THE CHEMISTRY OF THE LIPIDS OF TUBERCLE BACILLI LII. THE COMPOSITION OF THE ACETONE-SOLUBLE FAT OF BACILLUS LEPRa* BY R. J. ANDERSON, R. E. REEVES,t AND J. A. CROWDERf (From the Department of Chemistry, Yale University, New Haven) (Received for publication, September 17, 1937) The present report deals with the chemical composition of the acetone-soluble fat of the leprosy bacillus. The fat which had been isolated by Uyei and Anderson (1) proved to be by far the most complex mixture that we have encountered in our investi- gations of lipid products from acid-fast bacilli; for this and other reasons the completion of the work, which was begun in 1933, has been delayed. Even yet much remains unknown concerning certain components of the fat. The unsaponifiable matter, except for the isolation of LY-and Pleprosol (2), has not been examined. The fat was highly pigmented, being of deep reddish brown color, but no work has been done on this pigment. We could not find any phthiocol (3) in the leprosy bacillus fat. The fat was not a glyceride since in place of glycerol we were able to isolate the disaccharide trehalose. Of particular interest was the isolation of a seriesof new dextro- rotatory, branched chain, saturated fatty acids which, however, all differed from tuberculostearic acid (4, 5) and phthioic acid (4, 6). These acids belong apparently to the C&,, C19, and C&2 series and they formed lead salts which were easily soluble in ether. * The present report is a part of a cooperative investigation on tubercu- losis; it has been supported partly by funds provided by the Research Com- mittee of the National Tuberculosis Association. t Holder of a National Tuberculosis Association Fellowship at Yale University, 1936-37. $ Holder of a National Tuberculosis Association Fellowship at Yale University, 193334. 669 by guest on July 21, 2020 http://www.jbc.org/ Downloaded from
Transcript
THE CHEMISTRY OF THE LIPIDS OF TUBERCLE BACILLI
LII. THE COMPOSITION OF THE ACETONE-SOLUBLE FAT OF BACILLUS LEPRa*
BY R. J. ANDERSON, R. E. REEVES,t AND J. A. CROWDERf
(From the Department of Chemistry, Yale University, New Haven)
(Received for publication, September 17, 1937)
The present report deals with the chemical composition of the acetone-soluble fat of the leprosy bacillus. The fat which had been isolated by Uyei and Anderson (1) proved to be by far the most complex mixture that we have encountered in our investi- gations of lipid products from acid-fast bacilli; for this and other reasons the completion of the work, which was begun in 1933, has been delayed.
Even yet much remains unknown concerning certain components of the fat. The unsaponifiable matter, except for the isolation of LY- and Pleprosol (2), has not been examined. The fat was highly pigmented, being of deep reddish brown color, but no work has been done on this pigment. We could not find any phthiocol (3) in the leprosy bacillus fat.
The fat was not a glyceride since in place of glycerol we were able to isolate the disaccharide trehalose.
Of particular interest was the isolation of a series of new dextro- rotatory, branched chain, saturated fatty acids which, however, all differed from tuberculostearic acid (4, 5) and phthioic acid (4, 6). These acids belong apparently to the C&,, C19, and C&2 series and they formed lead salts which were easily soluble in ether.
* The present report is a part of a cooperative investigation on tubercu- losis; it has been supported partly by funds provided by the Research Com- mittee of the National Tuberculosis Association.
t Holder of a National Tuberculosis Association Fellowship at Yale University, 1936-37.
$ Holder of a National Tuberculosis Association Fellowship at Yale University, 193334.
Other higher acids, some of which were optically active, were isolated but could not be identified. Since none of the optically active acids could be obtained in a state approaching chemical purity, the further study of these products must be deferred until larger quantities of material become available.
EXPERIMENTAL
The acetone-soluble fat had been isolated in 1931 as described by Uyei and Anderson (l).’ In addition to the 289.5 gm. of fat mentioned in the former paper, a further quantity of fat weigh- ing 85.5 gm. was included in the present study. The second fat fraction had been isolated in the same manner as the first from a smaller lot of Bacillus lepra?. The two fractions had been com- bined and had stood in a sealed glass container under COS. The material was a dark red mass which was partly liquid.
