Synthesis, conformational analysis and biological studies of cyclic cationic antimicrobial peptides containing sugar amino acids

Article abstract of DOI:10.1021/jo801123q

(Chemical Equation Presented) Sugar amino acid based 24-membered macrocyclic C2-symmetric cationic peptides were designed and synthesized. The cationic group was introduced in the sugar amino acids. The conformation of these cyclic compounds was ascertain

Full text of DOI:10.1021/jo801123q

Synthesis, Conformational Analysis and Biological Studies of Cyclic
Cationic Antimicrobial Peptides Containing Sugar Amino Acids
Tushar Kanti Chakraborty,*,† Dipankar Koley,† Rapolu Ravi,† Viswanatha Krishnakumari,‡
Ramakrishnan Nagaraj,‡ and Ajit Chand Kunwar*,†
Indian Institute of Chemical Technology and Centre for Cellular and Molecular Biology,
Hyderabad 500607, India
chakraborty@iict.res.in
ReceiVed May 28, 2008
Sugar amino acid based 24-membered macrocyclic C2-symmetric cationic peptides were designed and
synthesized. The cationic group was introduced in the sugar amino acids. The conformation of these
cyclic compounds was ascertained through NMR techniques, which proved they were amphipathic in
nature. All the compounds were bacteriolytic, showed good activity against the Gr+ve and Gr-ve bacteria,
and exhibited low hemolytic activity.
Introduction
through pore formation.4 CAPs have either R-helical (e.g.,
magainins, mellitin, etc.)5 or ꢀ-sheet (e.g., gramicidin S,
tachyplesins, etc.)6 structures and are amphiphilic in nature.
However, as a result of the hemolytic activity of these natural
antibiotics toward human blood cells, their uses as therapeutic
agents are restricted.7 Medicinal chemists are thus very keen to
have new CAP antibiotics8 either with new scaffolds or by
mimicking their natural counterparts. Gellman et al. reported9
a series of compounds with unnatural ꢀ-amino acids to obtain
helical cationic peptides whose bactericidal activities are
comparable to that of magainin. Reports of Overhand10 and
Wipf11 on the synthesis of cyclic CAPs as gramicidin S mimics
In our continuous combat against virulent pathogenic bacteria,
development of new antibiotics with novel modes of action
assumes great significance today.1 Easily curable bacterial
diseases are nowadays becoming life-threatening owing to the
increasing resistance of the pathogens to the established drugs.
Cationic antimicrobial peptides (CAPs) are long considered as
potential alternative antibiotics.2 Both linear and cyclic cationic
peptides have been found as part of the innate immune response
of many vertebrates including humans.3 These compounds
exhibit a primary defense system of the host and are believed
to be fighting against bacteria by disrupting their cell membrane
† Indian Institute of Chemical Technology.
‡ Centre for Cellular and Molecular Biology.
(4) (a) Christensen, B.; Fink, J.; Merrifield, R. B.; Mauzerall, D. Proc. Natl.
Acad. Sci. U.S.A. 1988, 85, 5072–5076. (b) Kagan, B. L.; Selsted, M. E. Proc.
Natl. Acad. Sci. U.S.A. 1990, 87, 210–216.
(1) Chu, D. T. W.; Plattner, J. J.; Katz, L. J. Med. Chem. 1996, 39, 3853–
3874.
(2) (a) Pereira, H. A. Curr. Pharm. Biotechnol. 2006, 7, 229–234. (b) Brown,
K. L.; Hancock, R. E. W. Curr. Opin. Immunol. 2006, 18, 24–30. (c) Zasloff,
M. Nature 2002, 415, 389–395. (d) Hancock, R. E. W.; Lehrer, R. TIBTECH
1998, 16, 82–87.
(3) (a) Mookherjee, N.; Hancock, R. E. W. Cell. Mol. Life Sci. 2007, 64,
922–933. (b) Brogden, K. A. Nat. ReV. Microbiol. 2005, 3, 238–250. (c) Hancock,
R. E. W.; Falla, T.; Brown, M. H. AdV. Microb. Physiol. 1995, 37, 133–175. (d)
Boman, H. G.; Hultmark, D. Annu. ReV. Microbiol. 1987, 41, 103–126.
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Bechinger, B.; Zasloff, M.; Opella, S. J. Biophys. J. 1992, 62, 12–14.
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Nature 1978, 275, 206–207. (b) Nakamura, T.; Furunaka, H.; Miyata, T.;
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10.1021/jo801123q CCC: $40.75
Published on Web 10/21/2008
2008 American Chemical Society
J. Org. Chem. 2008, 73, 8731–8744 8731

Chakraborty et al.
SCHEME 1. Cyclic CAPs (1-3) Containing Sugar Amino
Acid 4
FIGURE 1. Graphical representation of a cyclic CAP molecule in
amphiphilic conformation showing segregation of hydrophobic and
hydrophilic residues on separate surfaces of an ordered cyclic frame.
also encouraged us to use suitably functionalized unnatural
amino acids in creating new antibacterial compounds.
Sugar amino acids (Saa), an important class of peptidomimetic
scaffolds, have been exploited by us and others to create ordered
structures.12 In our initial approach,13 we synthesized well-
defined intramolecularly hydrogen-bonded tetrahydrofuram
amino acid containing rigid cyclic peptides as basic framework.
Our next target was to impose amphiphilic nature to those
secondary structured cyclic peptides by distributing the hydro-
phobic and hydrophilic residues onto separate surfaces of the
molecules (Figure 1), which is a prerequisite for peptides to
act as CAPs.14 Knowing the conformational features of our
earlier reported cyclic peptides,13 we anticipated that tuning the
sugar amino acids with suitable functionalities could lead to
the generation of novel amphiphilic properties in our cyclic
compounds. We report here the synthesis and conformational
analysis of a set of novel cyclic peptides (1-3) that are
potentially active against bacteria.
SCHEME 2. Synthesis of Sugar Amino Acids 4a and 4b
(8) (a) Chongsiriwatana, N. P.; Patch, J. A.; Czyzewski, A. M.; Dohm, M. T.;
Ivankin, A.; Gidalevitz, D.; Zuckermann, R. N.; Barron, A. E. Proc. Natl. Acad.
Sci. U.S.A. 2008, 105, 2794–2799. (b) Yang, Z.; Liang, G.; Guo, Z.; Guo, Z.;
Xu, B. Angew. Chem., Int. Ed. 2007, 46, 8216–8219. (c) Ishitsuka, Y.; Arnt, L.;
Majewski, J.; Frey, S.; Ratajczek, M.; Kjaer, K.; Tew, G. N.; Lee, K. Y. C.
J. Am. Chem. Soc. 2006, 128, 13123–13129. (d) Liu, Z.; Deshazer, H.; Rice,
A. J.; Chen, K.; Zhou, C.; Kallenbach, N. R. J. Med. Chem. 2006, 49, 3436–
3439. (e) Rennie, J.; Arnt, L.; Tang, H.; Nu¨sslein, K.; Tew, G. N. J. Ind.
Microbiol. Biotechnol. 2005, 32, 296–300. (f) Ilker, M. F.; Nu¨sslein, K.; Tew,
G. N.; Coughlin, E. B. J. Am. Chem. Soc. 2004, 126, 15870–15875. (g)
Fernandez-Lopez, S.; Kim, H. S.; Choi, E. C.; Delgado, M.; Granja, J. R.;
Khasanov, A.; Kraehenbuehl, K.; Long, G.; Weinberger, D. A.; Wilcoxen, K. M.;
Ghadiri, M. R. Nature 2001, 412, 452–455. (h) Li, C.; Peters, A. S.; Meredith,
E. L.; Allman, G. W.; Savage, P. B. J. Am. Chem. Soc. 1998, 120, 2961–2962.
(9) (a) Porter, E. A.; Wang, X.; Lee, H.-S.; Weisblum, B.; Gellman, S. H.
Nature 2000, 404, 565. (b) Raguse, T. L.; Porter, E. A.; Weisblum, B.; Gellman,
S. H. J. Am. Chem. Soc. 2002, 124, 12774–12785. (c) Porter, E. A.; Weisblum,
B.; Gellman, S. H. J. Am. Chem. Soc. 2002, 124, 7324–7330. (d) Schmitt, M. A.;
Weisblum, B.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 417–428. (e)
Mowery, B. P.; Lee, S. E.; Kissounko, D. A.; Epand, R. F.; Epand, R. M.;
Weisblum, B.; Stahl, S. S.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129, 15474–
15476.
It was envisaged that placing cationic charges on the
tetrahydrofuran rings that occupy one side of the “tennis ball
seam” type structure13 of the cyclic framework would ensure
that the hydrophilic side chains remain opposite to the other
side chains of hydrophobic Phe, Leu residues.
The functionally tuned building blocks 4a and 4b were used
to synthesize these cyclic compounds. Introduction of an extra
amino group in the C-4 position of Saa ring was our interest
from a synthetic point of view. We were also interested to devise
a common strategy to synthesize these two diastereomeric sugar
amino acids from the same starting material.
(10) (a) Grotenbreg, G. M.; Timmer, M. S. M.; Llamas-Saiz, A. L.; Verdoes,
M.; van der Marel, G. A.; van Raaij, M. J.; Overkleeft, H. S.; Overhand, M.
J. Am. Chem. Soc. 2004, 126, 3444–3446. (b) Grotenbreg, G. M.; Buizert,
A. E. M.; Llamas-Saiz, A. L.; Spalburg, E.; van Hooft, P. A. V.; de Neeling,
A. J.; Noort, D.; van Raaij, M. J.; van der Marel, G. A.; Overkleeft, H. S.;
Overhand, M. J. Am. Chem. Soc. 2006, 128, 7559–7565.
(11) Xiao, J.; Weisblum, B.; Wipf, P. Org. Lett. 2006, 8, 4731–4734.
(12) For some review articles on sugar amino acids, see: (a) Risseeuw,
M. D. P.; Overhand, M.; Fleet, G. W. J.; Simone, M. I. Tetrahedron Asymmetry
2007, 18, 2001–2010. (b) Jensen, K. J.; Brask, J. Peptide Sci. 2005, 80, 747–
761. (c) Chakraborty, T. K.; Srinivasu, P.; Tapadar, S.; Mohan, B. K. J. Chem.
Sci. 2004, 116, 187–207. (d) Gruner, S. A. W.; Locardi, E.; Lohof, E.; Kessler,
H. Chem. ReV. 2002, 102, 491–514. (e) Schweizer, F. Angew. Chem., Int. Ed.
2002, 41, 230–253. (f) Gervay-Hague, J.; Weathers, T. M. In Pyranosyl Sugar
Amino Acid Conjugates: Their Biological Origins, Synthetic Preparations and
Structural Characterisation; Dekker: New York, 2001.
(13) Chakraborty, T. K.; Roy, S.; Koley, D.; Dutta, S. K.; Kunwar, A. C. J.
Org. Chem. 2006, 71, 6240–6243.
(14) (a) Langham, A. A.; Ahmad, A. S.; Kaznessis, Y. N. J. Am. Chem.
Soc. 2008, 130, 4338–4346. (b) Leontiadou, H.; Mark, A. E.; Marrink, S. J.
J. Am. Chem. Soc. 2006, 128, 12156–12161. (c) Straus, S. K.; Hancock, R. E. W.
Biochim. Biophys. Acta 2006, 1758, 1215–1223.
Results and Discussion
Synthesis of Compounds 1-3. Synthesis of the building
block 4 started from 6, which was prepared from D-xylose (5)
following literature procedure,15 as shown in Scheme 2. The
1,2-O-isopropylidene group of 6 was treated with dry HCl in
MeOH to yield both the anomers of methyl-3,5-di-O-benzyl-
D-xylofuranoside 7a,b in a ratio of 2:3, respectively. The C-2
hydroxyl groups of 7a,b were etherified using NaH, MeI, and
(15) Dyatkina, N. B.; Azhayev, A. V. Synthesis 1984, 961–963.
8732 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
SCHEME 3. Synthesis of Cyclic CAPs 1a and 1b
SCHEME 4. Synthesis of Cyclic CAPs 2a,b and 3a,b
a catalytic amount of TBAI. Next, the resulting mixture of
anomers 8a,b was treated with 20% 1N HCl in AcOH and
heated at 60 °C for 2 h. The intermediate hemiacetal was
acylated to 9 using Ac2O and Et3N. The inseparable anomers
were treated with TMSCN16 in presence of BF3-Et2O to furnish
10 as an anomeric mixture. Conversion of CN to aldehyde using
DIBAL-H,17 oxidation and treatment with CH2N2 resulted in
the formation of 11a,b (5:4) in 72% yield. The anomers could
easily be separated at this stage through silica gel column
chromatography. The next reactions of these isomers were
performed separately.
Complete debenzylation followed by selective TBDPS pro-
tection of the primary hydroxyl group yielded 13a,b. Our next
target was to introduce an azido group in the C-4 center.
Following Fleet’s procedure,18 the C-4 hydroxyl group was
reacted with Tf2O. The resulting triflate, after flash chromatog-
raphy, was treated with 6 equiv of NaN3 in dry DMF at -5 °C
to furnish the γ-azido ester 14a,b. The azido group was
hydrogenated in the subsequent step and the resulting amino
group was protected in situ using Boc2O to yield 15a,b.
Little elimination was observed in the R-anomer that was
inseparable from 14a. However, the eliminated product could
be separated in 8% yield in its saturated form in the next step.
Primary silyl deprotection using TBAF afforded 16a,b. Tosy-
lation of the primary hydroxyl group and heating of the tosylated
intermediate with NaN3 in DMF at 65 °C yielded the Saa’s 4a,b.
Synthesis of the cyclic framework of the target molecules is
described in Scheme 3. Saponification of Saa’s 4a,b followed
by coupling with TFA·H-Phe-Leu-OMe using EDCI and HOBt
in the presence of DIPEA afforded 17a,b in 85% yield. Stepwise
saponification of 17a,b followed by hydrogenation of the azido
group to amine yielded the crude tripeptide, which on the
subsequent step were cyclodimerized using FDPP19 in CH3CN
under dilute condition (2 × 10-3 M) to furnish 18a,b in 60-65%
yield. Similar reactions were carried out by us earlier in the
cyclodimerization of sugar amino acid containing peptides,13
in the cyclooligomerization of sugar amino acids,20 and in the
cyclotrimerization of furan-amino acids.21 One-pot cyclotrim-
erization of five-membered oxazole and thiazole ring containing
amino acids using various reagents has also been reported by
others.22 Theoretical calculations were carried out by us to
explain the preferential formation of cyclic trimers of furan
amino acids.23 However, this has still remained a trial and error
method in our hands, and we are not yet able to predict the
success of similar cyclodimerization reactions with other
peptides. Finally, removal of the Boc-protection using TFA in
DCM afforded 1a,b in quantitative yield.
