pathogen was examined by
a tryptophan fluorescence quenching study and
Antifungal peptides
Microwave
transmission electron microscopy. These studies indicate that the peptides plausibly interact with
the mimic membrane of pathogen by direct insertion, and results in disruption of membrane of
pathogen.
Ecofriendly peptide synthesis
C. neoformans
Alkylation
2009 Elsevier Ltd. All rights reserved.
Invasive fungal infections represent a growing threat, and over
the past two decades the incidence and diversity of fungal
infections has increased enormously. As secondary infections,
fungal pathogens are global threat for mankind, especially in
resource limited countries.1 The invasive mycoses of Candida,
Aspergillus and Cryptococcus origin have drastically decreased
the life expectancy of HIV/AIDS, cancer and immuno-
compromised patients.2 In addition to Candida and Aspergillus
spp., Cryptococcus neoformans is becoming a growing danger to
human health.3 Opportunistic fungi like C. neoformans is long
known to cause fungal meningitis and encephalitis.4 There are
over one million new cases each year and 625,000 deaths due to
this facultative pathogen.5 In regions like sub-Saharan Africa and
Southeast Asia, the annual mortality due to fungus has exceeded
tuberculosis.6 The recommended treatment of fungal infections
by the World Health Organization (WHO) is amphotericin B
(intravenous) and flucytosine or fluconazole for two to four
weeks (induction therapy).7 The side effects of amphotericin B in
the form of severe toxicity to kidney, central nervous system and
gastrointestinal intolerance have been observed.8 Since
flucytosine is commonly combined with amphotericin B, the
renal impairment caused by amphotericin B further increases
flucytosine hepatotoxicity. Also flucytosine is losing
effectiveness and reliability due to emergence of resistant
strains.9 Thus, there is currently an urgent need for the discovery
and development of new antifungal agents.
Relatively new antifungal echinocandins (e.g. capsofungin,
and micafungin) represent the most recent class of antifungal
drugs that reached the market in the last decade, and act on a
novel target.10 They are semi-synthetic derivatives of the natural
fermentation products and their total synthesis is difficult, due to
the complex structure and presence of large number of reactive
functional groups.11 Moreover, they are not active towards C.
neoformans and this is a limitation for their use.12
The other promising future candidates with potent antifungal
activity are cationic and amphipathic peptides, isolated from
diverse sources like plants, bacteria, fungi and animals.13
Selective targeting of microbes is a key feature with these
compounds, and multiple modes of action offers minimal or no
chance of resistance to occur.14 There are a number of reports,
which describe various classes of antifungal peptides (AFPs) and
their mode of action.15-16 One group of peptides, that act by lysis
of cell wall, which occurs by binding to the membrane surface
and disrupting it, includes maganin, lactoferricin, dermaseptin
etc.17 Lipodepsipeptide syringomycin E, and iturins; defensins
isolated from rabbit granulocytes NP-1; and mammalian origin
HNP-1, HNP-2, HNP-3 are potent fungicidal agents against C.
albicans and C. neoformans, which act by selective aggregation
and form variable size aqueous pores on the cell wall, resulting in
the passage of ions and solute through them, and finally cell
death.18 Peptides of another group act by interfering with the
biosynthesis of chitin and glucan mainly by inhibiting membrane
integrase enzyme (1,3)-β-D-glucan and examples include,

aculeacins, echinocandin, mulundocandin, and pneumocandin A
(it should be noted that echinocandins are not active against C.
neoformans).19
In this study, we first synthesized 2-alkylated-L-histidines and
later incorporated them in the designed peptide, with the
intention of increasing the lateral amphipathicity of the peptides,
and evaluated the effect on the antifungal and antibacterial
activity. In this direction, we designed two series of histidine-rich
tripeptidomimetics by keeping 2-alkyl-L-histidine residues at the
end terminals and tryptophan residue at the center. The rationale
behind these substitutions is that histidine is itself a cationic
amino acid, and the placement of bulkier groups at the 2-position
of the imidazole ring would impart hydrophobicity. On the other
hand, tryptophan has the unique property of binding in the
interfacial region of a membrane, thereby anchoring the peptide
to the bilayer surface. Another important factor is the extensive
π–electron system of the aromatic side chain that provides
significant quadrupole moment.31 The C-terminus in the designed
peptides is converted to an ester or an amide linkage to further
increase the overall lipophilicity. At the same time, lipophilicity
at the N-terminus was modulated by a free amino group or by
keeping Boc group intact (Fig. 1).
