Antimicrobial β-peptoids by a block synthesis approach

Article abstract of DOI:10.1016/j.bmcl.2005.11.075

Antimicrobial peptides and their mimetics offer a potential new disinfective tool. β-Peptoids (oligo-N-substituted β-alanines) were synthesized and investigated for antimicrobial activity. A block approach whereby di- and tri-β-peptoids were first prepared and then ligated via amide bond formation to synthesize larger β-peptoids was developed. The β-peptoids were found to possess moderate activity in an Escheridchia coli assay.

Full text of DOI:10.1016/j.bmcl.2005.11.075

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1245–1248
Antimicrobial b-peptoids by a block synthesis approach
Steven W. Shuey,* William J. Delaney, Mukesh C. Shah and Mark A. Scialdone
DuPont Central Research and Development, PO Box 80328, Experimental Station, Wilmington, DE 19880-0328, USA
Received 7 September 2005; revised 18 November 2005; accepted 21 November 2005
Available online 27 December 2005
Abstract—Antimicrobial peptides and their mimetics offer a potential new disinfective tool. b-Peptoids (oligo-N-substituted b-ala-
nines) were synthesized and investigated for antimicrobial activity. A block approach whereby di- and tri-b-peptoids were first pre-
pared and then ligated via amide bond formation to synthesize larger b-peptoids was developed. The b-peptoids were found to
possess moderate activity in an Escheridchia coli assay.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Antimicrobial peptides are found in most living organ-
isms and are part of the innate immune system in hu-
mans. There has been longstanding interest in these
peptides due to their interesting structures, and the fact
that they possess a different mode of action from most
commercial antibiotics.
Several classes of antimicrobial peptidomimetics have
been studied. These include b-peptides,8 a-peptoids,9
and several types of polymers.10 Another class of poten-
tial peptidomimetics, which has been much less studied,
are the b-peptoids (poly-N-substituted b-alanine). We
became interested in studying these molecules as poten-
tial antimicrobials, since they may have an interesting
combination of properties; they are isosteric to b-pep-
tides yet potentially easier to prepare since no chiral
b-amino acids need be made, they, like a-peptoids, are
expected to be protease resistant, and they may be more
tunable than synthetic polymers.
One common motif among antimicrobial peptides is a
cationic amphiphilic structure in which the peptides con-
tain a high number of cationic residues which are segre-
gated from a lipophilic domain. These peptides are
thought to act by interaction with, and disruption of,
the bacterial cell membrane, leading to pore formation.1
Peptides can obtain an amphipathic structure in a vari-
ety of ways including formation of amphiphilic helices,
where one face is cationic and the other lipophilic,
formation of hydrophobic and hydrophilic patches or
formation of b-sheets which segregate the hydrophobic
and cationic domains. Many natural, a-helical, and
b-sheet antimicrobial peptides are known including
cecropin,2 magainin,3 and defensins.4 Both helical and
b-sheet cationic amphipathic peptides have been
designed from first principles and shown to be antimi-
crobial.5 Although many helical antimicrobial structures
are known, it is clear that a pre-formed helix is not
necessary for activity. Indeed, it has been shown that
many natural peptides adopt amphiphilic structure only
in the presence of a membrane,6 and compounds which
cannot adopt helical structures can still be good
antimicrobials.7
Scheme 1 shows the synthesis of b-peptoids, first
described by Hamper.11 Reaction of a solid support with
Scheme 1. Reagents and conditions: (a) acryloyl chloride (2.0 equiv),
DIEA (3.0 equiv), THF 3 h; (b) RNH2 (20 equiv), 2-propanol, 50 ꢁC,
48 h; (c) acryloyl chloride (2.0 equiv), DIEA (3.0 equiv), THF 3 h;
(d) RNH2 (20 equiv), 2-propanol, 50 ꢁC, 48 h; acryloyl chloride
(2.0 equiv), DIEA (3.0 equiv), THF 3 h; RNH2 (20 equiv), 2-propanol,
50 ꢁC, 48 h.
Keywords: Antimicrobial; b-Peptoid; Peptide mimetic.
Corresponding author. Tel.: +1 302 695 4554; fax: +1 302 695
8412; e-mail: Steven.W.Shuey@usa.dupont.com
*
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.11.075

