Putative Probes of Intracellular Signaling - American Chemical Society

Chapter 14

Syntheses of 2-Modified Phosphatidylinositol 4, 5Bisphosphates: Putative Probes of Intracellular Signaling

Downloaded by IMPERIAL COLLEGE LONDON on May 2, 2017 | http://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch014

R. Aneja and S. G . Aneja Nutrimed Biotech, Cornell University Research Park, Langmuir Laboratory, Ithaca, N Y 14850 The first syntheses of phosphatidyl-myo-inositol 4,5-bisphosphates (PtdIns-4,5-P ) modified at the 2-OH are described. Complementary procedures are presented for modification of 2-OH by replacement or derivatization, exemplified by the 2-deoxy-2-fluoro epimer and the 2-O -acetyl derivative, respectively. The products optionally incorporate an ω-aminoalkyl-type residue for conjugation to photoaffinity or other reporter group or solid matrix. These analogues retain the core PtdIns4,5-P structure and stereochemistry, but lack the nucleophilic 2-OH deemed essential for substrate hydrolysis by the mammalian phosphoinositide-specific phospholipase C (PI-PLC). Therefore, the 2modified analogues are putative structure- and mechanism-based competitive inhibitors of PI-PLC, suitable as comparative probes of PtdIns-4,5-P -binding to PI-PLC and cellular regulatory proteins. 2

2

2

Multifarious roles emerging for cellular phosphoinositides as vital participants in intracellular signaling and allied processes, created a need for new probes of enzymes and regulatory proteins involved in phosphoinositide metabolism (i). For instance, PtdIns-4,5-P (1) functions as the preferred substrate of PI-PLC (2) and phosphoinositide 3-kinase (PI 3-kinase) (1) enzyme families, and as allosteric activating factor of cellular regulatory proteins with and without pleckstrin homology (PH) domains (3). The action of PI-PLC on PtdIns-4,5-P causes its hydrolysis to the two intraceUular second messengers myoinositol-1,4,5-trisphosphate (Ins-1,4,5-P ) and jn-l,2-diacylglycerol (DAG) (4). Evidence is accumulating that a critical early step in catalyzed hydrolysis involves intramolecular nucleophilic attack by the 2-OH on the 1-phosphodiester phosphorus, resulting in concomitant formation of the inositol 1,2cyclic phosphate intermediate and D A G , followed by further hydrolysis of the cyclic phosphate to Ins-1,4,5-P (5). However, with some isoforms of PI-PLC a small proportion of the cyclic phosphate survives. The two-step mechanism is supported, and the mode of binding of the myoinositol-phosphate residue at the catalytic site is revealed, by x-ray crystal structure analyses of the deletion mutant of PI-PLC-ôj in ternary complexes with calcium ions and Ins-1,4,5-P as a substrate-mimic (6), and DL-myoinositol-2-methylene-l,2-cyclic-monophosphate as an analogue of the putative 2

2

3

3

3

222

©1999 American Chemical Society

Bruzik; Phosphoinositides ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

223 R^OO—CH I

2

2

R COO**CH C

H

2

1: X = OH, Y = H; 2a: X = H , Y = F; 2b: X = OAc,Y = H.

-\4° OP0 '23


2-

cyclic phosphate intermediate (7). New probes incorporating both the inositol phosphate and the glycerolipid moieties could provide a comprehensive view and understanding of the interactions between the phosphoinositides, and, the enzymes and regulatory proteins involved in signaling. In this chapter, we describe the syntheses of 2-modified PtdIns-4,5-P s as putative probes of intracellular signaling mediated by PI-PLCs and allied regulatory proteins. The molecular design of these PtdIns-4,5-P analogs is based on the tenet that the participation of the axial 2-OH as an intramolecular nucleophile is essential for catalyzed hydrolysis; conversely, the intramolecular nucleophilic action is precluded in analogues lacking the 2-OH. The core PtdIns-4,5-P structure and absolute stereochemistry are retained to ensure efficient interaction with the catalytic as well as the non-catalytic P H domain binding sites. Thus, the PtdIns-4,5-P analogues modified at the 2-position by replacement or derivatization of the axial 2-OH are potential competitive inhibitors of PI-PLC enzymes, suitable as comparative probes of protein-binding and enzyme action. An essential caveat is that the modifying groups be small, preferably isosteric with OH, for a good fit at the catalytic site. In this context, we selected 2-deoxy-2-fluoro epimers 2a and 2-Oacetyl derivatives 2b as examples of modification at 2-OH by replacement and derivatization, respectively. 2

