Structural studies by exciton coupled circular dichroism over a large distance: Porphyrin derivatives of steroids, dimeric steroids, and brevetoxin B

Article abstract of DOI:10.1021/ja960126p

The present study (see ref 1) delineates the scope and limitations of porphyrin chromophores for structural studies by the exciton coupled circular dichroic (CD) method. A distance dependency of the porphyrin coupling was investigated in the range between

Full text of DOI:10.1021/ja960126p

5198
J. Am. Chem. Soc. 1996, 118, 5198-5206
Structural Studies by Exciton Coupled Circular Dichroism over
a Large Distance: Porphyrin Derivatives of Steroids, Dimeric
Steroids, and Brevetoxin B
Stefan Matile,† Nina Berova,† Koji Nakanishi,*,† Jo1rg Fleischhauer,‡ and
Robert W. Woody*,§
Contribution from the Department of Chemistry, Columbia UniVersity,
New York, New York 10027, Institut fu¨r Organische Chemie, RWTH Aachen,
52074 Aachen, Germany, and Department of Biochemistry and Molecular Biology,
Colorado State UniVersity, Fort Collins, Colorado 80523-1870
ReceiVed January 16, 1996X
Abstract: The present study (see ref 1) delineates the scope and limitations of porphyrin chromophores for structural
studies by the exciton coupled circular dichroic (CD) method. A distance dependency of the porphyrin coupling
was investigated in the range between 10 and 50 Å. Over short interchromophoric distances, significant changes in
the conformational distribution introduced by the bulky porphyrin chromophores were observed. Over longer distances,
the porphyrins showed ca. 10-fold sensitivity increase over commonly used chromophores, and an effective direction
for the interacting porphyrin transition moments was assigned by comparison. Porphyrins at the termini of dimeric
steroids and brevetoxin B exhibited exciton coupling over interchromophoric distances up to 50 Å. These results
represent the porphyrins as promising reporter chromophores for extending the exciton coupled CD method to structural
studies of biopolymers.
Introduction
mophores with intense absorbance (ꢀ 31 000-58 000) at longer
wavelengths (360-410 nm). Their applications to natural
products, such as taxinine (λmax 262 nm) and chromomycin A3
(λmax 270 nm),4c provided exciton split bands free of overlapping
effects, thus leading to unambiguous analysis of the couplets.
The amplitudes of split Cotton effects are inversely proportional
to the square of interchromophoric distance5 and proportional
to the square of extinction coefficients6 of the coupled chro-
mophores. Therefore, exciton coupling over a long distance
can be achieved only with strongly absorbing chromophores.
So far, the long distance effects have been examined with rigid
homosteroid7 and norsteroid8 derivatives where the interacting
transition moments of para-substituted benzoates attached to
rings A and D with a C-C distance of 18 Å showed CD
amplitudes or A values (distance between extrema of couplets
in ∆ꢀ) up to ca. 20. While a distance of 18 Å practically covers
all common natural products, the moderate A value of ca. 20
observed for benzoate chromophores is insufficient when dealing
with samples available only in minuscule amounts, or samples
in which the distance between stereogenic centers is much
longer. Consequently, chromophores with more intense absorp-
tions therefore with increased sensitivities are needed.
The exciton coupled circular dichroic (CD) method is a
versatile and sensitive method for determining the absolute
stereochemistry of organic molecules in solution.2 In this
procedure, two or more chromophores are attached to functional
groups of the molecule of interest. The interaction between
the excited states of these chromophores in chiral environments
gives rise to bisignate CD curves, i.e., CD with split Cotton
effects. The signs and shapes of the characteristic CDs are
determined by the absolute skewness of interacting chro-
mophores and, for relatively weak interactions, the principle of
pairwise additivity.3 When the original substrate already
contains a chromophore, the newly introduced chromophore may
be selected so that its absorption maximum will be close to
that of the preexisting chromophore. However, in most cases
overlap between the substrate and introduced chromophores
complicate analyses of the CD curves. In order to avoid such
interactions, the concept of “red-shifted” chromophores was
developed.4 Recently we have examined new acylating chro-
† Columbia University.
‡ Institut for Organische Chemie.
§ Colorado State University.
Although the exciton coupled CD method has proved to be
a sensitive tool in elucidating the absolute configuration and
detection of even small conformational changes,2 its applicability
in structural studies of biopolymers such as proteins or nucleic
Abbreviations: BTX: brevetoxin; DBU: 1,8-diazobicyclo[5.4.0]undec-
7-ene; DDQ: 2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DMAP: N,N ′-
dimethyl-4-aminopyridine; dmab: dimethylaminobenzoate; DPMS: diphe-
nylmethylsilyl; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide;
TBA: tetrabutylammonium.
X Abstract published in AdVance ACS Abstracts, May 15, 1996.
(1) Matile, S.; Berova, N.; Nakanishi, K.; Novkova, S.; Philipova, I.;
Blagoev, B. J. Am. Chem. Soc. 1995, 117, 7021.
(4) (a) Verdine, G. L.; Nakanishi, K. J. Chem. Soc., Chem. Commun.
1985, 1093. (b) Berova, N.; Gargiulo, D.; Derguini, F.; Nakanishi, K.;
Harada, N. J. Am. Chem. Soc. 1993, 115, 4769. (c) Cai, G.; Bozhkova, N.;
Odingo, J.; Berova, N.; Nakanishi, K. J. Am. Chem. Soc. 1993, 115, 7192.
(d) Gargiulo, D.; Ikemoto, N.; Odingo, J.; Bozhkova, N.; Iwashita, T.;
Berova, N.; Nakanishi, K. J. Am. Chem. Soc. 1994, 116, 3760.
(5) Harada, N.; Chen, S.-M. L.; Nakanishi, K. J. Am. Chem. Soc. 1975,
97, 5345.
(6) Heyn, M. P. J. Phys. Chem. 1975, 79, 2424.
(7) Chen, S. L.; Harada, N.; Nakanishi, K. J. Am. Chem. Soc. 1974, 96,
7352.
(8) Canceill, J.; Collet, A.; Jacques, J. J. Chem. Soc., Perkin II 1982,
83.
(2) (a) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy-Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill
Valley, CA, 1983. (b) Nakanishi, K.; Berova, N. In Circular Dichroism-
Principles and Applications; Nakanishi, K., Berova, N., Woody, R. W.,
Eds.; VCH Publishers, Inc.: New York, NY, 1994; p 361.
(3) (a) Liu, H. W.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 5591.
(b) Wiesler, W. T.; Vazquez, J. T.; Nakanishi K. J. Am. Chem. Soc. 1987,
109, 5586. (c) Wiesler, W. T.; Berova, N.; Ojika, M.; Meyers, H. V.; Chang,
M.; Zhou, P.; Lo, L.-C.; Niwa, M.; Takeda, R.; Nakanishi, K. HelV. Chim.
Acta 1990, 73, 509.
S0002-7863(96)00126-6 CCC: $12.00 © 1996 American Chemical Society

Porphyrin DeriVatiVes of Steroids
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5199
acids with covalently attached reporter chromophores has not
been explored. One obstacle was the strong interfering absorp-
tion of the biopolymers themselves below ca. 300 nm, which
therefore required suitable CD chromophores with an absorption
above 400 nm. More importantly, the distances encountered
in biopolymers need a CD chromophore which can couple over
a long distance, namely, a chromophore with a very high
extinction coefficient. The cyanine dye chromophores exhibit
intense, red-shifted absorptions around 520 nm with narrow half-
band widths of 1520 cm-1; however, chemical instability makes
them unsuited for practical applications.4b
metric disubstitution on the magnitude and direction of the Soret
transition moments have been uncertain.17 The effects of
substitution by one or two vinyl or formyl groups have been
clarified by recent theoretical studies.18
The Soret band consists of two perpendicularly polarized
transitions, Bx and By (Figure 1A). In symmetrically substituted
metalloporphyrins, which have fourfold symmetry, these transi-
tions are degenerate and have identical intensities. In the free-
base form of the porphyrin, with their lower symmetry, these
transitions are not exactly degenerate and they will in general
differ in intensity. However, most experimental and theoretical
evidence argues against any sizable splitting or intensity
difference in the components of the Soret band. Rimington et
al.19a found a splitting of 250 cm-1 in the Soret band of free-
base porphin at low temperatures. Polarized reflection spectra19b
of porphin single crystals show different polarization on the
long-wavelength and short-wavelength sides of the Soret
maximum, but these data have not been analyzed quantitatively,
so the extent of the splitting and the imbalance in oscillator
strengths is not known. The spectrum of the free-base form of
TPP is very similar to that of the Zn complex in the Soret
transition, except for a shift in λmax, some broadening and a
concomitant decrease in ꢀmax in the free-base form. In particular,
there is no detectable splitting of the strong major peak in the
free-base form, and both forms show the shoulder about 1200
cm-1 above the main peak. It is well-known that free-base
porphyrins show strong splitting of the visible (Q) bands, but
as pointed out by Gouterman20 this does not necessarily imply
a substantial deviation from fourfold symmetry. Explicit MO
calculations on free-base porphin give splittings of 25-250 cm-1
for the Bx and By bands, depending on the parameters. These
calculations also predicted similar intensities of the Bx and By
bands, with the ratio of transition moments predicted to range
from 1.03 to 1.16. Recently, further semiempirical MO
calculations on porphin in its free-base and dianion form have
been reported.18 These calculations predict a rather different
picture for free-base porphin, in which the two Soret components
are split by 2100 cm-1 (30 nm) and the ratio of the transition
moments is ca. 1.5. Although these calculations are more
sophisticated and give predictions for the splitting between the
Soret and the visible bands, the splitting of the x and y
components of the Q band, and the intensities of the Q bands
that agree better with experiment than the earlier calcula-
tions,20,21 the results for the Soret band appear to be less
satisfactory. First, if the splitting of the Soret components were
as large as predicted, one should be able to see two Soret peaks,
contrary to observation. Second, the calculations also predict
bands that are about twice as intense as the Soret band in the
310-320 nm region, where the absorption is in fact quite weak.
