Available online at www.sciencedirect.com ScienceDirect Synthesis of modified proteins via functionalization of dehydroalanine Jitka Dadova´ , Se´ bastien RG Galan and Benjamin G Davis Dehydroalanine has emerged in recent years as a non- selectivity is a major limitation in applications requiring proteinogenic residue with strong chemical utility in proteins for greater homogeneity and precision. While some site- the study of biology. In this review we cover the several selectivity can be achieved using the natural rarity of methods now available for its flexible and site-selective Cys (1% of Cys on average in proteins [9]), a ‘tag-and- incorporation via a variety of complementary chemical and modify’ [10] approach can be generally used as a site- biological techniques and examine its reactivity, allowing both selective protein labelling method that exploits position- creation of modified protein side-chains through a variety of ing of pre-determined functional groups, ‘tags’ bond-forming methods (C–S, C–N, C–Se, C–C) and as an (Figure 1a). It relies on the selective introduction of a activity-based probe in its own right. We illustrate its utility with ‘tag’ as a reactive handle to each site of interest followed selected examples of biological and technological discovery by chemoselective reaction to ‘modify’/graft on to that and application. site the function of interest. In the past decades, the range of non-proteinogenic, reactive tags (e.g. azide, alkyne, Address tetrazine) has been greatly expanded by biochemical and Department of Chemistry, University of Oxford, Chemistry Research cellular methods (auxotrophic replacement, nonsense Laboratory, Mansfield Road, Oxford OX1 3TA, United Kingdom codon suppression [11–13]). Many such bioconjugation methods now enable effective site-selective labelling. Corresponding author: Davis, Benjamin G ([email protected]) However, for the attachment linkage leaves a ligation ‘scar’ in the protein, often larger than the amino residue Current Opinion in Chemical Biology 2018, 46:71–81 itself, precluding precise or subtle functional study or This review comes from a themed issue on Synthetic biomolecules biomimicry [14,7]. Edited by Richard J. Payne and Nicolas Winssinger The amino acid dehydroalanine (Dha), as a biocompati- ble ‘tag’ in proteins, shows intriguing and varied reactivity with typically minimal (e.g. single b,g-C–X bonds) https://doi.org/10.1016/j.cbpa.2018.05.022 attachment marks/’scars’ that therefore allows striking flexibility in, for example, the installation of natural 1367-5931/ã 2018 The Authors. Published by Elsevier Ltd. This is an PTMs (or mimics) and chemical mutagenesis to a broad open access article under the CC BY-NC-ND license (http://creative- commons.org/licenses/by-nc-nd/4.0/). variety of natural and unnatural amino acids. The inser- tion of Dha tag into proteins proceeds under mild con- ditions via various complementary methods and can now be robustly scaled up to milligram protein quantities. In Background and motivation this review, we aim to provide an overview of approaches Modern chemical biology relies increasingly on protein to modify proteins via dehydroalanine. We describe chemistry, which (ideally) allows precise positioning of methods of Dha incorporation into a wide range of pro- labels, cargoes and post-translational modifications teins and illustrate that Dha functionalization is driven by (PTMs) in the contexts of complex protein structures its chemical properties. Finally, we discuss applications of [1–3]. The resulting modified proteins prove useful in Dha chemistry as a broadly applicable tool by highlight- therapeutic applications, the probing and modulating of ing recent achievements ranging from creating modified function, as well as their tracking and (un)caging in nucleosomes to preparing better therapeutics. cells [4–6]. Various methods have been developed for the convergent Introduction of dehydroalanine to proteins construction of site-selectively modified proteins [6,7]. Dehydroalanine is a naturally occurring amino acid, which Traditionally, the non-site-selective chemical modifica- is formed by serine (Ser) dehydration or phosphoserine tion of proteins has relied on the nucleophilicity of the (pSer) elimination in peptides [15] and proteins [16]. Its side-chains of natural amino acid residues lysine (Lys) formation is observed during lanthipeptide natural prod- and cysteine (Cys), as well as protein N-terminus through uct biosynthesis in prokaryotes [17]. Excretion of phos- direct acylation, alkylation and arylation with a wide array phothreonine lyases (OspF, SpvC and HopAI1) in path- of electrophiles [6–8]. However, while these techniques ogenic bacteria (Shigella, Salmonella and Pseudomonas have been extensively