Three challenges toward the assignment of absolute configuration of gymnocin-B

Article abstract of DOI:10.1021/ja0512677

The absolute configuration of gymnocin-B has been determined to be (S)-10 and (S)-37. Three challenges toward the configurational assignment of this largest of the polyether marine toxin include (i) introduction of p-(meso-triphenylporphyrin)-cinnamate gr

Full text of DOI:10.1021/ja0512677

Published on Web 06/11/2005
Three Challenges toward the Assignment of Absolute
Configuration of Gymnocin-B
Katsunori Tanaka,† Yasuhiro Itagaki,† Masayuki Satake,‡ Hideo Naoki,‡
Takeshi Yasumoto,‡ Koji Nakanishi,† and Nina Berova*,†
Contribution from the Department of Chemistry, Columbia UniVersity, 3000 Broadway,
New York, New York 10027, and Okinawa Health Biotechnology Research DeVelopment Center,
12-75 Suzaki, Gushikawa, Okinawa 904-2234, Japan
Received February 28, 2005; E-mail: ndb1@columbia.edu
Abstract: The absolute configuration of gymnocin-B has been determined to be (S)-10 and (S)-37. Three
challenges toward the configurational assignment of this largest of the polyether marine toxin include (i)
introduction of p-(meso-triphenylporphyrin)-cinnamate group (TPPcinnamate) on sterically hindered 10-,
37-hydroxyls under mild conditions, (ii) conformational analysis in the presence of TPPcinnamates at C-10
and C-37 positions on the flexible seven-membered rings embodied in a large polyether ladder-like scaffold
structure, and (iii) determination of the chirality at C-10 and C-37 on the basis of porphyrin/porphyrin circular
dichroism exciton-coupled interaction over a large distance. The experimentally obtained positive exciton
couplet by CD and FDCD of the bis-TPPcin derivative of gymnocin-B is in good agreement with that of
theoretically calculated CD of the MMFF optimized structures, by employing DeVoe’s coupled oscillator
approach, thus establishing the full absolute configuration of gymnocin-B.
including gymnocin-A (Chart 1).5 On the basis of extensive
NMR and MS analysis, the structure and the relative configu-
Introduction
Gymnocins are a series of cytotoxic marine polycyclic ethers,
produced by the red tide dinoflagellate Karenia (formerly
Gymnodinium) mikimotoi. The dinoflagellate Karenia mikimotoi
is one of the most notorious red tide species that cause
devastating damages to aquaculture and marine ecosystems
worldwide, yet the mechanism of the toxic effect remains
unknown.1,2 Recently, gymnocin-A, which exhibits potent
cytotoxicity against mouse lymphoide P388 cells, has been
isolated from K. mikimotoi (Chart 1).3 The structure of
gymnocin-A, characterized by 14 contiguous saturated ether
rings and a 2-methyl-2-butenal side-chain at 5-position of A
ring, was elucidated by 2D-NMR analysis and collision-induced
MS/MS experiments.3 Furthermore, the absolute configuration
of gymnocin-A was also determined by applying the modified
Mosher method at the 50-OH group. Very recently, an elegant
total synthesis of this complex molecule has been achieved by
Sasaki and co-workers.4
ration of gymnocin-B, shown in Chart 1, were recently
elucidated.5 Similar to gymnocin-A, this new toxin contains 15
contiguous polyether skeletons and a 2-methyl-2-butenal side-
chain at the C-5 position. The number of contiguous ether rings
is the largest among the polycyclic ethers hitherto known.
However, the absolute configuration of gymnocin-B remains
unsolved, since the three sterically hindered pseudoaxial hy-
droxyls, namely, 10-, 37-, and 54-hydroxyls hampered straight-
forward application of the Mosher method which relies on
esterification with MTPA reagents.
The CD exciton chirality method based on the coupled
oscillator theory has been widely used for determining the
absolute configurations of various organic compounds.6,7 The
presence of two hydroxyl groups at C-10 and C-37 in gymnocin-
B, separated by 20 Å, leads to the possibility of applying the
exciton chirality method by introducing potent CD reporter
groups at these hydroxyl moieties. However, this method faces
the following three challenges. First, since the amount of
gymnocin-B available from the natural source is very limited,
an efficient microscale protocol is required to derivatize the
sterically hindered 10- and 37-hydroxyls with strong UV/vis
absorbing CD reporter groups, which are commonly bulky π-π
electron systems. Second, a precise conformational analysis of
In our continuous effort to isolate and characterize new
gymnocin congeners, we recently isolated a new toxin,
gymnocin-B, from the dinoflagellate K. mikimotoi collected at
Kushimoto, Wakayama, in Japan, in addition to other congeners
† Columbia University.
‡ Okinawa Health Biotechnology Research Development Center.
(1) Honjo, T. ReV. Fish Sci. 1994, 2, 225-253.
(2) Jenkinson, I. R.; Arzul, G.; Xunta de Galicia and IOC of UNESCO: France,
1998.
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T. Tetrahedron Lett. 2005, 46, 3537-3540.
(6) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill
Valley, CA, 1983.
(7) Berova, N.; Nakanishi, K. In Circular Dichroism: Principles and Applica-
tions; 2nd ed.; Berova, N., Nakanishi, K., Woody, R. W., Eds.; Wiley-
VCH: New York, 2000; pp 337-382.
(3) Satake, M.; Shoji, M.; Oshima, Y.; Naoki, H.; Yasumoto, T. Tetrahedron
Lett. 2002, 43, 5829-5832.
(4) (a) Tsukano, C.; Sasaki, M. J. Am. Chem. Soc. 2003, 125, 14294-14295.
(b) Tsukano, C.; Ebine, M.; Sasaki, M. J. Am. Chem. Soc. 2005, 127, 4326-
4335.
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J. AM. CHEM. SOC. 2005, 127, 9561-9570
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A R T I C L E S
Tanaka et al.
Chart 1. Structures of Gymnocin-A and Gymnocin-B
this large polyether structure including the critical 10-OH, 37-
OH is required because of the five flexible seven-membered
rings. Third, difficulties are expected in the measurement of an
exciton couplet over a large interchromophoric distance of ca.
30 Å. In this contribution, we describe our strategy for
overcoming these difficulties and successful assignment of
absolute configuration of gymnocin-B.
model not only to test the simultaneous introduction of the two
TPPcin at the hindered hydroxyls but also to evaluate the
efficiency of long-range chromophoric interaction of new
TPPcin chromophore, and conformational preference of the
cinnamate moiety, i.e., s-cis or s-trans.
