Synthesis of selectively 13C-labelled bilin compounds

Article abstract of DOI:10.1002/ejoc.200600677

A series of open-chain tetrapyrroles, each carrying one or more ¹³C-labels in its three ring-bridging methine groups, were synthesized. These compounds - [5-, [10-, and [15-¹³C]- phycocyanobilins (PCB), [10-¹³C]-phytochrom

Full text of DOI:10.1002/ejoc.200600677

FULL PAPER
DOI: 10.1002/ejoc.200600677
13
Synthesis of Selectively C-Labelled Bilin Compounds
Yevgen Makhynya,[a] Zakir Hussain,[a] Tanja Bauschlicher,[a] Pascale Schwinte,[b]
Friedrich Siebert,[b] and Wolfgang Gärtner*[a]
Keywords: Isotope labelling / Bilin / Phytochrome / Infra-red spectroscopy / Tetrapyrrole chromophore / Phycocyanobilin
A series of open-chain tetrapyrroles, each carrying one or
more 13C-labels in its three ring-bridging methine groups,
were synthesized. These compounds – [5-, [10-, and [15-13C]-
phycocyanobilins (PCB), [10-13C]-phytochromobilin, and
[10,15-13C2]-PCB – were each obtained by a convergent
route, starting with the four pyrrole building blocks, with the
initial formation of the left and the right halves of the tetra-
pyrrole separately, followed by a final condensation of the
two dipyrrole units to yield the target bilin compound. The
13C-labels were all inserted as C1- or C2-units prior to the
appropriate condensation. The substitution pattern of these
bilins on ring A (ethylidene substituent at position 3) allows
covalent attachment to the apoprotein of the plant photo-
receptor phytochrome. The isotope shift produced by inser-
tion of a 13C-isotope is demonstrated in the FT-IR spectra of
phytochrome-incorporated [10-13C]-phytochromobilin.
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2007)
the pyrrole rings C and D; see Figure 1). This Z Ǟ E pho-
toisomerization is assumed to be followed by various single-
bond rotations and slight changes in the dihedral angles
between the other rings, in order to compensate for steric
hindrance to the surrounding protein after the double bond
rotation.
Introduction
Biological photoreceptors gain their exceptional proper-
ties from intimate interactions between the chromophore
and the protein moieties, which allow transmission of the
dynamic, light-induced changes in the chromophore to the
surrounding protein and the initiation of signal transduc-
tion. This is particularly the case for the covalently bound,
highly flexible bilin chromophores of the red-/far-red-sensi-
tive phytochromes, found ubiquitously in higher and lower
plants and in many prokaryotes.[1–3] In addition to the in-
vestigation of the holoprotein by time-resolved absorption
spectroscopy,[4] the light-induced conformational changes in
the chromophore itself can be probed directly by several
methods, such as vibrational (FT infra-red or Raman) spec-
troscopy or also by various NMR techniques. The highly
complex spectra obtained from the parent states of these
photoreceptors (Prs), from their final photoproducts (Pfrs)
or from potentially stabilizable intermediates are extremely
difficult to interpret, which in the case of vibrational spec-
troscopy is due to the extended conjugation of the π system,
giving rise to strongly coupled conformational modes.
For understanding of biological photoreceptors, and in
particular for phytochromes, it is thus of particular impor-
tance to identify selectively certain positions that are as-
sumed to be suggestive of the investigated conformational
changes. The bilin chromophores of the phytochromes are
known to undergo – in the instant light-induced process –
a photoisomerization of the 15,16-double bond (connecting
Figure 1. Structural formula of phycocyanobilin (1), covalently
bound to cysteine321 of oat phytochrome. Attachment is ac-
complished through position 3Ј in the A-ring, which in unbound
form is part of an ethylidene substituent. The chromophore is
shown in ZZZ,asa form. Phytochromobilin differs from PCB only
in the substituent at position 18 (vinyl vs. ethyl). The photoisomer-
ization of the 15–16 double bond is indicated by an arrow.
