Deoxygenated phosphorothioate inositol phosphate analogs: Synthesis, phosphatase stability, and binding affinity

Article abstract of DOI:10.1016/j.bmc.2007.10.032

Inositol phosphates, such as 1d-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], are cellular second messengers with potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single inositol phosphate because they are rapidly metabolized by phosphatases and kinases. In this study, we have designed stable analogs of myo-inositol 4,5-bisphosphate [Ins(4,5)P₂] and Ins(1,4,5)P₃ that retain the cyclohexane scaffold, but lack hydroxyl groups that might be phosphorylated and have phosphate groups replaced with phosphatase-resistant phosphorothioates. An Ins(1,4,5)P₃ analog, 1d-2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothioate, was synthesized from (-)-quebrachitol, and an Ins(4,5)P₂ analog, 1d-1,2,3-trideoxy-myo-inositol 4,5-bisphosphorothioate, was prepared from cyclohexenol. The Ins(1,4,5)P₃ analog was recognized by Ins(1,4,5)P₃ receptor with a binding constant (K_d) of 810 nM, compared with 54 nM for the native ligand Ins(1,4,5)P₃, and was resistant to dephosphorylation by alkaline phosphatase under conditions in which Ins(1,4,5)P₃ is extensively hydrolyzed. Analogs developed in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated by eliciting specific and prolonged activation of the Ins(1,4,5)P₃ receptor.

Full text of DOI:10.1016/j.bmc.2007.10.032

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3419–3427
Deoxygenated phosphorothioate inositol phosphate analogs:
Synthesis, phosphatase stability, and binding affinity
Xiaodan Liu,a Emily C. Moody,b Stephen S. Hechta,b and Shana J. Sturlaa,b,
*
aDepartment of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA
bThe Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA
Received 27 August 2007; revised 4 October 2007; accepted 11 October 2007
Available online 14 October 2007
Abstract—Inositol phosphates, such as 1D-myo-Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], are cellular second messengers with
potential roles in cancer prevention and therapy. It typically is difficult to attribute specific pharmacological activity to a single ino-
sitol phosphate because they are rapidly metabolized by phosphatases and kinases. In this study, we have designed stable analogs of
myo-inositol 4,5-bisphosphate [Ins(4,5)P2] and Ins(1,4,5)P3 that retain the cyclohexane scaffold, but lack hydroxyl groups that might
be phosphorylated and have phosphate groups replaced with phosphatase-resistant phosphorothioates. An Ins(1,4,5)P3 analog, 1D-
2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothioate, was synthesized from (-)-quebrachitol, and an Ins(4,5)P2 analog, 1D-1,2,3-tri-
deoxy-myo-inositol 4,5-bisphosphorothioate, was prepared from cyclohexenol. The Ins(1,4,5)P3 analog was recognized by
Ins(1,4,5)P3 receptor with a binding constant (Kd) of 810 nM, compared with 54 nM for the native ligand Ins(1,4,5)P3, and was resis-
tant to dephosphorylation by alkaline phosphatase under conditions in which Ins(1,4,5)P3 is extensively hydrolyzed. Analogs devel-
oped in this study are potential chemical probes for understanding mechanisms of inositol phosphate actions that may be elucidated
by eliciting specific and prolonged activation of the Ins(1,4,5)P3 receptor.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
rapidly dephosphorylated to lower inositol phosphates
that participate in signal transduction pathways and af-
fect the cell cycle.9 Preclinical studies indicate that the
combination of inositol with InsP6 further enhances che-
mopreventive and anticancer effects and it has been sug-
gested that this combination disproportionates to
Ins(1,4,5)P3, which normalizes the rate of cell division
thus preventing tumor formation. The resulting hypoth-
esis is that inositol phosphates with fewer than six phos-
phate groups, such as the biochemical second messenger
Ins(1,4,5)P3, may be responsible for the biological activ-
ities observed for inositol and InsP6.10–13
Myo-Inositol and its corresponding hexaphosphate
(InsP6, Chart 1) are widely distributed in plants and ani-
mals. Both have been shown to possess cancer preven-
tive and therapeutic activity in vitro and in animal
studies.1–8 In chemoprevention studies, these com-
pounds were effective in reducing tumor number and
size when given simultaneously with or after carcinogen
exposure.4–8 The biochemical mechanisms underlying
the chemopreventive activity of inositol compounds
are not known, but exogenously administered InsP6 is
There have been extensive studies of the roles of inositol
and inositol phosphates in signal transduction and of
the enzymes that catalyze the conversions between dif-
ferent inositol phosphates.14–21 Among the various ino-
sitol phosphates, Ins(1,4,5)P3 is the most understood; it
is established as an important second messenger in-
volved in many signaling pathways. After cleavage from
the membrane-bound phosphatidylinositol trisphos-
phate, Ins(1,4,5)P3 binds to its corresponding receptor
and stimulates the release of stored Ca2+ into the
cytoplasm. This event triggers an array of cellular re-
sponses including glycogen breakdown, muscle contrac-
Abbreviations: Ins, myo-inositol; InsP1, myo-inositol-monophosphate;
InsP2, myo-inositol-bisphosphate; Ins(4,5)P2, 1D-myo-inositol 4,5-bis-
phosphate; Ins(1,4,5)P3, 1D-myo-inositol 1,4,5-trisphosphate; InsP6,
myo-inositol hexaphosphate; Kd, dissociation constant; CID, collision-
induced dissociation; DTT, DL-dithiothreitol; MCPBA, meta-chloro-
perbenzoic acid; AMP, adenosine 20-monophosphate; AMPS,
adenosine 50-O-thiomonophosphate; TEAB, triethylammonium bicar-
bonate; MS, mass spectrometry; pNPP, p-nitrophenyl phosphate.
