Chemical synthesis of tripeptide thioesters for the biotechnological incorporation into the myxobacterial secondary metabolite argyrin via mutasynthesis

Article abstract of DOI:10.3762/bjoc.15.286

The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their structure–activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising approach where simpler and fully synthetic intermediates of the natural product’s biosynthesis can be biotechnologically incorporated. Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroalanine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence D-Ala-Dha-Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was formed before coupling to the Dha-containing dipeptide.

Full text of DOI:10.3762/bjoc.15.286

Chemical synthesis of tripeptide thioesters for the
biotechnological incorporation into the myxobacterial
secondary metabolite argyrin via mutasynthesis
David C. B. Siebert‡1,2, Roman Sommer‡1,2, Domen Pogorevc‡2,3,4,
Michael Hoffmann2,3,4, Silke C. Wenzel2,3,4, Rolf Müller2,3,4 and Alexander Titz*1,2,4
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2019, 15, 2922–2929.
1Chemical Biology of Carbohydrates, Helmholtz Institute for
Pharmaceutical Research Saarland (HIPS), Helmholtz Centre for
Infection Research (HZI), D-66123 Saarbrücken, Germany,
2Deutsches Zentrum für Infektionsforschung (DZIF), Standort
Hannover-Braunschweig, Germany, 3Microbial Natural Substances,
Helmholtz Institute for Pharmaceutical Research Saarland (HIPS),
Helmholtz Centre for Infection Research (HZI), D-66123 Saarbrücken,
Germany and 4Department of Pharmacy, Saarland University,
D-66123 Saarbrücken, Germany
doi:10.3762/bjoc.15.286
Received: 18 July 2019
Accepted: 20 November 2019
Published: 05 December 2019
Associate Editor: N. Sewald
© 2019 Siebert et al.; licensee Beilstein-Institut.
License and terms: see end of document.
Email:
Alexander Titz* - alexander.titz@helmholtz-hzi.de
* Corresponding author ‡ Equal contributors
Keywords:
antibiotic; argyrin; mutasynthesis; NRPS; peptide synthesis
Abstract
The argyrins are secondary metabolites from myxobacteria with antibiotic activity against Pseudomonas aeruginosa. Studying their
structure–activity relationship is hampered by the complexity of the chemical total synthesis. Mutasynthesis is a promising ap-
proach where simpler and fully synthetic intermediates of the natural product’s biosynthesis can be biotechnologically incorporated.
Here, we report the synthesis of a series of tripeptide thioesters as mutasynthons containing the native sequence with a dehydroala-
nine (Dha) Michael acceptor attached to a sarcosine (Sar) and derivatives. Chemical synthesis of the native sequence ᴅ-Ala-Dha-
Sar thioester required revision of the sequential peptide synthesis into a convergent strategy where the thioester with sarcosine was
formed before coupling to the Dha-containing dipeptide.
Introduction
Resistance to antibiotics is currently a major threat to public prising drugs active against Pseudomonas aeruginosa, one of
health. Especially Gram-negative bacterial pathogens are of the currently most problematic bacterial pathogens [1]. Espe-
concern, due to their widespread development of resistance cially among the quinolones, cephalosporins and carbapenems
mechanisms. To address this general antimicrobial resistance new compounds have been identified. In addition, the develop-
problem, new variants of known antibiotics are being de- ment of new β-lactamase inhibitors is ongoing and may restore
veloped and were approved in the last few years, also com- the activity of known β-lactams against β-lactamase-producing
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strains [2]. Unfortunately, most of these antibiotics rely on tion as basis for further structure-based optimization [11]. Jones
known modes of action and do not target novel binding sites. and colleagues analyzed possible resistance mechanisms to
To circumvent established resistances in clinically used bacteri- argyrin and established argyrin B as an efflux substrate. Further,
al targets, innovative antiinfective strategies comprising new they showed that in susceptible Stenotrophomonas maltophilia,
antibiotic classes and virulence-attenuating compounds have resistance appeared by mutational inactivation of the gene
been developed [3,4].
