OPTICAL ISOMERS O F CYSTINE AND THEIR ISOELECTRIC SOLUBILITIES BY JAMES
c. ANDREWS
AND EDWIN J.
DEBEER*
The comparative physical properties of the possible optical isomers of cystine have never been clearly defined although it has been observed that cystine samples of different optical activities have different isoelectric solubilities in water. It has been suggested that some of the anomalies in the chemical behavior of this substance may also be the result of varying proportions of different optical isomers and furthermore, the peculiar selective action which biological agents frequently show for one optical isomer as opposed to the other makes desirable a more minute acquaintance with these isomers. The present work was undertaken to determine the solubilities in water of isoelectric samples of cystine of different 1.1~ values with a greater accuracy than heretofore and to use these figures in their relation to the [ a ] ~ values to deduce what isomers might be present in samples of various activities. It was hoped that the work might ultimately make possible a more exact separation and characterization of the optical isomers of this compound. The usual formula for the number of optical isomers possible (znl where n = the number of asymmetric carbon atoms) points to four compounds in addition to racemic compounds; but here, owing t o the symmetry of the whole molecule, two of the isomers are identical and only three configurations of the molecule are possible. To illustrate this more clearly, the two asymmetric carbon atoms may be designated as X and Y, a prefixed plus or minus sign indicating dextro- or laevo-activity.
-x
+X
-Y
+Y
-X -Y
+X
-Y (1) (2) (3) (4) Combination ( I ) would indicate laevo, (2) dextro, and (3) and (4), being identical since X and Y are, in this compound, interchangeable, both represent the internally compensated or meso form. There is also the possibility of an optically inactive compound being formed of equimolar parts of dextro and laevo, according to the equation, d+l+dl this equibrium being controlled chiefly by the prevailing temperature.. Each of these phases is a chemical entity and, as such, has its own physical properties such as solubility, melting point, etc. which, for two enantiomorphous substances are regarded as being identical. If then a solution is saturated with respect to one phase, the introduction of another phase should give increased solubility which would be the sum of the solubility values of the two De artment of Physiological Chemistry, School of Medicine, Cniversity of Pennsylvania, Pbadelphia.
JAMES C. ANDREWS A S D E D W I S J. DEBEER
1032
individuals provided the sample be sufficiently large to maintain both solid phases. The possibility that the presence of one phase may affect the solubility of the other seems, in such dilute solutions, to be negligibly slight. Although solubility figures for cystine are few in number and not in good agreement, they do show definite variations with varying [ a ]values. ~ Inactive samples seem to be more soluble than laevo and hlorner obtained figures which indicate that the dextro variety is also more soluble than the laevo. His figures as well as those of other investigators are given in Table I.
TABLE I Specific Rotation [ab
Solubility in gm. per 1
(‘Laevo” “Laevo”
0.19
-224.3’
0.111
-66.0”
0,333
17.0
-5.5’ “Inactive” “Inactive”
0.2jo
17.0
0.490 0.326
20.0
+47.
0.344
17.0
0’
Temperature ‘ Centigrade. 20.0
0.113 17.0
Hoffman and Gortner’ Xeuberg and Meyer? ;\lorner3 ?vlOrner3 hlorner? Hoffman and Gortner’ Xeuberg and Meyer2 Rlorne?
