Structure and Metabolism of Natural and Synthetic Bilirubins

Original Article &&&&&&&&&&&&&& Structure and Metabolism of Natural and Synthetic Bilirubins

David A. Lightner studies.2,3 Yet, although the ridge-tile shape is far more stable than Antony F. McDonagh the conventional linear representation, or even a porphyrin-like shape, the bilirubin ridge tile is not rigid. It may flex and bend, and flutter lepidopterously — and even invert to the mirror image ridge tile — all the while maintaining most, if not all, of its 3 The secondary structure of bilirubin, with a ridge-tile shape and six intramolecular hydrogen bonds. And, just as knowledge of intramolecular hydrogen bonds, is more stable than any other conformation stereochemistry, especially the handedness of molecules, has recently and perhaps the most important determinant of its solubility and properties become the sine qua non for current studies in the efficacy and in solution and its hepatic metabolism/excretion. Uncoupling the metabolism of pharmacophores, so it, too, is having an impact on intramolecular hydrogen bonding, increasing the acidity of the propionic our understanding of bile pigment metabolism. acid, or even increasing its length can decrease the pigment’s It is apparently the tight embrace of propionic acid and hydrophobicity and permit its excretion intact across the liver into bile; less dipyrrinone, by hydrogen bonding, that dictates the hydrophobicity of intuitively obvious, so can subtle modifications at C(10), the pivotal carbon bilirubin-IX (or simply bilirubin) and its inability to be secreted in the ridge tile. (Taken from a talk given by D.A.L. at the International intact, across the liver from blood to bile. In striking contrast, the Congress on Neonatal Jaundice, Hong Kong, March 18–21, 2001.) IX (Figure 2A) IX , and isomers of bilirubin, none of which can Journal of Perinatology 2001; 21:S13–S16. engage in intramolecular hydrogen bonding (as in Figure 1), are far more polar and less lipophilic than the IX , and they are excreted mainly intact by the liver.4 This stark difference in polarity is not due to the presence or absence of methyl or vinyl groups (or ethyl groups, INTRODUCTION as in mesobilirubins, Figure 1D); it is directly linked to the location Some 60 years ago, Hans Fischer, the father of porphyrin chemistry of the propionic acid groups. Their location at positions 8 and 12, as and a Nobel Prize awardee, was the first to characterize the in the IX isomer, is indispensable for a lipophilic bilirubin. constitutional structure of bilirubin (Figure 1A). This was A healthy, adult human produces approximately 250 mg of accomplished by a long, degradative analysis followed by an bilirubin each day from degradation of heme. However, bilirubin is ingenious total synthesis.1 Although the Fischer structure brought too lipophilic to be excreted intact. Normally, bilirubin is converted about a major advance in the bile pigment area, certain details of by enzymes in the liver to mono- and diglucuronide conjugates that structure that we now know to be highly important remained are efficiently eliminated in bile5,6 by a series of complex, poorly unknown for nearly another 40 years. These details, particularly the understood steps. Glucuronidation alters the intrinsic lipophilicity of geometry of the carbon–carbon double bond exocyclic to the end the pigment by attaching polar, water-soluble groups. rings (Figure 1B) and the extraordinary propensity to fold into a Studies of the molecular mechanism of neonatal jaundice ridge-tile shape stabilized by a network of intramolecular hydrogen phototherapy showed that by simply breaking the intramolecular bonds (Figure 1C), have turned out to be of paramount importance hydrogen bonds to just one propionic acid, the lipophilicity of the for understanding the properties and metabolism of the pigment. We pigment decreases and its transport (intact) across the liver into bile now know that in the crystal or in solution, whether the propionic is rapid.7 The key, light-induced alteration in bilirubin structure is groups are in the acid form or are ionized to propionate ions, simple: a C=C bond geometry change in one dipyrrinone, from cis to intramolecular hydrogen bonding prevails and the ridge-tile trans (Z to E), concomitantly disengages the lactam group from its structure predominates — as confirmed by careful spectroscopic carboxylic acid hydrogen bonding partner (Figure 2B), thereby decreasing its hydrophobicity.7,8 The profound consequences of this simple structural change in Department of Chemistry ( D.A.L. ), University of Nevada, Reno, NV; and GI Unit and the Liver Center S - 357 ( A.F.M. ), University of California, San Francisco, CA. bilirubin, so essential to the success of jaundice phototherapy, are mimicked by our chemically designed, synthetic analogs of bilirubin. We thank the US National Institutes of Health ( grants HD - 17779, DK 26307, and GM 36633 ) for support of this research. Like those in nature, our syntheses typically start from small