2

2

2

Retrosynthetic Analysis and Strategy for Synthesis The strategy for synthesis is based on the retrosynthetic disconnection of the 2modified PtdIns-4,5-P structure into .sn-3-phosphatidic acid (sn-3-PA) and protected chiral myoinositol phosphate fragments shown in Scheme I. Thus, the essential stages entail (i) preparation of the key materials: an optically resolved Oprotected myoinositol-4,5-bisphosphate with a free 1-OH as the inositol synthon, and 1,2-di-O-fattyacyl-jn-glycero-3-phosphoric acid (sn-3-PA) as the lipid synthon; (ii) coupling of the inositol 1-OH and the lipid phosphoric acid by phosphodiester condensation; (iii) deprotection of the condensation product to generate the target 2-modified PtdIns-4,5P . Two tactically distinct options, comprising modification of the 2-OH either prior to, or after the phosphodiester condensation stage, are illustrated in the syntheses of the 2-deoxy-2-fluoro 2a and the 2-0-acetyl 2b series. 2

2

Inositol and Lipid Starting Materials and their Coupling Chiral natural products have been chemically modified and converted to lD-myo inositol derivatives, however, myoinositol is an inexpensive and competitive starting material for synthesis. Its use mandates an optical resolution, but resolution via diastereomeric esters with chiral acids is facile and efficient for several selectively Oprotected derivatives suitable as starting materials for syntheses (£). The choice of sn-PA as the lipid synthon and method of its incorporation into the target structure distinguish our approach from related syntheses, albeit of unmodified phosphoinositides, which all utilize D A G as the lipid synthon (9). This is Bruzik; Phosphoinositides ACS Symposium Series; American Chemical Society: Washington, DC, 1998.

224 advantageous because the sn-3-PA is stable in contrast with D A G which has a strong propensity to isomerize and racemize via 1,2- and 2,3-acyl migration. Chiral my ο-Inositol Derivatives. In an earlier synthesis of PtdIns-4,5-P , we employed 3-0-benzyl-l,2:4,5-di-0-cyclohexylidene-myo-inositol (10). This method is inefficient because regioselective 3-O-benzylation of l,2:4,5-di-0-cyclohexylidenemyoinositol concomitantly produces 6-O-benzyl and 3,6-di-O-benzyl derivatives as by-products. In the present syntheses, we employed lD-l,2:4,5-di-0-cyclohexylidenemyoinositol as the chiral inositol starting material. The compound had been described earlier, albeit in inadequate optical purity, and the ID- absolute configurations were assigned to both the (-)- and the (+)- enantiomers. We developed protocols for its preparation in an optically pure form, and by correlation with a reference of configuration, established unequivocally that (-)-l,2:4,5-di-0-cyclohexylidene-myo inositol belongs to the ID- absolute configuration series (77). In our experience, the enantiomeric l,2:4,5-di-0-cyclohexylidene-myoinositols are versatile starting materials for synthesis of the phosphoinositides. Specifically, complete benzylation of the (-)enantiomer gave a quantitative yield of lD-3,6-di-0-benzyl-l,2:4,5-di-0cyclohexylidene-myoinositol 3 which was utilized as the key chiral synthon as shown in Schemes Π and IV.


