200 RATNERAND H.T.

CLARKE
SARAH Vol. 59
[CONl'RIBUTION FROM TRIE D E P ~ T H E OF
N TBIOLOGICAL
CIiEMISTRY, COLUMBIA UNkVERSITY]

The Action of Formaldehyde upon Cysteine

BY SARAH AND H. T. CLARKE
RATNER~

The reaction of mercaptans with carbonyl com- Meyer6-a result characteristic of secondary, in
pounds has received extensive study, but little contrast to primary, a-amino acids.
attention has, until recently, been paid to the case The sulfur is present as a thio ether, for the
of cysteine. In a study of the effect of formalde- nitroprusside reaction is negative; oxidation of
hyde on the titration curves of amino acids, Birch the acetyl derivative by hydrogen peroxide leads,
and HarrisZ observed, on the addition of formal- according to the conditions, to a sulfoxide or a
dehyde to cysteine, the disappearance of the buf- sulfone. The unsubstituted acid, on treatment
fering action typical of the sulfhydryl group. with hydrogen peroxide or iodine, is converted,
Shinohara* in an investigation of the action of with loss of formaldehyde, into cystine; with
cysteine upon phosphotungstic acid, noted that bromine water, cysteic acid is produced.
the reducing power is inhibited completely by ad- Among the compounds described by Schubert'
dition of formaldehyde. During the progress of some are formed by addition and others by con-
the present investigation, Schubert4has described densation. It therefore seems probable that the
the formation of compounds of cysteine with reaction of cysteine with formaldehyde occurs in
various aldehydes, among them formaldehyde, two steps, firstly, addition of the aldehyde, and
and has produced evidence that the latter reacts secondly, ring closure by removal of a molecule of
with cysteine to form a thiazolidinecarboxylic water. The formaldehyde may, theoretically,
acid. The present report covers a more extended react primarily with either the sulfhydryl or the
investigation of this compound, its reactions, and amino group of cysteine. A decision betweefi
its manner of formation. these alternative possibilities can be reached by
The reaction between formaldehyde and cys- noting the ease of reaction of formaldehyde with
teine can take place over a wide range of hydro- acetylcysteine on the one hand, and S-ethylcys-
gen ion activity. The product, which is remark- teine on the other. Experiment shows that the
ably stable toward both acid and alkali, possesses rotation of S-ethylcysteine a t PH 5.12' is not
amphoteric properties, as is shown by the titra- changed by the presence of formaldehyde, while
tion curve (Fig. 3). The empirical formula, that of acetylcysteine is. At this PH, the rate of
CIH~O~NS, and the conditions of formation indi- change is extremely rapid, both with cysteine
cate that the compound is formed from equimo- and with acetylcysteine, but can be measured at
lecular quantities of cysteine and formaldehyde; higher hydrogen ion concentrations @I3 14).
its simple molecular character is indicated by the With cysteine (Fig. 1) the rotation reaches the
observed molecular weight of the methyl ester. theoretical value; with acetylcysteine (Fig. 2), on
The nitrogen exists in the form of a secondary the other hand, the levo-rotation is distinctly
amino group, as is shown by the formation of an lower than that observed for pure acetylthiazoli-
acetyl derivative and by the behavior with ai- dinecarboxylic acid under the same conditions.
trous acid in the Van Slyke procedure.s The This may be interpreted as indicating the forma-
product, moreover, displays no tendency to race- tion of a less strongly levorotatory addition com-
mize when its sodium salt is treated with acetic pound of the type RSCHaOH. By analogy, the
anhydride by the method of du Vigneaud and first stage in the reaction between cysteine and
(1) This report is from a dissertation submitted by Sarah Ratner formaldehyde may be regarded as taking place
in partial fulfilment of the requirements for the degree of Doctor of at the sulfur atom.
Philosophy in the Faculty of Pure Science of Columbia University.
(2) Birch and Harris, Biochem. J . , 24,1080 (1930). In determining the rates of the reaction at dif-
(3) Shinohara, J . B i d . Chem., 110,263 (1935). ferent PH levels, advantage was taken of the small
(4) Schubert, ibid., 111,671 (1935);114,341(1936).
(5) In this, 20 to 30%, according to the conditions, of the equiva- optical rotation of cysteine and the conveniently
lent amount of nitrogen is set free, but this anomaly may be ascribed, large one of thiazolidinecarboxylic acid by follow-
as in the case of cystine [Laugh and Lewis, ibid., 104, 601 (1934)],to
oxidative side reactions; under suitable conditions as much as 10% (6) Du Vigneaud and Meyer, ibid., 98, 295 (1032); 99, 143 (1932).
of the sulfur is converted into sulfuric acid by the reaction of nitrous (7) Formaldehyde does not influence the titration curve at this
acid. point.
Jan,,1937 THEACTIONOF FORMALDEHYDE
UPON CYSTEINE 20 1

