SUBSTITUTED HYDRAZINE DERIVATIVES OF THE HEXURONIC … · 2003-03-12 · previously pointed out, the method of drying greatly influences the melting point of the cinchonine and brucine

SUBSTITUTED HYDRAZINE DERIVATIVES OF THE HEXURONIC ACIDS*

PHENYLHYDRAZINE AND p-BROMOPHENYLHYDRAZINE DERIVA- TIVES OF d-GALACTURONIC ACID AND p-BROMO-

PHENYLHYDRAZINE DERIVATIVES OF d-MANNURONIC ACID

BY CARL NIEMANN, EUGENE SCHOEFFEL, AND KARL PAUL LINK

(From the Biochemistry Research Laboratory, Department of Agricultural Chemistry, University of Wisconsin, Madison)

(Received for publication, April 1, 1933)

INTRODUCTION

Progress in the chemistry and biochemistry of t,he hexuronic acids has been inhibited in part because well defined derivatives are lacking or difficult to prepare. The cha.racterization and identification of the uranic acids (especially from reaction mixtures in which they may be present in small amounts) would be placed on a firmer basis if more suitable derivatives were available. This need is especially illustrated by the difficulties usually encountered in the identification of the aldehyde sugar acid components of the complex polyuronide substances, since an inevitable destruction of the free uranic acids liberated accompanies their hydrolysis (1) .I

The limitations of the methods available at present can be illustrated by a review of the most important papers involving the isolation and identification of a uranic acid. Only some of the more salient points need be mentioned.

* Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

Supported in part by grants from the University Research Fund. 1 This paper gives references to the observations of various investigators

on this question. It is important to note here that Dr. P. A. Levene deals at some length on the difficulties involved in establishing the presence of d-glucuronic acid in chondrosin (2). The difficulties referred to by Levene in his studies on the constitution of the mucoproteins parallel in a general way our observations and those of others on the polyuronide substances.

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338 Derivatives of Hexuronic Acids

In the researches on the aldobionic acids obtained from the soluble specific polysaccharide substances of certain microorgan- isms (3, 4) the presence of the uranic acid was usually not estab- lished by direct isolation. In most cases it was necessary to oxi- dize the sugar acid component to the corresponding dicarboxylic acid, which was in turn isolated and characterized. The same method has frequently been employed to identify the uranic acid present in various plant gums and mucilages (5-10) and t,he alginic acids of marine algae (11, 12). The limitations of the oxida- tion procedure were pointed out by Butler and Cretcher (7) in their study on the composition of commercial cherry gum. The quantitative analysis of the cherry gum acid showed a uranic acid content of 30.0 per cent, yet they were unable to establish the nature of the uranic acid present.

Ehrlich (13) in an extensive review article on the biochemistry of galacturonic acid and the pectin substances, has pointed out that in certain cases the reported occurrence of uranic acids in hemicelluloses and other cell wall substances must be questioned. This dubiety is justified since the characterizations were frequently inferred from color reactions and the formation of derivatives that were not well defined.

It is significant to note that the supposed occurrence of d- glucuronic acid in the alginic acid of various species of brown alga? is still an open question, due to variations in the physical constants exhibited by some of the alkaloidal salts of d-glucuronic and d- mannuronic acid in the hands of different investigators (12,14-17).

The selection of the derivative to be used for the purposes of isolating the uranic acid is determined in part by the chemical nature of the accompanying substances. Thus in the presence of other unrelated acidic substances the use of a derivative involving combination with the carboxyl group would not lead to an easy isolation of the hexuronic acid.

The hexuronic acids have three functional groups, the carboxyl, t,he alcoholic hydroxyls, and the aldehydic or ketonic carbonyl. Reactions directed to combination with the carboxyl group resulting in salt formation have found considerable application. Thus in the preparation of d-glucuronic, d-galacturonic, and d-mannuronic acid from natural sources with a high uranic acid content, the formation of inorganic salts, particularly the barium and cal-


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Niemann, Schoeffel, and Link 339

cium salts, has proved to be quite successful (15, 16, 18-23). This procedure, however, cannot usually be employed for isolation and characterization purposes when small quantities are involved since the barium and calcium salts have so far not been obtained in a distinctly crystalline condition.

The characterization of the uranic acids through the use of alkaloidal salts has been successful in some investigations. On the other hand, confusion has arisen in certain instances, particularly in the case of d-mannuronic acid (12, 14-17).2

The identification of d-mannuronic acid as a constituent of various marine alga! has been attempted in the majority of cases, through the use of alkaloidal salts (11, 12, 14, 17). Nelson and Cretcher (14, 24) have reported the isolation of d-mannuronolactone from the alginic acid of several species of marine algae. Scho- effel and Link have obtained the (Y and 6 forms of the free acid from the alginic acid of Macrosystis pyrifera and Fucus serratus (15). The validity of the values assigned to the physical constants of d-mannuronolactone and its brucine and cinchonine salt by Nelson and Cretcher (14) has been corroborated by the synthesis of d- mannuronolactone from d-mannose by Niemann and Link (16). In all cases the physical constants of the lactone, the brucine and cinchonine salts of the naturally occurring acid, were in agreement with the constants exhibited by the same compounds prepared from the acid obtained by synthesis.

The variation in the melting points and rotations of the alkaloidal salts of d-mannuronic acid reported by various investigators (11, 12, 14, 16, 17) may be due to several factors. As has been previously pointed out, the method of drying greatly influences the melting point of the cinchonine and brucine salts (16). Fur- ther, as Nelson and Cretcher have suggested, when polymers of d-mannuronic acid are present alkaloidal salts with higher melting points are obtained (14).

