Solid-phase synthesis of lysobactin (katanosin B): Insights into structure and function

Article abstract of DOI:10.1021/ol300926d

The solid phase synthesis of the cyclic depsipeptide antibiotic lysobactin is described. The natural product was synthesized via a linear approach using mostly an Fmoc-strategy solid phase peptide synthesis (SPPS) with a single purification. A lysobactin analog has also been synthesized displaying nanomolar membrane disruption activity not seen with the natural product.

Full text of DOI:10.1021/ol300926d

ORGANIC
LETTERS
2012
Vol. 14, No. 11
2730–2733
Solid-Phase Synthesis of Lysobactin
(Katanosin B): Insights into Structure
and Function
Edward A. Hall, Erkin Kuru, and Michael S. VanNieuwenhze*
Department of Chemistry, Indiana University, 800 East Kirkwood Avenue, Bloomington,
Indiana 47405-7102, United States
mvannieu@indiana.edu
Received April 12, 2012
ABSTRACT
The solid phase synthesis of the cyclic depsipeptide antibiotic lysobactin is described. The natural product was synthesized via a linear approach
using mostly an Fmoc-strategy solid phase peptide synthesis (SPPS) with a single purification. A lysobactin analog has also been synthesized
displaying nanomolar membrane disruption activity not seen with the natural product.
Lysobactin (katanosin B) is an 11 amino acid cyclic
depsipeptide antibiotic that was isolated by groups at the
Shionogi Research Institute1 and the Squibb Institute of Medi-
cal Research.2 The 28-membered macrocycle contains six non-
proteinogenic amino acids including four that are β-hydroxy-
lated. Lysobactin shows extensive antibacterial activity against a
wide range of aerobic and anaerobic Gram-positive bacteria,
in some trials displaying 2- to 4-fold greater activity than
vancomycin.3 Very strong growth inhibition of methicillin-
resistant Staphylococcus aureus (MRSA) and vancomycin-
resistant enterococci (VRE) has been shown with minimum
inhibitory concentrations ranging from 0.39 to 0.78 μg/mL.2
There have been two syntheses to date of this natural
product that employed a modular, solution phase ap-
proach. The first employed an approach with minimal
use of protecting groups and utilized a crystal structure to
guide selection of a site for macrolactamization.4,5 The
second employed a more classical approach involving a
macrolactamization via activation of a C-terminal Gly
residue that allowed epimerization-free cyclization.6 While
both of these syntheses provided efficient routes to gen-
erate lysobactin, solution phase chemistry greatly restricts
the ability to rapidly access structural analogs. Thus, a
solid phase approach was desired in order to facilitate our
goal of identifying the key structural moieties that are
responsible for the excellent antibacterial activity of
lysobactin.
(1) Shoji, J.; Hinoo, H.; Matsumoto, K.; Hattori, T.; Yoshida, T.;
Matsuura, S.; Kondo, E. J. Antibiot. 1988, 41, 713–718.
(2) O’Sullivan, J.; McCullough, J. E.; Tymiak, A. A.; Kirsch, D. R.;
Trejo, W. H.; Principe, P. A. J. Antibiot. 1988, 1740–1744.
(3) Bonner, D. P.; O’Sullivan, J.; Tanaka, S. K.; Clark, J. M.;
Whitney, R. R. J. Antibiot. 1988, 1745–1751.
As is standard with most solid phase peptide syntheses
(SPPS), we chose to synthesize the linear precursor
to lysobactin in the C- to N-direction. We selected the
same site for macrocyclization that we exploited for our
synthesis in solution.6 This enabled epimerization-free
€
(4) von Nussbaum, F.; Anlauf, S.; Benet-Buchholz, J.; Habich, D.;
€
€
Kobberling, J.; Musza, L.; Telser, J.; Rubsamen-Waigmann, H.; Brunner,
N. A. Angew. Chem., Int. Ed. 2007, 2039–2042.
(5) (a) von Nussbaum, F.; Anlauf, S.; Koebberling, J.; Telser, J.;
Haebich, D. (Aicuris G.m.b.H. & Co. K.-G., Germany). Procedure for
synthesis of cyclic depsipeptides such as Lysobactin via peptide fragment
assembly and intramolecular cyclization. DE102006018250A1, 2007. (b)
von Nussbaum, F.; Brunner, N.; Endermann, R.; Fuerstner, C.; Hartmann, E.;
Paulsen, H.; Ragot, J.; Schiffer, G.; Schuhmacher, J.; Svenstrup, N.; Telser, J.;
Anlauf, S.; Bruening, M.-A. (Bayer Healthcare AG). Synthesis of lysobactin
derivs. for use as antibacterial agents in the treatment or prevention of disease.
DE102004053407A1, 2006.
(6) Guzman-Martinez, A.; VanNieuwenhze, M. S. J. Am. Chem. Soc.
2007, 129, 6017–6021.
(7) (a) Rich, D. H.; Bhatnagar, P.; Mathiaparanam, P.; Grant, J. A.;
Tam, J. P. J. Org. Chem. 1978, 43, 296–302. (b) 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. (c) Birch, D.; Hill, R. R.;
Jeffs, G. E.; North, M. Chem. Commun. 1999, 941–942.
r
10.1021/ol300926d
Published on Web 05/21/2012
2012 American Chemical Society

