Synthesis and biological study of a flavone acetic acid analogue containing an azido reporting group designed as a multifunctional binding site probe

Article abstract of DOI:10.1016/j.bmc.2005.02.035

Flavone-8-acetic acid (FAA) is a potent immunomodulatory small molecule that is uniquely characterized as being active on mouse but not human cells. Although FAA is a potent inducer of murine cytokine, chemokine and interferon gene expression, its mode of action remains unknown. In this report, we describe the synthesis of a new flavone acetic acid (FAA) analogue, (2-[2-(4- azidophenyl)-4-oxochromen-8-yl-]acetic acid (compound 2). We demonstrate that compound 2 is equally active as the parent FAA in inducing chemokine gene expression and that the azide functional group is capable of reacting with a reporter molecule, such as the FLAG peptide-phosphine, under mild conditions. This reaction will be useful for detecting the drug-bound protein active complex utilizing an anti-FLAG antibody.

Full text of DOI:10.1016/j.bmc.2005.02.035

Bioorganic & Medicinal Chemistry 13 (2005) 2717–2722
Synthesis and biological study of a flavone acetic acid
analogue containing an azido reporting group designed as
a multifunctional binding site probe
Krishnan Malolanarasimhan,a Christopher C. Lai,a James A. Kelley,a Lynn Iaccarino,b
Della Reynolds,b Howard A. Youngb and Victor E. Marqueza,*
aLaboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute—Frederick,
Center for Cancer Research, Frederick, MD 21702-1201, USA
bLaboratory of Experimental Immunology, Center for Cancer Research, National Cancer Institute—Frederick,
Center for Cancer Research, Frederick, MD 21702-1201, USA
Received 8 December 2004; revised 17 February 2005; accepted 17 February 2005
Abstract—Flavone-8-acetic acid (FAA) is a potent immunomodulatory small molecule that is uniquely characterized as being active
on mouse but not human cells. Although FAA is a potent inducer of murine cytokine, chemokine and interferon gene expression, its
mode of action remains unknown. In this report, we describe the synthesis of a new flavone acetic acid (FAA) analogue, (2-[2-(4-
azidophenyl)-4-oxochromen-8-yl-]acetic acid (compound 2). We demonstrate that compound 2 is equally active as the parent FAA
in inducing chemokine gene expression and that the azide functional group is capable of reacting with a reporter molecule, such as
the FLAG peptide–phosphine, under mild conditions. This reaction will be useful for detecting the drug-bound protein active com-
plex utilizing an anti-FLAG antibody.
Published by Elsevier Ltd.
1. Introduction
creases production of interferon,14 tumor necrosis fac-
tor-a (TNF-a),14 and nitric oxide and other cytokines
from activated mouse but not human macrophages.15
One of the main effects of FAA is TNF-a-mediated tu-
mor vascular collapse16–18 followed by tumor necrosis.19
More recently, the reported effects of FAA on endothe-
lial cell proliferation demonstrates that this flavone pos-
sesses antiangiogenic properties.20 Therefore, FAA is
capable of engaging the immune system by acting as a
biological response modifier.
Almost two decades ago, a synthetic flavonoid, flavone-
8-acetic acid (1, FAA),1 was widely investigated when its
ability to reduce tumor growth in a number of murine
solid tumors was discovered.2–6 Unfortunately, the anti-
tumor activity observed in murine models did not trans-
late to humans and FAA failed in early clinical trials.7,8
Despite extensive studies, the mechanism of FAA re-
mains unknown and to date no specific biological target
has been identified. Its antitumor activity appears to be
the result of indirect effects rather than direct cytotoxic-
ity, and it differs from most conventional chemothera-
peutic agents in that it does not induce
myelosuppression.9 The compound is able to activate
macrophages10 and natural killer (NK) cells;11–13 it in-
The immunomodulatory activity of FAA in murine sys-
tems has been linked to its ability to rapidly induce cyto-
kine gene expression. Because the drug fails to do the
same in humans,21 knowledge of its mechanism of ac-
tion in mice would be critically important to provide
new leads for drug development to treat human cancers.
