DNA-dependent protein kinase (DNA-PK) inhibitors: Structure-activity relationships for O-alkoxyphenylchromen-4-one probes of the ATP-binding domain

Article abstract of DOI:10.1016/j.bmcl.2010.12.047

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H- chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC₅₀ = 8 nM).

Full text of DOI:10.1016/j.bmcl.2010.12.047

Bioorganic & Medicinal Chemistry Letters 21 (2011) 966–970
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
DNA-dependent protein kinase (DNA-PK) inhibitors: Structure–activity
relationships for O-alkoxyphenylchromen-4-one probes
of the ATP-binding domain
Kate M. Clapham a, Julia Bardos b, M. Raymond V. Finlay c, Bernard T. Golding a, Edward J. Griffen c,
Roger J. Griffin a, Ian R. Hardcastle a, Keith A. Menear b, Attilla Ting c, Paul Turner c, Gail L. Young c,
Céline Cano a,
⇑
a Newcastle Cancer Centre, Northern Institute for Cancer Research, School of Chemistry, Bedson Building, Newcastle University, Newcastle Upon Tyne NE1 7RU, United Kingdom
b KuDOS Pharmaceuticals Ltd, 410 Cambridge Science Park, Milton Rd., Cambridge CB4 0PE, United Kingdom
c AstraZeneca Oncology iMed, Mereside, Alderley Park, Macclesfield SK10 4TG, United Kingdom
a r t i c l e i n f o
a b s t r a c t
Article history:
Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one
pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be
probed further. Structure–activity relationships have been elucidated for inhibition of DNA-PK and PI3K
(p110a), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-mor-
pholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC50 = 8 nM).
Received 15 November 2010
Revised 7 December 2010
Accepted 8 December 2010
Available online 13 December 2010
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
DNA repair
DNA-PK
Kinase inhibitors
Anticancer drugs
Structure–activity relationship studies
DNA-dependent protein kinase (DNA-PK), a member of the
phosphatidylinositol (PI) 3-kinase related kinase (PIKK) family, is
a multi-component serine/threonine protein kinase that plays a
key role in the repair of mammalian DNA double-strand breaks
(DSBs) via the non-homologous end joining pathway of DNA re-
pair.1,2 Human cell lines defective in DNA-PK function are hyper-
sensitive to agents that elicit DNA DSBs.3,4 By impeding DNA DSB
repair, selective DNA-PK inhibitors have potential application as
radio- and chemo-potentiators in the treatment of cancer.5–9 In
the absence of structural biology information for the enzyme, we
have conducted extensive structure–activity relationships studies
(SARs) using homology modelling based on the known crystal struc-
ture of PI3Kc10 and employing 2-morpholino-8-phenyl-4H-chro-
men-4-one (1, LY294002) as a template for inhibitor design.
Interestingly, a number of potent DNA-PK inhibitors have been
developed from this structural class.11–15 Previously we have
described the incorporation of a dibenzothiophen-4-yl substituent
at the 8-position of 2-morpholino-4H-chromen-4-one, which con-
a human tumour cell line to both ionising radiation and the topoiso-
merase II inhibitor etoposide in vitro and in vivo.16 Further refine-
ment of this template has provided the highly potent DNA-PK
inhibitor KU-0060648 (3; IC50 = 5 nM).17
hydrophobic
pocket
R
S
O
S
O
O
O
O
N
8
O
N
O
O
N
O
4
NEt
1
2; R = H
3; R =
O
N
HN
O
O
N
N
R
O
O
N
O
ferred excellent inhibitory activity against DNA-PK (2, IC50
=
O
O
N
28 nM). Crucially, chromenone 2 has been demonstrated to sensitise
O
5; R = OTf
O
O
B
6; R =
⇑
7
Corresponding author. Tel.: +44 191 2227060.
