Inositol tetrakisphosphate from chicken eggshell

Article abstract of DOI:10.1016/j.tet.2019.130853

Unlike our previous study that identified D-myo-inositol 4,5-bisphosphate (Ins(4,5)P₂, 1) from ostrich eggshell, the compound myo-inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P₄, 2) was isolated as an almost racemic mixture from t

Full text of DOI:10.1016/j.tet.2019.130853

Journal Pre-proof
Inositol tetrakisphosphate from chicken eggshell
Taku Ito, Hajime Itokawa, Takanori Miyaki, Miho Kamimura, Mariko Hamano, Masaya
Nakata, Yoko Saikawa
PII:
S0040-4020(19)31261-X
https://doi.org/10.1016/j.tet.2019.130853
TET 130853
DOI:
Reference:
To appear in: Tetrahedron
Received Date: 15 November 2019
Revised Date: 28 November 2019
Accepted Date: 29 November 2019
Please cite this article as: Ito T, Itokawa H, Miyaki T, Kamimura M, Hamano M, Nakata M, Saikawa
Y, Inositol tetrakisphosphate from chicken eggshell, Tetrahedron (2019), doi: https://doi.org/10.1016/
j.tet.2019.130853.
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Inositol tetrakisphosphate from chicken eggshell
a
a
a
a
a
a
Taku Ito, Hajime Itokawa, Takanori Miyaki, Miho Kamimura, Mariko Hamano, Masaya Nakata, and
a
Yoko Saikawa *
a
Department of Applied Chemistry, Faculty of Science and Technology, Keio University, 2238522 Japan
Abstract: Unlike our previous study that identified
D-myo-inositol 4,5-bisphosphate (Ins(4,5)P , 1) from
2
ostrich eggshell, the compound myo-inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P , 2) was isolated as an
4
2
almost racemic mixture from the internal region of chicken eggshell. Furthermore, 2, 2-[ H]-2, and amorphous
CaCO were prepared as tools for assessment about transformation of CaCO during bone formation.
3
3
*
Corresponding author. Faculty of Science and Technology, Keio University, 2238522 Japan
E-mail address: saikawa@applc.keio.ac.jp (Y. Saikawa)
Keywords: inositol phosphate • eggshell • labeled compound • amorphous calcium carbonate
1
. Introduction
Approximately 250,000 tons of chicken eggshell waste is produced annually worldwide after the
yolk and albumen are processed for food. About 80% of the eggshells are simply burned and the rest are used
1
,2
as fertilizer and supplements, or in investigations of new applications. Eggshell calcium, in particular,
exhibits a unique feature as a calcium supplement which is easier to digest than pure calcium carbonate, as
3
demonstrated by reports involving piglet feeding experiments and suppression of bone loss in mice after
4
ovariectomy by feeding eggshells to mice. These results are not surprising since some eggshell calcium is
5
6
resorbed by a chick embryo during its bone formation. As a representative biomineral, eggshell is regarded
as a hybrid of inorganic (CaCO ) and organic materials, although chicken eggshell consists of mostly CaCO
3
3
7
(
97%). The rest of eggshell components are mainly composed of proteins whose roles for biomineralization
are known to be nucleation of CaCO precipitation, promotion of faster mineralization, and regulation of the
3
8
size of calcite crystals. However, a key molecule which regulates the structure and composition of CaCO for
3
smooth dissolution as the chick embryo resorbs eggshell during embryogenesis was unknown. Therefore, we
have focused on the eggshell components particularly in the internal region of eggshells where dissolution of
CaCO occurs. In our previous paper, we reported that -myo-inositol 4,5-bisphosphate (Ins(4,5)P , 1) was
D
3
2
identified in ostrich eggshell and 1 stabilized amorphous calcium carbonate (ACC), a soluble polymorph of
9
CaCO (Figure 1). In addition, 1 was turned out to be localized in an internal region of the ostrich eggshell,
3
suggesting the organophosphate as a regulator of CaCO polymorph to assist smooth dissolution of ostrich
3
eggshell. To evaluate the contribution of the above organophosphate to resorption of calcium ion by the
1

