NO170890B - CHAINED OIGONUCLEOTIDES WITH DEFINED SEQUENCE, RELATIONSHIPS FOR USE AS INTERMEDIATE IN THE PREPARATION OF THE OIGONUCLEOTIDES AND PROCEDURES FOR CHEMICAL SYNTHESE SEAVED WITH A SINGLE OLIGONUCLEOTIDE WITH - Google Patents

Description

The present invention relates to single-stranded oligonucleotides consisting mainly of an exactly identical sequence of no more than 200 nucleotides and containing one or more nucleotide units where a linker arm is attached to the base to which one or more detectable markers are attached; since such oligonucleotides can be used in the identification, localization and detection of interesting, complementary nucleic acid sequences in cellular or cell-free systems.

The enzymatic production of labeled double-stranded deoxypolynucleotides has been carried out using previously known techniques which include the incorporation of radioisotopes into double-stranded DNA using the method of "nick" translation according to P. Rigby et al, J. Mol. Biol. 113; 237-251 (1977), or the gap-filling reaction described by G. Bourguignon et al, J. Virol. 20: 290-306

(1976). Specifically, a cut is introduced via DNase and then translated along the DNA chain using a DNA polymerase. During the process of nick translation, DNA polymerase from E. coli (Pol I), in the presence of added deoxynucleoside triphosphates, will condense nucleotides to the 3'-hydroxyl end of a single-stranded nick region in double-stranded DNA. At the same time, the enzyme will remove nucleotides from the 5' end of the incision. If one or more of the added triphosphates

32

is marked, e.g. with a - P-phosphate, the marker will be incorporated into the new chain of Pol I. By following the opening-refilling procedure, ends where an incision has formed after restriction endonuclease cutting can be filled again with Klenow fragments from DNA polymerase Pol I or T4.

Both the nick-translation method and the gap-filling method will yield double-stranded, labeled DNA. The length of the product depends on how much DNase I is added to the reaction. This is usually optimized so that 30% of the marker is incorporated and the chains are 400 to 800 nucleotide units long. The product length is unpredictably heterogeneous within the range of 400 to 800 units. To preserve the enzyme as well as the labeled nucleotide, usually only 1 µg of DNA is labeled

in each reaction vessel.

Double-stranded polynucleotides comprising pyrimidine bases modified by carbon chains at the C-5 position have been prepared enzymatically in a similar manner. This has been done by enzymatic extension of a homopolymer primer template as reported by J. Sagi et al, Biochem. Biophys. Acta., 606; 196-201 (1980), or by nick-translation/gap-replenishment with DNA polymerases using 2<1->deoxyuridine-5<1->triphosphate covalently bound to biotin as reported by P. Langer et al, Pro. Nat. Acad. Pollock. United States 78; 6633-6637 (1981), in that biotin is capable of acting as a recognition site for avidin.

Enzymatic methods described by Rigby et al, Bourguignon et al. and Langer et al., provide products with similar physical characteristics. Such enzymatically produced polynucleotides have a length of 400-800 units, require double-stranded polynucleotides as starting materials,

gives double-stranded polynucleotides in all cases and does not allow labeling in pre-selected sites.

In addition, all enzymatic methods modify both chains in the polynucleotide, and such product chains cannot be isolated from each other. Under such methods, enzymes either replace all of the units with modified units or, when provided with a mixture of modified and naturally occurring nucleoside triphosphates, introduce modified units at random. Furthermore, the enzymatic method described by Langer et al. is unable to produce polynucleotides comprising such detectable markers as fluorescent, luminescent or antigenic detectable markers. None of this type is capable of providing oligonucleotides of defined length, defined sequence or single-chain character, either with or without detectable markers. In the previously known methods, further modified bases with detectable markers attached to them cannot be incorporated

in a polynucleotide in preselected locations.

Double-stranded polynucleotides incorporating adenine bases modified at the C-8 position have also been prepared enzymatically. This has been done by adding 8-aminohexyl-amino-ATP (a ribonucleotide) to DNA fragments, as reported by C. Vincent et al., Nucl. Acids Res. 10: 6787-6796 (1982). However, the method is limited in scope as it only allows 3'-end labeling with triphosphates of adenine ribonucleotides. No modified pyrimidine nucleosides can be incorporated. As with other enzymatic methods, both chains of a double-stranded polynucleotide are further labeled, and no short (< 100 units) oligonucleotides with defined sequence can be produced.

The previously known enzymatic methods referred to above require the chemical synthesis of a substituted nucleoside-5'-triphosphate, and then require the following enzymatic recognition and incorporation of the non-natural substrate into double-stranded DNA where incisions have been made. Such methods are unable to provide polynucleotides of any preselected length or sequence,

and the polymerases and DNases used are expensive. Furthermore, these methods are time-consuming, inefficient, require substantial enzymatic activity and are limited to double-stranded DNA. Only small amounts, i.e. <y>ug, of ill-defined polynucleotides, usually restriction fragments, are produced, and these must be slowly isolated from natural sources. Furthermore, the extent of modifications that can be achieved in the polynucleotide product is severely limited as the DNA polymerase cannot recognize or incorporate potentially useful detectable markers - such as fluorescein or dinitrophenyl.

Attachment of a fluorescent molecule to the 3' end of long polyribonucleotide (RNA) molecules for limited biological application is described by J.G.J. Bauman et al.,

J. Histochem. Cytochem. _2j3: 238 (1981). This method also used very small amounts (^ug quantities) of RNA that were slowly isolated from natural sources using enzymatic methods, and cannot be applied to DNA as both the 2<1-> and 3<1->hydroxyls are required for this . The much greater chemical instability of RNA compared to DNA also makes the scope of application for the polyribonucleotide produced by this method minimal.

The non-enzymatic synthesis of oligonucleotides of defined sequence comprising naturally occurring nucleic acid bases has been reported or disclosed by S.A. Narang et al., Meth. Enzymol. 65: 610 (1980),

R. Letsinger, J. Org. Chem., 45_: 2715 (1980), M. Matteucci

et al., J. Amer. Chem. Soc. 103: 3185 (1982) and G. Alvarado-Urbina et al., Science, 214: 270 (1981). Such syntheses usually involved chain extension by linking an activated nucleotide monomer and a free hydroxyl end unit into a growing nucleotide chain. The connection is effected through a phosphorus-containing group such as in the phosphate-triester method described by Narang et al., or one of the phosphite-triester methods. Among the latter, used in the method according to Letsinger et al. and Alvarado-Urbina et al. phosphochloridite, and in the method according to Matteucci et al., phosphoamidite.

The previously mentioned chemical syntheses of oligonucleotides have only included unmodified or naturally occurring nucleic acid bases, and the end product thereof exists in short fragments and resembles unmodified or naturally occurring RNA or DNA. It is also worth noting that the final product in such syntheses does not include any detectable markers.

Direct modification of homopolymeric polynucleotides has been reported in polyuridylic acid systems [Bigge, et al., J. Carb., Nucleosides, Nucleotides 8:259 (1981)]. The reported method is limited in scope and does not provide useful products. Treatments described cause extensive polynucleotide cleavage and degradation, result in products that are irreversibly contaminated with metal ions and are capable of modifying only cytosine residues in a DNA polynucleotide. The method is unable to provide oligonucleotides of specific length and of defined sequence, cannot modify thymine or purine bases and cannot modify in previously selected sites.

Based on the above, it will be obvious that labeled polynucleotides with defined sequence have so far only been produced using enzymatic methods which have the disadvantages emphasized here, especially those relating to time, cost, product length, sequence and yield. Furthermore, such methods are only effective on double-chained products. Such double-stranded polynucleotides can be denatured with alkali or heat to cause spontaneous short-term separation of chains in solution. However, the individual single chains cannot be physically isolated from each other, and removal of the denaturing conditions results in rapid renaturation to the double-chain form. Since conditions that induce hybridization also induce renaturation, subjecting the denatured polynucleotide to hybridization conditions results in the return of the polynucleotide to its original double-stranded configuration where hybridization of either strand of a target polynucleotide is limited by competition from the other strand .

Modification of nucleosides has been carried out e.g. in the synthesis of antiviral C-5-substituted pyrimidine nucleosides described by Bergstrom et al., J. Amer. Chem. Soc. 98;1587-1589 (1976); Ruth et al., J. Org. Chem., 43:2870-2876 (1978) and Bergstrom et al., US Patent Nos. 4,247,544 and 4,267,171 or in the synthesis of C-8-substituted adenine derivatives described by Zappelli et al., US Patent No. 4 336,188 and 4,199,498. These nucleosides and others reported by Langer et al. and D. Ward, EP Patent Application No. 0063879, cannot be used in the method according to the present invention. Such reported nucleosides are highly reactive at undesired sites and, if used in oligonucleotide synthesis by chemical methods, would result in undesired by-products, uncontrollable synthesis and no desired product. Furthermore, such reported nucleosides do not contain any suitable sites in the substituent group, cannot be modified by the binding of detectable labels, nor do they contain blocked, reactive groups. No such nucleosides can be used in the method according to the present invention.

No chemical synthesis of oligonucleotides with a defined sequence comprising modified bases of any kind, either with or without detectable markers, has been described in the prior art.

There is an urgent need for labeled single-chain oligonucleotides with a defined sequence and of high quality that do not include dangerous and unstable radioisotopes, and satisfying this need is a main purpose of the present invention. This purpose is achieved by means of a chemical, i.e. non-enzymatic, method which provides a predictable, excellent product in high yield. More specifically, the method according to the present invention carries out the chemical incorporation of nucleotides modified with a wide variety of selected, detectable detectable markers into oligonucleotides of defined sequence, such oligonucleotides being useful, e.g. for the identification, localization, isolation and/or quantification of complementary sequences of interest.

Another object of the invention is to provide new nucleosides which are useful in the chemical synthesis of labeled, single-stranded oligonucleotides with defined sequence.

The method according to the present invention achieves the de novo chemical synthesis of labeled oligonucleotides with defined sequence and is superior to the previously known enzymatic methods in a number of respects. More specifically, the method of the present invention enables the synthesis of labeled, single-stranded oligonucleotides of defined sequence and of homogeneous, predictable defined length of no more than 200 nucleotides, as opposed to the preparation of heterogeneous, non-predictable stocks of 400 to 10,000 base units of double-stranded character produced by previously known enzymatic methods.

The product yield produced by the method

according to the present invention, is of the order of several hundred to several tens of thousands <y>ug, in contrast to the yield of a few <y>ug provided by the previously known enzymatic methods. Furthermore, the oligonucleotide products are according to

present invention single-chain, instead of the double-chain products of enzymatic methods. The single-stranded configuration of the product oligonucleotides avoids the competition from a complementary strand that naturally accompanies rehybridization of double-stranded polynucleotides.

The invention comprises a single-stranded oligonucleotide consisting essentially of an exactly identical sequence of no more than 200 nucleotides, which is characterized in that the nucleotides have the formula:

where

B is a pyrimidine or purine base,

R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and

Rt = blocking group, and

R5 = reactive phosphorus-containing group or

= H if the 5'-OH group in the 5'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group or

R5 = blocking group, and

R4 = reactive phosphorus-containing group or

= H if the 3<1->OH group in the 3'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, and

R8 = H or blocked hydroxyl group,

and that at least one of the nucleotides has a linker arm bound to the base in the nucleotide and where the linker arm is further bound to a compound selected from the group consisting of a reactive functional group, a blocked functional group, a detectable marker and a fixed surface.

