THE OXIDATION OF CYSTINE IN NON-AQUEOUS MEDIA

I. THE SOLUBILITY AND STABILITY OF CYSTINE IN NON-AQUEOUS ACID-BASE SYSTEMS

BY GERRIT TOENNIES AND THEODORE F. LAVINE*

(From the Research Institute of the Lankenau Hospital, Philadelphia)

(Received for publication, January 7, 1933)

The biological investigations of Hammett and collaborators (l-4) from this Institute, indicating a functional antagonism between the -SH group and its oxidation products, stimulated the present studies directed toward cystine derivatives intermediate between cystine and cysteic acid. Of the formally possible sulfur deriva- tives (Table I) only three, cysteine (I), cystine (II), and cysteic acid (X), are known.

In considering the possibilities of experimental approach to the other hypothetical derivatives of I-cystine, preparation of the sulfinic acid over the sulfonyl chloride did not appear very feasible.

Interception of the intermediate acids in an aqueous oxidation offers little promise since it is known that in compounds of the cystine type the combination of hydrolysis and oxidation equilibria leads to the sulfonic acid as the final product (5-13). On the other hand, if it were possible to effect oxidation without hydrolysis of the -S-S- linkage, this should lead into the series of “anhy- dride” oxidation products. Rather than render the cystine mole- cule more soluble by acylation or esterification, it was considered preferable to use cystine as a salt in a non-hydrolyzing medium. If this condition could be realized, perbeneoic acid might be a suitable oxidizing agent. To see whether this peroxide has an action on disulfide sulfur similar to its action on monosulfides investigated by Lewin (14-17), the oxidation of dithiodiglycolic acid in chloroform solution was attempted.

Oxidation of Dithiodiglycolic Acid by Perbenxoic Acid-10 mM of

* Daniel Wentz Fellow of the Lankenau Hospital Research Institute. 463

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464 Non-Aqueous Oxidation of Cystine

dithiodiglycolic acid (1.82 gm.) were dissolved in 200 cc. of chloro- form and 25 cc. of absolute alcohol and at 0” 20 mM of perbensoic acid in 70 cc. of chloroform (solution of perbenzoic acid prepared according to Lewin (16)) were added. Samples were taken at intervals and the peroxide concentration was determined by titrat- ing the iodine liberated from KI solution (Lewin (14)) with the following results.

Time, min.. . . . . . . . . . (0) 33 63 93 123 153 O2 consumed per mol

disulfide, gm.-atoms.. . . . (0.00) 1.33 1.63 1.86 1.93 1.95

TABLE I

Sulfur Derivatives of Cystine (R = HOOC-CH(NHJ--CHz--) Arranged According to Level of Oxidation

Acids I Anhydrides

Symmetrical

R-S H (I) R-S -S -R (II)

R-SO H (III) R-SO -SO-R (V)

R-SOzH (VII) R-SOpS02-R (IX)

R-SOsH (X)

Asymmetrical

R-S -SO-R (IV) R-S -SOY-R (VI) R-SO-SOz-R (VIII)

After 1 day the nitroprusside test for -S-S- was negative. Isolation of the oxidation product was abandoned on account of the difficulties presented by the similarities of benzoic acid and the other reaction product, both being acids and both being soluble in organic solvents.

Proceeding to investigate the solubility of salts of cystine cation in non-aqueous media, we found that cystine dissolves easily in methyl alcoholic solutions of HCl and that the optical rotation of such a solution is nearly identical with that of an aqueous HCI solution of equal concentration.1 A rather rapid decrease, how- ever, of the rotation that follows an exponential curve when plotted against time indicates that, even at low temperatures, cystine

1 This confirms an earlier impression (18) that the state of ionization rather than the solvent is the predominating factor in the optical rotation of the cystine molecule.

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G. Toennies and T. F. Lavine 465

undergoes esterification in the alcoholic solution. Besides, it was observed that perbenzoic acid oxidizes the HCl to CIZ in the alco- holic solution.

Wystine in Methyl Alcoholic HCZ-0.1006 gm. of cystine (0.42 mM) was dissolved in 10 cc. of freshly prepared 1 N HCl (10 InM)

in methyl alcohol. The following optical rotations, calculated as specific rotations for the amount of cystine used, were obtained at 25-27’.

