Rapid and efficient routes to phosphatidylinositol 3,4,5-trisphosphates via myo-inositol orthobenzoate

Article abstract of DOI:10.1016/j.tetlet.2007.01.095

Efficient routes to two phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃] analogues with different acyl chains have been developed by using cheaply available myo-inositol as the starting material. The high yield of the orthobenzoate derivative, preferential formation of the required protected inositol diastereomer in its desymmetrization and ease of separation make the synthesis expedient, economical and high yielding. Due to the inherent problem of racemization of diacylglycerol (DAG), the synthesis of phosphatidylinositol phosphates [PIPns] with unambiguous stereochemical purity has always been difficult. Our methodology excludes the possibility of racemization in the DAG unit and thus provides access to PtdIns(3,4,5)P₃ of high optical purity. Since the acyl functionalities are introduced last, the methodology reported is amenable to the synthesis of PtdIns(3,4,5)P₃ with any acyl chain (or even a library of analogues).

Full text of DOI:10.1016/j.tetlet.2007.01.095

Tetrahedron Letters 48 (2007) 1923–1926
Rapid and efficient routes to phosphatidylinositol
3,4,5-trisphosphates via myo-inositol orthobenzoate
Kana M. Sureshan, Andrew M. Riley and Barry V. L. Potter*
Wolfson Laboratory of Medicinal Chemistry, Department of Pharmacy and Pharmacology,
University of Bath, Claverton Down, Bath BA2 7AY, UK
Received 6 December 2006; revised 11 January 2007; accepted 17 January 2007
Available online 23 January 2007
Abstract—Efficient routes to two phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3] analogues with different acyl chains
have been developed by using cheaply available myo-inositol as the starting material. The high yield of the orthobenzoate derivative,
preferential formation of the required protected inositol diastereomer in its desymmetrization and ease of separation make the syn-
thesis expedient, economical and high yielding. Due to the inherent problem of racemization of diacylglycerol (DAG), the synthesis
of phosphatidylinositol phosphates [PIPns] with unambiguous stereochemical purity has always been difficult. Our methodology
excludes the possibility of racemization in the DAG unit and thus provides access to PtdIns(3,4,5)P3 of high optical purity. Since
the acyl functionalities are introduced last, the methodology reported is amenable to the synthesis of PtdIns(3,4,5)P3 with any acyl
chain (or even a library of analogues).
Ó 2007 Elsevier Ltd. All rights reserved.
The chemistry and biology of phosphorylated inositols
has become an intense area of research during the last
two decades due to their involvement in various cellular
signalling processes.1 Among the seven known phospha-
tidylinositol phosphates (PIPns), phosphatidylinositol
3,4,5-trisphosphate [PtdIns(3,4,5)P3, 1], (Fig. 1) has
attracted much attention due to its various biological
roles2 in Akt signalling, signal transduction3 non-capaci-
tative calcium influx,4 cell regulation,5 etc. The fact that
PtdIns(3,4,5)P3 interacts with many targets, namely,
protein kinases, phospholipases, G-nucleotide exchange
factors, etc, suggests that its role in cell signalling is par-
ticularly complex and there are many unsolved puzzles
regarding the function of this lipid. Considering the cen-
tral role of PtdIns(3,4,5)P3 in these various phenomena
and the fact that it occurs in nature in very small quan-
tities, it is not surprising that there have been several
attempts to synthesise this lipid.6 As a part of our ongo-
ing programme to synthesise various phosphoinositols,
we were interested in developing an efficient synthetic
protocol for PtdIns(3,4,5)P3.
