Use of lantibiotic synthetases for the preparation of bioactive constrained peptides

Article abstract of DOI:10.1016/j.bmcl.2008.01.062

Stabilization of biologically active peptides is a major goal in peptide-based drug design. Cyclization is an often-used strategy to enhance resistance of peptides toward protease degradation and simultaneously improve their affinity for targets by restricting their conformational flexibility. Among the various cyclization strategies, the use of thioether crosslinks has been successful for various peptides including enkephalin. The synthesis of these thioethers can be arduous, especially for longer peptides. Described herein is an enzymatic strategy taking advantage of the lantibiotic synthetase LctM that dehydrates Ser and Thr residues to the corresponding dehydroalanine and dehydrobutyrine residues and catalyzes the Michael-type addition of Cys residues to form thioether crosslinks. The use of LctM to prepare thioether containing analogs of enkephalin, contryphan, and inhibitors of human tripeptidyl peptidase II and spider venom epimerase is demonstrated.

Full text of DOI:10.1016/j.bmcl.2008.01.062

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3025–3028
Use of lantibiotic synthetases for the preparation
of bioactive constrained peptides
Matthew R. Levengood and Wilfred A. van der Donk*
Department of Chemistry, University of Illinois, 600 S. Mathews Avenue, Urbana, IL 61801, USA
Received 15 November 2007; revised 13 January 2008; accepted 15 January 2008
Available online 19 January 2008
Abstract—Stabilization of biologically active peptides is a major goal in peptide-based drug design. Cyclization is an often-used
strategy to enhance resistance of peptides toward protease degradation and simultaneously improve their affinity for targets by
restricting their conformational flexibility. Among the various cyclization strategies, the use of thioether crosslinks has been success-
ful for various peptides including enkephalin. The synthesis of these thioethers can be arduous, especially for longer peptides.
Described herein is an enzymatic strategy taking advantage of the lantibiotic synthetase LctM that dehydrates Ser and Thr residues
to the corresponding dehydroalanine and dehydrobutyrine residues and catalyzes the Michael-type addition of Cys residues to form
thioether crosslinks. The use of LctM to prepare thioether containing analogs of enkephalin, contryphan, and inhibitors of human
tripeptidyl peptidase II and spider venom epimerase is demonstrated.
Ó 2008 Elsevier Ltd. All rights reserved.
Interest in peptide-based materials for use in human
therapeutics has greatly increased in recent years, and
fully synthetic peptide drugs have increasingly reached
the clinic.1 The proteolytic instability of peptides still
presents a limitation, however, for widespread utiliza-
tion of peptide therapeutics. An often employed strategy
for the design of peptide-based drugs with improved
selectivity and decreased proteolytic susceptibility in-
volves cyclization to constrain their conformational flex-
ibility.2 A notable example features the stabilization of
enkephalin by the introduction of a thioether crosslink
between two alanines (Fig. 1), which increased the bio-
activity of the compound by several orders of magnitude
due to increased biostability.3 Other studies have also
shown the increased stability of peptides and proteins
by thioether crosslinks.4,5
OH
NH
O
N
NH2
NH
H
O
N
O
H2N
O
S
H
N
O
HN
O
NH
O
N
H
O
O
Ph
HN
N
HO
H2N
H
S
N
HN
X
O
N
O
H
O
O
R
O
R
Contryphan Sm (X = S, R = NH2)
Thioether analog 2 (X = CH2, R = Ala)
Enkephalin Analogs:
(1) (R = OH)
(1a) (R = Ala)
ValAla
PheAla
ArgAlaHN
GlyLeuHN
O
O
Spider Venom
Thioether crosslinks between two alanine residues are
called lanthionines and their synthesis has received much
attention.6 Despite important recent advances,7–12
at present it is still difficult to introduce these structures
efficiently into synthetic peptides, especially for large pep-
tides. One promising route to these structures is through
Epimerase Inhibitor (3)
Tripeptidyl Peptidase II Inhibitor (4)
Figure 1. Compounds prepared using lacticin 481 synthetase.
the biosynthetic machinery for lantibiotics. These com-
pounds are ribosomally synthesized and post-translation-
ally modified antimicrobial peptides.13,14 The first step in
the modification process of class II lantibiotics involves
phosphorylation of Ser and Thr residues and subsequent
elimination of the phosphate group to generate dehydro-
alanines and Z-dehydrobutyrines, respectively (Fig. 2).15
Keywords: Cyclic peptides; Enkephalin; Conotoxin; Lantibiotic;
Dehydroalanine.
*
Corresponding author. Tel.: +1 217 244 5360; fax: +1 217 244
8024; e-mail: vddonk@uiuc.edu
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.01.062

