Total synthesis of lysobactin

Article abstract of DOI:10.1021/ja067648h

Antibiotic resistance has become a significant public health concern. Antibiotics that belong to new structural classes and manifest their biological activity via novel mechanisms are urgently needed. Lysobactin, a depsipeptide antibiotic has displayed very strong antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) as well as vancomycin-resistant enterococci (VRE) with minimum inhibitory concentrations (MICs) ranging from 0.39 to 0.78 μg/mL. The MIC values against VRE were more than 50-fold lower than those reported for vancomycin itself. Lysobactin was found to inhibit nascent peptidoglycan formation; however, this activity was not antagonized in the presence of N-acyl-L-Lys-D-Ala-D-Ala, the binding domain on the cell wall precursors that is utilized by vancomycin. Thus, lysobactin represents a promising agent for the treatment bacterial infections due to resistant pathogens. We describe a convergent synthesis of lysobactin that relies upon a highly efficient macrocyclization reaction to assemble the 28-membered cyclic depsipeptide. This synthesis provides the foundation for further study of the mode of action utilized by lysobactin and its analogues.

Full text of DOI:10.1021/ja067648h

Published on Web 04/14/2007
Total Synthesis of Lysobactin
Aikomari Guzman-Martinez, Ryan Lamer, and Michael S. VanNieuwenhze*
Contribution from the Department of Chemistry and Biochemistry, UniVersity of California at
San Diego, La Jolla, California 92093
Received October 25, 2006; E-mail: msv@ucsd.edu
Abstract: Antibiotic resistance has become a significant public health concern. Antibiotics that belong to
new structural classes and manifest their biological activity via novel mechanisms are urgently needed.
Lysobactin, a depsipeptide antibiotic has displayed very strong antibacterial activity against methicillin-
resistant Staphylococcus aureus (MRSA) as well as vancomycin-resistant enterococci (VRE) with minimum
inhibitory concentrations (MICs) ranging from 0.39 to 0.78 µg/mL. The MIC values against VRE were more
than 50-fold lower than those reported for vancomycin itself. Lysobactin was found to inhibit nascent
peptidoglycan formation; however, this activity was not antagonized in the presence of N-acyl-L-Lys-D-Ala-
D-Ala, the binding domain on the cell wall precursors that is utilized by vancomycin. Thus, lysobactin
represents a promising agent for the treatment bacterial infections due to resistant pathogens. We describe
a convergent synthesis of lysobactin that relies upon a highly efficient macrocyclization reaction to assemble
the 28-membered cyclic depsipeptide. This synthesis provides the foundation for further study of the mode
of action utilized by lysobactin and its analogues.
Introduction
ics that belong to new structural classes or that function via
novel mechanisms are less likely to show cross-resistance with
Inhibition of bacterial cell wall (peptidoglycan) biosynthesis
is a mechanism exploited by many commonly used antibiotics,
including the â-lactams and glycopeptides.1 The peptidoglycan
biosynthetic pathway has proven to be a very popular target
for the development of chemotherapeutic agents for the treat-
ment of bacterial infections.1 One very important attribute of
this biosynthetic pathway as a target for antibacterial agents is
that it is unique to bacterial cells; there is no mammalian
counterpart. Thus, an agent that inhibits any step in this pathway
could be expected to show selective toxicity toward bacterial
cells, compromise the integrity of the bacterial cell wall, and
result in cell death.2 The historical popularity of the â-lactam
antibiotics is in part due to the fact that their molecular target
is displayed on the cell surface, thus cellular penetration is not
required to achieve their antibiotic effect. The glycopeptide
antibiotics (e.g., vancomycin), the antibiotic of last resort for
treatment of serious Gram-positive infections, also target a
reaction in the cell wall biosynthetic pathway that occurs on
the cell surface.
existing antibiotics. Unfortunately, in the period between 1962
and 2000, only one such drug (linezolid) was introduced
clinically; all other antibiotics that entered the market during
this period were analogues of existing drugs.5 Thus, antibiotics
belonging to new structural classes with novel modes of action
are urgently needed.
