Sequential activation and deactivation of protein function using spectrally differentiated caged phosphoamino acids

Article abstract of DOI:10.1021/ja2028074

Photolabile caging groups, including the 1-(2-nitrophenyl)ethyl (NPE) group, have been applied to probe many biological processes, including protein phosphorylation. Although studies with NPE-caged phosphoamino acids have provided valuable information, these investigations have been limited to the use of only one caged species in a single experiment. To expand the scope of these tools, we have developed an approach for sequentially uncaging two different phosphopeptides in one system, enabling interrogation of multiple phosphorylation events. We present the synthesis of [7-(diethylamino)coumarin-4- yl]methyl (DEACM)-caged phosphorylated serine, threonine, and tyrosine building blocks for Fmoc-based solid-phase peptide synthesis to allow convenient incorporation of these residues into peptides and proteins. Exposure of DEACM- and NPE-caged phosphopeptides to 420 nm light selectively releases the DEACM group without affecting the NPE-caged peptide. This then enables a subsequent irradiation event at 365 nm to remove the NPE group and liberate a second phosphopeptide. We demonstrate the versatility of this general sequential uncaging approach by applying it to control Wip1 phosphatase with two wavelengths of light.

Full text of DOI:10.1021/ja2028074

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pubs.acs.org/JACS
Sequential Activation and Deactivation of Protein Function Using
Spectrally Differentiated Caged Phosphoamino Acids
Brenda N. Goguen, Andreas Aemissegger, and Barbara Imperiali*
Department of Chemistry and Department of Biology, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge,
Massachusetts 02139, United States
S Supporting Information
b
and proteins containing these residues have been successfully
exploited for the investigation of many systems,8À12 the current
ABSTRACT: Photolabile caging groups, including the
1-(2-nitrophenyl)ethyl (NPE) group, have been applied to
caging approach is limited to the study of only one caged
probe many biological processes, including protein phos-
phosphopeptide or protein in an experiment. The development
phorylation. Although studies with NPE-caged phosphoa-
of orthogonal caging groups that can be independently released
mino acids have provided valuable information, these
would significantly expand the scope of these tools by enabling
investigations have been limited to the use of only one
interrogation of two phosphorylation pathways within one
caged species in a single experiment. To expand the scope of
system using different wavelengths of light. Selective photolysis
these tools, we have developed an approach for sequentially
uncaging two different phosphopeptides in one system,
enabling interrogation of multiple phosphorylation events.
We present the synthesis of [7-(diethylamino)coumarin-4-yl]-
requires caging groups with distinct photophysical properties,
and the few studies that have addressed this challenge have utilized
caging groups from the o-nitrobenzyl (NB) and coumarinyl
families.13À15 Kotzur et al.13 caged the sulfhydryl side chain of
methyl (DEACM)-caged phosphorylated serine, threonine,
cysteine with derivatives from these two families to enable
and tyrosine building blocks for Fmoc-based solid-phase
sequential deprotection in model peptide studies. Another report
peptide synthesis to allow convenient incorporation of
used NB-caged protein kinase G and coumarinyl-caged cAMP,
these residues into peptides and proteins. Exposure of
which stimulates protein kinase A upon uncaging, to sequentially
DEACM- and NPE-caged phosphopeptides to 420 nm
activate these protein kinase pathways.14 Finally, selective two-
light selectively releases the DEACM group without affect-
ing the NPE-caged peptide. This then enables a subsequent
irradiation event at 365 nm to remove the NPE group and
liberate a second phosphopeptide. We demonstrate the
photon uncaging of dinitroindolinyl-caged glutamate and cou-
marinyl-caged γ-aminobutyric acid was employed to modu-
late action potentials.15 All of these studies demonstrate
the value of a sequential uncaging approach; however, they
versatility of this general sequential uncaging approach by
describe the synthesis of specialized molecules and do not
applying it to control Wip1 phosphatase with two wave-
provide methods that can be readily applied to other systems.
lengths of light.
To provide further advancement of the tools available to probe
the role of phosphorylation in vitro and within cellular systems,
we sought to develop a general sequential uncaging strategy that
rotein phosphorylation is a ubiquitous mechanism mediating
protein function and dynamic signaling interactions. Regulated
can be conveniently applied to diverse systems. Herein we detail
the synthesis of coumarinyl-caged phosphorylated amino acid
building blocks for Fmoc-based SPPS that can be used together
with our previously reported NPE-caged phosphorylated residues
to create peptides that can be sequentially uncaged. We then
present the application of this system for photochemical control
over the activity of wild-type p53 induced phosphatase (Wip1)
(Figure 1).
