Stereospecific Syntheses of Inositol Phospholipids and Their

Chapter 11

Stereospecific Syntheses of Inositol Phospholipids and Their Phosphorothioate Analogs 1

Robert J. Kubiak, Xiangjun Yue, and Karol S. Bruzik

Downloaded by UNIV OF TEXAS AT DALLAS on July 11, 2016 | http://pubs.acs.org Publication Date: December 15, 1998 | doi: 10.1021/bk-1999-0718.ch011

Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago, Chicago, IL 60612

Following receptor stimulation, phosphatidylinositol phosphates (PIP ) undergo numerous processes involving cleavage of the phophate group. Earlier results obtained with phosphorothioate analogs of biophosphates and our recent finding of the resistance of the phosphorothioate analog of PI to phosphatidylinositol-specific phospholipase C suggest that analogs of PIP could be valuable research tools in studying interdependence of various pathways of inositol metabolism, and could be used as mechanistic probes and inhibitors of such enzymes as PI-PLC, PI kinases and PIP phosphatases. This work describes synthesis of a range of phosphorothioate analogs of PIP in which sulfur atom substitutes the oxygen atom in the nonbridging position of the phosphodiester and monoester functions, as well as in the bridging position of the phosphodiester function. n

n

n

n

Inositol signaling pathways are extremely complex and employ a large number of inositol phosphates and phospholipids (1). Due to their multiplicity it is difficult to understand their spatio-temporal relationships unless some signaling branches are eliminated or inhibited. This can be realized by way of gene mutations, where the specific functions of some proteins are abolished, and the effect of the protein modification on the cellular physiology is examined (2). Such an effect can also be accomplished by using synthetic analogs of inositol phospholipids or phosphates with properties altered in such a way, as to increase their resistance to enzymatic cleavage, inhibit synthesis or modify cell membrane permeability. These avenues have been explored in many other fields, and should be especially useful in the area of inositol signaling. Interconversion between inositol phosphates and phospholipids following signal stimulation employs almost exclusively reactions involving nucleophilic displacement at phosphorus, such as phosphodiester cleavage at the inositol 1-position and/or addition/removal of phosphomonoester residues at the inositol 3-, 4- and 5positions (1, 3, 4). In all, six phosphatidylinositols with the inositol residue bearing different number of phosphomonoester groups are known (1,3,4), five of which (Figure 1, 1-5) have been recently synthesized in our Laboratory (5, 6). A similar progress has also been achieved in other laboratories (8-16). Phosphatidylinositol (PI) phosphorylation and dephosphorylation reactions result in vastly different recognition 'Corresponding author.