Lysobactin 1 is a cyclic depsipeptide antibiotic produced by
a species of Lysobacter (ATCC53042) that was isolated by a
group at the Squibb Institute.6,7 The structure of lysobactin
(Figure 1) was determined through a series of chemical and
enzymatic transformations and subsequent spectroscopic analy-
sis.8 The core depsipeptide is a 28-membered macrocycle
composed of nine amino acid residues with an ester linkage
between the hydroxyl group of a â-hydroxyphenylalanine
residue and the carboxyl group of a serine residue. A L-Leu-
D-Leu dipeptide side chain is appended to the N-terminus of
the core depsipeptide. Of the nine amino acids comprising the
core structure of lysobactin, five are nonproteinogenic, including
four that are â-hydroxylated.6,8 Three of these residues are
â-hydroxylated variants of proteinogenic amino acids (Phe, Leu,
and Asn), while the fourth is L-allo threonine (aThr).
Bacterial resistance to these agents, as well as other classes
of antibacterials, has created an urgent need for new chemo-
therapeutic agents with novel modes of action.3 According to a
recent estimate, 20% of people admitted to hospitals have or
will develop an infection, and 70% of the bacteria that give
rise to these infections are resistant to one or more of the
common antimicrobial agents used to fight infections.4 Antibiot-
In work that just preceded the disclosure of lysobactin,
Shionogi and Company reported the structure of katanosin B,
(4) Coates, A.; Hu, Y.; Bax, R.; Page, C. Nat. ReV. Drug DiscoVery 2002, 1,
895-910.
(5) Walsh, C. Nat. ReV. Microbiol. 2003, 1, 65-70.
(6) O’Sullivan, J.; McCullough, J. E.; Tymiak, A. A.; Kirsch, D. R.; Trejo,
W. H.; Principe, P. A. J. Antibiot. 1988, 41, 1740-1744.
(7) Bonner, D. P.; O’Sullivan, J. O.; Tanaka, S. K.; Clark, J. M.; Whitney, R.
R. J. Antibiot. 1988, 41, 1745-1751.
(8) Tymiak, A. A.; McCormick, T. J.; Unger, S. E. J. Org. Chem. 1989, 54,
1149-1157.
(1) Gale, E. F.; Cundliffe, E.; Reynolds, P. E.; Richmond, M. H.; Waring, M.
J. The Molecular Basis of Antibiotic Action, 2nd ed.; Wiley-Interscience:
New York, 1981.
(2) Bugg, T. D. H.; Walsh, C. T. Nat. Prod. Rep. 1992, 199-215.
(3) Chu, D. T. W.; Plattner, J. J.; Katz, L. J. Med. Chem. 1996, 39, 3853-
3874.
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A R T I C L E S
Guzman-Martinez et al.
Figure 1. Structure and retrosynthetic analysis of lysobactin.
Figure 2.
a cyclic depsipeptide antibiotic that was isolated from a
producing organism (PBJ-5356) related to the genus Cytophaga.9
Interestingly, the structure reported for katanosin B was identical
to that reported for lysobactin, save for the absolute configu-
ration of the allo threonine residue. In their initial isolation paper,
the Shionogi group assigned the allo-threonine residue the
D-absolute configuration10 via a protocol that entailed L-
leucylation of the amino acid followed by HPLC comparison
to reference standards. In a subsequent report, the absolute
configuration of the allo-threonine residue katanosin B was
changed from D to L.11 Thus, katanosin B and lysobactin have
identical structures.
