Synthesis of modified proteins via functionalization of dehydroalanine

Dehydroalanine has emerged in recent years as a non-proteinogenic residue with strong chemical utility in proteins for the study of biology. In this review we cover the several methods now available for its flexible and site-selective incorporation via a variety of complementary chemical and biological techniques and examine its reactivity, allowing both creation of modified protein side-chains through a variety of bond-forming methods (C-S, C-N, C-Se, C-C) and as an activity-based probe in its own right. We illustrate its utility with selected examples of biological and technological discovery and application

Synthesis of modified proteins via functionalization of dehydroalanine

Dehydroalanine has emerged in recent years as a non-proteinogenic residue with strong chemical utility in proteins for the study of biology. In this review we cover the several methods now available for its flexible and site-selective incorporation via a variety of complementary chemical and biological techniques and examine its reactivity, allowing both creation of modified protein side-chains through a variety of bond-forming methods (C-S, C-N, C-Se, C-C) and as an activity-based probe in its own right. We illustrate its utility with selected examples of biological and technological discovery and application

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors

Characterisation of utrophin modulator SMT C1100 as a non-competitive inhibitor of firefly luciferase

Firefly luciferase (FLuc) is a powerful tool for molecular and cellular biology, and popular in high-throughput screening and drug discovery. However, FLuc assays have been plagued with positive and negative artefacts due to stabilisation and inhibition by small molecules from a range of chemical classes. Here we disclose Phase II clinical compound SMT C1100 for the treatment of Duchenne muscular dystrophy as an FLuc inhibitor (KD of 0.40 ± 0.15 µM). Enzyme kinetic studies using SMT C1100 and other non-competitive inhibitors including resveratrol and NFκBAI4 identified previously undescribed modes of inhibition with respect to FLuc's luciferyl adenylate intermediate. Employing a photoaffinity strategy to identify SMT C1100's binding site, a photolabelled SMT C1100 probe instead underwent FLuc-dependent photooxidation. Our findings support novel binding sites on FLuc for non-competitive inhibitors

Post-translational site-selective protein backbone alpha-deuteration

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N–H/D) labile sites only. The development of postbiosynthetic, C–1H → C–2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a ‘carba-Swern’ mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C–D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine

Post-translational site-selective protein backbone α-deuteration

Isotopic replacement has long-proven applications in small molecules. However, applications in proteins are largely limited to biosynthetic strategies or exchangeable (for example, N-H/D) labile sites only. The development of postbiosynthetic, C-1H → C-2H/D replacement in proteins could enable probing of mechanisms, among other uses. Here we describe a chemical method for selective protein α-carbon deuteration (proceeding from Cys to dehydroalanine (Dha) to deutero-Cys) allowing overall 1H→2H/D exchange at a nonexchangeable backbone site. It is used here to probe mechanisms of reactions used in protein bioconjugation. This analysis suggests, together with quantum mechanical calculations, stepwise deprotonations via on-protein carbanions and unexpected sulfonium ylides in the conversion of Cys to Dha, consistent with a 'carba-Swern' mechanism. The ready application on existing, intact protein constructs (without specialized culture or genetic methods) suggests this C-D labeling strategy as a possible tool in protein mechanism, structure, biotechnology and medicine

LanCLs add glutathione to dehydroamino acids generated at phosphorylated sites in the proteome

Enzyme-mediated damage repair or mitigation, while common for nucleic acids, is rare for proteins. Examples of protein damage are elimination of phosphorylated Ser/Thr to dehydroalanine/dehydrobutyrine (Dha/Dhb) in pathogenesis and aging. Bacterial LanC enzymes use Dha/Dhb to form carbon-sulfur linkages in antimicrobial peptides, but the functions of eukaryotic LanC-like (LanCL) counterparts are unknown. We show that LanCLs catalyze the addition of glutathione to Dha/Dhb in proteins, driving irreversible C-glutathionylation. Chemo-enzymatic methods were developed to site-selectively incorporate Dha/Dhb at phospho-regulated sites in kinases. In human MAPK-MEK1, such "elimination damage" generated aberrantly activated kinases, which were deactivated by LanCL-mediated C-glutathionylation. Surveys of endogenous proteins bearing damage from elimination (the eliminylome) also suggest it is a source of electrophilic reactivity. LanCLs thus remove these reactive electrophiles and their potentially dysregulatory effects from the proteome. As knockout of LanCL in mice can result in premature death, repair of this kind of protein damage appears important physiologically

Posttranslational mutagenesis: A chemical strategy for exploring protein side-chain diversity

Posttranslational modification of proteins expands their structural and functional capabilities beyond those directly specified by the genetic code. However, the vast diversity of chemically-plausible (including unnatural but functionally relevant) side-chains is not readily accessible. We describe C (sp3)–C (sp3) bond-forming reactions on proteins under biocompatible conditions, which exploit unusual carbon free radical chemistry, and use them to form Cβ–Cγ bonds with altered side chains. We demonstrate how these transformations enable a wide-diversity of natural, unnatural, posttranslationally-modified (methylated, glycosylated, phosphorylated, hydroxylated) and labeled (fluorinated, isotopically-labeled) side-chains to be added to a common, readily-accessible dehydroalanine precursor in a range of representative protein types and scaffolds. This approach, outside of the rigid constraints of the ribosome and enzymatic processing, may be modified more generally for accessing diverse proteins