Keywords: Myo-inositol 1,4,5-trisphosphate; Ins(1,4,5)P3 analog; Bind-
ing affinity; Ins(1,4,5)P3 receptor; Phosphatase-stable analogs.
*
Corresponding author. Tel.: +1 612 626 0496; fax: +1 612 624
0139; e-mail: sturl002@umn.edu
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.032

3420
X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
minor/unessential
2-
OH
OH
OPO3
enhancing effect
OH
2-
2-O3PO
2-O3PO
OPO3
HO
HO
OH
OH
2-
HO
OPO3
1
important H-bond donor
2-
OPO3
2-O3PO
OH
2-
4
2-
5
OPO3
OPO3
1,myo-Inositol
2, InsP6
critical
OH
OH
Figure 1. Previously established structure–activity relationships for
Ins(1,4,5)P3-receptor binding and calcium releasing.
2-
HO
OH
HO
OPO3
2-O3PO
OH
OPO3
2-O3PO
OH
OPO3
2-
2-
dideoxy-Ins(1,4,5)P3,39–41 which retain biological activ-
ities including receptor binding and Ca2+ mobilization.
By contrast, analogs deoxygenated at the 6-position,
such as 1D-2,3,6-trideoxy-Ins(1,4,5)P3 and 1D-6-deoxy-
Ins(1,4,5)P3, displayed significantly lower-binding
affinity to the Ins(1,4,5)P3 receptor.39,42
3, Ins(4,5)P2
4, Ins(1,4,5)P3
2-
OP(S)O2
2-O2(S)PO
OH
2-O2(S)PO
OH
2-
2-
OP(S)O2
OP(S)O2
Deoxygenation at specific positions of the inositol
framework eliminates kinase phosphorylation sites,39,43
and replacement of phosphate groups with phosphoro-
thioates retains key interactions between the molecules
and Ins(1,4,5)P3 receptor, but imparts phosphatase
resistance.44–46 We report here the synthesis of two novel
inositol phosphate analogs, 5 and 6, which on the basis
of these design principles are expected to be enzyme-sta-
ble receptor-binding analogs. An evaluation of their
chemical and biochemical properties, including binding
affinities for the Ins(1,4,5)P3 receptor and resistance to
enzymatic hydrolysis, is described.
5
6
Chart 1. Structures of D-myo-inositol (1), D-myo-inositol hexaphos-
phate (2), 1D-myo-inositol 4,5-bisphosphate (3), and 1D-myo-inositol
1,4,5-trisphosphate (4), 1D-1,2,3-trideoxy-myo-inositol 4,5-bisphosp-
horothioate (5), 1D-2,3-dideoxy-myo-inositol 1,4,5-trisphosphorothio-
ate (6).
tion, and the release of neurotransmitters.22–24
Ins(1,4,5)P3 is rapidly deactivated by stepwise dephos-
phorylation to Ins(1,4)P2, then to Ins-4-P1 and finally
myo-inositol.25,26 Alternatively, phosphorylation by
Ins(1,4,5)P3 3-kinase produces Ins(1,3,4,5)P4.27,28 The
metabolic instability and rapid clearance of Ins(1,4,5)P3
make it difficult to probe directly the role of Ins(1,4,5)P3
in biological events, such as those that may underlie
mechanisms of inositol and InsP6-mediated cancer che-
moprevention. Thus, the overall benefits of Ins(1,4,5)P3
in cancer prevention and therapy remain controversial.
2. Results
2.1. Chemistry
Ins(4,5)P2 analog 5 is a deoxygenated analog bearing two
phosphorothioate groups in the 4- and 5-positions and
retaining the 6-hydroxyl group, which is critical for the
binding of Ins(1,4,5)P3 to its receptor (Fig. 1 and Chart
1).36,37 While 5 is a putative Ins(4,5)P2 analog, its synthe-
sis was pursued primarily as a synthetic model and bio-
chemical control substrate for investigating the
Ins(1,4,5)P3 analog 6. Initially, we were interested in
establishing chemistry needed to prepare and purify
trans-vicinal phosphorothioates with a neighboring free
alcohol. The synthesis of 5 is outlined in Scheme 1. Race-
mic diol 8 was prepared from cyclohexene-2-ol by the
method of Savelli and co-workers.47 First, cyclohexene-
2-ol was protected as the benzyl ether 7 and subjected to
trans-dihydroxylation using MCPBA (Scheme 1). The
1H NMR analysis of the resulting white solid matched
the published spectrum48,49 and indicated that the result-
ing diol was obtained as a 1:1 mixture of diastereomers. A
procedure for installation of the phosphorothioate
groups was adapted from a phosphoramidite-based
method reported by Potter and co-workers for the prepa-
ration of DL-myo-Ins(1,4,5)P3.46 Diol 8 was treated with
excess bis(2-cyanoethyl)-N,N-diisopropyl phosphorami-
dite50 and the resulting crude phosphate was allowed to
react with sulfur to provide protected phosphorothioate
ester 9. Because 9 is the final compound bearing a chromo-
Inositol phosphate chemical analogs resistant to enzy-
matic degradation have potential for enhanced chemo-
preventive activity and mechanistic information
obtained could serve as a starting point for medicinally
focused approaches to develop improved agents for che-
moprevention.29–31 Furthermore, these compounds
could serve as synthetic probes useful for investigating
the role of inositol phosphates in biochemical signaling
pathways that cannot be tested using current methods.
