Contemporary approaches to site-selective protein modification

Abstract

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...

Selective Modification of Ribosomally Synthesized and Post‐Translationally Modified Peptides (RiPPs) through Diels–Alder Cycloadditions on Dehydroalanine Residues

“…[10] The electrophilic nature of dehydroamino acids has made them attractive functionalities for biorthogonal reactions. [11][12][13][14][15][16][17][18][19][20] In recent years, these dehydroamino acids have emerged as interesting targets for the late-stage modification of RiPPs, throughM ichael additions, [21][22][23][24] hydrogenations, [25] cross-coupling reactions, [26,27] photoredox catalysis, [28] cyclopropanations, [29] and 1,3-dipolar cycloadditions. [30] These studies have highlighted the potential of dehydroamino acid modification in RiPPs, but also illustrate the challenge of achieving selectivity due to the high structural complexity of RiPPs and the difficulties of discriminating between the various dehydroamino acids present.…”

“…due to solubility-and stabilityi ssues of nisin at pH > 5. In addition to the inevitable, but well-documented addition of water to Dha in nisin, [35] a5 2% conversion to single Diels-Alder modified product was observed after 16 ho fm icrowavei rradiation at 50 8C (SI- [19][20]. For nisin Z, which bears one Dhb and two Dha residues, the site selectivity could not be determined due to poor separation of isomerso nL C-MSa nd HPLC.…”

Cysteine-specific protein multi-functionalization and disulfide bridging using 3-bromo-5-methylene pyrrolones

“…Recently, the need to prepare homogeneous antibody-drug conjugates has inspired a wave of interest in exploring cysteine-specific bioconjugation reagents with improved reactivity, specificity, stability, and biocompatibility 6,7 . To this end, maleimide derivatives [8][9][10] , dehydroalanine 11,12 , perfluoroaromatic reagents [13][14][15][16] , organometallic palladium reagents 17,18 , and many others [19][20][21][22][23] have been developed, and each of these reagents shows unique merits. However, only a few of them permit multifunctionalization of a single Cys residue 22,24,25 , which would be advantageous in certain situations.…”

Modifying Methionine on Proteins

“…Protein‐modifying chemical reactions usually require high selectivity and yield, excellent biocompatibility, and fast kinetics. Thiol (cysteine), amino (lysine) and phenolic hydroxyl (tyrosine) consist of most easily modified residues so far . Besides, these above‐mentioned functional groups are usually related to the proteins’ function, and their alteration could permanently destroy the activity.…”