Protonated 2,3-dihydrobilindiones - Models for the chromophores of phycocyanin and the red-absorbing form of phytochrome

Article abstract of DOI:10.1002/(SICI)1521-3765(19980904)4:9<1653::AID-CHEM1653>3.0.CO;2-K

New structural and thermodynamic data on the protonation of 2,3-dihydrobilindiones are presented with respect to the ionic aspects of the chromophore-protein interactions in biliproteins. When intermolecular protonation with stoichiometric quantities of s

Full text of DOI:10.1002/(SICI)1521-3765(19980904)4:9<1653::AID-CHEM1653>3.0.CO;2-K

FULL PAPER
Protonated 2,3-DihydrobilindionesÐModels for the Chromophores of
Phycocyanin and the Red-Absorbing Form of Phytochrome
Michael Stanek and Karl Grubmayr*
Abstract: New structural and thermo-
dynamic data on the protonation of 2,3-
dihydrobilindiones are presented with
respect to the ionic aspects of the
chromophore ± protein interactions in
biliproteins. When intermolecular pro-
tonation with stoichiometric quantities
of strong (sulfonic) acids was investigat-
ed by NMR spectroscopy, it was found
that the positive charge is localized at
the nitrogen atom of the azafulvenic ring
B moiety. Complete protonation by
weaker (carboxylic) acids can be ach-
ieved only intramolecularly at low tem-
perature. The (2R,3R,3'R,CysR)-cys-
teine adduct of phycocyanobilin dimeth-
yl ester was synthesized to mimic the
acid ± base chemistry between protein
and chromophore. Thermodynamic data
for the equilibrium between its neutral
form 1 and zwitterion 1Æ were calculated
from the temperature dependence of the
sensus sequences necessary for the ef-
fective protonation of the chromophores
in various light-harvesting biliproteins
such as phycocyanin. On the other hand,
the highly negative value of DS8 indi-
cates the possibility of the reprotonation
of the protein in case of steric interfer-
ence. The geometrical changes of the
protonated Pr chromophore of phyto-
chrome as a result of the Z !E photo-
isomerization may trigger proton trans-
fer back to the protein and thus initiate
the sequence of dark reactions that lead
to the physiologically active Pfr form of
phytochrome.
1
visible spectra (DH8 ˆ 20.8 kJmol ,
1
DS8 ˆ 71 Jmol 1 K ). These data are
in accord with others from intramolec-
ular proton transfers, explaining the
perfect order of highly conserved con-
Keywords: chromophores ´ phyco-
cyanin ´ phytochrome ´ proteins ´
zwitterionic states
form of the various phytochromes that absorbs red light,
commonly named the Pr form, the chromophores are also
protonated by acidic residues of the protein. Protonation is
indicated best by significant bathochromic shifts of the long-
wavelength absorption maxima of phytochromobilin and
phycocyanobilin during self-assembly with recombinant apo-
phytochromes.[11]
Introduction
The photoactivity of the phycocyanins[1] and phytochromes[2]
depends on the unique structure of the 2,3-dihydrobilindione
chromophore,[3] which enables perfect tuning of their absorp-
tion spectra by distinct chromophore ± protein interactions.
Among these the covalent linkage of the chromophores to the
apoproteins by thioethers and the formation of ion pairs
between the chromophores in the protonated state and the
corresponding carboxylates of the proteins are thought to be
essential for photophysical and photochemical functionality:
for instance, excitation energy transfer in the light-harvesting
complexes of cyanobacteria or red algae,[4] and the regulation
of plant gene expression by light and phytochrome.[5]
From crystal structures of phycocyanin,[6] as well as other
phycobiliproteins,[7] such as allophycocyanin,[8] phycoerythro-
cyanin,[9] and phycoerythrin,[10] a common principle for the
protonation of their chromophores becomes evident: proto-
nation always takes place at the dipyrrin moiety of rings B and
C and is caused exclusively by aspartic acid residues. In the
In spite of all that is known about the holoproteins,
structural details of their protonated chromophores remain
uncertain. With respect to the distribution of the positive
charge three distinct structures can be taken into consider-
ation (I ± III, Figure 1b). In structure I the symmetry of the
protonated dipyrrin moiety[12] principally remains resulting in
charge delocalization as found in protonated bilindiones of
symmetric substitution patterns,[13] whereas in structures II
and III the different oxidation state of the two lactam
substituents, the 2,3-dihydropyrrolinone of ring A and the
pyrrolinone of ring D, may cause a localization of the positive
charge on either the nitrogen of ring B or that of ring C. In
addition to this the question arises as to whether protonation
by aspartic acid residues can be achieved quantitatively. At
first glance the formation of stable ion pairs seems to be
unlikely comparing the acidities of protonated 2,3-dihydrobi-
lindiones (pKa ˆ 4.6)[14] and aliphatic carboxylic acids, for
instance, acetic acid (pKa ˆ 4.8).