As a step toward these goals, the primary aim of this
study was to develop an enzyme-stable Ins(1,4,5)P3 ana-
log. The structural basis for its design involved struc-
ture–activity relationships previously established for
Ins(1,4,5)P3 receptor binding and Ca2+ release activity
(Fig. 1).23,32–37 Much of the information known regard-
ing receptor-binding specificity has been garnered from
studies involving synthetic deoxy-analogs. Strategies
for the syntheses of these compounds generally involve
lengthy (i.e., of the order of 7–15 steps) modification
of oxygenated carbohydrate precursors, including queb-
rachitol and D-galactopyranose.38–40 The influence of
deoxygenation at the 2- and 3-positions of the inositol
framework was examined with 1D-3-deoxy and 1D-2,3-

X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
3421
tection reaction and followed by extensive deprotection.
While we found the reported strategy to be a reliable
and reproducible method to obtain 10 on an approxi-
mately half-gram scale, drawbacks included its lengthi-
ness, the need to separate an intermediate mixture of
diastereomers (previous reports indicate that these inter-
mediates are separable by column chromatography, but
in our hands preparatory HPLC was required for satis-
factory separation), and the stench/toxicity (thiol/tin)
associated with deoxygenation reagents. Minor modifi-
cations that were made to published methods, including
a procedure for HPLC separation of precursors to 10,
are indicated in the experimental section. Triol 10 was
converted to the corresponding phosphates by treatment
with bis(2-cyanoethyl)-N,N-diisopropyl phosphorami-
dite, followed by reaction with sulfur, and deprotection
(Scheme 2). The final product was purified by anion ex-
change chromatography and analysis by HRMS dis-
played a molecular ion of 434.8963, consistent with the
presence of three phosphorothioate moieties. Analysis
by 31P NMR displayed three resonances (46.58, 48.80,
and 49.45), also consistent with the trisphosphorothio-
ate structure. No 31P resonances were observed in the
7.71–6.19 region,39,41 which would be expected for phos-
phate groups, and no mass corresponding to phosphate
(rather than phosphorothioate) groups were observed.
The synthesis involved 13 steps and the separation of
a 1:1 mixture of isomers; 6 was obtained in 4% overall
yield.
i
ii, iii
HO
OBn
OBn
OH
7
8
iv
5
(RO)2(S)PO
OBn
OP(S)(OR)2
, R = CH CH CN
9
2
2
Scheme 1. Synthesis of analog 5. Reagents: (i) MCPBA/H2SO4; (ii)
tetrazole, bis(2-cyanoethyl)-N,N-diisopropyl phosphoramidite; (iii)
sulfur, pyridine; (iv) Na, NH3.
phore in the synthetic sequence, and considering difficul-
ties encountered in the subsequent purification, we found
it important to carefully purify the phosphorothioate es-
ter by preparative reverse phase HPLC (UV detection).
By this method the corresponding diastereomers could
be resolved and separated if desired, but were routinely
isolated as a mixture. Both the cyanoethyl and benzyl pro-
tecting groups were removed under basic reducing condi-
tions (Na/NH3), generating 5. This material was isolated
as the triethylammonium salt after a short DEAE-Sepha-
dex anion exchange column, eluting with an increasing
gradient of triethylammonium bicarbonate (TEAB),
and visualized by Ellman’s reagent.51
Analog 6 was purified and isolated as the triethylammo-
nium salt using the same procedures employed in the
preparation of 5, as discussed above. The presence of
6 in chromatography fractions was determined using
Ellman’s reagent, which is used routinely as a sulfhydryl
indicator (i.e., a visible yellow color is produced). Stor-
The synthesis of 6 was accomplished using analogous
methods to those described above for the preparation
of 5 from 10 (Scheme 2). Triol 10 was prepared in enan-
tiomericaly pure form from (ꢀ)-quebrachitol using an
11-step linear sequence previously reported by Kozi-
kowski et al.39,41 Kozikowski’s overall strategy for the
preparation of 10 involves a series of selective protection
steps in which selected pairs of vicinal hydroxyl groups
are protected by exploiting their cis/trans and axial/
equatorial relationships. Deoxygenation reactions are
carried out on remaining free hydroxyl groups using
Barton-type deoxygenation conditions. Each of two
deoxygenation processes is preceded by a selective pro-
age of aqueous
6 solutions at low temperature
(ꢀ20 ꢁC) for extended time (months) resulted in its par-
tial decomposition, as indicated by MS analysis of aged
samples in which a new peak with a negative ion m/z 433
was observed. This decomposition product could be re-
moved by repurification on a DEAE-Sephadex anion
exchange column. Furthermore, it was found that when
treated with DL-dithiothreitol (DTT), a reagent useful
for reducing disulfide bonds, the impurity with m/z 433
was completely re-converted to 6. This relationship
was confirmed by MS analysis as indicated in Figure
2. These data suggest that the impurity resulted from
the formation of a disulfide bond between vicinal
phosphorothioates. The corresponding equilibrium
relationships and proposed structure 12 are illustrated
in Scheme 3, and this material appeared to be stable
to alkaline phosphatase-mediated hydrolysis (Fig. 4c).
OH
OH
O
OH
OH
H3C
HO
11 steps
i., ii.
HO
OBn
OH
OH
(-)-quebrachitol
10
OP(S)(OR)2
OBn
2.2. Biochemistry
iii.
