WO1997005156A1 - A new achiral linker reagent for the incorporation of multiple amino groups into oligonucleotides - Google Patents

Abstract

The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide with the linker in conventional phosphoamidite or H-phosphonate DNA syntheses. Directly, or via a post modification step, an oligonucleotide is labelled with one or more reporter moieties, e.g. dansyl (5-dimethylamino)-1-naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazolidine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and in vitro mutagenesis.

Description

A NEW ACHIRAL LINKER REAGENT FOR THE INCORPORATION OF MULTIPLE AMINO GROUPS INTO OLIGONUCLEOTIDES

The present invention relates to a new functionalized achiral linker reagent for the incorporation of multiple primary amino groups or reporter groups into oligonucleotides. The linker reagent as well as its immobilized analogue is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and it has been shown that the linker can be incorporated into oligonucleotides in good yield. Furthermore, the invention relates to modified oligonucleotides where one or more of such linker groups, which may include reporter groups, have been incorporated.

BACKGROUNDOFTHEINVENTION

Specific labelling of DNA-oligomers is of major importance in the construction of DNA-probes (Goodchild, J. Bioconjugate Chem., 1990, 1, 165). The hazardous nature associated with the traditional radio-isotopic labelling has prompted the search for alternative non-radioactive labelling methods. Particular attention is paid to photo-detectable fluorophores (Smith, L. M. et al., Nature 1986, 321, 674-679; Agrawal, S.; Christodoulou, C; Gait, M. J. Nucleic Acids Res. 1986, 14, 6227-6245; and Shinozuka, K.; Seto, Y.; Kawata, H.; Sawai, H. BioMed. Chem. Lett. 1993, 3(12), 2883-2886), or other reporter molecules such as biotin (Chu, B. C. F.; Orgel L. E. DNA 1985, 4, 327-331). Therefore, effort has been focused on routes to synthesize oligo¬ nucleotides, furnished with amino or thiol groups, wliich can function as handles for incorpo- ration of the desired reporter molecule (Gaur, R. K. Nucleosides & Nucleotides 1991, 10, 895-909; Connolly, B. A. Nucleic Acids Res. 1985, 13, 4485-4502; Douglas, M. E.; Beijer, B.; Sproat, B. S. BioMed. Chem. Lett. 1994, 4(8), 995-1000; and Ono, A.; Dan, A.; Matsuda, A. Bioconjugate Chem. 1993, 4, 499-508).

Several methods have been developed for the synthesis of oligonucleotides contaimng primary aliphatic amino groups. The variety of methods currently available ranges from enzymatic or chemical post modification of fully deprotected oligonucleotides (Kempe, T.; Sundquist, W. I.; Chow, F.; Hu, S.-L. Nucleic Acids Res. 1985, 13, 45-57), to the use of reagents compatible with the phosphoramidite based solid phase oligomerization technique (WO 91/17169; Gibson, K. J.; Benkovic, S. J. Nucleic Acids Res. 1987, 15, 6455-6467).

In the conventional phosphoramidite methodology, protected deoxynucleotide phosphoramidites are reacted under acid catalysis (tetrazole) with solid phase bonded δ'-nucleoside hydroxy groups to form phosphite triesters. These are subsequently oxidized with iodine to form phosphate triesters. Partial removal of the 5'-hydroxy protecting group (DMT) then leaves a new un- protected hydroxy group ready for the next coupling. Protecting groups on the heterocycles (e.g. benzoyl, isobutyryl), and on the phosphate group (e.g. 2-cyanoethyl), are removed by treatment with ammonia in methanol, which at the same time cleave the oligonucleotide from the support.

The use of phosphoramidite reagents probably offers the easiest route to amino modified oligonucleotides, as no operation is needed apart from the standard synthesis protocol. Simple amino alkanols of the general form NH₂-(CH₂)_n-OH, after protection of the primary amine and conversion of the hydroxyl group into the phosphoramidite group, have been widely used in this respect (Gaur, R. K. Nucleosides & Nucleotides 1991, 10, 895-909; Bischoff, R; Coull, J. M.; Regnier, F. Anal. Biochem. 1987, 164, 336-344; Connolly, B. A. Nucleic Acids Res. 1985, 13, 4485-4502; Connolly, B. A. Nucleic Acids Res. 1987, 15, 3131-3139; and Agrawal, S.;

Christodoulou, C; Gait, M. J. Nucleic Acids Res. 1986, 14, 6227-6245). However, having only one hydroxyl group, chain elongation is not possible, and these linkers therefore can only be used for the incorporation of a single 5'-terminal amino group.

Recently, Nelson et al. (Nelson, P. S.; Kent, M.; Muthini, S. Nucleic Acids Res. 1992, 20, 6253-6259) have reported the synthesis of 2-(4-aminobutyl)-l,3-propanediol, which in few steps can be converted into a Unker compatible with the phosphoramidite method. Incorporation of amino groups in both the 3'- and 5'-end of the oligonucleotide has been demonstrated. However, the reagent is produced as a racemic mixture of the 2R and 2S-form, thereby leading to 2ⁿ diastereomers of the modified oligonucleotide upon incorporation of the reagent n times. Since purification and characterisation of such a mixture of diastereomers can be difficult, there is a need for achiral linkers which allow multiple incorporation.

DESCRIPTION OF THE INVENTION

The present invention provides a simple achiral Unker reagent compatible with standard phosphoramidite oligonucleotide synthesis protocols. The new achiral linker reagent can be used to incorporate an amino functional linker or a Unker functionalized with a reporter molecule into an oUgonucleotide. Thus, in one aspect, the invention relates to a Unker reagent of the formula I

wherein n is an integer from 1 to 3, preferably from 1 to 2, such as 1; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionally interrupted or terminated by 1-5 heteroatom(s), such as oxygen and/or nitrogen atom(s), preferably oxygen atom(s) so as to form a (poly)- alkylene glycol, e.g. ethylene glycol, propylene glycol, butylene glycol, polyethylene glycol (e.g. -0-CH₂CH₂-0-CH₂CH₂-0-CH₂CH₂-0-CH₂CH2-0-CH₂CH₂- when a 10-carbon chain is inter¬ rupted and terminated by a total of five oxygen atoms), or polypropylene glycol; R¹ is a group selected from phosphoramidite groups and H-phosphonate; R² is selected from amino protecting groups and reporter groups; and R³ is selected from hydrogen and hydroxy protecting groups.

The group Z preferably designates a single bond or a chain of 1-4 carbon atoms, optionally interrupted or terminated by a nitrogen atom or an oxygen atom, preferably an oxygen atom; more preferably Z designates a single bond or an methylene or ethylene group, most preferably a methylene group. By the expression "interrupted or terminated by heteroatom(s)" is meant that heteroatoms are inserted in the carbon chain ("interrupted") or between the aromatic ring and the carbon chain ("terminated").

In the present context the term "reporter group" is intended to mean a group which allows detection, e.g. photodetection and binding, and optionally quantification, e.g. by photospectrometrical means, of a modified oUgonucleotide, wherein the reporter group is included. The reporter group may include a spacer element not contributing to the detectabiUty of the group in order for the reporter group to be attached to the nitrogen atom of the Unker.

Examples of such reporter groups are fluorescein, dansyl (5-(dimethylamino)-l- naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazoUdine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrroUdine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine, dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-l-diazole (NBD), and pyrene. EspeciaUy preferred are the photodetectable groups fluorescein and dansyl due to their high fluorometric sensitivity, and biotin due to its strong binding to avidin in the avidin-phosphatase assay (Chu, B. C. F.; Orgel, L. E.; DNA 1985, 4(4), 327-331). When used herein the term "hydroxy protecting group" is intended to mean any hydroxy protecting group known in the art. Preferred hydroxy protecting groups compatible with the phosphoramidite reaction conditions are 4,4'-dimethoxytrityl (DMT), 9-phenylxanthen-9-yl (pixyl), and 9-fluorenylmethoxycarbonyl (Fmoc).

