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ABSORPTION, LUMINESCENCE, SOLVENT-INDUCED CIRCULAR DICHROISM AND 'H NMR STUDY OF

BILIRUBIN DIMETHYL ESTER: OBSERVATION OF DIFFERENT FORMS IN SOLUTION*

ALIRED R . HOLZWARTH. ELISABETII LASGIIKt. HARALD h i l l N l X t

and KURT SCHAFFNERS lnstitut fur Strahlenchemie im Max-Planck-lnstitut fur Kohlenforschung.

D-4330 Miilheim a. d. R u h r , W. Germany

(Rrcriiwl 2 I Airgust 1979: accepted I5 January 1980)

Abstract-Bilirubin dimethyl ester ( I ) has been studied by absorption. Ruorescence and fluorescence excitation. solvent-induced circular dichroism, and 'H NMR techniques. The existence of two forms of the solute involving either or both syti-atiri dipyrromethenone and lactam- lactim equilibria was estab- lished. and the occurrence of even more forms remains possible. The relative population is strongly influenced by the nature of the solvent and by temperature. Thus, the population of one species predominates in ethanol. What is presumed to be the same species increases on cooling in all solvents. while the population of another species is strongly preferred in dichloromethane. In other solvents appreciable populations of both forms persist over the temperature range 77-300 K.

INTRODUCTION

Profound knowledge of the photophysics of bilirubin is prerequisite for the understanding of its photo- chemistry which. e.g. plays an important role in the phototherapy of neonatal jaundice. In order to eluci- date the excited state properties, the luminescence of bilirubin has been studied in organic solvents (Bon- nett er d., 1975). in water and organic solvents with added detergent (Cu el al.. 1975). and in basic EPA and ethanol (Matheson et af.. 1975). More recently. phosphorescence has been attributed to bilirubin at 77K on the basis of lifetime and flash photolytic measurements (Barber and Richards, 1977). These results were questioned, however, by Dalton et al. (1979) who found the emission to decay on a nano- second time scale.

1

Several of the above-mentioned authors as well as Jirsa et al. (1968) and Kuenzle et al. (1973) have pointed out the possibility that different forms of

*Part of these results have been presented at the 14th European Congress on Molecular Spectroscopy, Frankfurt, September, 1979; cf. Holzwarth er al. (1980).

tlnstitut f ir Organische Chemie der Universitat Wien. :To whom correspondence should be addressed.

bilirubin might be present in solution. However, con- clusive proof for such a heterogenous composition and characterization of its components are as yet lacking. In order t o gain more insight into the excited-state and ground-state properties of this bio- molecule, we have now extensively studied the absorption, solvent-induced circular dichroism. emission. and ' H NMR spectra of bilirubin dimethyl ester (1) (the conformation shown in this formula is arbitrary). The ester 1 rather than the free acid has been chosen for several reasons. The better solubility in a wide range of solvents renders 1 more readily amenable to a concerted study with a variety of spec- troscopic methods. Furthermore, a comparison of the data from 1 and from bilirubin and its dianion should eventually reveal the influence of the propionic side chains on the tetrapyrrole backbone properties.

MATERIALS AND METHODS

Bilirubin dimethyl ester ( I ) was prepared with diazo- methane from bilirubin (cryst. p. a. from Serva, Heidelberg) in chloroform solution (cf. Kiister. 1924). followed by chromatography on silica gel and repeated crystallization from chloroformxthanol. The purity of the sample, and in particular the absence of isomers and overmethylated products, is in evidence by the constant mp (ISCrl8I'C. uncorrected) upon further recrystallization, 270 MHz 'H-NMR spectra, infrared and mass spectra (M' m/e 612). Significantly different mp's have been reported, e.g. 168-169 and 198-200°C by Kiister (1924), 201-202°C by Jirsa er a / . (1968). and 198-200°C by Hutchinson et a/. (1973). and it appears that the mp per se is not a reliable criterion for the purity of 1 (nor is HPL chromatography: see Braslavsky et al., 1980).

