10Proteins constitute the majority of nature's worker biomolecules. Designed for 11 specific functions, complex tertiary structures make proteins ideal candidates for analyzing 12 natural systems and creating novel biological tools. Due to both large size and the need for 13 proper folding, de novo synthesis of proteins has been quite a challenge, leading scientists to 14 focus on modifying protein templates already provided by nature. Recently developed 15 methods for protein modification fall into two broad categories: those that can modify the 16 natural protein template directly and those that require genetic manipulation of the amino 17 acid sequence prior to modification. The goal of this review is to provide not only a window 18 through which to view the many opportunities created by novel protein modification 19 techniques, but also to act as an initial guide to help scientists find direction and form ideas 20 in an ever-growing field. In addition to the highlighting methods reported in the past five 21 years, we aim to provide a broader sense of the goals and outcomes of protein modification 22 and bioconjugation in general. While the main body of the paper comprises reactions directly 23 criteria is the need for modifications to occur under mild reaction conditions, in an aqueous 47 environment, and in the presence of multiple unprotected, chemical entities that can promote cross-48 reactions. Moreover, promoting such reactions under natural biological conditions while also 49 maintaining structural and functional integrity adds an extra level of difficulty. Nevertheless, 50 different methods have been developed that take advantage of reactive, endogenous AA 51 sidechains.. The nucleophilicity, solvent accessibility, and relative abundance of lysine (Lys) and 52 cysteine (Cys) residues have encouraged scientists to target these sidechains using maleimides, N-53 hydroxysuccinimide (NHS) esters, and a-halocarbonyls as electrophiles for modification. 6,7 54 Michael addition, activated ester amidation, and reductive amination have become particularly 55 popular ( Figure 1). 8 Each method presents particular advantages and disadvantages, but common 56 motivations for the continued search for optimized protein modification methods centre on 57 improving reaction rate and product homogeneity. 58Given that the available chemical functional groups are naturally limited to the canonical 59 AAs, different strategies have been pursued to increase selectivity and improve kinetics. 9 To do 60 so, researchers have employed creative solutions that take advantage of strategies within the realm 61 of nature (for example, enzymatic tags/recognition sites and acknowledgement of the various 62 microenvironments within a protein's structure), genetic engineering for the introduction of natural 63 or abiotic functional groups (e.g. genetic sequence insertions and subsequent chemical reactions), 64 or even previously unexplored chemistry or reaction optimizations (e.g. controlled reaction 65 conditions or metal-catalyzed/direc...

Protein modification has entered the limelight of chemical and biological sciences, since, by appending small molecules into proteins surfaces, fundamental biological and biophysical processes may be studied and even modulated in a physiological context. Herein we present a new strategy to modify the lysine's ε-amino group and the protein's N-terminal, based on the formation of stable iminoboronates in aqueous media. This functionality enables the stable and complete modification of these amine groups, which can be reversible upon the addition of fructose, dopamine, or glutathione. A detailed DFT study is also presented to rationalize the observed stability toward hydrolysis of the iminoboronate constructs.

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein–antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

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