An electron spin resonance investigation of radical intermediates in

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J . Phys. Chem. 1986, 90, 2963-2968

2963

An Electron Spin Resonance Investigation of Radical Intermediates in Cholesterol and Related Compounds: Relation to Solid-state Autoxidatlon Cynthia L. Sevilla, David Becker, and Michael D. Sevilla* Department of Chemistry, Oakland University, Rochester, Michigan 48063 (Received: October 1 1 , 1985)

This work reports an investigation of the free-radical chemistry of cholesterol and a number of cholesterol derivatives and analogues. The initial radicals formed in solid oxygen-free cholesterol samples after y-irradiation at 77 K are found to be a tertiary side-chain radical and an allylic radical in the cholesterol A and B rings. At 300 K only the allylic radical is found. The structure of the allylic radical is confirmed by experiments on two analogues of cholesterol, 7-OH cholesterol, and the selectively deuterated 7-D-7-OH cholesterol, both of which produce the allylic radical after y-irradiation by loss of the hydroxyl group. Cholesterol samples with oxygen present provide evidence for formation of two distinct peroxy radicals originating with the two carbon radicals found in the oxygen-free samples. These peroxy radicals are suggested to have different reactivities resulting from the different motional freedom each possesses. We suggest that the products found after radiation-induced autoxidation at or near room temperature are consistent with the different reactivities of the peroxy radicals. It is found for four cholesterol esters, one cholesterol derivative, and one sterol that the only radical stable at room temperatures in oxygen-free samples is the allylic radical. In only one ester, cholesterol chloroacetate, is the allylic radical not the stable room temperature radical.

Introduction Cholesterol and its esters are biologically important compounds which are major components of mammalian membranes. Cholesterol and its derivatives easily undergo autoxidation and it is known that some of its autoxidation products possess potent physiological activity.’ Autoxidation process are predominantly free radical in nature. In air-saturated y-irradiated samples the process begins when triplet oxygen reacts with a carbon-centered radial to produce a peroxy radical; through a hydrogen abstraction reaction, the peroxy radical then produces a hydroperoxide and a new carbon-centered radical which can further propagate the autoxidation cycle. In the case of cholesterol, Smith and coworkers, using product analysis techniques over a period of years, have established a detailed framework for understanding the autoxidation of cho1esterol.l The work by Smith, by its nature, did not involve direct observation of the intermediate radicals involved in the autoxidation process, and although the nature of the products formed in the reactions involved is suggestive of the actual pathway taken, confirmatory evidence by direct observation of the intermediate radicals is desirable. Previous ESR investigations undertaken have provided some valuable information in this regard;*” however no investigation to date has investigated the low-temperature irradiation of cholesterol and its derivatives in order to observe the variety of carbon-centered radicals which can be initially formed and the subsequent reaction of these species to more stable species. This study reports an ESR investigation of the free-radical chemistry of cholesterol and a number of its derivatives induced by cobalt-60 irradiation at low temperature. In this work we have verified the structure of the room temperature allylic radical (cholest-5-en-3-01(3B)-7-y1)in neat cholesterol through deuteration experiments on an analogue and have shown that the allylic radical formed in cholesterol is common to a variety of cholesterol esters and other related compounds. Samples with oxygen present provide evidence for the formation of two types of peroxy radicals which possess different reactivities due to their different positions on the cholesterol structure. Materials and Methods The compounds studied are cholesterol (I), two analogues of cholesterol, including one deuterated analogue (11, 111), five esters (1) Smith, L. L. Cholesterol Autoxidation; Plenum: New York, 1981, and references therein. (2) Rexroad, N. H.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 256. ( 3 ) Gordy, W. Radiat. Res., Suppl. I 1959, 491. (4) Ehrenberg, A,; Ehrenberg, L.; Lofroth, G. RISOReport I6 1960,21. ( 5 ) Hellinger, 0. Biophysik 1969, 6, 63. (6) Hellinger, 0; Heusinger, H.; Hug, 0. Biophysik 1970, 6, 193.

