Synthesis, antibacterial activity and cytotoxicity of novel Janus peptide dendrimers

Article abstract of DOI:10.1055/s-0031-1290403

In an attempt to find new antibacterial agents, a series of well-defined Janus peptide dendrimers, which feature multiple anionic groups and amphiphilic structure, were synthesized and characterized in detail. The antibacterial activities of all the synthesized dendrimers were tested and screened by using the two-fold serial dilution method. Several compounds showed activity against E. coli, S. aureus, methicillin-resistant S. aureus, and E. faecalis. Further cytotoxicity assays showed that the antibacterial dendrimers were nontoxic against HEK293. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1290403

LETTER
▌1937
lSetyternthesis, Antibacterial Activity and Cytotoxicity of Novel Janus Peptide
Dendrimers
Synthesis and Antibacterial Activity of Peptide Dendrimers
Junzhu Pan,a Li Guo,*a Liang Ouyang,b Dongqin Yin,c Yi Zhaoa
a
Key Laboratory of Drug Targeting and Drug Delivery System of Education Ministry, Department of Medicinal Chemistry, West China School
of Pharmacy, Sichuan University, Chengdu 610041, P. R. of China
Fax +86(28)85502609; E-mail: rosaguoli2000@yahoo.com.cn
b
c
State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu 610041, P. R. of China
Laboratory of Stem Cell Biology, West China Hospital West China Medical School, Sichuan University, Chengdu 610041, P. R. of China
Received: 13.04.2012; Accepted after revision: 06.05.2012
drimers with negatively charged biological membranes in
vivo.10
Abstract: In an attempt to find new antibacterial agents, a series of
well-defined Janus peptide dendrimers, which feature multiple an-
ionic groups and amphiphilic structure, were synthesized and char-
acterized in detail. The antibacterial activities of all the synthesized
dendrimers were tested and screened by using the two-fold serial di-
lution method. Several compounds showed activity against E. coli,
S. aureus, methicillin-resistant S. aureus, and E. faecalis. Further
cytotoxicity assays showed that the antibacterial dendrimers were
nontoxic against HEK293.
Janus dendrimers,11 which contain two different function-
alized segments on opposite sides, have been widely stud-
ied for their self-assembly properties,12 thermal
behavior,13 and their application to drug delivery.14 Re-
cently, Grinstaff et al. reported a series of anionic Janus
dendrimers that are composed of myristic acid and multi-
valent anions, and several of these dendrimers exhibited
antibacterial activity with minimal eukaryotic cell cyto-
toxicity.15 Clearly different from cationic antibacterial
dendrimers, these anionic dendrimers exhibited striking
selectivity in their cytotoxicity toward a prokaryotic bac-
terium compared to a eukaryotic human cell. Although the
specific mechanism of the antibacterial activity of these
anionic dendrimers is still unknown, it is generally con-
sidered to involve imitating detergent activity.3d Accord-
ingly, studies with this kind of anionic dendrimer
expanded the understanding of dendrimers as antibacteri-
al agents. Subsequently, Tulu et al. also reported anionic
dendrimers that displayed antibacterial activity against
both Gram-positive and Gram-negative bacteria.16
Key words: Janus dendrimer, peptide dendrimer, amphiphilic, an-
tibiotics, antibacterial activity
Dendrimers are artificial macromolecules that have
monodisperse and highly branched structures with well-
defined three-dimensional architecture. With the develop-
ment of macromolecules, dendrimers have been widely
applied in medical and biomedical fields such as drug
delivery1 and gene delivery.2 Moreover, some dendrimers
have inherent pharmacological activities such as antibac-
terial, antiviral, and antitumor activities.3 Because of the
dramatic structural differences between dendrimers and
traditional small-molecule drugs, dendrimers provide new
and distinct molecules that might be used to address prob-
lems of drug resistance, especially in the antibacterial
drug field.