The invention further comprises a compound for use as an intermediate in the chemical synthesis of a single-chain oligonucleotide, which is characterized by having the formula:

where

B is a pyrimidine or purine base,

R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and

R4 = blocking group, and

R5 = reactive phosphorus-containing group or

= H if the 5'-OH group in the 5'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, or

R5 = blocking group and

R4 = reactive phosphorus-containing group or

= H if the 3'-OH group in the 3'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, and

R8 = H or blocked hydroxyl group.

The invention also includes a method for the chemical synthesis of a single-chain oligonucleotide with a detectable residue which is characterized by the addition of an intermediate compound to a reactive nucleotide or reactive oligonucleotide under reactive conditions, where the intermediate compound has the formula:

where B is a pyrimidine or purine base,

R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and

R4 = blocking group and

R5 = a group containing reactive phosphorus, or = H if the 5' -OH group in the 5' -end nucleotide of a growing oligonucleotide contains a group containing reactive phosphorus, or R5 = is a blocking group and

R4 = a group containing reactive phosphorus, or H if the 3' -OH group in the 3' end nucleotide of a growing oligonucleotide contains a group containing reactive phosphorus, and

R8 = H or blocked hydroxyl group, and

where the reactive conditions comprise a temperature in the range from -20 to 50°C for a period of time in the range of 1 to 60 minutes,

the blocking group is removed and then a detectable marker is added which can react with the unblocked linker arm under reactive conditions, the reactive conditions comprising a temperature in the range of -20 up to 50°C for a period of time in the range of 1 to 24 hours.

The invention also includes a method for the chemical synthesis of a single-chain oligonucleotide with a detectable residue which is characterized by the fact that an intermediate compound is added to a reactive nucleotide or reactive oligonucleotide under reactive conditions, where the intermediate compound has the formula:

where B is a pyrimidine or purine base,

R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and

R4 = blocking group and

R5 = containing reactive phosphorus, or

H if the 5' -OH group in the 5' end nucleotide

in a growing oligonucleotide contains a group containing reactive phosphorus, or R5 = blocking group and

R4 = a group containing reactive phosphorus, or = H if the 3' -OH group in the 3' end nucleotide of a growing oligonucleotide contains a group containing a reactive phosphorus, and R8 = H or blocked hydroxyl group,

wherein the detectable marker does not prevent oligonucleotide synthesis, and

where the reactive conditions comprise a temperature in the range -20 up to 50 °C for a period of time in the range 1-60 minutes.

The link arms R which are bound to the bases B and

which are capable of binding detectable labels can generally be characterized as those which exhibit nucleophilic properties towards such labels. Examples of such linker arms are those containing primary and aromatic amines, carboxylic acids, hydroxyl groups and the like.

The bases in the nucleotide monomer and end unit are selected

to provide the predetermined sequence of nucleotide

units that are desired in the final product oligonucleotide. Such bases can be made up of the purines adenine (A), guanine (G) or hypoxanthine (H), or of the pyrimidines uracil (U), cyto-

sin (C) or thymine (T).

A sterically tolerant site on a nucleotide unit can be defined as a position on the nucleic acid base of the unit where modification of the unit can be effected by binding of the linker arm without causing any significant interaction with hybridization of the product oligonucleotide and complementary nucleic acid constituents, and without sterically hindering the linker arm in binding one or more detectable markers. Sterically tolerant sites are found at the C-8 position in purines and at the C-5 position in pyrimidines. As the oligonucleotides according to the present invention are particularly useful as hybridization probes, modifications that bind substituents and/or detectable markers should not be at sites on the pyrimidine or purine bases that are necessary for specific hybridization. Sites that should not be modified include N"*" and N in adenine bases, N^, N<2> and 0^ in guanine bases,

N 3 and N 4 in cytosine bases. In general, substitution should by

all heteroatoms (N or 0) are avoided.

A detectable marker can be defined as a chemical group which may be aromatic and/or polycyclic and which has a physical or chemical characteristic which can be easily measured or detected by suitable physical or chemical detection systems or methods. Detectable markers that can be used in oligonucleotides according to the present invention are easily detectable. Easy detectability can be provided by such characteristics as color change, luminescence, fluorescence or radioactivity, or it can be provided by the ability of the label to serve as a ligand recognition site. Such groups are functionally referred to as coiorimetric, luminescent, fluorescent, radioactive or ligand recognition groups. Among such groups are those suitable for rapid detection using commonly known detection techniques, such as colorimetric, spectrophotometric, fluorometric or radioactive detection, as well as those capable of participating in the formation of specific ligand-ligand complexes containing groups that are detectable using such commonly known detection methods.

A detectable marker can be characterized as a marker that has physical, chemical or other characteristics such as light . can be measured or detected using appropriate measurement or detection procedures. A detectable marker includes,

as defined herein, also ligand recognition groups capable of initiating one or more mutual ligand-ligand interactions that ultimately yield a reaction product or complex with physical, chemical, or other characteristics that can be readily measured or detected using appropriate measurement or detection procedures.

Examples of such measurable or detectable characteristics that such groups, reaction products or complexes can exhibit or induce are a color change, luminescence, fluorescence or radioactivity. Such characteristics can be measured or detected using commonly known colorimetric, spectrophotometric, fluorometric or radioactivity-sensing instrumentation.

The interactions usefully initiated by the detectable label defined herein include suitably specific and selective ligand-ligand interactions caused by groups or complexes that are readily detectable, e.g. by colorimetric, spectrophotonimetric, fluorometric or radioactive detection methods. Such mutual influences can be constituted by reactions between protein and ligand, enzyme and substrate, antibody and antigen, carbohydrate and lectin, protein and cofactor, protein and effector, nucleic acid and nucleic acid or nucleic acid and ligand. Examples of such mutual influences between ligand and ligand are the reaction between dinitrophenyl and dinitrophenyl antibody, biotin and avidin, oligonucleotide and complementary oligonucleotide, DNA and DNA, RNA and DNA and NADH and dehydrogenase. Either one or the other in each such ligand-ligand pair can serve as a detectable marker of ligand recognition type.

In the method according to the present invention, a selected, detectable marker or markers can optionally be bound to the nucleotide monomer before connecting the monomer and the terminal unit in the nucleotide chain, or it can be bound to the product oligonucleotide after its formation. The sequence of nucleotide units in the product oligonucleotide is selected in advance to provide such an oligonucleotide with precise specificity for its final application. The oligonucleotides according to the present invention are useful tools in recombinant DNA and other methods that include nucleic acid re-hybridization techniques. Among such applications are the identification, localization, isolation and/or quantification of complementary sequences of interest in cellular or cell-free systems. More specifically, such applications may include diagnostic applications or any fundamental biological event involving hybridization of nucleic acid constituents, or purification of complementary sequences by affinity chromatography when the product oligonucleotide is bound to a solid support through modifications of a sterically tolerant site, with or without subsequent detection.

The nucleotide units in the product oligonucleotide may be purine- or pyrimidine-based and may comprise units with naturally occurring bases mixed together with units with modified bases. Such units can be ribonucleotides or deoxyribonucleotides. The coupling step preferably comprises coupling of a monomer unit activated in the 3' position with a free 5'-hydroxyl group in the terminal unit of the growing nucleotide chain. Optionally, such coupling may comprise coupling of a monomer unit activated in the 5<1->position with a free 3'-hydroxyl group in the end unit of the nucleotide chain. The terminal unit may be the first or only unit in the growing nucleotide chain at the time of attachment of the nucleotide monomer, or it may be the terminal of a series of nucleotide units.

The method according to the present invention produces oligonucleotides with a defined sequence and with the following general formula:

where n is 1 to 199, preferably 5 to 60, and most preferably 10 to 40, R' is hydrogen or hydroxy and B is any of the naturally occurring purine or pyrimidine bases adenine, guanine, cytosine, uracil or thymine, the nucleotide units with naturally occurring bases being independently mixed together with one or more nucleotide units with modified bases (B<m>). The modified pyrimidine bases (Py<m>) are substituted in the C-5 position, and typical examples of these are the uracil and cytosine bases illustrated with the following general formulas:

The modified purine bases (Pu<m>) are substituted in the C-8 position, and typical examples of these are the modified adenine and guanine bases illustrated by the following general formulas:

The link arm R is characterized by its ability

to bind one or more detectable markers. In the modified pyrimidine bases the linker arm R comprises two or more carbon atoms, while in the modified purine bases R comprises one or more carbon atoms. In this context, R preferably takes the form of one of the following functionalized carbon chains:

where R-j^ is hydrogen or alkyl, R2 is alkyl, alkenyl, aryl or functionalized alkyl, alkenyl, aryl where functional groups comprise one or more amines, amides, nitriles, carboxylic acids or esters, hydroxy, dinitrophenyl, amino-

benzene sulphonates or the like, and Z is a polyvalent heteroatom such as nitrogen, oxygen or sulphur. In addition, R2 can be bound to a fixed carrier or to one or more detectable markers that act e.g. as a colorimetric, fluorescent, luminescent, radioactive or ligand recognition group. Functionally fluorescent groups include fluoresceins, rhodamines and the like or adducts thereof, functionally luminescent groups include luminols, acridines, luciferins, dioxetanes, dioxamides and the like or adducts thereof. Ligand recognition groups include vitamins (such as biotin or adducts thereof, including iminobiotin and desthiobiotin), antigens such as dinitrophenols, carbohydrates and other functional groups or adducts of such groups which can be recognized by ligand-like reactions with proteins, or from which such ligand-like reactions can be triggered. Another oligonucleotide capable of reacting with nucleic acids is illustrative of a group from which a ligand-like reaction can be triggered. Ligand recognition moieties can also serve as functional colorimetrically detectable markers when recognition results in color formation. E.g. when dinitrophenyl is used as a detectable marker, known detection systems using an anti-dinitrophenyl antibody coupled to peroxidase as a detection system are used, resulting in a color change. Functional radioactive groups comprise a radioactive element in the selected, detectable marker.

When reference is made here to the use of purine or pyrimidine bases, such expressions are intended to include analogues of such bases. Among such analogs are the analogs of purine bases, such as deazaadenosines (tubercidins, formycins, and the like), and the analogs of pyrimidine bases, such as deaza-uracil, deazacytosine, azauracils, azacytosines, and the like.

Oligonucleotides of formula I are best prepared by chemical synthesis from analogous units of monomeric nucleotide of the formula:

where R 3 is trityl (triphenylmethyl), dimethoxytrityl or another suitable blocking group for 5'-hydroxyl,

B and R' are, if necessary, blocked, and (?) is a phosphorous group suitable for internucleotide bond formation during chain extension in synthesis of a product oligonucleotide. The phosphorus-containing groups (<p>) suitable for internucleotide bond formation are preferably alkylphosphomonochloridites or alkylphosphomonoamidites. Optionally, phosphate triesters can be used for this purpose. The monomer unit may optionally have R 1 bound to the 3'-hydroxyl group and (<p>) bound to the 5'-hydroxyl group.

