Characterization of d-boroAla as a novel broad-spectrum antibacterial agent targeting d-Ala-d-Ala ligase

Article abstract of DOI:10.1111/j.1747-0285.2011.01210.x

d-boroAla was previously characterized as an inhibitor of bacterial alanine racemase and d-Ala-d-Ala ligase enzymes (Biochemistry, 28, 1989, 3541). In this study, d-boroAla was identified and characterized as an antibacterial agent. d-boroAla has activity against both Gram-positive and Gram-negative organisms, with minimal inhibitory concentrations down to 8μg/mL. A structure-function study on the alkyl side chain (NH₂-CHR-B(OR')₂) revealed that d-boroAla is the most effective agent in a series including boroGly, d-boroHomoAla, and d-boroVal. l-boroAla was much less active, and N-acetylation completely abolished activity. An LC-MS/MS assay was used to demonstrate that d-boroAla exerts its antibacterial activity by inhibition of d-Ala-d-Ala ligase. d-boroAla is bactericidal at 1× minimal inhibitory concentration against Staphylococcus aureus and Bacillus subtilis, which each encode one copy of d-Ala-d-Ala ligase, and at 4× minimal inhibitory concentration against Escherichia coli and Salmonella enterica serovar Typhimurium, which each encode two copies of d-Ala-d-Ala ligase. d-boroAla demonstrated a frequency of resistance of 8×10^-8 at 4× minimal inhibitory concentration in S. aureus. These results demonstrate that d-boroAla has promising antibacterial activity and could serve as the lead agent in a new class of d-Ala-d-Ala ligase targeted antibacterial agents. This study also demonstrates d-boroAla as a possible probe for d-Ala-d-Ala ligase function.

Full text of DOI:10.1111/j.1747-0285.2011.01210.x

ª 2011 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2011.01210.x
Chem Biol Drug Des 2011; 78: 757–763
Research Article
Characterization of D-boroAla as a Novel
Broad-Spectrum Antibacterial Agent Targeting
D-Ala-D-Ala Ligase
Sandeep Putty1, Aman Rai1, Darshan
This study also demonstrates D-boroAla as a possi-
ble probe for D-Ala-D-Ala ligase function.
Jamindar1, Paul Pagano2,†, Cheryl L.
Quinn2,‡, Takehiko Mima3,§, Herbert P.
Schweizer3 and William G. Gutheil1,*
Key words: alanine branch, antibacterial, broad spectrum, cell wall,
D-Ala-D-Ala ligase
1Division of Pharmaceutical Sciences, University of Missouri, Kansas
Received 15 April 2011, revised 15 July 2011 and accepted for publica-
City, 2464 Charlotte Street, Kansas City, MO 64108, USA
tion 2 August 2011
2PharmOptima, 6710 Quality Way, Portage, MI 49002, USA
3Department of Microbiology, Immunology and Pathology, Colorado
State University, IDRC at Foothills Campus, Campus Delivery 0922,
Fort Collins, CO 80523, USA
Bacterial infections were the major cause of death and morbidity
prior to the development of modern antibiotics, and the increasing
resistance of pathogenic bacteria to commonly used antibacterial
agents is of major public health concern. Methicillin-resistant
Staphylococcus aureus (MRSA) and several Gram-negative patho-
gens such as Pseudomonas aeruginosa, Burkholderia sp., Acineto-
bacter baumannii, and Klebsiella pneumoniae are of particular
concern (1–7). Further heightening concern about existing and
emerging bacterial drug resistance is the fact that, although a num-
ber of new antibacterial agents from known antibacterial classes
are under development, only two new class of antibacterial agents
have been introduced into clinical practice in the last 40 years –
the oxazolidinones as represented by linezolid (8) and the lipopep-
tides as represented by daptomycin (9,10). There is therefore an
urgent need to identify new classes of antibacterial agents, espe-
cially agents that act through novel mechanisms and for which
mechanisms of resistance are not yet known (6).
*Corresponding author: William G. Gutheil, gutheilw@umkc.edu.
Present address: Micromyx, LLC, 4717 Campus Drive, Kalamazoo,
MI 49008, USA.
àPresent address: QnA Pharma Consulting LLC, 11227 Cedar Pointe,
Minnetonka, MN 55305, USA.
§Present address: Department of Microbiology, Kitasato University
Medical School, 1-15-1 Kitasato, Minami-ku, Sagamihara,
Kanagawa 252-0374, Japan.