E-mail address: celine.cano@ncl.ac.uk (C. Cano).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.047

K. M. Clapham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 966–970
967
With a view to optimising the biological and pharmaceutical
properties of NU7441 (2), and to expand SARs, both the core
chromenone scaffold and the dibenzothiophen-4-yl moiety have
been systematically modified. To this end, we have recently re-
ported the identification of 8-biarylchromen-4-ones (e.g., 4;
IC50 = 18 nM), which offered the opportunity to further probe re-
gions of the ATP-binding domain of the enzyme and exhibited good
potency against DNA-PK.18 Interestingly, subsequent homology
modelling studies suggested that the heteroaryl substituent may
occupy a putative hydrophobic pocket that could be further
exploited. In this communication, we report studies designed to
probe this region of the ATP-binding site of the kinase, through
the synthesis and biological evaluation of a focused library of chro-
men-4-one DNA-PK inhibitors bearing O-alkoxyphenyl substitu-
ents at the 8-position.
Additional derivatives were synthesised using a modified ap-
proach (Scheme 2). Commercially available 4-bromo-2-methoxy-
aniline (12) was converted into the boronic ester 13, followed by
a Suzuki–Miyaura cross-coupling with chromenone triflate 5 to
afford the corresponding arylamine 14. Acylation of 14 with chlo-
roacetyl chloride, followed by reaction with morpholine, provided
the methoxy-variant 15 and subsequent deprotection gave the
phenol 16. The key intermediate 16 was also accessed using an
alternative synthetic route (Scheme 2). Commercially available
1-bromo-4-nitrobenzene (17) was converted into 5-bromo-2-nitro-
phenol (18) in 79% yield, using cumene hydroperoxide and tert-
BuOK in the presence of ammonia.21 Again, Suzuki–Miyaura
cross-coupling with 5 followed by reduction of the nitrophenol
intermediate gave aniline 20. Finally, phenol 16 was obtained in
78% yield by chloroacetylation followed by reaction with morpho-
line. The effect upon biological activity of alkylation of hydroxyphe-
nylchromen-4-one 16 was investigated through the preparation of a
small series of O-alkoxyphenyl derivatives 21a–j (Scheme 2 and
Table 1).22,23
To enable further SAR studies, the 2-hydroxyethoxy (23) and
aminoethoxy (26) derivatives were also synthesised (Scheme 3).
Alkylation of phenol intermediate 16 with 2-(tert-butyldimethylsi-
lyloxy)ethyl bromide24,25 proceeded in 92% yield, followed by
TBDMS removal giving the desired alcohol 23 in quantitative yield.
Tosylation of 23 and treatment with NaN3 in DMF at 60 °C gave
azide 25, which was reduced with triphenylphosphine in the pres-
ence of water to afford the desired amine 26 in 34% yield.
Our previous SAR studies have highlighted the importance of an
appropriate 8-aryl substituent for chromenone-based inhibitors
(e.g., dibenzothiophen-4-yl in 2 or a 3-arylphenyl moiety in 4).
The overall objective of this study was to investigate the effect
upon DNA-PK inhibitory activity of replacing the planar dibenzo-
thiophenyl group by a conformationally flexible and less lipophilic
O-alkoxyphenyl system, to probe the hydrophobic pocket of the
ATP-binding site. In addition, the analogues were chosen to inves-
tigate whether alkyl substituents were tolerated at this position,
given that all other known derivatives are substituted with an aryl
or heteroaryl group at C-8. The chemical structures and inhibitory
Our previous studies have utilised the chromenone triflate
5
11–13 or boronic ester 619 as key intermediates for the preparation
of 8-substituted chromenone libraries, employing Suzuki–Miyaura
cross-coupling reactions. This strategy was also amenable for the
synthesis of the target O-alkoxyphenylchromen-4-ones (11a–b,
15 and 21a–j). Introduction of the 8-phenyl substituent was
achieved by coupling of 6, formed in situ by reaction of 5 with
bis(pinacolato)diboron, with 4-bromo-2-fluoronitrobenzene to
give the chromen-4-one 8 in 58% yield (Scheme 1). The 3-alkoxy
side chain was then introduced in good overall yield by a fluoro-
displacement reaction, using an alkoxide generated through treat-
ment of the respective alcohol with sodium hydride in DMF.
