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Synthesis of 2,4-Di-O-methyl-L-erythrose Barlay, Andrew Ervin 1961

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(i) SYNTHESIS OP 2,i^-DI-O-METHYL-L-ERYTHROSE by ANDREW ERVIN BARLAY D i p l . Chem., University of Science, Budapest, Hungary, 19£5 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS" FOR THE DEGREE OF MASTER OF SCIENCE i n the Department of Chemistry We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1961 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make i t freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department The University of British Columbia, Vancouver 8 , Canada. Date ( i i) ABSTRACT 2., 4-Di-0_-methyl-L-eiythrose is thought to occur in the hydrolysis and periodate oxidation products of polysaccharides. This sugar has now been synthesized from methyl-L-arabofuranoside by tosylation, methylation, detosylation and periodate oxidation. The free sugar was obtained as a sirup and was characterized by the preparation of a crystalline phenylhydrazone and a crystalline p nitrobenzoate. (vi) ACKNOWLEDGEMENT I wish to express my sincere thanks to Dr. G.G.S. Dutton for his stimulating and patient guidance throughout the course of this work* ( i i i ) TABLE OF CONTENTS Page H I STOR ICAL INTRODUCTION 1 DESCR IPT ION AND RESULTS OF PRESENT" RESEARCH ii EXPERIMENTAL" 8 BIBL IOGRAPHY 35 (iv) LIST OF TABLES Page I Preparation of methyl-L-arabofuranoside - - - - - - 12 II Separation of methylated L-arabinose f r a c t i o n s on cellulose-hydrocellulose column - - 19 III P i l o t scale periodate oxidation of 3,5-di-O-methyl-L-arabinose - - - - - - - - 23 IV P i l o t scale periodate oxidation of 3,5-di-0-methyl-L-arabinose- - - - - - - - - 2 5 V P i l o t scale p u r i f i c a t i o n of 2,i^-di-0-methyl-L-erythrose on a F l o r i s i l column- - - 29 VI Large scale p u r i f i c a t i o n of 2,4-di-O-methyl-L-erythrose on a F l o r i s i l column- - - 31 (v) LIST OF FIGURES Page I Flow sheet showing synthesis of 2,I|-di-0-metbyl-L/-erythrose- - - - - - - - - - -10 II Curve of specific rotation versus time for preparation of methyl-L-arabofuranoside- - - - - 13 III Curve of periodate consumption versus time for oxidation of 3>5-<ii-0-methyl-L-arabinose- - -2li IV Curve of periodate consumption versus time for oxidation of 3,5-di-O-methyl-L-arabinose- - -26 (1) HISTORICAL INTRODUCTION Within recent years so many sugars previously prepared by synthetic methods in the laboratory have been found playing an important role in proof of structure of polysaccharides and in natural processes, that new attention has been focused upon the necessity of f i l l i n g those gaps which s t i l l remain in this f i e l d of carbohydrate chemistry. Particularly does this necessity apply to sugars of lower carbon atom number which could be encountered as intermediates of degradation of poly-saccharides. The tetroses and their derivatives are s t i l l among the least known members of the sugar group, despite the fact that the f i r s t efforts toward their preparation were made seventy years ago (1). These are two parent tetroses, erytbrose and threose, both have two asymmetric carbon atoms therefore four possible structures exist. CHO CHO CHO CHO l I I I HCOH HOCH HOCH HCOH I I I ' I HCOH HOCH HCOH HOCH 1 I I I CH2OH CH20H CH20H CHgOH a : D-erythrose b : L-erythrose c : g-threose d : L-tbreose (2) There are two general methods of synthesizing tetroses (a) by degradation of sugars with greater numbers of carbon atoms. (b) by building up from simple organic compounds. The latter method is not used very often, as tbe product of the synthesis is usually a mixture of the optically active sugars, which are d i f f i c u l t to separate. The methods of degrading sugars devised by Wohl (2,3>ll) and Ruff seemed at f i r s t to furnish a very promising means of preparing tetroses and from 1899 to 1902 several attempts were made to synthesize them, (5>6,7»8«9) but the results proved disappointing and very few crystalline compounds were prepared and few data recorded. Except for the later application of Weerman's (10) method, which resulted in l i t t l e actual new success the f i e l d was abandoned u n t i l 1935* In 1935 Hockett (11) synthesized D-threose by using Ruff's degradation. A crystalline derivative, D-threose triacetate (11) was obtained. In 1930 a new method of degradation, periodate oxidation, advanced the synthesis of