ing the change in rotation. Six buffered reaction TABLE I

mixtures between PH 1.9 and $33 12 were investi- DIS~OUTION CONSTA~WS w Ammo A-, T-ZOLIDINE.
CARBOXYLZC ACIDAND R r s w m COXPOUNDS
gated at 25'. The velocity curves (Fig. 2) dis- #Kl #K:
play a measurable rate of reaction which increases 2.61 9.72
rapidly on raising the $H, and above 5 becomes Cysteineb 1.96 8.18
too fast to follow. S-Ethylcysteine 2.03 8.60
Proline' 1.09 10.60
Thiazolidine-4-carboxylic acid 1.51 6.21
Acetylthiazolidine-4-carboxylicacid 2.96 ..
Methyl thiazolidine-4-carboxylate a . 4.00
Thiazolidine .. 6.31
Bjerrum, 2. physik. Chem., 106, 219 (1923). '
Can-
nan and Knight, Biochem. J., 21, 1384 (1927). e McCay
and Schmidt, J . Gen. Physlol., 9,336 (1926).

basic dissociation, of the inhibition of the ionizing

function of one group (Figs. 5 and 6).

0 40 80 120 160 200 240

Minutes.
Fig. 1.-Rate of thiazolidinecarboxylicacid formation.

Thiazolidinecarboxylic acid behaves as an

ampholyte (Fig. 3) ; the acidic dissociation ($XI
1.51) is strong, but the basic didsociation (@&
6.21) extremely weak in comparison with those
of a-amino acids in general, including proline
(Table I).

0.8 0.4 0 0.4 0.8

NaOH C- Equivalents --3 HCI.
Fig. 3.-Titration of thlamlidinecar-
boxylic acid 0-0 in wates and 0-0 in 18%
formaldehyde.

In the presence of a large amatnt of formalde-

hyde, the basic dissociation of thiazolidinecar-
boxylic acid is decreased (fig, 3) but to a smaller
extent thatl in proline and in S-ethylcysteine
0 20 40 60 80 100 120 140 (Fig. 4). The presence of the -CHzS- group in
Minutes.
the thiazolidine ring presumably exerts approxi-
Fig. 2.-Rate of reaction of acetylcysteine and for-
maldehyde.
mately the same effect on the basic dissociation
constant as the introduction of a single hydroxy-
Replacement of the labile hydrogen atoms in methyl group into a primary amino acid. The
thittzolidinecarboxylic acid produces changes in effectof formaldehyde upon the basic dissociation
dissociation constants which are similaa in direc- of thiazolidinecarboxylic acid, namely, depres-
tion to those which accompany similar substitu- sion by about one @K unit, is probably much the
tions in other amino acids.8 The constants for same as that of the second hydroxymethyl group
the methyl ester and the acetyl derivative demon- in a compound of two molecules of formaldehyde
strate the weakening effect, on both acidic and with one of a primary amino acidag
(8) Cohn, Ergebnisse Physiol , 88, 781 (1931). (9) Levy, J B i d Chem , 79, 767 (1933).
202 SARAH
RATNERAND H. T. CLARKE VOl. 59

Acetylation or esterification of thiazolidinecar- removal of formaldehyde. A solution of thiazoli-

boxylic acid has no tendency to open the ring, dinecarboxylic acid in N hydrochloric acid was
and the corresponding derivatives are obtained in heated in a sealed tube at 100' for twenty-four
excellent yields. hours; the rotation remained essentially un-
changed. On the other hand, hydrolysis was de-
tected by distilling the solution a t constant vol-
ume. Formaldehyde was found in the distillate
in amounts roughly equivalent to the cysteine
produced.
Oxidation of the sulfur atom exerts a progres-
sive effect in increasing carboxyl dissociation.
Acetylthiazolidinecarboxylic acid has a pK of
2.96, its sulfoxide 2.50 and its sulfone 2.23 (Fig. 6).
10

0.8 0.4 0 0.4 0.8

NaOH f- Equivalents +HCl.
Fig. 4.-Titrationof S-ethylcysteine0-0 6
in water and 0-0 in 18% formaldehyde.
Fi
Hydrolytic cleavage in N hydrochloric acid oc-
curs only to a very small extent, and can be de-
tected only when equilibrium is disturbed, as by 4

