l-Stercobilin-HCl and d-Urobilin-HCl. Analysis of Their Chiroptical and

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l-Stercobilin-HCl and d-Urobilin-HCl. Analysis of Their Chiroptical and Conformational Properties by VCD, ECD, CPL Experiments and MD, DFT Calculations Simone Ghidinelli, Sergio Abbate, Stefan E Boiadjiev, David A. Lightner, and Giovanna Longhi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b07954 • Publication Date (Web): 29 Nov 2018 Downloaded from http://pubs.acs.org on November 30, 2018

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The Journal of Physical Chemistry

l-Stercobilin-HCl and d-Urobilin-HCl. Analysis of their Chiroptical and Conformational Properties by VCD, ECD, CPL Experiments and MD, DFT Calculations § Simone Ghidinelli1, Sergio Abbate1, Stefan E. Boiadjiev2, David A. Lightner3, Giovanna Longhi*1 1Dipartimento

di Medicina Molecolare e Traslazionale, Università di Brescia, Viale Europa 11, 25123 Brescia, Italy

2Department

of Chemistry and Biochemistry, Medical University-Pleven, 1 St. Kl. Ohridski Str., 5800 Pleven, Bulgaria

3Department

of Chemistry, University of Nevada, 1664 North Virginia Street, Reno, 89557-0020, Nevada, USA

Corresponding Author *Giovanna Longhi: [email protected] § Work

partially presented at VOA-6 International Conference, Brescia, September 2018.

Abstract Vibrational circular dichroism (VCD) and IR spectra of dichloromethane solutions of lstercobilin and d-urobilin hydrochlorides have been recorded in the mid-IR region. The spectra are best interpreted by combining molecular dynamics (MD) calculations and DFT calculations within the QM/MM ONIOM-type framework, and the combined predicted results are better and more informative than the more standard analysis provided by DFT calculations. The same approach also sheds light on the Cotton effect sign inversion of room temperature versus low temperature electronic CD (ECD) spectra of the same compounds in methanol-glycerol solution. Finally, circularly polarized luminescence (CPL) spectra for lstercobilin in chloroform solution provide information on the excited state geometry of this molecule. Introduction Bile pigments have a long and fascinating history in chemistry and medicine1. Characterized by their green to yellow to red colors and found in bile, urine and feces, high levels of such pigments in human metabolism have been diagnostic for metabolic disorders such as primary biliary cirrhosis and viral hepatitis. In this work we focus on two of these pigments, durobilin2,3 and l-stercobilin3,4 [Fig. 1] by measuring and analyzing their VCD (vibrational circular dichroism) and CPL (circularly polarized luminescence) spectra, reinvestigating their ECD (electronic CD),5 and correlating the spectra to conformations defined through MD 1

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(molecular dynamics) and DFT (density functional theory) calculations. These bright orangered (UV-vis max approx. 490 nm) substances are found (especially) in pathologic urine (hence the name urobilin)2,6,7 and feces (hence stercobilin)4,8,9 following microfloral reduction in situ of transhepatic bilirubin,10-14 the yellow pigment of mammalian bile and the end product of heme metabolism13,15,16 [Fig. 1]. First detected in 1869-71 6,8 by their intense green fluorescence in the presence of Zn2+, the crystalline pigments were not isolated until six to seven decades later, and typically as their hydrochloride salts.2,4,17

Figure 1 Schematic representation of heme catabolism. At that time the constitutional structures of bilirubin1,16 and the urobilinoid pigments1,3 had been largely elucidated, leaving but a few important aspects remaining to be resolved. Such as i) favoring the end ring lactam tautomer,1,3 rather than the often represented lactim2,4,10-15,17 promoted by Hans Fischer17; ii) elucidating the exocyclic carbon-carbon double bond Z/E stereochemistry in favor of Z1,16,18 as opposed to the more sterically crowded E1,3,14,16 or 2


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noncommittal;2,4,10,13,17 and iii) learning the R/S stereochemistry at the stereogenic centers, especially at C-4 and C-165 [Fig. 1] of both d-urobilin and l-stercobilin - as well as the remaining stereochemistry in the pyrrolidone rings of the latter.

The last was solved

following chromic acid oxidation of l-stercobilin to afford trans-(+)-(2R)-methyl-(3R)ethylsuccinimide,19 which indicated the (2R, 3R, 17R, 18R) configuration in the pigment’s end rings. By correlating that as trans to the pigment’s stereochemistry at C-4 and C-16 by NMR,19 the latter was assigned (4S, 16S). However, long before the absolute configurations of the bile pigments were known, their observed intense molecular rotations at 589 nm begged explanation.2,20 When asked in 1964 by Cecil J. Watson (Prof. Med. at the University of Minnesota) to explain bile pigment specific optical rotations of the order of + or - 4000 [deg.cm3.dm-1.g-1] at 589 nm in CHCl3, Albert Moscowitz (Prof. Chem.) also at the Univ. Minnesota and the leading authority on the theory of optical activity, recognized immediately that the large rotations could not be reconciled by an inherently symmetric chromophore perturbed by dissymmetric vicinal action, i.e. a planar dipyrrylmethene (dipyrrin) perturbed by neighboring stereogenic centers. Rather, the large rotations pointed to an inherently dissymmetric chromophore21,22 as twisted dipyrrin cores of opposite helicity in d-urobilin and lstercobilin.5,23 In each pigment, as Moscowitz explained,23 the helicity was preserved by intramolecular hydrogen bonding between the lactam rings and the dipyrrin core, with the sense of helicity being determined by the stereochemistry at C-4 and C-16, i.e. the (4S, 16S) configuration favors a P-helical dipyrrin chromophore,21,23,24 as seen in space-filled molecular models.3,24 However, at that time there was no firm basis for relating the sign of the pigment’s Cotton effect (CE) to its absolute helicity, e.g. that a (+) CE might correspond to a P-helicity dipyrrin - except by computation: as in studies carried out by Lightner and Moscowitz in 1965 in a π-electron SCF-CI calculation on the dipyrrin chromophore and unpublished. Given the suspected importance of H-bonding, Moscowitz suggested to Watson that the intense CEs seen in CHCl3 solvent, as well as the molecular rotation would be greatly diminished by the addition of agents that disrupted the intramolecular hydrogen bonds, such as trichloroacetic acid or methanol. Exactly as was observed.21 We now know,5 thanks to the directed syntheses of Plieninger25 and Gossauer,26 the crystallographic studies by the latter,26 and the NMR-based stereochemical work,19 it can be 3