Address correspondence to David A. Lightner, Department of Chemistry, University of Nevada, molecules; but unlike the natural process, they require 15 to 30 steps Reno, NV 89557 - 0020. or reactions. Thus, mesobilirubin-VIII (Figure 2C), with one

intramolecularly hydrogen-bonded propionic acid and a second incapable of intramolecular hydrogen bonding, has two essential structural components in common with the bilirubin E isomers of phototherapy. Like the E isomers of bilirubin, mesobilirubin-VIII is excreted both intact and as a monoglucuronide in bile after administration to rats. Careful studies in the rat have shown that only the intramolecularly hydrogen-bonded propionic acid group is glucuronidated, whether in the two different bilirubin E isomers, or in (4Z,15Z) mesobilirubin-VIII . Thus, the presence of a single, nonhydrogen-bonded propionic acid group is sufficient to permit intact excretion by the liver. The overwhelming evidence currently points to the presence of two propionic acid groups and their location on the bilirubin as uniquely important to the pigment’s lipophilicity and its metabolism. In contrast, whether vinyl groups are present (as in bilirubins; Figure 1C), or replaced by ethyls (as in mesobilirubins; Figure 1D), or even their exact location on the end rings were found to be of little importance. The unique microstructure/macroscopic properties relationship of the bilirubin and mesobilirubin molecules is slowly becoming evident. Focusing on the microstructure, one might ask whether the intrinsic acidity of the carboxylic acid group is important. Would more acidic analogs change the behavior of the Figure 2. (A) Bilirubin-IX cannot engage in conformation-stabilizing pigment? One might also question whether the three-carbon intramolecular hydrogen bonding, but may adopt a ridge-tile shape. (B) propionic acid groups per se are important, or whether they might be (4E,15Z)-Bilirubin-IX showing the absence of intramolecular hydrogen replaced with acid chains of differing length — or even with bonds in the E-dipyrrinone half. (C)(4Z,15Z)-Mesobilirubin-VIII with the same degree of hydrogen bonding as in (B), and similar solution and sulfonic acids. Would such alterations perturb the pigments’ excretion properties. lipophilicity and metabolism? Given the current controversy over the acidity (pKa) of bilirubin and the influence of pKa on bilirubin

transmembrane transport,9,10 it was important to focus attention on the acid groups.

RESULTS AND DISCUSSION Generally, increasing the chain length of carboxylic acids (toward fatty acids) increases their lipophilicity. We synthesized analogs of mesobilirubin-XIII with both propionic acids replaced by butanoic acid chains, expecting these new mesobilirubins to be increasingly lipophilic. Intramolecular hydrogen bonding was also expected, as molecular modeling computations clearly indicated that butanoic acid chains (Figure 3A), and even acid chains as long as 20 carbon acids, are capable of intramolecular hydrogen bonding to the dipyrrinone components. Although the bis-butanoic analog behaves in solution very much like mesobilirubin-XIII , we were surprised to discover that slightly longer chain analogs were, counterintuitively, more polar. But very long chain analogs were quite lipophilic. Figure 1. (A) Bilirubin structure proposed by Fischer et al.1 (B) Linear representation of bilirubin-IX composed of two dipyrrinones and the Spectroscopic studies confirmed that intramolecular hydrogen 4Z,15Z geometry. (C) The most stable three-dimensional structure of bonding occurs in bilirubins with extended acid side chains, but bilirubin-IX , shaped like a ridge tile and intramolecularly hydrogen- solubility studies suggest that it is weaker in analogs with side chains bonded. (D) (4Z,15Z)-Mesobilirubin-XIII , a synthetic bilirubin analog longer than butanoic acid. with vinyls replaced by ethyls and full hydrogen bonding and a ridge-tile After intravenous administration to homozygous Gunn rats shape. (which are deficient in the UGT1A1 enzyme), the bis-butanoic

