Enzyme-assisted total synthesis of the optical antipodes D-myo-inositol 3,4,5-trisphosphate and D-myo-inositol 1,5,6-trisphosphate: Aspects of their structure-activity relationship to biologically active inositol phosphates

Article abstract of DOI:10.1021/jm981113k

Unambiguous total syntheses of both optical antipodes of the enantiomeric pair D-myo-inositol 3,4,5-trisphosphate (Ins(3,4,5)P₃) and D- myo-inositol 1,5,6-trisphosphate (Ins(1,5,6)P₃) are described. The ring system characteristic of

Full text of DOI:10.1021/jm981113k

1262
J . Med. Chem. 1999, 42, 1262-1273
En zym e-Assisted Tota l Syn th esis of th e Op tica l An tip od es D-m yo-In ositol
3,4,5-Tr isp h osp h a te a n d D-m yo-In ositol 1,5,6-Tr isp h osp h a te: Asp ects of Th eir
Str u ctu r e-Activity Rela tion sh ip to Biologica lly Active In ositol P h osp h a tes†
Stephan Adelt,‡ Oliver Plettenburg,§ Rolf Stricker,| Georg Reiser,| Hans-J osef Altenbach,§ and Gu¨nter Vogel*,‡
Institute fu¨r Biochemie und Organische Chemie, Bergische Universita¨t Wuppertal, Gaussstrasse 20, 42097 Wuppertal, FRG,
and Institute fu¨r Neurobiochemie, Otto-von-Guericke-Universita¨t Magdeburg, Medizinische Fakulta¨t, Leipziger Strasse 44,
39120 Magdeburg, FRG
Received November 10, 1998
Unambiguous total syntheses of both optical antipodes of the enantiomeric pair D-myo-inositol
3,4,5-trisphosphate (Ins(3,4,5)P3) and D-myo-inositol 1,5,6-trisphosphate (Ins(1,5,6)P3) are
described. The ring system characteristic of myo-inositol was constructed de novo from
p-benzoquinone. X-ray data for the enzymatically resolved (1S,2R,3R,4S)-1,4-diacetoxy-2,3-
dibromocyclohex-5-ene enabled the unequivocal assignment of the absolute configuration.
Subsequent transformations under stereocontrolled conditions led to enantiopure C2-sym-
metrical 1,4-(di-O-benzyldiphospho)conduritol B derivatives. Their synthetic potential was
exploited to prepare Ins(3,4,5,6)P4 and Ins(1,4,5,6)P4 in three steps. With a recently identified
and partially purified InsP5/InsP4 phosphohydrolase from Dictyostelium discoideum, these
enantiomers could be converted to the target compounds, Ins(3,4,5)P3 and Ins(1,5,6)P3, on a
preparative scale. An HPLC system employed for both purification of the inositol phosphates
and analytical runs ensured that the products were isomerically homogeneous. The sensitivity
of detection achieved by a complexometric postcolumn derivatization method indicates that
the complexation properties of Ins(3,4,5)P3/Ins(1,5,6)P3 resemble those of Ins(1,2,3)P3, a
compound with antioxidant potential. The set of inositol phosphates synthesized was used to
clarify structural motifs important for molecular recognition by p42IP4, a high-affinity Ins-
(1,3,4,5)P4/PtdIns(3,4,5)P3-specific binding protein from pig cerebellum.
In tr od u ction
in membrane recruitment of proteins, thereby enabling
a coordinated link between components of the signal-
transduction pathway. In the case of inositol phosphates
as possible ligands, PH domains derived from various
proteins showed distinct specificities.5 The main prob-
lem is to unravel interactions that are indeed of physi-
ological significance.
In a wide variety of eukaryotic cells intracellular
calcium mobilization in response to agonist stimulation
is mediated by the interaction of the second messenger
D-myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) with a
specific receptor, a ligand-gated calcium channel.1 The
action of this short-lived signaling molecule is modu-
lated by an extensive metabolism.2 Kinases and/or
phosphatases produce many other inositol polyphos-
phates, and some of them also appear to participate in
cellular signaling.3 Although their precise functions
have yet to be elucidated, the discovery of potential sites
for interaction supports this idea.4 Proteins such as
diacylglycerol kinase, RAC-protein kinase B, and phos-
pholipase C possess a pleckstrin-homology domain (PH;
abbreviation: platelet and leukocyte C kinase substr ate
protein ),5-7 a region of approximately 100 amino acids
that can form an electrostatically polarized tertiary
structure.7 Common ligands of this domain seem to be
inositol phosphates/phosphoinositides, the â/γ-subunits
of heterotrimeric G-proteins and protein kinase C.8-10
It has been proposed that the interaction plays a role
Despite the natural occurrence of a plethora of inositol
phosphate isomers in eukaryotic cells, e.g., about 25
compounds in Dictyostelium discoideum, the microor-
ganism used in our studies,11 it is obvious that not all
of them fulfill essential cellular functions. One feature
of a signaling molecule is the response of the intracel-
lular level to an appropriate stimulus. The levels of the
inositol tetrakisphosphates Ins(1,3,4,5)P4 and Ins-
(3,4,5,6)P4/Ins(1,4,5,6)P4 could be stimulated by agonists
in several cell lines,12-14 and high-affinity binding sites
are known15,16 especially for Ins(1,3,4,5)P4 (p42IP4 17
,
GAP1,18 centaurin-R19). Although some of these ”recep-
tors” have been purified, cloned, and sequenced, their
cellular functions remain elusive.
The isomers Ins(1,3,4,5)P4 and Ins(3,4,5,6)P4 share as
a common structural motif three adjacent phosphate
esters in positions C-3, C-4, and C-5. Thus the enanti-
omers Ins(3,4,5)P3 and Ins(1,5,6)P3 could be useful tools
for intervention in their metabolism or for binding
studies. This assumption encouraged us to synthesize
both optical antipodes of the enantiomeric pair Ins-
(3,4,5)P3/Ins(1,5,6)P3. We describe here a new approach
based on a straightforward chemical synthesis of Ins-
† According to the recommendation of IUPAC, all given names of
inositol phosphates refer to D-enantiomers; see: Nomenclature Com-
mittee of the International Union of Biochemistry (NC-IUB) Number-
ing of Atoms in myo-Inositol. Recommendations 1988. Eur. J . Biochem.
1989, 180, 485-486.
* Author for correspondence: fax, +49 (202) 439-2790; e-mail,
vogel@uni-wuppertal.de.
‡ Institute fu¨r Biochemie.
§ Institute fu¨r Organische Chemie.
| Institute fu¨r Neurobiochemie.
10.1021/jm981113k CCC: $18.00 © 1999 American Chemical Society
Published on Web 03/20/1999

Synthesis of Optical Antipodes Ins(3,4,5)P3, Ins(1,5,6)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1263
Sch em e 2. Enzymatic Resolution of Diacetate 2a
Sch em e 1. Enzyme-Assisted Synthesis of the Target
Compounds 9a and 9b
a
Conditions: (i) Br2, CHCl3, 0 °C (98%); (ii) NaBH4, Et2O, -20
°C to rt (88%); (iii) pyridine, acetic anhydride, overnight (68%);
(iv) PPL, phosphate buffer (pH 7), 4 days (38% each).
compounds. Therefore an unambiguous synthesis of the
pure enantiomers was needed, the more so as these
tetrakisphosphates are interesting objects for biological
investigation themselves.
Most myo-inositol phosphate syntheses start from
achiral myo-inositol and require separation of diaster-
eomers to yield enantiomerically pure compounds in the
end. In addition to the expenditure associated with this
step, errors made here may lead to confounding the
enantiomers. As knowledge of the correct stereochem-
istry is absolutely necessary for biological studies, we
put special emphasis on this aspect. A de novo synthesis
allows the employment of defined starting materials.
Carrying out the following steps under stereocontrolled
conditions ensures the identity of the product.
Previous work led us to believe that the known
dibromocyclohexenediol 3,27 which can easily be ob-
tained from p-benzoquinone in two steps, would be an
appropriate starting material. Enantiopure compounds
could be received by acetylation and subsequent enzy-
matic resolution by pig pancreas lipase28 in a phosphate
buffer (Scheme 2). The absolute configuration of the
resulting products 3a and 2b was determined by
synthesis of optically pure conduritol-B and (+)-pinitol28a
and was confirmed by an X-ray structure of the diac-
etate 2b. The crystals examined possess a chiral space
group, and the absolute structure parameter of 0.01(4)
shows the (+)-isomer 2b to be the (1S,2R,3R,4S)-
enantiomer.
The resolution procedure reported offers many ad-
vantages. For example, hydrolysis stops after 50%
conversion, so no reaction control is required. Further-
more, the application of relatively small quantities of
enzyme is sufficient to resolve up to 100 g of racemic
diacetate. The resulting products can be separated
simply by their different solubility in nonpolar solvents
such as dichloromethane: Diacetate 2b dissolves easily,
while dibromocyclohexenediol 3a is almost insoluble in
dichloromethane.
