Novel photocleavable universal support for oligonucleotide synthesis

Article abstract of DOI:10.1081/NCN-120022996

A novel photocleavable universal support for the automated solid phase synthesis of oligonucleotides is described. The linker between the growing oligonucleotide chain and CPG support contains a nucleophilic amine protected with a photo-cleavable group. On exposure to UV light, this group is detached and the free amine affords cleavage of the oligonucleotide from the support. The use of long wavelength UV light avoids damage to the DNA.

Full text of DOI:10.1081/NCN-120022996

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Novel Photocleavable Universal Support for
Oligonucleotide Synthesis
a c
b
a
Emma Anderson
, Tom Brown & Douglas Picken
a
Link Technologies, Strathclyde Business Park, Bellshill, Lanarkshire, UK
b
c
Department of Chemistry, University of Southampton, Highfield, Southampton, UK
Link Technologies Limited, 3 Mallard Way, Strathclyde Business Park, Bellshill, Lanarkshire,
ML4 3BF, UK
Published online: 31 Aug 2006.
To cite this article: Emma Anderson , Tom Brown & Douglas Picken (2003) Novel Photocleavable Universal Support for
Oligonucleotide Synthesis, Nucleosides, Nucleotides and Nucleic Acids, 22:5-8, 1403-1406, DOI: 10.1081/NCN-120022996
To link to this article: http://dx.doi.org/10.1081/NCN-120022996
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NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 22, Nos. 5–8, pp. 1403–1406, 2003
Novel Photocleavable Universal Support for
Oligonucleotide Synthesis
1,*
2
Emma Anderson, Tom Brown, and Douglas Picken
1
1
Link Technologies, Strathclyde Business Park,
Bellshill, Lanarkshire, UK
2
Department of Chemistry, University of Southampton,
Highfield, Southampton, UK
ABSTRACT
A novel photocleavable universal support for the automated solid phase synthesis
of oligonucleotides is described. The linker between the growing oligonucleotide
chain and CPG support contains a nucleophilic amine protected with a photo-
cleavable group. On exposure to UV light, this group is detached and the free
amine affords cleavage of the oligonucleotide from the support. The use of long
wavelength UV light avoids damage to the DNA.
Key Words: Photocleavable support; Universal support; Oligonucleotide
synthesis.
INTRODUCTION
0
Conventionally, a succinyl linker has been utilised to attach the 3 -OH of a
[
nucleotide to amino CPG support. This has the disadvantage that before
1]
*
Correspondence: Emma Anderson, Link Technologies Limited, 3 Mallard Way, Strathclyde
Business Park, Bellshill, Lanarkshire, ML4 3BF UK; Fax: þ44 1698 849922; E-mail: emma.
anderson@linktech.co.uk.
1
403
DOI: 10.1081/NCN-120022996
Copyright # 2003 by Marcel Dekker, Inc.
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com

1404
Anderson, Brown, and Picken
automated synthesis, the support must be pre-derivatised with the first nucleotide in
the desired sequence, which can be one of the four DNA bases. Use of a universal
support would solve this problem. Rapid or clean cleavage should then leave no
0
trace of the linker left on the 3 -OH as this free hydroxyl is essential for applications
such as sequencing and PCR.
[
Our approach is based on a substituted aminopropanediol, where the amine
2]
[
3]
protecting group is a photolabile o-nitrobenzyl group. The advantages of this type
of group are its stability under the synthesis conditions and that no reagents are
required for deprotection, only UV radiation. The nitroso by-product from the
UV treatment is then easily removed by washing of the CPG support.
RESULTS AND DISCUSSION
The synthetic route is shown in Sch. 1. The aldehyde 1 was converted to methyl
alcohol 2 in 92% yield. Disuccinimidyl carbonate provided an activated carbonate,
Scheme 1. Synthesis of the photocleavable supports.

Universal Support for Oligonucleotide Synthesis
1405
which was used crude in the next step. Coupling of this to 3-amino-1,2-propanediol
resulted in 51% of diol 3 over 2 steps after chromatography. Tritylation of the
primary alcohol and addition of succinic anhydride afforded the desired material 4
in 54% yield after chromatography. Coupling of this acid to amino CPG support
was carried out with TOTU and HOBt to give a loading of 25 mmol=g for 6. An
alternative linker utilised a sarcosine residue to provide a tertiary amide bond.
Amino-CPG was functionalised with Fmoc-sarcosine using standard conditions,
then deprotected and coupled to acid 4 to give the support 7 with a loading of
44 mmol=g.
Initial experiments on support 6 in MeCN or 0.1 M TEAA suggested that
UV absorbance could be used to follow the cleavage of the nitroso ketone.
Supports 6 and 7 were used in standard oligonucleotide synthesis to provide
solid supported T6 and mixed 19 mer G GGT GAA TTA CAA GCT CCG. For sup-
port 6 þ T6, photocleavage at 365 nm for 1–3 h at a distance of 5 cm from the light
source was attempted while the material was still in the oligo synthesis column with a
small quantity of MeCN. Cleavage of the oligonucleotide from the support was
achieved using 3.5% NH in 0.1 M TEAA to give an HPLC purity of 51%, while
3
0
MS data confirmed that no other 3 -modified products were observed.
Research was then performed in open topped fritted columns, whose frits were
around 3.5 cm from the UV source. Trials were carried out with dry support, dry
support with agitation, dry support with wash cycle, MeCN, MeCN with agitation
and 20% 0.1 M TEAA in MeCN over various time periods. Optimal results for
support 6 were from the dry support with 30 min wash cycle over 3 h and 20%
TEAA in MeCN over 6 h, so these conditions were applied to the supported oligo-
nucleotides.
Moving on to study photocleavage of T6 and 19 mer oligonucleotides, the func-
tionalised support was removed from the synthesis column and added to a fritted
open topped column. For support 6 þ T6: photocleavage was attempted in MeCN,
20% 0.1 M TEAA in MeCN and dry support with a wash cycle at regular time inter-
vals. Following cleavage from the support, the best HPLC result (72% desired pro-
duct) came from the dry support that was washed every 30 mins over 3 h. For
support 7 þ T6: photocleavage and oligonucleotide cleavage was carried out as
above and the MeCN for 2 h or the wash cycle over 3 h both gave 61% by HPLC.
For support 7 þ 19 mer: all the attempted conditions gave similar results ꢀ42% by
HPLC.
CONCLUSIONS
These novel photocleavable supports can be utilised to provide oligonucleotides
0
with no 3 -modification. The photocleavage has been shown to occur under a variety
of conditions and will be further optimised.
ACKNOWLEDGMENTS
We would like to thank Reuben Carr and the University of Southampton for the
initial synthesis work towards this support.

1406
Anderson, Brown, and Picken
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1
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Guillier, F.; Orain, D.; Bradley, M. Linkers and cleavage strategies in solid-
phase organic synthesis and combinatorial chemistry. Chemical Reviews 2000,
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Lyttle, M.H.; Hudson, D.; Cook, R.M. A new universal linker for solid phase
DNA synthesis. Nucleic Acids Research 1996, 24 (14), 2793–2798.
Hasan, A.; Stengele, K.; Giegrich, H.; Cornwell, P.; Isham, K.R.; Sachuben,
R.A.; Pfleider, W.; Foote, R.S. Photolabile protecting groups for nucleosides:
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3