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Conformational aspects in the studies of organic compounds by electronic circular dichroism Gennaro Pescitelli,a Lorenzo Di Bari*a and Nina Berova*b Received 8th February 2011 DOI: 10.1039/c1cs15036g The electronic circular dichroism (ECD) spectra of flexible molecules include the contributions of all conformers populated at the working temperature. ECD spectra of chiral substrates depend on their stereochemistry in terms of both absolute configuration, as reflected in the sign of the spectrum, and molecular conformation, which dictates the overall spectral shape (possibly including the sign) in a very sensitive manner. The unique high sensitivity of ECD towards conformation, as well as of other chiroptical spectroscopies, renders these techniques a useful alternative or complement to standard spectroscopic tools for conformational investigations, such as NMR. This tutorial review provides first a brief discussion of the main principles of ECD spectroscopy and related methods for interpretation of spectra, with special reference to conformational aspects. The review focuses on the common problems encountered in the application of ECD for assignments of absolute configuration of flexible molecules. These problems can be handled either by taking into account the whole conformational ensemble or by considering rigid derivatives prepared ad hoc. Finally, the review presents the relatively less common but very interesting application of ECD spectroscopy for conformational analyses of organic compounds. a b

Dipartimento di Chimica e Chimica Industriale, Universita` di Pisa, via Risorgimento 35, I-56126 Pisa, Italy. E-mail: [email protected] Department of Chemistry, Columbia University, 3000 Broadway, 10027 New York, USA. E-mail: [email protected]

Gennaro Pescitelli received his BSc and PhD (2001) degrees in Chemistry from University of Pisa under the supervision of Professor Piero Salvadori, and spent a postdoctoral fellowship at Columbia University, New York, in the group of Professor Koji Nakanishi and Nina Berova. He was appointed lecturer at the University of Pisa in 2006 working in collaboration with Prof. Di Bari. He is co-author of about 80 publications Gennaro Pescitelli including reviews and book chapters. His research is focused on spectroscopic and computational investigations of chiral organic molecules, especially natural products, catalysts and electro-active polymers.

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The Royal Society of Chemistry 2011

Lorenzo Di Bari received BSc and PhD from the Scuola Normale Superiore in Pisa, developing new tools for the analysis of conformational distributions of organic molecules in solution. After two experiences abroad (as graduate student in Lausanne, CH, with G. Bodenhusen, and as postodoc in Stockholme, SE, with J. Kowalewski), he returned to Pisa University, where he started a long collaboration with P. Salvadori on the appliLorenzo Di Bari cation of CD to stereochemistry of flexible molecules. His current interests extend from detailed studies of static and dynamic stereochemistry of small molecules to structural issues of large (bio)macromolecules, where he applies a combination of experimental techniques, mainly Circular Dichroism and NMR, including a special expertise on lanthanide compounds and adducts. He collaborated to the organization of three International Conferences on Circular Dichroism and related techniques (as co-chair of the 2009 edition), as well as of several other international meetings. Chem. Soc. Rev., 2011, 40, 4603–4625

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1. Introduction For a long time the high sensitivity of Electronic Circular Dichroism, ECD, to conformational factors has been explored and acknowledged in the field of biopolymers. It is well known that ECD spectra of proteins and nucleic acids are a rich source of structural information especially for determining secondary structures and following its evolution under the effect of various conditions.1 On the contrary, in the context of organic or organometallic compounds, ECD may appear restricted to the assignment of absolute configuration (AC) and there are relatively few applications of ECD spectroscopy for conformational investigations. Notable exceptions are described in the book by Lightner and Gurst, devoted to conformational analysis with a special focus on the carbonyl chromophore,2 and only a handful of reviews, among them those by Sandstro¨m.3 More recent reviews on the link between ECD and conformational analysis, especially with the aid of theoretical simulations, are missing, a gap which we hope to fill in the following. One of the reasons that encouraged us to write this review is the belief that the potentiality of ECD for conformational analysis of organic compounds still remains underestimated and largely underexploited by the scientific community. The same is true for other chiroptical spectroscopies such as vibrational CD (VCD) and Raman optical activity (ROA). Although the present tutorial review deals exclusively with ECD, it must be stressed that the three main chiroptical spectroscopies, namely ECD, VCD and ROA, may offer specific advantages in the investigation of different stereochemical aspects,4 and can usefully complement each other whenever needed.5,6 It is well-known that electronic circular dichroism, as any other chiroptical property, is opposite for a pair of enantiomers,7 which renders ECD an ideal candidate for their characterization and recognition. There are methods for correlating the sign of ECD data with the absolute arrangement of atoms and

Before joining the Columbia University as a research professor in 1988, Nina Berova carried out a teaching and research on stereochemistry at Univ. Sofia and Inst. Organic Chemistry, Bulgaria, from where she also received her PhD in chemistry. Her research is focused on organic stereochemistry and structural analysis, in particular, by chiroptical spectroscopy. She has been a recipient of many scholarships and visiting Nina Berova professorship from England, Germany, France, Italy, Spain and Japan, and of a few awards specifically for her studies on chirality. Since 1998present she is a co-Editor of Journal ‘‘Chirality. She was co-editor and co-author of two monographs ‘‘Circular Dichroism: Principles and Applications’’ (1994 & 2000), and of two-volume monograph ‘‘Comprehensive Chiroptical Spectroscopy’’ (in press 2011) by John Wiley & Sons. 4604

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groups in space, that is, with the absolute configuration, as reviewed elsewhere8 and very briefly summarized in the following; our present focus will rather be on conformational issues. Often, conformer interconversion implies relatively small energy barriers (compared to kT)w and sometimes modest differences between the energies of the local minima. This is usually in contrast to configurational isomerism, which involves (formal) breaking and reforming covalent bonds. Consequently, conformations may appear kinetically labile, and give rise to temperature-dependent manifolds of interconverting structures, while configurations often appear inert. It is important to note that in a molecule that contains stereogenic elements of defined configuration, such as stereogenic carbon atoms, any variation in conformational features leads to a structure which is not symmetry-related to the starting one. This means that for chiral molecules conformational isomers are in a diastereomeric relation. Only in cases of atropisomeric axial chirality,9 arising from a hindered rotation about a single bond, like in some biaryls, two conformers may appear as enantiomers. Among all spectroscopies, ECD and other chiroptical methods can be particularly sensitive to stereochemistry and able to distinguish diastereomers, and to recognize and quantify conformational manifolds. By shedding some light on the origin of such a marked sensitivity, we aim to provide some insights not only into the impact of conformational aspects on ECD spectra but also on the methods and procedures for using these spectra for accurate conformational analysis. We shall first recall that ECD can only be observed for chiral non-racemic compounds that contain one or more groups (chromophores) with electronic absorptions in the broad range of 170–1300 nm. Most commonly, the range used by UV-vis spectropolarimeters is that between 170–800 nm. For some special applications, the analysis can be extended to the Near IR (NIR) from 800 to about 1300 nm. Therefore the presence of chromophores, i.e. chemical moieties where the electronic transitions are mainly localized, is most essential for the observation of ECD. An electronic transition, which normally gives rise to an absorption band in the absorption spectrum, may be associated to a positive or a negative ECD band, which is called a Cotton effect (sometimes abbreviated CE).z A molecule that does not contain chromophores can often be transformed into a good candidate for ECD, by suitable chemical modifications. Such a modified substrate is called a Cottonogenic derivative, because it gives rise to nonvanishing Cotton effects. ECD results from the interference between electric and magnetic transition dipole moments li and mi, each CE being the result of non-negligible rotational strength Ri plimi. Note, that both the magnitude of each of these transition moments and their orientation contribute to determine Ri. Therefore, ECD is largely a question of magnitude and angles between transition moments and, at least in w When this is not the case, one deals with atropisomers, i.e. with kinetically inert conformational isomers that can be isolated. z Although the terminology ‘‘Cotton effect’’ historically refers to the anomalous optical rotatory dispersion (ORD) effect, it is currently (and here) used to refer to the accompanying ECD band (see Glossary in ref. 9).

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multichromophoric systems, between chromophores. This should clarify that ECD is not only opposite for enantiomers, but more importantly it is different for diastereomers. ECD offers at least three very relevant positive aspects: it is very sensitive and can be used with minute amounts of material, down to the mg scale; it allows for changing the cell path-length b by at least 3 orders of magnitude (from 0.01 cm to 10 cm), which means spanning a very large concentration c range (the product bc must be controlled to reach an absorbance around 0.8–1); it may be regarded as a fast technique, because the timescale of an electronic (or vibrational) transition is much shorter than a ps (1012 s), a reasonable lower boundary for a conformational rearrangement. In the context of stereochemical analysis, timescale is of a particular interest, because very often the activation energies for conformational exchange are small (compared to kT) and consequently the lifetimes of individual conformers are very short: to gain an idea, following the Eyring equationy an activation energy of 3 kcal mol1 corresponds at 300 K to a lifetime of ca. 20 ps, and 10 kcal mol1 correspond to ca. 2 ms. For conventional NMR even this upper limit very often falls in the fast exchange regime. In such cases, the observed average spectrum contains one set of resolved lines lacking any information about the presence of different conformers. In ECD the situation will be just opposite since the species with a lifetime of a ps would give its own contribution to the observed spectrum, which must thus be regarded as a sum or superposition of terms arising from individual conformers (weighted by their mole fractions). 1.1.

Case (2) deserves special mention because it concerns socalled exciton-coupled systems (see Section 2.2 below) where all spectral features can be related to the orientation between chromophores, and ultimately to the molecular conformation. When only one CE can be recognized (case 1), its usefulness for conformational analysis is in most cases limited to variable temperature (VT) studies. 1.2.

Merits and limits of ECD for conformational analysis

The dependence of ECD on chromophores constitutes at the same time its major limitation and its advantage with respect to other chiroptical techniques, like VCD and ROA (or optical rotation, OR). These latter techniques, with no particular requirement for the presence of UV-vis chromophores, have broader application than ECD. However, generally speaking, VCD or ROA spectra are global molecular properties. For this reason it can be more difficult to extract specific information about a selected molecular portion from vibrational spectra than from ECD. In fact, ECD primarily reflects the electronic structure and chiral environment of the chromophores, as well as their orientation and interaction, in strong dependence on configuration and conformation, and with insensitivity to molecular portions far from the chromophores. As it is said,

Sensitivity of ECD towards conformation

Before we discuss in detail the available methodologies for conformational analysis through ECD, let us consider a few different situations that can lead to the appearance of ECD spectra. We can distinguish three main cases: (1) only one transition is apparent in the ECD spectrum as for example in near-UV spectrum of saturated ketones; (2) two transitions provide distinct and oppositely signed Cotton effects, in case they are involved in exciton coupling; (3) ECD may contain multiple bands when the chromophore(s) have a manifold of electronic transitions, as e.g. is the case of some transition metals and lanthanide complexes. Often, in case (3) one may treat the ECD spectrum as a fingerprint of molecular stereochemistry, in the sense that signs and relative magnitudes of a sequence of CE’s change from one conformer to the other. A structured ECD profile can be of fundamental relevance when the stereochemical assignment is based on the (visual) comparison between spectra, a situation encountered either in the case of a purely empirical correlation, or by comparison between experimental and computer-generated data. In both cases, featureless and monotonous spectra are poorly suited for the analysis of conformational manifolds. y Interconversion rate (in s1) k E 2 1010T exp(DGz/2T), with activation energy DGz in cal mol1; life-time (in s) t= (ln 2)/k (from ref. 9).

