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Rapid Sodium Periodate Cleavage of an Unnatural Amino Acid Enables Unmasking of a Highly Reactive Α-Oxo Aldehyde for Protein Bioconjugation

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Rapid Sodium Periodate Cleavage of an Unnatural Amino Acid Enables Unmasking of a Highly Reactive Α-Oxo Aldehyde for Protein Bioconjugation This is a repository copy of Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation. White Rose Research Online URL for this paper: https://eprints.whiterose.ac.uk/161006/ Version: Published Version Article: Brabham, Robin, Keenan, Tessa, Husken, Annika et al. (5 more authors) (2020) Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive α-oxo aldehyde for protein bioconjugation. Organic and Biomolecular Chemistry. ISSN 1477-0520 https://doi.org/10.1039/d0ob00972e Reuse This article is distributed under the terms of the Creative Commons Attribution (CC BY) licence. This licence allows you to distribute, remix, tweak, and build upon the work, even commercially, as long as you credit the authors for the original work. More information and the full terms of the licence here: https://creativecommons.org/licenses/ Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing [email protected] including the URL of the record and the reason for the withdrawal request. [email protected] https://eprints.whiterose.ac.uk/ Organic & Biomolecular Chemistry View Article Online COMMUNICATION View Journal Rapid sodium periodate cleavage of an unnatural amino acid enables unmasking of a highly reactive Cite this: DOI: 10.1039/d0ob00972e α-oxo aldehyde for protein bioconjugation† Received 11th May 2020, Accepted 14th May 2020 Robin L. Brabham, ‡a Tessa Keenan, ‡a Annika Husken,a Jacob Bilsborrow, a DOI: 10.1039/d0ob00972e Ryan McBerney,b Vajinder Kumar,b,c W. Bruce Turnbull b and a rsc.li/obc Martin A. Fascione * The α-oxo aldehyde is a highly reactive aldehyde for which many use of amber stop codon suppression, site-specific protein protein bioconjugation strategies exist. Here, we explore the aldehyde modification could now move beyond such sequence genetic incorporation of a threonine-lysine dipeptide into proteins, limitations. harbouring a “masked” α-oxo aldehyde that is rapidly unveiled in Aldehydes can differ vastly in their electronic properties, fi 9 Creative Commons Attribution 3.0 Unported Licence. four minutes. The reactive aldehyde could undergo site-speci c dictated by the method used for their installation. The α-oxo protein modification by SPANC ligation. aldehyde is a highly reactive aldehyde for which many reliable protein bioconjugation methodologies have been Genetic code expansion has revolutionised our ability to site- established.9,12 Previously restricted to the N-terminus of a 1 selectively install unnatural functionality into proteins. The protein and requiring an exposed seryl, threonyl or glycyl use of the pyrrolysine (Pyl) tRNACUA/pyrrolysyl-tRNA synthetase residue for its formation, the site-specific incorporation of a (RS) pair has proven to be a highly successful platform for this thiazolidine-protected α-oxo aldehyde into a protein was purpose, with the introduction of reactive handles such as recently demonstrated.13 We recently reported a biocompatible 2 3–5 6–8 azides, alkenes and alkynes into proteins, opening up a method of unmasking a genetically encoded thiazolidine-pro- This article is licensed under a wide range of site-specific chemical bioconjugation strategies. tected α-oxo aldehyde in a protein, using stoichiometric allyl- The aldehyde is a particularly versatile functional group owing 14 palladium(II) chloride. However, this procedure requires to its unique reactivity, stability and relatively low abundance some optimisation for individual proteins, in order to balance Open Access Article. Published on 14 May 2020. Downloaded 5/20/2020 9:21:19 AM. in nature, however few examples of the site-specific installation the reactivity of the palladium complex to open the thiazoli- of aldehydes into proteins by amber stop codon suppression dine with potential side-reactions including protein precipi- have been reported, despite the many bioorthogonal method- tation. In this work, we explore an alternative route to the site- 9 ologies available that utilise these functional handles. specific installation of a α-oxo aldehyde, through the genetic Notably, through the use of a mutant Pyl tRNA-RS pair from incorporation of a threonine-lysine derivative, and its rapid Methanocaldococcus janaschii, an aldehyde-containing phenyl- unmasking within four minutes (Fig. 1). The reactivity of the alanine analogue was previously shown to be incorporated into α-oxo aldehyde in site-selective protein modification was sub- 10 a protein, facilitating modification by oxime ligation. This sequently demonstrated by Strain-Promoted Alkyne-Nitrone finding