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.
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