The advantage of incorporating the reporter group directly into the Unker reagent (see, e.g., compound 9, Scheme 3) is that no post modification and consequently no additional work-up procedures are necessary. When the reporter group is incorporated prior to couphng it may, however, be necessary or desirable to protect the reporter group during the soUd phase oUgomerization process, e.g. in the case of the reporter molecule being fluorescein where the free hydroxy groups reacts with phosphoramidites if they are unprotected. This is usually not necessary when using the post modification method.

In the case where the reporter molecule is incorporated into the modified oUgomer after the Unker itself has been incorporated (post modification, see, e.g., Scheme 5), the amino group of the Unker is preferably protected during soUd phase oUgomerization in order to avoid compUcations, e.g. acetylation with acetic anhydride (see, e.g., compounds 9 and 10, Scheme 1). A large variety of the amino protecting groups known for the person skiUed in the art may be used. Examples of such amino protecting groups compatible with the phosphoramidite strategy are urethane protecting groups, such as benzyloxycarbonyl, tert-butoxycarbonyl, 9-fluorenyl- methoxycarbonyl, and aUyloxycarbonyl, acyl groups, such as trifluoroaceryl, and phthaloyl (which of course involves two valences of the nitrogen atom whereby R²NH- in Formula I should be replaced by R -N-), among which trifluoroaceryl and 9-fluorenylmethoxycarbonyl are preferred.

Since the use of phosphoramidite functionalized hydroxy groups offers a convenient and efficient way of forming phosphate Unkages between individual sugar moieties in oUgonucleotides, this type of functionaUty is a natural choice for the Unker reagents according to the invention.

In the present context, the term "phosphoramidite" is intended to mean a group of the formula -P(OR')-NR"R'", wherein R' designates an optionaUy substituted alkyl group, e.g. methyl, 2-cyanoethyl, or benzyl (phenylmethyl), R" and R"' designate optionaUy substituted alkyl groups, e.g. ethyl or isopropyl, or the group -N(R"R'") forms a morphoUno group (-N(CH₂CH₂)2θ). R' preferably designates 2-cyanoethyl and R" and R"' are preferably identical and designate isopropyl. Thus, an especially relevant phosphoramidite in conventional DNA- synthesis is

(N,N-dusopropyl-0-(2-cyanoethyl)phosphoramidite).

As an alternative to the phosphoramidite functionaUty, the corresponding H-phosphonate may be used. In a preferred embodiment of the present invention, the Unker according to the invention has the formula Y

corresponding to compound 10 in Scheme 1.

The Unker, with or without the reporter group attached, can substitute any of the nucleotide units in an oUgonucleotide. It is beUeved that, in order to obtain an acceptable spatial relationship between the nucleotides adjacent to the Unker, the value of n should preferably be 1 or 2, in particular 1.

In the present context, the term "nucleotide" is intended to cover ribonucleotides comprising a ribose moiety as weU as deoxyribonucleotides comprising a deoxyribose moiety. With respect to the bases of such "nucleotides", it should be understood that this may be any of the naturaUy occurring bases, e.g. adenine, guanine, cytosine, thymine, an uraril, as weU as any modified variants thereof or any possible unnatural bases. The term "nucleotide unit" is intended to cover a nucleotide incorporated in a oUgonucleotide.

By the term "oUgonucleotide" is meant a successive chain of "nucleotides", however, in the present context it should be understood that one or more of such nucleotide units in an oUgonucleotide may have be modified, e.g. by substitution with a Unker such as in the case where a further Unker group is introduced into an oUgonucleotide.

FoUowing the phosphoramidite strategy, the Unker reagent is coupled to the 5'-end of a nucleotide or an oUgonucleotide. Thus, in the conventional DNA-synthesis, e.g. using the standard phosphoramidite protocol described herein, the Unker reagent simply replaces a protected deoxynucleoside phosphoramidite.

By using a variant of the phosphoramidite strategy, the protected deoxynucleoside derivatized lcaa-CPG column may be replaced by a Unker reagent derivatized soUd support material, i.e. an immobilized Unker reagent, e.g. compound 9 in Scheme 1. In this way a protected oUgonucleotide phosphoramidite or a protected nucleotide phosphoramidite can be Unked to a soUd support material, e.g. a CPG-material, by means of a soUd support material functionalized with the Unker reagent. This usually impUes coupUng of the Unker to the 3'-end of the oUgonucleotide or nucleotide, i.e. the immobilized Unker reagent has a free hydroxy group and the nucleotide component is in the form of the phosphoramidite.

The Unker part of the immobilized Unker reagent need not necessarily to be attached directly or through a functional spacer to the soUd support material, thus, it is envisaged that one or more nucleotide units may be present between the Unker and the soUd support material. In this way a predetermined sequence of nucleotides (an oUgonucleotide) may be synthesized on the soUd support material prior to coupUng with a protected Unker reagent, e.g. a Unker reagent of formula I, for the formation of a protected immobilized Unker reagent of formula II (see below), Le. an immobilized Unker reagent of formula II where R³ is a hydroxy protecting group.

Thus, in a further aspect the present invention relates to a immobilized Unker reagent of the formula II immobilized to a soUd support material

wherein n is an integer from 1 to 3, preferably from 1 to 2, such as 1; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionaUy interrupted or terminated by 1-5 heteroatom(s); X is selected from a single bond, nucleotides, and oUgonucleotides; FS is a functional spacer; SSM is the soUd support material to which the Unker reagent is immobilized; R² is selected from amino protecting groups and reporter groups; and R³ is selected from hydrogen and hydroxy protecting groups.

The terms "amino protecting group", "reporter group", and "hydroxy protecting group", and the symbol Z have the same meanings as defined above for the Unker reagent of formula I.

In a preferred embodiment, X is either a single bond or a single nucleotide.

The soUd support material (SSM) may, in order for the synthesized modified oUgonucleotide to be cleaved from the soUd phase, comprise a functional spacer between the core of the material and the Unker, when X designates a single bond, or the core of the material and the (first) nucleotide unit, when X designates an oUgonucleotide or a nucleotide. Examples of such a functional spacer, FS, are a succinyl moiety, a glutaryl moiety, and a adipinyl moiety which all are often used in conventional DNA-synthesis.

With respect to the soUd support material, SSM, it is preferably selected from long chain alkyl amine CPG Gcaa-CPG), 3-amino-propyl sUica gel, aminomethyl polystyrene resin, and TentaGel™ (polyethylene glycol-TENTAcles grafted on low cross-Unked GELatineous polystyrene matrix).

In a preferred embodiment of the immobilized Unker reagent according to the invention, n is 1; Z designates a methylene group; X is a single bond; FS designates a succinyl spacer; SSM designate a ControUed Pore Glass (CPG); R² is a reporter group (see, e.g., compound 19, Scheme 3, or compound 22, Scheme 4) or an amino protecting group (see, e.g., compound 9, Scheme 1); and R³ is hydrogen or a hydroxy protecting group.

In the above-mentioned examples, the core of the Unker consists of benzene. However, a variety of other heteroaromatic systems may in principle be used to estabUsh the necessary geometry of the Unker reagent. Examples of such alternative aromatic cores (AC) include pyrrole, triazole, pyridine, pyrimidine, and sym-triazine.

The Unker reagent can be used for multiple incorporations of Unkers functionalized with reporter groups, either directly connected in a row by repetitive coupUngs during oUgo¬ merization, or in various selected places along the oUgonucleotide sequence (see Examples, "Preparation of various modified oUgodeoxyribonucleotides"). This opens the possibiUty for signal ampUfication, which can be useful when reporter groups with low sensitivity are used.

