Mechanism-based inactivation by aromatization of the transaminase BioA involved in biotin biosynthesis in mycobaterium tuberculosis

Article abstract of DOI:10.1021/ja204036t

BioA catalyzes the second step of biotin biosynthesis, and this enzyme represents a potential target to develop new antitubercular agents. Herein we report the design, synthesis, and biochemical characterization of a mechanism-based inhibitor (1) featuring a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the pyridoxal 5′-phosphate (PLP) cofactor of BioA through aromatization. The structure of the PLP adduct was confirmed by MS/MS and X-ray crystallography at 1.94 A resolution. Inactivation of BioA by 1 was time- and concentration-dependent and protected by substrate. We used a conditional knock-down mutant of M. tuberculosis to demonstrate the antitubercular activity of 1 correlated with BioA expression, and these results provide support for the designed mechanism of action.

Full text of DOI:10.1021/ja204036t

ARTICLE
pubs.acs.org/JACS
Mechanism-based Inactivation by Aromatization of the Transaminase
BioA Involved in Biotin Biosynthesis in Mycobaterium tuberculosis
†
‡
§
†
||
||,#
Ce Shi, Todd W. Geders, Sae Woong Park, Daniel J. Wilson, Helena I. Boshoff, Orishadipe Abayomi,
||
§
‡
,†
Clifton E. Barry, III, Dirk Schnappinger, Barry C. Finzel, and Courtney C. Aldrich*
†
Center for Drug Design, Academic Health Center, University of Minnesota, Minnesota 55455, United States
Department of Medicinal Chemistry, University of Minnesota, Minnesota 55455, United States
‡
§
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, United States
Tuberculosis Research Section, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892, United States
S Supporting Information
b
ABSTRACT: BioA catalyzes the second step of biotin biosynthesis,
and this enzyme represents a potential target to develop new anti-
tubercular agents. Herein we report the design, synthesis, and bio-
chemical characterization of a mechanism-based inhibitor (1) featuring
a 3,6-dihydropyrid-2-one heterocycle that covalently modifies the
0
pyridoxal 5 -phosphate (PLP) cofactor of BioA through aromatization.
The structure of the PLP adduct was confirmed by MS/MS and X-ray
crystallography at 1.94 Å resolution. Inactivation of BioA by 1 was time-
and concentration-dependent and protected by substrate. We used a
conditional knock-down mutant of M. tuberculosis to demonstrate the
antitubercular activity of 1 correlated with BioA expression, and these
results provide support for the designed mechanism of action.
’
INTRODUCTION
7,8-diaminopelargonic acid (DAPA) using S-adenosylmethionine
6bꢀd
(
SAM) as the amino donor.
BioD, dethiobiotin synthetase,
Mycobacterium tuberculosis (Mtb), the etiological agent of
carboxylates DAPA to form the imidazolidin-2-one ring of
tuberculosis (TB), has plagued mankind for millennia and i1s
4a
dethiobiotin (DTB) in the third step. Finally, BioB, an ironꢀ
presently the leading cause of bacterial infectious disease mortality.
sulfur cluster enzyme, catalyzes the CꢀH activation and insertion
Rifampin, the last approved first-line drug for TB chemotherapy was
introduced in 1968. Duringthelastfourdecades resistance to all of
the first- and second-line antitubercular agents has been re-
ported. Consequently, new drugs that inhibit novel targets in
Mtb are needed. Biotin (vitamin H) is an essential cofactor
responsible for activation of carbon dioxide in acyl-CoA carbox-
ylases (ACCs) involved in fatty acid metabolism and pyruvate
carboxylase (PCC) in gluconeogenesis. Biotin starvation of an
Mtb biotin auxotroph led to cell death, suggesting that biotin
biosynthesis represents a very attractive pathway for develop-
ment of new antitubercular agents. Significantly, conditional
6e,f
2
of sulfur in DTB to afford biotin. Although the source of
pimelate in Mtb is unknown, Cronan and co-workers have
recently shown that E. coli co-opts the fatty acid synthase type
II pathway along with BioC (Mtb ortholog: Rv0089) and BioH
(
Mtb ortholog: Rv2715) for synthesis of pimeloyl-ACP, which is
7
proposed as the physiologically relevant substrate for BioF.
Amiclenomycin (ACM) isolated from Streptomyces lavendulae
subsp. amiclenomycini and its simplified amino-alcohol analogue
ACMꢀOH are potent inhibitors of BioA, and we have shown
that ACMꢀOH possesses better whole cell activity against Mtb
3
4
8,10c
(
Figure 1).
Synthetic studies indicated the stereochemistry of
silencing of bioA, which encodes for the second enzyme in the
biotin biosynthetic pathway, after establishment of an infection,
demonstrated that BioA is essential to maintain a chronic
the 1,4-cyclohexadiene ring is cis rather than trans as originally
9
assigned. Amiclenomycin is a mechanism-based inhibitor
5
through covalent modification of the PLP cofactor of BioA via
infection in a murine model of TB.
10
aromatization. Remarkably, ACM exhibits selective antituber-
In analogy to the well-studied pathway in E. coli, biotin
biosynthesis in Mtb begins from pimeloyl-CoA, which is elabo-
rated to biotin in four steps catalyzed, respectively by BioF, BioA,
BioD, and BioB (Scheme 1). The first step is carried out by BioF
that converts pimeloyl-CoA to 7-keto-8-aminopelargonic acid
8b
cular activity among 40 bacterial strains evaluated. Chemical com-
plementation of Mtb cultures with DAPA, but not KAPA,
antagonizes the activity of ACM, thus confirming the mechanism
6
8b
of action. Although amiclenomycin has served as an excellent
6a
(KAPA) through decarboxylative condensation with L-alanine.
0
In the second step, BioA, a pyridoxal 5 -phosphate (PLP)
dependent enzyme, catalyzes the transamination of KAPA into
Received: May 2, 2011
Published: October 11, 2011
r 2011 American Chemical Society
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|J. Am. Chem. Soc. 2011, 133, 18194–18201

Journal of the American Chemical Society
ARTICLE
Scheme 1. Biosynthesis of Biotin in Mtb
Scheme 2. Synthesis of 1
Figure 1. Structure of amiclenomycin (ACM), ACMꢀOH, and dihy-
dropyrid-2-one inhibitors 1 and 2.
ization of a mechanism-based inhibitor of BioA with improved
chemical stability.
