1926
H. TANIGUCH~, E(. FUKUI, S. OHNISHI,H. HATANO, H. HASEGAWA, AND T. MARUYAMA
B is based on the thermal conductivity at 0" for the gases involved. Mixtures of hydrogen and helium were made by measuring definite volumes of the two gases into the calibrated buret, taking into account the 4.8 ml included between points A and B of Figure 1, which was determined by PV measurements. Experience showed that the thermal conductivity of the mixture became constant (the bridge output did not change) after raising and lowering the mercury levelling bulb about 75 times, which indicated that the mixture was uniform. The bridge output for a given mixture was measured first with hydrogen as reference, E H ,and then , constant value of the with helium as reference, E H ~the sum, E H EHe, serving as a check on the accuracy of the observations. Approximately 200 observations were made over the entire range of concentrations, and the thermal conductivity of the mixture, K,, calculated by means of eq 1, was plotted against the measured per cent of
+
hydrogen. A smooth curve drawn between the points of the graph established average values of conductivity against the per cent of hydrogen, by means of which it was possible to calculate the values of constants NH and in eq 1. Taking &H = 41.8 X lo6 cal sec-l cm-l deg-l and O K ~=e 34.4 X lo5 cal sec-l cm-I deg-l1 it is found that N H = 0.0796 and NHe = 0.048. When these values are inserted into eq 1, calculated values of K,, which are plotted in Figure 2, can be obtained from the relation
K , = 41.8(1
+ 0.0796 In p ) p + 34.4[1 + 0.048 In (1 - p)](l - p )
(3) A few of the observations are plotted in Figure 2 in order to show the precision of the measurements. It can be stated, however, that the greatest deviation of any observation from the calculated graph was less than =4=0.5%, with the majority of the observations lying between 0.1 and 0.2% from the calculated average.
Free-Radical Intermediates in the Reaction of the Hydroxyl Radical with Amino Acids by Hitoshi Taniguchi, Katsuji Fukui, Shun-ichi Ohnishi, Hiroyuki Hatano, Department of Chemistry, Faculty of rScience, Kyoto University, Kyoto, Japan
Hideo Hasegawa, and Tetsuo Maruyama Japan Electron Optics Laboratory Company, Akishima, Tokyo, Japan
(Received January 3,1968)
Intermediate radicals formed in the reaction of the hydroxyl radical with some carboxylic acids, amines, and amino acids have been studied by esr spectroscopy using a continuous-flow method. A titanous chloridehydrogen peroxide system is employed as a source of the hydroxyl radical in most experiments and Fenton's reagent is also used in the case of alanine. Some good esr spectra are obtained for glycine, a-alanine, 0alanine, serine, threonine, valine, leucine, and isoleucine. The structures of the radicals deduced from analysis of the spectra and the hyperfine coupling constants are tabulated. It is found that the hydroxyl radical abstracts hydrogen atom preferentially from the CH bonds distant from the protonated amino group, those adjacent to the methyl group, and those distant from the carboxyl group. This is consistent with the electrophilic character of the hydroxyl radical. Hyperfine coupling data of various protons and 14N nuclei give information concerning steric conformations of the radicals and freedom in the internal rotations.
Introduction The flow method in esr studies first developed by Dixon and Norman' has been successfully used to study unstable intermediate free radicals in many organic redox reactions, and much knowledge on the nature and structure of these free radicals has been accumulated. With this technique, we have investigated the interThe Journal of Physical Chemistry
mediates in the reaction of the hydroxyl radical with amino acids and also with some related carboxylic acids and amines. Such knowledge might be basically important in understanding radiation-biochemical reactions in aqueous solutions, since the OH radical formed (1) W. T. Dixon and R. 0. C. Norman, J . Chem. Soc., 3119 (1963).
