Title: A novel and convenient method for the synthesis of free 5thiol modified oligonucleotides
1A 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_at_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 (pH7.0) - 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 (pH7.0)
- 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) 11
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 (pH7.0)
- 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 3trichloroacetic 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)
MH
3264.0
1600000
M-HK
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-2H2K
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.