group where it participates in a glycosyl transfer reaction
mediated by MurG. This reaction adds an N-acetylglucosamine
residue and provides lipid II 5, the final monomeric intermediate
utilized in the peptidoglycan biosynthetic pathway. Lipid II is
then translocated to the extracellular surface of the cell
membrane where it is a substrate for the transglycosylation and
transpeptidation reactions. The transglycosylation utilizes the
lipid II intermediate in a polymerization reaction that provides
the carbohydrate backbone of peptidoglycan. The transpepti-
dation reaction results in the installation of a cross-link between
peptide chains displayed on adjacent glycan strands.2
Although the mode of action of lysobactin is not precisely
understood, the available data suggest that its antibacterial
activity may be due to inhibition of the transglycosylation
reaction and/or steps in the biosynthetic pathway that precede
it (e.g., the MurG reaction). This activity may be the result of
binding to the lipid intermediates, a notion supported by
antagonism of its antibacterial activity by a cell wall-membrane
particulate.12 In this respect, its mode of action would be quite
similar to that utilized by ramoplanin, a cyclic depsipeptide
antibiotic currently in phase III trials for the treatment of VRE
infections.13 As a result of its impressive antibiotic activity,
lysobactin possesses significant promise for the treatment of
infections owing to vancomycin-resistant organisms.
In a recent evaluation, lysobactin displayed very strong
antibacterial activity against methicillin-resistant Staphylococcus
aureus (MRSA) as well as vancomycin-resistant enterococci
(VRE) with minimum inhibitory concentrations (MICs) ranging
from 0.39 to 0.78 µg/mL.12 The MIC values against VRE were
more than 50-fold lower than those reported for vancomycin
itself. Lysobactin inhibited the incorporation of isotopically
labeled N-acetylglucosamine (GlcNAc) into staphylococcal cell
wall peptidoglycan with a 50% inhibitory concentration (IC50
) 0.8 µg/mL) very close to its MIC.12 Lysobactin was also
found to inhibit nascent peptidoglycan formation; however,
unlike vancomycin, its activity was not antagonized in the
presence of N-acetyl-L-Lys-D-Ala-D-Ala.12 Thus, if lysobactin
achieves its inhibition of the peptidoglycan biosynthetic ma-
chinery through binding to the lipid intermediates (Figure 2) or
nascent peptidoglycan, it does so at a site on these precursors
that is not utilized by vancomycin itself.
Several efforts have been directed toward the synthesis of
various fragments of the lysobactin core structure.14-16 In
addition, a solid-phase synthesis of a structurally simplified
lysobactin analogue17 has been reported. Recently, a group at
Bayer AG reported the total synthesis and X-ray crystal structure
of lysobactin.18 This disclosure has prompted us to report our
effort that has culminated in the total synthesis of lysobactin.19
The latter stages of the peptidoglycan biosynthetic pathway
are illustrated in Figure 2. The lipid I intermediate 4 is anchored
to the cytoplasmic surface of the cell membrane by a lipid head
(13) Walker, S.; Chen, L.; Hu, Y.; Rew, Y.; Shin, D.; Boger, D. L. Chem. ReV.
2005, 105, 449-475.
(9) Shoji, J.; Hinoo, H.; Matsumoto, K.; Hattori, T.; Yoshida, T.; Matsuura,
S.; Kondo, E. J. Antibiot. 1988, 41, 713-718.
(14) Armaroli, S.; Cardillo, G.; Gentilucci, L.; Gianotti, M.; Tolomelli, A. Org.
Lett. 2000, 2, 1105-1107.
(10) Kato, T.; Hinoo, H.; Terui, Y.; Kikuchi, J.; Shoji, J. J. Antibiot. 1988, 41,
719-725.
(15) Cardillo, G.; Gentilucci, L.; Gianotti, M.; Tolomelli, A. Eur. J. Org. Chem.
2000, 2489-2494.
(11) Kato, T.; Hinoo, H.; Terui, Y.; Kikuchi, J.; Shoji, J. J. Antibiot. 1989, 42,
C-2.
(16) Palomo, C.; Oiarbide, M.; Ganboa, I.; Miranda, J. I. Tetrahedron Lett. 2001,
42, 8955-8957.
