Synthesis and Bioactivity of Diastereomers of the Virulence Lanthipeptide Cytolysin

Article abstract of DOI:10.1021/acs.orglett.6b03246

Cytolysin, a two-component lanthipeptide comprising cytolysin S (CylL_S″) and cytolysin L (CylL_L″), is the only family member to exhibit lytic activity against mammalian cells in addition to synergistic antimicrobial activity. A subset of the thioether cross-links of CylL_S″ and CylL_L″ have ll stereochemistry instead of the canonical dl stereochemistry in all previously characterized lanthipeptides. The synthesis of a CylL_S″ variant with dl stereochemistry is reported. Its antimicrobial activity was found to be decreased, but not its lytic activity against red blood cells. Hence, the unusual ll stereochemistry is not responsible for the lytic activity.

Full text of DOI:10.1021/acs.orglett.6b03246

Letter
pubs.acs.org/OrgLett
Synthesis and Bioactivity of Diastereomers of the Virulence
Lanthipeptide Cytolysin
Subha Mukherjee, Liujie Huo, Gabrielle N. Thibodeaux, 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: Cytolysin, a two-component lanthipeptide
comprising cytolysin S (CylLS″) and cytolysin L (CylLL″), is
the only family member to exhibit lytic activity against
mammalian cells in addition to synergistic antimicrobial
activity. A subset of the thioether cross-links of CylLS″ and
CylLL″ have LL stereochemistry instead of the canonical DL
stereochemistry in all previously characterized lanthipeptides.
The synthesis of a CylLS″ variant with DL stereochemistry is
reported. Its antimicrobial activity was found to be decreased,
but not its lytic activity against red blood cells. Hence, the
unusual LL stereochemistry is not responsible for the lytic activity.
anthipeptides belong to the class of ribosomally synthe-
sized and post-translationally modified peptides (RiPPs)
and bear lanthionine (Lan) and methyllanthionine (MeLan)
structures as well as dehydroalanine (Dha) and dehydrobutyr-
ine (Dhb) residues (Figure 1).1 Two-component lanthipeptides
isolates of Enterococcus faecalis.5 Compared with most
lanthipeptides, cytolysin exhibits unusual stereochemistry.
This class of molecules generally contain Lan and MeLan
with DL stereochemistry, i.e., (2S,6R)-lanthionine and
(2S,3S,6R)-methyllanthionine.6 In the case of cytolysin, the B
ring of CylLS″ and the C ring of CylLL″ contain Lan with the
canonical stereochemistry, but LL stereochemistry (i.e.,
(2R,6R)-Lan and (2R,3R,6R)-MeLan) was observed for the A
ring of CylLS″ and the A and B rings of CylLL″ (Figure 1).7
These observations suggested a possible correlation between
the unique stereochemistry and the unusual lytic activity against
mammalian cells. To test this hypothesis, we report the total
synthesis of a diastereomer of cytolysin to investigate the effect
of the stereochemistry of the thioether cross-links on the
biological activity.
L
Syntheses of just five lanthipeptides or their variants have
been reported to date: nisin, lactosin S, both components of
lacticin 3147, epilancin 15x, and lacticin 481.8 Four of these
have been completed on solid phase utilizing an orthogonal
protection scheme that allows on-demand cyclization (Figure
2A). Cytolysin synthesis poses several challenges not present in
these previously synthesized compounds. First, the sequences
of CylLS″ and CylLL″ are extremely hydrophobic, with only a
single charged residue in each peptide (Figure 1). Hydrophobic
peptides are prone to incomplete coupling during solid-phase
peptide synthesis (SPPS) because of inaccessibility of the
reagents to the N-terminus of the elongating peptide chain.9
Second, the structures of cytolysins contain a dehydro amino
acid in the second position within the thioether rings. Dehydro
amino acids cannot be incorporated via the usual elongation
Figure 1. Structures of cytolysin S and L. Lan and Dha (both derived
from Ser) are shown in red, and MeLan and Dhb (both derived from
Thr) are shown in blue.