6
(RO)2(S)PO
A competition-binding assay was carried out to deter-
mine the binding affinities of 5 and 6 with respect to
the Ins(1,4,5)P3 receptor. Equilibrium binding was
determined relative to [3H]Ins(1,4,5)P3 using protein
extract prepared from bovine adrenal cortex.52
Ins(1,4,5)P3, 5, and 6 replaced receptor-bound [3H]-
Ins(1,4,5)P3 in a concentration-dependent manner as
OP(S)(OR)2
11, R = CH2CH2CN
Scheme 2. Synthesis of the analog 6. Reagents: (i) tetrazole, bis(2-
cyanoethyl)-N,N-diisopropyl phosphoramidite; (ii) sulfur, pyridine;
(iii) Na, NH3.

3422
X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
Figure 3. Effects of 5, 6, and Ins(1,4,5)P3 on equilibrium competition
binding of [3H]Ins(1,4,5)P3 to bovine adrenal cortical protein extracts.
tion mixture were used to guide the anticipated product
ions that would result from hydrolysis of 6, for which
authentic hydrolysis co-injection standards were not
available. Thus, the products of the Ins(1,4,5)P3 incuba-
tion mixture were selected for collision-induced dissoci-
ation (CID), and fragment ions for Ins(1,4,5)P3 and
InsP2 of m/z 321 and 241, respectively, were observed
(Fig. 4a). These fragment ions correspond to loss of
orthophosphoric acid (H3PO4). The fragment ion gener-
ated from InsP1 (m/z 179) corresponds to phosphate
loss, and water loss for inositol (m/z 161). Data from
the LC/MS/MS analysis of incubates of 6 with alkaline
phosphatase under identical conditions as the positive
control are shown in Figure 4b. The m/z values for the
parent and fragment [MꢀH]ꢀ ions for 6 are 435 and
401, respectively. This fragmentation pattern is consis-
tent with loss of SH2. A product resulting from hydroly-
sis of 6 is expected to display m/z 339 and a fragment ion
m/z 305, corresponding to loss of one phosphorothioate
group. As indicated in Figure 4b, there is no evidence
for the formation of this product under the phosphatase
reaction conditions. As a further conformation of the
validity of the selected fragment, evidence for the puta-
tive hydrolyzed product (i.e., observation of a new MS
peak with m/z 339 that fragments to m/z 305) was ob-
served by carrying out the same MS analysis of 6 after
prolonged storage (several months). Furthermore, the
same behavior (i.e., the formation of hydrolysis prod-
ucts) was observed for stored Ins(1,4,5)P3. Finally, to
determine whether 6 inhibits alkaline phosphatase, en-
zyme activity was monitored using a colorimetric assay
with p-nitrophenyl phosphate (pNPP) as a substrate.53
Phosphatase activity was determined at 37 ꢁC by mea-
suring the increase in absorbance at 405 nm that accom-
panies the hydrolysis of pNPP, and these data indicated
Figure 2. Full-scan MS of 12 after storage of 6 at ꢀ20 ꢁC for a month
(a, m/z 432.9); and full-scan MS of the same material after treatment
with excess of DTT (b, m/z 434.9).
illustrated in Figure 3. The strongest binding was dem-
onstrated by Ins(1,4,5)P3 (Kd 54 nM). The binding con-
stant for 6 was in the high nanomolar range (Kd
810 nM), while binding of 5 was over three orders of
magnitude higher. The displacing activities of adenosine
20-monophosphate (AMP) and adenosine 50-O-thio-
monophosphate (AMPS) were also investigated to verify
the specificity for Ins(1,4,5)P3 and the analogs; neither
demonstrated a measurable binding affinity for the
receptor (data not shown).
The resistance of 6 to enzymatic degradation was ad-
dressed by reacting with alkaline phosphatase and
analyzing the resulting mixture by LC/MS/MS using
an electrospray ionization source (Fig. 4b). As a positive
control, Ins(1,4,5)P3 (m/z 419) was extensively
hydrolyzed by alkaline phosphatase (30 min, 37 ꢁC),
producing myo-inositol-bisphosphate (InsP2), myo-ino-
sitol-monophosphate (InsP1), and inositol (Fig. 4a) with
negatively charged [MꢀH]ꢀ ions m/z 339, 259, and 179,
respectively. The identities of the inositol phosphates
and inositol were verified by co-injection with authentic
standards. To evaluate the stability of 6, fragmentation
data obtained for the products of Ins(1,4,5)P3 incuba-
OP(S)(OH)2
OH
OH
OP(S)(OH)2
OP(S)(OH)2
O2
hydrolysis
O
HO O
P
HO O
P
O
OH
OH
OP(SH)(OH)
O
(HO)(HS)PO
OH
(HO)2(S)PO
O
O
OH
O
OH
DTT
S
S
OP(S)(OH)2
P
O
P
O
S
S
6
12
13
Scheme 3. Proposed decomposition pathway observed for 6 and proposed hydrolysis pathway. DTT, DL-dithiothreitol.

X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
3423
Intensity
6000
Intensity
4000
a
4000
2000
0
6000
2000
161
179
Ins; SRM: m/z 179
0
4000
4000
2000
0
6000
2000
InsP1; SRM: m/z 259
0
4000
4000
2000
2000
241
321
InsP2; SRM: m/z 339
0
4000
0
6000
4000
2000
2000
0
Ins(1,4,5)P3; SRM: m/z 419
0
0
5
5
5
10
10
10
15
20
20
20
25
25
25
30
30
30
0
5
10
15
20
25
30
1200
800
1000
600
b
c
400
305
401
SRM: m/z 339
200
1000
1200
800
600
400
6; SRM: m/z 435
200
0
0
0
15
0
5
10
15
20
25
30
2000
1000
1000
600
216
367
13; SRM: m/z 337
200
1000
2000
1000
0
600
12; SRM: m/z 433
200
0
0
15
0
5
10
15
20
25
30
Time (min)
Time (min)
Figure 4. SRM chromatograms of reactions of Ins(1,4,5)P3, 6, or 12 with alkaline phosphatase. (a) Ins(1,4,5)P3 control (left, unreacted Ins(1,4,5)P3;
right, Ins(1,4,5)P3 hydrolyzed to InsP2, InsP1, and inositol), a small amount of InsP1 is detected with rt 5.77 min. (b) 6 incubated with alkaline
phosphatase. (Left, unreacted 6; right, sample after incubation indicates that starting compound remains intact, and there is no evidence for a
hydrolysis product.) (c) 12 incubated with alkaline phosphatase. (Left, unreacted 12; right, sample after incubation indicates that starting compound
remains intact, and there is no evidence for 13.) Monitored SRM transitions are indicated on the right for each channel.
