Structure and total synthesis of lysobactin (katanosin B)

Article abstract of DOI:10.1002/anie.200604232

(Chemical Equation Presented) Tackle-resistant bacteria: Determination of the 3D structure of the antibiotic lysobactin has led to its total synthesis and resulted in a high-yielding macrolactamization step. The minimal use of protecting groups allowed pr

Full text of DOI:10.1002/anie.200604232

Angewandte
Chemie
DOI: 10.1002/anie.200604232
Antibiotic Resistance
Structure and Total Synthesis of Lysobactin (Katanosin B)**
Franz von Nussbaum,* Sonja Anlauf, Jordi Benet-Buchholz, Dieter Häbich,
Johannes Köbberling, Lµszló Musza, Joachim Telser, Helga Rübsamen-Waigmann, and
Nina A. Brunner
Bacterial infections increasingly evade standard treatments as
resistance to multiple antibiotics is growing. Multiresistant
pathogens are no longer a problem restricted to hospitals.[1]
New antibacterial lead structures[2] are urgently needed to
guarantee future therapeutic efficacy. With advanced tech-
nologies in screening,chemistry,and genomics (the genomics
revolution),[3] we possess new tools to transform natural
products into valuable antibacterial drugs (chemical post-
evolution).[4] A flexible and efficient total synthesis is among
the most powerful means to foster this process.
The cyclodepsipeptide[5] lysobactin (1,also known as
katanosin B) was isolated independently from bacteria
(Lysobacter sp.)[6] and from a strain related to the genus
Cytophaga (Scheme 1).[7] The natural depsipeptides pose a
significantly greater challenge for synthesis than regular
peptides because of their nonribosomal motifs,such as
hydroxylated or d-configured amino acids.[8] In particular,
macrocyclic depsipeptides are demanding and often sensitive
synthetic targets.[9] For many years,various research groups
have tried to gain synthetic access to the natural lariat
structure 1,[10,11] but,to the best of our knowledge,a complete
total synthesis has not been published so far.
Lysobactin has excellent in vitro activity against a wide
range of Gram-positive bacteria which are increasingly
associated with life-threatening infections,including multi-
resistant variants such as methicillin-resistant Staphylococcus
Scheme 1. Lariat structure of lysobactin (1), consisting of a dipeptidic
linear segment (d-Leu1–Leu2) anda nonapeptidic cyclic segment
(HyPhe3–Ser11). The depsipeptide 1 contains various nonribosomal
amino acids. Atom-description indices based on amino acid residue
andthen atom number. HyLeu =threo-hydroxyleucine;[12] aThr=allo-
threonine; HyAsn=threo-b-hydroxyasparagine;[13] HyPhe=threo-b-
hydroxyphenylalanine, Boc=tert-butoxycarbonyl, Cbz=benzyloxycar-
bonyl.
aureus (MRSA) and vancomycin-resistant enterococci
(VRE).[14] Substantial in vivo efficacy was shown in a mouse
sepsis model.[7] The bacterial cell wall precursor lipid II has
been assumed to be the target of 1. Inhibition of the bacterial
transglycosylation process would be based on molecular
recognition of 1 and lipid II,which are binding partners of
comparable molecular weights.[14]
[*] Dr. F. von Nussbaum, S. Anlauf, Dr. J. Benet-Buchholz,[+]
Dr. D. Häbich, Dr. J. Köbberling, Dr. L. Musza,[+] Dr. J. Telser
Bayer HealthCare AG
Medicinal Chemistry Europe
42096 Wuppertal (Germany)
Fax: (+49)202-36-9694797
E-mail: franz.nussbaum@bayerhealthcare.com
We envisioned lysobactin to be a biologically promising
but synthetically demanding lead structure. Although gram
amounts of 1 were accessible through fermentation,[15] for the
purpose of comprehensive chemical postevolution,[4] full
synthetic control of the whole molecule well beyond semi-
synthesis was our goal. The feasibility of a synthetic de novo
approach in this unexplored class should be proven with a
flexible and efficient total synthesis.
Until now,as is the case for the majority of sensitive
macrocyclic depsipeptides,no crystal structure has been
reported for 1,and two reported structures of 1 have been a
matter of debate.[7,16] Although total synthesis is a classical
way to confirm the structure of a natural product,[17] the
15 stereogenic centers left too many unaccountable options.
Thus,unambiguous clarity of the structure of 1 was necessary
prior to any attempt at synthesis.
Prof. Dr. H. Rübsamen-Waigmann, Dr. N. A. Brunner
AiCuris GmbH & Co. KG
Bayer Pharma- undChemiepark
Friedrich-Ebert Strasse 475
42117 Wuppertal (Germany)
[+] J.B.-B.: X-ray crystallography. L.M.: NMR solution structure.
