Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications

Article abstract of DOI:10.1002/cmdc.201500589

The natural product staurosporine is a high-affinity inhibitor of nearly all mammalian protein kinases. The labelling of staurosporine has proven effective as a means of generating protein kinase research tools. Most tools have been generated by acylation of the 4′-methylamine of the sugar moiety of staurosporine. Herein we describe the alkylation of this group as a first step to generate a fluorescently labelled staurosporine. Following alkylation, a polyethylene glycol linker was installed, allowing subsequent attachment of fluorescein. We report that this fluorescein-staurosporine conjugate binds to cAMP-dependent protein kinase in the nanomolar range. Furthermore, its binding can be antagonised with unmodified staurosporine as well as ATP, indicating it targets the ATP binding site in a similar fashion to native staurosporine. This reagent has potential application as a screening tool for protein kinases of interest.

Full text of DOI:10.1002/cmdc.201500589

DOI: 10.1002/cmdc.201500589
Full Papers
Alkylation of Staurosporine to Derive a Kinase Probe for
Fluorescence Applications
Alexander J. M. Disney, Barrie Kellam, and Lodewijk V. Dekker*[a]
The natural product staurosporine is a high-affinity inhibitor of
nearly all mammalian protein kinases. The labelling of stauro-
sporine has proven effective as a means of generating protein
kinase research tools. Most tools have been generated by acy-
lation of the 4’-methylamine of the sugar moiety of staurospor-
ine. Herein we describe the alkylation of this group as a first
step to generate a fluorescently labelled staurosporine. Follow-
ing alkylation, a polyethylene glycol linker was installed, allow-
ing subsequent attachment of fluorescein. We report that this
fluorescein–staurosporine conjugate binds to cAMP-dependent
protein kinase in the nanomolar range. Furthermore, its bind-
ing can be antagonised with unmodified staurosporine as well
as ATP, indicating it targets the ATP binding site in a similar
fashion to native staurosporine. This reagent has potential ap-
plication as a screening tool for protein kinases of interest.
Introduction
The natural product staurosporine, first described in 1977,[1]
has been demonstrated to be a high-affinity inhibitor of nearly
all mammalian protein kinases.[2,3] Staurosporine binds to the
ATP binding site of the kinase, making multiple contacts with
the hinge region and the N- and C-terminal lobes of the cata-
lytic domain. Staurosporine itself is not used as a therapeutic
agent, but has proven invaluable in the discovery of novel an-
ticancer drugs based on kinase inhibition.[4,5] Furthermore, it
has been modified to generate functionalised molecular tools
such as immobilised staurosporine to capture kinases,[6] a staur-
osporine-based photoaffinity probe,[7] a cell-permeable affinity-
based probe for kinome labelling,[8] a highly selective bivalent
staurosporine-tethered peptide ligand,[9] and a fluorescent
staurosporine conjugate.[10]
tions of this group have been implicated in binding, with dif-
ferent kinases engaging it in a different fashion.[17] For exam-
ple, cAMP-dependent protein kinase (PKA) is predicted to
make a hydrogen bond as well as an ionic interaction, whilst
the EGF receptor kinase relies on an ionic interaction and Fyn
kinase on a hydrogen bond interaction with this group.
To derivatise staurosporine, most strategies have relied upon
modification of this 4’-methylamine group, usually involving an
acylation step. The resulting amide may be predicted to dis-
play decreased basicity with respect to the nitrogen atom, af-
fecting its ability to act as hydrogen bond acceptor. As such,
the acyl-based chemical modification may result in some loss
of affinity of the probe relative to staurosporine itself. This has
indeed been suggested as one of the reasons why, compared
with staurosporine, a probe obtained by acylation of the sec-
ondary amine showed decreased affinity to the kinase ASK1.[10]
However, acylated tools have been shown to retain kinase
binding and in some cases even showed a slight increase in af-
finity (e.g., Ref. [8]). As an alternative to the acylation modifica-
tion, we have sought to derivatise staurosporine using alkyla-
tion of the 4’-methylamine, which does not lead to formation
of an amide. We report a high-affinity fluorescent ligand, con-
sisting of staurosporine linked to fluorescein via a polyethylene
glycol linker and show that this can be used to interrogate the
ATP binding site by simple fluorescence polarisation detection.
We show that the ligand is useful to predict compound inter-
actions at the ATP binding site of PKA and that the ligand is
viable for the exploration of a wide range of different kinases.
The molecular interactions between staurosporine and nu-
merous protein kinase catalytic domains have been deter-
mined in great detail in co-crystallisation experiments.[11–16] The
main polar interaction points on staurosporine involve the
lactam oxygen and nitrogen atoms, the tetrahydropyran
moiety, as well as the 3’-methoxy and 4’-methylamine side
groups (see Supporting Information for numbering). Interac-
tions at the 4’-methylamine contribute to binding, and the
number of interactions taking place at this group appears to
correlate with affinity. Both hydrogen bond and ionic interac-
[a] Dr. A. J. M. Disney, Prof. B. Kellam, Dr. L. V. Dekker
School of Pharmacy, Centre for Biomolecular Sciences
University of Nottingham, Nottingham NG7 2RD, Nottinghamshire (UK)
E-mail: lodewijk.dekker@nottingham.ac.uk
Supporting information and the ORCID identification number(s) for the
author(s) of this article can be found under http://dx.doi.org/10.1002/
cmdc.201500589.
