Discovery of a Membrane-Active, Ring-Modified Histidine Containing Ultrashort Amphiphilic Peptide That Exhibits Potent Inhibition of Cryptococcus neoformans

Article abstract of DOI:10.1021/acs.jmedchem.7b00481

The new structural classes of ultrashort peptides that exhibit potent microbicidal action have potential as future drugs. Herein, we report that C-2 arylated histidines containing tripeptides His(2-Ar)-Trp-His(2-Ar) exhibit potent antifungal activity against Cryptococcus neoformans with high selectivity. The most potent peptide 12f [His(2-biphenyl)-Trp-His(2-biphenyl)] displayed high in vitro activity against C. neoformans (IC₅₀ = 0.35 μg/mL, MIC = MFC = 0.63 μg/mL) with a selectivity index of >28 and 2 times higher potency compared to amphotericin B. Peptide 12f exhibited proteolytic stability, with no apparent hemolytic activity. The mechanism of action study of 12f by confocal laser scanning microscopy and electron microscopy indicates nuclear fragmentation and membrane disruption of C. neoformans cells. Combinations of 12f with fluconazole and amphotericin B at subinhibitory concentration were synergistic against C. neoformans. This study suggests that 12f is a new structural class of amphiphilic peptide with rapid fungicidal activity caused by C. neoformans.

Full text of DOI:10.1021/acs.jmedchem.7b00481

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Article
Discovery of a Membrane-Active, Ring-Modified
Histidines Containing Ultra Short Amphiphilic Peptide that
Exhibits Potent Inhibition of Cryptococcus neoformans
Krishna K Sharma, Indresh K. Maurya, Shabana I. Khan, Melissa
R Jacob, Vinod Kumar, Kulbhushan Tikoo, and Rahul Jain
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00481 • Publication Date (Web): 11 Jul 2017
Downloaded from http://pubs.acs.org on July 11, 2017
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Discovery of a Membrane-Active, Ring-Modified Histidines
Containing Ultra Short Amphiphilic Peptide that Exhibits Potent
Inhibition of Cryptococcus neoformans
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Krishna K. Sharma,a Indresh Kumar Maurya,b Shabana I. Khan,c Melissa R. Jacob,c Vinod Kumar,d
Kulbhushan Tikoo,d and Rahul Jaina*
aDepartment of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research,
Sector 67, S. A. S. Nagar, Punjab, 160 062, India
bDepartment of Microbial Biotechnology, Panjab University, Sector 25, Chandigarh, 160 014, India
cNational Center for Natural Products Research, School of Pharmacy, The University of Mississippi,
University, Mississippi 38677, USA
dDepartment of Pharmacology and Toxicology, National Institute of Pharmaceutical Education and
Research, Sector 67, S. A. S. Nagar, Punjab, 160 062, India
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ABSTRACT
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The new structural classes of ultra short peptides that exhibit potent microbicidal action have
potential as future drugs. Herein, we report that Cꢀ2 arylated histidines containing tripeptides, His(2ꢀ
Ar)ꢀTrpꢀHis(2ꢀAr) exhibit potent antifungal activity against Cryptococcus neoformans with high
selectivity. The most potent peptide 12f [His(2ꢀbiphenyl)ꢀTrpꢀHis(2ꢀbiphenyl)] displayed high in
vitro activity against C. neoformans (IC50 = 0.35 µg/mL, MIC = MFC = 0.63 µg/mL) with a
selectivity index of >28, and two times higher potency compared to amphotericin B. Peptide 12f
exhibited proteolytic stability, with no apparent hemolytic activity. The mechanism of action study
of 12f by confocal laser scanning microscopy and electron microscopy indicate nuclear
fragmentation and membrane disruption of C. neoformans cells. Combinations of 12f with
fluconazole and amphotericin B at sub inhibitory concentration were synergistic against C.
neoformans. This study suggests that 12f is a new structural class of amphiphilic peptide with rapid
fungicidal activity caused by C. neoformans.
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Introduction
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In recent years, the incidences of invasive fungal infections such as cryptococcosis, candidiasis and
aspergillosis have increased rapidly and caused morbidity and mortality in immunoꢀcompromised
situations such as burn, acquired immune deficiency syndrome (AIDS) cancer and organ
transplant.1ꢀ2 The infections caused by the yeast Cryptococcus neoformans is a human opportunistic
fungal pathogen, responsible for approximately 600,000 deaths per year worldwide.3 In the past two
decades, rapidly increasing antifungal drug resistance has emerged as a serious threat to public
health, and a major challenge in search of novel antifungals with distinct mode of actions. In
disparity to the steep rise in invasive fungal infections, there is a dearth of novel and new classes of
clinically approved antifungal activity specific drugs.4 The invasive antifungal treatments relied on
five main classes of marketed drugs: azoles (fluconazole), polyenes (amphotericin B),
fluoropyrimidines (5ꢀfluorocytosine), allyamines (terbinafine) and echinocandins (caspofungin).5
Along with the limited repertoire of the marketed antifungals, the drawbacks such as the
development of resistance to the pathogen, acute or chronic side effects, low clinical efficacy owing
to poor pharmacokinetics, complex drug interactions with the host cells, poor bioavailability and
low therapeutic index created a need for the discovery of new structural classes of synthetic
antifungal agents.6 The need is to discover new antifungal agents with potent fungicidal activity,
target specificity, rapid time kill, unique mechanism of action, and absence of crossꢀresistance with
the clinically available antifungal drugs.7
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The antifungal drug discovery is considered more challenging compared to antibacterial drug
discovery because fungi are metabolically similar to mammalian cells except cell wall, resulting in
few pathogen specific targets.8 The quest for new antifungal development is important because
extensively used antifungal drugs (amphotericin B and fluconazole) have developed resistance
against the prevalent fungal pathogens such as Candida albicans and C. neoformans.9 In such a
scenario, cationic antimicrobial peptides (CAMPs) offer a promising alternative for the development
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of synthetic antifungals.10 In the past decade, several types of CAMPs have been reported to show
efficacy against various fungal infections. The CAMPs are generally six to fifty amino acid residues
in length and exist in structural conformations, including helix, sheet extended and looped
structures. They possesses two common structural features, a net positive charge and
amphiphilicity.11 The positive charge is known to facilitates interaction with the negatively charged
microbial surface, while amphiphilicity allows an amphipathic secondary structure that permits the
incorporation of CAMPs into the lipid bilayer of microbial membranes.12 It has been well
established that CAMPs could alter the cell membrane functions through increased permeability, but
it is not their sole mode of action.
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Peptides due to their large size have limited clinical potential because of high commercialization
cost. This drawback has drawn attention towards the development of short synthetic peptides as
clinically viable drugs.13 The regioselective functionalization of DNAꢀencoded amino acids leading
to the synthesis of enantiomerically pure modified amino acids is receiving attention recently. These
modified amino acids forms ideal scaffolds in the synthesis of new structural classes of short
synthetic peptides with high acceptability in drug discovery.14,15 The advent of ringꢀmodified
histidines as a core amino acid residue in short peptides have opened new avenues in the
development of potential proteostable antimicrobial pharmacophores.15 In nutshell, the short
synthetic cationic peptides are ideally suited in search of novel synthetic antimicrobial agents
because of their size, cost, and microbicidal mode of action.
As a part of our ongoing interest in developing ultra short CAMPs against infectious diseases, we
investigated the minimum prerequisite features in reported antifungal peptides.16 It was observed
that peptide motif bearing +2/+3 charge with sufficient lipophilicity results in promising antifungals.
A number of histidineꢀrich natural CAMPs such as histatins (Histꢀ3, and Histꢀ5) and clavanins, and
tryptophanꢀrich CAMPs such as indolicidin and tritrpticin are known to exhibit potent antifungal
activity.17,18 Because of the presence of imidazole side chain that displays buffering properties at
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physiological pH and amphiphilic character, heterocyclic amino acid histidine emerges as an
obvious choice in the design of synthetic antifungal peptide motif.19 The tryptophan residue on the
other hand provide lipophilicity and cell membrane interaction characteristics to the synthetic
peptides.19
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Figure 1. Generalized structure of the synthesized peptides
Upon the basis of aboveꢀmentioned observations, we envisioned to use amphiphilic ringꢀmodified Lꢀ
histidine and lipophilic tryptophan as core amino acids to develop a new antifungal structural motif.