’
RESULTS
Synthesis of Inhibitors 1 and 2. The enantioselective synth-
eses of 1 and 2 were accomplished in a convergent fashion
employing allylic amine 12 and vinylglycine 13 building blocks as
depicted in Scheme 2. The allylic amine (R)-12, with high
enantiopurity, was efficiently prepared starting with pentynol 8,
which was converted to propargylic alcohol 9 by sequential
silylation with TBSCl and addition to para-formaldehyde. Red-
Al mediated reduction of 9 furnished exclusively the trans-allylic
alcohol and treatment with trichloroacetonitrile and DBU
afforded trichloroacetimidate 10. Asymmetric Overman rearran-
gement of 10 employing the cationic Pd(II)-catalyst [(R)-COP-
Figure 2. Proposed aromatization mechanism of 1. Transamination of
1
with enzyme-bound PLP 3 affords the substrate PLP aldimine 4.
Subsequent 1,3-prototropic shift via 5 mediated by Lys283 provides
ketimine 6. Tautomerization of 6 to 7 is irreversible and leads to
formation of a stabile PLP adduct.
Cl] provided the trichloroacetyl protected allylic amine (R)-11
2
12
in 93% yield and 96:4 enantiomeric ratio (er). Subsequent
deprotection with DIBAL-H yielded the desired allylic amine
(
R)-12. Due to the facile isomerization as well as racemization of
tool compound, it suffers from poor chemical stability due to
spontaneous aromatization.
vinylglycine derivatives under conventional peptide coupling
conditions, we employed DEPBT as the coupling reagent and
NaHCO as base, a very mild condition proven to minimize
10
Inspired by the unique aromatization mechanism of inhibition
3
1
0
14
13a,b
by amiclenomycin and the extensive previous work by
racemization, for the coupling of vinylglycine (S)-13
R)-12 to afford diene 14 in 71% yield. A variety of ring closing
metathesis (RCM) catalysts were screened for the RCM reaction
with
11
Silverman and co-workers on related PLP-dependent mechan-
ism-based inhibitors, we hypothesized that the introduction of a
heteroatom into the 1,4-cyclohexadiene ring scaffold would
decrease the aromatic stabilization energy and correspondingly
would improve the chemical stability of the inhibitor while
maintaining the same aromatization mechanism of inhibition
(
15
of 14, and Hoveda-Grubbs-II catalyst was found to be most
effective, furnishing (3S,6R)-dihydropyridone 15 in 83% yield.
Finally, the desired (3S,6R)-dihydropyridone 1 was obtained by
sequential deprotection of the TBS and Boc groups with HF Py
3
(Figure 2). Therefore, a pair of enantiomeric inhibitors based
and HClꢀdioxane, respectively. (3R,6S)-Dihydropyridone 2 was
on the simplified ACMꢀOH structure, containing the 3,6-
prepared analogously in 10 steps employing [(S)-COP-Cl] and
2
13c
dihydropyridone heterocycle, was designed to test our hypothesis
(R)-13.
Dihydropyridones 1 and 2 showed substantially
(Figure 1). Herein, we report the synthesis and kinetic character-
improved chemical stability compared to ACM, displaying no
1
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Journal of the American Chemical Society
ARTICLE
Figure 4. Observed fragments by MS/MS of the m/z 400 ion.
Figure 3. (A) Residual activity of BioA as measured by initial velocity, v,
versus preincubation time in the presence of increasing concentrations
of 1, showing time dependent inactivation of BioA. (B) Plot of the
observed rate of inactivation constants (kobs) versus the concentration of
1
, from which the kinetic parameters kinact, K
I
and the second-order rate
constant kinact/K were determined. (C) Scheme showing two-step
I
mechanism of inactivation of BioA by 1.
Table 1. Kinetic Parameters for Inhibition of BioA by ACM
and Analogues
ꢀ
1
ꢀ1
ꢀ1
Inhibitor
kinact (min
)
K
I
(μM)
kinact/K
I
(M min
)
Figure 5. Final 2F
prereaction complex and (B) postreaction complex.
o
ꢀF
c
electron density contoured at 1.0 σ for the (A)
2
1
2
0.18 ( 0.01
<0.01
520 ( 72
>2000
(3.5 ( 0.5) ꢁ 10
<5
fit by linear regression analysis to provide the k /K value of
inact
I
a
4
4
ACM
0.35 ( 0.05
0.56 ( 0.05
12 ( 2
20 ( 2
(2.9 ( 0.6) ꢁ 10
(2.8 ( 0.4) ꢁ 10
ꢀ1
ꢀ1
3
7 M min .
a
ACMꢀOH
Reference 6c.
MS analysis. To confirm the formation of the aromatic adduct
a
proposed in Figure 2, MS analysis was performed to identify the
formation of the aromatic adduct. We incubated 1 with BioA
(360 μM 1, 26 μM BioA, 50 mM Tris pH 8.0, 10 mM
2-mercaptoethanol and 100 μM PLP) at 37 °C for 2 h, which
led to complete enzyme inactivation. BioA was subsequently
isolated by gel filtration to remove excess 1 and PLP, then
analyzed using electrospray ionization tandem mass spectro-
metry. The PLP-adduct 7 was found to dissociate from the
protein complex and the quasimolecular ion was detected at m/z
400, which corresponds to the expected aromatized PLP adduct
(Figure 4). The MS/MS spectrum of this ion displayed two
degradation after 60 days at room temperature under aerobic
conditions in DMSO as measured by H NMR.
1
Biochemical Evaluation. The ability of 1 and 2 to irreversibly
inhibit BioA was evaluated by preincubation of 0.03ꢀ1 mM
inhibitor with 2 μM BioA in the absence of substrates (KAPA and
SAM). Aliquots were removed at time t and assayed for enzyme
activity using a coupled assay employing BioD that measures
production of DTB from (S)-KAPA. Dihydropyridone 1
exhibited time-dependent inhibition while 2 displayed no activ-
ity. Fitting the observed initial velocity as a function of incubation
time t for 1 provided the pseudo first-order rate constants of
inactivation, kobs at a given inhibitor concentration [I] (Figure 3A
and B). The hyperbolic relationship between kobs and [I] is
consistent with a two-step mechanism of inactivation (Figure 3C).
16
+
fragment ions at m/z 302 [M + H ꢀ 98] and 169 [M + H ꢀ
+
ꢀ
231] . The peak at m/z 302 corresponds to the loss of H O PO
3
3
from the parent molecule suggesting structure 16 while the peak
at m/z 169 is consistent with the aromatized inhibitor ion 17
(Figure 4). These results demonstrate formation of the aromatic
PLP-adduct and hence provide strong support for the proposed
aromatization mechanism.
