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(d, J=9 Hz, 2H), 4.02 (t, J=6 Hz, 2H), 3.51 (t, J=6.5 Hz, 2H), 2.16–
1.92 ppm (m, 4H); 13C NMR (75 MHz, CDCl3): d=190.62, 157.58,
152.21, 138.23, 131.41, 131.06, 129.03, 122.18, 116.09, 67.30, 33.47,
29.55, 27.99 ppm; MS (ES+): m/z: 453.0.
Experimental Section
General: All chemicals were purchased from Sigma–Aldrich or
Acros Organics and were used as received. All other chemicals
were used as received from the appropriate suppliers. Preparative
flash chromatography was carried out with Merck silica gel (Si 60,
35–70 mm). NMR spectra were recorded with a Bruker Advance 300
spectrometer; 1H NMR spectra were recorded at 300 MHz and
13C NMR spectra at 75 MHz. Chemical shifts are reported in ppm
downfield from TMS (d=0.00) for 1H NMR and from the central
CDCl3 resonance (d=77.16) or [D6]DMSO resonance (d=39.52) for
13C NMR. HPLC was carried out with a Waters Alliance instrument
equipped with a photodiode array detector and a Xterra MS
column with a linear gradient of acetonitrile versus water contain-
ing 0.1% FA ranging from 10 to 55% over 5 min and then to
100% over 6 min at a flow rate of 1 mLminÀ1. Mass spectrometry
was performed at the Institut Curie for Electrospray Ionization (ESI).
HRMS was performed at the Institut de Chimie des Substances Na-
turelles (ICSN) in Gif-sur-Yvette.
4’,4’’-Bisformyl-4-(4-maleimidobutoxy)triphenylamine
(6):
3a,4,7,7a-Tetrahydro-2H-4,7-epoxyisoindole-1,3-dione ([6253–28–7],
487 mg, 2.95 mmol, 2 equiv) and potassium carbonate (407 mg,
2.95 mmol, 2 equiv) were added under argon to a solution of dial-
dehyde 4 (667 mg, 1.47 mmol, 1 equiv) in anhydrous DMF (40 mL).
The resulting mixture was stirred for one hour at room tempera-
ture and overnight at 408C. Solvents were removed under reduced
pressure. Water (20 mL) was added to the residue, and the ob-
tained aqueous phase was extracted twice with CH2Cl2. The organ-
ic phase was washed twice with water, dried over MgSO4 and con-
centrated. After inspection by LC-MS, the protected maleimide 5
was dissolved in toluene (50 mL) and stirred at 1208C overnight.
After concentration, purification by flash chromatography using
CombiFlash (CH2Cl2/EtOH 100 to 98:2) afforded the title compound
(354 mg, 0.75 mmol, 51%) as a yellow oil. 1H NMR (300 MHz,
CDCl3): d=9.87 (s, 2H), 7.75 (d, J=8.5 Hz, 4H), 7.15 (d, J=8.5 Hz,
4H), 7.09 (d, J=9 Hz, 2H), 6.91 (d, J=9 Hz, 2H), 6.71 (s, 2H), 3.99
(m, 2H), 3.61 (m, 2H), 1.80 ppm (m, 4H); 13C NMR (75 MHz, CDCl3):
d=190.66, 170.98, 157.61, 152.22, 138.15, 134.26, 131.42, 131.02,
129.02, 122.17, 116.12, 67.53, 37.62, 26.60, 25.47 ppm; MS (ES+): m/
z: 469.2; HRMS (ESI+): m/z calcd for C28H24N2O5: 469.1763; found
469.1768
4,4’-Bisformyl-4’’-methoxytriphenylamine (2): POCl3 (10 mL,
16.7 g, 109 mmol, 15 equiv) was added dropwise, at 08C under Ar,
over 15 min to DMF (9 mL, 7.9 g, 109 mmol, 15 equiv). The solution
was stirred at 08C for 1 h. 4-Methoxytriphenylamine (2 g,
7.26 mmol) was added, and the solution was stirred at 958C for 4 h
30 min. The dark mixture was poured into an ice bath and stirred
for 30 min. The solution was basified with NaOH (1m). The aque-
ous phase was extracted with CH2Cl2, and the organic phase was
washed twice with water, dried over MgSO4 and concentrated
under reduced pressure to give a black oil. Purification by flash
chromatography using CombiFlash (AcOEt/cyclohexane 10:80 to
70:30) afforded the title compound (1.72 g, 5.19 mmol, 71%). CAS
TP-2Rho-maleimide: Dialdehyde 6 (180 mg, 0.38 mmol), rhoda-
mine-3-acetic acid (162 mg, 0.84 mmol, 2.2 equiv) and ammonium
acetate (119 mg, 1.53 mmol, 4 equiv) were dissolved in acetic acid
(50 mL). The solution was stirred at 110 8C overnight. The reaction
mixture was allowed to cool to room temperature and concentrat-
ed under vacuum until approximatively 10 mL of acetic acid re-
mained. Ether was then added to the solution, and a dark red pre-
cipitate formed. The solid was filtered, washed with ether and
dried to afford the expected product (142 mg, 0.17 mmol, 46%) as
1
number [149676-16-4]; H NMR (300 MHz, CDCl3): d=9.87 (s, 2H),
7.75 (d, J=9 Hz, 4H), 7.16 (d, J=9 Hz, 4H), 7.11 (d, J=9.0 Hz, 2H),
6.94 (d, J=9.0 Hz, 2H), 3.84 ppm (s, 3H).
