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Elucidating the structure of chiral molecules by using amplified vibrational circular dichroism: from theory to experimental realization Article
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Domingos, S. R., Hartl, F., Buma, W. J. and Woutersen, S. (2015) Elucidating the structure of chiral molecules by using amplified vibrational circular dichroism: from theory to experimental realization. ChemPhysChem, 16 (16). pp. 33633373. ISSN 14397641 doi: https://doi.org/10.1002/cphc.201500551 Available at http://centaur.reading.ac.uk/46520/
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Elucidating the Structure of Chiral Molecules by usingAmplified Vibrational Circular Dichroism: From Theory toExperimental RealizationS�rgio R. Domingos,[a, c] Frantisek Hartl,*[b] Wybren Jan Buma,*[a] and Sander Woutersen*[a]
1. Introduction
Chirality plays a key role in chemistry and biology. The discov-ery of molecular chirality emerged from a series of observa-tions of optical activity by Argo and Biot (1811), Herschel(1822), Pasteur (1848), van ‘t Hoff and Le Bel (1874) and LordKelvin (1904). Since then, the connection between molecularhandedness related to mirror-image molecules and optical ac-tivity has been well established. Two non-superimposablemirror-image isomers of a chiral molecule, similar to our rightand left hands, are referred to as enantiomers. Most biomole-cules are chiral and thus have a stereoselective bias for specificbiochemical interactions. Because of this bias, different enan-tiomers may exhibit completely different biological activities. Itis precisely for this reason that the unambiguous assignmentof enantiomers is a key aspect of stereochemistry, with in-creased importance in pharmaceutical research, even more sobecause of the stringent rules imposed nowadays by regulato-ry agencies on specifications of enantiomeric identity andpurity.
Vibrational circular dichroism (VCD), that is, the differentialabsorption, DA = AL�AR, for left and right circularly polarizedinfrared light is a powerful technique for this purpose becauseit makes it possible to determine the absolute configuration
and conformation (or distribution of conformations) of a chiralmolecule without reference to empirical rules. Due to the pio-neering work of Stephens, Nafie, Keiderling, and many othersduring the past two decades, VCD has emerged as a tool forstructure elucidation of chiral molecules in the condensedphase and is nowadays an established technique used routine-ly in many chemical and biochemical laboratories.[1–3] The ap-plication of VCD as a probe of chiral molecular structure has inrecent years received a boost due to significant experimentaland theoretical progress. Instrumental advances have led tothe development of commercial VCD spectrometers while theformulation and implementation of the theoretical expressionsfor calculating VCD intensities in quantum-chemical packagessuch as Gaussian[4] and ADF[5] allow for a direct comparison ofexperimentally recorded and theoretically predicted spectra.Such comparisons have by now become a robust method todetermine the absolute structure and conformational hetero-geneity of even complex mixtures of chiral molecules.
The applicability of VCD is nevertheless still limited in manycases by its signal intensities, which are typically four to sixorders of magnitude smaller than that of the infrared absorp-tion itself. One is thus required to work with highly concentrat-ed samples, which may not be possible because of low solubil-ity and aggregation of the sample. This limitation has seriouslyimpeded extensive application of VCD, in particular for systemswith biological relevance such as amino acids, peptides andproteins. In this paper, we show that one can overcome thisdrawback, not by improving the detection electronics, but byoptimizing the “electronics” of the molecules, resulting in theamplification of VCD signals.
In the following we will first briefly review the physics that isat the basis of VCD, present the theoretical expressions for theintensities in a VCD spectrum, and rationalize why these inten-sities are generally very small. We will then consider underwhich conditions one may expect enhanced VCD intensities.
Recent experimental observations of enhanced vibrational cir-cular dichroism (VCD) in molecular systems with low-lying elec-tronically excited states suggest interesting new applicationsof VCD spectroscopy. The theory describing VCD enhancementthrough vibronic coupling schemes was derived by Nafie in1983, but only recently experimental evidence of VCD amplifi-cation has demonstrated the extent to which this effect can be
exploited as a structure elucidation tool to probe local struc-ture. In this Concept paper, we give an overview of the physicsbehind vibrational circular dichroism, in particular the equa-tions governing the VCD amplification effect, and review thelatest experimental developments with a prospective view onthe application of amplified VCD to locally probe biomolecularstructure.
[a] Dr. S. R. Domingos, Prof. Dr. W. J. Buma, Prof. Dr. S. WoutersenVan‘t Hoff Institute for Molecular Sciences, University of AmsterdamScience Park 904, 1098 XH Amsterdam (The Netherlands)E-mail : [email protected]
[b] Prof. Dr. F. HartlDepartment of Chemistry, University of ReadingWhiteknights, Reading RG6 6AD (United Kingdom)E-mail : [email protected]
[c] Dr. S. R. DomingosCurrent address : Max Planck Institute for the Structure and Dynamics ofMatter at the Center for Free-Electron Laser Science and The HamburgCentre for Ultrafast Imaging, Universit�t HamburgLuruper Chaussee 149, 22671 Hamburg (Germany)
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We will show that low-lying electronically excited states playa crucial role in this context, and review experimental observa-tions from the past that already indicated that low-lying elec-tronically excited states can lead to enhancement of VCD in-tensities. We will then proceed to more recent studies in whichthe electronic manifold of molecules is explicitly manipulatedso as to optimize amplification of VCD intensities. From thesestudies it has become clear that the VCD enhancement is a lo-calized phenomenon that offers a unique potential to serve asa local probe of chiral structure.
