Faulknerynes A-C from a Bahamian Sponge Diplastrella sp.: Stereoassignment by Critical Application of Two Exciton Coupled CD Methods

Article abstract of DOI:10.1021/jo102188q

Long-chain polyacetylene alcohols, faulknerynesA-C, alongwith known compounds diplynesA,C and E, were isolated from two specimens of the encrusting sponge, Diplastrella sp., collected from the surface of coral in the Bahamas. Two CD methods were critically evaluated for their suitability to terminal propargylic glycols and applied to assignment of configurations of faulkneryne A and diplyne C.

Full text of DOI:10.1021/jo102188q

pubs.acs.org/joc
Faulknerynes A-C from a Bahamian Sponge Diplastrella sp.:
Stereoassignment by Critical Application of Two Exciton
Coupled CD Methods
Jaeyoung Ko,† Brandon I. Morinaka,† and Tadeusz F. Molinski*,†,§
§
†Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Pharmaceutical Sciences,
University of California, San Diego, La Jolla, California 92093-0358, United States
tmolinski@ucsd.edu
Received November 2, 2010
Long-chain polyacetylene alcohols, faulknerynes A-C, along with known compounds diplynes A, C and
E, were isolated from two specimens of the encrusting sponge, Diplastrella sp., collected from the surface
of coral in the Bahamas. Two CD methods were critically evaluated for their suitability to terminal
propargylic glycols and applied to assignment of configurations of faulkneryne A and diplyne C.
Introduction
related hydrocarbons, from Callyspongia truncata5 and si-
phonodiol (3) from Siphonochalina truncata.6,7 In a screen
for compounds with antiproliferative activity, extracts of
two specimens of the Bahamian sponge Diplastrella sp. were
found to elicit moderate in vitro cytotoxicity against the
cultured tumor cell line HCT-116. We describe new bromi-
nated long-chain PAAs, faulknerynes A-C (4a-c), from
these extracts and assignment of the absolute configuration
of 1 and 4a using two microscale methods based on exciton
coupling circular dichroism (ECCD). We also establish that 1
obtained from Diplastrella sp. from the Bahamas and the
Philippines are both (R).1
Long chain polyacetylenic alcohols (PAAs) are charac-
teristic natural products of Haplosclerid and Spira-
strellid sponges, including the genera Diplastrella,1 Petrosia,
Siphonochalina, Haliclona,2 and Cribochalina.3 Among the
first to be reported were the highly cytotoxic duryne3 and
the HIV reverse-transciptase inhibitor petrosynol4 from
Cribochalina and Petrosia, respectively. Many PAAs are
exceedingly cytotoxic toward tumor cell lines (IC500s e
30 ng/mL), a biological property that has been correlated
with the presence of a terminal propargylic alcohol.2 Faul-
kner and co-workers reported C16 brominated diynes A, B, C
(1), and E (2) and three related sulfate half-esters from the
Philippines sponge Diplastrella sp.1 Fusetani and co-workers
described the C23 polynols, callyspongiols A-E, along with
Results and Discussion
A sample of the thinly encrusting Diplastrella sp., recov-
ered from small patches laboriously scraped from coral
substrate with a knife, was exhaustively extracted with
(1) Lerch, M. L.; Harper, M. K.; Faulkner, D. J. J. Nat. Prod. 2003, 66,
667–670.
(2) Zhou, G.-X.; Molinski, T. F. Mar. Drugs 2003, 1, 46–53.
(3) Wright, A. E.; McConnell, O. J.; Kohmoto, S.; Lui, M. S.; Thompson,
W.; Snader, K. M. Tetrahedron Lett. 1987, 28, 1377–1379.
(4) Isaacs, S.; Kashman, Y.; Loya, S.; Hizi, A.; Loya, Y. Tetrahedron
1993, 49, 10435–10438.
(5) Tsukamoto, S.; Kato, H.; Hirota, H.; Fusetani, N. J. Nat. Prod. 1997,
60, 126–130.
(6) Tada, H.; Yasuda, F. Chem. Lett. 1984, 779–780.
(7) Fusetani, N.; Sugano, M.; Matsunaga, S.; Hashimoto, K. Tetrahedron
Lett. 1987, 28, 4311–4312.
894 J. Org. Chem. 2011, 76, 894–901
Published on Web 01/10/2011
DOI: 10.1021/jo102188q
r
2011 American Chemical Society

Ko et al.
JOCArticle
MeOH. The MeOH extract was progressively adjusted in water
content and partitioned against solvents of increasing polarity
(hexane, CHCl3, and n-BuOH). The CHCl3-soluble fraction
was further purified to give the known diplyne C (1, identified
from NMR data1) and new natural products, faulknerynes
A-C (4a-c).8 The formula C16H21BrO2 for 4a was secured
from HRMS measurements ([M þ Na]þ, 347.0633, Δmmu =
TABLE 1. NMR Data (CDCl3) for Faulkneryne A (4a)
no. δC, multa δH, mult (J in Hz)b
COSYc
HMBC (H f C)
1
66.4, CH
66.4, CH
64.0, CH
80.1, C
3.72, dd (11.4, 6.2) H2
3.78, dd (11.4, 3.8) H2
4.57, dd (6.2, 3.8) H1, H7
C2, C3
C3
C1, C3, C4, C5
2
3
4
5
6
7
8
9
71.1, C
76.8, Cd
76.5, Cd
1
1.6). Examination of the H NMR spectrum of 4a (Table 1)
readily revealed chemical shifts of the two chain termini: a 1,2-
disubstituted vinyl bromide (δ 6.01, d, J =13.5 Hz; 6.17, dt,
J 13.5, 7.3 Hz) and a mutually coupled ABX system corre-
sponding to a 1,2-glycol (δ 3.72, dd, J= 11.4, 6.2 Hz, H1a;
δ3.78, dd, J=11.4, 3.8 Hz, H1b; 4.57, dd, J= 6.2, 3.8, H2). The
formula required an additional five degrees of unsaturation,
which were assigned to a conjugated (Z)-1,3,5-ene-diyne sys-
tem, based on a red-shifted UV spectrum [4a; λmax 271 nm]
compared to 1 and 3 [3; λmax 228 (ε 15,100)6], the presence of
four acetylenic 13C NMR sp signals (δ 71.1 s, 76.5 s, 76.8 s, 80.1
107.8, CH 5.49, d (10.9)
149.5, CH 6.11, dt (10.9, 7.6) H7, H9
30.9, CH2 2.33, (7.6) H7, H8, H10
H2, H8, H9
C3, C4, C5, C8, C9
C6, C7, C9
C6, C7, C8, C11
C8, C9, C11
C12
e
10 28.7, CH2 1.41, m
e
11 28.8, CH2 1.30, m
H9,H11
H10, H12
H11, H13
H12, H14
12 28.9, CH2 1.31, m
13 28.6, CH2 1.39, m
14 33.0, CH2 2.03, m (7.3)
C11
C14, C15
H13, H15, H16 C12, C15, C16
C13, C14, C16
C14, C15
15 138.3, CH 6.17, dt (13.5, 7.3) H14, H16
16 104.3, CH 6.01, d (13.5) H14, H15
a125 MHz. b600 MHz. cMultiplicity assigned from HSQC. dPeaks
may be interchanged. ePeaks may be interchanged.