Enantiomeric excess after recrystallization of the
crude products was determined by chiral HPLC on a
Whelk S,S column to be >99% in each case. For this
analysis, 3a was converted into the corresponding
diacetate 2a with pyridine and acetic anhydride. Treat-
ment of diol 3a with potassium hydroxide and powdered
molecular sieves in tetrahydrofuran, a slight modifica-
tion of Farkas’s method,29 leads to anti-benzene dioxide
(trans-1,2:3,4-diepoxycyclohex-5-ene) 4a in good yields.
Conversion of the diacetate 2b under the same condi-
tions was unsatisfactory. It stopped after formation of
one epoxide ring and cleavage of the remaining acetate
function. Longer reaction times or employment of ad-
ditional base did not influence the course of the reaction.
(3,4,5,6)P4 (8a ) and Ins(1,4,5,6)P4 (8b) in combination
with enzymatic conversion of the resulting compounds
to Ins(3,4,5)P3 (9a ) and Ins(1,5,6)P3 (9b).
Conventional inositol phosphate synthesis usually
requires extensive separation of diastereomers to yield
enantiopure compounds.20 We avoid this by using clearly
defined precursors easily accessible from enzymatic
resolution of the racemic diacetate 2. Stereospecific
conversion leads in only two steps to C2-symmetric
conduritol-B derivatives, which can readily be trans-
formed into Ins(3,4,5,6)P4 (8a ) or Ins(1,4,5,6)P4 (8b).
In the course of our investigations of the inositol
phosphate metabolism in D. discoideum, we have previ-
ously found a cytosolic phosphatase activity that prefers
InsP5 and InsP4 isomers as substrates and produces
inositol trisphosphates. It exhibits characteristics quite
different from those of published inositol phosphate-
specific phosphatases.21 One remarkable feature is the
combination of high regiospecificity with low stereospec-
ificity. Mirror positions of enantiomerically pure sub-
strates are thus converted with nearly equal rates. This
is of especially practical value for the synthesis of
enantiomeric pairs, and the conversion of Ins(3,4,5,6)-
P4 (8a ) and Ins(1,4,5,6)P4 (8b) is presented in Scheme
1 as an example. The ability of the purified target
compounds Ins(3,4,5)P3 (9a ) and Ins(1,5,6)P3 (9b) to
compete with Ins(1,3,4,5)P4 in a displacement experi-
ment has been investigated with a recently character-
ized Ins(1,3,4,5)P4/PtdIns(3,4,5)P3-specific receptor from
pig cerebellum (p42IP4),17 a 42-kDa protein containing
two PH domains.22
Resu lts a n d Discu ssion
Ch em ica l Syn th esis. Knowledge of the regiospeci-
ficity of the InsP5/InsP4 phosphohydrolase examined
suggested to us that Ins(3,4,5,6)P4 (8a ) and Ins(1,4,5,6)-
P4 (8b) should be suitable precursors of the desired
inositol phosphates (Scheme 1). 8a and 8b have been
synthesized by several groups before;23-25 there is,
however, some confusion in the assignment of the
absolute configuration26 as is apparent from the opposite
signs of the optical rotation reported for the same

1264 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Adelt et al.
Sch em e 3. Preparation of Phosphorylated Conduritol-B
Derivatives 5a and 5ba
Sch em e 4. Preparation of Ins(1,4,5,6)P4 (8b)a
a
Conditions: (vii) 3-diethylamino-2,4,3-benzodioxaphosphep-
ane, 1H-tetrazole, CH2Cl2 (83%); (viii) RuCl3, NaIO4, ethyl acetate/
acetonitrile/water, 7 min (87%); (ix) Pd/C, H2, ethanol/water (95%).
ruthenium trichloride and 1.5 equiv of sodium metape-
riodate in acetonitrile/ethyl acetate/water were em-
ployed, gave good results. In the work cited, it was
shown that reaction times longer than 3 min often lead
to oxidative fission and/or glycol cleavage, because of
the presence of metaperiodate in the aqueous layer.
Examination of 1,4-disubstituted conduritol-B deriva-
tives with large substituents showed a low tendency
toward cleavage by sodium metaperiodate, though the
four phosphate groups make the compounds very polar.
Thus longer reaction times of 7-15 min to achieve
complete conversion of the starting material could be
employed. Removal of the remaining protecting groups
by treatment with hydrogen and Pd/C yields the desired
tetrakisphosphate 8b, already pure by NMR spectro-
scopic standards.
Purification of the resulting inositol phosphates by
HPLC ensures purities suitable for biological experi-
ments. Following this independent route, Ins(3,4,5,6)-
P4 (8a ) was identified as the (+)-enantiomer, which
corresponds to the data published in refs 23 and 24.
En zym e P r ep a r a tion a n d P r elim in a r y Ch a r a c-
ter iza tion . D. discoideum is a cellular slime mold that
shares features of animal and plant cells. Molecular
phylogenetic analysis has revealed that it is possibly
more closely related to the animal-fungal branch of the
eukaryotic tree than green plants are.31 The microor-
ganism naturally occurs in soil, where the unicellular
ameboid form feeds on bacteria. For laboratory use there
exist axenic mutants that can grow on fluid media with
doubling times of about 8 h,32 so it is possible to obtain
sufficient quantities within an acceptable time.
The partial purification of the cytosolic InsP5/InsP4
phosphohydrolase usually proceeded from 1010 cells
(strain AX-2). Tests with crude Dictyostelium homoge-
nates kept on ice had shown that the activity declined
with a half-life of about 3-4 h. On the assumption that
this was due to proteolytic degradation, the cells were
incubated prior to lysis in a Mes buffer containing the
nonmetabolizable sugar sucrose. Under these conditions
the cells remain intact but secrete a great part of their
lysosomal enzymes.33 Cells were lysed by freezing/
thawing and an additional passage through a 5-µm
Nucleopore filter. A slightly alkaline lysis buffer, which
was originally optimized to stabilize membranes, was
used.34 Cell debris was removed by ultracentrifugation,
and the yellow-colored cytosolic extract was applied to
a
Conditions: (v) KOH, powdered molecular sieves (4 Å), 4 °C
(77%); (v′) KOH, powdered molecular sieves (4 Å), methanol, 4 °C
(80%); (vi) dibenzyl phosphate, abs CH2Cl2, 8 h (55%).
However, by addition of limited amounts of ethanol to
the reaction mixture, complete reaction to 4b could be
achieved in less than 30 min (H.-J . Altenbach and A.
Kaffee, unpublished results).
Double allylic epoxide opening of 4a and 4b by
dibenzyl phosphate gives access to the phosphorylated
conduritol-B derivatives 5a and 5b (Scheme 3). The
inherent C2-symmetry of these compounds makes them
ideal for myo-inositol phosphate synthesis: Cis-dihy-
droxylation leads only to the myo-configuration, regard-
less of the direction of attack by the hydroxylating
reagent. As modifications at the free hydroxyl groups
can be carried out before the cis-dihydroxylation, the
method described here thus allows pairwise differentia-
tion of hydroxyl groups in myo-inositol systems, so that
a flexible approach to a wide variety of inositol phos-
phates is possible. Further work on this topic is in
progress and will be published elsewhere.
Ins(1,4,5,6)P4 (and Ins(3,4,5,6)P4 from enantiomer 5a
instead) was synthesized as shown in Scheme 4. The
introduction of two additional phosphate functionalities
was achieved by treatment with 3-diethylamino-2,4,3-
benzodioxaphosphepane in the presence of 1H-tetrazole
in dichloromethane. After thin layer chromatography
showed absence of the starting material, the resulting
phosphite was oxidized with m-CPBA. 31P-1H-coupled
NMR spectra allowed differentiation between the two
different phosphate groups. The dibenzyl phosphates
generate a pseudosextet (3J (P,H) ) 7.2 Hz) at -0.8 ppm,
while the other phosphates appear as a multiplet at
-2.2 ppm, as a result of the magnetic inequivalence of
the two fixed methylene groups.
Dihydroxylation of the resulting tetraphosphocondu-
ritol 6b with osmium tetraoxide was very slow and led
to unsatisfactory results. The flash dihydroxylation
method,30 in which catalytic amounts (10 mol %) of

Synthesis of Optical Antipodes Ins(3,4,5)P3, Ins(1,5,6)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1265
F igu r e 1. Synthetic potential of the partially purified InsP5/InsP4 phosphohydrolase. Defined inositol trisphosphates accessible
from the listed InsP5 and InsP4 isomers (dephosphorylated positions are marked in bold and italics): A, Ins(1,2,6)P3/Ins(2,3,4)P3;
B, meso-Ins(1,2,3)P3; C, Ins(3,4,5)P3 (9a )/Ins(1,5,6)P3 (9b).
a heparin agarose column. The column was eluted
stepwise with buffered NaCl solutions.
phate groups. The list of compounds tested is completed
by some isomers toward which little or no activity could
be measured. We can detect no conversion of InsP6 and
only negligible conversion of Ins(1,3,4,5,6)P5. Slow de-
phosphorylation of Ins(1,2,4,5)P4 and Ins(1,3,4,6)P4 was
observed.
