THE REACTION OF IODOACETIC ACID ON MERCAPTANS AND AMINES

BY L. MICHAELIS AND MAXWELL P. SCHUBERT

(From the Laboratories of The Rockefeller Institute for Medical Research, New York)

(Received for publication, May 10, 1934)

Since Lundsgaard (1) discovered the effect of iodoacetic acid on the chemical processes attending muscle contraction, various other remarkable effects of this substance in biochemical processes have been encountered. The interpretation of this effect, especially after Dickens’ publication on this matter, amounts to assuming a chemical interaction of iodoacetic acid and sulfhydryl groups ac- cording to the scheme

R-SH + ICHzCOOH = R-SCH#ZOOH + HI

Dickens (2) was the first to describe the preparation of the S- carboxymethyl compounds of cysteine and glutathione, which we also had just prepared when Dickens’ paper was published.

There can be no doubt that on the interaction of a sulfhydryl compound with iodoacetic acid this reaction occurs with great ease, and compounds of this kind will be described in the experi- mental part beside those prepared by Dickens.

It seems, however, not to be generally appreciated that iodoacetic acid also reacts quite easily with amino groups according to the equation

RNHz + 2ICH&OONa + 2NaOH --) RN(CH2COONa)2 + 2NaI + 2HtO

This reaction occurs with both aliphatic and aromatic amines and products of the action of iodoacetic acid on glycine, alanine, cystine, and p-phenylenediamine will be described. The product obtained from glycine and 2 moles of iodoacetic acid has also been obtained from ammonia and 3 moles of the halogen acid. This particular compound has already been prepared by Heintz (3). The remark-

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332 Reaction of Iodoacetic Acid

able feature of these reactions is the ease with which complete substitution of all the hydrogen of amino groups can be accom- plished. The products are polycarboxylic acids as an example of which may be mentioned N(CHZCOOH)~ obtained from either ammonia or glycine. Evidence is also presented that this can exist as a zwitter ion.

CH&OO- +/

HN-CH&OOH \

CH&OOH

When an aqueous solution of this compound is potentiometrically titrated with standard alkali, two carboxyl groups are titrated in succession behaving as rather strong acids and a 3rd equivalent of NaOH is used up in the alkaline range to deionize the ammonia group. The third carboxyl group is ionized from the beginning and does not show up during titration with alkali. There is so far no evidence that quaternary ammonium salts are formed in any of these reactions.

The reaction described has been carried out with halogen acids other than iodoacetic acid and is probably general with halogenated fatty acids, though very likely the iodo compounds are more reac- tive than bromo or chloro compounds. In many of the prepara- tions to be described other halogen fatty acids are used. With chloroacetic acid or a-bromopropionic acid on the one hand and glycine or alanine on the other, the following series of compounds has been made.

(CH,COOHh (CH,COOH) N(CH&OOH)a; N ;N ; N(CHCH,.COOH)z

(CHCH$ZOOH) (CHCH,COOH)z

Of these the tricarboxymethylamine is quite outstanding, differing from the others in its insolubility and the ease with which it crys- tallizes. It also forms an insoluble, easily crystallizable barium salt. All the others are extremely water-soluble as are also their barium salts; so more round about methods had to be used for their separation. An attempt was made to use this characteristic of tricarboxymethylamine to determine quantitatively the glycine content of proteins. Gelatin or silk was hydrolyzed with acid,

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L. Michaelis and M. P. Schubert 333

the hydrolysate made alkaline, and treated with excess of chloro- acetic acid and acidified to the turning point of Congo paper with HCl; the tricarboxymethylamine derived from glycine alone is precipitated, in the course of several days, with a yield, amounting to about 75 per cent of the theoretical yield calculated on the gly- tine content of the protein.