2. Theory of Vibrational Circular Dichroism
2.1. Classical Picture of CD
In a simple classical explanation of circular dichroism (CD),[6]
the chiral molecule is modelled as a conducting coil (seeFigure 1). In the scheme, the light wave propagates out of thepaper and thus perpendicular to the coils. The electric field~E(t)and the magnetic field ~B(t) both generate a current in thewire, the latter because of the magnetic flux through the coilchanging with time (Lenz’s Law). For right circularly polarized(RCP) light, the two currents have the same direction, whereasfor left circularly polarized (LCP) light, they have opposite di-rections. Hence, a larger current is generated, and moreenergy dissipated (“light absorbed”) in the coil for RCP lightthan for LCP light. Although this is a simplified explanation, itmay be noted that a box filled with 1 cm long copper coils ex-hibits strong optical activity in the microwave region.[7]
2.2. VCD Intensity
VCD is the extension of CD to the infrared region of the elec-tromagnetic spectrum, and is associated with the vibrationaltransitions of chiral molecules. The infrared absorbance can berelated to the extinction coefficient e(v) by the Beer–Lambertlaw as Abs ¼ �log10ð I
I0Þ ¼ eðnÞ � l � C, where l and C are the
length and concentration of the sample; I and I0 designatetransmitted and incoming light, respectively. The experimentalparameter e(v) is related to the Einstein’s coefficient for stimu-lated absorption between states i and f, Bif, by the followingexpression [Eq. (1)]:
A ¼Z
eðnÞdn ¼ hnfi
cNABif ð1Þ
where NA is the Avogadro number, c the velocity of light invacuum, and nfi ¼ DEfi=h. The quantity A on the left side ofEquation (1) is the integrated molar absorption coefficient, thatis, the area under the experimental infrared band. The VCD ofa transition is defined as the differential absorption of left andright circularly polarized (LCP and RCP, respectively) infraredlight passing through the sample, that is, the difference be-tween the integrated molar absorption coefficients for LCPand RCP light. We can thus write the differential absorption co-efficient DA as [Eq. (2)]:
DA ¼ ALCP � ARCP ¼ hnfi
cNAðBLCP
if � BRCPif Þ ð2Þ
The designations LCP and RCP indicate the handedness ofthe electric and magnetic field vector components as + and�, respectively. Introducing the expressions for Einstein’sstimulated emission coefficients[8] into Equation (2) leads toEquation (3):
DA ¼ ð32p3
3ÞðnfiNA
hcÞIm hY ij~meljY f i � hY f j~mmagjY ii� � ð3Þ
where ~mel and ~mmag are the electric and magnetic transition-dipole moment operators, respectively, and jY ii and jY f i arethe total wave functions for the initial and final states. The dotproduct of the two transition moments in Equation (3) is calledthe rotational strength, Rif, being thus defined as the imaginarypart of the dot product between the electric and magnetictransition-dipole moments [Eq. (4)]:
Rif ¼ Im hY ij~meljY f i � hY f j~mmagjY ii� �
ð4Þ
The differential absorption of LCP and RCP infrared light istherefore proportional to the rotational strength. For totalwave functions jY ii and jY f i that are both real, Rif is a realquantity since ~mmag is a purely imaginary operator. Hence, toevaluate the optical activity of a molecular system, one mustinclude the magnetic interaction to describe nonzero VCD in-tensities, since Rif has its origin in the interference betweenelectric and magnetic dipole transitions. Moreover, based onsymmetry considerations, it can be shown that two enantio-mers have rotational strengths of equal magnitude but oppo-site signs, the latter being determined by the angle betweenthe electric and magnetic transition-dipole moments.
Figure 1. Classical picture of circular dichroism. The conducting helices rep-resent the chiral molecules.
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2.3. Prediction of the Rotational Strengths
Theoretical prediction of rotational strengths for infrared tran-sitions in chiral molecules requires the calculation of the elec-tric and magnetic transition-dipole moments associated withthe operators ~mel and ~mmag. For a transition between two vibra-tional states (g!e) of a non-degenerate electronic state G, theelectric and magnetic transition-dipole moments of a molecularsystem are given in the Born–Oppenheimer approximation by[Eqs. (5) and (6)]:
hYGgj~meljYGei¼ hcGgjhyGj~meljyGijcGei¼ hcGgjhyGj~me
eljyGi þ~mneljcGei
ð5Þ
hYGgj~mmagjYGei¼ hcGgjhyGj~mmagjyGijcGei¼ hcGgjhyGj~me
magjyGi þ~mnmagjcGei
ð6Þ
where jyGi is the electronic ground-state wave function, whichhas the electronic coordinates as variable and depends para-metrically on the nuclear coordinates, and jcGki is the nuclearwave function of the kth vibrational level in electronic state G.