Results
Synthesis
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This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in
any medium, provided the original work is properly cited.
Staurosporine (1) (Scheme 1) was 4’-N alkylated with a variety
of alkyl halides of which methyl bromoacetate was found to
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Scheme 1. Reagents and conditions: a) methyl bromoacetate, potassium carbonate, THF, RT, 48 h; b) ethylenediamine, RT, 48 h; c) 1. succinic anhydride, MeCN,
RT, 12 h, 2. Fmoc-Cl, NaHCO3, MeCN, H2O, RT, 12 h; d) HBTU, DIPEA, DMF, RT, 12 h; e) diethylamine, THF, RT, 4 h; f) fluorescein isothiocyanate, DIPEA, DMF, RT,
48 h.
afford the N-acetyl methyl ester 2 in quantitative yield. Com-
pound 2 was sufficiently pure to use without further purifica-
tion so was aminolysed directly using ethylenediamine to give
compound 3 in quantitative yield. Compound 3 provided
a suitable moiety for further derivatisation of staurosporine
with dyes, linkers or potentially other functionalities.
overall yield of 43% was somewhat disappointing, so the final
step was repeated as a two-step one-pot procedure. Com-
pound 6 was deprotected and then immediately coupled with
fluorescein isothiocyanate to generate compound 8 with
a slightly improved yield of 52%. The overall yield for the prep-
aration of compound 8 from staurosporine (1) was 24%. NMR
analysis demonstrated that the tethering point for the linker
was indeed the 4’-methylamine.
To derivatise compound 3, we chose one of the shortest and
simplest commercially available polyethylene glycol linkers
available. Shorter linker strategies were attempted, but did not
yield product with viable solubility and optical properties (not
shown). Compound 3 was acylated using a fluorenylmethoxy-
carbonyl (Fmoc)-protected diamino-diethylene glycol linker
capped at the other amino terminus with a succinic acid
group (5) to afford 6. Compound 5 was originally prepared by
reacting commercially available 2,2-(ethylenedioxy)bisethyla-
mine (4) with succinic anhydride and Fmoc-Cl, with a yield of
41%.[18] Compound 5 was subsequently acylated to the pri-
mary amine of 3 using O-(benzotriazol-1-yl)-N,N,N’,N’-tetrame-
thyluronium hexafluorophosphate (HBTU) as the coupling
agent, followed by preparative HPLC, affording 6 in a yield of
47%.
Optical properties
Absorption and emission spectra of compound 8 and fluores-
cein isothiocyanate were compared. Figure 1 demonstrates
that 8 displayed similar absorbance and emission profiles to
those of fluorescein isothiocyanate itself. Thus the basic spec-
tral behaviour of the fluorophore was not affected by attach-
ment of the modified staurosporine. Next we evaluated the
anisotropic properties of 8 and compared these again to those
of fluorescein isothiocyanate. Fluorescein isothiocyanate pro-
voked a full depolarisation of incident polarised light at a con-
centration of 10 nm (Figure 1c). Compound 8 also depolarised
the incident polarised light; however, the concentration re-
quired was higher. The minimum concentration of 8 resulting
Compound 6 was deprotected using diethylamine, releasing
the free amine 7, which was again purified by RP-HPLC. The
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that the kinase bound the probe. Because this PKA preparation
is semi-pure, it could be hypothesised that the reversal was
not due to binding to PKA, but to nonspecific binding to non-
PKA proteins in the preparation. To assess this, a parallel incu-
bation was carried out with excess unmodified (’cold’) stauro-
sporine which would prevent specific binding of the probe. As
expected, excess staurosporine did not affect the polarisation
signal in the absence of PKA (as this is a signal derived from
the probe itself). In the presence of PKA, some reversal of the
polarisation signal was observed even in the presence of
excess ‘cold’ staurosporine (Figure 2a, ’nonspecific’), which we
attributed to a certain level of nonspecific interaction of the
probe. The difference between the total and nonspecific was
then taken as specific binding to PKA. This was used through-
out the study to assess probe binding. The specific binding of
8 to PKA was time dependent (Figure 2b). Increased incuba-
tion times were associated with a decrease in the variability of
the signal. To estimate the binding constant, the probe was in-
cubated with increasing concentrations of PKA. Specific bind-
ing of compound 8 to PKA was saturable in a way that is com-
patible with a one-site binding event at the kinase (Figure 2c).
The binding constant observed for binding of compound 8
was 44 nm. This is compatible with the Kd value recorded pre-
viously for unmodified staurosporine binding to PKA.[3,17,19]
To verify that 8 could be displaced from PKA, it was incubat-
ed at a concentration of 50 nm with PKA in the presence of in-
creasing concentrations of unmodified staurosporine. As
shown in Figure 2d, staurosporine inhibits the binding of com-
pound 8 to PKA in the low nanomolar range with a pIC50 value
of 8.0Æ0.1 (IC50 =11 nm). Similarly, binding of 8 to PKA was an-
tagonised by ATP in the presence of MgCl2, indicating it inter-
acted with the ATP binding site of PKA (Figure 2e).
