The Journal of Physical Chemistry, Vol. 83, No. 22, 7979 2893
ESR of N-Acetylamino Acid Containing Solutions
An Electron Spin Resonance Study of y-Irradiated Frozen Aqueous Solutions Containing N-Acetylamino Acids Mlchael D. Sevllla," James B. D'Arcy, and Klm M. Morehouse Department of Chemistry, Oakland University, Rochester, Michigan 48063 (Received May 3, 1979) Publication costs assisted by the U S . Army Natick Research and Development Center and the U S . Department of Energy
The results of y irradiation of a number of N-acetylamino acids at 77 K in frozen DzO and HzO solutions are reported. The radicals produced by the irradiation, their relative amounts, and their stability were studied as a function of temperature. The results found for ices of N-acetylalanine illustrate the reaction mechanisms found. At low temperatures (120 K) the peptide anion and decarboxylated species dominate the spectrum. Upon warming to 190 K the anion converts to the amide and fatty acid radical by secondary deamination: CH&O-NDCH(CH3)C02-+ D+ CH3CONDz+ CH(CH3)C0,. Further warming to 220 K results in abstraction of the intermediate radicals from the parent compound to form the a-carbon radical: CH3CHC02- + CH3CONDCH(CH3)C02- CH3CH2C02-+ CH3CONDCCH3C0f. Results found for N-acetylglutamic acid show similar reactions and also show evidence for electron attachment to the two carboxyl groups as well as the peptide linkage. An investigation as a function of pD shows that the site of electron attachment is strongly pD dependent with the carboxyl groups being favored at low pD and the peptide linkage favored at high pD.
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Introduction N-Acetylamino acids are of interest to radiation chemistry since they lack a primary amine group which is the fiial locus of electron attack in small peptides. They thus provide the simplest model for larger peptides. The radiation chemistry of N-acetylamino acids has been studied by a variety of technique~.l-~ Electron spin resonance investigations of irradiated single crystals of N-acetylglycine have provided the most detailed knowledge of radical mechanisms occurring after the primary ionization e ~ e n t . ~In- ~this case, both thedelectron and hole have been followed from initial production thru deamination and decarboxylation reactions to the final radical species. In this investigation we have continued our worklo with a study of irradiated frozen aqueous solutions of N acetylamino acids. We believe that in the case of Nacetylalanine we have elucidated the primary mechanisms of reaction. Experimental Section All N-acetylamino acids were obtained from Sigma. The N-acetylamino acids were y irradiated at 77 K with a dose of 0.5 Mrd in DzO. The radicals produced by the irradiation, their relative amounts, and their stability were studied as a function of temperature and pD. As in our previous work, all samples were annealed to 120 K to remove the signal due to the y irradiation of D20.l0 Dose studies showed that the number of radicals produced per unit dose of radiation was constant to well past 1 Mrd. Concentration studies showed that the radicals produced were proportional to the concentration of parent compound. The other experimental details are given in our previous report.1° Results and Discussion N-Acetyl-L-alanine. In Figure 1A we report the spectrum of N-acetyl-L-alanine (190 mg/mL of D20 at pD 8) a t 123 K after 0.5 Mrd of Co-60 y irradiation (77 K). After warming to 193 and 223 K, we observed the spectra in Figure 1B,C. From previous investigations in single crystal^^-^ and aqueous glassess-12 several radicals are expected to dominate these ESR spectra. They are the radicals produced by electron attachment (I and II), by 0022-3654/79/2083-2893$01 .OO/O
