The synthesis and reactivity of new 2-(N,N-diisopropylamino)-3-methylsulfonyl-1,3,2-benzoxazaphospholes. The utility of the 5-chloro analogue in the one-pot synthesis of oligothiophosphates: [ApsppA, ApspppA, ppp5′A2′ps5′A, m7GpsppA, Apspppp, Apspp]

Abstract The synthesis of 2-( N , N -diisopropylamino)-2,3-dihydro-3-methylsulfonyl-1,3,2-benzoxazaphospholes 22, 23 and 24 is reported. Their reactivities have been investigated using a variety of acid catalyst under conditions normally employed in phosphoramidite chemistry for oligonucleotide synthesis. The rate (k) of activation of 22, 23 and 24 by acid catalysis with N-methylanilinium hydrochloride (MAC) to their protonated species 25A, 26A and 27A , respectively, (Scheme 2) has been estimated to be 6.813 × 10 −7 mol −1 min −1 , 1.237 × 10 −6 mol −1 min −1 and 1.972 × 10 −7 mol −1 min −1 at 18°C with a ratio of the rates as 0.56 : 1 : 0.16. The 5-chlorobenzoxazaphosphole 23 selectively activated by MAC gave the intermediate 2-(N-methylanilinium)-5-chlorobenzoxazaphosphole 26A (∼90% by NMR), which was then reacted with alcohols (nucleosides) to generate the reactive 2-alkoxybenzoxazaphosphole 30 (Scheme 4) or 33–35 (Scheme 5) (∼70% by NMR). We have then shown that 33–35 (Scheme 5) react with binucleophilic reagents, ADP, ATP or pyrophosphate, to generate the corresponding P 1 -alkoxycyclometatriphosphite intermediate 36, 37, 50, 52 or 54 . These intermediates were then sulfurised to form the P 1 -alkoxy-1-thiocyclotriphosphate intermediates 38, 39, 51, 53 and 55 , which were ring-opened by hydrolysis. Thus these steps constituted a one-pot multicomponent reaction (MCR) leading to the synthesis of R P and S P mixtures of each of the mono-thioanalogues of naturally-occurring oligophosphates: Ap s ppA ( 43 ) (10%), Ap s pppA ( 45 ) (19%), ppp5′A2′p s 5′A ( 46 ) (24%), the cap structure m 7 Gp s ppA ( 47 ) (3%), Ap s pppp ( 42 ) (23%), Ap s pp ( 41 ) (33%) and Ap s p ( 40 ) (7%). The reaction of putative 34 and ADP gave the desired 43 (10%) along with pp5′A2′p s 5′A ( 44 ) (5%). The reaction sequences from 34 and ATP gave 45 (19%) and ppp5′A2′p s 5′A ( 46 ) (24%). The proposed reaction mechanism for the synthesis of 43, 45 and 47 proceeds via the corresponding (dinucleoside 5′)-cyclometatriphosphite intermediates 50, 54, 52 and the (dinucleoside 5′)-1-thiocyclotriphosphate intermediates 51, 55, 53 . The existence of these cyclic P(III) and P(V) intermediates were supported by 31 P- and 1 H-NMR spectroscopy. We here also demonstrate that 5-chlorobenzoxazaphosphole 23 can be used to synthesise a protected ribonucleoside 2′,3′-cyclic phosphorothioate block 28 , a protected ribonucleoside 3′-(O-(4-chloro-2-mesylsulfonamido)phenyl phosphorothioate diester block 29 and bis(2-deoxyadenosine-5′)-thymidine-3′-monophosphate 32 . The correct coupling of the nucleoside residues to the oligophosphate chain in 43, 45 & 47 has unequivocally been assessed by 2D 31 P- 31 P and 1 H- 31 P correlation spectroscopy.