1. Synthesis of 5’-thiol modifier containing oligonucleotides
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. *
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
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 *
MS analysis
Conclusions
References
[M+H]+
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)
3264.0
3302.0
[M-H+K]+
1. B. A. Conolly, P. Rider Nucleic Acids Res. (1985) 13, 2.
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,
Intensity
[M-2H+2K]+
3339.9
Acknowledgment
2500
m/z
4500
MALDI-TOF MS analysis of the 14 min peak of TMTr-seq 4 purified by the standard Poly-PakTM method (Calcd , Found: )
(Sequence of the seq 4 oligonucleotide: TTTGGGAAAC)
Financial support:
Travel grants: OTKA, OM Mecenatúra
FKFP No. 0597/1999
OTKA No. T
* Peaks between min were analyzed by nano-ESI and found to be the corresponding Tr/DMTr/TMTr protected oligonucleotides.