Total synthesis of plusbacin A3: A depsipeptide antibiotic active against vancomycin-resistant bacteria

Article abstract of DOI:10.1021/ja068455x

Plusbacin A₃ is a depsipeptide antibiotic isolated from Pseudomonas sp. Although the stereochemistry at the lactone stereocenter had not been determined, biological evaluation of this compound demonstrated it to have promising antibacterial act

Full text of DOI:10.1021/ja068455x

Published on Web 03/20/2007
Total Synthesis of Plusbacin A3: A Depsipeptide Antibiotic Active Against
Vancomycin-Resistant Bacteria
Aaron Wohlrab, Ryan Lamer, and Michael S. VanNieuwenhze*
Department of Chemistry and Biochemistry, UniVersity of California at San Diego, La Jolla, California 92093
Received November 24, 2006; E-mail: msv@ucsd.edu
Plusbacin A3 is a lipodepsipeptide isolated from a fermentation
broth of Pseudomonas sp. PB-6250 obtained from a soil sample
collected in the Okinawa Prefecture, Japan (Figure 1).1 The structure
of plusbacin A3 was established in 1992. It is a member of a family
of lipodepsipeptide natural products that differ either in the structure
of their respective fatty acid side chains or in substitution of
L-proline for 3-hydroxy-L-proline residues.2
In a recent evaluation, plusbacin A3 displayed strong antibiotic
activity against methicillin-resistant Staphylococcus aureus and
VanA-type vancomycin-resistant enterococci with minimum inhibi-
tory concentration (MIC) values from 0.78 to 3.13 µg/mL.3
Plusbacin A3 also inhibited incorporation of N-acetylglucosamine
into staphylococcal cell wall peptidoglycan with a 50% inhibitory
concentration (IC50) that was close to its MIC value. Like
vancomycin, plusbacin A3 was found to inhibit nascent peptidogly-
can formation; however, unlike vancomycin, plusbacin was also
found to inhibit the formation of the lipid intermediates utilized in
bacterial cell wall biosynthesis. Interestingly, the activity of
plusbacin A3 was not antagonized by the presence of N-acetyl-L-
Lys-D-Ala-D-Ala, a tripeptide mimic of the binding domain for
vancomycin,3 suggesting that if plusbacin A3 achieves its biological
activity through binding to the lipid intermediates or to nascent
peptidoglycan it does so at a site on these precursors that is not
Figure 1. Structure of plusbacin A3 and its retrosynthetic analysis.
utilized by vancomycin itself. As a result, plusbacin A3 possesses
significant promise for use in the treatment of vancomycin-resistant
infections.
could influence the intervening residues to adopt a conformation
that would facilitate the final macrocyclization event.
The amino acid sequences of the plusbacins were established
through Edman degradation of their deacylated products and
supported by mass spectrometric studies. Degradation experiments
also suggested a lactone linkage between an L-threo-â-hydroxyas-
partic acid residue and a 3-hydroxy fatty acid subunit. In the case
of plusbacin A3, the fatty acid component is reported to be
3-hydroxyisopentadecanoic acid, although the stereochemical con-
figuration at the hydroxyl stereocenter has yet to be assigned.
Plusbacin A3 also has several non-proteinogenic amino acids
embedded in its peptide backbone. In addition to the L-threo-â-
hydroxyaspartic acid residue mentioned above, other non-natural
amino acids contained in plusbacin A3 include D-threo-â-hydroxy-
aspartic acid, D-allo-threonine, and trans-3-hydroxy-L-proline. The
presence of these non-natural amino acids, coupled with the base
sensitivity of the lactone linkage, renders plusbacin A3 a challenging
target for total synthesis.
Our retrosynthetic analysis for plusbacin A3 with our selected
disconnections is presented in Figure 1. We chose to divide the
target molecule into four fragments of approximately equal
complexity. This was done in order to provide a measure of
flexibility over the amide bond to be made in the final macrolac-
tamization step since there was no solution conformation data
available to guide our selection. Our analysis was further guided
by a hypothesis that the hydroxyproline residues located at each
end of plusbacin A3 may enforce a â-turn type conformation that
Upon closer examination of the structure of plusbacin A3, one
is drawn to the possibility that the fatty acid side chain and the
arginine residue may play important roles in its biological activity.