The synthesis of open-chain tetrapyrroles has been de-
scribed for a large number of different derivatives, including
some work on the introduction of stable isotopes.[5] We have
recently presented a convergent synthesis, based on pre-
viously developed synthetic routes,[6] for an open-chain tet-
rapyrrole that, thanks to the separate synthesis of each ring,
allows virtually every position of the tetrapyrrole to be ad-
[a] Max-Planck-Institut für Bioanorganische Chemie,
45470 Mülheim, Germany
[b] Institut für Molekulare Medizin und Zellforschung, Sektion
Biophysik,
Hermann-Herderstr. 9, 79104 Freiburg, Germany
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dressed for requested changes.[7] We now describe the selec- reagent for condensation between the thioimide precursor
tive introduction of 13C-isotopes into the three methine of ring A and the ylide formed at ring B requires an elec-
bridge carbon atoms (positions 5, 10, and 15), together with tron-withdrawing substituent in order to prevent side reac-
one double labelling (positions 10 and 15), by application of tions such as deprotonation of the pyrrole nitrogen atom in
this concept and demonstrate the initial vibrational (FTIR) ring B, followed by electronic rearrangements.[11] On the
spectra of these bilins after their incorporation into recom- other hand, though, since the ethylidene substituent on ring
binant plant apo-phytochrome (phyA of oat). The same A is essential for covalent incorporation of a bilin into the
class of compounds allow detailed conformational analysis plant photoreceptor phytochrome, any electronic rearrange-
of the chromophore by 13C and 15N NMR spectroscopy, as ment has be avoided during the condensation reaction. The
the latter approach (15N NMR) has recently demon- compound usually employed is benzyl oxoacetate, formed
strated.[8]
by periodate-mediated oxidative splitting of dibenzyl tar-
trate. Benzyl oxoacetate was then attached to the free α-
position of the B-ring pyrrole 8, and the resulting hydroxy
function was converted by chlorination/triphenylphosphane
addition into a Wittig compound to connect rings A and
B. Several alternative C2-units for this condensation step in
Results and Discussion
Chemical Synthesis
Pyrroles to serve as precursors for rings A, B (which is bilin synthesis have been proposed, amongst others acetoni-
identical to ring C) and D of bilin compounds were synthe- trile, which would be excellently suited to support the reac-
sized as recently described.[9] In the convergent synthesis tion by virtue of its small size (see Figure 2). An attempted
described above, in which the left and right halves of a bilin removal of the nitrile moiety from the C-5 position of rings
were generated separately and finally condensed at its cen- A–B by reduction and retro-Mannich reaction was unsuc-
tral position, C1-synthons were used for the future positions cessful, however, so the very mild removal of a benzyl ester
10 and 15 (see Scheme 1). The bridging position between by hydrogenolysis and subsequent decarboxylation remains
rings A and B (position 5, Figure 1) was introduced as a the most preferable procedure. For the purpose of introduc-
C2-unit.[10] Isotope labelling at positions 10 and 15 has in ing a 13C-label the synthesis needs to be performed dif-
fact been described for several bilins,[5] thanks to the ready ferently from routine methods, since benzyl oxoacetate is
availability of the C1-units, but no isotope introduction at not available in isotope-labelled form, and so we employed
position 5 had previously been accomplished. The conden- [2-13C]-labelled acetic acid as the C2 building block. [2-13C]-
sation between rings A and B is one of the most challeng- 2-Bromoacetic acid was converted into the benzyl ester 6
ing. A number of attempts have established that the Wittig and subsequently into its 2-iodo analogue 7. The crucial
Scheme 1. Overview of the chemical synthesis of compounds 1–5. EWG: electron-withdrawing group.
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Synthesis of Selectively 13C-Labelled Bilin Compounds
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Figure 2. Retrosynthetic proposal for introduction of 13C-label into position 5 of PCB (1).
step in this reaction sequence was then the alkylation of a pricious and additionally has shown a strong light depen-
pyrrole at a free α-position. Only two possible procedures dency. When it was performed under daylight illumination,
were known – either by following a radical mechanism[12,13] only a 5% yield was obtained, whereas the high yield re-
or by performing the reaction in ionic liquids[14,15] – but ferred to above was obtained when the reaction vessel was
both of these turned out to be inefficient for the synthesis protected from light. This “left half” of the target bilin was
of an all-carbon-substituted pyrrole, due to low yields. then condensed in the routine manner with the “right half”
Nonetheless, we introduced benzyl [2-13C]-2-iodoacetic acid 14 to achieve the desired tetrapyrrole, still in its propionate
by a radical reaction similar to that described in ref.[12,13] dimethyl ester state. Hydrolysis with use of an ion exchange
and succeeded in improving the methodology, making it ap- resin then yielded [5-13C]-PCB 1 in an overall yield of 3.5%
plicable for the radical alkylation of mono-α-unsubstituted (relative to the starting amount of bromoacetic acid). The
pyrroles in average to good yields. The synthesis of 9 in developed methodology for the introduction of a C-5 13C-
70% isolated yield was thus accomplished (in addition, ca. label is a significant improvement in relation to previously
10% of starting material was recovered).
