Osazones mixed

Since the sugar osazones mutarotate in alcoholic pyridine solution (231) the classical formula for these substances may be questioned, and there is much evidence that they exist in cyclic as well as acyclic forms. The mutaro-tation has been ascribed to a partial hydrolysis of the osazones, and appreciable quantities of the sugar and hydrazine exist in the equilibrium solution (232). This explanation is also supported by the ease with which the hydrazine radicals of the osazones are exchanged with hydrazine molecules in the solvent (233). When the second hydrazine is different from that used in making the osazone, mixed osazones are formed (232, 234) ... 

Phenylhydrazine is usually dissolved in acetic acid for hydrazone formation if a salt of phenylhydrazine with an inorganic acid is used, it must be mixed with an excess of sodium acetate (see preparation of osazones, p. 137). 

It has recently been reported21 that a mixed osazone of 3,4-di-O-methyl-D-glucose can be converted, by treatment with p-nitrobenzaldehyde, into an osone which reacts with phenylhydrazine to give 3,4-di-O-methyl-D-glucose phenylosazone. Von Lebedev29 claimed to have obtained D-glu-cosone 6-phosphate, isolated as an amorphous lead salt, by the action of hydrochloric acid on the phenylosazone prepared from D-fructose 6-phosphate. 

The solutions were examined in a Varian model V-4548 aqueous solution sample cell. All experiments in solution required a trace of oxygen. Freshly prepared solutions of phenylhydrazones or osazones (0.1 to 3.5 mM) in methyl sulfoxide were mixed with base [1 to 3% (90 mM to 270 mM) potassium terf-butoxide in methyl sulfoxide] and exposed to air for about 30 sec. Initial ESR spectra were recorded in about 30 sec after mixing. 

The structure of epimelibiose was established58 through periodate oxidation, and by comparison of its osazone with that of melibiose (melting point, mixed melting point, and x-ray diffraction pattern). 

A solution containing 0.5 mg of D-mannose-C (uniformly labeled, specific activity 3.3 X 10 CPM//imole) was added to 50ml of a solution containing an unknown amount of unlabeled mannose. After mixing, the D-mannose was reisolated as the osazone. The osazone had a speciBc activity of 14,280 CPM//i,mole, Calculate the concentration of unlabeled D-mannose in the original solution. 

In order to confirm this conclusion, both D- and L-arabomethylose, which would give the same osazones as d- and L-ribomethylose, were prepared by ozonization of D- and L-rhamnal. Physical constants of derivatives of all pentomethyloses concerned in the argument are shown in Table III. From these data, as well as from mixed melting point... 

The Strv ture of the So-called Mixed A and Mixed B Osazones... 

The formazan reaction offers a means for clarifying the structure of the mixed A and mixed B osazones. The former was described by Voto6ek and Vondra6ek as acyclic n-glucose l-(iV -phenyl)-2-(Ar -methyl-A -phenyl)osazone, and the latter as acyclic n-glucose l-(A -methyl-i r -phenyl)-2-(V -phenyl)osazone. The Percivals assigned the same n-fruc-topyranose l-(Ar -phenyl)-2-(V -methyl-Ar -phenyl)osazone structure to the compounds, but differentiated them as probable syn and avli forms. 

Of these proposed structures, only that of the n-glucose l-(A -methyl-V -phenyl)-2-(A -phenyl)osazone, suggested by Voto6ek and Vondrafek for mixed osazone B, is theoretically unsuited for formazan formation since... 

Structure of the mixed A and mixed B osazones according to Voto k and Vondra6ek ... 

However, since mixed osazone A undergoes no formazan reaction, even in alkaline solution, it would appear that the n-glucose l-(iV -methyl-iV -phenyl)-2-(iV -phenyl)osazone structure, - hitherto thought to be incapable of coupling, should be assigned to this substance (instead of to the mixed osazone B). This view is fully confirmed by recent work of Henseke and Hautschel. ... 

Concerning the finer structure of the mixed osazone A, the following points merit mention. 

On the basis of these experimental facts and the above considerations concerning the structure of n-glucose phenylosazone, the chelate ring form VI of Fieser and Fieser appears to describe the finer structure of the mixed osazone A. 

The mixed osazones prepared by Henseke and Binte during studies on the osones and related compounds brought forth new facts regarding the mechanism of formation. They postulated a linkage of glycosyl to nitrogen, whereas the bond between a disubstituted hydrazine residue and C-2 is veiy labile and is subject to hydrazone wandering. 