COOBn
O
N
H
COOBn
COOBn
COOBn
Currently, a number of dendrimer classes (e.g., glycol-
dendrimers,4 m-terphenyl surfaced dendrimers5) have
been reported as antibacterial agents. However, research
on antibacterial dendrimers has mainly focused on cation-
ic dendrimers, such as PAMAM dendrimers and their
PEGylated derivates,6 quaternary ammonium functional-
ized PPI dendrimers,7 amine and ammonium terminated
carbosilane dendrimers,8 and peptide dendrimers that are
rich in Arg and/or Lys sequences.9 The cationic den-
drimers show antibacterial activity because they are able
to adhere to and damage the anionic bacterial membrane
so as to cause bacterial lysis.7a Meanwhile, the use of cat-
ionic dendrimers in biological systems is constrained be-
cause of the inherent toxicity, which is also attributed to
the interaction of the surface cationic charge of the den-
N
H2N
H
N
O
O
3a n = 1
3b n = 2
[G1]-dendron
COOBn
O
COOBn
NH
O
O
COOBn
H
O
H
N
N
N
O
COOBn
COOBn
N
H
N
H2N
O
O
N
N
H
O
COOBn
NH
O
COOBn
4a n = 1
4b n = 2
[G2]-dendron
SYNLETT 2012, 23, 1937–1940
Advanced online publication: 16.07.2012
COOBn
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6
DOI: 10.1055/s-0031-1290403; Art ID: ST-2012-W0323-L
© Georg Thieme Verlag Stuttgart · New York
Figure 1 [G1] and [G2] Dendritic Asp/Glu17

1938
J. Pan et al.
LETTER
In our previous work, dendritic L-aspartic acid (L-Asp) tween core 5 and myristic acid. Subsequently, activation
and L-glutamic acid (L-Glu) were prepared as drug deliv- of the focal point was achieved by removal of the benzyl
ery systems (Figure 1).17 We considered that the applica- group from 6 by catalytic hydrogenolysis, giving com-
tion of these peptide dendrons could be expanded to pound 7 (Scheme 1)
construct anionic dendrimers as antibacterial agents be-
The synthetic procedure used to access the target den-
cause (i) L-Asp and L-Glu are promising surface blocks to
drimers involved two steps: assembly of the two dendrons
supply multivalent anions; (ii) peptide dendrimers based
together, and removal of the protecting groups (Scheme
on natural amino acids are generally biocompatible and
1). Generally, the coupling of two different dendrons by
immunocompatible.18 In this study, we synthesized a se-
connecting their cores is challenging because of the steric
ries of Janus peptide dendrimers as Grinstaff type anionic
hindrance at dendron focal points, which may render the
dendrimers. The amphiphilic Janus dendrimers contained
coupling inefficient and then lead to low yields. In order
myristic acid as the lipophilic end, and negatively charged
to assemble the two dendrons effectively, several cou-
native amino acids as the hydrophilic end. The antibacte-
pling reagents, such as EDCI/1-hydroxy benzotriazole
rial activity and cytotoxicity of the new dendrimers were
(HOBt), o-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroni-
um hexafluorophosphate (HBTU)/DIPEA and isobutyl
chloroformate (IBCF)/NMM were tried; it was found that
evaluated through two-fold serial dilution method and
MTT assays, respectively.
First, the two types of the functional dendrons were pre- use of the IBCF/NMM system provided the best yields.
pared by a convergent approach. The [G1] and [G2] den- Finally, after removal of the protected groups (benzyl) by
dritic Asp/Glu were synthesized according to our previous catalytic hydrogenolysis, the target molecules 1a/b and
report.17 The [G1]-dendritic myristic acid 6 was prepared 2a/b were obtained. Quantitative coupling was proven by
by utilizing EDCI and DMAP as the coupling reagents be- 1H NMR spectroscopic analysis, which revealed reso-
O
O
OH
O
O
N
a
O
N
RO
BnO
O
O
b
O
OH
5
6 R = Bn
7 R = H
c
O
COOH
COOH
COOH
COOH
O
O
O
O
N
H
N
N
O
N
H
N
H
O
d
O
O
1a n = 1
1b n = 2
COOH
O
COOH
NH
O
O
O
COOH
H
O
O
N
H
N
N
N
O
O
O
COOH
COOH
N
N
O
O
H
N
N
H
O
O
N
H
O
COOH
NH
2a n = 1
2b n = 2
O
COOH
COOH
Scheme 1 Preparation of the Janus Dendrimer. Reagents and conditions: (a) myristic acid, EDCI, DMAP, CH2Cl2, r.t., 24 h, 82%; (b) H2, Pd/C
(10 wt%), CH2Cl2–MeOH, r.t., 8 h, 99%; (c) i. 3a/3b, IBCF, NMM, THF, –15 °C to r.t., 24 h; ii. H2, Pd/C (10 wt%), MeOH, r.t., 24 h (1a: 75%;
1b: 72% for two steps); (d) i. 4a/4b, IBCF, NMM, THF, –15 °C to r.t., 24 h; ii. H2, Pd/C (10 wt%), MeOH, r.t., 24 h (2a: 64%; 2b:61% for two
steps).