Over the past few years, our research focuses on the discovery
of short peptide based drugs for infectious diseases.20-21 In
literature there are an increasing number of peptides, both natural
and synthetic, which exhibit antifungal activity.22 These peptides
vary in length and amino acid sequences, but most of them have
a large number of common amino acids. The lack of sequence or
structural homology makes it challenging to design potent
synthetic antifungal peptides with the desired activities. One
particular class of peptides that exhibit strong antifungal activity
against various strains have histidine- and tryptophan-rich
sequences.23-24 A large number of natural and synthetic antifungal
peptides, rich in histidine and tryptophan containing are shown in
Table 1.25-30
Table 1. Sequence of natural and synthetic histidine-rich and
tryptophan containing AFPs
Peptide
Sequence
No. of
AAs
38
Histatin-1
Histatin-3
DSHEKRHHGYRRKFHEKHHSHREFPFYG
DYGSNYLYDN
DSHAKRHHGYKRKFHEKHHSHRGYRSN
YLYDN
Scheme 1. MW-assisted synthesis of His(2-alkyl)-Trp-His(2-
alkyl)-NHBzl 6a-f in water (series 1).
32
Histatin-5
P-113
P-113 amide
Dh5
H2K
DSHAKRHHGYKRKFHEKHHSHRGY
AKRHHGYKRKFH
AKRHHGYKRKFH-NH2
KRKFHEKHHSHRGY
KHKHHKHHKHHKHHKHHKHK
FKC1RRWQWRMKKLGAPSITC1VRRAF*
RRWQWRMKKLG
RRWQWR-NH2
RRKWFW
24
12
12
14
20
25
11
6
LfcinB
LfcinB4–14
LfcinB4–9
PAF-26
6
*Disulfide bonded cysteines are denoted by subscript numbers
Histatins are a distinct group of linear cationic peptides that are
isolated from human saliva and have potent and specific
biological activity against fungi.25 Histatin-1, -3, and -5 are
homologous and contain 38, 32, and 24 amino acids,
respectively. Histatin-5, a proteolytic fragment of histatin-3, is
potently fungicidal against several strains of fungi.26 There are
numerous examples of truncation of peptides or proteins to
smaller fragments that retain the activity or whose activities even
exceed that of the native peptide. P-113, a 12-mer fragment of
histatin-5, is the smallest peptide that retains fully original
anticandidal activity.27 P-113 when amidated on its C-terminus
has shown promising activity in comparison to its unamidated
form, thereby confirming the importance of capping of C-
terminus. A class of synthetic histidine-rich peptides, exemplified
by H2K (with 60% histidines in sequence) are potent inhibitors
of several Candida species in vitro.28 In addition to histidine-rich
peptides, other peptides such as LfcinB have several tryptophan
residues in the sequence.29 Smaller peptide sequences such as
LfcinB4–9 and PAF-26 containing tryptophan are also reported
with potent antifungal activity.30 Despite having promising
activities, these peptides have disadvantages in terms of high
cost, proteolytic instability, and cytotoxicity. One possible
method to overcome these shortcomings of larger peptides is to
synthesize peptides with shorter sequences keeping the bioactive
core intact.
For the synthesis of target peptides, a series of 2-alkylated-L-
histidine was synthesized using earlier reported methods.32-33The
peptides were synthesized by
a
recently developed
environmentally benign microwave (MW)-assisted peptide
synthesis protocol in neat water.34-35
Scheme 2. MW-assisted synthesis of His(2-alkyl)-Trp-His(2-
alkyl)-OMe 10a-f in water (series 2).
Using this protocol, synthesis of peptides of series 1 and 2 is
achieved as depicted in scheme 1 and 2. Key features of this
original synthetic protocol are the replacement of commonly used
toxic solvent like DMF, short reaction time, the use of side-chain
Figure 1.General structures of synthesized tripeptides (series 1 and 2).

unprotected histidine, and racemization-free peptide synthesis, in
high purity and yield. We also undertook the synthesis of Boc-L-
Trp-His-OMe, Boc-D-Trp-His-OMe, and Boc-D,L-Trp-His-OMe
under MW irradiation in water. The purified peptides upon
HPLC analysis confirmed the racemization-free synthesis of
peptide (see Supporting Information).