1
s) and H NMR signals ascribed to a (Z)-1,2-disubstituted
double bond, C7-C8 (δ 5.49, d, J = 10.9 Hz; δ 6.11, dt, J=
10.9, 7.6 Hz) conjugated to the diyne. HMBC crosspeaks from
H7 to the acetylenic carbon signals of C3-C5 corroborated
UV evidence that the (Z)-double bond was conjugated to the
diyne. The remaining signals were assigned to allylic CH2
groups and an unresolved methylene chain. Therefore, 4a is
(Z)-7,8-dehydro-1.
Faulknerynes B (4b) and C (4c) (C16H1779BrO2, m/z
343.0304 [M þ Na]þ Δmmu=0.5), obtained in amounts of
4 and 14 μg, respectively, are dehydro-derivatives of 2 and
isomeric with each other. Analysis of COSY spectrum and
vicinal J values of the vinyl proton signals reveals that the
two compounds differ only in the location of the (Z)-
carbon-carbon double bond; in 4b, it is positioned at
C-7-C-8 while compound 4c has a 11-Z double bond in
conjugation with the terminal ene-yne. Therefore, 4b is
C-13,14-didehydro faulkneryne A and 4c is the 11-(Z) geo-
metrical isomer of 11-E diplyne E (2).
With diplyne C (1) from Diplastrella sp. collected in two
different oceans, one from the Pacific and the other from the
Atlantic, an opportunity presented itself for configuration
analysis by chiroptical comparison, although not without
some challenges. The optical rotation of natural 1 was not
reported, although a total syntheses of (S)-(þ)-1 ([R]D
þ13.3)9 and unnatural (S)-(þ)-diplyne A ([R]D þ9.6)10 and
chiroptical comparison with levorotatory diplyne A ([R]D
-8.7)1 would suggest an (R) configuration for both diplynes
A and C (1). Insufficient amounts of 4a-c were available to
record rotations for reliable comparison of [R]D with 1 or 3,
so attention was turned to alternative methods. Configura-
tional assignment of secondary alcohols is frequently carried
out by NMR using the modified Mosher’s method;11 how-
ever, the limited amount of available sample necessitated
deployment of sensitive CD spectroscopy, a method well
suited to “nanomole-scale” natural products investigation.12
The stereochemistry of siphonodiol (R)-(-)-(3) was assigned
by Fusetani and co-workers using the dibenzoate exciton
coupling method; specifically, the observation of a negative
(8) The compounds are named in honor of the late D. John Faulkner
(1940-2002) a pioneer in marine natural products who described diplynes
A-E (ref 1), related polyacetylenic alcohols, and hundreds of other com-
pounds. Andersen, R. J.; Ireland, C. M.; Molinski, T. F.; Bewley, C. B.
J. Nat. Prod. 2004, 67, 1239–1251.
(9) Gung, B. W.; Gibeau, C.; Jones, A. Tetrahedron: Asymmetry 2005, 16,
3107–3114.
(10) Gung, B. W.; Gibeau, C.; Jones, A. Tetrahedron: Asymmetry 2004,
15, 3973–3977.
(11) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem.
Soc. 1991, 113, 4092–4096.
(12) (a) Molinski, T. F. Curr. Opin. Drug Discovery Dev. 2009, 12, 197–
206. (b) Molinski, T. F. Curr. Opin. Biotechnol. 2010, 21, 819–826.
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bisignate Cotton effect, CE [λ 324 nm (Δε -25.3); 303
(þ24.2)] in the exciton coupled CD spectrum of 3a, the
derived 4-dimethylaminobenzoate diester (DMB diester).7
From exciton coupling theory,13 the sign of the split CE is
predicted from the helicity (twist) of the electronic dipole
transition vectors associated with the prominent 1La charge-
transfer transition in degenerate benzoate chromophores.
These vectors are approximately aligned with the corre-
sponding C-O bond directions; a negative sign for the
long-wavelength band of the split CE for 3a indicates a
negative helicity of the two C-O vectors, and a positive split
CE indicates a positive helicity. The strength of the ECCD
signal depends upon several other factors and is conveniently
reported as the difference between maximum and minimum
of the bisignate split CE [e.g., for 3a, A=24.2 - (-25.3)=
49.5].13
Glycol dibenzoate ECCD is dependent upon the dihedral
angle of O-C-C-O around C1-C2. Because acyclic 1,2-
glycols have a freely rotating C1-C2 bond, the absolute
configurational assignment requires knowledge of the limit-
ing staggered conformers. We were concerned about two
implicit assumptions used in the assignment of 3 and, con-
sequently, our approach to 1 and 4a: a low barrier to rotation
of the C1-C2 bond and similar energies of staggered con-
formers due to the presence of the relatively sterically
unencumbered acetylenic group and additional exciton cou-
pling from the diyne chromophore with the propargylic
DMB chromophore. Dibenzoate esters of 1-4 would be
expected to exhibit both types of dichromophoric interac-
tions: yne-benzoate and benzoate-benzoate.14 Conse-
quently, room temperature CD measurements of pro-
pargylic benzoates would sample significant proportions of
populated staggered conformers a-c, with a and c giving rise to
opposing signs of ECCDs, and small structural differences may
even contribute nonstaggered conformers that further influence
the magnitude or even the sign of the ECCD. In order to
quantify some of these expected differences, we calculated the
eneriges of the conformers of propargylic 1,2-glycol dibenzo-
ates (Spartan, PM3) and ordered the lowest lying conformers
(Figure 1).