Compounds 1a,b were separately coupled with Boc-hGly-
OH and Boc-Lys(Boc)-OH to furnish 19a,b and 20a,b, respec-
tively, following the procedure stated earlier as shown in Scheme
4. Finally, global Boc-deprotection of each compound furnished
2a,b and 3a,b in quantitative yields.
Conformational Analysis of 18-20 in CDCl3. For all of
the protected cyclic peptides 18-20, NMR studies were carried
out in CDCl3 using 5-10 mM solutions in a 500 MHz
spectrometer. While peptides 18a, 18b, and 20b were investi-
1
gated at 300 K, 19a, 20a, and 19b were studied at 303 K. H
NMR spectra of peptides 18a, 19a, and 20a were very well
resolved. The 1H NMR chemical shifts and coupling constants
are given in Tables 1-6. For all of the peptides, the presence
of only one set of peaks for each type of amino acid residues
indicates 2-fold molecular symmetry in the NMR time scale.
For peptide 18a, amide proton chemical shifts (δ) > 7 ppm for
the Saa (7.31 ppm) and Phe (8.56 ppm) residues suggest their
participation in H-bonding. This was further confirmed by the
solvent titration studies, when sequential addition of 300 µL of
DMSO-d6 to 600 µL of CDCl3 solution of the peptide produced a
very insignificant change of <0.13 ppm in their chemical shifts
(∆δ) (see Supporting Information). Similar observations and
subsequent conclusions, regarding H-bonding of the Saa and Phe
amide protons, were made for peptides 19a and 20a. The coupling
constants and the nuclear Overhauser effect (NOE) correlation
involving the CεH protons allowed stereospecific assignments for
(16) Koert, U.; Stein, M.; Harms, K. Tetrahedron Lett. 1993, 34, 2299–2302.
(17) Kabota, I.; Takamura, H.; Yamamoto, Y. J. Am. Chem. Soc. 2004, 126,
14374–14376.
(18) Hungerford, N. L.; Fleet, G. W. J. J. Chem. Soc., Perkin Trans. 1 2000,
3680–3685.
(19) (a) Chen, S.; Xu, J. Tetrahedron Lett. 1991, 32, 6711–6714. (b) Leonard,
M. S.; Joullie, M. M. Encyclopedia of Reagents for Organic Synthesis; John
Wiley & Sons Ltd.: New York, 2002.
(20) Chakraborty, T. K.; Srinivasu, P.; Bikshapathy, E.; Nagaraj, R.;
Vairamani, M.; Kumar, S. K.; Kunwar, A. C. J. Org. Chem. 2003, 68, 6257–
6263.
(22) For some representative works, see:(a) Haberhauer, G.; Rominger, F.
Tetrahedron Lett. 2002, 43, 6335–6338. (b) Somogyi, L.; Haberhauer, G. H.;
Rebek, J., Jr. Tetrahedron 2001, 57, 1699–1708. (c) Bertram, A.; Hannam, J. S.;
Jolliffe, K. A.; de Turiso, F. G.-L.; Pattenden, G. Synlett 1999, 1723–1726. (d)
Sokolenko, N.; Abbenante, G.; Scanlon, M. J.; Jones, A.; Gahan, L. R.; Hanson,
G. R.; Fairlie, D. P. J. Am. Chem. Soc. 1999, 121, 2603–2604. (e) Wipf, P.;
Miller, C. P. J. Am. Chem. Soc. 1992, 114, 10975–10977. (f) Chen, S.; Xu, J.
Tetrahedron Lett. 1991, 32, 6711–6714.
(21) (a) Chakraborty, T. K.; Tapadar, S.; Raju, T. V.; Annapurna, J.; Singh,
H. Synlett 2004, 2484–2488. (b) Chakraborty, T. K.; Tapadar, S.; Kumar, S. K.
Tetrahedron Lett. 2002, 43, 1317–1320.
(23) (a) Sharma, M.; Kumar, P.; Singh, H.; Chakraborty, T. K. J. Mol. Struct.:
THEOCHEM 2006, 764, 111–117. (b) Singh, H.; Arora, K. S.; Tapadar, S.;
Chakraborty, T. K. J. Theor. Comput. Chem. 2004, 3, 555–566.
J. Org. Chem. Vol. 73, No. 22, 2008 8733

Chakraborty et al.
TABLE 1. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 18a (500 MHz, CDCl3, 300 K) and 1a (600 MHz, H2O/D2O
(9:1), 303 K)
residues
18a in CDCl3 solution
Phe
1a in H2O/D2O (9:1) solution
protons
NH
Saa
7.31 (m)
Leu
Saa
7.62 (dd)
J ) 4.8, 8.0
4.60 (d) J ) 4.2
Phe
Leu
8.57 (d) J ) 6.4
6.40 (d) J ) 7.1
8.55 (d) J ) 6.6
8.79 (d) J ) 7.1
CRH
4.45 (d) J ) 4.8
4.30 (ddd)
J ) 6.2, 6.4, 10.5
3.59 (dd)
J ) 10.5, 13.7
3.27 (dd)
3.44 (ddd)
J ) 4.2, 7.1, 10.5
1.78(ddd)
J ) 4.2, 9.7, 14.3
1.84 (ddd)
4.59 (ddd)
3.78 (ddd)
J ) 6.6, 7.1, 9.6
3.40 (dd)
J ) 4.6, 7.1, 10.6
1.48 (ddd)
CꢀH(pro-S)/CꢀH 4.02 (t) J ) 4.8
4.35 (dd)
J ) 4.2, 4.5
J ) 9.6, 13.7
3.29 (dd)
J ) 4.6, 9.1, 13.7
1.67 (ddd)
CꢀH(pro-R)
J ) 6.2, 13.7
J ) 4.4, 10.5, 14.3
J ) 7.1, 13.7
J ) 3.2, 10.6, 13.7
CγH
CδH
3.73 (ddd)
J ) 4.8, 9.5, 11.2
3.93 (m)
0.78 (m)
3.60 (dd)
J ) 4.5, 10.1
4.14 (ddd)
0.58 (m)
0.74 (d) J ) 6.2
0.63 (d) J ) 6.2
0.69 (m)
0.61 (m)
J ) 2.1, 9.4, 10.1
Cδ′H
CεH(pro-S)
3.93 (m)
3.89 (ddd)
J ) 2.1, 8.0, 14.5
CεH(pro-R)
others
2.57 (ddd)
J ) 4.5,10.0, 14.0
2.85 (ddd)
J ) 4.8, 9.4, 14.5
7.31-7.24 (m, 5H, phenyl), 4.92 (d, J ) 9.5, 1H,
Saa-CγNH), 3.37 (s, 3H, OCH3), 1.45 (s, 9H, Boc).
7.35-7.29 (m, 5H, phenyl), 3.37 (s, 3H, OCH3).
TABLE 2. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 19a (500 MHz, CDCl3, 303 K) and 2a (600 MHz, H2O:D2O
(9:1), 303 K)
residues
19a in CDCl3 solution
Phe
2a in H2O/D2O (9:1) solution
protons
NH
Saa
7.36 (dd)
J ) 4.6, 8.2
4.51 (d) J ) 4.4
Leu
6.37 (m)
Saa
7.53 (dd)
J ) 5.2, 8.3
4.66 (d) J ) 4.4
Phe
Leu
8.58 (d) J ) 6.6
8.55 (d) J ) 6.6
8.80 (d) J ) 7.3
CRH
4.31 (ddd)
J ) 6.4, 6.6, 10.0
3.59 (dd)
J ) 10.0, 13.7
3.27 (dd)
3.45 (m)
4.55 (ddd)
3.68 (ddd)
J ) 6.1, 6.6, 11.0
3.48 (dd)
J ) 4.1, 7.3, 11.7
1.43 (m)
CꢀH(pro-S)/CꢀH 4.05 (m)
CꢀH(pro-R)
1.77 (ddd)
J ) 4.3, 9.8, 14.2
1.84 (ddd)
4.20 (dd)
J ) 4.5, 4.6
J ) 11.0, 13.6
3.32 (dd)
J ) 4.1, 11.2, 13.8
1.61 (ddd)
J ) 6.4, 13.7
J ) 4.4, 10.6, 14.2
0.79 (m)
J ) 6.1, 13.6
J ) 4.1, 11.7, 13.8
CγH
CδH
4.06 (m)
3.97 (m)
4.09 (ddd)
J ) 4.5, 8.8, 10.1
3.92 (ddd)
0.39 (m)
0.73 (d) J ) 6.7
0.63 (d) J ) 6.7
0.64 (d) J ) 6.9
0.54 (d) J ) 6.5
J ) 1.6, 10.1, 11.1
Cδ′H
CεH(pro-S)
3.84 (dd)
J ) 8.2, 13.6
2.60 (ddd)
3.70 (m)
2.74 (m)
CεH(pro-R)
others
J ) 4.6, 10.1, 13.6
7.33-7.24 (m, 5H, phenyl), 6.09 (d, J ) 7.9, Saa-CγNH),
5.48 (m, 1H, h-Gly-NH), 3.42 (m, 1H, h-Gly-CꢀH),
3.37 (m, 1H, h-Gly-Cꢀ′H), 3.37 (s, -OCH3),
2.50 (m, 1H, h-Gly-CRH), 2.41(m, 1H, h-Gly-CRH),
1.44 (s, Boc)
8.30 (d, J ) 8.8, Saa-CγNH),
7.65 (m, 2H, NH2.TFA), 7.35-7.29 (m, 5H, phenyl),
3.26 (m, 2H, h-Gly-CꢀH), 3.38 (s, -OCH3),
2.74 (m, 2H, h-Gly-CRH)
the methylene protons. The assignments are consistent with
presence of two sugar rings having several constrained backbone
dihedral angles, we believe that the structure of the 24-
membered macrocycle is fairly rigid and the couplings involving
the backbone protons arise from predominantly a single rigid
conformation. Due to the presence of 2-fold symmetry, only
few medium range characteristic NOE correlations such as
NH(Phe)/CδH(Saa) and NH(Saa)/NH(Leu) were available for
deducing the structure of the molecule. The restraint MD
calculations were carried out using constraints on the dihedral
angles and distances derived from the ROESY data (Supporting
Information). The lowest energy MD structures of peptides
18a-20a are shown in Figure 2. The resulting structures show
that four H-bonds, PheNH-Phe′CO (Phe′NH-PheCO) and
SaaNH-Saa′CO (Saa′NH-SaaCO), stabilize the structure of the
24-membered macrocycle. The PheNH-Phe′CO (Phe′NH-
3JNH-C H(pro-S) ≈ 8.2, 3JNH-C H(pro-R) ≈ 4.6, 3JC H(pro-S)-C H ≈ 0, and
ε
ε
ε
δ
3JC H(pro-R)-C H ≈ 10.0 Hz and allowed us to recognize the
ε
δ
3
proximity of CεH(pro-R) with CRH. JNH-C H(pro-S) ≈ 8.2 Hz
ε
suggests that the NH and the CεH(pro-S) protons are anti-
periplanar, with dihedral angle C(O)-N-Cε-Cδ (φsugar) ≈
120°, which falls in the ꢀ-region of the Ramachandran plot.
3
The values of JNH-C H ≈ 6.6 and 7.1 Hz for Phe and Leu,
R
respectively, are not distinctive and could arise either due to
averaging between several conformations or from a single
structure. In either case it is not possible apriori to get unique
information on dihedral angle C(O)-N-CR-C(O) (φ) from
these couplings.
In view of the stabilization of the structure by involvement
of four of the amide protons in H-bonding and additionally the
8734 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
TABLE 3. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 20a (500 MHz, CDCl3, 303 K) and 3a (600 MHz, H2O/D2O
(9:1), 303 K)
residues
20a in CDCl3 solution
Phe
3a in H2O/D2O (9:1) solution
Phe
protons
NH
Saa
7.34 (dd)
J ) 4.6, 8.2
4.51 (d) J ) 4.7
Leu
6.37 (m)
Saa
7.56 (dd)
J ) 4.7, 8.2
4.70 (d) J ) 4.6
Leu
8.59 (d) J ) 6.6
8.57 (d) J ) 6.5
8.85 (d) J ) 7.3
CRH
4.31 (ddd)
J ) 6.1, 6.6, 10.5
3.60 (dd)
J ) 10.5, 13.5
3.27 (dd)
J ) 6.1, 13.5
3.44 (m)
1.82 (m)
1.82 (m)
0.74 (m)
4.58 (ddd) J ) 6.1, 6.5, 11.1 3.67 (ddd)
J ) 4.1, 7.3, 11.7
1.41 (ddd)
J ) 4.1, 11.2, 13.9
1.64 (ddd)
J ) 4.0, 11.7, 13.9
0.38 (m)
CꢀH(pro-S)/CꢀH 4.03 (m)
CꢀH(pro-R)
4.23 (t) J ) 4.6
3.50 (dd)
J ) 11.1, 13.5
3.36 (dd)
J ) 6.1, 13.5
CγH
CδH
4.08 (m)
4.01 (m)
4.17 (ddd)
J ) 4.6, 8.8, 10.6
0.73 (d) J ) 6.6 3.93 (ddd)
0.64 (d) J ) 6.5
0.55 (d) J ) 6.5
J ) 2.0, 10.6, 10.2
Cδ′H
CεH(pro-S)
0.62 (d) J ) 6.6
3.79 (dd)
3.64 (ddd)
J ) 8.2, 13.6
J ) 2.0, 8.2, 14.7
CεH(pro-R)
others
2.60 (ddd)
2.75 (ddd)
J ) 4.6, 9.9, 13.6
J ) 4.7, 10.2, 14.7
7.32-7.24 (m, 5H, phenyl), 6.24 (d, J ) 8.9 Hz,
1H, Saa-CγNH), 5.29 (t, J ) 6.0 Hz, 1H, Lys-CεNH),
5.08 (d, J ) 8.3 Hz, 1H, Lys-CRNH),
8.64 (d, J ) 8.8, 1H, Saa-CγNH), 7.48 (bs, 4H,
NH2.TFA), 7.37-7.30 (m, 5H, phenyl),
4.05 (d, J ) 8.3, 1H, Lys-CRNH), 3.39
(s, 3H, -OCH3), 2.98 (m, 2H, Lys-CεH, Cε′H),
1.93 (m, 2H, Lys-CꢀH, Cꢀ′H),
4.04 (m, 1H, Lys-CRH), 3.41 (s, 3H, -OCH3),
3.10 (m, 2H, Lys-CεH, Cε′H),
1.82 (m, 1H, Lys-CꢀH), 1.62 (m, 1H, Lys-Cꢀ′H),
1.52 (m, 2H, Lys-CδH, Cδ′H), 1.43,
1.70 (m, 2H, Lys-CδH, Cδ′H),
1.45 (m, 2H, Lys-CγH, Cγ′H)
1.41 (s, 18H, Boc), 1.40 (m, 2H, Lys-CγH, Cγ′H)
TABLE 4. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 18b (500 MHz, CDCl3, 300 K) and 1b (600 MHz, H2O/D2O
(9:1), 303 K)
residues
18b in CDCl3solution
1b in H2O/D2O (9:1) solution
protons
NH
Saa
7.39 (m)
Phe
Leu
6.67 (m)
Saa
Phe
Leu
7.56 (d) J ) 8.7
4.73 (dt)
8.05 (m)
4.61 (m)
8.46 (d) J ) 8.7
4.89 (ddd)
8.43 (d) J ) 6.8
4.37 (ddd)
CRH
4.27 (m)
4.63 (m)
J ) 6.1, 8.7
J ) 5.4, 8.6, 8.7
J ) 6.8, 7.6, 7.7
CꢀH(pro-S)/CꢀH
CꢀH(pro-R)
3.85 (d) J ) 4.3
3.26 (dd)
1.66 (td)
J ) 6.3, 13.5
1.45 (m)
4.50 (m)
3.32 (dd)
1.77 (td)
J ) 6.1, 14.2
3.10 (dd)
J ) 5.4, 14.5
3.10 (dd)
J ) 7.7, 13.4
1.60 (td)
J ) 8.7, 14.2
J ) 8.6, 14.5
J ) 7.6, 13.4
CγH
CδH
Cδ′H
3.90 (m)
3.90 (m)
1.52 (m)
0.89 (d) J ) 6.4
0.87 (d) J ) 6.4
3.45 (m)
3.97 (m)
1.55 (m)
0.96 (d) J ) 6.7
0.91 (d) J ) 7.0
CεH(pro-S)
CεH(pro-R)
others
4.05 (m)
3.17 (m)
3.82 (m)
3.54 (m)
7.30-7.20 (m, 5H, phenyl), 5.07 (d,
J ) 7.4, 1H, Saa-CγNH), 3.42 (s, 3H, OCH3),
1.43 (s, 9H, Boc)
7.45-7.26 (m, 5H, phenyl),
3.50 (s, 3H, OCH3)
PheCO) H-bond involves a 13-membered pseudo ring, while
the SaaNH-Saa′CO (Saa′NH-SaaCO) turn, a pseudo ꢀ-turn,
contains a 10-membered H-bonded ring around the Phe-Leu
very similar to an R-turn24 (φ ) -57°, Ψ ) -47°) with the
signs of the angles reversed, usually referred to as an R′-turn.
