J . Am. Chem. SOC.1992, 114, 10123-10133
10123
Absolute Configuration of Bilirubin Conformational Enantiomers Stefan E. Boiadjiev,’l Richard V. P e r s o n , l P Cisbert P u z i c h a , l a Carolyn Knobler,lb Emily Maverick,IbKenneth N. Trueblood,lband David A. Lightner*yla Contribution from the Department of Chemistry, University of Nevada, Reno, Nevada 89557-0020, and Department of Chemistry and Biochemistry, University of California, Los Angeles, California 90024. Received March 26, 1992 Abstract: Bilirubin, the yellow pigment of jaundice, is a bichromophoric tetrapyrrole formed in mammals by heme catabolism.
It readily adopts either of two enantiomeric folded conformations which are shaped like ridge tiles and are stabilized by a network of intramolecular hydrogen bonds. Interconversion of the conformational enantiomers is rapid at room temperature and may be displaced toward either enantiomer by complexation with chiral agents. Intramolecular steric effects may also affect enantioselection. Thus, introduction of a methyl group at each of the 8- and @’-carbonsof the propionic acid chains on the symmetric bilirubin analog, mesobilirubin XIIIa, can shift the conformational equilibrium toward either the M-or the P-chirality intramolecularly hydrogen-bonded conformer, depending only on the R or S stereochemistry at 0 and 0’. With the appropriate R or S configurations,intense bisignate circular dichroism (CD) may be detected for the -430-nm transition(s) that is characteristic of exciton coupling between the component dipyrrinone chromophores. The absolute configuration of the fi,B’-dimethylmesobilirubinXIIIa exhibiting a negative chirality CD exciton couplet (ACE:-337, ACE;+186 in CHC13) by X-ray crystallography of the brucine salt of a monopyrrole synthetic precursor, (+)is firmly established as @S,@’S (S)-3-[2,4-dimethyl-5-(ethoxycarbonyl)-lH-pyrrol-3-ylI butanoic acid (5). Molecular dynamics calculations on the j3S,@’S enantiomer confirm a strong preference (-20 kcallmol stabilization) for intramolecularly hydrogen-bonded conformational enantiomers in which the M-chirality is favored over the P-chirality (by -4 kcallmol). For the first time, the absolute configuration of a bilirubin ridge-tile conformational enantiomer has been unequivocally established.
Introduction Bilirubin (Figure l), the cytotoxic yellow-orange tetrapyrrole pigment of j a ~ n d i c econsists ~ , ~ of two dipyrrinone units conjoined by a -CH2- group at (2-10. As in structurally simpler molecular propellers, such as diphenylmethane? rotation of the dipyrrinones about the bonds to the central -CHI- generates a large array of conformations, one of which is unique in being near or at the global energy minimum5v6and, particularly, in positioning the propionic acid -COOH group of one dipyrrinone unit in an ideal location for intramolecular hydrogen bonding with the pyrrole and lactam N-H and C = O residues of the other dipyrrinone. Stabilization of this conformation by a complementary network of intramolecular hydrogen bonds was first detected in the solid (by X-ray crystallography),’ where bilirubin is folded into either of two ridgetileshaped enantiomers (Figure 1). Intramolecular hydrogen bonding has also been detected in solution by NMR,8,9where the bilirubin conformers are thought to persist as a pair of interconverting conformational enantiomers2J0 in solvents that do not strongly perturb the matrix of intramolecular hydrogen bonds. Although the situation is less clear for solvents or other agents that may disrupt the folded pigment’s hydrogen-bonding matrix, in the strong hydrogen bond acceptor (CD3)2S0, 13C-NMR analysis of segmental motion in