0.8 0.4 0
Equivalents of NaOH.
Fig. 6.-Titration of 0-0
6 acetylthiazolidinecarboxylicacid,
ti 0-0 acetylthiazolidmecarboxylic
acid sulfoxide and 0-0 acetyl-
thiazolidinecarboxylic acid sul-
fone.
4
With hot alkaline plumbite in an atmosphere of
nitrogen, lead sulfide is formed from thiazolidine-
carboxylic acid, but more slowly than from cys-
teine.1° The first evidence of its formation was
2I t 4 I I I I I I I
1
rfI
noticed after twenty-five minutes at 95'; after
twenty-four hours 97.5y0 of the theoretical
amount had precipitated.
0 0.4 0.8
Equivalents of HC1. With iodoacetic acid and benzyl chloride, which
Fig.5.-Titration of thiazolidine react with sulfhydryl groups,ll the corresponding
(10) Fruton and Clarke, J. Biol. Ckcm., 106, 667 (1934); Blu-
0-0in water,0-0 in 18%formal-
menthal and Clarke, ibid., 110,343 (1935).
dehyde and @-@ in methyl thiazoli- (11) Dickens, Biochem. J., 37, 1141 (1933); Michaelis and Schu-
dinecarboxylate. bert, J. B i d . Chcm., 106,331 (1934).
Jan., 1937 THEACTIONOF FORMALDEHYDE
UPON CYSTEINE 203

cysteine derivatives are formed from thiazolidine- in crystalline form. The free base is oxidized by
carboxylic acid in alkaline solution (PH 10-11) iodine to di-@-aminoethyldisulfide and by bro-
at room temperature. mine to taurine. The boiling point, 164-165', is
Ring opening may also be demonstrated at pH notably higher than that of thiazole (117'); that
10 by the action of air in the presence of a trace of of thiazoline (138-139') occupies an intermediate
ferric chloride; cystine is formed slowly. Other position.
oxidizing agents bring about ring opening more Experimental
readily: one equivalent of hydrogen peroxide or
Thiazolidme4-carboxylic Acid.-The cysteine hydro-
of iodine produces cystine and formaldehyde al- chloride from 30 g. of cystine was dissolved in 100 cc. of
most quantitatively, With bromine water, cys- water; 22 cc. of commercial 40% formaldehyde (1.1
teic acid is formed, six equivalents of the halogen mole) was added, and the mixture allowed t o stand over-
being consumed. night at room temperature. On the addition of 25 cc. of
pyridine, crystals soon separated. The whole was diluted
Thiazolidinecarboxylic acid reacts with sulfite with 50 cc. of alcohol and filtered. The product (28.2 g.)
apparently to form an equilibrium mixture. The was recrystallized from hot water. The resulting long,
progress of the reaction, which is rapid at PH 5 colorless needles, which melt with decomposition a t 196-
or above, can be measured at PH 4 by following 197", are insoluble in alcohol, somewhat soluble in cold
the change in optical rotation. Sulfhydryl is water, readily soluble in hot water, in acid and in alkali.
formed in the process, for an increasing power to Anal. Calcd. for C I H ~ O ~ N SC, : 36.06; H, 5.26; N,
10.52; S, 24.05. Found: C, 35.93; H, 5.27; N,'* 10.38;
reduce phosphotungstic acid is observed. Thia-
S, 24.09 [ u ] ~ @ D-141" in water; [ a ] 2 ' ~-1OOOin N hy-
7olidinecarboxylic acid alone does not reduce drochloric acid; [a] 2 2 -
~ 203 a in N sodium hydroxide.
phosphotungstic acid at PH levels below 11. The specific rotation was also determined a t a series of
All these observations may be interpreted as PH levels in suitable buffered solutions, the PH values of
indicating the existence of an equilibrium which were determined potentiometrically.
I1
TABLE
I
CH&H
CHNHp + CHtO e YHa->CHz
CH-NH 4-Ha0 PH 1 52 2 25 4 09 6.10 9 88
I I [a]*'D -120 -135 -142 -1i3 -214
COOH COOH
A solution in 10-15% hydrochloric acid, when allowed to
which lies so far to the right that sulfhydryl re- evaporate a t room temperature in WUCUO, deposited the
actions are ordinarily imperceptible. Disturb- hydrochloride as large quadrangular prisms, m. p. 184-
ance of this equilibrium by an irreversible process 185 O with decomposition, readily soluble in alcohol.
displaces it to the left. Anal. Calcd. for C4H702NS.HC1: N, 8.26; C1, 20.91.
Acetylation changes this behavior considerably. Found: N, 8.08; C1, 21.00.
Iodine does not oxidize acetylthiazolidinecarbox- When a concentrated aqueous solution is diluted with
water, the free acid crystallizes out.
ylic acid, and hydrogen peroxide in suitable or- Thiazolidinecarboxylic Acid Methyl Ester Hydrochlo-
ganic solvents yields the corresponding sulfoxide xide.-A solution of 10 g. of thiazolidinecarboxylic acid in
and sulfone, which are stable in aqueous solu- 100 cc. of methanol which had been saturated with dry
tion. The sulfoxide is reduced by hydrogen io- hydrogen chloride was warmed under reflux on a steam-.
dide, with liberation of two equivalents of iodine; bath for one hour. The solution was evaporated to a small
volume i n vacuo, ether was added, and the crystalline
the sulfone is not affected in this way. product chilled, filtered off and dried in wacuo over solid
The parent thiazolidine, prepared for purposes alkali: yield 13 g. (94%). I t was recrystallized by dis-
of comparison by the condensation of &amino- solving in methanol and precipitating with ether, and
ethyl mercaptan with formaldehyde, is a stable appears as shiny plates which decompose a t 164-165 O ; very
liquid of weakly basic character (Fig. 5 and Table soluble in water and alcohol, insoluble in ether.
I), and displays chemical properties analogous to Anal. Calcd. for CsHs02NS.HCl: C1, 19.31; S,17.46;
N, 7.63; CHsO, 16.89. Found: C1, 19.39; S, 17.74; N,
those of the carboxylic acid. Its basic dissocia- 7.60; CHsO, 16.76.
tion is depressed by formaldehyde (Fig. 5 ) to a Methyl Thiazo1idinecarboxylate.-To a suspension of
greater extent than is the case with the carboxylic 13 g. of ester hydrochloride in 3 - 4 cc. of water, covered
acid. With acetic anhydride it yields an acetyl with about 30 cc. of ether, anhydrous potassium carbon-
derivative which is not oxidized by iodine but on ate was added slowly in excess. The ether was decanted
treatment with two moles of hydrogen peroxide (12) As most of the compounds described in this paper gave low
in glacial acetic acid yields a crystalline sulfone; values for nitrogen by the micro-Dumas method, the micro-Kjelllahl
process was employed throughout. Grateful acknowledgment is
the corresponding sulfoxide could not be obtained made to Mr. William Saschek for the analytical results here reported.
204 SARAH RATNERAND H.T. CLARKE Vol. 59

