WO2008037568A2

WO2008037568A2 - Reversible terminators for efficient sequencing by synthesis

Info

Publication number: WO2008037568A2
Application number: PCT/EP2007/059207
Authority: WO
Inventors: Diana Katerina Knapp; Joachim W. Engels; Angelica Keller; Yangzhou Li; Julius Gagilas; Saulius Serva; Alina Stura; Andras Foldesi; Camilla Estmer Nilsson; Marek Kwiatkowski
Original assignee: Quiatech Ab
Priority date: 2006-09-04
Filing date: 2007-09-04
Publication date: 2008-04-03
Also published as: WO2008037568A3

Abstract

The invention relates to compounds of general structure (I) or salts thereof, wherein B is a nucleobase, X and Y independently are oxygen or sulphur, Z is a chemical group characterized by strong electron withdrawing properties, R1 is hydrogen, hydroxyl or a protected hydroxyl, R2 and R3 are together or separately hydrogen or a hydrocarbyl, R4 is hydrogen or a chemical moiety consisting of a linker molecule L, linking a detectable group to the rest of the structure (I), R5 is hydrogen or an additional electron withdrawing group. R5 can be identical with Z or different. R6 is hydrogen or a chemical moiety consisting of a linker unit L, a cleavable group and a detectable group joined together in the following order R6 = -L-cleavable group-detectable group. The detectable group is placed either on R4 or R6 or is absent, thus either R4 is hydrogen and the detectable group is placed on R6, or R6 is hydrogen and the detectable group is placed on R4, or both R4 and R6 are hydrogen. Numbers m and n are independently 0 or 1. Compounds of formula (I) are useful as reversible chain extension terminators. The invention also relates to the use of the compounds (I) in nucleic acid sequencing as well as to a method of preparing compounds of Formula (I).

Description

REVERSIBLE TERMINATORS FOR EFFICIENT SEQUENCING BY SYNTHESIS

Technical field

The present invention relates to novel nucleic acid chain extension terminators, their use in nucleic acid sequencing as well as a method for preparing such compounds.

Technical background

Currently the dominating method for DNA sequence determination is based on the dideoxy nucleotide chain termination (Sanger et al., Proc.Natl. Acad. Sci.74:5463-5467 (1977)). The terminated extension products are then separated electrophoretically on a polyacrylamide gel and detected by an optical system utilizing laserexcitation. Methods have also been proposed which detect the addition or removal either nucleotides or short nucleotide fragments from a DNA strand.

For example, Hyman E.D., Anal. Biochem., 174:423 (1988) discloses the addition of a nucleotide to a an immobilised DNA template/primer complex in the presence of a polymerase and determination of polymerisation reaction by detecting the pyrophosphate liberated as a result of the polymerisation.

Jett et al., J. Biomol. Struct. Dyn., I, p. 301, 1989 discloses a method wherein a single stranded DNA or RNA molecule of labelled nucleotides, complementary to the sequence to be determined, is suspended in a moving flow stream. Individual bases are then cleaved sequentially from the end of the suspended sequence and determined by a detector passed by the flow stream. EP-A-223 618 discloses the use of an immobilized DNA template, primer and polymerase exposed to a flow containing only one species of deoxynucleotide at a time. A downstream detection system then determines whether deoxynucleotide is incorporated into the copy or not by detecting the difference in deoxynucleotide concentrations entering and leaving the flow cell containing the complex of DNA template and polymerase.

WO 90/13666 proposes a method directly measuring the growth of the template copy rather than determining it indirectly from compositions in the flow medium. Only one of the four nucleotides is present at the same time, and the polymerization events reflecting the incorporation of a nucleotide or not are detected by spectroscopic means (evanescent wave spectroscopy, fluorescence detection, absorption spectroscopy) or by the individual nucleotides being labelled.

Similar methods employing labelled 3'-blocked deoxynucleotides where the blocking group is removable and which thus permit sequential deoxynucleotide addition/detection steps are disclosed in WO 91/06678, USA-5,302,509, DE-A-414 1178 and WO 93/21340. However, the necessary 3 '-bio eking groups were either not described in any detail, were not accepted by the required enzyme, or did not permit desired rapid deblocking of the growing template copy strand after each polymerization event. To our best knowledge, only two chemical functions for the temporary protection of the 3'-O- position in the nucleotide triphosphates have been disclosed, together with evidences of their incorporation to the primer in a template-based manner. Thus US6664079 suggest 3'-O-allyl group as an example of ethers removable upon treatment with palladium salts. This protecting group is well known in the literature and frequently used for the protection of other hydroxyls. The inventor of this patented protection has later on shown that at least one cycle of incorporation/deprotection can be performed. The researchers from Solexa has applied for a patent (WO 2004/018497), describing 3'-O-CH2-N3 as a protecting moiety for their reversible terminators. This function similarly to the allyl group was known earlier, and was even described as a moiety located on the 3'-0 position (Zavorodny et al. 1991, Tet. Lett. 32, 7593-7596). Interestingly it was suggested that this azidomethyl group may work as a mild labile protecting group as it can be reduced efficiently by phosphines. This is exactly what Solexa suggest in the mentioned patent application describing one cycle of incorporation/deprotection. Our own experience from work with similar type of compounds, suggest that the second step in Solexas deprotection procedure may be problematic.