Reversible binding with purified PKA catalytic subunit
The above-mentioned experiments were conducted using
a semi-pure preparation which used the whole PKA tetramer
of two catalytic subunits and two regulatory subunits bound
together. The binding characteristics of compound 8 indicate
binding to a bona fide ATP binding site. However, as shown in
Figure 2a, a degree of nonspecific binding decreased the an-
tagonisable window. We measured the binding of compound
8 to a purer PKA catalytic subunit preparation. The binding
window for specific binding (as assessed using the procedure
in Figure 2a) increased from 10–20 mP units using semi-pure
PKA tetramer to ~50 mP units for the purer PKA catalytic subu-
nit (Figure 3a).
Figure 1. Spectral behaviour of compound 8 in comparison with fluorescein
isothiocyanate. Solutions of a) fluorescein isothiocyanate and b) compound
8 were prepared at a concentration of 10 mm. Absorption and emission
spectra were recorded by measuring emission at 650 nm with excitation
over 300–600 nm, and by measuring emission over 400–700 nm with excita-
tion at 350 nm, respectively. c) Fluorescence polarisation in response to fluo-
rescein isothiocyanate and compound 8. A set of serially diluted wells were
prepared in triplicate for each of these, and fluorescence polarisation was
measured as described in the Experimental Section.
in near complete depolarisation was 50 nm (Figure 1c). This
concentration was used to measure kinase binding in subse-
quent assays.
The above data indicate that compound 8 can be used to
identify compounds that bind to the ATP binding site of PKA.
To confirm this further, H-7, a known ATP binding site blocker
unrelated to staurosporine was tested in the fluorescence po-
larisation binding assay described in Figure 3a. As shown in
Figure 3b, H-7 antagonised the binding of compound 8 to
PKA in a concentration-dependent fashion, with an IC50 value
of 3.9 mm. This value is similar to the Ki reported for antago-
nism of PKA enzyme activity.[20]
Evaluation of kinase binding
It is predicted that if compound 8 can bind to PKA, the depo-
larisation observed above (Figure 1c) will be reversed. Fig-
ure 2a shows that the fluorescence polarisation signal of
50 nm compound 8 was reversed upon incubation with a prep-
aration of tetrameric PKA enzyme (Figure 2a, ’total’), indicating
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Figure 2. Binding of compound 8 to PKA. a) PKA (200 nm) was incubated with compound 8 (50 nm) for 120 min at 208C in the presence (nonspecific) or ab-
sence (total) of excess unmodified staurosporine, after which the fluorescence polarisation signal was determined (meanÆSEM, n=2–3). b) Time dependence
of specific binding of compound 8 to 200 nm PKA (meanÆSEM, n=3). c) Concentration dependence of specific binding of PKA to 50 nm compound 8
(meanÆSEM, n=3); nonlinear regression (one-site binding site algorithm) was performed in Prism (version 6.04, GraphPad Software, La Jolla, CA, USA).
d) Competition of binding of compound 8 (50 nm) to 200 nm PKA with unlabelled staurosporine (meanÆSEM, n=3); nonlinear regression [log(agonist) vs. re-
sponse, variable slope (four parameters)] was performed in Prism. e) Compound 8 binds to the ATP binding site of PKA: compound 8 (50 nm) was incubated
with 200 nm PKA in the presence or absence of MgCl2 [1 mm] with ATP [1 mm], and specific binding was measured as described in panel a) (meanÆSEM,
n=2–3).
Figure 3. Antagonism of compound 8 binding to PKA by known kinase inhibitor H-7. a) Binding of compound 8 to pure PKA catalytic domain. Three concen-
trations of the catalytic subunit from bovine PKA were incubated with 50 nm compound 8 for 120 min in the presence or absence of excess unmodified
staurosporine, after which the specific fluorescence polarisation signal was determined as described in Figure 2a (meanÆSEM, n=2–3). b) Compound 8
(50 nm) was incubated with 200 nm PKA in the presence of increasing concentrations of H-7, and specific binding was measured as described in Figure 2a
(meanÆSEM, n=6). Data were fitted in GraphPad Prism (version 6.04) [log(agonist) vs. response, variable slope (four parameters)].
Interaction of compound 8 with other kinases
tivity, we tested this notion by evaluating the inhibition of
Whilst the experiments above indicate that compound 8 is
a useful probe to identify binding of compounds to the ATP
binding site of PKA, the promiscuous nature of staurosporine
from which compound 8 was derived suggests that compound
8 could be a more universal kinase probe. Using the ability of
the staurosporine moiety of compound 8 to inhibit kinase ac-
a range of kinases by compound 8 and comparing this with
PKA inhibition.
Table 1 shows the inhibition of 50 kinases (including PKA) by
compound 8 at both 100 nm and 1 mm. It can be observed
that compound 8 at a concentration of 100 nm inhibited PKA
by about 60%. Given that compound 8 is a good fluorescence
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Discussion
Table 1. Inhibitory activity of compound 8 against a range of serine/
threonine and tyrosine kinases.[a]
A fluorescein-labelled staurosporine derivative (8) has been
prepared by alkylation in five steps with an overall yield of
24%. Fluorescein is widely used as fluorophore in fluorescence
polarisation assays. The coupling of staurosporine to the fluo-
rophore did not affect the basic fluorescent characteristics of
the fluorophore, and the resulting probe was capable of depo-
larising plane-polarised light, indicating its potential applica-
tion in fluorescence polarisation assays. Using fluorescence po-
larisation, compound 8 was shown to bind to PKA in the nano-
molar range. Compound 8 binding was successfully antagon-
ised using unmodified staurosporine as well as ATP and H-7,
indicating it interacted with the ATP binding site of the kinase
and could monitor inhibitor binding.
