extfieldsofstudy: Chemistry Medicine

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Charge-transfer excitation: unconventional yet practical means for controlling stereoselectivity in asymmetric photoreactions

In chiral donor–acceptor (D–A) systems, irradiation wavelength plays vital roles in determining the photochemical consequences. Selective excitation of a D–A complex at the charge-transfer (C-T) band affords an excited C-T complex (ECT), while the local-band excitation of D or A may lead to the formation of a conventional exciplex (EX) upon subsequent interaction with the D–A partner. These two excited species, generated from the same D–A pair, may be categorized formally as excited complexes or exciplexes, but should be distinguished, provided that they significantly differ in structure and reactivity. Indeed, ECT and EX exhibit distinctly different temperature-dependent photophysical and photochemical behaviours, which are assignable to the differences in relative stability, conformational flexibility and/or solvation properties. Fine-tuning excitation wavelength further enabled us to discriminate stereoisomeric intramolecular C-T complexes through preferential excitation, as C-T complexes are generally composed of an ensemble of various geometries. Besides temperature and solvent polarity, the excitation wavelength was shown to be employed as an unconventional yet practical tool for critically controlling the chemo-, regioand stereoselectivities in molecular and supramolecular photochemistry.

Introduction
An aromatic electron donor (D) and acceptor (A) often form a weakly interacting complex in the ground state 1-3 with accompanying new absorption at longer wavelengths assignable to the C-T band.
Compared with the other weak interactions, such as hydrogen bonding, 4 the C-T interaction is less robust and less directional. Hence, its use for controlling chemical reaction is often discouraged. It is also true however that a pre-equilibrium complex of C-T character, or more forthrightly a C-T complex, plays crucial roles in such (thermal) bimolecular reactions as electrophilic aromatic substitution and addition reactions. 5,6 Accordingly, the properties of C-T complexes, as well as the structure-reactivity relationship, have been extensively investigated over the past three decades. In contrast, the importance of (ground-state) C-T interaction is not significantly appreciated or taken into account in general in the excited-state chemistry (including the photochemical reaction of D and A), although there has been a substantial dispute about its significance (vide infra). Photoirradiation of a C-T complex at its C-T band leads to the formation of an excited C-T complex (ECT), which may undergo electron transfer, addition, cleavage, rearrangement and/or other reactions, or revert to the ground state. Upon direct or local-band excitation of one component of a D-A pair at its local band, the excited D or A generated diffuses to encounter with the ground-state counterpart (A or D), affording a conventional exciplex (EX), 7 which may also undergo some net reaction(s). By definition, these two excited-state species should be categorized as exciplexes in a broader sense, as they are undoubtedly excited-state complexes. We will however employ a more precise definition in this review, whenever it is feasible. Thus, ECT refers to an excited-state species or an ensemble formed upon selective excitation of a ground-state complex at the C-T band, while EX denotes a conventional excited-state complex formed through the diffusion of an excited D or A to encounter with a ground-state partner. Throughout the discussion to follow, we will not specifically discern the two situations that arise from the difference in the ensemble or in structure, since they lead to the distinctly different photochemical behaviours in any event. More precise discrimination remains to be elucidated in the future study.
In the last decade, we have shown that the C-T interaction plays decisive roles in a variety of asymmetric photoreactions.
Despite the low effective concentration (as a consequence of the generally small association constants), the selective C-T excitation was readily achieved by carefully choosing the irradiation wavelength. Remarkably, the ECT species thus generated exhibits the photochirogenic and temperature-dependence behaviours significantly different from those displayed by the EX species. In this review, we will emphasize with ample examples that such a simple, readily attainable, change as excitation wavelength can dramatically alter the relative contribution of the ground-and excited-state conformer ensembles of a C-T complex, allowing us to favourably modulate the photochemical consequences. As for the longstanding debate on the identity of ECT, we will unambiguously demonstrate that ECT differs from EX both in structure and reactivity.

Ground-state charge-transfer interaction
Before getting into the details of the photochemistry of the C-T complex, we briefly summarize the nature of the C-T complex in the ground state. C-T complexes do not involve covalent or any other strong interactions, but are formed upon association of D and A molecules and hence equilibrated with free D/A. Consequently, they are, in general, extremely sensitive to the alteration in temperature, concentration, and solvent. According to Nobel laureate Robert Mulliken, the ground and excited states of a 1 : 1 C-T complex formed between D and A can be described as a resonance of a pair of wavefunctions as follows: 8 where the wavefunctions C 0 (D,A) and C 1 (D + ,A À ) designate the two canonical forms of a complex with non-bonded and dative structures. 1,3 The degree of C-T (r) is a measure of the resonance contribution of the non-bonded and dative states, and can be estimated by measuring dipole moment or by theoretical calculations. In weak molecular complexes, one electron moves from D to A in the excited state, which means r = 1. The transition energy of the (mostly coloured) characteristic C-T band observed in the absorption spectrum is determined as C E À C N , which is directly related to r. The energy gap for C-T transition is proportional to the ionization potential of D and the electron affinity of A, and is also altered slightly by the Coulombic term. The extinction coefficient of the C-T band is moderate to high in general, but widely varies with the degree of association, which is evaluated by the C-T association constant K CT . By analysing the intensity change of the C-T band as a function of the concentration of D or A upon titration (Benesi-Hildebrand), or by using the other related methods (Scott and so forth), the K CT values have been evaluated for a large number of C-T complexes. 9 The K CT values thus obtained are generally small, typically ranging from 0.01 to 10 M À1 cm À1 for aromatic D-A pairs. [10][11][12][13][14] In the C-T complex, D and A stack on each other generally in a cofacial manner with an inter-facial distance of typically E3.5 Å, which is appreciably shorter than the van der Waals contact. Despite the relatively weak association, such a cofacial D/A alignment is repeatedly and effectively utilized in biological systems and processes, imaging applications, and photoelectric devices. [15][16][17] Tadashi Mori

Selective excitation at the C-T band
Even when the ground-state C-T interaction is not apparent, the excited-state donor or acceptor (D* or A*), generated upon localband excitation of D or A, may have stronger interaction with the ground-state partner to form a conventional exciplex (EX) in a diffusive manner within the lifetime of the excited state (typically 1-100 ns). It is to be noted that due to the possible spectral overlap with the local bands of D and/or A, the C-T band would also be excited to a certain extent upon direct excitation of a local-band.

EX EX
An apparently similar, but conceptually different, excited-state complex may also be generated by selectively exciting the groundstate C-T complex. Thus, the irradiation at the C-T band leads to the formation of singlet, and triplet (after intersystem crossing), excited state(s) of the C-T complex (ECT), which subsequently undergo the intra-complex electron transfer to give a geminate radical ion pair (RIP). ECT and RIP may decay through back electron transfer and/or undergo some chemical processes, but generally dissociate to yield free radical ions, which eventually lead to net chemical reaction.
The photophysical processes of such reactive ECTs have been studied by time-resolved (picosecond) spectroscopies for a number of C-T systems. 18,19 Upon selective photoexcitation at the C-T band, ECT immediately (r0.1 ns) undergoes intra-complex electron transfer to give a singlet RIP. A recent femtosecond stimulated Raman spectroscopic study on the photoinduced charge-separation dynamics of a C-T complex of pyromellitic dianhydride and hexamethylbenezene showed that the structural relaxation is facilitated by solvation dynamics during the charge separation process and that the ECT already possesses the significant charge-separated character and spontaneously relaxes. 20

Conformational variety of ECT
The conformational variety of ECT, as well as the multidimensionality of the reaction coordinate, have become widely recognized recently, which may cause additional complexity in understanding the excited-state behaviour of the C-T complex. For instance, the transient absorption-stimulated emission spectroscopic studies on the relaxation dynamics of excited donor-acceptor auramine dyad revealed the existence of two distinct excited-state conformers which differ in geometry and dynamics. 21 Another investigation on an excited intramolecular D-A system underlined two distinct C-T excited states, in addition to the locally excited state. 22 Besides the intramolecular C-T systems (spectroscopically) distinguishable excited-state complexes have also been detected in the coupled electron-proton transfer process of hydrogen-bonded acid-base systems, for which the femtosecond transient absorption and coherent Raman spectroscopies confirmed the presence of two distinct excited states with different dynamic signatures. 23 Furthermore, the vibrational quantum state was also utilized recently in controlling the excited-state behaviour and the product distribution upon oxidation of acetylene under the atmospheric conditions. 24 Spectroscopic discrimination of ECT from EX Under the (thermally) fully equilibrated conditions, a common excited-state complex (or ensemble) is reached from ECT and EX through rapid relaxation. However, these species can exist as separate species if not freely interconverted within the lifetimes of these excited states due to the energy barrier or potential surface crossing (Fig. 1).
Accordingly, the similarities and dissimilarities of ECT and EX have been intensively discussed for a number of D-A pairs. However, the results of the foregoing studies are rather controversial or opposed, probably because the photophysical and photochemical behaviours observed are highly systemdependent. Fluorescence spectra in solution are generally broad and structureless, and thus the difference in fluorescence should be difficult to detect or very small between ECT and EX, if exists. Nevertheless, several investigations disclosed that ECT and EX display appreciably different fluorescence spectra. For instance, the EX emission (l EX max = 530 nm) was observed upon fluorescence quenching of 1,2,4,5-tetracyanobenzene by 2-phenylpropene. Interestingly, additional weak fluorescence, assignable to ECT with a geometry different from EX, was observed at shorter wavelengths (l ECT max = 480 nm) upon selective C-T excitation. 25 For the 9,10-dicyanoanthracene-naphthalene pair, diverse fluorescence from ECT was observed at 490 nm in non-polar solvent at low temperatures, which is somewhat higher in energy than the EX fluorescence observed at 540 nm, and the lifetimes of these species showed different temperature-dependence behaviour. 26,27 At higher temperatures, the difference in lifetime becomes diminished due to the faster solvent reorientation. Isomeric forms of the excited-state complexes have also been identified for anthracene-N,N-dimethylaniline and perylene-N-methylaniline pairs, but not for the anthracene-aniline or perylene-N,N-dimethylaniline pair for reasons not specified. 28

