Phosphorothioate Analogues of - American Chemical Society

Chapter 14

Phosphorothioate Analogues of D-myo-Inositol 1,4,5-Trisphosphate

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Chemistry and Biology

Barry V. L. Potter School of Pharmacy and Pharmacology, University of Bath, Claverton Down, Bath BA2 7AY, England D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P ] is a second messenger that mediates mobilization of i n t r a c e l l u l a r Ca . Chemical modification of Ins(1,4,5)P can yield analogues with novel properties towards the Ca2+-mobilizing receptor and the metabolic enzymes Ins(1,4,5)P 5-phosphatase and 3-kinase. We have synthesized phosphorothioate analogues of Ins(1,4,5)P3 which are finding many biological applications as potent non-hydrolysable agonists and enzyme inhibitors. The chemical synthesis and biological properties of these compounds are reviewed. 3

2+

3

3

D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P (1), Figure 1]* is a second messenger c o u p l i n g agonist s t i m u l a t i o n of c e l l surface receptors to the r e l e a s e of i n t r a c e l l u l a r Ca " " (1). Because of the fundamental importance of the polyphosphoinositide s i g n a l l i n g system in c e l l biology i t is essential to have access to chemical t o o l s which can f a c i l i t a t e pharmacological i n t e r v e n t i o n at I n s ( l , 4 , 5 ) P receptors and the metabolic enzymes which act upon t h i s second messenger (2). Indeed, Ins(l,4,5)P antagonists and compounds which block s i g n a l t r a n s d u c t i o n by the polyphosphoinositide pathway may have therapeutic value as potential drugs (3), provided they can gain i n t e r n a l access to 3

2

1

3

3

* Unless otherwise myo-isomer

indicated,

x

inositol'

refers

to

0097-6156/91/0463-0186$06.00/0 © 1991 American Chemical Society In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

the

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D-myo-Inositol 1,4,5-Trisphosphate

cells. Heparin has been found to act as an I n s ( l , 4 , 5 ) P antagonist (4), but few such t o o l s have yet been identified and there are significant difficulties intrinsic to a drug design strategy based upon Ins(l,4,5)P . Now t h a t problems with i n o s i t o l phosphate s y n t h e s i s have e s s e n t i a l l y been overcome, i t i s p o s s i b l e to envisage the r a t i o n a l design and chemical s y n t h e s i s of I n s ( l , 4 , 5 ) P analogues (5-7). Few s t r u c t u r a l l y - m o d i f i e d compounds possessing b i o l o g i c a l a c t i v i t y have, however, yet been prepared. The first example of such a compound was myo-inositol 1,4,5-trisphosphorothioate [Ins(l,4,5)PS (2), Figure 1] (8). Other phosphoro thioate analogues of Ins(l,4,5)P have now been synthesized i.e. inositol 1,4-bisphosphate-5phosphorothioate [ I n s ( l , 4 , 5 ) P - 5 S , (3)] (9), i n o s i t o l 1phosphorothioate-4,5-bisphosphate [Ins(1,4,5)P -1S, ( 4 ) ] (10) and inositol l-phosphate-4,5-bisphosphorothioate [Ins(l,4,5)P -4,5S, ( 5 ) ] (N. J . Noble & Β. V. L. P o t t e r , unpublished). Extensive s t u d i e s have demonstrated that the unique p r o p e r t i e s of i n o s i t o l phosphorothioates, especially their resistance to degradation by intracellular phosphatases (11,12), will make them v a l u a b l e t o o l s f o r i n v e s t i g a t i o n of the a c t i o n s and metabolism of i n o s i t o l phosphates. T h i s chapter w i l l address only i n o s i t o l phosphorothioates. For d e t a i l s of other i n o s i t o l and i n o s i t o l phosphate analogues which have been synthesized the reader i s d i r e c t e d to recent reviews (5-7). 3