Ethanol (EtOH; Merck UV grade) was distilled over potassium hydroxide and then repeatedly dried over molecular sieve (Merck, 10 A, perlform 2 mm). 2-Methyltetra-

i n ALFRED R. HOLzwARTtt el a / .

hydrofurane (MTHF) (Merck "zur Synthese") was fraction- ally distilled over metallic potassium. CH,CI2 (Merck U V grade) and C2H2CI, and C'HCI, (both Mcrck) were passed through alumina (Woelm. basic, act. I ) immediately prior to use: ('H,)THF (Merck) was used as purchased. CHCI, (Merck U V grade) was used either as purchased (stabilized with I " , , EtOH) or after filtration through alumina. Ethyl (Sk( - klactate. (R) - ( -)-butan-2-ol and (R.R)- ( - )-butane-2.3-diol (all three from Fluka) were vacuum distilled. Solutions for optical measurements were prepared by ultrasonication for ca. 30 min with concomi- tant slight warming.

Molecular weight determinations were carried out by Dornis and Kolbe. Miilheim a. d. Ruhr (vapor pressure osmometer 301 A Mechrolab) in stabilized and destabilized CHCI,. and in MTHF. Ultrasonication with warming was necessary to obtain reproducible results. Without this pre- caution the average mol wt were generally higher, ranging up to uniform dimer values. The osmometric method used precluded measurements in CH,CI,.

Absorption spectra at room temperature ( I O p V ] . 183 K and 77 K (20 1tM at the low temperatures) were measured on a Cary 17 absorption spectrometer. In M T H F and CH'CI,. small .deviations from Beer's law (see Lightner vr a/ . . 1979b) were still observed at these concentrations. owing to < lo";, dimer formation (Kdim : 1500). Solvent- induced circular dichroism (SICD) spectra were run on a Jobin-Yvon Mark 111 instrument, and the 'H-NMR spectra on a Bruker W H 270 spectrometer in the FT mode with 100 500 sweeps (concentrations 8 16mM). The assignments of the vinyl AMX systems. including the attachment of these groups to rings A and D. are based on decoupling experiments (using long-range couplings between the methyl and vinyl protons for the vinyl localiz- ations). and are in agreement with the assignments made by Manitto (1973).

Emission spectra were measured on a P D P I I compu ter-controlled Spex Fluorolog luminescence spectrometer. For details of the instrument and calibration pro- cedures see Holzwarth C I a/. (1978). All necessary correc- tions and the conversion into wavenumbers were per- formed by the computer.

Fluorescence lifetime measurements were carried out with a single-photon-counting Ruorimeter of Photo- chemical Research Associates Inc. (PRA). with the kind assistance of Dr R. Lyke of PRA and Mr I . Adam of AMKO GmbH who made the instrument available to us. I t was equipped with a gated hydrogen lamp/427 nm inter- ference filter (bandwidth 14 nm) combination for excitation and a monochromator in the emission path. The data were evaluated with a MINC-II computer by a least-squares fitting procedure. The lifetime values depended somewhat on the time range evaluated; estimated error range for short-lived components f long-lived components 10°,w The reason for this dependence is probably due to the presence of additional minor components with different lifetimes. it . , the decays measured did not exactly fit the assumed double exponential model.

For room temperature luminescence spectra, sample solutions were placed in 0.5cm square cuvettes. Concen- trations were 20pM at room temperature and 2 p M at 77K. The position of the cuvette and the optical paths were arranged in such a way that the penetration depth of the exciting radiation was ca. 1-2 mm. The relatively high concentration at room temperature. imposed by the ex- tremely low emission quantum yields. was at the upper limit for excitation measurements. Possible errors were largely compensated (and certainly did not reach the order of magnitude of the solvatochromic effect observed) by arranging for a minimum penetration depth which pro- vided for an effective average absorbance of ca. 0.1 in the observation volume (viz. the absorbance of ca. I .0 cm- I ) .

Emission spectra at 77 K were measured in an optical

quartz dewar with the sample tubes (4 mm id) directly im- mersed into liquid nitrogen. All solvents were checked for impurity emission in the relevant wavelength regions.

For SICD and emission measurements sample solutions were degassed by freeze-pump-thaw-cycles.

In order to prevent photochemical conversion (Mc- Donagh er a/.. 1979; Lightner er a/.. 1979a) all solutions were prepared and handled in red safety light. Each room temperature emission spectrum was run on a fresh sample for the same reason.

RESLLTS

Osmometric determinations for CHCl3 and MTHF solutions of 0.01 M I gave an average mol wt of I 1 0 0 & lo:<. This shows that bilirubin dimethyl ester (1) exists practically fully in an aggregated form at the concentrations which were used for the 'H-NMR studies. O n the other hand, a t the much lower concentrations used for the optical measurements the dimer content is < lo'?;.

