European Journal of Medicinal Chemistry 223 (2021) 113635

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European Journal of Medicinal Chemistry

journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Modified histidine containing amphipathic ultrashort antifungal

peptide, His[2-p-(n-butyl)phenyl]-Trp-Arg-OMe exhibits potent
anticryptococcal activity
Krishna K. Sharma a, Ravikant Ravi a, Indresh Kumar Maurya b, Akshay Kapadia a,
Shabana I. Khan c, Vinod Kumar d, Kulbhushan Tikoo d, Rahul Jain a, *
a
Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S. A. S. Nagar, Punjab 160 062, India
b
Department of Microbial Technology, Panjab University, Sector 25, Chandigarh 160 014, India
c
National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, University, Mississippi 38677, United States
d
Department of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S Nagar 160 062, Punjab India

a r t i c l e i n f o a b s t r a c t

Article history: In pursuit of ultrashort peptide-based antifungals, a new structural class, His(2-aryl)-Trp-Arg is reported.
Received 22 January 2021 Structural changes were investigated on His-Trp-Arg scaffold to demonstrate the impact of charge and
Received in revised form lipophilic character on the biological activity. The presence and size of the aryl moiety on imidazole of
31 May 2021
histidine modulated overall amphiphilic character, and biological activity. Peptides exhibited IC50 of 0.37
Accepted 6 June 2021
Available online 12 June 2021
e9.66 mg/mL against C. neoformans. Peptide 14f [His(2-p-(n-butyl)phenyl)-Trp-Arg-OMe] exhibited two-
fold potency (IC50 ¼ 0.37 mg/mL, MIC ¼ 0.63 mg/mL) related to amphotericin B, without any cytotoxic
effects up to 10 mg/mL. Peptide 14f act by nuclear fragmentation, membranes permeabilization,
Keywords:
Antifungal peptides (AFPs)
disruption and pore formations in the microbial cells as determined by the mechanistic studies
Cryptococcus neoformans employing Trp-quenching, CLSM, SEM, and HR-TEM. The amalgamation of short sequence, presence of
Membrane-disruption appropriate aryl group on L-histidine, potent anticryptococcal activity, no cytotoxicity, and detailed
Membrane selectivity mechanistic studies directed to the identification of 14f as a new antifungal structural lead.
Amphiphilicity © 2021 Elsevier Masson SAS. All rights reserved.
Cytotoxicity

1. Introduction unwanted drug-drug interactions, poor solubility, and importantly

Global incidences of fungal infections are increasing at an
alarming rate and pose an urgent necessity to discover new classes of
antifungals. Invasive fungal infections being potentially pathogenic,
takes a huge toll on human population mainly targeting immuno-
compromised individuals suffering from AIDS, cancer and organ
transplant receivers [1e3]. Cryptococcosis is one of the most
worldwide distributed infection responsible for more than 600,000
deaths per year and presents a substantial therapeutic challenge due
to increasing resistance cases to clinically used antifungal medica-
tions [1e5]. Clinical therapeutic interventions are mainly depend on
azoles (fluconazole), fluoropyrimidines (5-fluorocytosine), allyl-
amines (terbinafine), echinocandins (caspofungin) and macrocyclic
polyenes (amphotericin B). Clinically available treatments have
shortcomings such as side effects, non-optimal pharmacokinetics,
* Corresponding author.
E-mail address: rahuljain@niper.ac.in (R. Jain).
drug resistance that makes management a difficult and challenging
task [6e8]. A palliative solution to this conundrum is the discovery
and development of newer classes of antifungal agents with alter-
native mechanism of action [9,10].
Cationic antimicrobial peptides (CAMPs) bid encouraging pros-
pects for the medicinal chemistry research of newer antifungal
agents [11e13]. Optimal balance of cationic charge vs lipophilicity
ratio is considered to be important for a potent CAMP [14e16].
Their mechanisms of action are still debatable, though, its activity is
attributed to membrane infiltration and disruption of the pathogen
cells [17]. In terms of selectivity, CAMPs being positively charged,
preferentially bind to negatively charged microbial membranes,
and composition of cell surface of susceptible cells also played
importantly role. In case of fungal strain compared to bacteria, cell
wall composition can also play important role for its susceptibility
[18,19]. Therefore, unique mechanism of action makes this class of
compound less likely to develop resistance. There has been an
evolution in designing short synthetic peptide-based derivatives
from the long sequence natural AMPs with a view to improve

https://doi.org/10.1016/j.ejmech.2021.113635
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.
K.K. Sharma, R. Ravi, I.K. Maurya et al.
proteolytic stability and enhanced bioactivity [20,21]. A compara-
tive sequence analysis of natural as well as synthetically derived
antimicrobial peptides show that cationic amino acids (Arg, His and
Lys), and lipophilic amino acids (Trp, Phe, Ile and Val) have been
repeatedly present in randomized manner [22,23]. This gives a
clear idea of the necessity of incorporating amino acid residues that
optimally balance both the charge and lipophilicity in the designed
peptides [24e26].
In our quest to develop ultrashort peptide/mimetics-based
novel lead antimicrobial probes, we examined the prerequisite
features in the naturally occurring and synthetic peptides [27e32].
Further, peptide scaffolds possessing þ2/þ3 sites and tuned with
lipophilic character imparts an improved antifungal and antibac-
terial activity [27,29e32]. Naturally occurring cationic peptides
such as the histidine-rich histatins & clavanins, and tryptophan-
rich indolicidin & tritrpticin scaffolds display potent antifungal
activity, encouraging us to design ultrashort peptide sequence(s) by
judiciously using histidine and tryptophan residues [33e38]. It is
also known that naturally occurring cationic peptides containing
cationic amino acids such lysine and arginine at the end terminus
display activity [14e16]. So, we decided to use histidine, tryptophan
and arginine amino acids in the designed sequence by placing
lipophilic tryptophan centrally and arginine at the C-terminus.
Histidine was placed at the N-terminus to tune amphiphilicity of
the designed sequence by using imidazole ring derivatization.
Structural modifications on the aromatic ring of histidine was also
envisioned to generate amphiphilicity and stability in a sterically
complex functional architecture [31,39]. Recently, ring-modified
histidine scaffolds proposed themselves as medicinally important
the building blocks for peptide/mimetic-based design and discov-
ery [40e43]. It was envisioned that placement of C-2 substituted
aromatic moieties on the imidazole ring of histidine would
generate lateral amphiphilicity in the membrane-active peptide
sequence, which could possibly penetrate deeper into the lipid
chains of the fungal cell membranes. On the other hand, tryptophan
residue provides a lipophilic bulk and cell membrane interaction
properties to the His-Trp-Arg sequence [44]. Next, C-terminus
modification of arginine was carried out to mask the anionic
character of the carboxyl group with methyl ester (less lipophilic)
and benzylamide (higher lipophilicity) groups to modulate the
lipophilicity of the designed peptide scaffolds.
We designed His(aryl)-Trp-Arg-OMe/NHBzl scaffolds by
reasoning that arginine methyl ester (cationicity with masked
anionic character), arginine benzylamide (cationicity with masked
anionic character and lipophilicity) and tryptophan (lipophilicity)
along with ring-arylated histidine derivatives (to tune amphiphi-
licity) would create a milieu of balanced charge-to-bulk ratio in
quest of potential ultrashort antimicrobials (Fig. 1). Position of the
amino acids in the designed peptide scaffolds was assigned using
the nature of amino acids and to tune the characteristics of
membrane-active antimicrobial peptides. To systematically allocate
the charge and bulk distribution, the lipophilic tryptophan was
centrally placed with cationic modified arginine at the C-terminus
and amphiphilic ring-modified histidine at the N-terminus (Fig. 1).
Additionally, the use of terminally placed modified arginine and
histidine provide hindrance towards the peptidases to develop
shorter bioactive His(aryl)-Trp-Arg-OMe/NHBzl peptides. Here, we
report the design, synthesis, biological and cytotoxicity evaluation
of ring-modified histidine, tryptophan and arginine containing
His(aryl)-Trp-Arg class that exhibit potent antifungal activities
against C. neoformans and moderate antibacterial activities against
a type of staph bacteria, methicillin-resistant Staphylococcus aureus
(MSRA),and vancomycin-resistant Enterococci (VRE). The initial
biological activity assessment resulted in the identification of a lead
peptide 14f. Detailed mechanistic investigations were performed
2
European Journal of Medicinal Chemistry 223 (2021) 113635
using tryptophan quenching, confocal laser scanning microscopy
(CLSM), and electron microscopic examinations clearly demon-
strate the membrane-disruption and intracellular localization
mechanism of action of the peptide 14f.
2. Results and discussion
2.1. Chemistry and synthesis
The synthetic strategy employed to prepare the designed tri-
peptides has been summarized in Schemes 1-3. Initially, N-a-Boc-
2-aryl-L-histidines (4a-e) were synthesized in four steps (Scheme
1) using a previously reported protocol [40].
The method employed for arylboronic acids mediated arylation
of the imidazole moiety of the histidine, proceeds through a direct
regioselective homolytic mechanism, which involves the addition
of an aryl radical to electron deficient position of a histidine ring
followed by re-aromatization, affording the arylated derivatives
(2a-e) in 42e55% yields. Acidolysis by refluxing with methanolic
HCl provided the deprotected 2-aryl-L-histidine dihydrochloride
salts (3a-e) in 92e96% yields. N-a-Boc protection using di-tert-
butyl dicarbonate in basic reaction medium provided by 4 N NaOH,
furnished the desired N-a-Boc-2-aryl-L-histidine scaffolds (4a-e) in
91e95% yields for incorporation in the designed tripeptide skel-
eton. Next, to synthesize the starting material for Series 1, L-Arg-OH
was reacted with methanolic HCl to afford methyl ester derivative
(6). Similarly for Series 2, L-Arg-OH after reacting with benzylamine
using 1,10 -carbonyldiimidazole (CDI) in H2O afforded L-Arg-NHBzl
(7) [45]. This reaction is stereo conservative and the side chain
functional groups remained unaffected under the employed re-
agents and reaction conditions (Scheme 2).
The synthesis of the tripeptides was accomplished in two steps.
Initially, Boc-L-Trp-OH was coupled with L-Arg-OMe/NHBzl (6 or 7)
using a racemization free and microwave-mediated solution phase
peptide synthesis protocol [46,47], by employing DIC and HOBt as
coupling combination in anhydrous DMF to provided Boc protected
dipeptides derivatives (8 and 9).
These dipeptides were further subjected to acidolysis using 6 N
methanolic HCl to furnish Boc-deprotected L-Trp-L-Arg-OMe/
NHBzl derivatives (10 and 11). The coupling of respective arylated L-
histidine scaffolds (4a-f) were carried out with the deprotected
dipeptides using solution phase peptide synthesis to afford the
respective Boc-L-His(2-aryl)-L-Trp-L-Arg-OMe/NHBzl derivatives
(12a-f and 13a-f). Acidolysis using 6 N methanolic HCl furnished
desired Boc-deprotected L-His(2-aryl)-L-Trp-L-Arg-OMe/NHBzl
peptides (14a-f and 15a-f) (Scheme 3).
2.2. In vitro antimicrobial activity
In the synthesized tripeptides, three structural manipulations
on the lead skeleton His-Trp-Arg were performed to demonstrate
effect of charge and lipophilic character imparted through synthetic
derivatization on the biological activity of the peptides. C2 position
of imidazole ring was substituted by phenyl, p-tolyl, p-ethylphenyl,
biphenyl and p-(n-butyl)phenyl to investigate the effect of
increasing lipophilicity with constant positive charge residues.
Next, to evaluate the effect of bulkiness at the C-terminus, two
different series of tripeptides with C-terminus methyl ester and
benzylamide groups were synthesized. To get more insight of the
structure activity relationship (SAR) study, it was also planned to
biologically investigate Boc moiety containing peptides 12a-f and
13a-f to observe the effect of presence of aliphatic bulk and
decreased cationicity at the N-terminus along with final peptides
14a-f and 15a-f. Synthesized peptides (12a-f, 13a-f, 14a-f &15a-f)
were tested for in vitro antimicrobial activity against various fungal
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Fig. 1. Generalized scaffolds of the designed and synthesized peptides.