(12) Maki, H.; Miura, K.; Yamano, Y. Antimicrob. Agents Chemother. 2001,
45, 1823-1827.
(17) Egner, B. J.; Bradley, M. Tetrahedron 1997, 53, 14021-14030.
9
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Total Synthesis of Lysobactin
A R T I C L E S
Scheme 1. Synthesis of Fragment 2
Scheme 2. Synthesis of Fragment 3
Figure 3. Possible O,N-acyl migration of lysobactin core depsipeptide
during Edman degradation.
Our retrosynthetic analysis for lysobactin is presented in
Figure 1. We chose to construct the depsipeptide macrocycle
via an intramolecular coupling between L-HyAsn5 and Gly.6
Coupling reactions employing activated esters of glycine
residues are generally very efficient. In addition, since glycine
lacks an R-substituent, any possibility of epimerization during
the course of the activation/coupling sequence would be avoided.
The linear depsipeptide would be assembled via coupling
reaction between Leu12 and D-Arg11 on the basis of the literature
precedent that suggests amide bond formation reactions between
D and L coupling partners generally proceed more efficiently
than those employing L,L or D,D coupling partners.20,21,22 Thus
our target fragments for the assembly of lysobactin were
depsipeptide 2, incorporating the L-Leu-D-Leu dipeptide side
chain, and tetrapeptide 3.
in 6) that would be poised to undergo O,N-acyl migration; a
reaction frequently observed upon N-deprotection of linear
peptides containing N-terminal serine and threonine residues
bearing O-acyl protective groups. Adding to our concern, we
noted during the earlier work on the structure determination of
lysobactin, that two cycles of the Edman degradation reaction
provided clean phenylthiohydantion products for cleavage of
the first two N-terminal amino acid residues (D-Leu and L-Leu).8
During the third cycle, however, only a portion of the
didesleucyl peptide reacted with phenyl isothiocyanate suggest-
ing that structural constraints provided by the macrocycle may
at most only partially suppress the propensity for O,N-acyl
migration upon generation of a free N-terminal amino group8
(Figure 3).
Ideally, we would have preferred to introduce the dipeptide
side chain onto an N-terminal amino group at a late stage of
the synthesis since this is an obvious site for future modification.
This would require generation of a free amino group (e.g., as
(18) von Nussbaum, F.; Anlauf, S.; Benet-Bucholz, J.; Ha¨bich, D.; Ko¨bberling,
J.; Musza, L.; Telser, J.; Ru¨bsamen-Waigmann, H.; Brunner, N. A. Angew.
Chem., Int. Ed. Engl. 2007, 46, 2039-2042.
(19) Initial disclosure of the work presented in this manuscript: Guzman-
Martinez, A.; Lamer, R.; VanNieuwenhze, M. S., 232nd ACS National
Meeting, San Francisco, CA, September 10-14, 2006.
Results and Discussion
(20) Rich, D. H.; Bhatnagar, P.; Mathiaparanam, P.; Grant, J. A.; Tam, J. P. J.
Org. Chem. 1978, 43, 296-302.
Fragment Synthesis. Our synthesis of fragment 2 (Scheme
1) began with the methyl ester of â-hydroxyphenylalanine 8,
protected as the 2-(trimethylsilyl)ethyl carbamate (Teoc), that
(21) Brady, S. F.; Varga, S. L.; Freidinger, R. M.; Schwenk, D. A.; Mendlowski,
M.; Holly, F. W.; Veber, D. F. J. Org. Chem. 1979, 44, 3101-3105.
(22) Birch, D.; Hill, R. R.; Jeffs, G. E.; North, M. Chem. Commun. 1999, 941-
942.
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A R T I C L E S
Guzman-Martinez et al.