The synthesized peptides (5-6 and 9-10) were evaluated for in
vitro activity against fungal C. albicans, C. glabrata, C. krusei,
A. fumigatus and C. neoformans and bacterial (E. coli, S. aureus,
Table 2. In vitro antifungal activities of peptides
MRSA, M. intracellulare and P. aeruginosa) strains, and the
results are summarized in Table 2 and Table 3. All the peptides
were found to be inactive against Candida, Aspergillus, E. coli,
M. intracellulare and P. aeruginosa (data not shown). The
minimum inhibitory concentration (MIC) was measured using a
protocol suggested by the Clinical and Laboratory Standard
Institute (previously known as the National Committee for
Clinical Laboratory Standards, NCCLS).36 Amphotericin B,
served as a positive control in these studies.37
Peptide
R
R1
R2
C. neoformansd (µg/mL)
a
IC50
MICb
MFCc
CTXe (µg/mL)
H
0.80
NA
NA
20
1.25
NA
NA
NA
NA
2.5
10
1.25
NA
NA
NA
NA
5
5a
5b
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
NHBzl
OMe
Boc
Boc
Boc
Boc
Boc
Boc
H
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
CH(CH3)2
C(CH3)3
c-C5H9
5c
5d
c-C6H11
adamantan-1-yl
H
NA
2.12
8.1
5e
5f
6a
10
CH(CH3)2
C(CH3)3
c-C5H9
6.84
11.63
NA
1.02
0.60
NA
8
20
20
6b
6c
H
20
20
H
NA
1.25
0.63
NA
NA
NA
NA
10
NA
1.25
0.63
NA
NA
NA
NA
10
6d
6e
H
c-C6H11
adamantan-1-yl
H
H
6f
9a
H
Boc
Boc
Boc
Boc
Boc
Boc
H
CH(CH3)2
C(CH3)3
c-C5H9
9b
9c
OMe
OMe
8
8.24
5.78
2.44
9.81
11.34
16.14
5
9d
9e
9f
10a
OMe
OMe
OMe
OMe
c-C6H11
adamantan-1-yl
H
5
5
20
20
CH(CH3)2
C(CH3)3
c-C5H9
NA
NA
10
NA
NA
10
10b
10c
OMe
OMe
H
H
10d
10e
OMe
OMe
OMe
H
c-C6H11
adamantan-1-yl
2.28
0.68
5
5
H
1.25
1.25
10f
Amphotericin B
H
0.69
1.25
1.25
aIC50 is the concentration (µg/mL) that affords 50% inhibition of growth; bMIC (Minimum Inhibitory Concentration) is the lowest test concentration (µg/mL)
that allows no detectable growth; MFC (Minimum Fungicidal Concentration) is the lowest test concentration (µg/mL) that kills 100% of the organism; dHighest
c
e
tested concentration was 20 µg/mL; CTX (Cytotoxicity) Highest tested concentration was 10 µg/mL. NA, not active.
Peptides (9f and 10f) exhibit most potent activity against C.
neoformans with IC50 values of 2.44 and 0.68 µg/mL,
In general, the alkyl group placed at the C-2-position of the
respectively. Other peptides of this series 9b-9e and 10a-10e also
imidazole ring greatly influenced the overall antifungal activity
showed promising IC50 values against C. neoformans in the range
of the peptides. Also, the nature of groups present at the N- and
of 2-16 µg/mL. These results clearly demonstrate the selectivity
C-terminus played a significant role in activity. Peptide 5a with
of the designed peptides for Cryptococcus.
Boc and NHBzl groups at end terminus was equipotent to
standard drug as antifungal. Thus it appeared that a combination
of Boc and NHBzl group at the end terminus along with an alkyl
group at the C-2 position is detrimental for activity (peptides (5b-
f). In series 1, peptide 6f (R = adamantan-1-yl, R1 = NHBzl, R2 =
H) was the most potent fungicidal compound against C.
neoformans, with an IC50 and MFC value of 0.60 and 0.63 µg/mL
as compared to 0.69 and 1.25 µg/mL, respectively for standard
drug AMB. Peptide 6e (R = c-C6H11, R1 = NHBzl, R2 = H) also
showed potent activity with an IC50 and MFC value of 1.02 and
1.25 µg/mL, respectively. At the same time, peptides 6a, 6b and
6c produced modest antifungal activities with IC50sin the range of
9.81-16.14 µg/mL. Peptides 5f and 6f also exhibited weak
antibacterial activity against S. aureus and MRSA with IC50values
in the range of 3.45-4.94 and 5.55-8.81 µg/mL and MIC of 20.00
µg/mL (Table 3). In series 2, a similar trend is observed with
peptides (9f and 10f) having adamantan-1-yl group at the C-2
position.
