Benzyl Mono-P-Fluorophosphonate and Benzyl Penta-P-Fluorophosphate Anions Are Physiologically Stable Phosphotyrosine Mimetics and Inhibitors of Protein Tyrosine Phosphatases

Article abstract of DOI:10.1002/chem.201701204

α,α-Difluoro-benzyl phosphonates are currently the most popular class of phosphotyrosine mimetics. Structurally derived from the natural substrate phosphotyrosine, they constitute classical bioisosteres and have enabled the development of potent inhibitors of protein tyrosine phosphatases (PTP) and phosphotyrosine recognition sites such as SH2 domains. Being dianions bearing two negative charges, phosphonates, however, do not permeate membranes and thus are often inactive in cells and have not been a successful starting point toward therapeutics, yet. In this work, benzyl phosphonates were modified by replacing phosphorus-bound oxygen atoms with phosphorus-bound fluorine atoms. Surprisingly, mono-P-fluorophosphonates were fully stable under physiological conditions, thus enabling the investigation of their mode of action toward PTP. Three alternative scenarios were tested and mono-P-fluorophosphonates were identified as stable reversible PTP1B inhibitors, despite of the loss of one negative charge and the replacement of one oxygen atom as an H-bond donor by fluorine. In extending this replacement strategy, α,α-difluorobenzyl penta-P-fluorophosphates were synthesized and found to be novel phosphotyrosine mimetics with improved affinity to the phosphotyrosine binding site of PTP1B.

Full text of DOI:10.1002/chem.201701204

DOI: 10.1002/chem.201701204
Full Paper
&
Fluorophosphates
Benzyl Mono-P-Fluorophosphonate and Benzyl Penta-P-
Fluorophosphate Anions Are Physiologically Stable
Phosphotyrosine Mimetics and Inhibitors of Protein Tyrosine
Phosphatases
Stefan Wagner, Matteo Accorsi, and Jçrg Rademann*[a]
Abstract: a,a-Difluoro-benzyl phosphonates are currently
the most popular class of phosphotyrosine mimetics. Struc-
turally derived from the natural substrate phosphotyrosine,
they constitute classical bioisosteres and have enabled the
development of potent inhibitors of protein tyrosine phos-
phatases (PTP) and phosphotyrosine recognition sites such
as SH2 domains. Being dianions bearing two negative charg-
es, phosphonates, however, do not permeate membranes
and thus are often inactive in cells and have not been a suc-
cessful starting point toward therapeutics, yet. In this work,
benzyl phosphonates were modified by replacing phospho-
rus-bound oxygen atoms with phosphorus-bound fluorine
atoms. Surprisingly, mono-P-fluorophosphonates were fully
stable under physiological conditions, thus enabling the in-
vestigation of their mode of action toward PTP. Three alter-
native scenarios were tested and mono-P-fluorophospho-
nates were identified as stable reversible PTP1B inhibitors,
despite of the loss of one negative charge and the replace-
ment of one oxygen atom as an H-bond donor by fluorine.
In extending this replacement strategy, a,a-difluorobenzyl
penta-P-fluorophosphates were synthesized and found to be
novel phosphotyrosine mimetics with improved affinity to
the phosphotyrosine binding site of PTP1B.
Introduction
ed phosphotyrosine bioisosteres and have been used for the
specific deactivation and covalent labeling of phosphotyrosine
recognition domains[18] and photo-deactivation of PTP.[19] Other
chemically reactive inhibitors blocking and deactivating the
catalytic site of the enzyme in a mechanism-based reaction
have been reported.[20,21] In addition, the development of cova-
lent modifiers targeting the active sites of PTPs as “activity-
based probes” has been pursued.[22–25]
Phosphorylation and dephosphorylation of proteins at serine,
threonine, and tyrosine residues are major natural mechanisms
of activation or deactivation of biomacromolecules. Not sur-
prisingly, protein tyrosine, serine, and threonine phosphatases
have been postulated as valuable pharmacological targets.[1–3]
However, no phosphatase inhibitors have been admitted as
drugs for clinical use so far. For these reasons, novel chemical
tools for the detection and modulation of phosphatase activity
are still in high demand.[4]
Mono-P-fluorophosphonate esters have been described first
in the 1930s on the search for insecticides and some com-
pounds of this class were found to be highly toxic to humans.
