Synthesis of red-shifted 8-hydroxyquinoline derivatives using click chemistry and their incorporation into phosphorylation chemosensors

Article abstract of DOI:10.1021/jo901369k

(Chemical Equation Presented) Protein phosphorylation is a ubiquitous post-translational modification, and protein kinases, the enzymes that catalyze the phosphoryl transfer, are involved in nearly every aspect of normal, as well as aberrant, cell functio

Full text of DOI:10.1021/jo901369k

pubs.acs.org/joc
Synthesis of Red-Shifted 8-Hydroxyquinoline Derivatives Using Click
Chemistry and Their Incorporation into Phosphorylation Chemosensors
†
Juan A. Gonzalez-Vera, Elvedin Lukovic, and Barbara Imperiali*
,†,‡
ꢀ
ꢀ †
†Department of Chemistry, and ‡Department of Biology, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139
imper@mit.edu
Received June 26, 2009
Protein phosphorylation is a ubiquitous post-translational modification, and protein kinases, the
enzymes that catalyze the phosphoryl transfer, are involved in nearly every aspect of normal, as well
as aberrant, cell function. Here we describe the synthesis of novel, red-shifted 8-hydroxyquinoline-
based fluorophores and their incorporation into peptidyl kinase activity reporters. Replacement of
the sulfonamide group of the sulfonamido-oxine (1, Sox) chromophore, which has been previously
used in kinase sensing, by a 1,4-substituted triazole moiety prepared via click chemistry resulted in a
significant bathochromic shift in the fluorescence excitation (15 nm) and emission (40 nm) maxima
for the Mg2þ chelate. Furthermore, when a click derivative was incorporated into a chemosensor for
MK2, the kinase accepted the new substrate as efficiently as the previously reported Sox-based
sensor. Taken together, these results extend the utility range of kinase sensors that are based on
chelation-enhanced fluorescence (CHEF).
Introduction
a wide variety of diseases, from cancer to inflammation,2-6
they have also emerged as attractive targets for drug dis-
covery. Thus, tools that allow for facile monitoring of kinase
activity are in great demand in both pharmaceutical and
academic settings. Recently, our laboratory reported versa-
tile peptide sensors for the continuous fluorescence-based
assays of Ser/Thr and Tyr kinase activities that utilized the
sulfonamido-oxine (Sox) chromophore, which was intro-
duced into peptides either as the Sox7,8 or C-Sox9 residue
The ubiquitous process of protein phosphorylation is
central to signal transduction and regulation in all living
organisms. By catalyzing transfer of the γ-phosphoryl group
of ATP to the side chains of serine, threonine, and/or
tyrosine, protein kinases play an important role in regulating
many aspects of cellular function in eukaryotes, including
proliferation, cell cycle, metabolism, transcription, and
apoptosis.1 Since many protein kinases are associated with
(5) Johnson, L. N.; Lewis, R. J. Chem. Rev. 2001, 101, 2209–2242.
(6) Yarden, Y.; Sliwkowski, M. X. Nat. Rev. Mol. Cell Biol. 2001, 2, 127–
(1) Hunter, T. Cell 2000, 100, 113–127.
137.
(2) Adams, J. A. Chem. Rev. 2001, 101, 2271–2290.
(3) Manning, G.; Whyte, D. B.; Martinez, R.; Hunter, T.; Sudarsanam, S.
Science 2002, 298, 1912–1934.
(7) Shults, M. D.; Imperiali, B. J. Am. Chem. Soc. 2003, 125, 14248–
14249.
(8) Shults, M. D.; Carrico-Moniz, D.; Imperiali, B. Anal. Biochem. 2006,
352, 198–207.
(4) Skorski, T. Nat. Rev. Cancer 2002, 2, 351–360.
DOI: 10.1021/jo901369k
r
Published on Web 09/02/2009
J. Org. Chem. 2009, 74, 7309–7314 7309
2009 American Chemical Society

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SCHEME 1. 1,3-Dipolar Cycloaddition Reactions of 8 with
9a-v
FIGURE 1. Derivatives of 8-hydroxyquinoline-based amino acids
used to report kinase activity through CHEF.
