General method for the synthesis of caged phosphopeptides: Tools for the exploration of signal transduction pathways

Article abstract of DOI:10.1021/ol0262587

(figure presented) An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.

Full text of DOI:10.1021/ol0262587

ORGANIC
LETTERS
2002
Vol. 4, No. 17
2865-2868
General Method for the Synthesis of
Caged Phosphopeptides: Tools for the
Exploration of Signal Transduction
Pathways
Deborah M. Rothman, M. Eugenio Va´zquez, Elizabeth M. Vogel, and
Barbara Imperiali*
Department of Chemistry, Massachusetts Institute of Technology,
77 Massachusetts AVenue, Cambridge, Massachusetts 02139
imper@mit.edu
Received May 29, 2002
ABSTRACT
An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has
been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding
phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation
and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino
acids.
Kinase-mediated phosphorylation of tyrosine, serine, and
threonine residues in polypeptides and proteins represents a
central mechanism of cell regulation. As such, it is an area
of intense study in which there is considerable potential for
the development of new chemical tools.1 The study of protein
kinases involved in cell migration is of particular interest in
biological and medicinal studies. The present tools available
for the study of phosphorylation-dependent signal transduc-
tion pathways are limited when information in real time is
desired. For example, chemical inhibition, gene knockout,
or point mutation studies can often confirm the significance
or essentiality of a protein kinase; however, such methods
make it difficult to assess the biochemical role of the protein
in real time.
A caged compound includes a photocleavable-protecting
group that masks an essential functionality; upon removal
by photolysis, the functionality is revealed, generating a
biologically active molecule.2 This strategy allows research-
ers spatial and temporal control over the release of effector
molecules in living systems.3 There are several characteristics
required of a caging group to be used in biological systems.
The caged compound, as well as the byproducts of photoly-
sis, should be inert in the cell, neither agonizing nor
antagonizing the receptor molecule. Only the uncaged
effector molecule should alter cell activity. The caged
compound must be stable to hydrolysis and enzymatic
(2) Corrie, J. E. T.; Trentham, D. R. In Biological Applications of
Photochemical Switches; Morrison, H., Ed.; Wiley & Sons: New York,
1993; Chapter 5.
(1) Woodgett, J. R. Protein Kinase Functions, 2nd ed.; Oxford University
Press: New York, 2000.
(3) Park, C.-H.; Givens, R. S. J. Am. Chem. Soc. 1997, 119, 2453-
2463.
10.1021/ol0262587 CCC: $22.00 © 2002 American Chemical Society
Published on Web 07/30/2002

