Photochemically-activated probes of protein-protein interactions

Article abstract of DOI:10.1021/ol070238t

The activity of light-activatable ("caged") compounds can be temporally and spatially controlled, thereby providing a means to interrogate intracellular biochemical pathways as a function of time and space. Nearly all caged peplides contain photocleavable groups positioned on the side chains of key residues. We describe an alternative active site targeted strategy that disrupts the interaction between the protein target (SH2 domain, kinase, and proteinase) and a critical amide NH moiety of the peptide probe.

Full text of DOI:10.1021/ol070238t

ORGANIC
LETTERS
2007
Vol. 9, No. 12
2249-2252
Photochemically-Activated Probes of
Protein Protein Interactions
−
Sandip K. Nandy, Richard S. Agnes, and David S. Lawrence*
Department of Biochemistry, The Albert Einstein College of Medicine of YeshiVa
UniVersity, 1300 Morris Park AVenue, Bronx, New York 10461-1602
dlawrenc@aecom.yu.edu
Received January 30, 2007 (Revised Manuscript Received May 1, 2007)
ABSTRACT
The activity of light-activatable (“caged”) compounds can be temporally and spatially controlled, thereby providing a means to interrogate
intracellular biochemical pathways as a function of time and space. Nearly all caged peptides contain photocleavable groups positioned on
the side chains of key residues. We describe an alternative active site targeted strategy that disrupts the interaction between the protein target
(SH2 domain, kinase, and proteinase) and a critical amide NH moiety of the peptide probe.
Living cells have been referred to as the test tubes of the
21st century.1 Indeed, a host of reagents have been described
for inhibiting, manipulating, or visualizing a wide variety
of intracellularly relevant processes. Nonetheless, key chal-
lenges remain before the cell-as-a-test-tube analogy can be
fully realized. A particularly confounding attribute that
differentiates living cell biochemistry from its counterpart
in the test tube is that the cell, not the investigator, controls
where and when a given transformation occurs. Light-
activatable (“caged”) compounds allow the investigator to
retain control over the activity of the bio-reagent, even after
it has entered the cell.2 In this regard, a number of caged
derivatives of small molecules, including ATP, glutamate,
NO, and many others, has been described.2c Furthermore,
recent interest in defining the temporal and spatial dynamics
of signaling pathways, biochemical cascades largely driven
by protein-protein interactions, has led to the construction
of caged peptide derivatives.3 The latter compounds are
designed to engage specific protein recognition motifs, but
only upon photoactivation. Several examples of peptide-
based protein kinase sensors3e,g and inhibitors3b as well as
14-3-33d and SH23c,f domain-targeting species have been
reported. The general strategy for the preparation of these
peptidic species is based on the side-chain modification (with
photolabile groups) of key residues required for bio-
recognition, such as Ser or Tyr for protein kinases or
phosphorylated Ser or Tyr for 14-3-3 and SH2 domains,
(2) (a) Lawrence, D. S. Curr. Opin. Chem. Biol. 2005, 9, 570-575. (b)
Rothamn, D. M.; Shults, M. D.; Imperiali, B. Trends Cell Biol. 2005, 15,
502-510. (c) Goeldner, M., Givens, R., Eds. Dynamic Studies in Biology;
Wiley: New York, 2005.
(1) Hansen, P. M.; Oddershede, L. B. Proc. SPIE 2005, 5930, 593003-
1-9.
10.1021/ol070238t CCC: $37.00
© 2007 American Chemical Society
Published on Web 05/17/2007

