Biochemical Studies on Carbohydrates. LXXXVII. Chondrosin.

Transcription

1 The Tohoku Journal _??_, Nos. 1 & 2, 1947 Biochemical Studies on Carbohydrates. LXXXVII. Chondrosin. Sixth Report. Primary Oxidation- and Reduction-Products of Chondrosin and N-Acetylchondrosin. Tetu By Kobayasi. (From the Medico-Chemical Institute, Tohoku Imperial University, Sendai. Director: Prof. H. Masamune.) (Received for publication, May 11, 1943) Oxidation by bromine of chondrosin and N-acetylchondrosin ethyl ester gave "chondrosinic acid" (glucuronidochondrosaminic acid) and N acetylehondrosinic acid.."chondrosinite" (glucuronidochondrosaminol) and N-acetylchondrosinite were prepared from chondrosin and N-acetyl chondrosin by the use of calcium amalgam. As a product from chondrosin was separated in the latter case glucuronic acid besides, reminding us of Levene and La Forge's reduction method by which they could demonst rate the presence of this acid in chondrosin for the first time.1 Furthermore, since the aldehyde equivalent (iodine use) of acetylchondrosin, was found about 2.0, the molecular wieght of chorkdroitinsulfuric acid (acid sodium salt) is assumed to reach beyond 20,000 in Masamune and Osaki's calculation2. EXPERIMENTAL. Chondrosin and N-acetylchondrosin ethylester were prepared accord ing to Hebting3 and to Masamune, Hiyama and Kobayasi4 respec tively. N-acetylchondrosin was obtained by hydrolysis of the latter. (1) N-Acetylchondrosin.B arium salt. 2 21mgm. of N-acetylchondrosin ethylester were taken up in 4cc. of water and after addition of 2cc. of 0.313n. baryta water, stood at 0 Ž for 24hours. Then 0.1n. sulfuric acid was added to neutralize the-solution 83

2 84 T. Kobayasi (indicator, bromothymolblue). The quantity of the acid which was required showed that 97.5% acetylchondrosin had been liberated. The supernatant liquid after centrifugation was condensed to about 2cc. by distillation at a low temperature ( ƒ40 ) and 20cc. of acetone were added. The deposit was washed with alcohol and ether and dried in vacuo (P2O5) at room tempe rature. The white amorphous powder amounting to 210mgm. was reprecipi tated twice from a little volume of water and after washing similar to above dried in vacuo at first at room temperature and afterwards at It was very hygroscopic and very soluble in water and reduced hot Fehling and Tollens solutions. The naphthoresorcinol test for hexuron ic acid resulted in positive. Ba (Pregl): 4.156mgm. substance mgm. BaSO4. C14H22O12N Ba/2. Calc., 14.79%;. Found, 15.15%. N (micro Dumas): 4.475mgm. substance, 1.15cc. N2 (762mm., 19.8 ) Calc., 3.01%; Found, 3.00%. CH3CO (H. Suzuki): 7.06mgm. substance, 0.602mgm. CH3CO. Calc., 9.25%; Found, 8.58%. Aldehyde equivalent (Cajori): 8.80mgm. of the substance reduced 0.790cc. of 0.ln. iodine solution, so the aldehyde equivalent is calculated at NH2-N was not present (micro Van Slyke). Cinchonidine salt. 220mgm. of the barium salt were dissolved in 10 cc. of water. Barium was removed with just a sufficient quantity of sulfuric acid (1n. and 0.1n. sulfuric acid solutions were employed), 250mgm. of cinchonidine in alcohol were added, and after condensation nearly to dryness and addition of about 10cc. of water, the excess of cinchonidine was separated off on a funnel. The filtrate and washing were further condensed together (vacuum distillation) to about 1.5cc., whereupon crystallization in microscopic needle began to occur. On the next day the crystals were collected, washed with ice-cooled water and with alcohol crystallized from water. Yield, 120mgm. M. p., They were re Crystalline water: 4.012mgm. substance, 0.243mgm. H2O ( in vacuo).. C14H22O12N EC19H22ON2+2 1/2 H2O. Calc., 6.11%; Found, 6.05%. N (micro Dumas): 3.692mgm. substance (anhydrous) H22O12N EC19H22ON2. Calc., 6.07%; Found. Suzuki): 8.672mgm. substance -N was not present (micro Van Slyke), 0.477mgm. CH3CO. Calc., 5.84% Found., 0.196cc. N2 (755.3mm., 21.8 ).C14, 6.10%.CH3CO (H,, 5.35%..NH2 O ptical rotation in water. [ƒ ]21.5 D=0.38 ~3.002/ ~0.999 ~1=-69.2.