CH3C(0D)NDCHCH2CO2IA CH3C(O-)NDCHCH3C02IB CH,C(=O)NDCHCH,C(O-)OD I1 CH3C(=O)NDCCH3C02- CHCH coy I11 I$ CH3C(=O)NDCHCH3 V abstraction from the a carbon (III), by secondary deamination of the anion (IV),and by decarboxylation (V). Each of these species or species of similar structure have been observed in our previous work in aqueous glasses."12 Radical IB has been observed after electron attachment in alkaline glasses of N-a~etylalanine.~ Radicals I11 and IV have been observed after electron attachment in neutral (12 M LiC1-D20) glasses containing N-acetylalanine.* Radicals IA and/or I1 have also been observed in this medium.s Radical V was not directly observed, however, photolysis of dipeptides containing an aromatic residue and an alanyl residue have been found to produce radicals of the formll VI. In polycrystalline matrices the spectra of ND3+CHRCONDCHCH3 VI V and VI should be nearly identical. Radical IV has been produced in a separate experiment in which DL-alanine (220 mg/mL of DzO) was y irradiated (Figure 2A). This spectrum is found to be identical with one of the components of Figure 1B. Using this spectrum and computer subtraction methods, we resolved the spectra in Figure 1A-C into four component spectra which we believed are due to the individual radicals. These spectra are shown in Figures 1C and 2B,C. A comparison of Figures 1C and 2A-C with spectra reported previously for radicals I-IV shows that we have excellent agreement for the decarboxylated radical V (22 G, 4 H, g = 2.0029, Figure 2B), the deaminated radical IV (24 G, 4 H, plus resolved anisotropy, Figures 1B and 2A), and the abstracted radical I1 (18.7 G, 3 H, g = 2.0029, Figure 1C). The spectrum we associate with the anion (I or 11) is the final result of the subtraction process and must be considered with some uncertainty. 0 1979 American Chemical Society
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The Journal of Physical Chemlstry, Vol. 83, No. 22, 1979
H
Sevilla, D'Arcy, and Morehouse
though the doublet was only 22 G in spread. Finally, we note it is also likely from previous work by Sinclair and Codella3on N-acetylglycine single crystals that this species is a sum of radicals IA and 11. The relative fraction of each species as a function of temperature is estimated as follows: (1) 123 K, 45% I and 11,15% 111,40% V; (2) 193 K, 45% IV, 20% 111,35% V; (3) 223 K, 100% 111. These results can be explained as follows. At the low temperature (Figure 1A) we find anion radicals (I and 11) and decarboxylated radical (V) (reactions 1 and 2) as well as some abstracted radical (111). The anion CH&(=O)NDCHCH&02-
(-0
-+ -+
__*
Y
I1 K
CH3C(=O)NDCHCH,CO2*. CH3C(=O)NDCHCH3 V e-
C02 (1)
I1K + CH3C(=O)NDCHCH3C02- (+D+)
CH3C(OD)NDCHCH3C02CH,C(=O)NDCHCH&(OD)OH (2) Flgure 1. First derivative ESR spectrum of a y-irradlated (77 K) frozen aqueous solution of N-acetylalanine (190 mg/mL of D20at pD 8) after annealing to various temperatures: (A) 123 K, (B) 193 K, (C) 223 K. The spectrum in C is due to radical 111. The spectrum in A is analyzed in terms of radicals I, 11, 111, and V; that in B is analyzed in terms of radicals 111, IV, and V. The markers in the spectra represent the components of the Fremy satt spectrum. They are separated by 13.09 G with g = 2.0056.
B
Flgure 2. (A) ESR spectrum of the CH,CHCO,- produced by y irradiation of a frozen aqueous solution of m-alanine (220 mg/mL of D20)at 150 K. This spectrum is identical with that found for one component of Figure 1B. (B) ESR spectrum produced by the subtraction of 20% of radical I11 (Figure 1C) and 45% of radical I V (Figure ?A) from Figure 1B. This spectrum is assigned to radlcal V, CH3CONDCHCH3. (C) ESR spectrum produced by the subtraction of 15% of radical 111 (Figure IC) and 40% of radical V (Figure 2B) from Figure 1A. This spectrum is assigned to the anion radicals I and 11.