after shaking. Extraction with ether was repeated several Kjeldahl nitrogen determinations and the formaldehyde
times. The combined ethereal solution was dried with stock solution was analyzed by iodine titration.
barium oxide and the ether removed; the residual crude The values plotted in the curves were those of X calcu-
oil was distilled in vacuo; b. p. 75' (1.0 mm.); yield 7.85 g. lated from the formula
(75%). a0 (1 - x)+0r-X = %bad.
Anal. Calcd. for CsHpOzNS: CHaO, 21.08; S, 21.79;
mol. wt., 147. Found: CHaO, 21.14; S,21.82; mol. wt.,
In the experiments with acetylcysteine, the value for am
was based upon the final steady value observed in the reac-
149.2, 146.6 (cryoscopic in CeHs). [ a I z 3-83.0
~ in water;
tion mixture.
[.rlZ1D -106.3 in benzene; [orIz1~ -94.7" in CHaOH; dZ34
Titration Curves (Figs. 3-6).-Except in the formalde-
1.324; nab 1.527.
hyde titrations, 0.1 M solutions were titrated with N
The ester showed no tendency to condense with itself sodium hydroxide and hydrochloric acid. The #I meas-
even on long standing. urements, made with a glass electrode,18 are reliable to
Acetylthiazolidinecarboxylic Acid.-To a suspension of *0.03. The values reported (pKI for the acid constants
9.3 g. of thiazolidinecarboxylic acid in 30 cc. of water, 30
cc. of acetic anhydride was added slowly a t 90" with con-
-
and ~ K =zpKb pK, for the basic constants) each repre-
sent the average of the values calculated from five points in
stant stirring over a twenty-minute period. The clear the curve. In the formaldehyde titrations, 5 cc. quantities
solution was heated for forty minutes longer. Water and of 0.1 M solution were added to 100 cc. of 18% formal-
acetic acid were removed in vacuo, the residue dissolved in dehyde and titrated with 0.1 N sodium hydroxide. A11
30 cc. of hot water. On cooling, crystals appeared as six- curves were corrected for water blanks.
sided prisms which were filtered off after chilling; yield Hydrolysis of Thiazolidinecarboxyliplic Acid.-A solution
10.0 g. (82%), m. p. 143.5-144.5'; very soluble in water, of 0.3021 g. of the acid in 25 cc. of N hydrochloric acid was
somewhat less so in alcohol, acetone and ether. distilled slowly for nine hours with addition of water at the
deal. Calcd. for CeHpOaNS: C, 41.11; H, 5.18; N, approximate rate of distillation. The distillate, which
8.00. Found: C,40.92; H, 5.07; N, 7.85. {a]%-133.5 amounted to 100 cc., was collected under alcoholic dimedon
in water (PH2.1); [u]'OD -159.8Oin an equivalent quan- solution; it yielded 65 mg. of the formaldehyde derivative,
tity of dilute sodium hydroxide. m. p. 187'.
Acetylation of Sodium Salt of Thiazolidinecarboxyac The residual solution was made up to 25 cc.; it gave a
Acid.-Into a 10-cc. volumetric flask was weighed 0.875 g. strong reaction with nitroprusside. The observed rotation
of thiazolidinecarboxylic acid (5 mml.); 5 cc. of N sodium (2 dcm. tube) of the solution before distillation was -2.47';
hydroxide and 3.0 cc. of acetic anhydride (30 mml.) was after distillation - 2 . 0 4 O . The fall in rotation corresponds
added and the volume made up to 10 cc. with water. The to a conversion of 15.3% of the thiazolidinecarboxylic acid
h s k was kept at 37" and 2-cc. samples were withdrawn into cysteine having14 [a]D +7.6".16 The cysteine con-