As mentioned above, it has been noted frequently (14, 15, 23, 24) that the barium salt of d-mannuronic acid obtained by the hydrolysis of the complex polymannuronides (the alginic acids) are invariably contaminated with the barium salts of lower polymers. When the alkaloids are used to characterize d-mannuronic

2 Specific reference to the cases in point are given in these papers.


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acid, bhe presence of salts of the lower polymers cannot be revealed with certainty by elementary analysis. On the other hand, if the lower polymers represent complexes wherein the d-mannuronic acid units are linked in part through the aldehyde group, the use of p-bromophenylhydrazine would yield discriminating derivatives. The presence of a polymer or polymers would be detected by an elementary analysis of the hydrazine compound. In general, reactions directed t,oward combination with the carboxyl group alone do not permit ready and exact characterization of a hexuronic acid.

Reactions with the alcoholic hydroxyl groups are restricted to relatively pure compounds in the sugar acid group. While these are of t.he highest importance in constitutional studies (25-27) they are at present of limited value for direct characterization and identification purposes.

The property of the aldehydic or ketonic carbonyl of the sugar group to form hydrazones or under certain conditions osazones, as illustrated by the classical studies of Emil Fischer (28), has in the past been extensively and fairly successfully applied to one naturally occurring member of the hexuronic acids, namely d-glucuronic acid (29-32). In contrast to the numerous substituted hydrazine derivatives of d-glucuronic acid the lit’erature lists only two corresponding compounds of d-galacturonic acid (29, 30). Ohle and Berend (33) described the phenylhydrazine salt of the phenylosazone of d-galacturonic acid and indicated the existence of the phenylhydrazine salt of the phenylhydrazone.3 In a recent article Neuberg and Collatz (35) mentioned the 2,4-dinitrophenyl- hydrazone of d-galacturonic acid but did not report their experimental procedure.

In their first note on the constitution of vitamin C (originally called hexuronic acid, now ascorbic acid (36)) Hirst and Reynolds (37) stated that a p-bromophenylhydrazine derivative of d-galacturonic acid, comparable to the p-bromophenylosazone barium salt of d-glucuronic acid was formed, by following the procedure

3 Ohle and Berend (33) were the first to record that d-galacturonic acid yields a colored lead salt with basic lead acetate. Ehrlich (34) has used this color reaction as the basis for a qualitative test for d-galacturonic acid. Ehrlich apparently was not aware of Ohle and Berend’s observation, since he did not cite their work.


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of Goldschmiedt and Zerner (38). The compound isolated was not described in detail. In the light of the experimental results presented in this paper we are of the opinion that Hirst and Reynolds most likely did not have the p-bromophenylosazone barium salt in hand, but that the “yellow powder” mentioned is probably t,he highly insoluble barium p-bromophenylhydrazone d-galacturonate.

Bird and Haas (12) failed to prepare the p-bromophenylhydrazone of d-mannuronic acid and to the best of the authors’ knowl- edge, no other attempts have been reported.

In the extensive program on the chemistry and biochemistry of the free and combined uranic acids under way in this laboratory, we have included a study of the substituted hydrazine derivatives. The investigations have been directed toward the objective of finding derivatives of the various naturally occurring hexuronic acids that can be prepared with ease and that also possess desirable physical and chemical properties. The first incentive to explore the field of uranic acid derivatives more thoroughly, arose through the observations of one of us (K.P.L.) that the corn seedling (Zea mnys) contains small quantities of uranic acids in the cel1 wall, combined in the pectin fraction and possibly also to other polysaccharide subst’ances. In addition some uncombined uranic acid (possibly d-glucuronic acid) occurs in the cell sap (39). These observations were originally put forward with reserve, since it was not possible to establish definitely, with the derivatives of the uranic acids available at the time, which uranic acid was involved.

In this communication we describe the preparation and properties of t’he following derivatives: Section I, (a) barium phenylhydrazone d-galacturonate, (b) phenylhydrazine phenylhydrazone d-galacturonat’e, (c) d-galacturonic acid phenylhydrazone, (d) phenylhydrazine phenylosazone d-galacturonate, (e) barium phenylosazone d-galacturonate; Section II, (a) barium p-bromophenylhydrazone d-galacturonate, (b) p-bromophenylhydrazine p-bromophenylhydrazone d-galacturonate, (c) p-bromophenylhydrazone d-galacturonic acid, (d) p-bromophenylhydrazide p-bromophenylhydrazone d-galacturonic acid; SeeCon III, (a) barium p-bromophenylhydrazone d-mannuronate, (b) p-bromophenylhydrazone d-mannuronolactone, (c) p-bromophenylhydrazine p-bromophenylhydrazone d-mannuronate, and (d) p-bromophenylhydrazide p- bromophenylhydrazone d-mannuronic acid.


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In the light of t’he results embodied in this communication it is interesting to note that Ehrlich, the first investigator to obtain d-galacturonic acid in a crystalline condition, stated in his original publication (40) that in contrast to d-glucuronic acid, no difficultly soluble (or relatively insoluble) crystalline derivatives of d-galacturonic acid could be obtained with phenylhydrazine or p-bromophenylhydrazine. As far as we are able to ascertain from Ehrlich’s numerous later publications, he has not explored further the preparation of substituted hydrazine derivatives of d-galacturonic acid.4 This investigation is being continued and the preparation and properties of the alkaloidal salts of several substituted phenyl- hydrazones will bereported in a future communication.