[**] We are grateful to our colleagues C. FehØr, S. Heald, P. Schmitt, and
S. Seip for NMR analyses. G. Schiffer determined the minimal
inhibitory concentrations. A. Mayer-Bartschmidt and M.-A. Brüning
carriedout the fermentations. W. Schröder helpedus with small-
scale peptide analyses. J. Ragot resynthesized compound 2. We
thank A. Göller, J. Schamberger, andM. Lobell for fruitful
discussions about the 3D structure. We are indebted to H. Wild and
H. Haning for their continuous support of this project andto H.
Schirok andN. Griebenow for a critical revision of the manuscript.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Communications
Parallel crystallization studies were carried out with
Preorganization was expected to result in favorable cycliza-
tion kinetics,provided that most groups that would affect the
conformational constraint were in place.[19] Therefore,unpro-
tected side chains were expected to exert a beneficial
influence. Avoiding protection coincided well with our aim
to design a short and economic synthesis.
The biosynthesis of several cyclic depsipeptides proceeds
through a macrolactonization step.[20] With natural material 1
at hand,it was straightforward to evaluate this cyclization
method in a semisynthetic way (Scheme 2). We tried to
recyclize the ring-opened lysobactin 4 as well as the corre-
sponding N2.1-Boc derivative 5 under a number of esterifica-
tion conditions (4!1, 5!2). Although anhydro species were
observed,HPLC analysis of our reaction mixtures by co-
injection with 1 and semisynthetic 2[21]
natural 1 in aqueous solvent systems. Even at this stage,
opening of the macrolactone ring turned out to be the
omnipresent threat in lysobactin chemistry. Under basic or
neutral conditions,cleavage of the ester group readily
occurred (1!4,pH 7, t1/2 ꢀ 10 h). The inherent need to use
acidic conditions restricted the choice of applicable chemical
manipulations and purification protocols throughout the
entire project. However,a solution of 1 in acidic methanol
(pH ꢀ 3) proved to be stable over a prolonged period of time.
[18]
Fortunately,a crystal structure
was obtained that unques-
tionably verified the sequence originally published by Tymiak
et al.[16] For the first time,the instructive 3D architecture of 1
was at our disposal (Figure 1) and could serve as a basis for
showed that condensation had not
occurred between the C1.11 and O3.3
sites. Too many hydroxy groups had
competed with the rather unreactive
secondary alcohol at HyPhe3. Accord-
ingly,macrolactamization rather than
macrolactonization appeared to be the
method of choice: the superior reac-
tivity of an amine should outrival com-
peting nucleophilic groups.
For
our
macrolactamization
approach,all fragments were synthe-
sized de novo. The overall strategy had
to take into consideration the partic-
ular requirements posed by the func-
tionally crowded northern region of 1.
Figure 1. Crystal structure of 1, stereoview. Hydrogen atoms have been removed for clarity; O
red, N blue.
3
ꢁ
prospective chemical operations. Held in place by a rigid
cyclic peptide backbone that is stabilized by multiple hydro-
gen bonds,the side chains give 1 a highly organized,globular
appearance. Hydrophobic and hydrophilic domains can
clearly be distinguished. A transannular charge–p interaction
clamps the aromatic moiety of HyPhe3 and the d-Arg6
guanidinium group. While the lariat-type 2D structure of 1
suggests a flexible linear segment that points into the solvent,
in fact,the two N-terminal leucin moieties are in tight contact
with the rigid peptidic backbone fixed by four hydrogen
bonds and several van der Waals interactions.
Special attention was given to the sensitive Ser11 HyPhe
ester bond and to the nonribosomal amino acid HyAsn10 with
its multiple functionalities. In view of our unsuccessful
biomimetic cyclization experiments (Scheme 2),we chose to
form the ester bond early in the synthesis. This would allow
easier differentiation of the alcohols and reduce steric
To complement the biological and the chemical relevance
of the solid-state structure,an NMR solution structure was
determined. Conformational flexibility was only observed
near the N-terminal d-Leu1 residue. ROESY and NOESY
1
experiments gave the through-space H–1H distances,which,
together with torsion angles from coupling constants,dem-
onstrated that the solution structure of 1,particularly for the
large cyclic segment,was basically the same as in the solid
state.
At that point our target structure was defined. Yet,several
hurdles remained to be overcome en route to 1,which
included: 1) mode and site of cyclization,2) ester formation,
and 3) ester preservation in the presence of a plethora of
nucleophilic functional groups. Cyclization of the 28-mem-
bered ring was expected to be the most delicate task.
Fortunately, 1 is a rigid molecule with little conformational
flexibility in solution and in the solid state (Figure 1).
