Journal of Natural Products
NOTE
Biospin, Inc.) operating at 100 MHz for 13C. One-dimensional high-
resolution 13C NMR spectra for pahayokolide B (2) were acquired in
50:50 CD3OD/D2O on a Bruker Avance II 700 MHz spectrometer
equipped with a TCI cryoprobe operating at 176 MHz for 13C. All data
were collected at a temperature of 298 K. Chemical shifts were referenced
to the deuterium CD3 lock reference at 3.30 ppm using an absolute
referencing scheme based on nuclear gyromagnetic constant ratios; this
gives the center peak of the 13C CD3OD septet a chemical shift of
47.603 ppm. The NMR data were processed with Topspin software.
All mass spectra were acquired on a single-quadrupole mass spectro-
meter (ThermoQuest Finnigan Navigator) in ESI negative ion mode.
High-performance liquid chromatography (HPLC) was performed
using a Thermo Finnigan Spectra System HPLC (model P4000 pump;
model AS3000 autosampler; model UV6000LP PDA UV detector)
with an Apollo C18 (250 ꢀ 4.6 mm i.d., 5 μm, Alltech) column. Stable
isotope precursors were purchased from Cambridge Isotope Laboratories.
Culture of Lyngbya sp. Lyngbya sp. strain 15-2 was maintained
in 2 L cultures in BG11 medium, buffered with 2-(N-morpholino)-
ethanesulfonic acid buffer (2.6 mM) at pH 7.2. Cultures were supple-
mented with 150 mg of 13C-labeled L-leucine (50 mg each on days 30,
34, and 38) or 1.3 g of 13C-labeled sodium acetate (430 mg each on days
30, 34, and 38). Cultures were harvested after six weeks of growth.
Pahayokolides A (1) and B (2) were isolated from the biomass as
previously described.5
’ ASSOCIATED CONTENT
S
Supporting Information. Marfey’s analysis of pahayokolide
b
A and 13C NMR spectra for natural abundance pahayokolides A and
B, 13C-enriched pahayokolide A [1-13C]leucine, [1,2-13C]leucine,
and 13C-enriched pahayokolide B, [1-13C]acetate. This material is
’ AUTHOR INFORMATION
Corresponding Author
*Tel: þ1-305-348-6682. Fax: þ1-305-348-3772. E-mail: reink@
fiu.edu.
’ ACKNOWLEDGMENT
This work was supported by the National Institute of Envir-
onmental Health Sciences (NIEHS) Grant S11 ES11181. We
acknowledge the support of the Hollings Marine Laboratory
NMR Facility. Commercial equipment or materials are identified
in this paper to specify adequately the experimental procedure.
Such identification does not imply recommendation or endorse-
ment by NIST, nor does it imply that the materials or equipment
identified are necessarily the best available for the purpose.
Preparation of FDLA Derivatives. To 40 μL of a 2 μM solution
of each amino acid standard was added 20 μL of 1 M sodium bicarbonate
and 80 μL of 1% (w/v) L- or D-FDLA in acetone as previously
described.12,13 After incubating at 40 °C for 60 min the reactions were
quenched by the addition of 10 μL of 2 M HCl and stored at 4 °C. As
N-Me-D-Leu was not available, the D-FDLA derivative of N-Me-L-Leu
was used as a standard in lieu of its enantiomer, the L-FDLA-N-Me-D-Leu
derivative. A 100 μg amount of pahayokolide A was hydrolyzed at 110 °C
for 14 h with 500 μL of 4 M HCl. This solution was divided into two
portions and dried under a stream of N2. Each portion was derivatized
with either L- or D-FDLA as described above and stored at 4 °C.
HPLC/PDA Conditions. The mobile phase used for the separation
of the L- and D,L-FDLA derivatives of pahayokolide A was CH3CN/0.01
M TFA, step gradient [4:6 for 24 min; ramp to 1:1 from 24 to 34 min;
7:3 after 34 min] at a flow rate of 0.4 mL/min. The FDLA derivatives
were detected with a PDA UV detector at 340 nm. The retention times
for the L-FDLA derivatives of N-Me-L-Leu and L-homoPhe were very
close, at 47.3 and 47.7 min, respectively. In separate experiments, the
L-FDLA-derivatized pahayokolide A hydrolysate was spiked with the
L-FDLA derivatives of N-Me-L-Leu or L-homoPhe, confirmingthe presence
of N-Me-L-Leu and the absence of L-homoPhe. Similarly, the retention
times for the L-FDLA derivatives of D-Phe and N-Me-D-Leu were very
close, at 50.0 and 50.2 min, respectively. In separate experiments, the
L-FDLA-derivatized pahayokolide A hydrolysate was spiked with the
L-FDLA-D-Phe derivative and the D-FDLA-N-Me-L-Leu derivative, con-
firming the presence of N-Me-D-Leu and the absence of D-Phe. When
pahayokolide A was hydrolyzed in 6 M HCl, the L:D ratio of
N-Me-Leu was 2.75:1. However, hydrolysis of pahayokolide A in 4 M
HCl resulted in a L:D ratio of 5.5:1. We conclude that the N-Me-D-Leu
arose from epimerization of N-Me-L-Leu during hydrolysis. A similar
observation was made for the tychonamides.8 The FDLA derivative of
Athmu was observed onlywhenthe hydrolysis was performed in 4 M HCl.