published results[11] (which reported only a 25% yield for
The resulting 5-{benzyloxycarbonyl([13C]methyl)}pyrrole that particular condensation), and represents a versatile
derivative 9 was chlorinated at the 5Ј-position (10) and then method for the construction of bilirubins, biliverdins and
converted into the triphenylphosphonium chloride deriva- cyclic tetrapyrroles.
tive and finally into the active ylide 11 by treatment with
The synthesis of [10-, [15- and [10,15-13C2]-PCB (2, 3 and
sodium carbonate. This Wittig reagent was condensed with 4) followed the routine synthesis scheme (Scheme 1). Inser-
thioimide 12 (A ring) without basic activation, to yield the tion of a 13C atom at position 10 was performed by for-
[5-13C]-dipyrrole compound 13. This reaction is rather ca- mylation (trimethyl orthoformate, formyl 13C) of position
Scheme 2. Synthesis of 5-13C PCB. Bn: benzyl.
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11 of the dipyrrole 14 constituting the “right half” (num- vestigations. The effect of isotope incorporation can clearly
bering according to open-chain tetrapyrroles; see Figure 1), be seen in a Pr/Pfr FTIR difference spectrum recorded with
followed by condensation with the “left half” and final sa- a 10-13C-PΦB incorporated into apo-phyA (Figure 3). Dif-
ponification of the dimethyl esters at rings B and C. Inser- ference spectra are commonly presented as photoproduct
tion of a 13C label at position 15 was accomplished by for- minus parent state (i.e., Pfr – Pr state). As can be seen in
mylation of the α-free pyrrole 8 with 13C-labelled DMF the difference spectrum, most difference bands remain at
(formyl 13C) and POCl3, followed by condensation with the identical positions, but others (indicated by small arrows)
D-ring building block. This compound was then formylated exhibit characteristic shifts in the band position. Even with
in a routine way, to give the “right half” of the desired the very complex structure of the spectrum, one can clearly
tetrapyrrole, and this was treated with the “left half” as identify band patterns that can be ascribed to the single
described above. The tetrapyrrole compound was again incorporated isotope label. A detailed analysis of infrared
finally saponified, yielding [15-13C]-PCB. A combination of and of Raman spectra, also including spectra of the various
the described isotope label insertion reactions also finally photoprocess intermediates, should allow a deeper view
yielded [10,15-13C2]-labelled PCB (Scheme 2).
into the conformational changes that take place during the
In the synthesis of 5, the left half of the target tetrapyr- Pr to Pfr and Pfr to Pr conversion of phytochrome and will
role was prepared as described in the cases of 1–4. The right be described elsewhere.
half, though, unlike in the procedure described for 1–4, was
obtained by the cleavage of biliverdin IXa diethyl ester with
Experimental Section
thiobarbituric acid and subsequent formylation of the C-D
half with 13C1-trimethyl orthoformate.
Abbreviations: PCB: phycocyanobilin, Pr, Pfr: red-, far-red absorb-
ing forms of phytochrome, PΦB, phytochromobilin.
All five isotope-labelled bilin compounds 1–5 were ob-
tained in mg amounts and were identified by their spectral
properties, which were identical to those of their unlabelled
counterparts. The insertion of single 13C labels – or, in the
case of compound 4, a double 13C label – was unambigu-
ously verified by their mass spectra. Bilins prepared in the
manner described here are always racemic at position 2 in
the A-ring. However, since assembly with the apoprotein is
always performed in the presence of an excess of chromo-
phore, due to its instability under the incubation conditions,
no special effort to generate an enantiomerically pure tetra-
pyrrole was made.
After HPLC purification, the labelled PCBs were incu-
bated with the recombinantly expressed apo-protein of oat
phyA (N-terminal 65 kDa half). In each case the expected
Pr- and Pfr-specific absorption spectra were obtained, indi-
cating that this material could be used for spectroscopic in-
Chemical Synthesis: For an overview on the synthetic routes see
Scheme 1. IUPAC nomenclature is only used for the isolated ring
compounds.
Benzyl [2-13C]-2-Bromoacetate (6):[16] [2-13C]-2-Bromoacetic acid
(1 g, 7.15 mmol, 99% isotope purity, Aldrich, Germany) and ben-
zyl alcohol (0.79 g, 7.3 mmol) were dissolved in dichloromethane
(30 mL), and dicyclohexyldicarbodiimide (DCC; 1.51 g,
7.33 mmol) was added, followed by a catalytic amount (75 mg,
0.6 mmol) of 4-(dimethylamino)pyridine (DMAP). The mixture
was stirred overnight and finally chromatographed on a silica gel
column with dichloromethane. Concentration in vacuo gave the
corresponding benzyl ester 6 as a colourless liquid (1.48 g, 90%).