fusA1 and overexpression of the alternative elongation factor
FusA2 [13]. Due to the dual activity spectrum, cytotoxicity and
The argyrins 1–8 are a family of 8 naturally occurring cyclic antibiotic activity, the selectivity profile of this class of com-
peptides isolated by Sasse, Höfle and co-workers from the pounds should be considered for optimization of future antibac-
myxobacterium Archangium gephyra (Figure 1) [5,6]. These terials.
cyclic peptides have interesting biological activities such as
cytotoxic activity presumably via proteasome inhibition and The first total synthesis was reported by Ley and co-workers
immunomodulatory effects, and they also show good antibiotic [14] for argyrin B in 18 linear steps, followed by an alternative
effects against P. aeruginosa [7-9]. The structure–activity rela- strategy towards argyrin F (6) by the Kalesse group [15]. In
tionship for the natural argyrins A–H revealed argyrin B as 2011, Wu et al. reduced the length to 14 linear steps for the total
most potent derivative (2, IC50 0.08 µg/mL). In 1996, two synthesis of argyrins A and E [16]. Finally, Chen et al. de-
cyclic peptides with a similar sequence but a proposed regio- scribed a synthesis yielding several derivatives of argyrin A
isomer of the methoxytryptophan were reported as antibiotics with modification of the 4’-methoxytryptophan residue [17].
A21459A and B [7,10]. Later, it was shown that A21459A and Changing the 4’-methoxy group to halogens or other substitu-
B are identical to argyrin A and B, respectively, and their struc- ents in different positions led to a loss of antibacterial activity,
ture was revised with respect to the position of the methoxy whereas the 5’-methoxytryptophan regioisomer largely retained
substituent [6]. Argyrin A demonstrated a high efficacy against activity against P. aeruginosa PAO1.
a panel of P. aeruginosa multidrug resistant (MDR) strains and
the argyrins were shown to bind to elongation factor G (EF-G, Inspection of the crystal structure of argyrin in complex with
encoded by the gene fusA1) as their target [11,12]. The the bacterial elongation factor G reveals that the antibiotic is
co-crystal structure of argyrin B (2) and P. aeruginosa EF-G1 deeply buried inside the protein’s binding pocket and only a
provides structural information of the complex at atomic resolu- few sites of the molecule can possibly be modified without
introducing steric clash with the binding site. Besides the
methoxytryptophan which has been addressed by chemical syn-
thesis, one appealing possibility resides in the dehydroalanine-
sarcosine motif and modification seems possible as deduced
from the crystal structure [11] of argyrin with elongation factor
G1.
Despite the successful implementation of total syntheses of
argyrin derivatives, a more rapid access towards diversity in the
argyrins and the resulting structure–activity relationship know-
ledge is desirable. Besides the chemical total synthesis, argyrins
can be obtained from the producer organism in sufficient
amounts and purity but lack the diversity desired for an extend-
ed structure–activity relationship to develop lead candidates. It
has been observed in many cases that exogenous substrates can
be incorporated by bacteria into biosynthesis cascades of
natural products. The use of substrates which lead to non-
natural derivatives of the natural product coined the field of
mutasynthesis, e.g., siderophore analogue biosynthesis by
P. aeruginosa [18]. Biotechnological engineering of producer
strains aims to shutdown the natural substrate production and
thereby increase the usually poor yields of the mutasynthesis
Figure 1: Chemical structures of naturally occurring argyrins with po-
tent antipseudomonal activity.
products [19,20]. For bacterial natural products that originate
from a polyketide synthase (PKS) or a nonribosomal peptide
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synthetase (NRPS), mutasynthons often carry thioesters to Results and Discussion
mimic the natural phosphopantetheinyl conjugate [20].
