Deuteration exchange experiments

A fully automated system for performing detailed studies has been developed to improve the reproducibility and throughput (Fig. 12.2) [8]. It consists of two functional components a sample-deuteration device and a protein processing unit. The preparation operations (shown at the top of Fig. 12.2) are performed by two robotic arms equipped with low volume syringes and two temperature-controlled chambers, one held at 25 °C and the other held at 1 °C. To initiate the exchange experiment, a small amount of protein solution is mixed with a deuter-ated buffer and the mixture is then incubated for a programmed period of time in the temperature-controlled chamber. This on-exchanged sample is immediately transferred to the cold chamber where a quench solution is added to the mixture. 

In principle, a D-H back-exchange experiment does not necessarily need to be performed since the required structural information can also be deduced from the mass spectrum recorded under H-D exchange conditions (deuterated eluent), which is of advantage in terms of the ready distinction of hydrogen equivalents. However, it has to be noted that this approach is not compatible with the extensive use of existing MS data libraries (data bases) and it thus depends on the individual problem as to whether such spectra can be used to advantage [1]. 

To avoid the expensive use of deuterated solvents for H/D exchange experiments, Tolonen et al. [21] have described the postcolumn infusion of D2O to facilitate the LC-MS detection and identification of labile protons in a column eluant. Whilst acknowledging the potential limitations with respect to a reduced level of exchange, and hence sensitivity, compared to the use of deuterated mobile-phase solvents, they optimized the column effluent flow rate (via a splitting connector) with the infused D2O flow rate to enable the very useful determination of up to four labile protons. The method was exemplified by the differentiation of hydroxylated metabolites of the alkaloidal drugs imipramine and omeprazole (Figure 13.5) from the N-oxide and sulfone metabolites, respectively [21]. This was a differentiation that could not be achieved by high-resolution mass measurements. 

A study of the MS" fragmentation of resveratrol and piceatannol was performed by deuterium exchange experiments and accurate mass measurements (Stella et al., 2008). The product ion spectrum of the [M-H] ion of tram-rcsvcratrol at m/z 227, is reported in Fig. 6.6, and that of piceatannol at m/z 243 are shown in Fig. 6.7. Fragmentation patterns of the [M-H] ion of two compounds are reported in Fig. 6.8. Fragmentations were confirmed with deuterium labeling experiments by dissolving the standard compounds in deuterated methanol The... 

The version of the selective-deuteration experiment most widely practiced involves the use of deuterium oxide as the solvent for n.m.r. measurements, instead of water without this procedure, the interpretation of n.m.r. spectra of sugars in aqueous media is generally diflBcult. Even so, prior deuteration of labile protons (such as those of hydroxyl and amino groups) by a series of exchange experiments is necessary before the residual-water peak can be lessened to an acceptable intensity. Other, potentially acidic protons can be similarly exchanged by deuterium through repeated lyophilization from deuterium oxide. 

Fig. 9.25 A static exchange experiment recorded for a sample of methyl-deuterated isotactic polypropylene (iPP) at T = 387 K. The observed elliptical ridges are characteristic of...

In a standard hydrogen exchange experiment (see Section 2.3.4 for variations in experimental workflow), the sample is deuterated for a predetermined time (milliseconds and longer times), and the reaction is slowed down several orders of magnitude by the addition of an acidic quench solution. Quenched reactions can be subjected to direct mass spectrometry analysis or immediately flash frozen in liquid nitrogen and stored at -80°C in multiple aliquots for future analysis. These flash-frozen samples need to be thawed prior to mass spectrometry. It is important to note that freeze-thaw cycles contribute to signal loss due to back-exchange (this is described further in Section 2.3.8), which... 

A different approach to the problem of hydrocarbon acidity, and hence carbanion stability, is that of Shatenshtein and Shapiro, who treated hydrocarbons with deuterated potassium amide and measured the rates of hydrogen exchange. The experiments did not measure thermodynamic acidity, since rates were measured, not positions of equilibria. They measured kinetic acidity, that is, which compounds... 

A flow cell-procedure was then applied. The experiment consisted of (a) adsorption of methanol (in a solution containing deuterated methanol and light hydrogen base electrolyte), (b) solution exchange with base electrolyte and (c) application of two potential steps, one of short duration to oxidize the adsorbed residue and then a second one in the negative direction to reduce the ions H+ and/or D+ formed. During this time the mass intensity signals for HD, (m/e = 3) and for COz (m/e = 44) were... 

The atom % 2H values of the solvent-fractionated products are also shown in Table I. In previous hydrogenation experiments conducted without the use of a donor solvent (1,18), deuterium incorporation increased from the most soluble oil fraction to the insoluble residue. In E10 and El9, contact of the coal with Tetralin resulted in a uniform incorporation of deuterium in almost all of the four product fractions. In El9, the BMI fraction s high value of 61 atom % 2H may be due to direct gas-phase exchange and deuteration. 

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