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Fig. 1 Top: stable conformations of bilirubin (1) and its derivatives such as 2. Bottom: ECD spectra of 2 in various solvents. The four bonds indicated by the arrows are double in 1 and single in 2 (adapted from ref. 10, with permission; copyright 1997 American Chemical Society).

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ECD is ‘‘used to look at the stereochemistry of the molecule through the eyes of the chromophore’’.7 Thus, one may get selective stereochemical information on specific molecular sites, where chromophores are already present or can be introduced. This local sensitivity and also the interplay between configuration and conformation can be exemplified by two cases. The first example is provided by bilirubin (1, Fig. 1), the vertebrate bile pigment which exists in solution as a ridge-tile structure held by intramolecular hydrogen bonds. Two enantiomeric conformations (M and P, Fig. 1) are possible for bilirubin which interconvert rapidly at room temperature by rotation around the two C-9/C-10 and C-10/C-11 torsions. Chiral analogs of bilirubin, such as (bR,b 0 R)-dimethylmesobilirubin-XIIIa (2), show intense ECD spectra due to the exciton coupling (see Section 2.2) between the two dipyrrole units. In this case, the two M and P conformations are diastereomeric, and one or the other may prevail depending on the conditions. As a consequence, the shape of the ECD spectrum of 2 may change completely and even invert its sign upon changing the solvent because of different ratios between the two conformers (Fig. 1).10 Individual conformers of truly flexible molecules are hardly encountered experimentally, unless by suitable low-temperature measurements or upon conversion into conformationallylocked derivatives. On the contrary, when dealing with

theoretical models, i.e. with molecules in silico, they are the norm rather than the exception, because calculations are necessarily run on one asset of atoms, that is, on single conformers. This leads to the second example: the natural compound dioncophylline (3, Fig. 2).11 It contains three stereodefined elements: two stereogenic centers and one axis, of (P,R,R)-configuration. One can recognize two more or less independent chromophores, namely, the substituted naphthalene and phenol rings, connected through a single bond, whose conformation can be described by the dihedral angle yABCD. The ECD spectrum of this compound is rather insensitive to the configuration of the two stereogenic centers or to the conformation of the saturated ring. In fact, it is largely dominated by the stereochemistry of the aryl junction, and possibly very sensitive to even minor changes in yABCD. AM1 calculations predict a very shallow energy potential minimum for yABCD = 60–1201. Therefore, the observed ECD spectrum at room temperature is a Boltzmann average over this wide ensemble. It is particularly relevant that for two local minima (I and II, Fig. 2) with yABCD = 1061 and 781 (actually this second minimum is an optimization artifact), practically mirror-image ECD spectra have been calculated. These two examples clearly demonstrate the dramatic dependence of ECD on certain conformational features and call for a particular caution when ECD is employed for configurational assignments of chiral substrates which may exist as a mixture of conformers with significantly different characteristics.

2. Survey of methods for ECD spectra interpretation We shall discuss in the following a few commonly used methods for interpreting ECD data in their particular connection to conformational analysis. Deeper and more exhaustive descriptions can be found elsewhere.2,8,12–16 2.1.

Fig. 2 CNDO/S-calculated ECD spectra for two energy minima I and II of dioncophylline A (3) found by AM1 geometry optimizations (adapted from ref. 11, with permission; copyright 2009 Elsevier).

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Octant rule

This is a well-known semi-empirical approach for predicting the sign of the Cotton effect of the n - p* transition of saturated ketones around 300 nm,2 which is isolated from the rest of the spectrum and can be regarded as belonging to case (1) mentioned in Section 1.1. This rule is based on the simple fact that a carbonyl has two local symmetry planes (it belongs to the point-group C2v) and any atom or group occupying the space outside these planes breaks the symmetry and possibly leads to a chiral structure. This is well represented by an equatorial group in position b to the carbonyl in cyclohexanone derivatives. If we arbitrarily choose clockwise sense in atom numbering, 3 and 5 positions are enantiotopic and a substituent in one or the other leads to a molecule of opposite chirality. By watching (R)-3-methylcyclohexanone along the O–C direction, with the carbonyl plane horizontal, as represented in Fig. 3, we may say that the symmetrybreaking methyl group Me is in the upper left sector, or octant. Every other atom in the molecule either lays on the C2v planes, or, if it is in the space between them, it is exactly ‘‘compensated’’ by a symmetry-related equal one. Only Me faces a hydrogen atom, which introduces an unbalance. This journal is

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Fig. 3 Conformational inversion equilibrium (ring commutation) of (R)-3-methyl cyclohexanone and projections defining rear octants considered in the ketone octant rule.

This means that the magnitude of the Cotton effect is strongly dependent on temperature and that |Deobs (T)| increases on lowering the temperature, which is exactly what was found experimentally.2 Thus, for 3-methylcyclohexanone the sign of the ECD band is a reporter of the absolute configuration, while variable temperature trends provide conformational information. Use of vibrational spectroscopy would be preferred in such circumstances where different conformers give rise to distinct VCD or ROA bands identifiable in the average spectrum so that relative populations may be more easily quantified.17,18 2.2.

It is observed that, for the methyl group occupying the upper left octant, or the equivalent lower right one, De (300 nm) > 0. This also leads to the conclusion that had the methyl group occupied the enantiotopic lower left or the upper right sectors one would obtain a negative Cotton effect. This molecule lends itself to introducing one of the first cases when ECD was used for conformational analysis.2 (R)-3-Methylcyclohexanone has two main conformers: by ring commutation between the two pseudo-chair conformations, Me can occupy an equatorial or an axial position (Fig. 3). In the latter case, by inspecting molecular models, one finds that it occupies the upper right octant. As we have seen before, we can ignore the rest of the backbone, which is symmetric, and we can fix our attention on the Me group only: its exact locations in the two isomers with respect to CQO are not really symmetry-related, but one can assume that they are almost enantiotopic. We can expect that for the axial isomer De (300 nm) o 0. We are in the case where two different conformers lead to opposite ECD spectra (relative to the sole n - p* transition). We made this prediction on the basis of a symmetry argument and by assuming that the interactions by which we observe a Cotton effect of the n - p* transition occur through space and e.g. not through bonds. One can estimate the enthalpy difference between the two conformers with Me in axial or equatorial position to about 1.74 kcal mol1.9 At room temperature (300 K), this corresponds to an equilibrium constant Keq$ax = 0.053 and ultimately to a composition of about xax = 5% and xeq = 95%. The observed ECD around 300 nm can be written as a sum of contributions due to the methyl in equatorial (Deeq) and in axial (Deax) positions, the former being positive and the latter negative, according to the octant rule. Thus we can estimate

Exciton coupling

The ECD exciton coupling results from through space interaction between chromophores which are not conjugated, are located nearby in space and constitute a chiral array. Such ECD response provides a unique opportunity for determination of AC according to the basic concept known currently as the ‘‘exciton chirality method’’.12 Fig. 4 illustrates the basic principle of the method by the coupling between two identical steroidal 2,3-bis-p-substituted benzoate chromophores. When two individual chromophores with intense p–p* absorptions and identical or similar excitation energies are also in close spatial proximity to one another, they cannot be excited independently. Each chromophoric excited state will delocalize over all chromophores within the system and becomes an exciton. The excitons interact and couple with each other, thus giving rise to a characteristic pair of intense ECD bands with opposite signs and comparable band areas at shifted wavelengths, called an ECD exciton couplet, located in correspondence with chromophore transition wavelength l0. In the example shown in Fig. 4, the intense intramolecular charge transfer (CT) band originates from the 1La transition of para-substituted benzoates; the through-space coupling between these CT bands leads to exciton-split ECD and may serve for the determination of AC of the two stereogenic centers. In the past few decades the method has been established as one of the most sensitive and convenient spectroscopic tools

Deobs E xaxDeax + xeqDeeq and because of the opposite sign of Deax and Deeq, |Deobs|o|Deeq| that is, in absolute value the observed Cotton effect is smaller than it should be for the pure equatorial conformer. On lowering the temperature, we expect that the mole fraction of the less enthalpically stable isomer decreases in favor of the more stable one; ultimately xax - 0; xeq - 1. This journal is

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Fig. 4 Exciton coupling of two identical p-substituted benzoate chromophores forming a positive chiral twist between their La transitions; the carbinol hydrogens are represented syn to the carbonyls and the ester group is in preferred trans conformation. ACD is the exciton couplet amplitude in M1 cm1, defined as a difference between the peak and trough of the split ECD curve.

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for AC analysis of chiral synthetic compounds or natural products of great structural diversity. It is usually employed in solution, although there are recent reports on its successful application in solid state as well.15 The exciton coupled ECD approach is rather unique in the context of the interpretation of chiroptical properties, because it follows the coupled oscillator theory and, at least in simpler cases, the very characteristic bisignate ECD curve can be rigorously rationalized in terms of AC, by direct visual inspection of molecular models, whereupon the sign of an ECD couplet is a faithful reporter of the sense of chiral twist between two interacting transition moments. Because the basic principles of exciton chirality method have been a subject of many recent reviews8,19,20 we will specifically focus here on the conformational aspects which affect the scope and limitations of this method and are particularly critical for the straightforward application to flexible substrates. If the substrate is rigid and the chromophores orientation is known with certainty, the conclusion based on the exciton coupled ECD is comparable to X-ray analysis. Even though the two different methods provide information for solution or solid state conformation, respectively, they naturally reveal the same absolute configuration. Fig. 4 contains the partial structure of 5a-cholestane-2b,3bdiol bis-p-substituted benzoate and illustrates a rigid substrate where there is no conformational uncertainty about the steroidal scaffold. There is some free rotation around the C–O bonds, but the ester bonds are known to adopt usually a s-trans conformation. Furthermore, X-ray crystallographic data, molecular modeling, as well as other data have shown that the ester CQO is syn with respect to the carbinol or methine hydrogens. Thus, the intramolecular CT transition indicated by 1La and polarized along the long axis of the benzoate chromophore is almost parallel to the alcoholic C–O bond. Hence, the positive exciton ECD couplet, which

represents the absolute chirality of the 1La transition moments, also represents the chirality between two hydroxyl groups and allows for the unambiguous assignment of AC at C-2 and C-3. Many other examples of conformationally defined cyclic derivatives can be found in refs. 12 and 20. It is worth mentioning that the syn-orientation of CQO/methine hydrogen found in esters (Fig. 4) similarly holds for secondary amides, where trans OQC–N–H is the most favored conformation. Tertiary N-methyl amides, however, may also adopt a cis OQC–N–CH3 form which minimizes steric repulsion. Depending on the substituent size the latter conformation may predominate in solution, which leads to an opposite ECD exciton couplet. For this reason it is highly recommended that the ECD analysis of tertiary amides is supported by additional spectroscopic data and theoretical conformational analysis.21 Conformational ambiguity that can render application of exciton chirality method less straightforward than for steroidal diols may be of two kinds. The first one occurs when the cyclic system (or in general the skeleton to which the two chromophores are appended) adopts different conformations. Fig. 5 provides such an example. The 1,2-trans-cyclohexanediol diesters are usually the least complicated substrates when common chromophoric ester groups, such as p-substituted benzoates, are present. In the case of standard 1,2-dibenzoates, the two ester moieties adopt exclusively a diequatorial orientation and the 6-member ring is in a chair conformation (framed structure in Fig. 5). Interestingly, when the bulky tetraphenylporphyrin (TPP) chromophores are employed (as in 4) instead of benzoates, two TPP groups come in close 1,2-trans vicinal proximity to each other, and instead of a very intense exciton couplet within the region of porphyrin Soret band around 420 nm (due to two orthogonal quasi-degenerate p–p* transitions), a much weaker split ECD with amplitude ACD = 400 was observed (see definition of ACD in Fig. 4).22 For comparison

Fig. 5 The five most stable conformers of bisporphyrin derivative of (1R, 2R)-trans cyclohexane diol 4 with their calculated energy differences in kcal mol1 with molecular mechanics (MacroModel, force field not reported).22 Framed: most stable conformer for a ‘‘standard’’ dibenzoate of (1R, 2R)-trans cyclohexane diol.