used, for instance, for antibody– syringae, respectively) converts pSer to Dha in activation drug conjugate (ADC) manufacturing, their lack of loops of host mitogen-activated protein kinases (MAPK) www.sciencedirect.com Current Opinion in Chemical Biology 2018, 46:71–81 72 Synthetic biomolecules Figure 1 (a) "tag" "modify" position R of interest site-selective modification R = natural amino reactive handle modified protein acid side chain This review: = = Y' = Y Dha Y' = SH, NH2, SeH, CH2Br, CH2I Y = S, NH, Se, CH2 (b) site-directed Dha mutagenesis formation Dha R or protein X -HX semisynthesis R = natural amino Dha precursor acid side chain O O- Dha precursors P Ph O O- X O SH SeH Se Se N OBn = H N N N N N N H H H H H H O O O O O O pSerCys SeCys PhSeCys Cbz- SeLys O R1 R2 Dha precursor Conditions for Dha formation S O X X O NH2 pSer lyases (OspF, SpvC, HopAI1), Ba(OH)2 2-4 MSH (1) Cys MSH (1), alkylating reagents (2-4) Alkylating X R R reagent 1 2 SeCys DBHDA (3), NaIO4 DIB (2) I H H PhSeCys H2O2 DBHDA (3) Br CONH2 CONH2 Cbz- SeLys H2O2 MDBP (4) Br COOCH3 H Current Opinion in Chemical Biology (a) Site-selective covalent protein modification by a two-step ‘tag-and-modify’ approach. (b) Methods to introduce dehydroalanine (Dha) as a ‘tag’ to proteins. The position of interest is typically activated by conversion to a Dha precursor followed by its elimination to Dha. Protein taken from PDB: 1N2E [89]. [16]. Finally, Dha is formed by spontaneous non-enzy- position of interest, are first transformed into leaving matic elimination of pSer as a consequence of protein groups, which upon elimination, yield Dha (Figure 1b). aging in human cells [18,19], a process which in some Historically, the first attempts to form Dha on peptides and proteins may be accelerated chemically [20]. proteins reliedon Sersulfonylation followed by elimination underconditionstypically tooharshformostproteinsandin Ser and other natural amino acids Cys and selenocysteine a manner that is applicable only to activated (e.g. catalytic (SeCys or Sec) can be used as controllable Dha precursors triad) Ser [21]. Following phosphorylation, in vitro treat- by protein chemists. These residues, inserted at the ment of the resulting pSer with barium hydroxide at Current Opinion in Chemical Biology 2018, 46:71–81 www.sciencedirect.com Synthesis of proteins via dehydroalanine Dadova´ , Galan and Davis 73 ambient temperature can yield Dha [20]. However, despite [39], by native chemical ligation [38,40 ,41], SeCys-medi- progress in amber codon suppression technology and semi- ated expressed protein ligation [42], or in modified-forms synthetic methods, fully selective incorporation of pSer (e.g. phenyl-selenocysteine (PhSeCys) [43] or selena-Lys into larger proteins [22–26] remains a challenge and may variants [44,45]) by amber codon suppression. These can not always provide a flexible precursor. be converted to Dha via the corresponding selenoxides (oxidation/Cope-type elimination) using hydrogen perox- Therefore, methods to transform more rare (allowing gen- ide or sodium periodate [43–46]; however, undesired erality) and more reactive (allowing milder conditions) side-oxidations of susceptible amino acids (Met and amino acid Cys to Dha were developed to achieve com- Cys) are also observed. Therefore, DBHDA 3 can be patibility with sensitive protein structures as well as selec- used with SeCys, which, when coupled with the greater tivity [27 ,28 ]. Contrary to pSer, SeCys and its derivatives acidity of SeCys cf Cys (pKa(SeCys) = 5.2) allows some (see below), Cys may be introduced to a target protein quite selectivity over Cys [40 ,42]. simply by site-directed mutagenesis. Although early pio- neering work on Dha formation from Cys was complicated Together these techniques have now allowed the selective by associated protein cleavage [29], in 2008 an oxidative incorporation of Dha into several sites of many proteins, for amidation/Cope-type elimination protocol using O-mesi- example, GFPs [47], ubiquitins [48 ,49–51,52 ,53], his- tylenesulfonylhydroxylamine (MSH, 1) was reported on tones (H2A [54], H2B [54], H3 [55] and H4 [56 ]) anti- model protein subtilisin [27 ]. Whilst applicable to many bodies (cAbs [28 ,57] and so-called ‘ThioMabs’ [33]) proteins without side-reaction, low level undesired reac- kinases (Aurora A [34] and p38a [58]), as well as N-acetyl tivity of MSH as an oxidative reagent with nucleophilic neuraminic acid lyase [59], AcrA and annexin V [56 ] and amino acids (Met, Lys, His, Asp and Glu) under certain Npb [60], pantothenate synthetase [32], protease SBL conditions was observed[28 ].Thisled todevelopmentofa [27 ], phosphatase PTPa [35], and keratin hydrogels [61]. series of milder and more selective reagents (Figure 2b, 2– 4) that