Chemical synthesis of the lantibiotic lacticin 481 reveals the importance of lanthionine stereochemistry

Article abstract of DOI:10.1021/ja4014024

Lantibiotics are a family of antibacterial peptide natural products characterized by the post-translational installation of the thioether-containing amino acids lanthionine and methyllanthionine. Until recently, only a single naturally occurring stereoche

Full text of DOI:10.1021/ja4014024

Communication
pubs.acs.org/JACS
Chemical Synthesis of the Lantibiotic Lacticin 481 Reveals the
Importance of Lanthionine Stereochemistry
Patrick J. Knerr and Wilfred A. van der Donk*
Howard Hughes Medical Institute and Roger Adams Laboratory, Department of Chemistry, University of Illinois at
Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, United States
S
* Supporting Information
ABSTRACT: Lantibiotics are a family of antibacterial
peptide natural products characterized by the post-
translational installation of the thioether-containing
amino acids lanthionine and methyllanthionine. Until
recently, only a single naturally occurring stereochemical
configuration for each of these cross-links was known. The
discovery of lantibiotics with alternative lanthionine and
methyllanthionine stereochemistry has prompted an
investigation of its importance to biological activity.
Here, solid-supported chemical synthesis enabled the
total synthesis of the lantibiotic lacticin 481 and analogues
containing cross-links with non-native stereochemical
configurations. Biological evaluation revealed that these
alterations abolished the antibacterial activity in all of the
Figure 1. Sequence and ring topology of the lantibiotic lacticin 481 (1)
and chemical structures of its post-translational modifications.
analogues, revealing the critical importance of the
enzymatically installed stereochemistry for the biological
activity of lacticin 481.
importance of the cross-link stereochemistry to the biological
activity and mirrors a growing appreciation of stereoisomeric
natural product biosynthesis.17
To engineer lantibiotics for therapeutic use, a variety of in vivo
and in vitro platforms have been developed to produce analogues
ibosomally synthesized and post-translationally modified
Rpeptides (RiPPs) have become recognized as a major class
of natural products.1,2 The structural and functional diversity of
for the exploration of structure−activity relationships and
mechanisms of action.8,18,19 Of these approaches, total chemical
RiPPs has expanded greatly in recent years because of the
growing availability of sequenced genomes and the application of
synthesis presents the opportunity to remove any dependence on
bioinformatic analyses to the discovery of new compounds.3−7
the biosynthetic machinery of the producing organism, thus
opening up a wider chemical space for potential exploration.20
One of the largest and best-studied classes of RiPPs are the
The recent solid-supported total syntheses of lactocin S,21 both
lanthipeptides, which are polycyclic peptides with complex
peptides of lacticin 3147,14 and analogues of epilancin 15X22
topologies enforced by the thioether-containing cross-links meso-
lanthionine (Lan) and (2S,3S,6R)-3-methyllanthionine
(MeLan) (Figure 1).8 Many members of this family, termed
lantibiotics, possess potent antimicrobial activity against a variety
of human pathogens, and have therefore garnered substantial
attention for clinical applications.9−12 The biosynthesis of
lanthipeptides involves the enzymatic dehydration of select
serine and threonine residues in a linear, ribosomally synthesized
precursor peptide to yield 2,3-didehydroalanine (Dha) and (Z)-
2,3-didehydrobutyrine (Dhb), followed by enzymatic cyclization
via intramolecular Michael-type anti addition of cysteinyl thiols
to afford the Lan/MeLan structures.8 Until recently, all
characterized Lan and MeLan structures were reported to
possess D stereochemistry at the newly formed α-stereocenter
and thus an overall DL configuration (Figure 1);13−15 this
enforced the assumption that all lanthipeptide cross-links possess
this configuration because they are made by homologous
enzymes. The very recent discovery of lanthipeptides containing
cross-links with an LL configuration16 has called into question the
have demonstrated that complex lantibiotics, including those
containing overlapping topologies and MeLan cross-links, are
feasible synthetic targets. This approach has also been used by
Vederas and co-workers to produce lantibiotic analogues
containing non-thioether-based cross-links,23 an achievement
inaccessible to the biosynthetic system. Other recent synthetic
advances have focused on individual rings in various lanti-
biotics.24−27 However, the role of Lan/MeLan stereochemistry
in antibacterial activity has not been addressed to date. In this
study, chemical synthesis was used to construct the lantibiotic
lacticin 481 (1) (Figure 1). Systematic replacement of each DL-
Lan/MeLan cross-link with its LL stereoisomer enabled the first
assessment of the effect of the cross-link stereochemistry on the
antibacterial activity. While synthetic 1 possessed biological
activity comparable to the authentic natural product, all of the
Received: February 7, 2013
Published: April 26, 2013
© 2013 American Chemical Society
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a
stereochemical analogues were found to be inactive, highlighting
the importance of the natural, enzymatically installed Lan/
MeLan stereochemical configuration for biological activity.
