3814
J. fhys. Chem. 1980, 84, 3614-3619
Studies on Spin-Trapped Radicals in y-Irradiated Aqueous Solutions of Glycylglycine and Glycyl-L-alanine by High-Performance Liquid Chromatography and ESR Spectroscopy Furnlo Morlya,* Keisuke Maklno, Nobuhlro Suzukl, SouJIRokushika, and Hlroyukl Hatano Department of Chemistry, Faculty of Science, Kyoto University, Kyoto, 606 Japan (Received: June 19, 1980)
Aqueous solutions of glycylglycine and glycyl-L-alanine were y-irradiated in the presence of a spin trap, 2-methyl-2-nitrosopropane. Stable spin adducts produced in the y-irradiated solutions were analyzed by means of high-performanceliquid chromatographyand ESR spectroscopy. Five spin adducta were found and identified, as follows: t-BuN(O-)CH2CONHCHzCOO-(I)and NH,'CHzCONHCH(COO-)N(O~)-t-Bu (IIb)from glycylglycine; t-BuN(0.)CH2CONHCH(CH,)COO(111)) NH,+CH2CONHC(CH3)(COO-)N(0.)-t-Bu (IV) and NH3'CH2CONHCH(COO-)CH2N(0.)-t-Bu (V) from glycyl-L-alanine. It was found that spin adduct I11 exhibits ESR spectra with unequal splittings of the two P hydrogens while spin adduct I does not. This fact revealed that the asymmetry of the 6 carbon in spin adduct I11 causes the magnetic nonequivalence through a peptide bond. It was demonstrated that ESR spectra of spin adducts IIb and V changed remarkably with pH through the acid-dissociation equilibria of the carboxyl or amino groups. The pK, values for the dissociation have been determined to be 2.0 for the carboxyl group of spin adduct IIb, and 3.0 for that of V.
Introduction Concerning radiation effects on proteins, radical intermediates produced in aqueous solutions of many peptides by ionizing radiation have been observed by ESR spectroscopy during in situ radiolysis1s2or UV p h o t o l y ~ i and s~~~ in frozen glasses." The optical absorption of the transient species of peptides during pulse radiolysis has also been reported.8-10 Recently, the short-lived radicals produced in the aqueous solutions of peptides and proteins by y radiolysis and UV photolysis have been studied by the spin-trapping methodl1-l3using 2-methyl-2-nitrosopropane (MNP) as a spin trap.l4-I7 In most of these systems, ESR spectra obtained from the irradiated solutions are complicated because of overlaps of signals of several spin adducts. From such complicated ESR spectra it is not easy to investigate the structures of individual spin adducts only by ordinary computer simulation. Liquid chromatography has recently been combined with ESR spectroscopy for separation and detection of radical mixtures.l* This technique has been applied to separate and identify the spin adducts in the y-irradiated aqueous solutions of - * ~ MNP as several nucleotide^^^*^^ and amino a ~ i d s ~ ' with a spin trap, and also to investigation of y radiolysis of MNP itself in an aqueous solution.25 In the present work the spin-trapping method using MNP was applied to y-irradiated aqueous solutions of Gly-Gly and Gly-L-Ala. Short-lived radicals (Re) in the irradiated solutions were converted into the fairly stable nitroxide radicals (spin-trapped radicals or spin adducts) through reactions with MNP as represented by eq 1. f-Bu-NSO
+
R.
-
f-Bu-N-R
-
I 0.
t-Bu--+-R
From the ESR spectroscopic point of view, each atom in a spin adduct is designated cy, p, y, etc., as follows:
-N -C -C8-
Nr-
I I a I I 0.
