Total Syntheses of Bulgecins A, B, and C and Their Bactericidal Potentiation of the β-Lactam Antibiotics

Article abstract of DOI:10.1021/acsinfecdis.8b00105

The bulgecins are iminosaccharide secondary metabolites of the Gram-negative bacterium Paraburkholderia acidophila and inhibitors of lytic transglycosylases of bacterial cell-wall biosynthesis and remodeling. The activities of the bulgecins are intimately

Full text of DOI:10.1021/acsinfecdis.8b00105

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Featured Article
Total Syntheses of Bulgecins A, B and C and Their
Bactericidal Potentiation of the #-Lactam Antibiotics
Shusuke Tomoshige, David A Dik, Masaaki Akabane-Nakata, Chinedu
Madukoma, Jed F. Fisher, Joshua D. Shrout, and Shahriar Mobashery
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.8b00105 • Publication Date (Web): 01 May 2018
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Total Syntheses of Bulgecins A, B and C and Their Bactericidal
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Potentiation of the β-Lactam Antibiotics
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Shusuke Tomoshige,† David A. Dik,† Masaaki AkabaneꢀNakata,† Chinedu S. Madukoma, ‡ Jed F. Fishꢀ
er,† Joshua D. Shrout, ‡,¶ Shahriar Mobashery†,*
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† Department of Chemistry & Biochemistry, University of Notre Dame, 352 McCourtney Hall, Notre
Dame, Indiana 46556, United States
‡ Department of Civil & Environmental Engineering & Earth Sciences, University of Notre Dame, 156
Fitzpatrick Hall, Notre Dame, Indiana 46556, United States
¶ Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, United States
* mobashery@nd.edu
The bulgecins are iminosaccharide secondary metabolites of the Gramꢀnegative bacterium Para-
burkholderia acidophila and inhibitors of lytic transglycosylases of bacterial cellꢀwall biosynthesis and
remodeling. The activities of the bulgecins are intimately intertwined with the mechanism of a coꢀ
biosynthesized βꢀlactam antibiotic. βꢀLactams inhibit the penicillinꢀbinding proteins, enzymes also critꢀ
ical to cellꢀwall biosynthesis. The simultaneous loss of the lytic transglycosylase (by bulgecin) and penꢀ
icillinꢀbinding protein (by βꢀlactams) activities results in deformation of the septal cell wall, observed
microscopically as a bulge preceding bacterial cell lysis. We describe a practical synthesis of the three
naturally occurring bulgecin iminosaccharides and their mechanistic evaluation in a series of microbioꢀ
logical studies. These studies identify potentiation by the bulgecin at subꢀminimum inhibitory concenꢀ
trations of the βꢀlactam against three pathogenic Gramꢀnegative bacteria, and establish for the first time
that this potentiation results in a significant increase in the bactericidal efficacy of a clinical βꢀlactam.
Keywords: Bacteria, Cell Wall, Lytic Transglycosylases, Antibiotic Resistance, βꢀLactam Antibiotics
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The appearance of extensively drugꢀresistant bacterial infections in both the hospital and the
community has sustained debate as to whether the era of successful monochemotherapy of bacterial inꢀ
fections is fading. An alternative approach, with precedent in cancer chemotherapy, is the combination
of complementary agents. The combination of an antibiotic with another potentially synergistic antibiꢀ
otic, or with a compound that would potentiate the activity of the antibiotic, is attractive in the light of
the dearth of clinical options. While such combinations can have extraordinary medical value (as exemꢀ
plified by the combination of βꢀlactams with βꢀlactamase inhibitors),1–4 every aspect to the selection and
implementation of successful combination therapy is a scientific challenge.5–10 With respect to this chalꢀ
lenge, useful guidance may be provided by Nature.
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Imada et al.,11, 12 of the antibiotic discovery group at Takeda, reported the isolation of bulgecins
A–C (1–3 of Figure 1) in 1982. Each is a simple variation on a glycosylated iminosaccharide, and each
biosynthesized by a Gramꢀnegative bacterium (annotated then as Pseudomonas mesoacidophilia, but
recently reassigned as Paraburkholderia acidophila).13, 14 Additional studies with this bacterium by
Imada et al. led to the isolation of a monobactamꢀclass βꢀlactam antibiotic, sulfazecin (4).15, 16 Combinaꢀ
tion of bulgecin (itself bereft of antibacterial activity) with sulfazecin significantly improved the latter’s
antibacterial activity toward other Gramꢀnegative bacteria.17, 18 All doubts that this potentiation was forꢀ
tuitous were dispelled by the observation of an intimate connection of the genes encoding the biosynꢀ
thetic enzymes for sulfazecin and bulgecin.14, 19, 20 Hence, Nature selected coꢀproduction of an antibacteꢀ
rial and a potentiator within the same producer organism.