Scheme 1. Reagents and conditions: (i) 1 (1 equiv.), AreB(OH)2 (2 equiv.), (NH4)2S2O8 (2 equiv.), AgNO3 (0.2 equiv.), TFA (1.5 equiv.), CH2Cl2/H2O, rt, 12e18 h; (ii) 6 N HCl, 100  C,
12 h; (iii) (Boc)2O (3 equiv.), 4 N NaOH, 1,4-dioxane/H2O (3; 2), 24 h.

Scheme 2. Reaction and conditions: (i) HCl gas in MeOH, rt, 2 h; (ii) 5 (1 equiv.), benzylamine (1.5 equiv.), CDI (1.5 equiv.), H2O, rt, 12 h.

strains such as C. albicans, A. fumigatus, C. neoformans, and bacterial
strains such as MRSA, E. coli, K. pneumonia, P. aeruginosa, and VRE
up to 20 mg/mL, and beyond that peptides were considered as
inactive (NA) [48,49]. The synthesized peptides exhibited prom-
ising activity against C. neoformans and activity values have been
presented in Table 1, whereas a few peptides showed moderate
activity against MRSA and VRE and results are summarized in
Table 2. Synthesized peptides were inactive against C. albicans, A.
fumigatus, and K. pneumonia.
From the bioactivity results, it could be inferred that the tested
peptides showed preferential biological action against yeast,
C. neoformans of IC50 values of 0.37e9.66 mg/mL (Table 1) in
comparison to bacterial strains (Table 2). The results depict that the
variation in the substituents at the aryl ring of the terminally placed
L-histidine, resulting in varied lipophilicity greatly influenced the
potency. In series 1, Boc-protected peptide 12a without any sub-
stitution on histidine residue was found inactive while peptides
12b-f containing ring-arylated histidines displayed IC50 values in
the range of 1.99 mg/mL to 9.66 mg/mL. Peptide 12f also exhibited
MIC value of 2.50 mg/mL against C. neoformans.
At the same time, Boc-deprotected peptides 14a-f exhibited
high antifungal activity as compared to its Boc-protected pre-
cursors 12a-f. Peptide 14a with additional charge (þ3) at the N-
terminus due to Boc-deprotection of 12a exhibited IC50 and MIC

3
K.K. Sharma, R. Ravi, I.K. Maurya et al.
Scheme 3. Reagents and conditions: (i) 6 or 7 (1 equiv.), Boc-L-Trp-OH (1.5 equiv.), DIC (1.5 equiv.), DIEA (4 equiv.), HOBt (1.5 equiv.), DMF, 60  C, MW, 30 min; (ii) 6 N HCl in MeOH,
rt, 20 min; (iii) 10 or 11 (1 equiv.), Boc-L-His(2-aryl)-OH (1.5 equiv.), DIC (1.5 equiv.), DIEA (4 equiv.), HOBt (1.5 equiv.), DMF, 60  C, MW, 30 min.
values of 2.41 mg/mL and 10 mg/mL, respectively. Peptide 14f con-
taining a bulkier p-(n-butyl)phenyl aliphatic-aromatic pendant
attached to histidine ring exhibited the most potent and promising
activity with an IC50 value of 0.37 mg/mL and MIC of 0.63 mg/mL.
Peptide 14f was twice as potent related to the clinical agent,
amphotericin B (IC50 and MIC of 0.69 and 1.25 mg/mL, respectively).
Peptides 14d (p-ethylphenyl) and 14e (biphenyl) also showed high
antifungal activities with IC50 values of 1.85 and 1.51 mg/mL,
respectively and identical MIC value of 2.50 mg/mL. Hence, a
obvious pattern demonstrating the role of lipophilicity and cationic
charge sites was observed in the antifungal activity for peptides
12a-f and 14a-f having Boc/H and methylester at the N- and C-
terminus, respectively.
In series 2, peptide 13a (R1 ¼ H, R2 ¼ NHBzl) with Boc-group
intact was inactive while other Boc-protected peptides 13b-f
showed a linear trend of increase in activity with lipophilicity
provided by ring-modification of histidine. The peptide 13b-f
exhibited IC50 values ranged between 1.5 mg/mL to 7.93 mg/mL with
MIC values ranged in 2.5 mg/mL to 5 mg/mL. Peptide 13d (p-ethyl-
phenyl) emerged as most potent amongst 13a-f with IC50 and MIC
values of 1.5 and 2.5 mg/mL, respectively. In contrast, all Boc-
deprotected peptides 15a-f showed activity with IC50 values
ranged between 1.17 mg/mL to 4.92 mg/mL and MIC values between
5 mg/mL to 10 mg/mL. Herein, peptides with bulkier aryl moiety 15d-
f [p-ethylphenyl, biphenyl and p-(n-butyl)phenyl] exhibited less
potency at MIC values compared to their Boc-protected derivatives
(13d-f). Activity results suggests that both NHBzl and Boc moiety
exhibited more potent antifungal activity as compared to the
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European Journal of Medicinal Chemistry 223 (2021) 113635
combination of OMe and Boc group of series 1 peptides, possibly
due to better tuning of charge versus lipophilicity. In peptides 14a-f,
without Boc moiety and an increase in lipophilic character modu-
lated by the aryl moiety of histidine lead to higher antifungal ac-
tivity compared to the Boc group containing peptides 12a-f,
possibly due to better tuning of amphiphilicity and results in the
identification of the most promising peptide 14f. In the nutshell,
peptides 12f, 13e, 13f, 14e, 14f, 15e and 15f produced higher activity
and selectivity against C. neoformans.
These SAR results as depicted in Fig. 2 steered this study to infer
that the bulky substitution at the C-2 position of histidine residue
has a more obvious role in modulating the amphiphilicity and the
potency of the peptides.
The tested peptides also produced moderate activity against
MRSA (IC50 values ranged in 4.44e17.5 mg/mL and MIC values be-
tween 5 and 20 mg/mL) and VRE (IC50 values between 7.38 and
15.4 mg/mL and MIC values ranged in 10e20 mg/mL). Specifically,
peptides 13e and 13f showed IC50 values of 4.55 and 9.13 mg/mL,
respectively, with identical MIC value of 10 mg/mL against MRSA.
The same peptides also displayed IC50 values of 8.82 and 7.38 mg/
mL, respectively and identical MIC value of 10 mg/mL for VRE
(Table 2). Furthermore, peptide 15e displayed IC50 and MIC value of
4.44 and 5 mg/mL against MRSA, while producing noticeable IC50
and MICs of 8.33 and 20 mg/mL, respectively against VRE. Some
peptides also indicated low activity against E. coli and P. aeruginosa.
Aromatic lipophilic bulky groups such as ethylphenyl, biphenyl and
butylphenyl at the imidazole ring of histidine also imparted anti-
bacterial activity against MRSA and VRE. (Table 2). The results of
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Table 1
In vitro anticryptococcal activities of synthetic peptides against C. neoformans.

Entry R1 R2 R3 C. neoformans CTXa (mg/mL)

IC50 MIC

12a H Boc OMe >20 e >10

12b Phenyl Boc OMe 8.85 >20 >10
12c p-Tolyl Boc OMe 7.66 >20 >10
12d p-Ethylphenyl Boc OMe 9.66 >20 >10
12e Biphenyl Boc OMe 8.06 >20 >10
12f p-(n-Butyl)phenyl Boc OMe 1.99 2.5 >10
13a H Boc NHBzl >20 e >10
13b Phenyl Boc NHBzl 7.93 >20 >10
13c p-Tolyl Boc NHBzl 4.34 5 >10
13d p-Ethylphenyl Boc NHBzl 1.5 2.5 >10
13e Biphenyl Boc NHBzl 2.15 2.5 >10
13f p-(n-Butyl)phenyl Boc NHBzl 1.98 2.5 >10
14a H H OMe 2.41 10 >10
14b Phenyl H OMe 9.23 >20 >10
14c p-Tolyl H OMe 8.98 >20 >10
14d p-Ethylphenyl H OMe 1.85 2.5 >10
14e Biphenyl H OMe 1.51 2.5 >10
14f p-(n-Butyl)phenyl H OMe 0.37 0.63 >10
15a H H NHBzl 4.92 10 >10
15b Phenyl H NHBzl 5.31 10 >10
15c p-Tolyl H NHBzl 3.36 5 >10
15d p-Ethylphenyl H NHBzl 2.26 10 >10
15e Biphenyl H NHBzl 1.17 5 >10
15f p-(n-Butyl)phenyl H NHBzl 2.27 5 >10
Amphotericin B 0.69 1.25

IC50 and MIC values in mg/mL.

a
Highest tested concentration of 10 mg/mL.

antibacterial activities provides a starting designed structural with methyl ester (less bulky) and hydrophobic benzylamide (more
template for further fine tuning in quest of new scaffolds against bulky) motifs at the C-termini exhibited a linear increases in ac-
pathogenic and challenging VRE and MRSA strains [50,51]. tivity with increasing aromatic bulk. In general, increase in lipo-
philic bulk [p-(n-butyl)phenyl > biphenyl > p-ethylphenyl > p-
2.3. Cytotoxicity assay tolyl > phenyl > H] results in incremental increase in bioactivity
against C. neoformans. The results of this study further reinforces
The cytotoxicity is one of the most important parameters for the the essentiality of optimal charge-to-bulk ratio (ideal balance of
determination of the potential of membrane disruptive antifungal amphiphilicity) for promising antifungal activity.
peptides as both fungal cells and humans are eukaryotes. Peptides
(series 1e2) were evaluated for in vitro cytotoxicity against
noncancerous mammalian cell line, VERO using the neutral red Table 2
uptake assay [52]. The results as summarizied in Table 1 confirms In vitro anti-bacterial activities of peptides.
that all peptides were exhibited no toxicity up to 10.0 mg/mL. These
Entry MSRA VRE E. coli P. aeruginosa
results indicate that antifungal activity is exhibited by peptide-
mediated effects and not due to the cell cytotoxicity. IC50 MIC IC50 MIC IC50 MIC IC50 MIC

12e 16.3 >20 e e >20 e >20 e

2.4. Molecular properties and activity correlation analysis 12f 9.29 10 7.82 20 17.88 >20 >20 e
13c 17.5 20 >20 e >20 e >20 e
13d 8.93 20 14.9 >20 >20 e >20 e
For a better understanding at the molecular level and to eval- 13e 4.56 10 8.82 10 >20 e 19.78 e
uate the effect of lipophilic changes on the antifungal action of the 13f 9.13 10 7.38 10 >20 e >20 e
peptides, and a relationship was formed between the retention 14d >20 e >20 e 17.36 >20 >20 e
14e 7.81 10 13.2 20 >20 e >20 e
times (tR), measured by analytical C-18 RP-HPLC and predicted
14f 8.74 10 15.4 20 15.57 >20 >20 e
ClogP values using ACD software [53]. 15d 18.4 20 13.5 20 >20 e >20 e
The study was conducted on peptides 14a-f and 15a-f. The 15e 4.44 5 8.33 20 >20 e 18.7 >20
correlation of activity (IC50 values against C. neoformans) and lip- 15f 4.46 10 9.99 20 16.26 >20 17.22 >20
ophilicity (tR and ClogP) of the tested peptides are summarized in Cpx 0.09 0.25 0.15 0.63 0.007 0.031 0.09 0.50

Table 3. The results reveal that peptides 14a-f and 15a-f substituted Cpx: Ciprofloxacin, IC50 and MIC values in mg/mL.

