J. Biol. Chem.-carb Profiling 2

8/2/2019 J. Biol. Chem.-carb Profiling 2

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CARBOHYDRATE CHARACTERIZATION*I. TE E OXIDATION OF ALDOSE S BY HYPOIODITE IN METHANOLII. TH E IDENTIFICATION OF SEV EN ALDO-MONOSACCHARIDES

AS BENZIMIDAZOLE DER IVATIV ESBY STANFORD MOOREt AND KARL PAUL LINK

(From the Department of Biochem istry, College of Agriculture, Universityof Wisco nsin, Madison)

(Received for publication, November 15, 1939)In the course of work in this laboratory on several benzimida-zoles of the carbohydrate series n 1936, observation was made oftheir potential value as derivatives for carbohydrate identification.The general chemical and physical properties of the aldo-benzim-idazoles appeared to be remarkably superior, from the characteriza-

tion standpoint, to those of hydrazones and osazones. Thefollowing procedure for aldose identification has been developedto utilize the favorable properties inherent in this type of carbo-hydrate derivative.The general developmental investigation of methods for thepreparation of aldo-benzimidazoles is to be treated separately inorder to present concisely in this publication the working proce-dures for the identification of seven aldo-monosaccharides. Pre-liminary work has shown, in brief, that the direct oxidativecondensation of aldoses with o-phenylenediamine, as first studiedin 1887 by Griess and Harrow (I), even with the introduction ofmore recent approaches, fails to give satisfactory yields. The

* Published with the approval of the Director of the Wisconsin Agri-cultural Experiment Station.Abstracted from a thesis submitted by Stanford Moore to the GraduateFaculty of the University of Wisconsin in partial fulfilment of the require-ments for the degree of Doctor of Philosophy, June, 1938; Summaries ofdoctoral dissertations, Madison, 3, 191 (1938).t Stanford Moore is indebted to the Wisconsin Alumni Research Founda-tion for a fellowship in 1935-36 and to the Graduate Research Fund forassistantships for 1936-39.

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294 Carbohydrate Characterizationcondensation of carboxylic acids with o-phenylenediamine, how-ever, offers a path which proves practical. It is from t,his approachthat the following methods for the preparation of the carbohydratederivatives in good yields have been realized.

The procedure is based on the combination of (a) a potassiumhypoiodite-methanol oxidation, and (b) the use of o-phenylenedi-amine as reagent for the characterization of the resulting aldonicacids as benzimidazole derivatives according to the followingequations.

HCL0

(HAOH).--) CH

POH(CHOH)

C!H~OHAldose Aldonic acid o-Phenylenediamine

---+ CH,OH( CHOH),C + 2H&

Aldo-benzimidazoleI. The Oxidation of Aldoses by Hypoiodite in Methanol

For the efficient and rapid preparation of the aldonic acid saltsto serve as intermediates in the formation of benzimidazoles, aprocedure in which potassium hypoiodite in methanol solution isemployed has been developed. In identification work on syrupsfrom natural products we have found these conditions to providethe most convenient laboratory method for obtaining the desiredoxidation. However, the products of the bromine-barium ben-zoate procedure given by Hudson and Isbell (2), the bariumhypoiodite oxidation of Goebel (3), or, in general, any oxidationwhich yields aldonic acids may be identified as benzimidazolederivatives.The potassium hypoiodite-methanol procedure has been de-

1 The presence of inorganic salts and organic impurities in aqueoussyrups from natural sources generally renders it impossible to take ad-vantage of the efficient electrolytic method for pure aldoaes developed byIsbell and Frush (4).


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S. Moore and K. P. Linkveloped as a simplification of existing methods by the eliminationof several operations inherent in other techniques for carrying outthe oxidation. The preparation of the potassium salt of gluconicacid in a single operation has been realized by introducing meth-anol as the reaction solvent for the hypoiodite oxidation, aldose+ IZ + 3KOH + 2KI + K aldonate + 2Hz0. The glucose,iodine, potassium hydroxide, and potassium iodide are al l soluble.As the reaction proceeds, the desired potassium gluconate precipi-tates in the form of anhydrous needles (yield 92 per cent, t ime 50minutes). Under the same conditions arabinose and galactosegive good yields of the corresponding potassium aldonates. How-ever, when mannose, xylose, rhamnose, or lyxose is oxidized, littleor no precipitate is formed because of the appreciable solubilityof the potassium salts in the reaction solvent (methanol withabout 2 per cent water). Practically complete precipitation ofthe aldonic acids which thus remain in solution can be accom-plished by the addition of a solution of barium iodide in methanol.The barium salts obtained are amorphous, basic, and containiodide. As intermediates for benzimidazoles, these crude basicsalts, rapidly obtained in almost quantitative yields, have provedto be equally as satisfactory as pure barium or calcium salts pre-pared by longer procedures.