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concluded that d-urobilin has the (4R, 16R) configuration, a (+) long wavelength CE and the M-helicity of its dipyrrin chromophore5,26 in a solvent that promotes intramolecular hydrogen bonding. Therefore, the observed long wavelength (-) CE of l-stercobilin in CHCl3 is dictated by its (4S, 16S) configuration and corresponds to P-helical dipyrrin. Given that intramolecular hydrogen bonding preserves M or P helical conformations, what, however, is the most stable dipyrrin conformation in the absence of intramolecular hydrogen bonding?24 And why should the ECD CE reverse sign at low temperature in hydroxylic solvents? To the questions of multiple conformations for a single helical sense a possible answer may be elicited from IR and VCD spectra, coupled with calculations representing the molecule conformational space. VCD, which originated in 1974-1976,27,28 has become a handy way of analyzing conformational and configurational aspects of chiral molecules.29-38 Sometimes VCD spectra may be overcrowded with closely spaced bands, from which it is difficult to extract useful information. Generally, however, especially in some important regions of the spectra, the information is quite precise and appropriately sheds light on the conformations of some parts of the molecule. Bilirubin and biliverdin, which are intrinsically achiral, were investigated by VCD either in the presence of chiral complexing agents, such as peptides, cyclodextrin and metals,16,39-41 or as appropriate chemical derivatives where chiral pendant groups had been chemically inserted.42,43 However, to the best of our knowledge, no VCD study exists for the title metabolites l-stercobilin and d-urobilin. Given that computational analysis is an essential aspect of the VCD spectral interpretation, with these molecules one cannot be content with DFT calculations alone because interaction with the solvent may play a non-negligible role. For this reason we report here our results for MD investigations. Previously, bilirubin conformational analysis was conducted by MM (Molecular Mechanics) and DFT-PCM methods.16,44,45 Recently, the MD approach was used for the interpretation/calculations of ECD spectra45-52 and for VCD and ROA (Raman Optical Activity)53-60 spectra in conjunction with DFT calculations. One last chiroptical property presented herein is CPL, which allows one to characterize configuration and conformation of the molecule in the first electronic excited state.61,62

Materials and Methods. 4


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Materials As in previous studies of urobilinoids,3,24,63-65 samples of d-urobilin hydrochloride and lstercobilin hydrochloride were made available by the Dr. Cecil J. Watson laboratory at the University of Minnesota and Northwestern Hospital and provided by Ms. Mary Weimer of the Watson lab. These well-preserved samples correspond to those used for the studies of refs. 24, 63-65. Spectroscopic and chiroptical measurements All the ECD, UV, fluorescence and CPL spectra were measured for 10-5 M solutions in dichloromethane. A Jasco 815SE CD apparatus was employed for UV absorption and ECD measurements; the experimental conditions were: 2 mm quartz cuvette, 200 nm/min scanning speed, 1 scan per measurement. CPL measurements were taken with a home-built apparatus,61,66 excitation radiation (473 nm) was brought to the sample through an optical fiber interfaced with a Jasco 8200 Fluorimeter. The experimental conditions were: 2 mm-fluorescence semi-micro cuvette (2 mm path in the excitation side, 10 mm path in the emission direction), 30 nm/min scanning speed, 10 scans per measurement, 2 sec time constant, 90° geometry. The VCD spectra were taken with a JASCO FVS6000 FTIR spectrometer equipped with a VCD module and MCT detector. 8 mM CD2Cl2 solutions of l-stercobilin and 5 mM solutions of d-urobilin were prepared and 5000 scans at 4 cm-1 resolution were taken in 200 μm BaF2 cells for l-stercobilin and 500 μm BaF2 cells for d-urobilin. Solvent IR and VCD spectra in the same experimental conditions were subtracted. Computational methods a) Molecular dynamics simulations For dichloromethane and methanol solvents we used the parameters described in the work of Fox and Kollman.67 The General Amber Force Field (GAFF)68 force field was used for all simulations, charges have been calculated at HF-6.31G* level following the RESP69 protocol as implemented in the Antechamber module of the AmberTools1670 package. The molecule, treated as cation, was solvated using the tLeap70 module with molecules of CH2Cl2 or