FREE-RADICAL INTERMEDIATES in water by irradiation is supposed to be an important intermediate species.a I n titanous (Ti”) chloride-hydrogen peroxide system, Dixon, et al.,a investigated previously intermediate radicals of a few carboxylic acids and amines, and Smith, et u Z . , ~ investigated also those of some carboxylic acids. As for the amino acid radicals, Borg6 reported an esr spectrum due to L-tyrosine radical produced by potassium permanganate oxidation.B Experimental Section The flow and mixing equipment used were made by Japan Electron Optics Laboratory Co. (JEOL) (Model JESSM-1) , The reactant solutions were pressurized by putting various weights on the top of two syringes. The flow rate was measured at the end of the flow. The volume between’ the mixing point of the two streams and the center of the cavity is about 0.015 ml. The usual flow rate was 0.5-2 ml/sec. Titanous chloride and hydrogen peroxide were used as a source of the OH radical. These solutions were acidified with dilute sulfuric acid. Organic substrate compounds were added to both components in 0.2-2.0 M concentrations, according to their reactivity and solubility. Electron spin resonance spectra were recorded on an X-band spectrometer (JEOL Model JES-3BS) at room temperature. Hyperfine coupling constants were measured using manganous ion as a reference (splitting between the two central peaks, 87.5 G,) and for the estimation of g values, LiTCNQ (lithium tetracyano-pquinodimethane), g = 2.0026, was used. Some esr spectra were simulated giving appropriate coupling constants with a spectrum accumulator (JEOL Model JRA-1). All materials were obtained from commercial sources and used without further purification : titanium trichloride solution (17% wt/wt extra-pure reagent grade), from Kanto Chemical Co., and hydrogen peroxide (30% wt/wt guaranteed reagent grade), from Mitsubishi E,dogawa Chemical Co. Amino acids were kindly offered by the Kyowa Hakko Kogyo Co. and the Tanabe Seiyaku Co. On mixing titanous chloride with hydrogen peroxide, two peaks are generally obtained in the esr spectra, one at g = 2.0134 and the other at g = 2.0120. At a concentration of 6.1 M of hydrogen peroxide, the observed spectra are only the low-field peak for titanous concentrations lower than 0.01 M . As the latter concentration becomes larger, the high-field peak begins to appear and both peaks become nearly equal a t a 0.05 M of titanous concentration. When the concentration reaches 0.1 M , the high-field peak becomes predominant. The decay curve of the OH radical was measured by the stopped-flow method and the half-life was found to be about 7 see. Sicilio, et ale,’ have suggested that the low-field peak was due to the OH radical and the highfield peak was due to OH radical associated with titanic (Ti4+) ion. Chiang, et a1.,8 more recently, have
1927 proposed that these two peaks were due to OH radical or perhydroxyl (OzH) radical complexed with titanic ion and substrate species. In the presence of organic substrates, both peaks become smaller and the peaks due to substrate radicals appear. The low-field peak seems to be more effective for the formation of substrate radicals. To find optimum conditions for obtaining high concentrations of the radicals, we examined effects of pH and concentrations of titanous chloride, hydrogen peroxide, and methmol on the signal intensity of the radical. Keeping the concentration of methanol 0.1 M , the esr signal due to methanol radical was most intense at about pH 2 and for 0.008 M Ti9+ and 0.1 M hydrogen peroxide. In Figure 1, the peak height of the methanol signal is plotted as a function of pH. I n the case of ~y-alanine,~ the esr signal due to substrate radical showed similar pH dependence. We used these conditions throughout the
8.3 mseo 10.0 msec
20.0 msec
33.3 msec
0
1
2
3
PH*
Figure 1. The peak height of the methanol signal plotted as a function of pH. The time in this figure indicates the time after mixing two reactants. (2) See, for example, Z. M. Bacq and P. Alexander, Ed., “Fundamentals of Radiobiology,” 2nd ed, Pergamon Press Inc., New York, N. Y . , 1961. (3) W. T.Dixon, R. 0. C. Norman, and A. L. Buley, J . Chem. Soo., 3625 (1964). (4) P.Smith, J. T. Pearson, P. B. Wood, and T. C. Smith, J . Chem. Phys., 43, 1535 (1965). ( 5 ) D. C. Borg, Nature, 201, 1087 (1964). (6) L. H. Piette, et al. (private communication), are also studying
amino acid radicals. (7) F. Sioilio, R. E. Florin, and L. A. Wall, J. Phys. Chem., 70, 47 (1966). (8) Y. S. Chiang, J. Craddock, D. Mickewioh, and J. Turkevioh, {bid., 70, 3509 (1966). (9) The designations CI and CZrefer to the position relative to the oarbon atom which bears the unpaired electron, while a and p refer to the position relative to that of the carboxyl group.
Volume 7% Number 6 June 1968
H. TANIGUCHI, K. FUKUI, S. OHNISHI,H. HATANO, H. HASEGAWA, AND T. MARUYAMA
1928
Table I : Structures and Hyperfine Coupling Constants of the Intermediate Radicals from Carboxylic Acids" Radical
Substrate
Acetic acid Propionic acidb n-Butyric acid Isobutyric acid Isovaleric acid a
Ratio
CH~COOH C H ~ C H ~ C O O(I) H CHsCHCOOH (11) CHsCHCHzCOOH CH~CH~CH~COOH CH&HzCHCOOH CHs(CH8)CCOOH CHz(CH3)CHCOOH (CH~)ZCCH~COOH CHz(CHa)CHCH&OOH
See ref 9 for the notations in column 4.
b
CI-H
Coupling constant, G Ci-CHs
21.4 22.2 20.1 22.2 22.3 20.3
2.5 1 5.2 1.9 1 1.0 1 2.0 1
CrH
27.0 25.3 26.1
22.2 28.0 24.1
21.9 22.6
25.9 16.4 27.7
23.7 22.2
Radical I1 has a higher g value (by 0.0007) than that of radical I.