The aldonic acids formed in the oxidation are therefore, inpractice, isolated in two separate fractions. The insoluble po-tassium salts are obtained first. Addition of barium iodide to thefiltrate from the potassium salts yields barium salts of the remain-ing aldonic acids. The partitioning of a 2 gm. sample of a givenaldo-hexose (1.7 gm. of pentose) into potassium and barium saltsis shown in Table I. It will be noted that the division is relativelysharp between glucose, galactose, and arabinose, and the sugarswhich precipitate predominantly in the barium salt fraction. Inthe identification of the aldoses as benzimidazole derivatives, useis made of this preliminary fractionation accomplished in theoxidation step.

In order to determine the optimum experimental conditions forthe oxidation, a series of trials on glucose was made involving thevariables (a) aldose concentration, (b) ratio of iodine to aldose,(c) rate of potassium hydroxide addition, and (d) temperature. Aglucose concentration of 1 to 2 per cent was adopted, together


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296 Carbohydrate Characterizationwith the use of twice the theoretical amount of iodine and alkali,dropwise addition of the alkali over a period of about 20 minutes,and a temperature of 40.

The hypoiodite oxidation of aldose to aldonic acid is strictlyquantitative only in solutions of lower sugar concentration thanwas used in this case. The increased carbohydrate concentrationresults in a small sacrifice in the accuracy of oxidation, as alsoTABLE I

Aldo-Monosaccharides

C&OH KOZBaZr

K Salt Fraction Ba Salt FractionFractional Distribution of Aldonic Acids *

Aldose I Weight ofSsmph

Glucose. .......Galactose .......Arabinose ......Mannose .......Xylose .........Lyxose .........Rhamnose + Hz0

am .2.02.01.72.01.71.72.0

ICeaham .

2.42.21.90.90.20.00.0

per cm:92858330

8

am .0.40.80.52.53.43.73.9

* The amount of water in the starting syrup, the presence of impurities,and the temperature of the solution at the time of filtration affect theexact extent to which the potaskium salts crystallize.

reported by Goebel (3) for the barium hypoiodite method. It hasnot been possible to detect any interference in the benzimidazolemethod from the trace of overoxidized aldoses present in the po-tassium and barium salt fractions. It has been readily possible,however, by means of the benzimidazole procedure to detect thesmall amount of overoxidation by hypoiodite in the case of aketose (fructose), as will be noted in the next section.Acetone-free methanol is preferable as solvent but the content


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S. Moore and K. P. Link 297of this impurity in commercial absolute methanol is frequentlylow enough (0.1 per cent) to cause no serious interference. Ablank oxidation (no aldose present) on the given experimentalscale should cause little or no precipitation. Under normal condi-tions a slight precipitate may form (50 to 70 mg.), a part of whichis potassium or barium carbonate. Methanol may also containreducing impurities which consume hypoiodite but give no pre-cipitable reaction products. The most practical control on thereagents is an oxidation on crystalline glucose which should givea 90 per cent yield of potassium gluconate.The operations involved in this oxidation can be convenientlyperformed on a smaller scale than given here for 2 gm. samples.On small quantities the reaction itself can be run in a centrifugetube to minimize transfer losses.

EXPERIMENTALSample for Oxidation-In testing the procedure on known

crystalline sugars, prepare a concentrated sugar solution by dis-solving 2 gm. of aldo-hexose (1.7 gm. of pentose)* in 3 cc. of water.In the oxidation of samples from natural products, concentratethe carbohydrate fraction to a syrup and make a Willstiitter-Schudel titration (5) on a few tenths of a gm. of this syrup.Weigh out a sample equivalent to 2 gm. of aldo-hexose. If thesyrup is thick, add 1 or 2 cc. of water to the sample with warminguntil it flows readily. As an alternate procedure with dilutecarbohydrate solutions, a volume of solution equivalent to 2 gm.of aldo-hexose can be taken and concentrated directly underreduced pressure to about 4 cc.

Reagents-Absolute methyl alcohol (acetone-free).Solution of potassium hydroxide in methanol (40 gm. per liter).Resublimed iodine.Apparatus-A 3-necked flask (500 cc.), equipped with a stirrer,thermometer, and dropping funnel, is half immersed in a waterbath warmed by steam (hood). For filtration, a Buchner funnelis used in the center neck.Oxidation-Add 5.7 gm. of iodine and 80 cc. of methanol tothe flask and stir for a few minutes. Warm the water bath to2 The method of oxidation is also applicable to uranic acids and disaccharides.


