A water-soluble synthetic bilirubin with carboxyl groups replaced by sulfonyl moieties

Article abstract of DOI:10.1007/s007060170035

The first symmetrical bilirubin analog with CO²H groups replaced by SO³H, 8,12-bis-(2-sulfo-ethyl)-3,17-diethyl-2,7,13,18-tetramethyl- (10H,21H,23H,24H)-bilin-1,19-dione, was synthesized from methyl (2,4-dimethyl-5-ethoxycarbonyl-1H-pyrrol-3-yl) acetate in nine steps via the sulfonic acid analog of xanthobilirubic acid (XBR) and isolated as its disodium salt. The sulfonic acid group was introduced at an early stage of the synthesis by reaction of an intermediate, ethyl 4-(2-bromoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate, with sodium sulfite. The disodium bilirubin disulfonate exhibits NMR spectroscopic properties rather similar to those of the parent carboxylic acid, mesobilirubin-XIIIα; however, its UV/Vis spectra are blue-shifted and broadened relative to those of the parent compound. Like mesobilirubin, the disulfonate displays a positive exciton chirality circular dichroism spectrum, albeit with weaker Cotton effects, in a buffered aqueous solution (pH = 7.4) containing a 2:1 molar ratio of human serum albumin.

Full text of DOI:10.1007/s007060170035

Monatshefte f uÈ r Chemie 132, 1201±1212 (2001)
A Water-Soluble Synthetic Bilirubin with
Carboxyl Groups Replaced by Sulfonyl Moieties
Stefan E. Boiadjiev and David A. LightnerÃ
Department of Chemistry, University of Nevada, Reno, Nevada 89557-0020, USA
Summary. The ®rst symmetrical bilirubin analog with CO2H groups replaced by SO3H, 8,12-bis-(2-
sulfo-ethyl)-3,17-diethyl-2,7,13,18-tetramethyl-(10H,21H,23H,24H)-bilin-1,19-dione, was synthe-
sized from methyl (2,4-dimethyl-5-ethoxycarbonyl-1H-pyrrol-3-yl) acetate in nine steps via the
sulfonic acid analog of xanthobilirubic acid (XBR) and isolated as its disodium salt. The sulfonic acid
group was introduced at an early stage of the synthesis by reaction of an intermediate, ethyl 4-(2-
bromoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate, with sodium sul®te. The disodium bilirubin
disulfonate exhibits NMR spectroscopic properties rather similar to those of the parent carboxylic
acid, mesobilirubin-XIIIꢀ; however, its UV/Vis spectra are blue-shifted and broadened relative to
those of the parent compound. Like mesobilirubin, the disulfonate displays a positive exciton chirality
circular dichroism spectrum, albeit with weaker Cotton effects, in a buffered aqueous solution
(pH ˆ 7:4) containing a 2:1 molar ratio of human serum albumin.
Keywords. Pyrrole; Sulfonic acid; Spectroscopy; CD.
Introduction
Bilirubin-IXꢀ (Fig. 1A), the yellow pigment of jaundice, is formed from heme during
normal metabolism in humans and other mammals [1, 2] and owes its water
insolubility and other properties to a persistent tendency to tuck its polar carboxylic
acid and amide groups inward, linking them by hydrogen bonds [3, 4]. The most
stable conformation, shaped like a ridge-tile and secured by six intramolecular
hydrogen bonds (Fig. 1B), has been found in crystals of bilirubin [5] and its
dicarboxylate dianion salt [6] as well as in solution [7, 8]. The ridge-tile conformation
is believed to be important in the transport and metabolism of bilirubin [2, 9]. Analogs
with vinyl groups reduced to ethyl (as in mesobilirubin-XIIIꢀ), with alkyl substituents
on the propionic acids [10], or even with electronegative substituents such as OCH3
[11] or F [12] on the propionic acid chains all apparently retain the conformation-
determining intramolecular hydrogen bonding motif, which determines the pigment's
shape and properties. However, analogs with propionic acid groups transposed from
ring carbons 8 and 12 to other sites on the pigment backbone (e.g. to 7 and 13 in
mesobilirubin-IVꢀ [13]) are much more polar. They cannot engage in intramolecular
hydrogen bonding and behave completely differently in solution [13].
à Corresponding author. E-mail: lightner@unr.edu

1202
S. E. Boiadjiev and D. A. Lightner
Fig. 1. Bilirubin-IXꢀ shown in a linear representation (A) and in its most stable ridge-tile
conformation (B); the ridge-tile is stabilized by a network of 6 intramolecular hydrogen bonds, only
one of two enantiomers is shown; mesobilirubin-XIIIꢀ with both CO2H groups replaced by SO3H
groups (C)
The key elements required for intramolecular hydrogen bonding are thus a
dipyrrinone and a partner carboxylic acid group, which must be tethered to ring
carbons 8 and 12 (Fig. 1A, 1B). Carboxylic acid and dipyrrinone moieties form a
strongly attractive complementary hydrogen bonding pair [14]. Even hydrogen
bonding of the dipyrrinone to a carboxylate ion seems to be quite effective in
retaining the pigment's folded, hydrogen-bonded conformation [6±8]. Although the
carboxylic acid group appears to be a perfect match for a dipyrrinone receptor (and
in bilirubin, the tight intramolecular hydrogen bonding between them renders the

