12588
J. Am. Chem. Soc. 2000, 122, 12588-12589
Scheme 1. Preparation of the R-Carboxy-2-nitrobenzyl Caged
Leucyl Leucine Methyl Ester 4, Methyl Amide 5, and
Dendritic Caged Compounds 6-10a
New Dendritic Caged Compounds: Synthesis, Mass
Spectrometric Characterization, and Photochemical
Properties of Dendrimers with
r-Carboxy-2-nitrobenzyl Caged Compounds at Their
Periphery
Soichiro Watanabe, Mika Sato, Shigeru Sakamoto,†
Kentaro Yamaguchi,† and Michiko Iwamura*
Department of Biomolecular Science
Faculty of Science, Toho UniVersity
2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan
Chemical Analysis Center, Chiba UniVersity
1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan
ReceiVed May 22, 2000
a a: p-nitrophenyl chloroformate, DMAP, CHCl3, 1 d, 98%; b:
LeuLeuOMe‚TFA, DMAP, CHCl3, 2.5 d, 83%; c: KMnO4, CH3COOH-
H2O, 16 h, 98%; d: MeNH2, EEDQ, MeOH, 15 h, 36%.
Caged compounds1 are biologically inert molecules which can
release bioactive compounds upon photolysis. In the course of
our study on caged compounds for medicinal use, we designed a
dendritic caged compound, which may be able to carry a large
number of biomolecules without any loss during transport in an
inactive form.
Dendrimers, which are hyperbranched artificial polymers with
a monodisperse molecular weight and well-controlled shape, have
received considerable attention over the past decade.2 Although
dendritic structures are used as the fundamental skeletons of newly
developed functional materials, it is often difficult to characterize
them by mass spectrometry. Efficient methods for mass spectro-
metric characterization have to be developed for further progress
in dendrimer chemistry.
In this communication, we report the first synthesis, photo-
chemical properties of dendritic caged compounds,3 and a new
efficient method for preparing an R-carboxy-2-nitrobenzyl caged
compound4 of a leucyl leucine methyl ester, which induces
apoptosis in immunological cells,5 as a model substrate. We also
describe the mass spectrometric characterization of newly devel-
oped dendritic caged compounds with a high molecular weight
and a hydrophobic surface.
An R-carboxy-2-nitrobenzyl caged leucyl leucine methyl ester
4 was synthesized by the method shown in Scheme 1.6 The
terminal olefin moiety of 3 was oxidized by reaction with KMnO4
to give 4 as a diastereomeric mixture.7 Compared with the
previously reported method,8,9 the present method is unique in
that R-carboxy-2-nitrobenzyl caged compounds were obtained
from a 1-(2-nitrophenyl)allyl alcohol with no chemically sensitive
functional groups such as a carboxylic acid or an ester.
Methyl amide 5 was prepared as a model compound. Com-
mercially available PAMAM dendrimers were used as a dendritic
core. PAMAM dendrimers of generation 0-4, which have 4-64
primary amino groups at their periphery, were treated with 4 in
the presence of 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline
(EEDQ) in methanol (Scheme 1). The yields of the dendritic caged
compounds were high enough for use in further chemical and
biological applications (57-96%). The structures of dendritic
1
molecules 6-10 were confirmed by H NMR, 13C NMR, UV/
vis, and ESI-MS10 (vide infra).11
These dendritic caged compounds have a hydrophobically
functionalized surface and a high molecular weight compared with
those in the dendritic core (e.g., PAMAM dendrimer generation
2: MW 3256 vs 8: MW 10660), which made it difficult to
generate ionized species suitable for ESI-MS detection. In the
present case, both positive and negative molecular ion peaks of
6-8 were observed (Figure 1).12 As for 9 and 10 (molecular
weights of 21716 and 43823, respectively), molecular ion peaks
could not be observed.
None of the dendritic caged compounds obtained here gave
significant fluorescent adducts after reaction with fluorescamine,
suggesting the absence of primary amino groups which were
present on the surface of PAMAM dendrimers. The caged
compounds obtained above were used for the following experi-
ments as mixtures of diastereomers, since we did not expect the
diastereomers to have significantly different photochemical
properties.