TABLE 1. Relevant Spectroscopic Data for Oxn Derivatives That Form
Fluorescent Complexes with Mg2þ
complex
R1
R2
λex (nm)
λem (nm)
Φa,b
1c
2
3
4
5
6
7
SO2N(CH3)2
CHO
CN
COCH3
COCH2Cl
CO2H
CH3
CH3
CH3
CH3
CH3
CH3
H
360
373
362
370
373
362
375
485
460
485
465
460
508
460
0.342
0.597
0.276
0.168
0.034
0.004
0.002
CO-Ph
aSpectra were acquired in 150 mM NaCl, 50 mM HEPES (pH 7.4),
25 °C with 10 μM 1-7 and 10 mM MgCl2. bExcitation of all species is
provided at λmax (355-425 nm). Quantum yields were calculated with
reference to a quinine sulfate standard (in 0.05 M H2SO4). cExtinction
coefficient: ε355 = 8247 cm-1 M-1
.
excitation (15 nm) and emission wavelengths (40 nm) are
reported through the substitution of p-bromophenyl triazole
into the 5 position of Oxn (Clk). The Clk fluorophore was then
incorporated into peptidyl kinase substrates through the cysteine
residue side chain (C-Clk). Moreover, the C-Clk-based chemo-
sensor for MK2 was shown to be an efficient reporter of MK2
activity when compared to the existing C-Sox-based probe.
(Figure 1). The Sox-containing peptide substrates show low
fluorescence, but upon phosphorylation, the chromophore
binds to Mg2þ with greater avidity and undergoes chelation-
enhanced fluorescence (CHEF). Such probes have been
successfully used to monitor various kinases both with
recombinant enzymes and in crude cell lysates.10 To expand
the scope of these sensors in order to visualize kinase
activities in living cells, the photophysical properties of the
chemosensor should be improved by modulating the excita-
tion and the emission wavelengths of the quinoline reporter
to longer, lower energy wavelengths. The use of lower energy
to irradiate such fluorophores would minimize photodam-
age to biological systems and would mitigate problems with
high background signals that commonly complicate in cel-
lulo studies. Finally, the addition of another distinctly
colored chromophore could allow simultaneous visualiza-
tion of activities among multiple kinases.
Results and Discussion
Synthesis and Screening of Oxn Derivatives. In the first
phase of studies, a selection of substituted hydroxyquinoline
derivatives related to the original sulfonamide derivative (1),
but bearing different acceptor groups at position 5 (2-7),
was prepared using previously described methods (see the
Supporting Information). These analogues were screened for
fluorescence in the presence of excess Mg2þ and under
buffered aqueous conditions.
Toward that goal, herein we report studies on the chemical
modification of the 8-hydroxyquinoline (Oxn) moiety in order
to develop it as a building block for the assembly of new
phosphorylation sensors. In particular, shifts in fluorescence
Table 1 lists key spectroscopic data for compounds 1-7.
Notably, the excitation and emission wavelengths of the
magnesium-bound complexes are similar to those of the
original Sox derivative (1). However, the quantum yields,
measured by standard methods,11 are poorer. Indeed, only
(9) Lukovic, E.; Gonzalez-Vera, J. A.; Imperiali, B. J. Am. Chem. Soc.
2008, 130, 12821–12827.
(10) Shults, M. D.; Janes, K. A.; Lauffenburger, D. A.; Imperiali, B. Nat.
Methods 2005, 2, 277–283.
(11) Montes, V. A.; Pohl, R.; Shinar, J.; Anzenbacher, P. Jr. Chem. Eur. J.
2006, 12, 4523–4535.
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FIGURE 2. Qualitative comparison of emission wavelengths of 10a-v. The colors shown here do not represent the true fluorescent
wavelengths (shown in the Supporting Information). The fluorophores (10 μM) were illuminated (λex = 365 nm) in 50 mM HEPES (pH
7.4), 15% DMSO, 150 mM NaCl, 10 mM MgCl2 at 25 °C.
TABLE 2. Fluorescence Data for Magnesium Chelates of Selected
Triazole-Substituted Oxn Derivatives
For this purpose, azide 8 was prepared using previously
reported methods (Scheme 1).16 As expected, 8 showed no
fluorescence due to the quenching effect of the electron-rich
azido group.13 In the presence of catalytic Cu(I) and ascorbic
acid, 8 reacts readily at room temperature with 1-ethynylcy-
clohexene (9a) in DMF/4-methylpiperidine (8:2) to afford the
cycloaddition product 10a in excellent yield. In the presence of
10 mM MgCl2, quinoline 10a has a λex of 371 nm and a λem of
522 nm due to the elimination of azide quenching after
formation of the triazole ring. Despite the poor quantum
yield of 10a (Φ = 0.033), this initial result in the shift of the
emission wavelength encouraged us to screen additional
triazole-substituted quinolines by investigating the spectro-
scopic properties of products from the 1,3-dipolar cycloaddi-
tion reaction of 8 with 21 additional alkynes (9b-v).