that 6 does not inhibit alkaline phosphatase activity at
high micromolar range (data not shown).
the 6-hydroxyl group is important for receptor-binding
and Ca2+ release,37,42 and it is therefore present in 6.
Inositol phosphate multikinases catalyze phosphoryla-
tion of the 3- and 6-hydroxyl groups of Ins(1,4,5)P3,
but in cells 3-phosphorylation is the dominant prod-
uct.58 Although known enzyme profiles indicate that 6
is expected to be kinase-stable, further studies are re-
quired to explicitly address the susceptibility toward
phosphorylation by specific kinases. As a preliminary
test of enzyme stability carried out in this study, we used
alkaline phosphatase, which efficiently hydrolyzes
Ins(1,4,5)P3. In cells, Ins(1,4,5)P3 5-phosphatase is the
specific enzyme that hydrolyzes Ins(1,4,5)P3, but it has
low substrate-specificity compared with Ins(1,4,5)P3 3-
kinase.59 Many synthetic inositol phosphates are high-
Ki Ins(1,4,5)P3 5-phosphatase inhibitors,39,41,55,59 such
as myo-inositol 1,4,5-trisphosphorothioate, suggesting
that 6 is a potential Ins(1,4,5)P3 5-phosphatase inhibi-
tor. The current studies indicate that 6 is stable to gen-
eral enzyme-mediated hydrolysis, but further studies
with specific kinases and phosphatases and in the com-
plex environment of the cell are required to determine
the biological stability of 6.
3. Discussion
The design of the inositol phosphate analogs described in
this study relied on known structure–activity relation-
ships for Ins(1,4,5)P3 (Fig. 1), indicating that vicinal
4,5-phosphate groups are required for Ins(1,4,5)P3
Ca2+-releasing activity, and that the 1-phosphate acts as
an enhancer.33,54 Replacing Ins(1,4,5)P3’s phosphate
groups by phosphorothioate groups has been found to
provide resistance to phosphatase-catalyzed hydrolysis
without greatly affecting binding properties,46,55 but the
analog used in the study may still be phosphorylated at
the 2- and 3-positions. Therefore, deoxygenation at these
positions was a strategy utilized to block phosphoryla-
tion. 1D-2,3-dideoxy-Ins(1,4,5)P3 maintains a relatively
high affinity for the Ins(1,4,5)P3 receptor and is a full ago-
nist, releasing Ca2+ from permeabilized SH-SY5Y cells.41
In human cells, phosphorylation of Ins(1,4,5)P3 has been
observed at the 3-position,56 and the substrate of the
known 6-kinase is not Ins(1,4,5)P3, but Ins(1,3,4)P3,
which is formed by the hydrolysis of Ins(1,3,4,5)P4.57 In
addition, 1D-6-deoxy-myo-Ins(1,4,5)P3 was a full agonist,
but is 70-fold less potent than Ins(1,4,5)P3, indicating that
Thiophosphate 6 has a significant binding affinity
(810 nM) for the Ins(1,4,5)P3-receptor despite its struc-

3424
X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
tural modification. However, the presence of only the
4,5-phosphorothioates in 5 results in a dramatic de-
crease in binding affinity, consistent with the specificity
of the ligand–receptor interaction. The X-ray structure
of the Ins(1,4,5)P3-binding domain of mouse type-I
Ins(1,4,5)P3 receptor with bound Ins(1,4,5)P3 indicates
interactions of specific Arg and Lys residues with the
three phosphate groups,60 and replacement by phosp-
horothioates therefore is expected to influence the
interaction. Compared to other phosphatase-stable
Ins(1,4,5)P3 analogs, including 1L-(+)-myo-Ins(1,4,5)P3,55
5. Experimental
5.1. General details
THF was obtained anhydrous in sure-seal bottles from
Aldrich and used without further purification. Flash
chromatography was performed using 70–230 mesh Sil-
ica gel purchased from Aldrich Chemical Co. (Milwau-
kee, WI). Silica gel 60 mesh TLC plates were obtained
from Merck (Lawrence, KS). Tetrazole was obtained
from Glen Research (Sterling, VI) as a saturated solu-
tion in anhydrous acetonitrile and used as provided.
Ammonia was used from a lecture bottle, 99.9% pure,
obtained from Aldrich. DEAE-Sephadex A-25 anion ex-
change resin was obtained from Sigma Chemical Co.
(St. Louis, MO) and was packed in a glass column with
water. Alkaline phosphatase was supplied from Sigma in
a 50% glycerol solution with 5 mM MgCl2 and 0.1 mM
ZnCl2; activity was assessed at 4900 U/mg protein with
11.2 mg protein/mL. Amicon Centricon YM-3 centri-
fuge filters were obtained from Millipore and were
rinsed with 2 mL H2O for 3 h at 7000g prior to use.
