Facile incorporation of a phosphatase activity-dependent quinone methide generating motif into phosphotyrosine

Article abstract of DOI:10.1055/s-0029-1217025

A novel phosphotyrosine analogue that incorporates a phosphatase activity-dependent quinone methide generating motif is synthesized from tyrosine. Following orthoformylation of the phenol moiety of methyl N-Cbz-tyrosinate, the Fmoc-protected 3-difluoromethyl analogue of phosphotyrosine is obtained by functional group transformations. Georg thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-0029-1217025

PAPER
3765
Facile Incorporation of a Phosphatase Activity-Dependent Quinone Methide
Generating Motif into Phosphotyrosine
3-Difluoromethyl
A
u
nalogue of
i
Phosphotyros
S
ine hen,* Lixin Qi, Mohini Ravula
Northern Illinois University, Department of Chemistry and Biochemistry, DeKalb, Illinois 60115, USA
Fax +1(815)7534802; E-mail: kshen@niu.edu
Received 23 April 2009; revised 20 July 2009
phatase probe into a specific phosphopeptide substrate.4
The integration is made possible by the synthesis of a
Fmoc-protected 3-difluoromethyl analogue of phosphoty-
rosine 1 (Figure 1), a novel phosphotyrosine analogue that
Abstract: A novel phosphotyrosine analogue that incorporates a
phosphatase activity-dependent quinone methide generating motif
is synthesized from tyrosine. Following orthoformylation of the
phenol moiety of methyl N-Cbz-tyrosinate, the Fmoc-protected
3-difluoromethyl analogue of phosphotyrosine is obtained by func- incorporates a phosphatase activity-dependent quinone
methide generating motif.5
tional group transformations.
Key words: amino acids, fluorine, hydrolyses, phosphates, quino-
ne methides, quinomethanes
Fmoc NH
HO2C
CHF2
OPO3H2
1
Protein tyrosine kinases (PTKs) and protein tyrosine
phosphatases (PTPs) are key enzymes that regulate cellu-
lar signal transduction by adding or removing a phosphate
to/from a tyrosine residue of a protein, respectively.1 It is
not surprising that, like their counteracting enzymes
PTKs, PTPs are involved in normal regulation as well as
disease development. Investigators have used a variety of
methods in characterizing the role of PTPs. Genetic ap-
proaches such as overexpression and knockout have
helped to assess the role of several PTPs in cellular signal-
ing, but may cause compensatory responses and other ar-
tifacts. DNA microarrays can provide information on
gene expression at the transcription level but not necessar-
ily at the protein expression or protein activity level, con-
sidering that posttranslational modifications such as
phosphorylation can vary protein activities. While the
standard (phospho)proteomic techniques do address pro-
tein expression and protein phosphorylation levels, they
are often masked by abundant (phospho)proteins and not
optimized for analysis of PTP activities. In terms of direct
partnerships between PTPs and phosphoproteins, the sub-
strate-trapping mutants of PTPs have achieved some suc-
cess in identifying physiologically relevant substrates.2
While PTPs can be promising biomarkers or therapeutic
targets, no methods are previously available to allow di-
rect identification of physiologically relevant PTPs for
phosphoprotein substrates. In this regard, recent progress
in the emerging techniques of activity-based protein pro-
filing (ABPP) is encouraging.3 Such techniques utilize
mechanism-based inactivators, or activity-based probes,
to label enzymes including phosphatases, proteases, and
demethylases. Using a new variant of ABPP, our group
has recently achieved activity-based selective labeling of