Working with dithiomethyl protection O-CH2-SS-R for the 3'-O- in deoxynucleosides (US 6309836), we found that the transient hemithioformacetal formed upon reduction of the dithiobond is much more stable than the corresponding oxy hemiformacetal. Search in literature shows that similar nitrogen analogue of this formacetal (O-CH2-NH2) would be even more stable than the previously listed analogue hemiacetals, thus it may create a substantial problems with achieving the complete deprotection.

Searching for the optimal protection for the 3'-O-position in reversible terminators we turn our attention to two publications describing novel methods for RNA synthesis. The authors used 1- (2-cyanoethoxy)ethyl (CEE) (Tet. Lett. 2004, 45, 9529-9531), and cyanoethoxy methyl (O-CH2- O-CH2-CH2-CN) (CEM) (Org. Lett. 2005, 7, 3477-3480) for protection of the 2'-O- position during chemical synthesis of RNA. These authors found that both groups show amazing stability against even strong bases, but are readily removable upon treatment with tetrabutylammonium fluoride (TBAF) in waterfree tetrahydrofuran (THF). This is slightly unexpected as the suggested mechanism for deprotection involves proton abstraction and beta elimination, thus it would be expected that much stronger base DBU would perform the process much better. In reality TBAF where F- can also be seen as a base (although weaker) works much more efficient. This observation raises a question if the beta elimination is the only mechanism that may be acting in this process.

We asked the question if the mentioned groups could be used even for the protection of 3'-O- position in the deoxynucleotides, and further, if fully deprotected oligonucleotide primers containing such function on the 3 '-end, and often present as a hybride with the template DNA could be deprotected under such environment, so different from what would be called biological conditions. After making a series of experiments described in the Example part we found the answer for this question to be positive. It become then interesting if a simpler structure than cyanoethoxymethyl, namely cyanoethyl (CE) could also be used for the same protecting purpose. Recently this function was used by Sekine et. al. (J. Org. Chem. 2005, 70, 10453-10460) for its incorporation at 2'-OH of ribonucleotides, in order to achieve a permanent protection of this position. Reports describing cleavage of cyanoethyl protected hydroxyl groups are very rare, and there is no one showing that this function can be cleaved by means of a fluoride anion. In fact, Sekine is using successfully another than TBAF but related fluor containing reagent, to remove other protecting group than CE from his nucleotides. In our experiments we established conditions for introduction of this group also on the 3'- position and discovered that even this function can be removed from the 3'-0 protected nucleoside triphosphate, as well as from oligonucleotides possessing this group as the sole modifying function.

Yet another interesting functional group has been employed for the purpose of protection of the 2'-OH in the ribonucleosides. This function - nitrobenzyloxymethyl O-CH2-O-CH2PhNO2 was found to be also labile in the presence of TBAF (Gough et al. Tet. Lett. 1996, 37, 981-982). However, in this case the beta elimination mechanism cannot take place since the benzyl group contains only single CH2 function and not two which is necessary for the elimination pathway. Obviously, in this case a nucleophilic attack of the fluoride on the benzylic methylene group must take place with oxygen atom of the formacetal group acting as a leaving group. Since the bulky nitrophenyl group plays no other role than an efficient electron withdrawing group, it is logical to expect that reduction of the large nitrophenyl group to the small cyano function will result with minimal protecting group. We can further expect that the cyano group placed directly on the single methyl group ( -O-CH2-O-CH2CN) (cyanomethoxymethyl - CMM) will have even stronger tendency to withdraw electrons making the process of the deprotection even faster. We expect that the same logic which allowed us for squeezing of the original CEM group and obtaining the smaller CE protecting function will be possible if applied for the above CMM group and allow for its further miniaturizing to the cyanomethyl protecting group CM (-0- CH2CN). The Cm group can be easily introduced at a selected position in nucleosides upon introduction of the methylthiomethyl group at this position, its activation (eg by means of sulfuryl chloride) and reacting of obtained chloromethyl derivative with potassium cyanide in DMF, similarly to the method described by us in US 6309836. It is important to remember that the cyano group mentioned here acts only as a one of many possible electron withdrawing groups, thus other groups characterised by similar ability can be used instead in all of the above listed compounds. It is, however, natural that due to the size restriction enforced by most of polymerases the size of the alternative electron withdrawing groups should be kept to the necessary minimum. With respect to the listed cyanomethyl- and cyanoethyl- containing protecting groups it is noteworthy to mention a patent of Hiatt (US 5763594). This document is exemplified by one phosphate group for the protection of the 3 '-OH.