0022-3654/86/2090-2963$01.50/0

of cholesterol (IV-VIII), cholesteryl bromide (IX), and dehydroisoandrosterone (X).

HO A1

I: R, = H, R2 = H, cholesterol or cholest-5-en-3-ol(3(3)

&

HO

R1

11,111

11: R, = OH, R2 = H, 7-OH cholesterol or cholest-5-ene-3,7-diol(3&7[) 111: R, = D, R2 = OH, 7-D-7-OH cholesterol or

cholest-5-ene-3,7-diol(3(3,7.9-7-d

R-C-0 O II

R

IV-VIII

IV: R = H, cholesteryl formate or cholest-5-en-3-ol(3(3)formate V: R = (CH3)*-CH, cholesteryl isobutyrate or cholest-5-en-3-ol(3@)2-methylpropanoate cholesteryl palmitate or VI: R = CH3(CH2)14, cholest-5-en-3-ol(3(3)hexadecanoate VII: R = CH3(CH2),2,cholesteryl myristate or cholest-5-en-3-ol(3(3) tetradecanoate VIII: R = CH2C1, cholesteryl chloroacetate or cholest-5-en-3-ol(3(3) chloroacetate

0r

# IX

IX: cholesteryl bromide or cholest-5-ene-3-bromo(3(3)

&

HO

X

X: dehydroisoandrosterone or androst-5-en-3-01(3(3)17-one

0 1986 American Chemical Society

2964

Sevilla et al.

The Journal of Physical Chemistry, Vol. 90, No. 13, 1986

TABLE I: Hyperfrne Coupling Constants and g Values for Cholesterol, 7-OH Cholesterol, and 7-D-7-OH Cholesterol radicals radicals compound (oxygen-free samples) (oxygen present) I, cholesterol IA ID

N

#

HO

HOO & --

HO

30 G (lH), 23 G (6H), 5 G (1 H); g = 2.0029, 11 3 K

g , =

IB

IE

# &

HO

HO

24.5 G (2 H), 14.5 G (1 H); g = 2.0022, 305 K 11, 7-OH cholesterol

HO

N

IIA

H

g, = gs

2.018," 268 K

Jd??

2.031: g2 = 2.0091,* =2.0041, 268 K

HO

24 G (2 H), 14 G (1 H); g = 2.0025; 293 K 111, 7-D-7-OH cholesterol

IIIA

HO

HO

D

&

23.5 G (2 H); g = 2.0024, 333 K "This value corresponds to the population of ID that is undergoing incomplete motional averaging. It is not possible to distinguish the line components of the nonmotionally averaged population of ID from the line components of IE. bThese values are temperature-sensitive due to the motion of the radical. cis-Decalin (XI) and chloroacetic acid (XII) were also investigated in order to interpret the spectra of I and IX, respectively. 7-OH cholesterol was synthesized by reduction of 7-keto chowith sodium borohydride lesterol (cholest-5-en-3-01(3~)-7-one) in methanol solvent. Extraction of the reaction mixture with methylene chloride and crystallization from the methylene chloride was used to purify the product. Thin layer chromatography on silica gel with a 60/40 (v/v) mixture of benzene/ethyl acetate followed by development with 50% H 2 S 0 4and heating at 80 OC resulted in two blue spots confirming the presence of the a and /3 forms of the 7-OH cholesterol.' The 7-D-7-OH cholesterol was prepared in a similar fashion by using sodium deuteriohydride. All other compounds were purchased from Sigma and used without further purification. Samples were y-irradiated in Spectrosil quartz tubes at 77 K for doses of ca. 0.2 Mrad. Evacuated samples were degassed by pumping on the solid sample for extended times and sealing; heating of the sample itself was avoided to prevent unwanted reactions from occurring. A Varian Century ESR spectrometer with dual cavity was employed. Hyperfine splittings and g values were measured vs. Fremy's salt with AN = 13.09 G and g = 2.0056. Results Cholesterol. y-Irradiation of neat oxygen-free cholesterol at 77 K results in an ESR spectrum at 77 K which consists of an octet of poorly resolved doublets extending over 173 G.Annealing the sample to 113 K improves the resolution of the doublet structure and results in the spectrum shown in Figure 1A. Further warming of the sample to 223 K results in an irreversible change in the spectrum. The spectrum after this irreversible change, taken at 305 K after annealing to 328 K,is shown in Figure 1B. The intensities of the spectral lines at 113 K (and 77 K) suggest that two radicals are present at this temperature, and that one of these radicals is the species which remains on annealing to 223 K. Subtraction of the spectrum of Figure lB,as 60% of the total intensity, from the spectrum of Figure 1A results in the spectrum