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the 5a-cholestane-3b,6a-diol derivative with more remote porphyrins (with A/B ring in chair conformation and porphyrins in 3,6-diequatorial orientation) showed a positive exciton couplet amplitude ACD = +675. NMR data for compound 4 suggested the presence of a mixture of cyclohexane conformers in which the porphyrin protons are exposed to different ring current effects of the other porphyrin group. Molecular modeling found that the most stable conformer has diaxial bulky porphyrins and the less stable one has two equatorial porphyrins (Fig. 5). While the exciton theory correctly predict a negative couplet for the known (1R,2R)-diol derivative, this prediction was based on the assumption that two porphyrins adopt a 1,2-trans diequatorial orientation in a cyclohexane chair conformation. Obviously in this case the negative couplet appears fortuitous as result of slight distortion from co-linearity of 1,2-diaxially oriented porphyrins in the lowest energy chair conformation, or of the contribution of higher-energy conformers. It is clear that any attempt to assign the AC when the interacting chromophores are very bulky and in a vicinal position would be rather hazardous without a prior careful conformational analysis. The application of exciton chirality method for assignment of AC of sterically hindered and very remote C-10 and C-37 hydroxyl groups on the flexible B and J seven membered rings, part of 15 polyether ring system of gymnocin B (5), was found to be particularly challenging (Scheme 1).19 The NOE correlations revealed that the two hydroxyl groups are pseudoaxially (syn) oriented in 5a. The chances to reach a clear-cut exciton couplet upon derivatization by triphenylporphyrin(cinnamic acid) (TPPcinn) to afford derivative 5b (Scheme 1) were therefore very small if the porphyrin transition moments at a distance of over 30 A˚ from each other remained also parallel. However, the overall flexibility of this very large polyether structure poses additional challenges. It also opens the possibility that some steric interactions due to bulky porphyrin chromophores may alter the conformations of ring B and/or J, so that the 10-OTPPcin/ 37-OTPPcin adopt a chiral twist desirable for exciton coupling.

Scheme 1

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Surprisingly, the observed ECD spectrum of 5b did show a relatively weak but clear-cut positive couplet. Yet, the AC could not be assigned without an extensive conformational analysis. Monte Carlo/MMFF94s calculations on 5b and some of its truncated analogs revealed that in the most stable conformations the TPPcin at C-10 still adopts an axial orientation, while that at C-37 adopts an equatorial orientation, so that the chiral interchromophoric twist is positive, when the calculations are performed for the arbitrarily chosen (10S,37S) configuration. The outcome of this extensive conformational and experimental ECD analyses were fully confirmed by ECD calculations using the De Voe’s coupled oscillator method (see Section 2.3 below). The possibility of free rotation of the ECD chromophore at the point of attachment to the stereogenic center is the second aspect which may require an additional conformational and spectroscopic analysis. The following example illustrates such situation. The conversion of (S,S)-trans-1,2-diaminocyclohexane (6a) into neutral biscyanine dye derivative 6b (Fig. 6) leads to a strong negative ECD couplet between the p–p* transitions of the pentacyanine chromophores (405 nm, De 92, and 358 nm De +71). The bis-protonated cyanine dye derivative shows also a negative but more intense couplet with very large bathochromic shift (546 nm, De 232, and 475 nm, De +231).23 Note that the chiral twist of the N–C–C–N moiety in the starting diaminocyclohexane is positive. The rotation around the cyclohexyl C–N bond may lead to two different (syn and anti) conformations, one where the imino-C hydrogens Hb are syn to the axial hydrogens Ha at C-1 and C-2 of the cyclohexane ring, and one where they are anti. Only in this latter conformation the long axis of each cyanine chromophore is parallel to the C–N bond as in the starting diaminocyclohexane (Fig. 6). Molecular mechanics studies with MMP1 and MMP2 force fields (see ref. 23) have shown that the syn conformation is more stable, in agreement with observed ECD couplets for neutral and protonated forms. However, no data were reported whether the anti-conformer

Gymnocin B (5a) and its derivative 5b.

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Fig. 6 (1S, 2S)-trans diaminocyclohexane (6a) and the more stable syn-conformation (left) of negative helicity and anti-conformation with positive helicity of corresponding bis-Schiff base 6b prepared by reaction with 7-(piperidin-1-yl)hepta-2,4,6-trienal.

Fig. 7 Neutral bis-Schiff base of (1R, 2R)-1,2-trans-dimethylamino cyclohexane with p-(dimethylamino) cinnamalaldehyde (7a) and bisprotonated form after addition of TFA at time 0 (7b) and after 45 min (7c) in MeOH.

also contributed to the observed couplet. This example calls again for a need of prior careful examination if a conformational instability of the chromophores is suspected, in particular when the goal is determination of AC. The bis-Schiff base of (R,R)-trans-1,2-diaminocyclohexane with p-(dimethylamino) cinnamalaldehyde is another example about the effect of stereoelectronic factors on the mutual chromophoric disposition (Fig. 7). The neutral Schiff base (7a) shows an expected negative couplet (383 nm, De 70.2, and 344 nm, De +58.3). However, after protonation by TFA, a firstly observed more intense and bathochromically shifted negative couplet (508 nm, De –151, and 448 nm, De +144) (7b) decreases over the time, and after 45 min becomes positive (506 nm, De +54, and 449 nm, De –52) (7c). The NOESY spectrum revealed an E/Z-isomerization of CQN bond at one of the interacting Schiff base chromophores (Fig. 7), presumably resulting from an electrostatic repulsion between the two positively charged side chains and facilitated by the decrease of the double bond character of the CQN group upon protonation.20 The protonated Schiff bases have been 4610

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described to have many attractive spectroscopic properties and their application for AC analysis of chiral amines, aminoalcohols, aminosugars and other related natural products is highly recommended. Even so, this example warns about possible uncertainty, which may lead to an erroneous AC assignment, if left unchecked by other methods. 2.3.

DeVoe and related methods

In the previous section, we have seen how electric dipole transitions on two different chromophores can couple to give rise to non-vanishing ECD and how can one predict the sign of an exciton couplet in simple systems. When the number of chromophores and/or of transitions to be taken into account becomes larger or when one wants a more quantitative estimation of position and amplitude of the Cotton effects due to this mechanism, simple structural inspection is no longer sufficient, but one can resort to computational methods. Classical electromagnetic theory allows one to exactly evaluate the energy of any number of interacting dipoles and consequently This journal is

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the precise wavelength corrections; moreover, it allows one to evaluate the magnetic moment induced from one oscillating electric dipole to another one. This is what can be achieved by the so-called De Voe approach24,25 or by the matrix method. We have recently defined them as hybrid methods,13 in the sense that they rely on a good quantum-mechanical (QM) description of the isolated chromophores in terms of transition dipoles, but then treat their interactions by means of classical methods. Current computational power, available on any computer, makes De Voe calculations easily viable for practically any number of interacting dipoles. Moreover, one can repeat the calculation on a very large number of conformations. A computer routine due to Hugz uses De Voe equations and calculates Abs and ECD spectra resulting from dipolar interactions, by taking as input parameters: (a) Molecular geometry (Cartesian atomic coordinates); (b) Location and direction of electric dipole transition moments with respect to the above defined structure; (c) Spectroscopic data (lmax, oscillator strength, bandwidth). The main limitation or drawback consists in the dipole approximation, i.e. in saying that a transition charge distribution spans an area small compared to the distances between chromophores. With extended conjugation in large chromophores or when the two interacting moieties are nearby, this approximation may no longer be valid and the results unsatisfactory. 2.4.

QM calculations

‘‘Direct’’ or ‘‘full’’ ECD calculations with quantum-mechanical (QM) methods are nowadays very frequently employed, because the development of computer technology has made them a fully practicable option for increasingly large molecules. The theoretical quantity related to ECD is rotational strength Ri (referred to the electronic transition from the ground state to an electronic excited state i). To calculate rotational strengths, use of QM methods capable of describing excited states (in terms of wavefunctions, transition energies and moments) must be employed. To obtain reliable results, these methods must also take electron correlation into account and use large basis sets (mathematical functions whose combinations are used to describe atom-centered orbitals). The combination of the last two factors imply long computational times. Practical methods used for ECD calculations fall into the two main families identified as high-level ab initio and semiempirical methods, and will be briefly recalled here. Readers are referred to textbooks on computational methods26 and reviews on quantum-mechanical ECD calculations for a deeper understanding.13,14,16 Time-dependent density functional theory (TDDFT) has emerged in the last years as the high-level methodology leading perhaps to the best accuracy/cost compromise in ECD calculations.14,16 Especially reliable are the so-called ‘‘hybrid’’ DFT functionals, like the popular B3LYP, BH&HLYP and PBE0.27 Some DFT functionals have well-known drawbacks, including a poor description of loosely localized states, such as charge-transfer, diffuse and Rydberg states, which may be alleviated using ad hoc functionals such as CAM-B3LYP. z Available from the authors on request.