complex
λex (nm)
λem (nm)
Φa,b
10a
10b
10c
10d
10i
371
370
367
365
375
375
522
525
523
525
525
525
0.033
0.041
0.043
0.071
0.067
0.111
10uc
The cycloaddition reactions were generally complete in
12 h at room temperature and were monitored by TLC and
mass spectrometry. The formation of the fluorescent triazole
compounds could be easily established upon exposure to a
hand-held UV lamp. The fluorophores were then qualita-
tively compared in a 96-well plate format on a transillumi-
nator (λex,max = 365 nm) to identify promising compounds
(Figure 2) and were further subjected to quantitative analysis
in a fluorescence plate reader (Table 2 and the Supporting
Information). The excitation and emission wavelengths of
aSpectra were acquired in 150 mM NaCl, 50 mM HEPES (pH 7.4), 25
°C with 10 μM 10 and 10 mM MgCl2. bExcitation of all species is
provided at λmax (355-425 nm). Quantum yields were calculated with
reference to a quinine sulfate standard (in 0.05 M H2SO4). cExtinction
coefficient: ε355 = 7905 cm-1 M-1
.
the aldehyde-substituted chromophore (2) showed an improved
quantum yield. Nonetheless, this derivative was not appropriate
for further studies because it exhibited a shorter λem, and the
aldehyde group can be easily oxidized in biological systems. In
light of these findings, we focused on 8-hydroxyquinoline
derivatives with substitution at position 5 that extended the
conjugation of the quinoline ring system. In the extended
aromatic systems, the π-π* gap would be reduced,12 thereby
resulting in longer excitation and emission wavelength maxima.
Since the triazole ring is a versatile and readily installed linkage
thathasbeenusedtoextendtheconjugationofdiversearomatic
systems,13,14 we envisioned that installation of this moiety into
the Oxn core could potentially provide red-shifted derivatives.
The azide substitution at position 5 of the hydroxyquinoline
would afford a nonfluorescent intermediate that could be
readily subjected to 1,3-dipolar cycloaddition with a variety of
terminal alkynes using the Cu(I)-catalyzed Huisgen reaction.15
the magnesium-bound triazole products (10b-v, λex
=
360-375 nm, λem = 510-530 nm) were improved compared
to those for Sox (1, λex = 360 nm, λem = 485 nm, Φ = 0.342,
ε355 = 8247 cm-1 M-1). On the basis of the preliminary
screening, selected targets were then synthesized in larger
quantities and the quantum yields of the corresponding
hydroxyquinoline derivatives were determined (Table 2).
Compared to 10a (Φ = 0.033), a 3.5-fold improvement in
quantum yield was obtained in the case of the bromide 10u
(λex = 375 nm, λem = 525 nm, Φ = 0.111, ε355 = 7905 cm-1
M-1). In view of the fluorescent properties of this derivative,
we chose to use it as a chelation-sensitive fluorophore to
prepare peptide-based probes for the MAPK-activated pro-
tein kinase-2 (MK2)17 and sarcoma kinase (Src),18-20 used as
models of Ser/Thr and Tyr kinases, respectively. For the
(12) Ghedini, M.; La Deda, M.; Aiello, L.; Grisolia, A. Inorg. Chim. Acta
2004, 357, 33–40.
(13) Sivakumar, K.; Xie, F.; Cash, B. M.; Long, S.; Barnhill, H. N.;
Wang, Q. Org. Lett. 2004, 6, 4603–4606.
(16) Dunkin, I. R.; ElAyeb, A. A.; Gallivan, S. L.; Lynch, M. A. J. Chem.
Soc., Perkin Trans. 2 1997, 1419–1427.
(14) Sawa, M.; Hsu, T. L.; Itoh, T.; Sugiyama, M.; Hanson, S. R.; Vogt,
P. K.; Wong, C. H. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 12371–12376.
(15) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004–2021.
(17) Roux, P. P.; Blenis, J. Microbiol. Mol. Biol. Rev. 2004, 68, 320–44.
(18) Martin, G. S. Nat. Rev. Mol. Cell Biol. 2001, 2, 467–475.
(19) Frame, M. C. Biochim. Biophys. Acta 2002, 1602, 114–130.
(20) Schlessinger, J. Cell 2000, 100, 293–296.