Similarly, for the pseudo ꢀ-turn containing Phe-Leu motif, the
backbone φ and Ψ dihedral angles for the two residues are
-66°, 116° and 56°, 42°, which are close to those for a type II
ꢀ-turn for the first residue, while for the second residue they
resemble those for the R′-turn. The backbone, with four
H-bonds, resembles the tennis ball seam, corresponding to a
distorted “ꢀ-ꢀ corner” motif.13
residues. Also the dihedral angles φ for the Phe and Leu residues
3
are about -66° and 56°, which are consistent with JNH-C H
≈
R
7 Hz for these residues, justifying the consideration that a single
rotamer about the N-CR is responsible for these couplings as
well as highly constrained molecular geometry of the macro-
cycle. It was interesting to note that for the 13-membered pseudo
ring, φ and Ψ values for the first residue (Leu) are about 56°
and 42° respectively, whereas for the second residue, a sugar
moiety, being a dipeptide isostere, the corresponding angles are
C(O)-N-Cε-Cδ (φ′) and N-Cε-Cδ-O (θ1), with values of
69° and 55°, respectively. Thus the three residue turn appears
The structure for these peptides is very similar to that
observed for cyclic hexapeptides containing t-tetrahydrofuran
amino acids without substituents at the Cꢀ and Cγ.13 The MD
(24) Nataraj, D. V.; Srinivasan, N.; Saudhamini, R.; Ramakrishnan, C. Curr.
Sci. 1995, 69, 434–447.
J. Org. Chem. Vol. 73, No. 22, 2008 8735

Chakraborty et al.
TABLE 5. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 19b (500 MHz, CDCl3, 303 K) and 2b (600 MHz, H2O/D2O
(9:1), 313 K)
residues
19b in CDCl3 solution
Phe
2b in H2O/D2O (9:1) solution
protons
NH
Saa
7.47 (dd)
J ) 4.4, 7.4
4.22 (m)
Leu
6.97 (m)
Saa
Phe
Leu
8.46 (m)
7.44 (d) J ) 8.4
7.93 (m)
8.39 (m)
4.76 (m)
3.21 (m)
CRH
4.71 (ddd)
J ) 6.0, 8.3, 8.4
3.23 (dd)
J ) 6.0, 14.1
3.12 (dd)
J ) 8.3, 14.1
4.48 (m)
4.19 (m)
3.88 (m)
4.22 (m)
CꢀH(pro-S)/CꢀH
CꢀH(pro-R)
CγH
3.85 (d) J ) 6.0
1.77 (td)
J ) 6.8, 13.7
1.44 (m)
1.71 (td)
J ) 6.9, 13.9
1.46 (m)
2.93 (dd)
J ) 7.1, 14.6
4.16 (ddd)
J ) 6.0, 7.9, 9.6
3.85 (m)
1.57 (m)
4.19 (m)
3.76 (m)
3.69 (m)
3.33 (m)
1.40 (m)
CδH
Cδ′H
CεH(pro-S)
0.90 (d) J ) 6.6
0.88 (d) J ) 6.6
0.84 (d) J ) 7.0
0.75 (d) J ) 6.8
3.97 (dd)
J ) 7.4, 14.8
3.13 (m)
CεH(pro-R)
others
7.29-7.19 (m, 5H, phenyl), 6.07 (d, J ) 7.9, Saa-CγNH),
5.30 (t, J ) 6.4, 1H, h-GlyNH), 3.42 (s, -OCH3),
3.39 (m, 2H, h-Gly-CꢀH, Cꢀ′H), 2.40 (m, 2H, h-Gly-CRH, CRH),
1.42 (s, Boc)
7.29-7.04 (m, 5H, phenyl), 8.16
(m, Saa-CγNH), 3.39 (s, -OCH3),
3.22 (m, 2H, h-Gly-CꢀH, Cꢀ′H),
2.68 (m, 2H, h-Gly-CRH, CRH)
TABLE 6. 1H NMR Chemical Shifts (δ, ppm) and Coupling Constants (J, Hz) of 20b (500 MHz, CDCl3, 300 K) and 3b (600 MHz, H2O/D2O
(9:1), 303 K)
residues
20b in CDCl3 solution
3b in H2O/D2O (9:1) solution
protons
NH
CRH
Saa
Phe
Leu
Saa
Phe
Leu
7.95 (d) J ) 10.4
7.63 (d) J ) 8.3
4.81 (ddd)
J ) 4.7, 6.2, 8.3
9.62 (d) J ) 8.3
8.64 (m) 8.13 (d) J ) 8.0
4.26 (m) 4.74 (ddd) J ) 4.4, 7.0, 8.0 4.19 (m)
8.70 (d) J ) 7.1
4.38 (s)
4.76 (m)
CꢀH(pro-S)/CꢀH 4.13 (d) J ) 4.7
3.20 (dd)
1.77 (ddd)
J ) 6.2, 7.9, 13.8
3.34 (m) 3.19 (dd)
1.71 (m)
1.36 (m)
J ) 4.7, 14.1
3.13 (dd)
J ) 4.4, 14.5
CꢀH(pro-R)
1.54 (m)
2.93 (dd)
J ) 6.2, 14.1
J ) 7.0, 14.5
CγH
CδH
Cδ′H
4.02 (m)
4.13 (m)
1.38 (m)
0.86 (d) J ) 6.5
0.78 (d) J ) 6.5
3.82 (m)
4.23 (m)
1.36 (m)
0.84 (d) J ) 6.2
0.72 (d) J ) 5.9
CεH(pro-S)
CεH(pro-R)
others
4.15 (m)
3.27 (m)
3.92 (m)
3.80 (m)
7.44 (dd, J ) 2.5, 7.5, 1H, Lys-CεNH), 7.18-7.07 (m, 5H, phenyl),
5.89 (d, J ) 8.8, 1H, Saa-CγNH), 5.16 (d, J ) 8.2, 1H, Lys-CRNH),
4.05 (ddd, J ) 3.4, 8.2, 8.9, 1H, Lys-CRH),
3.38 (s, 3H, -OCH3), 3.32 (m, 1H, Lys-CεH), 2.95 (m, 1H, Lys-Cε′H),
1.85 (m, 1H, Lys-CꢀH), 1.52 (m, 1H, Lys-CδH),
1.50 (m, 1H, Lys-Cꢀ′H), 1.47, 1.42 (s, 18H, Boc),
1.38 (m, 1H, Lys-Cδ′H),
7.53 (bs, 4H, NH2 ·TFA), 7.30-7.04 (m, 5H, phenyl),
7.57 (m, 1H Saa-CγNH), 3.98 (m, 1H, Lys-CRH),
3.37 (s, 3H, -OCH3), 3.02 (m, 2H, Lys-CεH, Cε′H),
1.87 (m, 2H, Lys-CꢀH, Cꢀ′H),
1.71 (m, 2H, Lys-CδH, Cδ′H),
1.41 (m, 2H, Lys-CγH, Cγ′H)
1.23 (m, 2H, Lys-CγH, Cγ′H)
TABLE 7. Antibacterial Activity (in Lethal Concentration) against
Gram-Positive and Gram-Negative Bacteria and Hemolytic Activity
of the CAPs
side chains on one side of the ring providing a hydrophobic
surface, while the polar chains occupy the hydrophilic face of
the ring.
lethal concentration (µM)
These molecules interestingly display very restricted rotations
3
Gram-negative
E. P.
compound coli aeruginosa aureus subtilis
Gram-positive
of the side chains. For Phe in peptide 18a, JC H-C H(pro-S)
)
R
ꢀ
10.5 and 3JC H-C H(pro-R) ) 6.2 Hz, suggesting large populations
S. B.
% of RBC lysis
R
ꢀ
of rotamers with ꢁ1 (N-CR-Cꢀ-CPhe) ≈ 180° (t) with
significant populations of rotamers with ꢁ1 ≈ 60° (g+) and -60°
(g-). Using the relations from the literature,25 we found
populations of t, g+ and g- isomers to be about 77%, 13% and
10%, respectively. For 19a and 20a, the couplings are very
similar to those for 18a and thus provide the t and g- populations
about ꢁ1 as 72% (t) and 12% (g-) for 19a and 77% (t) and
10% (g-) for 20a, respectively. For Leu residue in peptide 18a
at 80 µM
1a
2a
3a
1b
2b
3b
35
10
18
460
30
18
80
40
35
925
60
25
23
3
9
70
20
18
23
3
9
70
20
9
0
67
20
0
0
25
pictures, for these cyclic peptides do show very clearly the
amphiphilic nature of the macrocycle, with the Leu and Phe
(25) Demarco, A.; Llinas, M.; Wu¨thrich, K. Biopolymers 1978, 17, 617–
636.
8736 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
However, a unique structure satisfying these observations and
the experimental NOEs, could not be derived.
Conformational Analysis of 1-3 in Water. NMR studies
of all the deprotected peptides 1-3 were carried out at 303 K
(2b at 313 K) in H2O-D2O (9:1) solutions (5-10 mM) at 500
and 600 MHz. The 1H NMR chemical shifts and coupling
constants are given in Tables 1-6. It was found that for 1a-3a
the spectral parameters (coupling constants), H-bonding infor-
mation, and medium range NOE correlations (Supporting
Information) are very similar to those obtained for their Boc-
protected precursors in CDCl3. In peptide 1a-3a, the temper-
ature coefficients of the amide proton chemical shifts (∆δ/∆T)
for the Phe and Saa residues are much smaller (<4.8 ppb/K)
than that for Leu resideue (>10.8 ppb/K), confirming their
participation in H-bonding. The stereospecific assignments for
methylene protons were obtained like those for 18a-20a. For
3
3
1a and 3a, the values of JNH-C H(pro-S) ≈ 8.0, JNH-C H(pro-R)
≈
ε
ε
4.7, 3JC H(pro-S)-C H ≈ 2.1, and 3JC H(pro-R)-C H ≈ 10.0 Hz, as well
ε
δ
ε
δ
as the CεH(pro-R)/CRH NOE correlation, are consistent with
the assignments. These data further suggest that the NH and
the CεH(pro-S) protons are antiperiplanar, with dihedral angle
C(O)-N-Cε-Cδ (φsugar) ≈ 120°. For 2a, due to overlap, some
of the couplings could not be obtained, and therefore the stereospe-
cific assignments were made on the basis of the similarity of
the chemical shifts of the methylene protons in this family of
peptides. The presence of four H-bonds and two sugar rings
renders the macrocycle fairly rigid. Like in 18a-20a, only few
medium range NOE correlations such as NH(Phe)/CδH(Saa) and
NH(Saa)/NH(Leu) were available for deducing the structure.
The restraint MD calculations were carried out using dihedral
angles constraints and distance constraints derived from the
ROESY data (Supporting Information). The resulting lowest
energy structures are shown in Figure 3. The structures are very
similar to those for 18a-20a in CDCl3, generating a distorted
“ꢀ-ꢀ corner” motif. The rigidity of 24-membered cyclic
frameworks of these peptides may be responsible for the
similarity of the structure in both nonpolar and polar environ-
ments. The amphiphilic nature of the macrocycle of these cyclic
peptides, with the Leu and Phe side chains providing a
hydrophobic surface on one side of the ring, and the polar chains
on the other side forming the hydrophilic face of the ring, is
very vividly displayed in the structures (Figure 3).
FIGURE 2. Lowest energy MD structure (left) and its backbone
highlighted with pseudo-Connolly surface using Insight II (right) of
peptides 18a, 19a, and 20a.
and 19a, 3JC H-C H(pro-R) ≈ 10.5 and 3JC H-C H(pro-S) ≈ 4.2 Hz again
R
ꢀ
R
ꢀ
represent the large populations of rotamers with ꢁ1
(N-CR-Cꢀ-Cγ) ≈ -60° (g-) with substantial populations of
rotamers with ꢁ1 ≈ 180° (t). The calculated populations of g-
and t are about 77% and 17%, respectively. It was also
interesting to note that for both these peptides, there are
significant restrictions even about ꢁ2, with 3JC H(pro-R)-CγH ≈ 4.4
ꢀ
and 3JC H(pro-S)-CγH ≈ 9.7 Hz, which corresponds to a predomi-
ꢀ
nance of a rotamer with CꢀH(pro-S) in trans disposition with
respect to CγH. For 20a, due to overlap of Cꢀ and Cꢀ′ protons
of Leu residue, it was not possible to obtain the information on
ꢁ1 and ꢁ2. We also observed that CγH resonances have unusually
small δ values of about 0.7-0.8 ppm for peptides 18a, 19a,
and 20a. MD studies very clearly bring out the fact that the
Leu residues are located just above the plane of aromatic ring
of Phe, which results in a substantial upfield shift for CγH.