Kinase
[8]/m
Kinase
[8]/m
10À6
10À7
10À6
10À7
MKK1
JNK1
p38a MAPK
RSK1
PDK1
PKBa
SGK1
S6K1
PKA
ROCK 2
PRK2
44Æ1
83Æ3
121Æ6
3Æ1
64Æ2
82Æ4
98Æ11
6Æ3
CK2
117 Æ1
10Æ1
82Æ35
3Æ2
115Æ10
55Æ12
84Æ12
7Æ2
DYRK1A
NEK6
TBK1
PIM1
SRPK1
EF2K
HIPK2
PAK4
MST2
MLK3
TAK1
IRAK4
RIPK2
TTK
Src
Lck
BTK
JAK2
SYK
EPHA2
HER4
IGF-1R
TrkA
4Æ1
24Æ8
75Æ2
77Æ9
59Æ16
40Æ2
54Æ3
98Æ16
40Æ0
59Æ6
49Æ13
60Æ2
87Æ6
91Æ7
56Æ5
47Æ11
101Æ4
24Æ2
116Æ2
1Æ0
20Æ1
25Æ1
97Æ12
74Æ2
12Æ6
4Æ1
70Æ3
85Æ4
94Æ1
93Æ1
40Æ15
10Æ4
23Æ5
53Æ9
37Æ3
93Æ9
54Æ5
20Æ14
40Æ1
47Æ1
4Æ0
42Æ1
34Æ1
5Æ1
8Æ0
4Æ0
35Æ19
8Æ1
13Æ1
4Æ1
PKCa
PKD1
MSK1
16Æ1
6Æ1
13Æ0
90Æ2
18Æ0
2Æ1
The most frequently employed method for attachment to
staurosporine has been acylation of the 4’-methylamine. Al-
though the 4’-methylamine is oriented towards solvent-acces-
sible space, it is involved in binding to the protein. In many
crystal structures the 4’-methylamine can be seen to be bind-
ing to acidic side chain residues in the kinase; for example,
Glu127 and Glu170 in the case of PKA,[21] and Asp807 in the
case of ASK1.[22] For kinases that hydrogen bond via aspartate
or glutamate to the 4’-methylamine nitrogen of staurosporine,
a 7- to 80-fold decrease in affinity was observed upon acyla-
tion of this moiety to introduce a PEG linker.[9] However, acyla-
tion did not affect the inhibition of c-Src by a recently devel-
oped covalently binding affinity probe.[8] The role of the 4’-
methylamine and tetrahydropyran functionality of staurospor-
ine may be inferred when comparing the IC50 values for stauro-
sporine and K252c (staurosporine aglycone) which for PKC
were 9 and 680 nm, respectively.[23] A similar but larger differ-
ence was observed for PKA (40 and >10000 nm).[23] This differ-
ence in IC50 suggests the importance of the tetrahydropyran
functionality that bears the 4’-methylamine with respect to re-
taining its bonding interactions with the target protein. The
tetrahydropyran ring of staurosporine is documented as being
flexible, and conformational changes have been observed
which include movement of the 4’-methylamine.[24] This flexi-
bility of the tetrahydropyran may be partly responsible for the
broad-ranging affinity of staurosporine.
CAMKKb
CAMK1
SmMLCK
CHK2
GSK3b
PLK1
Aurora B
LKB1
AMPK
MARK3
CK1d
6Æ0
18Æ2
49Æ2
20Æ0
11 Æ0
101Æ1
11 Æ1
101Æ1
1Æ0
13Æ2
13Æ2
3Æ0
26Æ5
91Æ8
88Æ8
56Æ5
4Æ0
49Æ9
101Æ5
92Æ16
116Æ5
6Æ1
1Æ0
13Æ11
118 Æ10 109Æ2
VEGFR
7Æ1
25Æ4
[a] Compound 8 was tested at 10À6 and 10À7 m, and values are the per-
cent kinase activity remaining. Data are the average of duplicate observa-
tions, with error bars indicating the spread between the duplicates. A
repeat experiment was conducted with the same results.
polarisation probe for PKA, this suggests that a kinase that is
inhibited by compound 8 to a similar extent could be detected
by compound 8 using fluorescence polarisation.
Kinases that were inhibited by compound 8 to a similar
extent as PKA include RSK1, PDK1, ROCK 2, PKCa, MSK1,
GSK3b, Aurora B, AMPK (hum), MARK3, TBK1, PAK4, MST2,
MLK3, IRAK4, Src, Lck, BTK, JAK2, TrkA, and VEG-FR. Therefore,
compound 8 would be predicted to be suitable to assay these
kinases by fluorescence polarisation. Kinases that were clearly
not inhibited by compound 8 at the concentrations tested in-
clude JNK1, p38a MAPK, PLK1, LKB1, CK1d, CK2, EF2K, NEK6,
HIPK2, EPH-A2, and HER4; therefore, this probe would not
appear to be suitable to assay these kinases. Some of these
kinases (p38a MAPK, CK1d, NEK6, CK2, EPH-A2, EF2K and to
a large extent JNK1) are, in fact, not sensitive to staurosporine
itself (not shown), and for others it appears that the modifica-
tion has led to a loss of binding. A number of kinases ap-
peared to be inhibited by compound 8 but to a lesser extent
than PKA, and for these kinases it would need to be estab-
lished individually whether compound 8 would make a viable
tool to interrogate the ATP binding site using fluorescence po-
larisation.