Table I1 : Structures and Hyperfine Coupling Constants of the Intermediate Radicals from Amines" Substrate
Monoethylamine n-Prop ylamine Isopropylamine n-Butylamine Isobutylamine Monoethanolamine Tris(hydroxymethy1)aminomethane Diethylamine
Rad ica 1
CH~CHZN +Ha CH2CH2CHzN+Ha CHz(CH8)C" +Ha CHaCHCHzCHzN+Ha CHZCH~CH~CHZN +Ha (CH3)zCCHzN+Ha CHz(CHa)CHCHzN'Ha H3 +NCHzCHOH Ha +NC(CHzOH)&HOH CH~CH~NHCH~CH~
Ratio
2.8 1 2.5 1
Ci-H
22.7 22.3 22.5 22.0 22.1
Coupling constant, G Ci-CHa CrH CrN
25.6 24.1
22.4 18.1 17.1 22.1
26.6 26.7 24.9 23.7 28.0 5.8 24.1 11.8 19.2
OH
5.1 5.0 9.4 10.4 6.1
1.3 0.8
5.7
" See ref 9 for the notations in column 4.
present study, although such conditions may be altered for different substrates.
Results and Discussion (1) Carboxylic Acids and Amines. Structures of the intermediate radicals and the hyperfine coupling constants are summarized in Tables I and 11. The concentration ratio is also given for the substrates where two or three kinds of radicals were observed. In carboxylic acids, the abstraction of hydrogen atom occurs generally at every CH bond with different reactivities. The CH bonds adjacent to the methyl group are more reactive with the OH radical, while those adjacent to the carboxyl group are less reactive. In amines, the radicals produced by hydrogen abstraction from the CH bonds adjacent to the protonated amino group were not observed. In the cases of nbutylamine and isobutylamine where two different kinds of radicals were observed, hydrogen abstraction occurs more at the CH bond adjacent to the methyl group than at the CH bonds in the methyl group, in the same way as in n-butyric acid, etc. The observed different reactivities can be reasonably explained by considering the electrophilic character of The Journal of Physical Chemistry
the OH radical and the strong inductive effect of the protonated amino group, the inductive effect of the carboxyl group, and the induced effect of the methyl group. The CH bonds in acetic acid and methylamine, therefore, have very little reactivity with the OH radical and we need more substrate molecules to observe an esr signal (2 M for acetic acid). Methylamine at 5 M did not give any esr spectrum, owing to its intermediate radical. In the case of ethylamine, a good esr spectrum was obtained with an 0.8 M substrate solution at pH 2.1. (2) Amino Acids. Several typical esr spectra for the intermediate radicals of amino acids are reproduced in Figures 2-5. The structures of the radicals deduced from analysis of the spectra and the estimated hyperfine coupling constants are summarized in Table 111. It is demonstrated that the hydrogen abstraction occurs at the CH bonds in the side chain distant from the protonated amino group and the carboxyl group, in agreement with the results for carboxylic acids and amines. In leucine, however, the abstraction occurs mainly a t the CH bonds in the methyl group. This is a different result from the cases of other substrates having isopropyl groups.
FREE-RADICAL INTERMEDIATES
1929
Table I11 : Structures and Hyperfine Coupling Constants of the Intermediate Radicals from Amino Acidsa RadicalC
Substrate
Glycine
H~NCHCOOH
a-Alanine ,%Alanine L-Serine DL-Threonine
RCH~ H~+NCH~CHCOOH
DL-, L-,
RC(CHa)OH (I) RCH(CH~)OH(11) R C ( C H ~(TI ~ RCH(CHa)CHa JII) RCHzCH (CHa)CHz RCH (CHa)CHCHa
L-Leucine DbIsoleucineb
See ref 9 for the notations in column 4.
Hg+NCH(COOH)-,
U N ~ .
Coupling oonstant, G-CrCHa CPH CrN
Ci-H
12.4 22.8 21.4 17.6
RCHOH
L-Valine
a
Ratio
1.2 1 1.1 1
20.3 22.6 23.9 22.5 21 - 7 21.9
25.9
5.5d 26.6 24.5 8.8 7.9 27.0 7.1 29.5 21.7 19.7
6.6' 3.6 3.3 8.1 6.2
OH
1.3
7.1
More than two kinds of radicals exist, but only one can be assigned.
