Synthesis of 3,5-difluorotyrosine-containing peptides: Application in substrate profiling of protein tyrosine phosphatases

Article abstract of DOI:10.1021/ol801868a

(Chemical Equation Presented) Fully protected 3,5-difluorotyrosine (F ₂Y), Fmoc-F₂Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual peptides and combinatorial peptide libraries. The F

Full text of DOI:10.1021/ol801868a

ORGANIC
LETTERS
2008
Vol. 10, No. 20
4605-4608
Synthesis of 3,5-Difluorotyrosine-
Containing Peptides: Application in
Substrate Profiling of Protein Tyrosine
Phosphatases
Bhaskar Gopishetty, Lige Ren, Tiffany M. Waller, Anne-Sophie Wavreille,
Miguel Lopez, Amit Thakkar, Jinge Zhu, and Dehua Pei*
Department of Chemistry and Ohio State Biochemistry Program, The Ohio State
UniVersity, 100 West 18th AVenue, Columbus, Ohio 43210
pei.3@osu.edu
Received August 14, 2008
ABSTRACT
Fully protected 3,5-difluorotyrosine (F2Y), Fmoc-F2Y(tBu)-OH, is efficiently prepared by a chemoenzymatic process and incorporated into individual
peptides and combinatorial peptide libraries. The F2Y-containing peptides display kinetic properties toward protein tyrosine phosphatases
(PTPs) similar to their corresponding tyrosine-containing counterparts but are resistant to tyrosinase action. These properties make F2Y a
useful tyrosine surrogate during peptide library screening for optimal PTP substrates.
Replacement of a hydrogen with fluorine results in a small
increase in the molecular size but often dramatically different
physical, chemical, and biological properties of the mol-
ecule.1 As such, fluorinated compounds provide useful
mechanistic probes of enzyme-catalyzed reactions and other
biological processes. For example, ring fluorinated analogues
of tyrosine have been used to examine the catalytic mech-
anisms of tyrosinase,2 tyrosine phenol-lyase,3 protein tyrosine
kinase and phosphatase,4,5 ∆5-3-ketosteroid isomerase,6 and
ribonucleotide reductase.7
family of enzymes that catalyze the hydrolysis of phospho-
tyrosine (pY) in proteins back to tyrosine and inorganic
phosphate. A major challenge in the PTP field is to determine
the physiological substrates and cellular function of these
enzymes. We recently developed a combinatorial library
method to profile the substrate specificity of PTPs and
subsequently used the specificity information to predict the
protein substrates of the PTPs.8 A key element of the
technique involved selective derivatization of the reaction
product (i.e., tyrosine) with a chemical tag (e.g., biotin). This
was accomplished by first oxidizing the tyrosine side chain
into an orthoquinone with O2 and tyrosinase, followed by
conjugate addition with biotin-hydrazide. To prevent false
positives from unreacted substrates, tyrosine was excluded
from the library. However, PTPs may require tyrosine
residues for optimal binding and catalysis. Thus, we envis-
aged the inclusion of a fluorinated tyrosine into the peptide
Our own interest in fluorotyrosines stems from our ongoing
studies on protein tyrosine phosphatases (PTPs), a large
(1) Hagmann, W. K. J. Med. Chem. 2008, 51, 4359–4369.
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10.1021/ol801868a CCC: $40.75
Published on Web 09/18/2008
2008 American Chemical Society