2

s/t-3-Glycerolipids. Natural sn-3-phosphatidylcholine, the derived jn-glycero-3phosphocholine and l-acyl-sn-glycero-3-phosphocholine have integral chiral glycerol3-phosphate, and are convenient starting materials for glycerophospholipid synthesis (72). These were utilized as the main starting materials for the lipid synthons exemplified by sn-3-PAs with identical or different fatty acyls at the sn-\ and sn-2 positions. Some sn-PAs are available from commercial sources, and were used in appropriate cases. An alternative material, 3-O-benzyl-i/i-glycerol, was employed for synthesis of the diether analogues. Phosphodiester Coupling of the Key my ο-Inositol and Lipid Synthons. The phosphodiester condensation procedure for coupling a phosphatidic acid with an alcohol was previously developed by us as a general method for the synthesis of glycerophospholipids (13). This method was modified and adapted in the present syntheses for conjugating the key myoinositol and lipid synthons. In the modified general protocol, the appropriately protected myoinositol, sn-3-PA and triisopropylbenzenesulfonyl chloride (TPSC1) in the molar ratio 1:1:2, were allowed to react in anhydrous pyridine solution at room temperature for 1 to 3 hr. The reaction mixture was treated with water to decompose excess TPSC1 and activated phosphate species. The crude product was obtained by evaporation under reduced pressure, and was further purified by chromatography. Deprotection of Condensation Products. The temporary protection of alcohol and phosphate ester functions was provided by benzyl, and that of aminoalkyl by benzyloxycarbonyl (Cbz) groups. The simultaneous removal of all protecting groups by Pd-catalyzed hydrogenolysis of the condensation products formed the target Ptdlns4,5-P analogues. 2

Synthesis of the Target Analogues 1

2

The 2-deoxy-2-fluoro analogues 2a (R = R = C H ) formally belonging to lO-scyllo configuration series are accessible more easily than the lD-myo series, and the preparation of 13 serves to illustrate our synthetic approach (Schemes Π and IV). 15

31



225 lD-l-[l,2-Di-0-hexadecanoyl-5ii-glycero-3-phospho]-2-deoxy-2-fluoroscy//o-inositol-4,5-bisphosphate (13). In this example, the replacement of the 2-OH with inversion by the isosteric and isopolar fluoro residue is carried out in a key inositol synthon prior to phosphodiester condensation. The chiral myoinositol 3, selected as the general starting material, was converted into the key myoinositol synthon lD-3,6-di-0-benzyl-2-deoxy-2-fluoro^cy//oinositol-4,5-bis(di-0,0-benzylphosphate) (10) as outlined in Scheme II. Complete deketalization of 3 by hydrolysis with acetic acid-water (90:10) at 95°C gave lD-3,6-di-0-benzyl-myoinositol (4). The treatment of 4 in DMSO with cyclohexanone dimethylketal catalyzed by p-toluenesulfonic acid at 40-45°C under reduced pressure (used to distill out methanol) provided kinetic control resulting in 3:1 selective reaction at the 4,5- versus the 1,2-OHs to produce an acceptable yield of the critical novel synthon lD-3,6-di-0-benzyl-4,5-0-cyclohexylidene-myoinositol (5). Reaction of 5 with Bu SnO in toluene with azeotropic removal of H 0 , rotary evaporation, solvent change to DMF and treatment with 4-methoxybenzyl chloride/CsF (14) at 80°C for 2 hr provided high regioselectivity and gave, after purification by HPLC, pure equatorial 1-0- substituted derivative 6. Mixing 6 in CH C1 with DAST (15) at 0-5°C followed by reaction at 35-40°C gave the 2-deoxy-2-fluoro derivative 7 formed by substitution with inversion. The structure of a second, albeit minor, product is being ascertained by independent syntheses via fluorination of the 2-epimer of 6 and related structures. Transketalization of 7 with ethylene glycol/catalytic ptoluenesulfonic acid at 35-40°C yielded the 4,5-diol 8. Dibenzylphosphorylation (16) of 8 using dibenzyl diisopropylphosphoramidite and l//-tetrazole in CH C1 followed by 3-chloroperoxybenzoic acid yielded the 4,5-bis-0-(dibenzylphosphate) derivative 9. The oxidation of 9 in CH C1 solution with DDQ at room temperature removed the methoxybenzyl group and gave 10. 2