Perhaps not unexpectedly, the potential energy surface of
the model propargylic 1,2-benzoate was found to be fairly
shallow. Nevertheless, for the dibenzoates of (R)-1, the three
lowest energy conformers depicted in Figure 1d,e comprising
47% of the Boltzmann distribution, subtended the same
negative helicity corresponding to the idealized geometry
(a). The torsional angle, θ, ranged from -55° to -84.1°
about the C-O vectors of the (CdO)O groups and differed
only in minor changes in orientation about the benzoate ester
bonds. Surprisingly, the nearest conformers with antiperi-
planar dispositions (θ ∼ 180°) of the two benzoate groups
and expected to give zero contribution to the ECCD (cf.
idealized geometry of Figure 1a) were higher in energy and
ranked fifth and sixth; together, they contribute only 13.2%
to the population [E = -0.54 kcal mol-1 (7.0%); E = -0.27
FIGURE 1. Conformers of propargylic 1,2-glycol O-dibenzoate
analogues of 5. (a-c) Idealized geometries, helicities, and signs of
contributions to ECCD (Δε). (d-f) Calculated conformers of
propargylic 1,2-glycol O-dibenzoate analogues of 5 ranked in
relative energy, E (kcal mol-1); θ, the dihedral angle of the C-O
bond vectors; and % Boltzmann populations (Spartan 08, semi-
empirical PM3, gas phase): (d) 0, -55.6°, (22.3%); (e) 1.29, -84.1°,
(13.3%); (f) 1.67, -55.0° (11.4%). Only the three best ranked
conformations are shown (see text). For ease of calculation, the
chain is truncated to a 3,5-diyne.
kcal mol-1 (6.2%)]. The gauche-gauche conformers (not
shown, but cf. Figure 1b), which are expected to give ECCD
of opposing sign to Figure 1c, were found to be of even lower
ranking and less stable by more than 2.8 kcal mol-1 than the
favored conformers; they represented less than 3% in the
conformer population.
ECCD of arenecarboxylate esters (acylates) of allylic and
propargylic alcohols arise from electronic coupling of arene
and C-C multiple bond π-π* transitions. Reliable assign-
ments of secondary allylic alcohols have been made from
ECCD of the corresponding allylic O-benzoates15 O-
naphthoates,16 and O-naphthoates of conjugated dienes.17
The model used for interpretation of ECCD in allylic benzo-
ates/naphthoates15 follows from well-understood considera-
tions of allylic strain18 leading to a dominant conformation
that is supported by NMR. A large number of examples
of chiral allylic benzoates were studied, which consistently
revealed large vicinal coupling constants (J ≈ 9 Hz) for
vicinal protons on the adjacent sp2-sp3 carbons that imply
the vinyl H is eclipsed by the allylic methine H.15,17 In turn,
this sets the torsional angle between the CdC bond and the
benzoate C-O vector and the relative helicity of the respec-
tive chromophoric transition dipoles. The allylic ECCD
method should be extendable to propargylic acylates; how-
ever, conformations of the latter are expected to exhibit subtle
differences (Figure 2a,b). In both double and triple carbon-
carbon bonds the π-π* transition dipoles are polarized along
the C-C direction. Unlike the planar CdC bonds in allylic
acylates, the radially symmetrical triple bond in a propargylic
acylate is essentially a free rotor. A consequence of this, as we
(13) Harada, N.; Nakanishi, K. Circular Dichroic Spectroscopy: Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill Valley,
CA, 1983; p 162.
(15) Gonnella, N. C.; Nakanishi, K.; Martin, V. S.; Sharpless, K. B.
J. Am. Chem. Soc. 1982, 104, 3775–3776.
(14) Presumably, for this reason, DMB diesters were chosen for the
dibenzoate assignment of 2 to move the bisignate split CE to longer
wavelengths (λ ∼310 nm) and diminish potential interference from diyne-
benzoate interactions (ref 7).
(16) Molinski, T. F.; Brzezinski, L. J.; Leahy, J. W. Tetrahedron: Asym-
metry 2002, 13, 1013–1016.
(17) Schneider, C.; Schreier, P.; Humpf, H. U. Chirality 1997, 9, 563–567.
(18) Johnson, F. Chem. Rev. 1968, 68, 375–413.
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FIGURE 2. Conformers of propargylic (R)-2-O-(20-naphthoate)
esters of terminal propargylic 1,2-glycols. (a, b) Idealized geometries
(two views) (cf. 6) and the predicted sign of Δε in the ECCD
spectrum. (R=CH2OAc, R0=alkenyl, alkynyl; Nph=20-naphthoyl;
shaded bar is 2-naphthyl (C10H11). (c, d) Two views of the energy
minimized model (MMFF, Spartan 08) (cf. 6; however, the chain is
truncated to a 3,5-diyne, R0=ethynyl,) showing a syn-clinal arrange-
ment of the conjugated diyne- and naphthoate plane with a positive
helicity (dihedral angle of C3-C2-C20-C60, θ=þ33.4°).
showed from molecular mechanics calculations (Spartan,
MMFF94, Figure 2c,d), is that the dominant conformer
places HR almost syn-coplanar with the conjugated ester
Ar(CdO)O group and an angle of (20° is subtended between
the synclinal arenecarboxylate plane and the acetylenic vector.
For propargylic esters of (R)-1 (Figure 2c), the respective tran-
sition dipoles have a calculated positive helicity (θ=þ33.4°),
where the dihedral θ is approximated by the dihedral angle
C3-C2-C20-C60 (C20, C60 are the ipso and para carbons of
the first benzenoid ring of the naphthoate). In turn, this
predicts that (R)-1 will exhibit a positive Δε in the CD
spectrum. Harada has described similar arguments and as-
signments of alk-1-yn-3-ols by ECCD after a two step-deriva-
tization: Sonogashira coupling with a terminal acetylene with
an aryl iodide followed by esterification of the secondary
alcohol to the corresponding arene-yne benzoate and mea-
surement of CD spectra.19 The sign of the resulting split CEs
conform to the rule; however, the major limitation of the
method is its applicability to terminal acetylenes only.
An advantage of allylic O-naphthoates over O-benzoates
is the very intense Cotton effects (A values of up to ∼20017)
associated with the 1Bb transition along the skew long axis of
the naphthalene ring system, closely parallel with the C-O
ester bond, which is better suited for subnanomole CD mea-
surements. Unlike vicinal propargylic dibenzoates, interpre-
tation of CD in propargylic naphthoates benefits from the
simplicity of only two participant chromophores in exciton
coupling.
FIGURE 3. CD spectra (EtOH, 23 °C) of (a) 5 and 16 and (b) 6.