1-1. Derivatization. An efficient method for introducing a
CD reporter group at sterically hindered -OH groups under
mild conditions, especially bulky porphyrinoids for the purpose
of observing long-range chromophoric interactions, would
greatly expand the versatility of the exciton-coupled CD method
for stereochemical analysis of large natural products.13-16 Thus
far, stereochemical analysis of sterically hindered hydroxyls has
been achieved by introducing (i) benzyl chromophores and/or
(ii) benzoate chromophores generated directly by oxidation of
benzyl ether derivatives.17-21 However, the benzyl/benzoate
approach is not suitable for the analysis of gymnocin-B for
several reasons; the simple benzyl/benzoate chromophores
cannot provide exciton coupling over a long range due to their
weak UV absorbance. Furthermore, the benzylation conditions,
namely, the reaction with para-substituted benzyl chloride in
the tetramethylurea/DMF/THF solvent system using sodium
hydride as a strong base, is not suited for minuscule amounts
of natural products, in particular, for complex molecules
exemplified by gymnocins. In addition, there is the conforma-
tional ambiguity arising from the rotation of benzyloxy moiety.
The harsh oxidation conditions (RuCl3/NaIO4) of the benzyl
ether to benzoate derivatives is also not applicable to substrates
containing sensitive functional groups, such as polyether
moieties and/or the oxidation-sensitive porphyrin derivatives.
For such reasons, we investigated a new approach to introduce
Results and Discussion
1. Model Study with Sterically Hindered Secondary 17-
OH of 3-Oxo-5R-androstan-17-ol (1) and Bis-steroid (5)
(Scheme 2). The extremely small amount of gymnocin-B
available was the first challenge for determination of its absolute
stereochemistry. Therefore, we carried out several model studies
aimed at selecting the appropriate CD reporter groups capable
of long-range exciton coupling8,9 and determining optimal
reaction conditions for gymnocin-B derivatization. We have
already shown that p-(meso-triphenylporphyrin)-carboxylic acid
(TPP-CO2H) introduced at the hydroxyl or amino groups via
acylates represents one of the most potent CD reporter groups
especially suited for the observation of long-range exciton
coupling.7,10 On the basis of this precedence, we investigated a
new tetraphenylporphyrin chromophore, p-(meso-triphenyl-
porphyrin)-cinnamate (TPPcin, 419 nm, ꢀ 387,000 in CH2Cl2,
and 415 nm, ꢀ 362,000 in MeOH),11 which was introduced at
the secondary 17-OH of 3-oxo-5R-androstan-17-ol 1 and bis-
steroid 5.12 Since the secondary 17-OH group present in
templates 1 and 5 is sterically hindered due to the neighboring
C-18 methyl group and C-13 quaternary carbon, introduction
of porphyrin chromophores in 1 and 5 is challenging. Further-
more, since the bis-steroid 5 has quite a rigid steroid skeleton
with two remote pseudoequatorial hydroxyls at the 17 and 17′
positions separated by ca. 19 Å, a distance similar to that
between gymnocin-B 10- and 37-hydroxyls (ca. 20 Å) (Figures
1a and 4a), this template represents a particularly important
(13) Yasumoto, T.; Murata, M. Chem. ReV. 1993, 93, 1897-1909.
(14) Satake, M.; Murata, M.; Yasumoto, T. J. Am. Chem. Soc. 1993, 115, 361-
362.
(15) Faulkner, D. J. Nat. Prod. Rep. 1999, 16, 155-198.
(16) Faulkner, D. J. Nat. Prod. Rep. 2002, 19, 1-48.
(17) 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, 509-51.
(18) Takeda, R.; Zask, A.; Nakanishi, K.; Park, M. H. J. Am. Chem. Soc. 1987,
109, 914-915.
(19) Takeda, R.; Park, M. H.; Nakanishi, K. Isr. J. Chem. 1989, 29, 239-242.
(20) Nakanishi, K.; Kuroyanagi, M.; Nambu, H.; Oltz, E. M.; Takeda, R.;
Verdine, G. L.; Zask, A. Pure Appl. Chem. 1984, 56, 1031-1048.
(21) Chang, M.; Meyers, H. V.; Nakanishi, K.; Ojika, M.; Park, J. H.; Park, M.
H.; Takeda, R.; Vazquez, J.; Wiesler, W. T. Pure Appl. Chem. 1989, 61,
1193-1200.
(8) Chen, S.-M. L.; Harada, N.; Nakanishi, K. J. Am. Chem. Soc. 1974, 96,
7352-7354.
(9) Canceill, J.; Collet, A.; Jacques, J. J. Chem. Soc., Perkin II 1982, 83.
(10) Matile, S.; Berova, N.; Nakanishi, K.; Fleischhauer, J.; Woody, R. W. J.
Am. Chem. Soc. 1996, 118, 5198-5206.
(11) Tanaka, K.; Pescitelli, G.; Nakanishi, K.; Berova, N. Monatsh. Chem. 2005,
136, 367-395.
(12) Cerny, I.; Pouzar, V.; Budesinsky, M.; Drasar, P. Collect. Czech. Chem.
Commun. 2000, 65, 1597-1608.
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Assignment of Configuration of Gymnocin-B
A R T I C L E S
Figure 1. (a) Most stable conformation of bis-TPPcin 7 calculated by Monte Carlo/MMFF94s with Spartan 02. (b) CD and FDCD spectra of 7 in CH2Cl2.
Gray line: CD at 1.1 × 10-6 M. Black line: FDCD at 1.1 × 10-8 M, light-cul filter, 520 nm, HT voltage, 586 V using Jasco J-465 devices with 4.0 mm
balancing masks.
Scheme 1. Introduction of Chromophores at Sterically Hindered Hydroxyl
the porphyrin-cinnamate chromophore at the sterically hindered
secondary -OH groups.
a variety of CdC double bonds with the styrene group in high
yield under mild conditions. Furthermore, we investigated the
application of this transition metal-catalyzed reaction toward
the derivatization of sterically hindered hydroxyls. This new
protocol involves the combined method of acylation of such
hydroxyls with the small and reactive acryloyl chloride reagent
(toxicity data shown in Experimental Section)29-33 followed by
cross metathesis with vinyl-porphyrins (Scheme 1). Thus, a
Recently, we reported that the olefin cross metathesis
reaction22,23 can be applied successfully for introducing styrene
chromophores as CD reporter groups, and based on this, a
chemical/chiroptical method for configurational assignment of
olefin containing natural and synthetic compounds was devel-
oped.24,25 Usage of the recently developed Grubbs’ second-
generation ruthenium catalyst26-28 leads to the replacement of
(27) Scholl, M.; Ding, S.; Lee, C. W.; Grubbs, R., H. Org. Lett. 1999, 1, 953-
956.