an individual PTP by integrating a nonspecific phos-
Figure 1 Fmoc-Protected 3-difluoromethyl analogue of phosphoty-
rosine 1
Potential routes to the synthesis of the 3-difluoromethyl
analogue of phosphotyrosine 1 include convergent routes
and divergent routes. Potential convergent routes involve
initial synthesis of an o-(difluoromethyl)phenyl phos-
phate and its subsequent integration into amino acid
framework via carbon–carbon coupling, whereas poten-
tial divergent routes involve only synthetic modifications
of tyrosine. On one hand, a potential convergent route
may start with 2-hydroxy-5-methylbenzaldehyde (2a) or
2-hydroxy-5-iodobenzaldehyde (3a), which can undergo
successive functional group transformations; phosphory-
lation (hydroxy) and fluorination (aldehyde), and mono-
bromination (methyl) for 2a only (Scheme 1). The
resulting benzyl bromide 2b/aryl iodide 3b can then be
converted into an amino acid using established carbon–
carbon coupling strategies, such as alkylation with carb-
anionic amino acid synthons or Negishi or Suzuki cou-
pling with amino acid synthons.6 However, the instability
of these nucleophile-sensitive phosphotriester intermedi-
ates under acidic as well as basic conditions could be trou-
blesome in the subsequent multistep processes.7 On the
other hand, a divergent route (Scheme 2) starting with ty-
rosine may be more advantageous since the potentially
poor yield and expensive fluorination step is placed later
in the reaction sequence and the need for the amino acid
OH
OPO3Et2
CHF2
CHO
1
Y
X
2b Y = CH2Br, when X = Me
3b Y = I, when X = I
2a X = Me
3a X = I
SYNTHESIS 2009, No. 22, pp 3765–3768
Advanced online publication: 23.09.2009
x
x.
x
x
.
2
0
0
9
DOI: 10.1055/s-0029-1217025; Art ID: M01909SS
© Georg Thieme Verlag Stuttgart · New York
Scheme 1 Potential convergent routes to the synthesis of 1

3766
K. Shen et al.
PAPER
Cbz NH
MeO2C
NH2
Cbz NH
MeO2C
O
iii
iv
i, ii
HO2C
OH
OH
O
OH
4
6
5
Cbz NH
MeO2C
Cbz NH
CHF2
OPO3Et2
Fmoc NH
HO2C
CHF2
v
vi, vii, viii
MeO2C
OPO3Et2
OPO3H2
8
7
1
Scheme 2 A simple, divergent route to the synthesis of 1 from tyrosine. Reagents and conditions: (i) 2,2-dimethoxypropane, 36% HCl, r.t.
overnight; (ii) Cbz-NHS, DMF, r.t., 3 h, 78% (from 4); (iii) anhyd MgCl2, anhyd Et3N, paraformaldehyde, anhyd MeCN, reflux, 15 h, 43%;
(iv) anhyd Et3N, ClPO3Et2, anhyd CH2Cl2, ice-water bath, 1 h then r.t. overnight, 85%; (v) DAST, anhyd CH2Cl2, 0 °C, 1 h then r.t., 18 h, 64%;
(vi) TMSBr, anhyd CH2Cl2, 0 °C, 1 h then r.t. 15 h; (vii) Fmoc-NHS, 1,4-dioxane–aq NaHCO3, r.t., 3 h; (viii) LiOH, CaCl2, THF–H2O, r.t., 8
h, 72% (from 8).
backbone-forming cross-coupling step is obviated. Once To be useful for peptide synthesis, the protective groups
the difluoromethyl moiety is introduced into the structure, in the compound 8 were then removed and its amino
the diethyl phenyl phosphate is deprotected so that the in- group was Fmoc-protected. Bromotrimethylsilane treat-
stability of the phosphotriester is no longer a concern. We ment, which removed Cbz and ethyl protection, followed
report herein the details of the synthesis of 1 by a simple, by Fmoc-NHS treatment and subsequent methyl removal
divergent route from tyrosine.
by lithium hydroxide, provided the peptide-synthesis-
ready, Fmoc-protected 3-difluoromethyl analogue of
phosphotyrosine 1. The identity of 1 was confirmed by
1H, 13C, 19F, and 31P NMR as well as by ESI-MS.
We started the divergent synthesis with orthoformylation
of the tyrosine phenol moiety, followed by routine func-
tional group transformations5 (Scheme 2).