SCHEME 1. Derivatization of Polyacetylenic Alcohols to
Chromophoric Derivatives 5, 6, and 16
In order to assess the relative merits of the dibenzoate
method and the propargylic O-naphthoate method, we chose
to determine the configuration of 1 and 4a using both
approaches. Diplyne C (1) was acylated (Scheme 1) (N-(4-
dimethylaminobenzoyl)imidazole, DBU, CH3CN, 25 °C) to
provide the DMB diester 5 (∼34 μg),20 which was purified by
HPLC. The CD spectrum of 5 (Figure 3a) exhibited a
pronounced ECCD [λ 301 nm (Δε þ22.4), 326 (-21.5)] of
the same sign and similar magnitude observed for the DMB
diesters of 3,7 indicating the same C2 configuration. Indepen-
dently, 1 was converted to a C2-O-(20-naphthoate) monoester
6 as follows (Scheme 1): the primary hydroxyl group was
selectively acetylated in the presence of a lipase (Novozyme
(19) Naito, J.; Yamamoto, Y.; Akagi, M.; Sekiguchi, S.; Watanabe, M.;
Harada, N. Monatsh. Chem. 2005, 136, 411–445.
(20) The yields of 5 and 6 were estimated by UV-vis assuming λmax 313
nm (ε = 56,000) and λmax 239 (ε = 65,000), respectively.
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SCHEME 2. Preparation of En-yne and Di-yne Propargylic 2-Naphthoate Esters (S)-7 and (S)-8a
aNote: CIP priorities of 7-15 differ from those of 1-4.
435, vinyl acetate, CH3CN), and the free secondary OH group
was subsequently acylated, as above, to give diester 6 (∼24 μg)
after HPLC purification. The CD (EtOH) spectrum of 6
(Figure 3b) revealed a positive long wavelength band CE [λ
238 nm (Δε þ25.3)]21 that was interpreted as arising from a
positive helicity between the diyne and naphthoate chromo-
phores.24 The negative component of the ECCD was observed
clearly when the CD spectrum of 6 was recorded in CH3CN
[λ 238 nm (Δε þ29.7), 188 nm (-50.5)]. Both methods lead to
the same answer, and we assign the (R) configuration to 1, 5,
and 6.
secondary alcohol (S)-14, corresponding to (S)-7 and lacking
a dichromophoric exciton coupling, shows essentially only
baseline CD (Figure 3). Because the π-π* transition of the
conjugated diyne occurs at lower wavelenths [λ 214 nm
(ε 37,300), Figure 5],1 the short-wavelength component of
the exciton couplet of (S)-8 [λ 188 nm (Δε -37.8)] is at the
edge of instrument detection limits; however, in (S)-7 this
high energy component reveals itself readily [λ 225 nm
(Δε -24.6)] due to the presence of a red-shifted extended
en-yne chromophore.24 Nevertheless, as noted by Nakanishi
To validate the outcome of assignment of 1 by the
propargylic acyclate ECCD method, CD spectra were ob-
tained of 2-naphthoyl esters (S)-7 and (S)-8 prepared from
the known (S)-9 propargylic alcohol22 (note the change in
CIP priorities) as shown in Scheme 2. Desilylation of (S)-9 to
alcohol (S)-10 was followed by protection as the acetate ester
and Sonogashira coupling with (E)-iododecene in the pre-
sence of bis(triphenylphosphine)palladium(II) dichloride
(11) and CuI to give ene-yne acetate ester 13. Exchange of
the O-acyl groups (acetyl for 2-naphthoyl) through the
intermediary alcohol 14 was achieved in two steps (NH3,
MeOH; 2-naphthoic acid, EDC, DMAP, 76% over two
steps) to provide en-yne model ester (S)-7.23 In parallel,
(S)-10 was acylated to the naphthoate ester 15, which was
desilylated (TBAF, 86%) to give the propargylic O-naptho-
ate 15a and transformed into the diyne naphthoate (S)-8
(CuCl, NH2OH HCl, n-BuNH2, 1-bromoheptyne).
3
The CD spectra of (S)-7 and (S)-8 (CH3CN) showed
strong bisignate CEs [(S)-7 λ 188 nm (Δε -37.8); (S)-8 225
(-24.6), 242 (þ35.3)] (Figures 4 and 5). Note that the
(21) Note that the remote C10-C14 diyne chromophore is “CD silent”.
(22) (a) Skepper, C. K. Marine Derived Heterocycles. Ph.D. Dissertation,
University of California, San Diego, La Jolla, CA, November, 2009. (b)
Supporting information of Skepper, C. K.; MacMillan, J. B.; Zhou, G. X.;
Masuno, M. N.; Molinski, T. F. J. Am. Chem. Soc. 2007, 129, 4150–4151.
(23) The configuration of the secondary alcohol 14 was verified using the
modified Mosher’s method.11
(24) Generally, the short-wavelength component of the ECCD is ob-
scured (λ < 200 nm) in most allylic benzoates with mono- and disubstituted
double bonds. However, this component is red-shifted and may be obser-
vable at λ ∼180-200 nm in allylic 2-naphthoates (see ref 16) with tri- and
tetra-alkyl substituted double bonds and, of course, in allylic acylates of
conjugated dienes (λ>220 nm).
FIGURE 4. CD spectra at 25 °C of (a) (S)-7 in CH3CN, (b) (S)-7 in
MeOH, and (c) (S)-14 in hexane.
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FIGURE 6. CD spectrum of (S)-15a (CH3CN, 25 °C).
posed ene-diyne benzoate ECCD interaction (cf. 6, Figure 3b)
are of the same sign (positive)25 and reinforce at ∼λ 302 nm.26
Therefore, the configurations of 4a and 16, are revealed to be
(R). Assuming congeneric 4b,c are of the same configuration as
4a, it would appear the biosynthesis of propargylic diols 1-4
from sponges across two different oceans conserves the C2
stereochemistry.27
Diplyne C (1) exhibited cytotoxicity against cultured hu-
man colon tumor cells (HCT-116; LD50 3.6 μg/mL); how-
ever, insufficient amounts of 4a-c were available for
biological evaluation.
FIGURE 5. CD spectra for (S)-8 in (a) CH3CN, (b) MeOH (λ <
200 nm, obscured by solvent end-absorption), and (c) hexane at
25 °C.
and co-workers,15 only the sign of the long-wavelength
component is necessary and sufficient to assign allylic
benzoates which also applies for propargylic naphthoates.
