Substituted MCM-41 Molecular Sieves - American Chemical Society

teristics of the matrix.4-6,12,13,17 In Scheme 1 are given the structures of lutein (I), which is a carotenoid with OH substit- uents that can hydroge...
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J. Phys. Chem. B 2008, 112, 5449-5457

5449

Pulsed Electron Nuclear Double Resonance Studies of Carotenoid Oxidation in Cu(II)-Substituted MCM-41 Molecular Sieves Jesse Lawrence,† A. Ligia Focsan,† Tatyana A. Konovalova,† Peter Molnar,‡ Jozsef Deli,‡ Michael K. Bowman,† and Lowell D. Kispert*,† Department of Chemistry, The UniVersity of Alabama, Box 870336, Tuscaloosa, Alabama 35487, and Department of Biochemistry and Medical Chemistry, UniVersity of Pe´ cs, H-7624, Szigeti u´ t 12, Pe´ cs, Hungary ReceiVed: NoVember 29, 2007; In Final Form: February 4, 2008

Carotenoid (Car) radical intermediates formed upon catalytic or photooxidation of lutein (I), 7′-apo-7′,7′dicyano-β-carotene (II), and lycopene (III) inside Cu(II)-MCM-41 molecular sieves were studied by pulsed electron nuclear double resonance (ENDOR) spectroscopies. The Davies and Mims ENDOR spectra (15-20 K) were simulated using the hyperfine coupling constants predicted by density functional theory (DFT) calculations. The DFT calculations revealed that upon chemical oxidation, carotenoid radical cations (Car•+) are formed, whereas carotenoid neutral radicals (#Car•) are produced by proton loss (indicated by #) from the radical cation. This loss is to first order independent of polarity or hydrogen bonding for carotenoids I, II, or III inside Cu(II)-MCM-41 molecular sieves.

Car + Matrix (Si-O) f Car•+ + Matrix-

Introduction Carotenoids are intrinsic components of reaction centers and pigment-protein complexes in photosynthetic membranes. They play a photoprotective role in plants, algae, and cyanobacteria by quenching reactive photoinduced radicals and dissipating the excess energy. Photooxidation of the isolated PS II reaction center leads to selective oxidation and irreversible bleaching of β-carotene by electron transfer to the primary donor P680•+ to form the carotenoid radical cation.1 The radical cation of β-carotene has been detected numerous times upon illumination in PS II by various workers, and its possible roles in photoprotection have been discussed.2,3 Examining the electrontransfer reactions of carotenoids within artificial matrixes is important for understanding the electron transfer and energy transfer processes of carotenoids. Large pore sizes of MCM-41 molecular sieves permit reactions involving bulky carotenoid molecules that are not capable of entering the channels of microporous zeolites. Incorporating metal ions into siliceous MCM-41 enhances electron-transfer efficiency between embedded carotenoid molecules and the MCM-41 framework.4-6 Development of practical devices to better utilize solar energy will benefit from understanding the mechanisms by which the host lattice affects the electron donor, as studied here through the charge transfer and proton loss properties of carotenoids when carotenoid radicals are formed. Previously, published reports7-9 have shown that carotenoids can transfer electrons to the silicate-alumina matrices because the matrix serves as a Lewis acid according to eq 1. In addition, the presence of protons on the silica matrix serves as a Bronsted acid site. If the pH of the matrix is less than the pKa of the radical cation,7 the radical cation is stabilized by the Bronsted acid because the presence of excess H+ drives the equilibrium (eq 2) to the left. * To whom correspondence should be addressed. E-mail: lkispert@ bama.ua.edu. † The University of Alabama. ‡ University of Pe ´ cs.

hydrolysis hV

Car•+ [\] #Car• + H+

(1) (2)

The presence of water causes the Car•+ to lose a proton, forming a neutral radical, #Car• (loss of a proton is indicated by #); thus, the requirement for the absence of water in the silicates. Nafion, a perfluorinated polymer, has also been used as an electronacceptor matrix on which carotenoid radical cation has been formed.8 It has been recently shown10,11 that upon irradiation with an unfiltered Xe lamp, carotenoid neutral radicals are formed by proton loss from Car•+ from carotenoids supported on a silica-alumina matrix. Carotenoid radical cations (Car•+) can also be produced in MCM-41 molecular sieves4-6,12,13 upon electron transfer to the matrix. In the absence of metals,5,12 the photoyield is dependent on oxidation potential. The photoyield is 4 times larger for incorporated β-carotene5,12 (E1/2 ) 0.6 V vs SCE)14,15 than for canthaxanthin5,12 (E1/2 ) 0.8 V vs SCE)14,15 or 7′-apo-7′,7′dicyano-β-carotene5 (E1/2 ) 0.8 V vs SCE).14,16 Incorporation of Ti,5,17 Fe,6 Cu,12,13 Ni,4 or Al4 greatly increases the overall yield of radicals up to a factor of 5. In addition, for iron-substituted MCM-41,6 the relative photoyield of radicals changed to 1:2.5:6.7 for β-carotene to canthaxanthin to 7′-apo-7′,7′-dicyano-β-carotene, respectively. Electron nuclear double resonance (ENDOR) measurements of canthaxanthin inside Fe-MCM-41 showed the relative formation of canthaxanthin neutral radicals to radical cation as 1.3:1, whereas this ratio increased to 2:1 for 7′-apo-7′,7′-dicyano-β-carotene. In TiMCM-41,5 the photoyield of β-carotene to canthaxanthin to 7′apo-7′,7′-dicyano-β-carotene radicals was 4.5:1:2.5. ENDOR measurements showed the ratio of neutral radicals to that of the radical cations as 1:1.3. An ENDOR powder spectrum of UV-irradiated samples of carotenoid on silica gel9 showed equal yields of neutral radicals and radical cations for 7′-apo-7′,7′-dicyano-β-carotene (E1/2 )