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molecules of methanol and one Cl- counterion was added to neutralize the system. A cubic box of 950 molecules of CH2Cl2 and one of 2900 molecules of methanol was employed. All MD simulations were performed using the GROMACS-5.1.2 package71 under periodic boundary conditions with 2 fs integration time. All bonds containing hydrogen atoms were constrained using the LINCS algorithm.72 A 10 Å cut-off value was adopted for non-bonded interactions, and long-range electrostatic interactions were handled with the PME scheme73. The stochastic velocity rescaling thermostat74 with 0.1 ps time constant and the ParrinelloRahman pressure coupling75 with 2 ps time constant were used. After minimization (steepest descent method for 5000 steps), and equilibration in NVT and NPT ensembles (for 20 ps and 120 ps respectively), the simulation in dichloromethane was performed at 300 K and at 1 bar for 2 µs for l-stercobilin-HCl. Preliminary tests have been conducted considering initial conditions with the chloride ion placed in various positions in the box: within the equilibration phase the ion gets close to the propionic acid groups. Snapshots were extracted every 10 ps from MD simulations and were clustered using gromos algorithm76. Different RMSD cut-off values were tested from 0.02 to 0.1 nm using a 0.01 nm step. Finally, a 0.05 nm RMSD cut-off was chosen. Larger RMSD values do not allow one to differentiate structures with different orientations in a single dihedral angle; smaller RMSD values give raise to multiple clusters with similar structures. The averaged structure and 8 other frames distant enough in simulation time (to average over the contribution of solvent layer) of each cluster were used for ONIOM77 calculations. Further, the simulations in methanol were performed in NPT at 1 bar and different temperatures: 300 K and 185 K, in order to be able to compare with experimental result of ref. 24. Snapshots were extracted every 10 ps from MD simulations and were clustered using gromos algorithm76 with a RMSD cut-off of 0.04 nm taken on the backbone of the molecule.

b) DFT calculations Optimization and frequency calculations were performed at the DFT B3LYP/6-31G** level of theory in the IEF-PCM78 approximation as implemented in the Gaussian0979 program. Dipole and rotational strengths were calculated according to the method defined by Stephens29 to obtain the IR and VCD spectra as a sum of Lorentzian bands, bandwidth 8 cm-1. A 0.985 scaling factor is applied to the PCM-DFT calculated spectra, a 0.975 scaling factor is 6


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applied to the ONIOM calculated spectra. The scaling factor values were chosen to maximize the similarity index as defined in ref 80, 81. Dipole and rotational strengths in the UV-VIS range for the first 40 electronic transitions were calculated by means of TDDFT at the CAM-B3LYP/6-31G** level of theory in the IEF-PCM approximation. The calculated spectra were generated using Gaussian functions with bandwidth of 0.16 eV, as expected82, 83 for long range corrected functionals, 45 nm red shift has been applied to calculated spectra. The most populated conformer was optimized in the ground state and first excited state at CAM-B3LYP/6-31G** level of theory in the IEF-PCM approximation to evaluate fluorescence and CPL spectra.

c) ONIOM calculations The two-layer ONIOM method77, implemented in GAUSSIAN09, was used to optimize the structures obtained by a cluster analysis of MD simulation of l-stercobilin-hydrochloride in dichloromethane. l-Stercobilin, Cl- ion and solvent molecules within a cutoff distance of 6 Å from l-stercobilin were extracted and their relative positions were optimized. l-Stercobilin (durobilin) and Cl- ion were treated at DFT level (B3LYP/6-31G**), the whole system, i.e. molecule, Cl- and solvent layer, was considered at MM level (GAFF force field). After optimization, a frequency calculation in ONIOM method was performed. Calculations were carried out with electronic embedding and polarizable continuum model (PCM) to take care of long-range solvent effects.

Results and Discussion Figure 2 shows the experimental spectra obtained with ECD and VCD spectroscopy.

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Figure 2 - A: top left) UV and ECD spectra of l-stercobilin. B: top right) IR and VCD spectra of l-stercobilin. C: bottom left) UV and ECD spectra of d-urobilin. D: bottom right) IR and VCD spectra of d-urobilin. UV and IR spectra are shown with dashed lines; ECD and VCD spectra are shown with solid lines. Data were obtained in CD2Cl2 solvent. ECD spectra of d-urobilin and l-stercobilin, which we repeated, have oppositely signed CEs, as shown previously in ref. 24. VCD spectroscopy shows that most, even if not all, features of the two molecules invert. Taking a closer look at the VCD spectra, before DFT and ONIOM calculations are presented, it is possible distinguish four main features: 1) In correspondence to the broad and intense IR absorption band at ca. 1690 cm-1, one finds for l-stercobilin two features of opposite sign, the lower frequency one being negative and the higher frequency one being positive. Similar features are observed in reverse order for durobilin. These are followed by a weak positive VCD feature at higher wavenumbers in durobilin, in close correspondence of a IR shoulder of the main absorption band. 2) In correspondence to the strongest and narrow IR feature at ca. 1610 cm-1 one finds a mono-signate negative VCD feature for l-stercobilin and a positive feature for d-urobilin. According to previous studies on peptides31,33 and on bilirubin derivatives39-43, the two regions

