Enantioselective syntheses of (13)C-labeled (2R)- and (2S)-phytochromobilin dimethyl ester.

Article abstract of DOI:10.1021/ol006977q

(2R)- and (2S)-phytochromobilin dimethyl ester have been prepared in enantiomerically pure form, specifically (13)C-labeled at C(10) or C(15).

Full text of DOI:10.1021/ol006977q

ORGANIC
LETTERS
2001
Vol. 3, No. 6
827-830
Enantioselective Syntheses of
13C-Labeled (2R)- and
(2S)-Phytochromobilin Dimethyl Ester
Peter A. Jacobi* and Douglas Pippin
Burke Chemical Laboratory, Dartmouth College, HanoVer, New Hampshire 03755
peter.a.jacobi@dartmouth.edu
Received December 8, 2000
ABSTRACT
(2R)- and (2S)-phytochromobilin dimethyl ester have been prepared in enantiomerically pure form, specifically 13C-labeled at C10 or C .
15
Phytochrome (Pr; 1) is a member of the biliprotein family
of chromophores, which are made up of linear tetrapyrrole
derivatives covalently bonded to the protein N-C (Figure
1).1 In green plants, Pr functions as the “on-off” switch for
photomorphogenesis, the process by which light transmits
growth regulatory information to a cell’s genetic apparatus.
Information of this type is crucial to the timing of seasonal
phenomena, such as seed germination, flowering and fruiting,
and chlorophyll production.1 In comparison to photosynthesis
(the other major light-induced process in nature), relatively
little is known about photomorphogenesis at the molecular
level. In part this is due to the difficulty of isolating 1 from
natural sources, where it is present in only trace amounts.
suggested that Pfr (2) might be derived from Pr by photo-
isomerization about the C15-C16 bond, with retention of a
“semiextended” conformation.2 According to this model, Z,E-
In the native state 1 can adopt at least two distinct forms:
an inactive red-absorbing species (Pr) and an active, far red
absorbing form designated as Pfr.1 These species are readily
interconverted upon irradiation at 665 and 730 nm, a
photoreversible/photochromic behavior that reflects the
stabilization of select geometries within the microenviron-
ment of the protein N-C. At present, only the structure of
the inactive Pr form of phytochrome is known with some
degree of certainty. Among other theories, it has been
(1) (a) Statter, R. L.; Galston, A. W. In Chemistry and Biochemistry of
Plant Pigments; Goodwin, T. W., Ed.; Academic Press: New York, 1976;
Vol. 1, p 680. (b) Phytochrome and Photoregulation in Plants; Furuya,
M., Ed.; Academic Press: New York, 1987. (c) Ru¨diger, W.; Thu¨mmler,
F. Angew. Chem., Int. Ed. Engl. 1991, 30, 1216.
Figure 1. Interconversion of Pr (1), Pfr (2), and PΦB (3).
10.1021/ol006977q CCC: $20.00 © 2001 American Chemical Society
Published on Web 02/21/2001

