A nonhydrolyzable analogue of phosphotyrosine, and related aryloxymethano- and aryloxyethano-phosphonic acids as motifs for inhibition of phosphatases

Article abstract of DOI:10.1016/j.bmcl.2004.09.008

Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl and aryloxy (or thio) ethyl phosphonic acids of the general formula RX-(CH₂)_n-PO ₃H₂ (where X = O or S and n = 1 or 2), have been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases (two alkaline phosphatases, a protein-tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs. The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties toward the protein-tyrosine phosphatase from Yersinia. The K_i for the d isomer is 33-fold lower than that of the l isomer, and is more than an order of magnitude lower than the reported K_m of the substrate l-phosphotyrosine.

Full text of DOI:10.1016/j.bmcl.2004.09.008

Bioorganic & Medicinal Chemistry Letters 14 (2004) 5931–5935
A nonhydrolyzable analogue of phosphotyrosine, and related
aryloxymethano- and aryloxyethano-phosphonic acids as motifs
for inhibition of phosphatases
Subashree Iyer, Jarod M. Younker, Przemyslaw G. Czyryca* and Alvan C. Hengge*
Department of Chemistry and Biochemistry, Utah State University, Logan, UT 84322-0300, USA
Received 19 June 2004; revised 2 September 2004; accepted 3 September 2004
Available online 5 October 2004
Abstract—Nonhydrolyzable analogues of both stereoisomers of phosphotyrosine, and a series of related aryloxy (or thio) methyl
and aryloxy (or thio) ethyl phosphonic acids of the general formula RX–(CH2)n–PO3H2 (where X = O or S and n = 1 or 2), have
been tested as nonhydrolyzable mimetics of phosphatase substrates. These compounds were tested against a panel of phosphatases
(two alkaline phosphatases, a protein–tyrosine phosphatase, and two serine/threonine phosphatases) with different active site motifs.
The compounds exhibit competitive inhibition toward all enzymes tested, with the best inhibition expressed toward the Ser/Thr
phosphatases. The stereoisomers of the phosphotyrosine analogues exhibited an unexpected difference in their inhibitory properties
toward the protein–tyrosine phosphatase from Yersinia. The Ki for the D isomer is 33-fold lower than that of the L isomer, and is
more than an order of magnitude lower than the reported Km of the substrate L-phosphotyrosine.
Ó 2004 Elsevier Ltd. All rights reserved.
The phosphorylation of certain amino acid residues,à
controlled by protein phosphatases and kinases, regu-
lates a number of processes in livingorganisms, includ-
ingmetabolic pathways, membrane transport, egne
transcription, and motor mechanisms. The therapeutic
potential of phosphatase inhibition creates an interest
in the design of potent and selective inhibitors, in mech-
anistic studies, and structural investigations. We have
sought nonhydrolyzable substrate analogues for use in
structural studies with phosphatases, particularly
metallophosphatases.
pH as their salts, called phosphonates) continue to be
the focus of recent active academic research2–4 and pat-
ents5–8 involvingthe potential use of such compounds in
several diseases, includingcancer and HIV.
However, structural studies show that phosphonates
bind in unusual manners. Structures have been reported
for EcAP with bound mercaptomethylphosphonic acid
(Ki = 600lM) and with bound phosphonoacetic acid
(Ki = 5.5mM).9 In the former case, the inhibitor binds
with the sulfur atom coordinated to zinc, and the phos-
phonate group is directed away from the active site.