are an interesting subclass of lanthipeptides in which two
peptides synergistically act to provide antibacterial activity.2
Cytolysin is a two-component lanthipeptide and comprises
cytolysin S (CylLS″) and cytolysin L (CylLL″).3 In addition to
exhibiting synergistic antimicrobial activity, cytolysin is the first
and thus far only lanthipeptide shown to potently lyse
mammalian cells.4 Cytolysin is responsible for the enhancement
of enterococcal virulence and is produced by many clinical
Received: October 29, 2016
© XXXX American Chemical Society
A
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(Figure S1).7 Purified CylLS″-Dhb2Dha was found to have very
similar antimicrobial activity as native CylLS″ (Figure S2), thus
making it a good target for synthesis. Hence, we set out to
make both native CylLS″-Dhb2Dha and its diastereomer with a
DL-MeLan A ring instead of an LL-MeLan. The desired
stereochemistry of the thioether cross-links was preset in
building blocks 3, 4, and 5 (Figure 3).8b,e,11
Figure 3. Building blocks used in SPPS of cytolysin analogues.
To aid in the synthesis of the hydrophobic peptide,
poly(ethylene glycol) (PEG)-based ChemMatrix resin was
employed instead of traditionally used polystyrene (PS) resin.
ChemMatrix resin offers improved chemical stability, and
because of its polar nature, this resin does not interact as much
with the side-chain-protected peptides,12 which we felt was
important given the hydrophobic nature of CylLS″. Addition-
ally, ChemMatrix resin has enhanced swelling properties in a
wide range of solvents, including solvents that minimize peptide
self-association on the resin.12a A trityl-group-containing linker
to the resin was employed to prevent racemization of the C-
terminal Lan residue. The bulky linker was also envisioned to
improve the stability of the C-terminal Lan building block, as
C-terminal-protected Cys residues often suffer from base-
catalyzed elimination and subsequent β-piperidylalanine
formation.13 The entire peptide was successfully synthesized
on-resin (Scheme 1). In place of the dehydroalanine residue in
the second position within the MeLan A ring of CylLS″-
Dhb2Dha, a cysteine was incorporated as a convenient
precursor to Dha.14 After cleavage of the peptide from the
resin and purification, the peptide was reacted with 2,5-
dibromohexanediamide, resulting in the formation of a cyclic
sulfonium intermediate at Cys2. As reported by Davis and co-
workers,14a under basic conditions, elimination generated the
desired dehydroalanine (Scheme 1, inset). The purity of the
final compound 11 was confirmed by analytical high-perform-
ance liquid chromatography and mass spectrometry (MS)
(Figure S3). The desired stereochemistry of the thioether cross-
links in compound 11 was confirmed by gas chromatography
coupled to MS analysis employing a chiral stationary phase
(Figure S4). For direct comparison in bioactivity assays,
CylLS″-Dhb2Dha with the natural LL-MeLan A ring and DL-
Lan B ring (compound 12) was also synthesized (Scheme 1).
This synthetic compound was expected to be identical to the
biosynthetically accessed CylLS″-Dhb2Dha and thus was
envisioned as a good control compound to assess the success
of the synthetic procedure. For the synthesis of compound 12,
similar synthetic steps were employed using synthetic building
blocks 3, 5, and 6.
Figure 2. (A) Orthogonal protecting groups on a (methyl)lanthionine
building block (DL-MeLan here) allow elongation and subsequent
cyclization of a peptide. For alternative protecting group schemes, see
ref 10. (B) Introduction of short oligopeptides containing dehydro
amino acids (Dhx). (C) If the amine coupling partner for cyclization is
a dehydro amino acid (Dha here), the low reactivity of the enamine
promotes hydrolysis to the ketone, preventing cyclization.
methods of Fmoc SPPS because the enamine liberated upon
Fmoc deprotection is very unreactive and would tautomerize to
the imine followed by hydrolysis to the ketone, preventing
further peptide coupling. Therefore, the traditional SPPS routes
to lanthipeptides rely on the preparation of short oligopeptides
containing preinstalled dehydro amino acids (e.g., Figure
2B).8b−e Unfortunately, this strategy does not work with a
dehydro amino acid incorporated at the second position of a
Lan/MeLan-containing ring because the dehydro amino acid is
the point of cyclization (Figure 2C). The synthesis of such a
structure has not been accomplished to date. Here we describe
the synthesis of cytolysin S as well as a diastereomer by
introduction of the dehydro amino acid after cyclization.