EXPERIMENTAL

The procedures used below are simple adaptations of those originally described by Fischer (28) and later recompiled by van der Haar (42). In all experiments freshly distilled phenylhydrazine was used. The p-bromophenylhydrazine, and the phenylhydrazine hydrochlorides were also recrystallized immediately before they were used. These precautions are indispensable. It is possible that the unsuccessful attempts of other investigators in this field have been due to the failure to observe these condit’ions.

The preparations of p-bromophenylhydrazine (either the free base or the hydrochloride) offered by the Eastman Kodak Com- pany, or Akatos, Inc., New York (agents for Kahlbaum), are invariably too impure to be used directly. However, we have experienced no difficulty in purifying the preparations marketed by these companies.

The barium d-mannuronate and the d-mannuronolactone used in this study were prepared after the procedure of Schoeffel and Link (23). The d-galacturonic acid and t,he barium salt were prepared from a polygalacturonide preparation obtained from citrus pectin by using the rapid practical procedure of Link and Nedden (19) .5

4 Ehrlich’s most recent review article wherein reference is made to all of his publications is in the book by Klein (41).

5 Ehrlich and Guttmann (43) commented adversely on our method for the preparation of d-galacturonic acid (19, 20). They are apparently unaware of the fact that the commercially available citrus pectin preparation which they have recommended as the starting material is identical


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All analyses were conducted through the use of the Pregl micro- methods. The individual preparations were dried for 5 hours at 60” on the Pregl block prior to the analysis. Rotations were determined with a Franz Schmidt and Haensch (Berlin) quartz wedge saccharimeter equipped with the Ventzke scale. The new electric sodium lamp made by the same firm was used as the source of light. The melting points were made with the Thiele apparatus with an inclosed scale thermomet,er. In every case t,he temperature was raised at the rate of 6” per minute.

I. Phenylhydraxine Derivatives of d-Galacturonic Acid

(a) Preparation of Barium Phenylhydrazone d-Galacturonate. Procedure i-O.58 gm. of phenylhydrazine was dissolvedina mixture of 12 cc. of water and 3.5 cc. of 50 per cent acetic acid. A solution of 1.45 gm. of barium d-galacturonate and 6.0 gm. of barium acetate in 15 cc. of water was then added to the hydrazine reagent. Within 10 minutes a heavy granular precipitate was formed which remained unchanged on the addition of 30 cc. of 95 per cent ethyl alcohol. After standing for 3 to 4 hours the precipitate was filtered off, washed successively with water, absolute ethyl alcohol, and ether. The product was finally dried over PZOs under 15 mm. pressure at 60”. 0.90 gm. of the derivative was obtained, whereas a theoretical yield requires 1.95 gm.

Procedure g-0.50 gm. of phenylhydrazine phenylhydrazone d-galacturonate ((Section I, b) see below) was dissolved in 25 cc. of 60 per cent ethyl alcohol and the resulting solution titrated to a phenolphthalein end-point with 0.2 N barium hydroxide. The precipitated barium salt was filtered off, washed, and dried in the manner previously described. The yield was practically quantitative.

Procedure 5-1.40 gm. of barium d-galacturonate were treated with 2.50 gm. of phenylhydrazine hydrochloride, 6.0 gm. of barium acetate, 100 cc. of water, and 3 cc. of glacial acetic acid after the

with the product that we had introduced in our first publication (20). They have likewise failed to realize that the intermediate Pectolxbre which they go to the trouble to prepare from this pectin is essentially the polygalacturonide that we recommended as the starting material in our second publication (19). This polygalacturonide can be obtained commercially at a fraction of the cost of laboratory preparation.


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procedure of Goldschmiedt and Zerner (38). The yellow precipitate obtained was filtered off, extracted wit,h ether in a continuous extractor, and finally dried in the usual manner. 1.0 gm. of the hydrazone barium salt was obtained, which is equivalent to 53 per cent of that required theoretically.

Melting Point-The compound chars but does not melt or de- compose wit.h evolut,ion of gas below 250’.

Analysis-Calculated for (C12H1606N2)2Ba. Ba 19.53, N 7.96 Found. Procedure 1. “ 19.46, “ 8.08

“ 2. “ 20.01, “ 8.06 “ 3. “ 19.64, “ 8.31

(b) Preparation of Phenylhydrazine Phenylhydraxone d-Galactu- ronate (93)---8.2 gm. of phenylhydraeine were dissolved in 8.2 gm. of 50 per cent acetic acid and 50 cc. of water. To the hydrazine reagent 4.50 gm. of d-galacturonic acid dissolved in 25 cc. of water were then added. Precipitation began within 10 to 15 minut’es and after standing overnight in the ice chest the impure salt was filtered off and washed with cold water and 30 per cent ethyl alcohol. To purify the compound it was dissolved in 50 per cent ethyl alcohol and the solution clarified with activated blood charcoal. It was then allowed to crystallize from the alcoholic solution. The freshly prepared crystals possessed a slight yellow color. The yield was 5.5 gm. or 60 per cent of that required for a theoretical yield.

Melting Point-The derivative melted at 133-134’ (uncorrected) with decomposition.

Rotation-[cr]2,2 = -10.4” f. 0.5” (initial rotation in methyl alcohol, c = 1.1 per cent).

Analysis Calculated for C18H2d06N4. N 14.28, N. E.* 25.47 cc. 0.1 N alkali Found. “ 14.33, “ 25.69 “ 0.1 “ “

* N. E. represents the neutralization equivalent.

