Synthesis and characterization of peptide-cationic steroid antibiotic conjugates

Article abstract of DOI:10.1021/ol048845t

(Chemical Equation Presented) New cationic steroid antibiotics have been prepared by conjugating tripeptides to a triamino analogue of cholic acid. These compounds were synthesized on a solid phase in an indexed library that was screened for antibacterial

Full text of DOI:10.1021/ol048845t

ORGANIC
LETTERS
2004
Vol. 6, No. 20
3433-3436
Synthesis and Characterization of
Peptide Cationic Steroid Antibiotic
−
Conjugates
Bangwei Ding, Uale Taotofa, Thomas Orsak, Matthew Chadwell, and
Paul B. Savage*
Department of Chemistry and Biochemistry, Brigham Young UniVersity,
ProVo, Utah 84602
paul_saVage@byu.edu
Received June 17, 2004
ABSTRACT
New cationic steroid antibiotics have been prepared by conjugating tripeptides to a triamino analogue of cholic acid. These compounds were
synthesized on a solid phase in an indexed library that was screened for antibacterial activity against Gram-negative and Gram-positive
bacteria. Selected compounds were synthesized on a larger scale, and minimum inhibition concentrations were measured. These results
correlated very well with the screening of the indexed library of antibiotics. The most active antibiotics demonstrate a marked improvement
over a related and well characterized cationic steroid antibiotic.
Cationic steroid antibiotics (CSAs) were developed to mimic
the antibacterial behavior of endogenous peptide antibiotics.1
This behavior includes selective association of the antibiotics
with and disruption of bacterial membranes.2,3 This activity
results in rapid bactericidal activity with a minimal potential
for causing the emergence of resistance. These antibiotics,
in general, adopt cationic, facially amphiphilic4 conforma-
tions (e.g., see 1 in Figure 1), which appears to be the key
requirement for antibacterial activity, and membrane selec-
tivity is primarily derived from ionic recognition of nega-
tively charged bacterial membranes. However, bacterial
membrane components present more that anionic groups, and
it is anticipated that additional, associative noncovalent
interactions would increase affinity for membrane-active
antibiotics for bacterial membranes and increase antibacterial
activity. Consequently, we have explored means of increasing
the functionality presented by CSAs. Due to the relatively
rigid and preorganized forms of CSAs, it is clear that
(1) For reviews see: (a) Savage, P. B.; Li, C.; Taotafa, U.; Ding, B.;
Guan, Q. FEMS Lett. 2002, 217, 1-7. (b) Savage, P. B. Eur. J. Org. Chem.
2002, 759-768.
(2) Ding, B.; Guan, Q.; Walsh, J. P.; Boswell, J. S.; Winter, T. W.;
Winter, E. S.; Boyd, S. S.; Li, C.; Savage, P. B. J. Med. Chem. 2002, 45,
663-669.
(3) Li, C.; Lewis, M. R.; Gilbert, A. B.; Noel, M. D.; Scoville, D. H.;
Allman, G. W.; Savage, P. B. Antimicrob. Agents Chemother. 1999, 43,
1347-1351.
(4) (a) Cheng, Y.; Ho, D. M.; Gottlieb, C. R.; Kahne, D. J. Am. Chem.
Soc. 1992, 114, 7319-7320. (b) Barrett, D. G.; Gellman, S. H. J. Am. Chem.
Soc. 1993, 115, 9343-9344.
Figure 1. Structure of CSA 1 and a schematic representation of a
CSA-peptide conjugate (2).
10.1021/ol048845t CCC: $27.50
© 2004 American Chemical Society
Published on Web 09/09/2004