The porphyrin chromophore, i.e., 5-substituted 5,10,15,20-
tetraphenylporphyrins, e.g., 1, was therefore investigated.1 With
their very intense, sharp Soret band at 414 nm, ꢀ 350 000,9 they
should provide powerful CD chromophores for configurational
and conformational analysis of natural products with remote
stereogenic centers and for conformational studies of biopoly-
mers.
Analyses of CD of porphyrin monomers induced by proteins10
or nucleic acids11 is a well established procedure to investigate
complex formation. The importance of this analytical technique
has led to model CD studies of oligopeptides covalently linked
to a porphyrin monomer.12 Investigations of heme/heme
interactions in multiheme proteins by CD are complicated by
effects induced by amino acids surrounding each porphyrin
chromophore. However, comparison of CD spectra of mono-
and multiheme proteins suggested heme/heme interactions;
calculations based on coordinates of isolated heme moieties
taken from protein X-ray structures confirmed the possibility
of long distance heme/heme interaction.13 The unique physical
properties of the photosynthetic reaction center gave rise to the
synthesis of several model compounds, i.e., aryl- and alkyl-
bridged porphyrin dimers.14 Shifts and/or splitting of the Soret
band in the UV-vis spectra of such achiral bridged porphyrin
dimers have been attributed to exciton coupling strongly
dependent on angle and distance. Chlorophyll dimers15 and a
dipeptide-bridged porphyrin dimer16 exhibit exciton coupled
Cotton effects in the CD. However, the nature of the observed
CD remained obscure, because the effects of mono-and asym-
(9) (a) Falk, J. E. Porphyrins and Metalloporphyrins; Elsevier: Am-
sterdam, 1964. (b) Gouterman, M. In The Porphyrins Vol. 3, Physical
Chemistry, Part A; Dolphin, D., Ed.; Academic: New York, NY, 1978.
(10) (a) Beychok, S.; Blout, E. R. J. Mol. Biol. 1961, 3, 769. (b) T.
Samejima, T.; Yang, J. T. J. Mol. Biol. 1964, 8, 863. (c) Urry, D. W. J.
Am. Chem. Soc. 1967, 89, 4190. (d) Myer, Y. P. Biochim. Biophys. Acta
1970, 214, 94. (e) Sugita, Y.; Nagai, M.; Yoneyama, Y. J. Biol. Chem.
1971, 246, 383. (f) Hsu, M.-C.; Woody, R. W. J. Am. Chem. Soc. 1971,
93, 3515. (g) Keinan, E.; Benory, E.; Sinha-Bagchi, A.; Eren, D.; Eshar,
Z.; Green, B. S. Inorg. Chem. 1992, 31, 5433. (h) Nezu, T.; Ikeda, S. Bull.
Chem. Soc. Jpn. 1993, 66, 18. (i) Woody, R. W. in ref 2b; p 473.
(11) (a) Carvlin, M. J.; Fiel, R. Nucl. Acid Res. 1983, 11, 6121. (b)
Carvlin, M. J.; Mark, E.; Fiel, R.; Howard, J. C. Nucl. Acid Res. 1983, 11,
6141. (c) Pasternack, R. F.; Garrity, P.; Ehrlich, B.; Davis, C. B.; Gibbs, E.
J.; Orloff, G.; Giartosio, A.; Turano, C. Nucl. Acid Res. 1986, 14, 5919.
(d) Gibbs, E. J.; Maurer, M. C.; Zhang, J. H.; Reiff, W. M.; Hill, D. R. F.
J. Inorg. Biochem. 1988, 32, 39. (e) Foster, N.; Singhal, A. K.; Smith, M.
W.; Marcos, N. G.; Schray, K. J. Biochim. Biophys. Acta 1988, 950, 118.
(f) Marzili, L. G.; Petho¨, G.; Lin, M.; Kim, M. S.; Dixon, D. W. J. Am.
Chem. Soc. 1992, 114, 7575. (g) Pasternack, R. F.; Bustamante, C.; Collings,
P. J.; Giannetto, A.; Gibbs, E. J. J. Am. Chem. Soc. 1993, 115, 5393. (h)
Petho¨, G.; Elliot, N. B.; Kim, M. S.; Lin, M.; Dixon, D. W.; Marzili, L. G.
J. Chem. Soc., Chem. Commun. 1993, 1547.
(12) (a) Nishino, N.; Mihara, H.; Hasegawa, R.; Yanai, T.; Fujimoto, T.
J. Chem. Soc., Chem. Commun. 1992, 692. (b) Mihara, H.; Nishino, N.;
Hasegawa, R.; Fujimoto, T. Chem. Lett. 1992, 1805.
(13) Woody, R. W. in Optical Properties and Structure of Tetrapyrroles;
Blauer, G., Sund, H., Eds.; Walter de Gruyter & Co.: Berlin, 1985; p 239.
(14) (a) Osuka, A.; Maruyama, K. J. Am. Chem. Soc. 1988, 110, 4454.
(b) Wasielewski, M. R. Chem. ReV. 1992, 92, 435.
(15) Bucks, R. R.; Boxer, S. G. J. Am. Chem. Soc. 1982, 104, 340.
(16) Tamiaki, H.; Suzuki, S.; Maruyama, K. Bull. Chem. Soc. Jpn. 1993,
66, 2633.
(17) Hsu and Woody10f inferred that the Soret components in myoglobin
are polarized along the methine carbon axes (Bx′, By′ in Figure 1A) because
this orientation gave the best agreement between theory and experiment
for the shape of the Soret CD band. Similar considerations led Fuhrhop et
al. (Fuhrhop, H.-J.; Demoulin, D.; Boettcher, C.; Ko¨nig, J.; Siggel, U. J.
Am. Chem. Soc. 1992, 114, 4159) to the same assignment of transition
moment directions in theoretical analyses of the CD spectra of self-
assembled protoporphyrin IX 13,17-bis-(glucosamides). Osuka and
Maruyama14a assumed a 5-15-direction of the Soret transition in a C(5)-
substituted porphyrin and estimated its magnitude (footnote 10 in ref 14a).
However, as noted in the text of this paper, the interpretation of the fine
structure of the Soret band made in ref 14a is not correct.
(18) (a) Masthay, M. B.; Findsen, L. A.; Pierce, B. M.; Bocian, D. F.;
Lindsey, J. S.; Birge, R. R. J. Chem. Phys. 1986, 84, 3901. (b) Bocian, D.
F.; Masthay, M. B.; Birge, R. R. Chem. Phys. Lett. 1986, 125, 467.
(19) (a) Rimington, C.; Mason, S. F.; Kennard, O. Spectrochim. Acta
1958, 12, 65. (b) Anex, B. G.; Umans, R. S. J. Am. Chem. Soc. 1964, 86,
5026.
(20) Gouterman, M. J. Chem. Phys. 1959, 30, 1139.
(21) Weiss, C.; Kobayashi, H.; Gouterman, M. J. Mol. Spectrosc. 1965,
16, 415.

5200 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Matile et al.
Scheme 1
to the Bx transition and of the weak shoulder near 400 nm to
the By transition are in error. The fine structure of the Soret
band must instead be attributed to vibronic fine structure in the
combined Bx, By bands: a 0 f 0 band near 420 nm and a 0 f
1 band near 400 nm. Thus, we cannot expect the porphyrin
chromophore to behave like a linear oscillator as previously
assumed. Instead, it must be treated as a nearly circular
oscillator, with two nearly degenerate, orthogonal transition
moments of approximately the same magnitude (Figure 1A).