5
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Fig. 2. Structure activity relationship study of the synthesized peptides.

Table 3 membranes (EYPC/cholesterol) and Stern-Volmer plots were

Correlation between molecular properties and anticryptococcal activity of peptides. generated (Fig. 3) [55]. The fluorescence of the indole ring of
Entry CLogPa tR (min) C. neoformans IC50 (mg/mL) tryptophan decreased due to quenching in a concentration reliant
approach in the absence and presence of liposomes, using acryl-
14a 2.19 15.013 2.41
14b 0.10 17.892 9.24
amide to the peptide solution, and without any other effects. The
14c 0.39 18.652 8.98 values for the Stern-Volmer quenching constant (Ksv) in the
14d 0.92 19.982 1.85 absence of liposomes was 153.40 for 14f. For bioactive peptide,
14e 1.78 21.909 1.51 Stern-Volmer quenching constant was decreased in the presence of
14f 1.98 22.383 0.37
EYPC/EYPG (7:3, w/w Ksv ¼ 6.68 for 14f) and remained almost
15a 1.17 20.202 4.92
15b 0.92 20.749 5.31 similar in the presence of EYPC/cholesterol (10:1, w/w Ksv ¼ 145.69
15c 1.42 20.955 3.36 for 14f). These results suggest that the tryptophan fluorescent
15d 1.95 22.837 2.26 indole ring was submerged in the bilayers, and therefore became
15e 2.81 23.438 1.17
unavailable for quenching. These results illustrate that peptide af-
15f 3.00 24.533 2.27
finity for liposomes was EYPC/EYPG > EYPC/cholesterol, and clearly
NA: not active. demonstrated that the peptides gets effectively implanted in EYPC/
b
Retention times (tR) are mentioned using a C-18 column.
a EYPG than EYPC/cholesterol, and that indicating higher selectivity
ClogP values using ACD/LogP software.
ratio of the peptides 12f, 13d, 14e, 14f and 15e towards mimics of
bacterial cell membranes in comparison to mammalian cells.
By using in vitro activity, cytotoxicity results, molecular prop-
erties and activity correlation analysis, we chose peptide 14f for 2.6. Peptide architecture study
thorough evaluation, including membrane selectivity study, pep-
tide architecture study, and mechanism of action investigations. Bioactivity of the amphiphilic antimicrobial peptides sometimes
depends upon the mixed interplay of hydrophobic and hydrophilic
2.5. Tryptophan quenching study interactions between the bioactive peptide and the membranes
[56]. Recently, it has been discovered that intermolecular in-
The correlation between antimicrobial activity and cytotoxicity teractions among the peptides themselves play an important role in
is vested on host versus microbe membrane selectivity. If peptides determining their activity. Hydrophobic interactions involving pp
show activity by membrane-targeting then membrane selectivity is stacking and hydrophilic interactions between the cationic groups
of utmost importance [54]. The cationic property of peptides present within the peptide skeleton are responsible for molecular
potentially contributes to cell selectivity because the surface of self-assembling, supramolecular aggregation and gel-formation
pathogenic membranes is more negatively charged in comparison properties [57].
to that of mammalian cells. The amphipathic character of a peptide
is the main component driving the interaction with microbial cell 2.6.1. Dynamic light scattering study
membranes compared to that of the mammalian membranes. We To investigate the nature of intermolecular interactions of 14f,
noted antibacterial activity is some of the compounds (Table 2). we decided to study the particle size and dispersion of the peptide
Upon the basis of activity profile of peptides, we selected five most in solution state using Zetasizer by performing dynamic light
promising antibacterial peptides 12f, 13d, 14e, 14f and 15e for scattering experiments (Fig. 4) [58e60]. For comparative purposes,
tryptophan fluorescence quenching study and to elucidate their we also conducted the same experiment on the inactive peptide
interactions with synthetic mimics of membrane. The interactions 12a.
of the selected peptides towards pathogen was evaluated to A time dependent particle size analysis of peptides 14f and 12a
differentiate between the interaction with mammalian mem- was carried out using dynamic light scattering experiments. Hy-
branes, and the outer layer of bacterial surface. drodynamic radius of 90% peptide population D(90) in PBS was
Quenching of the fluorescence of tryptophan by acrylamide was considered, and the results revealed that the particle size of 14f
studied using synthetic small unilamellar vesicles (SUVs) i.e. increases slightly from 2073 nm to 2792 nm whereas the particle
negatively charged (EYPC/EYPG) and the zwitterionic model size of 12a decreased from 802 nm to 108 nm on incubation from
6
K.K. Sharma, R. Ravi, I.K. Maurya et al.
Fig. 3. Stern-Volmer plots for the Trp fluorescence quenching. A) In the absence of
vesicles, B) Plots in the presence of EYPC/EYPG (7:3 w/w), and C) In the presence of
EYPC/cholesterol (10:1 w/w).
0 to 4 h (Fig. 5). The polydispersity index (Ð) for 14f sparingly in-
creases from 0.333 to 0.584, whereas for 12a it increases from 0.383
to 0.922. The experiments indicate higher dispersion, homogeneity
and uniformity within the solution for 12a in comparison to 14f.
However, inactive peptide 12a is better distributed within the so-
lution. To further understand peptide architecture, fluorescence
spectroscopic and electron microscopy techniques were employed.
2.6.2. Fluorescence spectroscopic study
Tryptophan fluorescence emission study is a useful method for
investigating the interaction of the synthetic peptides and their
assemblies [29]. One plausible interaction within the molecule
could be due to the formation of p-stacked aggregates of the
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European Journal of Medicinal Chemistry 223 (2021) 113635
tryptophan residue, which were evaluated by monitoring the
fluorescence emission spectra (Fig. 5) [58e60]. Upon investigation,
initially, peptides 14f and 12a displayed a prominent emission peak
around 335 nm representing the tryptophan residue on excitation
at 280 nm.
After 24 h, 14f displayed increase in emission in fluorescence,
which is consistent with extended p-stacked aggregate formation
of tryptophan residue possibly due to the presence of ring-arylation
at the histidine residue resulting in additional p-stacked aggrega-
tion. In the case of 12a, the increase in fluorescence was less
compared to 14f due to the absence of ring-arylation.
2.6.3. Scanning electron microscopy (SEM) study
To have a microscopic insight of the peptide assembly, we
investigated the morphology of the active (14f) and control peptide
(12a). SEM study revealed that the peptide 14f upon incubation for
24 h formed unorganized aggregates (Fig. 6A and B) in comparison
to that of 12a (Fig. 6C), which was quite homogenously dispersed in
the phosphate saline buffer (PBS) solution. The SEM results and
fluorescence emission sepctroscopic study indicate that peptide 14f
forms random aggregates.
The peptide architecture studies indiactes that the synthetic
amphiphilic peptide 14f exhibits self-aggregation properties at the
pathogen surface. For better understanding, we designed other
microscopic experiments to further investigate mechanism of ac-
tion studies.
2.7. Mechanism of action study
We investigated the thorough mechanism of action by
employing various microscopic approaches. We anticipate that
determination of mechanism of action of 14f could guide in further
design of potent analogues with defined mode of action in
combating microbes. The CLSM examination was conducted to
understand the various actions of 14f on the fungal permeabiliza-
tion, intracellular localization and binding to genetic materials in
the fungal cells. The SEM study was carried out to study the effect of
active peptide on the cellular morphological changes, and the high-
resolution transmission electron microscopy (HR-TEM) study hel-
ped to observed the ultrastrucural actions of active peptide 14f on
cell surface of fungi.
2.7.1. Confocal laser scanning microscopic study
Red fluorescent propidium iodide (PI) is not permeable to intact
cell membrane, and DNA binding dye. If interacting peptide per-
meabilizes cell wall and membrane, causing in compromised fungal
membrane permeability thereby permitting PI to enter inside the
dead cells and provide red fluorescence. Under confocal micro-
scopic visualization, the uptake of PI after treatment by fungal cells
with 14f at MIC (1 h) showed red fluorescence due to per-
meabilization of cell surface (Fig. 4B), although the untreated cells
did not possess red fluorescence due to undamaged cell surface
(Fig. 4A).
These results indicate that PI entered intracellularly within the
cells after treatment with 14f as treated cells were unable to
regulate the chemical movement from its cell surface.
To study intracellular localization of 14f in C. neoformans, we
prepared FITC-labeled peptides (fluorescein isothiocyanate; Ex
480 nm) 14f and 12a. The confocal investigation of FITC-labeled 12a
revealed that it can not cross the cell surface due to the intact cell
surface as confirmed by the absence of green fluorescence (Fig. 7C).
FITC-labeled 14f permeated from cell membrane barriers via per-
meabilization and disruption after treatment with fungal cells, and
able to enter inside fungal cells by exhibiting the green fluorescence
(Fig. 7D). These results confirm the ability of 14f to cross membrane
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Fig. 4. Dynamic light scattering studies (A) time dependent particle size distribution showing D(90) of the peptides 14f and 12a in PBS at pH 7.4, and (B) polydispersity index (Ð)
analysis.

Fig. 5. Fluorescence emission spectra of 10 mM samples of 14f and 12a, respectively at 24 h overlaid with emission at 0 h.

