α-peptide/β-peptoid chimeras

Article abstract of DOI:10.1021/ol070316c

Equation presented Minimum Inhibitory Concentrations (MICs) Ecoli = 15.6 μg/mL B. subtilis = 15.6 μg/mL S. aureus = 125 μg/mL We describe the synthesis and characterization of the first generation of oligomers consisting of alternating repeats of α-amino

Full text of DOI:10.1021/ol070316c

ORGANIC
LETTERS
2007
Vol. 9, No. 8
1549-1552
r
-Peptide/â-Peptoid Chimeras
Christian A. Olsen,*,†,§ Gitte Bonke,† Line Vedel,† Anne Adsersen,† Matthias Witt,‡
Henrik Franzyk,† and Jerzy W. Jaroszewski†
Department of Medicinal Chemistry, Faculty of Pharmaceutical Sciences,
UniVersity of Copenhagen, UniVersitetsparken 2, DK-2100 Copenhagen, Denmark, and
Bruker Daltonik GmbH, Fahrenheitstrasse 4, D-28359 Bremen, Germany
cao@farma.ku.dk
Received February 7, 2007
ABSTRACT
We describe the synthesis and characterization of the first generation of oligomers consisting of alternating repeats of
chiral N-alkyl- -alanine ( -peptoid) residues. These chimeras are stable toward proteolysis, non-hemolytic, and possess antibacterial activity
comparable to well-known antimicrobial agents. Moreover, the chimeras exhibit length-dependent, concentration-dependent, solvent-dependent,
r-amino acids and
â
â
and ion-strength-dependent ellipticity, indicating the presence of a secondary structure in solution. Thus,
a promising novel peptidomimetic backbone construct for biologically active ligands.
r-peptide/â-peptoid oligomers represent
Natural antimicrobial peptides (AMPs) have been developed
through evolution as host-defense systems of animals and
plants1,2 as well as fungi,3 providing immunity to attack by
a broad spectrum of microorganisms. Many AMPs adopt
amphipathic secondary structures displaying positively charged
domains believed to bind to negatively charged sites, which
are more abundant on the outer surface of microbial
membranes as compared to cytoplasmic membranes of higher
animals, thus facilitating selectivity for microbes.4-7
Peptides with a therapeutic potential such as plectasin may
be produced on an industrial scale by a fungal expression
system.3 Low molecular weight octapeptide lead compounds
have been discovered by high-throughput synthesis and
screening of libraries based on the natural AMP bactenecin.8
Nevertheless, an inherent weakness associated with antimi-
crobial peptides constructed from natural R-amino acids (1,
Figure 1) is their instability toward proteases, which severely
diminishes their bioavailability. This intrinsic obstacle has
been circumvented by the design of synthetic foldamers, i.e.,
biomimetic oligomers that exhibit a high propensity for
adopting specific secondary structures,9,10 for example,
† University of Copenhagen.
§ Current e-mail: caolsen@scripps.edu.
‡ Bruker Daltonik GmbH.
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10.1021/ol070316c CCC: $37.00
© 2007 American Chemical Society
Published on Web 03/13/2007

