A protected l-bromophosphonomethylphenylalanine amino acid derivative (BrPmp) for synthesis of irreversible protein tyrosine phosphatase inhibitors

Article abstract of DOI:10.1016/j.bmc.2010.09.040

Protein tyrosine phosphatases (PTPs) are important therapeutic targets for medicinal chemists and biochemists. General strategies for the development of inhibitors of these enzymes are needed. Several modular strategies which rely on phosphotyrosine mimics are known for PTP inhibitors. Previous strategies include phosphonomethylphenylalanine (Pmp) derivatives which act as competitive inhibitors. Pmp amino acid derivatives have been used to develop specific inhibitors by incorporation into sequences recognized by the PTP of interest. We report the synthesis of a new phosphonotyrosine analog, l- phosphonobromomethylphenylalanine (BrPmp), which acts as an inhibitor of PTPs. The BrPmp derivative was prepared as an Fmoc-protected amino acid which can be used in standard solid phase peptide synthesis (SPPS) methods. The synthesis of the protected amino acid derivative requires 11 steps from tyrosine with a 30% overall yield. Enzyme inhibition studies with the PTP CD45 demonstrate that BrPmp derivatives are irreversible inhibitors of the enzyme. A tripeptide which incorporated BrPmp had increased inhibitory potency against PTP relative to BrPmp alone, confirming that the incorporation of BrPmp into peptide sequences provides additional context to improve enzyme binding.

Full text of DOI:10.1016/j.bmc.2010.09.040

Bioorganic & Medicinal Chemistry 18 (2010) 8679–8686
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
A protected L-bromophosphonomethylphenylalanine amino acid derivative
(BrPmp) for synthesis of irreversible protein tyrosine phosphatase inhibitors
⇑
Naresh S. Tulsi, A. Michael Downey, Christopher W. Cairo
Alberta Ingenuity Centre for Carbohydrate Science, Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2G2
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 16 July 2010
Revised 10 September 2010
Accepted 16 September 2010
Available online 29 September 2010
Protein tyrosine phosphatases (PTPs) are important therapeutic targets for medicinal chemists and bio-
chemists. General strategies for the development of inhibitors of these enzymes are needed. Several mod-
ular strategies which rely on phosphotyrosine mimics are known for PTP inhibitors. Previous strategies
include phosphonomethylphenylalanine (Pmp) derivatives which act as competitive inhibitors. Pmp
amino acid derivatives have been used to develop specific inhibitors by incorporation into sequences rec-
ognized by the PTP of interest. We report the synthesis of a new phosphonotyrosine analog, L-phospho-
Keywords:
Phosphatases
CD45
nobromomethylphenylalanine (BrPmp), which acts as an inhibitor of PTPs. The BrPmp derivative was
prepared as an Fmoc-protected amino acid which can be used in standard solid phase peptide synthesis
(SPPS) methods. The synthesis of the protected amino acid derivative requires 11 steps from tyrosine
with a 30% overall yield. Enzyme inhibition studies with the PTP CD45 demonstrate that BrPmp deriva-
tives are irreversible inhibitors of the enzyme. A tripeptide which incorporated BrPmp had increased
inhibitory potency against PTP relative to BrPmp alone, confirming that the incorporation of BrPmp into
peptide sequences provides additional context to improve enzyme binding.
PTP
Peptide synthesis
Irreversible inhibitor
Phosphopeptide
Peptide analog
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
tyrosine (pTyr) mimics, such as phosphonomethylphenylalanine
(Pmp) which act as competitive inhibitors of the enzyme.8 Modifi-
Protein tyrosine phosphatases (PTPs) are important for the reg-
ulation of signaling pathways, acting as a biochemical counterbal-
ance to kinases.1,2 This mechanism plays diverse roles in biological
systems, including the regulation of T cell antigen recognition and
activation.3 As a result, PTPs are an important target for both
medicinal chemistry and biochemical research.4,5 However, few
general strategies have been elucidated for the determination of
PTP specificity or inhibitor design. CD45 is a well studied human
immune cell receptor-like PTP (RPTP), and the most prevalent
membrane-associated PTP in T cells.6 Misregulation of CD45 re-
sults in severe combined immunodeficiency (SCID), and the recep-
tor is implicated in autoimmune diseases such as lupus and
arthritis. Currently, the primary strategies to examine CD45 speci-
ficity rely on the use of phosphotyrosine-specific antibodies, or the
synthesis of phosphopeptide substrates.7 Methods that allow for
detection, labeling, or inhibition of active PTP enzymes could com-
plement or improve these strategies to expand our understanding
of PTP signaling.
cation of Pmp to
a phosphonodifluoromethylphenylalanine
(F2Pmp) improves the potency of these derivatives ( Fig. 1).9
A
key advantage of these compounds is their adaptability for solid
phase peptide synthesis (SPPS) when prepared with the appropri-
ate
a-amino and phosphonate protection. Reported derivatives of
Pmp include fluoro, difluoro, chloro, and dichloro derivatives.9–13
These strategies have been successfully applied to develop compet-
itive inhibitors of a variety of PTPs.14
F
F
O
O
O
O
P
P
HO
P
OH
OH
OH
HO
HO
H2N
CO2H
pTyr
H2N
CO2H
Pmp
H2N
CO2H
F2Pmp
Br
Br
O
P
O
P
The design of specific PTP inhibitors remains a challenge, and
new strategies that provide enhanced activity or reduce develop-
ment time are of continued interest.4 A classic strategy for
designing PTP inhibitors has exploited non-hydrolyzable phospho-
OH
OH
HO
HO
S
H
HN
H2N
CO2H
NH
H
O
HN
BrPmp
O
1
2
⇑
Corresponding author. Tel.: +1 780 492 0377; fax: +1 780 492 8231.
E-mail address: ccairo@ualberta.ca (C.W. Cairo).
Figure 1. Synthetic phosphotyrosine mimics.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.09.040

8680
N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
There has been sustained interest in identifying covalent inhib-
were PBr3,27 PPh3/NBS,25 and MsCl28 followed by treatment with
a bromide nucleophile (triethylammonium bromide or sodium
bromide) (see Supplementary data, Table SI1). We only observed
significant formation of product 5 after treatment with thionyl
bromide and pyridine.29 The reduced reactivity of the substrate
may be due to the sterically congested environment of the hydro-
xyl. After optimization of the thionyl bromide conditions, we were
able to achieve excellent yields for bromination of 4 (88%). With
access to 5, we then confirmed that deprotection of the
dimethoxyphosphonate proceeded in good yield with the use of
trimethylsilylbromide,30 for both the hydroxy and bromo deriva-
tives to give the model hydroxy(phenyl)dimethyl, 6, or bromo(phe-
nyl)dimethyl, 7, respectively. Based on these results, we proceeded
to develop a method for generating the desired phenylalanine
targets.
itors of PTPs. In addition to improved potency, covalent inhibitors
(sometimes referred to as suicide substrates) can be of interest for
developing enzyme labeling strategies. For example, covalent
inhibitors when attached to fluorophores or affinity tags have been
employed as activity-based protein probes (ABPP).15,16 Some of the
known covalent inhibitors for PTPs include quinone methides,17–20
aryl vinyl sulfonates,21 nitrostyrene,22 and
a-bromobenzylphosph-
onate (BBP) derivatives.23 Other notable strategies have included
fluorogenic substrates of PTP enzymes, which allow improved as-
say, detection, and imaging applications.24
Taylor et al. were the first to test the activity of
a-bro-
mobenzylphosphonate (BBP) analogs as inhibitors of PTPs,25 and
this was later used as a labeling strategy with biotin-tagged deriv-
ative 2.23 In contrast to the fluoro, difluoro, and chloro-Pmp deriv-
atives, the BBP analogs were found to form covalent adducts with
PTPs, forming the basis of proteomic strategies for PTP identifica-
tion. Compound 2 was shown to covalently label a variety of PTPs,
limiting its application as an inhibitor of specific enzymes. We con-
sidered that PTPs often recognize specific amino acid sequences,26
and that additional functional groups could potentially impart
2.2. Synthesis of Fmoc-L-BrPmp(OMe2)-OH (16)
We set out to generate a protected tyrosine derivative with a
benzylic aldehyde suitable for condensation to the phosphonate.
