LIBRARY 
 
 New York State Veterinary College 
 
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 Levene, P. A, 
 
 Hexosaraines, Their Derivatives, and Mucins 
 and Mucoids 1922 
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 321 
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LIBRARY 
 NEW YORK STATE VETERINARY COLLEGE 
 
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 "lSmA^S.±i'J^?.?, and muci 
 
 
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 http://www.archive.org/details/cu31924000946792 
 
MONOGRAPHS OF 
 
 THE ROCKEFELLER INSTITUTE 
 
 FOR MEDICAL RESEARCH 
 
 No. 18 July 7, 1922 
 
 HEXOSAMINES, THEIR DERIVATIVES, AND MUCINS AND 
 
 MUCOIDS 
 
 By 
 P. A. LEVENE 
 
 ' NEW YORK 
 The RodKEFEtLER iNSrrryTE For Medicai research 
 ■■ 1922 
 
, GopskiGht, 1922, by ' 
 
 The ROCKEFELME-toSTlWra rOK, MeDICAI RESEAXciH 
 
 LIBRARY 
 
 NEW YORK STATE VETERINARY COLLEGE 
 
 ITHACA, N. Y. 
 
 , . WAVERLY PRESS 
 
 The WnxuMs & Waziss CowAire 
 Baliuiqiie, v. S. a. ' 
 
MONOGRAPH No. 18 
 
 HEXOSAMINES, THEIR 
 
 DERIVATIVES, AND 
 
 MUCINS AND 
 
 MUCOIDS 
 
 By 
 P. A. LEVENE, M.D. 
 
 NEW YORK 
 
 The Rockefeller Institute for Medical Research 
 
 1922 
 
A T r. • 
 
 
 
PREFACE. 
 
 The purpose of this monograph is not to offer a complete review of 
 all the literature on the subject, but to bring together that part of 
 it which has appeared from this laboratory from time to time in 
 the course of many years. Many of the earlier observations received 
 a clear explanation only in the light of subsequent work. To corre- 
 late aU the details might be a taxing enterprise for one who has not 
 been personally engaged in the work. 
 
 The work collected in this monograph was done with the coopera- 
 tion of Drs. F. B. La Forge, J. L6pez-Suirez, G. M. Meyer, I. Matsuo, 
 and Mr. E. P. Clark. The cooperation of Dr. La Forge was par- 
 ticularly valuable in the earlier part of thework;Dr. Lopez-Sudrez 
 has devotedly and skillfully cooperated in the part of mucins and 
 mucoids; Dr. Meyer took part in the work on the optical rotation 
 of hexonic acids; Dr. Matsuo assisted in the synthesis of 3-amino- 
 heptonic acids, and to Mr. Clark we are indebted for the preparation 
 of the pentoses which were required for the synthetic part of the work. 
 
 P. A. L. 
 
!.. 1 
 
MONOGRAPH OP THE ROCKEPELtEE rNSTITUTE FOR MEDICAL RESEARCH, NO. 18, 
 
 JULY 7, 1922 
 
 HEXOSAMINES, THEIR DERIVATIVES, AND MUCINS 
 AND MUCOIDS. 
 
 By p. a. LEVENE. 
 (From the Laboratories of The Rockefeller Institute for Medical Research.) 
 
 (Received for publication, July 11, 1921.) 
 
 CONTENTS. 
 
 Part I. 
 
 2-Aminohexoses and Their Derivatives, 
 
 Theoretical. 
 
 1. Conhguration of Carbon Atom 2 5 
 
 2. STRUCTinuE of the Ammonia Derivatives of Sugars 8 
 
 3. Preparation and Equilibrium of Epimers of Hexosaminic Acids 9 
 
 4. The Relation of Optical Rotation of Monocarboxylic Sugar 
 
 Acids and of Hexosaminic Acms 10 
 
 5. 2-5-Anhydrotetroxyadipic Acids and the Walden Inversion in the 
 
 Hexosaminic Series 13 
 
 6. Configuration of Chitose and Epichitose 16 
 
 7. 3-Aminoheptonic Acms 16 
 
 Experimental. 
 
 1. Conversion of Hexosaminic Acids into Their Epimers 16 
 
 a. Epichitosaminic Acid 18 
 
 2. Preparation of Hexosaminic Acid from /-Aminopentosides 19 
 
 a. Chondrosaminic and Epichondrosaminic Acids 20 
 
 b. Xylohexosaminic Acid 22 
 
 c. Ribohexosaminic Acid 25 
 
 3. Synthesis of Hexosamines 30 
 
 a. Chitosamine 30 
 
 b. Chondrosamine 32 
 
 c. Epichitosamine 36 
 
 d. Dextro-Xylohexosamine 37 
 
 4. 2-S-Anhydropentoxycaproic Acids 39 
 
 a. Anhydromannonic and Anhydrogluconic Acids 39 
 
 b. 2-5-Anhydrotalonic and Anhydrogalactonic Acids. Prepared 
 
 Only in the Form of Their Brucine Salts 40 
 
2 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 5. 2-S-Anhvdrotetroxyadipic Acids 43 
 
 a. 2-S-Anhydro-i-Saccharic Acid 43 
 
 b. 2-S-Anhydromaimosaccharic Add 45 
 
 c. 2-S-Anhydro4-Saccharic Acid 45 
 
 d. 2-5-Anhydroidosaccharic Acid 48 
 
 e. 2-5-Aiihydromucic Acid 48 
 
 f. 2-S-Anhydrotalomucic Acid 49 
 
 g. 2-5-AnhydroaUomucic Acid 53 
 
 6. CmTOSE AND Epichitose 54 
 
 7. 3-Aminoheptonic Acids 54 
 
 a. Chitosaminoheptonic Acid 54 
 
 b. Chondrosaminoheptonic Acid 58 
 
 Part II. 
 
 Mucins and Mucoids. 
 Theoretical. 
 
 1. General Considerations 61 
 
 2. Structure of Chondroitin Sulfuric Acid 63 
 
 3. Structure of Mucoitin Sulfuric Acid 65 
 
 4. Distribution of the Acids of Various Types 66 
 
 Experimental. 
 
 1. Group I. Chondroitin Sulfuric Acid 66 
 
 I. From Chondromucoid 66 
 
 a. Preparation of Chondroitin Sulfuric Acid 66 
 
 b. Estimation of Acetyl Groups 68 
 
 c. Preparation of Chondrosin 69 
 
 d. Preparation of Chondridin 70 
 
 e. Preparation of Desaminochondrosin 72 
 
 f. Identification of Glucuronic Acid 72 
 
 g. Nitric Acid Oxidation of the Products of Hydrolysis of 
 
 Chondrosin 75 
 
 h. Nitric Acid Oxidation of Chondrosin and Subsequent 
 
 Hydrolysis 75 
 
 i. Identification of Chondrosamine 77 
 
 j. Phenylosazone of Chondrosamine 79 
 
 k. Preparation of Chondrosaminic Acid from Chondrosamine. 80 
 
 1. Preparation of 2-S-Anhydrotalonic Acid 81 
 
 m. Oxidation of 2-5-Anhydrotalonic to 2-5-Anhydrotalomucic 
 
 Acid 81 
 
 n. Oxidation of Chondrosamine to 2-5-Anhydrotalomucic 
 
 Acid 81 
 
p. A. LEVENE 3 
 
 o. Preparation of 2-5-Anhydrogalactonic Acid 82 
 
 p. Conversion of 2-S-Anhydrotalomucic into Anhydromucic 
 
 Acid 82 
 
 q. Conversion of 2-S-Anhydrotalomucic into Pyromucic Acid. 82 
 
 II. From Tendomucoid 82 
 
 a. Preparation of TendomucoiB. and the Chondroitin Sulfuric 
 
 Acid Contained in it 83 
 
 b. Chondrosin 84 
 
 c. Chondrosamine Hydrochloride 84 
 
 d. Glucuronic Acid Osazone Hydrazide 85 
 
 III. From Aorta Mucoid 85 
 
 a. Preparation of Chondroitin Sulfuric Acid 85 
 
 b. Preparation of Chondrosamine 86 
 
 c. Preparation of Chondrosin 86 
 
 IV. From Sclera Mucoid 87 
 
 a. Preparation of the Chondroitin Sulfuric Acid 87 
 
 b. Preparation of Chondrosamine 88 
 
 2. Grotjp II A. MucoiTiN SuLrmuc Acids 89 
 
 I. From Funis Mucin 89 
 
 a. Preparation of Mucoitin Sulfuric Acid 89 
 
 b. Preparation of Mucosin 90 
 
 c. Preparation of Chitosamine 91 
 
 d. Identification of Glucuronic Acid 92 
 
 e. Oxidation with Nitric Acid with Subsequent Hydrolysis 93 
 
 f. Estimation of the Number of Acetyl Groups 94 
 
 II. From Vitreous Mucoid 94 
 
 a. Preparation of Mucoitin Sulfuric Acid 94 
 
 b. Preparation of Chitosamine 95 
 
 c. Furfural Distillation 95 
 
 III. From Cornea Mucoid 96 
 
 a. Preparation of Mucoitin Sulfuric Acid 96 
 
 b. Preparation of Chitosamine 96 
 
 3. Group II B. Mucoitin Sulfuric Acids 97 
 
 I. From Mucin of the Gastric Mucosa 97 
 
 a. Preparation of Mucoitin Sulfuric Acid 97 
 
 b. Preparation of Chitosamine 98 
 
 c. Oxidation of Chitosamine 99 
 
 d. Preparation of Mucosin 100 
 
 e. Furfural Distillation 100 
 
 f. Hydrolysis by Sodium Amalgam 100 
 
 g. Acetyl Estimation 101 
 
 II. From Serum Mucoid 101 
 
 a. Preparation of Mucoitin Sulfuric Acid 101 
 
 b. Preparation of Chondrosamine 102 
 
PART I. 
 
 2-AMINOHEXOSES AND THEIR DERIVATIVES. 
 
 THEORETICAL. 
 
 1. CONFIGURATION OF CARBON ATOM 2. 
 
 The present work was undertaken with the object of procuring 
 data for the purpose of identification of 2-aminohexoses. When 
 this work was undertaken only one representative of this group 
 was known, namely chitosamine (glucosamine). All the details 
 of the configuration of chitosamine have been explained save one, 
 namely that of the configuration of carbon atom 2. The difficulties 
 which were in the way of solving the problem have as yet not been 
 overcome. They are brought about principally by the phenomenon 
 of Walden inversion. From the work of Fischer it was known that 
 when glucosamine was deaminized into chitose and this further 
 oxidized to the corresponding acid, chitonic acid formed. On the 
 other hand, when chitosamine was first oxidized to chitosaminic 
 acid and this subsequently deaminized, chitaric acid formed. 
 Fischer (1911) surmised that the acids were epimeric, but the config- 
 uration of either one of them was not disclosed by him. Irvine and 
 his coworkers (1911, 1912, 1914) have shown that from chitosamine 
 may be derived either glucose or mannose, depending on the condi- 
 tion of the experiment. The difficulties which lay in the way of the 
 discovery of the configuration of carbon atom 2 in chitosamine 
 could be foreseen to recur whenever the question arose as to the con- 
 figuration of carbon atom 2 of any other 2-aminohexoses. 
 
 Thus the problem of identification of any one of the still unknown 
 2-aminohexoses fell into two parts, one dealing with the configura- 
 tion of carbon atoms 3, 4, and 5, and the other dealing with the 
 configuration of carbon atom 2. A priori, the way towards the 
 solution of the first part of the problem seemed clear, whereas great 
 difficulties were foreseen in connection with the problem of the config- 
 uration of carbon atom 2. 
 
 S 
 
6 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Since the configuration of carbon atom 2 is identical in a sugar 
 and in the corresponding hexonic acid, the solution of the problem 
 of the configuration of one is in itself a solution of the configuration 
 of the other. The configuration of carbon atoms 3, 4, and 5 of any 
 natural hexosamine can be easily disclosed as soon as the synthesis 
 of the entire series of the 2-aminohexonic acids and the corresponding 
 anhydrotetroxyadipic acids is accomplished. 
 
 The question as to the configuration of carbon atom 2 cannot as 
 yet be answered by direct chemical evidence. Hence, one is justified 
 in the use of indirect evidence. 
 
 The entire series of <i-2-aminohexonic acids has now been prepared 
 synthetically. Certain analogies in the reactions leading to their 
 synthesis and in the properties of the corresponding hexonic and 
 hexosaminic acids were looked into. These analogies were then 
 
 TABLE I. 
 
 From. 
 
 Predominating form. 
 
 From. 
 
 Predominating form. 
 
 Arabinose. 
 Lyxose. 
 Xylose. 
 Ribose. 
 
 Mannonic acid. 
 Galactonic acid. 
 Gulonic acid. 
 Altronic acid. 
 
 Arabinoside. 
 Lyxoside. 
 Xyloside. 
 Riboside. 
 
 ChitosamTTiic acid. 
 Epichondrosaminic acid. 
 Dextro-xylohexosaminic acid. 
 Levo-ribohexosaminic acid. 
 
 utilized for a provisional explanation of the configuration of carbon 
 atom 2 in the 2-aminohexonic acids and hence in the corresponding 
 sugars. 
 
 The first analogy to which attention was drawn is the parallelism 
 in the equilibria of the two epimers which form on the condensation 
 of prussic acid with pentoses on the one hand and with amino 
 pentosides on the other. This is shown in Table I. 
 
 This parallelism, of course, is in itself no proof that the predominat- 
 ing forms in both series have an analogous configuration of their 
 carbon atom 2. It is, however, permissible to make this assump- 
 tion, and then to inquire to what extent other properties of hexo- 
 saminic acids are consistent with the assumption. 
 
 Attention was then directed to the optical rotation of the hexonic 
 and hexosaminic acids. First it was discovered that in a pair of 
 
p. A. LEVENE 
 
 hexonic acids the rotation of carbon atom 2 was to the right when 
 its hydroxyl was in the same position as in gluconic, and to the left 
 when its position was as in mannonic. Analyzing the optical rota^ 
 tions of the hexosaminic acids, it was observed that also in each 
 pair of epimers of this series carbon atom 2 rotated to the right in 
 one epimer and to the left in the other. A comparison of the pre- 
 dominating forms from the viewpoint of the rotation of their carbon 
 atom 2 gives the results shown in Table II. 
 
 The rotation of carbon atom 2 determines the direction of the 
 rotation of the hexonic acids. If the same rule were applicable also 
 to aminohexonic acid, one would observe that the conclusions reached 
 on the basis of the equilibrium of the two epimers are identical with 
 those based on the direction of the rotation of carbon atom 2. The 
 fact that the two assumptions are mutually supporting adds weight 
 to the original assumption. 
 
 TABLE II. 
 
 From. 
 
 Rotation of carbon atom 2 
 in predominating form. 
 
 From. 
 
 Rotation of carbon atom 2 
 in predominating form. 
 
 Arabinose. 
 Lyxose. 
 Xylose. 
 Ribose. 
 
 Levo. 
 
 u ^ 
 
 Dextro. 
 Levo. 
 
 Arabinoside. 
 Lyxoside. 
 Xyloside. 
 Riboside. 
 
 Levo. 
 (I 
 
 Dextro. 
 Levo. 
 
 E. Fischer and his coworkers in their work on the Walden inversion 
 have observed that when an amino-acid and its ester are acted upon 
 by nitrous acid, the inversion as a rule occurs in the acid but not in 
 the ester. Admitting that this rule holds also for hexosaminic acids, 
 one would expect that on deamination they will form acids rotating 
 in the direction opposite to that of the original amino-acid. This 
 expectation was realized for every one of the eight hexosaminic acids. 
 On the other hand, when not the acid but the sugar or the acid lac- 
 tone was deaminized, as a rule no change in the direction of the rota- 
 tion was observed. Thus also the observations on the Walden 
 inversion are consistent with the original assumption based on the 
 equilibrium of the epimeric forms. It follows that on the basis of 
 every one of the three assumptions, the configuration of the 2-amino- 
 hexonic acids is as shown in Table III. If eventually by direct 
 
8 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 evidence the configuration of carbon atom 2 in any one of these 
 acids should be found the reverse of that postulated above, then 
 our conception of the configuration of the same carbon atom in 
 every other hexosaminic acid will have to be reversed. In other 
 words, the knowledge of the configuration of any one of the 2-amino- 
 hexonic acids is sufl&cient to explain the configuration of every mem- 
 ber of the entire series. 
 
 TABLE III. 
 
 Chitosaminic acid 2-ammomaimonic acid. 
 
 Epichitosaminic acid , 2-aminogluconic acid. 
 
 Chondrosaminic acid 2-aminotaloiuc acid. 
 
 Epichondiosaminic acid 2-aminogalactonic add. 
 
 Dextro-ii-xylohexosaminic acid 2-amiiiogulomc acid. 
 
 Levo-i-xylohexosaminic acid 2-aminoidomc acid. 
 
 Dextro-<2-ribohexosaminic acid 2-aminoaUomc acid. 
 
 Levo-d-ribohexosaminic acid 2-aminoaltromc add. 
 
 2. STRUCTURE OF THE AMMONIA DERIVATIVES OF SUGARS. 
 
 Three alternative structures have been suggested for these deriva- 
 tives. 
 
 CH20H(CHOH)2 CH CH OH (J) 
 
 NH 
 CH8(0H) CHOHCH (CH0H)2CH OH (II) 
 
 NH 
 CHiOHCHOH CH (CHOH)j CH NH, (HI) 
 
 O 
 
 Structure (I) is advanced by Lobry de Bruyn, (II) by Wohl, 
 and (III) by Irvine, Thomson, and Garret. Hitherto none of 
 these theories was supported by experimental evidence. This was 
 furnished in the course of the present work (Levene, 1916, b); 
 namely, it was demonstrated that the nitrogen in these compounds 
 is present in the form of a primary amino group (Table IV). To 
 
p. A. LEVENE 
 
 the butylene ring, preference is given over the ethylene structure 
 because of the analogy with the structure of other glucosides. 
 
 TABLE IV. 
 
 Relation of Total Nitrogen to Amino Nitrogen in Aminopentosides. 
 Theory =9. 40 per cent. 
 
 
 Total N 
 
 NH.N 
 
 Aminoriboside 
 
 fer cent 
 
 9.73 
 9.06 
 9.64 
 
 ter cent 
 9.39 
 
 Aminolyxosidc 
 
 8.78 
 9.16 
 
 
 
 3. PREPARATION AND EQUILIBRIUM OF EPIMERS OF HEXOSAMINIC 
 
 Acms. 
 
 When the natural hexosamine is readily accessible, the oxidation 
 of it with bromine or with mercuric oxide often leads to the hexo- 
 saminic acid (Pringsheim and Ruschmann, 1915). Conditions of 
 oxidation have to be adapted to each individual sugar. The meth- 
 od sometimes fails. Of the three crystalline hexosamines prepared 
 up to the present date, two were oxidized readily; the third, epi^ 
 chitosamine, could not be oxidized to the corresponding acid. 
 
 When one 2-aminohexonic acid is obtained from a natural sugar, 
 its epimer is readily prepared by heating with pyridine in the same 
 manner as in the case of the formation of talonic from galactonic 
 acid. Epichitosaminic acid was prepared in this way (Levene, 
 1917, b). 
 
 The synthesis of 2-aminohexonic acids from aminopentosides is 
 accomplished by its condensation with prussic acid. Fischer and 
 Leuchs have applied the process for the synthesis of chitosaminic 
 acid from the aminoarabinoside. The yield, in their experience, 
 was 10 per cent of the riboside. It was observed in the course of 
 the present work that the conditions of the reaction must be 
 adapted to each pentoside. The temperature and duration of the 
 reaction are the conditions which determine the success of the opera- 
 tion. When the optimal conditions are found, the yield of the acid 
 is about 55 per cent of the employed pentoside. 
 
10 HEXOSAMINES, MUCINS, AJSTD MUCOIDS 
 
 The separation of the two epimers formed by the synthesis can 
 be accomplished by fractional crystallization. For final purification, 
 whenever possible the acids are converted into their crystalline 
 derivatives. The behavior of the acids towards benzaldehyde was 
 exploited for this purpose. Individual acids react differently with 
 this reagent. Often one epimer forms a benzilidene derivative, 
 whereas the other epimer under identical conditions does not com- 
 bine with the aldehyde and in its place a non-substituted lactone is 
 formed. Thus chitosaminic acid forms a benzilidene-chitosaminic- 
 ethyl-ester; its epimer forms a lactone. Dextro-J-xylohexosaminic 
 gave a dibenzilidene-xylohexosaminic-ethyl-ester; its epimer, a mono- 
 benzilidene lactone. Dextro-J-ribohexosaminic gave the dibenzili- 
 dene lactone; its epimer, the lactone. 
 
 Lyxo-<i-hexosaminic acids do not form crystalline lactones, esters, 
 or benzilidene derivatives, but the levo-rf-xylohexosaminic on reduc- 
 tion forms a crystalline hydrochloride of the sugar, whereas the 
 epimer does not do so. 
 
 Thus it was possible to obtain sufiicient proof of the purity of each 
 individual epimer of four pairs of (i-hexosaminic acids. The equilib- 
 ria of the two epimers derived on the one hand from each pentose, 
 and on the other from the corresponding aminopentoside, are com- 
 pared in Table I. 
 
 4. RELATION OF OPTICAL ROTATION OF EPIMERIC MONOCARBOXYLIC 
 SUGAR ACIDS AND OF HEXOSAMINIC ACIDS. 
 
 According to van't HoflE's theory, substances containing several 
 asymmetric carbon atoms possess a rotatory power equal to the 
 algebraic sum of the rotations of each group. Hudson (1909) has 
 successfully applied van't Hofi's theory to the study of the con- 
 figuration of the terminal carbon atom in glucosides. In applica- 
 tion to the monocarboxylic acids, van't Hoff's rule could be made 
 use of in the following way (Levene, 1915; Levene and Meyer, 1916, 
 1917). Given two epimeric acids differing in the configuration of 
 their carbon atom 2, such as 
 
p. A. LEVENE 11 
 
 1 COOH COOH 
 
 2 OHCH HCOH 
 
 3 OHCH OHCH 
 
 4 HCOH HCOH 
 
 5 HCOH HCOH 
 
 6 CHjOH CHsOH 
 
 the part of the molecule containing cafbon atoms 1 and 2 may be 
 
 designated by the letter B, and the part containing carbon atoms 3, 
 
 4, 5, and 6 by the letter A. The algebraic sum of one epimer is 
 
 A 4- B, and of the other A - B. From this it follows that if 
 
 . _. , . m + n 
 A + B = m, and A — B = n, then A = — 
 
 ^ 2m — m — n m — n 
 
 B = m - A = - 
 
 B = n- A = 
 
 2 2 
 
 2n — m — n m— n 
 
 2 2 
 
 In this manner the optical rotation of carbon atom 2 was calcu- 
 lated in derivatives of several pairs of sugar acids. The results are 
 presented in Tables V and VI. 
 
 From these data it is evident that there exists a constant correla- 
 tion between the configuration and the direction of the optical 
 rotation of carbon atom 2. In the acids with an allocation of the 
 carbon atom as in gluconic add, carbon atom 2 rotates to the right, 
 and in those with the carbon atom as in mannonic, to the left. 
 
 Later, Hudson and his coworkers (1917, 1918, 1919), and also 
 Weerman, calculated the optical rotation of carbon atom 2 in a series 
 of sugar acids, and found that the direction of the rotation of carbon 
 atom 2 determines the direction of the rotation of the acid. The 
 rule of Hudson holds for most sugar acids, but is not as general as 
 the first rule of the present writer. For the 2-S~anhydro acids the rule 
 of Hudson does not hold, whereas the original rule holds. Whether 
 the rule of Hudson or of the present writer is applied, the identical 
 conclusion is reached regarding the correlation of the configura- 
 tion of carbon atom 2 and the optical rotation of the acid. In the 
 dextrorotatory acids, the configuration of this carbon is as in glu- 
 conic, and vice versa in the levorotatory as in mannonic. 
 
12 
 
 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 
 TJ 
 
 VBLE V. 
 
 
 
 Inorganic salts. 
 
 C 
 
 [«]" 
 
 Rotation of 
 carbon 
 atom 2, 
 
 M" 
 
 rf-Na gluconate 
 
 20 
 10 
 10 
 10 
 10 
 10 
 10 
 10 
 10 
 10 
 
 +11.78 
 
 - 8.82 
 +12.68 
 
 - 2.52 
 + 0.40 
 
 Not determined. 
 + 4.30 
 + 4.05 
 +70.29 
 +33.65 
 
 +10.29 
 -10.29 
 + 7.60 
 
 - 7.60 
 
 + 4.20 
 
 - 4.20 
 +18.32 
 -18.32 
 
 +22.42(102) 
 
 
 -22.42 (102) 
 
 " gulonate 
 
 +16.55 (102) 
 
 
 -16.55 (102) 
 
 " galactonate 
 
 
 
 
 " alienate 
 
 + 9.15(102) 
 
 " altronate 
 
 - 9.15(102) 
 
 
 +39.72 (102) 
 
 " chitonate 
 
 -39.72 (102) 
 
 
 
 TABLE VI. 
 
 Brucine salts. 
 
 M.P. 
 
 C 
 
 Hn 
 
 Rotation of 
 carbon 
 atom 2. 
 
 M" 
 
 rf-Gluconate 
 
 'C. 
 155 
 212 
 162-4 
 188 
 170 
 13? 
 160 
 158 
 195 
 222 
 
 2.5 
 2.5 
 2.5 
 2.5 
 2.5 
 2.5 
 2.5 
 2.5 
 5.0 
 5.0 
 
 -15.95 
 -25.70 
 -19.59 
 -25.79 
 -21.01 
 -26.15 
 -21.28 
 -23.82 
 
 - 2.96 
 
 - 8.47 
 
 +4.85 
 
 -4.85 
 
 +3.1 
 
 -3.1 
 
 +2.57 
 
 -2.57 
 
 +1.28 
 
 -1.28 
 
 +2.72 
 
 -2.72 
 
 +28.50 (102) 
 
 
 -28.50 (102) 
 
 
 +18.20 (102) 
 -18.20 (102) 
 +15.10(102) 
 
 d-ldonsXe 
 
 
 
 -15.10(102) 
 + 7.51 (102) 
 - 7.51 (102) 
 +14.15(102) 
 -14.15(102) 
 
 d-Allonate 
 
 (f- Altronate 
 
 Chitarate .. . . 
 
 
 
 TABLE VII. 
 
 Add. 
 
 [«]n°f 
 carbon 
 atom 2. 
 
 M" 
 
 Phenyl- 
 hydrazide. 
 
 carbon 
 atom 2. 
 
 M" 
 
 Epichitosaminic 
 
 +12.5 
 
 -12.5 
 
 + 12.5 
 
 -12.5 
 
 +12.5 
 
 -12.5 
 
 +19.12 
 
 -19.12 
 
 +24.37 (102) 
 -24.37 (102) 
 +24.37 (102) 
 -24.37 (102) 
 +24.37 (102) 
 -24.37 (102) 
 +37.28(102) 
 -37.28 (102) 
 
 Gluconic. 
 
 Mannonic. 
 
 Gulonic. 
 
 Idonic. 
 
 Galactonic. 
 
 Talonic. 
 
 AUonic. 
 
 Altronic. 
 
 + 14.25 
 -14.25 
 +14.25 
 -14.25 
 + 8.25 
 - 8.25 
 +20.8 
 -20.8 
 
 +42.18(102) 
 -42.18(102) 
 +42.18(102) 
 -42.18(102) 
 +24.42 (102) 
 -24.42 (102) 
 +61.56(102) 
 -61.56(102) 
 
 Dextro-xylohexosaminic 
 Levo-xylohexosaminic. . 
 
 Epichondrosaminic 
 
 Chondrosaminic 
 
 Dextro-ribohexosaminic 
 Levo-ribohexosaminic. . 
 
p. A. LEVENE 13 
 
 Configuration and Rotation of Epimeric Hexosaminic Acids. — All 
 of the eight possible d-hexosaminic acids have been prepared. Ap- 
 plying the above method for calculating the value and the direction 
 of the rotation of carbon atom 2, it is observed that in the 2-am- 
 inohexonic acid also the rotation of carbon atom 2 is to the right in 
 one of the epimers and to the left in the other. Comparing the 
 numerical value of the rotation of this carbon atom in hexosaminic 
 adds and the value of the rotation of the same carbon atom in the 
 phenylhydrazides of the corresponding hexonic acids, one is struck by 
 the identity of the values in the two series. The exception in the pair 
 of galactonic-talonic epimers is undoubtedly due to the fact that 
 talonic acid has not yet been prepared in pure state. The values are 
 identical for the specific, but not for the molecular rotations. 
 
