Insights into the Structures of Bilirubin and Biliverdin from Vibrational and Electronic Circular Dichroism: History and Perspectives.

In this work we review research activities on a few of the most relevant structural aspects of bilirubin (BR) and biliverdin (BV). Special attention is paid to the exocyclic C=C bonds being in mostly Z rather than E configurations, and to the overall conformation being essentially different for BR and BV due to the presence or absence of the double C=C bond at C-10. In both cases, racemic mixtures of each compound of either M or P configuration are present in achiral solutions; however, imbalance between the two configurations may be easily achieved. In particular, results based on chiroptical spectroscopies, both electronic and vibrational circular dichroism (ECD and VCD) methods, are presented for chirally derivatized BR and BV molecules. Finally, we review deracemization experiments monitored with ECD data from our lab for BR in the presence of serum albumin and anesthetic compounds.

Biliverdin chiral derivatives as chiroptical switches for pH and metal cation sensing.

A series of six optically active derivatives of the bile pigment biliverdin, namely (ßS,ß'S)-dimethylmesobiliverdin-XIIIα, cyclic esters of linear diols [HO(CH2)nOH] where n = 1-6, have been investigated by vibrational circular dichroism (VCD) and density functional theory (DFT) calculations. The results were correlated with the length (n) of the diester belt, the verdin helicity and an M ⇄ P conformational equilibrium - as previously shown by electronic circular dichroism (ECD). Furthermore, ECD spectra have been found to be quite sensitive to solvent nature and pH. TD-DFT calculations of the protonated/deprotonated verdins with n = 1 and 2 diester belts respectively have allowed one, moreover, to explain the spectroscopic data in terms of a change in the M ⇄ P equilibrium. Finally, the set of investigated compounds, together with other chirally functionalized "non-belted" biliverdin analogs, has also been found to be sensitive to the presence of metal ions, with which the verdins chelate. On the basis of ECD and VCD data, we propose that the spectroscopic changes observed are consistent with self-association (dimerization) of the verdin molecules promoted by the metal cations, as bolstered by DFT calculations, and for which a dimerization constant of 73 000 M-1 is evaluated. We envision the use of current chiroptical spectroscopies in connection with chiral biliverdin derivatives as natural sensors or probes of the micro-environmental conditions, such as pH or the presence of metal ions.

l-Stercobilin-HCl and d-Urobilin-HCl. Analysis of Their Chiroptical and Conformational Properties by VCD, ECD, and CPL Experiments and MD and DFT Calculations.

Vibrational circular dichroism (CD) and IR spectra of dichloromethane solutions of l-stercobilin and d-urobilin hydrochlorides have been recorded in the mid-IR region. The spectra are best interpreted by combining molecular dynamics calculations and density functional theory (DFT) calculations within the quantum mechanics/molecular mechanics 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 spectra of the same compounds in methanol-glycerol solution. Finally, circularly polarized luminescence spectra for l-stercobilin in chloroform solution provide information on the excited-state geometry of this molecule.

On the aggregation of bilirubinoids in solution as evidenced by VCD and ECD spectroscopy and DFT calculations.

Vibrational and electronic circular dichroism (VCD and ECD) spectra of 3 optically active bilirubin analogs with propionic acid groups replaced by (1) 1-(S)-methylpropyl groups, (2) 3-acetoxy-1-(S)-methylpropyl groups, and (3) 1-(S)-2-(R)-dimethyl-2-(methoxycarbonyl)ethyl groups have been recorded at different concentrations in chloroform. The aliphatic chains attached to C-8 and C-12 of the 3 chosen mesobilirubins were modified so as to possess no OH group. The variation of the VCD spectra with concentration is consistent with the formation of dimers at high concentration. Density functional theory and time-dependent density functional theory calculations on monomeric and dimeric forms support such a conclusion. Comparing with previous VCD (ECD) and IR (UV) studies of other mesobilirubin molecules, it is concluded that here, the key feature for aggregation is the missing OH groups on the propionic acid chains. The latter, in synergy with the polar groups of lactam moieties, appear to be involved in intramolecular phenomena and thus favor monomeric forms. Investigation of ECD and UV spectra of the same compounds in mixed DMSO/chloroform solutions provide further clues to the proposed picture.

pH Dependent Chiroptical Properties of (1R,2R)- and (1S,2S)-trans-Cyclohexane Diesters and Diamides from VCD, ECD, and CPL Spectroscopy.