embryo, we planned to feed the ACC prepared in the presence of the organophosphate to an embryo in
5
shell-less culture, where an embryo is cultured in an artificial vessel and requires a calcium source. Although
large size of ostrich eggshells allowed chemical and analytical approaches, the shell-less culture of ostrich is
experimentally difficult. Therefore, we planned to use chicken embryo. We examined the interior of chicken
eggshell and identified myo-inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P , 2) as a major organophosphate
4
(
Figure 1).
Fig. 1.
D
-myo-inositol 4,5-bisphosphate (Ins(4,5)P , 1) and myo-inositol 1,4,5,6-tetrakisphosphate
2
(
Ins(1,4,5,6)P , 2).
4
2
2
. Results and Discussion
.1. Identification of an organophosphate in chicken eggshell
The chicken eggshell components were extracted according to our previous report for ostrich
9
eggshell. The inside of the eggshell was extracted for one hour by adding 10% acetic acid into the eggshell.
The acid etching dissolved CaCO in the eggshell, thereby removing the attached outer shell membrane. The
3
crude extract contained abundant calcium acetate, which could be removed by cation exchange
chromatography. Treatment of the extract with weakly acidic IRC-76 partially removed the calcium ions to
form white precipitates. Since phosphates strongly bind to calcium ion, the precipitates were expected to
contain calcium organophosphates. Thus, the precipitates were filtered and subjected to a second cation
1
exchange chromatography using strongly acidic SK1B resin. The H NMR spectrum of the resulting yellow
1
3
syrup contained doublets at around 2.0 ppm assigned to citric acid, and some polyol signals. Its C NMR
spectrum also contained major signals corresponding to citric acid and several peaks at 69–80 ppm. As
31
expected, the P NMR spectrum showed a major broad signal corresponding to phosphoric acid. This singlet
signal, however, was slightly asymmetrical, suggesting that another minor phosphorus compound existed in
1
31
the extract. To determine this, the H- P HMBC spectrum was obtained, which revealed the existence of a
3
1
1
minor phosphorus compound as four characteristic cross-peaks. P-correlated H signals were observed near
ppm that were similar to those of Ins(4,5)P . After continuous liquid-liquid extraction using diethyl ether to
4
2
remove the abundant citric acid and phosphoric acid, anion exchange chromatography was performed with an
1
0
Accell QMA Sep-pak, which was used to separate inositol phosphates. The extracts were loaded on the
Sep-pak cartridge, and successively washed with aqueous ammonium formate at different concentrations. The
1
major component eluted with 0.3 M ammonium formate and was recovered after lyophilization. The H NMR
spectrum of the major component showed six signals at around 4 ppm and four signals that were correlated to
2

1
31
phosphorus by the above H- P HMBC spectrum. A combination of DQF-COSY to analyze the coupling
constants and HMBC to determine the carbon skeleton suggested that the structure of this component was
1
1,12
myo-inositol 1,4,5,6-tetrakisphosphate (Ins(1,4,5,6)P , 2)
whose structure was confirmed by synthetic
4
1
2
methods (vide infra). Curiously, the optical rotation of the isolated compound was nearly zero. The reported
absolute value of the optical rotation of myo-inositol 1,4,5,6-tetrakisphosphate is small and is dependent on
1
3
the pH of the solution; the maximum absolute value reported was 10.2 in aqueous NaOH solution. However,
that of the isolated 2 in the basic solution prepared by adding NaOH was -0.77.
1
4
Both enantiomers,
D-myo-inositol 1,4,5,6-tetrakisphosphate ((−)-2)
and
D-myo-inositol
1
5
3
,4,5,6-tetrakisphosphate ((+)-2), have been identified in mixtures of intracellular metabolic products of
1
6
inositol 1,4,5-trisphosphate (Ins(1,4,5)P ) and inositol 1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6,)P ). In
3
5
3
these reports, 2-[ H] inositol was pre-incubated with the cells and the labeled metabolites were separated and
detected. In addition, the absolute configuration of the labeled 2 was determined by using enzymatic
1
4,15
conversion of one enantiomer into Ins(1,3,4,5,6)P5
or multistep degradation followed by enzymatic
17
oxidation. In spite of the subtle experiments, stereochemical complexities as seen in the present study were
often reported. The enantiomeric intricacy of the previously-isolated metabolite 2 was rationalized by the
following possibilities: acid-mediated interconversion of both enantiomers via phosphate migration in the
1
7
purification process (e.g. during solid phase extraction using 0.5 M HCl),
multi-enzymatic
1
6
phosphorylation-dephosphorylation process via Ins(1,4,5)P3 or Ins(1,3,4,5,6)P5, or enzymatic direct
1
8
conversion of enantiomers. However, the reason why Ins(1,4,5,6)P 2 was isolated as a racemic form in our
4
case is not clear.
In regard to the other possibilities, we believe that (−)- and (+)-2 in chicken eggshell are
metabolites of Ins(1,3,4,5,6)P , which is the major component that binds to hemoglobin in the erythrocytes of
5
1
9
birds, except for ostriches. . Eggshell forms in the oviduct surrounded by the blood vessels; through the blood
2
0
21
vessels, calcium ions and cuticle pigments (the degradation products of blood erythrocytes) are supplied
for shell formation. Similar to these components, Ins(1,4,5,6)P is expected to be supplied from the blood of
4
the mother bird. It is also expected that both enantiomers 2 are supplied through dephosphorylation of
Ins(1,3,4,5,6)P . We assume the reason for the difference in eggshell components (Ins(4,5)P 1 from ostrich
5
2
eggshell and Ins(1,4,5,6)P 2 from chicken eggshell) as follows. Most avian erythrocytes have a large quantity
4
1
9
of Ins(1,3,4,5,6)P5 whereas inositol tetrakisphosphate, whose entire structure was unknown, was isolated as
2
2
a major organophosphate from ostrich erythrocytes. The difference in inositol phosphates between ostrich
and chicken eggshells might stem from the difference in inositol phosphates in mother erythrocytes.
2
.2. Synthesis of racemic Ins(1,4,5,6)P 2 and ACC preparation
4
3