However, as is seen in Figure 2C, it appears as a triplet. This is believed to be an overlap of a 38-G doublet due to radical IA and a singlet due to 11. Later experiments under basic conditions show radical I is unquestionably formed (see below). In our previous work in 12 M LiCl for Nacetylalanine we also found a three-line spectrum,* al-
converts to the deaminated radical IV upon warming to 193 K (reaction 3). Further warming to 223 K results in
193 K
CH3C(=O)ND2 + CHCHBCOz (3) IV a nearly complete conversion to radical I11 (reaction 4). In
IA (and 11)
225 K
IV and V + CH3C(=O)NDCHCH3C02CH3C(=O)NDCCH3C02- + CH3CH2C02+ I11 CH3CONDCH2CH3 (4) experiments where the total spins were determined at each temperature we found that over 90% of the spin present originally converted to radical 111. This shows both IV and V undergo reaction 4. Further warming simply resulted in a loss of signal, presumably by dimerization and disproportionation. We note that the scheme proposed in reactions 1-4 is analogous to that found by Sinclair and Codella for Nacetylglycine single crystal^.^ The presence of some abstraction radical at low temperatures may suggest attack by matrix radicals (.OD and D-). However, the fact that the cation product and electron adduct predominate the signal and are present in nearly the same concentration shows the direct effect predominates. Experiments were performed with the solutions of N-acetylalanine adjusted to various pDs from 2 to >12. In each case the same set of radicals in different ratios are formed as at pD 8 with the exception of the spectra found at pD >12 for the anion radical, the precursor to IV. We show the spectrum of N-acetylalanine (pD >12) at 120 and 150 K in parts A and B of Figure 3. The change in the center of spectrum is found to be reversible. Subtraction of the spectra of radicals IV and V from Figure 3B gives the spectrum in Figure 3C. This spectrum is a 13.1-G quartet due to three protons. This, combined with the observation that it decays to the spectrum of radical IV (Figure 3D), clearly shows that it is due to radical IB. In our previous work in alkaline glasses this species gave a 13.5-G quartet and showed the same reversible temperature dependence. This phenomenon has been treated fully e1~ewhere.l~ The difference in the spectrum of the electron adduct at pD 8 and >12 is attributed to an increase in radical IB
The Journal of Physlcal Chemistty, Vol. 83,No. 22, 7979 2805
ESR of N-Acetylamino Acid Containing Solutions
spectra produced by y irradiation (77 K) of basic frozen aqueous solution of N-acetylalanine (180 mg/mL of D 2 0 at pD 12). (A) Initial spectrum at 120 K. (B) Spectrum at 150 K which shows the conversion of the central components to a quartet. This conversion is found to be reversible and is evidence for the anion radical IB. Fractions of radical V and radical IV are also found. (C) Spectrum of radical IB at 150 K resultlng from the computer subtraction of 15% of radical IV (Figure 1C) and 35% of radical V (Figure 28) from Figure 38. (D) The spectrum of the sample after further warming to, 200 K. The anion radical IB has completely converted to radlcal IV, CH,CHCO,. Figure 3. ESR
over 11. Thus the spectrum at pD 8 is thought to be principally that of 11. At lower pD (3) at 120 K there is a significant increase in the amount of I11 (to 30%) and a decrease in the amount of I and I1 (to 30%) relative to that found at pD 8. The decrease in anion I is corroborated by a decrease in deaminated radical IV (20%) found on warming to 193 K. These shifts are believed due to the conversion of electrons or electron adducts to deuterium atoms with a subsequent abstraction reaction (reaction 5). D.
+ CHSC(=O)NDCHCH3COzD
Flgure 4. ESR spectra produced by the y irradlatiin of a frozen aqueous solution of N-acetylglycine (80 mg/mL of D20, pD 4). (A) Initial spectrum at 105 K. (B) Spectrum found at 166 K. The conversion from doublet (A) to quartet (B) is found to be reversible and thus provides excellent evidence for the presence of radical VII. (C) The spectrum after warming to 233 K. This 17.9-G doublet is assigned to radical X and results from abstraction from the parent molecule.
4
CH3C(=O)NDCCH3C02D + D2 (5)
N-Acetylglycine. In Figures 4 and 5 we report the spectra found after y irradiation of N-acetylglycine (80 mg/mL of DzO) at pD e 4 and 13, respectively. In Figure 4A we find an -33-G doublet spectrum at 105 K which converts reversibly to the quartet of 13.5-G spacing shown in Figure 1B at 166 K. This is clear evidence for the presence of radical VII. CH&( 0D)NDCHZCOz- CH3C (O-)NDCH2COZVI1 VI11 CHZC02- CH,C(=O)NDCHCO,IX X CH,C(O-)=NCHCO,CH3C(=O)NDCHy XI1 XI The lack of resolution of further components in parts A and B of Figure 4 does not allow for the identification of further species expected from single crystal work or by analogy to N-acetylalanine such as the carboxylate anion or XII. Further warming to 233 K results in the gradual conversion to a 17.9-G doublet seen in Figure 4C. From previous work there is little doubt that this spectra is due to radical X. There is some evidence for IX as an intermediate between VI1 and X. At higher pD this species is quite clearly found. The results at pD 13 show the same reversibility in spectra at low temperatures (Figure 5A shows a spectrum intermediate between doublet and quartet). Upon
Flgure 5. ESR spectra produced by the y irradiation of a frozen basic aqueous solution of N-acetylglyclne (200 mg/mL of D20, pD 13). (A) Initial spectrum at 105 K. (B) Spectrum at 168 K. Radlcals Identifled in this spectrum are VI11 and IX. (C) Spectrum of radlcal XI produced by warming to 210 K. (D) Spectrum of -CH,C02-(radical IX) at 140 K produced by the y irradiation of glycine (220 mg/mL of D20) at 77 K.