Absorption, SICD mid emission spectra of 1

The spectral data from the absorption, SICD. emission and emission excitation measurements are compiled in Tables 1 and 2.

Rooni femperarure. The absorption spectrum of bilirubin dimethyl ester (1) in EtOH at room temperature shows a maximum at 448nm and a shoulder around 408 nm (Fig. la). In MTHF, and even more so in CH,Cl,, the band intensities are reversed (Fig. Ib and Ic) showing short-wavelength maxima at 397 and 396nm, respectively. The spectra in ethyl (S)-(-)- lactate, ( R H - )-butan-2-ol and (R,R)-( - )-butane-2,3- diol are intermediate between these two extremes. They are shown in Fig. 2z-c together with the SICD spectra in these solvents. The SICDs exhibit bisignate Cotton effects. While the wavelength positions of the SICD maxima are almost independent of the solvent, their relative intensities strongly respond to solvent changes as do the absorptivities.

At room temperature 1 shows a weak fluorescence in EtOH (Fig. 3a), MTHF (Fig. 3b) and ethyl lactate (Fig. 4). The emission maximum in MTHF is at 518 nm and the spectrum is almost independent of the excitation wavelength. Thus, excitations at 396 and 448 nm give rise to the same emission as shown in Fig. 3b for i.,,, = 410mm. The fluorescence excitation spectrum (A,,, = 438 nm) differs strongly from the corresponding absorption spectrum. The 396 nm absorption band is entirely absent. A reasonably close resemblance between absorption and excitation is found only in the long-wavelength range where the 438 nm excitation band overlaps with the corresponding absorption in MTHF (shoulder) and EtOH (main band).

The fluorescence in EtOH is quite similar to that in MTHF, and it is again almost independent of the excitation wavelength (Fig. 3A and 3B). Excitation and absorption spectra in EtOH are in much closer agreement than in MTHF.

Spectroscopy of bilirubin 19

Table I . Absorption and SICD maxima of I

SICD Lk

Absorption

( M - cm-I) (nm) ( M - I c m - ' ) Solvent Temperature L a x . nm/emu A,,,

EtOH* 298 K 408 (sh). 448/57500

MTHF' 298 K 397/70300. 445 (sh) 77 K 454 (sh),t 480

183 K 397.448 77 K 420. 470

CHZCIZ* 298 K 396/75700

(R) - ( - )-butan-2-ol$ 298 K 417 (sh), 451/54000 400 - 0.4 54OOO 450 + 0.9

460 i- 2.7

464 - 6.5

183 K 398

Ethyl (S)-( - )-lactate$ 29R K 415 (sh). 445/49900 405 -3.1

(R.R)-( - tbutane-2.3-diol: 298 K 420 (sh). 453/333oOjj 405 + 2.2

*See Fig. I . tThis shoulder is a vibrational band as judged from the mirror image relationship with emission (Fig. 5). $See Fig. 2. $This strikingly low E value may be due to the experimental difficulty of dissolving I completely in the very viscous solvent without partial decomposition, i.e. in a relatively short time at moderate temperature.

Low temperature. In MTHF 1 shows a pronounced thermochromism. The absorption maxima at 77 K are strongly red-shifted with respect to the room temperature spectrum, and the relative intensities are inverted (Fig. I). By comparison, the absorption band in EtOH is narrower but less red-shifted at 77K and bears greater similarity to the spectrum at room temperature. Cooling to 183K did not introduce a strong change in CH2CI,.

Emission and excitation spectra of 1 at 77 K were measured in MTHF and EtOH (Fig. 5). In contrast to EtOH, in MTHF a striking dependence on the excitation and emission wavelengths is found. The spectra appear to consist of at least two largely o\rerlapping emissions. Excitation in the long-wavelength part of

the absorption (it,, = 480nm; Fig. 5A) yields an emission similar to that in EtOH glass (Acxc = 475) whereas excitation at 420 nm (Fig. 5B) gives rise to a mixed spectrum containing a red-shifted emission with a maximum in the region 53C540 nm. The excitation spectra taken at different wavelengths (it,,, = 495 and 540 nm) show a corresponding behaviour.

