Versatile reagents to introduce caged phosphates

Article abstract of DOI:10.1016/S0040-4039(02)02832-0

Three novel reagents have been prepared to introduce photoactivatable o-nitrobenzyl phosphate esters. The use of fluorenylmethyl and t-butyl protecting groups allowed for a wide range of chemical transformations after phosphorylation. In addition, the use of S-acetylthioethyl and acyloxymethyl groups resulted in photo- and bioactivatable phosphate triesters of phosphatidic acid.

Full text of DOI:10.1016/S0040-4039(02)02832-0

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 1153–1155
Versatile reagents to introduce caged phosphates
Carlo Dinkel, Oliver Wichmann and Carsten Schultz*
European Molecular Biology Laboratory, Meyerhofstraße 1, 69126 Heidelberg, Germany
Received 20 September 2002; revised 14 October 2002; accepted 14 December 2002
Abstract—Three novel reagents have been prepared to introduce photoactivatable o-nitrobenzyl phosphate esters. The use of
fluorenylmethyl and t-butyl protecting groups allowed for a wide range of chemical transformations after phosphorylation. In
addition, the use of S-acetylthioethyl and acyloxymethyl groups resulted in photo- and bioactivatable phosphate triesters of
phosphatidic acid. © 2003 Elsevier Science Ltd. All rights reserved.
Photoactivatable derivatives of biologically active sub-
stances, usually referred to as caged compounds, are
important tools in biomedical research.1,2 The major
advantage is that the inactive compound can be deliv-
ered to living cells without significantly effecting intra-
cellular processes. Only after flash photolysis liberates
the active compound within a few seconds, effects are
monitored on a physiologically relevant time scale.
Therefore, caged compounds allow for intracellular
manipulations with a maximum of temporal resolu-
tion.3 Nitrobenzyl esters are by far the most commonly
used caging groups for phosphates, although cou-
marinylmethyl esters were also successfully attached.4
Very recently, it was shown that an amido-substituted
nitrobenzyl groups could be introduced by direct alkyl-
ation of cyclic nucleotides.5 In the past, however, cag-
ing groups were usually linked to phosphates via
diazomethane or diazoethane reagents. In case of sphin-
gosine 1-phosphate, Qiao and co-workers turned to the
phosphite reagent bis(2-nitrophenylethyl) N,N-diiso-
propylphosphoramidite to introduce a phosphate moi-
ety with two photosensitive groups.6 Another method
involving P(III) reagents was used for the preparation
of caged myo-inositol hexakisphosphate derivatives that
relied on an o-nitrobenzyl allyl phosphoramidite.7
However, the authors reported harsh conditions for the
removal of the allyl ester, which might not be generally
accepted by biomolecules. Finally, 1-(2-nitrophenyl)-
ethyl-caged phosphopeptides were prepared with the
help of a phosphoramidite reagent that proved suitable
for solid phase synthesis.8
Herein, we report several novel phosphoramidite
reagents that are suitable to prepare caged derivatives
of AMP, mannose 1-phosphate, and phosphatidic acid
(PA). The nitroveratryl phosphoramidites9 containing
fluorenylmethyl (Fm) or t-butyl protecting groups or
the bioactivatable S-acetylthioethyl (SATE) group (1–
3) were prepared in 64–78% total yield from the chloro-
or the t-butyloxy phosphorbis(amidite), respectively, as
shown in Scheme 1. The reagents were stable for many
months when kept cold and at least for weeks when
kept at room temperature. First targets to test the
feasibility of the reagents were AMP and phosphatidic
Scheme 1. Reagents and conditions: (a) 9-Fluorenylmethyl (Fm) alcohol (1 equiv.), TEA, n-pentane, 0°C to rt, 3 h; (b)
AcSCH2CH2OH (1 equiv.), TEA, Et2O, 0°C, 3 h; (c) DMNBnOH (1 equiv.), diisopropylamine tetrazolide (0.5 equiv.), DCM, 0°C
to rt, 16 h.
Keywords: photoactivatable; caged compounds; membrane-permeant; bioactivatable; phosphorylation; phosphatidic acid; AMP; mannose
1-phosphate.
* Corresponding author. Tel.: +49-(0)6221-387-210; fax: +49-(0)6221-387-206; e-mail: schultz@embl.de
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02832-0

