Periodic acid/phenylhydrazine

D. Volz, P. E. Reid, C. M. Park, D. A. Owen, and W. L. Dunn, Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its side chain O-acyl variants or O-sulfate ester. I. Methods based upon the selective periodate oxidation of sialic acids, Histochem. J., 19 (1987) 249-256 Histochemical procedures for the simultaneous visualization of neutral sugars and either sialic acid and its O-acyl variants or O-sulfate ester. II. Methods based upon the periodic acid-phenylhydrazine-Schiff reaction, Histochem. J., 19 (1987) 257-263. 

Octyl acrylate Periodic acid Phenylhydrazine Propylcyclohexane Propylcyclopentane Sodium persulfate 1,1,2,2-Tetrachloroethane 1,3,5-Trichlorobenzene 2,2,3-Trimethyl butane 1,1,2-Trimethylcyclopentane 1,1,3-Trimethylcyclopentane n-Undecane Xylidine... 

In the periodic acid/phenylhydrazine/Schiff (PAPS) procedure of Spicer (1961) a treatment with phenylhydrazine is interposed between the periodic acid oxidation and Schiff reagent steps of the standard PAS. As phenylhydrazine reacts readily with aldehydes it would be expected to block all subsequent reaction with Schiff reagent. It was found, however, that when this sequence was employed with sections of mouse salivary glands and other tissues rich in sialomucins, PAS staining was reduced but not abolished. When, however, the salivary glands were pre-treated with a neuraminidase the positive Schiff staining in the PAPS... 

Periodic acid oxidation of cyclic phenylhydrazine derivatives [125] (Eq. 66). 

Block the endogenous enzyme with 0.3% H202 for 30 min (alternatively, incubate in 2.3% periodic acid for 5 min, wash with water, rinse in 0.03% potassium borohydride and wash), or incubate for 5 min in 0.1% phenylhydrazine in PBS)... 

Several mechanisms34 have been proposed for this reaction. That of Weygand,36 in which an Amadori rearrangement is proposed, has considerable merit.37 Illustrations of unusual osazone formation are described by Bonner and Drisko.38 When phenyl /S-D-xylopyranosyl sulfone (XXII) or /J-D-glucopyranosyl sulfone (XXIV) is oxidized by periodic acid, a dialdehyde oxidation product (XXIII or XXV), which is susceptible toward further oxidation, is obtained. The reaction of XXIII or XXV with phenylhydrazine yields glyoxal phenylosazone and benzenesulfinic acid. Surprisingly, both XXII and XXIII react with phenylhydrazine to form D-xylosazone and D-glucosazone, respectively. 

To determine the incorporation of C-labeled phenylhydrazine in the LO protein, a sample of the desalted, radiolabeled two-banded material was analyzed by SDS-PAGE (16). The separating gel contained 10.5% acrylamide using N,N -diallyltartardiamide as crosslinking reagent. The protein gel was stained with Coomassie Brilliant Blue G - Colloidal (Sigma) and subsequently cut into 16 slices according to the position of the protein bands. The gel slices were then dissolved in 1 to 5 ml of 2% periodic acid solution, and the radioactivity in each gel slice was quantitated by liquid scintillation. 

To an ice-cooled (0°C to-—10°C) stirred flask containing 300 ml of water and 55.5 ml of concentrated hydrochloric acid is added dropwise over a 10 min period 33.5 gm (0.31 mole) of freshly distilled phenylhydrazine. Phenyl-hydrazine hydrochloride crystals precipitate as they are formed. Addition of... 

L-Mannitol does not occur naturally but is obtained by the reduction of L-mannose or L-mannonic acid lactone (80). It can be synthesized from the relatively abundant L-arabinose through the L-mannose and L-glucose cyanohydrins, conversion to the phenylhydrazines which are separated, liberation of L-mannose, and reduction with sodium borohydride (81). Another synthesis is from L-inositol (obtained from its monomethyl ether, quebrachitol) through the diacetonate, periodate oxidation to the blocked dialdehyde, reduction, and removal of the acetone blocking groups (82). 

Treatment of SXIA, after the consumption of one mole of periodate per mole, with phenylhydrazine, followed by acidic hydrolysis, yields a sugar phosphate which, on enzymic dephosphorylation, gives glucose. A galacto-sylerythritol is obtained from a Smith degradation of reduced, alkali-degraded SXIA. 

The product crystallises from ethyl acetate as small, hard plates, M.pt. 152° C., easily soluble in chloroform, less soluble in alcohol or ether. It reduces Fehling s solution on heating, is stable towards alkah, but with acids yields phenylhydrazine and phenylphosphmous acid when boiled for a long period with water a complex of the two latter compounds separates. 

MWI of dehydro-L-ascorbic acid or -o-isoascorbic acid generated by oxidation of 702 or 703 with iodine, o-phenylenediamine, and phenylhydrazine in ethanol and in the presence of acetic acid for 3.5 min gave quinoxalinones 704 in 80-86% yields. Similarly, thiosemicarbazones 705 were obtained within 5 min in 69-79% yields by using thiosemicarbazide instead of phenylhydrazine (Scheme 136). Cyclization of 704 to the pyrazolyl-quinoxalinone 706 (88-95%) was also achieved by MWI for 7 min by using acetic anhydride, and bentonite as a support MWI of 705 with acetic anhydride in pyridine for 4-5 min afforded the L-threo and o-erythro thiadiazolyl-quinoxalinones 707 and 708 in 80% and 79% yields from the two epimers of 705, respectively. Periodate oxidation of 704 under MWI gave 709 in 90-92% yields (Scheme 136) (05UP4). 

---