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just described are determined by normal modes containing C=O stretching and NH bendings. These two regions could well be important for the study of intramolecular H-bonds. 3) In correspondence to the strong IR band at ca. 1270 cm-1 one sees a strong negative monosignate VCD feature for l-stercobilin and a positive VCD feature for d-urobilin. (For lstercobilin a strong positive structured VCD feature has been registered at ca. 1350 cm-1). Since VCD spectra in that region are generally determined by C*H-bending,84,85 we expect this region to provide some information on the role of stereogenic carbon atoms. 4) Finally, similar considerations as above may be applied to the VCD features in correspondence to the medium IR band at ca. 1150 cm-1: a negative mono-signate structured VCD feature for l-stercobilin and a positive well-defined VCD feature for d-urobilin. Conformational analysis of urobilinoids in dichloromethane. In the following we examine the conformational distribution for l-stercobilin hydrochloride in dichloromethane, considering also a possible role of the chloride ion as well as intramolecular H-bonding stability, which obviously depends on the solvent used. In this instance, explicit solvent has been considered through MD simulations. Analogous analysis was also conducted for d-urobilin. In order to simulate the chiroptical spectra, the common procedure is to perform a preliminary conformational search with the aid of appropriate MM software packages. This approach could be less efficient if the molecule has many degrees of freedom and/or if the geometry is particularly folded and stabilized with intramolecular interactions. This is the case of urobilinoids. Given the fundamental importance of the Moscowitz studies on the subject,23,24 we chose as an initial condition for MD simulations a conformation based on reference 23. This required, however, some tests to confirm that the hypothesized structure is stable in the solvent considered. Examining the results from MD simulations, we can make the following observations: i) The chloride ion is not completely solvated in CH2Cl2. When placed initially in different positions within the solvation box, far from the molecule (see Fig S1A), it moves close to the two propionic acid groups within the equilibration time, interacting with OH groups. Simulations of counterions in organic solvents are not common in the literature, though a similar behavior was observed for the example in ref. 86. ii) The dynamics is quite slow (see Figure 3). Particularly in organic solvents favoring intramolecular interactions, one needs quite long simulation times in order to be able to explore all (or the most) energetically accessible conformers. For this reason we simulated the l-stercobilin system for more than 2 9



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s. Time evolution of significant dihedral angles is reported in Fig. 3, see Scheme 1 for atom numbers. The orientation of the propionic acid groups (Figure 3 A-D) is influenced by the chloride ion, which stabilizes specific orientations of the two propionic groups such that dihedral angles change in time in a concerted way (a change in 1 implies a change in 1 and also in 2 and 2 so that Cl- always interact with both groups). The central dihedral angle  is positive with an average value of about 30° during the simulation (see in the following a discussion regarding the dipyrrylmethene chromophore). iii) During the simulation lstercobilin or d-urobilin remains folded/coiled, stabilized by intramolecular hydrogen bonds among the four rings. As shown in Figure 3F, more than 90% of the MD snapshots present at least 2 intramolecular hydrogen bonds among the rings. iv) As is often the case for folded structures (peptides for example) it is easier to test whether a structure is stable in the solvent, than to reach the appropriate geometry starting from a generic uncoiled one. Nevertheless, we could in fact confirm that starting from an uncoiled initial condition; the intramolecularly Hbonded folded structure was reached after 180 ns (see Fig S1B). In all cases the chloride ion does not play a role in stabilizing the helical conformation.

Scheme 1 - Chemical structure and atom numbering of l-stercobilin-HCl: atoms numbers given for definition of dihedral angles discussed in the text.

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Figure 3 - Representation of the time-evolution up to 2 s of some molecular dihedral angles as predicted by MD simulation of l-stercobilin in CH2Cl2. A) Dihedral angle α1 (8-30-31-32). B) Dihedral angle α2 (12-35-36-37). C) Dihedral angle β1 (30-31-32-33). D) Dihedral angle β2 (35-36-37-38). E) Dihedral angle τ (22-9-11-23). F) Distribution of the number of hydrogen bonds averaged over the entire simulation time. Hydrogen bonds parameters: distance cutoff = 3.5 Å; angle donor-acceptor cutoff: 30 deg. (For the definition of atom numbering see Scheme 1) 11



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Vibrational Circular Dichroism spectra Once MD has provided evidence as to whether the folded structure is maintained (and in this case it is), one may undertake one of two possible approaches to analysis by VCD. Approach I) The first approach is to run calculations in the IEF-PCM approximation for the folded/coiled structure, taking into account all possible conformations of the propionic acid pendant groups and disregarding the chloride ion. This procedure recalls the standard approach, apart from giving priority to the folding proposed in the literature and confirmed herein by MD. We report in Fig. 4 the lowest energy coiled conformers of l-stercobilin (conformers 1-5 populated more than 1%). For comparison, other unfolded structures (numbered 6-10) were obtained from a global conformational search (see also Table S1 for a quantitative definition of the structures). We also report therein the Boltzmann averaged calculated spectra. The most populated conformer obtained in this way (48%) has not only the four H-bonds responsible for the folded structure hypothesized by Moscowitz but it also has H-bonds between the propionic acid groups (see conformer 1 of Fig. 4). However the corresponding calculated spectrum does not correctly reproduce the negative VCD feature at about 1690 cm-1 and the band at 1270 cm-1 is also not well reproduced (see Fig. 4), which renders this approach to the VCD analysis not completely satisfactory. Also the neglect of any influence of chloride ion on propionic acid conformation appears problematic. In all cases the ECD spectra have also been calculated: for the folded structures (1-5) of Fig. 4 ECD spectra look quite similar to those observed, upon unfolding some structures (6 and 7 of Fig. 4) give a negative band, but others have oppositely signed ECD with respect to the experimental. Similar considerations apply to the calculated VCD spectra of d-urobilin, presented in Fig. S2 for sake of conciseness. Conformers 1,2,3,4 are similar to the lowest energy folded/coiled conformers l-stercobilin. Two other uncoiled structures 5 and 6 are presented for comparison.

