Tivity of guanosine with the coumarins is decreased and uridine is the prominent reaction partner. C) Reaction of all 6 differently substituted coumarins considering the nucleoside abundance (factor cra) by analysis of tRNA E. coli composition. The upper graph is for reaction conditions 1, the graph below for reaction conditions 2. The processed data clearly indicates a preference for 4-thiouridine of all tested coumarins which is most pronounced for reaction conditions 1 compared to conditions 2.doi: 10.1371/journal.pone.0067945.gmajor reaction product and comparison with the left graph reveals and overrepresentation of G-BMB due to an outstanding detection efficiency by the MS and therefore needs correction by rf. The determination of rf is achieved bycomparison of the MS signal with the UV signal of the conjugate. Akin to extinction coefficients for UV absorption, the response factor rf reflects differential detection efficiency by MS, e.g. as a consequence of differential ionization efficienciesSpecific Alkylation of Modified NucleosidesFigure 4. The left diagram shows the nucleoside selectivity calculated for pseudouridine by dividing the U-conjugate abundance by the -conjugate abundance. All numbers below 1 present selectivity for uridine over pseudouridine and vice versa for all numbers above 1. In case of the main conjugates with 4-thiouridine the calculation includes the sum of all conjugates. In all cases conditions 1 lead to the highest overall selectivity for 4-thiouridine BIBS39 supplier except for compound 3 where conditions 2 give the best selectivity.doi: 10.1371/journal.pone.0067945.Eliglustat chemical information grooted in the chemical structure of the analytes. Of central importance, rf values allow relative quantification for amounts of coumarin conjugate that are too low to produce UV signals. Since absolute values can only be obtained by using macroscopic amounts of conjugates as standards, we have determined relative values by comparison with UV absorption traces. Thus obtained response factors are based on the approximation that the coumarin moiety displays a comparable extinction coefficient in each conjugate. This implies that peaks with identical absorption at 320 nm originate from comparable amounts of coumarin conjugate. In that case, comparison of the MS signal of those same peaks is a measure of their relative detection efficiency by MS. Correspondingly, rf values were extracted in multiple (n=3) runs by extracting quantitative correlation data of an MS signal to the corresponding coumarin signal at 320 nm in the UV chromatogram (Table S1 in File S1). The third correction factor concerns (iii) the relative abundance of nucleosides in the starting material RNA preparation, which is accounted for by application of cra ; a correction factor for relative abundance. The impact of cra for BMB is illustrated on the right of Figure 3A. Although U-BMB is the major reaction product, 4-thiouridine is the major reaction partner with the coumarin after consideration of general nucleoside abundance in the tRNA preparation. After application of all three correction factors it becomes apparent, that the selectivity of BMB towards pseudouridine 23977191 is comparable to uridine, and factor 5 lower than for 4-thiouridine. Because these findings are in clear contradiction to the previously published results [36], and because they do not offer a basis for selective labeling of minor or major nucleotides in RNA, we decided to explore if, in principle,bromomethylcoumarins can be dev.Tivity of guanosine with the coumarins is decreased and uridine is the prominent reaction partner. C) Reaction of all 6 differently substituted coumarins considering the nucleoside abundance (factor cra) by analysis of tRNA E. coli composition. The upper graph is for reaction conditions 1, the graph below for reaction conditions 2. The processed data clearly indicates a preference for 4-thiouridine of all tested coumarins which is most pronounced for reaction conditions 1 compared to conditions 2.doi: 10.1371/journal.pone.0067945.gmajor reaction product and comparison with the left graph reveals and overrepresentation of G-BMB due to an outstanding detection efficiency by the MS and therefore needs correction by rf. The determination of rf is achieved bycomparison of the MS signal with the UV signal of the conjugate. Akin to extinction coefficients for UV absorption, the response factor rf reflects differential detection efficiency by MS, e.g. as a consequence of differential ionization efficienciesSpecific Alkylation of Modified NucleosidesFigure 4. The left diagram shows the nucleoside selectivity calculated for pseudouridine by dividing the U-conjugate abundance by the -conjugate abundance. All numbers below 1 present selectivity for uridine over pseudouridine and vice versa for all numbers above 1. In case of the main conjugates with 4-thiouridine the calculation includes the sum of all conjugates. In all cases conditions 1 lead to the highest overall selectivity for 4-thiouridine except for compound 3 where conditions 2 give the best selectivity.doi: 10.1371/journal.pone.0067945.grooted in the chemical structure of the analytes. Of central importance, rf values allow relative quantification for amounts of coumarin conjugate that are too low to produce UV signals. Since absolute values can only be obtained by using macroscopic amounts of conjugates as standards, we have determined relative values by comparison with UV absorption traces. Thus obtained response factors are based on the approximation that the coumarin moiety displays a comparable extinction coefficient in each conjugate. This implies that peaks with identical absorption at 320 nm originate from comparable amounts of coumarin conjugate. In that case, comparison of the MS signal of those same peaks is a measure of their relative detection efficiency by MS. Correspondingly, rf values were extracted in multiple (n=3) runs by extracting quantitative correlation data of an MS signal to the corresponding coumarin signal at 320 nm in the UV chromatogram (Table S1 in File S1). The third correction factor concerns (iii) the relative abundance of nucleosides in the starting material RNA preparation, which is accounted for by application of cra ; a correction factor for relative abundance. The impact of cra for BMB is illustrated on the right of Figure 3A. Although U-BMB is the major reaction product, 4-thiouridine is the major reaction partner with the coumarin after consideration of general nucleoside abundance in the tRNA preparation. After application of all three correction factors it becomes apparent, that the selectivity of BMB towards pseudouridine 23977191 is comparable to uridine, and factor 5 lower than for 4-thiouridine. Because these findings are in clear contradiction to the previously published results [36], and because they do not offer a basis for selective labeling of minor or major nucleotides in RNA, we decided to explore if, in principle,bromomethylcoumarins can be dev.