= 427 nm) confirmed the dual nature of the 77K fluorescence in MTHF, with T~ = 2.511s and T~ = 9.711s. The shorter-lived component predominates at both 500 nm (ca. 8077 of the total intensity) and 530nm monitoring wavelengths (ca. 70%). Furthermore, a similar composition of the emission in EtOH was revealed, with T~ = 1.911s (SOOnrn, close to 100%) and T~ = 11.911s

Lifetime measurements

Table 2. Emission and excitation spectra of I

Fixed wavelength Amax

(nm) (nm) Solvent Temperature i.,,, 4, Emission Excitation

EtOH 298 K* 465 515

395 512 500 448. 436t

540 45 I

495 477. 450

540 480, 454

530 438. 409 (sh)

495 473. 448. 421 (sh)

540 473, 448, 421

77 K $ 475 503, 534 (sh)

420 504. 534 (sh)

MTHF 298 K* 410 518

77 KS 480 495. 526 (sh)

420 504. 535 (sh)

Ethyl 298 K§ 420 526. 552 (sh) lactate 450 523

490 446 530 447, 413 (sh)

*See Fig. 3. t f h i s maximum probably represents a Raman peak from the solvent. $See Fig. 5. §See Fig. 4.

20 ALFRFI) R. HOLZWARTH er a/.

L r m 33c 35c -3c 5CC 633 730

' - 7 l

Room Temperature

a

. ' ? C -

.... -

77 K

3500C 32333 ?50:', ?C;33 '533:

Figure I . Absorption spectra of I in EtOH (a). MTHF (b) and CH2C12 (c) at room temperature (a. b. c). I83 K (b. c). and 77 K (a. b) (see Table I ) . The low temperature spectra are not corrected for solvent

contraction.

(530nm. ca. IO",). Emission lifetimes at room temperature were too short for measurements.

' H . V M R spectra of I

NMR spectra were measured in C'HCI, at room temperature. and in C'H,CI, (Figs 6 and 7) and (*H8)THF (Figs 8 and 9) in the temperature range 2W302 K. The three spectra are quite similar at 302 K. Contrary to the claims by Kuenzle (1970) and Chedekel and Crist (1975). and in accordance with the comments by Hutchinson er a / . (1973). the spectrum in C'HC13 appears to be that of a homogeneous solute. as do the spectra in the other solvents. In C'H2C12 (Fig. 6) the signal of the C-I0 methylene protons which are isochronous at 302 K, broadens on cooling and at 200K an AB quadruplet emerges ( v A B = 50 Hz. JAB = 15 Hz). Concomitantly, two of the four N-H singlets broaden slightly (Fig. 7). while all other resonances remain reasonably sharp.

In ('H8)THF the CHI-I0 signal shows a similar trend with temperature although the A B quadruplet is not yet attained even at 183 K (Fig. 8), and the two N-H singlets broaden now even more strongly than in C2H2C12 (Fig. 9). In addition, also the A M X sys-

tem of the vinyl group attached to ring D is distinctly affected by cooling. Coalescence appears to be at around 183 K. A simultaneous study of the C-CH, signals was precluded by the residual 'H resonance of the ('H,)THF solvent.

DISCUSSION

Solraroclirornistn The solvatochromism of 1 points to the presence of

at least two species in solution, with individual absorption maxima at about 395 and 450 nm at room temperature. In hydrogen-bonding solvents the '450nm' species is more strongly populated. The Occurrence of two SICD bands with variable intensity ratios (Fig. 2) clearly indicates that these bands must be attributed to different species rather than an exciton splitting in one molecular form. This interpretation is supported by the parallel change of the corresponding absorptivities in the chiral solvents.

The fluorescence results provide further and independent proof of the coexistence of different molecular species of 1 in solution. Despite the striking dependence of the absorption spectrum on the solvent

A, nm

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22 ALFRED R. HOLZWARTH CI a/ .

Figure 4. Corrected room temperature fluorescence and fluorescence excitation spectra of 1 in ethyl lactate (with

different i,,, and I,,,: A. B) (see Table 2).

nature (Fig. I ) , the weak fluorescence at room temperature is almost identical in all solvents and independent of the excitation wavelength (Fig. 3). The excitation spectra of this emission, however. deviate to various degrees from the absorptions. This difference is most pronounced in solvents where the 395 nm band is strong in absorption.