unknown sugars, but even with this new method the number of crystalline tetroses was very small. As a preparatory method oxidation with periodic acid is of particular importance for the preparation of short chain sugars. The mild conditions of the oxidation are well suited for sensitive carbohydrate structures and the selectivity (12) of the oxidation gives the desired oxidation product. As mentioned before, the number of crystalline tetrose derivatives is relatively small and especially the number of (3) methylated tetrose derivatives Cl3»lli) is very small. The purpose of this research was to synthesize a di-O-methyl tetrose in the L series and to identify i t by preparation of crystalline derivatives. The choice f e l l upon 2,li-di-0-metbyl-L-erythrose. (U) DESCRIPTION AND RESULTS OF PRESENT RESEARCH To synthesize 2,lt-di-0-methyl-L-erythrose, L-arabo-furanose was chosen as starting material. The main problem was to achieve selective methylation at the 3 and 5 position and leaving the 1 and 2 position unsubstituted, vulnerable to periodate oxidation, which preferably attacks 1,2 glycols. The most useful path for blocking the 5 position of the £-arabo-furanose was to prepare at f i r s t the methyl L-arabofuranoside and then react i t with p-toluenesulpbonyl chloride. p-Toluene-sulphonyl chloride selectively esterifies carbohydrates under proper conditions and this property was used to block the primary hydroxyl at the 5 position (lit). The preparation of methyl L-arabofuranoside yields not only the required product, but as a byproduct methyl Ji-arabo-pyranoside forms too in significant amounts. Some unchanged L-arabofuranose can also be found in the reaction mixture. MeOJJ MCI In OH [H,0C„5 The mixture of L-arabofuranoside and L-arabopyranoside can be tosylated directly as only L-arabofuranoside has a free primary hydroxyl group. n Oil '|U,0CW3 ft OH (5) When methyl 5^p-toluenesulphonyl-L-arabofuranoside i s reacted with absolute acetone in the presence of hydrogen chloride, tbe glycosidic methoxyl group i s hydrolysed and the free hydroxyl groups at the 1 and 2 positions w i l l form a 1,2 isopropylidene derivative. The product of this reaction is crystalline and easily Identified. As the 1 and 2 positions are blocked, the methylation of the only free hydroxyl group at the 3 position can proceed. In this research Purdie's method was used for methylation, which employs methyl iodide and silver oxide, as this method is mild, and sensitive carbohydrates do not suffer decomposition. The product of this methylation is crystalline 1,2 isopropylidene-3-0-methyl-5-p-toluenesulphonyl-L-arabofuranose (17)• To obtain a free hydroxyl group at the 5* position for further methylation, tbe p-toluenesulphonyl group was removed by sodium-amalgam reduction (17)• The progress of tbe reaction can be visually followed as the starting material is only sparingly soluble in aqueous methanol and the reduced 1,2-isopropylidene-3-O-methyl-L-arabofuranose is soluble. 15 CU3 \U 3 (6) Tbe 1 ,2 isopropylidene-3-O-metbyl-L-arabofuranose was methylated by Purdie's method. Daring the methylation some side reactions occured and the isolated product was not chromatographically pure, tbe presence of a tri-O-methyl-L-arabofuranose was detected. The presence of this component was unexpected as the methylation was done in a non-acidic medium and tbe hydrolysis of the isopropylidene group should not have occured. To explain this hydrolysis the following reaction can be postulated: 2. U c - O t t + U U 3 I + A^O V-UC-0CH5 + a WW Ao^O \ I R R 2 U 4 * lV\ A « H ° * where the momentary formation of hydrogen iodide can induce some hydrolysis. No experimental work was done to establish the f e a s i b i l i t y of this theory. After acid hydrolysis of l , 2-isopropylidene - 3 , 5-di - 0 -L-arabinose, the hydrolysate which contained mainly 3~5-di-O-methyl-L-arabinose and traces of a tri-O-methyl-L-arabinose was put on a cellulose hydrocellulose column and the impurities separated from the required product. The chromatographically pure 3>5-di-0-methyl-L-arabinose, which had two bydroxyl groups adjacent was then oxidised with sodium metaperiodate. Tbe oxidation of this compound yields one mole of 2,ii-di-0-methyl-L-erythrose and one mole of formic acid. Before a large scale (7) periodate oxidation was done, several p i l o t scale oxidations were completed to find the necessary experimental conditions. After finding suitable experimental