Ribosomal synthesis of dehydrobutyrine- and methyllanthionine-containing peptides

Article abstract of DOI:10.1039/b904314d

We report here the ribosomal synthesis of methyllanthionine-containing cyclic peptides involving a site-specific incorporation of vinylglycine under the reprogrammed genetic code, followed by the isomerization of the vinylglycine to dehydrobutyrine, and t

Full text of DOI:10.1039/b904314d

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Ribosomal synthesis of dehydrobutyrine- and
methyllanthionine-containing peptidesw
Yuki Goto,ab Kazuhiro Iwasaki,ac Kohei Torikai,a Hiroshi Murakamia and Hiroaki Suga*abc
Received (in Cambridge, UK) 3rd March 2009, Accepted 31st March 2009
First published as an Advance Article on the web 30th April 2009
DOI: 10.1039/b904314d
We report here the ribosomal synthesis of methyllanthionine-
containing cyclic peptides involving a site-specific incorporation
of vinylglycine under the reprogrammed genetic code, followed
by the isomerization of the vinylglycine to dehydrobutyrine, and
the subsequent intramolecular Michael addition of a cysteine
residue placed at a downstream position of the vinylglycine.
precursor peptides bearing vinylglycine (Vgl) residue. We
conceived that Vgl could be useful as a key non-proteinogenic
amino acid based on the following considerations: (1) Vgl
could be site-specifically incorporated into the desired peptide
sequence by means of the genetic code reprogramming,4 and
Cys could also be incorporated into a downstream position of
the Vgl residue in the peptide chain. (2) Heating of the
Vgl-containing peptide at nearly neutral pH could induce
isomerization from Vgl to Dhb, and simultaneously, the
resulting Dhb residue could accept the intramolecular Michael
addition by Cys side-chains. Our report herein includes the
ribosomal synthesis of MeLn-containing cyclic peptides that
mimic the B- and C-ring segments of nisin,2c one of the
members of lantibiotics.
Dehydrobutyrine (Dhb), one of the family of dehydroamino
acids, is frequently found in naturally occurring peptides.1 For
instance, lantibiotics, a class of bacteriocin, have Dhb residues
that are generated by enzymatic dehydration of a threonine
residue in the precursor peptides expressed by the translation
machinery.2 In addition, lantibiotics also contain thioether-
bridged cyclic residues, referred to as methyllanthionine
(MeLn). These unique residues are generated from Dhb
residues by nucleophilic attack of the sulfhydryl group in
cysteine (Cys) residues via enzymatic intramolecular Michael
addition, forming cyclic structures.2 The thioether bonds
formed at MeLn residues are non-reducible, thereby providing
physiological stability of their active structures. This suggests
that MeLn-bridged cyclic structures provide excellent peptidic
frameworks for the discovery of new drug candidates.3
However, since the formation of Dhb and MeLn residues
from the precursor peptides relies on the actions of specific
enzymes,2 the biosynthetic method would not be synthetically
versatile enough to generate diverse kinds of such peptides. On
the other hand, direct incorporation of Dhb into the nascent
peptide chain is very likely intractable due to the hydrolytic
instability of the 2-amino group on Dhb itself (decomposing to
the 2-oxoacyl group) and its inefficient elongation by the
translation machinery. Hence, a generic and simple method
that facilitates the synthesis of Dhb- and MeLn-containing
peptides, particularly which enables the construction of a
library is needed.