Reduction of the nitro intermediates 9a and 9b to the required
arylamines (10a, 10b) proceeded in excellent yields using zinc in
acetic acid. Finally, in a one-pot two-step process, acylation of
10a–b with chloroacetyl chloride was followed by reaction with
morpholine to provide inhibitors 11a–b (Table 1). In this series,
the choice of a morpholine-substituted side chain was based upon
previous studies which demonstrated that the parent biaryl
derivative 7 exhibited good inhibitory activity against DNA-PK
(IC50 = 14 nM) and potentiated the DNA damage elicited by
ionising radiation at 2 Gy (HeLa cells, DMR (0.5
lM) = 8 and DMR
(0.1
l
M) = 11).20
NO2
RO
NO2
F
O
O
a
b
O
B
O
O
O
N
O
O
N
O
N
O
9a; R =
9b; R =
O
6
8
O
O
O
N
NH2
HN
RO
RO
c
d
O
O
O
N
O
O
N
O
10a-b
11a-b
Scheme 1. Reagents and conditions: (a) bis(pinacolato)diboron, PdCl2(dppf), dppf, KOAc, 1,4-dioxane, reflux, 18 h,19 then 4-bromo-2-fluoronitrobenzene, Pd(PPh3)4, Na2CO3,
H2O, reflux, 3 h, 58%; (b) cyclopropanemethanol (for 9a) or 3-methyl-3-oxetanemethanol (for 9b), NaH, DMF, 25 °C, 15 min, then 8, 100 °C, 20 min, 9a 89%, 9b 58%; (c) Zn
powder, AcOH, 25 °C, 4 h, 10a 82%, 10b 71%; (d) (i) chloroacetyl chloride, Et3N, DMA, 25 °C, 30 min; (ii) morpholine, 25 °C, 18 h, 11a 15%, 11b 65%.

968
K. M. Clapham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 966–970
NO2
Br
NH2
R
R
MeO
17; R = H
18; R = OH
12; R = Br
; R = B(pin)
a
e
13
f
b
O
O
O
O
R
NH2
N
N
HN
HN
HO
MeO
MeO
RO
c
d
h
O
O
O
O
O
N
O
O
N
O
O
N
O
O
N
O
14
19; R = NO2
20; R = NH2
15
16; R = H
21a-j; R = alkyl
g
i
Scheme 2. Reagents and conditions: (a) bis(pinacolato)diboron, PdCl2(dppf), dppf, KOAc, DMSO, 80 °C, 2 h, 36%; (b) 5, Pd(PPh3)4, Cs2CO3, 1,4-dioxane, DMA, MW, 140 °C,
15 min, 43%; (c) (i) chloroacetyl chloride, Et3N, DCM, 25 °C, 3 h; (ii) morpholine, 25 °C, 18 h, 74%; (d) BBr3, DCM, ꢀ78–25 °C, 1 h, 13%; (e) cumene hydroperoxide, tert-BuOK,
NH3, THF, ꢀ78 °C to reflux, 15 min, 79%; (f) 5, Pd(PPh3)4, 2 M Na2CO3, 1,4-dioxane, MW, 150 °C, 1 h, 59%; (g) Zn powder, AcOH, 25 °C, 3.5 h, 92%; (h) (i) chloroacetyl chloride,
DCM, 30 °C, 3 h; (ii) morpholine, 25 °C, 18 h, 78%; (i) R–X or R-OTs, K2CO3, DMF, MW, 100 °C, 15 min, 43–90% (general procedure in accordance with Ref. 22).
O
O
O
O
O
O
N
N
N
HN
HN
HN
a
d
HO
O
O
RO
R
O
O
O
O
O
N
O
O
N
O
O
N
16
22; R = TBDMS
23; R = H
24; R = Ts
25; R = N3
26; R = NH2
b
e
c
Scheme 3. Reagents and conditions: (a) 2-(tert-butyldimethylsilyloxy)ethyl bromide,24,25 K2CO3, DMF, MW, 100 °C, 15 min, 92%; (b) TBAF, THF, 25 °C, 1.5 h, quantitative; (c)
TsCl, Et3N, DMAP, DCM, 25 °C, 18 h, 48%; (d) NaN3, DMF, 60 °C, 2.5 h; (e) PPh3, H2O, 25 °C, 2.5 h, 34% over two steps.
activities of the library compounds are summarised in Table 1.