conditions a larger amount of 3>5-di-0-metbyl-L-arabinose was oxidised and the reaction product was Isolated. The oxidation product 2,4-di-O-methyl-L-erytbrose was purified by column chromatography, but could not be obtained in crystalline form. To identify this compound and characterize i t , a portion was reduced with sodium borohydride and the 2,14-di-O-methyl-L-erythritol was prepared. The 2 , i | - d i - 0 -methyl-L-erytbritol was not obtained in crystalline form, but by reacting i t with p-nitro-benzoyl chloride a crystalline derivative was obtained and tbis was characterized. • To another portion of 2,1^-di-O-methyl-L-erythrose, 2 , l j -dinitrophenylhydrazine was added and tbe reaction resulted in a crystalline derivative, which was also characterized. (8) EXPERIMENTAL Melting points* A l l melting points were taken by means of a L e i t z e l e c t r i c a l l y heated melting point block and are corrected. Optical rotations. Optical rotations were taken on a O.G. Rudolph & Sons, Caldwell N.Y. polarimeter using a sodium lamp (General E l e c t r i c ) and the s p e c i f i c rotations were calculated according to the formula (15) . where[cc] i s the s p e c i f i c r o t a t i o n at 20 C measured with the D l i n e of sodium, OC the observed rotation, c the concent-of the polarimeter tube expressed i n decimeters. Chromatography. 1. Paper p a r t i t i o n chromatography A l l paper p a r t i t i o n chromatography was done on Whatman No.l chromatography paper by means of the descending method. A l l chromatograms were a i r dried a f t e r development and a f t e r o spraying the chromatograms were oven dried at 110 C. 2. Column chromatography (a) Column chromatography was done on a cellulose-hydro-c e l l u l o s e ( l t l ) column (2.8 x i|2 cm), which was kept at a o r a t i o n expressed i n g per 100 ml. solution, and ^  the length ( 9 ) constant temperature (1|0°C) by means of a thermostat* Fractions were collected on an automatic fraction collector (Time/Flow). (b) F l o r i s i l columns were also used for separation and purifications. Electrophoresis. A high voltage apparatus (made at the Dept. of Chem, U.B.C) was used, aqueous sodium borate (O.OJp M.) served as electrolyte. Evaporations. o A l l evaporations were done in vacuo at 1(0 C. Metboxyl group determinations. A l l methoxyl analyses were carried out by tbe method of Viebock and Schwappach as described by Clark (16). Nitrogen analysis by Mrs. A. Aldricb of this department. ( 1 0 ) Figure 1# Flow sheet showing synthesis of 2,4-di-0-methyl-L-erythrose P-ios.yl c h l o n c U dr>y pyrfcUne IV V Na/U, (10a) VI VII VIII E I: L-arabofuranose I I : methyl L-arabofuranoside I I I : methyl 5-p-tosyl-L-arabofuranoside IV: 1,2 isopropylidene-5-tosyl-L-arabofuranose V: 1,2 isopropylidene-5-tosyl - 3 - 0-methyl-L-arabofuranose VI: 1,2 isopropylidene -3-0-methyl-L-arabofuranose VII: 1,2 isopropylidene -3»5-di -0-methyl-L-arabinose VIII: 3 ,5-di-0-metbyl-L-arabinose IX: 2,i|-di-0-methyl-L-erythrose ( i i ) Preparation of methyl L-arabofuranoside (17)» o r lz0 o To commercial L-arabinose (20 g,m.p.159-60 C , L 0 ^ -105 ) absolute methanol (600 ml) was added, containing hydrogen chloride (6 g). The mixture was shaken by a mechanical shaker and the reaction followed by polarimeter a f t e r a l l the L-arabinose was dissolved. When the maximum negative r o t a t i o n was obtained,>the hydrogen chloride was neutralised with lead carbonate. A f t e r f i l t r a t i o n the pr e c i p i t a t e was extracted several times with absolute methanol and the fractio n s united with the f i l t r a t e . The., combined solutions were evaporated i n vacuo and yielded a colourless s i r u p . Theoretical y i e l d 21.8 g, actual y i e l d 19.7 g (90.3$). Paper p a r t i t i o n chromatography was c a r r i e d out on the sirup and a f t e r developing with butanone-water azeotrope and detecting with p-anisidine spray three spots were found having: R J P O.Oli corresponding to L-arabinose Rp 0.09 corresponding to methyl L-arabopyranoside R R A 0.33 corresponding to methyl L-arabofuranoside No attempt was made to separate the methyl L-arabofuranoside from the mixture. (12) Table I PREPARATION OP METHYL L-ARABOFURANOSIDE In minutes oc 120 - 0 . 4 5 0 - 1 5 . 0 0 150 - 1 . 0 0 0 - 3 3 . 3 ° 180 -1.31+0 -Ml. 7° 210 - l . i t 5 o . - 4 8 . 3 ° 2i;0 -1 .532 - 5 1 . 0 0 300 -1 .550 - 5 1 . 7 ° 360 -1 .550 - 5 1 . 7 ° (13) F I G . 