[*] a. Univ.-Prof. Dr. K. Grubmayr, Dipl.-Ing. M. Stanek
Institut für Chemie, Johannes Kepler Universität Linz
Altenbergerstrasse 69, A-4040 Linz (Austria)
Fax : (‡ 43)732-2468-747
E-mail: karl.grubmayr@jk.uni-linz.ac.at
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1653

K. Grubmayr and M. Stanek
FULL PAPER
Here, we report on the structure and
the spectral properties of the synthetic
model compound 1, which is in equili-
brium with its zwitterionic species 1Æ,
representing the adduct of cysteine and
phycocyanobilin in its (2R,3R,3'R,CysR)-
configuration. With respect to the ex-
tended chromophores of phycocyanin
and the Pr form (Figure 1a) the main
difference is the helical conformation of 1
and 1Æ, respectively, adopted by bilin-
diones in solution as the most stable one.
Nevertheless, 1 is able to mimic the acid ±
base chemistry between protein and
chromophore. The protonation of the
dipyrrin moiety by the cysteinic carboxyl
group proceeds intramolecularly. Stereo-
chemical influences can be quantified in
comparison with the (2S,3S,3'S,CysR)-
diastereomer 2 and 2Æ, respectively. In-
vestigations are based on the results of
the intermolecular protonation of the 2,3-
dihydrobilindione models 3 and 4 (Fig-
ure 2). They will be presented first.
Figure 1. a) Comparison of the extended (5anti,10syn,14anti) conformation of the protein-bound
chromophores of phycocyanin and the Pr form and the helical (5syn,10syn,14syn)-conformation of
model compound 1 in its zwitterionic form 1Æ. b) Hypothetical chromophore structures of protonated
2,3-dihydrobilindiones.
Abstract in German: Neue strukturelle und thermodynami-
sche Daten zur Protonierung von 2,3-Dihydrobilindionen
werden im Hinblick auf die ionischen Anteile der Chromo-
phor ± Protein Wechselwirkung in Biliproteinen vorgestellt.
Die intermolekulare Protonierung wurde mit stöchiometri-
schen Mengen starker Säuren (Sulfonsäuren) NMR-spek-
troskopisch untersucht. Die positive Ladung wird am Stick-
Figure 2. Structural formula of the diastereomeric model compounds 3
and 4 in the (4Z,9Z,15Z,5syn,10syn,14syn)- and (4Z,9Z,15E,5syn,10syn,14-
syn) geometry.
stoffatom der Azafulveneinheit von Ring B lokalisiert. Die
vollständige Protonierung mit schwachen Säuren (Carbonsäu-
ren) erfolgt nur intramolekular bei tiefen Temperaturen. Das
(2R,3R,3'R,CysR)-Cystein-Addukt des Phycocyanobilindime-
thylesters wurde synthetisiert um die Säure ± Base-Chemie
zwischen Protein und Chromophor nachzuahmen. Die ther-
modynamischen Daten des Gleichgewichts zwischen der Neu-
tralform 1 und dem zugehörigen Zwitterion 1Æ wurden aus der
Temperaturabhängigkeit der UV/Vis-Spektren ermittelt
Results and Discussion
Intermolecular protonation: The results on the structure of
protonated 2,3-dihydrobilindiones are based predominantly
on NMR spectroscopic investigations. At first, model com-
pounds 3 and 4 were selected on account of their clear NMR
1
1
(DH8 ˆ 20.8 kJmol , DS8 ˆ 71 Jmol 1 K ). Die Überein-
stimmung mit den Daten anderer intramolekularer Protonen-
transfer-Reaktionen erklärt die Notwendigkeit für einen hohen
Ordnungszustand aus streng konservierten Konsensussequen-
zen für die Protonierung der Chromophore in Biliproteinen
mit Lichtsammelfunktion, beispielsweise dem Phycocyanin.
Andererseits deutet der stark negative DS8-Wert auf die
Möglichkeit einer Reprotonierung des Proteins nach sterischer
¾nderung hin. Demnach vermag die Geometrieänderung des
protonierten Chromophors in der Pr-Form des Phytochroms
nach erfolgter Z !E-Photoisomerisierung den Protonentrans-
fer zum Protein hin auszulösen und damit die Sequenz jener
Dunkelreaktionen einzuleiten, die zur physiologisch aktiven
Pfr-Form des Phytochroms führen.