As it turned out that Ins (1,4,5,6)P 2 was the major organophosphate in chicken eggshell, we next
4
forward to synthesis of 2 and preparation of ACC in the presence of 2 as a calcium source to be fed shell-less
1
2
culture. Synthesis of myo-inositol 1,4,5,6-tetrakisphosphate has already been reported. This synthetic method
was modified to make it shorter and more efficient. A vicinal cis-diol moiety of myo-inositol (3) were
23
converted selectively to an acetal with 1,1-dimethylcyclohexane and p-toluene sulfonic acid in DMSO.
When the crude mixture was treated with triethylamine and CHCl , a white precipitate formed. The precipitate
3
was collected by filtration and washed with a mixture of CHCl , methanol, and water. The washings were
3
concentrated to give the desired acetal 4. Then, the other four hydroxy groups of acetal 4 were phosphorylated
with freshly prepared amidite reagent 5 activated by tetrazole followed by mCPBA oxidation to give
2
4
tetrakisphosphate 6 in 54% yield. Finally, phosphate 6 was subjected to hydrogenation catalyzed by
palladium on carbon to cleave the benzene dimethanol ester, and the resulting phosphoric acid acted as an acid
1
3
catalyst to hydrolyze the acetal moiety to afford myo-inositol 1,4,5,6-tetrakis phosphate (2) as a free acid.
Scheme 1. Synthesis of 2.
9
In our previous report, ACC preparation in the presence of Ins(4,5)P 1 was successful in Tris-HCl
2
2
5
buffer, according to literal method; 100 mM NaHCO with 2 mM of 1 in the buffer was mixed with 100 mM
3
CaCl were mixed to give white nano-sized precipitates. Similarly, we succeeded in preparing ACC by
2
addition of Ins(1,4,5,6)P 2 (2 mM) by same method as above; the ability to stabilize ACC was comparable to
4
those of 1. However, the method to prepare ACC required modification since the ACC prepared by this
procedure would contain other ions such as sodium, ammonium, and chloride ion, the latter of which exerts a
2
6
fatal toxic effect on chick embryos. Thus, we adopted a method with bubbling CO into Ca(OH) solution.
2
2
Through several investigation of conditions such as concentration of Ca(OH) and timing of bubbling, the
2
4

ACC without the above ions was obtained by the following procedure; Ins(1,4,5,6)P 2 was dissolved in water
4
and bubbled CO and then to the solution was added a cold solution of 13 mM Ca(OH) . The resulting white
2
2
suspension was directly evaporated to give the ACC precipitates whose polymorph was determined by X-ray
diffraction (XRD). As similar to the previously reported ACC, the peaks corresponding to calcite, the most
stable polymorph of CaCO , was not observed (Fig. 2), indicating formation of the desired amorphos particles.
3
Fig. 2. XRD spectra of the CaCO precipitates in absense (upper) and in presence (lower) of 0.1 mM 2.
3
2
2
.3. Synthesis of 2-[ H]-Ins(1,4,5,6)P 2
4
D
1
6
Since inositol phosphates are responsible for various physiological cellular responses, our interest
lies on whether the inositol phosphates in avian eggshell are resorbed by chick embryo concomitant with
3
calcium resorption, and how the compounds are metabolized. As mentioned above, permeation of H-labeled
inositol into various cells followed by recovery of labeled metabolites were well investigated to examine
metabolic pathway of inositol phosphates. These labeling studies have been replaced by LC-MS
metabolomics analysis and ELISA (Enzyme-Linked Immunosorbent Assay) so far, probably due to limitation
9,27
of radioisotopes usage. We envisioned mass-imaging analysis and NMR method to detect the metabolite of
2
2
in shell-less culture, therefore H-labeled 2 was designed. At first, we investigated the late-stage
introduction of deuterium; such as deprotection of acetal 6 followed by conversion of the resulting 2,3-diol.
2
However, any conversions after phosphorylation were unsuccessful. Thus, synthesis of 2 via 2-[ H]-inositol
(
3D) was attempted (Scheme 2). The report for the synthesis of 3D involves deuteride reduction of
2
8
scyllo-inosose which is prepared by microbiological oxidation of 3, but the direct reduction of scyllo-inosose
2
9
provides a mixture of diastereomers. As a practical synthesis of 3 , the afore-mentioned acetal 4 was used
D
and converted to benzyl myo-inosose 7 via transformation of functionalities with combination of the known
30,31
procedures.
Reduction of the resulting benzyl scyllo-inosose 7 with NaBD required a modification. In the
4
reported procedure, NaBH reduction was conducted at 0 ºC to give the protected myo-inositol with its epimer
4
5

3
1
32
in a ratio of ~4:1. We performed NaBD reduction at −78 ºC to provide 8 as a single isomer. The
4
D
following hydrogenation catalyzed by Pd(OH) in a mixed solvent afforded 3 quantitavely. The obtained 3
D
2
D
2
was converted to 2-[ H]-Ins(1,4,5,6)P 2 by similar manner to the route for the synthesis of 2 (Scheme 1). In
4
D
the acetalization, the amount of 3 , much more costly than inositol (3), was reduced and the product was
D
recovered as precipitates by addition of hexane. The cyclohexylidene 4 was phosphorylated with 5 and the
D
resulting phosphate 6 was subjected to hydrogenation to give the desired 2 .
D
D
Scheme 2. Synthesis of 2 via deuteride reduction.
D
3
. Conclusion
In summary, we identified myo-inositol 1,4,5,6-tetrakisphosphate (2) as a racemic mixture from
chicken eggshell. The difference in inositol phosphates between ostrich and chicken eggshells was revealed
and the result implies that these inositol phosphates in avian eggshell might be supplied from erythrocytes of
mother birds and that the inositol phosphate might be resorbed by chick embryo during dissolution of eggshell.
2
Now that ACC prepared in presence of 2 is ready for assay and 2-[ H]-myo-inositol 1,4,5,6-tetrakisphosphate
(
2D) is available, we will discuss contribution of inositol phosphates in avian eggshell during embryonic
resorption of calcium ion in shell-less culture of chicken embryo, in near future.
6