Diesters of (1R,2R)- and (1S,2S)-cyclohexanediols and diamides of (1R,2R)- and (1S,2S)-diaminocyclohexane with p-hydroxycinnamic acid have been known for some time to exhibit intense bisignate electronic circular dichroism (ECD) spectra in CH3OH. It was also known that added NaOH causes a bathochromic shift of ∼50 nm in CH3OH, and an even higher one in DMSO. We have measured vibrational circular dichroism (VCD) spectra both for neutral compounds and in the presence of NaOH and other bases. The VCD and IR spectra in the mid-IR region for CD3OD and DMSO-d6 solution exhibit high sensitivity to the charged state for the diesters. They possess two strong bisignate features in the presence of bases in the mid-IR, which are interpreted in terms of vibrational exciton couplets, while this phenomenon is less evident in diamides. VCD allied to density functional theory (DFT) calculations allows one to shed some light on the spectral differences of diesters and diamides by studying their conformational properties. Optical rotatory dispersion (ORD) curves confirm the ECD data. Circularly polarized luminescence (CPL) data have been also acquired, which are rather intense in basified solution: the CPL spectra are monosignate and are as intense in the diester and in the diamide case.

Bicamphor: a prototypic molecular system to investigate vibrational excitons.

Vibrational circular dichroism (VCD) and IR spectra have been recorded in the fingerprint and carbonyl stretching regions for endo,endo-bicamphor (1), exo,exo-bicamphor (2), endo,exo-bicamphor (3), 3,3'-bicamphorylidene (4), and exo,exo-bis-thiocamphor (5). The C2 symmetry possessed by these molecular systems (except in one case), as well as their limited conformational mobility associated with well-defined degrees of freedom, allow for optimal test of the vibrational circular dichroism exciton chirality (VCDEC) rule introduced by Taniguchi and Monde. Density functional theory calculations are employed not only to predict the entire aspect of the VCD and IR spectra but also to study how the VCDEC rule may be impacted by the coupling between C═O stretchings and from C═O stretchings with other vibrational modes and by the rotation about the C-C bond connecting the two camphors. Comments are provided about the limitations and potentialities of the VCDEC method and about the manifestation of different vibrational excitons in other regions of the VCD spectra, either in the mid-IR or in the CH-stretching regions.

Vibrational and electronic circular dichroism of dimethyl mesobilirubins-XIIIα.

The vibrational circular dichroism (VCD) spectra of (αR,α'R)-, (αS,α'S)-, (ßR,ß'R)-, and (ßS,ß'S)-dimethylmesobilirubin-XIIIα have been recorded in the range of 1800-900 cm(-1) in CDCl3 solution and in mixed DMSO-d6/CDCl3 solutions. Ab initio density functional theory (DFT) calculations predict IR vibrational absorption (VA) and VCD spectra in excellent to good correspondence with observed data. The same calculations confirmed the ridge-tile conformation that has been known for a long time. Assignment of vibrational normal modes (NMs) sheds light on the relative importance of local moieties and groups in determining conformational properties of the molecules, as well as their interaction with solvent molecules. Time-dependent DFT (TDDFT) calculations were also performed to provide an understanding of electronic circular dichroism (ECD) spectra and confirm the well-known interpretation based on the exciton model.

Slipping past UGT1A1 and multidrug resistance-associated protein 2 in the liver: effects of steric compression and hydrogen bonding on the hepatobiliary elimination of synthetic bilirubins.

The hepatobiliary metabolism and excretion of three isomeric bilirubin analogs with propanoic replaced by benzoic acid side-chains were studied in the rat. Despite their isomeric relationship and similar constitutions, the three analogs were metabolized quite differently from each other and from bilirubin. In the di-o-benzoic compound, steric hindrance involving the phenyl groups reinforces intramolecular hydrogen bonding of the two carboxyl groups. This compound is considerably less polar than bilirubin on reverse-phase high-performance liquid chromatography and, like bilirubin, was not excreted in bile in UGT1-deficient (Gunn) rats. But, quite unlike bilirubin, it was not glucuronidated or excreted in bile in normal rats. In contrast to both bilirubin and the di-o-benzoic isomer, the more polar m- and p-isomers, in which intramolecular hydrogen bonding of carboxyl groups is sterically difficult, were excreted rapidly in bile in unchanged form in both normal and Gunn rats. However, only one of them, the di-m-isomer, was excreted rapidly and unchanged in bile in rats (TR(-) rats) congenitally deficient in the canalicular ATP-binding cassette transporter Mrp2. The marked differences in hepatobiliary metabolism of the three isomers studied can be rationalized on the basis of their computed three-dimensional structures and minimum-energy conformations and the remote effects of steric compression on intramolecular hydrogen bonding.

Converting 9-Methyldipyrrinones to 9-H and 9-CHO Dipyrrinones.

