EDWARD J. KING [CONTRIBUTION FROM THE
VOI. 7 s
DEPARTMENT O F CHEMISTRY, BARNARD COLLEGE, COLUMBIA UNIVERSITY]
The Thermodynamics of Ionization of Amino Acids. 111. The Ionization Constants of Some N-Carbamoylamino Acids1 BY EDWARD J. KING RECEIVED AVGUST20, 1956 The thermodynamic ionization constants of the Xi-carbamoylderivatives of glycine, DL-alanine, DL-a-amino-n-butyric acid, DL-or-aminoisobutyric acid, p-alanine and y-aminobutyric acid were obtained a t ten different temperatures from measurements on cells without liquid junction. Hydantoic acid (N-carbamoylglycine) is a slightly weaker acid than Nacetylglycine because the dipole moment of the terminal NH2CONH- group of the former is slightly further away from the ionizing proton and is tilted a t a larger angle than the moment of the CHzCONH- group. The carbamoylamino group is more effective than the acetylamino group in orienting water molecules, so that the entropies of ionization of the carbamoyl amino acids are less negative than those of the acetylamino acids. The substitution of alkyl groups for an a-hydrogen atom is associated with decreases in the enthalpy and entropy of ionization that are generally similar to those previously reported.* The negative logarithm of the ionization constant varies linearly with the reciprocal of the number of carbon atoms that separate the polar and carboxyl groups. The entropy and enthalpy of ionization may vary in an analogous way if the behavior of the first acid in a series of a-substituted acids is regarded as anomalous.
The important role played by water in the ionization of carboxylic acids has been emphasized in a recent paper.2 Orientation of water molecules about the ions and molecules of acids is reflected more sensitively in the entropy of ionization than in the free energy of ionization or ionization constant. Attention was called in the earlier discussion to entropy effects associated with branching and lengthening of the alkyl chain attached to the carboxyl group and with shifts in the location of a polar group like the peptide linkage. These effects were illustrated by the behavior of some N-acylamino acids. The present paper is a report of an analogous study of some N-carbamoylamino acids. These acids differ from the acyl derivatives in having a polar, hydrogen-bonding amino group rather than an alkyl group a t the end of the chain. From another point of view the carbamoylamino acids are substituted ureas. The charge distribution in the polar group will be somewhat different from that in the peptide linkage because the terminal amino group can participate in the resonance structures
o
+
//
P-
HzN=C-NHCH?COOH,
I-T?SC--SHCHzCOOH, 0/ +
H?X-C=XHCH2COOH
It may be expected that the polar group on this account will have a greater order-producing effect on neighboring water molecules than the peptide linkage does but not as large an effect as the combination of peptide linkage and positively charged ammonium group found in the N-glycyl peptides. The ionization constants and related thermodynamic properties of N-carbamoyl derivatives of glycine, DL-alanine, DL-a-amino-n-butyric acid, DL-a-aminoisobutyric acid, /%alanine and y-aminobutyric acid have been obtained from measurements on the cell3 ( P t ) Hz IHA(ml), KaA(my), NaCI(m3) (?~gC1-~4g( I ) (I) This investigation was supported b y a research g r a n t , H-1651, f r o m t h e National Heart I n s t i t u t e o f t h e National Institutes of Health, U. S. Public H e a l t h Service. ( 2 ) E. J . King and G . W. King, THISJ O U R N A L , 78, 1089 (195G). (3) H . S . Harned and R . B. Owen, "The Physical Chemistry of Electrolytic Solutions," 2 n d . e d . , Keinhold Pul,l. Corp., New York, X . Y . 1060, p p , 407-5.1G.
where HA stands for any of the acids and ml, mz and These acids have been chosen to illustrate the effects of chain branching a t the a-carbon atom and of shifts in the position of the polar group. Measurements were made a t 5" intervals from 5 to 50" in order that accurate values of the thermodynamic properties associated with the ionization reaction could be obtained.
ma are the stoichiometric molalities.