This implies that the simple exciton chirality method cannot be
applied to these bisporphyrin systems. However, in all cases
we have examined in which the bisporphyrin derivative can be
compared with the bis(p-dimethylaminobenzoyl) derivative, the
signs of the exciton effects are identical.1 This suggests that
an effective transition moment direction can be derived for these
circular oscillators that gives the same sign as the normal exciton
chirality rule for linear oscillators (Figure 1B). In addition,
theoretical exciton calculations that include the doubly degener-
ate Bx and By transitions in each porphyrin have been performed
for a number of conformers of 6b-9b with dihedral angles at
each stereogenic center varied about the minimum energy
conformation deduced for benzoate esters.2a,5 The four rota-
tional strengths that result occur in pairs, corresponding to a
strong inner couplet and a weak outer couplet, the latter
generally opposite in sign to the former. In all cases, when
Gaussian bands with the half bandwidth observed in the
absorption spectrum are used to calculate the CD spectrum, a
single couplet is predicted for the Soret region. Although
individual conformers of a given isomer can vary in the sign of
the predicted couplet, the sign of the unweighted average for
each of the molecules 6b-9b agrees with experiment, and the
magnitude is in reasonable agreement. Further theoretical
studies are in progress that will provide predicted CD spectra
properly weighted by conformational energies and will further
test the relationship between the correct Bx, By representation
and the simplified model of one effective Soret transition
moment per porphyrin.
Figure 1. A: Two choices of transition moment directions for the
porphyrin Soret transition. The directions labeled Bx and By are directed
along the N-H/N-H and the N/N axes, respectively. However, because
of the near degeneracy and approximate equivalence of these transitions,
any linear combination of Bx and By can also be used. One such
combination, designated Bx′ and By′, is shown as dashed lines and is
directed between opposite methine carbons. B: Indication of the
direction of the effective transition moment in 6b. C: CD data of 6b
and UV-vis data of 6b and 1 (dotted) in CH2Cl2, c ) 1.0 µM. In all
measured CD spectra no bands were detectable in the visible region
above 500 nm, and the exciton CD spectra are only slightly altered
when the free-base porphyrins are converted to the Zn complexes or
are protonated, thus making them four-fold symmetric.
Results and Discussion
Porphyrin Synthesis and Derivatization. Condensation of
p-(carboxymethyl)benzaldehyde (2), benzaldehyde (3), and
pyrrole (4) in propionic acid in the presence of zinc acetate,22
followed by removal of zinc, gave ester 5, which was hydrolyzed
to porphyrin 1 in 6.5% overall yield (Scheme 1). Derivatization
of all glycols (6a-11a) was performed under mild conditionss
room temperature, overnightsin CH2Cl2/EDC/DMAP,23 and
afforded bisporphyrin derivatives 6b-11b in 60-80% yield
(22) (a) Anton, J. A.; Loach, P. A. J. Heterocyclic Chem. 1975, 12, 573.
(b) Sta¨ubli, B.; Fretz, H.; Piantini, U.; Woggon, W.-D. HelV. Chim. Acta
1987, 70, 1173.
(23) Dhaon, M. K.; Olsen, R. K.; Ramasamy, K. J. Org. Chem. 1982,
47, 1962.
In light of this evidence, previous assignments of the main
Soret absorption feature in tetraphenylporphyrins, near 420 nm,

Porphyrin DeriVatiVes of Steroids
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5201
Table 1. CD Data of Bisporphyrins and Bis(p-dimethylaminobenzoates (dmab)) of Various Glycols, Measured at Concentrations between 0.8
and 1.2 µM
a Distance between the stereogenic centers, measured in a energy minimized structure (MacroModel 4.5).37 b Distance between the chromophoric
centers (see Figure 1B). c All distances are given in Å. d Due to the mixture of conformers, the average value is given. e Data already published in
the preliminary communication.1 f Data from ref 5. g Due to the semiflexibility of the brevetoxin bridge, the given distances will vary upon
conformational changes.
(Table 1). The harsher conditions required to convert 1R,2R-
cyclohexanediol (12a) as well as the lower yield (54%) of
bisester 12b can be explained by the conformational change of
the cyclohexane ring which must precede the second esterifi-
cation in order to minimize steric interaction of the porphyrin
chromophores. Although the mono(13c)- and trisesters have
been isolated, selective esterification of the allylic alcohols of
the brevetoxin B derivative 13a was already completed after 2
h giving bisporphyrin 13b as the main product in 32% yield.
Derivatization of steroids 7a, 9a, and 11a with dimethylami-
nobenzoyl triazole/DBU4d gave bisester 7c, 9c, and 11c. CD
data are listed in Table 1.
13-24 Å Interchromophoric Distance. Conformational
flexibility does not need to be considered when dealing with
rigid steroid skeletons, which enable unambiguous investigations
of distance and angle dependency of exciton coupled CD to be
performed.1,2,7 Of the known steroidal bis-chromophoric sys-
tems, 3,6-bis(dimethylaminobenzoate) 6c exhibited the strongest
coupling (A ) +89) (Table 1).7 Compared to 6c, the CD of
3,6-bisporphyrin ester 6b exhibited a bisignate CD with a >7-
fold stronger amplitude of +675 (Figure 1C); however, this
comparison is not a true measure of the increase in sensitivity,
since despite the constant C-C distance of 3.9 Å between the
chiral centers, the interchromophoric distance is 8 Å longer in
bisporphyrin 6b (Rij ) 13.0 Å) compared to bisbenzoate 6c (Rij
) 5.0 Å).
Because of the known syn orientation of the ester carbonyl
with respect to the methine hydrogen, the positive sign of the
Cotton effect in 6c reflects the chiral twist between the transition
moments of dimethylaminobenzoate which lies in the direction
of the phenyl 1,4-axis or the C(3)-O/C(6)-O bonds (Table
1). That the signs of the Cotton effects of bis(dimethylami-
nobenzoate) 6c and bisporphyrin ester 6b are the same indicate
that the direction of the effective coupling transition moments
of the porphyrins run in the same direction, i.e., 5,15-axis of
the porphyrin ring, as shown in 6b, Figure 1B; same is true for
7b/7c and 9b/9c. Therefore, the chiral sense of twist between
the porphyrin chromophores attached to the stereogenic centers
can be correctly simulated by the thick arrows depicted in
structure 6b, which in turn represents the chirality between the
two C-O bonds in question as in the case of other symmetric
chromophores, e.g., para-substituted benzoates.24,25
Comparisons of the bisporphyrin series 7b, 8b, and 9b,
(24) The possibility exists that due to hydrophobic interactions, the
porphyrin chromophores fold over the steroid skeleton so that straightfor-
ward analysis of the sign of the couplet cannot be correlated with the
absolute stereochemistry. However, such porphyrin-steroid interactions
would be reflected in shifts of the steroid protons in the 1H NMR spectrum,
which is not the case.
(25) Calculation of rotational strengths R1 and R2 from the experimental
CD spectrum of 6b in Figure 1 lead to an almost conservative coupling.
However, in all measured CD spectra of porphyrin dimers the first Cotton
effect has a higher ∆ꢀ value than the second which also bears a shoulder
around 395 nm.

5202 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Matile et al.
Scheme 2
interchromophoric distances 22-24 Å, with the corresponding
bis(dimethylaminobenzoates) 7c, 9c, and bisbenzoate 9d, show
the dramatic increase in sensitivity in the porphyrin series.1
29-34 Å Interchromophoric Distance. The dimeric ste-
roids26 10a and 11a were synthesized from stanolone 14 in order
to explore the extent of long distance exciton coupling with
the porphyrin chromophores (Scheme 2). Silylation (15)
followed by McMurry-coupling27 afforded dimers 16 and 17,
which were deprotected to 1,18-glycols 10a and 11a. The
Z-isomer 11a, mp >300 °C, was separated from the E-isomer
10a by crystallization from CH2Cl2. Both isomers were
derivatized to the corresponding porphyrin dimers 10b and 11b;
11a was also converted to the bis(p-dimethylaminobenzoate)
11c.
CD spectra of 11b (A ) +32) and 10b (A ) +11) are shown
in Figure 2. The spectrum of bis(p-dimethylaminobenzoate)
11c, the chromophore of choice in many applications of exciton
coupled CD,2 shows no coupling due to the smaller extinction
(ꢀ 28 200 for the isolated chromophore). Energy calculation
of Z-isomer 11b with two parallel steroid backbones leads to a
favorable projection angle between the 17-O/17′-O-bonds and
an interchromophoric distance of ca. 34 Å; a positive couplet
with A ) +32 is the outcome. The bent geometry of the steroid
moieties in 10b, in contrast, results in the two chromophores
being close to colinear, the outcome of which is a much smaller
CD amplitude of +11. Although similarities in spectroscopic
data of 10a/11a and other similar dimeric steroids, i.e., synthetic
cephalostatin analogs,26c preclude simple structural differentia-
tion, the long distance coupling between the porphyrins can
readily distinguish dimers 10b/11b.
Compared to the CD spectra of steroids, the CD couplet for
the Z-isomer 11b is clearly nonconservative (first ∆ꢀ > second
∆ꢀ), while the E-isomer 10b exhibits only the positive band of
the corresponding couplet; in both cases, the first CD Cotton
effect is red-shifted with respect to the UV maximum (418 nm)
(Figure 2). We found that independent of the spatial arrange-
ment of the chromophores, such strong nonconservativity was
not observed in the CD spectra of various 3,17-bisporphyrin
steroid derivatives 7b, 8b, and 9b (Table 1).