Synthesis of a Sulfonated Bilirubin
1203
pigment hydrophobic) when electronegative atoms such as F, or groups such as
CH O are located adjacent to the CO H group, the profound increase in carboxylic
3
2
acid acidity renders the pigment more polar and even water-soluble [11, 12]. In
the following, we have extended our studies of acidity, hydrogen bonding, and
solubility to a new bilirubin analog with CO H groups replaced by SO H (Fig. 1C).
2
3
Results and Discussion
Synthesis aspects
In view of its ease of synthesis in high yield, we prepared monopyrrole 9 (Scheme I)
as starting material for the eventual introduction of the sulfonic acid group in
monopyrrole 4. Introduction of the SO H group at a late stage of the synthesis
3
seemed unattractive because the potential rubin intermediates are much less robust
than their ꢀ-carboethoxypyrrole counterparts. Our approach was thus designed to
�
introduce the SO H group early by displacing Br from the ꢁ-(2-bromoethyl)-
3
pyrrole 5 with sodium sul®te. Synthesis of 5 was accomplished in a straightforward
way by selectively saponifying 9 in aqueous-ethanolic NaOH to give 8 in 97%
yield. Reduction of the free CO H group of 8 was achieved smoothly, selectively,
2
ꢀ
and in high yield (96%) by treatment with BH -THF at � 30 C. Tosylation of the
3
resulting ꢁ-(2-hydroxyethyl)-pyrrole (7) afforded 6 in 82% yield, and 6 was
converted to its ꢁ-(2-bromoethyl) derivative (5) by reaction with NaBr in DMSO.
Reaction of 5 with Na SO in aqueous dioxane led to a 79% yield of sulfonate 4.
2
3
Attempts to isolate the sulfonic acid parent of 4 proved to be rather dif®cult, but 4
could be converted to its methyl ester (4-Me), which was also isolated and
characterized, by reaction with CH N .
2
2
Conversion of 4 to dipyrrinone 3 was achieved in a conventional [15] way: 4
was saponi®ed to the corresponding carboxylic acid, which was reacted with 5-
bromomethylene-4-ethyl-3-methyl-3-pyrrolin-2-one [16] to give a 59% yield of 3,
isolated as its sodium salt. As above, treatment of 3 with CH N afforded a low
2
2
yield of the methyl ester 3-Me. p-Chloranil-promoted oxidative self-coupling [17]
of 3 led to biliverdin 2, and the isolated verdin disodium salt was reduced to rubin 1
by sodium borohydride. The isolated yields of salts 1 and 2 were low because it was
dif®cult to remove inorganic salts.
Properties
As a measure of the relative polarity of sulfonic acids 1±3 and their carboxylic acid
parents, we compared their behavior in reverse phase HPLC using 0:1 M di-n-
octylamine acetate as eluent [18]. In this system, we observed that xanthobilirubic
acid (XBR), mesobilirubin-XIIIꢀ, and mesobiliverbin-XIIIꢀ gave the same retention
times as their corresponding sodium salts. We observed retention times of 6.56 min
for 1 and 14.15 min for mesobilirubin-XIIIꢀ; 4.77 min for 2 and 6.26 min for
mesobiliverdin-XIIIꢀ; and 3.67 min for 3 and 4.13 min for XBR. The long retention
time of mesobilirubin-XIIIꢀ relative to its verdin and XBR is due to its much greater
lipophilicity and intramolecular hydrogen bonding. The shorter retention times of
the ionized sulfonic acids relative to the ionized carboxylic acids are consistent with
the former being more polar than the latter, and the longer retention time of 1

1204
S. E. Boiadjiev and D. A. Lightner
Scheme 1
relative to 2 and 3 is qualitatively consistent with 1 being less polar than either 2 or
, probably due to intramolecular hydrogen bonding in 1.
3
13
1
The C and H NMR spectra of dipyrinnone 3, verdin 2, and rubin 1 compare
favorably with their carboxylic acid analogs (Table 1). The chemical shifts are very
similar and differ signi®cantly only at carbons (and hydrogens) in the propionic acid
chains. The presence of the ±SO Na group causes the ꢀ-carbons to become very
3
strongly deshielded (by ꢁ17 ppm) relative to the CO H analog, but the ꢁ-carbons
2
0
and hydrogens are not affected much. A similar behavior was noted with ꢀ,ꢀ -
di¯uoromesobilirubin-XIIIꢀ [12]. Other than this noticeable difference, the chemical
shifts of the remaining carbons and hydrogens match well with their carboxylic acid
analogs.
The UV/Vis spectroscopic data (Table 2) of 2 and 3 differ only slightly from
those of their parent carboxylic acid analogs in polar solvents: DMSO (Fig. 2A, B),