Although it was known that treatment of N-benzoyl-Vgl
esters under acidic or basic conditions in non-aqueous media,
resulted in isomerization from Vgl to Dhb residue,5 the
chemical behaviour of Vgl at neutral or near-neutral pH in
aqueous media was unknown in the literature. Therefore, at
the first stage of this series of experiments, we synthesized two
esters, N-acetyl-Vgl methyl ester and Vgl 3,5-dinitrobenzyl
ester (1 and 2 in Fig. 1A and B, respectively), and their
chemical behaviours in D2O buffered at pH 7.4 were
monitored by 1H-NMR. It should be noted that 1 was a
mimic of Vgl in a peptidic structure6 and 2 was a substrate
for our acylation RNA catalyst (flexible tRNA acylation
ribozyme; flexizyme)4c that would be used in the subsequent
experiment. The summary of the observations was as follows
(see more detailed observations and discussions in the ESIw):
(1) The a-proton of 1 and 2 was deuterated at 37 1C over 2.5 h
to yield 3a and 4, respectively, but no isomerization to Dhb
was observed. (2) Upon heating 1 at 95 1C for 0.5 h, the
isomerization of 1 occurred to yield 5 with a single deuteration
on the g-methyl group, along with a byproduct 3b generated
through the ester hydrolysis. The characteristic triplet at
d 6.83 ppm assigned to the b-olefinic proton of Dhb has a
similar chemical shift to that of (Z)-Dhb found in lantibiotics.7
Based on the above results, we predicted that 2 would
We report here a novel methodology for the ribosomal
synthesis of Dhb-containing peptides and MeLn-containing
cyclic peptides without enzymatic assistance by expressing the
a Research Center for Advanced Science and Technology, The
University of Tokyo, 4-6-1, Komaba, Meguro, Tokyo 153-8904,
Japan. E-mail: hsuga@rcast.u-tokyo.ac.jp; Fax: +81 3 5452 5495;
Tel: +81 3 5452 5495
b Department of Advanced Interdisciplinary Studies, Graduate School
of Engineering, The University of Tokyo, 4-6-1, Komaba, Meguro,
Tokyo 153-8904, Japan
c Department of Chemistry and Biotechnology, Graduate School of
Engineering, The University of Tokyo, 4-6-1, Komaba, Meguro,
Tokyo 153-8904, Japan
w Electronic supplementary information (ESI) available: Discussion
about the model reactions for isomerization of Vgl and the
configuration of MeLn, NMR spectra of the model reactions, and
the experimental methods. See DOI: 10.1039/b904314d
Fig. 1 Model reactions for the isomerization of Vgl to Dhb. Each
1
reaction was monitored by H-NMR. See ESI for more details.w
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remain intact under mild conditions, i.e. Vgl would be likely
intact during flexizyme-catalyzing aminoacylation without
isomerization to Dhb, while at elevated temperatures Vgl in
peptide chain would isomerize to (Z)-Dhb.
At the second stage, the ribosomal synthesis of a model
peptide containing Vgl at a specific site was performed
(Fig. 2A, P-vgl). We used the genetic code reprogramming
by means of a withdrawn PURE (protein translation using
recombinant elements)8 (wPURE) system integrated with the
flexizyme.4c–e In this particular experiment, a wPURE system
lacking tryptophan (Trp) was prepared to make the cognate
codon (UGG) vacant; Vgl-tRNAAsnE-2
4d was prepared by
CCA
the flexizyme and added to the wPURE system for the
reassignment of UGG to Vgl. Tricine-SDS-PAGE analysis
of the peptide expressed under three different conditions
(Fig. 2B) revealed that P-vgl was expressed only in the
presence of Vgl-tRNAAsnEÀ2CCA in the wPURE system (lanes
2 vs. 3) and its expression level was 44% relative to the
wildtype peptide (P-wt) expressed in the wPURE system plus
Trp (lanes 1 vs. 3). MALDI-TOF mass spectrometry of P-vgl
showed a single major peak consistent with the desired
molecular mass (Fig. 2C). Upon heating P-vgl at 95 1C, we
expected that the Vgl would isomerize to Dhb residue. To
confirm this Vgl - Dhb isomerization, P-vgl was incubated
with an excess amount of 2-mercaptoethanol (ME), by which
the generated Dhb was trapped to yield the corresponding
Michael-adduct (P-vgl-ME). In fact, the molecular weight of
the resulting peptide increased by 78 Da that was consistent
with that of ME (Fig. 2D). This result clearly demonstrates
that the isomerization of Vgl to Dhb occurs under heating and
the resulting Dhb residue is readily reactive with a thiol via
intermolecular Michael addition to afford thioethers.
Fig. 3 Synthesis of B-ring (A) and C-ring (B) segments of nisin.