Herein, we report the synthesis of ringꢀmodified histidine and tryptophan (Trp) residues containing
tripeptide motif as promising antifungal agents bearing +2/+3 charge, where additional lipophilicity
was provided by the aromatic moiety placed at the Cꢀ2 position of histidine. We observed that
incorporation of 2ꢀarylꢀLꢀhistidines in the tripeptide scaffold has greatly influenced the potency of
synthetic peptides. The most potent antifungal peptide 12f was further examined for hemolysis,
stability, time kill kinetics, mechanism of action and its synergistic combination with marketed
drugs against clinical isolates of human fungal pathogen C. neoformans. This investigation
demonstrates that the presence of a lipophilic aromatic moiety at the Cꢀ2 position of the imidazole
ring of histidine imparts desired amphiphilicity to His(2ꢀAr)ꢀTrpꢀHis(2ꢀAr) class of peptides,
thereby increasing potency and cell membrane disruption properties.
Results and Discussion
Synthesis
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Two structural types of peptides, one with a benzylamide group and another with a methyl ester
group at the Cꢀterminus were synthesized (Fig. 1). The synthesis required availability of building
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block amino acids in the suitably derivatized form. NꢀαꢀTrifluoroacetylꢀ2ꢀarylꢀ
Lꢀhistidine methyl
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esters (2a-e) were synthesized from Nꢀαꢀtrifluoroacetylꢀ ꢀhistidine methyl ester (1) utilizing a
L
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regiospecific CꢀH arylation reaction in the presence of arylboronic acids under ambient conditions in
moderate yields (Scheme 1).20 2ꢀArylꢀ
Lꢀhistidines (3a-e) possessing dual hydrophobicꢀhydrophilic
character were obtained by deprotection of 2a-e under acidic conditions at 100 oC for 12 h (Scheme
1).
Scheme 1. Synthesis of 2-aryl-L-histidines (3a-e)
The latter compounds 3a-e upon reaction with diꢀtertꢀbutyl dicarbonate under basic conditions for
24 h afforded Bocꢀ ꢀHis(Ar)ꢀOH (4a-f) in excellent yields (Scheme 2). The Bocꢀprotected
L
derivatives 4a-f upon reaction with benzylamine in the presence of coupling reagent 1,3ꢀ
diispropylcarbodiimide (DIC) and auxiliary nucleophile Nꢀhydroxyꢀ5ꢀnorborneneꢀ2,3ꢀ
o
dicarboximide (HONB) at 60 C for 30 min under microwave (MW) irradiation produced key
intermediates BocꢀLꢀHis(Ar)ꢀNHBzl (5a-f) in high yields (Scheme 2).
Scheme 2. Synthesis of His(2-Ar)-Trp-His(2-Ar)-NHBzl (8a-f)
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The designed peptides (series 1 and 2) were synthesized by adapting a rapid and highly efficient
microwave (MW)ꢀmediated solution phase peptide synthesis protocol.21 First, Bocꢀ
LꢀHis(Ar)ꢀ
NHBzl derivatives (5a-f) were deprotected in the presence of 6N HCl in methanol (MeOH) for 15
min at ambient temperature. The resulting intermediates were neutralized using N,Nꢀ
diisopropylethylamine (DIEA) and subsequently coupled with BocꢀTrpꢀOH in the presence of
o
coupling reagent combination of DIC and HONB at 60 C for 30 min under MW irradiation to
produce dipeptides (6a-f) in high yields. The dipetides (6a-f) were deprotected using a solution of
6N HCl in methanol (MeOH) for 15 min at ambient temperature. The deprotected dipeptides were
neutralized in situ using DIEA, and allowed next coupling step immediately with BocꢀLꢀHis(Ar)ꢀOH
(4a-f) in the presence of DIC and HONB under MW irradiation at 60 oC for 30 min to afford first set
of desired tripeptides (7a-f). The protected tripeptides (7a-f) upon acidolysis reaction in the presence
of 6N HCl in MeOH for 15 min at ambient temperature produced the second set of peptides (8a-f)
(Scheme 2).
For the peptides of series 2, 2ꢀarylꢀ
Lꢀhistidines (3a-f) upon esterification with MeOH in the presence
of HCl gas afforded 2ꢀarylꢀ ꢀhistidine methyl esters (9a-f) in high yields (Scheme 3). The latter
L
compounds 9a-f were neutralized using DIEA and subsequently reacted with BocꢀTrpꢀOH in the
o
presence of DIC and HONB in DMF under MW heating at 50 C for 30 min to produce dipeptides
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10a-f, which were deprotected under acidolysis conditions and subsequently neutralized in situ (in
the presence of DIEA) and subjected to next coupling step immediately.
Scheme 3. Synthesis of His(2-Ar)-Trp-His(2-Ar)-OMe (12a-f)
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The deprotected dipeptides upon reaction with BocꢀHis(2ꢀaryl)ꢀOH (4a-f) in the presence of DIC
and HONB in DMF under MW heating afforded Boc group protected tripeptides 11a-f, which under
acidolysis conditions produced free peptides 12a-f. All dipeptides and tripeptides were purified on
neutral alumina using a fully automated flash purification system.
In vitro antimicrobial activity
For a better understanding of the structure activity profiles of the synthetic peptides, both Boc
protected peptides (7a-f and 11a-f) and free peptides (8a-f and 12a-f) were evaluated for in vitro
activity against a panel of fungal (C. albicans, C. glabrata, C. krusei, Aspergillus fumigatus, and C.
neoformans) and bacterial (E. coli, S. aureus, and MRSA) strains. The results are summarized for C.
neoformans (table 1), S. aureus, and MRSA (table 2). The peptides were found to be inactive against
Candida, Aspergillus, and E. coli (data not shown). The MIC (minimum inhibitory concentration)
values of evaluated peptides were determined by using a protocol suggested by the Clinical and
Laboratory Standard Institute (CLSI) guidelines.22 The marketed drug amphotericin B, which is
used for the treatment of C. neoformans infections but has toxicity toward mammalian cells, was
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used as a positive control in antifungal evaluation, while ciprofloxacin was used as a standard drug
for antibacterial activity evaluation.23
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A clear pattern confirming the role of lipophilicity in antifungal activity was observed for peptides
7a-f that contained Boc and NHBzl groups at the Nꢀ and Cꢀterminus, respectively. We noted that an
incremental increase in the overall lipophilicity in the designed peptides is proportional to decrease
in activity. Peptide 7a (R = H) and peptide 7b (R = C6H5) exhibited IC50s of 0.80 ꢀg/mL and 1.40
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ꢀg/mL, respectively. At the same time, peptides 7c-f containing bulkier aryl groups produced IC50
values ranged between 12.61 – 14.0 ꢀg/mL or no inhibition of C. neoformans at the highest tested
concentration of 20 ꢀg/mL. This trend indicates that a combination of NHBzl and Boc group at both
termini and the presence of bulkier aryl group at the Cꢀ2 position of the imidazole ring results in
dramatic reduction in antifungal activity of this class of peptides. In contrast, peptides 8a-f obtained
directly from 7a-f by removal of Boc group at the Nꢀterminus exhibited a reverse trend. In these
cases, the absence of Boc group at the Nꢀterminus and an increase in lipophilicity affected by the
introduction of bulkier aryl group at the Cꢀ2 position of imidazole ring results in enhanced
antifungal activity.
Table 1. In vitro Antifungal Activities of Peptides
Peptide
no.
