Chemical modification of proteins is a rapidly expanding area in chemical biology. Selective installation of biochemical probes has led to a better understanding of natural protein modification and macromolecular function. In other cases such chemical alterations have changed the protein function entirely. Additionally, tethering therapeutic cargo to proteins has proven invaluable in campaigns against disease. For controlled, selective access to such modified proteins, a unique chemical handle is required. Cysteine, with its unique reactivity, has long been used for such modifications. Cysteine has enjoyed widespread use in selective protein modification, yet new applications and even new reactions continue to emerge. This Focus Review highlights the enduring utility of cysteine in protein modification with special focus on recent innovations in chemistry and biology associated with such modifications.

Olefin metathesis has become a mainstay in organic synthesis. 1 Cross-metathesis (CM), however, is largely underdeveloped compared to ring closing metathesis (RCM) and ring opening metathesis polymerization since CM does not have the entropic driving force of RCM and is complicated by self-metathesis. 2 Our group has a long-term interest in site-selective chemical modification of proteins in an effort to study and modulate their function. 3 Olefin metathesis is an attractive way to install these protein modifications through a stable carbon-carbon bond. Indeed, incorporation of olefins into proteins has been possible for nearly a decade, 4 but metathesis at such residues has not been realized. Despite recent reports of olefin metathesis in water, the current benchmark for homogeneous aqueous CM is the self-metathesis of simple unsaturated alcohols such as allyl alcohol. 5,6 The limited examples revealed to date highlight the challenges for aqueous CM and the gap in substrate complexity that must be bridged to carry out metathesis on protein surfaces.To determine the viability of CM on protein surfaces, simple amino acid models were investigated. Substrates were selected on the basis of potential incorporation into proteins. A reasonable starting point was homoallylglycine (Hag) since its in ViVo incorporation by methionine auxotrophic Escherichia coli is known. 4 Hoveyda-Grubbs second generation catalyst 1 7 was selected since it is phosphine free and therefore more likely to be compatible with protein disulfides than other conventional catalysts. A simple test metathesis with allyl alcohol 2 was carried out to assess the reactivity of Hag derivative 3. At the outset, we limited ourselves to temperatures generally compatible with proteins (e37°C ) and made no effort to exclude oxygen. Since 1 is not freely soluble in water, it was added as a solution in t BuOH. Unfortunately, despite repeated attempts, only starting material 3 was recovered (Table 1, Entry 1). We turned next to cysteine derivatives since incorporation into proteins should be possible by either chemical or genetic means if they proved reactive in CM. Remarkably, S-allylcysteine (Sac) derivative 4 underwent metathesis with allyl alcohol (Entry 2), affording the CM product in 56% isolated yield (74% based on recovered 4). This result was noteworthy given the number of instances where thioethers were detrimental to rutheniumbased metathesis catalysts. 8 The metathesis was also efficient with allyl homocysteine 5 and bisamide Sac derivative 6. Yet when the alkene was extended by one or two methylene units from the sulfur center, only allyl alcohol self-metathesis was observed along with recovered starting material (Entries 5 and 6). Other allylheteroatom substrates were screened, but allyl sulfides remained the most efficient metathesis substrates under the conditions employed (Entries 7-13).While the self-metathesis of allyl sulfides has been carried out in organic solvents, the efficiency relative to other heteroatom or hydrocarbon analogues was...

Olefin metathesis has recently emerged as a viable reaction for chemical protein modification. The scope and limitations of olefin metathesis in bioconjugation, however, remain unclear. Herein we report an assessment of various factors that contribute to productive cross-metathesis on protein substrates. Sterics, substrate scope, and linker selection are all considered. It was discovered during this investigation that allyl chalcogenides generally enhance the rate of alkene metathesis reactions. Allyl selenides were found to be exceptionally reactive olefin metathesis substrates, enabling a broad range of protein modifications not previously possible. The principles considered in this report are important not only for expanding the repertoire of bioconjugation but also for the application of olefin metathesis in general synthetic endeavors.

Cross-metathesis (CM) has recently emerged as a viable strategy for protein modification. Here, efficient protein CM has been demonstrated through biomimetic chemical access to Se-allyl-selenocysteine (Seac), a metathesis-reactive amino acid substrate, via dehydroalanine. On-protein reaction kinetics reveal a rapid reaction with rate constants of Seac-mediated-CM comparable or superior to off-protein rates of many current bioconjugations. This use of Se-relayed Seac CM on proteins has now enabled reactions with substrates (allyl GlcNAc, N-allyl acetamide) that were previously not possible for the corresponding sulfur analogue. This CM strategy was applied to histone proteins to install a mimic of acetylated lysine (KAc, an epigenetic marker). The resulting synthetic H3 was successfully recognized by antibody that binds natural H3-K9Ac. Moreover, Cope-type selenoxide elimination allowed this putative marker (and function) to be chemically expunged, regenerating an H3 that can be rewritten to complete a chemically enabled “write (CM)–erase (ox)–rewrite (CM)” cycle.

For a reaction to be generally useful for protein modification, it must be site-selective and efficient under conditions compatible with proteins: aqueous media, low to ambient temperature, and at or near neutral pH. To engineer a reaction that satisfies these conditions is not a simple task. Olefin metathesis is one of most useful reactions for carbon-carbon bond formation, but does it fit these requirements? This minireview is an account of the development of olefin metathesis for protein modification. Highlighted below are examples of olefin metathesis in peptidic systems and in aqueous media that laid the groundwork for successful metathesis on protein substrates. Also discussed are the opportunities in protein engineering for the genetic introduction of amino acids suitable for metathesis and the related challenges in chemistry and biology.