2H labelling

Hampel, D., Mosandl, A., and Wnst, M., Biosynthesis of mono- and sesqniterpenes in strawberry fmits and fohage 2H labeling stndies, J. Agric. Food Chem. 54, 1473, 2006. 

A common feature of the reactions described in (1) is that the hydrogen migration can be traced directly from appropriate mass shifts in the spectra of suitably 2H labelled precursor ions. 

As mentioned above, methyl loss from ionized methoxymethyl derivatives of methyl benzoate is not restricted to the ortho-isomet 93. 2H labelling has shown that the mita and para compounds 96 also eliminate CH3 specifically from the methoxymethyl group. Interestingly, neither from the unsubstituted benzyl methyl ether 97 (X = H) nor from the X-substituted analogues 97 (X = CH2OH, NQ2) loss of CH3 is observed (21). Thus, the earbomethoxy function seems to play a decisive role in the dissociation process. 

The mechanistic details of the cleavage of (S—C) bonds in ionized sulfides, such as 111-+113 constitute a challenging problem. Some insight was obtained from a CA spectroscopy study49 and from the investigation of 2H-labelled precursors50,51 For ionized ethyl methyl sulfide 111 it was concluded from the CA spectra that in... 

A mechamism closely related to that of (29) can be viewed for the loss of methyl radicals from terminal positions of ionized n-alkanes54. For example, based on experiments with 2H-labelled hydrocarbons and on careful analysis of energetic data it was shown convicingly that CD3 loss from 128 gives only the secondary cation 130. From the fact that 131 is not found to be formed at all, it must be concluded... 

OH elimination from ortho substituted aldoximes 179 (X = CH2, NH, O) may be at least partially the result of a hydrogen migration/cyclization/elimination process, whereby the heterocycles 182 are formed72 (46). A metastable peak shape analysis, the investigation of 2H-labelled derivatives and the study of positional isomers indicate that in addition to 182 the protonated isocyanide 183 is formed via a mechanism which is not fully understood. However, it is known that the generation of 183 occurs without any detectable interaction with the XH ortho substituent. 

Fig. 1. Typical values for the scalar couplings in perdeuterated proteins, which can be used for the coherence transfer through the spin system (a). Strategies for obtaining resonance assignment in 15N/13C/2H labelled proteins discussed in this chapter (b). The boxes, circles, and triangles indicate correlations established in the corresponding pulse scheme.

Fig. 3. HNCA (a) and two implementations of HNCA-TROSY (b-c) experiments for recording intraresidual HN(/), 15N(/), 13C"(i) and sequential 1 HN(7), l5N(/), 13Ca(i — 1) correlations in 13C/15N/2H labelled proteins. Narrow and wide bars correspond to 90° and 180° flip angles, respectively, applied with phase x unless otherwise indicated. Half-ellipse denotes water selective 90° pulse to obtain water-flip-back.88,89 All 90°...

Fig. 4. The HNCO-TROSY experiment for recording solely interresidual 1HN, 15N, 13C correlations in 13C/15N/2H labelled proteins. All 90° (180°) pulses for the 13C and 13C spins are applied with a strength of 2/ /l5 (p/ /3), where 2 is the frequency difference between the centres of the 13C and 13Ca regions. All 13Ca pulses are applied off-resonance with phase modulation by Q. A = 1/(4/hn) Tn = l/(4/NC ) S = gradient + field recovery delay 0 < k < TN/z2,max- Phase cycling i = y 4>2 = x, — x + States-TPPI 03 = x 0rec = x, — x.

Figure 13 shows expansion of the HN(CO)CANH (panel a) and HN(CO)CA (panel b) spectra, recorded from a 30.4 kDa (286 amino acid residues), uniformly 15N, 13C, and 2H labelled, protein Cel6A from the thermophilic soil bacterium Thermobifida fuscaso at 800 H MHz at 4°C. Both spectra were recorded under identical conditions. As can be observed, several sequential cross peaks are missing in the HN(CO)CA-TROSY spectrum whereas they are clearly visible in the HN(CO)CANH-TROSY spectrum. On the contrary, some of the correlations are more intense in the HN(CO) CA-TROSY spectrum than in the HN(CO)CANH-TROSY spectrum. 

Fig. 16.— 13C-N.m.r. Spectra of Polysaccharides (in DsO) Containing (1— 3)- and (1—>4)-Linked /3-D-Mannopyranosyl Residues, Obtained from [2H]-Labelled D-Glu-coses. (Temperature, 70° chemical shifts expressed as 8C, relative to external tetra-methylsilane the number marked with a superscript asterisk refers to the position of labelling in each D-glucose derivative.)...

DNA can be isotopically labeled not only using the enzymatic approach mentioned above but also using solid-state phosphoamidite synthesis. This methodology, developed in the 1980 s and modified recently to incorporate the isotope labels, is of advantage when residue- and site-specific patterns of 13C and/or 15N and/or 2H labeling is needed [3]. 

Figure 10.1 shows a two-dimensional [15N, H]-TROSY correlation spectrum of the 15N,2H- labeled 110 kDa homo-octameric protein 7,8-dihydroneopterin aldolase from Staphylococcus aureus (DHNA) measured with the pulse sequence of Fig. 10.4 [13]. The gain in spectral resolution and sensitivity is readily apparent from comparison with the corresponding conventional experiment. The optimal sensitivity is achieved by adjusting the polarization transfer r in Fig. 10.4 (3 ms <2r<5.4 ms [3]). For an optimal suppression of the non-TROSY components, the so-called Clean TROSY might be used [19]. Similar signal and spectral resolution enhancements are achieved for 15N,2H-labeled or 13C,15N,2H-...

The three-dimensional X-ray structure of the enzyme [19] reveals that several Thr residues occur in both the NADH cofactor and substrate binding sites (Fig. 21.5 A see p. 463). A Met residue (Metl7) is also present at the interface between the cofactor NADH and a substrate analog pyridine-2,6-dicarboxylate (PDC) (Fig. 21.5 A). Therefore, we prepared a sample of DHPR that was selectively labeled in these amino acid residues as follows 13C /1H Met, 13C /1H lie, 13C/1H Thr and uniformly 2H-labeled elsewhere ([MIT]-DHPR). This labeling can be achieved by supplementing the media with appropriate commercially available labeled amino acids, 12C/2H-labeled glucose and DzO [20] (see also the caption to Fig. 21.5 for details). 

Fig. 1.2 El mass spectra of derivatized phenylalanine, isolated from 5. obliquus a unlabeled, b random 50% 2H-, c random 70% 2H-, d 100% 2H-, e 3C, 5N-, and f 13C, 5N/random 75% 2H-labeled.

Selective Protonation of Methyl Croups in 2H-Labeled Proteins... 

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