Oct., 1963
E.s.R.STUDIES O F IRRADIATED SINGLE CRYSl'ALS O F SUGARS
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ELECTRON SPIN RESONANCE STUDIES OF IRRADIATED SINGLE CRYSTALS OF SUGARS BY HISASHI UEDA~ Department of Physics, Duke University, Durham, Yorth Carolina Received April 5, 196.9 Single crystals of lactose hydrate, sucrose, methyl-D-glucoside, glucoronolactone, n-ghcosamine-HC1, and diacetone sorbose were irradiated at 77°K. and their e.8.r. spectra were observed immediately after irradiation or after annealing a t 193°K. These sugars also were irradiated a t room temperature, and their e m . spectra were observed a t this temperature. The position of the substituted functional group in a substituted sugar molecule is more accemible to radiation damage than other positions in the molecule. Therefore, the positions are selectively damaged by irradiation. For this reason, the e.s.r. spectra of irradiated single crystals of sugar derivatives differ greatly from those found in the unsubstituted parent sugars. The free radicals formed in sugars by irradiation a t 77°K. are transformed by subsequent annealing. These transformation processes can be explained by a change in the configuration of the radical species in most instances. However, there are a few cases where the transformation includes migration of the free-radical site.
intensities and the widths of the lines change a little Introduction with the crystal orientation. The possible structures Several authors have studied the radiation damage of the free radicals are as shown in Fig. 2. The free induced by ionizing radiation in sugars by means of rqdical shown in Fig. 2.4 gives rise to the double triplet electron spin r e ~ o n a n c e . ~ -Some ~ of these s t ~ d i e s ~ - ~ shown in Fig. 1A. The interaction of the unpaired were made on polycrystalline materials. The author electron with the two protons indicated by the dotted has studied the e.s.r. spectra of irradiated single crystals a double triplet, while the further splitarrows produces of sucrose,'j L-sorbose, and ~-fructose.' I n the case of ting is due to the proton in the OH group a t the left end sorbose and fructose, the e.s.r. spectra depended reThe spin density, calculated from this in Fig. 2A.9 markably on the irradiation temperature. In the configuration and the triplet splitting of 25 gauss, is present work, therefore, single crystals of other sugars approximately 0.6 at the carbon atom indicated by a were studied in order to observe a similar effect of temlobe. The spin density calculated from the formula perature. (splitting factor)/2& cos20 using the same value of cos26 Experimental (0.91) and Q (25 gauss) as were used for fructose and The paramagnetic resonance was observed with an x-band sorbose' is about 0.6 a t the carbon atom indicated by a spwtrometer a t 77 and 293°K. The e.8.r. signal was recorded lobe. The free radical shown in Fig. 2B gives rise as the second derivative of the absorption curve. to the doublet shown in Fig. 1A. The interaction The single crystals of Bucrose and glucuronolactone were preof the unpaired electron with the proton indicated pwed by slow evaporation of aqueous solutions. This method by the dotted arrow accounts for the doublet. was not successful in the other cases. The single crystals of i~iethyl-i~..glucositlewere grown by slowly cooling a methanol The spin density calculated from this configurasolution in a dewar flask. In a. similar way, single crystals of Dtion and the doublet spacing of 25 gauss is also 0.6 glucosamine-HCl were prepared by slowly cooling a solution in a t the carbon atom indicated by a lobe. diluted HC1. The single crystals of lactose hydrate were pseWhen the crystal irradiated a t 77'K. was warmed to pared by slowly cooling an aqueous solution. The single crystals of diacetone sorbose werle grown by the slow evaporation of a 193'K. and then cooled to 77'11., the e.s.r. spectrum solution in the mixed solvent, two parts of acetone: three parts of observed was similar to that in Fig. 1A. However, the methanol . splittings for the double doublet were 31 and 6.5 gauss, The irradiations, both a t 77 and 293"K., were made. using aiid the splittings for the doublets (the doublet in Fig. y-rays from a 6oCosource. Some of the crystals, after irradiation 1A further split into four lines a t this temperature), a t '7'7°K. and before measurements were made, were annealed in a mixture of Dry Ice and petroleum ether and then cooled to 77°K. were 26 and 5.0 gauss, respectively. This means that again. the