3026
M. R. Levengood, W. A. van der Donk / Bioorg. Med. Chem. Lett. 18 (2008) 3025–3028
Xn = n amino acid peptide
Xn
well documented and several approaches have been re-
ported to overcome their rapid proteolytic degradation,
poor receptor subtype selectivity, and relatively poor
bioavailability. Goodman and co-workers prepared
and tested a series of lanthionine containing enkephalin
analogs and determined their relative in vivo activity.
Structure 1 (Fig. 1) displayed an ED50 of 0.0018 nM
compared to a value of 15 nM for morphine as an ago-
nist for the l- and d-opioid receptors in mouse models.
Degradation studies attributed the subnanomolar anal-
gesic potencies to increased biostability.3 In this work
we pursued an in vitro enzymatic approach in which
the enkephalin peptide was attached to the C-terminus
of the lacticin 481 leader peptide with a Lys inserted
right before the enkephalin peptide (Fig. 4). This Lys al-
lows proteolytic removal of the leader peptide and isola-
tion of the enkephalin analog.
Xn
O
O
dehydratase
ATP
HN
HS
O
HN
O
R
R
HN
HN
2-
OH
OPO3
SH
R = H, Me
dehydratase
Xn
2-
HPO4
Xn
O
cyclase
O
HN
R
NH
HN
O
HN
O
HS
R = H, Dha
R = Me, Dhb
S
R
R = H, lanthionine
R = Me, 3-methyllanthionine
Figure 2. Post-translational modifications in lantibiotic biosynthesis.
The preparation of the substrate for modification by
LctM was achieved using a ligation strategy that relied
on the Cu(I)-catalyzed [2+3] cycloaddition25 of an al-
kyne moiety appended to the C-terminus of the leader
peptide17 (5) and an azide attached to the N-terminus
of a peptide comprising a linker peptide (underlined),
the aforementioned Lys (green), and the cyclic enkepha-
lin precursor (Fig. 4, 6). All peptides were prepared by
standard Fmoc-based solid phase peptide synthesis.
Upon generation of the ligation product, it was incu-
bated with lacticin 481 synthetase in the presence of
ATP and MgCl2. Subsequent analysis by MALDI mass
spectrometry demonstrated that the peptide had been
dehydrated (Supporting Information). Furthermore,
treatment with the thiol derivatizing agent p-hydroxy-
mercuribenzoic acid (PMBA)26,27 revealed the absence
of a thiol group in the product indicating cyclization
had taken place. This conclusion was further supported
by the absence of an adduct upon treatment of the enzy-
matic product with 2-mercaptoethanol, demonstrating
that a reactive Michael acceptor was not present. Based
on our previous studies as well as unsuccessful in vivo
attempts to cyclize this peptide using the nisin biosyn-
thetic machinery,21 it is very likely that the cyclization
In a succeeding step, intramolecular Michael-type addi-
tion by Cys residues to the dehydroamino acids forms
the lanthionine (from Ser) and methyllanthionine (from
Thr) crosslinks. Lacticin 481 synthetase (LctM) carries
out both steps in a regio- and stereoselective fashion as
illustrated in Figure 3.16 The enzyme only carries out
post-translational modification of the structural region
of its peptide substrate LctA. Although not absolutely re-
quired for activity and not modified itself, the leader pep-
tide greatly enhances the efficiency of LctM.17 In previous
studies we have shown that LctM tolerates variants of the
LctA substrate peptide, including substitution of Ser,
Thr, and Cys residues with non-proteinogenic ana-
logs.18–20 Furthermore, we17,18 and Moll and co-work-
ers21–24 have shown that non-lantibiotic peptides
attached to the leader sequence of lantibiotics are sub-
strates for the biosynthetic enzymes, and that the leader
and structural peptide can be linked by non-peptidic
structures. We here report the use of LctM for the prep-
aration of a series of peptides with diverse biological
properties including the previously mentioned lanthio-
nine-containing enkephalin derivative, a thioether analog
of the conotoxin contryphan (2), and inhibitors of spider
venom epimerase (3) and human tripeptidyl exopeptidase
II (4) (Fig. 1).
O
H
N
S
t
Bu
N3
leader peptide
GGSGVKYSGFCA
Enkephalins are thought to constitute the physiological
ligand for the human d-opioid receptor. The disadvan-
tages of enkephalins as therapeutic agents have been
5
6
1) CuSO4, hydrazine, TBTA
2) 2-mercaptoethanol
H
N
GGSGVKYSGFCA
leader peptide
structural region
leader peptide
leader peptide
N
N
24 25
1
51
N
N
O
T S
S
T
MKEQNSFNLLQEVTESELDLILGA
KGGSGVIH I HECNMN WQFVF CCS
7
LctA
LctM, ATP, Mg2+
LctM, ATP
S
leader peptide
Lys
Gly
S
H
N
NH
D
GGSGVKYAlaGFAlaA
H
N
O
B
N
N
Gly
Ser
Gly
Val
A
O
S
His
Glu
Met
Asn
Ile
O
NH
D
Ser
O
HN
His
HN
O
8
S
NH
Asn
O
Me
C
D
endoprotease Lys-C
O
NH
Phe
O
Ile
Val
Trp
Phe
Gln
Lacticin 481
1a
Figure 3. Biosynthetic pathway for the formation of lacticin 481 by the
bifunctional enzyme LctM.
Figure 4. Enzymatic route toward enkephalin analog 1a.