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298 Carbohydrate Characterizationbring the methanol solution to about 40 and apply no more heatduring the oxidation.Warm the sample to be oxidized (aldo-hexose content of 2 gm.,volume of about 4 cc.) on a steam bath and dissolve in 25 cc. ofmethanol. Filter the solution (if not perfectly clear) by suctionthrough asbestos directly into the iodine solution in the flask.Use decolorieing carbon if the sugar solution is dark. Rinse ontothe filter with two 10 cc. portions of methanol. With pure aldosesthe addition of methanol causes no precipitation and, the solutionis poured directly into the flask without filtration.As soon as the sugar has been added to the iodine and methanol,replace the stirrer and start the dropwise addition (over a periodof 10 to 15 minutes) of 65 cc. of 4 per cent potassium hydroxide

TABLE IIComposition of Crude Potassium Salts

M.p. I Il, PotassiumCalculated Found*c. degrua per cent pa centI-Arabonic.. 211 (with decom position) +4.5 19.1 20.3d-Ga lactonic .. 164 +4.3 16.7 16.9d-Glu conic.. 180 +11.0 16.7 16.8

solution. Stir for 10 minutes and add dropwise 50 cc. more ofpotassium hydroxide solution. The final color of the reactionmixture should be a light straw-yellow, indicating removal ofnearly all of the free iodine. A few more cc. of alkali may beadded if the iodine color is still prominent. Stir for a final 10minutes.Potassium Salt.-Remove the flask from the water bath andcool to room temperature before filtering the potassium salts

onto a thick paper on a Buchner funnel under gentle suction.Wash twice with methanol and once with ether. Air-dry. Inthe characterization procedure, in order to eliminate the possibleloss of a component acid in the process of recrystallization, thepotassium salt fraction is used directly, without purification forbenzimidazole preparation. The constants shown in Table II


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S. Moore and K. P. Link 299indicate the composition of the crude salts from arabinose,galactose, and glucose. Potassium carbonate is the principalinorganic impurity.s

Barium Salts-Return the filtrate from the potassium saltprecipitation to the reaction flask and add a solution of 5 gm. ofbarium iodide (dihydrate) in 25 cc. of methanol dropwise withstirring.4 Centrifuge the precipitated basic barium salts, washtwice with CHIOH on the centrifuge, suspend in ether, andtransfer to a Buchner funnel under slight suction. Airdry forabout 10 minutes and remove the final traces of solvents in avacuum desiccator. If left in the air for too long a period thesalts occasionally become hygroscopic. Analysis of the crude

TABLE IIIComposition of Crude Barium Salts

Crude barium ealta I Barium

d-lyxonic. ........................d-Mannonic .......................I-Rhamnonic. ......................d-Xylonic .........................

parant37.935.739.339.6

mdss0.900.951.020.95

salts (Table III) shows them to contain approximately 1 moleculeof barium per molecule of aldose represented by the salt (calcu-lated in conjunction with the data on fractional distribution).

3 For preparatory purposes the pure potassium aldonates may be ob-tained by recrystallization of the unpurified salts from water (a minimum)and methanol or ethanol.* I f no appreciable potassium salt precipitation occurs, filtration isomitted and the barium iodide is added directly to the oxidized solution.Also the barium iodide can be added before the oxidation if no potassiumsalt fraction is desired, as is the case when the oxidation is being used forpreparative purposes on a known aldose such as mannose. With thebarium added at the start, 2 gm. of mannose yield 4.1 gm. of barium salt.The barium precipitates obtained in this manner are fairly granular andfilter faster than the somewhat gelatinous salts formed by the addition ofbarium iodide at the end of the reaction. The salts obtained by bothmethods show essentially the same composition.


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300 Carbohydrate CharacterizationII. The Identification of Seven Aldo-Monosaccharides As

Benzimidazole DerivativesThe 2-(aldo-polyhydroxyalkyl)benaimidazole derivatives6 arestable white crystalline solids, with sharp melting points andgeneral chemical properties which make them specially suitablefor identification work.0 The fundamental operations involvedin their preparation and isolation are simple and rapid. They

can be prepared in 50 to 80 per cent yields by heating the aldonicacid with o-phenylenediamine, preferably in the presence of acidcatalysts.7 The condensation is best carried out in most casesat an oil bath temperature of 135 with the use of hydrochloricand phosphoric acids. (Xylonic acid requires a higher tempera-ture of 180 in the presence of zinc chloride.) In the isolation ofthe derivatives, advantage is taken of their slight solubility inwater and dilute ammonium hydroxide, high solubility in aqueoushydrochloric acid, and insolubility in acetone and ether. Aqueousacid is also conveniently employed as the solvent in determiningthe specific rotations.In contrast to the behavior of many of the derivatives of carbo-hydrates, the benzimidazoles crystallize readily. Individualderivatives may be separated by fractional crystallization.Practical examples are the rapid identification of glucose andarabinose in the same sample, or mannose, galactose, and glucoseoccurring simultaneously.Two properties to be desired in an organic characterization

r The imidazole structure for the carbohydrate derivatives was estab-lished by Hinsberg and Funcke (6) in 1893 and confirmed by Ohle (7) in1934.6 It is interesting to note that Schilling (8) in 1901 wrote of the possiblesuperiority of carbohydrate benzimidazoles over osazones as characteriza-tion derivatives but he was unable to utilize the idea because of the lowyields.