180

©1999 American Chemical Society

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

181 by the receptor or enzymatic proteins. For example, the presence of the phosphate groups at the 4- and 5-positions of inositol makes phosphatidylinositol 4,5bisphosphate (PI-4,5-P ) a preferred substrate for phosphatidylinositol-specific phospholipase C (PI-PLC) (77, 18), while the addition of the phosphate group at the 3-position makes phosphatidylinositol 3,4,5-trisphosphate completely resistant to this enzyme (79, 20). The ability to intervene into metabolic phosphate addition/removal steps can therefore be useful in understanding the overall phosphoinositide metabolism. This chapter describes synthesis of saturated, fatty acid-bearing phosphatidylinositol phosphates 1-5, as well as their analogs 6-17 (Figure 1) in which phosphate functions have been replaced by the phosphorothioate groups, at either or bom the phosphodiester and phosphomonoester positions (6). The trademark of these analogs is that the phosphate-phosphorothioate replacement is a rather minor structure modifications, which should ensure the same mode of their interaction with the target proteins as those of the natural compounds. These modifications are expected to impose, however, selective resistance to hydrolytic enzymes involved in the removal of phosphate groups of phosphoinositides. The effect of structure modification by sulfur substitution in the phosphate group (Figure 2) brings about three effects, briefly discussed below. (i) Most of the differences in the hydrolytic behavior of phosphorothioates, as compared to phosphates, arise from an impaired hydrogen bonding ability of the sulfur atom as compared to oxygen (27). This has a general effect of slowing down the reactions catalyzed by phosphoryl transfer enzymes, such as phosphodiesterases and phosphatases, where such hydrogen bonding to the phosphate group is an important activating catalytic factor (22). Furthermore, sulfur interacts only weekly with such metal ions as magnesium and calcium, which are also frequent elements of active sites of phosphotransferases (23). As a result, sulfur substitution at the nonbridging position of the phosphate group of oligonucleotides renders the phosphodiester group partially or completely resistant to nucleases (24). In the area of inositol enzymology, it has been shown that the 5-phosphorothioate analog of inositol 1,4,5-trisphosphate (IP ) is resistant to the 5-phosphatase (25). This leads to slower clearance of this analog as compared to IP , and a prolonged calcium-mobilizing effect of this analog (26). We have also shown earlier that sulfur substitution of the pro-S nonbridging oxygen in the phosphate group of PI brings about an almost complete resistance of the Sp-diastereomer (Sp-DPPsI, Sp-6) to the cleavage by the bacterial PI-PLC (27-30). TTie loss of the hydrogen bonding to the phosphate is the most likely reason for the very slow cleavage of this analog (see the following chapter by Hondal et al.). The Sp-diastereomer of the phosphorothionate analog of PI is probably the structurally closest, cleavage-resistant analog of PI. These findings have suggested that sulfur modification at various positions in phosphatidylinositol polyphosphates could provide useful research tools in phosphoinositide enzymology. (ii) Sulfur modification at the nonbridging position creates a stereogenic center at the phosphorus atom, and enables studying steric courses of reactions catalyzed by phosphotransferases (22). Application of the P-chiral, sulfur or oxygen-isotope modified analogs, allowed us in the past to determine that the cleavage of the phosphodiester bond in PI by both the bacterial and mammalian phospholipases C occurs by an analogous double-displacement mechanism, with the inositol 1,2-cyclic phosphate as an intermediate (31-33). (hi) Phosphatidylinositol analogs modified by sulfur substitution at the bridging position of the leaving group generate a thiol product upon their cleavage by PI-PLC, instead of the usual diacylglycerol, and hence constitute convenient substrates for quantitation of enzyme activity based on the amount of the thiol released (34, 35). Furthermore, the comparison of cleavage kinetics of the natural substrate and


2

3

3


182

Β D

RCOO \sn-l RCOO--(jn-2 )sn-33 O HO l =

Χ

Iy ρ=ο

H

9 R 0^>sû !

2

R o"

0

R0 3

R V ^ f % H R0

R

4

C O

o \

3

4


R COO-< 1

2

3

R = R = R = H,PI(1)

DP: dipalmitoyl DO: dioctanoyl

/ R^-R^ = R3 H,X = 0 , Y = S;DPPsI(6) =

3

η Γ o ~ » 2 » 3R ™ » / T » S ol o 22 , Î33 po " 2^. ρ?ι .S3 Λ 5;. 7ρ 3Î( 5^) ( 4 ) =

R

=

;

R

R

3

=

1

2

3

R = R = R = Η, Χ = S, Y = Ο; DOsPI (7), DPsPI (8) R^ - P S O ^ ; R = R = H,X = 0 , Y .= .S;DPPI-3-Ps(9) !

2

R i =

3

r 2 = r 3 = H χ = 0

γ =S;DppsI

3

ps (10)

R = R = PSO2 ", R = H, Χ = O, Y = O; DPPI-3.4-PS2 (11) 1

R R R R R

1

2

2

3

= H; R = R = PO3 -, Χ = S, Y = O; DPsPI-4,5-P (12) = H; R = R = P 0 \ Χ = O, Y = S; DPPsI-4,5-P (13) = H; R = R = PSÔ \ Χ = O, Y = O; ϋ Ρ Ρ Ι - ^ - Ρ ^ (14) = R = R = PSC^ ", Χ = O, Y = O; DPPI-3,4,5-Ps (15) = R = R = PSCh ", Χ = O, Y = S; DPPsI-3,4,5-Ps (16) 2

3

2

3

2

2

1

2

3

1

2

2

3

2

2

1

2

3

2

2

3

2

3

1

3

1

2

3

R = R = R = H, Χ = 0, Y = S; NPIPs (17)