These molecules also display very restricted rotations of the
side chains. For Phe, 3JC H-C H(pro-S) ≈ 10 and 3JC H-C H(pro-R)
≈
R
ꢀ
R
ꢀ
6 Hz suggest large populations of t rotamers. It was found that
the populations of t and g+ isomers for 1a-3a are about 69%
and 13%, 82% and 17%, and 82% and 18%, respectively.
3
For Leu residue in peptide 1a-3a, JC H-C H(pro-R) ≈ 11 and
R
ꢀ
3JC H-C H(pro-S) ≈ 4 Hz represent large populations of rotamers
R
ꢀ
with ꢁ1 (N-CR-Cꢀ-Cγ) ≈ -60° (g-). The calculated popula-
tions of g- and t are about 78% and 21%, 84% and 16%, and
79% and 16%, respectively, while the populations of g+
rotamers are very small. As seen earlier for 18a and 19a (not
well resolved in 20a), in 1a-3a also there are significant
restrictions even about ꢁ2, with 3JC H(pro-R)-CγH ≈ 4 and 3JC H(pro-
The peptides 18b, 19b, and 20b containing a cis orientation
of the 2,5-substituents in the furanoside ring also display 2-fold
symmetry. Though for 18b and 19b the amide proton chemical
shifts for Saa and Phe residues are >7 ppm, a large value of
∆δ (∼ 0.7 ppm) in the solvent titration studies seems to disfavor
their involvement in H-bonding. In addition, the absence of
distinctive couplings involving the backbone protons as well
as very few unique medium range NOEs, due to 2-fold
molecular symmetry, did not permit us to characterize the
geometry of the macrocycle. For 20b, all of the backbone amide
protons appear to be H-bonded, with large δ and small ∆δ
values, and the couplings involving amide protons are >8.3 Hz.
ꢀ
ꢀ
S)-CγH ≈ 10 Hz, which corresponds to a predominance of a
rotamer with CꢀH(pro-S) in trans disposition with respect to CγH.
It was also noticed that CγH resonances of Leu residues have
unusually small δ values of about 0.4-0.6 ppm for these
peptides, which is further supported by the MD studies, which
show that the Leu residues are located just above the plane of
aromatic ring of Phe, resulting in substantial upfield shifts.
J. Org. Chem. Vol. 73, No. 22, 2008 8737

Chakraborty et al.
bacteria (Table 7). Among Gram-negative bacteria, activity is
more pronounced against E. coli. They exhibited greater activity
against Gram-positive bacteria as compared to Gram-negative
bacteria. Even the antibacterial activity of the least active
compound 1b appears to be more specific toward Gram-positive
bacteria. Compound 2a was the most active against both the
Gram-positive and Gram-negative strains, whereas 2b was less
potent than the former though the net cationic charges (+2)
and hydrophobic residues are same for the two. Compounds
3a and 3b exhibited comparable activity against E. coli and B.
subtilis, while 3b was more active than 3a against P. aeruginosa
and less active against S. aureus. Interestingly, 3a was less active
than 2a, though the increase in net positive charge was expected
to contribute to a greater extent toward activity. On the contrary,
3b had better activity than 2b. Compound 1a also displayed
far better activity than 1b. Thus, it indicates that the relative
hydrophobicity/hydrophilicity produced by the change in con-
formation of the sugar ring has an important role for the activity.
From the results described here, it should be noted that the 2,5-
trans sugar amino acid residues containing cyclic compounds
were more active than their cis congeners.
The compound exhibiting greatest antimicrobial activity (2a)
showed hemolytic activity of 67% at a concentration ∼8-fold
greater than the lethal concentration for E. coli. At equivalent
concentrations, 3a and 3b showed only ∼25% hemolysis. Others
did not show any hemolytic activity at concentrations at which
they exhibited antibacterial activity.
Conclusion
In summary, we have demonstrated the design and synthesis
of sugar amino acid based CAPs. Structurally, these peptides
were found to resemble the dumbbell-shaped loloatin cyclo-
peptides27 more than the typical ꢀ-sheet structures of gramicidin
S6a or tachyplesins.6b,28 The encouraging biological activities
of these compounds may be useful to generate new CAP-based
drugs. Experimental manipulations through the syntheses of
various sugar amino acids and their incorporation to these
interesting cyclic frames, varying in the ratio of hydrophobicity
versus hydrophilicity, and finally their biological performance
could be the prospect of tomorrow’s antibiotic.
FIGURE 3. Lowest energy MD structure (left) and its backbone
highlighted with pseudo-Connolly surface using Insight II (right) of
peptides 1a, 2a, and 3a.
Just like their Boc-protected precursors, the spectra of the
2,5-cis-compounds 1b-3b in water failed to provide any
substantial structural information.
Circular Dichroism Studies of Peptides 1-3. The structures
of the cyclic molecules were examined by circular dichroism
(CD) studies in 5 mM HEPES buffer (pH 7.4) and TFE, which
provides a membrane-like environment and has been used
extensively to delineate conformations of peptides in the
membrane environment. The characteristics of the CD spectra
of sugar amino acid containing peptides have not been docu-
mented yet. They can only be compared with the spectra of
natural amino acid containing peptides. The CD spectra of
compounds 1a-3a, where the tetrahydrofuran rings have 2,5-
trans orientation, showed minima at ∼204 nm both in buffer
and TFE (Supporting Information). The spectra suggest the
population of a distorted turn structure26 in both polar and
nonpolar medium in agreement with what was observed in NMR
studies.
The CD studies of 1b-3b in TFE and buffer show spectra
characteristic of ꢀ conformation with a rigid structure in both
solvents.
Biological Studies. As expected, all of our designed amphi-
pathic peptides, with the exception of 1b, showed pronounced
membranolytic activity against Gram-negative (E. coli and P.
aeruginosa) and Gram-positive (S. aureus and B. subtilis)
Experimental Section
Synthesis of 7a and 7b. To a stirred solution of dry MeOH (125
mL) at 0 °C was added acetyl chloride (20 mL, 270 mmol) slowly
in dropwise manner under nitrogen atmosphere. After 30 min,
compound 6 (25 g, 67.5 mmol), dissolved in dry MeOH (50 mL),
was added to the reaction mixture at room temperature and stirred
for 3 h. The reaction mixture was quenched at 0 °C with liquor
ammonia. MeOH was evaporated under reduced pressure, and the
product was extracted with EtOAc, washed with brine, dried
(Na2SO4), filtered and concentrated in vacuo. Purification by silica
gel column chromatography (SiO2, 18-25% EtOAc in petroleum
ether eluant) afforded compound 7a (9.5 g, 41%) and compound
7b (11.5 g, 50%) as colorless oils. Data for 7a (R anomer): Rf )
0.5 (silica gel, 30% EtOAc in petroleum ether); [R]29 ) +39.63
D
(c 2.4, CHCl3); IR (neat): νmax 2923, 2867, 1452, 1046, 698 cm-1
;
1H NMR (200 MHz, CDCl3): δ 7.30-7.22 (m, 10H, ArH), 4.93
(d, J ) 4.4 Hz, 1H, C1-H), 4.76-4.45 (m, 4H, two -OCH2Ph),
(27) Krachkovskii, S. A.; Sobol, A. G.; Ovchinnikova, T. V.; Tagaev, A. A.;
Yakimenko, Z. A.; Azizbekyan, R. R.; Kuznetsova, N. I.; Shamshina, T. N.;
Arseniev, A. S. Russ. J. Bioorg. Chem 2002, 28, 269–273.
(28) Kawano, K.; Yoneya, T.; Miyata, T.; Yoshikawa, K.; Tokunaga, F.;
Terada, Y.; Iwanaga, S. J. Biol. Chem. 1990, 265, 15365–15367.
(26) Perczel, A.; Hollosi, M. In Circular Dichroism and the Conformational
Analysis of Biomolecules; Fasman, G. D., Ed.; Plenum Press: New York, 1996;
pp 285-380.
8738 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
4.32 (ddd, J ) 5.9, 5.8, 4.4 Hz, 1H, C4-H), 4.20 (dd, J ) 4.4, 3.6
Hz, 1H, C2-H), 3.94 (dd, J ) 5.8, 3.6 Hz, 1H, C3-H), 3.70 (dd, J
) 10.2, 4.4 Hz, 1H, C5-H′), 3.58 (dd, J ) 10.2, 5.9 Hz, 1H, C5-
H′′), 3.48 (s, 3H, -OCH3), 2.58 (d, J ) 7.3 Hz, 1H, C2-OH); 13C
NMR (75 MHz, CDCl3): δ 138.1, 137.9, 128.3, 128.2, 127.7, 127.6,
127.5, 127.4, 101.7, 83.5, 77.3, 76.9, 73.4, 71.8, 69.0, 55.7; MS
(ESI): m/z (%) 345 (5) [M + H]+, 362 (10) [M + NH4]+, 367
(100) [M + Na]+. HRMS (ESI): calcd for C20H24O5Na [M + Na]+
367.1521, found 367.1509. Data for 7b (ꢀ anomer): Rf ) 0.45
temperature. The reaction mixture was then stirred at 65 °C for 2 h.
Acetic acid was evaporated under reduced pressure. The reaction
mixture was quenched at 0 °C by slow addition of saturated aqueous
NaHCO3 solution (100 mL). The product was extracted with EtOAc,
washed with water and brine, dried (Na2SO4), filtered and concentrated
in vacuo to obtain the hemiacetal, which was used for the next reaction
without further purification.
To a stirred solution of the crude hemiacetal in dry dichlo-
romethane (50 mL) at 0 °C was added dry Et3N (8.1 mL, 58.5
mmol). After 5 min, dry acetic anhydride (2.2 mL, 23.4 mmol)
was added slowly to the stirred reaction mixture at 0 °C followed
by the addition of DMAP (238 mg, 1.95 mmol). After stirring for
5 min at room temperature, the reaction mixture was quenched at
0 °C by slow addition of saturated aqueous NH4Cl solution (10
mL). The reaction mixture was extracted with EtOAc, washed with
water and brine, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by silica gel column chromatography (SiO2, 14-16%
EtOAc in petroleum ether eluant) afforded compound 9 (7 g, 95%)
as an inseparable mixture of anomers. Following the same
procedure, compound 9 was also prepared from 8b. Rf ) 0.45 (SiO2,
25% EtOAc in petroleum ether); IR (neat): νmax 2929, 2867, 1738,
(SiO2, 30% EtOAc in petroleum ether); [R]28 ) -44.6 (c 2.9,
D
CHCl3); IR (neat): νmax 2924, 2866, 1453, 1054, 699 cm-1; 1H NMR
(300 MHz, CDCl3): δ 7.30-7.20 (m, 10H, ArH), 4.72 (d, J ) 2.3
Hz, 1H, C1-H), 4.63-4.49 (m, 4H, two -OCH2Ph), 4.4 (ddd, J )
6.7, 6.0, 5.2 Hz, 1H, C4-H), 4.13 (dd, J ) 3.0, 2.3 Hz, 1H, C2-H),
3.9 (dd, J ) 6.0, 3.0 Hz, 1H, C3-H), 3.73 (dd, J ) 10.5, 5.2 Hz,
1H, C5-H′), 3.63 (dd, J ) 10.5, 6.7 Hz, 1H, C5-H′′), 3.36 (s, 3H,
-OCH3); 13C NMR (75 MHz, CDCl3): δ 138.0, 137.7, 128.3, 128.2,
127.7, 127.6, 127.5, 109.4, 83.2, 79.9, 79.3, 73.3, 72.2, 69.8, 55.6;
MS (ESI): m/z (%) 345 (5) [M + H]+, 362 (95) [M + NH4]+, 367
(100) [M + Na]+; HRMS (ESI): calcd for C20H24O5Na [M + Na]+
367.1521, found 367.1521.
1
1094, 697 cm-1; H NMR (300 MHz, CDCl3): δ 7.31-7.19 (m,
Synthesis of 8a and 8b. Sodium hydride (1.8 g, 60% dispersion
in oil, 46 mmol) was added portionwise to a stirred solution of 7a
(8 g, 23 mmol) in dry THF (60 mL) at 0 °C under nitrogen
atmosphere. After the additions were completed, the reaction
mixture was stirred at 0 °C for 15 min. Then MeI (3 mL, 46 mmol)
was added slowly to the stirred reaction mixture followed by the
addition of TBAI (848 mg, 2.3 mmol). After stirring for 2 h at
room temperature, the reaction mixture was quenched at 0 °C by
slow addition of saturated aqueous NH4Cl solution. THF was
removed under reduced pressure, and the product was extracted
with EtOAc, washed with water and brine, dried (Na2SO4), filtered
and concentrated in vacuo. Purification by silica gel column
chromatography (SiO2, 16-18% EtOAc in petroleum ether eluant)
afforded compound 8a (8 g, 96%) as a colorless oil. (R-anomer):
total 20H), 6.31 (d, J ) 4.5 Hz, 1H), 6.04 (s, 1H), 4.70-4.37 (m,
total 9H), 4.15 (dd, J ) 6.8, 6.0 Hz, 1H), 3.99-3.93 (m, total 2H),
3.83-3.75 (m, total 4H), 3.71-3.64 (m, 1H), 3.56 (dd, J ) 10.6,
5.3 Hz, 1H), 3.37 (s, 3H), 3.35 (s, 3H), 2.09 (s, 3H), 2.02 (s, 3H);
13C NMR (mixture of diastereomers) (75 MHz, CDCl3): δ 169.9,
138.0, 137.9, 137.7, 137.6, 128.3, 128.2, 127.5, 127.4, 127.3, 99.8,
93.8, 87.2, 85.2, 81.8, 80.4, 77.9, 73.3, 73.2, 72.5, 72.0, 68.6, 58.8,
57.6, 21.1, 21.0; MS (ESI): m/z (%) 410 (100) [M + Na]+; HRMS
(ESI): calcd for C22H26O6Na [M + Na]+ 409.1627, found 409.1621.