We first considered a common strategy for modification of phenyl-
alanine via iodotyrosine,31 which could be a good substrate for
oxidation to the corresponding aldehyde.32 We selected carboxyb-
specificity to the
a-bromophosphonate group. Additionally, we de-
sired an efficient route to an
a-bromophoshonate that could be
used for both labeling and inhibitor studies. We report here a con-
venient and efficient route to a phosphonobromomethylphenylala-
nine (BrPmp, 1), and derivatives appropriately protected for SPPS
enzyl (Cbz) as the a-amino protecting group for the first part of the
synthesis, since other common protecting groups would be incom-
patible with intermediate steps of the synthesis. The use of a fluo-
ride catalyst required for the initial phosphonylation would
prevent the use of Fmoc, and bromination conditions with thionyl
bromide would be incompatible with Boc.
(Fmoc-L-BrPmp(OMe2)-OH, 16). We found that amino acid deriva-
tives, which incorporated BrPmp, act as irreversible PTP inhibitors.
Importantly, we find that inclusion of BrPmp in a peptide sequence
significantly improved its potency, suggesting that the context of
the BBP functional group can improve inhibitor activity.
In our first attempt at the route, phenylalanine was converted
to iodotyrosine in good yield and subsequently protected as a
methyl ester via acid-catalyzed esterification and a-amino protec-
tion as the Cbz amino acid (8) as previously reported.33 We at-
tempted conversion of 8 to the protected aldehyde 9 (Scheme 2).
However, this strategy gave unreliable results in our hands (see
Supplementary data, Table SI2).32 Although we could achieve mod-
erate yields, we found that the reaction was not scalable, with
decreasing yields at larger scale. We, therefore, chose to explore
alternative routes.
We chose to access the benzylic aldehyde through the conver-
sion of the protected tyrosine 10 to the triflate followed by carbon-
ylation (Scheme 3). Compound 10 was converted to triflate 11
using N-phenyl-bis-trifluoromethane sulfonamide in quantitative
yield.13 The triflate was converted to the carboxylic acid using
Pd(OAc)2.34,35 The crude acid was then reduced to the benzyl alco-
hol (12) with BH3–DMS complex.36 Oxidation of the purified ben-
zyl alcohol to the aldehyde using IBX was done using standard
conditions.37 We observed that prolonged storage of the interme-
diate aldehyde even at ꢀ20 °C resulted in partial conversion to
the hydrate. To avoid this issue, the aldehyde was used immedi-
ately in the subsequent cesium fluoride-catalyzed condensation38
reaction with dimethyl phosphite to generate compound 13.39 Ini-
tially, potassium fluoride was used to catalyze the condensation;
however, significant amounts of starting material were still pres-
ent after 24 h. We found that employing cesium fluoride gave com-
plete conversion within 20 min, likely due to the cesium salt acting
2. Results and discussion
2.1. a-Bromobenzylphosphonate analogs
Before synthesizing the desired amino acid targets, we first
undertook the synthesis of model BBP analogs based on benzalde-
hyde (Scheme 1). Condensation of benzaldehyde with dimethyl
phosphite gave the desired hydroxy(phenyl)dimethyl phosphonate
4 in quantitative yield.25 Initial attempts to convert 4 to the corre-
sponding bromide were unsuccessful using triphenylphospine
dibromide.23 We then explored a variety of reported bromination
conditions for related substrates. Among the conditions tested
O
P
O
P
OMe
OMe
OMe
OMe
HO
Br
O
HP(O)(OMe)2, KF
100%
SOBr2, pyr
88%
3
4
5
TMSBr
70%
TMSBr
85%
O
O
O
OH
OH
OH
OH
HO
P
Br
P
I
CO, Pd(OAc)2, ligand
R3SiH, NEt3
Cbz
N
H
CO2Me
Cbz
N
H
CO2Me
6
7
8
9
Scheme 1. Synthesis of model
a-bromophosophonates.
Scheme 2. Carbonylation of iodotyrosine.

N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
8681
1. dppf, Pd(OAc)2,
CO, K2CO3, DMF
2. BH3-DMS, THF
OH
OH
OTf
OH
1. SOCl2, MeOH
2. Cbz-Cl, H2O
PhN(Tf)2, AcCN
99% from Tyr
62%
H2N
CO2H
Cbz
N
H
CO2Me
CO2Me
CO2H
Cbz
N
H
CO2Me
10
13
16
11
14
1
OH
O
O
1. H2, Pd/C, MeOH
2. Fmoc-OSuc, AcCN/H2O
1. IBX, DMSO
2. HP(O)(OMe)2, CsF
OH
P
MeO
P
MeO
SOBr2, pyr, DCM
78%
OMe
OMe
85%
90%
Cbz
Cbz
N
H
CO2Me
N
H
Fmoc
N
H
CO2Me
12
15
Br
Br
Br
O
O
O
1. piperidine, DCM
2. TMSBr, AcCN
3. propyleneoxide, EtOH
P
MeO
P
MeO
P
OMe
OMe
OH
LiOH, H2O/THF
91%
HO
H
H
-
65%
+N
CO2
Fmoc
Fmoc
N
H
CO2Me
N
H
H2N
Scheme 3. Synthesis of Fmoc-L-BrPmp-OH.
as a more readily dissociated fluoride source. To make a protected
amino acid derivative suitable for automated SPPS, we chose to re-
place the Cbz group with Fmoc using standard conditions, to pro-
vide compound 14.
The key bromination step followed the conditions tested in our
model studies. Treatment of 14 with thionyl bromide in the pres-
reside at the C-terminal side—both of which are features
commonly found in phosphopeptide substrates of CD45.26 The
peptide was generated using standard solid phase methods, based
on the Fmoc protecting group strategy. For reactions using com-
pound 16 we employed HBTU as a coupling agent with DIPEA as
a base. Couplings were performed with a 2:1.96:4 molar ratio of
16/HBTU/DIPEA, in two cycles. To cleave the peptide from resin,
dry resin was treated with 95% TFA and 5% water for 1 h, followed
by treatment with TMSI for 100 min.40 Chromatograms of the
cleavage products suggested that the coupling reactions were effi-
ence of pyridine converted the a-hydroxyphosphonate to the fully
protected bromide, 15. Subsequent cleavage of the carboxylate
methyl ester with lithium hydroxide provided compound 16 in
good yield. The overall yield from tyrosine to 16 was 30% in 11
steps, with only five chromatographic steps. Compound 16 was
deprotected for use in enzyme assays by treatment with 20% piper-
idine in DMF to remove Fmoc, followed by cleavage of the phos-
phonomethyl groups with TMSBr.40 Propylene oxide was added
to remove residual HBr, allowing isolation of compound 1 as the
piperidine salt in 65% yield. It has not escaped our attention that
the final compound is isolated as a mixture of diastereomers at
cient. The isolated peptides were confirmed by HR-MS, 1H and 31
P
NMR and analytical HPLC. We initially chose to avoid commonly
employed thiol scavengers in the cleavage of the methyl ester
phosphonate protecting groups in anticipation of displacement of
the
a-bromide. However, we confirmed by NMR that compound
7 was stable to dithiothreitol (DTT) and thioanisole, suggesting
that scavenger nucleophiles should not interfere with peptide
cleavage and deprotection.23,25
the a-bromo-phosphonate, and that there may be potential differ-
ences in activity between the two isomers. However, there is a
large body of previous work with non-hydrolyzable phosphonate
derivatives which have been successfully used for inhibition stud-
ies with diastereomeric mixtures.41 We intend to investigate the
activity of the separated diastereomers in future work.
