Labelling acetals

The overall biosynthetic pathway to the tetracychnes has been reviewed (74). Studies (75—78) utilising labeled acetate and malonate and nmr analysis of the isolated oxytetracycline (2), have demonstrated the exclusive malonate origin of the tetracycline carbon skeleton, the carboxamide substituent, and the folding mode of the polyketide chain. Feeding experiments using [1- 02] acetate and analysis of the nmr isotope shift effects, led to the location of... 

Even though acetate units, such as those obtained from fatty acid oxidation, cannot be used for net synthesis of carbohydrate in animals, labeled carbon from " C-labeled acetate can be found in newly synthesized glucose (for example, in liver glycogen) in animal tracer studies. Explain how this can be. Which carbons of glucose would you expect to be the first to be labeled by "Relabeled acetate ... 

In 1952, Konrad Bloch and Robert Langdon showed conclusively that labeled squalene is synthesized rapidly from labeled acetate and also that cholesterol is derived from squalene. Langdon, a graduate student of Bloch s, performed the critical experiments in Bloch s laboratory at the University of Chicago, while Bloch spent the summer in Bermuda attempting to demonstrate that radioactively labeled squalene would be converted to cholesterol in shark livers. As Bloch himself admitted, All I was able to learn was that sharks of manageable length are very difficult to catch and their oily livers impossible to slice (Bloch, 1987). 

Terpenoid substances are of broad distribution and diverse function in insects. One set, elaborated by the mandibular glands of Acanthomyops claviger, acts both as a defensive secretion and as an alarm releaser. When fed Cu-labeled acetate or mevalonate, laboratory colonies of these ants produce radioactive citronellal and citral, providing unambiguous evidence for de novo synthesis of these terpenes by the ant. The incorporations of these precursors implicate the mevalonic acid pathway as the likely biosynthetic route. 

Ketene dimer (28), made from [ " Cl-labelled acetic acid, has been used to make doubly labelled mevalonic lactone (29) for studies on the biosynthesis of terpenes. Note... 

In another experiment, [l,2- C2-2-dJ double-labeled acetate was fed. First we observed a complete loss of deuterium atoms. In a short incubation, however, we obtained neosaxitoxin partially retaining a deuterium atom (40% equivalent of incorporated acetate molecule). The location of the deuterium atom was on C-5, which was originally the carboxyl carbon of acetate, suggesting that it migrated from the adjacent methyl-derived carbon C-6. 

We started feeding experiments using C-labeled acetate long before the establishment of the toxin structures with a hope that the analysis of the C-NMR... 

Methylphenol is carboxylated to 2-methyl-4-hydroxybenzoate by a methanogenic-enrich-ment culture before degradation to acetate (Figure 8.44a) (Roberts et al. 1990) C-labeled bicarbonate prodnced carboxyl-labeled acetate, while C-methyl-labeled 3-methylphenol yielded methyl-labeled acetate. However, 2-methylbenzoate formed by dehydroxylation of 2-methyl-4-hydroxybenzoate was not further metabolized (Figure 8.44b). A similar reaction occnrs with a sulfate-reducing mixed culture (Ramanand and Suflita 1991). 

Evidence for de novo synthesis of pheromone components was obtained by showing that labeled acetate and mevalonate were incorporated into ipsdienol by male Ips pini [103,104]. Similarly, labeled acetate and other labeled intermediates were shown to be incorporated into frontalin in a number of Dendroctonus species [105]. Possible precursors to frontalin include 6-methyl-6-hep-ten-2-one, which was incorporated into frontalin by D. ruffipennis [106]. The precursor 6-methyl-6-hepten-2-one also was shown to be converted to bre-vicomin in the bark beetle, Dendroctonus ponderosae [107]. In addition, the expression patterns of HMG-CoA reductase and HMG-CoA synthase are tightly correlated with frontalin production in Dendroctonus jeffreyi [108, 109]. A geranyl diphosphate synthase cDNA from I. pini was also isolated, functionally expressed, and modeled [110]. These data indicate that the de novo isoprenoid biosynthetic pathway is present in bark beetles. A variety of other monoterpene alcohols such as myrcenol, pityol, and sulcitol are probably synthesized through similar pathways [111]... 

In suberizing potato tuber disks, labeled oleic acid was incorporated into co-hy-droxyoleic acid and the corresponding dicarboxylic acid, the two major aliphatic components of potato suberin [73]. Exogenous labeled acetate was also incorporated into all of the aliphatic components of suberin, including the very long chain acids and alcohols in the wound-healing potato slices. The time-course of incorporation of the labeled precursors into the suberin components was consistent with the time-course of suberization. The biosynthetic pathway for the major aliphatic components of suberin is shown in Fig. 8a. 

The unique suberin components that are not found as significant components of cutin are the very long chain molecules and the dicarboxylic acids. Therefore, chain elongation and conversion of co-hydroxy acids to the corresponding dicarboxylic acids constitute two unique biochemical processes involved in the synthesis of suberin. Incorporation of labeled acetate into the very long chain components of suberin was demonstrated and this ability developed during suberization in potato tuber disks [73]. The enzymes involved... 

Further studies by Spenser demonstrated that l,2-13C-labeled acetate (13) was incorporated into lycopodine but gave a distribution of the labels that did not account for the pelletierine-route that was hypothesized (Scheme 6.2) [11]. An intact 3-carbon unit was desired for testing, but labeled acetoacetate (l,2,3,4-13C-acetoacetate (14), which could undergo decarboxylation to provide an intact 3-carbon unit) was found to give the same incorporation pattern as acetate (and therefore must have been cleaved to acetate prior to uptake). In addition, feeding studies using deuterated, 13C-labeled acetate provided a loss or washout of deuterium at the C16 methyl group. This could only occur if an intermediate had formed that would provide for facile enolization. Both the equal distribution of the 13C labels and loss of the deuteriums led the researchers to propose that the intermediate was symmetric, such as acetone dicarboxylic acid (15). 