Table 3. In vitro antibacterial activity of peptides
Peptide
S. aureus (µg/mL)
MRSA (µg/mL)
IC50
4.94
3.45
4.22
8.76
0.08
MIC
20.00
5.00
MBC
20.00
5.00
IC50
MIC
MBC
20.00
10.00
NA
8.81
5.55
11.28
16.04
0.09
20.00
10.00
NA
5f
6f
10.00
20.00
0.25
20.00
NA
9f
NA
NA
10f
Cipro
0.50
0.25
0.50
MBC (Minimum Bactericidal Concentration) is the lowest test concentration
(µg/mL) that kills 100% of the organism. NA, not active.
All synthesized peptides were also evaluated for cytotoxicity in
a panel of mammalian cell lines to determine their safety profile.

The in vitro cytotoxicity of analogs was determined against four
human cancer cell lines (SK-MEL, KB, BT-549, and SK-OV-3)
and two noncancerous mammalian cells (VERO and LLC-PK1)
using an earlier reported protocol.38 The results demonstrated that
the synthesized peptides were non-toxic up to a concentration of
10.00 µg/mL indicating
anticryptococcal activity (Table 2).
a
higher selectivity index of
A plausible correlation between lipophilicity and activity was
obtained for all compounds. To verify whether lipophilicity and
therefore cell penetration of peptides (6a-f and 10a-f) is
correlated to the antifungal activity, the degree of lipophilicity
was expressed as the ClogP value and shown in Fig. 2. The
ClogP values were calculated using the ACD/CLogP software
and are indicated as ClogP. The values of ClogP are an indicator
of cell penetration potential of the synthesized inhibitors and the
number increases with an increase in hydrophobicity.
Figure 3. Stern-Volmer plots for the quenching of Trp fluorescence of the
peptides in the presence of acrylamide.
The fluorescence of tryptophan decreased in a concentration-
dependent manner by the addition of acrylamide to the peptide
solution both in the absence and presence of liposomes, without
any other effects. Compared to the measurements in the absence
of liposomes (Tris HCl buffer, Ksv = 18.0), the values for the
Stern-Volmer quenching constant (Ksv) were decreased in the
presence of EYPC/EYPG (7:3, w/w Ksv = 11.3 for 6f) and
EYPC/cholesterol (10:1, w/w Ksv = 14.0 for 6f), suggesting that
the tryptophan was buried in the bilayers, thereby becoming
inaccessible for quenching by acrylamide. The comparison of
Ksv values for EYPG containing pure EYPC liposomes indicates
that the binding of peptide to liposomes is enhanced by EYPG.
The data thus suggest that affinity of peptide for liposomes
increased in the order EYPC/cholesterol<EYPC/EYPG. This
indicates that the peptide effectively embedded the negatively
charged membrane, but not the zwitterionic membrane,
suggesting that the selectivity of the tested peptide towards
mimics of microbial cell membrane is associated with a
preferential interaction with the negatively charged
phospholipids.
Figure 2. Correlation between lipophilicity and biological activity of peptides
6a-f and 10a-f (∆ represents peptide, IC50 and ClogP values respectively)
Peptide 6f, most potent against C. neoformans having most
hydrophobic substituent adamantan-1-yl showed ClogP and IC50
values of 6.06 and 0.60 µg/mL, which are in agreement. The
correlation of lipophilicity with ClogP for peptides 5a-f and 9a-f
is included in the supporting information.
To elucidate the interaction of peptides with synthetic mimics
of membrane, we performed tryptophan fluorescence quenching
and transmission electron microscopy studies on analogs that
may be of future interest.39 This requires the synthesis of the
small unilamellar vesicles (SUVs), which were prepared using
the reported method in the literature.40 We selected three most
promising peptides 6e, 6f and 10f for the tryptophan fluorescence
quenching study. To investigate whether the selectivity of
peptides (6e, 6f and 10f) towards microbial pathogen is related to
the differences in the interaction with the outer monolayer of
membranes of microbial and mammalian cells, tryptophan
fluorescence quenching study was performed in the presence of
negatively charged (EYPC/EYPG) and zwitterionic model
membranes (EYPC/cholesterol), respectively. The Stern-Volmer
plots of the tryptophan quenching study, in the absence and
presence of lipid vesicles are shown in Figure 3.