10.1021/jp711310u CCC: $40.75 © 2008 American Chemical Society Published on Web 04/08/2008

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Figure 2. The DFT calculated unpaired spin density distribution for the 7′-apo-7′,7′-dicyano-β-carotene radicals.

Figure 1. The DFT calculated unpaired spin density distribution for lutein radicals.

SCHEME 1: Structures of Carotenoids

0.8V vs SCE)14,16 and the diphenyl-substituted apo-carotenoid (E1/2 ) 0.65V vs SCE).14,16 Apparently the yield of radicals in metal-substituted MCM-41 and silica gel is not in general dependent on the oxidation potential, but rather is dependent

on the location of the carotenoid. For example, it has been shown12,13 that β-carotene coordinates to Cu2+ in Cu-MCM41, but canthaxanthin does not, and instead is attached to the exterior of the MCM-41 matrix. In this situation, the yield of radicals for β-carotene, canthaxanthin, and 7′-apo-7′,7′-dicyanoβ-carotene occurs in the ratio of 5:2:1, being highest for the carotenoid with the lowest oxidation potential, but unequal for the latter two carotenoids, which exhibit similar oxidation potential. In this case, the radical yield follows the ionization potential for β-carotene because it is coordinated to the metal ion. The aim of this manuscript is to uncover the principles behind the loss of a proton from a carotenoid radical cation incorporated in an electron acceptor matrix. This is attempted by examining the relative yield of radicals formed from the hydrogen-bonded carotenoid, as compared to that of a dicyano-substituted polar carotenoid or that of a nonpolar carotenoid, lycopene. Such information will provide improved design of artificial photoconversion systems in which hydrolysis, electron transfer, and deactivation of excited states is possible. A copper-substituted MCM-41 was chosen to increase the electron acceptor characteristics of the matrix.4-6,12,13,17 In Scheme 1 are given the structures of lutein (I), which is a carotenoid with OH substituents that can hydrogen bond to the Si-O matrix; 7′-apo-7′,7′dicyano-β-carotene (II), which is polar; and lycopene (III), a nonpolar molecule.

Pulsed ENDOR Studies of Carotenoid Oxidation

Figure 3. The DFT calculated unpaired spin density distribution for the lycopene radicals.

Experimental Synthesis of MCM-41 and Cu-MCM-41. The siliceous material (MCM-41) was previously prepared in our lab following the procedure of Beck et al.18 Cu-MCM-41 was prepared by liquid-state ion exchange according to the method of Xu et al.19 Fifty milliliters of aqueous 1 mM Cu(CH3COO)2‚H2O (98 wt %, Fisher) solution was added to 2 g of MCM-41. The resulting mixture was stirred for 24 h at room temperature and then filtered and washed with hot deionized water (90 °C) to remove any ions adsorbed on the external surface. This was designated as fresh Cu-MCM-41. SBA-15 was prepared according to a procedure described elsewhere,20 except the mixture was made in a Pyrex container instead of a Teflon container, and the batch was 20 times larger. Cu-SBA-15 was prepared by liquid-state ion exchange following a procedure similar to that used to prepare Cu-MCM41. Fifty milliliters of aqueous 2 mM Cu(CH3COO)2‚H2O (98 wt %, Fischer) solution was added to 2 g of SBA-15. The resulting mixture was stirred for 48 h at room temperatures and then filtered and washed with hot deionized water (80 °C) to remove any ions adsorbed on the external surface. Carotenoids Preparation. Lutein was isolated by preparative classic chromatography from the mixture of phytoxanthins which was isolated from maple leaves (Acer campestre). The synthesis and characterization of 7′-apo-7′,7′-dicyano-β-carotene have been described previously.21 Lycopene was extracted from a 6 oz. can of Thrifty Maid tomato paste using methylene chloride. The filtered solution was added to a silica gel column with a bit of sand at the top. The dark red portion of the column

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Figure 4. (a) (red) Experimental powder Mims ENDOR spectrum of lutein radicals in Cu-MCM-41. T ) 15 K, ν ) 9.8394 GHz, B ) 3505.0 G, MW π/2 pulse ) 32 ns, RF π pulse ) 14 µs, τ ) 200 ns, repetition time ) 2999.82 µs. (violet) Simulated spectrum of I•+ using DFT proton hyperfine coupling tensors in Table 1. (b) (red) Experimental powder Davies ENDOR spectrum of lutein radicals in CuMCM-41 measured at ν ) 9.7281 GHz and B ) 3466.7 G using the standard Davies sequence with the initial τ ) 664 ns, MW pulse ) 800 ns corresponding to a turning angle of ∼16.7 π, RF π pulse ) 25 µs, repetition time ) 2040 µs. (brown) Simulated spectrum of I•+ using DFT proton hyperfine coupling tensors in Table 1.