CDAP reaction of each peptide is shown in the grey box. Asterisks
indicate stereogenic centers newly produced by the intramolecular
Michael addition. See ESI for the MALDI-TOF mass spectra of the
respective peptides and the stereochemistry.w
peptides as synthetic targets based on the sequences of
B- and C-ring segments of nisin (Fig. 3A and B, P-MeLn1
and P-MeLn2). We designed two mRNAs encoding the
MeLn-containing cyclic peptide sequences along with the
C-terminal Flag-tag appendix, in which the MeLn residue was
replaced with Vgl and Cys assigned by UGG and UGC codons
(mR2 and mR3). MALDI-TOF mass spectrometry of the
expressed peptides showed the consistent mass values with those
of Vgl-containing peptides (P-preMeLn1 and P-preMeLn2,
respectively; ESI Fig. S1A and E).w We then performed thermal
isomerization of the Vgl to Dhb residue and simultaneous
intramolecular Michael addition of the Cys thiol. Unlike
the intermolecular reaction demonstrated in Fig. 2, the
molecular weight of the peptide before and after the tandem
isomerization-cyclization would not change; therefore we
verified if the Cys thiol in the products reacted with 1-cyano-
4-dimethylaminopyridinium tetrafluoroborate (CDAP), which
is known as a thiol-selective cyanylation agent9 suitable for
monitoring a loss of the free thiol group. As expected,
when both precursor peptides were treated with CDAP, their
respective mass values increased by 25 Da corresponding to the
cyano group (ESI Fig. S1B and F).w In contrast, upon the
CDAP treatment of the heated peptides (MnLn1 and MnLn2),
their mass values did not change (ESI Fig. S1D and H).w This
concluded that the desired cyclization took place smoothly to
afford the MeLn-containing cyclic peptides.10,11
At the final stage of our experiments, we synthesized
MeLn-containing cyclic peptides. We chose two cyclic
Fig. 2 Ribosomal synthesis of a Dhb-containing peptide. (A) mRNA
and the expressed peptide sequences used in this study. Conversions
from Vgl to Dhb, and to the ME adduct was illustrated in the grey
box. Flag in parentheses indicates the RNA sequence encoding a Flag
peptide (DYKDDDDK). (B) Tricine-SDS PAGE analysis of the
translation products. Lane 1, wild type expression in wPURE system
plus Trp; lane 2, wPURE system with tRNAAsnE-2CCA, lanes 3,
wPURE system with Vgl-tRNAAsnE-2CCA. The band indicated by
the asterisk corresponds to the remaining [14C]-Asp that was not
incorporated into the peptide. (C) MALDI-TOF mass spectrum of
the expressed peptide in wPURE system before ME treatment, and (D)
that after. The calculated (Calc) and observed (Obs) mass values are
shown in each spectrum. Each peak labelled with asterisk denotes a
sodium adduct of the major product.
Here we have reported a novel methodology for the
ribosomal synthesis of MeLn-containing cyclic peptides
without assistance of modification enzymes. The key chemistry
utilized in this method is the incorporation of Vgl into a
specific site designated by a reprogrammed genetic code and
the thermal isomerization of the Vgl residue to Dhb followed
by simultaneous intramolecular Michael addition of the
sulfhydryl group of a Cys residue present in the peptide chain.
The virtue of this method requires no reagents that potentially
damage the amino acid residues after the incorporation of Vgl.
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Moreover, the peptide sequence is readily programmable by
the mRNA template sequence. Seebeck et al. recently reported
a method to generate dehydroalanine-containing peptides
from the selenalysine-containing precursor peptides expressed
by translation.12 This elegant method relies on oxidative
elimination of selenium by hydrogen peroxide to afford a
dehydroalanine residue. However, strong oxidative reagents
such as hydrogen peroxide could also oxidize the thiol in Cys
(as well as the sulfide group in methionine) exisiting in the
peptide chain, so that Cys may be no longer reactive with Dha
as a Michael donor. In contrast, the method reported here
resulted in a clean formation of peptides bearing intact Dhb
and Cys residues, which can readily be transformed into MeLn
residues by heating. Hence, a coupling of ribosomal peptide
library synthesis with this methodology opens a new avenue
for the discovery of novel MeLn-containing cyclic peptides
with not only antimicrobial but also other biological activities
when it is integrated with an appropriate screening technique.
This work was supported by grants of the Japan Society for
the Promotion of Science Grants-in-Aid for Scientific
Research (S) (16101007) to H. S., Grants-in-Aid for JSPS
Fellows (18-10526) to Y. G., and a research and development
projects of the Industrial Science and Technology Program in
the New Energy and Industrial Technology Development
Organization (NEDO).
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