Figure 4. TEM images of SUVs with 6f; (a) Untreated EYPC/EYPG; (b)
EYPC/EYPG treated with 6f; (c) Untreated EYPC/cholesterol; (d)
EYPC/cholesterol treated with 6f. The peptide was used at 10 µg/mL
concentrations.
5. Jarvis, J. N.; Boulle, A.; Loyse, A.; Bicanic, T.; Rebe, K.; Williams, A.;
Harrison, T. S.; Meintjes, G. AIDS (London, England) 2009, 23, 1182-
1183.
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Disease
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http://www.cdc.gov/fungal/cryptococcosis-neoformans/ (Dec, 2013).
7. Harrison, T. S. Clin Infect Dis. 2009, 48, 1784-1786.
8. Pathak, A.; Pien, F. D.; Carvalho, L. Clin. Infect. Dis. 1998, 26, 334-338.
9. Schwarz, P.; Dromer, F.; Lortholary, O.; Dannaoui, E. Antimicrob.
Agents Chemother. 2006, 50, 113-120.
From TEM study, we observed the morphology of the SUVs in
the presence and absence of the peptide 6f by depositing a sample
of the treated and untreated SUVs, on to a carbon coated copper
grid, and negatively staining the sample with 2% (w/v)
phosphotungstic acid solution. The results indicated that the
untreated SUVs were uniformly shaped, with intact morphology
(Fig. 4a) whereas SUVs treated with 6f resulted in the destruction
of morphology (Fig. 4b), in case of EYPC/EYPG. Whereas in
case of EYPC/cholesterol, the SUVs treated with 6f retains the
integrity of membrane (Fig. 4d). From these studies, we conclude
that the peptide does not lyse mammalian membranes at the
concentrations, at which it destroys mimic of the pathogenic
membranes further confirming the results of cytotoxicity
experiments.
In summary, two series of synthetic histidine-rich
tripeptidomimetics with varying lipophilicity were synthesized
and evaluated in vitro against fungal and bacterial strains.
Peptides 6f and 10f substituted with a bulky adamantan-1-yl
group at the C-2-position of the imidazole ring were found to be
the most active against C. neoformans. To study the interaction
of these peptides, tryptophan fluorescence quenching and TEM
studies were performed using synthetic mimics of membrane.
The results indicate that tested peptides possibly interact with
membrane, followed by the non-specific disruption of the cell
membrane.
10. Denning, D. W. J. Antimicrob. Chemother. 2002, 49, 889-891.
11. Leonard, W. R.; Belyk, K. M.; Conlon, D. A.; Bender, D. R.; DiMichele,
L. M.; Liu, J.; Hughes, D. L. J. Org. Chem. 2007, 72, 2335-2343.
12. Maligie, M. A.; Selitrennikoff, C. P. Antimicrob. Agents Chemother.
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13. Zasloff, M. Nature 2002, 415, 389-395.
14. Theis, T.; Stahl, U. Cell. Mol. Life Sci.2004, 61, 437-455.
15. Hegedüs, N.; Marx, F. Fungal Biol. Rev. 2013, 26, 132-145.
16. Ribeiro, S. M.; Porto, W. F.; Silva, O. N.; Santos, Mde. O.; Dias, S. C.;
Franco, O. L. In Handbook of Biologically Active Peptides; Kastin, A. J.;
Academic Press: Waltham, Massachusetts, 2013; pp 169-179.
17. Morton, C. O.; Hayes, A.; Wilson, M.; Rash, B. M.; Oliver, S. G.; Coote,
P. Antimicrob. Agents Chemother. 2007, 51, 3948-3959.
18. Ganz, T. Nat. Rev. Immunol. 2003, 3, 710-720.
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21. Mahindra, A.; Bagra, N.; Wangoo, N.; Khan, S. I.; Jacob, M. R.; Jain, R.
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S. T.; Mixson, A. J. Drugs Future 2010, 35, 197.
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3905.