To minimize the problem of the high hydrophobicity of the
cytolysins, we chose CylLS″ rather than CylLL″ as our synthetic
target. In addition, the dehydrobutyrine in ring A of CylLS″ was
substituted with a dehydroalanine (CylLS″-Dhb2Dha), which
we envisioned could be accessed from a Cys. We first verified
that this change would not alter the bioactivity of the peptide
by preparing CylLS″-Dhb2Dha biosynthetically via coexpres-
sion of the precursor peptide CylLS-T2S with the lanthipeptide
synthetase CylM in Escherichia coli using previously described
methodology.7 Characterization of the product by tandem mass
spectrometry and GC/MS analysis of derivatized amino acids
after acid hydrolysis of CylLS″-Dhb2Dha demonstrated an LL-
MeLan A ring and a DL-Lan B ring, identical to native CylLS″
B
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Scheme 1. Synthesis of (A) CylLS″-Dhb2Dha (DL A ring, DL B ring) and (B) CylLS″-Dhb2Dha (LL A ring, DL B ring)
Antimicrobial activity was tested in combination with wild-
type (WT) CylLL″ against Lactococcus lactis HP and L. lactis
CNRZ 481. None of the peptides displayed antimicrobial
activity without its partner, and all of the CylLS″ peptides were
active in combination with CylLL″ (Table 1). Isobolograms
Table 1. Minimal Inhibitory Concentrations of Cytolysin
and Derivatives against L. lactis 481
individual MIC
entry
CylLL″
CylLS″
(μM)
combined with CylLL″ MIC (μM)
a
>50
>50
>50
>50
>50
n.a.
0.05
1.0
11
synthetic 12
biosynthetic 12
0.1
Figure 4. Antimicrobial activity assays of WT cytolysin against
Lactococcus lactis 481. The isobologram demonstrates that the MIC of
the combination of CylLL″ and CylLS″ is reached with each
component at 0.05 μM, suggesting a 1:1 stoichiometry in the active
species.
0.1
a
Not applicable.
demonstrated that WT CylLL″ and CylLS″ act in 1:1
stoichiometry with a minimal inhibitory concentration (MIC)
of 0.05 μM for each component (Figure 4 and Table 1).
Expressed CylLS″-Dhb2Dha also acted with 1:1 stoichiometry
but with a 2-fold decrease in MIC (0.1 μM). Synthetic 12
exhibited antimicrobial activity identical to that of biosynthetic
CylLS″-Dhb2Dha, confirming the fidelity of the synthesis
(Table 1 and Figure S5). Conversely, diastereomer 11 exhibited
decreased antimicrobial activity (Figure S5) when combined
with CylLL″, as illustrated by a markedly smaller zone of growth
inhibition and a MIC in liquid culture that was increased 10-
fold (Table 1).
The cytolysin S peptides were also tested in combination
with CylLL″ for synergistic hemolytic activity against rabbit red
blood cells. WT CylLS″ and expressed CylLS″-Dhb2Dha
exhibited very similar hemolytic activities. Surprisingly, 11
with a DL-MeLan A ring and DL-Lan B ring exhibited no
decrease in hemolytic activity (Figure S6). This result shows
C
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(b) Knerr, P. J.; van der Donk, W. A. Annu. Rev. Biochem. 2012, 81,
479.
(2) Lawton, E. M.; Ross, R. P.; Hill, C.; Cotter, P. D. Mini-Rev. Med.
that the influence of stereochemistry is different on the two
activities of cytolysin. Similar findings were reported for
mutants of cytolysin that affected antimicrobial and lytic
activities differently.15
Chem. 2007, 7, 1236.
(3) Gilmore, M. S.; Segarra, R. A.; Booth, M. C.; Bogie, C. P.; Hall, L.
R.; Clewell, D. B. J. Bacteriol. 1994, 176, 7335.