On comparing the relative rates of reaction of iodoacetic acid with -SH and with --NH, groups the one with -SH seems to be the more rapid. In the case of cysteine for example, which con- tains both -SH and --NH, groups, it is possible to obtain the S- ether compound with the -NH2 group unaffected provided too large an excess of iodoacetic acid is avoided. In all cases investi- gated the reaction with -NH, groups is very rapid when carried out under the conditions established for purposes of preparation of the products. At about 80” and in quite alkaline solution the reactions appear to be complete in 15 minutes. However, all these reactions also occur at room temperature and many of the prepa- rations have actually been carried out in this way. The reaction also occurs slowly in the physiological pH range as was determined for the case of glycine. A glycine solution made just alkaline to phenolphthalein, when mixed with an exactly neutralized solu- tion of iodoacetic acid, liberated at room temperature hydriodic acid as was determined by observing the progressive decrease in standard acid required to bring a sample of the mixture to the turning point of methyl red and also by the progressive appearance of iodide ion as determined by silver nitrate. A control solution containing no glycine did not show this effect, so it could not have been due merely to the hydrolysis of iodoacetic acid. This libera- tion of hydriodic acid continued even after the mixture, as a result of the reaction, had become acid to phenolphthalein. Another experiment in which a solution of glycine and iodoacetic acid was neutralized with excess sodium bicarbonate in such a way that the mixture was always in the NaHC03-H&O3 buffer range (acid to phenolphthalein) showed that the reaction had reached half com- pletion in about 8 hours at 30’.

Some experiments were also run to determine the relative rates of reaction of iodoacetic acid with the -SH groups of different compounds, cysteine, thioglycolic acid, and thioglycolic acid anilide being chosen. The rates were followed by noting the decrease in

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334 Reaction of Iodoacetic Acid

iodine consumption of samples of the mixture after various time intervals. Enough sodium carbonate was added in each case to be just equivalent to the initial acids present. The reaction was 70 per cent complete in the case of cysteine in about 10 minutes, with thioglycolic acid anilide in about 30 minutes, and with thio- glycolic acid in about 3 hours. This latter example shows that not all -SH compounds react with the same ease as cysteine. Glutathione belongs to the very rapidly reacting -SH compounds.

With these reactions of iodoacetic acid, shown to occur under conditions approaching the physiological, the interpretation of results obtained with this acid will have to be made cautiously. One may say, if the effect of some agent such as an enzyme is des- troyed by iodoacetic acid at a pH of 7 to 8, the point of attack of this acid may be considered as a -SH group, provided this is confirmed by some other evidence. If the effect of iodoacetic acid occurs only at pH > 7 or 8, the point of attack may be just as well an amino group. The reaction of iodoacetic acid on hydroxyl groups is probably negligible under conditions of physiological experiments.

EXPERIMENTAL

Iodoacetic acid was prepared from chloroacetic acid and potas- sium iodide, and recrystallized three or four times from carbon tetrachloride. It was perfectly white and free from free iodine.

Tricarboxymethylamine-Although this compound has already been described by Heintz, the preparation which has been worked out is so much simpler that it seems worth presenting. 1.5 gm. of glycine and 7.5 gm. of iodoacetic acid are dissolved in 20 cc. of water and 16 cc. of 6 M KOH are added. The mixture is warmed in a water bath at 80” and then cooled. HCI is added cautiously until the mixture is just acid to Congo red paper. The product crystallizes out, on scratching a little, as large plates and after drying weighs 3.1 gm. It can easily be recrystallized from hot water. 2.5 gm. are obtained or 66 per cent of the theoretical.

N(CH&OOH),. Calculated, N 7.33; found, N 7.42

The crystals melt with decomposition at 230-235’. Fig. 1 shows a potentiometric titration of an aqueous solution of this acid at the glass electrode. 3 equivalents of NaOH are used up; 2

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L. Michaelis and M. P. Schubert 335

within the acid range. The pK values of these two overlapping steps are somewhere around 3. The third constant is about 10 and should be interpreted as the acidic constant (in Brijnsted’s sense) of the amino group.

The rest of the carboxymethyl- and carboxyethylamines which were prepared could not be isolated directly because of their great

I I I I 2 4 6 8

Cc. NaOH

FIG. 1. Curve A, tri(carboxymethyl)amine, 0.0525 gm., titrated with 0.0993 M NaOH. The arrows at the top of the figure show where the 3 equivalents of alkali are used up, as read from the graph, the abscissa of the big jump being taken to calculate the other two steps. Curve B, di- carboxymethylcysteic acid, 0.0647 gm., titrated with 0.0993 M NaOH. The arrows at the bottom of the figure correspond to the successive consumption of 4 equivalents of alkali as deduced from the jump in the curve at, the 3rd. These curves were run with a glass electrode, the E.M.F. values being trans- lated to pH by comparison with the E.M.F. read in standard acetate buffer. In each case the end-point as read from the graph is between 3 and 4 per cent short of the end-point as calculated from the weight of substance and the formula deduced from analysis of the compound.

