Antibacterial and anti-TB tat-peptidomimetics with improved efficacy and half-life

Article abstract of DOI:10.1016/j.ejmech.2018.04.039

Non-natural antimicrobial peptides are ideal as next-generation antibiotics because of their ability to circumvent the problems of drug resistance and in vivo instability. We report novel all-α- and α,γ-mixed Tat peptide analogues as potential antibacterial and anti-TB agents. These peptides have broad spectrum antibacterial activities against Gram-positive (MICs 0.61 ± 0.03 to 1.35 ± 0.21 μM with the peptide γTatM4) and Gram-negative (MICs 0.71 ± 0.005 to 1.26 ± 0.02 μM with γTatM4) bacteria and are also effective against active and dormant forms of Mycobacterium tuberculosis, including strains that are resistant to rifampicin and isoniazid. The introduction of the non-natural amino acids of the study in the Tat peptide analogues results in increased resistance to degradation by proteolysis, significantly increasing their half-life. The peptides appear to inhibit bacteria by a membrane disruption mechanism, and have only a low cytotoxic effect on mammalian cells.

Full text of DOI:10.1016/j.ejmech.2018.04.039

Accepted Manuscript
Antibacterial and anti-TB tat-peptidomimetics with improved efficacy and half-life
Govind S. Bhosle, Laxman Nawale, Amar M. Yeware, Dhiman Sarkar, Moneesha
Fernandes
PII:
S0223-5234(18)30373-8
10.1016/j.ejmech.2018.04.039
EJMECH 10388
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 December 2017
Revised Date: 15 March 2018
Accepted Date: 18 April 2018
Please cite this article as: G.S. Bhosle, L. Nawale, A.M. Yeware, D. Sarkar, M. Fernandes, Antibacterial
and anti-TB tat-peptidomimetics with improved efficacy and half-life, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.039.
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ACCEPTED MANUSCRIPT
Graphical Abstract

ACCEPTED MANUSCRIPT
Antibacterial and anti-TB Tat-peptidomimetics with
improved efficacy and half-life
[
a,c]
[b]
[b,c]
[b,c]
Govind S. Bhosle, Laxman Nawale, Amar M. Yeware,
Dhiman Sarkar
and
[
a,c]
Moneesha Fernandes*
[
a]
Organic Chemistry Division, CSIR-National Chemical Laboratory (CSIR-NCL), Dr. Homi
Bhabha Road, Pune 411008, India. E-mail: m.dcosta@ncl.res.in
[
b]
CombiChem-Bio Resource Center, Organic Chemistry Division, CSIR-National Chemical
Laboratory (CSIR-NCL), Dr. Homi Bhabha Road, Pune 411008, India
[
c]
Academy of Scientific and Innovative Research (AcSIR), CSIR-NCL Campus, Pune, India
Keywords: Non-natural amino acid • Tat-peptide • anti-bacterial • anti-TB • Mycobacterium
tuberculosis
Abstract: Non-natural antimicrobial peptides are ideal as next generation antibiotics because
of their ability to circumvent the problems of drug resistance and in vivo instability. We
report novel all-α- and α,γ-mixed Tat peptide analogues as potential antibacterial and anti-TB
agents. These peptides have broad spectrum antibacterial activities against Gram-positive
(MICs 0.61±0.03 to 1.35±0.21 µM with the peptide γTatM4) and Gram-negative (MICs
0
.71±0.005 to 1.26±0.02 µM with γTatM4) bacteria and are also effective against active and
dormant forms of Mycobacterium tuberculosis, including strains that are resistant to
rifampicin and isoniazid. The introduction of the non-natural amino acids of the study in the
Tat peptide analogues results in increased resistance to degradation by proteolysis,
significantly increasing their half-life. The peptides appear to inhibit bacteria by a membrane
disruption mechanism, and have only a low cytotoxic effect on mammalian cells.
Introduction
Antimicrobial peptides (AMPs) act either by interacting with the cell membrane and causing
lysis and ultimately cell death, or after penetrating the cell membrane, upon interaction with
1
intracellular components, in contrast to conventional drugs that typically act through
inhibition of metabolic processes. Such peptides thus offer an opportunity to overcome the
problem of resistance development, which is increasingly being encountered, and that makes
diseases such as those caused by Mycobacterium tuberculosis (Mtb) and other pathogenic
bacteria difficult to treat. To improve the stability to enzymatic hydrolysis and to increase
2
3
bioavailability, several synthetic, non-natural AMPs and mimetics have been reported.
Many AMPs have a helical component that has been shown to be necessary for their
4
antimicrobial activity. Among these, recently, Nepal et al. described cationic and
amphiphilic proline-rich polyproline type II (PPII) helices for effective cell penetration of
5
macrophages that also exerted potent antibacterial activity. PPII and other helical peptides
such as 12-helical β-peptides have been reported by several groups to have varying degrees of
6-8
antimicrobial activities, including against some species that are resistant to common

ACCEPTED MANUSCRIPT
8
9
antibiotics such as vancomycin or penicillin. Several other groups have reported peptoids,
1
0
11,12
13,14
α-aminoxy-peptides, α/β-peptides,
or azapeptides,
as synthetic mimics of AMPs.
We chose to study the effect of non-natural amino acid surrogates on the antibacterial
properties of the Tat(48-57) peptide. The basic translocating region (48-60) of the trans-
1
5
activating transcriptional activator (TAT) peptide,
derived from the human
immunodeficiency virus 1 (HIV-1) tat protein is a positively charged peptide, rich in
arginine, and is highly studied for cell penetration and delivery of a variety of cargo
1
6-19
molecules.
The presence of several arginine residues, in addition to lysine residues,
makes it particularly susceptible to the degradative action of enzymes, precluding its possible
application in biological systems. Several reports of modifications in the Tat peptide have
2
0
21,22
appeared in literature, where non-natural amino acids as well as backbone linkages
have
been explored in relation to cell penetration. They have, however, not been studied for
2
3-28
antimicrobial activity.There have appeared few reports
of the antibacterial properties
23
involving the cell-penetrating Tat peptide that involved the use of Tat peptide dimers, Tat
peptide-porphyrin conjugates, D-Tat (with D-amino acids instead of L-amino acids) and
cholesterol-conjugated Tat peptides, besides the Tat peptide itself, that were shown to
form nanoparticles and one report of its antifungal activity against human pathogenic fungi.
2
5
26
2
7
26
2
8
2
3,24
Studies with model membranes
suggested that membrane disruption was the mode of
antibacterial action of the cell-penetrating Tat peptide. By using transmission electron
microscopy, we show that this is indeed the case. To our knowledge, there are no reports yet
demonstrating the activity of the Tat peptide against mycobacteria, specifically
Mycobacterium tuberculosis (Mtb). We report herein, the antibacterial and anti-TB effects of
novel synthetic analogues of the Tat(48-57) peptide (GRKKRRQRRR). Their microcidal
effect was envisioned considering the amphipathicity, overall charge and possible interaction
with bacterial cell membranes. The antimicrobial properties of the peptides are a consequence
of the novel non-natural lysine or arginine surrogates. Their inclusion in the Tat peptide leads
to enhanced antibacterial activity for many of the derived peptides and significantly superior
anti-TB properties in all of the derived peptides. Moreover, we show that the Tat peptide
analogues are effective against both active and dormant forms of Mtb and MDR-Mtb, making
this a promising class of candidates for future development in the therapy of tuberculosis.
The effect of the chiral monomers on structural pre-organization of the derived peptides is
also investigated to determine if this, in turn, influences their antimicrobial activity. An
additional advantage includes increased protease resistance, extending their half-life and
enhancing their application potential in biological systems. The novel Tat peptide analogues
may also be envisioned to overcome the limitation of bacterial resistance development that is
29
frequently encountered in the use of antibiotics, since AMPs are less likely to lead to
3
0
26
development of resistance, being known to act by targeting the microbial membranes,
3
1,32
rather than metabolic processes, as in the case of small molecule drugs.
Results and Discussion