4
4
. Experimental
.1. General
Optical rotations were measured on a P-2100 polarimeter (JASCO, Tokyo). IR spectra were recorded on an
ALPHA FT-IR spectrometer (Bruker, Billerica, USA). The NMR spectra were recorded on an ECA-500
spectrometer (JEOL, Tokyo, Japan), an ECS-400 spectrometer (JEOL, Tokyo, Japan), and ECZ800R (JEOL,
Tokyo, Japan) at ambient temperature. The high-resolution electron spray ionization (ESI) mass spectra were
recorded on a LCT Premier XE (Waters, Milford, USA) by direct injection with solvent (50% MeCN–0.1%
aqueous formic acid). X-ray diffraction (XRD) was measured on an AXS D8 ADVANCE X-ray powder
diffractometer (Bruker, Billerica, USA) with Cu-Kα radiation at 40 kV and 40 mA.
4
.2. Materials
Chicken eggshells: For chicken eggs, commercial ones were used. The eggs were cut at the broad pole by an
eggshell cutter to form a hole of approximately 3 cm in diameter. After removal of yolk, albumen, and inner
shell membrane by exfoliating, the eggshells with outer shell membrane were stored at 4 ºC until use.
4
.3. Extraction and purification of organophosphate in chicken eggshell
The inside of the eggshell (2.04 kg, 410 eggshells) was filled with 10% aqueous acetic acid for 1 h at room
temperature. The solution was filtered through filter paper (No. 5A, Kiriyama, Tokyo, Japan) under suction.
Each filtrate from 25-30 eggshells were decalcified by cation exchange chromatography (Amberlite IRC-76,
+
4
00 mL, H form, Organo, Tokyo, Japan) and the resin was filtered off and washed with water (2 L). The
above resin treatment was repeated until all of the filtrate was treated. The filtrate and washings were
combined and concentrated in vacuo. Then H O (3 L) was added to the residue, and white precipitates (1.77 g)
2
were collected by centrifugal separation. The precipitates were decalcified by cation exchange resin (DIAION
+
SK1B, 40 mL, H form), and the resin was filtered off and washed with water. The filtrate and washings were
combined and concentrated in vacuo. The residue (950 mg) was dissolved in H O (3 mL) and continuously
2
extracted with diethyl ether for 14 days by using Soxhlet’s apparatus. The H O layer was concentrated in
2
vacuo, and the residue was obtained (76.6 mg). A part of the residue (18.1 mg) was subjected to anion
–
exchange chromatography (Accell QMA SEP-PAK, HCO2 form, Waters, Milford, USA). The resin was
successively washed with water (Fr. 1, 14.6 mL), 0.1 M aqueous NH HCO (Fr. 2, pH 4.60, 7.3 mL), 0.2 M
4
2
aqueous NH HCO (Fr. 3, pH 4.78, 7.3 mL), 0.3 M aqueous NH HCO (Fr. 4, pH 4.82, 7.3 mL), 0.4 M
4
2
4
2
aqueous NH HCO (Fr. 5, pH 4.73, 7.3 mL), and 1.0 M aqueous NH HCO (Fr. 6, pH 4.75, 7.3 mL). Fr. 4 was
4
2
4
2
lyophilized to give the residue (16.5 mg) which contained (±)-myo-inositol 1,4,5,6-tetrakisphosphate (2).
4
.3.1. Analytical data of (±)-myo-inositol 1,4,5,6-tetrakisphosphate (2): a colorless foam; [α] −0.77 (c 0.84,
D
1
3
1
H O, sodium salt) (
2
D
-myo-inositol 1,4,5,6-tetrakisphosphate lit. [α] −10.2 (c 2.46, H O, sodium salt); H
D
2
NMR (500 MHz, D O, DSS = 0.00 as an external standard): δ 3.76 (dd, J = 9.3, 2.5 Hz, 1H, H-3), 4.13 (ddd, J =
2
9
.5, 9.5, 2.5 Hz, 1H, H-1), 4.17 (ddd, J = 9.5, 9.5, 9.3 Hz, 1H, H-5), 4.25 (br dd, 1H, H-2), 4.35 (ddd, J =9.3, 9.3,
7