separation was collected, and the solvent was removed at 35 °C using the rotavap. The purity of the carotenoids was checked by TLC analysis and by 1H NMR (360 MHz, CDCl3). The carotenoids were stored at -14 °C in a desiccator containing drierite. Sample Preparation. Cu-MCM-41 and Cu-SBA-15 were dried in air at room temperature for 24 h. One hundred milligram amounts of the dried sample were activated at 200 °C for 24 h in air, then immediately transferred to a N2 drybox and allowed to cool to room temperature. The solvent, CH2Cl2 (Aldrich, anhydrous), used for the preparation of the EPR samples was stored under nitrogen in a dry box and used without further purification. Approximately 0.3 mL of a 1 mM of carotenoid CH2Cl2 solution was allowed to diffuse into the Cu-MCM-41 and Cu-SBA-15. The EPR tube was sealed with a ground glass joint, taken out of the drybox, and freeze-pumped-thawed at the vacuum line to remove gases. After sitting for ∼15 min, the rest of the solvent was gently pumped off over ∼6 h, keeping the solution at or just above 77 K. Samples were kept in liquid nitrogen and irradiated with a 200-W Xe lamp. Pulsed ENDOR. Pulsed ENDOR experiments were carried out with a Bruker ELEXSYS E-680W/X FT/CW pulse X-band EPR spectrometer. CW X-band EPR measurements were carried out with an X-band (9.5 GHz) Varian (Palo Alto, CA) E-12

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SCHEME 2: H+ Loss upon the Photolysis of the Carotenoid Radical Cationa

a Here, as example, is given lutein for proton loss from C4 and C6′ positions and C5-, C9-, and C13-methyl groups. See 7′-apo-7′,7′-dicyanoβ-carotene (Scheme S1) and lycopene (Scheme S2) in the Supporting Information.

EPR spectrometer equipped with a rectangular cavity. The magnetic field was measured with a Bruker (Billerica, MA) EPR 035 M gaussmeter, and the microwave frequency was measured with a HP 5245L frequency counter. Previous studies10,11 for the carotenoid radical cations and neutral radicals have shown that density functional theory (DFT) calculations accurately predict the isotropic β-methyl proton couplings and the anisotropic R-proton coupling tensors used to simulate the experimental powder ENDOR spectrum. In this paper, couplings predicted by DFT are used to simulate pulsed ENDOR spectra. This analysis yields the type of neutral radicals formed upon proton loss from the radical cation. Pulsed ENDOR simulations are performed using the SimBud/SpecLab programs.22 DFT Calculations. All calculations were done with the Gaussian 03 program package23 on the Cray XD1 computer at the Alabama Supercomputer Center. Geometries were optimized

at the B3LYP/6-31G** level,24,25 which we have previously shown10 to be suitable for predicting the geometry of β-carotenebased radicals. Single point calculations on these geometries were used to predict ENDOR hyperfine couplings at the B3LYP level with the TZP basis set from the Ahlrichs group.26 This basis set has been shown to give good NMR chemical shifts27 and EPR parameters that agree well with the experimental data for carotenes.10,11 The hyperfine proton couplings calculated with this method are within 0.5 MHz of the experimental deduced couplings, whereas other levels of theory give values that differ by as much as a factor of 2.10 The unpaired spin densities were obtained using the AGUI interface28 from the wave functions and spin densities produced by Gaussian 03. The spin density is defined as the difference in the alpha and beta spin densities.

Pulsed ENDOR Studies of Carotenoid Oxidation

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TABLE 1: Isotropic β-Methyl Proton and Anisotropic r-Proton Couplings (MHz) of Lutein (I) Radicals Obtained by DFT Calculationsa for Radical Cation and Neutral Radicals Formed by Proton Loss from C6′ and C4 Positions I•+ position

AXX

C1 C1′ C2 C2′ C3 C3′ C4 C4′ C5 C5′ C6′ C7 C7′ C8 C8′ C9 C9′ C10 C10′ C11 C11′ C12 C12′ C13 C13′ C14 C14′ C15 C15′

1.26 -0.18 1.52 -0.31 -0.50 -0.165 0.004 -0.08 3.10 14.48 0.08

5.84 -14.66 -13.4 0.70 1.08

0.59 0.61 -10.45 -12.95 -0.71 0.86 -3.93 -1.69 -3.30 -6.60

AYY

#I• (6′) AZZ

-0.11 0.04 0.04 0.02 1.36 1.88 -0.06 0.69 1.65 2.08 -0.20 0.39 -0.42 0.57 -0.154 0.325 0.05 0.43 -0.08 0.19 3.18 4.76 14.93 16.28 0.10 0.54 6.20 0.10 5.87 8.19 -10.2 -4.15 -9.1 -3.42 1.70 4.45 2.02 4.32 8.04 8.94 0.28 2.56 1.39 3.97 -7.82 -2.94 -9.38 -3.81 0.02 2.37 1.33 4.41 4.31 6.44 -3.71 -0.26 -1.22 1.32 -3.20 -0.10 -5.43 -1.50