Thus, the Unker can be incorporated into the oUgonucleotide at any position using conventional phosphoramidite chemistry. Apart from the necessity of using tert-butyl hydroperoxide in the oxidation step as iodine, which is frequently used, is known to cleave benzyUc phosphate esters (Perich, J. W.; Johns, R. B. Tetrahedron Let. 1987, 28, 101-102), the Unker is fuUy compatible with the standard synthesis protocol for automated DNA-synthesis, and can been used in repetitive coupUng cycles. The Unker can be incorporated in good overaU yield and provides access to oUgonucleotides with labeUed or easily labelled and highly reactive amino groups. Thus, besides being achiral, the Unker offers a good alternative to the already existing reagents of this type for the preparation of modified oUgonucleotides. Thus, in a further aspect, the present invention relates to a modified oUgonucleotide comprising at least one Unker fragment of the formula LTI

R²

wherein n is an integer from 1 to 3, preferably from 1 to 2, such as 1; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionaUy interrupted or terminated by 1-5 heteroatom(s); each Y independently is selected from hydrogen, hydroxy protecting groups, nucleotide units, and further Unker fragments; and R² is selected from amino protecting groups, and reporter groups, preferably reporter groups; with the proviso that at least one Y is a nucleotide unit.

The term "further Unker fragments" is intended to mean an adjacent fragment similar or identical to the one of formula in, e.g. as is the case for items 6, 9, and 10 of Table I below (Examples).

Standard phosphoramidite protocol

As an example, the foUowing general procedure for the synthesis of modified oUgonucleotides may be used (KeUer, G. H.; Manak, M. M.; "DNA Probes", 2.ed. MacmiUan PubUshers Ltd, 1993 αSBN 0-333-57384-6) 89-90):

The standard synthesis protocol for automated DNA-synthesis, uses protected deoxynucleoside phosphoramidites, protected deoxynucleoside derivatized lcaa-CPG columns, 0.45 M tetrazole in acetonitrile, 1 M acetic anhydride and 1 M 2,6-lutidine in tetrahydrofuran, 2.4 M N-methyUmidazole in tetrahydrofuran, 3 % dichloroacetic acid in dichloromethane, tert-butyl hydroperoxide (1.1 M in acetone/CH₂Cl₂ 1:1), and concentrated aqueous ammonia.

Packing of the column

An appropriate amount of soUd support is weighted out (for a 1 μmol scale, 30 mg support with a loading of 33-35 μmol/g is used). The support is transferred to the synthesis column and a filter cap is attached. The column is sealed with an aluminium seal, attached to the DNA synthesizer via Luer fittings, and checked for leaks by flowing acetonitrile in both directions. Assembly Cycle

Reagent bottles on the DNA synthesizer is filled with protected deoxynucleoside phosphor¬ amidites and protected Unker reagent phosphoramidite (e.g. a Unker reagent of Formula I). The DNA-sequence including the modified Unker reagent is entered on the keypad, and the synthesis cycle is started. The cycle starts with deprotection of the DMT-group of the protected deoxynucleoside derivatized lcaa-CPG using 3 % dichloroacetic acid in dichloromethane. Protected deoxynucleoside phosphoramidites or protected Unker reagent phosphoramidite is loaded to the column, and aUowed to react for 1-2 min. using 0.45 M tetrazole in acetonitrile as catalyst. Unreacted alcohol groups is then capped with 1 M acetic anhydride and 1 M 2,6-lutidine in tetrahydrofuran. The newly formed phosphotriester is then oxidized to its phosphatetriester using tert-butyl hydroperoxide (1.1 M in acetone/CH₂Cl₂; 1:1), to finish up the synthesis cycle.

Cleavage from CPG-support

When the sequence is completed, the oUgonucleotide is cleaved from the CPG-support using concentrated aqueous ammonia (8-16 h at 60°C).

Thus, the present invention also relates to a method for labelling of oUgonucleotides, Le. preparation of oUgonucleotides comprising one or more fragments of the formula UI, wherein an oUgonucleotide fragment is reacted with a Unker reagent of formula I above in order to obtain an oUgonucleotide wherein an achiral Unker carrying a reporter group, where appropriate after post modification, have been incorporated.

This may also be accomplished by using a method for labeUing of oUgonucleotides, wherein an immobilized Unker reagent or formula U is reacted with a phosphoramidite functionalized nucleotide or a phosphoramidite functionalized oUgonucleotide.

A stiU further aspect the present invention relates the use of a Unker reagent of the formula I or a immobilized Unker reagent of formula LT for the labeUing of an oUgonucleotide.

With the Unker reagent bearing a reporter molecule or a free amino group, either in free or immobilized form, in hand, modified oUgonucleotides with useful properties with respect to, e.g., diagnostic or manipulation purposes, may easily be prepared.

Thus, the present invention also relates to the use of a Unker reagent of Formula I or a immobilized Unker reagent of formula U for preparing labeUed oUgonucleotides for detection and manipulation purposes; for preparing oUgonucleotides for use as hybridisation probes (KeUer, G. H.; Manak, M. M.; "DNA Probes", 2.ed. MacmiUan Publishers Ltd, 1993 (ISBN 0-333-57384-6)); for preparing oUgonucleotides for the capture of nucleic acids onto soUd support matrices resulting from soUd phase or solution phase hybridisation reactions (Bischoff, R.; CouU; J. M.; Reigner, F. E.; Anal. Biochem 164, 336-344 (1987) and Yehle, C. O.; Patterson, W. L.; Boguslawski, S. J.; AlbareUa, J. P.; Tip, K. F.; Carrico, R. J.; Mol. CeU. Probes 1; 177-193 (1987)); for preparing oUgonucleotides as a primer in the polymerase chain reaction (PCR) (Gibbs, R. A.; Anal. Chem. 62, 1202-1214 (1990) and ErUch, H. A.; Gefland, D.; Sninsky; J. J.; Science 252, 1643-1651 (1991)); for preparing oUgonucleotides as a primer in nucleic acid sequencing reactions (Maxam, A. M.; Gilbert, W.; Methods Enzymol., 65, 499-560 (1980),

Sanger, F.; Science, 214, 1205-1210 (1981), and Sanger, F.; Ann. Rev. Biochem., 57, 1-28 (1988)); for preparing oUgonucleotides in the production of affinity matrices for the purification of DNA binding proteins and other biomolecules (Rosenfeld, P. J.; Kelly, T. J. ; J. Biol. Chem., 262, 1398-1408 (1986) and Kadonaga, J. T.; Tjian, R; Proc. Natl. Acad. Sci. USA, 83, 5889-5893 (1986)); for preparing oUgonucleotides in the production of affinity matrices for the detection of nucleic acid sequences (Urdea, M. S.; Warner, B. D.; Running, J. A.; Stempien, M.; Clyne, J.; Horn, T., Nucleic Acid Res. 16, 4937-4956 (1988), and Bischoff, R; CouU; J. M.; Reigner, F. E.; Anal. Biochem 164, 336-344 (1987)); for preparing oUgonucleotides as a means of monitoring incorporation reactions; for preparing oUgonucleotides in the production of a random selection of labeUed probes for the detection of the total nucleic acid content of samples by hybridisation; for preparing oUgonucleotides in a sandwich hybridisation system where one labeUed probe acts as a capture and a probe with an alternative label acts as a reporter (Urdea, M. S.; Warner, B. D.; Running, J. A.; Stempien, M.; Clyne, J.; Horn T.; Nucleic Acid Res. 16, 4937-4956 (1988)); for preparing oUgonucleotides for providing a biotinylated or haptenylated oUgonucleotide for use in any DNA manipulation protocol (KeUer, G. H.; Manak, M. M.; "DNA Probes", 2.ed. MacmiUan PubUshers Ltd, 1993 (ISBN 0-333-57384-6)); for preparing oUgonucleotides in cloning recombinant DNA (Itakura, ; Miyake, T.; Kawashima, E. H.; Ike, Y.; Ito, H.; Morin, C; Reyes, A. A.; Johnson, M. J.; Schold, M.; WaUace, R. B. Recomb. DNA, Proc. Cleveland Symp. Macromol., 3rd (1981), 273-89, Editor(s): Walton, Alan G. PubUsher: Elsevier, Amsterdam, Neth.); and for preparing oUgonucleotides in vitro mutagenesis (Higuchi, R.; Krummel, B.; Saiki, R K.; Nucleic Acids Res., 16(15), 7351-7367 (1988) and Johnson, D. L.; Reid, T. M.; Lee, M.-S.; King, C. M.; Romano, L. J., Biochemistry, 25(2), 449-456 (1986)). EXPERIMENTAL