Acylation of the amine introduces an electron-withdrawing
carbonyl group that greatly decreases the basicity of the nitro-
gen. In alkylating rather than acylating the 4’-methylamine, the
basicity of the nitrogen is predicted to be preserved, while
also maintaining the nitrogen atom’s ability to act as a hydro-
gen bond acceptor.[25] In the work presented herein, alkylation
of the 4’-methylamine has successfully generated a functional
probe that has retained high affinity. As compound 8 has been
demonstrated to bind reversibly to the ATP binding site of
PKA, it could be employed to demonstrate whether a known
inhibitor binds in a competitive or allosteric manner, as only
competitive blockers are expected to displace compound 8.
This was confirmed using H-7, a competitive blocker of the
ATP binding site on PKA. Additionally, the fact that H-7 dis-
placed the probe from PKA indicates that the fluorescence po-
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made up in phosphate-buffered saline (PBS) 0.01m phosphate
buffer, 0.0027m potassium chloride, and 0.137m sodium chloride,
pH 7.4, at 258C with 2% DMSO final well concentration. The wells
were prepared in triplicate or quadruplicate.
larisation format used for compound 8 could be readily used
in the screening of ATP-competitive inhibitors.
Conclusions
Kinase profiling (Table 1) was performed in the Express Screen at
the University of Dundee International Centre for Kinase Profiling
using a radiolabel filter binding assay.[26]
Staurosporine was derivatised successfully via a five-step alky-
lation route to yield a fluorescein-based fluorescent tool. The
linking and labelling strategy could be readily modified to in-
troduce a wide range of other fluorophores or other moieties
to staurosporine. The tool developed here interacted with PKA
at the ATP binding site and was shown to be useful to monitor
inhibitor binding at this site. We established for a large
number of kinases whether or not the tool developed in this
study is predicted to interact. This suggests that it is useful as
probe for the identification of ATP-competitive blockers for nu-
merous kinases.
Methyl
N-((5R,7R,8R,9S)-8-methoxy-9-methyl-16-oxo-
6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-
methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-
yl)-N-methylglycinate (2). Staurosporine (LC Labs; 5 mg, 1.07”
10À5 m) was dissolved in tetrahydrofuran (THF, 0.5 mL) and toÀt4his
solution was added potassium carbonate (30 mg, 2.17”10 m,
20 equiv). Methyl bromoacetate (20 mL, 32 mg, 2.11”10À4 m,
20 equiv) was then added, and the mixture was stirred at room
temperature (RT) for 48 h. The solvent was removed under
vacuum, and the resulting solid was resuspended in THF, and the
mixture was centrifuged (5 min, 8100xg). The supernatant was
then removed under vacuum to afford a white amorphous powder
(5.6 mg, 97% yield). Purity: >99% by HPLC, tR =16.85 min; 1H
(400 MHz, [D6]DMSO, 348 K): d=9.32 (1H, d, J=7.7 Hz, H4), 8.24
(1H, s, H6), 8.05–8.00 (2H, m, H1 and H11), 7.57–7.55 (1H, m, H8),
7.51–7.46 (2H, m, H2 and H10), 7.37–7.27 (2H, m, H3 and H9), 6.82
(1H, dd, J=4.76, 3.15 Hz, H6’), 4.98 (2H, s, H7), 4.34 (1H, d, J=
1.6 Hz, H3’), 3.71 (1H, m, H4’), 3.56 (3H, s, H4’’) 3.23 (2H, m, H1’’),
2.89 (1H, m, H5’), 2.77 (3H, s, OCH33’), 2.45 (1H, m, H5’), 2.40 (3H,
s, NCH34’), 2.18 ppm (3H, s, CH32’); 13C (101 MHz, [D6]DMSO, 293 K):
d=171.9, 138.3, 136.3, 132.3, 129.8, 126.3, 125.7, 125.2 125.1,
123.9, 122.6, 121.5, 120.4, 119.5, 114.7, 114.0, 113.30, 113.27, 108.9,
93.6, 81.3, 79.3, 67.0, 59.0, 53.9, 51.9, 45.4, 34.4, 30.4, 27.9 ppm;
HRMS: calcd for C31H30N4O5 [M+H]+ 539.2250, found 539.2250.