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g
Synthesis of precursors X and Z to amino acid surrogates r, k and P respectively
Our laboratory has earlier reported the use of N-(aminoalkyl)proline-derived compounds
3
3
34
towards the synthesis of peptide nucleic acids, cell-penetrating oligomers and as anti-
3
5
glycation agents. The amino acid surrogates, (2S,4S)-4-amino-N1-(3-guanidinopropyl)-
proline (r), (2S,4S)-4-amino-N1-(3-aminopropyl)-proline (k) and (2S,4S)-4-amino-proline
g
(
P ) were derived from (2S,4R)-4-hydroxyproline through the orthogonally protected
precursors X and Z depicted in Figure 1. The incorporation of precursor X in combination
with α-amino acids during peptide synthesis leads to α,γ-peptides containing the residues r or
g
k , while the incorporation of precursor Z leads to all-α-peptides containing the residue P
respectively.
g
Figure 1. Amino acid surrogates- r, k and P , of the study.
Scheme 1 summarises the synthesis of protected non-natural amino acid X. The monomer
was synthesized from commercially available trans-L-4-hydroxyproline. The hydroxyproline
methyl ester 1 was N-alkylated upon reaction with 3-((tert-butyloxycarbonyl)amino)propyl
35
methylsulfonate in the presence of triethylamine and catalytic amount of DMAP in DMF.
The 4-hydroxyl group in compound 2 was then converted to the azide 3 by mesylation,
followed by S 2 displacement of 4-O-mesyl group using NaN in DMF. Catalytic
N
3
hydrogenation of the azide group in 3 to the amine, followed by hydrolysis of the ester
function and subsequent protection of the free amine using Fmoc-Cl and NaHCO in
3
dioxane:H O (1:1) yielded orthogonally protected non-natural amino acid X. A similar series
2
of reactions was used to synthesize the orthogonally protected precursor Z to arginine
g
surrogate P (Scheme S1, SI).
Scheme 1. Synthesis of orthogonally protected non-natural amino acid X. Reagents and
conditions: (i)MsO-(CH ) -NH-Boc, Et N, DMAP, DMF, 70°C, 62 % (ii) MsCl, Et N,
2
3
3
3
CH Cl , 73% (iii) NaN , DMF, 65°C, 67 % (iv) (a) H , Pd/C, MeOH, (b) LiOH, then HCl,62
2
2
3
2

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+
%
for (a) and (b) (v) (a) Fmoc-Cl, NaHCO , H O-dioxane, (b) Dowex H resin, 58 % for (a)
3 2
and (b).
Synthesis of Tat peptide analogues
The protected non-natural amino acids X and Z were used in the solid phase synthesis of Tat
peptide derivatives. Peptides were assembled on MBHA resin by either Fmoc- or Boc-
chemistry protocols. β-Alanine was coupled as the first amino acid as a spacer, followed by
the coupling of other amino acids. All the coupling steps were done in dry DMF in presence
of DIPEA, with HOBt and TBTU as coupling agents. Removal of the Boc-protecting group
was achieved using 50% TFA in CH Cl , while the Fmoc group was removed using 20%
2
2
piperidine in DMF. A phenylalanine residue was added at the N-termini of the peptides in
order to facilitate concentration calculation from the UV absorbance. Guanidinylation of
g
monomer Z to arginine surrogate P was performed on the solid support after N-acetylation
of the phenylalanine residue at N-terminus, and removal of the Fmoc protecting group, using
1
H-pyrazole-1-carboxamidine hydrochloride and DIPEA in dry DMF. The synthesized
peptides were cleaved from the resin by the TFA-TFMSA cleavage protocol and purified by
RP-HPLC on a C18 column. Their purity was re-checked by analytical HPLC and all the
peptides were characterized by MALDI-TOF analysis (SI). Table 1 lists the peptides
synthesized, while the chemical structures are depicted of representative peptides in Figure 2
and of all peptides in the SI, pages S16 & S17. The peptides γTat1M and γTat1C (Table 1,
entries 1 and 2 respectively) contain one r unit, either in the middle of the oligomer, or at the
'
C'-terminal. Likewise, the αTat1M and αTat1C peptides (Table 1, entries 7 & 8 respectively)
g
contain one P unit in the middle and 'C'-terminal positions respectively. γTatM3, γTatM3wg
g
and αTatM3 peptides contain three units each of r, k or P respectively, distributed across the
oligomer (Table 1, entries 3, 4 & 9 respectively), while γTatM4, γTatM4wg and αTatM4
g
peptides contain four units each of r, k or P respectively, distributed across the oligomer
(
(
Table 1, entries 5, 6 & 10 respectively). A control Tat peptide (ctrlTat) was also synthesized
Table 1, entry 11) for comparison.
Figure 2. Representative Tat peptides of the study- (A) γTatM4 and (B) αTatM4.
#
Table 1. Peptides of the study .
Entry
Code
Peptide sequence

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1
2
3
4
5
6
7
8
9
γTat1M
γTat1C
Ac-FGRKKrRQRRR-βAla-NH
Ac-FGRKKRRQRRr-βAla-NH
2
2
γTatM3
γTatM3wg
γTatM4
γTatM4wg
αTat1M
αTat1C
αTatM3
αTatM4
ctrlTat
Ac-FGrKKRrQRRr-βAla-NH
2
Ac-FGkKKRkQRRk-βAla-NH
2
Ac-FGrKKRrQrRr-βAla-NH
2
Ac-FGkKKRkQkRk-βAla-NH
2
g
Ac-FGRKKP RQRRR-βAla-NH
2
2
g
Ac-FGRKKRRQRRP -βAla-NH
g
g
g
Ac-FGP KKRP QRRP -βAla-NH
2
g
g
g
g
10
Ac-FGP KKRP QP RP -βAla-NH
2
11
Ac-FGRKKRRQRRR-NH
2
#
r = (2S,4S)-4-amino-N-(3-guanidinopropyl)-proline, k =
(2S,4S)-4-amino-N-(3-
g
aminopropyl)-proline, P = (2S,4S)-4-guanidinoproline.
Antibacterial activity of the Tat peptide analogues of the study
The novel Tat peptides of the study were tested for antimicrobial activity against Gram-
positive (Staphylococcus aureus, Bacillus subtilis, Staphylococcus epidermidis) and Gram-
negative (Escherichia coli, Pseudomonas aeruginosa, Enterobacter spp.) bacteria. The data
obtained are summarized as IC or MIC values in Table 2. The IC values are listed in the SI
9
0
50
(Table S1).
Among all the peptides, the γTatM4 peptide displayed the best activity, with the lowest IC50
SI, Table S1) and MIC values against Gram-positive as well as Gram-negative bacteria.
(
Specifically, the MIC values for γTatM4 against E. coli, P. aeruginosa, Enterobacter spp., S.
aureus, B. subtilis and S. epidermidis were 1.12, 0.61, 1.35, 1.26, 0.71 and 1.12 µM
respectively (Table 2).
Activity against Mycobacterium tuberculosis
The activity of the peptides of the study against Mtb and MDR-Mtb are depicted as their MIC
values in Figure 3 and listed in Table 3. The corresponding IC values are listed in the SI,
5
0
Table S2. Against Mtb, γTat1M was the most active against the dormant and active forms
MIC 13.8 and 20.1 µM respectively). Against MDR-Mtb, the γTatM4wg and αTatM3
(
peptides showed the best activity, with MIC values of 6.6 µM and 6.0 µM respectively
against the dormant form and 12.8 µM and 15.0 µM respectively, against the active form.
The γTat1M peptide also showed moderate activity against MDR-Mtb, with MIC values of
1
0.6 µM and 13.0 µM against the dormant and active forms respectively.
Circular Dichroism studies
The CD spectra of the Tat peptides of the study were recorded in water as well as in presence
of the secondary structure-inducing solvent, trifluoroethanol (TFE). The spectra are shown in
Figure 5. The ctrlTat peptide study displayed a random coil structure in water, as reported