R
R1
R2
Cytotoxicity Selectivity
C. neoformans (ꢀg/mL)
(CTX)
Index (SI)
(ꢀg/mL)
IC50
0.80
1.40
14.0
12.61
>20
>20
8.1
MIC
1.25
2.50
>20
>20
–
–
10
–
>20
10
MFC
1.25
2.50
>20
>20
–
–
10
–
>20
10
H
C6H5
NHBzl Boc
NHBzl Boc
NHBzl Boc
NHBzl Boc
NHBzl Boc
NHBzl Boc
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>12.5
>7.1
–
–
–
–
>1.2
–
>1.4
>1.8
7a
7b
7c
7d
7e
7f
8a
8b
8c
8d
4ꢀOCH3ꢀC6H4
4ꢀCH3ꢀC6H4
4ꢀC(CH3)3ꢀC6H4
4ꢀC6H5ꢀC6H4
H
C6H5
4ꢀOCH3ꢀC6H4
4ꢀCH3ꢀC6H4
NHBzl
NHBzl
NHBzl
NHBzl
H
H
H
H
>20
7.2
5.56
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4ꢀC(CH3)3ꢀC6H4
4ꢀC6H5ꢀC6H4
H
NHBzl
NHBzl
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
H
2.60
1.40
>20
9.14
15.21
2.68
1.27
>20
9.81
3.71
2.47
1.67
0.83
0.35
0.69
5
2.50
–
>20
>20
5
5
–
20
10
5
2.50
–
>20
>20
5
5
–
20
10
5
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>3.8
>7.1
–
>1.1
–
>3.7
>7.9
–
>1.0
>2.7
>4.0
>6.0
>12.0
>28.6
8e
8f
Boc
Boc
Boc
Boc
Boc
Boc
H
H
H
H
H
11a
11b
11c
11d
11e
11f
12a
12b
12c
12d
12e
12f
C6H5
4ꢀOCH3ꢀC6H4
4ꢀCH3ꢀC6H4
4ꢀC(CH3)3ꢀC6H4
4ꢀC6H5ꢀC6H4
H
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C6H5
4ꢀOCH3ꢀC6H4
4ꢀCH3ꢀC6H4
4ꢀC(CH3)3ꢀC6H4
4ꢀC6H5ꢀC6H4
5
2.50
1.25
0.63
1.25
5
1.25
0.63
1.25
H
Amphotericin B
IC50, the concentration (µg/mL) that affords 50% inhibition of growth; MIC, 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 the organism; selectivity index
(CTX/IC50)
For example, peptide 8f (R = 2ꢀbiphenyl) exhibited inhibition of C. neoformans at IC50 = 1.40
ꢀg/mL, MIC = MFC = 2.50 ꢀg/mL, and was the most promising of series 1. These observations
confirms that a delicate balance of bulk to charge ratio is essential to retain and enhance antifungal
activity (Table 1).
In complete reversal of the results obtained for Boc protected peptides 7a-f (series 1), peptides 11a-f
(series 2) containing Boc and OMe groups at the Nꢀ and Cꢀterminus exhibited a linear increase in
activity with the increased lipophilicity induced by the aryl substitution on both histidine residues
with the exception of peptide 11f. The activity profiles of Bocꢀdeprotected peptides 12a-f follow the
trajectory of 8a-f, wherein an increase in lipophilicity results in gradual increment in activity.
Peptide 12a (R = H) produced IC50 of 9.81 ꢁg/mL, but peptide 12e containing a bulky 4ꢀtertꢀ
butylphenyl group at the Cꢀ2 position of the imidazole ring was found to be a promising inhibitor of
C. neoformans with IC50 value of 0.83 ꢀg/mL compared to 0.69 ꢁg/mL for amphotericin B. The
MIC and MFC values of 12e were 1.25 ꢁg/mL similar to that for amphotericin B. The peptide 12f
containing a biphenyl group at the Cꢀ2 position of the imidazole ring exhibited even better inhibition
of C. neoformans (IC50 = 0.35 ꢁg/mL or 0.40 ꢁM, MIC = MFC = 0.63 ꢁg/mL or 0.72 ꢁM) and
emerged as the most active peptide across both series. The in vitro activity data show that in
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addition to the substituents pattern at the Cꢀ2 position of the imidazoles ring, the nature of group at
both termini also greatly influenced the antifungal potency. For the Bocꢀprotected peptides, the
incorporation of a methyl ester group (peptides 11a-f) offer optimal tuning of lipophilicity compared
to a NHBzl group (peptides 7a-f) at the Cꢀterminus. For Bocꢀdeprotected free peptides (8a-f and
12a-f), increase in lipophilicity by the introduction of C2ꢀAr group in histidine residue results in
enhanced activity. In general, peptides with free Nꢀterminus (8a-f and 12a-f) demonstrated higher
potency compared to Boc protected peptides (7a-f and 11a-f).
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The peptides 7b, 8e, 12e and 12f also displayed antibacterial activity against S. aureus and
methicillinꢀresistant Staphylococcus aureus (MRSA) (Table 2). The peptide 12e exhibited IC50
value of 4.33 ꢁg/mL and MIC of 10 ꢁg/mL against S. aureus, while 12f showed promising IC50,
MIC, and MBC values 1.93, 5, and 10 ꢁg/mL, respectively against MRSA.
Table 2. In Vitro Antibacterial Activity of Peptides
Peptide no.
CTX
SI
CTX
SI
S. aureus (ꢀg/mL)
MRSA (ꢀg/mL)
(ꢀg/mL)
(ꢀg/mL)
IC50 MIC MBC
IC50
MIC MBC
7.60 >20
3.72 10
4.33 10
2.56 >20
>20
20
>20
>20
>10
>10
>10
>10
>1.3
>2.68 6.70
>2.3
>3.9
10.50 >20 >20
>10
>10
>10
>10
–
7b
8e
12e
12f
>20 >20
>20 >20
>1.49
>1.49
>5.18
6.71
1.93
0.09
5
10
Ciprofloxacin 0.08 0.25 0.50
0.25 0.50
IC50, the concentration (µg/mL) that affords 50% inhibition of growth; MIC, minimum inhibitory concentration is the
lowest test concentration (µg/mL) that allows no detectable growth; MBC, minimum bactericidal concentration is the
lowest test concentration (µg/mL) that kills the organism; CTX, cytotoxicity, SI, selectivity index (CTX/IC50)
Cytotoxicity evaluation
The absence of cytotoxic effects is one of the most essential criteria to determine the potential of
membrane targeted antimicrobial peptides. The selectivity towards antimicrobial activity by
synthetic peptides is a prerequisite as both humans and microbes belongs to sister clades in the tree
of eukaryotes. To determine the safety profile, synthesized peptides were evaluated for cytotoxic
study in a panel of cell lines. The in vitro cytotoxicity of the synthetic peptides was determined
against four human cancer cell lines (SKꢀMEL, KB, BTꢀ549, and SKꢀOVꢀ3) and two noncancerous
mammalian cells (VERO and LLCPK1) by neutral red uptake assay.24 The results shows that all
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synthetic peptides (series 1 and 2) were nontoxic upto a highest tested concentration of 10.0 ꢁg/mL,
which indicates a higher selectivity index (>1ꢀ to >28ꢀfold) for anticryptococcal activity and a
selective index (>1.3 to >5.8ꢀfold) for antibacterial. activity Therefore, it could be concluded that
activity exhibited by peptides is not due to a general cytotoxic effect of synthetic peptides.
Upon the basis of in vitro activity and cytotoxicity results, we selected peptide 12f for detailed
evaluation, including hemolytic effect, timeꢀkill kinetics, proteolytic stability, mechanism of action
investigations and synergistic action with known drugs.
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Hemolytic study
The hemolytic activity of the antifungal peptides is considered as a marker of selectivity towards
fungal cells, and determined upon the basis of amount of hemoglobin released from human red
blood cells (hRBCs). We reꢀexamined peptide 12f against a new C. neoformans strain (ATCC350),
wherein it exhibited MIC value of 0.39 µg/mL or 0.44 ꢁM. All subsequent studies were conducted
on C. neoformans strain (ATCC350). Varied concentrations of peptide 12f was incubated with
hRBCs and the release of hemoglobin was measured. Triton Xꢀ100 was used as a positive control
for this study.
Figure 2. Hemolytic activity of 12f against human RBCs. The 12f peptide was incubated with 0.4%
hRBCs in PBS. The standard deviation from three independent experiments is plotted.
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The peptide 12f was tested upto a highest test concentration of 100 µg/mL. The peptide 12f did not
exhibit any hemolysis at its MIC (Figure 2). At the highest test concentration of 100 µg/mL, peptide
12f exhibited exhibited only 17.74% hemolysis. It can be concluded that 12f has a nonꢀhemolytic
nature at its MIC value, and high selectivity to C. neoformans cells. Several earlier reports have
established a link between increases in hydrophobicity to increase in hemolytic effects, resulting in
assumption that the presence of amphiphilic histidine residues and tryptophan in the case of 12f
results in no hemolytic effects.25
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Time kill kinetics
There is always a need to discover rapid fungicidal agents to combat antifungal resistance. Peptide
12f showed potent fungicidal activity against C. neoformans and time kill kinetics is an important
method to judge the efficacy of investigational antimicrobials. For better understanding of fungicidal
nature, time kill kinetics experiments with 12f, amphotericin B (Amp B) and control peptide 11a
(inactive peptide) were performed.