configurations of the free radical shown in Fig. 2A and 2B have changed slightly. The increment in the Results OH proton splitting may be interpreted either as an A. Lactose hydrate is a monoclinic crystal.8 When increment of the spin density 01-1 the carbon atom a t it was irradiated a t 77"K., the spectrum observed at which the unpaired electron is mainly located or as a t'he same temperature consisted of a double triplet and change of the location of OH proton. When the crystal a doublet as shown in Fig. 1A. The positions and the was irradiated at room temperature, the observed spacings of t'hese lines are the same a t any orientation spectra were isotropic in their spacings. These spectra of the crystal in the magnetic field. The ratios of the also consist of two kinds of lines, as shown in Fig. lB, a (1) This work was supportsed by the Air Force Office of Scientific Redoublet with a splitting of 22.5 gauss and a double search, Air Research and Development Command, Grant No. AF-AFOSRdoublet with splittings of 20 and 29 gauss. It can be 62-327. (2) h-ishina Memorial Fellow. concluded that double doublet in Fig. 1B and the (3) .J. D. Williams, J. E. Gensic, AX. L. Wolfram, and L. J. McGabe, double triplet in Fig. 1A arise from the interaction of I'roc. Natl. Acad. Sei. U . S., 44, 1128 (1958). the unpaired electron with the same two protons. (4) J. D. Williams, €3. Schmidt, .M. L. Wolfram, A. JIichaelakis, and L. J. McGabe, ibid., 46, 1'740 (19 The triplet component in Fig. 1A is due to the coupling ( 5 ) Z. Kuri, Y . Fujiwara, eda, and S. Shida, J . Chem. P h p . , 33, 1884 of two equivalent protons. Now, if these two protons (infin). become nonequivalent, on account of a slight change in (fi) 1% Ueda, Z. Kuri, and S. Shida, ihid., 36, 2145 (1961). (7) H. Ueda, J. I'hys. Chem., 67, 966 (1963). the configuration of the free radical when the crystal
( 8 ) P. Grotli, "Chemisclie Krystallograpliie," Vol. 3, Engelmann, Leipzig, 1908 D. 440.
(9) W. Derbyshire, MoE. Phzls., 6 , 225 (1962).
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t50G suss/
t29G4 Fig. l.-E.s.r. spectra of irradiated lactose hydrate. A: irradiation and observation a t 77%, H//a; the vertical lines indicate the grouping of the lines; the arrow indicates the position for g = 2.0036; B: irradiation and observation a t room temperature, H//b.
Vol. 67
Fig. 3.-E.s.r. spectra of irradiated methyl-n-glucoside, irradiation and observation a t 77"K., low microwave power; A: L a H = 105", L b H = 15"; B: L c H = 15', (110)//H.
B. Sucrose.-The spectra from sucrose irradiated a t room temperature have been reported.6 Therefore, this work is mainly concerned with the results observed with sucrose irradiated a t 77°K. The spectra of sucrose irradiated a t 77°K. and observed a t 77°K. are the same type as Fig. lB, with splitting factors as shown in Table I. In this case, as in the case of lactose hydrate, the spacings are constant. When this crystal is warmed to 193'K. and cooled to 77"K., the spectrum observed shows a broadening, but it is essentially the same as that before annealing. The g-factors of the two spectra are a little smaller than that of DPPH. When the crystal is warmed to room temperature, the splitting factors change as shown in Table I and the lines have small line widths. VALUESOF
THE
TABLEI SPLITTING FACTORS AT VARIOUSTEMPERATURES
Lactose hydrate
@ - - - CH201-I --H
0 ---c ---0
6 ---OH
Fig. 2.-The structure of the free radicals produced in lactose hydrate irradiated at 77°K. The dotted lines show the nuclei with which the unpaired eleetron, indicated by the lobe, interacts.
temperature is raised, these two protons will give rise to two doublets with different spacings. At room temperature, there is some distortion in the pyranose ring' when the bond scission, a t the positions indicated in Fig. 2, takes place and the original configuration with sp3 tetrahedral angles changes to one with sp2 angle^.^ The original configuration of the pyranose ring remains substantially unaltered when the crystal is irradiated a t 77°K. If the crystal is warmed to room temperature or if it is irradiated a t room temperature, a displacement of the hydrogen atom occurs to compensate for the ring deformation. Owing to the asymmetry of the molecule in the neighborhood of the two carbon atoms, indicated by lobes in Fig. 2A and 2B, the displacement of the two neighboring hydrogens can be different. Similarly, there can be a displacement of the H atom of the OH group and 110 splitting by this H is observed at room temperature.