Scheme 3. Final Assembly of Lysobactin
was readily available via Sharpless asymmetric aminohydroxy-
lation of (E)-methyl cinnamate.23 Cleavage of the Teoc protect-
ing group was efficiently achieved upon exposure of 8 to TFA
in dichloromethane and provided the free amine 9. Coupling
of 9 with Boc-D-Leu-L-Leu-OH was achieved through carboxyl
activation with 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-
4(3H)-one (DEPBT)24,25 and provided tripeptide 10 in 80%
overall yield. DEPBT was the reagent of choice for all peptide
coupling reactions on the basis of its demonstrated ability to
suppress racemization at the adjacent R-stereocenter of activated
carboxylates.24,26
With tripeptide 10 in hand, we now needed to install a
dipeptide fragment at its carboxy terminus and acylate its
â-hydoxyl group. In his seminal report of the synthesis of
ramoplanin A2 and the ramoplanose aglycon, Boger noted that
carboxylate activation of substrates containing â-acyloxy sub-
stituents typically resulted in preferential â-elimination, ir-
respective of the reagents used for carboxyl activation.27,28 Given
this precedent, we chose to install the dipeptide at the carboxy
terminus of the tripeptide prior to introduction of the backbone
ester. Thus, hydrolysis of methyl ester 10 (LiOH, THF/H2O,
quantitative) followed by coupling with H-â-OTBS-Leu-Leu-
OAll (DEPBT, DIPEA, DMF, 92%) provided pentapeptide 12.
Installation of the backbone ester was achieved through coupling
of 12 with Fmoc-L-Ser-OtBu (EDCI, DMAP, CH2Cl2) and
provided depsipeptide 13 in 77% yield. The N-terminal Fmoc
protective group was cleanly removed (piperidine, DMF, 99%)
and the resulting free amine was coupled with Fmoc-HyAsn-
(Trt)-OH29 (DEPBT, DIPEA, DMF, 87%) to provide 15. Finally,
cleavage of the C-terminal allyl ester (Pd(PPh3)4, PhSiH3, THF,
99%) provided fragment 2.
Our synthesis of fragment 3 (Scheme 2) began with D-allo-
Thr-OH 16 that was easily prepared from D-threonine via the
Elliott protocol.30 Conversion of the carboxylic acid to its methyl
ester (SOCl2, MeOH) followed by coupling with Boc-L-Ile-OH
(DEPBT, DIPEA, THF) provided dipeptide methyl ester 17 in
92% overall yield. Hydrolysis of the methyl ester (LiOH, THF/
H2O, quantitative) followed by coupling with H-Gly-OAll
(DEPBT, DIPEA, THF) provided tripeptide 19 in 95% yield.
Removal of the Boc protective group (4N HCl, dioxane)
followed by coupling with Fmoc-D-Arg(Boc)2-OH (DEPBT,
DIPEA, THF) provided tetrapeptide 20 in 96% overall yield.
A final Fmoc cleavage (10% piperidine, DMF, 98%) provided
fragment 3.
Macrocyclization and End Game. The final stages of our
lysobactin synthesis began with coupling of the C-terminal
carboxyl group of 2 with the N-terminal amino group of 3
(Scheme 3). Coupling of carboxylic acid 2 with the free amine
3 (DEPBT, DIEA, DMF, 0 °C to room temp) provided
depsipeptide 21 in 94% yield. Unmasking of the C-terminal
carboxyl group was readily achieved through treatment of 21
with Pd(PPh3)4 and phenylsilane in DMF and provided the free
carboxylic acid 22 in 93% yield. The N-terminal Fmoc
protective group was cleanly removed upon exposure to
piperidine in DMF. Macrocyclization was very efficiently
achieved by exposure of the amino acid intermediate to DEPBT
(DIPEA, DMF) and provided cyclic depsipeptide 23 in 83%
overall yield. A final global deprotection (TFA/H2O, 95:5)
provided lysobactin 1 in 33% isolated yield after reverse-phase
HPLC purification. The synthetic material was identical in all
respects to an authentic sample kindly provided by Shionogi
and Company.
(23) Tao, B.; Schlingloff, G.; Sharpless, K. B. Tetrahedron Lett. 1998, 39, 2507-
2510.