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C. neoformans
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Negative control
8
6
4
2
0
Amp B
12f
0
4
8
12
16
20
24
Time (hours)
Figure 3. Time kill kinetic of the 12f, Amp B and negative control peptide 11a. The standard
deviation from three independent experiments is plotted.
In the time kill assay, the colony forming units (CFUs) of the C. neoformans were rapidly reduced
after treatment with 12f at the concentration of 0.39
ꢀg/mL (MIC). The complete killing of C.
neoformans cells in the presence of 12f was observed within 4 h. The known antifungal drug, Amp
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B at the concentration of 0.89 ꢀg/mL (MIC) also completely killed C. neoformans cells after 4 h of
incubation (Figure 3). However, the negative control peptide 11a did not show any growth
inhibition and exhibits the growth trajectory similar of the untreated control. Therefore, it could be
interpreted that peptide 12f successfully inhibited the growth of C. neoformans cells.
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Figure 4. RPꢀHPLC chromatograms of 12f untreated (a) and after 2 h of incubation with trypsin (b).
Proteolytic Stability
Proteolytic stability is one of the most crucial parameters in the discovery of peptideꢀbased
therapeutics.26 In general, natural peptides due to long sequences are highly suseptible to proteolytic
degradation as compared synthetic peptide containing modified amino acids and modified termini.
In order to examine the proteolytic stability, peptide 12f was digested with proteolytic enzyme
trypsin in a ratio of 100:0.5 mol/mol, and analyzed by RPꢀHPLC after 2h (Figure 4). There was no
observed change in the retention time and peak intensity of the trypsin treated 12f (Figure 4a) and
untreated 12f (Figure 4b). Hence it may be assumed that incorporation of modified amino acids and
modified terminus resulted in the stability of 12f against proteolysis.
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Mechanism of action investigations
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High fungicidal potency, no apparent cytotoxicity, proteolytic stability and rapid killing of C.
neoformans cells by 12f promoted us to examine its mechanism of action. The amphipathic peptides
are presumed to exhibit activity due to their microbial membranes disruptive properties, and thus
mechanism of action was investigated by using various microscopic visualization techniques. We
conducted the confocal laser scanning microscopy (CLSM) investigation to known the effect of 12f
on the membrane permeability and intracellular localization in fungal cells. The scanning electron
microscopy (SEM) study was performed to examine the effect of 12f on the C. neoformans cells
morphology, and the high resolution transmission electron microscopy (HRTEM) study allowed us
to visualize the effects of 12f on both cell wall and cell membrane of C. neoformans cells.
Confocal laser scanning microscopy.
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Propidium iodide (PI) is a red fluorescent DNA binding dye when excited at 480 nm. In the
presence of membrane active molecules, it permeabilizes cell wall of microbial cells resulting in
compromised membrane permeability. Under confocal microscopic examination, uptake of PI after
1 h by C. neoformans cells in the presence of 12f at its MIC revealed red fluorescence due to
permiabilization of cell membrane when excited by the PI filter at 488 nm laser (Figure 5B),
whereas the cells without 12f treatment did not show any fluorescence due to intact membranes
(Figure 5A). This confirms that PI enters inside the fungal cells treated with 12f possibly because C.
neoformans cells were not able to control the chemical traffic from the surface.
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Figure 5. Confocal micrographs of C. neoformans cells to assess the effect of 12f at 0.39 ꢀg/mL
(MIC) inside the treated cells. (i) membrane permeabilization assay using propidium iodide (PI)
after 1h; (ii) intracellular localization of FITCꢀlabelled 12f after 1 h and FITCꢀlabelled 8b (control
peptide); (iii) nuclear fragmentation effect of DAPI after 1 h. Images (A) and (E) fungal cells
incubated alone, and image (C) obtained with FITCꢀlabeled 8b, images (B), (D) and (F)
corresponding to 12f peptides treated cells after 1 h of incubation.
In order to understand the localization of 12f inside the C. neoformans cells, we synthesized FITCꢀ
labeled (fluorescein isothiocyanate; green fluorescent dye, excitation at 480 nm) 12f and control
peptide 8b. The examination of control peptide 8b suggests that it was not able to internalize inside
the cells as seen from the intact cell surface (Figure 5C). FITCꢀlabelled 12f upon incubation with C.
neoformans cells translocated inside Cryptococcus cells, after cell membranes permiabilization and
disruption as evident from the increased green fluorescence (Figure 5D). It could be presumed that
12f interacts with the intracellular targets and the killing of C. neoformans might be the result of
multiple effects.
DAPI (4',6ꢀdiamidinoꢀ2ꢀphenylindole) is a fluorescent dye commonly used to detect the nuclear
fragmentation of genetic materials in microbial cells under confocal microscopic examination. The
DAPI dye binds selectively to the DNA (minor groove of adenineꢀand thymineꢀrich sequence) so
that any changes in fluorescence intensity reflect the extent of damage to DNA caused by the
membrane disruptive peptides. DAPI staining of C. neoformans cells treated with 12f showed a high
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split blue fluorescence intensity (Figure 5F) within the cells, as compared to untreated cells (Figure
5E). Therefore, these results indicated that 12f interact with genetic material and causes nuclear
fragmentation resulting in the fast killing of C. neoformans cells after membrane damage.
Electron Microscopy.
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Scanning electron microscopy (SEM) study was performed to understand the cell surface
morphological changes in the C. neoformans upon treatment with 12f at its MIC (0.39 ꢀg/mL). The
untreated cryptococcal cells have clean smooth surface (Figures 6AꢀB) as compared to cells
treatment with 12f (Figures 6CꢀD). Peptide 12f induced cell wall breakage was visible with an
increase in the coarseness on the cells surface (Figures 6CꢀD). It is assumed that positively charged
peptide bind with the negatively charged cell surface of C. neoformans cells by electrostatic
interactions. The other possibility is that amphiphilic peptide may aggregates over cell surface,
resulting in its distortion. It is concluded that 12f treated C. neoformans cells were unable to sustain
under the collective effects induced via nonꢀspecific electrostatic interactions and amphiphilicity,
resulting in severe disruption of cell surface integrity and membrane permeabilization.
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Figure 6. Surface morphological study of C. neoformans cells with 12f and untreated fungal cells by
SEM. Images A and B represents morphological of untreated cells (7000X, 25mm; 5000X, 24mm);
images C and D represents fungal cells (8000X, 24mm; 3700X, 24mm) treated with 12f. The
peptide was used at its MIC.
In the TEM examinations, 12f treatment resulted in severe effect on the C. neoformans cell wall and
membrane as compared to untreated cells showing intact cell structure. Untreated fungal cells show
intact cell membranes under TEM examination (Figures 7AꢀC). The Figure 7A showed intact cell
morphology of many cells. On the other hand, Figures 7BꢀC focused on single cell visualization and
showed defined and intact cell wall and plasma membrane. The peptide 12f treated TEM images
show severe distruption in cell wall and cytoplasmic membrane resulting in fungal cell lysis
(Figures 7DꢀF).
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Figure 7. Morphological study of C. neoformans cells treated with 12f and untreated fungal cells by
high resolution transmission electron microscopy. Images A, B, and C represents untreated cells;
images D, E and F represents cells treated with 12f. The peptide was used at its MIC. The scale bar
for images A and D is 0.5 µm and for other images is 0.2 µm.
The confocal microscopic study using PI revealed that the peptide 12f permeabilizes cell membranes
of C. neoformans cells. Cellular uptake of FITCꢀlabeled 12f peptide inside the fungal cells indicated
that peptide binds with the multiple intracellular sites. The DAPI experiment demonstrates that 12f
after permiabilization inside C. neoformans cells binds with the fragmented genetic material cells,
resulting in inhibition. The SEM results indicate that peptide 12f interacts with negatively charged
cell surface of C. neoformans cells via electrostatic interactions, resulting in the disruption of their
surface integrity. Finally, the TEM results show that 12f act by severe disruption of both cell wall
and plasma membrane, which results in permeabilization inside cells and interaction with
intracellular sites, responsible for its rapid fungicidal nature. These mechanistic studies reveals that
the high antiꢀcryptococcal effect of potent peptide 12f is probably a multistep process involving
initial membrane permeabilization, cell wall and plasma membrane disruption, interaction with
genetic materials and other intracellular components of the targeted fungi cells, ultimately leading to
rapid cell death.
