Avoiding Antibiotic Inactivation in Mycobacterium tuberculosis by Rv3406 through Strategic Nucleoside Modification

Article abstract of DOI:10.1021/acsinfecdis.8b00038

5′-[N-(d-biotinoyl)sulfamoyl]amino-5′-deoxyadenosine (Bio-AMS, 1) possesses selective activity against Mycobacterium tuberculosis (Mtb) and arrests fatty acid and lipid biosynthesis through inhibition of the Mycobacterium tuberculosis biotin protein ligase (MtBPL). Mtb develops spontaneous resistance to 1 with a frequency of at least 1 × 10^-7 by overexpression of Rv3406, a type II sulfatase that enzymatically inactivates 1. In an effort to circumvent this resistance mechanism, we describe herein strategic modification of the nucleoside at the 5′-position to prevent enzymatic inactivation. The new analogues retained subnanomolar potency to MtBPL (K_D = 0.66-0.97 nM), and 5′R-C-methyl derivative 6 exhibited identical antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an isogenic Rv3406 overexpression strain (minimum inhibitory concentration, MIC = 1.56 μM). Moreover, 6 was not metabolized by recombinant Rv3406 and resistant mutants to 6 could not be isolated (frequency of resistance <1.4 × 10^-10) demonstrating it successfully overcame Rv3406-mediated resistance.

Full text of DOI:10.1021/acsinfecdis.8b00038

Article
Cite This: ACS Infect. Dis. XXXX, XXX, XXX−XXX
pubs.acs.org/journal/aidcbc
Avoiding Antibiotic Inactivation in Mycobacterium tuberculosis by
Rv3406 through Strategic Nucleoside Modification
†
‡
†
†
‡
Matthew R. Bockman, Curtis A. Engelhart, Surendra Dawadi, Peter Larson, Divya Tiwari,
David M. Ferguson,† Dirk Schnappinger, and Courtney C. Aldrich*
‡
,†
†Department of Medicinal Chemistry, University of Minnesota, 308 Harvard Street SE, Minneapolis, Minnesota 55455, United States
‡Department of Microbiology and Immunology, Weill Cornell Medical College, 1300 York Avenue, New York, New York 10021,
United States
*
S Supporting Information
ABSTRACT: 5′-[N-(D-biotinoyl)sulfamoyl]amino-5′-deoxyadenosine (Bio-
AMS, 1) possesses selective activity against Mycobacterium tuberculosis (Mtb)
and arrests fatty acid and lipid biosynthesis through inhibition of the
Mycobacterium tuberculosis biotin protein ligase (MtBPL). Mtb develops
spontaneous resistance to 1 with a frequency of at least 1 × 10− by
overexpression of Rv3406, a type II sulfatase that enzymatically inactivates 1. In
an effort to circumvent this resistance mechanism, we describe herein strategic
modification of the nucleoside at the 5′-position to prevent enzymatic
inactivation. The new analogues retained subnanomolar potency to MtBPL
7
(
KD = 0.66−0.97 nM), and 5′R-C-methyl derivative 6 exhibited identical
antimycobacterial activity toward: Mtb H37Rv, MtBPL overexpression, and an
isogenic Rv3406 overexpression strain (minimum inhibitory concentration,
MIC = 1.56 μM). Moreover, 6 was not metabolized by recombinant Rv3406
and resistant mutants to 6 could not be isolated (frequency of resistance <1.4 ×
−10
1
0
) demonstrating it successfully overcame Rv3406-mediated resistance.
KEYWORDS: biotin protein ligase, Mycobacterium tuberculosis, tuberculosis, metabolism, adenylation, bisubstrate inhibitor
espite great advances in human medicine over the last
immune responses by complementary mechanisms while
glycolipids and lipoproteins have been implicated in modulating
adaptive immunity. Fatty acid degradation is also believed to
D
century, tuberculosis (TB), caused by Mycobacterium
1
5−7
tuberculosis (Mtb), remains a global health problem. In 2015,
TB overtook HIV as the leading source of infectious disease
mortality worldwide. Treatment of simple drug-susceptible TB
is very challenging, compared to most bacterial infections,
requiring 6−9 months of a four-drug regimen comprised of
isoniazid, rifampicin, ethambutol, and pyrazinamide. These
drugs were discovered over 40 years ago and remain the
cornerstone of TB chemotherapy. The underlying origin of the
persistence and drug tolerance of Mtb in vivo that necessitates
this long treatment course is still not fully understood but is
likely multifactorial. Considering the unique challenges posed
by drug susceptible TB, the emergence and dissemination of
multidrug resistant TB (MDR-TB) and extensively drug
resistant TB (XDR-TB), which are minimally resistant to the
two most effective antitubercular agents, isoniazid and
rifampicin, are a global health crisis.
be important, and several lines of evidence support the
hypothesis that mycobacteria are primarily lipolytic in vivo,
deriving sugars needed for nucleotide and cell wall biosynthesis
8
by breakdown of host fatty acids via gluconeogenesis. Biotin
protein ligase (BPL) in Mtb represents an extremely attractive
metabolic target since it globally regulates lipid metabolism
through the post-translational biotinylation of the three
nonredundant acyl CoA carboxylases (ACC1, ACC2, and
ACC3) involved in the synthesis of different malonyl coenzyme
A (CoA) building blocks used in mycobacterial lipid synthesis
as well as pyruvate CoA carboxylase, which catalyzes the first
9−11
step in gluconeogenesis.
Genetic silencing of Mycobacte-
rium tuberculosis biotin protein ligase (MtBPL) eliminated Mtb
from mice during both acute and chronic infections
demonstrating MtBPL’s essential role in Mtb survival and
Mycobacteria produce a tremendously diverse array of
12
persistence. Moreover, partial genetic inactivation of Mtb’s
ability to synthesize biotin was found to significantly enhance
clearance of Mtb from lungs and spleen by rifampicin serving to
lipophilic molecules ranging from simple short-chain fatty
2
,3
acids to the very long-chained mycolic acids. The mycolic
acids are essential components of the mycobacterial cell
envelope where they shield the bacterium from host-induced4
oxidative stress and contribute to intrinsic drug resistance.
Other lipids such as phthiocerol dimycocerosates (PDIMs),
phenolic glycolipids, and trehalose dimycolates limit innate
12
validate MtBPL as a vulnerable target.
Received: February 10, 2018
©
XXXX American Chemical Society
A
DOI: 10.1021/acsinfecdis.8b00038
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Figure 1. (A) Rv3406-mediated inactivation of Bio-AMS. (B) Proposed analogues to overcome metabolism by Rv3406.
Bisubstrate inhibitors have been described for the structurally
oxidation of commercially available 2′,3′-O-isopropylideneade-
24
related BPL from Staphylococcus aureus including alkylphos-
nosine 10 to give carboxylate 11, which was converted to 5′-
1
3
14
15,16
25
phates, β-ketophosphonates, and triazole nucleosides,
methyl ester 12 with diazomethane (Scheme 1). Grignard
as well as analogues wherein the adenosine nucleobase was
1
7,18
19
replaced with benzoxazoline
and aryl moieties. We have
Scheme 1. Synthesis of 5′-C,C-Dimethyladenosine
reported 5′-[N-(D-biotinoyl)sulfamoyl]amino-5′-deoxyadeno-
sine (Bio-AMS, 1, Figure 1A) is a potent subnanomolar
bisubstrate inhibitor of the mycobacterial biotin protein ligase
2
0
(
MtBPL). Bio-AMS possesses excellent antitubercular activity
against Mtb H37Rv and MDR/XDR-TB strains with minimum
inhibitory concentrations (MICs) ranging from 0.16 to 0.625
μM and is not affected by changes to the primary carbon
1
2,20−22
source.