Benzyl [2-13C]-2-Iodoacetate (7): Benzyl [2-13C]-2-bromoacetate
(1.48 g, 6.47 mmol) was dissolved in acetone (20 mL). NaI (1.32 g,
8.8 mmol) was added and the mixture was stirred at room tempera-
ture for 1 h, during which a voluminous white precipitate formed.
The solvent was evaporated, and the remaining solid was separated
between water and ether. The ether phase was separated, dried with
Na2SO4 and concentrated in vacuo to yield benzyl [2-13C]-2-
1
iodoacetate (7, 1.72 g, 97%) as a yellow oil. H NMR (400 MHz,
CDCl3): δ = 7.37 (m, 5 H, CH-benzyl), 5.16 (s, 2 H, CH2-benzyl),
3.70 (d, J = 153 Hz, 2 H, CH2-2) ppm.
tert-Butyl
5-{Benzyloxycarbonyl([13C]methyl)}-3-[2-(methoxycar-
bonyl)ethyl]-4-methyl-1H-pyrrole-2-carboxylate (9): One third of
the calculated amount (0.2 g, 2.06 mmol) of H2O2 (35% solution
in water, the whole amount being 6.17 mmol, 0.6 g) was added
dropwise to a stirred solution of α-free pyrrole 8 (1.1 g, 4.11 mmol),
benzyl [2-13C]-2-iodoacetate (0.57 g, 2.07 mmol; 1/3 of the total
amount) and FeSO4·7H2O (0.23 g, 0.82 mmol; 1/3 of the total
amount) in DMSO (40 mL). The reaction was slightly exothermic,
so a water bath was used to keep the reaction at room temperature.
After the mixture had been stirred for one hour, further portions
(again 1/3 of the total amount) of benzyl [2-13C]-2-iodoacetate,
FeSO4·7H2O and H2O2 were added. Finally, after the mixture had
been allowed to react for another hour, the last third of the reagents
was added. The colour of the reaction mixture changed from light
yellow to brown-red during the addition of H2O2, but turned back
to yellow during the course of the reaction. After the mixture had
been stirred for an additional 3 h, the reaction was quenched by
Figure 3. FTIR difference spectrum (Pfr – Pr) of 10-13C-labelled
PΦB, incorporated into apo phyA of oat (65 kDa N-terminal do-
main). Positive bands are assigned to the photoproduct (Pfr)
whereas negative bands are related to the Pr form. Changes due to
the isotope shift are indicated by arrows.
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Synthesis of Selectively 13C-Labelled Bilin Compounds
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the addition of a saturated brine solution and subsequently ex-
tracted with diethyl ether (3ϫ50 mL). The combined organic
phases were washed with brine, dried with Na2SO4 and concen-
trated in vacuo. Column chromatography with a pentane/diethyl
ether gradient (2:1 Ǟ 1:1) as eluent resulted in the target alkylated
pyrrole (9, 1.2 g, 70% isolated yield), initially obtained as a very
viscous yellow oil, which slowly crystallized to yield light yellow
crystals. Additionally, 135 mg (12% of the starting amount) of the
α-free pyrrole 8 was recovered. 1H NMR (400 MHz, CDCl3, 1H/
1H-COSY): δ = 9.08 (s, 1 H, NH), 7.29–7.37 (m, 5 H, CH-phenyl),
get benzyl ester 13 was obtained as a yellow foam (53 mg, 30%).