M. R. Levengood, W. A. van der Donk / Bioorg. Med. Chem. Lett. 18 (2008) 3025–3028
3027
step is performed by the enzyme but we cannot rule out
that some of the cyclic product arises from non-enzy-
matic background reaction. If so, it would not affect
the stereochemistry of cyclization because both the
non-enzymatic and enzymatic cyclization exhibit high
stereoselectivity for the D-isomer for peptides in which
the Dha and Cys are separated by two amino acids,10–
mercaptoethanol and the resulting peptide 13 was incu-
bated with LctM and ATP/MgCl2. Analysis of the assay
product by MALDI mass spectrometry demonstrated
that the peptide had been dehydrated although signifi-
cantly more enzyme was required for complete conver-
sion. Treatment with PMBA showed that the thiol of
homocysteine was not present in the final product, con-
sistent with the formation of a homolanthionine ring
structure 14 (Supporting Information). Proteolysis with
trypsin resulted in cleavage behind an Arg that had been
inserted intentionally.
12,28
including previous studies on the enkephalin
peptide.29 After having established that peptide 8 was
cyclized, it was incubated with endoproteinase Lys-C,
and the product was purified by HPLC providing a pep-
tide with the expected molecular weight for 1a as
determined by ESI MS (Supporting Information).
The venom peptide of the funnel web spider Agelenopsis
aperta contains a D-serine residue that is incorporated
by epimerization of an L-serine. Tanner and co-workers
reported a dehydroalanine containing peptide 3 as a po-
tent inhibitor of the epimerase, prepared by oxidative
elimination of a phenylselenocysteine precursor.11,38
Based on our previous work, we anticipated that the
dehydropeptide could be prepared rapidly using an
enzymatic dehydration. Although the precursor peptide
can be prepared using molecular biology techniques be-
cause unlike contryphans, it does not contain any non-
proteinogenic amino acids, we again used a purely syn-
thetic approach as this allows the possibility for intro-
duction of non-natural residues and therefore more
synthetic flexibility. The strategy to prepare peptide 3
followed the scheme depicted in Figure 6. As expected,
peptide 15 was a substrate for LctM resulting in the
dehydrated product 16. The target dehydropeptide
inhibitor 3 was obtained following incubation with
endoproteinase Lys-C and HPLC purification, which
provided peptide 3 as determined by ESI MS (Support-
ing Information).
Conotoxins are constrained, cyclic, post-translationally
modified peptides found in the venom of carnivorous
cone snails. They usually contain one or more disulfides
and have high affinities for various cell surface recep-
tors. As such they have received much attention as po-
tential therapeutics.30 Several approaches have been
reported to stabilize the reduction-sensitive disulfide
bridges, including the use of diselenide analogs (9)31
or thioethers (10)32 (Fig. 5). We decided to explore the
use of LctM for the preparation of analogs of conotox-
ins in which the disulfide bond would be replaced by a
thioether linkage of the same length (Fig. 5, 11). We
envisioned this could be achieved by an enzyme cata-
lyzed Michael-type addition of a homocysteine to a
dehydroalanine. The contryphans are a family of pep-
tides containing a single disulfide as well as other
post-translational modifications such as D-Trp and 4-
trans-hydroxyPro (O).33–37 The precursor peptide for
contryphan-Sm was prepared by standard Fmoc-based
SPPS. At the site of the C-terminal Cys of contry-
phan-Sm, a homocysteine was incorporated protected
as an ethyldisulfide. Upon linking peptide 12 to the lea-
der peptide using the Cu(I)-catalyzed [2+3] cycloaddi-
tion, the homocysteine residue was deprotected with 2-
In a final application of the use of lacticin 481 synthe-
tase, we focused on human tripeptidyl peptidase II from
erythrocytes, a serine peptidase belonging to the subtili-
sin class. Previous studies have reported the dehydroala-
nine containing pentapeptide 4 as a potent inhibitor
(Ki = 20 nM). The precursor peptide was again prepared
by SPPS and Cu(I)-catalyzed ligation, and upon treat-
ment with LctM in the presence of ATP and MgCl2,
complete dehydration was observed (Supporting
Information).
Figure 5. Enzymatic preparation of a thioether analog of contryphan-
Sm.
Figure 6. Enzymatic preparation of a snake venom epimerase
inhibitor.