Both nuclear and electronic components of the electric tran-sition-dipole moment [Eq. (5)] can be calculated using Born-Oppenheimer (BO) wave functions. The expressions for the in-dividual terms in Equation (5) are well understood and a com-plete derivation can be found elsewhere.[1] However, the elec-tronic contribution to the magnetic transition-dipole moment[Eq. (6)] is identically zero within the BO approximation.[9] Thisfollows from the hermitian and imaginary nature of the ~me
mag
operator together with the non-degeneracy of G.[8] Hence,hyGj~me
magjyGi ¼ 0, leading toa scenario where only the nucleiwould contribute to the magnet-ic transition-dipole moment.Hence, to evaluate the contribu-tion of the electrons to the mag-netic transition-dipole moment,an expansion of the theorybeyond the BO approximation isrequired.
2.3.1. A Vibronic CouplingMechanism
To obtain more accurate wavefunctions, we employ first-orderperturbation theory using theterms of the nuclear kineticenergy operator that are ne-glected in the BO approximationas the perturbation Hamiltonianand the BO wave functions aszero-order expansion functions.With these corrected wave func-tions, a nonzero electronic con-tribution to the magnetic transi-
tion-dipole moment associated with the transition YGg ! YGe
is obtained, which is given by Equation (7):[10]
hYcorGg j~mel
magjYcorGe i ¼
hcGgjXK 6¼G
hyGj~melmagjyKi
W0K �W0
G
ðhyKjT ð1Þn jyGi � hyGjT ð1Þn jyKiÞjcGeið7Þ
where W0K is the electronic energy for the Kth state and T ð1Þn is
a nuclear kinetic energy operator. The vibronically inducedmixing of BO wave functions expressed in Equation (7) is theessence of the mechanism through which VCD signals gain in-tensity. It shows that electronic magnetic transition-dipolemoment can be “borrowed” from electronic transitions due tomixing of BO states. In terms of the classical picture of Sec-tion 2.1, a larger current is generated in the coil, since the vi-brating nuclei induce a change in the electronic wave function.Equation (7) also shows that this electronic contribution de-pends strongly on the excitation energies of states from whichmagnetic transition dipole moment is borrowed. In fact, thetheory as expressed in Equation (7) leads one to expect that insystems with low-lying electronically excited states an en-hancement of VCD signal intensities might occur comparedwith analogous systems in which these low-lying electronicstates are absent.
2.4. Quantum-Chemical VCD Calculations
Although formally correct, Equation (7) has considerable draw-backs for practical purposes, as it can only be evaluated if thesum over the electronically excited states is truncated at some
Figure 2. Left : The IR absorption (upper curves) and circular dichroism spectra (lower curves) of [D3]chloroformsolutions of [Co(l-sp)Cl2] (full curves), [Ni(l-sp)Cl2] (dashed curves), and [Zn(l-sp)Cl2] (dash-dot curves). The absorp-tion spectrum of [Zn(l-sp)Cl2] is distinct from those of the CoII and NiII analogues, which are identical on the scalepresented, only at the band edges outside the C�H stretching vibration region. Reproduced by permission fromRef. [13] . Copyright 1980 of Elsevier. Right: Observed VCD spectra of Zn(sp)Cl2, Co(sp)Cl2, and Ni(sp)Cl2 in the3600–950 cm�1 region, 0.1 mm path length cell, 5 h collection for the sample and solvent, with the instrument op-timized at 3000 cm�1. The asterisk indicates an artifact arising from atmospheric CO2. Reproduced by permissionfrom Ref. [17] . Copyright 2001 of the American Chemical Society.
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point. This problem was tackled successfully by P. J. Ste-phens[11] who has shown that the sum over states expressioncan be rewritten to an expression involving the derivative ofthe ground state wave function with respect to nuclear dis-placement and the derivative of the ground state wave func-tion with respect to a magnetic field perturbation. This mag-netic field perturbation (MFP) theory is the theory that hasbeen implemented in Gaussian and ADF quantum chemicalpackages for the calculation of VCD intensities.
In deriving Equation (7) and the MFP theory it is assumedthat electronic wave functions vary slowly with nuclear dis-placements from the equilibrium geometry. This is reasonablefor closed-shell organic molecules with non-degenerateground states that are well-separated from the lower electroni-cally excited states. However, if we consider metal-organic sys-tems with open-shell configurations that can give rise to manylow-lying electronically excited states, these approximationsmay very well break down. Also, in both cases it is assumedthat vibronic energies (that is, electronic plus vibrational ener-gies) can be replaced by elec-tronic excitation energies. Forsystems in which the lower elec-tronically excited states have en-ergies comparable to vibrationalenergies, this is clearly not thecase. Equation (7) thus providesonly a qualitative assessment ofthe role of the various electronicexcited states, but a correct de-scription would require an ex-tension of the theory includingcorrection terms that accountfor such level of vibronic detail,as has been derived by Nafie.[12]
An implementation of theseequations in quantum-chemicalprograms is presently inprogress.