ACCEPTED MANUSCRIPT
3
6
earlier, but showed a significant change towards the α-helical structure in the presence of
TFE (Figure 4 (a) and 4 (b)). A similar trend was observed for the all-α-peptides of this study.
The α,γ-peptides, on the other hand, displayed differing CD signatures. The γTatM4,
γTatM4wg and γTatM3wg peptides were found to display markedly different CD signatures
from the other α,γ-peptides. This trend was also found in these peptides in the presence of
TFE. The γTat1C peptide was found to display a CD signature very similar to that of the
3
7
ctrlTat peptide in TFE, and tending towards the signature observed for α-helices in the case
of α-peptides. Coincidentally, this peptide also had very similar antibacterial activity to the
ctrlTat peptide against the bacteria tested, as evident from Figure 2.
Table 2. MIC data for peptides of the study against Gram-positive and Gram-negative
bacteria.
a
Entry
MIC (µM) of peptides with SD (±)
Gram-negative
Gram-positive
S. aureus
E. coli
P. aeruginosa
2.67 ± 0.04
3.35 ± 0.03
1.82 ± 0.005
2.32 ± 0.047
0.61 ± 0.03
2.2 ± 0.02
Enterobacter spp.
4.70 ± 0.49
18.85 ± 1.28
8.01 ± 0.58
4.65 ± 0.23
1.35 ± 0.21
4.51 ± 0.44
nd
B. subtilis
S. epidermidis
8.95 ± 1.2
4.92 ± 0.5
9.44 ± 0.82
7.25 ± 0.64
1.12 ± 0.23
3.94 ± 0.54
nd
γTat1M
γTat1C
γTatM3
γTatM3wg
γTatM4
γTatM4wg
αTat1M
αTat1C
αTatM3
αTatM4
ctrlTat
7.25 ± 0.06
5.71 ± 0.04
7.58 ± 0.03
3.52 ± 0.08
1.12 ± 0.01
2.34 ± 0.05
13.49 ±0.02
6.73 ± 0.03
11.8± 0.018
27.73 ±0.04
6.27 ± 0.05
4.17± 0.036
3.3 ± 0.024
5.25 ± 0.11
8.47 ± 0.17
9.05 ± 0.33
3.02 ± 0.05
1.26 ± 0.02
3.02 ± 0.03
8.65 ± 0.18
10.95 ±0.66
20.54 ±0.85
5.59 ± 0.42
9.91 ± 0.25
2.8 ± 0.01
14.1 ± 0.47
3.56 ± 0.04
2.81 ± 0.02
4.16 ± 0.06
0.71 ± 0.005
3.79 ± 0.08
29.85 ± 1.15
7.96 ± 0.12
13.2 ± 0.54
15.13 ± 0.73
3.34 ± 0.04
29.5 ± 0.3
6.53 ± 0.42
10.26 ± 0.03
12.62 ± 0.06
25.98 ± 0.79
7.46 ± 0.69
12.5 ± 0.09
1.01 ± 0.06
nd
nd
nd
nd
nd
nd
21.60 ± 1.95
nd
5.78 ± 0.51
nd
b
Ampicillin
b
Kanamycin
nd
>61.9 ± 0.6
2.8 ± 0.027
nd
a
The values are expressed as the mean of triplicates. Antibacterial activity of each peptide
b
was determined by serial dose dependent dilutions. Standard antibacterial drug and positive
control. SD (±) = standard deviation. nd = not determined.

ACCEPTED MANUSCRIPT
Figure 3. Activity of the peptides of the study against Mycobacterium tuberculosis.
Table 3. MIC data (µM) for peptides of the study against Mycobacterium tuberculosis in
vitro.
MIC (µM) values of peptides with SD (±)
Mtb H37Ra (ATCC 25177)
Dormant
MDR-Mtb
Dormant
Peptide
Active
Active
γTat1M
13.8 ± 0.001
18.4 ± 0.001
22. 8 ± 0.003
26.3 ± 0.003
36.3 ± 0.003
18.3 ± 0.003
33.9 ± 0.0042
23.9 ± 0.003
21.1 ± 0.0045
29.4 ± 0.004
0.91 ± 0.005
> 100
20.1 ± 0.00076
27.6 ± 0.00064
26.1 ± 0.003
43.0 ± 0.0029
43. 9 ± 0.0026
34.4 ± 0.003
28.6 ± 0.0042
32.10 ± 0.003
36.3 ±0.0047
53.9 ± 0.0036
0.97 ± 0.005
> 100
10.6 ± 0.0015
12.0 ± 0.001
10.4 ± 0.0014
14.0 ± 0.001
11.7 ± 0.001
6.6 ± 0.001
14.5 ± 0.0026
12.9 ± 0.002
6.0 ± 0.0034
21.5 ± 0.003
>12.1 ± 0.0037
> 100
13 ± 0.0035
22.1 ± 0.003
25.3 ± 0.0014
23.5 ± 0.001
24.6 ± 0.0023
12.0 ± 0.002
15.1 ± 0.0026
22.5 ± 0.0025
15.0 ± 0.0034
26. 4 ± 0.0026
>12.1 ± 0.0043
> 100
γTat1C
γTatM3
γTatM3wg
γTatM4
γTatM4wg
αTat1M
αTat1C
αTatM3
αTatM4
Rifampicin
a
a
Pyrazinamide
Anti-TB activity of each peptide was determined by serial dose dependent dilutions. Data are
a
expressed as the mean values of triplicates. Standard anti-TB drug and positive control.
(
a)
ctrlTat;
TatM3wg;
γ
Tat1M;
γ
Tat1C;
γTatM3
ctrlTat;
α
Tat1M;
TatM4
α
Tat1C
γ
γTatM4;
γTatM4wg
2
1
1
0000
5000
0000
α
TatM3;
α
2
0000
5000
0000
000
1
1
5
000
5
0
0
-
5000
10000
15000
20000
-
5000
10000
15000
20000
-
-
-
-
-
-
200
220
240
260
280
200
220
240
260
280
Wavelength / nm
Wavelength / nm
(
b)
ctrlTat;
TatM3wg;
γ
Tat1M;
γ
Tat1C;
γTatM3
ctrlTat;
α
Tat1M;
α
Tat1C
γ
γTatM4;
γTatM4wg
α
TatM3;
α
TatM4
20000
5000
0000
000
2
1
1
0000
1
5000
0000
1
5
5
000
0
0
-
5000
-5000
-10000
-15000
-20000
-
10000
15000
20000
-
-
200
220
240
260
280
200
220
240
260
280
Wavelength / nm
Wavelength / nm

ACCEPTED MANUSCRIPT
Figure 4. CD spectra of the all-α-Tat peptides and α,γ-Tat peptides of the study in water (a)
and in 90% TFE-water (b).
Fluorescence- and transmission electron microscopy images
Fluorescence microscopy images were recorded using a dual staining method
[
LIVE/DEAD™ BacLight™ Bacterial Viability Kit, (Invitrogen)] employing SYTO 9 and
propidium iodide (PI) dyes. As seen in Figure 5, significantly increased staining with PI is
observed in cells treated with a representative peptide of the study in comparison to untreated
cells.
Figure 5. Fluorescence micrographs of S. aureus. A and C are green channel (SYTO 9)-; B
and D are red channel (PI)- images captured for untreated cells (I) and cells treated with
γTatM4 (II) under 60X magnification respectively. Scale bar = 50 µ.
Representative Tat-peptides that displayed relatively higher antibacterial activity were
studied by TEM to examine their effect on the bacterial cell walls. Specifically, the effect of
the peptides γTatM4 and γTat1C were studied on cells of S. aureus and E. coli as Gram-
positive and Gram-negative bacteria respectively and are depicted in Figures 6 and 7 and on
Mtb (Figure 8). Extensive damage to the bacterial membranes was observed.
Figure 6. TEM images of S. aureus untreated and after treatment with representative peptides
of the study. Scale bar: upper panel = 200 nm; lower panel = 100 nm.