* To whom correspondence should be addressed. Telephone/Fax: +81-
† Chiba University.
(1) For leading reviews on caged compounds, see: (a) Kaplan, J. H. Annu.
ReV. Physiol. 1990, 52, 897. (b) Adams, S. R.; Tsien, R. Y. Annu. ReV. Physiol.
1993, 55, 755. (c) Bioorganic Photochemistry; Morrison, H., Ed.; John Wiley
& Sons: 1993; Vol. 2.
(2) For leading reviews on dendrimers, see: (a) Newkome, G. R.;
Moorefield, C. N.; Vo¨gtle, F. Dendritic Molecules: Concepts, Syntheses,
PerspectiVes; VCH: Weinheim, 1996. (b) Fischer, M.; Vo¨gtle, F. Angew.
Chem., Int., Ed. 1999, 38, 884-905. (c) Bosman, A, M.; Janssen, H. M.;
Meijer, E. W. Chem. ReV. 1999, 99, 1665-1688.
(3) Recent report on photolabile dendrimers, see: Smet, M.; Liao, L.-X.;
Dehaen, W.; McGrath, D. V. Org. Lett. 2000, 2, 511-513.
(4) Haugland, R. P., Ed. Photoactivable (Caged) Probes. In Handbook of
Fluorescent Probes and Research Chemicals, 6th ed.; Molecular Probes:
Eugene, OR, 1996; pp 447-456.
(5) (a) Sakauchi, H.; Furuta, T.; Kobayashi, Y.; Iwamura, M. Biochem.
Biophys. Res. Commun. 1997, 233, 211-215. (b) Watanabe, S.; Iwamura,
M. J. Org. Chem. 1997, 62, 8616-8617.
(6) The allyl alcohol 1 was prepared as reported, see: Marriot, G.; Ottl, J.
Methods Enzymol. 1998, 291, 155-175.
(7) R-Carboxy-2-nitrobenzyl caged leucyl leucine methyl ester 4: pale
yellow crystals; mp: 82.0-84.0 °C; 1H NMR (270 MHz, CD3OD): δ 0.60-
1.05 (m, 12H), 1.34-1.87 (m, 6H), 3.67 and 3.69 (s, 3H), 4.00-4.34 (m,
1H), 4.34-4.55 (m, 1H), 6.71 and 6.74 (s, 1H), 7.36-7.63 (m, 1H), 7.63-
7.80 (m, 2H), 7.90-8.10 (m, 1H), 8.30 (br s, 1H); UV/vis (MeOH): 257 nm
(ꢀ 4040). Anal. Calcd for C22H31N3O9: C, 54.88; H, 6.49; N, 8.73. Found:
C, 55.17; H, 6.73; N, 8.33%.
The molar extinction coefficients of dendrimers 6-10 as well
as those of monomers 4 and 5 at both λmax and 350 nm are almost
(8) Caged compounds derived from R-carboxy-2-nitrobenzylamine, see:
(a) Cohen, B. E.; Stoddard, B. L.; Koshland, Jr., D. E. Biochemistry 1997,
36, 9035-9044. (b) England, P. M.; Lester, H. A.; Davidson, N.; Dougherty,
D. A. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 11025-11030 and references
therein.
(9) Caged compounds derived from R-carboxy-2-nitrobenzylbromide,
see: (a) Curley, K.; Lawrence, D. S. J. Am. Chem. Soc. 1998, 120, 8573-
8574. (b) Niu, L.; Gee, K. R.; Schaper, K.; Hess, G. P. Biochemistry 1996,
35, 2030-2036 and references therein.
(10) Although molecular ion peaks of dendritic caged compounds 6 and 7
could be observed by MALDI-TOF MS, those of 8-10 were not observed in
this method.
(11) See Supporting Information.
(12) Detection of negative molecular ion peaks of dendritic caged
compounds were carried out as a method described in the previous report,
see: Cheng, X.; Gao, Q.; Smith, R. D. J. Org. Chem. 1996, 61, 2204-2206.
10.1021/ja001768m CCC: $19.00 © 2000 American Chemical Society
Published on Web 11/30/2000