RESUMO
Biomolecules containing adenosine di- or triphosphate (ADP or ATP) are crucial for diverse biological processes. Synthesis of these biomolecules and development of their chemical probes are important to elucidate their functions. Enabling reproducible and high-yielding access to these ADP- and ATP-containing molecules via conventional P(III)-P(V) and P(V)-P(V) coupling reactions is challenging owing to water content in highly polar phosphate-containing substrates. Herein, we report an efficient and reliable method for protecting-group-free P(V)-P(V) coupling reaction through inâ situ activation of phosphates using hydrolysis-stable 2-[N-(2-methylimidazoyl)]-1,3-dimethylimidazolinium chloride (2-MeImIm-Cl), providing the corresponding electrophilic P(V) intermediates for subsequent nucleophilic attack using their coupling partners. This P(V)-P(V) coupling reaction proceeded even in a wet reaction medium and showed a broad substrate scope, accommodating protecting-group-free synthesis of ADP-ribose and nicotinamide adenine diphosphate analogs, ATP-containing biomolecules, and ADP-ribosyl peptides.
Assuntos
Adenosina Difosfato Ribose , Trifosfato de Adenosina , Trifosfato de Adenosina/química , Adenosina Difosfato Ribose/química , Hidrólise , Difosfato de Adenosina/química , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Estrutura MolecularRESUMO
Adenylate kinase (AK) plays a crucial role in the metabolic monitoring of cellular adenine nucleotide homeostasis by catalyzing the reversible transfer of a phosphate group between ATP and AMP, yielding two ADP molecules. By regulating the nucleotide levels and energy metabolism, the enzyme is considered a disease modifier and potential therapeutic target for various human diseases, including malignancies and inflammatory and neurodegenerative disorders. However, lacking approved drugs targeting AK hinders broad studies on this enzyme's pathological importance and therapeutic potential. In this work, we determined the effect of a series of dinucleoside polyphosphate derivatives, commercially available (11 compounds) and newly synthesized (8 compounds), on the catalytic activity of human adenylate kinase isoenzyme 1 (hAK1). The tested compounds belonged to the following groups: (1) diadenosine polyphosphates with different phosphate chain lengths, (2) base-modified derivatives, and (3) phosphate-modified derivatives. We found that all the investigated compounds inhibited the catalytic activity of hAK1, yet with different efficiencies. Three dinucleoside polyphosphates showed IC50 values below 1 µM, and the most significant inhibitory effect was observed for P1-(5'-adenosyl) P5-(5'-adenosyl) pentaphosphate (Ap5A). To understand the observed differences in the inhibition efficiency of the tested dinucleoside polyphosphates, the molecular docking of these compounds to hAK1 was performed. Finally, we conducted a quantitative structure-activity relationship (QSAR) analysis to establish a computational prediction model for hAK1 modulators. Two PLS-regression-based models were built using kinetic data obtained from the AK1 activity analysis performed in both directions of the enzymatic reaction. Model 1 (AMP and ATP synthesis) had a good prediction power (R2 = 0.931, Q2 = 0.854, and MAE = 0.286), while Model 2 (ADP synthesis) exhibited a moderate quality (R2 = 0.913, Q2 = 0.848, and MAE = 0.370). These studies can help better understand the interactions between dinucleoside polyphosphates and adenylate kinase to attain more effective and selective inhibitors in the future.
Assuntos
Adenilato Quinase , Fosfatos de Dinucleosídeos , Relação Quantitativa Estrutura-Atividade , Humanos , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/farmacologia , Fosfatos de Dinucleosídeos/metabolismo , Cinética , Estrutura Molecular , Adenilato Quinase/metabolismo , Adenilato Quinase/antagonistas & inibidores , Relação Dose-Resposta a Droga , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/químicaRESUMO
Dinucleoside 5',5'-polyphosphates (DNPs) are endogenous substances that play important intra- and extracellular roles in various biological processes, such as cell proliferation, regulation of enzymes, neurotransmission, platelet disaggregation and modulation of vascular tone. Various methodologies have been developed over the past fifty years to access these compounds, involving enzymatic processes or chemical procedures based either on P(III) or P(V) chemistry. Both solution-phase and solid-support strategies have been developed and are reported here. Recently, green chemistry approaches have emerged, offering attracting alternatives. This review outlines the main synthetic pathways for the preparation of dinucleoside 5',5'-polyphosphates, focusing on pharmacologically relevant compounds, and highlighting recent advances.