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Figure 7. TEM images of E. coli untreated and after treatment with representative peptides of
the study. Scale bar: upper panel = 200 nm; lower panel = 100 nm.
Figure 8. TEM images of M. tuberculosis untreated and after treatment with γTatM4, a
representative peptide of the study. Scale bar: upper panel = 0.5 µ; lower panel = 50 nm.
Cytotoxicity studies
The cytotoxicity of the Tat peptides of the study on mammalian cells was estimated by the
standard MTT cell viability assay. HeLa cells were treated with the peptides of the study at
varying concentrations for 24 h and the cell viability was estimated. The results are depicted
in Figure 9. As seen in the plot, the peptides of the study show very low cytotoxicity to
mammalian cells. The cell viability is ≥80 % for almost all the peptides even at the higher
concentrations studied. The cytotoxicity of the peptides was also evaluated in HCT 116 and
HUVEC cells (SI).

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Figure 9. MTT cell viability assay upon treatment with Tat peptides of the study with HeLa
cells.
The hemolytic activity of a representative Tat peptide analogue, γTatM4, in comparison to
the ctrlTat peptide, was measured as an indication of its effect on the mammalian cell
membrane. This in vitro assay is an indicator of red blood cell lysis and evaluates the
hemoglobin released in the plasma spectrophotometrically at 540 nm, after exposure to the
test peptides. An increase in absorbance at this wavelength, is therefore, indicative of
increased hemolysis and toxicity. The γTatM4 peptide showed lower hemolytic activity
compared to ctrlTat peptide (Figure 10). Treatment with Triton X-100 and phosphate
buffered saline were used as positive and negative controls respectively. The results are in
agreement with the cytotoxicity data obtained by the MTT assay (Figure 9).
Figure 10. Hemolytic activity of representative peptides.
Stability to enzymatic hydrolysis
The stability of the Tat peptides of the study to hydrolytic digestion by enzymes was studied
by treating the peptides with trypsin, a commercially available protease, and estimating the
percent intact peptide remaining over time by HPLC analysis. The results of such a study
with γTatM4 as a representative example, in comparison to the control Tat peptide (ctrlTat) is
shown in Figure 11. It is evident that the introduction of the arginine surrogate r within the
Tat peptide sequence conferred the derived peptide with significant resistance to digestion by
trypsin. In particular, the t1/2 of γTatM4 was found to be 16 h in comparison to 4 h for the
ctrlTat peptide. After 24 h, 30% of the γTatM4 peptide still remained intact, while only 4% of
the ctrlTat peptide was found to be intact at the same time point. The fragments obtained after
digestion with trypsin were analysed by MALDI-TOF. The results are tabulated in the SI. In
the case of the ctrlTat peptide, cleavage was observed at the C-terminal sites of most of the
lysine and arginine residues. Five fragments, in addition to the intact peptide were observed
for the ctrlTat peptide even after 0.5 h, while the γTatM4 peptide was completely intact at
this time, with no observed fragmentation. The molecular ion of the intact γTatM4 peptide
was observed even after 24 h, and cleavage was observed to occur only at the C-termini of
the K1 and R2 residues, while the other cleavage sites were blocked by the presence of the
arginine analogues of the study. On the other hand, the molecular ion peak of the ctrlTat

ACCEPTED MANUSCRIPT
peptide was completely absent after only 8 h. Exposure to the enzymes chymotrypsin and
pepsin also confirmed the superior stability of γTatM4 to proteolytic digestion in comparison
to ctrlTat (SI).
Figure 11. Stability of the γTatM4 and ctrlTat peptides to proteolytic digestion by trypsin.
Discussion
Gram-positive and Gram-negative bacteria differ in the structure and composition of their cell
walls, which could lead to differential interactions on their surface. However, both types of
bacterial cell surfaces are negatively charged. The negative charge in Gram-positive bacteria
is mainly due to the techoic acid polymeric chains which bear anionic phosphates in the
glycerolphosphate units, while in Gram-negative bacteria, this comes mainly from the
phosphates and carboxylates of sugar acids. The overall negative charge makes them
amenable to interaction with positively charged ligands, such as the Tat-peptides of the
present study. The antibacterial effect of the α,γ-peptides of the study is far superior to that of
the all-α-peptides. Among all the peptides, the γTatM4 peptide displayed the best activity.
The MIC values of the γTatM4 peptide (0.61 to 1.35 µM) are in the range or even better than
those of some of the most effective antibiotics, e.g., ampicillin and kanamycin, currently used
against the tested bacteria. This activity is also significantly more than reported for some
natural antimicrobial peptides such as indolicidin, defensin, mellitin and magainin, against
3
8
some of the tested bacteria. The α,γ-peptides were also more active than the all-α-peptides
against Mtb. It is indeed noteworthy that while the control Tat peptide showed insignificant
activity against active and dormant forms of Mtb and MDR-Mtb (MIC> 150 µM), all the
peptides of the study were active to different extents. Although the MIC values for the Tat
peptides of the study were not as low as for rifampicin against Mtb, it is noteworthy that
many of the peptides are active against both, active and dormant forms of MDR-Mtb. It may
be pointed out that most of the currently used drugs against Mtb, including rifampicin, act
less effectively against the dormant forms. Importantly, the peptides reported herein act to a
greater extent against MDR-Mtb than against Mtb itself. This is a particularly significant
finding, since the treatment of MDR-Mtb is known to be increasingly challenging.
The observed antimicrobial properties of the Tat peptide analogues of this study could be a
consequence of their increased amphipathic character, caused by the inclusion of the non-

ACCEPTED MANUSCRIPT
natural amino acid surrogates. The increased hydrophobicity in comparison to ctrlTat could
4
2
partly contribute to the cell membrane disruption and hence antimicrobial properties of the
Tat peptide analogues described herein. The presence of the guanidine group rather than the
amino group was also shown to be favourable for increased activity. The ability of the
guanidine group to partake in bidentate hydrogen-bonding makes oligomers bearing this
function more potent than those bearing simple amines. Thus, the peptide γTatM4 was more
active than the peptide γTatM4wg. The highest activity was observed for the γTatM4 peptide
that bears four units of the γ-amino acid, r, while the peptides with fewer r units or bearing
g
the α-amino acid, P , displayed lower antimicrobial activity. In general, the non-natural
amino acid r, presenting the guanidino group on a relatively flexible alkyl spacer had a more
positive effect on the antimicrobial properties of the resulting peptide, rather than the amino
g
acid P , where the guanidino group is attached directly to the pyrrolidine ring in a more rigid
disposition. The flexibility associated with the alkyl spacer could be the reason for more
favourable interactions of the guanidines with the cell membrane in the derived α,γ-peptides,
thus leading to their enhanced activity. The potent antimicrobial activity of Tat-derived
AMPs indicated that they have potential to be used alone for the treatment of infections
caused by both Gram-positive and Gram-negative bacteria. Although the use of a single agent
to treat infections is the most commonly used practice, combination treatment can potentially
eliminate drug resistant strains, delay the evolution of drug resistance and reduce the size of
the dose, which can circumvent the side-effects.
The Tat47-58 peptide is reported to display a CD signature commensurate with a random coil
3
6,20
structure, even in the presence of TFE,
while the longer Tat47-72 peptide was reported to
3
6
display a significant increase in the alpha-helical content in 80% TFE. The ctrlTat peptide
3
6
in our study displayed a random coil structure in water, as reported earlier, but showed a
significant change towards the α-helical structure in the presence of TFE. The CD studies
suggest a possible correlation between the secondary structure of the peptides and their
antimicrobial activity. Further, our studies indicate that the peptides that lack a defined
secondary structure as seen for α-peptides, even in presence of the secondary structure-
inducing solvent, trifluoroethanol (TFE), are more effective antibacterial agents in
comparison to those that show a signature similar to that of an α-helix in the case of α-
peptides, in presence of TFE. Thus, our findings suggest that though the structure of the Tat
peptide analogues does seem to play a role in deciding their antibacterial activity, the helical
component in the structure may not be as important as previously suggested in several
4
,6-8
literature reports.
A helical predisposition may prove beneficial in terms of antimicrobial
activity in the case of some peptides, but is not an absolute requirement, and potent activity
may still be achieved with peptides that are not helically predisposed.
Of the two dyes used in the fluorescence microscopy studies, SYTO 9 can penetrate the cell
membrane and bind strongly to DNA; it can be used to stain both live and dead cells. When
bound to double-stranded DNA, its green fluorescence emission at 503 nm is enhanced
(
excitation maximum = 483 nm). PI, on the other hand, is membrane impermeant and is
generally excluded from viable cells. It is therefore, commonly used to identify dead cells
where the cells are ruptured, with a release of the intracellular constituents into the
(