(22) Connon, S. J.; Blechert, S. Angew. Chem., Int. Ed. 2003, 42, 1900-1923.
(23) Chatterjee, A. K.; Choi, T.-L.; Sanders, D. P.; Grubbs, R. H. J. Am. Chem.
Soc. 2003, 125, 11360-11370.
(28) Scholl, M.; Trnka, T. M.; Morgan, J. P.; Grubbs, R. H. Tetrahedron Lett.
1999, 40, 2247-2250.
(29) Olsson, T.; Stern, K.; Westman, G.; Sundell, S. Tetrahedron 1990, 46,
2473-2482.
(24) Tanaka, K.; Nakanishi, K.; Berova, N. J. Am. Chem. Soc. 2003, 125,
10802-10803.
(30) Mizutani, H.; Watanabe, M.; Honda, T. Tetrahedron 2002, 58, 8929-8936.
(31) Langer, P.; Albrecht, U. Synlett 2002, 1841-1842.
(32) Honda, T.; Namiki, H.; Kaneda, K.; Mizutani, H. Org. Lett. 2004, 6, 87-
89.
(25) Tanaka, K.; Pescitelli, G.; Di Bari, L.; Xiao, T. L.; Nakanishi, K.;
Armstrong, D. W.; Berova, N. Org. Biomol. Chem. 2004, 2, 48-58.
(26) Chatterjee, A. K.; Toste, F. D.; Choi, T.-L.; Grubbs, R. H. AdV. Synth.
Catal. 2002, 344, 634-637.
(33) Wang, C.; Russell, G. A. J. Org. Chem. 1999, 64, 2066-2069.
9
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Scheme 2. Introduction of TPP and TBP at 17-OH of 5R-Androstan-3-one Derivatives
variety of the cinnamate-type porphyrin chromophores, such as
tetraphenylporphyrin or tetrabenzoporphyrin, shown in Scheme
2, can be introduced at hindered hydroxyls under mild condi-
tions.
metathesis equilibrium toward the thermodynamically more
stable trans-CdC double bonds. The stereoselective construction
of the (E)-double bond by reaction with excess vinyl-TPP 3a
makes this approach attractive since the geometry of the
cinnamate double bond may occasionally affect the sign of CD
exciton chirality. Noteworthy, the vinyl-tetrabenzoporphyrin
(vinyl-TBP) 3b could also be introduced by cross metathesis
under equilibrium conditions to provide 4b as a single (E)-
stereoisomer in 65% yield; the intense deep-colored porphyrin
derivatives are also readily detectable by thin-layer chromatog-
raphy (TLC). Thus, the entire procedure including reaction
monitoring and product isolation can be performed at the
microgram-scale down to 100 µg.
The acylation of model substrate 1 with tetraphenylporphyrin
carboxylic acid (TPP-CO2H) under EDC coupling conditions
requires more than 10 equiv of the carboxylic acid reagent and
a long reaction time (24 h in dichloromethane under reflux
conditions). Furthermore, the EDC coupling between 1 and the
sterically congested tetrabenzoporphyrin carboxylic acid (TBP-
CO2H) due to phenyl groups at â,â′-positions of porphyrin34
failed to give the corresponding ester derivatives, while the
reactions at elevated temperature led to decomposition of the
sensitive benzoporphyrin chromophore.
The simultaneous derivatization of the two secondary 17-
OHs in bis-steroid 5 was next attempted. Our attempts for the
simultaneous bis-acylation of 5 at the two secondary 17-
hydroxyls by TPP-CO2H was not successful; the product
obtained under several different acylation conditions, e.g., with
EDC and DMAP in dichloromethane, surprisingly gave only
the monoacylate.
According to the procedure reported here, the 17-OH of
steroid 1 was first acylated with acryloyl chloride in the presence
of diisopropylethylamine in THF to give 2 in almost quantitative
yield. Since the reaction was also achieved with a weaker base,
such as triethylamine and DMAP, the acryloylation can also
be performed with base-sensitive substrates. The vinyl group
in 2 was then subjected to cross metathesis with 6 equiv of
vinyl-tetraphenylporphyrin (vinyl-TPP) 3a in the presence of
Grubbs’ second-generation ruthenium catalyst to provide 4a in
72% yield (see detailed conditions in the Experimental Section).
As reported by Grubbs and others,22-25 the (E)-stereoisomer is
the sole product under conditions of excess styrene derivatives
and presence of the second-generation catalyst which shifts the
However, two TPPcin chromophores could be efficiently
introduced at both hindered hydroxyls by a new approach; bis-
steroid 5 was first reacted with the reactive acryloyl chloride,
77% yield, and the two vinyl groups introduced in 6 were then
functionalized by “double cross metathesis” with 12 equiv of
vinyl-TPP 3a. This method yielded the bis-porphyrin derivative
7 with (E)-stereochemistry at both locations in ca. 50% yield
from 5.
(34) Giraud-Roux, M.; Proni, G.; Nakanishi, K.; Berova, N. Heterocycles 2003,
61, 417-432.
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Assignment of Configuration of Gymnocin-B
A R T I C L E S
Scheme 3. Derivatization of Gymnocin-B
1-2. Long-Range Chromophoric Interaction of Bis-TPPcin
Derivative (7). The lowest-energy conformation of bis-
TPPcinnamate 7 obtained by a MMFF94s/Monte Carlo con-
formational analysis is shown in Figure 1a. For both TPPcin
groups introduced at 17 and 17′ hydroxyls, the cinnamate moiety
possesses an s-cis conformation, in agreement with the preferred
conformation of regular methyl cinnamates; the s-cis conforma-
tion is 0.97 kcal/mol lower in energy than the corresponding
s-trans isomer, as evaluated by DFT/BLYP/6-31G* level
calculations. In the case of bis-TPPcin 7 with known absolute
configuration, the most stable conformation as calculated by
MC/MMFF94s with Spartan 02 led to a negative twist of -47
° between the porphyrin effective transition moments (see later)
along the 5-15 and 5′-15′ axes. Note that the CD spectrum of
7 (Figure 1b) shows a clear negative exciton couplet with an
amplitude of -37, reflecting the negative twist orientation
between the remote stereogenic centers at 17 and 17′ positions
over a distance of 42 Å. Furthermore, this relatively weak long-
range exciton coupling was measured by FDCD at a 2-fold
higher sensitivity, namely, 10-8 M, due to the favorable
fluorescent properties of TPPcinnamate (fluorescent quantum
yield Φf ) 0.13).11 Thus, the highly sensitive FDCD analysis
should prove very useful for detection of weak coupling resulting
from long-range interactions and/or limited sample amount.