Yellow 9-methyldipyrrinones can be converted readily and in high yields to symmetric linear tetrapyrroles, blue biliverdinoids, which are cleaved in half, smoothly at room temperature to afford yellow 9-H dipyrrinones, and 9-CHO dipyrrinones as their violet to orange colored adducts with the carbon acid used for the scission: thiobarbituric acid (TBA), N,N'-diethylthiobarbituric acid, barbituric acid, N,N'-dimethylbarbituric acid and Meldrum's acid. The adducts, usually only of passing interest, are formally Knövenagel condensation products of a 9-CHO dipyrrinone with TBA and other carbon acids of this work, and a reverse Knövenagel reaction of such adducts leads to 9-CHO dipyrrinones. Under a set of improved reaction conditions the sequence thus efficiently converts 9-CH(3) dipyrrinones to 9-H and 9-CHO dipyrrinones.

Synthesis and hepatic transport of strongly fluorescent cholephilic dipyrrinones.

A new class of highly fluorescent (phi(F) 0.3-0.8) low molecular weight water-soluble cholephilic compounds has been synthesized in two steps from dipyrrinones. The dipyrrinone nitrogens are first bridged by reaction with 1,1'-carbonyldiimidazole to form an N,N'-carbonyldipyrrinone (3H,5H-dipyrrolo[1,2-c:2',1'-f]pyrimidine-3,5-dione) nucleus, and a sulfonic acid group is then introduced at C(8) by reaction with concd H(2)SO(4). The resulting sulfonated N,N'-carbonyl-bridged dipyrrinones ("sulfoglows") are isolated as their sodium salts. When the alkyl substituents of the lactam ring are lengthened from ethyl to decyl, sulfoglows become increasingly lipophilic while maintaining water solubility. Low molecular weight sulfoglows were rapidly excreted intact in both bile and urine after intravenous infusion into rats, but higher molecular weight sulfoglows were excreted more selectively in bile. Hepatobiliary excretion of sulfoglows was partially, but not completely, blocked in mutant rats deficient in the multidrug-resistance associated transport protein Mrp2 (ABCC2). These observations point to the feasibility of developing simple sulfoglows with clinical diagnostic potential that are normally excreted in bile but appear in urine when hepatic elimination is impaired by cholestatic liver disease.

Exciton chirality in trans-1,2-diamidocyclohexanes: fluorescent chromophores.

N,N'-Carbonyl-bridged dipyrrinones constitute a new class of highly fluorescent chromophores suitable for investigations of stereochemistry and absolute configuration. Xanthoglow (N,N'-carbonylxanthobilirubic acid) diamides of trans-1,2-diaminocyclohexane are strongly fluorescent (phiF=0.37, lambdaem=500 nm, lambdaex=419 nm in CHCl3) but exhibit only weak exciton circular dichroism (CD). In contrast, the diamide of (1R,2R)-diaminocyclohexane from the xanthoglow analogue whose propionic acid has been replaced by benzoic acid (N,N'-carbonyl-8-(4-carboxyphenyl)-3-ethyl-2,7,9-trimethyl-(10H)-dipyrrin-1-one) exhibits even stronger fluorescence (phiF=0.62, lambdaem=495 nm, lambdaex=422 nm in CHCl3) and UV-visible absorption (epsilon=41,600 dm3.mol-1.cm-1 at 424 nm) in organic solvents. Its exciton CD (Deltaepsilon=-13 dm3.mol-1.cm-1, lambda=432 nm; Deltaepsilon=+2 dm3.mol-1.cm-1, lambda=382 nm) correlates with the exciton chirality rule.

Readily synthesized novel fluorescent dipyrrinones.

A new, highly fluorescent (phiF up to 0.85) rigid anti-Z-dipyrrinone chromophore has been synthesized in high yield in a one-pot reaction by condensing two monopyrroles in the presence of DBU to form the pyrrolo[3,2-f]indolizine-4,6-dione nucleus.

pKa and aggregation of bilirubin: titrimetric and ultracentrifugation studies on water-soluble pegylated conjugates of bilirubin and fatty acids.

A water-soluble conjugate (1) with intact carboxyl groups was prepared by addition of poly(ethylene glycol) thiol (MPEG-SH) regiospecifically to the exo vinyl group of bilirubin. (1)H and (13)C NMR and absorbance spectroscopy in CDCl(3) and DMSO-d(6) confirmed the assigned structure and showed that pegylation did not disrupt the hydrogen-bonded ridge-tile conformation of the pigment moiety. Aqueous solutions of 1 were optically clear, but NMR signals were seen only from the MPEG portion and none from the tetrapyrrole, consistent with dissolved assemblies containing aggregated bilirubin cores within mobile polyether chains. On alkalinization (pH >12), signals from the pigment moiety reappeared. Titrimetric measurements on 1 in water showed the pK(a)'s of the two carboxyl groups to be similar (average 6.42). Control studies with pegylated half-esters of succinic, suberic, brassylic, thapsic, and 1,20-eicosanedioic acid showed that pegylation per se has little, if any, effect on carboxyl ionization. However, aggregation increases the apparent pK(a) by approximately 1-2 units. The molecularity of bilirubin in solution was further characterized by ultracentrifugation. Over the pH range 8.5-10 in buffer, bilirubin formed multimers with aggregation numbers ranging from approximately 2-7. Bilirubin is monomeric in DMSO or CHCl(3) at approximately 2 x 10(-)(5) M, but aggregation occurred when the CHCl(3) was contaminated with trace adventitious (perhaps lipoidal) impurities. These observations show that aggregation increases the pK(a)'s of aliphatic carboxylic acids relative to their monomer values in water. They are consistent with earlier (13)C NMR-based estimates of approximately 4.2 and approximately 4.9 for the aqueous pK(a)'s of bilirubin and similar studies of bilirubin in micellar bile-salt solutions. Together with earlier work, they confirm that the pK(a)'s of bilirubin are about normal for aliphatic carboxyls and suggest that the high (>7.5) values occasionally reported, including those based on CHCl(3) partitioning, are artifacts of aggregation or technique.