Bio-AMS also has potent bactericidal activity6
similar to isoniazid and sterilized an Mtb culture of 5 × 10
1
2
colony forming units (CFU) within 3 weeks. Tiwari,
Schnappinger, and co-workers recently demonstrated the
most frequent mechanism of Bio-AMS resistance is caused by
overexpression of Rv3406, a type II sulfatase involved in the
scavenging of inorganic sulfates for metabolic and biosynthetic
2
2
purposes. Rv3406 was shown to oxidize the 5′-methylene
carbon of Bio-AMS to hemiaminal 2, which disproportionates
into biotinoyl sulfamide 3 and adenosine 5′-aldehyde 4 (Figure
1
A). Herein, we report the synthesis and biochemical and
biological evaluation of four Bio-AMS analogues designed to
circumvent Rv3406-mediated chemical inactivation. Our first
series of analogues sterically block oxidation by incorporation
of methyl groups at the 5′-C position of Bio-AMS (5−7, Figure
addition to 12 using methylmagnesium iodide at 40 °C
afforded 5′-C,C-dimethyl adenosine 13. Unfortunately, sulfa-
moylation of the tertiary alcohol under a variety of conditions
1
B) while our second set of compounds were conceived to
26
led to either no reaction or β-elimination (Table S3).
prevent disproportionation through replacement of the 5′-
amino group in Bio-AMS with a carbon atom (8, 9, Figure 1B).
We therefore opted to incorporate a single methyl group at
the 5′ position, which introduces a stereocenter, but we
expected may be sufficient to sterically block Rv3406-mediated
oxidation while not being susceptible to the undesired β-
elimination observed with tertiary alcohol 13. The 5′R-C-
methyl and 5′S-C-methyl analogues of 1 were conveniently
synthesized employing an asymmetric transfer hydrogenation
RESULTS
■
Our initial strategy focused on the synthesis of a 5′-gem-
dimethyl analogue of 1 lacking a C5′-hydrogen atom and thus
intrinsically resistant to Rv3406 metabolism. The synthesis
began by (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)
2
7
(ATH) reaction developed by Bligh and co-workers.
B
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Reduction of ketone 16 with chloro{[(1R,2R)-(−)-2-amino-
step, starting from protected nucleoside 21 in a two-step one-
pot procedure through oxidation and subsequent Horner-
Wittig olefination using N-Boc-diphenylphosphorylmethanesul-
fonamide to provide 22. Chemoselective deprotection of the
three tert-butyloxycarbonyl (Boc) protecting groups in the
presence of the isopropylidene was accomplished with 20%
1
,2-diphenylethyl](4-toluenesulfonyl)amido}(p-cymene)-
ruthenium(II) ((R,R)-TsDPEN) and chloro{[(1S,2S)-(−)-2-
amino-1,2-diphenylethyl](4-toluenesulfonyl)amido}(p-
cymene)ruthenium(II) ((S,S)-TsDPEN) catalysts afforded 5′R-
27
C-methyl 17 and 5′S-C-methyl 18, respectively (Scheme 2).
2
1
TFA in CH Cl2 to yield the vinyl sulfonamide 23.
2
21
Scheme 2. Synthesis of Both 5′R-C-Methyl-BioAMS (6) and
′S-C-Methyl-BioAMS (7) Analogues
Biotinylation and deprotection furnished 9 (Scheme 3).
5
Scheme 3. Synthesis of Vinyl Sulfonamide Bio-AMS (9)
Analogue
Both reactions yielded the secondary alcohols in 99%
diastereomeric purity, as confirmed by reverse-phase high
performance liquid chromatography (HPLC) analysis and
Each of the inhibitors was evaluated by isothermal titration
calorimetry (ITC) in a direct titration experiment with MtBPL.
However, the binding affinity was too high to determine, and
we simply observed stoichiometric titration of the protein. We
thus employed displacement ITC experiments, in which the
ligands were titrated into a solution of MtBPL prebound to
1
27
comparison to the reported H NMR spectra. Optimization
28
of our previously described sulfamoylation conditions using
sodium hydride as the base and freshly synthesized sulfamoyl
chloride in a 2:1 mixture of 1,2-dimethoxyethane (DME) and
acetonitrile provided 5′R-C-methyl 19 and 5′S-C-methyl 20
29
biotin to measure the apparent dissociation constants. The
true dissociation constants (K ) were subsequently determined
D
(Scheme 2). Biotinylation of sulfamates 19 and 20 employing
as described in the experimental section. The K values of 6−9
D
D-(+)-biotin N-hydroxysuccinimide ester (Biotin-NHS) medi-
ated by cesium carbonate furnished the corresponding
biotinylated adducts, which were sequentially deprotected
using saturated aqueous ammonia and 80% aqueous trifluoro-
acetic acid (TFA) to remove the N-benzoyl and isopropylidene
groups. Preparative reverse-phase HPLC purification afforded
the final biotinylated analogues 6 and 7 as the triethylammo-
nium salts in 33% and 22% yields, respectively, over the final
three steps. Both 6 and 7 were stable due to the increased steric
bulk at C-5′ and did not undergo cyclonucleoside formation
through nucleophilic attack of the N3 adenine atom onto C-5′,
which we have observed is a major decomposition pathway for
the corresponding nonmethylated sulfamate analogue (wherein
were nearly identical ranging from 0.66 to 0.97 nM (Table 1).
The results were further evaluated using the X-ray cocrystal
structure of 1 in MtBPL (PDB: 3rux). Model building
techniques were applied to examine the fit of the added
methyl groups of 6 and 7 within the active site of MtBPL
(Figure S6). The model shows the 5′-C-methyl groups project
toward Arg72 and Gly73 of an exterior loop region (Arg67−
Ala75). Given the experimental binding affinities of 1, 6, and 7
are nearly equivalent, it is fair to conclude that these potential
contacts have a minimal impact on ligand binding affinity,
which presumably is a result of the inherent flexibility of the
exterior loops.
The whole-cell antitubercular activity of each compound was
evaluated against wild-type (WT) Mtb (H37Rv), two Mtb
strains which overexpress MtBPL to different degrees (hereafter
referred to as MtBPL-H and MtBPL-M, described in our
1
2
20,21
R = R = H).
Bio-AMS avoids cyclonucleoside formation
by virtue of the poor nucleofugacity of the sulfamide compared
to the sulfamate at C-5′.
20
The synthesis of sulfonamide analogue 8 has been reported
previous work, Figure 2), and a Mtb mutant that highly
overexpresses Rv3406 (hereafter referred to as Rv3406-
overexpressor or Rv3406-OE). The parent compound 1 was
20,21
in our previous work.
The vinyl sulfonamide 9 was
synthesized in an analogous route avoiding the hydrogenation
C
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Table 1. MIC90 and Binding Data for Analogues 6−9
resistance. The activities of diastereomeric analogue 5′S-C-
methyl 7 and sulfonamide 8 both decreased 8-fold in potency
relative to 1 and, similar to 6, were equally active against WT
Mtb and Rv3406-OE. We have shown that subtle changes to
the ribose moiety of Bio-AMS resulted in decreased
mycobacterial accumulation, and we similarly expect the
reduced activities of 7 and 8 may also be due to this
phenomena. Among the four analogues studied, only vinyl
sulfonamide 9 was inactive against Rv3406-OE. Additionally, to
verify these compounds overcame Rv3406-mediated resistance,
22
we attempted to isolate resistant mutants to 6 by plating 7 ×
9
1
0
colony forming units (CFU) of Mtb on agar plates
containing concentrations of 6 at 4- and 10-fold its agar MIC.
We were unable to detect any colony growth after 9 weeks,
indicating the frequency of resistance (FOR) for 6 is less than
.4 × 10− at both 4- and 10-fold its agar MIC.
10
1
To biochemically verify the whole-cell results, each of the
analogues were incubated with 3 μM recombinant Rv3406 as
12
described and the parent molecules and potential metabolites
were analyzed by liquid chromatography tandem mass
spectrometry (LC-MS/MS). Under these conditions, the
time-dependent formation of 3 was readily observed. Rv3406
did not metabolize analogues 6−8. For 6 and 7, oxidation at the
5′ position was expected to provide the breakdown products N-
biotinoylsulfamic acid (S3) and 5′-adenosine-methyl ketone
a
Competitive and direct ITC experiments to determine KD and ΔG
were performed in triplicate. All analogues possessed n values of 1.0 ±
0
inhibitory concentrations (MIC) resulting in >90% growth inhibition
of M. tuberculosis H37Rv. MIC resulting in >90% growth inhibition of
M. tuberculosis Rv3406-OE. Experiments were performed twice
independently in triplicate. The MIC is defined as the lowest
concentration of inhibitors that prevented growth, as determined by
measuring the end point OD580 values. Based on a concentration ten
times the agar MIC of the compound tested. Approximately 10 cells
b
.2. The standard error of the K was ≤6.7% of the mean. Minimum
D
(
S4); however, neither of these were detected using authentic
c
standards of the metabolites (Scheme S1). Rv3406 catalyzed
oxidation of 8 was anticipated to produce a 5′-hydroxylsulfo-
namide Bio-AMS derivative (S5, Scheme S2) incapable of
disproportionation, but a metabolite relating to an M + 16 peak
was not identified. Compound 9 was the only analogue that we
expected could undergo oxidation. Indeed, incubation of 9 with
Rv3406 resulted in a time-dependent formation of an M + 16
metabolite tentatively assigned as epoxide S6 (Figure S3).