1
spectra: their H NMR spectra only consist of singlets, and
because of the peripheral methyl substituents the assignment
of all quaternary carbons by gradient-enhanced heteronuclear
multiple bond correlation (HMBC) is unambiguous even for
the protonated chromophores. Compounds 3 and 4 are Z/E
diastereomers with respect to the exocyclic double bond of
ring D representing those species, which are believed to be
important for the photochemistry of phytochrome.
Because of its high acid strength p-toluenesulfonic acid (p-
TsOH) was used for protonation forming the stable hydro-
‡
‡
tosylates 3 ´ H OTs and 4 ´ H OTs . Compared with the salts
of weaker acids, for instance hydrodichloroacetates, hydro-
tosylates are advantageous to NMR experiments by slowing
down intermolecular proton exchange rates and thus prevent-
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Chem. Eur. J. 1998, 4, No. 9

Protonated 2,3-Dihydrobilindiones
1653±1659
1
ing the formation of averaged signals. This is shown in
Figure 3 for mixtures of 3 and its hydrodichloroacetate as well
as its hydrotosylate within the range of the methine proton
signals: addition of dichloroacetic acid to solutions of 3
resulted in the formation of shifted averaged signals, whereas
NH signals cannot be seen in the H NMR spectra of the
hydrodichloroacetates, but under similar conditions the
hydrotosylates show four NH singlets slightly broadened
within the range d ˆ 9 ± 12. On protonation only the signal of
the methine proton in position 10 is shifted significantly
‡
‡
‡
the use of p-TsOH enables detection of the signals of 3 ´ H as
well as those of 3.
downfield (Dd (ppm) ˆ 0.416 [3 ´ H
3]; 0.686 [4 ´ H
4])
indicating protonation at the dipyrrin substructure. The Dd
values of all other 1H NMR signals are less than 0.25 ppm and
cannot be used for a detailed structure determination.
However, a comparison of the Dd values of all 13C NMR
signals belonging to the quaternary carbons of the chromo-
phore is helpful (Figure 4, Table 1). In particular, the upfield
‡
shifts of the C-6 signal (Dd (ppm) ˆ 13.57 [3 ´ H
3];
‡
14.07 [4 ´ H
4]) and the C-9 signal (Dd (ppm) ˆ 17.36
‡
‡
[3 ´ H
3]; 17.84 [4 ´ H
4]) are not only remarkable in
value but also conclusive. They exceed all other Dd values and
belong to both carbons in the a-position to the nitrogen of
ring B where the positive charge must be localized. Concern-
ing signal shifts, this finding is in accordance with 13C NMR
data of pyridine and pyridinium ions.[15] In addition, different
chemical shift values of the carbons positioned quasisym-
metrically within the dipyrrin skeleton, in particular the d
‡
‡
values of C-6 (d ˆ 152.4 [3 ´ H ]; 152.8 [4 ´ H ]) and C-14 (d ˆ
‡
‡
142.1 [3 ´ H ]; 142.0 [4 ´ H ]), argue against charge delocaliza-
tion and exclude structure I (Figure 1b). Thus, protonated 2,3-
dihydrobilindiones correspond to structure III.
Stereochemical properties of the chromophores are not
changed on protonation. According to the ROESY spectra
the (4Z,9Z,15Z,5syn,10syn,14syn)-geometry of 3 remains in
Figure 3. 1H NMR spectra of the methine proton region of 3 in CDCl3 at
258C. a) 3 (6.4 Â 10 3 m), not protonated. b) 3 (6.4 Â 10 3 m) on addition of
0.6 equiv dichloroacetic acid showing averaged signals of
3 and its
‡
hydrodichloroacetate. c) 3 (2.6 Â 10 3 m) on addition of 0.4 equiv p-TsOH
showing separated signals of 3 and its hydrotosylate.
3 ´ H OTs . The same applies to the (4Z,9Z,15E,5syn,10-
‡
syn,14syn) geometry of 4 and 4 ´ H OTs . The extension of the
‡
‡
Figure 4. Correlation of 13C NMR signals in the region of the quaternary carbons of a) 3 ´ H OTs and 3 and b) 4 and 4 ´ H OTs . Signals of TsO are
‡
indicated by crosses, those of the o- and m-carbons below d ˆ 130 are truncated for clarity. Complete dissolution of 4 ´ H OTs at 28C in CDCl3 necessitates
the addition of a small amount of TFA. The corresponding quartet of the carbonyl carbon near d ˆ 160 is indicated by circles.
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1655

K. Grubmayr and M. Stanek
FULL PAPER
Table 1. 13C chemical shifts (d) in CDCl3 assigned by HMQC and HMBC
spectroscopy.