Despite the reported syntheses of a number of ana-
logues, none of these compounds have been designed
to identify any of the possible molecular targets.22–25
To achieve such a goal, we decided to synthesize a com-
pound with a close structural resemblance to the parent
FAA, but modified with a functional group capable of
Keywords: Immunomodulatory small molecules; Flavone acetic acid;
Azide-modified flavone acetic acid; Affinity label; Staudinger reaction.
*
Corresponding author. Tel.: +1 3018465954; fax: +1 3018466033;
e-mail: marquezv@dc37a.nci.nih.gov
0968-0896/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmc.2005.02.035

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K. Malolanarasimhan et al. / Bioorg. Med. Chem. 13 (2005) 2717–2722
reacting in a biological milieu with a specific peptide tag.
With the use of a recently developed rapid tissue culture
screen, we have been able to dissect the molecular struc-
ture of FAA and determine, which chemical variations
enhance, maintain or abrogate its functional activity.
Using a mouse macrophage cell line we were able to
measure the direct induction of the CCL5 chemokine
(Rantes) within 6 h by FAA and its derivatives utilizing
100,000 target cells in a 96 well format (data not shown).
This assay led to the identification of a substitution
(X = N3) of the molecule (2-[2-(4-X-phenyl)-4-oxochro-
men-8-yl-]acetic acid, 2) that preserved the full biologi-
cal activity of FAA.
2. Results
2.1. Synthesis of 2-[2-(4-azidophenyl)-4-oxochromen-8-yl-]-
acetic acid (2)
Diketone 6 was readily formed by O-acylation of the
known 1-(2-hydroxy-3-prop-2-enylphenyl)ethan-1-one
(5) in the presence of K2CO3 (Scheme 2). The ensuing
intramolecular rearrangement resulted in the migration
of the p-nitrobenzoyl moiety from oxygen to carbon
according to the well-known Baker–Venkataraman
rearrangement.29 Cyclization to chromone 7 occurred
readily in acid, and conversion of the nitro group to
the azide was performed in two simple steps via the
amine intermediate to give 9. The allyl group was finally
oxidized with potassium permanganate22 in a mixture of
acetic acid, water, and acetone to give the desired target
2. Oxidation with NaIO4/RuO4 provided no decisive
advantage despite the excellent yields reported with this
methodology by Sharpless and co-workers.30
A major objective of this project is to ultimately be able
to identify a target macromolecule bound by FAA by
using the N3 group as an affinity label, which can be
activated either photochemically26 or through conven-
tional chemistry.27 In view of the technical difficulties
anticipated with photochemical activation, our attention
was directed to the use of the Staudinger reaction to lo-
cate the binding site of our azide-containing FAA by
reacting it with a FLAG peptide–phosphine (Scheme
1).27,28 Detection of the drug-bound complex could then
possibly be achieved by Western blot or immunoprecip-
itation with a mouse anti-FLAG antibody in a manner
similar to the elegant work of Bertozzi and co-work-
ers.27,28 The conditions for the Staudinger reaction be-
tween the phosphine and the azide are very mild
and ligations could be performed in a cell supernatant
maintained between pH 7.0 and 7.4 after FAA
treatment.28
2.2. Staudinger reaction between 2-[2-(4-azidophenyl)-4-
oxochromen-8-yl-]acetic acid (2) and the FLAG peptide–
phosphine probe (3)
A sample of the FLAG peptide–phosphine probe, syn-
thesized according to Ref. 27 was kindly provided by
Dr. Carolyn Bertozzi. The Staudinger ligation depicted
in Scheme 1 was carried out on a microscale in aqueous
media at pH 7.2 to mimic as closely as possible the con-
ditions of a potential biological experiment. Since mass
spectrometric analysis was used to follow and confirm
formation of the expected FAA–FLAG peptide conju-
gate (4), a volatile ammonium carbonate buffer was em-
ployed to maintain pH and yet allow lyophilization
prior to analysis in order to minimize inorganic salt con-
tamination of the sample. MALDI MS indicated com-
In order to reach our goal of developing a new reagent
that will permit us to identify the molecular target of
FAA, we needed to (1) synthesize (2-[2-(4-azidophe-
nyl)-4-oxochromen-8-yl-]acetic acid, 2), (2) confirm that
it was biologically equivalent to FAA, and (3) demon-
strate that the Staudinger reaction occurred readily
between the FLAG peptide–phosphine probe and 2.