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Figure 4 - Comparison of experimental IR (A), VCD (B), UV (C) and ECD (D) spectra (black lines) of l-stercobilin in deuterated dichloromethane (for IR and VCD) with the respective calculated spectra for different folded/coiled (1-5) and uncoiled conformers (6-10), as well as the Boltzmann average thereof. The conformer geometries (B3LYP/6-31G** IEFPCM) are illustrated in the pictures below the spectra (see Table S1 for a geometrical description of the structures).

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Approach II) Our second approach relies to a larger extent on MD results and consists of performing two-layer ONIOM calculations on the structures obtained after clustering MD simulated frames, as explained in the computational methods section. This method allows one to examine the effect of the chloride ion on conformational stabilization87 and consequently on the calculated spectra under the influence of explicit solvent. There are several different methods in the literature concerning the number of snapshots needed to represent the experimental data. Urago et al.56 have selected a large number of snapshots taken every constant time fraction for the QM/MM calculations. Recently, Mutter et al 57,58 used a reduced number of frames from the simulation and were guided in the choice of the frames by the study of the distribution of some significant dihedral angles. In order to best represent the different conformations and fluctuations caused by the solvent inside the clusters, we propose a method based on the clustering of the MD frames and the ONIOM calculation of eight frames within each cluster. This method requires some preliminary tests to choose the threshold for clustering (see computational method section). With the chosen value, we succeeded in differentiating propionic acid conformers; thus, in this way we obtained eight main clusters as reported in the Table S2. As stated above, during the MD simulation lstercobilin maintains the coiled structure with four intramolecular hydrogen bonds between pyrrole NH and lactam CO–NH (see Figure S1). The possible conformational changes of the propionic acids are limited by the presence of the chloride ion; thus, the chloride ion has an indirect effect on the VCD spectrum. In fact its presence in the proximity of the propionic acids hinders the conformations in which the propionic acids directly bind each other, which seems the reason why Approach I) does not accurately reproduce the spectrum. We report in Fig. 5 the IR and VCD calculated spectra for l-stercobilin (left) and for durobilin (right) for the representative structures of the eight main clusters, as obtained with the ONIOM method. The shape of all spectra is rather similar and is also similar to the experimental curve. An accurate evaluation of each cluster population (calculated as proportional to the number of frames within each cluster) requires a very long MD simulation time, especially for dichloromethane solvent that stabilizes intramolecular H-bonds. In the Table S2 the population of each cluster is reported. Alternatively, we also used the energy of the optimized conformer (with chloride ion and implicit dichloromethane PCM solvent) at DFT level, to obtain the Boltzmann population. The results are not identical: on one hand the long residence time within a cluster, i.e. the slow conformational change, may suggest the need of longer simulation times, on the other hand PCM treats the solvent effect only 14


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approximately. However, the similarity of the calculated spectra of Fig. 5 makes the error in population less crucial.

Figure 5 - Left: l-stercobilin Right: d-urobilin. Comparison of experimental IR (A and B) and VCD (C and D) spectra in CD2Cl2 solvent (black traces) and the calculated spectra of eight main representative clusters in CH2Cl2 solvent from ONIOM method (Approach II, see text). Recalling the four main regions of the VCD spectra, based on the ONIOM calculation, we can determine which normal modes are responsible of the main bands of the spectra. The features at ca. 1690 cm-1 host the C=O stretching normal mode transitions of the lactam rings, as shown for bilirubin systems.40-43. The different number of nearby stereogenic carbon atoms may justify the non perfect mirror image of this region in the two molecules studied herein. The calculated C=O stretchings of propionic acids do not give appreciable VCD contributions but it is clear that the corresponding absorption band is calculated at higher wavenumbers (about 1780 cm-1) with respect the experimental ones: a problem already encountered in other bilirubinoids.42,43 15



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The mono-signate negative VCD feature for l-stercobilin and the positive feature for durobilin at 1610 cm-1 are determined by complex normal mode transitions, involving NH- and C(10)-H in-plane bendings and ring-vibrations. This band, as well as the bands in the previously described spectroscopic region, is affected by the number and nature of intramolecular hydrogen bonds, for this reason they are common to all spectra obtained by MD/ONIOM method of Fig. 5. By comparison with Figure 4, one can appreciate how unfolded structures do not bear the same spectroscopic signature, and how the signal at 1690 cm-1 is not well reproduced when propionic acids are directly bound to each other. It is interesting to note that the observed and calculated sign reversal in the two VCD features of lstercobilin and d-urobilin noted above correspond to a mirror-image arrangement of the central portion (dipyrrin core) of the molecules determined by the four intramolecular hydrogen bonds: pyrrole NH• • •O=C–NH (lactam) and the lactam NH also participates in Hbond to opposing C=O. As anticipated, the mono-signate VCD feature at ca. 1270 cm-1, which is negative for lstercobilin and positive for d-urobilin, is determined by a C*H-bending mode mixed with inplane bending of NH and C(10)-H. The larger VCD intensity of this band in both experimental and calculated spectra for l-stercobilin is thus justified by the participation of a larger number of C*H bending modes. Finally, the mono-signate VCD structured feature at ca. 1150 cm-1, which is negative for lstercobilin but positive and well-defined for d-urobilin, is determined by quite delocalized normal modes, and thus it is hard to decipher to which structural motif it may be attributed. We report in Figure 6 a comparison between the experimental IR and VCD spectra of the two molecules with the corresponding PCM calculated spectra obtained after a conformational search, absent the chloride ion (Approach I) and MD/ONIOM calculations after weighted average over middle cluster structures (Approach II). We reiterate here that for l-stercobilin the MD runs were for 2 s, whereas d-urobilin was simulated for a shorter time (200 ns) with the scope of confirming the stability of the folded/coiled form and the role of chloride ion in determining propionic acid conformations. In both cases we can conclude that Approach II gives a better correspondence to the experimental data as confirmed by similarity indices (SI)80,81, in addition to providing a more detailed picture of the molecule’s behavior. The obtained results confirm this statement: for l-stercobilin: Approach I VCD: SI= 0.49, IR: SI= 0.92; Approach II VCD: SI= 0.62, IR: SI= 0.87, and for d-urobilin: Approach I VCD: SI= 0.34, IR: SI= 0.86; Approach II VCD: SI= 0.60, IR:0.75. 16


