Enantioselective synthesis and application of the highly fluorescent and environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine (DANA).

Article abstract of DOI:10.1039/b205224e

6-(2-Dimethylaminonaphthoyl) alanine (DANA) was prepared via an enantioselective synthesis and incorporated into the S-peptide of RNase S establishing the large changes in fluorescence that can occur upon peptide-protein interaction.

Full text of DOI:10.1039/b205224e

Enantioselective synthesis and application of the highly fluorescent and
environment-sensitive amino acid 6-(2-dimethylaminonaphthoyl) alanine
(DANA)†
Mark Nitz, Adam R. Mezo, Mayssam H. Ali and Barbara Imperiali*
Department of Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA
02139, USA. E-mail: imper@mit.edu
Received (in Cambridge, UK) 30th May 2002, Accepted 4th July 2002
First published as an Advance Article on the web 29th July 2002
6-(2-Dimethylaminonaphthoyl) alanine (DANA) was pre-
pared via an enantioselective synthesis and incorporated
into the S-peptide of RNase S establishing the large changes
in fluorescence that can occur upon peptide–protein inter-
action.
Several strategies have been developed for the enantiose-
lective synthesis of unnatural a-amino acids.11 One of the most
effective of these is the phase transfer catalysis mediated
asymmetric alkylation of electrophiles with glycine benzophe-
none imines as first introduced by O’Donnell et al. and
subsequently refined by Corey et al.12,13 More than 25 different
a-amino acids have been synthesized by this route but the
electrophiles used have largely been derivatives of primary,
allylic, or benzylic halides. Only one electrophile was an a-halo
tert-butyl ester and conflicting values of the yield and ee have
been reported.14 This report demonstrates that this method can
be extended to the implementation of aromatic a-halo ketones
as the electrophilic species; both high yield and excellent
enantioselectivity are achieved in the synthesis of DANA
(Scheme 1).
Fluorescent probes are exceptionally powerful tools for the
investigation of biological events.1 The utility of a fluorescent
probe is amplified when the fluorescence emission spectrum is
sensitive to environment. This allows the real time monitoring
of binding events and conformational changes of the labeled
molecule of interest. 6-Propionyl-2-(dimethylamino)naphtha-
lene 1 (PRODAN), first introduced by Weber and Farris2, is
ideal for monitoring biological events as the fluorophore
displays dramatic changes in fluorescence intensity and emis-
sion wavelength maxima with changes in environment. The
fluorophore is also excited at wavelengths longer than those of
most intrinsic biological chromophores and has a high quantum
yield. Although, the source of the unique solvatochromism of
PRODAN is a subject of debate, recent direct measurements of
the excited state dipole moment have determined it to be likely
caused by direct hydrogen bonding interactions with the
solvent.3
PRODAN has been used as a probe in many biological
systems, perhaps most notably in the study of lipid bilayers4 and
in proteins which have a fortuitous binding site for the
fluorophore.5 Derivatives of PRODAN have also been attached
covalently to proteins via reaction with amino acid side chain
thiols with 6-acryloyl-2-(dimethylamino)naphthalene6,7 or
Scheme 1 (a) tert-Butylglycinate benzophenone imine, O(9)allyl-N-
9-anthracenylmethylcinchonidium bromide (25 mol%), 40% KOH,
CH2Cl2, 225 °C, 83% yield, 87% ee, (b) i) 6 M HCl reflux, ii) Fmoc-OSu,
DMF, NaHCO3, 96%.
6-bromoacetyl-2-dimethylaminonaphthalene
(BADAN).8
BADAN (2) was synthesized via literature reported methods,
with minor modifications, from commercially available 6-ace-
tyl-2-methoxynapthalene.15,16 BADAN served as the electro-
phile for the key enantioselective alkylation reaction yielding
the desired amino acid derivative in 83% yield and 87% ee. It
was found that the choice of temperature was critical to the
reaction, as at temperatures below 230 °C the alkylation
reaction did not occur, and the a-bromoketone decomposed
under the reaction conditions. At higher temperatures the
enantioselectivity of the reaction eroded. Potassium hydroxide
proved to be a superior base to cesium hydroxide in this reaction
allowing a liquid/liquid biphasic mixture at the desired
temperature.
The resulting benzophenone imine 3 was hydrolyzed and
derivatized as the Fmoc carbamate to give the desired Fmoc-
DANA (N-Fmoc-6-(2-dimethylaminonaphthoyl) alanine) in
preparation for solid phase peptide synthesis. The enantiomeric
excess of the amino acid was determined by reaction of the free
amino acid with Marfey’s reagent followed by reverse phase
HPLC analysis.16,17
These methods provided useful information about the macro-
molecule of interest but are limited to systems with uniquely
reacting amino acid side chains. Furthermore, the labeling of
amino acid side chains results in significant distances and many
degrees of freedom between the fluorophore and the protein
backbone, thus reducing the environmental sensitivity of the
probe.
We sought to develop an enantioselective synthesis of
6-(2-dimethylaminonaphthoyl) alanine (DANA), a fluorescent
amino acid, in a form amenable to incorporation during solid
phase peptide synthesis, simplifying the specific labeling of
peptide substrates and minimizing the number of atoms between
the fluorophore and the peptide backbone. The syntheses of
many fluorescent amino acids have now been published
including those containing the 7-nitrobenz-2-oxa-1,3-diazol-
4-yl chromophore (NBD), but none have the same degree of
sensitivity to the environment as PRODAN. 9,10
† Electronic supplementary information (ESI) available: experimental
details. See http://www.rsc.org/suppdata/cc/b2/b205224e/