 Table VII contains the values of the rotations of carbon atom 2 
 in the hexosaminic acids and in the corresponding phenylhydrazides 
 of hexonic acids. 
 
 5. 2-5-ANHYDROTETEOXYADIPICl ACIDS AND THE WALDEN INVERSION 
 IN THE HEXOSAMINIC SERIES. 
 
 Acting with the same reagent, nitrous acid, on a given hexosa- 
 mine, it is possible to arrive at two epimeric acids. It was men- 
 tioned in the introductory chapter that Fischer obtained from chito- 
 samine either chitonic or chitaric acid, depending on the order of 
 the two reactions which are required to accomplish the transforma- 
 tion. When chitosamine was first deaminized into chitose and this 
 subsequently oxidized into an acid, chitonic acid was the product. 
 When chitosamine was first oxidized into chitosaminic acid and 
 subsequently deaminized, chitaric acid was formed. Since the 
 configuration of neither of the two acids was known, it remained 
 impossible to determine in which of the two the direction of the 
 rotation of carbon atom 2 was reversed from that of the original 
 hexosamine. Chitonic and chitaric acids are 2-S-anhydrohexonic 
 acids. It became necessary to establish the configuration of the 
 series of these acids in order to establish in which of the two hydroxy 
 acids formed from a hexosaminic acid, the rotation of carbon atom 2 
 was in a direction opposite to that of carbon atom 2 in the parent 
 hexosaminic acid. 
 
 ' The carbon atoms are numbered as in the corresponding hexoses. 
 
14 
 
 HEXOSAMINES, MTTCINS, AND MUCOIDS 
 
 The configuration of carbon atoms 2, 3, 4, and 5, remains the same 
 in hexoses and in the corresponding hexonic and tetroxyadipic acids. 
 Similarly, it remains the same in anhydrohexonic and anhydro- 
 tetroxyadipic acids. The configuration of the series of hexoses was 
 made clear by Fischer on the basis of the mutual relationship of 
 tetroxyadipic acids. In a similar way the configuration of 2-5- 
 anhydrohexonic acids was established by the mutual relationship 
 of the 2-5-anhydrotetroxyadipic acids. 
 
 The trend of thought which led to the solution of the problem is 
 seen from the following structures. 
 
 HCNHj 
 
 ohch\ 
 
 I I 
 
 HCOH O 
 
 I I 
 
 HC 1 
 
 CH,OH 
 
 J-Aminoarabinoside 
 
 /I 
 / 
 
 COOH COOH 
 
 HC- 
 
 1 
 OHCH 
 
 I 
 HCOH 
 
 Hi- 
 
 o 
 
 o 
 
 COOH 
 
 — CH 
 
 OHCH 
 
 I 
 HCOH 
 
 I 
 HC 
 
 COOH 
 
 2-S-Anhydro- 2-S-Anhydro- 
 <2-Saccharic (f-Manuosac- 
 charic 
 
 V VI 
 
 HCNHj 
 HCOH^i 
 OHCH O 
 
 HC ! 
 
 I 
 CH2OH 
 
 ^-Aminoxyloside 
 
 /"\ 
 
 COOH COOH 
 
 HC : 
 
 I 
 
 HCOH I 
 
 OHCH 
 
 I 
 HC- 
 
 O 
 
 CH 
 
 I 
 HCOH 
 
 I 
 OHCH 
 
 O I 
 HC 
 
 COOH 
 
 COOH 
 
 2-5-Anhydro- 2-5-Anhydro- 
 i-Saccharic (f-Idonic 
 
 HCNHj 
 
 ohch\ 
 
 I I 
 
 OHCH O 
 
 i— ! 
 
 I 
 CH2OH 
 
 (2-Anuiiolyxoside 
 ''IH^ 
 
 O 
 
 VII 
 
 VIII 
 
 COOH 
 
 I 
 HC 1 
 
 I I 
 OHCH 
 
 I 
 OHCH 
 
 I 
 HC- 
 
 I 
 COOH 
 
 2-5-Anhydro- 2-S-Anhy- 
 
 <J-Mucic dro-i-Talo- 
 
 mucic 
 
 IX X 
 
 COOH 
 
 I 
 CH 
 
 I 
 OHCH 
 
 I 
 OHCH 
 
 I 
 HC— 
 
 COOH 
 
 HCNHj 
 
 HCOH^ 
 HCOH O 
 
 HC ! 
 
 I 
 CH2OH 
 
 d-Aminoriboside 
 
 ^iv\ 
 
 COOH COOH 
 
 I I 
 HC r — CH 
 
 I 
 
 O 
 
 HCOH 
 
 1 
 HCOH 
 
 HCOH I 
 I O 
 
 HCOH I 
 I 
 
 HC ! 
 
 I 
 COOH 
 
 2-5-Anhy- 2-S-Aiihy- 
 dro-d-AUonic dro-d-Talo- 
 
 HC— 
 
 I 
 COOH 
 
 XI 
 
 mucic 
 
 xn 
 
 It is seen that Structures (V) and (VI) are antipodal anliydro- 
 saccharic acids, hence (VI) is mannosaccharic, and (VIII) idosac- 
 charic. Of the pair of anhydro acids derived from the lyxoside, 
 the inactive form is dihydromucic (IX) and the other is identical 
 with (XII) derived from the riboside and has the structure of dihydro- 
 d-talomucic. The second epimeric form from the riboside has the 
 configuration of anhydro-(i-allomucic. Thus the configuration of the 
 anhydrotetroxy acids was established and simultaneously the config- 
 
p. A. LEVENE 
 
 15 
 
 uration of the corresponding 2-S-anhydrohexonic adds and of the 
 2-S-anhydrohexoses was made clear. 
 
 Since it is permissible to refer to the anhydrotetroxyadipic instead 
 of to the anhydrohexonic acid, it is possible to compare the rota- 
 tion of carbon atom 2 in these and in the hexosaminic acids from 
 which they are derived. 
 
 From Table VIII it is seen that the direction of the rotation of 
 carbon atom 2 is reversed when the 2-aminohexonic acid is converted 
 
 TABLE vin. 
 
 
 
 Rotation 
 
 
 Rotation 
 
 
 Add. 
 
 of carbon 
 atom 2. 
 
 Obtainable from. 
 
 of carbon 
 atom 2. 
 
 Anhydro-(i-gluconic* 
 
 Right. 
 
 Chitosaminic acid. 
 
 Left. 
 
 
 (chitaric). 
 
 
 
 
 u 
 
 -if-mannonic 
 (chi tonic). 
 
 Left. 
 
 Chitosamine. 
 
 tt 
 
 
 tc 
 
 tt 
 
 Epichitosaminic acid. 
 
 Right. 
 
 
 it 
 
 It 
 
 Epichitosamimc lactone. 
 
 (t 
 
 
 -d-gulonic. 
 
 Right. 
 
 Levo-xylohexosaminic acid. 
 
 Left. 
 
 
 tt 
 
 tt 
 
 Dextro-xylohexosaminic lactone. 
 
 Right. 
 
 
 -(f-idonic. 
 
 Left. 
 
 Dextro-xylohexosaminic acid. 
 
 ft 
 
 
 -d-galactonic. 
 
 Right. 
 
 Chondrosaminic acid. 
 
 Left. 
 
 
 -d-talonic. 
 
 Left. 
 
 Chondrosamine . 
 
 it 
 
 
 tt 
 
 It 
 
 Epichondrosaminic acid. 
 
 Right. 
 
 
 -d-altronic. 
 
 tt 
 
 Dextro-ribohexosaminic acid. 
 
 te 
 
 
 -(2-allonic. 
 
 Right. 
 
 Levo-i-ribohexosaminic acid. 
 
 Left. 
 
 * Epichitosamine ought to form the same anhydro acid; however, it is the only 
 substance of this group that does not form the anhydro substance. On oxidation 
 it gives rise to saccharic acid. From the viewpoint of configuration the result 
 is as expected. 
 
 into the 2-5-anhydrohexonic acid. In the three instances when the 
 amino sugar was deaminized prior to oxidation, the direction of the 
 rotation of carbon atom 2 remained unaltered. Of the two occasions 
 when lactones were deaminized, in one the direction of the rotation 
 of carbon atom 2 was reversed, in the other not. 
 
 Thus, starting from the hexosamine, it is possible to arrive at two 
 epimeric 2-S-anhydrohexonic acids. Consequently a Walden inver- 
 sion has taken place on one occasion, probably when the hexosaminic 
 acid was deaminized. 
 
16 
 
 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 6. CONFIGURATION OF CHITOSE AND EPICHITOSE. 
 
 It has been known for a long time that on treatment with nitrous 
 acid a nitrogen-free 2-5-anhydrohexose was formed. The configura- 
 tion of the sugar remained unknown. On the basis of the present 
 work (Levene and La Forge, 1915, b; Levene, 1919), namely in view 
 of the fact that chitose is oxidized to 2-5-anhydromannonic acid, 
 it is evident that chitose has the configuration of 2-5-anhydro- 
 mannose. 
 
 Similarly the new anhydrohexose prepared in the course of this 
 work from epichitosamine has the structure of 2-5-anhydroglucose. 
 
 HC- 
 
 I 
 HC- 
 
 / 
 
 OH 
 
 / 
 
 OH 
 
 HC- 
 
 OHCH 
 
 O 
 
 HC- 
 
 I 
 HC- 
 
 -CH 
 
 CHjOH 
 
 2-5-Aiihydroglucose 
 (Epichitose) 
 
 OHCH 
 
 I 
 HC— 
 
 I 
 HC— 
 
 CH2OH 
 
 2-5-Aiihydromaimose 
 (Chitose) 
 
 7. 3-AMINOHEPTONIC ACIDS. 
 
 In conclusion, mention may be made of the synthesis of the two 
 pairs of epimeric 3-aminoheptonic acids, namely 3-aminochitohep- 
 tonic and 3-aminochondroheptonic acids. These acids were pre- 
 pared with the object of determining the configuration of their 
 deaminized derivatives, as these in their turn may be of service in 
 connection with further work on the configuration of the corre- 
 sponding 2--aminohexonic acids. 
 
 EXPERIMENTAL. 
 1. CONVERSION OF HEXOSAMINIC ACIDS INTO THEIR EPIMERS. 
 
 This change can be accomplished by heating with an aqueous 
 solution of pyridine at 100-105°C. for 4 hours (Levene, 1918, a; 
 1916, e). 
 
p. A. LEVENE 17 
 
 In a preliminary way the formation of the epimer was shown by 
 the fact that from both chitosaminic and chondrosaminic acids on 
 heating with pyridine, fractions were obtained with a distinctly 
 different optical rotation, thus: 
 
 Add. 
 
 Original. 
 
 After treatment with pyridine. 
 
 Chitosaminic 
 
 -15.02 
 -16.15 
 
 +2.11°, +6.75°, +7.72°, +8.57°' 
 
 Chondrosaminic 
 
 -4.83° 
 
 
 
 Later by this process from pure chitosaminic, epichitosaminic 
 acid was obtained. The details are as follows: 
 
 100 gm. of chitosaminic acid are taken up in 1,000 cc. of water to 
 which 100 gm. of pyridine are added and heated in an autoclave at 
 105°C. for 4 hours. The solution is then evaporated under dimin- 
 ished pressure to a small volume, so that the greater part of chito- 
 saminic acid crystallizes in the distillation flask. The residue is 
 again brought into solution with a minimum amount of boiling water. 
 To the solution while still hot an equal volume of 95 per cent alcohol 
 is added. The solution then becomes filled with crystals of chito- 
 saminic acid. To complete the crystallization the material is 
 allowed to stand from 8 to 12 hours. 
 
 The filtrate is then concentrated under diminished pressure 
 (the temperature of the water bath should not exceed S0°C.) to a 
 small volume. To this, hot 95 per cent alcohol is added until the 
 solution begins to show opalescence, and enough hot water is then 
 added to clarify the solution. On standing, more readily after some 
 scratching, a crystalline substance begins to deposit. The deposit 
 reaches its maximum in about 24 hours. This substance is dissolved 
 in a minimum amount of hot water, boiled with charcoal, and the 
 fiJtrate treated with 95 per cent alcohol in the manner just described. 
 The crystalline substance obtained is at times nearly colorless, and 
 at times of very light tan. It consists of a mixture of varying pro- 
 portions of chitosaminic and epichitosaminic acids. On one occa- 
 sion it consisted of pure chitosaminic acid. The optical rotation of 
 the substance varied from [a]" = -f-3.0° to +S.0°, and on one oc- 
 casion it had [a]o = 4-10.0°. 
 
18 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 For further purification the epichitosaminic acid was converted 
 into its lactone. This was accomplished in the following way: 
 to 3 gm. of the acid 2 cc. of 99.5 per cent alcohol and ice. of benzalde- 
 hyde were added, and dry hydrochloric acid gas was passed. The 
 acid first goes into solution and subsequently a crystalline deposit 
 begins to reappear. The treatment with the gas is continued for 
 approximately 7 minutes. After standing 8 to 12 hours, 3 cc. of 
 dry ether (dried over sodium) were added, and the material was 
 allowed to stand an additional 12 or 24 hours in order to obtain the 
 maximum yield of the lactone hydrochloride. The addition of 
 benzaldehyde has for its purpose the conversion of the chitosaminic 
 acid into the benzal of its ethyl ester. Under these conditions it 
 remains in solution and permits the crystallization of the epichito- 
 saminic acid lactone. In the absence of benzaldehyde an amorphous 
 precipitate is formed which probably consists of a mixture of the 
 two lactones. 
 
 The 'lactone is crystallized out of methyl alcohol. For this pur- 
 pose it is advisable to dissolve the crude substance in a large excess 
 of hot methyl alcohol, to decolorize the solution with charcoal, and 
 then to concentrate it to a small volume. The lactone crystallizes 
 in the form of colorless prismatic needles. The substance had a 
 melting point of 203°C. (with gas evolution). Its composition was 
 as follows: 
 
 0.1040 gm. substance: 0.1286 gm. CO2 and 0.1040 gm. H2O. 
 0.1000 " " required (Kjeldahl) 4.65 cc. 0.1 n acid. 
 
 0.1000 " " " (Volhard) 4.50 " 0.1 n AgNOs. 
 
 CeHuNOsCl. Calculated. C 33.71, H 5.67, N 6.55, CI 16.25. 
 Found. " 33.72, " 5.47, " 6.51, " 16.00. 
 
 The optical rotation of the substance was 
 
 , ,« +0.90X100 , .-no 
 
 a. Epichitosaminic Acid. 
 
 2 gm. of the lactone were dissolved in 20 cc. of distilled water and 
 the solution was rendered alkaline by means of a solution of barium 
 hydroxide. The solution was allowed to stand over night, then the 
 
p. A. LEVENE 19 
 
 barium was removed quantitatively by means of sulfuric acid, the 
 hydrochloric acid by silver carbonate, and the excess of the latter 
 reagent by hydrogen sulfide. The mother liquor of the silver sulfide 
 was concentrated to a small volume; on addition of a little alcohol 
 the epichondrosaminic acid crystallized immediately in prismatic 
 needles. The substance had a melting point of 198°C. (uncorrected) 
 with gas evolution. The composition of the substance was the 
 following: 
 
 0.0992 gm. substance: 0.1336 gm. CO2 and 0.0606 gm. H2O. 
 
 CeHisNOs. Calculated. C 36.92, H 6.66. 
 Found. " 36.80, " 6.72. 
 
 The optical rotation of the substance was 
 
 Initial. Equilibrium. 
 
 , ,» +0.20 X 100 , ,-„ . .M +0.78X100 , _„ 
 
 2. PREPARATION OF HEXOSAMINIC ACIDS FROM ^AMINOPENTOSIDES. 
 
 The optimal concentrations of pentosides and prussic acid were 
 found identical for all; namely, 30 gm. of the pentoside dried first 
 over soda lime under diminished pressure at room temperature and 
 finally at 50°C. also at diminished pressure, are taken up in 50 cc. 
 of water to which 40 cc. of 80 per cent prussic acid and 5 cc. of am- 
 monia water are added (Levene, 1918, a). The solution is warmed 
 and kept at constant temperature until the reaction is ended. The 
 colorless solution turns at first very light brown and gradually 
 darkens. The originally thin solution gradually turns viscous. 
 After some experience the end of the reaction can be determined by 
 the appearance of the solution. The temperature and duration of 
 the experiment varies with the configuration of the pentoside. 
 Lyxoside is warmed to 35°C. and maintained at that temperature 
 for 8 to 10 minutes. Xyloside is warmed to about 30°C. and 
 maintained at that temperature for 15 to 20 minutes. Subsequent 
 treatment is the same in every instance and is as follows : 
 
 The flask is immersed in a cooling mixture and the temperature 
 of the solution allowed to fall to 0°C., and is poured into 300 cc. of 
 
20 HEXOSAMINES, MTJCINS, AND MUCOIDS 
 
 concentrated hydrochloric acid previously cooled to the same tem- 
 perature. The solution is then saturated with hydrochloric acid 
 gas and allowed to stand not less than 1 hour; it may be allowed to 
 stand over night. The solution is concentrated under diminished 
 pressure, the temperature of the water bath not exceeding 75°C. 
 In the course of the concentration considerable ammonium chloride 
 crystallizes out in the flask. When the solution becomes quite 
 viscous the concentration is interrupted and the contents of the 
 flask are poured into 2 liters of 95 per cent alcohol. Generally a 
 small quantity of tar separates out which is removed by filtration 
 through cotton wool.^ The alcoholic filtrate is concentrated under 
 diminished pressure, the residue is dissolved in about 1 liter of water, 
 200 gm. of moist barium hydroxide are added, and the mixture 
 is repeatedly evaporated nearly to dryness, the water being renewed 
 at this phase. When all ammonia is removed the evaporation is 
 interrupted and the barium is removed quantitatively by means of 
 sulfuric acid, the hydrochloric acid by means of lead and silver 
 carbonate. The final solution is concentrated under diminished 
 pressure to a volume of about SO cc. To this solution hot methyl 
 alcohol is added gradually in small portions and the solution is kept 
 on a hot water bath until a crystalline deposit begins to form. 
 
 a. Chondrosaminic and Epichondrosaminic Acids. 
 
 The specific rotation of the crude material was + 3.0°. Since 
 the specific rotation of chondrosaminic acid is — 17.0°, it is evident 
 that the material is a mixture of the two epimers (Levene, 1918, a; 
 Levene and La Forge, 1915, c). 
 
 Separation of the Epimers. — 250 gm. of mixed acid having a rota- 
 tion of [aT° = +3.0° were dissolved in 750 cc. of hot water and 
 allowed to stand over night at room temperature. By far the greater 
 part of the material crystallized out. The rotation of this substance 
 (No. 379) was [a]" = 0.00°. The mother liquor was concentrated 
 under diminished pressure to dryness and the residue again crystal- 
 lized out of a minimum amount of water. A crystalline deposit 
 formed on standing over night (No. 532) with a rotation of [a]'^ = 
 -1-2.5°. The mother liquor was concentrated to dryness, taken up 
 in a minimum amount of water, and hot methyl alcohol was added 
 
 ^ Treatment with alcohol may be omitted. 
 
p. A. LEVENE 21 
 
 to opalescence. Soon a crystalline deposit formed (No. 533) which 
 had a rotation of [a]" = +8.0°. This material was again dissolved 
 in a minimxmi amount of hot water and allowed to stand at room 
 temperature over night. A crystalline deposit formed (No. 541) 
 with a rotation of [a]n = +7.75°. The mother liquor was con- 
 centrated under diminished pressure to dryness. The residue was 
 taken up in a minimum amount of water, and hot methyl alcohol 
 was added until the appearance of a slight opalescence; the solution 
 soon turned into a crystalline mass. The substance (No. 542) had 
 a rotation of [a]^ = + 8°, thus showing that the final treatment did 
 not accomplish any further fractionation. 
 
 The progress of fractionation is represented in the following 
 diagram. 
 
 +3.0° 
 
 +0.00° (No. 379) + 5.0° (No. 380) 
 
 \ 
 
 +2.5° (No. 532) + 8.0° (No. 533) 
 
 +7.75° (No. 541) + 8.0° (No. 542) 
 
 The substance with the rotation [a]'° = 0.00° was continually re- 
 crystaUized out of water until a fraction was obtained with a 
 rotation [a]'° = -17.5°. The dextro epimer had the following 
 composition: 
 
 0.1004 gm. substance: 0.1358 gm. CO2 and 0.0624 gm. H2O. 
 
 CeHisNO.. Calculated. C 36.92, H 6.66. 
 Found. " 36.89, " 6.96. 
 
 The melting point was 206°C. (uncorrected) with gas evolution. 
 The optical rotation was 
 
 , ,M +0.16X100 , 5J0 
 
 [«]„ lyj +8 
 
 The synthetic chondrosaminic acid is identical with that prepared 
 on oxidation of the natural chondrosamine. 
 
22 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 b. Xylohexosaminic Acids. 
 
 The optical rotation of the mixed acids was (Levene, 1918, a; 
 Levene and La Forge, 1915, b) 
 
 . ,M +0.07 X 100 , , „ 
 
 Separation of the Epimers. — 280 gm. of the mixed acids were 
 dissolved in a minimum amount of water, and methyl alcohol was 
 added until a crystalline precipitate began to form. This was 
 filtered off. It had a rotation of [0]^ = + 5.0°. The mother liquor 
 was concentrated to dryness under diminished pressure. The resi- 
 due was then taken up in a minimum amount of water and on addi- 
 tion of alcohol a precipitate formed, which was removed by filtration 
 and had the rotation [«]„ = 4-2.00° (No. 615). 
 
 180 gm. of this substance were dissolved in 300 cc. of hot water, 
 and the solution was allowed to cool. A precipitate (No. 630) thus 
 formed had a rotation of [d^^ = — 1 .0°. 1 10 gm. of No. 630 were 
 dissolved in 400 cc. of hot water and the solution was allowed to 
 stand over night and a crystalline deposit (No. 641) formed having 
 a rotation of [a]'' = —1.0°. On further fractionation of No. 641 
 the rotation of the substance did not change. The substance melted 
 at 200°C. (uncorrected) with gas evolution. The composition was 
 the following: 
 
 0.1000 gm. substance: 0.1354 gm. CO2 and 0.0590 gm. H2O. 
 0.1000 " " required (Kjeldahl) 5.25 cc. 0.1 n acid. 
 
 CeHiaNOs. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 36.92, « 6.60, " 7.35. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . ,» _ -0.22 X 100 ,,.„ , ,!o -0.63X100 „ „ 
 
 The progress of the fractionation of the epimers is given in the 
 following chart: 
 
p. A. LEVENE 23 
 
 Mixed acids [a]" = +3.5° 
 
 +5° (No. 614) +2.0° (No. 615) 
 
 i i 
 
 + 14.0 °(No. 627) -1.0° (No. 630) 
 
 +14.0° (No. 628) -1.0° -11.0° (No. 641) 
 
 The substance with rotation [a]'' = +5° was dissolved in a mini- 
 mum amount of boiling water, allowed to stand 30 minutes, and while 
 still warm was filtered. The precipitate, thus formed had a rota- 
 tion of [«]„= +14.0°. The yield of the substance was 87 gm. This 
 was dissolved in 400 cc. of boiling water. On cooling, a precipitate 
 was formed which was removed by filtration. The mother liquor 
 was concentrated to dryness under diminished pressure. The 
 residue was taken up in a minimum amount of water, and hot methyl 
 alcohol was added to opalescence. The crystallization began 
 immediately. The optical rotation of both substances was identical, 
 [a]': = +14.0°. 
 
 d-Dextro-Xylohexosaminic Lactone Hydrochloride.- — 10 gm. of amino- 
 acid were suspended in 100 cc. of 99.5 per cent ethyl alcohol into 
 which hydrochloric acid gas was passed, without cooling, until the 
 alcohol was saturated. At first the substance passed into solution 
 and immediately afterwards a small amount of a white precipitate 
 began to form. This in turn redissolved as more hydrochloric 
 acid was passed in. Finally, after the solution had become 
 saturated, crystallization of the lactone began and was complete 
 after the solution had stood in the cold for a few hours. The crys- 
 tals were then filtered off and washed with absolute alcohol. The 
 yield was practically quantitative. The substance crystallizes 
 in compact aggregates of prismatic needles grouped together in the 
 form of spheres. It was recrystallized from an excess of methyl 
 alcohol. The substance had a melting point of 205°C. (uncorrected) 
 with gas evolution. The composition was as follows : 
 
 0.1000 gm. substance: 0.1234 gm. CO2 and 0.0504 gm. H2O. 
 0.2000 " " required (Kjeldahl) 9.31 cc. 0.1 n acid. 
 
 0.1000 " " " (Volhard) 4.5 cc. 0.1 n AgNOs. 
 
 CsHuNOeCl. Calcukted. C 33.71, H 5.67, N 6.S5, CI 16.25. 
 Found. " 33.65, " 5.64, " 6.52, " 16.00. 
 
24 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Conversion of the Lactone Hydrochloride into the Parent Acid. — 
 5 gm. of the lactone were dissolved in 50 cc. of water. An excess 
 of barium hydroxide was added, and the solution was allowed to 
 stand over night. The barium and the chlorine were then removed 
 quantitatively. The filtrate was concentrated, and the acid crystal- 
 lized on the addition of hot methyl alcohol. The substance had the 
 melting point of 224° C. (uncorrected) with gas evolution. The 
 substance had the following composition : 
 
 0.1070 gm. substance: 0.1372 gm. CO2 and 0.0620 gm. H2O. 
 0.010 " " : 1.27 cc. N2 in the Van Slyke micro apparatus at 26°C. 
 
 and 759 mm. 
 
 CeHiaNOs. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 37.04, " 6.86, " 6.99. 
 
 The optical rotation of the substance was 
 
 W: = ^^^$^ = +14.0= 
 
 Monobenzal-d-Lew-Xylohexosaminic Lactone Hydrochloride. — 2.5 
 gm. of the J-levo-hexosaminic acid were taken up in a solution of 15 
 cc. of 99.5 per cent alcohol and 3 cc. of benzaldehyde. Hydrochloric 
 acid gas was passed through the solution. The amino-acid went 
 .gradually into solution and after some time the entire solution 
 solidified instantaneously. After 1 hour 2 cc. of ether (dried over 
 sodium) were added and the material was allowed to stand an addi- 
 tional 2 hours. The lactone was then filtered and recrystallized 
 out of methyl alcohol. The melting point was 206° C. (uncorrected) 
 with gas evolution. 
 
 0.1023 gm. substance: 0.1938 gm. CO2 and 0.0508 gm. H2O. 
 0.1000 " " required (Kjeldahl) 3.34 cc. 0.1 n acid. 
 
 CijHisNOsHCl (molecular wt. 301.6). Calculated. C 51.72, H 5.35, N 4.69. 
 
 Found. " 51.81, " 5.57, " 4.68. 
 
 The optical rotation of the substance in 50 per cent alcohol was 
 r ,!. -1.21X100 „ „ 
 
 f"^° — r3<2 ^°-^ 
 
p. A. LEVENE 25 
 
 Conversion of the Lactone into the Free Acid. — 10 gm. of the dry 
 monobenzal lactone hydrochloride were dissolved in 50 cc. of water, 
 and to the solution were added 12.5 gm. of barium hydroxide con- 
 taining 5 gm. of Ba. The solution was placed on a boiling water 
 bath. It was soon noticed that on wanning the solution turned 
 yellow and ammonia was formed. The experiment was therefore 
 interrupted after 7 minutes. The solution was then rendered acid 
 with sulfuric acid. The filtrate from the barium sulfate precipitate 
 was extracted repeatedly with ether, and the aqueous solution was 
 freed quantitatively from sulfuric and hydrochloric acids. The 
 filtrate was concentrated and the amino-acid crystallized on addition 
 of hot methyl alcohol. The melting point of the acid was 200°C. 
 (uncorrected) with gas evolution. The substance had the following 
 composition: 
 
 0.1000 gm. substance: 0.1354 gm. CO2 and 0.6000 gm. H2O. 
 0.0100 " " : 1.36 cc. N2 in the Van Slyke micro apparatus at 3S°C. 
 
 and 762 mm. 
 
