Hydrolytic reactions of the R? and Sp diastereomers of the phosphoromonothioate analog of uridylyl-(3',5')uridine (3',5'-UpU), having a nonbridging oxygen replaced with sulfur, have been followed by HPLC over a wide pH range at 363.2 K. Under neutral and acidic conditions three reactions compete: (i) desulfurization to an equilibrium mixture of 3',5'-and 2',5'-UpU, (ii) hydrolysis to uridine 2'-and 3'-monophosphates with release of uridine (either via a 2',3'-cyclic phosphoromonothioate, or a desulfurized cyclic triester), and (iii) isomerization to the 2',5'-dinucleoside phosphoromonothioate. With both diastereomers, desulfurization predominates over hydrolysis and migration at pH 1-8. Migration proceeds by retention of configuration at phosphorus and is most pronounced in very acidic solutions (Hq < 0.2, i.e., [HC1] > 0.5 mol Lr1), representing 20-30% of the total disappearance of the starting material. At pH 3-6, the proportion of this reaction is less than 10%. In the latter pH range, all the reactions are pH-independent. At lower pH, first-order dependence on acidity is observed, but at Ho < 0.2 desulfurization becomes slower than the competing reactions. The R? diastereomer is at pH < 7 up to three times as reactive as the Sp isomer. Under alkaline conditions (pH > 9), only base-catalyzed hydrolysis to uridine 2'-and 3'thiophosphates with release of uridine takes place. At pH < 1, the thioate analogs are more than 1 order of magnitude more stable than UpU, while at higher pH the reactivities are comparable.
Base-catalyzed intramolecular transesterification in uridine
3‘-(aryl phosphorothioate)s (Up(S)Ar) has been
studied with respect to the dependence on the acidity of the conjugate
acid of the leaving aryloxy group (pK
a
7.1−10) as well as on the basicity of the catalyst
(pK
a(BH) = 7−10.2). The synthesis
of the studied phosphorothioates
was accomplished by using a method based on condensation of a protected
uridine 3‘-H-phosphonate with the
appropriate phenols. The rate constants for hydroxide and
imidazole catalysis (25 °C, 0.25 M ionic strength) obey
Brønsted linear free energy relationships and the obtained
βleaving
group
(βlg) values are −0.55 and −0.63,
respectively.
General-base-catalyzed release of 4-nitrophenoxide from the
corresponding phosphorothioate also obeys a Brønsted
relationship with respect to the basicity of the catalyst (β =
0.59). Rates of reactions of the phosphorothioates
are
somewhat lower than for the corresponding phosphates
(k
(UpAr)/k
(
S
P
-Up(S)Ar)
≈ 1.7−3.6 and
k
(UpAr)/k
(
R
P
-Up(S)Ar)
≈ 1.2−2.6 (the spatial arrangement of phosphorus ligands in the
S
P isomer of Up(S)Ar are the same as
in the R
P isomer of
a dinucleotide). Leffler α values of 0.59 for proton abstraction
and 0.36 for bond breaking to the leaving group in
the transition state do not balance (imbalance in α = 0.23),
indicating some negative charge buildup on the central
group of atoms in the transition state. The intramolecular
transesterification in uridine 3‘-(aryl phosphorothioate)s
is considered to be a concerted associative process with a mechanism
that is similar to that in the corresponding
phosphate esters, the difference being that the transition state
appears to have a slightly more dissociative character
for the phosphorothioates.
The first method for solid support synthesis of all-Rp-oligo(ribonucleoside phosphorothioate)s is presented as well as attempts to increase the stereoselectivity of the key step in this approach. The synthetic strategy consists of (i) a solid support synthesis procedure, using 5'-O-(4-methoxytriphenylmethyl)-2'-O-tert-butyldimethylsilyl-ri bon ucleoside 3'-H- phosphonates, that due to stereoselectivity in the condensation step, gives oligomers with mostly Sp-H-phosphonate diesters (72-89% under standard conditions), (ii) stereospecific sulfurization with S8 in pyridine to produce oligo(ribonucleoside phosphorothioate)s enriched with internucleosidic linkages of Rp configuration, (iii) treatment of the deprotected oligonucleotides with the enzyme Nuclease P1 from Penicillium citrinum, that specifically catalyses cleavage of Sp-phosphorothioate diester linkages, which leaves a mixture of oligomers having all internucleosidic linkages as Rp-phosphorothioates, and finally (iv) isolation and HPLC purification of the full length all-Rp oligomer. Mixed sequences containing the four common nucleosidic residues up to the chain length of a heptamer were synthesized. Change of N-4-protection on the cytidine building block from propionyl to N-methylpyrrolidin-2-ylidene gave a slightly improved diastereoselectivity in H-phosphonate diester formation. Increased selectivity up to 99+% was obtained with the guanosine building block when the amount of pyridine in the coupling step was reduced.
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