Protein modification via alkyne hydrosilylation using a substoichiometric amount of ruthenium(ii) catalyst† †Dedicated to Professor Stuart L. Schreiber on the occasion of his 60th birthday. ‡ ‡Electronic supplementary information (ESI) available. See DOI: 10.1039/c6sc05313k Click here for additional data file.

The development of site-specific modification of alkyne-functionalized proteins using dimethylarylsilanes and substoichiometric or low-loading of Ru(ii) catalysts is reported. Furthermore, the resultant gem-vinylsilane can undergo further targeted chemical modifications, highlighting its potential for single-site, dual-modification applications.


Introduction
The chemical modication of biomolecules has emerged as a powerful tool to study cellular systems. [1][2][3] Alongside recombinant methods, 4-7 advances in organic chemistry have fueled the development of an increasing number of chemical reactions capable of modifying proteins at both genetically and chemically predened sites. [8][9][10][11][12][13] These bioorthogonal reactions have transformed our ability to visualize cellular processes, and have enabled the development of new therapeutic strategies to treat diseases. [14][15][16] Within this "toolbox" of bioorthogonal reactions, transition metal-mediated reactions are arguably the most underdeveloped. [17][18][19][20] This is likely due to transition metals' propensity for unproductive chelation within the biological milieu, resulting in the need for high catalyst loadings to achieve acceptable reaction rates and labeling efficiency (Fig. 1a). Previous examples of transition metal-mediated reactions include Cu(I) azide-alkyne cycloaddition, 21,22 Ru(II) crossmetathesis, [23][24][25][26] Pd(II) cysteine bioconjugation, 27 Suzuki-Miyaura 28,29 and some of their intracellular variants. 30,31 However, most published reaction conditions utilize high catalyst loading and the development of a truly catalytic transition metalmediated bioconjugation strategy has received little attention.
Here we report a new Ru-catalyzed alkyne hydrosilylation reaction for protein modication. Using the water-soluble Ru catalyst [Cp*Ru(MeCN) 3 ]PF 6 (1) and dimethylaryl hydrosilane derivatives, this methodology enables the efficient labeling of multiple protein targets modied both stochastically and site- specically with an alkyne-containing moiety (Fig. 1b). In addition, hydrosilylation has orthogonal chemical reactivity to ketone-hydrazine condensation reaction in vitro, and the resultant gem-disubstituted vinylsilane product can be further modied via thiol-ene coupling and uoride-induced protodesilylation, demonstrating the potential of this methodology for use in both orthogonal dual labeling and single-site, multiple-probe imaging applications. To the best of our knowledge, this represents the rst example of a C-Si bond formation on protein substrates using substoichiometric or low-loading of transition metal catalystsa feature that we hope will reinstate this mode of catalysis as a viable avenue for future research in the eld.

Results and discussion
Although hydrosilylation has gained widespread utility in organic synthesis and in the industrial production of organosilicon compounds, 32-35 aqueous alkyne hydrosilylation is largely underdeveloped. Inspired by the development of a cationic ruthenium catalyst [Cp*Ru(MeCN) 3 ]PF 6 1 by Trost and Ball, 36,37 we examined the catalyst's ability to catalyze hydrosilylation under biocompatible, aqueous conditions. We started our investigation by reacting 3,6,9,12-tetraoxapentadec-14-yne 2 as a model alkyne and a variety of water-soluble hydrosilanes with 1 (5 mol%). Despite previously reported reactivities of trialkoxy and trialkyl silanes, no vinylsilane products were observed under the reaction conditions tested ( Table 1, entries 1-3).
Gratifyingly, hydrosilylation of dimethylaryl hydrosilane 4 with 2 proceeded smoothly ( Table 1, entry 4), achieving full conversion with 92% isolated yield in less than 5 min. This apparent high reactivity may be attributed to the strong affinity for Cp*Ru complexes to coordinate with aromatic rings, 37 allowing hydrosilylation to proceed rapidly in aqueous solution and open air, thus reinforcing the use of aryldialkyl hydrosilanes in further experiments. The reaction proceeded with a 2 nd order rate constant k 2 $ 1.0 M À1 s À1 (see ESI, Fig. S20 ‡), which is comparable to Ru(II) cross-metathesis and strainpromoted alkyne-azide cycloadditions. Furthermore, 4 was found to be stable in buffered conditions at neutral pH, with a half-life (t 1/2 ) > 1 week (see ESI, Fig. S21 ‡).
One of the side reactions of aqueous hydrosilylation is the hydrolysis of hydrosilane to form silanol (Si-OH). In an effort to reduce silanol formation, we installed substituents adjacent to the Si-H bond with varying degree of steric hindrance (compounds 5-7) in the hope to increase selectivity for hydrosilylation over silanol formation. However, none of the tested analogues gave better selectivity or reaction rates (Table 1, entries 5-7). In particular, 6 and 7 showed incomplete conversion despite prolonged reaction times (Table 1, entries 6 and 7).