1
3
9
.0 Hz, 1H, H-4), 4.47 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H, H-6); C NMR (200 MHz, D O, DSS = 0.00 as an external
2
3
1
standard): δ 72.8 (C3), 73.7 (C2), 77.2 (C1), 79.0 (C6), 79.7 (C4), 80.3 (C5); P NMR (202 MHz, D O, H PO
4
2
3
3
1
=
0.00 as an external standard): δ −0.13 (P-1), 0.58 (P-6), 0.66 (P-4), 0.73 (P-5); P chemical shifts were
1
31
determined by H- P HMBC.
4
.4. Synthesis of (±)-Ins(1,4,5,6)P4
2
3b
4
.4.1. 1,2-O-Cyclohexylidene-myo-inositol (4).
A mixture of inositol (3) (1.00 g, 5.56 mmol),
1
,1-dimethoxycyclohexane (0.42 mL, 2.8 mmol), TsOH•H O (25.7 mg, 0.132 mmol), and DMSO (1.1 mL) was
2
stirred at 120 ºC for 3 h under Ar atmosphere. After cooling to rt, CHCl (16.6 mL) and Et N (0.002 mL) were
3
3
added with stirring to give precipitates. The precipitates was filtered off and the filtrate was concentrated. The
resulting residue was suspended in 5:3:0.1 CHCl –MeOH–H O and the precipitates was filtered and washed to
3
2
1
give 4 (0.337 g, 47%, based on 1,1-dimethoxycyclohexane); R = 0.73 (5:3:0.1 CHCl –MeOH–H O); H NMR
f
3
2
(
400 MHz, D O, HOD = 4.79): δ 1.36–1.74 (m, 10H, cyclohexylidene), 3.23 (dd, J = 11.5, 9.6 Hz, 1H, H-5),
2
3
.55 (dd, J = 11.5, 8.0 Hz, 1H, H-6), 3.62 (dd, J = 9.6, 9.6 Hz, 1H, H-4), 3.82 (dd, J = 9.6, 4.2 Hz, 1H, H-3), 4.02
(
dd, J = 8.0, 4.8 Hz, 1H, H-1), 4.45 (dd, J = 4.8, 4.2 Hz, 1H, H-2).
4
.4.2.
2,3-O-Cyclohexylidene-1,4,5,6-tetrakis-O-(1,5-dihydro-3-oxido-2,4,3-benzodioxaphosphepin-3-yl)
myo-inositol (6). To a stirred solution of 4 (800 mg, 3.08 mmol) in dry CH Cl (20.7 mL) were added at 0 ºC
2
2
24
1
H-tetrazole (1.94 g, 27.7 mmol) and N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (5)
3.99 mL, 18.5 mmol). After 1 h at rt, a solution of mCPBA (6.83 g, 27.7 mmol, contains ca. 30% water) in
dry CH Cl (31.9 mL) was added dropwise at –78 ºC before the mixture was warmed to rt and stirred for 1 h
(
2
2
at rt. Then the mixture was diluted with CH Cl and washed consecutively with aqueous NaHSO , saturated
2
2
3
aqueous NaHCO , and saturated aqueous NaCl, dried over Na SO , and concentrated in vacuo. The residue
3
2
4
was purified by column chromatography on silica gel (230 g, 3:1 CHCl –acetone) followed by precipitation in
3
EtOAc to give 6 (1.73 g, 58%) as white solids; R = 0.40 (2:1 EtOAc–benzene); mp: 205 ºC (dec); IR (KBr,
f
−
1
1
νmax cm ) 3483, 2938, 2898, 1462, 1371, 1294, 1225, 1020, 849, 733; H NMR (400 MHz, CDCl , CHCl =
3
3
7
4
.26): δ 1.40–1.95 (m, 10H, C H ), 4.35 (dd, J = 7.8, 4.6 Hz, 1H, H-3), 4.79 (dd, J = 4.6, 4.4 Hz, 1H, H-2),
6 10
.87 (dd, J = 13.2, 11.6 Hz, 1H, CH Ph), 4.90 (ddd, J = 9.6, 9.4, 9.4 Hz , 1H, H-5), 4.94 (ddd, J = 9.4, 9.2, 7.8
2
Hz, 1H, H-4), 5.01 (overlapped, 1H, H-1), 4.99–5.21 (m, 7H, CH Ph), 5.33 (ddd, J = 9.4, 9.2, 9.2 Hz , 1H,
2
1
3
H-6), 5.36 (dd, J = 13.6, 12.4 Hz, 1H, CH Ph), 5.45–5.72 (m, 7H, CH Ph), 7.29–7.43 (m, 16H, C H ); C
2
2
6
4
NMR (125 MHz, CDCl , CHCl = 77.00) δ 23.68, 24.78, 29.62, 35.30, 37.54, 68.62 (d, J = 6.5 Hz), 69.08 (d,
3
3
J = 7.8 Hz), 69.14 (d, J = 7.3 Hz), 69.25 (d, J = 7.8 Hz), 69.36 (d, J = 8.0 Hz), 69.43 (d, J = 6.2 Hz), 69.50 (d,
J = 7.8 Hz), 69.84 (d, J = 7.8 Hz), 73.74 (C-2), 74.19 (bd, C-1), 76.31 (C-3), 76.41 (bd, C-6), 77.00 (C-4),
7
1
9.11 (C-5), 112.49, 128.81, 128.99, 129.11 (3C), 129.20 (4C), 129.28, 129.36 (2C), 129.45 (3C), 129.51,
31
35.17, 135.21, 136.46 (2C), 135.60, 135.69, 135.74, 135.81; P NMR (202 MHz, CDCl , external reference:
3
8