shown in Figure 1C.Thus, the spectrum in Figure 1A consists of approximately 60% of that in Figure 1B plus 40% of that in 1 C. An isotropic simulation of the spectrum 1 C,using hyperfine couplings of 30 G (1 H),23 G (6 H), and 5 G (1 H), matches spectrum 1C quite well, except in the center portion of the spectrum where a signal due to the irradiated quartz sample tube interferes; this simulation is presented in Figure 1D. The only radical derived from cholesterol that can show couplings to eight hydrogens is IA, and we therefore attribute ca. 40% of the 1 1 3 K spectrum to IA. It is evident, due to presence of the six equivalent hydrogens with 23-Gcouplings, that the methyl groups are rotating even at 113 K (Table I). 30G,

,,5G

HO HO IA

IB

The above results indicate that the predominant radical species present a t 77 K is also the most stable radical formed upon annealing to higher temperatures. Analysis of the ESR spectrum of this species (Figure 1B) yields hyperfine couplings of 24.5 G (2 H),and 14.5 G (1 H). We assign this spectrum to the allylic radical IB. The two hydrogens with 24.5-Gcoupling are assigned to positions 4 and 8. These large couplings are associated with axial protons which have a small dihedral angle with the p-orbitals on carbons 5 and 7. If we assumed typical spin densities for allylic radicals of 0.58 for these carbons, the 24.5-Gcouplings are quite reasonable. The coupling of 14.5 G is typical of an a-proton in allylic radicals as well as the allyl radical itself.' The hydrogen (7) Carrington, A.; McLachlan, A. D. Introduction To Magnetic Resonance; Harper and Row: New York, 1967.

Radical Intermediates in Cholesterol

The Journal of Physical Chemistry, Vol. 90, No. 13, 1986 2965

I /

li

305Kl320K

C

303K

1'

Figure 2. (A) ESR spectrum of air-saturated cholesterol, recorded at 123 K after y-irradiation at 77 K. The spectrum is attributed to a mixture of peroxy radicals and carbon-centered radicals. (B) ESR of air-saturated cholesterol at 268K. This spectrum is characteristic of peroxy radicals; the central broad line component originates with a population of the radicals which are undergoing some type of motional averaging. (C) Spectrum at 303 K. The central line component has largely disappeared. The small sharp line at g = 2.001 is due to the quartz sample tube.

Figure 1. (A) First-derivatives ESR spectrum of evacuated cholesterol sample, recorded at 113 K after y-irradiation at 77 K. This spectrum