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Figure 1. Chemical structures of the bulgecins and of sulfazecin. Bulgecins are glycopeptides comꢀ
prised of a common βꢀsulfoꢀGlcNAc linked to a disubstituted Lꢀproline. Bulgecin A is the most abunꢀ
dant of the bulgecin natural products. Sulfazecin is a monobactam βꢀlactam antibiotic that is structurally
distinguished by the sulfamate functional group.
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The morphological effect resulting from the complementarity of these two agents is a characterꢀ
istic midꢀcell bulge (hence “bulgecin”) that precedes lysis of the bacterium. A biochemical basis for this
effect was provided by Templin et al.,21 who confirmed the original observations of Imada et al. that the
bulgecin effect extended to other βꢀlactam antibiotics, and determined that the bulge was a result of
bulgecin inhibition of a periplasmic enzyme, the Slt lytic transglycosylase. As optimal potentiation with
bulgecin was found with βꢀlactams that inactivated the penicillinꢀbinding proteins (PBPs) of septal cellꢀ
wall formation, the hypothesis was presented that the simultaneous loss of the catalytic activity of these
PBPs by βꢀlactams, and of the lytic transglycosylase(s) (LTs) by bulgecin, results in a structurally deꢀ
fective cellꢀwall septum. This hypothesis has experimental support.22
The lytic transglycosylases of Gramꢀnegative bacteria share key attributes with the PBPs. Both
are found as families of enzymes within Gramꢀnegative bacteria. For example, the Gramꢀnegative pathꢀ
ogen Pseudomonas aeruginosa has eight different PBPs and eleven distinct LTs. Within each family
there is redundancy of function, but inability to tolerate overall loss of all function. In comparison to the
PBPs (where there is a credible hypothesis for the function of each family member) the LT family is
both more structurally and more functionally diverse. At present within the LT family there are few
guiding hypotheses as to the function of its individual members.23, 24 The understanding of LT function
is (even 25 years after their discovery) a frontier at the nexus of bacterial shape, bacterial cellꢀwall bioꢀ
synthesis, and antibiotic mechanism.
Given our ambitions to explore critically this nexus,24–28 access to bulgecins (which have proven
invaluable for LT study)29–32 was required and we opted for total synthesis. We report here practical
syntheses of bulgecins A, B and C. Bulgecin A was accomplished in 34 steps and 3% overall yield from
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Dꢀserine as the starting material. Furthermore, we revisit the biological activities of these compounds by
modern methodology to disclose the unique potentiating activity of bulgecins.
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RESULTS and DISCUSSION
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Synthesis of the Bulgecins. Bulgecin A (1) is a glycopeptide comprised of three segments: a sulfoꢀ
GlcNAc saccharide, a 3Rꢀhydroxyꢀ4Sꢀ(hydroxymethyl)ꢀLꢀproline (numbering from Cα) core (named
bulgecinine), and a taurine in amide linkage to the bugecinine core. Bulgecin A is the most abundant of
the three bulgecins found as natural products (Figure 1: bulgecin B has a βꢀalanine amide replacing the
taurine; bulgecin C lacks amide functionalization of its proline carboxylate: structures 2 and 3, respecꢀ
tively). While several syntheses of bulgecinine were reported in the decades following the discovery of
the bulgecins, only two syntheses of bulgecins have been reported. The first synthesis reported was that
of bulgecin A by a group from Osaka University (without experimental details).33 Their synthesis was
followed by the synthesis (with experimental details) of bulgecin C by Barrett and Pilipauskas.34, 35 The
key decision points for our own synthetic plan (as guided by the useful disclosures in these previous
syntheses) were the selection of a concise and highꢀyielding preparation of a protected bulgecinine, and
the choice of a protected and activated GlcNAc glycosyl donor for the βꢀselective glycosylation. In both
respects our own choices diverged from those of Wakamiya et al.33, 36 and Barrett and Pilipauskas.34, 35
For the synthesis of protected bulgecinines, we were drawn to a classical method for the stereꢀ
oselective synthesis of substituted pyrrolidines, that of a intramolecular aminomercuration of a γꢀalkene
to a carbamateꢀprotected amine, followed by the retentive O2ꢀinduced oxidative cleavage of the interꢀ
mediate carbonꢀmercury bond to give a secondary alcohol.37 The value of this methodology for the synꢀ
thesis of bulgecinine was established by Khalaf et al.38 and confirmed by Wang et al.39 In this route the
single stereocenter of the Dꢀserine starting material sets the absolute stereochemistry of the two addiꢀ
tional stereogenic carbons of the pyrrolidine. We therefore envisioned 5 as the key intermediate, disasꢀ
sembled retrosynthetically into the known GlcNAc donor 6 and the protected bulgecinine 7, which in
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turn would be synthesized from Dꢀserine (9) through Wang’s trisubstituted pyrrolidine 8. The synthetic
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plan is shown in Scheme 1.
