Design and synthesis of a fluorescent reporter of protein kinase activity

Article abstract of DOI:10.1021/ja017530v

There is widespread interest in developing fluorescent reporters of protein kinase activity, species that can furnish a visual readout of both when and where intracellular kinases are activated in response to a stimulus. We have constructed and identified

Full text of DOI:10.1021/ja017530v

Published on Web 03/22/2002
Design and Synthesis of a Fluorescent Reporter of Protein Kinase Activity
Chien-An Chen,* Ren-Hwa Yeh,* and David S. Lawrence*
Department of Biochemistry, The Albert Einstein College of Medicine, 1300 Morris Park AVenue,
Bronx, New York 10461
Received November 13, 2001
Cells recognize and respond to environmental stimuli via
activation of intracellular biochemical pathways primarily composed
of protein kinases. These enzymes catalyze the phosphorylation of
serine, threonine, or tyrosine residues on protein substrates.
Members of this large enzyme family have been the objects of
intense scientific scrutiny due to their role in disease onset and
progression. In addition, they participate in the pathways that drive
a variety of important processes that range from cell division1 to
apoptosis.2 Consequently, there has been widespread interest in
developing sensors of protein kinase activity, species that could
furnish a visual readout of both where and when specific intrac-
ellular kinases are activated in response to a stimulus. A variety of
approaches have been described,3 including small peptide substrates
that possess an appended fluorophore positioned near the site of
phosphorylation.4 Unfortunately, the phosphorylation-induced change
in fluorescence intensity for these peptides is modest, at best
(∼20%). We have developed a strategically different approach for
the construction of a fluorescent reporter of protein kinase activity.
generate a M2+ receptor site composed of two carboxylates and
the newly introduced phosphate (4). Upon divalent metal ion
coordination, a fluorescence change should transpire via a mech-
anism analogous to that described for the Ca2+ indicators.
The strategy outlined above is made possible by the observation
that protein kinases will phosphorylate alcohol-containing residues
attached to the N- or C-terminus of appropriately designed pep-
tides.6,7 This structural feature allows the fluorophore to be directly
attached to the phosphorylatable residue (e.g., 3). With the latter
in mind, our initial synthetic target was compound 2, which contains
the requisite functionality in protected form, along with a free
carboxylate that can be activated and condensed with the N-terminus
of the peptide H2N-Ser-Phe-Arg-Arg-Arg-Arg-resin. The latter
sequence serves as a substrate for protein kinase C (PKC),7 an
enzyme family implicated in a variety of physiologically critical
processes, including cell division.8
The xanthene half of 2 was synthesized from the xanthone
precursor 7 (Scheme 1). The latter was prepared via the Friedel-
Crafts acylation of 6 9 with 5. The product was subsequently heated
in a sealed tube to furnish the xanthone 8 and the phenol moieties
then protected as 2-methoxyethoxymethyl (MEM)10 ethers (9). The
aromatic precursor (10) to the “northern” half of compound 2 was
prepared in three steps from commercially available o-aminophenol
(see Supporting Information). Compound 10 contains a doubly
protected iminodiacetic acid moiety and a benzylated phenol. The
latter will ultimately be debenzylated so that it can serve as the
attachment site for the peptide (vide infra). Compound 10 was
lithiated at -105 °C and coupled to 9, to furnish adduct 11 in 69%
yield. The benzylated phenol in 11 was transformed in three steps
to the desired carboxylic acid (2) and then coupled to H2N-Ser-
(O-tBu)-Phe-[(Arg)Mtr]4-resin (prepared via standard Fmoc solid-
phase peptide synthesis on the Rink resin). Finally, exposure of
the resin-appended fluorophore-peptide to 95% CF3CO2H resulted
in the simultaneous cleavage of the peptide from the resin, MEM
ether deprotection, and complete aromatization of the tricyclic
nucleus via loss of the tertiary hydroxyl moiety to yield 3.