warming to 168 K the spectrum of radical IX builds in (Figure 5B) as well as the quartet due to the anion VIII. The characteristic end components in Figure 5B verify the presence of IX. We show for comparison in Figure 5D the spectrum of radical IX produced by y irradiation of glycine (220 mg/mL of D20). Further warming to 210 K results in the appearance of the well-resolved spectrum shown in Figure 5C. This spectrum is associated with radical XI. The difference in resolution in radicals X and XI seems great for such similar radicals. We have found that in aqueous glasses that radicals of structures X and XI show a-proton couplings of 18 and 14 G, respectively.* However, in those matrices the resolution was not as good as found in Figure 5C. Since single crystal parameters are available for radical X,2-4we proceeded to simulate the spectrum of XI by a reduction in the a-proton tensor and an increase
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The Journal of Physical Chemistry, Vol. 83, No. 22, 1979
Sevilla, D'Arcy, and Morehouse
TABLE I single crystal parameters for X
parameters used in simulation of XI
A,, = 8.1G g,, = 2.0031 A,, = 7.0 G g, = 2.0029 A,, = 27.2 G g,, = 2.0045 A,, = 23.3 G g,, = 2.0042 A,, = 16.8 G g,, = 2.0020 A,, = 14.5 G g,, = 2.0020 a C H , = 2.7 G a C H , = 5.0 G line width = 3.5 G
Figure 7. ESR spectra of y-irradiated frozen aqueous solutions of N-acetyl-L-glutamic acid (180 mg/mL) at various pDs. (a) pD 2 (140 K), (b) pD 4 (1 15 K), (c) pD 6 (120 K), (d) pD 10 (120 K). The change in spectra with pD is associated with three possible sltes for electron attack on the parent molecule. The central singlet which Is associated with electron attack on site B is seen to decrease in relative abundance as the solution is made more basic.
Flgure 6, (A) ESR spectrum of radical XI (same as Figure 5C). (B) First-order computer simulation of the spectrum in A with the anisotropic parameters described in the text.
in the methyl group splitting. The values employed are shown in Table I and are consistent with isotropic values found for X and XI in aqueous solution at room temperature.14 The result of a simple first-order computer simulation is shown in Figure 6B. A comparison between parts A and B of Figure 6 confirms that Figure 6A arises from radical
XI. From the above we see that for N-acetylglycine we have been able to follow the anion pathway in these frozen aqueous solutions. It is likely that the cation or hole also follows the chemistry found here for N-acetylalanine and that found in irradiated single crystals of N-acetylglycine but due to poor resolution it is not observed in the ice matrix. N-Acetyl-L-glutamic Acid. N-Acetylglutamic acid (180 mg/mL of DzO) was investigated at various pDs (2,4,6, 10, and 12) of the original solution. The spectra formed a t low temperature show a marked dependence on pD (Figures 7A-D and 8A). The structure of N-acetylglutamic acid (XIII) has three possible sites for electron
,
L--..' q----yA CH,CH, ;CO,H;
L---J
XI11
attack, labeled A, B, and C. The sites labeled A and B have pKs near 4 and 2, respectively. The state of deuteration of N-acetylglutamic acid is expected to change in an analogous manner to the state of protonation. We interpret the spectra at low temperature as follows. Our work in aqueous glasses has shown that electron attach-
Figure 8. ESR spectra of a y-irradiated frozen solution of N acetyld-glutamic acid (200 mg/mL of D20, pD 12). (A) Initial spectrum at 120 K. (B)Spectrum at 150 K. The change in spectra between A and B is reversible and is excellent evidence for electron attack on site C. (C) Spectrum found at 200 K. This spectrum is chiefly due to radical XIV and contains a small fraction of XV. Radical XIV results from deamination of the anion (formed by electron addition to site C of structure XIII). (D) Spectrum of radical XV produced by warming sample to 215 K.
ment to site B will produce an unresolved singlet whereas attack on site A will produce a 24.7-G doublet. Attack at site C will produce an -30-G doublet at low temperature which reversibly converts to a 13.5-G quartet at high temperature. The singlet at pD 2 is thus indicative of electron attachment a t site B. Some doublet is also present. At pD 4 a mixture of singlet and doublet is found. A little reversible temperature effect is noted which is indicative of a small fraction of attack at site C. At pD 12 the singlet is lost and the substantial reversible conversion in spectra noted (Figure 8A,B). These results suggest that sites A, B, and C are all reactive toward the electron. The fraction of attack a t each site depends on the pH and gradually shifts from B A -,C as the pD is increased. Other experiments performed on y-irradiated glutamic acid in ice matrices and by electron attachment to glutamic acid in neutral aqueous glasses give results expected for attack only on sites A and B. These results support the analysis of anions reported here.