In the practice, a protection of only 3'-OH is often not enough. In order to read the information about which nucleotide become incorporated and at which position of the sequencing array, it is necessary to have an indicator - a detectable function that can be detected with high sensitivity, and with a specificity allowing for discrimination between all nucleobases. Before next round of incorporation this detectable group has to be eliminated to be not confused with the next coming label or to stop interaction of the old label with the newly coming label. This can done by degradation of the old label, and in the case of fluorescent labels is often performed as bleaching. It is however more temping to introduce a cleavable group between the label and the rest of the nucleotide and to have a possibility for the chemical cleavage of the detectable moiety that will be followed by washing of the array. In theory, the simplest version of such system would be made by introduction of the label directly on the 3'-O- protecting group as it was suggested in US 6255475 where the cleavable function was an acid labile acetal moiety protecting this hydroxyl. Thus cleavage of the acetal would simultaneously result in the cleavage of the detectable moiety making the primer ready for a next coupling. Unfortunately, most of nucleotide polymerases do not tolerate large substituents at this position so most of the work was concentrated around location of the label, together with an additional cleavable unit on the nucleobase and to keep the 3'- function at a minimal, but still functional size. Thus EP 1291354 discloses a linker between the nucleobase and a detectable group that can be cleaved by a reducing reagent. The similar type of linkers can be found in WO 2004/018497. This linker containing a dithio- unit could be cleaved by means of phosphines or dithiotreitol. US6664079 uses photolabile linkers or linkers cleavable by means of palladium ions. Any of these linkers could be used together with the present method of 3'-0 protection, and would be equivalent to two steps procedure for complete deprotection of incorporated nucleotide. It would be desirable, however, to perform these cleavages in one single step. Thus the function connecting the detectable moiety should contain a unit cleavable by means of the same reagent as is necessary for the cleavage of the 3'-O- protecting group. A silyl group or a disyloxyl linker (US 6291669) could be a working alternative as both are fluoride-labile. We found also that cleavable functions, analogues to the presented 3'- blocking units can be constructed, used for connection of detectable group to the nucleobase, and conveniently removed under the same conditions as are used for the removal of the 3-O-protection.

The methodologies presented here by us solve several important problems related to the Sequencing By Synthesis. We provide a closely related group of chemical functionalities that can be used for the crucial protection of 3'-hydroxyl in reversible terminators. We are proving that these structures are accepted by certain polymerases and incorporated into the primers in a specific manner. We are further showing that these functionalities are stable during the enzymatic reaction - with other word they are a truly terminating. These two criteria differentiate our structures from most of other structures suggested earlier as vast majority of the later is not accepted by any polymerase or is very efficiently cleaved of thus disintegrating the whole process. It should be mentioned here that even small conversion of the 3 '-protected triphosphate to the native 3'-OH form will always result with selective incorporation of the hydro lyzed one resulting in full extension and no sequencing data. Contrary to the other mentioned earlier prior arts, our methodology of deprotection utilizes only non-expensive reagents and has been proven to work quantitatively in a reasonable short time. Most of chemical structures presented by us were never used for protection of the 3'-0 position in nucleosides. None of these structures was anticipated to be used for protection of the 3-OH in nucleotides triphosphates, and none of them has been ever considered for the sequencing application. Finally, we envisioned situations with sequencing performed in an array format, despite the usual opinion that fluoride based reagents should never be in contacts with any array.

Summary of the invention

One object of the present invention is to provide nucleotide derivatives which are useful as chain terminators and which upon removal of the cleavable 3 '-protecting group may readily be converted into nucleotides or nucleotide analogues that may be further extended.

Another object of the present invention is to provide a method for nucleotide sequence determination using the novel chain terminators.

Another object of the present invention is to provide a process of preparing novel chain terminators according to the invention.

A still further object of the invention is to provide a kit comprising, in separate containers, the novel chain terminators of the invention, and necessary enzymes and/or nucleotides and/or reagents for preparing oligo- or polynucleotides.

Yet another object of the invention is to provide a kit comprising, in separate containers, the novel chain terminators of the invention and necessary enzymes and/or nucleotides and/or reagents, for determining the sequence of a nucleic acid.

The invention provides a method for sequencing a nucleic acid including the steps of : a) providing a target nucleic acid array including a plurality of target nucleic acids; b) contacting a sequencing primer with the target nucleic acids thereby forming target-primer complexes between complementary portions of the sequencing primers and the target nucleic acids; c) incorporating a first 3'-O-modifϊed nucleotide into at least one sequencing primer portion of the target-primer complexes, the first 3'-O-modified nucleotide being complementary to the target nucleic acid; and d) detecting the incorporation of the first 3'-0 -modified nucleotide, wherein the first 3'-0 -modified nucleotide is complementary to the target sequence at the first 3'-0 - modified nucleotide's site of incorporation. As with other methods described herein, the detecting step can be performed before or after removing a 3'-0 moiety. This sequencing method is effective for producing a plurality of nucleotide sequences wherein the nucleotide sequences correspond to overlapping nucleotide sequences of the target nucleic acid. Alternatively, the array may be composed with the plurality of sequencing primers forming primer-target complex upon contacting with a mixture containing plurality of target nucleic acids.