3028
M. R. Levengood, W. A. van der Donk / Bioorg. Med. Chem. Lett. 18 (2008) 3025–3028
10. Zhou, H.; van der Donk, W. A. Org. Lett. 2002, 4, 1335.
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13. Chatterjee, C.; Paul, M.; Xie, L.; van der Donk, W. A.
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In summary, this work demonstrates the remarkable
versatility of lacticin 481 synthetase. The enzyme effi-
ciently catalyzed the dehydration of Ser residues that
vary greatly in their flanking residues as well as the dis-
tance to the leader peptide. Furthermore the enzyme cat-
alyzed the formation of thioether containing cyclic
peptides, an approach that can be readily extended to
the preparation of libraries of compounds. Although
some of the products prepared in this study, notably
peptides 3 and 4, could have been prepared more readily
using straightforward peptide synthesis, the strength of
the methodology described herein is in the preparation
of long peptides that are still difficult to prepare by SPPS
in non-specialized laboratories. Lantibiotic synthetases
have been shown to process Ser/Thr residues as far as
42 residues C-terminal from the leader peptide in de-
signed peptides.23 Therefore, these enzymes can be used
to prepare peptides containing thioether rings and/or
dehydro amino acids in large peptides that are not read-
ily amenable to synthetic chemistry. The use of a prote-
ase cleavage site then allows the removal of the leader
peptide and triazole linker. We note that when a target
peptide contains a Lys or Arg, the use of endoproteinase
Lys-C or trypsin is prohibited, but we have previously
demonstrated that other proteases with more defined
recognition sites such as Factor Xa can be used as
well.39 Therefore, the use of lantibiotic synthetases offers
much potential for preparing designer peptides.
21. Kluskens, L. D.; Kuipers, A.; Rink, R.; de Boef, E.;
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Acknowledgments
This work was supported by the National Institutes of
Health (GM58822) and a Ruth-Kirschstein National
Research Award T32 (GM008276 to M.R.L.).
Supplementary data
Experimental procedures and mass spectrometric char-
acterization of all peptides. Supplementary data associ-
ated with this article can be found, in the online
version, at doi:10.1016/j.bmcl.2008.01.062.
32. Bondebjerg, J.; Grunnet, M.; Jespersen, T.; Meldal, M.
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