Distinction between ECT and EX by product analysis
Photocycloaddition behaviour of (E)-stilbene (ST) with dimethyl fumarate has been compared at different excitation wavelengths. 29 Excitation of the weak ground-state C-T complex resulted in the same chemical reactions as those obtained upon local-band excitation of (E)-ST. Fluorescence from the complex was also studied but indistinguishable from that of EX formed via the encounter of excited ST with dimethyl fumarate. As such, ECT was concluded to be indistinguishable from EX for this D-A pair. Contradictory observations have been reported for other D-A pairs. For instance, photoirradiation of acenaphthylene in the presence of tetracyanoethene led to the formation of a cycloadduct through the singlet excited state of the donor. However, the C-T excitation of the groundstate complex led to a complete recovery of the starting materials even upon prolonged irradiation in various solvents. [30][31][32] It has been reported also that the adducts' yields were considerably dependent on the excitation wavelength in the photocyclization of acenaphthylene or indene derivatives with a variety of acceptors. 33,34 These observations may be explained in terms of the geometry difference between EX and ECT. Such wavelength-dependent reactivities reported in the literature are indicative that ECT is an excited-state complex species different from EX in structure and reactivity, but how and to what extent they differ is an arduous question remained to be answered.

Asymmetric photoreactions of the intermolecular C-T complex upon local-band versus C-T excitations
Diastereoselective photocyclization of (E)-or (Z)-stilbene with chiral fumarate As discussed above, ECT and EX resemble each other and are difficult to differentiate. Oftentimes, the excitation mode (i.e., local-band versus C-T) does not appreciably affect the net photochemical outcomes (vide supra). Since the difference in free energy is relatively small in general for a pair of diastereomeric transition states leading to the end products, the relative rate constant, or diastereoselectivity, is likely to critically respond to the change in nature and geometry of the excited species. Hence, employing asymmetric photoreaction is expedient for experimentally differentiating ECT from EX by observing the change in the stereochemical outcome. We employed a chiral version of the well-established photocycloaddition of (E)-ST to alkyl fumarate, 29 where (R)-1-methylpropyl was chosen as the simplest chiral alkyl group. 35,36 In this system, the intervention of ECT versus EX was unambiguously sorted out by analysing the diastereoselectivities of the cyclobutanes obtained upon excitation at different wavelengths, as detailed below.
Photoirradiation of a mixture of (E)-ST and bis((R)-1-methylpropyl) fumarate ((R)-F) afforded a mixture of cycloadducts (Scheme 1), irrespective of the excitation mode (local-band or C-T). Thus, besides the geometrical isomers of both substrates and a small amount (B1%) of stilbene dimer, two stereoisomeric cyclobutanes, mand d-truxinates (m-and d-CB), were obtained in fair to good yields (typically 10-60%), along with a considerable amount (40-60%) of butadiene products most likely derived from the oxetanes produced by the Paternó-Büchi reaction. Despite the substantial efforts, the diastereoselectivities of these oxetanes were not able to be determined, due to the instability (ready rearrangement to the butadienes) under the GC conditions employed. We therefore concentrated on the analyses of the product ratio (m-CB/d-CB) as well as the diastereoselectivity. Although the photoreaction of (Z)-ST with dimethyl fumarate was reported to proceed much slower than that of (E)-ST, we noticed that (Z)-ST reacts with (R)-F in nearly comparable efficiencies, and more interestingly the diastereoselectivity of oxetane was inverted. As a consequence of concurrent E-Z photoisomerization of stilbene, the observed diastereoselectivities were gradually decreased with increasing irradiation time, irrespective of the geometry of the starting material, (E)-or (Z)-ST. Therefore, comparisons were made by using the values extrapolated to zero irradiation time, to facilitate the direct assessment of selectivities caused upon distinct excitation. Table 1 compares the product ratios and diastereomeric excesses (de's) obtained in the photocycloaddition of (E)-or (Z)-ST to (R)-F in toluene at various temperatures. 35,36 Under all the reaction conditions employed, the same products were formed, but in varying ratios and de's. Upon cycloaddition to (R)-F, (E)-ST gave more m-truxinate than d-truxinate at all the examined temperatures, while (Z)-ST afforded more d-truxinate. inversion of de. Crucially, both the product ratio and diastereoselectivity were highly dependent on the excitation mode, clearly demonstrating that ECT differs from EX in structure and reactivity. Temperature plays another important role in determining the (dia)stereoselectivities. Thus, the C-T excitation of the [(E)-STÁ(R)-F] complex yields the cyclodimers in consistently high de's of Z95% independent of the temperature, whereas the de significantly decreased at lower temperatures upon local-band excitation. The contrast in photobehaviour became even more apparent at higher temperatures upon local-band versus C-T excitation in the photoreaction of (Z)-ST with (R)-F. Intriguingly, this contrasting behaviour was strongly solvent-dependent, being observed in ether and toluene but not in methylcyclohexane, which implies the positive role of solvation in the excited-state relaxation dynamics of the ECT and EX species. Thus, in the local-band excitation, the product's de became more positive or less negative (favouring the formation of the same specific diastereomer) by raising the temperature, irrespective of the geometry of stilbene used. In contrast, the de value was kept high upon C-T excitation throughout the temperature range employed. Such contrasting temperaturedependence behaviours may be explained in terms of the differences in relative energy, equilibrium, and/or the cyclization rate of the corresponding diastereomeric transition states or precursor complexes. However, as the photoreaction of stilbene with fumarate gives rise to other products such as oxetane, the competition with the additional reaction channel(s) would also be taken into account in more detailed mechanistic elucidations.
The C-T complexation was substantially enhanced by applying hydrostatic pressure to give the cyclodimers at faster rates and in better yield upon excitation. Nevertheless, the diastereoselectivity did not show any appreciable changes, 37 indicating that the molecular and activation volumes of the diastereomeric C-T complexes and transition states are equally reduced or compensated with each other.
Theoretical investigations of the ground-and excited-state structures of a diastereomeric complex pair of (E)-stilbene with chiral fumarate Theoretical calculations provide us with valuable insights into the nature of the C-T complex in its ground and excited states. Thus, the geometries of the diastereomeric C-T complexes between (E)-ST and (R)-F in the ground state were theoretically optimized by the dispersion-corrected DFT method at the DFT-D3-B-LYP/TZVP level, 38 which is known to properly incorporate the non-bonded interactions. Fig. 2 shows the most stable diastereomeric re,si-and si,re-stacked pair of the C-T complex, in which the reacting double bonds are stacked face-to-face and the fumarate's double bond is s-cis to both of the carbonyls. For simplicity purpose, the isomeric s-cis, s-trans-and s-trans, s-trans-rotamers will not be separately examined in the following discussion, but note that they are only slightly higher in energy (by 0.1-0.2 or 0.3-0.4 kcal mol À1 ) than the s-cis, s-cisrotamer and should also contribute significantly as an ensemble to the overall diastereoselectivity. Compared with the parallel conformer, the other conformers with crossed double bonds were found to be considerably less stable (by >5 kcal mol À1 ) and hence ignored. Note however that the cross conformer, possessing the correct geometry, is a potential precursor to d-truxinate, if the photocycloaddition is concerted. The most stable si,re-[STÁF] complex, in which the ST's si-and F's re-faces are confronting, differs in alkyl orientation from the diastereomeric re,si-[STÁF], but these two share essentially the same geometry around the reacting double bonds. The energy difference in gas phase was evaluated as 0.43 kcal mol À1 by the most accurate SCS-MP2/TZVPP calculations, 39 indicating that the si,re-and re,si-complexes are populated in ca. 2 : 1 ratio (equivalent to 35% de) at 25 1C. The fact that a much higher diastereoselectivity of 95% de was achieved upon C-T excitation (Table 1) reveals that the product's de is not an immediate function of the ground-state diastereomer equilibrium but is also governed by the excitation efficiency and the excited-state Table 1 Product ratios and diastereomeric excesses (de's) obtained in the diastereoselective photocycloaddition of (E)-and (Z)-ST with (R)-F upon C-T versus local-band excitation a