Lacticin 481 is a tricyclic lantibiotic produced by Lactococcus
lactis subsp. lactis. This natural product exerts its antibacterial
activity via inhibition of transglycosylation involved in the
biosynthesis of peptidoglycan, likely via binding to the
peptidoglycan precursor lipid II.28 The in vitro reconstitution
of its biosynthesis in 200429 has led to the development of a
chemoenzymatic platform to produce analogues containing
nonproteinogenic amino acids,30,31 several of which display
improved antimicrobial activity compared with the parent
compound. However, as this approach relies on the biosynthetic
machinery to install the desired post-translational modifications,
alteration of the Lan/MeLan stereochemistry cannot be
achieved. Indeed, attempts to produce MeLan stereoisomers
biosynthetically from peptides containing allo-threonine were
unsuccessful, as allo-threonine was not accepted as a substrate.32
Therefore, we drew upon previous total syntheses of lantibiotics
via 9-fluorenylmethoxycarbonyl-based solid-phase peptide syn-
thesis (Fmoc-SPPS)14,22 to construct 1 and the desired
analogues bearing cross-link stereoisomers. Our approach
involved the solid-supported construction of the peptide
backbone incorporating orthogonally protected Lan/MeLan
building blocks, each of which could be selectively deprotected
and cyclized with the N-terminus of the growing peptide to yield
the desired cross-links. For 1, three such building blocks are
necessary: an orthogonal pair of Lan building blocks (2 and 3) for
the overlapping B and C rings, and one MeLan building block (4)
for the A ring (Figure 2).
Scheme 1. Synthesis of DL-2
a
Reagents and conditions: (a) pNzCl, Na2CO3, H2O, 1,4-dioxane; (b)
pNbBr, NaHCO3, DMF, 94% (two steps); (c) CF3CO2H, Pr3SiH,
CH2Cl2, 88%; (d) D-6, NaHCO3, Bu4NBr, EtOAc, H2O, Bu3P, 78%;
(e) CF3CO2H, PhSiH3, CH2Cl2, 95%. Abbreviations: pNz, p-
nitrobenzyloxycarbonyl; pNb, p-nitrobenzyl; Trt, trityl.
i
Following the successful syntheses of these three diastereo-
meric pairs of building blocks, we approached the construction of
1 containing only the natural DL configuration of the cross-links
via Fmoc-SPPS (Scheme 2). Preloaded Wang resin with a low-
density substitution of 0.1 mmol/g was utilized, effectively
preventing intermolecular side reactions during the solid-
supported cyclization reactions. Fmoc deprotection was
performed using piperidine, and amino acids were activated for
coupling using N,N′-diisopropylcarbodiimide (DIC) and 1-
hydroxybenzotriazole (HOBt) or 1-hydroxy-7-azabenzotriazole
(HOAt). To install the Dhb residue at position 24, the dipeptide
Fmoc-Phe-(Z)-Dhb-OH22 was synthesized in solution and
coupled under these standard conditions. After completion of
intermediate 9, the nitrobenzyl-based protecting groups of the
Lan building block were removed by treatment with 6 M SnCl2
and 5 mM HCl in N,N′-dimethylformamide (DMF), leaving the
allyl-based groups unaffected. After removal of the Fmoc group
from the N-terminus, cyclization was promoted using two 3 h
treatments with (7-azabenzotriazol-1-yloxy)tripyrrol-
idinophosphonium hexafluorophosphate (PyAOP), HOAt, and
2,4,6-collidine to give 10 bearing the C ring of 1. Further SPPS
gave 11, and subsequent removal of the allyl-based protecting
groups with Pd(PPh3)4 and phenylsilane, Fmoc removal, and
cyclization as described above yielded bicyclic intermediate 12.
Isoleucine coupling and installation of the A ring via the same
chemical sequence used for 10 gave tricyclic intermediate 13.
After further SPPS to complete the amino acid sequence,
cleavage from the resin and global deprotection were achieved
using trifluoroacetic acid, water, and triisopropylsilane to give
synthetic 1.
Figure 2. Structures of Lan/MeLan building blocks 2−4.
