Robust Synthesis and Crystal‐Structure Analysis of 7‐Cyano‐7‐deazaguanine (PreQ₀ Base) and 7‐(Aminomethyl)‐7‐deazaguanine (PreQ₁ Base)

Abstract

We describe robust and efficient synthetic methods for the synthesis of the preQ 0 and preQ 1 bases, which are the biosynthetic precursors of the hypermodified RNA nucleoside queuosine. The X-ray crystal-structure analysis of preQ 1 is also described.Introduction. -10 -25% of all nucleotides present in tRNA are modified [1]. Today, more than 80 different nucleotide modifications are known [2]. The nature of the observed nucleotide modifications varies strongly. Frequently, methylations of the nucleobase or of the sugar moieties are found. Sometimes, however, also strongly altered nucleotide structures, so called 'hypermodifications' of the canonical bases, are observed. The biological impact of the modified bases in tRNA is unknown but it is generally assumed that the modifications are needed for the fine tuning of the translational process at the ribosome [3]. The biosynthesis of the hypermodified bases is also only rudimentarily understood. To gain insight into the biosynthesis and the function of the modified bases in tRNA, the chemical synthesis of the modified bases via robust, efficient, and high-yielding methods is required.One of the most significant hypermodifications is the nucleoside queuosine (Q; (= 7-{[(3,4-trans-4,5-cis-4,5-dihydroxycyclopent-1-en-3-yl)amino]methyl}-7-deazaguanosine), which is generally found in the anticodon loop of the tRNAs encoding for the amino acids aspartate, asparagine, histidine, and tyrosine [4]. The nucleoside Q was initially discovered by Harada and Nishimura in 1972 [5]. The proposed biosynthesis of this unusual nucleoside via its biosynthetic precursors preQ 0 and preQ 1 is depicted in Scheme 1 [6] [7]. Today, it is clear that bacteria produce Q-nucleobase-modified tRNA directly by using the unmodified precursor tRNA as the substrate. The enzyme tRNA-guanine transglycosylase (TGT) removes in a first step a specific guanine from the tRNA by cleaving the glycosidic bond between the ribose and the nucleobase [8]. The abasic site, which is thus created, reacts in a second step with the preQ 1 nucleobase, which is biosynthesized by a recently discovered enzyme QueF from preQ 0 (= 7-cyano-7-deazaguanine) to generate a preQ 1 (= 7-(aminomethyl)-7-deazaguanine)-containing tRNA [9]. The tRNA containing preQ 1 base is subsequently further modified to give first epoxyqueuosine-containing tRNA by the action of the S-adenosylmethioninedependant enzyme QueA. Final conversion of epoxyqueuosine to queuosine is carried out by an unknown enzyme in a possibly vitamin B 12 -dependant reaction [10].

Kinetic Analysis and Probing with Substrate Analogues of the Reaction Pathway of the Nitrile Reductase QueF from Escherichia coli

“…VNMRJ 2.2D software was used to process the spectra. The spectra of coenzymes, preQ 0 and preQ 1 , were compared with the literature-known spectra of these compounds (20,30,31). Considering the possibility of deuterium labeling at the C4 of NADP ϩ , reported in the literature to occur in reactions of the T. brockii alcohol dehydrogenase under the conditions used here (32,33), we checked carefully with LC-MS the (4R)-[ 2 H]NADPH preparation used.…”

Structure of a class II preQ1 riboswitch reveals ligand recognition by a new fold

“…Fig. 2a) using starting coordinates derived from the small-molecule crystal structure 31 . Cs + ions were included in the refined model based on their anomalous diffraction signal (Supplementary Fig.…”

Folding of a transcriptionally acting PreQ 1 riboswitch

“…Its aptamer comprises a stretch of only 34 nucleotides in the 5′ untranslated leader region of the respective messenger RNA and specifically recognizes 7-aminomethyl-7-deazaguanine (preQ 1 ), which is an intermediate in queuosine biosynthesis (11). The minimal sequence and structure consensus refer to a hairpin comprising a five base-pair stem (P1) and a loop of eleven to thirteen nucleotides (L1) together with a 3′ singlestranded nucleoside overhang as originally proposed by Breaker and coworkers (10).…”