Assuntos
Fosfatos de Dinucleosídeos/síntese química , Agonistas do Receptor Purinérgico P2Y/síntese química , Nucleotídeos de Desoxicitosina/agonistas , Nucleotídeos de Desoxicitosina/química , Nucleotídeos de Desoxicitosina/farmacologia , Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/isolamento & purificação , Síndromes do Olho Seco/tratamento farmacológico , Química Verde , Humanos , Soluções Oftálmicas , Fosforilação , Polifosfatos/síntese química , Polifosfatos/química , Agonistas do Receptor Purinérgico P2Y/química , Agonistas do Receptor Purinérgico P2Y/isolamento & purificação , Receptores Purinérgicos/metabolismo , Nucleotídeos de Uracila/química , Uridina/agonistas , Uridina/análogos & derivados , Uridina/química , Uridina/farmacologiaRESUMO
Hydrogen-rich DNA dinucleotide cation radicals (dGG + 2H)+â¢, (dCG + 2H)+â¢, and (dGC + 2H)+⢠represent transient species comprising protonated and hydrogen atom adducted nucleobase rings that serve as models for proton and radical migrations in ionized DNA. These DNA cation radicals were generated in the gas phase by electron-transfer dissociation of dinucleotide dication-crown-ether complexes and characterized by UV-vis photodissociation action spectra, ab initio calculations of structures and relative energies, and time-dependent density functional theory calculations of UV-vis absorption spectra. Theoretical calculations indicate that (dGG + 2H)+⢠cation radicals formed by electron transfer underwent an exothermic conformational collapse that was accompanied by guanine ring stacking and facile internucleobase hydrogen atom transfer, forming 3'-guanine C-8-H radicals. In contrast, exothermic hydrogen transfer from the 5'-cytosine radical onto the guanine ring in (dCG + 2H)+⢠was kinetically hampered, resulting in the formation of a mixture of 5'-cytosine and 3'-guanine radicals. Conformational folding and nucleobase stacking were energetically unfavorable in (dGC + 2H)+⢠that retained its structure of a 3'-cytosine radical, as formed by one-electron reduction of the dication. Hydrogen-rich guanine (G + H)⢠and cytosine (C + H)⢠radicals were calculated to have vastly different basicities in water, as illustrated by the respective p Ka values of 20.0 and 4.6, which is pertinent to their different abilities to undergo proton-transfer reactions in solution.