Fig. 6. SEM micrographs showing aggregated state of 14f in comparison to homogenously dispersed 12a after 24 h incubation. Scale of image A (10.4 mm x 7.50 k, 5 mm), image B
(10.4 mm x 8.50 k, 5 mm), image C (10.6 mm x 7.50 k, 5 mm).

barriers and sebsequent interaction with the intracellular targets
resulting in the death of cells.
Blue fluorescnent DAPI (40 ,6-diamidino-2-phenylindole) re-
leases fluorescence after selective binding to the AT regions of DNA
while entering in live cells. The DAPI dye is utilized for nuclear or
chromatin fragmentation of DNA, and alterations in fluorescence
intensity indicate the magnitude of DNA damage triggered by the
active peptides. Treated fungal cells upon DAPI staining and treat-
ment with 14f showed the split and less blue fluorescence intensity
in the cryptococcal cells compared with untreated cells (Fig. 7E and
F). From these results, killing of fungal cells by 14f is also contrib-
uted to its interaction with genetic material such as DNA after
membrane permeabilization causing the nuclear fragmentation.
2.7.2. Electron microscopic study
The detailed mechanistic investigation of the potent peptide 14f
was investigated by SEM and HR-TEM with a view to gain addi-
tional understandings about the mechanism of action. SEM exam-
ination was carried out to observe changes in the cryptococcal cell
surface morphology after treatment with 14f at its MIC. The un-
treated cells have smooth outer cell surface when compared with
treated cells with 14f (Fig. 8A). Peptide 14f caused prominent cell
wall breakage or cell surface damage, and permeabilization and
hole(s) formation was evidently noticeable with the release of
intracellular material outside cells (Fig. 8BeD). Possibly, a cationic
amphiphilic peptide first interact with the negatively charged
fungal cells by electronic interactions and gets aggregated over

8
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Fig. 7. Confocal fluorescence microscopic imagesto examine the impact of 14f at MIC: (A and B) membrane permeabilization examination using PI after 1 h; (C and D) FITC-labeled
14f after 1 h and FITC-labeled control peptide; (E and F) peptide treatment using DAPI after 1 h. Images in (A) and (E) are fungal cells (ATCC350) incubated alone, and image in (C)
FITC-labeled control. Images in (B), (D), and (F) correspond to active peptide treatment after 1 h of incubation.

fungal surface in comparison to zwitterionic mammalian mem-

Fig. 8. Morphological examination of cells with 14f and untreated cells using SEM.
Images (A and B) represent untreated cells (10000  , 10 mm; 7500  , 10.1 mm, 5 mm).
Images (C and D) represent treated cells treated with 14f (7500  , 10.1 mm; 7500  ,
10.5 mm, 5 mm).
branes, thereby exhibiting preferential selectivity to fungal strains.
Further, amphiphilic peptide form pores over cell surface, resulting
in hole formation thereby increasing permiabilization. It is inter-
pretatedthat 14f treated fungal cells can not withstand the various
effects exerted by peptides, leading to extensive disruption of cell
surface chracterstics, and permeability through pore(s) formation.
In the HR-TEM examination of cells, 14f treatment (at MIC
concentration) resulted in disruptive effects on capsule, cell wall
and the plasma membrane.
Untreated fungal cells exhibit undamaged membranes including
capsule under the HR-TEM investigation. Fig. 9A showed intact cell
morphology of a group of fungal cells, while Fig. 9B and C centered
on a single cell imagining and exhibited undamaged cell surface
including capsule, cell wall and plasma membrane under untreated
conditions. Fig. 9DeF exhibits the rainbow images of the untreated
fungal cells. The peptide 14f treated fungal cells demonstrate se-
vere disruption of capsule (Fig. 9F and L), cell wall [(Fig. 9C and F
(intact) and Fig. 9I and L (disrupted)] and plasma membrane
resulting in membrane permeabilization leading to fungal cell lysis
(Fig. 9GeI). This examination clearly reveals that 14f peptide act by
interacting with cell surface as cryptococcus capsule was disrupted
followed by other membranes (please see marked arrows, Fig. 9I

9
K.K. Sharma, R. Ravi, I.K. Maurya et al. European Journal of Medicinal Chemistry 223 (2021) 113635

Fig. 9. Morphological examination of cells treated with 14f and untreated using HR-TEM. Images (AeF) represent untreated cells; rainbow images D-F generated from A-C. Images
(GeL) represent cells treated with 14f; rainbow images J-L generated from G-I. The scale bar of images A and G is 0.5 mm, images B and H is 0.2 mm and for other images it is 100 nm.