Structural Characterization. To provide structural insight on
adduct formation in the context of the protein active site, we
soaked yellow, PLP-loaded crystals of BioA with 1 and observed
1 concentrate rapidly to within the crystal affording red crystals
before apparently reacting to yield colorless crystals (Figure S2,
Supporting Information). Crystals soaked with 1 were frozen in
liquid nitrogen when visibly red or colorless. Diffraction data to
1.95 and 1.94 Å resolution were collected from a red, prereaction
crystal and a colorless, postreaction crystal, respectively
(Table S1, Supporting Information).
The kinetic parameters kinact, K (Table 1) were determined from
versus [I] data to eq 2 (see Experimental
Section). While the kinact of 1 compares favorably with ACM
I
fitting the k
obs
1
7
and ACMꢀOH, the K is approximately 26-fold higher than
I
4a
ACMꢀOH. The second-order rate constant for inactivation
ꢀ1
ꢀ1
k
/K of 1 is 346 M min , which is 80-fold lower than
I
inact
ACMꢀOH. We also showed that addition of the substrate (S)-
KAPA to the initial incubation mixture of BioA and 1 protected
against inactivation as expected for a mechanism-based inhibitor.
To evaluate potential biochemical selectivity, 1 was also evaluated
against two commercially available mammalian PLP-dependent
enzymes including alanine aminotransaminase and aspartate ami-
notransaminase. Inhibitor 1 was inactive against aspartate amino-
transaminase, but did show time-dependent inhibition against
alanine aminotransaminase. The inhibition was not saturable with
up to 2 mM 1; however, the slope of the kobs versus [I] could be
In the prereaction complex, the electron density describes a
well-defined PLP covalently bound to Lys283 in a manner
identical to previous reports (Figure 5A). New positive
4
difference density accumulated adjacent to PLP, presumably
1
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Journal of the American Chemical Society
ARTICLE
polar amino acid moiety on the C-4 side-chain was replaced with
6c
a simple neutral hydroxy group. We independently synthesized
ACMꢀOH and demonstrated that this structural simplification
10c
did not adversely impact antitubercular activity. Consequently,
we elected to use this modification in our designed 3,6-dihydro-
pyridone analogs. Although the X-ray structure of BioA from Mtb
4a
was reported, simple molecular docking studies were unable to
reveal whether 1 or 2 would be more active, thus both enantio-
mers were prepared.
Antitubercular Activity. Park and co-workers recently devel-
5
oped Mtb conditionally regulated knockdown strains of BioA.
Significantly, it was shown that BioA activity must be reduced by
5
approximately 99% to prevent bacterial growth. It should be
noted that these were among the first experiments to address the
extent of inactivation, or vulnerability, required to halt replication
against any target in Mtb. Based on these results, BioA is not
particularly vulnerable to inhibition since nearly complete in-
activation is required. Consequently, irreversible inhibitors or
tight-binding inhibitors with slow dissociation rates are likely to
Figure 6. Anti-TB activity of 1. Mtb bioA-TetON-1 was grown in biotin-
free Sauton’s medium containing 0 ng/mL or 10 ng/mL ATC and the
indicated concentration of compound 1. Growth was measured as
optical density at 580 nm and normalized to growth without 1. Error
bars represent the standard error of the mean of triplicate measurements.
2
9
be more effective than simple rapidly reversible inhibitors.
The Mtb strains that enable conditional BioA expression as
shown herein are also extremely useful to assess on-target activity
of compounds, since these permit the correlation of antituber-
cular activity with enzyme expression levels. In the present case,
overexpression of BioA in strain Mtb bioA-TetON-1 renders Mtb
resistant to 1, presumably by stoichiometric titration of the
inhibitor. This result is readily reconciled, since the amount of
BioA produced in this conditional strain is approximately 100-
fold greater in the presence of the small molecule anhydro-
tetracycline (ATC), versus in its absence.
due to the binding of 1 during soaking (Figure S3, Supporting
Information). In the postreaction complex, Lys283 is very
clearly no longer covalently bound to PLP and the planar
electron density is consistent with the aromatized adduct 7
(
Figure 5B, Figure S4B, Supporting Information). The ob-
served adduct possesses small differences in conformation
between the two crystallographically unique molecules in the
BioA dimer. In the second molecule in the asymmetric unit,
electron density for the aromatized adduct is less well-defined
and positioned on the opposite side of a flipped Trp64
Biochemical Activity and Mechanism. The kinact values for 1
is only 3-fold lower than ACMꢀOH showing that replacement
of the 1,4-cyclohexadiene warhead with the dihydropyridone has
little impact on the rate of inactivation. Other described mecha-
nism-based inhibitors of PLP dependent enzymes are typically
(Figure S4, Supporting Information). Nevertheless, degrada-
tion of the internal aldimine appears complete in both
molecules in the postreaction structure.
In vivo Antitubercular Activity. Lastly, we evaluated the
antitubercular activity of 1 in biotin-free medium against a mutant
strain of M. tuberculosis H37Rv termed Mtb bioA-TetON-1 that
ꢀ
1 11
on the order of ∼1 min
.
The slow rate of inactivation is
commensurate with the relatively slow turnover rate of PLP-
5
dependent enzyme due to slow transamination between the
enables conditional expression of BioA. In the presence of 10 ng/
30
internal and external aldimines.
mL anhydrotetracycline (ATC), this strain overexpresses BioA,
whereas in the absence of ATC, this strain expresses approxi-
mately 7% BioA compared to wt Mtb. Overexpression of BioA
conferred resistance to 1 while underexpression of BioA restored
sensitivity (Figure 6). The correlation between antitubercular
activity of 1 and BioA expression level provides support for the
designed mechanism of action. Additionally, the active enantio-
mer 1 was evaluated against two mammalian cell lines (Vero and
CHO-K1) to provide an initial assessment of potential toxicity
and 1 showed no inhibition of cell viability up to 1 mM, the
maximum concentration employed.
Mechanism-based inhibitors of PLP-dependent enzymes that
operate through an aromatization mechanism are fascinating and
have examples beyond amiclenomycin and 3,6-dihydropyridone
1
, including the natural product gabaculine, isogabaculine,
4
-amino-4,5-dihydro-2-furancarboxylic acid, 4-amino-4,5-dihy-
dro-2-thiophenecarboxylic acid, D-cycloserine, and gostatin.
1
1,34
Interestingly, D-cycloserine is a second-line antitubercular agent,
whose mechanism of action is due to irreversible inactivation of
2
the mycobacterial PLP-dependent enzyme D-alanine racemase.