In order to establish the mutasynthesis of argyrins, we designed
the native sequence mutasynthon 14 and various synthetic
For the argyrins, the Müller group identified the corresponding analogs of this initiating tripeptide for argyrin mutasynthesis
biosynthetic gene cluster from Cystobacter sp. SBCb004 [21], with relatively small structural changes to probe the biosyn-
studied the biosynthesis (Figure 2) and established a hetero- thetic machinery: the native sequence ᴅ-alanine-dehydroala-
logous expression system for the entire pathway in a derivative nine-sarcosine was varied in a small library by replacing dehy-
of Myxococcus xanthus DK1622 [22]. On this basis, a mutant droalanine (Dha) with ᴅ- or ʟ-alanine and sarcosine with
strain for the mutasynthesis approach (M. xanthus DK1622 glycine (Figure 3). The synthesis of these biosynthesis analogs
∆mchA-tet::pArg345-V1) lacking the genes arg1 and arg2 was 9–14 was performed following solution phase Boc-protected
constructed. This was designed to enable a biosynthetic produc-
tion of argyrin derivatives upon incorporation of synthetically
provided tripeptide thioester intermediates, the so called muta-
synthons. Mutasynthons are synthesized as SNAc thioesters
which mimic the phosphopantetheine (PPant) moiety normally
present on the PCP domain of the NRPS. It has been shown by
several mutasynthesis studies [20], that SNAc thioesters can
serve as a mimic of PPant and thus get accepted by the subse-
quent C domain which forms the peptide bond with the down-
stream building block. Because it lacks arg2, the carrier-associ-
ated tripeptide produced by the first NRPS subunit is not avail-
able for biosynthesis. This fact provides the opportunity to
chemically substitute the initial tripeptide thioester accepted by
module 4 on the Arg3 subunit and replace the natural construct
in biosynthesis. By feeding the block mutant with synthetic
analogs, derivatives should be accessible without the need for
Figure 3: Designed mutasynthons 9–14 for argyrin biosynthesis.
Peptides are based on three amino acids and additionally bear the
thioester moiety mimicking the native phosphopantetheinyl arm of the
peptide carrier protein (CP).
full total synthesis of this complex natural product (Supporting
Information File 1, Figure S1).
Figure 2: The biosynthetic pathway for argyrin production in Cystobacter sp. SBCb004 (Arg1, radical SAM-dependent methyltransferase; Arg2/Arg3,
nonribosomal peptide synthetases; Arg4, O‑methyltransferase; Arg5, tryptophan 2,3-dioxygenase). The initial tripeptide of the biosynthesis of the
argyrins, i.e., the target for the mutasynthons of this work, is color coded as product of the Arg2 synthetase and in the resulting final argyrin molecule.
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peptide coupling and functional group interconversion ʟ-alanine, acid-mediated deprotection of the Boc group and
(Scheme 1).
final coupling to Boc-ᴅ-alanine. Fully protected tripeptides
were transformed by base hydrolysis into the free carboxylic
The central alanine carrying nonnatural derivatives were acids, followed by activation of the unprotected C-terminus as a
sequentially synthesized from C- to N-terminus, using ester pro- SNAc thioester. Subsequent cleavage of the N-terminal Boc
tected glycine or sarcosine and coupling to Boc-protected ᴅ- or protecting group gave the unnatural analogs containing ᴅ- or
Scheme 1: Synthesis of tripeptide thioesters. Reagents and conditions: (a) SOCl2, EtOH, 78 °C; (b) IBCF, NMM, THF, −20 °C (for 20a, 20b, 30a);
(c) PyBOP, DIPEA, CH2Cl2, 0 °C (for 20c, 20d, 30b); (d) 4 M HCl/dioxane, rt; (e) NaOH, dioxane, 0 °C; (f) KOH, H2O, 0 °C to rt; (g) (for 26a)
HSCH2CH2NHAc (HSNAc), IBCF, NMM, THF, −20 °C; (for 26b–d, 35a,b) HSNAc, TFFH, DIPEA, CH2Cl2, 0 °C; (h) Pd/C, H2, MeOH, rt; (i) EDC,
CuCl, CH2Cl2, rt; (j) 0.5 M LiOH, THF/MeOH/H2O (4:1:2), 0 °C to rt.
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Beilstein J. Org. Chem. 2019, 15, 2922–2929.