ACCEPTED MANUSCRIPT
extracellular space) in a mixed population of dead and viable cells. When intercalated in
nucleic acids, its emission maximum is at 617 nm (excitation at 535 nm). The relatively
higher level of PI-staining in relation to SYTO 9-staining in the case of the γTatM4 peptide
analogue (Figure 5) is therefore, indicative of their higher ability to disrupt bacterial
membranes in comparison to untreated. The TEM images (Figures 6 and 7) clearly illustrate
the extensive damage that is caused to the bacterial cell walls, as previously suggested by
2
4
studies with model membranes, upon treatment with the Tat-peptides of the study. Further,
the disruptive effects of the peptides are seen to affect both, Gram-positive as well as Gram-
negative bacteria, with similar efficacy. The permeation of the cell wall can be seen in several
cases in the form of pores or holes, causing the leakage of intracellular contents and leading
to the formation of empty or dead cells. A similar devastating effect was observed with Mtb,
when both active and dormant forms were significantly found to be affected (Figure 8),
leading to permeation of the cell membranes and cell death.
The cytotoxicity of the Tat peptides of the study on mammalian cells (HeLa) was estimated
by the MTT cell viability assay. It is a colorimetric assay that is based on the reduction of the
tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to a purple
formazan derivative, that is achieved by enzymes present in viable cells. A decreased colour
intensity is therefore indicative of low cell viability as a result of increased cytotoxicity. The
peptides of the study showed low cytotoxic effects, with viability ≥ 70-80 % at elevated
concentrations (Figure 9). The low cytotoxic effect was also estimated and confirmed by the
hemolysis assay, when the γTatM4 peptide showed lower hemolytic activity than the ctrlTat
peptide (Figure 10). The difference in deleterious effects observed with bacteria/ Mtb vis-a-
vis mammalian cells could be attributed to the difference in composition of the bacterial and
mammalian cell membranes, which may be expected to influence the interaction properties of
the peptides with the respective membranes.
A major limitation of antimicrobial peptides is their inactivation by proteases. Poor protease
stability severely limits the clinical use of potentially therapeutic peptides. The Tat-peptide
contains multiple arginine and lysine residues, which constitute the proteolytic sites (at their
4
0
C-termini) of the enzyme trypsin, thus making it an ideal enzyme for a study of their
protease resistance. The non-natural amino acids used in the present work confer the derived
peptide with improved protease resistance properties, increasing their application potential in
biological systems, by increasing their half-life significantly. MALDI-TOF analysis of the
fragments obtained after trypsin digestion reaffirmed the protective effect the amino acid
surrogates.
Conclusions
The Tat peptide has been reported to have some antifungal activity against human pathogenic
2
8
fungi, but so far, there are only few reports concerning its antibacterial effects and no
reports of its anti-TB effects, this being the first. Further, in contrast to the antifungal effect,
where no disruption of fungal membranes was observed, and the antifungal effect was found
to be due to the arrest of the G1 phase of the fungal cell cycle after penetration into the

ACCEPTED MANUSCRIPT
2
8
nucleus, the antibacterial and anti-TB effects were found to be due to the interaction with
2
3,24
and disruption of microbial membranes, leading to cell lysis and death, as earlier reported.
Both electrostatic as well as hydrophobic interactions are possibly involved in bringing about
this effect. The detailed mechanism of action against Mtb is not known as yet. From our TEM
results, the anti-TB effect appears to be by membrane disruption, as with Gram-positive and
Gram-negative bacteria. The applicability of the peptides reported herein could be enhanced,
perhaps synergistically, by combination with known antimicrobial agents such as
41
antibiotics. This possibility is actively being pursued in our laboratory.
On the whole, the Tat peptide analogues reported herein are shown to posses good activity
against Gram-positive and Gram-negative bacteria and also Mtb The fact that they are active
particularly against the dormant forms of Mtb, in addition to the active form, makes them
promising for further study and development. Their low potential for resistance development
combined with their higher bio-stability, owing to the presence of non-natural amino acids,
further adds to their potential towards therapy.
Experimental Section
General Information
All the reagents used were obtained commercially and were of ≥ 95 % purity and used
without further purification. DMF, CH Cl , pyridine were dried over P O , CaH , KOH
2
2
2
5
2
respectively. DMF, CH Cl were stored by adding 4 Ǻ molecular sieves and pyridine by
2
2
adding KOH. Column chromatography was performed for purification of compounds on
silica gel (100- 200 mesh or 60-120 mesh, Merck). TLCs were performed on pre-coated with
silica gel 60 F254 (Merck) aluminium sheets. TLCs were performed using petroleum ether-
ethyl acetate and ethyl acetate-methanol solvent systems. TLCs were visualised after
1
13
spraying with ninhydrin reagent and heating. H and C NMR spectra were recorded on a
Bruker AV 200 or AV 400 or AV 500 spectrometer fitted with an Aspect 3000 computer and
1
13
all the chemical shifts (ppm) are referred to internal TMS, chloroform-d for H and/or C
1
NMR. H NMR data are reported in the order of chemical shift, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; br, broad; br s, broad singlet; m, multiplet and/ or multiple
resonance), number of protons. HRMS mass spectra were recorded on a Thermo Scientific Q-
Exactive, Accela 1250 pump MALDI-TOF spectra were obtained from a Voyager-De-STR
(Applied Biosystems) and CHCA (α-Cyano-4- hydroxycinnamic acid) matrix was used to
analyze MALDI-TOF samples. All final compounds and oligomers were ≥ 95% pure, as
1
13
determined by H NMR, C NMR, HRMS, HPLC and/or MALDI-TOF analysis, as
applicable. UV absorbance was performed on a Varian Cary 300 UV-VIS spectrophotometer.
Experimental procedures and spectral data
(
(
2S,4R)-methyl-1-(3-(tert-butyloxycarbonyl-amino)propyl)-4-hydroxypyrrolidine-2-carboxylate
2)
To a 250 mL round bottom flask containing trans-4-hyroxy-L-proline methyl ester 1 (5 g,
4.5 mmol) dissolved in dry DMF (15 mL), triethylamine (14.4 mL, 103.0 mmol) and
3