the propionic acid chains indicates that the -COOH residues are tethered to the dipyrrinones via bound solvent molecules.” Although it is probably folded,I2 the (1) (a) University of Nevada. (b) University of California. (2) Lightner, D. A.; McDonagh, A. F. Acc. Chem. Res. 1984,17,417-424. (3) For leading references, see: Gollan, J. L. (Guest Ed.). Seminars in Liver Disease 19%8,8, 103-199, 272-283. (4) Barnes, J. C.; Paton, J. D.; Damewood, J. R., Jr.; Mislow, K. J . Org. Chem. 1981, 46, 4975-4979. ( 5 ) For leading references, see: Falk, H. The Chemistry of Linear Oligopyrroles and Bile Pigmenfs; Springer-Verlag: Wien, New York, 1989. (6) Lightner, D.; Person, R.; Peterson, B.; Puzicha, G.; Pu, Y.-M.; Bojadziev, S. In Eiomolecular Spectroscopy II; Birge, R., Nafie, L., Eds.; Proc. SPIE-The International Society for Optical Engineering, Bellingham, WA, 1432; 1991; pp 2-3. (7) (a) Bonnett, R.; Davies, J. E.; Hursthouse, M. B.; Sheldrick, G. M. Proc. R . SOC.London, E 1978, 202, 249-268. (b) LeBas, G.; Allegret, A,; Mauguen, Y.; DeRango, C.; Bailly, M. Acta Crystallogr., Sect. E 1980,836, 3007-301 1. (8) Trull, F. R.; Ma, J.-S.;Landen, G. L.; Lightner, D. A. I s r . J . Chem. 1983, 23, 211-218. (9) Kaplan, D.; Navon, G. Isr. J . Chem. 1983, 23, 177-186. (10) (a) Manitto, P.; Monti, D. J . Chem. SOC.,Chem. Commun. 1976, 122-123. (b) Navon, G.; Frank, S.; Kaplan, D. J. Chem. SOC.,Perkin Trans 2 1984, 1145-1 149.
0002-7863192115 14-10123$03.00/0
precise conformation of the pigment in (CH3)2S0is uncertain.13 Molecular dynamics calculations on bilirubin confirm the importance of intramolecular hydrogen bonding. The conformational energy map (Figure 2) for rotations of the dipyrrinones about C-10 reveals a collection of isoenergetic global minima, which correspond to either identical or mirror image structures represented by the M-and P-chirality conformers of Figure 1. Interestingly, aside from differences due to enantiomerism, the global energy minimum conformation of bilirubin is essentially the same, whether hydrogen bonding is present or absent. Thus, with full hydrogen bonding, the global energy minimum for the P-chirality conformer lies at 4, = $2 = 64”;in the absence of hydrogen bonding, it lies at t$I = N 70”. However, the stabilization due to intramolecular hydrogen bonding is potentially considerable, which we estimate to be -20 kcal/mol. This suggests that other conformers are essentially absent and that studies of bilirubin conformation should, as a starting point, focus on intramolecularly hydrogen-bonded structures. The “internal” stereochemistry and nonbonded steric interactions in the intramolecularly bonded conformers are quite revealing. Close inspection of the steric environment of each of the diastereotopic hydrogens in the -CHI-CHI- fragment of the intramolecularly hydrogen-bonded propionic acid groups suggests a way to displace the M P equilibrium of Figure 1. Thus, when folded into the M-chirality ridge-tile enantiomer, the pro-R j3hydrogen (but not the pro-S) is brought into close nonbonded contact with the central -CH2- group a t C-10, as illustrated in Figure 3 for the symmetric bilirubin analog, mesobilirubin XIIIa. HOOC
COOH
Mesobillrubin-Xlllo
On the other hand, in the P-chirality enantiomer, it is the pro-S j3-hydrogen that is buttressed against the C-10 -CH2-. group. Consequently, when mesobilirubin or bilirubin adopts either of the thermodynamically preferred intramolecularly hydrogen(11) Kaplan, D.; Navon, G. Eiochem. J. 1982, 201, 605-613. (12) Gawrofiski, J. K.; Polonski, T.; Lightner, D. A. Tetrahedron 1990, 24, 8053-8066.
(13) Hsieh, Y.-Z.; Morris, M. D. J. Am. Chem. SOC.1988, 110, 62-67.