Figure 1. (A) Structures of synthetic phosphatidylinositol phosphates (PIP ) 15. A l l synthesized PIP are 1,2-dipalmitoyl (DP) glycerides except the compound 4 which was also synthesized as 1,2-dioctanoyl derivative (DO). (B) Structures of synthesized phosphorothioate analogs of PIP 6-17. A l l analogs were synthesized as 1,2-dipalmitoyl (DP) glycerides, except the analog 7 which was synthesized as 1,2-dioctanoyl (DO) derivative. n

n

n

RCOQ

X = S;Y = 0

- steric course of phosphodiester cleavage - estimation of phosphate activation toward nucleophilic attack - resistance to phosphodiesterases

Y = S, X = Ο

- assay substrate - estimation of activation of the leaving group toward phosphodiester cleavage

RCOO Y HP

y

I

ν P=X

o\r*>SÔIl 0

X = Y = 0

»

Z= S

" resistance to phosphatases before and after phosphodiester cleavage

Ζ

Figure 2. Summary of potential applications of the synthesized phosphorothioate analogs of phosphatidylinositol phosphates.


183 phosphorothiolate analogs offers an interesting possibility of estimating the overall contribution made by PI-PLC to stabilize the negative charge on the leaving group in the nucleophilic displacement reaction at the phosphorus center (27, 30). Applications of the phosphorothioate analogs of PIP will vary depending on which phosphate group the sulfur atom is positioned. We have synthesized three types of phosphorothioate analogs: (i) with the sulfur atom in the nonbridging position in the phosphodiester function (analogs 6,13 and 17); (ii) with the sulfur atom in the bridging position of the phosphodiester function (analogs 7, 8 and 12); (iii) with the sulfur atom in the nonbridging position in the phosphomonoester function (analogs 9, 11, 14 and 15), and (iv) analogs with sulfur in both the nonbridging diester and monoester positions (analogs 10 an 16). The analogs 6-17 should be useful in addressing various issues in the metabolic turnover of inositol phospholipids and in studying the mechanisms of such enzymes as Pi-specific phospholipases, PI kinases and PI phosphate phosphatases. In addition, the cleavage resistant substrate analogs may prove useful in solving structures of enzyme-substrate complexes. Finally, analogs which selectively inhibit the above enzymes could be potential drug candidates. The two potential enzymatic targets of such analogs could be the 5phosphatase of phosphatidylinositol 3,4,5-trisphosphate, and β- and γ-isozymes of the mammalian PI-PLCs.


n

Synthesis of Inositol Precursors: General Strategies Precursors of PI-4-P and PI-4,5-P . Synthesis of precursors of inositol phospholipids and phosphates has been a subject of intense effort in the last decade resulting in the development of numerous efficient pathways. These methods have been a subject of several recent reviews and monographs (36-38). The major synthetic strategies are: (i) the use of diastereomeric derivatives of inositol for separations of enantiomeric forms (38, 39), (ii) the use of enzymic esterification reaction for separation of enantiomers of inositol derivatives (40, 41)', (iii) the use of chiral noncyclitol precursors for synthesis of asymmetrically substituted inositol derivatives (13, 15, 42-45). In this Laboratory we have adopted the first approach, whereby we combine regioselective protection of inositol hydroxyl groups with the chiral separation of enantiomers (46). This is achieved by employing a chiral bornanediyl moiety derived from D-camphor as an acetal protective group for inositol 2- and 3hydroxyl groups (Scheme 1). This approach has resulted in developing the acetal 18a as a key starting intermediate for all our syntheses. We then systematically explored ways to achieveregioselectiveprotection of the remaining four hydroxyl groups, by examining a variety of reagents (47). The first important finding was that high regioselectivity (>20:1) of the mono-protection of the 1,4,5,6-tetrol 18a at the 1position can be achieved using sterically bulky silyl and acyl groups to give the derivatives such as 19. This finding was of the paramount importance, in view of the fact that most phosphoinositides have a phosphate group at this position. The disadvantage of using the acyl and silyl groups for protection of vicinal polyalcohols is that these groups tend to migrate under a variety of conditions used to introduce further hydroxyl-protective groups. Of the acyl and silyl protective groups examined, we have found that the tert-butyldiphenylsilyl (TBDPS) group affords the best combination of regioselectivity in its introduction, and sufficient stability during further protection of the 4,5,6-triol moiety (47). For the reason stated above, further manipulation of the 4,5,6-triol 19 is somewhat restricted, and has to be performed under mild conditions. The second important finding was that the 4,5,6-triol 19 can be highly selectively derivatized at the 4-position using low temperature acylation or silylation (47). The triol 19 can be also regioselectively derivatized at the 4- and 5-positions simultaneously using low 2