after 46, 92 and 168 hours. To each sample 12.7 CC. of tent, determined by the Lugg modification10 of the method
N sodium hydroxide was added, followed after three hours of Folin and Marenzi, was 11.0%.
by 6.85 cc. of 2 N sulfuric acid. The solution was taken Reaction with Iodoacetic Acid.-A solution of 0.66 g. of
to dryness in vacuo; the residue was extracted with 20 CC. thiazolidinecarboxylic acid (5 mml.), 0.93 g. of iodoacetic
of hot absolute ethyl alcohol, the alcohol was evaporated acid (5 mml.) and 1.04 g. of potassium carbonate (15
and the residue so obtained recrystallized from a small m. eq.) in 10 cc. of water w a s allowed to stand overnight a t
amount of water. [cx]*~D-133.2Oafter46 hours; -134.4" room temperature. Upon the addition Of 10 CC. of N
after ninety-two hours; -134.4' after one hundred and hydrochloric acid crystals slowly appeared. The yield of
sixty-eight hours. S-carboxymethyl cysteine'l was 0.77 g. (87%) ; thin trape-
Phenylalanine when treated in exactly the same way zoidal plates from water, m. p. 200' (dec.).
was found to be completely racemized after twenty-four Anal. Calcd. for CiHgOrNS: H, 5.06; C, 33.51; N,
hours. 7.82. Found: H, 4.88; C, 33.33; N, 7.87 (Van Slyke
Effect of pH on Velocity of Formation (Figs. 1, 2).-All amino N).
solutions were made up so that 25 cc. contained 1.70 mml.
Reaction with Benzyl Chloride.-A mixture of 0.66 g. of
each of cysteine hydrochloride (or acetylcysteine) and for-
thiazolidinecarboxylic acid, 0.63 g. of benzyl chloride and
maldehyde, 15 cc. of the appropriate 0.1 N buffer (or so-
0.69 g. of potassium carbonate in 10 cc. of water was stirred
dium chloride solution of equivalent ionic strength) with for twenty-four hours a t room temperature. S-Benzyl-
appropriate amounts of acid or alkali. The initial rota-
cysteine crystallized out during the reaction; m. p. 210"
tion for each curve was determined by omitting formalde- (dec.), unchanged by admixture with an authentic samp1e;l'
hyde from the solution, and the h a 1 values were deter- DI.[ +23.8' in N sodium hydroxide.
mmed from equimolar solutions of thiazolidinecarboxylic
Action of Oxygen in Alkaline Solution.-A solution mn-
acid. The final value of the cysteine reaction mixture
taining 0.266 g. of thiazolidinecarboxylk acid ( 2 mrnl.), 2
agreed with the rotation calculated for the eqd-product.
cc. of N sodium hydroxide, 5 cc. of half-neutralized 0.2 M
The PH of all solutions were determined with the glass
electrode. Dapite buffering, a change in p H (about 0.1) (13) A rnodi5cation by F. Rosebury of the apparatus described by
occurs during the reaction owing to the large difference him in I n d . Eng. Chem., Anal. Ed.,4,398 (1932).
(14) Assuming [ a ]+15.Q0 ~ (Vickery and Leavenworth, J . Bioi.
between the PK's of cysteine and thiazolidinecarborylir
Chcm., 86,129 (1930)] the conversion would be 14.8%.
acid. The initio1 and final pH values are recorded on the (15) Toennies and Bennet, J . B i d . Chcm., 11%499 (1936).
curves. The concentrations of the acetylcysteine and cys- 116) Lugg, Biochem. J . , 36, 2180 (1932).
teine hydrochloride stock solutions were calculated from (17) Clarke and Inouye, J . Biol. Chum., 84,549 (1932).
Jan., 1937 UPON CYSTEINE
THEACTIONOF FORMALDEHME 205