Due to the stronger oscillator strength of the 1Bb transition,
even acyclic nonconjugated propargylic O-naphthoates
should be amenable to chiroptical analysis, unlike the corre-
sponding O-benzoates that show only weak or nondetectable
ECCD from the shorter wavelength acetylene chromophore
(<λ 190 nm).19 This is demonstrated in the CD spectrum of
15a (Figure 6), which displays a weaker yet still prominent
positive component [λ 240 nm (Δε þ4.6)] of the biphasic
Cotton effect.
Since both the dibenzoate and the propargylic naphthoate
ECCD methods lead to the same configurational assignment
in 1, it can be concluded that both can be reliably used for
stereoassignments of other long-chain propargylic alcohols
under appropriate conditions, although the one-step di-
benzoate method is attractive for its operational simplicity.
A simple extension of the glycol benzoate method was
applied to faulkneryne A (4a). Conversion of 4a, by the
aforementioned procedure, gave the di-DMB ester 16 which
showed an CD spectrum (EtOH, Figure 3a) with a negative
split CE [λ 302 nm (Δε þ17.4), 327 (-9.0)], similar to that
of 5. As with 5, it is fortuitous that both the short-wavelength
component of the dibenzoate ECCD of 16 and the super-
Conclusions
The complete structures of new long-chain polyacetylenic
diols, faulknerynes A-C (4a-c), were elucidated with the
aid of microcryoprobe NMR spectroscopy. The configura-
tions of 4a and the known compound diplyne C (1) were
assigned the (R) configuration by two different microscale
ECCD methods that converged upon the same answer. The
ECCD spectrum of propargylic en-yne naphthoates are nicely
suited for observation of both halves of the exciton couplets for
microscale configurational analysis of polyacetylenic alcohols
without the necessity for additional synthetic elaboration of the
acetylenic chromophore.
Experimental Section
General Experimental Procedures. See Supporting Informa-
tion.
Animal Material. The sponge Diplastrella sp., Family
Spirastrellidae (Ridley & Dendy 1886) (08-05-026) was collected
on 31 May 2008 at Sweetings Cay, Bahamas (26° 34.0800 N, 77°
53.4070 W) at a depth of -19 m using scuba and kept in EtOH
at -20 °C until extraction. The tissue was pale pink to red in life,
thinly encrusting on coral in small patches. Microscopic spicule
analysis (SEM) showed tylostyles with rounded heads and
microscleres with a preponderance of diplasters over spirasters
(25) In fact, this is evident from the relative asymmetry of the split CEs in
16 and 5 due to differences of superposed paired propargylic O-DMB-alkyne
ECCDs arising from the diyne π-π* transition in 5 versus the red-shifted
7-en-3,5-diyne π-π* transition in 16.
(26) Insufficient sample of 4a-c remained to apply the ene-diyne
naphthoate ECCD method.
(27) The unnatural (S) enantiomer of siphonodiol 2 is depicted in ref 1.
J. Org. Chem. Vol. 76, No. 3, 2011 899

Ko et al.
JOCArticle
as is typical for Diplastrella.28 A second sponge, Diplastrella sp.
(08-07-038), was collected in the same vicinity at similar depths.
Voucher samples for all animal material are archived at UCSD.
Extraction and Isolation. The EtOH extract of the sponge
Diplastrella sp. (08-05-026, wet sponge vol ∼2-5 mL) was con-
centrated and redissolved in MeOH. The MeOH extract was
sequentially extracted with solvents of increasing polarity with
adjustment of the H2O content at each stage and removal of
solvent from the organic layer: hexane, 0% H2O (5.2 mg), CHCl3,
40% H2O (12.4 mg) The aqueous phase was concentrated
under reduced pressure to remove MeOH and then extracted with
n-BuOH (15.6 mg); the latter was shown to contain diplyne E.1
Finally, the aqueous phase was concentrated under reduced
pressure (106.9 mg). The CHCl3-soluble phase was separated
by flash chromatography (SiO2, 1-100% MeOH-CHCl3 step
gradient) to give a diyne-containing fraction (2.1 mg), which was
further separated by RP HPLC (C18, 5 μm, 10 mm ꢀ 250 mm,
CH3CN-H2O, 2 mL/min) to give diplyne C1 (1, 500 μg) and
faulkneryne A (4a, 400 μg).
The EtOH extract of a second specimen of Diplastrella sp.
(08-07-038; wet sponge vol ∼2-5 mL) was concentrated and
redissolved in MeOH. The MeOH extract was sequentially
extracted with solvents of increasing polarity with adjustment
of the H2O content at each stage and removal of solvent from
the organic layer: hexane, 0% H2O (9.8 mg), CHCl3, 40% H2O
(19.5 mg) The aqueous phase was concentrated under reduced
pressure to remove MeOH and then partitioned against with
n-BuOH, which was concentrated to give the n-BuOH-soluble
fraction (13.3 mg). Finally, the aqueous phase was concentrated
under reduced pressure (73.9 mg). The CHCl3-soluble phase was
separated by RP HPLC (C18, 5 μm, 10 mm ꢀ 250 mm, CH3CN-
H2O) to give faulknerynes A-C (4a, 1 mg), (4b, 4.7 μg29), (4c,
14.1 μg29), diplyne A1 (3 mg), and diplyne E (2, 100 μg).1
Faulkneryne A (4a). Colorless glass; UV (MeOH) λmax 271
nm. 1H NMR, 13C NMR, see Table 1. HRMS m/z 347.0633 [M þ
Na]þ calcd for C16H2179BrNaO2 347.0623.
crude product. The latter material was redissolved in anhydrous
CH3CN (100 μL) and treated with N-(20-naphthoyl)imidazole30
(137 μg, 0.61 μmol) and DBU (93 μg, 0.61 μmol) and allowed to
stir at rt for 6 h. The reaction mixture was concentrated under
reduced pressure to give the crude product, which was purified
by normal phase HPLC (9:91 EtOAc in hexane) to give pure (R)-
6. CD (EtOH) λ 238 nm (Δε þ25.3, see Figure 2b. CD (CH3CN)
λ 238 nm (Δε þ29.7), 188 nm (-50.5). 1H NMR (600 MHz) 8.63
(s, 1H), 8.05 (d, 1H, J = 8.5 Hz), 7.97 (d, 1H, J = 8.1 Hz), 7.90
(d, 1H, J = 8.6 Hz), 7.89 (d, 1H, J = 8.1 Hz), 7.62 (m, 1H), 7.56
(m, 1H), 6.15 (dt, 1H, J = 13.8, 7.3 Hz), 5.99 (d, 1H, J = 13.5
Hz), 5.95 (dd, 1H, J = 7.2, 4.1 Hz), 4.47 (dd, 1H, J = 11.8, 4.0
Hz), 4.44 (dd, 1H, J = 11.8, 7.4 Hz), 2.27 (t, 2H, J = 7.0 Hz),
2.08 (s, 3H), 2.02 (q, 2H, J = 7.2 Hz). HRMS m/z 545.1298 [M þ
Na]þ calcd for C29H3179BrNaO4 545.1303.