 CtHiaNOe. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 36.98, " 6.71, " 7.16. 
 
 The optical rotation of the substance was 
 
 Initial. Equilibrium, 
 
 . ,M -0.22 X 100 ,.-0 r 1" -0.60 X 100 „„ 
 
 c. Ribohexosaminic Acids. 
 
 The optical rotation of the mixed acids was (Levene and Clark, 
 1921) 
 
 . ,« -0.16 X 100 „ no 
 
 Separation of the Two Acids. 
 
 Levo-d-Ribohexosaminic Acid. — This is the more insoluble form 
 and is prepared without much difficulty. Two experiments are here 
 reported. 
 
 Experiment 1. — 57 gm. of the mixed acids were dissolved in 70 cc. 
 of boiling water and allowed to crystallize over night. The yield 
 
26 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 of the crystalline deposit was 22 gm. This substance had the 
 following optical rotation (No. 63820): 
 
 . .M -0.52 X 100 ,,0 
 
 This rotation indicates that the substance was the pure levo form. 
 On recrystallization from water two fractions were obtained (Nos. 
 640 and 641a), each having the same rotation, and, as will be shown 
 later, the substance prepared from the pure crystalline lactone pos- 
 sesses the same optical rotation. 
 
 Experiment 2. — 142 gm. of the mixed acids were dissolved in 300 cc. 
 of boiling water. 35 gm. of the levo form crystallized over night. 
 The rotation of the substance was as follows (No. 8831) : 
 .. -0.52 X 100 
 
 On further recrystallization the rotation of the substance did not 
 change. 
 
 Properties of the Levo-d-Ribohexosaminic Acid. — The substance 
 crystallizes in thin plates resembling those of cholesterol. It is 
 soluble in water and insoluble in the usual organic solvents. The 
 melting point of the substance is 212°C. (uncorrected) with 
 decomposition. 
 
 Lactone Hydrochloride of the Levo-d-Ribohexosaminic Acid. — 
 12 gm. of the acid carefully dried and pulverized were suspended 
 in 600 cc. of alcohol (99.5 per cent) and dry hydrochloric acid gas 
 was passed in for 15 minutes. The acid dissolved almost immie- 
 diately. The solution was concentrated under diminished pressure at 
 room temperature until crystallization took place in the distillation 
 flask. The contents were then transferred to an evaporating dish 
 and allowed to stand over night in a desiccator over sulfuric acid. 
 The yield of the lactone was 12 gm. The melting point of the sub- 
 stance was 188°C. (uncorrected). It had the following composition 
 (No.24i): 
 
 0.1076 gm. substance: 0.1304 gm. CO2 and 0.0S92 gm. H2O. 
 0.2000 " " required (Kjeldahl) 8.74 cc. 0.1 N acid. 
 
 0.2000 " " " (Volhard) 9.00 " 0.1 n AgNOa. 
 
 CsHuNObCI. Calculated. C 33.71, H 5.67, N 6.S5, CI 16.25. 
 Found. " 33.05, " 6.17, " 6.12, " 16.00. 
 
p. A. LEVENE 27 
 
 The substance still contained 0.46 per cent of mineral impurity. 
 The peculiarity of this la,ctone was that by the Van Slyke method 
 only 4.71 per cent of nitrogen was obtained, even when the reaction 
 was allowed to proceed for 30 minutes. 
 
 The rotation of the substance was 
 
 , ,« -0.22 X 100 ., no 
 
 Conversion of the Lactone into Free Acid. — 5 gm. of the lactone 
 hydrochloride were dissolved in 50 cc. of water. The solution was 
 treated with an excess of barium hydroxide and allowed to stand 
 over night. Barium and chlorine were removed from the solution 
 and the free acid was crystallized on concentration of the aqueous 
 solution. For analysis it was recrystalUzed out of water on addi- 
 tion of a little alcohol. After three recrystallizations the substance 
 was analyzed. The melting point was 212°C. (uncorrected). It 
 analyzed as follows: 
 
 0.1010 gm. substance: 0.1364 gm. COj and 0.1010 gm. HaO. 
 0.1990 " " required (Kjeldahl) 10.10 cc. 0.1 n acid. 
 
 CeHisNOs. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 36.83, " 7.04, " 7.10. 
 
 The rotation of the substance was 
 
 , ,,0 -0.52 X 100 ,,„ 
 
 Dextro-d-Ribohexosaminic Acid. — The separation of the dextro 
 form was found more difficult than that of its epimer, and was ac- 
 companied with considerable loss of material. The following proce- 
 dure was finally adopted. All fractions with the optical rotation 
 above 0.0" were combined, dissolved in about 5 to 8 volvimes of hot 
 water, and methyl alcohol was added to initial opalescence. The 
 solution was then placed on a boiling water bath and allowed to crys- 
 tallize. The crystalline deposit was filtered off while the mother 
 liquor was still hot. The operation was repeated until a constant 
 rotation was obtained. This was found to be 
 
 .. +0.25 X 100 _ , „ „ 
 ^< 1X2 +^" 
 
28 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 After three recrystallizations the rotation remained unchanged. 
 As will be seen later the substance obtained from the lactone pos- 
 sessed the same optical activity. The substance had a melting 
 point of 186°C. (uncorrected) and analyzed as follows: 
 
 0.1057 gm. substance: 0.1422 gm. CO2 and 0.0626 gm. H2O. 
 0.2000 " " required (Kjeldahl) 10.20 cc. 0.1 N acid. 
 
 CbHisNOb. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 36.70, " 6.63, " 7.14. 
 
 Lactone Hydrochloride of Dextro-d-Ribohexosaminic Acid. — 5 gm. 
 of the acid, carefully dried under diminished pressure at SO°C. and 
 pulverized, were suspended in 300 cc. of absolute alcohol (99.5 per 
 cent), and dry hydrochloric acid gas was passed through the alcohol. 
 Solution was accomplished almost immediately. The gas was passed 
 for 7 minutes. The solution was then concentrated under diminished 
 pressure until a considerable sediment began to form in the distilling 
 flask. The material was then transferred to an Erlenmeyer flask. 
 The sediment on standing increased in volume, but was found to be 
 amorphous. On heating, however, the sediment redissolved and on 
 prolonged standing on the water bath with stirring a sediment of 
 heavy crystals of the lactone settled out. The substance had a 
 melting point of 150°C. (uncorrected) and analyzed as follows: 
 
 0.0977 gm. substance: 0.1212 gm. CO2 and 0.0546 gm. H2O. 
 0.1872 " " required (Kjeldahl) 8.85 cc. 0.1 N acid. 
 
 GHbNOsCI. Calculated. C 33.71, H 5.67, N 6.55. 
 Found. " 33.83, " 6.25, " 6.62. 
 
 The rotation of the substance was 
 
 , ... +0.43 X 100 , ,, „ 
 ^"1° 1X2 = +21.5= 
 
 Conversion of the Lactone into the Dextro-d-Ribohexosaminic Acid. — 
 3 gm. of the lactone hydrochloride were dissolved in 25 cc. of water. 
 The solution was rendered alkaline by means of barium oxide and 
 allowed to stand over night. The barium and the hydrochloric 
 acid were then removed and the remaining aqueous solution was 
 concentrated to a small volume. To the concentrated solution 
 
p. A. LEVENE 29 
 
 alcohol was added to opalescence, and the solution was allowed to 
 digest on a boiling water bath until a heavy crystalline deposit 
 formed. The yield of this substance was 2 gm. The melting point 
 of the substance was 186°C. (uncorrected). 
 The rotation was 
 
 , ,» +0-25 X 100 , „ „ 
 
 Thus the melting point and the optical rotation of the substance 
 purified through conversion into its lactone and reconversion of this 
 into the acid remained identical with those of the original material. 
 
 Dibenzal-Dextro-d-Ribohexosaminic Ethyl Ester Hydrochloride. — 
 The dextro form differed from its epimer in that it formed the above 
 compound under the same condition in which the levo form gave 
 rise to its lactone hydrochloride. 2 gm. of the carefully dried and 
 pulverized acid were suspended in 20 cc. of absolute alcohol (99.5 per 
 cent) to which 2 cc. of redistilled benzaldehyde were added, and dry 
 hydrochloric acid gas was passed through the solution. The acid 
 dissolved rapidly, but the treatment with acid was continued for 
 7 minutes. The slightly turbid solution was allowed to stand over 
 night. A crystalline deposit consisting microscopically of long 
 needles was formed. It was filtered off, washed with alcohol and 
 ether, dried, and analyzed. The substance (No. 232) had a melting 
 point of 221°C. (uncorrected) and the following composition: 
 
 0.1069 gm. substance: 0.3270 gm. CO2 and 0.0602 gm. H2O. 
 0.1978 " " required (Kjeldahl) 4.60 cc. 0.1 N acid. 
 
 CssHssNOtCl. Calculated. C 60.65, H 6.34, N 3.20. 
 Found.. " 60.48, « 6.31, " 3.2S. 
 
 The rotation of the substance dissolved in methyl alcohol was 
 
 , ,.0 -0.26 X 100 „,o 
 
 Wb Y^i 26 
 
 From the mother liquor of the dibenzal derivative on standing a 
 second crop of crystals formed, which once recrystaUized had the 
 composition and the physical properties of the lactone hydrochloride 
 of the dextro-d-ribohexosaminic acid. This observation is important 
 
30 HKXOSAMnsTES, MUCINS, AND MUCOIDS 
 
 inasmuch as it offers additional evidence of the purity of the dextro- 
 (i-ribohexosaminic acid. The substance analyzed as follows: 
 
 0.0942 gm. substance: 0.1174 gm. CO2 and 0.0512 gm. H2O. 
 0.0918 " " required (Kjeldahl) 4.5 cc. 0.1 n acid. 
 
 CeHuNOjCl. Calculated. C 33.71, H S.67, N 6.55. 
 Found. " 33.99, " 6.08, " 6.86. 
 
 The optical rotation of the substance in 2.5 per cent hydrochloric 
 acid was 
 
 , ,.0 +0.43 X 100 _ , ,, -o 
 
 3. SYNTHESIS OF HEXOSAMESTES. 
 
 a. Chitosamine. 
 
 For the preparation of all lactones of hexosaminic acids it is im- 
 portant to start with very pure material. The glucosaminic acid 
 used in these experiments was recrystallized several times out of 
 water. 5 gm. lots of the acid were taken up in 50 cc. of 99.5 per 
 cent alcohol. Dry hydrochloric acid gas was passed in, in a lively 
 stream. The acid soon dissolved. After a while a flocculent pre- 
 cipitate began to appear. If the solution was actively shaken during 
 this phase nearly all the flocculent precipitate of the esters disap- 
 peared. For a while the solution remained clear and then turned 
 quite opaque. At this stage it was found advisable to immerse the 
 flask in a cooling mixture and to continue passing the gas. Soon a 
 precipitate of the lactone appeared. This was allowed to stand over 
 night in the refrigerator. One can wash the lactone by decantation 
 with 99.5 per cent alcohol, and with dry ether, and finally filter the 
 lactone and dry it in a vacuum desiccator. It is then possible to 
 continue the work on the pure lactone. However, it was found that 
 this mode of procedure had no advantage over the less troublesome 
 method of Fischer and Leuchs. In most of the experiments the 
 latter method was followed (Levene, 1916, c, d; 1917, a). 
 
 Isolation of Glucosamine Hydrochloride. — 5 gm. of the lactone 
 hydrochloride were reduced with 40 gm. of potassium amalgam 
 (2 per cent), hydrochloric acid being used for neutralization. The 
 
p. A. LEVENE 31 
 
 reduction was carried out in a vessel immersed in a cooUng mixture 
 and provided with a mechanical stirrer. At the end of the experi- 
 ment the solution tested with Fehling's reagent showed a reduction 
 eqmvalent to 1.9 gm. of glucose. The reaction product was con- 
 centrated nearly to dryness. The residue was taken up in about 
 30 cc. of methyl alcohol, and concentrated hydrochloric acid was 
 added drop by drop. The flask was carefully warmed over a flame 
 until the syrupy material was all in solution and the salt had the 
 appearance of a white crystalline powder. The salt was filtered. 
 It showed the presence of considerable sugar, judging by its reducing 
 power towards Fehling's solution. 
 
 The mother liquor on standing in the refrigerator formed a pre- 
 cipitate of practically pure glucosamine hydrochloride. 
 
 0.1030 gm. substance: 0.1246 gm CO2, 0.0600 gm. H2O, and 0.0026 gm. ash 
 (2.SS per cent). 
 
 CsHuNOsHCl. Calculated. C 33.40, H 6.S4. 
 Found. " 33.85, " 6.69. 
 
 In a few other experiments the sugar contained more salt. This 
 experiment shows that favorable conditions can be found for the 
 direct isolation of glucosamine hydrochloride from the reaction 
 product. 
 
 Preparation of the Pentabenzoyl Derivative. — The material used for 
 this preparation was obtained from several reduction experiments. 
 Potassium and sodium amalgam were used for reduction, and hydro- 
 chloric acid for neutralization. The reaction product was treated 
 as in the above experiment. The fraction containing only sugar and 
 salt was used for benzoylation. 
 
 6.5 gm. of the sugar (estimated by means of Fehling's solution) 
 were taken up in 140 cc. of water, 25 cc. of benzoyl chloride, and 
 40 cc. of a 50 per cent sodium hydroxide solution. It was shaken for 
 about IJ hours with constant cooling, and then placed in a shaking 
 machine over night. The benzoyl derivative, which had the appear- 
 ance of dry white balls, was washed with water, then dissolved in 
 chloroform, and shaken with water in a separatory fimnel. Finally 
 the chloroform solution was dried over sodium sulfate, filtered, and 
 concentrated to dryness. The residue was recrystaUized out of 4 
 
32 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 liters of 98 per cent alcohol. On cooling, the substance crystallized 
 in long colorless needles. The mother liquor on concentration gave 
 a second precipitate. The substance melted at 216°C. and had the 
 following composition: 
 
 0.1012 gm. substance: 0.2606 gm. CO2 and 0.0453 gm. H2O. 
 0.2530 " " required (Kjeldahl) 5.05 cc. 0.1 n acid. 
 
 CeHsNOsCCeHjCOs. Calculated. C 70.38, H 4.73, N 2.00. 
 Found. " 70.23, " S.OO, " 1.99. 
 
 The optical rotation of the substance in pyridine was 
 
 „ ^ +0.58X1.9992 ^ 
 '^° 0.5 X 0.0522 ^ 
 
 The benzoyl derivative had been converted into the hydrochloride 
 of glucosamine by previous workers; hence it was not considered 
 important to repeat it on this occasion. 
 
 b. Chondr OS amine. 
 
 IS gm. of lyxohexosaminic acid were taken up in three small flasks, 
 each containing 5 gm. of the acid and SO cc. of 99.5 per cent alcohol 
 (Levene, 1916, c; 1917, a). Into each a lively stream of dry hydro- 
 chloric acid gas was passed. The formation of the lactone proceeded 
 in the same way as with the giucosaminic acid. The flasks were 
 allowed to stand over night, and the mixture was then transferred 
 to a distilhng flask by the aid of distilled water. 150 gm. of 2.5 per 
 cent sodium amalgam were used for reduction. At the end of the 
 experiment the solution tested with Fehling's reagent showed a 
 reduction equivalent to 6.5 gm. of glucose. 
 
 The product of reaction was concentrated under diminished pres- 
 sure nearly to dryness. Care was taken to keep the temperature of 
 the water bath at 40°C. The residue was taken up in 75 cc. of 
 methyl alcohol, and concentrated hydrochloric acid added drop by 
 drop until all the syrup dissolved and sodium chloride appeared in 
 the form of white crystalline powder. This first precipitate showed 
 no reducing power. 
 
 The filtrate was again concentrated nearly to dryness and taken 
 up in 99.5 per cent ethyl alcohol and hydrochloric acid as before. 
 
p. A. LEVENE 33 
 
 On standing, an oily mass separated out. In the course of the night 
 this turned to a crystalline mass. The supernatant liquor was de- 
 canted, and the crystalline mass triturated with methyl alcohol. 
 It consisted of long heavy prisms with needle-shaped points. The 
 substance burned as carbohydrates do and left behind a small amount 
 of ash. The liquid which had been decanted off the crystalline mass 
 on standing began to form on the walls and on the bottom of the 
 flask a crystalline deposit consisting of long prisms of the same 
 appearance as those of the crystalline mass. On burning, it left 
 practically no ash. 
 
 The substance was redissolved in a little water, and 99.5 per cent 
 alcohol saturated with hydrochloric acid gas was added in small por- 
 tions until on scratching the sugar began to crystallize. The crystals 
 had the same appearance as on first crystallization. The substance 
 melted at 185°C. (corrected). 
 
 The rotation was 
 
 Initial. Equilibrium. 
 
 , ,» +1.50 X 2.0392 « -h 2.18X2.0392 
 
 '"'■> = 1.0X0.0616 = +^^-^ ^< = 1.0X0.0516 = +^^-^ 
 
 0.1050 gm. substance dried in a vacuum desiccator over sulfuric acid at 25°C.: 
 0.1284 gm. CO2 and 0.0604 gm. H2O. 
 
 0.0244 gm. substance: 2.94 cc. N2 at 22° and 762 mm. 
 0.0820 " " required 3.7 cc. 0.1 NAgNOa solution. 
 
 CijHiaOsN-HCl. Calculated. C 33.40, H 6.54, N 6.51, CI 16.45. 
 Found. " 33.35, " 6.39, " 6.74, " 16.00. 
 
 35 gm. of the synthetic sugar were prepared. 
 
 The mother liquor of the crystalline mass was concentrated under 
 diminished pressure. It contained 3 gm. of sugar and was taken up 
 in 400 cc. of water; 8 gm. of phenylhydrazine dissolved in glacial 
 acetic acid were added, and the flask containing the solution was 
 immersed in a boiling water bath for 4 hours. The osazone began 
 forming while the solution was still heated. On cooling, a volu- 
 minous precipitate of osazone deposited. The osazone was taken up 
 in 400 cc. of boiling water which was kept boiling while pyridine 
 was added gradually until the solution was complete. After 
 three recrystaUizations microscopic slides showed perfectly formed 
 
34 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 elongated plates with pointed ends, and an absolute absence of oily 
 droplets. The osazone was then filtered on a suction funnel, trans- 
 ferred into a mixture of equal parts of alcohol and ether, and filtered. 
 It then had the appearance of light orange glistening plates. 
 
 It melted at 201°C. and decomposed at 202° (corrected). Mixed 
 with osazone from chondrosamine and with galactosazone it showed 
 the same melting and decomposition points. 
 
 0.1006 gm. substance: 14.2 cc. Nz at 29°C. and 757 mm. 
 
 Ci8H24N40<. Calculated. H 15.64. 
 Found. " 15.8. 
 
 For the rotation of the substance 0.05 gm. was dissolved in 5 cc. 
 of Neuberg's pyridine and alcohol mixture. The initial rotation 
 was + 0.36°; after 24 hours, + 0.03°. (It was not followed further.) 
 
 CL and /3 Forms of Chondrosamine Hydrochloride. — ^A sample of 
 chondrosamine recrystallized out of water and ethyl alcohol saturated 
 with hydrochloric acid. 
 
 Initial. Equilibrium. 
 
 , o _ +1.54 X 2.0385 _ , „ ,„ , ,=. +2.28 X 2.0392 
 ^< - 1X0.0516 - +"-^ f"'» = 1X0.0514 = +^°-* 
 
 A sample was dissolved in a minimum amount of water, and 
 glacial acetic acid was added until the substance began to crystal- 
 lize, the mixture was brought to a boil, and filtered. 
 
 Initial. Equilibrium, 
 
 o +3.04 X 2.5 K +4.5 X 2.5 , „_ .„ 
 
 ^< = 1X0.125 = +^°-^ [< = 1X0.125 = +^°-° 
 
 A sample crystallized out of a minimum amount of aqueous hydro- 
 chloric acid. 
 
 Initial. Equilibriimi. 
 
 o +2.60 X 2.5 « +4.80 X 2.5 ... „„ 
 
 ^< = 1X0.1272 = +"-^° ^< = 1X0.1272 = +^*-20 
 
 No attempt was made to explain the slight discrepancies in the 
 solutions since the principal object was to determine conditions 
 controlling the formation of either one of the forms. As all attempts 
 to obtain a sample with the original rotation failed, the rotation of 
 the original material was redetermined.' 
 
 ' Dr. J. L6pez-Suarez and Dr. G. M. Meyer controlled the reading. 
 

 P. A. 
 
 LEVENE 
 
 Initial. 
 
 
 Equilibrium. 
 
 +2.50 X 2.5 
 1 X 0.0500 
 
 = +125°" 
 
 , ,« +1.90X2.0 
 '"^° 1 X 0.0500 
 
 35 
 
 = +95° 
 
 Calculating on the basis of Hudson's formula, the molecular 
 
 , , (12S.0-51S.0) 
 rotation of the end carbon atom = 215.S = 8,400. 
 
 The value found by Hudson for hexoses was in the neighborhood 
 of 8,000. The original form is to be regarded as the a and the new 
 as the (3 form. 
 
 Pentacetyl Derivative of the Synthetic Chondrosamine. — The sub- 
 stance was prepared from the natural chondrosamine by Hudson 
 and Dale. Practically the same conditions were followed in this 
 work. 
 
 5 gm. of zinc chloride were dissolved in 30 cc. of acetic anhydride. 
 To this solution 5 gm. of the hydrochloride were added, and the mix- 
 ture was warmed gently until a lively reaction developed. The 
 reaction was kept up for 2 minutes, then the reaction product was 
 poured into 100 cc. of water cooled to 0°C. The mixture was neutral- 
 ized with potassium bicarbonate, transferred to a separatory funnel, 
 and extracted with chloroform. The chloroform extract was washed 
 with water. Over the chloroform a layer of crystals appeared which 
 were insoluble in water. The crystals consisted of the more insoluble 
 fraction of the pentacetates. The chloroform extract was evaporated 
 to dryness under diminished pressure, the residue was recrystaUized 
 out of alcohol, and from the mother liquor a second crop of crystals 
 was obtained. The top fraction consisted of the pure a form, while 
 the more soluble form was practically the pure /3 form. The a form 
 turned slightly brown at 232° and melted with decomposition at 
 237°C. (corrected). The optical rotation in chloroform solution was 
 
 . « _ +0.07 X 20.0 _ 
 ^"^° 2 X 0.0802 "^ ' 
 
 A similar fraction from natural chondrosamine had a melting 
 point of 235°C. The optical rotation was 
 
 , ,» +0.09 X 20.0 , .,o 
 ["'■> = 2X0.075 = +^2 
 
36 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Hudson and Dale found for their ^ form the melting point 235° 
 (with decomposition) [a]" = + 11.00°. 
 
 The /3 form was apparently not quite pure, but taking into con- 
 sideration the small quantity of starting material it is rather sur- 
 prising that each form could be separated with so httle difficulty. 
 The melting point of the ;8 form was very sharp at 197°C. The 
 optical rotation in chloroform solution was 
 
 , ,=. -h0.90 X 20.0 _ „ 
 '"^>' = 2 X 1.000 - +^° 
 
 Hudson and Dale found for the a form the melting point 
 182-183°C. and H" = 101.3°. The composition of the pentacetyl 
 derivative was the following: 
 
 0.1014 gm. substance: 0.1834 gm. CO2 and 0.0560 gm. H2O. 
 
 C»H8N06(CH3CO)5. Calculated. C 49.49, H 5.96. 
 Found. " 49.32, " 6.18. 
 
 c. Epichitosamine. 
 
 The reduction of the lactone into the sugar was carried out in the 
 same maimer as on previous occasions (Levene, 1919). Lots of 10 
 gm. of lactone hydrochloride were taken up in 60 cc. of water and 
 reduced with 125 gm. of a 2 per cent sodium amalgam. The reaction 
 of the solution was kept on the acid side by adding at short inter- 
 vals small quantities of hydrochloric acid. 
 
 The filtrate from the mercury is concentrated at diminished pres- 
 sure and at room temperature. Care has to be taken to carry out 
 the concentration in such a manner that the material remains color- 
 less. The sodium chloride is separated by fractional precipitation 
 first with methyl alcohol and subsequently with ethyl alcohol. 
 After the product was obtained in such a state of purity that on 
 ignition no ash was visible, the sugar was recrystallized once or 
 twice out of water. 
 
 The analysis of the hydrochloride gave the following results: 
 
 0.1042 gm. substance: 0.1298 gm. CO2 and 0.0614 gm. HjO. 
 
 0.0100 " " in Van Slyke's micro apparatus: 1.18 cc. N2 at 21°C. 
 
 and 765 mm. 
 
 0.0200 gm. substance in Van Slyke's micro apparatus, in 5 minutes: 2.43 cc. N2 
 at 25°C. and 764 mm. 
 
p. A. LEVENE 37 
 
 The melting point of the substance was 187°C. (corrected). 
 
 CHisNOsHCl. Calculated. C 33.40, H 6.54, N 6.S1, CI 16.45. 
 
 Found. I. (No. 328H) " 6.73 
 
 II. ( " S75U) C 33.97, H 6.61, 
 
 III. ( " mil) CI 6.78 
 
 The rotation of the substance in 5 per cent HCl solution was 
 
 Initial. Eqidlibrium. 
 
 .. ^ -0.47 X 100 ^ _ « ^ -0-47X100 ^ _ 
 
 ' '" 1X10 •' ^"'o 1X10 ' 
 
 Preparation of the Osazone. — 4.6 gm. of the sugar hydrochloride 
 were taken up in 200 cc. of water, the solution was neutralized with 
 sodium acetate, and a solution of 8 gm. of phenylhydrazine in 5 cc. 
 of glacial acetic acid was added and the entire solution allowed to 
 stand on the boiling water bath for 1 hour. An osazone formed which 
 was recrystallized from methyl alcohol. The melting point at rapid 
 heating was 205 °C. It had the following composition: 
 
 0.1000 gm. substance: 0.2202 gm. CO2 and 0.5800 gm. H2O. 
 
 Ci8HsiN404. Calculated. C 60.33, H 6.14. 
 Found. " 60.05, " 6.49. 
 
 0.1000 gm. substance dissolved in 10 cc. Neuberg's alcohol-pyridine solution, 
 had an initial rotation, —0.31° and equilibrium —0.15°, which is in accord with 
 the rotation found for glucosazone. 
 
 d. Dexiro-Xylohexosamine. 
 
 The lactone was prepared in the same manner as that of chito- 
 saminic acid (Levene, 1916, d). Also in the reduction the general 
 plan was followed. However, all the attempts to obtain a fraction 
 free from the lactone were for the present unsuccessful. When 
 hydrochloric acid was used for neutralization the salt always sep- 
 arated out free from sugar, and when sulfuric acid was employed for 
 the same purpose, salt, lactone, and sugar crystallized in the same 
 fraction. 
 
 A solution containing about 6 gm. of the amino sugar and 1 gm. 
 of amino nitrogen was taken up in 500 cc. of water to which 80 cc. 
 
38 HEXOSAMINES, MUCINS, AND MUCOmS 
 
 of benzoyl chloride and 110 cc. of a 50 per cent sodium hydroxide 
 solution were added. The benzoylation proceeded as in the gluco- 
 samine experiment. The final product was taken up in methyl 
 alcohol, and ether was added as long as it caused the precipitation 
 of an oil. The supernatant liquid was decanted and allowed to 
 evaporate spontaneously. A precipitate of white needles formed. 
 It was filtered ofiE and recrystallized once out of 99.5 per cent alcohol. 
 It melted at 162°C. and had the following composition: 
 
 0.1012 gm. substance: 0.2632 gm. CO2 and 0.0416 gm. H2O. 
 
 CHsNOsCGHsCOt. Calculated. C 70.38, H 4.73. 
 Found. " 70.93, " 4.60. 
 
 The rotation of the substance was 
 
 , ,« + 1.0 X 1.9422 _ , „ ,„ 
 ^"^» ~ 0.5 X 0.0550 -+"-^ 
 
 Osazone of Xylokexosamine. — ^A solution containing 2.5 gm. of 
 the amino sugar was taken up in 250 cc. of water, and 6 gm. of 
 phenylhydrazine dissolved in glacial acetic acid were added. The 
 flask containing the solution was then placed in a boiling water 
 bath for 4 hours. On cooling, the osazone settled out. It was 
 recrystallized 4 times out of water and pyridine. It then consisted 
 uniformly of curved needles. The substance was taken up in a very 
 little alcohol and ether. It dissolved and crystallized on evaporation 
 of the solvent in the form of a bright lemon-yellow precipitate. It 
 melted at 173° (corrected) and decomposed at 185°C. The substance 
 had the following composition: 
 
 0.1000 gm. substance: 0.2208 gm. CO2 and 0.0560 gm. H2O. 
 