D-boroAla was previously characterized as an inhib-
itor of bacterial alanine racemase and D-Ala-D-Ala
ligase enzymes (Biochemistry, 28, 1989, 3541). In
this study, D-boroAla was identified and character-
ized as an antibacterial agent. D-boroAla has activ-
ity against both Gram-positive and Gram-negative
organisms, with minimal inhibitory concentrations
down to 8 lg / mL. A structure–function study on
the alkyl side chain (NH2-CHR-B(OR’)2) revealed that
D-boroAla is the most effective agent in a series
including boroGly, D-boroHomoAla, and D-boroVal.
L-boroAla was much less active, and N-acetylation
completely abolished activity. An LC-MS / MS assay
was used to demonstrate that D-boroAla exerts its
antibacterial activity by inhibition of D-Ala-D-Ala
ligase. D-boroAla is bactericidal at 1· minimal
inhibitory concentration against Staphylococcus
aureus and Bacillus subtilis, which each encode
one copy of D-Ala-D-Ala ligase, and at 4· minimal
inhibitory concentration against Escherichia coli
and Salmonella enterica serovar Typhimurium,
which each encode two copies of D-Ala-D-Ala
During our efforts to develop transition-state analog inhibitors for
bacterial cell wall–synthesizing enzymes (11,12), we observed that
D-boroAlanine (D-Ala with the -COOH group replaced with a -B(OH)2
group) had effective antibacterial activity. While D-boroAla has pre-
viously been described as an inhibitor of alanine racemase and
D-Ala-D-Ala ligase (DDL) (13), it has not previously – to our knowl-
edge – been reported as an antibacterial agent. In this report, we
describe the antibacterial properties of D-boroAla, structure–activity
correlation among several D-boroAla homologs, and determination
of the biochemical mechanism for D-boroAla's antibacterial activity.
This study demonstrates that D-boroAla has broad-spectrum antibac-
terial activity and targets DDL in the alanine branch of bacterial cell
wall biosynthesis (Figure 1).
ligase. D-boroAla demonstrated
a frequency of
resistance of 8 · 10)8 at 4· minimal inhibitory con-
centration in S. aureus. These results demonstrate
that D-boroAla has promising antibacterial activity
and could serve as the lead agent in a new class of
D-Ala-D-Ala ligase targeted antibacterial agents.
This study suggests that suitably designed derivatives and analogs
of D-boroAla could provide unique new antibacterial agents for use
in countering drug-resistant pathogenic bacteria.
757

Putty et al.
UDP-GlcNAc
O
O
OiPr
OiPr
i
ii
(-)-Pd
R
B
B(OiPr)3+R-Li
R
B
MurA
PEP
UDP-GlcNAc-enolpyruvate
NADPH
2
1
iii
MurB
MurC
MurD
Li+
R
R
O
O
O
iv
UDP-MurNAc
(-)-Pd
B
*
(-)-Pd
Cl
B
Cl2HC
4
O
L
-Ala
3
v
UDP-MurNAc-
-Glu
-Ala-γ-
-Lys
-Ala-γ- -Glu-
L-Ala
L
-Ala
D
R
R
O
vi
Alanine
racemase
O
O
UDP-MurNAc-
L
D-Glu
*
(-)-Pd
[(H3C)3Si]2N
B
(-)-Pd
(-)-Pd
*
H2N
B
O
5
MurE
MurF
L
D
-Ala
-Ala-
ligase
-Ala
6
D
D
-Ala
UDP-MurNAc-
L
D
L-Lys
CH3
CH3
O
O
O
O
D
-Ala-
-Ala
undecaprenyl (C55)-P
-Ala-γ- -Glu- -Lys- -Ala-
UDP-GlcNAc
-Ala-γ- -Glu-
5× Gly-t-RNA
-Glu- -Lys(Gly)5-
- C55-PP
Nascent peptidoglycan
D
vii
(-)-Pd
*
*
H2N
B
H3C
N
H
B
O
UDP-MurNAc-
MurY
L
-Ala-γ-D-Glu-L-Lys-D-Ala-D
7
6a
CH3
CH3
OH
OH
O
O
viii
Lipid I = C55-PP-MurNAc-
MurG
L
D
L
D
D-Ala
*
(-)-Pd
*
H2N
B
H2N
B
8
6a
Lipid II = C55-PP-(GlcNAc)-MurNAc-
FemABX
L
D
L-Lys-D-Ala-D-Ala
HO
HO
a; R = -CH3
b; R = -CH2CH3
c; R = -CH(CH3)2
(-)-pinanediol =
C
55-PP-(GlcNAc)-MurNAc-
Transglycosylases
L
-Ala-γ-
D
L
D-Ala-D-Ala
Scheme 1: Reagents and conditions: (i) a-ether, )72 ꢀC, 16 h.