J. Org. Chem. Vol. 74, No. 19, 2009 7311

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SCHEME 2. Synthesis of the RDF Chemosensors
TABLE 3. Substrate Sequences of the RDF Chemosensors and Their Fluorescence Increases
entry
name
target kinase
location of the chromophorea
peptide sequenceb
fold fluorescence increasec
1
2
3
4
P1
P2
P3
P4
MK2
MK2
Src
C
C
N
N
Ac-AHLQRQLS*I-C(Sox)-HH-CONH2
Ac-AHLQRQLS*I-C(Clk)-HH-CONH2
Ac-AEE-C(Sox)-IY*GEFEAKKKK-CONH2
Ac-AEE-C(Clk)-IY*GEFEAKKKK-CONH2
4.4 ( 0.2
2.1 ( 0.3
2.0 ( 0.1
2.4 ( 0.2
Src
aLocation determined in reference to the chromophore. C denotes C-terminus and N stands for N-terminus. bAsterisk (*) denotes the residue that is
phosphorylated. In cases where it has been determined, residues important in kinase recognition are underlined. cMeasured in triplicate as a quotient of
fluorescence intensity at 485 nm (for Sox peptides) or 525 nm (for Clk peptides) of phosphopeptide and substrate in 20 mM HEPES (pH 7.4), 10 mM
MgCl2, and 10 μM peptide (for Sox peptides λex = 360 nm; for Clk peptides λex = 375 nm).
purposes of comparison, the analogous fluorescent peptides
containing the original Sox chromophore were also prepared
as previously described.9
Table 3 shows the substrate sequences of the C-Sox- and
C-Clk-based RDF probes for MK2 and Src kinases, as well
as the fluorescence increases that were observed with the
corresponding phosphopeptides. The difference in fluores-
cence was determined by comparison of the fluorescence
intensity at the maximum emission wavelength (485 nm
for Sox and 525 nm for Clk) of phosphorylated and
unphosphorylated peptides in the presence of Mg2þ. The
fluorescence increases of the click peptides (2- to 2.5-fold) are
significant changes that are useful in enzymatic assays
(vide infra). More specifically, the click-based RDF peptides
exhibited larger fluorescence increases than the Sox-based
RDF peptides in the case of Src (entries 3 and 4), while this
trend is reversed in the case of MK2 (entries 1 and 2).
Synthesis and Evaluation of Clk-Based Peptidyl Kinase
Substrates. The synthesis of the probes, outlined in Scheme 2,
was performed using a strategy similar to that used for the
preparation of the Sox-based recognition domain focused
(RDF) chemosensors.9 Diazotization of 5-amino-2-methyl-
quinolin-8-ol (12, prepared from 8-hydroxyquinaldine using
literature methods)21 followed by treatment of the diazo-
nium salt with NaN3 gave the corresponding azide 13 (66%
yield). Protection of the phenolic hydroxyl group as a tert-
butyldiphenylsilyl ether produced 14 (98%), which was then
brominated under free-radical conditions to afford the bro-
mide 15 (30%). To avoid dibromination, the reaction was
stopped after 20 min, thereby providing a mixture of the
desired product (15) and the starting material (14), which
could not be separated using standard chromatographic
methods and was used in the next step without purification.
Fmoc-based solid phase peptide synthesis (SPPS) was uti-
lized to assemble the intact peptide that included an appro-
priately placed cysteine residue protected with mono-
methoxytrityl (Mmt), which is a hyper acid-labile protecting
group (Scheme 2). After selective on-resin sulfhydryl depro-
tection, the free thiol was alkylated with 15. Then a 1,3-
dipolar cycloaddition reaction with 1-bromo-4-ethynylben-
zene in the presence of catalytic Cu(I) gave the corresponding
triazole-substituted peptide. Standard TFA cleavage from
the resin and concomitant removal of all side-chain protect-
ing groups revealed the desired chemosensor with excellent
conversion to the final product (>95%).