Figure 2. Above: Steroid backbone of Z-isomer 11b (A) including
side-view (B) and E-isomer 10b (C), calcd by MacroModel 4.5.37
Ellipsoids denote porphyrins. Distances between the chiral centers and
the centers of chromophores are indicated. Below: CD- and UV/vis
data of 11b (solid, c ) 0.9 µM) and 10b (dashed, c ) 0.9 µM).
(see below) are long-distance exciton couplings or not. Jacques
and co-workers explained the nonconservative CD couplets in
2R(or â),17â-A-nor-5R-androstanes dimethoxybenzoates by
nonexciton coupled Cotton effects overlapping on the weak
exciton couplet.8 Since the CD spectra of steroid monopor-
phyrin derivatives exhibited only negligible Cotton effects under
the dilute concentrations we employed for measuring the bis-
porphyrin derivatives, it is clear that the chiral perturbation of
the porphyrin chromophore by the steroidal nucleus is negligible.
The reason for the reduced intensity of the second Cotton
effect and especially in the bisporphyrin derivatives with large
interchromophoric distances is still not understood. If the Soret
components were only interacting among themselves, the two
components of the exciton couplet should have rotational
strengths of equal magnitude. The observed imbalance indicates
that the Soret transitions are coupling with other transitions to
a significant extent. All measured CD spectra, e.g., those of
6b (Figure 1C), do not show detectable intensity in the visible
region or in the ultraviolet down to 250 nm. This excludes
coupling of the Soret band with the Q, N, and L bands of the
porphyrins.20,21 The absence of significant Soret rotational
strength in the monoporphyrin derivatives excludes the pos-
sibility of Soret coupling with high-energy transitions in the
steroid skeleton. Coupling of the Soret transitions in one
porphyrin with the high-energy transitions in the other porphyrin
has not been excluded experimentally and is being investigated
The question thus arises whether the Cotton effects observed
in 10b and 11b, and in the brevetoxin bridged porphyrin dimers
(26) Dimeric steroids have attracted attention because of the high
antitumor activity of cephalostatins isolated from marine animals
Cephalodiscus: (a) Pettit, G. R.; Inoue, M.; Kamano, Y.; Herald, D. L.;
Arm, C.; Dufresne, C.; Christie, D. N.; Schmidt, J. M.; Doubek, D. L.;
Krupa, T. S. J. Am. Chem. Soc. 1988, 110, 2006. (b) Jeong, J. U.; Fuchs,
P. L. J. Am. Chem. Soc. 1994, 116, 773. (c) Heathcock, C. H.; Smith, S. C.
J. Org. Chem. 1994, 59, 6828.
(27) McMurry, J. E. Chem. ReV. 1989, 89, 1513.

Porphyrin DeriVatiVes of Steroids
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5203
Chart 1
In nonpolar solvents such as CH2Cl2 or toluene, the CD of
bisporphyrin ester 13b is devoid of Cotton effects. However,
a bisignate CD is observed in the more polar and protic solvent
MeOH/water 4/1, A ) -7.0 (Figure 3). The fact that the
porphyrin monoester 13c exhibited no CD under the same
conditions shows that the chiral centers are too remote from
the chromophores; more importantly, it also demonstrates that
the bisignate CD observed in bis ester 13b is not due to stacking.
Thus the couplet observed for the porphyrin dimer 13b must
be caused by exciton coupling over a distance of up to 50 Å
between the porphyrin chromophores. The appearance of
exciton coupling in MeOH/water 4/1 shows that this solvent
system leads to conformational changes which favor coupling
between the porphyrin electric transition moments. The am-
plitude is increased to -10.5 by saturating the solution with
CsCl (Figure 3); this increase is possibly caused by complexation
of the large, polarizable Cs+ to multiple oxygens in the toxin
backbone, resulting in a shift from a cigar to a bent shaped
conformer of the skeleton.36
theoretically. For practical and theoretical reasons, it would
be interesting to see whether the trends observed in these
nonconservative coupling arising from the weak porphyrin/
porphyrin interaction over a long distance is a general feature
or not.28
Short Interchromophoric Distance. Due to the short
interchromophoric distance and favorable dihedral angle, a most
intense couplet would have been expected for the 1R,2R-
cyclohexanediol bridged porphyrin dimer 12b (Figure 4).
Surprisingly, however, the amplitude A ) -400 is significantly
smaller than that of 1,4-bisester 6b (A ) +675) (Table 1). Split
Up to 50 Å Interchromophoric Distance. The interchro-
mophoric distance is ca. 50 Å in the brevetoxin B (BTX-B)-
bridged porphyrin dimer 13b, which was prepared by selective
reduction29 of BTX-B 18 (red-tide toxin) followed by selective
esterification (Chart 1). The absolute configuration of BTX-B
was determined by exciton coupled CD of the bis-p-bromoben-
zoate of the vicinal 27,28-diol (obtained by oxidation of 27-
ene),30 by X-ray31 and more recently by total synthesis.32
Molecular modeling of 18 showed that the cigar-shaped skeleton
can be bent at two flexible moieties in the backbone, the eight-
membered ring H and the two seven-membered rings D and
E.33 These flexible moieties in the middle of the rigid trans-
fused backbones are a common feature of brevetoxins and
ciguatoxins and are suspected to play key roles in the bioac-
tivities of these toxins;34,35 however, as yet no conformational
studies in solution have been performed.
1
porphyrin signals in the H NMR spectrum of 12b, measured
at 25 °C in DMSO-d6, suggested the presence of a mixture of
cyclohexane conformers in which the porphyrin protons are
differently exposed to the ring current effects of the two
porphyrin cores (Figure 4a). This assumption was confirmed
by the enhanced signal resolution in the spectrum measured at
100 °C due to fast equilibrium between cyclohexane conformers
(Figure 4b). According to calculations by MacroModel,37 the
most stable conformer is 12c with axial bulky substituents; this
is followed by boat conformers 12d, 12e, then 12f, and finally
the normal chair form 12g (+34 kJ/mol) with two eq s-trans
esters. The twist of the effective transition moments (Figure
1B) give rise to a negative couplet in 12d, 12e, 12g, a positive
couplet in 12f, and no Cotton effect in 12c. Conformational
changes of the cyclohexane ring were also reflected in the
difficulties encountered in derivatising 1,2-glycol 12a. Thus,
for compounds with vicinal chiral centers, porphyrins should
be used with caution because of possible conformational
anomalies.38
(28) In weakly coupled chromophores, i.e., those with interchromophoric
dipole-dipole coupling energy weaker than the half band width of the
monomer absorption, one of us (R.W.) has demonstrated that calculations
using polarizability theory instead of exciton theory may provide additional
insight in the shape of bisignate curves.13 These calculations further predicted
the possibility of porphyrin/porphyrin interactions covering a distance of
40 Å or more. Since the analysis of CD spectra of multiheme proteins is
not straightforward due to the dominating effects from the protein
environment, the long distance porphyrin/porphyrin interactions observed
with the dimeric steroid and brevetoxin bridges favor the possibility of heme/
heme long distance interactions in multiheme proteins as well. A theoretical
analysis of the results presented here will be published elsewhere.
(29) Qin, G.-W.; Nakanishi, K. Unpublished results.
(30) (a) Lin, Y.-Y.; Risk, M.; Ray, S. M.; Van Engen, D; Clardy, J.;
Golik, J.; James, J. C.; Nakanishi, K. J. Am. Chem. Soc. 1981, 103, 6773.
(b) Lee, M. S.; Repeta, D. J.; Nakanishi, K.; Zagorski, M. G. J. Am. Chem.
Soc. 1986, 108, 7855.
(31) Shimizu, Y.; Bando, H.; Chou, H.-N.; Van Dyne, G.; Clardy, J. C.
J. Chem. Soc., Chem. Commun. 1986, 1656.
Conclusions
General distance screening of the exciton coupled CD by
porphyrin reporter groups exemplified by 1 show substantial
coupling even at a distance of ca. 50 Å. In all cases exciton
(36) Addition of TBA chloride to the MeOH/water 4:1 solution of 13b
decreases the CD amplitude. The effect of Cs+ is therefore likely to be
caused by complexation of the metallic ion rather than increase in medium
polarity.
(32) (a) Nicolaou, K. C.; Theodorakis, E. A.; Rutjes, F. P. J. T.; Tiebes,
J.; Sato, M.; Untersteller, E.; Xiao, X.-Y. J. Am. Chem. Soc. 1995, 117,
1171. (b) Nicolaou, K. C.; Rutjes, F. P. J. T.: Theodorakis, E. A.; Tiebes,
J.; Sato, M.; Untersteller, E. J. Am. Chem. Soc. 1995, 117, 1173.
(33) (a) Rein, K. S.; Baden, D. G.; Gawley, R. E. J. Org. Chem. 1994,
59, 2101. (b) Rein, K. S.; Lynn, B.; Gawley, R. E.; Baden, D. G. J. Org.