(c) Preparation of d-Galacturonic Acid Phenylhydraxone-9.2 gm. of barium phenylhydrazone d-galacturonate (Section I, a) were suspended in 30 cc. of 95 per cent ethyl alcohol. To this suspen- sion 35 cc. of 1 N sulfuric acid were added during the course of 20 minutes. The gelatinous mass which formed was then heated on the steam bath for 15 minutes, along with 5 gm. of activated blood charcoal. On cooling the filtered solution, the hydrazone crystal-


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lized out in fine yellow needles, which were collected and dried in the usual manner. The mother liquor can be reworked to increase the yield but this effort is not profitable. 2.0 gm. of the hydrazone were obtained which represent 27 per cent of the theoretical yield.

Melting Point-The compound melted at 140-141’ (uncorrected) with decomposition.

Rotation-[a12,2 = +l.O” =t 0.5’ (initial rotation in methyl alcohol, c = 1.0 per cent).

Analysis Calculated for ClzH,,06Nz. N 9.86, N. E. 35.20 cc. 0.1 N alkali Found. “ 9.96, “ 35.31 “ 0.1 “ “

(d) Preparation of Phenylhydrazine Phenylosazone d-Galacturo- nate (.%?)-2.25 gm. of d-galacturonic acid and 4.10 gm. of phenylhydrazine were dissolved in 38 cc. of water. After heating for 2 minutes on t,he steam bath, 4.1 cc. of glacial acetic acid were added to the solution. The reaction mixture was then heated again on the steam bath for 20 to 25 minutes. At the end of this period the precipitate that had formed was filtered off while the solution was st,ill hot. The filtrate was reheated for an additional 30 minutes to induce further precipitation. The second precipitate obtained was combined with the original fraction. The combined crude frac- tions were dissolved in hot 35 per cent ethyl alcohol, decolorized with activated carbon, and allowed to crystallize. The purified product was obtained as a light yellow semicrystalline mass. For analysis the compound was dried in the previously described manner. Only 0.25 gm. of the derivative was obtained, whereas a theoretical yield requires 5.56 gm.

Melting Point-The compound melts at 130-131" (uncorrected) with decomposition.

Rotation- [o(]i2 = +2.40” & 0.5” (initial rotation in 95 per cent methyl alcohol, c = 1.0 per cent).

Analysis Calculated for Cz4HzsOsNs. N 17.50, N. E. 20.82 cc. 0.1 N alkali Found. “ 17.00, “ 21.35 “ 0.1 “ “

(e) Prepara.tion of Barium Phenylosaxone d-Galacturonate-The mother liquor that remained after the recrystallization of the


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Derivatives of Hexuronic Acids

phenylhydrazine phenylosazone d-galacturonate (Section I, d) was titrated with 0.2 N barium hydroxide to a phenolphthalein end- point and after standing overnight the precipitated barium salt of the osazone was filtered off, washed wit.h carbon dioxide-free water, alcohol, ether, and finally dried in the above described manner. Only 0.5 gm. of the barium salt was obtained by this procedure.

Melting Point-The compound chars but does not melt or de- compose with evolution of gas below 250“.

Analysis-Calculated for (C18H1905N&Ba. Ba 15.62, N 12.73 Found. “ 15.42, “ 12.84

II. p-Bromophenylhydrazine Derivatives of d-Galacturonic Acid

(a) Preparation of Barium p-Bromophenylhydrazone d-Galactu- ronate. Procedure 1-1.00 gm. of p-bromophenylhydrazine was dissolved in a mixture of 12 cc. of warm water and 3.5 cc. of 50 per cent acetic acid. To this reagent a solution of 1.45 gm. of barium d-galacturonate and 6.00 gm. of barium acetate in 15 cc. of water was added. Within 10 minutes a heavy granular precipitate settled out which remained unchanged on the addition of 30 cc. of 95 per cent ethyl alcohol. After standing for 3 to 4 hours the precipitate was filtered off, washed successively with water, abso- 1ut.e ethyl alcohol, and ethyl ether. The product was finally dried at 60” over calcium chloride under 15 mm. pressure. The yield was 1.30 gm. or 55.0 per cent of the theoretical amount.

Procedure ,Z%-0.50 gm. of p-bromophenylhydrazine p-bromophenylhydrazone d-galacturonate (Section II, b) was dissolved in 30 cc. of warm 60 per cent ethyl alcohol. This solution was then titrated with 0.2 N barium hydroxide to a phenolphthalein end- point. The precipitated barium salt was filtered off, washed, and dried in the manner described above. A practically quantitative yield was obtained.

Procedure 5-1.40 gm. of barium d-galacturonate were treated with 4.00 gm. of p-bromophenylhydrazine,6 6.00 gm. of barium acetate, 100 cc. of water, and 3 cc. of glacial acetic acid after the procedure of Goldschmiedt and Zerner (38). The yellow precipitate obtained was extracted cont,inuously with ether and then dried in the usual manner. 1.30 gm. of the derivative were obtained which represent 55 per cent of the theoretical yield.

6 The hydrochloride may be used in place of the free base.


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Melting Point-No true melting point was observed below 250’.

Analysis-Calculated for (C&H1406NzBr)~Ba. Ba 15.95, N 6.51 Found. Procedure 1. “ 16.03, “ 6.34

“ 2. “ 15.97, “ 6.53 “ 3. “ 15.62, “ 6.71

(?I) Preparation of p-Bromophenylhydrazine p-Bromophenyl- hydraxone cl-Galacturonate (SS). Procedure I-3.00 gm. of p- bromophenylhydrazine were dissolved in a mixture of 30 cc. of water and 10.5 cc. of 50 per cent acetic acid. 1.07 gm. of d-galacturonic acid dissolved in 5.0 cc. of water were then added to the hydrazine solution. Within 10 minutes precipitation began, and after standing overnight at room temperature the precipitate was filtered off and washed with cold water and a small quantity of cold 70 per cent ethyl alcohol. The product obtained was redis- solved in hot 60 per cent ethyl alcohol, the solution decolorized, filtered, and allowed t,o crystallize. The recrystallized product was isolat.ed in the form of slightly yellow prisms having a tendency to form aggregates. The yield was 1.10 gm. or 36 per cent of that required for a theoretical yield.