Interconversion of the neutral and the zwitterionic forms is
temperature-dependent and can be followed by reversible
changes of their H NMR and UV/Vis spectra.
‡
‡
1
3 (298 K)
3 ´ H OTs
4 (298 K)
4 ´ H OTs
(278 K)
(275 K)
On cooling, the formation of the zwitterion can be observed
in the UV/Vis spectra by the batho- and hyperchromic shifts
of the long-wavelength absorption band (Figure 5). The
equilibrium is shifted almost quantitatively towards 1Æ at
578C and towards 2Æ at 338C. At these temperatures the
protonation of the chromophores is practically complete and
the absorption maxima remain quite constant despite further
cooling.
The temperature dependence of the equilibrium between
neutral and zwitterionic species is also characterized by
isosbestic points. For the calculation of the thermodynamic
data the extinction values of the pure neutral species 1 and 2,
which cannot be measured directly from the absorption
spectra, were set equal to those of carboxyl-deprotonated 1
and 2 (Table 2). The fit of their absorption spectra to the
isosbestic points was sufficient and differences in absorbance
compared with 1 and 2 were therefore neglected. The negative
values of DH8 and DS8, which are typical of intramolecular
proton transfer reactions,[16] regulate the equilibrium.
C1
C2
175.9
44.2
177.9
44.0
174.7
44.5
178.5
43.8
C3
39.5
40.9
39.3
41.1
C3'
C4
C5
29.4
159.3
89.9
28.5
161.1
87.4
28.9
158.4
89.9
28.4
160.8
88.0
C6
C7
C7'
C8
C8'
C9
C10
C11
C12
C12'
C13
C13'
C14
C15
C16
C17
C17'
C18
C18'
C19
166.0
131.0
9.9
139.5
9.9
149.4
111.3
132.7
128.6
9.5
124.2
9.4
133.5
96.6
136.3
141.7
10.0
127.6
8.7
152.4
127.5
9.8
143.6
10.6
132.0
114.8
130.3
139.4
10.4
127.1
9.8
142.1
97.5
140.2
142.1
9.8
128.6
8.2
166.9
130.9
9.7
139.8
9.7
149.5
112.3
130.1
128.0
9.5
152.8
127.7
9.6
144.5
10.5
131.7
116.0
128.5
139.4
10.3
122.7
9.7
126.4
10.1
131.4
101.3
139.2
138.3
12.6
132.5
8.8
170.9
142.0
104.6
142.1
139.7
11.8
132.7
8.4
172.6
Table 2. Data for the proton transfer equilibria.
173.6
173.5
lmax [nm]
lmaxÆ [nm] DG8
[kJmol
DH8
[kJmol
DS8
1
1
1
]
]
[Jmol 1 K
]
1 > 1Æ
2 > 2Æ
584
581
641
650
‡ 0.2
1.3
20.8
26.8
71
86
chromophores to the (anti,syn,anti) conformation as found in
phycobiliproteins seems to be unlikely for 2,3-dihydrobilin-
diones intermolecularly protonated in solution.
1
Depending on the temperature, the H NMR spectra of 1
and 2 show similarities with the spectra of the dichloroace-
Intramolecular protonation: Dissolved in chloroform the
chromophores of the compounds 1 and 2 are protonated
quantitatively at low temperature by the carboxyl group of the
covalently attached cysteine moiety. In contrast to carboxyl-
protected derivatives, for instance, the trimethylsilylethyl
esters 1 ´ Tmse and 2 ´ Tmse, the chromophores cannot be
protonated under similar conditions by acetic acid, not even in
large excess. Assuming that the pKa values of acetic acid and
the carboxyl groups in 1 and 2 are more or less equal, the
proton transfer in 1 and 2 must proceed intramolecularly
resulting in the formation of the zwitterions 1Æ and 2Æ.
‡
‡
tates as well as the tosylates of 3 ´ H and 4 ´ H . On cooling,
the averaged signal of the methine proton in position 10 is
shifted downfield and no NH signals are detectable. However,
below 508C all four NH signals can be observed in the case
of compound 2Æ, resembling the signal pattern of the hydro-
tosylates of 3 and 4 at room temperature. Thus, the intra-
molecular protonation by the cysteinic carboxyl group at low
temperature seems to be equivalent to intermolecular proto-
nation by p-TsOH at room temperature. Consequently, we
conclude that protonation of 2,3-dihydrobilindione chromo-
Figure 5. UV/Vis spectra showing the temperature dependence of the proton transfer equilibrium of 1 and 1Æ in chloroform. Spectra were recorded at
578C for 1Æ (black), and at 45, 30, 15, 0, 15, 30, and 588C for equilibrium mixtures of 1 and 1Æ (gray). All values of e are corrected to room
temperature. The spectrum indicated by 1 originates from the diisopropylethylammonium carboxylate of 1 at room temperature, because in chloroform the
equilibrium cannot be shifted completely towards 1 below the boiling point of the solvent.