The realization of these three objectives is described
below.
O
O
O
a
OH
OH
NO2
O
5
6
O
HO2C KDDDDKYD
PPh2
N
b
O
H
CO2CH3
+
HO2C
X
O
O
3
1, X = H
2, X = N3
e
for X = N3
O
O
X
HO2C
N3
O
O
2
HO2C KDDDDKYD
7, X = NO2
8, X = NH2
9, X = N3
PPh2
N
c
H
N
H
CO2H
d
O
O
O
Scheme 2. Reagents and conditions: (a) p-nitrobenzoyl chloride,
K2CO3, butanone, D (62%); (b) H2SO4, MeOH, D (90%); (c) Na2S2O4,
AcOH/acetone/H2O; (d) NaNO2, HCl, NaN3, CH2Cl2/H2O (biphasic)
(57%, two steps); (e) KMnO4, AcOH/acetone/H2O (18%).
4
Scheme 1.

K. Malolanarasimhan et al. / Bioorg. Med. Chem. 13 (2005) 2717–2722
2719
plete reaction of the FLAG peptide–phosphine 3 with 2
to form expected FAA–FLAG peptide conjugate 4 after
1 h at room temperature (25 ꢀC). The product mass
spectrum exhibited the correct mass (m/z 1638.52) and
appropriate isotopic distribution for the expected pro-
tonated molecule (MH+) with minimal cationization
by Na+ and K+. No indication of the presence of FLAG
peptide–phosphine 3 starting material (m/z 1359.48,
MH+) was observed in the product spectrum. Similar
MALDI MS analysis of a control sample containing
only the FLAG peptide–phosphine 3 indicated the
appropriate mass and isotopic distribution for this com-
pound and showed only the minor presence of the oxi-
dized phosphine (m/z 1375, MH+).
2.3. Biological activity
The biological activity of azido-FAA (2) relative to
FAA was determined by treating a mouse C57BL/6
macrophage cell line with the two compounds (at vary-
ing concentrations) for 6 h, isolating total cellular RNA
from the treated cells, and analyzing the extracted RNA
with the chemokine Multiprobe RNAse Protection
assay. As shown in Figure 1, the azido-FAA (2) deriva-
tive was equally as active as FAA in inducing chem-
okine gene expression in the mouse macrophage cell
line.
3. Discussion and conclusion
The synthesis of the target azido compound 2 proceeded
uneventfully according to Scheme 2. As demonstrated in
Figure 1, the azido group did not interfere with the de-
sired biological activity and the compound essentially
behaved as the parent FAA (1). The Staudinger reaction
between 2 and the FLAG peptide–phosphine probe oc-
curred under mild conditions that were compatible with
those of potential biological experiments (Scheme 1),
and the formation of the product was unequivocally
confirmed by MALDI MS analysis. One possible limita-
tion of this reaction in a biological system is the accessi-
bility of the azido group to react with the FLAG
peptide–phosphine probe after compound 2 binds to
its target protein. If the azido group finds itself in a deep
pocket at the active site, the Staudinger reaction will
probably fail. In this case, the reactive azido group
could be photoactivated and the resulting nitrene would
react with a proximal functional group on the target
macromolecule forming a covalent bond.25 The tagged
protein could then be identified and degraded to identify
the binding site. Whatever approach is to be followed,
we propose that compound 2 should be a useful probe
to define the active binding site of FAA. As FAA was
a potent immunomodulatory that demonstrated signifi-
cant activity in murine tumor models, the identification
of its molecular target will permit us to identify the hu-
man counterpart and develop FAA analogues that may
show equivalent biological responses when tested on hu-
man cells in vitro and in vivo. The approach described
here represents an important model for identifying the
molecular targets of novel small molecules whose mech-
anism of biological activity is unknown.