It is obvious that all dephosphorylatable positions in
the substrates investigated possess at least one vicinal
trans-hydroxyl group, possibly a structural motif re-
quired for enzymatic activity. In summary, the synthetic
potential of the enzyme preparation is such that all
inositol trisphosphates phosphorylated at three adjacent
positions are accessible, with the exception of Ins(4,5,6)-
P3.
There are only a few InsP5- or InsP4-specific phos-
phatases described in the literature.40-42 Typically, the
activities cited are measured for only one substrate, so
it is impossible to reach any firm conclusions about the
specificity of the enzymes. The research group of Mayr
recently presented the first report of an Ins(1,2,3,4,5)-
P5 5-phosphatase preparation from calf thymus, which
seems to be capable of dephosphorylating the enanti-
omer at the same position.43 Whether both enzymatic
activities are combined in a single protein remains to
be examined.
Figure 1C shows that Ins(3,4,5,6)P4 (8a ) and Ins-
(1,4,5,6)P4 (8b) are converted at their mirror positions
C-6 and C-4. These reactions occur under substrate
saturation of the enzyme(s) with almost equal rates
(e.g., 400 µM 8a , 10.2 mU/mL, or 400 µM 8b, 9.7 mU/
mL). To obtain further information as to whether both
reactions are catalyzed by a single enzyme, the concen-
trated enzyme preparation was subjected to size-exclu-
To rule out the possibility of misinterpretation, the
fractions were analyzed for acid phosphatase activity,35
Ins(1,4,5)P3 5-phosphatase activity,36 and InsP5/InsP4
phosphohydrolase activity. Acid phosphatase and the
5-phosphatase are two enzymes that could probably
utilize InsP5 or InsP4 isomers as substrates under the
assay conditions employed.37,21 These two activities were
detected in the 100 mM fraction, whereas the InsP5/
InsP4 phosphohydrolase was clearly separated and
appeared in the 500 mM fraction, with an approximately
100-fold enrichment over the original cytosolic extract.
The protein preparation is devoid of phosphate and
InsPx metabolites, a prerequisite for the determination
of the enzymatic activity by measuring liberated phos-
phate38 and analyzing the products by HPLC-MDD
(m etal-d ye d etection).39 We established a microplate
assay with 100 µM Ins(1,2,5,6)P4 as substrate to opti-
mize reaction conditions and found the highest activity
in the pH range 5.5-6.5 and in the presence of 1-2 mM
Mg2+. To conserve the activity for further studies and
the enzyme-assisted synthesis presented below, the
fraction from the heparin column was concentrated,
stabilized by the addition of 25% (v/v) glycerol, and
stored frozen (-83 °C).
The enzymatic activity exhibited an unusually broad
substrate tolerance, releasing phosphate from various
InsP5 and InsP4 isomers. Examination of the intermedi-
ates and products by HPLC-MDD and comparison with
authentic standards led to the results illustrated in
Figure 1. It is remarkable that all inositol trisphos-
phates identified as products bear three adjacent phos-

1266 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Adelt et al.
F igu r e 2. Gel filtration of the partially purified InsP5/InsP4
phosphohydrolase on Superdex-200 HR. The assay for phos-
phatase activity shows the coelution of the C-6 and C-4
position-hydrolyzing enzyme(s). Retention times for stan-
dards: aldolase (158 kDa), 42.13 min; BSA (66.2 kDa), 45.8
min; ovalbumin (45 kDa), 49.2 min; chymotrypsinogen A (25
kDa), 56.7 min; cytochrome c (12.5 kDa), 60.03 min.
sion chromatography. The major part of the activity
eluted as a peak with an apparent molecular weight of
120 kDa (see Figure 2), determined by comparison with
standard proteins run on the same column. Although a
loss in total activity with little gain in specific activity
was observed after chromatography, the enzyme assay
clearly demonstrates that the C-6- and C-4-hydrolyzing
activities coelute with exactly the same pattern (the use
of Ins(1,2,4,5,6)P5 and Ins(1,2,5,6)P4 as substrates in-
stead gave analogous results, not shown). This does not
prove definitely that only one enzyme is responsible for
the different catalytic activities observed. Nevertheless,
it is an intriguing thought that the active center of a
single enzyme could possibly bind and catalyze the
conversion of enantiomers at their mirror positions.
Syn th etic Ap p lica tion . In the synthetic section we
noted the obvious discrepancies in reported optical
rotations for Ins(3,4,5,6)P4 (8a ) and Ins(1,4,5,6)P4 (8b).
The unambiguous total synthesis presented here may
help to resolve this confusion. Unfortunately, one of the
very precursors that caused the misinterpretation men-
tioned above was used for the previously reported
synthesis of Ins(1,5,6)P3 (9b).44 A different approach was
exploited for the total synthesis of Ins(3,4,5)P3 (9a ),45
but no value for the optical activity was quoted. We
therefore set out to synthesize both optical antipodes,
employing the partially purified InsP5/InsP4-phospho-
hydrolase as an enzymatic tool.
F igu r e 3. 1H-31P COSY NMR spectrum of D-myo-inositol
3,4,5-trisphosphate (9a ).
led to salt-free samples. Because of remaining water, it
is not appropriate to determine the yield of the proto-
nated form by weight. Total phosphate determination
after sulfuric acid hydrolysis provides a reliable quan-
tification of phosphorylated substances. We calculated
an overall yield of 70-80% for the enantiomers Ins-
(3,4,5)P3 (9a ) and Ins(1,5,6)P3 (9b), equivalent to 10-
12 µmol of each enantiomer in the experiments de-
scribed here.
The identity of 9a and 9b was confirmed by one- and
two-dimensional NMR spectroscopy. It was possible to
assign all the 1H, 13C, and 31P signals. The NMR spectra
showed no traces of other inositol trisphosphate isomers
(see, for example, Figure 3). The chemical shifts of
inositol ring protons are highly dependent on pH, and
overlapping signals sometimes make interpretation
difficult. After adjustment of the pH to 5 or 6 with ND4-
OD, complete resolution was achieved. The detection
limit of the NMR method for isomeric impurities is so
high that amounts up to 5% of the total may be
overlooked. However, for biological investigations any
uncertainty about the purity of the compounds has to
be precluded.
For this purpose the assay initially designed for the
kinetic evaluation of the reaction was scaled up. The
enzymatic activity remained stable at room temperature
for more than 6 h. To follow the progress of the reaction,
inorganic phosphate generated was measured according
to the method of Lanzetta et al.38 Usually the enzymatic
conversion was stopped after 7 h by the addition of
hydrochloric acid. As a consequence of denaturation, the
majority of proteins coagulated and could be removed
by centrifugation. The supernatant was neutralized,
diluted, and applied to a Mono Q column. Separation
was achieved with a linear hydrochloric acid gradient
just as in the analytical runs. This has the following
benefits: (a) 9a /9b elutes as an individual peak not
superimposing any other inositol trisphosphate cur-
rently known39 and (b) freeze-drying the pooled fractions
Small differences in the net charge of inositol tris-
phosphates, caused by varying interactions between
hydroxyl and phosphate groups, suffice for separation
of up to seven regioisomers on a Mono Q column with a
modified, slightly alkaline elution system (see Figure
4A).46 In an analogous experiment, we injected amounts
of Ins(1,5,6)P3 and Ins(3,4,5)P3 more than 60 times the
detection limit (∼0.15 nmol, mean value) and were
unable to observe any of the possible isomeric impurities
(see, for example, 9b in Figure 4B). Purities of more
than 99% are thus assured.
Com p lexa tion P r op er ties. In the past decade re-
search on the topic of inositol phosphates has mainly
been focused on functions related to signal transduction.
Besides this, there is an inherent chemical property of
some myo-inositol polyphosphates that is reflected by

Synthesis of Optical Antipodes Ins(3,4,5)P3, Ins(1,5,6)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1267
F igu r e 5. Calibration curves for the HCl elution system
(HPLC-MDD). Sensitivity of detection reflects the complex-
ation properties of the compounds. Regression curves calcu-
lated for the mean values of two independent experiments:
Ins(1,2,3)P3 (0), y ) 4.799*x - 0.038, r ) 0.999; Ins(3,4,5)P3
(9a )/Ins(1,5,6)P3 (9b) (4), y ) 4.531* x - 0.131, r ) 0.998; Ins-
(1,2,6)P3/Ins(2,3,4)P3 ()), y ) 2.918* x - 0.269, r ) 0.999; Ins-
(1,4,5)P3/Ins(3,5,6)P3 (O), y ) 1.252*x - 0.028, r ) 0.998.