Time, hrs.. . . . . . . . . . , 0 (3 0.67 1 2 3.25

Iffl Hg, degrees.. . . . . . . -245 -225 -215 -193 -171

Time, hrs.. . . . . . 9 27.5 29.5 42.5 48.5 67

lffl Hg, degrees. , . . -101 -30.2 -26.8 -20.5 -17.6 -18.9

Perbenzoic Acid, Methyl Alcohol, and HC&(a) 5 cc. of 0.285 M

perbenzoic acid (1.425 InM) in chloroform combined with 25 cc. of methyl alcohol, kept at 0” for 24 hours, consumed after addition of 30 cc. of an aqueous 20 per cent KKI (36 mM) solution 28.30 cc. of NaZSzOs solution; whereas, 5 cc. of the same perbenzoic acid solu- tion (without alcohol) used 28.38 cc. of NazSz03. (b) 10 cc. of 0.252 M perbenzoic acid (2.52 mM) in chloroform and 3 cc. of 1 N

methyl alcoholic HCI (3 mM) were made up to 25 cc. with methyl alcohol and kept at -15”. 5 cc. samples consumed immediately, 29.4 cc. ; after 19 hours, 27.0 cc. ; after 44 hours, 22.9 cc. ; after 68 hours, 19.1 cc. of NazSz03. After the 18 hour period the escape of CL from the flask was noticed by the odor,

In the search for an acid-solvent system that would not be sub- ject to oxidation or esterification, the following strong organic acids in methyl alcoholic solution were tried but found to be incapable of dissolving significant amounts of cystine: picric acid (I), o- nitrobenzenesulfonic acid (II), 2-nitrobromobenzene-4-sulfonic acid (III), and 2,4-dinitro-1-naphthol-7-sulfonic acid (IV). After standing overnight with the solutions of (II), (III), and (IV) some cystine had dissolved, apparently due to a secondary reaction (esterification?).

A separate paper (19) will deal with some observations relating to cystine and picric acid. The system perchloric acid in glacial acetic acid (20,21) was studied next.

bCystine in Acetic Acid Solutions of Perchloric Acid-68 per cent

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Non-Aqueous Oxidation of Cystine

aqueous perchloric acid and acetic anhydride equivalent to the water of the HC104 solution are cooled to below 0’ and carefully, with strong cooling, combined. When the reaction is over, the solution is made up to the desired concentration with glacial acetic acid. To 0.417 mM of cystine (100 mg.) 10 mM of IIC104 in acetic acid (6.66 cc., 1.5 N) were slowly added; the solution was made up to 10 cc. with acetic acid. lcy]ng, calculated for cystine, = +19”; for cysteic acid, + 13.5”. After dilution of the solution to 10 times its volume with aqueous 1 N HCl, [o~]n~, calculated for cystine, = +20”; for cysteic acid, + 14”.

In order to effect solution at lower temperature, without freezing of the medium, an excess of acetic anhydride was used and finally dilution with acetone. To an ice-cooled solution of 40 mM of HCIOI in acetic acid and acetic anhydride (16 cc. of 2.5 N HC104 in acetic acid + 4 cc. of 90 per cent acetic anhydride) cystine was added in small portions. As the dissolving reaction slowed down, the temperature was allowed to go up. As soon as a turbidity began to form (to be distinguished from excess cystine) the mixture was filtered through a dry asbestos filter. After addition of 12 cc. of 90 per cent acetic anhydride to the filtrate, the mixture was left in the refrigerator for 1 or 2 days, until the precipitate had settled. The supernatant liquid was decanted and the precipitate was washed by decantation with acetic acid and ether. The yield was 1.47 gm. About 2.1 gm. (8.75 ITIM) of cystine had been used. Yield, calculated as cysteic acid, 49.5 per cent.

C3H7NS06. Calculated. N 8.28, S 18.93 Found. “ (Van Slyke amino, with cystine coefficient)

8.12, 8.04, S (Parr oxygen bomb) 18.89

With strong cooling 0.417 mM of cystine (100 mg.) were dis- solved in a 1.5 N solution of HC104 in acetic acid that had been diluted to twice its volume with acetone. About 6 mM of HClOh (8 cc.) were required. After the mixture was made up to 10 cc. with 1.5 N HC104 in acetic acid, [arIng = - 210’. The solution was deeply colored and soon became too dark for further readings.