Figure 1. Ribonuclease protection assay (1 = no treatment; 2 = FAA
(100 lg/mL); 3 = azido-FAA, 10 lg/mL).
4. Experimental
4.1. General
All chemical reagents were commercially available. Col-
umn chromatography was performed on silica gel 60,
230–240 mesh (E. Merck), and analytical TLC was per-
formed on Analtech Uniplates silica gel GF. Routine
IR, 1H NMR and 13C NMR spectra were recorded
using standard methods. Elemental analyses were per-
formed by Atlantic Microlab, Inc., Norcross, GA.

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K. Malolanarasimhan et al. / Bioorg. Med. Chem. 13 (2005) 2717–2722
4.2. (2Z)-3-Hydroxy-1-(2-hydroxy-3-prop-2-enylphenyl)-
3-(4-nitrophenyl)prop-2-en-1-one (6)
2H), 7.48 (dd, J = 8.9, 1.6 Hz, 1H), 7.33 (t, J = 7.6 Hz,
1H), 6.76 (dm, J = 8.8, 2H), 6.66 (s, 1H), 6.03–6.10
(m, 1H), 5.11–5.16 (m, 2H), 3.72 (d, J = 6.4 Hz, 2H);
13C NMR (CDCl3, 100 MHz) d 178.70, 163.60, 154.00.
149.90, 135.50, 133.70, 129.30, 127.95, 124.63, 124.00,
123.83, 116.85, 114.75, 104.83, 34.00; FAB MS m/z (rel-
ative intensity) 278 (MH+, 100). Crude 8 was then dis-
solved in cold (0 ꢀC) CH2Cl2 (5 mL), treated with cold
(0 ꢀC) 6 N HCl (10 mL), and stirred. Aqueous sodium
nitrite (0 ꢀC) was added slowly and stirred for 20 min.
Sodium azide was then added slowly and stirred for an
additional 25 min. The reaction mixture was diluted
with CH2Cl2 (25 mL) and the layers separated. The
aqueous layer was further washed with CH2Cl2
(2 · 25 mL) and the collected organic extracts were com-
bined, dried (MgSO4), and concentrated under reduced
pressure. Purification by column chromatography
(SiO2, 3:1 hexanes/EtOAc) afforded 9 (800 mg, 57% for
two steps); mp 153–155 ꢀC; 1H NMR (CDCl3,
400 MHz) d 8.02 (dd, J = 8.0, 1.6 Hz, 1H), 7.81 (d,
J = 8.9 Hz, 2H), 7.47 (br d, J = 8.0 Hz, 1H), 7.27 (t,
J = 7.8 Hz, 1H), 7.07 (d, J = 8.9 Hz, 2H), 6.68 (s, 1H),
5.98–6.05 (m, 1H), 5.08–5.14 (m, 2H), 3.67 (d,
J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz) d
178.38, 161.88, 154.03, 143.42, 135.21, 134.10, 129.34,
128.36, 127.71, 124.97, 123.92, 123.88, 119.61, 117.03,
106.83, 33.97; IR (neat) 2093, 1635 cmÀ1; HRMS
(FAB) calcd for C18H14N3O2 (MH+): 304.1086. Found:
304.1084.
