Improved synthesis of myo-inositol 1-(4-nitrophenyl hydrogen phosphate), a chromogenic substrate for phosphatidylinositol-specific phospholipase C

Article abstract of DOI:10.1016/S0009-3084(97)00069-8

This paper describes an improved procedure for the synthesis of racemic myo-inositol 1-(4-nitrophenyl hydrogen phosphate) (NPIP) a useful substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific phospholipase C (PI-PLC).

Full text of DOI:10.1016/S0009-3084(97)00069-8

Chemistry and Physics of Lipids
89 (1997) 153–157
Short Communication
Improved synthesis of myo-inositol 1-(4-nitrophenyl hydrogen
phosphate), a chromogenic substrate for
phosphatidylinositol-specific phospholipase C
Aleksey V. Rukavishnikov a, Tatiana O. Zaikova a, O. Hayes Griffith a,b,*,
John F.W. Keana a
a Department of Chemistry, Uni6ersity of Oregon, Eugene, OR 97403-1229, USA
b Institute of Molecular Biology, Uni6ersity of Oregon, Eugene, OR 97403-1229, USA
Received 12 June 1997; accepted 15 August 1997
Abstract
This paper describes an improved procedure for the synthesis of racemic myo-inositol 1-(4-nitrophenyl hydrogen
phosphate) (NPIP) a useful substrate for the continuous spectrophotometric assay of phosphatidylinositol-specific
phospholipase C (PI-PLC). © 1997 Elsevier Science Ireland Ltd.
Keywords: Phosphoinositide-specific phospholipase C; Substrate analog; Enzyme assay; 4-Nitrophenol; p-Nitrophenol
1. Introduction
Chromogenic substrate analogs based on 4-ni-
trophenol are widely used in enzymology because
Abbre6iations: BzCl, benzoyl chloride; d, doublet; DMF,
N,N-dimethylformamide;
Ins(1:2cyc)P, -myo-inositol-1,2-cyclic-phosphate; m, multi-
plet; NMR, nuclear magnetic resonance; NPIP,
tol 1-(4-nitrophenyl hydrogen phosphate);
DMSO,
dimethyl
sulfoxide;
of the ease of detection of the highly colored
nitrophenylate anion (Walsh, 1979; Price and
Stevens, 1989). In order to make available the
corresponding substrate for phosphatidylinositol-
specific phospholipase C (Scheme 1) (PI-PLC), the
synthesis and characterization of D,L-myo-inositol
1-(4-nitrophenyl hydrogen phosphate) (NPIP) was
carried out in our laboratory several years ago
D
D,L
-myo-inosi-
PI-PLC,
phosphatidylinositol-specific phospholipase C; PPTs, pyri-
dinium p-toluenesulfonate; Py, pyridine; q, quartet; s, singlet;
t, triplet; THF, tetrahydrofuran; TsOH, p-toluenesulfonic
acid.
* Corresponding author. Fax: +1 541 3464643; e-mail:
griffith@oregon.uoregon.edu
0009-3084/97/$17.00 © 1997 Elsevier Science Ireland Ltd. All rights reserved.
PII S0009-3084(97)00069-8