[3H]Ins(1,4,5)P3 was obtained from American Radiola-
beled Chemicals (St. Louis, MO) and used without fur-
ther purification. Radioactivity counting was done using
Eco Scint liquid scintillant from National Diagnostics
(Atlanta, GA). The phosphoramidite coupling reagent,
1D-6-deoxy-Ins(1,4,5)P3S3, and L-ch-Ins(2,3,5)P3S3,61
6
retains a notably high affinity for the Ins(1,4,5)P3
receptor. Three closely related Ins(1,4,5)P3-receptor
subtypes have been characterized and they are differen-
tially expressed and involved in physiological functions
such as regenerating Ca2+ signals.16 The previously re-
ported analog 2-deoxy-1,4,5-Ins(1,4,5)P3 had a slightly
greater affinity for type 2 and type 3 receptors, whereas
3-deoxy-1,4,5-Ins(1,4,5)P3 showed greater selectivity for
type 3 receptor.62 To define specific ligand–receptor-
binding interactions for 6, further studies are needed
to determine the selectivity of
Ins(1,4,5)P3 receptors.
6
for different
Techniques to assess the stabilities of inositol phos-
phates and analogs have included NMR, that is, mon-
itoring 31P signals for enzymatic reactions performed
in an NMR tube,63 radioflow HPLC after incubation
with [3H]Ins(1,4,5)P3,37 or using a functional assay
(i.e., measuring Ca2+ mobilization).22 The requirement
for large amounts of material and low sensitivity and
accuracy are drawbacks for NMR analysis, and prep-
arations of isotope-labeled analogs and tedious sample
preparation are generally required for the radioflow
methods. Finally, functional assays do not necessarily
transfer for the analysis of structural analogs. To
overcome limitations in these methods, we developed
a new LC/MS/MS analytical method for inositol phos-
phate analog-enzyme incubations. This method is sen-
sitive and fast, and sample preparation is easy. This
method was used here to investigate analog stability
toward phosphatase activity, but it has potential
applications for further studies of inositol phosphate
kinase activity and analysis of complex inositol phos-
phate mixtures.64
bis(2-cyanoethyl)-N,N-diisopropyl
phosphoramidite,
1
was prepared as previously described50 and the H and
31P NMR of the material obtained matched the pub-
lished spectra.65 1D-6-O-Benzyl-1,2,3-trideoxy-myo-ino-
sitol 8 was prepared by the method of Savelli and co-
workers47 and the 1H NMR matched the published
spectrum.48,49 Ellman’s reagent was prepared and used
to detect phosphorothioate compounds in fractions after
anion exchange chromatography as described
previously.51
Preparative HPLC was carried out in all systems
using two Waters Associates pumps (Waters Division,
Millipore, Milford, MA), with a UV detector at
254 nm (Shimadzu model SPD-10A). System 1: at a flow
rate of 5 mL/min, a Phenomenex Luna 5 lm C18
250 · 21.2 mm column was eluted isocratically for
5 min with 5% CH3OH/95% aqueous 40 mM NH4OAc,
then with a gradient from 5% to 100% CH3OH over a
course of 20 min. An isocratic flow of 100% CH3OH
was then maintained. System 2: at a flow rate of 5 mL/
min, a Phenomenex Luna 5 lm C18 250 · 21.2 mm col-
umn was eluted isocratically with 9:1 MeOH/H2O.
4. Conclusion
In conclusion, we have synthesized 5 and 6, two novel
deoxygenated phosphorothioate analogs of the inositol
phosphates Ins(1,4,5)P3 and Ins(4,5)P2. Phosphorothio-
ate 6 is stable to enzymatic hydrolysis, but a tendency of
the vicinal phosphorothioates to form disulfide bonds
was observed indicating limitations in its chemical sta-
bility. This analog specifically binds to Ins(1,4,5)P3
receptor with a Kd of 810 nM, which compares favor-
ably with previously reported Ins(1,4,5)P3 phosphoro-
thioate analogs.55,61 Future studies will be aimed at
characterizing cellular responses to 6 and using it to
probe the role of Ins(1,4,5)P3 and its receptor in physio-
logical processes.
Direct injection mass spectrometry analysis was per-
formed for compound characterization on an Agilent
1100 LC/MSD ion trap instrument (Agilent Technolo-
gies, Inc., Wilmington, DE) operating in negative-ion
mode.
5.2. Synthesis of compounds 5–11
5.2.1. Benzyl 2-cyclohexen-1-yl ether (7). 2-Cyclohexen-
1-ol (2 mL, 20 mmol) was added to a slurry of NaH
(1 g, 42 mmol) in THF at 25 ꢁC under an atmosphere
of nitrogen. After 1 h, benzyl chloride (6 mL, 52 mmol)

X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
3425
was added and the mixture was stirred for 12 h. Excess
NaH was quenched by addition of MeOH. Solvents
were removed by rotary evaporation and the resulting
residue was dissolved in diethyl ether (80 mL). This or-
ganic solution was transferred to a separatory funnel,
extracted three times with water, and dried over
Na2SO4. The solution was filtered and solvents removed
by rotary evaporation. The residue was concentrated
under high vacuum to remove any methyl benzyl ether
and then purified by column chromatography (hexanes,
followed by Ethyl Acetate/hexanes 1:9 v/v) to provide
1.7 g (45% yield) of the title compound as a colorless
0.75 mM TEAB, finishing with 1 M TEAB. The pres-
ence of phosphorothioate was detected by Ellman’s re-
agent in column fractions. Solvent was removed and
the title compound isolated as its triethylammonium salt
1
(91 mg, 0.11 mmol, 7.2% yield). H NMR (300 MHz,
D2O, mixture of diastereomers) d 1.35–1.60 (m, 4H),
1.70–1.80 (m, 1H); 1.95–2.05 (m, 1H); 3.40–3.50 (m,
1H); 3.85–4.00 (m, 2H); 4.15 (m, 0.5H); 4.35 (m,
0.5H). 31P NMR (300 MHz, D2O)
d
48.3 (d,
J = 146 Hz), 46.7 (d, J = 230 Hz); HRMS calculated
for [C6H13O7P2S2] 323.2407. Found: 322.9584.