The spin adducts were separated by high-performance 0022-3654/80/2084-3614$01 .OO/O
liquid chromatography and identified by ESR spectroscopy. The spin adducts of the dipeptides were characterized from their ESR spectra. Experimental Section Gly-Gly was purchased from Protein Research Foundation, Osaka; Gly-L-Ala was from Sigma Chemical Co., Missouri. MNP was synthesized and purified according to the method of Stowell.2e All other chemicals were of reagent grade. Aqueous solutions of MNP (5 mg/mL) were prepared in the dark by stirring for 1 h at 45 0C.27The concentration of the dipeptides in MNP solutions was 0.1 M. In the air the sample solution was cooled by ice and irradiated with 6oCoy-rays at a dose rate of 6.0 X lo6 rd/h to a total dose of 3.0 X lo5 rd. Immediately after the irradiation, 2 M phosphate buffer ([Na2HP04]/[NaH2P04] = l / J was added to the solution. Then 1.0 mL of the mixed solution was loaded on a 0.95 X 60 cm cation exchange column (IEX-21OSC of Toyo Soda Manufacturing Co., Tokyo) equilibrated with the starting eluent. The eluate was passed through UV and ESR detectors and collected into fraction tubes. The chromatographic system was illustrated in the previous paper.lg The high-performance liquid chromatograph was a Toyo Soda HLC-803. The UV detector was a JASCO UVIDEC 100 (10-mm light path), which was tuned to 240 nm. The ESR spectrometer was a JEOL PE-SX, which was operated at 100 kHz modulation frequency in the X band. For detection of the radicals during chromatography, the magnetic field was fixed at the positions indicated by the vertical arrows in Figures 1 and 6. The magnetic-field modulation was applied at a high amplitude as 10 G to cover a wide range. The eluents were degassed aqueous solutions and were used sequentially for the chromatography. The conditions of the chromatography were as follows: pressure, ca. 70 kg/cm2; flow rate, ca. 0.2 mL/min; temperature, ca. 25 "C. The ESR spectra were recorded at ca. 25 "C in the dark for the y-irradiated samples, and the fractions found to have radicals. Some of the fractions were mildly bubbled with nitrogen gas in 2 min in order to exclude a lowering of the ESR spectral resolution because of dissolved molecular oxygen. Hyperfine splitting constanta (hfsc's) were 0 1980 American Chemlcal Society
The Journal of Physical Chemistry, Vol. 84, No. 26, 1980 3615
Spin-Trapped Radicals of Gly-Gly and Gly-L-Ala
ii7
10 G
Flgure 3. ESR spectrum of the separated spin adduct obtained from the fractions giving peak A in Figure 2 at pH 6.0. Flgure 1. ESR spectrum of an aqueous solution of Gly-Gly with MNP observed just after y irradiation (at pH 5.6). During chromatography the magnetic field was fixed at the position lndlcated by the vertical arrow. 0.4
0.3 0.2 0.1
8 E:
W
0
cu) C c
E I
0
E
.-m
UI
a
m W
0
10
20 Elution
Eluent
1
30 40 50 Volume (mL)
--JIc
Eluent
2
4
Figure 2. Chromatogram of the y-irradiated aqueous solution of Gly-Gly with MNP drawn by UV (dashed line) and ESR (solid line) detections. Eluent l; 0.25 M sodium phosphate buffer, pH 6.0. Eluent 2; 0.2 M Na,HP04-NaOH buffer, pH 11.5.
measured by using Mn2+in MgO as a reference. The pH values of the solutions were adjusted by addition of dilute HC1 or NaOH and read in a Hitachi-Horiba pH meter M-5. The UV absorption spectra were recorded with a Cary 17 spectrophotometer of Varian Co., California. In order to detect intact dipeptides, we heated each fraction for color development with ninhydrin reagent in a water bath. Ninhydrin-positive fractions were analyzed by using an amino acid analyzer (Hitachi KLA-3B).