ʟ-alanine at the dehydroalanine site and sarcosine or glycine at To overcome the high reactivity of the Dha moiety towards
the C-terminus (9–12) in generally acceptable yields.
nucleophiles, we engaged in an alternative synthetic strategy
towards the tripeptide thioester carrying the native sequence
The synthesis of the dehydroalanine containing analogs 13 ᴅ-Ala-Dha-Sar by designing a convergent synthesis to mutasyn-
and 14 was performed following the same strategy with thon 14 (Scheme 2). First, Boc-ᴅ-Ala (16) was coupled to
incorporation of a benzyl protected serine at the Dha site. O-benzylserine ethyl ester 37 to give dipeptide 38,
Following hydrogenolytic deprotection, in situ produced serine hydrogenolytic debenzylation gave the substrate 39 for the
EDC adduct was subjected to copper(I) chloride-mediated copper(I)-mediated elimination of the carbodiimide formed in
elimination to give the dehydroalanine derivatives 34a situ using EDC, and then yielded the dipeptide Boc-ᴅ-Ala-Dha
and 34b in good yields. Deprotection of the ester protecting ester 40 in good yields. Lithium hydroxide deprotection of the
group also proceeded in good yields. The subsequent activation ester in compound 40 provided the free acrylate 41. In parallel,
as thioesters (→35a and 35b, respectively) proved difficult Boc-protected sarcosine 42 was transformed into the SNAc
due to the reactivity of the dehydroalanine moiety. Therefore, thioester 43 and the Boc group was removed to give 44. Then,
only moderate yields were obtained for the glycine analog both building blocks, 44 and 41, were coupled to give the pro-
13, whereas no product of the analog 14 carrying the tected tripeptide thioester 35b in good yields. The mutasynthon
native sequence containing the sarcosine moiety could be ob- 14 was obtained after Boc-deprotection from this convergent
tained.
synthetic approach in good yields and sufficient quantities for
Scheme 2: Improved synthesis of the tripeptide thioester 14. Reagents and conditions: (a) SOCl2, EtOH, 78 °C; (b) IBCF, NMM, THF, −20 °C;
(c) Pd/C, H2, MeOH, rt; (d) EDC, CuCl, CH2Cl2, rt; (e) 0.5 M LiOH, THF/H2O (4:1:2), 0 °C to rt; (f) HSCH2CH2NHAc, TFFH, DIPEA, CH2Cl2, 0 °C;
(g) BOP-Cl, DIPEA, CH2Cl2, 0 °C.
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testing its biotechnological incorporation into argyrin the ᴅ-Ala-Dha-Sar thioester 14 was performed. Surprisingly,
(Figure 4).
also in this experiment, incorporation could not be detected. In
general, advanced mutasynthesis studies relying on complex
biosynthetic intermediates are quite challenging, e.g., as
shown in previous studies on polyketide chain engineering in
α-pyrone antibiotic biosynthesis [23]. We assumed that the
insufficient/missing processing of the SNAc precursors by the
NRPS subunit Arg3 is the main culprit for unsuccessful restora-
tion of the argyrin production. As previously mentioned, one
reason for this could be high specificity of the biosynthetic ma-
chinery which might require a full length phosphopantetheine
moiety or even a PCP bound substrate, for condensation to take
place.
To verify the functionality of the truncated argyrin biosynthesis
operon (arg3-arg4-arg5) gene arg2 was integrated into the host
chromosome at a different locus. The obtained mutant strain
M. xanthus DK1622 ∆mchA-tet::pArg345-V1-nptII-arg2 was
shown to produce argyrins indicating that the engineered
operon is functionally expressed via the integrated pArg345-V1
construct.
One further possibility for the lack of successful incorporation
into argyrins is an intrinsically limited chemical stability of
compound 14 which possesses a thioester function and a
Michael acceptor motif. Stability of the ᴅ-Ala-Dha-Sar thioester
14, was evaluated by measuring its degradation kinetics under
the standard conditions in the cultivation medium. The
HPLC–MS analysis revealed a rather rapid degradation of the
analysed mutasynthon, as after only 2 h of incubation time the
majority was degraded and after 4 h of incubation almost com-
plete degradation of the compound was observed (Supporting
Figure 4: Analysis of mutasynthon 14 obtained via the convergent syn-
thetic route by HPLC on a HILIC stationary phase and UV detection
(A) and mass spectrometry (B).
