ANALYTICAL CHEMISTRY
1554
the reaction by converting excess metaperiodate (as well as formed iodate) to iodine. The latter was then titrated to colorless with thiosulfate solution; 1.5 and 2.0 ml. were required. The use of potassium iodide and thiosulfate as described was found necessary because the colorimetry depends on the formation of 2,4dinit~ophenylhydrazones of the products of periodate oxidation. However, if an excess of either periodate or thiosulfate was present, the addition of 2,4dinitrophenylhydrazine solution resulted in the formation of a heavy precipitate. The procedure was thus designed to ensure that no reagent is present in excess a t time of addition of 2,Pdinitrophenylhydrazine solution. Colorimetry. Each tube next received 0.1 ml. of 2,Cdinitrophenylhydrazine solution, following which all tubes were diluted to 5.0-ml. volume with water. Fifteen t o 20 minutes a t room temperature were allowed for hydrazone formation. One milliliter of sodium hydroxide solution was then added to each tube a t 30-second intervals. Fifteen minutes were allowed t o elapse, during which time the rate of color-fading decreased to its minimum: the tubes were then read in a Klett colorimeter a t 30second intervals. The colorimeter was adjusted to 100% trans-
mittance with an appropriate reagent blank and was used with the green (No. 54) filter. The results obtained with two different compounds are shown in Figure 1. Paper blanks have been subtracted in each case; blanks gave Klett values from 15 to 45, averaging about 30. This compares with a Klett value of about 125 for 0.1 micromole of tartaric acid, and about 80 for 0.1 micromole of a,@-dihydroxyp-methylbutyric acid. ACKNOWLEDGMENT
This work was supported by a contract between the Office of Kava1 Research and the regents of the University of California. LITERATURE CITED
(1) Adelberg, E. A., Bonner, D. M., and Tatum, E. L., J . BWZ. Chem.. 190. 837 (1951). (2) Buchanan, J: G., Dekker, C. A,, and Long, A. G., J . Chem. SOC.,1950, 3162. (3) Lugg, J. W. H., and Overell, B. T., Nature, 160, 87 (1947). (4) Tatum, E. L., and Adelberg, E. A., J . Bid. Chem., 190, 843
(1961). RECEIVED for review .4pril 10, 1953. Accepted June 22, 1953.
Titration of N-Carboxy-alpha-amino Acid Anhydrides in Nonaqueous Solvents ARIEH BERGER, MICHAEL SELA, AND EPHRAIM KATCHALSKI The Weizmann Znstitute of Science, Rehovoth, Israel
Figure 1 no special steps were needed, as carbon dioxide is practically insoluble under the experimental conditions used. The products of the titration of S-carboxyamino acid anhydrides with sodium methylate in the presence of methanol were investigated in order to elucidate the course of the reaction. For example, LV-carbox?rphenylalanine anhydride( I ) ( 5 ) b a y react with sodium methylate and methanol to give either the sodium salt of aV-carboxyphenylalanine methyl ester(II), or the sodium salt of S-carbomethoxyphenylalanine(II1).
to the interest in ,V-carboxy-a-amino acid anhydrides as 0 starting materials in the synthesis of poly-a-amino acids (8) WING
and low for their reported solvents
molecular weight peptides ( 1 , 7 ) , a convenient method quantitative determination was required. It has been that acid anhydrides could be titrated in nonaqueous with sodium methylate, using thymol blue as indicator (6, 11). This method was adapted for the quantitative determination of AT-carhoxy-a-amino acid anhydrides in various organic solvents. The color of the indicator changes sharply
CaHsCHZ-CH
COOCHB SH-CO
f
-
HC1 CeHoCHt-CH
\
\
(11)
SHCOOCHa CsHbCH2-CH
/ \
HCl CeH6CHt-CH
/
--L
‘COOH
(VI from yellow to deep blue upon the addition of an equimolar quantity of the titrant. From 0.1 to 0.5 millimole of the amino acid anhydrides could be estimated with an accuracy of +t2Y0 by the procedure described below. When using a micrometer syringe, from 0.003 t o 0.03 millimole could be estimated with the same accuracy. This titration seems to be particularly useful in polymerization studies of N-carboxy-a-amino acid anhydrides, since the amount of unreacted anhydride present a t any instant may be determined conveniently. The course of a typical polymerization reaction followed by this method is given in Figure 1. As the carbon dioxide evolved during polymerization interferes with the titration, it should be removed. In the particular case shown in
By determining the amounts of phenylalanine methyl ester hydrochloride(1V) ( 4 ) and N-carbomethoxyphenylalanine(V) (1.3) formed on acidification of the reaction mixture, it was found that 1 mole of ( I ) yields 0.96 mole of (11) and 0.03 mole of (111). Under similar conditions 1 mole of e,N-carbobenzoxy-a,-V-carboxylysine anhydride (3) gives 0.86 mole of the sodium salt of e,N-carbobenzoxy-a,N-carboxylysine methyl ester and 0.11 mole of the sodium salt of c,N-carbobenzoxy-a,N-carbomethoxylysine. REAGENTS AND MATERIALS
Solvents. Benzene, purified grade, dried over sodium;
methanol, absolute; dioxane and butylamine, refluxed with po-
V O L U M E 25, NO. 10, O C T O B E R 1 9 5 3 Table I.