3 Chondrosin \Sixth Report 85 (2) Chondrosinic Acid and N-Acetylchondrosinic Acid. Chondrosinic Acid. Crude barium salt. 1gm. of chondrosin in 30cc. of water was stood under occasional shaking with 0.7cc., a larger quantity than the calculation, of brominee as stoppered in a 100cc. flask at room temperature (23-25 ). After the lapse of 3 weeks5, the excess of bromine was removed by aeration, hydrobromic acid with lead carbonate, and finally lead with H2S. The solution (acid) obtained was condensed at a low temperature at first to about half the volume, and after neutralization with barium carbonate and filtration, furtheron to about 4cc. White precipitates appearing on addi tion of 5 volumes of alcohol were collected and washed with alcohol and ether. The amorphous powder (560mgm.) was very easily soluble in water. It reduced Fehling solution, though not markedly. N was detected (Lassaigne) I t contained barium less than the theoretical. Ba (Preg1): 4.326mgm: of the substance dried ( in vacuo) gave 1.829mgm. of BaSO4. C12H21O12NBa. Calc., 27.07%; Found, 24.94%. Cinchonidine salt. 500mgm. of the barium salt were taken. and barium was replaced with cinchonidine by the procedure adapted in (1). The crude product weighed 200mgm. and melted at 166 under gas evolution. It was soluble in water and insoluble in alcohol and ether. Fehling and Tollens reagents were not reduced at all. The naphthore sorcinol and aniline acetate reactions of hexuronic acid were given. Hav ing been recrystallized twice from water, it showed a constant melting point ( ) and analyzed: Crystalline water: 3.512mgm. substance, 0.063mgm. H2O ( in vacuo). C12H21O12N(C19H22ON2)2+H2O(977). Calc., 1.84%; Found, 1.79% N (micro Dumas)., 3.073mgm substance, 0.195cc. N2 (756.4mm., 21 ). Calc., 7.17%; Found, 7.32%. Neutral equivalent: 5.860mgm. of the substance were dissloved in 10cc. of water and titrated with 0.05n. NaOH, taking phenolphthalein as an indicator cc. of the alkali was required ~1000/0.246 ~0.05=475. Optical rotation in water: [ƒ ]25.5 D=-0.67 ~ / ~0.999 ~1 = N-Acetylchondrosinic Acid. B arium salt. N-acetylchondrosin ethylester6 was taken as a material. 500mgm. of it were dissolved in 20cc. of water ane stood with bromine (0.6cc. in,total \ 0.2cc. was added every week) for 3weeks7 (air temp., 17=21 ) under frequent shaking.

4 86 T. Kobayasi The remaining bromine and occurring hydrobromic acid; were taken off and the barium salt precipitated as in the case of chondrosinic acid. The white powder (265mgm.), that was obtained, reduced hot Fehling and Tollens reagents only with difficulty. The naphthoresorcinol and Goldschmiedt tests for hexuronic acid were positive. Nitrogen was detected (Lassaigne), but no nitrogen gas was evolved by Van Slyke procedure. It contained barium (calculated from the quantity of sulfuric acid requisite to precipitate out all the barium) as much as corresponds to acetylchondrosinic acid. Cinchonidine salt. 230mgm. of the barium salt were changed into the cinchonidine salt similar to above by using 600mgm. of this alkaloid. Recrystallization was carried out twice from water. Yield, 170mgm. M. P., 175. It showed the positive naphthoresorcinol and Goldschmiedt resactions of hexuronic acid but reduced neither hot Fehling solution nor hot ammoniacal silver solution. A nalysis. Crystalline water: 3.263mgm. substance, 0.138mgm. H2O ( in vacuo). C14H24 O13N(C19H22ON2)2+2 1/2 H2O. Calc., 4.27%; Found, 4.23%. N (micro Dumas): 3.050mgm. substance (anhydrous), 0.189cc. N2 (757.5mm., 26.8 ). C14H24O13N(C19H32ON2)2. Calc., 6.98%; Found, 7.07%. Neutral equivalent: ~1000/0.513 ~0.05=515. [ƒ ]25.5 D= ~ / ~0.999 ~1=-90.6, ' (3) Chondrosinite and N-Acetylchondrosinite. Chondrosinite and Glucuronic Acid. 1gm. of chondrosin was taken up in 40cc. of water (in a 150cc. round bottom flask) and immersed in ice, and under agitation fresh 3% calcium amalgam was added in small portions. During the whole course CO2 gas was passed to maintain the solution faintly alkaline. CaCO3 and mercury were separated off on a Buchner funnel occasionally. The solution show ed a reducing force on Feh1ing solution even when 200gm. of the amalgam had been employed. The reaction was stopped at this stage and the solu tion was filtered and evaporated to about 2cc. under, a reduced pressure. The substance thrown down by the aid of 20cc. of alcohol was collected, washed with alcohol and ether and dried in vacuo (P2O5). The amorphous white powder weighed 400mgm. Lassaigne test for nitrogen and the naphthoresorcinol and aniline acetate tests for hexuronic acid were positive. It reduced hot Fehling and Tollens solutions to about half the extent as chondrosin. The cinchonidine salt was prepared from 350mgm. of the crude calcium salt above. These were dissolved in 10cc. of water and calcium was pre-