Procedure 2-1.00 gm. of d-galacturonic acid, 3.60 gm. of p- bromophenylhydrazine in 3.0 cc. of glacial acetic acid, and 25 cc. of water were heated on the steam bath. A yellow precipitate formed almost instantaneously which persisted on continued heating. After being heated for 30 to 40 minutes the precipitate was filtered off and washed with dilute acetic acid and warm water. The crude product was dissolved in hot 70 per cent ethyl alcohol, the solution decolorized, filtered, and allowed to crystallize. The recrystallized derivative was washed with cold 70 per cent ethyl alcohol and dried at 30” over calcium chloride under 15 mm. pressure. 1.60 gm. of the derivative were obtained which are 56 per cent of the theoretical yield.

Melting Point-The compound melted at 145-146” (uncorrected) with decomposition.

Rotation- [CX] “,” = f9.0” =t 2.0’ (initial rotation in methyl alcohol, c = 0.7 per cent).

Analysis Calculated for C1sHz106NaBrz. N 10.19, N. E. 18.18 cc. 0.1 N alkali Found. Procedure 1. “ 10.31, “ 17.79 “ 0.1 “ “

“ 2. ‘6 10.12, ‘( 18.48 “ 0.1 “ “


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(c) Preparation of p-Bromophenylhydrazone of d-Galacturonic Acid-7.50 gm. of barium p-bromophenylhydrazone d-galacturonate (Section II, a) were suspended in 20 cc. of water and 40 cc. of 95 per cent ethyl alcohol. 17.5 cc. of N sulfuric acid were added in the course of 15 minutes and the reaction mixture heated on the steam bath for 20 to 25 minutes. After the addition of a small amount of blood charcoal the insoluble matter was filtered off. On cooling, the hydrazone of the free acid crystallized out in a short time. It was filtered off, washed with cold 50 per cent ethyl alcohol, and dried at 30’ over calcium chloride under 15 mm. of pressure. The yield can be increased by concentrating the mother liquor but the resulting product is usually somewhat colored. The yield was 1.20 gm. or 19.5 per cent of the theoretical yield.

Melting Point-The derivative melted at 150-151” (uncorrected) with decomposition.

Eotation- [(.y] “,” = +11.5’ f 3.0’ (initial rotation in met,hyl alcohol, c = 1.36 per cent).

Analysis Calculated for C12H1506N2Br. N 7.72, N. E. 27.54 cc. 0.1 N alkali Found. “ 7.74, “ 27.77 “ 0.1 “ “

(d) Preparation of p-Bromophenylhydrazide p-Bromophenyl- hydraxone of d-Galacturonic Acid-O.97 gm. of d-galacturonic acid, 3.35 gm. of p-bromophenylhydrazine hydrochloride, 1.23 gm. of sodium acetate, and 2 cc. of glacial acetic acid were dissolved in a mixture of 10 CC. of water and 15 cc. of pyridine. After the reaction mixture had been heated on the steam bath for 40 minut,es, 2 gm. of activated carbon were added and the solution filtered. The filtrate was poured into 400 to 500 cc. of cold water, the precipitate collected and washed copiously with water. The crude product was pressed dry on the filter and suspended in warm 95 per cent ethyl alcohol for a few minutes and again filtered. After being washed with 95 per cent ethyl alcohol, it was dissolved in a small amount, of pyridine and precipitated by pouring into 400 cc. of cold water. The purified derivat.ive was filtered off, pressed dry on the funnel, washed with 95 per cent ethyl a,lcohol, and dried at 60” over phosphorus pentoxide in a vacuum. 0.65 gm. of the derivative was obtained, representing 23.7 per cent of the theoretical yield.


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Niemann, Schoeffel, and Link

Melting Point-The derivative m&ed at. 1744175” (uncorrected) with decomposition.

Rotation- Lo11 “,” = +17.7’ i 1.0” (initial rotation in pyridine, c = 0.8 per cent).

Analysis-Calculated for Ci8H1905N4Br2. N 10.53, Br 30.03 Found. “ 10.62, “ 30.00

.III. p-Bromophenylhydrazine Derivatives of d-Mannuronic Acid

(a) Preparation of Barium p-Bromophenylhydrazone d-Mannur- onate. Procedure l-l.45 gm. of barium d-mannuronate and 6.00 gm. of barium acetate were dissolved in 15 cc. of water. A sufli- cient amount of a 7.5 per cent p-bromophenylhydrazine acetate solution was then added so that 1.00 gm. of t,he free base was present. After shaking for 1 to 2 hours a granular precipitate began to form. The precipitation was complehe in 4 hours. 30 cc. of 95 per cent ethyl alcohol were then added and the precipitate filtered off, washed with water, 95 per cent ethyl alcohol, and ethyl ether. The product was finally dried over calcium chloride at 60” under 15 mm. pressure. 1.40 gm. were obtained, represent,ing 59 per cent of that, required by theory.

Procedure g-1.40 gm. of barium d-mannuronate were treated with 4.10 gm. of p-bromophenylhydrazine hydrochloride, 6.00 gm. of barium acetate, 100 cc. of wa,ter, and 5 cc. of glacial acetic acid after the procedure of Goldschmiedt and Zerner (38). A yellow precipitate was formed immediately and persisted throughout the prescribed period of heating. The precipitate was filtered off, washed with hot water, 95 per cent ethyl alcohol, and ethyl ether. The preparation was dried as above. 1.30 gm. were obtained whereas the theoretical yield required 2.30 gm.