Subsequently, the mono-P-fluoromethylphosphonate esters
sarin, cyclosarin, and soman were developed as chemical war-
fare agents.[26,27] Toxicity of these fluorinated phosphonate
esters has been attributed to the inhibition of synaptic acetyl-
choline esterase.[28] Related P-fluorophosphate and phospho-
nate esters have been studied and found application as unspe-
cific inhibitors and activity-based probes of serine hydrolases
including proteases, lipases, and esterases.[29–32]
Most protein tyrosine phosphatase (PTP) inhibitors compete
with the phosphate substrate for the active site of the en-
zymes and thus contain phosphate bioisosteres, which can be
developed to highly specific inhibitors by extension with frag-
ments targeting specific secondary binding sites.[5–7] Bioisos-
teres of the phosphotyrosine residue include benzyl phospho-
nates,[8] difluorobenzyl phosphonates,[9,10] sulfonates,[11,12] trifly-
lamides,[13] carboxylic acids,[14,15] and isothiazolidinones.[16,17] Re-
cently, benzoyl phosphonates were discovered as photoactivat-
Results and Discussion
[a] Dr. S. Wagner, M. Accorsi, Prof. Dr. J. Rademann
Institute of Pharmacy, Medicinal Chemistry
Freie Universitꢀt Berlin
Despite the numerous reports on bioactivities of mono-P-fluo-
rophosphonate esters, no results on the bioactivity or on the
chemical stability of P-fluorinated benzyl phosphonates have
been published so far. Therefore, starting from the well-known
phosphotyrosine mimetics 1–3, we decided to investigate the
synthesis and the bioactivity of a series of mono-P-fluoro-
Kçnigin-Luise-Str. 2+4, 14195 Berlin (Germany)
E-mail: joerg.rademann@fu-berlin.de
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under https://doi.org/10.1002/
chem.201701204.
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Scheme 1. Synthesis of benzyl mono-P-fluorophosphonate anions 4–6. Reac-
tion conditions: a) (COF)2, acetonitrile; b) 1. H2O, 2. Na-Amberlite.
Although the protonated, acidic forms of 4–6 were only par-
tially stable in aqueous solution, ion exchange to the sodium
salts yielded molecules that were significantly more stable in
this environment and could be stored as solids for at least sev-
eral months at 48C. The sodium mono-P-fluorophosphonates
were also stable under HPLC-MS conditions (0.1% formic acid
in acetonitrile/water mixtures) and are retained by C-18 re-
verse-phase significantly stronger than their phosphonic acid
derivatives (e.g., 4.0 min for 1 vs. 5.6 min for 4, see Figure S1
in the Supporting Information).
Figure 1. Benzyl-mono-P-fluorophosphonates 4–6 are phosphotyrosine mim-
etics derived from phosphonates 1–3. They were investigated for three alter-
native modes of action toward PTP1B: A) as covalent, irreversible inhibitors,
B) as substrates, or C) as reversible, non-covalent inhibitors.
In buffer solution at pH 7, no degradation of these com-
pounds could be observed, whereas strongly basic conditions
like 1% (w/v) NaOD led to immediate hydrolysis. The mono-P-
fluorophosphonate anions 4–6 were completely stable for sev-
eral hours in buffer under mildly acidic and basic conditions at
pH 5 and 9. Importantly, benzyl mono-P-fluorophosphonate 4
was entirely stable in the presence of 1 mm of dithiothreitol
(DTT), the reducing agent conventionally used for the stabiliza-
tion of protein tyrosine phosphatases in buffer and which is
known to react with strong electrophiles (see Figure S2 in the
Supporting Information)
phosphonates 4–6 (Figure 1).[8–10] We presumed these com-
pounds to bind to the active site of the model enzyme PTP1B
in a similar way as displayed in published crystal structures of
this protein in complex with phosphonate inhibitors.[33–35] Nu-
merous predicted charge and hydrogen bond interactions be-
tween compounds 4–6 and amino acid residues of the active
site suggested non-covalent and eventually covalent interac-
tions between the fragments and the target enzyme, possibly
resulting in biological activity. Aim of our experiments was to
evaluate the nature of this hypothetical bioactivity by testing
three alternative scenarios (Figure 1A)–C)). According to sce-
nario A) compounds 4–6 could act as irreversible covalent in-
hibitors of PTP1B similar to mono-P-fluorophosphonate esters.
They react with the active site of the enzyme, presumably by
the attack of the thiolate of Cys215 at the phosphorus atom
resulting in the cleavage of the PÀF bond. This scenario is sup-
ported by the experimental findings demonstrating that thio-
esters of phosphonate 1 are chemically stable entities (see Fig-
ure S3 in the Supporting Information). In scenario B) mono-P-
fluorophosphonates might act as substrates of PTP1B forming
a covalent intermediate with the active site of the enzyme,
which is subsequently hydrolyzed as part of a catalytic cycle. In
scenario C) compounds 4–6 are stable phosphotyrosine mim-
etics acting as reversible, non-covalent inhibitors.
1
All compounds were fully characterized by H, 13C, 19F, and
31P NMR spectroscopy. Characteristic signals of compound 6 in
the 19F NMR include the P-bonded fluorine at À75.6 ppm [rela-
1
tive to the standard trifluoroacetic acid (TFA)] with a JP-F cou-
pling of 1035 Hz, and the phosphorus at 2.7 ppm in the
2
31P NMR with a JP-F coupling of 114 Hz resulting in a doublet-
triplet signal (Figure 2).
Synthesis of benzyl mono-P-fluorophosphonates
For a practical synthesis of the target compounds, we estab-
lished that mono-P-fluorophosphonates were accessible direct-
ly from the phosphonic acids 1–3 using oxalyl fluoride as a
powerful and efficient reagent (Scheme 1).[36] Oxalyl fluoride
was superior to other fluorinating reagents like diethylamino-
sulfur trifluoride (DAST) and fluoride–chloride exchange reac-
tions because it delivered pure products by evaporation of the
excess of volatile reagent and byproducts. Intermediary di-P-
fluorophosphonates were then converted to the targeted
monofluorides 4–6 by the addition of water (Scheme 1).