These data are again most plausibly interpreted in terms of at least two species. The room temperature emission in EtOH and MTHF seems to originate exclusively from one such species ('450 nm') by comparison of its excitation spectrum with the absorption in EtOH (where the overlap with the emission excitation is closest). One can therefore attribute this emission to the '450nm' species even in the solvents where this

A n n 3cc 350 LOO 500 600 700

35000 30ooC 25000 20000 !5000 j C r n I

Figure 5. Corrected 77 K fluorescence and fluorescence excitation spectra of I in M T H F (-)and in EtOH (. . .). A : I,,, = 480 (MTHF) and 475 nm (EtOH), I.*,,, = 495 nm (MTHF and EtOH); B: A,,, = 420 nm, ,Ic,,, = 540 nm (both

for M T H F and EtOH) (see Table 2).

LL-- 7 6 5 4 6

Figure 6. ' H NMR spectra of 1 in C2H,CI, at 200. 240 and 302 K. showing the signal of the methylene protons at C-10, the two AMX systems of the vinyl protons. and the two singlets of the olefinic methine protons at C-5 and

C- 15.

I -

I . . j l l , l , l l l l l . l , , , , 11 10 6

Figure 7. ' H NMR spectra of 1 in C2H2CI, at 200, 240, 270 and 302 K, showing the N-H signals.


I I I I 1

A 1 I , , I . . . I I . I . . 7 6 5 6

Figure 8. ‘ H NMR spectra of 1 in (’H,)THF at 183. 203, 223 and 302 K. showing the signal of the methylene protons at C-10, the two singlets of the olefinic methine protons at C-5 and C-15, and the two A M X systems of the

vinyl protons.

form is only weakly populated. A relatively weak emission of the ‘395nm’ species can only be recognized in ethyl lactate solution from a shoulder at 413 nm on the main excitation band (Fig. 4).

The fluorescence quantum yields of 1 can now be expected to depend also markedly on the solvent. The yields in EtOH have recently been determined by Holzwarth er al. (1978) at room temperature, GF = 7.6 x and at 77K, GF = 0.31. On the basis of these values and the reasonable assumption that any contribution of > 5% to the emission by any species other than the principal emitting species (‘450nm’) should have been recognized as such, we estimate an upper limit of ca. 5 x lo-’ for the emission quantum yield of such a second species in EtOH and MTHF at room temperature (and a corre- spondingly shorter lifetime than that of ‘450 nm’).

The fluorescence results at 77 K are even more con-

clusive (Fig. 5). Emission from at least two species can now be observed clearly in MTHF. whereas in EtOH a second species is only detected in the lifetime measurements.

Excitation at long wavelengths (j.,,, = 480nm) at 77 K leads to similar emissions in EtOH and in MTHF, which exhibit reasonable mirror-image re- lationships with the excitation spectra except for the maximum at 420 nm in MTHF. This emission therefore represents practically one single component. The emission yield appears to be generally higher at 77 K than at room temperature in agreement with the above EtOH results. Comparison of the excitation spectrum (j.=,,, = 540 nm) with the absorption at 77 K shows that the excitation peak at 420 nm corresponds to the absorption maximum at the same wavelength. Excitation at these wavelengths results in a composite emission spectrum with a new contribution at 53&540nm in MTHF. In EtOH this effect is very small. One may plausibly assume that the 420nm maximum at low temperature represents the first absorption band of a second species responsible for this red-shifted emission. I t is presently not clear why this latter species should emit at longer wavelength than the former one with a first absorption band at 481 nm (in EtOH). The assignment of the red-shifted emission to a phosphorescence is excluded by the short lifetime. Also, the energy of this emission is significantly higher than the lowest triplet energy of 150 kJ/mol which Land (1976) has estimated for bilirubin. Incidentally, the emission appears in the same wavelength region as the luminescence zt 77 K which Barber and Richards (1977) had interpreted as bilirubin phosphorescence (see Introduction). We note in this connection that literature data already indicate that ‘dual emission at 77K similar to that of 1 is encountcred also with bilirubin. It Seems to have escaped the attention of Dalton er al. (1979; compare the spectrum given with the discussion) that the emission maximum of bilirubin in ammoniacal meth- anol shifts from 525nm (recorded with CW excitation) to 540nm when the time-resolved emission is recorded 10 ns after the excitation pulse. This experimental finding by Dalton is, in fact, in keeping with our lifetime data and spectra for the two major species of 1.