3. First Observations ofEnhanced VCD
In 1980, Barnett et al.[13] reportedthe VCD spectra of complexesconsisting of a transition-metalion and the chiral chelatingligand (�)-sparteine in the C�Hstretching range (Figure 2, left).Comparison of the VCD spectrafor the ZnII, CoII and NiII com-plexes reveals small VCD peakintensities for the zinc complex,but extraordinarily large signalsfor the cobalt and nickel com-plexes. The explanation of largeVCD intensities was tentativelyformulated in terms of a Fano-
type coupling mechanism,[14] based on the overlap of vibra-tional transitions with the d-d electronic transitions of CoII andNiII. In 1992, a similar VCD enhancement was observed by Bor-mett et al.[15] for the antisymmetric stretch of azide-ligated site-directed mutant hemoglobins and myoglobins (see Figure 3).In Figure 3 we show IR and VCD spectra in the N3 stretchingregion for a series of heme protein complexes. The bisignateIR absorption is due to both the ionically bound high-spinazide and the covalently bound low-spin azide with differencesin intensities reflecting the differences in the spin state equilib-rium between the various proteins. The VCD intensity of the N3
stretching mode varies depending on heme and bound azideligand interactions with the distal heme pocket. The large VCDsignals observed for some complexes suggests that the sourceof the enhanced VCD could be due to a vibrationally inducedcurrent by the delocalized electrons in the heme plane, but inthe absence of further experimental support this explanationremains tentative. This study was followed by a report thatdemonstrated similar effects in non-heme metalloenzymes.[16]
Figure 3. IR absorption (8 cm�1 resolution) and VCD (6 cm�1 resolution) spectra of : A) 11.5 mm horse MbN3, and7.1 mm mutant (Asn E11) human MbN3, with 2.7 mm unbound N3
� ; B) 6.0 mm elephant MbN3 ; C) 8.0 mm humanHbN3; D) 6.0 mm CTT III HbN3, pH 7 with 0.01 m phosphate buffer adjusted to an ionic strength of 0.7 with KCl. Allspectra were measured in a 26-mm CaF2 cell, and contributions from uncomplexed azide were numerically re-moved. Reproduced by permission from Ref. [15] . Copyright 1992 of the American Chemical Society.
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In 2001, Nafie and co-workers[17] accurately reproduced theresults of Barnett et al. in the C�H stretching region (Figure 2,right) of the sparteine complexes but at the same time extend-ed their investigation studies to the 6 mm region, where theyalso observed large signal enhancements for fingerprint vibra-tional transitions. Only then a full explanation for the VCD en-hancement in terms of low-lying electronically excited stateswas provided and the pertaining theoretical expressions forthe VCD intensities were developed. Since then, Johannessenand Thulstrup,[18] Sato et al.[19] and Merten et al.[20] have report-ed similar VCD amplification effects in chelating chiral ligandscoordinated to transition metals, and rationalized them in partby invoking vibronic coupling with low-lying electronically ex-cited states. As an example, Figure 4 shows the IR and VCDspectra of the spin-triplet bis(biuretate) cobalt(III) complex re-ported by Johannessen and Thulstrup.[18] Comparison of the IRand VCD spectra in the 6 mm (Figure 4, upper panel) and 3 mm(Figure 4, lower panel) regions for both the free ligand and thecobalt complex reveals a clear enhancement of the intensitiesin the VCD spectrum of the complex. In the 6 mm region anoverall amplification is observed, while in the 3 mm regiona broad electronic absorption band is observed, which is as-signed to an electric dipole-forbidden, but magnetic dipole-al-lowed, low-lying d-d transition. Here, the cobalt(III) complexexhibits a square planar coordination geometry with an open-shell electronic configuration and low-lying electronically excit-ed states. This result further suggested that the enhancementof VCD signals caused by coupling of the vibrational transitionsto electronic transitions to low-lying electronically excitedstates.
4. On the Manipulation of the ElectronicManifold and the Amplification of VCD
Previously reported cases of enhanced VCD intensity were ob-served for molecular systems with rather exotic molecules andrigid molecular coordination spheres of chiral chelating ligandsbound to open-shell transition-metal ions. In the following sec-tions, we will show that the amplification of VCD is not limitedto such systems, and more importantly, that one can induceamplification of VCD intensities by manipulating the electronicstructure of the molecules in a controlled manner, such thatlow-lying electronic states are “created” that are prone to leadto strong vibronic coupling and thereby to an amplified VCDresponse.
4.1. Electrochemically Generated Radical Anions
Aromatic compounds undergo one-electron reduction if anelectron is transferred to an unoccupied p* orbital. The result-ing radical anions have an open-shell electronic configurationwith lower-lying electronically excited states compared withthat of their neutral counterparts. Based on this idea, we havedeveloped a novel approach to amplify VCD signals by modu-lating the energies of the excited-state manifold in a controlledmanner using spectroelectrochemistry.[21] For this purpose, anoptically transparent thin-layer electrochemical cell (OTTLE)
was designed and constructed to perform spectroelectrochem-ical-VCD measurements using a commercially available VCDspectrometer. The technical details of the experimental setupcan be found elsewhere.[22]
To investigate the effect of electrochemical reduction on theVCD response, compounds (S)- and (R)-methyl 2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propanoate ((S)-1 and (R)-1 re-spectively, see Figure 5) were investigated in both the neutraland radical anion forms, using UV/Vis, IR and VCD spectrosco-py. As expected, the UV/Vis absorption spectrum (Figure 6) ofthe radical anion shows absorption bands at much longerwavelengths than the corresponding spectrum of the neutral
Figure 4. Upper scheme, left : Molecular structure of the (6S,7S)-1,3,5,8,10,12-hexaaza- 2,4,9,11-tetraoxo-6,7-diphenyl-dodecanato(4-)cobaltate(III) anion.Right: The free ligand, stabilized by intramolecular hydrogen bonding. IR ab-sorption and VCD spectra of the coordination compound (c) and freeligand (a) in the 6 mm region (middle panel), and in the 3 mm region(lower panel). Adapted with permission from Ref. [18] . Copyright 2007 ofthe Royal Society of Chemistry.
