Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-09-10
2003-04-22
Siew, Jeffrey (Department: 1637)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S022100, C536S063000, C536S024300, C536S024310, C536S024320, C536S024330, C435S006120, C435S007100, C435S091100, C435S091200, C435S287200
Reexamination Certificate
active
06552182
ABSTRACT:
The present invention relates to a method for the specific photolytic deprotection of nucleoside derivatives that are immobilized on a substrate, especially in the photolithographic production of DNA chips.
For reasons of parallelization and miniaturization, DNA chips for analytic and diagnostic applications in molecular biology, medicine and related fields are commonly produced by means of photolithographic techniques. In these techniques the nucleoside derivatives are immobilized with photo labile protective groups on suitable substrates. Then the deprotection is specifically carried out by photolysis. Due to the lack of post-synthetic purifying methods, the requirements made of the chemical compositions of the protective groups are very high.
In correspondence with prior art, two methods are available for carrying out the photolytic deprotection of the protective groups. In the first method, the DNA chips are exposed by means of a suitable solvent or mixture of solvents in a flow chamber (cf. G. H. McGall, A. D. Barone, M. Diggelmann, S. P. A. Fodor, E. Gentalen, N. Ngo, J. Am. Chem. Soc. 1997, 119, 5081-5090). In this method, the substrate (e.g. in the form of a glass substrate) with the immobilized nucleoside derivatives is mounted in a flow chamber. Throughout the irradiation, a suitable mixture of solvents is pumped through the flow chamber so as to wet the synthesis side of the substrate such that the immobilized growing DNA chains are quasi present in dissolved form. Hence, the participation of the solvent or the mixture of solvents during the photo controlled deprotectioning operation is ensured in any case. Owing to its structure, the chip surface is exposed from the “wrong side”, i.e. from the rear side through the substrate (e.g. in the form of glass substrates).
This method entails some disadvantages. For example, the diffusion of light on the glass substrate gives rise to a bad optical resolution. Moreover, the heating of the substrate as well as an insufficient wetting of the substrate surface may result in thermal and secondary photolytic reactions. As the photo labile protective group to be separated is quasi located on the other end of the optical path, the oligo nucleotide chain ahead of it may have the function of a light filter, which involves, on the one hand, the inherent risk of secondary photolytic reactions and, on the other hand, is apt to give rise to an extension of the exposure time.
In the second known method for the photolithographic production of DNA chips, the chips are exposed from the “correct” side, i.e. from the front side, without using a solvent (cf. M. C. Pirrung, L. Fallon, G. McGall, J. Org. Chem. 1998, 63, 241-246). Experience has shown that a particular disadvantage in this method is the poor quality of the synthesized oligo nucleotides, which must be attributed to a slow and incomplete deprotection of the nucleoside derivatives as well as to secondary thermal or photolytic reactions.
The present invention was therefore based on the problem of developing a method for the specific photolytic deprotection of nucleoside derivatives immobilized on a substrate, particularly of protective groups common in the production of DNA chips, which does not present the aforementioned disadvantages of prior art but rather permits the rapid and complete deprotection.
This problem is solved in accordance with the present invention by the provision that prior to photolysis a layer of a gel or a viscous liquid of polymer compounds in water, a water/C
1
-C
4
alcohol mixture and/or a polar aprotic solvent is applied onto the substrate with the nucleoside derivatives to be deprotected.
It was a surprise to find that in this manner secondary thermal and photolytic reactions are largely repressed so that the synthesized nucleoside or nucleotide sequences present the required purity.
In the context of the inventive method, the expression “specific photolytic deprotection” is to be understood to denote the specific photolytic deprotection of the protected nucleoside derivatives. Within the scope of the present invention, it is therefore possible to separate only part of the photo labile protective groups, for instance by means of masks, in addition to the complete deprotection.
In the method according to the present invention, a layer of a gel or a viscous liquid of one or more polymer compounds in water, a mixture of water/C
1
-C
4
alcohol and/or a polar aprotic solvent is applied to the substrate surface, i.e. the substrate with the immobilized nucleoside derivatives consisting of nucleosides, nucleotides or oligo nucleotides before the exposure of the nucleoside derivatives commences, preferably from the front side. The thickness of the gel layer or the layer of the viscous liquid, respectively, may be varied within wide limits, but it has been found to be of advantage for an optimum optical resolution to set the thickness of the layer to a value between 0.1 &mgr;m and 5 mm, more preferably 10 &mgr;m to 5 mm.
Preferably the fraction of polymer compounds should amount to 0.1 to 40% by weight, more particularly 1 to 20% by weight, relative to the total weight of the gel or viscous liquid, respectively. In accordance with a preferred embodiment, such polymers are used for the build-up of gels presenting a sol/gel transition temperature of 15 to 90° C., particularly 30 to 50°C. The advantage of these gels resides in the fact that they are quasi solid at room temperature and can be converted into the liquid state by heating them slightly so that after the photolytic deprotection the corresponding gels may be separated very easily from the substrate.
The used gels should preferably have a gel concentration in the range from 20 to 10,000 g/cm
2
, especially 100 to 1,000 g/cm
2
. The gel concentration is usually measured by compression tests common to those skilled in the art. In the event that gelatin is used the gel concentration may also be determined by applying the Bloom technique. There, the gel concentration corresponds to the force—in gram—that must be created by a defined cylindrical piston on the surface of a 6.67% gelatin gel (obtained after 17 hours at 10° C.) in order to achieve a depth of depression of 4 mm. The gel concentration so determined then corresponds preferably to a value between 5 and 300 g for gelatin gels in the inventive method.
In the event of application of viscous liquids these liquids should preferably present a dynamic viscosity in the range of 5 to 40,000 mPa·s, particularly 50 to 15,000 mPa·s (measured at 25° C. and for the respective concentration). The type of the polymer compounds is largely uncritical, which means that they are merely expected to result in the desired gels or viscous liquids in the presence of water or the respective solvent. It is hence possible in the inventive method to use a number of synthetic or natural polymers. Among the synthetic polymers polyvinyl alcohol (PVA), polyvinyl acetal, polyacryl amide, polyvinyl pyrrolidone (PVP), polyvinyl acetate, polyethylene imine and Novolake (poly condensation products of phenol and formaldehyde) have been found to be particularly of advantage. According to the invention, gelatins, agarose, agar-agar, pectin, galactomannans, carragheenans, scleroglucans, xanthans and alginates are preferably used among the natural polymers.
In the inventive method, water, a water/C
1
-C
4
alcohol mixture and/or a polar aprotic solvent is used as a solvent for the gel or the viscous liquid. The alcohols, which may be linear or ramified, are used in the mixture with water in a preferred weight ratio of 90/10 to 19/90. The alcohols may contain one or more OH groups and may be selected, in particular, from the group including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-methyl-1-propanol, 2-butanol, 2-methyl-2-propanol, ethylene glycol, 1,3-propandiol, 1,2-propandiol, glycerin, 1,2-butandiol, 1,3-butandiol, 1,4-butandiol and 2,3-butandiol. The polar aprotic solvents preferably consist of dimethyl sulfoxide (DMSO), dimethyl formamide (DMF), dimethyl acetamide (DMA), aceto
Giegrich Heinrich
Stengele Klaus-Peter
Nigu Chemie GmbH
Siew Jeffrey
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