Preparation of L-α-phosphatidyl-D-myo-inositol 3-phosphate (3-PIP) and 3,5-biphosphate (3,5-PIP2)

Article abstract of DOI:10.1016/S0960-894X(00)00315-2

Practical, asymmetric total syntheses of the title phospholipids from a readily available myo-inositol derivative as well as short chain and cross-linkable aminoether analogues are described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Full text of DOI:10.1016/S0960-894X(00)00315-2

Bioorganic & Medicinal Chemistry Letters 10 (2000) 1711±1713
Preparation of L-ꢀ-Phosphatidyl-D-myo-inositol 3-Phosphate
(3-PIP) and 3,5-Bisphosphate (3,5-PIP2)
J. R. Falck,a,* U. Murali Krishnaa and Jorge H. Capdevilab
aDepartment of Biochemistry, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
bDepartments of Medicine and Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
Received 10 January 2000; accepted 30 May 2000
AbstractÐPractical, asymmetric total syntheses of the title phospholipids from a readily available myo-inositol derivative as well as
short chain and cross-linkable aminoether analogues are described. # 2000 Elsevier Science Ltd. All rights reserved.
Phosphoinositide (PI) 3-kinases1 constitute a critical link
in membrane trac and agonist-activated intracellular
signalling in eukaryotes.2,3 Recent, intensive investiga-
tions of these pathways at the molecular level have
revealed a novel PI 3-kinase metabolite, l-a-phosphati-
dyl-d-myo-inositol 3,5-bisphosphate (2; 3,5-PIP2), in a
wide variety of cells including lymphocytes, platelets,
®broblasts, COS-7, yeast, and p1ants.4 10 In select cases,7 9
a speci®c PI 3-phosphate 5-kinase that transforms 3-PIP
(1) to 3,5-PIP2 (2) has been characterized, although
alternative pathways for the biogenesis of 2 are possible.9
Despite considerable speculation,11 no de®nitive role(s)
for 1 and 2 have been elucidated to date.7 To expedite
current investigations of the physiological signi®cance of
these metabolites and their biochemical interconversions,12
we report herein practical, asymmetric total syntheses of
3-PIP (1) and 3,5-PIP2 (2), as well as some useful glyceryl
lipid analogues.13
unsuccessful attempts to resolve 4 into its enantiomers via
esterase/lipase di€erentiation and via derivatization with
chiral acids, the hydroxyls were masked as MPM ethers to
give 5 from which Æ-6 was obtained by removal of the
cyclohexylidene. The diastereomeric bis-camphanoyl
esters of the latter, however, were readily separable by
SiO2 chromatography (Et2O/CH2C12 (5/95), Rf ꢁ0.32
and 0.35).17 Saponi®cation of the more polar isomer pro-
vided ( )-6, mp 122±24 ꢀC, [a]d23 23.5ꢀ (c 0.69, CHCl3).
Regioselective O-allylation of the C(1)-alcohol of 6 by
way of the in-situ generated tin ester,18 benzylation of the
remaining C(2)-hydroxyl, and Rh-mediated deprotec-
tion19 of the allylic ether gave rise to 7. The lipid side chain
was introduced by phosphatidylation of 7 with freshly
prepared 1,2-di-O-hexadecanoyl-sn-glycerobenzyl (N,N-
diisopropylamino)phosphoramidite12c (10a) and low
temperature, in situ peracid oxidation; subsequent DDQ
cleavage of the MPM ethers led to the key intermediate
diol 8a.20 Treatment of 8a with excess O,O-dibenzyl-
N,N-diisopropylphosphoramidite21 and adjustment of
the phosphorus oxidation level a€orded tris-phosphate
9a. Exhaustive catalytic debenzylation of 9a in the pre-
sence of NaHCO3 led to 3,5-PIP2 (2a), isolated as an
amorphous solid by sequential trituration of the residue
with EtOAc, Et2O, and MeOH, and characterized by
1H/31P NMR.
Mild acidic hydrolysis of orthoformate 3 (mp 123±25 ꢀC),
obtained in two steps from myo-inositol as described by
Lee and Kishi,14 and ketalization with cyclohexanone
a€orded diol 415,16 (Scheme 1). Following several
*Corresponding author. Tel.: +1-214-648-2406; fax: +1-214-648-6455;
e-mail: jfalck@biochem.swmed.edu
0960-894X/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(00)00315-2