isomerization induces a change in the tertiary structure of
the surrounding protein N-C, providing a molecular basis
for transduction of the light signal to the cell’s genetic
regulatory apparatus. Similar theories have been postulated
involving isomerization about C4-C5 and C10-C11.
rac-8 and 9 (path a, Scheme 1).5 Several closely related
syntheses of rac-4 have appeared since. The versatility of
Scheme 1
Thermolysis of phytochrome (1) affords the tetrapyrrole
chromophore phytochromobilin (PΦB, 3), along with the
apoprotein N-C (Figure 1). Cleavage is believed to take
place via a concerted E2 elimination to selectively generate
the 3E-double bond in ring A. Remarkably, the reverse of
this process has also been demonstrated in vitro, employing
recombinant apophytochrome (N-C) and its native substrate
3.3 The in vitro adduct 1 exhibited a difference spectrum
identical to that of native oat phytochrome (Pr, 1). The ability
of phytochrome (1) to self-assemble in the dark may be
related to its role in etiolated plants as a first light sensor. In
principle, this process also provides a means for studying
the molecular events involved in the Pr T Pfr interconver-
sion.
Various spectroscopic techniques have been employed in
attempts to confirm the site of Z,E-isomerization. Perhaps
the most powerful tool is resonance Raman spectroscopy
(RS), which is readily adaptable to the study of prosthetic
groups in vivo.4 Using this technique it is possible to directly
observe the hydrogen out-of-plane (HOOP) wagging vibra-
tions of the methine hydrogens at C5, C10, and C15, for both
Pr and Pfr. Significant differences in these absorptions,
consistent with Z,E-isomerization, were noted as early as
1990 by Mathies and Lagarias. However, thus far it has
proven difficult to confirm specific assignments. One means
of accomplishing this would be by employing 13C-isotopic
substitution, which induces a characteristic shift in the HOOP
vibration frequency. In this paper we describe enantioselec-
tive syntheses of the 13C-labeled PΦB esters 5, 6, and ent-
5,6, as a first step toward achieving this goal (Figure 2).
this “AB + CD” approach to tetrapyrroles derives from its
highly convergent nature, although the precursor pyrro-
methenones can be difficult to prepare. A number of
alternative strategies have recently been reported,5b,c,h in
addition to improvements on the syntheses of both rac-8 and
9 and analogues.5d-g Despite these improvements, however,
we had several concerns about employing path a for the
synthesis of enantiomerically pure, 13C-labeled PΦB deriva-
tives. One of these pertained to the strongly acidic conditions
required for decarboxylative condensation (neat TFA), which
might cause epimerization at the labile C2-position in 8. Also,
path a offered little flexibility for the introduction of 13C at
the requisite meso-positions (vide infra). Finally, we hoped
(2) (a) Thu¨mmler, F.; Ru¨diger, W. Tetrahedron 1983, 39, 1943. (b)
Ru¨diger, W.; Thu¨mmler, F.; Cmiel, E.; Schneider, S. Proc. Natl. Acad.
Sci. U.S.A. 1983, 80, 6244. (c) Farrens, D. L.; Holt, R. E.; Rospendowski,
B. N.; Song, P.-S.; Cotton, T. M. J. Am. Chem. Soc. 1989, 111, 9162. (d)
Fodor, S. P. A.; Lagarias, J. C.; Mathies, R. A. Biochemistry 1990, 29,
11141. (e) Mizutani, Y.; Tokutomi, S.; Kitagawa, T. Biochemistry 1994,
33, 153. (f) Bischoff, M.; Hermann, G.; Rentsch, S.; Strehlow, D.; Winter,
S.; Chosrowjan, H. J. Phys. Chem. B 2000, 104, 1810. (g) Andel, F., III.;
Murphy, J. T.; Haas, J. A.; McDowell, M. T.; van der Hoef, I.; Lugtenburg,
J.; Lagarias. J. C.; Mathies, R. A. Biochemistry 2000, 39, 2667 and
references therein.
(3) (a) Lagarias, J. C.; Lagarias, D. M. Proc. Natl. Acad. Sci. U.S.A.
1989, 86, 5778. (b) Deforce, L.; Tomizawa, K.-I.; Ito, N.; Farrens, D.; Song,
P.-S.; Furuya, M. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10392. (c)
Wahleithner, J. A.; Li, L.; Lagarias, J. C. Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 10387. (d) Li, L.; Lagarias, J. C. J. Biol. Chem. 1992, 267, 19204.
(4) Biological Applications of Raman Spectroscopy; Spiro, T. G., Ed.;
Wiley-Interscience: New York, 1987. See also refs 2c-e.
Figure 2. 13C-Labeled Me esters of PΦB and ent-PΦB.
(5) (a) Weller, J.-P.; Gossauer, A. Chem. Ber. 1980, 113, 1603. (b) Jacobi,
P. A.; Guo, J.; Rajeswari, S.; Zheng, W. J. Org. Chem. 1997, 62, 2907. (c)
Jacobi, P. A.; Buddhu, S. C.; Fry, D.; Rajeswari, S. J. Org. Chem. 1997,
62, 2894. (d) Lindner, I.; Knipp, B.; Braslavsky, S. E.; Ga¨rtner, W.; Schaffer,
K. Angew. Chem., Int. Ed. 1998, 37, 1843. (e) Jayasundera, K. P.; Kinoshita,
H.; Inomata, K. Chem. Lett. 1998, 1227. (f) Takashi, K.; Kinoshita, H.;
Inomata, K. Synlett 1999, S1, 901. (g) Jayasundera, K. P.; Kinoshita, H.;
Inomata, K. Bull. Chem. Soc. Jpn. 2000, 73, 497. (h) Jacobi, P. A.; Coutts,
L. D.; Guo, J.; Hauck, S. I.; Leung, S. J. Org. Chem. 2000, 65, 205.
Incorporation studies with both the 2R- and 2S-enantiomers
of PΦB should also serve to confirm the assigned absolute
stereochemistry in Pr.
Racemic PΦB dimethyl ester (rac-4) was first prepared
in 1980 by Gossauer et al.,5a who obtained a ∼40% yield of
rac-4 by decarboxylative condensation of pyrromethenones
828
Org. Lett., Vol. 3, No. 6, 2001

