2'/3'-O-peptidyl adenosine as a general base catalyst of its own external peptidyl transfer: implications for the ribosome catalytic mechanism.

Although the presence of a 2’-OH in the 3’-terminal adenosine of peptidyl tRNA has long been known to be crucial for protein biosynthesis, the nature of its rate-enhancement effect is still poorly understood. A hydrogen-bond donation to the adjacent acyl group, a hydrogen-bond acceptance from the attacking amine nucleophile, a role in positioning the ribosome catalytic group (N3 of A2486), and even no participation at all in the elongation step have all been suggested. These hypotheses, however, have never been tested experimentally because of the supramolecular character of the ribosome and its complex with aminoacyl tRNA and peptidyl tRNA. The approaches of modern chemical biology, however, permit the supramolecular complexity to be reduced to a level that allows understanding of the chemical mechanism of this 2’-OH assistance on a fully chemical basis. Hence, we used a linear free-energy relationship (LFER) of the Brønsted type and kinetic isotope effect to study the external peptidyl transfer within a series of 2’/3’-O-peptidyl adenosine derivatives as peptidyl tRNA mimics. Herein we report an intramolecular general base catalysis by the vicinal 2’/3’-oxyanion of 2’/3’-peptidyl adenosine. This finding implies that a similar catalytic role of the partially deprotonated 2’-OH of the peptidyl tRNA A76 is possible, provided this P-site substrate group partially protonates the adjacent 3’oxygen in a cyclic hexagonal transition state involved in a substrate-assisted catalytic mechanism of the ribosome. The study became possible after Velikian et al. demonstrated that the acidity of 2’/3’-OH in ribonucleosides is steered by the nucleobase structure. Hence, we carried out modifications including atomic and group substitutions in the adenin-9-yl group of adenosine in order to obtain adenosine derivatives with varying 2’/3’-OH pKa values. Then we probed the pKa dependence of the rate of external transesterification (ethanolysis) of the corresponding 2’/3’-O-benzyloxycarbonyl-l-p-nitrophenylalanyl 5’-O-trityl adenosine derivatives 1–11 (Scheme 1). The transesterification reaction is known to be catalyzed by the ribosome. The substrates were designed so as to be soluble in organic media (tritylated at 5’-OH as well as functionalized with a hydrophobic 2’/3’-O-peptidyl group), to possess a suitable signal for HPLC monitoring (p-nitrated phenylalanine) and to undergo a peptidyl transfer reaction (transesterification instead of aminolysis) with a measurable rate. The solubility in aprotic organic media is required in order to mimic the environment of the ribosome active site, which must be nonaqueous in order to prevent premature hydrolysis. 8] In hydrogen-bond-donor solvents such as water, the adjacent 2’/3’-OH group is preferentially solvated separately by water molecules, thus preventing its potential catalytic effect. External transesterification of the adenosine derivatives 1–11 in acetonitrile does not occur in the absence of an organic base. It is promoted, however, by non-nucleophilic tertiary amines like 1,8-diazabicyclo[5.4.0]-undec-7-en (DBU).The measured pseudo-first-order rate constants kobs are summarized in Table 1. It can be seen that this apparent rate constant strongly depends on the presence of both a free 2’/3’-OH and a nucleobase, as well as on the nucleobase’s structural integrity. Actually, the rate of transesterification of the adenosine derivative 1 is more than 300-fold faster than that of the 2’-deoxyadenosine (2), 3’-deoxyadenosine (3), or 2’-methyladenosine (4) derivatives; this is in agreement with the earlier observation that the presence of an intact 2’/3’-OH is crucial to the reactivity. The absence of a nucleobase (1-deoxy ribose derivative 5) results in a more than one order of magnitude decrease of the rate as compared to the parent nucleoside 1 but is still approximately ten times faster than that of the 2’or 3’-deoxy derivatives. Substitution in and of the nucleobase also affects the rate. N6-benzoylation of adenosine 1 (N6-benzoyl adenosine 6), substitution of adenin-9-yl by guanin-9-yl group (7) and uracil-1-yl (10) significantly reduce the kobs values. N3-methylation of uridine derivative 10 (N3-methyluridine derivative 11), however, increases kobs. The substitution of N3 for C3-H (3-deazaadenosine derivative 8) and deletion of the pyrimidine ring (1-imidazolyl riboside (Im) derivative 9), surprisingly have no effect on kobs. Since 8 and 9 were designed to probe the plausible participation of N3 in the peptidyl transfer, we tried to find out whether such an effect really does not exist or whether it is masked. The lack of reactivity of 2’/3’-O-peptidyl adenosine derivatives 1–11 in the absence of a strong base, such as DBU, suggests that DBU promotes the transesterification reaction [a] M. M. Changalov, Dr. G. D. Ivanova, M. A. Rangelov, Dr. I. B. Stoineva, Dr. V. M. Yomtova, Prof. Dr. D. D. Petkov Laboratory of BioCatalysis, Institute of Organic Chemistry Bulgarian Academy of Sciences, 1113 Sofia (Bulgaria) Fax: (+359)2-8700225 E-mail : [email protected]