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Chem. Eur. J. 1998, 4, No. 9

Protonated 2,3-Dihydrobilindiones
1653±1659
phores by carboxylic acids requires intramolecularity and low
temperature, as demonstrated for the quantitative formation
of 1Æ and 2Æ. In phycobiliproteins protonation by aspartic acid
residues is favored by the highly ordered arrangement of short
consensus sequences[17] enabling the correct geometry neces-
sary for effective ion-pair formation. On the other hand,
conversion of the zwitterions 1Æ and 2Æ into the neutral forms
1 and 2 by raising the temperature indicates that in
biliproteins proton transfer from the protonated chromo-
phore back to the aspartate could be induced by geometrical
strain on the ion pair. Regarding phytochrome, geometrical
change by Z !E photoisomerization[18] of the Pr chromo-
phore may induce deprotonation in this way. Some results
from resonance Raman spectroscpy on phytochrome[19] sug-
gest that such chemistry may be relevant in the sequence of
dark reactions towards the physiologically active far-red-
absorbing form of phytochrome.
charge is localized. This result, based on NMR spectroscopic
investigations of synthetic model compounds, contributes to a
better understanding of the ionic chromophore ± protein
interactions in biliproteins. With respect to phycocyanin, we
conclude that protonation of the chromophores by aspartic
acid residues is entropically controlled. In general, the
formation of ion pairs in biliproteins depends on perfect
order among chromophore and amino acid residues, but
interference by steric strain may result in the reverse reaction.
This may be relevant to the photochemically initiated
conversion of the red-absorbing form of phytochrome, when
geometrical changes upon the Z !E photoisomerization of
the protonated Pr chromophore are followed by proton
transfer back to the protein.
Experimental Section
Synthesis and stereochemistry: The synthesis of 1Æ and 2Æ
started with the addition of N-(benzyloxycarbonyl)-cysteine
trimethylsilylethyl ester (Cbz-Cys-OTmse) to the 3-ethyl-
idene double bond of phycocyanobilin dimethyl ester
(PCBDME) according to ref. [20], resulting in the formation
of the four diastereomeric Tmse esters: 1 ´ Tmse
(2R,3R,3'R,CysR), 2 ´ Tmse (2S,3S,3'S,CysR), and two Tmse
esters with the (2R,3R,3'S,CysR) and (2S,3S,3'R,CysR) con-
figuration. From this mixture 1 ´ Tmse was separated by thin-
layer chromatography on silica gel. After deprotection with
tetra-n-butylammonium fluoride (TBAF) in tetrahydrofuran
(THF), followed by chromatography and treatment first with
aqueous HCl and then with water, compound 1 was obtained
in equilibrium with 1Æ. Analogously, the remaining three
diastereomeric Tmse esters were transformed into their
carboxylic acids yielding optically pure 2 in equilibrium with
2Æ after separation by column chromatography on silica gel
followed by successive treatment with aqueous HCl and
water.
General techniques: All chemicals were reagent grade. Solvents were
generally distilled prior to use; THF was distilled from sodium benzophe-
none ketyl. p-TsOH ´ H2O was melted under high vacuum prior to use.
Column chromatography was performed on silica gel (E. Merck, silica
gel 60, 0.063 ± 0.200 mm). Preparative thin-layer chromatography was
performed on precoated glass-backed plates (E. Merck, silica gel F256
,
0.5 mm). NMR spectra were recorded on a Bruker Avance DRX-500
spectrometer. The assignment of 13C signals is based on gradient-enhanced
HMQC and HMBC experiments. IR spectra were recorded on a Perkin
Elmer FT-IR spectrometer Paragon 1000PC. UV/Vis and CD spectra were
recorded on a Hitachi U-3210 spectrometer and a Jobin-Yvon Mark V
circular dichrograph. Isosbestic points are indicated by lip. Electrospray
and electron-impact mass spectra were measured on Hewlett Packard MS-
Engines 5989API and 5989A.
(4Z,9Z,15Z,2R,3R,3'R,CysR)-3-(1-(N-benzyloxycarbonyl-cystein-S-yl)-
ethyl)-18-ethyl-2,3-dihydro-8,12-bis-(2-methoxycarbonylethyl)-2,7,13,17-t-
etramethyl-23H-bilin-1,19(21H,24H)-dione (1): TBAF (10 mg, 31.7 mmol)
was added to a solution of 1 ´ Tmse (11.5 mg, 11.8 mmol) in THF (2 mL ).