Donor
Temp. (1C) Local-band excitation (313 nm)  dynamics upon excitation, relaxation, deactivation, intersystem crossing and cycloaddition. The ST and F molecules are in close contact at average spacings of 3.50 and 3.45 Å for the si,re-and re,si-complexes, respectively, while the facing double bonds are not exactly overlaid but slid by B1.5 Å. The spacing is smaller than the sum of the van der Waals radii, confirming the strong C-T interaction in the ground state. The slid-out benzene ring of ST is tilted by 10 and 201 in the si,re-and re,si-complexes, respectively, avoiding the steric clash with the facing alky group of F, while the benzene ring on the opposite side remains cofacial to retain the p overlap. As anticipated, the calculated degree of C-T was fairly small, merely amounting to 2.0 and 2.5% for the si,re-and re,si-complexes, respectively. On the whole, the ground-state C-T complex avoids the complete p overlap due to the electrostatic and Pauli repulsion and is less symmetrical. In contrast, the excited-state structure becomes almost symmetrical to maximize the p overlap. The structures of a diastereomeric exciplex pair of (E)-ST with (R)-F were theoretically predicted at the TD-DFT-BH-LYP/ TZVP level (Fig. 2). 40,41 Interestingly, the centre-to-centre distance between the double bonds of ST and F is significantly reduced to 3.14 and 3.08 Å in the excited-state si,re-and re,si-complexes, which are balanced by adjusting the ST conformation to avoid the steric congestions at the peripheries. Analogous bent structures have been reported for the excimers with relatively large p-systems. 42 As a result of the steric repulsion, the interperiphery distance is much larger for the exciplex than for the C-T complex. This explains at least in part why the C-T, rather than local-band, excitation is more effective in manipulating the stereochemical course of the subsequent photocycloaddition. In this context, it is crucial that the diastereotopic face preference predicted for the exciplex becomes opposite to that for the ground-state C-T complex, favouring the re,si-complex in a 53 : 47 ratio (DE = À0.07 kcal mol À1 ).
Such structural differences between the ground-and excitedstate complexes have already been recognized in other D-A systems. For example, the X-ray crystallographic study of the [hexamethylbenzeneÁ1,2,4,5-tetracycnobenzene] complex revealed that the centres of the two benzene rings are not completely overlapped but slid by B0.3 Å, while the theoretical calculations for the exciplex of the same D-A pair predict good overlap of the p-MOs. [43][44][45] Diastereoselective Paternó-Büchi reaction of 1,1-diphenylethene with chiral p-cyanobenzoates Paternó-Büchi reaction 46,47 proceeds through the [2+2] photocycloaddition of excited carbonyl compound to ground-state alkene. Typically, the excited ketone triplet adds to an alkene to give an oxetane as the final product through a triplet 1,4-biradical intermediate. It has been reported that the C-T band excitation of the [STÁchloranil] complex affords a mixture of stereoisomeric oxetanes in a molar ratio identical to that obtained upon localband excitation of chloranil. 48 This suggests that differentiating ECT from EX becomes more arduous in the triplet manifold, due to a better chance to relax or equilibrate during the generally much longer lifetime of the triplet state. The [2+2] photocycloaddition of p-cyanobenzoate to 1,1-diphenylethene (DE) leads to the formation of an oxetane, which is however known to proceed rather exceptionally through a singlet exciplex. 49 This allowed us to expand the scope of the wavelength-control concept to the diastereoselective Paternó-Büchi reaction of chiral alkyl p-cyanobenzoates (Scheme 2). Indeed, the chiral version of the Paternó-Büchi reaction turned out to be valuable in differentiating ECT from EX. Unlike the ST-F system, the Paternó-Büchi reaction was clean, yielding a pair of diastereomeric oxetanes as the sole product, absolute configuration of which was unambiguously determined by X-ray crystal structural analysis. 50,51 Table 2 compares the de's obtained upon local-band versus C-T excitation in the photocycloaddition of DE to (S)-1-methylpropyl or (S)-1,3-dimethylbutyl p-cyanobenzoate ((S)-CB or (S)-CB 0 ) in various solvents at different temperatures. In nonpolar methylcyclohexane, the local-band excitation of benzoate gave the oxetane in good de's of 34-77% for (S)-CB and 68-74% for (S)-CB 0 , while the C-T excitation led to much lower 3-26% de for (S)-CB and 18-44% de for (S)-CB 0 . By increasing the solvent polarity, the diastereoselectivity was significantly decreased and the   chiral sense of the product was inverted in polar solvent(s). Nevertheless, the local-band and C-T excitations consistently afforded different de values, exhibiting distinct temperature-dependence behaviours. These results unambiguously corroborate the discrete nature of ECT and EX, also for this D-A pair. Remarkably, the absolute de value was almost always higher for local-band rather than C-T excitation, a result in sharp contrast to the ST-F system, where the C-T excitation gave better de's. The structures of the diastereomeric C-T complexes of DE with (S)-CB were simulated by the theoretical calculations at the DFT-D3-B-LYP/TZVP level. 38 For the sake of simplicity, only the most stable T g + conformation of the chiral group is shown for the diastereomeric C-T complexes in Fig. 3, although an ensemble of all possible conformers was considered in the calculation. 50,51 The calculated results revealed that the ground-state C-T complex is stabilized mostly by the p-p interaction between the benzene rings of DE and (S)-CB. Thus, the rather modest diastereoselectivity observed upon C-T excitation seems reasonable, since the chiral auxiliary is not only located at a remote position from the centre of interaction but also avoids effective contacts with the DE's another phenyl group. The preference for the (1 0 S,2R)isomer upon C-T excitation at lower temperatures, where the entropic contribution is reduced, was nicely reproduced by the diastereomer ratio predicted by the theoretical calculation.
The degrees of C-T calculated for the ground-state si-and re-[DEÁ(S)-CB] complexes (Fig. 3, left) were again as small as 0.3 and 1.0%, respectively. The structures of the diastereomeric si-and re-[DEÁ(S)-CB]* exciplexes (Fig. 3, right) were optimized at the TD-DFT-BH-LYP/TZVP level. At a glance, the exciplex structures, especially the re-exciplex, look only marginally different from those of the corresponding C-T complexes, but careful examinations of the non-bonded interaction distances revealed significant differences, providing some insights into the nature of the C-T complex and the exciplex. Thus, the facing CQC and CQO bonds were separated by 5.5 and 3.6 Å in the si-and re-C-T complexes, respectively, but by almost equal to 3.8 Å in the si-and re-exciplex. Quite interestingly, the facing benzene rings were nevertheless kept parallel at E3.7 Å in both C-T complexes. The first excited singlet state of (S)-CB calculated at the RI-CC2/TZVPP level revealed that the main contributor (MO #52 -#55), possessing significant n-p* character, amounts to nearly 65% (Fig. 4). Thus, the carbonyl group would actively contribute to the excited-state behaviour. This implies that the exciplex formation is more sensitive to the stereochemistry of the ester group and the chiral information is more effectively transferred in EX than in ECT, if both do not equilibrate in the excited state.