respectively. Unfortunately, many amino acids lack side
chain functionality necessary for modification whereas the
preparation of caged derivatives of those that can be modified
is typically a multistep, off-resin, process.
Scheme 1. On-Resin Synthesis of Caged Peptide 4, Structure
of the Alternatively Caged Peptide 5, and Structure of the
Parent SH2 Domain-Directed Peptide 6
Protein-protein interactions are often dependent upon one
or a few key amino acid residues. These residues must be
able to achieve the requisite contacts with the protein-binding
partner in order for recognition and/or catalysis to occur. In
many instances, the amide NH of the essential and/or
adjacent residue is crucial for proper orientation of the critical
side chain. We reasoned that the incorporation of a photo-
labile moiety on this key amide nitrogen could significantly
compromise recognition or catalysis, via loss of amide
hydrogen bond donating ability and/or the presence of a
sterically demanding light-cleavable substituent. This notion
has been examined via the design, synthesis, and character-
ization of caged peptides for three different protein interaction
domains.
To the best of our knowledge, there have only been two
reports of backbone-caged peptides. Darszon, Yumoto, and
their colleagues described a backbone-substituted glycine
residue that was prepared off-resin as an Fmoc(N-o-nitro-
benzyl) derivative and subsequently coupled to the growing
peptide chain.4 These investigators reported difficulties in
coupling the subsequent residue, a serine moiety, to the
N-alkylated glycine. However, they found that acyl fluorides
or diisopropylcarbodiimide furnished the desired material.
More recently, Johnson and Kent examined photoremovable
N-benzylated protecting groups as part of Boc chemistry
solid-phase peptide synthesis.5 These investigators likewise
found that the subsequent addition of a standard amino acid
to the caged residue could be problematic, particularly if
hindered amino acids were involved.
and then reduced with NaBH3CN to furnish the desired
N-benzylated derivative 2 (HPLC and electrospray mass
spectrometry).4,5 Unfortunately, as reported by Darszon,
Yumoto, and Kent,6,7 we found that addition of the subse-
quent amino acid to the N-benzylated residue was problem-
atic. For example, Darszon and Yumoto’s diisopropylcar-
bodiimide protocol was employed to couple Fmoc-Ser to (N-
o-nitrobenzyl)Gly-peptide resin, but failed to furnish the
desired product 3. This may be a consequence of the
significantly more sterically demanding nature of the side
chain phosphorylated Fmoc-Tyr residue to be coupled.
Ultimately, we discovered that the activating agent PyBroP8
provided the desired pTyr-derivatized peptide 3 (however,
see compound 10, Vide infra). Subsequent solid-phase
synthesis proceeded uneventfully to furnish 4.
Src homology (SH) 2 domains play a key role in
organizing, assembling, and activating signaling complexes.9
These domains bind to phosphorylated Tyr- (pTyr) containing
residues positioned within an appropriate amino acid se-
quence context on a peptide or protein.8 For example, the
Lck SH2 domain displays a moderate affinity (KD ≈ 1-5
µM) for peptides of the general form Ac-pTyr-Xaa-Xaa-Ile-
amide (Figure 1). Previous structural studies have reported
that the amide moiety linking the pTyr-Xaa dyad forms a
key hydrogen bond with the SH2 domain.9b The caged
derivative 4 was prepared via the Scheme 1 protocol. We
also prepared the corresponding analogue 5, which contains
We examined whether the caged residue could be prepared
directly on the resin, a strategy that could be immediately
applied to conventional commercially available side chain
protected amino acids. Solid-phase peptide synthesis was
performed using Fmoc chemistry on the Rink resin. The
Fmoc group was removed, via standard piperidine treatment,
from the residue to be caged (Scheme 1). The free N-terminal
amine of 1 was exposed twice to 4,5-dimethoxy-2-nitro-
benzaldehyde to ensure complete conversion to the imine
(3) (a) 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. (b) Wood, J. S.; Koszelak, M.; Liu, J.;
Lawrence, D. S. J. Am. Chem. Soc. 1998, 120, 7145-7146. (c) Zou, K.;
Miller, W. T.; Givens, R. S.; Bayley, H. Angew. Chem., Int. Ed. 2001, 40,
3049-3051. (d) Nguyen, A.; Rothman, D. M.; Stehn, J.; Imperiali, B.; Yaffe,
M. B. Nat. Biotech. 2004, 22, 993-1000. (e) Veldhuyzen, W. F.; Nguyen,
Q.; McMaster, G.; Lawrence, D. S. J. Am. Chem. Soc. 2003, 125, 13358-
13359. (f) Humphrey, D.; Rajfur, Z.; Vazquez, M. E.; Scheswohl, D.;
Schaller, M. D.; Jacobson, K.; Imperiali, B. J. Biol. Chem. 2005, 280,
22091-22101. (g) Wang, Q.; Dai, Z.; Cahill, S. M.; Blumenstein, M.;
Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 14016-14017. For an
alternative strategy to side chain modification, see (h) Taniguchi, A.; Sohma,
Y.; Kimura, M.; Okada, T.; Ikeda, K.; Hayashi, Y.; Kimura, T.; Hirota, S.;
Matsuzaki, K.; Kiso, Y. J. Am. Chem. Soc. 2006, 128, 696-697.
(4) Sasaki, Y.; Coy, D. H. Peptides 1987, 8, 119-121.
(6) Tatsu, Y.; Nishigaki, N.; Darszon, A.; Yumoto, N. FEBS Lett. 2002,
525, 20-24.
(7) Johnson, E. C.; Kent; S. B. Chem. Commun. 2006, 1557-1559.
(8) Coste, J.; Dufour, M.-N.; Panatloni, A.; Castro, B. Tetrahedron Lett.
1990, 31, 669-672.
(5) An alternative strategy, which we did not explore, is the direct
alkylation of an o-nitrobenzenesulfonyl-activated amine: (a) Miller, S. C.;
Scanlan, T. S. J. Am. Chem. Soc. 1997, 119, 2301-2302. (b) Biron, E.;
Chatierjee, J.; Kessler, H. J. Peptide Sci. 2006, 12, 213-219 and references
cited therein.
(9) (a) Bradshaw, J. M.; Waksman, G. AdV. Protein Chem. 2002, 61,
161-210. (b) Coordinates obtained from the Protein Data Bank (1LCJ);
Eck, M. J.; Shoelson, S. E.; Harrison, S. C. Nature 1993, 362, 87-91.
2250
Org. Lett., Vol. 9, No. 12, 2007