2.4. Inhibition of CD45
To examine the ability of the BrPmp derivatives to act as inhib-
itors of a PTP, we tested compounds 1, 7, and 17 against CD45. As-
says were conducted with the fluorogenic substrate, 6,8-difluoro-
4-methylumbelliferyl phosphate (DiFMUP), and are summarized
in Table 1 (see Supplementary data, Figs. SI1 and SI2). The Km for
DiFMUP with CD45 was first determined, and the experiment
was then repeated in the presence of compounds 7, 1, and 17 to
provide an apparent Km (Km, obs). We found significant inhibition
of CD45 for compounds 1 and 17 at micromolar concentrations;
however, compound 7 did not have a significant effect on enzyme
kinetics even at millimolar concentration. Previous reports of PTP
inhibition using BBP analogs observed irreversible enzyme inhibi-
tion.23 Evaluation of our kinetic data using linear transforms was
not consistent with pure competitive inhibition of CD45 by 1 or
17; however, this analysis is not conclusive on its own (see Supple-
mentary data, Fig. SI3). To provide additional insight into the
inhibition of these compounds, we obtained KI values using a
Kitz–Wilson analysis.42,43 Compound 7 did not give a line with
positive slope in this analysis, and therefore could not be analyzed
by this method, consistent with its failure to alter the rate of
2.3. Synthesis of peptide sequences
To test the compatibility of compound 16 with SPPS, we chose
to synthesize a short peptide sequence, Asp-BrPmp-Leu (17)
(Scheme 4). We chose a short sequence that contained an acidic
residue N-terminal to the phosphotyrosine site, and a hydrophobic
O
Br
OH
P
OH
O
O
H
H2N
N
N
H
OH
O
17
COOH
Scheme 4. Synthetic tripeptide Asp-BrPmp-Leu (17).

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N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
Table 1
Inhibition of CD45
a
b
Compd
Enzyme
Inhibitor (lM)
Km, obs
(
lM)
KI
(
lM)
k3 (minꢀ1
)
—
7
1
CD45
CD45
CD45
CD45
0
1500
150
35
90 20
99 14
141 18
141 34
na
na
40
16
—
—
8
4
0.041 0.001
0.048 0.003
17
a
Values were determined by non-linear regression of the observed rate of reaction in the presence of inhibitor using the Michaelis–Menten
equation.43 Error is reported as the relative error from the fit.
b
For compounds 1 and 17, KI was determined by Kitz–Wilson analysis. The rate of enzyme inactivation, k3, was also determined.42
reaction (vide supra). Compound 7 has been previously tested as
an inhibitor of the PTP Yop51, and exact kinetic constants were dif-
ficult to obtain, and we found similar difficulties for this determi-
nation with CD45.25 This result may be due to the more
hydrophobic nature of the compound. Using the Kitz–Wilson anal-
ysis, both compound 1 and 17 were found to have KI values of
chains contribute additional specificity to the inhibitor. The Kitz–
Wilson analysis estimated the rate of irreversible inhibition (k3)
of CD45 at 0.05 minꢀ1 for both compounds. To provide additional
support for the expected mechanism of inhibition, we measured
CD45 activity for an enzyme sample which was pre-incubated with
the inhibitor and compared it to an enzyme sample that was only
incubated with the inhibitor for a short period. We found that pre-
incubation of the enzyme reduced activity, and at long incubation
times completely inactivated the enzyme (see Supplementary data,
Fig. SI3), confirming that the inhibitors act irreversibly at long
incubation times.
40
8
l
M and 16
4 lM, respectively (Fig. 2). These results indi-
cate that the tripeptide was approximately fourfold more potent
than BrPmp alone, suggesting that the adjacent amino acid side
50
a
3. Conclusion
We present an efficient synthetic route for the preparation of
BrPmp analogs that can be readily used in automated SPPS. Our
method provides the Fmoc-L-BrPmp-OH derivative in 30% yield
over 11 steps from tyrosine, and required only five column separa-
tions. These compounds act as irreversible inhibitors of the PTP
CD45, supporting the previously observed selectivity of the a-bro-
40
30
20
10
0
mobenzylphosphonate functional group.23 Importantly, the syn-
thetic method reported here is scalable, and the products can be
easily modified using standard amino acid chemistry. The observa-
tion of improved potency for BrPmp within the context of a peptide
sequence suggests that these derivatives can act as specific inhib-
itors of PTPs. Future work in our group will address the specificity
of PTP inhibition by BrPmp derivatives, and their use as ABPP.
0
10000 20000 30000 40000 50000
1/Inh [M-1]
4. Experimental methods
4.1. Synthesis of dimethyl [hydroxy(phenyl)methyl]phosphonate
(4)
50
40
30
20
10
0
b
Dimethyl phosphite (1.86 mL, 20.3 mmol, 1.03 equiv) was added
to benzaldehyde, 3, (2 mL, 19.8 mmol, 1 equiv) and stirred for 5 min.
KF (5.75 g, 99.0 mmol, 5 equiv) was then added and the reaction
mixture was stirred until it solidified (ꢁ20 min). The crude product
was then dissolved in DCM, filtered, and the solvent evaporated
yielding 4 as a white powder (4.38 g, 100%). The product was dried
under high vacuum over P2O5. No further purification was required.
1H NMR (400 MHz, CDCl3) d 7.53–7.45 (m, 2H), 7.43–7.23 (m, 3H),
5.05 (d, J = 11.1 Hz, 1H), 3.69 (d, J = 10.4 Hz, 3H), 3.65 (d,
J = 10.4 Hz, 3H). 13C NMR (101 MHz, CDCl3) d 136.7, 128.6, 128.5,
127.3 (d, JC–P = 5.7 Hz), 70.9 (d, JC–P = 159.6 Hz), 54.3 (d, JC–P
=
7.0 Hz), 53.7 (d, JC–P = 7.0 Hz). 31P NMR (162 MHz, CDCl3) d 24.71.
ESIMS calcd for C9H13O4P [M+Na]+ 239.0444, found: 239.0441; mp
85–86 °C.
0
10000 20000 30000 40000 50000
1/Inh [M-1]
4.2. Synthesis of dimethyl [bromo(phenyl)methyl]phosphonate
(5)
Figure 2. Kitz–Wilson analysis of compounds 1 and 17. Compounds (a) 1 and (b) 17
were examined using a Kitz–Wilson analysis.42 The apparent rate constant (kobs
)
In a round bottom flask 4 (1 g, 4.63 mmol, 1 equiv) was dis-
solved in dry DCM (10 mL) and dry pyridine (0.47 mL, 5.78 mmol,
1.25 equiv) was added. Thionyl bromide (0.45 mL, 5.78 mmol,
was determined for a series of concentrations of the inhibitor.44 The data are plotted
as the linear transform based on Eq. 1. Values from the non-linear regression were
used for Table 1.

