Synthesis of antimicrobial cyclodextrins bearing polyarylamino and polyalkylamino groups via click chemistry for bacterial membrane disruption

Article abstract of DOI:10.1039/c3cc49543d

Cyclodextrin derivatives are synthesized as membrane-disrupting agents via a microwave-assisted Huisgen reaction. Their ability to permeabilize bacterial membranes depends on the amino substituents and an appropriate balance of hydrophobicity and hydrophilicity, thus enabling the preparation of derivatives with selective toxicity against bacteria. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c3cc49543d

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
Synthesis of antimicrobial cyclodextrins bearing
polyarylamino and polyalkylamino groups via click
chemistry for bacterial membrane disruption†
Hatsuo Yamamura,*a Yuuki Sugiyama,a Kensuke Murata,a Takanori Yokoi,a
Ryuji Kurata,a Atsushi Miyagawa,a Kenji Sakamoto,b Keiko Komagoe,b
Tsuyoshi Inoueb and Takashi Katsub
Cite this: Chem. Commun., 2014,
50, 5444
Received 17th December 2013,
Accepted 3rd February 2014
DOI: 10.1039/c3cc49543d
www.rsc.org/chemcomm
Cyclodextrin derivatives are synthesized as membrane-disrupting agents activity.6 Recently we reported that CD derivatives possessing poly-
via a microwave-assisted Huisgen reaction. Their ability to permeabilize amino groups (more than six on the molecule) strongly disrupted
bacterial membranes depends on the amino substituents and an appro- bacterial membranes, and the addition of benzyl moieties to the
priate balance of hydrophobicity and hydrophilicity, thus enabling the amino groups enhanced the membrane permeabilization and inhib-
preparation of derivatives with selective toxicity against bacteria.
ited bacteria proliferation in a manner similar to that of the natural
peptides.7 These results suggest that functional groups bearing
positively charged amino groups that interact with anionic phosphate
moieties and hydrophobic groups that interact with fatty acid moieties
of bacterial membranes are promising. Thus, we studied CD deriva-
tives possessing arylamino and alkylamino moieties to enable
peptide-like antimicrobial activity. However, while the synthetic yield
of octakis(6-benzylamino)-g-CD 1 prepared via the reaction of benzyl-
amine as a nucleophile with a per-tosylated CD derivative was
moderate,7 polyfunctionalization of CD using other arylamines and
alkylamines did not afford the desired compounds in satisfactory
yields (data not shown). Such polymodifications resulted in compli-
cated mixtures containing products with lower levels of functionaliza-
tion than expected. To solve these problems, therefore, we herein
report that polyfunctionalization of CD molecules via a versatile,
microwave (MW)-assisted Huisgen 1,3-dipolar cycloaddition reaction,8
known as the click chemistry reaction,9 affords a new series of
antimicrobial g-CD derivatives.
Antimicrobial peptides have been recognized as promising agents
against multidrug-resistant bacteria.1 They contain positively charged
hydrophilic moieties that interact with negatively charged bacterial
cell membranes, while their hydrophobic groups interact with fatty
acid moieties in the lipid membrane to trigger membrane disruption.
They do not act on a specific cellular enzyme or receptor, but on the
bacterial membrane itself, and therefore exhibit broad-spectrum
antibacterial activity. Therefore, unlike conventional antibiotics, the
development of resistance is difficult.1c,2 Our research on membrane
active antibacterial peptide gramicidin S and its analogues3 and a
review of the literature4 on other types of peptides gave us the idea
that a structure–activity relationship study could be used to design
much simpler compounds with the biological activity of antimicrobial
peptides. Thus, cyclodextrins (CDs), cyclic oligosaccharides consisting
of six, seven, or eight D-glucose residues (a-, b-, or g-CDs, respectively)
were selected for the study. CDs have a truncated cone structure in
which one side of the cone is rimmed by the primary 6-OH groups of
the glucose residues, and the other by secondary 2- and 3-OH groups.