attachment of functionality designed to interact with bacterial
membrane components should be on the polar face of the
molecules. Derivatization of cholic acid, and analogues, with
diverse functional groups has been very effective in the
development of receptors for a variety of small molecules.5
We have developed an effective means of preparing
combinatorial libraries of CSAs derived from a steroid
scaffolding and amino acids.6 We now report extension of
this methodology to generation of libraries of peptide-CSA
conjugates. Libraries of peptide-steroid conjugates have been
reported with enzyme-like activity and as receptors for other
peptides.7 These libraries have incorporated only two peptide
chains on the steroid nucleus and were not screened for
antibacterial activity. Using a triamino analogue reported
earlier8 and in a parallel synthesis format, we have prepared
an indexed library of tripeptide-containing CSAs, using six
different amino acids, yielding 216 different compounds (2
in Figure 1). The six amino acids used, histidine, lysine,
methionine, phenylalanine, tryptophan, and valine, were
selected for an initial library because this group contains basic
amino acids that will contribute to the facial amphiphilicity
of the CSAs and hydrophobic and aromatic amino acids that
may interact with the nonpolar components of bacterial
membranes. Following preparation of the library, we screened
the compounds for activity against Gram-negative and Gram-
positive bacteria. Tripeptides were used in these compounds
because modeling of peptide-appended cholic acid derivatives
suggested that peptide chains of up to three amino acids in
length were sufficiently short to maintain the facially
amphiphilic morphology necessary for antibacterial activity.
Following the screening, we prepared and purified relatively
large amounts of selected compounds characterized in the
screening process, and we have verified that the screening
process yields meaningful information about antibacterial
activity. From this effort we have found sequences of
tripeptides that yield CSA-peptide conjugates with good
antibacterial properties.
This procedure was repeated two additional times adding
AAâ and AAγ, giving tripeptide-containing CSAs on ap-
proximately 5 mg of beads in each group. The remaining
Boc groups were removed, followed by cleavage of the Fmoc
groups on lysine. The beads were then treated with a 0.1 M
solution of sodium methoxide in methanol to release the
CSAs from the beads. The beads were removed; the solutions
were neutralized with dilute hydrochloric acid in methanol,
and the solvent was removed. The resulting materials were
dissolved in 1 mL of water and serially diluted for use in
antibacterial screening assays. In larger scale synthesis of
compounds represented by 2, we found that we could isolate
approximately 200 mg of material from 1 g of beads.
Consequently, the concentrations of the libraries of CSAs
were expected to be 1 mg/mL before the dilutions described
below.
The indexed libraries were screened for antibacterial
activity against Gram-negative (Escherichia coli (ATCC
25922)) and Gram-positive bacteria (Staphylococcus aureus
(ATCC 25923) using a micro-broth dilution method.9 The
solutions of CSAs were introduced to bacterial suspensions
at fractions of the original concentrations (1/4, 1/16, 1/64,
1/256), and the lowest concentration of each CSA that
inhibited bacterial growth (24 h incubation) was used to
classify the antibiotic as weakly, moderately, or strongly
active. A number of the antibiotics were active against S.
aureus at a dilution of 1/64 (Figure 2); CSAs with AAR of
Generation of the tripeptide-containing library began with
3 immobilized on carboxylic acid-modified polystyrene beads
(Scheme 1). The Boc groups were removed, and the beads
were partitioned into six groups. Each group was reacted
with a different activated Boc-protected amino acid (δ-Fmoc
lysine) to give amino acids directly attached to the amine
groups on the steroid (AAR).
Scheme 1. Preparation of Indexed Library 2
Figure 2. Dilutions at which indexed CSA libraries inhibited the
growth of S. aureus.
F, M, or V did not show activity at or below dilution of
1/64 and are not described in Figure 2. The most active
compounds inhibited growth at a dilution of 1/256. These
antibiotics, containing the tripeptides FFK, MMK, MWK,
KHK, and MHK, as well as a majority of the CSAs active
at a dilution of 1/64, contain lysine attached directly to the
steroid. The requirement for lysine at the AAR position may
3434
Org. Lett., Vol. 6, No. 20, 2004