Aiso

1.50 0.15 1.75 -0.04 -0.12 -0.002 0.16 0.01 3.68 15.23 0.24

AXX

0.54 -0.07 0.31 -1.11 -0.24 -0.079 10.55 -1.11 0.75 -11.11

AYY

#I• (4) AZZ

0.02 0.16 -0.01 0.61 0.59 0.86 -0.01 0.33 0.59 1.93 -0.94 0.77 -0.19 0.23 -0.073 0.144 10.77 12.32 -0.43 0.78 0.76 1.45 -6.94 -2.31

Aiso

0.66 0.08 0.94 -0.44 -0.07 -0.003 11.21 -0.25 0.99 -6.78

AXX

0.41 -0.77 0.82 -0.27 6.32 -0.59 -0.05 -0.086 -7.59 0.11

1.78 -2.61 6.63 -9.67 -8.64 2.28 2.47

-9.65 3.99 1.44 -20.01

0.75 1.99 -7.07 -8.71 0.56 2.2

2.62 -20.68 -15.66 5.10 3.54 -22.11

-2.63 -0.53 -2.2 -4.51

4.36 -20.75 -21.11 4.93

-6.68 4.18 1.67 -14.24

-3.01 9.09 4.06 -6.09

7.13 -6.49 2.89 6.58 -15.21 -6.76 -10.77 -5.07 6.18 12.49 4.43 9.15 -15.98 -7.24 11.17 -7.93 5.67 11.25 -15.19 - 7.02 -14.76 -6.78 6.63 12.85

-6.44 5.75 2.39 -13.44

3.24 3.53 -13.01 -14.59 2.16

4.03 -14.22 -10.50 7.92 5.71 -15.11

-18.09 3.93 4.94 -20.9 -20.34 5.14

7.09 -14.32 -14.22 8.14

-23.56 5.93 6.26 -25.42

AYY

AZZ

0.42 -0.13 -0.004 -0.04 0.58 1.52 -0.64 0.56 0.91 1.31 -0.19 0.35 6.43 7.57 -0.39 0.60 -0.02 0.33 -0.074 0.167 -4.78 -1.58 0.12 0.50 -1.85 0.11 3.40 5.58 3.63 7.36 -8.87 -3.85 -10.72 -4.82 2.55 5.42 -5.22 9.57 -13.28 -6.01 4.44 9.26 6.00 11.57 -14.56 -7.09 -15.09 -7.07 6.40 12.37 -7.85 13.94 -17.19 -8.01 7.56 14.19 7.85 14.62 -17.98 -8.44

Aiso

0.84 -0.28 1.02 -0.03 6.77 -0.13 0.09 0.004 -4.65 0.24

4.07 4.84 -8.57 -10.04 3.38 -12.46 5.88 7.50 -14.19 -14.1 7.97 -16.2 9.23 9.57 -17.2

for Neutral Radicals Formed by Proton Loss from Methyl Positions #I•(5) position

AXX

C1 C1′ C2 C2′ C3 C3′ C4 C4′ C5 C5′ C6′ C7 C7′ C8 C8′ C9 C9′

0.56 -0.77 0.84 -0.27 -0.22 -0.11 -0.04 -0.08 0.15 -1.37 0.14 -1.80 -2.09 2.97 2.20 -13.31 -12.96 2.12

AYY

#I•(9) AZZ

0.52 -0.10 -0.04 -0.01 0.67 1.41 -0.42 0.61 0.93 1.35 -0.19 0.36 -0.15 0.46 -0.07 0.24 -0.01 0.35 -0.08 0.17 0.33 0.93 -1.31 -0.29 0.15 0.55 -1.07 -0.70 -1.12 0.09 0.12 3.14 5.38 2.63 5.04 -9.03 -3.88 -9.87 -4.11 2.56 5.49 -4.37 9.85

Aiso

0.88 -0.19 1.04 -0.03 0.03 0.02 0.10 0.004 0.47 -0.99 0.28 -1.19 -1.04 3.83 3.29 -8.74 -8.98 3.39

AXX

-0.17 -0.1 0.91 -0.29 -0.03 -0.027 -0.05 -0.08 -0.17 -0.87 0.14

3.28 0.25 -14.26 -2.96 2.34 -16.05 -14.55

AYY

#I•(13) AZZ

-0.003 -0.04 0.05 -0.01 -0.15 -0.02 -0.07 0.10 1.01 1.44 -0.21 0.38 -0.02 0.08 0.023 0.053 -0.01 0.36 -0.08 0.19 -0.16 -0.06 -0.82 -0.68 0.16 0.57 -0.36 0.12 3.46 5.83 1.00 3.34 -9.71 -4.20 -2.61 -1.23 2.77 5.92 -9.81 -3.42 -9.73 -3.64 10.46