Chem. 1994, 59, 2107.
(37) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440.
(38) During the preparation of this manuscript, an 1S,2S-cyclohexanedi-
amine-bridged porphyrin dimer was described (Ema, T.; Nemugaki, S.;
Tsuboi, S.; Utaka, M. Tetrahedron Lett. 1995, 36, 5905). The opposite sense
of the observed couplet is to be expected from the opposite absolute
configurations. The significantly smaller amplitude is in part due to a smaller
oscillator strength for mono-phenylporphyrin used by Ema et al. as compared
with the tetra-phenylporphyrin used here. Correcting for this effect, based
upon the dipole strength reported for a 5-(1′-naphthyl)porphyrin,14a the
magnitude of couplet observed by Ema et al. would be increased from 113
to 248, much closer to the magnitude of 400 observed in this work for
12b. The remaining discrepancy can probably be attributed to differences
in conformational averaging for the bisamide Vs the bisester. Ema et al. do
not discuss the possibility of multiple conformers.
(34) Yasumoto, T.; Murata, M. Chem. ReV. 1993, 93, 1897.
(35) Brevetoxin B is a voltage sensitive sodium channel opener.33
However, we have shown that brevetoxin B itself is capable of forming
cation specific “channels” in lipid bilayers: Matile, S.; Nakanishi, K. Angew.
Chem. 1996, 108, 812. The mono- and bisporphyrin derivatives 13b and
13c were employed in investigating the nature and properties of these
“channels” formed by brevetoxin B as well as differentiating between CD
couplets caused by “channel” formation and stacking: Matile, S.; Berova,
N.; Nakanishi, K. Submitted for publication.

5204 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Matile et al.
Figure 3. Structure and CD spectra of brevetoxin-bridged porphyrin dimer 13b in MeOH/water 4/1 in the presence (solid) and absence (dotted)
of CsCl, c ) 1.0 µM.
the porphyrin chromophores have extended the interchro-
mophoric coupling distance from ca. 15 Å bis(dimethylami-
nobenzoate) to at least 50 Å. They constitute the most powerful
chromophores available for the exciton coupled CD method so
far. Conformational studies of various porphyrin derivatives
of BTX and application of porphyrin reporter groups to structural
studies of biopolymers such as membranes, proteins, nucleic
acids, etc., are ongoing.40
Experimental Section
General Procedures. Solvents employed were reagent grade.
Anhydrous solvents were dried and distilled (THF from Na/benzophe-
none, CH2Cl2 from CaH2). Acetonitrile was dried over molecular sieves
(4 Å). Unless otherwise noted, materials were obtained from com-
mercial suppliers and were used without further purification. All
reactions were performed in dried glassware under argon. Reactions
were followed by thin-layer chromatography (TLC) using Whatman
alumina sheets, coated with silica gel (4 × 10 cm, 250 µm, UV254),
and reported as retention factors (Rf). Microscale preparative TLC
(PTLC) was performed using Whatman thin layer chromatography
plates (K5F, 20 × 20 cm, silica gel 150 Å, 250 µm, UV254). Column
chromatography (CC) was performed using ICN silica gel (32-63
mesh). HPLC purifications were performed using a Waters HPLC
system equipped with a Waters 600 E Multisolvent Delivery System,
a Waters 600 Controller and a Waters 996 Photodiode Array Detector
using the Millenium 2010 software for data processing. Melting points
were measured on a Thomas Hoover Capillary Melting Point Apparatus
Figure 4. The five most stable conformers of the bisporphyrin 12b,
1
calcd by MacroModel4.5;37 400 MHz H NMR of 12b, in DMSO-d6
(uncorrected). UV-vis spectra were recorded as CH2Cl2 solutions on
at (a) 25 °C and (b) 100 °C.
a Perkin-Elmer Lambda 4B spectrophotometer and reported as λmax
[nm] (ꢀmax [L mol-1 cm-1]). IR spectra were obtained as CCl4 solutions
coupled CD of bisporphyrin esters correctly represent the chiral
using a NaCl microcell on a Perkin Elmer 1600 FT-IR spectropho-
tometer. 1H-NMR spectra and 13C-NMR spectra were obtained on a
Varian VXR400 or Varian VXR300 and are reported in parts per million
(ppm) relative to TMS (δ), with coupling constants (J) in hertz (Hz).
Low-resolution and high-resolution FAB mass spectra were measured
on a JEOL JMS-DX303 HF mass spectrometer using a glycerol matrix
and Xe ionizing gas. CI mass spectra were measured on a NERMAG
R10-10 spectrometer with NH3 as ionizing gas. CD spectra were
recorded as CH2Cl2 solutions on a JASCO J-720 spectropolarimeter
driven by a JASCO DP700N data processor, respectively, and reported
as λmax [nm] (∆ꢀmax [L mol-1 cm-1]). Smoothing and other manipula-
sense of twist between the C-O bonds at the corresponding
stereogenic centers, thus leading to the conclusion that for
interpreting exciton coupled CD curves, the effective transition
moment can be regarded as being in the 5,15-direction of the
porphyrin skeleton as shown in Figure 1B.
Application of porphyrins to compounds with vicinal chiral
centers is restricted by possible conformational changes; on the
other hand, steric interactions (repulsion or stacking) of the bulky
chromophores may be useful to stabilize distinct conformers in
flexible (acyclic) compounds39 or to investigate higher energy
conformers such as 12c. Porphyrin chromophores attached to
dimeric steroids revealed that application of exciton coupled
CD to microscale assignment of absolute configurations can be
extended to compounds with a distance of 18 Å between
stereogenic centers. Furthermore, porphyrins attached to bre-
vetoxin B revealed that the conformational studies by exciton
coupled CD can be extended further to compounds with a
distance of at least 35 Å between the stereogenic centers. Thus
(40) One major concern which applies to every labeling technique is
the question of the extent to which the introduced labels disturb a
conformationally flexible system. As it has already been demonstrated in
the case of philanthotoxins, the enormous amount of information available
about interactions of porphyrins with themselves, proteins, nucleic acids,
and membranes could be very useful in order to avoid or reduce such
undesired effects (Huang, D.; Matile, S.; Berova, N.; Nakanishi, K.
Heterocycles 1996, 42, 732). However, while oligonucleotides forming
straight duplexes for example appear to be very attractive for studies of
polymorphism and drug binding by exciton coupled CD between ap-
propriately selected porphyrin chromophores, more precautions are necessary
in case of kinked and flexible duplexes (Mahtab, R.; Rogers, J. P.; Murphy,
C. J. J. Am. Chem. Soc. 1995, 117, 9099). Extensive model studies with
porphyrin-labeled polypeptides and oligonucleotides thus will be required
to delineate the scope and limitations of the porphyrin-reported exciton
coupled CD method applied to biopolymers.
(39) The attachment of 1 to acyclic chiral diol/diamines leads to
stereocontrolled porphyrin π,π-stacking, and the resulting CD spectra allows
the straightforward determination of the absolute configuration of single
chiral centers: Matile, S.; Berova, N.; Nakanishi, K. Enantiomer, in press.
Scope and limitations of this new concept are under ongoing investigation.

Porphyrin DeriVatiVes of Steroids
J. Am. Chem. Soc., Vol. 118, No. 22, 1996 5205
tions of spectra were carried out with a software developed in house:
DFT (discrete Fourier transform) procedure.
J ) 8.06 Hz), 8.42, 8.49 (2d, 4 H, J ) 8.06 Hz), 8.78-8.90 (m, 16 H);
FAB-MS m/z 1473 (M + H+); FAB-HRMS m/z for C109H89N8O4 calcd
1573.7010, found 1573.7030.
5-(4′-Carboxymethylphenyl)-10,15,20-triphenylporphin (5). To
a mixture of 4-carboxymethylbenzaldehyde (2, 4.104 g, 25 mmol),
benzaldehyde (3, 7.960 g, 75 mmol), and Zn(OAc)2 (5.480 g, 25 mmol)
in propionic acid (500 mL) was added pyrrole (4, 6.710 g, 100 mmol)
at 100 °C within 1 h under vigorous stirring. The resulting dark solution
was refluxed for further 4 h and then cooled to room temperature. The
solvent was evaporated (15 Torr, 80 °C ), and the solid residue was
subjected to CC (SiO2, 40 g, 7 × 45 cm, CH2Cl2). All product
containing fractions were collected. The volume was reduced to 100
mL (15 Torr), and pyridine (2 mL) and an excess of DDQ (2 g) were
added at room temperature. The resulting mixture was refluxed until
the oxidation was complete (1 h), monitored by the disappearance of
the absorption at 626 nm. Cooled to room temperature, the solution
was extracted with 18% aqueous HCl solution (3 × 100 mL),
neutralized with saturated aqueous NaHCO3 solution (3 × 100 mL),
washed with brine (1 × 100 mL), dried (Na2SO4), and evaporated (15
Torr) to give 11.5 g of black-purple crude product. Purification by
CC (SiO2, 30 g, 3.5 × 30 cm, CH2Cl2) yielded pure 5 (1.23 g, 7.5%)
as deep purple crystals. TLC (CH2Cl2) Rf 0.74; mp >300 °C; UV-
vis (MeCN) 645 (4600), 588 (4500), 545 (7000), 511 (15 900), 413
(353 000), 365 (sh, 33 000); UV-vis (CH2Cl2) 645 (4400), 589 (4450),
549 (6900), 514 (15 700), 418 (371 000), 365 (sh, 31 500); IR (CCl4)
5r-Androstane-3r,17â-diol Bis(p-dimethylaminobenzoate) (7c).
