Interactions of Protonated Guanidine and Guanidine Derivatives with Multiply Deprotonated RNA Probed by Electrospray Ionization and Collisionally Activated Dissociation

Abstract

Interactions of ribonucleic acid (RNA) with guanidine and guanidine derivatives are important features in RNA–protein and RNA–drug binding. Here we have investigated noncovalently bound complexes of an 8‐nucleotide RNA and six different ligands, all of which have a guanidinium moiety, by using electrospray ionization (ESI) and collisionally activated dissociation (CAD) mass spectrometry (MS). The order of complex stability correlated almost linearly with the number of ligand atoms that can potentially be involved in hydrogen‐bond or salt‐bridge interactions with the RNA, but not with the proton affinity of the ligands. However, ligand dissociation of the complex ions in CAD was generally accompanied by proton transfer from ligand to RNA, which indicated conversion of salt‐bridge into hydrogen‐bond interactions. The relative stabilities and dissociation pathways of [RNA+m L−n H]n− complexes with different stoichiometries (m=1–5) and net charge (n= 2–5) revealed both specific and unspecific ligand binding to the RNA.

Relative Strength of Noncovalent Interactions and Covalent Backbone Bonds in Gaseous RNA–Peptide Complexes

“…The peptide ligands other than RR showed weaker binding to RNA 1 (Figure S6), and besides fragments from RNA backbone cleavage, CAD also produced free RNA as illustrated for VR in Figure 1b. Just like in our previous study, 35 (M – 4H) 4– RNA ions were not observed in CAD of the complexes with a net charge of −3, consistent with PT from protonated ligand to the RNA prior to ligand dissociation. The collision energy required for 50% ligand dissociation by breaking of noncovalent bonds, E 50 ( nc ), in CAD of the (M + VR – 3H) 3– ions was 36.3 ± 0.1 eV, and the collision energy required for 50% fragment formation by breaking of covalent bonds, E 50 ( c ), was 52.1 ± 0.1 eV.…”

“…However, RNAs 1 – 4 could potentially form hairpin structures in the presence of ligands. 35 The CAD experiments were performed over a period of 18 months, and we did not observe any correlation between the order of experiments and the collision energy required for dissociation of the RNA or RNA–ligand complexes. Between 50 and 100 scans were added for each spectrum.…”

“…The only ligand PA values available (tmeGnd, 1032 kJ/mol; Gnd, 986 kJ/mol; R, 1051 kJ/mol) 45 did not correlate with complex stability, the order of which (tmeGnd < Gnd < R) cannot be attributed to differences in the number of DOF of the complexes either (tmeGnd, 843; Gnd, 798; R, 855). 35 However, the E 50 ( nc ) values increased linearly with the number of hydrogen bond donors of the ligands (Figure 2, Scheme S1). E 50 ( nc ) values for GR, VR, DR, and ER were similar, which suggests that the side chain carboxylic acid functionalities were not involved in RNA–ligand binding.…”

“…In a recent CAD study of RNA 1 in complexes with guanidine or guanidine derivatives including R, we found that PT from protonated ligand (L) to a deprotonated phosphodiester moiety of the RNA, which converts the intermolecular salt bridge into a far weaker hydrogen bond, generally preceded ligand dissociation. 35 Likewise, PT from a protonated guanidinium group of RR to a deprotonated phosphodiester moiety of a c or y fragment should precede loss of RR. However, protonated RR has a zwitterionic structure in the gas phase 44 (Scheme 1) and can form two salt bridges with the RNA, such that transfer of two protons is required for SB to HB conversion.…”

Replacing H+by Na+or K+in phosphopeptide anions and cations prevents electron capture dissociation

“… 52 , 54 To test this hypothesis, we isolated the (M – H + 4Na) 2+ ˙ ions (along with the 7.7% (M – 2H + 4Na) 2+ ions from H˙ loss) and subjected them to IRMPD, which is a slow heating method 101 that effects dissociation via the channels of lowest energy. Thus IRMPD generally cleaves noncovalent before covalent bonds, 102 unless the energy required for noncovalent bond cleavage exceeds that for covalent bond cleavage. 53 , 103 Using energies (25% laser power, 180 ms irradiation time) that were sufficient for covalent bond cleavage in 59% of the (M – 2H + 4Na) 2+ ions from ESI and 27% dissociation of the (M + 2H) + ˙ ions from ECD of (M + 2H) 2+ ions resulted in only ∼15% dissociation of the (M – H + 4Na) 2+ ˙/(M – 2H + 4Na) 2+ ions.…”