After the realization that D-myo-inositol 1,4,5-trisphos-
phate acts as a second messenger in cellular signal trans-
duction, much attention has been focused on the
development of efficient routes to various phosphoinos-
itols.1 Among various methods for the synthesis of
phosphoinositols, those starting from the cheaply avail-
able myo-inositol (2) are advantageous in terms of cost,
time and practicality. During the synthesis of phosphoi-
nositols, various strategies for the selective protection
and deprotection of inositol hydroxyl groups7 have been
developed. Initial protection of inositol hydroxyl groups
as orthoesters for synthetic purposes has often been
preferred over the more classical ketalisation methods
in recent years. While the ketalisation of inositol gives
a mixture of four ketals making the separation tedious
and reducing the yield of the required ketal (less than
25%), the orthoesterification gives a single product, a
myo-inositol 1,3,5-orthoester, in high yield (about
90%). Also, the known strategies for the selective protec-
tion of the remaining three free hydroxyl groups (2-OH,
O
OH
O
H2O3PO
H2O3PO
H2O3PO
O
O
O
P
O
HO
O
HO
1.(2-O-arachidonyl-1-O-stearoyl-sn-glyceryl) Phosphatidylinositol 3,4,5-trisphosphate
Figure 1.
*
Corresponding author. Tel.: +44 1225 386693; fax: +44 1225
386114; e-mail: b.v.l.potter@bath.ac.uk
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.01.095

1924
K. M. Sureshan et al. / Tetrahedron Letters 48 (2007) 1923–1926
4-OH, or 6-OH) and the selective partial cleavage of
the orthoester cage with reducing agents in protected
orthoesters (to regenerate the 1(3)-OH or 5-OH) make
myo-inositol 1,3,5-orthoesters versatile intermediates
for synthetic purposes. For instance, orthoesters 3–5
(Fig. 2) have been synthesized and 3 and 4 have been
used extensively for phosphoinositol synthesis.1 In addi-
tion to the above mentioned reactions of orthoesters,
acid hydrolysis of a 2-O-protected orthoester leaves
the corresponding ester functionality at the 1(3)-O-posi-
tion, except in the case of orthoformate as a formate
ester is too labile to hydrolysis.8 We envisioned that if
a stable ester that can act as a temporary protecting
group is introduced at the 1-O-position by this strategy,
this reaction can be exploited for the efficient synthesis
of phosphatidylinositol phosphates. We report herein
efficient, rapid and high-yielding syntheses of two
PtdIns(3,4,5)P3 analogues, exploiting myo-inositol
orthobenzoate, 8 (which will leave a stable benzoate
ester at the 1-O-position on opening) as the synthetic
precursor. Although the natural PtdIns(3,4,5)P3 (and
other PIPns) has an unsaturated arachidonyl chain at
the sn2 position of the lipid, the unsaturated lipids often
aggregate (due to their folding) making biological
studies difficult in aqueous solutions. For these reasons,
saturated and short chain lipids (more soluble in water)
are preferred for biological studies.6k,9 Thus, we have
chosen two different saturated acyl chains in our synthe-
sis of PtdIns(3,4,5)P3 analogues.
We have recently developed10 methodologies for the
synthesis and resolution of myo-inositol 1,3,5-ortho-
benzoate (8) and derivatives that relate to earlier work
on orthoester desymmetrization.11 Thus, the orthoester
triol 8 on desymmetrization by esterification with cam-
phanoyl chloride provided diastereomer 9 (Scheme 1)
preferentially, which was converted to dibenzyl ether
10 via protection of the 6-OH as the MIP acetal, deacyl-
ation, benzylation and deprotection of the MIP acetal.
Thus, optically active dibenzyl ether 10 could be ob-
tained in about 50% yield from myo-inositol. Acid
hydrolysis of 10 provided two synthetically useful iso-
meric triols 11 (41%) and 12 (48%) that could be very
easily separated. Triol 12 on phosphitylation with diben-
zyl N,N-di-isopropyl phosphoramidite followed by
in situ oxidation provided the protected trisphosphate
13. Attempts to remove the benzoyl group by methanol-
ysis (MeOH, Et3N) resulted in a mixture of products
presumably arising from phosphate migration under ba-
sic conditions. However, the treatment of 13 with the
Grignard reagent EtMgCl provided alcohol 14. The
phosphate triesters were stable under these conditions.12
R
OH
OH
O
O
O
H+
HO
HO
OH
OH
OH
3. R = H
4. R = Me
HO
2
OH
5. R = C4H9
R
O
O
OR1
OH
O
aq H+
R2O
HO
OR1
OCOR
OR2
OR3
R3O
7
6
Figure 2.