As defined herein, the term "label" or "detectable group" means a molecule, which is possible to detect in a suitable manner. In particular, the terms "dye", "label" or "dye- label" include fluorescent molecules such as fluorescein, cyanine dyes, like Cy3, Cy-5, Cy-7, Cy-9 disclosed in U.S. 5268486 (Waggoner et al.) or variants thereof, such as Cy3.5 and Cy5.5, but may also include molecules such as Rhodamine, BODIPY, ROX, TAMRA, Rl 10, R6G. Joe, HEX, TET, Alexa or Texas Red.

As defined herein, the term "labeled nucleotide" or "dye-labeled nucleotide" means a nucleotide, which is connected to a label or detectable group as defined above.

As used herein, the term "array" refers to a heterogeneous pool of nucleic acid molecules that is distributed over a support matrix. These molecules, differing in sequence, are spaced at a distance from one another sufficient to permit the identification of discrete features of the array. It may also refer to miniaturized surfaces comprising ordered immobilized oligonucleotides, DNA or RNA molecules.

In Formula I above, the nucleobase B may be natural or synthetic. Natural nucleobases include common nucleobases, such as adenine, guanine, cytosine, thymine and uracil, as well as less common nucleobases, such as xanthine, hypoxanthine or 2-aminopurine. Synthetic nucleobases B are analogues to the natural nucleobases and capable of interacting with other nucleobases in a specific, hydrogen bond determined way.

X and Y can independently be oxygen or sulphur atom, although the most preferred for both positions is oxygen. The ability of the 3'-O- protecting group to be cleaved is to large extent depending on the nature of function Z. Thus Z is a chemical group characterized by strong electron withdrawing properties. This prerequisite is valid no matter which chemical mechanism is considered to be operating at a particular case, since both beta-elimination mechanism and nucleophilic substitution demands the presence of the mentioned electron withdrawing function. As such, Z maid be chosen from a large group of functions known to those skilled in the art, and can be selected among others from the following groups: nitro, cyano, sulfono, sulfoxide, trihalogenomethyl, aldehydo, ketone, ester, phenyl or substituted phenyl. The substituent Rl denotes hydrogen, hydroxyl or a protected hydroxyl. Any known permanent or transient protecting group can be applied for this purpose, however the most preferred function for this protection is methyl group. R2 and R3 are together or separately hydrogen, an alkyl or a substituted alkyl group. Assuming that a cleaving reagent is operating as a base and the 3'- O- protecting group is eliminated by a process of beta-elimination, then the kinetics of deprotection is dependent on the leaving group ability to accept a pair of electrons. In this context it is highly probable that an electron accepting function located on R2 or R3 will increase the ratio of the 3'- O- protecting group removal. For the most common application, the most preferred version of R2 or R3 is however a hydrogen atom. The other way to modulate the acidity of the alpha hydrogen (hydrogen most closely to the group Z) is to increase the total electron withdrawing ability of surrounding functions. The presence of one Z-group is defined as the minimum necessary for the cleavage process, but a possibility of having two similar functions can not be excluded. Thus R5 can be hydrogen or an additional electron withdrawing group. R5 can be identical with Z or different.

From the practical point of view it would be ideal if the sequencing process could utilize a nucleotide triphosphate carrying the detectable label located right on the 3'-O-protecting group. Such nucleotide, if accepted by the polymerase and incorporated into the primer, could subsequently be 3'-0 deprotected by a single reaction with simultaneous removal of the detectable function.

Thus R4 is hydrogen or a chemical moiety consisting of a linker molecule L, linking a detectable group to the rest of the structure (I). There is a large amount of different linkers known to those skilled in art. These linkers can be of different length, consist of other atoms than just carbon and contain different proportions of the heteroatoms. Here, it is however important to notice that the linkers importance is only in connecting a detectable label to the rest of the structure without substantial increase of the molecules size or its solubility parameters.

Detectable moiety can be chosen from a vast number of such moieties known to those skilled in the art. Exemplary such moieties are radioactively labelled functions, luminescent, electroluminescent or fluorescent labels, and labels that absorb characteristic visible or infrared light. Preferably, the detectable group is a fluorescent label. A way of keeping the 3 '-protecting structure at the minimum size is to place the detectable group at the nucleobase. The most favoured position for location of any functional group at a particular base has been elaborated in details during the last decade. It is thus commonly accepted that 5-C atom for cytosine, 5-C-methyl thymine, 7-deazaguanine and 7-deazaadenine are the optimal positions for attachment a linker to the base to assure that the nucleotide will be recognized by the polymerase and the incorporated base will perform well in a base-pairing with a nucleotide from the opposite strand. Placing a detectable group on the nucleobase renders the presence of the R4 unnecessary, so in such case the R4 group can be downsized to hydrogen.