As expected, peptide 3 serves as a substrate for the Ca2+
-
dependent PKCR. 3 displays a 140% increase in fluorescence
intensity upon phosphorylation, nearly an order of magnitude greater
than that previously described in protein kinase monitoring
systems.3,4 In addition, the difluorofluorescein moiety in 3 is an
extremely bright fluorophore (ꢀ ) 78 000 cm-1 M-1 and Φ ) 0.60)
and, thus, is easily an order of magnitude more sensitive than
fluorophores that have been previously used to observe protein
kinase activity.9 Although the Km value (26.5 µM) for the PKCR-
catalyzed phosphorylation of 3 is quite good, the corresponding
Perhaps the preeminent example of a fluorescent sensor that sam-
ples biologically relevant processes is the family of Ca2+ indicators
(e.g., 1) developed by Tsien and his colleagues.5 Formation of the
Ca2+-fluorophore complex, via coordination to the two iminodi-
acetic acid moieties, is manifested by a dramatic fluorescence
change. Tsien has proposed that Ca2+ coordination induces a twist
about the arylamine bond, altering the coupling between the nitrogen
lone pair and the aromatic ring system.5 We have designed a
peptide-based species (3) that contains some of the structural
features present in 1. Specifically, phosphorylation of 3 should
Vmax (0.32 µmol/min‚mg) is an order of magnitude less than ideal.
The large, negatively charged fluorophore, which is positioned
adjacent to the site of phosphorylation, might interfere with the
ability of the PKC active site to accommodate the serine moiety.
* Address correspondence to any of these authors. E-mail: dlawrenc@
aecom.yu.edu.
9
3840
J. AM. CHEM. SOC. 2002, 124, 3840-3841
10.1021/ja017530v CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
Scheme 1. Synthesis of the Fluorescein Precursor 2
Table 1.
V
max, Km, and Fluorescence Change Associated with the
PKC-Catalyzed Phosphorylation of Peptides 14 (five different
linkers), 3, and AcSFRRRRK7
LINKER
Vmax (µmol/min‚mg)
Km (µM)
% change fluorescence
L-proline
D-proline
N-Me glycine
a
1.9
1.0
8.5
1.7
2.2
0.32
63.0
23.5
20.5
25.0
24.9
26.5
10
150%
156%
264%
164%
157%
140%
-
b
peptide 3
AcSFRRRRK
24
the magnitude and rate of fluorescence change. Several fluoro-
phore-LINKER-peptide analogues were identified that display
promising enzymological and photophysical properties (Table 1).
N-methyl glycine serves as the LINKER in the lead protein kinase
substrate. Phosphorylation of the latter generates a 264% enhance-
ment in fluorescence intensity and proceeds with a Vmax of 8.5 µmol/
min‚mg and a Km of 20.5 µM. Indeed, the Vmax is more than an or-
der of magnitude greater than that displayed by compound 3, which
lacks a LINKER residue between the fluorophore and the peptide.
Saturating [Ca2+] produces 1.2- and 2.0-fold fluorescence en-
hancements in 3 and 14 (N-Me Gly), respectively. By contrast,
enhancements of 5- and 23-fold were observed with the chemically
synthesized phosphorylated analogues of 3 and 14, respectively.
However, the large metal-induced fluorescence change in the
phosphorylated species appears to be partly offset by a reduction
in the inherent (i.e., metal-free) fluorescence of the phosphopeptides
(Supporting Information). In summary, we have prepared, via
rational design and library synthesis, a new family of protein kinase
substrates that respond to phosphoryation in a fluorescently sensitive
fashion. The ability of compounds 3 and 14 to sample PKC activity
in a variety of cell-based systems is under investigation.
Scheme 2. Synthesis of Library 14
Acknowledgment. We thank the NIH for financial support.
Supporting Information Available: Experimental details of the
synthesis, characterization, and enzymology of compound 3/library 14
(PDF). This material is available free of charge via the Internet at http://
pubs.acs.org.