Synthesis of a Sulfonated Bilirubin
1205
Table 1. Comparison of 13C NMR assignments (5 Â 10 to 1 Â 10 M) in DMSO-d6 at 25 C; ꢂ/ppm
of the sodium salts of the bis-sulfonic acid rubin (1) and verdin (2) and the sulfonic acid dipyrrinone (3)
with their carboxylic acid analogs mesobilirubin-XIIIꢀ (MBR), mesobiliverdin-XIIIꢀ (MBV), and
xanthobilirubic acid (XBR)
� 3
� 2
ꢀ
Position
1
MBR
2
MBV
3
XBR
1
2
2
3
3
3
4
5
6
7
7
8
,19-CONH
171.74
122.38
9.40
171.93
122.51
9.14
172.29
127.58
9.15
172.28
127.66
9.18
171.84
122.28
9.20
171.89
122.29
9.21
,18
,18-CH3
,17
147.09
17.18
14.83
127.16
98.00
121.79
119.21
8.07
147.18
17.15
14.81
127.81
97.69
121.95
119.24
8.07
146.31
17.00
14.45
139.84
95.90
149.46
127.20
8.13
146.32
16.99
147.15
17.14
14.83
127.11
97.70
121.60
118.96
8.05
147.19
17.15
14.84
127.25
97.62
121.67
118.74
8.06
,17-CH2CH3
,17-CH2CH3
,16
14.46
139.96
95.78
,15-CH=
,14
149.35
127.41
8.15
,13
,13-CH3
,12
0
122.44
19.88
51.38
±
122.91
19.25
34.34
173.97
130.32
23.46
138.12
20.37
52.43
±
137.95
19.24
122.52
20.10
52.31
±
122.63
19.45
34.95
174.04
129.43
10.97
ꢁ
,ꢁ -CH2
0
ꢀ
,ꢀ -CH2
35.24
0
ꢀ
,ꢀ -COOH
173.62
140.47
116.02
9
1
,11
0
131.70
21.96
139.98
114.83
129.13
10.91
�
5
� 5
Table 2. Comparison of UV/Vis data (2 Â 10 to 4 Â 10 M in the given solvent containing 2% (v/v)
�
1
3
� 1
DMSO; ꢃmax/nm ("max=mol Á dm Á cm )) of the sodium salts of the bis-sulfonic acid rubin (1) and
verdin (2) and the sulfonic acid dipyrrinone (3) with their carboxylic acid analogs mesobilirubin-XIIIꢀ
(
MBR), mesobiliverdin-XIIIꢀ (MBV), and xanthobilirubic acid (XBR)
Solvent
1
MBR
2
MBV
3
XBR
4
26
439
(59000)
sh 410
sh (47700)
438
636
(9200)
374
638
(11000)
372
411
411
DMSO
(50800)
sh 401
(29700)
(34400)
sh (46800)
(30400)
639
(35800)
641
4
26
411
413
MeOH
MeCN
(53100)
sh 397
(58200)
sh 416
sh (48900)
438
(7900)
366
(9400)
366
(30800)
(36200)
sh (45700)
(29800)
624
(35500)
641
420
403
402
(50400)
sh 398
(56800)
(7800)
362
(9700)
365
(29700)
(34100)
sh (47800)
(28500)
657
(36400)
656
421
432
406
411
Phosphate
(43600)
sh 391
(53400)
sh 415
(6900)
364
(8600)
366
(26800)
(31800)
buffer (pH ˆ 8:60)
sh (39300)
sh (50200)
(27100)
(33900)

Fig. 2. UV/Vis spectra of 3 (A), 2 (B), and 1 (C) in DMSO (solid lines) as compared with their
corresponding carboxylic acid analogs (dotted lines), xanthobilirubic acid, mesobiliverdin-XIIIꢀ,
�
5
and mesobilirubin-XIIIꢀ, respectively; concentrations: ca. 10
M

S. E. Boiadjiev and D. A. Lightner: Synthesis of a Sulfonated Bilirubin
1207
�
5
Fig. 3. Circular dichroism (CD) spectra of 4 Â 10 M solutions of 1 (ÐÐÐ) and mesobilirubin-
ꢀ
XIIIꢀ (Á Á Á Á Á Á) in pH 7.4 aqueous phosphate buffer containing human serum albumin at 22 C; the
4
39
max
392
max
molar ratio of pigment to protein is 1:2; CD data for 1: Á"
ˆ ‡18; Á"
ˆ � 24; CD data for
mesobilirubin-XIIIꢀ Á"444 ˆ ‡55; Á"
391
ˆ � 62
max
max
MeOH, H O, and MeCN. The slightly lower intensity of the long wavelength
2
absorption ("max) of the sulfonic acid salts than that of the parent carboxylic acid
may be attributed to the presence of inorganic salts in the former. Unlike 2 and 3, in
polar solvents the UV/Vis spectra of 1 (Fig. 2C) are hypsochromically shifted (by
1
1±12 nm) and slightly broadened. Both 1 and its parent, mesobilirubin-XIIIꢀ, are
molecular excitons [3], and their UV/Vis spectra clearly indicate (more clearly in 1
than in mesobilirubin-XIIIꢀ) a splitting of the long-wavelength band. Their spectra
are consistent with a ridge-tile conformation similar to that observed in carboxylate
salts of bilirubin and mesobilirubin [6, 7].
Interestingly, solutions of 1 in pH 7.4 phosphate buffered human serum albumin
(
HSA) prepared as described previously [19a] for bilirubin-HSA solutions give
bisignate induced circular dichroism Cotton effects of the exciton coupling type
Fig. 3) [3] as has been observed previously for mesobilirubin-XIIIꢀ [19b]. The signed
(
order of the Cotton effects remains the same in both 1 and mesobilirubin-XIIIꢀ,
indicating a preference for binding the same conformational enantiomer (Fig. 1) in
the primary binding site of HSA [3, 19]. With 1, the Cotton effect intensities are
only about one-third of the magnitude of those of mesobilirubin-XIIIꢀ, suggesting
either poorer enantio-selectivity by the HSA or a weaker association complex,
possibly due to the fact that 1 is water soluble, whereas mesobilirubin-XIIIꢀ is
0
highly insoluble. ꢀ,ꢀ -Di¯uoromesobilirubin-XIIIꢀ, which is also water-soluble,
gave a qualitatively similar result from its CD in the presence of HSA [12].
Experimental
Nuclear magnetic resonance spectra (NMR) were recorded on GE GN-300 or Varian Unity Plus
spectrometers operating at a proton frequency of 300 or 500MHz. Chemical shifts are reported in ꢂ