Page 17 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6 - Left: l-stercobilin Right: d-urobilin. Comparison of experimental IR (A and B) and VCD (C and D) spectra in CD2Cl2 solvent (black traces) with the average calculated ones obtained by the PCM-DFT Approach I (green traces) and by the ONIOM Approach II (blue traces), see text.

17



Page 18 of 31

ECD and CPL spectra

Figure 7 - Comparison of experimental (black traces) ECD (A), UV (C), CPL (B) and fluorescence (D) spectra of l-stercobilin in CH2Cl2 solvent with the respective calculated (blue traces) ones of a folded l-stercobilin structure at CAM-B3LYP/6-31g** IEFPCM level of theory. The wavelength of calculated spectra was red shifted by 45 nm. Another interesting property of these compounds, already evidenced in the literature6,8, is their fluorescence, whose chiral counterpart is CPL. In Figure 7, we present the CPL spectrum of l-stercobilin, along with its fluorescence spectrum recorded under the same conditions. Comparison may be drawn to the UV and ECD spectra. The dissymmetry ratio gabs= was determined to be about 8.8 x 10-4 while glum=I/I is about 3.5 x 10-3, which is quite a notable value for an organic molecule.61,88 The Stokes shift for d-urobilin is much smaller than that of l-stercobilin, preventing us from recording a reliable CPL signal of the former compound. TD-DFT calculations at the simple IEF-PCM level on the averaged structure of the most populated cluster of l-stercobilin allows one to reproduce quite well the ECD spectra in terms of sign and magnitude (see Figure 7A). From the same Figure one may see that the UV spectrum is also well predicted (and that propionic acid pendant conformations do not have a significant influence on the first electronic transition). Analogous good results are obtained 18


Page 19 of 31 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


for ECD and UV of d-urobilin (results not reported here). Optimization of the first excited state also allows us to calculate the CPL and fluorescence spectra of l-stercobilin (Fig. 7B). Looking at computational results, we notice that: (1) the gabs ratio is overestimated by a factor of 2 approximately, while the glum value is underestimated by a factor of 2 approximately; (2) the computed Stokes shift is underestimated; (3) from Figure 7 and from the previous ECD data of ref. 24 one may notice an evident shoulder at shorter wavelengths. It is worthwhile recalling ref. 24 that, by changing the solvent (from CH2Cl2 to CH3OH), and especially by moving to much lower temperatures (or, precisely, in a methanol-glycerol glass), the latter shoulder transforms to a positive band. The interpretation given in ref. 24 was that an essential requisite for the existence and stabilization of the inherently dissymmetric chromophore responsible for the ECD spectrum, namely a concerted network of H-bonds, was missing in methanol and the ECD spectrum changed significantly. We pursued the investigation of the temperature and solvent dependence of the ECD spectra by theoretical and computational means, and we report our results as follows. Analysis of the inherently dissymmetric chromophore hypothesized for urobilinoids In 1964, when quantum mechanical calculations were largely of the -electron type, Moscowitz et al.23 proposed the model of the inherently dissymmetric chromophore21,22 to explain the optical activity of urobilinoids (Optical rotation23 and ECD spectra24). The dipyrrylmethene (dipyrrin) chromophore plays an essential role in the optical activity of the urobilinoids, constituting the central moiety of such molecules. We investigated two requisites in order to explain the observed spectra: i) stabilization of the chiral conformation due to intramolecular H-bonds (which is evident from previous calculations); ii) contributions from “extrachromophoric” components of the molecule extending beyond the simple two-ring dipyrrylmethene unit. From TD-DFT calculations it appears evident that extending the dissymmetric unit to include peripheral lactams and beta-substituents favors larger rotational strengths in the UV-vis range (see Table 1 in the text), in addition to giving a red shift. Also the calculated g-ratios are closer to the observed values for the more extended chromophore. The computed increase in rotational strength is not only due to an increase in the magnitude of the magnetic dipole transition moment but is related to the angle between electric and magnetic dipole transition moments sensibly different from 90°.