Since a preliminary examination showed that a large amount of free fatty acids was present, it was decided to separate the free acids from the neutral fat. For this purpose the fat was dissolved in 500 cc. of ether and the solution was filtered from a small quantity of insoluble, brownish red particles. The ether-insol- uble pigment weighed 0.9 gm. The ethereal solution was ex- tracted with three portions of 2 per cent aqueous potassium hydroxide. The fatty acids recovered from the alkaline solution after acidification and extraction with ether weighed 95.5 gm., representing 26.2 per cent of the crude fat. The aqueous solution was examined for water-soluble constituents as will be described below.
The neutral fat recovered from the ethereal solution was a deep red-colored, semisolid mass and weighed 269 gm.
Examination of Aqueous Solution-The red-colored aqueous solution from which the free fatty acids had been extracOed was neutralized with potassium hydroxide and evaporated to dryness under reduced pressure. The residue was extracted with 80 per cent alcohol and the solution.was evaporated to a thick syrup in vacua. The syrup was dissolved in water and a solution of lead acetate was added until no further precipitate separated.
‘The Mycobacterium leprz used in this work is known as the Hygienic Laboratory Strain No. 370 (Apa case) and it was isolated from a case of
The precipitate was filtered off and discarded. From the filtrate, by means of basic lead acetate and ammonium hydroxide, a carbohydrate was isolated in the usual manner which, after being dehydrated with absolute alcohol, was obtained as a white powder weighing 3.1 gm.
The substance did not reduce Fehling’s solution directly, but after it had been boiled with dilute sulfuric acid for several minutes, the solution gave a heavy reaction for reducing sugar. The crude carbohydrate did not crystallize from 80 per cent alco- hol or from water; hence it was converted into the acetyl deriva- tive by refluxing with acetic anhydride and fused sodium acetate. The reaction product was isolated as described in a former paper (7) and crystallized from methyl alcohol. Long, colorless, needle- shaped crystals were obtained which weighed 0.5 gm. The crystals melted at about 80” and, when mixed with trehalose octaacetate which melted at the same temperature, there was no depression of the melting point.
Rotation-O.0856 gm. of substance dissolved in chloroform and diluted to 10 cc. gave in a 1 dm. tube a reading of +1.392’; hence [cY]~’ = +162.6”.
The crystal form, melting point, and rotation show that the substance was trehalose octaacetate. The isolation of trehalose would indicate that a portion of the fat had been hydrolyzed during storage or during the extraction with alkali.
Examination of Free Fatty Acids-The fatty acids were con- verted into lead salts, in the usual manner, and the latter were filtered off and dried in a vacuum desiccator. The filtrate from the lead salts was examined for acids giving water-soluble lead salts, as will be described below.
The lead salts were digested in ether and the ether-insoluble portion was filtered off and washed thoroughly with ether. The ether-insoluble lead salts yielded 38.5 gm. of crystalline solid acids, while from the ether-soluble lead salts 53.5 gm. of dark colored liquid acids were obtained. The liquid acids had an iodine number of 46.1.
Examination of Aqueous Filtrate from Water-Insoluble Lead Salts-The aqueous filtrate and washings, about 3 liters, from the lead salts were acidified with hydrochloric acid and extracted with ether. The ethereal extract was washed with water, dried
over sodium sulfate, and evaporated to dryness. The residue after drying in vacua was a light brown-colored oil of an unpleasant odor and it weighed 3.5 gm. This fraction was nearly all volatile with steam. After steam distillation the non-volatile portion weighed 0.1 gm., while from the distillate 3.4 gm. of an oil were recovered by ether extraction. The non-volatile portion was a dark oil which was not further examined.
The steam-volatile acid was found to consist mainly of caproic acid. The p-bromophenacyl ester was prepared according to Judehnd and Reid (8) from a portion of the acid. The ester on crystallization from alcohol containing a little water was obtained in colorless crystals which melted at 71-72”. There was no de- pression of the melting point when the crystals were mixed with an authentic sample of the p-bromophenacyl ester of caproic acid which also melted at 71-72”.