D-Cycloserine has also been shown to inhibit host PLP-depen-
dent enzymes including γ-aminobutyric acid aminotransferase
11a
32
(
GABA-AT) and serine palmitoyltransferase. To investigate
the potential selectivity of 1 toward other PLP-dependent
enzymes we screened it against porcine alanine aminotransami-
nase and aspartate aminotransaminase. 3,6-Dihydropyridone 1 was
inactive against aspartate aminotransaminase but showed weak
time-dependent, yet nonsaturable inhibition against alanine ami-
’
DISCUSSION
Rationale and Design of 1. The 1,4-cyclohexadiene warhead
of amiclenomycin (ACM) is both thermodynamically and kine-
tically unstabile due to the large aromatic stabilization energy of
approximately 36 kcal/mol. Replacement of one of the olefins in
ACM with an amide isostere provides a 3,6-dihydropyridone
heterocycle, which has an estimated aromatic stabilization of only
ꢀ
1
ꢀ1
notransaminase with a kinact/K value of 37 M min demonstrat-
I
ing a promising level of biochemical selectivity for BioA.
Several mechanisms of BioA inactivation are plausible includ-
ing an aromatization mechanism, an enamine mechanism and a
2
8
2
5 kcal/mol, and hence is predicted to be substantially more
31
stabile. Mann and Ploux reported on the synthesis of the
Michael addition mechanism. However, the isolation of the
simplified ACM analog that we term ACMꢀOH wherein the
PMP adduct 7 by mass spectrometry and X-ray analysis provides
1
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Journal of the American Chemical Society
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unequivocal support of the aromatization mechanism of inhibi-
tion. Interestingly, inactivation of GABA-AT by 4-amino-4,
singlet, d = doublet, t = triplet, q = quartet, p = pentet, m = multiplet, br =
broad), integration, coupling constant. Melting points were measured
on a B €u chi 535 melting point apparatus and uncorrected. High resolu-
tion mass spectra were obtained on an Agilent TOF II TOF/MS
instrument equipped with either an ESI or APCI interface. TLC analyses
were performed on TLC silica gel 60F254 from EMD Chemical Inc., and
were visualized with UV light, iodine chamber, 10% sulfuric acid or 10%
PMA solution. Optical rotations values were obtained on a Rudoldf
Autopol III Polarimeter. Purifications were performed by flash chroma-
tography on silica gel (Dynamic Adsorbents, 60A). Enzymatic activity,
kinetic parameters, and inhibition assays were performed on a molecular
devices (Sunnyvale, CA) M5e multimode plate reader. ESI MS/MS
experiments were performed on a 2000 QTRAP LC/MS/MS spectro-
meter (Applied Biosystems).
5
-dihydro-2-furancarboxylic acid does not operate through
an aromatization mechanism, but rather a Michael-addition
mechanism. The resulting adduct between GABA-AT and
4
-amino-4,5-dihydro-2-furancarboxylic acid is not stabile and
undergoes slow hydrolysis resulting in restoration of enzyme activity.
By contrast, the corresponding thiophene derivative 4-amino-4,5-
dihydro-2-thiophenecarboxylic acid inactivates GABA-AT via an
aromatization mechanism and the resulting adduct is stabile. The
divergence in the mechanisms of these two structurally related
inhibitors has been reconciled by consideration of the lower aromatic
stabilization energy of furan (16 kca/mol) relative to thiophene (29
33
kcal/mol). As noted earlier, the aromatic stabilization of 1 is 25
28
Materials. Chemicals and solvents were purchased from Sigma-
Aldrich Company or Acros Organic Fischer Company, and were used as
received. An anhydrous solvent dispensing system (J. C. Meyer) using 2
kcal/mol, which appears to be a perfect balance ensuring improved
chemically stability of the inhibitor, yet adequate to favor reaction via
an aromatization pathway.
packed columns of neutral alumina was used for drying THF, Et
2
O, and
Structural Characterization. Consistent with the mass spec-
trometry results, the crystal structure clearly shows the formation
of an adduct of 1 with PLP that is consistent with the proposed
structure 7 (Figure 5). Lys283 is positioned to mediate the 1,3-
prototropic shift (Figure 2). This adduct is conformationally
flexible and possesses few hydrogen bonds to the surrounding
protein, which likely explains the discontinuous electron density
for the length of the adduct (Figure 5B, Supplemental Figure 3B,
Supporting Information). The adduct forms in a time- and
concentration-dependent manner with diffraction data collected
from intermediate time points showing a mixture of pre- and
postreaction states (data not shown). The initial red color
observed upon soaking crystals of BioA with 1 likely corresponds
to a minor, transient build-up of intermediate 5 (Figure 2).
CH Cl while 2 packed columns of molecular sieves were used to dry
2
2
DMF and the solvents were dispensed under argon. (S)-N-Boc-vinyl-
13a,b
13c
glycine 13,
(R)-N-Boc-vinylglycine enan-13, (8S)-7-keto-8-amino-
16
pelargonic acid hydrochloride salt (KAPA HCl) and fluorescein-
tagged dethiobiotin (Fl-DTB) were synthesized according to the
3
16
reported procedures. BioA and BioD were cloned, overexpressed, and
16
purified following the reported protocols. For crystallization, the
published purification protocols were altered to replace Tris and DTT
4a
with HEPES and TCEP, respectively.
Chemistry Experimental Procedures for Inhibitor 1. tert-
Butyldimethhyl(pent-4-ynyloxy)silane (S1). To a solution of pent-4-yn-
1-ol 8 (8.4 g, 100 mmol, 1.0 equiv) and imidazole (10.2 g, 150 mmol, 1.5
equiv) in THF (200 mL) at 0 °C was added TBSCl (16.6 g, 110 mmol,
1.1 equiv) portionwise. The reaction was gradually warmed from 0 to
2
5 °C over 0.5 h and stirred for 2 h at 25 °C. The reaction was quenched
’
CONCLUSION
by addition of saturated aqueous NH Cl (100 mL), and the aqueous
4
layer was separated and extracted with Et O (3 ꢁ 50 mL). The
2
In conclusion, we have designed, synthesized and kinetically
combined organic layers were washed with saturated aqueous NaCl
and structurally characterized a mechanism-based inhibitor of
BioA. The dihydropyrid-2-one heterocycle of 1 is an isostere of
the 1,4-cyclohexadiene warhead of amiclenomycin (ACM) and
we demonstrated that it irreversibly inactivates the PLP cofactor
of BioA via an aromatization mechanism. The process is highly
selective as the enantiomeric inhibitor 2 is completely inactive.