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Scheme 1. Synthetic plan for bulgecin A (1)
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BnO
AcO
BnO
TCAHN
O
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HO
HO3SO
HO
BnO
AcO
BnO
Glucosamine — HCl
O
O
NH
CCl3
O
O
O
O
6
SO3H
CO2Me
NHAc
NHTCA
N
HN
N
HO
AcO
O
H
Cbz
CO2Me
OH
Bulgecin A (1)
OTBS
HO
OH
N
N
OH
5
NH2
DꢀSerine (9)
Cbz
Cbz
OTBS
OTBS
7
8
The GlcNAc donor 6 was prepared using a reported procedure.40 Allyl carbamate 10 (Scheme 2)
was prepared as described by Wang et al.39 Its transformation to the protected pyrrolidine 8 (with conꢀ
firmation of its structure by Xꢀray crystallographic analysis; see box in Scheme 2 and Figure S1), by
successive aminomercuration and oxidative demercuration with oxygen gas, used a modified adaptation
of Wang’s procedure. Selective oxidation of the primary alcohol of 8 to the carboxylic acid used the seꢀ
quential combination of DessꢀMartin and Pinnick oxidations, with the resulting carboxylic acid transꢀ
formed to the methyl ester (11) with TMSCHN2. Zemplenꢀtype removal of the Oꢀacetyl group afforded
the key protected bulgecinine derivative 7.
Scheme 2. Synthesis of 5 from D-serine (9)a
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AcO
RO
c. DMP
d. NaClO2
O
OAc OTBS
a. Hg(OAc)2
7 steps
g. 6, TMSOTf
CO2Me
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5
HO
OH
N
N
OH
b. O2, NaBH4
39% for 2 steps
e. TMSCHN2
63% for 3 steps
NH2
DꢀSerine (9)
45% for 6 steps
NHCbz
10
Cbz
8
Cbz
OTBS
OTBS
11: R = Ac
f. K2CO3
7: R = H (96%)
l. DMP
m. NaClO2
n. TMSCHN2
AcO
HO
OH OH
OAc OBn
j. Hg(OAc)2
p. 6, TMSOTf
h. BnBr
CO2Me
5
N
N
OH
71%
i. Ac2O
k. O2, NaBH4
o. K2CO3
NHCbz
NHCbz
Cbz
14
Cbz
15
OBn
OBn
85% for 2 steps
72% for 2 steps
46% for 4 steps
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13
9
(mixture of isomers)
(>96% purity)
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AcO
AcO
s. K2CO3
31%
q. TBAF
93%
r. BTCA
90%
CO2Me
N
CO2Me
11
15
N
Cbz
Cbz
OH
OBn
Xꢀray structure of 8
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aReagents and conditions: (a) Hg(OAc)2, MeCN, 0 ºC, 1.5 h; EtOAc, brine, 2 h; (b) NaBH4, O2, DMF, 3
h; (c) DMP, DCM, 0 ºC to rt, O/N; (d) 2 M 2ꢀmethylꢀ2ꢀbutene in THF, tꢀBuOH, NaClO2ꢀNaH2PO4 in
H2O, 3 h; (e) 2 M TMSCHN2 in hexane, toluene, MeOH, 2 h; (f) K2CO3, MeOH, 1.5 h, 96%; (g) 6,
TMSOTf, MS4A, DCM, 0 ºC, 3 h; (h) BnBr, Ag2CO3, toluene, O/N; (i) Ac2O, pyridine, 0 ºC to rt, O/N;
(j) Hg(OAc)2, MeCN, 0 ºC to rt, O/N; EtOAc, brine, 1.5 h; (k) NaBH4, O2, DMF, 3 h; (l) DMP, DCM, 0
ºC to rt, 1.5 h; (m) 2 M 2ꢀmethylꢀ2ꢀbutene in THF, tꢀBuOH, NaClO2ꢀNaH2PO4 in H2O, 1 h; (n) 2 M
TMSCHN2 in hexane, toluene, MeOH, 1.5 h; (o) K2CO3, MeOH, 1 h; (p) 6, TMSOTf, MS4A, DCM, 0
ºC, 3 h; (q) 1 M TBAF in THF, AcOH, O/N; (r) BTCA, TfOH, DCM, O/N; (s) K2CO3, MeOH, 3 h.
