Novobiocin Enhances Polymyxin Activity by Stimulating Lipopolysaccharide Transport

Article abstract of DOI:10.1021/jacs.8b02283

Gram-negative bacteria are challenging to kill with antibiotics due to their impenetrable outer membrane containing lipopolysaccharide (LPS). The polymyxins, including colistin, are the drugs of last resort for treating Gram-negative infections. These drugs bind LPS and disrupt the outer membrane; however, their toxicity limits their usefulness. Polymyxin has been shown to synergize with many antibiotics including novobiocin, which inhibits DNA gyrase, by facilitating transport of these antibiotics across the outer membrane. Recently, we have shown that novobiocin not only inhibits DNA gyrase but also binds and stimulates LptB, the ATPase that powers LPS transport. Here, we report the synthesis of novobiocin derivatives that separate these two activities. One analog retains LptB-stimulatory activity but is unable to inhibit DNA gyrase. This analog, which is not toxic on its own, nevertheless enhances the lethality of polymyxin by binding LptB and stimulating LPS transport. Therefore, LPS transport agonism contributes substantially to novobiocin-polymyxin synergy. We also report other novobiocin analogs that inhibit DNA gyrase better than or equal to novobiocin, but bind better to LptB and therefore have even greater LptB stimulatory activity. These compounds are more potent than novobiocin when used in combination with polymyxin. Novobiocin analogs optimized for both gyrase inhibition and LPS transport agonism may allow the use of lower doses of polymyxin, increasing its efficacy and safety.

Full text of DOI:10.1021/jacs.8b02283

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Communication
Novobiocin enhances polymyxin activity by
stimulating lipopolysaccharide transport
Michael D. Mandler, Vadim Baidin, James Lee, Karanbir S. Pahil, Tristan W. Owens, and Daniel Kahne
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b02283 • Publication Date (Web): 10 May 2018
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Novobiocin enhances polymyxin activity by stimulating lipopolysaccha-
ride transport
Michael D. Mandler, ◊ Vadim Baidin,◊ James Lee, Karanbir S. Pahil, Tristan W. Owens, Daniel Kahne
Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA
Supporting Information Placeholder
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ABSTRACT: Gramꢀnegative bacteria are challenging to kill with
antibiotics due to their impenetrable outer membrane containing
lipopolysaccharide (LPS). The polymyxins, including colistin, are
the drugs of last resort for treating Gramꢀnegative infections. Theꢀ
se drugs bind LPS and disrupt the outer membrane; however, their
toxicity limits their usefulness. Polymyxin has been shown to
synergize with many antibiotics including novobiocin, which
inhibits DNA gyrase, by facilitating transport of these antibiotics
across the outer membrane. Recently, we have shown that novoꢀ
biocin not only inhibits DNA gyrase, but also binds and stimulates
LptB, the ATPase that powers LPS transport. Here, we report the
synthesis of novobiocin derivatives that separate these two activiꢀ
ties. One analog retains LptBꢀstimulatory activity but is unable to
inhibit DNA gyrase. This analog, which is not toxic on its own,
nevertheless enhances the lethality of polymyxin by binding LptB
and stimulating LPS transport. Therefore, LPS transport agonism
contributes substantially to novobiocinꢀpolymyxin synergy. We
also report other novobiocin analogs that inhibit DNA gyrase
better than or equal to novobiocin, but bind better to LptB and
therefore have even greater LptB stimulatory activity. These comꢀ
pounds are more potent than novobiocin when used in combinaꢀ
tion with polymyxin. Novobiocin analogs optimized for both gyꢀ
rase inhibition and LPS transport agonism may allow the use of
lower doses of polymyxin, increasing its efficacy and safety.