The reaction mixture was stirred under an argon atmosphere for 30 min,
diluted with CH2Cl2 (40 mL), washed with H2O (3 Â 120 mL), with aqueous
NaHCO3 (0.2m, 60 mL), and dried over Na2SO4. After evaporation of the
solvent the residue was purified by thin-layer chromatography (silica gel,
CH2Cl2/MeOH ˆ 10/1). Elution with MeOH gave 1 (8.2 mg, 80%) as a blue
solid. Dissolution in CH2Cl2 and treatment with aqueous HCl (0.1n, 50 mL)
and H2O (3 Â 100 mL) afforded a solution of 1 in equilibrium with 1Æ at a
ratio near to 1:1 at room temperature. Rf (silica gel, CH2Cl2/MeOH ˆ 10/1):
0.3; 1H NMR (500 MHz, CDCl3; d corresponds to the top of the broad
single averaged signals of 1 and 1Æ at 278 K): d ˆ 7.28 (5H; H-C-Ph), 7.08
(1H; H-C10), 5.97 (1H; H-C15), 5.93 (1H; H-N-Cys), 5.66 (1H; H-C5),
4.98 (2H; H2-C-Bzl), 4.30 (1H; H-aC-Cys), 3.65 (6H; 2 Â CH3-O), 3.41
(1H; H-C3'), 3.16 (1H; H(1)-bC-Cys), 3.09 ± 2.80 (6H; H2-C8', H2-C12',
H(2)-bC-Cys, H-C3), 2.60 ± 2.48 (5H; H2-C8'', H2-C12'', H-C2), 2.32 (2H;
H2-C18'), 2.18, 2.08 (6H; H3-C17', H3-C13'), 2.03 (3H; H3-C7'), 1.39 (3H;
H3-C3''), 1.25 (3H; H3-C2'), 1.13 (3H; H3-C18''); (H,H)-ROESY NMR
(500 MHz, CDCl3, 278 K): 2' $ 3, 2 $ 3'', 3 $ (5,3''), 3' $ 5, 5 $ 7', 7' $
(8',8''), 10 $ (8',12'), 13' $ (12',12''), 15 $ (13',17'), 2,6-Ph $ CH2-Bzl; IR
(CHCl3): nÄ ˆ 3372, 3270, 3151, 2973, 2954, 2934, 2875, 1732, 1687, 1633,
1609 cm 1; UV/Vis (CHCl3, 298 K): lmax (e) ˆ 624 (22000), 352 (26600),
274 nm (15100); lip [1/1Æ] (e) ˆ 575 (15960), 442 (2050), 367 (24200), 334
(25100), 319 nm (19300); CD (CHCl3, 298 K): lmax (De) ˆ 593 ( 35), 350
(52), 275 nm ( 16); C46H55N5O10S; MS (ESIp): m/z (%) ˆ 870 (100)
The assignment of the relative configuration with respect to
the centers 2, 3, and 3' in 1, 1 ´ Tmse, and 2 is based on distinct
H,H-ROESY cross-peaks: 2 $ 3''; 2' $ 3 $ 3''; 3 $ 5 $ 3'.
The absolute configuration of center 2 was determined by a
comparison of the CD spectra of optically pure PCBDME.[21]
The (2R) enantiomer was obtained by thermal elimination of
Cbz-Cys-OTmse from 1 ´ Tmse in toluene at 838C.
Model compounds 3 and 4 were synthesized according to
the protocol of Gossauer[22] by condensation of (4Z)- or (4E)-
9-formyl-2,3,7,8-tetramethyldipyrrin-1(10H)-one with (4Z)-
2,3-dihydro-9-tert-butyloxycarbonyl-3,3,7,8-tetramethyldipyr-
rin-1(10H)-one[23] in trifluoroacetic acid (TFA). For spectro-
‡
‡
scopic investigations the tosylates of 3 ´ H and 4 ´ H were
prepared by adding stoichiometric quantities of p-TsOH to
the solutions of 3 and 4.
‡
‡
[M‡H] , 615 (6) [PCBDME‡H] .