3 - P R E P A R A T I O N O F methyl -L- oraboPuranoside S P E C I F I C R O T A T I O N v s . T I M E (11+) Preparation of Methyl 5-P-toluene-sulphonyl-L-arabofuranoside ( l l ) . The mixture of glycosides (19.7 g) was dissolved in dried and d i s t i l l e d pyridine (180 ml) by shaking at room temperature. Recrystallised p-toluene sulphonyl chloride (27 g, m.p.67.5-r, O O 68.5» l i t . m.p. 68-69 C was dissolved in dry pyridine (50 ml) and was added to the above solution. The reaction mixture was l e f t standing for 20 hours at room temperature in the dark. After 20 hours the excess pyridine was d i s t i l l e d off In vacuo and to the yellow slrupy residue chloroform (50 ml) was added. The chloroform solution was washed in a separatory funnel with hydrochloric acid (1 N,30 ml), then with saturated sodium hydrogen carbonate solution (30 ml), f i n a l l y with water (3 times 30 ml). The purified chloroform solution was dried with anhydrous sodium sulphate for 10 hours then f i l t e r e d and evaporated in  vacuo. A yellow sirup methyl 5-P-toluene-sulphonyl-L-arabofurano-side resulted. Theoretical yield 36.2 g, actual yield 16.8 g (1+6.1$). Preparation of 1,2 isopropylidene-5-p-toluene-sulpbonyl-L-arabofuranose. The sirupy methyl 5-P-toluene-sulphonyl-Ir-arabofuranoside (16.8 g) was dissolved In absolute acetone (300 ml) containing hydrogen chloride (3 g), anhydrous calcium sulphate was added and the reaction mixture was l e f t standing for 70 hours at room temperature. After 70 hours the hydrogen chloride was (15) was neutralised with silver carbonate. The precipitate was extracted several times with acetone and the extracts were combined with the f i l t r a t e . The combined solution was evapo-rated in vacuo and to the sirupy residue a small amount of methanol (3 ml) was added. Crystals formed immediately* The crystals were recrystallised from methanol-light petroleum ether mixture. Theoretical yield 18.2 g, actual yield 8.ii g (ii6.2$0. M.p. 129-130°C, l i t . m.p. 129.5-130°C (17), W&-3^ .5° (c 0.7t CHCI3). Preparation of 1,2 isopropylidene-3-0-methyl-5-p-toluenesulphonyl-Ir-arabof uranose • 1,2 Isopropylidene-5-P-toluenesulphonyl-L-arabofuranose (5*6 g) was dissolved i n acetone (30 ml) and methyl iodide (2li.l g) was added. The solution was kept gently boiling and by constant mechanical s t i r r i n g freshly prepared silver oxide (20' g) was added i n ten portions at half hour intervals. The heating and sti r r i n g was continued for 15 more hours. After a reaction time of 20 hours the excess methyl iodide was d i s t i l l e d and the residue extracted several times with boiling chloroform. The extracts were combined and evaporated i n vacuo. After evaporation the sirupy residue was dissolved in a small amount of acetone (3-ml) and light petroleum ether was added (2 ml). The solution was l e f t standing overnight and white needle like crystals formed* Theoretical yield 5.8 g, actual yield 5*5 g (9li.l$). M.p.99-100GC, o l i t . m.p. 99-100 C (17).Methoxyl group determination showed 15.3$ 0CH v theoretical 0CH, content 15.2$. (16) Preparation of 1,2 isopropylidene - 3 - 0-metbyl-L-arabofuranose. To 1,2 isopropylidene - 3 - 0-methyl - 5-p-toluenesulphonyl-L-arabofuranose (5 g) methanol (30 ml) and water (15 ml) were added. The suspension was mechanically s t i r r e d at high speed and i n four portions at half hour i n t e r v a l s sodium amalgam (k%» 60 g) was added. The reaction mixture was kept under constant s t i r r i n g at room temperature u n t i l the suspension cleared (5 hours) and the reduced 1,2 isopropylidene - 3 - 0 -methyl-L-arabofuranose, went into s o l u t i o n . The excess sodium amalgam was destroyed by adding water and the solution was decanted. The mercury was washed several times with water and then with methanol. The washings were united with the solu t i o n and to the combined solution s o l i d carbon dioxide was added to lower the highly alkaline pH. When pH 7«5 was reached the aqueous solution was evaporated i n vacuo. Prom tbe residue tbe o i l y 1,2 isopropylidene -3-0-metbyl-L-arabofuranose was extracted with cold chloroform. The chloroform extract was evaporated i n vacuo and a l i g h t yellow o i l was obtained.. Theoretical y i e l d 2.98 g, actual y i e l d 2.71 g (91.2$). Preparation of 1,2 isopropylidene - 3 . 5-di - 0-metbyl-L-arabinose. 