Glycosylation of 7 with GlcNAc donor 6 using TMSOTf activation gave a complex mixture of
byproducts. Presuming that the TBS protecting group is not preserved under the acidic conditions, we
decided to use the Oꢀbenzyl bulgecinine derivative 15 as the acceptor (Scheme 2). We derived the necꢀ
essary Oꢀbenzylated allylcarbamate 13 by selective benzylation of the primary alcohol, followed by
acetylation of diol 12. Aminomercuration and successive oxidative demercuration gave 14. The yield
(72%) was comparable to the yield reported previously.39 Oxidation and protecting group manipulation
of 14 (as described for 7) gave 15. To confirm that the stereochemistry of 15 is identical to that of 7, we
also synthesized 15 from 11 (desilylation with TBAF, followed by Oꢀbenzylation using benzyl trichloꢀ
roacetimidate with TfOH activation; third row of Scheme 2). In contrast to the glycosylation of 7, glyꢀ
cosylation of 15 was a clean reaction that proceeded with satisfactory yield (71%).
The reductive dechlorination (Bu3SnH, AIBN) of the nonꢀparticipating Nꢀtrichloroacetyl group
of 5 was uneventful, giving 18 in a 91% yield (Scheme 3). Mindful of the possibility of Cα epimerizaꢀ
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tion of the bulgecinine core, milder saponification conditions (compared to previous reports)35 were
used(aq KOH in iPrOH). The resulting carboxylic acid 19 was converted to Oꢀsuccinimidyl active ester
20 and its secondary alcohol sulfated to afford 21. The overall yield for these four steps from 5 was exꢀ
cellent (67%). The remaining two steps provided challenge. Acyl transfer to taurine occurred in moderꢀ
ate yield (52%), in part due to the difficult isolation and purification of 22.
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Scheme 3. Synthesis of bulgecin A (1)a
BnO
AcO
BnO
BnO
AcO
BnO
BnO
HO
BnO
a. Bu3SnH
AIBN
O
O
O
O
O
O
b. KOH aq.
87%
c. NHS, DIC
93%
CO2Me
CO2Me
CO2H
NHTCA
NHAc
NHAc
91%
N
N
N
Cbz
Cbz
Cbz
OBn
5
OBn
18
OBn
19
BnO
RO
BnO
BnO
HO3SO
HO
O SO
O
O
O
Na
f. HCO2NH4,
Pd
3
O
O
O
e. Taurine
52%
O
O
BnO
HO
CO2Su
SO3H
SO3 Na
NHAc
NHAc
OBn
22
NHAc
OH
N
N
N
H
HN
HN
89%
Cbz
Cbz
— 2TEA
OBn
20: R = H
21: R = SO3H (91%)
Bulgecin A (1)
d. SO3—py.
aReagents and conditions: (a) Bu3SnH, AIBN, toluene, rt to 90 ºC, O/N; (b) 4 M KOH aq., iꢀPrOH, 3 d;
(c) NHS, DIC, DCM, 0 ºC to rt, O/N, (d) SO3·py., DMF, 2 h; (e) taurine, TEA, H2O, MeCN, O/N; (f)
HCO2NH4, Pd black, MeOH, O/N.