Figure 1. Proposed mechanisms for polymyxin and novobi-
ocin. A) The polymyxins bind lipopolysaccharide (LPS), a moleꢀ
cule that is translocated from the inner membrane to the outer
membrane by the sevenꢀmembered Lpt complex. Novobiocin
The increase in antibioticꢀresistant bacterial infections poses a
binds gyrase and inhibits DNA supercoiling. It also stimulates
major public health threat.1 Gramꢀnegative bacteria are particularꢀ
LPS transport by binding LptB. B) Structures of polymyxin B and
ly difficult to treat because they have an outer membrane that
colistin. C) Structure of novobiocin and derivatives used in this
prevents the entry of many antibiotics that kill Gramꢀpositive
study. Conditions: a) 5 M NaOH/H2O, 80 °C, 45 min. b) 1 M
NaOH/H2O, 50 °C, 45 min. c) 5ꢀmethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylic
anhydride (1.5 eq.), 10 mol % Sc(OTf)3, MeCN, 23 °C, 3 h. d)
organisms.2 Due to the spread of antibiotic resistance and a lack
of antibiotics effective against Gramꢀnegative organisms,3 colistin
NRRLꢀBꢀ3652 heavy cell suspension13, water, N2 atmosphere, 16
and polymyxin B, members of the polymyxin class of antimicroꢀ
bial peptides, have seen increased clinical use as drugs of last
h. e) cyclopropanecarbonyl chloride (0.8 eq.), pyridine, –40 °C,
resort.4 Although they are efficacious against nearly all Gramꢀ
16 h. f) 3ꢀ(naphthalenꢀ2ꢀyl)benzoyl chloride (0.8 eq.), pyridine, –
negative bacteria, they can cause irreversible kidney damage at
40 °C, 16 h. g) PhNCO (1.3 eq.), 2 mol % MoO2Cl2, DCM, 23 °C,
therapeutic doses.5 Therefore, strategies to lower the dosage of
12 h.14 See supporting information for full experimental details.
polymyxin required to clear resistant Gramꢀnegative infections are
We wondered whether the ability of novobiocin to stimulate
being actively sought.
LPS transport plays a role in its synergy with polymyxin. As noꢀ
vobiocin itself does have gyrase activity, synergy between 1 and
polymyxin may reflect that activity as well as LPS agonism. We
needed to separate the two activities of novobiocin to address
whether LPS transport agonism is crucial for synergy with polyꢀ
myxin.
Polymyxins disrupt the outer membrane by binding lipopolyꢀ
saccharide (LPS),6 which is largely responsible for barrier funcꢀ
tion (Figure 1A, 1B). Consequently, polymyxin has been investiꢀ
gated in combination therapy to increase the penetrance of antibiꢀ
otics that normally cannot cross the outer membrane.4b, 7 Novobiꢀ
ocin (Figure 1C), which inhibits DNA gyrase but generally exꢀ
hibits poor activity against Gramꢀnegative organisms,8 is one
We prepared a novobiocin analog, 2 (Figure 1C) lacking the Lꢀ
noviose sugar of 1, which is known to make important contacts
antibiotic shown to synergize with polymyxin (Figure S1, Table
with GyrB.15 An in vitro gyrase supercoiling assay revealed that
S1).9 It has been proposed that polymyxinꢀmediated disruption of
the activity of 2 against Acinetobacter baumannii gyrase was at
least 260ꢀfold lower than that of 1 (Figure 2A, S2). We chose A.
baumannii as our model strain because it is considered the highest
the outer membrane barrier allows more novobiocin to penetrate
into the cell and inhibit DNA gyrase.10 However, the basis of the
bactericidal mechanism behind this combination is poorly underꢀ
stood.9b We recently reported that novobiocin binds LptB,11 the
priority Gramꢀnegative pathogen to counteract according to the
World Health Organization.16 Consistent with a loss of gyrase
ATPase that powers the sevenꢀprotein LPS transport (Lpt) maꢀ
chine that delivers LPS to the cell surface (Figure 1A).12
activity, 2 does not have significant antibiotic activity even
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against Gramꢀpositive organisms that are susceptible to novobiꢀ
ocin (Table S2).
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After decoupling gyrase activity from 2, we wondered whether
2 could bind the LPS transporter. We developed a 19F NMR bindꢀ
ing assay to measure the affinity of 1 and 2 to the purified fiveꢀ
protein complex, LptB2FGC,12a, 12b, 17 that extracts LPS from the
inner membrane (Figure 1A). We prepared fluorinated derivaꢀ
tives of 1 and 2 (Figure S3) and we monitored changes in the 19
F
linewidths of these derivatives in response to increasing concenꢀ
trations of the LptB2FGC (Figure 2B). Both compounds bind
LptB2FGC with singleꢀdigit micromolar KD values. Therefore, 2
lacks gyrase activity but maintains affinity for its second target in
the inner membrane complex.