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Synergistic effect study of 12f with fluconazole and amphotericin B
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The emergence of resistance against marketed antifungal agents has renewed interest in the
evaluation of the combination therapy of antifungals. The use of new structural class of antifungals
with existing antifungal drugs acting via different mode of actions is a significant step in developing
more efficacious synergistic antifungal combinations and offers ways to combat drug resistance.27
For the synergistic study, we selected fluconazole and Amp B by considering their antifungal
potency, toxicity and different mode of actions when compared with 12f. The study was performed
against C. neoformans (ATCC350) and as shown earlier 12f produced MIC of 0.39 µg/mL, while
fluconazole and Amp B exhibited MIC values of 9.57 and 0.89 µg/mL, respectively. The in vitro
synergy was determined using checkerboard dilution assay. The fraction inhibitory concentration
index (FICI) was determined for combination of 12f with fluconazole and Amp B in inhibiting C.
neoformans cells growth that showed synergy at subꢀMIC values (≤0.5 is considered to be
synergistic). The results listed in table 3 indicates that 12f showed significant synergism with both
fluconazole and Amp B. Peptide 12f at nonꢀcidal concentration resulted in potentiation of
fluconazole by 4ꢀfold (MIC from 9.57 µg/mL to 2.37 µg/mL). At the same time, MIC value of
peptide 12f was lowered by 16ꢀfold in this combination (MIC from 0.39 µg/mL to 0.024 µg/mL).
The MIC for Amp B was lowered by 16ꢀfold in combination with 12f (MIC from 0.89 µg/mL to
0.055 µg/mL), while the MIC of peptide 12f was lowered by 32ꢀfold in this combination (MIC from
0.39 µg/mL to 0.012 µg/mL). FICI values of 12f in combinations with fluconazole and Amp B were
found to be 0.308 and 0.092, respectively. These results indicates that the synergistic combinations
of 12f peptide with clinically used drugs fluconazole and Amp B represents a dynamic strategy to
improve antifungal potency. This also opens up a highly efficacious approach to combat antifungal
drug resistance in a cost effective manner using less toxic therapeutics.
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Table 3. Evaluation of synergistic effect of 12f peptide with fluconazole (FLC) and amphotericin B
(Amp B)
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S. No.
Antifungal drug
alone MIC (ꢀg/mL)
Peptide 12f
alone MIC
(ꢀg/mL)
Conc. of antifungal
drug (ꢀg/mL) and
conc. of 12f in
combination
2.37 (FLC) + 0.024
(12f)
Fraction
inhibitory
conc. (FICI)
Effect
1
2
9.57 (FLC)
0.39
0.39
0.308
0.092
Synergistic
Synergistic
0.89 (Amp B)
0.055 (Amp B) +
0.012 (12f)
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Conclusions
We have designed and synthesized two series of His(2ꢀAr)ꢀTrpꢀHis(2ꢀAr) classes of amphipathic
peptides. Several synthetic peptides of this scaffold have displayed high selectivity and potency
against C. neoformans, leading to the identification of most potent peptide 12f. Peptide 12f did not
display hemolytic effects at MIC and exhibited 2ꢀfold higher potency compared to clinical used drug
Amp B. Peptide 12f act rapidly in the time kill kinetics study and results are comparable with Amp
B. Peptide 12f was also found to be proteostable against proteolytic enzyme trypsin. The mechanism
of action studies by confocal microscopy revealed that the presence of 12f permeabilizes cell
surface, translocate intracellularly and causes nuclear fragmentation of genetic material resulting in
killing of C. neoformans cells. The TEM results indicate that 12f act by cell wall and plasma
membrane disruption of C. neoformans cells. Combinations of 12f with marketed antifungal first
line drugs such as Amp B and fluconazole were found be remarkably synergistic in nature. It can be
concluded that peptide 12f has great potential in peptideꢀbased antifungal drug development. The
discovery of histidineꢀrich peptideꢀbased antimicrobials also provide insights that rationally
modified histidines as building blocks offers a better choice in search of rapidly acting and potent
ultra short membrane disruptive antifungal agents.
Experimental
General Chemistry
All starting material chemicals were purchased from SigmaꢀAldrich, ChemꢀImpex, and AlfaꢀAesar
and used further without any additional purification. Thinꢀlayer chromatography (TLC) was
performed using aluminum plates precoated with silica gel (0.25 mm, 60 A poreꢀsize) impregnated
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with a fluorescent indicator (254 nm). Visualization on TLC was achieved by the use of UV light
(254 nm), and also stained with molecular iodine. Automated flash column chromatography was
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1
undertaken on silica gel (230ꢀ400 mesh size). H and 13C NMR spectra were recorded on Bruker
9
AVANCE III 400 MHz instrument using CD3OD as solvent and TMS as internal standard. Proton
and carbon chemical shifts are expressed in parts per million (ppm, δ scale), and were referenced to
NMR solvent CD3OD, δ 3.31, 49.0 ppm. The following abbreviations were used to describe peak
patterns when appropriate: br = broad, s = singlet, d= doublet, t = triplet, q = quadruplet, m =
multiplet. Coupling constants (J) were reported in Hertz unit (Hz). High resolution mass spectra
(HRMS) were obtained with MAXISꢀBruker using ESIꢀTOF method. HPLC analysis was
performed on the SHIMADZUꢀprominence using SupelcosilTM LCꢀ18 column (25 cm × 4.6 mm)
run for 40 min with a flow of 1 mL/min, using a gradient run of 95ꢀ5% (A:B) where buffer A and B
were 0.1% TFA in H2O and CH3CN and detection at 220 or 254 nm; all peptides exhibited ≥95%
purity. All MWꢀirradiation mediated peptide coupling reactions were carried out on a CEM
Discover® microwave syntheszier.
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General procedure for the synthesis of 2-aryl-
L-histidines (3a-e).
NꢀαꢀTrifluoroacetylꢀ ꢀhistidine methyl ester (1, 3.76 mmol, 1.0 equiv.) was dissolved in CH2Cl2 (10
L
mL), and CF3CO2H (1.5 equiv.). A solution of AgNO3 (0.2 equiv) in water (10 mL) was added,
followed by ArB(OH)2 (1.5 equiv.) and (NH4)2S2O8 (2.0 equiv). The reaction mixture was stirred at
ambient conditions for 12ꢀ14 h and progress of the reaction was monitored by TLC. After the
completion of reaction, the pH of the reaction mixture was adjusted to 10ꢀ12 by the addition of
aqueous ammonia solution. The extraction of the reaction mixture was carried out with ethyl acetate
(3×50 mL) and the organic layer was washed with brine solution (2×10 mL). The extract was
concentrated under reduced pressure and purified on an automated flash column chromatography
system to provide 2a-e. The protected amino acid (2a-e, 1 mmol) was dissolved in 6N HCl (5 mL)
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and reaction mixture was heated at 100 C for 12 h. The removal of solvent afforded 2ꢀarylꢀ
Lꢀ
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histidines (3a-e).
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General procedure for the synthesis of
N
-α-Boc-2-aryl-L-histidines (4a-f).
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2ꢀArylꢀ ꢀhistidine (3a-e, 1 equiv.) was dissolved in a mixture of H2Oꢀdioxane (2:3). The pH of the
L
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reaction mixture was adjusted to 12 by addition of aqueous solution of 4N NaOH followed by the
addition of diꢀtertꢀbutyl dicarbonate (2 equiv.). Reaction mixture was allowed to stir at ambient
conditions for 12 h. Reaction mixture was concentrated under reduced pressure and methanol (20
mL) was added and stirring continued for additional 12 h at ambient temperature. The reaction
mixture containing NꢀαꢀBocꢀHis(2ꢀaryl)ꢀOꢀNa+ was concentrated and treated with saturated aqueous
solution of KHSO4 to pH 4 to generate NꢀαꢀBocꢀ2ꢀarylꢀLꢀhistidine. The solvent was removed under
reduced pressure and the resulting residue was extracted with tertꢀbutanol (3 times) to afforded 4a-e.
General synthetic procedure for tripeptides (8a-f).