0 1992 American Chemical Society
Boiadjiev et al.
10124 J. Am. Chem. Soc., Vol. 114, No. 26, 1992 bonded ridge-tile conformations, one conformational enantiomer can be destabilized relative to the other through allosteric action by judicious replacement of hydrogens on the propionic acid chain by methyl groups. For example, insertion of a methyl group at the pro-S site on the j3-carbon of the propionic acid would be expected to destabilize the P-chirality intramolecularly hydrogen-bonded conformational enantiomer by introducing a severe nonbonded CH3(CH2steric interaction with the C-10 -CH?: q o u p (Figure 4). This would shift the conformational equilibrium toward M. In contrast, introduction of a methyl group at the prpR site would destabilize the M-chirality enantiomer and shift the equilibrium toward P. Introduction of such methyl groups might thus be expected to force the resolution of bilirubin through intramolecular steric interaction, assuming that intramolecular hydrogen bonding remains a potent conformation-stabilizing force. In order to examine this principle, we targeted the synthesis of a bilirubin analog, &3’-dimethylmesobilirubin XIIIa ( l ) , where we could achieve an optical resolution and determine the absolute configuration at an early step. HOOC
H
H
Results and Discussion Synthesis. A stereospecific total synthesis of the S S and R,R enantiomers of &3’-dimethylmesobilirubin XIIIa was devised in which enantiomeric resolution was achieved at the monopyrrole stage. The stereogenic centers (/3,j3’)found in the target pigment 1 are carried along by monopyrrole precursor 6 (Scheme I) and originate early in the synthesis, having been introduced by a Michael reaction of pentane-2,4-dione with methyl crotonate. When racemic (R,S)-6 is converted ultimately into 1, two diastereomers are obtained: the meso diastereomer (IC),which has the RS configuration a t the &@’-centers, and the racemic diastereomer ( l a + lb), which consists of the R,R and S,Senantiomers. Diastereomer ICamounts to 50%of the mixture and can be separated from ( l a lb) by chromatography. The target optically active compounds (@S,TS)-and (j3R,,9’R)dimethylmesobibm XIIIa (la and lb) were prepared from optically active precursors Sa and Sb, respectively (Scheme I), which were resolved to 100% and 80% enantiomeric excess (ee), respectively, by fractional crystallization of the brucine salts. Thus, Sa could be saponified to diacid 4a, which, upon heating with (bromomethy1ene)oxopyrrole 714in refluxing C H 3 0 H , gave dipyrrinone 38 ([q]20D+61.8’ (c 0.8, CHC13)). Chloranil-promoted oxidative coup1ingl5 of 3a afforded the mesobiliverdin dimethyl ester 2a ([aIm4%-2720’ (c 0.0037, CHCI,)). After saponification of 2a, NaBH, reduction yielded l a ( [aI2’D -4730O (c 0.0086, CHC13)). Synthesis of racemic 6 was accomplished by Fischer-Knorr type conden~ation’~ of nitrosated ethyl acetoacetate and methyl 4-acetyl-3-methyl-5-oxohexanoatein 20% yield. The atypically low yield was caused, presumably, by the introduction
+
~~~
~~
BILIRUBIN
\J
\ /
ti
H
In the following, we report on the total synthesis and circular dichroism of the bilirubin analog (-)-(bS,B’S)-dimethylmesobilirubin XIIIa (la) and on the X-ray crystallographic determination of the absolute configuration of its precursor (+)(S)-3- [2,4-dimethyl-5-(ethoxycarbonyl)lH-pyrrol-3-ylI butanoic acid (Sa), as the brucine salt. These results are important because (i) they present an unequivocal experimental determination of the absolute configuration as M-helicity for the negatively rotating bilirubin conformational enantiomer, (ii) they c o n f i i the presence and significance of intramolecular hydrogen bonding in stabilizing conformation, and (iii) they provide useful molecular models for assessing the structure of bilirubin in biological tissues and fluids.