In general, the term "blocking group" is a functional term that refers to the chemical modification or "blocking" of an essential functional group by the attachment of a second residue to hide the chemical reactivity of the functional group and prevent it from reacting to an undesired manner. Such modification is reversible and then allows subsequent conversion back to the original functional group by means of suitable treatment. In many cases, such blocking formally transforms groups mutually, e.g. becomes a primary amine that is blocked by acetylation, a substituted amide that can later be converted back to the primary amine by appropriate hydrolysis.

The compounds of formula I include the acceptable conjugate acid salts thereof. Conjugate acids that can be used to prepare such salts are those that contain non-reactive cations, and include e.g. nitrogenous bases such as ammonium salts, mono-, di-, tri- or tetra-substituted amine salts, and the like, or suitable metal salts such as those of sodium, potassium and the like.

The procedural steps according to the present invention will now be described in general and illustrated by means of diagrams. Next, the invention will be illustrated in more detail, and detailed examples will be given. As the invention relates to oligonucleotides comprising both pyrimidine-based and purine-based nucleotide units, the use of both pyrimidine- and purine-based compounds in the synthesis method will be illustrated. The particular pyrimidine- and purine-based compounds illustrated are only examples of the respective pyrimidine- and purine-based classes, and it should be understood that any other member of the respective classes may be substituted for those mentioned in the method and product oligonucleotide when appropriate or desired. While for the most part deoxyribonucleotide compounds have been shown, it should be understood that ribonucleotide compounds are also covered by the invention and can replace the deoxyribonucleotide compounds when ribonucleotide compounds are desired in the product oligonucleotide.

One of the most important aspects of the invention is the provision of a new class of nucleosides which are essential as intermediate products in the process for synthesizing the new oligonucleotides. Such nucleosides each have a base that is modified by a substituent group comprising a functionalized carbon chain and one or more amides, the nitrogen atom in the amides being bound to a sterically tolerant site on the base through the carbon chain. In the case of pyrimidine-based nucleosides, the carbon chain is attached to the C-5 position, and in the case of the purine-based nucleosides, the carbon chain is attached to the C-8 position through a polyvalent heteroatom, such as nitrogen, oxygen or sulfur. In addition, such nucleosides are chemically blocked at the 5<1->position (or 3'-position) with a group, such as dimethoxytrityl, which is useful for the chemical synthesis of oligonucleotides.

In the new class of nucleosides, the linker arm can be selected from -CH„CHR.1C H„ Y, -CH=CR, C H0 Y, -CHR,C H, Y,

2 1 n 2n 1 n 2n 1 n 2n '

and where R 1 is hydrogen or C 1-6 alkyl, n is 0 - 20 and Y contains a blocked amino or blocked carboxyl or blocked hydroxy or blocked thio group, or a detectable label or a solid support. More specifically, Y may comprise one or more where X is hydrogen, fluorine or chlorine. Synthesis of these nucleosides, as well as the blocked forms thereof, is described below in Examples 1, 2, 4, 6, 7, 8, 10, 12 and 13. Preferred nucleosides include substituents at C-5 of pyrimidine nucleosides where n = 3 to 12 and Y is

Most preferred are such

nucleosides where the pyrimidine base is uracil.

The method according to the present invention can start with the preparation of the selected nucleoside. Generally, the most preferred nucleosides are best prepared in the following manner. 5-(methyl-3-acrylyl)-2<1->deoxyuridine is prepared from 2'-deoxyuridine by the method according to Bergstrom and Ruth [J. Amer. Chem. Soc., 9j>:1587 (1976)]. The nucleoside is then treated with 1.05 equivalents of dimethoxytrityl chloride in pyridine for 4 hours to block the 5'-hydroxyl with dimethoxytrityl (DMT). The product obtained is purified by means of chromatography on silicon dioxide gel, where it is eluted with a gradient of 0-10% methanol in chloroform containing 2% triethylamine. The purified 5<1->DMT-5-(methyl-3-acrylyl)-2<1->deoxyuridine is treated with IN KOH for 24 hours at ambient temperature to hydrolyze the methyl ester. The resulting 5'-DMT-5-(3-acrylyl)-2'-deoxyuridine is treated with excess dicyclohexylcarbodiimide and hydroxybenztriazole in pyridine. After 4 hours, a 2-5 times excess of 1,7-diaminoheptane is added, and the reaction mixture is stirred overnight. After 12-20 hours, a 10-20 times excess of trifluoroacetic anhydride is added, and the reaction mixture is stirred at room temperature for 4 hours. The product is purified by means of chromatography on silicon dioxide gel where it is eluted with a gradient of 0-10% methanol in chloroform containing 2% triethylamine, followed by exclusion chromatography using Sephadex^LH-20 and elution with 1% triethylamine in methanol. Appropriate fractions are combined to give 5<1->DMT-5-[N-(7-trifluoroacetylamino-heptyl)-1-acrylamido]-2'-deoxyuridine; such a product is suitable for oligonucleotide synthesis by the phosphochloridite method described in examples 15 and 18. Optionally, such a compound can be prepared by the combination of methods described in examples 2 and 3. Replacement of diaminoheptane in this method with other diaminoalkanes (e.g. diaminopropane , diaminohexane/diaminododecane) give other compounds with varying substituent length where

n = 3, 6 or 12, and

Two such

nucleosides/ one pyrimidine (uracil)-based and the other purine (adenine)-based, are shown at the top of the diagram below which illustrates the procedure. Reactive sites on the bases of the nucleosides are then blocked as shown in reaction 1 by binding e.g. a benzoyl group (Bz) to the amine in the 6-position of the adenine-based nucleoside. Such blocking is generally described in "Synthetic Procedures in Nucleic Acid Chemistry", vol. 1, W. Zorbach and R. Tipson, Eds., (Wiley - Interscience, N.Y.) (1968). Unprotected amines on the substituent group are blocked, e.g. by binding trifluoroacetyl groups (Ac) to these, as also shown in reaction 1.

The selected 3'- or 5'-hydroxyl group in the nucleoside is then blocked by binding a dimethoxytrityl (DMT) group to it. In reaction 2 illustrated below, the 5'-hydroxyl group is blocked, while the 3'-hydroxyl group remains free or available for reaction. Alternatively, the 3'-hydroxyl group could be blocked, while the 5<1->hydroxyl group remained free.

The nucleoside is then converted to an activated nucleotide monomer, preferably by attaching a phosphorus-containing group comprising an activating residue to its 3'-hydroxyl group.

When the modified nucleoside is appropriately blocked, modifications of the methods described by Letsinger et al., Matteucci et al., or described by Narang et al., can be used for oligonucleotide synthesis. The use of phosphochloridite chemistry as described by Letsinger et al. is discussed in more detail in Examples 16 to 18. To use phosphoamidite chemistry, a modification of the method according to Matteucci et al. is used, where the protected, modified nucleoside is phosphitylated with methyl chlorine -(N,N-diisopropyl)-phosphoamidite or methylchlorophosphomorpholidite, as in the improved method of Dorper et al., [Nucleic Acids Res. 11:2575 (1983)]. Optionally, the protected, modified nucleoside can be phosphorylated with 1.2 equivalents of chlorophenyldichlorophosphate in trimethylphosphate at room temperature followed by quenching with water, thereby obtaining the 3'-chlorophenylphosphate adduct of the modified nucleoside, such adducts can be used in a modification of the phosphotriester method as follows the illustrative is explained by Narang et al. The diagram illustrates in reaction 3 the synthesis of activated monomer nucleotide units of formula II by binding a phosphomonochloridite group to the 3'-hydroxyl group in the nucleoside, where the chlorine atom acts as an activating residue.

Coupling or condensation of the selected activated nucleotide monomer, i.e. the uracil-based monomer or the adenine-based monomer, to the terminal unit of a growing nucleotide chain is illustrated in reaction 4 in the diagram. The nucleotide chain is shown at its right end with a nucleoside unit that has a naturally occurring base and with a fixed support or blocking group R^ attached to the 3'-hydroxyl group. The illustrated chain also comprises one or more (n<1>) nucleotide units with naturally occurring bases, the units being bound to the 5'-hydroxyl group in the nucleoside unit, and the terminal nucleotide unit having a free hydroxyl group in the 5<1->position . In the coupling reaction, the chlorine atom in the monomer reacts with the hydrogen atom in the free hydroxyl group in the end unit and is replaced so that the oxygen atom in the end unit is connected to the phosphorus atom in the monomer as shown, and the monomer thereby becomes the new end unit in the nucleotide chain.

The DMT 5<1>-blocking group is then removed to allow further extension of the nucleotide chain by sequentially attaching additional activated nucleotide monomer units. The nucleotide units added to the chain can be selected in advance and can have either naturally occurring or modified bases. The diagram shows in reaction 4a the further extension of the chain by the addition of one or more (n") nucleotide units with naturally occurring bases.

When an oligonucleotide of the selected length and sequence has been synthesized, the DMT group is removed from the terminal unit, and the blocked, reactive groups are unblocked. Examples of modified uracil and adenine bases with their reactive groups unblocked are also shown schematically in reaction 5. If the first nucleotide unit in the chain is bound to a solid support, the chain is then removed from the solid support. The appropriate unblocking order can be determined in advance.

Detectable markers Rj. which are appropriate for the intended use of the product oligonucleotide, can then be attached to such linker arms as exemplified in reaction 6 which illustrates the respective bases with detectable markers R 2 attached to the respective linker arms.

Illustration of synthesis procedure

where e.g. The Bm of the product oligonucleotide (page 23) is:

A variety of useful detectable labels (R₂) can be attached to the product oligonucleotide. For example:

PRODUCT OLIGONUCLEOTIDES WITH DETECTABLE MARKERS (where

Rc = biotin or fluorescein)

After the method according to the present invention has been described above in general terms and illustrated schematically, each of the reactions referred to will now be described in more detail.

As regards reaction 1, blocking of chemically 4 6 2 reactive amines such as N in cytosine, N in adenine, N in guanine and alkyl or aryl amines in the modified bases can be carried out in a commonly known manner with suitable blocking groups in suitable solvents such as alcohols, pyridines, lutidines, chloroform and the like, by reacting the nucleosides with an excess of suitable acid anhydrides for approx. 1 to 24 hours at temperatures ranging from 0°C to 110°C, typically 20°C to 80°C. Suitable acid anhydrides include acetic anhydride, trifluoroacetic anhydride, benzoyl anhydride, anisoyl anhydride and the like. Acetyl, trifluoroacetyl, benzoyl and isobutyryl anhydride are preferred.

Blocking of the 5<1->hydroxy group in reaction 2 can be carried out in a commonly known manner by reacting the nucleosides with a small excess of appropriate acid-labile blocking reagents such as trityl chlorides, monomethoxytrityl chloride, dimethoxytrityl chloride, (DMTC1), trimethoxytrityl chloride and the like. Dimethoxytrityl chloride is preferred. The reactions are usually carried out in suitable solvents, such as pyridine, lutidines, trialkylamines and the like, at temperatures in the range from -20°C to 120°C, usually 20°C to 100°C, for approx. 1 to 48 hours. The preferred reaction uses 1.1 equivalents of DMTC1 in pyridine at room temperature for 2 hours.