N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
8683
1.25 equiv) was then added to the round bottom flask under inert
atmosphere. The round bottom flask was sealed with a septum,
cooled in an ice bath and slowly allowed to come to room temper-
ature overnight. The solvent was then evaporated and the crude
product was dissolved in ethyl acetate. The organic layer was
washed with 1 M HCl, saturated NaHCO3, water, brine, then dried
over Na2SO4 and filtered. The solvent was evaporated and the
crude product was purified on a silica plug 1:4 (hexanes/ethyl ace-
tate) followed by 3:7 (hexanes/ethyl acetate) to give 12 as a vis-
cous oil (1.14 g, 88%). The product was dried under high vacuum
over P2O5. 1H NMR (400 MHz, CDCl3) d 7.56 (d, J = 7.8 Hz, 2H),
7.34 (d, J = 7.5 Hz, 2H), 7.26 (1H), 4.88 (d, J = 13.1 Hz, 1H), 3.85
(d, J = 10.8 Hz, 3H), 3.60 (d, J = 10.7 Hz, 3H). 13C NMR (101 MHz,
CDCl3) d 134.6 (d, JC–P = 3.3 Hz), 129.7 (d, JC–P = 6.7 Hz), 129.4 (d, J
C–P = 2.2 Hz), 129.0 (d, JC–P = 1.3 Hz), 55.1 (d, JC–P = 7.0 Hz), 54.7
(d, JC–P = 7.0 Hz), 41.1 (d, JC–P = 159.9 Hz). 31P NMR (162 MHz,
CDCl3) d 20.65. ESIMS calcd for C9H12BrO3P [M+Na]+ 300.9600,
found: 300.9596.
1.5 equiv) was then added and the reaction mixture cooled on
ice. Benzyl chloroformate (0.64 mL, 4.52 mmol, 1.3 equiv) in diox-
ane (5 mL) was added dropwise and the reaction was stirred for
18 h at room temperature. The reaction mixture was then washed
with Et2O, and the pH of the aqueous layer was adjusted to 2 using
1 N HCl. The crude product was extracted with ethyl acetate and
dried over Na2SO4. The solvent was evaporated to give 1.46 g of
the crude Cbz protected product as a viscous yellow oil. To dry
methanol (8 mL) cooled on ice, was slowly added SOCl2 (0.43 mL,
5.88 mmol, 2.5 equiv). The resulting solution was warmed to room
temperature and the crude Cbz protected product (1.00 g,
2.38 mmol, 1 equiv) was added and the mixture was stirred for
18 h at room temperature under argon. The solvent was then evap-
orated yielding 1.05 g of the methyl ester Cbz crude product as a
viscous yellow oil. This product was used in the next step without
further purification.
4.6. Synthesis of methyl 2-{[(benzyloxy)carbonyl]amino}-3-(4-
formylphenyl)propanoate (9)
4.3. Synthesis of [hydroxy(phenyl)methyl]phosphonic acid (6)
A
mixture of Cbz-N-L-4-iodophenylalanine methyl ester 8
Compound 4 (0.15 g, 0.69 mmol, 1 equiv) was dissolved in dry
DCM (5 mL). TMSBr (0.77 mL, 5.52 mmol, 8 equiv) was added to
the solution under an inert atmosphere. The reaction was stirred
for 20 h at room temperature. The solvent was then evaporated,
and MeOH (5 mL) was added to the reaction mixture and stirred
for 1 h. The solvent was evaporated and the crude product was dis-
solved in water (5 mL), filtered and freeze dried, yielding 6 as a
white powder (130 mg, 70%). 1H NMR (400 MHz, D2O) d 7.52–
7.31 (m, 5H), 4.99 (d, J = 12.3 Hz, 1H). 13C NMR (101 MHz, D2O)
d 137.4, 128.8 (d, JC–P = 2.3 Hz), 128.5 (d, JC–P = 2.9 Hz), 127.4
(d, JC–P = 5.7 Hz), 71.0 (d, JC–P = 158.3 Hz). 31P NMR (162 MHz, D2O)
d 20.98. ESIMS calcd for C7H9O4P [MꢀH]ꢀ 187.0166, found:
187.0162; mp 160–162 °C.
(439 mg, 1 mmol, 1 equiv), Pd(OAc)2 (11.67 mg, 0.05 mmol, 0.05
equiv), 1,30-bis(diphenylphosphino)propane (20 mg, 0.05 mmol,
0.05 equiv) and Et3N (0.35 mL, 2.5 mmol, 2.5 equiv) in dry DMF
(5 mL) was purged with CO for 10 min. Trioctylsilane (0.9 mL,
2 mmol, 2 equiv) was added in one portion and the mixture was stir-
red under a CO balloon for 8 h at 70 °C. The reaction mixture was then
diluted with H2O (20 mL), extracted with Et2O, washed with H2O, sat-
urated NaHCO3, H2O, dried over Na2SO4 and the solvent evaporated.
The compound was purified on a silica column (9:1 hexanes/ethyl
acetate followed by 4:1 hexanes/ethyl acetate) to give 9 (205 mg,
60%) as a colorless oil. 1H NMR (500 MHz, CDCl3) d 9.98 (1H), 7.78
(d, J = 7.6 Hz, 2H), 7.53–7.13 (m, 7H), 5.35–5.25 (m, 1H), 5.13–5.05
(m 2H), 4.75–4.67 (m, 1H), 3.73 (3H), 3.28–3.10 (m, 2H). ESIMS calcd
for C19H19NO5 [M+Na]+ 364.1155, found: 364.1170.
4.4. Synthesis of [bromo(phenyl)methyl]phosphonic acid (7)
4.7. Synthesis of methyl 2-{[(benzyloxy)carbonyl]amino}-3-(4-
hydroxyphenyl)propanoate (10)
Compound 5 (0.3 g, 1.08 mmol, 1 equiv) was dissolved in dry
DCM (5 mL). TMSBr (1.2 mL, 8.64 mmol, 8 equiv) was added under
an inert atmosphere. The reaction was stirred for 20 h at room
temperature. The solvent was then evaporated, and MeOH (5 mL)
was added to the reaction mixture and stirred for 1 h. The solvent
was evaporated and the crude product was dissolved in water
(5 mL), filtered and freeze dried yielding 7 as a white powder
(229 mg, 85%). 1H NMR (400 MHz, D2O) d 7.61–7.52 (m, 2H),
7.44–7.33 (m, 3H), 5.10–5.02 (m, 1H). 13C NMR (101 MHz, D2O) d
136.5 (d, JC–P = 3.3 Hz), 129.4 (d, JC–P = 6.0 Hz), 129.1, 43.54. 31P
NMR (162 MHz, D2O) d 15.44. ESIMS calcd for C7H7BrO3P [MꢀH]
Thionyl chloride (4.6 mL, 63.33 mmol, 2 equiv) was added drop-
wise to dry MeOH (65 mL) at 0 °C and stirred for 5 min. L-Tyrosine
(5.70 g, 31.5 mmol, 1 equiv) was then added and the reaction ves-
sel was fitted with a drying tube filled with drierite and slowly al-
lowed to come to room temperature over 21 h. The solvent was
then evaporated and crude product dried over high vacuum for
8 h. The crude product was then dissolved in a 1:1 mixture of ace-
tone (63 mL) and a 7% solution of Na2CO3 in water (63 mL). Benzyl
chloroformate (4.7 mL, 34.7 mmol, 1.2 equiv) was then added
dropwise and the reaction was stirred for 3 h at room temperature.
Ethyl acetate was then added (300 mL), and the organic layer was
washed with water (100 mL), brine (100 mL) and dried over
Na2SO4. The solvent was evaporated to give 10 as a viscous yellow
oil (10.37 g). The product was used in the next step without
purification.
ꢀ
248.9322, found: 248.9317 mp 158–60 °C.
