Novel phosphonate and thiophosphate analogues of Ap3A, diadenosine 5′,5′''-P1,P3-triphosphate.

TetrahedrmLaters.Vo1.31,No.39. PI, 5637-5644 1990 Rited in Great Briti

oo4o-4039/90

NOVEL PHOSPHONATE AND THIOPHOSPHATE ANALOGUES DIADENOSINE 5’,5”‘-Pl,@-TRIPHOSPHATE. G.Michael

Blackburn,’

Chemistry

Abstract

Department,

(2a-f)

morpholidate methylene

PI rP3-dithio-

diphenyl

of Sheffield,

from the condensation analogues

(3) of Ap3A is obtained acid

PI ,P2-Carbon-bridged

of ADP, the

adenosine,

of Ap3A are obtained

to condense

S3 7HF, UK.

of adenosine

from the reaction

and 5’-0-tosy/

(46) analogues

phosphorochloridate

Sheffield

of Ap3A have been prepared.

with a range of a,/%methylene

phosphinomethyl)phosphinic and

University

of Ap3A (1) result

analogue

OF APaA,

Mao-Jun Guo, Steven P. Langston, and Graham E.Taylor

Three types of analogue

analogues

53.00 + .oa

Pcrgamml Rcssplc

5’-phosphoro-

Pl,P2:P2,P3-bis-

between

bis(dihydroxy-

and the

PI-monothio-

by activating

AMPS with

with ADP and with phosphoric

(4a)

acid

respectively.

Diadenosine

5’,5”‘-Pt ,@-triphosphate,

ApsA, (1)

is a natural

nucleotide,

discovered

along with Ap4A in 1966 by Paul Zamecnik. 1 It is found both in eukaryotes prokaryotes involves

and also,

corresponding

HO

platelets

in

synthetase.

triphosphate

Cellular

and which

hydrolases,

operate with Km values

homo.2

adenylate

Its biosynthesis catalysedll3

by the

levels of Ap3A are held down

range4 as a result of the very efficient

dinucleoside

mammals,s

in blood

of ADP with an aminoacyl

aminoacyl-tRNA

to the micromolar specific

significantly,

the condensation

and in

which

degradation

are found

of Ap3A by

in plants5 and in

in the range 10-4-l O-%4. 1

OH

X=Y=O

2

(a) X = CH,, (b) X = CHF, X = CF,, (d) X = CHCI, (e) X = Ccl,, (f) X = C2H4; Y = 0. (c)

4;,X.;,Y.“pP Hi 00

db-

.&

NH2

As part of a programmer-11 dinucleoside

polyphosphate

HO

OH

to investigate

hydrolases

5’,5”‘-PI,--triphosphate

the mechanism

and to uncover

nucleoside polyphosphates, we here describe of Ap3A for evaluation of their acceptability diadenosine

3 X=Y=CH2 of such

role of di-

the synthesis of a range of analogues as substrates and/or inhibitors of

hydrolases. 5637

of action

the biological

5638

For full inhibition Ap3A

which

ence has shown bridges,rby

bridges

condensation

that this may be achieved the synthesis (2a-f).

1.

to require

analogues

or at P-2.

Previous

either by the use of substituted

of analogues

having

of carbon-bridged

methylene

of these.11 Accordingly,

route to these compounds

by the method’s

of experi-

a range of Pl:P2-substituted

S-phosphoromorpholidate

prepared

Characteristics

we expect at P-l(P-3)

or by a combination

The most effective

of adenosine

of ADP, conveniently Table

hydrolases,

attack either

the use of thiophosphates,*

we first explored carbon

of the ApsA

can resist enzyme

with

of Poulter (Scheme

analogues

involves

Pf:P2-methylene

the

analogues

1 and Table 1).

of ApsA

2e

I= 2f 3

I I

4a 4b

a Centre for mixed Yields

diastereoisomers

for the first step are generally

in Table 1. The structures (Table 1). with 2J

Typical

of the products

AMX phosphorus

couplings

good, those for the second (2a-f)

were established

spectra are observed

of normal magnitude.

The expected

for compounds P-C-F couplings

(60Hz) and (2~) (83Hz) are similar for both Pt and P2 nuclei. feature

of these spectra

tional.

The spectra

diastereoisomers ylene bridge.

for products

These isomers showed

All of the compounds invariably

provides

tri-sodium

salts

centre

also

for (2b)

at 79.2Hz,

expected,

is excep-

the presence

of

at the Pt-P2-halogenometh-

no signs of separation

on reverse phase hplc.

gave good FAD mass spectra, in which the [M+Na]+ ion almost (Table

1, column

8) with di- and

identifiable. analogue

at Pl and at P2.

It was prepared

0-isopropylidene

adenosine

(3) is designed by the reaction

5’4osylate

Crude (3) was obtained

being very difficult

reveal,as

the base peak of the spectrum

The bismethylene

phinic acid.