Assuntos
Fosfatos de Dinucleosídeos/química , Radicais Livres/química , Oligodesoxirribonucleotídeos/química , Éteres de Coroa/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/efeitos da radiação , Elétrons , Radicais Livres/síntese química , Radicais Livres/efeitos da radiação , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/síntese química , Oligodesoxirribonucleotídeos/efeitos da radiação , Fótons , Prótons , Espectrofotometria UltravioletaRESUMO
The 5' cap consists of 7-methylguanosine (m7G) linked by a 5'-5'-triphosphate bridge to messenger RNA (mRNA) and acts as the master regulator of mRNA turnover and translation initiation in eukaryotes. Cap analogues that influence mRNA translation and turnover (either as small molecules or as part of an RNA transcript) are valuable tools for studying gene expression, which is often also of therapeutic relevance. Here, we synthesized a series of 15 dinucleotide cap (m7GpppG) analogues containing a 5'-phosphorothiolate (5'-PSL) moiety (i.e., an O-to-S substitution within the 5'-phosphoester) and studied their biological properties in the context of three major cap-binding proteins: translation initiation factor 4E (eIF4E) and two decapping enzymes, DcpS and Dcp2. While the 5'-PSL moiety was neutral or slightly stabilizing for cap interactions with eIF4E, it significantly influenced susceptibility to decapping. Replacing the γ-phosphoester with the 5'-PSL moiety (γ-PSL) prevented ß-γ-pyrophosphate bond cleavage by DcpS and conferred strong inhibitory properties. Combining the γ-PSL moiety with α-PSL and ß-phosphorothioate (PS) moiety afforded first cap-derived hDcpS inhibitor with low nanomolar potency. Susceptibility to Dcp2 and translational properties were studied after incorporation of the new analogues into mRNA transcripts by RNA polymerase. Transcripts containing the γ-PSL moiety were resistant to cleavage by Dcp2. Surprisingly, superior translational properties were observed for mRNAs containing the α-PSL moiety, which were Dcp2-susceptible. The overall protein expression measured in HeLa cells for this mRNA was comparable to mRNA capped with the translation augmenting ß-PS analogue reported previously. Overall, our study highlights 5'-PSL as a synthetically accessible cap modification, which, depending on the substitution site, can either reduce susceptibility to decapping or confer superior translational properties on the mRNA. The 5'-PSL-analogues may find application as reagents for the preparation of efficiently expressed mRNA or for investigation of the role of decapping enzymes in mRNA processing or neuromuscular disorders associated with decapping.
Assuntos
Fosfatos de Dinucleosídeos/farmacologia , Endorribonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , RNA Mensageiro/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Compostos de Sulfidrila/farmacologia , Cristalografia por Raios X , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Relação Dose-Resposta a Droga , Endorribonucleases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Células HeLa , Humanos , Hidrólise , Modelos Moleculares , Estrutura Molecular , RNA Mensageiro/biossíntese , RNA Mensageiro/química , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Compostos de Sulfidrila/síntese química , Compostos de Sulfidrila/químicaRESUMO
The excited states involved in the main photodimerization paths in TpdC and dCpT are mapped by PCM/TD-M052X calculations, considering different dinucleotide conformers. As for TT steps, a cyclobutane pyrimidine dimer (CPD) is formed on the PES of the lowest energy exciton, delocalized over two stacked pyrimidines; 6-4 pyrimidine-pyrimidone (64-PP) adduct's formation involves instead a 5'-ter â 3'-ter charge transfer state. For dCpT, 64-PP dimerization occurs via a two-step reaction, which proceeds through an oxetane intermediate. For TpdC, instead, the final 64-PP product is obtained in a single step and it is as stable as the CPD photoproduct, explaining the relatively large yield of 64-PP found experimentally for TC steps in DNA.
Assuntos
DNA/química , Fosfatos de Dinucleosídeos/química , Simulação de Dinâmica Molecular , Sequência de Bases , Dimerização , Fosfatos de Dinucleosídeos/síntese química , Processos FotoquímicosRESUMO
The cyclic dinucleotides (CDNs) c-di-GMP, c-di-AMP, and c-AMP-GMP are widely utilized as second messengers in bacteria, where they signal lifestyle changes such as motility and biofilm formation, cell wall and membrane homeostasis, virulence, and exo-electrogenesis. For all known bacterial CDNs, specific riboswitches have been identified that alter gene expression in response to the second messengers. In addition, bacterial CDNs trigger potent immune responses, making them attractive as adjuvants in immune therapies. Besides the three naturally occurring CDNs, seven further CDNs containing canonical 3'-5'-linkages are possible by combining the four natural ribonucleotides. Herein, we have synthesized all ten possible combinations of 3'-5'-linked CDNs. The binding affinity of novel CDNs and GEMM riboswitch variants was assessed utilizing a spinach aptamer fluorescence assay and in-line probing assays. The immune-stimulatory effect of CDNs was evaluated by induction of type I interferons (IFNs), and a novel CDN c-AMP-CMP was identified as a new immune-stimulatory agent.