Phosphonoacetic acid binds with the phosphonate moi-
ety in the active site, but exhibits an unconventional
hydrogen bond between an arginine nitrogen atom and
a hydrogen of the methylene group.9
Simple phosphonic acids are nonhydrolyzable analogues
of phosphate esters in which the P–O ester bond is
replaced with a nonhydrolyzable P–C bond (RO–
PO3H2 vs R–PO3H2). Such compounds are generally
moderate inhibitors of phosphatases; for example, Ki
values of phosphonates toward E. coli alkaline phospha-
tase (EcAP) are in the high micromolar to the millimolar
range.1 Phosphonic acids (which exist at physiological
a-Fluorophosphonates have been widely examined as
inhibitors of phosphatases, and structural elaborations
of such compounds are potent inhibitors of a number
of phosphatases, particularly members of the protein–
tyrosine phosphatase (PTP) family.10–13 However, as
with other phosphonate inhibitors, X-ray structural
analyses have shown that such compounds bind in a
fashion different from that of a substrate. A good exam-
ple has been documented with PTP 1-B. The X-ray
structure of the substrate phosphotyrosine, bound to a
Keywords: Phosphatase; Inhibition; Substrate analogue.
*
Correspondingauthors. Tel.: +1 435 797 3442; fax: +1 435 797
3390; e-mail addresses: hengge@cc.usu.edu; pgc@sun.chem.usu.edu
These authors contributed equally to this work.
à For a recent review of the research on phosphatases and kinases see
Chemical Reviews Vol. 101, No. 8.
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.09.008

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S. Iyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5931–5935
catalytically disabled form of PTP 1-B in which the
nucleophilic Cys was mutated to Ser, revealed a normal
bindingmode. 14 In contrast, structures of the native en-
zyme complexed with a-fluoro phosphonates show that
the fluorine atoms result in altered conformations of
the active site residues that bind the phosphoryl group
(Asp 181 and Phe 182).12,13 As a result, the catalytic gen-
eral acid is in a position that would be nonfunctional in
a catalytic complex.
O
PO3H2
1
2
O
PO3H2
PO3H2
O
3
4
HO2C
These unusual binding modes suggest that the presence
of the ester (P–OR) oxygen atom in the substrate is
important for proper bindingin the catalytic complex.
The compounds studied here were designed as substrate
mimics, incorporatingthe presence of an electronegative
heteroatom (O or S) in a position approximatingthat of
the ester oxygen atom, that might afford sufficient inhi-
bition to be used for future structural analysis studies.
On this basis, we explored compounds of the general
structure RX–(CH2)n–PO23ꢀ (where X = O or S and
n = 1 or 2). We also synthesized and tested the D and
L isomers of the nonhydrolyzable analogues of phospho-
tyrosine, where R = D or L tyrosine and n = 1. Figure 1
shows the compounds that were synthesized and tested.
O
PO3H2
HO2C
CO2H
5
6
O
PO3H2
O
S
PO3H2
PO3H2
1S
2S
Another advantage of aryloxymethyl and aryloxyethyl
phosphonic acids over simple phosphonic acids is the
reduction in pKa values of the phosphonyl moiety. Sim-
ple aliphatic phosphonic acids exhibit second pKa values
about one-half unit higher (ꢁ7.0) than those of phos-
phate esters (ꢁ6.5). For those phosphatases that hydrol-
yze phosphate esters in their dianion form, which
includes all of those in this study, this change in pKa will
result in a greater proportion of the inhibitor present in
the dianion form at physiological pH. The second pKa
values of alkyl–O–CH2–PO3H2 or alkyl–S–CH2–
PO3H2 alkyl–S are lowered relative to those of alkyl
phosphonic acids, to values close to those of phosphate
esters.15 This was confirmed by measurement of the sec-
ond pKa from a plot of 31P NMR chemical shift versus
pH for one of our compounds, entry 5 in Figure 1,
which bears an aryl–O moiety. The second phosphonic
acid pKa of 5 was determined to be 6.52 0.01.
S
PO3H2
PO3H2
COOH
H2N CH
L: LPT
D: DPT
O
Figure 1. Inhibitors synthesized for this work.
PO3H2 start from appropriate phenolates or thiolates,
which are coupled to 1,2-dibromoethane in a phase-
transfer catalysis version of the Williamson synthesis.
