16
I. Güell et al. / Peptides 33 (2012) 9–17
active than the unsubstituted analog BP244. Interestingly, pep-
tidotriazoles BP245 (Lys6(Tr-Ahx)), BP246 (Lys9(Tr)) and BP247
(Lys9(Tr-Ahx)) exhibited higher activity than BP100 against X.
axonopodis pv. vesicatoria and E. amylovora (MIC of 1.6–3.1 M).
The analysis of the antibacterial activity of peptidotriazoles
BP248–BP250, in which the benzene ring of Phe4 in BP100 was
replaced by a triazole, pointed out that the hydrophobicity at this
position is crucial. In fact, the analog incorporating a triazole sub-
stituted with a 2-aminohexanoic acid (BP249) was the least active,
whereas the peptidotriazole bearing a benzyl group at the tria-
In summary, we have designed and synthesized BP100 analogs
containing a triazole ring. The introduction of this moiety at a
Lys or a Phe side chain has led to the identification of sequences
active against X. axonopodis pv. vesicatoria, E. amylovora, P. syringae
pv. syringae, and F. oxysporum with low hemolytic activity, high
stability to protease digestion and no phytotoxicity, being good
candidates to design new antimicrobial agents. Efficacy tests with
selected peptidotriazoles in pathogen inoculated host plants are
under progress to confirm their potential as plant protection prod-
ucts.
Acknowledgments
The antifungal activity of the peptidotriazoles correlated with
that previously reported for structurally related undecapeptides,
which showed that F. oxysporum was more susceptible than P.
expansum to these compounds [4]. As for antibacterial activ-
Imma Güell was recipient of a predoctoral fellowship from
the Generalitat de Catalunya. This work was supported by grant
AGL2009-13255-C02-02/AGR from MICINN of Spain. We also
acknowledge the Serveis Tècnics de Recerca of the University of
Girona for the ESI-MS analysis.
ity, the introduction of
a triazole at the peptide backbone
resulted in sequences not active against these fungi. Peptidotria-
zoles BP238–BP247, bearing a triazole at a Lys side chain, were
more active than those including a triazole ring at position 4
(BP248–BP250), being the best derivatives as active as BP100.
Moreover, the incorporation of a substituent in the triazole ring
did not influence the antifungal activity.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
Peptidotriazole cytotoxicity was also strongly influenced by
the amino acid (Lys or Phe) that was modified with a triazole
as well as by the absence or presence of a substituent in this
heterocyclic ring. Peptidotriazoles resulting from the modifica-
tion of the Phe4 were less hemolytic than those obtained from
the incorporation of a triazole at a Lys side chain. For the for-
mer (BP248–BP250), the replacement of the benzene ring by a
triazole, either substituted or unsubstituted, rendered sequences
with a lower hydrophobic character than that of BP100, being
not hemolytic even at 150 M. For the derivatives incorporating
a triazole at a Lys side chain (BP238–BP247), the least hemolytic
sequences were those bearing a 2-aminohexanoic acid substituent
(0.6–21% at 50 M). In this case, the presence of this substituent
also decreases the hydrophobic character of the triazole moi-
suggesting that the triazole ring confers a higher hydrophobic char-
acter than the free -amino group of a Lys. These results are in good
agreement with previous studies on antimicrobial peptides report-
ing that an increase of the peptide hydrophobicity is related to an
increase in the cytotoxicity [7,36]. Peptides with an optimal bal-
ance between antibacterial and hemolytic activities were BP238,
BP239, BP243, BP245, BP247, and BP250. Among them, BP238,
BP239, BP243, and BP245 also showed high antifungal activity. In
addition, we examined the toxicity of the peptidotriazoles against
tobacco leaves. In contrast to mellitin which severely damaged
the leaf mesophyll tissues, these compounds did not induce any
phytotoxicity.
Protease digestion stability is a desired property in antimicro-
bial peptides to assure a reasonable half-life of the molecule in the
plant environment. Proteases from epiphytic microorganisms or
intrinsic to the plant in internal tissues may degrade antimicro-
bial peptides [2,9]. The incorporation of a triazole moiety at the
peptide backbone strongly increased the stability to protease diges-
tion. The modification of Lys5 and Lys6 with an unsubstituted or
substituted triazole as well as the incorporation of a triazole substi-
tuted with a 2-aminohexanoic acid at Lys9 afforded the least stable
peptidotriazoles (BP242–BP245 and BP247). The rest of sequences
showed similar protease susceptibility than that of the parent pep-
tide BP100. Peptidotriazoles with a good biological profile, BP238,
BP239, and BP250, also showed good stability toward protease
degradation. Especially noteworthy is BP250 that is active against
the three bacteria, not hemolytic and more stable to proteases than
BP100.