4.5. Synthesis of methyl 2-{[(benzyloxy)carbonyl]amino}-3-(4-
iodophenyl)propanoate (8)
Iodine (5.24 g, 20.64 mmol) and sodium iodate (2.04 g,
10.32 mmol) were added to a solution of phenylalanine (8.52 g,
51.6 mmol) in acetic acid (47 mL) and concentrated sulfuric acid
(6.2 mL). The mixture was heated at 70 °C and stirred for 24 h. So-
dium periodate (0.4 g) was then added and the reaction mixture
was stirred for 30 min. Acetic acid was then evaporated and the
crude mixture was diluted with H2O (80 mL) and washed with
Et2O and DCM. The pH of the aqueous layer was adjusted to pH
5 with concentrated NaOH solution. The precipitate was filtered
under vacuum and washed with H2O (170 mL) and EtOH (65 mL)
to afford 13.09 g of the crude product 4-iodophenylalanine. The
crude 4-iodophenylalanine product (1.00 g, 3.44 mmol. 1 equiv)
was then suspended in water (5 mL), NaHCO3 (0.45 g, 5.35 mmol,
4.8. Synthesis of methyl 2-{[(benzyloxy)carbonyl]amino}-3-(4-
{[(trifluoromethyl)sulfonyl]oxy}phenyl)propanoate (11)
Compound 10 (10.37 g, 31.5 mmol, 1 equiv) and N-phenyl bis-
trifluoromethane sulfonamide (12.39 g, 34.65 mmol, 1.1 equiv)
were dissolved in acetonitrile (150 mL). Et3N (5.3 mL, 37.8 mmol,
1.2 equiv) was then added and the reaction was stirred for 3 h.
The reaction mixture was then diluted with ethyl acetate
(150 mL) and water (100 mL). The organic layer was washed with
brine, dried over Na2SO4 and the solvent was evaporated. The
product was purified on a silica column (3:2 hexane/ethyl acetate

8684
N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
followed by 2:3 hexanes/ethyl acetate) to obtain 14.38 g (99%
yield) as a white solid. 1H NMR (400 MHz, CDC13) d 7.47–7.07
(m, 9H), 5.32–5.24 (m, 1H), 5.12–5.05 (m, 2H), 4.66 (dd, J = 13.7,
6.1 Hz, 1H), 3.71 (3H), 3.22–3.05 (m, 2H). ESIMS calcd for
(87P), 11.64 (1P). ESIMS calcd for C21H26NO8P [M+Na]+ 474.1288,
found: 474.1288; mp 68–71 °C.
4.11. Synthesis of L-phenylalanine, 4-[(dimethyloxyphosphinyl)-
bromomethyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-, methyl
C
19H18F3NO7S [M+Na]+ 484.0648, found: 484.0645; mp 70–73 °C.
ester (14)
4.9. Synthesis of methyl 2-{[(benzyloxy)carbonyl]amino}-3-[4-
(hydroxymethyl)phenyl]propanoate (12)
Compound 13 (3 g, 6.65 mmol, 1 equiv) was dissolved in dry
MeOH and Pd/C (200 mg, 30 mg/mmol) was added. A three way
stopcock (connected to an H2 balloon and vacuum line) was fitted
to the reaction vessel. The reaction mixture was then flushed with
H2 (3ꢂ) and stirred at room temperature for 6 h. The reaction mix-
ture was filtered through a Celite pad and the solvent evaporated.
A mixture of the residue, Fmoc-succinimide (2.35 g, 6.98 mmol,
1.05 equiv) and NaHCO3 (2.34 g, 27.92 mmol, 4 equiv) in acetoni-
trile and water (1:1, 130 mL) was stirred at room temperature
overnight. The acetonitrile was evaporated under reduced pressure
and the crude product was extracted with ethyl acetate. The organ-
ic layer was dried over Na2SO4, evaporated under reduced pressure
and purified on a silica plug (diethylether, followed by 10:1 DCM/
MeOH) to give 14 as a white solid (3.59 g, 90%). 1H NMR (400 MHz,
CDCl3) d 7.76 (d, J = 8.0 Hz, 2H), 7.64–7.48 (m, 2H), 7.48–7.35 (m,
4H), 7.34–7.28 (m, 2H), 7.1 (d, J = 8.0 Hz, 2H), 5.33 (d, J = 7.8 Hz,
1H), 5.02 (d, J = 11.0 Hz, 1H), 4.70–4.60 (m, 1H), 4.45–4.29
(m, 2H), 4.21–4.18 (m, 1H), 3.70 (d, J = 7.0 Hz, 4H), 3.67–3.65
(m, 3H), 3.63–3.62 (m, 2H), 3.20–3.00 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 172.1, 155.8, 144.0 (d, JC–P = 7.5 Hz), 141.6,
136.2, 135.7–134.3 (m), 129.7, 128.0, 127.5 (d, JC–P = 5.8 Hz),
127.3, 125.3 (d, JC–P = 6.1 Hz), 120.2, 70.6 (d, JC–P = 159.5 Hz),
67.2, 55.0, 54.1 (d, JC–P = 7.1 Hz), 54.0 (d, JC–P = 7.1 Hz), 52.6, 47.4,
Compound 11 (7.70 g, 16.70 mmol, 1 equiv), Pd(OAc)2 (378 mg,
1.68 mmol, 0.1 equiv) and 1,10-Bis(diphenylphosphino)ferrocene
(dppf) (1.86 g, 3.34 mmol, 0.2 equiv) were dissolved in dry DMF
(40 mL). K2CO3 (11.54 g, 83.5 mmol, 5 equiv) was then added to
the reaction mixture and CO gas was bubbled through for
15 min. The reaction mixture was then heated at 60 °C for 8 h un-
der a CO balloon. The reaction mixture was then cooled and parti-
tioned between ethyl acetate and saturated NaHCO3. The aqueous
layer was acidified with a 10% aqueous solution of citric acid and
extracted with ethyl acetate (4 ꢂ 75 mL). The organic layer was
washed with brine, dried over Na2SO4 and the solvent was evapo-
rated to give the crude carboxylic acid as a tan colored solid
(4.89 g). The acid was dried over P2O5 and was used in the next
step without purification. The crude acid was dissolved in dry
THF (70 mL) and cooled in an ice bath. The reaction was charged
with BH3–DMS complex (10 M, 6.96 mL, 68.47 mmol, 4 equiv)
added dropwise. The reaction mixture was warmed to room tem-
perature over 2 h. A solution of saturated NaHCO3 was added drop-
wise until the bubbling ceased. Ethyl acetate (70 mL) was added
and the organic layer was separated and dried over Na2SO4 and
then reduced. The crude product was purified on a silica column
(3:2 hexane/ethyl acetate followed by 2:3 hexanes/ethyl acetate)
38.2. 31P NMR (162 MHz, CDCl3)
d
24.51. ESIMS calcd for
to give 12 (3.56 g, 62%) as a white solid. ½a D25
ꢃ
+50.56 (c 0.99, CHCl3).
C
28H30NO8P [M+Na]+ 562.1601, found: 562.1595; mp 65–70 °C.
1H NMR (500 MHz, CDCl3) d 7.41–7.23 (m, 7H), 7.08 (d, J = 8.0 Hz,
2H), 5.23 (d, J = 7.8 Hz, 1H), 5.12–5.05 (m, 2H), 4.65 (3H), 3.72
(3H), 3.17–3.02 (m, 2H), 1.76 (1H). 13C NMR (101 MHz, CDCl3)
d 172.2, 155.9, 140.0, 136.5, 135.3, 129.7, 128.8, 128.4, 128.3,
127.5, 67.2, 65.2, 55.0, 52.6, 38.1. ESIMS calcd for C19H21NO5
[M+Na]+ 366.1312, found: 366.1311; mp 74–77 °C.