A mixture of 1-(2-hydroxy-3-prop-2-enylphenyl)ethan-
1-one (5, 6 g, 34 mmol), 4-nitrobenzoyl chloride (6 g,
32.4 mmol), and anhydrous K2CO3 (10 g) in anhydrous
butanone (100 mL) was refluxed for 36 h. The reaction
mixture was decanted to remove inorganic salts and
evaporated to half its volume. Hexane (100 mL) was
added and the precipitate formed was filtered off. The
solid was re-dissolved in EtOAc (100 mL) and acidified
with 6 N HCl to pH 1. The organic layer was separated
and the aqueous layer was further washed with EtOAc
(2 · 50 mL). The combined organic layers were dried
(MgSO4) and concentrated under reduced pressure to
afford pure 6 as an orange solid product (8 g, 62%),
which was crystallized from EtOAc/hexanes; mp 177–
1
179 ꢀC; H NMR (CDCl3, 400 MHz) d 12.26 (s, 1H),
8.34 (d, J = 8.8 Hz, 2H), 8.09 (d, J = 8.8 Hz, 2H), 7.69
(d, J = 7.2 Hz, 1H),7.40 (dd, J = 7.2 Hz, 1H), 6.88–
6.93 (m, 2H), 6.00–6.07 (m, 1H), 5.10–5.14 (m, 2H),
3.47 (d, J = 6.4 Hz, 2H); 13C NMR (CDCl3, 100 MHz)
d 196.6, 173.27, 160.8, 149.7, 139.5, 136.6, 135.9,
130.0, 127.6, 126.7, 123.9, 118.8, 118.2, 116.1, 94.3,
33.5; IR (neat) 3077, 1636, 1438, 1244 cmÀ1; FAB MS
(negative ion) m/z 324 (MÀH)À. Anal. Calcd for
(C18H15NO5Æ0.33H2O): C, 65.25; H, 4.77; N, 4.23.
Found: C, 65.27; H, 4.57; N, 4.21.
4.3. 2-(4-Nitrophenyl)-8-prop-2-enylchromen-4-one (7)
4.5. 2-[2-(4-Azidophenyl)-4-oxochromen-8-yl]acetic acid
(2)
Five drops of concd H2SO4 were added to solution of 6
(2.1 g, 6.3 mmol) in MeOH (50 mL) and heated to reflux
for 2 h. The solution was concentrated under reduced
pressure, and the brown solid obtained dissolved in
CH2Cl2 (100 mL) and washed with water (2 · 25 mL).
The organic layer was dried (MgSO4) and concentrated
under reduced pressure. The product was purified by
crystallization from EtOAc/hexanes to afford 7 (1.4 g,
KMnO4 (938 mg, 5.94 mmol) was added in aliquots to a
stirred solution of 9 (360 mg, 1.19 mmol) in a mixture of
AcOH (2.5 mL), acetone (5 mL), and water (1 mL) that
was maintained at 0 ꢀC for a period of 5 h. The reaction
mixture was further stirred for 1 h at room temperature.
The reaction was quenched with H2O2 and concentrated
under vacuum to a residue. The solid obtained was
washed with warm EtOH/EtOAc (1:1) and filtered.
The solid was then dissolved in EtOAc and washed with
water. The organic layer was dried (MgSO4) and con-
centrated under reduced pressure. Pure product 2
(80 mg, 18%) was obtained as an amorphous yellow so-
lid by precipitation from a solution in EtOH/EtOAc
1
70%); mp 210–212 ꢀC; H NMR (CDCl3, 400 MHz) d
8.40 (d, J = 9.2 Hz, 2H), 8.09 (dd, J = 8.0, 1.7 Hz, 1H),
8.06 (d, J = 9.1 Hz, 2H), 7.57 (br d, J = 7.4 Hz, 1H),
7.37 (t, J = 7.6 Hz, 1H), 6.88 (s, 1H), 6.02–6.09 (m,
1H), 5.10–5.18 (m, 2H), 3.74 (d, J = 6.3 Hz, 2H); 13C
NMR (CDCl3, 100 MHz) d 178.3, 160.3, 154.2, 149.4,
137.9, 135.10, 134.80, 129.50, 127.20, 125.50, 124.30,
124.10, 123.97, 117.20, 109.40, 34.00; IR (neat) 1747,
1525, 1346 cmÀ1; FAB MS m/z (relative intensity) 308
(MH+, 100). Anal. Calcd for (C18H13NO4Æ0.2H2O): C,
69.54; H, 4.34; N, 4.51. Found: C, 69.49; H, 4.53; N,
4.23.