+
triphenyl phosphate = –17.3) δ –1.95, –4.16, –4.24, –4.99; HRMS (m/z): [M+H] calcd. for C H O P ,
4
4
48 18 4
9
89.1868; found 989.1869.
1
3
4
.4.3. 1,4,5,6-Tetrakis(dihydrogen phosphate) myo-inositol (2). To a stirred solution of 6 (19.9 mg, 0.0202
mmol) in 4:1 MeOH–H O (2.17 mL) was added at 0 ºC 10% Pd on carbon (26.9 mg). The mixture was
2
vacuum degassed three times and refilled with Ar before replaced by H . After 15.5 h at rt, the mixture was
2
filtered through filter paper under suction and the filtrate was concentrated in vacuo to afford 2 (quant); R =
f
1
0
.00 (3:1 CHCl –acetone); H NMR (500 MHz, D O, DSS = 0.00 as an external standard): δ 3.75 (dd, J = 9.3,
3
2
2
.5 Hz, 1H, H-3), 4.12 (ddd, J = 9.5, 9.5, 2.5 Hz, 1H, H-1), 4.16 (ddd, J = 9.5, 9.5, 9.3 Hz, 1H, H-5), 4.25 (br
1
3
dd, 1H, H-2), 4.34 (ddd, J =9.3, 9.3, 9.0 Hz, 1H, H-4), 4.46 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H, H-6); C NMR
(
125 MHz, D O, DSS = 0.00 as an external standard): δ 72.7 (C3), 73.6 (C2), 77.2 (C1), 79.0 (C6), 79.7 (C4),
2
3
1
8
0.3 (C5); P NMR (202 MHz, D O, H PO = 0.00 as an external standard): δ −0.13 (P-1), 0.58 (P-6), 0.58
2 3 4
3
1
1
31
(
P-4), 0.65 (P-5).; P chemical shifts were determined by H- P HMBC.
4
.5. Preparation of ACC in the presence of 2.
To a cooled (0 ºC), aqueous solution of 2 (0.40 mM in 50 mL water) was bubbled with CO until saturation.
2
Then a cooled solution of Ca(OH) (13 mM in 150 mL water) was dropped into the above CO -filled solution
2
2
over 0.5 min with stirring at 0 ºC. Then the suspension was filtered and the filtrate was dried in vacuo to give
a white ACC powder. The obtained ACC powder was put into the well of 25 mm in diameter of glass sample
holder and pressed flat with a glass slide. The XRDs were measured to determine the polymorph of the above
sample; the intensity of calcite signal compared with those of control sample (calcite calcium carbonate) is
assessed as it is inversely correlated with the content of ACC.
2
4
.6. Synthesis of 2-[ H]-Ins(1,4,5,6)P 2
4
D
3
1
4
.6.1. 1,2,3,4,5-Penta-O-benzyl-myo-inosose (7).
Benzyl scyllo-inosose 7 was synthesized from
30,31
31
1
cyclohexylidene 4 according to the known procedure.
7; R = 0.33 (5:1 hexane–EtOAc); H NMR (400
f
MHz, CDCl , CHCl = 7.26) δ 3.61 (t, J =9.2 Hz, 2H, H-4, H-6), 3.86 (t, J =9.2 Hz, 1H, H-5), 4.15 (d, J =9.2
3
3
Hz, 2H, H-1, H-3), 4.53 (d, J =11.2 Hz, 2H, CH Ph), 4.73–4.93 (m, 8H, CH Ph), 7.58–8.11 (m, 25H, C H ).
2
2
6
5
2
4
.6.2. 1,3,4,5,6-Penta-O-benzyl-[2- H] myo-inositol (8 ). Benzyl scyllo-inosose 7 (1.00 g, 1.59 mmol) was
D
dissolved in 4:1 CH Cl –MeOH (79.6 mL). To this solution sodium borodeuteride (141 mg, 3.18 mmol) was
2
2
added in one portion at –78 ºC and the reaction mixture was stirred at –78 ºC for 0.5 h. The reaction was
quenched by adding saturated solution of ammonium chloride (1 mL). The resulting mixture was concentrated
under reduced pressure and the resulting suspension was extracted with EtOAc. The extract was filtered
through a pad of SiO with EtOAc, and the filtrate was concentrated under reduced pressure. The residue was
2
recrystallized in hexane to give 8 (918 mg, 92%) as colorless solids; R = 0.47 (2:1 hexane–EtOAc); mp
D
f
9