Our established regiospecific method for the polyfunctionalization of
CD was useful for enabling the attachment of membrane active
functionalities onto the molecule.5 Notably, the diameter of CDs is
ca. 1 nm, which is nearly the same as that of gramicidin S. An
important advantage of CDs is the rigidity of the cyclic structure due
to hydrogen bonding between the residues. In peptides, substitution
and insertion of amino acid residue(s) often cause changes in the
conformation of the peptides that result in reduced antibacterial
a Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho,
Showa-ku, Nagoya 466-8555, Japan. E-mail: yamamura.hatsuo@nitech.ac.jp
b Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,
Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama 700-8530, Japan
† Electronic supplementary information (ESI) available: Syntheses of the com-
pounds and bioactivity assays. See DOI: 10.1039/c3cc49543d
First, benzylamino groups were introduced onto a CD molecule
via the MW-assisted Huisgen reaction (120 1C, 20 min) of octaazido-
g-CD derivative 28 with N-benzyl-N-2-propynylamine, affording the
desired derivative 3 in 77% yield. To estimate its antimicrobial
5444 | Chem. Commun., 2014, 50, 5444--5446
This journal is ©The Royal Society of Chemistry 2014

View Article Online
Communication
ChemComm
products were sufficiently hydrophobic for ordinary handling, such
as purification via silica gel chromatography using an organic
solvent; thus, this synthetic method described for compounds
8–10 was very versatile and developed as an alternative to using
unprotected azide 1 and an alkyne. With respect to their antibiotic
activities, the cyclopentyl and cyclobutyl compounds 8 and 9, respec-
tively, were promising (Fig. 1). The MICs of 8 were 4 mg cmÀ3 and
8 mg cmÀ3 against gram-positive and -negative strains, respectively,
while those of 9 were 8 mg cmÀ3, 8 mg cmÀ3, and 32 mg cmÀ3 against
gram-positive strains, E. coli, and S. typhimurium. In contrast, the
cyclopropyl compound 10 exhibited dramatically lower antimicrobial
Fig. 1 MIC values for CD derivatives: black bars, S. aureus; gray bars,
B. subtilis; white bars, E. coli; and light gray bars, S. typhimurium.
activity, the inhibition of bacterial proliferation was observed using activity (MIC 4 128 mg cmÀ3). These results suggest that there may
the liquid microdilution method.10 Values for the minimum be optimum ring size(s) for antimicrobial activity.
inhibitory concentration (MIC) of the HCl salt of 3 against the
Next, to determine whether the observed antimicrobial activity of
gram-positive strain Staphylococcus aureus and the gram-negative the CD derivatives was attributed to membrane disruption, K+ efflux
strain Escherichia coli were 16 and 32 mg cmÀ3, respectively (Fig. 1). from bacterial cells and cell viability were examined.7,10 Bacteria
Notably, these values are nearly equal to those of derivative 1, whose maintain large K+ concentrations inside their cells (approximate
benzylamino groups are directly bound to the C6 carbons.7 Com- concentration 100–500 mM), and the permeabilization of their
pound 3 was also active against both the gram-positive strain Bacillus membranes causes ion efflux and subsequent bacterial death. The
subtilis and the gram-negative strain Salmonella typhimurium. These K+ efflux from S. aureus cells as a function of the concentration of
results demonstrate that the methyltriazole moiety introduced via cyclohexyl derivative 6 was measured using a K+ electrode (Fig. 2); it
the Huisgen reaction does not adversely affect the antimicrobial was found that the increase in K+ permeability was proportional to
activity. Interestingly, 2-naphtylmethylamino derivative 4,8 bearing a the decrease in cell viability. These results provided direct evidence
larger aromatic substituent than that in 3, and phenylpentylamino that 6 disrupted the cytoplasmic membrane of S. aureus and
derivative 5, with the same number of carbons as in 4, exhibited inhibited its growth. The K+ efflux from E. coli was also observed; it
decreased antimicrobial activity (MIC 4 64–128 mg cmÀ3).
was determined that 6 disrupted not only the cytoplasmic membrane
Next, the aromatic benzene ring in 3 was replaced with an aliphatic of E. coli but also its outer membrane. However, the effect seemed to
cyclohexyl ring (compound 6). The MIC of 6 was 8 mg cmÀ3 against be smaller than that observed for S. aureus. The outer membrane of
the gram-positive strains S. aureus and B. subtilis and 16 mg cmÀ3 gram-negative strains is known to be a barrier for antibiotics and is
against the gram-negative strains E. coli and S. typhimurium (Fig. 1). strengthened by divalent cations, such as Ca2+ and Mg2+. Pretreat-
Thus, the replacement of the benzyl group with a cyclohexylmethyl ment of E. coli with ethylenediamine tetraacetic acid (EDTA) led to a
group (both six-membered rings) enhanced the antimicrobial activity drastic increase in the K+ efflux upon the addition of 6. EDTA
by a factor of two. On the other hand, changing the cyclic structure to removes the ions from the outer membrane, triggering the release
a linear chain remarkably reduced the antimicrobial activity; the MIC of lipopolysaccharides and the disruption of the membrane;11 there-
of 78 against S. aureus and E. coli was 4128 mg cmÀ3. These results fore, compound 6 could reach the cytoplasmic membrane.
suggested that an aliphatic ring moiety was promising.