The syntheses of DL-333 and DL-422 have been reported
previously. The construction of the p-nitrobenzyl (pNb)-
protected Lan DL-2 proceeded with full preservation of
stereochemical integrity via a phase-transfer condensation of
protected D-cysteine D-6 and bromoalanine 7 (Scheme 1).
Importantly, as the cross-link stereochemistry was preinstalled
into each building block, a simple exchange of the D-amino acid
starting material with the L isomer afforded the LL diastereomers
of all three building blocks (LL-2, LL-3, and LL-4) in similar overall
yields [see the Supporting Information (SI)]. In the case of LL-4,
the use of L-threonine as starting material generated a change in
two stereocenters, yielding an overall configuration of
(2R,3R,6R). Because of the anti addition observed during the
biosynthesis of all naturally occurring MeLan cross-links to date,
we chose to explore this stereoisomer rather than those that
would result from net syn addition [i.e., (2R,3S,6R) and
(2S,3R,6R)], which have not been found in natural lanthipep-
tides.13,16,32
To probe the importance of the cross-link stereochemistry for
the biological activity, each ring of 1 was systematically changed
from the natural DL configuration to the diastereomeric LL
configuration. This goal was accomplished simply by replacing
the DL-Lan/MeLan building block used in the synthesis of 1 with
its LL counterpart. In this way, three additional peptides
containing Lan/MeLan stereoisomers for the A ring (LL-A),
the B ring (LL-B), and the C ring (LL-C) of lacticin 481 were
constructed.
Reversed-phase high-performance liquid chromatography
(RP-HPLC) purification gave milligram quantities of the desired
peptides in average overall yields of 1.3%, corresponding to an
average yield per step of 92%. Analysis via analytical RP-HPLC
revealed that 1 coeluted with authentic lacticin 481 (Figure S1 in
the SI). Interestingly, the analogues LL-A, LL-B, and LL-C all
exhibited substantial deviations in retention time compared with
1, which may indicate changes in the overall three-dimensional
structure of the peptide (Figures S2−S4). The desired ring
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Communication
a b
,
Scheme 2. Solid-Supported Synthesis of 1
a
Reagents and conditions: (a) SPPS; (b) SnCl2, HCl, DMF; (c) piperidine, DMF; (d) PyAOP, HOAt, 2,4,6-collidine, DMF; (e) Pd(PPh3)4, PhSiH3,
b
i
DMF, CH2Cl2; (f) CF3CO2H, H2O, Pr3SiH. Prior to cleavage from the resin, all residues contained appropriate side-chain protecting groups for
Fmoc-SPPS: tert-butoxycarbonyl (Boc) for Lys and Trp, tert-butyl (tBu) for Ser, tert-butyl ester (OtBu) for Glu, and trityl (Trt) for Asn, Gln, and
His. For experimental details, see the SI.
topology of 1 was confirmed using tandem mass spectrometry by
comparison to authentic lacticin 481 (Figure S5). The entire
SPPS, cleavage, and purification sequence could be completed in
10−12 days.
The desired absolute stereochemical configurations of the
Lan/MeLan residues in each peptide were confirmed via chiral
gas chromatography−mass spectrometry (GC−MS) analysis of
the hydrolyzed and derivatized peptide using synthetic standards
(Figure S6).15,21 During the course of each synthesis, an
appreciable amount of epimerization in the Lan building blocks
was observed, possibly from repeated exposure of the ester-
protected building blocks to piperidine. Fortunately, these
epimerization byproducts could be separated from the desired
full-length peptides during HPLC purification and were isolated
in submilligram quantities. Only the desired product of each
synthesis, as identified by chiral GC−MS analysis, was used for
Figure 3. Evaluation of the antibacterial activities of authentic and
subsequent biological evaluation.
synthetic 1 and the analogues LL-A, LL-B, and LL-C against L. lactis subsp.
cremoris HP in liquid culture. Error bars represent standard deviations of
The antimicrobial activity of 1 and its diastereomers LL-A, LL-B,
and LL-C were assessed and compared to authentic lacticin 481
using growth inhibition assays in liquid culture. Cultures of the
indicator strain L. lactis subsp. cremoris HP were treated with a 2-
fold serial dilution of each peptide in a 96-well plate format. Half-
maximal inhibitory concentration (IC50) and minimal inhibitory
concentration (MIC) values were determined from plots of
culture optical density at 600 nm (OD600) versus peptide
concentration (Figure 3). As expected, the activity of synthetic 1
(IC50 = 300 70 nM; MIC = 625 nM) was the same as that of the
authentic natural product (IC50 = 250 50 nM; MIC = 625 nM)
within experimental error. However, none of the three
diastereomeric analogues possessed any activity up to the
highest concentration tested (10 μM). These observations
indicate that the natural DL configuration of each cross-link is
essential for the biological activity of 1.
