©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
On The Structure of Bilirubin in Solution
C{^1H} HETERONUCLEAR OVERHAUSER EFFECT NMR ANALYSES IN AQUEOUS BUFFER AND ORGANIC SOLVENTS (*)

(Received for publication, September 15, 1994; and in revised form, October 20, 1994)

Daniel Nogales David A. Lightner (§)

From the Departments of Chemistry and Biochemistry and the Cell and Molecular Biology Program, University of Nevada, Reno, Nevada 89557-0020

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
CONCLUDING COMMENTS
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Select hydrogen-carbon distances have been determined from C{^1H} heteronuclear Overhauser effects observed in a 99% carbon-13 enriched CO(2)H bilirubin analog, [8^3,12^3-C(2)]-mesobilirubin XIIIalpha. Analysis of the data confirms that the propionic acid carbonyl lies within hydrogen bonding distance to the dipyrrinone lactam and pyrrole N-H groups in chloroform and indicates, surprisingly, that those distances are only slightly longer in dimethyl sulfoxide solvent or when the carboxyl group is ionized in pH 7.4 aqueous buffered solutions of the pigment. The data supports the presence and persistence of folded, intramolecularly hydrogen-bonded bilirubin conformations in solution.


INTRODUCTION

Bilirubin (Fig. 1),) the cytotoxic yellow-orange pigment of jaundice is biosynthesized in normal metabolism of healthy adults at the rate of about 300 mg per day. This water-insoluble and lipophilic linear tetrapyrrole dicarboxylic acid is found characteristically in mammals, where it is produced in copious quantities by catabolism of heme, principally from the hemoglobin of red blood cells(1, 2) . Bilirubin is essentially unexcretable in its native form but is efficiently cleared by the body following conversion in the liver to water-soluble glucuronides that are promptly secreted into bile. Accumulation of bilirubin and its glucuronides in the body is caused by certain pathologic conditions and sometimes results in jaundice(3) . Although impaired excretion of the glucuronides occurs in many types of hepatobiliary disease, retention of native bilirubin is principally observed in newborn babies(4) . What limits the facile excretability of bilirubin is its poor water solubility (estimated K 3 times 10M for bilirubin in water at 37 °C)(5) , high lipid/water partition coefficient(6) , and proclivity to form association complexes with serum albumin and other proteins(1, 2, 3, 5, 6) , three interrelated properties that dominate the transport and metabolism of the pigment in vivo(1, 2, 3, 7) .


Figure 1: A, porphyrin-like and B, linear representations of bilirubin. C, linear representation of mesobilirubin-IValpha.



Although the structure of bilirubin has been well known since it was proved over 50 years ago(8) , the interesting biological and unusual solubility properties of bilirubin (6) do not correlate well with conventional linear representation (Fig. 1B) favored by Fischer (8) or the porphyrin-like structure (Fig. 1A) that would follow logically from the structure of the heme precursor. If bilirubin adopted such conformations, which we now know are sterically disfavored, it would be predictably polar, not lipophilic, and very likely excretable across the liver into bile without resort to glucuronidation(9) . The unusual properties of bilirubin are hardly changed in its analogs with vinyl groups reduced to ethyls as in mesobilirubin IXalpha, or even when they are symmetrically arranged as in mesobilirubin IIIalpha or XIIIalpha. But major changes attend relocating the propionic acid groups away from C-8 and C-12. For example, transposing the propionic acid groups with the adjacent methyl groups at C-7 and C-13 yields a pigment, mesobilirubin IValpha (Fig. 1C), that is more polar, and excretable without requiring glucuronidation or other structural modification(9, 10) .