 CigHstNiOi. Calculated. C 60.33, H 6.14. 
 Found. " 60.21, " 6.22. 
 
 The rotation of the substance was as follows: 
 
 0.1000 gm. substance dissolved in S.O cc. Neuberg's alcohol-pyridine solution 
 had an initial rotation of a = -|-0.07°, and after 40 hours a = -1-0.45°. 
 
p. A. LEVENE 39 
 
 4. 2-S-ANHYDROPENTOXYCAPROIC ACIDS. 
 
 a. 2-5-Anhydromannonic and Anhydrogluconic Acids. 
 
 Of the series only four had been prepared. Two, chitonic and 
 chitaric, were prepared by Tiemann and by Fischer. It will be seen 
 from the following chapter that chitonic is oxidized into 2-5-anhydro- 
 mannosaccharic and chitaric into 2-5-anhydrosaccharic; hence 
 chitonic has the configuration of the anhydromannonic, and chitaric 
 of the anhydrogluconic. On the basis of the experience on chito- 
 samine, epichitosamine and epichitosaminic acid should form 2-5- 
 anhydrogluconic and 2-S-anhydromannonic acid respectively. The 
 expectation regarding the epichitosaminic was realized. It formed 
 on deamination anhydromaimonic acid. Contrary to expectation 
 the lactone also gave rise to the anhydromannonic acid. 
 
 Deamination of Epichitosaminic Acid {Levene, 19T8, b). — ^The 
 mother liquor after deamination was reduced by means of aluminimi 
 ainalgam according to the method of Levene and Meyer, and the 
 resulting solution neutralized with calcixmi carbonate, and concen- 
 trated under diminished pressure to a smaU volimie. The composi- 
 tion of the Ca salt was the following: 
 
 0.1020 gm. substance: 0.1244 gm. CO2, 0.0482 gm. H2O, and 0.0134 gm. CaO. 
 
 (C»H,OB)2Ca+2Hi!0. Calculated. C 33.49, H S.12, Ca 9.30. 
 Found. " 33.26, " S.07, " 9.38. 
 
 The optical rotation of the substance was 
 
 , ,M +0.71 X 100 , ,5 .0 
 
 Under similar conditions the lactone gave a salt of the following 
 composition: 
 
 0.1030 gm. substance: 0.1282 gm. CO2, 0.0484 gm. H2O, and 0.0138 gm. CaO. 
 
 (C6H,0.)jCa+2HaO. Calculated. C 33.49, H S.12, Ca 9.30. 
 Found. " 33.94, " 5.26, " 9.56. 
 
 The rotation of the substance was 
 
 , ,« +0.71 X 100 _ , „ „ 
 
40 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 b. 2-5-Anhydrotalofdc and Anhydrogalactonic Acids. 
 Prepared only in the Form of Their Brucine Salts. 
 
 2-5-Anhydrotalonic acid was obtained from chondrosamine and 
 from epichondrosaminic acid (Levene, 1918, b). The brucine salt 
 was prepared in larger quantities from the natural sugar and from the 
 sugar obtained from lyxohexosaminic acid. Lots of 30 gm. of chon- 
 drosamine hydrochloride were dissolved in 150 cc. of water. To the 
 solution 30 gm. of silver nitrite and a few drops of hydrochloric acid 
 were added. The mixture was allowed to react for 6 hours. The 
 silver chloride was then removed by filtration, the filtrate was placed 
 on a boiling water bath for 5 minutes, then treated with a slight 
 excess of hydrochloric acid, and again filtered. To the filtrate 65 gm. 
 of bromine were added and the mixture was allowed to stand at room 
 temperature for 3 days. The remaining traces of bromine were 
 removed by shaking the solution with mercury; and the hydrobromic 
 acid, by means of lead carbonate and silver carbonate. Finally the 
 nitric acid stiU present in the solution was removed by the aluminimi 
 amalgam method. The resulting solution was transformed into the 
 brucine salt in the usual manner. The solution of the brucine salt 
 was concentrated to a small volume when, on cooling, the brucine 
 salt was crystalUzed out. When first obtained, the substance was 
 readily soluble in methyl alcohol. However, on repeated recrystal- 
 lization, its solubility diminished, so that towards the end it dissolved 
 only in a large volume of boiling methyl alcohol. The salt then 
 crystallized in large polygonal prisms. The melting point was 218°C. 
 
 The optical rotation was 
 
 » ^ - 0.63 X 100 ^_ 
 ^ '" 2X2.522 ^^-^ 
 
 The analysis of the substance gave the following results: 
 
 0.1063 gm. substance, on drying under diminished pressure at the temperature 
 of xylene vapor, lost 0.003 gm. H2O. 
 
 CjBHseNsOio + H,0. Calculated. HjO 3.11. 
 Found. " 3.58. 
 
 0.1025 gm. dry substance: 0.2284 gm. CO2 and 0.0569 gm. H2O. 
 
 CjsHsaNjOio. Calculated. C 60.80, H 6.10. 
 Found. " 60.78, " 6.15. 
 
p. A. LEVENE 41 
 
 From the synthetic epichondrosaminic acid the substance was 
 prepared in the following manner. 30 gm. of the acid were dissolved 
 in a solution of 200 cc. of water and 40 cc. of 10 per cent hydrochloric 
 acid. 40 gm. of silver nitrite were added. The following morning 
 10 gm. of silver nitrite and 10 cc. of 10 per cent hydrochloric acid were 
 added. After 30 hours from the beginning of the experiment the 
 silver chloride was removed by filtration, and the other remaining 
 silver by hydrogen sulfide. From the filtrate the nitric and nitrous 
 acids were removed by the aluminum amalgam method. The sub- 
 stance had the following composition: 
 
 0.1012 gm. substance, on drying in a zylene bath under diminished pressure 
 lost 0.0038 gm. HjO. 
 
 0.0974 gm. substance: 0.2158 gm. CO2 and 0.0562 gm. HjO. 
 
 C29H88N20ioHaO. Calculated. HO 3.11. 
 Found. " 3.75. 
 
 CasHssNjOio. Calculated. C 60.80, H 6.10. 
 Found. " 60.42, " 6.45. 
 
 The substance had a melting point of 218°C. and the rotation 
 was 
 
 « - 0.65 X 100 ,, ,0 
 
 ^< = 2 X 2.66 ^2.3 
 
 In order to prove its identity with 2-5-anhydrotalonic acid it was 
 oxidized by means of nitric acid into 2-S-anhydrotalomucic acid. 
 21 gm. of the brucine salt were freed from brucine by means of 
 bariimi hydroxide and chloroform. After the removal of barium, 
 the solution was concentrated to 40 cc, an equal volume of concen- 
 trated nitric acid was added, and the solution was boiled over a free 
 flame until a lively evolution of red fumes set in. The solution was 
 then transferred to a large clock glass and evaporated to dryness. 
 The residue was dissolved in water and again evaporated to dryness. 
 This operation was repeated. The reaction product was converted 
 into the calciiun salt. For purification this calcium salt was decom- 
 posed by a little less than the calculated amount of oxalic acid and 
 then reconverted with the calcium salt. This operation was repeated 
 twice. 
 
42 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 0.1008 gm. air-dry substance, on drying in a xylene bath under diminished 
 pressure, lost 0.0130 gm. H2O. 
 
 CBHsOrCa + SHjO. Calculated. HaO 12.68. 
 Found. " 12.90. 
 
 0.0878 gm. substance: 0.0936 gm. CO2 and 0.0274 gm. H2O. 
 0.0896 " " : 0.0206 gm. CaO. 
 
 CsHaOrCa + H2O. Calculated. C 29.03, H 3.22, Ca 22.58. 
 Found. " 29.07, " 3.49, " 22.99. 
 
 The optical rotation of the substance in a 10 per cent solution of 
 HCl was 
 
 , ,.0 - 0.30 X 100 - ,0 
 
 ^< 0^0:4 ^-5 
 
 Thus the anhydrohexonic acid obtained from lyxohexosamine 
 yields on further oxidation an optically active anhydrotetrahy- 
 droxyadipic acid; hence it possesses the structure of anhydrotal- 
 onic acid. 
 
 2-5-Anhydrogalactonic Acid.- — ^This acid (Levene, 1917, a) was 
 obtained from chondrosaminic acid. 10 gm. lots of chondrosaminic 
 acid were treated in the same manner as epichondrosaminic acid in 
 the above experiment. The brucine salt obtained in this manner 
 melted at 244°. 
 
 The optical rotation was 
 
 uf= -0.47X100 ^ y, 
 ' '» 2 X 2.S08 ^ ' 
 
 The composition of the substance was the following: 
 
 0.0988 gm. substance, dried in a xylene bath: 0.2196 gm. CO2 and 0.0576 gm. 
 H2O. 
 
 C2jHmNjOio. Calculated. C 60.80, H 6.10. 
 Found. " 60.73, " 6.53. 
 
 10 gm. of the brucine salt were freed from brucine as in the above 
 experiment. The brucine-free solution was evaporated to 25 cc, 
 diluted with an equal volume of concentrated nitric acid, and boiled 
 over a free flame until the volume was reduced to about 20 cc; 
 then 10 cc. of nitric acid were again added, and the solution was 
 
p. A. LEVENE 43 
 
 again boiled over free flame. When the solution was concentrated 
 to 20 cc. it was transferred to a clock glass and concentrated on a 
 boiling water bath to dryness. The substance immediately crystal- 
 lized. It was redissolved in water and again evaporated. The 
 operation was repeated once more. The final crystalline residue was 
 dissolved in hot acetone and a very little ether was added, when a 
 small amorphous precipitate formed. This was removed by filtra- 
 tion, and the filtrate was allowed to crystallize. The crystals were 
 filtered, redissolved in hot acetone, and allowed to crystallize. The 
 final product melted at 205°C. 0.1600 gm. of the substance dis- 
 solved in 2.5 cc. of water showed no optical activity. 
 
 5. 2-5-ANHYDROTETROXYADIPIC ACIDS. 
 
 a. 2-5-Anhydrosaccharic Acid {Levene and La Forge, 1915, a). 
 
 Acid Potassium Salt.— 10 gm. of chitosaminic acid were deaminized 
 with silver nitrite according to Fischer and Tiemann, the resulting 
 solution concentrated to about 25 cc. and an equal volimie of nitric 
 acid added in the cold. Oxidation was carried out under the same 
 conditions as already described for chondrosaminic acid. The 
 syrupy product was dissolved in 10 cc. of water, neutralized with 
 strong potassiiun hydroxide solution, and an equal volume of glacial 
 acetic acid added. Upon dilution to 50 cc. with alcohol the acid 
 potassiiun salt of the dibasic acid soon began to crystallize, and after 
 standing one-half hour in the refrigerator the yield of the substance 
 washed and dried amounted to about 4 gm. For analysis it was 
 recrystallized from about 2 parts of hot water. The product contains 
 0.5 of a molecule of crystal water, which can be removed in vacuum 
 at 115°. 
 
 0.1868 gm. substance: 0.00S8 gm. H2O. 
 0.1811 " dried substance: O.OSll gm. K2CO3. 
 
 CeHsOjK -f- O-SHjO. Calculated. H2O 3.7. 
 Found. " 3.10. 
 
 CeHsOsK. Calculated. K 15.73. 
 Found. " 15.94. 
 
 Lead Salt. — 2.5 gm. of the acid potassium salt were dissolved in 
 about 100 cc. of water and an excess of neutral acetate solution was 
 
44 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 slowly added in the cold. Crystallization began after a few minutes 
 and was complete after one-half hour. The lead salt crystallizes 
 under these conditions in large nearly square plates which are very 
 difficultly soluble in water. The yield was practically quantitative. 
 The salt contained 2 molecules of crystal water, which could be 
 removed by heating in vacuum at 150°. The dried preparation 
 analyzed for a neutral lead salt of an anhydrodicarboxylic hexonic 
 acid. 
 
 0.2418 gm. substance: 0.0204 gm. H2O. 
 
 CsHsOsPb + 2H2O. Calculated. HaO 7.99. 
 
 CeHiOjPb + 2H!0. " " 8.32. 
 
 Found. " 8.40. 
 
 0.2214 gm. dried substance: 0.1690 gm. PbS04. 
 
 CsHsOaPb. Calculated. Pb 49.88. 
 
 CJJ«07Pb. " " 52.18. 
 
 Found. " 52.10. 
 
 Free Acid. — 7.8 gm. of the lead salt were suspended in 150 cc. of 
 water, and slightly less than the calculated amount of sulfuric acid 
 was added. The suspension was wanned on the water bath for 
 1 hour and then diluted with an equal volume of alcohol, filtered, 
 and the filtrate concentrated in vacuum to a thick syrup. This was 
 dissolved in acetone, filtered, and concentrated on the water bath. 
 The resulting syrup crystallized in the desiccator to a semisolid cake. 
 The crude product was extracted with a small amount of a mixture 
 of 1 part amyl alcohol and 2 parts ether, filtered, and washed with 
 the same solvent mixture and finally with dry ether. It may be 
 recrystallized by dissolving in a small amount of acetone and allow- 
 ing the solution to evaporate nearly to dryness in the air. It crystal- 
 lizes in aggregates of large plates, the edges of which are usually 
 rounded off by the solvent action of the ether used for washing. The 
 acid is extremely soluble in the usual reagents, with the exception 
 of cold amyl alcohol and ether. The yield was about 2 gm. of pure 
 substance. The product contains 1 molecule of crystal water which 
 can be removed by heating under diminished pressure at 78°. The 
 dry substance melts at 160° (uncorrected). 
 
p. A. LEVENE 45 
 
 0.1278 gm. substance: 0.0107 gm. H2O. 
 
 CoHsOt + HjO. Calculated. H3O 8.57. 
 Found. " 8.38. 
 
 0.1159 gm. substance: 0.1598 gm. CO2 and 0.0467 gm. H2O. 
 
 QHsO;. Calculated. C 37.50, H 4.28. 
 Found. " 37.57, " 4.47. 
 
 0.0898 gm. substance containing crystal water required for neutralization 8.6 
 CO. 0.1 N NaOH (calculated: 8.4 cc). 
 
 The specific rotation in water was 
 
 ..^ +3.12X2.1478 ^ 
 ^ '" 1.033X0.1637 ^*^- 
 
 2-S-Anhydrosaccharic acid should also be formed on oxidation 
 of epichitosamine. In its place, however, saccharic acid was formed. 
 
 b. 2-5-Anhydromannosacckaric Acid. 
 
 This acid (Levene, 1918, b) was previously obtained by Tiemann 
 on oxidation of chitosamine. In view of the fact that epichito- 
 saminic acid and its lactone on deamination gave rise to chitonic 
 acids they should give on oxidation 2-S-anhydromannonic acid. In 
 this instance the acid and its lactone gave rise to the same anhydro 
 acid. The Walden inversion took place possibly in both. 
 
 c. 2-5-Anhydro-l-Saccharic Acid. 
 
 This acid (Levene and La Forge, 1915, b) is formed on oxidation 
 of dextro-J-xylohexosaminic lactone and of levo-J-xylohexosaminic 
 acid, thus showing that in one of the two the Walden inversion took 
 place, probably in the acid. 
 
 3.5 gm. of the hydrochloride of xylohexosaminic acid lactone were 
 dissolved in 50 cc. of water and deaminized with 4 gm. of silver 
 nitrite. The reaction mixture was kept at 0° for the first 5 hours and 
 then allowed to stand over night at room temperature. If silver 
 was present in the solution it was removed with a few drops of hydro- 
 chloric acid, the silver chloride filtered off, and the filtrate concen- 
 trated in vacuum to about 15 cc. An equal volvune of concentrated 
 
46 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 nitric acid was then added and the solution boiled over a small 
 flame for about 12 minutes. It was then evaporated in a flat dish 
 on the water bath to a syrup which was freed from most of the nitric 
 acid by repeating the evaporation after adding a small amount of 
 water. The reaction product was taken up in about 6 cc. of water, 
 neutralized with a strong solution of potassium hydroxide, and al- 
 lowed to stand for 15 minutes at room temperature, after which 
 an equal volume of glacial acetic acid and about 3 volumes of ab- 
 solute alcohol were added. Upon standing in the refrigerator from 
 4 to 5 hours, crystallization of the acid potassitmi salt was complete. 
 The yield was 1.6 gm. It was recrystallized from 2 parts of hot 
 water. 
 
 0.2314 gm. substance: 0.0797 gm. K2S0<. 
 
 C6H7O7K + HjO. Calculated. K 15.70. 
 Found. " 1S.4S. 
 
 The rotation of the air-dried substance was 
 
 , ,s. -2.54X2.1114 „,„ 
 
 t"^"' 1X0.1400 ^^-^ 
 
 4 gm. of the acid potassium salt were dissolved in about 150 cc. 
 of water and the calculated amount of a 5 per cent solution of lead 
 acetate was added. Crystallization of the lead salt began at once 
 and was complete after about 1 hour. The lead salt was filtered 
 off with suction, washed with water, and dried at 100°. The yield 
 was 6 gm. The lead salt was suspended in about 100 cc. of water, 
 and slightly less than the calculated amount of sulfuric acid added. 
 After standing about 1 hour on the water bath the lead sulfate was 
 filtered off and the filtrate concentrated in vacuum to a thick syrup 
 which was dissolved in dry acetone and again evaporated to dr)Tiess. 
 The syrupy residue, which contained lead salts, was extracted with 
 dry acetone, and the colorless filtrate evaporated in a dish on the 
 water bath to a thick syrup. By repeating the evaporation with 
 acetone a few times the syrup crystaUized on cooling without crystal 
 water. It was freed from a small amount of adhering syrup by wash- 
 ing with a mixture of 1 part of amyl alcohol and 2 parts of dry ether. 
 The jdeld was 1.5 gm. It may be recrystallized by dissolving in a 
 
p. A. LEVENE 47 
 
 small amount of dry acetone and allowing the solution to evaporate 
 in the air. The substance melts at 163°. 
 
 0.1092 gm. substance: 0.1514 gm. CO2 and 0.0410 gm. HjO. 
 
 CHsOt. Calculated. C 37.50, H 4.20. 
 Found. " 37.80, " 4.20. 
 
 The rotation of the substance was 
 
 . „ ^ -3.08X2.1547 _ _ 
 ^"J" 1.034X0.1657 --38-80 
 
 Deamination of d-Levo-Xylohexosaminic Acid (Levene, 1918, b). — 
 7 gm. of the acid were deaminized in the way described above. 
 The reaction product was brought, by distillation under diminished 
 pressure, to a volume of 30 cc. An equal volume of nitric acid was 
 added. The solution was heated over a free flame until the begin- 
 ning of the evolution of fumes; it was then gently heated for 7 minutes 
 after which the solution was transferred to a clock glass, and evapo- 
 rated on a water bath to dryness. The residue was again dissolved 
 in a solution consisting of 5 cc. of nitric acid and 5 cc. of water. The 
 final product was converted into the acid potassium salt in the 
 manner described by Levene and La"Forge. The yield of the crude 
 product was 3.05 gm. The salt was recrystaUized three times and 
 then had the following composition: 
 
 0.1000 gm. substance: 0.0366 gm. KsSOi. 
 
 C6H7O7K -1- HjO. Calculated. K 15.70. 
 . Found. " 16.03. 
 
 The optical rotation was 
 
 . ,M -0. 75 X 100 -, -o 
 
 f^J" — n<2 ^^-^ 
 
 The fact that this acid was the antipodal form to the acid obtained 
 from chitosaminic brought out the configuration of the corresponding 
 acids as anhydrogulonic and anhydroglu.conic respectively, and 
 therefore of their epimers as anhydromannonic and anhydroidonic 
 acids. 
 
48 HEXOSAMDSTES, MUCINS, AND MTJCOrDS 
 
 d. 2-5-Anhydroidosaccharic Acid. 
 
 This acid (Levene and LaForge, 1915, b) was obtained on oxida- 
 tion of dextro-<i!-xyloliexosaminic acid. 
 
 10 gm. of pure dextro-rf-xylohexosaminic acid were dissolved in 
 100 cc. of water containing 3 per cent of hydrochloric acid, and 10 
 gm. of silver nitrate were added. Twice in the course of 24 hours, 
 portions of 2 gm. of silver nitrite and 4 cc. of 10 per cent hydrochloric 
 acid were added. The excess of hydrochloric, acid was removed 
 quantitatively by means of silver nitrate. The filtrate was then 
 concentrated to 20 cc, an equal volume of nitric acid was added, 
 and the solution was kept boiling for 12 minutes. The solution was 
 then evaporated on a clock glass. Immediately on evaporation the 
 substance crystallized. It was taken up in a minimiun amount of 
 water and several volumes of acetone were added. On standing, 
 the substance crystallized. The yield of recrystallized substance 
 was 4.8 gm. The composition of the substance was the following: 
 
 0.1128 gm. substance lost on drying 0.0186 gm. 
 
 CeHaO? + 2H20. Calculated. H2O 15.80. 
 Found. " 16.49. 
 
 0.0942 gm. dry substance on combustion: 0.1282 gm. CO2 and 0.0362 gm. H2O. 
 
 CeHsO?. Calculated. C 37.50, H 4.20. 
 Found. " 37.11, " 4.30. 
 
 The rotation of the subject was 
 
 ^ » -1.56X100 ^_y 
 
 or calculated for the dry substance = — 93.4°. 
 
 The same substance was obtained by Levene and LaForge on 
 oxidation of the mixed xylohexosaminic acids. 
 
 e. 2-5-Anhydromucic Acid. 
 
 This acid was obtained originally from the natural chitosaminic 
 acid by Levene and LaForge and subsequently from the synthetic 
 levo-ci-lyxohexosaminic acid. 
 
p. A. LEVENE 49 
 
 9 gm. of lyxohexosaminic acid were dissolved in 100 cc. of 3 per 
 cent hydrochloric acid, and 15 gm. of silver nitrite added in portions 
 over a period of about 4 hours. A few cc. of 10 per cent hydrochloric 
 acid were then added and the reaction was allowed to proceed over 
 night at room temperature. The silver chloride was filtered off 
 and all but a slight excess of the hydrochloric acid removed from the 
 filtrate with silver nitrate. The clear solution was concentrated 
 under diminished pressure to about 25 cc, 30 cc. of concentrated 
 nitric acid were added, and the solution was warmed in an Erlen- 
 meyer flask over a small flame until a vigorous evolution of red 
 fumes began. The flame was then removed and the reaction al- 
 lowed to proceed with occasional warming. After 12 minutes the 
 solution was poured into two glass dishes and evaporated to a thick 
 syrup on the water bath. The contents of the dishes were then 
 diluted with a little water, combined, and again concentrated. 
 After evaporating once more with water, the syrup on cooling solid- 
 ified to a mass of white crystals which appeared under the micro- 
 scope as white oblong plates. After standing in the refrigerator for 
 a time the mass was triturated with a few cc. of a mixture of dry 
 acetone and ether, and filtered on a Biichner funnel. The process 
 was repeated once more to remove the adhering syrup. The crude 
 product after drying weighed about 2 gm., although more crystallized 
 from the acetone-ether mixture used for drying. For analysis it 
 was recrystaUized by dissolving in about 100 parts of boiling dry 
 acetone, and filtering and concentrating the solution to about one- 
 eighth of its volume. Anhydromucic acid crystallizes from acetone 
 without crystal water in long prisms which melt at 203-204° 
 (corrected). 
 
 0.1040 gm. substance required 10.7 cc. 0.1 N KOH; calculated, 10.8 cc. 
 0.1563 " " in 3 cc. H2O showed no rotation with D-light in a 1 dm. 
 tube, where a rotation of 0.02° would not have escaped detection. 
 0.1424 gm. substance: 0.1946 gm. CO2 and 0.0534 gm. H2O. 
 
 CeHsOr. Calculated. C 37.50, H 4.20. 
 Found. " 37.27, " 4.17. 
 
 /. 2-5-Ankydro-d-Talomucic Acid. 
 
 This acid was obtained first by Levene and LaForge on oxidation 
 of natural chondrosamine'(1918, a). Later it was obtained from 
 
50 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 the synthetic chondrosamine (Ledderhose, 1880), from synthetic 
 dextro-(i-lyxohexosaminic acid (Levene, 1918, 6) (epichondrosaminic 
 acid) and from dextro-J-ribohexosaminic acids (1921). 
 
 From Synthetic Sugar. — 6 gm. of the synthetic chondrosamine 
 hydrochloride were dissolved in 30 cc. of water containing 1 cc. of 
 hydrochloric acid. 8 gm. of silver nitrite were added and the mix- 
 ture was allowed to stand for 6 hours. It was then filtered. The 
 silver was removed from the filtrate by means of hydrogen sulfide. 
 The clear filtrate from the silver sulfide was concentrated to 20 cc, 
 cooled to 0°C., and diluted with 20 cc. of nitric acid also cooled at 
 0°C. The solution was allowed to stand over night. It was then 
 boiled over a free flame until a lively evolution of yellow fumes set 
 in. The solution was then transferred to a clock glass, evaporated 
 on a water bath, and the. calcium salt was then prepared in the usual 
 way. The salt was reprecipitated twice and had the following 
 composition: 
 
 0.1054 gm. substance, on drying in a xylene bath, lost 0.0130 gm. H2O. 
 
 CeHsOsCa + 2H2O. Calculated. H3O 12.68. 
 Found. " 12.3. 
 
 0.0924 gm. dry substance, on combustion: 0.0982 gm. COs, 0.0298 gm. H2O, 
 and 0.0924 gm. CaO. 
 
 CsHeOsCa + HiO. Calculated. C 29.03, H 3.22, Ca 22.58. 
 Found. " 28.98, " 3.60, " 23.05. 
 
 The optical rotation of the substance in 10 per cent HCl was 
 
 Initial. Equilibrium. 
 . ,« -0.32 X 100 „n„ , ,« -0.32 X 100 . -„ 
 
 f< = 1X4 = -^-^ ^< 1X1 ^-^ 
 
 Hence the substance is 2-5-anhydrotalomucic acid. It is iden- 
 tical with the substance previously obtained from natural chon- 
 drosamine. 
 
 From Dextro-d-Lyxokexosaminic Acid. — The solution obtained on 
 deamination of 6 gm. of the amino-acid was concentrated to 35 cc, 
 and an equal volume of concentrated nitric acid was added, the 
 solution heated over a flame until the evolution of red fumes, and 
 then the heating maintained for 7 minutes. The reaction product 
 
p. A. LEVENE 51 
 
 was rapidly evaporated Avith constant stirring on a clock glass placed 
 on a boiling water bath. The residue was redissolved in a solution 
 of 5 cc. of water and 5 cc. of nitric acid, and again evaporated to 
 dryness. The product was then evaporated twice with water in 
 order to remove the adhering nitric acid. Finally the aqueous 
 solution was converted into the calciuna salt. The yield was 3.5 gm. 
 of the crude calcium salt. The crude material was purified by re- 
 moving the calcium by means of oxalic acid and reconverting the 
 filtrate into the calcium salt. The dry substance had the following: 
 composition: 
 
 0.0988 gm. substance: 0.1042 gm. CO2, 0.0302 gm.- H2O, and 0.0222 gm. CaOi. 
 
 (QiHsOgCa). Calculated. C 29.03, H 3.22, CaO 22.58. 
 Found. " 28.76, " 3.44, " 22.47. 
 
 The rotation of the substance was 
 
 . ,10 -0. 45 X 100 ..„ 
 
 ^< r3<5 ^-^ 
 
 Under the same conditions of oxidation, chondrosaminic acid 
 yields the inactive anhydromucic acid. 
 
