Isotopic labeling deuteration

The most frequently encountered examples of cyclopropyl ring opening reactions in the steroid field are usually associated with angular or side chain methylation sequences. In fact, isotope labeling of the C-19 angular methyl group is the only reported application of this reaction for deuteration or tritiation pui poses. 

Isotope labeling by derivative formation with deuterated reagents is useful for the preparation of analogs such as dg-acetonides, da-acetates, da-methyl ethers, dg-methyl esters, etc. The required reagents are either commercially available or can be easily prepared. (The preparation of da-methyl iodide is described in section IX-F. Various procedures are reported in the literature for the preparation of dg-acetone, da-diazometh-ane57.i63.i73 and da-acetyl chloride. ) These reactions can be carried out under the usual conditions and they need no further discussion. A convenient procedure has been reported for the da-methylation of sterically hindered or hydrogen bonded phenolic hydroxyl functions by using da-methyl iodide and sodium hydroxide in dimethyl sulfoxide solution. This procedure should be equally applicable to the preparation of estradiol da-methyl ether derivatives. 

The present method is simple, proceeds easily and in good yield. The starting materials are readily available. The method is of particular value for the ready preparation of C-l deuterated aldehydes using the 2-deuterio-N,4,4-trimethyl-2-oxazolinium iodide.6 Also, since relabeled formic acid is routinely available, this provides easy access to isotopically labeled aldehydes. 

Ionization changes can be efficiently corrected with the use of an isotopically labeled IS, which possesses identical ionization response and fragmentation pattem. Therefore, deuterated IS can be used to correct both the overall method variability (e.g., sample preparation, injection, electrophoretic process, etc.) as well as matrix effects since the amount of suppression from interferents is expected to be similar. However, the total concentration of analyte and IS should be below the saturation of the ionization process. Guidelines to obtain a reproducible CE—MS method were published by Ohnesorge et al. and took into account the use of an isotopically labeled IS. 

Although DIOS-MS is mainly a tool for qualitative analysis, many examples have shown that quantitative analysis is possible when internal standards are used. These may either be isotope-labelled - mostly deuterated - compounds or structurally related analogues. For example, subsequent to electrospray deposition, amino acids such as phenylalanine and tyrosine have been successfully quantified by means of DIOS-MS using their deuterated analogues as internal standards. 

All of the compounds can be deuterated by repeated crystalhzation from D2O. For SQA, labelhng can be accomplished by heating the dissolved compound in about 20% 0-labelled H2O, in a closed vessel [26]. Both the labelled water and squaric acid should be refrigerated until used. The isotope labelling should be confirmed by mass spectrometry or NMR spectroscopy. In general, a 10% label is quite satisfactory for good NMR signal... 

Application of alumina-supported sodium borodeuteride under the reaction conditions described by Varma26 provided deuterated alcohols from aldehydes and ketones with a high degree of deuterium incorporation. The method is thus suitable for isotopic labelling procedures (Scheme 4.12)32. 

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