Results and Discussion Glycylglycine. An aqueous solution containing 0.1 M Gly-Gly and 5 mg/mL MNP, cooled in an ice-bath, was y-irradiated to a total dose of 3.0 X lo5 rd. The ESR spectrum of this solution is shown in Figure 1. The ESR signals asterisked in Figure 1 decayed relatively fast and disappeared within 1 h. High-performance liquid chromatography was applied to the solution, immediately after the irradiation and the subsequent addition of 2 M phosphate buffer. The chromatogram by UV and ESR detections is shown in Figure 2. The ESR chromatogram exhibited two peaks as shown by the solid line. Typical ESR spectra for the fractions of peaks A and B in Figure 2 are depicted in Figures 3 and 4A, respectively, which are characteristic patterns of the spin adducts from Gly-Gly. These signals are revealed in the ESR spectrum in Figure 1. Most of the spin adducts produced by the self-trapping of MNP25were adsorbed on the column and eluted very slowly with diffusion. On the UV chromatogram shown by the dashed line in Figure 2, the third peak was due to intact Gly-Gly detected by using an amino acid analyzer. The large fourth peak was mainly
Figure 4. ESR spectra of the separated spin adduct obtained from the fractions giving peak B in Figure 2. A, 8,and C were observed at pH 6.0, 1.9, and 1.0, respectively.
due to tert-butylnitrosohydroxylamine(t-BuN(OH)N=O) characterized by the pH dependence of UV spectraa2' Figure 3 shows the ESR spectrum obtained for the fraction of peak A, which can be analyzed as a tripletriplet. The primary triplet of 15.9-G splitting is due to the nitrogen nucleus in the nitroxyl group. The secondary triplet of 9.1-G splitting with an intensity ratio of 1:2:1is assigned to two equivalent 0 hydrogens. The hfsc's can be clearly distinguished from those of the deaminated glycine adduct previously reported (uN = 16.1 G and a,,(2H) = 8.45 G)." It is well-known that, when hydrated electrons react with peptides, a reductive deamination is caused by the addition of e, - to the carbonyl groups of the peptide bond^.^^^^^ The ASR signal pattern in Figure 3 was assigned to the deaminated Gly-Gly adduct, whose structure can be written as follows: ~-BU-N-CH~-CONH-CH~-COO-
I
0.
I
Parts of the ESR signals in Figure 3 coincided with the asterisked signals in Figure 1. The separated spin adduct I in the fraction of peak A was fairly stable in contrast to the unseparated one in the irradiated solution. Figure 4A shows the ESR spectrum for the fraction of peak B, which can be analyzed as a triplet of four lines with an apparent intensity ratio of 1;2:2:1, and which is apparently a major component of ESR signals in Figure 1. The primary hfsc of a nitrogen nucleus is 15.6 G, and the secondary splittings were assigned to a /3 nitrogen and a /3 hydrogen ( a g . ~= 2.4 G and U ~ . H= 2.2 G). This ESR signal pattern suggests that the hydrogen abstraction by OH radicals occurs from either methylene group of two glycine residues, and it results in the formation of a spin
3616
The Journal of Physical Chemistry, Vol. 84, No. 26, 1980
Moriya et ai.
r I
f 0.8 I
I 0
2
1
3
4
5
PH Flgure 5. The effect of pH on the difference of the /3 hfsc's for spin adduct IIb. Circles indicate experimental results. Best-fitted soild line was calculated by eq 3.
adduct which has a component part, t-BuN(O.)CHN, that is, structure IIa or IIb. In the neutral and alkaline soluNH
:-
CH -CONH-
t -Bu -N
I -0.
Figure 6. ESR spectrum of an aqueous solution of GIyd-Ala with MNP observed just after y irradiation (at pH 5.5). During chromatography the magnetic field was fixed at the position indicated by the vertical arrow.