Volemitol is also oxidized by A. xylinum, but the identity of the sirupy product, volemulose, remains obscure specificity requirements are again satisfied at both ends of the molecule. Thus, although Hudson and coworkers obtained only n-oZiro-heptulose from a slow and incomplete oxidation, it has been reported" that, in the later stages, o-madapted cultures, D-monno-heptulose predominated throughout." The melting point recorded for volemulose phenylosazone does not agree with either the individual, or mixed, melting points of the two heptulose osazones. ... 

The choice between Weygand s Schemes A and B proved difficult, with authors at one time favoring one Scheme and then the other. An early attempt to test the validity of the respective Schemes was made by Ruggli and Zeller, using diphenylacetoin (p-bromophenyl)hydrazone and di-phenylacetoin (p-nitrophenyl)hydrazone, which they treated with phenylhydrazine they obtained the bis(phenylhydrazone) in both cases. This result was taken by them to exclude Scheme B, which, under similar conditions, should yield a mixed osazone, but this conclusion is now invalidated by our knowledge of transhydrazonation, that is, the exchange of hydrazone residues, as in (20) (21), a possibility, which has caused... 

A large number of glycosulose l-(2-substituted) hydrazones have been prepared by Henseke and coworkers from the osazones by action of nitrous acid. These products, such as (89), were then treated with other substituted hydrazines, to give mixed osazones," or with hydrazine and other carbonyl reagents, to obtain ketazines and azines" hydrazone thiosemicarbazones, and hydrazone oximes. ... 

Hexosulose hydrazones and the mixed osazones react with diazotized aniline to give formazans, and with hydrogen peroxide to give pentonic acids. Unlike the mixed osazones obtained by transhydrazonation, the derivatives obtained from the glycosulose hydrazones are pure, uncontaminated compounds, and the positions of their hydrazone residues are well established. Thus, they afford remarkable possibilities, not only in the preparation of new types of compound, but also in enabling the reaction of osazones to be followed more closely, as in the case of triazole formation," and in studying the structure of anhydro-osazones. ... 

Furthermore, he used the reaction to obtain a number of mixed osazones these compounds are, however, best prepared from the glycosulose hydra-zones. A detailed study of transhydrazonation was made by Mandel, who confirmed VotoCek s finding that the reaction proceeds best when the products have low solubilities, as with the (nitro- and dinitro-phenyl)-osazones. 

Ethers.—Methylation of D-arabino-hexulose phenylosazone (174) under mild conditions yields preferentially the 6-methyl ether.430431 Vigorous methylation leads to a mixture of methylated products. Mixed 1-Ar-methyl-A-phenyl-2-phenylosazones are obtained either by direct methylation of the unchelated nitrogen432,433 or by transhydrazonation of bis(yV-melhyl-jV-phenyl)osazones with phenylhydrazine.434 Two mono-iV-methyl derivatives of D-arabino-hexulose phenylosazone, designated mixed osazones A and B, have been described in the literature. Mixed osazone A was later identified as l-(A-methyl-A-phenyl)-2-phenylosazone, whereas mixed osazone B proved to be a mixture of compound A and d-arubino-hexulose phenylosazone.157,317,320... 

These findings were confirmed by Henseke and Winter, who started with D-amfifno-hexosulose ( n-glucosone ) l-(phenylhydrazone) or with D-ara6mo-hexosulose 2-(phenylhydrazone) and treated them with different arylhydrazines to give a number of mixed osazones having either (a) the... 

Although advantageous for the identification of the sugars, the phenyl osazones are not applicable to the isolation of sugars. The phenylhydrazine groups are removed by treatment with benzaldehyde, concentrated hydrochloric acid, or particularly well by pyruvic acid (230), but the resulting product, a sugar osone, is a mixed ketose-aldose. 

Procedure Phenylhydrazine ( 200 mg) in a 10-ml test tube is mixed with 0.12 ml of glacial acetic acid and 50 mg of sodium acetate, and 100 mg of glucose dissolved in 4 ml of water are added to this mixture. The test tube is immersed into a boiling water bath for 30 min, 1 ml of water is added, and the mixture is allowed to cool in a refrigerator. The separated osazone is filtered on a filtration tube, washed with 1 ml of 5% acetic acid and 1 ml of water, and dried in a stream of dry air. Yield, 0.1 g mp, 208—209 °C. For crystallization the product is dissolved in 15 ml of hot methanol, and water is then added dropwise to the solution to incipient turbidity. The mixture is then heated and allowed to crystallize (85 mg mp, 208 °C). 

---