The methodology of Sequencing by Synthesis demands, however, that the incorporated, fluorescence scanned and read detectable group to be quantitatively released and washed away before the next round of incorporation. This creates a need for an efficient cleavable unit between the detectable moiety and the nucleobase. As the process of deprotection must necessarily involve the protecting group from the 3'- position, it is natural that a methodology involving simultaneous cleavage of both functions will be preferred. Thus it is desirable that the cleavable group located between nucleobase and detectable group would be deprotected by the same reagent that is used for the cleavage of the 3'-O-protecting group. In the very simplified form protecting groups that are used here for the protection of the 3'- OH can be divided into three subcategories: 1) provided R2, R3, R4, R5 are hydrogen, n = 1 and m = 1 then the structure is -CH2-O-CH2-CH2-Z, and this function is called cyanoethoxymethyl for Z = CN, 2) provided n = 0, R4, R5 are hydrogen and m = 1, then the structure is -CH2-CH2-Z, and this function is called cyanoethyl for Z = CN, 3) provided n = 0, m = 0 and R5 is hydrogen, then the structure is -CH2-Z, and this function is called cyano methyl for Z = CN,. Cyanoethoxymethyl group is known from the literature to be labile upon treatment with fluoride anions, especially if the reagent is tetrabutylamminium fluoride (TBAF) and dissolved in an aprotic colvent like tetrahydrofuran (THF). Cyanoethyl group, when exists as an ether at the 2'-O- position, is regarded as stable even in the presence of fluoride anion as was shown before by Sekine et al. ( J. Org. Chem. 2005, 70, 10453-10460). Contrary to these data we have shown that this function when present at the 3'-0 position, can be cleaved by TBAF ant thus can be used as a hydroxyl protecting group. Further, cyanomethyl group studied for 2'-0 protection was found completely stable to TBAF. Analysis of the related literature data and some preliminary experiments from our laboratory strength us in opinion that even this function can be cleaved by TBAF or perhaps other nucleophilic reagents. As all the listed subcategories of a 3'-O- protecting group can be cleaved by TBAF it would be thus desirable if the cleavable unit located on the base would have similar reactivity. One of possibilities is to introduce on the base a group which resembles a cleavable function located on the 3'-O. Thus we have exemplified our idea by synthesizing of the base substituted nucleotide triphosphate, where the cleavable unit on the base is an analogue to cyanoethyl function and therefore labile in the presence of TBAF.

In summary, R6 can be hydrogen or a chemical moiety consisting of a linker unit L, a cleavable group and a detectable group joined together in the following order R6 = -L-cleavable group- detectable group. The detectable group is placed either on R4 or R6 or is absent, thus either R4 is hydrogen and the detectable group is placed on R6, or R6 is hydrogen and the detectable group is placed on R4, or both R4 and R6 are hydrogens.

Numbers m and n are independently 0 or 1.

While the compounds of Formula I may be used as pure chain extension inhibitors, or chain terminators, for example, in DNA sequencing according to the chain termination method, as is per se known in the art, the advantages of the compounds are, of course, better benefited from when the convenient deprotection capabilities of the compounds are utilized. This is, for example, the case when the compounds I are used in nucleic acid sequencing methods based on the sequential incorporation and determination of individual nucleotides in a growing nucleic acid copy strand as described in, for example, the aforementioned WO 91/06678, US-A- 5,302,509, DE-A-414 1178 and WO 93/21340.

Such sequencing method that uses reversibly blocked nucleotides is known as Sequencing by Synthesis (SBS). SBS determines the DNA sequence by incorporating nucleotides and detecting the sequence one base at a time. To effectively sequence long stretches of a nucleic acid using SBS, it is advantageous to be able to perform multiple iterations of the single nucleotide incorporation. Accordingly, SBS-based methods require 3'-OH protecting groups that are removable under conditions that do not disrupt the primer and target DNA interactions. As such, there exists a need for nucleotide triphosphates that are reversibly blocked at the 3 'position and which are also effective substrates for DNA polymerases

Another aspect of the invention therefore provides a method for determining the sequence of a nucleic acid, which method comprises providing a single- stranded template comprising the nucleic acid to be determined, and at least partially synthesizing a complementary nucleic acid molecule in a stepwise serial manner by the addition of nucleotides in which the identity of each nucleotide incorporated into the complementary nucleic acid molecule is determined subsequent to its incorporation, wherein said nucleotides are compounds of Formula I as defined above, and wherein the 3 '-blocking group is removed from the nucleotide after its incorporation to permit further extension of the nucleic acid molecule.

In one embodiment, a method for determining the sequence of a nucleic acid comprises the following steps: (i) providing a single-stranded template comprising the nucleic acid to be sequenced, (ii) hybridising a primer to the template to form a template/primer complex, (iii) subjecting the primer to an extension reaction by the addition of compounds of Formula I with different nucleobases B corresponding the four bases A, C, T and G or analogues thereof, (iv) determining the type of the compound of Formula I added to the primer, (v) selectively hydro lysing the 3'-O- protective group, and (vi) repeating steps (iii) to (v) sequentially and recording the order of incorporation of compounds of Formula I.