4.12. Synthesis ofL-phenylalanine, 4-[(dimethyloxyphosphinyl)-
bromomethyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-, methyl
ester (15)
In a round bottom flask 14 (2 g, 3.70 mmol, 1 equiv) was dis-
solved in dry DCM (30 mL) and dry pyridine (0.38 mL, 4.63 mmol,
1.25 equiv) was added. Thionyl bromide (0.36 mL, 4.63 mmol,
1.25 equiv) was then added to the round bottom flask under an in-
ert atmosphere. The round bottom flask was sealed with a septum,
cooled in an ice bath and slowly allowed to come to room temper-
ature overnight. The solvent was then evaporated and the crude
product was dissolved in ethyl acetate. The organic layer was
washed with 1 M HCl, saturated NaHCO3, water, and brine, dried
over Na2SO4 and filtered. The solvent was evaporated and the
crude product was purified on a silica plug (diethylether, followed
by 10:1 DCM/MeOH) to give 15 as a white solid (1.14 g, 78%). Pur-
4.10. Synthesis of L-phenylalanine, 4-[(dimethyloxyphosphinyl)-
hydroxymethyl]-N-[(phenylmethoxy)carbonyl]-, methyl ester (13)
Compound 12 (3.56 g, 10.37 mmol, 1 equiv) was dissolved in
DMSO (22 mL) and 2-iodoxybenzoic acid (3.77 g, 13.48 mmol,
1.3 equiv) was added and the reaction mixture and stirred for
1 h. The reaction mixture was then diluted with water (60 mL)
and diethylether (60 mL) and filtered. The organic layer was sepa-
rated and washed with water (2 ꢂ 50 mL), brine, and dried over
Na2SO4. The solvent was evaporated and the crude aldehyde was
used immediately in the next step without purification. The alde-
hyde was dissolved in dimethyl phosphite (1.05 mL, 11.41 mmol
1.1 equiv) with mild heating. CsF (9.45 g, 62.22 mmol, 6 equiv)
was added and the reaction was stirred until it solidified. The crude
product was then dissolved in DCM (40 mL), filtered, and the sol-
vent evaporated. The crude product was purified on a silica plug
(first with diethylether, then 1:20 DCM/MeOH) to give 13 as a
ity (>95%) determined by analytical HPLC, C18, flow: 1 mL minꢀ1
,
k: 254 nm, eluent: acetonitrile/0.1% TFA in water 10:90 (2 min)
to 50:50 (22 min), retention time: 49.7 min. ½a D25
ꢃ
+34.72 (c 1.02,
CHCl3). 1H NMR (400 MHz, CDCl3) d 7.77 (d, J = 7.5 Hz, 2H), 7.57
(d, J = 7.4 Hz, 2H), 7.49 (d, J = 7.5 Hz, 2H), 7.41 (t, J = 7.4 Hz, 2H),
7.32 (t, J = 7.5 Hz, 2H), 7.17–6.98 (m, 2H), 5.32–5.23 (m, 1H), 4.86
(d, J = 13.1 Hz, 1H), 4.66 (dd, J = 13.4, 6.1 Hz, 1H), 4.49–4.30 (m,
2H), 4.20 (t, J = 6.9 Hz, 1H), 3.85 (d, J = 10.8 Hz, 3H), 3.72 (s, 3H),
3.59 (dd, J = 10.7, 3.8 Hz, 3H), 3.17–3.05 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 171.9, 155.7, 144.0 (d, JC–P = 6.5 Hz), 141.6,
137.3, 133.5, 130.0 (d, JC–P = 3.6 Hz), 129.9, 128.0, 127.3, 125.3
(d, JC–P = 7.6 Hz), 120.3, 67.2 (d, JC–P = 6.2 Hz), 54.9 (m), 52.7, 47.4,
40.8 (d, JC–P = 160.0 Hz), 38.2 (d, JC–P = 7.0 Hz). 31P NMR
(162 MHz, CDCl3) d 20.43. ESIMS calcd for C28H29NO7BrP [M+Na]+
624.0757, found: 624.0756; mp 68–71 °C.
white solid (3.98 g, 85%). ½a D25
ꢃ
+38.08 (c 1.55, CHCl3). 1H NMR
(400 MHz, CDCl3) d 7.40 (dd, J = 8.1, 1.9 Hz, 2H), 7.37–7.27 (m,
5H), 7.11 (d, J = 8.0 Hz, 2H), 5.39–5.26 (m, 1H), 5.07 (2H), 5.00 (d,
J = 11.0 Hz, 1H), 4.63 (d, J = 7.6 Hz, 1H), 3.69 (d, J = 2.8 Hz, 3H),
3.65 (d, J = 4.7 Hz, 3H), 3.62 (3H), 3.16–3.04 (m, 2H). 13C NMR
(101 MHz, CDCl3) d 172.1, 155.9, 136.3 (d, JC–P = 23.9 Hz), 135.5,
129.6 (d, JC–P = 2.1 Hz), 128.8, 128.4, 128.3, 127.5 (d, JC–P = 5.8 Hz),
70.6 (d, J = 159.4 Hz), 67.2, 55.0, 54.1 (d, JC–P = 7.1 Hz), 53.9 (d,
C–P
JC–P = 7.1 Hz), 52.6, 38.2. 31P NMR (162 MHz, CDCl3) d 24.53

N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
8685
4.13. Synthesis of
bromomethyl]-N-[(9H-fluoren-9-ylmethoxy)carbonyl]; Fmoc-
BrPmp(OMe2)-OH (16)
L
-phenylalanine, 4-[(dimethyloxyphosphinyl)-
and DIPEA in NMP for 3.5 h. Fmoc-Asp(tBu)-OH (5 equiv) was cou-
pled using HBTU (4.9 equiv) in the presence of DIPEA (10 equiv) in
NMP for 3.5 h. Fmoc deprotection was achieved with 20% piperidine
in NMP. The resin was washed with NMP, AcOH, DCM, and MeOH.
Immediately after washing the resin with CH3CN, DCM and MeOH,
a mixture of TFA/H2O/TIPS (95:2.5:2.5) was added and the resin
was shaken at room temperature for 3 h. The cleaved peptide was
precipitated in diethylether, filtered, dissolved in a mixture of H2O
and CH3CN and lyophilized. The lyophilized peptide was suspended
in CH3CN, and TMSI (20 equiv) was added under an inert atmo-
sphere and the reaction mixture shaken for 100 min at room tem-
perature. The CH3CN/TMSI solution was evaporated under reduced
pressure, and the crude product was dissolved in water and washed
with diethylether (3ꢂ) and the aqueous layer was lyophilized. The
peptide was purified by HPLC (C-18 semipreparative column) using
a linear gradient (CH3CN/H2O mobile phase containing 0.1% TFA).
4 mg of pure compound was recovered from 18 mg crude product
(33% of theoretical yield). Purity (>95%) determined by analytical
HPLC, C18, flow: 1 mL minꢀ1, k: 212 nm, eluent: 0.1% TFA in acetoni-
trile/0.1% TFA in water 10:90 (2 min) to 20:80 (22 min), retention
time: 22.4 min. 1H NMR (500 MHz, D2O) d 7.57 (d, J = 6.8 Hz, 2H),
7.30 (dd, J = 7.9, 2.2 Hz, 2H), 5.02 (d, J = 11.7 Hz, 1H), 4.75–4.69
(m, 1H), 4.43–4.32 (m, 1H), 4.32–4.22 (m, 1H), 3.23–3.06 (m, 2H),
2.99–2.84 (m, 2H), 1.67–1.59 (m, 2H), 0.92 (dd, J = 19.7, 4.7 Hz,
6H). 31P NMR (162 MHz, D2O) d 13.78 (d, J = 4.8 Hz). ESIMS calcd
for C20H28N3O9BrP [MꢀH]ꢀ 564.0752, found: 564.0732.