Temperature dependence of diastereoselectivity and activation parameters for ECT and EX
It is now clear that simply changing excitation wavelength can critically manipulate the net photochemical outcome of D-A pairs, especially the (dia)stereoselectivity of chiral photoreaction. The distinctly different photobehaviours observed upon local-band and C-T excitations indicate that ECT differs in structure and reactivity from the conventional EX. Then the subsequent question may follow: how and to what extent do the two species differ? The formation of EX is a dynamic process that involves structural relaxation of an encounter complex with accompanying solvent reorganization, while ECT is thought to retain some structural information of the groundstate C-T complex due to the p-p stacking interaction between D and A, and hence the solvent reorganization is anticipated to be less extensive. It is likely therefore that EX, rather than ECT, is more susceptible to such environmental variants as temperature and solvent. In order to gain further insights into the nature of ECT and EX, we examined the temperaturedependence behaviour of the product's de values obtained in the above-mentioned diastereoselective photocycloaddition reactions, which were then subjected to the Eyring analysis to determine the differential activation parameters. 52,53 Thus, the logarithm of the diastereomer ratio was plotted against the reciprocal temperature to afford good straight lines of different slopes and intercepts (Fig. 5), indicating that a single but discrete diastereodifferentiation mechanism is operative in each system, but the activation parameters depend on the system and the excitation mode. This again confirms that the difference between ECT and EX is not an incidental phenomenon, but a common feature specific to the excited state behaviour of D-A system. Note that the plot was somewhat scattered for the C-T excitation of the (E)-ST-(R)-F pair, due to the high de's of Z90%. The Eyring treatment (eqn (3)) of the relative rate constant for competitive diastereomeric processes leads to the following   (4)). From the slope and intercept of the regression line, the differential enthalpy (DDH ‡ ) and entropy (DDS ‡ ) of activation can be calculated. 54 Accordingly, the differential activation parameters for each D-A system were evaluated for the local-band and C-T excitation (Table 3).
Careful examinations of these activation parameters revealed the contrasting nature of the two excited species, EX and ECT, as briefly discussed below. First of all, there seems no general rule to globally explain all the obtained activation parameters, which critically depend on the combination of D and A as well as the mode of excitation. Furthermore, the diastereoselectivity is highly temperature-dependent in both excitation modes. This means that a simple comparison of the de values obtained from ECT and EX at a given temperature is not valid but rather misleading. Hence, the nature of the two distinct excited species can be elucidated only by using the differential activation parameters.
All of the C-T systems examined share a common feature that the absolute activation entropy |DDS ‡ | is consistently and significantly larger for local-band rather than C-T excitation. This indicates that the diastereotopic face selectivity upon photocycloaddition is more critically controlled by the entropic factors in EX formed via local-band excitation. This seems reasonable because EX is a species formed dynamically in the excited state with significant desolvation and/or solvent reorganization, while the C-T complex which is originally solvated in the ground state suffers only modest solvent reorganization after excitation. It is also reasonable that the absolute activation parameters become smaller to reduce the product's de in polar solvents, irrespective of the excitation mode. Thus, both ECT and EX are considered to be more polarized than the relevant ground-state species and therefore better to be solvated in polar solvents (but not to the same extent), as schematically illustrated in Fig. 6.
The enthalpic and entropic changes often compensate each other. Indeed, the DDH ‡ and DDS ‡ values obtained for the photocycloaddition of DE to (S)-CB in various solvents show the compensatory enthalpy-entropy relationship to afford distinct linear plots, passing through the origin, both for ECT and EX. 55,56 These observations reinforce our claim that a single diastereodifferentiating mechanism is operative in each excitation mode and ECT and EX differ in structure and reactivity. In particular, the higher isodiastereodifferentiating temperature for EX (177 K) than for ECT (275 K) could be Fig. 5 Eyring plots of the relative rate constant for the formation of the diastereomeric pairs in the photocycloaddition of (E)-ST to (R)-F (left), of (Z)-ST to (R)-F (middle), and of DE to (S)-CB (right) via EX generated upon local-band excitation and ECT generated upon C-T excitation in toluene. Table 3 Differential activation enthalpies (DDH ‡ ) and entropies (DDS ‡ ) determined for various C-T systems upon local-band and C-T excitation by applying the Eyring analyses to the de values a C-T system Solvent Local-band excitation C-T excitation À3.6 À13 Tetrahydrofuran +3.9 +23 À2.8 À11 Acetonitrile À0.2 À2.7 +1.0 +2.4 DE + (S)-CB 0 Methylcyclohexane +1.6 +21 À3.8 À9 a Calculated from the slope and intercept of the regression lines obtained by plotting the logarithm of the diastereomer ratio against the reciprocal temperature for each C-T system, as exemplified in Fig. 5. taken as experimental evidence in support of the greater flexibility of EX.

Asymmetric photoreaction of an intramolecular donor-acceptor system
Diastereoselective photocyclization of (S)-1,1-dicyano-2-methyl-4-phenyl-1-pentene In order to explore the general validity of the wavelength control and also to further elucidate the mechanistic details, we now discuss the photochemical behaviours of intramolecular C-T systems upon local-band versus C-T excitation. In the intermolecular C-T systems discussed above, the intervening ECT and EX species are complicated conformer mixtures of different spacing, orientation and solvation state, each of which contributes to the overall photochemical outcomes. This situation more or less hinders further mechanistic elucidations. In contrast, the conformational variation is much reduced in intramolecular C-T systems with a tethered D-A pair, which allows us to more closely analyse the conformational behaviours in the ground and excited states, with the aid of spectroscopic and theoretical tools. 4-Aryl-1,1-dicyano-1-butene derivatives are known to undergo competitive cyclization/rearrangement reactions upon irradiation. 57 This class of compounds shows a C-T band at longer wavelengths, indicating strong intramolecular C-T interactions in its gauche conformation. Accordingly, the excitation mode can be switched by shifting the irradiation wavelength. A chiral D-A compound, (S)-1,1-dicyano-2-methyl-4-phenyl-1-pentene ((S)-DP), was employed for elucidating the mechanistic details of the photocyclization to a diastereomeric pair of indan derivative upon local-band and C-T excitation (Scheme 3). 58,59 The effects of temperature and solvent on the cyclization/ rearrangement ratio and the de of the cyclization product were examined in the photoreaction of (S)-DP, and the results are summarized in Table 4. Upon local-band excitation (254 nm), the formation of the (1R,3S)-product was favoured in dichloromethane but the opposing isomer, i.e., the (1S,3S)-product, designated by a negative sign for de in Table 4, was favoured in polar acetonitrile. The former isomer was always favoured upon C-T excitation over the entire temperature range examined, irrespective of the solvent polarity. At a higher concentration (20 mM), the wavelength effect became less extensive, due to the participation of intermolecular (self) quenching. The temperature and solvent effects are mostly ascribed to the shift of the ground-state conformer equilibrium, but the excitedstate conformer equilibrium becomes competitive at higher temperatures. Whatever the reason, it is more crucial that the stereochemical outcome was dramatically manipulated by the mode of excitation also in this intramolecular C-T system. This implies that the C-T excitation leads to an excited-state conformer ensemble which is different from the one formed upon localband excitation. Such a difference in the excited-state conformer ensemble is likely to be a source of the distinct nature of ECT and EX in the intermolecular C-T system.

Wavelength effects upon C-T band excitation at different wavelengths
Another intriguing observation in the above system is that the diastereoselectivity was susceptive to the excitation wavelength even within the C-T band. As can be seen from Table 4, the de value became much higher when irradiated at the edge of the C-T band at 300 nm, rather than the middle of the C-T band at 280 nm. 58,59 The effects of excitation wavelength on the dynamics of C-T complexes have recently been investigated extensively by ultrafast time-resolved spectroscopy. 60 The study disclosed that the wavelength effect can be categorized into three distinct types. The difference in photophysical behavior upon low-and high-energy C-T band excitation was ascribed to the different Scheme 3 Diastereodifferentiating photocyclization and photorearrangement of (S)-1,1-dicyano-2-methyl-4-phenyl-1-pentene ((S)-DP). Table 4 Diastereoselective photocyclization and rearrangement of (S)-1,1dicyano-2-methyl-4-phenyl-1-pentene ((S)-DP) upon local-band and C-T excitation a View Article Online degree of borrowing local excitation in the overlapped C-T band and also to the different complex geometry. 61 For instance, a C-T complex of anthracene dimer with tetracyanoethene shows two well-resolved C-T bands, individual excitations of which lead to clean cycloreversion to anthracene at significantly different quantum efficiencies. This wavelength-dependent cycloreversion upon C-T excitation at different wavelengths was explained by the unequal cycloreversion rates for the two distinct excited states of the C-T complex, where the excitation at the high-energy C-T band leads to the formation of an excited-state anthracene cation in the geminate ion pair, accelerating the cycloreversion. 62 In addition, the relative contribution of the hot and relaxed (or thermally equilibrated) states also varies with irradiation wavelength. In the present system, such further wavelength effect was noticed upon excitation at the red edge of the C-T band. Thus, the different product ratios and de values (Table 4) obtained upon C-T excitations at 280 and 300 nm may be ascribed to the different spectral profiles of the two diastereomeric conformers in the ground-state, leading to the unequal excitation efficiencies. Accordingly, even a small change in excitation wavelength within the C-T band allows us to manipulate the relative contribution of the excited-state ensemble components and hence the stereochemical outcomes. Similar observation has been reported recently in the wavelength-controlled supramolecular photocyclodimerization of 2-anthracenecarboxylic acid mediated by native and modified g-cyclodextrins. 63 6. Spectroscopic detection of the excited C-T complex As mentioned above, the spectroscopic discrimination of ECT from EX has been controversial or at least inconclusive. Nevertheless, the obvious dissimilarities in photochemical behavior observed upon local-band versus C-T excitation of several D-A systems (vide supra) prompted us to further explore spectroscopic investigations. We reexamined the fluorescence behaviour of the representative inter-and intramolecular C-T complexes; i.e., the [(E)-STÁ(R)-F] complex and 1,1-dicyano-2-methyl-4-p-anisyl-1-butene (unfortunately, the other substrates, (Z)-ST, (S)-CB and (S)-DE, are not appreciably fluorescent).
Fluorescence spectra of a mixture of (E)-ST and (R)-F were measured at different excitation wavelengths (Fig. 7, left). Upon excitation at 350 nm, two fluorescence peaks were observed at ca. 400 and 420 nm, tentatively assignable to ECT and EX, respectively. The ECT fluorescence is slightly higher in energy than the EX fluorescence in consistent with the energy diagram ( Fig. 1) depicted in consideration of the nature of the two excited species. Fluorescence lifetimes (t) of these species (observed at the wavelength specific to each species) were in the same range, indicating that the two excited species resemble each other or the lifetime is relatively insensitive to the small conformational difference. The observed t value of EX was 0.8-1.2 ns in methylcyclohexane and 1.3-1.4 ns in toluene, while that of ECT was only slightly longer or indistinguishable 1.1-1.3 ns in methylcyclohexane and 1.3-1.5 ns in toluene.
Two excited species were also detected in the lifetime measurement of 1,1-dicyano-2-methyl-4-p-anisyl-1-butene, an achiral analog of DP. Thus, the short-lived component (t = 2.8-3.9 ns measured at 370 nm) appeared slightly higher in energy than the long-lived component (t = 7.6-12 ns measured at 400 nm) in an ethanol matrix (Fig. 7, right). All of these observations are consistent with the photochemical results, both jointly revealing that ECT differs from EX in photophysical and photochemical behaviours.