Aiso

-0.11 -0.02 1.12 -0.04 0.01 0.001 0.10 0.01 -0.13 -0.79 0.29

4.19 1.53 -9.39 -2.27 3.68 -9.76 -9.31

AXX

-0.13 -0.05 1.06 -0.34 0.003 -0.01 -0.05 -0.1 -0.77 -0.2 0.14

4.22 0.34 -16.67 -0.43 2.76

AYY

AZZ

-0.003 -0.03 -0.05 -0.01 -0.12 -0.08 -0.02 0.04 1.17 1.68 -0.24 0.44 0.01 0.02 -0.005 0.018 -0.01 0.42 -0.09 0.22 -0.69 -0.64 -0.13 -0.09 0.16 0.64 -0.27 0.14 4.41 7.18 0.95 1.65 -11.37 -4.93 -0.29 -0.26 3.26 6.94 -1.39 12.12

Aiso

-0.11 -0.01 1.30 -0.05 0.01 0.001 0.12 0.01 -0.70 -0.14 0.31

5.27 0.98 -10.99 -0.33 4.32

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TABLE 1: cont. #I•(5) position

AXX

C10 C10′ C11 C11′ C12 C12′ C13

-17.06 3.91 4.43 -21.42 -19.76 5.07

-23.42 5.78 5.99 -25.72

C13′ C14 C14′ C15 C15′ a

AYY

#I•(9) AZZ

Aiso

AXX

AYY

-12.61 -5.55 4.36 9.32 5.10 10.31 -14.89 -7.19 -14.72 -6.74 6.24 12.34 -7.39

-11.74 5.86 6.61 -14.5 -13.74 7.88

-20.85 4.23 3.96 -22.6 -19.66 5.45

14.27 -17.11 -7.84 7.27 14.01 7.34 14.07 -18.17 -8.42

-16.12 9.02 9.13 -17.44

-24.02 6.10 6.21 -26.50

#I•(13) AZZ

Aiso

AXX

-14.26 -6.02 4.75 9.98 4.57 10.74 -15.76 -7.66 -14.87 -6.66 6.76 13.17 -7.40

-13.71 6.32 6.42 -15.34 -13.73 8.46

-0.84 4.82 0.21 -25.59 -3.78 6.02 -26.81 24.78

14.87 -17.61 -8.08 7.67 14.64 7.58 14.50 -18.8 -8.76

-16.57 9.47 9.43 -18.02

-29.44 6.21 5.25 -27.53

AYY

AZZ

-0.75 -0.48 5.39 11.39 1.60 5.39 -17.88 -8.64 -3.44 -1.51 7.34 14.58 -16.48 -5.94 -16.37 -6.19 16.35 -20.07 -8.42 7.57 15.29 6.26 14.89 -19.83 -8.86

Aiso -0.69 7.20 2.40 -17.37 -2.91 9.31 -16.41 -15.78 -19.31 9.69 8.80 -18.74

The bold values are the isotropic coupling constants Aiso given by averaging the three anisotropic coupling tensors AXX, AYY, AZZ.

TABLE 2: Relative Energies for Loss of a Proton from the Radical Cationa lutein (I) 7′-apo-7′,7′-dicyano-β-carotene (II) lycopene (III) β-carotene10 zeaxanthin11 8′-apo-β-caroten-8′-al canthaxanthin violaxanthin11 a

∆E(4)

∆E(5)

∆E(9)

∆E(13)

∆E(4′)

∆E(5′)

∆E(6′)

∆E(9′)

∆E(13′)

6.68 0 0 0 0 0

6.70 5.34 5.47 4.92 3.15 4.97 0 19.43

15.16 11.11 11.12 10.29 8.39 10.43 4.32 0

17.06 12.85 12.35 12.18 10.21 12.84 5.83 1.78

45.44

22.69

0

14.72 13.81 11.12 10.29 8.39 10.44 4.32 0

17.04 13.99 12.35 12.18 10.21 12.38 5.83 1.78

15.33

0 0 0 15.33

5.47 4.92 3.15 0 19.43

∆E is calculated relative to the minimum energy (kcal/mol).

Results and Discussion DFT Calculations. Lycopene (III) is a symmetric molecule that has six methyl groups in three distinct positions: C5(5′), C9(9′) and C13(13′) (see Scheme 1). The primed positions of these molecules are equivalent by symmetry with the unprimed positions, so calculations of the neutral carotenoid radicals were carried out only for the proton loss at the unprimed positions. Calculations for lutein (I) and 7′-apo-7′,7′-dicyano-β-carotene (II), which are asymmetric, were done for both unprimed (shown in this paper) and primed positions (in Supporting Information). Calculations were carried out for the radical cations and for the neutral radicals formed by loss of the C4(4′) methylene proton and C5(5′)-, C9(9′)-, and C13(13′)-methyl protons for I, II, and III. In addition, calculations for loss of a proton from the C6′ position was performed in the case of asymmetric I. On the basis of the results for β-carotene10 and zeaxanthin/violaxanthin,11 these species were expected to occur, according to Scheme 2 for I, and Schemes S1 and S2 for II and III (see Supporting Information) upon light irradiation in activated silica-alumina. The optimized geometry parameters for the minimized structures of I (I•+, #I•(4), #I•(5), #I•(9), #I•(13), #I•(4′), #I•(5′), #I•(6′), #I•(9′), and #I•(13′)), II (II•+, #II•(4), #II•(5), #II•(9), #II•(13), #I•(9′), and #I•(13′)), and III (III•+, #III•(4), #III•(5), #III•(9) and #III•(13)) are given in the Supporting Information in Tables S4-S25. Loss of H+ from the radical cation is indicated by #. The unpaired spin density distributions for the radical cation and the neutral radicals formed by proton loss from the unprimed positions are shown pictorially in Figures 1, 2, and 3 for I, II, and III, respectively. Notably, when a proton is lost from the radical cation (as in Scheme 2), the unpaired spin density distribution increases at carbons on the other end of the chain. The carbons of the radical cations with excess R (blue) possess excess β (green) unpaired spin density in the neutral

