Base Catalysis and Leaving Group Dependence in Intramolecular Alcoholysis of Uridine 3‘-(Aryl phosphorothioate)s

Abstract

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.

Ribozyme speed limits

“…As a result, the 2Ј-oxyanion has been proposed to a better nucleophile than a hydroxyl by a factor of up to 10 10 (Oivanen et al 1998). This parallels the observation that cyclic and acyclic phosphorous compounds are 10 10 -fold more susceptible to nucleophilic attack by hydroxide than by water (Almer and Strömberg 1996). Therefore, the slope of one depicted in Figure 2A under basic pH is established by a rate-determining deprotonation of the 2Ј-hydroxyl group (Li and Breaker 1999).…”

“…The RNA linkage (1) passes through a pentacoordinate species (2) that degrades into fragments that carry either a 2Ј,3Ј-cyclic phosphate terminus (3) or a 5Ј-hydroxyl terminus (4). The four catalytic strategies that can influence the reaction are identified as follows: ␣, in-line nucleophilic attack (blue); ␤, neutralization of negative charge on a nonbridging phosphate oxygen (purple); ␥, deprotonation of the 2Ј-hydroxyl group (red); and ␦,age of phosphoanhydrides, phosphomonoesters, and phosphoramidates (Benkovic and Schray 1973;Guthrie 1977;Westheimer 1987;Thatcher and Kluger 1989;Admiraal and Herschlag 1995;Oivanen et al 1995;Almer and Strömberg 1996). RNA transesterification occurs spontaneously in the absence of any added catalyst with a first-order rate constant that varies with changing pH (Järvinen 1991;Li and Breaker 1999).…”

“…4; Westheimer 1968), has never been detected in alkaline solution (Perrault and Anslyn 1997). Likewise, the products of decomposition of RNA analogs indicate that a monoanion or neutral species is required for pseudorotation (Almer and Strömberg 1996). Furthermore, pseudorotation of a dianion would place an electron-donating group unfavorably in an apical position (Zhou and Taira 1998).…”

Mutation of Arg-166 of Alkaline Phosphatase Alters the Thio Effect but Not the Transition State for Phosphoryl Transfer. Implications for the Interpretation of Thio Effects in Reactions of Phosphatases

“…The loss of the guanidinium group and its hydrogen bonding interactions will be deleterious to the complementarity between the active site and the phosphoryl group in the transition state. However, the removal of this side chain will allow for better accommodation of the larger phosphorothio- (23,(40)(41)(42)(43)(44). ate group in the active site.…”

Ribonucleases: From Prototypes to Therapeutic Targets? (General Articles)

“…The lack of a significant R P thioeffect in RNase T 1 is actually the net result of counterbalancing unfavorable and favorable interactions in the transition state upon thiosubstitution. On one hand, R P thiosubstitution disrupts a catalytic hydrogen bond between the hydroxyl group of Tyr38 and the pro-R P oxygen, but on the other hand it makes the transition state slightly more dissociative, causing an increased charge build up on the apical oxygens and hence improved catalytic interactions with these oxygens [47,50].…”