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Synthesis Many s y n t h e t i c routes to the n a t u r a l second messenger, I n s ( l , 4 , 5 ) P have now been devised (5-7), but w i l l not be discussed here. Our i n i t i a l route (8) was based upon Ρ(III) methodology, used s u c c e s s f u l l y to prepare myo inositol 4,5-bisphosphate (13) and involved p h o s p h i t y l a t i o n of the protected precursor 1,2,4-tri-Obenzyl-myo-inositol (14) [Figure 2]. Conversion of the resulting trisphosphoramidite to the hexacyanoethyl t r i s p h o s p h i t e was followed by o x i d a t i o n t o the f u l l y protected trisphosphate t r i e s t e r and removal of a l l p r o t e c t i n g groups i n one step using sodium i n l i q u i d ammonia. The r e s u l t i n g Ins(1,4,5)P , which was p u r i f i e d by ion-exchange chromatography, was fully active biologically at mobilising intracellular Ca and binding to the c e r e b e l l a r I n s ( l , 4 , 5 ) P receptor. The phosphorothioate analogue Ins(1,4,5)PS could be prepared by m o d i f i c a t i o n of t h i s route by o x i d a t i o n of the t r i s p h o s p h i t e hexaester with a s o l u t i o n of s u l f u r i n p y r i d i n e followed by d e p r o t e c t i o n and p u r i f i c a t i o n as for Ins(l,4,5)P . Once the b i o l o g i c a l a c t i v i t y of I n s ( l , 4 , 5 ) P S had been e s t a b l i s h e d , the p o t e n t i a l importance of s i m i l a r 3

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3

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3

In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


188

INOSITOL

X=Y=Z=0

(1)

X=Y=Ζ=S

(2)

X = Y = 0;Z = S

(3)

Y = Z = 0;X = S

(4)

Y = Z = S;X = 0

(5)

Figure 1 . Structures of phosphorothioate analogues.

PHOSPHATES

AND

Ins(l,4,5)P D-isomers are 3

DERIVATIVES

and shown.


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D-myo-Inositol l 4 5-Trisphosphate

189

y

y

analogues possessing only single phosphorothioate s u b s t i t u t i o n was r e a l i z e d . In p a r t i c u l a r , s y n t h e s i s of the analogue Ins(1,4,5)P -5S (3) possessing only a 5phosphorothioate group seemed d e s i r a b l e , s i n c e this molecule would be nearer i n s t r u c t u r e t o I n s ( l , 4 , 5 ) P than I n s ( l , 4 , 5 ) P S and yet would enjoy the advantages of 5-phosphatase r e s i s t a n c e . Our s y n t h e t i c route to t h i s molecule was developed using a combination of Ρ(III) and P(V) chemistry (9) [Figure 3]. Thus, phosphorylation of 2,3,6-tri-0-benzyl-5,6-0-isopropylidene-myo-inositol with bis(2,2,2-trichloroethyl) phosphorochloridate followed by removal of the i s o p r o p y l i d e n e group and c a r e f u l phosphorylation of the r e s u l t i n g d i o l gave a mixture of the 1 , 4 and 1,5-bisphosphates. The 1,4bisphosphate was crystallised, phosphitylated and o x i d i z e d t o e i t h e r the f u l l y p r o t e c t e d t r i s p h o s p h a t e or the f u l l y p r o t e c t e d 1,4-bisphosphate-5-phosphorothioate. Reductive d e p r o t e c t i o n with sodium i n l i q u i d ammonia gave either Ins(l,4,5)P or Ins(1,4,5)P -5S respectively. 1-(2,2,2-Trichloroethyl) phospho-2,3,6tri-O-benzyl-myo-inositol was used to prepare myoinositol-l-phosphate-4,5-bisphosphorothioate in a similar fashion (N. J . Noble & Β. V. L. Potter, unpublished). The n u c l e o p h i l i c c h a r a c t e r of the s u l f u r atom of a phosphorothioate group makes t h i s atom a s u i t a b l e p o i n t of attachment for environmentally-sensitive reporter groups such as f l u o r e s c e n t , s p i n , a f f i n i t y l a b e l s e t c . This has been especially exploited in the oligonucleotide f i e l d . The f i r s t example of an i n o s i t o l trisphosphate analogue modified by a r e p o r t e r group involved the attachment of a fluorescent nitrobenzoxadiazole (NBD) moiety to the 1phosphorothioate of myo-inositol l-phosphate-4,5bisphosphate [Figure 4 , ( 4 ) ] v i a the s u l f u r atom (10). The r e s u l t i n g NBD-Ins(1,4,5)P analogue (6) was h i g h l y potent at r e l e a s i n g i n t r a c e l l u l a r C a [ 6 - f o l d weaker than Ins(l,4,5)P ] and bound to the Ins(l,4,5)P c e r e b e l l a r receptor with high a f f i n i t y . Syntheses of other i n o s i t o l phosphorothioates have been reported. I n o s i t o l 1-phosphorothioate has been prepared by t h i o p h o s p h o r y l a t i o n of 1,2,4,5,6-penta-Oa c e t y l - m y o - i n o s i t o l and deblocking (15). Endo- and exodiastereoisomers of the 5membered (1,2-cyclic) phosphorothioates have been prepared by thiophos p h o r y l a t i o n of 1,4,5,6-tetra-O-acetyl-myo-inositol (16) or by simultaneous mono-phosphitylation of 1,4,5,6tetra-O-benzyl-myo-inositol a t e i t h e r the 1or 2positions, cyclisation and oxidation to the d i a s t e r e o i s o m e r i c c y c l i c phosphorothioates, separation of d i a s t e r e i s o m e r s and deblocking (17). 3