1,2 Isopropylidene-3-0-metbyl-L-arabofuranose (2.7 g) was dissolved i n acetone (25 ml) and methyl iodide (19 g) was added. The solution was kept gently b o i l i n g and s i l v e r oxide (15 g) was added i n ten portions at half hour intervals with constant mechanical s t i r r i n g . A f t e r the l a s t portion of s i l v e r oxide was (17) added, the heating and st i r r i n g were continued for an additional forty hours. After forty-five hours the methy-lation was complete and the excess methyl iodide was d i s t i l l e d . The residue was extracted several times with bot chloroform and the extracts were combined, and evaporated in vacuo. A yellow o i l resulted. Theoretical yie l d 2.90 g, actual yield 2.ii5 g (81i.5$). Metboxyl determination showed 28.7$ OCH^ , theoretical OCH^  content 28.1$. Preparation of 3»5-Di-0-methyl-L-arabinose. 1,2 Isopropylidene-3>5-di-0-methyl-L-arabinose (2.li g) was dissolved in water (10 ml) and sulphuric acid (2 W, 10 ml) was added. The reaction mixture was boiled for three hours and tbe hydrolysis followed by polarimeter. After three hours tbe specific rotation was constant and equal to the reported value of C ^ l p -4-9° (1$) and the reaction was stopped. After cooling the sulphuric acid was neutralised with barium carbonate and the solution f i l t e r e d . The precipitate was washed several times with methanol and the washings combined with the f i l t r a t e , and evaporated in vacuo. A yellow o i l was obtained. Theoretical yield 1.96 g, actual yield l . f 5 g (89.3$)• Methoxyl determination showed 35*4$ OCUy theoretical GCH3 content 3i+.8$. Paper partition chromatography was carried out using as developer butanone-water azeotrope and p-anisidine as spray reagent. After developing and spraying two spots were detected with Rp 0.86 corresponding to 2,3,5-tri-O-methyl-L-arabinose and Rp C6I4 corresponding to 3»5-di-0-metbyl-L-arabinose. (18) The o i l was investigated by electrophoresis using 1000 volt and 1+0 mA current for forty minutes. After spraying with p-anisldine, two spots were detected. A migrating component with M Q value 0.74 correspong to 3.5-di-O-methyl-^-arabinose and a non-migrating component with value of 0 . 0 corresponding to 2,3.5-trI-O-methyl-L-arabinose. Separation of 3.5-di-O-methyl-L-arabinose from 2 , 3 > 5-tri - 0 -methyl-L-arabinose. A mixture of 3»5-di-0-methyl-L-arabinose and 2 , 3 . 5-tri-Q-methyl-L-arabinose ( 0 . 6 0 0 g) in the minimum amount of butanone-water azeotrope. Fractions were collected at seven minute intervals at a flow rate of forty ml per hour. After 150 fractions were collected no further carbohydrate material could be detected in the eluate by application of the Molisch test and fractionation was terminated. The fractions were Investigated systematically by paper chromatography. Two fractions were found to be chromatographically pure, tubes 8-35 inclusive contained the pure 2 ,3 ,5-tri -0-methyl-L-arabinose and tubes 55-120 inclusive contained the pure 3,5-tri-O-methyl-L-arabinose while an overlap of the two components was found in tubes 36-54 inclusive. The separation yielded 3,5-dI-O-methyl-L-arabinose (0.334 g) and 2 , 3 , 5 - t r i -O-methyl-L-arabinose (0.130 g) in chromatographically pure state. (19) Table II SEPARATION OP METHYLATED L-ARABINOSE FRACTIONS ON CELLULOSE-HYDROCELLULOSE COLUMN Classification Tube number Weight mg 2,3,5-tri-O-methyl-L- 8-35 arabinose 3,5-di-O-methyl-L- 55-120 arabinose mixture 36-54 total recovery 130 334 0.81i Q .6ii iiO 0.6ij. & 0 . 6 i i 50li (810.) (20) Characterization of 3»5-di-0-methyl-L-arabinose. 1. Preparation of 3>5-di-0-methyl-L-arabonolactone 3,5-Di-O-methyl-L-arabinose (113 mg) was dissolved i n water (2 ml) and excess bromine was added. The reaction mixture was kept in the dark at room temperature for li8 hours. After li8 hours the excess bromine was removed by means of aeration. When the solution turned colourless* excess sil v e r carbonate wa3 added to remove a l l bromide ion present. After f i l t r a t i o n the precipitate was. washed several times with methanol and the washings combined with the f i l t r a t e . This solution was saturated with hydrogen sulphide to remove a l l silv e r ion present. The reaction mixture was evaporated i n  vacao and the 3»5-di-0-methyl-L-arabonolactone was extracted with hot chloroform.The extract was evaporated in vacuo and a yellow sirup resulted. By treatment with small amounts of ethyl acetate and light petroleum ether long needle like crystals formed from the sirup. The crystals were recrystallized from a mixture of ethyl acetate and light petroleum ether.Theoretical yield 122 mg, actual yield 86 mg ( 7 0 . 