Experimental Section
General: Solvents were obtained from Fisher and used without fur-
ther purification. Reagents were obtained from Sigma–Aldrich
unless otherwise stated. 1H and 13C NMR spectra were obtained
with Bruker AV(I) 400 MHz, Bruker AV(III) 400 MHz, and Bruker
AV(III) 500 MHz instruments. Mass spectra (ToF ESÆ) were recorded
on a micromass LCT. Reversed-phase HPLC was performed on
a Waters 2525 gradient module coupled with a Waters 2487 dual
absorbance detector set at l 254 and 366 nm. A linear gradient
was run over 35 min, from 100/0 phase 1 (deionised and degassed
H2O with 0.05% trifluoroacetic acid) to 100/0 phase 2 (90% MeCN,
10% H2O with 0.05% trifluoroacetic acid). The analytical column
used was a Phenominex Luna C8, 150”4.6 mm, 5 mm, at a flow
rate of 1 mLminÀ1. The semipreparative column was a YMC C8
150”10 mm, 5 mm, at a flow rate of 3.00 mLminÀ1. The preparative
column was a Phenominex Luna C8, 150”30 mm, 5 mm, at a flow
rate of 20 mLminÀ1. Retention times (tR) are given in minutes.
N-(2-Aminoethyl)-2-(((5R,7R,8R,9S)-8-methoxy-9-methyl-16-oxo-
6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-
methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-
yl)À(m6 ethyl)amino)acetamide (3). Compound
2 (5 mg, 9.29”
10 m) was dissolved in ethylenediamine (1 mL, 899 mg, 1.50”
10À2 m, 1700 equiv), and the resulting reaction mixture was stirred
at RT for 48 h. Deionised water was added (2 mL), and the product
was extracted into ethyl acetate, washed with deionised water (2”
2 mL) and then dried using MgSO4 then concentrated under nitro-
gen. This afforded an off-white amorphous powder (5.2 mg, 99%
yield). Purity: >99% by HPLC, tR =12.53 min; 1H NMR (400 MHz,
[D6]DMSO, 363 K): d=9.34 (1H, d, J=8.0 Hz, H4), 8.22 (1H, s, H6),
8.07 (1H, d, J=8.0 Hz H11), 8.02 (1H, d, J=8.5 Hz, H1), 7.76 (1H,
brs, H3’’) 7.55–7.47 (3H, m, H2, H8 and H10), 7.37 (1H, t, J=7.5 Hz,
H9), 7.31 (1H, t, J=8.0 Hz, H3), 6.88 (1H, dd, J=5.29, 3.59 Hz, H6’),
4.99 (2H, s, H27), 4.42 (1H, d, J=0.3 Hz, H3’), 3.67–3.64 (1H, m,
H4’), 3.41–3.23 (4H, m, H21’’ and H24’’) 3.05–2.98 (1H, m, H5’), 2.86
(2H, t, J=6.2 Hz, H25’’), 2.51 (3H, s, OCH33’), 2.45 (3H, s, NCH34’),
2.41 (3H, s, CH32’) 2.40–2.33 ppm (1H, m, H5’); 13C NMR (101 MHz,
[D6]DMSO, 293 K): d=172.4, 158.6, 158.4, 138.4, 136.7, 132.7, 130.3,
126.7, 126.2, 125.8, 124.3, 123.1, 122.2, 121.1, 120.0, 115.2, 114.5,
113.3, 109.6, 94.6 82.3, 59.2, 45.8, 41.1, 40.8, 38.9, 36.8, 28.1,
27.3 ppm; HRMS: calcd for C32H34N6O4 [M+H]+ 567.2675, found
567.2733.
PKA was used in two forms: semi-pure PKA from bovine heart as
a whole tetramer (Sigma–Aldrich P5511), and PKA catalytic subunit
purified from P5511 by column chromatography (Sigma–Aldrich
P2645). PKA was added to the assay at the required number of
enzyme units, and this was converted into a molar value using the
specific activity of the P2645 preparation as a guide (10 units per
mg protein, one unit of PKA being equivalent to 2.3 pmol of
kinase).
Fluorescence polarisation assays were performed in 384-well
plates in a final volume of 50 mL at RT in 0.01m phosphate buffer,
0.0027m potassium chloride, and 0.137m sodium chloride, pH 7.4.
Fluorescence polarisation was measured on a PerkinElmer Wallac
Envision 2104 Multilabel plate-reading spectrophotometer using
485 nm excitation and 535 nm emission filters, suitable for mea-
surement of fluorescein. Fluorescence polarisation was determined
by measuring the parallel and perpendicular fluorescence emission
intensity with respect to the polarised excitation light and is ex-
pressed in millipolarisation (mP) units following Equation (1):
mP value ¼ 1000 Â ðSÀG Â PÞ=ðS þ G Â PÞ
ð1Þ
1-(9H-Fluoren-9-yl)-3,14-dioxo-2,7,10-trioxa-4,13-diazaheptade-
can-17-oic acid (5). 2,2,-(Ethylenedioxy)bisethylamine (1.48 mL,
10 mm) was dissolved in acetonitrile (50 mL). To this was added
dropwise a solution of succinic anhydride (1.00 g, 10 mm) in aceto-
nitrile (25 mL) over 90 min with stirring at RT.[18] The mixture was al-
lowed to stir for a further 90 min by which time a waxy solid had
in which S is the parallel and P is the perpendicular fluorescence
signal, and G is the gain factor, which was set prior to the assay so
that 1 nm fluorescein isothiocyanate gives a reading of 27 mP
units. For fluorescence spectra in Figure 1, all solutions tested were
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977
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Full Papers
precipitated out of solution. The solvent was decanted off, and the
solid was dissolved in a 1:1 mixture of acetonitrile and deionised
water (100 mL). The solution was cooled in an ice bath, and to this
was added dropwise a solution of Fmoc chloride (3.37 g, 13 mm,
1.3 equiv) in acetonitrile (25 mL) over 60 min. The solution was ad-
justed to pH 7–8 with a 5% aqueous solution of sodium hydrogen
carbonate. The mixture was left to stand overnight. The mixture
was concentrated under vacuum. The solids were dissolved in 5%
sodium hydrogen carbonate (100 mL) and washed with ethyl ace-
tate (4”75 mL). The aqueous phase was then acidified with hydro-
chloric acid 1m, to between pH 2 and 3. The aqueous phase was
then extracted with ethyl acetate (4”100 mL). The combined ethyl
acetate extracts were washed with deionised water (2”100 mL),
dried over MgSO4, filtered and concentrated under vacuum to
afford the product as a colourless oil (1.932 g, 41.1%). The oil was
further purified by loading onto Isolute sorbent and run on silica
using ethyl acetate as an eluent. This gave the light-yellow oil that
crystallised on standing (750 mg, 16.0% yield); mp 98–1008C.