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DERIVATIVES


190


In Inositol Phosphates and Derivatives; Reitz, A.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. /

/

F i g u r e 2. Synthetic routes to Ins(l 4 5)P3 and Ins(l,4,5)PS^. Reproduced with permission from r e f . 7. Copyright 1990 John Wiley & Sons Ltd.

x - Ο (1) Χ - S (2)


192

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BnO

Figure 3 . Synthetic routes t o I n s ( l 4 , 5 ) P .and Ins(l,4,5)P -5S. Reagents: i (a) C I P ( N P r ) OCH CH CN, (b) NCCH CH OH-tetrazole, (c) f o r X=0, Bu^OH; X=S, sulphur i n C H N; i v Na i n l i q . NH . Reproduced with permission from r e f . 9. Copyright 1988 Royal Society of Chemistry, London. /

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D-myoinositol 1,4,5-Trisphosphate

III


0 BnO

I

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-1

0-P(0R )

2

x=o x=s

IV

R s-CHjCa

3

Bn= benzyl R = -CH CH CN 2

HO

ϊ

° -

p

o

2

2

r

V ^ T »>

X = 0 (1)

Η 0 - 4 ^ - - - - - γ - Λ ^ 0 —PO.

X = S (3)

H

Figure 3. Continued.


194

PHOSPHATES

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DERIVATIVES


INOSITOL

R = -CH CH CN 2

2

Bn=benzyl Figure 4 . S y n t h e t i c route t o I n s ( l , 4 , 5 ) P - l S and NBD-analogue. Reagents: i (a) (RO) PNPr tetrazole. (b) Bu^OH, (c) HgO-HgCl ; i i (a) (RO) PNPr -tetrazole (b) sulphur i n C 5 H 5 N ; i i i Na in l i q . NH ; i v IANBD-EtOH. A l l compounds are racemic. Reproduced with permission from r e f . 10. Copyright 1990 Royal S o c i e t y of Chemistry, London. 3