ACCEPTED MANUSCRIPT
catalytic amount of DMAP were added. The above reaction mixture was stirred for 30 min at
room temperature and 2-((Boc)amino)propyl methanesulfonate (13.1 g, 51.5 mmol) dissolved
in dry DMF was added drop-wise. The reaction was heated at 70 °C for 10 h and monitored
by TLC. The solvent was evaporated in vacuo and the crude reaction mixture was taken up in
ethyl acetate and water. The layers were separated, and the aqueous phase extracted with
ethyl acetate. The combined organic extracts were washed with brine, dried over Na SO and
2
4
evaporated under vacuum. The crude product was purified by silica gel column
chromatography using a gradient of ethyl acetate in petroleum ether to give title compound 2
2
0
1
as a pale yellow gummy liquid (6.4 g, 62%). [α] -143.72 (c 0.1, CHCl ). H NMR (200
D
3
MHz, CDCl ) δ: 5.38 (br s, 1H), 4.45 (m, 1H), 3.72 (s, 3H), 3.54 (m, 1H), 3.36 (m, 1H), 3.19
3
1
3
(
m, 2H), 2.73 (m, 2H), 2.56 (m, 2H), 2.14 (m, 2H), 1.63 (m, 2H), 1.44 (s, 9H). C NMR (50
1
3
MHz, CDCl ) δ: 174.4, 156.3, 77.1, 70.0, 64.4, 60.9, 51.8, 39.3, 38.5, 28.4, 28.0. C DEPT
3
(
50 MHz, CDCl ) δ: 70.0, 64.4, 60.9, 51.9, 51.8, 39.3, 38.5, 28.4, 28.0. HRMS (ESI): m/z
3
+
+
calculated for C H N O : 302.1842, Observed [M +H]: 303.1915, [M +Na]: 325.1713.
1
4
26
2
5
(
2S,4S)-methyl-4-azido-1-(3-(tert-butyloxycarbonyl amino)propyl)pyrrolidine-2-carboxylate (3)
Dry triethylamine (10 mL) was added to a solution of compound 2 (5 g, 16.5 mmol) in dry
CH Cl . The reaction mixture was stirred for 30 min, mesyl chloride (3.8 mL, 49.6 mmol)
2
2
°
was added drop-wise by syringe and stirring continued for another 30 min at 0-5 C. The
reaction was monitored by TLC. Upon completion, excess of CH Cl and water (100 mL)
2
2
were added. The layers were separated, and the aqueous phase was extracted with CH Cl (3
2
2
x 100 mL). The combined organic extracts were washed with brine (100 mL), dried over
Na SO The solvents were evaporated in vacuo and the mesylate obtained was used in the
2
4.
further reaction. The mesyl compound was dissolved in dry DMF (10 mL) and sodium azide
2.56 g, 39.5mmol) was added portion-wise at room temperature with constant stirring. The
(
reaction was heated at 65 °C for the next 6 h and monitored by TLC. Upon completion of
reaction, solvent was evaporated in vacuo, followed by addition of ethyl acetate and water.
The layers were separated, and the aqueous phase was extracted with ethyl acetate. The
combined organic extracts were washed with brine, dried over Na SO and evaporated. The
2
4
crude residue was purified by silica gel column chromatography using a gradient of ethyl
acetate in petroleum ether to give the title compound 3 as a pale yellow solid (1.72 g, 67%).
2
0
1
[
1
α] -137.92 (c 0.1, CHCl ). H NMR (200 MHz, CDCl ) δ: 5.47 (br s, 1H, NH), 3.96 (m,
D 3 3
1
3
H), 3.77 (s, 3H), 3.25 (m, 4H), 2.88 (m, 1H), 2.53 (m, 3H), 2.15 (m, 1H), 1.44 (s, 9H). C
NMR (50 MHz, CDCl ) δ: 173.3, 156.2, 78.6, 64.8, 58.7, 58.0, 52.1, 51.7, 38.5, 35.4, 28.4,
3
13
2
7.8. C DEPT (50 MHz, CDCl ) δ: 64.8, 58.7, 58.0, 52.1, 51.7, 38.4, 35.4, 28.4, 27.7.
3
+
HRMS (ESI): m/z calculated for C H N O : 327.1907; observed [M +H]: 328.1977;
1
4
25
5
4
+
[
M +Na]: 342.2131.
(
2S,4R)-4-((((9H-fluoren-9 yl)methoxy)carbonyl)amino)-1-(3-(tert-butyloxycarbonyl
amino)propyl)pyrrolidine-2-carboxylic acid (X)
To a solution of azido compound 3 (1.5 g, 4.58 mmol) dissolved in dry MeOH (25 mL), 10%
palladium on charcoal (300 mg, 20% w/w) was added and the resulting reaction mixture
stirred under hydrogen gas at 50 psi for 3 h and the reaction was monitored by TLC. After

ACCEPTED MANUSCRIPT
completion of reaction, it was filtered through celite and water:methanol (1:1, 10 mL) were
added. 2N LiOH (15 mL) was further added and the reaction was stirred for 30 min.
Methanol was evaporated under reduced pressure and the resulting reaction mixture was
neutralised with dil. HCl. The solvents were evaporated and the residue was suspended in
methanol and filtered. The supernatant was evaporated in vacuo and dried in a desiccator to
give the corresponding amino acid. To a solution of the amino acid, dissolved in 1, 4-
dioxane:water (1:1, 5 ml), NaHCO (3.0 g, 36 mmol ) was added to maintain an alkaline pH.
3
The resulting reaction mixture was stirred at room temperature for 30 min and Fmoc-Cl (2.69
g, 10.4 mmol) was added slowly. Stirring was further continued overnight at room
temperature. The reaction was monitored by TLC. Upon completion of reaction, the reaction
+
mixture was made slightly acidic by addition of Dowex H resin, which was subsequently
filtered off. The dioxane was removed under vacuum and the crude compound extracted in
ethyl acetate. The combined organic extracts were washed with brine and dried over sodium
sulfate and evaporated in vacuo. The crude product was purified by silica gel column
chromatography to give the title compound X (1.02 gm, 58 %) as a solid yellowish foam.
2
0
1
[
α] -27.36 (c 0.1, CHCl ). H NMR (200 MHz, CDCl ) δ: 7.75 (m, 2H), 7.57 (m, 2H), 7.34
D 3 3
(
2
1
m, 4H), 4.61 (m, 1H), 4.38 (m, 2H), 4.21 (m, 2H), 3.77 (m, 1H), 3.13 (m, 3H), 2.80 (m, 1H),
1
3
.53 (m, 2H), 2.33 (m, 1H), 2.00 (m, 2H), 1.46 & 1.44 (s, 9H). C NMR (50 MHz, CDCl ) δ:
3
1
3
74.3. 155.9, 144.0, 141.3, 127.1, 120.0, 79.0, 66.6, 64.4, 52.2, 47.3, 36.8, 28.1. C DEPT
(
50 MHz, CDCl ) δ: 127.0, 124.7, 119.9, 66.6, 64.4, 52.2, 47.2, 36.8, 28.4. HRMS (ESI): m/z
3
+
+
calculated for C H N O : 509.2526; observed: [M +H] 510.2596, [M +Na] 532.2411.
2
8
35
3
6
(
2S,4R)-methyl-1-(tert-butyloxycarbonyl)-4-hydroxypyrrolidine-2-carboxylate (4)
To a solution of trans-4-hyroxy-L-proline methyl ester 1 (10 g, 68.9 mmol) dissolved in
dioxane:water (1:1, 25 ml), sodium hydroxide (8.26 g, 206.7 mmol) was added. The reaction
mixture was stirred for 30 min at room temperature, followed by drop-wise addition of di-
tert-butyl dicarbonate (19.54 g, 89.5 mmol) dissolved in dioxane:water (1:1). The reaction
was further stirred at RT for 4 h and monitored by TLC. Upon completion of reaction, the
dioxane was removed under vacuum and ethyl acetate was added. The layers were separated,
and the aqueous phase was extracted with ethyl acetate. The combined organic extracts were
washed with brine and dried over Na SO . The solvent was evaporated and the crude residue
2
4
was purified by silca gel column chromatography to give the title compound 4 as a white
2
0
1
crystalline solid (14.2 g, 84%). [α]
= -134.30 (c 0.080, CHCl ). H NMR (200 MHz,
D
3
CDCl ) δ: 4.43 (m,1H), 4.36 (m,1H), 3.70 (s,3H), 3.56 (d, J = 3.4 Hz,1H), 3.52 (d, J = 11.6
3
1
3
Hz,1H), 2.25 (m, 1H), 2.01 (m,1H), 1.42 (s, major) & 1.37 (s, minor), 9H. C NMR (50
MHz, CDCl ) δ: 173.7, 154.0, 80.4, 69.1, 57.9, 54.6, 52.0, 39.0, 28.2. C DEPT (50 MHz,
13
3
CDCl ) δ: 69.1, 57.9, 54.6, 52.0, 39.0, 28.2. HRMS (ESI): m/z calculated for C H NO :
3
11 19
5
+
2
45.1263; Observed: [M +Na] 268.1151.
(
2S,4S)-methyl-1-(tert-butyloxycarbonyl)-4-azidopyrrolidine-2-carboxylate (5)
Dry triethylamine (6 mL) was added to a solution of compound 4 (3.0 g, 12.2 mmol) in dry
CH Cl . The reaction mixture was stirred for 30 min and mesyl chloride (1.42 mL, 18.4
2
2
°
mmol) was added drop-wise and stirring continued for another 30 min at 0-5 C. The reaction