Ph
Ph
OH
OH
O
O
O
O
O
O
HO
b
a
OH
OH
O(-) camph
HO
OH
OH
O(-) camph
HO
2
8
OH
9
c,d,e,f
Ph
OBn
OH
OBn
OBz
O
O
O
HO
HO
+
g
OBz
HO
HO
OH
OBn
12
OH
BnO
11
BnO
10
OBn
h
OBn
OR
OBn
OR
O
i
RO
RO
OBz
RO
RO
OH
O
BnO
BnO
. R = PO(OBn)
13
(-) camph =
14. R = PO(OBn)2
O
2
Scheme 1. Reagents and conditions: (a) PhC(OMe)3, CSA, DMSO, 80 °C, 5 h, 84%; (b) (1S)-(À)-camphanoyl chloride, Et3N, DMAP, CH2Cl2, 0 °C–
rt, 66%; (c) 2-methoxypropene, PTSA, THF, 0 °C–rt; (d) LiOHÆH2O, THF, MeOH, H2O; (e) NaH, BnBr, DMF; (f) TFA, wet CH2Cl2 (85% for 4
steps); (g) 1 M HCl, EtOH (1:2), reflux, 5 h, 11 (41%), 12 (48%); (h) (1) (BnO)2PN(i-Pr)2, 1H-tetrazole, CH2Cl2, rt, 45 min, (2) m-CPBA, À78 °C to rt,
1 h, 94%; (i) EtMgCl, THF, À42 °C, 30 min, 96%.

K. M. Sureshan et al. / Tetrahedron Letters 48 (2007) 1923–1926
1925
chromatographic purification of diacyl glycerol.15 It is
also known that 1,2-diacylglycerol isomerises readily
to an equilibrium mixture of 1,2-, 1,3- and 2,3-diacyl-
glycerols.16 In addition, the formation of 1,3-diacyl
glycerol during the coupling of 1,2-diacylglycerol with
BnOP(Ni-Pr2)2 has been reported.17
O
O
OR
OR
OR
OR
OR
OR
DDQ or
CAN
PIPn
OH
OPMB
OH
18OP(OBn)N(i-Pr)
2
15
16
17
R = fatty acyl
Figure 3.
Due to these problems, synthesis of PIPns with unambig-
uous stereochemical purity has always been difficult. An-
eja et al. circumvented this problem by esterifying an
appropriately protected inositol derivative with natural
phosphatidic acid obtained by phospholipase D medi-
ated lipolysis of phosphatidylcholines.18 Although this
offers a brilliant solution to the above problems, this
method is limited to DAGs that occur in natural
phosphatidylcholines. To exclude the problem of racemi-
zation and hence to provide access to PIPn of unambig-
uous optical purity, we introduced the acyl groups after
the introduction of glycerophosphate. Furthermore,
since the fatty acyl groups are introduced towards the
end, a library of PIPns with different acyl chains can be
prepared easily from the highly advanced common
precursor.
The usual protocol for the synthesis of PIPns is the
phosphitylation of an appropriately functionalised
inositol derivative with an alkyl 1,2-di-O-acyl glyceryl
N,N-diisopropyl phosphoramidite (e.g., 18) prepared
from an optically pure diacylglycerol 17 (Fig. 3). How-
ever, diacylglycerol13 is a synthon prone to racemization
via acyl migration during its synthesis, purification,
storage and reaction. Usually, diacylglycerol is prepared
from optically pure 1,2-O-isopropylidene-sn-glycerol (15)
through a series of operations via PMB ether 16. How-
ever, under the (acidic) conditions for the deprotection
of the PMB group (CAN or DDQ), the acyl groups
are prone to migrate (racemization).14 Moreover, Prest-
wich and co-worker observed this acyl migration during
O
O
N
OBn
N
Cl
OBn
P
N
P
N
15, tetrazole
DCM, rt, 30 min
O
BnOH
Et3N
P
N
21
20
19
Scheme 2.