For example, the fatty acid side chain may be important for
membrane localization, while the terminal guanidine group of the
arginine residue could be important for binding interactions with
diphosphate or carboxylate groups present in the lipid intermediates
and/or nascent peptidoglycan. Thus, we wished to devise a synthetic
strategy that would easily accommodate variations in the lipophilic
side chain and arginine/ornithine residues in order to gain a greater
insight into the roles each may have in the biological activity of
the plusbacins.
The preparation of fragments 2-4 is presented in Scheme 1.
Dipeptide 2 was prepared via coupling of Boc-D-Ser(OBn)-OH
with the allyl ester obtained from commercially available trans-3-
hydroxy-L-proline (EDCI, DIEA, HOAt, 89%). The C-terminal allyl
ester was then cleanly removed (PdCl2(PPh3)3, Ph3SiH, CH2Cl2,
85%) under standard deprotection conditions to provide the
C-terminal acid 2 in high overall yield.
Synthesis of fragment 3 required an independent synthesis of
an orthogonally protected D-threo-hydroxyaspartic acid derivative
7. This compound was prepared in eight steps from the Garner’s
aldehyde derived from Boc-L-Ser.4 Coupling of this precursor with
Boc-L-Orn(Fmoc)-OH (EDCI, DIEA, HOAt, 93%) provided the
corresponding dipeptide. Cleavage of the N-terminal Boc protective
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C O M M U N I C A T I O N S
enantiomeric purities as determined by chiral HPLC analysis.9 An
alkene cross metathesis reaction between (S)-12 and 11-methyl-1-
dodecene employing the Grubbs second generation catalyst 1410
followed by reduction of the alkene intermediate (H2, Pd/C, MeOH)
provided â-hydroxyester 15 in good overall yield. Cleavage of the
tert-butyl ester (TFA/CH2Cl2) and esterification of the free car-
boxylic acid (allyl-Br, K2CO3, DMF) provided the allyl-protected
â-hydroxyester 16 in 90% overall yield. Finally, coupling of Boc-
L-threo-â-OH-Asp(OCy)-OH (EDCI, DMAP, CH2Cl2, 84%) and
allyl ester deprotection (PdCl2(PPh3)2, PPh3, PhSiH3, CH2Cl2, 98%)
cleanly provided the target fragment 5.
Scheme 1. Synthesis of Fragments 2-4
With the four target fragments in hand, the stage was now set
for their assembly and elaboration into plusbacin A3. Our fragment
assembly began with coupling of fragments 3 and 4. The coupling
reaction (EDCI, HOBt, DIEA, THF) cleanly provided pentapeptide
17 in 91% yield. Removal of the N-terminal Boc protective group
followed by coupling with carboxylic acid 5 (EDCI, HOBt, THF,
83%) provided ester 18. It is noteworthy that no â-elimination of
the chemically sensitive â-acyloxy substituent was observed during
the activation/coupling sequence. Boc cleavage (4 N HCl, dioxane,
quantitative) and coupling of fragment 2 (EDCI, HOBt, DIEA,
DMF, 72%) provided the fully assembled linear lipodepsipeptide
precursor 19 for the final macrocyclization reaction.
Our choice for coupling of the N-terminal D-serine residue with
the C-terminal â-hydroxyaspartic acid residue was driven by the
efficiency of the macrocyclization of the simplified model substrate
24 shown in eq 1. The linear precursor was assembled using
standard protection/deprotection and activation/coupling sequences
in analogy to those utilized for the assembly of depsipeptide 18.
Non-hydroxylated amino acid residues were incorporated into the
peptide backbone of the model substrate, and a â-alanine subunit
was introduced in place of the depsipeptide linkage. Cyclization
of 24 to 25 was cleanly (72% for R ) Bn; 65% for R ) Ph) with
EDCI/HOAt activation.
Scheme 2. Synthesis of Fragment 5
group (4 N HCl, dioxane, quantitative yield) provided the N-
terminal amine 3 as its hydrochloride salt.
