FART II SOME EXPERIMENTS ON THE CHEMISTRY OF OSAZONES. INTRODUCTION 103 SuKar Osazones. Sugar osazones were first prepared by Emil Fischer (Ber., 579) in the year 1884; since then, these compounds have found widespread application both as derivatives for the identification of reducing sugars and as intermediates in the preparation of a variety of sugar compounds. The fact that the free sugars are brought to crystallisation only with great difficulty, together with the lack of a reagent that forms easily crystallisable derivatives with all reducing sugars, renders their identification a tedious process, especially when dealing with sugar mixtures. The osazones are unique in the ease with which they crystallise yet suffer from the disadvantage that every particular osazone is formed from a number of sugars, so that the complete identification of a sugar requires the use of further derivatives. Osazones can be prepared from compounds having in the O.—position to a carbonyl group another carbonyl ' group as in the case of osones, a hydroxyl group as in aldoses and ketoses, an amino group as in amino sugars, or a methoxyl or thio—alkyl group; in the latter two cases osazone formation proceeds with difficulty and 104 yields are poor. Constitution of sugar osazones. Fischer (Ber., 1887, 20, 821) ascribed to glucose phenyl-osazone structure (I); the other sugar osazones were likewise given open chain structures differing only in the configuration of the hydroxyl groups in the sugar molecules and in the substitution of the hydrazine residue. Fischer's formula has been criticised by several authors and at the present time, after more than sixty years, the structure of sugar osazones is not definitely established. Investigations have been mainly concerned with glucose phenyl-osazone where two structures deserve serious consideration, viz. Fischer's open chain structure (I) and the cyclic structure (II) proposed by Percival (J. Chem. Soc., 1935, 1398):- CH =N-NHC6H5 CH = N-NHC6H5 C = N-NHC6 H 5 C-NHNHC6H5 HO C H HO - C H H - C - OH 0 H - C - OH H - C - OH H C - OH CH2OH CH2 105 Evidence in support of structure (II) was given by Percival (loc.cit.) who obtained, by treating glucose phenyl-osazone (It) with dimethyl sulphate in alkali, a tetra-methyl derivative (III) which on treatment with 1-nitro benzaldehyde gave a trimethylglucosone (IV); this was finally reduced to the known 34:5-trimethyl- D-fructopyranose (V) denoting that only three of the methoxyl groups in (III) were attached to oxygen, the fourth being attached to a nitrogen atom in the hydrazone residue. CH=N -NHC6H5 CH=N-NHC H I 6 5 ---- -C-NH-NHC H 0 -NH -N(CH )C H 6 5 3 6 5 1 1 HO-C-H CH 0 -C -H 1 (aH )SO 3 1 H -C -OH 3 4, 0 H -0 -CH alkali -6 3 H -C -OH H -C -0 -CH 3 ------ CH2 ------- CH2 (II) (III) CHO CH/ 2 OH C OH ? -OH 1 nitro- CH30 -C -H Zn CH7 0 -C -H benzaldehydet 0 -I H -C -0 -CH acetic acid) 0 H -C -0 -CH I 3 i 3 H-! -0 -CH H -C -0 -CH 3 1 3 ! I CH2 CH2 (Iv) ( V) 106 Percival argues that the fact that the primary hydroxyl group in C6 was unattached while the secondary ones in positions 3-, 4- and 5—were, shows that position 6- was involved in ring formation. This was confirmed by Diels (Ber., 1938, 71, 1189) who showed that glucose phenylosazone failed to react with trityl chloride, which is known to react with primary hydroxyl groups. On the other hand, Wolfrom, Kbnigsberg and Saltzberg (J. Amer. Chem. Soc., 1936, 58, 490) obtained by acetyla- tion a tetra-acetyl-glucose phenyl-osazone which they hydrolysed using a method developed by Kunz and Hudson (J. Amer. Chem. Soc., 1926, 48, 1982) which is claimed to hydrolyse 0-acetyl groups but not N-acetyl ones. This method consists in dissolving the acetate in acetone and adding 0.1N-KOH dropwise to the ice cooled solution; after two hours standing at room temperature the solution is back-titrated. All four acetate groups in the osazone were hydrolysed by this treatment and the authors concluded that glucose phenyl-osazone must have structure (I). Percival (J. Chem. Soc., 1937, 1320) doubted the significance of these results, as it is known, that N-acetyl groups are sometimes hydrolysed by the method of Kunz and Hudson, for example in D(;/3-diacetyl-phenyl-hydrazine, where an amount corresponding to about one acetyl group was removed under similar conditions. 107 The strongest argument for the open chain structure (I) of glucose phenyl-osazone was given by Chargaff and Magasnnik (J. Amer. Chem. Soc., 1947, 69, 1459); they showed that on oxidation with periodic acid one mot. of glucose phenyl-osazone yields 2 mols of formic acid, one mol. of formaldehyde and mesoxaldehyde phenyl osazone (VI) in 85% yield. They formulated their results as follows:- CHZ-NHC H 6 5 C H=N-NHO6H5 I LN-NHC6H5 C=N-NHC6H5 HO- NCHO 311104 ,. H-C-OH ..