By using an elegant 13C-labeling study that involved incubation of Phoma sp. with 1-13C and 1,2-13C acetate, Oikawa et al. [9], were able to isolate the proposed biosynthetic intermediate phomactatriene (or Sch 49026), with 13C incorporation from singly labeled acetate units as indicated by in Fig. 8.3. Phomactatriene is strikingly reminiscent of taxadiene, a biosynthetic intermediate for Taxol . The net biosynthesis for both involves geranylgeranyl diphosphate (GGDP) cyclization [9]. It is noteworthy that prior to isolation of phomactins, the only known related structure is cleomeolide, a diterpene from the herb Cleome viscosa [10] that remarkably resembles phomactin H. 

The ability of bivalve molluscs to synthesize sterols is questioned [106]. Approximately forty sterols have been identified from the oyster Crassostrea virginica and, since it appeared that many of the sterols identified must be of dietary origin, the ability of the oyster to incorporate injected radioactive acetate was studied [110]. Of the forty sterols naturally occurring in the oyster, only four were labelled by injection of labelled acetate cholesterol, desmosterol, 24-methylenecholesterol and fucosterol. However, when an oyster hearth tissue culture was grown aseptically with addition of labelled acetate, the sterols were found to be non-radioactive [111], This finding does not rule out the possibility... 

The radiochemical results obtained with the Pt(lll) electrode in the electrolyte containing C-14 labeled acetic acid are shown in Figure 4 (circles). The adsorption is negligible at 0.15 V, small at more negative potentials and very pronounced at potentials more positive than 0.15 V. In the potential range between 0.3 and 0.85 V, the radiochemical signal from the surface, i.e., the surface concentration of acetic acid, equation... 

The mechanism of the catalysis in the rearrangements of 4-acetoxyhepta-2,5-dienes was investigated by using the 170-labeled acetate 362 in the presence of Pd° and Pdn catalysts187. It was shown by 170 NMR spectroscopy that the reaction catalyzed by Pd° affords a 1 1 mixture of the dienes 363 and 364 which results from the Pd-coordinated pentadienyl species intermediate and 170-acetate (equation 132). By using two Pdu-catalysts, viz. (RCN PdCL (R = Me, Ph) [Pdn(l)] and (PhjP Pd [i.e. Pdn(2)], two rearrangement products 365 and 366, respectively, were obtained. The heterocyclic 1,3-dioxanium cations 367 and 368 were assumed to be intermediates of these isomerizations (equation 133). 

The situation is quite different in the case of an acetic acid-water system. The energy of acetic acid adsorption on platinum is low and therefore the voltammetric curves taken in the absence and in the presence of acetic acid in the supporting electrolyte are nearly the same. However, radiometric data show that C-labeled acetic acid is adsorbed on the electrode surface. Most likely the acetic acid molecules are adsorbed on the top of the water molecules populating the electrode surface. Simultaneously recorded voltammetric and counting rate data are shown in Fig. 8. 

A typical example that illustrates the method concerns the lipase- or esterase-catalyzed hydrolytic kinetic resolution of rac-1-phenyl ethyl acetate, derived from rac-1-phenyl ethanol (20). However, the acetate of any chiral alcohol or the acetamide of any chiral amine can be used. A 1 1 mixture of labeled and non-labeled compounds (S)- C-19 and (f )-19 is prepared, which simulates a racemate. It is used in the actual catalytic hydrolytic kinetic resolution, which affords a mixture of true enantiomers (5)-20 and (J )-20 as well as labeled and non-labeled acetic acid C-21 and 21, respectively, together with non-reacted starting esters 19. At 50% conversion (or at any other point of the kinetic resolution), the ratio of (5)- C-19 to (1 )-19 correlates with the enantiomeric purity of the non-reacted ester, and the ratio of C-21 to 21 reveals the relative amounts of (5)-20 and (J )-20 (98). 

The detailed structure of caldariellaquinone (12), a unique benzo[6]thiophen-4,7-quinone from Caldariella acidophila, has been elucidated. Feeding experiments with C-labelled acetate helped to define the nature and biosynthetic origin of the C30 isoprenoid chain. 

The biosynthesis of tenuazonic acid was studied using [l-14C]-labeled acetate. N-acetoacetyl-L-isoleucine (7) was detected by radioactive trapping, indicating that amide formation, rather than carbon-carbon bond formation is probably the first step. None of the simple tetramic acid (8)... 

Condensation of unlabeled oxaloacetate with carboxyl-labeled acetate would be expected to produce citrate labeled in one of the two primary carboxyl groups. Citrate is a symmetric molecule, its two terminal carboxyl groups being chemically indistinguishable. Therefore, half the labeled citrate molecules were expected to yield a-ketoglutarate labeled in... 

When hydrolysis was carried out in 50% dioxan-water 1 M in acetic acid-acetate buffer of pH 5.5, it was found that some 75% of the 180 derived from one of the oxygen atoms of the labelled acetate ion appeared in the benzoic acid produced. 

A similar result was obtained by Schowen and Behn280, for the methanolysis of p-nitrophenyl acetate in the presence of tritium-labelled acetate ion. In this case the intermediate anhydride is effectively symmetrical, and methanol will attack equally at the labelled and unlabelled acetyl group. As expected for nucleophilic catalysis, 50% (within experimental error) of the methyl acetate produced was labelled T3CCOOCH3. 

Scheme 6 Formation of pyrazofurin (24) and oxazinomycin (26) from labelled acetates...

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