We have shown using a photocrosslinking assay12a that novobiꢀ
ocin 1 stimulates LPS transport11a by accelerating extraction of
LPS from the inner membrane and transfer to LptA,18 the protein
that forms the bridge to the outer membrane translocon.12c, 19 We
used this same assay (Figure 2C) to test if 2, like 1, stimulates
LPS transport. Compared to the DMSO control, 2 showed inꢀ
creased photocrosslinking to LptA containing a UVꢀcrosslinkable
amino acid (Figure 2D). Taken together, these results show that
this novobiocin derivative lacks gyrase activity but maintains LPS
transport activity.
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We next sought to test whether 2 would show synergy with
polymyxin B even though it does not inhibit DNA gyrase and has
no antibiotic activity. We carried out a checkerboard assay using
A. baumannii as a test strain. At concentrations of 2 that clearly
stimulate LPS release (Figure 2D), the minimal inhibitory conꢀ
centration of polymyxin B is reduced by at least fiveꢀfold despite
the compound having no antibiotic activity (Figure 2E). This
effect is independent of gyrase inhibition because compound 2
showed comparable synergy against an A. baumannii strain conꢀ
taining a mutation in gyrase that makes it insensitive to novobiꢀ
ocin (Figure S1). As 2 has no activity on its own even against
permeabilized strains20 (Figure S4), these results suggest that 2
potentiates the lethal effects of polymyxin B by acting as an agoꢀ
nist of LPS transport. That is, 2 potentiates the antimicrobial efꢀ
fects of polymyxin rather than viceꢀversa.
Figure 2. Polymyxin B-mediated killing by novobiocin does
not depend on its gyrase activity. A) 2 displays no inhibitory
activity against gyrase. Compounds were incubated with purified
A. baumannii gyrase, buffer, and relaxed pBR322 plasmid for 30
minutes at 37 °C. The supercoiling reaction was then quenched
and the DNA topoisomers were separated by gel electrophoresis
and stained. At higher than 80 µg/mL, 1 and 2 precipitate in the
gyrase assay buffer. B) 1F and 2F both display similar binding
affinities to LptB2FGC. Error bars represent the mean ± S.E.M (n
= 3). C) Schematic of LPS transport assay with rightꢀsideꢀout
(RSO) vesicles overexpressing LptB2FGC and purified LptA* =
LptA(I36pBPA). D) 2 retains activity on LptB2FGC. Compounds,
ATP, and LptA* were incubated with the vesicles for 1 hour at 30
°C prior to UV irradiation at 365 nm for 5 minutes. LPS x LptA*
crosslinks were detected by LPS immunoblots. E) 2 synergizes
with polymyxin B in a gyraseꢀindependent fashion. Checkerboard
broth microdilution assay against wildtype and gyraseꢀresistant A.
baumannii. The black squares denote concentrations of 2 that
reduce the polymyxin B concentration by at least fiveꢀfold. The
data are representative of a minimum of two biological replicates.
If the LPS transport stimulation activity of novobiocin plays a
role in the synergy observed with polymyxin B, we reasoned that
novobiocin analogs with improved LPS transport stimulation
activity should also improve the synergy with polymyxin B. We
screened other aminocoumarins for stimulation of LPS release
and discovered that the natural product clorobiocin was excepꢀ
tionally active (Figure S5). We wondered whether the 5ꢀmethylꢀ
pyrrolylcarbonyl group21 is the key moiety in clorobiocin responꢀ
sible for increased stimulation.
To further probe whether LPS transport stimulation activity
plays a critical role in the observed synergy, we examined the
crystal structure of LptB in complex with novobiocin to identify
positions likely to affect LptB2FGC binding. Replacing the benꢀ
zamide in 1 with a cyclopropylamide (4) (Figure 1C, S6) was
predicted to perturb the interaction with LptB residue R91,11a, 17
which is known to play a role in transducing information from the
ATPase to the transmembrane proteins LptF and LptG.11b, 17, 24
Although the activities of 4 and 1 against gyrase were similar, the
fluorinated probe 4F did not bind LptB2FGC (Figure 3B), and 4
did not stimulate LPS release (Figure 3C). Therefore, the LPS
stimulatory activity of novobiocin can be separated from gyrase
inhibitory activity by changing structural features of the benꢀ
zamide. Compound 4 did not synergize with polymyxin B against
either the sensitive or resistant strains, but interpreting this result
was complicated because, unlike 1 and 3, the compound showed
no activity against A. baumannii on its own.