With view to delineating SARs and designing selective
DNA-PK inhibitor, all compounds were also tested against the
related enzyme PI3-kinase
For the series of arylchromenone-4-ones bearing small O-alk-
oxy substituents at the phenyl 3-position (15, 21a–21d), it is
evident that substitution improved neither activity nor selectivity
compared with the dibenzothiophenyl analogue 2. Overall, a
6- to 12-fold reduction in potency was observed, and with the
exception of the ethoxy derivative 21a, which showed approxi-
mately a twofold selectivity for DNA-PK, all compounds proved
and previous SARs around this position, that indicate a limited
steric tolerance in this region of the ATP-binding domain.18
Disappointingly, the incorporation of hydrogen bond donor and
acceptor groups onto the alkoxy side chain (e.g., 21e, 23 and 26)
was not beneficial for DNA-PK inhibitory activity, and resulted in
up to 117-fold reduction in potency compared with the parent
8-dibenzothiophenyl chromen-4-one 2. These compounds also
proved to be non-selective being equipotent for DNA-PK and
a
a
a.
PI3K
function (23) resulted in a modest improvement in activity, but
both compounds were non-selective for DNA-PK versus PI3K
a. Replacement of the methoxy group of 21f by a hydroxyl
a.
to be equipotent for DNA-PK and PI3K
a.
Interestingly, removal of the alkoxy side chain conferred good
Increasing the length and steric bulk of the side chain (e.g.,
DNA-PK inhibitory activity, with phenol 16 exhibiting a fivefold
21f–21i) proved detrimental to inhibitory activity for both DNA-
selectivity profile for DNA-PK (IC50 = 0.08
(IC50 = 0.43 M).
In light of the overall loss of activity and selectivity observed
with this series of arylchromenone-4-ones bearing small O-alkoxy
lM) versus PI3Ka
PK and PI3K
21i, a noticeable reduction in potency towards DNA-PK and PI3K
was observed. This is consistent with homology modelling studies
a
. For example, with the cyclopropylethoxy derivative
l
a

K. M. Clapham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 966–970
969
Table 1
DNA-PK and PI3K
potent and a modestly selective inhibitor, achieving selectivity
for DNA-PK over PI3K has proven challenging. Overall, this study
a inhibitory activity of O-alkoxyphenyl chromen-4-ones
a
has further elucidated our understanding of SARs around a puta-
tive hydrophobic region of the ATP-binding site, indicating a lim-
ited steric tolerance and hydrophobic complementarily. Further
studies are currently underway to elucidate the binding mode of
this class of chromen-4-one based DNA-PK inhibitors.
O
O
N
HN
RO
O
Acknowledgements
O
O
N
The authors thank Dr. Karen Haggerty and the AstraZeneca
Oncology iMed purification group for the analytical and semi-pre-
parative HPLC. The biochemical screening expertise of Marcus Pea-
cock, Nahida Parveen, William Deacon and the AstraZeneca
Oncology iMed biochemical screening team is appreciatively
recognised. The use of the EPSRC Mass Spectrometry Service at
the University of Wales (Swansea) and financial support from Can-
cer Research UK are also gratefully acknowledged.
a
Compound
R
DNA-PK IC50
(lM)
PI3Kab IC50
(lM)
1 (LY294002)
2
—
—
1.3
0.03
0.50
0.13
11a
0.008
0.07
O
References and notes
11b
0.69
0.30
15
16
21a
21b
Me
H
Et
0.29
0.08
0.28
0.33
0.35
0.43
0.47
0.29
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R. J. Bioorg. Med. Chem. Lett. 2008, 18, 4885.
O
21e
3.3
1.1
H2N
21f
2.6
1.5
4.4
MeO
N
21g
>10
21h23
21i
0.16
0.5
0.29
0.4
21j
23
0.11
0.27
1.0
12
1.6
3
HO
26
H2N
a
IC50 values were determined in accordance with Ref. 13 and are the means of
three separate determinations.