The alkyl bromides are then reacted with triethyl phos-
phite in a Michaelis–Arbuzov reaction to form phospho-
noesters, which are then deprotected with HCl. The
RO–CH2–PO3H2 compounds are synthesized usingthe
reagent diethyl 4-chlorophenylsulfonyloxymethylphos-
phonate developed by Conforth and Wilson.16 Figure 3
The general synthetic routes leading to the phosphonic
acids are shown in Figure 2. Syntheses of RX–(CH2)2–
Figure 2. General synthetic routes to aryloxyethyl (top) and aryloxymethyl (bottom) phosphonic acids.

S. Iyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5931–5935
5933
shows the synthetic schemes used to prepare the sulfur
analogues 1S and 2S. The phosphotyrosine analogues
were prepared by reactingthe protected tyrosine with
diethyl 4-chlorophenylsulfonyloxymethylphosphonate in
the presence of potassium t-butoxide in DMSO, followed
by deprotection with HCl. All compounds were fully
characterized by proton and phosphorus NMR, and had
elemental analyses within 0.4% of the calculated values.
initial rates were measured spectrophotometrically as
changes of absorbance at 400nm, associated with the
formation of p-nitrophenolate anion. Lineweaver–Burk
plots were used to assess the type of inhibition. Inhibi-
tion constant values were calculated by nonlinear least
squares fit to the competitive inhibition Michaelis–
Menten equation (Eq. 1).
17
v
½SŠ
ꢀ
ꢁ
¼
:
ð1Þ
The compounds in Figure 1 were tested as inhibitors
with human placental alkaline phosphatase (PLAP), E.
coli alkaline phosphatase (EcAP), protein–tyrosine
phosphatase from Yersinia (YOP), and serine/threonine
protein phosphatases 2C (PP2C) and k (kPP). These
phosphatases represent three different classes of active
sites and catalytic mechanisms. The alkaline phospha-
tases have two zinc ions at their active sites, and catalyze
phosphoryl transfer by a two-step mechanism with a
phospho-serine intermediate.19,20 The Yersinia enzyme
has the active site typical of PTPases, which lack a metal
cofactor, and accomplish phosphoryl transfer by a two-
step mechanism via a phospho-cysteine intermediate.21
The serine/threonine protein phosphatases utilize a
dinuclear metal center, and catalyze direct phosphoryl
transfer to a metal-bound water molecule.22
½IŠ
V max
Km 1 þ K þ ½SŠ
i
Conditions used for kinetic measurements were as
follows:
For PLAP and EcAP: TRIS (100mM), ZnCl2 (1mM),
MgCl2 (1mM), pH = 9.0. For YOP: succinate
(100mM), NaCl (150mM), pH = 6.6. For PP2C: TRIS
(100mM), MnCl2 (5mM), DTT (1mM), pH = 7.5. In
case of compounds that precipitated at [Mn2+] = 5mM,
the kinetics were carried out at [Mn2+] = 1mM. For
kPP: MOPS (100mM), MnCl2 (1mM), DTT (1mM),
pH = 7.3. The results are shown in Table 1. All inhibi-
tors in this study were found to exhibit competitive
inhibition.
After this project was underway, we became aware of a
previous, but more limited, study of aryloxymethyl
Inhibition experiments were conducted using p-nitro-
phenyl phosphate as the substrate at 25°C. Reaction
b, c
a
O
O
S
P
S
S
Cl
S
P
HO OH
EtO OEt
O
O
b, c
OH
OH
OEt
OEt
a
P
P
Br
S
S
Figure 3. Synthetic scheme used to prepare compounds 1S and 2S. Chloromethyl phenyl sulfide was obtained commercially; 1-bromo-2-phenyl
thioethane was prepared by a literature procedure.18 Reagents and conditions: (a) P(OEt)3, 125°C; (b) TMSI, CH2Cl2, 0°C to rt; (c) MeOH.