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The Journal of Physical Chemistry, Vol. 83,No. 22, 1979 2897
ESR of N-Acetylamino Acid Containing Solutions
proton is at an orientation so as not to interact strongly. This conformation of the side group was found to be that of radicals of similar structure in aqueous glas~es.~ In the present work an 18-G doublet is found for all N-acetylglutamic acid samples above pD 3. Below this pD no change is found on warming. N-Acetyl-L-methionine. N-Acetylmethionine was studied at pD 10 (185 mg/mL of DzO). Between 115 and 150 K the spectra show the reversible conversion in spectra which is indicative of electron attack at the peptide linkage, XVI (Figure 9A,B). No evidence for methyl radicals is
A
0
0I
CH,CNDCHCO,*
I
( CH2) XVI
ZSCH3
II
CH,CNDCCO,I
(CHJ ,SCH3 XVII
found as might be expected from electron attack on the SCH, portion of the mole~ule.~ Warming to 200 K results in an 18-G doublet associated with abstraction at the a-carbon site, XVII (Figure 9C). Flgure 9. ESR spectra of a y-irradiated frozen solution of Nacetyl-L-methionine (185 mg/mL of D,O, pD 10). (A) Initial spectrum at 115 K. (B) Spectrum after warming to 150 K. The change in spectrum was reversible and indicates that electron attack is at the peptide linkage to form radical XVI. (C) Spectrum found at 220 K arising from radical XVII.
Acknowledgment. The authors thank the Food Engineering Laboratory of the U.S. Army Natick Development Center and the US.Department of Energy for support of this work. The authors also thank Irwin Taub of the US. Army Natick Research and Development Center for helpful discussions.
In Figure 8C the spectrum of a N-acetylglutamic acid sample (pD 12) warmed to 200 K is shown. The spectrum extends over 88 G (1 H at 45 G, 2 H at 21.5 .G) and is characteristic of a radical of structure RCH2CHCO2-.l5 The spectrum is therefore assigned to the radical produced by secondary deamination, radical XIV. A fraction of the
References and Notes
0
co ,-CH,CH
,CHCO 2-
XIV
iI
CH,CNDCCO I
,-
CH,CH,CO
2-
xv doublet shown in Figure 8D is also present. The spectrum observed upon further warming to 220 K is shown in Figure 8D and is assigned to XV. The 18-G doublet arises from the interaction of one (3 proton with the unpaired electron at the a-carbon site. The other /3
I. A. Taub, J. W. Halliday, and M. D. Sevilla, Adv. Chem. Sei., in press. W. M. Garrison, Radiaf. Res. Rev., 3 , 305 (1972). J. Sinclair and P. Codella, J. Chem. Phys., 39, 1569 (1973). (a) G. Saxeboi, T. B. Melo, and T. Henriksen, Radiat. Res., 51, 31 (1972). (b) G. Saxebol, Int. J . Radiat. Bioi., 27, 293 (1975). H. C. Box, E. E. Budzinski, and K. T. Lilga, J . Chem. Phys., 57, 4295 (1972). P. Neta and R. W. Fessenden, J. Phys. Chem., 75, 738 (1971). S. Rustgi and P. Riesz, Int. J . Radiaf. Bioi., 34, 149 (1978). M. D. Sevllia and V. L. Brooks, J. Phys. Chem., 77, 2954 (1973). M. D. Sevilla, J. Phys. Chem., 74, 669, 2096, 3366 (1970). M. D. Sevilla, J. B. D’Arcy, and K. M. Morehouse, J . Phys. Chem., preceding article in this issue. M. D. Sevilla and J. B. D’Arcy, J . Phys. Chem., 82, 338 (1978). M. D. Sevilla, Radiat. Phys. Chem., 13, 119 (1979). D. Swyanarayana arid M. D. Sevilh, J. mys. Chem., 83, 1323 (1979). Y. Kirino and H. Taniguchi, J . Am. Chem. Soc., 98, 5089 (1976). M. D. Sevilla, J. B. D’Arcy, and D. Suryanarayana, J. Phys. Chem., 82, 2589 (1978).
