Dihydroxy-acetone

This cleavage is a retro aldol reaction It is the reverse of the process by which d fruc tose 1 6 diphosphate would be formed by aldol addition of the enolate of dihydroxy acetone phosphate to d glyceraldehyde 3 phosphate The enzyme aldolase catalyzes both the aldol addition of the two components and m glycolysis the retro aldol cleavage of D fructose 1 6 diphosphate... 

Anthrarufin [l,5-dihydroxy-9,10-anthraquinone] [117-12-4] M 240.1, m 280 (dec), pKj 9.90, pK 11.05. Purified by column chromatography on silica gel with CHCl3/Et20 as eluent, followed by recrystn from acetone. Alternatively recrystd from glacial acetic acid [Flom and Barbara J Phys Chem 89 4489 1985]. 

When 3a,17a-dihydroxy-5jS-pregnane-ll,20-dione is allowed to react at room temperature overnight with sodium borohydride in aqueous methanol, no crystals form and only 5j5-pregnane-3a,l ljS,17a,20j5-tetrol is isolated in good yield. If the reaction is halted at the end of 3 h y the addition of water and extraction with chloroform, it is possible xo obtain a 55% yield of 3a,17a,20jS-trihydroxy-5j5-pregnan-ll-one, mp 218-220°,after recrystallization of the chloroform residue from aqueous methanol. The analytical sample, crystallized once more, has mp 219.0-220.6° [a][, 36° (acetone), reported mp 220° [aJu 38°. 

Periodic Acid Degradation 17a,20 -Dihydroxy-4,4,6,16a-tetramethyl-pregn-5-en-3-one (0.3 g) is dissolved in 30 ml of methanol and treated with an aqueous solution of 0.25 g of periodic acid in 5 ml of water at room temperature for 17 hr. On dilution with water, the resultant crystals are collected by filtration, washed well with water, and dried to give 0.26 g mp 158-160°. Recrystallization from hexane-acetone gives 0.24 g (90%) of 4,4,6,16a-tetramethylandrost-5-ene-3,17-dione mp 160-161° [aj —6° (CHCI3). 

The enamine (1 g) in 30 ml of benzene and 5 ml of toluene is treated, with cooling, with a solution of 0.33 g (1.04 eq.) of perbenzoic acid in 2.1 ml of benzene. The reaction is complete in a few minutes. Ether (30 ml) is added, then the solution is washed with 2 N sodium hydroxide and with water to neutrality, dried over sodium sulfate and evaporated under reduced pressure. Crystallization from acetone gives 0.51 g of crude product mp 230°. One further crystallization from methanol gives 0.38 g (50%) of 3J5,17a-dihydroxy-5a-pregnan-20-one rap 265°. 

A solution of 0.38 g of the diazoketone in 2 ml of acetic acid is heated 0.5 hr at 100-105°. Removal of the solvent under reduced pressure and crystallization of the residue from acetone-ether gives 0.27 g of 1 la,21-dihydroxy-pregn-4-ene-3,20-dione 11,21-diacetate mp, 144-146° [a]o 156° (CHCI3). An additional 60 mg of this product is obtained by chromatography of the mother liquor (total yield 72%). 

Definitive identification of lysine as the modified active-site residue has come from radioisotope-labeling studies. NaBH4 reduction of the aldolase Schiff base intermediate formed from C-labeled dihydroxyacetone-P yields an enzyme covalently labeled with C. Acid hydrolysis of the inactivated enzyme liberates a novel C-labeled amino acid, N -dihydroxypropyl-L-lysine. This is the product anticipated from reduction of the Schiff base formed between a lysine residue and the C-labeled dihydroxy-acetone-P. (The phosphate group is lost during acid hydrolysis of the inactivated enzyme.) The use of C labeling in a case such as this facilitates the separation and identification of the telltale amino acid. 

Classical corticosteroids have in common an unsaturated ketone at the 3 position, oxygen ai the 11 position and a dihydroxy-acetone side chain at the 17 position (see, for example, 56). 