b-1 M HCl in ether, 0 ꢀC. (ii) ())-(Pd). (iii) CH2Cl2, BuLi, THF,
)100 ꢀC, 30 min. (iv) ZnCl2, 16 h. (v) hexamethyldisilazane, BuLi,
THF, )78 ꢀC, 16 h. (vi) 1 M HCl in ether, 0 ꢀC, 1 h. (vii) Triethyl-
amine, acetyl chloride. (viii) phenylboronic acid, H2O ⁄ ether (1:1) +0.2
eq 1 M HCl, 1 h.
Figure 1: Bacterial cell wall biosynthesis pathway in Staphylo-
coccus aureus.
O
O
i
Materials and Methods
Cl CH2
I
B(OiPr)
+
3
Cl CH2
B
Synthesis of boro-amino acid analogs
The synthesis strategy for alkyl side chain D-boro-amino acid ana-
logs was adapted from general procedures for L-boro analogs devel-
oped by Matteson, Kettner et al. (14–16) and described previously
(11) (Scheme 1). The synthesis strategy used for boroGly pinacol is
outlined in Scheme 2.
9
ii
O
O
O
iii
B
[(H3C)3Si]2N CH2
B
H2N CH2
())-Pinanediol (1S)-(1-aminoethyl)-1-boronate (D-boroAla-())-pinane-
diol) (R = CH3) (6a) was synthesized and characterized as described
previously (11).
O
10
11
(+)-Pinanediol (1R)-(1-aminoethyl)-1-boronate (L-boroAla-(+)-pinanedi-
ol), the opposite enantiomer to 6a, was synthesized as described
previously (11).
Scheme 2: Reagents and conditions: (i) a-ether, BuLi, )78 ꢀC,
1 h, 10 ꢀC, 30 min. b-pinacol, r.t., 16 h. (ii) hexamethyldisilazane,
BuLi, THF, )78 ꢀC, 16 h. (iii) 1 M HCl in ether, 0 ꢀC.
758
Chem Biol Drug Des 2011; 78: 757–763

D-boroAla as a Novel Broad-Spectrum Antibacterial Agent
())-Pinanediol (1S)-1-amino-propane-1-boronate (D-boroHomoAlanine-
())-pinanediol) (R = Et) (6b) was synthesized following the proce-
dure described by Vankatraman et al. for the L-isomer (17), substi-
tuting ())-pinanediol for (+)-pinanediol as the stereochemical
directing group, and using EtLi in place of EtMgBr as the alkyl
anion.
MIC determination assays onto agar media. After overnight (24 h)
incubation at 35 ꢀC, colonies were counted and used to calculate
cfu of the samples. The MBC was defined as the lowest concentra-
tion of drug, which killed 99.9% (‡3 log reduction) of the original
inoculum.
())-Pinanediol (1S)-1-amino-2-methylpropane-1-boronate (D-boroVa-
line-())-pinanediol) (R = iPr) (6c) was synthesized following the
procedure described by Kettner and Shenvi (16) for the L-isomer and
substituting ())-pinanediol for (+)-pinanediol as the stereochemical
directing group.
Frequency of resistance
A clinical isolate of methicillin-sensitive S. aureus (MSSA) was
grown for 16 h at 35 ꢀC in 100 mL Mueller Hinton broth, with
shaking at 250 rpm. Bacterial cells were concentrated from
50 mL of this saturated overnight culture by centrifugation at
2500 · g for 15 min and reconstituted into 5 mL media. Samples
of 0.4 mL (ꢀ1 · 109 CFU) were plated onto 150-mm agar
plates containing 75 mL of media with (6a) at concentrations of
2· and 4· MIC. As a reference, the cfu of the reconstituted
culture were also determined. At each concentration, 3–4 plates
were used. Inoculated plates were incubated for 48 h at
35 ꢀC, and each plate visually screened for growth of single
colonies. Representative subsets of observed colonies were
selected using a sterile inoculating loop and re-streaked onto
fresh media containing 6a (at selection concentration) to confirm
resistance. The frequency of resistance was calculated by divid-
ing the number of resistant colonies by the total number of cfu
plated.