1208
S. E. Boiadjiev and D. A. Lightner
1
13
(
ppm) and referenced to the residual CHCl3 signal at 7.26 ppm ( H) and CDCl3 at 77.00 ppm ( C). A
J-modulated spin-echo experiment was used to obtain carbon multiplicities. The UV/Vis spectra were
recorded on a Perkin Elmer Lambda 12 spectrophotometer. All solutions for UV/Vis measurements
contained 2% (by volume) of DMSO. GC-MS analyses were carried out on a Hewlett-Packard 5890A
capillary gas chromatograph (30 m DB-1 column) equipped with Hewlett-Packard 5970 mass
selective detector. Analytical thin layer chromatography was carried out on J.T. Baker silica gel IB-F
plates (Merck, 125 mm layer). Silica gel 70±230 mesh was employed for column chromatography.
Melting points were determined on a Mel-Temp capillary apparatus and are uncorrected. Combustion
analyses were carried out by Desert Analytics, Tucson, AZ, and the data were within Æ0.3% from the
calculated values. High-resolution FAB mass spectra were obtained at the Nebraska Center for Mass
Spectrometry, University of Nebraska, Lincoln, for samples which were >90% pure by NMR.
Commercial reagents and HPLC grade solvents (Aldrich or Fischer) were dried and puri®ed following
standard procedures [20]. The starting ethyl 3,5-dimethyl-4-(methoxycarbonyl)-methyl-1H-pyrrole-2-
carboxylate (9) was synthesized as previously described [21].
Ethyl 4-carboxymethyl-3,5-dimethyl-1H-pyrrole-2-carboxylate (8; C11H15NO4)
3
3
A mixture of 11.96 g (50 mmol) of 9, 2.20 g (55 mmol) of NaOH, 200cm of EtOH, and 25 cm of
H2O was stirred for 24 h at room temperature. Then the ethanol was evaporated under vacuum, the
3
ꢀ
residue was diluted with 350cm of H2O, and carefully acidi®ed by addition of 10% HCl at 0 C. The
3
precipitated product was collected by ®ltration, washed with H2O (2 Â 100 cm ), and dried under
vacuum to afford monoacid 8.
Yield: 10.97 g (97%); m.p.: 198±199 C (decomp.; Ref. [22]: 194±196 C); H NMR (CDCl3, ꢂ):
.34 (t, J ˆ 7:3 Hz, 3H), 2.23 (s, 3H), 2.28 (s, 3H), 3.41 (s, 2H), 4.29 (q, J ˆ 7:3 Hz, 2H), 9.12 (br s,
ꢀ
ꢀ
1
1
1
1
1
3
H), 11.44 (br s, 1H) ppm; C NMR (CDCl3, ꢂ): 10.63, 11.50, 14.52, 29.82, 59.93, 113.88, 117.22,
27.63, 131.25, 162.86, 177.11 ppm.
Ethyl 3,5-dimethyl-4-(hydroxyethyl)-1H-pyrrole-2-carboxylate (7; C11H17NO4)
3
ꢀ
To a solution of 11.26 g (50 mmol) of 8 in 250 cm of anhydrous THF cooled to � 30 C under dry
3
nitrogen, 65 cm of 1 M BH3-THF complex (65 mmol) were added during 1 h. After stirring for 2 h at
ꢀ
�
30 C, the mixture was allowed to warm slowly to room temperature over 22 h. The reaction was
3
quenched by addition of 20 cm THF: H2O (1:1 by volume), and the THF was evaporated under
3
vacuum. The residue was dissolved in 250cm of CH2Cl2 which was washed successively with
3
3
5
0 cm of 0:2 M NaOH and H2O (4 Â 50 cm ). The organic phase was dried over anhydrous MgSO4
and ®ltered. The solvent was evaporated under vacuum, and the residue was recrystallized from ethyl
acetate:hexane (ca. 1:3 by volume) to afford the pure hydroxy derivative 7.
ꢀ
ꢀ
‡Á
Yield: 10.18 g (96%); m.p.: 122±123 C (Ref. [22]: 121±123 C); MS m/z (rel. int.) ˆ 211 (M ,
1
1
2
1
1
4%), 180 (42%), 166 (8%), 134 (100%); H NMR (CDCl3, ꢂ): 1.35 (t, J ˆ 7:2 Hz, 3H), 2.23 (s, 3H),
.28 (s, 3H), 2.65 (t, J ˆ 6:3 Hz, 2H), 3.66 (t, J ˆ 6:3 Hz, 2H), 4.30 (q, J ˆ 7:2 Hz, 2H), 8.65 (br s,
1
3
H) ppm; C NMR (CDCl3, ꢂ): 10.58, 11.34, 14.44, 27.48, 59.63, 62.57, 117.03, 117.38, 127.31,
30.95, 161.94 ppm.
Ethyl 3,5-dimethyl-4-(2-p-toluenesulfonyloxyethyl)-1H-pyrrole-2-carboxylate (6; C18H23NO5S)
3
3
To a solution of 6.34 g (30 mmol) of 7 in 75 cm of anhydrous CH2Cl2 and 6.07 g (8.4 cm , 60 mmol)
ꢀ
of triethylamine cooled to � 5 C, 8.58 g (45 mmol) of p-toluenesulfonyl chloride were added during
ꢀ
1
h. The mixture was stirred for 2 h at 0 C and slowly warmed to room temperature over 14 h. Then
the mixture was diluted with 100 cm3 of CH2Cl2, washed with 2% HCl (50 cm ) and water
3
3
(
4 Â 50 cm ), dried (anhydrous MgSO4), and ®ltered. The solvent was evaporated under vacuum, and
the residue recrystallized from ethyl acetate:hexane (ca. 1:2 by volume) to afford pure tosylate 6.

