Photacid Generators for Oligonucleotide Synthesis
A R T I C L E S
Chart 1. Photolabile Esters and Their Photoproducts
etry, TLC, and HPLC analysis to follow photolysis. Quantum yields
were determined from the ratio of the amount of ester photolyzed to
the light energy absorption over a given time. Acid release was
measured using titration with alkali and an indicator. Other photoprod-
ucts were isolated by column chromatography on silicagel or HPLC
or both, and characterized by 1H and 13C NMR, HRMS, and UV
spectroscopy.
Two esters of particular promise were tested for their ability on
photolysis to detritylate DMTr-T-cpg. They were then examined for
their ability to replace TCA in automated solid-phase oligonucleotide
synthesis. The key issue was whether the stepwise synthetic yields
observed with the conventional method using directly added TCA could
be matched by a photodirected method using TCA that was photo-
generated in situ. For both methods, overall % yields of synthesized
5′-DMTr-oligonucleotides in the reaction products released from cpg
with ammonia and analyzed by reverse phase HPLC with spectro-
photometric flow detection at 254 and 280 nm were equated with the
% areas under each peak. Average stepwise yields for N-mers were
calculated as the Nth root of the overall yield. In comparing the yields
of photodirected to conventional synthesis we have used the ratios of
the two, thereby attaching less significance to the absolute values.
risk of free radical modification of freshly unblocked oligo-
nucleotide 5′-OH groups. We therefore chose to explore esters
based on 2-nitrobenzyl alcohol,11 a well-known photosensitive
blocking group that can be modified to give desired photo-
chemical properties.
Results and Discussion
There are alternative but less well-known photosensitive
groups that could also be used for photoacid generators, such
as 2,5-dimethylphenacyl12 and 2-(2-nitro-phenyl)ethyl.13 Never-
theless, whatever group is chosen, it must still meet the same
performance criteria, not least of which is a suitably high
extinction coefficient at 330 nm or greater (to be clear of the
absorption bands of the oligonucleotide bases) and a useful
quantum yield. Other design criteria include straightforward
synthesis in high yield, stability, solubility in organic solvents,
absence of basic sites in the ester or its photoproduct, formation
of preferably a single photoproduct and certainly none that may
modify oligonucleotides, and ability when used for photodirected
oligonucleotide synthesis to give the same stepwise yields as
obtained in conventional synthesis.
Evaluation of Photolabile Esters. Solutions of the synthe-
sized esters, in dichloromethane or acetonitrile,19 were irradiated
at 350 or 365 nm using either a semimicro photochemical reactor
with a 4-watt UV lamp, or spectrophotometer cuvettes (or flasks)
with a 100-W high-pressure mercury arc lamp. These experi-
ments demonstrated that each ester could be converted into the
corresponding photoproduct, substituted 2-nitroso-benzaldehyde
(R1 ) H), 2-nitrosoacetophenone (R1 ) CH3), or 2-nitroso-
benzophenone (R1 ) C6H5) with the formation of the appropriate
carboxylic acid (see Supporting Information and Chart 1).
We found that R-phenyl-4,5-dimethoxy-2-nitrobenzyltri-
chloroacetate (ester A: R1 ) C6H5, R2 ) OCH3, R3 ) OCH3,
R4 ) CCl3) and R-phenyl-4,5-dimethoxy-2,6-dinitro-benzyl-
trichloroacetate (ester D: R1 ) C6H5, R2 ) OCH3, R3 ) OCH3,
R4 ) CCl3, R5 ) NO2) gave the best fit to our design criteria.
Ester A is nonionic, has an extinction coefficient at 365 nm of
3500 cm‚M-1, an acceptable quantum yield of 0.14, stability,
ease of synthesis, and only one UV-absorbing product on
photolysis (4,5-dimethoxy-2-nitrosobenzophenone: R1 ) C6H5,
R2 ) OCH3, R3 ) OCH3) as analyzed by HPLC, TLC, MS,
and NMR. Irradiation of the ester released close to 1.0 proton
per molecule of ester photolyzed. Ester D is also nonionic and
has an extinction coefficient at 365 nm of 2000 cm‚M-1, a
higher quantum yield of 0.4, a similar stability and ease of
synthesis, and again only one UV-absorbing product on pho-
tolysis (4,5-dimethoxy-2-nitroso-6-nitro-benzo-phenone: R1 )
C6H5, R2 ) OCH3, R3 ) OCH3, R5 ) NO2).
Experimental Section
Full details are provided in the Supporting Information. Descriptions
given here are to indicate general strategy.
Synthesis of Photolabile Esters. Literature searches showed that
various photoacid generators based on 2-nitrobenzyl esters have been
synthesized,14 but only a few with carboxylic acids.15 Two general
features of 2-nitrobenzyl esters are (a) the quantum yield for photolysis
is enhanced by R-substitution with a methyl or phenyl group16 and also
by introduction of a second nitro group at the 6-position,17 and (b)
extinction coefficients in the near UV region can be increased by
suitable benzyl ring substitutions.18
We have therefore synthesized a series of substituted 2-nitrobenzyl
esters of trichloroacetic acid (TCA), using condensation of trichloro-
acetic anhydride with appropriate 2-nitrobenzyl alcohols. This method
can also be used for synthesis of mono- or di-chloroacetyl esters of
2-nitrobenzyl alcohol (see Supporting Information) (Chart 1). Esters
with useful extinction coefficients above 330 nm were tested for their
photosensitivity to light at 350 or 365 nm light, using spectrophotom-
Both the esters were assessed for their ability to effect
detritylation when irradiated in solution with DMTr-protected
nucleotides either attached or unattached to controlled porosity
glass. The experiments demonstrated that complete detritylation
occurred, given time, even with a relatively dilute solution of
the ester, 10-30 mM, and a weak source of UV light.
Protocols for Photodirected Oligonucleotide Synthesis. To
provide proof of principle for the use of photogenerated
trichloroacetic acid in oligonucleotide synthesis, we developed
protocols for automation with a Millipore Expedite DNA
(11) Amit, B.; Zehavi, U.; Patchornik, A. Isr. J. Chem. 1974, 12, 103-113.
(12) Klan, P., Zabadi, M., Heger, D. Org. Lett. 2000, 2, 1569-1571.
(13) Walbert, S.; Pfleiderer, W.; Steiner, U. E. HelV. Chim. Acta 2001, 84, 1601-
1611.
(14) (a) Reichmanis, E.; Smith, B. C.; Gooden, R. J. Polym. Sci.: Polym. Chem.
Ed. 1985, 23, 1-8. (b) Houlihan, F. M.; Xeenan, T. X.; Reichmanis, E.;
Kometani, J. M.; Chin, T. Chem. Mater. 1991, 3, 462-471.
(15) Chemical Abstracts Online Database. Beilstein Online Database.
(16) Reichmanis, E.; Smith, B. C.; Gooden, R. J. Polym. Sci. 1985, 23, 1-8.
(17) Reichmanis, E.; Gooden, R.; Wilkins, C. W., Jr.; Schonhorn, H. J. Polym.
Sci. 1983, 21, 1075-1083.
(19) Acetonitrile inhibits detritylation by trichloroacetic acid, and is best avoided.
(18) Barzynski, H.; Sa¨nger, D. Angew. Makromol. Chem. 1981, 93, 131-141.
Paul, C. H.; Royappa, A. T. Nucleic Acids Res. 1996, 24, 3048-3052.
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