The successful “TPPcinnamate” microscale approach per-
formed for the conformationally rigid dimeric steroid 5 provides
an intriguing protocol for the stereochemical analysis of many
large natural products carrying a variety of functional groups,
e.g., sterically hindered hydroxyl and amino groups as exempli-
fied by gymnocin-B.
2. Derivatization of Gymnocin-B. On the basis of these
successful model studies described above, we performed de-
rivatization of gymnocin-B (8) using the 300 µg amount of
available sample (Scheme 3). To differentiate the 10- and 37-
hydroxyls out of the four hydroxyls in 8, the 1,3-diol moiety in
the O ring was first protected as the acetonide. After aqueous
workup with saturated NaHCO3 solution and quick filtration
through silica gel, the product was immediately reacted with
acryloyl chloride in the presence of triethylamine and DMAP
in CH2Cl2 at 60 °C for 6 h. After direct purification of the
reaction mixture by silica gel chromatography, the resulting bis-
acryloyl derivative 10 was subjected to cross metathesis with
excess of vinyl-TPP 3a (10 equiv) under equilibrium conditions
to afford the red-colored bis-TPPcin derivative 11, ca. 10 µg,
as estimated from its UV-vis spectrum. Although the presence
of two chromophores is supported by observed exciton-coupled
CD, despite repeated attempts the molecular ion peak of 11
could not be observed. The highest peak we found was at 1846,
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A R T I C L E S
Tanaka et al.
Chart 2. Gymnocin-B Derivatives for Conformational Analysis
resulting from a loss of one TPPcinnamic acid group (see the
Experimental Section).
constant (1 Hz) between 10-H and 11-H and an NOE between
10-H/11-H in gymnocin-B (8) indicated that the 10-H/11-H
dihedral angle was close to 90° (Figure 2a).3,5 Therefore, in the
oxepane ring B this leads to 10-H pseudoequatorial and 10-OH
pseudoaxial orientations, similar to those of gymnocin-A. The
pseudoaxial disposition for 10-OH is corroborated by NOEs,
8-H/9â-H, 8-H/11-H, 9R-H/10-H, and 9â-H/10-H.
On the other hand, a 37-OH/39-H NOE indicates a similar
orientation for 37-OH and 39-H in ring J (Figure 2b). NOE
correlation was observed between 37-H/60-Me and 36â-H/60Me
but not between 35-H/37-H. Thus, similar to 10-OH, the NOE
data support a pseudoaxial orientation for 37-OH. The NOE
analysis of tetrabenzoate derivative 12 also led to a diaxial
conformation of 10-OBz and 37-OBz.37
3-2. Molecular Modeling. The complexity of such a large
polyether structure on one hand, and the difficulties in param-
etrization of such a large molecule by MMFF94s on the other,
necessitated the performance of a conformational analysis of
several simpler truncated models of gymnocin-B that carry the
simpler regular cinnamate moiety at 10-OH and 37-OH instead
of the bulkier TPPcinnamates (see structure 13 in Chart 2). The
molecular modeling results for the truncated models were then
compared with that of the gymnocin bis-cinnamate (13) and
with NOE analysis of gymnocin-B (8), and its tetrabenzoate
12. Finally, the molecular modeling of the real system 14, i.e.,
bis-TPPcin of 8, was undertaken to evaluate the projection angle
3. Conformational Analysis. The gymnocin-B bis-
TPPcinnamate (11) is a unique and large molecule (MW )
2530) consisting of 15 polyether ring systems, five of them being
conformationally flexible seven-membered rings (B, G, H, J,
and O rings). An extensive conformational analysis was
necessary to predict the dihedral angles between the transition
dipoles of two porphyrins present in such an unusual system as
11 (Chart 2). Especially critical was the precise conformational
analysis around the 10-TPPcinnamate and 37-TPPcinnamate
groups in rings B and J, respectively, since the pseudoaxial and/
or pseudoequatorial orientations of the hydroxyl groups could
sensitively affect the absolute sense of twist between the
porphyrins (vide infra). The conformational analysis started from
the NOE analysis of gymnocin-B (8) and its tetrabenzoate
derivative 12. Subsequently, the molecular modeling study was
applied to bis-cinnamate (13) and bis-TPPcinnamate (14),
(simplified model of 11), by Molecular Mechanics (MM) using
the Merck Molecular Force Field (MMFF94) developed by
Halgren35 in its “static” version MMFFs, which is the default
in Macromodel 7.1.36
3-1. NOE Investigation of Native Gymnocin-B (8) and
Tetrabenzoate Derivative (12) (Figure 2). A small coupling
(35) (a) Halgren, T. A. J. Comput. Chem. 1996, 17, 490-519. (b) Halgren, T.
A. J. Comput. Chem. 1996, 17, 520-552. (c) Halgren, T. A. J. Comput.
Chem. 1996, 17, 553-586. (d) Halgren, T. A.; Nachbar, R. B. J. Comput.
Chem. 1996, 17, 587-615. (e) Halgren, T. A. J. Comput. Chem. 1996, 17,
616-641.
(36) Mohamadi, F.; Richards, N. G. J.; Guida, W. C.; Liskamp, R.; Lipton, M.;
Caufield, C.; Chang, G.; Hendrickson, T.; Still, W. C. J. Comput. Chem.
1990, 11, 440-467.
Figure 2. NOE correlations at B,C and J,K rings of gymnocin-B.
(37) Satake, M.; Tanaka, Y.; Ishikura, Y.; Oshima, Y. Personal communication.
9
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Assignment of Configuration of Gymnocin-B
A R T I C L E S
Figure 3. Most stable conformations of bis-cinnamate (13) and monocinnamate truncated models: (a) A-C fragment: 10-cin (axial). (b) I-K fragment:
37-cin (axial). (c) A-O fragment 13: 10-, 37-bis-cin (diaxial). All confirmations were optimized by Monte Carlo/MMFF94s with Spartan 02. Similarly, the
fragments H-K, G-K, and F-K show the lowest-energy conformations with 37-cin axial.
between the 5-15 direction of the two porphyrins, which
according to theoretical studies38 is considered as the direction
of porphyrin Soret band effective transition moment.