10
K.K. Sharma, R. Ravi, I.K. Maurya et al.
and L). It is known that Cryptococcal cells produce a characteristic
polysaccharide capsule that provide protection against environ-
mental stress and predators. Capsule provides protection inside
host through reducing host immune responses by down regulating
inflammatory cytokines and inhibiting monocytes [18,19]. There-
fore, alteration in capsule can makes C. neoformans cells susceptible
for killing through various means, including drugs. The rainbow
images were generated from corresponding images to enhance
examination clarity (Fig. 9J-L).
The mechanism of action studies by using CLSM, SEM, and HR-
TEM indicate that 14f is a surface or membrane active peptide and
act by infiltrating the fungal cellular membranes, thus killing the
fungal pathogen. Selectivity of this class of peptides towards
C. neoformans cells might be due to disruption of specialized
capsule and cell wall composition. Additionally, the positively
charged bioactive peptide interacts with the negatively charged
fungal membrane by electrostatic interactions in comparison to
zwitterionic mammalian membranes and exhibit preferential
selectivity to cryptococcal strain.
3. Conclusions
In conclusion, design and synthesis of new scaffold of His(2-Ar)-
Trp-Arg of the amphipathic peptides composed of C-2 arylated
histidine along with cationic arginine and hydrophobic tryptophan
is reported. These peptides have demonstrated high potency
against C. neoformans fungal strain, take the lead to identification of
bioactive peptide 14f as the most potent, which is two-fold more
potent than amphotericin B. Some other peptides (13e, 13f, 15e and
15f) of this class also provide a starting template for desiging new
leads against pathogenic and challenging VRE and MRSA strains.
Short sequential size facilitates the ease of synthesis along with
lower production cost in this class of peptides. The peptides
exhibited no apparent mammalian cytotoxicity confirmed by Vero
Cells experiment. It is noted that variation in lipophilicity, partic-
ularly imparted by substituent at the imidazole ring played a vital
role in antifungal potency and selectivity. The potency is found to
be related to the bulkiness of the substituent attached to the
imidazole ring. Membrane selectivity study with tryptophan fluo-
rescence quenching and mechanistic investigations by electron
microscopy prove that 14f is selective to fungal cell surface and
disrupt the fungal membranes resulting in killing of pathogenic
cells. This study experimentally strengthen the concept of the role
of suitably modified amino acids, their sequence, and importance of
the synthetically tuned amphiphilicity in the antifunal peptide
design. Indentification of the lead peptide 14f provided insights in
the design, amino acids placement in a sequence, and role of the
modified amino acids. This study opens additional avenues for the
discovery of ultrashort, membrane-disruptive, and selective
peptide-based antifungal probes.
4. Experimental
4.1. General chemistry
All chemicals were purchased from commercial sources such as
Sigma-Aldrich, Acros Organics, Chem-Impex, Himedia, Avra and
Alfa-Aesar. Solution phase peptide synthesis was performed on
CEM Microwave Discover® using MW assisted solution phase
peptide synthesis protocol. 1H NMR were obtained on AVANCE III
400 Bruker (400 MHz) andchemical shifts are stated in parts per
million (ppm, d scale) and are referenced to residual solvent peak in
the NMR solvent or TMS. 13C NMR was recorded on AVANCE III 400
Bruker (100 MHz) and decoupled by broad band decoupling. The
following abbreviations were utilized to present peptides peak
11
European Journal of Medicinal Chemistry 223 (2021) 113635
patterns when suitable: br ¼ broad, s ¼ singlet, d ¼ doublet,
dd ¼ doublet of doublets, t ¼ triplet, q ¼ quadruplet, m ¼ multiplet.
Coupling constants (J) were reported in hertz unit (Hz). The final
peptides were purified through Shimadzu preparative HPLC using a
Luna 10 mM C-18 (250 mm  21.2 mm, 10 mm) column. The purity of
peptides was assessed on a Shimadzu SPD-M20A analytical HPLC
using a supelcosil™ LC-18 (25 cm  4.6 mm ID, 5 mm), and final
purity assessed, using a gradient run of 955% (1 mL/min; A, 0.1%
TFA in H2O/B, 0.1% TFA in CH3CN), and detection using PDA detector
at 220 or 254 nm; all biologically evaluated peptides exhibited
95% purity. The high-resolution mass of the synthesized peptides
were analyzed on a MAXIS Bruker spectrometer using ESI-TOF
method.
4.1.1. General synthesis of N-a-Boc-2-aryl-L-histidines (4a-e)
In a flask, starting material (1, 1.0 equiv.) was subjected in CH2Cl2
(5 mL), and CF3CO2H (1.5 equiv.) was added and stirred for 10 min.
Thereafter, AgNO3 (0.2 equiv.) dissolved in H2O (5 mL) was added in
flask and followed by addition of arylboronic acid (1.5 equiv.). Next,
(NH4)2S2O8 (2.0 equiv.) was added at ambient conditions for
12e18 h and reaction monitored through TLC. After completion, the
pH of the reaction was adjusted (pH ¼ 11e12) by the NH4OH, and
CH2Cl2 was evaporated under vacuum, and extraction of the reac-
tion mixture with EtOAc. The organic layer was washed with brine
and dried using Na2SO4and crude mixture purified by flash chro-
matography to isolate N-a-trifluoroacetyl-2-aryl-L-histidine methyl
esters (2a-f) in 42e55% yields [40]. 2-Aryl-L-histidine.2HCl salts
(3a-f) were produced by refluxing a solution of 2a-f in 6 N HCl for
12 h in 92e96% yields. Derivatives of 2-Aryl-L-histidine salts (3a-f, 1
equiv.) were suspended in water-dioxane (1:2) and pH of solution
adjusted to 12 with 4 N NaOH, and then (Boc)2O (1.5 equiv.) was
added to the reaction. After 15 min, pH was again adjusted to 12
and another (Boc)2O (1.5 equiv.) was added, and stirred (12 h, RT).
Solvent removed and methanol (10 mL) was added, and stirred
again for 12 h. The Reaction oncentrated under vacuum and fol-
lowed by pH adjustment of resulting N-a-Boc-L-His(2-aryl)-O-Naþ
with aq. KHSO4 to pH 3 gave free N-a-Boc-2-aryl-L-histidines. After
concentrated reaction under vacuum and the mixture was extrac-
ted through tert-butanol, afforded N-a-Boc-2-aryl-L-histidines (4a-
e) in 91e95% yields.
4.1.2. Synthesis of L-Arg-OMe (6)
L-Arg-OH (5, 1 equiv.) was purged with HCl gas in MeOH for 2 h
at RT and stirred for 8 h at 10  Cto afford pure L-Arg-OMe (6) in 95%
yields.
4.1.3. Synthesis of L-Arg-NHBzl (7)
In a dry flask, L-Arg-OH (5, 1 equiv.), CDI (1.5 equiv.) and ben-
zylamine (1.5 equiv.) was added and mixed, and allowed to stir at
ambient temperature for 12 h. The complete removal of solvent
under reduced pressure produced crude product, which was puri-
fied on basic alumina column to afford L-Arg-NHBzl (7) in 72%
yields [45].
4.1.4. Synthesis of N-a-Boc-L-Trp-L-Arg-OMe (8) and N-a-Boc-L-
Trp-L-Arg-NHBzl (9)
In a dry MW vial (10 mL) equiped with a magnetic stir bar, L-
Arg-OMe/NHBzl (1 equiv.) and DIEA (4 equiv.) was added togather.
Next, Boc-L-Trp-OH (1.5 equiv.), DIC (1.5 equiv.) and HOBt (1.5
equiv.) were added to vial in DMF (5 mL). The reaction mixture was
placed under MW heating (CEM Discover® microwave reactor) for
30 min at 60  C. The reaction mixture was diluted with methanol
for homogenization and purified through column chromatography
system to afford pure Boc-L-Trp-L-Arg-OMe/NHBzl (8 and 9) in 78%
and 71% yields, respectively.
K.K. Sharma, R. Ravi, I.K. Maurya et al.
4.1.5. General synthesis of L-Trp-L-Arg-OMe (10) and L-Trp-L-Arg-
NHBzl (11)
The dipeptides, Boc-L-Trp-L-Arg-OMe/NHBzl (8 or 9) and 6 N
methanolic HCl (5 mL) were stirred at ambient temperature for
20 min, and thenconcentrated under vacuum to get 10 and 11 in
92% and 93% yields, respectively.
4.1.6. General procedure of the synthesis of N-a-Boc-L-His(2-aryl)-
L-Trp-L-Arg-OMe (12a-f) and N-a-Boc-L-His(2-aryl)-L-Trp-L-Arg-
NHBzl (13a-f)
In a dry MW vial (10 mL) equiped with a magnetic stir bar, 10 or
11 (1 equiv.) was added and then DIEA (4 equiv.), DIC (1.5 equiv.),
HOBt (1.5 equiv.), dry DMF (3 mL) and N-a-Boc-2-aryl-L-histidines
(4a-f, 1.5 equiv.) were added, and MW irradiation was applied at
60  C (30 min). Solvent was evaporated under reduced pressure
and the crude peptide was precipitated using cold diethyl ether.
The peptides were purified on preparative-HPLC system using a C-
18 RP-column with CH3CN and water a solvent system containing
0.08% TFA to yield Boc-L-His(2-aryl)-L-Trp-L-Arg-OMe/NHBzl (12a-
f and 13a-f).
4.1.7. General synthetic procedure for L-His(2-aryl)-L-Trp-L-Arg-
OMe (14a-f) and L-His(2-aryl)-L-Trp-L-Arg-NHBzl (15a-f)
N-a-Boc-L-His(2-aryl)-L-Trp-L-Arg-OMe/NHBzl (12a-f or 13a-f)
upon reaction with 6 N HCl (5 mL) at 25  C for 15e20 min resulted
in the removal of Boc group to give the desired tripeptides (14a-f
and 15a-f).