It is generally preferred that the respective products from each reaction described above are separated and/or isolated before use as a starting material for a subsequent reaction. Separation and isolation can be carried out using any suitable purification method such as e.g. evaporation, filtration, crystallization, column chromatography, thin layer chromatography, etc. Specific illustrations of typical separation and isolation procedures may be obtained by reference to the appropriate examples described below; however, of course, other equivalent separation methods can also be used. It should also be understood that, when typical reaction conditions (eg, temperatures, molar ratios, reaction times) are given, conditions both above and below the usual ranges may also be used,

although it is generally less convenient.

Activation of the phosphite analog illustrated in reaction 3 can most conveniently be carried out by treating the nucleoside compounds with suitable phosphitylating agents in suitable solvents at temperatures in the range from -90°C to 60°C for 1 minute to 2 hours. Suitable phosphitylating agents include methylphosphodichloridite, o-chlorophenylphosphodichloridite, p-chlorophenylphosphodichloridite, methylphospho-(dialkylamino)-monochloridite, and the like. Suitable solvents include pyridine, lutidines, acetonitrile, tetrahydrofuran, dioxane, chloroform and the like containing 0-20% of appropriate base (usually 1-5 vol%) such as lutidines, collidines, trialkylamines and the like. Preferred phosphitylating agents are methylphosphodichloridite, o-chlorophenylphosphodichloridite and methylphospho-(diisopropylamino)-monochloridite. Preferred phosphitylation conditions are with 0.9 equivalents of methylphosphodichloridite in pyridine or acetonitrile containing 5% 2,6-lutidine for 5 to 10 minutes at room temperature or lower.

The chemical incorporation of the modified nucleotide analog monomers into a growing nucleotide chain to produce nucleotides of defined sequence is illustrated in reactions 4 and 4a. Common condensations are in suitable solvents at temperatures in the range from -20°C to 50°C, preferably at ambient temperature, for approx. 1 to 60 minutes. Suitable solvent mixtures include pyridine, lutidines, acetonitrile, tetrahydrofuran, dioxane, chloroform and the like containing 0-20% of appropriate base (usually 1 to 5% by volume) such as lutidines, collidines, trialkylamines and the like. The growing chain may be soluble, insoluble or bound to a suitable solid support by suitable chemical methods known in the art. Bonding to a solid carrier is preferred. Furthermore, the growing chain may or may not have previously been incorporated with one or more modified nucleoside analogues.

After condensation of the activated monomer to the growing chain in reaction 4, the starting product is treated with suitable reagents to achieve oxidation of the intermediate phosphite triester, optionally capping to block unreacted 5<1->hydroxyl groups on the oligonucleotide chain, and removal of the 5'-DMT group. Oxidation of the phosphite triester can be achieved by treatment with 0.1-5% by weight/volume of iodine in suitable solvents, e.g. tetrahydrofuran/water/lutidine mixtures. Chemical blocking of unreacted 5'-hydroxyl groups can be achieved by acetylation or acylation with e.g. acetic anhydride and 4-dimethylaminopyridine in tetrahydrofuran/lutidine mixtures. Removal of the 5'-blocking group, usually DMT, is most conveniently carried out by treatment with weak organic acids in non-protic solvents, such as weak acids comprising e.g. 1-5 vol% dichloroacetic or trichloroacetic acid in chloroform or dichloromethane. The growing nucleotide chain can, after removal of DMT, now serve as an acceptor for subsequent elongation by means of reaction in sequence with activated monomers to possibly produce the oligonucleotide of the desired length and sequence as shown in reaction 4a.

After an oligonucleotide of the desired sequence has been prepared, reaction 5 is carried out to provide the product oligonucleotide. To this end, thiophenol treatment is used to remove methyl blocking groups from phosphate triesters, and suitable aqueous alkali or ammonia treatment is used to remove benzoyl, acetyl, isobutyl, trifluoroacetyl or other groups from the protected amines and/or to remove the product from the permanent bearer. Removal of DMT from the oligonucleotide product is achieved by the appropriate treatment with a weak acid, such as aqueous acetic acid at ambient temperature to 40°C for 10 to 60 minutes. Such reactions can be carried out before or during final purifications. Final purification is achieved by suitable methods, such as polyacrylamide gel electrophoresis, high pressure liquid chromatography (HPLC), reverse phase or anion exchange on DEAE cellulose, or combinations of these methods.

The method described here for the synthesis of oligonucleotides can use modified deoxyribonucleosides (where R' is H) or modified ribonucleosides (where R' is hydroxyl). When ribonucleosides are used, the 2'-hydroxyl group is blocked by means of an appropriate blocking group such as e.g. that given off by silyl ethers. Other ribose analogues, including arabinose and 3'-deoxyribose, can also be helpful in providing the desired oligonucleotide in the first step.

The linker arm that modifies a nucleotide base must be

capable of binding one or more detectable labels either before or after the chain extension coupling reaction. In the latter case, selected product oligonucleotides are reacted with suitable means to bind such detectable markers. When modified bases are incorporated into the oligonucleotide and R2

in the substituent group contains one or more primary amines, gives e.g. coupling with amine-reactive groups such as isocyanate, isothiocyanate, active carboxylic acid conjugates, epoxides or active aromatic compounds using suitable, mild conditions, amide, uric acid, thio-uric acid, amine or aromatic amine linkages. E.g. As shown in the scheme for reaction 5, an oligonucleotide containing a uracil or adenine base modified by means of a linker arm with a primary amine can be reacted with a suitable reagent, such as fluorescein isothiocyanate or N-hydroxysuccinimidyl-6- biotinylaminocaproic acid, whereby a detectable marker R^ (respectively fluorescein or biotin) is obtained bound to the linker arm as shown in reaction 6. Other detectable markers that can be bound in a similar way include a wide range of organic residues such as fluoresceins, rhodamines, acridine salts, dinitrophenyls, benzenesulfonyls, luminols, luciferins, biotins,

vitamins, carbohydrates and the like. Suitable active, detectable markers are available commercially or can be synthesized e.g. by methods of the type that are generally

written in "Bioluminescence and Chemiluminescence", [M. DeLuca and W. McElroy, Prof., Acad. Press, New York (1981)], by D. Roswell et al., or H. Schroeder et al., [Meth. Enzymol. LXII, (1978)], and the literature references indicated there.

Binding of detectable markers is usually conveniently achieved in predominantly aqueous solvents by reaction of the linker arms in modified bases where R~ = C H~ NH„ with over-øs n zn 2.

shot of the selected, detectable marker at temperatures in the range from approx. -20°C to 50°C (preferably 20°C to 40°C) for 1 to 24 hours. Suitable solvents are an aqueous buffer and 0-50% organic solvents such as lower alcohols, tetrahydrofuran, dimethylformamide, pyridine and the like. Preferred detectable marker reactants include fluorescein isothiocyanates, dinitrophenyl isothiocyanates, fluorodinitrobenzene, N-hydroxysuccinimidyl biotin, N-hydroxysuccinimidyl dinitrobenzoate, isothiocyanates such as aminobutyl ethyl isoluminol isothiocyanate, active esters of carboxyfluorescein, rhodamine, biotin adducts, dioxetanes, dioxamides, carboxy -acridines, carbohydrates and the like.

When the product oligonucleotide comprises modified bases where R contains one or more carboxylic acids, mild condensations with e.g. primary alkylamines amide bonds. Typically, this is conveniently accomplished in predominantly aqueous solvents by reacting the oligonucleotide with an excess of detectable labels containing a primary amine in the presence of suitable condensing agents, such as water-soluble carbodiimides, at temperatures ranging from about -20°C to 50°C (preferably 20°C to 40°C) for 6 to 72 hours. Preferred detectable markers in this class include (aminoalkyl)aminonaphthalene-1,2-dicarboxylic acid hydrazides, aminofluoresceins, aminorhodamines, aminoalkylluminols, aminoalkylaminobenzenesulfonyl adducts, aminosugars, and the like. Furthermore, the chemical synthesis of the starting oligo-nucleotide product can be carried out with modified nucleotide monomers where such detectable markers are attached to the linker arm prior to the coupling reaction. If such a detectable marker adversely affects the coupling reaction, it is appropriately blocked to prevent such an adverse effect. On the other hand, certain other detectable markers are virtually unreactive with respect to the coupling reaction and therefore do not require blocking. E.g. nitrophenyl adducts can be attached to the linker arm prior to the coupling reaction and be present without blocking the modified nucleotide monomer during the coupling reaction without serious effect.

Detectable markers that can be used in the method according to the present invention usually comprise aromatic, polyaromatic, cyclic and polycyclic organic residues which are further functionalized by the incorporation of heteroatoms such as nitrogen, oxygen, sulfur and the like.

Product oligonucleotides may comprise more than one type of modification or more than one modified base. An illustrative example of an oligonucleotide of this type is one with the structural formula:

where Cm is 5-(3-aminopropyl)-cytosine, 5-[N-(4-aminobutyl)-1-acrylamido]-uracil and Am is 8-[6-2,4-dinitrophenyl)-aminohexyl]-aminoadenine . This product is further modified by reaction with fluorescein isothiocyanate, whereby a detectable fluorescein marker is obtained on Cm and U™.

Such a product oligonucleotide illustrates the variation in the selection of modified and unmodified nucleotide units in a product oligonucleotide made possible by the method according to the present invention. More specifically, such an oligonucleotide illustrates the use of more than one type of nucleotide unit where the base is modified with linker arms to which detectable markers are attached which give it the same or other types of detectable marker action. Also shown are units where the bases are modified by means of linker arms to which detectable markers are attached after the coupling reaction, i.e. Cm and U 111, while Am is illustrative of a unit where the base is modified with a linker arm to which it was attached a dinitrophenyl label prior to the coupling reaction. Such an oligonucleotide also shows that it can comprise more than one nucleotide unit of the same type, and that it can comprise units with unmodified bases interspersed with units with modified bases.

Instead of binding detectable markers to the primary amines in the linker arms as shown in reaction 6, such amine groups or other groups can optionally be connected with suitable activated, solid supports. This gives an oligonucleotide with a defined sequence which is covalently bound to such carriers via the modified bases. Such solid carriers can be used for the detection and isolation of complementary nucleic acid constituents. Optionally, the modified nucleoside monomers can be coupled with solid supports before the chain-extending coupling reaction 4 to thereby provide solid supports for such monomers during the coupling reaction.

The following specific examples are provided to enable those skilled in the art to practice the invention. The examples are illustrative and representative of the scope of the invention. To assist with the structural clarification, reference is made to the reactions illustrated in the aforementioned method core.

Example 1

This example illustrates the synthesis of a modified nucleoside precursor 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine.

5-chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml of methanol. N-allyl trifluoroacetamide (6.8 ml,

55 irimol) is added followed by the addition of 41 ml of 0.2N lithium tetrachloropalladate in methanol. After 18 hours of stirring at room temperature, the reaction mixture is gravity filtered to remove the black, solid palladium, and the yellow methanol-containing filtrate is treated with five 200 mg portions of sodium borohydride, then concentrated under reduced pressure to a solid residue. The residue is purified by rapid column chromatography on silicon dioxide gel, eluting with 15 vol% methanol in chloroform. Suitable pure product fractions are mixed and concentrated under reduced pressure, whereby crystalline 5-(3-trifluoroacetylaminopropenyl)-2*-deoxyuridine (2.4 g) is obtained. UV A max, 291 nm (£7800), Amin#

266nm, (£4400); TLC (silicon dioxide, elution with 15 vol% methanol in chloroform) Rf = 0.4.