F igu r e 4. HPLC-MDD chromatograms showing (A) standard
mixture including seven InsP3 regioisomers and (B) Ins(1,5,6)-
P3 (9b), the product of the phosphatase reaction starting from
Ins(1,4,5,6)P4 (8b).
ment experiments (see Figure 6A) confirm the relevance
of the phosphate groups in position C-3, C-4, and C-5,
as postulated in a previous study in which racemic
mixtures were employed.50 IC50 values for Ins(1,3,4,5)-
P4 and Ins(3,4,5)P3 (9a ) differ minimally (factor 2), so a
phosphate group in position C-1 is not an essential motif
for high-affinity binding. Like other Ins(1,3,4,5)P4 bind-
ing sites described, the protein exhibited relatively poor
stereoselectivity.18,51 Ins(1,5,6)P3 (9b) (identical to L-Ins-
(3,4,5)P3) was nearly one-seventh as effective as Ins-
(3,4,5)P3 (9a ) at displacing specific [3H]Ins(1,3,4,5)P4
binding from the receptor population. Searching for a
rational explanation, we note that a nearly complete
superposition could be achieved by a simple rotation of
Ins(1,5,6)P3 (see Figure 6B). On the precondition that
the orientations of the hydroxyl groups in positions C-2
(Ins(3,4,5)P3)/C-4 (Ins(1,5,6)P3) and in positions C-6 (Ins-
(3,4,5)P3)/C-2 (Ins(1,5,6)P3) have only minor influences
on the recognition, this could be the reason for the
observed lack of stereospecificity. The binding charac-
teristics of the inositol tetrakisphosphates indicate that
additional phosphate groups in positions C-6 (8a ) and
C-4 (8b) clearly reduce the interaction. This tendency
becomes more pronounced in going from Ins(3,4,5)P3 to
Ins(3,4,5,6)P4 than from Ins(1,5,6)P3 to Ins(1,4,5,6)P4.
their antioxidant potential.47 The protective effect of
dietary InsP6 against cancer is attributed in part to its
ability to chelate iron by occupying all of the coordina-
tion sites.48 This complete occupation seems to be the
reason InsP6 can effectively prevent free hydroxyl
radical formation catalyzed by iron in the Haber-Weiss
cycle. It has been demonstrated that not all six phos-
phates on myo-inositol are required for the inhibition
and concluded that the 1,2,3-trisphosphate grouping
contained the essential binding site.49
Complexation of transition-metal ions by myo-inositol
(poly)phosphates is not restricted to iron. The metal-
dye detection employed as a postcolumn derivatization
method is a kind of ”on-line complexometry”, based on
the ability of yttrium to reversibly bind both the cation-
specific dye PAR and the polyanion InsPx with high
affinity.39 Consequently, the sensitivity of detection
depends on the degree of complexation of yttrium by the
inositol phosphate of interest.
The employed HPLC system is routinely calibrated
for new inositol phosphate isomers, recording the peak
area versus injected mass. In Figure 5 the calculated
curves for Ins(1,2,3)P3, Ins(3,4,5)P3 (9a )/Ins(1,5,6)P3
(9b), and Ins(1,2,6)P3/Ins(2,3,4)P3 are depicted. The
inositol trisphosphates, all bearing three adjacent phos-
phate groups with different spatial orientations (see
Figure 1), were detected with the following sensitivity:
Ins(1,2,3)P3 ∼ 9a /9b > Ins(1,2,6)P3/Ins(2,3,4)P3. This
indicates that the compounds 9a /9b chelate yttrium
about as well as Ins(1,2,3)P3 does. It would thus be
interesting to examine their antioxidant potential. The
sensitivity decreases drastically if the block of neighbor-
ing phosphate groups is divided as is shown for Ins-
(1,4,5)P3 in Figure 5.
Biologica l Eva lu a tion /Recogn ition by a Sp ecific
Recep tor . The set of inositol phosphates (8a /8b and
9a /9b, Scheme 1) synthesized was used to gain ad-
ditional insights into structural requirements of ligand
binding to p42IP4, an Ins(1,3,4,5)P4/PtdIns(3,4,5)P3-
specific receptor from pig cerebellum,17 especially with
regard to stereospecificity. The results of the displace-
Con clu sion s
The major advantage of the approach presented here
for synthesizing myo-inositol phosphates de novo is its
versatility. As mentioned in the chemical section above,
the synthetic potential of the precursors 5a and 5b is
not restricted to the compounds described here.
A defined starting material, modified by stereocon-
trolled reaction steps, assured the correct absolute
configuration of the inositol tetrakisphosphates. The use
of a regiospecific InsP5/InsP4 phosphohydrolase as an
enzymatic tool increased significantly the number of
isomers obtainable. The unusual substrate specificity
of the enzymatic activity seems to allow the conversion
of a series of enantiomeric pairs (Figure 1). Since
dephosphorylation of the enantiomers occurs at mirror

1268 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Adelt et al.
(1,5,6)P3 (9b) is no longer limited to scarce material
isolated from natural sources, so it will be possible to
examine such phenomena in detail.
Exp er im en ta l Section
Chemicals were purchased from Fluka, Aldrich, or Acros.
Dichloromethane was dried over phosphorus pentoxide and
kept over 4-Å molecular sieves. TLC was performed on
precoated plates (Merck aluminum TLC sheets silica F254, art.
no. 5554), with detection by UV light or a solution of 1%
vanillin in sulfuric acid, followed by heating. Flash column
chromatography was performed on silica gel (63-200 µm;
Merck). 1H and 13C NMR spectra (internal Me4Si as reference)
and 31P NMR (external 85% phosphoric acid as reference) were
recorded with an ARX 400 spectrometer (Bruker). Chemical
shifts are given in ppm (δ) relative to the used reference;
coupling constants are in Hz. Assignments of 1H, 13C, and 31P
NMR resonances were aided by 2D COSY and DEPT experi-
ments. Optical rotation was determined on a Zeiss spectrom-
eter model.
IR spectra were recorded on a Perkin-Elmer spectrometer
model 1420. Melting points were determined on a Bu¨chi 510
heating block (not corrected). Mass analysis was carried out
on a Varian MAT 311 A (EI), Perkin-Elmer API 150 (ESI), or
a Finnigan MAT 90 (FAB) spectrometer. High-resolution mass
measurements were carried out on a Finnigan II-SQ 30.
Elemental analysis was carried out on a PE model 240 B
microelemental analyzer.
Inositol phosphates synthesized in our laboratories were
purified by HPLC. All reagents used were obtained in the
highest purity available. A membrane-associated, stereospe-
cific InsP6 phosphohydrolase from D. discoideum was exploited
for the synthesis of Ins(1,2,3,4,5)P5, Ins(1,2,4,5)P4, Ins(1,2,4,6)-
P4, and Ins(1,2,4)P3 (M. Knipp and M. Lammertz, manuscript
in preparation). Ins(1,2,4,5,6)P5 and Ins(1,2,5,6)P4 were syn-
thesized enzymatically using baker’s yeast as previously
described.55 The meso-compounds, Ins(1,3,4,5,6)P5, and Ins-
(1,2,3,4,6)P5 were prepared by acid-catalyzed dephosphoryla-
tion of InsP6.55 Ins(2,3,4,5,6)P5 was a generous gift from C.
Schultz (University of Bremen, FRG).43 Chemically synthe-
sized Ins(1,4,6)P3 and Ins(4,5,6)P3 were kindly provided by the
research group of M. Schneider (University of Wuppertal,
FRG). Commercially available inositol phosphates were pur-
chased from Sigma (InsP6, Ins(1,3,4,6)P4, and Ins(1,4,5)P3),
Boehringer Mannheim (Ins(1,3,4,5)P4), and Calbiochem (Ins-
(2,4,5)P3). The sources of other materials are described below.
Cell Cu ltu r e a n d P a r tia l P u r ifica tion of In sP 5/In sP 4
P h osph oh ydr olase. D. discoideum strain AX-2 (ATCC 24397)
was grown in axenic medium32 supplemented with 0.025%
dihydrostreptomycin sesquisulfate (w/v). Fernbach flasks were
filled with at most 500 mL of medium, and cells were cultured
at 22 °C on a rotary shaker (130 rpm). At a density of about
1×107 cells/mL, the cells were collected by centrifugation
(1500g, 10 min, 4 °C), washed once with 20 mM Mes/Na+ (pH
6.5), and resuspended to the same density in the Mes buffer
containing 0.1 M sucrose.33 After 3 h of incubation under the
conditions mentioned above, the cells were harvested by
centrifugation, washed once with ice-cold lysis buffer (5 mM
glycine (pH 8.5), 0.5 mM CaCl2, 0.5 mM MgCl2), and lysed at
a density of 1×108 cells/mL in the glycine buffer by freezing
(-83 °C)/thawing. Virtually complete lysis was achieved by
an additional passage through a 5-µm Nucleopore (Millipore)
filter.34 Resulting cell debris was removed by ultracentrifuga-
tion (140000g, 45 min, 4 °C). The following steps were
performed at 4 °C. The cytosolic supernatant from a total of
1010 cells was carefully decanted and applied to a 15-mL
heparin agarose (type II, Sigma) column, equilibrated with the
lysis buffer (about 310 mg of soluble protein; flow rate 60 mL/
h). The column was eluted stepwise with 50 mM Tris/HCl (pH
7.7), 1 mM EDTA containing increasing concentrations of
NaCl. The following distribution of protein was typically found
in the collected fractions: 210 mg (void volume/wash), 65 mg
(100 mM fraction), 3 mg (200 mM fraction), 7 mg (500 mM
fraction), and 2 mg (1 M fraction). The fractions were analyzed
F igu r e 6. A: [3H]Ins(1,3,4,5)P4 displacement experiment with
a membrane preparation from pig cerebellum containing p42IP4
(for details, see Experimental Section). B: Model for a possible
interpretation of the observed lack of stereospecificity.
positions, a further enantiomeric pair or a meso-
compound is accessible.