(4) Coburn, P. S.; Gilmore, M. S. Cell. Microbiol. 2003, 5, 661.
(5) Jett, B. D.; Huycke, M. M.; Gilmore, M. S. Clin. Microbiol. Rev.
1994, 7, 462.
(6) Chatterjee, C.; Paul, M.; Xie, L.; van der Donk, W. A. Chem. Rev.
2005, 105, 633.
(7) (a) Tang, W.; van der Donk, W. A. Nat. Chem. Biol. 2013, 9, 157.
In summary, it appears that CylLS″ with the LL stereo-
chemistry of the A ring has evolved for optimal complemen-
tarity with native CylLL″ with respect to antimicrobial activity.
In CylLS″ with DL stereochemistry of the A ring, the synergy
with native CylLL″ is clearly attenuated. Regarding the
hemolytic activity, the stereochemistry of the A ring of CylLS″
does not appear to be important. These findings further
reinforce previous conclusions that these two activities have
different structure−activity relationships.15 They are also
consistent with the proposal that cytolysin evolved predom-
inantly for its antimicrobial activity since E. faecalis is mostly a
commensal organism.16
́ ́
(b) Tang, W.; Jimenez-Oses, G.; Houk, K. N.; van der Donk, W. A.
Nat. Chem. 2015, 7, 57. (c) Tang, W.; Thibodeaux, G. N.; van der
Donk, W. A. ACS Chem. Biol. 2016, 11, 2438.
(8) (a) Fukase, K.; Kitazawa, M.; Sano, A.; Shimbo, K.; Fujita, H.;
Horimoto, S.; Wakamiya, T.; Shiba, T. Tetrahedron Lett. 1988, 29, 795.
(b) Ross, A. C.; Liu, H.; Pattabiraman, V. R.; Vederas, J. C. J. Am.
Chem. Soc. 2010, 132, 462. (c) Liu, W.; Chan, A. S. H.; Liu, H.;
Cochrane, S. A.; Vederas, J. C. J. Am. Chem. Soc. 2011, 133, 14216.
(d) Knerr, P. J.; van der Donk, W. A. J. Am. Chem. Soc. 2012, 134,
7648. (e) Knerr, P. J.; van der Donk, W. A. J. Am. Chem. Soc. 2013,
135, 7094.
(9) (a) Ludwick, A. G.; Jelinski, L. W.; Live, D.; Kintanar, A.; Dumais,
J. J. J. Am. Chem. Soc. 1986, 108, 6493. (b) Masuda, K.; Ooyama, H.;
Shikano, K.; Kondo, K.; Furumitsu, M.; Iwakoshi-Ukena, E.; Ukena, K.
J. Pept. Sci. 2015, 21, 454.
(10) (a) Carrillo, A. K.; VanNieuwenhze, M. S. Org. Lett. 2012, 14,
1034. (b) García-Reynaga, P.; Carrillo, A. K.; VanNieuwenhze, M. S.
Org. Lett. 2012, 14, 1030. (c) Bregant, S.; Tabor, A. B. J. Org. Chem.
2005, 70, 2430. (d) Mothia, B.; Appleyard, A. N.; Wadman, S.; Tabor,
A. B. Org. Lett. 2011, 13, 4216. (e) Narayan, R. S.; VanNieuwenhze,
M. S. Org. Lett. 2005, 7, 2655. (f) Martin, N. I. J. Org. Chem. 2009, 74,
946.
(11) (a) Pattabiraman, V. R.; McKinnie, S. M. K.; Vederas, J. C.
Angew. Chem., Int. Ed. 2008, 47, 9472. (b) Zhu, X.; Schmidt, R. R. Eur.
J. Org. Chem. 2003, 2003, 4069.
ASSOCIATED CONTENT
* Supporting Information
■
S
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.or-
glett.6b03246.