(S)-1-Cyclohexylprop-2-ynyl Acetate (12). (S)-9 (52.4 mg, 0.21
mmol) was dissolved in THF (2 mL), and TBAF (0.25 mL, 1 M
in THF) was added dropwise at 0 °C. The mixture was stirred
at 0 °C for 30 min, and the solvent was removed under reduced
pressure to give a crude product that was immediately subject to
acetylation (Ac2O, 30 μL) in anhydrous pyridine (500 μL) at
25 °C for 48 h. The volatiles were removed and the mixture
separated (silica cartridge, 3:7 hexane-CH2Cl2) to provide (-)-
12 (14.1 mg, 38%). [R]24 -66.6 (c 0.011, CHCl3) [lit. [R]24
D
D
-65.5 (c 1.5, CHCl3)].22 MS and NMR data were identical with
literature values.
En-yne (13). A mixture of 1-iododecene (43 mg, 0.12 mmol),
(Ph3P)2PdCl2 (5.5 mg, 7.8 μmol), CuI (6.0 mg, 0.032 mmol) and
Et3N (2.5 mL) were stirred at rt for 30 min. This solution was
then added dropwise to a solution of alkyne 12 in Et3N (1 mL)
and the mixture reaction stirred at rt for 1.5 h. The volatiles were
removed and the residue separated by flash chromatography
(silica cartridge, 3:97 EtOAc-hexanes) to yield acetoxy en-yne
13 as an oil (21.5 mg, 79%). [R]24D -88.0 (c 0.004, CHCl3); IR
(KBr) vmax 2925, 2854, 1743, 1451, 1369, 1229, 1017, 977, 955
1
cm-1; H NMR (400 MHz, CDCl3) 0.86 (t, J = 7.2 Hz, 3H),
Faulkneryne B (4b). Colorless glass: UV (MeOH) λmax 273 nm
1H NMR, see Table S1, Supporting Information. HREIMS: the
compound failed to give pseudomolecular ions.
1.04-1.36 (m, 21H), 1.61-1.84 (m, 6H), 2.06 (s, 3H), 2.06 (dq,
J = 1.6, 7.2 Hz, 5H), 5.30 (dd, J = 2.0, 6.4 Hz, 1H), 5.45 (dq, J =
2.0, 15.6 Hz, 1H), 6.15 (dt, J = 7.2, 15.6 Hz, 1H); 13C NMR (100
MHz, CDCl3) δ 14.1 (CH3), 21.1 (CH3), 22.7 (CH2), 25.7 (CH2),
25.8 (CH2), 26.2 (CH2), 28.1 (CH2), 28.6 (CH2), 29.1 (CH2), 29.3
(CH2), 29.4 (CH2), 29.5 (CH2), 29.6 (CH2), 31.9 (CH2), 33.1
(CH2), 42.0 (CH), 68.9 (CH), 74.0 (C), 83.9 (C), 84.7 (C), 108.6
(CH), 146.0 (CH), 170.2 (C). HREIMS m/z 346.2869 [M]þ,
calcd for C23H38O2 346.2866.
En-yn-ol (14). A solution of 13 in MeOH (100 μL) was treated
with anhydrous NH3 (2 M in MeOH, 300 μL), stirred overnight,
and then concentrated under a stream of N2. The crude mixture
was separated by chromatography (silica cartridge, 95:5
hexanes-EtOAc) to yield alcohol 14 as an oil (4.8 mg, 91%).
[R]24D þ5.3 (c0.02, CHCl3);1H NMR (400 MHz, CDCl3) δ0.86 (t,
J = 6.8 Hz, 3H), 1.02-1.38 (m, 21H), 1.49-1.86 (m, 7H), 2.07 (dq,
J = 1.2, 7.2 Hz, 5H), 4.22 (m, 1H), 5.47 (dq, J = 1.6, 15.6 Hz, 1H),
6.13 (dt, J = 7.2, 15.6 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
14.1 (CH3), 22.7 (CH3), 25.9 (CH2), 26.3 (CH2), 28.2 (CH2), 28.5
(CH2), 28.6, 29.1 (CH2), 29.3 (CH2), 29.4 (CH2), 29.5 (CH2), 29.6
(CH2), 29.7 (CH2), 31.9 (CH2), 33.1 (CH2), 44.3 (CH), 67.7 (CH),
84.5 (C), 87.5 (C), 108.8 (CH), 145.4 (CH). The configuration of
the carbinol center in 14 was confirmed by the modified Moshers’s
method.11 (R)-MTPA ester of compound 14: compound 14 (0.8 mg,
2.6 μmol) was dissolved DCM-pyridine (1:1 200 μL). (S)-MTPA-
Cl (5 μL, 0.27 mmol) was added and the mixture stirred for 1.5 h.
The volatiles were removed and the crude product separated by
chromatography (silica, 95:5 hexanes-EtOAc) to yield the (R)-
MTPA ester of compound 14 (1.3 mg, 95%). 1H NMR (400 MHz,
Faulkneryne C (4c). Colorless glass: UV (MeOH) λmax 272 nm.
1H NMR, see Table S1, Supporting Information. HRMS m/z
343.0304 [M þ Na]þ calcd for C16H1779BrNaO2 343.0309
Preparation of 1,2-O-Bis(40-dimethylaminobenzoyl)diplyne C
(5). A solution of 1 (100 μg, 0.305 μmol) in CH3CN (100 μL) was
treated with N-(4-dimethylaminobenzoyl)imidazole (197 μg,
0.917 μmol) and DBU (140 μg, 0.917 μmol) and the mixture
allowed to stir for 6 h, then concentrated to give a crude product,
which was purified by normal phase HPLC (17:83 EtOAc/
hexane) to give pure 5. UV-vis (EtOH), λmax 313 nm. CD
(EtOH) λ 301 nm (Δε þ22.4), 326 (-21.5); 1H NMR (600 MHz,
CDCl3) 7.91 (d, 2H, J = 8.8 Hz), 7.88 (d, 2H, J = 8.8 Hz), 6.62
(d, 2H, J = 8.8 Hz), 6.60 (d, 2H, J = 8.8 Hz), 6.15 (dt, 1H, J =
13.9, 7.3 Hz), 5.99 (d, 1H, J = 13.7 Hz), 5.96 (m, 1H), 4.56 (m,
2H), 3.03 (s, 6H), 3.02 (s, 6H), 2.25 (t, 2H, J = 7.1 Hz), 2.01 (q,
2H, J = 7.4 Hz); HRMS m/z 621.2330 [M þ H]þ calcd for
C34H4279BrN2O4 621.2328.