Solid Fatty Acids-The solid fatty acids were converted into methyl esters in the usual manner and yielded 39.1 gm. of solid crystalline esters. The esters were dissolved in 100 cc. of ether. On standing at room temperature overnight a considerable amount of thin, plate-shaped crystals had separated. The crystals were filtered off, washed with cold ether, and dried in vucuo. The crystals weighed 2.7 gm. The ethereal solution was evaporated to dryness and the esters were later combined with the similar fraction obtained on saponification of the neutral fat, and the material was examined as will be described later.
Examination of the Ester Which Crystallized from the Ethereal Solution-The crystalline ester which separated from the ethereal solution melted at 56”. The substance was twice recrystallized from 20 cc. of acetone and separated as thin, small plates. The product weighed 2.1 gm. and melted at 56-57”, solidified at 55.5”, and remelted at 56.5”.
Analysis - CMH~TO&H~ (382). Calculated. C 78.53, H 13.08 CZ~H~~O&H~ (396). “ “ 78.78, “ 13.13
Found. “ 78.61, “ 13.01 ‘I ” 78.52, “ 12.92
The values found agree approximately with the calculated composition of the methyl ester of a tetra- or a pentacosanoic acid.
The ester was saponified and the free acid was crystallized twice from 40 cc. of acetone and once from 10 cc. of benzene plus 20 cc. of methyl alcohol. The acid separated from the latter solvent in dense heavy particles consisting of fine crystals. The substance weighed 1.6 gm. and melted at 82”, solidified at 80”, and remelted at 82”. Two further crystallizations from ether yielded 1.13 gm. of acid which also melted at 82”.
Analysis - Cz,Hso02 (382). Calculated. C 78.53, H 13.08 Found. “ 78.63, “ 13.02
“ Mol. wt., 379, 379, 379
The values found agree best for a pentacosanoic acid. It is impossible, however, to state definitely whether the acid described above is a pure substance or a mixture of higher acids.
Liquid Fatty Acids-The liquid fatty acids were converted into methyl esters and the esters were distilled in uacuo. An attempt was made to reduce the esters of the unsaturated acids in ethereal solutions but without success, some substance being present which poisoned the catalyst. The esters were saponified and the free acids were isolated. The acids were easily reduced with hydro- gen in the presence of platinum oxide, after which the lead salt- ether separation was repeated.
The reduced acids recovered from the ether-insoluble lead salts were obtained as a white crystalline solid which weighed 30.9 gm. The ether-soluble lead salts yielded 8.8 gm. of slightly yellow, liquid saturated acids.
In the operations described above large losses were inevitable. The solutions of the esters and of the crude fatty acids were very dark in color and repeated treatments with norit were necessary to remove coloring matter. Highly colored products removed by the norit, after elution with chloroform, weighed 5.8 gm. Another serious loss occurred in the distillation of the methyl esters because a considerable non-volatile residue remained in the distillation flask.
Examination of Reduced Acids-The reduced acids on crystal- lization from acetone yielded fractions which were obviously mix- tures of several acids. They were reserved and combined with the reduced acids obtained from the neutral fat and examined as will be described later.
Liquid Saturated Fatty Acids--The liquid fatty acids isolated from the ether-soluble lead salts were saturated acids, because they did not absorb any bromine in chloroform solution. In ethereal solution this fraction showed optical activity, [a]:’ = +1.25”. The complete examination of the liquid saturated fatty acids was deferred until a larger quantity would be available from the neutral fat.
Neutral Fat-The following constants of the neutral fat were determined: melting point 19-20”, iodine number 47.9, Reichert- Meissl number 5.02, Polenske number 2.09, unsaponifiable matter 22.1 per cent, fatty acids after saponification 75.7 per cent.
The saponification number could not be determined owing to the dark color of the saponified solution.
SaponQication of Neutral Fat-A portion of the neutral fat weighing 251.7 gm. was saponified with alcoholic potassium hydroxide and the cleavage products were isolated in the usual manner and yielded the following fractions.
UnsaponiJiable Matter-The unsaponifiable matter formed a very thick, nearly black mass. On standing for some time at room temperature, a portion of the unsaponifiable matter sepa- rated as a solid. The solid material was isolated by treatment with petroleum ether and weighed 11.2 gm. From this substance Q- and p-leprosol were isolated as described in Paper XLV of this series (2). Lack of time has prevented further examination of the unsaponifiable matter.
