Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification

Joshi, Pralhad Namdev; Rai, Vishal

doi:10.1039/c8cc08733d

Cited by 46 publications

(43 citation statements)

References 24 publications

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“…Next, we selected ubiquitin (1 d) with one N a -NH 2 and seven N e -NH 2 groups. The LDM K-K reagents 4 a and 4 c-4 f led to 15,10,8,13, and 5 % conversion, respectively (Figures S47-S51). To our delight, the LDM K-K reagent 4 b proved successful in rendering single-site (K6) labeled ubiquitin 6 d (35 % conv., Figures 3 and S27).…”

Section: Angewandte Chemiementioning

confidence: 98%

“…After successful demonstration of LDM K-K with proteins bearing moderate number of primary amines, we selected a-lactalbumin, cytochrome C, and myoglobin with higher number of primary amines (13)(14)(15)(16)(17)(18)(19)(20). The vortexing of a-lactalbumin 1 e (one N a -NH 2 and twelve N e -NH 2 ) with 4 a renders mono-labeled product 6 e (37 % conv., Figures 3 and S28).…”

Section: Angewandte Chemiementioning

confidence: 99%

“…[8] We and others have demonstrated selective methods for labeling of low-frequency residues, [9] N-terminus a-amine, [10] or specific N-terminus residues. [11] Also, the labeling driven by ligand-protein interaction [12] or the unique nature of His [13] or Lys residue [14] inspired confidence for precision technologies. However, the off-target reactions in protein modification resulting in a heterogeneous mixture of products remains a significant concern.…”

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Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

et al. 2020

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The necessity for precision labeling of proteins emerged during the efforts to understand and regulate their structure and function. It demands selective attachment of tags such as affinity probes, fluorophores, and potent cytotoxins. Here, we report a method that enables single‐site labeling of a high‐frequency Lys residue in the native proteins. At first, the enabling reagent forms stabilized imines with multiple solvent‐accessible Lys residues chemoselectively. These linchpins create the opportunity to regulate the position of a second Lys‐selective electrophile connected by a spacer. Consequently, it enables the irreversible single‐site labeling of a Lys residue independent of its place in the reactivity order. The user‐friendly protocol involves a series of steps to deconvolute and address chemoselectivity, site‐selectivity, and modularity. Also, it delivers ordered immobilization and analytically pure probe‐tagged proteins. Besides, the methodology provides access to antibody‐drug conjugate (ADC), which exhibits highly selective anti‐proliferative activity towards HER‐2 expressing SKBR‐3 breast cancer cells.

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Section: Angewandte Chemiementioning

confidence: 98%

Section: Angewandte Chemiementioning

confidence: 99%

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confidence: 99%

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Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

et al. 2020

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“…Peptides are excellent scaffolds for biological studies, as they can be used to monitor highly specific molecular interactions with a broad range of biomolecules. Fluorescent peptides have traditionally been synthesized by coupling fluorophores to reactive groups (initially to amines, carboxylic acids and thiols 82 , but now also to imidazoles 83 or phenols 84 ) on the side chains of amino acids or in conveniently placed spacers. However, in some cases, this synthetic approach can alter the native conformation of the peptide or its biological properties (such as functional activity, binding affinity and/or subcellular localization) 85 .…”

Section: [H1] Bioactive Fluorescent Peptides [H2] Studying Biomoleculmentioning

confidence: 99%

Fluorescent amino acids as versatile building blocks for chemical biology

et al. 2020

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Fluorophores have transformed the way we study biological systems, enabling noninvasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids (FlAAs) have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein-protein interactions in situ or imaging nanoscopic events in real-time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of FlAAs and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using sitespecific genetic encoding and bio-orthogonal approaches, and their application to design novel artificial proteins as well as to investigate biological processes in cells by means of optical imaging.

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“…In this perspective, 4‐hydroxynon‐2‐enal (HNE) reagent and thiophosphorodichloridate exhibits high chemoselectivity for histidine . Recently, we demonstrated that 2‐cyclohexenone enables the single‐site labeling of His residue and allows late stage installation of probes (Scheme d) . The electrophiles in the previous two examples have been able to overcome the competition with lysine.…”

Section: Single‐site Labeling Of Native Proteinsmentioning

confidence: 99%

Chemical Methods for Selective Labeling of Proteins

et al. 2019

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Protein bioconjugation poses outstanding questions of selectivity to the organic transformations. Besides, the presence of a pool of functional groups in the structural outfit of a protein brings its own set of characteristics. In this minireview, we highlight the challenges faced by a chemical transformation to deliver selectivity in the modification of proteins. The examples of pre‐engineered proteins outline the attributes associated with chemoselectivity and chemical orthogonality. Building on this foundation, we discuss the complexity of site‐selectivity in the single‐site modification of native proteins. The gradual evolution of chemical methods while addressing the challenges associated with different amino acids are outlined. The modular methods for labeling a residue independent of its reactivity order closes the discussion.

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Single-site labeling of histidine in proteins, on-demand reversibility, and traceless metal-free protein purification

Abstract: A precision methodology distinguishes one His from all the nucleophilic residues and its multiple copies.

Cited by 46 publications

References 24 publications

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

Chemoselective and Site‐Selective Lysine‐Directed Lysine Modification Enables Single‐Site Labeling of Native Proteins

Fluorescent amino acids as versatile building blocks for chemical biology

Chemical Methods for Selective Labeling of Proteins

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