A solution of 7a (4.0 mg, 13.6 µmol), p-dimethylaminobenzoyl triazol4c
(20 mg, 92.6 µmol), and DBU (5.4 mg, 35.4 µmol) in absolute CH2-
Cl2 (1 mL) was stirred at room temperature for 24 h, diluted with CH2-
Cl2 (20 mL), extracted with a solution of saturated aqueous NH4Cl (3
× 20 mL), washed with brine (2 × 20 mL), dried (Na2SO4), and
evaporated (15 Torr) to give 10.0 mg (132%) of crude product, which
was purified by PTLC (SiO2, CH2Cl2, Rf 0.10, 2×) to yield pure 7c
(5.3 mg, 75%) as a slightly yellow powder. TLC (CH2Cl2/MeOH 20:
1) Rf 0.33; mp 218-220 °C; UV-vis (CH2Cl2) 310 (53 200); IR (CCl4)
2932, 2863, 1705, 1547, 1252, 1217, 1107, 1068, 1004, 978; 1H NMR
(400 MHz, CDCl3) δ 0.80-2.30 (several m, 22 H), 0.86, 0.93 (2s, 2
CH3), 3.04, 3.05 (2s, N(CH3)2), 4.80 (t, 1 H, J ) 9.07 Hz, H-C(17)),
5.22-5.24 (m, 1 H), 6.65, 6.67 (2d, 4 H, J ) 5.41 Hz), 7.91, 7.93 (2d,
4 H, J ) 5.41 Hz); FAB-MS m/z 586 (100, M+); FAB-HRMS m/z for
C37H50N2O4 calcd 586.3771, found 586.3775.
5r-Androstane-3â,17r-diol Bis(p-[10′,15′,20′-triphenyl-5′-por-
phyrinyl]benzoate) (8b). Following the procedure for 6b, 8a (3.6 mg,
12.33 µmol) was converted to 19.0 mg (130%) of crude product, which
was purified by CC (SiO2, 2 × 25 cm, 20 g, CH2Cl2, Rf 0.66) to yield
pure 8b (11.1 mg, 75%) as a deep purple powder. TLC (CH2Cl2) Rf
0.69; UV-vis (CH2Cl2) 645 (8200), 589 (8300), 549 (12 800), 514
1
1718, 1285; H NMR (400 MHz, CDCl3) δ -2.64 (s, 2 H, exchange
1
with D2O), 4.13 (s, 3 H), 7.75-7.80 (m, 9 H), 8.22-8.25 (m, 6 H),
8.32 (d, 2 H, J ) 8.14 Hz), 8.46 (d, 2 H, J ) 8.14 Hz), 8.85-8.89 (m,
8 H); FAB-MS m/z 673 (M + H+); FAB-HRMS m/z for C46H33N4O2,
calcd 673.2603, found 673.2598; CD (MeOH) -; CD (CH2Cl2) -.
5-(4′-Carboxyphenyl)-10,15,20-triphenylporphin (TPP, 1). To a
solution of 5 (1.10 g, 1.63 mmol) in EtOH (50 mL) was added 2 N
NaOH (100 mL), and the suspension was refluxed for 4 h. Cooled to
room temperature, the solvent was decanted, and the crude product
was washed with water (2 × 50 mL), MeOH (3 × 20 mL), and hexane
(2 × 50 mL). Evaporation (15 Torr and 0.01 Torr) gave anal. pure 1
(922 mg, 86%) as a deep purple powder. Mp >300 °C; UV-vis
(MeOH) 645 (4600), 588 (4500), 545 (7000), 511 (15 900), 413
(29 200), 418 (690 000); H-NMR (400 MHz, CDCl3) δ -2.67 (s, 2
H, exchange with D2O), 0.93-2.50 (several m, 22 H), 0.96, 1.02 (2s,
2 CH3), 5.09-5.16 (m, 1 H), 5.27 (d, 1 H, J ) 6.23 Hz), 7.74-7.81
(m, 18 H), 8.21-8.26 (m, 12 H), 8.29, 8.36 (2d, 4 H, J ) 8.04 Hz),
8.43, 8.49 (2d, 4 H, J ) 8.06 Hz), 8.79-9.00 (m, 16 H).
5r-Androstane-3â,17â-diol Bis(p-[10′,15′,20′-triphenyl-5′-porphy-
rinyl]benzoate) (9b). Following the procedure for 6b, 9a (1.1 mg,
7.6 µmol) was converted to 7.0 mg (118%) of crude product, which
was purified by PTLC (SiO2, CH2Cl2, Rf 0.35) to yield pure 9b (3.8
mg, 64%) as a deep purple powder. TLC (CH2Cl2) Rf 0.35; UV-vis
(CH2Cl2) 645 (8200), 589 (8300), 549 (12 800), 514 (29 200), 418
1
(690 000); H NMR (400 MHz, CDCl3) δ -2.63 (s, 2 H, exchange
1
(353 000), 365 (sh, 33 000); IR (CCl4) 3444, 1639, 1211; H NMR
with D2O), 0.93-2.50 (several m, 22 H), 0.97, 1.05 (2s, 2 CH3), 5.02
(t, 1 H, J ) 8.42 Hz), 5.11-5.19 (m, 1 H), 7.73-7.85 (m, 18 H),
8.16-8.22 (m, 12 H), 8.34, 8.36 (2d, 4 H, J ) 8.04 Hz), 8.44 (d, 8 H,
J ) 8.06 Hz), 8.80-9.00 (m, 16 H).
(400 MHz, CDCl3/CD3OD 4:1) δ -3.00 to -2.00 (br s, ca. 2 H), 7.74-
7.80 (m, 9 H), 8.21-8.24 (m, 6 H), 8.35 (d, 2 H, J ) 8.05 Hz), 8.51
(d, 2 H, J ) 8.05 Hz), 8.81-8.83 (m, 8 H); FAB-MS m/z 659 (M +
H+); FAB-HRMS m/z for C45H31N4O2, calcd 659.2446, found 659.2457.
5r-Cholestane-3â,6r-diol Bis(p-[10′,15′,20′-triphenyl-5′-porphy-
rinyl]benzoate) (6b). To a solution of 1 (24.4 mg, 37.1 µmol), EDC
(7.1 mg, 37.1 µmol), and DMAP (4.0 mg, 37.1 µmol) in absolute CH2-
Cl2 (1 mL) was added (room temperature) a solution of 6a (5 mg, 12.4
µmol) in CH2Cl2 (1 mL). The mixture was stirred (room temperature,
12 h), diluted with CH2Cl2 (20 mL), extracted with a solution of
saturated aqueous NH4Cl (3 × 20 mL), washed with brine (2 × 20
mL), dried (Na2SO4), and evaporated (15 Torr) to give 29 mg (139%)
of crude product, which was purified by CC (SiO2, 2 × 25 cm, 20 g,
CH2Cl2/hexane 3:1, Rf 0.36) to yield pure 6b (16.3 mg, 78%) as a deep
purple powder. TLC (CH2Cl2/hexane 3:1) Rf 0.36; mp >300 °C; UV-
vis (CH2Cl2) 645 (8200), 589 (8300), 549 (12 800), 514 (29 200), 418
(690 000); IR (CCl4) 2949, 2880, 1713, 1547, 1252, 1218, 1110, 1068,
5r-Androstane-3â,17â-diol Bis(p-dimethylaminobenzoate) (9c).
Following the procedure for 7c, 9a (4.0 mg, 13.6 µmol) was converted
to 7.5 mg (100%) of crude product, which was purified by PTLC (SiO2,
CH2Cl2/MeOH 40:1 Rf 0.62) to yield pure 9c (5.8 mg, 77%) as a slightly
yellow powder. TLC (CH2Cl2/MeOH 40:1) Rf 0.62; mp 221-224 °C;
UV-vis (CH2Cl2) 310 (53 200); 1H NMR (400 MHz, CDCl3) δ 0.80-
2.30 (several m, 22 H), 0.90, 0.93 (2s, 2 CH3), 3.05 (s, 2 N(CH3)2),
4.80 (t, 1 H, J ) 9.07 Hz), 4.85-4.94 (m, 1 H), 6.65, 6.67 (2d, 4 H,
J ) 5.41 Hz), 7.90, 7.93 (2d, 4 H, J ) 5.41 Hz).