 2-5-Anhydrotalomucic Acid From Dextro-d-Ribohexosaminic Acid. — 
 5 gm. of the substance were deaminized in the same manner as the 
 levo form. For oxidation the solution was diluted to 60 cc, to 
 which an equal volume of nitric acid was added. The resulting 
 solution was heated over a free flame for 13 minutes. The product 
 of the reaction was transferred to clock glasses and rapidly concen- 
 trated nearly to dryness. The residue was reoxidized once more with 
 a solution of equal parts of water and nitric acid, and finally 
 evaporated with water to remove nitric atfid. The residue was then 
 converted into Ca salt. The yield was 2.8 gm. For purification 
 the salt was reconverted into free acid by means of oxalic acid and 
 reconverted into the Ca salt. The pure salt crystallized partly 
 in plates and partly in prismatic needles. Heated under diminished 
 pressure at a temperature of xylene vapor, it lost 2 molecules of water. 
 The salt was levorotatory. It analyzed as follows: 
 
52 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 0.1216 gm. substance on drying lost 0.0160 gm. 
 
 CeHjOsCa + 2HaO. Calculated. H2O 12.68 
 Found. « 13.16 
 
 0.1056 gm. substance: 0.1120 gm. CO2, 0.0302 gm. H2O, and 0.0242 gm. CaO. 
 
 CeHeOsCa + H4O. Calculated. C 29.03, H 3.22, CaO 22.58. 
 Found. " 28.98, « 3.20, " 22.92. 
 
 The optical rotation of the substance was 
 
 . ,20 -0.18 X 100 -no 
 
 ^< rx2 ^-^ 
 
 Free Acid. — 6.8 gm. of the calcium salt prepared from the natural 
 sugar were introduced in portions into 250 cc. of boiling water con- 
 taining 3.2 gm. of oxahc acid. After boiling for one-half hour, the 
 solution was treated with a little animal charcoal and filtered; the 
 filtrate was concentrated to a syrup which was dissolved in about 
 30 cc. of acetone, filtered, and again concentrated to a syrup on the 
 water bath. On standing over night, large colorless prisms had 
 separated out. Upon completion of crystallization, the adhering 
 syrup was dissolved from the crystals with a few cc. of a solution of 
 equal parts of acetone and ether. The crystals were then filtered 
 off with suction and washed with a small amount of acetone-ether 
 solution. The crystals consisted of large parallelopipeds which 
 melted at 179-181° (uncorrected), and at this temperature slowly 
 decomposed with gas evolution. The pure substance is rather 
 difficultly soluble in acetone (about 1 to 40 at the boiling point of the 
 solvent). For analysis it was dissolved in acetone, which was 
 allowed to evaporate partially. The melting point of the recrystal- 
 lized product did not differ from that of the first. The substance 
 is easily soluble in water and alcohol, but nearly insoluble in ether. 
 The yield amounted to 2.2 gm. 
 
 0.1094 gm. substance: 0.1480 gm. CO2 and 0.0404 gm. H2O. 
 
 CsHsOj. Calculated. C 37.50, H 4.20. 
 Found. " 37.11, " 4.15. 
 
 1500 gm. substance required 15.9 cc. 0.1 n NaOH (calculated: 15.6 cc). 
 
 , 2, _ - 1.07 X 3.3839 _ _ • 
 ^"'° IX 0.2056 X 1.0262 ~ •*° 
 
p. A. lEVENE 53 
 
 g. 2-5-Anhydroallonmcic Acid. 
 
 This acid (Levene and Clark) was prepared from levo-<i-ribohexo- 
 saminic acid. Two 5 gm. portions of the amino-acid were dissolved 
 each in 35 cc. of water; to the solution 15 cc. of 10 per cent hydro- 
 chloric acid and 5 gm. of silver nitrite were added. The mixture was 
 allowed to react over night. Twice during the reaction 2 gm. portions 
 of the nitrite and 2 cc. of hydrochloric acid were added. The reac- 
 tion product was freed from excess of silver and the filtrate reduced 
 to a volume of 75 cc. by distillation under diminished pressure at 
 40-50°C. of the water bath. To this solution 50 cc. of nitric acid 
 were added, and the resulting solution was heated over a flame for 
 20 minutes and then rapidly concentrated to a thick mass on a water 
 bath. The thick residue was dissolved in 10 cc. of a solution con- 
 sisting of equal parts of water and concentrated nitric acid, then 
 evaporated once with water to remove nitric acid. The final residue 
 was then converted into the calcium salt of 2-5-anhydroallomucic 
 acid. For final analysis the salt was suspended in water and its 
 calcium removed by boiling in hot water containing a slight excess 
 over the required amount of oxalic acid. 
 
 The calcitmi salt of 2-5-anhydroallomucic differs from that of 
 2-5-anhydrotalomucic, first by being optically inactive and second 
 by the difference in behavior on heating. Both salts crystallize 
 with 3 molecules of crystal water. Heated under diminished pres- 
 sure at the temperature of xylene vapor, anhydroallomucic salt 
 loses all its 3 molecules of crystal water, whereas the corresponding 
 anhydrotalomucic salt loses only 2, retaining the third one. The 
 Ca salt of anhydroallomucic acid analyzed as follows : 
 
 0.1228 gm. substance on drying lost 0.0244 gm. 
 
 GftOTCa + 3H2O. Calculated. H2O 19.02. 
 Found. " 19.87. 
 
 0.0984 gm. substance on combustion: 0.1100 gm. CO2, 0.0270 gm. HjO, and 
 0.0984 gm. CaO. 
 
 CHsOrCa. Calculated. C 31.30, H 2.61, CaO 24.35. 
 Found. " 30.99, " 3.07, " 25.00. 
 
54 HKXOSAMINES, MUCINS, AND MUCOIDS 
 
 6. CHITOSE AND EPICHITOSE. 
 
 Chitose was obtained first by Ledderhose (1880) and by Tiemann. 
 Fischer and Andrea have recognized the substance as 2-5-anhydro- 
 hexose. In the light of the fact that on further oxidation the sub- 
 stance is converted into 2-5-anhydroniannosaccharic acid, it is 
 evident that it has the structure of anhydromannose. Epichitose, 
 being the epimer of chitose, has the configuration of 2-5-anhydro- 
 glucose. The substance was prepared in the following way. 
 
 Epichitose {Levene, 1919). — 15 gm. of ash-free epichitosamine 
 hydrochloride were taken up in 250 cc. of water and 80 gm. of mer- 
 curic oxide, and heated on a boihng water bath for 30 minutes, at . 
 the end of which time the greater part of the oxide turned from 
 orange to gray. Through the filtrate from the oxide, hydrogen 
 sulfide gas was passed and the filtrate from the sulfide was concen- 
 trated to a very thick syrup. This was taken up with a little methyl 
 alcohol. On standing over night a crystalline deposit formed. It 
 was nitrogen-free, it reduced Fehling's solution on heating, and 
 melted at 240° (corrected) with decomposition. The analysis of the 
 substance gave the following results : 
 
 0.1031 gm. substance: 0.1670 gm. CO2 and 0.0S70 gm. HjO. 
 
 CJIioOs Calculated. C 44.44, H 6.17. 
 Found. " 44.18, " 6.18. 
 
 The optical rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . ,» -1.92 X 100 ofio r i» -1.92X100 .,„ 
 
 7. 3-AMINOHEPTONIC ACIDS. 
 
 3-Aminoheptonic acids were prepared in the form of their amor- 
 phous copper salts by Neuberg, and Neuberg and Wold. These 
 were not pure. 
 
 a. Chitosaniinoheptonic Acids. 
 
 Preparation of Ckitosaminoheptonic Acids (Levene, 1916, a; Levene 
 and Matsuo). — The conditions for the preparation o : these acids are 
 
p. A. lEVENE 55 
 
 more uncertain than of any other substance of this group. At one 
 time (1915) good yields of the crystalline substance were obtained in 
 the following way. 50 gm. of chitosamine hydrochloride were taken 
 up in 100 cc. of water, 18 cc. of an 80 per cent aqueous prussic acid and 
 25 cc. of ammonium hydroxide were added, and the solution was 
 allowed to stand from 24 to 48 hours. The product was then trans- 
 ferred to water containing about 150 gm. of barium hydroxide, and 
 the solution was boiled over free flame as long as ammonia was still 
 evolved (about 48 hours) . The barium and hydrochloric acid were 
 removed in the usual way and the final solution was concentrated 
 under diminished pressure to a thick syrup. This was taken up 
 in a little water, hot 95 per cent alcohol was added as long as a 
 gimimy precipitate formed, and the supernatant liquid was decanted 
 and allowed to stand over night at 0°C. A crystalline deposit 
 formed, and from the mother liquor, on concentration and repeated 
 treatment with alcohol, etc., a second crop of crystals was gener- 
 ally obtained. The total yield was about 30 per cent of the 
 employed chitosamine. This material was slightly levorotatory. 
 
 In 1916 the laboratories were transferred to a new building, and 
 all attempts to prepare the material by apparently the same process 
 failed. After many experiments it was finally possible again to 
 obtain the substance under the following conditions. 35 gm. of 
 chitosamine hydrochloride were dissolved in 100 cc. of water; 35 gm. 
 of aqueous 80 per cent prussic acid and 20 cc. of ammonia were added. 
 The mixture was warmed to 30°C. and then allowed to stand at room 
 temperature for 1 hour. The temperature generally remained 
 constant and all the sugar was dissolved in the course of that time. 
 The solution was then transferred to an aqueous solution of barium 
 hydroxide and the further treatment proceeded as above. The 
 final solution was concentrated to a small volume and hot methyl 
 alcohol was added to slight opalescence. The solution was then 
 placed on a hot water bath until a crystalline deposit began to form. 
 The yield varied, the maximum being 15 per cent of the employed 
 chitosamine hydrochloride. The specific rotation of this substance 
 was [at = + 4.0°. 
 
 Dextro-d-Chitosaminoheptonic Acid. — 130 gm. of chitosamino- 
 heptonic acid were dissolved in 4 parts of hot water. The original 
 material had the following rotation in 2.5 per cent HCl solution: 
 
56 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Initial. Equilibriiim. 
 
 No. 225 
 . ,« +0.16 X 100 , -o r 1" -0.04 X 100 
 
 After standing over night no crystals separated, hence the solution 
 was concentrated under diminished pressure until a crystalline 
 deposit began to form and then allowed to stand over night. 74 gm. 
 of crystals were obtained which had the following rotation in 2.5 
 per cent HCl solution. 
 
 Initial. 
 No. 236 
 , ,« +0.23 X 100 ,.., 
 
 These 74 gm. of heptonic acid were dissolved in 150 cc. of hot 
 water; 43 gm. of crystals settled out which had the following rota- 
 tion in 2.5 per cent HCl solution. 
 
 Initial. Final f 24 hrs.). 
 
 No. 246 
 
 , ,» ^ +0.26 X 100 ^ „ _ +0.11 X 100 _ 
 
 ' ^" 1X4 +°-^ ^"Jo fxl - +2-^ 
 
 These crystals were dissolved again in 86 cc. of hot water and 35 
 
 gm. of crystals were obtained which had the following rotation in 2.5 
 
 per cent HCl solution. 
 
 Initial. 
 No. 247 
 
 , ,« +0.26 X 100 , , ,„ 
 l«iD = ][3^-4 = +6.5° 
 
 These 35 gm. were again dissolved in 70 cc. of hot water, and there 
 were obtained 28 gm. of crystals which had the following rotation 
 in 2.5 per cent HCl solution. 
 
 Initial, 
 f ,» +0.26 X 100 , . „ 
 
 From the mother liquor 37 gm. of the same substance were ob- 
 tained; the total yield was 65 gm. The substance crystallized 
 out of water in heavy prisms. Melting point was 192°C. with 
 decomposition. 
 
p. A. LEVENE 57 
 
 Levo-d-Chitosaminoheptonic Acid. — ^To the mother liquor of the 
 first precipitate, hot methyl alcohol was added to a slight opalescence 
 and the solution was allowed to stand for 4 hours. A precipitate 
 then separated and was filtered off; the mother liquor was concen- 
 trated under diminished pressure and hot alcohol was added. A 
 precipitate was again formed and filtered off, and to the mother 
 liquor more alcohol was added. 5 gm. of crystals separated which 
 had the following rotation in 2.5 per cent HCl solution. 
 
 Initial. 
 No.^307 
 , ,» -0.16X100 .„ 
 
 ^< rx4 ^ 
 
 These 5 gm. were dissolved in 5 cc. of hot water. After long stand- 
 ing there were obtained 1.85 gm. of crystals which had the follow- 
 ing rotation in 2.5 per cent HCl solution. 
 
 Initial. 
 No. 309 
 . ,» -0 .13 X 100 ,„ 
 
 ^< rx2 ^-^ 
 
 The mother liquor of No. 307 was allowed to stand. On long 
 standing, there crystallized 4.75 gm. of a substance with the follow- 
 ing rotation in 2.5 per cent HCl solution. 
 
 Initial. 
 , No. 261 
 
 , ,» -0.11 X 100 , ,0 
 
 ^< 0^2 ^-^ 
 
 These crystals were dissolved in 5 cc. of hot water and gave 1.6 gm. 
 of crystals which had the following rotation. 
 
 Initial. 
 No. 358 
 , ... -0.15 X 100 , „ 
 
 ^< — n<2 ^-^ 
 
 Nos. 309 and 358 were combined and dissolved in 4 cc. of hot 
 water. 1.5 gm. of crystals were obtained which had the following 
 rotation in 2.5 per cent HCl solution. 
 
58 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Inifial. Final (24 hrs.). 
 
 No. 380 
 
 , ,« -0. 15 X 100 , -„ r i» -0.24 X 100 „„ 
 
 [«]„ j-3^2 — " "^-^ ^ '° Txl ^^ 
 
 The 1.5 gm. were dissolved again in 1.5 cc. of hot water and gave 
 1.2 gm. of crystals which had the following rotation in 2.5 per cent 
 
 HCl solution. 
 
 Initial. 
 No. 381 
 , ,« -0.15 X 100 - -o 
 
 t"'» — no ^-^ 
 
 It crystallized out of 25 per cent alcohol solution in long prismatic 
 needles. The melting point was 139°C. (corrected) with decom- 
 position. 
 
 b. Chondrosaminoheptonic Acid. 
 
 Preparation of Chondrosaminoheptonic Acid (Levene, 1916, c; Levene 
 and Matsuo). — 35 gm. of chondrosamine hydrochloride were taken 
 up in 70 cc. of water; 35 cc. of 80 per cent aqueous solution of 
 hydrocyanic acid and 22 cc. of strong ammonia water were added. 
 This mixture was warmed at 42-45° for about 15 minutes. The solu- 
 tion turned dark in color and became viscous. It was cooled in an 
 ice-alcohol bath untU the temperature of solution came down to 0°C., 
 when it was transferred to an aqueous solution of 200 gm. of barium 
 hydroxide. The solution was allowed to stand about 1 hour;, then 
 boiled on free flame for about 3 hours and distDled under diminished 
 pressure to dryness. The residue was dissolved in 100 cc. of water 
 and the solution was again distilled to dryness; this operation was 
 repeated 10 times. By this procedure all ammonia was driven off. 
 
 Barimn was removed by means of a smaU excess of sulfuric acid, 
 and the excess of this was removed by means of lead carbonate. 
 The hydrochloric acid was removed by means of AgCOs. The excess 
 of lead and silver was removed by means of hydrogen sulfide gas. 
 The filtrate from silver sulfide was concentrated under diminished 
 pressure to a syrup, and seeded with chondrosaminoheptonic acid. 
 Hot methyl alcohol was added very cautiously until slight opales- 
 cence, and the solution was allowed to stand for about 72 hours at 
 
p. A. LEVENE 59 
 
 room temperature. (Sometimes the substance crystallized out of 
 aqueous solution.) 
 
 The crystalline deposit was filtered, washed with 50 per cent alco- 
 hol, 95 per cent alcohol, and absolute alcohol successively, and 
 finally with ether. The best yield was 11 gm. In this manner 
 443 gm. of the substance were prepared. 
 
 Levo-d-Chondrosaminokeptonic Acid. — 440 gm. of chondrosamino- 
 heptonic acid, which had the following rotation in 2.5 per cent HCl 
 solution, 
 
 Initial. 
 , ,25 -0.14 X 100 - .„ 
 
 t< TxT— =-^-^ 
 
 were dissolved in 800 cc. of hot water. On standing over night 
 crystals separated out; which had the following rotation: 
 
 Initial. Equilibrium (24hrs.). 
 
 , ,K -0.30 x'lOO - ,„ r i» -0.48 X 100 ,,„ 
 
 f"^° r3<^ = -^-^ t«]x, ^3^^- = -12 
 
 The yield was 344 gm. 
 
 The 344 gm. of heptonic acid were dissolved in 800 cc. of hot water, 
 and 391 gm. of crystals were obtained. 
 The rotation in 2.5 per cent HCl solution was 
 
 Initial. Final (24 hrs.). 
 , ,» -0.33X100 -o r i» -0.49 X 100 ,,„ 
 
 Wd 1X4 = ~* f"^" = Txl ^2 
 
 These crystals were dissolved again in 800 cc. of hot water, and 
 265 gm. of crystals were obtained which had the same rotation in 
 2.5 per cent HCl solution. 
 
 Initial. Final (24 hrs.). 
 
 , ,» -0.3 3 X 100 .„ r ,» -0.52 X 100 _ ,,o 
 
 In the mother liquor a further deposit of the same substance 
 settled out. The total yield of levo-(/-chondrosaminoheptonic acid 
 was 313 gm. It crystallized out of water in elongated prisms. 
 The melting point was 139°C. (corrected) with decomposition. 
 
60 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 Dextro-d-Chondrosaminoheptonic Acid. — The mother liquor of the 
 first crystallization was concentrated under diminished pressure and 
 a precipitate formed which was filtered off. To the filtrate an equal 
 volimie of 95 per cent alcohol was added and the solution was allowed 
 to stand for 24 hours. A second precipitate formed and was filtered 
 off again and the solution was poured into absolute alcohol under 
 constant stirring. A gimimy precipitate formed which was removed 
 and the solution was concentrated to a syrup under diminished 
 pressure at room temperature. Alcohol was then added and the 
 solution allowed to stand for several days. 
 
 Crystals separated which had the following rotation in 2.5 per cent 
 HCl solution: 
 
 Initial. Final (24 hrs.). 
 
 No. 351 
 . ,» +0.64 X 100 , ,,„ r ,» +1.02 X 100 
 
 Wd = — Yxi — " ''■^^ ^"1° " — r^i — = +2^ 
 
 The yield was 1.9 gm. 
 
 In the mother liquor a second deposit of 2.3 gm. of crystals formed 
 which had the following rotation in 2.5 per cent HCl solution: 
 
 Initial. Final (24 hrs.). 
 
 No. 311 
 
 r„1» - +0-50 X 100 _ « +0.85 X 100 , ._ 
 Wo 1X2 - +25 [«]„ = ^3^^ +42° 
 
 In the mother liquor a further deposit of crystals formed which 
 had the following rotation in 2.5 per cent HCl solution: 
 
 Initial. Final (24 hrs.). 
 
 No. 352 
 
 U]» = ±0:1^X100 ^ ^^^„ ^^j„ ^ +i,«xiOO. ^ ^^^„ 
 
 From the mother liquor of this substance there was obtained on 
 further crystallization a substance with the following specific rotation 
 in 2.5 per cent HCl solution: 
 
 Initial Final (24 hrs.). 
 
 No. 407 
 
 r„f = +0-85 X 100 _ „ +1.30X100 , ^„ 
 
PART II, 
 MUCINS AND MUCOIDS. 
 
 THEORETICAL. 
 
 1.. GENERAL CONSIDERATIONS. 
 
 The structure of mucin and allied substances has remained in 
 obscurity until recent years, although these substances interested 
 both the chemist and the physiologist. Knowledge was lacking, 
 particularly regarding the mode of union between the sugar and the 
 rest of the molecule. The information given in text-books was 
 generally limited to that of the solubilities and some other physical 
 properties. The substances were classified in three groups: mucins, 
 mucoids, and glucoproteins. More recently Irvine and Hynd 
 (1913) have suggested the following structure of this group of 
 substances: 
 
 CHj(OR,)CH(ORi)CH CH(0R3)CH CH 
 
 ii 
 
 <r— o 
 
 RiRiG 
 
 According to this conception, the amino group of the nitrogenous 
 sugars serves as a bridge for the linking of amino-acids to the sugar, 
 in a peptide as well as in 9, glycosidic union. The hypothesis sug- 
 gested itself to Irvine and Hynd on the ground of their work on the 
 glycosides of chitosamine, and was based entirely on speculative 
 evidence. Irvine expressed the view rather tentatively, realizing 
 the need of experimental proof. 
 
 On the other hand, evidence was accumulating tending to show 
 that all substances allied to mucins contained in their molecule a 
 complex acid which in its structure bore no similarity or relationship 
 to peptides. 
 
 For many years it was known that an acid of the tj^e of conjuga- 
 ted sulfuric acid existed in the molecule of chondromucoid (Morner). 
 Later its existence was revealed also in tendomucoid. The acid 
 
 61 
 
62 HEXOSAMnSTES, MUCINS, AND MUCOIDS 
 
 under the name of chondroitin sulfuric acid was discovered by 
 Morner, and received further attention through the efforts of 
 Schmiedeberg at a time when methods applied to the study of 
 carbohydrates were very imperfect. Later, in the course of the work 
 it was found that the acid could not be isolated with equal facility 
 from every mucin or mucoid (Levene, 1900-01; L6pez-Suarez). 
 In many instances the removal of the protein radicle was accom- 
 plished only after mmierous operations, and in the course of these 
 operations the conjugated sulfuric acid suffered partial decomposi- 
 tion. It is still not very clear whether the differences of individual 
 mucins lay in the protein or in the acid radicles. 
 
 Finally a chemical distinction was discovered between the con- 
 jugated sulfuric acids of different origin (Mandel and Levene, 
 Alzona, Levene and Lopez-Suarez, 1916, a). The difference was in 
 the components. Whereas one contained in its molecule the nitro- 
 genous hexose, chondrosamine, the other had in its place chitosa- 
 mine. Miiller had previously shown that this sugar could be ob- 
 tained on hydrolysis of saUvary mucin. For the former the name 
 chondroitin sulfuric acid was retained; the latter was named mucoitin 
 sulfuric acid. 
 
 The substances of the first group revealed a comparatively greater 
 resistance towards hydrolytic agents and could be obtained in a fair 
 degree of purity, whereas the substances of the second group, when 
 prepared free from protein, always appeared in the form of a mixture 
 of mucoitin sulfuric acid with mucoitin. Also the intermediary 
 substances showed a difference in their resistance towards hydrolytic 
 agents, so that chondrosin could be prepared in a practically pure 
 state whereas mucosin was found so labile that, under the conditions 
 which permitted the preparation of the former, the latter was nearly 
 completely hydrolyzed. On the other hand, when mucoitin sulfuric 
 acid was subjected to milder hydrolysis a mucosin resulted which 
 still contained undecomposed mucoitin. 
 
 Because of this instability of mucoitin sulfuric acid, the acid itself 
 and the products of its partial hydrolysis were not obtained in the 
 same degree of purity as chondroitin sulfuric acid and the corre- 
 sponding products derived from it. 
 
p. A. LEVENE 
 
 63 
 
 Furthermore, some differences were observed between various 
 members of the mucoitin sulfuric acid group. It is true that, since 
 the individual members were not always obtained in absolutely pure 
 state, there is also the possibility that these differences were brought 
 about by impurities. However, the impression still remains that the 
 acids of this group belong to two types. A representative of one 
 type is the substance derived from funis mucin. The acid of this 
 type is characterized by its gelatinous nature when precipitated by 
 glacial acetic acid, by the comparatively small quantity of glacial 
 acetic acid required for its precipitation, and by the readiness with 
 which it forms an insoluble barium salt. The bariiun salt is very 
 sparingly soluble in water, but is readily dissolved in the presence 
 of acetates. 
 
 The second type of mucoitin sulfuric acid is characterized by the 
 greater solubility of the barium salt, by the fact that the substance 
 is precipitated by glacial acetic acid in the form of a flocculent 
 precipitate, and by the fact that a large excess of the acid is required 
 in order to bring about the precipitation. A representative member 
 of this type is the substance obtained from gastric mucus. 
 
 2. STRUCTURE OF CHONDROITIN SULFURIC ACID. 
 
 The structure of chondroitin sulfuric acid is most satisfactorily 
 expressed by the following graphic formula: 
 
 C Hj - O - SOi - OH 
 I 
 
 C Hj - O - SOj - OH 
 
 OH CH 
 H-t- 
 
 OH CH 
 
 I 
 
 H— COH 
 
 I 
 H— C NH 
 
 I 
 H— C 
 
 H— C- 
 
 . COCH3 
 
 O 
 
 -0- 
 
 OH H 1 I 
 H-C-C-C-C-C- COOH 
 I H OH H H 
 i O 
 
 _J 
 
 H— COH 
 
 I 
 H— C NH ■ 
 
 I 
 H— C 
 
 I 
 
 O 
 OH H 
 
 H-C-C-C-C-C- COOH 
 H OH H H 
 
 COCH, 
 
64 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 In both formulas the position of the sulfuric acid radicle, the place 
 of union of sugar and glucuronic acid, and the position of the amino 
 groups are arbitrary. To the sulfuric acid is assigned a different 
 position in the two substances in order to indicate the existence of 
 a difference in their respective behavior towards hydrolytic agents. 
 
 The data which led to the formula were obtained by Levene and 
 La Forge, (1913, 1914o, 1915a) and by Levene. The formula postu- 
 lates that chondroitin sulfuric acid contains 4 components in equi- 
 molecular proportion: chondrosaminic, glucuronic, acetic and sulfuric 
 acids. Each one of these components has been isolated and identified. 
 
 The order of linking as expressed in this formula is the following: 
 Chondrosamine and glucuronic acid are linked into a disaccharide. 
 This was made evident by the discovery of Schmiedeberg, that on 
 partial hydrolysis of chondroitin sulfuric acid a disaccharide chon- 
 drosin is formed. The linking of the disaccharide is as expressed 
 in the formula and not as assumed by Schmiedeberg; namely, carbon 
 atom 1 of chondrosamine is combined in glucosidic linking with one 
 of the hydroxyls of the glucuronic acid. The experimental facts 
 leading to this conclusion are the following: 
 
 1. In chondrosin the carboxyl of glucuronic acid is free. 
 
 2. When chondrosin is oxidized by bromine and subsequently 
 cleaved, saccharic acid is formed. 
 
 3. In chondrosin the nitrogen is present in the form of a primary 
 amino group. This observation was made on amorphous chondrosin 
 hydrochloride and on chondridin. Chondridin is a crystalline 
 substance discovered by Hebting in Hofmeister's laboratory. 
 Levene and Lopez-Suarez have shown the substance to have the 
 structure of anhydrochondrogin. 
 
 The formula further postulates that the acetyl group is linked to 
 the amino group of the sugar. This assumption is based on the 
 observation that chondroitin sulfuric acid does not contain free 
 amino groups, whereas the nitrogen in chondrosin is present entirely 
 in this form. 
 
 The formula finally postulates that two units are combined one 
 to another in a glucosidic linking between the glucuronic acids. 
 This assumption is based on the fact that chondroitin sulfuric acid 
 does not reduce Fehling's solution, whereas chondrosin does. 
 
p. A. LEVENE 
 
 65 
 
 3. STRUCTURE OF MUCOITIN SULFURIC ACID. 
 
 Concerning the structure of the acids of this group, it seems per- 
 missible to assume the same hypothesis as was formulated for chon- 
 droitin sulfuric acid. On removal of sulfuric acid a substance is 
 formed which is analogous to chondroitin. It is non-reducing, and 
 does not contain free amino groups. On hydrolysis it forms mucosin, 
 which is a disaccharide composed of glucuronic acid and chitosamine. 
 Mucoitin, similarly to chondroitin, contains one acetyl group to 
 each molecule of chitosamine, glucuronic, and sulfuric acids. 
 
 The glucuronic acid was identified in all the acids of the mucoitin 
 sulfuric acid group but not with the same degree of exactness. 
 Whereas in the acids of the funis mucin type the glucuronic acid was 
 identified by the formation of furfural, by the analysis of the phenyl- 
 hydrazine derivatives, and by the analysis of the acid potassium salt 
 of saccharic acid formed on oxidation with nitric acid. In the acids 
 of the second grouff it was demonstrated only by the furfural distilla- 
 tion and by the formation of the phenylhydrazine, which, however, 
 was obtained in a quantity too small for purification and analysis. 
 