library as a tyrosinase-resistant tyrosine surrogate. Among
all of the known mono-, di-, and multiply fluorinated tyrosine
analogues, we reasoned that 3,5-difluorotyrosine (F2Y,
compound 1 in Scheme 1) should contain the minimal
phase by using standard Fmoc/HBTU chemistry (Table 1,
Table 1. Comparison of the Kinetic Constants of F2Y- and
Tyr-Containing Peptides against PTP1B (pH 7.4)
peptide
kcat (s-1 KM (µM) kcat/KM (µM-1 s-1
) )
Scheme 1. Synthesis of Fully Protected F2Y
DNLF2YpYWD-NH2 (4)
DNLYpYWD-NH2 (5)
DDTF2YDpYAA-NH2 (6) 36 ( 2
DDTYDpYAA-NH2 (7) 33 ( 1
REF2YEFpYAA-NH2 (8) 44 ( 2
REYEFpYAA-NH2 (9)
36 ( 1 3.6 ( 0.4
32 ( 1 5.8 ( 0.8
10.0
5.5
2.7
2.3
3.2
5.4
13 ( 3
14 ( 2
14 ( 2
33 ( 1 6.0 ( 0.7
compounds 4-9). Peptide 5 is derived from a known pY
motif of receptor protein tyrosine kinase erbB2,11 whereas
peptides 7 and 9 are analogous to the prefered substrates of
PTP1B (the prototypical PTP), previously identified from a
peptide library.8 These peptides contain F2Y (or Tyr) at the
pY-3, pY-2, or pY-1 position (relative to pY, which is
designated as position 0). Earlier studies have shown that
residues at the N-terminal side of pY are most critical in
defining the substrate specificity of PTPs.12 During peptide
synthesis, NR-Fmoc-F2Y(tBu)-OH was efficiently incorpo-
rated into peptides without incidence (as judged by ninhydrin
tests), despite that it was used at only 1.5 equiv (4 equiv
was used for all other amino acids). Reversed-phase HPLC
analysis of the crude peptides showed that in each case the
desired peptide was the major product (see Figure 1, for
number of fluorine substitutions to render it resistant to
tyrosinase action. Because F and H atoms have similar van
der Waals radii (1.35 Å for F and 1.10 Å for H), Tyr and
F2Y are essentially isosteric, although the side chain of F2Y
has a lower pKa value (7.2) than that of Tyr (9.9).9
F2Y has previously been synthesized both chemically10
and enzymatically.3,4,7 Synthesis of F2Y-containing peptides
employed side chain unprotected Fmoc-F2Y-OH, and the
resulting peptides were purified by HPLC.4,7 The reported
peptides were either very short or contained F2Y near their
N-termini (therefore no repeated coupling reactions after
incorporation of F2Y), and each contained only a single F2Y
residue. We felt that the unprotected F2Y side chain might
be problematic with peptide library synthesis, during which
more forcing coupling conditions are typically employed to
drive reactions to completion, some library members will
contain multiple F2Y residues, and HPLC purification is not
an option. In this report, we describe the synthesis of fully
protected F2Y, its incorporation into peptides and peptide
libraries, and its activity against PTPs in comparison with
the tyrosine counterparts.
Figure 1. HPLC analysis of peptide 4 on a C-18 column, eluted
with a linear gradient of 10-60% CH3CN in H2O containing 0.1%
We employed the enzymatic systhesis originally developed
by Phillips and co-workers3 to prepare multigram quantities
of F2Y from 2,6-difluorophenol, pyruvate, and NH3 (Scheme
1). Treatment of F2Y with N-(9-fluorenylmethylcarbonylox-
y)succinimide in 10% sodium carbonate gave NR-Fmoc-F2Y-
OH, which was subsequently converted into its methyl ester
2 using thionyl chloride in refluxing methanol. The phenol
group was next protected as a tert-butyl ether by treatment
with isobutylene and H2SO4. Finally, hydrolysis of the methyl
ester by LiOH in THF/H2O gave the desired NR-Fmoc-
F2Y(tBu)-OH (3) in 24% overall yield (from F2Y).
trifluoroacetic acid over 50 min.
example). Peptides 4-9 were purified by preparative HPLC,
and their kinetic activities toward PTP1B (i.e., kcat, KM, and
kcat/KM values) were determined at pH 7.4 (Table 1). The
data show that substitution of F2Y for tyrosine resulted in
minimal changes in kinetic constants (e2-fold, which is
within the margin of experimental error). Thus, F2Y is a good
functional mimic of tyrosine, in terms of binding to the active
site of PTPs.
Three F2Y-containing peptides and their corresponding
Tyr-containing counterparts were synthesized on the solid
(11) Yamamoto, T.; Ikawa, S.; Akiyama, T.; Nomura, N.; Miyajima,
N.; Saito, T.; Toyoshima, K. Nature 1986, 319, 230–234.
(12) (a) Zhang, Z.-Y.; Thieme-Sefler, A. M.; Maclean, D.; McNamara,
D. J.; Dobrusin, E. M.; Sawyer, T. M.; Dixon, J. E. Proc. Natl. Acad. Sci.
U.S.A. 1993, 90, 4446–4450. (b) Zhang, Z.-Y.; Maclean, D.; McNamara,
D. J.; Sawyer, T. K.; Dixon, J. E. Biochemistry 1994, 33, 2285–2290.
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4606
Org. Lett., Vol. 10, No. 20, 2008