Acetyl-D-boroAlanine-())-pinanediol (7) was synthesized from D-
boroAla-())-pinanediol as described previously (11).
(1S)-(1-aminoethyl)-1-boronate (D-boroAlanine) (8), the free acid of
D-boroAla, was prepared from the HCl salt of (6a) by treatment
with excess phenylboronic acid in a 1:1 water ⁄ ether mixture plus
0.2 equivalents of 1 M HCl, followed by extraction of the aqueous
phase 3· with an equal volume of ether (18) (Scheme 1). The pure
HCl salt of D-boroAla was obtained from the separated aqueous
phase after lyophilization.
Pinacol (chloromethyl)boronate (9) was synthesized following the
procedure of Sadhu and Matteson (19) and converted in situ to the
pinacol ester, pinacol (chloromethyl)boronate (9).
In vivo biochemical mechanism determination
Pinacol aminomethylboronate (boroGlycine-pinacol) (11) was synthe-
sized following the procedure described above for the conversion of
intermediates 4a–c to products 6a–c in Scheme 1, as described
by Martichonok and Jones (20).
Determination of in vivo intracellular L-Ala,
D-Ala, and D-Ala-D-Ala levels in response to
D-boroAla-())-pinanediol
Levels of L-Ala, D-Ala, and D-Ala-D-Ala were determined as
described in detail previously (22). Bacteria [Escherichia coli K12 or
MRSA (clinical isolate)] were grown to an OD at 600 nm of 0.6 in
either minimal media (E. coli) or Mueller Hinton broth (MRSA). To a
test culture was added D-boroAla-())-pinanediol (6a) to 4· MIC,
and to control cultures were added no antibiotic, or a control antibi-
otic (cycloserine, tetracycline, or vancomycin) at 4–8· their respec-
tive MICs. Growth inhibition was observed within 15 min. Once
growth inhibition was apparent, cultures were rapidly cooled in an
ice ⁄ water bath, four samples of 10 mL were moved from each flask
to ice-cold 15-mL centrifuge tubes, and the cells were pelleted by
centrifuge at 2500 · g for 10 min at 2 ꢀC. Cell pellets (ꢀ50 lL)
were resuspended in 100 lL of ice-cold M9 minimal medium and
treated with 200 lL of ice-cold 80% acetone spiked with 20 lM
13C3-D-Ala as an internal standard. Tubes were kept on ice with
occasional vortexing for 5 min. These tubes were again centrifuged,
and supernatants were collected into fresh ice-cold microcentrifuge
tubes. Samples (15 lL) were derivatized with Marfey's reagent and
analyzed by LC-MS ⁄ MS.
Antibacterial properties characterization
MICs and spectrum of activity
Minimal inhibitory concentrations (MICs) were determined by broth
microdilution following Clinical and Laboratory Standards Institute
guidelines [CLSI, formerly National Committee for Clinical Laboratory
Standards (21)]. Twofold serial dilutions of test agents were
prepared in 100 lL of Mueller Hinton broth (Difco, Sparks, MD,
USA) in the wells of microtiter plates. Wells were inoculated with
ꢀ1 · 104 colony-forming units (CFU) of the test bacteria, and
plates incubated for 16–20 h at 35 ꢀC. The plates were read for
turbidity either visually or at 600 nm in a Tecan SpectroFluor Plus
microtiter plate reader. The MIC was read as the lowest concentra-
tion of test compound for which no turbidity is apparent (trans-
mittance >90% of a media control well). Minimal inhibitory
concentrations were determined against several bacterial pathogens
(Table 1), including both Gram-positive and Gram-negative organ-
isms, to determine spectrum of activity. All MIC determinations
were performed in triplicate.
Results
Minimal bactericidal concentrations
Structure–activity correlation
Minimal bactericidal concentrations (MBCs) were performed by plat-
ing serially diluted samples from wells of microtiter plates from
A summary of antibacterial activities of the newly synthesized com-
pounds against MSSA (clinical isolate), MRSA (clinical isolate), and
Chem Biol Drug Des 2011; 78: 757–763
759

Putty et al.