be due to selection of compounds that are strongly facially
amphiphilic, whereas compounds with amine groups found
only at the N-termini of the peptide chains may not retain
the necessary amphiphilic morphology.
on a larger scale for comparison of minimum inhibition
concentrations (MICs). As the least active compound, we
prepared the CSA containing VKF; as a compound with
intermediate activity, we prepared the CSA containing VKW;
and as the most active compound, we prepared the CSA
containing FFK. In the screening experiments, these three
compounds inhibited growth of S. aureus at dilutions of 1/16,
1/64, and 1/256, respectively.
The antibiotics that were active against E. coli included
compounds that were active against S. aureus, but there were
some differences upon screening with Gram-positive vs
Gram-negative bacteria. As with S. aureus, compounds with
AAR of F, M, or V were not active at dilutions below 1/16,
and only compounds active at dilutions of 1/64 and 1/256
are described in Figure 3. The antibiotics active at a dilution
Efficient syntheses of the three targeted CSAs in the
relatively large scale necessary for characterization and MIC
measurements required modification of the synthetic proce-
dures used in preparing the indexed libraries. To maximize
the convergent nature of the synthesis, tripeptides were
prepared and purified before attachment to the steroid
scaffolding. The tripeptides were assembled using standard
peptide coupling procedures (Boc-protected amino acids,
diisopropylcarbodiimide, N-hydroxysuccinimide) beginning
with the methyl ester of the C-terminal amino acid. For
lysine, a δ-Cbz protecting group was used, and after
generation of the tripeptide, this group was replaced by a
Boc protecting group to allow deprotection of all amine
groups in one step. The methyl ester was hydrolyzed with
lithium hydroxide in methanol, and no epimerization was
1
observed in the corresponding H NMR spectrum of each
tripeptide. The resulting acid was coupled to the required
triamino analogue of cholic acid using diisopropylcarbodi-
imide and N-hydroxysuccinimide. Use of 4-(dimethylamino)-
pyridine as a catalyst in this step resulted in significant
amounts of epimerization as observed by 13C NMR, and
while the reaction was relatively slow without this catalyst,
epimerization was not observed via NMR. Removal of the
six Boc groups on each compound generated compounds
4-6 (Figure 4).
Figure 3. Dilutions at which indexed CSA libraries inhibited the
growth of E. coli.
of 1/256 were FFK, MMK, and WKK. In this case an even
stronger preference for lysine at the AAR position was
observed, a feature of active antibiotics possibly necessary
for effective interaction with the outer membranes of Gram-
negative bacteria.
To verify that the solid-phase synthesis and screening
procedures allowed identification of novel active antibiotics,
we selected three compounds with varied activities to prepare
Figure 4. Structures of tripeptide-containing CSAs prepared for
comparison of MIC values.
(5) For recent examples, see: (a) Boon, J. M.; Lambert, T. N.; Sisson,
A. L.; Davis, A. P.; Smith, B. D. J. Am. Chem. Soc. 2003, 125, 8195-
8201. (b) Koulov, A. V.; Lambert, T. N.; Shukla, R.; Jain, M.; Boon, J.
M.; Smith, B. D.; Li, H.; Sheppard, D. N.; Joos, J.-B.; Clare, J. P.; Davis,
A. P. Angew. Chem., Int. Ed. 2003, 32, 4931-4933. (c) Siracusa, L.; Hurley,
F. M.; Dresen, S.; Lawless, L. J.; Pe´rez-Paya´n, M. N.; Davis, A. P. Org.
Lett. 2002, 4, 4639-4642.
MIC values for compounds 4-6 with S. aureus were
measured using macro-broth dilution methods.9 These values
are >100, 40, and 8 µg/mL, respectively. As we had
anticipated, the trend of increasing antibacterial activity (4
to 5 to 6) is consistent with the screening results (Table 1).
In the screening assay, 4 and 5 inhibited growth of E. coli
at dilutions of 1/16, while 6 inhibited growth at a dilution
of 1/256. The respective MIC values of these compounds
with E. coli are >100, >100, and 8 µg/mL, again consistent
with the outcome of the screening experiments. Taken
together, these results suggest that solid-phase synthesis and
(6) Zhou, X.-T.; Rehman, A.; Li, C.; Savage, P. B. Org. Lett. 2000, 2,
3015-3018.
(7) For examples see: (a) Madder, A.; Li, L.; De Muynck, H.; Farcy,
N.; Van Haver, D.; Fant, F.; Vanhoenacker, G.; Sandra, P.; Davis, A. P.;
De Clercq, P. J. J. Comb. Chem. 2002, 4, 552-562. (b) Cheng, Y.; Seunaga,
T.; Still, W. C. J. Am. Chem. Soc. 1996, 118, 1813-1814.
(8) Li, C.; Rehman, A.; Dalley, N. K.; Savage, P. B. Tetrahedron Lett.
1999, 40, 1861-1864.
(9) National Committee for Clinical Laboratory Standards. Methods for
Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobi-
cally; ApproVed Standard M7-A4, 4th ed.; NCCLS: Villanova, PA, 1997.
Org. Lett., Vol. 6, No. 20, 2004
3435