4.1.8. Characterization of synthesized peptides
N-a-Boc-L-His-L-Trp-L-Arg-OMe (12a). Yield: 56%. 1H NMR
[400 MHz, CD3OD]: d 8.73 (s, 1H), 7.61 (d, J ¼ 7.84, 1H), 7.37 (d,
J ¼ 8.08, 1H), 7.19 (d, J ¼ 6.80, 2H), 7.12 (t, J ¼ 7.54, 1H), 7.04 (d,
J ¼ 7.44, 1H), 4.67 (t, J ¼ 6.90, 1H), 4.44 (t, J ¼ 6.68, 1H), 4.35 (t,
J ¼ 6.78, 1H), 3.6849 (s, 3H), 3.30e3.11 (m, 5H), 2.9607 (dd,
J ¼ 15.26, 8.34, 1H), 1.91e1.85 (m, 1H), 1.73e1.57 (m, 3H), 1.39 (s,
9H); 13C NMR [100 MHz, CD3OD]: d 172.7, 171.8, 169.7, 157.2, 156.2,
136.6, 133.4, 129.6, 127.4, 123.5, 121.1, 118.5, 117.8, 117.1, 111.0, 108.8,
79.9, 54.4, 53.5, 51.8, 51.5, 40.4, 29.3, 28.7, 28.2, 27.1, 24.6;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 612.3258, found:
612.3259; HPLC: tR ¼ 20.1 min, 95.3% purity.
N-a-Boc-L-His(2-phenyl)-L-Trp-L-Arg-OMe (12b). Yield: 63%.
1
H NMR [400 MHz, CD3OD]: d 7.93 (d, J ¼ 6.64, 2H), 7.65 (d, J ¼ 7.32,
3H), 7.58 (d, J ¼ 8.20, 1H), 7.35 (d, J ¼ 8.04, 1H), 7.27 (s, 1H), 7.20 (d,
J ¼ 8.16, 1H), 7.10 (t, J ¼ 7.48, 1H), 7.02 (t, J ¼ 7.24, 1H), 4.66 (t,
J ¼ 6.90, 1H), 4.44e4.38 (m, 2H), 3.65 (s, 3H), 3.28e3.17 (m, 3H),
3.13e3.03 (m, 3H), 1.83e1.80 (m, 1H), 1.68e1.64 (m, 1H), 1.57e1.52
(m, 2H), 1.37 (s, 9H); 13C NMR [100 MHz, CD3OD]: d 172.9, 171.7,
171.3, 157.1, 156.1, 144.5, 136.6, 132.1, 130.39, 129.4, 127.3, 126.4,
123.4, 122.9, 121.1, 118.5, 118.0, 117.8, 111.0, 108.8, 79.8, 54.4, 53.4,
51.9, 51.4, 40.3, 28.2, 27.3, 27.1, 24.6; HRMS(ESI-TOF), calculated for
m/z [M þ Hþ]: 688.3571, found: 688.3552; HPLC: tR ¼ 22.1 min,
99.8%.
N-a-Boc-L-His(2-tolyl)-L-Trp-L-Arg-OMe (12c). Yield: 65%. 1H
NMR [400 MHz, CD3OD]: d 7.80 (t, J ¼ 7.62,2H), 7.58 (d, J ¼ 7.04, 1H),
7.49e7.47 (m, 3H), 7.35 (d, J ¼ 8.16, 1H), 7.11 (t, J ¼ 7.14, 1H), 7.03 (t,
J ¼ 6.76, 1H), 4.66 (t, J ¼ 7.00, 1H), 4.42e4.39 (m, 2H), 3.67 (s, 3H),
3.27e3.19 (m, 2H), 3.15e2.99 (m, 4H), 2.47 (s, 3H), 1.86e1.81 (m,
1H), 1.69e1.65 (m, 1H), 1.57e1.52 (m, 2H), 1.38 (s, 9H); 13C NMR
[100 MHz, CD3OD]: d 174.3, 173.2, 172.8, 158.6, 157.9, 144.6, 131.9,
131.5, 131.5, 127.7, 127.6, 127.5, 124.9, 122.5, 121.6, 119.9, 119.2, 112.4,
110.2, 81.0, 54.0, 53.3, 53.1, 52.9, 41.8, 29.6, 28.7, 28.6, 26.0, 21.5;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 702.3727, found:
702.3724; HPLC: tR ¼ 23.3 min, 96.2%.
N-a-Boc-L-His(2-ethylphenyl)-L-Trp-L-Arg-OMe (12d). Yield:
61%. 1H NMR [400 MHz, CD3OD]: d 7.84 (t, J ¼ 6.88,2H), 7.58 (d,
12
European Journal of Medicinal Chemistry 223 (2021) 113635
J ¼ 7.84, 1H), 7.50 (d, J ¼ 7.16, 3H), 7.36 (d, J ¼ 8.04, 1H), 7.19 (s, 1H),
7.13e7.09 (m, 1H), 7.05e7.01 (m, 1H), 4.66 (t, J ¼ 7.02, 1H), 4.41e4.38
(m, 2H), 3.66 (s, 3H), 3.27 (d, J ¼ 6.44, 1H), 3.22 (t, J ¼ 6.52, 1H), 3.17
(d, J ¼ 9.28, 1H), 3.12 (t, J ¼ 7.10, 2H), 3.06e2.99 (m, 1H), 2.78 (q,
J ¼ 7.60, 7.52, 2H), 1.83e1.78 (m, 1H), 1.68e1.65 (m, 1H), 1.56e1.52
(m, 2H), 1.38 (s, 9H), 1.30 (t, J ¼ 7.60, 3H); 13C NMR [100 MHz,
CD3OD]: d 172.9, 171.8, 171.4, 157.2, 156.5, 149.5, 136.6, 130.4, 129.0,
128.9, 127.3, 126.5, 126.3, 123.5, 121.1, 118.5, 117.8, 111.0, 108.8, 79.9,
53.4, 51.9, 51.7, 51.5, 40.4, 28.3, 28.2, 27.3, 27.1, 27.0, 24.5, 14.2;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 716.3884, found:
716.3868; HPLC: tR ¼ 24.1 min, 96.2% purity.
N-a-Boc-L-His(2-biphenyl)-L-Trp-L-Arg-OMe (12e). Yield:
60%. 1H NMR [400 MHz, CD3OD]: d 8.00 (d, J ¼ 6.32, 2H), 7.86 (d,
J ¼ 6.80, 2H), 7.72 (d, J ¼ 7.52, 2H), 7.58 (d, J ¼ 7.40, 1H), 7.51 (t,
J ¼ 7.46, 2H), 7.42 (t, J ¼ 7.28, 1H), 7.35 (d, J ¼ 8.08, 1H), 7.19 (s, 2H),
7.10 (t, J ¼ 7.42, 1H), 7.02 (J ¼ 7.36, 1H), 4.66 (t, J ¼ 6.70, 1H), 4.42, (br.
s, 2H), 3.65 (s, 3H), 3.26 (t, J ¼ 7.30, 3H), 3.20e3.08 (m, 2H),
3.06e3.00 (m, 1H), 1.80 (t, J ¼ 6.54, 1H), 1.65e1.52 (m, 3H), 1.38 (s,
9H); 13C NMR [100 MHz, CD3OD]: d 172.9, 171.8, 171.7, 157.1, 145.9,
139.3, 136.6, 133.3, 130.5, 128.7, 127.9, 127.5, 127.4, 126.6, 126.5,
123.4, 121.0, 118.5, 117.8, 110.9, 108.8, 79.7, 54.4, 53.8, 51.8, 51.4, 40.4,
29.3, 28.2, 27.1, 24.5, 22.3; HRMS(ESI-TOF): calculated for m/z
[M þ Hþ]: 764.3884, found: 764.3884; HPLC: tR ¼ 26.1 min, 99.8%
purity.
N-a-Boc-L-His[(2-p-(n-butylphenyl)]-L-Trp-L-Arg-OMe (12f).
Yield: 59% 1H NMR [400 MHz, CD3OD]: d 7.87e7.80 (m, 2H), 7.55 (d,
J ¼ 7.76, 1H), 7.51e7.47 (m, 2H), 7.41 (d, J ¼ 8.12, 1H), 7.28 (s, 1H),
7.22 (d, J ¼ 4.32, 1H), 7.15 (t, J ¼ 7.12, 1H), 7.06 (t, J ¼ 7.08, 1H), 4.64 (t,
J ¼ 7.56, 1H), 4.43 (t, J ¼ 6.96, 1H), 4.33e4.29 (m, 1H), 3.64 (s, 3H),
3.25e3.18 (m, 3H), 3.10 (t, J ¼ 6.90, 2H), 2.72 (t, J ¼ 7.54, 2H),
1.80e1.74 (m, 1H), 1.67e1.59 (m, 3H), 1.49e1.44 (m, 2H), 1.39e1.32
(m, 11H), 0.93 (t, J ¼ 7.36, 3H); 13C NMR [100 MHz, CD3OD]: d 173.2,
172.9, 172.5, 156.8, 153.9, 148.4, 144.7, 136.3, 129.7, 129.7, 127.2,
126.9, 126.4, 123.9, 121.5, 119.9, 118.9, 117.8, 111.4, 108.4, 80.8, 54.6,
53.5, 52.3, 52.1, 40.4, 34.9, 32.9, 28.1, 27.2, 27.1, 27.1, 24.3, 21.7, 13.0;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 744.4197, found:
744.4180; HPLC: tR ¼ 27.1 min, 96.4% purity.
N-a-Boc-L-His-L-Trp-L-Arg-NHBzl (13a). Yield: 55%. 1H NMR
[400 MHz, CD3OD]: d 8.70 (s, 1H), 7.58 (d, J ¼ 7.72, 1H), 7.33 (d,
J ¼ 8.08, 1H), 7.30e7.26 (m, 2H), 7.23e7.18 (m, 4H), 7.15 (s, 1H), 7.10
(t, J ¼ 7.34, 1H), 7.02 (t, J ¼ 7.34, 1H), 4.63 (t, J ¼ 6.60, 1H), 4.31e4.23
(m, 4H), 3.26e3.20 (m, 2H), 3.10e3.06 (m, 3H), 2.96e2.90 (m, 1H),
1.85e1.78 (m, 1H), 1.63e1.48 (m, 3H), 1.36 (s, 9H) 13C NMR
[100 MHz, CD3OD]: d 172.9, 171.9, 171.6, 157.1, 156.3, 138.2, 136.6,
133.4, 129.6, 128.1, 127.2, 127.0, 126.8, 123.4, 121.3, 118.7, 117.9, 117.1,
111.1, 108.8, 79.9, 54.6, 53.6, 53.0, 42.6, 40.4, 28.5, 27.1, 27.0, 26.7,
24.7; HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 687.3731, found:
687.3725; HPLC: tR ¼ 22.2 min, 95.4% purity.
N-a-Boc-L-His(2-phenyl)-L-Trp-L-Arg-NHBzl (13b). Yield:
62%. 1H NMR [400 MHz, CD3OD]: d 7.92 (d, J ¼ 6.64, 2H), 7.68e7.62
(m, 3H), 7.57 (d, J ¼ 7.88, 1H), 7.34 (d, J ¼ 8.00, 1H), 7.30e7.27 (m,
3H), 7.23e7.21 (m, 3H), 7.17 (s, 1H), 7.11 (t, J ¼ 7.42, 1H), 7.03 (t,
J ¼ 7.14, 1H), 4.66 (t, J ¼ 6.60, 1H), 4.41 (t, J ¼ 6.78, 1H), 4.33e4.29 (m,
1H), 4.26e4.24 (m, 2H), 3.27e3.14 (m, 3H), 3.11e2.99 (m, 3H),
1.85e1.80 (m, 1H), 1.62e1.57 (m, 1H), 1.49 (d, J ¼ 3.68, 2H), 1.37 (s,
9H) 13C NMR [100 MHz, CD3OD]: d 172.9, 171.8, 171.6, 157.1, 156.4,
144.4, 138.2, 136.6, 132.1, 130.5, 129.5, 128.1, 127.2, 127.0, 126.8,
126.7, 126.4, 123.4, 122.8, 121.2, 118.7, 117.8, 111.1, 108.8, 79.8, 54.7,
53.6, 53.0, 42.6, 40.4, 28.5, 27.1, 26.7, 26.6, 24.7; HRMS(ESI-TOF):
calculated for m/z [M þ Hþ]: 763.4044, found: 763.4034; HPLC:
tR ¼ 24.2 min, 96.8% purity.
N-a-Boc-L-His(2-tolyl)-L-Trp-L-Arg-NHBzl (13c). Yield: 66%.
1
H NMR [400 MHz, CD3OD]: d 7.84e7.78 (m, 2H), 7.51e7.45 (m, 3H),
7.39 (t, J ¼ 7.02, 1H), 7.32e7.27 (m, 4H), 7.21e7.14 (m, 4H), 7.08 (t,
J ¼ 8.52, 1H), 4.66 (t, J ¼ 7.12, 1H), 4.40e4.36 (m, 1H), 4.34e4.21 (m,
K.K. Sharma, R. Ravi, I.K. Maurya et al.
2H), 4.10 (s, 1H), 3.50e3.45 (m, 1H), 3.26e3.25 (m, 1H), 3.21e3.16
(m, 2H), 3.07e3.02 (m, 2H), 2.45 (s, 3H), 1.79e1.53 (m, 4H), 1.33 (s,
9H); 13C NMR [100 MHz, CD3OD]: d 171.9, 171.7, 170.9, 156.5, 153.4,
145.5, 135.1, 131.4, 130.1, 129.4, 128.2, 127.0, 126.8, 126.8, 126.5, 126.1,
125.1, 123.7, 121.0, 119.0, 117.7, 111.4, 108.3, 79.6, 54.8, 54.6, 53.5,
42.5, 40.3, 28.1, 27.1, 26.7, 26.3, 24.2, 20.3; HRMS(ESI-TOF): calcu-
lated for m/z [M þ Hþ]: 777.4200, found: 777.4193; HPLC:
tR ¼ 25.2 min, 95.6% purity.
N-a-Boc-L-His(2-ethylphenyl)-L-Trp-L-Arg-NHBzl (13d).
Yield: 58%. 1H NMR [400 MHz, CD3OD]: d 7.83 (d, J ¼ 8.00, 2H), 7.57
(d, J ¼ 7.80, 1H), 7.47 (d, J ¼ 8.28, 2H), 7.34 (d, J ¼ 8.08, 1H),
7.30e7.26 (m, 2H), 7.23e7.18 (m, 5H), 7.11 (t, J ¼ 7.20, 1H), 7.03 (t,
J ¼ 7.40, 1H), 4.66 (t, J ¼ 7.20, 1H), 4.40 (t, J ¼ 7.10, 1H), 4.33e4.30 (m,
1H), 4.25 (d, J ¼ 2.40, 2H), 3.26 (d, J ¼ 6.52, 2H), 3.19e3.14 (m, 1H),
3.09 (t, J ¼ 6.76, 2H), 3.02e2.97 (m, 1H), 2.75 (q, J ¼ 7.60, 7.56, 2H)
1.84e1.78 (m, 1H), 1.64e1.57 (m, 1H), 1.52e1.46 (m, 2H), 1.36 (s, 9H),
1.28 (t, J ¼ 7.60, 3H); 13C NMR [100 MHz, CD3OD]: d 172.9, 171.9,
171.7, 157.1, 156.3, 149.5, 144.6, 138.1, 136.5, 130.1, 129.0, 128.1, 127.2,
127.0, 126.8, 126.4, 123.5, 121.3, 120.2, 118.8, 117.9, 117.6, 111.2, 108.7,
80.0, 54.7, 53.7, 53.1, 42.6, 40.4, 28.5, 28.3, 27.1, 26.1, 24.7, 14.2;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 791.4357, found:
791.4333; HPLC: tR ¼ 26.2 min, 95.8% purity.
N-a-Boc-L-His(2-biphenyl)-L-Trp-L-Arg-NHBzl (13e). Yield:
61%. 1H NMR [400 MHz, CD3OD]: d 8.00 (d, J ¼ 8.08, 2H), 7.90 (d,
J ¼ 8.32, 2H), 7.71 (d, J ¼ 7.32, 2H), 7.57 (d, J ¼ 7.92, 1H), 7.53 (t,
J ¼ 7.46, 2H), 7.45 (d, J ¼ 7.12, 1H), 7.33 (d, J ¼ 8.08, 1H), 7.30 (s, 1H),
7.26 (d, J ¼ 7.04, 2H), 7.21 (t, J ¼ 2.94, 2H), 7.19 (d, J ¼ 4.44, 2H), 7.10
(t, J ¼ 7.42, 1H), 7.02 (t, J ¼ 7.44, 1H), 4.66 (t, J ¼ 6.72, 1H), 4.42 (t,