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Another issue related to TDDFT calculations is that, because of the perturbative nature of the approach, they are intrinsically more accurate in the prediction of low-lying excited states. More rigorous (and demanding) alternatives to TDDFT for small and relatively rigid molecules are DFT/MRCI (multi-reference configuration interaction) and coupled-cluster (CC) theory.14 Predicted chiroptical properties are especially sensitive to the basis set used. Split-valence basis sets of triple-z (like Ahlrichs’ TZVP set) or at least double-z quality, with additional polarization functions and (whenever possible) a minimal set of diffuse functions usually represent good choices. When only low-lying excited states need to be evaluated, use of smaller basis sets such as SVP or 6-31G(d) may be sufficient.27 One question connected to the choice of the basis set is that related to the so-called gauge formulation used to express rotational strengths, which may be of the dipole-length or the dipole-velocity type. For this very technical (though with very practical consequences) problem we refer the reader to the already quoted literature.13,14 Semi-empirical quantum-mechanical methods rely on strong simplifications, which disregard the core electrons and neglect the differential overlap (NDO) in order to skip the explicit calculation of one- and two-electron integrals.26 These methods rely on the choice of a set of atom-related parameters and are very computationally fast. In the context of ECD, the two schemes known as CNDO/S (complete NDO) and ZINDO/S (Zerner’s intermediate NDO) have found large application and demonstrated to be quite successful at least for some specific cases, for example for describing p–p* transitions of aromatic and heteroaromatic chromophores. Two important simplifications are commonly (but not necessarily) made in ECD spectra calculations, namely, neglecting environmental (solvent) and vibrational effects. Ideally, they should both be taken into account in the generation of input geometries (Section 3.1.1 below) and in the excited states calculation. In particular, ECD spectra of conformationally flexible molecules can significantly be affected by the solvent. It is worth mentioning that recently developed strategies allow for introducing vibronic coupling28 and solvent models in ECD calculations,29,30 although they require a considerably longer computational effort. The usual output of ECD calculations is a list of rotational strengths Ri at discrete transition frequencies ni, or a stick plot, which should be converted in a true spectrum.31 This means to associate each rotational strength with a certain band-shape function (with intensity proportional to the Ri value), and then to sum all bands. The band-shape widths si can be decided on an empirical basis as those providing the best fit with the experimental spectra; reasonable values are s = 1500–3000 cm1 (corresponding to 6–12 nm at 200 nm and 24–48 nm at 400 nm). Expressing the rotational strengths in 1040 cgs units, the ECD spectrum calculated as sum of Gaussians in the wavenumbers (v˜ in cm1) domain is:31 " # X Ri v~i v~ v~i 2 Deð~ vÞ ¼ 0:0247 exp ð1Þ si si i where si are the exponential half-widths at 1/e height, related to the half-height full widths by Dv˜1/2 = 1.66s. If it happens Chem. Soc. Rev., 2011, 40, 4603–4625

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that computed transition frequencies are systematically shifted with respect to experimental ones, one may apply a so-called wavelength correction by shifting the whole spectrum by an appropriate value.


2.5.

NIR-ECD

Most of what discussed above is practically limited to organic chromophores, where strong electric dipole transitions are often located and also provide the most suitable objects for the hybrid or QM methods outlined. Complexes including metal ions bring about metal-centered transitions, some feature of which deserve to be briefly treated here.32 We shall neglect charge transfer or delocalized transitions, because they involve a deep perturbation of both the metal and the ligand, and consequently are very difficult to treat and describe. Both d- and f-metals give rise to intraconfigurational electronic transitions, which are electric-dipole forbidden (because of symmetry) and produce very weak absorption spectra. On the contrary, they may be characterized by large magnetic transition dipole moments and, as a consequence, they give rise to very large dissymmetry factors (ECD divided by Abs). Owing to a rich manifold of orbitals and to comparably small energy splittings, metal complexes often absorb in a field between near UV and near IR (or NIR). This feature is particularly interesting, because such a red-shifted spectral range is very well separated from other contributions, while being adequately covered by conventional ECD instrumentation (possibly with a modest wavelength extension in the NIR). A major difference between d- and f-metals consists in the fact that usually the former give rise to very broad spectra, possibly covering thousands of cm1, while the latter have very narrow bands made of atom-like lines. Hence, usually d-metals give weak and poorly structured ECD spectra, because the rotational strength is spread over a large spectral field. The opposite is true for lanthanides, which give multiplets of resolved lines, allied to Cotton effects of different signs and amplitudes. As discussed in the Introduction, this is a very desirable feature, because it offers a fingerprint of molecular stereochemistry as exemplified in Section 3.3.4.

3. AC assignment of flexible molecules By far the most common application of ECD spectroscopy in the field of organic chemistry is the assignment of absolute configurations (AC). As stressed in the Introduction, configurational and conformational factors must always be considered together when dealing with ECD spectra. In the very large majority of cases, the conformation (or conformational distribution) must be known independently and before the interpretation of ECD spectra in terms of AC. Independently means that tools other than ECD—either spectroscopic or computational—must provide a conformational picture in the state of measurement of the ECD spectrum (solution, or the solid state). Before means that any interpretation of the ECD spectrum regarding the AC must be based on such a picture. In the presence of several conformers, the ECD spectrum is a superposition of the component spectra related to the various conformers. The conformational analysis therefore has to identify correctly the molecular geometries and the relative 4612

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energies of all conformers present in solution having a sizable population at the working temperature (WT). Whichever approach for predicting the ECD is chosen, one must evaluate the spectra for each populated conformer at the same level of theory. Thereafter, all calculated spectra must be averaged and only the result of this operation may be compared to the experiment. Frequently, the molecular structure is too complex and the number of populated conformers too large to make high-level computational approaches practical, because molecular complexity goes hand in hand with molecular flexibility. Often, many steps must be followed before applying ECD spectroscopy to assign AC, most of which are time-consuming and prone to errors. In the following we shall outline the standard procedure to deal with flexible compounds whose AC has to be assigned by means of ECD spectra analysis. We shall describe in a following section (Section 3.4) some means to bypass the problems posed by conformational flexibility. 3.1.

Methods for conformational analysis

The aim of a conformational analysis is to elucidate the number and structure of all conformers present in the state of interest (usually, solution) at the working temperature WT (usually, room temperature, which may be taken as 298 K or 300 K). Apart from rigid or semi-rigid compounds, conformational analysis by spectroscopic methods (mostly NMR) must be complemented by computational methods, such as molecular modeling, in order to obtain a true conformational distribution and not just an indication of the most populated (or the most significant) conformer(s). 3.1.1. Conformational sampling and geometry optimizations. Conformational analysis locates local energy minima on a n-dimensional potential energy surface (PES). This may be achieved by several procedures well described in textbooks and in computational software manuals.26,33 In so-called step-wise systematic procedures, all rotatable bonds are systematically varied, and the resulting structure is optimized to an energy minimum and stored. Systematic procedures are very time consuming, since the number of conformations to be searched grows exponentially with the molecular size. This computational effort may be reduced through Monte Carlo and molecular dynamics simulations. In this case the conformational space is randomly sampled either by one or more rotations around single bonds or by ‘‘switching on’’ molecular motions allowed at a certain temperature (vibrations and rotations, which can be regarded as low-frequency vibrations). The molecule is first placed at high temperature to overcome high rotation barriers, then as an increasing number of minima are found, the temperature is lowered to let the procedure converge (simulated annealing). Both methods are usually very effective in exploring conformational ensembles for very flexible molecules. At the end of systematic and Monte Carlo procedures, all structures found are checked for acceptance on the basis of an energy criterion (let’s say, they must fall within a 2–10 kcal mol1 energy window from the lowest-energy or absolute minimum). Molecular dynamics trajectories are instead sampled at regular time intervals to produce a set of snapshots that would represent a statistical molecular ensemble. This journal is

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Although all the methods above may be performed at different levels of theory, for medium-size molecules they are practically limited to molecular mechanics methods (i.e., those based on empirical force fields).26,34 The main possible error associated with conformational searches is overlooking one or more significant conformations. Geometry optimizations or energy minimizations serve to obtain the molecular structure corresponding to an energy minimum.26,33,34 Geometry optimizations must both find ‘‘correct’’ geometries and assess their relative energies accurately. Although correlated ab initio methods such as MøllerPlesset MP2 provide excellent descriptions of equilibrium geometries and conformations,33 their computational cost is often too large (MP2 scales with N5, where N is the number of electrons). Currently, the most employed method for geometry optimizations is density functional theory (DFT). Since its computational cost is very limited (it scales with N3), DFT offers a very good accuracy/cost compromise, especially when employing B3LYP and other hybrid DFT functionals,27 although they are not free from some shortcomings. As for the choice of the basis set, the B3LYP/6-31G(d) combination may be taken as a ‘‘classic’’ for preliminary geometry optimizations, but more reliable results would require larger basis sets such as 6-311+G(d,p) or those belonging to Dunning’s family (aug-cc-pVDZ and on).27 Compared to quantum-mechanics (QM) methods, molecular mechanics (MM) methods are exceptionally faster and some force fields (such as MMFF and OPLS) perform surprisingly well for almost all classes of organic compounds. Geometry optimizations also afford absolute internal (potential) energies for all optimized structures. Taking the lowest-energy conformer as reference, these are converted in a list of relative internal energies to be employed for assessing populations at WT (see Section 3.2). In the case of ECD spectroscopy, it must be stressed that a relatively poorly populated conformer associated with a strong ECD spectrum may surmount a much more populated conformer with weak ECD. In practice, the major sources of error associated with geometry optimizations are the accuracy of the obtained geometries, and, above all, of relative energies of optimized conformers. Although geometry optimizations are very often performed in vacuo, inclusion of solvent effect is desirable whenever possible.29 Especially in the case of compounds with multiple populated conformers at WT, a solvent model such as the Polarizable Continuum (PCM) or the Conductor-like Screening model (COSMO) can profoundly affect relative energies, and have a deep impact on any calculated property. Inclusion of a solvent model will however unavoidably extend the computational time, but most often this is well repaid by the increased reliability of results. 3.1.2. Experimental conformational analysis. NMR plays undoubtedly a major role in the field of conformational analysis in solution. Particularly significant are NMR data sensitive to the three-dimensional structure such as NOE effects and scalar J-couplings (especially 3JHH and 3JCH).35 NMR data may complement molecular modelling results in two ways: (1) as post-calculation checks to support low-energy This journal is

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Scheme 2 Michelleamine A (8). The dashed line indicates the point of dissection into the two fragments 8a and 8b (obtained by adding H to the ‘‘broken’’ bond).

conformers and possibly to exclude some of them; (2) as effective calculation restraints used to bias the whole conformational search. This latter option is especially valid when paramagnetic complexes of some lanthanide ions are involved. In the presence of a paramagnetic nucleus, both chemical shifts and relaxation rates of surrounding nuclei are greatly affected (so-called Lanthanide-Induced Shift, LIS, and Relaxation, LIR). In some favourable cases (Yb3+, Pr3+, Eu3+ and so on) LIS and LIR values may be quantitatively related to the solution structures by using them as restraints in least-square fitting procedures.32 3.1.3. Dissection strategies (truncated models). It is a common view to look at flexible molecules as composed of several rigid fragments linked together by flexible bonds. Such a virtual dissection of molecular geometry may in fact help to simplify the problem of a thorough conformational analysis. If two (or more) torsional modes of a flexible compound are not effectively correlated to each other, they can be investigated separately through suitable models representing the various fragments. For example, michellamine A (8, Scheme 2) is composed of four aromatic moieties connected through three aryl–aryl junctions which, due to the relative meta arrangement, can be considered independent of each other. By cutting the geometry of michellamine A at the junction indicated in Scheme 2 one obtains two simpler biaryl systems (8a and 8b). The conformational analysis of the two biaryls can be performed independently, then the resulting energy minima are recombined to afford ‘‘intact’’ michellamine A in conformations corresponding to local energy minima which are re-optimized and subjected to ECD calculations.36 Dissection strategies lead to time-saving since several high-energy conformers which would be reached in a systematic conformational investigation on the whole molecule are effectively discarded. 3.2.