is attributed to two radicals, IA and IB. (B) ESR spectrum, recorded at 305 K, after annealing to 328 K. This is the spectrum of the allylic radical, IB. (C) Computer-generated spectrum of IA obtained by subtracting the spectrum of the allylic radical IB (as 60% of the total radical concentration) from the spectrum recorded at 113 K. Since this spectrum is generated by subtracting the (presumably) temperature-invariant spectrum of IB from the spectrum of the mixture of radicals at 113 K, this spectrum should be representative of the spectrum of IA at 113 K. (D) Isotropic computer simulation of spectrum C using the hyperfine coupling parameters given in Table I. The three hash marks indicate the line positions of a Fremy Salt standard, and are separated by 13.09 G. on carbon 6 should possess a small ca.4-G coupling, again typical of allylic radicals, which is too small to be resolved. Finally, the remaining hydrogen on carbon 4 is equatorial, and, therefore, lies nearly in the plane of the radical; its coupling is also too small to be resolved. The small splitting noticeable in the wings of the spectrum we believe is due to anisotropy in the coupling of the allylic proton rather than an additional isotropic coupling. The large line widths found in these spectra obscure most of the anisotropy in the splittings of the a-proton. &Protons typically show very little anisotropy in their hyperfine couplings; the highly isotropic nature of their spectra is expected.' Considering the complexity of this compound and the many possibilities for radical addition and hydrogen abstraction that exist, we do not consider the identification of IB a trivial matter. Additional results on I1 and 111, presented below, further confirm this assignment; however, additional comments on the assignment of IB to the spectrum of Figure 1B would seem to be in order. The lack of a 22-G coupling precludes the existence of an aliphatic a-proton; hence the only other choice for this radical would be one such as IC, formed (in this case) by radical addition to the

expect I C to display at least one ca. 38-41-G coupling, but no such coupling exists in the experimental spectrum. Also, the results of product analysis studies of the reaction of irradiated cholesterol with oxygen indicate that the allylic radical plays a part in the dominant reaction pathway.' These facts plus the well-known stability of allylic radicals and the isotopic substitution results presented below give us much confidence in the identification of the allylic radical. We also note that this assignment is in agreement with that reported in two earlier ESR studies of irradiated c h o l e ~ t e r o l . ~ ~ ~ When cholesterol is irradiated at 77 K in an open tube, with oxygen present, a different course of events is followed. The spectrum, at 123 K, that results is shown in Figure 2A. Comparison of this spectrum with that of Figure 1A suggests that some of IA and IB are both present, but also present is the spectrum characteristic of a peroxy radical-the peroxy radical spectrum shows no hyperfine coupling and a nearly axially symmetric g tensor.'0,'' As the sample is warmed further, the peroxy radical spectrum increases in intensity as the underlying specta of the carbon radicals IA and IB disappear. Figure 2B shows the peroxy radical spectrum at 268 K. This spectrum, which shows a (lowfield) parallel component, a nearly axial (high-field) perpendicular component, and a broad component in between, is typical of a peroxy radical which is undergoing partial motional averaging.I2J3 Warming the sample to 293 K results in the loss of the intermediates component, that component with corresponds to the motionally averaged peroxy radical; the spectrum that remains corresponds to a peroxy radical which is not undergoing motional averaging on an ESR time scale. This result can be understood in terms of the initial radicals formed, i.e., IA and IB. Formation of peroxy radicals results from direct reaction of a carbon radical with dissolved oxygen. In this case, there are two carbon radicals which react to form the two peroxy radicals, ID and IE.

co

HO

HO

ID

1E

XIA IC

double bond. However a radical (XIA) analagous to IC in decalin shows couplings variously reported as 41.5 G (3 H) and 10 G (2 H),* or as 38.5 G ( 3 H) and 9.5 G (2 H).9 Hence, we would (8) Becker, D; Herrington, J; Sevilla, M. D., unpublished results.

(9) Bichiashvili, A. D.; Tsomaia, N. N.; Nanobashvili, E. M. Soobshch. Akad. Nauk Gruz. SSR 1974, 73,617. (10) Chien, J. C. W.; Boss, C. R. J . Am. Chem. SOC.1967, 89, 571. (11) Hori, Y.; Aoyama, S.; Kashiwabara, H. J . Chem. Phys. 1981, 75, 1581. (12) (13)

Schlick, S.; Kevan, L. J . Phys. Chem. 1979, 83, 3424. Suryanarayana, D.; Kevan, L. J . Am. Chem. SOC.1982, 104, 6251.