The ability of each analogue to antagonize the antibacterial
activity of authentic lacticin 481 was also tested. Several
lantibiotics form multimeric complexes with lipid II;34,35 indeed,
haloduracin α, which contains the mersacidin-like lipid II binding
motif that is also present in lacticin 481, binds lipid II with 2:1
stoichiometry.36 It was therefore possible that the inactive
lacticin 481 analogues could still bind the same biological target
as wild-type lacticin 481 but lack the ability to form the
complexes necessary for full activity. If this were the case,
three replicates.
antagonism could result when active and inactive species are
supplied together. This possibility was investigated by applying
authentic lacticin 481 and each of the three analogues on agar
plates cultured with L. lactis HP. In each case, no antagonism was
observed (Figures 4 and S8), likely indicating that the inactive
analogues lack the ability to bind the biological target of lacticin
481.
In this work, both the first total synthesis of lacticin 481, a
lantibiotic possessing a complex tricyclic topology, and the first
investigation of the importance of the Lan/MeLan stereo-
chemistry for the antibacterial activity of a lantibiotic have been
reported. In the case of lacticin 481, substitution of any of the
three DL-Lan/MeLan residues with the corresponding LL
stereoisomers completely abolished the activity. As it has
recently been shown that some of the homologous enzymes
that determine the stereochemistry of the Lan/MeLan residues
can make both DL and LL isomers,16 the results provided herein
suggest that the stereochemistry of lacticin 481 evolved
specifically to optimize tight binding of its biological target and
not because its biosynthetic machinery is limited to generating
only the DL configuration. The substantial difference between the
RP-HPLC retention times of 1 and its stereoisomeric analogues
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Figure 4. Evaluation of antagonism between 1 and the analogue LL-C on
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(Figures S1−S4) indicates an alteration in the three-dimensional
structure that likely prevents binding of the target and thus
eliminates the activity, a conclusion reinforced by the lack of
antagonism when active and inactive species were applied
simultaneously. However, these findings leave unaddressed the
potential importance of the newly discovered LL stereo-
configuration of cross-links in several other natural lantibiotics,
including both peptides of the enterococcal cytolysin and the β-
peptide of haloduracin.16 Further synthetic efforts using this
solid-supported strategy may shed additional light on the
stereochemistry−activity relationships of these compounds.
ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, characterization of novel molecules,
and GC−MS results. This material is available free of charge via
the Internet at http://pubs.acs.org.
■
S
(22) Knerr, P. J.; van der Donk, W. A. J. Am. Chem. Soc. 2012, 134,
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AUTHOR INFORMATION
Corresponding Author
vddonk@illinois.edu
■
Notes
(24) Carrillo, A. K.; VanNieuwenhze, M. S. Org. Lett. 2012, 14, 1034.
(25) García-Reynaga, P.; Carrillo, A. K.; VanNieuwenhze, M. S. Org.
Lett. 2012, 14, 1030.
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(27) Mothia, B.; Appleyard, A. N.; Wadman, S.; Tabor, A. B. Org. Lett.
2011, 13, 4216.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The authors thank Ting Chen (University of Illinois) for support
with small-molecule synthesis, Dr. Juan Velas
■
́
quez (University of
Illinois) for providing authentic lacticin 481, and Weixin Tang
(University of Illinois) for providing synthetic standards for
GC−MS analysis. This work was supported by the National
Institutes of Health (GM58822 to W.A.v.d.D.) and an American
Heart Association Midwest Affiliate Predoctoral Fellowship
(11PRE7620039 to P.J.K.).
(28) Knerr, P. J.; Oman, T. J.; Garcia de Gonzalo, C. V.; Lupoli, T. J.;
Walker, S.; van der Donk, W. A. ACS Chem. Biol. 2012, 7, 1791.
(29) Xie, L.; Miller, L. M.; Chatterjee, C.; Averin, O.; Kelleher, N. L.;
van der Donk, W. A. Science 2004, 303, 679.
(30) Levengood, M. R.; Knerr, P. J.; Oman, T. J.; van der Donk, W. A. J.
Am. Chem. Soc. 2009, 131, 12024.
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Donk, W. A. J. Am. Chem. Soc. 2012, 134, 6952.
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