This strategy enabled us to synthesize two enantio-
meric pairs (8a /8b and 9a /9b) in a single experimental
sequence. The set of enantiopure compounds was suc-
cessfully exploited in a competition assay that revealed
the relevance of the phosphate groups in positions C-3,
C-4, and C-5 for high-affinity binding to p42IP4, a Ins-
(1,3,4,5)P4/PtdIns(3,4,5)P3-specific protein. These com-
pounds are expected to be of considerable use in
unraveling aspects of inositol phosphate binding to the
expanding number of eukaryotic proteins with identified
PH domains.52
Ins(1,5,6)P3 (9b), a product of the metabolism of Ins-
(1,3,4,5,6)P5 from avian erythrocytes,53 is one of a series
of isomers with an unknown function in mammalian
cells. Recently an inhibitory effect on the activity of SIP-
110, an Ins(1,3,4,5)P4/PtdIns(3,4,5)P3-specific 5-phos-
phatase, could be demonstrated in in vitro studies.54 It
is supposed that this enzyme is involved in the control
of the cellular level of PtdIns(3,4,5)P3. The supply of Ins-

Synthesis of Optical Antipodes Ins(3,4,5)P3, Ins(1,5,6)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1269
for acid phosphatase, Ins(1,4,5)P3 5-phosphatase, and InsP5/
InsP4 phosphohydrolase activity. The 500 mM protein fraction
was concentrated to the minimal volume attainable by the
ultrafiltration method employed (Centriprep-10, Millipore),
diluted 1:1 with the Tris buffer, aliquoted, and frozen (-83
°C) after the addition of 25% glycerol (v/v).
A 1.5-mg portion of this protein preparation was subjected
to size-exclusion chromatography on a Superdex 200 HR 10/
30 column (Pharmacia; fraction size 0.3 mL, flow rate 0.3 mL/
min). The column was equilibrated and eluted with 40 mM
Tris/HCl (pH 7.7), 50 mM NaCl, 1 mM EDTA. An identical
run with standard proteins allowed the molecular weight to
be estimated.
HPLC-MDD method described previously.39 The compounds
were separated by anion-exchange chromatography on a Mono
Q HR 10/10 column (Pharmacia). To distinguish between InsP4
and InsP5 isomers and to purify 8a /8b and 9a /9b , a linear
gradient of HCl was applied to elute the inositol phosphates
(0 min, 0.2 mM HCl; 70 min, 0.5 M HCl; flow rate 1.5 mL/
min). In analytical runs photometric detection at 546 nm was
achieved using a modified metal-dye reagent (2 M Tris/HCl
(pH 9.1), 200 µM 4-(2-pyridylazo)resorcinol (PAR), 30 µM YCl3,
10% (v/v) MeOH; flow rate 0.75 mL/min). Where on-line
detection was impossible (e.g., purification steps), an analogous
experiment could be carried out on a microplate. This allowed
fast screening for the presence of inositol phosphates. In brief,
a 0.2-10-µL portion of the sample (depending on the InsPx
concentration) was mixed with 100 µL of metal-dye reagent,
and the absorbance was measured at 540 nm.
The isomeric purity of the inositol trisphosphates 9a /9b was
controlled by use of a modified, slightly alkaline elution
system.46 The gradient composed of 50 mM Tris/HCl (pH 8.5;
solvent A) and 50 mM Tris/HCl, 0.4 M KCl (pH 8.5; solvent
B) had the following characteristics: 0 min, 30% B; 2 min, 40%
B; 16 min, 42% B; 20 min, 50% B; 38 min, 60% B; 48 min,
75% B; 50 min, 100% B; 60 min, 100% B (flow rate 1.5 mL/
min). The metal-dye reagent consisted of 2 mM NH4OAc/AcOH
(pH 5.0), 200 µM 4-(2-pyridylazo)resorcinol, 30 µM YCl3, and
10% (v/v) MeOH (flow rate 0.75 mL/min).
En zym e Assa ys. 1. In sP 5/In sP 4 P h osp h oh yd r ola se. A
microplate assay was established that allowed fast screening
of various substrates and provided kinetic data. A standard
incubation mixture contained 10 µL of assay buffer (200 mM
Mes/Na+ (pH 5.5), 4 mM MgCl2, 0.4% Triton X-100 hydr. (w/
v)), 10 µL of water, 10 µL of a protein fraction, and 10 µL of a
400 µM inositol phosphate solution. The samples were incu-
bated at room temperature, with the plate placed on
a
reciprocal shaker (80 rpm). The enzymatic reaction was
terminated after varying periods by adding 10 µL of 0.5 M HCl.
No acid-catalyzed, chemical dephosphorylation of inositol
phosphates could be detected after 2 h under these conditions.
It was thus possible to complete an experiment before the
mixtures were either analyzed for enzymatically released
inorganic phosphate or alternatively subjected to HPLC-MDD
analysis of inositol phosphates after neutralization.
2. In s(1,4,5)P 3 5-P h osp h a ta se. Activity was determined
by a modification of the method of van Lookeren Campagne
et al.36 The assay was carried out on a microplate in a solution
composed of the following components: 10 µL of buffer (160
mM Bis-Tris (pH 7.0), 800 mM sucrose, 1 mM EDTA, 20 mM
MgCl2), 10 µL of water, 10 µL of protein fraction, and 10 µL of
200 µM Ins(1,4,5)P3 solution. The samples were treated, the
reaction was stopped, and inorganic phosphate was analyzed
as reported for the assay of InsP5/InsP4 phosphohydrolase
activity.
Peaks were assigned to specific compounds by cochromatog-
raphy with commercially available standards or with prepara-
tively synthesized and purified inositol phosphates, which were
identified by NMR analysis.
Ch em ica l Syn th eses. (5RS,6RS)-5,6-Dibr om ocycloh ex-
2-en e-1,4-d ion e. Bromine (159.8 g, 1.0 mol) in 400 mL of
CHCl3 was added dropwise over 2 h to a cooled solution of
p-benzoquinone (1) (108.1 g, 1.0 mol) in 900 mL of CHCl3. The
reaction mixture was allowed to warm to room temperature
and stirred for another hour. The chloroform was evaporated
to give 262.5 g (98 %) of crude 2,3-dibromo-1,4-benzoquinone,
which was immediately used for reduction: mp: 82-83 °C
1
(lit.29 mp 82-84 °C, lit.56 mp 85.5-86 °C); H NMR (CDCl3,
3. Acid P h osp h a ta se. Activity was assayed essentially as
described in the literature.35 Volume activities were calcu-
lated from the linear regions of kinetic data plots. All assays
were tested for linearity with increasing enzyme concentration.
Protein was measured using an assay supplied by BIO-RAD
(catalog no. 500-0112) with bovine serum albumin as a
standard.
400 MHz) 4.80 (s, 2H, H-C5 and H-C6), 6.75 (s, 2H, H-C2
and H-C3); 13C NMR (CDCl3, 101 MHz) 45.15 (2 CH, C5 and
C6), 136.89 (2 CH, C2 and C3), 188.02 (2 C, C1 and C4); IR
(KBr) 3045 (dC-H), 3000, 2975 (-C-H), 1750, 1690 (CdO);
MS (EI, 70 eV, rel intensity) m/z ) 266/268/270 [(M+), 6], 187/
189 [(M+ - Br), 68], 159/161 [(M+ - Br - CO), 3], 108 [(M+
-
2 Br), 5].
In or ga n ic P h osp h a te Deter m in a tion . A colorimetric
assay was employed for the determination of nanomolar
amounts of inorganic phosphate.38 Reducing the volume to a
microplate scale made it possible to handle a great number of
probes simultaneously. The method was applicable to follow
the progress of the enzymatic phosphatase reactions and to
quantify the mass of purified inositol phosphates. In the former
case, the acidic solution (generally 50 µL; preparation de-
scribed under Enzyme Assays) was mixed with 100 µL of
malachite green reagent, and after 1 min 10 µL of 34% sodium
citrate‚2H2O (w/v) was added. For full color development the
mixture was incubated for 20 min at room temperature.