In both previous syntheses of bulgecins difficulties were encountered with the hydrogenolysis
required for the final deprotections.33–35 We confirmed this difficulty. In our hands as well, transferꢀ
hydrogenation conditions, as identified by Barrett and Pilipauskas, was the superior choice. However,
while this procedure in our hands achieved full deprotection to bulgecin A (1), the sample had 15% of a
byꢀproduct that could not be removed without substantial loss of product. Based on an observation that
ammonium formate was a superior reducing agent compared to formic acid under transferꢀ
hydrogenation conditions (increased yield of Oꢀbenzyl deprotection and reduced impurity levels),41 we
finally obtained pure bulgecin A (1) in excellent yield (89%) for this last step. We further observed that
use of Na+ꢀform cationꢀexchange resin in the reaction workꢀup was superior to the use of H+ꢀform
strongly acidic cationꢀexchange resin. This latter resin also gave an impurity. These observations sugꢀ
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gest that bulgecin A is not stable to strong acid. Bulgecin A was obtained from Dꢀserine as the starting
material in 34 synthetic steps and with a 3% overall yield. Syntheses of bulgecins B and C were accomꢀ
plished from the intermediates used in the synthesis of bulgecin A (Scheme 4).
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Scheme 4. Synthesis of bulgecin B (2) and C (3)a
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BnO
HO3SO
BnO
BnO
HO3SO
HO
NaO3SO
HO
b. HCO2NH4,
Pd
O
O
O
O
O
O
a. βꢀalanine
O
O
BnO
CO2Su
CO2H
CO2 Na
NHAc
NHAc
NHAc
100%
N
N
N
49%
HN
HN
H
Cbz
Cbz
— 2TEA
OBn
21
OBn
23
OH
Bulgecin B (2)
BnO
HO
BnO
BnO
HO3SO
BnO
HO
O3SO
HO
d. HCO2NH4,
Pd
O
O
O
O
c. SO3—py.
82%
O
O
CO2H
CO2H
CO2H
NHAc
NHAc
NHAc
N
N
N
79%
H2
Cbz
Cbz
OBn
19
OBn
OH
24
Bulgecin C (3)
aReagents and conditions: (a) βꢀalanine, TEA, H2O, DCM, O/N; (b) HCO2NH4, Pd black, MeOH, O/N;
(c) SO3·py., DMF, 3 h; (d) HCO2NH4, Pd black, MeOH, O/N.
Coupling of 21 with βꢀalanine gave precursor 23, which was converted quantitatively into
bulgecin B (2). Compound 24 was synthesized by Oꢀsulfation of 19. Transfer hydrogenation using amꢀ
monium formate gave bulgecin C (3) in good yield (79%).
Bulgecin A potentiation with a clinical β-lactam antibiotic as assessed by bacterial growth-curves.
We assessed bulgecin A, B, and C in microbiological assays. We performed growthꢀcurve assays14, 31
for P. aeruginosa. These experiments were conducted in the presence of 2ꢀ to 8ꢀfold below minimalꢀ
inhibitory concentration (subꢀMIC) of the βꢀlactam antibiotic ceftazidime, in the absence of ceftazidime,
in the presence of bulgecin (A, B, or C), and in the presence of bulgecin (A, B, or C) and subꢀMIC of
ceftazidime (Figure S2). Each synthetic bulgecin analog demonstrated comparable ability to potentiate
ceftazidime against P. aeruginosa. For this reason, we continued our additional analyses with bulgecin
A.
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Figure 2. Bacterial growthꢀcurve assays and bacterial bulgeꢀformation as visualized by microscopy. (a)
Growthꢀcurve assays of P. aeruginosa, E. aerogenes and A. baumannii. The results for no antibiotic
(yellow curve), 50 ꢁg/mL bulgecin A (green curve), ceftazidime (CAZ; blue curve) and 50 ꢁg/mL
bulgecin A + CAZ (red curve) are shown. The subꢀMIC concentrations of CAZ used for P. aeruginosa,
E. aerogenes and A. baumannii were 0.28 ꢁg/mL, 8 ꢁg/mL, and 2 ꢁg/mL, respectively. (b) Confocal
microscopy demonstrating that GFPꢀlabeled P. aeruginosa grown in the presence of subꢀMIC of CAZ
exhibits elongation without bulges (on the left) and those grown in the presence of CAZ and bulgecin A
exhibit elongation with bulges (on the right). The boxed area is expanded to highlight the bulges. A 10ꢀ
ꢁm scale bar is given in the top left corner. (c) In cellꢀwall homeostasis, lipid II is polymerized to the
nascent peptidoglycan chain, comprised of repeats of the Nꢀacetylglucosamine (NAG, lightꢀgreen hexaꢀ
gons)ꢀNꢀacetylmuramylpentapeptide (NAM, darkꢀgreen hexagons) disaccharide, by the transglycosylase
(TG). The transpeptidase (TP) crosslinks the nascent peptidoglycan to the growing edge of the cell wall.