The CD concentrations giving 50% K+ efflux (ED50) and those
Therefore, CDs 8, 9, and 10 containing cyclopentyl, cyclobutyl, giving a 50% decrease in bacterial cell survival (LC50) for antibiotics
and cyclopropyl aliphatic ring structures, respectively, were prepared. 3, 6, 8, 9, and 10 are summarized in Table 1. All derivatives showed a
Cyclopentylmethylamino derivative 8 was synthesized via the click good correlation between the K+ efflux and cell viability similar to
reaction of per-2,3-acetylated g-CD octaazide 118 with N-Boc-N- that of 6, which clearly demonstrates that the point of action of CDs
cyclopentylmethyl-N-2-propynylamine (yield 64%), followed by the is the cytoplasmic membrane of gram-positive and gram-negative
deprotection of the acetyl groups using NaOMe–MeOH and the Boc strains. Notably, CDs do not seem to disrupt the bacterial mem-
group using TFA (yield 92%). It should be noted that the click branes via transmembrane-pore formation, which results in the
Fig. 2 Dose-dependence curves for the K+ efflux and cell viability of bacteria upon addition of 6. (a) S. aureus, (b) E. coli (native), (c) E. coli (treated with
EDTA). Solid lines; K+ efflux; broken lines: cell viability. Cells were incubated with the CD derivative for 30 min at 37 1C. To determine the 100% level of
K+ efflux, melittin (10 mM for S. aureus) and polymixin B (200 mg cmÀ3 for E. coli) were used.
This journal is ©The Royal Society of Chemistry 2014
Chem. Commun., 2014, 50, 5444--5446 | 5445

View Article Online
ChemComm
Communication
Table 1 ED50 (CD concentration giving 50% K+ efflux, mM) and LC50 (CD
concentration giving 50% bacterial cell viability, mM) for CDs 3, 6, and 8–10a
derivative with excessive hydrophobicity, such as 4 with naphthyl
groups, did not disrupt the bacterial membrane, while CD 6 with a
cyclohexyl group and slightly lower hydrophobicity permeabilized
both bacterial and red blood cell membranes. Reducing the size of
the hydrophobic ring to phenyl, cyclopentyl, and cyclobutyl groups
led to retention of antibiotic activity with a dramatic decrease in
toxicity against red blood cell membranes, while further reduction in
size to a cyclopropyl ring led to no membrane activity. Thus, a
balance between hydrophobicity and hydrophilicity (or cationic
properties) may be important for expressing bioactivity.
E. coli
S. aureus
(Native)
(EDTA treated)
ED50/mM LC50/mM ED50/mM LC50/mM ED50/mM LC50/mM
3
6
8
9
20
20
450
50
50
450
450
100
450
50
50
450
450
—
50
20
10
50
450
10
50
20
10
50
450
—
5–10
5–10
20
5–10
5–10
20
450
5–10
10
450
GSb 5–10
In conclusion, CD derivatives possessing arylamino and alkyl-
a
Cells were incubated with a CD and peptide for 30 min at 37 1C. amino groups were prepared as novel membrane-disrupting
Gramicidin S, see ref. 12.
b
agents. The functionalization was accomplished via a versatile
release of inner ions in seconds.4 At the concentrations corre-
sponding to the MICs against S. aureus, the K+ efflux was only
20–30%, and therefore more than 60% of the bacteria were alive
when treated with CDs for 30 min under the experimental conditions
used in this study.
MW-assisted Huisgen reaction. Their bioactivity for the permeabi-
lization of bacterial and animal membranes depended on the
substituents on the amino group, leading to the possibility for
selective toxicity against bacteria. It is noteworthy that CD deriva-
tives possessing one kind of simple substituents exhibited activity
similar to that of peptides composed of a variety of amino acids.
The results presented herein demonstrate that our chemistry to
develop novel antimicrobial agents by use of CDs is promising.
Further studies are currently underway.
Next, the hemolysis of rabbit red blood cells by CDs 3, 6, and 8–10
was examined to determine whether they disrupt the membranes of
animal cells (Fig. 3). Focusing on the benzylamino group, compound
3 exhibited significantly less hemolysis at the experimental concen-
tration (B50 mM), while 1,7 with benzylamino groups bonded
directly to the C6 carbons, measurably permeabilized the red blood
cells (90% hemolysis at 50 mM), even though the MICs for the two
compounds were nearly the same. In addition, Fahmi et al.
described that the CD derivative for which the triazole group of 3
was replaced with an amido group exhibited reduced antimicrobial
activity.12 The triazole group, therefore, works very well not only for
versatile click functionalization but also for selective membrane
disruption. The cyclohexylmethylamino group of 6, however, per-
meabilized both bacterial and animal cell membranes. Notably,
removal of one of the methylene groups (cyclopentyl substituent in
compound 8) dramatically decreased the hemolytic activity. Its
activity below 20 mM was less than 10%, although its MIC against
S. aureus was 8 mg cmÀ3 (3 mM). Furthermore, the antimicrobial
cyclobutyl derivative 9 showed almost no hemolytic activity in the
dose range, while the cyclopropyl derivative 10 did not disrupt the
membrane. It is interesting that the membrane permeabilization
activity of the CD derivatives depends on their substituents. A CD
This work was supported by a Grant-in-Aid for Scientific
Research (KAKENHI 25460036) from the Japan Society for the
Promotion of Science.