Thermochromism

Comparison between the room and low temperature absorption spectra reveals a thermochromic change of 1 in the sense that the equilibrium is shifted at low temperature towards the species absorbing at longer wavelength (‘481 nm’). Since the cooling procedure was fast, the equilibrium between the different species must be relatively rapid in order to be consis- tent with the solvatochromism observed. The ’difference between EtOH and MTHF at 77 K is probably due to the fact that, in EtOH, what is presumed to be the same species (‘450/481 nm’) predominates at room and at low temperature, whereas in MTHF the popu-

24

302 K I

I 1

183 K

, - . -c4

1 . . . - I .._-. I 1.-_1-. 12 11 10 E

Figure 9, ' H NMR spectra of I in ('HH,)THF at 183. 203 and 301 K , showing the N H signals.

lation of this form still increases upon cooling. The high solvent viscosity at low temperature might prevent full thermodynamic equilibration.

The solvent and temperature influence on the relative population of the major species can be summar- ked as follows. In EtOH. one species ['450nm' and long-lived (relative to '395 nrn') at room temperature, '48 I nm' and short-lived (relative to '420 nrn') at 77 K] strongly dominates in the entire temperature range (see Fig. I for the continuous spectral changes with temperature). A small fraction of the other species ('395 nm' and relatively short-lived at room temperature, '420nm' and relatively long-lived at 77 K ) is detected only at 77 K. The inverse situation is found with CHzClz where almost exclusively the latter species ('395/420 nm') is seen from room temperature to I83 K. In MTHF, appreciable populations of both species persist at all temperatures. We emphasize that the absorption and emission data do not rigorously permit structural correlations between the room temperature species ('450 nm' and '395 nm') on one hand and thosc at 77 K ('48 I nm' and '420 nm') on the other hand. It appears plausible and justified, however, to presently work on the basis of such correlations as we have done above and in the following discussion. We find no contradictory evidence in our own work and in the literature.

Naturr qf the coc~sisririg species

The observation of at least two coexisting species of 1 in solution iaiscs the question about their nature. Changes of tautomeric. associative and conforrna- tional nature must a priori be taken into consider- ation. The possible lactam-lactim tautomerism in bilirubin has been investigated by several groups (von Dobeneck and Brunner, 1965; Falk el al., 1976; Hut- chinson PI al.: 1971 : Kuenzle. 1970; Kuenzle el al..

1973). and from their results lactim tautomers seem to be populated to a negligible extent only. Falk Y I a/. (1976) also ruled out the presence of any lactim tautomer for dicster I . However. we are presently unable to rigorously exclude, from our own results. a contribution of lactim tautomer(s) to the hetcrogcneitj of 1 observed by absorption. fluorescence and SICD.

A monomer-dimer equilibrium can be excluded on the basis of the thermochromic properties. Upon cooling the equilibrium is shifted towards the species with red-shifted absorption. This form is undoubtedly monomeric as judged from its preferred population in hydrogen-bonding solvents, from the mirror-image re- lation of excitation and emission. and from the Stokes shift. A monomer-dimer equilibrium would be expected to change with temperature in the opposite way than was observed. The osmometric data yielding Kdim : 500 k support these conclusions. The effects observed in the optical spectra are much larger than could be caused by a dimcr content of less than 10";) in these solutions. Still. such B dimer component might be responsible for the slight deviation from the double exponential decay found in the lifetime measurements (see Materials and Methods).

Kuenzle (1970) and Nichol and Morell (1969) had already recognized a solvatochromic behaviour of I in absorption, and the former author discussed another associative process. He proposed that 1 selectively forms strong aggregates with polar solvents only, and that the strength of these interactions alone determines the solvatochromism without any further molecular change. Largely varying strengths in inter- action between solute and polar vs apolar solvent molecules should causc a sudden change in the absorption spectrum when small amounts of a polar solvent, e.g. EtOH, are added to a solution of 1 in which the '395/420 nm' species predominates. How- ever. in agreement with Nichol and Morell (1969) our results d o not reveal such an effect. Rather. continuous slow changes with increasing amomts of added polar solvent are observed. We therefore conclude that the solvatochromism must originate from different molecular species.

The Occurrence of two distinct conformations and/or tautomers of 1 in solution therefore remains a plausible interpretation. A conformational isomerism could involve rotations around the central C-I0 methylene-ring and/or around the C-5 and C-15 methine-ring single bonds (for example 2: torsion angles and i+b, respectively). Variations of the angles w should be the least probable causes for the spectral changes observed. Bilirubin and its dimethyl ester 1 posscss the Z,Z configuration. and the former is believed to be stabilized in the .sjn conformation by internal carboxylic hydrogen bonds (Kaplan er a/. . 1977: Manitto et a/.. 1974). The diester 1 lacks such strong hydrogen bonds and any conformational stabiliaztion by other possible hydrogen bridging (e.g., CO,,,,, . . . . HN) is expected to be weaker.