When the hydrolysis was carried out in 6 M HCl, a derivative having
a molecular ion at m/z 668 was observed, corresponding to a loss of water
fromthe FDLA-Athmuderivative. Eitherone of thealcohols isdehydrated
to an alkene or the lactone is formed under these conditions.
’ REFERENCES
(1) Gademann, K.; Portmann, C. Curr. Org. Chem. 2008, 12,
326–341.
(2) Sivonen, K.; B€orner, T. The Cyanobacteria: Molecular Biology,
Genomics and Evolution; Herrero, A. Flores, E., Ed.; Caister Academic
Press, 2008; Chapter 7, pp 159ꢁ197.
(3) Tan, L. T. Phytochemistry 2007, 68, 954–979.
(4) Banker, R.; Teltsch, B.; Sukenik, A.; Carmeli, S. J. Nat. Prod.
2000, 63, 387–389.
(5) An, T.; Kumar, T. K. S.; Wang, M.; Liu, L.; Lay, J. O., Jr.;
Liyanage, R.; Berry, J.; Gantar, M.; Marks, V.; Gawley, R. E.; Rein, K. S.
J. Nat. Prod. 2007, 70, 730–735.
(6) Berry, J. P.; Gantar, M.; Gawley, R. E.; Rein, K. S. In Harmful Algae
2002, Xth International Conference; Steidinger, K. A., Landsberg, J. H.,
Tomas, C. R., Vargo, G. A., Eds.; Florida Fish and Wildlife Conserva-
tion Commission, Florida Institute of Oceanography, Intergovernmental
Oceanographic Commission of UNESCO: St. Petersburg, FL, 2004;
Vol. 1, pp 192ꢁ194.
(7) Berry, J. P.; Gantar, M.; Gawley, R. E.; Wang, M.; Rein, K. S.
Comp. Biochem. Physiol., C: Toxicol. Pharmacol. 2004, 139C, 231–238.
(8) Mehner, C.; Mueller, D.; Krick, A.; Kehraus, S.; L€oeser, R.;
G€uetschow, M.; Maier, A.; Fiebig, H.-F.; Brun, R.; K€oenig, G. M. Eur.
J. Org. Chem. 2008, 10, 1732–1739.
(9) Pergament, I.; Carmeli, S. Tetrahedron Lett. 1994, 35, 8473–8476.
(10) Le~ao, P. N.; Pereira, A. R.; Liu, W. T.; Ng, J.; Pevzner, P. A.;
Dorrestein, P. C.; K€onig, G. M.; Vasconcelos, V. M.; Gerwick, W. H.
Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 11183–11188.
(11) Zainuddin, E. N.; Jansen, R.; Nimtz, M.; Wray, V.; Preisitsch,
M.; Lalk, M.; Mundt, S. J. Nat. Prod. 2009, 72, 1373–1378.
(12) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem.
1997, 69, 5146–5151.
(13) Fujii, K.; Shimoya, T.; Ikai, Y.; Oka, H.; Harada, K. Tetrahedron
Lett. 1998, 39, 2579–2582.
(14) As a result of experiments reported here, we have revised our
original assignments of C-31 and C-64. The correct assignments are
C-31, 172.6, and C-64, 172.4.
ESI LC/MS Conditions. The sample probe was set at 400 °C and 4
kV with an entrance cone voltage of 10 V. Chromatographic condi-
tions were as described for the HPLC-PDA analysis with a flow rate of
0.5 mL/min.
(15) Sitachitta, N.; Marquez, B. L.; Thomas Williamson, R.; Rossi, J.;
Ann Roberts, M.; Gerwick, W. H.; Nguyen, V.-A.; Willis, C. L. Tetrahedron
2000, 56, 9103–9113.
(16) Carmichael, W. W. Sci. Am. 1994, 270, 64–72.
1537
dx.doi.org/10.1021/np200362q |J. Nat. Prod. 2011, 74, 1535–1538