Correlation of propionic acid group location and the chemical, biological, and physical properties of bilirubin is, however, only one important structural consideration. Other stereochemical determinants include configuration and conformation of the tetrapyrrole skeleton, elements of three-dimensional structure common to all bilirubin pigments. Conformation plays a key role, especially the changes in shape brought about by rotations of the dipyrrinone groups about the central CH(2) groups at C-10. Such rotations bring about major stereochemical changes, interconverting the linear and porphyrin-like structures of Fig. 1, A and B, inter alia, and generating a vast array of pigment shapes, of which one type is possessed of unique properties. In this conformation, the dipyrrinones are rotated about the C-9 to C-10 and C-10 to C11 carbon-carbon single bonds so as to create a ridge-tile shape where the propionic acid CO(2)H groups and dipyrrinone NH and C=O groups are brought into sufficiently close proximity to allow them to engage in intramolecular hydrogen bonding. Such intramolecular hydrogen bonding stabilizes the ridge-tile conformation (Fig. 2) to such an extent that it is thought to persist and have a dominating influence on conformation under a variety of solvation and ionization conditions. It is also thought to explain many properties of the molecule, such as its high oil-water partition coefficient(6) , its resistance to hepatobiliary excretion(9) , and, unusual for a carboxylic acid, its insolubility in aqueous sodium bicarbonate solution(6) .


Figure 2: Interconverting intramolecularly hydrogen-bonded enantiomeric conformers of bilirubin.



Stabilization of the bilirubin ridge-tile conformation by a complementary network of intramolecular hydrogen bonds was first detected by x-ray crystallography (11, 12) in the solid where bilirubin is folded into either of two ridge-tile-shaped enantiomers (Fig. 2). Intramolecular hydrogen bonding has also been detected by nuclear magnetic resonance (NMR) (^1)in solution(13, 14) , where the bilirubin conformers are thought to persist as a pair of interconverting conformational enantiomers (Fig. 2), and in organic solvents that do not strongly perturb the matrix of intramolecular hydrogen bonds. The conformational structure is less clear in solvents or other agents that may disrupt the folded pigment's hydrogen bonding matrix. In the strong hydrogen bond acceptor, (CD(3))(2)SO, C NMR analysis of segmental motion in the propionic acid chains indicates that the -COOH residues are tethered to the dipyrrinones via bound solvent molecules (15) , but the precise conformation of the pigment in (CH(3))(2)SO is uncertain(16) . In metabolism, bilirubin is found bound to albumin or other proteins, in membranes, micelles or to a lesser extent, in aqueous solution. But here again, the conformation of bilirubin is poorly understood. Yet, an intramolecularly hydrogen bonded conformation is thought to be essential for hepatic glucuronidation and excretion(9) . In order to understand whether intramolecular hydrogen bonding persists in solutions of bilirubin, we have prepared [8^3,12^3-C(2)]-mesobilirubin XIIIalpha, measured the NMR C{^1H} heteronuclear nuclear Overhauser effects (NOE) between its dipyrrinone N-H's and propionic acid CO(2)H groups, and calculated their non-bonded distances. These results are important because they help define a comprehensive picture of intramolecular hydrogen bonding and the probable shape and stereochemistry of bilirubin in solution, and also illustrate a method for determining the structure of bilirubin in biological tissues.


EXPERIMENTAL PROCEDURES

[8^3,12^3-C(2)]-Mesobilirubin XIIIalpha (99% C-enriched), its dimethyl ester and methyl [8^3-C]-xanthobilirubinate were prepared as described previously(17) . The tetra-n-butylammonium salt was prepared as described previously (18) by suspending [8^3,12^3-C(2)]-mesobilirubin XIIIalpha (5.0 mg, 7.5 times 10 mmol) in dichloromethane (2 ml) and adding excess methanolic tetra-n-butylammonium hydroxide (Aldrich) (15.0 µl of 1 M, 1.5 times 10 mmol). The solution was evaporated to dryness with a stream of dry N(2), then dried overnight under vacuum (0.1 torr) over P(2)O(5). NMR solutions ranging from 7.5 times 10 to 1.5 times 10M were prepared in deuterated solvents, CDCl(3) or (CD(3))(2)SO, obtained from Cambridge Isotope Labs. NMR solutions 3.5 times 10M in aqueous medium were prepared by dissolving the bis-tetra-n-butylammonium salt of CO(2)H-labeled mesobilirubin XIIIalpha in (CD(3))(2)SO, then diluting with pH 7.4 0.1 M phosphate buffer to give a final solution with 30% (volumes) (CD(3))(2)SO. The final pH is 7.4 and the concentration of (CD(3))(2)SO in the aqueous buffer is 3.8 M (for a molar ratio of 15:1, H(2)O:(CD(3))(2)SO).

