Concise syntheses of L-α-phosphatidyl-D-myo-inositol 3-phosphate (3- PIP), 5-phosphate (5-PIP), and 3,5-bisphosphate (3,5-PIP2)

Article abstract of DOI:10.1016/S0040-4039(00)00465-2

Highly efficient, asymmetric total syntheses of the title phospholipids as well as short chain and crosslinkable aminoether analogs were achieved in five to seven steps from a readily available myo-inositol derivative. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4039(00)00465-2

Tetrahedron Letters 41 (2000) 4271±4275
Concise syntheses of l-a-phosphatidyl-d-myo-inositol
3-phosphate (3-PIP), 5-phosphate (5-PIP), and
3,5-bisphosphate (3,5-PIP2)
J. R. Falck,a,* U. Murali Krishna,a Kishta Reddy Katipally,a
Jorge H. Capdevilab and Emin T. Ulugc
aDepartments of Biochemistry and Pharmacology, University of Texas Southwestern Medical Center, Dallas,
Texas 75390, USA
bDepartments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232, USA
cInstitute of Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA
Received 6 January 2000; revised 23 February 2000; accepted 27 February 2000
Abstract
Highly ecient, asymmetric total syntheses of the title phospholipids as well as short chain and cross-
linkable aminoether analogs were achieved in ®ve to seven steps from a readily available myo-inositol
derivative. # 2000 Elsevier Science Ltd. All rights reserved.
Keywords: cyclitols; phospholipids; inositols; phosphoramidites.
The minor cellular lipid l-a-phosphatidyl-d-myo-inositol (1; PI) and its phosphorylated progeny
have been implicated in a vast array of essential physiologic processes including mitogenesis,
calcium regulation, vesicle tracking, apoptosis, and cytoskeleton assembly.1 They function as
precursors2 to low molecular weight second messengers or act directly via regional recruitment
and/or regulation of macromolecules.3 Recent investigations by several laboratories, however,
have identi®ed l-a-phosphatidyl-d-myo-inositol 5-phosphate (3; 5-PIP) and l-a-phosphatidyl-d-
myo-inositol 3,5-bisphosphate (4; 3,5-PIP2) as potential new members of the PI cascade4 and
revealed additional pathways for the biosynthesis of PIPns in eukaryotes.5 The physiologic role(s)
of these novel PI metabolites and their interconversions, e.g. 2$4$3, are areas of intense,
worldwide scrutiny. As part of our continuing program6 to provide comprehensive access to all
components of the PI cycle, we report herein concise, total syntheses of 3-PIP (2), 5-PIP (3), and
3,5-PIP2 (4) as well as some useful glyceryl lipid analogs.7
* Corresponding author. Tel: 214-648-2406; fax: 214-648-6455; e-mail: jfalck@biochem.swmed.edu
0040-4039/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(00)00465-2

4272
Mild acidic hydrolysis of orthoformate 5 (mp 123±25ꢀC), readily available8 in two steps from
myo-inositol, followed by p-toluenesulfonic acid (PTSA) catalyzed exchange with (+)-camphor
dimethyl ketal9 provided convenient access to the chromatographically separable ketals 6 and 7
[TLC: Et2O:CH2Cl2 (5:95), Rf ꢁ0.32 and 0.35, respectively] in good overall yield (Scheme 1).10,11
Treatment of diastereomer 7 with excess O,O-dibenzyl-N,N-diisopropylphosphoramidite12 and in
situ peracid oxidation generated the corresponding bis-phosphate triester. Subsequent cleavage of
the camphor ketal at 0ꢀC using tri¯uoroacetic acid smoothly led to diol 8 that was converted to tris-
phosphate 9a13,14 utilizing Watanabe's pyridinium perbromide methodology15 for the activation
of 1,2-di-O-hexadecanoyl-sn-glyceryl dibenzylphosphite6 10a and regioselective phosphorylation
of the C(1)-alcohol.
Scheme 1. Reaction conditions: (a) MeOH:10N HCl (12.5:1), 65ꢀC, 0.45 h (87%); (b) (+)-camphor dimethyl ketal (3
equiv.), PTSA (2 mol%), CH2Cl2, 23ꢀC, 4 h (82%); (c) (iPr)2NP(OBn)2 (2.5 equiv.), 1H-tetrazole, CH2Cl2, 23ꢀC, 2 h;
m-CPBA, ^40ꢀC, 1 h (88%); (d) CF3CO2H:CH2Cl2:MeOH (1.ꢀ5:3:0.5), 0ꢀC, 0.5 h (77%); (e) phosphite 10a±c (2 equiv.),
ꢀ
.
py HBr3 (2.25 equiv.), CH2Cl2:py:Et3N (5:1:0.1), ^20 C to 0 C, 0.5 h (63%); (f) Pd black, H2 (52 psi), NaHCO3 (5
equiv.), EtOH:H2O (6:1), 23ꢀC, 6 h (79%)
Phosphoramidite6 11a was less satisfactory under a variety of conditions and often resulted in
mixtures of regioisomers and/or bis-derivatized products. Exhaustive debenzylation by catalytic
hydrogenation over Pd black in EtOH/H2O in the presence of NaHCO3 a€orded 3,5-PIP2 (4a),
isolated as its sodium salt.16