Notes and references
1 (a) J. Li, R. L. Nation, R. W. Milne, J. D. Turnidge and K. Coulthard,
Int. J. Antimicrob. Agents, 2005, 25, 11; (b) A. P. Zavascki, L. Z. Goldani
and R. L. Nation, J. Antimicrob. Chemother., 2007, 60, 1206;
(c) M. Zasloff, Nature, 2002, 415, 389; (d) R. E. W. Hancock and
H. G. Sahl, Nat. Biotechnol., 2006, 24, 155.
2 M. R. Yeaman and N. Y. Yount, Pharmacol. Rev., 2003, 55, 27.
3 (a) K. Yamada, M. Unno, K. Kobayashi, H. Oku, H. Yamamura, S. Araki,
H. Matsumoto and R. Katakai, J. Am. Chem. Soc., 2002, 124, 12684;
(b) M. Kawai, R. Tanaka, H. Yamamura, K. Yasuda, S. Narita,
H. Umemoto, S. Ando and T. Katsu, Chem. Commun., 2003, 1264.
4 W. C. Wimley, ACS Chem. Biol., 2010, 5, 905–917 and references therein.
5 For example, see M. V. Rekharsky, H. Yamamura, M. Kawai and Y. Inoue,
J. Am. Chem. Soc., 2001, 123, 5360; H. Yamamura, M. V. Rekharsky,
Y. Ishihara, M. Kawai and Y. Inoue, J. Am. Chem. Soc., 2004, 126, 14224.
6 (a) H. Urakawa, K. Yamada, K. Komagoe, S. Ando, H. Oku, T. Katsu and
I. Matsuo, Bioorg. Med. Chem. Lett., 2010, 20, 1771; (b) V. V. Kapoerchan,
A. D. Knijnenburg, M. Niamat, E. Spalburg, A. J. de Neeling,
P. H. Nibbering, R. H. Mars-Groenendijk, D. Noort, J. M. Otero,
A. L. Llamas-Saiz, M. J. van Raaj, G. A. van der Marel, H. S. Overkleeft
and M. Overhand, Chem.–Eur. J., 2010, 16, 12174; (c) E. J. Prenner,
M. Kiricsi, M. Jelokhani-Niaraki, R. N. A. H. Lewis, R. S. Hodges and
R. N. McElhaney, J. Biol. Chem., 2005, 280, 2002.
7 H. Yamamura, K. Suzuki, K. Uchibori, A. Miyagawa, M. Kawai,
C. Omizo and T. Katsu, Chem. Commun., 2011, 48, 892.
8 H. Yamamura, K. Shimohara, R. Kurata, Y. Fujita, K. Murata,
T. Hayashi and A. Miyagawa, Chem. Lett., 2013, 6, 643.
9 (a) S. Srinivasachari, K. M. Fichter and T. M. Reineke, J. Am. Chem. Soc.,
´
´
´
2008, 130, 4618; (b) A. Mendez-Ardoy, M. Gomez-Garcıa, C. O. Mellet,
´
´
´
N. Sevillano, M. D. Giron, R. Salto, F. Santoyo-Gonzalez and
J. M. G. Fernandez, Org. Biomol. Chem., 2009, 7, 2657; (c) A. Martı,
´
´
´
´
C. Bienvieu, J. J. Blanco, P. Vierling, C. O. Mellet, J. M. Garcıa-Fernandez
and C. D. Giorgio, J. Org. Chem., 2013, 78, 8143.
10 T. Katsu, H. Kobayashi and Y. Fujita, Biochim. Biophys. Acta, 1986,
860, 608–619; T. Katsu, H. Nakagawa and K. Yasuda, Antimicrob.
Agents Chemother., 2002, 46, 1073.
11 C. Ohmizo and T. Katsu, J. Microbiol. Methods, 2004, 59, 173.
12 N. Fahmi, F. W. Schmidtman and S. Hecht, PP Pat., WO2006/
083678, 2006.
Fig. 3 Dose-dependence curves for hemolysis induced by the CD derivatives.
Rabbit erythrocytes were incubated with a CD derivative at 37 1C for 30 min,
and hemolysis was estimated by measuring the absorbance at 540 nm.
Lysolecithin (50 mM) was used to determine the 100% level of hemolysis.
5446 | Chem. Commun., 2014, 50, 5444--5446
This journal is ©The Royal Society of Chemistry 2014