We expect the greater chemical stability of 1 compensates for the
attenuated inhibitor efficiency relative to ACM as measured by
(
4
50 mL), dried (MgSO ), and concentrated to afford the crude product
as colorless oil. Distillation under reduced pressure afforded the title
compound (18.8 g, 95%) as a colorless oil: bp 63ꢀ65 °C (P = 10 mbar);
1
3
H NMR (600 MHz, CDCl ) δ 0.01 (s, 6H), 0.85 (s, 9H), 1.63 (p, J =
7
(
2
.0 Hz, 2H), 1.81 (t, J = 2.7 Hz, 1H), 2.22 (td, J = 7.0, 2.7 Hz, 2H), 3.64
13
t, J = 6.0 Hz, 2H); C NMR (150 MHz, CDCl ) δ ꢀ5.4, 15.2, 18.5,
3
18
6.2, 30.9, 62.2, 68.3, 84.0. All data are consistent with reported values.
6-(tert-Butyldimethylsilyloxy)hex-2-yn-1-ol (9). To a solution of S1
the second order rate constant kinact/K . The inverse relationship
I
(1.98 g, 10 mmol, 1.0 equiv) in THF (50 mL) at ꢀ78 °C was added
between antitubercular activity of 1 and BioA protein expression
provides support for the designed mechanism of action. In view
of the importance of drug-target residence time to efficacy of new
drugs, selective mechanism-based inhibitors such as these may
n-BuLi (4.8 mL, 2.5 M in hexanes, 12 mmol, 1.2 equiv) dropwise over
1
5 min. The solution was stirred at ꢀ78 °C for 30 min then paraformalde-
hyde (1.5 g, 50 mmol, 5.0 equiv) was added portionwise over 1 h. The
mixture was gradually warmed up to 25 °C and stirred 16 h. The reaction
was quenched by addition of saturated aqueous NH Cl solution
29
have a higher likelihood of translating into useful therapeutics.
4
The postreaction complex structure will be used to guide the
generation of derivative compounds that will possess higher
specificity and activity.
(50 mL) and the aqueous layer was separated and extracted with Et O
2
(3 ꢁ 25 mL). The combined organic layers were washed with saturated
aqueous NaCl (50 mL), dried (MgSO ) and concentrated. Purification
4
by flash chromatograph afforded the title compound (2.01 g, 88%) as
1
colorless liquid: R
CDCl ) δ 0.03 (s, 6H), 0.87 (s, 9H), 1.68 (p, J = 6.0 Hz, 2H), 2.12 (br t,
J = 6.0 Hz, 1H), 2.27 (tt, J = 6.0, 2.4 Hz, 2H), 3.66 (t, J = 6.0 Hz, 2H),
f
0.30 (15% EtOAc/hexanes); H NMR (600 MHz,
’
EXPERIMENTAL SECTION
3
General Methods. All chemical reactions were performed under
1
3
an inert atmosphere of dry Ar or N
H and C NMR spectra were recorded on a Varian 600 MHz
2
in oven-dried (150 °C) glassware.
4.21 (t, J = 2.4 Hz, 2H); C NMR (150 MHz, CDCl
3
) δ ꢀ5.1, 15.4,
1
13
18.5, 26.1, 31.8, 51.4, 61.8, 78.7, 86.1. All data are consistent with
1
8
spectrometer. Proton chemical shifts are reported in ppm from an
internal standard of residual chloroform (7.26 ppm) or methanol (3.31
ppm), and carbon chemical shifts are reported using an internal standard
of residual chloroform (77.0 ppm) or methanol (49.1 ppm). Proton
chemical data are reported as follows: chemical shift, multiplicity (s =
reported values.
(E)-5-(tert-Butyldimethylsilyloxy)pent-2-en-1-ol (S2). To a solution
of 9 (1.14 g, 5 mmol, 1.0 equiv) in Et
2
O (50 mL) at 0 °C was added Red-
Al (3.75 mL, 65 wt %% in toluene, 12.5 mmol, 2.5 equiv) dropwise over
20 min. The solution was warmed to 23 °C over 1 h, then stirred 6 h at
1
8198
dx.doi.org/10.1021/ja204036t |J. Am. Chem. Soc. 2011, 133, 18194–18201

Journal of the American Chemical Society
ARTICLE
4
quenched by the addition of saturated NH Cl aqueous solution (20 mL).
2
3 °C. The reaction was cooled down to 0 °C and quenched by the
dropwise addition of MeOH (0.5 mL). The reaction mixture was then
The aqueous layer was separated and extracted with EtOAc (3 ꢁ 10 mL).
diluted with Rochelle’s salt solution (50 mL) and stirred for 30 min at
The combined organic layers were washed with saturated aqueous
2
3 °C. The aqueous layer was separated and extracted with Et
2
O
NaCl (30 mL), dried (MgSO
chromatograph afforded the title compound (877 mg, 71%) as a
4
) and concentrated. Purification by flash
(
3 ꢁ 25 mL). The combined organic layers were washed with saturated
2
3
aqueous NaCl (25 mL), dried (MgSO ) and concentrated. Purification
colorless oil: R 0.60 (40% EtOAc/hexanes); [α] = ꢀ7.5 (c 3.2,
4
f
D
1
by flash chromatograph afforded the title compound (1.00 g, 87%) as a
colorless oil: R
CDCl ) δ 0.05 (s, 6H), 0.88 (s, 9H), 1.56 (p, J = 6.0 Hz, 2H), 2.07 (q, J =
6
CH
2
Cl
2
); H NMR (600 MHz, CDCl
3
) δ 0.03 (s, 6H), 0.88 (s, 9H),
1
f
0.25 (15% EtOAc/hexanes); H NMR (600 MHz,
1.44 (s, 9H), 1.45ꢀ1.58 (br m, 3H), 1.61ꢀ1.67 (br m, 1H), 3.60 (br d,
J = 4.8 Hz, 2H), 4.44 (br s, 1H), 4.61 (br s, 1H), 5.11 (d, J = 9.6 Hz, 1H),
5.14 (d, J = 16.8 Hz, 1H), 5.28 (d, J = 9.6 Hz, 1H), 5.36 (br s, 1H), 5.39
3
.6 Hz, 2H), 3.56 (t, J = 6.6 Hz, 2H), 4.05 (d, J = 3.5 Hz, 2H), 5.50ꢀ5.75
13
(
6
m, 2H); C NMR (150 MHz, CDCl
2.5, 63.5, 129.5, 133.5. All data are consistent with reported values.