19



Page 20 of 31

Table 1. Ground state and excited state (e.s.) properties of l-stercobilin and other model molecules: dipyrrylmethene at different torsional angles; Gossauer’s urobilin analog (8) with only CH2CH3 and CH3 pyrrole beta substituents (ref. 26) to show that propionic acids do not contribute to the rotational strength; a folded/coiled l-stercobilin (averaged structure of the first cluster of Fig. 5).

a

Molecule



(nm)

|µ|a

|m|b

Rc

E-Md

De

gf

Dipyrrylmethene 1

0

413

3.6

1.2

-0.0

90.0

8.4E+5

0

Dipyrrylmethene 2

2.5

410

3.8

1.0

-39.5

87.4

9.2E+5

-0.17E-3

Dipyrrylmethene 3

5

410

3.8

1.0

-39.3

87.5

9.2E+5

-0.17E-3

Dipyrrylmethene 4

17

413

3.8

1.0

-39.0

87.5

9.1E+5

-0.17E-3

Dipyrrylmethene 5

23

414

3.8

1.0

-37.2

91.4

9.1E+5

-0.17E-3

Compound 8 in ref. 27

20

432

3.1

2.6

-294.5

99.3

6.3E+5

-1.9E-3

l-stercobilin

23

432

3.4

1.7

-233.2

100.0

7.5E+5

-1.3E-3

e.s. Dipyrrylmethene

25.5

474

3.9

0.9

-31.0

92.2

1.0E+6

-0.12E-3

e.s. l-stercobilin excited state

29.7

473

3.8

1.2

-224.6

102.6

9.1E+5

-0.98E-3

electric dipole moment (atomic units), b magnetic dipole moment (atomic units), c rotational strength

(10-40esu2cm2), d angle between electric and magnetic dipole moment (deg), e dipole strength (10-36esu2cm2), f

g-factor. Dihedral angle τ=22-9-11-23 (deg).

Molecular dynamics of l-stercobilin in methanol at 185 and 300K Lightner et al

24

measured the temperature dependence of the circular dichroism spectra of l-

stercobilin and d-urobilin in methanol-glycerol (9:1 v/v) glass. At 163 K l-stercobilin shows inversion of the ECD spectra (with just a little reminiscent negative component), while durobilin exhibits a bisignate spectrum. In contrast, the sign of the CE recorded at 300K is the same as in dichloromethane or chloroform, but with half the intensity. We have performed two MD simulations of l-stercobilin in methanol at two different temperatures: 185 K and 300 K (600 ns each). From these simulations, we make the following observations: i) the chloride ion is completely solvated, thus it does not affect in any way the orientation of the propionic acids, which are free to interact with the solvent; ii) methanol competes with the intramolecular hydrogen bonds of l-stercobilin. Thus, absent the conformation-determining intramolecular hydrogen bonds, the structure may unfold, as already hypothesized by Lightner et al

24

(see Fig. S3F and S4F). Thus, the conformational

mobility is then very high, as can be appreciated from Fig. S3 and S4. 20


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The conformation of the l-stercobilin “backbone”, i.e. the reciprocal orientation of the four rings, can be described approximately by three dihedral angles: the central one τ (τ=22-9-1123) within the dipyrrylmethene core and θ1 and θ2 (θ1=21-4-6-22 and θ2=23-14-16-24), see Scheme 1. In Figure S5 see the distribution of these dihedral angles in methanol at the two temperatures and in dichloromethane. Calculation of ECD spectra from simulated structures of a molecule with high conformational mobility requires some attention. Several approaches have been proposed in the literature51-53 testing different criteria to choose the structures to be considered for averaging, taking account of partial reoptimization and with explicit solvent contributing to spectral intensities. As a preliminary analysis, we adopt an approach similar to the Approach II previously described for calculating the VCD spectra in CD2Cl2. Briefly, we made a clustering of the MD frames based on the molecule’s backbone (excluding the propionic acids that do not contribute to ECD) and then we calculate the ECD spectrum of the representative structure of each cluster with IEF-PCM CAM-B3LYP/6-31G**. The number of clusters obtained with this analysis is quite high. The weighted averages, considering cluster populations, of the rotational strengths give a negative signal at 300 K (-38·10-40 esu2cm2 from Table S3, disregarding differences in wavelength), and a positive one at 185 K (69·10-40 esu2cm2 from Table S4, disregarding differences in wavelength). In Fig. 8 we present a reconstruction of the average spectra considering rotational strength of each cluster weighted with population.

Figure 8 - Gaussian-shaped representation of the weighted average rotational strengths of the l-stercobilin conformers at 300 K (left) and 185 K (right) in methanol. Gaussian bandwidths are 0.13 eV. Superimposed are stick representations of calculated rotational strengths for all cluster-conformers, each stick is weighted with cluster population. 21



Page 22 of 31

From the calculations of these unfolded conformations, it appears evident how the sign of the rotational strength for the 490 nm transition in l-stercobilin is not simply related to the value of the dihedral angle  (22-9-11-23) but the sign is related to the dissymmetry of the entire molecule as can be seen from the correlation between dihedral ω (N21-N22-N23-N24, see Scheme 1) and the sign of the rotational strength in Table S3 and S4 and in Figure 9.

