Xenobiotic conjugates methods

Dramatic improvements in instrumentation and methods used for the identification of xenobiotic conjugates (and other metabolites), notably proton nuclear magnetic resonance and mass spectroscopy, have significantly influenced the strategies for the isolation of conjugates. The recently developed soft ionization techniques make it possible to obtain useful mass spectral information for a wide variety of intact underivatized xenobiotic conjugates. The state of... 

Thereafter the mixtures of xenobiotic conjugates must be separated into individual fractions (separation) which in turn must be ultimately purified (purification) for spectroscopic identification. As the investigator moves from the enrichment via separation to purification steps the methods require less capacity and selectivity, but greater resolution. Suitable methods have to be selected and arranged in a meaningful sequence in order to obtain an adequate, (i.e. a successful and economical) Isolation strategy. 

The synthesis of model compounds to verify the structures of xenobiotic amino acid adducts (products of nonenzymatic reactions of electrophilic xenobiotics with amino acids In proteins) are Included. Methods for synthesis of some rarely observed conjugate types will be briefly Introduced. The major organic methods for synthesis are exemplified and xenobiotic conjugates prepared via these reactions are tabulated with references to the original publications. 

In addition to the examples discussed above, a number of other xenobiotics are measured by their phase I reaction products. These compounds and their metabolites are listed in Table 20.1. These methods are for metabolites in urine. Normally, the urine sample is acidified to release the phase I metabolites from phase II conjugates that they might have formed, and except where direct sample injection is employed, the analyte is collected as vapor or extracted into an organic solvent. In some cases, the analyte is reacted with a reagent that produces a volatile derivative that is readily separated and detected by gas chromatography. 

Botran was one of the first xenobiotics reported to be metabolized to an N-malonyl conjugate by this mechanism In plant tissues (120) (Equation 28). The N-malonyl conjugate of botran was the major metabolite In both soybean and soybean callus culture. It was Isolated by chromatographic methods and Identified by synthesis and by chemical and mass spectral methods. 

Chemical synthesis of glucuronic acid conjugates of poly-functlonal xenobiotics may yield undesirable side products enzymatic methods may be preferable for glucuronic acid... 

Even though the number of glycoside conjugates of xenobiotics that have been synthesized so far Is limited, the methods described here are convenient both for large and small scale operations. No specific restrictions In synthesis of Isotopically labeled conjugates should be encountered due to the synthetic methods. 

Conjugation of 0-, N- and S-contalnlng functional groups with sulfate Is very common In animal systems. Sulfate Is transferred from adenosine 3 -phosphate-5 -sulfatophosphate (PAPS) by a variety of transfer enzymes. The mechanisms for sulfation of xenobiotics (74,75) and the chemistry of sulfate esters and related compounds and the synthesis of these have been reviewed (75-77). Syntheses of 0-, N- and S-sulfate conjugates of xenobiotics may be carried out by the methods described below. 

Reactions with sulfur trloxlde. Sulfur trloxlde (SO3) may react violently with nucleophiles unless the conditions are well controlled and this route Is not commmonly used for syntheses of xenobiotic sulfate conjugates. However a method suitable for both macro and micro scale synthesis of ascorbic acid 2-P sj sulfate (Figure 6A) was recently reported where the SO3 was Introduced to the substrate dissolved In DMF at -180"C (7 ). The reaction was completed at -15 C In 30 min and gave a 74Z yield of the 2-0-sulfate. 

Reactions with aliphatic and aromatic epoxides. The metabolism of many alkenes and arenes Is known to proceed via epoxides formed by phase I reactions (166). Chemical methods for the preparation of epoxides will not be discussed here but valuable references (or references cited therein) for their synthesis are found In Table VII. The electrophilic character of the epoxides has been utilized In chemical synthesis of several PMAF metabolites. Differences In the reactlvltes of arene oxides are probably less Important In chemical synthesis of their conjugates but may Influence the ratio of positional and dlasteromers formed. The reaction of GSH with a xenobiotic epoxide gives the 1,2-dlhydro--l-hydroxy-2-glutathlonyl-xenoblotlc conjugate and with cysteine and N-acetyl-cystelne the corresponding dlhydro-hydroxy conjugates. The nucleophile (GSH, cysteine or N-acetyl-cystelne)... 

The formation of an amide bond by organic chemical methods Is usually accomplished by reacting the acid halogenlde and the amino acid unless the acid substrate contains other reactive groups. The acid chloride Is prepared directly with thlonyl chloride (244) or Indirectly via the formation of a mixed anhydride by use of another acid halogenlde (245,246). Isotoplcally labeled [(2,4-Dlchlorophenoxy)acetyl]valine was synthesized by a DCC catalyzed reaction between the acid and valine (247). A few examples of synthetic amino acid conjugates of xenobiotics are shown In Table X. Several taurine conjugates of both alkanolc and benzylic acids have been synthesized and their physical properties determined by Idle et al. (2 ). 

Perhaps the most common reason for the existence of hidden conjugates, especially in the past, is the failure of the analytical methods employed to detect total xenobiotic residues present in a sample. This problem has been solved to a great extent by the use of radiotracer techniques in xenobiotic investigations. Once its existence is established, the analytical chemist can usually isolate and identify the metabolite. This, however, does not mean that... 

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