7 In the preparation of a series of aldo-benzimidazoles for studies inthe resolution of racemic acids, Haskins and Hudson (9) in 1939, completelyindependently of our work, have employed the aldonic acid condensation(100 both with and without addition of HCl) to give good yields in a num-ber of cases. Their application of the benzimidazole from D-gluco-D-gulo-heptonolactone to the practical preparation of L-tartaric acidmakes use of the crystallization properties of the salts of this type of hetero-cyclic base.


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S. Moore and K. P. Link 301derivative are (1) a means for quantitative isolation and (2) thepossibility of secondary derivative formation. The aldo-benzimidazole possesses both of these properties. The fact thatbenzimidazoles in general can be precipitated as copper saltsfrom aqueous solution facilitates the isolation of the small amountsof derivatives which may remain in solution after direct crystalliza-tion. The benzimidazole is regenerated from the copper salt bythe use of hydrogen sulfide. The potential value of this propertyof the benzimidazole nucleus in identification work is evident. Itmeans, for example, that with a sample containing a small amount

TABLE IVBenzimidazole Derivatives of Alob-Monosacch arides

BenzimidamleCarbohydrate Hydrochloride Picrate m.p.

M.P. bl:: Imp.oc. degreea C. C.

l-Arabinose. 235t +49.2 230 158d-Galactose. 245t +43.3 202-204 217td-Glucose. . . 215 +9.6 180 m3 td-Lyxose 189 -12.8 191 95-99d-Mannose . . . . 227t -22.0 101-150 205tZ-Rhamnose . . 207 +27.4 173-175 168d-Xylose . . . . . 224 +64.8 200-202 191

l All melting points are uncorrected. The rotations were taken in 5per cent citric acid solution with c = 2 (approximately).

t With decomposition.

of glucose in the presence of a relatively large quantity of ara-binose, the gluco-benzimidazole in the mother liquors will be re-covered by copper salt precipitation and will not escape detection.Secondary derivatives of the benzimidazole nucleus can beprepared by making crystalline acid salts which provide third andfourth constants for identification. The hydrochlorides andpicrates are readily obtained and in general have characteristicmelting points which are lower than those of the parent base.When three or four of the constants have been taken on anyaldo-benzimidazole in Table IV, the identity of the derivative isknown beyond any question of doubt. This accuracy fills a needin carbohydrate chemistry which an osazone, or any other single


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Carbohydrate Characterizationderivative, does not satisfy. The two constants (m.p. and[a] D ) which can be taken on a hydrazone or osazone are limitedin accuracy, as those who have worked with them well know, byanomalous melting behavior and by mutarotation of the difficultlysoluble derivatives in the colored solutions which they give inpyridine and alcohol. The aldo-benzimidazoles are free fromthese disadvantages. In the case of the benzimidazole derivativesit is not necessary to resort to description of crystalline habits asa means of identification.The chemical properties of the benzimidazole nucleus whichcontribute to its value as a tool for characterization are bestmade apparent by the experimental detail of the identificationprocedure.

Aldo-monosaccharides*K s!lts Oxidation ( IS + KC)Ija,,

o-Phenylene- HCI-HsPO, HCl-HaPO,diamine 135 o-Phenylene-diamine 135I II I

Benzimidazoles Benzimtdazolesfrom glucose,arabinose, from mannose,galactose rhamnose,lyxoseHCl-ZnClz130IBenzimidazolefrom xylose

*This schematic presentation of the procedure is to be considered inconnection with Table I of the potassium hypoiodite-methanol oxidationmethod, which gives the partitioning of aldonic acids between the two saltfractions. Not indicated in the diagram, for instance, is the fact that while90 per cent of the bensimidazole from a pure glucose sample will appear inthe potassium salt fraction, the residual 10 per cent is also detectable in thebarium salt fraction.