The following examples and the reaction schemes 1 to 5 iUustrate the invention:

3,5-Dicarboxyaniline (2):

5-Nitroisophthalic acid 1 (9.98 g, 47.74 mmol) was dissolved in methanol (100 ml), and the solution was flushed with nitrogen. PaUadium, 10% on carbon (0.50 g), was added, and the mixture was hydrogenated at 1 atm., 20°C, for 1 h. The mixture was filtered, and the filter washed with hot methanol (2 x 50 ml). The combined extract was taken to dryness, and the residue used without further purification. Yield: 8.21 g (95%). Elemental analysis (calc.;found): C(53.04;53.26); H(3.87;3.80); N(7.73;7.68).

3,5-Bis(ethoxycarbonyl)aniline (3):

3,5-DicarboxyaniUne 2 (25 g; 0.14 mol) was dissolved in abs. ethanol (400 ml), and cone, sulfuric acid (20 ml) was added. After refluxing for 16 h the mixture was poured on ice-water containing sodium hydrogen carbonate (50 g). The aqueous solution was extracted with diethylether (3 x 100 ml). The organic phase was washed with saturated sodium chloride solution, and dried over anhydrous magnesium sulphate. Evaporation, in vacuo, foUowed by recrystaUisation of the residue from ethanol/water (1:1) yielded 26.8 g (82 %) of 3,5-bis(ethoxycarbonyl)aniUne in the form of needles. Elemental analysis (calc.;found): C(60.76;60.83); H(6.33;6.31); N(5.91;5.84). 1H-NMR (CDC1₃): d = 8.10 ppm (s, IH); 7.56 (s, 2H); 4.37 (q, 4H); 1.39 (t, 6H).

3,5-Bis(hydiOxymethyl)aniline (4):

Lithium aluminium hydride (6 g, 158 mmol) was added to a reaction flask charged with dry tetrahydrofuran (220 ml), and the resulting suspension heated to reflux. A solution of 3,5-bis(ethoxycarbonyl)aniUne 3 (10 g , 42.2 mmol) in dry tetrahydrofuran (100 ml), was added to the refluxing mixture over a period of 1 h. After reflux for an additional 2h, the mixture was cooled to room temperature, and methanol (8 ml), foUowed by water (12 ml), were added. The mixture was stirred for 2 h and filtered. The filtrate was evaporated to dryness, in vacuo, and the residue recrystallized from ethyl acetate. Yield 5.19 g (80.4 %). Elemental analysis (calc.;found): C(62.74;62.77); H(7.19;7.11); N(9.15;9.14). IH NMR (D₂0): d = 6.77 ppm (s, IH); 6.72 (s, 2H); 4.68 (s, 4H). MS: m/e = 153. 3,5-Bis(hydroxymethyl)benzonitrile (5):

3,5-Bis(hydroxymethyl)aniUne 4 (5 g, 32.7 mmol) was dissolved in hydrochloric acid (2N, 100 ml), cooled to -5°C, and stirred for 30 min. A cold sodium nitrite solution (2.48 g , 36 mmol in 50 ml water) was added, while the temperature was kept at -5°C. The solution was stirred for 30 min., and subsequently neutraUsed with soUd sodium carbonate (6.80 g). A solution of cuprous cyanide (3.20 g, 35.7 mmol) and sodium cyanide (3.52 g, 71.8 mmol) in water (50 ml) was prepared, and after coohng to 0°C, added in one portion to the stirred diazonium salt solution. The temperature was kept at 0°C for 2 h, while the solution changed colour from yellow to dark black. After stirring overnight, foUowed by nitration, the dark solution was extracted continuously with methylene chloride (230 ml) for 36 h. The organic phase was dried over anhydrous sodium sulphate and evaporated to dryness, in vacuo. The residue was recrystallized from ethyl acetate. Yield 3.52 g (65.7 %). Mp = 138-140°C Elemental analysis (calc.;found): C(66.26;66.24); H(5.52;5.65); N(8.59;8.59). Η-NMR (DMSO-d₆): d = 7.6 ppm (d, 3H); 4.7 (s, 2H); 2.6 (s, 4H). MS: m/z = 163. LR(KBr): v = 2230 cm^'1.

0³-DMT-3,5-bis(hydroxymethyl)benzonitrile (6):

3,5-Bis(hydroxymethyl)benzonitrile 5 (1.00 g; 6.13 mmol) was dissolved in anhydrous pyridine (25 ml), and 4,4'-dimethoxytrityl chloride (2.29 g; 6.76 mmol) was added. The mixture was stirred overnight at 20°C under a nitrogen atmosphere. Methanol (5 ml) was added, and the mixture stirred for another 10 min. Pyridine was removed by evaporation, in vacuo, and the residue suspended in ether. After filtration of unreacted starting material, the filtrate was concentrated to 4 ml, and appUed on a siUca gel column (25 x 4 cm). The column was eluted with n-hexane/ethyl acetate (800 ml; 3:1) foUowed by n-hexane/ethyl acetate (400 ml; 1:1). Fractions showing a single spot on TLC at R_f 0.6 (ethyl acetate/hexane; 1:1) were pooled, and the solvent removed by rotary evaporation to give 1.49 g (52.3 %) of a yeUow oU. The oD sohdified to an amorphous compound by evaporation to dryness from ethyl ether. Elemental analysis (calc.;found): C(77.41;77.07); H(5.80;5.95); N(3.01;3.03). IR(KBr): v = 2232 cm^'1.

0³-DMT-3,5-bis(hydroxymethyl)benzylaιnine (7):

0 -DMT-3,5-bis(hydroxymethyl)benzonitrile 6 (356.8 mg; 0.76 mmol) was dissolved in dry diethyl ether (50 ml), and Uthium aluminium hydride (90 mg, 2.40 mmol) was added. The mixture was stirred overnight, and then quenched with methanol (3.0 ml). Water (0.5 ml) was added, and the mixture stirred for a further 30 min. The inorganic salts were filtered off, and the solvent removed by evaporation, in vacuo. The hygroscopic foam obtained, was sufficiently pure to be used in later reactions. 1H-NMR (DMSO-d₆): d 3.54 ppm (broad s; 3H); 3.82 (s, 6H); 3.85 (s, 2H); 4.11 (s, 2H); 4.58 (s, 2H); 6.98-7.53 (m, 16H). ¹³C-NMR(DMSO-d₆): d = 55.08 ppm; 62.94; 65.21; 67.05; 85.91; 113.30; 123.41; 124.40; 124.57; 126.74; 127.70; 127.93; 129.67; 135.71; 138.28; 141.87; 142.60; 144.94; 158.13.