radicals. In Table 1 of this paper and Tables S1, S2, and S3 in Supporting Information, the DFT isotropic proton couplings for all radical cations and neutral radicals are given in bold type, and the anisotropic R-proton couplings are given in standard type. Previous CW-ENDOR measurements7,8 showed resolved proton ENDOR lines that were attributed to the presence of rapidly rotating methyl groups. Subsequently it was shown29 that the β-methyl protons in carotenoids undergo rapid rotational motion even at 5 K, which averages out the small dipolar anisotropy30 and results in the same value of isotropic proton coupling for all three protons in each methyl group. This is the reason that β-methyl protons in carotenoid radicals give rise to narrow, well-resolved and intense CW-ENDOR lines in powder spectra. According to our previous data10 on carotenoid radicals, CW-ENDOR lines from the methyl protons are well-resolved, whereas lines from R-protons are often broadened and not easily detected.7,9,31,32 The Davies pulsed ENDOR spectrum of I was obtained using a pulse length that does not record proton hyperfine couplings less than 1 MHz. The spectral lines denoted with A in the center of both Davies and Mims ENDOR spectra are due to proton couplings less than 2 MHz and to the matrix protons and are not considered in the spectral comparison. Simulations of Mims and Davies ENDOR spectra (Figure 4), assuming only the radical cation is formed, lack notable features seen in experimental spectra at B, C, D, and E. Other radicals are making a significant contribution to experimental spectra. For example, different neutral radicals have predicted Davies ENDOR spectra with features that coincide with features seen in the experimental spectrum but absent in the radical cation spectrum (Figure 5). The neutral radicals with loss of a proton at C6′ and C4 positions and C5-, C9-, and C13-methyl groups give rise to the simulated spectra in Figure 5b, c, d, e, and f respectively. Areas D and E

Pulsed ENDOR Studies of Carotenoid Oxidation

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Figure 5. (a) Experimental powder Davies ENDOR spectrum of lutein radicals in Cu-MCM-41 as in Figure 4b. (b) Simulated powder ENDOR spectrum using DFT proton hyperfine couplings listed in Table 1 for #I•(6′), (c) #I•(4), (d) #I•(5), (e) #I•(9), and (f) #I•(13).

Figure 7. (a) (red) Experimental powder Mims ENDOR spectrum of lutein radicals in Cu-MCM-41 as in Figure 4a. (blue) Simulated spectrum of I•+, #I•(6′), #I•(4), #I•(5), #I•(9), and #I•(13) in 1:1:1:1:1:1 ratio using DFT proton hyperfine coupling tensors in Table 1. (b) (red) Experimental powder Davies ENDOR spectrum of lutein radicals in Cu-MCM-41 as in Figure 4b. (blue) Simulated spectrum of I•+, #I•(6′), #I•(4), #I•(5), #I•(9), and #I•(13) in 1:1:1:1:1:1 ratio using DFT proton hyperfine coupling tensors in Table 1.

Figure 6. (a) (red) Experimental powder Mims ENDOR spectrum of lutein radicals in Cu-MCM-41 as in Figure 4a. (purple) Simulated spectrum of I•+ and #I•(6′) using DFT proton hyperfine coupling tensors in Table 1. (b) (red) Experimental powder Davies ENDOR spectrum of lutein radicals in Cu-MCM-41 as in Figure 4b. (purple) Simulated spectrum of I•+ and #I•(6′) using DFT proton hyperfine coupling tensors in Table 1.

require spectra from #I•(13), with the peaks at B, C and D contributed by #I•(6′), #I•(4), #I•(5), and #I•(9). If the ENDOR spectrum of the neutral radical with the lowest energy, #I•(6′), is simulated and added to the simulated Mims spectrum of the radical cation, then the match is improved at B, C, and D (Figure 6a). Similar improvement occurs at B, C, D, and E for the Davies ENDOR spectrum given in Figure 6b. Close inspection of the Mims ENDOR spectrum shows better agreement at (D)D′, indicating the presence of neutral radicals with larger couplings, such as #I•(13). Combining spectra in a 1:1:1:1:1:1 ratio gave an improved match at D′ (Figure 7a) due

