Synthesis of three enantiomeric pairs of scyllo-inositol phosphate and molecular interactions between all possible regioisomers of scyllo-inositol phosphate and inositol 1,4,5-trisphosphate 3-kinase

Article abstract of DOI:10.1016/S0960-894X(03)00629-2

scyllo-Inositol phosphates, which are among the stereoisomers of myo-inositol phosphate, can have 15 possible regioisomers including three enantiomeric pairs: scyllo-I(1,2)P₂, scyllo-I(1,2,4)P₃, scyllo-I(1,2,3,4)P₄. We her

Full text of DOI:10.1016/S0960-894X(03)00629-2

Bioorganic & Medicinal Chemistry Letters 13 (2003) 2981–2984
Synthesis of Three Enantiomeric Pairs of scyllo-Inositol Phosphate
and Molecular Interactions Between All Possible Regioisomers of
scyllo-Inositol Phosphate and Inositol 1,4,5-Trisphosphate 3-Kinase
Yong-Uk Kwon,a Jungkyun Im,a Gildon Choi,b Young-Soo Kim,a
Kwan Yong Choib and Sung-Kee Chunga,*
aDepartment of Chemistry, Division of Molecular and Life Sciences, Pohang University of Science & Technology,
Pohang 790-784, South Korea
bNational Research Laboratory of Protein Folding and Engineering, Division of Molecular and Life Sciences,
Pohang University of Science & Technology, Pohang 790-784, South Korea
Received 6 February 2003; accepted 21 March 2003
Abstract—scyllo-Inositol phosphates, which are among the stereoisomers of myo-inositol phosphate, can have 15 possible regio-
isomers including three enantiomeric pairs: scyllo-I(1,2)P2, scyllo-I(1,2,4)P3, scyllo-I(1,2,3,4)P4. We herein describe the facile syn-
thetic routes to the three enantiomeric pairs of scyllo-inositol phosphate and the molecular interactions between 15 regioisomers of
scyllo-inositol phosphate and inositol 1,4,5-trisphosphate 3-kinase. Geometry of the enzyme binding site is discussed.
# 2003 Elsevier Ltd. All rights reserved.
d-myo-Inositol 1,4,5-trisphosphate [I(1,4,5)P3], which is
generated from the phospholipase C catalyzed cleavage
of membrane bound phosphatidylinositol bisphosphate
(PIP2), plays a pivotal role as a second messenger by
inducing Ca2+ release from the intracellular storage.1 In
addition, nuclear inositol phosphates were also reported
to control mRNA export and transcription, thus sug-
gesting that activation of IPn signaling might regulate
gene expression as well.2 I(1,4,5)P3 is metabolized by
The catalytic domain of the enzyme has been mapped to
be in the C-terminus with the last 275 amino acid resi-
dues being indispensable forthe enzyme activity. It has
been proposed that in rat isoform A Lys-197 and Asp-
414 are involved in the ATP binding, whereas Lys-262 is
important for the I(1,4,5)P3 binding.6
Forthe moleculardesign of selective and potent inhibi-
tors of the enzyme as opposed to random screening, it
would be highly desirable to have some detailed geo-
metry information on the binding domain of the
enzyme.7 We previously proposed a binding site model
for IP3K on the basis of the correlations between the
distinct stereochemical disposition of all 38 regioisomers
of myo-inositol phosphates [IPn, where n=1–6] and the
enzyme.8 As an extension of this investigation, we now
have carried out the syntheses of all possible optically
active regioisomers of scyllo-inositol phosphates from
enzymatically resolved conduritol B derivatives, and
utilized them togetherwith the perviously synthesized
achiral scyllo-inositol phosphates as molecularprobes in
order to obtain a refined picture of the binding model of
IP3K.