1H NMR (400 MHz, CDCl3, 1H/1H-COSY): δ = 10.80 (s, 0.5 H,
NH), 10.70 (s, 0.5 H, NH), 9.12 (brs,0.5 H, NH), 8.90 (brs, 0.5 H,
NH), 7.14–7.24 (m, 5 H, CH phenyl), 5.29 (q, J = 6.93 Hz, 0.6 H,
HC-31), 5.19 (m, 0.4 H, HC-31), 5.00–5.16 (m, 2 H, benzyl CH2),
3.62 (s, 3 H, CO2Me), 3.09 (m, 1 H, HC-2), 2.99 (m, 2 H, CH2-81),
2.50 (m, 2 H, CH2-82), 1.77 (s, 3 H, CH3-71), 1.64 (m, 3 H, CH3-
32), 1.50 (s, 9 H, CO2tBu), 1.31 (m, 3 H, CH3 -21) ppm. 13C NMR
1
(100 MHz, CDCl3, BB, DEPT, H/13C-COSY): δ = 177.86 (Cq, C-
1), 173.48 (Cq, CO2Me), 168.70 (Cq, CO2Bn), 161.00 (d, J =
5.14 (s, 2 H, H2C-benzyl), 3.64 (s, 3 H, CO2Me), 3.60 (d, J = 12.7 Hz, Cq, CO2tBu), 152.88 (dd, J = 81.4, 25.8 Hz, Cq, C-5),
130 Hz, 2 H, H2C-51), 2.97 (m, 2 H, CH2-31), 2.50 (m, 2 H, CH2-
136.14 (Cq, Ar), 134.36 (d, J = 16.4 Hz, Cq, pyrrole), 133.68 (Cq,
32), 1.92 (s, 3 H, CH3-41), 1.53 [s, 9 H, 3 CH3 (t Bu)] ppm. 13C pyrrole), 132.52 (Cq, pyrrole), 128.33 (CH, Ar), 127.79 (CH, Ar),
NMR (100 MHz, CDCl3, BB, DEPT, 1H/13C-COSY): δ = 173.70 127.21 (CH, C-31), 127.11 (CH, Ar), 125.60 (dd, J = 69.7, 20.4 Hz,
(Cq, CO2Me), 169.59 (Cq, CO2Bn), 160.57 (Cq, CO2tBu), 135.40
Cq, pyrrole), 119.86 (Cq, pyrrole), 118.90 (Cq, pyrrole), 91.92 (m,
(Cq, Ar), 128.62 (CH, Ar) 128.52 (d, J = 2.5 Hz, Cq-3), 128.45 enriched meso), 80.84 (Cq, tBu), 65.92 (CH2, benzyl), 51.38 (CH3,
(CH, Ar), 128.29 (CH, Ar), 123.58 (Cq, C-5), 119.43 (d, J = 1.7 Hz, OMe), 37.82 (d, J = 8.9 Hz, CH, C-2), 34.24 (CH2,C-82), 28.30
Cq, C-2), 117.72 (d, J = 4 Hz, Cq, C-4), 80,63 (Cq, C-tBu), 67.03
(3ϫCH3, tBu), 21.06 (CH2,C-81), 16.40 (d, J = 36.7 Hz, CH3,C-
(CH2-benzyl), 51.41 (CH3, OMe), 34.98 (CH2, C-32), 31.56 (m, 21) 15.97 (d, J = 29.5 Hz, CH3,C-32), 9.04 (CH3,C -71) ppm. MS
CH2, C-51), 28.43 (CH3, tBu), 20.76 (CH2, C-31), 8.56 (CH3, C- (EI, 165 °C): m/z (%) = 537 (70) [M]+ (C2913CH36,N2O7), 481 (55)
41) ppm. MS (EI, 130 °C): m/z (%)
=
416 (43) [M]+
[M – C4H8(tBu)]+, 466 (12), 436 (10), 390 (23), 346 (100)
(C2213CH29,NO6), 360 (54) [M – C4H8(tBu)]+, 343 (5), 329 (14), [M – C4H8(tBu) – CO2Bn]+, 286 (8), 228 (6), 91 [54, C7H7+ (tropyl-
225 (100) [M – C4H8(tBu) – CO2Bn]+, 207 (12), 193 (19), 181 (16),
165 (11), 91 [77, C7H7+ (tropylium ion)], 57 [6, C4H9+(tBu)], 41 (3).
HRMS (ESI-pos): C2213CH29NNaO6 [M + Na]: theor: 439.192064;
found: 439.192369.
ium ion)], 57 [5, C4H9+(tBu)], 41 (3) HRMS (ESI-pos):
C2913CH36N2NaO7 [M
560.244888.
+
Na]: theor: 560.244827; found:
Removal of Benzyl Ester from 13: In the dark and under argon, the
benzyl ester 13 (53 mg, 0.097 mmol) was dissolved in absolute THF
(7 mL) and palladium on charcoal (10%, 34 mg, 0.032 mmol) was
added without stirring. Vacuum was applied, until the solvent
started to boil, and the reaction vessel was flushed with hydrogen.
This procedure was repeated five times. The reaction mixture was
then stirred at ambient temperature for 1.5 h (the reaction was
monitored by TLC). Finally, the mixture was filtered through a
double paper filter (blue band) and the filter was washed with THF.