E)-6-(tert-Butyldimethylsilyloxy)hex-2-en-1-yl 2,2,2-Trichloroaceti-
midate (10). To a solution of S2 (920 mg, 4 mmol, 1.0 equiv) in CH Cl
CN
0.6 mL, 6 mmol, 1.5 equiv) were sequentially added dropwise. The
3
) δ ꢀ5.1, 18.3, 25.9, 28.5, 32.2,
(br s, 1H), 5.71ꢀ5.77 (m, 1H), 5.83ꢀ5.88 (m, 1H), 5.89ꢀ5.97 (m,
9
13
1H); C NMR (150 MHz, CDCl
3
) δ ꢀ5.3, 18.3, 25.9, 28.3, 28.9, 31.2,
(
51.4, 57.3, 62.5, 80.1, 115.3, 118.2, 134.3, 137.9, 155.4, 169.2; HRMS
+
2
2
41 2 4
(APCI+): calcd for C21H N O Si [M + H] 413.2830, found 413.2847
(
(
20 mL) at 0 °C, DBU (0.72 mL, 4.8 mmol, 1.2 equiv) and CCl
3
(error 4.1 ppm).
tert-Butyl {(3S,6R)-6-[3-(tert-Butyldimethylsilyloxy)propyl]-2-oxo-
3,6-dihydro-pyridin-3-nonyl}carbamate (15). To a solution of diene
14 (82.4 mg, 0.20 mmol, 1.0 equiv) in CH Cl (20 mL) at 23 °C was
2 2
added the Hoveyda-Grubbs-II catalyst (6.2 mg, 0.01 mmol, 0.05 equiv).
The solution was then stirred at reflux for 12 h. The reaction was cooled
to room temperature and concentrated in vacuo. Purification by flash
chromatography afforded the title compound (63.7 mg, 83%) as a brown
reaction was stirred 10 min at 0 °C then warmed to 23 °C and stirred for
h. The reaction was concentrated in vacuo and directly purified by flash
chromatograph to afford the title compound (1.42 g, 95%) as colorless
2
1
oil: R
f
0.70 (10% EtOAc/hexanes); H NMR (600 MHz, CDCl
3
) δ 0.04
(s, 6H), 0.88 (s, 9H), 1.63 (p, J = 6.9 Hz, 2H), 2.14 (td, J = 6.9, 6.6 Hz,
2
6
H), 3.62 (t, J = 6.9 Hz, 2H), 4.73 (d, J = 6.6 Hz, 2H), 5.68 (td, J = 15.0,
2
3
1
3
solid: R
f
0.30 (50% EtOAc/hexane); mp 108ꢀ109.5 °C; [α]
D
= +23.2
.6 Hz, 1H), 5.88 (td, J = 15.0, 6.6 Hz, 1H), 8.26 (br s, 1H); C NMR
) δ ꢀ5.05, 18.5, 26.2, 28.8, 32.1, 62.6, 70.1, 91.8,
23.5, 136.7, 162.8; HRMS (ESI+): calcd for C H Cl NO Si [M +
1
2 2 3
(
c 0.4, CH Cl ); H NMR (600 MHz, CDCl ) δ 0.04 (s, 3H), 0.05 (s,
(150 MHz, CDCl
3
3
3
H), 0.88 (s, 9H), 1.45 (s, 9H), 1.55ꢀ1.63 (m, 3H), 1.66ꢀ1.68 (m, 1H),
.62ꢀ3.65 (m, 2H), 4.08 (br s, 1H), 4.59 (br s, 1H), 5.29 (br s, 1H), 5.72
1
14
27
3
2
+
H] 374.0871, found 374.0859 (error 3.2 ppm).
R)-N-[6-(tert-Butyldimethylsilyloxy)hex-1-en-3-yl]-2,2,2-trichloro-
acetamide (11). To a solution of 10 (1.12 g, 3 mmol, 1.0 equiv) in
CH Cl (15 mL) at 23 °C was added [(R)-COP-Cl] (219 mg, 0.15
mmol, 0.05 equiv). The reaction was stirred at 38 °C for 36 h, then
cooled to 23 °C and concentrated in vacuo. Purification by flash
chromatograph afforded the title compound (1.04 g, 93%) as light
13
(d, J = 12 Hz, 1H), 5.93 (d, J = 12 Hz, 1H), 6.65 (br s, 1H); C NMR
(
(
5
C
150 MHz, CDCl
3
) δ ꢀ5.39, ꢀ5.37, 18.3, 25.9, 28.0 28.3, 32.5, 49.1,
2.8, 62.5, 79.8, 126.8, 126.9, 155.9, 168.7; HRMS (APCI+): calcd for
2
2
2
+
19
H
37
N
2
O
tert-Butyl [(3S,6R)-6-(3-Hydroxypropyl)-2-oxo-3,6-dihydropyridin-
4
Si [M + H] 385.2517, found 385.2518 (error 0.3 ppm).
3-nonyl]carbamate (S3). To a solution of 15 (57.6 mg, 0.15 mmol)
2
3
yellow oil: R
f
0.66 (10% EtOAc/hexane); [α]
D
= ꢀ5.0 (c 0.5, CH
2
Cl
2
);
in THF (5 mL) at 0 °C was added HF Py solution (0.5 mL) dropwise
3
1
H NMR (600 MHz, CDCl
3
) δ 0.04 (s, 6H), 0.88 (s, 9H), 1.55ꢀ1.59
over10 min and thereaction mixture stirred at0 °C for anadditional1.5 h.
(
m, 2H), 1.61ꢀ1.69 (m, 1H), 1.73ꢀ1.79 (m, 1H), 3.61ꢀ3.67 (m, 2H),
The solution was diluted with EtOAc (20 mL) and saturated aqueous
4
1
1
6
.43 (dt, J = 13.8, 6.9 Hz, 1H), 5.18 (d, J = 10.8 Hz, 1H), 5.23 (d, J =
3
NaHCO (10 mL) was added slowly to neutralize the mixture. The
7.4 Hz, 1H), 5.80 (ddd, J = 17.4, 10.8, 6.9 Hz, 1H), 6.66 (br d, J = 6.0 Hz,
aqueous layer was separated and extracted with EtOAc (3 ꢁ 10 mL).