Rv3406 is a nonheme α-ketoglutarate-dependent Fe(II)
dioxygenase, and epoxidation by the Fe(IV)O active species
is highly plausible based on the established chemistry of these
d
8
9
were plated for 1 and 7 × 10 cells for 6. FOR was calculated as the
number of counted CFUs divided by the total number of bacteria
plated. Not determined.
e
active against WT Mtb possessing a MIC of 0.78 μM, but the
MIC increased more than 125-fold to >100 μM against
Rv3406-OE (Table 1). Compounds 1 and 6 showed a decrease
in the susceptibility of Mtb by more than 125-fold when MtBPL
was overexpressed at both moderate (MtBPL-M) and high
levels (MtBPL-H), demonstrating 1 and 6 both engage with
MtBPL in Mtb (Figure 2B,C). Consistent with their binding
affinity to MtBPL, analogues 6−9 maintained activity toward
WT Mtb, yet the MICs were reduced 2−8-fold relative to 1.
30,31
enzymes.
Unfortunately, we were unable to isolate the
metabolite S6 or synthesize an authentic standard. We
hypothesize epoxide ring-opening via nucleophilic water in
solution forming a sulfamoylated diol, S10, which could then
spontaneously decompose into N-biotinoylsulfuramidous acid
(
S7) and 5′-C-formyladenosine (S8) (Scheme S3B).
DISCUSSION
5
′R-C-Methyl 6 was the most potent analogue examined
■
against WT Mtb having a MIC of 1.56 μM. Remarkably,
Rv3406-OE was equally sensitive to 6 indicating introduction of
the 5′R methyl group fully overcame Rv3406-mediated
When examining Mtb clones resistant to 1, there was no
mutation in the target gene birA or to the promoter region.
Rather, the gene mutated in all resistant clones was rv3405c,
12
Figure 2. Susceptibility of Mtb [H37Rv (red circles), MtBPL-H (black triangles), and MtBPL-M (blue squares)] to (A) rifampicin (control), (B)
Bio-AMS (1), and (C) 5′R-C-methyl-Bio-AMS (6). Normalized growth was calculated as OD5 at the indicated concentration divided by OD580
80
without drug (DMSO). Experiments were run in triplicate.
D
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Figure 3. Proposed mechanism of Rv3406-mediate inactivation of 1. (Step 1) A molecule of α-ketoglutarate (α-KG) binds to the iron, displacing
two water molecules in the coordination sphere. (Step 2A) Substrate binds in the active site. (Step 2B) Substrate binding facilitates the coordination
of oxygen to the iron. (Step 3) A tandem oxidation/decarboxylation of the iron/α-KG occurs, respectively, forming a ferryl Fe(IV)O species.
(
Step 4) CO dissociates from the iron core, allowing for the highly reactive ferryl species to abstract a hydrogen atom from the substrate. (Step 5)
2
The ferryl Fe(IV)O species is consequently reduced to Fe(III). The alkyl radical reacts further with the iron species through hydroxyl radical
transfer, forming the sulfated hemiacetal or sulfamoylated hemiaminal intermediate and further reducing the iron core to Fe(II). (Step 6) Succinate
is displaced from the iron center by three water molecules. The sulfated hemiacetal or sulfamoylated hemiaminal intermediate dissociates (bold
arrow) and undergoes spontaneous disproportionation, affording an alkyl aldehyde and sulfate/sulfamide.
which is a member of the tetR family of DNA binding proteins.
Rv3405c and the orthologous protein in M. bovis have been
biochemically and biophysically characterized and shown to
bind to the intergenic region between rv3405c and rv3406
pathogenic bacteria are thought to utilize sulfatases in
scavenging inorganic sulfates from the environment, and
activity of sulfatases has been linked to several human
36
diseases.
32,33
leading to their repression.
Indeed, we observed the mRNA
Bertozzi and co-workers were the first to determine the
structure and function of Rv3406 and found Rv3406 was
essential for Mtb growth in sulfur-free media containing 2-
levels of rv3405c and rv3406 in the resistant isolates increased
12
1
0- and 190-fold, respectively. The Rv3406-OE strain (used
in this study) was constructed with an overexpression plasmid
pGMEH-Ptb38-rv3406) and found to have more than a 128-
2
3
ethylhexylsulfate (2-EHS) as the sole sulfur source.
(
Furthermore, the structure of Rv3406 shares a 54% sequence
fold increase in MIC to 1. Collectively, these data provide
strong evidence for the overexpression of Rv3406 as the
mechanism of resistance to 1.
identity and 66% sequence similarity with Pseudomonas putida
23
AtsK. The putative active sites of both enzymes are nearly
identical and share a nonheme, α-ketoglutarate-dependent
Fe(II) dioxygenases binding motif. Rv3406 possesses a typical
Rv3406 is a type II alkyl sulfatase. Currently, three known
classes of sulfatases have been identified. Type I sulfatases rely
on a post-translational modification to afford a catalytically
H-X-D/E-X -H iron-binding triad where X is any amino acid,
n
and an additional arginine residue is involved in α-ketoglutarate
34
37
active formylglycine residue for activity. These sulfatases are
binding. A proposed mechanism of Rv3406-mediated
−
known to cleave the RO−SO
bond, consuming one
cleavage of 2-EHS and 1 is shown in Figure 3.
3
equivalent of water through a sulfated hemiacetal intermediate.
Type III sulfatases utilize a Zn cofactor to activate a water
molecule for nucleophilic attack and cleave the same bond as
The oxidation mechanism is dependent on the proximity of
Fe(II) ion to the corresponding oxidized carbon. Molecular
dynamics simulations of 1, 6, and 7 in a model-built structure of
Rv3406 suggest this could be due to the failure of the enzyme
to turnover the 5′-C-methyl derivatives. On the basis of a
comparative analysis of MD simulations of all three ligands, it is
2
+
3
5
type I sulfatases. Curiously, type II alkyl sulfatases, which
utilize a Fe2 cofactor, are the only known enzyme in
+
−
23
prokaryotes capable of cleaving the R−OSO3 bond. Many
E
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evident that steric interactions of the added methyl group in 7,
with the coordinating residues of the Fe(II) ion (including the
conserved residue, histidine 97), forces the 5′ carbon to be
positioned further away from the reaction center (Figure 4).
Rv3406 are both nonessential proteins, which are co-opted by
Mtb to inactivate small molecules and illustrate the impressive
ability of Mtb to spontaneously develop antibiotic resistance.
CONCLUSION
■
We successfully synthesized three analogues of 1 that
maintained potent biochemical inhibition of MtBPL and were
not substrates for Rv3406 using an in vitro biochemical assay.
5
′R Methyl-Bio-AMS 6 emerged as the most potent compound
with an MIC of 1.56 μM against both WT Mtb and Rv3406-
OE. We were unable to isolate resistant mutants to 6 at neither
four nor ten times the agar MIC providing a frequency of
−10
resistance (FOR) of less than 1.44 × 10 compared to (0.1−
) × 10− for 1. These data along with the biochemical and
6
4
whole-cell activity, demonstrate modification of the nucleoside
at the 5′-position overcame Rv3406-mediated resistance.
Moreover, the low FOR of 6 indicates single step mutations
in Mtb are unlikely to confer resistance to 6. Overall, we believe
this work nicely illustrates how a detailed molecular under-
standing of antibiotic inactivation can be used to guide the
design of new analogues to overcome spontaneous resistance.
Figure 4. Simulated structure of 5′S-C-methyl Bio-AMS analog (7) in
Rv3406. (A) Protein surface of Rv3406 with ligand, Fe(II) ion, and α-
KG. Ligand 7 is solvent exposed and positioned unfavorably for
oxidation of the 5′ carbon to occur. (B) Close up view of active site.