Summary and outlook
By selectively exciting the C-T band of a ground-state complex, unconventional excited species ECT, which distinctly differs from EX, is formed. EX is usually formed upon encounter of an excited D or A with a ground-state A or D and hence dynamic in nature, while ECT more or less retains the original structure of the C-T complex pre-organized in the ground state mostly through orbital or p-p interactions. The two excited-state complex species could eventually equilibrate with each other or relax to a common geometry, but nevertheless have been proven to significantly differ in structure and reactivity as described above. Such differences may be better explained by considering an ensemble of excited-state conformers with different reactivities. Consequently, the distinction of ECT from EX has been a difficult controversial issue in the literature. By closely examining the diastereodifferentiating photoreactions that are expected to differ in activation parameter, we have sorted out the elusive ECT from the conventional EX for the representative inter-and intramolecular C-T systems. Thus, the diastereoselectivity was critically manipulated by alternating excitation wavelength to reveal the distinctly different nature of ECT versus EX, which were more specifically characterized by analyzing the activation parameters determined by the temperature-dependence studies. The Eyring plots obtained over a limited temperature range always gave good straight lines, indicating operation of a single diastereodifferentiation mechanism. Crucially, the obtained slopes and intercepts significantly differ for the photoreactions via ECT and EX, indicating the discrete reactivity specific to each Red and black lines denote the excitation spectra monitored at the ECT fluorescence (indicated by arrows) and the absorption spectra, respectively. species. The differential entropy of activation was consistently larger for EX than for ECT, reflecting the flexible nature of the former species, for which larger solvent reorganization in the excited state is necessary because no stabilization is expected in the ground state through the C-T interaction that is available to the latter. In this connection, it is important to examine the photochemical behaviour of ECT and EX at different temperatures, since the stereoselectivities obtained upon local-band and C-T excitation could incidentally become close at a given temperature. The theoretical calculations provided a wealth of insights into the structures and properties of C-T complexes, ECT and EX. The temperature-dependence behaviour may be more precisely understood or rationalized by theory if the entropic factors are appropriately treated in the calculation. Incorporating the solvent effect may also be encouraged in calculation, as solvation plays a vital role in bimolecular photochemical reactions, although such advanced theoretical issues remain to be elucidated in a future study.
In addition to the conventional variants such as temperature and solvent polarity, we can exploit the excitation wavelength as an extra tool for critically controlling the photochemical consequences, in particular the stereoselectivity in chiral (supramolecular) photochemistry. Such an unconventional, yet practical, means should be widely examined in other donor-acceptor systems not only for better controlling the stereochemical outcomes but also for expanding the regime of photochemistry.


text:
Polychlorinated dibenzo-p-dioxins and dibenzofurans formed from sucralose at high temperatures

Sucralose is a widely-used artificial high-intensity sweetener. Although doubts have been raised about the safety of sucralose by several researchers, it can still be found in a broad range of foods and beverages worldwide, including in baked goods. Sucralose may decompose at high temperatures, and participate in chlorination reactions, generating highly toxic compounds. Here, we demonstrate that heating sucralose at high temperatures in stainless steel or other metal utensils in the presence of rust (Al2O3, Fe2O3, and CuO) produces polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs). PCDD/Fs were found in smoke generated during the heating of sucralose and in the residues after heating. CuO enhanced the PCDD/F yield in comparison with Al2O3 and Fe2O3.

S ucralose (4,19,69-trichloro-4,19,69-trideoxygalactosucrose, TGS) is an artificial sweetener which results from the selective substitution of three of the sucrose hydroxyl groups with chlorine atoms, involving inverting the configuration at the 4-position from the gluco-to the galacto-analogue, resulting in a product 600 times sweeter than sugar 1,2 . Sucralose is used as a food additive in more than 80 countries, and is present in over 4,000 products, including baked goods [3][4][5][6] . However, several studies have shown that sucralose has metabolic effects that may negatively impact human health [7][8][9][10][11][12][13] . In addition, it has been found that sucralose is not stable at high temperatures 14,15 .
Using thermoanalytical techniques, Bannach et al. (2009) found that sucralose is thermally stable at temperatures up to 119uC, but above this temperature, and up to 550uC, thermal decomposition takes place in three steps without the sucralose melting 14 . Rahn & Yaylayan (2010) reported that the thermal degradation of sucralose at 250uC resulted in the generation of toxic chloropropanols 15 . Our previous studies showed that highly toxic polychlorinated aromatic compounds, such as polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/ Fs), dioxin-like polychlorinated biphenyls, and polychlorinated naphthalenes, could be generated during the heating of foodstuffs in the presence of sucralose [16][17][18] . It still remains unclear whether these highly toxic compounds could be produced when pure sucralose is heated, and the formation mechanisms for these compounds have not yet been elucidated.
The effects of temperature, utensil materials, and rusts (metal oxides) on the formation of PCDD/Fs when sucralose is heated were investigated in the study presented here. The concentrations of tetra-through octachlorinated PCDD/Fs and 17 toxic 2,3,7,8-substituted PCDD/F congeners, collected from the smoke generated by heated sucralose and from the residues after heating, were measured.

Results
Large amounts of sucralose (5 g) were used in the experiments, and the PCDD/Fs produced were quantified. PCDD/Fs were not detected in the smoke or residues when sucralose was heated in quartz, aluminium, or copper utensils (the metal utensils were polished and rust was removed before use) at temperatures between 200 and 400uC. However, PCDD/Fs were detected in both the smoke and the residues when sucralose was heated at 350uC in stainless steel utensils, and it should be noted that stainless steel is commonly used for cooking utensils.
The PCDD/F concentrations in the smoke and residues were calculated on the basis of the original weight of sucralose (5 g) used in the experiments. Figure 1 shows that the PCDD/F concentrations increased in both the smoke and the residues with increasing temperature. The PCDD/F concentrations were higher in the smoke than in the residues. The total PCDD/F concentrations produced from sucralose (5 g) heated at 400uC reached 3.1 3 10 4 pg g 21 , which was approximately 25 times higher than the concentrations produced at 350uC. The 17 toxic The surface of metal cooking utensils can become oxidized, so rust, which is composed of metal oxides, is commonly found on utensil surfaces. The effects of different rusts (Al 2 O 3 , Fe 2 O 3 , and CuO), chosen based on metals typically used for cooking utensils, on PCDD/F formation during the heating of sucralose at 400uC were investigated. Figure 2 shows the PCDD/F concentrations produced in the smoke and residues when sucralose (5 g) was heated in the presence of Al 2 O 3 , Fe 2 O 3 , and CuO (1 g) at 400uC. CuO enhanced the yield of PCDD/Fs in comparison with Al 2 O 3 and Fe 2 O 3 . The total PCDD/F concentrations reached 4.2 3 10 6 pg g 21 when sucralose was heated in the presence of CuO. Fe 2 O 3 also had a strong effect on PCDD/F formation (giving PCDD/F concentrations of up to 1.2 3 10 6 pg g 21 ) in comparison with Al 2 O 3 (which gave PCDD/F concentrations of up to 9.7 3 10 4 pg g 21 ). PCDD/F concentrations produced when sucralose was heated in the presence of Al 2 O 3 , Fe 2 O 3 , and CuO were significantly higher than the concentrations produced when sucralose was heated in stainless steel utensils (which gave PCDD/F concentrations of up to 3.1 3 10 4 pg g 21 ). No significant amounts of PCDD/Fs were detected when sucralose was heated in the presence of the metal oxides at 300uC. Sucralose (5 g) was heated in the presence of small amounts of CuO (1 g, 0.1 g, and 0.01 g) at 400uC to study the effects of the amount of metal oxide present on PCDD/F formation. The PCDD/F concentrations in the smoke and residues decreased as the amount of CuO present decreased. Smaller amounts of sucralose were also tested, and PCDD/F concentrations of approximately 3.0 3 10 6 pg g 21 were produced from 1 g of sucralose and 0.1 g of CuO at 400uC.