While lithium hydroxide has been used for carboxylate es-
ter hydrolysis in the presence of N-Fmoc and/or phos-
ph(on)ate ester protective groups,4,10 it could remove N-
Fmoc by b-elimination, even at low temperatures and for
a reduced experiment time, leading to a reduced total
yield. In our current procedure, we used calcium chloride
as an additive in methyl group removal to avoid such a
side reaction. According to Pascal and Sola,11 the effec-
tive hydroxide ion concentration (and the undesired com-
peting b-elimination reaction) could thus be suppressed
through solubility equilibrium of calcium hydroxide. Al-
though our modified procedure was not optimized, we in-
deed observed an increased yield (72%) upon using
calcium chloride additive at room temperature for a pro-
longed experiment time (8 h), as compared to using lithi-
um hydroxide only (60% at 0 °C for 1.5 h).4 Although in
our particular case the phosphate was not protected, lithi-
um hydroxide treatment has been known to tolerate phos-
ph(on)ate esters.10 This suggests that the lithium
hydroxide/calcium chloride combination could provide a
useful orthogonality in the protective group chemistry of
amino acids, phosph(on)ates and carboxylates. Moreover,
unlike phosphotriesters, the phenyl phosphate 1 was sta-
ble enough to use in peptide synthesis and HPLC.4
Skattebøl and co-workers recently reported orthoformyla-
tion of phenols using paraformaldehyde and magnesium
chloride where a series of other functional groups includ-
ing esters were tolerated.8 We therefore speculated that a
protected tyrosine like methyl N-Cbz-tyrosinate would be
a good substrate for such orthoformylation reaction,
which introduced 3-formyl group onto the tyrosine phenol
moiety. First of all, tyrosine 4 was methylated at the carb-
oxy group using a literature procedure9 and subsequently
protected at the amino group by Cbz-NHS to afford the
protected tyrosine 5, which showed NMR and MS data
identical to the commercially available form from
Bachem. Orthoformylation of 5 was carried out under re-
flux using anhydrous magnesium chloride, anhydrous tri-
ethylamine, and paraformaldehyde (dried over
phosphorus pentoxide) in anhydrous acetonitrile. The
presence of the aldehyde functional group was confirmed
by 1H and 13C NMR and ESI-MS. While the conditions for
the orthoformylation of 5 were not optimized, we found
that longer reaction times (15–20 h) were needed for the
protected tyrosine than for simple phenols (4 h).8
Orthoformylated tyrosine 6 was then phosphorylated us-
ing diethyl chlorophosphate to afford 7, which was con-
1
firmed by H, 13C, and 31P NMR and ESI-MS. Since
In summary, we have described in detail the synthesis of
a phosphotyrosine analogue that incorporates a phos-
phatase activity-dependent quinone methide generating
motif. This novel amino acid has been used in peptide
synthesis to generate a PTP activity-based probe that
showed the first specific labeling of an individual PTP
(PTP1B) in the presence of other PTPs.4 It is expected that
such amino acids, when incorporated into appropriate
peptides, or proteins (in combination with expressed pro-
compound 7 was sensitive to acidic conditions, its column
purification was performed with triethylamine as a co-sol-
vent.
The formyl group of 7 was subsequently converted into
the difluoromethyl group using diethylaminosulfur tri-
fluoride (DAST) to afford 8, whose column purification
also used triethylamine as a co-solvent. The presence of
difluoromethyl and phosphate groups in 8 was confirmed
by NMR and MS data including 19F and 31P NMR.
Synthesis 2009, No. 22, 3765–3768 © Thieme Stuttgart · New York

PAPER
3-Difluoromethyl Analogue of Phosphotyrosine
3767
tein ligation),12 will be useful in probing specific phos-
phoprotein-PTP interactions.
ice-water bath was added Et3N (0.60 mL, 4.3 mmol) and ClPO3Et2
(0.48 mL, 3.3 mmol). The mixture was kept in the ice-water bath for
1 h and then kept at r.t. overnight. The mixture was washed with
H2O and then brine and dried (anhyd Na2SO4). The mixture was
concentrated to an oily liquid and then purified by flash column
chromatography (20% EtOAc–CH2Cl2 containing 0.1% Et3N). The
desired orthoformylated phosphotyrosine 7 was obtained as color-
less oil (1.21 g, 2.5 mmol, 85%).