Purity: >99% by HPLC, tR =17.37 min; 1H NMR (400 MHz, CDCl3,
293 K): d=7.76 (2H, d, J=7.4 Hz), 7.56 (2H, d, J=7.4 Hz), 7.40 (2H,
t, J=7.4 Hz), 7.31 (2H, t, J=7.4 Hz), 4.52–4.39 (2H, m) 4.28–4.18
(1H, m), 3.61 (4H, s), 3.59–3.53 (2H, m), 3.49–3.41 (4H, m), 3.41–
3.31 (2H, m), 2.72–2.64 (2H, m), 2.53–2.45 ppm (2H, m); 13C NMR
(101 MHz, [D6]DMSO, 293 K): d=174.0, 171.3, 156.3, 144.0, 140.9,
127.2, 125.3, 120.2, 69.6, 65.5, 46.9, 38.7, 30.1, 29.3 ppm; HRMS:
calcd for C25H30N2O7 [M+H]+ 471.2087, found 471.2151, HRMS:
calcd for C25H30N2O7 [MÀH]À 469.1980, found 469.1860.
vacuum. The remaining solids were triturated with THF and petro-
leum ether and then dried under high vacuum for 6 h to afford an
off-white powder. Half of this material was then purified for analy-
sis by semi-preparative RP-HPLC to afford an amorphous white
powder 7 (0.7 mg, 45% yield). Purity: >99% by HPLC, tR =
12.03 min; 1H NMR (400 MHz, [D6]DMSO/D2O 9:1, 293 K): d=9.20
(1H, d, J=8.1 Hz, H4), 8.04–7.99 (2H, m, H1 and H11), 7.54–7.46
(3H, m, H2, H8 and H10), 7.36 (1H, t, J=7.4 Hz, H9), 7.31–7.27 (1H,
m, H3), 6.81–6.78 (1H, m, H6’), 4.98 (2H, s, H27), 4.22 (1H, s, H3’),
3.53 (2H, t, J=5.1 Hz, CH2), 3.50–3.46 (4H, m, 2CH2) 3.41–3.39 (1H,
m, CH), 3.35 (2H, t, J=5.9 Hz, CH2), 3.31–3.27 (1H, m, H4’), 3.15
(2H, t, J=5.8 Hz, CH2), 3.06–2.81 (8H, m, H5’, H21’’, H24’’, and 3CH),
2.53 (3H, s, OCH33’), 2.36 (3H, s, NCH34’), 2.33–2.28 (1H, m, H5’),
2.27–2.21 (4H, m, 2CH2), 2.00 ppm (3H, s, CH32’); 13C NMR
(101 MHz, [D6]DMSO, 293 K): d=139.2, 136.3, 132.3, 129.8, 128.0,
126.2, 124.9, 123.9, 122.7, 114.8, 94.2, 72.3, 69.6, 69.4, 69.0, 66.6,
60.0, 58.8, 38.6, 38.4, 34.4, 30.7, 30.5, 30.4, 21.0, 16.7 ppm; HRMS:
calcd for C42H52N8O8 [M+H]+ 797.39417, found 797.3945
2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-(3-(2-((5R,7R,8R,9S)-8-me-
thoxy-9-methyl-16-oxo-6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-
4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]-cyclopen-
ta[e]-as-indacen-7-yl)-4,9,12-trioxo-16,19-dioxa-2,5,8,13-tetraaza-
henicosan-21-yl)thioureido)benzoic acid (8). The remaining half
of the deprotected material described above for the preparation of
compound 7 was dissolved in DMF (100 mL) and added to a solu-
tion of fluorescein isothiocyanate (1 mg, 1.3 equiv) in DMF
(100 mL). This was left to stir at RT in the dark for 12 h. Monitoring
by mass spectrometry indicated unreacted precursor; therefore,
further fluorescein isothiocyanate (0.76 mg, 1 equiv) was added
and left to stir for a further 24 h. At this point there was little or no
detectable precursor remaining, so the solvent was removed under
vacuum and the material was purified by HPLC to afford an amor-
phous dark-orange powder (1.2 mg, 52% yield). Purity: >99% by
HPLC, tR-15.60 min; 1H NMR (500 MHz, [D6]DMSO/D2O 9:1, 298 K):
d=9.25 (1H, d, J=8.1 Hz), 8.26 (1H, brs), 8.07 (1H, d J=7.9 Hz),
8.02 (1H, d, J=8.3 Hz), 7.70 (1H, d, J=7.6 Hz), 7.52 (3H, quintet,
J=8.0 Hz), 7.31 (1H, t, J=7.7 Hz), 7.14 (1H, d, J=7.9 Hz), 6.93 (1H,
d, J=8.8 Hz), 6.68–6.67 (2H, m), 6.59–6.57 (2H, m), 6.56–6.53 (2H,
m), 4.98 (2H, s), 4.64 (1H, s), 3.65 (2H, s), 3.56 (2H, t, J=5.1 Hz),
3.50, (4H, dd, J=4.0, 10.6 Hz), 3.34 (2H, t, J=5.4 Hz), 3.13–3.11
(6H, m), 2.84 (3H, s), 2.45 (2H, s) 2.36 (1H, s) 2.27 (4H, q, J=
6.5 Hz), 2.19 (3H, s), 2.07–2.06 ppm (1H, m); HRMS: calcd for
C63H63N9O13S [M+H]+ 1186.4300, found 1186.3001.