two majorpathways which yield I(1,3,4,5)P
by
4
I(1,4,5)P3 3-kinase [IP3K] and I(1,4)P2 by I(1,4,5)P3
5-phosphatase. I(1,3,4,5)P4 has recently attracted a lot
of attention because of its roles in relation with
I(1,4,5)P3 as well as in its own right as second messenger
and regulator.3 IP3K, a memberof inositol polyphos-
phate kinase family, shows a remarkable stereo- and
regioselectivity toward the recognition of I(1,4,5)P3; in
some cases the binding selectivity appears to be higher
than that of eitherI(1,4,5)P
receptor or I(1,4,5)P3
3
5-phosphatase.1b,4 At the level of cDNAs, several iso-
forms (A, B, and C) of IP3K have been cloned: A-C
from human, A and B from rat, and A from chicken.5
Although scyllo-inositol and scyllo-inositol phosphates
have been found in nature, the understanding of their
*Corresponding author. Tel.: +82-54-279-2103; fax: +82-54-279-
3399; e-mail: skchung@postech.ac.kr
0960-894X/03/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00629-2

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Y.-U. Kwon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2981–2984
biological activities leaves a lot to be desired.9 scyllo-
Inositol phosphates, which are among the stereoisomers
of myo-inositol phosphate, can have 15 possible regioi-
somers, including three enantiomeric pairs: scyllo-
I(1,2)P2, scyllo-I(1,2,4)P3, scyllo-I(1,2,3,4)P4 (Fig. 1).
We previously synthesized for the first time all possible
12 regioisomers of scyllo-inositol phosphate in meso or
racemic forms.10 To ourknowledge, only L- scyllo-
I(1,2,4)P3 has been synthesized in optically pure form.11
Our synthetic approaches to homochiral regioisomers of
scyllo-IPn are based on the lipase-catalyzed resolution of
racemic conduritol B diacetate which we previously uti-
lized in the syntheses of optically active stereoisomers of
conduritol and inositol.12 Enantiomerically enriched
scyllo-inositol derivative [(À)-4] was obtained by the
Mitsunobu reaction of myo-inositol diol [(À)-3], which
was derived from enzymatically enriched conduritol B
diol [(+)-1] (Scheme 1). Compound (À)-4 might be
considered as a suitably protected intermediate for the
synthesis of chiral scyllo-IPn. The vicinal diol [(À)-5]
was easily prepared by debenzoylation of compound
(À)-4 with a catalytic amount of NaOMe in MeOH at
reflux (Scheme 2). Acid-catalyzed hydrolysis of com-
pound (À)-4 afforded the triol (À)-6, which could be
converted to l-scyllo-I(1,2,4)P3. Benzoylation of com-
pound (À)-4 underconventional conditions, followed
by hydrogenolysis in the presence of a small amount of
AcOH provided the vicinal dibenzoate (+)-8.
Scheme 2. Reagents and conditions: (a) NaOMe, MeOH, reflux, 96%;
(b) 80% aq AcOH, 100 ꢀC, quant; (c) BzCl, pyridine, 99%; (d) H2 (50
psi), Pd(OH)2/C, AcOH, EtOAc/MeOH, 96%; (e) (i) iPr2NP(OBn)2,
1H-tetrazole, CHCl3; (ii) mCPBA; 75% for( À)-9, 96% for( À)-10,
77% for(+)- 11; (f) (i) H2 (50 psi), Pd/C, AcOH, EtOH/MeOH; (ii) pH
10 (NaOH), 94%; (g) (i) H2 (50 psi), Pd/C, EtOH/MeOH; (ii) 1 N
LiOH; (iii) Dowex 50WX8-100 (H+); (iv) pH 10 (NaOH), 91% for
IIIL, 89% for IVD.
active scyllo-I(1,2,4)P3 (IIIL) and scyllo-I(1,2,3,4)P4
(IVD) were obtained after chromatography on Dowex
50WX8-100 (H+), pH adjustment to 10 with NaOH,
and then lyophilization. Identical series of reactions
were also carried out with (+)-4 to afford compounds
IIL, IIID and IVL. All products were fully characterized
with NMR spectroscopy, and their optical rotations are
listed in Table 1.