Materials and experimental procedures, synthesis and
characterization of small molecules and peptides, and
biosynthesis of cytolysin analogues and bioactivity assays
(PDF)
AUTHOR INFORMATION
■
Corresponding Author
*vddonk@illinois.edu
ORCID
Wilfred A. van der Donk: 0000-0002-5467-7071
Notes
(12) (a) García-Martín, F.; Quintanar-Audelo, M.; García-Ramos, Y.;
Cruz, L. J.; Gravel, C.; Furic, R.; Co
J. Comb. Chem. 2006, 8, 213. (b) García-Martín, F.; White, P.;
Steinauer, R.; Cote, S.; Tulla-Puche, J.; Albericio, F. Biopolymers 2006,
84, 566.
̂ ́
te, S.; Tulla-Puche, J.; Albericio, F.
The authors declare no competing financial interest.
̂
́
ACKNOWLEDGMENTS
■
(13) (a) Eritja, R.; Ziehler-Martin, J. P.; Walker, P. A.; Lee, T. D.;
Legesse, K.; Albericio, F.; Kaplan, B. E. Tetrahedron 1987, 43, 2675.
(b) Lukszo, J.; Patterson, D.; Albericio, F.; Kates, S. Lett. Pept. Sci.
1996, 3, 157.
(14) (a) Chalker, J. M.; Gunnoo, S. B.; Boutureira, O.; Gerstberger,
S. C.; Fernandez-Gonzalez, M.; Bernardes, G. J. L.; Griffin, L.; Hailu,
H.; Schofield, C. J.; Davis, B. G. Chem. Sci. 2011, 2, 1666.
(b) Morrison, P. M.; Foley, P. J.; Warriner, S. L.; Webb, M. E.
Chem. Commun. 2015, 51, 13470.
We thank Dr. Patrick Knerr (UIUC) for providing Boc-Phe-
Thr-OtBu and DL-Lan building block for initial studies. We
thank Dr. Weixin Tang (UIUC) for providing Lan/MeLan
synthetic standards for GC/MS analysis. We thank Dr.
Alexander V. Ulanov of the Metabolomics Center at UIUC
for assistance with GC/MS analysis. This study was supported
by the Howard Hughes Medical Institute. A Bruker Ultra-
fleXtreme MALDI-TOF/TOF mass spectrometer was pur-
chased in part with a grant from the National Institutes of
Health (S10 RR027109).
(15) Cox, C. R.; Coburn, P. S.; Gilmore, M. S. Curr. Protein Pept. Sci.
2005, 6, 77.
(16) Van Tyne, D.; Martin, M. J.; Gilmore, M. S. Toxins 2013, 5, 895.
REFERENCES
■
(1) (a) Arnison, P. G.; Bibb, M. J.; Bierbaum, G.; Bowers, A. A.;
Bugni, T. S.; Bulaj, G.; Camarero, J. A.; Campopiano, D. J.; Challis, G.
L.; Clardy, J.; Cotter, P. D.; Craik, D. J.; Dawson, M.; Dittmann, E.;
Donadio, S.; Dorrestein, P. C.; Entian, K.-D.; Fischbach, M. A.;
Garavelli, J. S.; Goransson, U.; Gruber, C. W.; Haft, D. H.;
Hemscheidt, T. K.; Hertweck, C.; Hill, C.; Horswill, A. R.; Jaspars,
M.; Kelly, W. L.; Klinman, J. P.; Kuipers, O. P.; Link, A. J.; Liu, W.;
Marahiel, M. A.; Mitchell, D. A.; Moll, G. N.; Moore, B. S.; Muller, R.;
Nair, S. K.; Nes, I. F.; Norris, G. E.; Olivera, B. M.; Onaka, H.;
Patchett, M. L.; Piel, J.; Reaney, M. J. T.; Rebuffat, S.; Ross, R. P.; Sahl,
H.-G.; Schmidt, E. W.; Selsted, M. E.; Severinov, K.; Shen, B.; Sivonen,
K.; Smith, L.; Stein, T.; Sussmuth, R. D.; Tagg, J. R.; Tang, G.-L.;
Truman, A. W.; Vederas, J. C.; Walsh, C. T.; Walton, J. D.; Wenzel, S.
C.; Willey, J. M.; van der Donk, W. A. Nat. Prod. Rep. 2013, 30, 108.
D
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