Synlett 2012, 23, 1937–1940
© Georg Thieme Verlag Stuttgart · New York

LETTER
Synthesis and Antibacterial Activity of Peptide Dendrimers
1939
nance signals that could be assigned to the two opposite
sides. For Asp-grafted dendrimers 1a and 2a, the struc-
tures were confirmed by the appearance of peaks at δ =
4.50 ppm (Asp-α-CH) and the peaks at δ = 0.85, 1.23, and
1.49 ppm (signals of myristic acid). For Glu-grafted den-
drimers 1b and 2b, the peaks of Glu-α-CH were over-
lapped with those of the dendritic skeleton protons; in this
case the peaks at δ = 1.75 ppm (Glu-β-CH2) could be used
to compare with the peaks of myristic acid, which con-
firmed the perfect joining. Furthermore, the formation of
well-defined dendrons and dendrimers was further veri-
fied by ESI MS analysis, and elemental analysis was also
in good agreement with those of the target structures.19
Table 1 Antibacterial Activity of Janus Dendrimers
Compounds
MIC (mM)
E. colia
S. aureusb MRSAc
E. faecalisd
1a
>0.256
>0.256
0.064
0.032
0.001
0.001
>0.256
>0.256
0.064
0.128
0.002
0.001
>0.256
>0.256
0.016
>0.256
>0.256
0.128
0.256
0.002
0.001
1b
2a
2b
0.128
Cefotaxime
Ampicillin
0.128
>0.256
The antibacterial activity of Janus peptide dendrimers
against strains of four pathogenic bacteria, including two
standard strains (E. coli and S. aureus) and two clinical
strains (methicillin-resistant S. aureus MRSA and E. fae-
calis), were determined by applying the two-fold serial di-
lution method.20 The minimum inhibitory concentration
(MIC) values are presented in Table 1. Dendrimers 1a and
1b were inactive even when the concentration was in-
creased to the highest level (0.256 mM), while 2a and 2b
showed moderate activity against most of the tested
pathogens. We supposed that the dramatically different
antibacterial activities between 1a/b and 2a/b were
caused by the difference of amphipathicity. For various
known antibacterial agents, either macromolecule (den-
drimer and polymer) or small molecular compounds, suit-
able amphipathicity was important for the antibacterial
activity because the amphiphilic structure acted through
perturbation and disruption of the prokaryotic mem-
brane.21 For the dendrimers studied in this paper, while the
lipophilic dendrons are the same, 2a/b (with [G2]-Asp/Glu
dendron) possessed a higher number and more condensed
hydrophilic groups than 1a/b (with [G1] -Asp/Glu den-
dron). Presumably, the hydrophilic/lipophilic ratio of 2a
and 2b provided appropriate amphipathicity for the anti-
bacterial activity. Moreover, 2a was the most active com-
pound; revealing an MIC value of 0.016 mM against
MRSA compared with 0.128 mM for Cefotaxime and
more than 0.256 mM for Ampicillin.
a Escherichia coliATCC25922.
b Staphyloccocus aureus ATCC29213.
c Methicillin-resistant Staphylococcus aureus. Clinical isolated strain
from West China Hospital, Chengdu, P. R. of China.
d Enterococcus faecalis. Clinical isolated strain from West China
Hospital, Chengdu, P. R. of China.