cH~-COO-
IIa NH,+-CH~-CONH-CH-COO-
I
t -Bu-N-O*
IIb
tions the spectral pattern was pH independent, which suggests the absence of an amino group near the nitroxyl group. However, in the acidic pH range, the spin adduct exhibited reversible pH-dependent shifts of the signals as shown in Figure 4. At pH 1.0 the hfsc's, a N , aPN, and a,H, are 15.2,2.6, and 1.6 G, respectively. The spectral changes are mainly due to the increase of the difference of the 6 hfsc's, and seem to be caused by the protonation of the carboxyl group in the vicinity of the nitroxyl group. From the observation of such reversible pH effects, the spin adduct was assigned not to structure IIa but to IIb derived from a short-lived radical which is produced by the hydrogen abstraction from the carbon adjacent to the carboxyl group of Gly-Gly by OH radicals. The differences - aPH,were plotted as a function of the /3 hfsc's, Aag = aBSN of pH in Figure 5. In the acid-dissociation equilibrium of the carboxyl group of adduct IIb, if the interchange between the acid form (A) and the base form (B) is rapid, which is the case here, the observed Aa, represents the weighted average of the two form^.^^^^^ For a given solution the acid dissociation constant can be calculated by eq 2, where AaBAand AagBare the differences of the /3 AagB- Aag
pKa = pH
+ log ff-AB = pH + log AaB- AagA
(2)
hfsc's for the acid and base forms, respectively, and fA and fB refer to the fraction of each form (fA + fB = 1). From eq 2 a difference of /3 hfsc's, Aag, is derived as a function of pH in eq 3, where AaBA= 1.1 G and AaBB= 0.2 G for AagA AaBBX 10pH-pKa Aag = (3) 1 + 10pH-pKa
+
adduct IIb. The best fit with the experimental points was obtained by substituting pKa = 2.0 to eq 3 as shown by the solid line in Figure 5. The pKcooD value for adduct IIb in D 2 0 has been determined to be 2.5 previou~ly.'~ The UV absorption spectrum obtained from the fractions of peak B exhibited a broad slope extended from the far UV range to ca. 340 nm with a gentle shoulder near 250 nm. Unsaturated bonds of Gly-Gly and di-tert-butyl nitroxide originally have intense absorption bands around
0
10
20
Elution Eluent
Volume 1
30
LO
( mL)
+-
Eluent
2
4
Flgure 7. Chromatogram of the y-irradiated aqueous solution of Gly-L-Ala with MNP drawn by UV (dashed line) and ESR (solld line) detections. Eluent 1; 0.1 M sodium phosphate buffer, pH 6.0. Eluent 2; 0.2 M Na,HPO,-NaOH buffer, pH 11.5.
185 nm ( t N 7 X 103)32and 240 nm ( t N 3 X 103),33 respectively. Therefore the obtained UV spectrum is speculated to be mainly an overlap of the two absorption bands, and spin adduct IIb seems to be isolated in a fairly high state of purity from other radicals and diamagnetic compounds in the y-irradiated solution. These inferences are also supported by the good correspondence of the peak on the UV chromatogram at 240 nm to peak B on the ESR chromatogram in Figure 2 and by the relationship between the absorption intensity of the UV spectrum and the concentration of adduct IIb. Glycyl-L-Alanine. Figure 6 shows the ESR spectrum of a y-irradiated aqueous solution of Gly-L-Ala with MNP. The ESR signals asterisked in Figure 6 decayed relatively fast and disappeared within 1 h. The chromatogram by UV and ESR detections is shown in Figure 7. The ESR chromatogram exhibited three peaks as shown by the solid line. Typical ESR spectra for the fractions of peaks A-C in Figure 7 are depicted in Figure 8A-C, respectively,which are characteristic patterns of the spin adducts from Gly-L-Ala. These signals are partly revealed in the ESR spectrum in Figure 6. On the UV chromatogram shown by the dashed line in Figure 7, the third peak was due to intact Gly-L-Ala and the large fourth peak was mainly due to tert-butylnitrosohydro~ylamine.~~ Figure 8A shows the ESR spectrum for the fraction of peak A, which can be analyzed as a triplet of four lines with an intensity ratio of 1:l:l:L The primary hfsc of a nitrogen
Spin-Trapped Radicals of Gly-Gly and Gly-L-Ala
nucleus is 16.0 G, and the secondary splittings were assigned to two nonequivalent fl hydrogens (agH= 10.0 and 8.2 G). This ESR signal pattern was assigned to the deaminated Gly-L-Ala adduct, whose structure can be written as follows:
t-Bu-7
--CH~-CONH-*CH-COO0.