Table 1: Spectrum of activity: MICs for D-boroAla-())-pinanediol, D-boroAla (no pinanediol), and controls
MIC (lg ⁄ mL)
Control antibioticsa
Tet
Strain
D-boroAla-())-Pd (6a)
D-boroAla (8)
D-Cycloser
Vanc
Amp
Cefoxitin
Enterococcus faecium (VRE, clinical)
MRSA (clinical)
MSSA (clinical)
Salmonella typhi (clinical)
Escherichia coli K12
Shigella sonnei (clinical)
G+
G+
G+
G)
G)
G)
G)
G)
16
16
8
>128
64
64
32
16
8
64
8
>512
1
8
0.125
0.0625
0.5
128
128
32
8
8
>256
>256
4
2
8
32
>512
>256
>512
NDb
NDb
2
2
2
32
64
128
64
256
>128
NDb
NDb
0.5
32
16
2
Pseudomonas aeruginosa (ATCC 27853)
Burkholderia pseudomallei (strain 1026b)
NDb
NDb
NDb
NDb
NDb
NDb
NDb
NDb
a
D-Cycloser, D-cycloserine; Vanc, vancomycin; Tet, tetracycline; Amp, ampicillin.
bND, not determined.
MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensitive S. aureus; MIC, minimal inhibitory concentrations.
E. coli K12 is given in Table 2. D-boroAla-())-pinanediol (6a) has
the most potent activity in this series.
Table 2: Structure–activity correlation
MIC: lM and (lg ⁄ mL)
MRSAa
D-boroHomoAla-())-pinanediol (6b), D-boroVal-())-pinanediol (6c),
and boroGly-())-pinacol (11) were found to have worse antibacte-
rial activity (higher MIC values) than D-boroAla-())-pinanediol (6a).
Removal of the pinanediol group also reduced antibacterial effec-
tiveness, which is presumably because of the lipophilic pinanediol
group facilitating transport across the bacterial membrane. Acetyl-D-
boroAla-())-pinanediol (7) was completely inactive, indicating that a
positively charged amino group is required for antibacterial activity.
Finally, L-boroAla-(+)- pinanediol (11) (the opposite enantiomer of
6a) had very weak activity.
Escherichia
coli K12
Compound
MSSAa
50 (10)
1000 (250)
200 (50)
6a
L-boroAla-(+)-Pd
6b
6c
7
50 (10)
100 (20)
400 (100)
1000 (250)
5000 (1250)
NAb
NDa
1000 (250)
5000 (1250)
NAb
5000 (1250)
NAb
8
11
200 (25)
Weak activity
200 (25)
400 (50)
NDb
NAb
aMSSA and MRSA were both clinical strains as described in the text.
bND, not determined; NA, no activity, minimal inhibitory concentrations (MIC)
>5000 lM.
Antibacterial activity
MRSA, methicillin-resistant Staphylococcus aureus; MSSA, methicillin-sensi-
tive S. aureus.
The spectrum of activity results is summarized in Tables 1 and 3. D-
boroAla demonstrated activity against several strains of both Gram-
positive and Gram-negative organisms.
Table 3: MICs and MBCs for D-boroAla-())-pinanediol against
several bacterial strains and correlation of MBC ⁄ MIC ratio with
genomic copies of DDL
D-boroAla-())-pinanediol (6a) was bactericidal against S. aureus
and Bacillus subtilis at 1· MIC and against E. coli and Salmonella
MIC
MBC
DDLs
Strain
(lg ⁄ mL) (lg ⁄ mL) MBC ⁄ MIC in genomea
100
80
60
40
20
0
Staphylococcus
G+ 16
G– 32
16
1
4
1
2
aureus (ATCC 29213)
Salmonella enterica
serovar Typhimurium
(ATCC 14028)
Bacillus subtilis
(ATCC 6633)
128
G– 16
G– 32
16
1
4
1
2
Escherichia coli K12
128
aBased on searches of annotated full genomes for S. aureus and E. coli at
the Welcome Trust Sanger Institute (http://www.sanger.ac.uk/), and the
annotated full genomes of B. subtilis and S. enterica serovar Typhimurium at
the Comprehensive Microbial Resource (http://cmr.jcvi.org/cgi-bin/CMR/
CmrHomePage.cgi).
½-Ala
—-Ala
—-Ala-—-Ala
Figure 2: Levels of L-Ala, D-Ala, and D-Ala-D-Ala in Escherichia
coli as a function of added agents.