bicarbonate buffer and 0.1 cc. of 0.01 Mferric chloride was acid solution, buffered t o $H 5.7, according to the direc-
made up t o a volume of 10 cc. Air was passed through the tions of Lugg." No appreciable color appeared. W h e n
solution for twenty-four hours at room temperature. The 1 cc. of molar sodium sulfite was incorporated in an other-
pH (10.2) was then brought to 6.4 by the addition of 2 cc. wise similar mixture, a blue color appeared; this continued
of N hydrochloric acid. After several days hexagonal to increase in intensity a t a rate considerably less than that
plates had separated. The product, weighing 8 mg. observed with cystine Comparison with cystine stand-
(3.3%), was identified as cystine. ards showed after thirteen hours a color intensity equiva-
Oxidation with Hydrogen Peroxide.-A solution of 0.266 lent t o 43% of that observed with an equimolar quantity
g. of thiazolidinecarboxylic acid in 30 cc. of water and 0.12 of cystine.
cc. (1.05 equiv.) of 30% hydrogen peroxide was allowed t o In another experiment, 50 cc. of 0.00832 molar thiaeoli-
stand a t room temperature for several days; regular dinecarboxylic acid was mixed with 20 cc. of molar sulfite,
hexagonal plates separated; yield 0.210 g. (87%) of cys- and diluted t o 100 cc. The color developed with phos-
tine; [ a ] -214.5'
~ in N hydrochloric acid. The filtrate photungstic acid was determined at intervals under the
was distilled into alcoholic dimedon; 0.408 g (70%) of the conditions described by Lugg. The color intensities, ex-
formaldehyde derivative was secured. pressed as percentages of that of the equimolar cystine
Oxidation with Iodine.-To 10 cc. of 0.1 molar thiazoli- standard, were
dinecarboxylic acid was added 10 cc. of 0.1 N iodine in
Hours 0.1 4 10 22
2.5% potassium iodide. The iodine was decolorized rap-
Color 11 27 28 28
idly and regular hexagonal plates soon separated. Pyri-
dine (0.2 cc.) was added and after two days 0.100 g. (83%) With twice the amount of sulfite, the maximum color de-
of cystine was obtained; [aIaaD -212" in N hydrochloric veloped after six hours was 47%; after ninety-three hours,
acid. it had fallen to 30%. No attempt was made further to in-
The filtrate yielded 0.141 g. (50%) of the dimedon de- vestigate the effect of variations in the proportion of sulfite.
rivative of formaldehyde, m. p. 187". Sulfoxide of Acetylthiazolidinecarboxylic Acid.-A solu-
On titration with iodine, 1.6-1.7 equivalents were con- tion of 0.876 g. (5 mrnl.) of acetylthiazolidinecarboxylic
sumed by thiazolidinecarboxylic acid, 1.0-1.2 by its acid in 40 cc. of acetone was treated with 0.6 cc. (5.25 mml.)
methyl ester. The consumption of iodine in excess of one of 30% hydrogen peroxide. After three days the solvent
equivalent is attributable to oxidation of cystine.18 was evaporated under reduced pressure. The residue
Oxidation with Bromine.-A solution of 0.2896 g. was recrystallized by adding acetone t o its solution in the
(2.175 mrnl.) of thiazolidinecarboxylic acid in 10 cc. of minimum quantity of hot alcohol: 0.690 g. of octagonal
water was titrated at 0' with N bromine in acetic acid. prisms, m. p. 188-190" (dec.); [ a l a 6 D -118" in water.
The end-point, taken when the yellow color persisted for Anal. Calcd. for CoHeOrNS: C, 37.67; H, 4.75; N,
thirty seconds, was reached with 6.45 mml. of bromine 7.33; neut. eq., 191. Found: C, 37.57; H, 4.90; N, 7.32;
(5 94 equivalents). The resulting solution was evaporated neut. eq., 190.
t o dryness in wcuo, and the crystalline residue purified
by repeated precipitation from water by absolute alcohol. The same product was obtained in substantially the
same yield by the use of twice the above proportion of
[cUIz4D $7.7' in water. The cysteic acid was identified as
the copper salt. hydrogen peroxide in acetone.
A weighed amount (30.6 mg.) of the sulfoxide was added
Anal. Calcd. for CaH,OoNSCu: N, 5.63, S, 12.89; Cu, to a solution containing 1 cc. of 5 N KI and 0.5 cc. of 10 N
25.56. Found: N, 5.57; S, 13.22; Cu, 25.77. HC1. After an hour 3 cc. of water was added and the
Action of Sulfite on Thiazolidinecarboxylic Acid.-To solution quantitatively transferred to a separatory funnel
a solution of 1.5 mml. was added 15 mml. of sodium sul- with 5 cc. more water. The iodine extracted with perox-
fite, suitably buffered and the volume made up t o 25 cc. ide-free ether and titrated with 0.1 N thiosulfate, required
The optical rotation of the resulting solution was deter- 3.23 cc. (2.02 equivalents).
mined a t intervals: Sulfone of Acetylthiazolidinecarboxylic Acid.-To a so-
lution of 0.876 g. (5 mml.) of acetylthiazolidinecarboxylic
OBSERVED ROTATIONS (2 DCM.) AFTER SPECIFIEDTIMES
acid in 20 cc. of glacial acetic acid (distilled over chromic
9H 0 5 min 15 min. 20 min.
anhydride) 1.5 cc. (11 mrnl.) of 30% hydrogen peroxide
4.03 -2.26' -2.02' -1.80'
was added. After seven days the acetic acid was re-
6.41 -2.80' -0.96"
moved in vucao. The crystalline residue (0.77 g.) was
fiH 30 min. 96 min. 8 hrs. twice recrystallized from 10 cc. of hot alcohol; trilateral
4.03 -1.66O -1.66' prisms, m. p. 190' (dec.); [CY]% -90.8" in water.
6.41 -0.92' Anal. Calcd. for CeHe06NS: C, 34.76; H, 4.38; N,
The zero values, determined in the absence of sulfite, were 6.76; neut. eq., 207. Found: C,35.10; H, 4.48; N, 6.70;
obtained by interpolation from the p H dependence data neut. eq., 205.
(Table 11). The compound liberated practically no iodine from acidi-
Action of Phosphotungstic Acid and Sulfite on Thiazoli- fied potassium iodide solution.
dinecarboxylic Acid.-To 2 cc. of 0.00832 molar thiazoli- Thiazo1idine.-Twelve grams of phthalimidoethyl mer-
dinecarboxylic acid was added 2 cc. of phosphotungstic captanl9 was hydrolyzed by boiling for twelve hours under
reflux with a mixture of 120 cc. of 20% hydrochloric acid
(18) Simonsen, J . R i d . Chcm., 101, 35 (19.73); Shinohara, i b i d . ,
96, 286 (1933). (19) Gabriel, Ber., 24, 1110 (1891).
206 L. P. KYRIDES VOl. 59