(d) βꢀLactam antibiotics inhibit the TP activity, resulting in the accumulation of nonꢀcrosslinked nascent
peptidoglycan, which serves as substrate for the lytic transglycosylase Slt in P. aeruginosa. Bulgecin A
inhibits the Slt activity. Dual inhibition of Slt and TP results in the formation of the bulge, leading to the
breached structural integrity of the cell wall.22
Next, we performed growthꢀcurve assays for the four Gramꢀnegative members of the ESKAPE
panel of bacterial pathogens (“KAPE”: Klebsiella pneumoniae, Acinetobacter baumannii, P. aerugino-
sa, and Enterobacter species). The ESKAPE panel members comprise the most problematic bacterial
pathogens.42–45 Figure 2 documents the potentiation of the ceftazidime activity for P. aeruginosa, Enter-
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obacter aerogenes, and A. baumannii. The results for K. pneumoniae indicated no potentiation (data not
shown), possibly implying lack of penetration of bulgecin A into the periplasmic space of this organism.
The bacteria grew in the absence of the antibiotic, in the presence of subꢀMIC of ceftazidime, or in the
presence of bulgecin A by itself. However, potentiation by bulgecin A was seen at subꢀMIC of
ceftazidime for these three bacteria (Figure 2A). In the case of P. aeruginosa, bulgecin A caused rapid
cell lysis once bacteria reached the midꢀlogarithmic phase of growth (approximately OD600 = 0.5) in the
presence of ceftazidime. In contrast, bulgecin A and subꢀMIC of ceftazidime caused sustained and early
inhibition of growth of E. aerogenes and A. baumannii.
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We visualized these events using a PAO1 strain of P. aeruginosa containing a miniꢀTn7 chroꢀ
mosomal, constitutive GFPꢀexpressing gene insertion, which fluoresces upon excitation at 488 nm with
light emission at 525 nm. We performed swarmꢀmotility assays (as described previously).46, 47 Bulgecin
A was combined with P. aeruginosa planktonic culture and spot inoculated at the plate center and
ceftazidime was placed at a second site 20 mm away. These placements allowed bacteria to be exposed
to bulgecin A prior to swarming towards ceftazidime. A control experiment omitted bulgecin A. Bacteꢀ
ria were imaged at the swarm colony edge. We saw the elongated phenotype in both cases due to bacteꢀ
rial exposure to subꢀMIC of ceftazidime.48, 49 However, in the presence of bulgecin A, we saw extensive
and periodic bulge formation in elongated bacteria. The bacteria in this group also demonstrated an inꢀ
crease in girth. In the control experiment in the absence of bulgecin A, these bulges were absent (Figure
2B). The formation of bulges was documented previously by Imada and Kintaka by scanning electron
microscopy and is the origin of the name of the natural product.11, 18 The bulges are at the sites of cell
division (midꢀcell), whereby failure of the organism to divide in the presence of ceftazidime is coupled
with the formation of the bulges (Figure 2B). We also showed that cells grown in the presence of
bulgecin A, but without antibiotic, displayed no alteration in the phenotype (not shown). This observaꢀ
tion is likely due to the multiplicity of LTs—11 are known in P. aeruginosa—with overlapping redunꢀ
dant activities.27 Not all of the 11 LTs are expected to be inhibited by bulgecin A.50
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Figure 3. Bulgecin potentiation of the bactericidal activity of ceftazidime. GFPꢀlabeled P. aeruginosa
was spotted at the center of a 100 mm swarm plate and 350 ꢁg bulgecin A and/or 5 ꢁg CAZ were/was
spotted at the red dot. Bacteria were imaged at (a) 14 hours and (b) 22 hours postꢀinoculation. The fluoꢀ
rescent images (100× magnification) were captured at the growing edge. As a representative example,
an arrow marks the imaging site for the top left plate. Red fluorescence indicates cell lysis. A 10ꢀꢁm
scale bar is given in the leftꢀmost panel.