Fluoren-9-ylmethyl
(2-((5R,7R,8R,9S)-8-methoxy-9-methyl-16-
oxo-6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-
methanodibenzo[b,h]cyclonona[jkl]cyclopenta[e]-as-indacen-7-
yl)-4,9,12-trioxo-16,19-dioxa-2,5,8,13-tetraazahenicosan-21-yl)-
carbamate (6). Compound 3 (9.40 mg, 1.66”10À5 m) was dissolved
in DMF (100 mL) and to this solution was added DIPEA (3.2 mL,
2 equiv). A separate solution of 5 (9.37 mg, 1.2 equiv), HBTU
(7.55 mg, 1.2 equiv), and DIPEA (3.2 mL, 2 equiv) was prepared in
DMF (100 mL). The two solutions were stirred for 1 min and then
combined. The resulting reaction mixture was stirred for a further
12 h. The solvent was removed under vacuum and the reaction
product purified by semi-preparative RP-HPLC to afford an amor-
phous white powder (8 mg, 47% yield). Purity: >99% by HPLC,
tR =18.12 min; 1H NMR (500 MHz, [D6]DMSO, 298 K): d=9.32 (1H,
d, J=8.0 Hz), 8.63 (1H, s), 8.09 (1H, d, J=8.0 Hz), 8.05 (1H, d, J=
8.5 Hz), 7.89–7.86 (4H, m), 7.68–7.63 (2H, m), 7.56–7.49 (3H, m),
7.39 (3H, t, J=7.1 Hz), 7.34–7.30 (3H, m), 6.99–6.97 (1H, m), 5.00
(2H, s), 4.29–4.27 (2H, m), 4.21–4.18 (1H, m), 3.47 (4H s), 3.40–3.34
(5H, m), 3.17 (6H, s), 3.15–3.10 (6H, m), 2.48 (2H, s), 2.32–2.28 (4H,
m), 2.25 ppm (2H, brs); 13C NMR (126 MHz, [D6]DMSO, 293 K): d=
206.5, 171.9, 171.8, 171.5, 156.2, 143.9, 140.7, 137.8, 136.3, 132.3,
129.7, 127.6, 127.0, 126.2, 125.8, 125.4, 125.4, 125.2, 124.9, 123.9,
122.7, 121.8, 120.8, 120.1, 119.8, 119.7, 118.1, 117.8, 115.5, 114.9,
114.2, 112.7, 109.2, 94.1, 81.6, 79.1, 69.5, 69.5, 69.1 65.3, 59.0, 54.7,
48.6, 46.7, 45.4, 40.5, 38.8, 38.5, 37.9, 30.8, 30.5, 29.1, 27.4, 26.1,
22.5, 1.2 ppm; HRMS: calcd for C57H62N8O10 [M+H]+ 1019.4622,
found 1019.4639.
Acknowledgements
A.J.M.D. was funded by a Biotechnology and Biological Sciences
Research Council studentship (grant ref. BB/F016867/1). The au-
thors are grateful for additional financial support from the
School of Pharmacy, University of Nottingham.
Keywords: alkylation
kinases · screening
· fluorescein · fluorescent probes ·
N1-(2-(2-(2-Aminoethoxy)ethoxy)ethyl)-N4-(2-(2-(((5R,7R,8R,9S)-8-
methoxy-9-methyl-16-oxo-6,7,8,9,15,16-hexahydro-5H,14H-17-
oxa-4b,9a,15-triaza-5,9-methanodibenzo[b,h]cyclonona[jkl]-cy-
clopenta[e]-as-indacen-7-yl)(methyl)amino)acetamido)ethyl)suc-
cinimide (7). Diethylamine (200 mL) was added to a stirred solution
of 6 (4 mg, 3.93”10À6 m) in THF (800 mL) and the mixture was
stirred for 4 h; completion was assessed by mass spectrometry.
The solvent and excess diethylamine were removed under
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Published online on March 23, 2016
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