Ketoses chromatography

Aromatic amines that have been used include o-toluidine, p-aminosali-cylic acid, p-aminobenzoic acid, diphenylamine and p-aminophenol. Their ability to react preferentially with a particular carbohydrate or class of carbohydrate is often useful, e.g. p-aminophenol, which shows some specificity for ketoses compared with aldoses and is useful for measuring fructose. These reagents have proved particularly useful for the visualization and identification of carbohydrates after separation of mixtures by paper or thin-layer chromatography, when colour variations and the presence or absence of a reaction aid the interpretation of the chromatogram. 

Thus, the 2-nitroethanol synthesis results in the addition of two carbon atoms to an aldose sugar to produce two higher-carbon ketose sugars epimeric at carbon 3. While the synthesis is general in nature, the two ketoses produced from any one aldose are seldom as readily separable as is fortunately the case with D-mannoheptulose and D-gluco-heptulose. However, the newly developed techniques of adsorption and partition chromatography will undoubtedly be of service for the more difficult separations. 

Anhydro-C-(p-methoxyphenyl)aldoses and anhydro- 1,3-dideoxy-ketoses were found to be compounds usually produced on heating free and partially protected aldoses with p-methoxybenzoyl- and acetyl-methylenetriphenylphosphoranes, respectively, in N,N-di-methylformamide. Many of them were isolated by thin-layer chromatography on alumina, although, on several occasions, these compounds were available only as mixtures of the five- and six-membered anhydro derivatives, because of their similar chromatographic mobilities. Five-membered anhydro derivatives are favored, but exclusive formation of six-membered anhydro derivatives was observed when five-membered ring-closure was impossible. Only L-rhamnose on reaction with acetylmethylenephosphorane formed a six-membered derivative, 196b. 

The conversion of aldoses to ketoses is also effected by dilute sodium hydroxide solutions. However, these reactions occur very slowly and are not observed in the time period necessary for the chromatography. 

The unknown compound was isolated by means of ion displacement chromatography. A crystalline product was obtained which was identified as 2-hydroxy-2-(r(S), 2 (R), 3 -trfliydroxypropyl)-tetrahydro-2H-l,4-thiazine-5(S)-carboxylic acid (Figure 2, compound 9a, n=3)). This tetrahydro-l,4-thiazine is the cyclic hemithioacetal form of the Amadori compound of cysteine and xylose (D-xylulose-L-cysteine). In water, the Amadori compounds of cysteine (ketose-L-cysteines) exist in two isomeric forms with the oxygen atom at C2 in either cis or trans position to the carboxyl group at C5. On the basis of NMR analysis. 

Often, purification of the acetylated diazomethyl ketoses cannot be achieved by crystallization, and resort is then had to chromatography on Magnesol/Celite, as described in the following typical preparation. 

We have developed preparative enzymatic syntheses of several unusual hexoketoses using fructose-1,6-diphosphate aldolase (FDP-aldolase, E.C.4.1.2.13) as catalyst and dihydroxyacetone phosphate (DHAP) and an aldehyde as substrates (15). The enzyme appears to be very specific for DHAP but will accept a variety of aldehydes as acceptors. The ketose-1-phosphates prepared are converted to the phosphate free ketoses after removal of the phosphate group by acid- or phosphatase-catalyzed hydrolysis. The ketoses can be isomerized stereospecifically to aldoses catalyzed by glucose isomerase (E.C.5.3.1.5.) from Flavobacteriuum arborescens. The equilibrium mixtures of aldoses and ketoses are then separated by chromatography on Dowex 50 (Ba ) or Dowex 1 (HSO "). Figure 1 illustrates the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose... 

Some of the ketoses prepared in the FDP aldolase reactions can be converted to the corresponding aldolases catalyzed by glucose isomerase ( ) (from Miles), the enzyme used for the manufacture of high fructose com syrup. Figure 5 summarizes some of the isomerization reactions. The equilibrium mixture can be separated by Dowex-50-Ba column chromatography using water as the mobile phase. This isomerization step provides a new entry to several interesting aldolases. 

Comments Oshima et al. [121] achieved resolution of 24 monosaccharides, which included ketoses and N-acetylamino sugars, by gas chromatography using the procedure described above. 

Adachi [9] examined silica gel G impregnated with 0.1 M sodium bisulfite as an adsorbent for thin-layer chromatography of sugars (Table 198) and has reported hi /-values for the sugars, including various ketoses, in several solvent systems. 

The individual 2-C-(hydroxymethyl)aldoses were obtained after a combined separation of the epimers via their phenylhydrazones followed by chemisorption chromatography on a cation-exchange resin in the Ba form [50]. Then, on treatment with molybdic acid at increased temperature, 2-C-(hydroxymethyl)-D-mannose (16c) rearranged to D- /Mco-hept-2-ulose (15 c) as expected. Under similar conditions, 2-C-(hydroxymethyl)-D-glucose (15 d) was transformed to d-ma o-hept-2-ulose (16d). The analysis of the reaction equilibria by NMR spectroscopy revealed the presence of small quantities of the respective starting branched-chain aldoses 15d (3.5%) and 16c (8%). The same equilibria were also obtained from the side of the model ketoses 15 c and 16 d. Thus, for example, the equilibrium of the sugars 15c and 16c at their total 2% concentration in a 0.2%... 

Supercritical fluid chromatography on polar columns (diol- and cyano-bonded silica) with C02-MeOH mobile phases and light scattering detection has been investigated for the separation of aldoses, glycosides, desulphoglucosinolates, di- and tri-saccharides, cyclitols and ketoses. ... 

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