Synthesis of peptides containing overlapping lanthionine bridges on the solid phase: An analogue of rings d and e of the lantibiotic nisin

Article abstract of DOI:10.1021/ol201548m

A methodology for the solid-phase synthesis of the overlapping lanthionine bridges found in many lantibiotics has been developed. A novel Teoc/TMSE-protected lanthionine derivative has been synthesized, and this lanthionine, and an Aloc/allyl-protected la

Full text of DOI:10.1021/ol201548m

ORGANIC
LETTERS
2011
Vol. 13, No. 16
4216–4219
Synthesis of Peptides Containing
Overlapping Lanthionine Bridges on the
Solid Phase: An Analogue of Rings D and
E of the Lantibiotic Nisin
Begum Mothia,† Antony N. Appleyard,‡ Sjoerd Wadman,‡ and Alethea B. Tabor*,†
Department of Chemistry, UCL, 20, Gordon Street, London WC1H 0AJ, U.K., and
NovactaBiosystemsLtd., BioPark Hertfordshire, Broadwater Road, WelwynGarden City,
Hertfordshire AL7 3AX, U.K.
a.b.tabor@ucl.ac.uk
Received June 9, 2011
ABSTRACT
A methodology for the solid-phase synthesis of the overlapping lanthionine bridges found in many lantibiotics has been developed. A novel Teoc/
TMSE-protected lanthionine derivative has been synthesized, and this lanthionine, and an Aloc/allyl-protected lanthionine derivative, have been
incorporated into a linear peptide using solid-phase peptide synthesis. Selective deprotection of the silyl protecting groups, followed by
sequential cyclization, deprotection of the allyl protecting groups, and further cyclization, enabled the regioselective formation of an analogue of
rings D and E of nisin.
The rapid rise of antibiotic resistant bacteria makes it
imperative that chemical biologists explore the synthesis
and mode of action of natural products which exert
antibiotic activity through novel pathways. Lantibiotics
such as nisin have a unique mode of antibiotic action,
which involves binding to lipid II, a key intermediate in the
biosynthesis of the cell walls of Gram-positive bacteria.1,2
They may therefore represent important new leads in the
search for new antibacterial agents to treat antibiotic-
resistant infections. These peptides have very complex
structures, with multiple thioether bridges between side
chains, making them a formidable challenge for synthetic
chemists.3,4
We have previously developed a flexible approach to the
solid-phase synthesis of lanthionine-containing peptides,
using orthogonally protected lanthionine derivative 1.5
This approach has been used by ourselves and others to
synthesize fragments and analogues6ꢀ8 of lantibiotics. The
€
(3) (a) Polinsky, A.; Cooney, M. G.; Toy-Palmer, A.; Osapay, G.;
† UCL.
Goodman, M. J. Med. Chem. 1992, 35, 4185. (b) Toogood, P. L.
Tetrahedron Lett. 1993, 34, 7833. (c) Burrage, S.; Raynham, T.;
Williams, G.; Essex, J. W.; Allen, C.; Cardno, M.; Swali, V.; Bradley,
M. Chem.;Eur. J. 2000, 6, 1455. (d) Okeley, N. M.; Y. Zhu, Y.; van der
Donk, W. A. Org. Lett. 2002, 4, 1335.
‡ Novacta Biosystems Ltd.
(1) Chatterjee, C.; Paul, M.; Xie, L.; van der Donk, W. A. Chem. Rev.
2005, 105, 633.
(2) (a) Breukink, E.; de Kruijff, B. Biochim. Biophys. Acta 1999, 1462,
223. (b) Hsu, S.-T. D.; Breukink, E.; Tischenko, E.; Lutters, M. A. G.; de
Kruijff, B.; Kaptein, R.; Bonvin, A. M. J. J.; van Nuland, N. A. J. Nature
Struct. Mol. Biol. 2004, 11, 963. (c) Hasper, H. E.; Kramer, N. E.; Smith,
J. L.; Hillman, J. D.; Zachariah, C.; Kuipers, O. P.; de Kruijff, B.;
Breukink, E. Science 2006, 313, 1636.
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(4) (a) Osapay, G.; Goodman, M. J. Chem. Soc., Chem. Commun.
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1993, 1599. (b) Osapay, G.; Prokai, L.; Kim, H.-S.; Medzihradszky,
K. F.; Coy, D. H.; Liapakis, G.; Reisine, T.; Melacini, G.; Zhu, Q.;
Wang, S. H.-H.; Mattern, R.-H.; Goodman, M. J. Med. Chem. 1997, 40,
2241.
r
10.1021/ol201548m
Published on Web 07/18/2011
2011 American Chemical Society