Procedure z-0.300 gm. of p-bromophenylhydrazone d-mannuronolactone (Section III, 6) was dissolved in 25 cc. of warm (60”) 95 per cent ethyl alcohol and the resulting solution titrated to a phenolphthalein end-point with 0.2 N barium hydroxide. The precipitated barium salt was allowed to stand overnight and was then filtered off, washed, and dried in the usual manner. A practically quantitative yield was obtained.

Procedure 4-0.100 gm. of p-bromophenylhydrazine p-bromophenylhydrazone d-mannuronate (Section III, c) was dissolved in


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15 cc. of warm 95 per cent ethyl alcohol and converted into the hydrazone barium salt by treatment in the cold with barium hydroxide. A practically quantitative yield was obtained.

Melting Point-No true melting point was found below 250’.

Analysis-Calculated for (ClzHlrOsNnBr)zBa. Ba 15.95, N 6.51 Found. Procedure 1. “ 15.76, “ 6.36

“ 2. “ 15.99, “ 6.58 “ 3. “ 16.35, “ 6.43 “ 4. “ 15.44

(b) Preparation of p-Bromophenylhydrazone d-Mannuronolac- tone-O.87 gm. of d-mannuronolactone was dissolved in 10 cc. of water and added to a solution of 1.12 gm. of p-bromophenylhydrazine hydrochloride and 0.411 gm. of anhydrous sodium acetate in 30 cc. of water. A white crystalline precipitate began to form immediately. It was collected on a filter after the solution was allowed to stand for 3 hours in a closed flask in the ice chest. The crude hydrazone was recrystallized from 40 per cent ethyl alcohol and after being washed with cold 30 per cent ethyl alcohol was dried at 30” over calcium chloride under 15 mm. pressure. 1.00 gm. of the hydrazone was obtained, representing 58.6 per cent of that required for a theoretical yield.

Melting Point-The compound melted at 160” (uncorrected) with decomposition.

Rotation-[a]2,2 = +64.5” f 1.0” (initial rotation in methyl alcohol, c = 2.3 per cent).

Analysis Calculated for C12H1305N2Br. N 8.12, N. E. 28.98 cc. 0.1 N alkali Found. “ 8.05, “ 29.09 “ 0.1 “ “

(c) Preparation of p-Bromophenylhydrazine p-Bromophenylhydra- zone d-Mannuronate-0.338 gm. of p-bromophenylhydrazone d-mannuronolactone (Section III, b) and 0.188 gm. of p-bromophenylhydrazine were dissolved in 15 cc. of hot 40 per cent ethyl alcohol and heated on the steam bath for 15 minutes. A small quantity of activated charcoal was added to the hot solution and upon filtration the salt crystallized out from the filtrate in colorless aggregates. After filtering off the derivative, it was washed with a small quantity of cold 30 per cent ethyl alcohol and dried at 30” over calcium chloride under 15 mm. pressure. 0.10 gm. of the salt was obtained, whereas 0.54 gm. is required for a theoretical yield.


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Melting Point-The derivative melted at 143-144” (uncorrected) with decomposition.

Rotation-[al2,2 = +48.5’ f 1.5” (initial rotation in methyl alcohol, c = 1.4 per cent).

Analysis Calculated for C18H2106N4Br2, N 10.19, N. E. 18.18 cc. 0.1 N alkali Found. “ 10.39, “ 18.52 “ 0.1 “ “

(d) Preparation of p-Bromophenylhydrazide p-Bromophenyl- hydrazone of d-Mannuronic Acid. Procedure l-0.88 gm. of d-man- nuronolacetone, 3.35 gm. of p-bromophenylhydrazine hydrochloride, and 1.23 gm. of anhydrous sodium acetate were intimately mixed and introduced into a small flask. 40 cc. of boiling water and 1 cc. of glacial acetic acid were then added and the reaction mixture heated on the steam bath for 30 to 40 minutes. A white voluminous precipitate is formed upon the addition of the water, which dissolves after heating for a few minutes. Before the first precipitate has completely dissolved a yellow precipitate begins to appear and continues to form throughout the period of heating. Upon the termination of the heating the precipitate was filtered off and sucked dry. The partially dried, crude derivative was suspended in 15 to 20 cc. of cold 95 per cent ethyl alcohol, filtered off, and washed with a small quantity of cold 95 per cent ethyl alcohol. The product thus obtained was then recrystallized from boiling 95 per cent ethyl alcohol, filtered off, washed with 95 per cent ethyl alcohol and ether, and dried at 30” over calcium chloride under 15 mm. pressure. 0.50 gm. of the derivative was obtained, representing 18.8 per cent of that required for a theoretical yield.

Procedure 2-A solid mixture of 0.44 gm. of d-mannuronolactone, 1.68 gm. of p-bromophenylhydrazine hydrochloride, and 0.61 gm. of anhydrous sodium acetate was treated with 20 cc. of boiling 95 per cent ethyl alcohol and 1 cc. of glacial acetic acid. The reaction mixture was heated on the steam bath for 30 to 40 minutes and then cooled in the ice chest overnight. The crystalline precipitate was filtered off and recrystallized from hot 95 per cent ethyl alcohol. The derivative was washed and dried as described in Pro- cedure 1. 0.60 gm. of the compound was obtained, which is 45.2 per cent of that required for a theoretical yield.

Melting Point-The compound melted at 174-175” (uncorrected) with decomposition.


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352 Derivatives of Mexuronic Acids

ROtUtiOn-[Cg2 = +18.5” f 1.0’ (initial rotation in pyridine, c = 0.75 per cent.).