Preparation of (R)-1-O-Acetyl-(20-O-naphthoyl)diplyne C (6).
A solution of 1 (100 μg, 0.305 μmol) in CH3CN (100 μL) was
treated with Novozyme 435 (7 mg) and vinyl acetate (263 μg,
3.05 μmol, 10 equiv), and the mixture stirred at 40-45 °C. After
2 h the reaction was quenched, the enzyme was filtered off, and
the filtrate was concentrated under reduced pressure to give the
€
(28) Rutzler, K., Family Spirastrellidae Ridley & Dendy, 1886. In Sys-
tema Porifera; Hooper, J., Van Soest, R. W. M., Eds.; Kluwer/Plenum: New
York, 1994; Vol. 1.
(29) Submilligram yields were determined using quantitation by solvent
13C satellite (QSCS). Dalisay, D. S.; Molinski, T. F. J. Nat. Prod. 2009, 72,
739–744.
(30) Ikemoto, N.; Lo, L.-C.; Nakanishi, K. Angew. Chem., Int. Ed. 1992,
31, 890–891.
900 J. Org. Chem. Vol. 76, No. 3, 2011

Ko et al.
JOCArticle
CDCl3) δ 0.86 (t, J = 6.8 Hz, 3H), 1.05-1.36 (m, 21H), 1.63-1.74
(m, 5H),1.81 (brd, J = 12.4 Hz, 1H), 2.08 (dq, J = 1.2, 6.8 Hz,
2H), 3.54 (s, 3H), 5.42 (d, J = 5.6 Hz, 1H), 5.43 (dq, J = 1.6, 15.6
Hz, 1H), 6.12 (dt, J = 6.8, 15.6 Hz, 1H). (S)-MTPA ester of
compound 14: The same procedure was applied to compound 14
(1.0 mg, 3.3 μmol) with (S)-MTPA-Cl (5 μL, 0.27 mmol) to yield
the corresponding (S)-MTPA ester (1.4 mg, 99%) yield. 1HNMR
(400 MHz, CDCl3) δ 0.86 (t, J = 6.8 Hz, 3H), 0.99-1.38 (m, 21H),
1.60-1.71 (m, 5H),1.71 (m, 1H), 2.09 (dq, J = 1.6, 7.2 Hz, 2H),
3.57 (d, J = 1.2 Hz, 3H), 5.42 (d, J = 5.6 Hz, 1H), 5.46 (dq, J=1.6,
16.0 Hz, 1H), 6.15 (dt, J = 6.8, 16.0 Hz, 1H).
(silica cartridge, 8:1, hexanes-diethyl ether) to give diyne (S)-
8 as an oil (8.2 mg, 80%). [R]23D þ58.8 (c 1.24, CHCl3); 1H NMR
(400 MHz, CDCl3) δ 8.60 (bs, 1H), 8.05 (dd, J = 8.4, 1.2 Hz,
1H), 7.95 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.55 (m,
2H), 5.55 (d, J = 6 Hz, 1H), 2.25 (t, J = 6.8 Hz, 2H), 1.67-1.99
(m, 6H), 1.50 (p, J = 7.6 Hz, 2H), 1.17-1.37 (m, 4H), 0.87 (t,
J = 7.2 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 165.6 (C),
135.6 (C), 132.4 (C), 131.3 (CH), 129.4 (CH), 128.4 (CH), 128.2
(CH), 127.8 (CH), 127.1 (C), 126.7 (CH), 125.3 (CH), 81.8 (C),
72.0 (C), 71.2 (C), 69.2 (CH), 64.5 (C), 42.2 (CH), 31.0 (CH2),
28.6 (CH2), 28.3 (CH2), 27.8 (CH2), 26.1 (CH2), 25.8 (CH2), 25.7
(CH2), 22.1 (CH2), 19.2 (CH2), 13.9 (CH3). HREIMS m/z
386.2238 [Mþ]þ calcd for C37H30O2, 386.2240.
Naphthoate Ester (S)-7. A solution of enyn-ol 14 (2 mg,
6.57 μmol) in CH2Cl2 (200 μL) was treated with 2-naphthoic
acid (2.8 mg, 16.4 μmol), EDC (3.0 mg, 19.7 μmol), and a small
crystal of DMAP. The resulting mixture was stirred for 48 h,
concentrated, and separated by flash chromatography (SiO2) to
Preparation of (R)-1,2-O-Bis(40-dimethylaminobenzoyl)faul-
kneryne (16). Using the procedure described above for 1,
faulkneryne A (3, 100 μg, 0.307 μmol) was converted into the
corresponding DMB diester (R)-16 (21 μg). UV-vis (EtOH),
afford naphthoate ester (S)-7 (2.4 mg, 82%). [R]24 þ20.2
D
(c 0.94, CHCl3); 1H NMR (400 MHz, CDCl3) δ 8.61 (bs, 1H),
8.07 (dd, 8.4, 1.2 Hz, 1H), 7.95 (d, 8.0 Hz, 1H), 7.86 (d, 8.4 Hz,
2H), 7.55 (m, 2H), 6.17 (dt, 16.0, 7.2 Hz, 1H), 5.62 (dd, 5.6, 1.2
Hz, 1H), 5.49 (dq, 16.0, 0.8 Hz, 1H), 2.07 (dq, 6.8, 1.6 Hz, 2H),
1.66-1.98 (m, 4H), 1.22 (m, 20H); HREIMS m/z 458.3176 [Mþ]
calcd for C32H42O2, 458.3179.