1H NMR (200 MHz, CDCl3): d = 10.37 (s, 1 H), 7.64 (s, 1 H), 7.43–
7.31 (m, 7 H), 5.28 (d, J = 7.7 Hz, 1 H), 5.11 (s, 2 H), 4.66 (m, 1 H),
4.33–4.18 (m, 4 H), 3.75 (s, 3 H), 3.21 (dd, J1 = 14.1 Hz, J2 = 5.6
Hz, 1 H), 3.09 (dd, J1 = 14.1 Hz, J2 = 6.1 Hz, 1 H), 1.37 (m, 6 H).
Chemicals were obtained from commercial suppliers and used with-
out further purification. MgCl2, anhydrous beads (10 mesh,
99.99%) was purchased from the Aldrich Chemical Company, Inc.
Dry solvents were used for reactions wherever appropriate. NMR
spectra were recorded using Bruker Avance DPX 200 or 500 spec-
trometers. Mass spectra (ESI) were recorded on a Thermo LCQ Ad-
vantage LC-MS spectrometer by direct injection. Column
chromatography was performed using EM silica gel 60 (230–400
mesh).
13C NMR (50 MHz, CDCl3): d = 188.3, 171.4, 155.5, 152.0 (d),
136.5, 136.1, 133.4, 129.3, 128.6, 128.3, 128.1, 127.2 (d), 121.3,
67.1, 65.2 (d), 54.7, 52.6, 37.4, 16.1 (d).
31P NMR (81 MHz, CDCl3): d = –6.4.
Methyl (S)-2-(Benzyloxycarbonylamino)-3-(4-hydroxyphe-
nyl)propionate (5)
To L-tyrosine (4, 3.30 g, 18 mmol) suspended in 2,2-dimethoxypro-
pane (50 mL), 36% HCl (5 mL) was added. The resulting mixture
was stirred overnight at r.t. and then concentrated to dryness to af-
MS (ESI): m/z [M] calcd for C23H28NO9P: 493.1; found: 494.1
[M + H]+.
1
ford crude methyl tyrosinate, which was confirmed by H NMR
showing the formation of methyl ester. The mixture was then dis-
solved in a soln of DMF (20 mL) containing i-Pr2NEt (8.0 mL, 46
mmol). This soln was chilled in an ice-water bath, mixed with CBz-
NHS (4.78 g, 19 mmol) in DMF (10 mL) and stirred at r.t. for 3 h
before concentration by rotary evaporation. The residue was dis-
solved in EtOAc and H2O, acidified to pH 3 and extracted with
EtOAc. The extract was washed with brine, dried (anhyd Na2SO4),
and rotary evaporated to dryness. Flash column purification afford-
ed a white solid; yield: 4.65 g (14 mmol, 78%).
1H NMR (500 MHz, CDCl3): d = 7.40–7.34 (m, 5 H), 6.96 (d,
J = 7.8 Hz, 2 H), 6.73 (d, J = 7.8 Hz, 2 H), 5.28 (s, 1 H), 5.14 (d,
J = 12.4 Hz, 1 H), 5.10 (d, J = 12.4 Hz, 1 H), 4.65 (m, 1 H), 3.70 (s,
3 H), 3.09 (dd, J1 = 13.8 Hz, J2 = 5.5 Hz, 1 H), 3.02 (dd, J1 = 13.8
Hz, J2 = 5.7 Hz, 1 H).
Methyl (S)-2-(Benzyloxycarbonylamino)-3-[4-(diethoxyphos-
phoryloxy)-3-(difluoromethyl)phenyl]propionate (8)
To 7 (540 mg, 1.1 mmol) in anhyd CH2Cl2 (2 mL) at 0 °C, DAST
(1.5 mL, 11 mmol) was added. The mixture was kept at 0 °C for 1
h and allowed to rise to r.t. After 18 h, the mixture was diluted with
CH2Cl2 and added to sat. NaHCO3 soln (10 mL) at 0 °C. The aque-
ous phase was then extracted with CH2Cl2 (2 × 10 mL). The com-
bined organic phases were washed with brine, dried (anhyd
Na2SO4), concentrated to an oily liquid, and purified by flash col-
umn chromatography (10% EtOAc–CH2Cl2 containing 0.1% Et3N).