Sevilla et al.

2966 The Journal of Physical Chemistry, Vol. 90, No. 13, 1986

B

_-

B

t

-

t

180K

,

/-

C Figure 3. (A) ESR spectrum of evacuated 7-OH cholesterol at 293 K after y-irradiation at 77 K, attributed to the allylic radical IIA. (B) ESR spectrum of 7-D-7-OH cholesterol at 333 K after irradiation at 77 K, attributed to the partially deuterated allylic radical IIIA. The spectrum shows the expected absence of the 1 4 4 coupling present in IIA.

Radical ID is apparently able to undergo rotation of the side chain which produces the motional averaging seen in the ESR spectrum. This increased freedom of motion, over that of IE, allows this radical to undergo reactions which ultimately lead to radical recombination. Radical IE, on the other hand, is more constrained due to the fact that it is in the cholesterol ring system. Its ESR spectrum does not show significant motional averaging, and, concomitantly it cannot react as easily. Therefore, at higher temperatures, the only peroxy radical present is IE. This result is in reasonable agreement with the work of Smith and co-workers, who find the 7-hydroperoxide as the predominant product in irradiated air-saturated cholesterol samples at temperatures near room temperature.' However, we might also expect to find a relatively low yield of product(s) of some type originating from ID (see Discussion). 7-OH Cholesterol and 7-D-7-OH Cholesterol. For each of these compounds, the radical formed at 77 K is the only major one observed throughout the temperature range employed, from 77 K to above room temperature. Figure 3A is a spectrum at 293 K of the radical found in 7-OH cholesterol, after y-irradiation at 77 K and annealing of oxygen-free samples. The spectrum, which is consistent with hyperfine couplings of 24 G (2 H ) and 14 G (1 H ) is virtually identical with that found for the allylic radical in cholesterol. For this compound the radical is IIA, which

277Kl323K -

_-

7

Figure 4. (A) ESR spectrum of evacuated cholesteryl formate at 173 K after y-irradiation at 77 K. This spectrum shows an acyloxy radical singlet and electron attachment radical anion doublet. (B) FSR spectrum of cholesteryl formate after annealing to 188 K. This spectrum is attributed to a mixture of the acyloxy singlet at g = 2.0006, an allylic radical analogous to IA, and a peroxy radical. The two-line peroxy radical spectrum line positions are indicated by arrows. ( C ) ESR spectrum of cholesteryl formate allyl radical, recorded at 277 K after annealing to 323 K.

is identical in structure with IB. The loss of an OH group in this compound is in agreement with the results for y-irradiated and UV-irradiated neat allyl alcohol, in which it is found that the allyl radical is formed at 77 K.'4315 The spectrum of the radical resulting after irradiation of 7-D7-OH cholesterol at 77 K and annealing of oxygen-free samples to 333 K is shown in Figure 3B. It consists of a 1:2:1 triplet, consistent with hyperfine couplings of 23.5 G (2 H) due to two protons. This spectrum is precisely that expected from radical IIIA, in which the expected deuterium coupling of 2.1 G (= 14.5/6.514) is too small to be resolved. We consider this result an excellent confirmation of the allylic radical proposed for cholesterol itself. It is quite interesting that we do not observe, in this case, the side-chain tertiary radical analogous to IA. The loss of the 7-hydroxy group (probably as OH or HzO) is a clearly favorable process, even at 77 K. Cholesterol Esters. The initial radicals produced on irradiating cholesterol esters at 77 K and their subsequent reactions depend

