Recent highlights on photolithic oligonucleotide array in situ synthesis

Article abstract of DOI:10.1081/NCN-200059241

Light directed synthesis of high-density oligonucleotide microarrays is currently performed using either ortho-nitro-benzyl-type [MeNPOC] (Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, T.M.; Holmes, C.P.; Fodor, S.P.A. Proc. Natl. Acad. Sci. U.S.A. 1994

Full text of DOI:10.1081/NCN-200059241

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RECENT HIGHLIGHTS ON PHOTOLITHIC
OLIGONUCLEOTIDE ARRAY IN SITU SYNTHESIS
Klaus-Peter Stengele a , Jochen Bühler a , Sigrid Bühler a , Evgueni Kvassiouk a , Roland
Green b , Tamara Prykota c & Wolfgang Pfleiderer c
a Chemogenix GmbH , Waldkraiburg, Germany
b NimbleGen Systems Inc , Madison, Wisconsin, USA
c University of Konstanz , Konstanz, Germany
Published online: 15 Nov 2011.
To cite this article: Klaus-Peter Stengele , Jochen Bühler , Sigrid Bühler , Evgueni Kvassiouk , Roland Green , Tamara
Prykota & Wolfgang Pfleiderer (2005) RECENT HIGHLIGHTS ON PHOTOLITHIC OLIGONUCLEOTIDE ARRAY IN SITU SYNTHESIS,
Nucleosides, Nucleotides and Nucleic Acids, 24:5-7, 891-896, DOI: 10.1081/NCN-200059241
To link to this article: http://dx.doi.org/10.1081/NCN-200059241
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Nucleosides, Nucleotides, and Nucleic Acids, 24 (5–7):891–896, (2005)
Copyright D Taylor & Francis, Inc.
ISSN: 1525-7770 print/ 1532-2335 online
DOI: 10.1081/NCN-200059241
RECENT HIGHLIGHTS ON PHOTOLITHIC OLIGONUCLEOTIDE ARRAY
IN SITU SYNTHESIS
Klaus-Peter Stengele, Jochen B¨uhler, Sigrid B¨uhler, and Evgueni Kvassiouk
5
Chemogenix GmbH, Waldkraiburg, Germany
5
Roland Green
NimbleGen Systems Inc, Madison, Wisconsin, USA
5
Tamara Prykota and Wolfgang Pfleiderer
University of Konstanz, Konstanz,
Germany
5
Light directed synthesis of high-density oligonucleotide microarrays is currently performed using
either ortho-nitro-benzyl-type [MeNPOC] (Pease, A.C.; Solas, D.; Sullivan, E.J.; Cronin, T.M.;
Holmes, C.P.; Fodor, S.P.A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 6333.) or ortho-nitrophenyl-
ethyl-type [NPPOC] (Hasan, A.; Stengele, K.P.; Giegrich, H.; Cornwell, P.; Isham, K.R.; Sachleben,
R.A.; Pfleiderer, W.; Foote, R.S. Tetrahedron 1997, 53, 4247.) protecting groups as the 5’-O-
carbonate ester of the phosphoramidite building block. The synthesis cycle uses a combinatorial approach
attaching one specific base per cycle, thus as many as 100 cycles need to be run to make an array of
25-mers. Time needed for deprotection/activation of the growing oligo chain determines overall
manufacturing time and consequently also cost. In this report we demonstrate the development of
photoprotected phosphoramidite monomers for light directed array synthesis with increasing sensitivity to
the UV light used. If combined with maskless array synthesis, this technology allows for synthesis of
arrays with >780,000 different 25-mer oligonucleotides in about one hour and allows for high
flexibility in array design and reiterative redesign. The arrays synthesized show high quality and
reproducibility in our standard hybridization based assay.
INTRODUCTION
High-density oligonucleotide microarrays are preferably synthesized by UV
light-directed photolithic synthesis by either a mask bearing or micro-mirror
device[1] approach, the latter enabling highly flexible iteration of design, synthesis,
hybridization, evaluation, and redesign.
Financial support of parts of this work from NIGU Chemie and Proligo Biochemie is greatfully
acknowledged.
Address correspondence to Klaus-Peter Stengele, Chemogenix GmbH, Waldkraiburg, Germany.
891
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K.-P. Stengele et al.
FIGURE 1 Nitrobenzyl-type (as in MeNPOC) and nitrophenylethyl-type (as in NPPOC) photolabile protec-
tive groups.
With combinatorial light-pattern projections, a typical synthesis of an array
consisting of as many as 780,000 35-mer oligonucleotides may need up to 140
consecutive chemistry and illumination cycles. It is therefore important top cut
down on synthesis time per cycle, especially if only one array is made at a time per
instrument. In addition, the recent increase in interest in experiments requiring
long-mers, 60 to 70 nucleotides,[2] make faster chemistry cycles a very attractive
target for development.
RESULTS
We have reported earlier[3] on improvements of NPPOC-type photolabile
protecting groups for photolithic synthesis mainly by mechanistic driven hypotheses
of improving either molar absorption coefficient for the (n-pi*)-transition of the
aromatic nitro group or enhancing quantum yield for the photocleavage reaction
from the excited triplet state[4] (Figure 1).
Briefly, a stabilization of the benzylic radical intermediate of NPPOC by
appropriate substituents on the phenyl ring led to favorable radical recombination,
benzylic anion formation, and fragmentation to the nitro styrene, carbon dioxide
and release of the nucleosidic alcohol. At the same time, the oxidation of the
benzylic position was repressed, which would lead to a non-cleavable photoproduct
in the case of NPPOC.
FIGURE 2 Preparation of benzoyl-NPPOC-type photolabile groups. a) KOH/methanol, then hydrogen
peroxide; b) propylene glycol, toluene sulfonic acid (kat.), toluene (azeotropic dist.); c) paraformaldehyde,
TRITON-B, DMSO; d) HCl(aq)/methanol.