154
A.V. Ruka6ishniko6 et al. / Chemistry and Physics of Lipids 89 (1997) 153–157
Scheme 1. The cleavage of NPIP to yield Ins(1:2cyc)P and the highly colored nitrophenolate anion
(Shashidhar et al., 1991). The continued interest in
the use of NPIP as a substrate for biophysical
studies on bacterial PI-PLCs (Ryan et al., 1996;
Martin and Wagman, 1996; Bruzik and Tsai, 1994;
Leigh et al., 1992) has prompted us to look for
ways to improve the original protocol. Here we
report procedures that simplify the synthesis and
increase the yield of NPIP (Scheme 2).
imidazole (1.27 g, 18.9 mmol) in pyridine (Py) (30
ml) tert-butyldimethylsilyl chloride (3.74 g, 13.6
mmol) was added while stirring and cooling at
−15°C. The mixture was left at −20°C for 48 h.
Methanol (2 ml) was added to the solution, the
solvents were then evaporated in a vacuum. The
residue was treated with water (20 ml). The
product was extracted with ether (3×20 ml). The
combined ether extracts were washed with brine,
dried over Na2SO4 and evaporated to dryness by
azeotropic evaporation with toluene (2×15 ml)
to give alcohol 4 as a viscous oil (5.04 g, 83%)
that solidified at room temperature (RT). Melting
point (m.p.) was 143–146°C (from ethyl acetate).
This compound was essentially pure by NMR
(greater then 90%) and was used for the next step
as received. Literature m.p. 142–145°C (Ward
and Young, 1988). 1H NMR (CDCl3): 1.07, s, 9H;
1.27, s, 3H; 1.41, s, 3H; 1.48, s, 3H; 1.54, s, 3H;
3.13, dd (J 8.7, 9.9 Hz), 1H; 3.66–3.76, m, 1H;
3.80–3.96, m, 2H; 3.97–4.10, m, 2H; 7.20–7.56,
m, 6H; 7.80, d (J 9.0 Hz), 2H; 7.84, d (J 9.0 Hz),
2H.
2. Experimental
2.1. General methods
Commercial reagents were used without further
purification. Melting points were taken on a Uni-
Melt Thomas Hoover melting point apparatus and
uncorrected. Dichloromethane was distilled from
P2O5. Di-iso-propylidene-myo-inositol 3 was pre-
pared from myo-inositol 2 as described (Gigg et al.,
1985). Column chromatography was performed on
Mallinckrodt silica gel 150 (60–200 mesh). Analyt-
ical thin layer chromatography was performed on
aluminum-backed silica gel 60 F254 plates and
visualization was effected with an ultraviolet lamp
or by spraying with alcohol solution of vanillin
(5%) and H2S04 (5%) followed by heating at 60–
2.3. 3-O-[(1,1-dimethylethyl)diphenylsilyl]-6-O-
(D
,L
-1-ethoxyethyl)-1,2:4,5-bis-O-
1
(1-methylethylidene)- -myo-inositol 5
D,L
70°C. H NMR spectra were recorded on a 300
MHz Varian Inova instrument; chemical shifts are
reported in delta units (l) referenced to residual
proton signals of the deuterated solvents (CHCl3,
l7.26; CH3SOCH2D, l2.49; HOD, l4.80).
To a solution of alcohol 4 (9.0 g, 18 mmol) and
ethyl vinyl ether (34.57 ml, 0.36 mol) in dry
methylene chloride (90 ml), pyridinium p-toluene-
sulfonate (PPTs) (1.97 g, 7.88 mmol) was added
while stirring at RT. The mixture was kept at RT
for 30 min. Then the solution was diluted with
ether (120 ml), washed with 5% NaHCO3 (40 ml),
brine (30 ml) and dried over Na2SO4. The solvents
2.2. 3-O-[(1,1-dimethylethyl)diphenylsilyl]-1,2:
4,5-bis-O-(1-methylethylidene)-D,L-myo-inositol 4
To a solution of diol 3 (3.17 g, 12.2 mmol) and

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155
Scheme 2. Synthesis of NPIP; i – (MeO)2CMe2/TsOH/DMF; ii – B2Cl/Py/DMF; iii – NaOH/MeOH; iv – tBuPh2SiCl/imidazole/
Py; v – ethyl vinyl ether/PPTs/CH2Cl2; vi – nBu4NF/THF; vii – H2O/Py; viii – AcOH/H2O.
were evaporated in vacuo to give the silyl deriva-
tive 5 as an oil.
ing solution was kept at RT for 20 h, then THF
was evaporated. The residue was partitioned be-
tween ethyl ether (30 ml) and water (20 ml). The
ether layer was removed, and the aqueous solution
was washed with ether (2×15 ml). The combined
organic extracts were washed with brine, dried
over Na2SO4 and evaporated to yield alcohol 6 as
a mixture with tert-butyldiphenylsilyl fluoride that
was used for the next step without any purifica-
The crude product was purified by column chro-
matography on 250 g of silica gel. The impurities
were eluted with 10:1 hexane/ether mixture. Silyl
derivative 5 was eluted with 10:2 hexane/ether
mixture. The eluate was evaporated in vacuum to
give desired product 5 (diastereomeric mixture) as
a clear oil, solidified at RT (6.8 g, 66%). M.p.
1
1
146–147°C, H NMR (CDCl3): 1.06, s, 18H; 1.16,
tion. H NMR (CDCl3): 1.18, t (J 6.9 Hz), 3H;
t (J 6.9 Hz), 3H; 1.17, t (J 6.9 Hz), 3H; 1.23–1.30,
m, 12H; 1.36, s, 3H; 1.38, s, 3H; 1.43, s, 3H; 1.44,
s, 3H; 1.54, s, 3H; 3.06–3.16, m, 2H; 3.42–3.57, m,
2H; 3.62–4.08, m, 12H; 4.93–5.01, m, 2H; 7.20–
7.43, m, 12H; 7.78–7.85, m, 8H.
1.19, t (J 6.9 Hz), 3H; 1.32–1.37, m, 12H; 1.40, s,
3H; 1.41, s, 3H; 1.43, s, 3H; 1.44, s, 3H; 1.53, s,
3H; 1.54, s, 3H; 3.26–3.36, m, 2H; 3.40–4.12, m,
12H; 4.43, t (J 4.5 Hz), 1H; 4.44, t (J 4.5 Hz), 1H;
t
4.97–5.05, m, 2H. (signals of BuPh2SiF: 1.06, s;
7.32–7.37, m, 7.72–7.75, m).
2.4. 6-O-(
(1-methylethylidene)-
D
,
L
-1-ethoxyethyl)-1,2:4,5-bis-O-
-myo-inositol 6
D
,L
2.5. 6-O-(
(1-methylethylidene)-D,L
D
,
L
-1-ethoxyethyl)-1,2:4,5-bis-O-
-myo-inositol 3-(4-nitro-
To a solution of silyl derivative 5 (6.8 g, 12
mmol) in THF (7 ml), was added 1 M solution of
tetrabutylammonium fluoride (17 ml). The result-
phenyl hydrogen phosphate) ammonium salt 7
4-Nitrophenyl phosphorodichloridate (9.16 g,