J ¼ 6.72, 1H), 4.34e4.30 (m, 1H), 4.25 (s 2H), 3.27e3.15 (m, 3H),
3.11e3.03 (m, 3H), 1.85e1.79 (m, 1H), 1.66e1.44 (m, 4H), 1.36 (s,
9H); 13C NMR [100 MHz, CD3OD]: d 173.0, 171.8, 171.6, 157.1, 156.3,
148.0, 145.0, 144.2, 139.0, 138.2, 136.6, 130.4, 128.8, 128.2, 128.1,
127.8, 127.2, 126.9, 126.9, 126.8, 126.7, 123.4, 121.5, 121.2, 118.7, 118.0,
117.8, 111.1, 108.7, 79.8, 54.7, 53.6, 53.0, 42.6, 40.4, 30.5, 29.3, 28.5,
27.1, 24.7; HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 839.4357,
found: 839.4339; HPLC: tR ¼ 27.5 min, 99.8% purity.
N-a-Boc-L-His[(2-p-(n-butylphenyl)]-L-Trp-L-Arg-NHBzl
(13f). Yield: 64%. 1H NMR [400 MHz, CD3OD]: d 7.93e7.82 (m, 2H),
7.56 (d, J ¼ 7.68, 1H), 7.50e7.45 (m, 3H), 7.34e7.31 (m, 1H),
7.28e7.18 (m, 6H), 7.10 (t, J ¼ 7.54, 1H), 7.02 (t, J ¼ 7.08, 1H), 4.65 (t,
J ¼ 6.74, 1H), 4.40 (t, J ¼ 6.62, 1H), 4.33e4.29 (m, 1H), 4.24 (d,
J ¼ 7.72, 2H), 3.42e3.36 (m, 1H), 3.25 (d, J ¼ 6.68, 1H), 3.18e3.08 (m,
3H), 3.03e2.97 (m, 1H), 2.75e2.70 (m, 2H), 1.82e1.78 (m, 1H),
1.66e1.61 (m, 3H), 1.42e1.41 (m, 2H), 1.37 (s, 9H), 1.29 (s, 2H),
0.98e0.93 (m, 3H); 13C NMR [100 MHz, CD3OD]: d 173.3, 172.9,
171.8, 157.1, 156.4, 148.1, 144.6, 138.2, 136.5, 130.1, 129.5, 128.1, 127.0,
126.8, 126.3, 126.2, 123.4, 121.2, 120.2, 118.7, 117.8, 117.6, 111.1, 108.7,
79.8, 54.7, 53.0, 51.7, 42.6, 40.4, 35.0, 33.0, 29.3, 28.5, 27.1, 24.7, 21.9
12.8; HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 819.4670,
found: 819.4665; HPLC: tR ¼ 28.4 min, 99.8% purity.
L-His-L-Trp-L-Arg-OMe (14a). Yield: 58%. 1H NMR [400 MHz,
CD3OD]: d 8.85 (br. s, 1H), 7.64e7.44 (m, 2H), 7.35e7.26 (m, 2H),
7.10e7.02 (m, 2H), 4.49 (br. s, 2H), 4.27 (br. s, 1H), 3.68 (s, 3H),
3.48e3.45 (m, 2H), 3.25e3.19 (m, 4H), 2.02e1.66 (m, 4H); 13C NMR
[100 MHz, CD3OD]: d 171.8, 168.7, 167.4, 157.1, 132.1, 127.8, 126.2,
123.8, 121.1, 119.0, 118.5, 117.8, 111.0, 52.3, 51.7, 51.7, 40.7, 29.3, 27.4,
26.4, 24.8; HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 512.2734,
found: 512.2726; HPLC: tR ¼ 15.0 min, 95.8% purity.
L-His(2-phenyl)-L-Trp-L-Arg-OMe (14b). Yield: 65%. 1H NMR
[400 MHz, CD3OD]: d 7.85 (t, J ¼ 7.48, 2H), 7.71e7.64 (m, 3H)
7.50e7.40 (m, 3H), 7.18 (t, J ¼ 7.28, 2H), 7.08 (t, J ¼ 7.16, 1H), 4.61 (t,
J ¼ 7.80, 1H), 4.37e4.33 (m, 1H), 4.00e3.96 (m, 1H), 3.52 (s, 3H),
3.38e3.34 (m, 2H), 3.21e3.13 (m, 2H), 2.98e2.89 (m, 2H), 1.50e1.39
(m, 2H), 1.25e1.16 (m, 2H); 13C NMR [100 MHz, CD3OD]: d 171.2,
170.9, 166.0, 154.9, 143.9, 134.5, 131.2, 128.1, 125.4, 125.2, 124.6,
13
European Journal of Medicinal Chemistry 223 (2021) 113635
122.9, 120.8, 120.2, 117.8, 117.6, 116.3, 110.2, 106.4, 53.4, 51.1, 50.8,
50.2, 38.7, 26.4, 25.4, 24.8, 22.3; HRMS(ESI-TOF): calculated for m/z
[M þ Hþ]: 588.3047, found: 588.3029; HPLC: tR ¼ 17.8 min, 96.1%
purity.
L-His(2-tolyl)-L-Trp-L-Arg-OMe (14c). Yield: 67%. 1H NMR
[400 MHz, CD3OD]: d 7.89 (br. s, 2H), 7.57e7.55 (m, 2H), 7.46 (br. s,
2H), 7.30 (d, J ¼ 7.56, 1H), 7.23e7.20 (m, 1H), 7.06 (t, J ¼ 6.56, 1H),
6.98 (t, J ¼ 5.34, 1H), 4.73e4.71 (m, 2H), 4.30 (br. s, 1H), 3.63 (s, 3H),
3.14e2.83 (m, 6H), 2.45 (s, 3H), 1.67e1.50 (m, 4H); 13C NMR
[100 MHz, CD3OD]: d 172.9, 172.4, 171.6, 157.5, 149.3, 143.5, 136.6,
134.1, 130.2, 128.0, 127.4, 127.2, 127.1, 118.5, 117.1, 116.9, 111.0, 108.6,
58.7, 53.3, 52.4, 51.8, 40.8, 30.5, 29.3, 29.1, 22.2, 20.4; HRMS(ESI-
TOF): calculated for m/z [M þ Hþ]: 602.3203, found: 602.3198;
HPLC: tR ¼ 18.6 min, 95.6% purity.
L-His(2-ethylphenyl)-L-Trp-L-Arg-OMe (14d). Yield: 64%. 1H
NMR [400 MHz, CD3OD]: d 7.95e7.88 (m, 2H), 7.61e7.51 (m, 4H),
7.34 (d, J ¼ 8.00, 1H), 7.24 (s, 1H), 7.10 (t, J ¼ 7.40, 1H), 7.01 (t, J ¼ 7.30,
1H), 4.54 (t, J ¼ 6.00, 1H), 4.32 (br. s, 1H), 3.65 (s, 3H), 3.53e3.42 (m,
3H), 3.23e3.17 (m, 1H), 3.09e3.03 (m, 2H), 2.81e2.74 (m, 2H)
1.76e1.65 (m, 2H), 1.56e1.50 (m, 1H), 1.45e1.37 (m, 1H), 1.32e1.26
(m, 3H); 13C NMR [100 MHz, CD3OD]: d 173.3, 171.7, 167.4, 157.1,
149.7, 145.6, 136.6, 129.1, 129.0, 127.1, 126.7, 123.7, 121.1, 119.6, 118.5,
117.8, 111.0, 108.5, 54.8, 53.1, 52.3, 51.7, 40.7, 29.2, 28.5, 28.2, 26.7,
24.7, 14.4; HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 616.3360,
found: 616.3357; HPLC: tR ¼ 19.9 min, 97.0% purity.
L-His(2-biphenyl)-L-Trp-L-Arg-OMe (14e). Yield: 56%. 1H NMR
[400 MHz, CD3OD]: d 8.10 (br. s, 2H), 7.93 (br. s, 2H), 7.73 (br. s, 2H),
7.64e7.59 (m, 2H), 7.50e7.42 (m, 3H), 7.32e7.25 (m, 2H), 7.07 (t,
J ¼ 7.32, 1H), 6.98 (br. s, 1H), 4.48e4.45 (m, 1H), 4.33 (br. s, 2H), 3.62
(s, 3H), 3.21e3.16 (m, 3H), 3.04e3.01 (m, 3H), 1.72e1.51 (m, 4H); 13C
NMR [100 MHz, CD3OD]: d 176.2, 172.5, 171.1, 155.0, 146.8, 140.1,
138.8, 137.4, 131.3, 129.1, 129.0, 128.3, 127.6, 127.5, 127.5, 127.4, 126.7,
125.8, 123.6, 121.7, 119.0, 117.7, 111.7, 107.8, 54.5, 53.3, 52.5, 52.2,
40.1, 30.2, 29.7, 27.7, 25.0; HRMS(ESI-TOF): calculated for m/z
[M þ Hþ]: 664.3360, found: 664.3345; HPLC: tR ¼ 20.9 min, 96.8%
purity.
L-His[(2-p-(n-butylphenyl)]-L-Trp-L-Arg-OMe (14f). Yield:
55%. 1H NMR [400 MHz, CD3OD]: d 7.93 (d, J ¼ 7.68, 2H), 7.59 (d,
J ¼ 7.76, 1H), 7.45 (s, 1H), 7.49 (d, J ¼ 7.64, 2H), 7.35 (d, J ¼ 8.00, 1H),
7.26 (d, J ¼ 6.80, 1H), 7.09 (t, J ¼ 7.38, 1H), 7.01 (t, J ¼ 7.32, 1H), 4.72
(t, J ¼ 7.12, 1H), 4.33e4.30 (m, 2H), 3.63 (s, 3H), 3.54e3.43 (m, 2H),
3.23e3.18 (m, 1H), 3.09e3.02 (m, 2H), 2.74 (t, J ¼ 7.38, 2H),
1.73e1.71 (m, 1H), 1.69e1.63 (m, 3H), 1.53e1.49 (m, 2H), 1.41e1.35
(m, 2H), 0.95 (t, J ¼ 7.48, 3H); 13C NMR [100 MHz, CD3OD]: d 173.3,
171.8, 167.5, 157.0, 148.3, 145.6, 136.5, 129.5, 127.1, 126.9, 126.5, 123.7,
121.1, 120.1, 119.4, 118.5, 117.8, 111.0, 108.5, 54.88, 52.4, 51.7, 51.6,
40.5, 35.1, 33.1, 28.1, 27.3, 26.5, 24.6, 21.8, 12.9; HRMS(ESI-TOF):
calculated for m/z [M þ Hþ]: 644.3673, found: 644.3660; HPLC:
tR ¼ 22.3 min, 97.1% purity.
L-His-L-Trp-L-Arg-NHBzl (15a). Yield: 62%. 1H NMR [400 MHz,
CD3OD]: d 8.81 (br. s, 1H), 7.64 (d, J ¼ 7.04, 1H), 7.49e7.43 (m, 2H),
7.33e7.23 (m, 6H), 7.08 (t, J ¼ 7.16, 1H), 7.00 (t, J ¼ 7.10, 1H),
4.41e4.24 (m, 5H), 3.78e3.63 (m, 1H), 3.43 (br. s, 1H), 3.15 (br. s,
3H), 2.81 (s, 1H), 1.84e1.58 (m, 4H); 13C NMR [100 MHz, CD3OD]:
d 171.6, 170.0, 167.4, 157.1, 138.3, 136.6, 134.6, 128.1, 127.1126.8, 121.3,
118.6, 118.1, 111.0, 108.7, 54.8, 51.7, 42.6, 29.3, 28.7, 27.4, 26.4, 24.9;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 587.3206, found:
587.3196; HPLC: tR ¼ 20.2 min, 95.4% purity.
L-His(2-phenyl)-L-Trp-L-Arg-NHBzl (15b). Yield: 66%. 1H NMR
[400 MHz, CD3OD]: d 7.92e7.85 (m, 2H), 7.75e7.63 (m, 4H),
7.49e7.31 (m, 6H), 7.18e7.09 (m, 4H), 4.36 (br. s, 1H), 4.05 (s, 2H),
3.93e3.87 (m, 2H), 3.50e3.46 (m, 1H), 3.24e3.06 (m, 3H), 2.91 (br.
s, 2H), 1.52e1.34 (m, 2H), 1.21e1.13 (m, 2H); 13C NMR [100 MHz,
CD3OD]: d 172.8, 171.9, 167.7156.4, 145.4, 137.6, 135.9, 132.7, 129.7,
128.6, 127.4, 127.1, 127.0, 126.6, 126.3, 124.3, 122.3, 122.1, 119.5, 119.3,
K.K. Sharma, R. Ravi, I.K. Maurya et al.
118.0, 111.9, 108.0, 54.9, 53.6, 51.9, 42.9, 40.4, 28.3, 26.9, 26.4, 24.1;
HRMS(ESI-TOF): calculated for m/z [M þ Hþ]: 663.3519, found:
663.3510; HPLC: tR ¼ 20.7 min, 96.8% purity.
L-His(2-tolyl)-L-Trp-L-Arg-NHBzl (15c). Yield: 63%. 1H NMR
[400 MHz, CD3OD]: d 7.87 (d, J ¼ 7.76, 2H), 7.51e7.44 (m, 4H), 7.34
(d, J ¼ 8.00, 1H), 7.29e7.22 (m, 4H), 7.16e7.09 (m, 3H), 7.02 (t,
J ¼ 7.78, 1H), 4.32 (t, J ¼ 6.14, 2H), 4.20e4.12 (m, 3H), 3.49e3.39 (m,
2H), 3.16 (d, J ¼ 6.92, 2H), 3.06 (t, J ¼ 6.76, 2H), 2.44 (s, 3H),
1.75e1.72 (m, 2H), 1.63e1.59 (m, 2H); 13C NMR [100 MHz, CD3OD]:
d 174.9, 171.0, 168.8, 156.1, 145.6, 139.0, 138.2, 130.1, 128.1, 128.0,
126.9, 126.8, 126.7, 126.5, 123.6, 121.2, 118.6, 117.9, 117.7, 111.0, 108.6,
57.0, 55.4, 53.5, 42.6, 40.6, 31.7, 29.3, 28.7, 24.7, 20.0; HRMS(ESI-
TOF): calculated for m/z [M þ Hþ]: 677.3676, found: 677.3670;
HPLC: tR ¼ 20.9 min, 96.1% purity.
L-His(2-ethylphenyl)-L-Trp-L-Arg-NHBzl (15d). Yield: 61%. 1H
NMR [400 MHz, CD3OD]: d 7.93 (br. s, 2H), 7.54e7.47 (m, 4H),
7.31e7.18 (m, 7H), 7.06e6.98 (m, 2H), 4.56 (br. s, 1H), 4.41e4.20 (m,
4H), 3.31 (br. s, 2H), 3.20e2.92 (m, 4H), 2.74 (br. s, 2H), 1.77e1.65
(m, 2H), 1.53e1.42 (m, 2H), 1.25 (s, 3H); 13C NMR [100 MHz,
CD3OD]: d 174.2, 173.0, 172.6, 157.2, 155.3, 149.7, 138.3, 136.6, 129.1,
128.1, 127.0, 126.8, 121.3, 111.1, 108.5, 57.1, 53.5, 51.9, 42.7, 40.9, 28.7,
28.5, 27.4, 27.1, 24.9, 21.5, 14.6; HRMS(ESI-TOF): calculated for m/z
[M þ Hþ]: 691. 3832, found: 691.3826; HPLC: tR ¼ 22.8 min, 95.6%
purity.
L-His(2-biphenyl)-L-Trp-L-Arg-NHBzl (15e). Yield: 58%. 1H
NMR [400 MHz, CD3OD]: d 7.75 (d, J ¼ 7.92, 2H), 7.55 (d, J ¼ 5.68,
2H), 7.43 (d, J ¼ 6.76, 3H), 7.29 (d, J ¼ 6.76, 3H), 7.16e7.07 (m, 6H),
6.92e6.87 (m, 3H), 6.73 (t, J ¼ 7.34, 1H), 4.63 (t, J ¼ 7.90, 1H), 4.33 (t,
J ¼ 6.76, 1H), 3.94 (t, J ¼ 6.76, 1H), 3.78 (s, 2H), 3.50e3.42 (m, 1H),
3.22e3.11 (m, 2H), 2.96 (br. s, 1H), 2.81 (br. s, 2H), 1.51e1.50 (m, 1H),
