Design and synthesis of an activity-based probe template for protein kinases

Article abstract of DOI:10.1055/s-0029-1218543

A potential activity-based probe for protein kinases was designed around mechanism-based inhibitors of insulin receptor kinase. The probe was synthesized by a chemoselective conjugate addition of ATPγS to an epoxyenone. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0029-1218543

LETTER
521
Design and Synthesis of an Activity-Based Probe Template for Protein Kinases
D
esign and Sy
a
nthesis of
a
n
n AB
P
for
P
t
rotein
K
o
inases sh Keshipeddy, Yu Shi, Nathan Hnatiuk, Martha Morton, Xudong Yao, Amy R. Howell*
Department of Chemistry, University of Connecticut, Storrs, CT 06269-3060, USA
Fax +1(860)4862981; E-mail: amy.howell@uconn.edu
Received 28 August 2009
Dedicated to Gerry Pattenden in celebration of his 70th birthday
teases,9 metalloproteases,10 kinases,11 phosphatases,4,12
Abstract: A potential activity-based probe for protein kinases was
designed around mechanism-based inhibitors of insulin receptor
kinase. The probe was synthesized by a chemoselective conjugate
addition of ATPgS to an epoxyenone.
phosphorylases,13 and carbohydrate-related enzymes.14
This work reports the design and synthesis of an activity-
based probe (1) for protein tyrosine kinases (Figure 1).
Key words: activity-based probes, addition reactions, regioselec- Our probe was patterned around the potent, mechanism
tivity, enones, epoxides
based, bisubstrate inhibitors 2 for insulin receptor ki-
nase.15 These inhibitors have two elements responsible for
high binding affinity. One is the correct distance, defined
by the spacing in the dissociative transition state between
the ATP unit and the tyrosine unit. The second feature is
the hydrogen bonding between Asp-1132 and the amide
hydrogen.14 In probe 1, the amide NH was changed into an
epoxyethylene group in order to target a conserved car-
boxylate group in the protein tyrosine kinase active site to
form covalent adducts between the probe and protein
tyrosine kinases. The azide moiety is incorporated for
coupling with a reporter group through ‘click’ chemis-
try,16 which can be used for displaying or separating the
probe-modified enzymes.
Proteins with phosphotyrosine, although few (ca. 0.05%
of proteins in human cells),1 play key roles in cellular sig-
naling pathways that regulate cell proliferation, differen-
tiation, adhesion, and motility.2 Phosphotyrosinyl
proteins are the net products of the delicate balance be-
tween enzymatic reactions that are catalyzed by protein
tyrosine kinases and phosphatases. In order to understand
the cellular dynamics of tyrosine phosphorylation, inves-
tigations have focused on analyzing the sites of tyrosine
phosphorylation across numerous proteins in proteomes,
simultaneously, as well as sequentially, over time.1,3
However, these analyses are indirect measurements of the
enzyme activities. Tyrosine kinases and phosphatases that
control the phosphorylation and dephosphorylation of
proteins have rarely been studied for their activities on a
proteome scale.4
(a)
R
O
NH2
N
N
N
O
O
O
H
Small molecules are used in studies of proteome-wide cat-
alytic and binding events in cells. These compounds are
often referred to as activity-based probes (ABPs), and
analysis of probe–protein adducts, mainly by mass spec-
trometry, is referred to as activity-based protein profiling
or, sometimes, chemical proteomics.5 This approach pro-
vides a means to measure proteome-wide enzyme activity
directly. ABPs have a reactive moiety, a linker and a re-
porter. Some form covalent adducts at enzyme active sites
that can be analyzed by mass spectrometry-based pro-
teomics. The design of the reactive moieties takes cues
from organic compounds that are used for affinity label-
ing, suicide inhibition and mechanism-based inhibition of
enzymes, where covalent bonds are formed with protein
targets.6 The pool of these molecules has been enriched by
new small molecules that have been used in chemical ge-
netics and functional proteomics.7 ABPs have recently
been utilized for profiling the activity of various enzymes
in proteomes, including serine proteases,8 cysteine pro-
S
P
O−
O
P
O−
O
P
O−
O
N
N
O
O
O
HOOH
D1132
2
(b)
N3
O
NH2
N
N
O
O
O
O
S
P
O−
O
P
O−
O
P
O−
O
N
O
N
O
O
D1132
HOOH
1
Figure 1 Illustration of protein binding-site interactions with ABPs
for protein tyrosine kinases. (a) Interactions between tyrosine kinase
and bisubstrate analog inhibitors 2, drawn based on reference 15; (b)
Proposed interactions between tyrosine kinase and ABP 1. Kinase
binding sites are represented with blue lines. D1132 represents Asp-
1132 on the kinase.