largely to the contribution from #I•(13). However, an inspection of Figure 7b indicates a poor fit at E. Unfortunately, ENDOR is a nonlinear spectroscopy that depends on relaxation, thus making it impossible to quantify the concentration of different radicals. However, the presence of an ENDOR peak requires the presence of a species with such a peak. Davies and Mims ENDOR have different intensities for a given radical so that different ratios of spectra are to be expected when matching experimental data from radical mixtures. The pulse settings used for Mims ENDOR has made it possible to observe the ENDOR lines at D(D′). Including the proton couplings for #I•(9′) and #I•(13′) would not change the simulation due to the similarity in couplings (see Table S1 in the Supporting Information). The large proton couplings from the C3′, C4′, and C5′ positions of #I•(5′) would give resolved EPR lines, but they are not experimentally observed. The small couplings for the #I•(4′) would contribute to the center of the ENDOR spectrum. In Figure 8 are the Mims ENDOR spectra for I (Figure 8a), II (8c) and III (8d) in Cu-MCM-41; Figure 8b has the spectrum of I in Cu-SBA-15. In these four spectra, the concentrations of the neutral radicals (noted by areas C and D) are shown as a function of hydrogen bonding (Figure 8a) vs polarity (Figure 8c and d) and in a different matrix (Figure 8b). The absolute concentration of radicals by integration of the EPR spectra is greater in Cu-MCM-41 than in Cu-SBA-15. However, intensity at B, C, and D is relatively the same in Figure 8a, c, and d. The pulse times used have eliminated any 5 MHz couplings in the Mims ENDOR spectrum (Figure 8). However,

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Figure 8. Mims ENDOR spectra of the carotenoid radicals measured using a sequence of pulses and delays (π/2-τ-π/2-T-π/2-τ-echo). (a) T ) 15 K, ν ) 9.8394 GHz, B ) 3505.0 G, MW π/2 pulse ) 32ns, RF π pulse ) 14 µs, τ ) 200 ns, repetition time ) 2999.82 µs, as in Figure 4a. (b) T ) 20 K, ν ) 9.8330 GHz, B ) 3505.0 G, MW π/2 pulse ) 32 ns, RF π pulse ) 14 µs, τ ) 200 ns, repetition time ) 3999.42 µs. (c) T ) 10 K, ν ) 9.8439 GHz, B ) 3506.0 G, MW π/2 pulse ) 32 ns, RF π pulse ) 14 µs, τ ) 220 ns, repetition time ) 3999.42 µs. (d) ν ) 9.8357 GHz, B ) 3505.0 G, MW π/2 pulse ) 20 ns, RF π pulse ) 8 µs, τ ) 200 ns, repetition time ) 3999.82 µs.

the only couplings of interest are the 12-16 MHz couplings due to neutral carotenoid radicals marked by the letters C and D. Although it is clear that the presence4-6 of a metal ion increases the photoyield of carotenoid radicals, it is not possible to explain the variation in yield as a function of metal ion. While such data are lacking, integration of the EPR spectra (Figure 8) shows that as the carotenoid varies from the hydrogen-bonded lutein to the polar 7′-apo-7′,7′-dicyano-β-carotene to the non-

Lawrence et al. polar lycopene in Cu-MCM-41, the photoyield for the production of the carotenoid radicals varies considerably: ∼15:5:2 and then to 1.5 for lutein in Cu-SBA-15. On the basis of our previous studies,4-6,12,13 this variation is due to the distance and location of the carotenoid with respect to the metal ion to the location of the carotenoid on the surface or in the cylindrical pores. However, the relative concentration of the #Car• neutral radical (lines C and D of Figure 8) is similar for the three carotenoids, despite the difference in polarity, hydrogen bonding, and coordination to Cu. An important feature can be deduced by revisiting previously reported5,6,12 CW-ENDOR spectra. If a comparison is made between an ENDOR spectrum of canthaxanthin in Ti-MCM415 (Figure 9 of reference 5; compare the peak at 22.5 MHz (#Car•) to that at 17.5 MHz (Car•+)), the apparent concentration of the neutral radical is approximately equal to that of Car•+. In the presence of Fe-MCM-416 (Figure 10 of reference 6) and Al-MCM-4112 (Figure 7 of reference 12; compare the peak at 22 MHz to that at 17 MHz), the apparent concentration of the neutral radical exceeds that of Car•+ by almost factors of 2 or 4 respectively, whereas that of the 7′-apo-7′,7′-dicyano-βcarotene compound is almost 4 times as great. In Table 2 are given the relative DFT energies for the loss of a proton from the methylene position C4 (∆E(4)) and from the methyl groups attached to either C5 (∆E(5)), C9 (∆E(9)), or C 13 (∆E(13)), as well as for the primed positions for I, II, III, β-carotene, zeaxanthin, 8′-apo-β-caroten-8′-al, canthaxanthin, and violaxanthin. In addition, DFT minimum energy for loss of a proton from position C6′ (∆E(6′)) of I is listed. Notably, ∆E for the loss at 4, 5, 9, or 13 for II, III, β-carotene, and 8′apo-β-caroten-8′-al are all about the same value (0, 5.0 ( 0.5, 11.0 ( 0.5, and 12.5 ( 0.5 kcal/mol) but higher than zeaxanthin by ∼2.5 kcal/mol (with a 0.01 kcal/mol value for ∆E(5) of zeaxanthin) and higher than canthaxanthin by 7.0 kcal/mol. Despite this energy difference, the ENDOR-deduced concentration of #Car•(4), #Car•(5), #Car•(9), and #Car•(13) does not reflect a greater barrier for formation of #Car•(5), #Car•(9), and #Car•(13) (or the primed #Car•(4′), #Car•(5′), #Car•(9′) and #Car•(13′)), as the concentrations of #Car•(4) should greatly exceed that of #Car•(5), #Car•(9), and #Car•(13). Mims and Davies ENDOR simulations (Figure 7a and b, respectively) of I showed similar concentrations (1:1:1:1:1:1) for I•+, #I•(6′), #I•(4), #I•(5), #I•(9), and #I•(13), respectively. The contribution of #I•(4), #I•(5), #I•(9), and #I•(13) to the spectra are essential, even though they are energetically more unfavorable than #I•(6′) (see Table 2). Even though ∆E(9) and ∆E(13) for I are higher than that of II (Figure 8c) and III (Figure 8d), a similar concentration ratio also occurs for II and III (compare Mims intensity of peak D at 23-24 MHz (#Car•(13)) to the peak B at 18-19 MHz (#Car•+ + #Car•(4) + #Car•(5) + #Car•(9)). However, the most significant observation is that there is little variation in the apparent concentration of #Car•(4), #Car•(5), #Car•(9), and #Car•(13), whereas energetically, only #Car•(6′) in I should be present and the same, small variation in #Car•(5), #Car•(9), and #Car•(13) and only #Car•(4) in II and III should be observed. This same behavior was also observed for zeaxanthin11 and β-carotene,10 in which concentration ratios 1:1:1:1:1 and 5:3:1:1, respectively, were observed. The similar amounts suggest that the reaction products are kinetically rather than thermodynamically controlled; the same result was found for other carotenoids.11 Conclusion The relative yield of the neutral carotenoid radical, #Car•, formed by loss of a proton from the carotenoid radical cation,