The combined organic phases were concentrated and the residue
was subjected to HPLC separation (water/acetonitrile/TFA, gradi-
ent from 97.95:2:0.05 to 0:99.95:0.05) to give the corresponding
acid (17.4 mg, 40% isolated yield, 75% considering recovered start-
ing material). Some benzyl ester 13 (24 mg, 45% from the starting
material) was also recovered.
tert-Butyl
5-{Benzyloxycarbonylchloro([13C]methyl)}-3-[2-(meth-
oxycarbonyl)ethyl]-4-methyl-1H-pyrrole-2-carboxylate (10): The la-
belled pyrrole 9 from the previous step (503.2 mg, 1.2 mmol) was
added to a suspension of anhydrous K2CO3 (1.66 g, 12 mmol) in
dichloromethane (50 mL) and the system was cooled to –78 °C
with a dry ice/acetone bath. SO2Cl2 (162.3 mg, 1.2 mmol) was
added dropwise at this temperature and the reaction mixture was
allowed to stir for a further 3 h, the temperature being maintained
below –70 °C. Afterwards the cooling was removed and the reac-
tion mixture was allowed to warm to room temperature. The colour
of the mixture changed from light yellow to yellow-brown. Potas-
sium carbonate was filtered off and the solvent was removed in
vacuo. The crude product was transferred to a next step without
further purification.
[5-13C]-Labelled A–B Ring Dipyrrole (13): The crude product 10
from the previous step was dissolved in ether (15 mL) and a solu-
tion of triphenylphosphane (349 mg, 1.33 mmol) in ether (6 mL)
was added dropwise. The reaction mixture was stirred at room tem-
perature overnight. After this stirring, a voluminous white precipi-
tate had formed, and this was removed by filtration. A second por-
tion was obtained from the mother liquid. The combined portions
of the phosphonium salt were dissolved in dichloromethane and
washed with saturated NaHCO3, and the organic phase was dried
with sodium sulfate and concentrated to give a yellow, very viscous
oil. This oil was dried in vacuo to give the target phosphorus ylide
11 as a yellow voluminous foam (527 mg, 65%). This foam had a
UV spectrum and an Rf value identical to those of the unlabelled
compound,[10,11] so no further analysis was performed.
Synthesis of [5-13C]-Labelled Phycocyanobilin Dimethyl Ester (1-Di-
methyl Ester): The coupling was performed in the dark and under
constant argon flow. The A–B rings compound (17.4 mg,
0.039 mmol), the left half of the target molecule, was dissolved in
TFA (1 mL) and the system was stirred for 30 min at room tem-
perature. The reaction mixture was cooled to –15 °C with an ace-
tone/dry ice bath. The right half (C–D rings 14, prepared and puri-
fied by the already published procedure,[18] 13.5 mg, 0.041 mmol)
was dissolved in TFA (4 mL) and added dropwise. The reaction
was allowed to proceed for an additional 8 h, and the temperature
was kept between –10 and –15 °C. Finally, methanol (2 mL) was
added at this temperature. The cooling bath was removed and the
mixture was stirred for another 30 min. During this time the mix-
ture warmed to room temperature and a colour change from red-
dish to dark blue was observed. The solvent was removed carefully
under a strong argon flow and the resulting residue was dissolved
in dichloromethane (30 mL). The organic phase was washed with
diluted NaHCO3 (30 mL) and subsequently with water until neu-
tral pH, dried and concentrated under reduced pressure. The re-
maining residue was purified by HPLC (methanol/water 5:1). After
removal of the solvent, the [5-13C]-labelled phycocyanobilin di-
methyl ester was obtained as a blue solid (12.2 mg, 55%). This
The coupling to yield 13 was performed in the dark and under
argon. The phosphorus ylide 11 (227 mg, 0.335 mmol; ring B) and
the thioimide 12 (52 mg, 0.335 mol; ring A, prepared by the litera-
ture procedure[17]) were dissolved in dry toluene (30 mL). This reac-
tion mixture was heated at reflux for 24 h and the colour changed
from yellow to reddish. The solvent was evaporated and the re-
sulting residue was purified by HPLC (pentane/ethyl acetate, 2:1).
After removal of the solvents and further drying in vacuo, the tar-
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product was then transferred to the next step without any charac-
terisation.
by Lindner,[9] to yield the right and the left halves of the desired
molecule separately. The C–D ring building block (32 mg,
97.4 µmol) was formylated with trimethyl orthoformate (for-
myl 13C, 1.52 mmol) in trifluoroacetic acid (3 mL) and condensed
with the “left half”, to yield [10-13C]-phytochromobilin as the
methyl ethyl ester in 45% yield.[9] [10-13C]-Phytochromobilin was
obtained upon ester hydrolysis, as described for the PCB counter-
parts, in 89% yield. HRMS (ESI – pos. + neg.): C3213CH36N4O6
[M – H]: 584, 13C incorporation Ͼ 95%.