These experiments seem to indicate that the primary reaction is solution of the cystine by simple salt formation, but that more or less rapidly, according to concentration and temperature, a second reaction takes place in which at least half of the cystine is con-

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G. Toennies and T. F. Lavine

verted into cysteic acid by the oxidizing decomposition of per- chloric acid. The possibility of preventing the second reaction by working in another medium and at low temperature, was further studied. Of various solvents tried, highly purified acetonitrile, a hydroxyl-free solvent of relatively high dissociating power (dielec- tric constant 36) showed the most promising results, which led to its use as the sole solvent. Disturbing experiences with aceto- nitrile of insufficient purity led to the development of certain minimum requirements.

Standard of Purity of Acetonitrile. 1. Freedom from H&--The CH&N, predried by anhydrous K&O3 (not CaC12, as this gives rise to HCl in the distillate from Pz05), should be refluxed for 1 hour with an excess of Pz06 and then distilled under protection from humidity.

2. Boiling Point-The boiling point of CH&N was found to be 81.75” f 0.05” at 760 mm. of Hg (reduced t.o O’), with a correction value of 0.046” per 1 mm. of Hg near 760 mm. of Hg. The total boiling range should be within f0.2” of the theoretical boiling point.

3. Neutrality-5 cc. of CH,CN should become yellow, not red, on addition of thymol blue (alkali salt) indicator, and not more than 1 drop of 0.1 N NaOCH3 in CHBOH should be required to turn the solution blue. The presence of HCN or other weak acids can be detected by this test.

4. Miscibility with HzO-On gradual addition of Hz0 to CH&N, up to 5 volumes, no turbidity should appear at any stage. A turbidity indicates the presence of CGH6 (b.p. 80.4”).

5. Reducing Power-When 1 drop of 0.15 per cent aqueous KMn04 solution is added to 5 cc. of CH&N, the pink color should be definitely present after 1 hour. Also, when 5 cc. of CHSCN, after addition of 3 drops of 70 per cent aqueous HC104 solution, are heated to the boiling point and allowed to cool, no color whatso- ever should appear. Insufficient purity is indicated by formation of a faint pink tinge.

l-Cyst&e in Acetonitrile Solutions of Perchloric Acid-The cal- culated amounts, accurately weighed out, of aqueous HClOh and of acetic anhydride, both of the highest commercial concentrations, at least 68 and 90 per cent respectively, are separately dissolved in acetonitrile. With careful cooling the solutions are combined and

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468 Non-Aqueous Oxidation of Cystine

made up to the required concentration with acetonitrile. The solution is kept at 0’.

A 0.4 N solution of HCIO, in CH&N (containing about 13.7 gm. of acetic acid per 100 cc.) was diluted, 18 hours after it had been prepared, with CH&N, to give solutions 0.3 N, 0.2 N, and 0.1 N in HC104. Each solution was shaken with excess cystine for 10 minutes at 25”. The results of polarimetric readings, together with approximate values for the specific rotations of cystine in the four acid concentrations, obtained on solutions of weighed amounts (approximately 100 mg. per 10 cc.), are given in Table II.

TABLE II

Cystine and Perchloric Acid in Acetonitrile

HCIOh M., . . . . . . . .

4, of saturated solu-

tions, 1 dm. tube, degrees..............

Iffl&, 0.1 gm. cystine per 10 cc., degrees.. .

Calculated concentra- tion of saturated solution of cystine, M . . . . . . . . . . . .

Molar ratio, HClOh: cystine.. . . . . . . . .

* Extrapolated value.

7-

0.10

-3.40

-298

0.0475

2.11

0.20 0.30

-6.39 -9.28

-284 -268

0.094 0.144

2.13 2.08

-

t

-

0.40

-12.38

I--254)*

0.2035

1.97

When the saturated solutions had stood for 7 days at room tem- perature (above 30”) with excess cystine, no decrease in rotation and no trace of discoloration had occurred. Full stoichiometrical equivalence between cystine and HCIO, was obtained when cystine was dissolved in the acetonitrile solution of hydrated HClO, before addition of acetic anhydride (see below). By the addition of an organic base, such as pyridine, the cystine, essentially unchanged, is reprecipitated.

