Chemoenzymatic Synthesis of Thiazolyl Peptide Natural
Products Featuring an Enzyme-Catalyzed Formal [4 + 2]
Cycloaddition
Walter J. Wever
†
, Jonathan W. Bogart
†
, Joshua A. Baccile
‡
, Andrew N. Chan
§
, Frank C.
Schroeder
‡
, and Albert A. Bowers
*,†
†
Division of Chemical Biology and Medicinal Chemistry, Eshelman School of Pharmacy,
University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States
‡
Boyce Thompson Institute and Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14853, United States
§
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
27599, United States
Abstract
Thiocillins from Bacillus cereus ATCC 14579 are members of the well-known thiazolyl peptide
class of natural product antibiotics, the biosynthesis of which has recently been shown to proceed
via posttranslational modification of ribosomally encoded precursor peptides. It has long been
hypothesized that the final step of thiazolyl peptide biosynthesis involves a formal [4 + 2]
cycloaddition between two dehydroalanines, a unique transformation that had eluded enzymatic
characterization. Here we demonstrate that TclM, a single enzyme from the thiocillin biosynthetic
pathway, catalyzes this transformation. To facilitate characterization of this new class of enzyme,
we have developed a combined chemical and biological route to the complex peptide substrate,
relying on chemical synthesis of a modified C-terminal fragment and coupling to a 38-residue
leader peptide by means of native chemical ligation (NCL). This strategy, combined with active
enzyme, provides a new chemoenzymatic route to this promising class of antibiotics.
Thiazolyl peptides are a growing class of highly modified, peptide-derived natural products
with potent activity against antibiotic resistant bacteria, including methicillin-resistant
Staphylococcus aureus and vancomycin-resistant Enterococci.
1,2
Despite their potential
therapeutic value, thiazolyl peptides have gone largely underdeveloped because of their
generally poor solubility and bioavailability. Moreover, efforts to improve on these limiting
properties by means of synthesis or chemical modification are hindered by the complex
macrocyclic architecture of the compound class: thiazolyl peptides feature multiple azoline
© 2015 American Chemical Society
*
Corresponding Author abower2@email.unc.edu.
The authors declare no competing financial interest.
Supporting Information
Experimental details, synthetic schemes, and figures. This material is available free of charge via the Internet at http://pubs.acs.org.
HHS Public Access
Author manuscript
J Am Chem Soc. Author manuscript; available in PMC 2016 March 18.
Published in final edited form as:
J Am Chem Soc. 2015 March 18; 137(10): 3494–3497. doi:10.1021/jacs.5b00940.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
heterocycles alternating with modified peptide side chains and cyclized on a trisubstituted
pyridine (or piperidine) core (Figure 1).
Although a number of total syntheses have tackled the task of modified thiazolyl peptides, to
date the most substantive improvements have come by means of semisynthetic strategies.
3–7
Researchers at Novartis installed new solubilizing functionalities at the C-terminus of
Streptomycete-derived GE2270A, yielding LFF571, a derivative currently in phase-II
clinical trials for treatment of Clostridium difficile.
8
Nevertheless, the key central
macrocycle, constituting the majority of the drug pharmacophore, remains largely
intractable to semisynthesis, limiting LFF571 solubility and efficacy to infections of the
upper gastrointestinal tract.
Thiazolyl peptides are ribosomally synthesized and posttranslationally modified peptide
natural products (RiPPs).
9
They are biosynthesized from a precursor peptide containing an
N-terminal leader and a C-terminal core peptide motif. Knockout studies in Bacillus cereus
ATCC 14579 have demonstrated the importance of the putative enzyme TclM in late-stage
biosynthetic cyclization of this group of thiazolyl peptides.
10–12
TclM presents a potentially
unique enzymatic activity: the long-hypothesized formal [4 + 2] cycloaddition between two
dehydroalanines (Dhas) to generate the substituted pyridine core of the thiocillins.