4273
Alternatively, phosphorylation of the C(3)-alcohol in 7 using a limited amount of reagent and trityl
cation mediated17 benzylation of the remaining C(5)-hydroxyl with benzyltrichloroacetimidate
gave rise to phosphate 12 (Scheme 2). The latter's transformation to 3-PIP (2a) via 13 and 14a
proceeded as described above in comparable yields.
Scheme 2. Reaction conditions: (a) (iPr)2NP(OBn)2 (1 equiv.), 1H-tetrazole, CH2Cl2, 23ꢀC, 2 h; m-CPBA, ^40ꢀC, 1 h
(71%); (b) PhCH2OC(NH)CCl3, Ph3CBF4 (5 mol%), Et2O, 23ꢀC, 36 h (73%); (c) CF3CO2H:CH2Cl2:MeOH
ꢀ
ꢀ
.
(1.5:3:0.5), 0 C, 0.5 h (77%); (d) phosphite 10a±c (2 equiv.), py HBr3 (2.25 equiv.), CH2Cl2:py:Et3N (5:1:0.1), ^20 C to
0ꢀC, 0.5 h (63%); (e) Pd black, H2 (52 psi), NaHCO3 (5 equiv.), EtOH:H2O (6:1), 23ꢀC, 6 h (79%)
The remaining acetal 6 was exploited for the preparation of 5-PIP (3) via regioselective etheri®cation
of the in situ generated stannyl ester of the C(1)-hydroxyl and phosphorylation of the residual
C(5)-alcohol (Scheme 3). The resultant phosphate triester 15 yielded the di€erentially protected
inositol 16 when subjected to acid ketal hydrolysis and trichloroacetimidate benzylation. Liberation
Scheme 3. Reaction conditions: (a) (n-Bu3Sn)2O (1 equiv.), PhH, 80ꢀC, 6 h with Dean±Stark; MPM-Cl (1.1 equiv.), CsF
(4 equiv.), DMF, 23ꢀC, 12 h (71%); (b) (iPr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, 23ꢀC, 2 h; m-CPBA, ^40ꢀC, 1 h (81%);
(c) CF3CO2H:CH2Cl2:MeOH (1.5:3:0.5), 0ꢀC, 0.5 h (77%); (d) PhCH2OC(NH)CCl3, Ph3CBF4 (5 mol%), Et2O, 23ꢀC,
36 h (73%); (e) DDQ, CH2Cl2:H2O (9:1), 23ꢀC, 4 h (80%); (f) phosphoramidite 11a±c, 1H-tetrazole, CH2Cl2, 23ꢀC, 2
h; m-CPBA, ^40ꢀC, 1 h (81%); (g) Pd black, H2 (52 psi), NaHCO3 (5 equiv.), EtOH:H2O (6:1), 23ꢀC, 6 h (79%)