Reprecipitation of I-Cystine from Acetonitrile Solution-A solution of cystine perchlorate in acetonitrile, prepared by saturating the CH&N solution of hydrated HCIOI with cystine and subsequently adding the calculated amount of acetic anhydride, was used. Ac- cording to the amount of cystine dissolved, the concentration was

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G. Toennies and T. F. Lavine 469

2.4105 gm. of oystine per 100 co. ; [CX& = -311’. After 27 days standing at room temperature [a]g, = -303”, a decrease of 2.5 per cent. Now to a weighed amount of the solution, correspond- ing to 2.031 gm. (8.46 mM) of cystine, according to the original concentration, 2.22 cc. (28 mM) of pyridine were added. After 2 hours the precipitate was filtered, washed with CHsCN, and dried. The resulting amorphous gel-like mass had a strong pyridine odor; it was carefully pulverized and washed again with CH&N. Now a pure whit,e powder resulted on drying. Drying at 115’ in vacua was continued up to 11 hours as the odor of pyridine persisted and weight continued to be lost. When evidence of beginning decom- position (discoloration, formation of H,S) appeared, drying was discontinued. Total weight, 2.096 gm. [(Y]%~ (1 gm. per 100 cc. with 1 N HCI) found, -228.5”; cystine used, -241.9”. S (oxida- tion by alkaline KMnOJ found, 24.98, 25.05 per cent; calculated, 26.67 per cent. Difference in [cY], 5.5 per cent; difference in S, 6.3 per cent; average difference, 5.9 per cent. Recovery, cor- rected for pure cystine, 2.096 gm. -5.9 per cent correction = 1.972 gm. Starting material, 2.031 gm., or if corrected according to 2.5 per cent decrease of original rotation, 2.031 gm. -2.5 per cent correction = 1.980 gm. The recovered substance was re- precipitated by neutralization with NH, from HCl solution and gave [a]& = -237.3”, corresponding to a difference from the starting material of 1.9 per cent.

These observations show that here is a system that dissolves more cystine per mol of acid than any dilute aqueous acid solution does. A study of the physicoohemical literature on the newer developments in the conception of acids and bases (22, 23) gives a better understanding of these findings. If we compare a saturated solution of cystine, on the one hand in an aqueous “strong” acid and on the other hand in the strong acid HC104 in acetonitrile, it appears that in the aqueous solution less cystine is dissolved because of the basicity of Hz0 which causes practically complete ionization of the strong acid.

ClOlH + Hz0 -+ ClOJ- + H30+ (1)

Thus the acid HClO, is replaced by the acid H30+. The isoelectric solubility of oystine may be represented, in ac-

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470 Non-Aqueous Oxidation of Cystine

cordance with the Zwitter Ion conception of amino acids estab- lished by Bjerrum (24) by the equilibrium

+H&J coo- Cystine solid e R (2)

+HSN coo-

The extent to which cystine will be dissolved by an aqueous acid then depends essentially on the further reaction of the Zwitter Ion with &O+.

+H,N coo- +HaN COOH R f HO+ S R + Hz0 (3)

+HaN COO- +HsN coo-

+HJV COOH +HJ9 COOH R + H,O+ $ R + He0 (4)

+H,N coo- +HaN COOH

The extent to which the reactions of Equations 3 and 4 will proceed is determined by the relation between the proton-binding powers (i.e. basicity, according to Bronsted’s definition (23)) of Hz0 on the one hand and

+HaN coo- +H3N COOH R and R

+HsN coo- +H,N coo-

on the other. The solubility of cystine in aqueous acids then is practically independent of the individual acid used as long as we are dealing with one of the “strong” acids; i.e., those which are almost completely converted into HaO+ as represented by Equa- tion 1.