13–15
This
putative “hetero-Diels–Alderase” activity is unique from both enzymatic and purely
synthetic standpoints. In terms of enzymology, very few such “Diels–Alderases” are known,
let alone members effecting a heteroannulative version of the chemistry. Enzymes that have
been characterized as potential “Diels–Alderases”
16
often enhance spontaneous chemical
cyclizations (e.g., SpnF
17
and VstJ
18
), yet the proposed transformation of TclM is
demonstrably nonspontaneous: Moody and co-workers have induced this chemistry on
simple substrates by severe microwave irradiation.
19
From a synthetic standpoint, TclM
could provide a mild, late-stage route to the thiazolyl peptide core and allow access to new
soluble derivatives. Herein we develop a combined chemical and biological strategy to
access a leader-peptide-conjugated, doubly dehydrated substrate for TclM and demonstrate
in vitro the ability of TclM to catalyze a formal [4 + 2] cycloaddition to form the mature
pyridine ring.
Previous experiments in our lab suggested that the leader peptide is necessary for TclM
modification of the putative substrate, but solid-phase peptide synthesis (SPPS) of a full 52-
residue substrate with thiazolyl and dehydroamino acid residues seemed daunting, even with
current SPPS technology. SPPS of the full substrate would also necessitate complete
resynthesis of C-terminal variants for structure–activity probing of TclM. We therefore
devised a strategy involving separate preparations of the C-terminal-modified component
and the N-terminal leader peptide (Figure 2a). The fully modified C-terminal fragment could
be prepared by total chemical synthesis, with cysteine side chains as surrogates for the
required dehydroalanine residues. The use of cysteine at the N-terminus of the modified
fragment would allow coupling to an intein-derived thioester of the leader peptide via native
chemical ligation (NCL). Upon coupling, cysteine side chains could be converted to Dhas by
a bisalkylation/elimination protocol.
20
Wever et al.
Page 2
J Am Chem Soc. Author manuscript; available in PMC 2016 March 18.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
We chose to prepare a simplified C-terminal fragment containing all six thiazoles of the
natural thiocillin but with alanines used in place of threonines 4 and 6 and valine 8 and an
ethyl ester in place of the two modified threonines at the C-terminus. Previous efforts had
indicated that TclM would be permissive to alanine substitution at these sites, although these
analogues would likely not be antibiotically active.
12
This hexathiazole precursor could be
built by adapted protocols from previous total syntheses by Bagley, Ciufolini, and Nicolaou
(see the Supporting Information (SI)).
3–5,7,21
Although we anticipate that this route will be
readily amenable to the solid phase, we employed a block solution-phase synthesis. The
desired product was obtained in good yield over seven steps (longest linear sequence from
the building blocks; see the SI) and could then be deprotected to give compound 12.
The 38-residue leader peptide was cloned into vector pETXSH (P. Loll lab) containing a C-
terminally His
6
-tagged Mycobacterium xenopi intein.
22
This could be expressed and purified
by Ni affinity chromatography and cleaved to afford the 2-mercaptoethanesulfonate (Mesna)
thioester. Subtractive purification gave the pure thioester, which was buffer-exchanged and
coupled to an excess of hexathiazole 12. Elimination to give compound 16 could be carried
out under conditions published by Davis and co-workers.
20
The structure of the final
precursor was confirmed by LC/MS (Figure 2c) and rigorously characterized by MS/MS
fragmentation (see the SI).
To evaluate the ability of TclM to effect the formal [4 + 2] cycloaddition of the two Dhas,
the tclM gene was amplified from genomic DNA isolated from B. cereus ATCC 14579 and
subcloned for translation as the N-terminal His
6
MBP-tagged fusion protein. Soluble protein
was obtained in over 90% purity after Ni-NTA affinity and size-exclusion chromatography
followed by tobacco etch virus (TEV) cleavage of the tag (Figure 3a and the SI). Although
TclM does not have any obvious predicted cofactor binding pocket, we note that the purified
protein did not appear to have any unusual absorbance in the visible-light range. Assays for
the putative cycloaddition were conducted in a HEPES buffer at pH 7.2 and room
temperature. Time points were taken at 4 h intervals and analyzed by high-resolution QTOF
LC/MS and UV–vis (Figure 3b and the SI). Over course of the reaction, a new peak grew in
at 350 nm, consistent with the known, distinctive absorbance of the trisubstituted pyridine
core in the thiazolyl peptides.