Similarly, the MMFF94s calculation of I-K fragment showed
an axially oriented 37-cinnamate on the J ring being the most
stable conformation (Figure 3b). Again, only chairlike confor-
mations of seven membered J ring and pseudoaxial 37-
cinnamate were found for all other three conformations obtained
within a 2 kcal/mol energy window.
Furthermore, the continuous conformational analysis of the
H-K, G-K, and F-K truncated models (structures not shown)
with an enlarged polyether ring system similarly exhibited
preference for the pseudoaxial 37-cinnamate conformations
among the lowest-energy conformations obtained within 2 kcal/
mol. These results indicate that the presence of F, G, and H
ring systems attached to the I-K fragment have nil, or only
negligible, effects on the axial conformation of 37-cinnamate.
Thus, the molecular modeling of these truncated models showed
a strong preference for the diaxial conformation of both 10-
and 37-cinnamates.
The conformational analysis was performed by molecular
mechanics calculations using the MMFF94s force field with
Monte Carlo conformational search using a Spartan ‘02 package
(see the Computational Section for details). The main degrees
of conformational freedom for both truncated models and
gymnocin-B bis-cinnamate derivatives are (i) the C10-O and/
or C37-O rotamerisms, (ii) the s-cis/s-trans CdC-CdO bond
isomerism of the cinnamate moiety, and (iii) seven-membered
ring conformations, i.e., chair- or boatlike conformation of B,
G, H, J, and O rings. To simplify the calculations, the
unsaturated aldehyde side chain at C-5 of A ring was replaced
by the methyl group (structures 13 and 14 in Chart 2). The (10S,
37S) absolute configurations of gymnocin-B shown in Chart 2
were chosen for all calculations.
3-2-1. Truncated Models of 13; Monocinnamate Models.
While only the A-C ring system of 13 was considered as the
truncated model for obtaining the 10-cinnamate conformation,
more extensive molecular modeling was required for obtaining
geometrical information of the 37-cinnamate on the J ring. For
this purpose, four truncated models I-K, H-K, G-K, and F-K
fragments were studied, expecting that an enlargement of the
ring system from the I-K fragment to the F-K fragment may
provide more precise data for the seven-membered J ring and
the 37-cinnamate, located at the right and suspected more
flexible side of gymnocin-B (Chart 2).
The conformational analysis of the A-C truncated system
revealed four conformations within 2 kcal/mol from the most
stable conformation. In the lowest-energy conformation the 10-
cinnamate is pseudoaxial with an s-cis orientation between the
carbonyl and CdC double bond as shown in Figure 3a. The
slight difference between four minima arises from the s-cis and
s-trans rotamerism of the cinnamate, but importantly, only the
chairlike conformation for B ring and the axial conformation
for 10-cinnamate were found.
3-2-2. Bis-cinnamate (13). The lowest-energy conformation
resulting from conformational analysis of the whole structure
of 13 with A-O ring system (Chart 2) is shown in Figure 3c.
In this structure, both 10- and 37-cinnamates also adopt
pseudoaxial orientation, consistent with the stable conformations
obtained for the truncated monocinnamate models. Furthermore,
the ten lowest-energy conformations within a 2 kcal/mol energy
window, all show the 10-cinnamate with a pseudoaxial orienta-
tion. The 37-cinnamate also shows a preference for pseudoaxial
orientation, except in one case where the orientation is equatorial
at a 1.7 kcal/mol higher energy level than the most stable
conformation (Boltzman-weighted population of about 5%). As
described previously, the NOE study of gymnocin-B (8) and
the tetrabenzoate derivative 12 (Figure 2) has predicted the
pseudodiaxial conformation for both the 10- and 37-benzoates.
This is in good agreement with the MMFFs molecular modeling
results of the bis-cinnamates 13 conformation, thus validating
the MMFFs conformational analysis of a large polyether
gymnocin-B.
3-2-3. Bis-TPPcinnamate (14). Finally, an MMFF94s
conformational analysis was performed on gymnocin-B bis-
TPPcinnamate (14) (Chart 2). Due to the complexity of structure
(38) Pescitelli, G.; Gabriel, S.; Wang, Y.; Fleischhauer, J.; Woody, R. W.;
Berova, N. J. Am. Chem. Soc. 2003, 125, 7613-7628.
9
J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005 9567

A R T I C L E S
Tanaka et al.
Table 1. Conformational Analysis of
Gymnocin-B-Bis-TPPcinnamate (14)a
∆
G
orientation of
10-TPPcin
orientation of
37-TPPcin
τ
12 degreesc and predicted
interporphyrin twist
conforlmation
(kcal/mol)b
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
0
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
ax
eq
eq
ax
eq
eq
ax
eq
eq
eq
eq
eq
eq
eq
eq
eq
eq
29 (+)
33 (+)
-9 (-)
35 (+)
76 (+)
12 (+)
35 (+)
81 (+)
109 (+)
34 (+)
31 (+)
95 (+)
33 (+)
48 (+)
66 (+)
16 (+)
1.35
1.72
2.21
2.59
3.34
3.96
4.10
4.12
4.12
4.25
4.82
4.98
5.93
6.24
6.42
a Conformational analysis was performed by molecular mechanics
calculations using the MMFF94s force field with Monte Carlo conforma-
tional search by Spartan02 package. b Relative free energy: Boltzman-
weighted population at 298 K consists of 1 (86%), 2 (9%), and 3 (5%)
conformation, respectively. c Projection angle between 5 and 15 directions
of porphyrins (see Figure 4).
14 containing a large gymnocin polyether skeleton with two
large TPPcinnamate moieties, it was safer to take into consid-
eration the conformations obtained within a rather large energy
window. Therefore, we considered a 7 kcal/mol energy window
as well as the distributions of structures characterized by the
sign and the dihedral angle between the transition dipole
moments of the two porphyrins. A total of sixteen main
conformations were found for bis-TPP cinnamate (14) within
7 kcal/mol (Table 1). The most stable conformation with s-cis
orientation of both 10- and 37-TPPcin is shown in Figure 4a.