Now finding that in the non-aqueous HC104 solution an amount of cystine equivalent to the HClOd is dissolved, we may conclude that the acidity of HClO., is so much higher than that of H30+ and the basicity of CH&N is so much lower than that of HZO, that in effect a complete proton transfer takes place from perchloric acid to cystine,

+EI,IT coo- +HsN COOH R + 2HC104 + R + 2 ClO,- (5)

+JW coo- +HaN COOH

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G. Toennies and T. F. Lavine 471

neither HC104 nor CH&N-H+ being stable in the presence of basic cystine ions.

While according to the above experiments saturated solutions are quite stable, it was noticed that an excess of free HClOd causes a decomposition to take place. These observations are summarized in Table III.

The decomposition appears to be similar to that observed in the case of acetic acid solutions, with the difference that in the latter oxidation seems to take place more rapidly and begins before complete neutralization of the HClOd by cystine can be attained.

Stability of LCystine in Presence of Excess of Hydrated Perchloric Acid-A solution 0.05 M in cystine, 0.2 M in HClOJ, and 0.53 M

in HzO, gave, in a 1 dm. tube, &, = - 13.63” and after 11 days

TABLE III

Cystine in Presence of Excess HClOd

Total HC104

N

0.1 0.1 0.2 0.3

0.4

Cystine Excess HClOd Stability of solution

M N

0.042 0.016 Stable 0.0375 0.025 15 days at 30”; OL unchanged 0.042 0.116 30 hrs.; half value of initial CY 0.042 0.216 Turns brown, precipitates within

15 min. 0.042 0.316 Turns brown, precipitates imme-

diately

25 = -13660 -g . . No acetic anhydride had been added. The ex- cess of perchloric acid, while leading to decomposition when it is anhydrous, does not affect the stability of the cystine solution as long as it is present in combination with water as in the commercial 70 per cent HClO, (about 1HC104 + 23H20).

Solubility of &Cystine in Hydrated Perchloric Acid-I.0 cc. of a freshly prepared solution of aqueous HClO( in CH&N, 0.500 M in HClOd and 1.3 M in HzO, were saturated with cystine by shaking for 18 hours. By weighing the excess cystine, 99.5 per cent of the equivalent amount was found to have dissolved. Similarly, the saturation of 200 cc. of a solution 0.400 M in HC104 and 1.06 M in Hz0 consumed 100.3 per cent of the equivalent amount. The presence of 2.5 mols of Hz0 per mol of HCIOl does not prevent

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472 Non-Aqueous Oxidation of Cystine

completion of the dissolving reaction (Equation 5) between HCIO, and cystine.

The picture then is that 23H20 per molecule of HC104prevent the oxidizing action of HCIO, without preventing quantitative neutralization of cystine. We may assume the following equi- libria2 in our solutions.

2HC104 + CLOT + Hz0 (6)

HClOa + Hz0 S H,O+ + Clod- (7)

2HC104 + R(NHS)2(COO-)z = R(NHS)z(COOH)z + 2C104- (8)

With small Hz0 concentrations, the basicity of Hz0 then appears negligible compared with that of cystine, and HCIOI practically disappears from the reactions (Equations 6 and 7) in the presence of equivalent cystine. In the case of excess HCIOI, it depends on the Hz0 concentration how far the concentration of free HClO, will be reduced by the reaction indicated in Equation 7, which in turn de- termines the possible C&O7 concentration according to Equation 6.

Instability of Anhydrous Acetonitrile Solutions of Perchloric Acid-A summary of the solubilities of I-cystine in various 0.1 N

HClOd solutions in CH&N, made anhydrous by addition of acetic anhydride, as was described above, at various periods after the solutions had been prepared, is as follows :

Age of solution at O”, days.. . 0 1 1 2 2 3 3 10 12 13 Per cent of theoretical amount

of cystine dissolved. . . . . . . 100 95 83 83 81 79 75 75 79 59

The solutions were shaken, either for 3 to 1 hour by hand or for 24 hours and longer by machine. In no case was the dissolved amount increased by more than 1 per cent by shaking longer than 1 hour. The solutions showed, on standing, a gradually increasing yellow color that disappeared on addition of some HzO. In the last case where only 59 per cent of the expected amount of cystine went into solution, a precipitate containing cysteic acid formed immediately after the cystine had been dissolved, similar to the reaction in the case of HCIOl in acetic acid. This reaction, together with knowledge gained later about the reaction between acetonitrile and water in the presence of acid, points to the follow-

s Regarding equilibrium (Equation 6) see Ephraim (25).