Figure 2. 31P NMR spectra of the phosphonic acid 3 (162 MHz, [D6]DMSO,
left) and the mono-P-fluorophosphonate 6 (162 MHz, D2O, right) indicating
the additional ipso JP-F coupling and the modified geminal JP-CF2 coupling in
6.
1
2
Biochemical evaluation of benzyl mono-P-fluorophospho-
nates
Next, the biological activity of the P-monofluorides was investi-
gated. Enzymatic activity of PTP1B was measured using 4-nitro-
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phenylphosphate (pNPP) as a substrate. Progress of the enzy-
matic reaction was monitored at 405 nm by recording the ab-
sorption of the 4-nitrophenolate, the product of pNPP cleav-
age. The sodium salts of 4–6 showed no time-dependent in-
hibition at concentrations up to 20 mm, suggesting that the
mono-P-fluorophosphonates did not act as irreversible inhibi-
tors. Time-dependent inhibition of PTP1B, however, was ob-
served when the acid forms of 1–6 were investigated in the
same assay. This inhibitory effect disappeared at doubled
buffer concentration (50 mm) and could be traced back to the
deactivation of PTP1B at reduced pH values (see Figures S4–S6
in the Supporting Information). All measured fragments
showed inhibitory constants in mm-range (Table 1).
Table 1. Inhibition of PTP1B with the phosphonates 1, 2, 3, 7, 9, 11, and
the corresponding mono-P-fluorophosphonates 4, 5, 6, 8, 10, and 12. All
compounds measured as mono sodium salts.
Cmp.
KI [mm][a]
Ratio KI(PÀF)/KI(PÀOÀ)
–
1
n.i.[b]
2
3
4
ꢀ5
–
–
–
1.910Æ0.228[c]
n.i.[b]
Figure 3. Synthesized mono-P-fluoro probe 8 incubated with PTP1B to eval-
uate a covalent modification of PTP1B; Lane 1: PTP1B incubated with 8
(c=100 mm), Lane 2 and 3: PTP1B incubated with 8 (c=500 mm), Lane 4:
blank, Lane 5: STAT5B-MBP photo-crosslinked with peptide CF-K(Bio-
tin)GpcFLSLPPW-NH2 as a positive control.[18]
5
6
7
8
ꢀ10
ꢀ2
2.02
-
5.87
–
1.96
–
1.36
3.855Æ0.349
0.101Æ0.011
0.593Æ0.047
0.164Æ0.032
0.321Æ0.063
0.060Æ0.007[d]
0.081Æ0.011
9
10
11
12
As a second potential mode of action, we assumed a cova-
lent reaction with the active cysteine residue followed by the
hydrolysis of the putative instable thioester intermediate. This
should result in the formation of the starting material, the
phosphonic acid, which could be monitored via 19F NMR spec-
troscopy. For testing the substrate character we selected a 1:1
mixture of compound 6 and the corresponding phosphonic
acid 3 to identify a hydrolysis reaction via the change in its
ratio. The mixture was incubated with and without the active
protein tyrosine phosphatase PTP1B in the very same assay
buffer that is conventionally used for enzyme assays. NMR
spectra were recorded after 14 h to detect enzyme-induced hy-
drolysis (Figure 4). No enzymatic cleavage of various tested
compounds could be observed, not even with changing the
buffer to HEPES and the protein tyrosine phosphatase to SHP2.
Thus, the experiments revealed that mono-P-fluorophospho-
nate 6 acted as reversible and (bio)-chemically stable inhibitor
of PTP1B. Considering that the model compounds displayed
only weak binding interactions to the protein, we decided to
synthesize more potent inhibitors to evaluate the effect of the
substitution of a negatively charged oxygen atom by fluorine
over a broader range of binding affinities. To gain a representa-
tive view, two structures that differ by two magnitudes of affin-
ity were selected (Scheme 2).[37,38] First, the naphthyl-substitut-
ed phosphonic acid 9 was synthesized, starting from 2-(bromo-
methyl)-naphthalene, which was reacted with triethyl phos-
phite, followed by electrophilic fluorination and hydrolysis
after treatment with bromotrimethylsilane (TMSBr) (see the
Supporting Information).
[a] Calculated with Cheng–Prusoff equation; error in standard deviation.
[b] No inhibition. [c] Lit.: KI =2.5 mm Æ0.120.[40] [d] Reproducible value; re-
ported in literature with an IC50 =2.0 mm.[38]
To exclude the irreversible covalent reaction, we synthesized
the mono-P-fluoro derivative of the fluorescein labeled phos-
phonate 7. Considering that the direct fluorination of 7 would
have affected the fluorophore as well, we synthesized the
mono-P-fluorophosphonate 8 by prefluorination of the alkyne
followed by CuI-catalyzed cycloaddition with a biotin azide and
subsequent ion exchange. Potential covalent modifications of
the phosphatase PTP1B after incubation with mono-P-fluoro
phosphonates 8 were examined by gel electrophoresis looking
for fluorescent protein bands (Figure 3).