The precursors (À)-5, (À)-6 and (+)-8 were phos-
phorylated by successive treatments with the phosphor-
amidite reagent and 1H-tetrazole, and then mCPBA to
give compounds (À)-9, (À)-10 and (+)-11 all in good
yields. In the final steps, all protecting groups of com-
pound (À)-9 were removed by hydrogenolysis in the
presence of a small amount of AcOH, thus giving the
Na salt of d-scyllo-I(1,2)P2 (IID) afterpH adjustment
with NaOH. Hydrogenolysis of compounds (À)-10 and
(+)-11, followed by treatment of LiOH removed all
protecting groups. The target compounds, the optically
Table 1. Optical rotations of three enantiomeric pairs of scyllo-IPn
scyllo-IPn
[P=P(O)(ONa)2]
[a]2D5 (in H2O)
d-form
l-form
scyllo-I(1,2)P2
scyllo-I(1,2,4)P3
+24.8 (c 0.34, pH 9.5) À20.2 (c 0.45, pH 9.2)
+25.9 (c 0.91, pH 9.5) À20.6 (c 0.57, pH 9.2)a
scyllo-I(1,2,3,4)P4 +16.9 (c 2.23, pH 9.2) À15.1 (c 1.79, pH 9.2)
aLit.11 [a]2D1 À24.1 (c 0.4, CHCl3).
Bioassays with IP3K
IP3K that catalyzes the ATP-dependent phosphoryla-
tion of I(1,4,5)P3 to generate I(1,3,4,5)P4, is one of the
key metabolic enzymes in the phosphoinositide signal
transduction system. Though we could get much useful
information on the binding mode of IP3K from our
previous results using myo-IPns,8 all 15 synthetic scyllo-
IPn regioisomers were tested for their binding affinity
toward IP3K to obtain more detailed information. We
have constructed recombinant plasmids for the rat brain
Figure 1. Three enantiomeric pairs of scyllo-inositol phosphate.
IP3K, overexpressed them in Escherichia coli, and pur-
13
ified the enzyme to nearhomogeneity.
Assays were
performed with 1 mM [3H]-d-myo-I(1,4,5)P3 by increas-
ing the concentration of each scyllo-IPn regioisomer in
the absence of Ca2+.8 The IC50 values of 15 scyllo-IPn
Scheme 1. Reagents and conditions: (a) BnBr, NaH, DMF, 96%;
(b) OsO4, NMO, aq acetone, 94.4%; (c) BzOH, Ph3P, DEAD, toluene,
80 ꢀC, 79%.

Y.-U. Kwon et al. / Bioorg. Med. Chem. Lett. 13 (2003) 2981–2984
2983
regioisomers have been determined by inhibition assays
at various concentrations and are listed in Table 2.
Table 2. Inhibition of IP3K by 15 scyllo-IPn regioisomers
scyllo-Inositol phosphates
IC50 (mM)
d-myo-I(1,4,5)P3
scyllo-IP1
4 scyllo-IP2s
2.6Æ0.3
N.D.a
N.D.a
d-scyllo-I(1,2,4)P3
l-scyllo-I(1,2,4)P3
scyllo-I(1,2,3)P3
scyllo-I(1,3,5)P3
d-scyllo-I(1,2,3,4)P4
l-scyllo-I(1,2,3,4)P4
scyllo-I(1,2,3,5)P4
scyllo-I(1,2,4,5)P4
scyllo-IP5
43.7Æ7.2
6.3Æ0.7
27.4Æ2.0
90Æ16
>100
>100
27.8Æ0.1
39.4Æ2.1
N.D.a
N.D.a
Figure 2. Geometry comparison between d-myo-I(1,4,5)P3 and com-
pounds with inverted C-2 configuration.