I
CH3
I11
The Journal of Physical Chemistry, Vol. 84, No. 26, 1980 3617
10 G
Parts of the ESR signals of adduct I11 coincided with the asterisked signals in Figure 6. The separated spin adduct I11 in the fraction was fairly stable in contrast to the unseparated one in the irradiated solution. Spin adduct I11 exhibits ESR spectra with unequal splittings of the two fl hydrogens while adduct I does not. It has already been known that two fl-H hfsc's are usually unequal for a tert-butyl nitroxide radical which has both an LY methylene and an asymmetric fl carbon in the molecule, that is, tBuN(O-)-CH2-*CXYZ (X # Y # Z # X).34The origin of the nonequivalence of the two 0 hydrogens in such a radical has been interpreted by assuming, for instance, interconvertion between two minimum energy conformers which have mutual1,y different sets of dihedral angles between the plane of HP-C, and C,-N bonds and the plane including the C,-N bond and the p orbital on nitrogen.34 In the present case the spectrum shown in Figure SA reveals that even the ,%symmetryof the 6 carbon in adduct I11 causes the magnetic nonequivalence of the two fl hydrogens. Generally a peptide bond forms a fairly rigid planar structure because of a partial double bond character of the C-N bond as represented by the two resonant structures in eq 4.35 Consequently the strong asymmetric
Figure 8. ESR spectra of the separated spin adducts. A-C represent the spectra obtained from the fractions giving peaks A-C in Figure 7, respectively. A and B, pH 6.0; C, pH 8.4. Stick dlagram is added for C.
effect of the 6 carbon in adduct I11 seems to be attributable to the medium of the planar structure. To our knowledge a radical having such1 characteristics has been found by the chromatographic separation for the first time. Figure 8B shows the ESR spectrum for the fraction of peak B, which can be analyzed as a triplet-triplet, and which is apparently a major component of ESR signals in Figure 6. The primary hfsc of a nitrogen nucleus is 15.2 G, and the secondary 3.4-G triplet with equal intensities was assigned to a p nitrogen. This ESR signal pattern was assigned to structure IV derived from a short-lived radical NH:-Cti2-CONH-C(CH3)-COO-
I
t-Bu-N-0.
IV
which is produced by the hydrogen abstraction from the carbon adjacent to the carboxyl group of Gly-L-Ala by OH radicals. Each line of MI = 0 and +1 components in Figure 8B slightly splits into several lines. Figure 9A shows the enlarged spectrum of MI = 0 component, which appears that the slight y hfs consists of equally spaced even lines. At least eight lines of the y hfs were confirmed by the observation of a second-derivative spectrum. In spin adduct IV, y nuclei having spins of I I1/2 are nine hydrogens in the tert-butyl group from MNP, three in the methyl group of the L-alanine side chain, and one in the peptide bond, and no 6 nuclei have spins of 1 I1/2. On the basis of this fact the spectiral simulation of Figure 9A was made by considering various cases. The simulated spectrum shown in Figure 9B was computed with a p nitrogen of
Flgure 9. (A) Enlarged ESR spectrum of M, = 0 component in Figure 8B. (B) Computer-simulated spectrum for A. Parameters used for spectrum simulation are specified in the text.