As far as can be determined, no account was taken, in any of the above determinations, of the possible effect of variations in size of solid sample. It is impossible to say, except in the light of further experiments, how many phases were really in equilibrium with the solution. Experimental Cystine samples of different optical activities were purified by making a t least five isoelectric precipitations from HC1 solution, using dilute S a O H for neutralization. I n all cases, a ‘‘c.P.” cystine, giving a negative Millon test, was used as the starting point. Particular care was always taken in the neutralization not to carry the solution to a pH even as high as 7. The precipitate was then washed repeatedly with distilled water until the washings were electrolyte free, as determined by testing for chloride ion. The purified substance was kept in a desiccator. Xtrogen determinations by the Kjeldahl method gave almost theoretical results and sulfur determinations, when made, have always given results corresponding very closely to the purity obtained by Kjeldahl. After washing the sample electrolyte-free, it was always examined minutely under the microscope for the purpose of detecting any irregularity in crystalline form. The standard method, recommended by Xndrew4, was followed in determining the specific rotation. Readings were made with a Schmidt and Haensch polariscope, sensitive to *o.oI’, using a four decimeter tube. The solubility measurements were made by placing weighed amounts of the samples with definite quantities of twice-distilled water in Pyrex Kjeldahl flasks, which were then tightly stoppered and deeply immersed with frequent
SOLUBILITIES OF OPTICAL ISOMERS O F CYSTISE
I033
shaking for a t least 24 hours (in some casBs for weeks) in a Freas water thermostat kept a t 25OC i o.ojo. The pH of the water used, as checked by the indicator method, was what would result from equilibrium with atmospheric carbon dioxide (about pH 6) and after saturating with the cystine, no detectable change in pH could be observed. The solution was then filtered a t z j°C through one or more layers of hardened filter paper and the amount dissolved was determined by making four or five duplicate analyses, using the Folin-Looney colorimetric method. Each analysis was checked against a separate and independently measured standard. In order to place the solubility figures on a more fundamental basis, micro-Kjeldahl determinations were occasionally run against ammonium sulfate standards. The agreement with the Folin-Looney results was excellent and the Folin-Looney method was then used for most of the work because of its greater speed and convenience. a rule the Folin-Looney procedure gave more consistent results with less trouble. The observed rotation of the solution was also read in a 4 dm. tube at 2 9 O C . These readings could he duplicated to within =to 01’. Table I1 shows a suiiimary of the results obtained. The individual figures for each determination are tabulated, as well as the average figure adopted. I t will be noted that all “.1”samples consist of o . o j gm. cystine per I O O cc. water [a figure only slightly above the solubility previously reported for 1cystine), “B” samples consist of ten times the weight of solid in “A,” “C,” ten tinies that of “B,” and “D,” where necessary, twice that of “C.” Thus in the samples with greater excess of solid, an opportunity is given for phases present in comparatively small amounts to attain saturation. The necessity for this is amply shown in the table. Since the determinations were carried out using ordinary distilled water, it -xas thought well to determine if dissolved C O , and the resulting acidity produced any change in the solution figures, although the very flat isoelectric range determined by Merrillj made it seem unlikely. Analyses were therefore made of solutions which had been constantly swept in the thermostat with a slow stream of very pure nitrogen. KO appreciable difference in results could be detected. While the actual isoelectric point of cystine is on the acid side of pH 7 , the difference between the presence or absence of such amounts of carbon dioxide as could result from contact with ordinary air evidently has no detectable effect. Theoretical These data present several interesting features. The presence of a t least two phases is indicated in all samples by the fact that large proportions of solid were required to give plateau solubility figures. The 204.2’ lot gives a low solubility value of 0.083 gm. per liter in the “A” sample, with a residue consisting of hexagonal plates as shown on microscopical examination, and a plateau figure of 0.163 gm. per liter for the “B” and “C” samples. The observed rotation (not the specific rotation) of all the samples was about - 0 13’ indicating a constant concentration of the laevo phase. The fact that the usual hexagonal crystals made up the residue of the “A” sample confirms
JAMES C. ANDREWS AND EDWIN J. DEBEER
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SOLUBILITIES OF OPTICAL ISOMERS OF CYSTINE
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1036
J A M E S C. ANDREWS AND EDWIN J. DEBEER
the prevalent idea that these crystals represent the laevo form, with which even the "A" solution has been saturated. The - 148.7' lot gives a much higher plateau, 0.1I j gm. per liter but practically the same observed rotation. This increase in total solubility must be ascribed to the appearance of a new phase. The - I I 5.0" lot gave the same plateau figure as the previous lot indicating that, although racemization had proceeded further no new phase had appeared. The inactive sample gave the highest value of all, 0.635 gm. per liter. The one dextro lot had a plateau figure which was slightly lower, 0 . j j j gm. per liter, indicating the absence of one of the phases which was present in the inactive sample. These figures obviously indicate an increase in solubility as optical activity decreases, such that increased proportions of solid to liquid are required to give plateau solubility figures. Even in the case of the sample of highest activity ( - 204.2') there is evidently a very small proportion of an inactive phase which required more than the minimum ("A") proportion of solid to furnish enough for saturation. I n racemizing such a compound as cy'stine, containing two asymmetric carbon atoms of identical configuration, a situation is encountered analogous to that of tartaric acid. To racemize any sample, the configuration of some of the carbon atoms of some of the molecules must be reversed. It seems improbable and contrary to past experience that both asymmetric carbon atoms are affected simultaneously, forming the optical antipode a t one step. On the other hand, if they are reversed one a t a time, the only other alternative, the result can only be the formation of the meso form as an intermediate step in the racemization process, followed by later reversal of the group on the other asymmetric atom. Thus the sequence is: laevo to meso, meso to dextro, and depending on conditions, dextro plus laevo to racemic compound.