Titration of N-Carboxy-a-amino Acid Anhydrides in Dioxane
a,.V-Carboxy Anhydride of
(6)
Equivalent Weight Found CalcuMacroMicrolated titration titration 101.1 102; 104 ... 191.2 192; 193 191
(0) (8)
341.3 249.3
336; 339 244; 245
340 249
(9)
306.4
302; 305
305
(10)
292.3
291; 295
...
(18)
264.3
265; 266
264
Prepared hccording to
Glycine DL-Phenylalanine o-Carbobenzoxy-L-tyrosine 6-Benzyl-L-aspartate e,N-Carbobenzoxy-Llysine 6,A'-Carbobenzoxy-DLornithine @,N-Carbobenzoxy-La,@-diaminopropionic acid
(6)
i+
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1555
0 7
1
'I
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2
0
I
1
I
I
I
5
10
15
20
25
e ' 0 0 m
TIME, HOURS Figure 1. Determination of s,N-Carbobenzoxy-a,Ncarboxy-L-lysine Anhydride during Polymerization
tassium hydroxide and distilled from sodium; acetone and chloroforni, purified grade; ether and pyridine, dried over sodium and distilled; ethyl acetate, purified according t o Wade and Merriman (14); acetophenone, dried over phosphorus pentoside, anhydrous potassium carbonate, and fractionated in vacuo; and dimethyl formamide, obtained from D u Pont Co. 0.1 N Sodium Methylate Solution. Prepared according to Fritz and Lisicki ( 6 ) , but using equal volumes of methanol and benzene. Thymol Blue Solution. Thymol blue (0.5 gram) was dissolved in 100 ml. of methanol; for microtitrations 0.05 gram of thymol blue was dissolved in 100 ml. of methanol. N-Carboxy-a-amino Acid Anhydrides. Freshly prepared as given in Table I and recrystallized from ethyl acetate. PROCEDURE AND EWERIMENTAL RESULTS
Titrations. A sample of 0.1 to 0.5 millimole of N-carboxy-aamino acid anhydride was dissolved in 10 ml. of solvent. Three drops of indicator were added, and the solution was titrated to a deep blue end point with 0.1 N sodium methylate, using a 5-ml. buret. Upon the addition of the indicator, the solutions of the anhydrides in neutral organic solvents remained colorless, but turned yellow on the addition of the first drop of the titrant. When basic solvents were used, the yellow color appeared a t once. The appearance of a red color indicates contamination of the anhydride with free acids. Data for the titration of various N-carboxy-a-amino acid anhydrides in dioxane are given in Table I. Equivalent weights of 190 to 195 were obtained for N-carboxyphenylalanine anhydride titrated (macro) in the following solvents: methanol, benzene-methanol (1 to 1 by volume), acetone, ether, chloroform, ethyl acetate, acetophenone, dimethylformamide, pyridine, aniline, and butylamine. When the titrations were carried out in basic solvents, pfovision wras made for protecting the solution from carbon dioxlde in the atmosphere. The titration could not be carried out in ethanolamine or ethylene diamine, as thymol blue gave a greenish color in these solvents. Microtitrations. A sample of 0.003 t o 0.03 millimole of anhydride was weighed into a small test tube (75-mm. length, 8-
mm. inner diameter), dissolved in 0.5 ml. of solvent, and 1 drop thymol blue was added. The solution was titrated of 0.0E1~~ with 0.1 N sodium methylate with magnetic stirring. The titrant was delivered from an .4gla micrometer syringe (Burroughs Wellcome & Co., London), the tip of which dipped into the solution. A piece of iron wire, 5 mm. in length, made from a paper clip, served as stirrer. Efficient stirring was obtained when the test tube was held somewhat off center of the magnetic stirrer. The rather close fit between syringe and test tube gives sufficient protection from the carbon dioxide in the atmosphere. Data for the microtitration of various N-carboxy-a-amino acid anhydrides in dioxane arc given in Table I. Equivalent weights from 190 to 192 were obtained for S-carboxyphenylalanine anhydride in benzene, dioxane, butylamine, and dimethylformamide. Isolation of the Titration Products of N-Carboxyphenylalaninnine Anhydride. One-hundred-five milliliters of 0.1 N sodium meth>latewere added to 2 grams of N-carboxyphenylalanine anhydride