Boltzmann averaging of component spectra

The simulation of the ECD spectrum of a flexible molecule requires a few additional steps after obtaining a set of low-energy conformers with relative energies (Section 3.1) Chem. Soc. Rev., 2011, 40, 4603–4625

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and estimating a single ECD feature or a full spectrum for each of them (Sections 2.1–2.4). They consist in: (1) evaluating the conformer populations at WT; (2) weighting the component spectra with respective populations; (3) summing all weighted spectra in order to afford a so-called weightedaverage spectrum. This latter operation is justified by the property of ECD being a fast spectroscopy as discussed in the Introduction. As for the second point, according to Boltzmann distribution a wavelength-dependent weighted-average ECD spectrum can be expressed as: P P w Decalc (l) = aw Dew(l) = De (l) exp(Ew/RT) where w is a conformer whose relative energy is Ew with respect to the lowest-energy conformer whose Ew 0, Dew(l) is the calculated component spectrum for conformer w, aw is the conformer population, and the sum runs on all populated conformers at the working temperature T. Such a familiar expression is actually the result of several implicit assumptions. First, it is strictly valid only if the conformers are well separated from each other on the potential energy surface, that is, they reside in sharp energy wells with barriers high enough to keep vibrational states confined to each well (not delocalized). Moreover, the predicted property (i.e., ECD spectrum) must be equivalent for all vibrational states within the energy well. These approximations tend to be imprecise for compounds with very low-frequency vibrational motions (corresponding to torsional modes), and their consequences may be especially significant when predicting chiroptical properties such as ECD and OR. Crawford et al. checked the validity of Boltzmann averaging in OR calculations of 3-chloro-1-butene and concluded that in this case the Boltzmann approach is reliable enough even in the presence of low conformational barriers (E500 cm1), perhaps owing to fortuitous cancellation of errors associated with the various approximations.37 Finally, what ‘‘energy’’ Ew should be used in the Boltzmann formula? Simple geometry optimizations afford internal energies, equivalent in relative terms to enthalpies, corresponding to the true energy well minimum. To include zero-point vibrational (ZPV) correction and afford free energies rather than internal energies, a frequency calculation is necessary for each populated conformer at the same level of theory of the geometry optimization. Frequency calculations also offer a useful check of the true minimum-energy nature of the optimized geometry. In fact, calculated frequencies are all real for an energy minimum, while e.g. a saddle point (an energy maximum with respect to one coordinate, such as a transition state) will have one imaginary frequency. 3.3. Examples of AC assignments of compounds with multiple conformers In this section we exemplify some problems in assigning the AC of flexible compounds by ECD. The examples, far from being exhaustive, were chosen with the intention: (1) to cover as wide as possible areas of research; (2) to illustrate as many as possible types of approaches used for ECD interpretations (described in Section 2); (3) to present various degrees of 4614

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complexity posed by conformational issues and show how the various methods should be considered. 3.3.1. Exciton analysis of polyols, aminopolyols etc.. The 1,2 terminal diols and aminoalcohols, and related internal 1,2-, mixed 1,2/1,3 polyols and aminoalcohols perhaps are the best examples of acyclic compounds where the elucidation of AC is complicated and hampered by the presence of multiple conformers in solution. The AC analysis by ECD exciton chirality method of these acyclic molecules, mainly derived from biologically important molecules, such as sugars and various glycoconjugates and polyenemacrolides, is often very complicated since requires consideration of the main conformer(s) responsible for observed ECD couplet. This is because many factors, such as the chemical structure and relative configuration of starting diol or polyol, the nature of covalently introduced chromophores, as well as the solvent used in ECD measurements can profoundly affect the ECD spectra.20,38 The AC’s of acyclic 1,2-diols and polyols have been usually determined upon derivatization into corresponding p-substituted dibenzoates and biscinnamates, less often to bis 2-naphthoates, bis 2-anthroates and other strongly absorbing acylates, or by using two different chromophores (‘‘bichromophoric exciton chirality method’’) so that the ECD curve becomes more characteristic in shape, and covers a broader spectral region.20 Fig. 8 depicts 1,2-syn and 1,2-anti internal diols whose OH groups have been converted to ester groups by one-step derivatization protocol.38,39 Since the acyclic bis p-methoxy cinnamates can rotate around the C–C bond connecting the two ester chromophores, the appearance of the ECD spectrum depends on the conformational equilibrium. For example, the 1,2-syn diester with the AC as shown in Fig. 8 adopts three rotational conformers S-1, S-2, and S-3, among which the conformers S-2 and S-3 are unstable because of three gauche relations between four bulky groups, while the conformer S-1 has only two gauche relations, and hence it is more stable and dominant in the equilibrium. The two chromophores in conformers S-1 and S-2 have negative and positive twists, respectively, while in the conformer S-3 the two chromophores are in an anti relation, and therefore, no exciton chirality is generated. It has been found that for most 1,2-syn diesters the 1 H-NMR coupling constant (Jtrans = 6.1–8.7 Hz) supports the preference of the S-1 conformer. Therefore the ECD spectrum of 1,2-syn diester 9a reflects the negative chirality of this particular conformer S-1. The significant difference in ECD couplet intensity of 1,2-syn and 1,2-anti bis p-methoxy cinnamates is not surprising. In the case of the 1,2-anti derivative both A-1 and A-2 conformers contain three gauche interactions, but of opposite exciton chirality, therefore the weak ECD of 9b is the result of their partial cancellation depending on the equilibrium of A-1/A-2. The presence of the third conformer A-3 with no ECD contribution can only diminish the overall couplet intensity. This situation makes the AC determination of anti-1,2-diols much more difficult or even impossible without further conformational analysis by other methods, e.g., NOE.38,39 Furthermore, if the syn-1,2-diol has polar or extremely bulky groups (R1 and R2), the conformational equilibrium This journal is

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Fig. 8 Left: staggered conformations of 1,2-syn and 1,2-anti-diol derivative. Right: ECD spectra of (2R,3S,4R)-1,2-diacetoxy-3,4-syn-bis-pmethoxycinnamate-6-heptene-1,2,3,4-tetrol 9a (solid line) and (2R,3R,4R)-1,2-diacetoxy-3,4-trans-bis-p-methoxycinnamate-6-heptene-1,2,3,4tetrol 9b (dotted line) in MeCN.

may change. In such a case, the two groups may adopt a trans relation to decrease the electric or steric repulsive forces, and therefore the conformer S-2 (Fig. 8) becomes dominant, which leads to a couplet inversion. For example, the preference of S-2 type conformer in case of (2R,3R)-diethyl tartrate bis(p-Br-benzoate) (a mirror image of S-2 shown in Fig. 8) has been supported by the 1H-NMR coupling constant (Jgauche = 2.9–4.1 Hz). Accordingly, the ECD spectrum in which the two polar ethyl ester groups adopt a trans-relation shows a negative exciton couplet.40 In case of 1,2-syn diols additional difficulties arise when the groups R1 and R2 are identical, since the 1H-NMR vicinal coupling constant between the two isochronous methine protons cannot be obtained from the routine NMR spectrum. In such a case, the 1H–13C satellite band method is useful to determine the J value.40,41 In summary, there is no doubt that clarifying the conformational status of Cottonogenic derivatives of flexible 1,2-diols, as well as of higher polyols and related aminoalcohols, is extremely critical for the proper interpretation of ECD on the way to their AC analysis. The acyclic 1,3-diols and 1,3 skipped polyols, found in many natural products, such as polyene macrolides and antibiotics, have a characteristic preference for zigzag conformation, which favored the development of an efficient chiroptical/ chemical strategy for their AC assignment.42 Upon conversion into chromophoric acylates (for example, p-Br-benzoates or p-methoxycinnamates) the acyclic anti-1,3-bis(acylate) 10a adopts a planar zigzag form in the most stable conformation and exhibits a typical ECD exciton couplet corresponding to the sign of the screw sense between the two gauche oriented chromophores.20 The syn-analog 10b, also in most stable zigzag conformation, has almost parallel orientation of corresponding interacting electric transition moments, and therefore exhibits an extremely weak ECD spectrum, so that the syn-a,a or syn-b,b-configuration of simple 1,3-diols cannot be discriminated by it (Fig. 9). The stable zigzag conformation of 1,3-diols holds also for higher polyols, so that the ECD This journal is

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contribution of anti-1,3-acylate array (11a) reflects sensitively (in additive manner) the chiral twist of the stable gauche conformation over the long chain, while the arrays with 1,3 syn-relationship (11b) remains almost ECD-silent. If the 1,3-polyol contains multiple stereogenic centers in syn/anti relation the overall ECD spectrum follows the additivity principle of the pair-wise interactions (Fig. 9, right).20 3.3.2. Quantum mechanical calculations. Since full ECD calculations are ‘‘quantitative’’ by definition, the problems related to obtaining an accurate set of input structures are especially crucial when this approach is employed. These calculations require in advance a reliable conformational picture obtained as described above (Section 3.1). A recent example of flexible compounds is presented by macrolides 12 and 13 (Scheme 3), a family of odorous natural products.43 In the case of 12, after a Monte Carlo/AM1 conformational search, 100 conformers were isolated with energies within 10 kcal mol1, which were first optimized with B3LYP/6-31G(d) affording 22 conformers within 4 kcal mol1, then re-optimized with B3LYP/TZVP leading to 18 conformers within 4 kcal mol1. The most stable structures were found to be in agreement with NMR observations. TDDFT calculations were then run with B3LYP/TZVP on all 18 structures, and component ECD spectra were Boltzmann-averaged affording the final calculated spectra. This case is relatively simple from the spectroscopic point of view, because ECD spectra of compounds 12 and 13 consist in a single ECD band around 215 nm due to n–p* lactone transition. Another favorable aspect is that all first 7 low energy conformers for 12 have a predicted n–p* ECD band of the same sign. In this situation, even a moderate error in the estimated conformer populations is unlikely to change the sign of the average predicted ECD band.43 Sometimes, the outcome of a supposedly accurate conformational analysis is contradicted by an experimental fact. For example, Mori et al. have recently reported on the donor–acceptor charge-transfer (CT) dyad 14 (Scheme 3).44 The conformational analysis was run using very reliable Chem. Soc. Rev., 2011, 40, 4603–4625

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Fig. 9 Discrimination between bis p-methoxycinnamate derivatives of 1,3-syn and 1,3-anti diols. In the 1,3-anti derivative 10a the two cinnamate chromophores are gauche oriented with a clockwise sense of helicity giving rise to a strong positive ECD couplet (black continuous line). In the 1,3syn derivative 10b the two cinnamates are almost parallel and exhibit negligible coupling (red dotted line). The ECD pattern of 1,3-syn and 1,3-anti conformations are preserved even when more than two stereogenic centers are present (right panel). While 1,3-synsyn 11b has two almost negligible contributions (red dotted line), the ECD of 1,3-synanti 11a is a summation of one significant contribution due to 1,3-anti pair with a positive exciton chirality and one close to zero due to the 1,3-syn pair (black continuous line); all ECD spectra were taken in methylcyclohexane.