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B i o l o g y and a p p l i c a t i o n s o f i n o s i t o l phosphates Several studies on biological applications of Ins(l,4,5)PS and Ins(1,4,5)P -5S have now been reported. Thus, i n o s i t o l 1,4,5-trisphosphorothioate (2), binds t o Ins(l,4,5)P^ receptor s i t e s i n b r a i n (12,18) and l i v e r (19) and i s only s l i g h t l y l e s s potent than I n s ( l , 4 , 5 ) P . Moreover, i t i s a f u l l and potent agonist f o r the r e l e a s e of i n t r a c e l l u l a r C a in a v a r i e t y of systems, such as i n Xenopus oocytes (20,21), permeabilised Swiss 3T3 c e l l s (20,22), GH c e l l s (22), hepatocytes (23), p a n c r e a t i c (24,25) and p a r o t i d (26) acinar cells, skeletal muscle traids (27), mouse l a c r i m a l c e l l s (28) and SH-SY5Y neuroblastoma c e l l s ( 2 ) , being only some 3-4 f o l d l e s s potent than I n s ( l , 4 , 5 ) P ^ . As expected from the p r o p e r t i e s of phosphorothioates, i t is resistant to 5-phosphatase-catalyzed dephosphorylation (12,23) and can t h e r e f o r e produce a sustained calcium transient in cells (2,23). Ins(1,4,5)PS i s the most potent competitive i n h i b i t o r of 5-phosphatase y e t reported (29), but i t i s not bound by the 3-kinase and does not compete with I n s ( l , 4 , 5 ) P for this enzyme (23,30). Thus, Ins(1,4,5)PS is recognised with high affinity by Ins(1,4,5)PSj receptors. I t i s a f u l l agonist with respect t o C a * r e l e a s e and y e t i s r e s i s t a n t t o a l l known routes of I n s ( l , 4 , 5 ) P metabolism. The possible synergy between Ins(l,4,5)P and Ins(l,3,4,5)P i s promoting C a entry a t the plasma membrane has been the s u b j e c t of considerable debate. In s i n g l y i n t e r n a l l y perfused mouse l a c r i m a l a c i n a r c e l l s , using the patch clamp technique f o r whole c e l l current recording, monitoring a Ca -activated K c u r r e n t , Ins(1,4,5)PS alone §ives r i s e t o a s i n g l e transient response, typical of Ins(1,4,5)P , and independent of external Ca . Together with I n s ( l , 3 , 4 , 5 ) P , however , i t evokes a sustained response dependent upon e x t e r n a l Ca , suggesting t h a t the t r a n s i e n t response of I n s ( l , 4 , 5 ) P i s not a consequence of r a p i d metabolism, and t h a t Ins( 1,3,4,5)P i s not acting by protecting Ins(l,4,5)P against dephosphorylation by the common 5-phosphatase (28). Agonist-stimulated cells often give rise to oscillating internal C a l e v e l s , r a t h e r than a smooth r i s e , and the r o l e and mechanism of generation of such oscillations have been the s u b j e c t of s i g n i f i c a n t i n t e r e s t and controversy. In s i n g l y i n t e r n a l l y perfused mouse p a n c r e a t i c a c i n a r c e l l s Ins(1,4,5)PS , a p p l i e d through a patch p i p e t t e , evokes r e p e t i t i v e pulses of Ca -activated C l ~ current, which are s i m i l a r i n amplitude and frequency t o the response of such c e l l s t o acetylcholine, acting through muscarinic receptors. Thus, p u l s a t i l e C a r e l e a s e i s p o s s i b l e even a t a constant level of t h i s analogue, and presumably, 3

3

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3

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+

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therefore, of Ins(1,4,5)P . T h i s has been used to argue against a receptor-controlled oscillator in the generation of C a o s c i l l a t i o n s , as w e l l as any r o l e f o r the periodic phosphorylation or degradation of Ins(l,4,5)P (25). The polyphosphoinositide pathway is thought to mediate the a b i l i t y of light to release Ca f r o m ER within invertebrate microvillar photoreceptors, via the formation of I n s ( l , 4 , 5 ) P . Ins(1,4,5)PS has been used to investigate mechanisms that terminate the mobilisation of Ca in ventral photoreceptors of the horseshoe crab Limulus. It can generate sustained repetitive oscillations of Ca -dependent membrane p o t e n t i a l i n t h e Limulus p h o t o r e c e p t o r , w h e r e t h e a c t i o n of Ins(l,4,5)P is normally rapidly terminated by metabolism (31). Ins(1,4,5)PS is also capable of generating oscillations o f membrane p o t e n t i a l (20) a n d C a ~ - d e p e n d e n t C I " c u r r e n t (21) i n Xenopus o o c y t e s . In the oocyte, however, such oscillations are not sustained, i n d i c a t i n g that factors other than metabolism are important i n terminating the response. Oscillations of membrane potential caused by Ins(l,4,5)PS are different from those generated by Ins(l,4,5)P and r e s e m b l e more t h o s e from I n s ( 2 , 4 , 5 ) P . The exact reason for t h i s i s not y e t c l e a r , but d i f f e r e n t mechanisms for setting up C a oscillations may be possible (32). Oscillations in Ca -dependent C l ~ current, i n d u c e d by Ins(l,4,5)PS , however, appear to resemble those induced by I n s ( l , 3 , 4 , 5 ) P rather than I n s ( l , 4 , 5 ) P (33). 3

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Ins(l,4,5)PS has been used in rat pancreatic acinar cells to help distinguish functionally between Ins(1,4,5)P -sensitive and -insensitive nonmitochondrial MgATP-dependent Ca pools (24). Ins(l,4,5)PSQ was used to keep the Ins ( 1 , 4 , 5 ) P sensitive Ca- " " p o o l empty and C a reuptake occurred into the Ins(l,4,5)P insensitive pool. However, in experiments on Ca "^ m o b i l i s a t i o n i n permeabilised rat parotid acinar cells, evidence has been obtained for C a reuptake into an Ins(1,4,5)P and t h a p s i g a r g i n sensitive C a store i n the presence of Ins(1,4,5)PS 3