4274
of the C(1)-alcohol by DDQ induced deprotection, phosphatidylation with 11a, and oxidation
furnished 17a from which 5-PIP (3a) was obtained as its sodium salt by standard catalytic
hydrogenolysis.
Repetition of the ®nal condensations in Schemes 1±3 using 10/11b,c6 a€orded 4/2/3b,d as
appropriate. The dioctanoyl glyceryl analogs (b-series) are more water soluble than the fatty acid
versions (a-series) and have proven more tractable in some assays. The o-aminoalkyl analogs
(d-series) can be derivatized with ¯uorescent, radioactive, and anity labels; their application in
the isolation of several speci®c PIP binding proteins will be reported elsewhere.
Acknowledgements
Supported ®nancially by the Robert A. Welch Foundation and NIH (GM31278, GM37922,
CA58291).
References
1. For a review, see: Rameh, L. E.; Cantley, L. C. J. Biol. Chem. 1999, 274, 8347±8350.
2. Nishizuka, Y. Science 1992, 258, 607±614.
3. Cantley, L. C.; Auger, K. R.; Carpenter, C.; Duckworth, B.; Graziani, A.; Kapeller, R.; Solto€, S. Cell 1991, 64,
281±302. Martin, T. F. J. Ann. Rev. Cell Develop. Biol. 1998, 14, 231±264.
4. Auger, K. R.; Serunian, L. A.; Solto€, S. P.; Libby, P.; Cantley, L. C. Cell, 1989, 57, 167±175. Dove, S. K.; Cooke,
F. T.; Douglas, M. R.; Sayers, L. G.; Parker, P. J.; Michell, R. H. Nature 1997, 390, 187±192. Jones, D. R.;
Gonzalez-Garcõa, A.; Dõez, E.; Martinez-A., C.; Carrera, A. C.; Merida, I. J. Biol. Chem. 1999, 274, 18407±18413.
5. Rameh, L. E.; Tolias, K. F.; Duckworth, B. C.; Cantley, L. C. Nature 1997, 390, 192±196. Whiteford, C. C.;
Brearley, C. A.; Ulug, E. T. Biochem. J. 1997, 323, 597±601.
6. 3,4-PIP2: Reddy, K. K.; Rizo, J.; Falck, J. R. Tetrahedron Lett. 1997, 38, 4729±4730. Reddy, K. K.; Ye, J.; Falck,
J. R.; Capdevila, J. H. Bioorg. Med. Chem. Lett. 1997, 7, 2115±2116. 3,4,5-PIP3: Reddy, K. K.; Saady, M.; Falck,
J. R.; Whited, G. J. Org. Chem. 1995, 60, 3385±3390. 4,5-PIP2: Falck, J. R.; Krishna, U. M.; Capdevila, J. H.
Tetrahedron Lett. 1999, 40, 8771±8774.
7. Previous syntheses of 5-PIP and/or 3,5-PIP2: Peng, J.; Prestwich, G. D. Tetrahedron Lett. 1998, 39, 3965±3968.
Riley, A. M.; Potter, B. V. L. Tetrahedron Lett. 1998, 39, 6769±6772.
8. Lee, H. W.; Kishi, Y. J. Org. Chem. 1985, 50, 4402±4404.
9. Bruzik, K. S.; Tsai, M. D. J. Am. Chem. Soc. 1992, 112, 6361±6374.
10. Careful spectral and chromatographic analyses revealed 6 and 7 are each single stereoisomers. However, it was not
possible to assign the con®guration as either exo or endo. Cf., Grove, S. J. A.; Gilbert, I. H.; Holmes, A. B.;
Painter, G. F.; Hill, M. L. Chem. Commun. 1997, 1633±1634.
11. The absolute con®guration of ketal 6 was established by perbenzylation (BnCl, NaH), ketal cleavage with
methanolic HCl, and comparison with the standards reported by Ozaki, S.; Kohno, M.; Nakahira, H.; Bunya,
M.; Watanabe, Y. Chem. Lett. 1988, 77±80.
12. Tegge, W.; Ballou, C. E. Proc. Natl. Acad. Sci. USA 1989, 86, 94±98.
13. Intermediate 9 was somewhat labile and variable amounts of phosphate migration products were noted. This
could be minimized by rapid puri®cation at neutral pH and storage at low temperatures.

4275
14. Consists of an ꢂ1:1.6 diastereomeric mixture by 31P NMR analysis as a consequence of the newly created
tetrahedral phosphorus, but is typically used in the next step without separation.
15. Watanabe, Y.; Inada, E.; Jinno. M.; Ozaki, S. Tetrahedron Lett. 1993, 34, 497±500.
16. Spectral data for 7: 1H NMR (CDCl3, 400 MHz) ꢀ 0.87 (s, 3H), 0.88 (s, 3H), 1.04 (s, 3H), 1.20±1.27 (m, 2H), 1.42±
1.48 (m, 2H), 1.73±1.80 (m, 2H), 1.90±2.08 (m, 1H), 2.46 (d, J=3.1 Hz, 1H), 2.66 (d, J=2.8 Hz, 1H), 3.68 (ddd,
J=2.8, 6.1, 9.2 Hz, 1H), 3.73 (apparent t, J=5.8 Hz, 1H), 3.88 (dd, J=6.4, 9.2 Hz, 1H), 4.03±4.06 (m, 1H), 4.20±
4.27 (m, 2H), 4.66 (d, J=11.6 Hz, 1H), 4.72 (d, J=11.6 Hz, 1H), 4.81 (d, J=11.6 Hz, 1H), 4.89 (d, J=11.6 Hz,
1H), 7.21±7.39 (m, 10H). Compound 2b: 1H NMR (D2O, 400 MHz) ꢀ 0.84 (t, J=6.2 Hz, 6H), 1.14±1.38 (m, 16H),
1.48±1.62 (m, 4H), 2.12±2.39 (m, 4H), 3.38±3.42 (m, 1H), 3.75±3.82 (m, 2H), 3.94±4.01 (m, 2H), 4.06±4.13 (m, 2H),
4.25±4.31 (m, 1H), 4.37±4.46 (m, 2H), 5.29±5.36 (m, 1H); 31P NMR (121.4 MHz, D2O, 85% H3PO4 external
1
reference) ꢀ 1.82, ^3.0. Compound 3b: H NMR (D2O, 400 MHz) ꢀ 0.86 (t, J=6.8 Hz, 6H), 1.19±1.38 (m, 16H),
1.49±1.68 (m, 4H), 2.32±2.48 (m, 4H), 3.62 (dd, J=2.5, 9.6 Hz, 1H), 3.76±3.85 (m, 3H), 4.01±4.09 (m, 3H), 4.21 (t,
J=2.6 Hz, 1H), 4.23±4.29 (m, 1H), 4.41±4.49 (m, 1H), 5.29±5.36 (m, 1H); 31P NMR (121.4 MHz, D2O, 85%
H3PO4 external reference) ꢀ 1.90, ^3.0.
17. Nakajima, N.; Horita, K.; Abe, R.; Yonemitsu, O. Tetrahedron Lett. 1988, 29, 4139±4142.