approach allows both single rings and peptide sequences
with multiple thioether bridges in sequence to be prepared,
most recently demonstrated by Vederas and co-workers in
the total synthesis, on-resin, of lactocin S.9 However, until
now it has not been possible to synthesize lantibiotic
sequences having two overlapping thioether bridges using
solid-phase techniques. This motif occurs very frequently
in lantibiotics, for example in the C-terminus of nisin 2,
rings D and E, which is thought to be the pore-forming
region of this lantibiotic.
In this paper, we report the first solid-phase synthesis of
a bicyclic peptide containing two overlapping thioether
bridges, using a quadruply orthogonal protecting group
strategy to prepare an analogue of rings D and E of nisin.
We envisaged that the overlapping bridges of rings D and
E of nisin could be prepared from a linear resin-bound
peptide intermediate 3 (Figure 1). This contains two dis-
tinct lanthionine residues with protecting groups orthogo-
nal to each other and also to the transient (Fmoc) and
permanent (Boc/tBu) protecting groups used in Fmoc-
based solid-phase peptide synthesis. We had already de-
monstrated that the allyl ester and Aloc protecting groups
of 1 could be selectively removed with Pd(PPh3)4 without
loss of either Fmoc or Boc protecting groups.6,7 Of the
many other protecting groups available for amino acids,
we selected the β-(trimethylsilyl)ethoxycarbonyl (Teoc)10
and trimethylsilylethyl (TMSE)11 groups for the amino
and carboxylic acid, respectively, as these silyl-based pro-
tecting groups could be easily removed using TBAF under
mild conditions at neutral pH.
As a first step, it was necessary to synthesize the (Teoc,
TMSE)/Fmoc-protectedlanthionine4. Trt-D-Ser-OTMSE
5 was prepared from commercially available Boc-D-Ser-
(Bzl)-OH and then converted to iodoalanine 6 via a Mit-
sunobu reaction (Scheme 1).7 Coupling of 6 to Fmoc-Cys-
OTce12 gave lanthionine 7, which was converted to the
Teoc-protected amine 8 and then successfully deprotected
under neutral conditions13 to afford the desired protected
lanthionine 4.
Figure 1. Strategy for synthesizing nisin rings D and E.
Scheme 1. Synthesis of (Teoc, TMSE/Fmoc) Lanthionine 4
(5) (a) Mohd Mustapa, M. F.; Harris, R.; Bulic-Subanovic, N.;
Elliott, S. L.; Bregant, S.; Groussier, M. F. A.; Mould, J.; Schultz, D.;
Chubb, N. A. L.; Gaffney, P. R. J.; Driscoll, P. C.; Tabor, A. B. J. Org.
Chem. 2003, 68, 8185. For other recent syntheses of orthogonally
protected lanthionine and nor-lanthionine derivatives, see: (b) Zhu,
X.; Schmidt, R. R. Eur. J. Org. Chem. 2003, 4069. (c) Narayan, R. S.;
VanNieuwenhze, M. S. Org. Lett. 2005, 7, 2655. (d) Martin, N. I. J. Org.
ꢀ
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Chem. 2009, 74, 946. (e) Avenoza, A.; Busto, J. H.; Jimenez-Oses, G.;
Peregrina, J. M. Org. Lett. 2006, 8, 2855. (f) Cobb, S. L.; Vederas, J. C.
Org. Biomol. Chem. 2007, 5, 1031.
(6) Mohd Mustapa, M. F.; Harris, R.; Esposito, D.; Chubb, N. A. L.;
Mould, J.; Schultz, D.; Driscoll, P. C.; Tabor, A. B. J. Org. Chem. 2003,
68, 8193.
(7) Bregant, S.; Tabor, A. B. J. Org. Chem. 2005, 70, 2430.
(8) Pattabiraman, V. R.; McKinnie, S. M. K.; Vederas, J. C. Angew.
Chem., Int. Ed. 2008, 47, 9472.
(9) Ross, A. C.; Liu, H.; Pattabiraman, V. R.; Vederas, J. C. J. Am.
Chem. Soc. 2010, 132, 462.
(10) Carpino, L. A.; Tsao, J.-H.; Ringsdorf, H.; Fell, E.; Hettrich, G.
J. Chem. Soc., Chem. Commun. 1978, 358.
(11) (a) Sieber, P. Helv. Chim. Acta 1977, 60, 2711. (b) Marlowe,
C. K. Bioorg. Med. Chem. Lett. 1993, 3, 437.
(12) Boger, D. L.; Ichikawa, S.; Tse, W. C.; Hedrick, M. P.; Jin, Q.
In order to test whether 4 could be used in the solid-
phase synthesis of lanthionine-containing thioether-
bridged cyclic peptides, we first prepared an analogue of
ring D of nisin. The linear resin-bound peptide 9 was first
prepared by standard solid-phase peptide synthesis meth-
ods, incorporating 4 as the second amino acid, and sub-
stituting a Met residue as the third amino acid in place of
the other lanthionine (Scheme 2).
J. Am. Chem. Soc. 2001, 123, 561.
ꢀ
(13) Jou, G.; Gonzalez, I.; Albericio, F.; Lloyd-Williams, P.; Giralt,
E. J. Org. Chem. 1997, 62, 354.
Org. Lett., Vol. 13, No. 16, 2011
4217