In a first mutasynthesis experiment, the 5 nonnatural deriva- Information File 1, Figure S2).
tives 9–13 were tested for biotechnological incorporation into
argyin by using the arg1/arg2-deficient producer strain. Al- To exclude the possibility of inefficient mutasynthon perme-
though thioesters have been successfully used for incorporation ability through the bacterial membrane, we decided to repeat
in different mutasynthesis projects [20], the feeding experiment the mutasynthesis experiment in vitro using the cell lysate of
using 9–13 for argyrin biosynthesis unfortunately resulted in no the respective arg2 deletion mutant. The in vitro reconstitution
detectable incorporation of these nonnatural mutasynthons. The of nonribosomal peptide biosynthesis has previously been suc-
reasons for failure remain obscure and it could not be excluded cessfully performed using cell lysate, e.g., in case of grami-
that the argyrin biosynthetic machinery is highly specific and cidin [24], or with purified proteins, e.g., in case of myxochelin
requires the natural substrate analog, i.e., the sequence ᴅ-Ala- [25] and tilivalline [26]. To evaluate if in vitro formation of
Dha-Sar. Furthermore, it is not clear whether uptake of these argyrin is indeed possible, the reaction buffer was incubated
tripeptide-thioesters into the bacterial cell was insufficient or with the cell lysate mixture of M. xanthus DK1622 mutants
compound stability under microbiological growth conditions is expressing the Arg2-Arg5 argyrin biosynthetic proteins. To
insufficient.
ensure that no argyrin is present in the lysate prior to the incu-
bation, a lysate mixture of two different mutants harboring arg2
Mutasynthons representing mimics of the native biosynthetic and arg3-5 genes was used, respectively. Both of the mutants
intermediate can be used as proof of concept in mutasynthesis are incapable of argyrin production on their own, however,
studies, to show that production of the native compound can be together they express all the necessary proteins. Despite signifi-
restored [23]. Following this example, incorporation using cant efforts and evaluation of various incubation conditions, in
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Beilstein J. Org. Chem. 2019, 15, 2922–2929.
vitro reconstitution of argyrins could never be achieved. The Financial support by the Helmholtz Association (HGF, grant
exact reason for this is unknown, however, it is very likely that no. VH-NG-934), the European Research Council (ERC StG,
one or more of the biosynthetic proteins are not active under the grant no SWEETBULLETS) and the German Centre for Infec-
applied conditions. Experimental details are provided in Sup- tion Research (DZIF) is acknowledged.
porting Information File 1.
Author Contributions Statement
Conclusion
D.C.B.S. and R.S. designed and performed peptide synthesis.
In summary, we have designed and successfully synthesized a D.P. and S.C.W. designed and performed microbiological ex-
library of tripeptide thioesters for the use in the mutasynthesis periments. M.H. analysed mutasynthesis experiments by
of argyrin derivatives. In the initial strategy, the sequential syn- LCMS. A.T., S.C.W., R.M. conceived and supervised the study.
thesis of the peptides followed by activation as a SNAc ester D.C.B.S., R.S., D.P. and A.T. wrote the manuscript. All authors
successfully yielded five out of six desired library members. reviewed the manuscript.
Following this strategy, the mutasynthon carrying the native se-
quence ᴅ-Ala-Dha-Sar could not be transformed into the desired Competing Interests Statement
thioester probably due to the higher reactivity of the Michael The authors declare no competing interests.
acceptor system. Revision of the synthetic approach into a
ORCID® iDs
convergent synthesis with initial formation of the thioester at
the amino acid level and subsequent coupling to a dipeptide
finally yielded the desired mutasynthon carrying the native se-
quence in high amounts and purity. Reasons for failure to incor-
Roman Sommer - https://orcid.org/0000-0003-0592-0810
Rolf Müller - https://orcid.org/0000-0002-1042-5665
Alexander Titz - https://orcid.org/0000-0001-7408-5084
porate these mutasynthons even at analytical scale remain
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