caged fluorescent substrate for chymotrypsin that contains
a C-terminal coumarin at the scissile position. Previous
studies have demonstrated that the free coumarin, generated
upon proteolysis, is highly fluorescent.12 The peptide sub-
strate 7 (Scheme 2) was synthesized using an Fmoc solid-
Scheme 2. Chymotrypsin Substrate 7, its Caged Counterpart 8,
and the Caged PKA Substrate 10
Figure 1. (A) pTyr peptide (ball and stick) bound to the SH2
domain of Lck. The key NH amide moiety linking the pTyr (red
bar) and Glu residues is highlighted (white arrow) and shown within
hydrogen-bonding distance of the His-180 carbonyl (space filling).9b
(B) PKA substrate peptide (ball and stick) complexed with an ATP
transition state mimic (ADP/AlF3; yellow space filling). The
phosphoryl acceptor Ser (red arrow) and the key amide NH (white
arrow) are highlighted.14
the photolabile group on the pTyr-amide moiety. Kd values
were acquired via a competition assay using a previously
described dapoxyl-labeled peptide,10 which displays a 4-fold
fluorescence enhancement upon binding to the Lck SH2
domain (Supporting Information). The caged derivative 4
has a significantly lower SH2 domain affinity (127 ( 6 µM)
than the parent peptide 6 (2.6 ( 0.2 µM). By contrast, the
difference in affinity between 5 (43 ( 10 µM) and 6 is
somewhat more modest. The latent ligand 4 is converted to
the high affinity form 6 upon photolysis. Furthermore, longer
photolysis times generate larger yields of the active peptide
6 (Supporting Information). The latter property represents
one of the distinct advantages associated with caged com-
pounds, namely the ability to control both the timing and
the quantity of the photoactivated species.
Proteinases participate in a diverse array of biochemical
processes that range from the generation of biologically
active proteins from their immature counterparts to the
degradation of aged or otherwise defective proteins. Like
many other proteins that act on protein substrates, proteinases
are capable of utilizing short peptides of the appropriate
amino acid sequence as substrates. Chymotrypsin’s distinct
preference for Phe at the scissile bond is most noteworthy
within the context of caged peptides ligands. The aromatic
side chain lacks a modifiable substituent and is therefore not
susceptible to the standard side chain caging strategy.
Chymotrypsin substrate recognition and subsequent hy-
drolysis is dependent upon the amide moieties of the scissile
bond and of adjacent residues. Indeed, N-methyl substitution
at the scissile amide (and at the amide moieties at nearby
residues) renders chymotrypsin substrates resistant to pro-
teolysis.11 N-methylation not only results in the loss of a
key hydrogen bond required for substrate recognition, but
also introduces a steric factor that compromises active site
incorporation. With these features in mind, we prepared a
phase protocol (Supporting Information). The corresponding
caged derivative 8 was prepared by interrupting the standard
protocol at the Phe-courmarin stage with the Scheme 1
caging strategy. Peptide 7, and its caged analogue 8, were
examined as chymotrypsin substrates. Compound 7 is an
efficient substrate (Km ) 6.3 ( 1.3 µM; Vmax ) 7.2 ( µmol/
min-mg) and exhibits a 6-fold enhancement in fluorescence
upon complete hydrolysis. By contrast, the caged derivative
8 is inactive as a chymotrypsin substrate (Figure 2). As in
the case of the caged SH2 ligand 4, increasing photolysis
times converts increasing amounts of caged analogue (8) to
the active substrate (7). To the best of our knowledge,
compound 8 is the first example of a caged proteinase sensor.
Hydrogen bonds play a critical role in orienting the
phosphorylatable Ser of Leu-Arg-Arg-Ala-Ser-Leu-Gly-
amide (9) into the active site of the PKA. Indeed, N-
methylation of the Ala-Ser-amide moiety reduces the PKA-
catalyzed efficiency (kcat/Km) of phosphorylation by 7 orders
of magnitude.13 Crystallographic studies revealed that the
amide NH of Ser is within hydrogen-bonding distance of
the side chain hydroxyl and is also oriented toward the
incoming phosphoryl group from ATP (Figure 1B).14 Con-
sequently, we anticipated that steric bulk, positioned at this
sensitive site (10), could have a deleterious effect on
(12) (a) Zimmerman, M.; Ashe, B.; Yurewicz, E. C.; Patel, G. Anal.
Biochem. 1977, 78, 47-51. (b) Backes, B. J.; Harris, J. L.; Leonetti, F.;
Craik, C. S.; Ellman, J. A. Nat. Biotechnol. 2000, 18, 187-193.
(13) Bramson, H. N.; Thomas, N. E.; Kaiser, E. T. J. Biol. Chem. 1985,
260, 15452-15457.
(14) Coordinates, obtained from the Protein Data Bank (1L3R);
Madhusudan Akamine, P.; Xuong, N.-H.; Taylor, S. S. Nature Struc. Biol.
2002, 9, 273-277.
(10) Wang, Q.; Lawrence, D. S. J. Am. Chem. Soc. 2005, 127, 7684-
7685.
(11) Haviv, F.; Fitzpatrik, T. D.; Swenson, R. E.; Nichols, C. J.; Mort,
N. A.; Bush, E. N.; Diaz, G.; Bammert, G.; Nguyen, A.; Rhutasel, N. S.;
Nellans, H. N.; Hoffman, D. J.; Johnson, E. S.; Greer, J. J. Med. Chem.
1993, 36, 363-369.
Org. Lett., Vol. 9, No. 12, 2007
2251

