Universal Support for Oligonucleotide Synthesis
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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.