cleavage, and the rate of uncaging must be fast in order to
give a concentration burst of the photolysis product on a
µs-ms time scale.4 Derivatives of the o-nitrobenzyl group
are commonly used to cage biologically active molecules.
For example, caged analogues of divalent calcium,5 ATP,2
cAMP,2 peptides,6,7 and proteins8 have been prepared. The
photochemical release of these species occurs with reasonable
quantum efficiencies at wavelengths above 300 nm.2 Increas-
ing the concentration of a reducing agent such as glutathione
in the medium is often necessary to prevent the nitroso
photobyproduct from reacting with free amines in the cell.18
Therefore, caging biological mediators is an attractive
method for introducing specific concentrations of compounds
into cells and controlling their release in order to observe a
cellular response. Thus, to observe the consequences of
phosphorylation in vivo, the controlled release of a physio-
logical concentration of phosphopeptide can be effected by
benign photolysis. To date, there are no general strategies
for the synthesis of caged phosphopeptides. Bayley and co-
workers have recently reported syntheses of nitrobenzyl- and
p-hydroxyphenacyl-caged thiophosphotyrosine peptides.9 The
preparation of these analogues involves a Hck kinase-
mediated thiophosphorylation of tyrosine-containing peptides
with ATP(γ)S in the presence of divalent cobalt. The
incipient thiophosphate can then be chemically modified with
2-nitrobenzyl bromide or p-hydroxyphenacyl bromide due
to the intrinsic reactivity of the thiophosphate sulfur. While
the resultant caged thiophosphopeptides show good bio-
chemical utility, a disadvantage to this method is that the
kinase-mediated thiophosphorylation must be optimized for
each substrate and enzyme and the thiophosphate residue
unveiled upon photolysis may have altered functionality in
downstream events when compared with native phosphor-
ylated substrates.
(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and
-tyrosine residues. The synthesis of three representative
kinase target sequences (Figure 1) is presented to demonstrate
Figure 1. Target caged phosphopeptides, including modified serine,
threonine, and tyrosine.
the versatility of the method. The caged phosphoserine
peptide, Ac-PL(cpS)PAKLAFQFP-CONH2 (cpErk),12 in-
cludes the core recognition motif for the ERK kinase
(PLSP)13 as well as an ERK docking motif (FXFP).14 The
ERK kinases make up a group of mitogen-activated protein
kinases, MAPKs. ERK has been implicated in multiple
pathways, including cell motility, differentiation, and pro-
liferation. The caged phosphothreonine peptide is Ac-
MARHFD(cpT)YLIRR-CONH2 (cpChk2). The corre-
sponding uncaged peptide would be an antagonist of
Chk2, a homologue of the human Rad53p checkpoint
kinase.15 The caged phosphotyrosine peptide Ac-
EEEHV(cpY)SFPNKQK-CONH2 (cpPax) mimics a section
of human paxillin, which is thought to be involved in focal
adhesions during cell movement. Paxillin can be phos-
phorylated at two sites, Tyr31 and Tyr118: Tyr118 has been
identified as the major phosphorylation site of Paxillin by
FAK (Focal Adhesion Kinase) in vitro.16 cpPax corresponds
to residues 113-125 of paxillin.
Herein, we report a general route for the synthesis of
peptides containing 2-nitrophenylethyl-caged phosphoserine,
-threonine, and -tyrosine residues. The synthesis is based on
an interassembly approach,10,11 integrated into Fmoc solid-
phase peptide synthesis (SPPS). The method is general and
enables the preparation of various peptides containing 1-
(4) Gurney A. M.; Lester, H. A. Physiol. ReV. 1987, 67, 583-617.
(5) Ellis-Davies, G. C. R.; Kaplan, J. Proc. Natl. Acad. Sci. U.S.A. 1988,
85, 6571-6575.
(6) Wood, J. S.; Koszelak, M.; Liu, J.; Lawrence, D. S. J. Am. Chem.
Soc. 1998, 120, 7145-7146.
(7) Walker, J. W.; Gilbert, S. H.; Drummond, R. M.; Yamada, M.;
Sreekumar, R.; Carraway, R. E.; Ikebe, M.; Fay, F. S. Proc. Natl. Acad.
Sci. U.S.A. 1998, 95, 1568-1573.
The caged oligopeptide sequences derived from the current
methodology will also be amenable to integration into full-
length proteins via native chemical ligation methods.17
(8) Curley, K.; Lawrence, D. S. Curr. Opin. Chem. Biol. 1999, 3, 84-
88.
Synthesis of Caged Peptides. The general scheme for the
assembly of caged phosphopeptides is illustrated with a
serine-containing peptide in Scheme 1.
(9) Zou, K.; Miller, W. T.; Givens, R. S.; Bayley, H. Angew. Chem.,
Int. Ed. 2001, 40, 3049-3051.
(10) McMurray, J. S.; Coleman, D. R.; Wang, W.; Campbell, M. L.
Biopolymers 2001, 60, 3-31.
(11) Meutermans, W. D. F.; Alewood, P. F. Tetrahedron Lett. 1996, 37,
4765-4766.
The basic strategy involves the introduction of a 1-(2-
nitrophenyl)ethyl-caged phosphate via the intermediacy of
a trivalent phosphitylating agent, which is subsequently
oxidized. The phosphitylating agent, O-1-(2-nitrophenyl)-
ethyl-O′-â-cyanoethyl-N,N-diisopropylphosphoramidite (1),
was prepared as follows. The precursor 1-(2-nitrophenyl)-
ethanol was derived by reduction of 2-nitroacetophenone with
sodium borohydride using a method modified from Kaplan
(12) “cp” designates caged phospho.
(13) Adams, J. A. Chem. ReV. 2001, 101, 2271-2290.
(14) Jacobs, D.; Glossip, D.; Xing, H.; Muslin, A. J.; Kornfield, K. Genes
DeV. 1999, 13, 163-175.
(15) Yaffe, M. B.; Smerdon, S. J. Structure 2001 9, R33-R38.
(16) Giacontti, F. G. Nature Cell Biol. 2000, 2, E13-E14.
(17) Cotton, G. J.; Muir, T. W. Chem. Biol. 2000, 6, R247-R256.
(18) Kaplan, J. H.; Forbush, B., III; Hoffman, J. F. Biochemistry 1978,
17, 1929-1935.
2866
Org. Lett., Vol. 4, No. 17, 2002