scyllo-IP6
substituent at that position.14 It is intriguing that the
equatorially oriented, negatively-charged phosphate
group at C-1 of scyllo-I(1,2,3)P3 and scyllo-I(1,2,3,5)P4
is well tolerated, even though d-myo-I(1,3,4,5)P4, the
product of IP3K, is known to be a very weak inhibitor
presumably due to the repulsive collision between the
C-3 phosphate and the negative charge and/or steric
tightness on the enzyme.1b,8 This observation is con-
sistent with the prior observation that d-myo-
I(1,2,4,6)P4 showed a good inhibitory activity, and these
results suggest that the inverted 2-OH causes somewhat
twisted ortilted binding of the scyllo-IP3 and-IP4 to
IP3K, and the C-1 phosphate group is now directed
away from the usual orientation of C-3 phosphate of
d-myo-I(1,3,4,5)P4. This in turn suggests that IP3K has
a pocket in the C-3 axial direction of d-myo-I(1,4,5)P3
(Fig. 2). It has been previously speculated that the
empty space in the C-3 axial direction is to be occupied
by ATP.8 scyllo-I(1,2,3,4)P4 with the bulky phosphate
group at the C-6 position of d-myo-I(1,4,5)P3 showed a
very weak inhibitory activity, strongly indicating that
the enzyme binding pocket does not have as much lee-
way in the direction of the equatorial 6-OH of d-myo-
I(1,4,5)P3 as compared to 2-OH and 3-OH.
aIC50 values were not determined for the compounds that showed very
weak ornegligible inhibition effect even at 200 mM.
All regioisomers of scyllo-inositol monophosphate,
scyllo-inositol bisphosphate, scyllo-inositol pentaki-
sphosphate, and scyllo-inositol hexakisphosphate were
found to be very weak or negligible in inhibiting the
IP3K activity even at 200 mM. dl-scyllo-I(1,2,4)P3 was
previously reported to be
a good substrate for
IP3K.11,14 As expected, l-scyllo-I(1,2,4)P3 which may be
visualized as having the geometry of d-myo-I(1,4,5)P3
with inverted 2-OH, showed comparable inhibitory
activity to that of d-myo-I(1,4,5)P3, the natural sub-
strate of IP3K. d-scyllo-I(1,2,4)P3 was also found to be
a good inhibitoralthough it was about 7 times less
potent than l-scyllo-I(1,2,4)P3. However, it is surprising
to find that scyllo-I(1,2,3)P3 shows a potent inhibitory
activity against radiolabeled d-myo-I(1,4,5)P3 even
though it does not have the usual structural requirement
of the three phosphate groups, that is the d-1,4,5-tri-
sphosphate motif, and also in view of the prior obser-
vations that its positional analogues, dl-myo-I(3,4,5)P3
and myo-I(4,5,6)P3 did not have any meaningful inhibi-
tory activities.8 scyllo-I(1,2,3,5)P4 and scyllo-I(1,2,4,5)P4
also significantly inhibited the phosphorylation of
d-myo-[3H]-I(1,4,5)P3 by IP3K. But scyllo-I(1,3,5)P3,
d-scyllo-I(1,2,3,4)P4 and l-scyllo-I(1,2,3,4)P4 displayed
less potent inhibitory activities than the other regio-
isomers of scyllo-IP3 and scyllo-IP4.
In sum, the binding interaction data between the 15
regioisomers of scyllo-IPn and IP3K are in good accord
with the binding site model previously proposed, and we
are now proceeding to design effective inhibitors of
IP3K as well as attempting to crystallize the enzyme.
These observations imply that all three phosphate
groups of d-myo-I(1,4,5)P3 play important roles for the
efficient binding to IP3K, but the 1-phosphate group
appears less critical than the other two. It is apparent
that IP3K has a vacant space in the C-2 axial direction
of d-myo-I(1,4,5)P3 and negative charges near the
vacant space surrounding the C-2 position.8,15 In addi-
tion, inhibitory activities of l-scyllo-I(1,2,4)P3 and
scyllo-I(1,2,4,5)P4 indicate that not only IP3K does have
a vacant space in the C-2 equatorial direction but also
the negative charges in the C-2 equatorial direction are
well-tolerated. d-myo-I(1,4,5)P3 derivatives with a bulky
substituent such as 3-benzoyl or3-methylbenzoyl group
are known to be ineffective in inhibiting the IP3K
activity, implying that IP3K does not tolerate a bulky
Acknowledgements
This work was financially supported by the Ministry of
Education/Basic Science Research Institute fund,
POSTECH/POSCO, and the KISTEP/NRL program.
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