The methyl sterically interferes with His97 and the Fe(II) ion.
This results in an unfavorable distance for hydrogen atom
abstraction (Figure 3, step 4). In the case of the 5′R-C-methyl
stereoisomer 6, the results are not as clear. Average distances
from the Fe(II) ion and the 5′ carbon were comparable for 1
and 6, suggesting the 5′ carbon is accessible to oxidation.
However, the simulations show the R-configured methyl of 6
METHODS
■
M. tuberculosis MIC Assays and MtBPL/Rv3406 Over-
expression Strain Cloning. MICs were experimentally
determined as previously described using M. tuberculosis
H37Rv grown in GAST medium [0.3 g/L Difco Bacto
Casitone, 4.0 g/L K HPO , 2.0 g/L citric acid, 1.0 g/L L-
alanine, 1.2 g/L MgCl ·6 H O, 0.6 g/L K SO , 2.0 g/L NH Cl,
8 mM NaOH, 1% (v/v) glycerol, and 0.005% (v/v) Tween-80
or 0.05% Tyloxapol] with an initial inoculum of 10 CFU/mL
in a 96- or 384-well plate. The plates were incubated at 37 °C
2
4
crowds the active site, which may block entry of the O , ending
2
2
2
2
4
4
the catalytic cycle. It is intuitive that further additions in the 5′
carbon region of 6 or 7 would only exacerbate this issue.
Interestingly, Cole and co-workers have reported over-
expression of Rv3406 conferred resistance to an unrelated
1
5
20
3
8
under 5% CO , and OD580 was measured after 14 or 18 days of
2
carboxyquinoxalines series of DprE1 inhibitors in Mtb.
−6
incubation to monitor growth. MIC90 was defined as the
concentration of the compound at which roughly 90% growth
inhibition was observed compared to the no drug control. To
study the resistance of these analogues toward degradation by
Rv3406, the MICs were determined using the previously
reported Mtb Rv3406 overexpression strain employing the
assay conditions described above. To confirm the engage-
ment of 1 and 6 by MtBPL, MICs were determined with the
MtBPL overexpression strain described previously using the
same assay as mentioned above.
MtBPL and Rv3406 Expression and Purification.
MtBPL and Rv3406 were cloned, overexpressed, and purified
as previously described.
Frequency of Resistance (FOR). The FOR for 6 was
performed as previously described for 1. Briefly, the MIC of 6
Mutants were recovered at frequency of 1 in 10 that is
1
2
approximately 10-fold greater than observed with 1. Whole-
genome sequencing mapped resistant mutants to different
nonsynonymous single nucleotide polymorphisms (SNP) or a
single base deletion in the rv3405c gene, which led to a 30- to
38
4
7-fold increase in transcription levels of rv3406. The
12
mechanism of inactivation of the carboxyquinoxalines by
Rv3406 occurs through decarboxylation leading to a bio-
logically inactive keto derivative. The carboxyquinoxalines thus
mimic the α-ketoglutarate substrate, whereas 1 mimics the alkyl
sulfate. The carboxyquinoxalines were inactivated at rates
20
−1
similar to 1 with rates ranging from 0.02 to 1.07 min versus
2
0,23
.015 min−1 for 1. The observation that overexpression of
0
Rv3406 leads to oxidative destruction of two unrelated
antitubercular agents is intriguing and further highlights the
importance of TetR family transcriptional regulators (TFTR) in
1
2
6
on agar plates was determined by spotting approximately 10
39−41
bacteria onto 7H10 agar plates supplemented with OADC and
varying drug concentrations. The lowest drug concentration
where no growth was observed upon visual inspection after
incubation at 37 °C for 3 weeks was used as the agar MIC. To
antibiotic resistance in mycobacteria.
Although a variety of resistance mechanisms have been
discovered for antitubercular agents, only a few examples of
transcriptional dysregulation have been described leading to
antibiotic inactivation. One recent example is Nathan and co-
workers’ discovery of a novel resistance mechanism involving
N-methylation by the SAM-dependent methyltransferase
9
isolate resistant mutants, approximately 7 × 10 bacteria were
plated onto 7H10 agar plates supplemented with OADC and
containing 6 at a concentration of 4× or 10× the agar MIC.
After incubation at 37 °C for 3 weeks, colonies were counted.
Plates without colonies were incubated for a further 6 weeks,
and a final count was done at the end of 9 weeks of incubation.
The initial number of bacteria was determined by plating
dilutions of the original culture. FOR was calculated as the
4
2
Rv0560c. Mutation to the transcriptional repressor rv2887,
a member of the MarR family of DNA binding proteins, led to a
more than 400-fold induction of rv0560c. Overexpression of
Rv0560c completely ablated the biochemical and whole-cell
activity of a pyrido-benzimiazole DprE1 inhibitor. Rv0560c and
F
DOI: 10.1021/acsinfecdis.8b00038
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
number of CFUs divided by the total number of bacteria plated
for each concentration of 6.
in 1 mL of 95:5 water/MeCN + 0.1% formic acid, and 100 μL
aliquots were used for LC-MS/MS analysis.
Isothermal Titration Calorimetry (ITC). All ITC experi-
ments were conducted on an automated microcalorimeter
Malvern Instruments). The experiments were performed at 25
C in ITC buffer (10 mM Tris, pH 7.5, 200 mM KCl, 2.5 mM
Incubations of 8 and 9 with Rv3406. All reactions were
performed in 100 μL at 37 °C. Complete assay contained 40
mM Tris acetate buffer, pH 7.5, 50 mM NaCl, 0.2% triton, 100
μM iron(II) chloride, 1 mM α-ketoglutarate (α-KG), 2 mM
ascorbate, and 250 μM substrate (either 8 or 9). Reactions were
started by the addition of 6.6 μL of Rv3406 (stock 1.517 mg/
mL), making the concentration of Rv3406 exactly 3 μM.
Reaction time points were run at 0 and 60 min and immediately
quenched with 300 μL of 10% aqueous TCA (trichloroacetic
acid) + 200 nM biotin (as an internal standard) to precipitate
the protein. All reactions were run in triplicate. Samples were
then centrifuged for 10 min at 15 000 rcf. The supernatant was
(
°
MgCl ). MtBPL was exchanged (2 × 10 mL) into ITC buffer
2
using an Amicon Ultra concentrator, and the final filtrate was
used to prepare a solution of the analogues from a 10 mM stock
in DMSO. Protein concentrations were 10 μM MtBPL
(
determined by active site titration with 1), 100 μM analogue,
and 100 μM biotin (determined by weighing sample on an
ultramicrobalance [Mettler Toledo] accurate to 0.001 mg). In
the direct titration experiments, the analogue was injected into
a solution of the enzyme. In the competitive titration
experiments, the analogue was injected into a solution of the
enzyme and biotin. Titrations were carried out with a stirring
speed of 750 rpm and 200 s interval between 4 μL injections.
The first injection for each sample was excluded from data
fitting. Titrations were run past the point of enzyme saturation
to correct for heats of dilution. The experimental data were
fitted to a theoretical titration curve using the Origin software
package (version 7.0) provided with the instrument to afford
diluted 1:20 in 95:5 H O/MeCN + 0.5% formic acid, and 100
2
μL aliquots were used for LC-MS/MS analysis.
LC-MS/MS Analysis. Samples were analyzed by LC-MS/
MS (Shimadzu UFLC XR-AB SCIEX QTRAP 5500). Reverse-
phase LC was performed on a Kinetix C18 column (50 mm ×
2.1 mm, 2.6 μm particle size; Phenomenex, Torrance, CA).