Example 2

This example illustrates the synthesis of a modified nucleoside precursor 5-[N-(trifluoroacetylaminoheptyl)-1-acrylamido]-2<1->deoxyuridine.

5-chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml of methanol. N-(7-trifluoroacetylaminoheptyl)-acrylamide (55 mmol) is added followed by the addition of 41 ml of 0.2N lithium tetrachloropalladate in methanol. After stirring for 18 hours at room temperature, the reaction mixture is gravity filtered to remove the black, solid palladium, and the yellow methanol-containing filtrate is treated with five 200 mg portions of sodium borohydride, then concentrated under reduced pressure to a solid residue. The residue is purified by rapid column chromatography on silicon dioxide gel, where it is eluted with 10 volume% methanol in chloroform. Suitable pure fractions of the product are mixed and concentrated under reduced pressure, whereby crystalline 5-[N-(7-trifluoroacetylaminoheptyl)-1-acrylamino]-2'-deoxyuridine (2.8 g) is obtained. UV ^ t, 302 nm (£, 18000), A. „ 230 nm, 280 nm;

TLC (silicon dioxide gel, elution with 15 vol% methanol in chloroform) R 1 = 0.3.

Example 3

This example illustrates blocking of 5'-hydroxyl

to prepare 5<1->dimethoxytrityl-5-(3-trifluoroacetylamino-propenyl)-2*-deoxyuridine as illustrated in reaction 2.

5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuridine

(2.4 g) is completely evaporated twice from pyridine, then stirred in 40 ml of pyridine. Dimethoxytrityl (DMT) chloride (2.3 g, 6.6 mmol) is added and the mixture is stirred at room temperature for 4 hours. After thin-layer chromatography (TLC) on silicon dioxide gel eluting with 10 volume% methanol in chloroform indicates that the reaction is complete, the reaction mixture is concentrated to a solid residue. This residue is purified by means of column chromatography on silicon dioxide gel, where it is eluted with chloroform until all impurities that pass quickly through the column have been eluted, then the product is brought out with 5 vol% methanol in chloroform. The residue is then concentrated to give 5<1->dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-21-deoxyuridine as a white, fluffy, solid (4 g). The product decomposes after heating; UV A^^g 291 nm, ^min 266 nm; TLC R^ 0.6 on silicon dioxide with 10 volume% methanol in chloroform as eluent.

Example 4

This example illustrates exocyclic double bond hydrogenation and 51 -hydroxyl blocking to prepare 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine.

By repeating the synthesis of nucleoside precursor and the procedure for blocking the 5'-hydroxyl according to Examples 1

and 3, but subjecting the purified 5-(3-trifluoroacetylamino-propenyl)-2'-deoxyuridine to two atmospheres of hydrogen pressure while stirring at room temperature in methanol over 10% palladium-on-carbon catalyst prior to the addition of the DMT chloride, is obtained the 5<1->dimethoxytrityl-5-(3-trifluoroacetyl-aminopropyl)-2'-deoxyuridine.

Examples 5 to 8 illustrate the synthesis of further modified uracil nucleosides, followed by blocking of 5'-hydroxyl groups as shown in reaction 2.

Example 5

By repeating the nucleoside precursor synthesis and the procedure for blocking 5 1-hydroxyl group according to examples 1 and 3, but by replacing N-allyltrifluoroacetamide with compounds numbered 1 to 8^ below, it is obtained respectively the connections numbered 1' to 8' below, i.e. insertion of

1 N-(3-butenyl)-trichloroacetamide

2 N-(5-hexenyl)-trifluoroacetamide

3_ N-(2-methyl-2-propenyl)-trifluoroacetamide

A_ N-(4-ethenylphenylmethyl)-trifluoroacetamide

5_ N-(1-methyl-3-butenyl)-trifluoroacetamide

6^ N-(12-trichloroaminododecyl)-acrylamide

1_ N-(pertrifluoroacetylpolylysyl)-acrylamide

8 N-(3-trifluoroacetylamidopropyl)-acrylamide gives

1' 5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten-1-yl)-2'-deoxyuridine

2' 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxyuridine

3' 5 * -dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-propen-1-yl)-2<1->deoxyuridine

4_" 5'-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethyl-phenyl)-ethen-1-yl]-2*-deoxyuridine

5<_>' 5' -dimethoxytrityl-5-(4-trifluoroacetylamino-4-methyl-buten-1-yl)-2<1->deoxyuridine

6<*> 5'-dimethoxytrityl-5-[N-(12-trichloroacetylaminododecyl)-1-acrylamino]-2<1->deoxyuridine

2' 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-2*-deoxyuridine

8' 5'-dimethoxytrityl-5-[N-(3-trichloroacetylaminopropyl)-acrylamido]-2'-deoxyuridine

Example 6

By repeating the blocking procedure for the 5'-hydroxyl group according to example 3, but replacing 5-(3-trifluoroacetyl-aminopropenyl)-2'-deoxyuridine with the 5-substituted-2'-deoxyuridines numbered 9_ to 18_ below, the products numbered respectively 9/ to 18</> below, i.e. insertion of

9 5-(propen-1-yl)-2'-deoxyuridine

10 5-(carbmethoxyethyl)-2'-deoxyuridine

11 5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

12 5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

13 5-(3-cyanopropen-1-yl)-2'-deoxyuridine

14 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

15 5-[2-(4-carbmethoxyphenyl)-ethen-1-yl]-2'-deoxyuridine

16 5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

17 5-(4-acetoxybut-1-yl)-2'-deoxyuridine

18 5-[4-(2,4-dinitrophenyl)-butyl]-2'-deoxyuridine gives the following 5'-dimethoxytrityl-5-alkyl-2'-deoxyuridines 9/ 5<1->dimethoxytrityl-5-(propene -1-yl)-2'-deoxyuridine

10' 5'-dimethoxytrityl-5-(2-carbmethoxyethyl)-2'-deoxyuridine

11' 5'-dimethoxytrityl-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

12' 5'-dimethoxytrityl-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

13' 5'-dimethoxytrityl-5-(3-cyanopropen-1-yl)-2'-deoxyuridine

14' 5'-dimethoxytrityl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

15' 5'-dimethoxytrityl-5-[2-(4-carbmethoxyphenyl)-ethen-1-yl]-2'-deoxyuridine

16' 5'-dimethoxytrityl-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

17' 5'-dimethoxytrityl-5-(4-acetoxybut-1-yl)-2'-deoxyuridine

18' 5'-dimethoxytrityl-5-[4-(2,4-dinitrophenyl)-butyl]-2'-deoxyuridine

Example 7

Repeating the nucleoside precursor synthesis and 5'-hydroxyl blocking procedures of Examples 1 to 6, but replacing 5-chloromercuri-2<1->deoxyuridine with 5-chloromercuri-uridine, gives the corresponding 5<1->dimethoxytrityl-5-substituted uridines .

Examples 8 to 11 illustrate the synthesis of modified cytosine nucleosides. As cytosine nucleosides as well as adenosine nucleosides have reactive groups on their bases that differ from the uracil nucleosides, such reactive groups are blocked to prevent unwanted reactions with them. These examples illustrate blocking of reactive groups on the cytosine base residue as in reaction 1, as well as blocking of the 5<1->hydroxyl group as in reaction 2.

Example 8

5-( 3- trifluoroacetylaminopropenyl)- N 4- benzoyl- 2'- deoxycytidine

Repeating the procedure for the nucleoside precursor synthesis according to Example 1, but replacing 5-chloromercury-2<1->deoxyuridine with 5-chloromercury-2<1->deoxycytidine gives 5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine (UV /\ max 287 nm). Purified 5-(3-trifluoroacetylaminopropenyl)-2<1->deoxycytidine (1.3 g, 4.6 mmol) is stirred in 80 mL of anhydrous ethanol, benzoyl anhydride (1.5 g, 7 mmol) is added, and the reaction mixture is refluxed . A further five 1.5 g portions of benzoyl anhydride are added every hour. After the reaction is judged to be complete using

thin-layer chromatography (silica gel plates and elution with n-butanol/methanol/concentrated NH4OH/H20 (60:20:1:20))

for 6 to 10 hours, the reaction mixture is cooled and concentrated under reduced pressure to a semi-solid. The solid is triturated with ether three times, decanted and

be dried. The crude product is crystallized from water whereby chromatographically pure N<4->benzoyl-5-(3-trifluoroacetylamino-propenyl)-2'-deoxycytidine is obtained as a white solid. The product decomposes above 120°C; UV A max 311 nm.

Example 9 51-dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-N 4-benzoyl-2'-deoxycytidine

Repeating the procedure for 5'-hydroxyl masking according to Example 3, but replacing 5-(3-trifluoroacetyl-aminopropenyl)-2 1 -deoxyuridine with 5-(3-trifluoroacetylamino-propenyl)-N 4-benzoyl-21-deoxycytidine gives 5 <1->dimethoxytrityl-5-(3-trifluoroacetylaminopropenyl)-N 4-benzoyl-2'-deoxycytidine.

Example 10

Repeating the nucleoside precursor synthesis and the 5'-hydroxyl blocking procedure of Examples 8 and 9, but replacing N-allyltrifluoroacetamide with the N-alkyltrifluoroacetamides of Example 5, gives the corresponding 5<1->dimethoxytrityl-5-(trifluoroacety1)-aminoalkyl) -N 4-benzoyl-2'-deoxy-cytidines, ie: 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl)-N 4-benzoyl-2'-deoxycytidine

5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-N 4-benzoyl-2'-deoxycytidine

5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methylpropen)-1-yl)-N 4-benzoyl-2'-deoxycytidine

5'-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethylphenyl)-ethen-1-yl]-N 4-benzoyl-2'-deoxycytidine

5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-methylbuten-1-yl)-N 4-benzoyl-21-deoxycytidine

5<1->dimethoxytrityl-5-[N-(12-trifluoroacetylaminododecyl)-1-acrylamido]-N 4-benzoyl-21-deoxycytidine

5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-N 4-benzoyl-21-deoxycytidine

Example 11

Synthesis of 5'-dimethoxytrityl-N 4-benzoyl-5-(2-carbmethoxy-ethenyl)-2'-deoxycytidine

5-(2-carbmethoxyethenyl)-2'-deoxycytidine (0.82 g,

2.6 mmol) is stirred in 50 ml of anhydrous ethanol. Benzoic anhydride (500 mg, 2.2 mmol) is added, and the reaction mixture

heated until reflux. A further five 500 mg portions of benzoic anhydride are added every hour. After the reaction is judged to be complete by thin layer chromatography (typically 6-8 hours), the reaction mixture is cooled and evaporated under reduced pressure to a yellow semi-solid. Chromatography on silica gel eluting with a linear 1:19 to 1:3 methanol/chloroform mixture followed by complete evaporation of appropriately mixed fractions gives N 4-benzoyl-5-(2-carbmethoxyethenyl)-2*-deoxycytidine as an amorphous, white solid. UV Amax 296 nm, A • 270 nm. The solid is thoroughly dried and dissolved in 20 ml of pyridine. Dimethoxytrityl chloride (1.1 equivalent) is added and the reaction mixture is stirred at ambient temperature for 6 hours. Concentration to a solid followed by column chromatography on silica gel eluting with 10% methanol in chloroform gives 5'-dimethoxytrityl-N 4-benzoyl-5-(2-carbmethoxyethenyl)-21-deoxycytidine as a fluffy, yellowish-white solid.