Synthesis of a Sulfonated Bilirubin
1209
ꢀ
ꢀ
1
Yield: 8.95 g (82%); m.p. 129±130 C (Ref. [22]: 126±128 C); H NMR (CDCl3, ꢂ): 1.35 (t,
J ˆ 7:2 Hz, 3H), 2.11 (s, 3H), 2.14 (s, 3H), 2.42 (s, 3H), 2.71 (t, J ˆ 7:1 Hz, 2H), 3.99 (t, J ˆ 7:1 Hz,
2
H), 4.29 (q, J ˆ 7:2 Hz, 2H), 7.27 (d, J ˆ 8:0 Hz, 2H), 7.67 (d, J ˆ 8:0 Hz, 2H), 8.67 (br s, 1H)
13
ppm; C NMR (CDCl3, ꢂ): 10.23, 11.01, 14.36, 21.31, 23.83, 59.56, 69.73, 115.33, 116.90, 126.77,
27.45, 129.47, 131.06, 132.75, 144.40, 161.74 ppm.
1
Ethyl 4-(2-bromoethyl)-3,5-dimethyl-1H-pyrrole-2-carboxylate (5; C11H16BrNO2)
3
A mixture of 3.65g (10 mmol) of 6, 60 cm of anhydrous DMSO, and 10.29 g (100 mmol) of NaBr
ꢀ
was heated brie¯y to 60 C to achieve dissolution of all tosylate and then stirred for 16 h at room
3
temperature. Ice cold H2O (ꢁ250cm ) was slowly added to precipitate the product which was
®ltered, washed with H2O, and dried. Recrystallization from ethyl acetate:hexane (ca. 1:2 by volume)
afforded pure bromide 5.
Yield: 2.53 g (92%); m.p.: 145±146 C; MS: m/z (rel. int.) ˆ 275, 273 (M , 32%), 230, 228 (11%),
ꢀ
‡Á
1
1
94 (23%), 180 (80%), 148 (25%), 134 (100%); H NMR (CDCl3, ꢂ): 1.35 (t, J ˆ 7:1 Hz, 3H), 2.24
(
(
s, 3H), 2.28 (s, 3H), 2.93 (t, J ˆ 7:8 Hz, 2H), 3.36 (t, J ˆ 7:8 Hz, 2H), 4.29 (q, J ˆ 7:1 Hz, 2H), 8.78
13
br s, 1H) ppm; C NMR (CDCl3, ꢂ): 10.52, 11.59, 14.57, 28.22, 32.42, 59.76, 117.29, 118.98,
26.88, 130.16, 161.52 ppm.
1
Ethyl 3,5-dimethyl-4-(2-ethanesulfonic acid sodium salt)-1H-pyrrole-2-carboxylate
4; C11H16NNaO5S)
(
3
To a solution of 2.74 g (10 mmol) of 5 in 30 cm of puri®ed 1,4-dioxane, a solution of 6.30 g
3
(
50 mmol) of Na2SO3 in 30 cm of H2O was added, and the mixture was heated at vigorous re¯ux for
h. After stirring for 16 h at room temperature, the supernatant was decanted and evaporated under
9
3
vacuum to about 3 cm . Then, concentrated HCl was added until pH < 7. The precipitate was
3
collected by ®ltration, washed with ice cold H2O (2 Â 5 cm ), and dried under vacuum to afford
sulfonate 4.
Yield: 2.34g (79%); m.p.: 284±286 C (decomp.); H NMR (DMSO-d6, ꢂ): 1.24 (t, J ˆ 7:0 Hz,
H), 2.09 (s, 3H), 2.14 (s, 3H), 2.41 (m, 2H), 2.60 (m, 2H), 4.16 (q, J ˆ 7:0 Hz, 2H), 11.04 (br s, 1H)
ꢀ
1
3
1
3
ppm; C NMR (DMSO-d6, ꢂ): 10.34, 10.76, 14.55, 19.85, 52.15, 58.66, 115.79, 119.57, 125.69,
130.24, 160.80 ppm; LRMS (FAB, 3-NBA) m=z ˆ 298:3, 297.3.
Ethyl 3,5-dimethyl-4-(methyl 2-ethanesulfonate)-1H-pyrrole-2-carboxylate (4-Me; C12H19NO5S)
A solution of 297mg (1 mmol) of 4 in 15 cm3 of MeOH was treated with excess of ethereal
diazomethane until a yellow color persisted. Excess CH2N2 was destroyed by addition of 2±3 drops of
3
3
acetic acid. Then the mixture was diluted with 50 cm of CHCl3, washed with H2O (2Â50 cm ), and
dried (anhydrous Na2SO4). After ®ltration and evaporation of the solvent under vacuum, the residue
was puri®ed by radial chromatography (eluent: 0.5±1.0% MeOH in CH2Cl2, v/v) to afford methyl
sulfonate 4-Me after recrystallization from ethyl acetate:hexane ˆ 1:3.
ꢀ
1
Yield: 143mg (49%); m.p.: 118±119 C; H NMR (CDCl3, ꢂ): 1.35 (t, J ˆ 7:1 Hz, 3H), 2.24 (s,
0
0
0 0
3
2
1
H), 2.28 (s, 3H), 2.93 (ABA B , m, 2H), 3.15 (ABA B , m, 2H), 3.88 (s, 3H), 4.30 (q, J ˆ 7:1 Hz,
13
H), 8.68 (br s, 1H) ppm; C NMR (CDCl3, ꢂ): 10.47, 11.27, 14.49, 18.40, 49.76, 55.03, 59.84,
16.91, 117.41, 126.42, 130.12, 161.63 ppm.
8-(2-Ethanesulfonic acid sodium salt)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one
(
3; C16H21N2NaO4S)
3
3
A mixture of 1.49 g (5 mmol) of 4, 1.00 g (25 mmol) of NaOH, 15 cm of EtOH, and 7.5 cm of H2O
was heated at vigorous re¯ux for 3.5 h. The solvents were evaporated until dryness under vacuum. To