IC50 values were determined in accordance with Ref. 26 and are the means of
three separate determinations.
b
substituents, we were pleased to identify a potent and relatively
selective inhibitor 11a, exhibiting IC50 values of 0.008
0.07 M for DNA-PK and PI3K , respectively. The fact that 11a is
approximately ninefold selective for DNA-PK versus PI3K and
fourfold more potent than the parent 8-dibenzothiophenyl chro-
men-4-one 2, suggests that the cyclopropylmethoxy group of 11a
might be making productive interactions with a hydrophobic
region of the ATP-binding domain of DNA-PK. It is notable that
compared to 11a, the sec-butyl analogue 21c is ca. 20-fold less
potent against DNA-PK but only ca. twofold less potent against
lM and
l
a
a
19. Cano, C.; Golding, B. T.; Haggerty, K.; Hardcastle, I. R.; Peacock, M.; Griffin, R. J.
Org. Biomol. Chem. 2010, 8, 1922.
20. DMR clonogenic assay: HeLa cells were seeded per well into a 6-well tissue
culture treated dish and incubated overnight at 37 °C/5% CO2. Following a 1 h
pre-treatment with either vehicle or compound the plates were exposed to
2 Gy ionising radiation using a Faxitron 43855D X-ray source and incubated
overnight at 37 °C/5% CO2. The media was replaced with fresh media in the
absence of compound or vehicle and incubated for a further 6–8 days. The
media was removed and the cell colonies were fixed and stained with Giemsa
and scored with an automated colony counter (Oxford Optronics Ltd, Oxford,
United Kingdom).
PI3Ka.
In summary, we have identified a novel series of O-alkoxyphe-
nyl chromen-4-ones that exhibit a range of potencies against
DNA-PK. These compounds represent the first exemplified chrome-
none-based DNA-PK inhibitors that lack an aryl substituent
directly attached at the C-2 position of the phenyl ring. With the
exception of the cyclopropylmethoxy derivative 11a, the most
The dose modification ratio (DMR) is defined as the ratio of the number of cells
that survive a single 2 Gy dose of IR to that of the number of cells that survive
the same dose in combination with a given concentration of DNA-PK inhibitor.
This value provides an indirect measure of the ability of a particular compound
to potentiate the DNA damage elicited by IR, and also indicates whether or not
the compound in question is cell permeable. IR alone at the dose used (2 Gy)

970
K. M. Clapham et al. / Bioorg. Med. Chem. Lett. 21 (2011) 966–970
caused 40–60% survival. The DNA-PK inhibitor alone, at the concentrations
used (0.5 and 0.1 M), was not cytotoxic and hence the dose modification
observed represents radiopotentiation and not additive toxicity. The Dose
Modification Ratio (DMR) at 2 Gy irradiation was calculated as follows:
112.2, 119.3, 122.3, 123.6, 124.9, 125.1, 127.4, 130.3, 132.0, 133.7, 147.8, 150.8,
l
162.8, 168.3, 177.4; HRMS calcd for C30H36N3O6 [M+H]+ 534.2599, found
534.2589.
24. Borbas, K. E.; Mroz, P.; Hamblin, M. R.; Lindsey, J. S. Bioconjugate Chem. 2006,
17, 638.
% survival ꢀ compound= þ 2 Gy
DMR ¼
25. Vader, J.; Sengers, H.; de Groot, A. Tetrahedron 1989, 45, 2131.
26. PI3K alpha enzyme assay: To generate IC50 values, compounds were solubilised
in 100% DMSO at 10 mM, and then serially-diluted in 100% DMSO using a
VPrep liquid handler (Agilent Technologies UK Limited, Stockport, Cheshire,
UK) to give four 1:100 dilutions in a Labcyte acoustic-certified 384-well plate
(Labcyte Incorporated, Sunnyvale, California, USA). A Labcyte Echo acoustic
% survival þ compound= þ 2 Gy
21. Makosza, M.; Sienkiewicz, K. J. Org. Chem. 1998, 63, 4199.
22. General procedure for the library synthesis (21a–21j):
A mixture of N-(2-
hydroxy-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-
morpholinoacetamide 16 (1.0 equiv), K2CO3 (1.0 equiv), alkyl halide for 21a–g
(2.0 equiv), or alkyl tosylate for 21h–j (2.0 equiv; alkyl tosylates were
synthesised from the alkyl alcohol, using TsCl, Et3N, DMAP, DCM, 25 °C, 18 h)
in anhydrous DMF was heated under microwave irradiation at 100 °C for
15 min. Water was added to the resulting mixture and the product was
extracted into EtOAc. The combined organic layers were washed with brine
and dried over Na2SO4. The solvent was removed in vacuo to give the crude
product which was subsequently purified by medium pressure flash
chromatography.