2

2

2

2

2

2

2

The structure of the inositol synthon 10 may be varied by replacing reaction of 6 with DAST in Scheme Π by other reagents to produce 2-deoxy, O-acyl, 0-alkyl, deoxyhalo or deoxydihalo analogues. As a special case, benzylation of 6 yielded the 2O-benzyl analogue of 7. Subsequent transformations exactly as in Scheme Π gave 1D2,3,6-tri-0-benzyl-myoinositol-4,5-bis(dibenzylphosphate), the inositol synthon for unmodified PtdIns-4,5-P s. l,2-Di-0-hexadecanoyl-sn-glycero-3-phosphoric acid (11, Scheme ΙΠ) was prepared by partial synthesis from natural sn-3-phosphatidylcholine by methods developed previously (12). In early stage exploratory experiments, a reagent grade material from a commercial source was employed. The preparation of sn-PA incorporating useful variation in structure is illustrated later. The phosphodiester condensation reaction of the protected 2-deoxy-2-fluoro derivative 10 and the sn-3-PA 11 by the general protocol, yielded lD-l-[l,2-di-0-hexadecanoyl-5n-glycero-3phospho]-2-deoxy-2-fluoro-3,6-di-0-benzyl-5 cy//oinositol 4,5-bis(0,0-dibenzylphosphate) (12). Hydrogenolysis using H ^ d gave the target 2-deoxy-2-fluoro PtdIns-4,5-P 13. 2

,

2

lD-l-[l,2-Di-0-hexadecanoyl-5n-glycero-3-phospho]-2-0-acetyl-ntyoinositol-4,5-bisphosphate (18). This analogue illustrates an example of modification wherein the 2-OH is derivatized by esterification, specifically acetylation, subsequent to the phosphodiester condensation. The chiral myoinositol 3 used in Scheme II was converted in 2 steps into the


226

1

R COO-CH

2 1

R COO-CH2

2

R COO^CH I CH \

2

R COO-CH I

2

oCH

0

\

2

OP0 "

2

OBn OP(OXOBn) OP(0)(OBn)

BnO X

3

2

PtdIns(4,5)P 2-Modified PtdIns(4,5)P

2


2

2

1

R COO-CH

2

2

R COO>CH I CH \

X OH OBn OP(0)(OBn)

2

OH ^40

+

2

OP(0)(OBn)

2

î

O-Protected 1 D-znyo-Inositol

j/i-3-Phosphatidic Acid OH Scheme I. Retrosynthetic analysis of 2-modified PtdIns(4,5)P

Bu SnO, PMBCl,CsF

2

2

H Η

81% ^

n υ

DAST

OPMB \ wr ivijj V-^ï^rOBn

•

78%

F

OPMB urMD --^l^VOBn

O 2

O

F

^X3^SH

Β η 0 ^ ^ ϊ Τ 8

Ο

Η

•

92%

O

(/-Pr) NP(OBn) , m-CPBA • .^^x^T-ORn 8 8 % ^ï^^%0)(OBn) ^ W ) ( B n ) 2

OPMB

EG,/>-TSA

F

P

M

p

n

0

0

9

2

Q OH , * F-^T-^^TOBn ^^\.OP(0)(OBn) OP(0)(OBn) w

Q

-

B

p

R

9 2

3

0 /

%

F

n

0

2

10

Scheme II. Synthesis of lD-3,6-di-0-benzyl-2-deoxy-2-fluoro-5cy//o-inositol 4,5-bisdibenzylphosphate (10).