Conclusion
(24) Li, H.; Jiang, X.; Ye, Y.; Fan, C.; Romoff, T.; Goodman, M. Org. Lett.
1999, 1, 91-93.
In summary, we have developed a convergent synthesis of
the depsipeptide antibiotic, lysobactin. Key to the success of
our synthetic route was a two-step deprotection/coupling
sequence that provided a 28-membered cyclic depsipeptide in
(25) Fan, C.-X.; Hao, X.-L.; Ye, Y.-H. Synth. Commun. 1996, 26, 1455-1457.
(26) Ye, Y.-H.; Li, H.; Jiang, X. Biopolymers 2005, 80, 172-178.
(27) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L. J. Am.
Chem. Soc. 2002, 124, 5288-5290.
(28) Carboxyl activation with DEPBT was the only exception to this general
trend.
(29) Guzman-Martinez, A.; VanNieuwenhze, M. S. Unpublished results.
(30) Elliott, D. F. J. Chem. Soc., Chem. Commun. 1949, 589-594.
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Total Synthesis of Lysobactin
A R T I C L E S
83% yield. The efficiency of this transformation is noteworthy
when viewed in the context of previously reported results for
depsipeptide macrocyclization.31,32 The relatively unhindered
structure of the glycine electrophile is one factor that may
contribute to the efficiency of the macrocyclization. A second
factor may involve preorganization of the cyclization precursor
resulting in more favorable cyclization kinetics.33 This attribute
has been used to great advantage in the synthesis of ramoplanin
A2,13,27,34 tyrocidine A,35 and gramicidin S36 and is believed to
be due to a â-sheet structure, or minimally a structural element
that enforces a â-turn, adopted by the cyclization precursors
that entropically enhances the rate of head-to-tail cyclization.
Lysobactin is a promising depsipeptide antibiotic with activity
against vancomycin-resistant organisms. Although little is
known regarding the mode of action utilized by lysobactin, the
available data implicate a target in the bacterial cell wall
biosynthetic pathway possibly involving binding/sequestration
of the lipid intermediate(s).12 Future work will focus on
elucidating the molecular target(s) of lysobactin, identification
of the key structural attributes responsible for its biological
activity, and structural studies of the complexes lysobactin forms
with the lipid intermediates. In addition, spectroscopic studies
designed to probe the role that substrate preorganization may
play in the overall efficiency of the lysobactin macrocyclization
are currently in progress. Results from these studies will be
presented in due course.
Acknowledgment. Financial support provided by the National
Institutes of Health (Grant AI 059327), The Hellman Founda-
tion, and the Regents of the University of California, is gratefully
acknowledged. M.S.V. thanks Eli Lilly and Company for
support in the form of an Eli Lilly and Company New Faculty
Award. A.G.M. thanks the University of California at San Diego
for support in the form of GAANN Fellowship. We thank
Shionogi and Company for kindly providing a sample of
lysobactin (katanosin B).
(31) Davies, J. S. J. Peptide Res. 2003, 9, 471-501.
Supporting Information Available: Experimental procedures
and spectroscopic data for compounds 1-3, 10-15, and 17-
23. This material is available free of charge via the Internet at
http://pubs.acs.org.
(32) Hamada, Y.; Shioiri, T. Chem. ReV. 2005, 105, 4441-4482.
(33) Blankenstein, J.; Zhu, J. Eur. J. Org. Chem. 2005, 1949-1964.
(34) Jiang, W.; Wanner, J.; Lee, R. J.; Bounaud, P.-Y.; Boger, D. L. J. Am.
Chem. Soc. 2003, 125, 1877-1887.
(35) Bu, X.; Wu, X.; Xie, G.; Guo, Z. Org. Lett. 2002, 4, 2893-2895.
(36) Bu, X.; Wu, X.; Ng, N. L. J.; Mak, C. K.; Qin, C.; Guo, Z. J. Org. Chem.
2004, 69, 2681-2685.
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