Our previous work focused on the NPE group over other
commonly used NB derivatives for application in a biological
environment because photolysis at 365 nm is efficient and the
nitrosoacetophenone photobyproduct is less reactive than the
nitrosobenzaldehyde byproduct of NB groups.16 To establish a
method for sequential uncaging of two different phosphopep-
tides in the same solution, we investigated the photolysis of
the [7-(diethylamino)coumarin-4-yl]methyl (DEACM) caging
group together with the NPE group. The DEACM group has
been reported to be an effective cage for cyclic nucleotides17
P
by a complex balance of kinases and phosphatases, phosphoryla-
tion governsfundamentalphysiologicalprocessesandcancontribute
to human disease.1 Although traditional methods for interrogat-
ing the role of phosphorylation within the cell have provided
valuable information,2,3 these approaches do not offer insight
into the spatial and temporal dynamics of the modification, which
are critical for proper cellular function. A complementary approach
that overcomes this limitation is the use of photolabile protecting
groups, or “caging groups”.4 These groups are covalently attached
to an essential phosphorylated amino acid residue of a peptide or
protein, masking the active component and rendering the
molecule inactive within the biological system. Upon irradiation,
the caging group is released, liberating the active phosphorylated
species. The timing and spatial localization of uncaging are defined
by the investigator, allowing precise control over the modifica-
tion.5 We previously reported the synthesis of 1-(2-nitrophenyl)-
ethyl (NPE)-caged phosphorylated amino acid building blocks for
Fmoc-based solid-phasepeptidesynthesis(SPPS).6,7 While peptides
Received: March 28, 2011
Published: June 21, 2011
r
2011 American Chemical Society
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Scheme 1. Synthesis of DEACM-Caged Phosphoserinea
a Reagents and conditions: (a) 2-cyanoethyl N,N-diisopropylchlorophos-
phoramidite, triethylamine, CH2Cl2 (quantitative); (b) NaHCO3/
H2O, Aliquat 336, allyl bromide, CH2Cl2 (91%); (c) 5, 4,5-dicyano-
imidazole, THF; (d) tert-butyl hydroperoxide, 0 °C (75% over two
steps); (e) Wilkinson’s catalyst, 9:1 EtOH/H2O (73%).
Figure 1. Sequential uncaging to control the activity of Wip1 phospha-
tase. The DEACM-caged phosphopeptide substrate enables the first
irradiation at 420 nm to initiate the Wip1 reaction. A subsequent
irradiation at 365 nm releases the NPE-caged phosphopeptide inhibitor
to attenuate the Wip1 activity.
Wip1-catalyzed reaction in a wavelength-dependent and enzyme-
independent fashion (Figure 1).
We synthesized DEACM-caged phosphorylated serine (1),
threonine (2), and tyrosine (3) amino acid building blocks for
Fmoc-based SPPS (Figure 2 and Scheme 1) to enable efficient
incorporation of these caged residues into peptides and proteins.
First, a DEACM-modified trivalent phosphitylating agent (5) was
generated by reaction of DEACMÀOH23 (4) with 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite. Each commercially avail-
able Fmoc-amino acid was protected as the corresponding allyl
ester, and the side-chain hydroxyl group of each derivative was
treated with 5 to afford a DEACM phosphite intermediate.
Subsequent in situ oxidation with tert-butyl hydroperoxide
yielded the corresponding caged phosphoamino acid, and final
allyl deprotection with Wilkinson’s catalyst furnished the free-
acid building blocks (1, 2, and 3) for peptide synthesis. Peptides
were generated by standard SPPS employing PyBOP or HBTU/
HOBt as coupling agents. The caged amino acid was coupled
onto the amino terminus of the growing peptide using slightly
modified conditions (HATU/HOAt and collidine) to prevent
β-elimination of the phosphotriester, which can be a deleterious
side reaction with serine and, to some extent, with threonine.
