Isotopic labelling/tracing

Another widely used approach to the elucidation of metabolic sequences is to feed cells a substrate or metabolic intermediate labeled with a particular isotopic form of an element that can be traced. Two sorts of isotopes are useful in this regard radioactive isotopes, such as and stable heavy isotopes, such as or (Table 18.3). Because the chemical behavior of isotopically labeled compounds is rarely distinguishable from that of their unlabeled counterparts, isotopes provide reliable tags for observing metabolic changes. The metabolic fate of a radioactively labeled substance can be traced by determining the presence and position of the radioactive atoms in intermediates derived from the labeled compound (Figure 18.13). 

Much useful information has been obtained by using molecules that have been isotopically labeled and tracing the path of the reaction in that way. For example, in the reaction... 

Choosing a method to determine isotope effects on rate constants, and selecting a particular set of techniques and instrumentation, will very much depend on the rate and kind of reaction to be studied, (i.e. does the reaction occur in the gas, liquid, or solid phase , is it 1st or 2nd order , fast or slow , very fast or very slow , etc.), as well as on the kind and position of the isotopic label, the level of enrichment (which may vary from trace amounts, through natural abundance, to full isotopic substitution). Also, does the isotopic substitution employ stable isotopes or radioactive ones, etc. With such a variety of possibilities it is useless to attempt to generate methods that apply to all reactions. Instead we will resort to discussing a few examples of commonly encountered strategies used to study kinetic isotope effects. 

One alternative would be to use isotopically labeled intermediates to trace incorporation of groups in to the catalyst. In spite of the drawbacks I feel that the solid-phase approach will be used successfully in the synthesis of catalysts with enzyme-like properties. 

A measure of the amount of a stable isotopic label that exceeds its natural abundance in unlabeled tracee. This is most directly accomplished using an ion ratio mass spectrometer to measure the ratio of ion currents for isotopomers such as C02 at mass 44 and 2 at mass 45. From the difference between the ion current ratio for a sample (Zsampie) and the ion current ratio for a reference gas (Zreference), the atom percent excess (APE) can be estimated. See Tracer/Tracee Ratio Compart-mental Analysis Isotope Exchange Kinetics... 

The precision of MS assays is in the range typical of most clinical assays (i.e., under 5-15%). The best choice of internal standard is the stable-isotope-labeled form (preferably 13C) of the compound of interest (e.g., P-hydroxy myristic acid or muramic acid). Specific trace detection of chemical markers in complex matrices requires appropriate negative controls. Procedures are often described that do not employ the mass spectrometer and false positives are often reported. The mere analysis of blank filters or water blanks is not satisfactory since chemical noise contributed by the sample is much greater and is not accounted for with this form of control. 

The method of isotopic labelling is employed to determine the presence of traces of copper with precision. An ICP/MS installation was employed. The results of the experiment rely upon the ratio of the intensities of the peaks corresponding to the masses 63 and 65 of the two isotopes 63Cu and 65Cu of elemental copper. The experiment is described below. First, an injection of a small quantity of the unknown solution was injected. The intensities of 63Cu and 65Cu are 82 908 and 37 092 respectively (arbitrary units). 

The benefits of initial product studies, followed by a more detailed examination using isotopic labelling, are well illustrated by the amination of halobenzenes, Scheme 2.7. The reaction of p-bromomethoxybenzene (15) with lithium diethylamide in ether gives a 1 1 mixture of m- and p-diethylamino-substituted products (16 and 17), with no trace of the o-isomer. One possible mechanism for these and many related reactions was that a normal direct displacement reaction to give 17 was in competition with an abnormal displacement,... 

Isotopic labelling and substitution Less commonly encountered isotopes of an element (trace isotopes) provide a useful way to follow chemical reactions as they... 

As demonstrated, different time-resolved FTIR techniques allow to study the complete photocycle of bacteriorhodopsin in the entire range from picoseconds to several milliseconds. Infrared difference spectra trace reactions which take place in different parts of the protein molecule. Isotopically labeled proteins or proteins with mutations at specific sites... 

Tracer techniques have revolutionized biochemistry and molecular biology. For example, the availability of isotopically labeled compounds made it possible to demonstrate that macromolecules such as proteins, nucleic acids, and complex lipids are synthesized from simple cellular metabolites and provided many insights into the mechanisms and control of the synthetic events. The utility of radiochemical techniques is afforded by (1) their great sensitivity compared to other analytical methods (Table 3-1) and (2) the fact that they label the atoms of molecules without significantly altering their chemical properties, thus allowing them to be traced or followed from one molecule to another. 

Insertion of SO3 into the OF bond of F2O forms FOOSO2F. Isotopic labeling indicates that the reaction takes place via the intermediacy of radicals. Insertion into AsFj yields products containing the ASOSO2F linkage, but SeOj yields only polymers that do not result from insertion". Similar results are obtained with SbF3 except that traces of insertion products are obtained with SeOs. Reactions of SO3 with metal halides (e.g., CrFs, NaCl, CsAgF4) form halosulfonates . 

The remark has been made that compounds of tin can be studied by more techniques than those of any other element. The fact that it has more stable isotopes that any other element gives it very characteristic mass spectra, and isotopic labelling can be used to interpret vibrational spectra, and for spiking samples in trace analysis two of the isotopes have spin 1/2 and are suitable for NMR spectroscopy, and their presence adds information to the ESR spectra of radical species. Further, the radioactive isotope 119mSn is appropriate for Mossbauer spectroscopy. The structural complications that are referred to in the previous chapter have therefore been investigated very thoroughly by spectroscopic and diffraction methods, and structural studies have always been prominent in organotin chemistry. 

Amongst the earliest applications of labelling was the use of doubly labelled [1, 2- C2] acetate to trace the mode of incorporation of intact acetate units into a wide range of metabolites. This has been one of the major recent developments in biosynthetic methodology and permits information to be obtained which would have been impossible or at best extremely difficult to obtain by classical radio-isotope labelling techniques. The basic concept can be illustrated by a model polyketide system (Fig. 1). 

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