1712
J. R. Falck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1711±1713
References and Notes
1. Review: Rameh, L. E.; Cantley, L. C. J. Biol. Chem. 1999,
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2. Signal transduction web site: http://www.stke.org
3. Review: Martin, T. F. J. Annu. Rev. Cell Develop. Biol.
1998, 14, 231.
4. Tolias, K. F.; Rameh, L. E.; Ishihara, H.; Shibasaki, Y.;
Chen, J.; Prestwich, G. D.; Cantley, L. C.; Carpenter, C. L. J.
Biol. Chem. 1998, 273, 18040.
5. Ban®c, H.; Downes, C. P.; Rittenhouse, S. E. J. Biol. Chem.
1998, 273, 11630.
6. Cantley, L. C.; Auger, K. R.; Carpenter, C.; Duckworth,
B.; Graziani, A.; Kapeller, R.; Solto€, S. Cell 1991, 64, 281.
7. Jones, D. R.; Gonzalez-Garcia, A.; Diez, E.; Martinez-A,
C.; Carrera, A. C.; Merida, I. J. Biol. Chem. 1999, 274, 18407.
8. Whiteford, C. C.; Brearley, C. A.; Ulug, E. T. Biochem. J.
1997, 323, 597.
9. Dove, S. K.; Cooke, F. T.; Douglas, M. R.; Sayers, L. G.;
Parker, P. J.; Michell, R. H. Nature 1997, 390, 187.
10. Meijer, H. J. G.; Divecha, N.; van den Ende, H.; Mus-
grave, A.; Munnik, T. Planta 1999, 208, 294.
11. Hinchli€e, K.; Irvine, R. Nature 1997, 390, 123.
12. For syntheses of other metabolites of the PI cycle, see: (a)
(3,4-PIP2) Reddy, K. K.; Rizo, J.; Falck, J. R. Tetrahedron
Lett. 1997, 38, 4729. (b) Reddy, K. K.; Ye, J.; Falck, J. R.;
Capdevila, J. H. Bioorg. Med. Chem. Lett. 1997, 7, 2115; (c)
(3,4,5-PIP3) Reddy, K. K.; Sandy, M.; Faick, J. R.; Whited,
G. J. Org. Chem. 1995, 60, 3385; (d) (4,5-PIP2) Falck, J. R.;
Krishna, U. M.; Capdevila, J. H. Tetrahedron Lett. 1999, 40,
8771.
Scheme 1. (a) MeOH/10 N HCl (12.5/1), 65 ꢀC, 0.45 h (87%); (b)
cyclohexanone (2 equiv), PTSA, DMF/PhCH3 (1/4), 140 ꢀC, 12 h
(68%); (c) MPM-Cl, NaH, DMF, 23 ꢀC, 6 h (76%); (d) MeOH/
CH2Cl2, 12 N HCl (4/1/1), 23 ꢀC, 1 h (83%); (e) S-( )-camphanic
chloride, DMAP, EI3N, CH2Cl2, 23 ꢀC. 12 h (91%); (f) MeOH, KOH,
23 ꢀC, 14 h (88%); (g) n-Bu2SnO, PhH, 85 ꢀC, 6 h; CH2ˆCHCH2Br,
CsF, DMF, 23 ꢀC, 12 h (86%); (h) BnBr, NaH, DMF, 23 ꢀC, 4 h
(86%); (i) Rh(Ph3P)Cl, DABCO, EtOH, 78 ꢀC, 4 h; 1 N HCl/acetone
(1/9), 0.5 h (88%); (j) Phosphoramidite 10, 1H-tetrazole, CH2Cl2,
23 ꢀC, 2 h; m-CPBA, 40 ꢀC, 1 h (81%); (k) DDQ, CH2Cl2/H2O (9/1),
23 ꢀC, 4 h (80%); (l) (iPr)2NP(OBn)2, 1H-tetrazole, CH2Cl2, 23 ꢀC, 2
h; m-CPBA, 40 ꢀC, 1 h (81%); (m) Pd black, H2 (52 psi), NaHCO3 (5
equiv), EtOH/H2O (6/1), 23 ꢀC, 6 h (79%).
13. Previous syntheses of 3-PIP and/or 3,5-PIP2: Peng, J.;
Prestwich, G. D. Tetrahedron Lett. 1998, 39, 3965. Riley, A.
M.; Potter, B. V. L. ibid. 1998, 39, 6769. Bruzik, K. S.;
Kubiak, ibid 1995, 36, 24 15. Wang, D.-S.; Chen, C.-S. J. Org.
Chem. 1996, 61, 5905.