Absorbance was measured at 595 nm. The assay was linear
in the range 0-3 nmol of phosphate.
Covalently bound organic phosphate had to be liberated by
treatment with hot sulfuric acid. In brief, a 10-µL aliquot of
an inositol phosphate solution (approximately 1-2 mM InsP3
or InsP4) was pipetted into 50 µL of 10 N H2SO4, mixed, and
heated for 5 h at 170 °C (borosilicate glass tubes of hydrolysis
class 1 are required; Macherey-Nagel). The residue was diluted
100-fold with 1 N H2SO4, and 50-µL portions of the solution
were assayed for inorganic phosphate. Mass determinations
for each substance were done in triplicate, and the results were
validated by HPLC-MDD analysis (HCl gradient). On injection
of the same amounts of enantiomers in two independent
experiments, the recorded peak areas differed less than 5%.
P u r ifica tion a n d An a lysis of In ositol P h osp h a tes.
Inositol phosphates were purified and analyzed using the
(1RS,2SR,3SR,4RS)-2,3-Dibr om ocycloh ex-5-en e-1,4-d i-
ol (3). A solution of 5,6-dibromocyclohex-2-ene-1,4-dione (200
g, 0.75 mmol) in 2.5 L of diethyl ether was cooled to -15 °C.
During 1 h a solution of sodium borohydride (60 g, 1.6 mol) in
1 L of water was added to the vigorously stirred solution. The
reaction mixture was allowed to warm to room temperature
and stirred for another 2 h. The ether phase was separated,
and the aqueous layer was extracted five times with ethyl
acetate. The combined organic layers were evaporated to yield
178.6 g (0.66 mol, 88%) of 3: mp 149 °C (lit.27 mp 149 °C); 1H
NMR (400 MHz, MeOD) 5.72 (2 H, s, H-C5 and H-C6), 4.42
(2 H, m, H-C1 and H-C4), 4.11 (2 H, m, H-C2 and H-C3);
13C NMR (101 MHz, MeOD) 61.22 (CH, C2 and C3), 74.23 (CH,
C1 and C4), 131.42 (CH, C5 and C6); IR (KBr) 3300 (O-H),
2960, 2890 (C-H), 1055 (C-O); MS (EI, 70 eV, rel intensity)
m/z ) 191/193 [(M+ - Br), 8], 173/175 [(M+ - Br - H2O), 8],
111 [(M+ - 2 Br - 1), 100]. Anal. (C6H8O2Br2) C, H.
(1RS,2SR,3SR,4RS)-1,4-Diacetoxy-2,3-dibr om ocycloh ex-
5-en e (2). Compound 3 (178.6 g, 0.66 mol) was dissolved in a
cooled mixture of 200 mL of pyridine and 200 mL of acetic
anhydride. The reaction mixture was stirred for 12 h. Ice (300
g) was added, and after stirring for 15 min dichloromethane
(300 mL) was added. The layers were separated, and the
aqueous layer was extracted four times with dichloromethane.
The combined organic layer was washed with saturated
aqueous NaHCO3 (3 × 200 mL), 0.75 N HCl (3 × 200 mL),
and brine (200 mL). After evaporation, the resulting residue
was recrystallized from ethanol to yield 2 (159.0 g, 0.45 mol,

1270 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7
Adelt et al.
68%): mp 94 °C (lit.57 mp 91-92 °C); 1H NMR (CDCl3, 400
MHz) 5.61 (s, 2 H, H-C5 and H-C6), 5.68 (2 H, mc, H-C1
and H-C4), 4.26 (2 H, mc, H-C2 and H-C3), 2.20 (2 CH3,
COCH3); 13C NMR (CDCl3, 101 MHz) 20.70 (CH3, COCH3),
52.65 (CH, C2 and C3), 73.29 (CH, C1 and C4), 128.12 (CH,
C5 and C6), 169.68 (C, CdO); IR 2980, 2940 (-C-H), 1750
(CdO), 1220, 1040 (C-O-C), 715 (C-Br); MS (EI, 70 eV, rel
intensity) m/z ) 295/297/299 [(M+ - OC(O)CH3), 1], 257/277
[(M+ - Br), 2], 173/175 [(M+ - Br - 2 OC(O)CH3), 33], 153
[(M+ - 2 Br - OC(O)CH3), 33]. Anal. (C10H12Br2O4) C, H.
6.6 g (10 mmol, 55%) of a colorless, voluminous solid: mp 157
°C; [R]20 +56.6° (c ) 1.1, CHCl3); 1H NMR (CDCl3, 400 MHz)
D
3.75 (dd, 2H, J ) 2.4 Hz, 5.2 Hz, H-C2 and H-C3), 3.97 (s-
b, 2H, OH), 4.85 (m, 2H, H-C1 and H-C4), 5.00-5.14 (m,
8H,-O-CH2-), 5.62 (s, 2H, H-C5 and H-C6), 7.37-7.28 (m,
20H, -C6H5); 13C NMR (CDCl3, 101 MHz) 69.91 (m, CH2, CH2-
C6H5), 74.25 (d, CH, J ) 3.8 Hz, C3 and C4), 78.80 (d, CH, J
) 5.1 Hz, C1 and C4), 127.95, 127.99, 128.51, 128.56, 128.59
(5 CH, C6H5), 135.53, 135.60 (d, 2C, J ) 3.1 Hz, C6H5); 31P
NMR (CDCl3, 162 MHz) 0.58 (2P); IR (KBr) 3380 (O-H), 3065,
3020 (dC-H), 2940, 2880 (-C-H), 1250 (PdO), 1135 (C-O-
C), 1020 (P(O)-OR), 730, 690 (dC-H); MS (ESI, rel intensity)
m/z ) 667.7 [(M + H)+, 100]. Anal. (C34H36O10P2) C, H.
(1R,2R,3R,4R)-1,4-Bis(d i-O-b en zylp h osp h o)con d u r i-
tol-B (5b). The (+)-enantiomer 4b (1.5 g, 13.6 mmol) was
treated as described for 4a and yielded 5b as a colorless solid
(1S,2R,3R,4S)-Dia ceta te 2b a n d (1R,2S,3S,4R)-Diol 3a .
Powdered racemic diacetate 2 (100.0 g, 0.28 mol) and pig
pancreas lipase (54.7 g) were suspended in 1.2 L of 0.1 M
phosphate buffer (pH 7) and 120 mL of diethyl ether. The
mixture was stirred vigorously for 4 days. Ethyl acetate (500
mL) was added, and the enzyme was filtered over a pad of
Celite. The residue was washed with ethyl acetate (4 × 150
mL) and water (4 × 150 mL). The combined aqueous phase
was extracted with ethyl acetate (3 × 150 mL). The organic
phase was evaporated, and the resulting solid was suspended
in dichloromethane (600 mL). Diol 3a remained insoluble as
a white, crystalline solid, while diacetate 2b dissolved totally.
The solid was filtered off and recrystallized from toluene to
yield 3a in 38% (28.9 g, 0.1 mol). The dichloromethane was
evaporated, and the resulting solid was recrystallized from
ethanol to give 2b as colorless needles (37.9 g, 0.1 mol, 38%).
(5.0 g, 7.5 mmol, 55%): mp 157 °C; [R]20 -55.2° (c ) 1.0,
D
CHCl3); NMR, IR, and MS data were identical to those for the
other enantiomer 5a .
(1S,2S,3S,4S)-1,4-Bis-O-(d i-O-b en zylp h osp h o)-2,3-b is-
O-(2-oxo-5,6-b en zo-1,3,2-d ioxa p h osp h ep -2-yl)con d u r i-
tol-B (6a ). To a solution of 5a (300 mg, 0.45 mmol) and 1H-
tetrazole (508 mg, 3.6 mmol) in anhydrous dichloromethane
(30 mL) was added 3-diethylamino-2,4,3-benzodioxaphosphep-
ane (432 mg, 1.8 mmol), and the mixture was stirred at room
temperature for 2 h. The mixture was cooled to -60 °C, and a
solution of m-CPBA (350 mg) in dichloromethane (5 mL) was
added. Stirring was continued for 30 min at -60 °C and for
another hour at room temperature. The reaction mixture was
diluted with 100 mL of dichloromethane and washed consecu-
tively with aqueous sodium bisulfite (50 mL), saturated
aqueous NaHCO3 (2 × 40 mL), and brine (40 mL). After
evaporation of dichloromethane the resulting oil was purified
by flash chromatography (CH2Cl2/MeOH, 97:3) to yield 6a in
2b: mp 110 °C (lit.28b mp 107-109 °C); [R]20 +11.3° (c ) 5.1,
D
CH2Cl2), lit.28b [R]20 +11.7° (c ) 1.1, CH2Cl2); HPLC (Whelk,
D
S,S; heptane/2-propanol, 90:10, flow 0.8 mL/min) t ) 9.08 min
(other enantiomer 2a , t ) 10.24 min); for spectral data see
racemic compound 2. 3a : mp 164 °C (lit.28b mp 164-166 °C),
[R]20 +41.8 ° (c ) 5.0, MeOH), lit.28b [R]20 +45.8° (c ) 1.2,
D
D
acetone); for spectral data see racemic compound 3.