Preparation of 9, 11 -Epoxy-17a-21 -Dihydroxy-16 -Methyl-4-Pregnene-3 0-Dione 21-Acetate To a stirred solution of 100 mg of the 9a-bromo-11(3,17a,2Ttrihydroxy-16 3-methyl-4-pregnene-3,20-dione 21-acetate in 3 ml of tetrahydrofuran and 1 ml of methanol under nitrogen was added 1.02 ml of 0.215 N methanolic sodium methoxide. After 10 minutes at 25°C, 0.2 ml of acetic acid was added and the methanol removed in vacuo. The residue was acetylated with 1.00 ml of pyridine and 0.5 ml of acetic anhydride at 60°C for 70 minutes. The mixture was taken to dryness in vacuo, water added, and the product extracted into chloroform. The residue was crystallized from ether-acetone to give pure 9(3,11 (3-epoxy-17a,21-dihydroxy-16(3-methyl-4-pregnene-3,20-dione 21-acetate. 

Preparation of 9a-Fluoro-110,17a,21-Trihydroxy-160-Methyl-4-Pregnene-3,2O-Dione 21-Acetate To a solution of 200 mg of 9(3,11(3-epoxy-1 7a,21-dihydroxy-16(3-methyl-4-pregnene 3,20-dione 21-acetate in 2 ml of chloroform and 2 ml of tetrahydrofuran in a polyethylene bottle at -60°C was added 2 ml of a 2 1 (by weight) mixture of anhydrous hydrogen fluoride and tetrahydrofuran. After 4 hours at -10°C the mixture was cooled to -60°C and cautiously added to a stirred mixture of 30 ml or 25% aqueous potassium carbonate and 25 ml of chloroform kept at -5°C. The aqueous phase was further extracted with chloroform and the latter phase washed with water and dried over magnesium sulfate. The residue on crystallization from acetone-ether gave pure 9a-fluoro-11(3,17a,21-trihydroxy-16(3-methyl-4-pregnene-3,20-dione 21-acetate. 

A solution of 90 -fluoro-11/3-hydroxy-16/3-methyI-170 ,21-(1 -ethyl-1 -ethoxymethylenedioxy) pregna-1,4-dlene-3.20-dione (538 mg) in acetic acid (20 ml), containing 2 drops of water, was allowed to stand at room temperature for 5 hours. Dilution of the mixture with water gave a white solid (457 mg) which, after being filtered off and dried, was recrystallized from acetone to afford 90 -fluoro-11/3,21 -dihydroxy-16/3-methy 1-170 -propionyloxypregna-1,4-diene-3, 20-dione (361 mg), MP 230°-235°C. 

The acetic acid solution was poured into water (100 ml) and extracted with chloroform. The chloroform extracts were washed in turn with water, saturated sodium bicarbonate solution and water, dried and evaporated in vacuo. The residual gum was triturated with ether and a white crystalline solid (1.16 grams) isolated by filtration. Recrystallization from ether (containing a small amount of acetone)-petroleum ether gave 9a-fluoro-110,21-dihydroxy-160-methyl-1 7a-valeryloxypregna-1,4-diene-3,20-dione (871 mg) as fine needles. 

A solution of 1.0 g of A -3,11-diketo-20-cyano-21-acetoxy-pregnene in 10 cc of benzene is treated with 1.0 g of osmium tetroxide and 0.43 g of pyridine. After standing at room temperature for 18 hours, the resulting solution is treated successively with 50 cc of alcohol, and with 50 cc of water containing 2.5 g of sodium sulfite. The mixture is stirred for 30 hours, filtered, and the filtrate acidified with 0.5 cc of acetic acid and concentrated to small volume in vacuo. The aqueous suspension is then extracted four times with chloroform, the chloroform extracts are combined, washed with water and concentrated to dryness in vacuo. Recrystallization of the residue from acetone gives 3,11,20-triketo-17(a)-21-dihydroxy-pregnane MP 227° to 229°C. This compound is then treated with acetic anhydride and pyridine for 15 minutes at room temperature to produce 3,11,20-triketo-17(a)-hydroxy-21-acetoxy-pregnane or cortisone acetate. 

Fractions 11 through 24 inclusive were combined, evaporated and twice recrystallized from acetone to give pure 6a-methyl-9a-fluoro-11(3,17a-dihydroxy-1,4-pregnadiene-3,20-dione of melting point 292° to 303°C. 

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