Pulsed ENDOR Studies of Carotenoid Oxidation to that of the carotenoid radical cation is to first order independent of the polarity or hydrogen bonding of the carotenoid for Cu-MCM-41. By revisiting previously published CW-ENDOR spectra, it is now clear that the concentration ratio of the neutral radical to the radical cation does depend on the metal, being greater for Fe- and Al-substituted MCM-41 than for Ti- or Cu-substituted MCM-41. In the absence of metals, the formation of carotenoid radicals upon light illumination of carotenoids in MCM-41 is dependent on the ionization potential of the carotenoid, being greatest for the carotenoid with the lowest ionization potential. Acknowledgment. This work was supported in part by the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Sciences, U.S. Department of Energy, Grant DE-FG02-86ER-13465 and the National Science Foundation for EPR Instrument Grants CHE-0342921 and CHE-0079498 to U.A. This study, on the part of the Hungarian authors, was supported by a grant from OTKA K 60121 (Hungarian National Research Foundation). This work was carried out in part using the resources provided by the Alabama Supercomputer Center in Huntsville, AL. For computer facilities, we thank David A. Dixon, who is indebted to the Robert Ramsay Endowment of the University of Alabama and to the Catalysis Center Program of the Department of Energy. Supporting Information Available: Schemes of H+ loss upon the photolysis of the carotenoid radical cations of 7′-apo7′,7′-dicyano-β-carotene (II) and lycopene (III); isotropic βmethyl proton and anisotropic R-proton tensors (MHz) of lutein (#I•(4′), #I•(5′), #I•(9′), #I•(13′)), 7′-apo-7′,7′-dicyano-β-carotene (II•+, #II•(4), #II•(5), #II•(9), #II•(13), #II•(9′), #II•(13′)), and lycopene (III•+, #III•(4), #III•(5), #III•(9), #III•(13)) obtained by DFT calculations; Optimized x, y, z coordinates at B3LYP/631G** level for I•+, #I•(4), #I•(5), #I•(9), #I•(13), #I•(4′), #I•(6′), #I•(5′), #I•(9′), #I•(13′), II•+, #II•(4), #II•(5), #II•(9), #II•(13), #II•(9′), #II•(13′), III•+, #III•(4), #III•(5), #III•(9), and #III•(13). This information is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Telfer, A.; De Las Rivas, J.; Barber, J. Biochim. Biophys. Acta 1991, 1060, 106. (2) Frank, H. A.; Brudvig G. W. Biochemistry 2004, 43, 8609. (3) Tracewell, C. A.; Vrettos, J. S.; Bautista, J. A.; Frank, H. A.; Brudvig, G. W. Arch. Biochem. Biophys. 2001, 385, 61. (4) Konovalova, T. A.; Gao, Y.; Schad, R.; Kispert, L. D.; Saylor, C. A.; Brunel, L-C. J. Phys. Chem. B 2001, 105, 7459. (5) Gao, Y.; Konovalova, T. A.; Xu, T.; Kispert, L. D. J. Phys. Chem. B 2002, 106, 10808. (6) Konovalova, T. A.; Gao, Y.; Kispert, L. D.; van Tol, J.; Brunel, L.-C. J. Phys. Chem. B 2003, 107, 1006. (7) Jeevarajan, A. S.; Kispert, L. D.; Piekara-Say, L. Chem. Phys. Lett. 1993, 209, 269.

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