Discussion
The PCDD/F concentrations were higher in the smoke than in the residues (Fig. 1). The PCDD/F distribution agreed with that described in previous reports, the PCDD/Fs produced being mainly in the gas phase 17,18 . The PCDD/F homologue and congener profiles in the smoke and residues were similar (Fig. 3), but they were different from those observed in previous studies 17 . This indicates that the PCDD/F formation pathways when sucralose is heated could be altered if foodstuffs (e.g., beef and vegetable oil) are added. Foodstuffs could provide a carbon source for the PCDD/F backbone formation. Sucralose was found to mainly act as a chlorine source in previous studies, but it acted as both a chlorine source and a carbon source in   the study presented here. Furthermore, the addition of foodstuffs could cause PCDD/Fs to be produced at lower temperatures 18 .
CuO was more effective than the other metal oxides as a catalyst for PCDD/F formation 19 . Sucralose thermally decomposes at 119uC, liberating HCl 14 . Hisham and Benson (1995) investigated several metal oxides and found that they could absorb HCl and release Cl 2 (the Deacon reaction) 20 , and they found that CuO was the most efficient of the metal oxides that were studied in releasing Cl 2 at approximately 375uC. This reaction would allow Cl 2 to be able to participate in the chlorination of aromatic structures, which could then be followed by the formation of dual ring structures in a second metal-catalysed reaction 21,22 . Figure 2 also shows that higher concentrations of PCDD/Fs were found in the residues than in the smoke when CuO was present. However, the PCDD/F distributions produced in the presence of Al 2 O 3 and Fe 2 O 3 were different, the PCDD/F concentrations being higher in the smoke than in the residues. We concluded, therefore, that the metal-catalysed PCDD/F formation reaction occurred on the metal oxide surfaces 19 . The PCDD/F homologue and congener profiles produced from sucralose heated in the presence of metal oxides were similar to the PCDD/F profiles formed from sucralose heated in stainless steel utensils (Fig. 4). This suggests that the metal oxides acted as catalysts, promoting PCDD/F formation, but did not alter the PCDD/F formation pathways.
The PCDFs were found at higher concentrations than the PCDDs in all cases (Figs. 3 and 4), suggesting that PCDD/Fs might be produced when a carbon matrix is broken down, in a series of oxidation and chlorination reactions, called de novo pathways 23 . The de novo synthesis of PCDD/Fs mostly occurred between 200 and 400uC, giving a PCDF/PCDD ratio of more than one 24 . It has also been reported that the oxidation of chlorobenzenes (CBzs) produces more PCDFs than PCDDs 25 . Polycyclic aromatic hydrocarbons (PAHs) and CBzs are considered to be precursors for PCDD/F formation 19,26 . Although no PAHs or CBzs were detected in the sucralose before it was heated, both PAHs and CBzs were found, along with PCDD/Fs, in the smoke. CBzs were also detected in the residues. This indicates that compounds containing benzene rings can be formed, chlorinated, and oxidized when sucralose is heated in the presence of metal oxides.
Our data indicate that sucralose may produce significant amounts of PCDD/Fs when heated in stainless steel utensils or in metal utensils in the presence of rust (Al 2 O 3 , Fe 2 O 3 , or CuO) at high temperatures. Occupational exposure to PCDD/Fs has attracted concern 27 . The improper use of sucralose at high temperatures may result in human exposure to PCDD/Fs.
The experimental system and heating procedures used have been described previously by Dong et al. (2011Dong et al. ( , 2013 16,18 . The tetra-to octa-chlorinated PCDD/F homologue groups and 17 individual 2,3,7,8-substituted PCDD/F congeners in the smoke and the residues were analysed following the U.S. Environmental Protection Agency method 1613. The sucralose residues after the heat tests were treated with 1 mol L 21 HCl and left to equilibrate overnight, washed with deionized water until the washings had a neutral pH, then freeze-dried. The sample extraction, clean-up, and instrumental analysis for the PCDD/Fs were conducted following the methods published by Wu et al. (2011) 17 . The ions monitored in the gas chromatograph-high resolution mass spectrometer analysis of PCDD/Fs are given in the Supplementary  Table S1.
The smoke samples were analysed for PAHs following the Californian Environmental Protection Agency method 429. Known amounts of d-PAHs were added to the sample extracts, cleaned using gel permeation chromatography and then using a multilayer column filled with silica gel and neutral alumina. The extracts were then analysed for PAHs using an Agilent 6890/5973 N gas chromatograph-mass spectrometer (Agilent Technologies Inc., Santa Clara, CA, USA) equipped with an HP-5MS capillary column (30 m long, 0.32 mm i.d., 0.25 mm film thickness; Agilent Technologies Inc.) and operated in selected ion monitoring (SIM) mode. The ions monitored in the gas chromatograph-mass spectrometer analysis of PAHs are given in the Supplementary Table S2.
The samples were extracted and cleaned-up for the analysis of the tetra-, penta-, and hexa-chlorinated CBzs following a procedure for ''dioxin-like'' polychlorinated biphenyl analysis that has been published previously 9 , and known amounts of 13 Clabelled CBzs were added to the samples before being extracted. The CBzs were analysed using an Agilent 6890/5973 N instrument equipped with an HP-5MS capillary column (30 m long, 0.32 mm i.d., 0.25 mm film thickness) and operated in selected ion monitoring mode, monitoring the two most abundant ions in the molecular ion clusters. The CBz isomers were quantified using the 13 C-labelled CBzs as isotope-dilution internal standards. The ions monitored in the gas chromatographmass spectrometer analysis of CBzs are given in the Supplementary Table S3.
All of the experiments were run at least in duplicate. The relative percentage differences between the target compound concentrations in the duplicate experiments were less than 37%. The recoveries of the 13 C-labelled PCDD/F internal standards were 60-110%, the d-PAH recoveries were 68-92%, and the 13 C-labelled CBz recoveries were 60-85%. The limits of detection (LODs; defined as the concentration giving a signal-to-noise ratio of 3) for the PCDD/Fs, PAHs, and CBzs were 0.01-0.4 pg g 21 , 4-30 pg g 21 , and 4-15 pg g 21 , respectively. The concentrations of all of the analytes in the blanks and the unheated sucralose were near or below the LODs, so no blank correction was performed.


s2fieldsofstudy: Chemistry Physics Biology

text:
A phosphorylation map of the photosystem II supercomplex C2S2M2

PS II PHOSPHORYLATION It was 36 years ago when John Bennett first discovered chloroplast protein phosphorylation (Bennett, 1977). The most conspicuous chloroplast phosphoprotein was the apoprotein of the Light Harvesting Complex II (LHC II) of photosystem II (PS II). Other phosphoproteins included the reaction center protein CP43 and the quinone-binding proteins D1 and D2 of PS II. These earlier studies also identified the PsbH protein, the minor subunit of PS II, as another chloroplast phosphoprotein (Allen, 1992). Nearly all of the first-known chloroplast phosphoproteins belong to PS II, the water-oxidizing photosystem of oxygenic photosynthesis. With more than 25 protein subunits and 350 kDa in size, the PS II core monomer is a massive structure (Umena et al., 2011). This enormous size is in addition to the peripheral light harvesting complexes. The major peripheral antenna of PS II is LHC II, which is a trimer. Some, so-called minor, LHC are monomeric and act as linkers in connecting the trimeric LHC II to the core. The core is composed of the reaction center proteins D1 and D2 and an internal antenna made up of CP43 and CP47. The PS II core with its peripheral antenna forms supercomplexes, which mainly differ in the number of LHC II bound to the core (Caffarri et al., 2009). The core itself is dimeric (C2) to which the LHC II could strongly (S), or moderately (M), or loosely (L) bind. The monomeric linker associated with the Sform is CP26, and for the M-form are CP29 and CP24. A dimeric core (C2) with 2 S and 2 M LHC II (C2S2M2) is the most abundant supercomplex in plant photosynthetic membranes (Kouril et al., 2012). PS II phosphorylation is light dependent. It turned out, however, light acts through the redox state of the plastoquinone (PQ) pool (Allen et al., 1981; Bennett, 1991). PS II phosphorylation can be divided into the core phosphorylation (D1, D2, CP43, and PsbH) and the peripheral antenna phosphorylation (LHC II). In plants, the protein kinases that phosphorylate the core and the LHC II have been identified as Stn8 and Stn7, respectively, (Bellafiore et al., 2005; Bonardi et al., 2005). PS II phosphorylation is only observed in green algae and plants, and not in cyanobacteria and red algae where the light harvesting antenna is phycobilisomebased (Pursiheimo et al., 1998). Core phosphorylation is responsive to light intensity with increasing levels of phosphorylation observed in high light (Elich et al., 1992; Tikkanen and Aro, 2012). On the contrary, LHC II phosphorylation was restricted to low light condition specific to PS II (Rintamaki et al., 2000). At higher light intensities, the activity of the Stn7 is inhibited by the reduced stromal electron carrier thioredoxin (Rintamaki et al., 2000; Puthiyaveetil, 2011). It was also noted that only the L-form of the LHC II trimer is phosphorylated, while the LHC II isoforms comprising the Sand M-trimers are not phosphorylated or do not contain phosphorylation sites (Galka et al., 2012). LHC II phosphorylation is the basis of state transitions, an acclimatory response to changes in light quality (Bonaventura and Myers, 1969; Murata, 1969). The precise function of PS II core phosphorylation, however, is still unclear. PS II undergoes damage in light, which results in photoinhibition—light-induced loss of photosynthetic activity (Aro et al., 1993; Long et al., 1994). In plants and green algae, PS II is mainly found in the stacked granal regions of the thylakoid membrane where it forms almost immobile supercomplexes (Kirchhoff et al., 2008; Mullineaux, 2008). The centrally located reaction center protein D1 is the main target of photodamage in PS II (Kyle et al., 1984). To maintain photosynthetic efficiency in high light, chloroplasts have evolved a robust repair process known as the PS II repair cycle, wherein the damaged D1 protein is degraded and replaced with a newly synthesized copy (Melis, 1999; Nixon et al., 2010). The PS II repair cycle operates through many constraints (Kirchhoff, 2013b), chief among them are how the repair machinery located in the unstacked regions of the thylakoid membrane accesses the damaged photosystems in the stacked, crowded granal membranes and how the damaged photosystems are mobilized in the rather “immobile” grana. The proposed functions of PS II phosphorylation include “marking” the damaged photosystems for degradation (Aro et al., 1993), increasing the mobility of photosystems (Goral et al., 2010; Herbstova et al., 2012), disassembly of PS II (Tikkanen et al., 2008; Fristedt and Vener, 2011), decreasing the granal diameter (Herbstova et al., 2012; Kirchhoff, 2013a), and for maintaining electron flow in high light (Harrison and Allen, 1991). Some of these proposals are not mutually exclusive. Functions such as increased mobility and disassembly are especially supported but how precisely phosphorylation produces these effects are unclear.