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(26). By virtue of their properties as potent 5phosphatase inhibitors, Ins(l,4,5)P and the related monophosphorothioate analogue, inositol 1,4bisphosphate-5-phosphorothioate Ins(1,4,5)P -5S (9), have been employed to i n h i b i t I n s ( l , 4 , 5 ) P breakdown i n electrically-permeabilized SH-SY5Y human n e u r o b l a s t o m a cells (34). Inhibition of 5-phosphatase-mediated metabolism of exogenously added 5 [ P ] - I n s ( l , 4 , 5 ) P was ca. 10 t i m e s g r e a t e r t h a n t h a t o f c e l l m e m b r a n e - d e r i v e d [ H]-Ins(l,4,5)P , indicating the possibility of Ins(l,4,5)P compartmentation, i.e. that homogenous mixing of exogenously-added and endogenously-generated Ins(l,4,5)P does not o c c u r . 3

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197

Another a p p l i c a t i o n of I n s ( 1 , 4 , 5 ) P S has been i n the i n v e s t i g a t i o n of *quantal' r e l e a s e of i n t r a c e l l u l a r Ca by I n s ( l , 4 , 5 ) P i n permeabilized hepatocytes (35), where the s i z e of the Ins( 1 , 4 , 5 ) P o - s e n s i t i v e C a pool is apparently dependent upon the c o n c e n t r a t i o n of Ins(l,4,5)P . The f a i l u r e of sub-maximal c o n c e n t r a t i o n s of I n s ( l , 4 , 5 ) P o r I n s ( 1 , 4 , 5 ) P S t o empty the C a store completely was not due t o d e a c t i v a t i o n of the stimulus or r e c e p t o r d e s e n s i t i s a t i o n . The metabolic s t a b i l i t y of Ins(l,4,5)P allowed Ca efflux experiments t o be performed a t a high c e l l d e n s i t y where degradation of Ins(l,4,5)P would normally have posed significant problems. T h i s s t a b i l i t y of I n s ( l , 4 , 5 ) P S was a l s o c r u c i a l f o r the o b s e r v a t i o n t h a t an analogue of I n s ( 1 , 4 , 5 ) P S can a c t i v a t e a novel voltage-dependent K conductance i n r a t CA1 hippocampal pyramidal c e l l s (36). Ins(l,4,5)PS i n h i b i t e d a c t i o n p o t e n t i a l f i r i n g when i n j e c t e d into these c e l l s . I n s ( l , 4 , 5 ) P i t s e l f d i d not e l i c i t t h i s conductance, presumably because of i t s r a p i d breakdown i n these c e l l s . Thus, use of I n s ( 1 , 4 , 5 ) P S may uncover activities of Ins(l,4,5)P^ which may not be experimentally observable using the n a t u r a l messenger because of r a p i d metabolism or slow d i f f u s i o n of exogenously-added messenger. Ins(l,4,5)PS r e l e a s e d up t o 20% of an a c t i v e l y loaded C a pool i n t r i a d s from r a b b i t s k e l e t a l muscle, although a c t i v a t i o n of ryanodine r e c e p t o r C a channels was zero or minimal (27), r a i s i n g the p o s s i b l i l i t y t h a t the C a m o b i l i s i n g a c t i v i t y may be mediated by other channels or mechanisms. The k i n e t i c s of C a r e l e a s e by Ins(l,4,5)PS i n the sarcoplasmic r e t i c u l u m of s k e l e t a l muscle was s u r p r i s i n g l y f a s t (20-100 ms, depending upon agonist concentration), i n d i c a t i n g that I n s ( l , 4 , 5 ) P r e c e p t o r s of s k e l e t a l muscle are k i n e t i c a l l y competent to s i g n a l the r a p i d e l e v a t i o n of c y t o s o l i c C a that precedes muscle c o n t r a c t i o n (37). Although p o l y p h o s p h o i n o s i t i d e s t u r n over i n human red blood c e l l s , a r o l e f o r I n s ( l , 4 , 5 ) P has y e t t o be established. In permeabilised human r e d blood c e l l s Ins(l,4,5)P evokes sustained release of C a and i r r e v e r s i b l e shape changes and d i s o r g a n i s a t i o n of the s p e c t r i n network, as measured by immunofluorescence, whereas I n s ( l , 4 , 5 ) P evokes r e v e r s i b l e shape changes (38). The p o l y p h o s p h o i n o s i t i d e signalling pathway evidently plays an important role i n the shape maintenance of r e d blood c e l l s . 3