K.-P. Stengele et al.
893
FIGURE 3 Synthesis of nitrothioxanthonepropanols. A = H; B = Nitro-; C = Ethyl-.
Preferred substituents and positions were reported in Bu¨hler et al.[3]
Furthermore, we now have introduced a benzoyl-group meta- to the benzylic
position, which according to[5] led to a very strong benzylic effect.
Synthesis of the new benzophenones was achieved by coupling of appropriate
substituted benzyl cyanides with ortho-nitro ethylbenzene and subsequent oxidative
decarboxylation according to Davis et al.[6] in moderate yields for electron rich and
good yields for electron deficient benzylcyanides. Subsequent hydroxymethylation
of the ethyl group led to the parent alcohol of the photolabile group. In most cases
we could improve the yield for the hydroxymethylation reaction by intermittently
protecting the benzophenone by ketalization with propylene glycol (Figure 2).
Another previously reported photolysis improvement, triplet sensitization[7] of
NPPOC chromophore with thioxanthone, led us to the design of an entity having
both meta-benzoyl- and thioxanthone structure elements in an NPPOC molecule.
We synthesized diphenylsulfide molecules by Heck type reaction of bromo
nitro ethylbenzene with thiosalicylic acid under copper catalysis,[8] which
underwent cyclization upon heating in polyphosphoric acid with little to no
stereoselectivity and moderate yield, in most cases less than 20% for a single isomer.
Hydroxymethylation with paraformaldehyde then led to the parent alcohol in again
only about 10–20% yield due to the strong activation and presumably oligo-
merization under the basic reaction conditions (Figures 3 and 4).
Photolysis studies of newly protected nucleosides analyzed by HPLC indicated
the extremely fast photocleavage kinetics (half-lives) of NTXPOC (0.65 s) over the
current gold standard MeNPOC and NPPOC (6–7 s) groups. Benzoyl-NPPOC
(3.5 s) also showed favorable kinetics over NPPOC but was significantly slower than
NTXPOC and slower than previously reported Phenyl-NPPOC (2 s) in our
analytical test set-up, essentially similar to[9] but with higher light intensity
(approximately 300 mW/cm2) (Figure 5).
FIGURE 4 Structural isomers of nitrothioxanthonepropanols.

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K.-P. Stengele et al.
FIGURE 5 Photolysis kinetics of different NPPOC-type photolabile groups.
Nucleotide-5’-carbonates of o-nitrobenzylalcohol, o-nitroveratryl-1-ethanol
(MeNPOC) o-nitrophenyl-2-propanol (NPPOC), o-nitro(m-benzoylphenyl)-2-propa-
nol, and o-nitrothioxanthone-2-propanol were then synthesized according to
published methods by first reacting the alcohols with diphosgen and then the
properly functionalized nucleosides generally in good yields. Further conversion to
the beta-cyanoethyl-N-diisopropyl phosphoramidites furnished the array synthesis
monomers, e.g., NTXPOC-amidites, as shown in Figure 6.
Arrays of a test oligonucleotide were then synthesized on a NimbleGen
maskless array synthesizer as previously described.[7,10] Briefly, a 24-mer
oligonucleotide complementary to a stretch of the calmodulin gene of rat was
made in an arrangement where multiple groups of 20 Â 20 mirror subsets 10 Â 10
positive features (separated by one dark feature in each direction) were made with
increasing light dose per each irradiation step for the full-length oligo from right to
left columns, resulting in 10 identical lines per square of a dose-response curve
based on hybridization signals obtained from complementary synthetic Cy-3-
labeled oligonucleotide. It is herewith possible to determine optimum irradiation
time for each chemistry chosen in a very straightforward and direct manner not
having to extrapolate from ‘‘off-array’’ experiments. Best irradiation time at a given
light intensity corresponds to the maximum of the hybridization signal (Figures 7–9).
FIGURE 6 5’-[3-Nitrothioxanthone-2-(propyl-2-oxycarbonyl)]-nucleoside-3’-phosphoramidite.

K.-P. Stengele et al.
895
FIGURE 7 Dose matrix (3–21 s) synthesis using NTXPOC-amidites: 5 s best mode.
FIGURE 8 Dose matrix (15–60 s) benzoyl-NPPOC-amidites: 35 s best mode.
FIGURE 9 Dose matrix (35–80 s) MeNPOC-amidites: 60 s best mode.

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K.-P. Stengele et al.
SUMMARY
In summary we have developed new photolabile protective groups chemistry
that will allow for about 10 times faster deprotection rates in the MAS synthesis
cycle. The overall machine time for making a high-density array consisting of
70-mers may thus be reduced by up to 4 h, essentially cut in half. Experiments are
now under way to improve synthesis of the new building blocks, optimization of
chemistry cycles and validation in a number of different applications. Finally, we
will qualify the new synthesis chemistry for routine production in the MAS systems.
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