PS II PHOSPHORYLATION
It was 36 years ago when John Bennett first discovered chloroplast protein phosphorylation (Bennett, 1977). The most conspicuous chloroplast phosphoprotein was the apoprotein of the Light Harvesting Complex II (LHC II) of photosystem II (PS II). Other phosphoproteins included the reaction center protein CP43 and the quinone-binding proteins D1 and D2 of PS II. These earlier studies also identified the PsbH protein, the minor subunit of PS II, as another chloroplast phosphoprotein (Allen, 1992). Nearly all of the first-known chloroplast phosphoproteins belong to PS II, the water-oxidizing photosystem of oxygenic photosynthesis.
With more than 25 protein subunits and 350 kDa in size, the PS II core monomer is a massive structure (Umena et al., 2011). This enormous size is in addition to the peripheral light harvesting complexes. The major peripheral antenna of PS II is LHC II, which is a trimer. Some, so-called minor, LHC are monomeric and act as linkers in connecting the trimeric LHC II to the core. The core is composed of the reaction center proteins D1 and D2 and an internal antenna made up of CP43 and CP47. The PS II core with its peripheral antenna forms supercomplexes, which mainly differ in the number of LHC II bound to the core (Caffarri et al., 2009). The core itself is dimeric (C2) to which the LHC II could strongly (S), or moderately (M), or loosely (L) bind. The monomeric linker associated with the Sform is CP26, and for the M-form are CP29 and CP24. A dimeric core (C2) with 2 S and 2 M LHC II (C2S2M2) is the most abundant supercomplex in plant photosynthetic membranes (Kouril et al., 2012).
PS II phosphorylation is light dependent. It turned out, however, light acts through the redox state of the plastoquinone (PQ) pool (Allen et al., 1981;Bennett, 1991). PS II phosphorylation can be divided into the core phosphorylation (D1, D2, CP43, and PsbH) and the peripheral antenna phosphorylation (LHC II). In plants, the protein kinases that phosphorylate the core and the LHC II have been identified as Stn8 and Stn7, respectively, (Bellafiore et al., 2005;Bonardi et al., 2005). PS II phosphorylation is only observed in green algae and plants, and not in cyanobacteria and red algae where the light harvesting antenna is phycobilisomebased (Pursiheimo et al., 1998). Core phosphorylation is responsive to light intensity with increasing levels of phosphorylation observed in high light (Elich et al., 1992;Tikkanen and Aro, 2012). On the contrary, LHC II phosphorylation was restricted to low light condition specific to PS II (Rintamaki et al., 2000). At higher light intensities, the activity of the Stn7 is inhibited by the reduced stromal electron carrier thioredoxin (Rintamaki et al., 2000;Puthiyaveetil, 2011). It was also noted that only the L-form of the LHC II trimer is phosphorylated, while the LHC II isoforms comprising the S-and M-trimers are not phosphorylated or do not contain phosphorylation sites (Galka et al., 2012). LHC II phosphorylation is the basis of state transitions, an acclimatory response to changes in light quality (Bonaventura and Myers, 1969;Murata, 1969). The precise function of PS II core phosphorylation, however, is still unclear. PS II undergoes damage in light, which results in photoinhibition-light-induced loss of photosynthetic activity (Aro et al., 1993;Long et al., 1994).
In plants and green algae, PS II is mainly found in the stacked granal regions of the thylakoid membrane where it forms almost immobile supercomplexes (Kirchhoff et al., 2008;Mullineaux, 2008). The centrally located reaction center protein D1 is the main target of photodamage in PS II (Kyle et al., 1984). To maintain photosynthetic efficiency in high light, chloroplasts have evolved a robust repair process known as the PS II repair cycle, wherein the damaged D1 protein is degraded and replaced with a newly synthesized copy (Melis, 1999;Nixon et al., 2010). The PS II repair cycle operates through many constraints (Kirchhoff, 2013b), chief among them are how the repair machinery located in the unstacked regions of the thylakoid membrane accesses the damaged photosystems in the stacked, crowded granal membranes and how the damaged photosystems are mobilized in the rather "immobile" grana. The proposed functions of PS II phosphorylation include "marking" the damaged photosystems for degradation (Aro et al., 1993), increasing the mobility of photosystems (Goral et al., 2010;Herbstova et al., 2012), disassembly of PS II (Tikkanen et al., 2008;Fristedt and Vener, 2011), decreasing the granal diameter (Herbstova et al., 2012;Kirchhoff, 2013a), and for maintaining electron flow in high light (Harrison and Allen, 1991). Some of these proposals are not mutually exclusive. Functions such as increased mobility and disassembly are especially supported but how precisely phosphorylation produces these effects are unclear.

MAPPING PHOSPHORYLATION SITES ONTO THE PS II SUPERCOMPLEX C2S2M2
Significant advances in massspectrometry-based phosphoproteomics approaches have enabled the mapping of phosphorylation sites in PS II proteins (Turkina et al., 2006;Lemeille and Rochaix, 2010;Reiland et al., 2011). To gain insight into the function of PS II core phosphorylation, we projected known phosphorylation sites on the PS II supercomplex C2S2M2. The available PS II atomic coordinates, however, are for cyanobacteria (Umena et al., 2011) and not for plants where PS II phosphorylation actually occurs. Furthermore, the mapped phosphorylation sites are for Arabidopsis and Chlamydomonas proteins. For the monomeric antenna CP29, however, structure from a plant source is available (Pan et al., 2011). LHC II also has a structure solved from spinach (Liu et al., 2004). In our approach, we therefore, aligned the amino acid sequence of the Arabidopsis PS II core phosphoproteins with that of cyanobacteria and then projected the positions of phosphorylation sites on the cyanobacterial sequence and structure. For PS II core proteins, phosphorylation sites are located usually on their stromaly-positioned N-termini. Even though homology exists between Ntermini of cyanobacterial and Arabidopsis proteins and the information on phosphorylation site positions can safely be transferred between them, the structure of their N-termini has not been resolved to their entire length for some cyanobacterial proteins (Umena et al., 2011). Under these circumstances, the nearest amino acid on the N-terminus, for which structural information exists, is taken as the approximate position of the phosphorylation site.
For the Arabidopsis D1 and D2 proteins, threonine 2 is the phosphorylation site and for Arabidopsis CP43, threonine 15 (Reiland et al., 2011). All three sites are conserved in Chlamydomonas and cyanobacterial proteins. For PsbH, multiple phosphorylation sites have been noted as threonines 3 and 5 (Reiland et al., 2011), both of which are conserved in Chlamydomonas but not in cyanobacteria. The monomeric antenna CP29 has at least five high light-dependent phosphorylation sites, only one of which, threonine 37 in Lhcb4.2 isoform, is catalyzed by the Stn8 kinase (Fristedt and Vener, 2011), while others are catalyzed by Stn7.
Atomic coordinates for Thermosynechococcus vulcanus PS II core proteins D1, D2, CP43, CP47, and PsbH are rendered in a space-filling model using the Chimera program (Pettersen et al., 2004) (Figure 1). The monomeric and trimeric antenna coordinates are rendered by the ribbon diagram, and modeled around the core from positions determined by the single particle analysis (Caffarri et al., 2009). The phosphorylation sites of PS II proteins are then projected onto the supercomplex structure to reveal the distribution of phosphosites in relation to PS II proteins (Figure 1). FIGURE 1 | Phosphorylation map of the C2S2M2 supercomplex as catalyzed by the Stn8 kinase. C2S2M2 rendered by the Chimera visualization program where the view is from the stromal side looking down. Coordinates for PS II core were taken from the pdb structure 3ARC (Umena et al., 2011), LHC II from 1RWT (Liu et al., 2004), and CP29 from 3PL9 (Pan et al., 2011). Since no structures were available for CP26 and CP24, CP29 coordinates were used instead. Supercomplex structure was modeled as in Caffarri et al. (2009). Major subunits of the core and the complete peripheral antenna are shown. Individual subunits of the core have been differently colored and labeled. The peripheral antenna is shown in silver and labeled. Approximate positions of phosphorylation sites of D1, D2, CP43, PsbH, and CP29 are shown. For CP29, up to five high light-dependent phosphorylation sites are known (Fristedt and Vener, 2011), but only the Stn8 phosphosite is shown for consistency. Phosphosites have also been reported for the other two monomeric antennas CP24 and CP26, but the identity of their kinases and the light condition in which they become phosphorylated are uncertain (Reiland et al., 2011), hence not included in our analysis.