The different compounds of Formula I in step (iii) may be added in sequence, in which case the four different compounds I may carry the same detectable group (label). Alternatively, the different compounds I may have different labels and may added at the same time.

In a preferred embodiment of the method of the invention, the template/primer complex is bound to a solid-phase support, such as a sequencing chip, or porous and non-porous beads for example. The template may be attached to the solid support via a binding linker, which, for instance, is ligated to the 5 '-end of the template or incorporated in one of the ends of the template by polymerase chain reaction (PCR). The binding linker may then be attached to the solid support for instance by use of chemical covalent bond or using non-covalent interactions exemplified by a streptavidin coupling system. Alternatively, the primer may be attached to the solid support.

The compounds of Formula I may, of course, also conveniently be used in so-called mini- sequencing (Syvanen A-C et al., Genomics 8:684-692 (1990).

In the following, the invention will be illustrated by some non-limiting examples. Reference to a respective chemical structure will be related the accompanying drawings, which at least in this provisional description are placed in direct proximity to the respective Example.

Examples

Example 1 Synthesis of 3'-O-(2-cyanoethyl)-5 '-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite)- thymidine (8).

(Similar synthesis utilizing a transient protection of O-6 position in dG can be applied for the synthesis of 3 '-protected deoxy guanosine derivatives. These derivatives can further be converted to the 5'-O-phosphoramidite or 5'-O-triphosphate))

5'-O-Dimethoxytrityl-3'-O-t-butyldimethylsilyl-thymidine (2) 5'-O-DMTr-thymidine (1) (5.34 g, ~10 mmol) was co-evaporated with dry pyridine (2x) then re-dissolved in the same solvent (~80 ml). Imidazole (2.72 g, 40 mmol) was added followed by tert-butyldimethylsilyl chloride (3.01g, 20 mmol). Stirring at room temperature was maintained for -3.75 h, then the reaction mixture was poured into saturated aqueous NaHCO3 and extracted with dichloromethane (3x). The pooled organic phase was dried over MgSO4, filtered and evaporated. Co-evaporations with toluene (3x) removed traces of pyridine, the resulting oil was purified by column chromatography (cyclohexane-ethyl acetate+2 pipettes of TEA) to afford compound 2 (5.21 g, 7.91 mmol, 81%) as a yellowish foam.

3'-O-t-Butyldimethylsilyl-thymidine (3) Compound (2) (5.21 g, 7.91 mmol) was dissolved in 2% toluenesulfonic acid in dichloromethane/methanole (7:3, 100 ml). The reaction mixture was stirred for 5 min at room temperature when tic showed full deprotection of the starting material. Triethylamine was added until the disappearance of the DMTr colour. The whole mixture was transferred into a shaking- funnel containing sat. aqeous NaHCO3 and extracted with dichloromethane (3x). The organic phases were pooled and dried over MgSO4, filtered and evaporated. The residue was purified by short-column chromatography (slow gradient of methanol in dichloromethane) to yield compound 3 (2.54 g, 7.13 mmol, 90%).

5'-O-Benzoyl-3'-O-t-butyldimethylsilyl-N3-benzoylthymidine (4) After thorough drying on an oil-pump overnight, thymidine derivative 3 (2.54 g, 7.13 mmol) was dissolved in dry pyridine (70 ml). Benzoyl chloride (3.31 ml, 28.5 mmol) was added and the mixture was stirred for 30 at room temperature under argon. Diisopropylethylamine was then added and stirring was maintained for 3 h. Methanol was added to the reaction and stirring was continued for additional 30 min. The mixture was poured into saturated aqueous NaHCO3, extracted with CH2C12, the organic phase was dried over MgSO4, filtered and evaporated. After repeated co-evaporation with toluene, the residue was purified by column chromatography (cyclohexane-ethyl acetate) to give compound 4 (3.8 g, 6.73 mmol, 94%).

5'-O-Benzoyl-N3-benzoylthymidine (5) The fully protected thymidine derivative 4 (3.8 g, 6.73 mmol) was dissolved in dry tetrahydrofurane (ca 60 ml) and tetrabutylammonium fluoride (IM in dry THF, 6.7 ml) was added. The reaction was stirred for 40 min at room temperature.

Volatile matters were evaporated, and the residue was separated by column chromatography (cyclohexane-dichloromethane-methanol) to afford compound 5 as a foam (2.27 g, 5.04 mmol, 75%).

5'-O-Benzoyl-3'-O-(2-cyanoethyl)-N3-benzoylthymidine (6) Thymidine derivative 5 (1.71 g, 3.8 mmol) was dissolved in dry t-butanol. To the stirred solution acrylonitrile (1.31 ml, ) was added followed by cesium carbonate (350 mg) and the resulting inhomogeneous mixture was stirred under argon for 1 h. The reaction was filtered through a Celite pad, the pad was washed with dichloromethane. The organic phase was evaporated and the residue was separated by short column chromatography (cyclohexane-ethyl acetate) to give the fully protected nucleoside 6 (1.24 g, 2.46 mmo 1, 65%).