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[ S ] - L a b e l l e d myo-inositol

phosphorothioates

The wide range of applications of inositol phiosphorothioates d e t a i l e d above has been extended by the commercial availability of [ -S]-labelled I n s ( 1 , 4 , 5 ) P S , which has been shown t o be a v a l u a b l e 3 5

3


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INOSITOL PHOSPHATES AND DERIVATIVES

m e t a b o l i c a l l y s t a b l e r a d i o l i g a n d with high a f f i n i t y f o r the Insfl,4,5)P3 receptor (39). [ S ] - L a b e l l e d D-Ins(1,4,5)PS was prepared by an adaptation of the chemical s y n t h e s i s of u n l a b e l l e d material (8). Experiments to c h a r a c t e r i z e the interaction of this ligand with the c e r e b e l l a r Ins(l,4,5)P receptor have shown that D-[ S]I n s ( l , 4 5 ) P S binds with high a f f i n i t y ( K , 31±2.8 nM) t o c e r e b e l l a r membranes, r e v e a l i n g a high d e n s i t y of Ins(l 4 5)P r e c e p t o r s t o be present (B 16.9±0.3 pmol/mg p r o t e i n ) . I d e n t i c a l r e s u l t s are obtainable by saturation a n a l y s i s of the isotope dilution data obtained by competition with u n l a b e l l e d Ins(1,4,5)PS . These results agree with previous estimates of Ins(l,4 5)P receptor d e n s i t y i n t h i s t i s s u e (18) and suggest t h a t [ S ] - I n s ( l 4 , 5 ) P S has only a s l i g h t l y lower a f f i n i t y f o r t h i s b i n d i n g s i t e , c o n s i s t e n t with a study on the u n l a b e l l e d l i g a n d (12). Further s t u d i e s suggest t h a t the a s s o c i a t i o n r a t e of [ S ] - I n s ( l , 4 , 5 ) P S with the c e r e b e l l a r receptor s i t e i s r a p i d , with e q u i l i b r i u m b i n d i n g being a t t a i n e d w i t h i n 10 min. a t 4°C and t h a t the s t e r e o - and p o s i t i o n a l displacement of s p e c i f i c a l l y bound [ S]-Ins(l,4,5)PS by D-Ins(l,4,5)P , L-Ins(1,4,5)P and D-Ins(2,4,5)P have the same rank order compared t o previous s t u d i e s using D-[ H]-Ins(l,4,5)P as the r a d i o l i g a n d (18). The r e a l p o t e n t i a l value of t h i s r a d i o l i g a n d l i e s with i t s metabolic stability, allowing evaluation of the p r o p e r t i e s of the I n s ( l , 4 , 5 ) P receptor a t 37°C and with a p h y s i o l o g i c a l i o n i c environment. The p r e p a r a t i o n of r a d i o l a b e l l e d phosphorothioate analogues of I n s ( l , 4 , 5 ) P has a l s o been achieved by phosphorylation of the polyphosphoinositide lipids Ptdlns and PtdIns(4)P using human e r y t h r o c y t e ghost kinases and A T P 7 S . [ S ] - R a d i o l a b e l l e d m a t e r i a l can be synthesized by employing [ S ] - A T P 7 S , and i n c u b a t i o n of [ S]-ATP7S with e r y t h r o c y t e ghosts produced [ S]labelled PtdIns(4,5)P analogues, with [ S]-label uniquely i n the 5 - p o s i t i o n or i n both the 4- and 5positions [Figure 5 ] . These modified lipids were cleaved by the endogenous Ca -activated phosphoinositidase C t o g i v e a mixture of i n o s i t o l 1,4bisphosphate-5J* S]-phosphorothioate (7) and i n o s i t o l 1phosphate-4,5[ S]-bisphosphorothioate (8), which was demonstrated t o be r e s i s t a n t t o 5-phosphatase. (40). Chemical s y n t h e s i s of [ S ] - i n o s i t o l phosphorothioates as d e s c r i b e d above, however, o f f e r s a more d i r e c t and reproducible strategy. In summary, i n o s i t o l phosphorothioates are proving t o be v a l u a b l e pharmacological and biochemical t o o l s . Clearly, the commercial availability of both DI n s ( l , 4 , 5 ) P S and D - [ S ] - I n s ( 1 , 4 , 5 ) P S w i l l now provide even greater opportunities for the biological e x p l o i t a t i o n of these novel analogues. 3 5