The affinity of 7 and 8 was determined with KI values of
101 mm for 7 and 593 mm for compound 8 respectively. The flu-
orescently labeled mono-P-fluorophosphonate 8 was then in-
cubated at a concentration close to its binding constant with
the protein for 1.5 h. No signs of covalent attachment of the
probe 8 were detected in SDS-PAGE analysis. As a positive con-
trol STAT5B was photo-crosslinked with the fluorescein labelled
benzoyl phosphonate peptide CF-K(Biotin)GpcFLSLPPW-NH2
containing 4-phosphono-carbonyl phenylalanine (pcF) as the
photoactive component.[18] A subsequently synthesized biotin-
labeled mono-P-fluorophosphonate showed no response in
anti-biotin Western blots. Thus, an irreversible covalent reac-
tion was excluded.
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affinities of the five mono-P-fluorophosphonates 5, 6, 8, 10,
and 12 were compared with those of the respective phos-
phonic acids 2, 3, 7, 9, and 11, affinity ratios of 2, 2.02, 5.87,
1.96, and 1.36 were determined (Table 1). Most pairs displayed
an affinity ratio close to 2, both for the simple fragments 5
and 6 and for the stronger binding compounds 10 and 12,
suggesting that in these cases, the affinity constant of the fluo-
ride is twice as high as those of the phosphonate. Only the flu-
orescently labeled probes 7 and 8 deviated from this regularity
displaying an affinity ratio of 5.87. This observation could cor-
respond to an increased binding of phosphonate 7, possibly
due to aggregation of this of this fluorescein-labeled, amphi-
philic compound.
Considering the relation between the binding affinities and
the free binding energies in a pair of compounds (ÀRT ln(KI1/
KI2)=DG1ÀDG2), the observed affinity ratio of 2 corresponds at
208C to the difference in free binding energy DDG of
1.7 kJmolÀ1 between a bound phosphonate and its respective
mono-P-fluoro-derivative. This difference in binding energy re-
flects the change in Coulomb interactions between a PÀOÀ res-
idue and a PÀF substituent and the cost in binding energy for
the substitution of a dianionic species by that of a monoanion
in this individual case. Although hydrogen bond pairing in pro-
tein–ligand interactions and its influence on binding affinities
is a widely discussed and not well-understood topic,[41–45] a loss
of free binding of only 1.7 kJmolÀ1 for replacing a PÀOÀ resi-
due by a PÀF functionality appeared to be a moderate price
and suggested the possibility of favorable interactions for the
PÀF residue with the phosphotyrosine binding pocket.
Figure 4. Evaluation of the enzymatic turn-over of mono-P-fluorophospho-
nate 6 as a substrate of PTP; 19F NMR spectra of the mixture (c=4 mm each)
of 3 and 6 without (top) and with (bottom) incubation with PTP1B.
Scheme 2. Synthesis of the mono-P-fluorophosphonates 9 and 11 as stable
phosphotyrosine mimetics.[37–38] Reaction conditions: a) (COF)2, acetonitrile;
b) 1. H2O, 2. Na-Amberlite.
Synthesis and evaluation of a,a-difluorobenzyl-penta-P-fluo-
rophosphate 13
Considering that the substitution of an OÀ functionality with a
fluorine atom resulted in stable and reversible inhibitors of the
protein tyrosine phosphatase PTP1B with only a 2-fold de-
crease in affinity, we wondered whether the incorporation of
additional fluorine substituents at the phosphorus might yield
another phosphotyrosine mimetic. During attempts of synthe-
sizing the mono-P-fluorinated phosphonate 6 via the treat-
ment of the activated di-P-chloro phosphonate of 3 with alka-
line fluorides, we observed the formation of a compound that
showed a more complex 31P–19F-coupling. These signals corre-
sponded to the heptafluorinated derivative 13, a,a-difluoro-
benzyl-penta-P-fluorophosphate, which initially could not be
isolated in a pure form and was obtained in a mixture with
other organophosphorus species. NMR spectra indicated the
coupling pattern of an octahedral coordination at the phos-
phorus atom with one axial and four equatorial fluorine atoms.
HRMS measurements confirmed the pentafluorination at the
phosphorus center. Placement of compound 13 in the active
site of a published structure of the protein PTP1B with refer-
ence inhibitor 3[35] led after energy minimization to a binding
mode of the ligand 13 with numerous interactions exceeding
those of the phosphonate 3 and of the mono-P-fluorophosph-
onate 6 (Figure 5). According to the molecular model, binding
of 13 was accomplished by H-bonds from Cys215, Ser216,
The mono-P-fluorinated phosphonate 10 was obtained after
difluorination with oxalyl fluoride followed by hydrolysis and
treatment with sodium-loaded Amberlite resin. Secondly, the
phosphonic acid 11 based on a phenylacetophenone scaffold
was synthesized (see the Supporting Information). Fluorination
and ion exchange furnished the mono-P-fluorophosphonate
12.