NꢀαꢀBocꢀ2ꢀarylꢀHisꢀOH (4a-f, 1 equiv.) was dissolved in DMF (2 mL) in a microwave (MW)
reaction vessel. Benzylamine (1.2 equiv.), DIC (1.2 equiv.) and HONB (1.2 equiv.) was added to the
vessel and reaction mixture was heated under MW irradiation at 60 oC for 30 min. The solvent was
removed and residue was purified using a solvent combination of hexane:EtOAc (3:2), on a fully
automated flash column chromatographic system to afford BocꢀHis(2ꢀaryl)ꢀNHBzl (5a-f). The latter
compounds 5a-f were dissolved in a solution of 6N HCl in MeOH (5 mL), and reaction mixture was
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stirred at 25 C for 15 min. The removal of solvent afforded the Bocꢀdeprotected amino acids. The
latter compounds were neutralized with DIEA (3 equiv.) in DMF (2 mL) followed by the addition of
BocꢀTrpꢀOH (1.2 equiv.), DIC (1.2 equiv.) and HONB (1.2 equiv.). The reaction mixture was
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subjected to MWꢀheating at 60 C for 30 min. The solvent was removed and crude product was
purified using a solvent combination of CHCl2:MeOH (98:2) on neutral alumina to afford dipeptides
6a-f. The Bocꢀdeprotections were carried out as described above and to the residue was added DIEA
(3 equiv.), NꢀαꢀBocꢀHis(2ꢀaryl)ꢀOH (4a-f, 1.2 equiv.), DIC (1.2 equiv.) and HONB (1.2 equiv.) in
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in DMF (2 mL). The reaction mixture was subjected to MWꢀheating at 60 C for 30 min. The
solvent was removed and crude product was purified using a combination of CHCl2:MeOH
(97.5:2.5) on neutral alumina to afford 7a-f. The latter compounds 7a-f upon acidolysis with 6N
HCl for 15 min at ambient temperature cleanly afforded final tripeptides 8a-f.
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General synthetic procedure for tripeptides (12a-f).
2ꢀArylꢀLꢀhistidine (4a-f, 1 equiv.) was dissolved in dry methanol and HCl gas pass through the
solution for 2h and followed by stirring at ambient temperature for 12 h. Reaction mixture was
concentrated under reduced pressure to afford 9a-f. Dipeptides (10a-f) were synthesized by coupling
9a-f (1 equiv.) with BocꢀTrpꢀOH (1.2 equiv.) in the presence of DIEA (3 equiv.), DIC (1.2 equiv.),
and HONB (1.2 equiv.) in DMF (2 mL) under MWꢀheating at 60 oC for 30 min. The peptides were
purified using a solvent combination of CHCl2:MeOH (98:2) on neutral alumina to afford 10a-f.
Dipeptides 10a-f upon Bocꢀdeprotection in the presence of 6N HCl in MeOH (5 mL) at 25 oC for 15
min produced the free dipeptides. The latter dipeptides upon coupling with NꢀαꢀBocꢀHis(2ꢀaryl)ꢀOH
(4a-f, 1 equiv.) in the presence of DIEA (3 equiv.) DIC (1.2 equiv.) and HONB (1.2 equiv.) in DMF
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(2 mL) under MWꢀheating at 60 C for 30 min produced crude tripeptides. The tripeptides were
purified using a solvent combination of CHCl2:MeOH (97:3) on neutral alumina afforded Bocꢀ
protected tripeptides 11a-f. The Boc latter peptides (1 mmol) in 6N HCl (5 mL) was stirred for 15
min at ambient temperature to produce peptides 12a-f.
Characterization data of synthesized peptides
Boc-His-Trp-His-NHBzl (7a): Yield = 60%; 1H NMR (400 MHz, CD3OD): δ 7.66 (s, 2H), 7.47 (d,
J = 7.7 Hz, 1H), 7.37 (d, J = 8.0 Hz, 1H), 7.30ꢀ7.24 (m, 3H), 7.16ꢀ7.04 (m, 5H), 6.83 (s, 1H), 6.71
(s, 1H), 4.61ꢀ4.54 (m, 2H), 4.27ꢀ4.15 (m, 3H), 3.20ꢀ3.17 (m, 2H), 2.95ꢀ2.71 (m, 4H), 1.31 (s, 9H);
13C NMR (100 MHz, CD3OD): δ 173.3, 172.6, 171.5, 156.7, 137.7, 136.3, 135.2, 134.0, 132.6,
128.3, 127.1, 127.0, 123.8, 121.6, 119.1, 117.8, 116.8, 111.5, 108.4, 80.6, 54.8, 54.6, 53.6, 42.7,
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28.7, 28.5, 27.3, 26.7; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for 668.3309, found 668.3309 ;
HPLC: tR= 21.9 min, 97.4% purity.
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Boc-His(2-phenyl)-Trp-His(2-phenyl)-NHBzl (7b): Yield = 61%; 1H NMR (400 MHz, CD3OD): δ
7.93ꢀ7.82 (m, 3H), 7.75ꢀ7.52 (m, 5H), 7.48ꢀ7.29 (m, 6H), 7.24ꢀ6.98 (m, 8H), 4.69ꢀ4.65 (m, 2H),
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4.39 (br.s., 1H), 4.22 (br.s., 2H), 3.19ꢀ3.07 (m,3H), 3.02ꢀ2.91 (m, 3H), 1.37 (s, 9H); C NMR (100
MHz, CD3OD): δ 174.2,173.6, 171.9, 157.8, 146.7, 144.2, 140.9, 139.4, 137.9, 133.4, 131.4,130.3,
130.1, 129.5, 129.1, 128.7, 128.4, 128.2, 127.9, 127.4, 127.0, 124.9, 122.6, 120.1, 119.2, 112.5,
110.2, 81.1, 56.3, 55.8, 54.3, 43.8, 29.8, 29.1, 28.6, 28.4; HRMS (ESIꢀTOF): m/z [(M+H)+]
calculated for 820.3935, found 820.3927; HPLC: tR= 26.3 min, 95.7% purity.
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Boc-His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]-NHBzl (7c): Yield = 66%; H NMR
(400 MHz, CD3OD): δ 7.69 (s, 4H), 7.48 (d, J = 6.3 Hz, 1H), 7.28ꢀ7.08 (m, 12H), 6.96 (s, 1H), 6.76
(s, 2H), 4.63 (br.s., 2H), 4.22 (br.s., 3H), 3.21 (d, J = 12.1 Hz, 2H), 3.03ꢀ2.89 (m, 3H), 2.78 (d, J =
8.2Hz, 1H), 2.33 (s, 3H), 2.30 (s, 3H), 1.31 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 174.3, 173.9,
172.6, 157.8, 147.9, 140.0, 139.9, 139.5, 137.9, 135.5, 134.7, 130.5, 129.4, 128.7, 128.5, 128.4,
128.3, 128.1, 126.4, 124.7, 122.6, 120.3, 120.1, 119.2, 112.4, 110.3, 80.9, 56.1, 54.8, 52.2, 43.8,
30.3, 29.9 28.6, 28.4, 21.3; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for 848.4248, found
848.4226; HPLC: tR= 27.9 min, 97.8% purity.
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Boc-His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]-NHBzl (7d): Yield = 63%; H
NMR (400 MHz, CD3OD): δ 7.76ꢀ7.73 (m, 4H), 7.50 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H),
7.22ꢀ7.16 (m, 3H), 7.11ꢀ7.05 (m, 4H), 6.98ꢀ6.92 (m, 5H), 6.75 (d, J = 6.4 Hz, 2H), 4.64 (t, J = 6.6
Hz, 2H), 4.28ꢀ4.24 (m, 3H), 3.81 (s, 3H), 3.78 (s, 3H), 3.22 (d, J = 6.3 Hz, 2H), 3.02ꢀ2.89 (m, 3H),
2.79ꢀ2.74 (m, 1H), 1.33 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 173.0, 172.4, 171.2, 160.2, 160.1,
156.4, 146.5, 138.0, 136.5, 128.0,127.3, 126.9, 126.7, 126.4, 123.3, 122.7, 122.6, 121.2, 118.7,
117.8, 113.9, 113.8, 111.0, 108.8, 79.5, 54.7, 54.4, 54.4, 53.5, 42.5, 31.3, 28.6, 27.2, 26.9; HRMS
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(ESIꢀTOF): m/z [(M+H)+] calculated for 880.4146, found 880.4093; HPLC: tR= 27.4 min, 97.2%
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purity.
