ARTICLE https://doi.org/10.1038/s41467-021-20992-0 OPEN Posttranslational chemical installation of azoles into translated peptides ✉ ✉ Haruka Tsutsumi1,2, Tomohiro Kuroda1,2, Hiroyuki Kimura 1, Yuki Goto 1 & Hiroaki Suga 1 Azoles are five-membered heterocycles often found in the backbones of peptidic natural products and synthetic peptidomimetics. Here, we report a method of ribosomal synthesis of azole-containing peptides involving specific ribosomal incorporation of a bromovinylglycine 1234567890():,; derivative into the nascent peptide chain and its chemoselective conversion to a unique azole structure. The chemoselective conversion was achieved by posttranslational dehydro- bromination of the bromovinyl group and isomerization in aqueous media under fairly mild conditions. This method enables us to install exotic azole groups, oxazole and thiazole, at designated positions in the peptide chain with both linear and macrocyclic scaffolds and thereby expand the repertoire of building blocks in the mRNA-templated synthesis of designer peptides. 1 Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo, Tokyo, Japan. 2These authors contributed equally: Haruka Tsutsumi, ✉ Tomohiro Kuroda. email: [email protected]; [email protected] NATURE COMMUNICATIONS | (2021) 12:696 | https://doi.org/10.1038/s41467-021-20992-0 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-20992-0 zoles, such as oxazoles and thiazoles, are five-membered heterocycles often found in the backbone of peptidic UCAG DNA template U A 1,2 Phe Tyr Cys C natural products . Such azole-containing natural pep- U Ser O Leu A tides exhibit a variety of bioactivities, including antitumor, anti- Trp G – His U H2N 3 9 C Leu Pro Arg C OH fungal, antibiotic, and antiviral activities . Incorporation of Gln A G Br azoles into the polyamide backbone generally enhances structural U H Ile Asn Ser C A BrvG A rigidity, which can create conformationally preorganized Lys Arg C H 10,11 Met G 3 7 scaffolds , thereby resulting in potent binding to the targets as U Asp C BrvG 12 G Val Ala Gly A well as better membrane permeability . The local azole moieties FIT Glu G system themselves often act as key building blocks in the bioactivities, as reprogrammed genetic code they can interact with nucleic acids13, proteins14, and metals15. Thus, azoles are attractive entities to have in not only natural – R O products but also artificial peptidomimetics16 19 in devising R O H H therapeutically valuable molecules. N N N TBAF N Since the translation apparatus allows for the facile synthesis of H H O Br O various peptides in an mRNA template-dependent manner, our H group and others have engaged in the use of in vitro translation C3H7 AkyG systems to express nonstandard peptides containing diverse and BrvG-containing peptide C3H7 exotic building blocks, such as β/γ-amino acids20–23, amino carbothioic acids24, and even non-amino acids25–29. However, base installation of an azole ring(s) into the peptide backbone by translation remains a major challenge. To the best of our R O R H O knowledge, the only adequate approach reported in the literature N N N N is the use of a mutated ribosome that incorporates azole- H H O containing dipeptide units into the nascent peptide chain30–34. O C3H7 + C H Unfortunately, the incorporation of such azole-containing azole-containing peptide H 3 7 dipeptide units into the nascent peptide chain was very modest where only 1–7% efficiency was observed for the expression of Fig. 1 Schematic illustration of the chemical posttranslational green fluorescent protein (GFP) mutants compared to the wild- modification for in vitro ribosomal synthesis of azole-containing type GFP expression even using the dedicated mutant ribosome peptides. For posttranslational installation of azoles into translated (i.e., the conventional wildtype ribosome could not do the job at peptides, a 4-bromovinylglycine derivative (BrvG, shown in blue) is all)31,32. This implies that direct incorporation of azole moiety via incorporated into the nascent peptide chain via genetic code such a peptidyl-transfer reaction is yet an arduous task. reprogramming. Subsequent treatment of the expressed peptide with Posttranslational chemical modification of peptides could offer tetrabutylammonium fluoride (TBAF) induces dehydrobromination to give an alternative approach to generate azole-containing peptides. the corresponding β,γ-alkynylglycine derivative (AkyG, shown in green), Several biomimetic conversions of Cys/Ser/Thr analogs into azole which spontaneously undergoes isomerization to produce an azole ring – moieties have been reported35 41, where the 2-step enzymatic (shown in red) in the peptide backbone. conversion (ATP-mediated cyclodehydration42–44 followed