 As regards the details of the structure, all the arguments which 
 
 suggested the structural formula of chondroitin sulfuric acid may be 
 
 repeated in the formulation for mucoitin sulfuric acid. This is as 
 
 follows: 
 
 CH2OH CHsOH 
 
 I I 
 
 OH-SOj - O - C H OH-SOj - O - C H 
 
 -CH 
 
 O 
 
 HCOH 
 
 I 
 HCNH-COCH3 
 
 -CH 
 
 HCOH 
 O I 
 
 HCNH-COCH, 
 
 -0- 
 
 CH 
 
 I 
 
 O 
 OH H I 1 
 HC-C-C-C-C- COOH 
 H OH H H 
 O 
 
 -CH 
 
 ■0- 
 
 L 
 
 HC 
 
 O 
 
 OH H 
 
 C-C-C-C- COOH 
 H OH H H 
 
 Several details of the graphic formula are entirely arbitrary. 
 They are the position of the sulfuric acid radicle, the carbon atom in 
 glucuronic acid which is combined with chondrosamine, and the 
 allocation of the amino group in the carbon atom 2 of the sugar. 
 
66 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 4. DISTRIBUTION OF THE ACIDS OF VARIOUS TYPES. 
 
 The distribution of the acids in various organs and tissues was 
 found to be the following: 
 
 I. Chondroitin sulfuric acid. 
 
 1 Cartilage. 
 
 2 Tendons. 
 
 3 Aorta. 
 
 4 Sclera. 
 
 n. Mucoitin sulfuric acid. 
 
 A. I Funis mucin 
 
 2 Humor vitreous. 
 
 3 Cornea. 
 
 B. 1 Mucin of gastric mucosa. 
 
 2 Serum mucoid. 
 
 3 Ovomucoid. 
 
 4 Ovarian cysts. 
 
 Table DC contains a summary of the analytical data obtained on 
 the individual substances. 
 
 EXPERIMENTAL. 
 1. GROUP I. CHONDROITIN SULFURIC ACID. 
 
 I. From Chondromucoid. 
 
 a. Preparation oj Chondroitin Sulfuric Acid Barium Salt. 
 
 Nasal septums of cattle were freed from bone and other extraneous 
 material, and ground through a meat chopper (Levene and La Forge, 
 1913; 1914, a; 1915, a). Portion^ of 5 kilos each were allowed to 
 stand for 2 days with 10 liters of 2 per cent potassium hydroxide 
 solution. The extract was strained through a cloth and the residue 
 again subjected to the same treatment with 5 liters of potassium 
 hydroxide solution and finally washed once with water. The united 
 extracts were acidified with acetic acid and concentrated on the steam 
 bath with an excess of barium carbonate to about one-half of their 
 volume. The clear liquid was then poured off and the residue 
 thrown on a folded filter and allowed to drain off. This filtrate was 
 united with the decanted liquid and the whole acidified with acetic 
 acid and evaporated as before with barium carbonate to about 2 liters. 
 
p. A. LEVENE 
 
 67 
 
 
 
 
 S3 
 
 ■- 
 
 lO vo ■* 
 
 
 
 
 
 
 
 
 
 g§ 
 
 s 
 
 fS o\ »-t 
 
 
 
 
 
 
 
 
 
 ,?J 2 
 
 fe 
 
 o -^ c> 
 
 
 
 
 
 
 
 
 m 
 
 C/] g 
 
 
 CN 
 
 
 
 
 
 
 
 
 
 1^ 00 
 
 
 
 
 CN 
 
 
 
 
 .s »; 
 
 a 
 
 ■* ■* 
 
 O 00 
 
 
 
 tH 
 
 r* 
 
 
 
 
 ■^ 3 
 
 S 
 
 PO ^H 
 
 to t^ 
 
 
 + 
 
 cs 
 
 LO* 
 
 
 B 
 
 
 Ji 
 
 
 
 
 
 
 
 cs 
 
 + 
 
 V 
 
 
 a 
 
 "5 
 
 PO CN CN O -^ 
 
 
 
 
 
 
 a 
 
 
 a 
 
 S 
 
 CO CN VO S <-l 
 
 
 
 
 
 
 c 
 
 g 
 
 
 o 
 
 fe 
 
 J--^ VO ■>* C^ •*' 
 
 
 
 
 
 
 to 
 
 O 
 
 
 U 
 
 ■w. 
 
 CO ^ 
 
 
 
 
 
 
 o 
 
 3^ 
 
 e 
 
 0\ N « c«5 O 
 
 
 
 
 
 
 
 -^ 
 
 jl_ 
 
 A 
 
 ro r>. 0\ vo lo 
 
 o 
 
 CO 
 
 cs 
 
 
 
 
 
 
 
 
 0\ OO fO JT^ 
 
 
 1 
 
 lO o ■<* 
 
 
 
 
 1-2 
 
 a 
 S 
 
 ro fO O O 
 ro lO -"^ Th 
 
 
 O 
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 + 
 
 cs oo cs 
 
 00 lO 1 CO 
 
 
 
 
 
 ;«S 
 
 % 
 
 CO 
 
 tH 
 
 •* 
 
 
 CO 
 
 cs 
 
 
 
 
 *, 
 
 
 
 1 
 
 
 
 + 
 
 
 
 
 ■- 
 
 t-^ VO "O .t^ CO 
 
 
 
 
 
 
 
 
 i 
 
 w 
 
 cs e» \o lo oo 
 
 
 
 i 
 
 
 
 
 
 •3 
 
 1.. 
 
 ■^ Tj< lO -^ 00 
 
 
 
 
 
 
 
 
 W 
 
 «. 
 
 CO CM 
 
 
 
 
 
 
 6 
 
 .9 
 'E 
 
 ^ 
 
 k. 
 
 csi esi 
 
 
 
 I 
 
 CO TtH 
 
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68 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 The separated protein and barium carbonate were removed by 
 centrifugalization and the clear yellow liquid dropped into 8 times 
 its volume of glacial acetic acid and kept agitated by a turbine. 
 The acid potassium salt thus obtained was filtered by suction, 
 washed with glacial acetic acid, and finally with alcohol and ether. 
 
 200 gm. of this product, which gave a slight biuret test, were dis- 
 solved in 10 liters of water and while the solution was kept stirred 
 with a turbine, a solution of basic lead acetate was dropped in until 
 complete precipitation had taken place. The lead salt, after having 
 been washed several times by grinding in a mortar with water and 
 filtering with suction, was suspended in 5 liters of water containing 
 100 gm. of barium acetate and 50 qc. of acetic acid and decomposed 
 by long treatment with hydrogen sulfide with constant stirring. 
 After standing for 12 hours the lead sulfide was filtered off and the 
 slightly turbid solution of the barium salt precipitated by the addi- 
 tion of about one-third of its volume of 95 per cent alcohol. After 
 filtering and washing with 50 per cent alcohol, then with 95 per 
 cent alcohol, absolute alcohol, and finally with ether, the product after 
 drying was a pure white powder, showing no trace of biuret. 
 
 This product is a mixture of the barium salts of chondroitin and 
 chondroitin sulfuric acid. It showed no reduction of Fehling's 
 solution, and in the apparatus of Van Slyke no amino nitrogen. 
 
 0.5070 gm. substance: 7.75 cc. 0.1 n NH3. 
 0.4650 " " : 7.40 " 0.1 n NHj. 
 
 0.4166 " " : 0.1125 gm. BaSO^. 
 
 C18H27NSO17. Calculated. 'N 2.01, S 4.60. 
 
 Found. " 2.14,2.23, " 3.72. 
 
 A simpler way for the preparation of free chondroitin sulfuric 
 acid is to dissolve the lead salt in a 10 per cent hydrochloric acid. 
 The filtrate from lead chloride is then treated with glacial acetic 
 acid until all the chondroitin sulfuric acid is precipitated. This is 
 then washed with glacial acetic acid, alcohol, and finally with ether. 
 
 b. Estimation of Acetyl Groups. 
 
 25 gm. of the barium salt of chondroitin sulfuric acid were re- 
 purified by dissolving in 2 liters of distilled water with the addition 
 
p. A. LEVENE 69 
 
 of 10 gm. of pure barium chloride, and precipitating by the addition 
 of 1 liter of 95 per cent alcohol. The precipitate was washed chlorine- 
 free with 50 per cent alcohol, absolute alcohol, and finally with ether, 
 and dried in vacuum. About 10 gm. of this material were dissolved 
 in 10 cc. of water. 
 
 The acetic acid determinations were carried out in the following 
 • manner. The solution of the chondroitin sulfuric acid barium salt 
 was subjected to simultaneous hydrolysis with 25 per cent sulfuric 
 acid and distillation at atmospheric pressure. The distillate was col- 
 lected in a receiver cooled with ice, and protected from the atmos- 
 pheric carbon dioxide by soda lime, while the volume of the solution 
 in the reaction flask was kept constant by the addition of water 
 through a dropping funnel. 
 
 5 cc. 
 
 solution 
 
 7.40 cc. 0.1 N NHa (Kjelda] 
 
 5 " 
 
 te 
 
 7.60 " 0.1 N acetic acid. 
 
 5 " 
 
 u 
 
 7.80 " 0.1 N " 
 
 5 " 
 
 t( 
 
 7.55 « 0.1 N " 
 
 5 cc. of another solution, which corresponded to 8.50 cc. of 0.1 N 
 NHg (Kjeldahl) were hydrolyzed in the same apparatus with 20 per 
 cent barium hydroxide until no more -ammonia was given off. The 
 solution was then acidified with sulfuric acid, and the acetic acid 
 determination carried out as above. 
 
 5 cc. solution: 9.75 cc. 0.1 n acetic acid. 
 
 c. Preparation of Chondrosin. 
 
 50 gm.of the barium salt of chondroitin sulfuric acid were dissolved 
 in ISO cc. of equal parts of concentrated hydrochloric acid and water 
 and heated for 1 hour on the water bath. Barium sulfate began to 
 separate at once, and after 1 hour the solution, which was only slightly 
 colored, showed its maximum reduction of Fehling's solution, and all 
 the nitrogen was present as amino nitrogen. The filtered solution was 
 evaporated in vacuum to a very thick syrup and this was taken up 
 in about 40 cc. of hot water and poured into 500 cc. of absolute 
 alcohol. Partial precipitation of the chondrosin hydrochloric acid 
 salt as a nearly colorless flocculent precipitate takes place. After 
 standing over night, 2 volumes of absolute ether were added and 
 
70 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 the precipitate was filtered with suction, and again thoroughly washed 
 with absolute ether. For a final purification the product thus ob- 
 tained is dissolved in about its own weight of water, and precipitated 
 and washed again as above described. It was a quite colorless powder, 
 which when properly washed is not hygroscopic. The yield of the 
 first product, dried over calcium chloride for 2 days in vacuum, was 
 27 gm. 
 
 0.1966 gm. substance, dried to constant weight at 100°: 12.7 cc. amino N 
 at 21°, 764 mm. N = 3.67 per cent. 
 
 0.3319 gm. chondroitin sulfuric acid barium salt: 5.8 cc. 0.1 N amino N = 
 2.44 per cent. 
 
 0.5044 gm. substance hydrolyzed for 1 hr. with 1 part HCl and one part H2O: 
 18.3 cc. N at 16°, 760 mm. (Van Slyke). N = 2.11 per cent. 
 
 d. Preparation of Chondridin. 
 
 Mode of Preparation of the Substance. — This differed in its details 
 from that of Hebting. The analytical data published by this author 
 for chondridin seemed to agree with those required by theory for 
 chondrosin oxalate. At the outset of the work it was planned to 
 test this possibility. Chondrosin chlorohydrate was prepared follow- 
 ing the conditions employed in the earlier work by Levene and 
 La Forge. The chondrosin chlorohydrate obtained in this manner 
 was freed from hydrochloric acid. To the aqueous solution of chon- 
 drosin a slight excess over 1 equivalent of oxalic acid was added, 
 and to the resulting-solution alcohol was added to opalescence. On 
 standing there was no evidence of crystallization. However, when 
 the solution was allowed to stand on a boiling water bath for 1 hour 
 prior to the addition of alcohol, crystallization did take place. 
 Alcohol was added to marked opalescence and the solution was 
 allowed to remain on the water bath until it clarified. On scratching 
 along the walls of the beaker a crystalline deposit soon began to 
 form. The substance was recrystallized by dissolving in hot water, 
 adding to the aqueous solution 99.5 per cent alcohol to opalescence, 
 and boiling the solution until it clarified. After 3 or 4 recrystalliza- 
 tions the substance contained only traces of mineral impurities. 
 
 In later experiments the preparation of the substance was some- 
 what simplified; namely, no attempt was made to isolate the chon- 
 
p. A. LEVENE 71 
 
 drosin hydrochloride before the digestion with oxalic acid. The 
 procedure was as follows: Portions of 50 gm. of the barium salt of 
 chondroitin sulfuric acid were hydrolyzed by heating for 1 hour on 
 the water bath in a solution of 150 cc. of 20 per cent hydrochloric 
 acid. From the product of reaction, barium and hydrochloric acid 
 were removed and the solution was concentrated to a small volume 
 under diminished pressure. The subsequent treatment was as 
 above described. 
 
 Properties of the Substance. — The lactone is u white crystalline 
 powder. It does not melt, but contracts and turns dark at 200°C. 
 The analysis of the substance was as follows: 
 
 No. 43. 0.1176 gm. substance, dried to constant weight at temperature of 
 water vapor and under diminished pressure, lost 0.0078 gm. 
 No. 81. 0.1130 gm. substance under the same conditions, lost 0.0075 gm. 
 
 C12H21NOH + IJHjO. Calculated. H2O 7.20. 
 
 Found. 1. " 6.63. 
 
 2. " 6.63. 
 
 No. 43. 0.1098 gm. of the above substance on combustion: 0.1626 gm. CO2 
 and 0.0580 gm. H2O. 
 
 0.1867 gm. substance used for Kjeldahl nitrogen estimation, required for 
 neutralization 5.30 cc. of 0.1 n acid. 
 
 0.0500 gm. substance for the amino estimation was dissolved in 5 cc. of water. 
 
 2 cc. solution in the Van Slyke micro apparatus: 1.36 cc. N2 at 20°C., and 
 764 mm. 
 
 No. 81. 0.1055 gm. substance: 0.1564 gm. CO2 and 0.0568 gm. H2O. 
 
 CuHijNOio + HaO. Calculated. C 40.54, H 5.96, N 3.94, NHj-N 3.94- 
 
 Found. 1. C 40.43, H 5.98. 
 
 2. " 40.11, " S.92, N 3.97, NHj-N 3.89. 
 
 The rotation of the air-dry substance was 
 
 , ,M + .97 X 100 , ,10 
 t«J-> = 1.6X1 = + ^^ 
 
 Titration oj the Substance with Alkali. — 0.1000 gm. of the sub- 
 stance was dissolved in 25 cc. of water and titrated with 0.1 N sodium 
 hydroxide. Alizarin was used as indicator. After the addition of 
 the first drop, the'solution reacted neutral. 0.1000 gm. of the sub- 
 stance was dissolved in 25 cc. of water. 15 cc. of 0.1 N alkali were 
 
72 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 added and the solution was allowed to stand over night. It required 
 12.45 cc. of 0.1 N add to titrate the solution to neutral. 
 
 C12H19NO10 + 2JH2O. Calculated. Molecular weight 382.17. 
 Found. " " 392. 
 
 Furfural Estimation. — 0.2000 gm. of the substance was distilled 
 in the usual way with hydrochloric acid having a specific gravity of 
 1.06. The yield of the phloroglucide was 0.0248, which corresponds 
 to 0.00744 gm. of glucuronic acid. 
 
 CuHisNOio + 2IH2O. Calculated. Glucuronic acid 50.77. 
 Found. " " 37.2. 
 
 Taking into consideration the limit of error of the method, the 
 result is not unsatisfactory. 
 
 e. Preparation of Desaminochondrosin. 
 
 3 gm. of chondrosin hydrochloride in 50 cc. of water were treated 
 with the calculated amount of silver nitrite (1.1 gm.). After stand- 
 ing for several hours at room temperature the reaction mixture 
 was warmed on the water bath with occasional shaking. After the 
 solution had been allowed to stand over night at room tempera- 
 ture it was again warmed on the water bath for about 2 hours, after 
 addition of 0.3 gm. of silver nitrite and about 5 cc. of diluted hydro- 
 chloric acid. The excess of silver was then removed with a slight 
 excess of hydrochloric acid and the solution evaporated in vacuum 
 to a syrup which was taken up in a very small quantity of water and 
 poured into dry acetone. The gummy precipitate hardened quickly, 
 and was then ground with more dry acetone and washed with ether. 
 The product was a white amorphous powder resembling chondrosin 
 in all its physical properties and in its power to reduce Fehling's 
 solution, and gave the same amount of furfural. 
 
 0.3710 gm. substance, dried at 100° in vacuum: 0.0575 gm. phloroglucoside 
 corresponding to 0.1725 gm. glucuronic acid. 
 
 /. Identification of Glucuronic Acid. 
 
 Cleavage of Chondrosin with Sodium Amalgam". — 12 gm. of chon- 
 drosin hydrochloride in 100 cc. of water were allowed to stand with 
 
p. A. LEVENE 73 
 
 100 gm. of 2.5 per cent sodium amalgam. After about 20 minutes 
 at room temperature the solution took on a bright yellow color 
 and at the same time evolution of ammonia began. The solu- 
 tion is then neutralized with sulfuric acid, and 100 gm. of sodium 
 amalgam were again added, the temperature always being kept at 
 about 25°. After about 1 hour the solution was again acidified with 
 sulfuric acid and allowed to stand over night, after the addition of a 
 third 100 gm. portion of amalgam. The solution is then separated 
 from the mercury and filtered from the sodium sulfate with the 
 addition of some animal charcoal. 
 
 Preparation of the Phenylhydrazine Compound. — The solution ob- 
 tained by the above treatment was diluted to about 200 cc. and after 
 the addition of 15 gm. of phenylhydrazine in 50 per cent acetic acid 
 allowed to stand on the water bath. After about 20 to 30 minutes 
 a dark tarry material separates together with a small amount of 
 solid material. At this point the solution is quickly filtered with 
 suction on a hot funnel into a hot flask, and the filtrate allowed to 
 stand from 2 to 3 hours on the water bath. After this time the 
 solution was filled with long yellow needles to which very little of the 
 light-colored oil adhered. The crystals were filtered and washed with 
 warm water and then with cold absolute alcohol until no more oil 
 drops could be discerned under the microscope. When dried in 
 vacuum the product melts with decomposition at about 115°C. 
 Attempts to recrystallize did not effect a purification, and therefore 
 the first product was used for the analysis. 
 
 0.1188 gm. substance: 0.2484 gm. CO2 and 0.0634 gm. H2O. 
 0.1278 " " : 19.2 cc. N2, 17°, 758 mm. 
 
 CMHs,N.04+liH20. Calculated. C 58.93, H 5.93, N 17.17. 
 Found. " 58.80, " 6.12, " 17.3. 
 
 0.0599 gm. substance in 5 cc. pyridine-alcohol mixture rotated in a 0.5 dm. 
 tube with D-light - 0.32°. 
 
 Phenylhydrazine Compound from Glucuron. — 1 gm. of glucuron was 
 warmed on the water bath for 2 hours with a little more than the 
 required amount of normal sodium hydroxide. The solution was neu- 
 tralized with acetic acid, and 4 gm. of phenylhydrazine in 50 per 
 cent acetic acid and 4 gm. of sodium acetate were added. After a 
 
74 HEXOSAMESTES, MUCINS, AND MUCOIDS 
 
 short time crystallization of the phenylhydrazine compound in long 
 yellow needles began, and after 3 hours their amount had reached 
 1.6 gm. The material was purified by washing with cold alcohol 
 and ether. It decomposed at about 115°C. 
 
 0.0598 gm. substance in 5 cc. pyridine-alcohol mixture rotated in a 0.5 dm. 
 tube with D-light - 0.32°. 
 
 By prolonged heating in vacuum at 100° the substance lost 
 weight, but before becoming constant, decomposition sets in, while 
 at lower temperatures no loss of weight was observed. 
 
 Parabromopkenylhydrazine Compound from Chondrosin. — 20 gm. of 
 chondrosin hydrochloride were treated in the usual way with sodium 
 amalgam, and the resulting solution, after acidifying with acetic 
 acid, was heated on the water bath with 4 gm. of parabromophenylhy- 
 drazine hydrochloride. After about 1 hour the solution was filtered 
 from the separated tarry material and allowed to stand for 3 hours 
 longer on the water bath. The impure phenylhydrazine compound 
 obtained was washed with alcohol until the impurities had been 
 removed, and then with ether. The substance may be recrystallized 
 by dissolving it in as small a quantity as possible of a mixture of 
 1 part SO per cent acetic acid and 1 part alcohol, and then precipi- 
 tating by the addition of 2 parts of hot water. 
 
 0.0568 gm. substance in 5 cc. pyridine-alcohol mixture rotated with D-light in 
 a 0.5 dm. tube - 0.8°. 
 
 0.0614 gm. substance twice recrystallized under the same conditions, rotated 
 -0.75°. 
 0.1454 gm. substance: 12.5 cc. Nj, 22°, 762 mm. 
 0.1126 " " : 0.0118 gm. AgBr. 
 
 Calculated. Br 28.95, N 10.15. 
 Found. " 27.0, " 9.7. 
 
 Parabromopkenylhydrazine Compound from Glucuron. — 1 gm. of 
 glucuron in 100 cc. of water was heated on the water bath with 
 2.5 gm. of parabromophenylhydrazine hydrochloric acid salt, which 
 had been purified by twice recrystallizing from dilute hydrochloric 
 acid and washing with ether, and 2.5 gm. of sodium acetate. After 
 about 1 hour 0.3 gm. of a yellow crystalline substance had separated. 
 The mother liquor filtered from the first crystallization gave upon 
 
p. A. LEVENE 75 
 
 further heating 0.2 gm. more of the same substance. After recrystal- 
 lization from 50 per cent acetic acid and alcohol it had the following 
 composition: 
 
 , 0.1436 gm. substance: 13 cc. N2 at 22°, 758 mm. 
 0.1268 " " : 0.0824 gm. AgBr. 
 0.1338 " " : 0.0124 " Na2S04. 
 
 Br2C,8Hi706N4Na(Ci2Hi7N207Br). 
 
 Calculated. Br 28.95, Na 4.17, N 10.15. 
 
 Found. " 27.65, " 3.01, " 10.20. 
 
 0.0653 gm.'substance in 5 cc. pyridine-alcohol mixture rotated in a 0.5 dm. 
 tube with D-light - 0.90°. 
 
 g. Nitric Acid Oxidation of the Products of Hydrolysis of Chondrosin. 
 
 25 gm. of chondrosin hydrochloride were treated with sodium 
 amalgam in exactly the same manner as described in the previous 
 experiment. The solution, after having been freed from inorganic 
 salts by precipitation with alcohol, was evaporated to a syruj). 
 This syrup was quickly evaporated in a flat dish with dilute nitric acid 
 composed of 1 part of nitric acid, specific gravity of 1.42, and 1 part 
 of water. The residue was then evaporated several times with water, 
 and finally taken up in 15 cc. of water and neutralized with potassium 
 hydroxide. Upon addition of glacial acetic acid the crystallization 
 of the acid potassium saccharate began after a short time. After 2 
 days the yield amounted to 1.1 gm. For analysis it was recrystallized 
 from water. 
 
 0.1253 gm. substance: 0.0427 gm. KsS04. 
 
 Calculated. K 15.72. 
 Found. " 15.32. 
 
 h. Nitric Acid Oxidation of Chondrosin and Subsequent Hydrolysis. 
 
 10 gm. of chondrosin hydrochloride were evaporated in a flat 
 dish on a water bath with 10 cc. of nitric acid and 10 cc. of water. 
 The residue was dissolved in 10 cc. of water and 5 cc. of nitric acid 
 and again evaporated to dryness. The final residue was then 
 dissolved in 10 cc. of water and the solution divided into 2 parts of 7 
 and 3 cc. each and neutralized in the cold with potassium hydroxide. 
 
76 HEXOSAMTNES, MUCINS, AND MUCOIDS' 
 
 The larger portion, after addition of 2 cc. of 50 per cent potassium 
 hydroxide, was allowed to stand for 2 hours on the water bath and then 
 acidified "with acetic acid. After several hours the acid potassium 
 saccharate began to separate. The yield amounted to 0.5 gm. after 
 2 days. From the smaller portion, after addition of acetic acid, 
 only a trace of the same substance separated after long standing. 
 
 0.1276 gm. substance: 0.0440 gm. K2SO4. 
 
 Calculated. K 15.72. 
 Found. " 15.46. 
 
 Brom Oxidation of the Products of Hydrolysis of Chondrosin. — ^A 
 solution of 25 gm. of chondrosin hydrochloride was treated in the 
 usual way with 2.5 per cent sodium amalgam. The solution was 
 acidified with hydrochloric acid and allowed to stand for 5 days at 
 ordinary temperature with an excess of bromine. It was then 
 concentrated in vacuum to about 100 cc. and the principal amount 
 of the salt separated by pouring the substance into hot absolute 
 alcohol. The alcoholic solution was concentrated in vacuum to a 
 syrup, taken up in water, and the halogen determined in an aliquot 
 part. The requisite amount of lead acetate was then added to the 
 remainder of the solution and the lead chloride and bromide were 
 removed by filtration. The excess of lead was then removed by 
 hydrogen sulfide and the solution evaporated in vacuum to about 
 30 cc. It was then neutralized with potassium hydroxide and after 
 the addition of 10 cc. of glacial acetic acid allowed to stand for 2 days 
 in the refrigerator. The separated crystals were filtered by suction 
 and the product recrystallized from water. After drying it amoimted 
 to 1.6 gm. 
 
 0.1209 gm. substance: 0.0419 gm. K2SO4. 
 
 0.1210 " " : 0.0466 " H2O and 0.1242 gm. CO2. 
 
 CeHsOsK. Calculated. H 3.65, C 27.90,* K 15.72. 
 Found. " 4.28, " 27.98, " 15.55. 
 
 * Considering that 1 atom of carbon is contained in the ash as K2CO3. 
 
 Furfural from Chondrosin, after Oxidation with Nitric Acid. — 
 0.4219 gm. of chondrosin (calctilated from the nitrogen content) 
 was evaporated to dryness with 5 cc. of concentrated nitric acid and 
 
p. A. LEVENE 77 
 
 5 cc. of water. After repeated evaporation with water the solution 
 of the residue was distilled in the usual way with hydrochloric acid 
 of specific gravity 1.06 until no more furfural was given o£f. Upon 
 addition of 0.1 gm. of phloroglucin 0.0076 gm. of phloroglucoside was 
 obtained, corresponding to 0.0218 gm. of glucuronic acid, or about 
 one-tenth of the amount present in chondrosin. 
 
 i. Identification of Ckondrosamine. 
 
 The aminohexose of chondroitin sulfuric acid was identified as 
 2-amino-(Z-lyxohexose by the identity of the physical constants of 
 the natural sugar with those of the synthetic prepared from amino- 
 lyxoside, by the identity of the phenylosazone with the phenyl- 
 galactosazone, and by the fact that on oxidation the sugar gives rise 
 to chondrosaminic acid which is identical with the synthetic levo-d- 
 lyxohexosaminic acid. 
 
 The amino sugar on deamination and subsequent oxidation gave 
 2-S-anhydrotalonic and 2-5-anhydrotalomucic acids. As was to 
 be expected, 2-S-anhydrotalomucic acid gave anhydromucic and 
 pyromucic acids. 
 
 Preparation of Ckondrosamine from Chondroitin Sulfuric Acid. — 
 75 gm. of chondroitin sulfuric acid barium salt were hydrolyzed for 
 about 7| hours with 400 cc. of 20 per cent hydrochloric acid, with the 
 addition of 15 gm. of stannous chloride. Barium sulfate began to 
 separate at once, and the solution soon began to take on a yellow 
 color which passed rapidly through brown to black with the separa- 
 tion of dark particles due to decomposition of glucuronic acid. 
 Upon completion of the reaction the solution was diluted with twice 
 its volume of warm water, and, without filtering, the tin removed 
 with hydrogen sulfide. The sulfides of tin were separated by filtra- 
 tion with suction, leaving a clear, almost colorless filtrate which, 
 without further treatment, was concentrated in vacuum to about 
 35 cc. This syrup-like residue was at once taken up in 75 to 80 cc* 
 of absolute alcohol, poured into a beaker, and the hydrochloride of 
 the amino hexose caused to crystallize by adding about 100 cc. of 
 absolute ether slowly in portions of about 10 cc. with constant 
 
 *The presence of too much alcohol or water causes the product to separate 
 oily at first. 
 