Next, a pair of Tyr- and F2Y-containing peptides (peptides
8 and 9 in Table 1) were tested for activity against tyrosinase.
The peptides were individually treated with tyrosinase in the
presence of atmospheric O2 and excess biotin hydrazide.
Reversed-phase HPLC/MS analysis of the reaction mixtures
revealed that the Tyr-containing peptide REYEFpYAA was
quantitatively converted into a new species, which had an
increased retention time (from 32.0 to 34.5 min) and
molecular mass (from m/z 1168.4. to 1440.7) (Figure S1 in
Supporting Information). The increase of 272.3 amu in
molecular mass is consistent with the addition of a single
biotin hydrazide molecule to the tyrosine side chain. As
expected, the F2Y-containing peptide (8) was completely
resistant to tyrosinase action.
Finally, to test whether F2Y is compatible with peptide
library synthesis, screening, and postscreening sequence
identification, we designed a pY peptide library containing
five random positions immediately N-terminal to pY
[SAXXXXXpYAABBRM-resin, where B is ꢀ-alanine and
X is F2Y, norleucine (Nle), or any of the 17 proteinogenic
amino acids excluding Met, Cys, and Tyr]. The library was
synthesized on polyethylene glycol polyacrylamide (PEGA)
resin13 by the split-and-pool method.14 A portion of the
resulting one-bead-one-compound (OBOC) library (∼40 000
beads) was treated with 1.0 nM PTP1B for 20 min (at pH
7.4), followed by incubation with 1.2 µM mushroom
tyrosinase in the presence of atmospheric O2 and 6 mM
3-methyl-2-benzothiazolinonehydrazone (MBTH). Under
these conditions, only beads that carried the most preferred
substrates of PTP1B were dephosphorylated (usually only
partially). The exposed tyrosine side chain was subsequently
oxidized by the excess tyrosinase activity into an ortho-
quinone, which was trapped by reaction with MBTH to form
a dark pink pigment (Figure 2a).15 Thus, as a result of this
reaction cascade, a bead carrying a preferred PTP1B substrate
would turn pink/red, whereas beads carrying poor substrates
would not. This was indeed the case (Figure 2b). The pink/
red colored beads were removed from the library and
individually sequenced by partial Edman degradation/mass
spectrometry16 to reveal the most preferred PTP1B substrates
(Table 2). Figure 2c shows an example of the MS spectrum
derived from one of the pink beads (bead No. 2 in Table
2), carrying the sequence of SAVITDF2YpYAABBRM.
Our results are in agreement with earlier reports that
PTP1B prefers acidic residues at the N-terminal side of
pY, especially at the pY-2 position.8,12,17 A control
Figure 2. (a) Reactions involved in library screening against PTP
and bead coloration. (b) A portion of the OBOC pY library after
treatment with PTP1B (1.0 nM) and tyrosinase (1.2 µM) (viewed
under a disecting microscope). (c) MALDI-TOF spectrum of peptide
SAVITDF2YpYAABBRM* and its truncation products after partial
Edman degradation (derived from a single PEGA resin bead). M*,
homoserine lactone.
Table 2. Partial List of the Selected PTP1B Substratesa
bead no.
peptide sequence
(13) Meldal, M. Tetrahedron Lett. 1992, 33, 3077–3080.
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Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82–84. (b) Houghten,
R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo,
J. H. Nature 1991, 354, 84–86. (c) Furka, A.; Sebestyen, F.; Asgedom, M.;
Dibo, G. Int. J. Pept. Protein Res. 1991, 37, 487–493.
1
2
QDVDApYAA
VITDF2YpYAA
LQF2YDNpYAA
ITMDQpYAA
WGTDSpYAA
SSFDVpYAA
SRHEWpYAA
ETDFApYAA
NDLF2YEpYAA
FTSGLpYAA
3
4
5
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(16) Thakkar, A.; Wavreille, A.-S.; Pei, D. Anal. Chem. 2006, 78, 5935–
5939.
6
7
8
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A.; Vlattas, I.; Sytwu, T.; Li, Y.-C.; Wennogle, L. P. Biochemistry 1998,
37, 15598–15606. (b) Vetter, S. W.; Keng, Y.-F.; Lawrence, D. S.; Zhang,
Z.-Y. J. Biol. Chem. 2000, 275, 2265–2268. (c) Walchli, S.; Espanel, X.;
Harrenga, A.; Rossi, M.; Cesareni, G.; van Huijsduiinen, R. H. J. Biol.
9
10
a M, norleucine.
Chem. 2004, 279, 311–318
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Org. Lett., Vol. 10, No. 20, 2008
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experiment without the PTP1B treatment resulted in no pink/
red colored beads at all (data not shown). Since ∼25% of
the library members contain at least one F2Y residue in the
random region, the lack of any pink/red beads in the control
provides further evidence that F2Y is completely resistant
to tyrosinase action. Most importantly, F2Y is compatible
with every aspect of library synthesis, screening, and
sequence identification.
been developed by employing MBTH as the coupling
reagent. Because the pink/red pigment is only formed on
the beads that have undergone PTP reaction, the current
method does not generate any false positives and is opera-
tionally straightforward. Application of the methodologies
to systematically profile the substrate specificity of PTPs is
currently ongoing in our laboratory and will be reported
elsewhere in due course.
Acknowledgment. This work was supported by the
National Institutes of Health (GM062820).
In summary, we have found that F2Y is a functional,
tyrosinase-resistant mimetic of tyrosine for applications such
as defining the substrate specificity of PTPs through com-
binatorial library screening. An efficient method has been
developed for the chemoenzymatic synthesis of fully pro-
tected F2Y and its incorporation into peptides and peptide
libraries. In addition, an improved PTP screening assay has
Supporting Information Available: Detailed experimen-
tal procedure and spectral data. This material is available
free of charge via the Internet at http://pubs.acs.org.
OL801868A
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Org. Lett., Vol. 10, No. 20, 2008