O
OBn
OR
OBn
OR
O
O
RO
RO
a
O
OH
RO
RO
P
R = PO(OBn)2
BnO
BnO
O
BnO
22
14
b
OR1
OR1
OBn
OR
OH
OH
OBn
OR
O
RO
RO
O
O
RO
c or d
O
P
RO
P
BnO
O
BnO
23
BnO
O
BnO
. R1 = C H15CO
24
25
7
. R1 = C17H35CO
e
OR1
OR1
OH
H2O3PO
O
H2O3PO
H2O3PO
O
. R1 = C H15CO
26
27
P
7
. R1 = C17H35CO
HO
O
HO
Scheme 3. Reagents and conditions: (a) (1) 21, 1H-tetrazole, DCM, rt, 1 h; (2) m-CPBA, À78 °C; (b) CHCl3, TFA, MeOH (1:1:1, v/v/v), 0 °C,
20 min; (c) C7H15COOH, DCC, DMAP, DCM, rt, 12 h, 85%; (d) C17H35COOH, DCC, DMAP, DCM, rt, 12 h, 100%; (e) H2 (60 psi), Pd(OH)2 on
carbon, t-BuOH, H2O (5:1, v/v), 12 h, 26 (97%), 27 (88%).

1926
K. M. Sureshan et al. / Tetrahedron Letters 48 (2007) 1923–1926
6. Natural PtdIns(3,4,5)P3: (a) Watanabe, Y.; Nakatomi, M.
Tetrahedron 1999, 55, 9743–9754; (b) Gaffney, P. R. J.;
Reese, C. B. J. Chem. Soc., Perkin Trans. 1 2001, 192–205;
(c) Kubiak, R. J.; Bruzik, K. S. J. Org. Chem. 2003, 68,
960–968; PtdIns(3,4,5)P3 with saturated acyl chain: (i)
racemic: (d) Watanabe, Y.; Hirofuji, H.; Ozaki, S.
Tetrahedron Lett. 1994, 35, 123–124; (ii) Optically pure:
(e) Gou, D.-M.; Chen, C.-S. J. Chem. Soc., Chem.
Commun. 1994, 2125–2126; (f) Bruzik, K. S.; Kubiak, R.
J. Tetrahedron Lett. 1995, 36, 2415–2418; (g) Reddy, K.
K.; Saady, M.; Falck, J. R.; Whited, G. J. Org. Chem.
1995, 60, 3385–3390; (h) Chen, J.; Profit, A. A.; Prestwich,
G. D. J. Org. Chem. 1996, 61, 6305–6312; (i) Grove, S. J.
A.; Holmes, A. B.; Painter, G. F.; Hawkins, P. T.;
Stephens, L. R. Chem. Commun. 1997, 1635–1636; (j)
Shirai, R.; Morita, K.; Nishikawa, A.; Nakatsu, N.;
Fukui, Y.; Morisaki, N.; Hashimoto, Y. Tetrahedron Lett.
1998, 39, 9485–9488; (k) Jiang, T.; Sweeney, G.; Rudolf,
M. T.; Klip, A.; Traynor-Kaplan, A.; Tsien, R. Y. J. Biol.
Chem. 1998, 273, 11017–11024; Phosphorothioate ana-
logue: (l) Kubiak, R. J.; Bruzik, K. S. Bioorg. Med. Chem.
Lett. 1997, 7, 1231–1234.