λ
max 312 nm. CD (EtOH) λ 302 nm (Δε þ17.4), 327 (-9.0); 1H
NMR (600 MHz, CDCl3) 7.92 (d, 2H, J = 8.8 Hz), 7.89 (d, 2H,
J = 9.0 Hz), 6.63 (d, 2H, J = 9.6 Hz), 6.60 (d, 2H, J = 9.4 Hz),
6.15 (dt, 1H, J = 13.3, 7.5 Hz), 6.09 (dt, 1H, J = 10.8, 7.7 Hz),
6.02 (m, 1H), 5.98 (m, 2H), 5.48 (m, 1H), 4.60 (m, 1H), 2.35(m,
2H), 2.03(m, 2H). HRMS m/z 619.2141 [M þ H]þ calcd for
C34H3979BrN2O4, 619.2172.
Naphthoate Ester (15). Alkynol (S)-9 (10.4 mg, 0.041 mmol)
was converted into naphthoate ester 15 (13.5 mg, 82%) using the
Molecular Modeling. An analogue of compound 5 (truncated
to the 3,5-diyne) was mimimized using molecular mechanics
(MMFF94, Spartan 08, gas phase). Energies were calculated
(semiempirical, PM3) and Monte Carlo conformational search-
ing applied to obtain the lowest 25 models ranging in energy
from -1.06 to 25 kcal mol-1. For the three most stable con-
formers, see Figure 1d-f. An analogue of dibenzoate ester 6
(truncated to the 3,5-diyne) was minimized using semiempirical
methods (PM3, Spartan 08) and the 25 lowest energy confor-
mers determined by Monte Carlo methods. See Figure 2.
Cytotoxicity Assay. Cytotoxicity of 1 against cultured human
colon tumor cells (HCT-116) incubated under 5% CO2 in the
presence and absence of compound, followed by colorimetric
measurement of growth inhibition by the MTS method using a
microplate reader as described elsewhere.2 Diplyne C (1) ex-
hibited an LC50 of 3.6 μg/mL (etoposide was used as a positive
control).
1
procedure described above. [R]24 þ12.6 (c 1.93, CHCl3); H
D
NMR (400 MHz, CDCl3) δ 8.61 (bs, 1H), 8.07 (dd, J = 8.4, 1.2
Hz, 1H), 7.95 (d, J = 8.0 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.55
(m, 2H), 5.56 (d, J = 6.0 Hz, 1H), 1.67-1.99 (m, 6H), 1.13-1.32
(m, 4H), 0.98 (t, J = 8.0 Hz, 9H), 0.59 (q, J = 8.0 Hz, 6H); 13
C
NMR (100 MHz, CDCl3) δ 165.7 (C), 135.5 (C), 132.4(C), 131.2
(CH), 129.4 (CH), 128.3 (CH), 128.1 (CH), 127.7 (CH), 127.4
(C), 126.6 (CH), 125.4 (CH), 103.0 (C), 88.6 (C), 69.3 (CH), 42.1
(CH), 28.7 (CH2), 28.1 (CH2), 26.3 (CH2), 25.8 (CH2), 25.7
(CH2), 7.8 (CH3), 4.2 (CH2); HREIMS m/z 406.2330 [Mþ] calcd
for C26H34O2Si, 406.2323.
Diyne Naphthoate Ester (S)-(8). Naphthoate ester 15
(13.5 mg, 0.033 mmol) was dissolved in THF (1 mL), and the
solution was cooled to 0 °C. TBAF (1 M in THF, 33.2 μL) was
added dropwise, and the mixture was stirred for 10 min before
removal of the volatiles. The residue was purified by flash
chromatography (SiO2, 9:1 hexanes-ether) to afford essentially
pure propargyl O-naphthoate 15a (8.1 mg, 86%). [R]23D -12.8
(c 1.19, CHCl3); FTIR (KBr) vmax 3298, 2929, 2853, 1719, 1281,
1225, 1195, 1129, 1088, 973, 777, 761 cm-1; 1H NMR (400 MHz,
CDCl3) δ 8.61 (bs, 1H), 8.07 (dd, J = 8.4, 1.2 Hz, 1H), 7.95 (d,
J = 8.0 Hz, 1H), 7.86 (d, J = 8.4 Hz, 2H), 7.55 (m, 2H), 5.51 (dd,
J = 6.0, 2.4 Hz, 1H), 2.48 (d, J = 2.4 Hz, 1H), 1.68-2.00 (m,
6H), 1.18-1.32 (m, 4H); 13C NMR (100 MHz, CDCl3) δ 165.
(C), 135.6 (C), 132.4 (C), 131.3 (CH), 129.4 (CH), 128.4 (CH),
128.2 (CH), 127.8 (CH), 127.1 (C), 126.7 (CH), 125.3 (CH), 80.3
(C), 74.3 (CH), 68.6 (CH), 41.8 (CH), 28.5 (CH2), 28.2 (CH2),
26.2 (CH2), 25.8 (CH2), 25.7 (CH2).
Acknowledgment. We thank C. K. Skepper for assistance
sponge collection and preparation of S-(þ)-9. We’re grateful
to S. Zea and M-K. Harper (University of Utah) for helpful
discussions on sponge taxonomy, J. Siegel (University of
€
Zurich) for stereochemical advice, J. R. Pawlik (University
of North Carolina, Wilmington), and the captain and crew of
the RV Seward Johnson for logistical support during collect-
ing expeditions. The NSF Biological Oceanography Pro-
gram (OCE-0095724, 0550468 to J.R.P).) is acknowledged
for ship time. The 500 MHz NMR spectrometers were
purchased with a grant from the NSF (CRIF, CHE0741968).
This work was supported by grants from NIH (CA122256
and AI039987 to T.F.M.) and a Ruth L. Kirschstein Na-
tional Research Service Award NIH/NCI (T32 CA009523 to
B.I.M).
A mixture of 15a (7.8 mg, 27 μmol), CuCl (0.5 mg, 5.3 μmol),
and NH2OH HCl (2.8 mg, 40 μmol) was suspended in MeOH
3
(150 μL) at 0 °C under N2 and treated dropwise with neat
n-butylamine (250 μL). The mixture was stirred for 10 min and
treated dropwise with a solution of 1-bromoheptyne (4.6 mg,
26.7 μmol) in MeOH (100 μL). The mixture was stirred at 0 °C
for 1 h and then poured into ice-water (2 mL) before acidifica-
tion with 5% H2SO4 and extraction with ether (3 ꢀ 4 mL). The
combined ether extracts were washed with brine, dried with
anhydrous MgSO4, and separated by flash chromatography
Supporting Information Available: 1H, 13C NMR and 2D
1
NMR spectra of 3, and H, 13C NMR of 4 and all synthetic
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
J. Org. Chem. Vol. 76, No. 3, 2011 901