3'-O-(2-Cyanoethyl)-thymidine (7) Thymidine derivative 6 (1.54 g, 3.06 mmol) was dissolved in methanol and aqueous ammonia (32%, 20 ml) was added followed by dioxane (~5 ml). The mixture was stirred at room temperature for 6.5 h. Then the solvents were evaporated and the residue co-evaporated with methanol (3x) and after that with dichloromethane (2x). The resulted glass was separated by short column chromatography (dichloromethane-methanol) to give the fully protected nucleoside 7 (828 mg, 2.8 mmol, 92%).

3'-O-(2-Cyanoethyl)-thymidine 5'-O-(2-cyanoethyl N,N-diisopropylphosphoramidite) (8) The 5 '-hydroxy block 7 (428 mg, 1.45 mmol) was dissolved in dry THF (15 ml) and DIPEA (1.01 ml, 5.8 mmol) was added followed by 2-cyanoethyl N,N-diisopropylchlorophosphoramidite (650 1). The mixture was stirred under argon at room temperature for 1.5 h. Dry methanol was added and stirring was maintained for additional 30 min. Then the mixture was poured into saturated NaHCO3 and extracted with dichloromethane (3x). The collected organic phase was dried over MgSO4, filtered and evaporated. The residue was separated by short column chromatography (cyclohexane-dichloromethane then dichloromethane-acetone with ca. 3 ml triethylamine/100 ml eluant) to give the phosphoramidite 8 (490 mg, 0.99 mmol, 68%). Synthesis of 3 '-O-(2-cyanoethyl) thymidine-5 '-O-tri-phosphate (9).

The reaction was carried out according to the protocol of Ludwig and Eckstein (JOC 1989, 54,

631-635).

3'-O-(2-cyanoethyl)thymidine (7) (55 mg, 0.19 mmol, 1.0 eq.) was co-evaporated two times with dry pyridine (3 ml) and dried overnight in vacuo at the rotary vane |>um|). The flask was filled with argon and closed with a septum. The following steps were carried out under a slight positive argon pressure.

The nucleoside was dissolved in anhydrous pyridine (190 μl) and anhydrous dioxane (570 μl). Afterwards 190 μl (0.19 mmol, 1.0 eq.) of a freshly prepared 1 M solution of the phosphitylating agent 2-chloro-4H-l,2,3-benzo-dioxaphosphorin-4-one in anhydrous dioxane were added to the well stirred solution of the nucleoside. After 10 min a mixture of 570 μl (0.28 mmol, 1.5 eq.) 0.5 M solution of bis(tri-n-butylammonium)pyrophosphate in anhydrous DMF and 190 μl (0.80 mmol, 4.3 eq.) of tri-n-butylamine was added quickly. The reaction mixture again was stirred for 10 min and then a 1% iodine solution in pyridine: water = 98/2 (v/v) (3.8 ml) was added. After stirring for 15 min a 5% aqueous solution of NaHSO3 was added until the brown solution turned light yellow to destroy excess of iodine. The solution was evaporated to dryness in vacuo. The residue was dissolved in 20 ml water and left at room temperature for 30 min. Then 38 ml concentrated ammonia were added and after stirring for 60 min at room temperature the solution was evaporated to dryness in vacuo.

The residue (330 mg light yellow solid) was dissolved in water (about 12 ml), filtered through a 0.2 μm syringe filter and purified in four portions over a DEAE-Sepharose fast flow column (1.6 cm diameter, 15 cm length) at 4°C to obtain 28.3 mg (16.2%) of the HPLC-purified triphosphate as the tetrakis(triethylammonuium) salt (9).

Example 2

Synthesis of 3'-O-(2-cyanoethoxy)methyl-thymidine triphosphate (6).

5'-O-Benzoyl-thymidine (2) Thymidine (1) (2.42 g, 10 mmol) was co-evaporated with dry pyridine (3x) and dissolved in the same solvent (100 ml). The solution was placed in an ice-bath and benzoyl chloride (1.28 ml, 11 mmol) dissolved in dry dichloromethane (10 ml) was added drop wise (ca 10 min). Stirring at ice-bath temperature was maintained for ~3.5 h, and then the reaction mixture was poured into saturated aqueous NaHCO3 and extracted with dichloromethane (3x). The pooled organic phase was dried over MgSO4, filtered and evaporated. Co-evaporations with toluene (3x) removed traces of pyridine, the resulting off-white material was purified by column chromatography (dichloromethane-methanol) to afford compound 2 (3.0 g, 8.7 mmol, 87%) as a white amorphous solid.