solubility. They are formed by warming together 40 mM of either glycine or alanine and 80 rnM of chloroacetic or cr-bromopropionic acid with enough potassium hydroxide to neutralize the original acids as well as the halogen acid which splits out as a result of the

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Reaction of Iodoacetic Acid

reaction. These mixtures are all warmed at about 80” for half an hour, 6 M HCI is added until the solution is acid to Congo red paper, and then it is evaporated in vacua to dryness. The residue is extracted with about 50 cc. of hot glacial acetic acid containing about 4 gm. of potassium acetate and the hot acetic acid filtered off quickly with suction. After cooling the filtrate it may be neces- sary to filter again if potassium bromide separates. About 400 to 500 cc. of absolute alcohol are then stirred into the filtrate and the mixture set on ice. In the case of the two unsymmetrical amines precipitation is immediate but the products are not crystalline, while with the two symmetrical amines complete separation may take 4 days to a week but the products are crystalline. After fil- tering off the deposits, washing with absolute alcohol, and drying in vacua, these analytical results were obtained.

Calculated. N 5.76, K 16.04

[ N<:EZZaZO,] HZK. Found. “ 5.48, “ 14.59

The first two of t’hese compounds are extremely hygroscopic and difficult to obtain in a form suitable for analysis.

Tetrapotassium Tetracarboxymethylcystine Diacetate-A solution of 10.7 gm. of cystine in 20 cc. of water and 13.7 cc. of 6.4 M KOH is mixed with one of 51.8 gm. of iodoacetic acid in 20 cc. of water and 43 cc. of 6.4 M KOH. To the mixture 35 cc. more 6.4 M KOH are added and the clear solution either heated in a water bath at 90” for 30 minutes or allowed to stand at room temperature for an hour or two, after which it is filtered if necessary. To the clear solution 750 cc. of alcohol are added and the mixture set on ice for 2 hours. A heavy liquid separates at the bottom of the beaker. To obtain an analyzable pure substance from the liquid, the fol- lowing procedure was adopted. The main body of liquid is de- canted and the heavy liquid residue is washed by decantation with three 25 cc. portions of alcohol and two 25 cc. portions of acetone. The remaining acetone is then removed by gentle warming. On

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L. Michaelis and M. P. Schubert 337

cooling, the residue often crystallizes at this point. It is dissolved in 150 cc. of glacial acetic acid containing in solution 18 to 20 gm. of potassium acetate. When it is completely dissolved, which process requires considerable stirring, the solution must be per- fectly clear; if it is not, it is filtered. 600 cc. of absolute alcohol are added, producing a heavy white precipitate. The mixture is now set in a water bath at 70” for an hour and then cooled. The precipitate becomes somewhat sandy though not definitely crys- talline. It is filtered off and washed with a large volume of abso- lute alcohol and then dried over HzS04 in a vacuum. 33.0 gm. of product are obtained, a yield of 88 per cent. The formula appears to be

[

-S.CHz.CH.COOH

I 1 SCH,COOH N(CHrCOOK)z 2

Calculated. S 8.60, N 3.76, K 20.98 Found. “ 8.34, (‘ 3.84, “ 21.55

The product is perfectly white and is extraordinarily easily solu- ble in water. This property may make it of some interest, being an easily obtainable derivative of cystine which is soluble in both the disulfide and the sulfhydryl forms. The aqueous solution is acid in reaction. As to the formulation of this compound, one way is, as shown above, to regard the acetic acid as analogous to the water of hydrates. Another way is to regard the compound as an amphi-salt in the sense described by Pfeiffer (4), that is

[

-S-CH,-CH-COOH

I (CH,COO)- H+N(CHz.COO-)z2K+ 1 1

No decision between these formulations being yet possible, it suffices to classify this compound in the group of crystalline com- pounds consisting of a molecule of an ampholyte plus 1 molecule of a neutral salt.

Tetracarboxymethylcystine-Attempts to prepare the free acid itself were largely unsuccessful. The barium salt was precipitated as an amorphous solid from a slightly alkaline solution of the po- tassium salt just described. This barium salt is filtered off, washed well with water, resuspended in water, and sulfuric acid added in the cold until t,here is just a very slight excess of the acid. The

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338 Reaction of Iodoacetic Acid

barium sulfate is filtered off, the clear solution evaporated in vucuo to dryness, and the residue ground up with acetone. The white sandy mass that is left is filtered off, washed with acetone, and dried. Most preparations gave values very low in sulfur, indicat- ing some kind of decomposition. Only one preparation gave analytical figures which began to appear reasonable.