Table 1. Inhibition constants (mM) for the phosphotyrosine analogues LPT and DPT, compounds 1–6, and the thio analogues 1S and 2S, with the
phosphatases examined in this study
Alkaline phosphatases
PTPase
Ser/Thr PPases
PLAP (pH = 9.0)
EcAP (pH = 9)
YOP (pH = 6.6)
PP2C (pH = 7.5)
kPP (pH = 7.3)
1
0.9 0.1
No inhibition
0.102 0.003
5.1 0.6
5
2.17 0.22
1
0.32 0.05*
0.40 0.09*
0.13 0.01
0.23 0.02
0.067 0.005
Precipitation
0.17 0.03*
0.3 0.1*
0.27 0.03
0.05 0.01
0.19 0.03
0.12 0.06
0.037 0.002
Precipitation
0.33 0.06
0.14 0.09
0.022 0.009
0.033 0.006
2
0.118 0.004
No inhibition
0.14 0.03 (pH = 8.0)
3.4 0.5
3
12
19
1
3
4
0.6 0.1
4.2 0.8
5
2.2 0.2
6.3 0.7
4.6 0.5
6
1.1 0.1
4.0 0.8
0.04 0.04
0.9 0.2
1S
2S
LPT
DPT
0.131 0.007
0.69 0.03
1.7 0.3
10
21
2
4
5
4.8 0.9
1
0.76 0.05
0.34 0.04
1.85 0.02
0.63 0.06
ÔNo inhibitionÕ indicates that no reduction in rate was observed in the presence of 5mM of the inhibitor. For PP2C, [Mn2+] = 5mM except where
noted with asterisks, where [Mn2+] = 1mM. The Km for the substrate pNPP is the same within experimental error for PP2C whether [Mn2+] = 5mM
or 1mM (see text), thus, the phosphonic acids are not expected to show appreciably different affinities for PP2C as a function of [Mn2+].

5934
S. Iyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5931–5935
phosphonic acids (ROCH2PO3H2) as inhibitors of pro-
tein–tyrosine phosphatases.23 In that study, several com-
pounds were tested as inhibitors of PTP1B and VHR,
which share the PTPase catalytic site motif. The
reported inhibition constants were generally in the milli-
molar range,23 similar to those found in this study with
YOP. The present study differs in several significant
ways. The prior study tested only two enzymes, both
PTPases; no metallophosphatases were examined. No
amino acid analogues were prepared, while in the pre-
sent study, the synthesis and inhibition properties of
novel nonhydrolyzable analogues of both the D and L
forms of phosphotyrosine are described. In fact, an
unexpected difference between the inhibition properties
of these stereoisomers provided one of the most intrigu-
ingresults obtained in this study. The prior study had no
chiral compounds of any kind, and the results in this
study are the first report of this structural motif incorpo-
rated into an amino acid and tested as a phosphatase
inhibitor. Finally, the results presented here show that
aryloxymethano- and aryloxyethano-phosphonic acids
provide significantly better inhibition for metallophos-
phatases than for PTPases; to our knowledge, these data
are the first regarding the inhibition of metallophospha-
tases by compounds of this type. Only two of the 10
compounds in the present study were examined in the
prior one, and none of the phosphatases used in this
study are in common.
insoluble polymeric complexes with a number of diva-
lent cations, and a number of compounds in Figure 1
precipitate in the presence of 5mM Mn2+. Compounds
substituted with the carboxyl group (3, 4, 5) did not pre-
cipitate under these conditions, but the manganese con-
centration was lowered to 1mM for the inhibition
kinetics of PP2C with 1, 1S, 2, and 2S. Complexes of
the naphthyl phosphonic acid 6 precipitated even under
these conditions, precluding Ki measurements.
The carboxynaphthyl moiety provided the best discrimi-
nation for the two Ser/Thr phosphatases relative to the
other enzymes in this study, and was also the best inhibi-
tor of both PP2C and kPP. Comparisons of the Ki values
of 1 with 2, and of 3 with 4, show that the length of the
spacer (methylene or ethylene) does not make a signifi-
cant difference in inhibitory ability. The substitution of
sulfur for oxygen also results in negligible changes in
inhibition with the Ser/Thr metallophosphatases, in con-
trast to the alkaline phosphatases.