1210
S. E. Boiadjiev and D. A. Lightner
the residue, 1.08 g (5 mmol) of 5-bromomethylene-4-ethyl-3-methyl-2-oxo-1H-pyrrole [16] and
3
1
7.5 cm of anhydrous EtOH were added, and the mixture was acidi®ed to pH < 3 with concentrated
ꢀ
HNO3. Then it was heated at re¯ux for 3 h and chilled overnight at � 20 C. The precipitate was
3
3
®
ltered, washed with 20 cm of CH2Cl2, 2 Â 10 cm of cold H2O, and dried in vacuum to afford
dipyrrinone 3.
ꢀ
1
Yield: 1.06 g (59%); m.p.: >320 C (decomp.); H NMR (DMSO-d6, ꢂ): 1.06 (t, J ˆ 7:5 Hz, 3H),
.76 (s, 3H), 1.99 (s, 3H), 2.15 (s, 3H), 2.41 (m, 2H), 2.51 (q, J ˆ 7:5 Hz, 2H), 2.58 (m, 2H), 5.91 (s,
1
1
5
1
3
H), 9.74 (s, 1H), 10.26 (s, 1H) ppm; C NMR (DMSO-d6, ꢂ): 8.05, 9.20, 10.91, 14.83, 17.14, 20.10,
2.31, 97.70, 118.96, 121.60, 122.28, 122.52, 127.11, 129.13, 147.15, 171.84 ppm; LRMS (FAB, 3-
NBA): m=z ˆ 361:3, 360.3; HRMS (FAB, 3-NBA): calcd.: 360.1120, found: 360.1114, Á ˆ 0:6 mDa,
error: 1.7 ppm; UV/Vis (DMSO): ꢃmax ꢂ"† ˆ 411 (29700) nm; UV/Vis (CH3CN): ꢃmax ꢂ"† ˆ 403
(
29700) nm; UV/Vis (H2O): ꢃmax ꢂ"† ˆ 406 (27200) nm.
8-(Methyl 2-ethanesulfonate)-3-ethyl-2,7,9-trimethyl-1,10-dihydro-11H-dipyrrin-1-one
(
3-Me; C17H24N2O4S)
3
A solution of 180 mg (0.5 mmol) of 3 in 10 cm of MeOH was treated with an excess of ethereal
CH2N2 during 10 min. Then the excess CH2N2 was destroyed by addition of 3±4 drops of acetic acid,
3
3
and the solution was diluted with 100 cm of CHCl3 and washed with H2O (2 Â 100 cm ). The CHCl3
layer was dried over anhydrous Na2SO4. After ®ltration and evaporation of the solvent, the residue
was puri®ed by radial chromatography eluting with 2±4% MeOH in CH2Cl2 (v/v) to yield the methyl
sulfonate (3-Me) after recrystallization from MeOH:CH2Cl2 ˆ 5:1.
ꢀ
1
Yield: 39 mg (22%); m.p.: 249±251 C; H NMR (CDCl3, ꢂ): 1.18 (t, J ˆ 7:7 Hz, 3H), 1.95 (s,
0
0
0 0
3
2
9
1
3
3
1
1
H), 2.15 (s, 3H), 2.43 (s, 3H), 2.56 (q, J ˆ 7:7 Hz, 2H), 2.97 (ABA B , m, 2H), 3.18 (ABA B , m,
13
H), 3.89 (s, 3H), 6.12 (s, 1H), 10.41 (br s, 1H), 11.23 (br s, 1H) ppm; C NMR (CDCl3, ꢂ): 8.53,