Assuntos
GMP Cíclico/análogos & derivados , Fosfatos de Dinucleosídeos/imunologia , Geobacter/imunologia , GMP Cíclico/síntese química , GMP Cíclico/química , GMP Cíclico/imunologia , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Geobacter/química , Conformação MolecularRESUMO
Protected dinucleoside-2',5'-monophosphate has been prepared to develop a prodrug strategy for 2-5A. The removal of enzymatically and thermally labile 4-(acetylthio)-2-(ethoxycarbonyl)-3-oxo-2-methylbutyl phosphate protecting group and enzymatically labile 3'-O-pivaloyloxymethyl group was followed at pH 7.5 and 37 °C by HPLC from the fully protected dimeric adenosine-2',5'-monophosphate 1 used as a model compound for 2-5A. The desired unprotected 2',3'-O-isopropylideneadenosine-2',5'-monophosphate (9) was observed to accumulate as a major product. Neither the competitive isomerization of 2',5'- to a 3',5'-linkage nor the P-O5' bond cleavage was detected. The phosphate protecting group was removed faster than the 3'-O-protection and, hence, the attack of the neighbouring 3'-OH on phosphotriester moiety did not take place.
Assuntos
Monofosfato de Adenosina/síntese química , Fosfatos de Dinucleosídeos/síntese química , Pró-Fármacos/síntese química , Monofosfato de Adenosina/química , Cromatografia Líquida de Alta Pressão , Dimerização , Fosfatos de Dinucleosídeos/química , Pró-Fármacos/químicaRESUMO
A 3'-N,5'-S-bridging thiophosphoramidate analogue of thymidylyl-3',5'-thymidine was synthesised under aqueous conditions. (1)H NMR conformational measurements show that the 3'-N-substituted deoxyribose ring is biased towards the 'north', RNA-like conformation. Rate constants for hydrolysis of the analogue were measured at 90 °C in the pH range 1.3-10.9. The pH-log kobs profile displays a pH-independent region between approximately pH 7 and 10 (t1/2 â¼13 days). Under acidic conditions, kobs displays a first order dependence on [H3O(+)].
Assuntos
Fosfatos de Dinucleosídeos/química , Compostos Organofosforados/química , Cromatografia Líquida de Alta Pressão/métodos , Desoxirribose/análogos & derivados , Desoxirribose/química , Fosfatos de Dinucleosídeos/síntese química , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Espectroscopia de Ressonância Magnética , Conformação Molecular , Compostos Organofosforados/síntese químicaRESUMO
The fundamental roles of nucleoside triphosphates and nucleotide cofactors such as NAD(+) in biochemistry are well known. In recent decades, continuing research has revealed the key role of 5'-capped RNA and 5',5'-dinucleoside polyphosphates in the regulation of vitally important physiological processes. Last but not least, the commercial potential of nucleoside triphosphate synthesis can hardly be overestimated. Nevertheless, despite decades of investigation and the obvious topicality of the research on the chemical synthesis of the nucleotide compounds containing phosphate anhydride linkages, none of the existing procedures can be considered an up-to-date "gold standard". However, there are a number of fruitful synthetic approaches to forming phosphate anhydride linkages in satisfactory yield. These are summarized in this concise review, organized by the type of active phosphorous intermediate and reagents used.
Assuntos
Nucleotídeos/química , Anidridos/química , Carbodi-Imidas/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Organofosfonatos/química , Fosfatos/químicaRESUMO
Lysyl-tRNA synthetases efficiently produce diadenosine tetraphosphate (Ap4A) from lysyl-AMP with ATP in the absence of tRNA. We characterized recombinant class II lysyl-tRNA synthetase (LysS) from Myxococcus xanthus and found that it is monomeric and requires Mn(2+) for the synthesis of Ap4A. Surprisingly, Zn(2+) inhibited enzyme activity in the presence of Mn(2+). When incubated with ATP, Mn(2+), lysine, and inorganic pyrophosphatase, LysS first produced Ap4A and ADP, then converted Ap4A to diadenosine triphosphate (Ap3A), and finally converted Ap3A to ADP, the end product of the reaction. Recombinant LysS retained Ap4A synthase activity without lysine addition. Additionally, when incubated with Ap4A (minus pyrophosphatase), LysS converted Ap4A mainly ATP and AMP, or ADP in the presence or absence of lysine, respectively. These results demonstrate that M. xanthus LysS has different enzymatic properties from class II lysyl-tRNA synthetases previously reported.