The hydrophobicity of chemical probes and modications oen require the use of organic co-solvents in the reaction mixture. Alcohol-based solvents were found to be tolerated as co-solvents in aqueous hydrosilylation and achieved similar reaction rates to those using pure water (see ESI, Fig. S22 ‡). Thus, the hydrosilylation of 2 with triethylene glycol hydrosilane derivative 8 in 50% t-BuOH in phosphate buffered saline (PBS) at pH 7.4 gave the corresponding vinylsilane in 99% isolated yield (Table 1, entry 8). In the presence of human plasma, the reaction initially proceeded extremely slowly and gave only trace of product. It was suspected that 1 is inactive in hydrosilylation due to nonproductive chelation to ruthenium in 10% human plasma. Remarkably, the addition of hippuric acid (BzNHCH 2 CO 2 H) as an additive/ligand helped to stabilize the Ru(II) complex from rapid exchange processes with, for example, histidine 38,39 and aspartic acid 40 residues in plasma protein and restored the activity of 1, with the corresponding vinylsilane product isolated in a good yield (Table 1, entry 9). This result demonstrates that our novel hydrosilylation methodology for protein modication can proceed under physiological conditions.
The scope of this reaction was further evaluated using a variety of small molecule alkynes representative of amino acids, carbohydrates, and hydrophobic drugs such as alkynes 9, 11-13 that may be considered substructural motifs of 3-Omethyl-DOPA (3-OMD), which is one of the most important metabolites of L-DOPA. We rst investigated whether nearby chalcogens on the terminal alkyne group could increase the rate of hydrosilylation. With no nearby coordinating groups, the reaction with alkyne 9 proceeded slowly, requiring a long reaction time to reach 68% yield ( Table 2, entry 1). Contrary to the reported chalcogen effect in protein cross-metathesis, 23,26 Spropargyl 11 and Se-propargyl 12 inhibited hydrosilylation and the respective vinylsilane products were not detected, despite extended reaction times ( Table 2, entries 2 and 3). Surprisingly, O-propargyl 13 showed the most promise, affording vinylsilane 14 in 91% isolated yield ( Table 2, entry 4). This is likely due to the intricate balance between ruthenium-coordination (X ¼ O) and inhibition (X ¼ S, Se). This observation was further conrmed by the decreasing isolated yields when reacting PhMe 2 SiH 15 with O-propargylserine 16, S-propargyl-cysteine 18, and Se-propargylselenocysteine 20 derivatives (Table 2, entries 5-7). Nonetheless, hydrosilylation proceeded smoothly on amino acids 21 and 23, as well as alkyne-sugar derivative 25, affording the corresponding products in excellent isolated yields ( Table 2, entries [8][9][10]. These examples are of particular importance, as strategies for the in vivo incorporation of such moieties into proteins and cell-surface glycans have been developed. [41][42][43] Full conversion was also achieved on a model peptide 27 with biotinylated hydrosilane 29, demonstrating the potential for Ru(II) aqueous hydrosilylation protocol to modify more complex biomolecules (Scheme 1). Furthermore, the stability of the resulting gem-disubstituted vinylsilane moiety was assessed under physiological conditions and in the presence of biological thiols, with no observable degradation at 37 C for up to 24 h (see ESI, Fig. S24 ‡).
Biocompatible chemical transformations are oen most powerful when used in conjunction with each other, allowing for the site-specic incorporation of multiple chemical modications into a single biomolecule. 44 Encouragingly, hydrosilylation was found to be compatible with the widely used asubstituted amine/carbonyl condensation, [45][46][47][48] where O-propargyl 13 reacted smoothly with hydrosilane 15 in the presence of Most site-selective dual-labeling efforts require the incorporation of two unique bioorthogonal functional groups 49,50 or the use of bifunctional substrates, 51,52 which can be a synthetic challenge and limits the wide adoption of such methods. Moreover, it would be advantageous to have the ability to selectively remove synthetically incorporated chemical modications, allowing for potential "switch on/off" applications. To address these issues, we sought to further elaborate the gemdisubstituted vinylsilane linkage via radical thiol-ene 53-55 and uoride-induced protodesilylation reactions 56,57 (Scheme 2b). To illustrate the dual-labeling methodology, we incubated model vinylsilane substrate 31 with benzyl mercaptan, 10 mol% 2,2-dimethoxy-2-phenylacetophenone (DMPA) and irradiated at 365 nm to give doubly-modied derivative 34 in excellent isolated yield (81%). Furthermore, the gem-disubstituted vinylsilane linkage can be cleaved by treatment with TBAF to give the corresponding O-allyl 35, demonstrating the potential for chemical Si-modications installed via hydrosilylation to be selectively removed.