on the specific ester involved. However, for all of the simple esters studied (cholesterol formate, isobutyrate, palmitate, and myristate) there is a consistency of results; Le., initially upon irradiation at 77 K, the anion radical due to electron attachment to the carbonyl in the ester functional group and a number of other radicals are formed; as the sample is annealed, the final stable radical is always the allylic radical. Cholesteryl Formate. The spectra of cholesterol formate at various temperatures after y-irradiation at 77 K are shown in Figure 4. The spectrum at 123 K is dominated by a singlet at g = 2.0006 and a 17-G doublet. There is also evidence for at least one underlying radical. This compound appears to be following the well-understood chemistry of irradiated small esters.I6 The doublet is assigned to the anion radical formed by electron attachment to the carbonyl, and the singlet at g = 2.0006 is characteristic of an acyloxy radical, most likely formed by deprotonation of the cationic primary radical. The acyloxy radical can also be formed through hydrogen abstraction of an adjacent acyl hydrogen by the anion radical; however, this reaction generally takes place at higher temperatures which allow for increased radical mobility. The major underlying radical spectrum has a spectral width of approximately 170 G and many lines coincident with the spectrum of IA; thus it is likely due to the cholesterol formate radical analogous to IA. As the sample is annealed the anion doublet disappears-by 165 K it is no longer visible. The singlet due to the acyloxy radical also decays, but more slowly. As these signals decay, a peroxy radical signal becomes evident owing to a small residue of molecular oxygen left in the degassed sample. This signal decays by 273 K leaving predominantly the familiar allylic radical spectrum (Figure 4C). Cholesteryl Zsobutyrate. The spectrum of 7-irradiated cholesteryl isobutyrate, irradiated and run at 77 K, results in the anion radical formed by electron attachment to the carbonyl plus a number of other radicals. Photobleaching removes a large central component due to the anion radical and results in the spectrum shown in Figure 5A. Figure 2B shows the spectrum of a photobleached sample after annealing briefly in a low-temperature Dewar and then recooling to 77 K. Figure 5C is the spectrum of a n unbleached sample after annealing to 361 K. It is quite clear, from inspection of the spectra, that the spectrum in Figure

(14) Maas, K. A.; Volman, D. H. Trans. Faraday SOC.1964, 60, 1202. (15) Smaller, B.; Matheson, M. S. J . Chem. Phys. 1958, 28, 1169.

(16) BOX,H. C.Radiation Effects, ESR and ENDOR Analysis; Academic: New York, 1979.

HO

HO

IIA

IllA

The Journal of Physical Chemistry, Vol. 90, No. 13, 1986 2961

Radical Intermediates in Cholesterol

Figure 5. (A) ESR spectrum of evacuated cholesteryl isobutyrate at 77 K after y-iradiation and photobleaching at 77 K. This spectrum is

attributed to a mixture of the two radicals VA and VB. (B) ESR spectrum at 77 K of cholesteryl isobutyrate after y-irradiation at 77 K and brief warming in low-temperature dewar. The septet is attributed to VA. (C) ESR spectrum of cholesteryl isobutyrate after annealing to 361 K, attributed to the allylic radical VB. 5A is due largely to the radicals responsible for the spectra of Figure 5B,C. The spectrum in Figure 5B, which consists of a 22.2-G septet, is easily assigned to VA; the couplings to the six

V

Figure 6. (A) ESR spectrum of evacuated cholesteryl chloroacetate at 251 K after y-irradiation at 77 K. (B) ESR spectrum of evacuated chloroacetic acid at 270 K after y-irradiation at 77 K. The line positions

(although not the intensities) of the two spectra are identical, indicating analogous radicals are responsible for the spectra. These radicals are VIIIA and XIIA.