complete deprotection of the N-terminus. Cyclization on-
resin15 then gave the resin-bound peptide 10. Finally, Fmoc-
Lys(Boc)-OH was added to the free N-terminus and the
cyclic peptide 11 cleaved from the resin. Purification by
HPLC gave 11, which was characterized by NMR.
We also used the (Aloc, allyl)/Fmoc-protected lanthio-
nine 17 to prepare an analogue of ring E of nisin. Incor-
poration of 1 into a linear resin-bound precursor 12 was
carried out using standard coupling conditions, again sub-
stituting a Met residue for the lanthionine at the fourth
amino acid (Scheme 3). Deprotection of the allyl and Aloc
groups was carried out with Ph(PPh3)4 using N0,N-dimethyl-
barbituric acid (NDMBA) as an allyl group scavenger.16
Removal of the Fmoc group was again followed by on-
resin cyclization to give 13; the sequence was then extended
with Fmoc-Ala-OH before cleavage from the resin. Pur-
ification by reversed-phase HPLC gave 14, which was also
characterized by NMR.
Scheme 2. Synthesis of an Analogue of Ring D of Nisin
Scheme 4. Synthesis of an Analogue of Rings D and E of Nisin
Scheme 3. Synthesis of an Analogue of Ring E of Nisin
Treatment with TBAF successfully removed the Teoc
and TMSE groups;10,11 although the Fmoc group was
probably also removed during the reaction,14 the resin-
bound peptide was then treated with piperidine to ensure
With the two individual rings successfully synthesized and
characterized, we then tackled the synthesis of the bicyclic
peptide with two overlapping lanthionine bridges.
(14) Ueki, M.; Amemiya, M. Tetrahedron Lett. 1987, 28, 6617.
(15) Albericio, F.; Cases, M.; Alsina, J.; Triolo, S. A.; Carpino, L. A.;
Kates, S. A. Tetrahedron Lett. 1997, 38, 4853.
€
(16) (a) Kunz, H.; Marz, J. Angew. Chem., Int. Ed. Engl. 1988, 27,
1375. (b) Machauer, R.; Waldmann, H. Chem.;Eur. J. 2001, 7, 2940.
4218
Org. Lett., Vol. 13, No. 16, 2011

