Phospholipases are metalloenzymes which cause esterolysis of 3 sn-phosphoglycerides. The rate determining esterolysis of phospholipids by PLA2 is the gateway to further metabolites which mediate inflammation. Thus effective inhibition of PLA2 is a route to alleviation of chronic physiological effects of inflammation. Information on the detailed structural and dynamic interactions of inhibitors with the active site of the enzyme would point out characteristics for more effective inhibitors. Since PLA2 stereospecifically attacks the R isomer of the substrate, a mono-unsaturated phospholipid was synthesized as a precursor to a site-specific tritium labelled analog of very high specific activity and known label position and stereochemistry. The labelled compound will be used in investigations of phospholipase A2 (PLA2) competitive enzymatic inhibition by Fisons Pharmaceuticals. The combined techniques of using a tritiated ligand and 3H NMR to study the active site regions of proteins of biological importance is still novel, but promises to provide high quality, unambiguous data using standard experimental NMR methods. A number of labelling regimes were investigated, namely: (i) heterogeneously catalyzed double bond reduction with tritium gas; (ii) diimide reduction and (iii) homogeneously catalyzed reduction using Wilkinson's catalyst, tris(triphenylphosphine)chlororhodium. Regime (i) was very efficient but the specificity of label addition was low, giving a complex mixture of isotopomers which would not give precise geometrical information of the PLA2 active site. Conversely, regime (ii) resulted in the addition of tritium specifically across the double bond yet suffered from a high level of isotopic dilution. Since NMR as a detection method is characterized by its insensitivity, a high level of tritium incorporation was required. Regime (iii) was most suited to our application, and the reduction product was labelled only across the double bond, with known stereochemistry, to yield a pair of diastereomers of absolute configuration (R,R,R)- and (S,S,R)- at maximum tritium incorporation. The diastereomers were readily resolved in 1D 3H NMR spectroscopic analysis. The labelled Phospholipid was isolated from the homogeneous catalyst by column chromatography. Preliminary 3H/1H NMR experiments using this material were conducted at Fisons Pharmaceuticals, Loughborough, England. These experiements are being extended and improved at the NTLF in Berkeley. Extended aims of the project Through the use of an amide substrate analogue inhibitor, site specifically tritium and fluorine labelled at various and identical positions along the length of the sn-2 acyl chain, it is hoped to obtain selective information of the non-bonding, hydrophobic interactions between the alkyl chain of the inhibitor and the various amino acid residues of the active region of bovine pancreatic extracellular PLA2. The extent of information available would hopefully include structure (heteronuclear NOE's) and rate information (principally from T1 data) in order to describe the interaction processes occurring. With these data, it may then be possible to establish how significant the sn-2 alkyl chain/enzyme interaction is to the overall binding of this inhibitor to the active region. Also, a direct comparison may be made between the data obtained from 19F and 3H NMR approaches.