Synthesis of 10. To a stirred solution of 9 (10 g, 25.9 mmol) in
dry acetonitrile (70 mL) was added TMSCN (5.2 mL, 38.8 mmol)
was added at 0 °C followed by BF3 ·Et2O (3 mL) and stirring was
continued for 30 min at room temperature. The reaction mixture was
quenched at 0 °C by slow addition of saturated aqueous NaHCO3
solution (10 mL). Acetonitrile was evaporated under reduced pressure,
and the reaction mixture was extracted with EtOAc, washed with water
and brine, dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by silica gel column chromatography (SiO2, 10-12% EtOAc in
petroleum ether eluant) afforded compound 10 (8.3 g, 91%) as colorless
oil and as a mixture of anomers. Rf ) 0.45 (SiO2, 20% EtOAc in
Rf ) 0.5 (SiO2, 25% EtOAc in petroleum ether); [R]28 ) +88.0
D
(c 1.6, CHCl3); IR (neat): νmax 2918, 1453, 1033, 698 cm-1; H
1
NMR (300 MHz, CDCl3): δ 7.33-7.22 (m, 10H, ArH), 4.91 (d, J
) 4.5 Hz, 1H, C1-H), 4.64 (d, J ) 12.0 Hz, 1H, HA of -OCH2Ph),
4.57 (d, J ) 12.0 Hz, 1H, HB of -OCH2Ph), 4.53 (d, J ) 12.0 Hz,
1H, HA of another -OCH2Ph), 4.50 (d, J ) 12.0 Hz, 1H, HB of
another -OCH2Ph), 4.32 (ddd, J ) 6.7, 6.0, 4.5 Hz, 1H, C4-H),
4.19 (dd, J ) 6.7, 6.0 Hz, 1H, C3-H), 3.82 (dd, J ) 6.0, 4.5 Hz,
1H, C2-H), 3.68 (dd, J ) 10.5, 4.5 Hz, 1H, C5-H′), 3.55 (dd, J )
10.5, 6.7 Hz, 1H, C5-H′′), 3.43 (s, 3H, -OCH3), 3.36 (s, 3H, -OCH3);
13C NMR (75 MHz, CDCl3): δ 138.1, 137.9, 128.3, 128.2, 127.7,
127.6, 127.5, 100.2, 86.3, 81.3, 76.1, 73.4, 72.6, 69.2, 58.2, 55.2;
MS (ESI): m/z (%) 359 (25) [M + H]+, 376 (96) [M + NH4]+,
381 (100) [M + Na]+; HRMS (ESI): calcd for C21H26O5Na [M +
Na]+ 381.1677, found 381.1674. Compound 8b (9.5 g, 91%) was
prepared as colorless oil from 7b (10 g, 29 mmol) following the
same procedure as described for the synthesis of 8a. (ꢀ-anomer):
Rf ) 0.45 (SiO2, 20% EtOAc in petroleum ether); [R]28D ) -70.38
petroleum ether). IR (neat): νmax 2931, 1791, 1244, 1071, 678 cm-1
;
1H NMR (300 MHz, CDCl3): δ 7.34-7.21 (m, total 20H), 4.83 (d, J
) 4.5 Hz, 1H), 4.70-4.46 (m, total 9H), 4.35-4.29 (m, 1H),
4.23-4.17 (m, 1H), 4.10-4.08 (m, 1H), 4.04 (dd, J ) 3.8, 2.3 Hz,
1H), 3.98 (dd, J ) 3.8, 1.5 Hz, 1H), 3.89 (dd, J ) 4.5, 2.3 Hz, 1H),
3.76 (dd, J ) 9.8, 6.0 Hz, 1H), 3.70 (dd, J ) 6.0, 1.5 Hz, 1H),
3.68-3.50 (m, total 2H), 3.41 (s, 3H), 3.32 (s, 3H); 13C NMR (mixture
of diastereomers) (75 MHz, CDCl3): δ 137.7, 137.0, 129.6, 128.5,
128.4, 128.3, 128.1, 127.9, 127.7, 127.4, 117.2, 115.6, 87.4, 84.5, 81.7,
80.5, 80.2, 73.4, 72.6, 72.2, 69.6, 67.7, 67.3, 58.6, 57.8; MS (ESI):
m/z (%) 354 (5) [M + H]+ 376 (100) [M + Na]+; HRMS (ESI): calcd
for C21H23NO4Na [M + Na]+ 376.1524, found 376.1508.
1
(c 1.8, CHCl3); IR (neat): νmax 2920, 1450, 1059, 668 cm-1; H
Synthesis of 11a and 11b. To a stirring solution of compound 10
(8 g, 22.6 mmol) in dry dichloromethane (130 mL) at -78 °C was
added DIBAL-H (1.4 M, 16.1 mL) very slowly in a dropwise manner
under nitrogen atmosphere. After being stirred for 8 h at -78 °C, the
reaction mixture was quenched with saturated aqueous solution of
sodium potassium tartrate (50 mL). The reaction mixture was stirred
at room temperature until two layers got separated (1 h). Dichlo-
romethane was decanted and the remaining aqueous layer was extracted
with EtOAc. The combined organic layers were washed with H2O
and brine, dried (Na2SO4), filtered, concentrated in vacuo and used
for the next reaction without further purification.
NMR (300 MHz, CDCl3): δ 7.30-7.22 (m, 10H, ArH), 4.76 (d, J
) 1.5 Hz, 1H, C1-H), 4.60 (d, J ) 12.0 Hz, 1H, HA of -OCH2Ph),
4.57 (d, J ) 12.0 Hz, 1H, HB of -OCH2Ph), 4.52 (d, J ) 12.0 Hz,
1H, HA of another -OCH2Ph), 4.51 (d, J ) 12.0 Hz, 1H, HB of
another -OCH2Ph), 4.34 (ddd, J ) 6.7, 6.0, 5.2 Hz, 1H, C5-H),
3.94 (dd, J ) 6.0, 3.0 Hz, 1H, C4-H), 3.75 (dd, J ) 9.8, 5.2 Hz,
1H, C6-H′), 3.69 (dd, J ) 3.0, 1.5 Hz, 1H, C3-H), 3.64 (dd, J )
9.8, 6.7 Hz, 1H, C6-H′′), 3.38 (s, 3H, -OCH3), 3.31 (s, 3H, -OCH3);
13C NMR (75 MHz, CDCl3): δ 138.2, 137.6, 128.4, 128.3, 127.7,
127.5, 107.8, 88.8, 81.1, 80.0, 73.4, 72.2, 69.6, 57.6, 55.6; MS
(ESI): m/z (%) 359 (4) [M + H]+ 376 (100) [M + NH4]+, 381
(20) [M + Na]+. HRMS (ESI): calcd for C21H26O5Na [M + Na]+
381.1677, found 381.1673.
To a solution of the crude aldehyde, Rf ) 0.3 (SiO2, 30% EtOAc
in petroleum ether), in t-BuOH/2-methyl-2-butene (2.5:0.3, 63 mL) at
room temperature were addedNaClO2 (6.1 g, 67.8 mmol) and NaH2PO4
(10.5 g, 67.8 mmol) by dissolving in a minimum amount of water.
Synthesis of 9. To a stirring solution of compound 8a (7 g, 19.5
mmol) in acetic acid (50 mL) was added 1 N HCl (10 mL) at room
J. Org. Chem. Vol. 73, No. 22, 2008 8739

Chakraborty et al.
After being stirred for 1 h, the solvent was removed in rotary
evaporator, 1 N HCl was added to the mixture at room temperature to
raise the pH up to 5, and the residue was extracted with EtOAc. The
combined organic extracts were washed with brine, dried (Na2SO4)
and concentrated in vacuo. To the resulting crude acid in ether was
added diazomethane dropwise at 0 °C until the acid was converted to
the methyl ester (as shown by TLC). The solvent was concentrated
and silica gel column chromatographic purification (SiO2, 12-14%
EtOAc in petroleum ether eluant) afforded compound 11a (3.6 g, 40%)
and 11b (2.9 g, 32%) as liquids. Data for 11a: Rf ) 0.40 (SiO2, 20%
EtOAc in petroleum ether); [R]28D ) -30.52 (c 5.1, CHCl3); IR (neat):
+ NH4]+, 911 (100) [2 M + Na]+; HRMS (ESI): calcd for
C24H32O6NaSi [M + Na]+ 467.1865, found 467.1871.
Synthesis of 14a. To a stirring solution of compound 13a (935
mg, 2.1 mmol) in dry dichloromethane (5 mL) at -40 °C was added
dry pyridine (4 mL). After 5 min, triflic anhydride (0.52 mL, 3.15
mmol) was added to the reaction mixture. After being stirred for
30 min at 0 °C, the reaction mixture was quenched with saturated
aqueous NH4Cl, extracted with EtOAc, washed with water, saturated
aqueous cupric sulfate solution, water, and brine, dried (Na2SO4),
filtered and concentrated in vacuo. Flash column chromatography
afforded quantitative yield of the triflate intermediate, which was
used for the next reaction without further characterization.
To a stirring solution of the triflate in dry DMF (5 mL) at -5
°C was added sodium azide (819 mg, 12.6 mmol). After being
stirred for 6 h at -5 °C, the reaction mixture was quenched with
water (3 mL) and extracted with EtOAc, washed with water and
brine, dried (Na2SO4), filtered and concentrated in vacuo. Purifica-
tion by silica gel column chromatography (SiO2, 8-10% EtOAc
in petroleum ether eluant) afforded compound 14a (690 mg, 70%)
as a colorless oil. Rf ) 0.45 (SiO2, 15% EtOAc in petroleum ether);
ν
max 2925, 2859, 1738, 1090, 699 cm-1; 1H NMR (300 MHz, CDCl3):
δ 7.30-7.22 (m, 10H, ArH), 4.70 (d, J ) 4.5 Hz, 1H, C2-H),
4.60-4.48 (m, 4H, two -OCH2Ph), 4.40 (ddd, J ) 6.8, 5.2, 3.8 Hz,
1H, C5-H), 4.01 (dd, J ) 4.5, 2.3 Hz, 1H, C3-H), 3.98 (dd, J ) 5.2,
2.3 Hz, 1H, C4-H), 3.76-3.63 (m, 2H, C6-H′, C6-H′′), 3.73 (s, 3H,
-COOCH3) 3.26 (s, 3H, -OCH3); 13C NMR (75 MHz, CDCl3): δ 170.1,
138.1, 137.5, 128.4, 128.3, 127.9, 127.7, 127.6, 127.5, 85.3, 80.1, 79.9,
79.8, 73.4, 72.5, 67.4, 58.4, 51.9; MS (ESI): m/z (%) 409 (10) [M +
Na]+, 795 (100) [2 M + Na]+; HRMS (ESI): calcd for C22H26O6Na
[M + Na]+ 409.1627, found 409.1620. Data for 11b: Rf ) 0.45 (SiO2,
20% EtOAc in petroleum ether); [R]28D ) -36.11 (c 1.6, CHCl3); IR
(neat): νmax 2928, 2858, 1727, 1098, 699 cm-1; 1H NMR (300 MHz,
CDCl3): δ 7.34-7.22 (m, 10H, ArH), 4.60 (d, J ) 12.0 Hz, 2H,
-OCH2Ph) 4.54 (s, 1H, C2-H), 4.48 (ABq, J ) 11.5 Hz, 2H, -OCH2Ph),
4.35 (ddd, J ) 6.0, 5.8, 4.1 Hz, 1H, C5-H), 4.17 (t, J ) 1.5 Hz, 1H,
C3-H), 3.96 (dd, J ) 4.1, 1.5 Hz, 1H, C4-H), 3.83 (s, 1H, C6-H′), 3.81
(s, 1H, C6-H′′), 3.66 (s, 3H, -COOCH3) 3.39 (s, 3H, -OCH3); 13C NMR
(75 MHz, CDCl3): δ 171.0, 138.1, 137.5, 128.3, 128.2, 127.5, 127.4,
127.3, 87.1, 81.1, 80.8, 80.3, 73.3, 71.7, 67.9, 57.2, 52.1; MS (ESI):
m/z (%) 409 (100) [M + Na]+; HRMS (ESI): calcd for C22H27O6 [M
+ H]+ 387.1807, found 387.1795.
[R]32 ) +20.2 (c 0.5, CHCl3); IR (neat): νmax 2932, 2859, 2105,
D
1739, 1104, 702 cm-1; 1H NMR (300 MHz, CDCl3): δ 7.69-7.64
(m, 4H, ArH), 7.47-7.35 (m, 6H, ArH), 4.73 (d, J ) 5.2 Hz, 1H,
C2-H), 4.31 (td, J ) 8.3, 3.0 Hz, 1H, C5-H), 4.24 (dd, J ) 5.2, 4.5
Hz, 1H, C3-H), 4.02-3.95 (m, 2H, C4-H and C6-H′), 3.82 (s, 3H,
-COOCH3), 3.79 (dd, J ) 11.3, 3.0 Hz, 1H, C6-H′′), 3.56 (s, 3H,
-OCH3), 1.05 (s, 9H, tBu); 13C NMR (100 MHz, CDCl3): δ 169.4,
135.6, 135.5, 133.0, 132.8, 129.9, 129.8, 127.8, 127.7, 83.4, 81.0,
79.9, 62.8, 61.0, 60.5, 52.1, 26.8, 19.3; MS (ESI): m/z (%) 493
(100) [M + Na]+; HRMS (ESI): calcd for C24H31N3O5NaSi [M +
Na]+ 492.1930, found 492.1927.
Synthesis of 15a. To a solution of 14a (500 mg, 1.06 mmol) in
MeOH (3 mL) was added Pd on C (10%, 60 mg) and the mixture
was hydrogenated under atmospheric pressure using a H2 balloon
for 15 min. The reaction mixture was then filtered through a short
pad of Celite, and the filter cake was washed with MeOH. The
filtrate and the washings were combined and concentrated in vacuo
and were used for the next reaction without further purification.
To a stirring solution of the crude amine in dry dichloromethane
(3 mL) was added Boc2O (0.38 mL, 1.58 mmol) at room
temperature. After being stirred for 1 h, the solvent was evaporated
in vacuo. Purification by silica gel column chromatography (SiO2,
8-10% EtOAc in petroleum ether eluant) afforded compound 15a
(510 mg, 88%) as colorless gummy liquid. Rf ) 0.3 (SiO2, 25%
Synthesis of 12a. To a solution of 11a (3 g, 7.4 mmol) in MeOH
(15 mL) was added Pd on C (10%, 600 mg) and the mixture was
hydrogenated under atmospheric pressure using a H2 balloon for
12 h. The reaction mixture was then filtered through a short pad of
Celite, and the filter cake was washed with MeOH. The filtrate
and the washings were combined and concentrated in vacuo.
Purification by silica gel column chromatography (SiO2, 90%
EtOAc in petroleum ether eluant) afforded compound 12a (1.3 g,
85%) as colorless gummy liquid. Rf ) 0.35 (silica gel, EtOAc).
[R]28 ) -18.8 (c 0.7, CHCl3); IR (neat): νmax 2924, 2855, 1742,
D
1085, 702 cm-1; H NMR (300 MHz, CDCl3): δ 4.89 (d, J ) 4.9
1
Hz, 1H, C2-H), 4.42-4.40 (m, 1H), 4.27-4.23 (m, 1H), 4.17 (dd,
J ) 12.4, 3.8 Hz, 1H), 4.04-3.98 (m, 2H), 3.78 (s, 3H, -COOCH3),
3.40 (s, 3H, -OCH3); 13C NMR (75 MHz, CDCl3): δ 170.3, 87.9,
80.0, 79.9, 75.9, 61.6, 58.7, 52.0; MS (ESI): m/z (%) 207 (20) [M
+ H]+, 224 (100) [M + NH4]+; HRMS (ESI): calcd for C8H14O6Na
[M + Na]+ 229.0688, found 229.0680.