All experiments were performed on a General Electric GN-300 NMR spectrometer. All samples were degassed with argon and shielded from light. T(1) measurements of the C-labeled pigments were performed by the inversion recovery method. All 75.58-MHz C spectra were collected using a 90° pulse width of 18.0 s. The C spectra was obtained coupled, with a minimum of 256 scans and a pulse delay of 20 s (4 times T(1)). The printed carbon resonances were digitized using Un-Plot-It and the areas were determined by the Un-Plot-It software. No further data manipulation was performed on the raw data.

C{^1H} Heteronuclear NOEs were determined by pulse techniques developed previously(19, 20, 21, 22, 23, 24) and involve irradiating selected hydrogens (^1H) while observing signal enhancement of the COOH C NMR resonance. The semiquantitative NOE values (an average of three to five independent determinations and reported as average percent NOE) were determined by subtracting integrated areas of the COOH off-resonance from the COOH resonance observed during selective ^1H saturation irradiation then divided by the resonance peak area. For NOE determinations in aqueous buffer, a 1331 water suppression experiment was used in order to reduce the ^1H NMR signal due to H(2)O(25) . Normal ^1H saturation experiments could not be used due to the exchange of the N-H's with solvent. The 1331 technique does not reduce the magnitude of the H(2)O signal in the NMR; rather, it applies a wave to the spectrum where no data points are taken in the region of the H(2)O resonances. Data points are taken on each side of the H(2)O resonance.

Approximate non-bonded ^1H to C interatomic distances were calculated from the NOE results by calibration to a known ^1H to C reference distance: in our work, the C to alpha-H distance (2.12 Å) in the propionic acid segment, -C(beta)H(2)-C(alpha)H(2)-COOH. The formula (20) used for relating NOE to distance is,

where r is the non-bonded distance between the COOH carbon and the proton of interest, (r) (ref.) = 2.12 Å and C{^1H} (ref.) = 38% between the alpha-H to carboxylic acid carbon. The alpha-H to CO(2)H carbon distance is constant for either alpha-H and is fixed by the H-C and C-C(=O) bond distances and the H-C-C angle. These vary only insignificantly in propionic acid. In CDCl(3) solvent, the two alpha-hydrogens have distinctly different chemical shifts. Irradiation of either gives exactly the same C{^1H} NOE (38%), and that value was used for estimating the N-H bulletbulletbullet C(=O) non-bonded distances of this study according to the equation above.


RESULTS AND DISCUSSION

In NMR spectroscopy, the NOE occurs when the net intensities of resonances change while a selected resonance is perturbed by saturation or inversion. Its importance lies primarily in the fact that the resonances which change their intensities are those from spin systems that lie close to those directly affected by the perturbation(19, 26) . This means that when the non-bonded distance between two hydrogens is small, irradiation of one hydrogen can lead to an enhanced signal for the other. With appropriate calibration non-bonded interatomic distances may be calculated from NOE measurements(19, 27) . Such homonuclear NOE's have been seen in bilirubin between the dipyrrinone pyrrole and lactam hydrogens(13) , which lie some 2 Å apart and between the C-5 H and C-7 CH3, and between the C-15 H and C-13 CH(3) which lie some 3 Å apart. They have provided evidence for the syn-Z configuration in the dipyrrinones of bilirubin in solution (28) and for conformational enantioselection forced by non-bonded steric effects in bilirubin analogs with methyl groups on the alpha or beta sites on the propionic acid chains(29, 30) .

