Gorovoy et al.
Staphylococcus aureus (ATCC 25923), Escherichia coli
(ATCC 25922), Streptococcus pyogenes (ATCC 19615),
and Pseudomonas aeruginosa (ATCC 27853) were
included. The liquid media used for growth were Luria-
Bertani (Becton Dickinson, Sparks, MD, USA) and Mueller-
Hinton (bio-Merieux, Paris, France) broths and Middle-
brook 7H9 medium (Difco) for mycobacteria.
(14) into the molecule and second, by making use of
boronic acids as isosters for the carboxylic acid
(Figure 1) (15).
In parallel to earlier observations on (naturally occurring)
amino acids, the first dipetides synthesized did not exhibit
any, or only minor, effect against S. aureus, E. coli, S. py-
ogenes, or P. aeruginosa while being active against
The strains were grown at 37 ꢀC, and after suitable cell
concentrations had been reached, 100 lL of each cell
suspension was added to a tube with growth media and
test compound. Cultivation of the bacteria was then car-
ried out in the presence of each test compound at 37 ꢀC,
and the tubes examined for visible growth. The com-
pounds were tested at concentrations of 500, 50, or
5 mg ⁄ L. This assay was repeated twice.
M. tuberculosis with MIC values ranging from
500 lg ⁄ mL (Table 1).
5 to
It was therefore decided to explore this new class of
compounds and investigate the influence on tuberculosis
activity by extended synthesis. Parameters to screen
included what amino acid was incorporated, the effect of
the substituent a to the boron, and whether there was a
difference in activity between the boronic acids and
esters. In addition, the effect of the stereochemistry on
either side of the amide bond was also included as a
parameter.
Mycobacterium tuberculosis (strain H37Rv) was cultivated
in Middlebrook 7H9 medium (Difco) or on Middlebrook
7H10 agar plates at 37 ꢀC. For testing, M. tuberculosis
was cultivated in 4 mL of broth until the culture reached a
concentration of approximately 1 · 108 cfu ⁄ mL and then
diluted 10 times in PBS to yield a suspension with minimal
viscosity and a concentration of bacteria of approximately
1 · 107 cfu ⁄ mL, and 100 lL of this suspension was
added to a tube with media with tested agent in concen-
trations 500.0, 50.0, or 5.0 mg ⁄ L. After cultivation at
37 ꢀC from the tube where growth was determined, the
aliquots were plated on Middlebrook 7H12 agar to deter-
mine the presence of bacterial growth and identification of
bactericidal or bacteriostatic effect of tested peptide.
Matteson homologation is an efficient procedure for the
synthesis of enantiomerically pure a-chloroalkylboronates,
and stereochemistry is controlled by the use of a diol as
chiral director (16). Subsequent nucleophilic substitution of
the chlorine atom by nucleophiles proceeds with stereo-
control (17), and there are a number of publications
devoted to applications of chiral a-chloroalkylboronates in
asymmetric synthesis. Combining Matteson homologation
and nucleophilic substitution thus appeared interesting as
a method for the synthesis of substituted derivatives of b-
amino boronic acids (Scheme 1). In our studies, both (+)-
and ())-pinanediol were used as chiral directors and were
obtained from the corresponding pinene according to a
well-known procedure (18).
In a further experiment, a sample of cells from the test
tubes, in which the above broth activity was assayed,
were plated on agar to determine the presence of bacterial
growth. The CFUs were counted.
The starting material, 1,3-propandiol-bromomethyl boro-
nate, was obtained by a procedure analogous to a pub-
Results and Discussions
lished procedure (19) with
a
yield of 57% and
reesterification by pinanediol gave 1 in 93% yield. (This
approach is one step longer than direct esterification with
pinanediol, but loss of precious pinanediol is avoided.) In
the next step, bromine was substituted by azide, and the
Our interest in antimicrobial peptides stems from the dis-
coveries regarding lactoferrin ⁄ lactoferricin and their role
as anti-infective agents in breast-feeding children (8). Fur-
ther studies led to the identification of the active region
of the peptides, which was further developed into results
about minimum requirements for peptide structure to
preserve antibiotic activity (9). Synthetic peptides were
then investigated to improve activity as well as a means
to improve uptake and to avoid side effects, for example
enzymatic degradation (10,11). From these results, it was
clear that the smallest peptidomimetics still exhibiting
desired effect were tripeptides with certain features, that
is, two bulky side-groups and two charged side chains
(12).
Figure 1: General scaffold for the target compounds library,
where R1= H, methyl, phenethyl, benzyl, 4-(F)-benzyl, 4-(CF3O)-
benzyl, 2-naphthylmethyl, phenyl. R= remaining portion of (l)- or
(D)-lysine, (L)-phenylalanine, (L)-alanine, (L)-arginine.
It was therefore decided to modify the C-terminal end of
the peptides: first, by employing b-amino acids as this
introduces more flexibility (13) and enzymatic stability
410
Chem Biol Drug Des 2013; 81: 408–413
Gorovoy, Alexey S.