The synthesized phosphonates 1, 2, 3, 7, 9, and 11 and their
corresponding mono-P-fluorophosphonates 4, 5, 6, 8, 10, and
12 were investigated as inhibitors of PTP1B using the pNPP
assay. All compounds were reversible inhibitors. IC50 values
were recorded and converted to the inhibitory constants KI
using the Cheng–Prusoff equation (Table 1).[39]
A measurable inhibitory effect of the benzyl-substituted
compounds 1 and 4 was observed only at concentrations
>30 mm. However, the data did not allow the determination
of an inhibition constant for these compounds. The a-C-mono-
fluorinated bioisosteres 2 and 5 inhibited PTP1B with KI values
around 5 and 10 mm, respectively, although the determination
of KI values at such high concentrations lacks precision. KI
values of the compound 3 and 6–12 were measured with high
reproducibility and acceptable statistical deviation. When the
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Figure 5. Calculated interactions of phosphonate 3, the mono-P-fluorophosphonate 6 and PhÀCF2ÀPF5À 13 (from left to right) PTP1B.[46] Based on the crystal
structure from reference, PDB code: 4Y14 (Dimer B).[35]
Ile219, Gly220, and Arg221. Moreover, the protonated side
chain of Arg221 was involved in charge interactions, while
Tyr46, Val49, and Phe182 contributed to hydrophobic interac-
tions.
teracted strongly with the column material, resulting in elution
after 9 min at the very end of the default gradient (5 to 95%
CH3CN in H2O, see Figure S2 in the Supporting Information).
Additionally, no absorbance of 13 at the wavelength 210 nm,
254 nm and 290 nm was observed. These properties hampered
chromatographic purifications like reversed-phase MPLC and
HPLC. Crystallization of 13 from the product fractions resulted
in an overall yield of 21%. Subsequent NMR analysis clearly
showed the octahedral coordination around the phosphorus
nucleus (Figure 6).
On the basis of these favorable binding predictions, further
effort was invested into developing a protocol for the synthe-
sis and isolation of compound 13.
As mentioned above, treatment of phosphonate 3 with
oxalyl chloride followed by metal fluorides yielded mixtures of
difluorobenzyl penta-P-fluorophosphate 13 with other com-
pounds such as difluorobenzyl mono-P-fluorophosphonate 6.
This synthetic route, however, showed unsatisfying reproduci-
bility in product formation and the isolation of compound 13
could not be accomplished. A much more reliable preparation
of the PhCF2PF5-anion 13 was realized when tetramethylam-
monium fluoride (NMe4F) was used as the source of fluoride
(Scheme 3).[47]
In accordance with the elution properties on RP-HPLC, the
tetramethylammonium a,a-difluorobenzyl pentafluoro phos-
phate 13 is significantly more lipophilic compared to phospho-
nate 3, thus being more soluble in acetonitrile than in water.
The stability of pentafluorophosphate 13 was investigated
over the pH range from 2 to 12 in aqueous solution using
19F NMR spectroscopy in D2O and no degradation or hydrolysis
could be observed, also confirming the stability of 13 under
physiological buffer conditions. Initial investigations of the in-
hibitory potential towards the protein tyrosine phosphatase
PTP1B showed a significant affinity but insufficient reproduci-
bility. In order to enhance the solubility in aqueous systems
and to exclude any interfering effects of the counter ion
NMe4+, we transferred compound 13 into its sodium salt 13-
Na. While showing the same characteristics during HPLC meas-
urements, ion exchange resulted in an increased solubility of
13-Na in water up to 200 mm without any loss in stability.
Using the very same assay, the detection of the chromogenic
para-nitrophenolate at 405 nm, we determined the inhibition
constant KI of product fractions from several synthetic batches
in a final set of 187 data points as a value of 1.81 mm (Fig-
ure 7A).
Formation of a,a-difluorobenzyl-penta-P-fluorophosphate 13
was confirmed by HPLC-MS analysis displaying very uncom-
mon elution properties both on C8 and C18 reverse-phase
silica. Although the phosphonic acids 3 and 6 eluted at 3.4
and 5.2 min, respectively on RP-18 material, displaying sym-
metric peaks and characteristic UV spectra, compound 13 in-
Scheme 3. Synthesis of the NMe4-a,a-difluorobenzyl-penta-P-fluorophos-
phate 13. Reaction conditions: a) 1. (COCl)2, [DMF], DCM; 2. NMe4F, acetoni-
trile.
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Figure 6. 19F NMR (D2O) (left) and 31P NMR ([D6]DMSO) (right) spectra of the penta-P-fluorophosphate 13.
forded a heptafluorinated product 14-Na which was stable
and soluble in the biochemical buffer. The KI of 14-Na was de-
termined as 0.026 mm, more than 6 times lower than the re-
spective phosphonate 9 (0.164 mm).
These results corroborate that difluoro-methyl-pentafluoro-
phosphate fragments are indeed bioisosteres and biomimetics
of phosphate residues.
Conclusion
In this work, novel ligands of protein tyrosine phosphates were
derived from known phosphonate-based PTP inhibitors
through substitution of a negatively charged O-functionality
bound to the phosphorus center by a fluorine atom. Benzyl-
mono-P-fluorophosphonates were subsequently examined for
their mode of action toward the protein tyrosine phosphatase
PTP1B. Three different scenarios were hypothesized and
tested: first, benzyl-mono-P-fluorophosphonates might under-
go a covalent reaction with the enzyme acting A) as irreversi-
ble inhibitors of PTP1B or B) as substrates resulting in the hy-
drolysis of the covalent intermediate. Alternatively, they might
interact with the enzyme non-covalently as chemically stable
and reversible inhibitors (C).