(4Z,9Z,15Z,2S,3S,3'S,CysR)-3-(1-(N-benzyloxycarbonyl-cystein-S-yl)-eth-
yl)-18-ethyl-2,3-dihydro-8,12-bis-(2-methoxycarbonylethyl)-2,7,13,17-tetra-
methyl-23H-bilin-1,19(21H,24H)-dione (2): TBAF (60 mg, 230 mmol) was
Conclusion
added to
a solution of 2 ´ Tmse and its diastereomers with the
In summary, we have shown that the chromophores of 2,3-
dihydrobilindiones are protonated without change of their
overall geometry at the nitrogen of ring B where the positive
(2R,3R,3'S,CysR) and (2S,3S,3'R,CysR) configuration (90 mg, 93 mmol) in
THF (4 mL). The reaction mixture was stirred under an argon atmosphere
for 40 min, diluted with CH2Cl2 (80 mL), washed with H2O (3 Â 120 mL),
Chem. Eur. J. 1998, 4, No. 9
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1657

K. Grubmayr and M. Stanek
FULL PAPER
with aqueous NaHCO3 (0.2m, 60 mL), and dried over Na2SO4. After
evaporation of the solvent the residue was subjected to column chroma-
tography (silica gel, CH2Cl2/MeOH ˆ 15/1) separating 2 from its diaster-
eomers. Fractions containing 2 were treated with aqueous HCl (0.1n,
50 mL) and H2O (3 Â 100 mL). The ratio of 2 and 2Æ was near 1:2 at room
temperature. Rf (silica gel, CH2Cl2/MeOH ˆ 10/1): 0.4; 1H NMR (500 MHz,
CDCl3; d corresponds to the top of the broad averaged signals of 2 and 2Æ at
273 K): d ˆ 7.30 (5H; H-C-Ph), 7.23 (1H; H-C10), 5.89 (2H; H-C15, H-N-
Cys)), 5.69 (1H; H-C5), 5.07 (2H; H2-C-Bzl), 4.41 (1H; H-aC-Cys), 3.66,
3.65 (6H; 2 Â CH3-O), 3.55 (2H; H-C3', H(1)-bC-Cys), 3.10 ± 2.80 (7H; H2-
C8', H2-C12', H(2)-bC-Cys, H-C3, H-C2), 2.55 (4H; H2-C8'', H2-C12''), 2.32
(2H; H2-C18'), 2.13, 2.04, 2.02 (9H; H3-C17', H3-C13', H3-C7'), 1.43 (3H;
H3-C3''), 1.30 (3H; H3-C2'), 1.07 (3H; H3-C18''); (H,H)-ROESY NMR
(500 MHz, CDCl3, 299 K): 2' $ 3, 2 $ 3'', 3 $ (5, 3''), 3' $ (5, aC-Cys), 5 $
(7', aC-Cys), 7' $ (8', 8''), 10 $ (8', 12'), 13' $ 12', 15 $ (13', 17'), 2,6-Ph $
CH2-Bzl; IR (CHCl3): nÄ ˆ 3294, 2973, 2954, 2934, 2875, 1732, 1690, 1630,
1602 cm 1; UV/Vis (CHCl3, 298 K): lmax (e) ˆ 638 (24100), 350 (24800), 330
(25400), 277 nm (12300); lip [2/2Æ] (e) ˆ 581 (15800), 432 (2500), 376
(18700), 333 (24500), 318 nm (19500); CD (CHCl3, 298 K): lmax (De) ˆ 623
(38), 349 ( 56), 273 nm (19); C46H55N5O10S; MS (ESIp): m/z (%) ˆ 914
3H; H3-C18'), 1.33 (s, 6H; 2H3-C3'), tosylate: 7.54 (d, 3J ˆ 8 Hz, 2H; H-
C2,C6), 7.05 (d, 3J ˆ 8 Hz, 2H; H-C3,C5), 2.29 (s, 3H; H3-C4); (H,H)-
ROESY NMR (500 MHz, CDCl3, 280 K): 2 $ 3', 5 $ (7', 3'), 10 $ (8', 12'),
15 $ (13'), 17' $ 18'; UV/Vis (CHCl3): lmax (e) ˆ 610 (34000), 353 (23400),
304 nm (12900).
Acknowledgments: This work was supported by the Fonds zur Förderung
der wissenschaftlichen Forschung in Austria (FWF-Project P-9166-CHE).
We thank Dr. G. Niessner and Dipl.-Ing. W. Ahrer for mass spectral
measurements. K.G. thanks Prof. W. Wehrmeyer for drawing his attention
to ref. [17].
Received: January 14, 1998 [F963]
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‡
‡
‡
(22) [M H‡2Na] , 892 (100) [M‡Na] , 870 (15) [M‡H] .