compounds, use of 20 common amino acids will yield 8000.
Using micro-broth dilution screening, it will be possible to
screen this size of library in a reasonable amount of time. In
these libraries, the peptides at C3, C7, and C12 are identical,
and because we have developed means of sequentially adding
different amino acids at each of these positions on the steroid
scaffolding, there is enormous potential for generation of
chemical diversity (use of 20 amino acids would yield over
500 billion combinations).
Table 1. Comparison of the Dilutions at Which Compounds
4-6 from Solid-Phase Synthesis Inhibited Bacterial Growth and
the MIC Values of These Compounds Measured Independently
compounds
4
5
6
S. aureus
active dilutiona
MIC (µg/mL)
1/16
>100
1/64
40
1/256
8
We have demonstrated that peptide-containing CSAs can
be effectively prepared in indexed libraries and rapidly
screened for antibacterial activities. MIC values measured
with CSAs characterized in the screening process correlate
very well with screening results. From this work, we have
identified key requirements for peptide-containing CSAs and
compounds that appear to offer an improvement in antibacte-
rial activity over simpler CSAs. While a limited number of
amino acids were used in preparing the libraries, wide
variation in antibacterial activities was observed. It is
anticipated that expansion of the libraries and screening will
result in discovery of compounds with improved antibacterial
activities against both Gram-negative and Gram-positive
bacteria.
E. coli
active dilutiona
1/16
>100
1/16
>100
1/256
8
MIC (µg/mL)
a Dilution of the initial solutions from the library (see text) that inhibit
bacterial growth.
micro-broth dilution screening for antibacterial activity yield
meaningful information about the types of amino acids that
can be incorporated into active CSAs.
We have demonstrated that the nature of the group at the
C24 position of CSAs (see Figure 1 for steroid numbering)
strongly influences their antibacterial activities.10 For ex-
ample, 1 has an MIC of 36 µg/mL against E. coli, and if the
hydroxyl group at C24 is replaced by an octylamine group,
the MIC of the resulting CSA is 3 µg/mL. As compared to
1, CSA 6 has a relatively low MIC value. It is anticipated
that incorporation of an octylamine or polyamine chain at
C24 will similarly result in a dramatic decrease in MIC
values. Before pursuing this line of research, however, it
would be advantageous to expand the screening of peptide-
containing CSAs. While the libraries reported contain 216
Acknowledgment. Generous support from the National
Institutes of Health (NIGMS) is gratefully acknowledged.
Supporting Information Available: Detailed description
of the micro-broth antibacterial screening method and
experimental procedures for the preparation of 4-6. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(10) Li, C.; Budge, L. P.; Driscoll, C. D.; Willardson, B. M.; Allman,
G. W.; Savage, P. B. J. Am. Chem. Soc. 1999, 121, 931-940.
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