Photoaffinity labeling scheme

Scheme 2. The three levels of identification of biopolymers provided by photoaffinity labeling...

Scheme 2 Photoaffinity Labeling and Identification of a Ligand-Contact Site...

We have developed a new photoreactive analogue of paclitaxel, 3 -N-BzDC-3 -N-debenzoylpaclitaxel (109) and its ditritiated derivative ([3H]-109) has been evaluated for its ability to photolabel tubulin and P-glycoprotein.66 Radiolabeled photoreactive analogue [3H]-109 was synthesized by N-acylation of 3 -N-deben-zoyl-2, 7-bis(0-TES)paclitaxel (108) with N-(2,3-ditritio-3-(4-benzoyl-phenyl)propanoyloxy)succinimide ([3H]-107), followed by purification on a reversed phase semipreparative HPLC using a C-18 column (Scheme 21).66 Photoaffinity label [3H]-109 was assessed to possess >99.9% radiochemical purity and a high specific radioactivity (34 Ci/mmol). 

The first compound of this class (39) was synthesized to test its applicability in photoaffinity labeling of sugar-binding proteins. Cyclocondensation of the or-D-galactopyranosylmethyl ketone (37) with ammonia and hydroxyl-amine (9-sulfonic acid followed by oxidation with iodine in the presence of triethylamine gave 39 (85MI3) (Scheme 5). 

To study the relation between structure and photoaffinity labeling of sugar-binding proteins, a number of diazirine acyclo C-nucleosides (40-42) have been synthesized using a similar sequence to that shown in Scheme 5 (93MI5). 

The synthesis of myo-inositol derivative (83) by esterification of racemic 2,3,4,5,6-pentabenzyl myo-inositol as a potential inhibitor of PLC has been reported." The synthesis of a phosphodiester derivative of D,L-myo-inositol 1,3,4,5-tetraphosphate has been described (Scheme 14)." The ester was subsequently linked via the amine to an affinity resin and a photoaffinity label in order to probe the IP4 receptor. 

The result of a standard photoaffinity experiment can provide an output in three levels [8] as shown in Scheme 2. The photolytically induced targeted introduction of a (radio )label onto the target biopolymer helps to identify any specific binding proteins (first level of identification) responsible for a particular action in the signaling cascade or to identify the binding domain within the target protein (second level of identification) or allows one to identify the amino acid sequence of the binding protein (third level of identification). 

In addition to the attempts at unraveling the modification site, Dive and coworkers also constructed two biotinylated A/BPs, 22 and 23 (Scheme 3c), and used these to study the difference in affinity- and photoaffinity MMP enrichment from a complex proteome [57]. For this, tumor extracts were spiked with hMMP-12 and hMMP-8, after which compounds 22 and 23 were applied, followed by streptavidin-coated magnetic beads for MMP pull-down. Affinity-based labeling with 23 appeared superior to photoaffinity-based labeling with 22 in terms of quantity of captured MMPs and identification of the tryptic fragments by mass spectrometry. 

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