3

35

3

/


/

/

3

d

3

m a x

3

/

3

3 5

/

3

35

3

35

3

3

3

3

3

3

3

3

3 5

3 5

3 5

3 5

3 5

?

2 +

35

35

3 5

35

3

3



14.

POTTER


3 5

ATP 7 [ S ] Erythrocyte Ghosts ' S - l a b e l l e d Phospholipid 2

R-, - P 0 ' ; R = P ^ S O j ^ l P I R S ) ] 3

2

and R

t

- R

35

2

2

- P S0 "[PIP(S) ] 2

2

2 +

Ca -Activated Phosphodiesterase

DAG +

3 5

F i g u r e 5. Preparation of [ S ] - i n o s i t o l phosphorot h i o a t e s from Ptdlns and PtdIns(4)P. A f t e r Folk e t al. (40). Reproduced with permission from r e f . 7. Copyright 1990 John Wiley & Sons L t d .


199

200

INOSITOL PHOSPHATES A N D DERIVATIVES

Acknowledgments Research on m y o - i n o s i t o l phosphorothioates was supported by t h e Science & Engineering Research C o u n c i l (U.K.). [ S]-labelled jnyo-inositol phosphorothioates were synthesized by Dupont NEN (Boston, U.S.A.). Biological e v a l u a t i o n s were c a r r i e d out i n c o l l a b o r a t i o n with S. R. Nahorski. We thank Susan Alston f o r manuscript preparation. Β. V. L. P o t t e r i s a L i s t e r I n s t i t u t e Research P r o f e s s o r . Downloaded by NORTH CAROLINA STATE UNIV on August 1, 2012 | http://pubs.acs.org Publication Date: May 30, 1991 | doi: 10.1021/bk-1991-0463.ch014

3 5

Literature cited 1. 2. 3. 4. 5. 6. 7.

8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

Berridge, M. J. Annu. Rev. Biochem. 1987, 56, 159193. Nahorski, S. R.; Potter, Β. V. L. Trends Pharmacol. Sci. 1989, 10, 139-144. Chilvers, E. R.; Kennedy, E. D.; Potter, Β. V. L. Drug News & Perspectives 1989, 2, 342-346. Ghosh, T. K.; Eis, P. S.; Mullaney, J . M.; Ebert, C. L . ; Gill, D. L. J. Biol. Chem. 1988, 263, 1107511079. Billington, D. C. Chem. Soc. Rev. 1989, 18, 83-122. Potter, Β. V. L. Nat. Prod. Reports 1990, 7, 1-24. Potter, Β. V L. In Transmembrane Signalling, Intracellular Messengers & Implications for Drug Development, Nahorski, S. R., ed.; Wiley, Chichester, UK., 1990, pp207-239. Cooke, A. M.; Noble, N. J.; Gigg, R.; Payne, S.; Potter, Β. V. L. J. Chem. Soc. Chem. Commun. 1987, 1525-1526. Cooke, A. M.; Noble, N. J.; Gigg, R.; Payne, S.; Potter, Β. V. L. J. Chem. Soc. Chem. Commun. 1988, 269-271. Lampe, D.; Potter, Β. V. L. J. Chem. Soc. Chem. Commun. 1990, 1500-1501. Hamblin, M. R.; Flora, J . S.; Potter, Β. V. L . ; Biochem. J. 1987, 246, 771-774. Willcocks, A. L.; Potter, Β. V. L.; Cooke, A. M.; Nahorski, S. R. Eur. J. Pharmacol. 1988, 155, 181183. Hamblin, M. R.; Gigg, R.; Potter, Β. V. L. J. Chem. Soc. Chem. Commun. 1987, 626-627. Gigg, J.; Gigg, R.; Payne, S.; Conant, R. J. Chem. Soc. Perkin Trans. I 1987, 423-429. Metschies, T . ; Schulz, C . ; Jastorff, B. Tet. Lett. 1988, 29, 3921-3922. Schulz, C . ; Metschies, T . ; Jastorff, B. Tet. Lett. 1988, 29, 3919-3920. Lin. G.; Tsai, M. -D. J. Amer. Chem. Soc. 1989, 111, 3099-3101. Willcocks, A. L.; Cooke, A. M.; Potter, Β. V. L . ; Nahorski, S. R. Biochem. Biophys. Res. Commun. 1987, 146, 1071-1078.