Mobile phase A was 0.1% aqueous formic acid while mobile
phase B was 0.1% formic acid in acetonitrile. Initial conditions
were 5% B from 0 to 0.5 min, after which the %B was increased
to 95% from 0.5 to 3 min. The column was washed in 95% B
for 2 min, returned to 5% over 0.2 min, and allowed to re-
equilibrate for 2.8 min in 5% B to provide a total run time of 8
min. The flow rate was 0.5 mL/min, and the column oven was
maintained at 40 °C. The injection volume was 10 μL. All
analytes from analogues 6 and 7 were analyzed by MS in
negative ionization mode by multiple reaction monitoring
app
values of KA (the apparent binding constant of the ligands in
−1
the presence of biotin in M ), n (stoichiometry of binding),
and ΔH (the binding enthalpy change in kcal/mol). The K
A
app
values for each ligand was obtained from the KA value using eq
1
:
app
B
A
KA = K (1 + K [B])
(1)
A
(
MRM), while all analytes from analogues 8 and 9 were
where [B] is the concentration of biotin, and KB is the
A
analyzed by MS in positive ionization mode by MRM. The
mass spectrometry settings were optimized by direct infusion of
authentic standards for 8, 9, S3, and biotin. The collision
energy ranged from +20 to +36 for positive mode and −40 for
negative mode. The declustering potential was +35 for positive
mode and −35 for negative mode. The following transitions
association constant for biotin experimentally determined to be
5
7
.29 × 10 . The thermodynamic parameters (ΔG and ΔS) were
calculated from K determined from the displacement titration
and ΔH from the direct binding titration using eq 2.
ΔG = −RTln(K) = ΔH − TΔS
(2)
+
were utilized: m/z 571.1 [M + H] → m/z 227.1 [biotin −
+
+
where ΔG, ΔH, and ΔS are the changes in free energy,
enthalpy, and entropy of binding, respectively; R = 1.98 cal
H O + H] , m/z 571.1 [M + H] → m/z 244.1, m/z 571.1 [M
2
+
+
+
+ H] → m/z 136.1 [adenine + H] for 8; m/z 571.1 [M + H]
−1
−1
+
+
mol K ; T is the absolute temperature (298 K). The affinity
of the ligands for the protein is given as the dissociation
constant (K = 1/K ). ITC experiments were run in triplicate
→ m/z 227.1 [biotin − H O + H] , m/z 571.1 [M + H] →
2
+
−
m/z 136.1 [adenine + H] for 9; m/z 322.1 [M − H] → m/z
+
196.1 for S3; m/z 587.1 [M + H] → m/z 227.1 [biotin − H O
2
D
A
+
+
and analyzed independently, and the thermodynamic values
were averaged.
+ H] , m/z 587.1 [M + H] → m/z 244.1, m/z 587.1 [M +
+
+
+
H] → m/z 136.1 [adenine + H] for S5; m/z 585.1 [M + H]
→ m/z 227.1 [biotin − H O + H] for S6; m/z 307.1 [M +
H] for S7; m/z 295.1 [M + H] for S8; m/z 603.1 [M + H]
→ m/z 227.1 [biotin − H O + H] for S10; m/z 245.1 [M +
H] → m/z 227.1 [M − H O + H] , m/z 243.1 [M − H] →
+
Incubations of 6 and 7 with Rv3406. Assay conditions
2
12
+
+
+
were adapted and modified from previous conditions with 1.
+
All reactions were performed in 100 μL at 37 °C. Complete
assay contained 40 mM Tris acetate buffer, pH 7.5, 50 mM
NaCl, 0.2% triton, 100 μM iron(II) chloride, 1 mM α-
ketoglutarate (α-KG), 2 mM ascorbate, and substrate (either 6
or 7). The concentrations of substrate were tested at 4, 2, 1, 0.5,
2
+
+
−
2
m/z 200.0 for biotin. Analyte and internal standard peak areas
were calculated (MultiQuant, version 2.0.2, AB SCIEX).
Analyte peak areas were normalized to internal standard peak
areas, and the analyte concentrations were determined with an
appropriate standard curve.
0
.25, 0.125, 0.0625, and 0 mM. Reactions were started by the
addition of 6.6 μL of Rv3406 (stock 1.517 mg/mL), making the
concentration of Rv3406 exactly 3 μM. Negative control
reactions with respect to protein lacked Rv3406 while positive
controls employed Bio-AMS as substrate. Both test and control
reactions were run in duplicate. Reactions incubated in a 37 °C
water bath; aliquots (30 μL) were withdrawn at 0 and 60 min
and immediately quenched with 300 μL of 4:1 MeOH/acetone
150 nM biotin (as an internal standard) to precipitate the
protein. Samples were then centrifuged in vacuo for 30 min at
000 rcf. The residue leftover after centrifugation was dissolved
General Materials and Methods. Chemicals and solvents
were purchased from Acros Organics, Alfa Aesar, Sigma-
Aldrich, and TCI America and were used as received. The
nucleoside 2′,3′-O-isopropylideneadenosine 10 was obtained
6
from Carbosynth (Berkshire, UK). N -Benzoyl-2′,3′-O-isopro-
43
pylideneadenosine, 15, N-Boc-diphenylphosphorylmethane-
4
4
45
+
sulfonamide, sulfamoyl chloride, D-(+)-biotin N-hydrox-
46
ysuccinimide ester, and 5′-[N-(D-biotinoyl)sulfamoyl]amino-
5′-deoxyadenosine triethylammonium salt (Bio-AMS), 1,
20
5
G
DOI: 10.1021/acsinfecdis.8b00038
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
Article
1
3
were prepared as described. An anhydrous solvent dispensing
system using two packed columns of neutral alumina was used
for drying THF and CH Cl , while two packed columns of
8.71 (s, 1 H); C NMR (100 MHz, CDCl ) δ 25.2, 26.0, 26.8,
3
82.7, 84.2, 91.6, 93.2, 114.5, 123.0, 127.9, 128.9, 133.0, 133.3,
142.5, 149.7, 151.0, 152.3, 164.5, 204.9; HRMS (ESI+) calcd
for C H N O Na [M + Na] , 446.1435; found, 446.1472.
2
2
+
molecular sieves were used to dry DMF, and the solvents were
dispensed under argon (Ar). Anhydrous grade MeOH, MeCN,
pyridine, and DMA were purchased from Aldrich. EtOAc and
hexanes were purchased from Fisher Scientific. All reactions
were performed under an inert atmosphere of dry argon (Ar) in
oven-dried (180 °C) glassware. TLC analyses were performed
on TLC silica gel plates 60F254 from EMD Chemical Inc. and
were visualized with UV light. Optical rotations values were
obtained on a polarimeter using a 1 dm cell. Purification by
flash chromatography was performed using a medium-pressure
flash chromatography system equipped with flash column silica
cartridges with the indicated solvent system. Preparative
reversed-phase HPLC purification was performed on a
Phenomenex Gemini 10 μm C18 250 × 20 mm column
operating at 21.0 mL/min with detection at 254 nm with the
indicated solvent system. Analytical reversed-phase HPLC was
performed on a Phenomenex Gemini 5 μm C18 250 × 4.6 mm
column operating at 1 mL/min with detection at 254 nm
employing a linear gradient from 5% to 50% MeCN in 50 mM
aqueous triethylammonium bicarbonate (TEAB) at pH 7.5 for
21
21
5
5
6
N -Benzoyl-2′,3′-O-isopropylidene-(5′R)-5′-C-methyl-
adenosine (17). A solution of sodium formate (6.8 g, 99.2
mmol) in H O (40 mL) was added to a 100 mL round-bottom
flask charged with 16 (1.00 g, 2.36 mmol, 1.0 equiv) and η -(p-
2
6
c y m e n e ) - ( R , R ) - N - t o l u e n e s u l f o n y l - 1 , 2 -
diphenylethylenediamine(1−)ruthenium(II) chloride (16 mg,
0.0236 mmol, 1 mol %). EtOAc (10 mL) was then added, and
reaction mixture was stirred for 2 d at 23 °C during which some
solid precipitated from the solution. After 2 d, reaction was
diluted with additional CH Cl (30 mL), and the aqueous layer
was separated. The organic layer was concentrated in vacuo to
afford a dark amber residue which was redissolved in EtOAc
(20 mL) and transferred to a beaker (100 mL). H O (10 mL)
was then added, and the layers were covered and stirred at 23
°C overnight. A white solid precipitated and was collected by
filtration, washed with MBTE (2 × 10 mL), and dried in vacuo
to afford the title compound (0.55 g, 55%) as a white solid: R =
0.33 (5% MeOH/CH Cl ); R = 0.12 (EtOAc); HPLC purity:
2
2
2
f
2
2
f
2
3
99.0%, t = 17.45 min, k′ = 3.30 (method B); [α] − 64.1° (c
R
D
1
30 min (Method A) or operating at 0.8 mL/min with detection
1.00, MeOH); H NMR (400 MHz, DMSO-d ) δ 1.04 (d, J =
6
at 260 nm employing a linear gradient from 30% to 70%
6.3 Hz, 3H), 1.35 (s, 3H), 1.56 (s, 4H), 3.67−3.82 (m, 1H),
3.98 (dd, J = 5.5, 2.7 Hz, 1H), 5.10 (dd, J = 6.3, 2.3 Hz, 1H),
5.18 (d, J = 4.3 Hz, 1H), 5.41 (dd, J = 6.3, 3.1 Hz, 1H), 6.26 (d,
J = 2.7 Hz, 1H), 7.55 (t, J = 7.4 Hz, 2H), 7.65 (t, J = 7.4 Hz,
1H), 7.98−8.14 (m, 2H), 8.68 (s, 1H), 8.77 (s, 1H), 11.25 (br
MeOH with 0.1% TFA in H O with 0.1% TFA for 29 min
2
1
13
(
Method B). H and C spectra were recorded on 400 or 500
MHz NMR spectrometers. Proton chemical shifts are reported
in ppm from an internal standard of residual chloroform (7.27),
methanol (3.31), or dimethyl sulfoxide (2.50); carbon chemical
shifts are reported in ppm from an internal standard of residual
chloroform (77.0), methanol (49.1), or dimethyl sulfoxide
s, 2H); 13C NMR (100 MHz, DMSO-d ) δ 19.7, 25.2, 27.1,
6
66.1, 80.4, 83.2, 89.4, 90.1, 113.1, 125.6, 128.45, 128.48, 132.4,
133.3, 143.2, 150.5, 151.8, 165.7; HRMS (ESI+) calcd for
+
(39.5). Proton chemical data are reported as follows: chemical
C H N O Na [M + Na] , 448.1591; found, 448.1585.