3.4-G hfs, 13 equivalent y hydrogens of 0.3-G hfs, and Lorentzian peak-to-peak line widths of 0.35 G. The good agreement of the simulated spectrum with the observed one suggests that the y hfs of spin adduct IV consists of all y hydrogens containing the tert-butyl group originated from MNP. It is a rare event that the 7-H hyperfine splittings due to the tert-butyl group are observable. The y hfs of adduct I V can be interpreted as 14 lines with an intensity ratio of a binominal coefficient set, 1:13:78:286:715:1f!87:1716:1716:1287:715:286:78:13:1. Pure spin adduct IV vvas isolated in the fractions of peak B. Figure 8C shovvs the ESR spectrum for the fraction of peak C, which apparently exhibits as a mixture of six
3618
The Journal of Physical Chemistry, Vol. 84, No. 26, 1980
Moriya et al.
TABLE I : Hyperfine Splitting Constants (G) of Spin Adducts from Dipeptides
i
substrate Gly-Gls
structurea pH
I IIb
1.0
Gly-L-Ala
I11
IV V
aN
ap-u
6.0 15.9 6.0 15.6 6.0 6.0 12.2
8.4 6.0 2.0
aO-N
a7-U
9.1b 2.2 1.6
15.2 16.0 15.2
10.0,8.2
16.4 16.4 16.4 16.4
13.9,10.1 14.2,9.9 14.6 9.7 13.7b
2.4 2.6 3.4
0.3c
0.7 0.7 0.7 0.7
a Shown as structures at pH 6.0. Two equivalent hydrogens. Thirteen equivalent hydrogens.
Figure 10. ESR spectra of spin adduct V observed at various pHs: (A) pH 12.2; (B) pH 6.0;(C) pH 3.2; (D) pH 3.0; (E)pH 2.8; (F) pH 2.0.
narrow and six broad lines. Each of the former lines further splits into a doublet. Since the narrow set and the broad set gave the same g value and N hfsc, the spectrum was inferred to be due to only one kind of spin adduct with line-width alternation. It can be analyzed as three groups of a 1:l:l:l quartet whose inner pair is selectively broad. The stick diagram is added to the bottom of Figure 8C in which the minor doublets are neglected. The primary hfsc of a nitrogen nucleus is 16.4 G, and the secondary splittings were assigned to two nonequivalent p hydrogens and a y hydrogen (up.H = 14.2 and 9.9 G, and uy.H = 0.7 G at pH 8.4). Such a spectrum as shown in Figure 8C is a typical pattern of a nitroxide radical, t-BuN(0.)-CH2-*CXYZ (X # Y # Z # X).34 The nonequivalence with line-width alternation is interpreted by assuming, for instance, insufficiently rapid interconversion between two rotational isomers which have mutually different sets of 0-H hfscs. The mechanism of such hfs patterns has been discussed in detail by Gilbert et al.34 The spin adduct was assigned to structure V derived from a short-lived radical which is N H ~+--CH~-CONH-*CH-COO-
I
rHz
t -Bu-N-0.
V
produced by the hydrogen abstraction from the methyl group of Gly-L-Ala by OH radicals. Spin adduct V ex-
hibited reversible spectral changes accompanying pH variation of the fraction as shown in Figure 10. In the alkaline pH ranging from ca. 7 to 9.5, small pH-dependent shifts of signals were observed, which were attributable to the proton-dissociation equilibrium of the amino group of adduct V. At pH 12.2 (Figure lOA), the P hfsc’s (2H) of the adduct having a deprotonated amino group are 13.9 and 10.1 G. The ESR signals were almost pH independent in the pH range ca. 4-7 (Figure lOB), which were assigned to the zwitterion form of adduct V. The P-H hfsc’s at pH 8.4 nearly represent the arithmetic means of the P-H hfsc’s at pH 12.2 and those at pH 6.0 (14.6 and 9.7 G). Therefore the pKa value for the amino group of adduct V was inferred to be -8.4. In the acidic pH range ca. 2-4, remarkable spectral changes were observed with pH-dependent broadening and shifts of the signals as shown in Figure 10C-F. It seems to be caused by the proton-dissociation equilibrium of the carboxyl group of adduct V. Figure 10F shows the ESR spectrum of adduct V having a nearly protonated carboxyl group at pH 2.0 whose two 0hydrogens gave equal hfsc’s of 13.7 G with line-width alternation. At pH 3.0 the broadest ESR signals were observed and situated near the averaged positions of the signals at pH 6.0 and 2.0. These facts suggest that the pK, value for the carboxyl group of adduct V is 3.0. The line broadening around pH 3 refers to moderate exchange rates not only between the conformers based on the asymmetric P carbon but also between the positive-charged and zwitterion form^.