cyclization, via a glycine-derived activated ester, and
avoided coupling residues of like chirality since these are
known to be less efficient than amino acid coupling
reactions employing partners having identical absolute
configurations.7
Scheme 1. Iterative Fmoc-SPPS of Key Intermediate 6
Figure 1. Synthetic Fmoc-protected building blocks.
All synthetic amino acid building blocks were orthog-
onally protected to enable trifluoroacetic acid (TFA)
promoted global deprotection (Figure 1). Fmoc-allo-
Thr(OTBS)-OH (1) was produced starting from Thr as
described by Elliott.8 Fmoc-D-Arg(Boc2)-OH (2) was gen-
eratedby couplingFmoc-D-Orn-OAllyl withN,N0-bis(tert-
butoxycarbonyl)-S-methylisothiourea.9 Fmoc-threo-HyAsn-
(CONHTrt)-OH (3) was synthesized via our previously
established protocol that relied upon a stereospecific
oxazoline cyclization to introduce the β-hydroxy stereo-
center.10 Fmoc-threo-HyLeu(OTBS)-OH (4) was gener-
ated using chemistry first reported by Hamada11 in which
RuCl2(binap)-catalyzed hydrogenation of the corre-
sponding β-keto-R-amino acid ester afforded a nearly
diastereo- and enantiopure product through dynamic
kinetic resolution. threo-Phenylserine was purchased
from a commercial source and protected as its corre-
sponding Fmoc derivative (5).
The preparation of intermediate 6 began with the
C-terminal glycine residue loaded on a 2-chlorotrityl resin
(Scheme 1). All peptide couplings were performed using
3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one
(DEPBT)12 and diisopropylethylamine (DIPEA) in DMF
followed by Fmoc deprotection using 20% piperidine in
DMF. In an effort to conserve precious materials, only 1.5
equivalents of synthetic amino acids were used during
coupling while 5 equivalents were used for commercially
available building blocks (excluding Fmoc-threo-phenyl-
serine-OH). Longer reaction times (up to 24 h) were
required for couplings using fewer equivalents, but no
epimerization was detected as determined by reverse-phase
HPLC.
With linear peptide 6 in hand, our attention was turned
to the esterification of the hindered secondary hydroxyl. A
variety of coupling reactions employing the use of carbo-
diimide activating reagents were investigated. It was found
that concentration, temperature, dry and distilled reaction
solvents, and the choice of R-amino protecting group were
critical to both the coupling efficiency and the elimination
of byproduct arising from epimerization. Initial attempts
at coupling Fmoc-Ser(tBu)-OH were plagued by low con-
versions and unacceptable amounts of epimerization.
Furthermore, we found that varying amounts of β-elim-
ination had occurred under standard Fmoc cleavage con-
ditions. To circumvent β-elimination, Alloc-Ser(tBu)-OH
was prepared to allow amine deprotection under neutral
conditions.13 To our delight, coupling of this building
block proceeded with complete conversion and no detect-
able epimerization when performed at 37 °C (Scheme 2). It
is worth noting that these conditions are very similar to
those used for macrolactonization of the simplified lyso-
bactin analog synthesized by Bradley.14 Analogous effects
of R-amino protecting groups on coupling efficiency have
also been reported for the coupling of N-methyl amino
acids in Rich’s studies on cyclosporine.15,16
Cleavage of the intermediate allyl carbamate with cata-
lytic Pd(PPh3)4 unfortunately gave a 2:1 mixture of of
identical mass while extended reaction times provided a
complex mixture of compounds. However, reducing the
reaction time to 10 min afforded complete deprotection
without any appearance of any undesired side product(s).
The remaining β-HyAsn (3) was installed under standard
conditions (DEPBT, DIPEA, THF) and cleanly deprotected
(8) Elliott, D. F. J. Chem. Soc. 1949, 589–594.
(9) Markowski, P. J. J. Pept. Sci. 2005, 11, 60–64.
(10) Guzman-Martinez, A.; VanNieuwenhze, M. S. Synlett 2007, 10,
1513–1516.
(13) Albericio, F.; Thieriet, N.; Alsina, J.; Giralt, E.; Guibe, F.
Tetrahedron Lett. 1997, 38, 7275–7278.
(14) Egner, J. E.; Bradley, M. Tetrahedron 1997, 53, 14021–14030.
(15) Tung, R. D.; Rich, D. H. J. Am. Chem. Soc. 1985, 107, 4342–
4343.
(16) Colucci, W. J.; Tung, R. D.; Petri, J. A.; Rich, D. H. J. Org.
Chem. 1990, 55, 2895–2903.
(11) Makino, K.; Okamoto, N.; Hara, O.; Hamada, Y. Tetrahedron:
Asymmetry 2001, 12, 1757–1762.
(12) (a) Li, H.;Jiang, X.;Ye, Y.-h.;Fan, C.-X.;Romoff,T.;Goodman,
M. Org. Lett. 1999, 1 (1), 91–93. (b) Ye, Y.-H.; Li, H.; Jiang, X. Bio-
polymers 2005, 80, 172–178.
Org. Lett., Vol. 14, No. 11, 2012
2731

