Sugar with complex mutarotation

The majority of the many methods used to study the composition of equilibrium solutions of carbohydrates examine the mixture without separating the individual components. With the discovery that the anomeric forms of sugars could be readily separated by gas chromatography of their tri-methylsilyl ethers, a new approach to the problem was found. A protocol was developed for the direct gas chromatographic analysis of the amount of each anomer present in an aqueous solution. The protocol can be used on the micro scale and can be used in enzyme assays such as that for mutarotase. The method has been made more effective by combining gas chromatography with mass spectrometry. It is shown how mass spectral intensity ratios can be used to discriminate anomers one from another. The application of these methods to the study of complex mutarotations is discussed. 

Some 40 years ago Isbell (16) observed complex formation between calcium ions and a sugar which possesses the ax-eq-ax sequence—i.e., a-D-gulopyranose. He observed that an equilibrated solution of D-gulose, CaCl2, underwent further mutarotation on dilution with water and he correctly interpreted the phenomenon by postulating that a-D-gulo-pyranose (10), but not -D-gulopyranose, forms a complex with calcium... 

With the establishment of the permease hypothesis, however, it was apparent that the mere formation of a complex with the mutarotase protein may be the necessary interaction in transport (15). The subsequent mutarotation could be considered to be a coincidental consequence of the complex formation. To support this idea, it was found that 1-deoxy glucose and a-methyl glucoside are excellent competitive inhibitors of the enzyme (16,61). Keston also showed that a number of cataractogenic sugars were inhibitors of lens mutarotase (62). It has since been shown that in all cases where a sugar is a substrate for the mammalian intestinal transport system it is also a competitive inhibitor of mutarotase. 

The rate of the interconversion may also be followed by measuring the change in volume or in refractive index. Such measurements give rate coefficients identical with those obtained by the polarimetric method. In Table XVIII, rate coefficients for the mutarotation of a number of sugars are listed. The rates of mutarotation of several sugars (for example, D-ri-bose, D-galactose, and all the ketoses) do not obey the first-order law. Their complex mutarotations result from the presence in solution, in appreciable concentrations, of more than two species. In addition to pyranoses, there must be present either furanoses or acyclic forms, or both. 

When the two modifications of a sugar showing a simple mutarotation are known, the equilibrium proportions may be calculated from the equilibrium rotation and the initial optical rotation of the known crystalline modifications. In the event that only one modification is known in the crystalline form, the proportion of the second modification may be obtained from the initial and maximum solubilities of the first, as described on page 18. With sugars that show complex mutarotation, the proportions of the a- and /8-pyranose modifications may be estimated roughly from the extent of the slow mutarotation as compared to the optical rotations of the known a- and... 

Although no mutarotation was observed with the first small sample of D-altrose, its [a]D value of +32.6° in water was in agreement with the equilibrium rotation —32.3° recorded by Austin and Humoller for L-altrose. When a larger amount of the sugar became available, D-altrose was found to exhibit a complex mutarotation. From calculations of the velocity coefficients it would appear that the mutarotation consists of a very rapid interconversion of furanose and pyranose modifications, followed by a slower interconversion of o and /3 pyranose modifications. 

D-Mannose forms an easily crystallizable compound (90) with calcium chloride of the formula C6Hi206 CaCl2 4H20, which exhibits a complex mutarotation with a maximum and which appears to contain the furanose modification of the sugar. 

Although the acetylated galactose hydrazones probably have acyclic structures, the hydrazones with free hydroxyls may exist in the ring forms. In solution, the sugar hydrazones show complex mutarotations which pass through a maximum or minimum 208 210, 211). The failure of the muta-rotation equation to follow the first-order equation indicates that three or more substances take part in the equilibrium. Three isomeric glucose eiO. H. Jacobi, Ann. 272, 170 (1892). 

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