21
23
5
5
6
shift, multiplicity (s = singlet, d = doublet, dt = doublet of
triplets, t = triplet, q = quartet, pentet = pent, m = multiplet, ap
N -Benzoyl-2′,3′-O-isopropylidene-(5′R)-5′-C-methyl-
5′-O-sulfamoyladenosine (19). To a solution of 17 (0.15 g,
0.35 mmol, 1.0 equiv) in dimethoxyethane (2 mL) cooled to 0
°C was added sodium hydride (21 mg, 0.53 mmol, 1.5 equiv,
60% in mineral oil) in one portion. The reaction was stirred at
=
apparent, br = broad, ovlp = overlapping), coupling
constant(s), and integration. High-resolution mass spectra
were obtained on an LTQ Orbitrap Velos instrument (Thermo
Scientific, Waltham, MA). All compounds were determined to
be >95% by analytical reverse-phase HPLC (purities for each
final compound are given in the experimental section below).
0 °C until H evolution ceased and the mixture became a thick
2
slurry (40 min). To the reaction was then added dropwise a
solution of freshly prepared sulfamoyl chloride (0.12 g, 1.1
mmol, 3.0 equiv) in MeCN (2 mL). After 16 h, the reaction
was quenched with triethylamine/MeOH (1:1, 1 mL) and
concentrated in vacuo to afford an off-white gel. The product
was extracted with EtOAc (30 mL), successively washed with
6
N -Benzoyl-5′-deoxy-2′,3′-O-isoproylideneadenosine-
5
′-methylcarbonyl (16). To a solution of S18 (4.0 g, 8.54
mmol, 1.0 equiv) in THF (55 mL) cooled to −20 °C (10%
EtOH/ethylene glycol, dry ice bath) was added a solution of
methylmagnesium bromide in THF (3 M, 3.41 mL, 10.2 mmol,
H O (30 mL), and saturated aqueous NaCl (30 mL), then
2
1.2 equiv). The reaction was stirred for 30 min, followed by
dried (MgSO ), and concentrated to afford an off-white foam.
4
addition of another equivalent of methylmagnesium bromide in
THF (3 M, 3.13 mL, 9.39 mmol, 1.1 equiv). The reaction was
stirred at −20 °C for 4 h and then quenched carefully with
Purification by flash chromatography (50−100% EtOAc/
Hexanes, linear gradient) afforded the title compound (0.11
1
g, 62%) as a white solid: R = 0.49 (EtOAc); H NMR (500
f
saturated aqueous NH Cl (30 mL; caution: methane gas
MHz, MeOH-d ) δ 1.37 (d, J = 6.4 Hz, 3H), 1.41 (s, 3H), 1.63
4
4
evolution). Organic products were diluted with EtOAc (50
mL). Combined organic layers were washed successively with
(s, 3H), 4.20 (dd, J = 6.0, 3.5 Hz, 1H), 4.83−4.86 (m, ovlp with
HDO, 1H), 5.33 (dd, J = 6.4, 3.4 Hz, 1H), 5.47 (dd, J = 6.4, 2.7
Hz, 1H), 6.34 (d, J = 2.7 Hz, 1H), 7.52−7.62 (m, 2H), 7.63−
7.70 (m, 1H), 7.98−8.20 (m, 2H), 8.57 (s, 1H), 8.76 (s, 1H);
H O (50 mL), and saturated aqueous NaCl (50 mL), then
2
dried (MgSO ), and concentrated in vacuo to afford an off-
4
13
white foam. Purification by flash chromatography (0−5%
C NMR (125 MHz, MeOH-d ) δ 17.8, 25.7, 27.7, 78.0, 82.3,
4
MeOH−CH Cl stepwise gradient) afforded the title com-
85.3, 89.8, 91.6, 116.0, 125.5, 129.6, 129.9, 134.1, 135.1, 145.0,
151.5, 153.1, 153.7; HRMS (ESI+) calcd for C H N O SNa
2
2
pound (3.18 g, 88%) as a white foam: R = 0.37 (5% MeOH/
f
21 25
6
7
2
3
+
CH Cl ); R = 0.35 (EtOAc); [α] − 38.0° (c 1.00, MeOH);
H NMR (400 MHz, CDCl ) δ 1.43 (s, 3H), 1.64 (s, 3H), 1.96
[M + Na] , 528.1398; found, 528.1411.
2
2
f
D
1
3
(5′R)-5′-O-[N-(D-Biotinoyl)sulfamoyl]-5′-C-methylade-
nosine Triethylammonium Salt (6). To a solution of 19
(100 mg, 0.198 mmol, 1.0 equiv) and Cs CO (161 mg, 0.500
(
(
7
s, 3H), 4.69 (d, J = 2.7 Hz, 1H), 5.41 (d, J = 5.9 Hz, 1H), 5.59
dd, J = 6.3, 2.3 Hz, 1H), 6.27 (s, 1H), 7.46−7.58 (m, 2H),
2
3
.62 (d, J = 7.4 Hz, 1H), 8.04 (d, J = 7.4 Hz, 2H), 8.18 (s, 1 H),
mmol, 2.5 equiv) in DMF (2 mL) at 0 °C was added D-
H
DOI: 10.1021/acsinfecdis.8b00038
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
+)-biotin N-hydroxysuccinimide ester (102 mg, 0.297 mmol,
Article
(
DMSO-d ) δ 19.2, 25.3, 27.2, 66.3, 81.4, 83.5, 89.3, 89.6, 113.1,
6
1
.5 equiv). The reaction mixture was stirred for 16 h at 23 °C
125.6, 128.5, 132.4, 133.3, 143.1, 150.4, 151.7, 151.9, 165.6;
+
during which time all starting material was consumed as
monitored by TLC. DMF was removed in vacuo, redissolved in
MeOH−CH Cl (1:9, 5 mL), filtered through Celite, and
concentrated in vacuo to afford crude biotinylated nucleoside.
The crude product was used in the next deprotection reactions
directly without further purification.
HRMS (ESI+) calcd for C H N O Na [M + Na] , 448.1591;
21
23
5
5
found, 448.1614.