Scheme 3 Macrolides 12 and 13; donor–acceptor CT dyad 14; molecules 15 and 16 whose AC has been revised based on ECD ab initio calculations.

computational tools, including DFT-D geometry optimizations.8 It was found that 14 can assume two different families of conformations: folded (directed by an intramolecular CT interaction) and unfolded. Folded conformations were calculated to be much more stable than unfolded ones, both in vacuo (ratio 8 : 2) and using COSMO (ratio 6 : 4). However, no ROESY signals in support of folded conformers in solution could be detected. In fact, the ECD spectrum of 14 in solution could be correctly reproduced by TDDFT calculations only by neglecting the contribution from folded conformers. It can be concluded that the stability of the folded conformers was overestimated by geometry optimizations, especially in the gas phase, and also the use of the solvent model was insufficient to solve the problem.44 8 DFT-D is a dispersion-corrected DFT method thought to describe non-bonding interactions such as Van der Waals (VdW) or CT interactions with high accuracy.

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As already seen in general discussion in Sections 3.1–3.2, the most frequent error sources in the procedure of assigning the AC of flexible compounds by computational conformational analysis are: (1) missing important conformers in the conformational search; (2) wrong estimation of conformer population; (3) insufficient accuracy in the prediction of ECD spectra. In a few cases (15 and 16, Scheme 3),45,46 one of these points, or a combination thereof, has been deemed responsible for initially incorrect AC predictions made on the basis of ECD or OR calculations. The correct solution could be finally reached by increasing the basis set size and/or adding a solvent model in geometry optimizations and/or ECD calculations. It must be borne in mind that the assignment of AC (an operation which has always 50% probability to succeed!) by chiroptical spectroscopies, especially of flexible molecules, is never completely free from some uncertainty. On the contrary, when properly run, the use of X-ray anomalous dispersion method is absolutely safe.47 3.3.3. DeVoe calculations of flexible compounds. The application of coupled-oscillator DeVoe calculations to assign the AC of organic compounds has been exhaustively reviewed.48 More recently, DeVoe calculations have been employed to reproduce the ECD spectrum of d-tocotrienol (18), an extremely flexible compound. A surprising feature in the ECD spectrum of 18 is the difference with the related d-tocopherol (17). The moderately intense positive ECD couplet in the spectrum of 18 between 190–220 nm is missing for 17 (Scheme 4).49 The difference was attributed to the assumed weak non-degenerate exciton coupling between the p–p* transitions of the aromatic chromophore and the closest alkene group. Exciton-coupled spectra were calculated with DeVoe’s method using a very large set of input structures (10 000) obtained by MD simulations with MM3 force field.49 The calculation method appeared the only reasonable choice This journal is

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Scheme 4 Structure and ECD data of d-tocopherol (17) and d-tocotrienol (18).

in view of the pronounced flexibility of the substrate. The same concept is demonstrated by other recent reports.50 3.3.4. Metal complexes and organometallic compounds. Unlike most of the systems treated above, metal complexes are most often synthetic and contain stereogenic elements of known configuration. Nonetheless, they can display relevant stereochemical aspects due to ligand conformation and to the dissymmetric arrangement of ligands about the metal. We shall describe a problem of ligand conformation, which can be easily solved by NIR-ECD of its Yb compound, by means of reference to conformationally-locked analogues. This can be regarded as a recent example of a classical procedure in the determination of the stereochemistry of organic compounds, which takes full advantage of the ECD fingerprint ensured by f–f transitions. DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid, 19, Fig. 10) is a ligand particularly well suited for lanthanides and Gd DOTA is one of the most commonly used Magnetic Resonance Imaging (MRI) contrast agents. It is based on a 12-membered macrocycle (cyclen), which upon metal coordination can assume two enantiomeric structures, depending on the regular gauche +/ conformations of the four ethylene bonds. Accordingly, they are called (llll) and (dddd) as depicted in Fig. 10(a). In the presence of other stereogenic elements on cyclen itself, on the acetate arms or even following interactions with other chiral entities, (llll) and (dddd) forms are no longer related by mirror symmetry, they become diastereomeric and differently populated. Because of the lack of chromophores on cyclen, they can hardly be distinguished by UV-vis Abs and ECD and even if paramagnetic NMR differentiates them, it may fail to provide an unambiguous assignment. Most lanthanide(III) ions are endowed with a rich manifold of intraconfigurational f–f electronic transitions, with weak electric dipole component, often falling at low energy in the vis or Near IR range. In a chiral environment, some of them are associated with a significant ECD spectrum, consisting of many Cotton effects of alternating signs, which is notably the case for the 2 F7/2 - 2F5/2 transition of Yb3+ around 980 nm. This ECD multiplet is extremely sensitive to the geometry of Yb3+ coordination polyhedron and can be used as a fingerprint of the complex structure, by visual comparison. Conformationally locked model compounds provide a secure reference, because the sequence of signs and intensities of Near IR ECD is completely different for diastereomeric compounds, as This journal is

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Fig. 10 (a) Schematic representation of two diastereomeric structures of Ln DOTA (19Ln), which are normally in dynamic exchange. (b) Near IR ECD spectra of conformationally locked derivatives. Continuous line: YbS(SSSS)-(p-nitrobenzyl) DOTMA (20), assuming a L(llll) conformation; dashed line: YbS(RRRR)-(p-nitrobenzyl) DOTMA, assuming a L(dddd) conformation; dotted line: normalized spectrum of the 1 : 4 mixture of Yb DOTA and g-CDex.

demonstrated in Fig. 10(b), and it falls in case (3) of Section 1.1. Once this is known, one can immediately assign the structure of unknown compounds. As an example, we can take the inclusion compound of Yb DOTA (19Yb) into g-cyclodextrin (g-CDex), where a preferential chirality is enforced onto the complex by the sugar through weak, non-covalent interactions. As shown in Fig. 10(b), one can recognize that the Near IR ECD of the adduct Yb DOTA/g-CDex is superimposable on the one of the conformationally locked compound YbS(RRRR)-(p-nitrobenzyl) DOTMA (20) and that the chirality of the cyclen ring must be (dddd).51 3.4.

Strategies for solving flexibility-related problems

3.4.1. Exciton coupled ECD spectra of chromophoric derivatives with reduced flexibility. It was already mentioned above (Section 3.3.1) that the exciton chirality approach cannot be applied in a straightforward manner to acyclic cases such as Chem. Soc. Rev., 2011, 40, 4603–4625

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Scheme 5 Formation of rigid derivatives of 1,2-diols (21–23) with diagnostic ECD spectrum. In cases (a) and (b), the derivatives show exciton-coupled ECD spectra reflecting the chirality shown by arrows; X and X 0 may be auxochromic substituents, and other aromatic chromophores may be present instead of p-substituted benzene. In case (c), the biphenyl moiety with shown preferred chirality (driven by steric interactions between R/R 0 groups and benzylic hydrogens) displays a diagnostic so-called biphenyl ‘‘A’’ band.

anti-1,2 diols, where several conformers with similar energy and opposite chirality between the interacting chromophores may exist. One possibility to avoid such difficulties consists in restricting the ‘‘free’’ rotation in a predictable, stereocontrolled manner. Depending on the presence of chromophores on the substrate and/or the number of available functional groups for derivatization, several different strategies may be employed all of which aim at obtaining a rigid bischromophoric derivative with clear-cut exciton-coupled ECD spectrum. Using 1,2-diols again as example (Scheme 5), the substrate may contain (a) two, (b) one, or (c) no pre-existing chromophores suitable for exciton coupling. In case (a), the compound only needs to be rigidified, e.g. by conversion into a 2,2-dimethyl-1,3-dioxolane (21). In case (b), one must also add one chromophore, e.g. by conversion into a 4-biphenylboronate (22). In the last case (c), two chromophores or an inherently chiral chromophore must be added with a diagnostic ECD spectrum, such as a biphenyl moiety included in a dioxolane structure (23). The three cases have been reported by Rosini and others to treat efficiently several 1,2-diols.52 Many chiral compounds with a single functional group suitable for chromophoric derivatization, such as various acyclic alcohols, amines, a-amino- and a-hydroxyacids, remain in general unsuitable for the exciton chirality approach. In order to deal with such situations a different concept must be employed that envisages a covalent attachment of an acyclic spacer moiety, which carries the second necessary 4618

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functional group (OH or NH2). The possible problems related to the increased conformational flexibility due to the spacer are avoided using tetraphenylporphyrins and metalloporphyrins instead of conventional chromophores for exciton coupling.20,53 The usefulness of porphyrin chromophore in this case is related to its unique properties, in particular, to its p-stacking ability. For example, when (S)-mandelic acid (24) is derivatized with 2-aminoethanol (the spacer) and converted to bisporphyrin derivative 25, this latter may undergo intramolecular p-stacking and adopt a predictable and stereocontrolled folded conformation (Fig. 11). If so, the ECD coupling between stacked porphyrins would provide a way to correlate this preferred conformation with the chirality of starting substrate. Ever since the concept was born53 many experimental results have confirmed its validity. The p-stacking is stereoselective in the sense that the terminal porphyrin preferably approaches the stereogenic center carrying the second porphyrin from the less hindered side having the hydrogen (small or S) group. This produces a preferred clockwise arrangement of the porphyrins transition moments and therefore results in a positive exciton split ECD. The other possibility with stacking from the side of the larger phenyl group (L) would be sterically less favorable. The p-stacking is confirmed by the split pattern of the 1H-NMR aromatic signals, the chemical shits of which do not vary with the concentration, thus supporting the intramolecular nature of stacking.54 Application of this ECD approach, where an intramolecular porphyrin/porphyrin p-stacking leads to a stereocontrolled transformation of the flexible chiral substrate into a preferred rigid conformation with clear-cut experimental ECD signature, has its limitation. The approach can be considered a reliable tool for AC assignment, provided unambiguous arguments and data are available for the particular substrate regarding the steric preference of the groups (L and S) at the stereogenic center, so that under their control a safe link can be made between the structure of preferred rigid conformation and the observed ECD exciton chirality. The method so far has been successfully applied to various flexible substrates, such as aminoalcohols, diols, a-hydroxy acids, including the biologically important ceramides. Application of this protocol on a microscale can facilitate the ceramides analysis.19 In the past their flexibility has been a big challenge and has therefore hindered the correlation with various sphingolipids, including gangliosides and cerebrosides as their biological precursors. 3.4.2. ECD of Cottonogenic metal complexes with reduced flexibility. An alternative strategy to the use of exciton-coupled rigidified Cottonogenic derivatives exists for some classes of compounds including 1,2- and 1,3-diols. Several mono- or dinuclear metal ions, such as Mo24+, Rh24+, Ni2+, Ln3+ (Ln is a lanthanide), which show distinct absorption bands in the UV-vis or NIR range of the spectrum, are able to form ECD-active complexes with chiral diols. The diol moiety acts as a bidentate ligand and chelates the metal core to form a cyclic complex. The sign of the ECD spectrum can be related with the AC of the chiral substrate, although such correlation is often empirical, that is, it is based on the consistency of the ECD spectra for several substrates with similar structure and This journal is