Although NOE studies are most common in ^1H{^1H} homo-NOE experiments, they are not limited to such and have been expanded to include more complicated heteronuclear experiments, especially those involving ^1H with C resonances (21) or ^1H and P resonances(21) . Recently, heteronuclear C{^1H} NOE measurements have played a major role in detecting hydrogen bonds (^1HbulletbulletbulletO=C) and determining the secondary structure of peptides and oligonucleotides(19, 20, 22) . In even simpler systems, (E)- and (Z)-stereoisomers of 4(alpha-arylethylidene)-2-phenyl-5(4H)-oxazolones could be distinguished by C{^1H} NOE measurements (23) , while hydrogen bonding of water to 2-pyridones has been studied by C{^1H} two-dimensional NOE spectroscopy (24) . In an especially relevant example, C{^1H} heteronuclear NOE measurements on valinomycin were used to identify the amide carbonyl and NH partners involved in the intramolecular hydrogen bonding that preserves its secondary structure(20) .

In our C{^1H} NOE study of CO(2)H-labeled mesobilirubin, in either deuterated chloroform (CDCl(3)) or deuterated dimethyl sulfoxide ((CD(3))(2)SO) solvents, saturation irradiation of either the pyrrole or lactam N-H's led to an enhancement of the COOH signal in the C NMR. The results are shown in Fig. 3and provide the first clear evidence that the N-H and CO(2)H groups of bilirubin in solution lie very close to one another. The non-bonded H to C (N-H to COOH) distances calculated from the observed NOE's in CDCl(3) are very nearly the same as those found in crystals of bilirubin, where the pigment has been shown to adopt an intramolecularly hydrogen bonded ridge-tile conformation(11, 12) . They are also very nearly the same as those found in the global minimum energy conformation of bilirubin and mesobilirubin XIIIalpha by molecular dynamics calculations (31) (Fig. 3). The pigment in CDCl(3) solvent is thus shown by C{^1H} NOE spectroscopy to adopt the hydrogen-bonded ridge-tile conformation.


Figure 3: A, C{^1H} NOE enhancements of the COOH resonance at 179.3 in CDCl(3) and 174.0 in (CD(3))(2)SO by saturation irradiating the pyrrole and lactam hydrogens. B, approximate non-bonded H to C distances (A) in the N-HbulletbulletbulletO=C hydrogen bond matrix calculated from the observed C{^1H} NOE enhancements are indicated on the structure. C, non-bonded H to C distances (in the hydrogen bond matrix found by molecular dynamics computations in the global energy minimum ridge-tile conformation are indicated on the structure. In B, the upper set of distances are for CDCl(3) solvent; the lower set are for (CD(3))(2)SO solvent. The HbulletbulletbulletO distances in C found by molecular dynamics are 1.6, as compared with 1.7 in the crystal ( (11) and (12) ).



The C{^1H} NOE's seen in (CD(3))(2)SO solvent are somewhat smaller than those found in CDCl(3) (Fig. 3A), indicating a slightly longer distance (Table 1) between pyrrole and lactam N-H's and the COOH. The data suggest that dimethyl sulfoxide does not completely break the matrix of intramolecular hydrogen bonds but only slightly weakens the hydrogen-bonded ridge-tile conformation, as suggested earlier by C NMR T(1) relaxation times of the carbons in the propionic acid chains (13, 15, 32) and by circular dichoism spectroscopy(33, 34) .



In strong contrast, no C{^1H} NOE's (<0.1) are found between the N-H's and CO(2)CH(3) groups of [8^3,12^3-C(2)]-mesobilirubin XIIIalpha dimethyl ester. The data suggest large distances between the dipyrrinone N-H groups and the propionic ester carbonyl carbon, providing no support for an intramolecularly hydrogen-bonded conformation in either chloroform or dimethyl sulfoxide. Although bilirubin dimethyl ester is thought to be monomeric in the latter solvent, in the former a dimer is favored, as shown by vapor phase osmometry(35) .