14. POTTER

D-myo-Inositol I4 5-Trisphosphate y

y


19.

201

Nunn, D. L . ; Potter, Β. V. L . ; Taylor, C. W. Biochem. J. 1990, 265, 393-398. 20. Taylor, C. W.; Berridge, M. J.; Brown, K. D.; Cooke, A. M.; Potter, Β. V. L. Biochem. Biophys. Res. Commun. 1988, 150, 626-632. 21. DeLisle, S.; Krause, K. -H.; Denning, G.; Potter, Β. V. L . ; Welsh, M. J . J. Biol. Chem. 1990, 265, 11726-11730. 22. Strupish, J.; Cooke, A. M.; Potter, Β. V. L . ; Gigg, R.; Nahorski, S. R. Biochem. J. 1988, 253, 901-905. 23. Taylor, C. W.; Berridge, M. J..; Cooke, A. M.; Potter, Β. V. L. Biochem. J. 1989, 259, 645-650. 24. Thevenod, F.; Dehlinger-Kremer, M.; Kemmer, T. P.; Christian, A. -L.; Potter, Β. V. L . ; Schulz, I. J. Membr. Biol. 1989, 109, 173-186. 25. Wakui, M.; Potter, Β. V. L . ; Petersen, Ο. H. Nature 1989, 339, 317-320. 26. Mennitti, F. S.; Takemura, H . . ; Thastrup, O.; Potter, Β. V. L . ; Putney, J . W., Jnr. J. Biol. Chem. 1991 submitted. 27. Valdivia, C.; Valdivia, H. H.; Potter, Β. V. L . ; Coronado, R. Biophys. J. 1990, 57, 1233-1243. 28. Changya, L . ; Gallacher, D. V.; Irvine, R. F.; Potter, Β. V. L . ; Petersen, Ο. H. J Membr. Biol. 1989, 109, 85-93. 29. Cooke, A. M.; Nahorski, S. R . ; Potter, Β. V. L. FEBS Lett. 1989, 242, 373-377. 30. Safrany, S. T.; Wojcikiewicz, R. J . H.; Strupish, J.; McBain, J.; Cooke, A. M.; Potter, Β. V. L . ; Nahorski, S. R. Brit. J. Pharmacol. Proc. Suppl. 1990, 99, 88P. 31. Payne, R. F.; Potter, Β. V. L. J. Gen. Physiol. 1991, in press. 32. Berridge, M. J.; Potter, Β. V. L. Cell Regulation 1990, 1, 675-681. 33. Ferguson, J . ; Potter, Β. V. L . ; Nuccitelli, R. Biochem. Biophys. Res. Commun. 1990, 172, 229-236. 34. Wojcikiewicz, R. J . H.; Cooke, A. M.; Potter, Β. V. L.; Nahorski, S. R. Eur. J. Biochem. 1990, 192, 459-467. 35. Taylor, C. W.; Potter, Β. V. L . ; Biochem. J. 1990, 266, 189-194. 36. McCarren, M.; Potter, Β. V. L . ; Miller, R. J . Neuron 1989, 3, 461-471. 37. Valdivia, C.; Vaughan, D.; Potter, Β. V. L . ; Coronado, R. Science 1991 submitted. 38. Strunecka, A . ; Kmonickova, E . ; El Desouki, N.; Krpejsova, L . ; Palacek. J . ; Potter, Β. V. L. Receptor 1991, in press. 39. Potter, Β. V. L . ; Challiss, R. A. J.; Nahorski, S. R. Dupont Biotech Updates 1990, 5, 85-90. 40. Folk, P.; Kmonickova, E.; Krpejsova, L . ; Strunecka, A. J. Labelled Comp. Radiopharm. 1988, 25, 793-803. RECEIVED February 11, 1991


Phosphorothioate Analogues of - American Chemical Society

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