The reported Km of the substrate pNPP with PP2C is
1mM, at pH7.0, 5mM Mn2+ 24 we obtained a very sim-
;
ilar value (1.1mM) at pH7.5 under the conditions of our
inhibition studies. This value was the same, within
experimental error, when Mn2+ concentration was low-
ered to 1mM. For the lambda phosphatase, at pH7.3,
the Km of pNPP is 5mM.25 Both the aryloxymethyl
and aryloxyethyl phosphonic acids exhibit Ki values sub-
stantially below this value, with those of the best inhib-
itors (2 and 5) more than two orders of magnitude
lower.
1. Alkaline phosphatases
For both of the alkaline phosphatases examined, the
aryloxyethyl phosphonic acids 2 and 4 are notably better
inhibitors than the aryloxymethyl compounds 1, 3, 5,
and 6. The most dramatic examples are 1, which shows
no inhibition of EcAP, while for the ethyl analogue 2,
Ki = 102lM; and 3, which shows no inhibition of
PLAP, while for the ethyl analogue 4, Ki = 140lM.
The nonhydrolyzable analogues of the D and L forms of
phosphotyrosine deserve special mention. They are poor
inhibitors of the alkaline phosphatases, but better for
PP2C and kPP. For kPP, they are the best inhibitors
tested. The results with YOP are particularly interesting.
Only with this phosphatase do the two stereoisomers ex-
hibit significantly different Ki values. YOP is the only en-
zyme in this study for which the Km for the
phosphotyrosine substrate has been reported to our
knowledge: 9.5mM at pH6.6 for L-phosphotyrosine;26
Km for pNPP is 1.7mM.26 The Ki for the analogue of
L-phosphotyrosine (LPT) is 21mM (Table 2), but the
value for the D-isomer is 33-fold lower, 0.6mM. Signifi-
cantly, the latter value is an order of magnitude below
the reported Km for the L-phosphotyrosine substrate,
despite the fact that it is the D isomer. This result sug-
gests that chirality can significantly affect the inhibi-
tion properties of even simple phosphonic acids, and,
by extension, the incorporation of such nonnatural
stereoisomers into more elaborate molecules or poly-
peptides may afford a way to control phosphatase
selectivity.
The substitution of sulfur for oxygen in the ether posi-
tion has opposite effects on inhibition dependingupon
its position in the molecule. Comparison of data for 1
and 2 with 1S and 2S show that with the methylene
spacer, sulfur substitution significantly improves inhibi-
tion for both alkaline phosphatases, while with the eth-
ylene spacer, inhibition is poorer for the sulfur analogue.
Under the conditions of the inhibitions studies
(pH = 9.0), the Km for pNPP was 15lM for EcAP and
25lM for PLAP. None of the inhibitors exhibited Ki
values this low, the closest beingthe 40 lM Ki exhibited
by 1S toward EcAP.
2. Ser/Thr phosphatases
In summary, we have found that aryloxymethano- and
aryloxyethano-phosphonic acids constitute a motif that
preferentially inhibits metallophosphatases over PTP-
ases. Incorporation of the methano-phosphonic acid
moiety into D and L tyrosine results in novel nonhydrol-
yzable phosphotyrosine analogues that maintain the
oxygen atom corresponding to the scissile P–O ester
The Ser/Thr phosphatases PP2C and kPP were the most
strongly inhibited of the phosphatases examined in this
study. These phosphatases have been most often studied
kinetically in the presence of Mn2+. With PP2C a Mn2+
concentration of 5mM has been shown to be necessary
for full activity. Phosphonic acids are known to form

S. Iyer et al. / Bioorg. Med. Chem. Lett. 14 (2004) 5931–5935
5935
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Acknowledgements
This work was supported by NIH Grant GM47297 to
A.C.H. and an Undergraduate Research and Creative
Opportunities (URCO) grant from USU to J.M.Y. We
thank Professor John Denu for PP2C, and Frank Rus-
nak (deceased) for kPP. The authors gratefully acknowl-
edge the use of equipment in the Shimadzu Analytical
Sciences Laboratory at Utah State University, created
with equipment donated by Shimadzu Scientific Instru-
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