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Fig. 11 Top: two-step conversion of (S)-mandelic acid (24) by attachment of ethanolamine spacer and acylation with 5-(p-carboxyphenyl)10,15,20-triphenylporphyrin (TPP, see Fig. 5) into bisporphyrin derivative 25. Bottom: stereocontrolled p-stacking of 25.

same AC. This is repaid by the fact that such methods do not require chemical derivatization, but just mixing two reagents, and are therefore very practical. The method based on dimolybdenum tetraacetate Mo2(OAc)4, developed by Snaztke and coworkers, and a short bibliography on similar ones is reported in ref. 55. 3.4.3. QM calculations of solid-state ECD spectra. Configurational assignments in the crystal state can provide great but still largely unexplored opportunities to overcome the problems encountered by flexible compounds, because their solid-state molecular conformation will be fixed and unequivocal. When crystals amenable to X-ray analysis are available, the solid-state structure can be determined with high accuracy. In addition, ECD spectra can be recorded in the solid state with various techniques, the most useful of which consists in using a translucent glassy pellet obtained by mixing the sample with excess KBr or KCl. In fact, solid-state ECD spectra are very useful source of structural information.15 In particular for assigning AC’s, one can use directly the X-ray geometry as input structure for TDDFT or other ECD calculations to be compared with the experimental solid-state spectrum. The procedure is extremely fast, because no conformational search or geometry optimization is needed, and it often leads to a good agreement between calculated and experimental quantities, because they are related to the very same geometry. This strategy, recently known as solid-state ECD/TDDFT approach, has been successfully applied to assign the AC of several chiral natural products,15 including e.g. viburspiran (26, Fig. 12).56 Extracted from the fungal endophyte Cryptosporiopsis sp., viburspiran has antifungal activity and an unprecedented skeleton for a maleic anhydride derivative. Should one try to reproduce its solution ECD spectrum, several low-energy conformers should be taken into account because of the flexible n-pentyl chain (which could perhaps be ‘‘dissected’’) and the eight-membered ring. However, 26 could This journal is

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Fig. 12 Top: Molecular and X-ray structure of viburspiran (26). Only one of two independent molecules per asymmetric unit, differing in the orientation of the n-pentyl chain, is shown. Bottom: ECD spectra of viburspiran: experimental solid-state spectrum, and average spectrum calculated with CAM-B3LYP/TZVP on the X-ray geometries for the two molecules in the crystal cell.

be obtained in crystalline form so that its X-ray geometry was determined (Fig. 12). According to the standard protocol for Chem. Soc. Rev., 2011, 40, 4603–4625

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the solid-state ECD/TDDFT approach, the X-ray geometry was refined by re-optimizing only hydrogen atoms,** to provide the input geometry for TDDFT calculations (with CAM-B3LYP/TZVP). The calculated spectrum, obtained with a minimal computational effort, was in very good agreement with the experimental solid-state spectrum (KCl pellet) and allowed AC assignment.56 Apart from the necessity of obtaining crystals amenable to X-ray measurement the method is quite general. For example it may be used for compounds devoid of ‘‘heavy’’ elements (which is instead a limitation of the anomalous scattering technique). An important requirement is that inter-molecular interactions (e.g., of the exciton type) between molecules closely packed in the crystals do not give rise to significant contributions to the solid-state ECD spectrum, which is indeed the most frequent situation encountered thus far. Finally, solid-state ECD measurements must be run carefully to prevent contamination by artifacts.15

4. Conformational investigations by ECD 4.1.

Qualitative or semi-quantitative applications

In this section we shall discuss two different families of approaches where ECD spectra are used as a source of structural information without the need for explicit calculations. In the first one, described in Section 4.1.1, the onset of an ECD spectrum of given shape and intensity is taken as qualitative proof of the occurrence of stereo-regular structures, for example helical polymers, and the ECD sign is possibly employed to determine the structure helicity. In the second situation, the shape and the sign of some ECD bands are used to draw semi-quantitative conclusions on solution conformation(s) on the basis of pre-existing models for ECD interpretation, such as the exciton chirality method and helicity or sector rules. Selected examples will be surveyed in Sections 4.1.2–4.1.4 concerning biaryls, disulfides and dienes. For these compounds, configurational and conformational factors are especially intertwined, because due to atropisomerism, the rotation around a torsional axis may not only change the conformation, but may also invert the configuration. The classical example of asymmetrically substituted biphenyls serves to illustrate this situation: if the rotation around the biphenyl axis (a stereogenic axis) is sufficiently fast, we speak of conformational isomerism and of conformers. If, however, it is slow enough, we speak of atropisomerism and possibly of enantiomers.9 In this latter case, if ECD is sensitive towards the rotation around the axis, the method can be employed either to study conformational or configurational aspects (rarely both of them at the same time). 4.1.1. Proof of stereo-regular structures (helices). Let’s consider the hypothetical case of two exciton-coupled chromophores linked together by a flexible non-chromophoric chain. If the relative arrangement between the two chromophores is completely random, it is intuitive that their average exciton coupling will vanish and produce a negligible ECD ** X–H bonds, with X = C, O, N, generated during X-ray refinement procedure are often too short.

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Scheme 6 General scheme for the transition of a chromophoric polymer from a random-coil to ordered helical conformations.

signal. This is because conformations giving rise to positive ECD couplets will be counter-balanced by conformations producing negative ECD couplets. Similarly, in the case of a polymeric chain containing chromophoric side groups, it is expected that a completely unordered conformation will average all possible exciton couplings to zero (Scheme 6). On the contrary, if the polymer assumes a stereo-regular conformation, for example a helical one, not only each pair-wise exciton coupling between every two chromophores will produce an ECD couplet, but all possible couplings will reinforce each other (because of a regular repeating arrangement) and will lead to an amplified exciton-coupled ECD signal. Moreover, for the two enantiomorphic right- and left-handed helices, the sign of the ECD signal will be opposite (Scheme 6). This is the reason why ECD spectra of helical macromolecules and biopolymers can be taken as an evidence for helix formation and as a signature of their helical (absolute) conformation. However, it must be stressed that the relation between the sign of the ECD spectrum and the sense of helical twist is never trivial: in particular, it is incorrect that a right-handed helix will always lead to a positive ECD band or couplet. In fact, it depends on many parameters including the helix geometry and the direction of polarization of the chromophoric transitions with respect to the helix axis. The most immediate example comes from biopolymers: protein a-helix and DNA B-form, although both are right-handed, give rise to a negative and a positive exciton couplet in their own transition ranges, respectively. There are countless examples of application of ECD spectroscopy of the type just described. In the most common situation concerning a covalent polymer or a supramolecular aggregate, the onset of a relatively intense ECD signal is taken as proof of formation of an ordered macromolecular or supramolecular structure, sometimes triggered by external stimuli. In favorable cases, the sign of the ECD spectrum gives indication of the sense of the twist of the polymer or the aggregate. These kinds of studies include a very broad range of substrates: (a) chiral polymers, such as polypeptides, nucleic acids but also synthetic polymers;1,9 (b) supramolecular aggregates of chiral molecules;57 (c) polymers formed by achiral monomers either polymerized in the presence of a chiral inducer or assuming a preferential chiral structure in the presence of a chiral inducer (this latter case is referred to as induced ECD);58 (d) supramolecular aggregates of achiral molecules formed in the presence of a chiral template, including compounds exhibiting memory effects.59,60 This journal is

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4.1.2. 1,1 0 -Binaphthyls and other biaryls. 1,1 0 -Binaphthyl derivatives, a family of chiral auxiliaries and enantioselective catalysts, have represented a sort of benchmark for conformational analysis by means of ECD, for a twofold reason. First, their structure is easily described in terms of a single key parameter, namely, the dihedral angle y defined by the two planes (Fig. 13). Second, their ECD is largely dominated by the exciton coupling between the strong p–p* transitions of the two naphthalene chromophores, and may be quantitatively predicted to a large extent by means of exciton theory and De Voe calculations. We have already pointed elsewhere at the structural information contained in the exciton couplet around 220–230 nm due to the coupling between the two naphthalene 1 Bb transitions.8,61 Inspection of the ECD spectrum of 1,1 0 -binaphthyl derivatives may be sufficient to reveal the absolute configuration around the stereogenic axis and the most probable value adopted by angle y (Fig. 13).61 We want to stress that for symmetrical 1,1 0 -binaphthyl derivatives there is no other simple means to assess the molecular conformation so directly and in such detail as offered by ECD. For example, the NOE measurements are useless because relevant nuclei such as H8 and H8 0 are isochronous.

Fig. 13 Top: definition of dihedral angle y for a generic 1,1 0 binaphthyl derivative. Bottom: ECD spectrum of (R)-4,5-dihydro3H-dinaphtho[2,1-c:1,2-e]oxepine (27) in the 1Bb couplet region deconvoluted in the two exciton-coupled components. Dlmax is wavelength splitting between the two components which correlates with y as shown in the inset. The experimental values represented by diamonds correspond to a series of derivatives of 2,2 0 -dimethyl-1,1 0 binaphthyl, including oxepine 27.61

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Scheme 7 Biaryl compounds 28–32 discussed in the text.

The binaphthyl derivative 6,6 0 -dibromo-1,1 0 -bi-2-naphthol (28, Scheme 7) offers a good example to emphasize the different, and sometimes complementary, structural sensitivity of ECD vs. VCD and ROA discussed in Section 1.2. While the ECD spectrum of 28 immediately reveals its absolute configuration and delivers quick information about the aryl–aryl dihedral angle, it is rather insensitive to the hydroxy groups conformation. Conversely, a full interpretation of the VCD spectrum of 28 needs quantum-mechanical calculations, which however will also provide the required information about the hydroxy groups.62 Similar to 1,1 0 -binaphthyls, several other biaryls have distinct ECD spectra that are informative of their absolute conformation. The specific ECD spectrum feature to be taken into account varies case by case; for example, for donor– acceptor binaphthyls dyads 29 and 30 (Scheme 7), it is the intensity of the CT band above 360 nm that correlates with the dihedral angle y.63 In 9,10-dihydrophenanthrenes (31, formally 1,1 0 -biphenyl derivatives, Scheme 7), the ECD spectrum reveals the M/P chirality of the biphenyl core together with the axial/equatorial arrangement of the 9,10 substituents Y/Y 0 , two aspects directly related to each other.64 A noteworthy class of compounds containing multiple aromatic chromophores is represented by tripodal complexes of Zn2+, Cu+ and Cu2+ with multidentate ligands of the type 32 (Scheme 7), formed by derivatization of chiral primary amines with 2-bromomethyl quinolines, as reported by Canary and coworkers. In the complexes, the aromatic moieties adopt a stable propeller-like conformation, which depends on the metal coordination geometry. Exciton coupling between quinolines leads to characteristic bisignate ECD curves reflecting the AC of the starting substrate.65 Interestingly, ECD can also be a diagnostic spectroscopic probe for the changes in propeller twist conformation as result of alteration in metal ion coordination number or hardness.66 4.1.3. Disulfides. The disulfide linkage offers an immediate example of a so-called inherently chiral chromophore,9 because the C–SSC dihedral angle j tends to assume values around 901. The disulfide bond SS has two characteristic n1,2s* transitions in the 220–300 nm region that give rise to two oppositely-signed ECD bands. The sign Chem. Soc. Rev., 2011, 40, 4603–4625

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the individual double bonds and extrinsic contributions play a role, which may easily encompass and mask the one due to diene chirality, which prevents any straightforward deduction. As a consequence, the ECD of dienes should be analyzed by means of more thorough computational approaches, which should explicitly take into account the role exerted by nearby substituents and cycles.68


4.2.