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Boc-His[2-(4-tert-butylphenyl)]-Trp-His[2-(4-tert-butylphenyl)]-NHBzl (7e): Yield = 57%; H
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NMR (400 MHz, CD3OD): δ 7.75 (t, J = 9.0 Hz, 4H), 7.52ꢀ7.42 (m, 5H), 7.30 (d, J = 8.0 Hz, 1H),
7.20ꢀ7.05 (m, 7H), 6.98 (t, J = 7.2 Hz, 1H), 6.79 (s, 2H), 4.66 (t, J = 6.0 Hz, 2H), 4.25 (br.s., 3H),
3.27ꢀ3.21 (m, 2H), 3.04ꢀ2.90 (m, 3H), 2.82ꢀ2.76 (m, 1H), 1.34 (s, 18H), 1.31 (s, 9H); 13C NMR (100
MHz, CD3OD): δ 174.4, 173.9, 172.7, 157.9, 153.1, 153.0, 147.9, 139.5, 137.9, 135.5, 129.4, 128.7,
128.6, 128.5, 128.3, 128.1, 126.8, 126.2, 124.7, 122.6, 120.1, 119.2, 112.5, 110.3, 80.9, 56.1, 54.9,
52.2, 43.8, 35.5, 31.6, 30.3, 30.0, 28.6, 28.4; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for
932.5187, found 932.5117; HPLC: tR= 32.3 min, 98.0% purity.
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Boc-His(2-biphenyl)-Trp-His(2-biphenyl)-NHBzl (7f): Yield = 53%; 1H NMR (400 MHz,
CD3OD): δ 7.86 (s, 4H), 7.65ꢀ7.56 (m, 9H), 7.45ꢀ7.29 (m, 7H), 7.21ꢀ6.99 (m, 8H), 6.82 (d, J = 7.0
Hz, 2H), 4.68 (s, 2H), 4.31ꢀ4.21 (m, 3H), 3.23 (br.s, 1H), 3.01ꢀ2.77 (m, 5H), 1.34 (s, 9H); 13C NMR
(100 MHz, CD3OD): δ 174.4, 173.9, 172.6, 157.8, 147.5, 142.5, 141.5, 139.5, 138.0, 135.5, 130.2,
130.1, 129.9, 129.9, 129.4, 128.7, 128.6, 128.6, 128.3, 128.1, 127.8, 126.8, 124.7, 122.6, 120.6,
120.1, 119.3, 112.5, 110.3, 80.9, 56.0, 54.8, 52.2, 43.8, 30.4, 30.0, 28.6, 28.4; HRMS (ESIꢀTOF):
m/z [(M+H)+] calculated for 972.4561, found 972.4128; HPLC: tR= 31.3 min, 97.5% purity.
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His-Trp-His-NHBzl (8a): Yield = 92%; H NMR (400 MHz, CD3OD): δ7.57ꢀ7.49 (m, 6H), 7.39ꢀ
7.24 (m, 4H), 7.16ꢀ7.12 (m, 3H), 7.05 (t, J = 7.4 Hz, 1H), 4.67 (t, J = 7.5 Hz 1H), 4.58 (t, J = 6.9
Hz, 1H), 4.49 (t, J = 6.9 Hz, 1H), 4.37ꢀ4.33 (m, 2H), 3.50ꢀ3.47 (m, 1H), 3.40ꢀ3.37 (m, 1H), 3.27ꢀ
3.16 (m, 3H), 3.08 (dd, J = 15.4, 7.4 Hz, 1H); 13C NMR (100 MHz, CD3OD): δ 173.0, 169.7, 167.7,
137.8, 136.2, 134.3, 133.2, 128.4, 127.2, 126.9, 126.3, 126.0, 124.1, 121.6, 118.7, 118.5, 117.9,
117.2, 111.5, 108.2, 55.3, 53.5, 51.8, 27.0, 26.4, 26.1; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated
for 568.2784, found 568.2782; HPLC: tR= 17.8 min, 95.8% purity.
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His(2-phenyl)-Trp-His(2-phenyl)-NHBzl (8b): Yield = 97%; H NMR (400 MHz, CD3OD): δ
8.01 (d, J = 8.4 Hz, 2H), 7.81ꢀ7.74 (m, 4H), 7.61ꢀ7.56 (m, 4H), 7.45ꢀ7.38 (m, 4H), 7.30ꢀ7.24 (m,
2H), 7.20ꢀ7.09 (m, 3H), 7.06ꢀ6.93 (m, 3H), 4.70 (t, J = 7.5 Hz, 1H), 4.55 (t, J = 7.0 Hz, 1H), 4.40 (t,
J = 6.6 Hz, 1H), 4.14 (d, J = 14.8 Hz, 1H), 3.99 (d, J = 9.1Hz, 1H), 3.48 (dd, J = 16.1, 5.7 Hz, 1H),
3.28ꢀ3.22 (m, 2H), 3.18ꢀ3.06 (m, 2H), 2.88 (dd, J = 12.8, 7.5 Hz, 1H); 13C NMR (100 MHz,
CD3OD): δ 173.0, 169.5, 167.6, 144.8, 144.5, 143.7, 138.4, 137.7, 136.2, 132.3, 129.6, 129.4, 129.0,
128.5, 128.3, 127.7, 127.3, 127.1, 127.0, 126.9, 126.6, 126.1, 124.0, 122.2, 121.4, 121.2, 119.5,
118.9, 117.7, 111.4, 108.1, 55.3, 54.4, 52.7, 42.5, 27.1, 26.7, 26.6; HRMS (ESIꢀTOF): m/z
[(M+H)+] calculated for 720.3410, found 720.3404; HPLC: tR= 23.4 min, 95.5% purity.
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His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]-NHBzl (8c): Yield = 96%; H NMR (400
MHz, CD3OD): δ 7.90ꢀ7.83 (m, 4H), 7.55ꢀ7.42 (m, 6H), 7.30ꢀ7.02 (m, 10H), 4.72 (br.s., 2H), 4.37ꢀ
4.19 (m, 3H), 3.68ꢀ3.36 (m, 3H), 3.22ꢀ3.08 (m, 3H), 2.43 (s, 6H); 13C NMR (100 MHz, CD3OD): δ
174.7, 171.1, 169.0, 146.7, 145.2, 144.9, 144.6, 139.5, 137.9, 131.5, 131.5, 131.1, 129.5, 128.5,
128.4, 128.3, 128.1, 128.0, 127.9, 127.6, 125.1, 122.7, 121.2,121.1, 120.7, 120.1, 119.2, 118.9,
112.5, 109.8, 56.8, 53.9, 53.1, 44.2, 28.6, 28.1, 28.0, 21.6; HRMS (ESIꢀTOF): m/z [(M+H)+]
calculated for 748.3723, found 748.3724; HPLC: tR= 24.7 min, 97.5% purity.
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His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]-NHBzl (8d): Yield = 91%; H NMR
(400 MHz, CD3OD): δ 7.85 (d, J = 8.9 Hz, 2H), 7.75 (d, J = 6.9 Hz, 2H), 7.41 (s, 1H), 7.33 (d, J =
8.0 Hz, 1H), 7.24ꢀ7.17 (m, 4H), 7.14ꢀ7.01 (m, 10H), 4.69ꢀ4.65 (m, 1H), 4.50 (t, J = 7.0 Hz, 1H),
4.32ꢀ4.29 (m, 1H), 4.15ꢀ4.01 (m, 2H), 3.87 (s, 3H), 3.82 (s, 3H), 3.39ꢀ3.35 (m, 1H), 3.27ꢀ3.02 (m,
4H), 2.88 (dd, J = 15.6, 7.1 Hz, 1H), 13C NMR (100 MHz, CD3OD): δ 173.0, 169.5, 167.8, 162.6,
145.4, 144.0, 137.7, 136.2, 128.8, 128.5, 128.3, 128.0, 127.2, 127.0, 126.9, 126.5, 124.0, 119.0,
118.7, 117.7, 117.2, 115.1, 115.0, 114.8, 111.5, 108.1, 55.4, 55.3, 55.2, 52.6, 51.8, 42.8, 29.9, 27.1,
26.7; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for 780.3622, found 780.3603; HPLC: tR= 24.0
min, 98.4% purity.