.57, 11.51, 15.00, 17.95, 18.64, 50.02, 55.03, 100.71, 116.05, 122.72, 122.91, 123.97, 127.72,
1
31.59, 148.55, 174.25 ppm; H NMR (DMSO-d6, ꢂ): 1.08 (t, J ˆ 7:6 Hz, 3H), 1.77 (s, 3H), 2.04 (s,
0
0
0 0
H), 2.19 (s, 3H), 2.50 (q, J ˆ 7:6 Hz, 2H), 2.73 (ABA B , m, 2H), 3.33 (ABA B , m, 2H), 3.84 (s,
1
3
H), 5.93 (s, 1H), 9.74 (s, 1H), 10.36 (s, 1H) ppm; C NMR (DMSO-d6, ꢂ): 8.04, 9.09, 10.86, 14.81,
7.11, 18.06, 48.18, 56.40, 97.40, 115.70, 121.79, 122.11, 122.91, 127.65, 129.84, 147.21,
71.89 ppm.
8,12-Bis-(2-ethanesulfonic acid sodium salt)-3,17-diethyl-2,7,13,18-tetramethyl-(21H,24H)-
bilin-1,19-dione (2; C31H36N4Na2O8S2)
3
3
A mixture of 720mg (2 mmol) of 3, 1.23 g (5 mmol) of p-chloranil, 500cm of CH2Cl2, and 24 cm of
3
formic acid was heated at re¯ux for 24 h. After cooling, MeOH (50 cm ) was added, and the mixture
3
was concentrated under vacuum to a volume of about 35 cm . This residue was chilled overnight at
ꢀ
�
20 C, and the precipitated yellow solid was separated by ®ltration and washed with cold MeOH to
afford after evaporation of the bright blue ®ltrate crude mesobiliverdin-XIIIꢀ-bis-sodium sulfonate
which was used without further puri®cation in the next step. A fraction (100 mg) of crude 2 from an
independent synthesis was passed through a 6 cm column with silica gel (deactivated with 5% of
water) and eluted with CH2Cl2:CH3OH:NH4OH ˆ 100:7.5:0 to 100:25:5. Evaporation of the most
polar bright blue fractions afforded a suitable sample for characterization by NMR.
ꢀ
1
Yield: 244 mg (35%); m.p.: >304 C (decomp.); H NMR (DMSO-d6, ꢂ): 1.11 (t, J ˆ 7:4 Hz, 6H),
.69 (s, 6H), 2.03 (s, 6H), 2.51 (q, J ˆ 7:4 Hz, 4H), 2.54 (m, 4H), 2.85 (m, 4H), 5.97 (s, 2H), 6.79 (s,
1
1
2
1
3
H), 9.85 (br s, 2H), 11.3 (very br s, 1H) ppm; C NMR (DMSO-d6, ꢂ): 8.13, 9.15, 14.45, 17.00,
0.37, 52.43, 95.90, 114.83, 127.20, 127.58, 138.12, 139.84, 139.98, 146.31, 149.46, 172.29 ppm;
‡
HRMS (FAB, 3-NBA): calcd.: 703.1848 (MH ), found: 703.1855, Á ˆ 0:7 mDa, error: 1.0 ppm.

