Photoaffinity labelling carbene

A highly reactive compound containing a neutral divalent carbon with two nonbondmg electrons (ie.,. CR2 or a substitution derivative). The nonbonding electrons can have parallel spins (triplet state) or antiparallel spins (singlet state). The parent species, iCRz, is also known as methylene. A number of carbene derivatives have been used as photoaffinity labels of proteins. Irradiation of 3 -0-(4-benzoyl)benzoyl-ATP will cause 70% inactivation of mitochondrial Fi-ATPase. ... 

Another general approach is the use of photoaffinity labels.9-11 A compound that is stable in the absence of light but that is activated by photolysis is reversibly bound to an enzyme and photolyzed. The usual reagents are diazo compounds that when photolyzed give highly reactive carbenes, or azides that give highly reactive nitrenes ... 

In the literature of photoaffinity labeling, much ado is made about the half-lifes of carbenes, nitrenes and other reactive intermediates. It is often implied that the half-life has a fixed value for each intermediate, but it is of course a function of the temperature and environment. 

Human corticosteroid binding globulin (CBG) has been the target for photoaffinity labeling by the carbene precursor, [6,7-3H]-21-diazo-21-deoxycorticosterone (Compound V)... 

Carbene rearrangement of a different type is preferred in the (pentamethyldisilanyl-diazomethyl) ketones (70) which are converted on irradiation into 1-oxa-2-silacyclobut-3-enes (71) and 1,5-dioxa-2,6-disilacyclo-octa-3,7-dienes (72) via the unstable 3-oxa-1-silaprop-1-enes (73). 3-Diazoacetylretinals have been synthesized for use in photoaffinity labelling. 

Westheimer and his associates have used carbenes in their studies (Singh et al. 1962 Shafer et al. 1966 Vaughan and Westheimer 1969 Browne et al. 1971 Hexter and Westheimer 1971). Carbenes are capable of reacting not only with polar residues but also with hydrophobic residues. Carbenes have been generated from diazoacetates and diazomalonate derivatives. Diazoacetates were initially employed since they may be photolyzed at 370 nm. This minimizes the potential for photochemical decomposition of the protein - one of the important limitations of photoaffinity labels. 

Nitrenes, the nitrogen analogs of carbenes have been introduced as a reactive group in photoaffinity labels for antibodies by Fleet et al. (1969). Nitrenes tend to be more selective in their reactions and possibly will provide fewer products than carbenes (but see also 6.2.3). The types of reaction that they can undergo (eq. 6.2, Knowles 1972) include insertions into C-H bonds to yield secondary amines, cycloadditions to double bonds to form cyclic 3-member imines and... 

These authors have emphasized that the best way to distinguish between these two possible mechanisms of labeling is by the addition of scavengers of carbenes, nitrenes or whatever the chemical nature of the activated form of the photoaffinity label is. A true photoaffinity label (i.e. one which reacts irreversibly with the same protein molecule to which it was bound at the instant of photolysis) will not be inhibited by added scavengers, whereas a pseudo photoaffinity label (i.e. one which accomplishes derivatization via a reactive intermediate which has been generated in solution) will be. In their studies with nitrenes, Ruoho et al. (1973) found that 10 M p-aminobenzoic acid was an effective scavenger. It should be emphasized that the two possible mechan-... 

Despite the fact that they can be much more reactive than nitrenes, carbene derivatives are less frequently cited in reports on photoaffinity labels. Two such derivatives of thyroid hormone have been described in which the amino group of the alanine side chain of 3,5,3 -triiodo-L-thyronine or 3,5,3, 5 -tetraiodo-L-thyronine was derivatized with 2-diazo-3,3,3-trifluoropropionate (246). Irradiation at 254 nm of these compounds with several cell lines yielded low incorporation of reagent (0.5-13.5%) into two forms of thyroid hormone receptor proteins. Low incorporation was also characteristic of early reports of photoaffinity labeling (e.g., 247) in which 0 -ethyl-2-diazomalonyl-cAMP and A, C)2. (ji(ethyl-2-diazomalonyl)-cAMP were used to generate carbenes which modify rabbit muscle phosphofructokinase. Similarly, only 3-14% of the potential sites of lactate dehydrogenase were labeled by the carbene derived from 3-(3//-diazirino)pyridine adenine dinucleotide (248). 

Esters and thioesters of (dansyldiazomethyl)methylphosphinic acid undergo carbene insertion reactions in high yield on irradiation their fluorescent properties make them suitable reagents for photoaffinity labelling studies. 

The triphosphate (102) has been prepared from the CDI-mediated condensation between pyrophosphate and corresponding nucleoside S -monophosphate and has been designed for the photoaffinity labelling of HIV-reverse transcriptase. The analogue is relatively stable but upon irradiation at 365 nm a highly reactive carbene moiety is generated. 

The carbene-generating biotinylated 2-acetamido-2-deoxy-D-glucopyranosyl-amide diazirine derivative 54 and the lactoside 55 have been synthesized for photoaffinity labelling of P-(l ->4)-galactosyl transferase and GMj synthase. ... 

The carbene and nitrene products of photoaffinity labels have been shown to be sensitive to most thiol protecting reagents except thioglycollate. ... 

Diazirines are simply cyclic isomers of diazo compounds (Eq. 16.74). In fact, the two forms can sometimes be interconverted photochemically. The photochemistry of a diazirine mirrors that of the analogous diazo compound—that is, loss of N2 to produce a carbene. In some circumstances the diazirine form is more desirable, especially in photoaffinity labeling applications. 

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