78 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 scratching of the sides of the vessel. The deposit of long white 
 prismatic needles thus obtained was filtered with suction, and 
 washed with absolute alcohol and ether. The first yield usually 
 amounts to about 16 gm. while, upon addition of 50 cc. more of ether 
 to the first filtrate, about 4 gm. more of equally pure product are 
 obtained. The total yield corresponds to about 90 per cent of the 
 theory. Upon recrystaUization under the above conditions or on 
 long keeping in a desiccator, the product tends to lose a small amoimt 
 of hydrochloric acid, since an analysis of a product twice so treated 
 gave the following figures : 
 
 0.1794 gm. substance required 14.6 cc. AgNOj solution (1 cc. = 0.00186 gm. CI). 
 0.1701 " " : 0.0977 gm. HjO and 0.2113 gm. COa. 
 
 CHisOsN-HCl. Calculated. C 33.40, H 6.S4, CI 16.45. 
 Found. " 33.93, " 6.SS, " 15.15. 
 
 The optical measurement was carried out at 0°. All apparatus 
 was cooled to this temperature. 
 
 0.3000 gm. substance in 3 cc. H2O, total weight of solution 3.2871 gm. 
 specific gravity 1.03S2, had the following rotation: 
 
 After 5 min. + 6.10°: [a]" = 129.° 
 " 24 hrs. + 4.44°: [a]^° = 94.° 
 
 The activity of pure glucosamine was determined for comparison 
 under the same condition. 
 
 0.3000 gm. substance in 3 cc. H2O, weight of solution 3.2876 gm., specific 
 gravity 1.0327, rotated 
 
 After 5 min. + 4.79°: [a]" = 102.° 
 " 24 hrs. + 3.50°: U" = 74.° 
 
 Later the process was simplified as follows: 400 gm. of the dry 
 lead salt were taken up in 1,600 cc. of a 20 per cent solution of 
 hydrochloric acid, 40 gm. of stannous chloride and 100 gm. of bariimi 
 chloride added, and all was heated with return condenser over a 
 free flame for 12 hours. When the hydrolysis was completed the 
 reaction product was filtered. The filtrate was concentrated under 
 diminished pressure to a thick syrup. This was taken up in 800 cc. 
 of water and the barium removed by means of sulfuric acid. The 
 final filtrate was concentrated under diminished pressure to a thick 
 
p. A. LEVENE 79 
 
 syrup. This was taken up in about 100 cc. of methyl alcohol, and 
 ether was added very cautiously until the sugar began to crystallize. 
 The crystallization progressed continuously for about 24 hours. The 
 yield was about 35 to 40 gm. of chondrosamine hydrochloride. 
 
 j. Pkenylosazone of Chondrosamine. 
 
 Osazone of Chondrosamine. — 4 gm. of chondrosamine hydrochloride 
 were dissolved in 400 cc. of water, and enough sodium acetate was 
 added to neutralize the hydrochloric acid. To this solution were 
 added 10 gm. of phenylhydrazine dissolved in glacial acetic acid. 
 The solution was then immersed for 5 hours in a boiling water bath. 
 A considerable precipitate of osazone appeared during the process 
 of heating. A voluminous precipitate formed on cooling over night. 
 The precipitate was then filtered, washed, and suspended in 500 cc. 
 of hot water. The flask was placed over a fl^me, and pyridine 
 added in small portions until osazone dissolved. The operation 
 was repeated once. Under the microscope the osazone consisted 
 of long plates with pointed ends. No impurity could be noticed on 
 the microscopic slide. The precipitate was filtered on a suction 
 funnel, washed with a very small portion of alcohol, and then trans- 
 ferred into a solution of equal parts of alcohol and ether, allowed to 
 stand for several hours in the refrigerator, and filtered. It had the 
 appearance of very light orange glistening plates. It melted at 
 201° and decomposed at 202°C. A mixed melting point with galac- 
 tosazone was identical with the original. Also a mixed melting 
 point with the osazone obtained from synthetic lyxohexosamine 
 was identical. 
 
 For the optical rotation only 0.0500 gm. were used, since it was 
 found that by employing 0.100 gm. in the Neuberg pyridine-alcohol 
 solution one was not certain of accomplishing rapidly a complete 
 solution. A parallel experiment was made with the osazone ob- 
 tained from the synthetic lyxohexosamine. 
 
 O.OSOO gm. substance in a 0.5 dm. tube had an initial rotation of a = +0.36° 
 
 After 24 hrs. 0.00° 
 
 « 40 " -0.10° 
 
 " 80 " -0.25° 
 
 « 96 " -0.30° 
 
 Observations were interrupted at this point. 
 
80 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 k. Preparation of Chondrosaminic Acid from Chondrosamine. 
 
 Chondrosaminic Acid from Natural and Synthetic Sugars. — ^This 
 acid was obtained from both the natural and synthetic sugar by 
 oxidation with mercuric oxide. The conditions given by Pringsheim 
 and Ruschmann for oxidation of glucosamine had to be modified. 
 
 4 gm. of sugar were dissolved in 62 cc. of water; 20 gm. of mercuric 
 oxide were added, and the mixture was warmed on the water bath 
 for 6 minutes. The reaction product was filtered immediately, the 
 filtrate was freed from mercury by means of hydrogen sulfide, the 
 filtrate from the sulfide concentrated to a small volume, under 
 diminished pressure, when the acid crystallized in the distilling flask. 
 The substance was recrystallized once. It did not melt, but turned 
 light brown at 190°, like the acid obtained from the natural sugar. 
 A parallel measurement of the rotation of each product gave the 
 following results: 
 
 Natural product. 
 Initial (in 2.5 per cent HCl). Equilibrium. 
 
 laf = -°-90X^-5 - _ ,g« r„l» _ -1-60X2.5 _ 
 
 ^ ^° 1 X 0.1254 - ^* l"l° - 1 X 0.1254 ' ~ ^^ 
 
 Synthetic. 
 
 Initial (in 2.5 per cent HCl). Equilibrium. 
 
 r„l« = -0-94X2.5 _ „ _ - 1.60 X 2.5 _ 
 
 '"J" 1 X 0.1256 ^^ '"'■> - 1X0.1256 " ~ ^^ 
 
 The analysis of the acid from the natural sugar was: 
 
 0.0992 gm. substance: 0.1326 gm. CO2 and 0.0568 gm. H2O. 
 0.1396 gm. " : (Van Slyke) 17.3 cc. N2 at 22°, 760 mm. 
 
 C«Hi,OiiN. Calculated. C 36.92, H 6.66, N 7.18. 
 Found. " 36.61, " 6.44, " 7.02. 
 
 The analysis of the synthetic acid gave the following results: 
 
 0.1050 gm. substance: 0.1426 gm. CO2 and 0.0618 gm. HjO. 
 
 CHuNO,. Calculated. C 36.92, H 6.66. 
 Found. " 37.03. " 6.58. 
 
 Thus the identity of the two chondrosaminic acids is established. 
 
p. A. LEVENE 81 
 
 /. Preparation of 2-5-Anhydrotalonic Acid* 
 m. Oxidation of 2-5-Anhydrotalonic to 2-5-Anhydrotalotnucic Acid** 
 n. Oxidation of Ckondrosamine to 2-5-Anhydrotalomucic Acid. 
 
 9 gm. of chondrosamine hydrochloride were deaminized with silver 
 nitrite, the resulting solution was concentrated to about 20 cc, mixed 
 with an equal volume of concentrated nitric acid, and allowed to 
 stand over night at room temperature. It was then rapidly evap- 
 orated in a shallow dish on a water bath and the syrup, after having 
 been again evaporated with water, was diluted to 250 cc, and boiled 
 with calcium carbonate until neutral (one-half hour). The filtrate, 
 upon standing for 2 days, deposited white prisms of the calcium 
 salt of a dibasic hexonic acid. The yield did not exceed 25 per cent 
 of the theory. For analysis it was recrystallized by dissolving in 
 50 parts of boiling water containing slightly over the theoretical 
 amount of oxalic acid and again transformed into the calcium salt 
 by boiling with calcium carbonate. The compound contained 
 2 molecules of crystal water which can be removed in vacuum at 
 108°. By heating for 16 hours at 138° no further appreciable loss 
 of weight was observed. 
 
 The dried substance analyzes best for the calcivmi salt of a normal 
 dibasic hexonic acid. 
 
 0.1S04 gm. air-dried substance: 0.0200 gm. H2O (108°). 
 
 0.1508 " " " : 0.0208 " " (108°). 
 
 0.1509 " " " : 0.0198 " " (108°). 
 
 0.1670 " " " : 0.0232 " " (140°). 
 
 CcHsOsCa -I- 2H2O. Calculated. H2O 12.68. 
 Found. I. " 13.3. 
 " n. " 13. 
 " m. " 13.1. 
 " IV. " 13.3. 
 
 0.1304 gm. dried substance: 0.0344 gm. H2O, 0.1376 gm. CO2, and 0.0302 gm. 
 CaO. 
 
 0.1312 gm. dried substance: 0.0380 gm. H2O, 0.1380 gm. CO2, and 0.0302 gm. 
 CaO. 
 
 0.1030 gm. dried substance: 0.0296 gm. H2O, 0.1083 gm. CO2, and 0.0243 gm. 
 CaO. 
 
 •* See page 40. 
 ** See page 41. 
 
82 HEXOSAMESTES, MUCINS, AND MUCOIDS 
 
 CjHsOsCa. Calculated. 
 
 C 29.03, 
 
 H 
 
 3.22, 
 
 CaO 22.6. 
 
 Found. I. 
 
 " 28.78, 
 
 It 
 
 2.95, 
 
 " 23.4. 
 
 11, 
 
 « 28.68, 
 
 a 
 
 3.24, 
 
 " 21.8. 
 
 " III. 
 
 " 28.67, 
 
 i( 
 
 3.22, 
 
 « 23.2. 
 
 " IV. 
 
 
 
 
 " 22.5. 
 
 0.1020 gm. substance, in 2 cc. 10 per cent HCl, rotated in 1 dm. tube: 
 
 After 10 min., -0.45°. 
 " IShrs., -0.37°. 
 
 o. Preparation of 2-5-Anhydrogalactonic Acid* 
 
 p. Conversion of 2-5-Anhydrotalomucic into Anhydromucic Acid. 
 
 1 gm. of 2-S-anhydrotalomucic acid in 1 cc. of concentrated hydro- 
 chloric acid plus 1 cc, of concentrated hydrobromic acid heated in 
 a sealed tube at 1S0°C. for 8 hours, according to Fischer gave 0.2 
 gm. of anhydromucic acid. 
 
 0.1002 gm. substance: 0.1684 gm. CO2 and 0.0276 gm. HjO. 
 CoHjOs. Calculated. C 46.16, H 2.57. 
 Found. " 45.84, " 3.08. 
 
 q. Conversion of 2-5-Anhydroialomucic into Pyromticic Acid. 
 
 1 gm. of 2-S-anhydrotalomucic acid was heated at about 200°C. 
 in a test-tube in an atmosphere of carbon dioxide for one-half hour. 
 The substance sublimed and separated out on the cooler parts of the 
 tube from which it was afterwards removed, extracted with ether, 
 and recrystallized by dissolving in a large amount of ether and allow- 
 ing the solution to evaporate. It melted at 13S°C. 
 
 0.1000 gm. substance: 0.1964 gm. CO2 and 0.0370 gm. H2O. 
 CsHia. Calculated. C 53.57, H 3.57. 
 Found. " 53.56, " 4.14. 
 
 II. From Tendomucoid. 
 
 The presence of a conjugated sulfuric acid in the molecule of 
 tendomucoid was first discovered by Levene. Later Mandel and 
 Levene showed the presence of a similar acid in many tissues and 
 even in leucocytes. 
 
 * See page 42. 
 
p. A. LEVENE 83 
 
 a. Preparation of Tendomucoid and Chondroitin Sulfuric Acid 
 
 Contained in It. 
 
 Owing to the nature of the combined protein the method of pre- 
 paring chondroitin sulfuric acid from tendons differs somewhat from 
 its preparation from cartilage (Levene, 1914, b). Portions of 50 
 achilles tendons from cattle were cleaned, passed through a hashing 
 machine and allowed to stand over night with 20 liters of two-thirds 
 saturated lime water. The liquid was strained off and the process 
 repeated once again on the residue. The combined filtrates were 
 just acidified with hydrochloric acid which produced a flocculent 
 precipitate of tendomucoid. The supernatant liquid is then siphoned 
 off and after addition of an equal volume of 95 per cent alcohol the 
 mucoid was filtered off on a folded filter. The moist product was 
 agitated for some time with 1 .5 liters of a 2 per cent potassiimi hy- 
 droxide solution. After standing over night the turbid brown solution 
 was acidified with acetic acid and the separated protein removed 
 by filtration on a folded filter. The filtrates from two such experi- 
 ments were neutralized with sodium hydroxide and the chondroitin 
 sulfuric acid was precipitated by a solution of basic lead acetate. The 
 Itad precipitate was repeatedly washed by triturating in a mortar 
 with distilled water and filtering with suction. The washed product 
 was suspended in about 2 Uters of water; 10 cc. of glacial acetic acid 
 and 20 gm. of barium acetate were added and decomposition was 
 effected by passing in hydrogen sulfide with constant stirring. The 
 lead sulfide was filtered off with suction, the filtrate concentrated to 
 about 350 cc, and the barium salt precipitated by the addition of 
 about 250 cc. of alcohol. It was then filtered with suction, washed, 
 first with 50 per cent, then with 95 per cent, with absolute alcohol, 
 and finally with ether. The yield amounts to about 12 to 15 gm. 
 
 0.2220 gm. substance 
 0.6136 " 
 0.6141 " 
 0.7002 " 
 
 0.2043 gm. CO2 and 0.0775 gm. H2O. 
 9.25 cc. 0.1 N NH3 (Kjeldahl). 
 9.50 " 0.1 N acetic acid. 
 0.2170 gm. BaS04. 
 
 Gi8H«N2S20j9. Calculated. C 27.80, H 3.48, N 2.32, S 5.30, B3 22.70. 
 Found. " 25.13, " 3.88, " 2.11, " 4.26, " 18.35. 
 
 N:C = 1:13.89. 
 
84 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 b. Chondrosin. 
 
 Chondrosin Hydrochloride. — This substance was prepared exactly 
 as described in a previous section (Levene and La Forge, 1913). 
 It analyzed as follows: 
 
 0.12S8 gm. substance: 0.16S3 gm. CO2 and 0.0685 gm. H3O. 
 0.2238 " " : 13.6 cc. N2 (Van Slyke) at 20°, 763 mm. 
 
 0.4024 " " : in 3 cc. water, weight of solution 3.3792 gm., rotated in 
 
 a 0.5 dm. tube at 20° with D-light, + 2.46°. 
 
 Chondrosin Hydrochloride from Cartilage. 
 
 0.1584 gm. substance: 0.2167 gm. CO2 and 0.0806 gm. H2O. 
 0.2085 " " : 12.5 cc. amino N at 18°, 765 mm. (Van Slyke). 
 
 0.6543 " " : in 3 " H2O, weight of solution 3.6481 gm., rotated in 
 
 a 0.5 dm. tube with D-light, + 3.90°. 
 
 Ci2H2,NOnHCl(390.5). Calculated. C 36.9, H 5.64, N 3.S8. 
 
 Found. (From tendons.) " 35.8, " 6.00, " 3.4S. 
 
 N:C = 1:12.1. [«]" =+41.5°. 
 Found. (From cartilage.) C 37.3, H 5.66, N 3.45. 
 N:C = 1:12.6. [a]" =+43.40. 
 
 c. Chondrosamine Hydrochloride. 
 
 This substance was prepared in exactly the same manner as de- 
 scribed in a previous section (Levene and La Forge, 1914, a). 
 
 From 18 gm. of the bariimi salt 3.5 gm. of amino hexose were 
 obtained. For analysis it was dissolved in 3 parts of water with the 
 addition of a few drops of hydrochloric acid, allowed to crystallize 
 by evaporation and dried in a desiccator. The melting point was 
 180°. 
 
 0.1596 gm. substance: 0.1930 gm. CO2 and 0.0945 gm. HjO. 
 0.1516 " " : 17.6 cc. N2 (Van Slyke) at 19°, 774 mm. 
 
 0.1932 " " : in 2 cc. of water, weight of solution 2.1904 gm., rotated 
 
 in a 1 dm. tube at 20° with D-hght: 
 
 After about 15 min., +10.75° 
 " 24hrs., + 8.5° 
 
 CoHisOsNHCl. Calculated. C 33.40, H 6.54, N 6.51. 
 Found. " 33.01, " 6.57, " 6.79. 
 
 Equilibrium (without consideration of specific gravity), 
 [a];" = +96.4°. 
 
p. A. LEVENE 85 
 
 d. Glucuronic Acid Osazone Hydrazide. 
 
 This substance was prepared exactly as described in a previous 
 section (Levene and La Forge, 1913). 
 
 From 4 gm. of chondrosin hydrochloride 0.1 gm. of the substance 
 was obtained. The melting point was exactly the same as the product 
 from glucuronic acid cartilage chondrosin, 122°. 
 
 0.0805 gm. substance: 12.1 cc. Nj (Dumas) at 24°, 766 mm. 
 
 C24H26N6O1 + 1| H2O. Calculated. N 17.17. 
 Found. " 16.9. 
 
 III. From Aorta Mucoid, 
 a. Preparation of Chondroitin Sulfuric Acid. 
 
 100 pounds of aorta freed from extraneous tissue were put through 
 a hashing machine, taken up in 20 liters of a 2 per cent solution of 
 sodium hydroxide, and allowed to stand for 36 hours (Levene and 
 Lopez-Sudrez, 1918). The extract was decanted and the residue 
 again extracted for another 36 hours. The combined solutions were 
 strained, neutralized, concentrated with an excess of barium car- 
 bonate, filtered, and finally precipitated with glacial acetic acid. 
 The precipitate had the appearance of chondroitin sulfuric acid. 
 The precipitate was washed with glacial acetic acid, then with alco- 
 hol, and dried. The yield was 40 gm. Of these, 8 gm. were used 
 for hydrolysis and the remaining material was purified in the follow- 
 ing manner. 
 
 The material was again dissolved in water and precipitated with 
 glacial acetic acid. The precipitate was washed with glacial acetic 
 acid and then with alcohol. The dry precipitate was dissolved 
 with the aid of potassimn hydroxide. To this solution a sHght ex- 
 cess over the required amount of barium chloride was added, followed 
 by the addition of an equal volume of 95 per cent alcohol. The 
 precipitate was washed by decantation with 50 per cent alcohol 
 until free from barium chloride. The washing was then continued 
 with alcohol of progressively increasing strength, finally with ether, 
 and dried. The yield of this material was 25 gm. The substance 
 analyzed as follows: 
 
86 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 0.2000 gm. substance required for neutralization 3.63 cc. 0.1 n acid. 
 
 0.2000 " " on fusion: 0.0354 gm. BaS04. . 
 
 0.0778 " " " combustion: 0.0818 gm. CO2 and 0.0386 gm. H2O. 
 
 CseHwOssNjSjBaj. Calculated. C 27.8, H 3.48, N 2.32, S 5.30. 
 Found. " 28.7, " 3.35, " 2.54, " 2.4. 
 
 b. Preparation of Chondrosamine. 
 
 8 gm. of the substance with 60 cc. of 20 per cent hydrochloric acid, 
 together with 1.5 gm. of barium chloride and 1.5 gm. of stannous 
 chloride, were hydrolyzed with a reflux condenser for 12 hours. 
 The product of hydrolysis was freed from barium and tin, and the 
 filtrate, concentrated under diminished pressure, warmed to about 
 50°C. in a water bath. On concentration the sugar crystallized in 
 long, microscopic, prismatic needles. These were transferred to a 
 flask by means of alcohol containing hydrochloric acid. The flask 
 was allowed to stand over night and the precipitate was then filtered 
 and washed with alcohol and ether. The yield was about 1 gm. 
 The melting point was 183° (uncorrected). 
 
 0.0200 gm. substance in the Van Slyke apparatus: 2.30 cc. Nj at 25"'C. 
 and 762.7 mm. 
 
 CJIisOsN-HCl. Calculated. N 6.51. 
 Found. « 6.40. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . .« _ +1.64X2.0593 _ +2.27 X 2.0593 _ 
 
 ^"^o 1 X 0.0508 '^^ 1 X 0.0508 ~ "*■ 
 
 c. Preparation of Chondrosin. 
 
 20 gm. of chondroitin sulfuric acid were hydrolyzed for 1 hour 
 in a boiling water bath with 60 cc. of 20 per cent hydrochloric acid. 
 The reaction product was filtered, concentrated under diminished 
 pressure, and precipitated by means of alcohol and ether. The 
 yield was 4 gm. The analysis of the substance gave the following 
 results: 
 
p. A. LEVENE 87 
 
 0.1000 gm. substance required for neutralization 3.10 cc. 0.1 N acid. 
 0.0200 " " in the Van Slyke micro apparatus: 1.2 cc. Ns at 2S°C. 
 and 759.6 mm. 
 
 CuHjiOiiN-HCl. Calculated. Total N 3.58, NHj-N 3.S8. 
 Found. " 4.34, " 3.41. 
 
 The rotation of the substance was 
 
 r„f = +1-04X2.0026 ^ 
 
 ^ '" 1 X 0.0496 +*'' 
 
 I'V. From Sclera Mucoid, 
 a. Preparation of Chondroitin Sulfuric Acid, 
 
 For the preparation of the substance (Levene and Lopez-Suarez, 
 1918), originally the sclera and cornea were worked up in one. The 
 hydrolysis of the substance, however, revealed the presence of two 
 sugars, chitosamine and chondrosamine. Because of this, in later 
 experiments the cornea was dissected out. The sample used for ul- 
 timate analysis was prepared from sclera and cornea combined. But 
 the sugar fraction still contained some glucosamine. On the other 
 hand, the mucoid from the cornea contained only glucosamine. 
 The sclera was the only tissue which yielded mucoid containing 
 both sugars, and the possibility is not excluded that one of them 
 (chitosamine) is derived from adhering extraneous tissues. 
 
 The procedure for the preparation of the conjugated sulfuric acid 
 was the following. The eyes were freed from adhering muscle and 
 connective tissue, then the humor vitreous, lens, and retina were 
 removed. Finally, the cornea and sclera were washed with running 
 water from all extraneous material. After this the corneas were 
 carefully dissected out, and the scleras were minced in the hashing 
 machine, and placed in a large volume of 3 per cent sodium hydroxide. 
 For the corneas of 1,000 eyes 20 liters of sodium hydroxide were 
 used. The extraction was continued 3 days, at the end of which 
 time the solution was strained through cheese-cloth and neutralized 
 with acetic acid. Barium carbonate was then added in excess and 
 the mixture was concentrated on a water bath to a small volume. 
 The product of the reaction was filtered on a suction funnel and then 
 
88 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 converted into the lead salt. The lead salt was treated in the usual 
 way. It was converted into the sodium salt for the analysis of 
 conjugated sulfuric acid. For the isolation of the sugar it was 
 hydrolyzed directly. The sodium salt analyzed as follows: 
 
 0.1903 gm. substance required for neutralization 7.69 cc. 0.1 n acid. 
 
 0.28SS " " : 0.0950 gm. BaS04. 
 
 0.0986 " " : 0.1239 " CO2 and 0.0428 gm. H2O. 
 
 CjsHwOasNjSjBaj. Calculated. C 27.8, H 3.48, N 2.32, S 5.30, Ba 22.70. 
 Found. " 34.3, " 4.86, " 5.66, " 4.57, " 23.83. 
 
 b. Preparation of Chondrosamine. 
 
 12 gm. of the lead salt with 2 gm. stannous chloride, 2 gm. of 
 barium chloride, and 60 gm. of 20 per cent hydrochloric acid were 
 heated with a reflux condenser for 10 hours over a free flame. The 
 reaction product was freed from lead, tin, and barium, and then 
 concentrated to syrup. This was dissolved in a minimal amount of 
 hot methyl alcohol. Soon crystals appeared which had the typical 
 appearance of chitosamine. These were filtered off and the mother 
 liquor was allowed to stand; from time to time a few drops of ether 
 were added. At the end of a week the maximum amount of chondro- 
 samine hydrochloride settled out. The best yield for 1 kg. was 
 0.5 gm. of the sugar. 
 
 The melting point of this was 182° (corrected). The analysis of 
 the substance was as follows: 
 
 0.0200 gm. substance: 2.39 cc. N2 at 19°C. and 745 mm. 
 0.0523 " " required for titration of the HCl 2.39 cc. AgNOaCl cc. = 
 
 0.003546 gm.). 
 
 0.1036 gm. substance: 0.1224 gm. CO2 and 0.0602 gm. H2O. 
 
 CsHiaOjN-HCl. Calculated. C 33.40, H 6.54, N 6.57, CI 16.45. 
 Found. " 33.18, " 6.90, " 6.38, " 16.2. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 r„l" = +1-42 X 2.0408 _ „„ +2.39 X 2.0408 _ , .,„ 
 
 ^ '" 1 X 0.0510 ~'^^' IX 0.0510 ~ "^^^ 
 
p. A. LEVENE 89 
 
 2. GROUP II A. MUCOITDSr SULFURIC ACIDS. 
 
 /. From Funis Mucin, 
 a. Preparation of Mucoitin Sulfuric Acid. 
 
 For the preparation of the substance (Levene and L6pez-Suirez, 
 1918; 1916, b), it is essential to free the cords from all adhering blood 
 clots and blood vessels, as otherwise the resulting substance is 
 contaminated with nucleic acid. The separation of the two is very- 
 troublesome. The purification, however, was accomplished by vig- 
 orous treatment with glacial acetic acid. The treatment with lead 
 acetate is a convenient step in order to free the substance from 
 adhering salt as well as from other impurities. 
 
 In removing the lead by means of hydrogen sulfide one has to 
 bear in mind the insolubility of the acid in water. Because of this, 
 it is necessary to carry out the separation of lead in a sHghtly alkaline 
 solution. 
 
 The details of the process as carried out at present are as follows : 
 About 100 cords, freed from blood vessels, either shredded or chopped 
 in a hashing machine, were taken up in 6 liters of 72 per cent NaOH 
 and allowed to stand for 3 days, then acidulated and centrifugalized 
 to remove the precipitate. The supernatant liquid was concentrated 
 with an excess of barium carbonate on a water bath. This operation 
 was continued for 24 hours and the product centrifugalized. The 
 supernatant liquid was allowed to stand on a hot water bath after 
 a second addition of barium carbonate. Water was added from time 
 to time. The operation was continued for about 2 days. The 
 resulting material was then centrifugalized, and the supernatant 
 liquid allowed to stand until part of the barium acetate had crystal- 
 lized out. The material was again centrifugalized and the clear 
 supernatant solution precipitated with glacial acetic acid. The 
 precipitate was redissolved in water on addition of barium acetate; 
 the substance was reprecipitated out of this solution with glacial 
 acetic acid. The crude material was washed with 95 per cent alcohol 
 to remove the excess of acetic acid. The material was dissolved in 
 water, and the solution was neutralized with a solution of barium 
 hydroxide until it reacted neutral to litmus. 
 
90 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 To the final solution enough 95 per cent alcohol was added to 
 precipitate the crude baritim salt. This was washed first with 
 50 per cent alcohol to remove adhering barium acetate, then with 
 alcohol of increasing concentration, and with 99.5 per cent alcohol 
 and then with ether. The final product analyzed as follows: 
 
 0.1000 gm. substance required for neutralization 2.88 cc. 0.1 N acid. 
 
 0.2000 " " on fusion: 0.0592 gm. BaS04. 
 
 0.0936 " " " combustion: 0.0450 gm.H20 and 0.1146 gm. CO2. 
 
 GeHwOagNj&Baj. Calculated. C 27.8, H 3.48, N 2.32, S 5.30. 
 Found. " 33.4, " 5.38, " 4.03, " 4.07. 
 
 The optical rotation of the substance was 
 
 . ,« _ -0.50 X 5.0641 _ ^„ 
 ^"'° 1 X 0.0555 ^ 
 
 b. Preparation of Miicosin. 
 