After incorporation of the residue into the peptide, Fmoc removal
with 20% 4-methylpiperidine in N,N-dimethylformamide con-
comitantly cleaved the β-cyanoethyl group to generate a mono-
protected phosphopeptide that is resistant to β-elimination,
thereby allowing peptide elongation to proceed under the standard
conditions. The DEACM-caged peptides were routinely stored
in the dark, but studies in which the caged peptides were
maintained under ambient light revealed that the DEACM-caged
residues are stable with half-lives ranging from 5À33 h, depend-
ing on the caged residue and peptide sequence (Table S1 in the
Supporting Information).
Figure 2. DEACM-caged phosphorylated serine, threonine, and tyrosine
building blocks for SPPS.
and ATP,18 and uncaging of this derivative is very efficient.17
Importantly, the peak of maximal absorbance of the DEACM
chromophore occurs at 390 nm,19 allowing it to be selectively
released in the presence of the NPE group, which has minimal
absorption at wavelengths above 380 nm (Figure S1 in the
Supporting Information). Additionally, this coumarinyl group is
advantageous in its own right because, relative to the NB family,
the 7-diethylamino-4-(hydroxymethyl)coumarin (DEACMÀOH)
photobyproduct is inert in solution, and cellular systems that are
sensitive to UV irradiation may benefit from long-wavelength
activation.
As a model system, we chose Wip1 phosphatase, a Ser/Thr
protein phosphatase central for attenuating the DNA damage
and repair response and p53 tumor suppressor activity. It
dephosphorylates and inactivates a number of proteins, including
p38 MAP kinase and proteins that stimulate p53. Wip1 is
involved in tumorogenesis, and the gene encoding it is amplified
in some cancers.20 A phosphopeptide substrate based on the
phosphorylation site of p38 has been used for in vitro studies of
Wip1 activity,21 and a cyclic phosphopeptide inhibitor of Wip1
has been identified.22 Thus, a DEACM-caged substrate and NPE-
caged inhibitor constitute an ideal system to test the ability of
the sequential uncaging scheme to initiate and then inhibit the
We then synthesized NPE- and DEACM-caged phosphopep-
tides to explore selective release of the DEACM group in the pre-
sence of the NPE group (Table S2). The peptide sequences were
based on a phosphopeptide substrate (Ac-TDDEMpTGpYVAT)
and cyclic phosphopeptide inhibitor [cyclic(MpSIpYVAC)] of Wip1.
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COMMUNICATION
Encouraged by the model uncaging studies, we tested the
caged peptides in a Wip1 phosphatase assay, in which dephos-
phorylation of the substrate peptide was monitored by quanti-
fication of the released inorganic phosphate. Initially, we eval-
uated the caged peptides before and after irradiation to confirm
that incorporation of the caging groups conferred the predicted
properties. We compared Wip1 activity with the DEACM-
caged substrate and the native, noncaged substrate (Figure S2).
As anticipated, no activity was observed before irradiation, but
exposure to 420 nm light for 3 min unmasked the pThr and restored
the phosphatase activity to a level similar to that observed with
the native substrate subjected to the same irradiation conditions
(0.52 ( 0.03 sÀ1 and 0.66 ( 0.06 sÀ1, respectively). On the basis
of MichaelisÀMenten kinetics with the reported KM of the
phosphopeptide substrate (13 μM),21 this activity corresponds
to release of 53% of the phosphorylated substrate and is consistent
with the time course of uncaging (Figure 3a).
We then turned our attention to the NPE-caged inhibitor
peptide (Figure S3). To assess the effectiveness of caging pSer
within the peptide, we added the caged inhibitor to an assay
solution containing Wip1 and the DEACM-caged substrate after
the reaction had first been initiated by irradiation at 420 nm.
Without exposure of the caged inhibitor to 420 nm light, the
activity was slightly reduced to 0.38 ( 0.04 sÀ1, demonstrating
that masking the pSer in the peptide largely eliminates the
inhibitory properties. Next, examination of Wip1 activity follow-
ing irradiation of both the DEACM- and NPE-caged peptides at
420 nm was performed to ensure that this treatment did not
significantly affect the NPE group. Indeed, irradiation of the two
caged peptides at 420 nm for 3 min yielded Wip1 activity similar
to that observed when the NPE-caged inhibitor was present but not
exposed to 420 nm irradiation (0.32 ( 0.01 sÀ1 and 0.38 (
0.04 sÀ1, respectively). The modest inhibition resulted from
minor release of the NPE-caged inhibitor under these conditions.