R represents
uC,-N.
a
a
-3
IO G Figure 2. Electron spin resonance spectra of the intermediate radicals from (a) 1 M DL-a-alanine and (b) 0.5 M p-alanine. Microwave power, 25 mW. Figure 4. (a) Electron spin resonance spectrum of the intermediate radical from 0.7 M L-valine. (b) Simulated spectrum assuming equal concentrations of radicals I and 11. The line shape is considered to be Lorentzian and the line width is taken to be 1.2 G.
1 -
10 G Figure 3. Electron spin resonance spectrum of the intermediate radical from 0.4 M L-serine.
It is interesting to note tha" the protonated amino group has more deactivating effect against the attack by the OH radical than the carboxyl group, as seen in the case of /?-alanine.a With a-alanine, we used a Fenton's reagent as well as a Ti3+-hydrogen peroxide system and obtained the same spectra at pH 5. The seven-line spectrum with further triplet splitting of glycine (Figure 5a) can be attributed to the H2NcHCOOH radical, with the following hyperfine coupling
constants: @,-H = 12.4 G, UN = 6.6 G, and UNH = 5.5 G. A spectral simulation (Figure 5b) based on these coupling constants gives a satisfactory reproduction of the observed spectrum. The spin densities pcl and PN are calculated by the simple LCAO-110 method, using appropriate parameters,'O and pcl = 0.53 and PN = 0.26 are obtained. The theoretical proton-coupling constant U C ~ - - H is, therefore, 23 X 0.53 = 12.2 G. Two different formulas, one proposed by Atherton, et ul.," and the other by Carrington and Santos-Veiga,l2 are used t o calculate theoretical U N values, and the obtained values are 6.59 G and 6.58 G, (10) B . Pullman and A. Pullman, "Quantum Biochemistry," Interscience Publishers, Inc., New Pork, N . Y., 1963,p 104. (11) N. M. Atherton, F. Gerson, and J. N. Murrell, MOL Phys., 5, 509 (1962). (12) A. Carrington and J. dos Santos-Veiga, ibid., 5, 21 (1962).
Volume 78, Number 6 June I068
1930
H. TANIGUCHI, E(. FUKUI, S. OHNISHI,H. HATANO, H. HASEGAWA, AND T. MARUYAMA
I 1
__f
IO G
0
10
20
30
40
60
60
Time after mixing, msec.
Figure 6. Second-order decay of valine radicals. Time scale is expressed by the time after mixing two solutions: X I radical I; 0, radical 11.
Figure 5 . (a) Electron spin resonance spectrum of the intermediate radical from 2.0 M glycine. (b) A part of the simulated spectrum assuming the Lorentzian line shape with the line width of 1.5 G.
respectively. These calculated values agree well with our observed one. At the low pH values of esr observation, the amino group of amino acids must be in the protonated form. The amino group of the glycine radical, however, was found t o be unprotonated. This difference may suggest a change of apparent pKb value of the amino group on going from glycine to the glycine radical, where the unpaired electron has been delocalized. I n the case of valine, where two different kinds of radicals were observed, we measured apparent decay processes of the radicals by changing the flow rate over the range 2.75-16.5 m1/10 sec. As shown in Figure 6, the reciprocal plots of the peak heights against the time from the mixing point to the cavity center gave straight lines with different slopes for radical I and 11. Assuming that the radical production is complete in far shorter than 9 msec, the linear dependence suggests the second-order decay of the radicals. L-Lysine monohydrochloride and L-arginine monohydrochloride gave very complex spectra, suggesting the formation of various kinds of radicals. L-Glutamic acid, L-histidine monohydrochloride, L-methionine, Lphenylalanine, and L-tyrosine gave signals with poor signal-to-noise ratios. (3) HyperJine Coupling Constants. It is well established that the C1-H coupling constant is proportional t o the unpaired spin density on the C1 atom, and the C2-H coupling comes from the hyperconjugative interaction between the unpaired 2pz orbital and the C2-H3-,R, group, The C2-H coupling constant, UC~-H, gives a measure whether the latter group freely rotates around the C1-C2 bond, and when the group is fixed in a certain conformation, it depends on the rotation angle of the Ca-H bond with respect to the 2pz orbital. The C2-N coupling probably comes from a The Journal of Physical Chemistry