ACCEPTED MANUSCRIPT
was monitored by TLC. Upon completion of reaction, excess CH Cl and water (100 mL)
2
2
were added. The layers were separated, and the aqueous phase was extracted with CH Cl (3
2
2
x 100 mL). The combined organic extracts were washed with brine (100 mL), dried over
Na SO The solvents were evaporated in vacuo and the mesylate obtained was used in the
2
4.
further reaction. To a flask containing the mesyl compound dissolved in dry DMF, was added
sodium azide (2 g, 6.19 mmol) portion-wise at room temperature with constant stirring. The
reaction was heated at 65 °C for 10 h, and monitored by TLC. Upon completion of reaction,
the solvent was evaporated in vacuo and ethyl acetate and water were added to the residue.
The layers were separated, and the aqueous phase was further extracted with ethyl acetate.
The combined organic extracts were washed with brine and dried over Na SO . The solvent
2
4
was evaporated under vacuum and the crude product was purified by silica gel column
2
0
chromatography to give the title compound 5 as a thick gummy liquid (1.12 g, 67%). [α]
-
D
1
7
4
2
1.13 (c 0.065, CHCl ). H NMR (200 MHz, CDCl ) δ: 4.41 (min) & 4.31 (maj) (m, 1H),
.15 (m, 1H), 3.73 (s, 3H), 3.66 (d, J = 4.0 Hz, 1H), 3.46 (d, J = 4.0 Hz, 1H), 2.44 (m, 1H),
.16 (dd, J = 1.0 & 4.0 Hz,1H), 1.45 (min) & 1.39 (maj) (s, 9H). C NMR (50 MHz, CDCl )
δ: 172.2, 153.0, 80.5, 59.2, 52.2, 51.2, 35.0, 28.1. C DEPT (50 MHz, CDCl ) δ: 59.2,
3
3
1
3
3
1
3
3
5
7.6,52.2, 51.2, 35.9,28.1. HRMS (ESI): m/z calculated for C H N O : 270.1328;
11 18 4 4
+
Observed: [M +Na] 293.1214.
(
2S,4S)-4-((((9H-fluoren-9 yl)methoxy)carbonyl)amino)-1-(tert-butoxycarbonyl)pyrrolidine-2-
carboxylic acid (Z)
To a solution of azido compound 5 (1.5 g, 5.5 mmol) dissolved in dry MeOH (25 mL), 10%
palladium on charcoal (300 mg, 20% w/w) was added, the resulting reaction mixture stirred
under hydrogen gas at 50 psi for 3 h and the reaction was monitored by TLC. After
completion of reaction, it was filtered trough celite and water and methanol (1:1, 10 mL)
were added. 2 N LiOH (15 mL) was further added to the reaction mixture and stirred for 30
min. Methanol was evaporated under reduced pressure and the resulting mixture was
neutralised with dil. HCl. Solvents were evaporated and the residue was suspended in
methanol and filtered. The supernatant collected was evaporated in vacuo to obtain the
corresponding amino acid. The amino acid obtained was dissolved in 1, 4-dioxane:water
(
1:1), and NaHCO (3.6 g, 43.4 mmol) was added to ensure an alkaline pH. The resulting
3
reaction mixture was stirred at room temperature for 30 min and Fmoc-Cl (1.33 g, 5.1 mmol)
was added in portions. The reaction mixture was stirred overnight at room temperature and
monitored by TLC. Upon completion of reaction, the reaction mixture was slightly acidified
+
by addition of Dowex H resin and the resin was subsequently filtered off. The dioxane was
evaporated under vacuum and ethyl acetate was added. The product was extracted in ethyl
acetate. The organic extracts were washed with brine and dried over sodium sulfate. The
solvents were evaporated in vacuo and the product was washed repeatedly by petroleum ether
2
0
and diethyl ether to give title compound Z (1.13 g, 58%) as a solid yellowish foam. [α]
-
D
1
6
4
1
1
9.60 (c 0.050, CHCl ). H NMR (200 MHz, CDCl ) δ: 7.74 (m, 2H), 7.57 (m, 2H), 7.40 (m,
H), 5.80 (m, 1H), 4.49 (m, 2H), 4.34 (m, 3H), 4.20 (m, 1H), 3.53 (m, 2H), 2.38 (m, 1H),
.50 & 1.44 (s, 9H). C NMR (50 MHz, CDCl ) δ: 174.5. 156.7, 156.0, 143.8, 142.6, 141.3,
27.8, 120.0, 82.4, 67.1, 58.4, 47.1, 28.3. C DEPT (50 MHz, CDCl ) δ: 125.8, 125.2,
3
3
1
3
3
13
3

ACCEPTED MANUSCRIPT
1
4
22.3, 118.1, 65.2, 56.5, 51.8, 45.2, 31.7, 26.4. HRMS (ESI): m/z calculated for C H N O :
52.1947; Observed: [M +Na] 475.1832.
2
5
28
2
6
+
Solid phase peptide synthesis of Tat analogues
All the peptides were synthesized using standard Boc- or Fmoc- chemistry protocol and
MBHA (4-methyl-benzhydrylamine) resin as the solid support (SI). Synthesis was carried out
on 50 µmol scale manually. Removal of the Fmoc-or Boc-group was carried out using 20%
piperidine in DMF or 50% TFA in CH Cl respectively. Boc-removal was followed by
2
2
neutralization using 5% DIPEA in DCM. Further coupling reactions were performed by use
of three equivalents each of monomeric amino acid, TBTU, HOBt and DIPEA in DMF for 6
h. Successive deprotection, coupling and washing steps were carried out as iterative cycles
until the desired length of peptide was synthesized. The N-terminal Fmoc- or Boc-protecting
groups in the completely synthesized peptides were finally cleaved using piperidine/ DMF or
TFA/CH Cl respectively as described above, and the resulting amines capped as acetates
2
2
using acetic anhydride in 50% pyridine/ DCM. All the deprotection and coupling reactions
were monitored by the Kaiser test. Guanidinylation of pendant amines on solid phase was
performed by using 10 equivalents of 1-H-pyrazole-1-carboxamidine hydrochloride reagent
and 10 equivalents (corresponding to each amino group) DIPEA in DMF overnight.
Cleavage and purification of resin bound peptide
After synthesis of the desired peptide sequences, they were cleaved from the solid support by
TFA-TFMSA cleavage protocol. In detail, 5 mg of dry peptide-bound resin was taken in a
glass sample vial. Thioanisole (10 µL) and 1, 2-dithian (4µL) were added to it. After 5 min, to
the ice-cooled sample vial, 80 µL of TFA was added. After another 10 min, 8 µL of TFMSA
was added with slow shaking and the resultant reaction mixture was kept for the next 2 h to
ensure removal of all the side chain protecting groups and complete detachment of the
peptide from the resin support. After 2 h, the residual resin was filtered off through a sintered
funnel and washed with neat TFA. The filtrate was evaporated on a rotary evaporator and
cold diethylether was added. The resulting precipitate was then separated by centrifugation
and water was added. This crude peptide was further purified by RP-HPLC on a C-18 column
using an increasing gradient of acetonitrile in water containing 0.1 % TFA. The peptides were
re-confirmed to be ≥ 95 % pure by analytical HPLC and MALDI-TOF analysis.
Anti-bacterial activity
°
All bacterial cultures were first grown in LB media at 37 C at 180 rpm. Once the culture
reached an absorbance of 1 OD, as measured at 620 nm, it was used for anti-bacterial assays.
Bacterial strains E. coli (NCIM 2688), P. aeruginosa (NCIM 2036), Enterobacter spp.
(NCIM 5392) as Gram-negative and B. subtilis (NCIM 2079), S. aureus (NCIM 2010), S.
epiderimidis (NCIM 5270) as Gram-positive were obtained from NCIM (CSIR-NCL, Pune)
and were grown in Luria Bertani medium (Himedia, India). 0.1 % of 1 OD culture at 600 nm
was used for screening. 0.1 % inoculated culture was added into each well of the 96-well
plate containing the compounds to be tested. Optical density for each plate was measured at
6
20 nm after 8 h for Gram-negative bacteria and after 12 h for Gram-positive bacteria.