[5-13C]-Labelled Phycocyanobilin (1): [5-13C]-Labelled phycocyano-
bilin dimethyl ester (12.2 mg, 0.02 mmol) was dissolved, in the dark
and under argon, in a mixture of trifluoroacetic acid and water
[1:1 (v:v), 30 mL], and acidic ion exchange resin DOWEX (12.3 g)
was added. The resin had been washed extensively with water and
dried in air prior to its addition. This suspension was stirred at
room temperature for 64 h, and the resin was finally filtered off
(ceramic filter). The residue was washed successively on a filter pa-
per, alternately with water (40 mL, five times each) and with a chlo-
roform/methanol mixture (40 mL, 49:1 v/v). The aqueous phase
was separated and extracted with a chloroform/methanol (49:1
mixture, four times, 40 mL each). The combined organic phases
were washed with brine until pH 6 and dried with Na2SO4. Evapo-
ration of the solvents resulted in the crude product as a dark blue
solid that was further purified by HPLC (7 m KH2PO4 buffer, pH
7, acetonitrile; gradient from 7:3 to 1:4). After removal of meth-
anol, chloroform (30 mL) and water (30 mL) were added to the
residue and the phases were separated. The water phase was ex-
tracted with chloroform until the aqueous layer remained colour-
less, and the combined organic phases were then dried with
Na2SO4. After filtration, the Na2SO4 cake was washed extensively
with a chloroform/methanol mixture (4:1 v/v), until the cake was
nearly colourless. Evaporation of the solvent resulted in the [5-13C]-
labelled phycocyanobilin 1 (8 mg, 70%) as a dark blue solid. Quali-
tative HPLC analysis revealed the product to be homogeneous. Par-
tial NMR: 1H NMR (400 MHz, CDCl3): δ = 5.96 (d, J =
158.67 Hz, CH-5) ppm. 13C NMR (100 MHz, CD3OD): δ = 88.92
(CH, C-5) ppm. HRMS (ESI – pos.): C3213CH39N4O6 [M + H]:
theor: 588.289767; found: 588.289637.
Biochemical Procedures: The N-terminal half of apo-phytochrome
of oat (Avena sativa), spanning positions 1–595, was heterologously
expressed in Hansenula polymorpha. A C-terminally attached His6-
tag allowed affinity purification on Ni-NTA (Serva, Heidelberg) as
described.[20] Assembly with 13C-labelled PCBs (1–5) was per-
formed with the crude cell lysate prior to purification. The isolated
chromoprotein was characterized by SDS-PAGE. The Pr and Pfr
states were produced by far-red and red irradiation of the chromo-
proteins, respectively. The extent of photoconversion was followed
by UV/Vis absorption spectroscopy (Shimadzu UV2401-PC).
FTIR Spectroscopy: For the FTIR measurements, the recently de-
scribed sandwich cuvette was used.[21] All other parameters – sam-
ple preparation, measuring conditions such as in-situ illumination,
number of scans, and spectral resolution, data collection and treat-
ment – were essentially identical to those recently described.[22]
Acknowledgments
This work was supported by the Volkswagen-Foundation (I79 976)
and the Deutsche Forschungsgemeinschaft (GA377/13).
[1] E. Kevei, F. Nagy, Physiologia Plantarum 2003, 117, 305–313.
[2] P. H. Quail, Nature Reviews Molecular Cell Biology 2002, 3,
85–93.
[3] B. L. Montgomery, J. C. Lagarias, Trends in Plant Science
2002, 7, 357–366.
[4] W. Gärtner, S. E. Braslavsky, in: Photoreceptors and Light Sig-
nalling (Ed.: A. Batschauer), Royal Society of Chemistry, Cam-
bridge, UK 2003, pp. 137–180.
[5] P. A. Jacobi, D. Pippin, Org. Lett. 2001, 3, 827–830.
[6] A. Gossauer, W. Hirsch, Justus Liebigs Ann. Chem. 1974, 1496–
1513.