Bulgecin A potentiation of a clinical βꢀlactam antibiotic as visualized by microscopy. A distinct
and reproducible feature of P. aeruginosa growth in the presence bulgecin A and subꢀMIC of
ceftazidime is that the rate of growth was largely unperturbed until the midꢀlog phase, when cell lysis
occurs. The reason for this lag, not seen with the other two organisms, is not known. We analyzed this
event using the swarm assays, with one exception. The bacteria grown to midꢀlog phase were placed at
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one position on an agar plate and a mixture of bulgecin A and ceftazidime was placed at a second posiꢀ
tion on the plate at a distance of 20 mm. This permutation was intended to mimic the effect of encounter
of bacteria with two drugs in combination (bulgecin A and ceftazidime). At the conclusion of the experꢀ
iment propidium iodide (λexci 561 nm, λemi 595 nm) was imaged to visualize DNA liberated by cell lyꢀ
sis.51 We imaged the swarm colony edge (white arrow, top left panel) for each case (Figure 3A) at 14
hours, and at 22 hours, after inoculation. In the case of control bacteria and that of bacteria in the presꢀ
ence of bulgecin A alone, we saw no effect. In the presence of ceftazidime alone, we saw an elongated
cell phenotype after 14 hours (Figure 3A). Cell lysis was not detected. We began to see cell lysis by 22
hours (Figure 3B) in the presence of ceftazidime alone. In contrast, when bulgecin A and ceftazidime
were present at 14 hours, the elongatedꢀbulged bacteria had already begun to lyse (Figure 3A, bottom
right corner). Significant cell death at 22 hours is seen only with the combination of the two (Figure 3B,
bottom right corner).
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CONCLUSIONS
The prescient discovery of Imada et al. of the bulgecins was made in an era when clinical opꢀ
tions for treatment of infections were not as limited as the present. Some infections by Gramꢀnegative
bacteria may be treated currently with only a single antibiotic and certain infections could be fatal by
more than 50%, such that a return to the preꢀantibiotic era has become subject of discussion.52–55 We
revisited bulgecins with this clinical backdrop. Our practical syntheses of the bulgecins, and our microꢀ
biological proof that their potentiation is bactericidal at sub-MIC of ceftazidime—a βꢀlactam used cliniꢀ
cally to treat Gramꢀnegative ESKAPE infections—merit a fresh look at bulgecins. We hasten to add that
the mixture of bulgecin A and subꢀMIC of ceftazidime results in early onset of bactericidal activity,
which is critical for rapid reduction of bacterial load in an infection. Bulgecins as potentiators of antiꢀ
bacterial activity hold great promise. This study is not a culmination for the bulgecins, but emphatic evꢀ
idence supporting their further mechanistic study.
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ASSOCIATED CONTENT
Supporting Information
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The Supporting Information is available free of charge on the ACS Publications website.
Figure S1–S4, Table S1 and S2, Xꢀray structure details of 8, experimental details of biological assays,
synthetic procedures and characterization data, NMR spectra of newly synthesized compounds and key
compounds (PDF)
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Crystallographic structure of 8 (CIF)
Abbreviations
PBP, penicillinꢀbinding protein; LT, lytic transglycosylase; GlcNAc, Nꢀacetyl glucosamine; BlgA,
bulgecin A; CAZ, ceftazidime; MIC, minimumꢀinhibitory concentration; NAG, Nꢀacetylglucosamine;
NAM, Nꢀacetylmuramic acid; GFP, green fluorescent protein.
AUTHOR INFORMATION
Corresponding Author
* mobashery@nd.edu
ORCID
Shusuke Tomoshige: 0000ꢀ0002ꢀ4948ꢀ5809
David A. Dik: 0000ꢀ0002ꢀ4700ꢀ3837
Jed F. Fisher: 0000ꢀ0002ꢀ7174ꢀ4352
Shahriar Mobashery: 0000ꢀ0002ꢀ7695ꢀ7883
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
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This work was supported by grants GM61629 (SM) and AI113219 (JDS) from the NIH. ST was supꢀ
ported by a postdoctoral fellowship from the Uehara Memorial Foundation. DAD was supported by
NIH Training Grant T32GM075762 and by the ECK Institute for Global Health.
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