Bifunctional phosphitylating reagent 20 was pre-
pared from bis(N,N-diisopropylamino)chlorophosphine
(Scheme 2).19 Treatment of 20 with 1,2-O-isopropylid-
ene-sn-glycerol (15) in the presence of tetrazole provided
phosphoramidite 21 (95%).20
Trisphosphate 14 was phosphitylated with phospho-
ramidite 21 in the presence of tetrazole to afford 22
(99%) as a diastereomeric mixture (Scheme 3). The iso-
propylidene group was cleaved with TFA following
Watanabe’s protocol21 to afford diol 23 (88%). Diol 23
is a highly advanced common precursor for the synthesis
of various PtdIns(3,4,5)P3 analogues with different acyl
chains or affinity probes. DCC-promoted esterification
of diol 23 with octanoic acid provided the fully pro-
tected lipid 24. We have not observed any phosphate
migration (cyclisation) under these conditions. Finally,
the benzyl protecting groups were removed by hydro-
genolysis to provide di-octanoyl (di-C8)-PtdIns(3,4,5)P3,
26 in good yield. Similarly distearoyl PtdIns(3,4,5)P3,
27 was synthesized via stearoylation of diol 23 followed
by hydrogenolysis. Thus, PtdIns(3,4,5)P3 analogues with
any acyl chain can be prepared from the diol 23. The
known method to deprotect the benzyl protecting
groups without affecting the unsaturated fatty acyl
chain,22 extends the scope of our strategy for unsatu-
rated lipid synthesis.
7. Sureshan, K. M.; Shashidhar, M. S.; Praveen, T.; Das, T.
Chem. Rev. 2003, 103, 4477–4503.
8. Garret, S. W.; Liu, C.; Riley, A. M.; Potter, B. V. L. J.
Chem. Soc., Perkin Trans. 1 1998, 1367–1368; But when
the 2-OH is free, the ester takes the 2-O-position. See:
Godage, H. Y.; Riley, A. M.; Potter, B. V. L. Chem.
Commun. 2006, 2989–2991.
9. Toker, A.; Meyer, M.; Reddy, K. K.; Falck, J. R.; Aneja,
R.; Aneja, S.; Parra, A.; Burns, D. J.; Ballas, L. M.;
Cantley, L. C. J. Biol. Chem. 1994, 269, 32358–32367.
10. Riley, A. M.; Godage, H. Y.; Mahon, M. F.; Potter, B. V.
L. Tetrahedron: Asymmetry 2006, 17, 171–174.
11. Riley, A. M.; Mahon, M. F.; Potter, B. V. L. Angew.
Chem., Int. Ed. 1997, 36, 1472–1474.
12. Watanabe et al. have selectively deprotected the ester
protecting groups leaving the silyl ethers unaffected using a
Grignard reagent. Watanabe, Y.; Fujimoto, T.; Shino-
hara, T.; Ozaki, S. J. Chem. Soc., Chem. Commun. 1991,
428–429.
In conclusion, we have reported efficient routes for the
syntheses of two PtdIns(3,4,5)P3 analogues. Our strat-
egy excludes the possibility of racemization in the lipid
chain and thus provides access to PtdIns(3,4,5)P3 ana-
logues with an unambiguous optical homogeneity. Since
the introduction of the acyl moiety is towards the end, a
library of PtdIns(3,4,5)P3 analogues with any acyl chain
can be synthesized in a short period of time. Moreover,
since the synthesis starts from a high yielding orthoeste-
rification of myo-inositol, and the desymmetrization of
the triol provides the required diastereomer preferen-
tially, the total yield of the lipid is much better than
previously reported methods.
13. Gaffney, P. R. J.; Reese, C. B. Tetrahedron Lett. 1997, 38,
2539–2542.
14. Although this transformation has been used for the
synthesis of various PIPns, in our hands, this transforma-
tion, at times, has resulted in acyl migration to some
extent.
15. Xu, Y.; Lee, S. A.; Kutateladze, T. G.; Sbrissa, D.;
Shisheva, A.; Prestwich, G. D. J. Am. Chem. Soc 2006,
128, 885–897.
16. Freeman, I. P.; Morton, I. D. J. Chem. Soc. 1966, 1710–
1714; Serdarevich, B. J. Am. Oil Chem. Soc. 1967, 44, 381–
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Acknowledgement
We thank the Wellcome Trust for Programme Grant
support (060544).
17. Aneja, S. G.; Parra, A.; Stoenescu, C.; Xia, W.; Aneja, R.
Tetrahedron Lett. 1997, 38, 803–806.
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