The difluorinated phosphotyrosine 8 was obtained as a colorless oil
(360 mg, 0.70 mmol, 64%).
1H NMR (500 MHz, CDCl3): d = 7.37–7.25 (m, 7 H), 7.20 (d,
J = 8.1 Hz, 1 H), 6.90 (t, JF = 55.1 Hz, 1 H), 5.61 (d, J = 7.8 Hz, 1
H), 5.08 (d, J = 12.5 Hz, 1 H), 5.05 (d, J = 12.5 Hz, 1 H), 4.61 (m,
1 H), 4.24–4.16 (m, 4 H), 3.69 (s, 3 H), 3.15 (dd, J1 = 13.8 Hz,
J2 = 5.3 Hz, 1 H), 3.06 (dd, J1 = 13.8 Hz, J2 = 6.4 Hz, 1 H), 1.32 (m,
6 H).
13C NMR (125 MHz, CDCl3): d = 172.2, 162.8, 155.7, 154.9, 136.2,
130.4, 128.6, 128.2, 128.1, 127.5, 115.5, 67.1, 55.0, 52.4, 37.5.
MS (ESI): m/z [M] calcd for C18H19NO5: 329.1; found: 330.1 [M +
H]+.
13C NMR (125 MHz, CDCl3): d = 171.8, 155.9, 147.4, 136.4, 133.9,
132.8, 128.3, 127.8, 127.7, 127.4, 125.2 (dt), 120.1, 111.2 (t,
JF = 237 Hz), 66.5, 64.9 (d), 55.0, 52.0, 36.8, 15.7 (d).
19F NMR (81 MHz, CDCl3): d = –115.3 (d, JH = 54.6 Hz).
31P NMR (188 MHz, CDCl3): d = –6.5.
Methyl (S)-2-(Benzyloxycarbonylamino)-3-(3-formyl-4-hy-
droxyphenyl)propionate (6)
To a soln of 5 (4.18 g, 13 mmol) in anhyd MeCN (100 mL) was add-
ed anhyd MgCl2 (1.5 g, 16 mmol) and anhyd Et3N (5 mL, 36 mmol)
followed by paraformaldehyde (3.52 g, 120 mmol). The mixture
was refluxed under argon for 15 h and allowed to cool down to r.t.
After acidification to pH 5 using 1 M HCl, the mixture was extract-
ed with Et2O (3 × 20 mL). The combined ether extracts were
washed with brine, dried (anhyd Na2SO4), and rotary evaporated to
a sticky liquid. The desired orthoformylated tyrosine 6 was obtained
as a white solid (1.97 g, 5.5 mmol, 43%) after purification by flash
column chromatography (2% EtOAc–CH2Cl2).
1H NMR (200 MHz, CDCl3): d = 10.87 (s, 1 H), 9.66 (s, 1 H), 7.27–
7.21 (m, 7 H), 6.82 (d, J = 9.1 Hz, 1 H), 5.83 (d, J = 8.2 Hz, 1 H),
5.08 (d, J = 12.3 Hz, 1 H), 4.99 (d, J = 12.3 Hz, 1 H), 4.62 (m, 1 H),
3.67 (s, 3 H), 3.12 (dd, J1 = 14.0 Hz, J2 = 6.9 Hz, 1 H), 2.99 (dd,
J1 = 14.0 Hz, J2 = 5.4 Hz, 1 H).
MS (ESI): m/z [M] calcd for C23H28F2NO8P: 515.1; found: 516.1
[M + H]+.