Linear resin-bound peptide 3 was first synthesized, incorpor-
ating both of the protected lanthionines 1 and 4 (Scheme 4).
Deprotection of the allyl and Aloc groups proceeded
smoothly using the same conditions as before, without
removal of the Teoc and TMSE groups. Removal of the
Fmoc group from the (Teoc, TMSE/Fmoc) lanthionine
residue then allowed ring E to be formed on-resin, giving
15 (Scheme 4). In order to install ring D, chain extension
with Fmoc-Ala-OH was then followed by removal of the
Teoc and TMSE groups and a second cyclization reac-
tion. Finally, Fmoc-Lys(Boc)-OH was again added to
the free N-terminus, and the cyclic peptide 16 was
cleaved from the resin. A two-stage purification proce-
dure using C18 SPE columns gave the pure peptide 16.
High-resolution mass spectrometry showed that the
desired peptide had been synthesized, with a mass of
1008.37 Da.17
In conclusion, we have developed an effective metho-
dology for the solid-phase synthesis of peptides containing
overlapping lanthionine bridges. We have demonstrated
the applicability of this approach in the solid-phase synth-
esis of rings D and E of nisin. The D and E segment of nisin
has been synthesized previously in solution by Shiba and
co-workers using a solution-phase desulfurization approach18
and has been coupled to other segments of nisin.19 How-
ever the solid-phase synthesis approach described here
avoids the pitfalls of segment synthesis20 and will allow
for the rapid preparation of all types of lanthionine con-
taining peptides. This quadruply orthogonal protecting
group approach will be a powerful additional tool for the
chemical synthesis of highly structurally complex lantibio-
tics, with the potential to prepare more potent and bioa-
vailable synthetic analogues, unrestricted in amino acid
sequence and composition, that could not be accessed via a
biosynthetic approach.21
Acknowledgment. We thank the Engineering and Phy-
sical Sciences Research Council (EPSRC) for an EngD
studentship (to B.M.) and Novacta Biosystems Ltd. for
financial support. We thank Daniel Nicolau (Department
of Chemistry, UCL) for the allyl/Aloc deprotection pro-
cedure using NDMBA, Lisa Haigh (Department of Chem-
istry, UCL) for assistance with the HRMS data, and John
Kirkpatrick (Research Department of Structural and Mo-
lecular Biology, UCL) for assistance with NMR data.
Supporting Information Available. Experimental pro-
cedures and characterization of all new amino acid and
lanthionine derivatives and of peptides 11, 14, and 16.
This material is available free of charge via the Internet at
http://pubs.acs.org
(17) Sequence characterisation by MS/MS of these bicyclic peptides
is not possible because of the crossed lanthionine bridges which do not
give a unique fragmentation pattern: (a) Kleinnijenhuis, A. J.; Duursma,
M. C.; Breukink, E.; Heeren, R. M. A.; Heck, A. J. R. Anal. Chem. 2003,
75, 3219. (b) Lavanant, H.; Derrick, P. J.; Heck, A. R.; Mellon, F. A.
Anal. Biochem. 1998, 255, 74. (c) Ghalit, N.; Kemmink, J.; Hilbers,
H. W.; Versluis, C.; Rijkers, D. T. S.; Liskamp, R. M. J. Org. Biomol.
Chem. 2007, 5, 924.
(19) Fukase, K.; Kitazawa, M.; Sano, A.; Shimbo, K.; Horimoto, S.;
Fujita, H.; Kubo, A.; Wakamiya, T.; Shiba, T. Bull. Chem. Soc. Jpn.
1992, 65, 2227.
(20) Kent, S. B. H. Chem. Soc. Rev. 2009, 38, 338.
(21) Ross, A. C.; Vederas, J. C. J. Antibiot. 2011, 64, 27.
(18) Fukase, K.; Oda, Y.; Kubo, A.; Wakamiya, T.; Shiba, T. Bull.
Chem. Soc. Jpn. 1990, 63, 1758.
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