to attain higher yields for the condensation of relatively
expensive 13C-labeled precursors. For this purpose path b
appeared to hold greater promise. The successful realization
of this strategy is outlined below.
Our synthesis of enantiomerically pure pyrromethenone
10 took advantage of the ready availability of chiral alkyne
acids 12 and ent-12 (Scheme 2).6 Recently we employed
elimination steps. We explored many conditions for effecting
this transformation without concomitant racemization or
isomerization (ethylidenes of type 10 are known to undergo
facile exo-endo double bond migration). In most cases we
observed either no reaction or under forcing conditions,
significant decomposition. However, we eventually found
that a two-phase system consisting of CHCl3 and 1 equiv of
12 N HCl accomplished the desired transformations at rtand
in >95% overall yield. Compound 10 thus prepared was
obtained as a single enantiomer, and with exclusively the
3E,5Z-double bond geometry. In identical fashion, but
beginning with alkyne acid ent-12, we also prepared pyrro-
methenone ent-10 as a single isomer.
Scheme 2
We have also developed an efficient synthesis of the C,D-
ring pyrromethenone 11 (Scheme 3). Here it was desirable
Scheme 3
these compounds in an asymmetric synthesis of the ring-
A,B portion of phytochrome (1),5c where it was necessary
to control the relative and absolute stereochemistry at C2,
C3, and C3′ In the case of 10, alkyne acids 12 and ent-12
proved to be equally efficient precursors for ring-A. The
joining of rings A and B was accomplished by a Pd0-initiated
coupling-cyclization reaction with iodopyrrole 13.7 This
reaction afforded an 86% yield of the enelactone 14 as a
single stereo- and geometric isomer.8b The structure of 14
was evident from its highly characteristic spectral data (cf.
Supporting Information) and confirmed by its eventual
conversion to both 10 and PΦB dimethyl ester (4). The first
step in this sequence involved aminolysis at -33 °C, a
procedure that we and others have employed for the
conversion of enelactones to cyclic enamides.8 Under these
conditions the lactone ring suffers rapid opening, followed
by keto-amide cyclization to give an essentially quantitative
yield of the amidoalcohols 15 as a mixture of diastereomers.
Normally this mixture was not separated, but rather was
directly carried on to the dehydration and benzyl ether
to avoid the strongly alkaline conditions typically employed
in aldol-like condensations with the formylpyrrole 17. A
convenient solution to this problem was realized in the form
of the silyloxypyrrole 18, which can be prepared on a
multigram scale from inexpensive starting materials.9 Pyrrole
18 is an excellent surrogate for ring D, since it exhibits strong
nucleophilic properties under mild Lewis acid catalysis.
Employing TiCl4, for example, condensation of 17 and 18
occurred rapidly at -78 °C, to afford a >95% yield of the
aldol products 19 (R ) OH, TBS). Initially these adducts
were isolated and characterized by analytical and spectral
data. However, once again it proved advantageous to carry
this mixture forward to the next step. This involved dissolu-
tion of 19 in TFA at rt, which initiated a remarkably clean
series of transformations. The first of these involved dehy-
dration to generate the meso-double bond, followed by
(6) Schreiber, S. L.; Klimas, M. T.; Sammakia, T. J. Am. Chem. Soc.
1987, 109, 5749. See also refs 5b,c,h.
(7) (a) Bishop, J. E.; O’Connell, J. F.; Rapoport, H. J. Org. Chem. 1991,
56, 5079. (b) Jacobi, P. A.; DeSimone, R. W.; Tetrahedron Lett. 1992, 33,
6239.
(8) (a) Micklefield, J.; Mackman, R. L.; Aucken, C. J.; Beckmann, M.;
Block, M. H.; Leeper, F. J.; Battersby, A. R. J. Chem. Soc., Chem. Commun
1993, 275 and references therein. (b) Jacobi, P. A.; Liu, H. J. Org. Chem.
1999, 64, 1778. See also ref 5h.
(9) Jacobi, P. A.; DeSimone, R. W.; Ghosh, I.; Guo, J.; Leung, S. H.;
Pippin, D. J. Org. Chem. 2000, 65, 8478.
Org. Lett., Vol. 3, No. 6, 2001
829