EtOAc in petroleum ether). [R]28 ) +28.97 (c 1.4, CHCl3); IR
D
1
(neat): νmax 2950, 2855, 1745, 1710, 1097, 698 cm-1; H NMR
(300 MHz, CDCl3): δ 7.68-7.64 (m, 4H, ArH), 7.39-7.32 (m,
6H, ArH), 5.12 (d, J ) 9.0 Hz, 1H, NHBoc) 4.69 (d, J ) 6.0 Hz,
1H, C2-H), 4.57-4.48 (m, 1H, C5-H), 4.21 (t, J ) 6.0 Hz, 1H,
C3-H), 4.06 (ddd, J ) 9.0, 6.0, 3.8 Hz, 1H, C4-H), 3.83 (dd, J )
11.3, 2.3 Hz, 1H, C6-H′), 3.80 (s, 3H, -COOCH3), 3.73 (dd, J )
11.3, 2.3 Hz, 1H, C6-H′′), 3.44 (s, 3H, -OCH3), 1.46 (s, 9H, tBu of
Boc), 1.06 (s, 9H, tBu of -OTBDPS); 13C NMR (75 MHz, CDCl3):
δ 170.7, 155.4, 135.7, 135.6, 133.0, 129.7, 129.6, 127.6, 84.1, 81.7,
80.2, 79.7, 63.9, 60.2, 53.5, 52.0, 28.3, 26.8, 19.2; MS (ESI): m/z
(%) 544 (70) [M + H]+, 561 (100) [M + NH4]+, 566 (60) [M +
Na]+; HRMS (ESI): calcd for C29H41NO7NaSi [M + Na]+
566.2550, found 566.2538.
Synthesis of 13a. Et3N (1.62 mL, 11.6 mmol) and TBDPS-Cl
(1.7 mL, 6.38 mmol) were added sequentially to a solution of
compound 12a (1.2 g, 5.82 mmol) in dry CH2Cl2 (14 mL) at 0 °C.
After stirring for 5 min at the same temperature, DMAP (71 mg,
0.58 mmol) was added to the reaction mixture. After being stirred
for 8 h at room temperature, the reaction mixture was quenched
with saturated aqueous NH4Cl, extracted with EtOAc, washed with
water and brine, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by column chromatography (SiO2, 10-12% EtOAc in
petroleum ether eluant) afforded compound 13a (2.1 g, 80%) as
Synthesis of 16a. To a stirring solution of compound 15a (400
mg, 0.74 mmol) in dry THF (2 mL) at 0 °C was added TBAF in
THF (1 M, 1.1 mL, 1.1 mmol). After being stirred for 1 h at 0 °C,
the reaction mixture was quenched with saturated aqueous NH4Cl,
extracted with EtOAc, washed with water and brine, dried (Na2SO4),
filtered and concentrated in vacuo. Purification by column chro-
matography (SiO2, 30-32% EtOAc in petroleum ether eluant)
afforded compound 16a (154 mg, 68%). Rf ) 0.35 (SiO2, 60%
colorless oil. Rf ) 0.5 (SiO2, 33% EtOAc in petroleum ether); [R]28
D
) -12.96 (c 6.4, CHCl3); IR (neat): νmax 3206, 2952, 2842, 1647,
1015 cm-1;1H NMR (200 MHz, CDCl3): δ 7.71-7.61 (m, 4H),
7.44-7.34 (m, 6H), 4.86 (d, J ) 5.5 Hz, 1H, C2-H), 4.43-4.38
(m, 1H, C5-H), 4.24-4.16 (m, 1H, C6-H′), 4.15-4.05 (m, 2H, C6-
H′′, C3-H), 4.03-3.98 (m, 1H, C4-H), 3.76 (s, 3H, -COOCH3), 3.43
t
(s, 3H, -OCH3), 1.07 (s, 9H, Bu); 13C NMR (75 MHz, CDCl3): δ
EtOAc in petroleum ether); [R]28 ) +20.9 (c 1.8, CHCl3); IR
D
1
(neat): νmax 3509, 3361, 2946, 1744, 1682, 1520, 1068 cm-1; H
170.3, 135.5, 135.3, 132.2, 131.8, 129.9, 127.8, 87.8, 79.9, 79.7,
75.4, 63.0, 58.5, 51.8, 26.6, 18.9; MS (ESI): m/z (%) 462 (66) [M
NMR (300 MHz, CDCl3): δ 5.07 (d, J ) 7.9 Hz, 1H, C4-NHBoc)
8740 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
4.78 (d, J ) 4.7 Hz, 1H, C2-H), 4.24-4.12 (m, 2H, C3-H and C5-
H), 3.94-3.84 (m, 2H, C4-H and C6-H′), 3.79 (s, 3H, -COOCH3),
3.70-3.63 (m, 1H, C6-H′′), 3.46 (s, 3H, -OCH3), 1.45 (s, 9H, tBu);
13C NMR (75 MHz, CDCl3): δ 169.9, 155.9, 82.9, 81.7, 80.3, 79.9,
61.3, 60.6, 52.7, 51.8, 28.1; MS (ESI): m/z (%) 206 (45) [M -
Boc + H]+, 328 (100) [M + Na]+; HRMS (ESI): calcd for
C13H23NO7Na [M + Na]+ 328.1372, found 328.1379.
Synthesis of 4a. To a stirring solution of compound 16a (150
mg, 0.49 mmol) in dry dichloromethane (2 mL) at 0 °C was added
dry Et3N (0.2 mL, 1.47 mmol). After 5 min, TsCl (187 mg, 0.98
mmol) was added to the reaction mixture followed by the addition
of DMAP (6 mg, 0.05 mmol). After being stirred for 2 h at room
temperature, the reaction mixture was quenched with saturated
aqueous NH4Cl, extracted with EtOAc, washed with water and
brine, dried (Na2SO4), filtered and concentrated in vacuo. The crude
product was used for the next reaction without further purification.
132.7, 129.8, 129.7, 127.7, 127.6, 84.2, 82.0, 80.0, 63.4, 61.1, 58.5,
52.3, 26.7, 19.1; MS (ESI): m/z (%) 470 (3) [M + H]+, 487 (55)
[M + NH4]+, 492 (100) [M + Na]+; HRMS (ESI): calcd for
C24H31N3O5NaSi [M + Na]+ 492.1930, found 492.1930.
Synthesis of 15b. Compound 14b (400 mg, 0.85 mmol) was
transformed into compound 15b (390 mg, 85%) following the same
procedure as described for the synthesis of 15a. Rf ) 0.35 (SiO2,
25% EtOAc in petroleum ether); [R]28 ) +8.47 (c 1.7, CHCl3);
D
IR (neat): νmax 2933, 2860, 1740, 1714, 1093, 703 cm-1; 1H NMR
(300 MHz, CDCl3): δ 7.73-7.69 (m, 4H, ArH), 7.43-7.33 (m,
6H, ArH), 4.93 (d, J ) 6.7 Hz, 1H, NHBoc), 4.52 (d, J ) 1.5 Hz,
1H, C2-H), 4.16-4.08 (m, 1H, C5-H), 3.99-3.92 (m, 2H, C3-H
and C4-H), 3.89-3.85 (m, 2H, C6-H′ and C6-H′′), 3.69 (s, 3H,
-COOCH3), 3.45 (s, 3H, -OCH3), 1.40 (s, 9H, tBu of Boc), 1.05 (s,
9H, tBu of -OTBDPS); 13C NMR (75 MHz, CDCl3): δ 170.7, 155.2,
135.7, 135.6, 133.4, 133.2, 129.6, 127.6, 83.4, 83.0, 80.4, 79.7,
64.7, 57.8, 53.2, 52.2, 28.2, 26.7, 19.2; MS (ESI): m/z (%) 544
(20) [M + H]+, 561 (93) [M + NH4]+, 566 (100) [M + Na]+;
HRMS (ESI): calcd for C29H41NO7NaSi 566.2550, found 566.2545.
Synthesis of 16b. Compound 15b (350 mg, 0.64 mmol) was
transformed into compound 16b (180 mg, 80%) following the same
procedure as described for the synthesis of 16a. Rf ) 0.35 (silica
To a stirring solution of the crude product in dry DMF (2 mL)
at room temperature was added sodium azide (96 mg, 1.47 mmol).
After being stirred for 2 h at 65 °C, the reaction mixture was
quenched with water (3 mL) and extracted with EtOAc, washed
with water and brine, dried (Na2SO4), filtered and concentrated in
vacuo. Purification by silica gel column chromatography (SiO2,
18-20% EtOAc in petroleum ether eluant) afforded compound 4a
(142 mg, 88%). Rf ) 0.5 (silica gel, 35% EtOAc in petroleum ether);
gel, 40% EtOAc in petroleum ether); [R]28 ) -1.07 (c 2.1,
D
1
CHCl3); IR (neat): νmax 2927, 1740, 1011 cm-1; H NMR (300
[R]28 ) +87.7 (c 1.6, CHCl3); IR (neat): νmax 3363, 2978, 2938,
MHz, CDCl3): δ 5.09 (d, J ) 7.5 Hz, 1H, C4-NHBoc), 4.53 (s,
1H, C2-H), 4.29-4.21 (m, 1H, C5-H), 3.93-3.85 (m, 3H, C3-H,
C4-H and C6-H′), 3.73 (s, 3H, -COOCH3), 3.70-3.63 (m, 1H, C6-
D
2101, 1763, 1708, 1099 cm-1; 1H NMR (300 MHz, CDCl3): δ 4.98
(d, J ) 8.3 Hz 1H, C4-NHBoc), 4.75 (d, J ) 5.2 Hz, 1H, C2-H),
4.24-4.16 (m, 1H, C5-H), 4.11-4.03 (m, 2H, C3-H and C4-H),
3.78 (s, 3H, -COOCH3), 3.68-3.60 (m, 1H, C6-H′), 3.45 (s, 3H,
-OCH3), 3.32 (dd, J ) 12.8, 2.3 Hz, 1H, C6-H′′) 1.45 (s, 9H, tBu);
13C NMR (75 MHz, CDCl3): δ 169.7, 155.3, 81.7, 81.4, 80.0, 60.6,
53.6, 52.0, 51.8, 28.2; MS (ESI): m/z (%) 353 (100) [M + Na]+;
HRMS (ESI): calcd for C13H22N4O6Na [M + Na]+ 353.1437, found
353.1442.
t
H′′), 3.45 (s, 3H, -OCH3), 1.42 (s, 9H, Bu); 13C NMR (75 MHz,
CDCl3): δ 171.4, 155.5, 83.6, 83.2, 80.0, 79.5, 61.2, 57.5, 52.5,
51.6, 28.0; MS (ESI): m/z (%) 306 (68) [M + H]+, 323 (100) [M
+ NH4]+, 328 (66) [M + Na]+; HRMS (ESI): calcd for
C13H23NO7Na 328.1372, found 328.1374.
Synthesis of 4b. Compound 16b (150 mg, 0.49 mmol) was
transformed into compound 4b (121 mg, 78%) following the same
procedure as described for the synthesis of 4a. Rf ) 0.5 (SiO2,
35% EtOAc in petroleum ether); [R]28D ) +80.51 (c 2.8, CHCl3);
IR (neat): νmax 2934, 2103, 1749, 1711, 1094, 751 cm-1; 1H NMR
(300 MHz, CDCl3): δ 4.98 (d, J ) 8.3 Hz, 1H, C4-NHBoc), 4.53
(d, J ) 0.7 Hz, 1H, C2-H), 4.15-4.02 (m, 1H, C5-H), 3.93-3.85
(m, 2H, C3-H, C4-H), 3.78 (s, 3H, -COOCH3), 3.53-3.50 (m, 2H,
C6-H′ and C6-H′′), 3.48 (s, 3H, -OCH3), 1.44 (s, 9H, tBu); 13C NMR
(75 MHz, CDCl3): δ 170.3, 155.2, 83.1, 81.9, 80.3, 80.1, 57.6, 53.5,
52.8, 52.5, 28.0; MS (ESI): MS (ESI): m/z (%) 331 (5) [M + H]+,
354 (100) [M + Na]+; HRMS (ESI): calcd for C13H22N4O6Na [M
+ Na]+ 353.1437, found 353.1431.
Synthesis of 17a. To a solution of 4a (113 mg, 0.34 mmol) in
THF/MeOH/H2O (1.5:0.5:0.5 mL) at 0 °C was added LiOH ·H2O
(36 mg, 0.85 mmol) and this mixture was stirred at room
temperature for 1 h. The mixture was then acidified to pH 2 with
1 N HCl. The reaction mixture was extracted with EtOAc, washed
with water and brine, dried (Na2SO4), filtered and concentrated in
vacuo to obtain the acid. The crude acid was used for the next
reaction without further purification.
Synthesis of 12b. Compound 11b (2.5 g, 6.2 mmol) was
converted into compound 12b (1.1 g, 88%) following the same
procedure as described for the synthesis of 12a. Rf ) 0.35 (SiO2,
EtOAc); [R]28 ) -28.42 (c 0.7, CHCl3); IR (neat): νmax 2925,
D
2859, 1738, 1090, 699 cm-1; 1H NMR (300 MHz, CDCl3): δ 4.62
(d, J ) 4.3 Hz, 1H, C2-H), 4.51(s, 1H), 4.35-4.31 (m, 1H),
4.28-4.12 (m, 2H), 4.05-3.97 (m, 1H), 3.79 (s, 3H, -COOCH3),
3.44 (s, 3H, -OCH3); 13C NMR (75 MHz, CDCl3): δ 172.7, 90.8,
81.2, 80.2, 76.4, 61.5, 57.5, 52.5; MS (ESI): m/z (%) 207 (90) [M
+ H]+, 224 (100) [M + NH4]+, 229 (35) [M + Na]+; HRMS (ESI):
calcd for C8H14O6Na [M + Na]+ 229.0688, found 229.0691.
Synthesis of 13b. Compound 12b (900 mg, 4.3 mmol) was
transformed into compound 13b (1.6 g, 84%) following the same
procedure as described for the synthesis of 13a. Rf ) 0.5 (SiO2,
30% EtOAc in petroleum ether). [R]28D ) -5.2 (c 2.3, CHCl3); IR
(neat): νmax 3206, 2952, 1650, 1109, 669 cm-1; 1H NMR (200 MHz,
CDCl3): δ 7.79-7.67 (m, 4H, ArH), 7.44-7.34 (m, 6H, ArH), 4.39
(d, J ) 1.5 Hz, 1H, C2-H), 4.22 (bs, 1H), 4.15-3.98 (m, 3H), 3.93
(d, J ) 1.5 Hz, 1H, C3-H), 3.75 (s, 3H, -COOCH3), 3.45 (s, 3H,
t
-OCH3), 1.07 (s, 9H, Bu); 13C NMR (75 MHz, CDCl3): δ 171.6,
To a stirred solution of Boc-Phe-Leu-OMe (200 mg, 0.51 mmol)
in dry dichloromethane (2 mL) at 0 °C was added trifluoroacetic
acid (1 mL) and the mixture was stirred for 2 h at room temperature.