Novel benzoic acid congeners of bilirubin.

Three regioisomeric bilirubins and biliverdins with propionic acids replaced by benzoic acids were synthesized from the corresponding xanthobilirubic acids by oxidative coupling. The rubins were found to exhibit widely varying polarity, spectroscopic properties, and stereochemistry. The isomer with ortho benzoic acids (1o) was much less polar than either the meta (1m) or para (1p) because 1o (but not 1m and 1p) can adopt a folded conformation with both carboxylic acids intramolecularly hydrogen bonded to the opposing dipyrrinones. The consequences of such conformational differentiation are found in the varying 1H NMR, UV-vis, and circular dichroism spectral properties.

Hepatobiliary excretion of dipyrrinone sulfonates in Mrp2-deficient (TR(-)) rats.

The biliary excretion of the sodium salts of 8-(2-ethanesulfonic acid)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one (xanthosulfonic acid) and a fluorescent analogue (8-desethyl-N,N'-carbonyl-kryptopyrromethenone-8-sulfonic acid) was compared in Mrp2-deficient (TR(-)) and normal rats. Both organic anions were excreted rapidly in bile in Mrp2-deficient rats, but the biliary excretion of the fluorescent sulfonate was impaired relative to normal controls. The rat clearly has efficient Mrp2-independent mechanisms for biliary efflux of these anions that are not used by bilirubin or its mono- and diglucuronides.

Altering the Acidity and Solution Properties of Bilirubin. Methoxy and Methylthio Substituents.

Substitution of electron-withdrawing groups at the alpha-position of an aliphatic carboxylic acid can be expected to increase the acidity of the acid. Methoxy and methylthio groups are especially effective; they increase the acidity of acetic acid by approximately 1.1 pK(a) units. Bilirubin, the water-insoluble pigment of jaundice, has two propionic acids, and an alpha-methoxy or alpha-methylthio substituent in each propionic acid can be expected to lower the pK(a) similarly and thus alter its solubility properties. (A previously synthesized analogue, alpha,alpha'-difluororubin (4), is soluble in water.) Two new analogues of bilirubin, alpha,alpha'-dimethoxyrubin (1) and alpha,alpha'-bis(methylthio)rubin (2), have been synthesized, separated into diastereomers, and analyzed. The isomers are shown by NMR to adopt intramolecularly hydrogen-bonded ridge-tile-shaped conformations. Like bilirubin, both 1 and 2 are insoluble in water. Unlike bilirubin, 1 is soluble in dilute aqueous bicarbonate, but 2 is insoluble, which would not be predicted from the expectation that 1 and 2 have the same pK(a). The data hint at a much larger steric size of SCH(3) relative to OCH(3).

Synthesis of the First Fluorinated Bilirubin.

A symmetrical difluorinated bilirubin analog, 8,12-bis(2-carboxy-2-fluoroethyl)-3,17-diethyl-2,7,13,18-tetramethyl-10H,21H,23H,24H-biline-1,19-dione (9), was synthesized from methyl 3-[2,4-dimethyl-5-(methoxycarbonyl)-1H-pyrrol-3-yl] propionate (1) in nine steps. Fluorine was introduced by reaction of an intermediate methyl 3-[1-(tert-butoxycarbonyl)-2,4-dimethyl-5-(methoxycarbonyl)-1H-pyrrol-3-yl]-2-hydroxypropionate (5), with (diethylamino)sulfur trifluoride (DAST). The fluorinated rubin exhibited the expected IR, UV-vis, and NMR spectroscopic properties, similar to those of the unfluorinated parent, mesobilirubin XIIIalpha. However, the solubility properties unexpectedly differed, with the fluorinated rubin being less soluble in organic solvents than its parent. While this phenomenon may be attributed to the much increased acidity of the carboxylic acid hydrogens in 9, it probably also arises from less effective intramolecular hydrogen bonding due to a decreased basicity of the propionic acid carbonyl groups.