N-Fmoc-0³-DMT-3,5-bis(hydroxymethyl)benzylamine (8):

0³-DMT-3,5-bis(hydroxymethyl)benzonitrile 6 (2.00 g; 4.30 mmol), was dissolved in anhydrous ether (40 ml), and added to a solution of Uthium aluminium hydride (0.75 g; 19.75 mmol) in anhydrous ether (50 ml) over a period of 1 h at 20°C. After stirring for 3.5 h at 20°C, the mixture was quenched with ethanol/water (30 ml; 9:1) and stirred for a further 30 min. The resulting white suspension was filtered, and the filter cake washed with hot ethanol (3 x 25 ml). The combined filtrates were evaporated to dryness, to give 1.65 g of a white amorphous soUd, almost pure on TLC (Rf 0.15, CH₂C1₂/CH₃0H; 3:2). A sample of the soUd (460 mg) was redis¬ solved in dry DMF (6 ml), and cooled on an ice bath. Dusopropylethylamine (153 g; 1.19 mmol) and 9-fluorenylmethyl chloroformate (280 mg; 1.08 mmol) was added, and the mixture stirred for 40 min., during which time the temperature reached 20°C. The mixture was poured out in a cold solution of 1 N sodium hydrogen carbonate (100 ml), and the precipitated soUd coUected by filtration.The soUd was appUed on a sihca gel column (20 g), which was eluted with n-hexane/ethyl acetate (400 ml; 1:1). Fractions showing one spot on TLC at R 0.23 (ethyl acetate/hexane; 1:1) were coUected and the solvent removed on a rotary evaporator. The resulting oil was sohdified by addition of water (5 ml). Yield 462 mg (68.2 % overaU). Mp = 68-72°C. Elemental analysis (calc.;found): C(78.14;76.84); H(5.95;6.40); N(2.03;1.99). -NMRφMSO-de): d = 3.80 ppm (s, 6H (MeO-)); 4.10 (s, 2H (CH₂ODMT)); 4.27 (t, 2H (CH₂OH)); 4.36 (d, 2H (OCH₂CH)); 4.39 (t, IH (OCH₂CH)) 4.56 (d; 2H (CH₂NHFmoc)); 5.26 (t, IH (CH₂NHFmoc)); 6.96-7.97 (m, 24 H(Aromatic)). ¹³C-NMR(DMSO-d₆): d = 43.96 ppm; 46.79; 55.16; 63.00; 65.22; 65.62; 113.41; 120.24; 123.58; 124.27; 125.28; 126.87; 127.16; 127.73; 128.05; 129.80; 135.81; 158.25. (only the strong signals is reported herein).

Procedure for preparation of modified CPG (9):

N-Fmoc-0³-DMT-3,5-bis(hydroxymethyl)benzylamine 8 (125 mg; 0.18 mmol) was dissolved in pyridine (2.0 ml). Succinyl anhydride (16.5 mg; 0.17 mmol) and 4-N,N-dimethylaminopyridine (10 mg; 0.08 mmol) was added, and the mixture was stirred at room temperature overnight. The solvent was stripped off, and the residue redissolved in ethyl acetate (10 ml). The organic phase was washed with brine (2 x 10 ml), dried with anhydrous sodium sulfate, and then taken to dryness by evaporation, in vacuo. Traces of water was removed by co-evaporation from pyridine (2 x 5.0 ml), and the oUy-residue was dissolved in anhydrous DMF (2.0 ml). Pyridine (200 μl), p-nitrophenol (43.1 mg; 0.31 mmol) and dicyclohexylcarbodumide (45.4 mg; 0.22 mmol) was added. The mixture was stirred for 4 h, at room temperature. Long chain alkylamine CPG (300 mg, 89 μmol/g) previously dried by evaporation from dry acetomtrile, was added. The mixture was placed in an orbital shaker overnight. The CPG was filtered off and washed with DMF (2 x 20 ml), methanol (3 x 15 ml), and diethylether (2 x 15 ml). The modified CPG was capped by treatment with a solution of acetic anhydride/pyridine/dichloromethane (6 ml, 1:2:2) for 2 h. After filtration, the CPG was washed with pyridine (20 ml), DMF (3 x 20 ml), water (3 x 20 ml), methanol (3 x 20 ml) and diethyl ether (5 x 20 ml) and finally dried, in vacuo. Loading was 25 μmol/g.

Procedure for preparation of 0³-(cyanoethyl-N,N-diisopropyl phosphoroamidyD-N'- Fmoc-O^δ-DMT-3,5-bis(hydroxymethyl)benzylamine (10):

N-Fmoc-0³-DMT-3,5-bis(hydroxymethyl)benzylamine 8 (500 mg; 0.72 mmol) was dissolved in dry chloroform (2.0 ml). 2-Cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (243.3 mg; 0.79 mmol), and a solution of tetrazole (56 mg; 0.79 mmol) in acetonitrile (2.0 ml) was added. The mixture was stirred for 1 h at room temperature, before dusopropylethylamine (0.7 ml) was added. The reaction mixture was diluted with dichloromethane (5.0 ml), and washed with a 5% solution of sodium bicarbonate in water (2 x 10 ml). The organic phase was dried with anhydrous magnesium sulfate, and evaporated to dryness, in vacuo. The residue was purified by column chromatography using sihca gel 60, and ethyl acetate/dichloromethane/triethylamine (50:45:5) as eluent. The foam obtained was dissolved in a minimum of ethyl acetate, and precipitated from cold hexane to give the analytical pure phosphoramidite 10 as a white powder. ³¹P-NMR (90 MHz, CDC1₃): d 148.5 ppm. ^XH-NMR (400 MHz, CDC1₃) d 1.19 (t, 12H ((CH₃)₂CH)₂N); 2.57 (t, 2H (CH₂CH₂CN)); 3.63 (dt, 2H (CH₂CH₂CN)); 3.78 (s, 6H (MeO-)); 3.82 (m, 2H ((CH₃)₂CH)₂N); 4.18 (s, 2H (CH₂ODMT)); 4.22 (t, IH (OCH₂CH)); 4.39 (d, 2H(CH₂NHFmoc)); 4.43 (d, 2H (OCH₂CH)); 4.71 (ddd, 2H (CH₂OP)); 5.15 (t, IH (CH₂NHFmoc)); 6.82-7.75 (m, 24H (Aromatic)). ¹³C-NMR (400 MHz, CDC1₃) d 20.34; 20.41; 24.60; 24.63; 24.67; 24.70; 43.16; 43.28; 47.28; 55.20; 58.41; 58.60; 65.24; 65.41; 66.80; 113.13; 119.91; 124.81; 125.03; 125.27; 126.75; 127.01; 127.63; 127.81; 128.16; 130.05; 136.19; 140.02; 158.47 (only the strong signals are reported). Elemental analysis, found: C, 72.65; H, 6.45; N, 4.70. C₆₄H₅₈N₃0₇P requires: C, 72.71; H, 6.55; N, 4.71.

3,5-Dimethylbenzyl bromide (12):

The method described by Herr et al. J. Am. Chem. Soc. 79 (1957) p. 4229, was used, with some modifications. Mesitylene 11 (118 ml, 848 mmol), N-bromosuccinimide (60.1 g, 337 mmol), carbon tetrachloride (150 ml), and benzoyl peroxide (0.50 g) were refluxed for 1.5 h. The mixture was cooled, filtered, and taken to dryness. The residue was distiUed, in vacuo, and the fraction boiUng at 106-109°C (9-11 mmHg) was coUected. Yield: 45.13 g (67.31%). Mp = 30-32°C. Elemental analysis (calc.;found): C(54.30;54.42); H(5.53;5.63); Br(40.17;39.00).

