Synthesis-driven chemical biology
why modify biomolecules?
In recent years, the Gaunt group has been branching out into the field of chemical biology, where we are developing a range of biomolecule functionalisation platforms. Currently we are working both to exploit the reactivity of our methionine diazo-sulfonium conjugate, and to target other unexplored amino acids as well as other biomolecules for conjugation.
Proteins are vital carriers of function not only naturally but also in pharmaceutical applications. To enable maximum site selectivity when functionalising proteins based only on native functional groups, it is highly desirable to target rare amino acids such as methionine. Our group has realised a protein functionalisation method based on methionine using our expertise in the area of iodonium salt chemistry.
For more information on our flagship publication on methionine conjugation, read the paper >
Nature routinely carries out site-selective modification of proteins, enabling a dramatic increase in functional diversity. In contrast, synthetic manipulation of proteins is restricted by the availability of suitable chemical transformations. However, access to synthetically modified proteins has become fundamentally important to chemical biology, molecular biology & medicine.
This has stimulated intensive research into the development of chemical transformations that are compatible with biological systems. Ideally, a reaction should be selective at a single site on a protein at a rate that is commensurate with the kinetic demands of complex molecules; should operate under ambient conditions to prevent disruption of the protein architecture or function; & provide homogeneous products in near perfect conversion. Despite these challenges, the past 20 years have seen a number of exciting methodologies emerge for executing transformations, both in vitro & in vivo, at natural & non-natural amino-acid residues in proteins. While most chemical methods have focused on expanding the toolkit for reaction at cysteine (Cys) & lysine (Lys) residues, there has been burgeoning interest in transformations at non-natural amino acids (via genetic encoding) that display side chain functionality with orthogonal reactivity to standard residues. Reagents that probe biological processes tend to rely on relatively simple reactions to circumvent problems with chemistry in complex environments. However, there is a need for complementary tools (to reactions at Cys & Lys) that selectively produce functional protein conjugates via previously unexplored amino acids.
While many elegant bioconjugation methods exist at cysteine, lysine and tyrosine, we reasoned that a method targeting a less explored amino acid would significantly expand the protein functionalization toolbox. We have developed of a multifaceted approach to protein functionalization based on chemoselective labelling at methionine residues. By exploiting the unique electrophilic reactivity of a bespoke hypervalent iodine reagent, one can target the S-Me group in the side-chain of methionine. The bioconjugation reaction is fast, selective, operates at low µM concentrations and is complementary to existing bioconjugation strategies. Moreover, the new reaction produces a protein conjugate that is, itself, a high energy intermediate with reactive properties that can serve as a platform for the development of secondary, visible-light mediated bioorthogonal protein functionalization processes. Taken together, the merger of these approaches provides a versatile platform for the development of distinct transformations that can deliver versatile, information-rich protein conjugates directly from the native biomacromolecules.
How it works
Methionine (Met) is a proteogenic amino acids & displays a number of features which make it potentially amenable as a bioconjugation target: For example, Met has a <2% abundance in proteins, is easily encoded, has a limited role in ancillary protein function (mainly protection against oxidative stress) & contains a possible reactive handle via its weakly nucleophilic S-atom. Until recently, practical Met-selective bioconjugation was unknown. Concurrent with our initial work, Chang et al reported a redox-activated tagging strategy that converted Met to sulfoximine-derivatives & successfully applying it in biology-driven applications. Our attention had been drawn to an unusual hypervalent iodine compound containing a diazo motif which reacts with Me2S to form a sulfonium salt. This reagent doesn’t look like a suitable bioconjugating agent due to a plethora of reactive functionality. However, we found that a derivative (Ar=2,4-F2C6H3, X=BF4, R=Et – we call these reagents MetSIS, mean-ing Methionine Selective Iodonium Salts) has a t1/2~100 h (0.05 M in H2O) & reacts selectively with polypeptides & proteins, often giving >95% conversion to a stable diazo-sulfonium conjugate at low µM concentration in H2O in <1 minute. The reaction is tolerant of Lys, Ser, Gln, Tyr & Trp, meaning that the Met-bioconjugation should be complementary with other strategies. Although MetSIS oxidizes sulfhydryls to disulfides, no labelling at Cys is observed; importantly MetSIS does not react with disulfides. The diazo-Met conjugates of polypeptides can be purified by HPLC and the exenatide conjugate has t1/2= 96 h at 0.05 M in H2O & pH 3 and is partially stable in 5 mM glutathione (GSH) solution.
We anticipated that multifaceted reactivity would be intrinsic to the high-energy methionine-derived conjugate. The electrophilicity of the diazo sulfonium-conjugate prompted us to investigate the single electron transfer (SET) chemistry of this reactive motif enabled by visible light photocatalysis. Addition of a single electron to the diazo sulfonium conjugate could result in an intermediate, which upon cleavage of the C–N2 bond would form a putative radical ylide. We envisaged a number of pathways through which we could exploit the reactivity of the previously unexplored radical ylide intermediate. Firstly, combining the radical ylide with photo-activated Hantzsch ester may lead to a reduction process resulting in the generation of a trialkylsulfonium motif, which would impart enhanced chemical stability to the protein-conjugate. When the sulfonium diazo conjugate was irradiated with a 30 W lamp in the presence of fac-Ir(ppy)3 and the Hantzsch ester, we observed the formation of the reduced trialkylsulfonium product in 95% yield. Using these conditions, we showed that a range of sulfonium-protein conjugates, including exenatide, glucagon and thioredoxin derivatives are reduced, with excellent conversions, to stable trialkylsulfonium species. Interestingly, reduction of a thioredoxin-derivative to its trialkylsulfonium-protein congener proceeds in high conversion without affecting its labile disulfide linkage, serving to highlight the mild nature of this protocol. Additionally, the methionine bioconjugation and photoreduction steps can be carried out in a one pot operation, significantly simplifying the overall process without compromising the yield or purity of the trialkylsulfonium product.