SYNLETT 2010, No. 4, pp 0521–0524
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Advanced online publication: 01.12.2009
DOI: 10.1055/s-0029-1218543; Art ID: D23809ST
© Georg Thieme Verlag Stuttgart · New York
We envisaged that potential ABP 1 could be synthesized
by the conjugate addition of ATPgS to epoxyenone 3

522
S. Keshipeddy et al.
LETTER
(Figure 2), which would be prepared from a vinylation/
oxidation sequence with epoxyaldehyde 4. Compound 4
would result from an initial Suzuki coupling of iodo-
propene 5 and 4-(hydroxymethyl)phenyl boronic acid,
followed by routine functional group manipulations.
OH
OH
1. CBr4, Ph3P
CH2Cl2, 98%
Pd(PPh3)4, Cs2CO3
+ 5
EtOH, 60 °C
88%
2. NaN3, DMF, 92%
B(OH)2
N3
OTBS
1.
The synthesis of epoxyenone 3 commenced with Suzuki
coupling of known,17 silyl-protected iodopropene 5 and
commercially available 4-(hydroxymethyl)phenyl boron-
ic acid (Scheme 1). Reaction of the coupled product 6
with carbon tetrabromide and triphenylphosphine, fol-
lowed by treatment of the resultant benzylic bromide with
sodium azide, gave 7 in 90% yield over two steps. Vari-
able amounts of desilylated product were observed in the
6
MgBr
1. TBAF, THF, 84%
2. DMDO, CH2Cl2, 100%
THF, 63%
3
4
2. DMP, CH2Cl2
51 %
3. DMP, CH2Cl2, 0 °C
to r.t., 67%
OTBS
7
bromination step with reaction times longer than three Scheme 1 Synthesis of epoxyenone 3
hours. Intermediate 7 was deprotected using TBAF; ep-
oxidation using dimethyldioxirane (DMDO) and Dess–
Martin periodinane oxidation18 furnished key epoxyalde-
hyde 4 in 56% yield over three steps. Subsequent nucleo-
philic addition of vinylmagnesium bromide afforded a
mixture (~2:1) of diastereomeric allylic alcohols. The fi-
nal oxidation to 3 proved to be problematic.
permeation chromatography, were seen at higher temper-
atures (ca. 45 °C and above). Recognizing the sensitivity
of 3 to even slightly elevated temperatures, we returned to
Dess–Martin periodinane mediated oxidation with care
taken in both the reaction and isolation (via quick flash
chromatography) so as not to allow the temperature of the
compound to exceed 25 °C. This provided epoxyenone 3
in moderate yields with good purity. Delayed purification
resulted in a very low recovery of the product, and extend-
ed storage of neat 3, even at –78 °C, resulted in the forma-
tion of oligomers. The lifetime of the epoxyenone could
be increased from days to weeks by storing it in CH2Cl2 in
the freezer.
N3
O
NH2
N
N
O
O
O
O
S
P
O
P
O
P
O
N
N
O
O–
O–
O–
Michael addition
To access 1 effectively, selective reaction of ATPgS with
the enone moiety of 3 would be required. Since epoxy-
enone 3 has multiple potentially reactive sites, model
studies using thiophenol as the nucleophile were conduct-
ed (Scheme 2). With one equivalent of thiophenol, com-
pound 3 was consumed within ten minutes, and the
conjugate addition adduct 8 was the sole product observed
1
HO OH
NH2
N3
N
N
N
O
O
O
O
+
–S
P
O
P
O
P
O
O
Li+
N
O–Li+ O–Li+ O–Li+
1
in the H NMR spectrum of the crude reaction mixture
O
ATPγS
3
vinylation/
oxidation
(75% isolated yield). With two or more equivalents of
thiophenol, clean conversion into 9 resulted, although the
rate of epoxide opening was considerably slower. These
model studies suggested that selective reaction between
the enone of 3 and ATPgS should be achievable.