6 $ ) . M.p. 75-76°C,lit. m.p. 75°C (17),78°C (19).M * ° - 8 5 ° (c 0.5,water). 2. Preparation of 3>5-di-0-methyl-L-arabonamide 3»5-Di-0-methyl-L-arabonolactone (ii8 mg) was dissolved in methanol (15 ml) containing ammonia and was kept at -5°C for 2li hours. After 2li hours the solution was evaporated i n vacuo. A yellow sirup was obtained from which upon addition of ethyl (21) acetate and light petroleum ether crystals formed. The crystals were recrystalllzed from ethyl acetate-light petroleum ether. Theoretical yield 1+7 mg» actual yield 1+5 mg ( 9 5 . 7 $ ) . M.p. 132-132.5°C, l i t . m.p. 132.5°C ( 1 7 ) . r i 2 0 o LocJp 10 (c 0 . 5 , water). Preparation of 2,l+-di-0-methyl-L-erythrose. 1. Pilot scale periodate oxidation of 3.5-di-O-methyl-L-arabinose Aqueous solutions containing 3.5-di-O-metbyl-L-arabinose (20 mg,0.112 mmole) and sodium metaperiodate (1+8 mg,0.221+ mmole) were mixed and immediately made up to 20 ml with d i s t i l l e d water o and maintained at 20 C in the dark. Aliquots (1 ml) were withdrawn at intervals and excess sodium hydrogen carbonate and a measured excess of 0 . 1 N sodium arsenite solution were added and tbe solutions were allowed to stand for 15 minutes to allow complete reduction of the excess sodium metaperiodate. Consumption of periodate was then determined by back titr a t i o n with 0 . 1 N iodine solution. When the periodate consumption had reached a constant value the solution was neutralized with barium carbonate and the precipitate was extracted several times with cold chloroform. The extracts and the f i l t r a t e were combined and evaporated in vacuo. A yellow o i l y residue was obtained. Paper partition chromatography was carried out. As developer butanone-water azeotrope was used and the chromato-gram was sprayed with p-anisidine. One spot was detected witb Rp value 0 . 7 1 . The periodate oxidation was followed by polari-metry, and optical rotations were taken at tbe time when aliquots (22) were withdrawn from the solution. The periodate oxidation was repeated by using a fourfold excess of sodium metaperiodate. The results were tabulated and graphically evaluated and are shown in Tables III and IV. (23) Table III PILOT SCALE PERIODATE OXIDATION OF 3,5-DI-O-METHYL-L-ARABINOSE Time Volume Net volume IO[^~ f minutes of Ig.ml of I 2 ml uptake/mole Lcc 0 3.900 - 4 7 . 0 ° 30. 3 .970 0.070 0.618 - 3 2 . 0 ° 60. 4 .000 0.100 0.883 0 - 1 0 . 0 90 4.010 0.110 0.972 0 - 6 .2 120 4.010 0.110 0.972 0 - 6 . 0 150 4.010 0.110 0.972 0 - 6 . 0 180 4.010 0.110 0.972 - 6.0° 900 4.013 0.113 1.003 - 6 .0° 2 Mole excess of sodium periodate was used F I G . 3- P E R I O D A T E O X I D A T I O N O F 3,5 — di — O — methyl — L ~ arabinose I O H U P T A K E vs. T I M E (25) Table IV PILOT SCALE PERIODATE OXIDATION OF 3,5-DI-O-METHYL-L-ARABINOSE Time minutes Volume of I 2 ml Net volume of I 2 ml uptake/mole 0 ii.360 -48.0° 30 4-450 0.090 0.795 -30.0° 60 4.J+60 0.100 0.883 0 -10.0 90 4.480 0.120 1.000 0 - 6.0 120 4.480 0.120 1.000 0 -6.0 150 4.480 0.120 1.000 - 6.0° 900 4-485 0.125 1.040 - 6.0° 4 mole excess of sodium periodate used. (26) 1.00-080--U P T A K E 0.G0- -0.20--4 F O L D E X C E S S O F 1 0 4 T E M P E R A T U R E S O # C 30 GO % T I M E IN M I N U T E S it .0 F I G . 4 - P E R I O D A T E O X I D A T I O N O F 3,5 - di - O - methyl - U ~ arabinose I O 4 U P T A K E V S " T ! M E (27) 2 . Large scale periodate oxidation of 3.5-di-Q-methyl-L-arabinose Chromatographically pure 3,5»-di-0-methyl-L-arabinose ( 2 . 0 g, 0.0112M) was dissolved in d i s t i l l e d water (100 ml) and a solution of 0.5 N sodium metaperiodate (99 .6 ml, O.OI4I48 M) was added. The reaction mixture was kept at room temperature in the dark for 2 hours and then barium carbonate was added to precipitate periodate and iodate and to neutralize formic acid formed. The reaction mixture was f i l t e r e d and the precipitate extracted several times with cold chloroform. The extracts and the f i l t r a t e were combined and evaporated i n vacuo. A yellow o i l was obtained. Paper partition chromatography was carried out dsing as developer 1. butanone-water azeotrope. 2 . butan~ 1-ol-ethanol-water-ammonia (i+ilrl+Jl:) • The chromatograms were sprayed with p-anisidine and two spots were detected on each cbromatogram. When developer 1. was used spots with Rj, value of 0.73 corresponding to the oxidised product and value 0 . 