EXPERIMENTALCondensation with G&conic Acid-A specific example of aldo-benzimidazole preparation is of illustrative value. Calciumgluconate is a convenient starting product. To 2 gm. ofCa gluconate.H20 (0.009 mole of gluconic acid) in a test-tube,add 1.1 gm. (0.01 mole) of o-phenylenediamine, about 4 cc. ofwater, 1 cc. of ethanol, and 1.7 cc. (0.02 mole) of concentrated


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S. Moore and K. P. Link 303hydrochloric acid.8 Warm until in solution, add a boiling chip,and heat for 2 hours on an oil bath kept at 135 f 5. The tubeshould be immersed to about the level of the contained solution.During the 1st hour water boils off to leave a thick syrup in whichbubbling gradually ceases during the 2nd hour. Remove thetube from the bath and while still warm dissolve the syrup inabout 10 cc. of water, add carbon, filter through asbestos, dilutethe filtrate to about 30 cc., and make alkaline with ammoniumhydroxide. Crystallization is usually rapid. When it is com-plete, filter and wash the crystals with water, acetone, and ether.The yield of gluco-benzimidazole (m.p. 215) is 1.7 to 1.9 gm.(70 to 80 per cent of theory).Condensation from Potassium Salts-For the identification ofthe aldonic acids present in the potassium salt fraction of thepotassium hypoiodite-methanol oxidation, 1 gm. of potassiumaldonate is added to 0.6 gm. of o-phenylenediamine, 0.8 cc. ofconcentrated HCl, 0.4 cc. of syrupy H3P04 (sp. gr. 1.7), 2 cc. ofwater, and 1 cc. of ethanol. Carry out the condensation as givenabove for Ca gluconate. Use 5 cc. of water to dissolve the syrupand crystallize from a volume of approximately 15 cc.If the tube is too large in reference to the size of sample, thesmall syrupy residue may spatter on the wall of the tube and failto undergo complete condensation. To avoid this, in experimentswith small quantities, the addition of a few tenths of a cc. ofdiethylene glycol at the start of the reaction has been found helpfulin insuring smooth condensation.When the starting products are relatively pure aldoses, crystal-lization of the resulting benzimidazoles is usually rapid. Theyield is 0.7 to 0.9 gm. (60 to 80 per cent of theory). If crystalliza-tion is slow, leave the flask in a refrigerator for 12 hours.9 In theabsence of crystals, filter off any trace of amorphous precipitate

8 With calcium salts good yields are obtained without the use of phos-phoric acid in addition to hydrochloric. I f phosphoric acid is used thecalcium must be removed first to prevent the formation of a calcium phos-phate precipitate when the reaction mixture is made basic.0 I f too large an excess of ammonium hydroxide has been added, am-monium phosphate may crystallize from the reaction mixture. I f removedby filtration, the crystals dissolve readily when washed with water andoffer no interference.


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304 Carbohydrate Characterizationwhich may have formed and extract the filtrate with ether threetimes to remove unreacted o-phenylenediamine. Mother liquorsfrom a first crystallization are also submitted to ether extraction.OEvaporate the extracted solutions nearly to dryness. Takeup in a few cc. of water and filter off any water-insoluble crystals.They may represent a first crop or a second crop from the motherliquors.

Copper Salt Precipitation-To the filtrates, from which excessammonia has thus been removed by the evaporation, add a cupricammonia solution prepared by suspending 10 gm. of cupricacetate (monohydrate) in water, adding sufficient NHdOH to givea clear solution, and diluting to 100 cc. Estimate approximatelythe amount of benzimidazole in the mother liquor and add about20 cc. of cupric ammonia solution per gm. of benzimidazole. If abenzimidazole is present, a heavy green precipitate forms. Warmthe mixture on a steam bath for 5 to 10 minutes with an air jetdirected on the surface of the solution to insure complete pre-cipitation (removal of excess NHdOH). Transfer to a tube andwash with water three times on the centrifuge. Suspend thewashed salt in 4 to 5 times its volume of 3: 1 water-alcohol,decompose with hydrogen sulfide, heat and aerate, centrifugewhile warm, and decant from the copper sulfide through a carbon-asbestos pad under suction.

The benzimidazoles in the filtrate (usually a few tenths of a gm.)thus are separated from practically al l contaminants and crystal-lize readily upon concentration of the solution by evaporation.After the evaporation add 1 or 2 drops of NHIOH to insurealkalinity.

Condensation from Barium Salts-Weigh out a sample ofbarium salt equivalent to about 1 gm. of aldo-hexose (approxi-mately 2 gm. of salt in the absence of contaminants), suspend in10 cc. of water in a centrifuge tube, add phenolphthalein, andneutralize by the addition of dilute HCI. To the neutral solution

10 With unknowns it is frequently of value to transfer the ether extractto a tared evaporating dish for determination of the weight of ether-extractable solids. The recovery of o-phenylenediamine thus estimatedgives an indication of the amount which has undergone reaction. A con-densation of potassium gluconate (1 pm.) as above yields an ether extractof 0.1 gm. (theoretical excess of o-phenylenediamine added, 0.14 gm.).


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S. Moore and K. P. Linkadd 0.8 cc. of concentrated HCl and precipitate the barium ion(approximately 0.5 cc. of 1: 1 concentrated sulfuric acid-water).On the basis of drop tests, adjust the solution until it shows aslight excess of barium. An excess of sulfuric acid is to be avoided.Centrifuge, decant through a filter, and evaporate the f&rate(steam and air) to about 4 cc. Transfer to a test-tube (usingethanol in washing) containing 0.7 gm. of o-phenylenediamine.Add 0.5 cc. of H3P04 and condense as in the case of the potassiumsalt. If foaming occurs during the early part of the condensation,a few drops of ethanol may be added to wash down the materialon the sides of the tube by reflux.