We addressed the possibility of fluorophore-mediated disruption
of the kinase-catalyzed reaction by preparing a small library of 22
derivatives of 3 using the synthetic strategy outlined in Scheme 2.
A series of turn-promoting/metal chelating LINKERs was inserted
between the peptide and the fluorophore (14), which might allow
the serine moiety to be more optimally accommodated within the
active site. Following phosphorylation, the turn-inducing/chelating
ability of the LINKER should enable the iminodiacetic acid carbox-
ylates to assume a position that promotes metal coordination. The
library of 22 compounds was prepared on a cystamine-derivatized
TentaGel resin,11 which contains a disulfide bridge between the
peptide and the resin (12). The side-chain protected peptide 12 was
split into 22 portions of 10 mg each and added to a solvent-resistant
multiwell filter plate. Twenty-two Fmoc-amino acids (“LINKER”s,
see Supporting Information) were added to individual wells and
condensed with 12. The Fmoc group was removed (13) and the
product coupled to compound 2. CF3CO2H (95%) was subsequently
employed to simultaneously deprotect the phenol and carboxylic
acid moieties and transform the xanthene nucleus into the fluores-
cein derivative. Finally, all 22 compounds were cleaved from the
Tentagel resin with PKC assay buffer, which contains dithiothreitol.
The library members (14) were filtered into a receiving plate and
then assayed under standard conditions with monitoring for both
References
(1) Pines, J. Nat. Cell Biol. 1999, 1 E73-79.
(2) Blatt, N. B.; Glick, G. D. Bioorg. Med. Chem. 2001, 9, 1371-1384.
(3) Ng, T.; Squire, A.; Hansra, G.; Bornancin, F.; Prevostel, C.; Hanby, A.;
Harris, W.; Barnes, D.; Schmidt, S.; Mellor, H.; Bastiaens, P. I. H.; Parker,
P. J. Science 1999, 283, 2085-2089; Lee, C. L.; Linton, J.; Soughayer,
J. S.; Sims, C. E.; Allbritton, N. L. Nat. Biotechnol. 1999, 17, 759-762;
Nagai, Y.; Miyazaki, M.; Aoki, R.; Zama, T.; Inouye, S.; Hirose, K.; Iino,
M.; Hagiwara, M. Nat. Biotechnol. 2000, 18, 313-316.
(4) McIlroy, B. K.; Walters, J. D.; Johnson, J. D. Anal. Biochem. 1991, 195,
148-152; Bowman, B. F.; Peterson, J. A.; Stull, J. T. J. Biol. Chem. 1992,
267, 5346-5354; Post, P. L.; Trybus, K. M.; Taylor, D. L. J. Biol. Chem.
1994, 269, 12880-12887.
(5) Grynkiewicz, G.; Poenie, M.; Tsien, R. Y. J. Biol. Chem. 1985, 260, 3440-
3450; Minta, A.; Kao, J. P. Y.; Tsien, R. Y. J. Biol. Chem. 1989, 264,
8171-8178; Tsien, R. Y. Ann. ReV. Neurosci. 1989, 12, 227-253.
(6) Kwon, Y.-G.; Mendelow, M.; Lawrence, D. S. J. Biol. Chem. 1994, 269,
16725-16729.
(7) Yan, X.; Curley, K.; Lawrence, D. S. Biochem. J. 2000, 349, 709-715.
(8) Varlamova, O.; Spektor, A.; Bresnick, A. R. J. Muscle Res. Cell Motil.
2001, 22, 243-250.
(9) Sun, W.-C.; Gee, K. R.; Klaubert, D. H.; Haugland, R. P. J. Org. Chem.
1997, 62, 6469-6475.
(10) Corey, E. J.; Gras, J.-L.; Ulrich, P. Tetrahedron Lett. 1976, 809-813.
(11) Lee, T. R.; Lawrence, D. S. J. Med. Chem. 1999, 42, 784-787.
JA017530V
9
J. AM. CHEM. SOC. VOL. 124, NO. 15, 2002 3841