N-(3,5-Dimethylbenzyl)phthalimide (13):

3,5-Dimethylbenzylbromide 12 (35.0 g, 176 mmol) and potassium phthaUmide (32.6 g, 176 mmol) were refluxed in dimethylformamide (230 ml) for 3.5 h. After cooUng, water (200 ml) was added, and the suspension was filtered. The crude product was recrystallized from ethanol/ethyl acetate (3:1) which gave the title compound in the form of fine needles. Yield: 35.37 g (76 %). Mp = 150-151°C. Elemental analysis (calcjfound): C(76.98;76.80); H(5.66;5.79); N(5.28;5.27). ^-NMR CDCl_g): d = 2.27 ppm (s, 6H); 4.76 (s, 2H); 7.02 (d (1:2), 3H); 7.75 (m, 4H).

N-[3,5-Bis(bromomethyl)benzyl]phthalimide (14):

N-(3,5-Dimethylbenzyl)phthaUmide 13 (4.5 g, 16.98 mmol), N-bromosuccinimide (6.31 g, 35.44 mmol), carbon tetrachloride (120 ml) and benzoyl peroxide (0.10 g) were refluxed for 1 h. The mixture was filtered hot, and the filtrate evaporated, in vacuo, to dryness. The residue was recrystallized in abs. ethanol (160 ml). The yield was 4.46 g (62 %). Elemental analysis (calcjfound): C(48.25;46.83); H(3.07;3.00); N(3.31;3.19); Br(37.80;37.31). ¹H-NMR(CDC1₃): d = 4.43 ppm (s, 4H); 4.82 (s, 2H); 7.37 (d (1:2), 3H); 7.65-7.91 (m, 4H). MS-FAB +: 424 (63 %); 423 (90 %); 422 (100%).

N-(3,5-Bis[acetoxymethyl]benzyl)phthalimide (15):

N-[3,5-Bis(bromomethyl)benzyl]phthaUmide 14 (500 mg; 1.2 mmol) and sUver nitrate (0.55 g; 3.24 mmol) was heated to reflux with acetic acid (25 ml). After 4 h, the mixture was filtered hot; the filtrate was added to a cold sodium hydrogen carbonate solution (50 ml; IN), and extracted with ethyl acetate (3 x 50 ml). After washing with HCl (2 x 50 ml; 0.5 N) and saturated sodium chloride solution, the organic phase was dried with anhydrous sodium sulphate and evaporated to dryness, in vacuo. The residue underwent chromatography on a smaU column of silica gel (25 g) eluted with 400 ml n-hexane/ethyl acetate (3:1). Fractions showing a single spot at R_f = 0.45 (ethyl acetate/hexane; 1:1) were pooled, and taken to dryness, by evaporation, in vacuo. Yield: 354 mg (78.5 %). Mp = 129-32°C Elemental analysis (calcjfound): C(66.14;66.26); H(4.98;4.97); N(3.67;3.76). -NMR CDCl_g): d = 2.08 ppm (s, 6H); 4.84 (s, 2H); 5.06 (s, 4H); 7.25 (s, 2H); 7.36 (s, IH); 7.69-7.86 (m, 4H). N-Fmoc-0³-DMT-3,5-bis(hydroxymethyl)benzylamine (8) from 15:

Ν-(3,5-Bis[acetoxymethyl]benzyl)phthaUmide 15 is dissolved in methanol and 3 equivalents of hydrazine hydrate is added. The solution is refluxed for several hours, and then taken to dryness by evaporation in vacuo. The residue is suspended in water, filtered, and extracted several times with ethyl acetate. The water phase, which now contains 3,5-bis(hydroxymethyl)benzyl amine, is taken to dryness, and redissolved in DMF. 1.1 equivalents of dusopropyl amine and 1.1 equivalents of 9-fluorenylmethyl chloroformate is added, and the mixture is stirred at ambient temperature, until completion. The mixture is taken to dryness once more, the residue dissolved in pyridine, and 1 equivalent of 4,4'-dimethoxytrityl chloride is added. When the reaction have reached completion, the solvent is removed and the residue appUed on a siUca gel column, which is eluted with hexane/ethyl acetate (1:1). Fractions showing one spot on TLC at R_f= 0.23 (hexane/ethyl acetate; 1:1) is coUected and solvent is removed on a rotary evaporator. The resulting oU is sohdified by addition of water, and the soUd is coUected by filtration.

Dipivaloylfluorescein isothiocyanate (17):

Fluorescein isothiocyanate 16 (200 mg; 0.51 mmol) was dissolved in dry THF (25 ml). Diisopropylethylamine (270 μl; 1.53 mmol) was added, and the solution was cooled to 0°C on an ice bath. Pivaloyl chloride (155 mg; 1.28 mmol) was added, and the mixture was stirred for 10 min. at 0^CC, and then for 12 h at room temperature. Methanol (5 ml) was added, and stirring was continued for an additional 30 min. Solvent was removed, in vacuo, and the residue purified by column chromatography, using Sihca gel 60, and dichloromethane as eluent. Yield: 217 mg (76 %) R_f = 0.6 (dichloromethane). ^XH-NMR (CDC1₃): d 1.35 ppm (s, 18 H); 6.79 (s, 4 H); 7.05 (s, 2 H); 7.14 (d, 1 H, J = 8.0 Hz); 7.47 (q, IH, J, = 8.0 Hz, J₂ = 1.7 Hz); 7.82 (d, IH, J_j = 1.7 Hz). ¹³C-NMR(CDC1₃): d 26.96 ppm; 39.11; 81.00; 110.36; 115.33; 117.75; 121.67; 125.31; 127.64; 128.53; 132.38; 133.94; 139.41; 150.85; 151.39; 152.67; 167.42; 176.31.

N-DipivaloylfluoresceinyI-N'-(0³-DMT-3,5-bis(hydroxymetb.yI)benzyl) thiourea (18):

0³-DMT-3,5-bis(hydroxymethyl)benzylamine 7 (789.6 mg; 1.68 mmol) was dissolved in Al₂0₃-dried tetrahydrofuran (20 ml), and 2 drops of triethylamine was added. The reaction flask was cooled on an ice bath, and a solution of dipivaloylfluorescein isothiocyanate 17 (937 mg; 1.68 mmol) in dry THF (30 ml) was added over 3 min. The mixture was stirred at 0^CC for 3 h, then taken to dryness by evaporation, in vacuo. The white foam obtained, was further purified by column chromatography (SiUca Gel 60/5% triethylamine/5% methanol/90 % dichloromethane), foUowed by precipitation from petrol ether, to give a yeUow powder. Yield: 1320.0 mg (78 %) Anal. (calc;found): C(71.33; 70.00); H(5.69; 6.46); N(2.73; 3.60). ^XH-NMR (DMSO-d₆): 1.33 ppm (s, 18H); 3.01 (bs, IH); 3.75 (s, 6H); 4.15 (s, 2H); 4.64 (d, 2H); 4.85 (bs, 2H); 5.26 (t, IH); 6.95-7.80 (m, 25H); 8.52 (s, IH). ¹³C-NMR (DMSO-d₆): 27.06 ppm; 55.21; 64.89; 65.36; 110.37; 113.16; 115.95; 117.75; 118.24; 124.60; 124.86; 125.72; 126.77; 126.99; 127.85; 128.12; 128.88; 130.05; 136.08; 158.46; 176.47; 181.08 (only the strong signals are reported).