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Anti-TB assay
All the synthetic peptides were screened for their in vitro activity against M. tuberculosis
H37Ra (Mtb) (ATCC 25177) and multidrug resistant M. tuberculosis H37Ra (MDR-Mtb).
MICs were determined in both these strains by using the standard XTT Reduction Menadione
4
2
Assay (XRMA) protocol previously described by Singh et al. Briefly, 2.5 µl aliquots of 2-
fold serially diluted peptides were mixed in 247.5 µl of Mtb/MDR Mtb inoculated cultures (~
5
5
3
x 10 cells/ml) in 96-well plates and sealed with plate sealer (Nunc Inc.). After incubation at
°
7 C, the XTT menadione assay was performed after 8 days for detection of active stage
inhibitors and 12 days for detection of dormant stage inhibitors respectively, by removing the
plate sealer. Absorbance was measured at 470 nm and percentage inhibition was calculated
using the formula: % inhibition = [(Control – Test) / (Control – Blank)] x 100 , where
‘Control’ is the absorbance in the absence of added compounds, ‘Test’ is the absorbance in
the presence of compounds and ‘Blank’ is the absorbance of the culture medium without
mycobacteria.
CD analysis
The secondary structures of the peptides in different environments were measured using a J-
8
15 spectropolarimeter (Jasco, Japan). The spectra were recorded at a scan speed of 100
nm/min at wavelengths ranging from 190 to 300 nm in water and 90 % TFE (Sigma). An
average of three scans was collected for each peptide. The final concentration of the peptides
was 500 µM. The acquired CD signal spectra were converted to the molar ellipticity using the
2
−1
equation: [θ]= (θ × 1000)/(c × l), where, [θ] is the molar ellipticity (deg.cm .dmol ), θ is the
observed ellipticity corrected for the buffer at a given wavelength (mdeg), c is the peptide
concentration (M), l is the path length (cm).
Fluorescence microscopy images
S. aureus was allowed to grow to the mid-log phase and then incubated with the peptides (10
µg/mL) at 37 °C for 4 h. The solution was centrifuged at 1000 g for 10 min. The supernatant
was removed and the bacterial pellets were washed with PBS three to four times. PI (5
µg/mL) was added and incubated for 15 min in the dark. Excess dye was removed by PBS
washes (×3). Next, the cells were incubated with SYTO 9 (10 µg/mL in water) for 15 min in
the dark at and excess dye was removed, followed by PBS washes (×3). The bacteria were
then
examined
under
oil-immersion
objective
(60
X)
by using
the
Invitrogen™ EVOS™ FL Cell Imaging Microscope.
Transmission Electron Microscopy images
E. coli and S. aureus were cultured to mid-log phase. The cells were harvested by
centrifugation at 1,000 × g for 10 min, washed thrice with 10 mM PBS and re-suspended to
an OD600nm of 0.2. The cell suspension was incubated at 37 °C for 60 min with different
peptides at their MIC. Following the incubation, the cells were centrifuged and washed with
PBS 3 times at 5,000 g for 5 min. Microbial cell pellets were then fixed overnight with 2.5 %
(v/v) glutaraldehyde in PBS at 4 °C and washed twice with PBS. After pre-fixation with 2.5

ACCEPTED MANUSCRIPT
%
glutaraldehyde overnight, the cell pellets were washed 3 times with PBS and post-fixed
with 2 % osmium tetroxide in PBS for 70 min. The samples were washed twice with PBS,
followed by dehydration for 9 min in a graded ethanol series (50%, 70%, 90% and 100%),
and incubated for 10 min each in 100% ethanol, a mixture (1:1) of 100% ethanol and acetone,
and absolute acetone. These samples were then transferred to a constant-temperature (37 °C)
incubator overnight. Finally, the specimens were observed using a transmission electron
microscope.
Cytotoxicity studies: MTT cell viability and hemolysis assays
The cytotoxicity of peptides against cells was determined by the MTT cell viability assay. All
cells were plated overnight in 96-well plates at a density of 10000 cells per well in 0.2 ml of
appropriate growth medium with 10 % FBS at 37 °C. Different concentrations of peptides up
to a maximum of 50 µM were incubated with the cell for 24 h, following which, the peptides
were removed from the media by replacing with fresh media. Cell culture medium alone or
with cells, both without peptides, were included in each experiment as controls. After 24 h
incubation, 10 µL of 5 mg/ml of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium
bromide (MTT) was added and incubated for 4 h. Conversion of MTT into purple formazan
product by metabolically active cells indicates the extent of cell viability. The crystals of
formazan were dissolved with isopropanol and the optical density was measured at 570 nm
pro5
using a Spectramax
viability.
plate reader (Molecular Devices Inc) for quantification of cell
In the hemolysis assay, freshly drawn human red blood cells (hRBCs) with additive K2
EDTA (spray-dried) were washed with PBS buffer several times and centrifuged at 1000 g
for 10 min until a clear supernatant was observed. The hRBCs were re-suspended in 1× PBS
to get a 0.5 % v/v suspension. Peptides dissolved in 1× PBS were added to a sterile 96-well
plate to make up to a total volume of 75 µL in each well. Then 75 µL of 0.5 % v/v hRBC
solution was added to make up a total volume of 150 µL in each well. The 0 % and 100 %
hemolysis points were determined in 1 × PBS and 0.2 % Triton-X-100 respectively. The
°
plate was then incubated at 37 C for 1h, followed by centrifugation at 3500 rpm for 10 min.
The supernatant (120 µL) was transferred to fresh wells and absorption was detected by
measuring the (optical density at 414 nm by VarioskanFlash (4.00.53) Microplate Reader.
Results were with respect to the positive Triton X-100) and negative (PBS) controls. %
hemolysis was determined by the equation: % hemolysis = (Abs.sample – Abs.PBS)/ (Abs.Triton
Abs.PBS) × 100.
–
Trypsin Digestion Assay
A reverse phase high-performance liquid chromatography (RP-HPLC) assay was used to
assess proteolytic susceptibility. Peptide concentration was calculated by measuring the
−
1
−1
absorbance at 260 nm and using the extinction co-efficient ε260nm = 200 M cm for
phenylalanine. Peptide stock solutions were prepared in Tris-EDTA buffer. Cell culture grade
trypsin from bovine pancreas was purchased from Thermo Fisher Scientific. Each
trypsinolysis experiment was carried out at a peptide concentration of 10 mM, and run in
triplicate. Following addition of trypsin (1 µL of 1X Tryple Express enzyme in 100 µL

ACCEPTED MANUSCRIPT
peptide solution), aliquots of the reaction were removed at different time intervals and
quenched by combining a 10 µL aliquot of the trypsinolysis mixture with 10 µL of 2 %
trifluoroacetic acid in acetonitrile. A portion (20 µL) of the quenched reaction mixture was
subjected to RP-HPLC and the peaks obtained were analyzed. The extent of peptide
trypsinolysis was determined by integrating the area of the peak corresponding to the intact
peptide. The peaks observed in the HPLCs were collected and analyzed by matrix-assisted
laser desorption/ionization–time of flight (MALDI-TOF) to identify the peptide fragments.
Acknowledgements
The Council of Scientific and Industrial Research, New Delhi, is gratefully acknowledged for
a research grant (BSC0302). GSB thanks UGC, New Delhi, for a research fellowship.
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Highlights
•
•
•
•
Non-natural lysine and arginine surrogates in Tat(48-57).
Increased resistance to enzymatic proteolysis.
Good anti-bacterial actvity-MICs between 0.61 to 1.26 µM for γTatM4.
MICs 6.6 & 6.0 µM for γTatM4wg & αTatM3 respectively against dormant M.
tuberculosis.
•
Very low cytotoxic effect on mammalian cells.