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A.V. Ruka6ishniko6 et al. / Chemistry and Physics of Lipids 89 (1997) 153–157
35.8 mmol) was dissolved in dry Py (100 ml), then
the solution of alcohol 6 (crude product from
above experiment, 3.95 g, 11.2 mmol) in Py (10 ml)
was added while stirring and cooling (ice/water
bath). The mixture was stirred for 16 h at ambient
temperature, then water (20 ml) was added while
cooling (ice/water bath). Stirring was continued for
approximately 1 h, then the reaction mixture was
diluted with chloroform (250 ml). The aqueous
layer was separated and the chloroform solution
was dried over anhydrous sodium sulfate. The
clear solution obtained was passed through a
column of silica gel and the column was further
eluted with ethyl ether to remove Py. The column
was then eluted with chloroform/methanol/ammo-
nium hydroxide (7:3:0.1) mixture. The solution
collected was evaporated in vacuum to obtain the
ammonium salt of phosphate 7 (diastereomeric
before, the diol 3 was prepared from commercially
available myo-inositol 2, using a procedure origi-
nally reported by Gigg et al. (1985). Next, instead
of protection with benzoyl chloride (BzCl) which
we employed previously, the hydroxyl group at the
first position was regioselectively protected using
tert-butyldiphenylsilyl chloride by a modification
of the procedure of Ward and Young (1988) to
produce the key intermediate 4 in 83% yield.
Silylation improved the regioselectivity, producing
a higher yield of the penta-protected inositol and
eliminating the necessity of chromatographic
purification at this step.
In order to protect the last free OH group of
alcohol 4, we selected the readily available and
inexpensive ethyl vinyl ether instead of 5,6-dihy-
dro-4-methoxy-2H-pyran which was used previ-
ously. If desired, the racemic alcohol 4 can be
prepared in enantiomerically pure form as reported
previously (Leigh et al., 1992) and converted into
1
mixture) as a white solid (5.0 g, 76%). H NMR
(dimethyl sulfoxide (DMSO)-d6): 0.94, s, 6H; 1.04,
t (J 7.2 Hz), 6H; 1.16, d (J 4.8 Hz), 3H; 1.17, d (J
5.1 Hz), 3H; 1.28, s, 12H; 1.31, s, 3H; 1.34, s, 3H;
3.99, t (J 5.7 Hz), 2H; 4.04–4.18, m, 2H; 4.35, t (J
4.5 Hz), 2H; 4.48–4.60, m, 2H; 4.82, q (J 5.4 Hz),
1H; 4.87, q (J 5.1 Hz), 1H; 7.34, d (J 9.0 Hz), 2H;
8.11, d (J 9.3 Hz), 2H.
the
procedure. However, it has been shown that B.
cereus PI-PLC is essentially stereospecific for the
enantiomer. The enantiomer is neither a sub-
D and L enantiomers of NPIP by the present
D
L
strate nor an inhibitor (Leigh et al., 1992). There-
fore, the racemic mixture can be used in many
situations without going to the extra effort of
separating the enantiomers.
2.6.
D,L-myo-Inositol 1-(4-nitrophenyl hydrogen
phosphate) ammonium salt 1
Continuing from 4, compound 5 was desilylated
with nBu4NF to produce the alcohol 6 in quantita-
tive yield. Alcohol 6 was phosphorylated with
4-nitrophenyl phosphorodichloridate as before to
yield phosphate 7. The final deprotection of phos-
phate 6 gave the desired final product 1 (NPIP). To
summarize, the procedure reported here is more
convenient, because it eliminates the chromato-
graphic separation step, utilizes a less expensive
reagent for protection of the 4-OH, and also results
in a higher yield of NPIP.
4-Nitrophenyl phosphate 7 (5.0 g, 9.0 mmol) was
suspended in 1:4 acetic acid/water solution (230
ml) and stirred at RT for 24 h. The clear solution
obtained was extracted with ether (3×40 ml) and
the aqueous solution was evaporated to dryness by
azeotropic evaporation with a 1:1 ethanol-toluene
mixture in vacuum to obtain the ammonium salt of
1
NPIP 1 as a white solid (4.5 g, 98%). H NMR
(D2O): 3.15, t (J 9.0 Hz), 1H; 3.38, dd (J 2.4, 9.9
Hz), 1H; 3.48, t (J 9.6 Hz), 1H; 3.61, t (J 9.6 Hz),
1H; 3.94, dt (J 2.4, 8.7 Hz), 1H; 4.10, br.s, 1H; 7.22,
d (J 9.0 Hz), 2H; 8.09, d (J 9.0 Hz), 2H.
Acknowledgements
We are pleased to acknowledge Drs Donald H.
LaMunyon, Miles P. Smith, M.S. Shashidhar,
and Margret Ryan for useful discussions. This
work was supported by NIH grants GM27137
and GM25698.
3. Results and discussion
In this revised procedure, two of the steps of the
original synthesis of NPIP were improved. As

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157
61, 8016–8023.
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