Anal
Biochem 1991 Aug 1;196(2):271-8
A
stable diazo photoaffinity label with high absorptivity and effective photoactivation
beyond 300 nm.
Hahn KM, Southwick PL
Department of Biological Sciences, Carnegie Mellon University, Pittsburgh,
Pennsylvania 15213.
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.
PMID: 1776676, UI: 92133737