HO OH
HO
N3
Suzuki coupling
O
OTBS
+
I
N3
N3
5
O
B(OH)2
epoxidation
4
PhSH, Et3N
PhSH, Et3N
Figure 2 Approach to the synthesis of probe 1
3
O
HO
CH2Cl2, 75%
CH2Cl2, 57%
O
O
Initial attempts to oxidize allylic alcohol 3 using MnO2 or
standard oxidants (i.e., Dess–Martin periodinane,18 PCC,
PDC, Swern conditions) did not provide clean conver-
sions. Although the alcohol was consumed, if any product
was isolated, the yield was low, and the product was not
pure. This outcome was not completely unexpected, con-
sidering that 3 is a highly reactive, a,b-unsaturated ketone
where the carbonyl group is also flanked by a styrene ox-
ide. Indeed, oligomers were observed at ca. 30 °C, and
cross-linked polymers, as confirmed by 1H NMR and gel
PhS
SPh
SPh
8
9
Scheme 2 Model studies of the reactivity of epoxyenone 3
An initial concern was identifying an appropriate binary
solvent system, considering the widely differing polarities
of the commercial ATPgS tetralithium salt and epoxy-
enone 3. Although such reactions can be conducted under
Synlett 2010, No. 4, 521–524
© Thieme Stuttgart · New York

LETTER
Design and Synthesis of an ABP for Protein Kinases
523
biphasic conditions,19 preliminary experiments had shown tage of a chemoselective reaction between ATPgS and the
that, under such conditions, hydrolysis of ATPgS oc- enone of epoxyenone 3. Probe 1 was found to have limited
curred over time. Consequently, we elected to convert the stability and, consequently, will not be used for the
ATPgS into a dichloromethane-soluble form.20
planned proteomic profiling. However, we believe that
the array of functionality and distinct reactivity of 3 make
it an excellent scaffold for coupling with either an alterna-
tive ATP surrogate or with other binding moieties for ac-
tivity-based profiling.
The ATPgS lithium salt was converted into the corre-
sponding tetra(tributylammonium) salt by first passing it
through an acidic ion-exchange resin, then treating the re-
sultant acid with tributylamine. This salt was reacted with
an excess of epoxyenone 3 (3 equiv) in CH2Cl2. The con-
sumption of the ATPgS, which occurred within four to six
hours, was monitored by LC-MS. MS analysis suggested
that product formation was occurring, however, the for-
mation of products of higher molecular weight was also
observed. After passing the crude reaction mixture
through an acidic resin in order to generate the acid form
of the product, LC-MS showed a decrease in the amount
1-{2-[4-(Azidomethyl)phenyl]oxiran-2-yl}-3-[adenosine-5¢-(3-thio-
triphosphate)]propan-1-one (1): Adenosine-5¢-(3-thiotriphos-
phate)trilithium salt (5 mg, 0.01 mmol) in H2O (0.8 mL) was added
to a solution of 1-{2-[4-(azidomethyl)phenyl]oxiran-2-yl}prop-2-
en-1-one (3; 11 mg, 0.05 mmol) in THF (1.6 mL). The reaction was
monitored by LC-MS. After 6 h, the solvent was removed under
vacuum, and H2O (0.5 mL) and CH2Cl2 (0.5 mL) were added to dis-
solve the resultant solid. The layers were separated and the aqueous
of desired product and the generation of new by-products. solution was washed with CH2Cl2 (3 × 0.5 mL) and purified using
hydrophilic/lipophilic balanced materials that were packed in-house
We therefore decided to avoid acidic conditions in both
in a spin column (0.8 mL). Deionized H2O was used as both the
the reaction and during purification and chose to work
equilibration and washing solution, and MeCN–H2O (70% v/v) was
with the trilithium salt.