but with different side chain positioning. This design extends
the structural space available for side chain topology of the
constructs. Moreover, additional stabilization of potential
secondary structures was expected due to the possibility of
forming internal hydrogen bonds, possibly resulting in
compounds with attractive physical and biological features.
In our initial studies we selected lysine as the R-amino acid
residue, since peptide oligomers with cationic sites have
previously proved to exhibit antimicrobial activity. Recently,
Gellman and co-workers reported that “scrambled” R/â-
peptide analogues, designed not to exhibit a global amphi-
pathic structure, gave rise to antimicrobials with high
membrane selectivity (i.e., low hemolytic activity toward
mammalian cell membranes compared to the antibacterial
MIC values).25 Interestingly, our chimeric design, which has
alternating repeats of lipophilic and cationic residues, is not
expected to adopt globally amphipathic secondary structures
either.
Figure 1. Backbone structures of natural R-peptides and main
peptidomimetic designs.
oligomers of â-amino acids11 or R-chiral N-alkylated gly-
cines, i.e., â-peptides (2) and peptoids (3), respectively
(Figure 1).11,12 Mimicry of the non-hemolytic antimicrobial
activity of the medium-sized natural host-defense peptide
magainin13 with â-peptides,14-16 peptoids,17 N,N′-linked
oligoureas,18 and arylamide oligomers19 has been reported.
Furthermore, cationic N-alkylated â-alanine oligomers (â-
peptoids, 4, Figure 1) showing a moderate inhibitory activity
against E. coli have recently been reported,20 and self-
assembling cyclic D,L-R-peptide nanotubes have proved to
be efficient antimicrobial agents in mice.21
Recently, the first synthesis and characterization of ho-
momeric â-peptoid oligomers with R-chiral phenethyl side
chains were described by Arvidsson’s group.22 Here, we
present the synthesis, CD characterization, and antimicrobial
activity of this chiral N-(S)-phenethyl-â-alanine (â-Nspe)
motif within a chimeric framework including R-amino acid
residues.
Initially, a strategy involving a submonomer SPS of the
â-peptoid residues was considered; however, preliminary
experiments showed that SPPS couplings involving the
sterically hindered â-peptoid nitrogen atom did not perform
satisfactorily. Hence, a versatile dimeric building block (9)
was prepared in solution in three steps on gram-scale
(Supporting Information, Scheme S1). The 2-(trimethylsilyl)-
ethoxycarbonyl (Teoc) group was chosen for side chain
protection to allow on-resin derivatization of the side chain
amino groups upon oligomer assembly. Attachment of the
dimer 9 to a Rink amide resin followed by elongation by
SPPS, N-terminal acetylation, and removal of the Teoc
groups, afforded resin 10 (Scheme 1). The possibility of post-
assembly on-resin side chain functionalization was demon-
strated by guanidinylation and acylation to give 12 and 13,
respectively. This approach indeed furnished good overall
isolated yields of the oligomers (27-41%).
The chimeras were evaluated by CD spectroscopy in
several solvents and at various concentrations (Figure 2). The
CD curves of 11-13 show mean residue molar ellipticities
(MREs) around 219 nm (-15000 to -20000 deg cm2
dmol-1) that are much higher than those observed for
unordered (random coil) peptides, but in the same range as
those found for â-peptides and peptoids with a high helix
content. In all these structures, the observed Cotton effects
are exhibited at very similar wavelengths, and hence are
expected to be due to a common structural feature, namely
a helical polyamide backbone interacting with the side chain
chromophores via exciton coupling. Interestingly, 11 exhib-
ited the same trend in solvent effect on the CD spectra as
that previously observed for water-soluble peptoids, where
the molar ellipticity in methanol was diminished as compared
to phosphate buffer (pH 7), while addition of 20% of 2,2,2-
trifluoroethanol (TFE) to the aqueous medium resulted in
Although the structures of the chiral â-peptoid homooli-
gomers appeared to exhibit insignificant chain-length-de-
pendent CD behavior according to Arvidsson’s preliminary
investigations,22 we decided to explore oligomers of alternat-
ing R-amino acid and â-peptoid monomers giving rise to a
heteromeric backbone resembling that of R/â-peptides,23-25
(11) (a) Cheng, R. P.; Gellman, S. H.; DeGrado, W. F. Chem. ReV. 2001,
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R. N. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 4303-4308.
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1550
Org. Lett., Vol. 9, No. 8, 2007

