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...

With a resurgence in interest in covalent drugs, there is need to identify new moieties capable of cysteine bond formation that are differentiated from commonly employed systems such as acrylamide. Herein, we report on the discovery of new alkynyl benzoxazine and dihydroquinazoline moieties capable of covalent reaction with cysteine. Their utility as alternative electrophilic warheads for chemical biological probes and drug molecules is demonstrated through site-selective protein modification and incorporation into kinase drug scaffolds. A potent covalent inhibitor of JAK3 kinase was identified with superior selectivity across the kinome and improvements in in vitro pharmacokinetic profile relative to the related acrylamide-based inhibitor. In addition, the use of a novel heterocycle as cysteine reactive warhead is employed to target Cys788 in c-KIT where acrylamide has previously failed to form covalent interactions. These new reactive and selective heterocyclic warheads supplement the current repertoire for cysteine covalent modification whilst avoiding some of the limitations generally associated with established moieties.

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Natural products that contain ortho-quinones show great potential as anticancer agents but have been largely discarded from clinical development because their redox-cycling behaviour results in general systemic toxicity. Here we report conjugation of ortho-quinones to a carrier, which simultaneously masks their underlying redox activity. C-benzylation at a quinone carbonyl forms a redox-inactive benzyl ketol. Upon a specific enzymatic trigger, an acid-promoted, self-immolative C–C bond-cleaving 1,6-elimination mechanism releases the redox-active hydroquinone inside cells. By using a 5-lipoxygenase modulator, β-lapachone, we created cathepsin-B-cleavable quinone prodrugs. We applied the strategy for intracellular release of β-lapachone upon antibody-mediated delivery. Conjugation of protected β-lapachone to Gem-IgG1 antibodies, which contain the variable region of gemtuzumab, results in homogeneous, systemically non-toxic and conditionally stable CD33+-specific antibody–drug conjugates with in vivo efficacy against a xenograft murine model of acute myeloid leukaemia. This protection strategy could allow the use of previously overlooked natural products as anticancer agents, thus extending the range of drugs available for next-generation targeted therapeutics.

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.

Antibody-drug conjugates (ADCs) are a class of targeted therapeutics used to selectively kill cancer cells. It is important that they remain intact in the bloodstream and release their payload in the target cancer cell for maximum efficacy and minimum toxicity. The development of effective ADCs requires the study of factors that can alter the stability of these therapeutics at the atomic level. Here, we present a general strategy that combines synthesis, bioconjugation, linker technology, site-directed mutagenesis, and modeling to investigate the influence of the site and microenvironment of the trastuzumab antibody on the stability of the conjugation and linkers. Trastuzumab is widely used to produce targeted ADCs because it can target with high specificity a receptor that is overexpressed in certain breast cancer cells (HER2). We show that the chemical environment of the conjugation site of trastuzumab plays a key role in the stability of linkers featuring acid-sensitive groups such as acetals. More specifically, Lys-207, located near the reactive Cys-205 of a thiomab variant of the antibody, may act as an acid catalyst and promote the hydrolysis of acetals. Mutation of Lys-207 into an alanine or using a longer linker that separates this residue from the acetal group stabilizes the conjugates. Analogously, Lys-207 promotes the beneficial hydrolysis of the succinimide ring when maleimide reagents are used for conjugation, thus stabilizing the subsequent ADCs by impairing the undesired retro-Michael reactions. This work provides new insights for the design of novel ADCs with improved stability properties.

An azanorbornadiene bromovinyl sulfone reagent for cysteine‐selective bioconjugation has been developed. Subsequent reaction with dipyridyl tetrazine leads to bond cleavage and formation of a pyrrole‐linked conjugate. The latter involves ligation of the tetrazine to the azanorbornadiene‐tagged protein through inverse electron demand Diels–Alder cycloaddition with subsequent double retro‐Diels–Alder reactions to form a stable pyrrole linkage. The sequence of site‐selective bioconjugation followed by bioorthogonal bond cleavage was efficiently employed for the labelling of three different proteins. This method benefits from easy preparation of these reagents, selectivity for cysteine, and stability after reaction with a commercial tetrazine, which has potential for the routine preparation of protein conjugates for chemical biology studies.