The reaction mixture was then concentrated in vacuo to obtain the
trifluoroacetate salt.
135.7, 135.5, 132.6, 132.3, 129.9, 129.8, 127.8, 127.7, 90.1, 81.3,
81.1, 75.4, 62.6, 57.5, 52.3, 26.6, 19.0; MS (ESI): m/z (%) 445 (5)
[M + H]+, 467 (100) [M + Na]+; HRMS (ESI): calcd for
C24H32O6NaSi [M + Na]+ 467.1865, found 467.1846.
Synthesis of 14b. Compound 13b (800 mg, 1.8 mmol) was
transformed into compound 14b (750 mg, 90%) following the same
procedure as described for the synthesis of 14a. Rf ) 0.5 (SiO2,
15% EtOAc in petroleum ether); [R]28D ) +15.76 (c 4.8, CHCl3);
IR (neat): νmax 2929, 2860, 2108, 1736, 1113, 705 cm-1; 1H NMR
(500 MHz, CDCl3): δ 7.74-7.65 (m, 4H, ArH), 7.43-7.33 (m,
6H, ArH), 4.47 (d, J ) 3.6 Hz, 1H, C2-H), 4.15-4.11 (m, 2H,
C3-H and C5-H), 3.92 (dd, J ) 6.3, 5.4 Hz, 1H, C4-H), 3.85 (dd, J
) 11.8, 3.6 Hz, 1H, C6-H′), 3.80 (dd, J ) 11.8, 3.8 Hz, 1H, C6-
H′′), 3.73 (s, 3H, -COOCH3), 3.52 (s, 3H, -OCH3), 1.06 (s, 9H,
tBu); 13C NMR (75 MHz, CDCl3): δ 170.5, 135.6, 135.5, 132.8,
To a stirring solution of the crude acid in dry dichloromethane
(3 mL) at 0 °C were sequentially added HOBt ·H2O (69 mg, 0.51
mmol) and EDCI (98 mg, 0.51 mmol). After 10 min, the above-
prepared trifluoroacetate salt was dissolved in dichloromethane (3
mL) and added to the reaction mixture followed by the addition of
DIPEA (0.16 mL, 0.9 mmol). After stirring for 12 h at room
temperature, the reaction mixture was diluted with EtOAc, washed
with 1 N HCl solution, saturated NaHCO3 solution, water, and brine,
dried (Na2SO4), filtered and concentrated in vacuo. Purification by
silica gel column chromatography (SiO2, 35-37% EtOAc in
petroleum ether eluant) afforded compound 17a (172 mg, 85%).
J. Org. Chem. Vol. 73, No. 22, 2008 8741

Chakraborty et al.
Rf ) 0.4 (SiO2, 50% EtOAc in petroleum ether); [R]28 ) -5.41
m/z (%) 592 (8) [M + H]+, 614 (100) [M + Na]+; HRMS (ESI):
calcd for C28H42N6O8Na [M + Na]+ 613.2961, found 613.2951.
Synthesis of 18b. Compound 17b (140 mg, 0.23 mmol) was
transformed into compound 18b (75 mg, 61%) following the same
procedure as described for the synthesis of 18a. Rf ) 0.5 (silica
gel, 40% acetone in petroleum ether); IR (neat): νmax 3285, 2925,
2856, 1649, 1502, 1460, 1089, 752, 666 cm-1; 1H NMR, see Table
4; 13C NMR (100 MHz, CDCl3): δ 173.0, 170.5, 169.7, 155.7,
136.9, 129.1, 128.6, 126.8, 83.4, 81.2, 80.6, 80.3, 57.2, 54.0, 52.1,
51.7, 41.9, 38.7, 37.5, 28.9, 24.8, 22.8, 22.3; MS (ESI): m/z (%)
1087 (100) [M + Na]+; HRMS (ESI): calcd for C54H80N8O14Na
[M + Na]+ 1087.5691, found 1087.5721.
D
(c 1.3, CHCl3); IR (neat): νmax 2958, 2103, 1742, 1707, 1676, 1518,
1
1167 cm-1; H MNR (300 MHz, CDCl3): δ 7.27-7.19 (m, 5H,
ArH), 7.11 (d, J ) 8.3 Hz, 1H, PheNH), 6.17 (d, J ) 7.5 Hz, 1H,
LeuNH), 4.84 (d, J ) 8.3 Hz, 1H, C4-NHBoc), 4.64 (td, J ) 8.3,
6.7 Hz, 1H, PheRH), 4.52 (d, J ) 3.8 Hz, 1H, C2-H), 4.50-4.42
(m, 1H, LeuRH), 4.21-4.12 (m, 1H, C5-H), 3.98 (t, J ) 3.8 Hz,
1H, C3-H), 3.82 (ddd, J ) 8.3, 4.5, 3.8 Hz, 1H, C4-H), 3.63 (s, 3H,
-COOCH3), 3.56-3.51 (m, 1H, C6-H′), 3.30-3.22 (m, 1H, C6-
H′′), 3.27 (s, 3H, -OCH3), 3.11 (dd, J ) 13.5, 6.7 Hz, 1H,
-CH′HPh), 2.99 (dd, J ) 13.5, 6.7 Hz, 1H, -CHH′′Ph), 1.52-1.41
(m, 3H, LeuꢀH′H, LeuꢀHH′′and LeuγH), 1.40 (s, 9H, tBu of Boc),
0.81 (d, J ) 2.3 Hz, 3H, one LeuCH3), 0.80 (d, J ) 2.3 Hz, 3H,
another LeuCH3); 13C NMR (75 MHz, CDCl3): δ 172.7, 169.9,
168.4, 155, 136.3, 129.4, 128.5, 126.9, 81.8, 81.7, 81.6, 80.4, 60.9,
54.5, 53.7, 52.2, 50.8, 41.3, 37.9, 28.2, 24.7, 22.6, 21.8; MS (ESI):
m/z (%) 491 (10) [M - Boc + H]+, 591 (3) [M + H]+, 613 (100)
[M + Na]+; HRMS (ESI): calcd for C28H42N6O8Na [M + Na]+
613.2961, found 613.2963.
Synthesis of 1a. To a stirred solution of 18a (20 mg, 0.018
mmol) in dry dichloromethane (2 mL) at 0 °C was added
trifluoroacetic acid (1 mL) and the mixture was stirred for 12 h at
room temperature. The reaction mixture was then poured into dry
diethyl ether (30 mL). White precipitate came out of the solution.
Centrifugation and filtration gave white amorphous solid 1a in
quantitative yield. IR (neat): νmax 3285, 3062, 2957, 1646, 1533,
1
1196, 1130, 1051, 699 cm-1; H NMR (300 MHz, DMSO-d6): δ
Synthesis of 18a. To a stirring solution of 17a (113 mg, 0.19
mmol) in THF/MeOH/H2O (1.5:0.5:0.5 mL) at 0 °C was added
LiOH.H2O (20 mg, 0.47 mmol) and the mixture was stirred at room
temperature for 1 h. The reaction mixture was then acidified to pH
2 with 1 N HCl. The reaction mixture was extracted with EtOAc,
washed with water and brine, dried (Na2SO4), filtered and concen-
trated in vacuo to obtain the crude acid, which was used for the
next reaction without further purification.
To a solution of the acid in MeOH (2 mL) were added Pd on C
(10%, 30 mg) and 1 N HCl (0.23 mL), and the mixture was
hydrogenated under atmospheric pressure using a a H2 balloon for
1 h. The reaction mixture was then filtered through a short pad of
Celite and the filter cake was washed with MeOH. The filtrate and
the washings were combined and concentrated in vacuo to obtain
the hydrochloride salt, which was used in the next step without
further purification.
9.10 (bs, 1H), 8.68 (bs, 1H), 8.38 (bs, 3H), 7.46 (bs, 1H), 7.35-7.19
(m, 5H), 4.63-4.50 (m, 2H), 4.20-4.12 (m, 1H), 4.07-3.96 (m,
1H), 3.93-3.81 (m, 1H), 3.16-3.07 (m, 1H), 1.84-1.72 (m, 1H),
1.61-1.48 (m, 1H), 0.92-0.76 (m, 1H), 0.68 (d, J ) 5.3 Hz, 3H),
0.63 (d, J ) 5.3 Hz, 3H); 13C NMR (150 MHz, DMSO-d6): δ 172.3,
172.1, 171.0, 137.2, 129.4, 128.7, 126.8, 81.4, 80.6, 77.5, 60.6,
56.0, 54.0, 53.0, 42.0, 36.9, 35.5, 24.0, 21.1; MS (ESI): m/z (%)
433 (10) [H + M + H]2+, 866 (100) [M + H]+; HRMS (ESI):
calcd for C44H65N8O10 [M + H]+ 865.4823, found 865.4851.
Synthesis of 1b. Compound 1b was prepared in quantitative yield
from compound 18b following the same procedure as described
for the synthesis of 1a. IR (neat): νmax 3283, 2960, 1741, 1675,
1658, 1136 cm-1; 1H NMR (400 MHz, DMSO-d6): δ 8.95 (d, J )
8.1 Hz, 1H), 8.33 (s, 3H), 8.08 (bs, 1H), 7.95 (bs, 1H), 7.33-7.16
(m, 5H), 4.54 (m, 1H), 4.46-4.35 (m, 2H), 4.13-4.05 (m, 1H),
3.67-3.51 (m, 2H), 3.45-3.31 (m, 2H), 3.30 (s, 3H), 3.24-3.17
(m, 1H), 3.02-2.92 (m, 1H), 1.66-1.45 (m, 3H), 0.92 (d, J ) 5.7
Hz, 3H), 0.89 (d, J ) 5.7 Hz, 3H); 13C NMR (100 MHz, DMSO-
d6): δ 172.4, 170.4, 169.5, 138.1, 129.0, 127.0, 126.3, 82.5, 80.6,
79.5, 57.3, 53.9, 52.6, 51.0, 41.8, 41.0, 36.6, 24.3, 22.9, 21.9; MS
(ESI): m/z (%) 434 (60) [H + M + H]2+, 866 (100) [M + H]+;
HRMS (ESI): calcd for C44H65N8O10 [M + H]+ 865.4823, found
865.4809.
To a stirring solution of the hydrochloride salt in dry acetonitrile
(38 mL, 5 × 10-3 M) at 0 °C was added FDPP (110 mg, 0.29
mmol). After 15 min, DIPEA (0.1 mL, 0.57 mmol) was added to
the reaction mixture. After being stirred for 72 h at room
temperature, the solvent was evaporated under reduced pressure,
and the product was dissolved in EtOAc, washed with 1 N NaOH,
water, and brine, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by column chromatography (SiO2, 30-32% Acetone
in petroleum ether eluant) afforded compound 18a (64 mg, 63%).
Rf ) 0.5 (SiO2, 50% acetone in petroleum ether); IR (neat): νmax
Synthesis of 19a. To a stirring solution of Boc-(h-Gly)-OH (28
mg, 0.15 mmol) in dry dichloromethane (3 mL) at 0 °C was
sequentially added HOBt ·H2O (20 mg, 0.15 mmol) and EDCI (29
mg, 0.15 mmol). After 10 min, TFA-salt 1a (20 mg, 0.018 mmol)
was added to the reaction mixture followed by the addition of
DIPEA (0.015 mL, 0.09 mmol). After stirring for 24 h at room
temperature, the reaction mixture was diluted with dichloromethane,
washed with 1 N HCl solution, water, saturated NaHCO3 solution,
water, and brine, dried (Na2SO4), filtered and concentrated in vacuo.
Purification by silica gel column chromatography (SiO2, 4.7%
MeOH in dichloromethane eluant) afforded compound 19a (18 mg,
80%). Rf ) 0.5 (silica gel, 10% MeOH in dichloromethane); IR
1
3298, 2956, 2931, 1679, 1644, 1533, 1089, 750 cm-1; H NMR,
see Table 1; 13C NMR (75 MHz, CDCl3): δ 172.4, 172.2, 171.3,
155.4, 136.8, 129.2, 128.3, 127.0, 81.6, 81.3, 80.0, 79.2, 61.0, 58.0,
55.8, 54.1, 43.0, 36.7, 36.0, 28.3, 24.1, 23.5, 20.6; MS (ESI): m/z
(%) 1087 (100) [M + Na]+; HRMS (ESI): calcd for C54H80N8O14Na
[M + Na]+ 1087.5691, found 1087.5668.
Synthesis of 17b. Compound 4b (100 mg, 0.3 mmol) was
transformed into compound 17b (135 mg, 75%) following the same
procedure as described for the synthesis of 17a. Rf ) 0.4 (SiO2,
50% EtOAc in petroleum ether); [R]D30 ) +14.94 (c 1.8, CHCl3);
IR (neat): νmax 3314, 2958, 2103, 1742, 1659, 1084, 699 cm-1; 1H
MNR (300 MHz, CDCl3): δ 7.56 (d, J ) 7.9 Hz, 1H, PheNH),
7.31-7.19 (m, 5H, ArH), 6.26 (d, J ) 7.9 Hz, 1H, LeuNH), 5.0
(d, J ) 8.3 Hz, 1H, C4-NHBoc), 4.63-4.50 (m, 2H, PheRH,
LeuRH), 4.34 (s, 1H, C2-H), 3.98 (ddd, J ) 9.6, 8.3, 3.4,1H, C5-
H), 3.87-3.81 (m, 2H, C3-H, C4-H), 3.71 (dd, J ) 13.0, 9.6 Hz,
1H, C6-H′) 3.70 (s, 3H, -COOCH3), 3.53 (dd, J ) 13.0, 3.4 Hz,
1H, C6-H′′), 3.49 (s, 3H, -OCH3), 3.17 (dd, J ) 13.9, 6.7 Hz, 1H,
-CH′HPh), 3.06 (dd, J ) 13.9, 6.6 Hz, 1H, -CHH′′Ph), 1.60-1.45
(m, 3H, LeuꢀH′H, LeuꢀHH′′and LeuγH), 1.44 (s, 9H, tBu of Boc),
0.94 (d, J ) 6.0 Hz, 3H, one LeuCH3), 0.90 (d, J ) 6.0 Hz, 3H,
another LeuCH3); 13C NMR (75 MHz, CDCl3): δ 172.7, 169.8,
169.5, 154.9, 136.1, 129.2, 128.5, 126.9, 83.4, 81.7, 81.1, 79.9,
57.2, 54.4, 52.1, 51.6, 50.6, 41.3, 37.6, 28.1, 22.5, 21.8; MS (ESI):
1
(neat): νmax 3303, 2931, 1648, 1532, 1062, 752 cm-1; H NMR,
see Table 2; 13C NMR (75 MHz, CDCl3): δ 172.3, 171.9, 171.3,
156.0, 136.7, 129.2, 128.9, 126.9, 81.6, 79.6, 60.9, 58.0, 54.1, 43.1,
37.1, 36.6, 36.4, 36.1, 28.5, 24.1, 23.5, 20.7; MS (ESI): m/z (%)
1229 (100) [M + Na]+; HRMS (ESI): calcd for C60H90N10O16Na
[M + Na]+ 1229.6433, found 1229.6440.