(1R,2R,3R,4R)-a n ti-Ben zen e Dioxid e (4a ). Compound 3a
(8.2 g, 30.4 mmol) was dissolved in 300 mL of absolute THF
under argon and cooled to 4 °C. During 30 min a mixture of
powdered KOH (20 g) and powdered 4-Å molecular sieves (13
g) was added in portions. The reaction mixture was stirred
for 1 h and allowed to warm to room temperature. To complete
conversion, another 5 g of the mixture (2 g KOH and 3 g
molecular sieves) was added. Stirring was continued for
another hour. Then 400 mL of diethyl ether was added, and
the solution was filtered. The residue was washed with diethyl
ether (3 × 100 mL), and the combined organic phase was
evaporated to yield 4a as a colorless solid (2.57 g, 23.3 mmol,
83% (385 mg, 0.37 mmol): mp 135 °C; [R]20 +19.8° (c ) 1.32,
D
CHCl3); 1H NMR (CDCl3, 400 MHz) 4.92-5.30 and 5.52-5.59
(m, 20H, CH2C6H5 and CH2C6H4, H-C1, H-C2, H-C3 and
H-C4), 5.93 (s, 2H, H-C5 and H-C6), 7.43-7.30 and 7.11-
7.16 (m, 28H, C6H5 and C6H4); 13C NMR (CDCl3, 101 MHz)
68.92, 69.21 (2 CH2, d, J ) 6.8 Hz, OCH2C6H5), 69.73, 69.94
(2 CH2, m, OCH2C6H4), 76.19 (CH, C2 and C3), 77.92 (CH, C1
and C4), 127.15 (CH, C5 and C6), 128.00, 128.16, 128.57,
128.61, 128.63 (5 CH, 4 C6H5), 128.94, 129.03 (2 CH, d, J )
4.1 Hz, 2 C6H4), 135.31, 135.44 (2C, 4 C6H5), 135.48, 135.50,
135.55, 135.57 (4 C, C6H4); 31P NMR (CDCl3, 162 MHz) -0.84
(2P, P-C1 and P-C4), -2.18 (2P, P-C2 and P-C3); IR (KBr)
3470 (O-H), 3065, 3035 (dC-H), 2960, 2930, 2895 (-C-H),
1290 (PdO), 1045, 1020 (C-O-C), 730, 695 (dC-H); MS (ESI,
77%): mp 59 °C; [R]20 -320.2° (c ) 0.75, CHCl3); 1H NMR
D
(CDCl3, 400 MHz) 3.03 (m, 2H, H-C2 and H-C3), 3.69 (m,
2H, H-C1 and H-C4), 6.02 (s, 2 H, H-C5 and H-C6); 13C
NMR (CDCl3, 101 MHz) 46.38 (CH, C2 and C3), 53.64 (CH,
C1 and C4), 129.48 (CH, C5 and C6); MS (EI, 70 eV, rel
rel intensity) m/z ) 1049.5 [(M+ + H2O), 37], 1031.5 [(M+
+
H), 100], 425.6 [10], 367.5 [8], 151.1 [12], 120.1[23], 79.0 [18];
MS m/z ) 1031.2125 [M + H]+ calcd for C50H51O16P4 1031.2128.
Anal. (C50H50O16P4): C, H.
intensity) m/z ) 111 [(M + H)+ 4], 110 [(M+), 79], 109 [(M -
,
H)+, 11], 82 [23], 81 [100], 68 [4], 55 [27], 54 [45], 42 [5]. Anal.
(C6H6O2) C, H.
(1R,2R,3R,4R)-1,4-Di-O-(d i-O-b en zylp h osp h o)-2,3-b is-
O-(2-oxo-5,6-b en zo-1,3,2-d ioxa p h osp h ep -2-yl)con d u r i-
tol-B (6b). The (-)-enantiomer 5b (500 mg, 0.75 mmol) was
phosphitylated as described for 5a . Oxidation and purification
(1S,2S,3S,4S)-a n ti-Ben zen e Dioxid e (4b). Compound 2b
(10.1 g, 28.4 mmol) was dissolved in 300 mL of absolute THF
under argon and cooled to 4 °C. Over 15 min a mixture of
powdered KOH (10 g) and powdered 4-Å molecular sieves (6.5
g) was added in portions, followed by addition of 2 mL of
absolute methanol and subsequently a mixture of powdered
KOH (10 g) and powdered 4-Å molecular sieves (6.5 g). The
reaction mixture was stirred for 15 min and allowed to warm
to room temperature. To complete conversion, stirring was
continued for 1 h. Then 400 mL of diethyl ether was added,
and the solution was filtered. The residue was washed with
diethyl ether (3 × 100 mL), and the combined organic layers
were evaporated to yield 4b as a colorless solid (2.50 g, 22.7
as before gave 6b (649 mg, 0.63 mmol, 84%): mp 135 °C; [R]20
D
-19.7° (c ) 0.98, CHCl3); NMR, IR, and MS data were identical
to those for the other enantiomer 6a .
D-m yo-3,6-Bis-O-(d i-O-ben zylp h osp h o)-4,5-bis-O-(2-oxo-
5,6-ben zo-1,3,2-d ioxa p h osp h ep -2-yl)in ositol (7a ). To a
vigorously stirred solution of 6a (400 mg, 0.38 mmol) in ethyl
acetate/acetonitrile (1:1, 20 mL) was added a solution of 125
mg (0.58 mmol) of sodium metaperiodate and ruthenium
trichloride (10 mg, 10 mol %) in 2 mL of water. The stirring
was continued until TLC showed absence of 6a (CH2Cl2/MeOH,
95:5) (about 10 min). The reaction was quenched by addition
of saturated Na2S2O3 (20 mL). The aqueous layer was sepa-
rated and extracted with dichloromethane (3 × 20 mL). The
combined organic layers were evaporated, and the product was
purified by flash chromatography (CH2Cl2/MeOH, 97:3) to give
7a as a white solid (348 mg, 0.33 mmol, 86%): mp 168 °C;
mmol, 80%): mp 53 °C (lit.58 mp 53-54 °C); [R]20 +320.6 ° (c
D
) 1.44, CHCl3), lit.58 +170° (c ) 0.3, CHCl3); NMR and MS
data were identical to those for the other enantiomer 4a .
(1S ,2S ,3S ,4S )-1,4-Bis(d i-O-b e n zylp h osp h o)con d u r i-
tol-B (5a ). To a solution of 4a (1.98 g, 18 mmol) in absolute
dichloromethane (150 mL) under argon was added dibenzyl
phosphate (10.5 g, 37.8 mmol). The solution was stirred for
12 h and evaporated. Crystallization from ethyl acetate gave
[R]20 -5.7° (c ) 4.5, CHCl3); 1H NMR (CDCl3, 400 MHz) 3.71
D
(dd, 1H, J ) 2.5 Hz, 9.3 Hz,H-C1) ) 4.39 (ddd, 1H, J ) 2.6
Hz, 10.0 Hz, 6.1 Hz, H-C3), 4.51 (t, 1H, J ) 2.5 Hz, H-C2),

Synthesis of Optical Antipodes Ins(3,4,5)P3, Ins(1,5,6)P3
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 7 1271
4.77 (m, 1H, H-C6), 4.82 (m, 1H, H-C5), 4.59-5.53 (m, 8H,
-CH2), 5.35 (q, 1H, J ) 9.2 Hz, H-C4), 7.40-7.22 and 7.05-
6.92 (m, 28H, C6H5 and C6H4); 13C NMR (CDCl3, 101 MHz)
70.24 (CH, C1 and C2), 76.07 (m, CH, C3), 76.88 (m, CH, C4),
77.83 (m, CH, C5), 79.83 (m, CH, C6), 69.92, 70.07(d, 2 CH2,
J ) 5.3 Hz, -OCH2C6H5), 69.10, 68.71 (m, 2 CH2, -OCH2C6H4),
128.05, 128.15, 128.22, 128.37, 128.56, 128.64, 128.73, 128.81,
128.88, 128.94 (10 CH, C6H5 and C6H4), 135.25, 135.30, 135.42,
135.51, 135.55, 135.58, 135.60, 135.68 (8 C, C6H5 and C6H4);
31P NMR (CDCl3, 162 MHz) 0.61 (P-C4), -1.42 (P-C3), -1.78
(P-C5), -1.93 (P-C6); IR (KBr) 3400 (O-H), 3080, 3057, 3035
(dC-H), 2950, 2910, 2885 (C-H), 1285 (PdO), 1050, 1025,
1015 (C-O-C), 730, 693 (CdC); MS (+ ion FAB, rel intensity)
m/z )1065.5 [(M+ + H), 100], 1084.8 [(M + H3O+), 45], 425.6
[80], 120 [61]; MS m/z ) 1065.2189 [M + H]+, calcd for
C50H53O18P4 1065.2182. Anal. (C50H52O18P4) C, H.