Scheme 1. SPPS of Oligomers 11-13
Figure 2. (a) CD spectra of compound 11 (60 µM) in various
solvents. (b) CD-spectra of chimera 11 in phosphate buffer (pH 7)
at various concentrations. The solutions were prepared from
lyophilized hexakis(trifluoroacetates) of the peptide. MRE ) mean
residue ellipticity.
(Supporting Information, Figure S3). No significant concen-
tration-induced changes were observed below 200 µM in
either of the two solvents, but at higher concentrations the
CD spectra changed in intensity as well as in shape,
suggesting that the compounds are monomeric at biologically
relevant concentrations (<200 µM). On the other hand, the
apparent aggregation at higher concentrations will severely
complicate NMR studies.31
increased molar ellipticity.26 The CD intensity in neat TFE
resembled that in the aqueous buffer, while the spectrum
obtained in neat acetonitrile was considerably less intense.
Compounds 12 and 13 gave CD spectra similar to those of
11, but 13 was only tested in the organic solvents due to its
insolubility in water.27 The normalized CD spectrum of an
N-acylated dipeptide building block did not by itself exhibit
the characteristic saddle-shaped curve (Supporting Informa-
tion, Figure S2).
Next, we wished to assess whether the chain length and
the â-peptoid chirality influenced the CD spectra. Hence,
compounds (14-20, Figure 3b) were obtained by oligomer-
The water-soluble oligomers retained remarkably strong
CD spectra in aqueous media as compared to the trend
observed for â3-peptides when going from structure-promot-
ing organic solvents (e.g., MeOH and TFE) to aqueous
solutions.16 This property has previously been observed for
peptoids26 and â-peptides with conformationally restrained
backbone elements16,28 or side chain macrodipoles.29 Also,
addition of 1 M NaCl to the phosphate buffer affected the
shape of the CD curve, diminishing the intensity of the saddle
section and increasing the Cotton effect around 195 nm
considerably (Figure 2a, orange). The salt effect observed
for this chimera parallels that observed for R-helical peptides
due to charge screening by counterions.30
The concentration effect on the CD behavior was inves-
tigated in phosphate buffer (Figure 2b) and in MeOH
Figure 3. (a) CD spectra of compounds 12, 15-17, 19, and 20
(60 µM) in MeOH. The solutions were prepared from lyophilized
trifluoroacetate salts of the peptides. (b) Structures of compounds
14-20.
(26) Sanborn, T. J.; Wu, C. W.; Zuckermann, R. N.; Barron, A. E.
Biopolymers 2002, 63, 12-20.
(27) See the Supporting Information (Figure S1) for normalized CD
spectra of 12 and 13 in various solvents.
(28) Appella, D. H.; Christianson, L. A.; Klein, D. A.; Powell, D. R.;
Huang, X.; Barchi, J. J., Jr.; Gellman, S. H. Nature 1997, 387, 381-384.
(29) Hart, S. A.; Bahadoor, A. B. F.; Matthews, E. E.; Qiu, X. J.;
Schepartz, A. J. Am. Chem. Soc. 2003, 125, 4022-4023.
(30) For examples, see: (a) Kojima, S.; Kuriki, Y.; Sato, Y.; Arisaka,
F.; Kumagai, I.; Takahashi, S.; Miura, K.-i. Biochem. Biophys. Acta 1996,
1294, 129-137. (b) Smith, J. S.; Scholtz, J. M. Biochemistry 1998, 37,
33-40.
ization on Rink amide resin with use of pre-guanidinylated
building blocks 24a,b (Supporting Information, Scheme S2).
The CD spectra showed that lack of â-peptoid chirality (as
in 14-17) resulted in a substantial drop in the ellipticity
amplitudes at 219 nm, which indicates a low degree of
Org. Lett., Vol. 9, No. 8, 2007
1551