1(R),2(R)-trans-Cyclohexanediol Bis(p-[10′,15′,20′-triphenyl-5′-
porphyrinyl]benzoate) (12b). To a solution of 1 (15 mg, 22.8 µmol),
EDC (7.1 mg, 37.1 µmol), and DMAP (4.0 mg, 37.1 µmol) in absolute
CH2Cl2 (2 mL), a solution of 12a (1.3 mg, 11.3 µmol) in CH2Cl2 (1
mL) was added (room temperature). The mixture was refluxed (4 h),
diluted with CH2Cl2 (20 mL), extracted with a solution of saturated
aqueous NH4Cl (3 × 20 mL), washed with brine (2 × 20 mL), dried
(Na2SO4), and evaporated (15 Torr) to give 15 mg (95%) of crude
product, which was purified by CC (SiO2, 2 × 25 cm, 20 g, CH2Cl2,
Rf 0.56) to yield pure 12b (8.5 mg, 54%) as a deep purple powder.
TLC (CH2Cl2) Rf 0.56; mp >300 °C; UV-vis (MeCN) 644 (7100),
593 (8000), 551 (20 600), 513 (26 700), 419 (690 500); IR (CCl4) 2918,
2857, 1717, 1598, 1550, 1348, 1269, 1251, 1110, 1097, 1001, 961;
1H-NMR (400 MHz, CDCl3) δ -2.70 (s, 2 H, exchange with D2O),
1.2-2.5 (several m, 8 H), 5.57-5.63 (br m, 2 H), 7.50-7.62, 7.69-
7.80 (2m, 18 H), 8.07-8.12, 8.18-8.22 (2m, 12 H), 8.29-8.54 (several
d-lk m, 8 H), 8.68-8.99 (several m, 16 H); FAB-MS m/z 1397 (M +
H+); FAB-HRMS m/z for C96H69N8O4 calcd 1397.5440, found 1397.5447.
5r-Androstane-17â-diphenylmethylsilyloxy-3-one (15). A solu-
tion of stanolone 14 (480 mg, 1.655 mmol), DPMSCl (385 mg, 1.67
mmol), and imidazole (113 mg, 1.67 mmol) in absolute CH2Cl2 (50
mL) was stirred at room temperature for 15 h, diluted with CH2Cl2 (50
1
1000, 978; H NMR (400 MHz, CDCl3) δ -2.64 (s, 2 H, exchange
with D2O), 0.74-2.55 (several m, 44 H), 5.23-5.34 (br m, 2 H), 7.67-
7.83 (2m, 18 H), 8.21-8.29 (m, 12 H), 8.31, 8.36 (2d, 4 H, J ) 8.06
Hz), 8.48, 8.50 (2d, 4 H, J ) 8.06 Hz), 8.85-8.89 (m, 16 H); FAB-
MS m/z 1685 (M + H+); FAB-HRMS m/z for C117H105N8O4 calcd
1685.8280, found 1685.8260.
5r-Androstane-3r,17â-diol Bis(p-[10′,15′,20′-triphenyl-5′-por-
phyrinyl]benzoate) (7b). Following the procedure for 6b, 7a (7.6 mg,
11.5 µmol) was converted to 12.0 mg (135%) of crude product, which
was purified by CC (SiO2, 2 × 25 cm, 20 g, CH2Cl2, Rf 0.69) to yield
pure 7b (6.5 mg, 73%) as a deep purple powder. TLC (CH2Cl2) Rf
0.69; mp >300 °C; UV-vis (CH2Cl2) 645 (8200), 589 (8300), 549
(12 800), 514 (29 200), 418 (690 000); IR (CCl4) 2940, 2875, 1710,
1547, 1252, 1218, 1110, 1068, 1000, 978; 1H NMR (400 MHz, CDCl3)
δ -2.64 (s, 2 H, exchange with D2O), 0.93-2.50 (several m, 22 H),
0.99, 1.12 (2s, 2 CH3), 5.02 (t, 1 H, J ) 8.42 Hz), 5.49-5.52 (m, 1
H), 7.74-7.79 (m, 18 H), 8.21-8.25 (m, 12 H), 8.29, 8.36 (2d, 4 H,

5206 J. Am. Chem. Soc., Vol. 118, No. 22, 1996
Matile et al.
mL), extracted with a solution of saturated aqueous NH4Cl (3 × 100
mL), washed with brine (1 × 100 mL), dried (Na2SO4), and evaporated
(15 Torr) to give 906 mg (100%) of crude product, which was purified
by CC (SiO2, CH2Cl2, Rf 0.31) to yield pure 15 (716 mg, 89%) as a
Z-Bis-5r-androstane-3,3′-ene-17â,17â′-diol Bis-(p-dimethylami-
nobenzoate) (11c). Following the procedure for 7c, 11a (1.0 mg, 1.82
µmol) was converted to 1.5 mg (99%) of crude product, which was
purified by PTLC (SiO2, CH2Cl2/MeOH 40:1, Rf 0.36) to yield pure
11c (0.7 mg, 45%) as a deep purple powder. TLC (CH2Cl2/MeOH
1
colorless oil. TLC (CH2Cl2) Rf 0.31; H NMR (400 MHz, CDCl3) δ
1
0.01 (s, SiCH3) 0.60-2.33 (several m, 22 H), 0.62, 0.85 (2s, 2 CH3),
3.67 (t, 1 H, J ) 8.34 Hz), 7.34-7.42, 7.58-7.61 (2m, 10 H).
Z-Bis-5r-androstane-17â,17â′-diphenylmethylsilyloxy-3,3′-ene (16)
and E-Bis-5r-androstane-17â,17â′-diphenylmethylsilyloxy-3,3′-ene
(17). To freshly distilled THF (15 mL), carefully degassed with Ar,
TiCl3 (3.39 g, 20.0 mmol) was added under Ar at room temperature.
To the resulting violet suspension LiAlH4 (362 mg, 9.53 mmol) was
added at room temperature under Ar, and the mixture was refluxed for
15 min. Then a solution of 15 (137 mg, 0.28 mmol) in dry and degassed
THF was added to the refluxing black suspension. Refluxing was
continued until the intermediate diol (TLC (CH2Cl2/MeOH 40:1) Rf
0.68) was completely converted (20 h). Cooled to room temperature,
the mixture was carefully poored into ice water (100 mL). Hexane
(100 mL) was added, and the organic layer was extracted with water
(4 × 100 mL), evaporated (15 Torr), dissolved in CH2Cl2 (100 mL),
washed with brine (2 × 100 mL), dried (Na2SO4), and evaporated (15
Torr) to give 150 mg (109%) of crude product. Purification by CC
(SiO2, CH2Cl2/hexane 1:4 Rf 0.42) afforded a 1:1 mixture of the pure
isomers 16/17 (30.9 mg, 24%) as a colorless solid. TLC (CH2Cl2/
hexane 1:4) Rf 0.31; 1H NMR (400 MHz, CDCl3) δ 0.01 (s, 2 SiCH3)
0.60-1.89 (several m, 40 H), 0.60, 0.82 (2s, 4 CH3), 2.24-2.31, 2.52-
2.62 (2 br d-lk m, 4 H), 3.65 (t, 2 H, J ) 8.32 Hz), 7.34-7.42, 7.58-
7.61 (2m, 20 H); CI-MS m/z 858 (M + NH4+).
40:1) Rf 0.36; UV-vis (CH2Cl2) 310 (53 200); H NMR (400 MHz,
CDCl3) δ 0.80-2.30 (several m, 40 H), 0.91, 0.94 (2s, 4 CH3), 2.24-
2.31, 2.55-2.64 (2 br d-lk m, 4 H), 3.05 (s, 2 N(CH3)2), 4.78 (t, 2 H,
J ) 8.97 Hz), 6.67 (d, 4 H, J ) 5.40 Hz), 7.91 (d, 4 H, J ) 5.40 Hz);
FAB-MS m/z 843 (M + H+); FAB-HRMS m/z for C56H78N2O4 calcd
842.5962, found 842.5947.