Experimental One of Apparatus.-This has been described the unsaturated standard cells was recalibrated by the National Bureau of Standards in November, 1955. Its electromotive force had decreased 0.12 mv. since the previous calibration. The calculation of the ionization constants has been made with working standard electromotive forces, E,,.o, appropriate to the cells and technique used in this Laboratory.* The values of these given in the previous paper should be decreased by 0.06 mv. for use in the calculations that follow. The maximum error in the previously reported values of PK, the negative logarithm of the ionization constant, should not exceed 0.0002. Materials.-The preparation and standardization of all of the chemicals except the acids themselves have been described before.2~~The acids were synthesized from the appropriate amino acid and potassium cyanate6 and were recrystallized several times from conductivity water and dried in air. Their purity was tested by titration with standard sodium hydroxide solution, by determination of their water content with Karl Fischer reagent, and by various semi-quantitative tests? The assays and water contents are, respectively: iS-carbamoylglycine (hydantoic acid), 99.7% pure, 0.20% water; ~-carbamo)rl-DL-alaniiie, 99.6 and 0.38%; N-carbamoyl-DL-a-amino-n-butyric acid, 100.1 and 0.17%; N-carbamoyl-DL-a-aminoisobutyric acid, 99.8 and 0.1870; N-carbamoyl-8-alanine, 98.8 and 0.30%); and N-carbamoyl-y-aminobutyric acid, 100.0 and 0.20' 0 . All of the acids contained less than 0 . 0 0 4 ~ oiron, chloride, ammonia, phosphate and heavy metals except the derivative of a-amino-n-butyric acid which contained not more than 0.007y0 chloride. The derivative of p-alanine, which has a low assay, was examined by paper chromatography. Butanol-water-acetic acid was used as the solvent and two chromatograms were prepared by the ascending technique. One was developed nith ninhydrin. KOfree p-alanine was found though O.lyo could have been detected under the conditions of the experiment. The second chromatogram was developed with brom cresol purple ant1 only one acid spot was obtained. Techniques.-These have been described b e f ~ r e . ~ , ~ Almost all of the cells had electromotive forces a t 25" after (4) E. J. King, THISJ O U R N A L , 73, 155 (1951). ( 5 ) E. J. King, ibid., 76, lOOF (1954). ( 8 ) H. D. Dakin, J . Chem. Sor., 107, 434 (1016). (7) h I . P. Stoddnrd and I f . S D u n n , J . f3i~iL. Chem.. 142, 320 (1942).
THERMODYNAMICS OF IONIZATION
Dec. 5, 1056
two days that were within 0.10 mv. of the initial reading, but cells containing N-carbamoyl-a-aminoisobutyric acid showed a drop in electromotive force of 0.7 to 1 mv. over this period. Free a-aminoisobutyric acid was detected by paper chromatography in a typical cell solution after the measurements but not in the original acid. The rate of decrease of electromotive force at the higher temperatures was noted. The total changes in electromotive force a t 25' that occurred overnight and over the first and second days were determined. Four of the cells were taken over the higher portion of the temperature range first, the other six over the lower. It has been possible on the basis of all of these observations to correct the electromotive forces to the time of preparation of the solutions with a maximum probable error of 1 0 . 1 2 mv., the equivalent of 420.0020 in pK. The electromotive forces of cell I, corrected to a hydrogen pressure of one atmosphere, can be represented as a function of temperature by
Et = Ezs
+ a(t - 25) f b ( t - 25)'
(1)
The parameters of this equation for electromotive forces in absolute volts and concentrations in moles per kilogram of water are given in Table I together with the standard deviations between the observed and calculated electromotive forces of each set.
OF
6021
AMINOACIDS
hT-CARBAMOYL-DL-a-AMINO-?Z-BUTYRIC ACID
ml = m 2 ;stand. dev. = 10.025 mv. I3.01009
,01998 ,02506 ,03008 .04008 .05007
0.01008 .02010 .02515 .03008 .04012 .05035
0.57145 ,55362 .54777 .54315 .53583 ,53001
598 538 515 500 476 454
30 30 25 20 25 20
m, = 0.5 m2: stand. dev. = 420 031 mv.
0.01004 .02007 .02508 .03008 ,04102 ,05017
0.01008 .02006 ,02512 .03037 ,04136 ,05110
0.58904 .57131 ,56555 ,56069 .55279 ,54741
Gk57 597 576 559 532 513
30 30 20 25 20 20
M-CARBAMOYL-DL-a-AMINOISOBUTYRIC ACID ml = m2; stand. dev. = f 0 . 0 8 0 mv
TABLE I PARAMETERS OF EQUATION 1 X-CARBAMOYLGLYCINE (HYDANTOIC ACID) ma
ma
E25
1080
m, = my; stand. dev. = 420.064 mv. 484 0.01018 0.57062 0.01018 423 .55254 .02025 ,02027 372 .53853 .03450 .03462 .53020 344 ,04775 ,04784 .52103 312 .06822 .06784 276 .lo072 .51103 .lo005 ml
0.01000 .02021 .03264 .05393 .06791 .lo31
-10sb
-15 -20 -28 -30 -20 -15
m2; stand. dev. = f0.050 mv. 0.55328 429 $6 0,01011 -12 .52553 424 ,02907 0 .52234 321 .03276 -9 .50832 274 ,05608 254 -7 .06827 ,50307 215 -7 .lo35 ,49258
= 2
ml = 3 mz; stand. dev. = 420.041 mv. 397 +12 0.01029 0.54337 0.01028 325 0 .02166 .52291 .02079 290 0 ,03153 .51305 .03146 265 0 .04102 .50614 .04059 0 .50025 247 ,05061 .05024 216 0 .07143 .49178 .07027
N-CARBAMOYL-DL-ALANINE ml = mz; stand. dev. = f0.048 mv. 0.57181 563 0.01004 0.01004 .55390 503 .01994 .01993 .54361 477 .02966 .02962 .53585 441 .04004 .04014 .52538 411 .06009 ,05992 380 .07996 .51807 .07990
30 30 60 30 40 15
ml = 2 mt; stand. dev. = 420.028 mv.