Procedure for preparation of fluorescein modified CPG (19):

N-Dipivaloylfluoresceinyl-N'-(0³-DMT-3,5-bis(hydroxymethyl)benzyl) thiourea 18 (230 mg; 0.22 mmol) was dissolved in pyridine (2.0 ml). Succinyl anhydride (20.0 mg; 0.20 mmol) and 4-N,N-dimethylaminopyridine (10 mg; 0.08 mmol) was added, and the mixture was stirred at room temperature overnight. The solvent was stripped off, and the residue redissolved in ethyl acetate (10 ml). The organic phase was washed with brine (2 x 10 ml), dried with anhydrous sodium sulfate, and then taken to dryness by evaporation, in vacuo. Traces of water was removed by co-evaporation from pyridine (2 x 5.0 ml), and the oily-residue was dissolved in anhydrous DMF (2.0 ml). Pyridine (200 μl), p-nitrophenol (43.1 mg; 0.31 mmol) and dicyclohexylcarbodiimide (45.4 mg; 0.22 mmol) was added. The mixture was stirred for 4 h, at room temperature. Long chain alkylamine CPG (300 mg, 89 μmol/g) previously dried by evaporation from dry acetonitrile, was added. The mixture was placed in an orbital shaker overnight. The CPG was filtered off and washed with DMF (2 x 20 ml), methanol (3 x 15 ml), and diethylether (2 x 15 ml). The modified CPG was capped by treatment with a solution of acetic anhydride/pyridine/dichloromethane (6 ml, 1:2:2) for 2 h. After filtration, the CPG was washed with pyridine (20 ml), DMF (3 x 20 ml), water (3 x 20 ml), methanol (3 x 20 ml) and diethyl ether (5 x 20 ml) and finaUy dried, in vacuo. Loading: 33.1 μmol/g.

Procedure for preparation of N-Dipivaloylfluoresceinyl-N'-(0 -[cyanoethyl-N",N"- diisopropyl-phosphoroamidyl]-0⁵-DMT-3,5-bis[hydroxymethyl]benzyl) thiourea (20):

N-Dipivaloylfluoresceinyl-N'-(0³-DMT-3,5-bis(hydroxymethyl)benzyl) thiourea 18 (580 mg; 0.56 mmol) was dissolved in dry chloroform (2.0 ml). 2-Cyanoethyl N,N,N',N'-tetraisopropyl- phosphorodiamidite (196 mg; 0.65 mmol), and a solution of tetrazole (35.3 mg; 0.50 mmol) in acetonitrile (2.0 ml) was added. The mixture was stirred for 1 h at room temperature, before dusopropylethylamine (0.7 ml) was added. The reaction mixture was diluted with dichloromethane (5.0 ml), and washed with a 5% solution of sodium bicarbonate in water (2 x 10 ml). The organic phase was dried with anhydrous magnesium sulfate, and evaporated to dryness, in vacuo. The residue was precipitated from cold hexane to give the fluorescein labeUed phosphoramidite 20 as a white powder. Yield: 610 mg (89 %) ³¹P-NMR (90 MHz, CDC1₃): d 148.4 ppm. N-Biotinyl-0³-DMT-3,5-bis(hydroxymethyl)benzylamine (21):

0³-DMT-3,5-bis(hydroxymethyl)benzylamine 7 (470 mg; 1.0 mmol), was dissolved in dry DMF (8.0 ml), dusopropylethylamine (196 μl) was added foUowed by biotinyl NHS ester (375 mg, 1.1 mmol). The resulting clear solution was stirred at room temperature for 48 h. The reaction mixture was partituated between water and dichloromethane, the phases separated, and the water phase extracted twice with dichloromethane. The combined organic phases were washed twice with saturated aqueous sodium bicarbonate solution and once with brine. After drying with anhydrous sodium sulfate, the solvent were stripped off to leave an ouy residue. Further purification by column chromatography using 5 % triethyl amine and 5 % methanol in dichloromethane gave 650 mg (92 %) of the title compound as a hygroscopic glass. ^-NMR

(DMSO-d₆): d 1.2-1.6 ppm (m, 6H); 2.15 (t, 2H); 2.56 (d, IH); 2.76 (dd, IH); 3.05 (m, IH); 3.74 (s, 6H); 4.02 (s, 2H); 4.08 (m, IH); 4.25 (s, 2H); 4.27 (s, IH); 4.48 (d, 2H); 5.19 (t, IH); 6.35 (s, IH); 6.40 (s; IH); 6.90-7.45 (m, 16H); 8.32 (t, IH). ¹³C-NMR (DMSO-d₆): d 25.39; 28.08; 28.30; 35.27; 42.05; 45.63; 55.11; 55.44; 59.26; 61.07; 62.90; 65.10; 85.91; 113.31; 123.36; 124.06; 124.27; 126.78; 127.70; 127.95; 129.69; 135.71; 138.43; 139.70; 142.70; 144.96; 158.15; 162.73; 172.02. Anal. (calc.;found): C(69.04; 66.00); H(6.51; 6.58); N(6.04; 5.98); S(4.60;4.76).

Procedure for preparation of biotin modified CPG (22):

N-Biotinyl-0³-DMT-3,5-bis(hydroxymethyl)benzylamine 21 (191 mg; 0.28 mmol) was dissolved in pyridine (2.0 ml). Succinyl anhydride (27.0 mg; 0.27 mmol) and 4-N,N-dimethylaminopyridine (10 mg; 0.08 mmol) was added, and the mixture was stirred at room temperature overnight. The solvent was stripped off, and the residue redissolved in ethyl acetate (10 ml). The organic phase was washed with brine (2 x 10 ml), dried with anhydrous sodium sulfate, and then taken to dryness by evaporation, in vacuo. Traces of water was removed by co-evaporation from pyridine (2 x 5.0 ml), and the ouy-residue was dissolved in anhydrous DMF (2.0 ml). Pyridine (200 μl), p-nitrophenol (43.1 mg; 0.31 mmol) and dicyclohexylcarbodnmide (45.4 mg; 0.22 mmol) was added. The mixture was stirred for 4 h, at room temperature. Long chain alkylamine CPG (400 mg, 89 μmol/g) previously dried by evaporation from dry acetonitrile, was added. The mixture was placed in an orbital shaker overnight. The CPG was filtered off and washed with DMF (2 x 20 ml), methanol (3 x 15 ml), and diethylether (2 x 15 ml). The modified CPG was capped by treatment with a solution of acetic anhydride/pyridine/dichloromethane (6 ml, 1:2:2) for 2 h.

After filtration, the CPG was washed with pyridine (20 ml), DMF (3 x 20 ml), water (3 x 20 ml), methanol (3 x 20 ml) and diethyl ether (5 x 20 ml) and finaUy dried, in vacuo. Loading: 22.3 μmol/g. Preparation of various modified oligodeoxyribonucleotides

The reagents 9 and 10 were used to incorporate 5-(aminomethyl)-l,3-benzenedimethanol into various oUgodeoxyribonucleotides (Table I, page 20). Siπύlar reagents 19 and 20 were used to incorporate N-fluresceinyl-(5-aminomethyl)-l,3-benzenedimethanol into various oligonucleotides (Table H, page 21). In both cases, standard soUd support phosphoramidite chemistry was used, apart from the oxidation step, where iodine/water was exchanged with tert-butyl hydroperoxide (Biosearch 8750 DNA-synthesizer, 0.2 or 1 μmol scale. Standard synthesis cycles and cyanoethyl phosphoramidites were used, but with tert-butyl hydroperoxide (1.1 M in acetone/CH₂Cl₂ 1:1, 3 min) as the oxidation reagent. For experimental details see Marugg, J. E.; Nielsen, J.; Dahl, O.; Burik, A.; van der Marel, G. A.; van Boom, J. H.; Reel. Trav. Chim. Pays-Bas. 1987, 106, 72-76.), since the benzyUc phosphate esters were not compatible with iodine (Perich, J. W.; Johns, R. B. Tetrahedron Lett. 1987, 28, 101-102). The isolated yield of modified oUgonucleotides were in the range 50-75 % after ethanol precipitation, calculated from optical density measurements.