1154
C. Dinkel et al. / Tetrahedron Letters 44 (2003) 1153–1155
In order to provide caged tools to study PA activity,16
we then prepared caged derivatives of di-O-octanoyl-
glycerol (8)17 and a fluorescent, nile red-tagged SN1-
ether derivative 9 thereof (Scheme 4). We considered
the latter a challenge, because the nile red group does
not comply with aggressive conditions. On the other
hand, nile red exhibits little absorbance at 365 nm and
is likely to be fairly resistant to bleaching under the
conditions needed to removed the cage. Compounds 8
and 9 were readily phosphitylated with 1.5 equiv. of
reagent 2 in the presence of tetrazole in 43% and 55%
isolated yield after oxidation to the phosphate, respec-
tively. Removal of the Fm group was quantitative and
gave the caged-PA derivatives 10 and 11. Both com-
pounds were alkylated with acetoxymethyl bromide
(AM-Br) to give the potentially bioactivatable triesters
12 and 13, respectively. Alternatively, 9 was treated
with reagent 3 to introduce cage and bioactivatable
S-acetylthioethyl group in a single reaction. The
diastereomeric phosphate triester 14 was isolated in
52% yield.
acid. A caged AMP (trinitrophenyl-AMP) was a valu-
able tool to investigate myosin movement in the past10
and PA was shown to mediate vesicle budding from the
Golgi, among other functions.11 The classical starting
material for the preparation of AMP derivatives is
2%,3%-O-isopropylidene adenosine. For a two-step reac-
tion to caged-AMP (4) the use of reagent 1 was desir-
able, because both acid-labile protecting groups could
be removed in a single step (Scheme 2). The Fm group
of reagent 2 allowed phosphorylation followed by
deprotection of the phosphate under mild basic condi-
tions and subsequent removal of the ketale. This per-
mitted additional modification of the phosphate diester,
as was demonstrated by the reaction with propi-
onoxymethyl iodide (PM-I), that yielded an uncharged,
potentially bioactivatable caged AMP derivative after
cleavage of the ketale (not shown). Acyloxymethyl
esters or S-acetylthioethyl groups of biologically active
phosphates were shown to facilitate cellular entry of
usually impermeant compounds.12–14 It should be men-
tioned that the quantitative nature of the Fm cleavage
reaction combined with the products’ large differences
in polarity allowed for very fast preparations.
The reagents presented here combine the possibility to
introduce phosphate and cage at the same time. In
addition, it was shown, that bioactivatable protecting
groups could be introduced in the same procedure or
after removal of the phosphate protecting groups. The
method might be generally suitable to replace diazo
compounds when total synthesis of caged phosphate
derivatives is required. An application in a more labori-
ous synthetic pathway is described in an additional
paper.18 The biological potential of the final products
described will be published elsewhere.
The next task was supposed to show the stability of the
caged phosphate derivatives under stronger basic condi-
tions. For this purpose, we prepared the caged 2,3,4,6-
tetra-O-acetyl-mannose 1-phosphate 6 in three steps
from penta-O-acetyl-mannose 5 (Scheme 3).15 Deacety-
lation at pH 13 in the presence of the nitroveratryl
group proceeded smoothly within 36 h to give caged
mannose-1-phosphate 7 in 95% yield.
Scheme 2. Reagents and conditions: (a) 1, tetrazole, MeCN, rt, 16 h, then t-BuOOH, rt; (b) TFA (95%), rt, 45% (two steps); (c)
2, tetrazole, MeCN, rt, 16 h, then t-BuOOH, rt, 50%; (d) pyrrolidine, DCM, rt, 5 min, quant.; (e) TFA (95%), rt, 95%.
Scheme 3. Reagents and conditions: (a) Porcine liver esterase, 10% DMF in phosphate buffer (0.2 M), pH 7, rt, 8 h;15 (b) 2,
tetrazole, MeCN, rt, 16 h, then t-BuOOH, rt, 61%; (c) pyrrolidine, DCM, rt, 5 min, quant.; (d) aq. KOH, pH 13, rt, 36 h, 95%.