References
[1] Agrios GN. Plant Pathology. 4th ed. California: Academic Press; 1998.
[2] Ali GS, Reddy ASN. Inhibition of fungal and bacterial plant pathogens by syn-
thetic peptides: in vitro growth inhibition, interaction between peptides, and
inhibition of disease progression. Mol Plant-Microbe Interact 2000;13:847–59.
[3] Aufort M, Herscovici J, Bouhours P, Moreau N, Girard C. Synthesis antibiotic
activity of a small molecules library of 1,2,3-triazole derivatives. Bioorg Med
Chem Lett 2008;18:1195–8.
[4] Badosa E, Ferre R, Francés J, Bardají E, Feliu L, Planas M, Montesinos E. Sporicidal
activity of synthetic antifungal undecapeptides and control of penicillium rot
of apples. Appl Environ Microbiol 2009;75:5563–9.
[5] Badosa E, Ferre R, Planas M, Feliu L, Besalú E, Cabrefiga J, Bardají E, Mon-
tesinos E. A library of linear undecapeptides with bactericidal activity against
phytopathogenic bacteria. Peptides 2007;28:2276–85.
[6] Bechinger B, Lohner K. Detergent-like actions of linear amphipathic cationic
antimicrobial peptides. Biochim Biophys Acta 2006;1758:1529–39.
[7] Blondelle SE, Lohner K. Combinatorial libraries:
a tool to design antimi-
crobial and antifungal peptide analogues having lytic specificities for
structure–activity relationship studies. Biopolymers 2000;55:74–87.
[8] Cantel S, Isaad AC, Scrima M, Levy JJ, DiMarchi RD, Rovero P, Halperin JA, D’Ursi
AM, Papini AM, Synthesis Chorev M. conformational analysis of a cyclic peptide
obtained via i to i + 4 intramolecular side-chain to side-chain azide–alkyne 1,3-
dipolar cycloaddition. J Org Chem 2008;73:5663–74.
[9] Cavallarin L, Andreu D, San Segundo B. Cecropin A-derived peptides are
potent inhibitors of fungal plant pathogens. Mol Plant-Microbe Interact
1998;11:218–27.
[10] Chakrabarty SP, Ramapanicker R, Mishra R, Chandrasekaran S, Balaram H.
Development and characterization of lysine based tripeptide analogues as
inhibitors of sir2 activity. Bioorg Med Chem 2009;17:8060–72.
[11] Chassaing S, Sani Souda Sido A, Alix A, Kumarraja M, Pale P, Sommer J. Click
chemistry in zeolites: copper(I) zeolites as new heterogeneous and ligand-free
catalysts for the Huisgen [3+2] cycloaddition. Chem Eur J 2008;14:6713–21.
[12] Evans RA. The rise of azide–alkyne 1,3-dipolar ‘Click’ cycloaddition and
its application to polymer science and surface modification. Aust J Chem
2007;60:384–95.
[13] Fan WQ, Katritzky AR. In: Katritzky AR, Rees CW, Scriven VEFB, editors. Com-
prehensive Heterocyclic Chemistry II, vol. 4. Oxford: Elsevier Science; 1996. p.
1–126.
[14] Ferre R, Badosa E, Feliu L, Planas M, Montesinos E, Bardají E. Inhibition of plant-
pathogenic bacteria by short synthetic cecropin A-melittin hybrid peptides.
Appl Environ Microbiol 2006;72:3302–8.
[15] Franke R, Doll C, Eichler J. Peptide ligation through click chemistry for
the generation of assembled and scaffolded peptides. Tetrahedron Lett
2005;46:4479–82.
[16] Güell I, Cabrefiga J, Badosa E, Ferre R, Talleda M, Bardají E, Planas M, Feliu L, Mon-
tesinos E. Improvement of the efficacy of linear undecapeptides against plant
pathogenic bacteria by incorporating d-amino acids. Appl Environ Microbiol
2011;77:2667–75.
[17] Hancock REW, Sahl HG. Antimicrobial and host defense peptides as new anti-
infective therapeutic strategies. Nature Biotechnol 2006;24:1551–7.
[18] Hein JE, Fokin VV. Copper-catalyzed azide–alkyne cycloaddition (CuAAC)
and beyond: new reactivity of copper(I) acetylides. Chem Soc Rev
2010;39:1302–15.