−
1
1
21.5–124.5 ºC; IR (KBr, νmax cm ) 3453, 3223, 3088, 3065, 3032, 2920, 2864, 1497, 1454, 1356, 1151,
1
1
133, 1067, 1030, 754, 721, 700.; H NMR (500 MHz, CDCl , CHCl = 7.26) δ 2.47 (s, 1H, OH) 3.38 (d, J
3
3
=9.5 Hz, 2H, H-1, H-3), 3.45 (dd, J =9.5, 9.5 Hz, 1H, H-5), 4.00 (dd, J =9.5, 9.5 Hz, 2H, H-4, H-6), 4.68–
1
3
4
.91 (m, 10H, CH Ph), 7.24–7.35 (m, 25H, C H ).; C NMR (125 MHz, CDCl , CHCl = 77.00) δ 66.94 (bt,
2 6 5 3 3
C2), 72.72 (CH Ph), 72.72 (CH Ph), 75.93 (CH Ph), 75.93 (CH Ph), 75.93 (CH Ph), 79.69 (C1, C3), 81.17
2
2
2
2
2
(
(
C4, C6), 83.13 (C5), 127.54 (Ph), 127.56 (Ph), 127.56 (Ph), 127.82 (Ph), 127.82 (Ph), 127.82 (Ph), 127.82
Ph), 127.85 (Ph), 127.85 (Ph), 127.85 (Ph), 127.85 (Ph), 127.85 (Ph), 127.99 (Ph), 127.99 (Ph), 127.99 (Ph),
1
1
27.99 (Ph), 128.33 (Ph), 128.33 (Ph), 128.33 (Ph), 128.33 (Ph), 128.33 (Ph), 128.45 (Ph), 128.45 (Ph),
28.45 (Ph), 128.45 (Ph), 137.91 (Ph), 137.91 (Ph), 138.64 (Ph), 138.69 (Ph), 138.69 (Ph).; HRMS m/z
+
(
M+Na) calcd for C H DO Na 654.2942, found 654.2957.
4
1
41
6
2
29a
4
.6.3. [2- H] myo-Inositol (3 ). 8 (350 mg, 0.554 mmol) and 20 wt% Pd(OH) (87.5 mg, 25 wt %) were
D D 2
suspended in 10:10:1 (v/v) CH Cl –MeOH–H O (8.0 mL). The suspension was stirred at rt under H
2
2
2
2
2
9a
atmosphere for 22 h. The catalyst (Pd(OH) ) was filtered off and the filtrate was evaporated to give 3
(105
2
D
1
mg, >100%) as white solids; R = 0.00 (3:1 hexane–EtOAc); H NMR (400 MHz, D O, HOD = 4.79) δ 3.27
f
2
(
dd, J =9.6, 9.6 Hz, 1H, H-5), 3.52 (d, J =9.6 Hz, 2H, H-1, H-3), 3.62 (dd, J =9.6, 9.6 Hz, 2H, H-4, H-6).
2
4
.6.4. 1,2-O-Cyclohexylidene-[2- H]myo-inositol (4 ). A mixture of 3 (40.9 mg, 0.266 mmol),
D
D
1
,1-dimethoxycyclohexane (0.0815 mL, 0.542 mmol), TsOH-H O (2.0 mg, 0.011 mmol), and DMSO (0.2
2
mL) was stirred at 120 ºC for 1.5 h under Ar atmosphere. After cooling to rt, CHCl (1.35 mL), Et N (0.002
3
3
mL, 0.011 mmol), and hexane (5 ml) were added to give precipitates. Filtration of the suspension and washing
with hexane afforded 4 (27.5 mg, 47%) as white solids; R = 0.73 (5:3:0.1 CHCl –MeOH–H O); mp 178.5–
D
f
3
2
−
1
1
1
82.0 ºC; IR (KBr, νmax cm ) 3376, 2935, 2859, 1447, 1366, 1279, 1253, 1231, 1165, 1151, 1121, 1067,
1
014, 962, 927, 894, 750, 716, 603.; H NMR (500 MHz, D O, TSP = 0.00) δ 1.39–1.76 (m, 10H,
2
cyclohexylidene), 3.27 (dd, J = 10.0, 10.0 Hz, 1H, H-5), 3.58 (dd, J = 8.0, 10.0 Hz, 1H, H-6), 3.65 (dd, J =
1
3
1
0.0, 10.0 Hz, 1H, H-4), 3.85 (d, J = 10.0 Hz, 1H, H-3), 4.06 (d, J = 8.0 Hz, 1H, H-1).; C NMR (125 MHz,
D O, TSP = 0.00) δ 26.08, 26.39, 27.10, 37.43, 40.27, 72.34, 75.09, 75.47, 77.90, 78.12 (t, J = 22.6 Hz),
2
+
8
0.90, 114.31.; HRMS m/z (M+H) calcd for C H DO 262.1401, found 262.1380.
1
2
20
6
4
.6.5. 2,3-O-Cyclohexylidene-1,4,5,6-tetrakis-O-(1,5-dihydro-3-oxido-2,4,3-benzodioxaphosphepin-3-yl)-
2
[
2- H]myo-inositol (6 ). To a solution of 4 (43.3 mg, 0.164 mmol) and 1H-tetrazole (104 mg, 1.48 mmol)
D
D
2
4
in dry CH Cl (1.5 mL) was added N,N-diethyl-1,5-dihydro-2,4,3-benzodioxaphosphepin-3-amine (5) (0.21
2
2
mL, 0.98 mmol), and the mixture was stirred for 1 h at rt under Ar atmosphere. The mixture was cooled to –78
ºC, and a solution of mCPBA (258 mg, 1.48 mmol) in dry CH Cl (2.6 mL) was added. Stirring was continued
2
2
for 1 h at rt. Then the reaction mixture was diluted with CH Cl (10 mL × 5) and washed consecutively with
2
2
aqueous NaHSO (10 mL × 5), saturated aqueous NaHCO (10 mL × 5), and brine (10 mL × 5). After
3
3
evaporation of CH Cl , the resulting oil was chromatographed on silica gel (16 g, 3:1 CHCl –acetone) to give
2
2
3
10