DDL, D-Ala-D-Ala ligase; MBC, minimal bactericidal concentrations; MIC,
minimal inhibitory concentrations.
760
Chem Biol Drug Des 2011; 78: 757–763

D-boroAla as a Novel Broad-Spectrum Antibacterial Agent
enterica serovar Typhimurium at 4· MIC (Table 3). A frequency of
resistance determination was performed for D-boroAla-())-pinanediol
(6a) against MSSA (clinical, Table 1) at 2· and 4· MIC (16 and
32 lg ⁄ mL, respectively). At 2· MIC, a frequency of resistance of
1 · 10)6 was observed, whereas at 4· MIC, a frequency of resis-
tance of 8 · 10)8 was observed.
that a positively charged amine group is required for antibacterial
activity.
Activity of D-boroAla was then tested against several Gram-negative
and Gram-positive pathogenic bacteria to determine spectrum of
activity (Table 1). Broad-spectrum activity against both Gram-positive
and Gram-negative bacteria was observed, with MICs ranging from
8 to 128 lg ⁄ mL. Bactericidal activity was apparent at 1· MIC
against S. aureus and B. subtilis and at 4· MIC against S. enterica
serovar Typhimurium and E. coli (Table 3).
Determination of biochemical mechanism
Treating E. coli with D-boroAla-())-pinanediol (6a) at 4·, MIC had a
profound effect on the intracellular levels of D-Ala-D-Ala (Figure 2).
The frequency of resistance of S. aureus at 4· MIC was 8 · 10)8
.
A similar result was observed in MRSA (Figure S1). Treating cells
with sub-MIC levels of 6a resulted in only a modest decrease in
D-Ala-D-Ala levels (data also not shown). This experiment verifies that
D-boroAla exerts its antibacterial activity through inhibition of DDL.
This is comparable to or lower than rifampicin resistance frequency
in several bacterial strains (24,25) and falls at the lower end of the
weakly hypermutable range (4 · 10)8–4 · 10)7), and just above
the normomutable range (8 · 10)9–4 · 10)8) (26,27).
Given these observations, an obvious question was: what is the
molecular target of D-boroAla? Several lines of evidence suggested
that D-boroAla would act in the alanine branch of the bacterial cell
wall biosynthesis pathway (Figure 1), including that bacterial cell
wall biosynthesis is unique in its requirement for D-Alanine, that
the antibacterial activity in this series of compounds is correspond-
ingly specific to D-boroAla (Table 2), and that D-boroAla has previ-
ously been described as an inhibitor of both alanine racemase and
DDL (13) – the two enzymes catalyzing the reactions in the alanine
branch (Figure 1). We have recently developed an assay for the
intermediates (L-Ala, D-Ala, and D-Ala-D-Ala) in the alanine branch
of bacterial cell wall biosynthesis (22). This assay was used to
determine whether D-boroAla had a significant impact on the early
cell wall intermediates in both E. coli and S. aureus and demon-
strate that D-boroAla has a substantial effect on the level of D-Ala-
D-Ala above its MIC in both E. coli (Figure 2) and S. aureus (Figure
S1). This effect is centered on the MIC for D-boroAla (Figure 2) –
D-boroAla exhibits little effect on D-Ala-D-Ala levels below its MIC,
but a pronounced effect above its MIC. It is also notable that the
control antibiotic cycloserine exerts its effect on both D-Ala and
D-Ala-D-Ala levels, consistent with its known mechanism of action
as an alanine racemase inhibitor. From these observations, we con-
clude that D-boroAla exerts its antibacterial activity through inhibi-
tion of DDL. As a further test of biochemical mechanism, it is
known that in S. aureus, the addition of D-Ala can antagonize the
antibacterial action of cycloserine (28,29). We have also observed
that D-Ala at 2.5 mM antagonizes the antibacterial activity of cyclo-
serine at 2· and 4· MIC, but does not antagonize the antibacterial
activity of D-boroAla at 2· and 4· MIC, which is an observation
also consistent with DDL as the molecular target of D-boroAla.