If the optical activity figures be temporarily neglected and the phases assumed t o appear in this order: laevo, meso, dextro which unites with the excess laevo to form the dl compound, and finally a dextro phase, a rough idea of the solubility of each phase may be gained by the following method of reasoning. Thc -204.2' lot is evidently not pure 1-cystine but contains some other phase, most probably the meso. Because a constant rotation of about -0.13~ was obtained with all samples of the -204.2~ lot, the solutions must be, in all cases, saturated with the 1-phase, and its solubility must be less than the lowest figure obtained. Therefore 0.083 gm. per liter may be taken as a maximum figure for the solubility of the laevo phase. Moreover, as stated above, a microscopic examination of the residue disclosed the presence of the hexagonal plates which commonly occur in the more laevo samples of cystine. The increase in total dissolved cystine from 0.083 to 0.163 gm. per liter may be attributed t o saturation with the other phase present, probably the meso. As racemization.progresses, it is found that a new plateau value is reached. The - 148.7' and the - I I 5.0' lots both give 0.3 I 5 gm. per liter as a saturation figure. This higher figure may be assumed to be due to the appearance of a new phase, which should be the dl compound, formed by the combination
'03 7
SOLUBILITIES O F OPTICAL ISOMERS O F CYSTINE
of.some of the newly formed dextro with some of the laevo. If then, the two plateau figures be subtracted, a solubility value for the third phase will be obtained. - 1 1 5 ,0 ' dl 0.31; gm. per 1. - 148.7' 1 m -204.3' 1 m 0.163 gm. per 1. dl 0 . 1 5 2 gm. per 1.
+ + +
The inactive sample gave a still higher plateau figure and a water solution which was dextro rotatory. These facts suggest the appearance of a dextro phase, more soluble than the laevo. The difference in solubility value bet,ween this sample and the - 115.0 lot should be due to the appearance of the dextro phase. 0.0'
1
11j.o"
1
+ m + dl + d + m + dl
0.630 gm. per 1. 0.315 gm. per 1. 0.320gm. per 1.
d The
+
12.0
lot may be assumed to have no laevo phase present. 0.0' +IZ.O'
m+dl+d+l m dl d
+ +
1.
0.630gm.perl. 0 . j j j gm. per 1. 0 , 0 7 5 gm. per 1.
The solubility of the meso can be obtained from the first or -204.2'
l+m 1 ni
-204.2'
lot.
0.163 gm. per 1. 0 . 0 ; j gm. per 1. 0.088 gm. per 1.
I t should be noted that the figure of 0.0;s g. per liter obtained above for the I-phase is very close to that obtained as a maximum value for its solubility in the A series of the - 2 0 4 . 2 ~sample.
If the solution is saturated with one or more phases the addition of a sample containing other phases should cause increased solubility values, even though the rotation of the HC1 solution of the total sample should be the same for some sample with fewer phases than the mixture. Thus the history of the sample should be considered when making solubility determinations. To test this point a mechanical mixture was made by grinding together in a mortar the proper proportions of the inactive and the - 2 0 4 . 2 ~ lots so as to give a rotation in HC1 solution of - I I j.0". The actual specific rotation obtained was - 112.5'. This mixture should have all four phases present while the - 115.0' sample, prepared by boiling a laevo sample with HC1, should have, according to the theory, only the 1-, m-, and dl forms. The solubility value of the mixture was slightly higher (see Table 11) than that obtained for the inactive, but showed no agreement with the - I I 5.0' lot. It is evident that there was no interaction during the time available between the various phases present since otherwise the dextro-phase assumed to be present in the
1038
JAMES C. ANDREWS AND EDWIN J. DEBEER
inactive sample would have combined with the excess of laevo phase from the - 204.2' lot, with the consequent removal of the dextro-and drop in solubility. However, there shouldalso be considered the possibilities of a readjustment of equilibrium conditions between the d- and 1- forms and the dl-compound. Since racemic compounds are usually regarded as being dissociated in solution, the following equilibrium would obtain: dl (solid)
e
d(solution)
+
1(solution)