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S. W. Shuey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1245–1248
acrylic acid or acryloyl chloride to generate acrylate res-
in 1, followed by a Michael-type addition of a primary
amine, gave secondary amines 2. Iterative reaction with
acryl chloride and primary amines then led to the b-pep-
toids 4. We found this chemistry to work well for several
cycles, but had difficulty generating longer b-peptoids,
as the yields fell off after approximately the pentamer
stage. Antimicrobial testing of these short oligomers
showed no measurable activity (data not shown).
described in Scheme 3 to be most efficient. Reaction of
a primary amine with t-butyl acrylate in a Michael-type
addition gave the N-substituted b-alanine 7. Reaction of
the crude mixture of 7 with excess acryloyl chloride gave
acrylamide 8 which could be purified by flash chroma-
tography. Repeating this cycle of Michael additions with
primary amines and acryoylation, dimers and trimers 9
were constructed. The intermediates were purified at
each acrylamide stage by flash chromatography. When
the desired block was complete, its amine terminus
was protected with an Fmoc group to give 10 and the
t-butyl group cleaved by formic acid treatment to pro-
vide the final building blocks, 6. In this way, multi-gram
quantities of the b-peptoid building blocks could be
accessed.
One hypothesis concerning the mechanism of antimicro-
bial peptides supposes that oligomers need to be able to
span the membrane and we would therefore need b-pep-
toids greater than 5 residues in length to obtain good
activity.12 Since we were unable to prepare longer oligo-
mers by the published route, we decided to explore the
use of a building block approach whereby we could syn-
thesize blocks of di- or tri-b-peptoids and then ligate
them together on solid phase via amide bond formation
to achieve the desired length b-peptoids. Our first ap-
proach utilized Hamper’s synthesis to prepare di- and
tri-b-peptoid blocks. The amine terminus of the b-pep-
toid blocks was then protected with Fmoc to give 5,
and the building blocks 6 released from the resin, as
shown in Scheme 2, by treatment with TFA.
To make an initial scouting set of b-peptoids, a group of
side chains was chosen in which hydrophobic and cat-
ionic groups were represented. Figure 1 shows the com-
position of the side chains for the scouting library. The
isobutyl and benzyl groups were chosen for hydrophobic
side chains, while the 2-aminoethyl, 4-aminobutyl, and
N,N-dimethyl-3-aminopropyl groups were chosen for
cationic groups. The dimethylaminopropyl side chains
could be incorporated directly into the b-peptoids,
whereas the aminobutyl and aminoethyl groups required
protection of the terminal amine to allow the blocks to
be synthesized.
While the solid phase synthesis of the building blocks
was convenient for smaller quantities, to enable synthe-
sis of longer oligomers by a block approach we required
a method to quickly produce larger quantities of the
building blocks. We explored several solution phase syn-
thetic routes including preparation and amide coupling
of N-substituted b-alanines. We found the method
Ten different building blocks were synthesized by the
solution and solid phase routes described above. The
composition of the building blocks is listed in Table 1.
The blocks were designed to probe the effects of the ratio
of cationic to hydrophobic groups, the importance of
the side-chain composition, and the effect of the relative
position of the cationic and hydrophobic groups. Our
initial hypothesis being that if the group has sufficient
conformational freedom, it could adopt an amphiphilic
structure in the presence of a bacterial membrane. The
blocks were designed to cover a hydrophobic content
range from 33 to 66% but because of the oligomer syn-
thesis limitations encountered, we are limited to 50–
66%, which is still within the optimal range found for
idealized helical peptides which has been found to be
50–60% Figure 2.13
Scheme 2. Protection of and cleavage from the resin of b-peptoid
blocks. Reagents and conditions: (a) Fmoc-Cl (2.0 equiv), DIEA
(3.0 equiv), NMP, 3 h; (b) 50% TFA/DCM, 1 h.
The final b-peptoids were constructed using a solid phase
synthesis analogous to peptide synthesis (Scheme 4).
Scheme 3. Solution synthesis of b-peptoid blocks. Reagents and conditions: (a) RNH2 5.0 equiv, 50 C MeOH 48 h; (b) acryloyl chloride, 1.2 equiv,
DIEA 2.0 equiv, DCM 3 h; (c) FMOC-Cl 1.3 equiv, DIEA 2.0 equiv, DCM 12 h; (d) HCOOH, 12 h.