In working up the condensate, at the time of addition of NHkOH,filter the solution rapidly through a carbon-asbestos pad within afew seconds after the addition of ammonia in order to remove theamorphous precipitate which may form. If the filtration is notperformed rapidly, the relatively insoluble benzimidazole ofmannose may begin crystallization during the filtration. Ondirect crystallization, the yields are 0.7 to 0.8 gm. (50 per cent oftheory) of benzimidazole per gm. of aldose involved. Extractthe mother liquors with ether and work up by the copper saltprocedure to obtain the derivatives remaining in solution.

Condensation at 180-Under the above conditions at 135xylonic acid yields little or no benzimidazole derivative. Comple-tion of the condensation can be accomplished at 180. Thedifference in reactivity of the individual aldonic acids towardo-phenylenediamine can therefore in this case be used for thepurpose of separation. The solution obtained by decompositionof the copper salt with H2S contains the water-soluble intermediatereaction product with o-phenylenediamine of any xylonic acidpresent. The solution is evaporated to a thin syrup as usual topermit crystallization of any benzimidazoles of other aldosespresent.

Transfer the syrup to a test-tube and, for an estimated 0.5gm. as the possible xylose content, add 0.5 cc. of concentratedHCl and 0.3 gm. of zinc chloride (preferable to H8P04 in this case).

11 This estimate (1, 0.5, 0.25, etc., gm.) of the xylose content, if any, forthe purpose of determining the approximate amounts of reagents to beadded, can be roughly made on the basis of the amounts of benzimidazolesof other aldoses already isolated and pentose tests on the original sample.


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Carbohydrate CharacterizationPlace on the oi l bath at 135 and raise the temperature over aperiod of 45 minutes to 180 f 5 and hold at that temperaturefor 1 hour. Take the condensed syrup up in water as usual anddecolorize. The addition of NH40H causes precipitation of zincbenzimidazole salts and zinc hydroxide, both of which are solublein excess NH,OH. But in this case it is preferable to add aminimum of NH.,OH, remove the excess by heat and air as in thecopper procedure, pass HGS directly into the resulting zinc saltsuspension, heat, and centrifuge while warm. Decant from thezinc sulfide through a filter, and free from hydrogen sulfide.Xylo-benzimidazole, if present, crystallizes during concentrationof the filtrate. Add 1 or 2 drops of NH40H at the end of theevaporation. In this procedure, 1 gm. of xylose in the originalsample will yield about 0.4 gm. of benzimidazole. The yield ishigher (0.7 gm., 50 per cent) if the 180 condensation is rundirectly on the barium salt without the intermediate steps in-volved above. In the absence of appreciable quantities of otheraldonic acids in the barium fraction, the direct high temperaturecondensation can be run on a separate sample of barium salt. Aweight equivalent to 1 gm. of aldose is taken, which is 2 gm. incase of the usual salt, neutralized, 1 cc. of concentrated HCl added,and the solution freed of barium and condensed with 0.7 gm. ofo-phenylenediamine and 0.5 gm. of zinc chloride, as given above,at 180 for 1 hour.12

Behavior of Fructose-This ketose has been carried through thebenzimidazole scheme to determine what reactions, if any, itundergoes. We do not present the following data as an efficientmethod for fructose identification but as information of value inthe application of the benzimidazole procedure for the identifica-tion of aldoses to a natural product also containing fructose. 2gm. of fructose, when put through the potassium hypoiodite-methanol oxidation, yield practically no precipitate in the potas-sium salt fraction. A barium salt precipitate of 2.9 gm. is ob-tained which gives a strong Seliwanoffs test for fructose. Con-densation of this barium salt fraction by the same procedure given

I* The benzimidazoles which form readily at 135 are not all stable atthe higher temperature of 180. Therefore xylo-benzimidazole is at pres-ent the only product of the HO0 condensation which possesses characteriza-tion value.


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S. Moore and K. P. Linkfor aldoses produces considerable charring during the period ofheating. The acidic filtrate from the first decolorization is almostblack. The addition of NH,OH precipitates the dark impuritiesas a tar which is removed by the filtration of the alkaline solutionto give a final clear filtrate. On standing, or during the processof ether extraction, the filtrate deposits a small amount (0.1 gm.)of crystals and concentration of the solution yields a second crop(0.1 gm.) of the same product (m.p. 235-236, [cx]~ = -51,hydrochloride m.p. 229, picrate m.p. 158). The compound thusproves to be d-arabo-benzimidazole.