similar hyperconjugative mechanism. There are, however, few published data, on the C2-N coupling constant, UO~--N, and the coupling mechanism is not well understood. Horsfield13 obtained a c Z - ~= 2.9 G and CCC*-N = 7.9 G for the (CH&CCH(N+H3)COO- radical produced by irradiation of avaline single crystal. Shields, et al.,14 have recently observed the same radical by irradiating a m-valine single crystal and obtained Q-H = 4 G at its maximum value and uC,-N = 7.7 G. Dixon, et aLJ3have reported UC~-H= 11.8 G and UC~-N = 10.3 G for the K3N+CH&HOH radical produced on mixing monoethanolamine with a Tia+-hydrogen peroxide system. Corvaja, et U L , ’ ~have obtained a number of UC~-N values for radicals of the type H2NCHX1cX2X3 produced in a flow system by conducting redox reactions in the presence of vinyl monomers and Tia+-hydroxylamine, The amino groups in the radicals appear to be protonated, considering the pH values of the solution. I n the present investigation, we have obtained series of C2-H and C2-N hyperfine coupling constants for various amines and amino acids. Looking a t the obtained coupling constants, we notice that in one group of amines and amino acids, the UC~-H values are around 26 G and the corresponding ac2-N values around 3-6 G, while in the other group, the former values become quite small, 6-12 G, and the latter values become correspondingly large, 7-10 G. We may think that, in the radicals belonging to the first group, the internal rotations around the C r C 2 axis are less hindered, considering larger UC~--H values and the structures of radicals. A typical example is eH,CH,N+H,, giving CQ-H = 26.6 G and m z - ~= 5.1 G. On the other hand, the internal rotation must be considerably restricted in the radicals belonging to the second group. (13) Unpublished results quoted by M. C. R. Symons in “Advances in Physical Organic Chemistry,” Vol. 1, V. Gold, Ed., Academic Press Inc., New York, N. Y.,1963, pp 323, 331. (14) H.Shields, P. Hamrick, and D. DeLaigle, J. Chem. Phys., 46, 3649 (1967). (15) C. Corvaja, H. Fischer, and G. Giacometti, Z . Phys. Chem. (Frankfurt), 45, 1 (1965).
FREE-RADICAL INTERMEDIATES In the serine radical, HO(H)CCH(N+H,)COOH, the OH group can make an intramolecular hydrogen bond with N+H3 and/or COOH, thus hindering the rotation. An nmr study by Ogura, et a1.,I6 showed that such a hydrogen bond played an important role in determining specific conformation of L-serine in aqueous acidic solution. In the valine radical, (CH,)&CH (N+H3)COOH, the bulky methyl groups will severely interfere with the rotation. (4) Some Remarks in Connection with Radiation Chemistry of Amino Acids. Many studies have been done on radiation chemistry of amino acids in dilute aqueous solutions, and it is known that deamination is a major reaction and the principal organic products are keto acids.17 The basic reaction resulting in the release of ammonia is supposed to be that of amino acids with the OH radical and the hydrogen atom produced by irradiation. Weeks and G a r r i ~ o n ’have ~ proposed the following reaction schemes for the case of glycine. Reductive deamination occurs by the attack of hydrogen atom, leaving CHZCOOH radical. The OH radical, on the other hand, first makes HzNcHCOOH radical which leads to ammonia after two steps. Since we found HzNcHCOOH radical as the intermediate between the OH radical from a Ti3+-hydrogen peroxide system and
1931 glycine, the present result seems to agree with their schemes. Hatano20has proposed that the OH radical generally abstracted an a-hydrogen of amino acids, and from the resultant radical, a-keto acids and ammonia were formed. Our general results of the present study, however, are that the OH radical abstracts a hydrogen atom from the CH bonds located a t the end of the side chains or close t o the end. Reactivity and other properties of the OH radical produced in a Tia+-hydrogen peroxide system and of that in an irradiation system should be made clear in order to apply the results in this study to the radiation chemistry of amino acids.
Acknowledgment. We are grateful to Kyowa Hakko Kogyo Co. and Tanabe Seiyaku Co. for providing many samples of amino acids. (16) H. Ogura, Y. Arata, and S. Fujiwara, J . Mol. Spectrosc., 23, 76 (1967). (17) A. J. Swallow, “Radiation Chemistry of Organic Compounds,” Pergamon Press Inc., New York, N. Y., 1960, pp 200-207. (18) A. M. Kuzin, “Radiation Biochemistry,” Israel Program for Scientific Translations, Jerusalem, 1964, Chapter 4. (19) B. M. Weeks and W. M . Garrison, Radiation Res., 9, 291 (1958). (20) H . Hatano, J . Radiation Res., 1, 28 (1960)
Volume 7W, Number 6 June 1968