and 20 cc. of glacial acetic acid. After cooling, phthalic 5 cc. of water to a faint permanent yellow (5.8 equivalents).
acid was filtered off, and the filtrate was concentrated i n The solution was taken to dryness i n vacuo, and the resi-
vacuo, filtered again, and finally taken to dryness in a vac- due recrystallized by adding alcohol to a concentrated
uum desiccator over potassium hydroxide. The crude solution in water. The product, taurine, formed fine
aminoethyl mercaptan hydrochloride20 was dissolved in needles, was neutral in reaction and did not melt below
20 cc. of water, treated with 4.6 cc. of formalin, allowed to 260"; yield, 0.430 g. (86%).
stand overnight, and taken to dryness i n vacuo. The Anal. Calcd. for C2H7OBNS: N, 11.19. Found: N,
residue was crystallized from alcohol; yield, 6.63 g. (91%) 10.95.
of long needles; m. p. 180" (dec.).
Oxidation of Thiazolidme with Iodine.-To 0.3358 g. of
Anal. Calcd. for CsHrNS.HC1: C, 28.66; H, 6.42; S, free thiazolidine (3.77 mml.) in 5 cc. of water was added
25.53; N, 11.15; CI, 28.24. Found: C, 28.86; H, 6.76; 37.7 cc. of 0.1 N iodine in 2.5% potassium iodide. A white
S, 25.77; N, 10.88; C1, 28.1. solid appeared which redissolved slowly. After twenty
The free base, liberated from the above hydrochloride hours the colorless solution was filtered to remove a small
by potassium carbonate, is readily volatile with steam, red precipitate, and 3.77 cc. of N sodium hydroxide was
miscible in all proportions with water, and can be salted added. A colorless solid which precipitated was filtered
out readily with potassium carbonate. It is a colorless off. Formaldehyde was identified in the filtrate. The
liquid, b. p. 164-165", d*S4 1.131, n J O1.551.
~ colorless precipitate, apparently an aldehydeammonia,
Anal. Calcd. for CaH7NS: N, 15.72; C, 40.39; H, 7.92. was dissolved in excess hydrochloric acid, when a strong
Found: N, 1.5.77; C, 40.40; H, 7.80. odor of formaldehyde was evolved. The solution was
Acety1thiazolidine.-An excess (2.5 cc.) of acetic anhy- taken to dryness in vacuo and the residue recrystallized
dride was added to 1.51 g. of the free base; the acetic acid from ethyl alcohol. The resulting needles, 0.250 g., m. p.
was removed in vucuo, and the residual liquid distilled un- 206 ', were identified as di-0-aminoethyl disulfide hydro-
der reduced pressure; b. p. 83-85' (0.7 mm.); yield 1.96 chloride, described by GabrieLel
g. (88%). Anal. Calcd. for C4H12N2S2.2HCI: N, 12.43. Found:
Anal. Calcd. for C~HBONS:C, 45.76; H, 6.91; N, N, 12.33.
10.68. Found: C, 44.66; H , 6.74; N, 10.24. Summary
Sulfone of Acetylthiazo1idme.-To a solution of 0.712 g.
of acetylthiazolidine in 10 cc. of glacial acetic acid, 1.5 cc. Formaldehyde reacts with cysteine over a wide
(2.3 mole) of hydrogen peroxide was added. After a week range of PH to form thiazolidine-4-carboxylic acid,
the solvent was removed in vucuo and the residue recrystal-
the mode of formation, constitution and proper-
lized from hot alcohol; stout hexagonal prisms or long
plates, m. p. 122 ". ties of which are here discussed.
Anal. Calcd. for CsHgOaNS: C, 36.78; H, 5.56; N, Thiazolidine, similarly prepared from formalde-
8.58. Found: C,36.72; H, 5.33; N, 8.65. hyde and 0-aminoethyl mercaptan, is also de-
Oxidation of Thiazolidine with Bromine.-N Bromine in scribed.
acetic acid was added to 0.354 g. (4 mrnl.) of thiazolidine in (21) Coblenz and Gabriel, ;bid., 24,1122 (1891).
(20) Gabriel, Bet.., 22, 1137 (1889). NEW Yo=, N. Y. RECEIVED
OCTOBER
31, 1936