by FMN-dependent oxidation45,46) found in natural product bio- custom-made cell-free translation, so-called flexible in vitro synthesis pathways47,48 is mimicked by appropriate chemical translation (FIT) system50 (Fig. 1). The expressed BrvG- reagents. But, the requirement of water-sensitive reagents under containing peptide can be subjected to tetrabutylammonium rather harsh conditions would prohibit achieving our goal for the fluoride (TBAF)-induced dehydrobromination51 conditions, posttranslational modification on peptides in aqueous media. In where the BrvG residue can be specifically converted to the another method, α-chloroglycine esters could be chemically corresponding β,γ-alkynylglycine derivative (AkyG). It has been modified to β,γ-alkynylglycine esters, undergoing base-induced reported that the β,γ-alkynyl group is prone to react with an cyclization to yield oxazoles in aqueous media49. However, as adjacent amide carbonyl group under mild basic conditions via such amino acid monomers are rather labile in water, the pre- an allene intermediate49,52 to yield a backbone oxazole moiety. paration of the corresponding aminoacyl-tRNA would be difficult Thus, we have envisioned that the AkyG-containing peptide to achieve and thereby inapplicable to ribosomal synthesis. generated in situ can be spontaneously converted to the corre- Altogether, combinations of contemporary modification reactions sponding peptide bearing oxazole. and artificial amino acids currently known in the literature To utilize BrvG in reprogrammed genetic codes, BrvG bearing seemed inapplicable to the translation system. 3,5-dinitrobenzyl ester (BrvG-DBE) was chemically synthesized Here, we design a translation-compatible artificial amino acid (Supplementary Fig. 1). To confirm that BrvG can be charged based on a 4-bromovinylglycine derivative (BrvG). Incorporation onto tRNA by means of the flexizyme technology, we used our of this amino acid into the nascent peptide chain enables us to conventional assay system using a tRNA analog, microhelix RNA execute the posttranslational installation of azoles (oxazole and (µhRNA)53. The BrvG-DBE substrate was incubated with µhRNA thiazole) at designated positions. Thus, this method allows for the in the presence of a flexizyme (dFx), and the resulting products synthesis of designer azole-containing peptides in an mRNA- were analyzed by acidic polyacrylamide gel electrophoresis dependent manner under the reprogrammed genetic code. (PAGE) to evaluate the aminoacylation efficiency (Supplementary Fig. 2a). The gel showed a mobility-shifted band corresponding to the BrvG-µhRNA, indicating that the flexizyme system allowed Results for the preparation of BrvG-tRNA under these conditions. To Design and in vitro ribosomal incorporation of BrvG.To ribosomally synthesize a model peptide bearing a BrvG residue fi AsnE2 achieve chemical posttranslational modi cation yielding azoles, (Pep1-Br), BrvG-tRNA GGU was added to a Thr-depleted FIT we have conceived the ribosomal incorporation of BrvG via system, in which the ACC codon was reprogrammed with BrvG genetic code reprogramming powered by flexizymes and a (Supplementary Fig. 2b). Tricine-SDS PAGE analysis of the 2 NATURE COMMUNICATIONS | (2021) 12:696 | https://doi.org/10.1038/s41467-021-20992-0 | www.nature.com/naturecommunications NATURE COMMUNICATIONS | https://doi.org/10.1038/s41467-021-20992-0 ARTICLE translation product revealed that BrvG was efficiently (86% of the dehydrobrominated product (Supplementary Fig. 2e). This compared with the wildtype expression) incorporated into the result suggested that BrvG could undergo dehydrobromination by nascent peptide chain (Supplementary Fig. 2c). In addition, TBAF and that no significant side reactions on other proteino- matrix-assisted laser desorption/ionization time-of-flight mass genic amino acids occurred. spectrometry (MALDI-TOF-MS) of the translation product As the molecular weight of the desired oxazole-containing showed a peak corresponding to the expected peptide (Supple- peptide (Pep1-Oxz) is identical to that of the expected mentary Fig. 2d), confirming that BrvG could be successfully intermediate (Pep1-AkyG), the MALDI-TOF-MS experiment utilized in the FIT system to yield Pep1-Br. above could not determine whether the oxazole moiety was generated as designed. Thus, to monitor the production of the intermediate and the desired oxazole-containing product at a Posttranslational chemical modification of BrvG to an oxazole. finer temporal resolution, the reaction was quenched after 0, 5, The expressed Pep1-Br peptide was then subjected to the che- 15, 30, and 120 min and analyzed by liquid chromatography- mical posttranslational modification to determine whether the