THE FUNCTION OF PS II CORE PHOSPHORYLATION
Our projections reveal strategic location of phosphosites around the periphery of the core PS II, where the monomeric antennas CP29, CP24, and CP26 attach and at the monomer-monomer interface (Figure 1). From this distribution, we suggest that the function of PS II core phosphorylation is antenna dissociation and monomerization of the dimeric core. Phosphorylationmediated antenna dissociation is likely to be the mechanism by which the S-and M-trimers are removed from the core. It remains to be determined how, in molecular terms, phosphorylation causes antenna dissociation and core monomerization.

PS II PHOSPHORYLATION IN PS II REPAIR CYCLE
The primary purpose of antenna dissociation will be to relieve the excitation pressure on PS II reaction centers and to prevent further damage to D1 in high light. The detached antenna, however, unless in a quenched state, can be a source of destructive reactive oxygen species. An antenna supercomplex dubbed as "band 4," comprising LHC II, CP29, and CP24 has been suggested as the site of protective non-photochemical quenching (NPQ) in high light (Betterle et al., 2009). Interestingly, this supercomplex is precisely what our model of phosphorylation-mediated antenna dissociation would generate during disassembly (Figure 1). However, the apparent correlation between phosphorylation and the band 4-mediated NPQ is yet to be investigated.
As discussed earlier, our model predicts that PS II core phosphorylation drives monomerization of the dimeric cores. Monomerization has also been suggested to result from the damage or rearrangement of the extrinsic PsbO subunit of PS II (Nixon et al., 2010). If this is indeed the case, phosphorylation might act alone or in conjunction with PsbO in causing monomerization. As a consequence of antenna dissociation and core monomerization, larger supercomplexes will be broken up into smaller PS II particles. The observation that most of the PS II supercomplexes and PS II core dimers remain intact in high light in the absence of phosphorylation is consistent with our proposal (Tikkanen et al., 2008;Nath et al., 2013).
During the PS II repair cycle, the following scenario can be envisioned. PS II is phosphorylated, whether they are damaged or not. This will break up supercomplexes into monomeric cores. Monomeric cores in the grana, with their smaller particle size, will be able to diffuse faster toward the stromaly-exposed regions of the thylakoid membrane where the repair machinery has access to the damaged photosystems. A swift migration of the monomeric cores may also result from the smaller diffusion area of the grana as they shrink in diameter in high light (Herbstova et al., 2012;Kirchhoff, 2013a). Following dephosphorylation and the partial disassembly of the PS II monomer into the so-called "RC47 complex," proteases recognize damaged PS II by conformational changes brought about by the damage and degrade them. The newly synthesized and processed D1 is then inserted, activated, and assembled by the repair machinery. The resultant PS II core monomer will diffuse back to the grana where they could dimerize and be fitted with their new antenna. Similarly, the phosphorylated but undamaged PS II core monomer, following dephosphorylation, could simply slip back to the grana, dimerize, and reassemble with their antenna.

ANTENNA DISSOCIATION IN STATE TRANSITIONS AND PS II REPAIR CYCLE: A COMMON REGULATORY THEME
State transitions correct imbalance in excitation of individual photosystems by redistributing excitation energy to the rate-limiting photosystem (Bonaventura and Myers, 1969;Murata, 1969;Allen, 2003). When PS II is preferentially illuminated, LHC II gets phosphorylated and the phospho-LHC II dissociates from PS II. It then associates with photosystem I (PS I) as its light harvesting antenna. When dephosphorylated, LHC II re-joins PS II from PS I. Phosphorylation of LHC II is thought to change its molecular recognition of PS II (Allen and Forsberg, 2001). The similarity of phosphorylation-mediated antenna dissociation in state transitions and PS II repair is thus unmistakable and supports antenna dissociation as a unifying function of PS II phosphorylation.
This functional similarity of LHC II and PS II core phosphorylation is even deeper. The protein kinases, Stn7 and Stn8, which underpin state transitions and PS II repair, are paralogs, sharing 43% identity at the amino acid sequence level. They both require reduced PQ for activation (Allen et al., 1981;Bennett, 1991). Studies of the kinase mutants reveal significant substrate overlap between the kinases (Depege et al., 2003;Bellafiore et al., 2005;Bonardi et al., 2005;Puthiyaveetil et al., 2012), suggesting their ancient origin from gene duplication and subsequent divergence. Even though the Stn7 kinase is inhibited in high light, its role in PS II disassembly through CP29 phosphorylation in the early stages of high light acclimation cannot be ruled out (Fristedt and Vener, 2011). Phosphorylation-mediated detachment of CP29 from PS II is also a feature of green algal state transitions (Kargul et al., 2005;Takahashi et al., 2006). Significant functional and amino acid sequence similarity exists also for the LHC II and PS II core phosphatases (Pribil et al., 2010;Shapiguzov et al., 2010;Samol et al., 2012). Minimizing PS II excitation pressure through antenna dissociation thus appears to be the defining feature of phosphorylation in state transitions and in PS II repair cycle.

AUTHOR CONTRIBUTIONS
Sujith Puthiyaveetil and Helmut Kirchhoff wrote the paper.


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text:
catena-Poly[di-μ3-bromido-bis[(1-ethyl-1H-imidazole-κN 3)disilver(I)]]

The asymmetric unit of the title coordination complex, [Ag2Br2(C5H8N2)2]n, comprises a monodentate 1-ethylimidazole ligand, an Ag+ cation and a μ3-bridging Br− anion, giving a distorted tetrahedral AgNBr3 stereochemistry about the Ag+ cation [Ag—N = 2.247 (2) Å and Ag—Br = 2.7372 (4)–2.7523 (4) Å]. Two bridging bromide anions generate the dimeric [Ag2Br2(C5H8N)2] repeat unit [Ag⋯Ag = 3.0028 (5) Å], while a third Br− anion links the units through corner sharing in an inversion-related Ag2Br2 association [Ag⋯Ag = 3.0407 (4) Å], generating a one-dimensional ribbon step-polymer structure, extending along the c axis.

Related literature
For general background to N-heterocyclic carbenes, see: Arnold (2002); Lin & Vasam (2004). For related structures, see: Wang & Lin (1998); Liu et al. (2003); Helgesson & Jagner (1990, 1991; Chen & Liu (2003 Data collection: APEX2 (Bruker, 2006); cell refinement: SAINT (Bruker, 2006); data reduction: SAINT; program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: SHELXTL (Sheldrick, 2008); software used to prepare material for publication: SHELXTL.  Arnold (2002) and Lin & Vasam (2004). The products differ depending upon reaction conditions and the imidazolium salt used. Deprotonation by use of Ag 2 O has been the most widely used method in the syntheses of Nheterocyclic carbene complexes of silver. The procedure can be accomplished using the reaction of Ag 2 O with the imidazolium salt in CH 2 Cl 2 solution. The 3-diethylbenzole N-heterocyclic carbene complexes of silver have been successfully synthesized by the reaction of the 1,3-diethylbenzolium salt with Ag 2 O in CH 2 Cl 2 (Wang & Lin, 1998). In an attempt to prepare similar N-heterocyclic carbene complexes of silver by the reaction of Ag 2 O with 1,2-dibromocyclohexane and 1-ethylimidazole in DMSO solution, we obtained the title compound, [(C 5 H 8 N) 2 Ag 2 Br 2 ] n , instead and the synthesis and crystal structure are reported herein. Although the stair polymers of [(C 5 H 5 N) 4 Ag 4 I 4 ] n  and 1-allyl-3-methylimidazole carbine silver iodide (Chen & Liu, 2003) have recently been reported, their structural features are different from that of the title complex being formed through triple and quadruple halide bridges with Ag···Ag interactions.
Experimental 1,2-Dibromocyclohexane (2.42 g, 10 mmol) was added to a solution of 1-ethylimidazole (1.92 g, 20 mmol) in DMSO (100 ml) at room temperature and stirred for 2 h, after which Ag 2 O (2.32 g, 10 mmol) was added and the mixture was refluxed for 3 h with stirring. The volume of the solution was reduced to 50 ml under vacuum, the residue was removed by filtration and the filtrate was kept at room temperature for a few days. Colorless crystals of the title compound were

Refinement
The H atoms attached to C atoms of the imidazole ring were positioned geometrically and allowed to ride on their parent atoms, with C-H = 0.95 Å and U iso (H) = 1.2U eq (C). Methylene and methyl H atoms were likewise positioned geometrically (C-H = 0.99 and 0.98 Å, respectively) and also refined as riding atoms, and U iso (H) = 1.2U eq (C)

Figure 2
The step-polymeric structure of the title complex, extending along the c axial direction.

catena-Poly[di-µ 3 -bromido-bis[(1-ethyl-1H-imidazole-κN 3 )disilver(I)]
Crystal data where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.38 e Å −3 Δρ min = −0.68 e Å −3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F 2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F 2 , conventional R-factors R are based on F, with F set to zero for negative F 2 . The threshold expression of F 2 > σ(F 2 ) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F 2 are statistically about twice as large as those based on F, and R-factors based on ALL data will be even larger.