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compound. The observed electronic structure was confirmedby time-dependent density-functional theory calculations.[23]
The IR and VCD spectra of the two forms are displayed inFigure 7. A peak to peak comparison of the VCD peak intensi-ties for neutral and radical-anion forms of 1 shows up to oneorder of magnitude VCD signal enhancements for the latterform. Interestingly, one has to conclude that these signal en-hancements are not the same for all bands. For the C=Ostretching modes of 1 a tenfold amplification is found, whereasfor the ring and methyl-ester modes the amplification is smallor negligible. These differences are directly related to the in-duced mixing of electronically excited states with the vibra-tional manifold of the molecules and thus provide valuable in-formation on the finer details of the influence of the lower-lying electronic states. In fact, time-dependent density-func-tional theory (TD-DFT) calculations predict that it is especiallythe D2 state that is expected to be involved in lending themagnetic transition dipole moment to vibrational transitions.Electron difference density plots between D0 and D2 show thatmajor changes occur in the electron density on the carbonylgroups and much less on other parts of the molecule. Onetherefore indeed expects amplification for the C=O stretchingmodes and much less for other modes.
The same calculations allow us to make a quantitative pre-diction of the amplification factor using the electronic energygap between the ground and lower electronically excitedstates for the neutral and radical-anion species and the magni-
tude of the magnetic transition-dipole moment for transitionsbetween the ground and electronically excited states [seeEq. (7)] . Using these quantities, the expected amplification ofthe VCD signals is predicted to be approximately one order ofmagnitude, which nicely reproduces the observed VCD en-hancements and provides further confirmation that the VCDenhancement is due to vibronically induced mixing of low-lying electronically excited states with the ground state.
4.2. Biomolecules Surrounding Paramagnetic Metal Ions
In the previous section, electrochemistry has been used tochange the electronic manifold of the compound of interestand optimize it for enhancing VCD signal intensities. Bearing inmind that key to this enhancement is the presence of low-lying electronically excited states, one could very well imagine
Figure 5. Chemical structure of compounds (R)-1 and (S)-1. Reproduced bypermission from Ref. [23] . Copyright 2012 of the Royal Society of Chemistry.
Figure 6. Optical absorption spectrum of (S)-1 in the neutral (blue) and radi-cal anion (red) forms. Reproduced by permission from Ref. [23] . Copyright2012 of the Royal Society of Chemistry.
Figure 7. A) Thin-layer cyclic voltammogram of (�)-1 obtained using the IROTTLE cell.[21] B) Potential-dependent steady-state IR spectra of 35 mm (�)-1(optical path = 200 mm). The blue and red curves represent the spectra ofthe initial (neutral) and final state (radical anion), respectively. C) VCD spectraof 7 mm (�)-1 (solid-line) and 7 mm (+)-1 (dashed-line) (opticalpath = 1.2 mm) for the neutral (blue) and radical anion (red) species. Forclarity, the spectra for the radical anion have been offset vertically. Repro-duced by permission from Ref. [23]. Copyright 2012 of the Royal Society ofChemistry.
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that such states can also be in-troduced by coupling the mole-cule of interest—which by itselfmight not have such states—toan auxilliary that does possesslow-lying electronically excitedstates. We have demonstratedthe effectiveness of such an ap-proach in experiments on aminoacids and peptides under biolog-ical conditions[24] for which nor-mally only weak VCD signals areobserved. To provide suitableconditions for VCD enhance-ment, the electronic manifoldwas altered by actively bindingamino acids and peptides toa paramagnetic metal ion (CoII).Interestingly, the thereby createdsituation closely resembles thatof a binding pocket of a proteinin which ligands are coordinatedaround the metal. These studiesshowed gigantic signal enhance-ments (up to two orders of mag-nitude) in the VCD spectra ofvarious amino acids such as alanine, proline and valine(Figure 8), and an approximately tenfold amplification for di-and tripeptides (Figure 9). As an example, we show in Figure 8the infrared absorption (A, upper panel) and VCD (A, lowerpanel) spectra of a [CoII(Pro)2(D2O)2] (Pro = proline) complex(colored lines) and of the uncomplexed proline molecule(black lines). The IR spectra confirm complexation of prolinewith CoII since a splitting is observed for the carboxylatestretching mode as a consequence of exciton coupling be-tween pairs of prolines in the complex. Of special interest inFigure 8 A is the inset which shows sharp vibrational bands,but also a much broader band spanning several hundreds ofwavenumbers. This band can be assigned to magnetic-dipoleallowed d-d transitions of CoII in agreement with previous ex-periments.[17, 18] Its observation strongly suggests that vibroniccoupling plays a dominant role in VCD amplification, but byitself does not yet provide conclusive evidence for it. Definiteproof is provided by the VCD spectra of proline–cobalt com-plexes with the metal center in its diamagnetic CoIII state. Inthis state, the electronically excited states of Co are at muchhigher excitation energies, and one would therefore expectsignificantly smaller VCD signals. This expectation is nicelyborne out in experiments (Figure 8 B) that show a completeabsence of VCD amplification under these conditions.