Synthesis of 3 and 4: A solution of (Z)-9-formyl-2,3,7,8-tetramethyldipyr-
rin-1(10H)-one[22] (60 mg, 235 mmol) in MeOH (200 mL) was irradiated
with a high-pressure mercury immersion lamp (Hanau, TQ 150 ± 1725)
under an argon atmosphere for 2 h in a Pyrex well. After evaporation of the
solvent under reduced pressure the residue was added at once to a stirred
solution of (Z)-2,3-dihydro-9-tert-butyloxycarbonyl-3,3,7,8-tetramethyldi-
pyrrin-1(10H)-one[23] (72 mg, 235 mmol) in TFA (5 mL). After stirring for
90 min at room temperature MeOH (15 mL) was added. The reaction
mixture was diluted with CH2Cl2 (200 mL), washed with H2O (3 Â 150 mL),
with aqueous NaHCO3 (0.5m, 100 mL), and dried over Na2SO4. After
evaporation of the solvent the residue was subjected to column chroma-
tography (silica gel, CH2Cl2/MeOH ˆ 30/1) separating 3 (80 mg, 61%)
from 4 (25 mg, 19%).
(4Z,9Z,15Z)-2,3-Dihydro-3,3,7,8,12,13,17,18-octamethyl-23H-bilin-1,19(21-
H,24H)-dione (3): M.p. 2448C; Rf (silica gel, CH2Cl2/MeOH ˆ 30/1): 0.5;
1H NMR (500 MHz, CDCl3): d ˆ 6.60 (s, 1H; H-C10), 6.01 (s, 1H; H-C15),
5.45 (s, 1H; H-C5), 2.35 (s, 2H; H2-C2), 2.18 (s, 3H; H3-C12'), 2.13 (s, 3H;
H3-C8'), 2.11 (s, 3H; H3-C17'), 2.09 (s, 3H; H3-C13'), 1.99 (s, 3H; H3-C7'),
1.84 (s, 3H; H3-C18'), 1.42 (s, 6H; 2 Â H3-C3'); (H,H)-ROESY NMR
(500 MHz, CDCl3): 2 $ 3', 5 $ (7', 3'), 10 $ (8', 12'), 15 $ (13', 17'), 17' $
18'; IR (CHCl3): nÄ ˆ 3430, 3006, 2921, 2862, 1738, 1716, 1688, 1635,
1596 cm 1; UV/Vis (CHCl3): lmax (e) ˆ 584 (14100), 347 (32500), 275 nm
‡
(17400); C27H32N4O2; MS (70 eV; EI): m/z (%) ˆ 444 (100) [M] .
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(4Z,9Z,15E)-2,3-Dihydro-3,3,7,8,12,13,17,18-octamethyl-23H-bilin-1,19(21-
H,24H)-dione (4): M.p. 2428C; Rf (silica gel, CH2Cl2/MeOH ˆ 30/1): 0.3;
1H NMR (500 MHz, CDCl3): d ˆ 7.56 (s, 1H; H-N24), 6.58 (s, 1H; H-C10),
6.23 (s, 1H; H-C15), 5.38 (s, 1H; H-C5), 2.33 (s, 2H; H2-C2), 2.15 (s, 3H;
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‡
276 nm (14500); C27H32N4O2; MS (70 eV; EI): m/z (%) ˆ 444 (100) [M] .
Hydrotosylates of 3 and 4: Hydrotosylates were prepared in solution by
adding stoichiometric quantities of p-TsOH dissolved in CDCl3 or CHCl3
to the corresponding solutions of 3 and 4.
‡
3 ´ H OTs : 1H NMR (500 MHz, CDCl3, 278 K): d ˆ 11.73, 11.46, 10.66,
9.92 (4 Â s, 4H; 4 Â H-N), 7.02 (s, 1H; H-C10), 6.03 (s, 1H; H-C15), 5.40 (s,
1H; H-C5), 2.31 (s, 3H; H3-C8'), 2.28 (s, 3H; H3-C12'), 2.27 (s, 2H; H2-C2),
2.08 (s, 3H; H3-C13'), 2.05 (s, 3H; H3-C7'), 1.91 (s, 3H; H3-C17'), 1.60 (s,
3H; H3-C18'), 1.27 (s, 6H; 2  H3-C3'), tosylate: 7.75 (d, 3J ˆ 8 Hz, 2H; H-
C2,C6), 6.97 (d, 3J ˆ 8 Hz, 2H; H-C3,C5), 2.26 (s, 3H; H3-C4'); (H,H)-
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15 $ (13', 17'), 17' $ 18'; IR (CHCl3): nÄ ˆ 3440, 3006, 2921, 1710, 1688, 1633,
1600 cm 1; UV/Vis (CHCl3): lmax (e) ˆ 624 (36900), 352 (28100), 277 nm
(11500).
‡
4 ´ H OTs : 1H NMR (500 MHz, CDCl3, 275 K): d ˆ 11.65, 11.51, 10.41,
9.45 (4 Â s, 4H; 4 Â H-N), 7.02 (s, 1H; H-C10), 6.42 (s, 1H; H-C15), 5.47 (s,
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