Analysis-Calculated for C1JSi905N4Br2. N 10.53, Br 30.03 Found. Procedure 1. “ 10.61, “ 30.33

“ 2. “ 10.40, “ 30.30

DISCUSSION

I. Phenylhydrazine Derivatives of d-Galacturonic Acid

In giving a r&urn6 of the properties of the phenylhydrazine derivatives of d-galacturonic acid described in this paper the preparation of the barium phenylhydrazone d-galacturonate (Section I, a) which can be obtained by three different methods is of unique interest. This compound is readily formed at low temperatures through the reaction of phenylhydrazine with barium d-galacturonate (Section I, a). It is not only insoluble in all of the commoner organic solvents but its solubility in boiling water is extremely low. The extreme insolubility in hot water is responsible for the insur- mountable difficulties encountered in the direct preparation of the barium salt of Ohe phenylosazone of d-galacturonic acid from the barium salt of the acid. Thus if an attempt is made to prepare barium phenylosazone d-galacturonate by following the procedure that Goldschmiedt and Zerner (38) devised for barium d-glucuronate the resulting product will invariably be the barium salt of the hydrazone. The insolubility of the hydrazone barium salt (Sec- tion I, a) coupled with its ease of formation and ready transforma- tion into a water- and alcohol-soluble crystalline product, i.e. the hydrazone of the free acid (Section I, c), suggests its potential value for t,he isolation of d-galacturonic acid from complex reaction mixtures.

The phenylhydrazine salt of the phenylhydrazone of d-galacturonic acid (Section I, b) is readily formed when the free acid is treated with phenylhydrazine (33). Ohle and Berend (33) failed in their attempts to recrystallize it from boiling water. Conse- quently they could not report the analytical data or physical constants. As shown above, the derivative can be recrystallized readily from 50 to 60 per cent ethyl alcohol to yield well defined crystals. This derivative of d-galacturonic acid possesses a sharp melting point, and is distinctly levorotatory. In addition it is


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soluble in methyl and ethyl alcohol. Its solublity in the common solvents is, however, rather limited at low temperatures.

As pointed out above, the phenylhydrazone of d-galacturonic acid (Section I, c) can be prepared readily by decomposition of t,he barium salt (Section I, a) with dilute sulfuric acid. It can be obtained in a well defined crystalline form and consequently is more suitable for purposes of identification than the hydrazone barium salt (Section I, a). The latter compound is, on the other hand,. more readily isolated from reaction mixtures because of its great insolubility.

The phenylhydrazine salt of the phenylosazone of d-galacturonic acid (Section I, d) reported by Ohle and Berend (33) had a melting point of 140°, whereas the same compound prepared by us melted at 130-131”. Attempts to recrystallize it from hot aqueous ethyl alcoholic mixtures led to higher values for the melting point (144- 145’). However, as the melting point of the preparation increased the neutralization equivalent? decreased. This possibly indicates that the procedure employed for its purification induced the forma- Con of a hydrazide. Because of this difficulty and in view of the low yield its use can be considered unsatisfactory for either isolation or characterization purposes. While the barium salt of the phenylosazone (Section I, e) is more readily isolated than the phenylhydrazine salt (Section I, d), the former possesses none of the qualities requisite for purposes of characterization. Further- more it (Section I, e) is inferior to the barium salt of the phenylhydrazone (Section I, a) for problems involving the isolation of d-galacturonic acid. These features greatly diminish its value for the identification of d-galacturonic acid.

II. p-Bromophenylhydrazine Derivatives of d-Galacturonic Acid

The outstanding features of the p-bromophenylhydrazine deriva- t.ives of d-galacturonic acid, in contrast to the corresponding phenylhydrazine derivatives (see above), are both their stability and their solubilities, which permit ready purification. In general when small quantities of d-galacturonic acid are to be isolated and characterized, the use of p-bromophenylhydrazine is more prac- t,ical than phenylhydrazine. It is difficult for us to understand

7 Titrated with 0.01 N sodium hydroxide in cold methyl alcohol.


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how Ehrlich could have overlooked these facts since he made a special point to the effect that d-galacturonic acid, in contrast to d-glucuronic acid, would not form insoluble derivatives of p-bromo- phenylhydrasine comparable to those of d-glucuronic acid (40).

The barium p-bromophenylhydrazone d-galacturonate (Section II, a) is produced under the same conditions which lead to the formation of the corresponding phenylhydrazine compound. The derivative is insoluble in all of the common solvents and consequently its value lies solely in its application to isolation problems. The Goldschmiedt and Zerner procedure for the preparation of barium p-bromophenylosazone d-glucuronate (38) when applied to barium d-galacturonate will not yield the barium p-bromophenylosazone d-galacturonate, but will invariably form the p-bromophenylhydrazone barium salt (Section II, a). The Goldschmiedt and Zerner conditions likewise did not yield the corresponding phenylosazone of barium d-galacturonate (see above).

The p-bromophenylhydrazine salt of d-galacturonic acid p-bromophenylhydrazone (Section II, b) is probably one of the most, desirable derivatives for isolation and characterization purposes. The compound possesses a melting point in a desirable range, has a definitely distinguishable dextrorotation, and possesses solubility characteristics that permit easy purification. Of the two procedures presented, Procedure 2 (Se&ion II, b) gives a better yield, but in some cases this factor may be sacrificed when heating might tend to form hydrazine derivatives of accompanying substances. Thus, in the presence of glucose, galactose, or arabinose Procedure 1 (Section II, b) would be decidedly more advantageous.