The synthesis of tripeptide 4 proceeded from Boc-protected
D-allo-threonine that was prepared according to the Elliot protocol.5
Coupling with H-D-Ala-OBn (EDCI, DIEA, HOBt, 87%) provided
dipeptide 9 in high yield. Hydrogenolysis of the C-terminal benzyl
ester (H2, 5% Pd/C, quantitative yield) followed by coupling of
the C-terminal carboxyl group with HCl•H-trans-â-OH-L-Pro-OBn
(EDCI, DIEA, HOBt, 90%) provided tripeptide 10. Hydrogenolytic
cleavage of the C-terminal benzyl ester (H2, Pd/C, MeOH,
quantitative yield) cleanly provided the target tripeptide 4.
Our synthetic route to fragment 5 is provided in Scheme 2. Given
the uncertainty regarding the stereochemistry at the lactone ste-
reocenter, we desired a synthetic method that would allow access
to a suitably protected â-hydroxyester derivative (e.g., 12) in both
enantiomeric forms. The alkene functional group would provide a
handle for an olefin cross metathesis reaction that could be used
for incorporation of the remainder of the isohexadecanoic acid side
chain, as well as numerous additional side chain variations.
Our sequence began with a lipase-mediated kinetic resolution
of racemic â-hydroxyester 11. The kinetic resolution utilized the
Amano PS lipase6-8 and provided enantioenriched â-acetoxyester
(S)-12 and (R)-allylic alcohol 13 in good yields and with very high
To set the stage for the final macrocyclization (Scheme 3), the
C-terminal carboxyl group was unmasked (PdCl2(PPh3)3, Ph3SiH,
CH2Cl2, 95%). Cleavage of the N-terminal Boc protective group
(4 N HCl, dioxane, quantitative) provided the amino acid cyclization
precursor 21. Carboxyl activation followed by cyclization (EDCI,
HOBt, DMF, 48 h) provided the cyclic depsipeptide 22 in 72%
yield. Finally, cleavage of the side chain Fmoc protective group
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C O M M U N I C A T I O N S
Scheme 3. Synthesis of Plusbacin A3sThe Final Stagesa
a (a) EDCI, HOBt, DIEA, THF, 91%; (b) 4 N HCl/dioxane, quantitative; then EDCI, HOBt, 5, DIEA, THF, 83%; (c) 4 N HCl, dioxane, quantitative; (d)
EDCI, HOBt, 2, DIEA, DMF, 72%; (e) PdCl2(PPh3)2, Ph3SiH, CH2Cl2, 95%; (f) 4 N HCl, dioxane, quantitative; (g) EDCI, HOBt, 0 °C, DMF, 48 h, 72%;
(h) 5% piperidine/DMF, quantitative; (i) N,N′-diBoc-N′′-triflylguanidine, DIEA, DMF, 70%; (j) HF, anisole, 20% after HPLC purification.
(5% piperidine/DMF, quantitative yield) provided depsipeptide 23.
Guanidinylation (N,N′-diBoc-N′′-triflylguanidine, DIEA, DMF,
70%) followed by cleavage of the cyclohexyl esters (HF, anisole)
provided target compound, plusbacin A3, possessing the (R)-
configuration at the lactone stereocenter.
the form of an Eli Lilly and Company New Faculty Award. We
thank the Shionogi Corporation for a gift of plusbacin A3.
Supporting Information Available: Experimental details and
spectroscopic data for all new compounds. This material is available
free of charge via the Internet at http://pubs.acs.org.
Our synthetic sample of plusbacin A3 was then compared with
an authentic sample of the natural product provided by the Shionogi
corporation and a synthetic sample of the plusbacin A3 diastereomer
that incorporated the (S)-3-hydroxyisopentanoic acid fragment in
the peptide backbone.11 The synthetic substance containing the (R)-
3-hydroxyisopentanoic acid residue in the peptide backbone was
identical in all respects to an authentic sample of the natural product,
thus establishing that the 3-hydroxyisopentanoic acid residue in
plusbacin A3 has the (R)-configuration.
In summary, we have described the first total synthesis of
plusbacin A3, a promising depsipeptide antibiotic activity against
vancomycin-resistant organisms. Future studies are aimed at the
study of the structure and function of this very promising antibacte-
rial agent. The results of these studies will be reported in due course.
References
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(11) The requisite fragment containing the (S)-3-hydroxyisopentanoic acid
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Acknowledgment. Financial support provided by the National
Institutes of Health (AI 059327), The Hellman Foundation, and
the Regents of the University of California is gratefully acknowl-
edged. M.S.V. also thanks Eli Lilly and Company for support in
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