1
oil. H NMR matched the published spectrum.66
5.2.4. 1D-6-O-Benzyl-2,3-dideoxy-myo-inositol (10). The
title compound was prepared by the method of Kozi-
kowski et al. with minor modifications as follows.39
Isomers of 1D-4,5-di-O-benzoyl-6-O-benzyl-3-deoxy-
1,2-O-isopropylidine-myo-inositol were separated by
preparatory HPLC, using System 2, and 1D-4,5-di-O-
benzoyl-6-O-benzyl-3-deoxy-1,2-O-isopropylidene-myo-
inositol (35–36 min) eluted after 1D-5,6-di-O-benzoyl-4-
O-benzyl-3-deoxy-1,2-O-isopropylidene-myo-inositol
5.2.2. 1D-6-O-Benzyl-1,2,3-trideoxy-myo-inositol (8). To
a stirred dispersion of 7 (0.9 g, 5 mmol) in water
(30 mL) was added MCPBA in small portions over a
period of 5–10 min at 0 ꢁC. The mixture was warmed
to 25 ꢁC and stirred for 4 h. To the mixture then was
added H2SO4 (1 mL of a 10% solution) and stirring
was continued for 12 h. NaOH (1 M solution) was
added to the reaction mixture to adjust the pH to 7. This
aqueous solution was saturated with NaCl and extracted
three times with ethyl acetate. The combined organic
fractions were dried over Na2SO4, filtered, and concen-
trated. Purification by flash chromatography (ethyl ace-
tate) afforded the title compound as a white solid
(29.5–30.5 min).
1D-4,5-Di-O-benzoyl-6-O-benzyl-3-
deoxy-1,2-O-isopropylidine-myo-inositol (1.13 g, 2.25
mmol) was dissolved in methanol (50 mL) and p-tolu-
enesulfonic acid (140 mg, 0.736 mmol) was added. The
reaction mixture was allowed to stir at 23 ꢁC. After
3.5 h, water (approx. 5 mL) was added to the reaction
mixture, and the resulting solution was extracted with
CH2Cl2 (3· 20 mL). The combined organic solutions
were washed sequentially with saturated sodium bicar-
bonate and brine, dried over Na2SO4, and solvent was
evaporated to yield 0.53 g of 10 (2.21 mmol, 98%) as a
1
(330 mg, 31% yield). H NMR matched the published
spectrum.48,49
5.2.3. 1D-1,2,3-Trideoxy-myo-inositol 4,5-bisphosphoro-
thioate (5). 1D-6-O-Benzyl-1,2,3-trideoxy-myo-inositol 8
(330 mg, 1.5 mmol) was dissolved in an acetonitrile
solution of tetrazole (40 mL, 18 mmol) under a N2
atmosphere. Bis(2-cyanoethyl)-N,N-diisopropyl phos-
phoramidite50 (2.3 g, 8.48 mmol) was added. The reac-
tion mixture was stirred at 23 ꢁC under N2 for 2 h,
and turned cloudy. Solvent was removed under vacuum
and the resulting residue was re-dissolved in pyridine
(10 mL). Sulfur (460 mg, 14.3 mmol) was added and
the reaction mixture was stirred for 24 h. Solvent was
evaporated and crude product was dissolved in MeOH
(ꢁ3 mL) and filtered through a nylon disk. The solution
was concentrated and purified by flash column chroma-
tography with a gradient from 25:75 EtOAc/hexanes to
50:50 EtOAc/hexanes. Subsequent purification by
HPLC from multiple injections using System 1 yielded
299 mg 9 (0.48 mmol, 32%). Retention time was at 47
and 52 min (ꢁ1:3 mixture of diastereomers). This mate-
rial was dissolved in dry dioxane (6 mL) and a portion
(2 mL) of this solution was added to liquid ammonia,
of which approximately 50 mL had been condensed in
a three-necked flask fitted with a dry ice/acetone-filled
condenser. To this mixture was added Na (290 mg,
13 mmol) in small pieces producing a deep blue solution.
The mixture was stirred at 25 ꢁC for 10 min. The reac-
tion mixture was quenched by adding ethanol (4 mL).
