Isotopic Label Incorporation Determination

Another application of LC-NMR in natural products chemistry concerns biosynthetic studies employing feeding experiments with stable isotope-labelled compounds. H NMR spectra allow the determination of the amount of isotopic label incorporated into metabolites, e.g. by observing signals that arise from /-couplings of protons to 13C-labelled nuclei [41,42],... 

Both absolute quantitation and relative quantitation of species in mixtures is of interest in some circumstances. Quantitation in a 5-minute analysis can be achieved by addition of an internal standard, ideally the target microorganism grown in special media to incorporate heavy isotopes92-95 and determination of the relative peak heights of pairs of proteins from the analyte and the standard. Isotope-labeled proteins or peptides, selected to match proteins or peptides characteristic of target microorganisms, can also serve as internal standards for isotope ratio measurement. The addition of unmatched proteins or peptides is less reliable for either ESI or MALDI measurements because of unpredictable suppression in the variable mixture. 

Much research has gone into raising the sensitivity and selectivity of immunosensors to the desired levels. Several labels have proved to ensure a high sensitivity, yet radioisotopic labels have essentially been avoided. Non-isotopic labels for immunosensors include various enzymes, catalysts, fluorophores, electrochemically active molecules and liposomes. Labelled immunosensors are basically designed so that immunochemical complexation takes place on the surface of the sensor matrix. There are several variants of the procedure used to form an immunocomplex on the matrix. In the final step, however, the label should always be incorporated into the immunocomplex for determination, as shown in Fig. 3.27.B. 

Scheme 14 Biosynthesis of the C5 unit of thiamin from isotopically labelled hexoses. Label from D-[l-14C]glucose and D-[l-uC]fructose label C-4 exclusively D-[2-14C]glucose labels C-4 and C-4 equally D-[6-14C]glucose labels C-4 (16% of 14C incorporated the precise location of the remaining 14C was not determined) d-[6-D2]glucose gives rise to both [4 -D2]- and [7-D2]-thiamin...

Quantitative analysis has become possible due to technical advances in synthesis of complex molecules with isotopic labels at any one of many specific position and measurements of KIE determined accurately and precisely by mass-spectrometry and radioactive methods. The most informative method for elucidation of the enzyme reaction limiting step and nature of transition-state is the competitive labeled method (Schramm, 1999). This method is based on the use of two labeled preparations of the same substrate, one with the labeled atom at a site expected to experience bonding changes at the TS and a second preparation with a different labeled atom at a site remote from the bond-breaking site. Many molecules of interest can be specifically labeled with radioactive atoms T or I4C and can be incorporated into substrates that also contain stable isotopes D, 15N and 180. 

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