cleavage of both the Boc protecting group and the tert-butyl
ester. The resulting pyrromethenone carboxylic acid could
be isolated if desired but upon decarboxylation gave a
virtually quantitative yield of 20. Finally it was required to
carry out the oxidative elimination of selenide 20 to give
the desired C,D-ring pyrromethenone 11. This presented
some initial difficulties, since the liberated p-chlorophenyl-
selenous acid reacted rapidly with the unsubstituted pyrrole
ring in 11 to give variable yields of adduct 21. However,
these difficulties were resolved by carrying out the elimina-
tion step in a two-phase system consisting of CH2Cl2 and
pH 12 buffer. Under these conditions the selenous acid was
rapidly removed from the organic layer and the production
of 21 was limited to trace amounts.
derived form. In identical fashion, but beginning with ent-
10, we have also synthesized the optical antipode ent-4.
For the purpose of preparing 13C-labeled PΦB derivatives
5 and 6 (and ent-5,6) (cf. Figure 2), it was necessary to
synthesize the appropriate B- and C-ring pyrroles 13C-13 and
13C-17 (Scheme 5). With only slight modifications, this was
Scheme 5
With ample quantities of both 10 and 11 in hand, we were
confident that sufficiently mild conditions could be developed
to effect their coupling to give PΦB dimethyl ester (4)
(Scheme 4; see also path b, Scheme 1). However, this was
Scheme 4
accomplished by following the same procedures as for the
nonlabeled species 13 and 17, but employing 13C-DMF (*)
as the source of 13C (Supporting Information).10 The syn-
theses of 5, 6, and ent-5,6 were then carried out as previously
described for PΦB (4) (Schemes 2-4).
Currently we are synthesizing the C4,5-labeled phyto-
chrome derivatives 7 and ent-7 (cf. Figure 2), beginning with
the A-ring precursors 13C-12 (13C-ent-12) (Scheme 5). Also,
we are investigating the hydrolysis of 5-7 to the corre-
sponding free carboxylic acids, which will be reconstituted
with recombinant apoprotein N-C. Studies employing these
13C-labeled phytochrome derivatives should help clarify the
role of 1 in photomorphogenesis.
not the case, since pyrromethenone 11 decomposed rapidly
under even mildly acidic conditions. Eventually this problem
was circumvented by reversing the order of selenoxide
elimination and coupling. Thus, pyrromethenones 10 and 20
underwent clean condensation to afford the tetrapyrrole 22
(76%), under conditions which cause little or no decomposi-
tion (HCl/Et2O/MeOH). Finally, selenoxide formation oc-
curred cleanly at -78 °C, followed by elimination using the
two-phase system described above for pyrromethenone 11
(cf. Scheme 3). PΦB dimethyl ester (4) thus synthesized was
obtained as the 2R-enantiomer exclusively, which we believe
is the first time this material has been prepared in its naturally
Acknowledgment. Financial support of this work by the
National Institutes of Health, NIGMS Grant GM38913, is
gratefully acknowledged.
Supporting Information Available: Experimental pro-
cedures and spectral data for all new compounds reported.
This material is available free of charge via the Internet at
http://pubs.acs.org.
OL006977Q
(10) Cambridge Isotope Laboratories, Andover, MA 01810.
830
Org. Lett., Vol. 3, No. 6, 2001