used as the elution solution. The eluant was lyophilized and further
When the reaction was conducted in a mixture of dichlo-
romethane and water, little conversion into the product
was observed by LC-MS. Longer reaction times did not
produce a significant increase in product formation and
resulted in the hydrolysis of ATPgS. Gratifyingly, the use
of a combination of THF and water (2:1) as solvent result-
ed in complete consumption of the ATPgS within six
purified using hydrophilic/lipophilic materials packed in-house in a
TopTipTM spin column (Glygen, MD; 10–200 mL) using the same
equilibration, washing and elution conditions. The eluant was ana-
lyzed by LC-MS. The separation was run at 50 mL/min with a binary
gradient [solvent A = 1% MeCN in 10 mM aq NH4OAc (pH 6.86);
solvent B = MeCN. Gradient: 1% B at 0 min → 1% B at 5 min →
55% B at 25 min → 70% B at 27 min → 70% B at 30 min → 1% B
at 30.1 min → 1% B at 35 min). Pure eluant was lyophilized to yield
hours with minimal levels of hydrolysis being observed. probe 1 (1:1 mixture of diastereomers) as a white, fluffy solid (2 mg,
1
30%): H NMR (500 MHz, D2O): d = 8.53 (s, 0.5 H), 8.52 (s, 0.5
H), 8.27 (s, 0.5 H), 8.26 (s, 0.5 H), 7.42 (m, 4 H), 6.13 (d, J = 5.6
Hz, 1 H), 4.75 (m, 1 H), 4.58 (m, 1 H), 4.44 (s, 1 H), 4.43 (s, 1 H),
4.40 (m, 1 H), 4.29 (m, 2 H), 3.46 (d, J = 4.7 Hz, 0.5 H), 3.45 (d,
J = 4.7 Hz, 0.5 H), 3.31 (d, J = 4.7 Hz, 0.5 H), 3.29 (d, J = 4.7 Hz,
0.5 H), 2.97 (m, 4 H); 31P NMR (202 MHz, D2O): d = 8.78 (d,
J = 29.3 Hz), 8.75 (d, J = 27.6 Hz), –11.48 (d, J = 21.5 Hz), –23.84
(m); HRMS (TOF): m/z [M – H]– calcd for C22H26N8O14P3S:
751.050; found: 751.045; HRMS (TOF): m/z [M + H]+ calcd for
C22H28N8O14P3S: 753.066; found: 753.060.
Over the course of the reaction, LC-MS showed one major
product peak with a mass corresponding to target kinase
probe 1.
Probe 1 was purified by spin column chromatography. Af-
ter purification, a single peak with m/z = 751.045 (theoret-
ical [M – H]– m/z = 751.050) and m/z = 753.060
(theoretical [M + H]+ m/z = 753.066) was detected in LC-
MS analysis. Considering that epoxyenone 3 is racemic, a
1:1 mixture of diastereomers of 1 should result, and this
was seen in the 1H NMR spectrum. Each of the expected
adenine proton singlets was doubled, although the chemi-
cal shift variation of the individual signals was small.
There were two sets of doublets for the epoxide protons
between d = 3.0 and 3.2 ppm, which were found in the
same region as those corresponding to the hydrogens in 3.
The remaining proton signals were not as well defined as
those in the corresponding reactants, which is not unex-
pected considering the more complex coupling patterns.
31P NMR analysis showed three signals with two of the
signals having increased splitting, reflecting the presence
of the two diastereomers. The probe was stable in aqueous
solution at neutral pH and room temperature for up to six
hours. Beyond this time, degradation of the probe was ob-
served by 1H NMR analysis, with an approximate half-life
of 12 hours (based on the appearance of a new anomeric
proton signal over that time-frame). The probe could be
stored in a frozen solution at –80 °C for only a few days.
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.
Acknowledgment
Helpful discussion with Professor Michael Stone, Vanderbilt Uni-
versity is gratefully acknowledged.
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Synlett 2010, No. 4, 521–524
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LETTER
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© Thieme Stuttgart · New York

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ADDENDA AND ERRATA
This article has been corrected as described in the following Erratum published on April, 15th:
Erratum
Design and Synthesis of an Activity-Based Probe Template for Protein Kinases
Santosh Keshipeddy, Yu Shi, Nathan Hnatiuk, Martha Morton, Xudong Yao, Amy R. Howell* Synlett 2010, 521.
The PDF and the print version of this article contained the wrong publication year (2009 instead of 2010). This mistake has
been corrected for the current online PDF version. We apologize profusely for this mistake.
SYNLETT 2010, No. 7, pp 1142–1142
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Advanced online publication: 15.4.2010
DOI: 10.1055/s-0029-1219805; Art ID: R10710ST
© Georg Thieme Verlag Stuttgart · New York
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