14. Lee, H. W.; Kishi, Y. J Org. Chem. 1985, 50, 4402.
15. All new compounds were fully characterized by
1H/13C/31P NMR, and MS analyses.
1
16. Spectral data for 4: H NMR(CDC13, 400 MHz) d 1.36±
1.80 (m, 10H), 2.54 (d, J=6.4 Hz, 1H), 2.64 (d, J=6.4 Hz, 1
H), 3.60 (ddd, J=2.7, 7.0, 9.3 Hz, 1H), 3.72±3.81 (m, 2H),
3.94 (app. quintet, J=3.9 Hz, 1H), 4.24 (app. t, J=6.6 Hz,
1H), 4.46 (dd,J=3.9, 6.4 Hz, 1H), 4.70 (d, J=11.4 Hz,1H),
4.74 (d, J=11.4 Hz, 1H), 4.89 (d, J=11.4 Hz, 1H), 4.94 (d,
J=11.4 Hz, 1H), 7.20±7.39 (m, 10H). 1a: 1H NMR (D2O, 400
MHz) d 0.84 (t, J=6.2 Hz, 6H), 1.14±1.38 (m, 48H), 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 reference) d 1.82, 3.0. 2b: 1H
NMR (D2O, 400 MHz) d 0.86 (t, J=6.2 Hz, 6H), 1.21±1.44
(m, 16H), 1.43±1.64 (m, 4H), 2.37±2.48 (m, 4H), 3.85±4.19 (m,
7H), 4.23±4.36 (m, 1H), 4.42±4.53 (m, 2H), 5.34 (bs, 1H); 31P
NMR (121.4 MHz, D2O, 85% H3PO4 external reference) d
3.0, 1.40, 2.13.
Scheme 2. (a) (iPr)2NP(OBn)2 (1 equiv), 1H-tetrazole, CH2Cl2, 23 ꢀC,
2 h; m-CPBA, 40 ꢀC, 1 h (55%); (b) Pd black, H2 (52 psi), NaHCO3
(5 equiv), EtOH/H2O (6/1), 23 ꢀC, 6 h (79%).
Alternatively, selective phosphorylation of the C(3)-alco-
hol in 8a utilizing a limited amount of reagent provided
convenient access to 3-PIP (la) following the standard
deprotection procedure described above (Scheme 2).
Repetition of the phosphorylation of 7 using 10b,c12c
and ®nal elaboration of the adducts 8b,c as outlined in
Schemes 1 and 2 a€orded lb,d and 2b,d, respectively.
The dioctanoyl glyceryl analogues (b series) are more
water soluble than the fatty acid versions (a series) and
have proven more tractable in some assays;19 both are
able to compete with natural material for binding to PIP
binding proteins. The o-aminoalkyl analogues (d series)
have been useful for the introduction of ¯uorescent,
radioactive, and anity labels.12c
17. The absolute con®guration of the less polar diastereomer
was established by the sequence outlined below and compari-
sons with standards reported by Ozaki, S.; Kohno, M.;
Nakahira, H.; Bunya, M.; Watanabe, Y. Chem. Lett. , 1988,
77.
Acknowledgements
Supported ®nancially by the Robert A. Welch Foundation
and NIH (GM31278, GM37922).
18. David, S.; Hannesian, S. Tetrahedron 1985, 41, 643.

J. R. Falck et al. / Bioorg. Med. Chem. Lett. 10 (2000) 1711±1713
1713
19. Wang, D. S.; Hsu, A. L.; Song, X.; Chiou, C. M.; Chen,
C. S. J. Org. Chem. 1998, 63, 5430.
phosphorus, but is typically used in the next step without
separation.
21. Tegge, W.; Ballou, C. E. Proc. Natl. Acad. Sci. USA 1989,
86, 94.
20. Consists of anꢁ1:1.6 diastereomeric mixture by 31P NMR
analysis as a consequence of the newly created tetrahedral