It can be stated, therefore, that the presence of fructose doesnot interfere with the isolation of aldo-benzimidazoles. It has noeffect on the potassium salt fraction and the unchanged fructosein the barium salt undergoes decomposition in the presence of themineral acids to give hydroxymethylfurfural and other productswhich, either as such or in condensed forms with o-phenylene-diamine, are readily removed from the benzimidazoles by thealkaline filtration. The only precaution to be taken is in theinterpretation of the isolation of d-arabo-benzimidazole. Sincel-arabinose is the form which usually occurs in nature, the casesfor possible confusion are rare. But if the d form of arabinose ispresent in appreciable quantity in a natural product, the corre-sponding derivative will be readily identif ied from the potassiumsalt fraction. The problem of whether a small amount of crystalsof d-arabo-benzimidazole present in the products from the bariumsalt condensation may have come from d-arabinose, d-fructose,or both, should prove solvable in conjunction with pentose andketose tests on the sample and the methylphenylhydrazinereaction.

The isolation of the pentose derivative from a ketose indicatesthat oxidation of the terminal CHtOH-CO grouping has takenplace to some extent during the hypoiodite oxidation. Purefructose (unoxidized), when submitted to the condensation condi-tions with o-phenylenediamine and HCI-HIPOd, gives no d-arabo-benzimidazole.

Recrystallization-The aldo-benzimidazoles can be recrystal-lized and fractionated from hot water. The relative solubilitiesmay be indicated by giving the approximate volume of hot waterrequired to recrystallize 1 gm. of the given derivative. For


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308 Carbohydrate Characterizationlyxo-benzimidaaole 15 cc. are required, for gluco- 20, rhamno- 30,xylo- 40, manno- 100, and arabino- and galacto- about 200. Inrecrystallizing the last three derivatives it is preferable to use 1: 1ethanol-water in which they are more than twice as soluble as inwater alone. With these less soluble derivatives it is also con-venient in some cases to suspend the derivative in a small volumeof hot water, to add dilute HCl until the benzimidazole is insolution, filter, and add NHhOH to the reheated filtrate. It isalways satisfactory to use HCl in the first recrystallization butfor a sample being crystallized for specific rotation or analysis noacid should be used, since this treatment may not remove smallamounts of acid-soluble, alkali-insoluble impurities.

Analyses-The rhamno, 1~x0, and xylo derivatives (Table V)have not been reported in the previous literature (1, 7, 9).

TABLE VAnalyses of Rhamno, Lyxo, and Xylo Derivatives

Benzimidamle

Z-Rhamno-. . .d-Lyxo-...........................d-Xylo- .

Nitrogen (Dumaa)Calculated Found

per cent per cent11.11 11.011.77 11.811.77 11.8

Preparation of Picrates-Suspend about 100 mg. of pure aldo-benzimidazole in 1 cc. of water and add an equal weight of picricacid (containing 15 per cent water). If the picric acid salt doesnot completely dissolve when the mixture is warmed, add ethanoldropwise. Cool, filter the crystals on a small Buchner funnel,and wash with water. When the starting benzimidazole is pure,the melting point of the picrate is usually correct without furtherpurification. If required, recrystallize from water and ethanol.In all cases, especially with semimicro quantities, a reasonablyaccurate weighing of the benzimidazole and its picric acid equiva-lent is desirable.Preparation of Hydrochlorides-To 15 mg. of pure aldo-benzi-midazole in a 3 cc. centrifuge tube, add 0.1 cc. of dry HCl inabsolute ethanol (about 10 per cent HCl by weight). On slightwarming the benzimidazole dissolves and may almost simul-


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S. Moore and K. P. Linktaneously crystallize out as the hydrochloric acid salt. If crystal-lization occurs at this point, stir for a few minutes to insure com-plete reaction. To the solution (or crystal suspension) add 0.5 cc.of acetone. If necessary, cool with scratching to induce crystal-lization. In some cases the further addition of 1 cc. of ether maybe required. Wash the hydrochloride (centrifuge) with 0.5 cc.of acetone followed by 0.5 cc. of ether, and air-dry. The hydro-chlorides can be recrystallized from ethanol and acetone. Themelting points of the hydrochlorides of most benzimidazoles aresharp and are usually lower than the melting point of the parentbase. The manno salt, as obtained, is hydrated and shows acharacteristically low melting range. Gluco-benzimidazole hydro-chloride, C&O~N&l, calculated, Cl 11.66; found, Cl 11.7.SpeciJic Rotations-Benzimidazoles dissolve in aqueous acidsto give colorless, non-mutarotating solutions (in contrast toosazones). Hydrochloric acid (1 to 5 N) has been used by others(7, 9) as solvent. In order to avoid repeated use of HCl withpolariscope tubes and accessories, we have considered it preferableto choose a non-corrosive acid. A 5 per cent aqueous solution ofcitric acid has been adopted for the benzimidazole rotationsreported here. The solution of citric acid is filtered throughsintered glass before being used as solvent for the rotations andit is checked (10~1~= O.OO) to be sure that it is free from opticallyactive impurities.