Synthesis of 20a. To a stirring solution of Boc-Lys(Boc)-OH
(52 mg, 0.15 mmol) in dry dichloromethane (3 mL) at 0 °C was
sequentially added HOBt ·H2O (20 mg, 0.15 mmol) and EDCI (29
mg, 0.15 mmol). After 10 min, TFA-salt 1a was added to the
reaction mixture followed by the addition of DIPEA (0.015 mL,
0.09 mmol). After stirring for 24 h at room temperature, the reaction
mixture was diluted with dichloromethane, washed with 1 N HCl
solution, water, saturated NaHCO3 solution, water, and brine, dried
8742 J. Org. Chem. Vol. 73, No. 22, 2008

Cyclic Cationic Antimicrobial Peptides Containing Sugar Amino Acids
(Na2SO4), filtered and concentrated in vacuo. Purification by column
chromatography (SiO2, 5.0% MeOH in dichloromethane eluant)
afforded compound 20a (22 mg, 80%). Rf ) 0.3 (silica gel, 6%
MeOH in dichloromethane); IR (neat): νmax 3305, 2933, 1672, 1657,
1518, 1099, 751 cm-1; 1H NMR, see Table 3; 13C NMR (75 MHz,
CDCl3): δ 172.3, 172.2, 171.9, 171.4, 156.0, 155.2, 136.8, 129.2,
128.8, 126.9, 81.7, 81.6, 79.6, 79.4, 61.0, 58.0, 54.6, 54.0, 53.8,
43.2, 40.0, 36.6, 36.1, 32.6, 28.7, 28.5, 24.2, 23.7, 23.0, 20.8; MS
(ESI): m/z (%) 784 (100) [Na + M + Na]2+; HRMS (ESI): calcd
for C76H120N12O20Na2 [Na + M + Na]2+ 783.4263, found 783.4260.
Synthesis of 2a. Compound 2a was prepared in quantitative yield
from compound 19a following the same procedure as described
for the synthesis of 1a. IR (neat): νmax 3284, 2957, 1645, 1531.
Synthesis of 20b. Coupling of 1b (20 mg, 0.018 mmol) with
Boc-Lys(Boc)-OH (52 mg, 0.15 mmol) using EDCI (29 mg, 0.15
mmol), HOBt ·H2O (20 mg, 0.15 mmol), and DIPEA (0.015 mL,
0.09 mmol), following the same procedure as described for the
synthesis of 20a, gave compound 20b (22 mg, 80%). Rf ) 0.4 (silica
gel, 4% MeOH in dichloromethane); IR (neat): νmax 2986, 1688,
1
1648, 1523, 1470, 1003 cm-1; H NMR, see Table 6; 13C NMR
(75 MHz, CDCl3): δ 174.0, 171.4, 170.8, 167.8, 157.0, 155.2, 136.0,
129.8, 128.1, 126.7, 82.3, 81.8, 80.3, 79.8, 79.4, 57.7, 54.1, 52.6,
51.9, 49.8, 42.0, 40.1, 37.7, 36.9, 33.3, 30.0, 28.4, 28.3, 23.3, 22.7,
21.4; MS (ESI): m/z (%) 1544 (100) [M+ Na]+; HRMS (ESI):
calcd for C76H120N12O20Na2 [Na + M + Na]2+ 783.4263, found
783.4260.
1
1196, 1132, 1066 cm-1; H NMR (500 MHz, DMSO-d6): δ 8.98
Synthesis of 3b. Compound 3b was prepared in quantitative yield
from compound 20b following the same procedure as described
for the synthesis of 1a. IR (neat): νmax 3269, 3067, 2959, 1674,
(bs, 1H), 8.67-8.48 (m, 1H), 8.17 (d, J ) 8.4 Hz, 1H), 7.76 (bs,
3H), 7.46 (bs, 1H), 7.30-7.19 (m, 5H), 4.68-4.58 (m, 1H),
4.55-4.48 (m, 1H), 4.07-3.99 (m, 1H), 3.94-3.83 (m, 2H),
3.59-3.51 (m, 1H), 3.21 (s, 3H), 3.16-3.10 (m, 1H), 3.04-2.97
(m, 3H), 2.59-2.53 (m, 2H) 1.84-1.76 (m, 1H), 1.61-1.53 (m,
1H), 0.92-0.77 (m, 1H), 0.69 (d, J ) 5.6 Hz, 3H), 0.63 (d, J )
5.6 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 171.8, 171.3,
171.2, 169.8, 136.8, 128.9, 128.0, 126.2, 81.4, 80.5, 77.6, 73.6,
59.9, 55.4, 54.0, 52.5, 42.4, 36.5, 35.0, 32.2, 23.4, 20.6; MS (ESI):
m/z (%) 505 (100) [H + M + H]2+, 1008 (20) [M + H]+. HRMS
(ESI) C50H76N10O12 [H + M + H]+2 504.2816, found 504.2828.
Synthesis of 3a. Compound 3a was prepared in quantitative yield
from compound 20a following the same procedure as described
for the synthesis of 1a. IR (neat): νmax 3283, 2959, 1675, 1533,
1
1532, 1463, 1198 cm-1; H NMR (300 MHz, DMSO-d6): δ 8.56
(d, J ) 6.8 Hz, 1H), 8.46-8.29 (m, 2H), 8.21 (s, 3H), 7.99 (s,
3H), 7.81-7.70 (bs, 1H), 7.28-7.14 (m, 5H), 4.60-4.50 (m, 1H),
4.44-4.33 (m, 2H), 4.06-3.87 (m, 2H), 3.86-3.74 (m, 1H),
3.74.3.53 (m, 2H), 3.27 (s, 3H), 3.18-2.93 (m, 3H), 2.87-2.74
(m, 2H), 1.74-1.25 (m, 9H), 0.98-0.77 (m, 6H); 13C NMR (150
MHz, DMSO-d6): δ 172.1, 170.4, 169.3, 168.8, 137.3, 129.3, 128.0,
126.4, 82.4, 81.0, 79.6, 57.1, 54.9, 52.8, 51.8, 51.4, 40.6, 38.5, 37.1,
30.7, 26.7, 24.1, 22.5, 22.0, 21.0; MS (ESI): m/z (%) 562 (100) [H
+ M + H]2+; HRMS (ESI): calcd for C56H90N12O12 [H + M +
H]2+ 561.3395, found 561.3370.
Circular Dichroism Studies. CD spectra were recorded in 5
mM HEPES buffer (pH 7.4) and trifluoroethanol (TFE) on a JASCO
J-815 automatic recording spectropolarimeter at 25 °C using a quartz
cell of 1 mm path length. The spectra are shown in Supporting
Information. Data are represented as molar ellipticities. Sample
concentrations were 200 µM in buffer and TFE.
Antibacterial Activity. Bacterial strains used were Escherichia
coli (MG1655), Pseudomonas aeruginosa (NCTC 6750), Staphy-
loccoccus aureus (NCTC 8530), and Bacillus subtilis (CCMB
collection). The antibacterial activity of the compounds was
examined in sterile 96-well plates in a final volume of 100 µL as
follows:29 Bacteria were grown in nutrient broth (Bacto Difco
nutrient broth) to mid-log phase and diluted to 106 colony-forming
units (cfu)/mL in 10 mM sodium phosphate buffer (pH 7.4).
Bacteria were incubated with different concentrations of samples
for 2 h at 37 °C, and suitably diluted aliquots were plated on nutrient
agar plates. After the plates were incubated at 37 °C for 18 h,
colonies formed were counted. The concentration of the sample at
which no viable colonies were formed was taken as lethal
concentration (LC). The average of three independent experiments
done in duplicate was determined for LC.
Hemolytic Activity. The hemolytic activities of the compounds
were determined using rat red blood cells (RBCs). The RBCs were
prepared as follows: 2-3 mL of rat blood was drawn into 12 mL
of heparinsed 5 mM HEPES buffer pH 7.4 containing 150 mM
NaCl and centrifuged at 4000 rpm for 5 min. The cell pellet was
washed three times with the buffer, and the buffy coat was removed.
A dilute stock of RBCs was made by diluting the pellet 0.5 to 15
mL with the buffer. Different volumes of each compound were
taken in 0.5 mL of 5 mM HEPES buffer pH 7.4 containing 150
mM NaCl. Diluted stock of RBCs containing 1 × 107 cells/mL
were added, and the tubes were incubated at 37 °C for 30 min in
a gentle shaking water bath. After incubation, the samples were
centrifuged at 4000 rpm for 5 min to remove unhemolysed cells.
The absorbance of the supernatant was measured at 540 nm. Buffer
containing RBCs suspension was taken as buffer blank (A0). The
lysis obtained with 0.1% of Triton X-100 was taken for 100% lysis
(A100). The percentage hemolysis was calculated using the following
equation: %H ) (Asample - A0) × 100/(A100 - A0), where A100 and
1
1198, 1135 cm-1; H NMR (500 MHz, DMSO-d6): δ 9.06 (bd, J
) 5.6 Hz, 1H), 8.69 (bs, 1H), 8.42 (d, J ) 8.4 Hz, 1H), 8.21 (bs,
3H), 7.77 (bs, 3H), 7.56-7.51 (m, 1H), 7.31-7.20 (m, 5H), 4.60(m,
1H), 4.57-4.51 (m, 1H), 4.14-4.07 (m, 1H), 3.96-3.88 (m, 2H),
3.86-3.80 (m, 1H), 3.51-3.44 (m, 1H), 3.42-3.35 (m, 1H), 3.30
(s, 3H) 3.17-3.11 (m, 1H), 2.86-2.78 (m, 2H), 2.50-2.42 (m,
2H) 1.89-1.81 (m, 1H), 1.76-1.70 (m, 2H), 1.59-1.51 (m, 3H),
1.44-1.36 (m, 2H), 0.83-0.76 (m, 1H), 0.68 (d, J ) 6.3 Hz, 3H),
0.61 (d, J ) 6.3 Hz, 3H); 13C NMR (100 MHz, DMSO-d6): δ 172.0,
171.9, 171.8, 168.9, 136.7, 128.9, 128.1, 126.3, 81.5, 80.9, 78.3,
60.1, 55.7, 53.7, 52.7, 51.6, 42.6, 38.4, 36.2, 30.8, 26.7, 23.5, 23.4,
21.1, 20.5; MS (ESI): m/z (%) 562 (100) [H + M + H]2+, 1122
(5) [M + H]+; HRMS (ESI): calcd for C56H90N12O12 [H + M +
H]2+ 561.3395, found 561.3388.
Synthesis of 19b. Coupling of 1b (20 mg, 0.018 mmol) with
Boc-(h-Gly)-OH (28 mg, 0.15 mmol) using EDCI (29 mg, 0.15
mmol), HOBt.H2O (20 mg, 0.15 mmol), and DIPEA (0.015 mL,
0.09 mmol), following the same procedure as described for the
synthesis of 19a, gave compound 19b (20 mg, 80%). Rf ) 0.4 (silica
gel, 6% MeOH in dichloromethane); IR (neat): νmax 3303, 2925,
2856, 1741, 1651, 1519, 1461, 1088 cm-1; 1H NMR, see Table 5;
13C NMR (75 MHz, CDCl3): δ 172.9, 171.9, 170.9, 169.7, 156.0,
136.9, 129.3, 128.7, 126.9, 83.0, 81.7, 81.3, 79.4, 57.2, 54.6, 52.4,
50.6, 40.8, 39.1, 37.9, 36.9, 36.4, 28.4, 24.9, 22.6, 22.5; (ESI): m/z
(%) 1229 (85) [M + Na]+; HRMS (ESI): calcd for C60H90N10O16Na
[M + Na]+ 1229.6433, found 1229.6445.
Synthesis of 2b. Compound 2b was prepared in quantitative yield
from compound 19b following the same procedure as described
for the synthesis of 1a. IR (neat): νmax 3270, 2959, 1680, 1650,
1
1533, 1132 cm-1; H NMR (300 MHz, DMSO-d6): δ 8.58 (bs,
1H), 8.61 (d, J ) 7.6 Hz, 1H), 8.06 (bs, 1H), 7.90 (bs, 1H), 7.75
(s, 3H), 7.28-7.16 (m, 5H), 4.59-4.47 (m, 1H), 4.46-4.35 (m,
1H), 4.30 (s, 1H), 4.04-3.95 (m, 1H), 3.93-3.85 (m, 1H),
3.60-3.35 (m, 4H), 3.22 (s, 3H), 3.21-3.09 (m, 2H), 3.03-2.90
(m, 3H), 1.65-1.50 (m, 2H), 1.47-1.35 (m, 1H), 0.86 (d, J ) 5.3
Hz, 6H); 13C NMR (150 MHz, DMSO-d6): δ 172.2, 170.4, 169.8
(two carbons), 137.9, 129.2, 128.0, 126.4, 82.4, 80.9, 79.8, 79.3,
57.0, 53.3, 52.1, 51.1, 40.9, 36.8, 35.2, 32.0, 24.1, 22.5, 22.2; MS
(ESI): m/z (%) 505 (100) [H + M + H]2+, 1008 (10) [M + H]+;
HRMS (ESI): calcd for C50H76N10O12 [H + M + H]2+ 504.2816,
found 504.2799.
(29) Krishnakumari, V.; Singh, S.; Nagaraj, R. Peptides 2006, 27, 2607–
2613.
J. Org. Chem. Vol. 73, No. 22, 2008 8743

Chakraborty et al.
A0 are the absorbance of 100% and 0% hemolysed cells, respec-
tively. The average of three independent experiments done in
duplicate was taken for calculations.
Supporting Information Available: General experimental
procedures, detailed protocols for NMR and MD studies,
NOESY/ROESY, TOCSY spectra of 18a,b-20a,b and
1a,b-3a,b and copies of 1H and 13C NMR spectra for all
compounds, and CD spectra of 1a,b-3a,b. This material is
available free of charge via the Internet at http://pubs.acs.org.
Acknowledgment. The authors wish to thank DST, New
Delhi for financial support (SR/S1/OC-01/2007), CSIR, New
Delhi (D. K.) and UGC (R. R.) for research fellowships.
JO801123Q
8744 J. Org. Chem. Vol. 73, No. 22, 2008