6
N -Benzoyl-2′,3′-O-isopropylidene-(5′S)-5′-C-methyl-
5′-O-sulfamoyladenosine (20). To a solution of 18 (0.20 g,
0.47 mmol, 1.0 equiv) in dimethoxyethane (2 mL) cooled to 0
°C was added sodium hydride (28 mg, 0.71 mmol, 1.5 equiv,
60% in mineral oil) in one portion. The reaction was stirred at
2
2
The crude material was dissolved in pyridine (1 mL), and
saturated aqueous ammonia (1 mL) was added. The reaction
was stirred at 23 °C in a sealed vessel. After 2 d, the reaction
was concentrated under high vacuum (P = 0.05 Torr). The
crude debenzoylated material was redissolved in 80% aqueous
trifluoroacetic acid (5 mL) and stirred for 2 h at 23 °C. The
crude material was then concentrated in vacuo and redissolved
in 1:1 MeCN/50 mM TEAB (10−20 mg/mL) and filtered to
remove insoluble solids. The resulting solution was purified by
preparative reverse phase HPLC with a Phenomenx Gemini
C18 (250 × 20 mm) column at a flow rate of 21.0 mL/min
employing a linear gradient of 5−50% acetonitrile (solvent B)
in 50 mM aqueous triethylammonium bicarbonate (TEAB) at
pH 7.0 (solvent A) for 30 min. The appropriate fractions were
pooled and lyophilized to afford the title compound (38 mg,
0 °C until H evolution ceased and the mixture became a thick
2
slurry (40 min). To the reaction was then added dropwise a
solution of freshly prepared sulfamoyl chloride (0.16 g, 1.4
mmol, 3 equiv) in MeCN (2 mL). After 3 h, the reaction was
quenched with triethylamine/MeOH (1:1, 1 mL) and
concentrated in vacuo to afford an off-white gel. The product
was extracted with EtOAc (30 mL), washed with H O (30
2
mL), saturated aqueous NaCl (30 mL), dried (MgSO ), and
4
concentrated to afford an off-white foam. Purification by flash
chromatography (50−100% EtOAc/Hexanes, linear gradient)
afforded the title compound (0.19 g, 80%) as a white solid: R =
f
1
0.49 (EtOAc); H NMR (500 MHz, MeOH-d ) δ 1.41 (s, 3H),
4
1.45 (d, J = 6.4 Hz, 3H), 1.63 (s, 3H), 4.36 (dd, J = 3.7, 3.1 Hz,
1H), 4.79 (qd, J = 6.5, 4.0 Hz, 1H), 5.16 (dd, J = 6.1, 2.7 Hz,
1H), 5.42 (dd, J = 6.1, 2.7 Hz, 1H), 6.41 (d, J = 2.7 Hz, 1H),
33% over 3 steps) as the triethylammonium salt (2.4 equiv of
Et N) as a white solid: HPLC purity: 99.0%, t = 13.30 min, k′
7.40−7.62 (m, 2H), 7.62−7.73 (m, 1H), 8.01−8.20 (m, 2H),
3
R
1
13
=
=
2.2 (method A); H NMR (500 MHz, MeOH-d ) δ 1.10 (t, J
8.59 (s, 1H), 8.74 (s, 1H); C NMR (125 MHz, MeOH-d ) δ
4
4
7.3 Hz, 28H, excess Et N), 1.20−1.24 (m, 1H), 1.42 (d, J =
17.4, 25.7, 27.7, 78.9, 83.4, 85.6, 89.9, 91.8, 115.6, 125.1, 129.6,
129.9, 134.1, 135.1, 144.4, 151.3, 153.51, 153.53, 168.4; HRMS
3
6
1
7
3
1
1
1
.4 Hz, 3H), 1.42−1.45 (m ovlp, 1H), 1.49−1.66 (m, 3H),
+
.66−1.79 (m, 1H), 2.19 (td, J = 7.5, 1.8 Hz, 2H), 2.69 (q, J =
(ESI+) calcd for C H N O SNa [M + Na] , 528.1398; found,
21
25
6
7
.2 Hz, 19H, excess Et N), 2.89 (dd, J = 12.7, 5.0 Hz, 1 H),
528.1411.
3
.16 (ddd, J = 8.3, 6.2, 4.7 Hz, 1H), 3.98 (dd, J = 4.7, 2.6 Hz,
H), 4.29 (dd, J = 7.9, 4.6 Hz, 1H), 4.46 (dd, J = 7.6, 4.3 Hz,
H), 4.49 (dd, J = 5.3, 2.6 Hz, 1H), 4.74 (dd, J = 6.9, 5.3 Hz,
(5′S)-5′-O-[N-(D-Biotinoyl)sulfamoyl]-5′-C-methylade-
nosine Triethylammonium Salt (7). To a solution of 20
(190 mg, 0.377 mmol, 1.0 equiv) and Cs CO (307 mg, 0.941
2
3
H), 6.03 (d, J = 7.0 Hz, 1H), 8.21 (s, 1H), 8.59 (s, 1H); 13
C
mmol, 2.5 equiv) in DMF (4 mL) at 0 °C was added D-
(+)-biotin N-hydroxysuccinimide ester (193 mg, 0.565 mmol,
1.5 equiv). The reaction mixture was stirred for 16 h at 23 °C
during which time all starting material was consumed as
monitored by TLC. DMF was removed in vacuo, and the
residue was redissolved in MeOH−CH Cl (1:9, 10 mL),
NMR (125 MHz, MeOH-d ) δ 10.9, 18.0, 27.3, 29.6, 30.1, 40.3,
4
4
1
1.2, 47.3, 57.1, 61.7, 63.4, 71.6, 75.6, 77.4, 88.5, 89.0, 120.4,
42.0, 151.2, 154.0, 157.4, 183.1; HRMS (ESI−) calcd for
−
−
C H N O S [M − Et NH] , 585.1555; found, 585.1516.
2
1
29
8
8
2
3
6
N -Benzoyl-2′,3′-O-isopropylidene-(5′S)-5′-C-methyl-
2
2
adenosine (18). A solution of sodium formate (6.8 g, 99.2
filtered through Celite, and concentrated in vacuo to afford
crude biotinylated nucleoside. The crude product was used in
the next deprotection reactions directly without further
purification.
mmol) in H O (40 mL) was added to a 100 mL round-bottom
2
6
flask charged with 16 (1.00 g, 2.36 mmol, 1.0 equiv) and η -(p-
cymene)-(S,S)-N-toluenesulfonyl-1,2-diphenylethylenediamine-
(
1−)ruthenium(II) chloride (16 mg, 0.0236 mmol, 1 mol %).
The crude material was dissolved in pyridine (1 mL), and
saturated aqueous ammonia (1 mL) was added. The reaction
stirred at 23 °C in a sealed vessel. After 2 d, the reaction was
concentrated under high vacuum (P = 0.05 Torr). The crude
debenzoylated material was redissolved in 80% aqueous
trifluoroacetic acid (5 mL) and stirred for 2 h at 23 °C. The
crude material was then concentrated in vacuo and redissolved
in 1:1 MeCN/50 mM TEAB (10−20 mg/mL) and filtered to
remove insoluble solids. The resulting solution was purified by
preparative reverse phase HPLC with a Phenomenx Gemini
C18 (250 × 20 mm) column at a flow rate of 21.0 mL/min
employing a linear gradient of 5−50% acetonitrile (solvent B)
in 50 mM aqueous triethylammonium bicarbonate (TEAB) at
pH 7.0 (solvent A) for 30 min. The appropriate fractions were
pooled and lyophilized to afford the title compound (56 mg,
22% over 3 steps) as the triethylammonium salt (1.5 equiv of
Et N) as a white solid: HPLC purity: 99.2%, t = 12.86 min, k′
EtOAc (10 mL) was then added, and the reaction mixture was
stirred at 23 °C during which some solid precipitated from the
solution. After 22 h, the reaction was diluted with additional
EtOAc (20 mL), and the aqueous layer was separated. The
organic layer was concentrated in vacuo to afford a dark amber
solid which was resuspended in EtOAc (12 mL) and heptane
(8 mL). The suspension was heated to 80 °C for 15 min and
slowly cooled to 23 °C with stirring during which a solid
precipitated. The solid was collected by filtration and dried in
vacuo to afford the title compound (0.61 g, 61%) as an off-white
solid: mp = 166−167 °C; R = 0.33 (5% MeOH/CH Cl ); R =
f
2
2
f
0
(
.12 (EtOAc); HPLC purity: 99.0%, t = 17.18 min, k′ = 3.23
R
23 1
method B); [α] − 44.9° (c 1.00, MeOH); H NMR (400
D
MHz, DMSO-d ) δ 1.11 (d, J = 6.3 Hz, 3H), 1.34 (s, 3H), 1.57
(
4
(
7
(
6
s, 3H), 3.71−3.89 (m, 1H), 4.07 (dd, J = 4.3, 3.1 Hz, 1H),
.97 (dd, J = 6.1, 2.9 Hz, 1H), 5.14 (d, J = 5.1 Hz, 1H), 5.34
dd, J = 6.3, 3.1 Hz, 1H), 6.28 (d, J = 3.1 Hz, 2H), 7.56 (t, J =
.6 Hz, 2H), 7.65 (t, J = 7.2 Hz, 1H), 7.99−8.11 (m, 2H), 8.75
3
R
1
= 2.5 (method A); H NMR (500 MHz, MeOH-d ) δ 1.15 (t, J
4
= 7.3 Hz, 9H, excess Et N), 1.23−1.30 (m, 1H), 1.46 (d, J = 6.4
3
1
3
s, 1H), 8.77 (s, 1H), 11.21 (s, 1H); C NMR (100 MHz,
Hz, 3H), 1.56−1.79 (m, 4H), 2.24 (td, J = 7.4, 2.6 Hz, 2H),
I
DOI: 10.1021/acsinfecdis.8b00038
ACS Infect. Dis. XXXX, XXX, XXX−XXX

ACS Infectious Diseases
.68 (d, J = 12.8 Hz, 1H), 2.79 (q, J = 7.3 Hz, 13H, excess
Article
2
AUTHOR INFORMATION
■
*
Et N), 2.86−2.98 (m, 2H), 3.15−3.23 (m, 1H), 4.10 (dd, J =
3
Corresponding Author
Phone: 612-625-7956. Fax: 612-626-3114. E-mail: aldri015@
2
7
1
8
9
6
.7, 1.8 Hz, 1H), 4.30 (dd, J = 7.9, 4.6 Hz, 1H), 4.46 (dd, J =
.9, 4.3 Hz, 1H), 4.50 (dd, J = 5.0, 2.9 Hz, 1H), 4.72−4.78 (m,
H), 4.85 (dd, J = 6.6, 1.7 Hz, 1H), 6.14 (d, J = 6.1 Hz, 1H),
umn.edu.