Synthesis of a Sulfonated Bilirubin
,12-Bis-(2-ethanesulfonic acid sodium salt)-3,17-diethyl-2,7,13,18-tetramethyl-
1211
8
(
10H,21H,23H,24H)-bilin-1,19-dione (1; C31H38N4Na2O8S2)
3
A mixture of 211 mg (0.3 mmol) of crude 2 and 80 cm of anhydrous MeOH was sonicated for 20 min
ꢀ
while purging it with N2. NaBH4 (1.02 g, 30 mmol) was added in small portions during 15 min at 0 C.
ꢀ
The blue mixture was stirred under nitrogen for 1 h at 0 C and for 30 min at room temperature when
3
the color changed to light green. H2O (100 cm ) was added, and the mixture was acidi®ed with
3
concentrated HCl to pH < 2. The yellow product was extracted with CHCl3 (5 Â 50 cm ) leaving a
blue aqueous phase. The solvent from the combined extracts was evaporated under vacuum to afford
mesobilirubin sulfonate 1. Passing the aqueous solution of this material through a 60 cm long (10 mm
ID) column packed with 20 g of Dowex 50W-X8 sulfonic acid resin (Baker, 50±100 mesh) and eluting
with water led to 50% recovery of an orange solid whose H and 13C NMR spectra in DMSO-d6 were
identical to those of the crude mesobilirubin 1.
1
ꢀ
1
Yield: 21 mg (10%); m.p.: >290 C (decomp.); H NMR (DMSO-d6, ꢂ): 1.07 (t, J ˆ 7:3 Hz, 6H),
.74 (s, 6H), 2.05 (s, 6H), 2.51 (q, J ˆ 7:3 Hz, 4H), 2.66 (t, J ˆ 6:2, 6.6 Hz, 4H), 2.73 (t, J ˆ 6:6
1
6
9
1
2
1
3
.2 Hz, 4H), 3.94 (s, 2H), 5.92 (s, 2H), 9.84 (s, 2H), 10.54 (s, 2H) ppm; C NMR (DMSO-d6, ꢂ): 8.07,
.40, 14.83, 17.18, 19.88, 21.96, 51.38, 98.00, 119.21, 121.79, 122.38, 122.44, 127.16, 131.70,
47.09, 171.74 ppm; HRMS (FAB, 3-NBA): calcd.: 704.1926, found: 704.1906, Á ˆ 2:0 mDa, error:
.9 ppm.
Acknowledgements
We thank the National Institutes of Health (HD-17779) for support of this research. S. E. Boiadjiev is
on leave from the Institute of Organic Chemistry, So®a, Bulgaria.
References
[
1] McDonagh AF (1979) Bile Pigments: Bilatrienes and 5,15-Biladienes. In: Dolphin D (ed) The
Porphyrins, vol 6. Academic Press, New York, p 293
[
[
[
[
2] Berk PD, Noyer C (1994) Seminars Liver Dis 14: 323
3] Person RV, Peterson BR, Lightner DA (1994) J Am Chem Soc 116: 42
4] Lightner DA, McDonagh AF (1984) Accounts Chem Res 17: 417 and references therein
5] a) Bonnett R, Davies JE, Hursthouse MB, Sheldrick GM (1978) Proc R Soc London Ser B 202:
249; b) LeBas G, Allegret A, Mauguen Y, DeRango C, Bailly M (1980) Acta Crystallogr Sect B
B36: 3007
[
6] Mugnoli A, Manitto P, Monti D (1983) Acta Crystallogr Sect C C38: 1287
7] a) D oÈ rner T, Knipp B, Lightner DA (1997) Tetrahedron 53: 2697; b) Nogales D, Lightner DA
[
(
1995) J Biol Chem 270: 73
8] Boiadjiev SE, Person RV, Puzicha G, Knobler C, Maverick E, Trueblood KN, Lightner DA
1992) J Am Chem Soc 114: 10123
[
[
(
9] a) McDonagh AF, Lightner DA (1991) The Importance of Molecular Structure in Bilirubin
Metabolism and Excretion. In: Bock KW, Gerok W, Matern S (eds) Hepatic Metabolism and
Disposition of Endo and Xenobiotics, chap 5, Falk Symposium No 57. Kluwer, Dordrecht, The
Netherlands, p 47; b) McDonagh AF, Lightner DA (1994) Cellular and Molecular Biology 40:
965
[
[
[
[
[
10] Boiadjiev SE, Lightner DA (2000) J Am Chem Soc 122: 11328
11] Boiadjiev SE, Lightner DA (1998) J Org Chem 63: 6220
12] Boiadjiev SE, Lightner DA (1997) J Org Chem 62: 399
13] Trull FR, Franklin RW, Lightner DA (1987) J Heterocyclic Chem 24: 1573
14] a) Huggins MT, Lightner DA (2001) Tetrahedron 57: 2279; b) Huggins MT, Lightner DA (2000)
J Org Chem 65: 6001; c) Huggins MT, Lightner DA (2000) Tetrahedron 56: 1797; d) Tipton AK,

1212
S. E. Boiadjiev and D. A. Lightner: Synthesis of a Sulfonated Bilirubin
Lightner DA (1999) Monatsh Chem 130: 425; e) Boiadjiev SE, Anstine DT, Lightner DA (1994)
Tetrahedron Asymm 5: 1945
15] Boiadjiev SE, Lightner DA (1994) Synlett 777
[
[
[
[
[
16] Shrout DP, Lightner DA (1990) Synthesis 1062
17] Shrout DP, Puzicha G, Lightner DA (1992) Synthesis 328
18] McDonagh AF, Palma LA, Trull FR, Lightner DA (1982) J Am Chem Soc 104: 6865
19] a) Lightner DA, Reisinger M, Landen GL (1986) J Biol Chem 261: 6034; b) Lightner DA,
Wijekoon WMD, Zhang MH (1988) J Biol Chem 263: 16669
th
[
20] Armarego WLF, Perrin DD (1997) Puri®cation of Laboratory Chemicals, 4 edn. Butterworth-
Heinemann, Oxford, England
[
21] Paine JB III, Dolphin D (1985) J Org Chem 50: 5598
22] Holmes DL, Lightner DA (1995) Tetrahedron 51: 1607
[
Received April 24, 2001. Accepted May 7, 2001