Obtaining the pyrroleꢀmodified noviose 3 required selectively
performing an acylation reaction on one of four hydroxyl groups,
a challenge others have faced with this scaffold.21ꢀ22 We found
that Yamamoto’s Sc(OTf)3ꢀcatalyzed acylation method23 convertꢀ
ed descarbamyl novobiocin to 3 with high regioselectivity for the
equatorial Lꢀnoviose alcohol and no detectable acylation of the
phenolic hydroxyls (Figure 1C, ESI). 3 showed both improved
activity against gyrase and increased LPS transport stimulation
activity (Figure 3A, C). Consistent with the latter finding, the
fluorinated NMR probe 3F bound the LPS transporter with higher
affinity than 1F (Figure 3B). We found that 3 is superior to 1 in
its ability to reduce the lethal concentration of polymyxin B (Fig-
ure 3D). Notably, 3 showed synergy with polymyxin B against
gyraseꢀresistant as well as gyraseꢀsensitive strains, whereas 1 only
showed synergy against the gyraseꢀsensitive strain at the concenꢀ
trations tested. This finding suggests that the LPS stimulatory
activity plays an important role in the synergy.
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Figure 4. Novobiocin induced LPS release enhances polymyx-
in-mediated killing. A) 1 and 5 share the same gyrase activity in
vitro against A. baumannii gyrase. B) 5 is between 10ꢀ100X more
potent than 2 at stimulating LPS transport. C) Checkerboard broth
microdilution assay of 1-5 against gyraseꢀresistant A. baumannii.
Both gyrase inhibition and LPS transport stimulation appear to
play an important role in the synergy.
We have demonstrated that it is possible to improve the LPS reꢀ
lease agonism of novobiocin by making structural changes to the
noviose sugar and replacing the benzamide with a suitable group.
We have also shown that novobiocin derivatives that maintain
gyrase activity but are better LPS release agonists show greatly
improved synergy with polymyxin. For example, at 0.016 µg/mL
(~0.1X MIC), 3 reduces the amount of polymyxin B needed to kill
A. baumannii by 30ꢀfold (Figure 3D). Why increasing LPS
transport enhances the antibacterial activity of polymyxin remains
unclear, but studies to address this question are underway. We
now have a rational framework for the design of novobiocin anaꢀ
logs with both improved gyrase inhibition and LPS transport
stimulation activity. Coꢀadministration of polymyxin with novoꢀ
biocin analogs optimized for both activities could represent an
effective strategy to reduce polymyxin toxicity in the treatment of
recalcitrant Gramꢀnegative infections.
Figure 3. A novobiocin derivative with improved LPS
transport stimulating activity reduces the lethal concentration
of polymyxin B. A) 1 and 4 inhibit gyrase supercoiling at 0.63
µg/mL; 3 inhibits at 0.16 µg/mL. B) 1F and 3F bind to
LptB2FGC, while 4F does not. NMR binding experiments were
performed as in Figure 2. N/D (not determined). C) Substitution
of the carbamate in 1 for 5ꢀmethylꢀ1Hꢀpyrroleꢀ2ꢀcarboxylate in 3
increases LPS transport activity relative to 1. 4 does not stimulate
LPS release. D) 3 potentiates polymyxin B activity at least eightꢀ
fold better than 1 does.
We synthesized additional compounds, focusing on improving
the LPS stimulatory activity without changing gyrase activity.
This effort led to compound 5, which contains an aryl substituent
on the noviose carbamate and a rigidified benzamide substituent.
Compound 5 showed no change in gyrase activity compared with
1 (Figure 4A), but a 10ꢀ to 100ꢀfold increase in LPS release (Fig-
ure 4B). Like 2 and 4, compound 5 also had no antibiotic activity
against A. baumannii, but unlike 4, it showed synergy with polyꢀ
myxin B against both gyraseꢀsensitive and gyraseꢀresistant strains
(Figure 4, ESI). These results provide additional evidence that
LPS transport stimulation contributes to the synergy of novobiꢀ
ocin and some of its derivatives with polymyxin B.
AUTHOR INFORMATION
Corresponding Author
kahne@chemistry.harvard.edu
Present Addresses
Author Contributions
◊These authors contributed equally to the work.
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10.
Ofek, I.; Cohen, S.; Rahmani, R.; Kabha, K.; Tamarkin, D.;
Funding Sources
Herzig, Y.; Rubinstein, E., Antimicrob. Agents Ch. 1994, 38, 374.