Water-Soluble Carbohydrate-This fraction was isolated from the aqueous solution, after the fatty acids had been extracted, by means of basic lead acetate and ammonium hydroxide. It gave no reduction with Fehling’s solution until it had been boiled for several minutes with dilute sulfuric acid. The substance, which probably contained trehalose, was dissolved in 2 cc. of water and the solution was diluted with 8 cc. of absolute alcohol. After the solution had stood in a refrigerator for some time, large, colorless, rhombic crystals separated. The dried, powdered crystals melted at 98-100” and gave no depression when mixed with pure trehalose which melted at the same temperature.
Trehalose was the only water-soluble compound that could be isolated. No glycerol could be found. In fact, we have not found glycerol in the fats of any of the acid-fast bacteria but trehalose has been isolated previously from the acetone-soluble fat of the human tubercle bacillus (7).
Separation of Fatty Acids-The fatty acids were converted into lead salts and separated into solid and liquid acids in the usual manner. The solid acids weighed 93.8 gm. and the liquid acids weighed 88.4 gm.
Isolation of a Phenolic Substance from Aqueous Solution after Fatty Acids Had Been Precipitated As Lead Salts-The aqueous solution, after the lead salts had been filtered off, was acidified with hydrochloric acid and extracted with ether. The ethereal solution, after being washed with water, dried over sodium sulfate, and filtered, was evaporated to dryness. The partly crystalline residue weighed 0.69 gm. The oily portion was re- moved by treatment with a mixture of benzene and petroleum ether, thus leaving 0.18 gm. of crystalline material. The sub- stance was soluble in warm water. The solution was neutral in reaction and gave no coloration with ferric chloride. The sub- stance was readily soluble in ether and could be removed fromthe ethereal solution by extraction with aqueous alkali. The alkaline solution was concentrated and acidified with hydrochloric acid, whereupon 0.1 gm. of an amorphous solid separated. When heated under reduced pressure, a portion of the substance sub- limed at lOO-130”, separating on the cool part of the apparatus in crystalline form. The crystals, which weighed 40 mg., were recrystallized three times from benzene by the addition of petro- leum ether. The purified substance weighed 14 mg. and melted sharply at 105-106”, solidified at 98”, and remelted at 105-106”.
Analysis2 - 2.497 mg. substance gave 2.10 mg. Hz0 and 6.46 mg. CO* CIaHz203 (238). Calculated. C 70.56, H 9.23
Found. “ 70.56, ‘I 9.41
Hydroxyl determination by the method of Stodola (9) indicated the presence of two OH groups.
Molecular Weight-O.448 mg. of substance dissolved in 4.506
2 We are indebted to Dr. F. H. Stodola for this and many other micro- analyses.
mg. of camphor gave a depression of 17”. Found, molecular weight 228.
The further investigation of this substance must be deferred until some future time when more material is available.
Solid Fatty Acids-The solid fatty acids were converted into methyl esters, the yield being 96.3 gm. This material was com- bined with the similar fraction obtained from the free fatty acids and separated by fractional distillation as will be described later.
Liquid Fatty Acids-The liquid fatty acids had an iodine number of 63.8 and were of dark brown color. The material was dissolved in alcohol and the solution was treated repeatedly with norit until most of the coloring matter had been removed, after which the acids were reduced with hydrogen in the presence
TABLE I
Fractionation of Methyl Esters of Solid Fatty Acids
Fraction No.
1 2
3 4 5
Residue
-
. --
-
Pressure Melting point
“C. mm. “C.
155-156 About 1 28-29
156-160 ‘I 1 28-29
160-175 (I 1 36-37
175-180 it 1 45-46 180-195 0.001 * 52-52.5
Weight
9m.
57.0 21.9 18.1
15.8 13.1
6.4
of platinum oxide. The lead soap-ether treatment was repeated and there were obtained 65.0 gm. of solid reduced acids and 18.8 gm. of liquid saturated acids.
Considerable losses were inevitable in the various operations with the fatty acids. The highly pigmented material removed from the fatty acids and from the methyl esters by treatment with norit, after elution with chloroform, formed a black, tarry mass weighing about 8.8 gm. This material, which was of such a na- ture that further chemical examination of it appeared fruitless, was set aside.