^"^^ The N hfsc and the y-H hfsc of adduct V were pH independent. N
Conclusions Five kinds of spin adducts in the y-irradiated aqueous solutions of Gly-Gly and Gly-L-Ala with MNP as a spin trap were separated and identified by high-performance liquid chromatography and ESR spectroscopy. Their structures and hfsc’s are summarized in Table I. This method indicates that most of the short-lived radicals previously reported in aqueous solutions of Gly-Gly1,3~6~6 and Gly-~-Ala~@,’ are spin trapped. Spin adducts I and I11 are derived from deaminated radicals produced by the addition of ea; to the peptide carbonyl groups of Gly-Gly and Gly-L-Ala,respectively. Spin adducts IIb and IV are derived from a radical produced by hydrogen abstraction by OH radicals from the carbon adjacent to the carboxyl group of Gly-Gly and Gly-L-Ala, respectively. Spin adduct V is derived from a radical produced by hydrogen abstraction from the methyl group of Gly-L-Ala by OH radicals. From these results on dipeptide systems, proposed y-radiolytic and spin-trapping mechanisms to produce spin adducts are summarized as follows:
The Journal of Physical Chemistry, Voi. 84, No. 26, 7980 3619
Spin-Trapped Radicals of Gly-Gly and Gly-L-Ala R’
R’
I NH~+-CHCO--Y
/vw-
Co., Ltd., Tokyo, for supplying a high-performance liquid chromatograph, HLC-803,-and a cation exchange column, IEX-21OSC.
MNF
.CHCO-Y
9’
References and Notes
I
(5)
t-Bu-N-CHCO-Y
I 0.
I
X-NHCCOO
- .on
n-X-NHCCOO
I
MNP
I
I
I HR3
X-NHCCOO
(6)
I
t-Bu-N--O.
ti X-NHCHCOO
P2 l -
-
-
-
.‘DH
nN\rX-NHCHCOO
-
MNP
X-NHCHCOO-
I R3
(7)
I
t-Bu-N-0.
I
where each of X and Y represents an amino residue, R1 and R2are H or side chains, HR3 is an aliphatic side chain, and the left, centrad, and right members of each equation are a dipeptide, a short-lived radical, and a spin adduct, respectively. The spin adducts in eq 6-7 were called a deamino adduct, a backbone adduct, and a side-chain adduct, respectively. Though the secondary deamination has been reported by the frozen method’ and the spin trapping with sodium formate,16J7“secondary deamino adducts” such as t-IBuN(O.)CH(CH,)COO-from Gly-iAla were not found. “Amino-terminal backbone adducts” such as t-BUN(0.)CH (NH3+)CONHCH(CH,) COO- from GlyL-Ala were not ascertained also. Spin adduct I11 exhibits ESR spectra with unequal splittings of the twlo /3 hydrogens while adduct I does not. This fact revealed that the asymmetry of the 6 carbon in adduct I11 causes the magnetic nonequivalence through a peptide bond. The y-H hyperfine splittings due to the tert-butyl group originated from MNP have been observed in ESR spectra of spin adduct IV. ESR spectra of spin adduct IIb and V changed with pH through the acid-dissociation equilibria of the carboxyl or amino groups in the molecules. The pK, values for the dissociation have been determined to be 2.0 for the carboxyl group of spin adduct IIb, and 3.0 for that of V. Acknowledgment, We express our sincere gratitude to Dr. Hitoshi Taniguchi, Yamaguchi University, for valuable discussion. We are indebted to Toyo Soda Manufacturing
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