Do intramolecularly hydrogen-bonded conformations of bilirubin persist when the propionic acid groups are ionized to carboxylate anions? The answer is apparently yes, as learned from C{^1H} NOE measurements on the bis-tetra-n-butylammonium salts of [8^3,12^3-C(2)]-mesobilirubin XIIIalpha (Fig. 4). In CDCl(3) solvent, the NOE enhancements found are comparably strong to those observed from the free acid; whereas, in (CD(3))(2)SO solvent the NOE enhancements are somewhat larger than in the free acid. It would appear that bilirubin carboxylate anions are just as tightly hydrogen-bonded in chloroform solution as the free acids. And in dimethyl sulfoxide solution the dicarboxylate anions are probably even more strongly hydrogen bonded than the free acids. The data suggest that in the free acid dimethyl sulfoxide solvent is more attracted to the carboxylic acid OH group than to the dipyrrinone N-H's.


Figure 4: A, C{^1H} NOE enhancements of the CO(2) resonance at 179.5 in CDCl(3) and 177.6 in (CD(3))(2)SO by saturation irradiating the pyrrole and lactam hydrogens. B, approximate H to C non-bonded distances (Å) in the N-HbulletbulletbulletO=C hydrogen bonded pairs calculated from the observed C{^1H} NOE enhancements are indicated on the structure. C, H to C non-bonded distances (Å) in the hydrogen bonding matrix found by molecular dynamics computation in the global energy minimum ridge-tile conformation are indicated on the structure. In B, the upper set of distances are from CDCl(3) solvent; the lower set are from (CD(3))(2)SO solvent.



Is bilirubin in pH 7.4 aqueous buffer intramolecularly hydrogen bonded? When [8^3,12^3-C(2)]-mesobilirubin XIIIalpha is dissolved in a small quantity of (CD(3))(2)SO and diluted with 0.1 M pH 7.4 phosphate buffer, the C NMR resonance for the propionic carboxyl ( 181.9 ppm downfield from (CH(3))(4)Si standard) is observed at the same position ( 181.9 ppm) as in a solution where the bis-tetra-n-butylammonium salt is similarly dissolved. The carboxyl resonance is thus apparently insensitive to the cation used (tetra-n-butylammonium versus sodium or potassium) for the carboxylate anion. When the bis-tetra-n-butylammonium salt is dissolved in H(2)O, 10% D(2)O (no (CD(3))(2)SO) at pH 8.0 or 11.0, the carboxyl resonance appears at essentially the same shielding, 182.5 and 182.4, respectively. Since the carboxyl C NMR resonances of carboxylic acids are known to be sensitive to ionization ( 181.5 for acetate ion versus 176.8 for acetic acid)(36) , the observed resonances for our bilirubin analog are consistent with predominantly ionized (carboxylate ion) species at pH 8, 11, and even 7.4.

The C{^1H} NOE's observed for [8^3,12^3-C(2)]-mesobilirubin XIIIalpha in pH 7.4 aqueous phosphate buffer containing 3.8 M (CD(3))(2)SO (Fig. 5) are significant and meaningful. Although smaller than those seen for the anion in either CDCl(3) or pure (CD(3))(2)SO, they are comparable to those seen for the acid in (CD(3))(2)SO. The weaker NOE's are consistent with a somewhat more open or flatter ridge-tile conformation, and the computed N-H to C=O distance is still within the range found for long hydrogen bonds (^1HbulletbulletbulletO=C). The data are summarized in Table 1.