Such an equilibrium would produce the two antipodes in equimolecular proportions and an inactive solution would result provided that exterior influences react equally on the activities of both forms. If such an equilibrium as the above holds, the solubility of the dl-compound should be decreased by the presence of excess quantities of either antipode, particularly of the more soluble d- form. The resulting adjustments should then cause progressive solution of both d- and 1- forms and removal as the dl- compound since the concentrations of both antipodes, indicated by their solubilities, are far in excess of that indicated by the "solubility product" of the dl- compound, as calculated from its solubility. The net result would be that a sample originally inactive in acid solution should produce only an inactive water solution since the inactive sample should contain equimolar amounts of both configurations, regardless of their form of combination, and the adjustment of the above described equilibrium results in the removal of equimolar proportions to form the dl-compound. Since the opposite is true, Le., an inactive sample actually produces a dextro-rotatory water solution, it is to be inferred that no such adjustment takes place. We have shown by experiment that the dextro rotation persists a t least for several weeks. That the inactive sample of cystine used for these determinations was really inactive seems fairly certain. It not only failed to show any detectable rotation under standard conditions but it also failed to show any when the concentration of the cystine and the acidity of the solution were both varied. Change in temperature also had no effect. When we consider the optical activity figures certain discrepancies are seen. The values for the observed rotation of the solutions obtained from the laevorotatory samples indicate constant saturation with the laevo phase, although the steady upward drift of these values suggests a slight increase in solubility as the proportion of the inactive phase increases. However, if we similarly credit the dextro phase with being the cause of the rotation obtained from solutions of the +x.zo0 lot, it is evident that while solubility figures suggest that the dextro phase is four times as soluble as the laevo, the observed rotations are respectively about-0.13~ and + 0 . 2 IO. I n the case of the inactive sample the dextro rotation observed could be explained by the difference in solubilities between the d- and 1- phase, the +O.IZ' value being regarded as the algebraic sum of the rotation of the saturated d- and 1- solutions. Assuming equal rotatory effects from equal
SOLUBILITIES O F OPTICAL ISOMERS OF CYSTINE
1039
concentrations of each, this would argue a rotation of a water solution of dThe dextro sample actually gave a form alone as about f o . 2 joor +0.26'. value of +0.21'. The artificial mixture of [(r]D = - 1 1 z . 5 ~ shows about the same positive rotation as the inactive sample for the same reason. The specific rotations of these isoelectric solutions are also of interest. The activities of the solutions in Table I1 are expressed in terms of observed rotation. I n calculating the specific rotation only the concentration of the active phase and not the total solubility of the sample should be considered. By using the solubility figures for 1- and d- cystine obtained above, it is found that 1- cystine has a specific rotation of -433 and d- of 164. X o explanation can a t present be offered as to why these two antipodes differ in specific rotation. It can only be pointed out that the rotation of cystine solutions has been shown to be highly influenced by the presence of other substances and this influence may not necessarily be identical on the d- and 1- forms. Such an influence may be exerted by the inactive phases. The above [ a ] D value for 1- cystine is, a t this high dilution, approximately what one would expect from previously published curves of cystine rotations.'" The conclusion that the two enantiomorphs may have different solubilities is obviously highly unorthodox. While the possibility of some other explanation for the above results cannot be excluded it seems justifiable to expect much more experimental proof than exists a t present in the literature before identical solubilities of such substances be regarded as a foregone conclusion. Of the many optical isomers known, exact solubilities have been determined for but a startlingly small number. Of these there are as many differences as identities recorded and the few identities concern substances of comparatively high solubilities such that slight diff erences-and one would expect such differences to be slight, if they exist a t all-would be easily lost sight of. summary The solubility of isoelectric cystine in water at 25'C was determined, using cystine samples of different specific rotations. The existence is indicated of the following optical isomers of cystine: laevo, dextro, meso and racemic. A solubility figure for each phase was calculated. The results suggest that the d- form may be about four times as soluble as the 1-form.
+
Bibliography W.F. Hoffman and R. A. Gortner: J. Am. Chem. Soc., 44,341 (1922). C.Xeuberg and P. Meyer: 2.zhysiol. Chern., 44,504 (1905). a
K. 8. H. Morner: Z. physiol. hem., 28,595 (1899).
* J. C. Andrews: J. Biol. Chem. 65,147 (1925). A. T.Merrill: J. Am. Chern. Sbc., 63,2686 (1921).
6