Assuntos
Aminoacil-tRNA Sintetases/química , Fosfatos de Dinucleosídeos/síntese química , Lisina-tRNA Ligase/química , Manganês/química , Myxococcus xanthus/enzimologia , Simulação por Computador , Ativação Enzimática , Estabilidade Enzimática , Cinética , Modelos Químicos , Modelos Moleculares , Especificidade por SubstratoRESUMO
4'-(Hydroxymethyl)uridylyl-3',5'-thymidine, an RNA model bearing an extra hydroxymethyl group at the 4'-position of the 3'-linked nucleoside, has been prepared and its cleavage and isomerization reactions studied over a wide pH range (from 0 to 12). Overall, the pH-rate profiles of these reactions were very similar to those of uridylyl-3',5'-uridine (UpU) - only a very modest acceleration was observed under acidic and neutral conditions. Evidently, hydrogen bond assistance by the additional hydroxymethyl function does not play a significant role.
Assuntos
Fosfatos de Dinucleosídeos/síntese química , Ésteres/química , Timidina/química , Timidina/síntese química , Uridina/química , Catálise , Cromatografia Líquida de Alta Pressão , Ligação de Hidrogênio , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Nucleosídeos/química , Organofosfatos/química , Fosforanos/química , RNA/química , Ribonucleosídeos/químicaRESUMO
The di-2'-α-fluoro analogue of thymidylyl(3',5')thymidine, synthesized to probe the effect of a minimum amount of S conformer on the photoreactivity of dinucleotides, is endowed with only 3% and 8% of S sugar conformation at its 5'- and 3'-end, respectively. This analogue gives rise to the (6-4) photoproduct as efficiently as the dithymine dinucleotide (74% and 66% at the 5'- and 3'-end, respectively) under 254 nm. Our results suggest that the 5'-N, 3'-S conformer gives rise to the (6-4) photoproduct.
Assuntos
Carboidratos/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Conformação Molecular , Processos FotoquímicosRESUMO
Chemical synthesis of N-acyl-aminoacyl-pdCpA and its ligation to tRNA(minus CA) is widely used for the preparation of unnatural aminoacyl-tRNA substrates for ribosomal translation. However, the presence of the unnatural deoxyribose can decrease incorporation yield in translation and there is no straightforward method for chemical synthesis of the natural ribo version. Here, we show that pCpA is surprisingly stable to treatment with strong organic bases provided that anhydrous conditions are used. This allowed development of a facile method for chemical aminoacylation of pCpA. Preparative synthesis of pCpA was also simplified by using t-butyl-dithiomethyl protecting group methodology, and a more reliable pCpA postpurification treatment method was developed. Such aminoacyl-pCpA analogues ligated to tRNA(minus CA) transcripts are highly active in a purified translation system, demonstrating utility of our synthetic method.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , RNA de Transferência/química , Ribossomos , Acilação , Técnicas de Química Sintética , Modelos Moleculares , Conformação MolecularRESUMO
Recombinant Escherichia coli lysyl-tRNA synthase (LysU) has been previously utilised in the production of stabile, synthetic diadenosine polyphosphate (ApnA) analogues. Here we report on the extended use of a new recombinant histidine residue-tagged LysU as a tool for highly controlled phosphatephosphate bond formation between nucleotides, avoiding the need for complex protecting group chemistries. Resulting high yielding tandem LysU-based biosynthetic-synthetic/synthetic-biosynthetic strategies emerge for the preparation of varieties of ApnA analogues directly from inexpensive natural nucleotides and nucleosides. Analogues so formed make a useful small library with which to probe ApnA activities in vitro and in vivo leading to the discovery of new, potentially potent biopharmaceuticals active against chronic pain and other chronic, high-burden disease states.