As an example, Figure 3a shows a comparison between the
fluorescence spectrum of the synthetically obtained phospho-
peptide MK2(Sox) [P1] and that from the analogous MK2-
(Clk) [P2]. In the case of the Src sensors, the result was the same
(see the Supporting Information). Neither species was fluor-
escent at pH 7.0 (50 mM HEPES, 150 mM NaCl) in the absence
of the metal ion. Upon addition of Mg2þ, the fluorescence
spectra of Mg2þ-bound P1 and Mg2þ-bound P2 exhibited
emission maxima of 485 and 525 nm, respectively, indicating
a red shift of 40 nm. In contrast, in the corresponding excitation
spectra, the Mg2þ-bound P1 and P2 reached maxima at 364
and 368 nm, respectively, representing a slightly decreased
bathochromic shift relative to that observed with the parent
chromophores. The change in absorption maxima (and thus
the excitation spectra) is most likely due to the difference in
coordination spheres. For the chromophore, this would be a 1:1
or 2:1 chelate with Mg2þ; in contrast, phosphopeptides bearing
the chromophore would exclusively form a 1:1 complex, enga-
(21) Gershon, H.; McNeil, M. W. J. Heterocycl. Chem. 1972, 9, 659–667.
7312 J. Org. Chem. Vol. 74, No. 19, 2009
ging the quinoline and phosphate groups with Mg2þ
.

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potential capabilities of the hydroxyquinoline-based kinase
sensing strategy.
Experimental Section
Synthesis of the Triazolyl Derivatives 10a-v. 5-Azido-8-hy-
droxy-2-methylquinoline (13, 50 mg, 0.26 mmol) and the corre-
sponding alkyne (9a-v) (0.26 mmol) were suspended in a
8:2 mixture of DMF/4-methylpiperidine (2 mL). Ascorbic acid
(7.1 mg, 0.04 mmol) and copper iodide (2.5 mg, 0.01 mmol) were
added suspended in a 8:2 mixture of DMF/4-methylpiperidine
(1 mL), and the heterogeneous mixture was stirred vigorously
overnight in the dark at room temperature. TLC analysis
indicated complete consumption of the reactants in 12 h.
The mixture was dissolved in ethyl acetate (40 mL), washed
with H2O (5 mL) and brine (5 mL), dried over Na2SO4,
and evaporated to yield the corresponding triazolyl deriva-
tives 10a-v.
Peptide Synthesis. All peptides were synthesized using the
standard Fmoc-based amino acid protection chemistry. Pep-
tides were synthesized on Fmoc-PAL-PEG-PS resin (Applied
Biosystems, 0.19 mmol/g) using on-resin alkylation. The resin
was swelled in CH2Cl2 (5 min) and then DMF (5 min) prior to
synthesis. All of the amino acids were coupled according to the
following procedure: Fmoc deprotection (20% 4-methylpiper-
idine in DMF, 3 ꢀ 5 min), rinsing step (DMF, 5ꢀ), coupling
step (amino acid/PyBOP/HOBt/DIEA, 6:6:6:6, 0.15 M in
DMF, 30-45 min), rinsing step (DMF, 5ꢀ; CH2Cl2, 5ꢀ).
The coupling was repeated if necessary as determined by the
TNBS test. At the end of the synthesis, the Fmoc group was
removed with 20% 4-methylpiperidine in DMF (3 ꢀ 5 min),
and the resin was rinsed with DMF (5ꢀ). The resin-attached
free amines were capped by exposure to Ac2O (20 equiv) and
pyridine (20 equiv) in DMF for 30 min. The resin was rinsed
with DMF (5ꢀ) and CH2Cl2 (5ꢀ) and subjected to 20%
4-methylpiperidine in DMF (3 ꢀ 5 min). The resin was finally
washed with DMF, CH2Cl2, and MeOH (5ꢀ each) and dried
under vacuum.
On-Resin Alkylation of Peptides with 15. Resin-attached
peptides (50 mg, 0.0095 mmol, 1 equiv) incorporating Cys-
(Mmt) were swelled in CH2Cl2 and then DMF. The Mmt
protecting group was removed from the resin-bound peptide
by bubbling N2 through a solution of 1% TFA and 5% TIS in
CH2Cl2 (4 ꢀ 20 min). The resin was washed with CH2Cl2 (5ꢀ)
and DMF (5ꢀ). Anhydrous DMF (200 μL) was added to
the resin followed by freshly distilled tetramethylguanidine
(5.96 μL, 0.0475 mmol, 5 equiv). The mixture was incubated
for 2-3 min. 15 (17 mg, 0.0285 mmol, 3 equiv) was dissolved
in anhydrous DMF (150 μL) and added to the resin. After
ca. 12 h of reaction time, the excess reagents were drained
and the resin was washed with DMF, CH2Cl2, MeOH, and
CH2Cl2 (5ꢀ).
