Synthesis and biochemical application of 2'-O-methyl-3'-thioguanosine as a probe to explore group I intron catalysis

Abstract

Oligonucleotides containing 3'-S-phosphorothiolate linkages provide valuable analogues for exploring the catalytic mechanisms of enzymes and ribozymes, both to identify catalytic metal ions and to probe hydrogen-bonding interactions. Here, we have synthesized 2'-O-methyl-3'-thioguanosine to test a possible hydrogen-bonding interaction in the Tetrahymena ribozyme reaction. We developed an efficient method for the synthesis of 2'-O-methyl-3'-thioguanosine phosphoramidite in eight steps starting from 2'-O-methyl-N(2)-(isobutyryl) guanosine with 10.4% overall yield. Following incorporation into oligonucleotides using solid-phase synthesis, we used this new analogue to investigate whether the 3'-oxygen of the guanosine cofactor in the Tetrahymena ribozyme reaction serves as an acceptor for the hydrogen bond donated by the adjacent 2'-hydroxyl group. We show that regardless of whether the guanosine cofactor bears a 3'-oxygen or 3'-sulfur leaving group, replacing the adjacent 2'-hydroxyl group with a 2'-methoxy group incurs the same energetic penalty, providing evidence against an interaction. These results indicate that the hydrogen bond donated by the guanosine 2'-hydroxyl group contributes to catalytic function in a manner distinct from the U(-1) 2'-hydroxyl group.

Scheme 1

Reagents and conditions: i) ^tBuPh₂SiCl, Py, rt, 20 h. ii) CF₃SO₂Cl, DMAP, CH₂Cl₂ 0 □ 2 h. iii) NaBr, CH₃COCH₃, reflux, 3 h. iv) KSAc, DMF, 60 °C, 24 h. v) Bu₄NF·3H₂O, AcOH, THF, rt, 36 h. vi) (MeO)₂TrCl, Py, rt, 24 h. vii) Guanidine hydrochloride-NaOMe (4:1), MeOH-CH₂Cl₂ (1:1), rt, 30 min. viii) (^i-Pr)₂ NP(Cl)OC₂H₄CN, ^i-Pr₂NEt, 1-methyl-1H-imidazole, rt, 1 h

Figure 1. A 3′-S-phosphorothiolate linkage.

Figure 2. Transition state model of phosphoryl transfer catalyzed by the Tetrahymena group I ribozyme

M_A, M_B, and M_C represent the catalytic metal ions in the active site. The red rectangle highlights the 2′-hydroxyl group of the guanosine nucleophile under investigation. Hatched lines indicate putative hydrogen bonds, and dots symbolize metal ion coordination. Figure taken by permission from reference 21(b).

Figure 3. Atomic mutation cycle analysis of an RNA 2′-hydroxyl group.

Figure 4. Ribozyme reactions with CUCG_2′OHA, CUCG_2′OCH3A, CUCG_3′S,2′OHA and CUCG_{3′S,2′OCH3}A

Reactions took place in the presence of saturating ribozyme and product to form the E•P complex. CUCG_XA substrates were 5′-³²P-labeled (denoted by the asterisk). For ribozyme reactions with the phosphorothiolate substrates, we observed that CUCG_{3′SH,2′OCH3} migrated slower than CUCG3′SH,2′OH. To confirm the presence of the free thiol, we treated the ribozyme-catalyzed products with iodoacetamide, an alkylating sulfhydryl reagent (data not shown). Both CUCG_{3′SH,2′OH} and CUCG_{3′SH,2′OCH3} were susceptible to iodoacetamide modification and, upon alkylation, comigrated (data not shown).