[-S.CHz.CHN(CH2COOH)2.COOH]2. Calculated. S 13.55, N 5.93 Found. “ 14.40, “ 5.98

Dicarboxymethylcysteic Acid-This is the only compound in this series which could be obtained in a distinctly crystalline condition. The potassium salt described above is dissolved in a little water, bromine added to a slight excess, the solution made alkaline, and the barium salt of the sulfonic acid is precipitated. This is washed with much water, decomposed with exactly an equivalent amount of sulfuric acid, and the filtered solution evaporated to dryness in vacua. The residue is ground up with acetone, filtered, and dried. It is then dissolved in a small quantity of absolute methyl alcohol, filtered, and about 10 volumes of acetone added. After standing on ice a few days a crystalline deposit of short needles separates. This is filtered off, washed with acetone, and dried.

HO~S~CH2~CHN(CH2COOH)z~COOH~Hz0. Calculated. S 10.55, N 4.62 Found. “ 10.36, “ 4.47

An acid titration curve for this substance is given in Fig. 1. This shows three strong acid constants (pK< 3) and a fourth in the neighborhood of 9.5. According to expectation, instead of the 3 equivalents of alkali used up in the case of the glycine compound plotted in Fig. 1, 4 equivalents are used up here due to the addi- tional presence of a SOjH group.

Dipotassium Dicarboxymethylcysteine Monoacetate-Attempts to prepare the corresponding -SH compound also failed to yield a crystalline product. By reduction of the above potassium com- pound with tin in 4 M HCI, neutralization of most of the acid, removal of tin with H2S, and evaporation of the resulting solution to dryness in vczcuo, an oily product is obtained which is extracted with a mixture of 59 cc. of glacial acetic acid containing about 5 gm. of potassium acetate. The acetic acid is filtered off and the product precipitated with 4 volumes of absolute alcohol. The

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L. Michaelis and M. P. Schubert 339

mixture is digested at 80” for an hour, which renders the precipitate somewhat sandy but not definitely crystalline. It is filtered off and washed with absolute alcohol.

HS-CHy CH-COOH

I CH,COO- H+N(CHaCOOK)z

Calculated. S 8.58, N 3.75, K 20.90 Found. “ 10.06, ‘( 3.99, “ 21.83

Titration of this compound with alcoholic iodine gave an equivalent weight of 338 instead of the theoretical 373, but the end-point is very unsatisfactory. That point is taken at which the iodine color suffuses the whole solution for a few seconds, but much more iodine may be added that is very slowly consumed probably caus- ing further oxidations such as investigated by Simonsen (5). There can be no doubt that the expected substance has been ob- tained, although not in a perfectly pure state owing to the lack of a well crystallized salt. In alkaline solution it gives oxidized com- plex compounds with cobalt just as cysteine itself does (6). Of interest also is the fact that it does not give the Sullivan reaction characteristic of cysteine.

In addition to these aliphatic amines the action of iodoacetic acid on aromatic amines has also been tried. The neutralized iodoacetic acid and p-phenylenediamine are warmed together with enough alkali to neutralize the HI which splits out. The reaction is very rapid, 15 minutes being enough for completion. Both the barium salt and the free acid are well crystallized compounds.

Tetracarboxymethyl-p-Phenylenediamine-This compound melts with decomposition at 165”. On partial oxidation with bromine water it develops a semiquinoid violet dyestuff analogous to Wur- ster’s blue, showing two distinct absorption bands, whose wave- lengths are somewhat different from all Wurster’s dyes known to us and may be described at another occasion (cf. (7)).

(HOOC~CH,),~N~C~H~~N(CHQ~COOH)~. Calculated. N 8.24 Found. “ 8.48, 8.40

Tetracarboxymethyl-p-Phenylenediamine Barium Xalt-

Ba2(00C.CH.J4Nz*CaH4. Calculated. N 4.59, Ba 45.04 Found. ‘I 4.38, “ 45.25

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340 Reaction of Iodoacetic Acid

Some products of the interaction of iodoacetic acid on -SH com- pounds had already been prepared when the work of Dickens ap- peared, describing those of cysteine and of glutathione. As the compound with cysteine we have prepared was made by a much simpler method, has been recrystallized, and gives a much higher melting point than the one reported by Dickens, it seems advis- able to describe its preparation.

