Synthesis of choline and ethanolamine phospholipids with thiophosphoester bonds as substrates for phospholipase C

Synthesis of sulfur-containing glycerophospholipids

“…One mode of sulfur protection is through disulfide formation. Cox et al [112] called on FeCl 3 /air to effect disulfide formation from 1-thioglycerol (2). Disulfide 95 could then be peracylated and reduction of 96 with dithiothreitol (DTT) in the presence of alkaline ethanol (pH = 9.6) regenerated the thiol (97) (scheme 27).…”

“…Although the chemistry was initially established for hexadecylthio chains [128], it has since been tailored to glycerol-based substrates, most notably 1,2-dimyristoyloxypropane-3-thiophthalimide. A room temperature reaction of the thiophthalimide (112) and the phosphite makes the requisite S-P bond of 114. Deprotection then affords the thioPI analog 1,2-dimyristoyloxypropane-3-thiophospho(1D-1-myo-inositol) (D-thio-DMPI, 115 (R,R = C 13 H 27 )) [115,129].…”

“…The thiophosphate group is prone to phospholipase C hydrolysis [111,112] and, in analogy with thiols released from other positions, can be used to evaluate the kinetics of this enzyme by functionalizing the sulfur. This technique has been applied mostly to phosphatidylinositols [113][114][115].…”

“…PC installation and eventual formation of 98 followed an established protocol [78] involving the sulfur of 97 selectively reacting at the phosphorus of 2-bromoethylphosphoryldichloride. Thiophosphatidyl ethanolamine analog 99 was accessed through transphosphatidylation of 98 by phospholipase D from green cabbage in the presence of ethanolamine (scheme 27) [112]. Various R groups are amenable to this chemistry, and the initial sulfur oxidation can also be effected with H 2 O 2 [111].…”

Phosphatidylcholine-Preferring Phospholipase C from B. cereus. Function, Structure, and Mechanism

“…Although using NPPC as a substrate allows for the facile evaluation of PLC Bc activity, NPPC is a very poor substrate for PLC Bc [29,30]; hence the sensitivity of this assay is low. Another non-natural substrate is 1,2-dioctanoyl-sn-(3-thioglycero)-phosphocholine [31], hydrolysis of which by PLC Bc liberates a free thiol that may be easily quantitated with 5,5¢-dithio-bis(2-nitrobenzoic acid) (DTNB, Ellman's Reagent) [32]. Unfortunately, PLC Bc processes this thiophospholipid 100 times slower than a natural phosphatidylcholine, so this assay is also of limited utility.…”