Figure 3. Caged peptide 10 (0 min) is not phosphorylated by PKA
as assessed by a fixed time point assay. Longer photolysis times
produce larger amounts of active substrate and therefore enhanced
reaction rates.
Figure 2. Caged peptide 8 (0 min) is not hydrolyzed by
chymotrypsin as assessed by a fluorescent continuous assay (λex
)
380 nm; λem ) 460 nm). Longer photolysis times produce greater
amounts of active substrate and therefore enhanced reaction rates.
In summary, we have described an on-resin, solid-phase
method that directly furnishes caged peptides in a straight-
forward fashion. These derivatives, represented by an SH2
ligand and substrates for a protein kinase and a protease,
provide a means to control both the timing of target protein
interaction as well as the amount of active material unleashed
even after the bio-agent has been delivered to the cell (e.g.,
via microinjection).
phosphoryl acceptor capability. Compound 10 was prepared
in a fashion analogous to that outlined in Scheme 1.
However, we discovered that Fmoc-Ala could not be coupled
to the reductively alkylated Ser residue using the PyBroP
protocol. Instead, coupling was achieved using the acid
chloride of Fmoc-Ala.15 The efficiency of PKA-catalyzed
phosphorylation of peptide 10 was assessed via a [γ-33P]-
ATP assay. In the absence of photolysis, peptide 10 is not
phosphorylated (Figure 3). Photolysis of 10 generates the
active substrate 9 (mass spectrometry) that, upon addition
of PKA, furnishes the phospho-product (scintillation count-
ing). Furthermore, longer photolysis times generate larger
quantities of radiolabeled product.
Acknowledgment. We thank the National Institutes of
Health for financial support (GM067198 and CA79954).
Supporting Information Available: Experimental details
of peptide synthesis and characterization and enzyme assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(15) Carpino, L. A.; Cohen, B. J.; Stephens, K. E., Jr.; Sadat-Aalaee,
Y.; Tien, J.-H.; Langridge, D. C. J. Org. Chem. 1986, 51, 3732-3734.
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