S14 Journal of Perinatology 2001; 21:S13 – S16 Bilirubins: Structure and Metabolism Lightner and McDonagh

both the Gunn rat and the Sprague-Dawley rat, with no evidence of glucuronide formation. The metabolic difference between bilirubin and the bis-fluororubin may be due to the resistance of the latter to become protonated at physiologic pH, especially in nonaqueous (lipid) environments. The total inability to conjugate bilirubin, which leads to severe hyperbilirubinemia and jaundice and often to neurologic damage, does not block bilirubin elimination completely. Thus, in some patients with Type I Crigler-Najjar syndrome, or in the homozygous Gunn rat, blood levels of bilirubin attain a steady state despite constant bilirubin production, a complete lack of bilirubin-conjugating activity in the liver, and total absence of bilirubin glycosyl conjugates in urine or bile. Crigler-Najjar patients and Gunn rats are apparently able to eliminate bilirubin via unknown pathways that do not involve glucuronidation.5,12,13 There is some evidence that these alternate pathways of bilirubin elimination may be partly oxidative in nature, and Galliani et al.14 have suggested that they might involve production of 10-oxo-bilirubin (Figure 3C). Recently, we Figure 3. Mesobilirubin-XIII analogs with (A) both propionic acid synthesized and fully characterized the related 10-oxo-rubin of chains replaced by butanoic acids and resulting in a more open ridge tile; mesobilirubin-XIII and observed that this interesting pigment, (B)a single fluorine located adjacent to the carboxylic acid group to lower which was rendered more polar by the seemingly trivial alteration at the pK by two pK units; and (C) bilirubin with C(10) oxidized from CH a 2 C(10) from CH2 to C=O, was excreted both intact and as a mixture to C=O. of mono- (mainly) and diglucuronides in Sprague-Dawley rats.15 Molecular dynamics calculations indicate that the presence of the carbonyl, and the change from tetrahedral geometry at C(10) in analog was not excreted, like bilirubin and mesobilirubin-XIII .In bilirubin and mesobilirubins to trigonal in the 10-oxo-rubin, at the contrast, in normal Sprague-Dawley rats, all three were excreted hinge region of the molecule, stress intramolecular hydrogen promptly as a mixture of mono- and diglucuronides. However, bonding sufficient to increase polarity and allow direct excretion into analogs with even longer acid chains, e.g., bis-hexanoic, were bile, yet not so much that glucuronidation is totally impaired. excreted both intact and as a mixture of mono- and diglucuronides Replacing the hinge CH2 group with a C=O group has a much in Sprague-Dawley rats, and excreted intact in Gunn rats. greater effect than replacement with a sulfur atom. We have recently Apparently, in the absence of ‘‘tight’’ intramolecular hydrogen synthesized the first thia-rubin in which the CH2 group of bonding, bilirubins can be excreted intact, but with even residual mesobilirubin-XIII was replaced with a sulfur atom.16 This thia- intramolecular hydrogen bonding, the carboxylic acid chains are rubin behaved in vivo in the rat almost exactly like bilirubin or also glucuronidated. mesobilirubin-XIII : it required conjugation for biliary excretion In order to explore the role of carboxylic acid pKa on the and was excreted in the form of a mono- and a diglucuronide. properties of bilirubin or mesobilirubin, we designed fluoro and other Molecular dynamics calculations show that when the C(10) CH2 derivatives of mesobilirubin-XIII predicted to exhibit strongly is replaced by a sulfur atom, the bilirubin ridge tile is forced into increased acidity. It is known from pKa measurements that somewhat toward a more closed shape (as opposed to more open fluoroacetic acid is 100 times more acidic than acetic acid. in the 10-oxo-rubin). This does not weaken the intramolecular Expecting similarly large increases in acidity in monofluorinated hydrogen bonding, and as a consequence, 10-thia-rubin, unlike propionic chains of synthetic rubins, we designed and synthesized the 10-oxo-rubin, is lipophilic. first bis-fluoromesobilirubin-XIII (Figure 3B)11 and discovered, much to our surprise that despite its possessing all of the necessary components and proper geometry for intramolecular hydrogen SUMMARY bonding, it is far more polar than mesobilirubin itself. So much so Bilirubin preferentially adopts an intramolecularly hydrogen-bonded that, unprecedented for a bilirubin, it is soluble in water. (Both ridge-tile conformation, which explains its surprising lipophilicity. bilirubin and mesobilirubin are extremely insoluble in water, and are This makes intact bilirubin unexcretable in normal hepatic not extracted from chloroform into 0.1 M bicarbonate.) In view of its metabolism. The liver makes and excretes glucuronide conjugates of extraordinary aqueous solubility, we were not surprised to discover bilirubin that are more polar than the intact pigment. There is, that this fluororubin is excreted unchanged (albeit rather slowly) in however, a fine line between intact excretion and necessitated

