3
3.2. Synthesis
We characterized all synthesized compounds by NMR and/or
MS. NMR spectra were recorded at 300 K and standard
parameters. A Bruker Avance 400 MHz spectrometer at 400
MHz (1H NMR) and 100 MHz (13C NMR) was used. High
resolution mass spectra were recorded by a direct injection ESI-
TOF mass spectrometer (Bruker micrOTOF, Bremen, Germany).
All of the synthetic work was carried out using standard
laboratory equipment. Chemicals were used without prior
purification, except methanol and ethanol which were dried with
sodium and distilled off.
4. Conclusions
The presented synthetic path offers the opportunity to produce
ageladine A in gram-scale batches with standard laboratory
equipment and making use of non-specialized chemicals. Using
column-chromatography when needed, the only limitation is the
volume of solvent that has to be used in some of the reaction
steps (both brominations). The overall yield (starting with methyl
vinyl ketone) is 6.6 % over 7 steps (schemes 1 and 2). The
overall yield for the end-sequence (scheme 2) is 18.0 % (3 steps).
A comparison of our yields with the related and previously
published yields of synthetic strategies by Ando and Terashima
(11.7 %, 9 steps)14,31,32 Ma et al. (10.0 %, last 3 steps)15 and
Karuso et al. (28.6 %, last 3 steps)16 reveal an average result33.
Figure 2. Emission spectra of three regions of interest during
excitation with a 405 nm diode laser (a) and during excitation with a
multiphoton laser at 780 nm (b). One region is outside of the animal
(Macrostomum lignano) the others are inside of the animal.
Emission spectra of published data of ageladine A in water3 (c,
black line), compared to the emission spectrum revealed inside a
living flatworm (green line). There is a shift of the maximum
amplitude of around 30 nm and the spectrum is broader, especially
at longer wavelength.
Due to our observations of yields and the purities of products
no reaction steps were combined, while the work-up process is
not necessary after every reaction. Chances to shorten this
synthesis are given by combining steps A and B or B and C (both
Scheme 2)2.
The altered Stokes shift in living marine flatworms offers the
opportunity to use common filter settings as e.g. DAPI filter to
take advantage of the broad and shifted emission spectrum.
Ageladine A therefore is useful in staining whole animals and
dissected tissues.
Figure 2 (a and b) shows the increased Stokes shift of
agedaline A in a living marine flatworm (Macrostomum lignano)
compared to water (Figure 2, c). The emission maximum shifts
30 nm from about 420 nm to 450 nm and the emission profile is
much broader, exceeding 550 nm. The comparison of a 405 nm
diode laser to multiphoton excitation using 780 nm revealed no
difference as could be expected as emission profiles do not
change with excitation wavelength29, but with the environment.
The altered Stokes shift in living tissue matches the filter settings
for the commercially applied dye DAPI.
Acknowledgments
This work was in part supported by the Helmholtz society
grant: HE-2012-03.
We would like to thank Dr. Matthew J. Slater for language
improvement.
3. Experimental section
References and notes
3.1. Fluorescence measurements using Macrostomum lignano
1. Fujita, M.; Nakao, Y.; Matsunaga, S.; Seiki, M.; Itoh, Y.;
Yamashita, J.; van Soest, Rob W. M.; Fusetani, N. J. Am. Chem.
Soc. 2003, 125, 15700-15701.
The culture of Macrostomum lignano30 was originally
received from Dita Vizoso and Lucas Schärer (Basel) and was
raised and maintained in 16/8 LD cycle in Petri dishes together
with the diatom Nitzschia sp. at 20 2 °C in our lab since 2011.
Worms were incubated in F/2 medium for 1 h with ageladine A,
washed with medium, and anesthetized with 7.18 % MgCl2.
2. Shengule, S.R.; Loa-Kum-Cheung, W.L.; Parish, C.R.; Blairvacq,
M.; Meijer, L,; Nakao, Y.; Karuso, P. A one-pot synthesis and
biological activity of ageladine A and analogues, J. Med. Chem.
2011, 54, 2492-2503.
3. Bickmeyer, U., Grube, A., Klings, K. W.; Köck, M. Biochem.
Biophys. Res. Commun. 2008, 3, 419-422.
4. Hong-Hermesdorf, A.; Miethke, M.; Gallaher, S. D.; Kropat, J.;
Dodani, S. C.; Chan, J.; Barupala, D.; Domaille, D. W.; Shirasaki,
D. I.; Loo, J. A.; Weber, P. K.; Pett-Ridge, J.; Stemmler, T. L.;
Chang, C. J.; Merchant, S.S. Nat. Chem. Biol. 2014, 10, 1034–
1042.
Fluorescence was monitored with a confocal laser scanning
microscope TCS SP5 (Leica, Wetzlar, Germany) equipped with a
multiphoton laser and other standard lasers. Wavelength scans
were performed from 390 nm to 750 nm with a slit width of 10
nm (MP-excitation) or 420 nm to 750 nm for 405 nm diode laser
excitation.
5. Obermann, D.; Bickmeyer, U.; Wägele, H. Toxicon 2012, 60,
1108-1116.
6. Bickmeyer, U. Mar. Drugs 2012, 10, 223-233.