184


Scheme 1

OH

X T 'OH

PI-4,5-P

OH 18a i (88%)

2

I Ri-o

Ri-o

HO OTBDPS iii (90%) _HO^kÔTBDPS iv ... (89%) R0
O "^0" S x

x

°3i

p

=44

ppm

43 1

44

2

R = M O M ; R = DPG; i : TMS-Cl/Me N; ii: aqueous buffer pH 7.0, iii: BFa/EtSH 3



1

2

R = C H C O or C H C O ; R = M O M or MEM; i: CH COSH; ii: C H COCl/Py; iii: EtOH/AgN0 ; iv: Cl-P(OMe)NiPr /iPr EtN; v: alcohol 28/tetrazole; vi: N Bu4, I0 "; vii: M e ^ ; viii: B F ^ t S H , ix: Bi^N*, I"; x: iPr NP(OBn) /tetrazole, xi: S 7

15

15

31

3

15

31

+

3

4

2

2

2

2

8



Scheme 6

] R0" 2

OMOM MOMO J ÔH s

OMOM Ϋ Μ Ο Μ Ο ^ Λ ^ Ο ' OCH V

v

R^^y^'OMOM OR

3

OMOM ΐ MOMO^^L/)' OCH V

R^^^'OMOM OR

1

1

_

Ν

Κ (^γ 0ΜΟΜ OR

1

1

3

1

1

28 (R = MOM) 34 (R*= -P(0)(OBn)

53 (R = MOM), (i-iii, 85%) 54 (R^ -P(0)(OBn) , (i-iii, 65%)

2

2

R0

R0

2

2

R 0»«(

R2O'

2

S' OMOM

>

S OH \ / ° ~ Λ

I/OCH3

MOMO< R^^'-'-OMOM OR

RVY''''OH

1

OR i V , V

55 ( R ^ MOM) 56 (R*= -P(0)(OBn)

2

iv

vi

v

»

' ' »

3

3

8(R = H),(iv-v,78%) 3

2

12 (R = -P0 -), (iv-vi, 83%) 3

R = C H C O or C H C O ; i: C^POCH^/PÊtN; ii: thiol 45/iPr EtN; iii: B114N+ I0 " iv: Me N; v: BFyEtSH, vi: H /Pd 2

7

15

15

31

2

3

2


4

194 phosphorothioites 53 and 54, respectively, in high yields. The subsequent oxidation of these thiophosphites with tetra-n-butylammonium periodate gave the fully protected phosphorothiolates 55 and 56, correspondingly. Ultimately, sequential deprotection of the triester 55 with trimemylamine and ethanethiol/BF -etherate gave the phosphorothiolate analog of PI (8, R = H). The deprotection of 56 was completed analogously, except that the complete removal of the benzyl group from the phosphates was achieved by hydrogenolysis over Pd-charcoal catalyst. Application of the analog 7 to studies of the mechanism of the bacterial PI-PLC has been reported earlier (27), and use of the analog 12 for elucidation of the mechanism of the mammalian enzymes will be reported elsewhere. 3


Summary In conclusion, we have accomplished a general, systematic synthesis of almost all naturally occurring phosphatidylinositols, and many of their phosphorothioate analogs which offer selective resistance towards enzymes involved in inositol phospholipid metabolism. Application of the phosphorothioate analogs to study the mechanism of the bacterial phospholipase C resulted in obtaining results which would have been difficult to obtain otherwise. We are currently studying the behavior of analogs of PI4,5-P2 with the mammalian phospholipase C-δ,, and application of other analogs to studies of PI kinases and PIP phosphatases will be reported elsewhere. n

Acknowledgements: This work was supported by the grant from the National Institutes of Health, GM30327. References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

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Stereospecific Syntheses of Inositol Phospholipids and Their

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