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G. Toennies and T. F. Lavine 473

ing explanation. As Hz0 disappears from the equilibrium indicated by Equation 7, due to its reaction with acetic anhydride, HClO( will dissociate according to Equation 6 with the formation of C&O~, causing the reversible yellow color of the solutions, and of H30+ by Equation 7. This causes a catalytic hydration of aceto- nitrile to ammonium acetate and neutralization of the ammonium acetate formed by HCIOI

CH,CN + 2H,O+ + 2C104- - CH3COONHa + 2HClO4 (9)

CH,COO- + NH,+ + HClOd - CH,COOH + NHd+ + ClOa- (10)

the net result (Equation 6 to Equation 10) being

5HClO., + CH,CN - 2C120, + CHaCOOH + NHd+ + Clod- (11)

i.e., decreased solubility of cystine caused by the disappearance of HClOd and increasing tendency toward oxidative decomposition caused by the accumulation of C12O7.3

Stability of Dehydrated Acetonitrile Solution of I-Cystine Per- chlorate-To 200 cc. of an acetonitrile solution 0.400 N in HCIOl and 1.06 M in HzO, and containing 100.3 per cent of the equivalent amount of cystine (cf. p. 471), the calculated amount of 90 per cent acetic anhydride was added, and the solution was made up to 400 cc. (0.200 N) at 25”. [a]$” = -311”; after 1 day [cr]gg = -310’; after 27 days, -303’; after 49 days, -300’. The solution had gradually acquired a very slight discoloration. Thus on adding the acetic anhydride after saturating with cystine, a comparatively stable solution results; the equilibrium shown by Equation 8 by being displaced to the right almost completely suppresses the reaction of Equation 6.

Decrease with Time of Xolubility of Wystine in Acetonitrile Xolu- tions of Hydrated Perchloric Acid-An acetonitrile solution, 0.500 N in HC104 and 1.3 M in Hz0 (cf. p. 471) dissolved, immediately after preparation, 99.5 per cent of the equivalent amount of cystine. After 4 days another portion of the same HClO, solution dissolved only 87.5 per cent, while after 8 days the dissolving capacity had decreased to 81.5 per cent. There was no discoloration or sub- sequent decomposition in this case. The decreasing dissolving

3 Just as in the case of HzSOd accumulation of the anhydride S03increases the oxidizing tendency.

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474 Non-Aqueous Oxidation of Cystine

power for cystine in this case,i.e. in the absence of acetic anhydride, is due to the reactions of Equations 9 and 10 alone, as will be shown in Paper II of this series.

Reaction between Water and Acetic Anhydride-when and if our solution is a truly anhydrous one depends on the velocity of the irreversible (26, 27) reaction between acetic anhydride and water. It was noticed that if in the preparation of the mixture an excess of acetic anhydride was used, a pink to brown color was formed accompanied by a drop in the optical rotation. With an excess of 25 per cent the rotation had decreased 10 per cent after 1 day. This reaction, which might involve acetylation and racemization (28), is mentioned merely as indirect evidence for a high degree of completeness of the anhydride-water reaction in our medium; if any considerable anhydride persisted, its reaction with cystine itself ought to become apparent by a change of color and optical rotation. A rapid rate of the acetic anhydride reaction seems likely according to the work of Orton and Jones (26) who found in the presence of a strong acid, an increase in the velocity constant of the hydration in glacial acetic acid of the order of 1000 times. In the case of a slow reaction rate the progress of the hydration might reveal itself by a change in the optical rotation or its tem- perature coefficient. The following experiment shows that no definite change was detectable over a period of 4 weeks. Similar evidence was obtained from a dilatometric experiment.

Polarimetry of LCystine Perchlorate-Acetonitrile Solution-An acetonitrile solution whose initial composition was cystine, 0.0452 M; HClO+ 0.0938 M; HzO, 0.247 M; (CH,CO),O, 0.248 M;

CH&OOH, 0.0467 M was used. Table IV gives a summary of the polarimetric results, with average errors. The readings (for technique cf. (29)) extend over the interval of 20-30’. The first readings were taken 1 hour aft,er removing the excess cystine which was 20 hours after the components were combined.