H-C3), 4.20 (t, 1H, J ) 3.0 Hz, H-C2), 4.37 (q, 1H, J ) 9.3
Hz, H-C4); 13C NMR (D2O, 101 MHz, pH adjusted to 6 (ND4-
OD)) 72.60 (d, CH, J ) 2.7 Hz, C1), 73.75 (s, CH, C2), 74.04
(d, CH, J ) 3.0 Hz, C6), 77.05 (dd, CH, J ) 2.0 Hz, 6.0 Hz,
C3), 78.42 (m, CH, C4), 80.76 (dd, CH, J ) 3.0 Hz, 6.1 Hz,
C5); 31P NMR (D2O, 162 MHz, pH adjusted to 6 (ND4OD)) 2.17
(P-C3), 2.77 (P-C5), 2.87 (P-C4); MS (+ionFAB, rel inten-
sity) m/z ) 420.9 [(M+) 16], 277.2 [5], 202.1 [22], 185.1 [80],
110.2 [100]; MS m/z ) 420.966 [M + H]+, calcd for C6H16O15P3
420.9702.
D-m yo-In ositol 1,5,6-Tr isp h osp h a te (9b). Dephosphory-
lation of 8b was carried out as described for 8a , yielding 9b
in 70-80%: [R]20 +2.2° (c ) 2.3, H2O, free acid), lit.44 [R]20
D
D
-2.8 ° (c ) 1.43, H2O, sodium salt); NMR and MS data were
identical to those for the other enantiomer 9a .
Bin d in g Assa ys. Membranes containing p42IP4 were pre-
pared from pig cerebellum in the presence of protease inhibi-
tors (0.2 mM Pefabloc SC, 1 mM EGTA, 1 µg/mL pepstatin, 1
µg/mL leupeptin, 1 mM benzamidin) as described previously.59
The binding assays were performed in a final volume of 400
µL in microcentrifuge tubes. Membranes (300 µg of protein/
assay) were incubated in 10 mM HEPES/KOH (pH 7.0)
containing 1.2 nM (22 500 dpm) [3H]Ins(1,3,4,5)P4 (777 gBq/
mmol; DuPont-NEN), 0.25% BSA, 1 mM EDTA, 20 mM NaCl,
and 100 mM KCl. The samples were incubated in the presence
of different concentrations of unlabeled Ins(1,3,4,5)P4 (Boe-
hringer Mannheim), Ins(3,4,5)P3 (9a ), Ins(1,5,6)P3 (9b), Ins-
(3,4,5,6)P4 (8a ), or Ins(1,4,5,6)P4 (8b) for 20 min on ice. Bound
ligand was separated from free ligand by addition of 100 µL
of γ-globulin (10 mg/mL) and 500 µL of 5% PEG-8000, further
incubation for 15 min, and then centrifugation (14000g, 15
min, 4 °C). The pellets were washed with 100 µL of binding
buffer and solubilized with 2% SDS; the bound radioactivity
was measured. Nonspecific binding was determined in the
presence of 1 µM Ins(1,3,4,5)P4. All determinations were made
in duplicate and were repeated at least twice with two different
membrane preparations.
D-m yo-1,4-Bis-O-(d i-O-ben zylp h osp h o)-5,6-bis-O-(2-oxo-
5,6-ben zo-1,3,2-d ioxa p h osp h ep -2-yl)in ositol (7b). The (-)-
enantiomer 6b (500 mg, 0.49 mmol) was cis-dihydroxylated
as described for 6a . Workup and purification as before gave
7b (437 mg, 0.42 mmol, 87%): mp 173 °C; [R]20 +5.5° (c )
D
4.5, CHCl3); NMR, IR, and MS data were identical to those
for the other enantiomer 7a .
D-m yo-In ositol 3,4,5,6-Tetr a k isp h osp h a te (8a ). To a
suspension of 7a (300 mg, 0.28 mmol) in ethanol/water (1:2)
was added 10% Pd/C (50 mg). The mixture was stirred at room
temperature under H2 overnight. The catalyst was filtered off,
and the filtrate was lyophilized to give 132 mg (0.27 mmol,
95%) of 8a . Further purification by HPLC assured purity
>99% for the following reaction: [R]20 +4.1° (c ) 2.7, H2O,
D
free acid), lit.24 [R]24D +9.8 ° (c ) 1.43, H2O, sodium salt), lit.25
[R]20 -5.6 ° (c ) 0.2, H2O), lit.23 [R]D +6.2 ° (c ) 2.15, H2O,
D
pH 9.5); 1H NMR (D2O, 400 MHz, pH adjusted to 5 (ND4OD))
3.75 (dd, 1H, J ) 3.1 Hz, 9.7 Hz, H-C1), 4.12 (dt, 1H, J ) 3.1
Hz, 9.7 Hz, H-C3), 4.14 (q, 1H, J ) 9.7 Hz, H-C4), 4.25 (t,
1H, J ) 3.1 Hz, H-C2), 4.33 (q, 1H, J ) 9.6 Hz, H-C6), 4.45
(q, 1H, J ) 9.5 Hz, H-C6); 13C NMR (D2O, 101 MHz, pH
adjusted to 5 (ND4OD)) 70.35 (d, CH, J ) 2.7 Hz, C1), 71,23
(s, CH, C2), 74.76 (m, CH, C3), 76.47 (m, CH, C4), 77.07 (m,
CH, C6), 77.71 (m, CH, C5); 31P NMR (D2O, 162 MHz, pH
adjusted to 5 (ND4OD)) 1.37 (P-C3), 2.28 (P-C4), 2.32 (P-
C6), 2.36 (P-C5); MS (ESI-neg, rel intensity) m/z ) 499.1 [(M
- H)-, 75], 419.0 [18], 321.0 [31] 158.9 [158.9]; MS m/z )
500.934 [M + H]+, calcd for C6H17O18P4 500.9365.
Ack n ow led gm en t. We are grateful to Dr. W. V.
Turner for stimulating discussions and critical reading
of the manuscript. We thank Ch. Lenz, Dr. H. Budzik-
iewicz, and H. Musche for recording the mass spectra
and Dr. W. S. Sheldrick and Dr. F. P. Ritter for
performing the X-ray study. We wish to thank O. Block,
A. Hansen, and A. Kaffee for sharing their unpublished
observations with us. This work was supported by the
Deutsche Forschungsgemeinschaft (Grant VO 348/2-2
and Sonderforschungsbereich 426/Reiser) and the Fonds
der Chemischen Industrie.
D-m yo-In ositol 1,4,5,6-Tetr a k isp h osp h a te (8b). Depro-
tection of 7b (200 mg, 0.19 mmol) as described for 7a led to
8b as a colorless foam (89 mg, 0.18 mmol, 96%): [R]20 -4.8°
D
(c ) 2.7, H2O, free acid), lit.24 [R]20 -10.2° (c ) 2.46, H2O,
D
sodium salt); NMR and MS data were identical to those for
the other enantiomer 8a .
D-m yo-In ositol 3,4,5-Tr isp h osp h a te (9a ). The enzymatic
reaction was carried out at room temperature in a volume of
12 mL on a reciprocal shaker (40 rpm). The final buffer
composition was identical to that used for the analytical assay
Su p p or tin g In for m a tion Ava ila ble: X-ray data for com-
pound 2b. This information is available free of charge via the
Internet at http://pubs.acs.org.
of InsP5/InsP4 phosphohydrolase activity. At
a substrate
concentration of 300 µM 8a and a volume activity of about
3-3.5 mU/mL (equivalent to 50-75% of the total activity
obtained from 1010 cells), it took approximately 1.5 h to convert
all the inositol tetrakisphosphate. Depending on the phosphate
release measured, new substrate was added three times from
a 30 mM stock solution. To verify complete conversion of the
substrate, an aliquot was subjected to HPLC-MDD (HCl
gradient: t(9a ) ) 30.0 min, t(8a ) ) 44.4 min) before the
reaction was stopped by addition of 3 mL of 0.5 M HCl. The
pH of the mixture was adjusted to 6, and the precipitate
formed was removed by centrifugation (6000g, 15 min, room
temperature). The supernatant was diluted 10-fold with water,
and the inositol phosphates were separated by HPLC. After
freeze-drying, overall yields were in the range of 70-80% (10-
12 µmol) for 9a (quantification based on inorganic phosphate
determination): [R]20D -4.6° (c ) 1.4, H2O, free acid); 1H NMR
(D2O, 400 MHz, pH adjusted to 6 (ND4OD)) 3.64 (dd, 1H, J )
3.0 Hz, 10.1 Hz, H-C1), 3.80 (t, 1H, J ) 9.6 Hz, H-C6), 3.98
(q, 1H, J ) 9.0 Hz, H-C5), 4.07 (dt, 1H, J ) 3.0 Hz, 9.8 Hz,
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