Figure 9 - Correlation diagrams between dihedral angle ω (N21-N22-N23-N24) value and rotational strength values (R) calculated for clusters obtained from molecular dynamics simulation in methanol at 300 K (left) and 185 K (right). Units: R x 10-40 esu2cm2. These calculations may appear oversimplified and preliminary because we made the simulation in methanol, without glycerol, and we adopted an average based on clustering. Yet our computational results agree with what was experimentally observed by Lightner et al.24 suggesting that in methanol there is an equilibrium between numerous uncoiled conformations, an equilibrium that can change rapidly as a function of the temperature. Conclusions l-Stercobilin hydrochloride and d-urobilin hydrochloride, though being not exactly enantiomers of each other, are known to exhibit almost opposite optical rotation and almost mirror images ECD spectra. In this work we have added two new chiroptical spectra, namely VCD, presenting opposite signals in the two molecules for majority of the vibrational features, and, CPL for l-stercobilin (whereas d-urobilin shows a lower Stokes shift and no CPL signal was recorded with our present instrumentation). The VCD spectra of the two samples in deuterated dichloromethane have been interpreted by combining MD with ab initio calculations, within a QM/MM procedure. In this way we have been able to i) monitor the importance of conformational aspects, ii) show that contributions from the chloride ion have a 22


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non-negligible role on propionic acid residue conformations, and iii) show that contributions from intramolecular H-bonds which stabilize the central dipyrrin unit chirality, constituting an inherently dissymmetric chromophore, as defined by Moscowitz. Solvation is an essential aspect of the VCD spectra and of the ECD spectra as well. In the latter case we interpreted the rather puzzling reversal of the ECD spectra from room temperature towards liquid N2temperature in methanol/glycerol solution, by simulating the molecule in methanol. The absence of intramolecular H-bonds allows of variety of structures generating different rotational strengths whose sign correlate with the helicity of the “backbone” of the four rings (dihedral angle defined by the four N atoms). Different temperatures establish different populations for the various structures and account for the observed experimental data. Conflicts of interest There are no conflicts to declare. Acknowledgements We thank Professor Sandro Fornili, University of Milan, for helpful discussion. Funding was provided by University of Brescia. We wish to acknowledge the use of computer and software facilities at CINECA-Bologna, Italy, and Regione Lombardia for the LISA grant “LI08p_ChiPhyto” and for support from Big&Open Data Innovation Laboratory (BODaI-Lab), University of Brescia, granted by Fondazione Cariplo.

Supplementary Information Supplementary information is available free of charge, it reports four tables and 5 figures with info about MD calculations and ONIOM results. References 1. Falk, H. The Chemistry of Linear Oligopyrroles and Bile Pigments; Springer-Verlag, Wien, Austria, 1989. 2. Schwartz, S.; Watson, C. J. Isolation of a Dextrorotatory Urobilin from Human Histula Bile. J. Prod. Soc. Exp. Biol. Med. 1942, 49, 641-643. 23



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3. For leading references and summary, see: Lightner, D. A. In The Porphyrins, Academic Press, New York, San Francisco, London, 1979, Vol. VIA, Ch. 8, 521-584. 4. Watson, C. J. Über Stercobilin, Kopromesobiliviolin und Kopronigrin. Hoppe-Seyler’s Z. physiol. Chem. 1932, 208, 101-118. 5. For a comprehensive review, see: Boiadjiev, S. E.; Lightner, D. A. Optical Activity and Stereochemistry of Oligopyrroles and Bile Pigments. Tetrahedron: Asymmetry 1999, 10, 607655. 6. Jaffe, M. Zur Lehre von den Eigenschaften und der Abstammung der Harnpigmente. Virchow’s Arch. Pathol. Anatom. Physiol. Klin. Med. 1869, 47, 405-427. 7. Fischer, H.; Meyer-Betz, F. Zur Kenntniss des Gallenfarbstoffs. II. Mittlg. Über das Urobilinogen des Urins und das Wesen der Ehrlich’schen Aldehyd-Reaktion. Hoppe-Seyler’s Z. physiol. Chem. 1911, 75, 232-261. 8. van Lair, C. F.; Masius, J. B. Über einen neuen Abkommling des Gallenfarbstoffes in Darminhalt Centralbl. Med. Wissensch. 1871, 9, 369-371. 9. Watson, C. J. The Origin of Natural Crystalline Urobilin (Stercobilin). J. Biol. Chem. 1936, 114, 47-57. 10. Watson, C. J. Recent Studies of the Urobilin Problem. J. Clin. Pathol. 1963, 16, 1-11. 11. Watson, C. J. In Chemistry and Physiology of Bile Pigments, Ed. Berk, P. D.; Berlin, 1977, N.I. Fogarty International Center Proceedings No. 35, DHEW Publication No. (NIH 771100). Conference held April 28-30, 1975. 12. Watson, C. J. Gold from Dross: The First Century of the Urobilinoids. Ann. Int. Med. 1969, 70, 839-851. 13. Gray, C. H. The Bile Pigments, John Wiley & Sons, Inc., New York, NY, US, 1953. 14. Stoll, M. S. In Bilirubin, Ed. Heirwegh, K. P. M.; Brown, S. B. CRC Press, Boca Raton, Florida, US, 1982, Vol II, Ch. 4, 103-132. 15. Bissell, D. M. In Bile Pigments and Jaundice, Ed. Ostrow, J. D. Marcel Dekker, Inc., New York, 1986, 133-156. 16. Lightner, D. A. Bilirubin: Jekyll and Hyde Pigment of Life, Prog. Chem. Org. Nat. Prod. Springer-Verlag, Wien, Austria, 2013. 17. Fischer, H.; Orth, H. Die Chemie des Pyrrols, Akademische Verlagsgesellschaft, G.m.b.H., Leipzig, Germany, 1937, Vol 2, 675-697. 18. Bonnett, R.; Davies, J. E.; Hursthouse, M. B.; Sheldrick, G. M. The Structure of Bilirubin. Proc. Roy. Soc. London 1978, B202, 249-268. 24


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l-Stercobilin-HCl and d-Urobilin-HCl. Analysis of Their Chiroptical and

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