2

227 key myoinositol synthon, lD-3,6-di-0-benzyl-myoinositol-4,5-bis(0,0-dibenzylphosphate) (16) (Scheme IV). Transketalization under kinetic control by reaction of 3 (Scheme Π) with ethylene glycol/catalytic p-toluenesulfonic acid in CH C1 at room temperature for 3 h effected selective removal of the 4,5-cyclohexylidene group and gave lD-l,2-0-cyclohexylidene-3,6-di-0-benzyl-myoinositol (14). Bisdibenzylphosphorylation of 14 using dibenzyl diisopropylphosphoramidite and lH-tetrazole in CH C1 followed by m-chloroperbenzoic acid yielded 1D-3,6-di-O-benzyl-1,2-0cyclohexylidene-myoinositol 4,5-bis(0,0-dibenzylphosphate) (15). The 1,2-0cyclohexylidene protecting group in 15 was removed by hot aqueous acetic acid resulting in the 1,2-diol 16 (10). The l,2-di-0-hexadecanoyl-5n-glycero-3-phosphoric acid (11) prepared for the synthesis of the 2-deoxy-2-fluoro analogue was employed as the lipid synthon. The condensation of the myoinositol synthon 16 and sn-3-PA 11 occurred smoothly by the general protocol. Both possible isomeric products were formed, the 1-phosphatidyl derivative 17 (Scheme V) was the predominant product obtained by chromatography in pure state in 55% yield. The observed regioselectivity is consistent with the greater reactivity of the equatorial 1-OH compared to the axial 2-OH flanked by two cisneighbors. For post-condensation modification of the 2-OH, reaction between A c 0 / D M A P and 17, without or with DCC, gave a low yield of the corresponding 2O-acetate derivative. A phosphoric-acetic mixed anhydride of 17, and a strained 5cyclic phosphotriester are plausible consecutive side-intermediates transformed by hydrolytic work-up into unacetylated starting 17 and its 2-phosphatidyl isomer which were isolated as by-products. Much improved yield of the 2-0-acetate derivative was obtained by acetylation using A c 0 / D M A P with an excess of NaOAc added to suppress the displacement of acetate from phosphoric-acetic mixed anhydride and the consequent formation of the 5-cyclic side-intermediate. Hydrogenolysis gave the title 2-0-Ac PtdIns-4,5-P analogue 18. 2


2

2

2

2

2

2

Matched Pairs of Normal and 2-Modified PtdIns-4,5-P Analogues 2

For comparative evaluation of the normal unmodified PtdIns-4,5-P and the 2-modified analogues, matched pairs with identical fatty acyl residues are essential. Both approaches to the 2-modified series are suitable equally for the synthesis of unmodified PtdIns-4,5-P s, and hence of the said matched pairs. Thus, condensation of sn-3-PA 11 and lD-2,3,6-tri-0-benzyl-myoinositol 4,5-bis-(0,0-dibenzylphosphate), prepared by benzylation of 6 and subsequent transformations as described for 7 in Scheme II, followed by hydrogenolysis, gave lD-l-[l,2-di-0-hexadecanoyl-.sn-grycero-3phospho]-myoinositol-4,5-bisphosphate to be paired with 13 or 18. Alternatively, the same dihexadecanoyl PtdIns-4,5-P was obtained by hydrogenolysis of 17 prepared as an intermediate in the synthesis of 18. Analogues with hexadecanoyl and other long chain fattyacyl residues are akin to the cellular PtdIns-4,5-P (1, R CO = stearoyl, R C O = arachidonyl) in so far as these form multimolecular aggregates on hydration. Analogues with short chain fatty acyls form clear solutions in water and are monomelic (8,10). Representative water soluble analogues of normal and 2-modified PtdIns-4,5-P s were synthesized by reaction of l,2-dihexanoyl-5/z-glycero-3-phosphoric acid with appropriate inositol synthons. The broad utility of the analogues as probes and reagents is enhanced by the incorporation of an ω-aminoalkanoyl residue into the analogue structure to obtain conjugands suitable for linking to fluorescent and other reporter groups, and to solid 2

2

2

l

2

2

2


228

Ci H iCOO-CH 5

3

2

C H COO^

Putative Probes of Intracellular Signaling - American Chemical Society

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