Assuntos
Nucleotídeos de Adenina/síntese química , Fosfatos de Dinucleosídeos/síntese química , Lisina-tRNA Ligase/química , Polifosfatos/síntese química , Nucleotídeos de Adenina/química , Fosfatos de Dinucleosídeos/química , Histidina/análogos & derivados , Histidina/química , Lisina-tRNA Ligase/metabolismo , Polifosfatos/químicaRESUMO
The synthesis of a pyrazolo[1,5-a]-1,3,5-triazine C-nucleoside (dA(PT)), designed to form two hydrogen bonds with a complementary dT residue, is reported. Oligonucleotides including this dA nucleoside analogue possess base-pairing properties similar to those of the parent oligonucleotide. This dA nucleoside analogue is more resistant to acid-catalyzed hydrolysis than dA.
Assuntos
Desoxiadenosinas/química , Fosfatos de Dinucleosídeos/síntese química , Nucleosídeos/química , Nucleosídeos/síntese química , Pirazinas/síntese química , Triazinas/síntese química , Pareamento de Bases , Fenômenos Bioquímicos , Fosfatos de Dinucleosídeos/química , Ligação de Hidrogênio , Hidrólise , Estrutura Molecular , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Pirazinas/química , Triazinas/químicaRESUMO
A new, straightforward, reliable, and convenient protection-free one-pot method for the synthesis of 2'-deoxynucleoside-5'-tetraphosphate and ribonucleoside-5'-tetraphosphate is reported. The present synthetic strategy involves the monophosphorylation of a nucleoside followed by reaction with tris-(tri-n-butylammonium) triphosphate and subsequent hydrolysis of the putative cyclic tetrametaphosphate intermediate to provide nucleoside-5'-tetraphosphate in moderate yield with high purity. A plausible mechanism is proposed to account for the formation of product.
Assuntos
Nucleotídeos de Adenina/síntese química , Fosfatos de Dinucleosídeos/síntese química , Guanosina Tetrafosfato/síntese química , Nucleotídeos de Uracila/síntese química , Nucleotídeos de Adenina/química , Fosfatos de Dinucleosídeos/química , Guanosina Tetrafosfato/química , Hidrólise , Fosforilação , Polifosfatos/química , Nucleotídeos de Uracila/químicaRESUMO
We designed a one-pot synthesis that allows CpG-functionalized AgNCs to be prepared, combining attractive features of enhanced immune response and intracellular imaging.
Assuntos
Citocinas/efeitos dos fármacos , DNA/farmacologia , Fosfatos de Dinucleosídeos/farmacologia , Nanopartículas Metálicas/química , Prata/farmacologia , Animais , Linhagem Celular , Citocinas/imunologia , DNA/química , Fosfatos de Dinucleosídeos/síntese química , Fosfatos de Dinucleosídeos/química , Humanos , Camundongos , Microscopia de Fluorescência , Tamanho da Partícula , Prata/química , Propriedades de SuperfícieRESUMO
A procedure for the synthesis of dinucleoside 5'-pentaphosphates (Np5N) and nucleoside 5'-tetraphosphates (Np4) is described. The procedure relies on the activation of cyclic trimetaphosphate followed by a reaction with a nucleoside 5'-monophosphate (NMP) to give intermediates of type 3. Reaction of 3 with water or an NMP gives the desired products in yields ranging from 77 to 86%.
Assuntos
Fosfatos de Dinucleosídeos/química , Fosfatos de Dinucleosídeos/síntese química , Nucleosídeos/química , Nucleotídeos/química , Polifosfatos/química , Espectroscopia de Ressonância Magnética , Estrutura MolecularRESUMO
The biotinylated c-di-GMP and c-di-AMP conjugates 10a/b were synthesized by a straightforward set of procedures from standard, commercially available phosphoramidites. Their availability should allow isolation and characterization of new protein and RNA receptors for these key bacterial signaling molecules.