A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays

Zhao, Yanjun; Pirrung, Michael C.; Liao, Jiayu

doi:10.1039/c2mb05471j

Cited by 5 publications

(4 citation statements)

References 40 publications

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“…Furthermore, the Fukuyama-Mitsunobu reaction was used to couple 6-acyl-2-naphthylamine to L-serine to produce 3-(6-acetylnaphthalen-2-ylamino)-2aminopropanoic acid (ANAP) (20), a building block with remarkable optical properties (λem.= 490 nm, quantum yield ~50%) that is widely used for fluorescent labelling of proteins 68 . NBDlike fluorophores have been conjugated by a nucleophilic aromatic substitution (SNAr) reaction to nucleophilic groups (21) in amino acid side chains (such as 3-amino-alanine) 58,69 , whereas phospholyl(borane) amino acids (22) have been produced by nucleophilic substitution of fluorescent phospholide anions with iodo-derivatized amino acids 70 , which enabled the synthesis of a range of phospholyl derivatives with fluorescence properties covering a broad spectrum of emission maxima wavelengths from ultraviolet to green (λem.= 340-530 nm).…”

Section: [H2] Non-natural Flaasmentioning

confidence: 99%

“…Furthermore, a 7-azatryptophan-containing peptide has been used for detection of haeme levels in cells 94 using FRET-based measurements based on the overlap between the emission spectrum of 7-azatryptophan (λem.= 400 nm) and the absorbance of the haeme group. Peptides containing other fluorophores have also been used in FRET assays to measure protease activity 69 , including benzoacridone amino acids to detect active caspase 3 (ref. 74 ), a key enzyme in the apoptotic pathway.…”

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

confidence: 99%

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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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Section: [H2] Non-natural Flaasmentioning

confidence: 99%

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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“…The controlled immobilization of biomolecules such as proteins and oligonucleotides on material surfaces is critical in biological research, as it underpins the advancement of various biotechnologies such as microarrays , biosensing, drug delivery, , and tissue engineering . Microarrays are particularly useful in bioanalytical research since they allow highly parallel, multiplex analysis of biomolecules in a rapid and miniaturized manner.…”

Section: Introductionmentioning

confidence: 99%

Selective C-Terminal Conjugation of Protease-Derived Native Peptides for Proteomic Measurements

et al. 2022

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Bottom-up proteomic experiments often require selective conjugation or labeling of the N- and/or C-termini of peptides resulting from proteolytic digestion. For example, techniques based on surface fluorescence imaging are emerging as a promising route to high-throughput protein sequencing but require the generation of peptide surface arrays immobilized through single C-terminal point attachment while leaving the N-terminus free. While several robust approaches are available for selective N-terminal conjugation, it has proven to be much more challenging to implement methods for selective labeling or conjugation of the C-termini that can discriminate between the C-terminal carboxyl group and other carboxyl groups on aspartate and glutamate residues. Further, many approaches based on conjugation through amide bond formation require protection of the N-terminus to avoid unwanted cross-linking reactions. To overcome these challenges, herein, we describe a new strategy for single-point selective immobilization of peptides generated by protease digestion via the C-terminus. The method involves immobilization of peptides via lysine amino acids which are found naturally at the C-terminal end of cleaved peptides from digestions of certain serine endoproteinases, like LysC. This lysine and the N-terminus, the sole two primary amines in the peptide fragments, are chemically reacted with a custom phenyl isothiocyanate (EPITC) that contains an alkyne handle. Subsequent exposure of the double-modified peptides to acid selectively cleaves the N-terminal amino acid, while the modified C-terminus lysine remains unchanged. The alkyne-modified peptides with free N-termini can then be immobilized on an azide surface through standard click chemistry. Using this general approach, surface functionalization is demonstrated using a combination of X-ray photoelectron spectroscopy (XPS), ellipsometry, and atomic force microscopy (AFM).

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“…For example, in our previous work, we showed the possibility of introducing fragments of 2,1,3benzoxadiazoles into nucleosides and oligonucleotides [5,6]. They are also used for the synthesis of fluorescent amino acids, glucosides or protein binders [7][8][9].…”

Section: Introductionmentioning

confidence: 99%

Halogenated 2,1,3-benzoxadiazoles as Potential Fluorescent Warheads for Covalent Protease Inhibitors

Kuznetsova

Klein

Opatz

2018

22nd International Electronic Conference on Synthetic Organic Chemistry

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Recently there has been a growing interest in covalent protease inhibitors in both industry and academia, caused by their longer residence times, their higher potency and their high ligand efficiency. Covalently reactive moieties which interact with activated amino acid residues such as serine or cysteine in enzymes like proteases or esterases mostly act through nucleophilic addition, substitution or ring opening. In contrast, nucleophilic aromatic substitution (SNAr) is rarely employed. In our previous work, we prepared and investigated electrophilic “warheads”, which contain aromatic, heteroaromatic or quinoid fragments. Some of them show potent inhibition constants for cathepsin L, cathepsin B, rhodesain or dengue-protease, and depending on the exact nature of the electrophile they exhibit reversible covalent or irreversible inhibition modes. In the present work, we demonstrate the synthesis of fluorescent “warhead” candidates based on 2,1,3-benzoxadiazoles and the investigation of their physicochemical and photophysical properties. These molecules shall serve as probes for the detailed analysis of association/dissociation mechanism and of the kinetic parameters of the bond forming event.

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A fluorescent amino acid probe to monitor efficiency of peptide conjugation to glass surfaces for high density microarrays

Cited by 5 publications

References 40 publications

Fluorescent amino acids as versatile building blocks for chemical biology

Fluorescent amino acids as versatile building blocks for chemical biology

Selective C-Terminal Conjugation of Protease-Derived Native Peptides for Proteomic Measurements

Halogenated 2,1,3-benzoxadiazoles as Potential Fluorescent Warheads for Covalent Protease Inhibitors

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