FIGURE 3. Spectral characterization and enzymatic evaluation of
Clk-based substrates. (a) Fluorescence excitation and emission
spectra of P1 (- - -) and P2 (;) with Mg2þ. Samples were prepared
in 50 mM HEPES (pH 7.4) and 150 mM NaCl. Spectra were
acquired at 25 °C and were baseline-corrected using a sample of
the buffer solution. (b) Percentage of turnover of the Clk-based (9,
entry 2) or Sox-based (0, entry 1) substrate with MK2 after 10 min.
Assays were performed in 20 mM HEPES (pH 7.4), 10 mM MgCl2,
0.1 mM EGTA, 0.01% Brij 35, 0.1 mg/mL BSA, 1 mM DTT, 1 mM
ATP, 5 μM substrate, and 10 ng MK2 at 30 °C. Plotted values
indicate the mean ( sem for triplicate measurements.
Evaluation of Clk-Based Substrates in Enzymatic Assays.
Having validated the utility of the new fluorophore through
photophysical characterization, we also evaluated its effi-
cacy in kinase reactions. Following established protocols,9
Sox- and Clk-based substrates (entries 1 and 2, respectively,
in Table 3) were subjected to MK2 under identical condi-
tions and then the overall turnover of each substrate was
compared. As shown in Figure 3b, MK2 phosphorylated
the Clk substrate just as efficiently as the Sox substrate,
indicating that the size of Clk chromophore does not
adversely influence reaction kinetics. Due to previous work,
which showed that Sox-based substrates had at least com-
parable, if not better, kinetics than parent peptides, we
believe that the Clk-based reporters will also follow the
same trend for Src and other kinases. However, kinase
substrate kinetics are highly empirical; thus, new sub-
strates will have to be experimentally evaluated and can be
improved using high-throughput mass-spectrometry-based
methods.22
Conclusions
In conclusion, this report presents the synthesis, peptide
incorporation, and characterization of new phosphorylation
chemosensors that contain 1,4-triazole-substituted 8-hydro-
xyquinolines and exhibit improved fluorescence properties
compared to Sox. This modification results in significant red
shifts in the excitation (15 nm) and emission maxima (40 nm)
of the chromophore when complexed to Mg2þ. Although the
Clk fluorophore exhibits a somewhat decreased excitation
shifts when incorporated into a peptide to monitor kinase
activity, the chromophore does not inhibit the ability of
MK2 to recognize and efficiently phosphorylate the Clk-
bearing probe. Together, these results effectively expand the
On-Resin Click Chemistry of Peptides with 9u. Resin-attached
peptides (50 mg, 0.0095 mmol, 1 equiv) incorporating 5-azido-
8-hydroxyquinoline were swelled in CH2Cl2 and then DMF
(5 min). A mixture of 1-bromo-4-ethynylbenzene (9u, 34.4 mg,
0.19 mmol), ascorbic acid (0.75 mg, 0.0043 mmol), and copper
iodide (0.27 mg, 0.0014 mmol) was added to the resin suspended
in a 8:2 mixture of DMF/4-methylpiperidine (1.5 mL). After
ca. 12 h of reaction time, the excess reagents were drained and
the resin was washed with DMF, CH2Cl2, MeOH, and CH2Cl2
(5ꢀ). The resin cleavage and protecting group removal was
achieved by exposing the resin-bound peptides to TFA/H2O/
TIS (95:2.5:2.5 v/v). The resulting solution was concentrated
under a stream of N2 and precipitated by addition of cold Et2O.
The pellet was triturated with cold Et2O, redissolved in water,
filtered, and lyophilized. The peptides were purified by prepara-
tive reverse-phase HPLC using UV detection at 228 nm (amide
(22) Gonzalez-Vera, J. A.; Lukovic, E.; Imperiali, B. Bioorg. Med. Chem.
Lett. 2009, 19, 1258–1260.
J. Org. Chem. Vol. 74, No. 19, 2009 7313

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bond absorption) and 316 nm (8-hydroxyquinoline absorption).
Only fractions showing a single peak of correct mass by analy-
tical HPLC were used in further experiments.
J.A.G.-V. We also thank the Department of Chemistry
Instrumentation Facility (NIH-1S10RR013886-01).
Supporting Information Available: Experimental proce-
dures, characterization of chromophores and peptides, fluores-
cence spectra, and enzyme assays. This material is available free
of charge via the Internet at http://pubs.acs.org.
Acknowledgment. This research was supported by the
NIH Cell Migration Consortium (GM064346) and the
A.M. Escudero Foundation postdoctoral fellowship for
7314 J. Org. Chem. Vol. 74, No. 19, 2009