1
with hexanes; H NMR (CD3OD/CDCl3, 400 MHz) d
7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.80 (br d, J = 8.9 Hz,
2H), 7.50 (br d, J = 7.2 Hz, 1H), 7.24 (t, J = 8.0 Hz,
1H), 7.01 (dd, J = 8.9, 1.6 Hz, 2H), 6.64 (s, 1H), 3.80
(s, 2H); 13C NMR (CDCl3, 100 MHz) d 179.20,
172.58, 162.95, 154.34, 143.73, 135.71, 127.91, 127.68,
125.10, 124.53, 124.43, 123.38, 119.45, 106.24, 35.64;
IR (neat) 3500–3073, 2094, 1698, 1632 cmÀ1; HRMS
(FAB) calcd for C17H12N3O2 (MH+): 322.0828. Found:
322.0841.
4.4. 2-(4-Azidophenyl)-8-prop-2-enylchromen-4-one (9)
A slurry of compound 7 (1.4 g, 4.5 mmol) and Na2S2O4
(8.3 g, 48.5 mmol) in a mixture of acetone (40 mL) and
water (20 mL) was at 50 ꢀC for 1 h. The reaction mixture
was concentrated to a yellow solid. The solid was
washed with EtOAc (100 mL) and the washing was con-
centrated under reduced pressure to afford intermediate
amine 8. This product was considered pure enough for
5. Experimental (mass spectrometry)
All fast-atom bombardment mass spectra (FABMS)
were obtained on a VG 7070E-HF double-focusing mass
spectrometer in positive ion mode, except where noted
1
the subsequent reaction: H NMR (CDCl3, 400 MHz)
d 8.06 (dd, J = 9.5, 1.6 Hz, 1H), 7.71 (dm, J = 8.8 Hz,

K. Malolanarasimhan et al. / Bioorg. Med. Chem. 13 (2005) 2717–2722
2721
otherwise. A sample matrix of 3-nitrobenzyl alcohol
(NBA) was employed, and ionization was effected by a
beam of xenon atoms generated in a saddle-field ion
gun at 1.2 mA 8.2 kV. Nominal mass MS were obtained
at a resolution of 1200, while accurate mass analysis
(HRMS) was carried out at a resolution of 4000. For
the latter, a limited-range V/E scan was employed under
control of a MASPEC-II32 data system for Windows
(MasCom GmbH). Matrix-derived ions were utilized
as the internal mass references for accurate mass deter-
minations. 1H and 13C NMR data were used to set con-
straints for the calculation of all possible elemental
compositions within 20 ppm of the measured accurate
mass. In all cases, a unique molecular formula could
be determined. MALDI mass spectra were used to fol-
low the Staudinger reaction and were obtained on a
Kratos-Axima-CRF mass spectrometer using a-cyano-
4-hydroxycinnamic acid as the sample matrix. Both
external and internal mass calibration was employed;
for internal calibration, the peptide mass reference stan-
dards (m/z 757–2465) were directly co-mixed with the
sample in the matrix.
CA). Total cellular RNA (5 lg) from each sample was
utilized for the Multiprobe RNAse Protection Assay.
5.3. Multiprobe RNAse protection assay
This assay was performed according to the manufac-
turerꢀs directions (Pharmingen, San Diego, CA) with
the following modifications.
5.4. Hybridization
Probes were synthesized with 33P UTP (70–80 lC/full
reaction) utilizing the Pharmingen In Vitro Transcrip-
tion kit and the mck-5b probe set. Following incuba-
tion, yeast tRNA and EDTA were added as described
by the manufacturer (Pharmingen), the reaction was
placed on Amersham-Pharmacia G25 Microspin col-
umns and the probe purified by centrifugation for
2 min at 3000 rpm. 0.5–1.0 · 106 cpm was added to each
RNA in a final hybridization volume of 10–20 lL (at
least 50% Pharmingen hybridization buffer).