Figure 7. Inhibition of PTP1B at various concentrations of 13-Na (A) and 14-
Na (B). In case of 13-Na a data point with 100% inhibition was added at
100 mm concentration without altering the calculated KI values.
Elucidation of the mode of action revealed that the benzyl-
mono-P-fluorophosphonates 5, 6, 8, 10, and 12 acted as rever-
sible, chemically stable inhibitors of PTP1B. No covalent modifi-
cation of the protein was observed and no hydrolysis took
place in the presence of active PTP1B. Pairwise comparison of
the binding constants of mono-P-fluorophosphonates with the
respective phosphonates revealed a reduction of the free bind-
ing energy DDG of 1.7 kJmolÀ1 by replacing a P-OÀ with a P-F
substituent. Considering that the P-OÀ contributes to the bind-
ing of phosphonates through charge interactions and H-
bonds, this moderate reduction of the free binding energy
suggests that the P-F residue contributes to the binding of the
mono-P-fluorophosphonates, presumably by establishing F–H-
bonds with the protein’s active site.
During the measurements of the inhibitory effect of 13-Na,
inhibitors with known inhibition constants were used in paral-
lel as positive controls to verify the reliability of the assay. The
inhibition constant of 1.81 mm suggests equal or even more
À
favorable protein-inhibitor interactions of the RÀPF5 moiety
than the phosphonate RÀP(O)O22À, which is present in com-
pound 3 (KI =1.910 mm).
Next, we wanted to extend these findings to other penta-P-
fluorophosphates. Pentafluorination of benzyl phosphonate 1
using a modified reaction protocol furnished a pentafluorinat-
ed product that decomposed at room temperature and thus
was not suitable for biochemical studies.[48] Pentafluorination
of the difluoro-naphth-2-yl-methylphosphonate 9, however, af-
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cooled to À208C. Oxalyl fluoride dissolved in CD3CN (1:3, 200 mL)
was added through a syringe and the reaction mixture stirred for
2 h at À208C. The solvent and the excess of oxalyl fluoride were
removed under a stream of nitrogen. Sodium loaded Amberliteꢁ
resin (Weakly Acidic Cation Exchanger, CG50) and water (1 mL) was
added and the mixture warmed to room temperature. Filtration
and lyophilization yielded the corresponding mono-P-fluoro-
phosphonate as off-white powders.
This observation prompted us to synthesize and test further
fluorine-substituted phosphorus compounds as potential new
phosphotyrosine mimetics. Difluoro-benzyl-penta-P-fluorophos-
phate 13 was prepared and found to be a stable compound in
aqueous solutions over a broad pH range. As hypothesized the
penta-P-fluorophosphate 13 was established as a novel phos-
photyrosine mimetic and identified as a reversible inhibitor of
the protein tyrosine phosphatase PTP1B with an affinity similar
to the reference compound, a,a-difluoro-benzyl phosphonate
3. In the case of the difluoro-naphth-2-yl-methyl phosphonate
9 this effect was even more pronounced. The pentafluorophos-
phate derivative 14-Na was found to be an inhibitor of PTP1B
that was more than six times more active than the respective
phosphonate 9 (0.026 mm vs. 0.164 mm).
Further research will show whether PTP inhibitors derived
from the novel phosphotyrosine mimetic 13 with their reduced
negative charge density and increased hydrophobicity will be
able to penetrate cells and are metabolically stable, possibly
leading to potent protein tyrosine phosphatase inhibitors with
activity in vivo.
Sodium
mono-P-fluoro-(fluoro-(phenyl)-methyl)-phosphonate
1
(5): m.p. >3158C; Yield: 17 mg, 73%; H NMR (400 MHz, D2O): d=
7.49 (m, 5H, Ar), 5.83 ppm (dd, J=44 Hz, J=8 Hz); 13C NMR
(126 MHz, D2O): d=133.8 (d, J=19 Hz), 129.2 (br m), 128.7 (br m),
126.7 (br m), 89.1 ppm (br m); 19F NMR (376 MHz, D2O): d=
À70.4 ppm (dd, J=1019 Hz, J=9 Hz, P-F), À196.2 (ddd, J=85 Hz,
J=44 Hz, J=9 Hz, CHF); 31P NMR (162 MHz, D2O): d=12.9 ppm
(ddd, J=1019 Hz, J=84 Hz, J=8 Hz); ESI-HRMS (m/z): [MÀH]À cal-
culated for C7H6F2O2PÀ: 191.00790 Da; found: 191.00848.