~
COOH
COOH
~,~‘-Dimethylmesobilirubin-Xllla
~~
HOOC
~
(14) Shrout, D. P.; Lightner, D. A. Synthesis 1990, 1062-1065. (15) Shrout, D. P.; hzicha, G.; Lightner. D. A. Synthesis 1992, 328-332. Lightner, D. A . J . Heterocycl. Chem. (16) Trull, F. R.; Franklin, R. W.; 1987, 24, 1573-1579.
Figure 1. (Upper) Porphyrin-like representation of (42,15Z)-bilirubin IXa, whose two dipyrrinone chromophores are connected by a C H I group. Rotation of the two dipyrrinones, like propeller blades, about the central -CH2- (rotation angles #I and b2) converts this nearly planar shape (bI = b2 = OD) into a folded shape in which the propionic acid groups are brought into sufficiently close nonbonded proximity (bI = b2 H 60° for P,and bI = b2 H d o D for M) to facilitate intramolecular hydrogen bonding to the opposing dipyrrinone lactam and pyrrole groups. (Lower) Three-dimensional representations for two enantiomeric intramolecularly hydrogen-bonded conformations, which are shaped like ridge tiles. These conformers interconvert by breaking a set of hydrogen bonds and then rotating one dipyrrinone about C-10 followed by re-forming the hydrogen bonds. (Inset) The chirality or helicity of the two enantiomers is shown as M or P,according to the relative helical orientation of the long wavelength (-430 nm) transition induced electric dipole moments that are oriented along the long axis of each of the two dipyrrinone chromophores of each molecule.
of steric ortho type hindrance between the propionic acid @methyl group and the pyrrole ring methyls at C-2 and C-4. A better alternative involved condensation of nitrosated diethyl malonate in place of nitrosated ethyl acetoacetate, and this reaction gave 6 in 60% yield. Diester 6 was selectively saponified to give racemic monoacid 5, which was resolved as the 1:l salt with brucine*4H20 by fractional crystallization from acetone until the acid (Sa) maintained a constant rotation, [.]20D +30S0 (c 0.8, ethanol). The absolute configuration of the highly resolved acid was determined by X-ray crystallography (below) of its 1:l salt with brucine. Highly resolved enantiomeric acid Sb, [al2’D -24.4O (c 1.3, ethanol), was obtained from the mother liquors by repeated fractional crystallization, and it was converted to (BR,TR)-dimethylmesobilirubin XI& (lb), [aI2’D = +4880° (c 0.0075, CHC13), as outlined in Scheme I. The synthesis of l b illustrates an interesting principle, which results in resolution by synthesis. When 5b of 80% ee is carried through the synthetic scheme, the coupling step that converts dipyrrole 3 to tetrapyrrole 2 presents an opportunity for increasing the ee of the desired product (lb). Assuming equal rates of coupled oxidation, 3b with 80% ee should give 2 containing the statistical mixture of isomers: 81% 2b, 1% 2a, and 18% 2c. In fact, this is essentially what we find, and since the meso diastereomer IC derived from 2c can be removed easily at the final product stage (l), the desired product (lb) can be obtained very simply in >97% ee starting with 5b of only 80%ee. Molecular Geometry and Absolute Configuration from X-ray Crystallography. In the 1:l salt of Sa with brucine, a water molecule links the cation and the anion via the brucine amide 0 and a pyrrole acid 0 (Figure 5 ) , with nearly linear H-bonds. The other acid 0 is H-bonded to the -NH+ of a second brucine cation,
J. Am. Chem. Soc.. Vol. 114. No. 26.1992 10125
Bilirubin Conformational Enantiomers
M
P
R p 3. Ball and stick conformational representations for the ridge-tilc shape M-and P-chirality intramolecularly hydrogen-bonded. interconverting enantiomers of mesobilirubin XIIlu. In the propionic acid side chains attached to pyrrole ring carbons C-8 and C-12. the hydrogens on the @ and @'