1.39e1.38 (m, 1H), 1.24e1.22 (m, 1H), 1.14e1.12 (m, 1H); 13C NMR
[100 MHz, CD3OD]: d 171.5, 170.2166.2154.7, 143.1, 142.5, 136.7,
135.9, 134.1, 127.5, 126.8, 126.0, 125.7, 125.6, 125.3, 125.2, 125.0,
124.7, 122.5, 120.1, 119.3, 118.0, 117.6, 116.1, 110.2, 106.2, 53.4, 52.0,
50.3, 41.1, 38.8, 26.6, 25.1, 25.0, 22.6; HRMS(ESI-TOF): calculated for
m/z [M þ Hþ]: 739.3832, found: 739.3827; HPLC: tR ¼ 23.4 min,
96.2% purity.
L-His[(2-p-(n-butylphenyl)]-L-Trp-L-Arg-NHBzl (15f). Yield:
67%. 1H NMR [400 MHz, CD3OD]: d 7.89 (d, J ¼ 8.04, 2H), 7.55e7.42
(m, 5H), 7.34 (d, J ¼ 7.46, 1H), 7.28e7.21 (m, 3H), 7.15e7.08 (m, 3H),
7.01 (t, J ¼ 7.44, 1H), 4.52 (t, J ¼ 6.72, 1H), 4.33 (t, J ¼ 6.12, 1H), 4.18 (t,
J ¼ 7.20, 1H), 4.10 (s, 2H), 3.52e3.45 (m, 2H), 3.20 (d, J ¼ 7.12, 2H),
3.06 (t, J ¼ 6.52, 2H), 2.74e2.67 (m, 2H), 1.76e1.70 (m, 1H),
1.61e1.56 (m, 4H), 1.36e1.30 (m, 3H), 0.91 (t, J ¼ 7.34, 3H); 13C NMR
[100 MHz, CD3OD]: d 174.6, 173.2, 169.1, 158.2, 149.8, 146.9, 139.3,
137.7, 131.1, 131.1, 129.7, 128.4, 128.2, 127.9, 125.2, 122.9, 121.3, 121.3,
120.7, 120.3, 119.3, 112.8, 109.6, 56.4, 55.0, 53.2, 44.0, 41.9, 36.4, 34.4,
29.7, 28.4, 27.9, 25.9, 23.2, 14.4; HRMS(ESI-TOF): calculated for m/z
[M þ Hþ]: 719.4145, found: 719.4131; HPLC: tR ¼ 24.5 min, 95.4%
purity.
4.2. FITC labelling of peptides
In a dry round bottom flask, pyridine/DMF/dichloromethane
(12:7:5, 1 mL), 14f or 12a (0.012 mmol, 1 equiv.) was added at
ambient temperature, and then FITC (1.2 equiv.) was added. The
reaction mixture was stirred for 15 h at room temperature. Reaction
mixture was conc under vacuum and purified using CH2Cl2:me-
thanol (95e92:5e8) using neutral alumina to get FITC-labeled
peptides (14f and 12a).
4.3. Assay for in vitro antimicrobial activity
The antimicrobial activities of all synthesized peptides (12a-f
and 15a-f) were evaluated against the fungal strains of Candida
14
European Journal of Medicinal Chemistry 223 (2021) 113635
albicans (ATCC 10231), Aspergillus fumigatus (ATCC 1022), Crypto-
coccus neoformans (ATCC 90113), and bacterial strains of MRSA
(ATCC 33591), Escherichia coli (ATCC 35218), Klebsiella pneumoniae
(ATCC BAA-1705), Pseudomonas aeruginosa (ATCC 27853), and VRE
(ATCC MP-1), at the highest concentration of 20 mg/mL. Suscepti-
bility of bacterial and fungal to the tested peptides was evaluated
according to a modified CLSI protocol. For pharmacological evalu-
ation, all tested peptides through serial dilution in 20% DMSO/sa-
line, added in duplicate to 96-well plates, and inocula were made
by correcting the OD630 of microbe suspensions in incubation
broth [Sabouraud Dextrose (Difco) for C. neoformans, cation
adjusted Mueller-20 Hinton (Difco) at pH 7.3 for non-mycobacterial
bacteria to afford: C. neoformans 1.5  103 CFU/mL, non-
mycobacterial bacteria 5  105 CFU/mL, and for detailed test pro-
cedure see SI [61].
4.4. Cytotoxicity assay
Cytotoxicity of tested synthetic tripeptides were analyzedin a
mammalian cell line to evaluate itssafety profile. All cells were
obtained from ATCC, and the in vitro mammalian cell cytotoxicity of
these synthetic peptides was carried out against non-cancerous
kidney cells VERO. 25,000 cells/well were seeded and incubated
for 24 h for checked for confluency. Tested synthetic peptide
samples were added, and the plates were again incubated for 48 h.
The number of viable cells were established according to the
modified version of neutral red assay, and doxorubicin and DMSO
were used as the positive and negative control, respectively, and for
detailed test procedure see SI [52].
4.5. Tryptophan quenching study
For quenching study, L-a-phosphatidylcholine (EYPC), L-a-
phosphatidyl-D,L-glycerol (EYPG), acrylamide and cholesterol were
procured from Sigma Aldrich. Briefly, SUVs were prepared consist
of EYPC, EYPG and/or cholesterol, and the bacterial SUVs (EYP-
C:EYPG, 7:3) and mammalian cell membranes SUVs (EYPC:choles-
terol, 10:1) developed by dissolving in chloroform, and followed by
air dying. The fluorescence quenching was calculated using Stern-
Volmer equation: F0/F ¼ 1 þ KSV [Q] and for detailed test pro-
cedure see SI [54,55].
4.6. Dynamic light scattering study
Hydrodynamic sizes of the synthetic peptides was assessed
through a Zetasizer nano-ZS (Malvern Instruments, UK). For size
analysis, peptides (1 mg) were dissolved in PBS pH 7.4 (1.0 mL) by
ultrasonic treatment and run for measurements at specified time
intervals. Experimental recordings were conducted at 25  C (90
and 60 s), and the viscosity and refractive index were considered of
water 0.89 cp and 1.330, respectively. All readings were taken in
triplicates and an average of the three was compiled as the final
reading for further observations.
4.7. Fluorescence spectroscopic study
A fluorescence quenching experiment for determining trypto-
phan aggregation and stacking was evaluated with the Cary Eclipse
spectrofluorometer a magnetically stirred at 25  C (300 mL cuvette,
1 cm) and thermostated cuvette using Ex 280 nm (bandwidth
5 nm). Here, 600 nm/min scan speed was used with the emission
scan from 300 to 600 nm (bandwidth 10 nm). and the average of
three scans was used with 5 min of equilibration period.
K.K. Sharma, R. Ravi, I.K. Maurya et al.
4.8. SEM study
Sample preparation for this study was carried out by using the
peptides 14f and 12a (1 mg). Peptides were suspended in PBSpH 7.4
(1.0 mL) by ultrasonic irradiation and incubated at 37  C for 24 h.
The peptide solutions were placed on the coverslips previously
attached to the SEM holder adhered by adhesive tape, and samples
were dried with carbon dioxide up to the critical point. In last step,
sample preparations used sputter gold coating, and finally peptides
were examined using SEM (Hitachi Se3400 N, Japan) [31].
4.9. CLSM and electron microscopic study
In confocal and electron microscopic imaging studies,
C. neoformans (ATCC350) cells (~1  104 CFU/mL) were used in a
RPMI-1640 medium containing peptide 14f at the MIC.
For PI study, after incubation (1 h, 30  C, shaking 200 rpm) with
14f, fungal cells were collected by centrifugation, and resuspended
in PBS (pH 7.4). Next, the funal cellsamples were observed by
confocal microscope at >560 nm wavelength (Olympus Fluoview
FV1000 SPD; Japan). Untreated cryptococcal cells served as a con-
trol in this study.
For DAPI study, after incubation, fungal cells were collected by
centrifugation and resuspended in PBS, and DAPI added (3.01 mM,
10 min). The untreated fungalcells used for confocal imaging with
DAPI used as a control. For confocal imaging with DAPI, samples
were examined with Ex/Em; 350/470 nm [31,62].
For FITC-labeled peptide study, MIC of FITC labeled peptides was
also considered same as its parent peptide. After incubation with
labeled peptides for 1 h, fungal cells were collected and resus-
pended in PBS, then examined under confocal microscope (Ex/Em;
488/515 nm). In this study, 12a served as control for examination of
fungal cells [31,63].
For morphological examination using SEM, the prepared sam-
ples were dehydrated, and then protected with coverslip with
hexamethyldisilizane (HMDS), and samples were dried at 25  C,
and then followed by gold coating and examined using the SEM
(JEOL 6100) [31,64]. For ultrastructure study, semithin and ultrathin
section was made using ultramicrotome (Ultramicotome Lecia EM
UC6), and then sections of treated and control samples were placed
on the copper grid of 3.05 mm diameter (200 mess). Staining was
done with uranyl acetate and lead acetate, and finally observations
made usingthe HRTEM (FEI Technai G2 F20, 120 kV) [31,65], for
detailed test procedure please see SI.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgements
K.K.S. thanks the University Grant Commission (UGC), New
Delhi (India) for the award of senior research fellowship (UGC-NET-
SRF).
Abbreviations
CAMP cationic antimicrobial peptide
AFP antifungal peptides
DIEA N,N-diisopropylethylamine
CDI 1,10 -Carbonyldiimidazole
DIC N,N0 -diisopropylcarbodiimide
HOBt 1-Hydroxybenzotriazole
15
European Journal of Medicinal Chemistry 223 (2021) 113635
MIC minimum inhibitory concentration
Amp B amphotericin B
CTX cytotoxicity
SI selectivity index
MRSA Methicillin-resistant S. aureus
VRE Vancomycin-resistant Enterococcus
PBS phosphate buffer saline
PI propidium iodide
FITC fluorescein isothiocyanate
DAPI 40 ,6-diamidino-2-phenylindole
Cpx ciprofloxacin
SEM scanning electron microscopy
CLSM confocal laser scanning microscopy
Ex exciataion
Em emission
HRTEM high resolution transmission electron microscopy
HMDS hexamethyldisilizane
RP-HPLC reverse-phase high pressure liquid chromatography
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113635.
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