S. W. Shuey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1245–1248
1247
in and to conserve the b-peptoid blocks. While coupling of
the secondary amines with carboxylic acids was more dif-
ficult than a standard peptide bond formation, adequate
results were obtained.14 When the desired b-peptoid was
complete, the final Fmoc group was removed and the b-
peptoid capped with acetic anhydride to give the N-termi-
nal acetamide. After cleavage from the resin by treatment
with TFA (which also removed the Boc from the lysine
linker), the Cbz-side-chain protection, if present, was re-
moved by hydrogenation to give the final b-peptoids.
Purification was achieved by prep HPLC, and all com-
pounds were identified by mass spectral analysis. The
purified b-peptoids were assayed for antimicrobial
activity.15
Figure 1. Side-chain composition for the b-peptoids.
Table 1. Composition of the di- and tri-b-peptoid building blocks used
to construct the final b-peptoids
The development of the block synthesis approach en-
abled synthesis of longer b-peptoids for this study.
Although the conditions developed for coupling the
blocks to form the tertiary amides which were generally
useful, when the side chain of the N-terminal group to
be coupled contained the dimethylaminopropyl group
(6d, 6e), the reaction failed to yield any recoverable
oligomers. A strategy in which blocks terminating in
an uncharged group were instead constructed and used
to prepare the oligomers. We were unable to prepare
blocks with two dimethylaminopropyl containing b-pep-
toid residues, presumably for the same reason. When
blocks containing two protected primary amine bearing
side chains were used, we were able to prepare the
blocks, but the synthesis failed for oligomers longer than
hexamers for reasons we don’t understand.
Compound
R1
R2
R3
6a
6b
6c
6d
6e
6f
Ibu
DMAP
Ibu
Ibu
Bz
Bz
DMAP
DMAP
Ibu
DMAP
Ibu
Ibu
DMAP
AE
AE
DMAP
Ibu
6g
6h
6i
Ibu
AE
AE
AB
Ibu
Ibu
Ibu
Ibu
6j
AB
When R3 is blank, this denotes a di-peptoid. The designations Ibu, etc.,
denote the side-chain content and are described in Figure 1.
We chose to synthesize b-peptoids from 9 to 18 mers to
demonstrate proof of principle for the synthesis method
and to get initial results of the efficacy of this class of
compounds as antimicrobials. Table 2 shows the MIC
values obtained for the b-peptoids.
Figure 2.
Maximal activity of 128 lg/mL was seen for the b-pep-
toids which is about an order of magnitude lower than
that of natural antimicrobials (For comparison, Magai-
nin and the idealized Leu-Lys helical peptides have MIC
Repetitive cycles of coupling to resin bound amine fol-
lowed by deprotection of the Fmoc group served to build
the desired b-peptoids. The resin was first reacted with
Fmoc(Boc)lysine to ensure high initial loadings of the res-
Scheme 4. Method for condensation of the b-peptoid blocks into oligo b-peptoids. Reagents and conditions: (a) HATU (1.6 equiv), DIEA
(1.3 equiv), NMP, 20 h; (b) 20% piperidine/DMF, 1 h; (c) Ac2O/TEA/DMF 1:2:1, 1 h; (d) 50% TFA/DCM, 1 h.