Interestingly, while the 10-TPPcin is found at the axial position
similar to previous cases of simple cinnamates, the 37-TPPcin
clearly adopts a pseudoequatorial orientation (Figure 4a). This
lowest-energy conformation with 10-axial and 37-equatorial
TPPcin results in a clockwise twist between the two porphyrin
effective transitions defined by the 5-15 and 5′-15′ directions
(dihedral angle of +29°), hence leading to positive exciton
chirality. Similarly, the survey of the other lowest-energy
structures obtained within 7 kcal/mol (Table 1) gave rise to a
preferred equatorial 37-TPPcin orientation, except for conforma-
tions 3 and 6, while 10-TPPcin always remains axial in all
calculated conformations. This reveals a preferred clockwise
twist between the porphyrin effective transitions in 15 of a total
of 16 conformations. From two conformations 3 and 6 with
axial 37-TPPcin, only 3 shows a negative interporphyrin twist
(dihedral angles of -9°, Figure 4b) and is of 1.72 kcal/mol
higher energy, i.e., 5% of the Boltzmann-weighted population,
therefore expecting to provide a negligible contribution to CD.
However, the second conformation found with axial 37-TPPcin,
e.g., 6, interestingly exhibits a positive interporphyrin helicity,
presumably as a result of a slight distortion of the J ring from
its usual chairlike conformation. This conformational analysis
therefore clearly predicts a positive CD exciton chirality in the
porphyrin region for the chosen absolute configurations of 10S,
37S. A more quantitative evaluation of exciton-coupled CD
based on the Boltzmann-weighted DeVoe calculation will be
described in the next section.
Figure 4. Two low-energy conformations of bis-TPPcinnamate (14)
obtained by Monte Carlo/MMFF94s with Spartan 02. (a) The lowest-energy
conformation of 14 with 10-axial, 37-equatorial. (b) Conformation with 10-,
37-diaxial of 1.72 kcal/mol higher energy.
It is not surprising that the introduction of the tetraphenyl-
porphyrins often results in an unusual conformational preference
due to their steric bulkiness, aromaticity, and hydrophobicity.
For example, it is known that the TPP-diester derivative of trans-
1,2-cyclohexanol exhibits an unusual 1,2-diaxial conformation
due to the severe porphyrin/porphyrin steric interaction of the
1,2-diequatorial conformation.10 Yet the equatorial preference
of 37-TPPcinnamate conformation in 14, in contrast to the
conformational preference of the regular axial cinnamate, was
an interesting finding. An additional molecular modeling of the
A-K fragment of 14 without the L-O ring system was
conducted. Similar to the case of 14, equatorial 37-TPPcin was
found as the lowest-energy conformation, but interestingly, also
axial 37-TPPcin was found only at a 0.19 kcal/mol higher energy
level. Furthermore, out of 18 energy minima obtained within 7
kcal/mol, eleven conformations were found with axial 37-TPPcin
orientation (see Supporting Information). The comparison of
these MMFFs molecular modeling results of 14 and its truncated
A-K fragment reflects some effects played by the L-O
polyether tail, the presence of which most likely changes the K
ring strain. This in turn affects the axial/equatorial position of
37-TPPcin and leads to some distortion of the J ring (cf. ref
39).
(39) Eliel, E. L.; Allinger, N. L.; Angyal, S. J.; Morrison, G. A. Conformational
Analysis; John Wiley & Sons: New York, 1965; pp 189-210.
9
9568 J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005

Assignment of Configuration of Gymnocin-B
A R T I C L E S
membered rings, and especially the investigation of critical 10-
and 37-axial and/or equatorial -OH orientation in the B and J
rings, and (iii) measurement of clear bisignate CD arising from
exciton-coupled interaction between very remote chromopores
(distance over ca. 30 Å) attached to a flexible polyether scaffold.
We have developed a microscale method for introducing a bulky
TPPcin chromophore into sterically hindered hydroxyls by
acryloylation/cross metathesis. This resulted in a clear positive
CD exciton coupling over a large distance. Extensive confor-
mational analysis of gymnocin-B and its truncated models by
MMFF94s/Monte Carlo calculation resulted in 10-axial and 37-
equatorial TPPcin conformation in 11, namely, prediction of a
positive interporphyrin twist for arbitrarily chosen 10(S),37(S)
configuration. Finally, the Boltzman-weighted calculated CD
by DeVoe’s coupled oscillator method was in full agreement
with the experimental CD of 11, thus establishing the absolute
configuration of gymnocin-B as 10(S),37(S), and hence, the
absolute configurations of remaining 31 stereogenic centers
based on the relative configuration of gymnocin-B previously
determined by NMR.5
Figure 5. Experimental and calculated CD spectra of compound 11. Solid
line: experimental, c ) 3.0 × 10-6 M in MeOH. Dotted line: Boltzman-
weighted (at 298 K) average CD calculated with DeVoe’s method and
porphyrin hybrid approach (D ) 77 D2 for 5-15 dipoles and 39 D2 for
10-20 dipoles, νmax ) 24.1 kK, Lorentzian band with ∆ν1/2 ) 0.75 kK
width) on lowest-energy MC/MMFF94s structures within 2 kcal/mol (see
Table 1).
4. CD Spectroscopic Analysis of Bis-TPP Derivative of
Gymnocin-B. The CD spectrum of bis-TPPcinnamate 11 in
MeOH at 3.0 × 10-6 M is shown in Figure 5 (solid line). A
clear almost conservative CD exciton couplet was observed with
extrema at 419 nm (∆ꢀ + 11) and 414 nm (∆ꢀ -15). The
positive exciton chirality led to the assignment of gymnocin-B
with (S)-10 and (S)-37 as depicted in this paper (see absolute
structure in Figure 4a).
To investigate the nature of the exciton coupling operating
at this large interchromophoric distance of ca. 30 Å (Figure
4a), quantitative CD calculations were also performed by the
DeVoe method.40-43 A hybrid approach was used to describe
the Soret transition,10,38 where the 5-15 component was given
full dipolar strength (77 D2, extrapolated from the absorption
spectrum of mono-TPPcin 4a, 415 nm, ꢀ 362,000 in MeOH),
while the 10-20 component was given half dipolar strength (39
D2). We have previously found that this approach is well
justified when a largely unrestricted libration around the
phenyl-porphyrin bonds exists.38 The MC/MMFF94s confor-
mational search revealed three conformations within the 2 kcal/
mol energy window (Table 1). The corresponding Boltzmann-
weighted calculated CD spectrum (Figure 5, dotted line) was
found in good agreement with the observed CD spectrum (solid
line). To summarize, both the experimental and calculated CD
data led to the assignment of absolute configuration of gym-
nocin-B as depicted in Chart 1.