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Fmoc-DANA proved to be an excellent amino acid for solid
phase synthesis using standard protocols. No loss in optical
activity was observed upon incorporation of the amino acid into
the peptide, elongation of the peptide, or cleavage of the peptide
from the solid phase.16 The solvent dependence of a short
peptide (A-(DANA)-A) was evaluated and found to be similar
to that of parent fluorophore, PRODAN, indicating that no
significant change of its fluorescence properties had occurred as
a result of its incorporation into a peptide scaffold (see ESI† for
figure).16
To outline the utility of this amino acid, the well-charac-
terized RNase S model system, consisting of a 15 amino acid S-
peptide which associates tightly with S-protein (Ka 106–107
M21) to give catalytically active ribonuclease S (RNase S), was
employed.18,19 The amino acid DANA was incorporated into
the S-Peptide and the changes in the fluorescence of DANA
were observed upon binding to the S-Protein. These changes led
directly to the dissociation constants for the newly synthesized
peptides.
binding mode could be favored over a 5–45 °C temperature
range suggesting both modes to be similar in energy. Analysis
of the titration data leads directly to an affinity constant of 3.0
3 106 M21
.
The first facile synthesis of Fmoc-DANA has been reported
in both high yield and high enantioselectivity. DANA was
incorporated into peptides using solid phase synthesis and
shown to retain similar fluorescent properties to PRODAN.
Given the recent advances in protein labeling using in vitro
biosynthesis22 and chemical ligation23 techniques this amino
acid should prove to be a valuable tool for the study of protein–
protein interactions.
This research was supported by the NSF (CHE-9996335) and
the NIH (GM64346 Cell Migration Consortium). The award of
an NSERC postdoctoral fellowship to M. N. is also gratefully
acknowledged.
Note added at proof: after submission of this manuscript
similar work appeared in Science web alerts 30/05/02.24
Two peptides which, based upon the crystal structure of
RNase S and literature examples, would place the DANA amino
acid in a highly hydrophilic (5)20 or hydrophobic environment
(6)21 were synthesized by standard solid phase synthesis.
Notes and references
1 Fluorescent and Luminescent Probes for Biological Activity, ed. W. T.
Mason, Academic Press, London, UK, 1999.
2 G. Weber and F. J. Farris, Biochemistry, 1979, 19, 3075.
3 A. Samanta and R. W. Fessenden, J. Phys. Chem. A, 2000, 104, 8972;
F. M. Cerezo, S. C. Rocafort, P. S. Sierra, F. García-Bianco, C. D. Oliva
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4 L.-G. P. Chong, Biochemistry, 1988, 27, 399; J. Zeng and L.-G. P.
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5 T. Hiratsuka, Biochemistry, 1998, 37, 7167–7176.
6 S. S. Lehrer and Y. Ishii, Biochemistry, 1988, 27, 5899.
7 K. G. Harikumar, D. I. Pinon, W. S. Wessels, F. G. Prendergast and J.
L. Miller, J. Biol. Chem., 2002, in press.
Peptide 5 KETAAAXFERQHLDS X = DANA
Peptide 6 KETAAAKFERQHLXS
Fig. 1 illustrates the large changes in fluorescence emission
spectrum of DANA upon binding to the S-protein. Peptide 5,
which places DANA in a solvent exposed position upon
binding, gave a 26% reduction in fluorescence intensity at 520
nm after saturation with Pro-S protein. Comparison of the
titration data to a literature S-peptide containing a fluorescein
labeled lysine at the equivalent position revealed DANA to have
twice as large a fluorescence intensity change upon binding but
10 times weaker affinity for the Pro-S protein (Ka 1.6 3 107
M21 vs. Ka 1.6 3 108 M21(lit)).20 The decreased affinity is
likely due to the closer proximity of the DANA fluorophore to
the peptide backbone which could lead to steric congestion
upon binding.
In contrast, peptide 6 exhibits a large increase in fluorescence
upon binding to the Pro-S protein leading to the development of
a fluorescence emission band at 450 nm that was completely
absent before binding. The presence of two bands in the Pro-S
protein saturated emission spectra may indicate that DANA is
binding in two modes to the Pro-S protein, one of which places
the fluorophore in a hydrophobic environment leading to the
450 nm emission band and a second band at 510–520 nm which
places DANA in a more solvent exposed environment. Neither
8 T. Hiratsuka, J. Biol. Chem., 1999, 274, 29159.
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11 For recent reviews see (a) M. J. O’Donnell, Aldrichim. Acta, 2001, 34,
3; (b) T. Abellán, R. Chinchilla, N. Galindo, G. Guillena, C. Nájera and
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16 Details contained in ESI†.
17 The absolute stereochemistry was deduced from the literature precedent
of
literature precedent of the
eluting before the -enantiomer when analyzed by reverse phase HPLC.
L
-Cinchonidine derived catalyst producing the
L-enantiomer and the
L
-Marfey diastereomer of most amino acids
D
K. Fugii, Y. Ikai, T. Mayumi, H. Oka, M. Suzuki and K. Harada, Anal.
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21 E. E. Kim, R. Varadarajan, H. W. Wyckoff and F. Richards,
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24 B. E. Cohen, T. M. McAnaney, E. S. Park, N. Y. Jan, S. G. Boxer and
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Fig. 1 Fluorescence emission spectra of peptides 5 and 6. Top: peptide 5 (2.8
mM) in absence (hashed) and presence (12 mM) (bold) of saturating Pro-S
protein. Bottom: peptide 6 (2.5 mM) in absence (hashed) and presence (16
mM) (bold) of saturating Pro-S protein. All spectra were collected in 10 mM
HEPES pH 7.0, 150 mM NaCl at 20 °C with excitation at 367 nm.
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