On the basis of the model for competitive inhibition with the
reported inhibitor Ki of 0.7 μM,22 over 94% of the NPE-caged
inhibitor was unaffected by the irradiation at 420 nm. This result
is consistent with the time course of uncaging and demonstrates
that photolysis of the DEACM group can occur independently of
the NPE group. We then investigated the effect of liberating the
NPE-caged inhibitor by irradiation at 365 nm. When the two
caged peptides and Wip1 were irradiated together first at 420 nm
and then at 365 nm immediately thereafter, the activity was
significantly reduced to 0.10 ( 0.02 sÀ1 and was comparable to
that resulting from the native noncaged inhibitor peptide
(Figures S4 and S5).
The aforementioned sequential uncaging results are summarized
in Figure 4, which shows the phosphatase activity before irradia-
tion (region A) and after exposure to 420 nm light (region B) and then
365 nm light (region C). The Wip1 reaction was initiated upon
irradiation at 420 nm and inhibited following exposure to 365 nm light.
These experiments demonstrate that the system provides effective
photocontrol over Wip1 activity by enabling two distinct biochemical
processes to be activated in a wavelength-selective manner.
The sequential uncaging system can be applied to diverse
systems to investigate the effects of multiple phosphorylation
events within the same protein or within a signaling cascade. For
instance, the scaffolding protein paxillin, which is essential for cell
migration, is phosphorylated at multiple sites throughout the
protein.24 Semisynthesis of full-length paxillin12 to incorporate
two caged phosphorylated residues should enable interrogation
of the influence of sequential phosphorylation at discrete sites.
Figure 3. Time course of uncaging of DEACM-caged substrate
and NPE-caged inhibitor peptides at 420 nm and then 365 nm.
(a) A solution of the DEACM- and NPE-caged peptides (113 μM each) in
10 mM HEPES (pH 7.1) with 70 μM inosine was irradiated at 420 nm
and then at 365 nm. The reactions were followed by analytical RP-HPLC
at 228 nm. Peptides were quantified on the basis of HPLC standard
curves of the caged and noncaged derivatives. The amount of each peptide
was normalized to the amount of inosine present, and the amounts of
the uncaged peptides are plotted relative to the initial amounts of the
corresponding caged species. (b) HPLC traces of the DEACM- and
NPE-caged peptides (labeled as 2 and 3, respectively) before (t = 0 min)
and after irradiation at 420 and 365 nm. The released substrate peptide,
the DEACMÀOH byproduct of uncaging, and the released inhibitor
peptide are denoted as 4, 5, and 6, respectively. The inset shows the
inosine peak (labeled as 1), which was used to normalize for the HPLC
injection volume, on the same scale as the peptide traces.
Wip1 catalyzes the dephosphorylation of phosphothreonine (pThr)
in the substrate peptide. Therefore, to prevent Wip1 activity until
irradiation, the substrate peptide was prepared with DEACM-
caged pThr. Additionally, because studies of Wip1 inhibition by
cyclic phosphopeptides have revealed that phosphoserine (pSer)
is necessary for inhibition, this peptide was synthesized with
NPE-caged pSer to enable 365 nm light to drive inhibitor
activation. Next, we used reversed-phase (RP) HPLC analysis to
examine the kinetics of photolysis after exposure to irradiation
from a hand-held light source equipped with a 420 nm cut-on
filter (Figure 3). Under these conditions, irradiation of a mixture
of the two peptides led to efficient release of the DEACM group
and generation of the free pThr substrate peptide. Importantly,
the NPE group was stable under these conditions, with only
negligible uncaging even after irradiation for 3 min. Following
this, irradiation at 365 nm released the NPE group to afford the
corresponding pSer inhibitor peptide. These studies demon-
strate that the two caging groups can be employed together to
enable two uncaging events within one system. Because the
DEACM group absorbs at 365 nm, it must be irradiated before
the NPE group to achieve wavelength-selective photolysis.
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building blocks. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
imper@mit.edu
’ ACKNOWLEDGMENT
This research was supported by the NIH Cell Migration
Consortium (GM064346). B.N.G. was supported by the NIGMS
Biotechnology Training Grant (T32-GM08334), and A.A. was
supported by a postdoctoral fellowship from the Swiss National
Science Foundation.
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Figure 4. Wip1 phosphatase activity. Wip1 was incubated with the
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’ ASSOCIATED CONTENT
S
Supporting Information. Supporting figures, experimen-
b
tal methods, and NMR characterization of the caged amino acid
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