Specific isotopic labelling

Benzoates. The selective debenzoylation of sucrose octabenzoate [2425-84-5] using isopropylamine in the absence of solvents caused deacylation in the furanose ring to give 2,3,4,6,1/3/6 -hepta- and 2,3,4,6,1/6 -hexa-O-benzoyl-sucroses in 24.1 and 25.4% after 21 and 80 hours, respectively (54). The unambiguous assignment of partially benzoylated sucrose derivatives was accompHshed by specific isotopic labeling techniques (54). Identification of any benzoylated sucrose derivative can thus be achieved by comparison of its C-nmr carbonyl carbon resonances with those of the assigned octabenzoate derivative after benzoylation with 10 atom % benzoyl—carbonyl chloride in pyridine. 

Solid-phase peptide synthesis offers a fast and convenient route for many peptides when isotope-enriched compounds are not required. Classical synthesis additionally permits the use of non-natural amino acids and allows site-specific isotope labeling. Although Fmoc protected 15N-labeled amino adds are commercially available, the cost of such synthesis is usually prohibitive, and the peptides from chemical synthesis require perdeuterated detergents and unfortunately exclude investigation of internal dynamics through measurement of 15N relaxation. 

With regard to isotopomers, tw o sub-categories arise. The first is the natural abundance isotopomer distribution of the organics and organometallics. The second is specific isotopic labeling. Normally, the pure component spectra recovered are lumped para-... 

Recently, Alia et al.m studied Yp site-specific isotopically labelled with 2H, 13C and 170 by EPR. The hyperfine couplings and the spin density distribution at all ring positions of the radical were obtained. A strong H-bond to the protein was detected and the bond length estimated to 1.5 A. Isotope labelling has also been used earlier by Babcock s group (reviewed in reference 385). 

One way by means of which the structural and dynamic information inherent to the anisotropic interactions can be probed in a site-selective fashion involves the investigation of samples where specific isotopic labels, e.g., 2H, 13C, 15N, are introduced. 2H is a spin 1= 1 nucleus, and the quadrupolar interaction dominates 2H NMR. By an analysis of onedimensional line shapes, fast dynamic processes (10-4 to 10-7 s) can be quantitatively probed, while two-dimensional exchange experiments allow the inves-... 

J Ellman, D Mendel, P Schultz, D Wemmer, B Volkman. Site-specific isotopic labeling of proteins for NMR studies. J Am Chem Soc 114 7959-7961, 1992. 

In a typical organic solid, experiments on abundant nuclei like are far too unselective one of the powers of NMR, the chemical selectivity or molecular resolution, is lost due to the strong dipolar interaction between the spins that broadens the spectra and prevents the extraction of the desired detailed information. This drawback was overcome by the introduction of specifically isotopically labeled samples where the nucleus ... 

Specific deuterium-labelled samples of poly(7-benzyl L-glutamates) were synthesized by Hiraoki et In general, specific isotope labelling is a very... 

The last example shows that chemical synthesis of proteins can be a useful tool to study pertinent questions in biochemistry. Its main advantage so far relates to its capability to introduce unnatural amino acids, specific isotope labels, biophysical monitors, or very important posttranslational modifications. The latter modification the introduction of a GPI anchor or a GPI anchor mimic onto PrP, has been the subject of further research. These experiments were not solely directed towards specific questions of PrP research but also towards development of a general method to modify proteins with a GPI anchor. 

The quality of NMR-derived information has been exclusively dependent on NOE. To compensate the prediction ambiguity of NOE (Section 8.1), many experimental methods have been studied as introduced below. Furthermore, to be applicable for large protein systems with a specific isotope label or a selective deuteration (see Section 12) that results in a remarkable loss of NOE quantities, minimal NOE-based methods are also required.75,76... 

In contrast to biological systems, the accuracy of the measured distance is less stringent for synthetic polymers, because they do not form high quality crystals and it is not easy to perform specifically isotope label. Local packing in a glassy polycarbonate has been examined in a homogeneous mixture of 5% [Carbonyl- C]polycarbonate and 95% [methyl-d6]polycarbonate using REDOR NMR. The distance from the carbonyl carbon of one... 