FTTC-labelling of modified oligonucleotides:

The procedure described for 5'-X-GTA-GAT-CAC-T-3' is typical (Scheme 5). The amino-modified oUgonudeotide (0.25 μmol) was dissolved in a 0.1 N aqueous Na2HP0₄-solution (180 μl), and a solution of fluorescein isothiocyanate (10 mg, ca. 100 fold excess) and triethyl amine (100 μl) in DMF (100 μl) was added. After 2 h the product was purified by size exclusion chromatography using a NAP-10-column (Pharmaάa) and 0.1 N aqueous Na₂HP0 -solution (1200 μl) as eluent. Reverse phase HPLC of the coUected fraction, gave one major product (84 % by integration at λ = 260 nm).

Multiple labeUing of 5' - XXX - GTA GAT CAC T - 3' has also been reaUsed, although after 16 h, the product was a mixture of mono-, bis-, and tris- fluorescein labeUed oUgomer.

Coupling efficiency

Sequence X = ^ Reagent

CHsNHs X^a average*⁵

5'-X-GTAGATCACT -3" 10 95% 97.5

5'-GTAGATCACT-X-3' 9 99.5

5' - TGT ACG TXA CAA CTA - 3' 10 99% 98.2

5' - TGT ACX TCA XAA CTA - 3' 10 98-99 % 98.5

5' - X TGT ACG TCA CAA CTA X - 3' 9 t-3 8- 10 98% 99.2

5'-T₁₀ XXT₁₀-3' 10 97-98 % 99.2

5' - X - CAT GAT CTG ACA GAG GGA ACC CAG T - 3' 10 90% 99.2

5' - CAT GAT CTG ACA GAG GGA ACC CAG T - X - 3' 9 99.3

5" -XXX -GTA GAT CAC T -3' 10 95-99 % 98.8

5'- GTA GAT CAC TXX A GT GAT CTA C -3' 10 97-99 % 99.2 a) Coupling effidency for reagent 10 (determined from the DMT-cation release). b) Average coupling efficiency for the sequence indicated, including X.

a) Average coupling efficiency for the sequence indicated, including F.

Claims

1. A Unker reagent of the formula I

wherein n is an integer from 1 to 3; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionaUy interrupted or terminated by 1-5 heteroatom(s); R¹ is a group selected from phosphoramidite groups and H-phosphonate; R² is selected from amino protecting groups and reporter groups; and R³ is selected from hydrogen and hydroxy protecting groups.

2. A Unker reagent according to claim 1, wherein R¹ is a phosphoramidite group.

3. A Unker reagent according to claim 2, wherein R¹ is -P(OCH₂CH₂CN)NPr¹ ₂ (Ν,N-dusopropyl- 0-(2-cyanoethyl)phosphoramidite).

4. A Unker reagent according to any of the claims 1-3, wherein R² is an amino protecting group selected from benzyloxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, aUyloxycarbonyl, trifluoroaceryl, and phthaloyl.

5. A Unker reagent according to claim 4, wherein R² is selected from 9-fluorenylmethoxycarbonyl and trifluoroacetyl.

6. A Unker reagent according to any of the claims 1-3, wherein R² is a reporter group selected from fluorescein, dansyl, biotin, digoxigenin, DOXYL (Ν-oxyl-4,4-dimethyloxazoUdine), PROXYL (N-oxyl-2,2,5,5-tetramethylpyrroUdine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine, dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-l-diazole (NBD), and pyrene.

7. A Unker reagent according to claim 6, wherein R² is selected from fluorescein, dansyl, and biotin.

8. A Unker reagent according to any of the claims 1-7, wherein R³ is a hydroxy protecting group selected from 4,4'-dimethoxytrityl, 9-phenylxanthen-9-yl, and 9-fluorenylmethoxycarbonyl.

9. A Unker reagent according to claim 1, which has the formula Y

10. An immobilized Unker reagent of the formula II immobilized to a solid support material

wherein n is an integer from 1 to 3; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionaUy interrupted terminated by 1-5 heteroatom(s); X is selected from a single bond, nucleotides, and oUgonucleotides; FS is a functional spacer; SSM is the soUd support material to which the Unker reagent is immobilized; R² is selected from amino protecting groups and reporter groups; and R is selected from hydrogen and hydroxy protecting groups.

11. An immobilized Unker reagent according to claim 10, wherein R² is an amino protecting group selected from benzyloxycarbonyl, tert-butoxycarbonyl, 9-fluorenylmethoxycarbonyl, aUyloxycarbonyl, trifluoroacetyl, and phthaloyl.

12. An immobilized Unker reagent according to claim 11, wherein R² is an amino protecting group selected from 9-fluorenylmethoxycarbonyl and trifluoroacetyl.

13. An immobilized Unker reagent according to any of the claims 10-12, wherein R² is a reporter group selected from fluorescein, dansyl, biotin, digoxigenin, proxyl, dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-l-diazole (NBD), and pyrene.

14. An immobilized linker reagent according to claim 13, wherein R² is selected from fluorescein, dansyl, and biotin.

15. An immobilized Unker reagent according to any of the claims 10-14, wherein R is a hydroxy protecting group selected from 4,4'-dimethoxytrityl, 9-phenylxanthen-9-yl, and 9-fluorenyl- methoxycarbonyl.

16. An immobiUzed Unker reagent according to any of the claims 10-14, wherein R³ is hydrogen.

17. An immobiUzed Unker reagent according to any of the claims 10-16, wherein FS is a succinyl moiety and SSM is a ControUed Pore Glass (CPG).

18. A method for labeUing of oUgonucleotides, wherein an immobiUzed oUgonucleotide fragment is reacted with a Unker reagent according to any of the claims 1-9.

19. A method for labeUing of oUgonucleotides, wherein an immobiUzed Unker reagent according to any of claims 10-17 is reacted with a phosphoramidite functionalized nucleotide or a phosphoramidite functionaUzed oligonucleotide.

20. A modified oUgonucleotide comprising at least one fragment of the formula HI

wherein n is an integer from 1 to 3; Z designates a single bond or a chain of 1 to 10 carbon atoms, optionaUy interrupted terminated by 1-5 heteroatom(s); each Y independently is selected from hydrogen, hydroxy protecting groups, nucleotide units, and further Unker fragments; and R is selected from amino protecting groups and reporter groups; with the proviso that at least one Y is a nucleotide unit.

21. A modified oligonucleotide according to claim 20, wherein R² is a reporter group.

22. The use of a Unker reagent as claimed in any of the claims 1-17 for the labeUing of an oUgonucleotide.

23. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides for use as hybridisation probes.

24. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides for the capture of nucleic acids onto solid support matrices resulting from soUd phase or solution phase hybridisation reactions.

25. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides as a primer in the polymerase chain reaction.

26. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides as a primer in nucleic acid sequencing reactions.

27. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in the production of affinity matrices for the purification of DNA binding proteins and other biomolecules.

28. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in the production of affinity matrices for the detection of nucleic acid sequences.

29. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides as a means of monitoring incorporation reactions.

30. The use of a linker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in the production of a random selection of labelled probes for the detection of the total nucleic acid content of samples by hybridisation.

31. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in a sandwich hybridisation system where one labeUed probe acts as a capture and a probe with an alternative label acts as a reporter.

32. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides for providing a biotinylated or haptenylated oUgonucleotide for use in any DNA manipulation protocol.

33. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in cloning recombinant DNA.

34. The use of a Unker reagent as claimed in any of the claims 1-17 for preparing oUgonucleotides in vitro mutagenesis.