At 20 h, all of the starting material 16 was consumed in the presence of TclM, and the new
peak at 350 nm ceased to increase. The observed peak at m/z 962.1171 matches within
instrumental error the value for [M + H
+
] of expected product 17 (m/z 962.1118) and
exhibits the distinctive MS/MS fragmentation pattern of thiazolyl peptides (see the SI).
11
We further scaled up the enzymatic reaction and were able to isolate milligram quantities of
product under unoptimized conditions to facilitate complete characterization via two-
dimensional NMR spectroscopy. For example, signals representing the newly formed
pyridine protons can be clearly seen in the aromatic region, confirming the formation of the
trithiazolylpyridine core (Figure 3d and the SI). This compound was absent from all
controls, including those with heat-denatured enzyme or prolonged (multiple days) solution
without enzyme.
Wever et al.
Page 3
J Am Chem Soc. Author manuscript; available in PMC 2016 March 18.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
A potential mechanism for the TclM reaction is illustrated in Figure 4. As drawn, the
reaction involves concerted cyclization via a stabilized tautomer of the amide carbonyl
(imidic acid tautomer). Alternatively, the mechanism could proceed in a stepwise manner,
allowing nucleophilic attack from a stabilized
α
-carbanion of the formal 2
π
component into
an imidocarbonyl intermediate. Regardless of the mechanism, stepwise or concerted, to
arrive at the final product, the putative cyclic intermediate would have to undergo
aromatization by elimination of water and/or the leader peptide carboxamide; both steps
may be spontaneous. We searched LC/MS traces for peaks that could represent any putative
intermediates but found none of the anticipated masses, suggesting that either these
intermediates are short-lived or else TclM itself catalyzes the aromatization.
This work provides direct evidence of the key role of TclM in the formal [4 + 2]
cycloaddition to form the trithiazolylpyridine core of the thiocillins and most thiazolyl
peptides. Our work further represents the first functional characterization of a member of
this new class of enzymes. TclM and its homologues are remarkable in that they show little
or no homology to members of other known enzyme classes. Even between members of the
family, homology is low (14/31% identity/similarity to the nearest homologue, TsrE).
10
Remarkably, TclM is capable of carrying out the formal addition by itself, without accessory
proteins or cofactors.
In conclusion, we have demonstrated that TclM catalyzes the formation of the trisubstituted
pyridine core of an artificial thiazolyl peptide from a leader-peptide-bound substrate.
Mechanistically, the reaction can be drawn as a formal [4 + 2] cycloaddition between two
Dhas with subsequent aromatization, but the exact mechanism, whether stepwise or
concerted, is still undetermined. Substrate specificity and enzyme efficiency as well as a
detailed mechanistic understanding of this new class of enzymes are subjects of ongoing
efforts in our lab. We have also demonstrated a chemoenzymatic route to this attractive class
of natural products; we anticipate that this route will allow the preparation of new and
improved analogues that are not accessible by synthetic or semisynthetic methods.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
The authors thank Scott Allen, Stephen Frye, Harold Kohn, Bo Li, Chris Neumann, and Chris Walsh for
informative discussions and careful reading of the manuscript. A.A.B. is a Beckman Young Investigator and
acknowledges support by the Arnold and Mabel Beckman Foundation. Additional support was provided by NIH
Grants R01GM112739 (F.C.S.) and T32GM008500 (J.A.B.).