5 Chondrosin \Sixth deport 87 cipitated out with just an equivalent quantity of oxalic acid. Cinchonidine applied was 500mgm. The crude cinchonidine salt (colorless needles; 100 mgm.); which was obtained, reduced hot Fehiing solution faintly and melted at 184. After recrystallizing twice from water it melted at (the constant melting point) and possessed no longer any reducing force. The naphthoresorcinol and aniline acetate reactions of hexuronic acid were given. Crystalline water: 3.945mgm. substance, 0.115mgm. H2O ( in vacuo). C12H23O11N EC19H22ON2+H2O(669). Calc., 2.69%; Found, 2,91%. N (micro Dumas): 3.820mgm. substance, 0.200cc. N2 (754.4mm., 22 ). Neutral equivalent: ~1000/0.099 ~0.05=643. [ƒ ]25.5 D=-0.10 ~2.9929/ ~0.999 ~1=-58.4 Calc., 6.27%; Found, 6.01%. On condensation of the mother liquid of chondrosinite cinchonidine salt in a vacuum desiccator (P2O5) came out crystals8 slowly in the form of soft thin needle. After standing in air overnight, they were collected and washed with ice-cooled water and ice-cooled alcohol. Yield, 40mgm, The product melted at 180 and showed an intensive reducing force on Fehiing solution and the colour reactions of hexuronic acid. But two times recry stallization from water raised its melting point to 191 and the mixed melting pointtest showed that it was identical with cinchonidine glucuronate (Masamune, Hiyama and Kobayasi4). A nalysis: Crystalline water: 3.000mgm. substance, 0.150mgm. H2O ( in vacuo). ` C6H10O7 EC19H22ON2+1 1/2 H2O(513). Calc., 5.25%; Found, 5.00%. N (micro Dumas): 2.819mgm. substance (anyhdrous), 0.147cc. N2 (754.4mm., 22 ). C6H10O7 EC19H22ON2.Calc., 5,74% Found, 5.99%. Neutral equivalent., ~1000/0.287 ~0.1=500. [ƒ ]21 D=-0.27 ~ / ~1.001 ~1= N-Acetylchondrosinite. 600mgm. of the barium salt of N-acetylchondrosin were treated similarly as above. 100gm, of the amalgam were used. The amount of the produced crude salt reached only 80mgm. It reduced hot Fehling and Tollens solutions but faintly. The naphthoresorcinol test for hexuro nic acid Was positive. Nitrogen (Lassaigne) was present but no amino nitrogen (Van Slyke). 64mgm. of the substance above were taken to prepare the cinchonidine salt. Cinchonidine used was 100mgm. The solution, which had been

6 88 T. Kobayasi removed from the excess of the alkaloid, was evaporated to about 1cc. On standing, colorless crystals in thick needle came out of it in a little while. After one day, the crystals were e separated and, washed with alcohol. 25 mgm. of the, twice recrystallized (from water), substance were yielded. M.p., (the melting point was not elevated by further recry stallization). Fehling solution was not reduced at all. The naphthore sorcinol reaction of hexuronic acid was shown. Crystalline water: 3.165mgm. substance, 0.199mgm. H2O ( in vacuo). C14H25O12N EC19H22ON2+2 1/2 H2O. Calc., 6.09%; Found, 6.02%. N (micro Dumas): 2.941mgm. substance (anhydrous), 0.149cc. N2 (759.8mm., 24.9 ). C14H25O12N EC19H22ON2. Calc., 6.06%; Found, 5.80%. [ƒ ]25.5 D = ~2.9948/ ~0.998 ~= From the mother liquid appeared colorless crystals in needle. The substance reduced Fehling solution and, after recrystallizing twice (the yield, 15mgm.), melted at , which nearly accords with the melting point of the cinchonidine salt of N-acetylchondrosin started with. The mixed melting point with this salt was the same9. The substance showed a similar reducing force as before the purification. We were enabled to carry out these experiments by a grant from the Foundation for Promotion of Science, and Industrial Research and that bestowed by the Grant Committee for Scientific Researches of the Educa tion Department, which is gratefully acknowledged. ordinary BIBLIOGRAPHY AND NOTES. 1 Levene and La Forge, J. Biol. Chem., 1913, 15, Masamune and Osaki, Tohoku J. Exp. Med., 1943, 45, Hebting, Bioch. Z.,.1914, 63, Masamune, Hiyama and, Kobayasi, Tohoku J. Exp. Med., 1942, 44, Oxidation with bromine takes an enormously long time for this compound contrary to disaccharides mgm. of the substance reduced 0.55cc. of 0.1n. iodine solution (Cajori), which corresponds,to aldelayde equivalent of The product, which was obtained after 2weeks of standing Tollens reagents markedly., still, reduced Fehling and 8 The crystals began to appear, when the liquid was reduced to about 2/3 in volume. 9 Mixed melting point tests demonstrated that the substance was, identical, neither with cinchonidine glucuronate nor the corresponding salt of N-acetylchondrosinite (the mixed melting points were 184 and 182 respectively).