Sucrose
Me-D-glucoside
Tempera tures" LN D I RT LN D I RT LX D I RT Doublet 25 26 23 28 28 19 31 30 20 Double 25' 31' 21 15 15 10 30' 32* 9 doublets 29 28 28 19 20 a LN signifies irradiation and measurement a t 77°K.; D I signifies irradiation a t 77"K., then heating to 193"K., cooling, and measurement a t 77°K.; and R T signifies measurement a t room temperature. In these cases the two splittings for the double doublets coincide; therefore they are the splittings for the triplet. The triplet observed in the spectrum from the single crystals of diacetone sorbose (see section F) when irradiated a t 77°K. has a splitting factor of 24 gauss.
C. Methyl-D-glucoside.-a-Methyl-D-glucoside is a rhombic crystal. When this crystal is irradiated at 77"K., the spectra obtained clearly show anisotropy. There are two groups of lines, the observation of which is dependent on the microwave power used. The one group of lines is of the same nature as that found in lactose hydrate when irradiated a t 77°K. (Fig. l a ) . These lines, therefore, are isotropic with respect to the g-factor and the spacings. The second group of lines, as shown in Fig. 3A is strongly anisotropic. These lines are observed only with a large microwave power, when the first group of lines becomes relatively weak due to saturation. On the basis of the unusually large splittings observed for the second group of lines and the anisotropic nature of their g-factor, it is suggested that the free radical present is .OCH,. When this crystal is warmed to 193'K., the second component disappears and the resulting spectrum, even a t a higher power, is of the same nature as that shown in Fig. 1A. When the crystal is irradiated a t room temperature, the spectrum observed is almost the same as that of siicrose.
E.s.R. STUDIESOF IRRADIATED SINGLECRYSTALS OF SUGARS
Oct., 1963 ,
2187
X Ll i
1 i!
YIH I8O-
+'I-+
1 Y Y
I
I
. 3 - 1 Fig. 4.-E.s.r. spectra of irradiated D-glucosamine-HC1. -4: irradiation and observation a t 77"K., low microwave porn-er; LaH = 4 5 O , L b H = 135"; B: irradiation a t 77'K., annealing a t 193OK., and observation at 77°K; L a H = 45', L b H = 135"; C: irradiation and observation a t room temperature, low microwave power; H//b.
D. D-Gluccisamine!-HCl is a monoclinic crystal.8 When the single crystal is irradiated a t 77"K., t,wo groups of lines are observed which are isotropic in their splittiiigs. The one is a triplet with 8-gauss spacings and the other is a dloublet with 31-gauss spacings, as shown in Fig. 4A. Under the above conditions all the spacings are k o tropic. However, when the crystal is warmed to room temperature, the spectrum shows remarkable anisotropy and possesses three unequal splitting factors. One of them (al) is almost isotropic, but the other two (as and as) are anisotropic. As the number of the molecule in the unit cell is not known, it is necessary to consider the plossibility that either these eight lines are two sets of quartets, or four sets of doublets. However, the outermost lines in Fig. 5 always remain outside the remaining lines of the spectrum a t any crystal orientation and therefore neither of the above two possibilities is correct. Further, there is the possibility that the spectrum arises from two or three differentfree radicals. However, this is not correct since five approximately isotropic lines are usually observed when the magnetic field is applied in the ( q x ) plane. The spectra of unsubstituted sugars irradiated a t low temperatures are isotropic both at low temperatures and a t room temperature. Therefore, the different behavior obsei-ved
Y
I
s
Fig. 5.-The angular variation of the e.s.1. lines from Dglucosamine-Hal; irradiation and observation a t room temperature. The conventional axes x, y, and z are: e, = cos 34' e, e,)/Z; e, = (eb - e , ) / & where e is a - sin 34" e,; ey = ( e , unit vector.