With these promising results in hand, we conducted proteinlabeling experiments via hydrosilylation on different protein systems. First, lysine residues on lysozyme (Lyz) were nonselectively modied with 36 to give O-propargyl modied Lyz (OP-Lyz) ( Fig. 2a and b). When treated with biotinylated hydrosilane 29 and only 10 mol% of 1, we were pleased to observe selective labeling of OP-Lyz over Lyz with negligible background labeling (Fig. 2c). Similarly, when the reaction time or concentration of 29 was held constant (1 h and 250 mM, respectively), dose-and time-dependent labeling was observed, even at very low catalyst loading (2 mol%) (see ESI, Fig. S1 ‡). Inductively coupled plasma-mass spectrometry (ICP-MS) determined that ruthenium content was <10 ppb aer purication when using 10 mol% catalyst (see ESI ‡ for details). To the best of our knowledge this result is the rst demonstration of a protein modication protocol mediated by a substoichiometric amount of transition metal catalyst.
Next, we incorporated O-propargyl groups site-specically into a super-folder GFP (sfGFP) protein (Fig. 2d). 58 Briey, PylRS/pylT pair, the wild-type orthogonal Methanosarcina barkeri pyrrolysyl-tRNA synthetase and tRNA CUA pair and Cterminally hexahistidine-tagged sfGFP containing an amber codon (TAG) at position 150 (sfGFP 150TAG His 6 ) were introduced into E. coli. Addition of 37 (5 mM) led to the amino acid dependent synthesis of full-length sfGFP-37 150 in good yield (15 mg L À1 of culture). A similar approach was used to obtain sfGFP-38 150 as a negative control for labeling experiments (Fig. S2 and S3 ‡). We subsequently incubated sfGFP-37 150 with uorescent hydrosilane 39 and 1 (5 mol%) in PBS (pH 7.4) at 37 C. A uorescent band was detected aer 24 h of incubation with limited background uorescence observed. This result is particularly noteworthy because no uorescence was observed when sfGFP-38 150 was reacted under the same conditions, highlighting the bioorthogonality and specicity of this reaction towards O-propargyl groups (Fig. 2e). The formation of the expected ligated protein was further conrmed by LC-MS (see ESI, Fig. S4 ‡).
As an alternative to recombinant techniques, we also site-specically incorporated the alkyne handle through chemical modications at cysteine. Using the methodology developed by Davis and co-workers, 59 the single cysteine mutant of the C2A domain of Synaptotagmin I C2Am (eukaryotic marker of apoptosis) was converted to OP-C2Am via the dehydroalaninetagged protein intermediate in >95% conversion (Scheme 3a). Gratifyingly, 1 successfully mediated hydrosilylation of OP-C2Am with 8 at 37 C for 1 h to afford VS-C2Am as detected by LC-MS (Scheme 3b). Compared to the high loading of transition metal complexes used in typical metal-mediated protein modication protocols, Ru(II) complex 1 mediated aqueous hydrosilylation offers a milder alternative and these results highlight its potential for site-specic chemical protein modi-cation using either substoichiometric or low-catalyst loading systems.
Having succinctly demonstrated the ability to carry out alkyne hydrosilylation on numerous protein systems, efforts were then directed towards ascertaining whether it was possible to modify the protein-incorporated vinylsilane through our earlier described radical thiol-ene and protodesilylation reactions. Vinylsilane-modied lysozyme (VS-Lyz) was chosen for initial studies. We found that treatment of VS-Lyz with a protected cysteine amino acid and DMPA under hn irradiation yielded Cys-Lyz, as detected by LC-MS (see ESI, Fig. S18 ‡). Similarly, treatment of VS-Lyz with TBAF$3H 2 O afforded Ene-Lyz (see ESI, Fig. S19 ‡). These proof-of-principle experiments show that the vinylsilane can be further modied aer its introduction on a protein through Ru(II)-catalyzed aqueous hydrosilylation.

Conclusions
In conclusion, we have demonstrated that O-propargyl groups and dimethylaryl hydrosilanes (HSiMe 2 Ar) are effective coupling partners for Ru(II) complex 1 catalyzed aqueous hydrosilylation, where alkyne-labeled small-molecules and peptides are site-specically modied in good to excellent yields. Furthermore, hydrosilylation is shown to have orthogonal reactivity to the widely used bioorthogonal hydrazine condensation reaction, giving rise to potential biomolecule dual-labeling applications. Furthermore, O-propargyl tagged proteins (via chemical and genetic strategies) successfully undergo site-specic hydrosilylation in the presence of substoichiometric or low loading of 1 to achieve the rst C-Si bond on protein substrates. Finally, the resultant gem-disubstituted vinylsilane linkage serves as a reactive chemical handle for thiol-ene coupling and protodesilylation, highlighting the potential for single-site dual-modication and the selective removal of vinylsilane modications. Hence, we believe this work greatly expands the reaction conditions and substrate complexity of hydrosilylation and complements the growing interest in metal-mediated protein modication strategies.