Figure 5: A, C{^1H} NOE enhancements of the CO(2) resonance at 181.9 by saturation irradiating the pyrrole and lactam hydrogens. B, approximate H to C non-bonded distances (Å) in the N-HbulletbulletbulletO=C hydrogen bonded pairs calculated from the observed C{^1H} NOE enhancements are indicated on the structure. C, H to C non-bonded distances (Å) found in the hydrogen bond matrix by molecular dynamics computations in the global energy minimum ridge-tile conformation.




CONCLUDING COMMENTS

The results of the C{^1H} NOE study on [8^3,12^3-C(2)]-mesobilirubin XIIIalpha and its bispropionate anion indicate that the carboxylic acid and carboxylate anion carbons are sufficiently close to the dipyrrinone pyrrole and lactam N-H groups to produce an NOE, but the carboxylic ester carbon is not. The larger NOEs are found in the more lipophilic solvent (CDCl(3)) as might be expected, where the calculated pyrrole/lactam N-H to COOH non-bonded H to C distances are essentially the same as those computed by molecular dynamics or found in crystals of bilirubin. As might also be expected, the smaller NOEs are found in the more polar solvents, (CD(3))(2)SO and H(2)O, where solvent is thought to interfere with the intramolecular hydrogen bonds. What is surprising, perhaps, is that the NOEs are found in all of these solvents. The data clearly indicate, however, that in the acid, and even in the propionate anion, the carboxyl group lies close to the dipyrrinone NH groups. In fact, the computed distance between the carboxyl carbon and dipyrrinone N-H's lies within the distance of a long hydrogen bond, either N-HbulletbulletbulletO=C(-OH) or N-HbulletbulletbulletO=C(-O) N-HbulletbulletbulletO-C(=O).

These results are important because they reveal the persistence of intramolecular hydrogen bonding in bilirubin and its anions, in organic (CDCl(3), (CD(3))(2)SO) solvents, and in water. They suggest a way to learn the conformation of bilirubin in lipids, on proteins, or in intact cells or other biological tissues using C{^1H} heteronuclear NOE measurements. They indicate that key steps in bilirubin metabolism, such as glucuronidation, might be followed by C NMR spectroscopy. They also suggest that bilirubin pK values in microenvironments might be determined from the CO(2)H-labeled pigment.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant HD-17779. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 702-784-4980; Fax: 702-784-6804.

(^1)
The abbreviations used are: NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect.


ACKNOWLEDGEMENTS

We sincerely thank Lewis Cary for help with the NMR experiments and D. Timothy Anstine for help in determining non-bonded distances by molecular dynamics calculations.