 An attempt was made to prepare mucosin under the same condi- 
 tions of hydrolysis as employed for the preparation of chondrosin. 
 However, the largest part of the substance underwent complete 
 hydrolysis, with the formation of free chitosamine, which was iden- 
 tified in the usual way. It was found subsequently, that a sub- 
 stance analogous to chondrosin could be obtained under the following 
 conditions. 
 
 4.5 gm. of the barium salt were dissolved in 100 cc. of 10 per cent 
 hydrochloric acid and heated on a water bath for | hour. The 
 solution then contained all its nitrogen in form of primary amino 
 nitrogen, and showed a reduction of Fehling's solution equivalent 
 to 1.12 gm. of glucose; the theory requires 1.26 gm. The solution 
 was freed quantitatively from barium, and concentrated to 3 cc. under 
 diminished pressure at a temperature of water bath not exceeding 
 45°C. This was then gradually poured into 200 cc. of alcohol, to 
 which 400 cc. of dry ether had been added. A white flocculent 
 precipitate then formed. It was allowed to stand over night, then 
 filtered, and dried. The yield was 1.5 gm. 
 
 0.0188 gm. substance, in the Van Slyke micro apparatus: 1 cc. Nj at 27° and 
 759.3 mm. pressure. 
 
 0.0918 gm. substance: 0.1288 gm. CO2 and 0.0476 gm. HjO. 
 
p. A. LEVENE 
 
 
 CuHsiOuN-HCl. Calculated. C 36.9, H S.64, 
 
 NHi-N 3.58. 
 
 Found. " 38.25, " 5.8, 
 
 " 3.2. 
 
 , ,« +0.66 X 2.0287 
 
 ^< ~ 1 X 0.0524 ~ +26 
 
 
 91 
 
 Later it was found possible to prepare mucosin by hydrolyzing 
 the barium salt for one-half hour on a water bath in an aqueous 
 solution of 1 per cent sulfuric acid. The excess of acid was then 
 removed by means of barium hydroxide and the filtrate concentrated, 
 
 0.1004 gm. substance: 0.1312 gm. CO2 and 0.0478 gm. HjO. 
 0.1371 " " : 3.50 cc. 0.1 n acid (Kjeldahl). 
 0.1247 gm. substance on fusion: 0.0214 gm. BaS04. 
 0.1315 " " : 0.0410 gm. BaSOi. 
 
 C,8HoNjS2029Ba2. Calculated. C 27.80, H 3.48, N 2.32, S 5.30, Ba 22.70. 
 Found. " 35.61, " 5.30, " 3.57, " 2.35, " 18.3. 
 
 c. Preparation of Chitosamine. 
 
 The material obtained for this experiment was prepared by the 
 first of the two procedures described above. SO cords were passed 
 through a meat chopper, and then extracted with 1,500 cc. of a 3 per 
 cent solution of sodium hydroxide. The solution was treated exactly 
 as the first sample. The yield was 15 gm. of the lead salt. 
 
 These 15 gm. were taken up in 100 cc. of 20 per cent hydrochloric 
 acid solution, 1.5 gm. stannous chloride were added, and the mixture 
 was heated with a reflux condenser for 10 hours. The barium sulfate 
 and pigment were removed by filtration, and the remaining clear 
 solution was concentrated under diminished pressure to small 
 volimie. The residue was dissolved in 100 cc. of distilled water, 
 and lead and tin were removed by means of hydrogen sulfide. The 
 filtrate from the sulfide was freed from bariiun quantitatively and 
 the clear solution was concentrated under diminished pressure to 
 a syrup. A perfectly white crystalline deposit formed in the dis- 
 tilling flask. This was transferred to a suction funnel by means of 
 methyl alcohol. It consisted of perfectly uniform crystals, in ap- 
 pearance unusual for glucosamine hydrochloride. Once recrystal- 
 lized out of dilute alcohol it assumed the crystal form t)^ical of 
 chitosamine hydrochloride. Heated in a sealed capillary to a tem- 
 
92 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 perature of 220°C. it contracted slightly, turned dark, but did not 
 melt. Dried over sulfuric acid under diminished pressure the sub- 
 stance gave the following analytical results: 
 
 0.0314 gm. substance in Van Slyke micro apparatus: 3.80 cc. N2 at 25° and 
 765 mm. 
 
 0.1577 gm. substance required 7.30 cc. 0.1 n AgNOj. 
 
 CeHisNOsHCl. Calculated. N 6.50, CI 16.45. 
 Found. " 6.45, " 16.4. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 « _ +4.08X2.1459 _ „ , ,« ^ +3.5 X 2.1459 ^ „„ 
 
 ^"'° ~ 0.5 X 0.2008 X 1.04 ^"'° 0.5 X 0.2008 X 1.04 
 
 d. Identification of Glucuronic Acid. 
 
 The presence of glucuronic acid was demonstrated by the forma- 
 tion of furfural on distillation of the mucoitin sulfuric acid with 
 hydrochloric acid, by the phenylhydrazine derivative of glucuronic 
 acid after the hydrolysis of mucosin with sodium amalgam, and 
 finally by the isolation of the acid potassium salt of saccharic acid 
 on oxidation of mucosin with nitric acid. 
 
 The estimation of the yield of furfural phloroglucide also per- 
 mitted an approximate estimate of the proportion of glucuronic in 
 the molecule of the mucoitin sulfuric acid. 
 
 Distillation with Hydrochloric Acid. — 1.5 gm. of barium salt were 
 distilled over a flame in 250 cc. of HCl (specific gravity 1.06) until 
 the distillate no longer gave a test with aniline acetate. To the 
 distDlate 0.3 gm. of phloroglucide was added and the solution al- 
 lowed to stand over night. The phloroglucide was filtered over 
 a Gooch crucible. The yield was 0.0870 gm., which corresponds to 
 0.2610 gm. of glucuronic acid. The theory requires 0.5000 gm. 
 
 Hydrolysis by Means of Sodium Amalgam. — 3.5 gm. of mucosin 
 hydrochloride were dissolved in 50 cc, and 150 gm. of 2 per cent 
 sodium amalgam were added in 25 gm. portions. After each addi- 
 tion the solution was neutralized with sulfuric acid. After the last 
 portion of amalgam had been added the flask was placed in a shaking 
 machine for 5 hours and then allowed to stand over night. The 
 
p. A. LEVENE 93 
 
 following day the solution was filtered and neutralized with sulfuric 
 acid. 5 gm. of phenyUiydrazine dissolved in 5 cc. of glacial acetic 
 acid were then added and the solution was warmed on a boiling 
 water bath for 30 minutes with a reflux condenser. On cooling over 
 night a crystalline deposit formed. This was filtered, suspended in 
 water, again filtered, and suspended in 99.5 per cent alcohol, filtered, 
 and dried in a vacuum desiccator over sulfuric acid. 
 
 The melting point of the substance was 125°C. and decomposition 
 with effervescence took place at 132°C. (corrected). A sample 
 prepared from chondrosin had exactly the same melting point. In 
 the conamunication of Levene and La Forge the melting point was 
 given at 115°. The manner of purification of the substance as 
 carried out at the later date was more rigorous, and the melting 
 point of 125°C. with decomposition at 132° is to be regarded as the 
 correct one. 
 
 0.0632 gm. substance on combustion: 9.4 cc. N2 at 28°C. and 767.5 mm. 
 
 CjiHjsOiN.+UHaO. Calculated. N 17.17. 
 Found. " 16.9. 
 
 e. Oxidation with Nitric Acid with Subsequent Hydrolysis. 
 
 10 gm. of mucosin hydrochloride were dissolved in 10 cc. of dis- 
 tilled water to which 10 cc. of nitric acid (specific gravity 1.40) were 
 added, and the solution was heated over a free flame until the evolu- 
 tion of nitrous acid fumes became very lively. The solution was 
 immediately transferred to a clock glass and evaporated with con- 
 stant stirring. The subsequent treatment was as usual. The 
 final solution was made up to 10 cc. Of this 1 cc. was used as 
 control, and 9 cc. were allowed to digest with 2 cc. of a 50 per cent 
 solution of potassium hydroxide on a boiling water bath for 2 hours. 
 The solution was then made acid with acetic acid and the acid potas- 
 sium salt was allowed to crystallize. The crude salt on fractionation 
 out of water yielded a sample of the salt which analyzed as follows: 
 
 0.1000 gm. salt: 0.0356 gm. K2SO4. 
 
 CeHjOrK. Calculated. K 15.70. 
 Found. " 15.95. 
 
94 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 /. Estimation of the Number of Acetyl Groups. 
 
 2 gm. of the barium salt of mucoitin sulfuric acid were dissolved 
 in 200 cc. of water containing 15 gm. of barium hydroxide, and hydro- 
 lyzed on a water bath for 5 hours. The product of the reaction was 
 rendered acid to Congo red by means of sulfuric acid, and filtered. 
 The solution was then distilled guarding the original volume (600 cc). 
 The distillate was received in a measured volume of 0.1 N sodium 
 hydroxide. 31.3 cc. of 0.1 N alkali were neutralized by the distillate. 
 Calculated for acetic acid the yield was 0.1878 gm. The theory 
 for one acetyl group requires 0.1944 gm. 
 
 Identification of Acetic Acid. — ^The entire distillate was concen- 
 trated under diminished pressure to 8 cc, rendered acid with sul- 
 furic acid, and extracted with ether. To the ethereal extract a few 
 drops of aqueous ammonia were added and the ether was allowed 
 to evaporate spontaneously. The residue was converted into the 
 silver salt. 
 
 0.10S2 gm. dry substance: 0.0681 gm. Ag. 
 
 CaH302Ag. Calculated. Ag 64.26. 
 Found. " 64.73. 
 
 //. From Vitreous Mucoid. 
 
 a. Preparation of Mucoitin Sulfuric Acid. 
 
 To vitreous humor of 1,000 eyes enough of a SO per cent sodium 
 hydroxide solution was added to make the concentration of alkali 
 3 per cent (Levene and L6pez-Suirez, 1918). The material was 
 allowed to stand 3 days; it was then acidulated and concentrated 
 on a water bath after addition of an excess of bariimi carbonate. 
 The final product was filtered on suction. To the filtrate enough 
 of basic lead acetate solution was added to precipitate all of the acid. 
 The crude lead salt was washed by decantation, then filtered, and 
 the precipitate was washed once with glacial acetic acid. The 
 precipitate was then filtered and washed with alcohol. After this 
 the substance was taken up in water, the mixture rendered 
 slightly alkaline by means of a solution of potassium hydroxide, 
 and the lead salt was decomposed by hydrogen sulfide. From the 
 
p. A. LEVENE 95 
 
 filtrate, hydrogen sulfide was removed by aeration and the solution 
 was poured into 2 liters of alcohol. A precipitate thus formed was 
 washed with alcohol and ether. The substance analyzed as follows : 
 
 0.1000 gm. substance required for neutralization 3.54 cc. 0.1 n acid. 
 
 0.2000 " " : 0.0528 gm. BaS04. 
 
 0.0958 " Ba salt: 0.1208 gm. CO2 and 0.0490 gm. H2O. 
 
 CjiiHM02.N2S2Ba». Calculated. C 27.8, H 3.48, N 2.32, S 5.3, Base 22.7. 
 Found. " 34.4, " 5.72, « 4.96, " 3.6, " 21.5. 
 
 b. Preparation of Chitosamine. 
 
 6 gm. of the substance were dissolved in 30 cc. of 20 per cent 
 HCl + 1 gm. of staimous chloride + 1 gm. of barium chloride heated 
 with a reflux condenser for 8 hours over a Babo funnel. The solu- 
 tion was filtered, decomposed with hydrogen sulfide, and freed from 
 barimn quantitatively. The final solution was concentrated under 
 diminished pressure to about 5 cc. The sugar began to crystallize 
 in the distilling flask. The entire residue was taken up in methyl 
 alcohol and allowed to crystallize at room temperature. The yield 
 of the substance was 1 gm. 
 
 0.0302 gm. substance: 3.47 cc. Nj at 19°C. and 745 mm. 
 
 C(JIis06N-HCl. Calculated. N 6.51. 
 Found. " 6.45. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . ,« _ +4.60 X 2.1248 ^ .„ +3.43 X 2.1248 ^ ^ 
 
 '"^° 1X0.1010X1.0018 "^ 1X0.1010X1.0018 '^' 
 
 c. Furfural Distillation. 
 
 1.5 gm. of the barium salt were distilled with 250 cc. of HCl (1.06) 
 as long as distillate showed the presence of furfural. Further treat- 
 ment was carried out as above, and 0.3 gm. of phloroglucine was 
 added. The yield was 0.1165 gm., which corresponds to 0.3495 gm. 
 of glucuronic acid. The theory required 0.500 gm. 
 
96 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 III. From Cornea Mucoid, 
 a. Preparation of Mucoitin Sulfuric Acid. 
 
 The corneas of 1,000 beef eyes were mechanically separated from 
 the sclera and placed in 1,500 cc. of 3 per cent sodium hydroxide 
 solution and allowed to stand for 3 days, then strained through 
 cheese-cloth and acidulated with acetic acid (Levene and Lopez- 
 Suarez, 1918). Barium carbonate was added in excess and the 
 mixture concentrated on a water bath to a thick syrupy mass con- 
 taining the coagulated protein and the barium carbonate. 
 
 The mass was filtered on suction, and to the filtrate sufficient lead 
 carbonate was added to precipitate all the acid. The purification 
 of the lead salt was carried out in the manner described above. The 
 final substance had the following composition. 
 
 0.1000 gm. substance required for neutralization 2.42 cc. 0.1 n acid. 
 0.2000 " " : 0.0330 gm. BaSOi. 
 
 A second precipitate was prepared as follows: The corneas were 
 treated with alkali in the same manner as in the former experiment. 
 The acidulated solution was concentrated in the presence of barium 
 carbonate, and the filtrate poured into an excess of glacial acetic 
 acid. The precipitate was washed repeatedly with glacial acetic 
 acid, then with alcohol, The dry substance was then redissolved in 
 a Kttle water with the aid of potassium hydroxide. The solution 
 was poured into a large excess of 99.5 per cent alcohol. The 
 potassium salt obtained in this manner was dried and analyzed. 
 
 0.2000 gm. substance required for neutralization 6.90 cc. 0.1 n acid. 
 
 0.2000 " " on fusion: 0.04S2 gm. BaS04. 
 
 0.09S0 " potassium salt of substance: 0.1318 gm. CO2 and 0.0528 gm. H2O. 
 
 CseHuOMNjSjBaj. Calculated. C 27.8, H 3.48, N 2.32, S 5.30, Base 22.70. 
 Found. I. " 3.93, " 2.27. 
 
 " II. C 37.83, H 6.32, " 4.62, " 3.10, Base 14.74. 
 
 h. Preparation of Chitosamine. 
 
 4.5 gm. of the substance were hydrolyzed in 60 cc. of 20 per cent 
 hydrochloric acid, together with 1 gm. of barium chloride and 1 gm. 
 of stannous chloride, and the solution was heated with a reflux 
 
p. A. LEVENE 97 
 
 condenser for 12 hours over a Babo funnel. The solution as 
 usual turned dark brown. It was diluted with an equal volume 
 of water and was then freed from tin by means of hydrogen 
 sulfide; and from barium by means of sulfuric acid. The solution 
 was concentrated to a thicK syrup. Chitosamine crystallized in 
 the distilling flask. It was taken up in methyl alcohol and kept 
 at room temperature in order to complete the crystallization. The 
 yield was 0.520 gm. The appearance of the crystals under the 
 microscope was typical for glucosamine. The substance turned 
 brown at about 200°C., and black at 220°C. It did not melt. 
 
 0.0200 gm. substance in the Van Slyke apparatus: 2.41 cc. of N2 at 27°C. 
 and 756.8 mm. 
 
 CsHisOaN-HCl. Calculated. N 6.51. 
 Found. " 6.80. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . >o _ +2.27 X 2.0390 _ +1.71 X 2.0390 _ , „„ 
 
 ^"^"^ ~ 1X0.0485 ""*■ 1X0.485 ~ '^' 
 
 3. GROUP II B. MUCOITm SULFITRIC ACIDS. 
 
 I. From Mucin of the Gastric Mucosa. 
 
 a. Preparation of Mucoitin Sulfuric Acid. 
 
 Mucus was removed from the gastric waU mechanically and a 
 concentrated solution of barium hydroxide was added to make the 
 total solution contain 3 per cent of the hydroxide (Levene and Lopez- 
 Suirez, 1916, a; 1918). The solution was allowed to stand for 3 days 
 at room temperature. At the end of this time the solution was 
 rendered acid to Congo red by means of sulfuric acid, then centri- 
 fugalized. The supernatant liquid was neutralized with a solution 
 of barium hydroxide until neutral to Congo red, but still acid to 
 litmus, and finally neutralized to litmus by means of barium carbo- 
 nate, then boiled for about 3 hours, and filtered. To the filtrate again 
 barium carbonate was added and the mixture was allowed to stand 
 on a water bath from 2 to 3 days until a sample of the filtrate showed 
 a negative biuret test. This was centrifugalized and the supernatant 
 
98 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 liquid precipitated by means of glacial acetic acid. The precipitate 
 was redissolved in water and reprecipitated by means of glacial 
 acetic acid. The precipitate thus formed was repeatedly washed 
 with 95 per cent alcohol until most of the glacial acetic acid was 
 removed. This material was then dissolved in a minimvim amount 
 of water, the solution was exactly neutralized with a solution of 
 barium hydroxide, and the barium salt of mucoitin sulfuric acid 
 precipitated by means of alcohol. The crude salt was repeatedly 
 washed with a 50 per cent solution of alcohol until most of the in- 
 organic impurities were removed, then with alcohol of progressively 
 increasing concentration. This salt was a mixture of mucoitin, 
 sulfuric, and nucleic acids. To separate the two, the mixture was 
 taken up in water and centrifugalized. The salt of the nucleic acid, 
 being insoluble, was removed in this manner. To complete separa- 
 tion it was necessary to repeat the operation several times. Finally 
 the clear solution was poured into an excess of alcohol, giving a 
 precipitate of the barium salt of the mucoitin sulfuric acid. A 
 sample of the material prepared in this manner had the following 
 composition: 
 
 0.1000 gm. substance required for neutralization 2.48 cc. 0.1 n acid. 
 0.1500 " " : 0.0162 gm. BaS04. 
 
 C!«HM02,NjSjBaj Calculated. N 2.32, S 5.30. 
 Found. " 3.47, " 1.48 
 
 . ,« _ -0.19 X 5.3035 _ 
 ^"^o ~ 1 X 0.0447 ~ 
 
 b. Preparation of Chitosamine. 
 
 20 gm. of the partially purified substance, taken up with 100 cc. 
 of 20 per cent hydrochloric acid and 4 gm. of stannous chloride, 
 were heated with a return condenser over flame for 7^ hours. The 
 solution was diluted with an equal volume of water, hydrogen sulfide 
 passed through the solution, and the filtrate concentrated under 
 diminished pressure (approximately 15 mm.) nearly to dryness. 
 A crystalline sediment formed in the flask. This was transferred to 
 a beaker by means of methyl alcohol. The substance was recrystal- 
 lized out of dilute methyl alcohol. Unlike chondrosamine hydro- 
 
p. A. LEVENE 99 
 
 chloride, the substance was insoluble in methyl alcohol, and crystal- 
 lized in plates resembling those of chitosamine hydrochloride. The 
 substance did not melt. It reduced Fehling's solution, and formed 
 a glucosazone. For analysis it was dried in a vacuum desiccator. 
 
 0.0200 gm. substance in the Van Slyke amino apparatus: 2.38 cc. N at 25° 
 and 757 mm. 
 
 0.1500 gm. substance by the Volhard method required 6.86 cc. 0.1 N AgNOs. 
 
 CeHisOsNHCl. Calculated. N 6.S1, CI 16.45. 
 Found. " 6.57, " 16.23. 
 
 The rotation of the substance was 
 
 Initial. Equilibrium. 
 
 . ■i.__^ +4.30 X 2.1862 ^ „ i. ^ +3.37X2.1862 ^ , -.o 
 
 ^"'" 0.5 X 0.2008 X 1.039 ■*" ^"'^ 0.5 X 0.2008 X 1.039 ■*" 
 
 c. Oxidation of the Chitosamine. 
 
 6 gm. of the sugar were dissolved in 25 cc. of water, 6 gm. of silver 
 nitrite were added, and the mixture was allowed to stand 6 hours; then 
 another portion of 3 gm. of silver nitrite and the equivalent quantity 
 of a 10 per cent hydrochloric acid solution were added. The mixture 
 was allowed to stand over night. The solution then contained 
 0.0027 gm. of amino nitrogen. The excess of silver was removed by 
 means of hydrogen sulfide. To the solution 15 gm. of bromine were 
 added and allowed to stand for 48 hours. The calcium salt of chi- 
 tonic acid was then prepared in the usual way. 
 
 For analysis the calcium salt was dried in a vacuum desiccator 
 at the temperature of water vapor. 
 
 0.0994 gm. substance: 0.1212 gm. CO2, 0.0472 gm. H2O, and 0.0134 gm. CaO. 
 (C6H,06)Ca + 2H20. 
 
 Calculated. 
 
 C 33.47, 
 
 H 5.11, 
 
 CaO 13.07. 
 
 Found. 
 
 " 33.25, 
 
 " 5.27, 
 
 « 13.6. 
 
 The optical rotation of the substance was 
 
 n _ +1.63 X 2.149 ^ »„ 
 
 "''° 1 X 1.000 X 1.010 ■*" 
 Fischer gives for the same substance 
 
 [a]" =+32.8" 
 
100 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 d. Preparation of Mucosin. 
 
 For the preparation of mucosin 14 gm. of the barium salt were 
 dissolved in 100 cc. of water and 15 cc. of concentrated hydrochloric 
 acid, and allowed to stand on the boiling water bath for 20 minutes. 
 The solution was concentrated under diminished pressure (the 
 temperature of the bath not exceeding 40°C.) to a volume of 5 cc. 
 The solution was poured into 2 liters of a 50 per cent mixture of 
 alcohol and ether. A precipitate formed which was removed 
 by filtration. The precipitate was then dissolved in about 3 cc. 
 of water and precipitated by 400 cc. of 99.5 per cent alcohol. To 
 the filtrate, an equal volume of ether was added and thus a second 
 precipitate was formed which reduced Fehling's solution, but was 
 not yet free from mucoitin. 
 
 0.0100 gm. in the Van Slyke apparatus: 0.38 cc. N at 22° and 753.7 mm. 
 
 CuHiiOiiN-HCl. Calculated. NHj-N 3.58. 
 Found. " 2.12. 
 
 The optical rotation of the substance was 
 
 . ,2. +0.23 X 5.8780 , .,„ 
 f"^°= 1X0.0525 =+2^ 
 
 e. Furfural Distillation. 
 
 The presence of glucuronic acid was demonstrated by furfural 
 distillation. 0.0065 gm. were distilled with 250 cc. of hydrochloric 
 acid (specific gravity 1.06). The yield of phloroglucine was 
 0.0440 gm. The theory requires 0.100 gm. 
 
 /. Hydrolysis by Sodium Amalgam. 
 
 7 gm. of mucosin prepared as above were dissolved in 100 cc. of 
 water, and 200 gm. of a 2 per cent amalgam were added in portions 
 of 25 gm. at intervals. The entire operation lasted 24 hours. 
 Before each new addition of amalgam the solution was neutralized 
 with sulfuric acid. The final product was separated from mercury 
 and filtered. To this solution were added 7 cc. of phenylhydrazine 
 dissolved in 7 cc. of glacial acetic acid, and the entire solution was 
 allowed to stand for 30 minutes with a reflux condenser on a boiling 
 
p. A. LEVENE 101 
 
 water bath. The reaction product was filtered from tar, and al- 
 lowed to stand at (fC. over night. A small crystalline deposit 
 formed. There was not suflnicient material for purification or 
 analysis. 
 
 g. Acetyl Estimation. 
 
 2 gm. of the barium salt with 300 cc. of water and IS gm. of barium 
 hydroxide, were allowed to hydrolyze for 5 hours, then neutralized, 
 and the acetic acid was distilled into a 0.1 N solution of sodium 
 hydroxide. The acid neutralized 26 cc. of the 0.1 N alkali, which 
 corresponds to 0.156 gm. of acetic acid. The theory for one acetyl 
 group requires 0.194 gm. The distillate was concentrated to a very 
 small volume. This was acidulated with sulfuric acid and extracted 
 with ether. From this the silver salt was obtained. It analyzed 
 as follows: 
 
 0.1032 gm. substance: 0.0662 gm. Ag. 
 
 CaHjOaAg. Calculated. Ag 64.14. 
 Found. " 64.3. 
 
 II. From Serum Mucoid, 
 a. Preparation of Mucoitin Sulfuric Acid. 
 
 The mucoitin sulfuric acid from this mucoid (Levene and L6- 
 pez-Suirez, 1918) was prepared on one occasion by treatment of the 
 entire serum, and on the other, by treatment of the protein obtained 
 from the serum on coagulation by boiling. 
 
 The first sample was prepared in the following way. To 12.5 
 liters of the sertim a 50 per cent solution of NaOH was added until 
 the solution contained 3 per cent of alkali. It was allowed to stand 
 for 3 days at 40°C., then rendered acid by means of acetic acid, and 
 concentrated on a water bath in the presence of excess of BaCOs. 
 The filtrate was converted into the lead salt. This was treated with 
 glacial acetic acid, dried with alcohol, freed from lead, and again 
 reprecipitated with lead acetate. The lead salt was repeatedly 
 washed in a mortar with glacial acetic acid. Finally it was washed 
 with alcohol and dried. The yield of the dry substance was 14 gm. 
 
102 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
 The substance contained 5.10 per cent nitrogen. This sample was 
 used for hydrolysis. 
 
 The second sample was prepared from the coagulum obtained 
 from 12.5 liters of beef serum. The process of preparation was ex- 
 actly as in the above experiment. The lead salt was converted into 
 the barium salt. The lead salt was suspended in water, and an 
 excess of barivim carbonate was added, and hydrogen sulfide gas 
 passed until all lead separated out. From the filtrate the hydrogen 
 sulfide was removed by aeration. The solution was finally precipi- 
 tated by means of 99.5 per cent alcohol. The precipitate was then 
 dissolved in a little water and the mixture was centrifugalized to 
 remove all insoluble Ba salts. This operation was repeated several 
 times. Finally the solution was precipitated by means of 99.5 per 
 cent alcohol. This substance was dried and yielded 1.5 gm. Analy- 
 sis gave the following values: 
 
 0.1000 gm. substance required for neutralization 3.75 cc. 0.1 n acid. 
 0.1961 " " on fusion: 0.0279 gm. BaS04. 
 
 0.1008 " " " combustion: 0.1098 gm. CO2 and 0.0400 gm. H2O. 
 
 0.1961 " " treated with sulfuric acid: 0.0971 gm. BaS04. 
 
 C26HM029N2S2Ba. Calculated. C 27.80, H 3.48, N 2.32, S 5.30, Ba 22.74. 
 Found. " 29.71, " 4.44, " S.2S, " 1.96, " 29.14. 
 
 b. Preparation of Chitosamine. 
 
 13 gm. of the first sample were taken up in 80 cc. of a 20 per cent 
 solution of hydrochloric acid. 2 gm. of barium chloride and 2 gm. 
 of staimous chloride were added. The solution was heated with a 
 refliix condenser for 13 hours. The dark brown solution was filtered 
 from the melanin and diluted with an equal volume of water. The 
 solution was freed from lead and tin by means of hydrogen sulfide 
 and from barium by sulfuric acid. The solution was concentrated 
 under diminished pressure nearly to drjTiess. The residue was 
 taken up in methyl alcohol and allowed to stand. Typical crystals 
 of glucosamine hydrochloride separated out. 
 
 0.0200 gm. in the Van Slyke apparatus: 2.23 cc. N at 25''C. and 759.6 mm. 
 
 CiHisOsN-HCl. Calculated. N 6.51. 
 Found. " 6.19. 
 
p. A. LEVENE 103 
 
 Initial. Equilibrium. 
 
 r-.i» _ +1-78 X 2.0355 ,„„„ . ,» +1.42X2.0355 ,,,^ 
 '"^» - 1 X 0.0405 +^' l"'» 1X0.0405 = +^^ 
 
 The substance began to turn brown at 200°C. and turned black 
 at 220°C. It did not melt. 
 
 BIBLIOGRAPHY. 
 
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104 HEXOSAMINES, MUCINS, AND MUCOIDS 
 
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