4.2.1. Mode-Selective VCD Enhancement
Many biomolecules, such as metalloproteins, contain transi-tion-metal ions as part of their active site. The spatial structureof these metal binding pockets is often directly related to thebiological functionality of the system; having access to this
structure is thus of primary importance to understand thisfunctionality. Interestingly, it turns out that the intensity andthe shape of the amplified VCD signals in combination withthe spectral information contained in the IR spectra is a power-ful means to retrieve the coordination geometries of polypep-tides with metal ions.[24] To illustrate this, we show in Figure 9IR absorption and VCD spectra of bare di- and tripeptides(dashed-black) and of the same peptides bound to the metalion auxiliary (full-colored). Comparison of the signal intensitiesof the bands in the VCD spectra show: 1) that VCD bands arestrongly amplified for the CoII-coordinated peptides and 2) thatthis amplification is strongly mode-dependent. We observe, forexample, for the dipeptide that the carboxylate mode at1580 cm�1 is not amplified at all in the VCD spectrum, whilethe amide I mode—which shifts from 1650 to 1610 cm�1 uponcomplexation—is amplified by more than an order of magni-tude. These observations demonstrate that binding of the di-peptide does not involve the carboxylate group and occurs insuch a way that the carbonyl stretch directly affects the elec-tronic manifold of the Co ion. The only binding configurationthat can be reconciled with these requirements is the one de-picted next to Figures 9 a and b. The IR and VCD spectra of tri-peptides (see Figure 9) show that the spatial resolving powerof this approach goes even further. From the IR spectra (seeFigure 9 c) it can be concluded that the two amide moieties ofthe tripeptide do not interact equally with the CoII center:upon coordination, the amide I band 1 is shifted by 40 cm�1 tothe red (band 1’), while the amide I band 2’ is only shifted by3 cm�1 from its non-complexed counterpart band 2. Apartfrom different frequency shifts, the two bands also exhibita very different amplification of their intensity in the VCD spec-
Figure 8. A) IR absorption and VCD spectra of l-proline (a,b) in D2O (black dashed lines) and of the[CoII(Pro)2(D2O)2] complex (solid filled lines). The sample was prepared in D2O (c�25 mm). The VCD spectra of theamino acid and of the complex were averaged with 4320 scans (1 hour) at a resolution of 4 cm�1. The VCD spectraof the amino acid (dashed lines in b) have been scaled for better comparison with the enhanced VCD spectrumof the complex. The inset b* displays an extension up to 2100 cm�1 of the spectrum depicted in b. B) Experimen-tal VCD spectra of [CoII(Pro)2(D2O)2] (in blue) and [CoIII(Pro)2(D2O)2] (in orange) complexes. C) Molecular structure of[CoII(Pro)2(D2O)2] . Reproduced by permission from Ref. [24] . Copyright 2014 of the American Chemical Society.
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trum. This observation indicates that the electronic manifold ofthe CoII ion is much more susceptible to the carbonyl stretchinvolved in modes 1 and 1’ than the one associated withmodes 2 and 2’. This, in turn, leads to the conclusion that thetripeptide is bound to the CoII ion in the configuration shownin Figure 9.
4.3. Probing Local Structure: Cutting through a CongestedVCD Spectrum
A detailed understanding of the relation between molecularstructure and functionality requires the ability to zoom in onspecific parts of a molecular system. The selective enhance-ment of VCD signals in the vicinity of metal ions discussed inthe previous section seems well suited for this purpose, butfor larger molecular systems, its usefulness rapidly deterioratesbecause all parts of the molecule contribute to the VCD spec-trum. As a result, very congested spectra are obtained fromwhich it is hard—if not impossible—to extract information onamplified modes. To address this problem, we have deviseda novel methodology based on the concept of a switchablelocal VCD amplifier.[25] The amplifier is in this case a molecularentity that can be covalently coupled to a user-defined part of
a molecule and that can beswitched on and off electro-chemically using the VCD-OTTLEcell. The first experimental dem-onstration of this methodologyhas been performed using ferro-cene (Fc) as the VCD amplifier. Inthe neutral form (FeII), ferrocenehas a closed-shell electronic con-figuration with electronically ex-cited states well separated fromthe electronic ground state. Theone-electron-oxidized ferroceni-um cation, in contrast, has anopen-shell configuration (FeIII)with low-lying electronically ex-cited states. Adjusting the elec-trochemical potential thusallows alternation between so-called ON and OFF configura-tions: the ON configuration gen-erates an electronic manifoldwith low-energy electronicallyexcited states that “activate”VCD signal amplification, where-as the OFF configuration turnsoff the amplification by return-ing the electronic manifold to itsoriginal configuration withoutlow-lying electronically excitedstates. Subtraction(DaON � DaOFF) of VCD spectranow directly isolates the normalmodes which undergo VCD am-
plification, and eliminates signals associated with regions out-side the spatial amplification range that would otherwise over-whelm the VCD spectrum. The validity of this concept hasbeen demonstrated on di- and tripeptides. Figure 10 (left) andFigure 11 (left) show infrared absorption and VCD spectra ofthe di- and tripeptides, respectively, in the OFF (solid lines) andON (dashed lines) configurations. An overall amplification inthe ON configuration is perceptible through comparison of itsVCD intensities with those recorded for the OFF configuration.Moreover, modes that fall outside the spatial range over whichthe amplifier is active are eliminated in the difference spec-trum.
