2Department of Organic Chemistry, Dóm tér 8, H-6720 Szeged, Hungary Synthesis and MS analysis of thiazolium and pyridinium derivatives of peptide nucleic acids (PNAs) and peptides Györgyi Kovács1*, Petra Pádár1*, Zoltán Kupihár1*, Zoltán Kele1, Péter Forgó2, Lajos Kovács1* University of Szeged, 1Department of Medicinal Chemistry, * Nucleic Acids Laboratory, 2Department of Organic Chemistry, Dóm tér 8, H-6720 Szeged, Hungary Intoduction In our laboratory PNA, peptide, oligonucleotide oligomers and their derivatives (e.g. conjugates) are synthesized and analysed with HPLC and ESI-MS (and MALDI-MS, if neccesary). Before purification it would be good to know whether the crude product, available only in small amount contains our oligomer and/or what kind of side reactions took place during synthesis and/or cleavage. Mass spectra of the crude products are usually not interpretable. Cheap and reactive labels synthesized by us (except for CMP, Mukaiyama reagent) were studied to solve these problems. During PNA synthesis using CMP as monomer activator intensive [Mx+93]+ values were obtained in MS (-t-Gly-NH2 (M1); -tt-Gly-NH2 (M2); -gtt-Gly-NH2 (M3); H-Gly-ggtt- Gly-NH2 (M)). Aims To enhance the sensitivity of MS analysis of crude PNAs and peptides, with introducing positive charge onto the oligomer by preparation of their pyridinium and thiazolium derivatives To indentify the molecular ion of the product easier, based on the isotope distribution of bromine-containing labels Results Synthesis of pyridinium and thiazolium derivatives of PNAs and peptides Three Br containing label molecules and their PNA and peptide derivatvives were synthesized and analysed. These labeles are reactive under the conditions of nucleobase acyl (isobutyryl, anisoyl and 4-tert-butylbenzoyl) group deprotection (NH3/ 50ºC/o.n.). In the case of labelling the free N-terminus (primary amino group) deprotonation took place but in the case of secondary amino group which does not contain removable proton (attaching sarcosine onto the N-terminus) a loss of label occured. In conclusion, these labels are not compatible with our protecting group strategy but in this context it is not important. In case of H-tn-X (n=1, 2, 3… and X=NH2/OH/amino acid) basic treatment is not needed, thus the syntesis can be carried out on acid labile resin e.g. Wang resin. The same problem is present in the case of oligonucleotides as the label is removed from 5’-OH during the oligomer cleavage and nucleobase deprotection with ammonia solution. These reagents can be used for the MS analysis of other hydroxy compounds. Br containing label molecules R1H NHR1R2=sarcosyl-PNA, R1=H R2={PNA, peptide} R3=oligonucleotide Base= DIPEA or lutidine Labelling and reactions during treatment with NH3 The study of labelled oligomers With ESI technique the labelled product can be identified also in the crude sample not only after purification. Usually the M+, but sometimes [M+H]2+ ion can be detected. Isotope distribution of bromine-containing label helps to identify the product. Furthermore, labelled oligomers are more apolar than the unlabelled product, thus the HPLC purification is easier as they are better separated from other oligomers (e.g. the capped ones). m/z 1665.0 1675.0 Intensity 40 1667.9 Magnified and calculated isotope distribution of peak 1667.9 of BET-t5 product ESI-MS of BEP-t5 MALDI-MS of MP-t5 and BET-t5 721.5 [Mlab3]+ 90 80 10.00 [Mlab2]+ 1567.9 70 40 1667.9 Relative Abundance 50 60 40 722.5 [Mlab1+H]2+ Intensity 811.68 [Mlab1]+ Intensity 30 848.76 20 740.0 832.3 1662.1 848.73 1476.8 1252.8 1587.9 1476.9 10 763.5 833.5 969.7 1044.5 1175.7 1296.0 1478.3 1587.2 1084.7 986.71 1042.8 800 1000 m/z 1200 1400 1600 1084.8 600 m/z 1600 800 m/z 1800 Study of side products by labelled oligomers In our examples side reactions are caused by incomplete protecting group removal (e.g. Pbf; M+ 252) , oxidation of methionin (M+16), cleavage of linker of resin (M+ 106 mass). If these reactions take place in a synthesis and, furthermore, if couplings are not quantitative and capped sequences are also present, then in the crude product a lot of peaks are present. In addition, one mass can belong to more molecules in the case of low resolution MS technique. Interpretation of these spectra or HPLC purification takes a long time. If the product is labelled then everything is easier as only the product peaks and their derivatives are changed with the same value and only these must be interpreted. Mlab=M-H+93 (as MP is the label) m/z 1000 1350 Intensity 90 1031.8 1045.8 1137.9 1283.9 1063.8 1153.9 1159.9 1299.9 1047.8 1400 80 1120.9 1138.9 1154.9 1228.9 1244.9 1251.0 1260.9 1267.0 1325.0 1365.0 1375.0 1122.9 H-RRMKWKK-OH MP-RRMKWKK-OH [M+H]+ [M+16+H]+ [M+252+H]+ [M+106+H]+ [Mlab]+ [Mlab+106]+ [Mlab+16]+ [Mlab+252]+ Labelling of non-polar peptides and their sequencing with nano-ESI-MS/MS technique [Mlab]+ 673.3 80 In apolar peptides the introduction of a charge simplifies the interpretation of their mass spectra. Peptides not containing amino acids with nucleophilic functional groups in their side chain (test pentamer: GPVYF) can be labelled in solution (1 eq labelling reagent, 2 eq lutidine in 50 μl DMF), otherwise on-resin labelling is needed. For solution phase labelling a purification with zip-tip is needed to remove the excess of reagent and base that can disturb the MS analysis. Intensive MP-GPVYF peak could be observed in the MS spectrum which was analysed with MS/MS and gave a clear spectrum that contained only an peaks. (Mlab=M-H+93 (M=581); a2-a1=98 (Pro); a3-a2=99 (Val); a4-a3=163 (Tyr); a5-a4=147 (Phe)) RelativeAbundance 50 a3 317.6 a5 a1 628.5 121.2 a2 345.2 a4 219.3 246.4 481.2 Synthesized and analysed PNA oligomers 150 200 250 300 350 400 450 500 550 600 650 m/z Abbreviations: t5=ttttt-Lys-OH; MP=N-methyl-pyridinium; CMP= 2-chloro-N-methyl-pyridinium; BEP=5-bromo-N-ethyl-pyridinium; BET= 5-bromo-N-ethyl-thiazolium; sar=sarcosine; Mlab1=M-H+186; Mlab2=M-H+93; Mlab3=M-H+192 *treated with 25% NH3/60ºC/2h HPLC conditions: 5-45% B, 40 min; LiChrospher RP select B, A:0.1% TFA/H2O, B:0.1%TFA/(ACN:H2O=8:2) Acknowledgement Financial support: OTKA, FKFP, Alapítvány a Magyar Peptid és Fehérjekutatásért Travel grants: Richter Gedeon Centenáriumi Alapítvány, OTKA, Mecenatura