5.5 gm. of cysteine hydrochloride and 3.6 gm. of chloroacetic acid are dissolved in 10 cc. of water, and 17 cc. of 6.7 M KOH are added. The heat of neutralization of the acids and the heat of reaction make the mixture quite hot. After 5 minutes it should still be alkaline to litmus. Add glacial acetic acid until the solution is just acid to litmus and let stand on ice 2 hours. Filter off the cystine. 6 M HCl is now added so the mixture becomes just blue to Congo red paper, and it is again set on ice for 2 hours. The crystalline precipitate which has separated is filtered off, dissolved in 200 cc. of boiling water, and the hot solution filtered and set on ice. 6-Sided plates separate. They differ from cystine in not, being regular hexagons as well as their easy solubility in hot water. 70 per cent of the theoretical yield is obtained after one recrystalli- zation.

Calculated. N 7.82, S 17.88, C 33.51, H 5.06 Found. “ 7.75, “ 17.81, “ 32.97, “ 5.24

The melting point is 175-176” with decomposition. The corresponding compound with thioglycolic acid anilide is

made similarly by warming together in solution equivalent quanti- ties of the anilide and of iodoacetic acid neutralized with NaLJ03. After 20 minutes the mixture is cooled, acidified with HCl, and set, on ice. The crystals may require some scratching before appearing. They can be recrystallized from a small volume of hot alcohol. After drying in vacua 80 per cent yields can easily be obtained. The corrected melting point is 99-100”.

C6Hs.NH.CO.CHg.S.CHz.COOH. Calculated. S 14.21, N 6.22, C 53.38, H 4.93 Found. “ 14.30, “ 5.77, “ 53.40, (‘ 5.11

The glutathione compound was prepared by allowing a solution of crystallized glutathione and iodoacetic acid just neutralized

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L. Michaelis and M. P. Schubert

with Na2C03 to stand in a heavy Verona1 buffer at room tempera- ture. The precipitated Verona1 is removed by filtration, carbonate is removed with barium, and the barium salt of the glutathione compound precipitated with alcohol. This is filtered, washed, dissolved in water, and the lead salt precipitated. From this the desired compound is obtained by treatment with H$S, filtration, and evaporation to dryness.

CI~H~~O~N~S.CH~COOH~HZO. Calculated. S 8.35, N 10.96, C 37.60, H 5.53 Found. “ 8.30, “ 10.73, ‘< 35.10, “ 5.34

Thiodiglycolic acid has also been isolated from the reaction of iodoacetic acid and thioglycolic acid.

SUMMARY

Iodoacetic acid reacts not only with sulfhydryl compounds but also with amino compounds both aliphatic and aromatic, particu- larly with amino acids. All H atoms of the -SH or --NH2 group are easily substituted by the radical -CH&OOH. For both these groups of reactions examples are described. The following compounds have been prepared: tri(carboxymethyl)amine,l di- (carboxymethyl) (or-carboxyethyl)amine potassium salt, carboxy- methyldi(cY-carboxyethyl)amine potassium salt, tri(ar-carboxy- ethyl)amine potassium salt, tetracarboxymethylcystine, dicar- boxymethylcysteic acid, dicarboxymethylcysteine acetate, tetra- carboxymethyl-p-phenylenediamine, S-carboxymethylcysteine,’ S- carboxymethylglutathione,l S-carboxymethylthioglycolic acid ani- lide.

BIBLIOGRAPHY

1. Lundsgaard, E., Biochem. Z., 217, 162 (1930); 220, 1 (1930). 2. Dickens, F., Biochem. J., 27, 1141 (1933). 3. Heintz, W., Ann. Chem., 122, 257 (1862); 146, 49 (1868). 4. Pfeiffer, P., Organische Molektilverbindungen, Stuttgart, 139 (1927). 5. Simonsen, D. G., J. Biol. Chem., 101, 35 (1933). 6, Schubert, M. P., J. Am. Chem. Sot., 63, 3851 (1931). 7. Michaelis, L., J. Am. Chem. Sot., 63, 2953 (1931).

1 These compounds had been prepared by different methods previously.

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L. Michaelis and Maxwell P. SchubertACID ON MERCAPTANS AND AMINES

THE REACTION OF IODOACETIC

1934, 106:331-341.J. Biol. Chem. 

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