[10-13C]-Labelled Phycocyanobilin (2): The α-free pyrrole was for-
mylated with DMF and POCl3 to afford the corresponding alde-
hyde, which was condensed with ring D (synthesized by the pub-
lished procedure[19]) to give the right half (C–D ring building
block). This was again formylated, this time with trimethyl ortho-
formate (formyl 13C) in trifluoroacetic acid as described for phy-
tochromobilin.[18] The latter compound was condensed with the left
half (A–B ring building block) in the routine manner to give the
[10-13C]-labelled PCB as its dimethyl ester. Hydrolysis afforded the
[10-13C]-labelled phycocyanobilin in 20% yield (for the last two
steps).
[7] U. Robben, I. Lindner, W. Gärtner, K. Schaffner, Angew. Chem.
Int. Ed. 2001, 40, 1048–1050.
[15-13C]-Labelled Phycocyanobilin (3): The α-free pyrrole was for-
mylated with labelled DMF (13C-formyl) and POCl3 to afford the
labelled aldehyde, which was condensed with the ring D as de-
scribed for [10-13C]-labelled PCB (2). Further formylation and sub-
sequent coupling with the A–B ring building block, followed by
hydrolysis, resulted in the [15-13C]-labelled PCB (24% yield for the
last two steps). HRMS (ESI – pos. + neg.): C3213CH39N4O6 [M +
H]: 588, 13C incorporation Ͼ 95%.
[10,15-13C2]-Labelled Phycocyanobilin (4): The α-free pyrrole was
formylated with labelled DMF (13C-formyl) and POCl3 to give the
labelled aldehyde, which was condensed with ring D and formyl-
ated with trimethyl orthoformate (formyl 13C) in trifluoroacetic
acid. Finally, this C–D ring building block was condensed with the
A–B ring segment to afford [10,15-13C2]-labelled phycocyanobilin
dimethyl ester in 35% yield. Hydrolysis gave [10,15-13C2]-labelled
phycocyanobilin in 80% yield. HRMS (ESI – pos. + neg.):
C3113C2H39N4O6 [M + H]: 589, 13C incorporation Ͼ 95%.
[8] T. Rohmer, H. Strauss, J. Hughes, H. de Groot, W. Gärtner,
P. Schmieder, J. Matysik, J. Phys. Chem. B 2006, 110, 20580–
20585.
[9] I. Lindner, B. Knipp, S. E. Braslavsky, W. Gärtner, K. Schaff-
ner, Angew. Chem. Int. Ed. Engl. 1998, 37, 1843–1846.
[10] J. E. Bishop, J. F. O’Connell, H. Rapoport, J. Org. Chem. 1991,
56, 5079–5091.
[11] A. R. Battersby, M. H. Block, C. J. R. Fookes, P. J. Harrison,
G. B. Henderson, F. J. Leeper, J. Chem. Soc., Perkin Trans. 1
1992, 2175–2187.
[12] F. Minisci, E. Vismara, F. Fontana, J. Org. Chem. 1989, 54,
5224–5227.
[13] E. Baciocchi, E. Muraglia, G. Sleiter, J. Org. Chem. 1992, 57,
6817–6820.
[14] Y. R. Jorapur, C. H. Lee, D. Y. Chi, Org. Lett. 2005, 7, 1231–
1234.
[15] M. Seki, K. Mori, Eur. J. Org. Chem. 2001, 503–506.
[16] C. C. Huang, J. Labelled Compounds Radiopharmaceuticals
1987, 24, 675–681.
[17] D. Sawamoto, H. Nakamura, P. Kinoshita, S. Fujinami, K.
Inomata, Chem. Lett. 2000, 1398–1399.
[18] A. Gossauer, D. Miehe, Justus Liebigs Ann. Chem. 1974, 352–
362.
Synthesis of [10-13C]-Labelled Phytochromobilin (5): Biliverdin IXa
diethyl ester (315 mg, 0.49 mmol) was treated with thiobarbituric
acid (95.4 mg, 0.66 mmol) in ethyl acetate (640 mL) as described
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Eur. J. Org. Chem. 2007, 1287–1293

Synthesis of Selectively 13C-Labelled Bilin Compounds
FULL PAPER
[19] A. K. Tipton, D. A. Lightner, Monatsh. Chemie 1999, 130,
425–440.
[20] D. Mozley, A. Remberg, W. Gärtner, Photochem. Photobiol.
1997, 66, 710–715.
[21] R. Vogel, F. Siebert, Biopolymers 2003, 72, 133–148.
[22] H. Foerstendorf, C. Benda, W. Gärtner, M. Storf, H. Scheer,
F. Siebert, Biochemistry 2001, 40, 14952–14959.
Received: August 2, 2006
Published Online: January 17, 2007
Eur. J. Org. Chem. 2007, 1287–1293
© 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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