Discussion
Previous biochemical studies (13) have identified D-boroAla as an
effective inhibitor of both alanine racemase (saturable time-depen-
dent inhibition with KI = 20 mM and kinact = 0.35 ⁄ min) and DDL (KI
under intracellular conditions against the S. enterica serovar
Typhimurium enzyme of 18 lM). There have however been no previ-
ous reports on the antibacterial activity of D-boroAla. During the
course of our investigations on peptide-D-boroAla derivatives as
inhibitors of the penicillin-binding proteins (11), we observed anti-
bacterial activity in some crude peptide-D-boroAla preparations,
which was lost on purification of the peptide-D-boroAla derivative.
A filter disk test of D-boroAla for antibacterial activity revealed sur-
prisingly good activity for D-boroAla-())-Pd against both E. coli and
S. aureus, indicating possible broad-spectrum activity, and it
seemed worthwhile to further characterize the antibacterial activity
of D-boroAla and its homologs.
A structure–activity study was first performed by synthesizing a
series of D-boroAla homologs. Three features of D-boroAla were
examined including (i) the length of the side chain alkyl group, (ii)
the effect of N-acylation, and (iii) the presence or absence of the
pinanediol protecting group. Pinanediol protecting groups are used
in amino boronic acid syntheses to control the stereochemical out-
come of the product (15,16,23). In aqueous solutions, the boro-
pinanediol ester is in equilibrium with the free boronic acid and
pinanediol. A control test of racemic pinanediol revealed no anti-
bacterial activity (data not shown). Among the compounds tested
in this study, D-boroAla-())-pinanediol was the most active, with
MICs against E. coli and S. aureus in the 8–32 lg ⁄ mL range
(Tables 1 and 2). Removal of the pinanediol group resulted in
higher MICs, likely due to the lipophilic pinanediol group facilitat-
ing membrane permeability. L-boroAla showed much lower antibac-
terial activity, demonstrating stereospecificity of antibacterial
activity. This observation indicates that D-boroAla likely acts on a
specific macromolecular target and not simply as a non-specific
membrane-disrupting agent. Longer and shorter side chain homo-
logs of D-boroAla (e.g., boroGly, D-boroHomoAla, and D-boroVal)
demonstrated greatly reduced antibacterial activity. Acetylation of
D-boroAla to give acetyl-D-boroAla abolished activity, demonstrating
The identification of DDL as the molecular target of D-boroAla's
antibacterial activity can be used to rationalize the MBC ⁄ MIC ratios
observed against several bacterial strains (Table 3). The difference
in MBC ⁄ MIC ratio between bacterial strains appears correlated
with the number of copies of DDL in the genomes of these organ-
isms (one in S. aureus and B. subtilis and two in E. coli and S. ty-
phimurium). Our working hypothesis is that at 1· MIC D-boroAla
inhibits the one DDL in S. aureus and B. subtilis and is bactericidal.
However, against E. coli and S. typhimurium which have two copies
of DDL, 1· MIC D-boroAla inhibits only one of the two DDLs that
Chem Biol Drug Des 2011; 78: 757–763
761

Putty et al.
inhibits grown, while the other DDL appears sufficiently active to
ensure viability of these organisms. At higher D-boroAla concentra-
tions (4· MIC), the second DDL in E. coli and S. enterica serovar
Typhimurium is also inhibited by D-boroAla, which then causes cell
death.
9. Abbanat D., Macielag M., Bush K. (2003) Novel antibacterial
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Conclusions and Future Directions
This study demonstrates that D-boroAla has broad-spectrum antibac-
terial activity, is bactericidal, and acts on DDL. There has recently
been considerable interest in DDL as a potential target for antibac-
terial agent development [recently reviewed in (30)], and the obser-
vations reported here further support DDL as a viable target for the
development of novel antibacterial agents. Future studies directed
toward characterizing D-boroAla against the two DDL variants found
in E. coli and S. enterica serovar Typhimurium will be of interest to
determine the relative affinities of these DDLs for D-boroAla and
correlation with D-boroAla's in vivo activity. This study also raises
the question of why some bacteria have two copies of DDL. One
explanation is that two different DDL enzymes with different kinetic
properties may be required to allow efficient bacterial cell growth
or survival under different growth conditions. D-boroAla and gene
knockout experiments could be used to address this question.
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Acknowledgments
Supported by the American Heart Association [Grant number
0650179Z (WGG)], NIH grant GM060149 (WGG), and by funds from
the University of Missouri [University Research Board Grant
K2303015 (WGG)]. HPS is supported by NIH-NIAID grant AI065357.
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Any queries (other than missing material) should be directed to the
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