1
3
ORCID
.22 (s, 1H), 8.56 (s, 1H); C NMR (125 MHz, MeOH-d ) δ
4
David M. Ferguson: 0000-0001-6294-8057
Courtney C. Aldrich: 0000-0001-9261-594X
Author Contributions
.1, 16.7, 25.8, 28.0, 28.4, 38.6, 39.7, 45.9, 47.8, 48.1, 55.5, 60.2,
1.8, 71.6, 75.0, 75.4, 87.1, 87.8, 139.5, 149.7, 152.5, 181.7;
−
−
HRMS (ESI−) calcd for C H N O S [M − Et NH] ,
2
1
29
8
8
2
3
5
85.1555; found, 585.1599.
E ) - N - B i o t i n o y l - C - ( 5 ′ - a d e n o s y l i d e n e ) -
methanesulfonamide Triethylammonium Salt (9). To a
solution of 22 (21 mg, 0.055 mmol, 1.0 equiv) and Cs CO (54
The manuscript was written with contributions of all authors.
All authors have given approval to the final version of the
manuscript.
(
2
3
Notes
mg, 0.165 mmol, 3 equiv) in DMF (3 mL) at 0 °C was added
D-(+)-biotin N-hydroxysuccinimide ester (38 mg, 0.11 mmol, 2
equiv). The reaction mixture was stirred for 16 h at 23 °C
during which time all starting material was consumed as
monitored by TLC. DMF was removed in vacuo, and the
residue was redissolved in MeOH−CH Cl (1:9, 5 mL), filtered
through Celite, and concentrated in vacuo to afford crude
biotinylated nucleoside. The crude product was used in the next
deprotection reactions directly without further purification.
The crude coupled product was dissolved in 80% aqueous
trifluoroacetic acid (5 mL) and stirred for 2 h at 23 °C. The
crude material was then concentrated in vacuo and redissolved
in 1:1 MeCN/50 mM TEAB (10−20 mg/mL) and filtered to
remove insoluble solids. The resulting solution was purified by
preparative reverse phase HPLC with a Phenomenx Gemini
C18 (250 × 20 mm) column at a flow rate of 21.0 mL/min
employing a linear gradient of 5−50% acetonitrile (solvent B)
in 50 mM aqueous triethylammonium bicarbonate (TEAB) at
pH 7.0 (solvent A) for 30 min. The appropriate fractions were
pooled and lyophilized to afford the title compound (19 mg,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by a grant (AI091790 to D.S.) from
the National Institutes of Health as well as the University of
Minnesota Bighley Fellowship. Isothermal titration calorimetry
was carried out using an ITC-200 microcalorimeter, funded by
the NIH Shared Instrumentation Grant S10-OD017982. Mass
spectrometry was carried out in the Center for Mass
Spectrometry and Proteomics, University of Minnesota. We
also gratefully acknowledge Bruce Witthuhn for his contribu-
tions and assistance using the LC-MS/MS instruments. We also
thank Carolyn Bertozzi for providing the Rv3406 over-
expression plasmid. Finally, we acknowledge the Minnesota
Supercomputing Institute (MSI) at the University of Minnesota
for providing resources that contributed to the research results
reported within this paper (http://www.msi.umn.edu).
2
2
ABBREVIATIONS
■
ACCs, acyl CoA carboxylases; Bio-AMS, 5′-[N-(D-biotinoyl)-
sulfamoyl]amino-5′-deoxyadenosine; Bio-NHS, D-(+)-biotin N-
hydroxysuccinimide ester; TEMPO, (2,2,6,6-tetramethylpiper-
5
2% over 3 steps) as the triethylammonium salt (1.1 equiv of
1
Et N) as a white solid: H NMR (500 MHz, DMSO-d ) δ 0.89
3
6
(
3
t, J = 7.1 Hz, 9H, Et N), 1.01−0.96 (m, 1H), 1.30−1.20 (m,
3
6
idin-1-yl)oxyl; Boc, tert-butyloxycarbonyl; (R,R)-TsDPEN, η -
H), 1.48−1.34 (m, 3H), 1.61−1.54 (m, 1H), 1.89 (t, J = 7.4
(
p - c y m e n e ) - ( R , R ) - N - t o l u e n e s u l f o n y l - 1 , 2 -
Hz, 2H), 2.39 (q, J = 7.1 Hz, 6H, Et N), 2.56−2.50 (m, 1H),
3
diphenylethylenediamine(1−)ruthenium(II) chloride; (S,S)-
2
1
5
.78 (ddd, J = 12.4, 7.2, 5.1 Hz, 1H), 3.04 (dq, J = 6.1, 4.0 Hz,
H), 4.13−4.06 (m, 2H), 4.28−4.23 (m, 2H), 4.38 (ddd, J =
.9, 4.6, 1.5 Hz, 1H), 4.56 (t, J = 5.2 Hz, 1H), 5.91 (d, J = 5.2
6
TsDPEN, η -(p-cymene)-(S,S)-N-toluenesulfonyl-1,2-
diphenylethylenediamine(1−)ruthenium(II) chloride; HPLC,
high performance liquid chromatography; LC-MS/MS, liquid
chromatography tandem mass spectrometry; ITC, isothermal
titration calorimetry; MIC, minimum inhibitory concentration;
Mtb, Mycobacterium tuberculosis; MtBPL, Mycobacterium tuber-
culosis biotin protein ligase; TB, tuberculosis; TFA, trifluoro-
acetic acid; MDR-TB, multidrug resistant tuberculosis; XDR-
TB, extensively drug resistant tuberculosis
Hz, 1H), 6.31 (br s, 1H), 6.38 (dd, J = 15.4, 6.0 Hz, 1H), 6.46
(
s, 1H), 6.68 (dd, J = 15.3, 1.4 Hz, 1H), 7.29 (s, 2H), 8.11 (s,
1
3
1
2
8
1
H), 8.28 (s, 1H); C NMR (125 MHz, DMSO-d ) δ 11.7,
6
5.9, 28.1, 28.4, 39.8, 45.7, 55.5, 59.2, 61.0, 72.9, 73.7, 82.4,
7.3, 119.0, 132.8, 135.6, 139.5, 149.5, 152.7, 156.1, 162.8,
−
78.1; HRMS (ESI−) calcd for C H N O S [M −
3
2
1
29
8
8 2
−
Et NH] , 567.1450; found, 567.1454.
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