Isotopic labelling studies synthesis

The study of the biosynthesis of vitamin B 2 is a saga whose resolution, due primarily to Battersby (80—83) and Scott (84,85), requited an effort on the same magnitude as the total synthesis. It was only when recent molecular biology tools became available to complement en2ymology, isotopic labeling, chemical synthesis, and spectroscopy that solution of this problem became possible. 

It is curious that there have been more fundamental developments in 1,3,5-triazine synthesis by this method than in any of the other sections, yet it is the oldest of all the synthetic routes. Cyanuric acid was originally prepared by the pyrolysis of uric acid (7) (see Section 2.20.1.2). The mechanism was proposed by Brandenberger and coworkers on the basis of isotopic labelling studies, and was reviewed by Modest (B-61MI22000, p. 706). The rearrangement of pyrimidines to 1,3,5-triazines is the most important advance in this area. 

Many biologically active substances and neuroleptic drugs have a seven-membered ring in their structure. Benzodizapines of extremely high specific activity used in receptor binding studies are isotopically labelled by synthesis. The specific activities of compounds 137-150 are sufficiently high for in vitro metabolic and radiotracer studies. 

More detailed isotopic labeling studies have also been performed. Hydroxycyclopropanation of trans-f.3-deutero-styrene 273 under Kulinkovich conditions furnishes m-2-phcny 1-1 -cyclopropanol, 274, indicating retention of configuration at the carbon bound to titanium and is consistent with frontside attack of the Ti-C bond on a titanium-bound carbonyl.220 For the related de Meijere cyclopropylamine synthesis, the opposite outcome has been observed where a 3 1 mixture of A, Wdimethyl-W(/ra t-3-deutero-/ra j-2-phenylcyclopropyl)amine 278 and -dim ethyl-. V-((7.i-dcutcro-t7r-2-phony Icyclopropy I )amine 277 is produced. These products require inversion of configuration at the carbon bound to titanium and are consistent with a W-shaped transition structure for ring closure (Scheme 46). 

Such mechanism has been postulated mainly on the basis of isotope-labelling studies. " Methanol is formed from CO2 that is produced during the water-gas shift reaction. Under normal operating conditions, carbon dioxide is added to the feed gas. There appears to be a limit to the amount of CO2 that can be present in the feed, since water produced during methanol synthesis from... 

The mechanism has involved considerable debate over the years [2289, 2304, 2305, 2315]. A number of approaches can be applied to the mechanistic question, including studies with simplified models [2291, 2318-2321], synthesis of potential intermediates for testing with the enzyme [2303, 2304], application of theory [2308, 2309], spectroscopy [2301, 2302], and isotopic labeling studies [2299,2305]. 

Isotopic labeling studies of phosphotranferase reactions culminated in the synthesis of ATP chiral at the y-phosphorus. Chirality was achieved by the synthesis of [y- 0, 0, 0]ATP of one configuration, and the analysis of its chirality was achieved by stereochenucally controlled transfer of the y-phosphoryl moiety to (S)-propane-l,2-diol where the absolute configuration was determined by a chemical/mass spectrometric sequence. The observation of inversion of configuration has been accepted as evidence of an in-line displacement mechanism at phosphorus by the two bound substrates the observation of retention of configuration was used to implicate the existence of a phosphoryl enzyme intermediate in the phosphoryl transfer process. For hexokinase, our case study, the finding is one of inversion, consistent with a direct transfer mechanism. 

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). 

In order to obtain a compound labeled with a radioactive isotope for studies about the pancreas, p-2- and /i-3-sclenienyl alanine were prepared. Initially the synthetic route shown in Scheme 14, which allowed insertion of radioactive selenium as late in the synthesis as possible, was designed for the ji-2 isomer (116).149... 

Probably, one of the most valuable advances in this field has dealt with the first chemoenzymatic synthesis of the stable isotope-enriched heparin from a uniformly double labelled 13C, 15N /V-acetylheparosan from E. coli K5. Heteronuclear, multidimensional nuclear magnetic resonance spectroscopy was employed to analyze the chemical composition and solution conformation of N-acety 1 hcparosan, the precursors, and heparin. Isotopic enrichment was found to provide well-resolved 13C spectra with the high sensitivity required for conformational studies of these biomolecules. Stable isotope-labelled heparin was indistinguishable from heparin derived from animal tissues and might be employed as a novel tool for studying the interaction of heparin with different receptors.30... 

Among the classic methods for the extension of the aldose chain by one carbon atom from the reducing end [9J, the Kiliani-Fischer cyanohydrin synthesis [10] is a milestone in carbohydrate chemistry. However after 110 years from discovery and numerous applications [11], including the preparation of carbon and hydrogen isotopically labeled compounds for mechanistic and structural studies [12], there are still several drawbacks that make the method impractical. These are the low and variable degree of selectivity and the harsh reaction conditions that are required to reveal the aldose from either the aldonic acid or directly from the cyanohydrin. Synthetic applications that have appeared in recent times confirmed these limitations. For instance, a quite low selectivity was registered [13] in the addition of the cyanide ion to the D-ga/acfo-hexodialdo-l,5-pyranose derivative 1... 

Sowden82 recently has prepared D-glucuronic acid isotopically labeled at carbon atom 6 for certain chemical and biochemical studies. In this novel synthesis, 5-aldehydo-l,2-isopropylidene-D-xylofuranose88 (XVI), prepared by periodate cleavage of 1,2-isopropylidene-a-D-glucofuranose (XV), was condensed with Cu-labeled sodium cyanide. Alkaline... 

Isotopically-labeled GAs and GA precursors are required for metabolic studies and as internal standards for quantitation by mass spectrometry. Numerous chemical methods have been used for the synthesis of labeled GAs from readily-available GAs. Labeled GAs have also been prepared biochemically using either fungal cultures or cell-free preparations from higher plants. 

In addition to information compiled elsewhere 1-7> the preparation of N-trimethylborazine has been described in Inorganic Syntheses 20> and the standard procedures have been used to make several isotopically labeled species of borazine 21> and N-methylborazine 22.28). Recently, the direct synthesis of N-organoborazines by the interaction of nitriles with diborane or alkali metal hydroborates, rather than the two-step procedures involving the reduction of the corresponding B-chloroborazi-nes, has received much attention. Besides the extensive study by Wade et al. 24>, it has been shown 25> that borazines can be prepared in high yield according to the reaction depicted in [Eq. (4)]. 

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