A novel and convenient method for the synthesis of free 5’-thiol modified oligonucleotides

1. A novel and convenient method for the synthesis of free 5’-thiol modified oligonucleotides Z. Kupihár,a, * G. Kovács,a Z. Kele,a Z. Darula,b L. Kovácsa aNucleic Acids Laboratory, Department of Medicinal Chemistry, University of Szeged, H-6720 Szeged, Dóm tér 8, Hungary and bMass Spectrometry Laboratory, Biological Research Center, Temesvári krt. 62. H-6726 Szeged, Hungary. *E-mail: kupi@ovrisc.mdche.u-szeged.hu Introduction Preliminary experiments Aims The synthesis of DNA bioconjugates often requires 5'-thiol modified oligonucleotides. The most frequently applied protecting group on thiol-modifier linkers is the trityl group. However, removal of this group is usually problematic: heavy metal cations (Ag+, Hg2+) are used [1] for removal and residual ion impurity may hamper the application of the desired DNA in biological systems. Moreover, metal ion precipitation may result in significant loss of the DNA product, too. • Applied 5’-thiol protecting group: Tr • Trityl deprotection method: a modified Poly-PakTM purification [2] • Purification procedure: • 1.Pretreatment and equilibration: acetonitrile, 1M TEAA (pH=7.0) • 2.Sample loading, washing : 3% aq. NH3 , water • 3.Tr cleavage: 2, 5 or 10% aq.TFA with different scavengers (thiophenol, thioanisol, (NH4)2S) • 4. Washing, elution: water, 20% aq. acetonitrile • Results: • - using 2% TFA: low yield of detritylated 5’-thiol modified oligonucleotide • - using 5 or 10 % TFA: loss of oligonucleotide (probably oligonucleotide degradation) • - undesired scavenger impurities in product • To develop a similarly simple purification and thiol deprotecting method for 5’-S-protected oligonucleotides as the Poly-PakTM purification procedure for native oligonucleotides without using scavengers applied in the standard protocol • Strategy • - to apply more acid labile protecting groups • - scavengers used in a different phase (organic phase or immobilized on solid-phase) Synthesis of 5’-thiol modifier linkers Synthesis of 5’-thiol modifier containing oligonucleotides Thiol deprotection and purification 1. Standard Poly-PakTM 2. Mixed column 3. Extraction • Applied column: Standard Poly-PakTM column • Purification procedure: Standard Poly-PakTM purification • 1.Pretreatment: acetonitrile • 2.Equilibration: 1M TEAA (pH=7.0) • 3.Sample loading • 4.Washing : 3% aq. NH3 , water • 5.Tr/DMTr/TMTr cleavage: 2% aq.TFA • 6.Washing: water • 7.Elution: 20% aq. acetonitrile • Applied column: Mixed Poly-PakTM and thiol-sepharose (as immobilized scavenger) 1:1 m/m • Purification procedure: Slightly modified Poly-PakTM purification: • 1.Thiol activation on sepharose: 1M aq. DTT • 2.Washing: EtOAc, water, acetonitrile • 3.Equilibration: 1M TEAA (pH=7.0) • 4.Sample loading, washing : 3% aq. NH3 , water • 5.Tr/DMTr/TMTr cleavage: 2% aq.TFA • 6. Washing, elution: water, 20% aq. acetonitrile • Extraction system 1.: • 0.5 ml thiol-protected oligonucleotide / water • 0.5 ml 3%trichloroacetic acid in dichloromethane • 0.1 ml triethyl silane • Extraction system 2.: • 0.25 ml thiol protected oligonucleotide / water • 0.25 ml acetic acid • 0.5 ml dichloromethane • 0.1 ml triethyl silane • Conditions: shaking for 30, 60 min at room temperature Results HPLC traces * Tr-seq 4 * DMTr-seq 4 free 5’-thiol modified oligonucleotide TMTr-seq 4 * Conclusions MS analysis References • Purification with extraction: • -based on HPLC study, the aqueous phase did not contain oligonucleotides (a possible explanation is oligonucleotide degradation – further studies are required to investigate this unexpected experience) • Purification on mixed columns: • -similar results as using standard Poly-PakTM method with somewhat lower yields • -DTT (used for activating the sepharose-linked thiol scavenger) impurities in product • Standard Poly-PakTM purification: • best results • only use of TMTr-group results in high yield of deprotection (80-90%) • does not require use of heavy metal ions and scavengers • in case of TMTr protecting group, product of deprotection can directly be used for conjugation reactions (residual protected oligonucleotide can be separated after conjugation if required) [M+H]+ 3264.0 [M-H+K]+ 1600000 1. B. A. Conolly, P. Rider Nucleic Acids Res. (1985) 13, 4485-4502 2. http://glenres.com/ 3. Z. Kupihár, Z. Schmél, L. Kovács Molecules(2000) 5, M144 4. Z. Kupihár, Z. Schmél, Z. Kele, B. Penke, L. Kovács Bioorg. Med. Chem.(2001) 9, 1241-1247 3302.0 Intensity [M-2H+2K]+ 3339.9 Acknowledgment 0 2500 4500 m/z MALDI-TOF MS analysis of the 14 min peak of TMTr-seq 4 purified by the standard Poly-PakTM method (Calcd. 3263.4 , Found: 3263.0) (Sequence of the seq 4 oligonucleotide: TTTGGGAAAC) Financial support: Travel grants:OTKA, OM Mecenatúra • FKFP No. 0597/1999 • OTKA No. T 030135 * Peaks between 30-35 min were analyzed by nano-ESI and found to be the corresponding Tr/DMTr/TMTr protected oligonucleotides.