liquid dispenser was used to generate a 12-step dose range from 200
lM down
to 0.2 nM (in a 6 L total reaction volume), dispensing a total of 120 nL
l
compound and/or DMSO into each well of a white low-volume 384-well plate
(Greiner Bio-One Limited, Stonehouse, UK). The maximum signal was
generated using 120 nL of a known inhibitor, and the minimum signal was
generated with 120 nL of 100% DMSO. Kinase reaction buffer was prepared in
deionised water to give 50 mM Tris (pH 7.4), 0.05% CHAPSO, 10 mM MgCl2 and
2.1 nM DTT. Enzyme solution was made up in kinase reaction buffer to give
40 nM PI3K alpha isoform enzyme (20 nM final concentration, in-house
enzyme preparation), and substrate solution was made up in kinase reaction
23. Synthesis of N-(2-(cyclobutylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-
yl)phenyl)-2-morpholinoacetamide (21h): Following the general procedure22
using 16 (40 mg, 0.086 mmol), cyclobutylmethanol O-toluene-4-sulfonate
(43 mg, 0.18 mmol), K2CO3 (12 mg, 0.086 mmol) and DMF (3 mL).
Purification by medium pressure chromatography on Si-NH using EtOAc gave
the title compound as a white solid (35 mg, 76%): mp 229–231 °C; kmax (EtOH)/
nm 209.0, 302.5; IR (cmꢀ1), 3309, 2851, 1687, 1626, 1568, 1525, 1404; 1H NMR
(500 MHz, CDCl3) d 1.88–1.94 (2H, m, cyclobutyl-CH2), 1.98–2.09 (2H, m,
cyclobutyl-CH2), 2.23–2.29 (2H, m, cyclobutyl-CH2), 2.66 (4H, t, J = 4.4 Hz,
NCH2-morpholine), 2.87 (1H, septet, J = 7.0 Hz, cyclobutyl-CH), 3.21 (2H, s,
N(H)C(O)CH2), 3.38 (4H, t, J = 4.8 Hz, NCH2-morpholine), 3.74 (4H, t, J = 4.8 Hz,
OCH2-morpholine), 3.81 (4H, t, J = 4.4 Hz, OCH2-morpholine), 4.06 (2H, d,
J = 7.0 Hz, Ar-OCH2cyclobutane), 5.52 (1H, s, C-3 chromenone), 7.03 (1H, d,
J = 1.6 Hz, H-Ar), 7.14 (1H, dd, J = 8.3 Hz, J = 1.6 Hz, H-Ar), 7.40 (1H, t, J = 7.6 Hz,
H-Ar), 7.55 (1H, dd, J = 7.6 Hz, J = 1.7 Hz, H-Ar), 8.17 (1H, dd, J = 7.7 Hz,
J = 1.7 Hz, H-Ar), 8.54 (1H, d, J = 8.3 Hz, H-Ar), 9.74 (1H, s, N(H)C(O)); 13C NMR
(125 MHz, CDCl3) d 18.9, 25.2, 34.7, 44.9, 54.0, 63.0, 66.1, 67.3, 73.0, 87.3,
buffer to give 16
concentration, respectively, PIP2 purchased from Cayman Chemical
Company, Ann Arbor, Michigan, USA). 3 L enzyme solution was added to
each well with an Aquamax liquid dispenser (Molecular Devices, Sunnyvale,
California, USA) and, after a 20 min incubation at room temperature, 3 L per
lM ATP and 160 lM PIP2 (8 lM and 80 lM final
l
l
well substrate solution was added. Plates were then covered and incubated at
room temperature for 80 min. At the end of the incubation period, the kinase
reaction was stopped by adding 4 l
L per well of Kinase-GloÒ Plus Reagent
(Promega Corporation, Madison, Wisconsin, USA, prepared as per
manufacturers instructions and brought to room temperature prior to
addition) with an Aquamax. Plates were incubated for at least 20 min at
room temperature, before being read with a Pherastar microplate reader (BMG
Labtech GmbH, Offenburg, Germany) with the standard luminescence filter
block, and the gain set at 3400.