I CH

;c-c-0

HC-C-0 / VA

VB

JrJr Jr"

equivalent &hydrogens are slightly lower than normal for alkyl radicals because of spin delocalization to the -C02- ?r-system. The presence of line components due to this radical is observed to 333 K. The spectrum of Figure 5C is that of the now familiar allylic radical, VB, for this compound; this is the only radical present at 343 K. Cholesteryl Palmitate and Cholesteryl Myristate. Both of these compounds behave quite similarly; their spectra at 113 K have identical line positions. Irradiation at 77 K and annealing to 113 K results in a mixture of radicals which we believe include the side-chain radical analogous to IA, the anion formed by electron attachment to the carbonyl, and the allylic radical. The final radical formed on warming for both the myristate and the palmitate is the allylic radical, with couplings of 26 G (2 H) and 15 G (1 H) for the myristate a t 339 K and the same couplings for the palmitate at 328 K. In a separate experiment y-irradiation of cholesteryl myristate (18 mol %) in tripalmitin at 298 K was performed to test for radical transfer between lipid components. The results showed only formation of the allylic radical, with couplings of 24 G (2 H) and 14 G (1 H). The majority of the radicals must, in this case, be initially formed in the tripalmitin, but they apparently attack the cholesteryl myristate specifically at the allylic site. This spin transfer shows the mobility of these radicals, even in the solid state. Cholesteryl Chloroacetate. Over the whole temperature range employed for this compound (77-368 K), only one radical is present after irradiation a t 77 K. A spectrum of this radical is shown in Figure 6A. This spectrum shows hyperfine coupling to chlorine and is assigned to VIIIA. The analogous radical

m

;c-c-o f: /

H

/rJy VlllA

\::

CI

;C-C-oH

/

-l++ Figure 7. (A) ESR spectrum of evacuated cholesteryl bromide at 137 K after y-irradiation at 77 K. The spectrum is attributed to the side-

chain radical IXA, but with methylene hydrogen couplings (from the two hydrogens on carbon 24) slightly different than those found in the analogous cholesterol radical IA. (B) ESR spectrum of cholesteryl bromide at 221 K, attributed to the allylic radical IXB. shown in Figure 6B; the spectra of VIIIA and XIIA are nearly identical insofar as line positions are concerned and support our identification of VIIA. The spectra are complicated not only by the presence of two chlorine isotopes, but also by a large nuclear quadrupole interaction which introduces line shifts and the appearance of lines due to normally forbidden transitions. As a consequence, the hyperfine couplings of the radical present are not easily determined. The single crystal analysis of XIIA yielded principal values of the chlorine hyperfine tensor of +20.0, -2.5, and -6.4 G;ls one principal value of the a-hydrogen tensor was determined to be 30.7 G.I7 However, even in the case of a single crystal, a complete analysis has not yet been reported. It is most notable that there is no indication of any formation of an allylic radical in irradiated cholesteryl chloroacetate, even a t 363 K. Clearly, in this particular case, radical VIIIA is thermodynamically more stable than the allylic radical of the parent compound. Cholesteryl Bromide. y-Irradiation of oxygen-free cholesteryl bromide at 77 K results in a spectrum similar to, but not identical with, that found for cholesterol. Figure 7A presents the spectrum

XllA

(XIIA) has been identified and studied in chloroacetic acid in detail in a single c r y ~ t a l . ' ~ JThe ~ powder spectrum of XIIA is

(17) Pooley, D.;Whiffen, D. H. Spectrochirn. Acro 1962, 18, 291. (18) Kohin, R. P . J . Chem. Phys. 1969, 50. 5536.

2968

The Journal of Physical Chemistry, Vol. 90, No. 13, 1986

a t 137 K, where resolution is improved. The spectrum appears to be due to two radicals. One, IXA, is similar to IA, but in this

X

Br

IXA

IXB

Br#

case the methylene couplings have changed, presumably due to a conformational change in the methylene group. Hyperfine couplings of 23 G (6 H ) , 15 G (1 H ) , and 11 G (1 H) are consistent with the spectrum shown. The large intensities of the central lines suggest the presence of the allylic radical IXB and we presume that some significant concentration of the allylic radical is present although in this case, an analysis to ascertain the amount of each radical present was not performed. Warming to 189 K results in a spectrum due largely to the allylic radical IXB; Figure 7B presents its spectrum at 221 K. IXB is stable to 243 K; by 278 K no ESR signal remains. Dehydroisoandrosterone. y-Irradiation at 77 K of dehydroisoandrosterone results in a spectrum which consists predominantly of a 1:2:1 triplet, with a separation of 23.5 G and is assigned to radical XA, formed by electron attachment to the carbonyl group.