BTX-D(1,1,42)-1,42-diol (13a). BTX-B (18, 2.0 mg, 2.2 µmol) was
dissolved in a solution of CeCl3 (0.8 mg, 2.2 µmol) and NaBD4 (0.4
mg, 10.0 µmol) in absolute MeOH (1 mL), stirred for 4 min at room
temperature, filtered off through Celite, dried (Na2SO4), and evaporated
(15 Torr) to give a crude product, which was purified by CC (CH2-
Cl2/MeOH 9:1, Rf 0.45) to yield pure 13a (0.7 mg, 37%) as a colorless
powder. (As a second main product, the selectively 42-reduced, 42-
D-labeled BTX (0.8 mg, 42%) was isolated (CH2Cl2/MeOH 9:1, Rf
0.73) and characterized and will be used for further BTX-membrane
studies.) TLC (CH2Cl2/MeOH 9:1) Rf 0.45; 1H NMR (400 MHz,
CDCl3) δ 1.02, 1.04 (d, 3 H), 1.17 (s, 3 H), 1.24 (s, 3 H), 1.29 (s, 9
H), 1.47-1.90, 1.95-2.14, 2.14-2.28, 2.30-2.44 (4m, ca. 28 H),
2.65-2.67 (m, 1 H), 2.95-3.14, 3.18-3.44, 3.50-3.62 (3m, ca. 13
H), 3.79-3.81 (m, 1 H), 3.84-3.89 (dd-lk m, 1 H) 3.91-3.97 (dd-lk
m, 2 H), 4.02-4.12 (m, 2 H), 4.37-4.39 (d-lk m, 1 H), 4.71-4.74 (m,
1 H), 4.91-4.95 (m, 1 H) 5.09-5.13 (m, 1 H), 5.76-5.80 (m, 2 H),
2
5.81-5.85 (m, 1 H); H NMR (46 MHz, CHCl3) 3.96-4.19; CI-MS
+
Z-Bis-5r-androstane-3,3′-ene-17â,17â′-diol (11a) and E-Bis-5r-
androstane-3,3′-ene-17â,17â′-diol (10a). A 1:1 mixture of the isomers
16/17 (31 mg, 33 µmol) was solved in a 1 M solution of TBAF in
THF (1 mL) and stirred at room temperature for 3 h. To the resulting
mixture were added brine (50 mL) and hexane (50 mL) . The water
layer was extracted with hexane (2 × 50 mL) and EtOAC (3 × 50
mL). The resulting EtOAc solution was reduced (50 mL), washed with
brine (1 × 50 mL), dried (Na2SO4), and evaporated (15 Torr) to give
906 mg (100%) of crude product. The two isomers were separated by
CC (SiO2, CH2Cl2, Rf 0.52 (11a), Rf 0.48 (10a)). Crystallization of
11a from CH2Cl2 afforded 11a (6.9 mg, 32%) as a white powder, 10a
(7.5 mg, 42%) as a highly viscous oil, and an oily mixture of 10a/11a
(2.2 mg, 12%). 11a: Because of the extremely low solubility of 11a
in all usual solvents, spectroscopic analysis is almost impossible. TLC
m/z 922 (M + NH4
)
BTX-D(1,1,42)-1,42-diol Bis(p-[10′,15′,20′-triphenyl-5′-porphy-
rinyl]benzoate) (13b) and BTX-D(1,1,42)-1,42-ol p-(10′,15′,20′-
triphenyl-5′-porphyrinyl)benzoate (13c). To a solution of 1 (3.0 mg,
4.6 µmol), EDC (0.9 mg, 4.6 µmol), and DMAP (0.5 mg, 4.6 µmol) in
absolute CH2Cl2 (1 mL), a solution of 13a (0.7 mg, 0.78 µmol) in CH2-
Cl2 (1 mL) was added (room temperature). The mixture was stirred
(room temperature, 1.5 h), until almost all 13a was consumed and four
purple spots were visible on TLC (CH2Cl2/MeOH 40:1, Rf 0.36, Rf
0.28, Rf 0.17, Rf 0.14, ratio ca. 10:10:2:3). Then, the reaction mixture
was diluted with CH2Cl2 (10 mL), extracted with a solution of saturated
aqueous NH4Cl (10 mL), washed with brine (10 mL), dried (Na2SO4),
and evaporated (15 Torr) to give crude product, which was purified by
PTLC (CH2Cl2/MeOH 40:1) to yield pure 13b (0.5 mg, 32%) and 13c
(0.2 mg, 16%) as a deep purple powder. (As another main product,
the 1,5,42-porphyrin trimer (0.7 mg, 32%) was isolated (CH2Cl2/MeOH
40:1, Rf 0.36) and characterized.) 13b: TLC (CH2Cl2/MeOH 40:1) Rf
0.28; UV-vis (CH2Cl2) 645 (8200), 589 (8300), 549 (12 800), 514
1
(CH2Cl2) Rf 0.52; mp >300 °C; H NMR (400 MHz, CDCl3/CD3OD
1:1) δ 0.6-2.4 (several m, 56 H), 3.61-3.64 (t-lk m, 2 H); FAB-, CI-
1
or DCI-MS: no signal. 10a: TLC (CH2Cl2) Rf 0.48; H NMR (400
MHz, CDCl3) δ 0.60-1.89 (several m, 40 H), 0.73, 0.86 (2s, 4 CH3),
2.24-2.31, 2.52-2.62 (2 br d-lk m, 4 H), 3.61 (t, 2 H, J ) 7.98 Hz);
CI-MS m/z 548 (M+).
1
(29 200), 418 (690 000); H NMR (400 MHz, CDCl3) δ -2.64 (s, 2
H, exchange with D2O), 1.02, 1.04 (d, 3 H), 1.17 (s, 3 H), 1.24 (s, 3
H), 1.29 (s, 9 H), 1.47-1.90, 1.95-2.14, 2.14-2.28, 2.30-2.44 (4m,
ca. 26 H), 2.65-2.67 (m, 1 H), 2.95-3.14, 3.18-3.44, 3.50-3.62 (3m,
ca. 13 H), 3.79-3.81 (m, 1 H), 3.84-3.89 (dd-lk m, 1 H) 3.91-3.97
(dd-lk m, 2 H), 4.02-4.12 (m, 2 H), 4.37-4.39 (d-lk m, 1 H), 4.71-
4.74 (m, 1 H), 4.91-4.95 (m, 1 H) 5.09-5.13 (m, 1 H), 5.76-5.80
(m, 3 H), 5.91-5.94 (m, 1 H); 7.82-7.88 (2m, 18 H), 8.07-8.11 (m,
12 H), 8.27 (d, 4 H, J ) 6.63 Hz), 8.40 (d, 4 H, J ) 8.06 Hz), 8.76-
Z-Bis-5r-androstane-3,3′-ene-17â,17â′-diol Bis(p-[10′,15′,20′-
triphenyl-5′-porphyrinyl]benzoate) (11b). Following the procedure
for 6b, 11a (1.0 mg, 1.82 µmol) was converted to 3.3 mg (100%) of
crude product, which was purified by PTLC (SiO2, CH2Cl2/MeOH 40:
1, Rf 0.65) to yield pure 11b (2.3 mg, 70%) as a deep purple powder.
TLC (CH2Cl2/MeOH 40:1) Rf 0.65; UV-vis (CH2Cl2) 645 (8200), 589
(8300), 549 (12 800), 514 (29 200), 418 (690 000); 1H NMR (400 MHz,
CDCl3) δ -2.80 (s, 2 H, exchange with D2O), 0.93-2.50 (several m,
44 H), 1.06, 1.10 (2s, 4 CH3), 5.04 (t, 2 H, J ) 7.69 Hz), 7.71-7.80
(m, 18 H), 8.11-8.27 (m, 12 H), 8.29 (d, 4 H, J ) 8.30 Hz), 8.42 (d,
4 H, J ) 8.30 Hz), 8.77-8.98 (m, 16 H); FAB-MS m/z 1828 (M+).
E-Bis-5r-androstane-3,3′-ene-17â,17â′-diol Bis(p-[10′,15′,20′-
triphenyl-5′-porphyrinyl]benzoate) (10b). Following the procedure
for 6b, 10a (1.0 mg, 1.82 µmol) was converted to 3.5 mg (106%) of
crude product, which was purified by PTLC (SiO2, CH2Cl2/MeOH 40:
1, Rf 0.62) to yield pure 10b (2.3 mg, 70%) as a deep purple powder.
TLC (CH2Cl2/MeOH 40:1) Rf 0.62; UV-vis (CH2Cl2) 645 (8200), 589
(8300), 549 (12 800), 514 (29 200), 418 (690 000); 1H NMR (400 MHz,
CDCl3) δ -2.80 (s, 2 H, exchange with D2O), 0.93-2.50 (several m,
44 H), 1.06, 1.10 (2s, 4 CH3), 5.04 (t, 2 H, J ) 7.69 Hz), 7.71-7.80
(m, 18 H), 8.11-8.27 (m, 12 H), 8.29 (d, 4 H, J ) 8.30 Hz), 8.42 (d,
4 H, J ) 8.30 Hz), 8.77-8.98 (m, 16 H); FAB-MS m/z 1828 (M+).
2
8.86 (m, 16 H); H NMR (46 MHz, CHCl3): 5.53-5.65; FAB-MS
m/z 1545 (100, M - 1 + 18 + H+). 13c: Rf 0.17, Rf 0.14; UV-vis
(CH2Cl2) 645 (4400), 589 (4450), 549 (6900), 514 (15 700), 418
(371 000), 365 (sh, 31 500); 1H NMR (400 MHz, CDCl3). Due to the
small amount and the product mixture the spectrum of 13c only
confirms the presence of porphyrin and BTX (1:1); FAB-MS m/z 1543
(M+).
Acknowledgment. This work was supported by NIH GM
34509 (to K.N.), Schweizerischer Nationalfonds (to S.M.), NIH
GM 22994 and NIH Fogarty Senior International Fellowship
F06 TW02122 (to R.W.W.), and Deutsche Forschungsgemein-
schaft Grant FI 142/3-2 (to J.F.).
JA960126P