Figure 2 Cytotoxicity of dendrimers against HEK293 cells over a 24
hour incubation period determined by MTT assay. Cell viability was
expressed as a percentage of the control cell culture. Values are rep-
resented as mean ± SD (n = 3).
ther 2a nor 2b exhibited significant cytotoxicity against
HEK293 cells at concentrations up to 1 mM. The informa-
tion presented here may be used to expand the understand-
ing of dendrimers as antibacterial agents and for the
design of new compounds with improved antibacterial po-
tential.
Cytotoxicity is one of the most important factors to be
considered in selecting dendrimers for biomedical appli-
cations. The toxicity of 2a and 2b against HEK293 cells
was evaluated by MTT assays20 and the results are shown Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett. Included are
detailed synthetic procedures and characterization data, as well as
methods for antibacterial and toxicity experiments.SnoIufproi
in Figure 2. The assays demonstrated that neither 2a nor
2b was significantly toxic against HEK293 cells at con-
centrations up to 1 mM. The synthesized anionic den-
drimers based on natural amino acids were nontoxic, as
expected.
m
tgioSrantnugIifoop
r
imaotrtn
References and Notes
In summary, we synthesized a series of Janus peptide den-
drimers that showed amphipathicity due to the multivalent
anions and alkyl chains. The antibacterial activity of these
dendrimers against strains of four pathogenic bacteria was
tested in vitro. It was found that 2a and 2b were moderate-
ly active against the majority of the tested pathogens. Es-
pecially, 2a revealed an MIC value of 0.016 mM against
MRSA. Moreover, cell viability studies showed that nei-
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2a: Compound 7 (313 mg, 0.5 mmol) dissolved in anhyd
THF (25 mL) was cooled to –15 °C, and NMM (10 mmol)
and IBCF (10 mmol) were added. After stirring for 10 min,
4a (900 mg, 0.5 mmol) dissolved in anhyd THF (10 mL) was
added dropwise. The reaction mixture was vigorously stirred
at r.t. for 24 h, then the solution was concentrated under
vacuum, and the residue was taken up in EtOAc (30 mL) and
washed with 1 M HCl (10 mL), 1 M NaHCO3 (10 mL), and
brine (10 mL). The organic layer was dried over anhyd
Na2SO4. After concentration, the residue was purified by
silica gel column chromatography (CH2Cl2–MeOH) to
obtain a white waxy solid. Pd/C (10%, 100 mg) was added
to a solution of the obtained white waxy solid in MeOH–
CH2Cl2 (30 mL, 3:1 v/v). The reaction mixture was stirred
under a hydrogen atmosphere for 24 h, filtered through a
membrane filter, and concentrated under reduced pressure to
afford 2a (539 mg, 64%) as a white foam. 1H NMR
(400 MHz, DMSO-d6): δ = 0.83–0.87 (t, J = 6.4 Hz, 6 H,
2 × CH3), 1.23 (br s, 40 H, myristic acid 20 × CH2), 1.48–
1.49 (m, 4 H, myristic acid-β-CH2 × 2), 2.24–2.35 (m, 8 H,
myristic acid-α-CH2 × 2 + Asp-β-CH2/2 × 4), 2.38–2.59 (m,
16 H, succinic acid CH2 × 2 + NCH2CH2CONH × 6), 2.63–
2.70 (m, 4 H, Asp-β-CH2/2 × 4), 3.41–3.60 (m, 16 H,
NCH2CH2O × 3 + NCH2CH2CONH × 3), 3.99 (br s, 6 H,
Gly-CH2 × 3), 4.05–4.18 (m, 4 H, NCH2CH2O × 2), 4.48–
4.54 (m, 4 H, Asp-α-CH × 4), 7.89 (br s, 1 H, CONH), 7.96
(br s, 1 H, CONH), 8.10 (br s, 1 H, CONH), 8.25 (br s, 1 H,
CONH), 8.27 (br s, 1 H, CONH), 8.39 (br s, 1 H, CONH),
8.41 (br s, 1 H, CONH), 12.64 (br s, 8 H, Asp-COOH × 8).
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N, 9.01; O, 29.31.
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