The strength of the 3′-gauche effect dictates the structure of 3′-O-anthraniloyladenosine and its 5′-phosphate, two analogues of the 3′-end of aminoacyl-tRNA

Abstract

Anthranilic acid charged yeast tRNA^Phe or E. coli tRNA^Val are able to form a stable complex with EF-Tu*GTP, hence the 2′- and 3′-O-anthraniloyladenosines and their 5′-phosphate counterparts have been conceived to be the smallest units that are capable of mimicking aminoacyl-tRNA. Since the 3′-O-anthraniloyladenosine also binds more efficiently to the EF-Tu*GTP complex compared to its 2′-isomer, we have herein delineated the stereoelectronic features that dictate the conformation of 3′-O-anthraniloyladenosine and its 5′-phosphate vis-a-vis their 2′-counterparts and we have also addressed how their structures and thermodynamic stabilizations are different from adenosine and 5′-AMP. It has been found that the electron-withdrawing anthraniloyl group exerts gauche effects of variable strengths depending upon whether it is at the 2′- or at 3′-position because of either the presence or absence of O2′–N9 gauche effect, [GE(O2′–C2′–C1′–N9)], thereby steering the pseudorotation of the constituent sugar moiety either to the North (N)-type (C3′-endo) or South (S)-type (C2′-endo) conformation. The 3′-O-anthraniloyladenosine 5′-phosphate has a relatively more stabilized S-type conformation ΔG° = –4.6 kJ mol^–1) than 3′-O-anthraniloyladenosine itself (ΔG° = –3.9 kJ mol^–1), whereas the ΔG° for 2′-O-anthraniloyladenosine and its 5′-monophosphate are respectively –0.9 and –1.8 kJ mol^–1, suggesting that the 3′-gauche effect of the 3′-O-anthraniloyl group is stronger than that of 2′-O-anthraniloyl in the drive of the sugar conformation. Since the EF-Tu can specifically recognize the aminoacylated-tRNA from the non-charged tRNA, we have assessed the free-energy (ΔG°) for this recognition switch to be the least ≈ –2.9 kJ mol^–1 by comparison of ΔG° of the N⇄pseudorotational equilibrium for 3′-O-anthraniloyladenosine 5′-phosphate and 5′-AMP. The 3′-O-anthraniloyladenosine and its 5′-phosphate are much more flexible than the isomeric 2′-counterparts as is evident from the temperature-dependent coupling constants analysis. The relative rate of the transacylation reaction of 2′(3′)-O-anthraniloyladenosine and its 5′-phosphate is cooperatively dictated by the two-state N⇄S pseudorotational equilibrium of the sugar, which in turn is controlled by a balance of the stereoelectronic 3′- and 2′-gauche effects as well as by the pseudoaxial preference of the 3′-O- or 2′-O-anthraniloyl group. The reason for the larger stabilization of the 2′-endo conformer for 3′-O-anthraniloyladenosine and its 5′-phosphate lies in the fact that the C3′–O3′ bond takes up an optimal gauche orientation with respect to the C4′–O4′ bond dictating the pseudoaxial orientation of the 3′-anthraniloyl residue, which can be achieved only in the S-type sugar conformation with adenin-9-yl and the 2′-OH groups in the pseudoequatorial geometry, compared to the preferred C3′-endo sugar with a pseudoaxial aglycone and 2′-OH found in the 3′-terminal adenosine moiety in the helical 3′-CCA end of uncharged tRNA.