Sodium mono-P-fluoro-(difluoro-(phenyl)-methyl)-phosphonate
1
(6): m.p. >3008C; Yield: 16 mg, 73%; H NMR (400 MHz, D2O): d=
7.55 ppm (m, 5H); 13C NMR (126 MHz, D2O): d=130.9 (br m), 130.3
(br m), 128.7 (br m), 128.4 (br m), 125.7 ppm (vbr m); 19F NMR
(376 MHz, D2O): d=À75.6 (d, J=1036 Hz, P-F), À108.4 ppm (d, J=
115 Hz, CF2); 31P{1H} NMR (162 MHz, D2O): d=2.7 ppm (dt, J=
1036 Hz, J=114 Hz); ESI-HRMS (m/z): [MÀH]À calculated for
C7H5F3O2PÀ: 208.99847 Da; found: 208.99873.
Experimental Section
Sodium mono-P-fluoro-(fluoresceinyl)-phosphonate (8): For syn-
thesis see the Supporting Information. ESI-HRMS (m/z): [M+H]+
calculated for C37H32F3N5O9P+: 778.18843 Da; found: 778.19087.
Materials and methods: All reactions were performed in typical
glassware and if appropriate, under dry conditions using Schlenk
technology. All chemicals were purchased from common suppliers
and were used without any further purification, if not mentioned
otherwise. NMR spectra were measured on the following spec-
trometers (Bruker, AV 300; Varian, Mercury 300 MHz; Varian, Mercu-
ry 400 MHz, JEOL, ECX 400; JEOL, ECP 500). Chemical shifts are
given in ppm relative to the signal of the used deuterated solvent
as internal and TFA as well as 85% H3PO4 as external standards.
ESI-HRMS were recorded with an ESI-Q-TOF spectrometer (Agilent
Technologies, 6550) coupled with a HPLC (Agilent Technologies, In-
finity II 1290).
Sodium mono-P-fluoro-(difluoro-(naphth-2-yl)-methyl)-phospho-
1
nate (10): m.p. >3158C; Yield: 9 mg, 99%; H NMR (500 MHz, D2O):
d=8.21 (br s, 1H, H-1), 8.03 (m, 3H, H-4, H-5, H-8), 7.70 ppm (m,
3H, H-3, H-6, H-7); 13C NMR (126 MHz, D2O): d=133.6, 132.2, 131.4
(td, J=22.1 Hz, J=12.6 Hz), 128.5, 128.3, 127.7, 127.5 (d, J=7.4 Hz),
126.8 (d, J=7.4 Hz), 126.0 (v br), 122.8 ppm (v br), the 19F and 31P
coupled 13C signal of -CF2- was not resolved; 19F NMR (376 MHz,
D2O): d=À75.2 (d, J=1037 Hz), À107.9 ppm (d, J=115 Hz);
31P{1H} NMR (162 MHz, D2O): d=3.5 ppm (dt, J=1037 Hz, J=
114 Hz); ESI-HRMS (m/z): [MÀH]À calculated for C11H7F3O2PÀ:
259.01412 Da; found: 259.01531.
Oxalyl fluoride: Oxalyl fluoride was prepared according to litera-
ture.[36] Deviant from the existing preparation instructions, multiple
distillation of oxalyl fluoride could not be accomplished without
significant loss of the product. Considering that the published boil-
ing points were given as ambiguous values and taken into account
the hazardous potential of this compound, we dissolved the con-
densate of the first distillation in deuterated acetonitrile (1:3). Ace-
tonitrile, besides toluene, was the only suitable solvent for a pro-
longed storage up to 2 weeks.
Sodium
mono-P-fluoro-4-((3-oxo-2,3-diphenylpropyl)-difluor-
methyl)-phosphonate (12): m.p. >3158C; Yield: 12 mg, 99%;
1H NMR (400 MHz, CD3CN/ D2O): d=7.64 (d, J=7.7 Hz, 2H, Hortho,Ph-
CO), 7.29 (d, J=8.0 Hz, 2H, Hortho,Ph-CF2), 6.99 (m, 8H), 6.85 (m, 2H),
4.79 (t, J=7.4 Hz, 1H, -CH-), 3.27 (dd, J=13.7 Hz, J=7.7 Hz, 1H, À
CH2À), 2.86 ppm (dd, J=13.6 Hz, J=7.8 Hz, 1H, ÀCH2À); 13C NMR
(176 MHz, D2O): d=203.3, 141.5, 138.4, 135.9, 133.8, 129.2, 129.0,
128.9, 128.7, 128.4, 127.4, 125.8, 120.0 (m), 54.7, 38.4 ppm; 19F NMR
(376 MHz, CD3CN/D2O, 1:1): d=À74.1 (d, J=1029 Hz),
À107.01 ppm (d, J=114 Hz); 31P{1H} NMR (162 MHz, CD3CN/D2O):
d=2.7 ppm (dt, J=1028 Hz, J=115 Hz); ESI-MS (m/z): [M+H]+ cal-
culated for C22H19F3O3P+: 419.10184 Da; found: 419.10190.