11.
(a) May, J. M.; Owens, T. W.; Mandler, M. D.; Simpson,
ACKNOWLEDGMENT
B. W.; Lazarus, M. B.; Sherman, D. J.; Davis, R. M.; Okuda, S.;
Massefski, W.; Ruiz, N.; Kahne, D., J. Am. Chem. Soc. 2017, 139,
17221; (b) Sherman, D. J.; Lazarus, M. B.; Murphy, L.; Liu, C.;
Walker, S.; Ruiz, N.; Kahne, D., Proc. Natl. Acad. Sci. USA 2014,
111, 4982; (c) Sperandeo, P.; Cescutti, R.; Villa, R.; Di Benedetto, C.;
Candia, D.; Deho, G.; Polissi, A., J. Bacteriol. 2007, 189, 244.
This research was supported by the NIH (R01 GM066174, U19
AI109764, and R01 AI081059 to D.K.; F31GM116210 to J.L.),
NSF GRFP (DGEꢀ1144152 to M.D.M.), and the Blavatnik Bioꢀ
medical Accelerator at Harvard University. We wish to thank the
Dana Farber Cancer Institute / Harvard Medical School joint
NMR Core. We thank Dr. ShaoꢀLiang Zheng for his help with the
Xꢀray data collection and structure determination for 3.
12.
(a) Okuda, S.; Freinkman, E.; Kahne, D., Science 2012,
9
338, 1214; (b) Narita, S.; Tokuda, H., FEBS Lett. 2009, 583, 2160; (c)
Sperandeo, P.; Lau, F. K.; Carpentieri, A.; De Castro, C.; Molinaro,
A.; Deho, G.; Silhavy, T. J.; Polissi, A., J. Bacteriol. 2008, 190, 4460.
13.
14.
2309.
15.
e107289.
16.
carbapenemꢀresistant Enterobacteriaceae, Acinetobacter baumannii
and Pseudomonas aeruginosa in health care facilities. World Health
Organization: Geneva, 2017; (b) Willyard, C., Nature 2017, 543, 15.
17.
M.; May, J. M.; Trauger, S. A.; Kahne, D.; Ruiz, N., MBio 2016, 7, 1.
18. Tran, A. X.; Trent, M. S.; Whitfield, C., J. Biol. Chem.
2008, 283, 20342.
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REFERENCES
Sebek, O. K.; Hoeksema, H., J. Antibiot. 1972, 25, 434.
Stock, C.; Bruckner, R., Adv. Synth. Catal. 2012, 354,
1.
2.
Fair, R. J.; Tor, Y., Perspect. Medicin. Chem. 2014, 6, 25.
(a) Nikaido, H., Microbiol. Mol. Biol. Rev. 2003, 67, 593;
Stanger, F. V.; Dehio, C.; Schirmer, T., Plos One 2014, 9,
(b) Richter, M. F.; Drown, B. S.; Riley, A. P.; Garcia, A.; Shirai, T.;
Svec, R. L.; Hergenrother, P. J., Nature 2017, 545, 299.
(a) Guidelines for the prevention and control of
3.
(a) Rabanal, F.; Cajal, Y., Nat. Prod. Rep. 2017, 34, 886;
(b) Dijkmans, A. C.; Wilms, E. B.; Kamerling, I. M.; Birkhoff, W.;
OrtizꢀZacarias, N. V.; van Nieuwkoop, C.; Verbrugh, H. A.; Touw, D.
J., Ther. Drug Monit. 2015, 37, 419; (c) Kadar, B.; Kocsis, B.; Nagy,
K.; Szabo, D., Curr. Med. Chem. 2013, 20, 3759.
Simpson, B. W.; Owens, T. W.; Orabella, M. J.; Davis, R.
4.
(a) Falagas, M. E.; Kasiakou, S. K., Clin. Infect. Dis. 2005,
40, 1333; (b) Zavascki, A. P.; Goldani, L. Z.; Li, J.; Nation, R. L., J.
Antimicrob. Chemother. 2007, 60, 1206; (c) Nation, R. L.; Velkov, T.;
Li, J., Clin. Infect. Dis. 2014, 59, 88.
19.
