Chemo-enzymatic synthesis of selectively ¹³C/¹5N-labeled RNA for NMR structural and dynamics studies.

RNAs are an important class of cellular regulatory elements, and they are well characterized by X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy in their folded or bound states. However, the apo or unfolded states are more difficult to characterize by either method. Particularly, effective NMR spectroscopy studies of RNAs in the past were hampered by chemical shift overlap of resonances and associated rapid signal loss due to line broadening for RNAs larger than the median size found in the PDB (~25 nt); most functional riboswitches are bigger than this median size. Incorporation of selective site-specific (13)C/(15)N-labeled nucleotides into RNAs promises to overcome this NMR size limitation. Unlike previous isotopic enrichment methods such as phosphoramidite, de novo, uniform-labeling, and selective-biomass approaches, this newer chemical-enzymatic selective method presents a number of advantages for producing labeled nucleotides over these other methods. For example, total chemical synthesis of nucleotides, followed by solid-phase synthesis of RNA using phosphoramidite chemistry, while versatile in incorporating isotope labels into RNA at any desired position, faces problems of low yields (<10%) that drop precipitously for oligonucleotides larger than 50 nt. The alternative method of de novo pyrimidine biosynthesis of NTPs is also a robust technique, with modest yields of up to 45%, but it comes at the cost of using 16 enzymes, expensive substrates, and difficulty in making some needed labeling patterns such as selective labeling of the ribose C1' and C5' and the pyrimidine nucleobase C2, C4, C5, or C6. Biomass-produced, uniformly or selectively labeled NTPs offer a third method, but suffer from low overall yield per labeled input metabolite and isotopic scrambling with only modest suppression of (13)C-(13)C couplings. In contrast to these four methods, our current chemo-enzymatic approach overcomes most of these shortcomings and allows for the synthesis of gram quantities of nucleotides with >80% yields while using a limited number of enzymes, six at most. The unavailability of selectively labeled ribose and base precursors had prevented the effective use of this versatile method until now. Recently, we combined an improved organic synthetic approach that selectively places (13)C/(15)N labels in the pyrimidine nucleobase (either (15)N1, (15)N3, (13)C2, (13)C4, (13)C5, or (13)C6 or any combination) with a very efficient enzymatic method to couple ribose with uracil to produce previously unattainable labeling patterns (Alvarado et al., 2014). Herein we provide detailed steps of both our chemo-enzymatic synthesis of custom nucleotides and their incorporation into RNAs with sizes ranging from 29 to 155 nt and showcase the dramatic improvement in spectral quality of reduced crowding and narrow linewidths. Applications of this selective labeling technology should prove valuable in overcoming two major obstacles, chemical shift overlap of resonances and associated rapid signal loss due to line broadening, that have impeded studying the structure and dynamics of large RNAs such as full-length riboswitches larger than the ~25 nt median size of RNA NMR structures found in the PDB.

Pyridine derivatives, especially 2,6-di-tert-butylpyridine, labeled with nitrogen-15.

Pyridine derivatives labeled with (15) N can be prepared by the reaction of the corresponding pyrylium salts with (15) NH4 Cl in close to a stoichiometric ratio, in a sodium acetate-acetic acid buffer. In particular, the reaction of 2,6-di-tert-butylpyrylium perchlorate gave 2,6-di-tert-butylpyridine with a conversion of 95%. The compound is valuable for studies of acid-base interactions on solid acid catalysts by (15) N nuclear magnetic resonance.

Synthesis of [1,3, NH2-(15)N3] (5'S)-8,5'-cyclo-2'-deoxyguanosine.

To facilitate NMR studies and low-level detection in biological samples by mass spectrometry, [1,3, NH2-(15)N3] (5'S)-8,5'-cyclo-2'-deoxyguanosine was synthesized from imidazole-4,5-dicarboxylic acid in 21 steps. The three (15)N isotopes were introduced during the chemo-enzymatic preparation of [1,3, NH2-(15)N3]-2'-deoxyguanosine using an established procedure. The (15)N-labeled 2'-deoxyguanosine was converted to a 5'-phenylthio derivative, which allowed the 8-5' covalent bond formation via photochemical homolytic cleavage of the C-SPh bond. SeO2 oxidation of C-5' followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH2-(15)N3] (5'S)-8,5'-cyclo-2'-deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography-mass spectrometry measurements.

15N magnetic resonance hyperpolarization via the reaction of parahydrogen with 15N-propargylcholine.

(15)N-Propargylcholine has been synthesized and hydrogenated with para-H(2). Through the application of a field cycling procedure, parahydrogen spin order is transferred to the (15)N resonance. Among the different isomers formed upon hydrogenation of (15)N-propargylcholine, only the nontransposed derivative contributes to the observed N-15 enhanced emission signal. The parahydrogen-induced polarization factor is about 3000. The precise identification of the isomer responsible for the observed (15)N enhancement has been attained through a retro-INEPT ((15)N-(1)H) experiment. T(1) of the hyperpolarized (15)N resonance has been estimated to be ca. 150 s, i.e., similar to that reported for the parent propargylcholine (144 s). Experimental results are accompanied by theoretical calculations that stress the role of scalar coupling constants (J(HN) and J(HH)) and of the field dependence in the formation of the observed (15)N polarized signal. Insights into the good cellular uptake of the compound have been gained.

(15)N-Labeled ionic probes for bioanalytical mass spectrometry.

An effective La-complex-based probe ionization method is reported. Novel stable isotopically labeled probes containing the (15)N-labeled 2,6-bis(oxazolin-2-yl)pyridine (pybox) ligand, succinimide-tetramethylpybox (NHS-TMpybox), maleimide-tetramethylpybox (Mal-TMpybox), and 4-(tetramethylpybox)-butyl bromoacetate (BrAc-TMpybox), have been synthesized and their value in analyzing large complex molecules has been studied. The value of the (15)N-labeled pybox-La complex in ionizing various compounds, including bioactive peptides by cold-spray ionization mass spectrometry is emphasized.

Syntheses of specifically 15N-labeled adenosine and guanosine.

This unit describes the specific incorporation of (15)N into the N7 and amino positions of adenosine, and conversion of the adenosine to guanosine labeled at the N1, N7, and amino positions. Two variations of the procedures are also presented that include either (12)C or (13)C at the C8 position of adenosine, and (13)C at either the C8 or C2 position of guanosine. These (13)C tags permit the incorporation of two (15)N-labeled nucleosides into an RNA strand while ensuring that their nuclear magnetic resonance (NMR) signals can be distinguished from each other by the presence or absence of C-N coupling. While the major application of these specifically (15)N-labeled nucleosides is NMR, the additional mass makes them useful in mass spectrometry (MS) as well. The procedures can also be adapted to synthesize the labeled deoxynucleosides. A support protocol describes the synthesis of 7-methylguanosine.