Fractionation of Esters of Solid Fatty Acids-The esters of the solid fatty acids obtained from the free acids, 36.2 gm., and those obtained from the neutral fat after saponification, 96.3 gm., were combined and fractionated through a modified Widmer (10) column into five fractions and a residue, as shown in Table I.
The five fractions were subjected to repeated refractionations until apparently pure esters were obtained. The pure esters were saponified and the free acids were recrystallized. In the interest of economy of space the various operations are not described in detail.
TABLE II
Solid Fatty Acids from Acetone-Soluble Fat
1
2 3 4 5
6
7
8 9
10 10-a
11
12
Weight
8Y7
“C.
4- 5
3.5 17-18 28.8 28-29 9.0 38-39 0.8 4546
1.6
6.2
0.8 0.4
1.6
52-53 1.4362, ‘I 55”
54-55
58-59 1.4360, ‘I 60” Ioc le No. 69.3
Ester fractions I
1.4325, at 24”
1.4230, ” 55” 1.4271, “ 55’ 1.4317, (‘ 55”
1.4350. ‘I 55O
<‘ “ 66.7 By catalytic reduction of (10)
I Ester residue [(Y]: =
+2.0° Ester residue [LY]: =
+4.6’
- .
- .
:
I
t
t
;
, 8
;
j
:
;
;
t
-
Mol. Melt- w$. iwz .b
point t;;;;-
~- “C.
$839
X-55 228 $2-63 256 38-69 284 72-73 316
75-76 346
77-78 356
15-86 369 $435 343
Oil 354 77 357
76-78 412
j4-65 510
Fatty acids
ReDU&S
Impure; containing some acid lower than myristic acid
and Czc acids Tetracosanoic acid Slightly impure erucic
acid Either tricosanoic acid
or mixture of Cez and Czr acids
New unidentified acid
“ I‘ ‘I
The properties of the esters and of the fatty acids are given in Table II. The esters having melting points higher than that of methyl stearate were purified not only by fractional distillation but by repeated crystallization from acetone until the melting point remained constant.
From the less volatile portions of Fractions 3, 4, and 5 a small amount of unsaturated esters was isolated which even after
numerous redistillations could not be separated into definitely pure fractions. The esters were therefore saponified and attempts were made to separate the free acids by means of crystallization from glacial acetic acid, by fractional precipitations of thepotas- sium salts, and by fractionation of the lead salts, but the results were not very satisfactory.
The purest acid obtained approached in properties those of erucic acid, m.p. 3435”, iodine number 69.3, molecular weight 343.
Many fractions were obtained, however, which had lower iodine numbers. One of these fractions was a thick oil, iodine number 66.7, molecular weight 354. This product on catalytic reduction yielded an acid which crystallized in thin colorless plates, m.p. 77’, molecular weight 357. Repeated recrystalliza- tions from acetone caused no change in properties.
Analysis - CssH,aOz (354). Calculated. C 77.96, H 12.99 Found. “ 77.94, “ 13.00
The above values agree with those of a tricosanoic acid. It is, however, more likely that we were dealing with a mixture of docosanoic and tetracosanoic acids.
First Ester Residue-The residue remaining after the first distil- lation of the esters, 6.4 gm., was a dark brown solid. The sub- stance was dissolved in 30 cc. of warm acetone and the solution was allowed to cool to room temperature, whereupon a fine globular powder separated. After three reprecipitations in the same manner, a slightly yellowish powder weighing 2.5 gm. was obtained, m.p. 52”.
The mother liquors on evaporation to dryness left a dark brown solid weighing 3.9 gm. which was not further examined.
The powder mentioned above was separated into a top and a bottom fraction as follows: The substance, after being decolorized with norit, was reprecipitated five times from acetone and gave 0.95 gm. of a white powder, m.p. 57-58”. Two further precipi- tations from acetone gave 0.9 gm. of the top fraction, m.p. 57-58”, [a];’ = $4.6’. The bottom fraction was obtained on the evapo- ration of the mother liquors from the top fraction.