Evidently there is no change in the temperature coefficient, beyond the limits of experimental accuracy, and no definite trend in the rotation values. The combined averages give [a]$ = -317” =t lo for the specific rotation and d[a]&dt = +2.3” f 0.2” for the temperature coefficient.

Dilatometry of l-Cystine Perchlorate-Acetonitrile Solution-An acetonitrile solution approximately 0.1 M in HClO+ 0.05 M in

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G. Toennies and T. F. Lavine 475

cystine, and 0.26 M in (CH&O),O was kept at 32.5” =t 0.005” in a 50 cc. dilatometer with a stem cross-section of 0.00238 sq. cm. The meniscus readings, reduced to 32.5”, are listed in Table V. The time is counted from the addition of the acetic anhydride.

Calculated by the contraction of the hydration of acetic anhy- dride in water determined by Kilpatrick (27), the total contraction in our experiment would correspond to 16 cm. on the dilatometer scale. Apparently after 1 hour the reaction is practically over. The final proof for the completeness and rapidity of this reaction

TABLE IV

Optical Rotation of Cystine Perchlorate in Acetonitrile

Time

0.04 1 3 7

13 26

No. of determina- tion groups at

separate tempera- ture levels

(cf. (29))

Temperature coefficient, da&at

0.052 f 0.004 0.050 f 0.006 0.047 f 0.007 0.051 f 0.001 0.049 f 0.007 0.050 i 0.002

TABLE V

Dilatometer Readings

Rotation at 25” az5 ’ Hg

-6.90 f 0.01 -6.85 f 0.005 -6.865 f 0.005 -6.89 f 0.005

-6.915 f 0.005

Time, min ............. 52 54 56 58 60 62 64 66 72 Meniscus, mm.. ........ 290 286 281.8278.4274.5272.5273.3272.1271.5

Time, hrs .............. 17 20 21 24 41 47 65 67 Meniscus, mm. ......... 271.2271.8271.9272.2272.1271.8271.6272.1

indicating at least 99 per cent completeness within the 1st hour was furnished by a titrimetric method, which will be described in Paper II of this series.

The cooperation and encouragement received from Dr. Stanley P. Reimann, Director of the Institute, as well as from fellow members are gratefully acknowledged. Credit is due Miss Mar- garet Elliott for her active assistance in various phases of the experimental work. It is a pleasure to express our appreciation

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476 Non-Aqueous Oxidation of Cystine

for the material aid given by Merck and Company, Inc., Rahway, New Jersey, who supplied I-cystine and acetonitrile of highest purity specially prepared for this work.

SUMMARY

In order to find a medium for the oxidation of cystine leading to compounds intermediate between cystine and cysteic acid the solubility of some cystine salts in non-aqueous solvents was studied. The main observations are:

1. Cystine is soluble in methyl alcoholic HCI but undergoes spontaneous esterification therein.

2. Cystine is soluble in solutions of perchloric acid in acetic acid, but undergoes spontaneous oxidation, leading to cysteic acid, in the solution.

3. Cystine is soluble, in equivalent amount, in solutions of perchloric acid in acetonitrile. These cystine solutions are stable at room temperature.

4. Cystine is oxidized by free HCIOJ while it is stable in the pres- ence of Clod- ion.

5. The acid concentration of a solution of perchloric acid in acetonitrile, dehydrated by the reaction of water with acetic anhydride, decreases on standing. No such decrease takes place when cystine equivalent to the perchloric acid is added before addi- tion of acetic anhydride. A solution of perchloric acid and water in acetonitrile also decreases in acidity. The mechanism of these reactions is discussed.

BIBLIOGRAPHY

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Gerrit Toennies and Theodore F. LavineACID-BASE SYSTEMS

CYSTINE IN NON-AQUEOUS SOLUBILITY AND STABILITY OF

NON-AQUEOUS MEDIA: I. THE THE OXIDATION OF CYSTINE IN

1933, 100:463-477.J. Biol. Chem. 

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