L-
Compound 15 (3.00 g, 5.00 mmol, 1 equiv) was dissolved in THF
(35 mL) and cooled in an ice bath. LiOH (240 mg, 10.00 mmol,
2 equiv) was dissolved in water (35 mL) and cooled in an ice bath.
The lithium hydroxide solution was then added to the reaction
mixture and stirred for 30 min. The THF was then evaporated,
and the aqueous layer was washed with diethylether (30 mL).
The aqueous layer was acidified to pH 2 with concentrated HCl
and was extracted with ethyl acetate (4 ꢂ 75 mL). The combined
organic layers were dried over Na2SO4, and concentrated to a white
solid (2.67 g, 91% yield). Purity (>95%) determined by analytical
HPLC, C18, flow: 1 mL minꢀ1, k: 254 nm, eluent: acetonitrile/0.1%
TFA in water 10:90 (2 min) to 50:50 (22 min), retention time:
35.9 min. ½a 2D5
ꢃ
+40.24 (c 1.31, CHCl3). 1H NMR (400 MHz, CDCl3)
d 7.76 (d, J = 7.2 Hz, 2H), 7.57 (d, J = 6.3 Hz, 2H), 7.48–7.35 (m,
4H), 7.30 (t, J = 7.2 Hz, 2H), 7.16 (2H), 5.52 (d, J = 13.1 Hz, 1H),
4.98–4.81 (m, 1H), 4.67 (1H), 4.47 (m, 1H), 4.33 (1H), 4.19 (1H),
3.82 (d, J = 10.7 Hz, 3H), 3.54 (t, J = 10.4 Hz, 3H), 3.18 (2H). 13C
NMR (101 MHz, CDCl3) d 155.98, 144.0 (d, JC–P = 10.5 Hz), 141.6,
137.8, 132.7, 130.3, 129.8 (d, JC–P = 5.5 Hz), 128.0, 127.3, 125.3 (d,
JC–P = 7.5 Hz), 120.2, 77.6, 77.3, 76.9, 67.1, 55.6–54.9 (m), 54.8,
47.4, 41.1 (d, JC–P = 7.6 Hz), 39.5 (d, JC–P = 7.6 Hz), 37.8. 31P NMR
(162 MHz, CDCl3)
d
20.59 (d, J = 38.6 Hz). ESIMS calcd for
C
27H27NO7BrP [M+Na]+ 610.0601, found: 610.0595; mp 84–88 °C.
4.16. Phosphatase inhibition assays
4.14. Synthesis of L-BrPmp-OH (1)
Enzyme assays were conducted using human CD45-cytoplasmic
Compound 16 (200 mg, 0.34 mmol, 1 equiv) was dissolved in a
solution of 20% piperidine in dry DCM (10 mL) and stirred at room
temperature for 30 min. The solvent was then evaporated and the
residue was dried on high vacuum over P2O5 overnight. The resi-
due was then dissolved in dry acetonitrile (10 mL) and TMSBr
(0.47 mL, 3.4 mmol, 10 equiv) was added under an inert atmo-
sphere and the reaction mixture was stirred overnight. The organic
solvent was evaporated and the crude residue was dissolved in
water and the aqueous layer was washed with diethylether
(5 mL) and freeze dried. The crude bromide salt was then dissolved
domain (Enzo Life Science; diluted to 4 mU/lL in 50 mM HEPES,
pH 7.2, 1 mM EDTA, and 0.1% nonidet P-40). Enzyme activity was
detected with a fluorogenic substrate (6,8-difluoro-4-methylum-
belliferyl phosphate; DiFMUP) (Invitrogen). Assays were per-
formed in black 96-well plates and read in a Spectra Max M2
plate reader (Molecular Devices). Stock solutions of inhibitors were
prepared and stored at ꢀ20 °C. Final solutions in microplate wells
contained a total volume of 100
zyme, inhibitor, and DiFMUP substrate diluted to 100
l
L consisting of 2
l
L of diluted en-
L in the
l
appropriate buffer. All wells were incubated for 10 min at 37 °C
in the plate reader prior to the addition of DiFMUP. After addition
of the substrate, the plate was read at an excitation maximum of
358 nm and an emission maximum of 450 nm. Competitive inhibi-
tion was determined by observing the initial velocity of CD45 over
a range of substrate concentrations to determine Km, or, in the
presence of the inhibitor, Km, obs. To determine the activity of irre-
versible inhibitors, the enzyme activity was monitored over 60 min
in the presence of inhibitor, and the curve was fit to obtain kobs as
described elsewhere.44 Values of kobs were then fit to
in anhydrous EtOH (3 mL), propylene oxide (36 lL, 0.51 mmol,
1.5 equiv) was added and the reaction mixture was stirred over-
night resulting in a white precipitate the next day. Water (5 mL)
was added and the EtOH was then removed under reduced pres-
sure. The aqueous layer was filtered and freeze dried. The crude
product was dissolved in water (1 mL) and passed through a C18
Sep-pak syringe column. The aqueous fractions were freeze dried
yielding 1 (94 mg, 65%) as a white solid of the piperidine salt.
½
a 2D5
ꢃ
ꢀ16.56 (c 1.36, CHCl3). 1H NMR (400 MHz, D2O) d 7.54 (d,
J = 7.8 Hz, 2H), 7.26 (d, J = 7.6 Hz, 2H), 4.95 (d, J = 11.6 Hz, 1H),
4.07–3.92 (m, 1H), 3.28 (d, J = 10.5 Hz, 1H), 3.16–3.00 (m, 5H),
1.77–1.68 (m, 4H), 1.67–1.57 (m, 2H). 13C NMR (101 MHz, D2O) d
135.6, 135.6 (d, J = 1.1 Hz), 129.9 (d, J = 5.8 Hz), 129.8 (d,
J = 1.4 Hz), 56.0, 46.2, 44.8, 36.2, 22.5, 21.7. 31P NMR (162 MHz,
D2O) d 13.47. ESIMS calcd for C10H12NO5BrP [MꢀH]ꢀ 335.9642,
found: 335.9640; decomp. 140 °C.
k3
kobs
¼
ð1Þ
1 þ KI=ðIÞ
by non-linear regression; where KI was the inhibition constant, I
was the concentration of inhibitor, and k3 was the rate of inactiva-
tion of the enzyme.42 Inhibitors which did not show a positive slope
by Kitz–Wilson analysis were treated as competitive inhibitors for
the determination of KI.
4.15. Synthesis of tripeptide Asp-BrPmp-Leu (17)
Acknowledgements
The tripeptide was assembled manually on Wang resin
(0.6 mmol/g), preloaded with an Fmoc protected Leu residue
This work was supported by Natural Sciences and Engineering
Research Council of Canada (NSERC) and the Alberta Ingenuity
Centre for Carbohydrate Sciences (AICCS). We would like to thank
the Canadian Foundation for Innovation for infrastructure support.
