A stable diazo photoaffinity label with high absorptivity and effective photoactivation beyond 300 nm.
The sulfosuccinimidyl active ester of 3-(3-carbethoxy-4-diazo-5-oxo-2-pyrrolin-1-yl)propanoic acid (DIAZOPY-SE) has been synthesized for use as a photoaffinity labeling reagent. This compound was obtained from commercial chemicals by a four-step synthesis requiring no complex procedures or special apparatus. The active ester efficiently derivatizes protein amino groups with the chromophore 3-carbethoxy-4-diazo-5-oxo-2-pyrroline (DIAZOPY, epsilon 8800 M-1 cm-1 at lambda max 330 nm), which on irradiation yielded products expected from formation of a reactive carbene intermediate. Brief irradiation of DIAZOPY in 2-propanol using wavelengths greater than 300 nm for photolysis yielded mainly an isopropyl ether resulting from insertion of the carbene into the O-H bond of the alcohol. Formed concurrently and to a somewhat lesser extent was an isopropyl ester, resulting from a ring-contracting Wolff rearrangement of the carbene and subsequent reaction with isopropanol. Analogous products were produced by photolysis in 2-propanol of DIAZOPY-PA (for DIAZOPY propanoic acid), the carboxylic acid precursor of DIAZOPY-SE. Facile protein derivatization by DIAZOPY-SE was demonstrated using actin and sheep IgG. Actin labeled with DIAZOPY-SE and irradiated while in the F-actin (reversibly polymerized) form was crosslinked to yield a covalently-linked dimer, illustrating the potential of the reagent in photoaffinity applications. Advantages of DIAZOPY-SE as a photoaffinity labeling reagent include ease of synthesis, chemical and photostability, efficient photolysis at wavelengths greater than 300 nm, and a capacity for crosslinking by carbene insertion processes.
Hahn
K M
KM
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
Southwick
P L
PL
D000199:Actins / Q000737:chemistry; D000345:Affinity Labels / Q000138:chemical synthesis* / Q000378:metabolism; D000818:Animals; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry; D003979:Diazonium Compounds / Q000138:chemical synthesis* / Q000737:chemistry; D004355:Drug Stability; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D010777:Photochemistry; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D000199:Actins; D000345:Affinity Labels; D003432:Cross-Linking Reagents; D003979:Diazonium Compounds; D007074:Immunoglobulin G