0.01003 .02005 .02503 .03012 .04000 ,04989
0.01004 .02007 .02513 .03019 .04016 ,04992
0.55441 .53621 .53039 .52562 .51878 .51257
506 444 424 406 383 360
30 35 30 20 15 12
0.01003 .02505 .02757 .03007
0.01004 .02513 .02760 ,03012
0.60513 ,58169 ,57853 .57694
608 529 519 513
65 65 60 60
ml = 0.9467 ms; stand. dev. = f0.034 mv. 0,00529 .01030 .01889 .02363 .02788 .03792
0.00541 ,01053 ,01930 .02415 ,02850 .03875
0.61975 ,60243 ,58694 ,58118 ,57694 ,56905
664 601 549 530 516 490
50 30 30 30 35 20
N-CARB AMOYL-&ALANINE ml = 0,00998 .01989 ,03493 ,04064 .06018 .08064
m2;
0.01018 ,02027 .03499 ,04064 .06047 .08119
ml = 2 0.01004 .02002 .02503 ,03005 .04008 .05000
stand. dev. = 10.026 mv.
m2;
0.60617 .58864 .57487 ,57105 .56117 ,55390
617 555 503 490 452 423
40 25 25 20 20 20
stand. dev. = 420.031 mv.
0.01003 .02001 .02604 .03005 .04009 ,05001
0.58888 ,57129 ,56468 ,56105 .55383 .54831
558 496 469 458 430 409
30 35 45 30 35 30
N-CARBAMOYL--~AMINOBUTYRIC Acrr> ml = mz; stand. dev. = =k0.031 mv. 0.01002 .01003 .01507 ,02007 .02509 .03003 .04022 ,05010
0.01007 ,01002 .01506 .02009 .02509 .03007 .04030
,05012
0,61803 ,61815 ,60784 ,60052 .59496 .59043 .58311 ,57769
700 699 664 637 617 60 1 574 553
40 35 35 40 40 40 35 30
EDIVAKU J . KING
(io22
'r.4BLE
PAKAME1.E:PS O F EQUATION
2
Vol. 7s
111
AND THEKMODYNAhllC kJ'KOPERTIES i\SSOCIATED ACIDS Al"oaia.ib.
Acid
A
CarLainoylglycine Carbarnoylnlariirie Cnrb3!noyl-or-amiIro-rr-butyric acid Carbatnoyl-or-aiiiiri~isubutpricacid
1364.94 1083.10 1018.G5 1126.63 1152.77 1074 .OG
C3rbnriioyl-p-alaiiiIi~
Carbairioyl-~~-aiiiiiiobutyric acid
cal. mole - 1
C
H
5.0676 0.014(i-lU .012S10 3.3079 ,012670 2.9285 ,012129 3.1036 .1112--1!10 2.6066 .0123G-1 3.5768
5287.3 5309.8 53(J2.0 G089.0 G121.2 6387.7
290 -233 -493 +217 19-1 -115
-16.76 -18.59 -19.43 -19.69 -19.88 -21.83
305.3 291.4 2SB.G :W.6 303.8 29-1.7
-40 -25 -35 -33 -31 -3-1
3.8729 3.8901 3.8772 4.4814 4.4851 1.G816
iiie,isureiiieiits a i d lor carbaiiioyl-oc-,tiiiiiioisobuCalculations and Results 'rhe iiietliod of calculating the ionizatioii coil- tyric acid because of its dcconipositioii a t the higher stants froin electromotive force data is well l i n o w ~ i ~teinperatures. The uncertainty in the PI< values and need not be described here. The negative loga- for the @-alaninederivative caused by its low purity rithms of the thermodynamic ionization con- should not exceed 0.002 unless the impurities are stants recorded in Table I1 are average values of acidic, and the chromatographic experiments irithe intercepts found by independent extrapolations dicate that such contaminants probably are abof the results a t different buffer ratios to zero ionic sent. strength. The precision of the extrapolations can Discussion be judged froin the standard deviations of each set Comparison with Other Acids.-The N-carof pk' values. bainoylamino acids are slightly weaker than the corresponding N-acyl derivatives and have larger TABLE I1 (less negative) entropies and enthalpies of ionizaTHENEca.rIve I,OGAKITHMS OF THE 1UNIZATlON CONSTANTS tion. This behavior is to be expected when the OF CARBAMOYLAMIXO ACIDS terminal alkyl group of the acyl amino acids is C a r b a n - CatbauiCarbarnreplaced by an amino group. The peptide linkage oyl; oyl: oyl~-:2m ,.l , c , -o-:,m,n