3.3
Scheme 2. Semisynthetic cyclization studies. Biomimetic C1.11
ꢁ
O
macrolactonization was not observed. Reagents and conditions:
a) EDC, DMAP, CH2Cl2/DMF; or 2,4,6-trichlorobenzoylchloride, DIPEA,
THF; then DMAP. EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodi-
imide; DIPEA=diisopropylethylamine; DMAP=4-dimethylaminopyri-
dine; DMF=N,N-dimethylformamide.
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interactions during the attack on the secondary alcohol of
HyPhe3. On the other hand,the labile ester would have to be
conserved throughout multiple synthetic steps and purifica-
tion procedures. Three different levels of protecting groups
were deemed to be essential (Scheme 3). The situation in the
southern region was different. Despite a strongly basic
guanidinium group in d-Arg6,minimal side-chain protection
seemed possible,and the unprotected functional groups were
envisioned to facilitate preorganization in the cyclization step.
For convergence,the cyclic segment of the lysobactin lariat
was cut into two equally long subfragments. The depsipeptidic
ester bond was concealed in the middle of the linear northern
fragment. Two major fragments 11 and 12 were the logical
consequence (Scheme 3).
avoided using toxic osmium salts,reduced the need for
protecting groups,and led to minimal epimerization during
aminolysis.
3
The formation of the depsipeptidic Ser11 HyPhe ester
ꢁ
bond was most critical. Direct couplings of tripeptide 8 with
Boc-Gly-HyAsn-(tBu)Ser-OH or even longer fragments gave
unsatisfactory results: low yields and unacceptable levels of
epimerization were observed in every case. Hence, 8 was
linked by reaction with a single amino acid 9. The missing
dipeptide 7 was subsequently attached to 10,thereby finishing
the northern fragment 11.
At this stage,we had to connect and cyclize the main
fragments 11 and 12. We chose the sterically less hindered
aThr8-Gly9 site for the first step. Coupling of segments 11 and
12 occurred with almost no epimerization. To obtain the full
potential of preorganization,all protective groups,with the
exception of the Cbz group,were then removed,which
liberated the open-chain precursor 13. Ring closure at a
concentration of 1.1 mm occurred smoothly with 72% yield
(13!3). Hydrogenolytic deprotection had to be carried out
under strictly controlled conditions to avoid over-reduction
(3!1). We obtained synthetic lysobactin,which was indis-
tinguishable from the natural material according to all
analytical and biological data (Table 1).
The southern fragment 12 was indeed synthesized with
standard liquid-phase peptide chemistry by using Boc-pro-
tection and HATU/NMM coupling strategy without protec-
tion of the side chains.
For the northern fragment 11,a 2-(trimethylsilyl)ethyl
(TMSE) ester was chosen as the third level of protection
(along with Cbz and Boc). The synthesis of a suitable HyAsn
precursor turned out to be troublesome until we established
the dipeptidic building block Boc-Gly-HyAsn-OH (7,
Scheme 3). The most efficient method to insert HyAsn10
into 11 started from 6[22] by using selective esterification of
the side chain. The free amino group of O4-methyl-HyAsn
was captured with an active ester of Boc-Gly followed by
aminolysis to yield the primary amide 7. This sequence
In summary,we have designed a convergent total syn-
thesis of lysobactin with a high-yielding macrolactamization
step. As a result of minimal use of protecting groups,the
present approach leaves ample room for postevolutive
Scheme 3. Convergent synthesis of lysobactin through macrolactamization. Reagents andconditions: a) conc. aqueous HCl, MeOH, 130 8C,
microwave, 3 min, 89%; b) Boc-Gly-OSu, DIPEA, DMAP, quant.; c) aqueous NH3 (25%), 52%; d) 9, DCC, DMAP, CH2Cl2, molecular sieves (4 Š),
84%; e) 20% TFA in CH2Cl2, 10 min, 83%; f) 7, HATU, NMM, DMF, 79%; g) 20% TFA in CH2Cl2, 10 min, 80%; h) 11, HATU, NMM, DMF, 67%;
i) TBAF, Na2SO4, molecular sieves (4 Š), THF, 89%; j) iPr3SiH, TFA, H2O, 86%; k) HATU, NMM, DMF, 72%; l) H2, 10% Pd/C, TFA (0.1%), 1,4-
dioxane, 88%. DCC=dicyclohexylcarbodiimide, HATU=O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluronium hexafluorophosphate; OSu=N-
oxysuccinimido, NMM=N-methylmorpholine, TBAF=tetra-n-butylammonium fluoride, TFA=trifluoroacetic acid.
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Communications
Table 1: Minimal inhibitory concentrations (MIC) [mgmLꢁ1] as deter-
mined according to CLSI conditions.
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about the 3D structure of 1 provides a firm basis for a future
understanding and rational tuning of the interactions of
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10
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Keywords: antibiotics · cyclization · drug resistance ·
natural products · total synthesis
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