The barium salt of the p-bromophenylhydrazone (Section II, a) is difficultly soluble and therefore readily isolated. Its high melting point, on the other hand, limits its value. However, with sulfuric acid it is easily converted into the hydrazone of the free acid (Section II, c) which may be characterized readily by its melting point, rotation, and neutralization equivalent. Because of the low yields that are obtained the hydrazone of the free acid (Section II, c) is not as practical a derivative as the hydrazine salt of the hydrazone (Section II, b).

The d-galacturonic acid p-bromophenylhydrazide p-bromophenylhydrazone (Section II, d) may find application where the hydrazine compounds of accompanying substances are readily


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soluble in 95 per cent ethyl alcohol. This derivative of galacturonic acid is but slightly soluble in warm 95 per cent ethyl alcohol, but is readily soluble in pyridine. It is practically inert to cold or warm dilute alkali. This property indicates the validity of the structure assigned since the osazone of the free acid or of the lactone would react with alkali under the above conditions. In general the hydraside-hydrazone (Section II, d) is inferior to the hydrazine salt of the hydrazone (Section II, b) because of lower yields, and greater difficulty of preparation. An attempt was made to prepare a p-bromophenylosazone derivative, but in all cases the resulting products were hydrazones contaminated with highly colored impurities.

III. p-Bromophenylhydrazine Derivatives of d-Mannuronic Acid

In view of the insolubility and stability of d-mannose phenyl- and p-bromophenylhydrazone (42,44), it was not surprising to find that the corresponding d-mannuronic acid derivatives possessed similar properties. The barium p-bromophenylhydrazone d-mannuronate (Section III, a) is readily formed by Procedure 1 at room temperature and is insoluble in all of the common solvents. Like the corresponding derivative of d-galacturonic acid (see above) the hydrazone barium salt is suitable only for purposes of isolation. It is of interest to point out that barium d-mannuronate, when treated according to the procedure of Goldschmiedt and Zerner for the preparation of barium p-bromophenylosazone d-glucuronate (42), will invariably yield the hydrazone barium salt.8

The p-bromophenylhydrazone of d-mannuronolactone (Section III, b) is an exceedingly useful derivative. It is readily formed at room temperatures and is insoluble in water. Purification can readily be accomplished by recrystallization from 40 per cent ethyl alcohol. Its melting point is within the desirable range and it is strongly dextrorotatory. The high rotation of the p-bromophenylhydrazone of d-mannuronolactone (Section III, b), and p-bromophenylhydrazine p-bromophenylhydrazone d-mannuronate (Sec- tion III, c) serves to distinguish these compounds from the corresponding derivatives of d-galacturonic acid (see above).

* Barium d-mannuronate and phenylhydrazine under these conditions also form the barium salt of the phenylhydraxone (unpublished data of the authors).


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The p-bromophenylhydrazine salt of d-mannuronic acidp-bromophenylhydrazone (Section III, c) will form when free d-mannuronic acid is present in solution. Thus in many cases the treatment of a crude product, e.g. a mixture of d-mannuronic acid la&one and d-mannuronic acid, will yield this derivative contaminated with the hydrazone of the lactone. In such cases it is desirable to convert the mixture completely into the hydrazine salt by heating with a sufficient amount of a 40 per cent alcohol solution of p-bromophenylhydrazine. The hydrasine salt of the hydrazone (Section III, c) possesses all of the desirable qualities shown by the hydrazone of the lactone (Section III, b) and, as pointed out above, is easily distinguished from the corresponding compound of d-galacturonic acid. The p-bromophenylhydrazide of d-mannuronic acid p-bromophenylhydrazone (Section III, d) is readily prepared and easily purified. The physical constants are well within an easily discernible range and suggest the value of this derivative for both isolation and characterization purposes.

In general all of the p-bromophenylhydrazine derivatives of d-mannuronic acid described herein are suitable for the identification of this acid. Several attempts t.o prepare the p-bromophenylosazone led to intractable products which could not be purified.

SUMMARY

The preparation and properties of the following derivatives of d-galacturonic and d-mannuronic acid have been described: Sec- tion I, barium phenylhydrazone d-galacturonate (a), phenylhydrazine phenylhydrazone d-galacturonate (b), d-galacturonic acid phenylhydrazone (c), phenylhydrazine phenylosazone d-galacturonate (d), barium phenylosazone d-galacturonate (e); Sec- tion II, barium p-bromophenylhydrazone d-galacturonate (a), p-bromophenylhydrazine p-bromophenylhydrazone d-galacturonate (b), d-galacturonic acid p-bromophenylhydrazone (c), the p-bromophenylhydrazide p-bromophenylhydrazone of d-galacturonic acid (d); Section III, barium p-bromophenylhydrazone d-mannuronate (a), p-bromophenylhydrazone d-mannuronolactone (b), p-bromophenylhydrazine p-bromophenylhydrazone d-mannuronate (c), and the p-bromophenylhydrazide p-bromophenylhydrazone d-mannuronic acid (d). Their value as suitable derivatives for purposes of isolation and identification has been discussed.


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The behavior of barium d-galacturonate and barium d-mannuronate in the Goldschmiedt and Zerner reaction has been pointed out for the first time.

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LinkCarl Niemann, Eugene Schoeffel and Karl Paul

ACIDDERIVATIVES OF d-MANNURONIC

p-BROMOPHENYLHYDRAZINEACID AND

DERIVATIVES OF d-GALACTURONICp-BROMOPHENYLHYDRAZINE

ACIDS: PHENYLHYDRAZINE ANDDERIVATIVES OF THE HEXURONIC

SUBSTITUTED HYDRAZINE

1933, 101:337-358.J. Biol. Chem.

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