(a) Freinkman, E.; Okuda, S.; Ruiz, N.; Kahne, D.,
Biochemistry 2012, 51, 4800; (b) Chng, S. S.; Gronenberg, L. S.;
Kahne, D., Biochemistry 2010, 49, 4565; (c) Wu, T.; McCandlish, A.
C.; Gronenberg, L. S.; Chng, S. S.; Silhavy, T. J.; Kahne, D., Proc.
Natl. Acad. Sci. USA 2006, 103, 11754; (d) Bos, M. P.; Tefsen, B.;
Geurtsen, J.; Tommassen, J., Proc. Natl. Acad. Sci. USA 2004, 101,
9417; (e) Sherman, D. J.; Xie, R.; Taylor, R. J.; George, A. H.;
Okuda, S.; Foster, P. J.; Needleman, D. J.; Kahne, D., Science 2018,
359, 798.
5.
(a) Kelesidis, T.; Falagas, M. E., Expert Opin. Drug Saf.
2015, 14, 1687; (b) Justo, J. A.; Bosso, J. A., Pharmacotherapy 2015,
35, 28.
6.
7.
Schindler, M.; Osborn, M. J., Biochemistry 1979, 18, 4425.
(a) Hogg, G. M.; Barr, J. G.; Webb, C. H., J. Antimicrob.
Chemother. 1998, 41, 494; (b) GiamarellosꢀBourboulis, E. J.;
Xirouchaki, E.; Giamarellou, H., Diagn. Microbiol. Infect. Dis. 2001,
40, 117; (c) Zusman, O.; Avni, T.; Leibovici, L.; Adler, A.; Friberg,
L.; Stergiopoulou, T.; Carmeli, Y.; Paul, M., Antimicrob. Agents
Chemother. 2013, 57, 5104; (d) Timurkaynak, F.; Can, F.; Azap, O.
K.; Demirbilek, M.; Arslan, H.; Karaman, S. O., Int. J. Antimicrob.
Agents 2006, 27, 224; (e) Gordon, N. C.; Png, K.; Wareham, D. W.,
Antimicrob. Agents Chemother. 2010, 54, 5316; (f) Cerny, G.;
Teuber, M., Arch Mikrobiol 1971, 78, 166; (g) Sud, I. J.; Feingold, D.
S., Antimicrob. Agents Ch. 1975, 8, 34; (h) Greenfield, S.; Feingold,
D. S., J Infect Dis 1970, 121, 555; (i) Rosenthal, K. S.; Storm, D. R.,
J. Antibiot. 1977, 30, 1087.
20.
Morris, C. M.; George, A.; Wilson, W. W.; Champlin, F.
R., J. Antibiot. 1995, 48, 67.
21.
C. T.; GarneauꢀTsodikova, S., Biochemistry 2007, 46, 8462.
22.
61.
23.
Fridman, M.; Balibar, C. J.; Lupoli, T.; Kahne, D.; Walsh,
Olson, S. H.; Slossberg, L. H., Tetrahedron Lett. 2003, 44,
Ishihara, K.; Kubota, M.; Kurihara, H.; Yamamoto, H., J.
Org. Chem. 1996, 61, 4560.
24.
(a) Ruiz, N.; Gronenberg, L. S.; Kahne, D.; Silhavy, T. J.,
Proc. Natl. Acad. Sci. USA 2008, 105, 5537; (b) Luo, Q.; Yang, X.;
Yu, S.; Shi, H.; Wang, K.; Xiao, L.; Zhu, G.; Sun, C.; Li, T.; Li, D.;
Zhang, X.; Zhou, M.; Huang, Y., Nat. Struct. Mol. Biol. 2017, 24,
469; (c) Dong, H.; Zhang, Z.; Tang, X.; Paterson, N. G.; Dong, C.,
Nat. Commun. 2017, 8, 222.
8.
Gellert, M.; O'Dea, M. H.; Itoh, T.; Tomizawa, J., Proc.
Natl. Acad. Sci. USA 1976, 73, 4474.
9.
(a) Jorge, P.; PerezꢀPerez, M.; Rodriguez, G. P.; Pereira,
M. O.; Lourenco, A., Int. J. Antimicrob. Agents 2017, 49, 668; (b)
MacNair, C. R.; Stokes, J. M.; Carfrae, L. A.; FiebigꢀComyn, A. A.;
Coombes, B. K.; Mulvey, M. R.; Brown, E. D., Nat. Commun. 2018,
9, 458.
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