Example 12

Repeating the procedure for nucleoside precursor synthesis according to Example 11, but replacing 5-(2-carb-methoxyethenyl)-2*-deoxycytidine with the following compounds numbered 19 to 27 below, gives the corresponding compounds numbered respectively 19_" to 27* below, i.e. insertion of:

19 5-(2-carbmethoxyethyl)-2'-deoxycytidine

20 5-(3-carbmethoxyprop-1-yl)-2<1->deoxycytidine

21 5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine

22 5-(3-cyanopropen-1-yl)-2<*->deoxycytidine

23 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine

24 5-[2-(4-carbmethoxyphenyl)-ethen-1-yl]-2'-deoxycytidine

25 5-(4-acetoxybuten-1-yl)-2<1->deoxycytidine

26 5-(4-acetoxybut-1-yl)-2<1->deoxycytidine

27 5-[4-(2,4-dinitrophenyl)-butyl]-2'-deoxycytidine gives the following 5'-dimethoxytrityl-N 4-benzoyl-5-alkyl-2'-deoxycytidines: 19' 5'-DMT- N 4-benzoyl-5-(2-carbmethoxyethen-1-yl)-2'-deoxycytidine

20' 5'-DMT-N 4-benzoyl-5-(3-carbmethoxyprop-1-yl)-2*-deoxycytidine

21' 5'-DMT-N<4->benzoyl-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine

22' 5'-DMT-N<4->benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxycytidine

23' 5'-DMT-N<4->benzoyl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine

24' 5'-DMT-N<4->benzoyl-5-[2-(4-carbmethoxyphenyl)-ethen-1-yl]-2'-deoxycytidine

25' 5'-DMT-N 4-benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine

26' 5'-DMT-N<4->benzoyl-5-(4-acetoxybut-1-yl)-2'-deoxycytidine

_27' 5'-DMT-N4-benzoyl-5-[4-(2,4-dinitroenyl)-butyl]-2'-deoxycytidine

Similarly, the use of the other acid anhydrides, e.g. acetic anhydride, anisoyl anhydride or tolyl anhydride, the corresponding N 4 -acyl- or N 4-acetyl-5-alkyl-2'-deoxycytidines according to examples 10 and 11 where benzoyl is replaced by acetyl or acyl.

Example 13

Repeating the procedures for nucleoside precursor synthesis and 5'-hydroxyl blocking according to Examples 8 to 10, but replacing 5-chloromercuri-2'-deoxycytidine with 5-chloromercuricytidine, gives the corresponding 5'-dimethoxytrityl-N 4-benzoyl-5 -substituted cytidines.

Example 14

This is a typical example of the blocking of reactive base residues and the blocking of 5'-hydroxyl in adenine nucleosides.

N -benzoyl-8-(6-aminohexyl)-amino-2'-deoxyadenosine

(4 mmol) is stirred in 60 ml of anhydrous ethanol. Trifluoroacetic anhydride (6 mmol) is added, and the reaction mixture is stirred at room temperature. Two additional portions of trifluoroacetic anhydride are added every hour. After 4 hours, the reaction mixture is concentrated to a solid residue, and it is lyophilized overnight. The crude N -benzoyl-8-(6-trifluoro-acety laminohexyl)-amino-2'-deoxyadenosine is thoroughly dried and concentrated to a solid residue twice from pyridine. The solid is stirred in 40 ml of pyridine, and dimethoxytrityl chloride (6.5 mmol) is added. After 4 hours, the reaction mixture is concentrated, whereby a solid residue is obtained. Purification by column chromatography on silica gel eluting with a multistep gradient of 0 to 15% methanol in chloroform affords 5'-dimethoxytrityl-N-benzoyl-8-(6-trifluoroacetylaminohexyl)-amino-2'-deoxyadenosine as a yellow-white, solid.

Examples 15 to 17 are typical of the activation of 5'-blockerbe 5-substituted and naturally occurring nucleosides, to their respective phosphomonochloridites, as illustrated in reaction 3 of the scheme.

Example 15

Preparation of 3'-phosphomonochloridite from 5*-DMT-5-(3-tri-fluoroacetylaminopropyl-1-yl)-2'-deoxyuridine.

Dry 5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine (1.54 g, 2.2 mmol) is lyophilized from 20 mL of benzene three times over 12 h each to remove residual water and solids. The resulting very fluffy white powder is transferred to a nitrogen atmosphere while still under vacuum and dissolved in anhydrous acetonitrile containing 5% by volume of 2,6-lutidine to a final concentration of the nucleoside of 30 mM. While vigorously swirling under nitrogen, a large lump of methylphosphodichloridite (1.0 equivalent) is quickly added using a syringe. The reaction mixture is swirled for approx. 1 minute under nitrogen. The resulting crude reaction solution of 5<*->DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2<1->deoxyuridine-3'-methylphosphomonochloridite is then used directly for deoxyoligonucleotide synthesis (Example 18) without any further purification , [ 31 P-NMR(CH^CN/CDCl^) usually indicates 40-70 mol% of the desired product (167.5 ppm);

the remainder is composed of bis-3',31-[5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridylyl]-methylphosphite (140 ppm) and 5'-DMT-5-( 3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine-3<1->methylphosphonate (9.5 ppm), the latter product being formed in amounts reflecting the presence of water in the reaction mixture.]

Example 16

Preparation of 3'-phosphomonochloridites from the naturally occurring 2'-deoxynucleosides

Repeating the procedure according to example 15, but replacing 5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-21-deoxyuridine with:

5<1->DMT-2'-deoxythymidine

5'-DMT-N 4-benzoyl-2'-deoxycytidine

5'-DMT-N c -benzoyl-21-deoxyadenosine

5'-DMT-N 2-isobutyryl-2'-deoxyguanosine

gives the corresponding phosphomonochloridites, i.e.: 5<1->DMT-2'-deoxythymidine-3'-methylphosphomonochloridite 5'-DMT-N 4-benzoyl-2'-deoxycytidine-3'-methylphosphomonochloridite

5'-DMT-N-benzoyl-2'-deoxyadenosine-31-methylphosphomonochloridite

5'-DMT-N 2-isobutyryl-2'-deoxyguanosine-3'-methylphosphomonochloridite

Example 17

Repeating the procedures for the phosphomonochloridite synthesis according to Examples 15 and 16, but replacing methylphosphodichloridite with o-chlorophenylphosphodichloridite, gives the corresponding 5'-DMT-nucleoside-3'-phosphomonochloridites, i.e.: 5'-DMT-5-(3- trifluoroacetylaminopropyl)-2'-deoxyuridine-3'-o-chlorophenylphosphomonochloridite 5'-DMT-2 *-deoxythymidine-3'-o-chlorophenylphosphomonochloridite 5'-DMT-N 4-benzoyl-21-deoxycytidine-31-o-chlorophenylphosphono- monochloridite

51-DMT-N -benzoyl-2'-deoxyadenosine-3'-o-chlorophenylphosphono-monochloridite

51-DMT-N 2-isobutyryl-2'-deoxyguanosine-31-o-chlorophenylphosphono-monochloridite

Similarly, the use of p-chlorophenylphosphodichloridite gives the analogous 3<1->p-chlorophenylphosphomonochloridite adducts.

[<32>P]NMR (CH3CNCDC13) of o-chlorophenylphosphomonochloridite products 160.7, 160.5 ppm (diastereomers).

Examples 18 to 24 are typical of the chemical synthesis of oligonucleotides comprising modified bases as illustrated by reactions 4 and 5 in the scheme.

Example 18

Synthesis of deoxyoligonucleotides containing 5-(3-amino-propyl)-uracil and naturally occurring nucleotide units

The phosphomonochloridite synthesis procedures of Examples 15 and 16 are carried out immediately prior to the deoxyoligonucleotide synthesis, and the resulting products are used directly as 30 mM crude 3<1->methylphosphomonochloridites in anhydrous acetonitrile/5 vol% 2,6-lutidine.

Solid carrier (5-DMT-N^-benzoyl-2'-deoxyadenosine-3<1->succinamidepropyl-silicon dioxide, 250 mg, 20y.equiv.) is placed in a suitable vessel for reaction flow (glass or (Po

Teflon^ column or funnel). The solid support is conditioned beforehand by successive treatments with acetonitrile/5% by volume lutidine, 2% w/v iodine in tetrahydrofuran/water/lutidine for 2 minutes, acetonitrile/5% lutidine, chloroform, 4% by volume % dichloroacetic acid in chloroform for 2.5 minutes and acetonitrile/5% lutidine, the treatments being total volumes of 5-15 ml in either 2 or 3 portions or by constant flow, as desired.

The deoxyoligonucleotide is synthesized according to reaction 4 by successive addition of the desired activated 5'-DMT nucleoside-3'-methylphosphomonochloridite monomer and linking this with the free 5'-hydroxyl group in the end unit of the growing nucleotide chain, which unit originally is the only unit in the chain, i.e. the debxy-adenosine-based unit comprising the fixed support. The additions take place by reaction of 10 ml of the crude 30 mM monochloridites selected from examples 15 and 16 with the now unprotected 5<1->hydroxyl group in the chain either in 2 or 3 portions or by constant flow through for 2 to 6 minutes. The first phosphomonochloridite addition followed by a complete reagent cycle consists of consecutive treatments with: 5-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine-3'-methylphosphomonochloridite

acetonitrile/lutidine wash

"capping" for 5 minutes with 0.3M 4-dimethylaminopyridine in acetic anhydride/lutidine/tetrahydrofuran (1:3:2) acetonitrile/5% lutidine wash

oxidation with 2% iodine in tetrahydrofuran/water/lutidine (6:2:1)

for 2 minutes

acetonitrile/5% lutidine wash

chloro form-va sk

removal of DMT by 2.5 minute treatment with 4 vol% dichloroacetic acid in chloroform

chloroform wash

acetonitrile/lutidine wash

The stepwise procedure above is repeated 13 times, each time replacing 5<1->DMT-5-(3-trifluoroacetylamino-propyl)-21-deoxyuridine-31-methylphosphomonochloridite with another of the following 3'-methylphosphomonochloridites: 5' -DMT-2<1->deoxythymidine-3<1->methylphosphomonochloridite 51-DMT-5-(3-trifluoroacetylaminopropyl)-21-deoxyuridine-31 - methylphosphomonochloridite

51-DMT-N g-benzoyl-2'-deoxyadenosine-31-methylphosphomonochloridite

5'-DMT-N 4-benzoyl-2'-deoxycytidine-31-methylphosphomonochloridite

51-DMT-N 2-isobutyryl-21-deoxyguanosine-3'-methylphosphomonochloridite

51-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine-31-methylphosphomonochloridite