4,4’-Bisformyl-4’’-hydroxytriphenylamine (3): BBr3 (2.7 mL, 7.21 g,
28.79 mmol, 3 equiv) was added dropwise at 08C to a solution of 2
(3.18 g, 9.6 mmol, 1 equiv) in CH2Cl2 (20 mL). The reaction mixture
was then allowed to warm slowly to RT and stirred for 3 h. The so-
lution turned from yellow to dark. The mixture was then slowly
poured into an ice bath. The aqueous phase was extracted twice
with CH2Cl2. The combined organic phases were washed twice
with water, dried over MgSO4 and concentrated to give a green
foamy solid. Purification by flash chromatography using Combi-
Flash (AcOEt/cyclohexane 10:90!60:40) afforded the title com-
pound (2.8 g, 8.82 mmol, 92%) as a yellow solid. CAS number
[336619-78-4]; 1H NMR (300 MHz, CDCl3): d=9.88 (s, 2H), 7.76 (d,
J=8.5 Hz, 4H), 7.17 (d, J=8.5 Hz, 4H), 7.07 (d, J=8.5 Hz, 2H),
6.89 ppm (d, J=8.5 Hz, 2H).
1
a dark red powder. M.p. 244–2468C; H NMR (300 MHz, DMSO): d=
7.75 (s, 2H), 7.60 (d, J=9 Hz, 4H), 7.14 (m, 6H), 7.01 (m, 4H), 4.54
(s, 4H), 4.00 (t, J=5 Hz, 2H), 3.48 (t, J=6 Hz, 2H), 1.72–1.64 ppm
(m, 4H); 13C NMR (75 MHz, DMSO): d=192.82, 171.12, 167.16,
166.62, 157.04, 148.51, 137.33, 134.45, 132.79, 132.56, 128.93,
126.75, 122.20, 119.22, 116.08, 67.13, 46.27, 36.82, 25.98,
24.74 ppm; HRMS (ESI+): m/z calcd for C38H30N4O9S4: 815.0974;
found 815.1003.
Peptide synthesis and labelling: Peptides used in this study were
purchased from GL Biochem (Shanghai, P.R. China), labelled on
their single cysteine residues with maleimide dye derivatives, in
the presence of a five- to tenfold excess of dye overnight, and
then purified from free dye on NAP-5 columns (GE Healthcare).
4’,4’’-Bisformyl-4-(4-bromobutoxy)triphenylamine (4): A solution
of caesium carbonate (4.1 g, 12.6 mmol, 5 equiv) and 3 (0.8 g,
2.52 mmol, 1 equiv) in anhydrous DMF (30 mL) was stirred at room
temperature for 15 min. 1,4-Dibromobutane (4.5 mL, 37.5 mmol,
15 equiv) was then added, and the resulting mixture was stirred at
room temperature for 2 h. The reaction mixture was then concen-
trated under reduced pressure. Water (20 mL) was added to the
residue, and the obtained aqueous phase was extracted twice with
CH2Cl2. The organic phase was washed twice with water, dried
over MgSO4 and concentrated to give a yellow oil. Purification by
flash chromatography using CombiFlash (AcOEt/cyclohexane 10:90
to 60:40) afforded the title compound (475 mg, 1.05 mmol, 42%)
PISTVRE peptide: GGGCPISTVREVALLRRL
a5 D1 peptide: SIRPEELLQMELLLVNKLKWNLC
Rb peptide: YKFCSSPLRIPG
Pep-PISTVRE peptide: KETWWETWWTEKK GGGCPISTVREVALLRRL
Protein expression and purification of CDKs: Recombinant CDKs
and cyclins were expressed in Escherichia coli by IPTG induction
and purified by chromatography as described previously.[9h] GST-
CDKs or cyclins were first purified on GSTTrap columns, followed
by gel filtration chromatography on HiLoad 16/600 Superdex 75
preparatory grade.
1
as a yellow oil. H NMR (300 MHz, CDCl3): d=9.88 (s, 2H), 7.76 (d,
Design and engineering of CDK4 mutant: The cDNA sequence of
J=8.5 Hz, 4H), 7.16 (d, J=8.5 Hz, 4H), 7.11 (d, J=9 Hz, 2H), 6.92
mouse CDK4 was cloned into the pGex6P1 vector (GE Healthcare)
ChemBioChem 2016, 17, 737 – 744
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