folding for these compounds. On the other hand, a compari-
son of the CD spectra of compounds 12 (dodecamer), 19
(tetradecamer), and 20 (hexadecamer) clearly revealed that
the minima at 219 nm increased in amplitude with increasing
chain length (Figure 3a). This trend contrasts the behavior
of â-peptoid homooligomers described recently by Arvidsson
and co-workers, where no significant chain-length effect on
CD spectra was observed. This suggests that hydrogen
bonding from the R-amino acid residues contributes to the
stabilization of a secondary structure apparently present in
solutions of the chimeras.
Since these compounds constitute a new backbone design,
no reference CD data are available, and thus no exact
structural properties can be derived from the CD measure-
ments. In addition, similar CD spectra may arise from very
different ensembles of folded secondary structures in solu-
tion.32 Nevertheless, the indications of the overall folding
propensity of the chimeras are important, as also reported
in the initial characterization of previous novel types of
oligomers.12,22
50, and 50 µg/mL, respectively. These results are remarkable
considering that these compounds represent the first genera-
tion of R-peptide/â-peptoid chimeras. The oligomer 15,
lacking chirality in the â-peptoid residue, was twice as potent
as the dodecamer 12, indicating that this chirality is not
essential for antimicrobial activity. This finding is significant,
as it extends the range of possible building blocks and
possibly simplifies the construct design in future structure-
activity investigations in search of chimeras with enhanced
potency and membrane selectivity. Compounds 11, 12, and
14-20 are currently undergoing extensive testing against a
broader range of bacteria, and second-generation compounds
with different ratios of cationic and lipophilic sites are in
preparation; the results of these studies will be reported in
due course.
Finally, stability of the compounds toward proteolytic
degradation was tested. Incubation of compound 11 with a
large excess of trypsin from porcine pancreas for up to 50 h
did not result in any detectable degradation (Supporting
Information, Figure S4) showing that the R-peptide/â-peptoid
chimeras are highly stable toward proteolysis.
In summary, the first generation of oligomers with an
alternating R-peptide/â-peptoid backbone construct is de-
scribed. The compounds were readily prepared in high yields
by combined solution- and solid-phase synthesis methods
and were stable toward proteolysis. Furthermore, the guani-
dinium-functionalized analogues proved to be relatively
potent antimicrobial agents. Thus, the data reported herein
show that â-peptoid residues may be valuable for diversifica-
tion of peptide analogues, thus opening new avenues in
peptidomimetic research.
In a preliminary evaluation, compounds 11-15 were tested
for antibacterial activity against one strain of Gram-negative
bacteria (Escherichia coli) and two strains of Gram-positive
bacteria (Bacillus subtilis and Staphyllococcus aureus). The
results are shown in Table 1. Compound 11 was only
Table 1. Antimicrobial Activities of Chimeras 11-15 and
Reference Compounds
MIC in µg/mLa
compd
E. coli
B. subtilis
S. aureus
Acknowledgment. We thank Ms. Dorte Brix, Ms. Katrine
Krydsfeldt, Ms. Uraiwan N. Adamsen, and Ms. Birgitte
Simonsen (all from University of Copenhagen) for excellent
technical assistance. This work was supported by the Danish
Medical Research Council (Grant No. 271-06-0126) and by
the Danish Technical Research Council (Grant No. 26-04-
0248).
11
12
13
14
15
125
15.6
n.s.c
15.6
7.8
62.5
15.6
n.s.c
15.6
7.8
n.a.b
125
n.s.c
125
31.5
n.a.b
62.5
magainin-2
streptomycin
62.5
15.6
62.5
15.6
Note Added After ASAP Publication: In the Supporting
Information, Figure S3 and the caption, there was an error
in the version published ASAP on March 13, 2007; the
correct version was published on March 14, 2007.
a MIC ) minimum inhibitory concentration. b Not active (MIC > 200
µg/mL). c Not soluble in the medium.
moderately active against E. coli and B. subtilis, but the
guanidinium-functionalized chimeras exhibited potencies
comparable to that of streptomycin and higher than that of
magainin-2, a well-known antimicrobial peptide. Importantly,
no significant hemolysis of human red blood cells was
observed with 11, 12, and 15 at concentrations up to 500,
Supporting Information Available: Experimental pro-
cedures, characterization data, CD spectra of 12, 13, and of
an N-acetylated chimeric building block as well as concen-
tration dependence data for 11 in MeOH, and proteolysis
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
(31) Thus far, structural investigations of the synthesized oligomers by
NOESY and ROESY experiments proved fruitless due to signal overlap.
Hence, efforts to prepare analogues suitable for NMR spectroscopic
investigation have been initiated.
OL070316C
(32) Gla¨ttli, A.; Daura, X.; Seebach, D.; van Gunsteren, W. F. J. Am.
Chem. Soc. 2002, 124, 12972-12978.
1552
Org. Lett., Vol. 9, No. 8, 2007