(S)-3-[3-(Difluoromethyl)-4-(dihydroxyphosphoryloxy)phe-
nyl]-2-[(9-fluorenylmethoxycarbonyl)amino]propionic Acid (1)
To 8 (244 mg, 0.47 mmol) in anhyd CH2Cl2 (5 mL) at 0 °C, TMSBr
(2 mL, 15 mmol) was added. The mixture was kept at 0 °C for 1 h
and allowed to rise to r.t. After 15 h, the mixture was rotary evapo-
rated to dryness and then dissolved in MeOH (2 mL) and rotary
evaporated again to dryness. After trituration with Et2O, removal of
1
the ethyl and Cbz protective groups was confirmed by H NMR
(D2O). The crude product was then dissolved in a soln of NaHCO3
(200 mg, 2.4 mmol) in H2O (5 mL). This soln was chilled in an ice-
water bath, mixed with Fmoc-NHS (170 mg, 0.05 mmol) in 1,4-di-
oxane (5 mL) and stirred at r.t. for 3 h. It was then diluted with sat.
NaHCO3 soln (10 mL) and washed with Et2O. The aqueous phase
was acidified to pH 3 and extracted with EtOAc. The combined ex-
tracts were washed with brine and dried (anhyd Na2SO4) and rotary
evaporated to dryness. The resulting mixture was dissolved in THF
(5 mL) and treated with LiOH (30 mg, 1.3 mmol) in aq 0.8 M CaCl2
(5 mL) at r.t. for 8 h to remove the methyl protective group, and then
acidified to pH 3 and extracted with EtOAc. The extract was rotary
13C NMR (50 MHz, CDCl3): d = 196.3, 171.7, 160.4, 155.7, 137.8,
136.3, 134.0, 128.4, 128.1, 127.9, 127.6, 120.5, 117.7, 66.8, 54.9,
52.3, 37.0.
MS (ESI): m/z [M] calcd for C19H19NO6: 357.1; found: 358.2 [M +
H]+.
Methyl (S)-2-(Benzyloxycarbonylamino)-3-[4-(diethoxyphos-
phoryloxy)-3-formylphenyl]propionate (7)
To a soln of 6 (1.03 g, 2.9 mmol) in anhyd CH2Cl2 (20 mL) in an
Synthesis 2009, No. 22, 3765–3768 © Thieme Stuttgart · New York

3768
K. Shen et al.
PAPER
evaporated to dryness and purified by RP-HPLC (100% MeCN–
H2O containing 0.05% TFA0) to afford the desired product 1 (184
mg, 0.34 mmol, 72%).
1H NMR (200 MHz, acetone-d6): d = 10.45 (s, 3 H), 7.84–6.81 (m,
12 H), 4.56 (m, 1 H), 4.31–4.15 (m, 3 H), 3.32 (dd, J1 = 13.9 Hz,
J2 = 4.7 Hz, 1 H), 3.11 (dd, J1 = 13.9 Hz, J2 = 9.3 Hz, 1 H).
13C NMR (50 MHz, acetone-d6): d = 172.1, 156.0, 147.8 (m), 144.0,
141.1, 134.6, 132.9, 127.6, 127.1, 127.0, 125.5 (dt), 125.1, 120.4,
119.8, 111.5 (t, JF = 236 Hz), 66.5, 55.1, 47.1, 36.6.
19F NMR (81 MHz, acetone-d6): d = –115.5 (d, JH = 54.6 Hz).
31P NMR (188 MHz, acetone-d6): d = –5.2.
Velculescu, V. E.; Wang, Z. J. Proc. Natl. Acad. Sci. U.S.A.
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MS (ESI): m/z [M] calcd for C25H22F2NO8P: 533.1; found: 532.1
[M – H]–; 1065.1 [2 M – H]–; 1597.7 [3 M – H]–.
Acknowledgment
We thank Dr. Narayan Hosmane for helpful discussions, Dr. Heike
Hofstetter for help with NMR experiments and Northern Illinois
University (Department of Chemistry and Biochemistry and Center
for Biochemical and Biophysical Studies) for financial support.
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Synthesis 2009, No. 22, 3765–3768 © Thieme Stuttgart · New York