Experimental Section
Acryloylation of Sterically Hindered Hydroxyls: Acryloylated
Derivative of Monosteroid (2). To a solution of 5R-androstan-17-â-
ol-3-one (1) (50 mg, 0.172 mmol) and diisopropylethylamine (60.0 µL,
0.344 mmol) in dichloromethane (1.0 mL) was slowly added acryloyl
chloride (28.0 µL, 0.344 mmol) at 0 °C (toxicity data of acryloyl
chloride: IHL-MUS LC50 (inhalation-mouse lethal concentration 50%
kill) ) 92 mg/m3/2 h). After the mixture was warmed to room
temperature and stirred for an additional 1 h, H2O and saturated aqueous
NH4Cl solution were added, and the resulting mixture was extracted
with ethyl acetate. The organic layers were combined, washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo to give
the crude products. Column chromatography on silica gel (gradually
9% to 25% ethyl acetate in hexane) gave 2 (60 mg, 100%) as a white
solid: 1H NMR (400 MHz, CDCl3) δ 0.73-1.80 (m, 16H), 0.84 (s,
3H), 1.02 (s, 3H), 1.99-2.04 (m, 1H), 2.07-2.12 (m, 1H), 2.16-2.43
(m, 4H), 4.68 (dd, 1H, J ) 8.8, 8.8 Hz), 5.81 (dd, 1H, J ) 10.4, 1.3
Hz), 6.12 (dd, 1H, J ) 17.3, 10.4 Hz), 6.38 (dd, 1H, J ) 17.4, 1.3
Hz); HRFABMS calcd for C22H33O3 [M + H]+ 345.2429, found
345.2435.
Cross Metathesis with Vinyl-TPP (3a): TPPcinnamate Derivative
of Monosteroid (4a). To a solution of 2 (3.0 mg, 8.71 µmol) in
dichloromethane (2.0 mL) were added Grubbs’ second-generation
ruthenium catalyst (1.7 mg, 1.99 µmol) and vinyl-TPP 3a (33.5 mg,
52.3 µmol) at room temperature, and the mixture was stirred at 80 °C
for 3 h. The reaction mixture was concentrated in vacuo to give the
crude product which was purified by column chromatography on silica
gel (ethyl acetate in hexane, gradually from 9% to 20%) to afford 4a
(6.0 mg, 72%) as a deep red-colored solid. The reaction was also
performed using 100 µg of 2: 1H NMR (300 MHz, CDCl3) δ 0.97 (s,
3H), 1.05 (s, 3H), 0.82-2.45 (m, 22H), 4.85 (dd, 1H, J ) 7.9, 7.9
Hz), 6.75 (d, 1H, J ) 16.0 Hz), 7.72-7.79 (m, 9H), 7.93 (d, 2H, J )
8.1 Hz), 8.00 (d, 1H, J ) 16.1 Hz), 8.20-8.26 (m, 8H), 8.83-8.87
(m, 8H); HRFABMS calcd for C66H61O3N4 [M + H]+ 957.4743, found
957.4766.
Conclusion
In conclusion, we have determined the absolute configuration
of gymnocin-B, a cytotoxic marine natural product containing
the largest 15-polyether skeleton isolated so far. The three
challenges encountered during the course of configurational
assignments of this intriguing natural product are the follow-
ing: (i) introduction of bulky porphyrinoid reporter groups at
the sterically hindered axial 10- and 37-hydroxyls, (ii) confor-
mational flexibility arising from the presence of five seven
Cross Metathesis with Vinyl-TBP (3b): TBPcinnamate Deriva-
tive of Monosteroid (4b). To a solution of 2 (693 µg, 2.01 µmol) in
dichloromethane (2.0 mL) were added Grubbs’ second-generation
ruthenium catalyst (513 µg, 0.604 µmol) and vinyl-TBP 3b (3.70 mg,
6.04 µmol) at room temperature, and the mixture was stirred at 80 °C
for 5 h. The reaction mixture was concentrated in vacuo to give the
crude product, which was purified by column chromatography on silica
gel (33% ethyl acetate in hexane) to afford 4b (1.2 mg, 64%) as a
(40) DeVoe, H. J. Chem. Phys. 1964, 41, 393-400.
(41) DeVoe, H. J. Chem. Phys. 1965, 43, 3199-208.
(42) Rosini, C.; Zandomeneghi, M.; Salvadori, P. Tetrahedron: Asymmetry 1993,
4, 545-54.
(43) Superchi, S.; Giorgio, E.; Rosini, C. Chirality 2004, 16, 422-451.
9
J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005 9569

A R T I C L E S
Tanaka et al.
green-colored solid. Due to benzoporphyrin aggregation in solution,
only TLC and MS analysis were possible for identification of the
product: Rf ) 0.40 (50% ethyl acetate in hexane); HRFABMS calcd
for C64H56O3N4 [M]+ 928.4352, found 928.4384.
5.85 (d, 1H, J ) 10.4 Hz), 6.12 (dd, 1H, J ) 17.4, 10.4 Hz), 6.13 (dd,
1H, J ) 17.4, 10.6 Hz), 6.38 (d, 2H, J ) 17.9 Hz), 6.49 (dd, 1H, J )
7.0, 7.0 Hz), 9.41 (s, 1H); FABMS calcd for C71H100O22 [M]+ 1306,
found 1306.
Bis-acryloylated Derivative of Bis-steroid (6). To a solution of
bis-steroid 5 (10 mg, 17.5 µmol) and diisopropylethylamine (6.08 µL,
34.9 µmol) in dichloromethane (2.0 mL) was slowly added acryloyl
chloride (3.12 µL, 38.4 µmol) at room temperature. After the mixture
was stirred 3.5 h at this temperature, H2O and saturated aqueous NH4Cl
solution were added, and the resulting mixture was extracted with
chloroform. The organic layers were combined, washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo to give the crude
products. Column chromatography on silica gel (gradually 17% to 25%
ethyl acetate in hexane) gave bis-acryloylated derivative 6 (9.1 mg,
77%) as a white solid: 1H NMR (300 MHz, CDCl3) δ 0.81 (s, 6H),
0.84 (s, 6H), 0.88-1.84 (m, 30H), 2.15-2.27 (m, 2H), 2.50-2.63 (m,
4H), 2.78 (dd, 2H, J ) 17.8, 5.2 Hz), 2.91 (d, 2H, J ) 16.7 Hz), 4.70
(dd, 2H, J ) 8.0, 8.0 Hz), 5.81 (dd, 2H, J ) 10.4, 1.6 Hz), 6.12 (dd,
2H, J ) 17.3, 10.4 Hz), 6.39 (dd, 2H, J ) 17.3, 1.6 Hz); HRFABMS
calcd for C44H61O4N2 [M + H]+ 681.4632, found 681.4631.
