13C labelling

The structures of PMs are trimeric, consisting of three molecules of PS and two molecules of methylamine that are condensed together (Table 9.5). In the structures of PMs, however, the bonds created by the condensation lack adjacent hydrogen atoms, making the connectivity assignment in 1H-NMR studies virtually impossible (see the 9-membered rings in Fig. 9.11). To circumvent this problem, a model compound of panal, K-l, having 13C-labeles at the C12 and C13 positions, has been synthesized at Kishi s laboratory (Harvard University) to make a model compound of PM (Stojanovic, 1995). 

MMA polymerization is one of the most studied systems and was thought to be explicable, within experimental error, in terms of Bemoullian statistics. Moad et ai.jb have made precise measurements of the configurational sequence distribution for PMMA prepared from 13C-labeled monomer. It is clear that... 

The novel highly substituted spiro[4.4]nonatrienes 98 and 99 are produced by a [3+2+2+2] cocyclization with participation of three alkyne molecules and the (2 -dimethylamino-2 -trimethylsilyl)ethenylcarbene complex 96 (Scheme 20). This transformation is the first one ever observed involving threefold insertion of an alkyne and was first reported in 1999 by de Meijere et al. [81]. The structure of the product was eventually determined by X-ray crystal structure analysis of the quaternary ammonium iodide prepared from the regioisomer 98 (Ar=Ph) with methyl iodide. Interestingly, these formal [3+2+2+2] cycloaddition products are formed only from terminal arylacetylenes. In a control experiment with the complex 96 13C-labeled at the carbene carbon, the 13C label was found only at the spiro carbon atom of the products 98 and 99 [42]. 

Other miscellaneous imines that underwent photoreaction with chromium alkoxycarbenes include iminodithiocarbonates [33],the mono-N-phenylimine of benzil and the bis-JV-phenyl imine of acetoin [20]. By preparing the chromium carbene complex from 13CO-labeled chromium hexacarbonyl, /J-lactams with two adjacent 13C labels were synthesized [34]. 

By a combination of synthetic approaches, isotopic labeling, using tocopherols with 13C-labeling at C-5a and C-7a, EPR spectroscopy, and high-level DFT computations, it was shown that there is no radical formation at either C-5a or C-7a and that chromanol methide radical 10 does not occur in tocopherol.11 EPR failed to detect... 

SCHEME 7.5 Generation of the prekinamycin quinone methide. The 13C label is designated with an asterisk ( ). 

The UV-Vis spectral detection of an intermediate in the catalytic reductive alkylation reaction provides only circumstantial evidence of the quinone methide species. If the bioreductive alkylating agent has a 13C label at the methide center, then a 13C-NMR could provide chemical shift evidence of the methide intermediate. Although this concept is simple, the synthesis of such 13C-labeled materials may not be trivial. We carried out the synthesis of the 13C-labeled prekinamycin shown in Scheme 7.5 and prepared its quinone methide by catalytic reduction in an N2 glove box. An enriched 13C-NMR spectrum of this reaction mixture was obtained within 100 min of the catalytic reduction (the time of the peak intermediate concentration in Fig. 7.2). This spectrum clearly shows the chemical shift associated with the quinone methide along with those of decomposition products (Fig. 7.3). 

Our studies of quinone methides and related species using 13C labeling and spectral global fitting started in the late 1990s and have continued to the present with two papers on prekinamycins slated for publication in 2009. In this section, we summarize what we learned from both our published and unpublished studies. 

SCHEME 7.7 Products of reductive activation of a simplified WV-15 analogue. The 13C labels are designated with asterisks ( ). 

The toxicity of 3-methylindole has been attributed to methyleneindolenine trapping of nitrogen and sulfur nucleophiles.57 60-62 Likewise, the ene-imine shown in Scheme 7.9 readily reacted with hydroquinone nucleophiles, resulting in head-to-tail products. Shown in Fig. 7.6 is the 13C-NMR spectrum of a 13C-labeled ene-imine generated by reductive activation. The presence of the methylene center of the ene-imine is apparent at 98 ppm, along with starting material at 58 ppm and an internal redox reaction product at 18 ppm. Thus, the reactive ene-imine actually builds up in solution and can be used as a synthetic reagent. 

Dithionite reduction of 13C-labeled WV-15 (structure in Scheme 7.6) was carried out in anaerobic D20 and the resulting products evaluated by quantitative 13C-NMR spectroscopy immediately after the reduction (Fig. 7.8).45 The spectrum indicates that both the sulfite ester and the sulfonate are formed in a 60 40 ratio. Also, no long-lived methide species are observed in this reaction. 

FIGURE 7.8 Quantitative 13C-NMR (peak areas proportional to concentration) of dithionite-reduced I ()a-13C-labeled WV-15 (structure in Scheme 7.6). 

Probing DNA Adduct Structures with 13C-Labeled Methides... 

Many of the contributors to this volume have addressed the reactions of quinone methides with DNA nucleophiles. The 13C-labeled methide center has the potential of identifying the type and number of such adducts using 13C-NMR. An obvious... 

FIGURE 7.9 13C-NMR spectrum of 10a-13C-labeled WV-15 reductively alkylated d (GGGCCC). 

FIGURE 7.10 13C-NMR spectrum of d(ATGCAT) reductively alkylated with the 3a-13C-labeled cleaving agent shown in the inset of Scheme 7.12. 

To distinguish adjacent 13C labels from natural abundance isotopes, proton-detected 13C-NMR spectra (HMBC) will show cross peaks associated with the double label that are split into doublets. In contrast, natural abundance 13C will show single cross peaks. 

The reductive activation reaction of the 13C-labeled pyrrolo[ 1,2-a]indole shown in Scheme 7.14 was carried out in methanol and a 13C-NMR spectrum was obtained for the crude organic extract. This 13C-NMR spectrum, shown in Fig. 7.14, reveals the presence of starting material as well as products with 13C-labeled alkene and alkane centers. We confirmed the 13C assignments shown... 

In a recent study, we showed that the more flexible pyrido[l,2-a]indole-based cyclopropyl quinone methide is not subject to the stereoelectronic effect.47 Scheme 7.17 shows an electrostatic potential map of the protonated cyclopropyl quinone methide with arrows indicating the two possible nucleophilic attack sites on the electron-deficient (blue-colored) cyclopropyl ring. The 13C label allows both nucleophile attack products, the pyrido[l,2-a]indole and azepino [l,2-a]indole, to be distinguished without isolation. The site of nucleophilic is under steric control with pyrido [1,2-a]indole ring formation favored by large nucleophiles. 

With a 13C label at the methide center, the presence of reactive methide intermediate can be verified and complex reaction products can be inventoried and eventually identified. The only limitations are the synthesis and cost involved in incorporation of the 13C label. As a rule we, only use 13C-labeled dimethylformamide and NaCN as starting materials because of their low cost and availability. Another limitation of enriched 13C-NMR monitoring is dilution of the enriched label to natural abundance levels. Currently, we are developing isotope-editing techniques that utilize unnatural 13C double labels to solve this problem. 

The discoveries made possible by the use of 13C labels are outlined below. A new ene-imine carbon-carbon bond coupling reaction was discovered that afforded... 

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