2

3

The '450/481 nm' species of I resembles the pre- dominant species of bilirubin. as judged from the spectral properties (Holzwarth et a/ . , 1979: Kuerv.le. 1970). The fluorescence quantum yield of this form at room temperature appears to be significantly higher than the yield of the '395/420nm' species. Only iv more viscous media such as ethyl lactate is the latter species observable at room temperature by fluorescence. This may be taken as an indication that the '395/420 nm' species is less rigid than the '450/48 I nm' form. The temperature dependence of the quantum yields falls in line with this conclusion, in agreement with results obtained previously on a pyrromethenone serving as a close model of rings A and B of I (Holz- warth ef a/ . . 197X). The rigidity could conceivably be reduced by the loss of hydrogen bonding associated with either a conformational (I$) or a lactim+ lactam change, which in turn could be compensated by pro- viding that molecule with a more viscous surround- ing. A similar explanation for the heterogeneity of 1 in solution, although evidently based on invalid conclusions from an NMR sample containing an impurity (see above). has already been given by Kuenzle (1970) and Kuenzle et a/ . (1973).

I t is of interest to note in this connection that the spectral properties of bilirubin and its diester 1 can- not be derived in a straightforward way from those of the individual partial chromophors. Thus, the absorption and fluorescence maxima of the above-mentioned pyrromethenone are significantly shifted (e.g., ilbs = 412 and i,, = 495nm in ethanol at room temperature) [Braslavsky et a/ . (1979): for similar data with related pyrromethenones see Falk and Neufingerl ( 1 979)].

The NMR results afford some more insight into the dynamic propertics of the conformers of 1, independent of the fact that these are spectra of aggregates rather than of monomeric 1. In the '395/420nm'

species present in C2H2C12 (Fig. 6). the methylene protons HA and H, at C-I0 selectively change from an isochronous nature at 302 K (average C, symmetry of 1) to non-equivalence at lower temperatures. This degenerate equilibrium indicates that the preferred conformation of this species is chiral (C, symmetry). An average rotational barrier around the C-10 methylene-ring single bonds of 40 kJ/mol is estimated from the NMR parameters and the coalescence temperature (ca. 220K). Manitto and Monti (1976) reported a considerably larger value for bilirubin (ca. 75 kJ/mol). This difference between the free diacid bilirubin and the diester 1 is attributable to stronger internal hydrogen bonding in the former.

Preliminary results from our laboratory (Bras- lavsky et a/ . . 1979) and literature data suggest that both the diacid bilirubin and its dianion behave in a qualitatively similar manner as does the diester 1. Specifically, the CD spectra of bilirubin serum albumin complexes (Beaven er a/.. 1973; Blauer and Wagniere. 1975) are strongly reminiscent of the SICD spectra of 1. This similarity suggests that the bisignate Cotton effect of these complexes is also due to two species rather than to an exciton splitting of a single species as proposed by Blauer and Wagniere (1975). A n analogy between bilirubin, its salt and its dimethyl ( I ) is further supported by a recent study of Dalton ef al. (1979) on the solvent dependence of the absorption of bilirubin. It revealed the presence of two bands with different solvent shifts. Although not claimed explicitly by the authors, this may also reflect the presence of two species [for solvatochromic effects of the salt see also Brodersen ( I 979) and Carey and Kor- etsky (1979)l. Finally, the 77 K fluorescence spectra of bilirubin in EPA and EPA/detergent (Cu et a/.. 1975) resemble tho* of 1 in MTHF (Fig. 5).

One may conclude, therefore, that the optical properties of the species observed reflect those of the tetrapyrrole backbone rather than a direct influence by the propionic side chains. The effect of the specific nature of the side chains (acid. anion or ester) appears to be restricted to influencing the relative population of the different forms in liquid solution and low temperature glasses. In summary, our results show that bilirubin dimethyl ester ( I ) in solutions can adopt various forms of aggregation and of monomeric species such as conformers and/or tautomers, depend- ing on concentration, solvent and temperature.

Ac!aiowlrdgerne,irs We thank Miss D. Kreft. Mr J. Bitter, Mr W. Riemer and Mr H.-V. Seeling for able technical assistance.

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