Bis-TPPcinnamate Derivative of Gymnocin-B (11). To a solution
of 10 obtained above in dichloromethane (1.0 mL) were added Grubbs’
second-generation ruthenium catalyst (137 µg, 0.161 µmol) and vinyl-
TPP 3a (491 µg, 0.766 µmol) at room temperature, and the mixture
was stirred at 80 °C for 9 h. The reaction mixture was concentrated in
vacuo to give the crude product which was purified twice by column
chromatography on silica gel (from 50% ethyl acetate in hexane to
6% MeOH in CHCl3) to afford 11 (ca. 10 µg) as a deep red-colored
solid: Rf ) 0.07 (50% ethyl acetate in hexane); UV/vis (MeOH) 415
nm, ꢀ ) 720,000; CD (MeOH) 419 nm (∆ꢀ ) +11), 414 nm (∆ꢀ )
-15); Despite repeated attempts, it was not possible to observe the
molecular ion peak of bis-TPPcinnamate derivative 11 (calculated
MW: 2531) either by ESI or MALDI. Instead an intense peak
corresponding to the loss of a TPPcinnamic acid fragment (McLafferty
rearrangement) was observed. MALDI-TOFMS calcd for C112H124O20N4
[M-C47H32O2N4 (TPPcinnamic acid)]+ 1846, found 1846.
Bis-TPPcinnamate Derivative of Bis-steroid (7). To a solution of
6 (3.0 mg, 4.41 µmol) and vinyl-TPP 3a (33.9 mg, 52.9 µmol) in
dichloromethane (2.0 mL) at 80 °C was gradually added a solution of
excess amount of Grubbs’ second-generation ruthenium catalyst (4.49
mg, 5.29 µmol) in dichloromethane (5.0 mL) over 5 h with vigorously
stirring. The reaction mixture was concentrated in vacuo to give the
crude product which was purified by column chromatography on silica
gel twice (ethyl acetate in hexane, gradually from 25% to 33%) to afford
7 (5.0 mg, 60%) as a deep red-colored solid: 1H NMR (400 MHz,
CDCl3) δ 0.82-2.30 (m, 44H), 2.51-2.59 (m, 4H), 2.77 (br s, 2H),
2.89-2.97 (m, 2H), 4.87 (dd, 2H, J ) 8.3, 8.3 Hz), 6.75 (d, 2H, J )
16.0 Hz), 7.73-7.80 (m, 18H), 7.93 (d, 4H, J ) 8.1 Hz), 8.01 (d, 2H,
J ) 16.3 Hz), 8.20-8.26 (m, 16H), 8.84-8.87 (m, 16H); FABMS calcd
for C132H116O4N10 [M]+ 1906.40, found 1906.91.
Computational Methods
Molecular modeling was performed by Spartan ‘02 (Wavefunction,
Inc., Irvine, CA), using default parameters and convergence criteria.
The conformations were calculated with Merck force field (MMFF94s)36
using the Monte Carlo conformational search method with 1000
iteration steps. The coupled oscillator CD calculations were performed
by DeVoe method using Fortran program developed by W. Hug.44
Spectral parameters for DeVoe calculation were extrapolated from the
experimental UV/vis spectrum of the mono-TPPcin chromophore 4a
(D ) 77 D2 for 5-15 dipoles and 39 D2 for 10-20 dipoles, νmax ) 24.1
kK, Lorentzian band with ∆ν1/2 ) 0.75 kK width). The conformations
optimized by MMFF94s were used as the input geometries and the
Boltzmann-weighted CD spectrum was calculated at 298 K.
Acetonide Derivative of Gymnocin-B (9). To a solution of
gymnocin-B (8) (300 µg, 0.259 µmol) in acetone (1.0 mL) was added
p-toluenesulfonic acid monohydrate (1.2 mg, 6.31 µmol) at room
temperature. After the mixture was stirred at room temperature for 10
h, saturated aqueous NaHCO3 solution was added, and the resulting
mixture was extracted with CHCl3. The organic layers were combined,
washed with brine, dried over MgSO4, filtered on silica gel (9% MeOH
in CHCl3), and concentrated in vacuo to give a crude product as white
solid, which was analyzed by MS and immediately subjected for
acryloylation without further purification: HRFABMS calcd for
C65H97O20 [M + H]+ 1197.6573, found 1197.6584.
Bis-acryloylated Derivative of Gymnocin-B (10). To a solution
of the crude 9, obtained as described above, in dichloromethane (1.0
mL) were added acryloyl chloride (4.21 µL, 51.8 µmol), triethylamine
(7.23 µL, 51.8 µmol), and (dimethylamino)pyridine (633 µg, 51.8 µmol)
at room temperature, and the mixture was stirred at 60 °C for 6 h. The
reaction mixture was concentrated in vacuo to give the crude product
which was purified by preparative TLC on silica gel (50% ethyl acetate
in hexane) and then by column chromatography on silica gel (from
50% ethyl acetate in hexane to 6% MeOH in CHCl3) to provide 10 as
a white solid: 1H NMR (400 MHz, CDCl3) representative signals: δ
5.35 (br s, 1H), 5.44 (d, 1H, J ) 10.6 Hz), 5.83 (d, 1H, J ) 10.2 Hz),
Acknowledgment. We are grateful for the discussions to Dr.
Gennaro Pescitelli and Prof. Lorenzo DiBari, University of Pisa,
Prof. Wolfgang Kreiser, University of Dortmund, and to Prof.
Carlo Rosini and Dr. Egidio Giorgio, University of Potenza,
who also made available to us the DeVoe program. We
acknowledge the Jasco Corporation for generously providing
us with a new FDCD J-465 device. This research is supported
by the National Institutes of Health, NIH Grant GM 34509 (K.N.
and N.B.). K.T. is grateful to the JSPS Postdoctoral Fellowships
for Research Abroad.
Supporting Information Available: Copies of COSY and
1
ROESY spectra of gymnocin-B, H NMR and MS spectra of
the compounds 2, 4a, 4b, 6, 7, and 9-11, as well as some
conformational data. This material is available free of charge
via the Internet at http://pubs.acs.org.
JA0512677
(44) Hug, W.; Ciardelli, F.; Tinoco, I., Jr. J. Am. Chem. Soc. 1974, 96, 3407-
3410.
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9570 J. AM. CHEM. SOC. VOL. 127, NO. 26, 2005