REFERENCES

  1. McDonagh, A. F. (1979) in The Porphyrins (Dolphin, D., ed) Vol. 6, pp. 293-491, Academic Press, New York
  2. Ostrow, J. D. (ed) (1986) Bile Pigments and Jaundice , Marcel-Dekker, New York
  3. Gollan, J. L. (1988) Semin. Liver Dis. 8(2) ; 8(3)
  4. Maisels, M. J. (1990) Clinics in Perinatology: Neonatal Jaundice, Vol. 17, W. B. Saunders, Philadelphia, PA
  5. Brodersen, R. (1986) in Bile Pigments and Jaundice (Ostrow, J. D., ed) p. 158, Academic Press, New York
  6. McDonagh, A. F., and Lightner, D. A. (1985) Pediatrics 75, 443-455 [Abstract]
  7. Tiribelli, C., and Ostrow, J. D. (1993) Hepatology 17, 715-736 [Medline] [Order article via Infotrieve]
  8. Fischer, H., Plieninger, H., and Weissbarth, O. (1941) Hoppe-Seyler's Z. Physiol. Chem. 268, 197-226
  9. McDonagh, A. F., and Lightner, D. A. (1991) in Hepatic Metabolism and Disposition of Endo and Xenobiotics (Bock, K. W., Gerock, W., and Matern, S., eds) pp. 47-59, Falk Symposium No. 57, Kluwer, Dordrecht, The Netherlands
  10. Trull, F. R., Franklin, R. W., and Lightner, D. A. (1987) J. Heterocycl. Chem. 24, 1573-1579
  11. Bonnett, R., Davies, J. E., Hursthouse, M. B., and Sheldrick, G. M. (1978) Proc. R. Soc. Lond. Ser. B 202, 249-268 [Medline] [Order article via Infotrieve]
  12. LeBas, G., Allegret, A., Mauguen, Y., DeRango, C., and Bailly, M. (1980) Acta Crystallogr. Sect. B B36, 3007-3011 [CrossRef]
  13. Kaplan, D., and Navon, G. (1983) Isr. J. Chem. 23, 177-186
  14. Trull, F. R., Ma, J. S., Landen, G. L., and Lightner, D. A. (1983) Isr. J. Chem. 23(2), 211-218
  15. Kaplan, D., and Navon, G. (1982) Biochem. J. 201, 605-613 [Medline] [Order article via Infotrieve]
  16. Hsieh, Y.-Z., and Morris, M. D. (1988) J. Am. Chem. Soc. 110, 62-67
  17. Nogales, D. F., and Lightner, D. A. (1994) J. Labelled Compds & Radiopharm. 34, 453-462
  18. Pu, Y. M., and Lightner, D. A. (1991) Tetrahedron 47, 6163-6170 [CrossRef]
  19. Neuhaus, D, and Williamson, M. (1988) The Nuclear Overhauser Effect in Structural and Conformational Analysis , VCH Publishers, New York
  20. Khaled, M. A., and Watkins, C. L. (1983) J. Am. Chem. Soc. 105, 3363-3365
  21. Yu, C., and Levy, G. C. (1984) J. Am. Chem. Soc. 106, 6533-6537
  22. Mukundan, S., Jr., Xu, Y., Zon, G., and Marzilli, L. G. (1991) J. Am. Chem. Soc. 113, 3021-3027
  23. Cativela, C., and Sánchez-Ferrando, F. (1985) Magn. Res. Chem. 23, 1072-1075
  24. Seba, H. B., and Ancian, B. (1990) J. Chem. Soc. Chem. Commun. 996-997
  25. Hore, P. J. (1983) J. Magn. Reson. 54, 539-542
  26. James, T. L., and Basus, V. J. (1991) Annu. Rev. Phys. Chem. 42, 501-542 [CrossRef][Medline] [Order article via Infotrieve]
  27. Liu, H., Kumar, A., Weisz, K., Schmitz, U., Bishop, K. D., and James, T. L. (1993) J. Am. Chem. Soc. 115, 1590-1591
  28. Kaplan, D., and Navon, G. (1981) J. Chem. Soc. Perkin Trans. II , 1374-1383
  29. Puzicha, G., Pu, Y-M., and Lightner, D. A. (1991) J. Am. Chem. Soc. 113, 3583-3592
  30. Boiadjiev, S. E., Person, R. V., Puzicha, G., Knobler, C., Maverick, E., Trueblood, K. N., and Lightner, D. A. (1992) J. Am. Chem. Soc. 114, 10123-10133
  31. Person, R. V., Peterson, B. R., and Lightner, D. A. (1994) J. Am. Chem. Soc. 116, 42-59
  32. Shout, D. P., and Lightner, D. A. (1993) Spectrosc. Lett. 26, 461-472
  33. Gawronski, J. K., Polonski, T., and Lightner, D. A. (1990) Tetrahedron 46, 8053-8066 [CrossRef]
  34. Trull, F. R., Shout, D. P., and Lightner, D. A. (1992) Tetrahedron 48, 8189-8198 [CrossRef]
  35. Falk, H. (1989) The Chemistry of Linear Oligopyrroles and Bile Pigments , Springer Verlag, New York
  36. Cistola, D. P., Small, D. M., and Hamilton, J. A. (1982) J. Lipid Res. 23, 795-799 [Abstract]

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