References
1. Walsh CT, Acker MG, Bowers AA. J Biol Chem. 2010; 285:27525. [PubMed: 20522549]
2. Just-Baringo X, Albericio F, Alvarez M. Angew Chem, Int Ed. 2014; 53:6602.
3. Nicolaou KC, Zou B, Dethe DH, Li DB, Chen DYK. Angew Chem, Int Ed. 2006; 45:7786.
4. Nicolaou KC, Zak M, Safina BS, Estrada AA, Lee SH, Nevalainen M. J Am Chem Soc. 2005;
127:11176. [PubMed: 16076225]
Wever et al.
Page 4
J Am Chem Soc. Author manuscript; available in PMC 2016 March 18.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript
5. Nicolaou KC, Safina BS, Zak M, Lee SH, Nevalainen M, Bella M, Estrada AA, Funke C, Zécri FJ,
Bulat S. J Am Chem Soc. 2005; 127:11159. [PubMed: 16076224]
6. Gross S, Nguyen F, Bierschenk M, Sohmen D, Menzel T, Antes I, Wilson DN, Bach T.
ChemMedChem. 2013; 8:1954. [PubMed: 24106106]
7. Ciufolini MA, Lefranc D. Nat Prod Rep. 2010; 27:330. [PubMed: 20179875]
8. LaMarche MJ, Leeds JA, Dzink-Fox J, Gangl E, Krastel P, Neckermann G, Palestrant D, Patane
MA, Rann EM, Tiamfook S, Yu D. J Med Chem. 2012; 55:6934. [PubMed: 22812377]
9. Bindman, NA.; van der Donk, WA. Natural Products: Discourse, Diversity, and Design. Osbourn,
A.; Goos, RJ.; Carter, GT., editors. Wiley-Blackwell; Oxford, U.K: 2014. p. 197-217.
10. Bowers AA, Walsh CT, Acker MG. J Am Chem Soc. 2010; 132:12182. [PubMed: 20707374]
11. Bowers AA, Acker MG, Young TS, Walsh CT. J Am Chem Soc. 2012; 134:10313. [PubMed:
22686940]
12. Bowers AA, Acker MG, Koglin A, Walsh CT. J Am Chem Soc. 2010; 132:7519. [PubMed:
20455532]
13. Mocek U, Zeng Z, O’Hagan D, Zhou P, Fan LDG, Beale JM, Floss HG. J Am Chem Soc. 1993;
115:7992.
14. Mocek U, Knaggs AR, Tsuchiya R, Nguyen T, Beale JM, Floss HG. J Am Chem Soc. 1993;
115:7557.
15. Bycroft BW, Gowland MS. J Chem Soc, Chem Commun. 1978:256.
16. Kelly WL. Org Biomol Chem. 2008; 6:4483. [PubMed: 19039353]
17. Kim HJ, Ruszczycky MW, Choi SH, Liu YN, Liu HW. Nature. 2011; 473:109. [PubMed:
21544146]
18. Hashimoto T, Hashimoto J, Teruya K, Hirano T, Shin-Ya K, Ikeda H, Liu HW, Nishiyama M,
Kuzuyama T. J Am Chem Soc. 2015; 137:572. [PubMed: 25551461]
19. Moody CJ, Hughes RA, Thompson SP, Alcaraz L. Chem Commun. 2002:1760.
20. Chalker JM, Gunnoo SB, Boutureira O, Gerstberger SC, Fernández-González M, Bernardes GJL,
Griffin L, Hailu H, Schofield CJ, Davis BG. Chem Sci. 2011; 2:1666.
21. Bagley MC, Bashford KE, Hesketh CL, Moody CJ. J Am Chem Soc. 2000; 122:3301.
22. Economou NJN, Nahoum VV, Weeks SDS, Grasty KCK, Zentner IJI, Townsend TMT, Bhuiya
MWM, Cocklin SS, Loll PJP. J Am Chem Soc. 2012; 134:4637. [PubMed: 22352468]
Wever et al. Page 5
J Am Chem Soc. Author manuscript; available in PMC 2016 March 18.
Author Manuscript Author Manuscript Author Manuscript Author Manuscript