Scheme 1. Interassembly Approach for the Synthesis of Caged Phosphopeptides
and co-workers.18 Next, 1 was synthesized by substituting
prevalence of arginine residues in many protein kinase22
recognition sequences makes this aspect of the synthesis
important.
1-(2-nitrophenyl)ethanol for chlorine in 2-cyanoethyl diiso-
propylchlorophosphoramidite using a procedure similar to
that of Kuphiar et al.19 The preparation of a caged phospho-
serine-containing peptide is illustrated in Scheme 1; the
method is generally applicable to the synthesis of the
corresponding phosphothreonine and phosphotyrosine-
containing peptides with noted modifications. In summary,
each peptide was synthesized by standard Fmoc SPPS, on a
PAL-PEG-PS solid support, up to the key serine, threonine,
or tyrosine residue; these three residues were incorporated
without side chain protection. For cpErk, the free serine
residue was phosphitylated with 5 equiv each of 1 and 1H-
tetrazole in 1:1 dichloromethane (DCM)/tetrahydrofuran
(THF) to afford the resin-bound phosphite 2.20 Oxidation of
2 with m-chloroperoxybenzoic acid (mCPBA) afforded the
more stable bisprotected phosphate 3. While phosphotriesters
are prone to â-elimination during the basic Fmoc deprotec-
tion steps of SPPS,11 treatment of 3 with 20% piperidine in
N,N-dimethylformamide (DMF) concurrently removed the
Fmoc group for chain elongation and the cyanoethyl group21
on the phosphate to give the caged phosphate 4. The resulting
phosphodiester was stable toward elimination yet suitably
protected from side reactions during chain elongation.11 Upon
completion of the synthesis, the peptides were cleaved under
various conditions. Notably, it was found that the 2-nitro-
phenylethyl group was stable to long treatments of 90% TFA
that are necessary to remove the relatively stable benzene-
sulfonyl protecting groups of the arginine precursors. The
The incorporation of threonine and tyrosine residues into
synthetic peptide modules necessitated some minor modifi-
cations of the above procedure. Since the secondary hydroxyl
group of threonine is less reactive than that of serine, 10
equiv of 1 and 1H-tetrazole were required to achieve efficient
phosphitylation. The cyanoethyl-protected phosphorylated
threonine (threonine version of 3) appeared as two separate
peaks in the HPLC chromatogram. These components may
result from the chiral phosphorus center adjacent to the chiral
center at the â-carbon of threonine since the two peaks
showed identical masses in the mass spectrometry analysis.
After deprotection of the cyanoethyl group with piperidine,
the HPLC chromatogram for the peptide was converted to a
single peak. Due to the potential for methionine oxidation,
the cpChk2 peptide was dissolved in deoxygenated water
and stored at -20 °C. It is important to note that residues
that are sensitive to oxidation11 such as methionine and
tryptophan can only be incorporated into the sequence of
caged phosphopeptides after the phosphite oxidation step.
The tyrosine hydroxyl also showed reduced reactivity in
the phosphitylation step; optimal conditions implemented 10
equiv of 1 and 1H-tetrazole together with dried 4 Å molecular
sieves. It was also found that 4,5-dicyanoimidazole may be
used in place of 1H-tetrazole with a similar yield. Oxidation
of the tyrosine phosphite with mCPBA resulted in several
side products. Therefore, oxidation was achieved with either
tert-butyl hydroperoxide solution in decane or hydrogen
peroxide as an aqueous solution in THF. No further
(19) Kupihar, Z.; Varadi, G.; Monostori, E.; Toth, G. K. Tetrahedron
Lett. 2000 41, 4457-4461.
(20) Barnwarth, W.; Trzeciak, HelV. Chim. Acta 1987, 70, 175-186.
(21) Wakamiya, T.; Saruta, K.; Yasuoka, J.; Kusumoto, S. Chem. Lett.
1994, 1099-1102
(22) Pinna, L.; Ruzzene, M. Biochim. Biophys. Acta 1996, 1314, 191-
225.
Org. Lett., Vol. 4, No. 17, 2002
2867