It is to be noted that the rotation observed in citric acid maynot be identical with the value obtained in a different acid solvent.Galacto-benzimidazole, for example, gives a specific rotation of43.3 in citric acid and 44.4 in hydrochloric. With gluco-benzimidazole the difference is negligible (in citric acid, 9.6;in hydrochloric, 9.4).

DISCUSSIONThe fact that the benzimidazole procedure is not now applicableto aldo-disaccharides and ketoses and has not yet been studied

with all aldo-monosaccharides places a limit on its present rangeof use. The practical value of the benzimidazole derivatives canbe util ized directly for the seven aldoses presented here and inother cases may prove of value in supplementary relationshipwith other characterization reactions in the sugar group. Ex-


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310 Carbohydrate Characterizationtension of the benzimidazole procedure is being made to includeadditional aldoses, members of the uranic acid series, and thecharacterization of naturally occurring aldonic acids in thepresence of aldoses. The operations in the procedure can beconveniently carried out on a smaller scale than that given hereand a description of the detail of semimicro application is inpreparation.

In the hydrazone and osazone reactions the purity of the sugarsample and of the hydrazine reagent is a major factor in deter-mining satisfactory results. It is of practical significance thatin general the benzimidazole reaction is much less sensitive to thepresence of impurities. We have not yet encountered crudealdose syrups from which the benzimidazoles so far studied couldnot be prepared in satisfactory yields. It may be mentioned, also,that the reagent for the benzimidazole reaction is more con-venient to handle than phenylhydrazine. o-Phenylenediamine isa solid, and commercial preparations can be used without furtherpurification.The separation of carbohydrate components from oxidizableand non-oxidizable materials which may be present in a naturalsample is accomplished in certain cases by the hypoiodite-methanoloxidation. In general, the addition of methanol and filtrationinto the reaction flask may remove methanol-insoluble impuritiesfrom the aldoses. As the oxidation proceeds, the precipitationof the salts of the aldonic acids, in turn, separates the carbohydrateportion from methanol-soluble material. Impurities may bepresent in some instances which will tend to come out as gums onthe salt fractions. It has been observed that lead acetate clarifica-tion of the original aldose sample may remove contaminants ofthis type. Organic acids, if present, may also precipitate aspotassium or barium salts. In the identification procedure, thebenzimidazole derivatives resulting from most aliphatic andaromatic acids are soluble in alcohol and ether and therefore canbe separated from the carbohydrate derivatives which are allrelatively insoluble in these two solvents.

SUMMARYA method has been developed for the hypoiodite oxidation ofaldoses to aldonic acids in methanol. High yields of the potas-


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S. Moore and K. P. Link 311sium or barium aldonates are obtained in one operation by pre-cipitation from the reaction mixture (yield 90 per cent or over,oxidation period 50 to 60 minutes). This oxidation procedurehas been developed not as a preparational method for chemicallypure aldonic acid salts but efficiently to provide satisfactory saltsto serve as intermediates in a method of aldose identification basedon the characterization of the component aldonic acids as benzi-midazole derivatives.The condensation of aldonic acids with o-phenylenediamine togive 2-(aldo-polyhydroxyalkyl)benzimidazoles has been developedas the basis for a systematic procedure for the identification ofseven aldo-monosaccharides in natural products. The chemicaland physical properties of the aldo-benzimidazole derivativeshave been found to be remarkably superior, from the charac-terization standpoint, to those of hydrazones and osazones. Adescription has been given of the preparation, properties, and useof these derivatives in the characterization of arabinose, galactose,glucose, lyxose (if naturally occurring), mannose, rhamnose, andxylose.

BIBLIOGRAPHY1. Griess, P., and Harrow, G., Ber. them. Ges., 20, 281, 2295, 3111 (1887).2. Hudson, C. S., and Isbell, H. S., Bur. Standards J. Research, 3, 57 (1929).3. Goebel, W. F., J. Biol. Chem., 72, 809 (1927).4. Isbell, H. S., and Frush, H. L., BUT. Standards J. Research, 6, 1145(1931).5. Hinton, C. L., and Macara, T., Analyst, 49, 2 (1924).6. Hinsberg, O., and Funcke, F., Ber. the m. Ges., 26, 3093 (1893).7. Ohle, H., Ber. them . Ges., 67, 155 (1934).8. Schilling, B., Ber. them. Ges., 34, 905 (1901).9. Haskins, W. T., and Hudson, C. S., J. Am. Chem. Sot., 61, 1266 (1939).


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J. Biol. Chem.-carb Profiling 2

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