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S. W. Shuey et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1245–1248
Table 2. Composition of the b-peptoid test compounds and their mic
values against E. coli
3. Zasloff, M. Proc. Natl. Acad. Sci. U.S.A. 1987, 84, 5449.
4. White, S. H.; Wimley, W. C.; Selsted, M. E. Curr. Opin.
Struct. Biol. 1995, 5, 521.
5. (a) DeGrado, W. F.; Kezdy, F. J.; Kaiser, E. T. J. Am.
Chem. Soc. 1981, 103, 679; (b) Haynie, S. L.; Crum, G. A.;
Doele, B. A. Antimicrob. Agents Chemother. 1995, 39, 301;
(c) DeGrado, W.F.; Pept.: Struct. Funct., Proc. Am. Pept.
Symp., 8th, Hruby, V. J., Ed. (1983); (d) Blondelle, S. E.;
Houghten, R. A. Biochemistry 1992, 31, 12688; (e) Minor,
D. L., Jr.; Kim, P. S. Nature 1994, 367, 660.
6. Bechinger, B.; Zasloff, M.; Opella, S. J. Protein Sci. 1993,
2, 2077.
7. Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55.
8. Porter, E. A.; Weisblum, B.; Gellman, S. H. J. Am. Chem.
Soc. 2002, 124, 7324.
9. (a) Patch, J. A.; Barron, A. E. J. Am. Chem. Soc. 2003,
125, 12092; (b) Ng, S.; Goodson, B.; Ehrhardt, A.; Moos,
W. H.; Siani, M.; Winter, J. Bioorg. Med. Chem. 1999, 7,
1781.
10. (a) Mandeville, W. H.; Neenan, T. X.; Holmes-Farley, S.
R. U.S. Patent 6,034,129, 2000; (b) Tew, G. N.; Liu, D.;
Chen, B.; Doerksen, R. J.; Kaplan, J.; Carroll, P. J.; Klein,
M. L.; DeGrado, W. F. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 5110; (c) Kuroda, K.; DeGrado, W. F. J. Am.
Chem. Soc. 2005, 127, 4128.
Compound
Peptoid
block (6)
Number of repeat
(n)
MIC
(lg/mL)
15a
15b
15c
15d
15e
15f
15g
15h
15i
6a
6a
6a
6b
6b
6b
6c
6c
6c
6f
6f
6f
6g
6g
6g
6h
6i
3
5
6
3
4
5
5
7
8
3
4
5
5
7
8
2
1
2
3
2
3
128
128
128
512
128
128
>512
>512
>512
>512
256
15j
15k
15l
256
256
15m
15n
15o
15p
15q
15r
15s
15t
15u
128
256
>512
>512
>512
>512
>512
512
6i
6i
6j
6j
11. Hamper, B. C.; Kolodziej, S. A.; Scates, A. M.; Smith, R.
G.; Cortez, E. J. Org. Chem. 1998, 63, 708.
12. Lee, S.; Mihara, H.; Aoyagi, H.; Kato, T.; Izumiya, N.;
Yamasaki, N. Biochim. Biophys. Acta 1986, 862, 211.
13. (a) Anzai, K.; Hamasuna, M.; Kadono, H.; Lee, S.;
Aoyagi, H.; Kirino, Y. Biochim. Biophys. Acta 1991, 1064,
256; (b) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.;
Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991,
354, 84(c) Houghten, R. A.; Blondelle, S. Int. Pat. App.
1992, W.O. 92/0146.
of 16 lg/mL in this assay). This initial library demon-
strates that b-peptoids can be prepared with antimicro-
bial activity. More compounds are needed to draw
conclusions as to the minimal length necessary for
activity, the ideal hydrophobic/cationic content, and
preferred side-chain composition.
14. Rink-Fmoc-Lys(BOC)-OH resin (0.042 mmol) was treated
with 3.0 mL of 25% piperidine/THF for 1.0 h. The resin
was then drained, washed, and dried under N2 pressure.
Added 6a–j (0.126 mmol) as a 0.5 mg/lL stock in NMP
and DIEA (0.168 mmol) to the resin. Prepared HATU
(0.210 mmol) in 1.0 mL NMP, added to the resin, and
agitated at 25 ꢁC for 20 h under N2.
15. The minimal inhibitory concentration (MIC) was deter-
mined in sterile microtiter plates in a final volume of
200 lL using trypticase soy broth (TSB) as the growth
medium. Serial 2-fold dilutions of the b-peptoid stock
solutions were made in the plate wells such that concen-
trations ranged from 1024 to 2 lg/mL in a volume of
100 lL. Each well was then inoculated with 100 lL of a
dilute solution of bacteria (E. coli ATCC 25922) in TSB
yielding a final concentration of 1 · 10À4 bacteria/mL. The
final b-peptoid concentrations ranged from 512 to 1 lg/
mL. The assay plates were incubated at 37 ꢁC for 24 h in a
bioscreen C microtiter plate reader. The optical density of
the medium at 600 nm was recorded every 20 min to
monitor cell growth. The lowest concentration of b-
peptoid preventing bacterial growth at 24 h was defined as
the MIC.
A method for preparing oligo-b-peptoids was developed
by using a block approach. The method is far more
efficient than synthesis by sequential sub-monomer
approach which failed in our hands after about the
pentamer stage. The method was successfully used to
prepare oligomers up to 18 mers which were tested in an
antimicrobial assay. This route opens the way for a more
detailed look at this class of peptide-mimetic oligomers.
More work is needed to fully elucidate the importance
of length, hydrophobic content, and side-chain composi-
tion on the SAR. In addition, elucidation of secondary
structure for these materials compared to that of peptides,
b-peptides and a-peptoids is now possible.
References and notes
1. For a review of the mechanism of antimicrobial peptides
see: Shai, Y. Biochim. Biophys. Acta 1999, 1462, 55.
2. Steiner, H.; Hultmark, D.; Engstrom, A.; Bennich, H.;
Boman, H. G. Nature 1981, 292, 246.