+
in the present case is caused by the amino group in this amino sugar. The following explanation of the observation is postulated. When D-glucosamine-HC1 is irradiated a t 77OK., the hydrogen atom, indicated by a solid arrow in Fig. 6, is removed and the resulting free radical gives rise to an e m . spectrum consisting of a triplet or a doublet. These two possibilities are explained in the following way. If there are two molecules in a unit cell, it is possible that one of them is in the configuration A and the other is in C, Fig. 6. When the hydrogen indicated by a solid arrow is removed, the resulting unpaired electron will interact either with the proton indicated by a dotted arrow in A or with the nitrogen indicated by a dotted arrow in C. The presence of the amino group weakens the ring structure and, when the crystal is warmed, the ring is ruptured in the neighborhood of the amino group. This rupture moves the radical site to the primary alcohol chain liberating the OH group and, thus, forming the RCH(R')CH2. radical, as shown in Fig. 6. The spin density a t the end carbon atom is approximately unity in this case. E. Glucurono1actone.-Crystals of this compound are monoclinic and the unit cell contains two molec u l e ~ . When ~ a crystal was irradiated a t 77"K., the isotropic spectrum observed (Fig. 7 ) consisted of approximately 12 lines. The lines in the spectrum overlap; therefore, it is difficult to determine the relative intensities. When this crystal was warmed to IY3°K.,
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yo]. 67
A
+40Guuss+
b \ \
Fig. 6.-The structure of the free radicals formed in irradiated n-glucosamine-HCl. The representation for the atoms used liere are the same as those used in Fig. 2. The solid arrows indicate the hydrogen atoms removed. The dotted arrows indicate the nuclei with which the unpaired electron interacts.
this spectrum still consisted of 12 lines. The spectrum was almost identical with that observed a t 77OK. However, when the crystal was warmed to room temperature the spectrum became simpler (Fig. 7B and C). When microwave power was intense it gave six lines, shown in Fig. 7B. The least saturated and weak lines are a doublet with a splitting of 16 gauss while the remaining lines are a quartet with two splitting factors of 9 and 15 gauss. The relative intensities of the lines in the quartet are 1: 16: 1: IC where lc > 1. Even though the spectrum is isotropic, the relative intensities of the lines vary with the orientation of the crystal in the applied magnetic field, as in the case with other sugars.' According to Keihn and King, lo glucuronolactone has a double ring structure, Fig. 8A. The scission of the C-H bond a t the C1 or Cg positions produces the radical which has a doublet spectrum, while the scission of the C-H bond a t Cz, Ce,or C4 positions give rise to a radical which has a quartet spectrum. The doublet splitting of 16 gauss indicates that a spin density of approximately 0.5 is to be expected on the C1 or Ce carbon atoms. A similar estimation from the splitting factors for the quartet is complicated by the fact that the angular relations of the C-H bonds are unknown, because the angle between two rings has not been determined. It is possible that the large number of lines observed a t 77'11., Fig. 711, is due to the complicated angular relations of the C-H bonds. F. Diacetone Sorbose.-When a single crystal is irradiated a t 77"K., an isotropic spectrum coiisistiiig of (10) F. G. ICeilin and A. J. Icing, Acta Cryst., 4, 473 (1951).
Fig. 7.--E.s.r. spectra of irradiated gIucuronolactone. A: irradiation and observation a t 77°K.; LaH = 89"12', LbH = 90"; B and C: irradiation and observation a t room temperature; LaH = 135", LbH = 90". B: microwave power = 30 mw. C: microwave power = 1 mw.
+
T43
T21
.
w s w s
Fig. %--A: The structure of the freeradicals formed in irradiated glucuronolactone. The representations for the atoms used here are the same as those used in Fig. 2. B: The energy levels of the unpaired electron. w and s signify the weak and strong transitions observed a t high microwave power.
a triplet and a quartet is observed (Fig. 9). The quartet has a splitting factor of 22.5 gauss and the triplet has a splitting factor of 24 gauss. The triplet is identified with the triplet found in lactose hydrate irradiated a t 77°K. The quartet, whose lines have a11 intensity ratio of 1 :3 :3 :1, has narrower line widths thaii the usual quartet observed in other sugars. Since the
2180 foi*mcrlilies are found only i n diacetoiie sorbose, tlicy (mi be assigned to thc isopropenyl group. The isotropic character and the equal coupling with the tlircc protons indicate that the free radical formed is that showii in lcig. 1012. 111this radical, thc methyl group should be rotating mid tlierefore the splitting factor, A , is calculated as a time avcrage value from the expression A = 2Q cos2 8 ( t ) , where t signifies time. Sirice cos2 6 call be written as cos2 10!jo 27' cos2 ut, where w is the angular velocity of the methyl group, the timc average value of cos2 6 is 0.450. Tlic cxpcriment'al value of A is 22.5 gauss mid, therefore, thc estimated value of '(2' is 24.5/(spin dciisity). When tlic crystal is mi-mcd to 193°1
