Specific Labeling of Streptavidin for Better Understanding of Ligand Modification in Modular Method for Affinity Labeling (MoAL)

Abstract

We studied the specific labeling of streptavidin using the modular method for affinity labeling (MoAL) that we developed based on a catalytic amide-forming reaction using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and a tertiary amine catalyst. The primary structures of avidin and streptavidin are significantly different from each other, and streptavidin does not possess an acidic amino acid equivalent to Asp108 of avidin, which is the target acidic amino acid that was labeled using MoAL. However, using biotinylated modular ligand catalysts (MLC) originally designed for labeling avidin, the labeling of streptavidin was found to successfully proceed at Glu51, which is located in a different region. The present study indicates that MoAL is readily applicable to protein labeling without a precise design for MLC. The most important factor for the design of MLC is to ensure that the linker is of sufficient length to connect the ligands to a catalytic site.

FRET-Created Traffic Light Immunoassay Based on Polymer Dots for PSA Detection

“…These above materials were finally assembled together and put inside the plastic cassettes. Green fluorescent coumarin derivatives, CA , were synthesized on the basis of the previously reported literature with revised procedures. − …”

“…After that, CH 2 Cl 2 was removed by the rotary evaporator and vacuum system to yield 400 mg (49%) of compound 3 as a colorless oil. 1 56 295 mg (0.52 mmol) of compound 4 was deprotected by trifluoroacetic acid (20%) in CH 2 Cl 2 at room temperature for 12 h. The reaction was terminated by extracting with sodium bicarbonate aqueous solution (10%) for 3 times. The organic solvent was then removed to obtain 217 mg (90%) of compound CA as a brown liquid.…”

“…, 4 56. In a round glass flask were injected 384 mg (1.2 mmol) of compound 3, 261 mg (1.00 mmol) of compound 2, 288 mg (1.50 mmol) of EDC, and 173 mg (1.50 mmol) of NHS in 2 mL of CH 2 Cl 2 .…”

Phototriggered Ketone Formation from an Aminocyclopropenone and a Carboxylic Acid

“…Recently a photoaffinity labeling method of a carboxylic acid using a tetrazole derivative was reported by Yao et al In this method, the tetrazole was used as a photolabile precursor of a nitrile imine, which selectively reacts with a carboxylic acid to form a stable acyl hydrazide. Because of the relatively high population of carboxylic acids on the surface area of proteins, a carboxylic acid is a promising target for a ligand directed specific functionalization method . In fact, the phototriggered functionalization of a carboxylic acid using a tetrazole derivative could yield superior results compared to classical photoaffinity labeling methods. , In addition to biological studies, the tetrazole-based phototriggered conjugation reaction has been applied to the modification of polymers .…”

Targeted Diazotransfer Reagents Enable Selective Modification of Proteins with Azides

“…A lysine (K121) is located near the biotin-binding site of wild-type core streptavidin . It has been shown that the ε-amine of this residue reacts with ligand-directed reagents, and it is therefore conceivable that it will also function as a diazotransfer acceptor, making streptavidin a good model protein . To prepare the DtBio 2 required for these studies, we synthesized p -nitrophenyl ester 4 from d -biotin and coupled it to histamine (Scheme ).…”