C. Dinkel et al. / Tetrahedron Letters 44 (2003) 1153–1155
1155
Scheme 4. Reagents and conditions: (a) 2, tetrazole, 5 h, then t-BuOOH, rt, 43–55%; (b) pyrrolidine, DCM, rt, 5 min, quant.; (c)
3, 4,5-dicyanoimidazole, rt, 0.5 h, then t-BuOOH, rt; (d) acetoxymethyl bromide, DIPEA, MeCN, rt, 3 h, 46–52%.
Acknowledgements
9. All compounds gave satisfactory analytical and spectro-
scopic data. 31P NMR (162 MHz) of 1 (in C6D6): l
(ppm) 138.28; 2 (in C6D6): l 148.31; 3 (in C6D6): l
148.4; 4 (in CD3OD/D2O, 5:1 v/v): l −0.41; 6 (in
CD3OD): l −2.76 (pyrrolidinium salt, pH 10); 7 (in
CD3OD): l −0.16 (free acid, pH 2); 10 (in CD3OD): l
−1.87 (pyrrolidinium salt, pH 10); 11 (in CD3OD): l
We thank Biolog Lifescience Institute, Germany, for
nucleosides, SiChem GmbH, Germany, for chlorophos-
phoramidites, Dr. Marc Gentzel for MS analysis, and
Ms. Nicole Heath for revising the manuscript. The
work was supported by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie.
−2.89; 12 (in C6H5CD3):
l −1.74, −1.91; 13 (in
C6H5CD3): l −2.63, −2.76; 14 (in CDCl3): l −1.60,
−1.64.
10. Fujita, X.; Nawata, T.; Yamada, K. J. Physiol. (London)
References
1999, 515, 869.
11. Siddhanta, A.; Backer, J. M.; Shields, D. J. Biol. Chem.
2000, 275, 12023.
1. Adams, S. R.; Tsien, R. Y. Annu. Rev. Physiol. 1993, 55,
755.
12. Schultz, C.; Vajanaphanich, M.; Harootunian, A. T.;
Sammak, P. J.; Barrett, K. E.; Tsien, R. Y. J. Biol.
Chem. 1993, 268, 6316.
2. Dorma´n, G.; Prestwich, G. D. Trends Biotechnol. 2000,
18, 64.
3. Li, W.-h.; Llopis, J.; Whitney, M.; Zlokarnik, G.; Tsien,
13. Meier, C. Synlett 1998, 233.
R. Y. Nature 1998, 392, 936.
14. Perigaud, C.; Girardet, J.-L.; Gosselin, G.; Imbach, J.-L.
In Advances in Antiviral Drug Design; De Clerq, E., Ed.;
JAI Press: Stamford, CT, 1996; Vol. 2, p. 147.
15. Hennen, W. J.; Sweers, H. M.; Wang, Y. F.; Wong, C.
H. J. Org. Chem. 1988, 53, 4939.
4. Hagen, V.; Bendig, J.; Frings, S.; Eckardt, T.; Helm, S.;
Reuter, D.; Kaupp, U. B. Angew. Chem., Int. Ed. 2001,
40, 1046.
5. Wang, L. J.; Corrie, J. E. T.; Wootton, J. F. J. Org.
Chem. 2002, 67, 3474.
16. Williger, B. T.; Reich, R.; Neeman, M.; Bercovici, T.;
Liscovitch, M. J. Biol. Chem. 1995, 270, 29656.
17. Martin, S. F.; Josey, J. A.; Wong, Y.-L.; Dean, D. W.
J. Org. Chem. 1994, 59, 4805.
6. Qiao, L. X.; Kozikowski, A. P.; Olivera, A.; Spiegel, S.
Bioorg. Med. Chem. Lett. 1998, 8, 711.
7. Chen, J. A.; Prestwich, G. D. Tetrahedron Lett. 1997,
38, 969.
18. Dinkel, C.; Schultz, C. Tetrahedron Lett. 2003, 44,
1157.
8. Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Impe-
riali, B. Org. Lett. 2002, 4, 2865.