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His[2(4-tert-butylphenyl)]-Trp-His[2-(4-tert-butylphenyl)]-NHBzl (8e): Yield = 94%; H NMR
(400 MHz, CD3OD): δ 7.84 (d, J = 8.5 Hz, 2H), 7.72ꢀ7.60 (m, 6H), 7.44 (t, J = 6.7 Hz, 2H), 7.33 (d,
J = 8.0 Hz, 1H), 7.22ꢀ7.16 (m, 4H), 7.12ꢀ7.04 (m, 5H), 4.67ꢀ4.63 (m, 1H), 4.52 (t, J = 7.1 Hz, 1H),
4.31ꢀ4.28 (m, 1H), 4.14ꢀ3.98 (m, 2H), 3.42 (d, J = 5.0 Hz, 1H), 3.26ꢀ3.07 (m, 4H), 2.97ꢀ2.91 (m,
1H), 1.30 (s, 9H), 1.28 (9H); 13C NMR (100 MHz, CD3OD): δ 175.9, 173.2, 169.4, 145.4, 144.0,
137.6, 136.1, 134.3, 129.2, 128.4, 127.2, 127.0, 126.9, 126.7, 126.4, 125.9, 124.1, 121.8, 119.8,
119.1, 117.7, 111.6, 108.1, 52.8, 51.8, 51.1, 42.6, 34.6, 31.4, 30.9, 30.1; HRMS (ESIꢀTOF): m/z
[(M+H)+] calculated for 832.4662, found 832.4632; HPLC: tR= 30.6 min, 95.5% purity.
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His(2-biphenyl)-Trp-His(2-biphenyl)-NHBzl (8f): Yield = 92%; H NMR (400 MHz, CD3OD): δ
7.99 (d, J = 8.4 Hz, 2H), 7.89 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.6 Hz, 3H), 7.63 (t, J = 9.0 Hz, 4H),
7.52ꢀ7.40 (m, 9H), 7.32 (d, J = 8.1 Hz, 1H), 7.25ꢀ7.11 (m, 7H), 7.06 (t, J = 7.5 Hz, 1H), 6.99 (t, J =
7.3 Hz, 1H), 4.73ꢀ4.66 (m, 2H), 4.36 (t, J = 6.5 Hz, 1H), 4.21ꢀ4.08 (m, 2H), 3.45ꢀ3.40 (m, 1H),
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3.29ꢀ3.16 (m, 4H), 3.20ꢀ2.97 (m, 1H); C NMR (100 MHz, CD3OD): δ 174.9, 169.6, 167.6, 144.9,
144.6, 143.5, 138.6, 137.9, 136.3, 134.2 129.7, 128.9, 128.4, 128.2, 127.7, 127.1, 127.1, 127.0,
126.7, 126.6, 123.8, 121.4, 121.1, 119.3, 118.9, 117.9, 117.7, 111.3, 108.3, 55.3, 52.5, 51.8, 42.5,
27.2, 26.7, 26.6; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for 872.4036, found 872.4026;
HPLC: tR= 28.9 min, 97.3% purity.
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Boc-His-Trp-His-OMe (11a): Yield = 65%; H NMR (400 MHz, CD3OD): δ 7.91ꢀ7.83 (m, 2H),
7.41 (t, J = 7.3 Hz, 1H), 7.18ꢀ7.05 (m, 4H), 6.97ꢀ6.85 (m, 2H), 4.66ꢀ4.57 (m, 2H), 4.26 (s, 1H), 3.63
(s, 3H), 3.24ꢀ3.15 (m, 2H), 3.05ꢀ2.94 (m, 3H), 2.86ꢀ2.78 (m, 1H), 1.30 (s, 9H); 13C NMR (100 MHz,
CD3OD): δ 175.4, 172.6, 171.9, 156.7, 136.3, 134.8, 133.4, 131.6, 130.3, 127.1, 123.9, 121.5, 119.0,
117.9, 116.8, 111.5, 108.4, 80.8, 54.5, 54.3, 52.6, 52.4, 28.3, 27.8, 27.3, 27.0; HRMS (ESIꢀTOF):
m/z [(M+H)+] calculated for 593.2836, found 593.2838; HPLC: tR= 19.3 min, 97.6% purity.
Boc-His(2-phenyl)-Trp-His(2-phenyl)-OMe (11b): Yield = 61%; 1H NMR (400 MHz, CD3OD): δ
7.85ꢀ7.80 (m, 4H), 7.53ꢀ7.49 (m, 7H), 7.31 (d, J = 8.1 Hz, 1H), 7.15ꢀ7.05 (m, 4H), 6.98 (t, J = 7.5
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Journal of Medicinal Chemistry
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Hz, 1H), 4.75ꢀ4.69 (m, 1H), 4.58 (t, J = 7.1 Hz, 1H), 4.36 (br.s., 1H ), 3.65 (s, 3H), 3.22ꢀ3.03 (m,
5H), 2.95ꢀ2.90 (m, 1H), 1.35 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 173.9, 173.8, 172.8, 157.7,
148.0, 137.9, 132.4,128.8, 127.9, 127.5, 124.8, 124.2, 124.1, 122.8, 122.4, 119.9, 119.2, 115.3,
115.2, 112.3, 110.3, 80.9, 56.0, 55.8, 54.1, 52.8, 30.7, 29.9, 28.6, 27.9; HRMS (ESIꢀTOF): m/z
[(M+H)+] calculated for 745.3462, found 745.3450; HPLC: tR= 24.9 min, 95.6% purity.
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Boc-His[2-(4-methylphenyl)]-Trp-His[2-(4-methylphenyl)]-OMe (11c): Yield = 58%; H NMR
(400 MHz, CD3OD): δ 7.68ꢀ7.66 (m, 4H), 7.51 (d, J = 7.7 Hz, 1H), 7.30 (d, J = 8.1 Hz, 1H), 7.22ꢀ
7.16 (m, 4H), 7.10ꢀ7.04 (m, 2H), 6.96 (t, J = 7.3 Hz, 1H), 6.98 (d, J = 6.8Hz, 2H), 4.66 (t, J = 6.9
Hz, 2H), 4.31ꢀ4.28 (m, 1H), 3.62 (s, 3H), 3.30ꢀ3.09 (m, 3H), 3.05ꢀ2.84 (m, 3H), 2.34 (s, 3H), 2.30
(s, 3H), 1.33 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 173.9, 173.8, 170.4, 157.3, 148.1, 147.2,
139.8, 139.7, 138.0, 130.5, 130.4, 128.8, 128.7, 128.6, 126.3, 124.7, 122.4, 119.8, 119.2, 112.3,
110.4, 80.8, 55.6, 54.8, 54.1, 52.7, 30.5, 29.9, 28.6, 28.3, 21.3; HRMS (ESIꢀTOF): m/z [(M+H)+]
calculated for 773.3775, found 773.3779; HPLC: tR= 26.1 min, 96.7% purity.
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Boc-His[2-(4-methoxyphenyl)]-Trp-His[2-(4-methoxyphenyl)]-OMe (11d): Yield = 61%; H
NMR (400 MHz, CD3OD): δ 7.76 (d, J = 8.4 Hz, 4H), 7.54 (d, J = 7.8 Hz, 1H), 7.33 (d, J = 8.1 Hz,
1H), 7.15 (s, 1H), 7.09 (t, J = 7.2 Hz, 1H), 7.03ꢀ6.94 (m, 6H),6.91 (s, 1H), 4.71ꢀ4.63 (m,2H), 4.36ꢀ
4.32 (m, 1H), 3.84 (s, 3H), 3.81 (s, 3H), 3.66 (s, 3H), 3.24ꢀ3.10 (m, 3H), 3.05ꢀ2.98 (m, 2H), 2.91ꢀ
2.88 (m, 1H), 1.36 (9H); 13C NMR (100 MHz, CD3OD): δ 174.5, 174.0, 172.2, 162.5, 157.7, 147.3,
143.3, 138.0, 133.4, 128.8, 128.5, 124.8, 122.9, 122.4, 119.9, 119.2, 115.5, 112.3, 110.3, 81.0, 55.9,
55.9, 55.5, 53.7, 52.9, 30.7, 30.1, 28.6, 27.9; HRMS (ESIꢀTOF): m/z [(M+H)+] calculated for
805.3673, found 805.3650; HPLC: tR= 24.7 min, 95.9% purity.
Boc-His[2-(4-tert-butylphenyl)]-Trp-His[(4-tert-butylphenyl)]-OMe (11e): Yield = 56%; 1H
NMR (400 MHz, CD3OD): δ 7.62 (t, J = 9.5 Hz, 4H), 7.49ꢀ7.41 (m, 3H), 7.35ꢀ7.19 (m, 3H), 7.01ꢀ
6.87 (m, 3H), 6.69 (d, J = 9.7 Hz, 2H), 4.57 (br.s., 2H), 4.20 (br.s., 1H), 3.53 (s, 3H), 3.10ꢀ3.04 (m,
2H), 2.98ꢀ2.70 (m, 4H), 1.21 (s, 18H), 1.17 (s, 9H); 13C NMR (100 MHz, CD3OD): δ 173.8, 172.7,
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