13
H); C NMR (150 MHz, CDCl
3
) δ ꢀ5.3, 18.3, 25.9, 28.7, 30.8, 53.3,
The combined organic extracts were washed with saturated aqueous
2.3, 92.9, 116.0, 136.6, 161.2; HRMS (ESIꢀ): calcd for C H Cl NO
NaCl (30 mL), dried (MgSO
chromatograph afforded the title compound (30.3 mg, 75%) as colorless
4
) and concentrated. Purification by flash
14
25
3
2-
ꢀ
Si [M ꢀ H] 372.0726, found 372.0734 (error 2.2 ppm).
2
3
(
R)-6-(tert-Butyldimethylsilyloxy)hex-1-en-3-amine (12). To a solu-
oil: R
f
0.30 (10% MeOH/CH
2 2 D 2 2
Cl ); [α] = +8.0 (c 0.8, CH Cl );
1
tion of 11 (935 mg, 2.5 mmol, 1.0 equiv) in toluene (10 mL) at ꢀ78 °C,
H NMR (600 MHz, CDCl
1.83 (m, 1H), 2.17 (br s, 1H), 3.68 (br s, 2H), 4.12 (br s, 1H), 4.60 (br s,
3
) δ 1.46 (s, 9H), 1.63ꢀ1.67 (m, 3H), 1.77ꢀ
was added a solution of DIBAL-H (12.5 mL, 1 M in toluene, 12.5 mmol,
5
equiv) over 15 min. The solution was stirred for 1 h at ꢀ78 °C, then
1H), 5.35 (br s, 1H), 5.73 (d, J = 12 Hz, 1H), 5.93 (d, J = 12 Hz, 1H),
1
3
quenched with MeOH (0.5 mL). Next, EtOAc (25 mL) and Rochelle’s
salt solution (25 mL) were added and the reaction mixture was stirred at
6.98 (br s, 1H); C NMR (150 MHz, CDCl
3
) δ 27.5, 28.3, 32.1, 48.8,
52.9, 61.9, 79.9, 126.3, 127.1, 156.0, 169.4; HRMS (APCI+): calcd for
+
2
3 °C for 30 min. The aqueous layer was separated and extracted with
EtOAc (3 ꢁ 10 mL). The combined organic extracts were washed with
saturated aqueous NaCl (30 mL), dried (MgSO ) and concentrated.
Purification by flash chromatograph afforded the title compound (384
C
13
H
(3S,6R)-3-Amino-6-(3-hydroxypropyl)-3,6-dihydropyridin-2(3H)-
23
N
2
O
4
[M + H] 271.1652, found 271.1649 (error 1.1 ppm).
one Hydrochloride Salt (1). A solution of HCl in dioxane (1.5 mL, 3 M)
was added to S3 (54 mg, 0.2 mmol) at 0 °C and the solution stirred for 1
h at 0 °C, then the reaction was concentrated in vacuo. Recrystallization
4
2
3
mg, 67%) as colorless oil: R
f
0.15 (10% MeOH/CH
); H NMR (600 MHz, CDCl ) δ 0.04 (s, 6H), 0.88 (s,
H), 1.56ꢀ1.61 (m, 2H), 1.71ꢀ1.76 (m, 2H), 3.56ꢀ3.64 (m, 3H), 5.26
d, J = 10.2 Hz, 1H), 5.36 (d, J = 17.4 Hz, 1H), 5.84 (ddd, J = 17.4, 12.5,
2
Cl
2
); [α]
D
= ꢀ13.2
1
(
9
(
c 0.4, CH
2
Cl
2
3
2
with 1:20 EtOH/Et O (6 mL) afforded the title compound (37 mg,
2
3
88%) as an off-white solid: mp 182ꢀ183.5 °C; [α]
D
= ꢀ32 (c 0.5,
OD) δ 1.55ꢀ1.63 (m, 2H), 1.77ꢀ
1.81 (m, 2H), 3.58ꢀ3.62 (m, 2H), 4.24 (br s, 1H), 4.43 (br s, 1H), 5.93
1
MeOH); H NMR (600 MHz, CD
3
1
3
10.2 Hz, 1H); C NMR (150 MHz, CDCl
3
) δ ꢀ5.31, 18.3, 26.0, 28.7,
1
3
3
1.0, 54.4, 62.5, 118.6, 136.2; HRMS (ESI+): calcd for C12
H
28NOSi
(dt, J = 10.8, 2.4 Hz, 1H), 6.14 (dt, J = 10.8, 2.4 Hz, 1H); C NMR (150
MHz, CD OD) δ 28.4, 32.6, 54.7, 62.6, 70.1, 120.7, 133.1, 166.8; HRMS
+
[
M + H] 230.1935, found 230.1941 (error 2.6 ppm).
tert-Butyl ((S)-1-{[(R)-6-(tert-Butyldimethylsilyloxy)hex-1-en-3-
yl]amino}-1-oxobut-3-en-2-yl)carbamate (14). To a solution of 12
3
+
8 15 2 2
(ESI+): calcd for C H N O [M + H] 171.1128, found 171.1124
(error 2.3 ppm).
3
(687 mg, 3.0 mmol, 1.0 equiv) and (S)-N-Boc-vinylglycine 13 (600 mg,
Anti-TB activities in BioA-suppressed Mtb Mutant (bioA-TetON-1).
Mtb bioA-TetON-1 was grown at 37 °C in Sauton’s medium containing
1 μM biotin to an OD580 of 0.2 to 0.4, harvested by centrifugation,
washed twice with biotin-free Sauton’s medium, and diluted in fresh
biotin-free Sauton’s medium to an OD580 of ∼0.07. 50 μL of the
3.0 mmol, 1.0 equiv) in THF (20 mL) at 0 °C, was added NaHCO (756
3
mg, 9.0 mmol, 3.0 equiv) and DEPBT (2.69 g, 9.0 mmol, 3.0 equiv)
sequentially. The mixture was stirred at 0 °C for 1 h then warmed to 23 °C
and stirred an additional 48 h. The reaction was cooled down to 0 °C and
1
8199
dx.doi.org/10.1021/ja204036t |J. Am. Chem. Soc. 2011, 133, 18194–18201

Journal of the American Chemical Society
ARTICLE
bacterial culture were used to inoculate 96-well plates and 150 μL of
compound 1 were added to give final concentrations of 500 to 1.95 mM
using 2-fold serial dilutions. When added, anhydrotetracycline (ATC)
wasused at a concentrationof 10 ng/mL. Wells containing no compound
complex, red crystals were obtained by soaking the yellow, PLP-loaded
crystals in reservoir solution containing 15% PEG 400 and 10 mM 1 for
5 s before freezing in liquid nitrogen. For the postreaction complex,
colorless crystals were obtained by soaking the yellow, PLP-loaded
crystals in 2 mM 1 for 30 min at 23 °C followed by a 3-s soak in reservoir
solution containing 2 mM 1 and 15% PEG 400 and immediately freezing
in liquid nitrogen.