0 corresponding to impurities were found. In developer 2 . spots with Rp value 0.70 corresponding to the oxidised product and Rp value 0 . 0 corresponding to impurities were found.;' Theoretical yield 1.66 g, actual yield l.i}3 g (86.0$).[cc]*°-3o.2°( c 1 .3 , water). Purification of 2,4-di-O-methyl-L^erythrose by column elution chromatography. 1. Pilot scale purification In a minimum amount of benzene, impure 2,2j-di-0-methyl-L-erythrose (50 mg) was placed on a F l o r i s i l column (10.5 x 1.1+ cm) which was previously wetted with petroleum ether (b.p 60-110°C). (28) The column was developed with 50 ml fractions of petroleum ether (b.p. 60-110°C), petroleum ether-benzene ( i p l * b.p. of benzene 79-80°C), petroleum ether-benzene (1:1), benzene, benzene-chloroform b.p. of chloroform 59-6l°C), benzene-chloroform (1:1) and chloroform. Fractions, each containing 25 ml were collected and individually evaporated in vacuo. The individual fractions were chromatographically investigated by means of paper partition chromatography. Chromatographically pure 2,li-di-0-methyl-L-erythrose was found in fractions 8-10 inclusive. The results are given i n Table V. (29) Table V PILOT SCALE PURIFICATION OF 2,4-DI-fi-METHYL-L-ERYTHROSE BY ELUTION CHROMATOGRAPHY ON A FLORISIL COLUMN Fraction Volume of Composition of developer Recovery fraction in ml in mg 1 25 petroleum ether O.G 2 25 petroleum ether 0.0 3 25 petroleum ether-benzene 0.0 (4:1) 4 25 petroleum ether-benzene 0.0 5 25 petroleum ether-benzene 0.0 (1:1) 6 25 petroleum ether-benzene 0.0 (1:1) 7 25 benzene 0.0 8 25 benzene 7.1 9 25 benzene-chloroform (4:1) 14•2 10 25 benzene-chloroform (4:1) 22.5 11 25 benzene-chloroform (1:1) 2.2 12 25 benzene-chloroform (1:1) 0.0 13 25 chloroform 0.0 Total recovery 46.0 (92.0$) (30) 2 . Large scale p u r i f i c a t i o n of 2,ii-di-0j-methyl-L-erythrose Impure 2,ii-di - 0-methyl-L-erythrose (1 .50 g) was dissolved i n minimum amount of benzene and was placed on a F l o r i s i l column (28 x 3*3 cm) which was previously wetted with petroleum ether. The column was developed with 200 ml f r a c t i o n s of petroleum ether, petroleum ether-benzene ( i p l ) petroleum ether-benzene ( 1 : 1 ) , benzene, benzene-chloroform ( i i : l ) , benzene-chloroform (1:1) and chloroform.* Fractions, each containing 100 ml were c o l l e c t e d and i n d i v i d u a l l y evaporated i n vacuo. The i n d i v i d u a l f r a c t i o n s were investigated by means of paper p a r t i t i o n chromatography. Chromatographycally pure 2 ,4-di -0-methyl-L-erythrose was found i n f r a c t i o n s 6-10 i n c l u s i v e , see table VI. These f r a c t i o n s were combined and checked f o r methoxyl content. Theoretical methoxyl content i l l . 9 $ , actual methoxyl content i l l . 8 $ . CocL - 3 2 . 7 ° (c 0 . 5 , water). The chromatographically pure 2,l|-di-0-methyl-L-erythrose gave p o s i t i v e S c h i f f and Benedict t e s t s . • B B o i l i n g p o i n t s o f d e v e l o p i n g advents were i d e n t i c a l . t o t h o s e , w h i c h were used i n t h e p i l o t s c a l e p u r i f i c a t i o n . ( 3 D Table VI LARGE SCALE PURIFICATION OF 2, li-DI-0-METHYL-^-ERYTHROSE BY ELUTION CHROMATOGRAPHY ON A FLORISIL COLUMN Fraction .. Volume of Composition of developer Recovery in mg fraction in ml 1 100 petroleum ether 0.0 2 100 petroleum ether 0.0 3 100 petroleum ether-benzene 0.0 (4:1) 4 100 petroleum ether-benzene 0.0 i k t l ) 5 100 petroleum ether-benzene 0.0 (1:1) 6 100 petroleum ether-benzene 110.0 (1:1) 7 100 benzene 424.5 8 f 100 benzene 676.4 9 100 benzene-chloroform 63.0 (4:D 10 100 benz one-c hloroform 21.2 (4:D 5.0 11 100 benzene-chloroform (1:1) 12 100 benzene-chloroform 0.0 (1:1) 13 100 chloroform 0.0 Total recovery i n mg 1300.1 ( 8 6 . 6 %) Optical rotation of fractions 6-10 inclusive was - 3 2 . 7 ° (32) Preparation of 2 , 4-di-O-methyl-L-erythritol. Chromatographically pure 2 ,4-di-O-methyl-L-erythrose (200 mg) was dissolved i n water (20 ml) and sodium borohydride (600 mg) was added. The reaction mixture was kept at room temperature f o r twenty hours. After twenty hours methanol containing hydrogen chloride was added and the pr e c i p i t a t e d sodium chloride was f i l t e r e d . The p r e c i p i t a t e was washed several times with absolute methanol and the washings were united with the f i l t r a t e , and evaporated i n vacuo. To the residue methanol was added, the evaporation was continued u n t i l no more boric a c i d was present i n the residue. The o i l y residue was kept under vacuum u n t i l constant weight was obtained. Theoretical y i e l d 202 mg, actual y i e l d 160 mg ( 7 8 . 