Carboxyl group labeling

Liu, P., Regnier, F. E. (2002). An isotope coding strategy for proteomics involving both amine and carboxyl group labeling. J. Proteome Res. 1, 4434-50. 

Scheme 19. The Barton reaction for carboxyl group labelling...

The top mechanism is an SmI on the alcohol the bottom mechanism is the standard addition-elimination on the carboxyl group. Labeling the alcohol with "O will distinguish them. The 0 will be lost in the top mechanism, but it will be retained in the bottom one. 

Fig. 14 Predicted low-energy conformers of proline and relative energies (MP2/6-311-H-G(d,p)) with respect to the global minimum. Conformers ate labeled as I, II, or III depending on the hydrogen bond established between the amino group and carboxylic groups. Labels aotb indicate the configuration endo (a) or exo (h) adopted by the ring. The detected conformers are encircled. (From [59, 128])...

The aldehyde or ketone needed for this reaction is not always readily available. TM 133, labelled with radioactive in one carboxyl group, was needed for a biochemical labelling experiment. How would you make it ... 

Labeling studies have shown that the 3-ketone provides the carboxyl group of (28). This led to the conclusion that attack of hydroxide ion occurs at C-3 to give (30), and subsequently the norsteroid (32) via the transition state (31). Attack at C-3 to give adduct (30) is favored over attack at C-4... 

Isotopic (2H, 180) labelling of the carboxylate groups has virtually no effect, as expected, on this band but produces shifts of 6-14cm-1 in bands at 321 and 338 cm-1, showing them to arise from Rh—O stretching [75]. 

For quantitative work, it is necessary to estimate the concentration of 5-amino-l-(P-D-ribofuranosyl)imidazole in aqueous solution. It seems that the only available method is the Bratton-Marshall assay, which was originally developed for the estimation of arylamines in biological fluids. The principle of the method is the spectrometric estimation of a salmon-pink colored dyestuff obtained by diazotation in situ, followed by coupling with /V-( 1 -naphthyl)ethyl-enediamine.65 The only remaining problem then is to know the molar extinction of this dye because pure samples of AIRs are not available. A value of 16800 at 520 nM was obtained for the dyes prepared from a model compound, 5-amino-l-cyclohexylimidazole-4-carboxylic acid (54), which is crystalline. A comparable molar extinction can be expected for the dye prepared from imidazole 55, if the carboxyl group does not exert too much influence on the chromophore. Actually, its influence is perceptible even with the naked eye, the dyestuff prepared from 53 having a somewhat different, wine-red color, with max>520 nM. The molar extinction for 55 is 17400 at 500 nM. When the decarboxylation of 54 was conducted under mild acidic conditions (pH 4.8, 50°C, 1 hour), estimation of 5-aminoimidazole 55 by the Bratton-Marshall method led to the conclusion that the reaction was almost quantitative.66 Similar conditions for the final decarboxylation were adopted in the preparation of samples of AIRs labeled with stable isotopes.58... 

Basic hydrolysis has been carried out on carboxylic esters labeled with O in the carbonyl group. If this reaction proceeded by the normal Sn2 mechanism, all the 0 would remain in the carbonyl group, even if, in an equilibrium process, some of the carboxylic acid formed went back to the starting material ... 

It may be noted that 16 are stable compounds hence it should be possible to prepare them independently and to subject them to the conditions of the von Richter rearrangement. This was done and the correct products are obtained. Further evidence is that when 15 (Z=C1 or Br) was treated with cyanide in H O, one-half the oxygen in the product was labeled, showing that one of the oxygens of the carboxyl group came from the nitro group and one from the solvent, as required by this mechanism. ... 

A catalytic mechanism, which is supported by deuterium-labeling experiments in the corresponding Ru-catalyzed procedure [146], is shown in Scheme 47. Accordingly, the reactive Fe-hydride species is formed in situ by the reaction of the iron precatalyst with hydrosilane. Hydrosilylation of the carboxyl group affords the 0-silyl-A,0-acetal a, which is converted into the iminium intermediate b. Reduction of b by a second Fe-hydride species finally generates the corresponding amine and disiloxane. 

Figure 1.71 Cystamine may be used to label protein carboxylate groups using the water-soluble carbodiimide EDC.

Diazomethane and other diazoalkyl derivatives long have been used to label carboxylate groups for analysis (Herriott, 1947 Riehm and Scheraga, 1965). A major application of such... 

AMCA may be coupled to amine-containing molecules through the use of the carbodiimide reaction using EDC (Chapter 3, Section 1.1). EDC will activate the carboxylate on AMCA to a highly reactive o-acylisourea intermediate. Attack by a nucleophilic primary amine group results in the formation of an amide bond (Figure 9.22). Derivatization of AMCA off its carboxylate group causes no major effects on its fluorescent properties. Thus, proteins and other macromolecules may be labeled with this intensely blue probe and easily detected by fluorescence microscopy and other techniques. 

AMCA-NHS, succinimidyl-7-amino-4-methylcoumarin-3-acetic acid, is an amine-reactive derivative of AMCA containing an NHS ester on its carboxylate group (Thermo Fisher). The result is reactivity directed toward amine-containing molecules, forming amide linkages with the AMCA fluorophore (Figure 9.23). Proteins labeled with AMCA show little-to-no effect on the isoelectric point of the molecule. 

The Derivative, 5-(biotinamido)pentylamine, contains a 5-carbon cadaverine spacer group attached to the valeric acid side chain of biotin (Thermo Fisher). The compound can be used in a carbodi-imide reaction process to label carboxylate groups in proteins and other molecules, forming amide bond linkages (Chapter 3, Section 1). However, the main use of this biotinylation reagent is in the determination of factor XHIa or transglutaminase enzymes in plasma, cell, or tissue extracts. 

---