5'-DMT-2'-deoxythymidine-3*-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine-3'-methylphosphomonochloridite

5'-DMT-deoxythymidine-3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine-3'-methylphosphomonochloridite

5'-DMT-N~o-isobutyryl-2'-deoxyguanosine-3'-methylphosphomonochloridite

5'-DMT-N-benzoyl-2'-deoxyadenosine-3'-methylphosphomonochloridite

5'-DMT-N 4-benzoyl-2*-deoxycytidine-3'-methylphosphomonochloridite

in respective order, omitting dichloroacetic acid treatment during the last reagent cycle. The support is transferred and treated with 2 ml of concentrated ammonium hydroxide for 4 hours at ambient temperature to release the product from the support. The supernatant is removed, the solid is washed three times with 0.5 ml of concentrated ammonium hydroxide, and the mixed supernatants are sealed and heated at 50°C overnight. The clear, yellow supernatant is thoroughly lyophilized. The first purification is performed by reverse phase high pressure liquid chromatography (HPLC) on an "RP-8 (C-8)" column eluting with a 60 minute linear gradient of 0 to 30% by volume acetonitrile in

25 mM ammonium acetate, pH 6.8. The 5<1->DMT-terminated product eluting as a sharp peak at ca. 40 minutes, collected; all shorter chains, both protected and unprotected, elute before 25 minutes. The collected product is evaporated to a solid residue, treated with 80% acetic acid at ambient temperature for 20 minutes (to remove DMT), lyophilized to a solid residue and dissolved in a small amount of aqueous buffer. The product, which is usually more than 90% homogeneous by HPLC, is further purified by conventional electrophoresis on 20% polyacrylamide gels (1 to 6 mm thick) by excision and ex-

stretching of the relevant product band (the product usually moves more slowly than unmodified deoxyoligonucleotides of equal length). The purified 5-aminopropyl-ureacil-containing pentadecadeoxyoligonucleotide product illustrated schematically below, where = 5-(3-aminopropyl)-uracil, is prepared in this manner.

Note that the usual deprotection of the oligonucleotide with ammonia has also removed the trifluoroacetyl blocking group on the substituent.

The length and sequence of this oligonucleotide can then

32

determined by P-kinase treatment and sequencing using suitable methods, e.g. the methods which have hitherto been used to determine the length and sequence of the previously known oligonucleotides where the bases of the nucleotide units are not modified.

Likewise, deliberate variation in the order and number of methylphosphomonochloridite additions used provides other 5-(modified) uracil-containing deoxyoligonucleotides that vary in chosen length and base order. In addition, replacing the nucleoside 3<1->methylphosphomonochloridite adducts according to examples 15 and 16 with the corresponding 3'-o- or p-chlorophenylphosphomonochloridite adducts according to example 17 and incorporating pyridinium oximate treatment to remove chlorophenyl blocking groups (at the end of the deoxyoligonucleotide synthesis and before treatment with concentrated ammonium hydroxide) the same deoxyoligonucleotide products.

Example 19

Repetition of the procedures for the phosphomonochloridite and deoxyoligonucleotide synthesis according to examples 15 to 18, but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with the 5'-DMT-5-alkyl-2'-deoxyuridines numbered 28_ to 36, they give the corresponding oligonucleotides with uracil bases U111 numbered respectively. 2_8' to 3_6* below, i.e. insertion of: 28 5 *-dimethoxytrityl-5-(3-trifluoroacetylaminopropen-1-yl)-deoxyuridine 29 51-dimethoxytrityl-5-(4-trifluoroacetylaminobut-1-yl)-21 - deoxyuridine 30 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-1-yl)-2'-deoxyuridine 31 5<1->dimethoxytrityl-5-(6-trifluoroacetylaminohex-1-yl)-2<1->deoxyuridine 32 5<1->dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxyuridine 33 5'-dimethoxytrityl-5-(2-trifluoroacetylaminoprop-2-yl)-2<1->deoxyuridine 34 5< 1->dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-propen-l-yl)-2<1->deoxyuridine 35 5<1->dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-prop- 1-yl)-2<1->deoxyuridine 36 5<1->dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethyl-phenyl)-ethen-1-yl]-2'-deoxyuridine 37 5<1->dimethoxytrityl -5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido]-2<1->deoxyuridine 38 5 *-DMT-5-[N-(7-trifluoroacetylaminoheptyl)-1-acrylamido]-2'-deoxyuridine

gives the deoxynucleotides corresponding to the product of Example 18, where Um is:

28' 5-(3-aminopropen-1-yl)-uracil

29' 5-(4-aminobut-1-yl)-uracil

30' 5-(4-aminobuten-1-yl)-uracil

31' 5-(6-aminohex-1-yl)-uracil

32' 5-(6-arainohexen-1-yl)-uracil

33' 5-(3-aminoprop-2-yl)-uracil

34' 5-(3-amino-2-methylpropen-1-yl)-uracil

35' 5-(3-amino-2-methylprop-1-yl)-uracil

36' 5-[2-(4-aminoethylphenyl)-ethen-1-yl]-uracil 37' 5-[N-(polylysyl)-1-acrylaraido]-uracil

38' 5-[N-(7-aminoheptyl)-1-acrylamido]-uracil

By using other 5'-DMT-5-(acylaminoalkyl)-2'-deoxyuridines, the analogous deoxyoligonucleotides are likewise prepared.

Example 20

Repeating the procedures for phosphomonochloridite and deoxyoligonucleotide synthesis of Examples 15 to 18, but replacing 5'-DMT-5-(3-acetylaminopropyl)-2'-deoxyuridine with 5-substituted 2'-deoxyuridines numbered 3_7 to 4_6 below, gives the corresponding oligonucleotides with the Um-ura-cil bases numbered respectively. 37' to 4_6', i.e. insertion of:

37 5'-DMT-5-(propen-1-yl)-2'-deoxyuridine

38 5'-DMT-5-(2-carbmethoxyethyl)-2'-deoxyuridine

39 5'-DMT-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine

40 5'-DMT-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine

41 5'-DMT-5-(3-cyanopropen-1-yl)-2'-deoxyuridine

4 2 5'-DMT-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine

43 5'-DMT-5-[2-(4-carbmethoxyphenyl)-ethen-1-yl)-2<1->deoxyuridine

44 5'-DMT-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine

45 5'-DMT-5-(4-acetoxybut-1-yl)-2'-deoxyuridine

46 5'-DMT-5-[4-(2,4-dinitrophenyl)-butyl]-2'-deoxyuridine gives the products where is:

37' 5-(propen-1-yl)-uracil

38' 5-(2-carboxyethyl)-uracil

39' 5-(3-carboxyprop-1-yl)-uracil

40' 5-(4-carboxy-2-methylbuten-1-yl)-uracil

41' 5-(3-cyanopropen-1-yl)-uracil

42' 5-(4-cyano-2-methylbuten-1-yl)-uracil

43' 5-[2-(4-carboxyphenyl)-ethen-1-yl]-uracil 44' 5-(4-hydroxybuten-1-yl)-uracil

45' 5-(4-hydroxybut-1-yl)-uracil

46' 5-[4-(2,4-dinitrophenyl)-butyl]-uracil

Note: In 4<_>6<*>, dinitrophenyl is a detectable marker of ligand recognition type, i.e. using anti-dinitrophenyl antibody as the ligand. By using other suitable 5'-DMT-5-alkyl-2<1->deoxyuridines, the analogous deoxyoligonucleotides are likewise prepared.

Example 21

Repeating the procedures according to examples 15 to 18, but replacing 5<1->DMT-5-(3-trifluoroacetylaminopropyl)-2<1->deoxyuridine with 5 *-DMT-N 4-benzoyl-5-(3-trichloroacetylamino -propyl)-2<1->deoxycytidine gives deoxyoligonucleotides such as in example 18 where Um [5-(3-aminopropyl)-uracil] is replaced by 5-(3-aminopropyl)-cytosines. E.g.

where Cm = 5-(3-aminopropyl)-cytosine.

Example 22

Repeating the procedure for the deoxyoligonucleotide synthesis according to Example 21, but replacing 5'-DMT-N 4-benzoyl-5-(3-trichloroacetylaminopropyl)-2*-deoxycytidine with compounds numbered 4_7 to 5_7 below, gives the corresponding oligonucleotides with the Cm-cytosine bases numbered or

47' to _57' below, i.e. insertion of:

47 5-DMT-N 4-benzoyl-5-(3-trifluoroacetylaminopropen-1-yl)-2'-deoxycytidine 48 5'-DMT-N 4-benzoyl-5-(4-trifluoroacetylaminobut-1-yl)-2 '-deoxycytidine 49 5'-DMT-N 4-benzoyl-5-(4-trifluoroacetylaminobuten-1-yl)-2<1->deoxycytidine 50 5'-DMT-N 4-benzoyl-5-(6-trifluoroacetylaminohex- 1-yl)-2'-deoxycytidine 51 5'-DMT-N 4-benzoyl-5-(6-trifluoroacetylaminohexen-1-yl)-2'-deoxycytidine 52 5'-DMT-N 4-benzoyl-5-( 3-trifluoroacetylaminoprop-2-yl)-2'-deoxycytidine 53 5'-DMT-N <4->benzoyl-5-(3-trifluoroacetylamino-2-methyl-propen-1-yl)-2'-deoxycytidine 54 5 '-DMT-N 4-benzoyl-5-(3-trifluoroacetylamino-2-methylprop-1-yl)-2'-deoxycytidine 55 5'-DMT-N 4-benzoyl-5-[2-(4-trifluoroacetylaminomethyl- phenyl)-ethen-1-yl]-2'-deoxycytidine 56 5'-DMT-N <4->benzoyl-5-[N-(pertrifluoroacetylpolylysyl)-1-acrylamido)-2'-deoxycytidine 57 5'-DMT -N 4-benzoyl-5-[N-(trifluoroacetylaminoheptyl)-acrylamido]-2'-deoxycytidine

gives the products where Cm is:

47' 5-(3-aminopropen-1-yl)-cytosine

48' 5-(4-aminobut-1-yl)-cytosine

49' 5-(4-aminobuten-1-yl)-cytosine

50' 5-(6-aminohex-1-yl)-cytosine

51' 5-(6-aminohexen-1-yl)-cytosine

52' 5-(3-aminoprop-2-yl)-cytosine

53' 5-(3-amino-2-methylpropen-1-yl)-cytosine

54' 5-(3-amino-2-ethylprop-1-yl)-cytosine

55' 5-[2-(4-aminomethylphenyl)-ethen-1-yl]-cytosine 56' 5-[N-(polylysyl)-1-acrylamido]-cytosine

57' 5-[N-(7-aminoheptyl)-1-acrylamido]-cytosine

By using other N 4-acyl-5-(acylaminoalkyl)-2'-deoxycytidines, the analogous deoxyoligonucleotides are likewise prepared.