- + I H-C-OH. 2H-000H 1 CH2OH + H-CHO (I) (VI) Engel (J. Amer. Chem. Soc., 1935, 2a, 2419) studied the ultra violet absorption spectra of sugar phenyl- osazones and found them similar to glyceraldehyde phenyl- osazone; this was taken to favour the open chain structure of sugar osazones since it is most unlikely that glyceraldehyde-phenyl-osazone has a ring structure. From the above review it is clear that there is strong evidence for both structures (I) and (II). This 108 contradiction could be explained if we assume an equilibrium between the two structures and that under methylation or tritylation conditions the cyclic structure (II) prevails while during periodic acid oxidation the equilibrium is shifted towards the open chain structure (I). An example of such a shift in structure is known in the case of free sugars which behave as open chain compounds towards periodic acid and as cyclic ones towards methylating agents. Reactions of sugar-osazones. 1. Reduction. Fischer (Ber., 1899, 22, 88) has shown that sugar- osazones when reduced with zinc in acetic acid yield 1-amino-ketoses (VII); later Maurer and Schiedt (Ber., 1935, 68, 2187) obtained the same products by catalytic hydrogenation of the osazones. CH=N-NHC6 H5 1 red. Cr.g-NHC6H5 (I) The importance of this reaction lies in the fact that it 109 offers a means of converting aldoses into ketoses; thus, by starting with glucose and reducing its osazone 1-amino-fructose is obtained which, on deamination with nitrous acid, yields fructose (Fischer and Tafel,. Bert, 1887, 20, 2566). 2. Oxidation with molecular oxygen in alkali. Diels (Ber., 1938, 71, 1189) oxidised glucose phenyl- osazone with oxygen in alkaline solution, and obtained a dehydro-osazone to which he ascribed structure (VIII):- CHZ.4THC H 6 5 CH =N`NO H C-NHNHC H CI-N ,/ 6 5 6 5 .---NHC6H5 HO-C-H (0) HO-6-H > 0 H-C-OH H-C-OH H-C-OH H-C-OH CH2 CH2 (II) (VIII) The structure of the dehydro-osazone was based on the following experiments:- Acetylation of the dehydro-osazone gave a triacetate denoting the presence of three hydroxyl- groups; trityl chloride failed to react with the dehydro-osazone showing that the primary hydroxyl group ).10 in C6 was engaged in ring-formation; lastly it was found that substituted osazones such as methyl-phenyl-osazones failed to give on oxidation a dehydro-osazone, denoting that the hydrogen atoms in the hydrazone residues were involved in the oxidation. In the light of this evidence Diels (loc.cit.) suggested structure (VIII) for glucose dehydro-phenyl-osazone. 3. Action of Alkali. Diels (Ann., 1936, 525, 94) has shown that sugar phenyl-osazones, when heated with 1% alcoholic alkalies, break up to form glyoxal phenyl-osasone (IX) thus CH=N-NH H C6 5 CH=N7NHC6H5 CZ--NHC H 6 5 CHZ-NHC6 5 (1) 4. Conversion of osazones into osones. Fischer (Ber., 1888, 21, 2631; 1889, 22, 88) found that on heating sugar osazones with concentrated hydrochloric acid the hydrazone residues are removed yielding dicarbonyl compounds, the osones. These. 111 can be reduced with zinc and acetic acid yielding ketoses, thus offering another route for the conversion of aldoses to ketoses (Fischer, Ber., 1889, 22, 188). Sugar osones have recently acquired great interest as intermediates in the preparation of ascorbic acid and its homologues (Haworth, Hirst, Jones and Smith, J. Chem. Soc., 1931+, 1192; Reichstein, GrUssner and Oppenauer, Hely. 1933, 16, 561, 1019; 1934, 17, 510). The yield of ascorbic acid was found to depend on the purity of the osone. This. led to a search for a method for the preparation of osones in a high state of purity; the old methods using aldehydes such as benzaldehyde and o-nitro benzaldehyde (Morell and Ballard, J. Chem. Soc., 1905, 87, 280) were found to produce the osones in poor yields; BrUll (Ann. Chim. applicata, 1936, 26, 415) obtained glucosone in 40% yield and in a high state of purity by treating glucose phenyl-osazone with pyruvic acid. During the course of the present study on sugar osazones, a method has been developed which yields the osone from a substituted osazone in a high state of purity and in a yield of 80%. This method will be . discussed in greater detail in the theoretical section. 5. Conversion of Osazones to Osotriazoles. The use of sugar osazones for the identification of 112 sugars has the disadvantage, among other things, that the osazones melt with decomposition and that their melting points depend to a great extent on the rate of heating; furthermore their optical rotations are not constant owing to mutarotation.