breathed fresh life into well-established protein carbo- Cycloaddition (SPANC) ligation. nyl chemical modification, which had been hampered by the To generate a protein α-oxo aldehyde, we explored the peri- limitations on the positioning of the required reactive alde- odate-mediated cleavage of a genetically encoded ε-lysine hydes. Previous methods to install protein aldehydes required dipeptide harbouring a 1,2-amino alcohol motif, arising from 11 an enzymatic tag, such as the use of FGE or an exposed a serine or threonine residue. In order to maximise the 9 N-terminal serine, threonine or glycine residue; through the chances of discovering a suitable substrate for amber stop codon suppression, four dipeptides (1–4) were synthesised, ff aDepartment of Chemistry, University of York, Heslington, YO10 5DD, York, UK. di ering in the absence/presence of a β-methyl group (serine/ E-mail: [email protected] threonine respectively) and the α-configuration (S/R, “natural” bSchool of Chemistry and Astbury Centre for Structural Molecular Biology, University vs. “unnatural” respectively) (Fig. 2). The modulation of of Leeds, Leeds LS2 9JT, UK α-configuration and β-methyl groups was considered to offer cAkal University, Talwandi Sabo, Punjab, India insight into the amber stop codon suppression process, with †Electronic supplementary information (ESI) available. See DOI: 10.1039/ d0ob00972e these changes subtly altering amino acid polarity, steric bulk, ‡These authors contributed equally. and positioning within the pylS active site. An activation-coup- This journal is © The Royal Society of Chemistry 2020 Org. Biomol. Chem. View Article Online Communication Organic & Biomolecular Chemistry Fig. 1 New route for the site-selective installation of a α-oxo aldehyde into proteins. threonine derivative being a superior PylRS substrate to a serine derivative, is that the extra methyl group can better occupy the hydrophobic space within the substrate binding pocket.20 Following the identification of a suitable dipeptide sub- strate for the M. mazei Pyl tRNA-RS pair to facilitate the site- specific installation of a α-oxo aldehyde into a protein, the 3 – Thr-Lys dipeptide was also introduced into sfGFP at the Fig. 2 Screening of lysine dipeptides 1 4 for their incorporation into 21 EGFP at position Y39 by the M. mazei Pyl tRNA-RS pair and analysis by surface exposed position N150, using a method of amber 14 SDS-PAGE. The full uncropped SDS-PAGE gel is included in the ESI stop codon suppression adapted for GFP expression. The (Fig. S7†). cells were cultivated in Terrific Broth medium supplemented 3 Creative Commons Attribution 3.0 Unported Licence. with 0.02% arabinose and 1.5 mM of Thr-Lys dipeptide ,pro- moting the expression of the full-length sfGFP(N150ThrK)-His6 ling-deprotection strategy was used to synthesise all four protein 6. Following purification by Ni2+ affinity chromato- dipeptides in three steps with cumulative yields of 39% (1), graphy, the purity and molecular mass of 6 was validated by 50% (2), 32% (3) and 56% (4) respectively (Schemes S1 and S2, SDS-PAGE and ESI-FTICR-MS (Fig. S2 and S3, ESI†). Following ESI†). Considering that the promiscuity of the wild type the successful incorporation of the Thr-Lys dipeptide 3, peri- M. mazei Pyl tRNA-RS pair had been shown to extend to several odate-mediated oxidation of the 1,2-aminoalcohol motif in the – lysine dipeptides,14 16 we chose to investigate this pair for the threonine residue was explored (Fig. 3). Whilst periodate- genetic incorporation of our dipeptides 1–4. To screen the suit- mediated oxidation of 1,2-aminoalcohols is generally fast, peri- This article is licensed under a ability of the dipeptides 1–4 as substrates for the M. mazei Pyl odate will also oxidise other amino acid residues in proteins, tRNA-RS pair, an expression trial using EGFP(Y39TAG)-His6 as including cysteine, methionine, tryptophan, tyrosine, and his- a reporter protein was carried out and the samples were ana- tidine, albeit more slowly and dependent on the experimental 12,22 Open Access Article. Published on 14 May 2020. Downloaded 5/20/2020 9:21:19 AM. lysed by SDS-PAGE (Fig. 2). EGFP(Y39TAG), containing an conditions. However, protein over-oxidation can be amber mutation at surface-exposed position Y39,17 was avoided by performing the reaction at neutral pH, controlling selected due to its ease of visualisation and highly optimised the reaction stoichiometry and minimising the reaction time. expression system for use with unnatural amino acid (UAA) Additionally, excess methionine or ethylene glycol should be mutagenesis. The known Pyl tRNA-RS pair substrate Nε-pro- added either during or after the reaction, to quench the 12 pargyloxycarbonyl-L-lysine 5 served as a positive control for the unreacted periodate. Based on conditions previously opti- expression
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