[CONTRIBUTION
FROM THE RESEARCH
LABORATORIES,
MONSANTO Co.]
CHEMICAL

Phthalyl Chloride
BY L. P. KYRIDES

Phthalyl chloride has been prepared by the peratures in presence of very small amounts of
interaction of phthalic anhydride and phos- anhydrous zinc ~ h l o r i d e . ~We also observed
phorus pentachloride.' Thionyl chloride con- that the reaction is reversible, since thionyl
verts phthalic acid into the anhydride, but is chloride is obtained when sulfur dioxide and
reported to have no action on the latter a t re- phthalyl chloride react at around 200°, also in
fluxing temperatures even in presence of cata- presence of zinc chloride. The reaction is, there-
lysts or pyridine.* We found that excellent fore, expressed as
yields of phthalyl chloride are readily obtained
if the reaction is carried out a t elevated tem- + SOClz C4H4/cocl + so2
(1) Bruehl, Ann., 385, 13 (1886). Cd%<I% \COCl
(2) Meyer, Monatsh., 22, 437 (1901); McMaster and Ahmann,
THISJOURNAL, SO, 146 (1928); Carre and Libermann, ComPf. rend.,
If no zinc chloride is present and thionyl chloride
199, 1422 (1934). (3) U. S. Patent 1,951,364.