Journal of Perinatology 2001; 21:S13 – S16 S15 Lightner and McDonagh Bilirubins: Structure and Metabolism

glucuronidation. When the propionic acid groups of bilirubin are disorders of bilirubin metabolism. In: Scriver CR, Beaudet AL, Sly WS, Valle lengthened to hexanoic, the pigment becomes more polar and is D, editors. The Metabolic and Molecular Bases of Inherited Disease. New York: McGraw-Hill; 1995, p. 2161–208. excreted intact. When the pKa of the propionic acids is lowered sufficiently by fluorine substituents, the pigment becomes very polar 6. McDonagh AF. Bile pigments: Bilatrienes and 5,15-biladienes. In: and is excreted intact. When C(10) is oxidized to (C10)-oxo (from Dolphin D, editor. The Porphyrins. vol. VI. New York: Academic Press; CH to C=O), the pigment becomes more polar and is excreted 1979, p. 293–491. 2 7. McDonagh AF, Lightner DA. Like a shrivelled blood orange. Bilirubin, intact and as glucuronides. The intramolecular hydrogen bonding is jaundice and phototherapy. Pediatrics 1985;75:443–55. weakened by mismatches with the opposing dipyrrinones and by 8. Cheng L, Lightner DA. A new photoisomerization of bilirubin. Photochem ridge-tile deformations. Even small deformations that open the ridge Photobiol 1999;70:941–8. tile can lead to excretion of intact rubins. Inducing or controlling 9. Tiribelli C, Ostrow JD. New concepts in bilirubin and jaundice: report of the such structural deformations in the jaundiced neonate might offer Third International Bilirubin Workshop, Trieste, Italy, April 6–8, 1995. new routes to treat physiologic jaundice of the newborn. Hepatology 1996;24:1296–311. 10. Trull FR, Boiadjiev SE, Lightner DA, McDonagh AF. Aqueous dissociation constants of bile pigments and sparingly soluble carboxylic acids by 13C- Acknowledgment NMR in aqueous dimethyl sulfoxide: Effects of hydrogen bonding. J Lipid Res We thank Wilma Norona for expert surgical and analytical assistance. 1997;38:1178–88. 11. Boiadjiev SE, Lightner DA. Synthesis of the first fluorinated bilirubin. J Org Chem 1997;62:399–404. References 12. Tiribelli C, Ostrow JD. New concepts in bilirubin chemistry, transport and 1. Fischer H, Plieninger H, Weissbarth O. U¨ ber die konstitution des bilirubins metabolism: Report of the Second International Bilirubin Workshop, Trieste, und u¨ber bilirubinoide farbstoffe. Z Physiol Chem 1941;268:197–226. Italy, April 9–11, 1992. Hepatology 1993;17:715–36. 2. Bonnett R, Davies JE, Hursthouse MB, et al. Structure of bilirubin. Proc R Soc 13. Schmid R, Hammaker L. Metabolism and disposition of C14 -bilirubin in London 1978;B202:249–68. congenital nonhemolytic jaundice. J Clin Invest 1963;42 1720–34. 3. Do¨rner T, Knipp B, Lightner DA. Heteronuclear NOE analysis of bilirubin 14. Galliani G, Monti D, Speranza G, et al. The interactions of biliverdin and its solution conformation and intramolecular hydrogen bonding. Tetrahedron dimethyl ester with superoxide ion. Tetrahedron Lett 1984;25:6037–40. 1997;53:2697–716. 15. Chen Q, Huggins MT, Lightner DA, et al. Synthesis of a 10-oxo-bilirubin: 4. Blanckaert N, Heirwegh KPM, Zaman Z. Comparisons of the biliary excretion Effect of the oxo group on conformation, transhepatic transport and of the four isomers of bilirubin-IX in Wistar and homozygous Gunn rats. glucuronidation. J Am Chem Soc 1999;121:9253–64. Biochem J 1977;164:229–36. 16. Tipton AK, Lightner DA, McDonagh AF. Synthesis and metabolism of the first 5. Chowdhury JR, Wolkoff AW, Chowdhury NR, et al. Hereditary jaundice and thia-bilirubin. J Org Chem 2001;66:1832–8.

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