NH3 was removed under a flow of N2 over the course
of ꢁ2 h. The remaining solution was concentrated under
vacuum and crude residue was purified by anion ex-
change chromatography on DEAE-Sephadex A-25 resin
with a stepwise gradient from water to 0.25 M triethyl-
ammonium bicarbonate (TEAB), 0.5 mM TEAB,
1
white solid. H NMR matched published data.39
5.2.5. 1D-6-O-Benzyl-2,3-dideoxy-myo-inositol 1,4,5-
tris[di-(2-cyanoethyl)phosphorothioate] (11). 1D-6-O-Ben-
zyl-2,3-dideoxy-myo-inositol 10 (52 mg, 0.218 mmol)
was dissolved in an acetonitrile solution of tetrazole
(8.5 mL, 3.825 mmol) under a N2 atmosphere. A solu-
tion of bis(2-cyanoethyl)-N,N-diisopropyl phospho-
ramidite50 (575 mg, 2.12 mmol) in CH2Cl2 (2.5 mL)
was added dropwise. The reaction mixture was stirred
at 23 ꢁC under N2 for 2.5 h. Solvent was removed under
vacuum and the resulting residue was re-dissolved in
pyridine (2.5 mL). Sulfur (107 mg, 3.34 mmol) was
added and the reaction mixture was stirred for 24 h. Sol-
vent was evaporated and crude product was purified by
flash column chromatography with a gradient from
20:80 EtOAc/hexanes to EtOAc. Subsequent purifica-
tion by HPLC using System 1 yielded 108 mg 11 (rt
28.5 min) (0.128 mmol, 57%). TLC Rf 0.33 (2:1 ethyl
1
acetate/hexanes). H NMR (300 MHz, CDCl3) d 7.26–
7.43 (m, 5H), 4.85 (s, 2H), 4.66 (m, 1H), 4.50 (m, 2H),
4.29–4.40 (m, 8H), 4.19 (m, 2H), 4.10 (m, 2H), 3.85
(m, 2H), 3.65 (dd, J = 8.7 Hz, 1H), 2.71 (m, 8H), 2.22–
2.44 (m, 4H), 2.15 (m, 1H), 1.65 (m, 2H), 1.28 (m,
3H), 0.85 (m, 1H); 31P NMR (300 MHz, CDCl3) d
66.26,
67.13,
67.96;
HRMS
calculated
for
[C31H39N6O10P3S3] 844.1103. Found: 844.1113.
5.2.6. 1D-2,3-Dideoxy-myo-inositol 1,4,5-trisphosphoro-
thioate (6). A 250 mL three-necked round bottom flask
fitted with a condenser was flushed with N2 and cooled

3426
X. Liu et al. / Bioorg. Med. Chem. 16 (2008) 3419–3427
to ꢀ78 ꢁC. Ammonia (50 mL) was condensed into the
flask at ꢀ78 ꢁC. A solution of 1-D-6-O-benzyl-2,3-dide-
oxy-myo-inositol 1,4,5-tris[di-(2-cyanoethyl)phosphoro-
thioate] 11 (21 g, 25 mmol) in anhydrous dioxane
(3 mL) was added dropwise while the solution was stir-
red. Na, stored in mineral oil, was slowly cut into small
pieces, rinsed with Et2O, and immediately added to the
reaction mixture. After 1–2 min, a color change to dark
blue was observed and this solution was stirred at
ꢀ78 ꢁC for 10 min. The reaction was quenched by drop-
wise addition of MeOH (approx. 3 mL) and solvent was
evaporated by warming to 23 ꢁC under a N2 purge.
Crude residue was purified by anion exchange chroma-
tography on DEAE-Sephadex A-25 resin with a step-
wise gradient starting with 0.3 mM triethylammonium
bicarbonate (TEAB) to 0.7 mM TEAB. The presence
of phosphorothioate in column fractions was detected
by Ellman’s reagent in column fractions, yielding
tion times and molecular weights observed for com-
pounds of interest are as follows: Ins(1,4,5)P3,
17.91 min, [Mꢀ1]ꢀ 419, Inositol, 4.44 min, [Mꢀ1]ꢀ
179; InsP1, 5.77 min, [Mꢀ1]ꢀ 259; InsP2, 16.42 min,
[Mꢀ1]ꢀ 339; 6, 20.36 min, [Mꢀ1]ꢀ 435; hydrolyzed 6,
16.79 min, [Mꢀ1]ꢀ 339; 12, 15.93 min, [Mꢀ1]ꢀ 433;
13, 6.34 min, [Mꢀ1]ꢀ 337. Negative ESI-MS/MS was
performed using the following parameters: spray voltage
5.0 kV; sheath gas pressure, 31; capillary temperature,
330 ꢁC; collision energy, 19 V; scan width, 0.3 amu; scan
time, 0.25 s; Q1 peak width, 0.7 amu; Q3 peak width,
0.7 amu; Q2 gas pressure, 1.5 mTorr; source CID,
ꢀ16 V; and tube lens offset, 59–69 V; MS/MS data were
acquired and processed by Xcaliber software version 1.4
(Thermo Electron).
Acknowledgments
1
5.45 mg 6 isolated (32%) as a yellowish white solid. H
NMR (D2O) d 4.21 (m, 2H), 3.63 (m, 1H), 2.21 (m,
2H), 1.47 (dd, J = 9.0, 2H); 31P NMR (D2O) d 46.6,
48.8, 49.4; HRMS calculated for [C6 H14 O10 P3 S3]:
434.8962. Found: 434.8963.
We acknowledge the American Cancer Society Bowman
Research Fund for support of this work, and thank Dr.
Peter W. Villalta for expert assistance with mass
spectrometry.
5.3. Binding assay
References and notes
Bovine adrenal cortical protein extract was prepared as
previously described.52 Protein concentration was deter-
mined to be 17.3 mg/mL by Bradford Assay.67 The bind-
ing assay was performed using the method of Challiss
et al.52 Samples contained protein solution (30 lL,
0.519 mg), Tris/HCl/EDTA buffer (30 lL, 100 mM
Tris/4 mM EDTA, pH 8.0), [3H]Ins(1,4,5)P3 (30 lL
aqueous solution, approx. 7000 dpm), and 30 lL aque-
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every 5 min through a 45-min incubation period at
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Whatman GF/B filters and washed 4· 2.5 mL with ice
cold buffer (25 mM Tris/HCl/5 mM NaHCO3/1 mM
EDTA, pH 8.0). Non-specific binding was determined
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