Saponi$cation of Top Fraction-The top fraction was saponified and the free acid after being isolated was precipitated from ace-
tone until the melting point remained constant at 64-65”. The acid was a white amorphous powder which weighed 0.8 gm. [a]E2 in CHCl, = +4.3”. The molecular weight by titration was 510.
Analysis - Found. C 79.16, H 12.86
This acid is no doubt an impure mixture, because the composi- tion does not agree with any acid having a molecular weight of 510.
Saponification of Bottom Fraction-The bottom fraction was saponified and the free acid was purified as described for the top fraction. The acid was a white amorphous powder which weighed 0.9 gm., m.p. 76-78”, [a]:* in CHCL = +2.0”. The molecular weight by titration was 412.
Analysis - Found. C 78.49, H 13.18
This acid was most probably a mixture. Although the acids from both the top and bottom fractions
were not obtained in definitely pure form, the results show, never- theless, that the ester residue yielded optically active acids on saponification. The optical activity would indicate that the acids had branched chains, since the analytical values suggest that they are not hydroxy acids. The high melting points of the acids show that the acids differ in constitution from phthioic acid (4, 6) obtained from the human tubercle bacillus, although the percentage composition is very similar.
Reduced Fatty Acids-The solid fatty acids obtained after cata- lytic reduction of the liquid fatty acids, 65.0 gm., were combined with the reduced acids, 30.9 gm., obtained from the free fatty acid fraction and the mixture was converted into methyl esters. The crude esters formed a dark colored, semisolid mass which weighed 97.6 gm. The esters were fractionated at a pressure of about 2 mm., as described under the solid fatty acids, into the fractions given in Table III.
Fractions 1 to 5 were repeatedly fractionated by distillation at a pressure of about 2 mm. through a modified Widmer column until apparently pure esters were obtained. The purified esters were saponified and the free acids were isolated and recrystallized.
During the redistillation of Fraction 1 a trace of an ester pos-
sessing a pleasant odor was collected in a trap cooled to -80"
inserted between the receiver and the vacuum line. The amount of this ester was so slight that no attempt at identification could be made.
The next fraction, 0.4 gm., was an oil which melted at about l”, hence it must have contained esters of acids lower than myristic. No pure acid could be isolated from this fraction.
From ester Fraction 5 ‘I I‘ residue on first distillation
The balance of Fractions 1, 2, 3, and 4 yielded methyl myri- state, palmitate, and stearate.
Fraction 5 was purified not only by distillation but also by repeated crystallization from acetone until the melting point remained constant at 52-53".
The ester residue from the first distillation was crystallized
repeatedly from acetone until the melting point was constant at 56-57”.
From the amounts of ester fractions obtained and their refrac- tive indices the composition of the total esters of the reduced acids was calculated as indicated in Table IV.
The acid Fractions 2, 3, and 4 (Table IV) were identified by means of melting points and refractive indices of the esters, and the free acids by melting points plus mixed melting point,s as well as by titration values.
The purity of acid Fractions 5 and 6 is doubtful. The data obtained agreed with the formulas suggested in Table IV. Acid Fraction 5 was obtained from the purified ester Fraction 5, m.p. 52-53”. The free acid crystallized from acetone in thin colorless plates, m.p. 75-76”; molecular weight by titration 329. Acid Fraction 6 was obtained from the purified ester residue from the first distillation. The ester crystallized from acetone in very thin colorless plates, m.p. 56-57’. The free acid separated from acetone in the form of small, rather dense particles which showed no crystalline structure, m.p. 80-Sl”, molecular weight by titra- tion 382.
Although the higher acids could not be definitely identified, the results show clearly that acids higher than stearic acid were present among the reduced acids.
Liquid Saturated Fatty Acids-The liquid saturated fatty acids from the neutral fat, 18.8 gm., were combined with the corre- sponding fraction isolated from the free fatty acids, 8.8 gm., and treated repeatedly with norit in alcoholic solution until nearly colorless. The acids were recovered and converted into methyl esters which after further treatment with norit yielded 22.6 gm. of esters.
The crude esters were separated by fractional distillation through a modified Widmer column at about 2 mm. pressure into three principal fractions; i.e., a lower (b.p. below loo”), a middle (b.p. 130-150’), and a higher boiling fraction (b.p. 160-200’). The esters were further purified by repeated fractional distilla- tions, those fractions having similar boiling points, optical ac- tivity, and refractive indices being combined. A considerable amount of very dark colored residue remained on the first distil- lation but was not further examined.