Scheme 2. End Game Synthesis
all HPLC conditions. Furthermore, 1H NMR, 13C NMR,
optical rotation, and high resolution ESI-TOF mass spec-
tral analysis correlated with synthetic lysobactin.18
With the ability to rapidly generate structurally diverse
lysobactin analogs in place, we chose to begin studies
focused on a potentially important cationꢀπ interaction
bridging the macrocycle. As seen in the crystal structure of
lysobactin reported by von Nussbaum, the guanidine moi-
ety of D-Arg lies in close proximity to the phenyl substituent
of phenylserine.3 If this structural attribute is critical for the
biological activity of lysobactin, we hypothesized that the
removal of this interaction would result in increased con-
formational flexibility and diminished antibacterial activity.
Our first target for synthesis was lysobactin analog 11
(Figure 2) for which our solid phase strategy only required
incorporation of of Fmoc-Thr-OH in place of threo-phe-
nylserine. The newly introduced methyl group would
eliminate the possibility of any cationꢀπ interaction.
Upon completion of the solid-phase synthesis of 11,
comparison of its circular dichroism spectrum with that
obtained from lysobactin revealed a distinct conforma-
tional change/relaxation.19 Minimum inhibitory concen-
trations for lysobactin and 11 were determined against
Bacillus subtilis. The MIC values were 0.06 and 2 μg/mL
for lysobactin and 11, respectively. Although the analog
(11) shows a 32-fold decrease in activity, the MIC still
remains at a respectable level meaning that either the
cationꢀπ interaction is not critical for antibacterial activ-
ity or 11 may be achieving its antibacterial activity via an
additional/different mechanism.19,20
Figure 2. Δ3-Thr-lysobactin.
(5% piperidine in DMF) withno evidence of epimerization
or β-elimination.
Cleavage from the 2-Cl-Trt resin with acetic acid/
trifluoroethanol/dichloromethane (1:1:3) yielded the linear
peptide in high purity (>90% by HPLC). No additional
peptide was collected after a second treatment of the resin
under the same conditions. The crude peptide was cyclized
using the conditions (DEPBT, DIPEA, DMF) from our
previous synthesis of lysobactin.5 The cyclized product was
submitted to global deprotection conditions (TFA/Et3SiH/
DCM; 90/5/5) that removed all protecting groups except
for a single TBDMS ether. Treatment with neat TFA for an
additional 9ꢀ12 h was required for full deprotection. Depro-
tection conditions used in our previous synthesis (TFA/H2O;
95/5) removed all protecting groups reproducibly in only 4 h;
however, there was no significant increase in yield when
compared to the best results under anhydrous conditions.
Reversed-phase preparatory HPLC purification yielded
lysobactin in 8.4% overall yield. Co-injection with an
authentic sample showed a single, symmetrical peak under
A bacterial cell membrane disruption assay was per-
formed in order to compare the activity of 11 with the
(18) Lysobactin derivative 11 was produced in 5.4% overall yield.
(19) See Supporting Information.
(20) Lysobactin is an inhibitor of the transglycosylation reaction in
the bacterial cell wall biosynthetic pathway: Maki, H.; Miura, K.;
Yamano, Y. Antimicrob. Agents Chemother. 2001, 45 (6), 1823–1827.
(17) Initial deprotection conditions omitted water with the intent of
avoiding any undesired hydrolytic ring-opening reactions.
2732
Org. Lett., Vol. 14, No. 11, 2012