9 $ ) , Preparation of 1 , 3-di-p-nitrobenzoyl, 2 ,4-di-0-methyl-L-e r y t h r i t o l . 2,4 Di-O-methyl-L-erythritol (150 mg) was dissolved In dry pyridine (20 ml) and p-nitrobenzoyl chloride (1+00 mg) i n pyridine (20 ml) was added. The reaction mixture was kept at 75° C f o r three hours and then a few drops of water and a saturated solution of sodium hydrogen carbonate (25 ml) were added. The reaction product, 1 , 3-di-p-nitrobenzoyl, 2 ,4-di-O-methyl-L-erythritol p r e c i p i t a t e d and was i s o l a t e d by f i l t r a t i o n . The p r e c i p i t a t e was washed several times with water and then i t was r e c r y s t a l l i s e d from methanol-water and dried (33) i n vacuo. T h e o r e t i c a l y i e l d l+i+8 mg, a c t u a l y i e l d 376 mg (83.9$). M.p. 217-219°C. M e t h o x y l d e t e r m i n a t i o n showed 13.0$ OCH-j, t h e o r e t i c a l OCH^  c o n t e n t 13.8$. T h e o r e t i c a l n i t r o g e n c o n t e n t 6.3 $ f o u n d 7»8$. These r e s u l t s show t h a t tbe d e r i v a t i v e i s a m i x t u r e , though the m e l t i n g p o i n t was s h a r p . P r e p a r a t i o n o f 2,1+ d i n i t r o p h e n y l h y d r a z o n e o f 2,i4-di-0-methyl-£-e r y t b r o s e . 2,l+-Di - 0-metbyl-L-erythrose (100 mg) was d i s s o l v e d i n e t h a n o l (10 ml) and 2,1+ d i n i t r o p h e n y l h y d r a z i n e (200 mg) I n e t h a n o l (10 ml) was added. The r e a c t i o n m i x t u r e was k e p t a t room tem p e r a t u r e o v e r n i g h t . D u r i n g t b i s time a c r y s t a l l i n e p r o d u c t s e p a r a t e d w h i c h was f i l t e r e d and washed w i t h c o l d m e t h a n o l . A f t e r d r y i n g i n vacuo tbe c r y s t a l s were d i s s o l v e d i n c h l o r o f o r m (2 ml) and were put on a F l o r i s i l column (10 .5 x 1.1+ cm), w h i c h was prewasbed w i t h c h l o r o f o r m . The column was e l u t e d w i t h c h l o r o f o r m and two bands were o b t a i n e d by the e l u t i o n . F r a c t i o n s were c o l l e c t e d and were e v a p o r a t e d i n vacuo. Each f r a c t i o n was checked by m e l t i n g p o i n t d e t e r m i n a t i o n and the i d e n t i c a l f r a c t i o n s were u n i t e d . Two main f r a c t i o n s were f o u n d , one whicb appeared to c o n t a i n 2,1+ d i n i t r o p h e n y l h y d r a z i n e the o t h e r f r a c t i o n c o n t a i n e d l i g h t y e l l o w c r y s t a l s w h i c h were assumed to be the 2,1+ d i n i t r o p h e n y l h y d r a z o n e o f 2,l+-di - 0-metbyl-L - e r y t h r o s e . T h e o r e t i c a l y i e l d 221 mg, a c t u a l y i e l d 168 mg (76 $ ) . M.p.ll+8-l50°C. M e t h o x y l d e t e r m i n a t i o n showed 18.9$ OCH^ , t h e o r e t i c a l OCH^  c o n t e n t 18 .1$. T h e o r e t i c a l n i t r o g e n c o n t e n t 17 .1$, f o u n d 18 .1$. (34) Tbe higber nitrogen content and tbe lower methoxy content indicates tbat some of the methoxy groups have been replaced by 2,1+ dinitrophenyl hydrazone. (35) BIBLIOGRAPHY 1. E. Fischer and H. Tafel, Ber., 20, 1090 (1887). 2. A. Wohl, Ber., 26, 7U3 (1893). 3. 0. Ruff and H. Meusser, Ber., £2, 3672 (1899). 1+. 0. Ruff, H. Meusser and K. Kohn, Ber., ^ k, 1362 (1901). 5. L. Maquenne, Compt. rend. 130, llji02 (1900). 6. G. Bertrand, Compt. rend. 130» 1330 (1900). 7. W.R. Fenton and R.F. Jackson, J . Chem. Soc. 1899, 1. 8. R.S. Morrell, J . Chem. Soc. 1902, 674. 9. R.F. Jackson, J . Chem. Soc. 1900, 130. 10. R.A. Weerman, Rec. trav. chim. J37_, 16 (1918). 11. R.C. Hockett, J . Am. Chem. Soc, 2260 (1935). 12. P. Fleury, Inst, intern, chim. Solvay, Counseil Chim. Brussel, 1950. 13. I.J. Goldstein, H. Sorger-Domenig and F. Smith, J. Am. Soc, 81, 1+1+1+ (1959). 11+. W.B. Baker and W.N. Haworth, J. Chem. Soc 1925, 365. 15* U.S. Dept. of Commerce, National Bureau of Standards. Polarimetry, Saccharimetry and the Sugars. U.S. Goverment Printing Office, Washington D.C, 191+2. 16. E.P. Clark, Semimlcro Quantitative Organic Analysis, Academic Press, Inc., New York, N.Y., 191+3* 17. J.K.N. Jones, J. Chem. Soc. 191+7» 1062. 18. T. Purdie and J.C. Irvine, J. Chem. Soc. 1905» 1022. 19. E.L. Hirst, J.K.N. Jones and K.T. Williams, J. Chem. Soc 191+7, 1062. 

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