The purified esters were next saponified and the free acids were converted into lead salts. The lead salts on treatment with ether left small amounts of ether-insoluble lead salts from which solid crystalline acids were obtained, apparently mixtures of palmitic and stearic acids that had not been completely removed in the first lead salt-ether separation.
The free acids recovered from the ether-soluble lead salts from the middle fraction, which represented the principal portion of the acids, were separated into several fractions by means of the Twitchell lead salt procedure. The alcohol-insoluble and the alco- hol-soluble lead salts obtained in this separation were easily soluble in ether and yielded acids that varied decidedly in optical rota- tion.
Some of these acids were further fract,ionated by means of their barium salts, but the only separation that could be effected con- cerned optical activity.
The results obtained with the branched chain fatty acids are summarized in Table V. A number of carbon and hydrogen determinations were made on the acids of the middle fractions. The results were in agreement with the formula C19H3802.
It was noted in every case that the rotations of the free acids were lower than those of the esters. No claim to purity of any of the acid fractions can be made; in fact, the properties ob- served indicate clearly that we were dealing with mixtures. Nevertheless the results are definite on certain points, as mentioned below.
1. The acetone-soluble fat of the leprosy bacillus contains a series of new acids that are optically active. These acids are saturated compounds and presumably possess branched chains.
2. All of these acids give lead salts which are very easily soluble in ether.
3. The acids can be separated by fractional distillation of their methyl esters into (a) a small amount of low boiling esters, (b) a larger quantity of a middle fraction, and (c) a small amount of a high boiling fraction.
4. The acids isolated from the low boiling ester possess a molec- ular weight close to that of palmitic acid, C~H3202.
5. The acids isolated from the middle fraction have the molec- ular weight corresponding to an acid having the formula C19H3802.
This acid can be separated into a number of fractions which show considerable variation in optical rotation. Those fractions hav- ing the higher rotation possess the higher melting point. It is therefore probable that a number of isomeric C19 acids are pres- ent and that the differences in dextrorotation and melting point depend upon the position of the branch in the chain.
TABLE V
Liquid Saturated or Branched Chain Optically Active Fatty Acids from Leprosy Bacillus Fat
Description ?&3igh
1. Acids from low boiling esters...................
2. Acids from middle esters (a) Acid from benzene-
3. Acids from higher boiling esters (a) Acid from alcohol-in-
soluble lead salt. .
(b) Acid from alcohol- soluble lead salt. . . .
-
gm.
0.52
1.2
2.0
2.0
1.2
0.9
1.6
It
--
,
-
Melting point
“C.
Oil
28-30
2930
Partly liquid
“ ‘I
I‘ ‘I
“ ‘I
300
297 .l
298
C19&309
I‘
298.: ‘I
312 Probably mix- ture of Cl9
and C22 acids
337.: CzHuOz
6. The acids isolated from the higher boiling ester fractions possess a higher molecular weight and correspond approximately to a docosanoic acid, C22H4402.
7. The optically active branched chain acids obtained from the leprosy bacillus fat are all different from the analogous acids previously isolated from the human tubercle bacillus.
1. An investigation has been made of the chemical composition of the acetone-soluble fat of Bacillus leprs.
2. The crude fat was found to be a complex mixture of free fatty acids and neutral fat.
3. The neutral fat consisted apparently of fatty acid esters of the disaccharide trehalose. No glycerol could be found.
4. The fatty acids were composed of ordinary solid saturated acids, unsaturated solid acids, unsaturated liquid acids, and satu- rated, optically active, branched chain acids.
5. Three different substances possessing phenolic properties were isolated.
6. The ordinary saturated fatty acids were represented by caproic, myristic, palmitic, stearic, arachidic, behenic, and tetra- cosanoic acids. Certain new optically active higher acids were also present but could not be definitely identified.
7. Among the unsaturated acids examples of CM, G,, Cl*, GO, C&z, and apparently CL and CX, were found.
8. A series of new dextrorotatory, branched chain, saturated acids apparently of the Crs, Cn,, and Czz series was isolated.
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