Corresponding Author
*E-mail: rahuljain@niper.ac.in; Fax: +91 (172) 2214692; Tel:
+91 (172) 2292024.
27. Rothstein, D. M.; Spacciapoli, P.; Tran, L. T.; Xu, T.; Roberts, F. D.;
Dalla Serra, M.; Buxton, D. K.; Oppenheim, F. G.; Friden, P. Antimicrob.
Agents Chemother. 2001, 45, 1367-1373.
Acknowledgments
28. Zhu, J.; Luther, P. W.; Leng, Q.; Mixson, A. J. Antimicrob. Agents
Chemother. 2006, 50, 2797-2805.
Amit Mahindra thanks the Council of Scientific and
IndustrialResearch (CSIR), New Delhi for the award of a Senior
Research Fellowship. Ms Marsha Wright and Mr John Trott are
acknowledged for excellent technical support in biological testing
at NCNPR.Antimicrobial studies were supported by the NIH,
NIAID, Division of AIDS, Grant No. AI 27094 (antifungal) and
the USDA Agricultural Research Service Specific Cooperative
Agreement No. 58-6408-1-603 (antibacterial).
29. Chan, D. I.; Prenner, E. J.; Vogel, H. J. Biochim. Biophys. Acta (BBA) -
Biomembranes 2006, 1758, 1184-1202.
30. Jose, F. M.; Mόnica, G.; Eleonora, H.; Lourdes, C.; Alberto, M. In Small
Wonders: Peptides for Disease Control; American Chemical Society:
Washington D.C., 2012; 1095, pp 337-357.
31. Minoux, H.; Chipot, C. J. Am. Chem. Soc. 1999, 121, 10366-10372.
32. Jain, R.; Cohen, L. A.; El-Kadi, N. A.; King, M. M. Tetrahedron 1997,
53, 2365-2370.
33. Jain, R.; Cohen, L. A.; King, M. M. Tetrahedron 1997, 53, 4539-4548.
34. Mahindra, A.; Sharma, K. K.; Jain, R. Tetrahedron Lett. 2012, 53, 6931-
6935.
Abbreviations
35. Mahindra, A.; Nooney, K.; Uraon, S.; Sharma, K. K.; Jain, R. RSC Adv.
2013, 3, 16810-16816.
36. Reference Method for Broth Dilution Antifungal Susceptibility Testing of
Yeasts: Approved Standard, 2nd ed.; NCCLS document M27-A2;
National Committee for Clinical Laboratory Standards: Wayne, PA,
2002.
AA, amino acid; Boc, tert-Butoxycarbonyl; DIC, 1,3-
Diisopropylcarbodiimide; DIEA, N,N-Diisopropylethylamine;
EYPC, Egg yolk L-a-phosphatidylcholine; DMF, N,N-
Dimethylformamide; EYPG, Egg yolk L-a-phosphatidyl-DL-
glycerol; TBTU,
tetramethyluronium
O-(Benzotriazol-1-yl)-N,N,N′,N′-
tetrafluoroborate; HOBt, 1-
37. Kagan, S.; Ickowicz, D.; Shmuel, M.; Altschuler, Y.; Sionov, E.; Pitusi,
M.; Weiss, A.; Farber, S.; Domb, A. J.; Polacheck, I. Antimicrob. Agents
Chemother. 2012, 56, 5603-5611.
Hydroxybenzotriazole; HONB, N-endo-Hydroxy-5-norbornene-
2,3-dicarboximide; IC50, The concentration (µg/mL) that affords
50% inhibition of growth; MeOH, Methanol; MFC/MBC,
38. Borenfreund, E.; Babich, H.; Martin-Alguacil, N. In Vitro Cell Dev. Biol.
1990, 26, 1030-1034.
39. Eftink, M. R.; Ghiron, C. A. Anal. Biochem. 1981, 114, 199-227.
40. Morrissey, J. H. 2001. Morrissey laboratory protocol for preparing
Minimum
Fungicidal/Bactericidal
Concentration;
MIC,
Minimum Inhibitory Concentration; TEM, Transmission electron
microscopy; Trp, Tryptophan.
phospholipid vesicles (SUV) by sonication
http://tf7.org/suv.pdf).
(please see:
References and notes
Supplementary Material
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Detailed synthetic procedures, characterization data, HPLC
chromatograms and details on the biological assays are available
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