the mixture including 6 . The mixture was recrystallized in EtOAc to give 6 (79.6 mg, 49%) as colorless
D
D
−
1
solids; R = 0.27 (3:1 CHCl –acetone); mp: >200 ºC (decomp.); IR (KBr, ν cm ) 3477, 3000, 2938, 2897,
f
3
max
2
861, 1500, 1462, 1371, 1295, 1225, 1196, 1196, 1164, 1124, 1050, 1019, 944, 848, 732, 665, 623, 603, 561,
1
4
61, 431.; H NMR (500 MHz, CDCl , CHCl = 7.26) δ 1.38–1.97 (m, 10H, C H ), 4.32 (d, J = 8.0 Hz, 1H,
3
3
6
10
H-3), 4.87 (dd, J = 13.5, 7.5 Hz, 1H, CH Ph), 4.88 (ddd, J = 7.5, 7.5, 7.5 Hz , 1H, H-5), 4.96 (ddd, J = 7.5,
2
7
1
.5, 7.5 Hz, 1H, H-4), 4.98 (overlapped, 1H, H-1) 4.93–5.21 (m, 7H, CH Ph), 5.34 (ddd, J = 7.5, 7.5, 7.5 Hz ,
2
1
3
H, H-6) 5.35 (dd, J = 13.5, 7.5 Hz, 1H, CH Ph), 5.45–5.72 (m, 7H, CH Ph), 7.30–7.42 (m, 16H, C H ).; C
2
2
6
4
NMR (125 MHz, CDCl , CDCl = 77.00) δ 23.72, 23.83, 24.82, 35.37, 37.57, 68.69 (d, J = 6.5 Hz), 69.17 (d,
3
3
J = 6.6 Hz), 69.22 (d, J = 5.9 Hz), 69.32 (d, J = 7.8 Hz), 69.47 (d, J = 8.0 Hz), 69.50 (d, J = 6.1 Hz), 69.56 (d,
J = 7.8 Hz), 69.92 (d, J = 7.1 Hz), 74.19 (br d, C-1), 76.23 (C-3), 76.14 (b, C-5, C-6), 79.36 (br d, C-4), 90.03
(
br t, C-2), 112.60, 128.84, 129.02, 129.16, 129.16, 129.23, 129.23, 129.27, 129.27, 129.30,129.30, 129.35,
31
1
29.43,129.43, 129.51, 129.51, 129.59, 135.16, 135.21, 135.48, 135.51, 135.66, 135.73, 135.76, 135.86.; P
NMR (202 MHz, CDCl , external reference: triphenyl phosphate = –17.3) δ –1.98, –4.15, –4.24, –4.99.;
3
+
HRMS m/z (M+H) calcd for C H DO P 990.1932, found 990.1910.
4
4
48
18 4
2
4
.6.6. 1,4,5,6-Tetrakis(dihydrogen phosphate)-[2- H]myo-inositol (2 ). To a suspension of 6 (41.0 mg,
D D
0
.0414 mmol) in 4:1 MeOH–H O (3.97 mL) was added 10% Pd–C (49.2 mg) at 0 ºC under Ar atmosphere.
2
The suspension was stirred at rt under H atmosphere for 5 h. The catalyst (Pd–C) was filtered off and the
2
−
1
filtrate was evaporated to give (±)-2 (21.1 mg, 100%); R = 0.00 (3:1 CHCl –acetone); IR (KBr, ν cm )
D
f
3
max
1
3
424, 2934, 2378, 2345, 1721, 1688, 1630, 1384, 1152, 1041, 966, 825, 497.; H NMR (500 MHz, D O, TSP
2
=
0.00) δ 3.81 (d, J = 9.5 Hz, 1H, H-3), 4.28 (dd, J = 9.5, 9.5 Hz, 1H, H-1), 4.31 (ddd, J = 9.5, 9.5, 9.5 Hz,
1
3
1
H, H-4), 4.44 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H, H-5), 4.57 (ddd, J = 9.5, 9.5, 9.5 Hz, 1H, H-6).; C NMR (125
MHz, D O, TSP = 0.00) δ 72.33 (C-3), 72.69 (br t, C2), 77.68 (br d, C-1), 79.45 (br d, C-6), 80.42 (br d, C-4),
2
3
1
8
0.57 (br d, C-5). P NMR (202 MHz, D O, external reference: triphenyl phosphate = –17.3): δ −0.25, 0.36
2
(
2P), 0.65.
Acknowledgements
We thank Hiroyuki Shidara (QP Corporation) for technical advice to prepare ACC. This work was
partially supported by a Grant-in-Aid for Challenging Exploratory Research (25560406) from the
Japan Society for the Promotion of Science (Y.S.) and Kieikai Research Foundation (Y.S.).
Conflicts of interest
There are no conflicts to declare.
Supplemental data
Supplemental data to this article can be found online at..
11

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2 To determine the stereochemistry of the product, ketone
condition as described. The resulting alcohol was identical with the precursor of
,3,4,5,6-pentabenzyl myo-inositol.
7 was reduced by NaBH under same
4
7
,
1
13

•
•
•
•
Myo-inositol 1,4,5,6-tetrakisphosphate was isolated from chicken eggshell.
The racemic inositol tetrakisphosphate and its deuterated analog were synthesized.
ACC as a calcium source for chick embryo was prepared.
The origin of inositol phosphates in eggshell were discussed.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: