De novo synthesis of the enantiomers Ins(1,2,3,4)P4 and Ins(1,2,3,6)P4-regiospecificity of their enzymatic dephosphorylation

Article abstract of DOI:10.1016/S0957-4166(00)00063-X

The first total synthesis of Ins(1,2,3,4)P₄ and Ins(1,2,3,6)P₄ is presented. Starting from p-benzoquinone, we took advantage of the C₂-symmetry of conduritol-B intermediates. The target compounds were dephosphorylated by s

Full text of DOI:10.1016/S0957-4166(00)00063-X

Tetrahedron: Asymmetry 11 (2000) 1057±1061
De novo synthesis of the enantiomers Ins(1,2,3,4)P4 and
Ins(1,2,3,6)P4Ðregiospeci®city of their enzymatic
dephosphorylation
O. Plettenburg, S. Adelt, G. Vogel and H.-J. Altenbach*
Institut fur Organische Chemie und Biochemie, Bergische Universitat Wuppertal, Gaussstrasse 20,
È
È
42097 Wuppertal, Germany
Received 22 December 1999; accepted 11 February 2000
Abstract
The ®rst total synthesis of Ins(1,2,3,4)P4 and Ins(1,2,3,6)P4 is presented. Starting from p-benzoquinone,
we took advantage of the C2-symmetry of conduritol-B intermediates. The target compounds were
dephosphorylated by several enzymes, and the resulting InsP3 isomers were identi®ed. Some of these
enzymatic conversions were found to be preparatively applicable and to allow the synthesis of Ins(1,2,3)P3,
Ins(2,3,6)P3 and Ins(1,2,4)P3. # 2000 Elsevier Science Ltd. All rights reserved.
Phytases (InsP6-phosphohydrolases) are enzymes capable of hydrolyzing phytic acid to yield
phosphate and a series of lower phosphorylated inositols, ultimately in many cases free myo-
inositol itself. Ins(1,2,3,4)P41 1a and Ins(1,2,3,6)P42 1b have each been identi®ed, along with other
InsP4 intermediates, as products of several phytases. In addition, either 1a or 1b has been detected
in rat pancreatoma cells.3 In the course of studies of the p42IP4 receptor,4 we obtained evidence
that 1a or 1b occurs in rat brain tissues as well. These compounds may function physiologically as
iron carriers5 and calcium absorption enhancers.6
We present here the ®rst total synthesis of the enantiomeric pair 1a and 1b (Fig. 1).
Figure 1.
* Corresponding author. E-mail: orgchem@uni-wuppertal.de
0957-4166/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0957-4166(00)00063-X

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Dibromodiacetate 4, which serves as starting material, can be prepared on a large scale in only
three steps from p-benzoquinone (Scheme 1).
Scheme 1.
Enzymatic resolution of racemic 4 can easily be achieved with pig pancreas lipase;7,8 it delivers
3b and 4a in good yields and excellent ee (>99% after recrystallization in each case).
Basic treatment of 4a (KOH in anhydrous THF and limited amounts of methanol) leads to the
formation of the diepoxide 5a, which on double allylic opening with dibenzyl phosphate gives
diphosphoconduritol-B 6a (Scheme 2). The enantiomer 5b can be prepared from 3b by treatment
with KOH in THF without addition of methanol and allows the synthesis of the enantiomeric
series 6b, 7b,8 etc.
Scheme 2.
Their C2-symmetry makes conduritol-B derivatives ideal precursors for myo-inositol synthesis.
Thus acetylation with subsequent ¯ash cis-dihydroxylation with RuCl3/NaIO4 (86% overall yield)
gives the protected myo-inositol derivative 8a, which can be phosphorylated by reaction with
3-diethylamino-2,4,3-benzodioxaphosphepane in the presence of 1H-tetrazole and subsequent
oxidation of the resulting phosphite with m-CPBA to give 9a in 80% yield. Hydrogenation
followed by cleavage of the acetate groups in aqueous NaOH leads quantitatively to the target
compound 1a.9

O. Plettenburg et al. / Tetrahedron: Asymmetry 11 (2000) 1057±1061
1059
One focus of our research is the establishment of new, preparatively applicable enzymatic
dephosphorylation reactions. The dephosphorylation of 1a and 1b by several phytases was
examined in this context.
As an example of the class of so-called 6-phytases10 the phytase from Aspergillus ®cuum was
used, and for the 3-phytases10 wheat phytase and a phytase11 partially puri®ed from Dictyostelium
discoideum were employed. In addition, the hydrolysis by an InsP5/InsP4-phosphohydrolase from
Dictyostelium was examined. This enzyme was previously utilized in enzyme-assisted synthesis of
the enantiomeric pair Ins(1,5,6)P3 and Ins(3,4,5)P3.8 The reactions were stopped after 80±90%
dephosphorylation of the substrate. The tendency toward further hydrolyzation to InsP2, esti-
mated from the recovery rates of InsP3 isomers, decreases in the following order: Aspergillus
phytase (approximately 30% InsP3 recovered)>wheat phytase (ꢀ65%)>Dictyostelium phytase
(ꢀ80%)>InsP5/InsP4-phosphohydrolase (>95%).
This sequence would indicate, that the Dictyostelium phytase and the InsP5/InsP4-phospho-
hydrolase would be suitable for isolation of InsP3 isomers. The resulting InsP3 isomers were
identi®ed by HPLC-mdd.12 The ®ndings of these investigations are summarized in Table 1.
Table 1
Speci®city of enzymatic dephosphorylationa
The commercially available phytases from wheat and Aspergillus show poor enantioselectivity,
the two enantiomers being dephosphorylated at mirror positions. The regiospeci®city of the
Aspergillus phytase was also quite poor. As can be seen in Table 1, however, in two cases the
conversions led to a single InsP3 isomer (Scheme 3).
The InsP5/InsP4-phosphohydrolase from Dictyostelium is suitable for converting 1a into
Ins(1,2,3)P3 10 on a preparative scale, while Ins(2,3,6)P3 11b can be obtained by dephosphorylation
of 1b with the phytase from Dictyostelium. Both 10 and 11b have isomeric purities of >99% and
can be isolated from the reaction mixtures by conventional anion-exchange chromatography.
Two other reactions can also be used for InsP3 synthesis, but the presence of byproducts
requires high-resolution anion-exchange chromatography for puri®cation. Thus, Ins(1,2,4)P3 11a
can be obtained by dephosphorylation of 1a with the phytase from Dictyostelium. Furthermore,
racemic 1 can be used in the InsP5/InsP4-phosphohydrolase reaction to give the meso-compound
Ins(1,2,3)P3 10, which makes enzymatic resolution of 4 super¯uous.

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O. Plettenburg et al. / Tetrahedron: Asymmetry 11 (2000) 1057±1061
Scheme 3.
In summary, we present a new synthetic approach to two inositol tetrakisphosphates and three
inositol trisphosphates. Their ready availability may contribute to the elucidation of their physio-
logical functions. Very recently, for example, d/l-Ins(1,2,4)P3 11 was found to be a relatively
potent inhibitor of Ins(3,4,5,6)P4 1-kinase.13
Acknowledgements
We are grateful to Dr. W. V. Turner for stimulating discussions and critical reading of the
manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (grant VO 348/2-2)
and the Fonds der Chemischen Industrie.
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20
D
20
D
J=9.2 Hz, H-5); ‰ꢁŠ
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20
D
with published data for 1b (‰ꢁŠ +1.5, Ref. 2).

O. Plettenburg et al. / Tetrahedron: Asymmetry 11 (2000) 1057±1061
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