Isotopically labeled esters

Since the imidazolide method proceeds almost quantitatively, it has been used for the synthesis of isotopically labeled esters (see also Section 3.2), and it is always useful for the esterification of sensitive carboxylic acids, alcohols, and phenols under mild conditions. This advantage has been utilized in biochemistry for the study of transacylating enzymes. A number of enzymatic transacylations (e.g., those catalyzed by oc-chymo-trypsin) have been shown to proceed in two steps an acyl group is first transferred from the substrate to the enzyme to form an acyl enzyme, which is then deacylated in a second step. In this context it has been shown[21] that oc-chymotrypsin is rapidly and quantitatively acylated by Af-fraw.s-cinnamoylimidazole to give /ra/w-cinnamoyl-a-chymotrypsin, which can be isolated in preparative quantities and retains its enzymatic activity (see also Chapter 6). 

Evidence for this mechanism comes from studies done with isotopically labeled esters. When ethyl propanoate labeled with in the ether-type oxygen of the ester (below) is subjected to hydrolysis with aqueous NaOH, all of the shows up in the ethanol that is produced. None of the 0 appears in the propanoate ion ... 

In an extension of the work described m the preceding section Bender showed that basic ester hydrolysis was not concerted and like acid hydrolysis took place by way of a tetrahedral intermediate The nature of the experiment was the same and the results were similar to those observed m the acid catalyzed reaction Ethyl benzoate enriched m 0 at the carbonyl oxygen was subjected to hydrolysis m base and samples were isolated before saponification was complete The recovered ethyl benzoate was found to have lost a por tion of Its isotopic label consistent with the formation of a tetrahedral intermediate... 

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 hydrazinolysis is usually conducted in refluxing ethanol, and is a fast process in many cases. Functional groups, that would be affected under hydrolytic conditions, may be stable under hydrazinolysis conditions. The primary amine is often obtained in high yield. The Gabriel synthesis is for example recommended for the synthesis of isotopically labeled amines and amino acids. a-Amino acids 9 can be prepared by the Gabriel route, if a halomalonic ester—e.g. diethyl bromomalonate 7—is employed as the starting material instead of the alkyl halide ... 

Yeom and Frei [96] showed that irradiation at 266 nm of TS-1 loaded with CO and CH3OH gas at 173 K gave methyl formate as the main product. The photoreaction was monitored in situ by FT-IR spectroscopy and was attributed to reduction of CO at LMCT-excited framework Ti centers (see Sect. 3.2) under concurrent oxidation of methanol. Infrared product analysis based on experiments with isotopically labeled molecules revealed that carbon monoxide is incorporated into the ester as a carbonyl moiety. The authors proposed that CO is photoreduced by transient Ti + to HCO radical in the primary redox step. This finding opens up the possibility for synthetic chemistry of carbon monoxide in transition metal materials by photoactivation of framework metal centers. 

Many mechanistic aspects of the hydrolysis of phosphate esters in protic media remain uncertain. In spite of predictions that racemization at phosphorus should be the final outcome if indeed the (hypothetical) metaphosphate intermediate is involved in the solvolysis of monoesters, the results of several studies on the methanolysis of appropriately O-isotopically labelled compounds are consistent with reactions proceeding with inversion of configuration, as observed for all enzymic and non-enzymic systems so far examined this has resulted in the suggestion that if metaphosphate is actually formed, then it must be in a masked form. 

Syntheses of Isotopically Labeled, Spin-labeled, and Photoreactive Esters... 

A reaction that has been much investigated is the hydrolysis of esters, e.g. (164), by aqueous base, i.e. eOH. It is found to be kinetically second order, and lsO isotopic labelling experiments on (164) have... 

The photorearrangement of enol esters is strictly intramolecular. Upon irradiation of two different steroidal enol acetates, one of them possessing a trideuterated acetyl group, the original isotope labels were fully retained in the products.105... 

A c2 pathway, 241, 384 A,4 1 pathway, 241, 380 acid-catalysed, 240, 378 acyl-oxygen fission, 88, 240, 242 alkyl-oxygen fission, 240 isotope labels in, 88, 241 steric effects in, 242 Esters... 

The corresponding reactions with [ieO] 2-propanol and [180]t-butyl alcohol show that the isotopic label is not incorporated in the product ion. This result clearly establishes that the alkyl—oxygen bond in the alcohol is cleaved in process (80). A similar situation is observed even for the case when the ester has a lower proton affinity than the alcohol as in the case of reaction (81). 

Acylcarnitine analysis of dried blood or bile spots is very similar to the analysis of plasma. A small disk (diameter typically 5 mm or less) is punched out of the blood spot and the acylcarnitines extracted by the addition of methanol and known concentrations of isotopically labeled acylcarnitines, which function as internal standards. The extract is dried under a stream of nitrogen, and derivatized by the addition of either n-butanol HC1 or n-methanol HC1. The acylcarnitines are measured as their butyl or methyl esters by MS-MS. The concentrations of the analytes are established by computerized comparison of ion intensities of these analytes to that of the internal standards. 

Isotope labeled standards a few aroma volatiles are commercially available from CDN Isotopes and Cambridge Isotope Laboratories (CIL) custom synthesis is available from Aspen Research Laboratories and Aldrich the standards may also be synthesized in-house Purified aroma compounds to be quantified Ester of high GC purity (e.g., methyloctanoate)... 

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