natural product, as lysobactin has previously shown lim-
ited amounts of membrane permeabilization at concentra-
tions above its MIC.1 To this end, exponentially growing
wild-type B. subtilis cells were treated with varying con-
centrations of antibiotic in the presence of propidium
iodide (PI). PI is a normally membrane impermeable
dye, which becomes up to 40 times more fluorescent upon
intercalation into membrane enclosed DNA. Thus, cells
with compromised membranes show a time dependent
enhancement of PI fluorescence compared to cells with
intact membranes (Figure 3).21,22 In agreement with pre-
vious reports, lysobactin showed poor PI influx until a
concentration of 64 to 128 times the MIC is reached
(Figure 3 and Supporting Information (SI), Figure 2).
Surprisingly, 11 caused a rapid PI influx even at concen-
trations 4 times lower than its MIC. This difference in rate
of PI influx relative to the respective MIC values for
lysobactin and 11 suggests that the antibacterial activity
for the lysobactin analog (11) may be the result of mem-
brane permeabilization.
This notion was further supported by a killing kinetics
experiment with B. subtilis, showing that cell death after
treatment with 11 occurred more rapidly than after treat-
ment with lysobactin, a characteristic result that is con-
sistent with known membrane disrupting antibiotics such
as nisin.23 At identical concentrations, lysobactin showed a
gradual decline to ∼50% cell survival while the analog
displayed an exponential decrease in viability with levels of
cell survival falling below 20% within 1 h (SI, Figure 4).
In B. subtilis, lysobactin began to show significant levels
of membrane disruption at concentrations approaching its
MIC values for some Gram-negative bacteria (i.e., ∼8μg/mL,
Figure 3b).1 We reasoned that the ability to permeabi-
lize bacterial membranes might, in part, contribute to
its moderate activity against Gram-negative bacteria.
However, repeating the membrane disruption assay
with a Gram-negative organism (E. coli) showed no sig-
nificant PI influx for 11 even at the highest concentration
tested (64 μg/mL), whereas lysobactin only caused moder-
ate levels of PI influx at high concentrations (32ꢀ64 μg/mL)
(SI, Figure 3).
In summary, we have completed a highly efficient solid
phase total synthesis of the cyclic depsipeptide antibiotic
lysobactin with only a single purification of the final
product. Using this route, we have synthesized Δ3-Thr-
lysobactin (11) that lacks the functionality required for the
cationꢀπ interaction that has been observed in the parent
natural product. Additional experiments revealed that
B. subtilis cells (a Gram-positive organism) treated with
11 showed significant membrane permeabilization at con-
centrations four times lower than its MIC suggesting that
membrane permeabilization may contibute significantly to
Figure 3. Propidium iodide bacterial membrane disruption
assay. Cell culture of B. subtilis PY79 treated with depsipeptide
concentrations of (a) 1 μg/mL and (b) 8 μg/mL.
its observed antibacterial activity, a notion also supported
by killing kinetics experiments. Interestingly, in E. coli (a
Gram-negative organism), only lysobactin showed any
evidence of membrane permeabilization, and that was at
concentrations significantly greater (32ꢀ64 μg/mL) than
its reported MIC value.1
Additional analogues will reveal the key structural attributes
that are responsible for the biological activity of lysobactin and
enable studies directed toward determining the conformation
of the natural product when bound to its cellular target.
Results of these studies will be reported in due course.
Acknowledgment. Financial support provided by the
National Institutes of Health (AI 059327) and Indiana
University is gratefully acknowledged.
Supporting Information Available. Experimental de-
tails and spectral data for all new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
(23) Ruhr, E.; Sahl, H. G. Antimicrob. Agents Chemother. 1985, 27,
841–845.
(21) Bhakdi, S.; Martin, E. Infect. Immun. 1991, 59, 2955–2962.
(22) Virto, R.; Manas, P.; Alvarez, I.; Condon, S.; Raso, J. Appl.
Environ. Microb. 2005, 71, 5022–5028.
The authors declare no competing financial interest.
Org. Lett., Vol. 14, No. 11, 2012
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