4.3.1. Distance Dependence of the Amplification Effect
The studies described above clearly indicate that the amplifica-tion of VCD signals is strongly dependent on the distance fromthe amplifying entity. To quantify this distance dependence—and thereby determine an effective amplification range—wehave measured amplification factors of localised modes thatare increasingly further away from the VCD amplifier. The AAdipeptide shown in Figure 10 features two amide I vibrationalmodes (Ala1 and Ala2) and one methyl ester C=O stretching
Figure 9. IR absorption (a) and VCD (b) spectra of l-Val-Val (black lines) and [CoII(l-Val-Val)(D2O)2] (green lines) andIR absorption (c) and VCD (d) spectra of l-Val-Val-Val (black lines) and [CoII(l-Val-Val-Val)(D2O)] (red lines). The num-bered IR bands in panels (a) and (c) correspond to the VCD bands in panels (b) and (d), respectively. The num-bered moieties in the molecular structures of the CoII bound valine dipeptide (high-spin) and CoII bound valine tri-peptide (high-spin) correspond to the numbered peaks in the VCD spectra. Reproduced by permission fromRef. [24] . Copyright 2014 of the American Chemical Society.
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mode (Ala3) whereas the APA tri-peptide shown in Figure 11 ex-hibits three amide I vibrationalmodes (Ala1, Pro2 and Ala3) andone methyl ester C=O stretchingmode (Ala4). The subscripts inthese designations correspondto the labeled groups and thecorresponding normal modes inthe IR and VCD spectra, andhave been chosen in order ofthe increasing distance from theamplifier. All modes have well-separated frequencies, whichallows for a better comparisonof signal intensities and amplifi-cation factors. A direct measureof the amplification factor foreach vibrational mode can beobtained by determining theanisotropy factor (g ¼ De=e) foreach individual normal mode inthe ON and OFF configurations.In Figure 12 the amplificationfactor, defined as g’/g (g!OFF,g’!ON), is plotted as a functionof the distance of the normalmode to the amplifier. A fit tothe data points by using thefunction 1þ Ae�x=R0 , where x isthe distance (in number ofbonds) between the chemicalgroup and the amplifier, showsthat the amplification is reducedby a factor of 1/e for normalmodes at a characteristic dis-tance, R0, of 2.0�0.3 bonds fromthe amplifier. In a simplified pic-ture all parts of the moleculethat are located further than twocovalent bonds away from theamplifier will thus not undergoany meaningful VCD-intensityamplification. Put in anotherway, one might say that theswitchable VCD amplifier allowsus to zoom in on a region inspace equivalent to a spherearound the amplifier witha radius of two bond lengths Al-though this result might vary de-pending on the type of bond orits spatial orientation, this resultclearly demonstrates the poten-tial of amplified VCD as a probeof local structure.
Figure 10. Left: IR (A) and VCD spectra (B) of 10�2 M Fc-(l/d)-Ala-Ala-Ester in CD3CN (10�1 M Bu4NPF6, 200 mm opti-cal pathlength). The VCD spectra of the ON configuration have been offset for clarity. Panel C displays the differ-ence spectrum of the VCD spectra in the ON and OFF configurations. Right: Molecular structure of Fc-(l)-Ala-Ala.A schematic ruler is plotted next to the peptide backbone to highlight the distance between the ferrocenemoiety, and the amide and ester groups. Reproduced by permission from Ref. [25] . Copyright 2014 of Wiley-VCH.
Figure 11. Left : IR (A,C) and VCD spectra (B,D) of 10�2 M Fc-(l)-Ala-Pro-Ala in CD3CN (10�1 M Bu4NPF6, 200 mm opti-cal pathlength), for the OFF and ON configurations, respectively. Panel E displays the difference spectrum of theVCD spectra in the ON and OFF configurations. Right: Molecular structure of Fc-(l)-Ala-Pro-Ala. A schematic ruleris plotted next to the peptide backbone to highlight the distance between the ferrocene moiety, and the amideand ester groups. Reproduced by permission from Ref. [25] . Copyright 2014 of Wiley-VCH.