ionization mass spectrometry (ESMS). The overall phos-
phorylation efficiency ranged from 75 to 95% as determined
by HPLC. The quantum yield for the uncaging of each
peptide was determined by a previously reported method.23
cpErk has a quantum yield of 0.26; cpChk2 has a quantum
yield of 0.33, and cpPax has a quantum yield of 0.28.24
31P Magic Angle Spinning NMR. In addition to cpErk,
cpChk2, and cpPax, a resin-bound tripeptide, cpSer-Pro-
Gly-resin, was prepared on an aminomethylated polystyrene
solid support. The higher loading provided by this resin
enabled magic angle spinning (MAS) 31P NMR experiments,
which could be used as an unambiguous method of following
the progression of phosphitylation, oxidation, and subsequent
cyanoethyl deprotection on-bead during the course of
developing the synthetic strategy. 31P MAS NMR spectra
of key intermediates in the synthesis of cpSer-Pro-Gly-resin
are illustrated in Figure 2.
Herein we have described the generally applicable solid-
phase syntheses of peptides containing 1-(2-nitrophenyl)-
ethyl-caged phosphoserine, -threonine, and -tyrosine residues.
For each representative hydroxyamino acid residue, a
biologically active phosphopeptide was synthesized and may
be used in real-time studies of kinases in vivo.
Acknowledgment. This research was supported by
the NIH (GM64346 Cell Migration Consortium) and
Merck Research Laboratories. We also acknowledge NIH
1S10RR13886-01 and NSF CHE-9808061 for support of the
NMR facility and Fundacio´n Ramo´n Areces and Human
Frontier Science Program for the award of postdoctoral
fellowships to M.E.V. We would also like to thank Professor
Graham Ellis-Davies (MCP Hahnemann) for helpful discus-
sions and for a gift of 1-(2-nitrophenyl)ethyl-phosphate.
Supporting Information Available: Details of the solid-
phase synthesis of cpErk, cpChk2, and cpPax, quantum
yield determination, and 31P MAS NMR experiments. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Figure 2. 31P Magic Angle Spinning NMR. Phosphorus NMR
taken on AM resin at 202 MHz. (a) Free hydroxyl on serine, (b)
phosphite on serine, (c) phosphate on serine, fully protected, and
(d) phosphate on serine after cyanoethyl removal.
OL0262587
modifications to the synthesis were necessary after the
oxidation step.
(23) Ellis-Davies, G. C. R.; Kaplan, J. Proc. Natl. Acad. Sci. U.S.A. 1994,
91, 187-191.
(24) See Supporting Information for data and calculations.
The peptides were characterized by reversed-phase high-
performance liquid chromatography (HPLC) and electrospray
2868
Org. Lett., Vol. 4, No. 17, 2002