1
were used as controls. 96-well plates were incubated in a humidified
incubator at 37 °C. Optical densities were measured after 6 to 28 days.
Coupled Continuous Assay to Evaluate the Time-depen-
dent Inhibition of BioA. A. Assay Procedure. BioA (2 μM) was
combined with compound 1 (0ꢀ1 mM) in 2ꢁ reaction buffer (200 mM
Structure Determination. Diffraction data were collected at 100 K on
GM/CA-CAT beamline 23ID-D at the Advanced Photon Source in the
Argonne National Laboratory (Argonne, IL). Data were integrated and
3 2
bicine pH 8.6, 100 mM NaHCO , 2 mM MgCl , 0.005% Igepal CA-630,
19
1
0 mM ATP, 200 μM PLP) at 0, 10, 15, 20, 25, and 27.5 min, which
correspond to preincubation times of 30, 20, 15, 10, 5, and 2.5 min. At
0 min, 2.5 μL of these BioAꢀ1 incubation mixtures were added to 47.5 μL
of a reaction solution, resulting in a final concentration of 100 nM BioA,
scaled with d*TREK. The structure was solvedbymolecular replacement
using the program Phaser within CCP4 and PDB code 3BV0 as a search
2
0,21
3
model.
REFMAC5 and Coot, respectively.
7 were generated using the PRODRG server. Final refinement was
Initial refinement and model building was done using
22,23
Topology and parameter files for
2
4
3
1
20 nM BioD, 12.5 μM KAPA, 20 nM Fl-DTB, 185 nM streptavidin,
mM TCEP, 1 mM SAM in 1ꢁ reaction buffer in duplicate. Addition of
25
carried out using Phenix. The structures were evaluated using the
MOLPROBITY server. Images and figures were prepared using
PyMOL.
2
6
the substrates KAPA and SAM at these concentrations was shown to
prevent measurable binding of 1 or 2 to BioA over the time period used
to analyze the data. Reactions were read in a 384 well black plate
1
9,27
Coordinates and structure factors have been deposited at
the Protein Data Bank with accession codes of 3TFT (prereaction) and
3TFU (postreaction).
(Corning #3575) using an excitation of 485 nm, emission at 530 nm and
cutoff also at 530 nm. The initial velocities were determined as the time
to produce 70 nM dethiobiotin.
B. Data Processing. Inhibitor 1 showed time dependent inhibition of
BioA while 2 was not active under the assay condition. The residual activity
’ ASSOCIATED CONTENT
S
Supporting Information. Completed citation for ref 25,
curve for 1 shown in Figure 3A, which describes the initial velocity, v
i
as a
b
function of preincubation time, t, was fit to eq 1 by nonlinear regression
analysis using Graphpad Prism 4.0 to obtain values for kobs at each inhibitor
concentration:
characterization data for 2 and all synthetic intermediates, general
description of the coupled enzyme assays for the other transaminases
evaluated, cell cytotoxicity assays, MS/MS data of PLP-inhibitor
adduct, data collection and refinement statistics for BioA structural
characterization, figures of pre- and postreaction complex, and
kobst
½
Pꢂ ¼ vieꢀ
ð1Þ
1
13
H NMR and C NMR spectra for all compounds. This material
where P is the concentration of product formed at time t. The secondaryplot
of kobs as a function of inhibitor concentration, [I], was fit to eq 2 to provide
is available free of charge via the Internet at http://pubs.acs.org.
values for kinact and K
.0.
I
by nonlinear regression analysis using Graphpad Prism
’
AUTHOR INFORMATION
4
Corresponding Author
aldri015@umn.edu
kinact
kobs ¼
ð2Þ
1
þ KI=½Iꢂ
Present Addresses
Chemistry Advanced Laboratory' Sheda Science and Technol-
ogy Complex, P. M. B. 186 Sheda-Kwali, Abuja, FCT, Nigeria
#
MS/MS Analysis of PLP-inhibitor Adduct. A. Sample Pre-
paration. A solution of inhibitor 400 μM 1, 26 μM BioA, 50 mM
TrisꢀHCl (pH 8.0), 10 mM 2-mercaptoethanol and 100 μM PLP was
incubated at 37 °C for 2 h in a total volume of 100 μL. Excess inhibitor
and PLP were removed by gel-filtration chromatography on a Zeba
desalting column (Thermo Fisher). The columns were washed 3 times
with 300 μL 1 mM TrisꢀHCl buffer pH 7.5, after which 100 μL of the
reaction mixture was applied to the column and eluted by centrifugation.
The purified protein solution was diluted 10-fold with deionized H O.
2
B. MS Procedure and Data Analysis. Enzyme samples were intro-
duced into the ESI source by infusion with a syringe pump (120 μL/h).
’ ACKNOWLEDGMENT
This research was supported by a grant from the Bill and
Melinda Gates foundation and the Wellcome Trust through the
Grand Challenges in Global Health Initiative (to Douglas Young,
Imperial College), the National Institutes of Health (AI091790
to D.S. and C.C.A.) and the Intramural Research Program of the
NIAID, NIH (to C.E.B.). We thank Dr. Lorraine Anderson for
assistance in MS/MS experiments, Kathryn Gustafson for assis-
tance in purification and crystallization experiments, and Dr.
Anja Meißner for performing cytotoxicity assays. We are grateful
for resources from the University of Minnesota Supercomputing
Institute.
Optimal ESI conditions (source temperature 250 °C; drying gas (N )
2
flow rate 7 mL/min; neblulizing gas 7 psi; capillary voltage 400 V;
capillary offset 70 V; skimmer 27 V; octopole RF 120 Vpp) were based
10b
on similar studies by Mann et al.
For MS/MS experiments, the
observed quasimolecular molecular ion at m/z 400.4 was selected and a
collision energy of 40 was applied. The resulting MS/MS spectrum is
shown in Figure S2 (Supporting Information).
Structural Characterization of BioA 1 Complex. Crystal-
lization of BioA. Yellow crystals of BioA were grown within 24ꢀ48 h at
3
’ REFERENCES
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1
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dx.doi.org/10.1021/ja204036t |J. Am. Chem. Soc. 2011, 133, 18194–18201

Journal of the American Chemical Society
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