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5. Outlook
Vibrational circular dichroism has come of age since its first ex-perimental observation in 1973. In particular, it has becomeclear that VCD has tremendous potential as a spectroscopictool in the investigation of molecular stereochemistry in gener-al, and of chirality in biomolecular systems in particular. Never-theless, it has also become clear that there is still much togain, primary targets in this respect being the ability to over-come the intrinsic small-signal limitations of VCD and the abili-ty to zoom in on user-defined regions of large molecular sys-tems. This is an ambitious goal, but one that nowadays iscoming within reach, as demonstrated by the studies on vi-bronically induced amplificationof VCD signals that we have de-scribed in this Concept article.These studies have shown thatas far as signal intensities areconcerned it is possible to ma-nipulate electronic manifolds insuch a way that circular dichro-ism associated with nuclearmotion is essentially coupled toelectronic circular dichroism,thereby putting VCD and ECDon equal footing. One aspectthat we have not been touchedupon so far is the comparisonbetween experimentally record-ed and theoretically predictedVCD spectra, which traditionallyhas been the stronghold for ex-
tracting the maximum amount of structural information possi-ble from experimental VCD spectra. VCD studies by us andothers show that the MFP-based theoretical description de-rived by Stephens, which is currently implemented in quantumchemical programs, does not suffice. In fact, these studiesclearly indicate that the stronger the influence of low-lyingelectronically excited states, the larger the differences betweenexperimental and theoretical spectra. To correctly describe VCDspectra under low-lying excited-state conditions, a different ap-proach needs to be adopted in which the theory is extendedas to include vibronic detail. The appropriate equations haverecently been derived.[12] Once these equations are implement-ed—and this is currently underway—it will be possible to useVCD signal amplification not only in a qualitative way, but alsoto derive a quantitative structural information, thereby open-ing a new research territory. The concept of a local VCD ampli-fier offers a plethora of possibilities to study binding pocketsand active sites of proteins and enzymes. Our initial studies onmodel systems indicate that implementation of such an ampli-fier by coupling it to user-defined location within a moleculeshould provide unprecedented structure elucidation at thelocal scale. As a perspective for the future, Figure 13 showsa schematic outlook for the application of locally amplifiedVCD for the investigation of site-specific chiral molecular tar-gets. The highlighted region of the molecule contains an elec-troactive group which is embedded within the molecule. Sub-tracting the VCD spectra in the ON and OFF states of the am-plifier then gives rise to signals that are exclusively associatedwith oscillators in the vicinity of the electroactive group, andeffectively enables us to turn VCD into a zero-backgroundtechnique (subtraction of the ON and OFF VCD spectra leadsto null signals for spatial regions not connected to the switch).Local VCD amplification thus paves the way towards a uniquemanner of spectrally resolving protein local structure in solu-tion.
In many aspects, the concept of a switchable local amplifierresembles that of a molecular beacon: a probe that can beplaced in a key location within the molecule, and be externally
Figure 12. VCD amplification factors (g’/g) as a function of the distance(number of covalent bonds) from the electroactive group to each of the in-dicated functional groups of Fc-(l)- Ala-Ala and Fc-(l)-Ala-Pro-Ala. Repro-duced with permission from Ref. [25] . Copyright 2014 of Wiley-VCH.
Figure 13. Schematic figure demonstrating amplified VCD as a zero-background technique using ON/OFF subtrac-tion to zoom in into specific regions of large biomolecular systems. This protein, catalytic antibody Fab 1345 (PDB1A3L), contains a ferrocene moiety in its active site.
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controlled to illuminate that specific part of the molecule andits surroundings. It should be emphasized that in principle anymolecular entity with a suitable electronic configuration canbe used as an auxiliary, thereby generating a local environmentwhere the necessary conditions for VCD amplification are atwork. Switchable amplifiers thus do not necessarily need to beelectroactive entities, but can also be based on other types ofswitches. Our findings regarding the localized nature of theVCD enhancement greatly extend the possible applications ofVCD to locally elucidate the chiral structure of functional bio-molecules under biologically relevant conditions which are in-accessible with other techniques under similar conditions.
Acknowledgements
The authors thank Professors Bas de Bruin and Larry Nafie fortheir insights and helpful discussions and Bert H. Bakker, MichielF. Hilbers, Hans J. Sanders, Henk Luyten, and Fred van Anrooij forthe synthetic work and technical support. S.R.D. acknowledges fi-nancial support from the Portuguese Foundation for Science andTechnology (FCT) under the fellowship SFRH/BD/48295/2008. S.W.acknowledges the European Research Council (ERC) for fundingthrough Grant No. 210999. F.H. thanks the University of Readingfor the support of the Reading Spectroelectrochemistry laboratory(Project D14-015).
Keywords: biomolecules · chirality · circular dichroism · localamplification · structure elucidation
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Manuscript received: July 12, 2015Final Article published: && &&, 0000
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S. R. Domingos, F. Hartl,* W. J. Buma,*S. Woutersen*
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Elucidating the Structure of ChiralMolecules by using AmplifiedVibrational Circular Dichroism: FromTheory to Experimental Realization
Zooming in on chirality is now possibleby controlled manipulation of the “elec-tronics” of a chiral molecule and obser-vation of its locally enhanced vibrationalcircular dichroism (VCD). We review thetheoretical foundations of amplifiedVCD and discuss its first experimentalobservations, with a prospective viewon future applications.
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