HO

&E & '

XA

HO

XB

The couplings are to two equivalent hydrogens on carbon 16. The nature of the radical formed and the couplings that result are in accord with those expected for a ketone anion.I6 The spectrum produced upon further annealing to 279 K corresponds to that of the allylic radical, XB in this case, with couplings of 24.5 G (2 H) and 14 G (1 H). The spectrum of the allylic radical persists until 253 K, at which point diamagnetic products are formed.

Discussion There are three results found in this work which we feel are important to cholesterol free-radical chemistry. The first is the definitive identification of the allylic radical in cholesterol by the use of deuteration techniques. The allylic radical was first proposed by Ehrenberg and co-workers4 in 1960 and later in 1969 by Hellir~ger.~ But considering the complexity of the cholesterol molecule and the knowledge developed in the last 15 years regarding the solid-state reactions that cholesterol may undergo, we felt a more definitive identification was desirable. Our current work together with that of earlier investigators clearly establishes the identity of and couplings in the allylic radical.

Sevilla et al. The second result is the most interesting ubiquitous appearance of the allylic radical in a large variety of sterols and related compounds. In the solid-state systems we investigated the allylic radical was almost always the predominant high-temperature stable radical. The only exceptions occurred in oxygenated cholesterol, in which a peroxide radical persisted, and in cholesterol chloroacetate, in which a chlorinated carbon radical remained. The allylic radical was found in several cases to be increased in concentration by abstraction reactions from other initial radicals present. Thus the allylic radical in cholesterol is evidently a thermodynamically stable species and will tend to form from other initial radicals in systems where some molecular mobility permits molecules to orient in a way that allows the abstraction to occur. The stability of the allylic radical compared to other species investigated in this study is due to the resonance stabilization provided by the conjugated system as well as the steric protection provided by the bulky ring system. Third, our results with irradiated air-saturated cholesterol samples indicate that both the 25-peroxy and 7-proxy cholesterol radicals are formed at low temperatures, but that at higher temperatures the 25-peroxy system has more freedom of motion and quickly reacts, probably by abstraction, leaving only the 7-peroxy species which is more rigidly held in the solid state. However, even the 7-peroxy radical decays at temperatures near room temperature (in air-saturated samples) while the carbon-centered allylic species is stable at these temperatures (in degassed samples). It appears that the relatively high mobility of oxygen in the cholesterol microcrystal plays an important role in the decay of the peroxy radical by expediting cyclic autoxidation. Thus, the peroxy radicals abstract adjacent allylic hydrogens forming allylic radicals which in turn react with oxygen to form a new peroxy radical. The mobility of the oxygen assures that it will be available at the proper site for reaction to occur, and the relatively high stability of the allylic radical at room temperature assures that it will not react through some other pathway before the oxygen can reach it. Through the autoxidation cycle, the unpaired spin is able to migrate through the solid and eventually undergo recombination. The autoxidation cycle would also have the effect of generating a large amount of products originating with the 7-peroxy site and explains the observation by Smith' that products from the 7-radical site are almost exclusively formed in radiation-induced autoxidation, despite the fact, as we have described in this work, that a significant amount of the 25-peroxy radical is initially formed upon irradiation.

Acknowledgment. We thank the US.Army Natick Research and Development Laboratory and the Office of Health and Environmental Research of the U S . Department of Energy for support of this research. The US.Department of Agriculture is also thanked for support which initiated this work.