Quantitative studies

By ‘‘quantitative’’ we mean studies where ECD spectroscopy has been employed to draw quantitative information regarding solution conformation of organic compounds and polymers, by means of comparison between the ECD spectra calculated for a more or less extended set of geometries of a flexible compound and the experimental data. In particular, as far as input geometries are concerned, we can distinguish between two limiting cases: (1) a large ensemble of geometries generated by molecular modeling; (2) a single geometry determined by geometry optimization or other methods. In the first case, ECD is used to establish the most probable solution structure(s), while in the second case it serves to confirm the results coming from other studies.

Fig. 14 Top: disulfide quadrant rule showing the sign of the first ns* ECD band as a function of dihedral angle j. Bottom: ECD spectra calculated for dimethyl disulfide (33) as a function of dihedral angle C–S–S–C. Calculations were run with TDDFT (B3LYP/TZVP) using DFT-optimized structures (B3LYP/6-311+G(d,p)).

and intensity of the first (long-wavelength) band follow a regular dependence on j.67 The correlation may be expressed through a quadrant rule (Fig. 14): the sign of the first ns* band is positive for 01 o j o +901 and –1801 o j o –901, and negative for +901 o jo +1801 and –901 o j o 01. Although useful, application of the rule is subject to an obvious restriction: the absolute helicity (sign of j) of a disulfide cannot be assigned by simple inspection of its ECD spectrum without an assumption of the value of the dihedral angle j, and vice versa. For example, a disulfide with P helicity (positive j) will have opposite ECD spectrum when it is cisoid (j o 901) or transoid (j > 901). Therefore the conformation may be assessed only if the absolute configuration of a disulfide is already known. 4.1.4. Dienes. A very similar situation to disulfides and biaryls is found for conjugated dienes. The planar (s-cis and s-trans) arrangements are usually preferred, but certain molecular backbones may enforce a skewed chiral conformation. In this case, the diene chromophore (which must be treated as a whole) becomes intrinsically dissymmetric; this provides a mechanism for justifying the sign of the Cotton effect of the low-energy transition, usually found around 220–240 nm: similar to disulfides, a positive angle (smaller than 901) may be associated to a positive ECD.9 Unfortunately, distortion of 4622

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4.2.1. Establishing the most probable structure from a conformational ensemble. A common assumption in the application of ECD spectroscopy in conformational analysis is that if a flexible molecule can assume a certain number of conformations in solution, the one whose calculated ECD spectrum is in the best agreement with the experimental ECD, is the most probable or the dominating in solution. The examples of dioncophyllin (Section 1.1), biaryls and disulfides (Section 4.1) discussed above imply that this approach would be better applicable to compounds that have already established AC but unknown conformation, because extracting both pieces of information from ECD spectra may be not straightforward. The flavonoid 3,3 0 ,4,4 0 ,7-flavanpentol (34, Scheme 8) offers an example of this kind of applications. This relatively small molecule has several degrees of conformational freedom, two of which (torsions o and t in Scheme 8) directly affect the reciprocal arrangement between the two aromatic chromophores. Cappelli et al. used ECD calculations and MD simulations to study its solution conformation.69 The chirality assumed by the ring (related to torsion o) was established as P based on the negative 1Lb ECD band. Furthermore, a set of TDDFT-calculated spectra was obtained by varying the torsion t systematically. Comparison with the experimental spectrum led the authors conclude that this latter is a result of contribution of several conformers with torsional angle t (Scheme 8) in the range 1201–1451.69

Scheme 8 Flavanpentol (34).

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the best fit with the experimental one (Fig. 15), such a conformation was taken as the most probable in solution.70 What is implicitly assumed in this kind of studies is a so-called ‘‘single-conformer approximation’’. This means that one seeks one single structure which provides the best fit between experimental and calculated spectrum. However, one cannot exclude the possibility that a combination of calculated spectra, for a few or several other structures, may provide an average spectrum similar to the experimental one too. Since we know that any observed ECD is actually an average, this uncertainty affects all conformational investigations based on ECD. 4.2.2. Validation of structures from other evidence. A further possible application of ECD spectroscopy in conformational analysis consists in the following. Let us assume that for a given compound a likely conformation has been established by means of other methods, as for example NMR, X-ray, molecular modeling or a combination thereof. Then, ECD calculations may be run on such a structure with a suitable method to be compared with the experimental spectrum and offer an independent confirmation of the structure obtained. Such an approach has for instance been successfully employed to investigate the solution structures of several metal complexes of 1,1 0 -bi-(2-naphthol), or BINOL, used as enantioselective catalysts. Especially significant are the cases of lanthanide complexes of BINOL (or its derivatives), where the structure for the major solution species has been established by quantitative use of LIS and LIR effects (Section 3.1.2) and used as input for De Voe calculations.71 The significance of this kind of applications is related to the extreme sensitivity of ECD to the whole molecular structure. 4.3.

Fig. 15 (a) Structure of 1,3-di-tert-butyl-1,3-diethynylallenes oligomers 35n and definition of dihedral angle y between buta-1,3-diynediyl moieties. (b) ECD spectra calculated for tetramer 354 with ZINDO as a function of y on AM1-calculated geometries. (c) Experimental ECD spectrum of 354 in n-hexane and spectrum calculated with TDHF/ 6-31G(d) for the HF/6-31G(d)-calculated geometry with y = 451.

A short discussion on monodisperse polymers offers a good chance to further highlight potentiality and limitations of conformational analyses based on ECD. Let us use as example allenoacetylenic oligomers (35n, Fig. 15) reported by Rivera-Fuentes et al.70 These compounds show an interesting phenomenon of chiral amplification by increasing the molecular size. It was found that OR and ECD values grow nonlinearly with the number of monomers n. To rationalize this observation, a conformational analysis was run using ECD and OR calculations. The torsion angle y across the buta-1,3diynediyl moieties was varied systematically in the 1351 to +1351 range, and the resulting structures used for ZINDO and TDHF/6-31G(d) calculations. For example, since for the tetramer 354 the ECD spectrum calculated for y = 451 offers This journal is

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Variable-temperature and solvent-dependent spectra

Variable temperature (VT) is one of the most important experimental tools at hand for studying thermodynamic parameters of equilibrium reactions and among them of conformational rearrangements. In order to simplify the discussion, we shall limit it to the case of the equilibrium between two forms, which we shall call A and B. For example, they may be a gauche or anti conformation of an ethylene bond, or the product of ring inversion in a cyclic compound. It is commonly assumed that the chiroptical properties of a single conformer are not temperature-dependent, which implies that any variation in the sign, position and intensity of the ECD bands will be related to a change in conformational distribution, i.e., in the relative population of the conformers present in solution. By lowering the temperature the equilibrium shifts toward the most enthalpically stable conformation. In the case of spectra dominated by a single Cotton effect, this will result in a change in its rotational strength, whose absolute value may increase or decrease upon cooling depending on the relative sign and intensity of the ECD of the forms A and B, as shown by the example of 3-methylcyclohexanone discussed in Section 2.1. In more complex cases, low temperature ECD spectra may be compared with computational results relative to the most stable conformer only, which constitutes a simplification of the procedure described in Section 3.3.2. Chem. Soc. Rev., 2011, 40, 4603–4625

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Changing the solvent may have a dramatic impact on ECD spectra, on account of two different effects: (1) the medium properties such as dielectric constant or hydrogen bond ability may influence strongly the conformational distribution; (2) the spectroscopic features may change by changing solvent polarity (solvatochromism). Disentangling the two effects may be difficult and this kind of investigations should be carried out with great care. Typically a change in pH is likely to strongly affect chromophores containing hydrogen bond donors or acceptors, and at the same time, by changing the charge on polarizable groups a relevant conformational rearrangement may follow (see example in Fig. 7). Another feature, which could be strongly modulated by changing solvent properties and other environmental factors and may have a deep impact on ECD is intermolecular aggregation, but this subject goes beyond the scope of the present tutorial review.

5. Conclusions Electronic circular dichroism spectra of compounds containing at least one absorbing unit in the UV-vis-NIR range are a very rich source of structural information which goes far beyond the correlation between the absolute configuration and the sign of the spectrum. Chromophore-lacking compounds, which are per se amenable only to vibrational chiroptical spectroscopies (VCD and ROA) or optical rotation measurements, may be analyzed by ECD after suitable derivatization. This procedure can permit one to focus on specific stereochemical problems, while neglecting other features associated with non-chromophoric molecular portions. Nowadays, classical means of interpretation are extended by readily available high-level calculations, which makes ECD a sensitive and faithful tool for providing deep details on molecular and supramolecular stereochemistry, including, most importantly, conformations. When ECD is employed solely for configurational assignments, molecular flexibility may be an undesired complication, but it can be overcome (e.g., by chemical derivatization into a rigid derivative) or properly taken into account (e.g., by conformational analysis and spectral averaging). At the same time, however, ECD, together with other chiroptical spectroscopies, should be viewed as the ideal tool to analyze chiral flexible molecules, which uniquely complement other techniques such as NMR.

References 1 G. D. Fasman, Circular Dichroism and the Conformational Analysis of Biomolecules, Plenum Press, New York, 1996. 2 D. A. Lightner and J. E. Gurst, Organic conformational analysis and stereochemistry from circular dichroism spectroscopy, Wiley, New York, 2000. 3 J. Sandstro¨m, Chirality, 2000, 12, 162–171. 4 An extensive coverage of all chiroptical spectroscopies and their applications can be found in the forthcoming monography, Comprehensive Chiroptical Spectroscopy, Eds. N. Berova, P. Polavarapu, K. Nakanishi and R. W. Woody, Wiley-VCH, vol. 1&2, 2011, in press. 5 P. L. Polavarapu, Chem. Rec., 2007, 7, 125–136. 6 P. L. Polavarapu, Chirality, 2008, 20, 664–672.

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