Example 23

Repeating the procedure for the deoxyoligonucleotide synthesis according to Example 21, but replacing 5'-DMT-N 4-benzoyl-5-(3-trifluoroacetylaminopropyl)-2<1->deoxycytidine with the compounds numbered 58 to j>8_ below, gives the corresponding oligonucleotides with the Cm<->cytosine bases numbered respectively.

58<1> to 6_8" below, i.e. insertion of:

58 5'-DMT-N 4-benzoyl-5-(propen-1-yl)-21-deoxycytidine

59 5'-DMT-N<4->benzoyl-5-(2-carbmethoxyethyl)-2'-deoxycytidine 60 5'-DMT-N<4->benzoyl-5-(2-carbmethoxyethen-1-yl)- 2'-deoxycytidine 61 5'-DMT-N <4->benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine 62 5'-DMT-N <4->benzoyl-5-(4 -carbmethoxy-2-methylbuten-1-yl)-2'-deoxycytidine 63 5'-DMT-N <4->benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxycytidine 64 5'-DMT -N<4->benzoyl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine 65 5'-DMT-N<4->benzoyl-5-[2-(4-carbmethoxyphenyl) )-ethen-1-yl]-2'-deoxycytidine 66 5'-DMT-N 4-benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine 67 5'-DMT-N 4-benzoyl -5-(4-acetoxybut-1-yl)-21-deoxycytidine 68 5'-DMT-N<4->benzoyl-5-[4-(2,4-dinitrophenyl)-butyl]-deoxycytidine

gives the products where Cm is:

58' 5-(propen-1-yl)-cytosine

59' 5-(2-carboxyethyl)-cytosine

60' 5-(2-carboxyethen-1-yl)-cytosine

61' 5-(3-carboxyprop-1-yl)-cytosine

62' 5-(4-carboxy-2-methylbuten-1-yl)-cytosine 63' 5-(3-cyanopropen-1-yl)-cytosine

64' 5-(4-cyano-2-methylbuten-1-yl)-cytosine

65' 5-[2-(4-carboxyphenyl)-ethen-1-yl]-cytosine 66' 5-(4-hydroxybuten-1-yl)-cytosine

67' 5-(4-hydroxybut-1-yl)-cytosine

68' 5-[4-(2,4-dinitrophenyl)-butyl]-cytosine

By using other suitable 5'-DMT-N4-acyl-5-alkyl-2*-deoxycytidines, the analogous deoxyoligonucleotides are likewise prepared.

Example 24

Repeating the procedures for the phosphomonochloridite and deoxyoligonucleotide synthesis according to examples 15 to 23, but replacing 5<1->DMT-5-(3-trifluoroacetylaminopropyl)-2<1->deoxyuridine with 5'-DMT-N -benzoyl-8- (6-trifluoroacetylaminohexyl)-amino-2'-deoxyadenosine gives deoxyoligonucleotides as in example 18, with the exception that u is replaced by A t and A = 8-(6-aminohexyl)-amino-2'-deoxyadenosine.

Examples 25 to 28 are typical of the binding of detectable labels to oligonucleotides containing appropriately modified bases as illustrated in reaction 6.

Example 25

Fluoresceinated deoxyoligonucleotides

A purified pentadecanucleotide (from Example 28) with the structural formula: [where Um is 5-[N-(7-aminoheptyl)-1-acrylamido]-uracil] dissolves at 25 A2gQ units per ml in aqueous 300 mM sodium borate or sodium carbonate buffer, pH 9.5, containing 30 mM sodium chloride. Solid fluorescein isothiocyanate (0.5 mg per ml) is added, and the mixture is sealed and shaken gently at 4°C to 25°C overnight. The reaction mixture is chromatographed directly on a column of G-50 Sephadex^ to separate unbound fluorescein adducts that are retained; the fluoresceined deoxyoligonucleotide adducts elute just before the amount of liquid runs out. Early fractions containing significant A2gQ units are pooled and lyophilized to a solid product with a structure corresponding to the starting pentadecadeoxyoligonucleotide where Um is now either

or unreacted 5-[N-(7-aminoheptyl)-1-acrylamido]-uracil.

*max. (H20) 262 m' 498 m-

Repeating the procedure on compounds described in examples 19, 21, 22 and 24 gives the corresponding fluoresceinated or polyfluoresceinated deoxyoligonucleotides in a similar manner.

Example 26

Binding of detectable markers other than fluorescein can be carried out by repeating the procedure of Example 25, but replacing fluorescein isothiocyanate with e.g.:

2,4-dinitrophenyl isothiocyanate

1-fluoro-2,4-dinitrobenzene

aminoethyl isoluminol isothiocyanate

aminoethylaminonaphthalene-1,2-carboxylic acid hydrazide isothiocyanate

N,N'-bis-(alkylsulfonyl)-N-aryl-N<1->isothiocyanatoaryl-dioxamide

m-sulfonyl-aniline isothiocyanate

N-hydroxysuccinimidyl-biotin

9-(N-hydroxysuccinimidyl-carboxy)-N-methylacridine

(s)

or cyanobromide-activated Sepharose^

give the corresponding adducts where the group that is attached is different from fluorescein.

Example 27

Binding of detectable markers containing isoluminol and free, primary amine

A purified pentadecanucleotide from example 18 with the structural formula: [where is 5-(2-carboxyethenyl)-uracil] dissolves in water at 30 A2gQ units per ml and dilute with 1 part by volume of pyridine. Aminobutyl-ethyl-isoluminol is added to a final concentration of 1 mg/ml, followed by the addition of a 5-fold molar excess of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The reaction mixture is sealed and shaken gently in the dark from 12 to 48 hours. The reaction mixture is concentrated under reduced pressure to a solid residue and chromatographed directly on a column of G-50 Sephadex^; the isoluminol-deoxyoligonucleotide conjugates elute just before the amount of liquid runs out. Early fractions containing significant amounts of A2gQ_units are pooled and lyophilized to solid product with a similar structure to the starting deoxyoligonucleotide where Um is now either

or unreacted 5-(2-carboxyethenyl)-uracil.

Repeating the procedure on compounds from examples 15 and 23 where R 2 contains carboxyl gives the corresponding deoxyoligonucleotide-isoluminol adducts in a similar manner.

Repeating the procedure but replacing the aminobutyl isoluminol with other detectable labels containing a free primary amine gives the corresponding deoxyoligonucleotide label adducts in a similar fashion.

Example 28

Binding of dinitrophenyl markers

A purified nonanucleotide with the structural formula:

where Am = 8-(6-aminohexyl)-aminoadenine, dissolves with 20 A2gQ units per ml in 250 mM sodium carbonate buffer, pH 9, and 1-fluoro-2,4-dinitrobenzene is added. The reaction mixture is shaken at ambient temperature overnight, then chromatographed directly on a Sephadex®G-50 column. Early fractions containing significant amounts of A2g0 units are pooled and concentrated, yielding an oligonucleotide product corresponding to the starting decanucleotide where Am is now either

or unreacted 8-(6-aminohexyl)-aminoadenine.

Repeating the procedure, but replacing 8-(6-aminohexyl)-aminoadenine with other modified bases containing a free, primary amine, also gives the corresponding dinitrophenylated oligonucleotide adducts.

Claims (10)

1. Single-stranded oligonucleotide consisting mainly of an exactly identical sequence of no more than 200 nucleotides, characterized in that the nucleotides have the formula: where B is a pyrimidine or purine base, R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and R4 = blocking group, and R5 = reactive phosphorus-containing group or = H if the 5'-OH group in the 5'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group or R5 = blocking group, and R4 = reactive phosphorus-containing group or = H if the 3'-OH group in the 3'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, and R8 = H or blocked hydroxyl group, and that at least one of the nucleotides has a linker arm bound to the base in the nucleotide and where the linker arm is further bound to a compound selected from the group consisting of a reactive functional group, a blocked functional group, a detectable marker and a fixed surface. 2. Single-stranded oligonucleotides according to claim 1, characterized in that R is where R-1 is H or alkyl, n is 0-20 and Y contains a blocked amino or blocked carboxyl or blocked hydroxy or blocked thio group, or a detectable label or a solid support. 3. Single-chain oligonucleotides according to claim 1, characterized in that the base is a pyrimidine and the linker arm is attached to the C-5 position. 4. Single-chain oligonucleotides according to claim 1, characterized in that the base is a purine and the linker arm is attached to the C-8 position. 5. Single-stranded oligonucleotides according to claim 1, characterized in that the sequence is between 10 and 40 nucleotides long. 6. Compound for use as an intermediate in the chemical synthesis of a single-chain oligonucleotide, characterized in that it has the formula: where B is a pyrimidine or purine base, R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and R4 = blocking group, and R5 = reactive phosphorus-containing group or = H if the 5'-OH group in the 5'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, or R5 = blocking group and R4 = reactive phosphorus-containing group or = H if the 3'-OH group in the 3'-end nucleotide of a growing oligonucleotide contains a reactive phosphorus-containing group, and R8 = H or blocked hydroxyl group. 7. Connection according to claim 6, characterized in that R is wherein R 1 is H or alkyl, n is 0-20 and Y contains a blocked amino or blocked carboxyl or blocked hydroxy or blocked thio group, or a detectable label or a solid support. 8. Connection according to claim 6, characterized in that R is bound in the C-5 position when B is a pyrimidine, or in the C-8 position when B is a purine. 9. Process for the chemical synthesis of a single-chain oligonucleotide with a detectable residue, characterized in that an intermediate compound is added to a reactive nucleotide or reactive oligonucleotide under reactive conditions, where the intermediate compound has the formula: where B is a pyrimidine or purine base, R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and R4 = blocking group and R5 = a group containing reactive phosphorus, or = H if the 5' -OH group in the 5' -end nucleotide of a growing oligonucleotide contains a group containing reactive phosphorus, or R5 = is a blocking group and R4 = a group containing reactive phosphorus, or H if the 3' -OH group in the 3' -end nucleotide in a growing oligonucleotide contains a group containing reactive phosphorus, and R8 = H or blocked hydroxyl group, and where the reactive conditions comprise a temperature in the range from -20 to 50°C for a period of time in the range of 1 to 60 minutes, the blocking group is removed and then a detectable marker is added which can react with the unblocked linker arm under reactive conditions, the reactive conditions comprising a temperature in the range of -20 up to 50°C for a period of time in the range of 1 to 24 hours. 10. Process for the chemical synthesis of a single-chain oligonucleotide with a detectable residue, characterized in that an intermediate compound is added to a reactive nucleotide or reactive oligonucleotide under reactive conditions, where the raw product compound has the formula: where B is a pyrimidine or purine base, R is a linker arm attached to a compound selected from the group consisting of a blocking group, a detectable label and a fixed surface, and in R4 = blocking group and R5 = containing reactive phosphorus, or = H if the 5' -OH group in the 5' -end nucleotide of a growing oligonucleotide contains a group containing reactive phosphorus, or R5 = blocking group and R4 = a group containing reactive phosphorus, or H if the 3' -OH group in the 3' -end nucleotide in a growing oligonucleotide contains a group containing a reactive phosphorus, and R8 = H or blocked hydroxyl group, wherein the detectable marker does not prevent oligonucleotide synthesis, and where the reactive conditions comprise a temperature in the range of -20 up to 50 °C for a period of time in the range of 3-60 minutes.