The authors would like to thank W. Moffat at the University of
(Fmoc-
L
-Leu-OH). Following
a
standard protocol: Fmoc-L-
BrPmp(OMe2)-OH 16 (2 equiv) was coupled to the resin using HBTU
(1.96 equiv) in the presence of DIPEA (4 equiv) in NMP for 3.5 h. The
reaction was monitored by Kaiser test. The coupling was repeated
using the same equivalents of Fmoc-L-BrPmp(OMe2)-OH, HBTU

8686
N. S. Tulsi et al. / Bioorg. Med. Chem. 18 (2010) 8679–8686
19. Shen, K.; Qi, L.; Ravula, M.; Klimaszewski, K. Bioorg. Med. Chem. Lett. 2009, 19,
3264.
20. Shen, K.; Qi, L. X.; Ravula, M. Synthesis 2009, 3765.
21. Liu, S.; Zhou, B.; Yang, H.; He, Y.; Jiang, Z. X.; Kumar, S.; Wu, L.; Zhang, Z. Y. J.
Am. Chem. Soc. 2008, 130, 8251.
Alberta Spectral Services Laboratory, and Professor D.R. Bundle for
peptide synthesizer access.
Supplementary data
22. Park, J.; Pei, D. Biochemistry 2004, 43, 15014.
23. Kumar, S.; Zhou, B.; Liang, F.; Wang, W. Q.; Huang, Z.; Zhang, Z. Y. Proc. Natl.
Acad. Sci. U.S.A. 2004, 101, 7943.
Supplementary data (enzyme kinetic data and spectral charac-
terization) associated with this article can be found, in the online
version, at doi:10.1016/j.bmc.2010.09.040.
24. Mitra, S.; Barrios, A. M. ChemBioChem 2008, 9, 1216.
25. Taylor, W. P.; Zhang, Z. Y.; Widlanski, T. S. Bioorg. Med. Chem. 1996, 4, 1515.
26. Barr, A. J.; Ugochukwu, E.; Lee, W. H.; King, O. N. F.; Filippakopoulos, P.; Alfano,
I.; Savitsky, P.; Burgess-Brown, N. A.; Muller, S.; Knapp, S. Cell 2009, 136, 352.
27. Swamy, K. C. K.; Kumar, K. V. P. P.; Suresh, R. R.; Kumar, N. S. Synthesis 2007,
1485.
References and notes
28. Creary, X.; Underiner, T. L. J. Org. Chem. 1985, 50, 2165.
29. Gross, H.; Costisella, B.; Ozegowski, S.; Keitel, I.; Forner, K. Phosphorus, Sulfur
Silicon Relat. Elem. 1993, 84, 121.
30. Su, D.; Guo, C. Y.; Willett, R. D.; Scott, B.; Kirchmeier, R. L.; Shreeve, J. M. J. Am.
Chem. Soc. 1990, 112, 3152.
31. Lei, H. Y.; Stoakes, M. S.; Herath, K. P. B.; Lee, J. H.; Schwabacher, A. W. J. Org.
Chem. 1994, 59, 4206.
32. Morera, E.; Ortar, G.; Varani, A. Synth. Commun. 1998, 28, 4279.
33. Lin, S.; Yang, Z. Q.; Kwok, B. H. B.; Koldobskiy, M.; Crews, C. M.; Danishefsky, S.
J. J. Am. Chem. Soc. 2004, 126, 6347.
34. Cacchi, S.; Ciattini, P. G.; Morera, E.; Ortar, G. Tetrahedron Lett. 1986, 27, 3931.
35. Cai, C. Z.; Breslin, H. J.; He, W. Tetrahedron 2005, 61, 6836.
36. Klein, L. L.; Li, L. P.; Chen, H. J.; Curty, C. B.; DeGoey, D. A.; Grampovnik, D. J.;
Leone, C. L.; Thomas, S. A.; Yeung, C. M.; Funk, K. W.; Kishore, V.; Lundell, E. O.;
Wodka, D.; Meulbroek, J. A.; Alder, J. D.; Nilius, A. M.; Lartey, P. A.; Plattner, J. J.
Bioorg. Med. Chem. 2000, 8, 1677.
37. Frigerio, M.; Santagostino, M. Tetrahedron Lett. 1994, 35, 8019.
38. Wroblewski, A. E.; Maniukiewicz, W.; Karolczak, W. J. Chem. Soc., Perkin Trans. 1
2000, 1433.
1. Petrone, A.; Sap, J. J. Cell Sci. 2000, 113, 2345.
2. Moorhead, G. B. G.; De Wever, V.; Templeton, G.; Kerk, D. Biochem. J. 2009, 417,
401.
3. Chan, A. C.; Desai, D. M.; Weiss, A. Annu. Rev. Immunol. 1994, 12, 555.
4. Burke, T. R., Jr.; Zhang, Z. Y. Biopolymers 1998, 47, 225.
5. Zhang, Z. Y. Curr. Opin. Chem. Biol. 2001, 5, 416.
6. Hermiston, M. L.; Xu, Z.; Weiss, A. Annu. Rev. Immunol. 2003, 21, 107.
7. Hegedus, Z.; Chitu, V.; Toth, G. K.; Finta, C.; Varadi, G.; Ando, I.; Monostori, E.
Immunol. Lett. 1999, 67, 31.
8. Hubbard, C. E.; Barrios, A. M. Bioorg. Med. Chem. Lett. 2008, 18, 679.
9. Burke, T. R., Jr.; Smyth, M. S.; Nomizu, M.; Otaka, A.; Roller, P. P. J. Org. Chem.
1993, 58, 1336.
10. Leung, C.; Grzyb, J.; Lee, J.; Meyer, N.; Hum, G.; Jia, C.; Liu, S.; Taylor, S. D. Bioorg.
Med. Chem. 2002, 10, 2309.
11. Li, P.; Zhang, M.; Peach, M. L.; Liu, H.; Yang, D.; Roller, P. P. Org. Lett. 2003, 5,
3095.
12. Qabar, M. N.; Urban, J.; Kahn, M. Tetrahedron 1997, 53, 11171.
13. Yokomatsu, T.; Yamagishi, T.; Matsumoto, K.; Shibuya, S. Tetrahedron 1996, 52,
11725.
14. Lee, K.; Gao, Y.; Yao, Z. J.; Phan, J.; Wu, L.; Liang, J.; Waugh, D. S.; Zhang, Z. Y.;
Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2003, 13, 2577.
15. Saghatelian, A.; Cravatt, B. F. Nat. Chem. Biol. 2005, 1, 130.
16. Sadaghiani, A. M.; Verhelst, S. H. L.; Bogyo, M. Curr. Opin. Chem. Biol. 2007, 11,
20.
17. Huang, Y. Y.; Kuo, C. C.; Chu, C. Y.; Huang, Y. H.; Hu, Y. L.; Lin, J. J.; Lo, L. C.
Tetrahedron 2010, 66, 4521.
18. Lo, L. C.; Chiang, Y. L.; Kuo, C. H.; Liao, H. K.; Chen, Y. J.; Lin, J. J. Biochem. Biophys.
Res. Commun. 2004, 326, 30.
39. Texierboullet, F.; Foucaud, A. Synthesis 1982, 165.
40. Larose, L.; Gish, G.; Pawson, T. J. Biol. Chem. 1995, 270, 3858.
41. Burke, T. R., Jr.; Lee, K. Acc. Chem. Res. 2003, 36, 426.
42. Kitz, R.; Wilson, I. B. J. Biol. Chem. 1962, 237, 3245.
43. Copeland, R. A. Enzymes: A Practical Introduction to Structure, Mechanism, and
Data Analysis; John Wiley & Sons: New York, NY, 2000.
44. Skorey, K.; Ly, H. D.; Kelly, J.; Hammond, M.; Ramachandran, C.; Huang, Z.;
Gresser, M. J.; Wang, Q. P. J. Biol. Chem. 1997, 272, 22472.