As mentioned above, the determination of atomic level structure, i.e., the backbone torsion angles for an oriented protein fiber, is possible by using both solid-state NMR method described here and specifically isotope labeling. This is basically to obtain the angle information. Another structural parameter is distance between the nuclei for atomic coordinate determination. The observation of Nuclear Overhauser Enhancements (NOEs) between hydrogen atoms is a well known technique to determine the atomic coordinates of proteins in solution [14]. In the field of solid-state NMR, REDOR (rotational echo double resonance) for detection of weak heteronuclear dipole interactions, such as those due to C and N nuclei [15, 16] or R (rotational resonance) for detection of the distance between homonuclei, are typical methods for internuclear distance determination [17,18]. The REDOR technique has been applied to structure determination of a silk fibroin model compound [19]. In general, this does not require orientation of the samples in the analysis, but selective isotope labeling between specified nuclear pairs in the samples is required which frequently becomes a problem. A review of these approaches has appeared elsewhere [16]. 

Spin contrast variation will probably bear fruit in in situ structure determinations of arbitrary parts of biological structures. For this purpose a selected region of the macromolecular structure has to have the usual hydrogen isotope H whereas the larger remainder is preferably completely deuterated. This technique of specific isotopic labelling is now well established with ribosomes and other subcellular particles which allow in vitro recombination of the constituents... 

Resonance Raman spectroscopy carried out on carbon-specific, isotopically-labeled carotenoids can provide an important means to assign the Raman spectral bands and, upon incorporation of the carotenoid into RCs, can aid greatly in elucidating structural features not evident from X-ray diffraction studies and in understanding the effect of the protein on the vibrational activity of the carotenoid. Kok et al. (1994,1997) have examined the resonance Raman spectra of several C- and H-labeled spheroidenes incorporated into Rb. sphaeroides R26.1 RCs. 14- H, 15- H, 15 - 14,15 -2H and 15,15 - H... 

The mass spectrum of l,6-anhydro-2,3-0-isopropylidene- 8-D-talo-pyranose has been studied by using specific, isotope-labelling techniques. The molecular ion was not observed, but (M — CHs) was an abundant fragment. The identity of the molecular ion was deduced from the metastable peak M 50.8 corresponding to the rearrangement process shown in Scheme 25. The study enabled the authors to formulate fragmentation pathways for all fragments produced on electron impact. 

Electron-nuclear double resonance (ENDOR) studies of PFL-AE complexed to specifically isotopically labeled AdoMets has revealed the details of the interaction between AdoMet and the cluster in this enzyme. Deuterium ENDOR spectra of PFL-AE in the [4Fe-4S]" state complexed with methyl-D2-AdoMet showed a pair of peaks centered at the deuteron Larmor frequency and split by the hyperfine coupling to the spin of the cluster. Examination of the field-dependence of the coupling showed that it was dipolar in nature, and gave an estimation of the... 

The proposal that the sulfonium of AdoMet interacts with a //3-bridging sulfide of the cluster left open the question of the role for the putative unique iron site. A unique iron site was in fact demonstrated in PFE-AE via specific isotopic labeling of the unique iron site with Fe, in the same way described previously for aconitase (see Section 8.27.2.3), which allowed probing of the unique site by Mossbauer spectroscopy. " The Mossbauer parameters for this unique site in the absence of AdoMet were typical for iron in [4Fe-4S] clusters (Table 2). Addition of AdoMet, however, dramatically altered the Mossbauer parameters (Table 2). The new parameters are inconsistent with coordination of a sulfur to the unique site, but are consistent with an increase in coordination number of the unique iron to 5 or 6, and/or coordination of an ionic ligand."" Thus it was proposed that the carboxylate of AdoMet coordinated to the unique iron site of the cluster."" ... 

The use of infrared to probe the details of interactions (especially in hydrogen-bonded systems) was demonstrated nicely by Keiderling s group [108]. Coupling the use of specific isotopic labeling with the aid of ab initio calculations for the analysis, they were able to establish the pattern of cross-strand coupling in a (3-hairpin peptide. 

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