5.5. RNAse inactivation
5.1. Formation of FAA–FLAG peptide conjugate 4
A master cocktail, containing 200 lL Ambion RNAse
inactivation/precipitation reagent III (Ambion, Inc.,
Austin, TX), 50 lL ethanol, 5 lg yeast tRNA, and
1 lL Ambion GycoBlue co-precipitate per RNA sample
was utilized to precipitate the protected RNA. After
adding the individual RNAse treated samples to
250 lL of the inactivation/precipitation cocktail, the
samples were mixed well, placed at À70 ꢀC for 15 min,
and subjected to centrifugation at 14,000 rpm for
15 min in a room temperature microcentrifuge. The
supernatants were decanted, a sterile cotton swab was
used to remove excess liquid and the pellet was resus-
pended in 3 lL of Pharmingen sample buffer prior to
gel electrophoresis and autoradiography.
To a screw-cap polypropylene Eppendorf tube of
1.5 mL capacity were added 125 lL of 0.24 mM FLAG
peptide–phosphine 3 (29 nmol) in pH 7.2, 10 mM
NH4CO3 buffer and 40 lL 1.0 mM FAA-azide 2
(40 nmol) in pH 7.2, 10 mM NH4CO3 buffer containing
2.5% DMSO. The resulting solution was mixed by vor-
texing and allowed to react for 1 h in an Eppendorf
ThermoStat plus constant temperature block main-
tained at 25 0.5 ꢀC. A second 125 lL aliquot of
0.24 mM FLAG peptide–phosphine 3 (29 nmol) in
pH 7.2 buffer was placed in an Eppendorf tube without
FAA-azide 2 to serve as a control. At the end of the
reaction period, each solution was diluted with an equal
volume (165 and 125 lL, respectively) of distilled H2O
and frozen in dry ice prior to an initial lyophilization.
The resulting residues were redissolved in 250 lL dis-
tilled H2O and lyophilized two more times. After the
third lyophilization, each residue was dissolved in
200 lL 4:1 CH3CN/H2O, and a 25 lL aliquot of each
solution was further diluted with an equal volume of
0.05% trifluoroacetic acid in 50% CH3CN/H2O. Multi-
ple 0.5 and 1.0 lL samples of these diluted solutions
were taken for analysis by MALDI MS.
Acknowledgements
We wish to thank Professor Carolyn R. Bertozzi, Uni-
versity of California, Berkeley, CA, and her student Jen-
nifer Prescher for kindly providing a generous amount
of the FLAG peptide–phosphine (3). We also wish to
thank Mike Sanford (LEI) for performing the Multip-
robe RNAse Protection Assay.
MALDI MS calcd for C78H85N11O27P (MH+):
1638.535. Found: 1638.522.
References and notes
1. Atassi, G.; Briet, P.; Berthelon, J.-J.; Collonges, F.
Synthesis and antitumor activity of some 8-substituted-4-
oxo-4H-1-benzopyrans. Eur. J. Med. Chem. Chim. Ther.
1985, 20, 393–402.
2. Plowman, J.; Narayanan, V. L.; Dykes, D.; Szarvasi, E.;
Briet, P.; Yader, O. C.; Paull, K. D. Flavone acetic acid: a
novel agent with preclinical anti-tumor activity against
colon adenocarcinoma 38 in mice. Cancer Treat. Rep.
1986, 70, 631–635.
MALDI MS (control, 3) calcd for C62H76N10O23P
(MH+): 1359.482. Found: 1359.473.
5.2. Cell culture
A mouse C57BL/6 macrophage cell line, immortalized
with the raf and myc oncogenes, was treated with azi-
do-FAA (10 lg/mL) or FAA (100 g/mL) for 6 h at
37 ꢀC. A that time the media was removed and total cel-
lular RNA extracted with Trisol (Invitrogen, Carlsbad,
3. Corbett, T. H.; Bissery, M.-C.; Wozniak, A.; Plowman, J.;
Polin, L.; Tapazoglou, E.; Dieckman, J.; Valeriote, F.

2722
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