(2b) and (2d)

from the new chiral

(2a, c, and e)

One major, unexpected

is the very large 3Jpccp for (2f) which,

resulting

step are listed

by 1H and 31 P NMR

and

to resist cleavage between

by ApsAases

a 2.6-fold

excess

bis(dihydroxyphosphinomethyl)phos-

in only 12% yield and, with an isomeric

to remove, the final yield of pure (3) was a meagre

It gives an A2X phosphorus

NMR spectrum

both

of 2’,3’product

3% (Table 1).

5639

Tos-0

0Ade

,O,p/X.P/O.P/O.*do Ado bv\ //\ 1\ 0 o- 0 o- 0 0

ii

i_

k HO OH

HO OH

I?%agmb: i, Ho3PXP%3m;

2 a, X = CH2; b, X I CHF; c, X = CF2; d, X = CHCI; 8, X = CC12; f, x = C2H4

ii, Ado-O-Pop-- NzO

In view of the fact that Ap4A hydrolases from both Ecoli and lupin have shown an unexpected capability in the case 7-s of certain phosphonate mimics of Ap4A to switch

their

target

the E.co/i enzyme phosphoryl

centre

for hydrolytic and P-l (P-l

attack

from the usual

for the lupin

for the Ecoli

hydrolase)

phosphorus

atom

to an alternative

(P-2 for

adjacent

and P-2 for the lupin hydrolase),

we also have

need of the PI-monothio-

and Pl ,Ps-dithio-analogues of ApsA to complete an investigation of These were obtained by condensation of adenosine the full range of ApsA hydrolases. S-thiophosphate adenosine

by diphenyl

phosphorochloridate

S-diphosphate

to give

(4a)

and with a limiting

respectively

(Scheme

2).

Ade

i, ii

to give (4b)

activated

o

k HO

This

process

and of three

necessarily

provides

context

phosphate either

it is noteworthy

chains

generally

as enzyme

is the difficulty enzymatic

is apparent

by reversed

in preparing

and its analogues,’

or inhibitors.

in internal

of dinucleoside

polyphosphates

Results on the use of these compounds ApgAases will be reported in due course.

in poly-

to hydrolysis

at internal and/or

for use

of this situation

the stereochemical

as substrates

of ApsA.

positions

One of the consequences

to determine

1

phase hplc.

the p-monothio-analogue

that thiophosphates

on attempts

for (4a) in the 31P

In view of our recent success

of Pl ,P4-dithio-Ap4A

have been found to be too unstable

substrates

imposed

hydrolysis

species (Table 1).

of resolution

Lastly, we have not succeeded In this

acid

(4b) if = S

of two diastereoisomers

and their existence

diastereoisomers

we expect these to be capable

(da) 2 -0;

a mixture

for (4b),

for the unseparated

of mixed

of

of phosphoric

OH

diastereoisomers

NMR resonances in separation

an excess

c

i, (Ph0)2POCl/pyridine; ii, ADP (Z = 0) or PI (Z = S)

Reaaents:

with

quantity

course

positions.13 inhibitors

for

of

5640

Acknowledgements work and Amersham Studentship

We thank the SERC for generous International

plc for their active

financial

support

subvention

through

of this

a CASE

(to G.E.T.).

References 1.

2. 3. 4. 5. 6. 7. 6. 9. 10. 11. 12. 13.

Zamecnik P., Stephenson M., Janeway CM., and Randerath K., Biochem.Biophys.Res. Commun., 1966,24, 91; Randerath K.. Janeway CM, Stephenson ML., and Zamecnik P., Biochem. Biophys. Res.Commun., 1966,24,98. Hurtado C, Rufz A., Sillero A., Sillero G., and Maria A., J.Bacterio/, 1987,169,1718; Lijthje J. and Ogilvie A., Biochem.Biophys. Res.Commun., 1983,115.253. Plateau P., Mayaux J-F., and Blanquet S., Biochemistry; 1981,20,4654. Ogilvie A. and Jakob P., AnalyLBiochem., 1983,134,382. Jakubowski H., and Guranowski A., J.Bio/.Chem., 1983,258,9982 Sillero M.A.G., Villalba FL, Moreno A., Quintanilla M., Lobat6n CD., and Sillero A., Eur. J. Biochem., 1977,76,33 1. Blackburn G.M., Thatcher G.R.J., Taylor G.E., Prescott M., and McLennan A.G., Nucleic Acids Res., 1987,15,6991. McLennan A.G. Taylor G.E., Prescott M., and Blackburn G.M., Biochemistry,l989,28,3868 . Guranowski A., Starzynska A., Taylor G.E., and Blackburn G.M., Biochem.J.,1989,262,241. Blackburn G.M., Guranowski A., Guo M-J., McLennan A.G., and Taylor G.E., Phosphorus and Sulphur, 1990,51,31. Blackburn GM. and Guo MJ., Tefrahedron Let& 1990,31,4371. Davisson V.J., Davis D.R., Dixit V.M., and Poulter C.D., J.Org.Chem., 1987,52,1794; and refs therein. Lowe,G., personal communication.

(Received in U& 20 July 1990)