Sodium benzyl-mono-P-fluorophosphonate (4): Benzyl-di-P-fluoro
phosphonate was prepared according to literature (see the Sup-
porting Information).[49] The solidified difluoride was hydrolyzed in
water followed by the addition of sodium-loaded Amberlite resin
(weakly acidic, acetate-based). The mixture was stirred for 10 min,
filtered and lyophilized to give 4 as a white solid in quantitative
Tetramethylammonium (difluoro-(phenyl)-methyl)-penta-P-fluo-
rophosphate (13): Compound 3 (460 mg, 2 mmol) and a catalytic
amount of dry DMF were dissolved in dry DCM (4 mL). Oxalyl chlo-
ride (428.8 mL, 5 mmol, 2.5 equiv.) was added dropwise and the
mixture heated to 408C and stirred for 2 hours at room tempera-
ture. The solvent and excess of (COCl)2 were removed under re-
duced pressure and the residue dissolved in dry acetonitrile.
NMe4F (763 mg, 8.2 mmol, 4.1 equiv.) was added and the reaction
was stirred 16 h at room temperature. The white precipitate was
filtrated off, the filtrate was evaporated and purified by RP-MPLC
(H2O/ ACN, 5–99% acetonitrile). Lyophilization of the product frac-
1
yield. H NMR (300 MHz, D2O): d=7.39 (m, 5H, Ar), 3.21 ppm (dd,
J=21.8 Hz, J=3.9 Hz, 2H, -CH2-); 13C NMR (75 MHz, D2O): d=133.6
(m), 129.7 (d, J=7.7 Hz), 128.9 (d, J=3.2 Hz), 126.7 (d, J=4.4 Hz),
33.4 ppm (dd, J=136 Hz, J=29.9 Hz); 19F NMR (282 MHz, D2O): d=
À63.5 ppm (dt, J=989 Hz, J=3.9 Hz); 31P{1H} NMR (162 MHz, D2O):
d=24.9 ppm (d, J=990 Hz); ESI-HRMS (m/z): [M+H]+ calculated
for C7H9FO2P+: 175.03187 Da; found: 175.03259.
General procedure for the fluorination with oxalyl fluoride: The
corresponding phosphonic acid 2, 3, 9, or 11 (10–20 mg) was dried
under high vacuum, dissolved in a minimal amount of CD3CN and
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tions afforded tetramethylammonium (difluoro-(phenyl)-methyl)-
penta-P-fluorophosphate 13 as colorless crystals with light brown
reflexes. Yield: 140 mg, 21%; m.p. 1078C; 1H NMR (400 MHz,
[D6]DMSO): d=7.55 (d, J=6.3 Hz, 1H), 7.44 (d, J=6.8 Hz, 1H), 7.32
(ddd, J=19.0 Hz, J=10.9 Hz, J=7.3 Hz, 3H), 3.09 ppm (s, 12H,
N(CH3)4); 13C NMR (101 MHz, [D6]DMSO): d=129.9, 128.3, 128.2,
127.6, 126.4, 125.7, 54.9 ppm; 19F NMR (470 MHz, CD3OD): d=
À67.7 (dp, J=700 Hz, J=46 Hz, 1F, Fax), À69.9 (ddt, J=858 Hz, J=
45 Hz, J=8.6 Hz, 4F, Feq), À97.3 ppm (ddt, J=121 Hz, J=17 Hz, J=
7.3 Hz, 2F, CF2); 31P NMR (162 MHz, [D6]DMSO): d=À144 ppm
(dtquin, J=858 Hz, J=700 Hz, J=120 Hz); ESI-HRMS (m/z): [M]À
calculated for C7H5F7PÀ: 253.0023; found: 253.0033.
Conflict of interest
The authors declare no conflict of interest.
Keywords: fluorine
chemistry
·
fluorophosphates
·
·
fluorophosphonates
· fragment-based drug discovery
phosphotyrosine mimetics · protein tyrosine phosphatases
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1
m.p. 1428C; H NMR (700 MHz, CD3OD/CD3CN (7:1 (v/v))): d=7.95
(s, 1H, Ar), 7.90 (dd, J=6.0, J=3.4 Hz, 1H, Ar), 7.87 (dd, J=6.1, J=
3.4 Hz, 1H, Ar), 7.83 (d, J=8.7 Hz, 1H, Ar), 7.62 (d, J=8.5 Hz, 1H,
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13C NMR (176 MHz, CD3OD/CD3CN): d=133.1, 132.5, 128.0, 127.2,
126.3, 125.8, 125.6, 124.0, 123.9, 116.9, 54.9 ppm; 19F NMR
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Fax), À73.1 (ddt, J=860.6, J=44.3, J=8.7 Hz, 4F, Feq), À99.7 ppm
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Version of record online: && &&, 0000
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FULL PAPER
&
Fluorophosphates
S. Wagner, M. Accorsi, J. Rademann*
&& – &&
Benzyl Mono-P-Fluorophosphonate
and Benzyl Penta-P-Fluorophosphate
Anions Are Physiologically Stable
Phosphotyrosine Mimetics and
Inhibitors of Protein Tyrosine
Phosphatases
Through decades, phosphonates like 3
have constituted classical inhibitors of
protein tyrosine phosphatases mimick-
ing the favorable interactions of phenyl
phosphate substrates while being non-
hydrolyzable. Despite their popularity,
phosphonates were not developed as
drugs, yet, mainly due to severe issues
in cell penetration. Here, modifications
of this traditional class of inhibitors are
presented by substituting P-bonded
oxygen atoms with one (6) or five fluo-
rine atoms (13).
&
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