5'-O-Benzoyl-3'-O-methythiomethyl-thymidine (3) Compound (2) (3.0 g, 8.7 mmol) was dissolved in dimethyl sulfoxide (23 ml). To this solution acetic acid (25 ml) was added followed by acetic anhydride (15 ml). The reaction mixture was stirred for 25 h at room temperature when TLC showed full consumption of the starting material. Potassium carbonate (55 g) was dissolved in water (ca. 400 ml), the solution was placed into an ice-bath. The reaction mixture was poured slowly with vigorous stirring into the carbonate solution; pH was checked at the end of the addition for neutrality. The whole mixture was transferred into a extracting- funnel and extracted with dichloromethane (3x). The organic phases were pooled and dried over MgSO4, filtered and evaporated, finally co-evaporated a few times with toluene. The residual oily material was purified by short-column chromatography (slow gradient of methanol in dichloromethane) to yield compound 3 (1.76 g, 4.33 mmol, 50%).

5'-O-Benzoyl-3'-O-(2-cyanoethoxy)methyl-thymidine (4) After thorough drying on an oil-pump overnight, thymidine derivative 3 (2.45 g, 6.03 mmol) was dissolved in dry dichloromethane (50 ml). Triethylamine (0.84 ml) was added followed by molecular sieves (3 A, ca 40 ml, pre- activated in an oven at ca 95 ⁰C), and the heterogeneous mixture was gently stirred for 2h at room temperature under argon. It was then placed into an ice-bath, and sulfuryl chloride (0.49 ml, 6.63 mmol) in dry dichloromethane (10 ml) was dropwise added over ~5 min. After stirring the reaction in the ice-bath for 1 h, 3-hydroxypropionitrile (906 1, 13.3 mmol) was added in dry dichloromethane (10 ml) dropwise still in the ice-bath (over ca 10 min). The bath was removed and stirring was maintained overnight. The mixture was filtered through a Celite pad, evaporated and purified by column chromatography (dichloromethane-methanol) to give compound 4 (1.43 g, 3.33 mmol, 55%).

3'-O-(2-Cyanoethoxy)methyl-thymidine (5) The protected thymidine 4 (1.43 g, 3.33 mmol) was dissolved in methanol (ca 20 ml) and aqueous ammonia (32%, 30 ml) was added. The reaction was stirred for 5 h at room temperature. Volatile matters were evaporated and the residue was co-evaporated with methanol. Compound was dissolved in methanol- water, silica was added and the slurry was dried on a rotary evaporator. It was then transferred in dichloromethane onto a top of a short silica column and compound 5 was separated as sticky foam (dichloromethane- methanol). Since HPLC showed a small fraction (1-2%) of a faster moving impurity, part of the compound was further purified by preparative HPLC (Kromasil C 18, 250x50mm, 10% acetonitrile in water, 70ml/min).

3'-O-(2-Cyanoethoxy)methyl-thymidine triphosphate (6) 3'-O-Cyanoethoxymethyl-thymidine (5) (90mg, 0.27mmol, leq) was co-evaporated three times with dry pyridine and dried in vacuo overnight. The starting material was then transferred into a reaction vessel equipped with a magnetic stirring bar and a septum and dissolved in anhydrous dimethylformamide (4 ml) and anhydrous pyridine (1 ml). A freshly prepared 1 M solution of 2-chloro-4H- 1,3,2- benzodioxaphosphorin-4-one in 1,4-dioxane (0.3 ml, 0.29 mmol, 1.1 eq) was added to the reaction mixture via syringe through the septum. The reaction was stirred at room temperature for 20 minutes when a pre-mixed solution of 0.5 M bis(tri-n-butylammonium)pyrophosphate in dry DMF (1.5 ml) and tri-n-butylamine (0.5 ml) was added (1.9 ml, 0.81 mmol, 3 eq). The resulting mixture was again stirred for 45 minutes at room temperature. Afterwards iodine- solution (1% in pyridine/water 98ml/2ml) (5.6 ml, 0.22 mmol, 0.8eq) was added and the reaction mixture turned to brown color. The mixture was stirred for 30 minutes at room temperature and then a few drops of an aqueous 5% NaHSO4 solution were added until the reaction mixture turned to yellow. All solvents were then evaporated via cooling distillation in vacuo. The oily residue was diluted with water (10 ml) and filtered with a Nalgene Syringe Filter in order to remove all insoluble particles. The water was evaporated and the residue was diluted again with Millipore-filtered water (5 ml). The product was purified twice on RP-HPLC to give the colorless triphosphate (6) as triethylammonium salt.

Example 3

Synthesis of 3 '-O-cyanoethyl-2 '-deoxyadenosine-5 '-triphosphate

(Similar straightforward procedure is used for the synthesis of 3 '-O-cyanoethyl-2 '- deoxyacytidine-5 '-triphosphate)

PTSA CH₂Cl₂1 EtOH

3'-O-Cyanoethyl-5'-O-(dimethoxyltrityl)-6-N-benzoyl-2'-deoxyadenosine (2). The commercial 5'-O-(dimethoxyltrityl)-6-N-benzoyl-2'-deoxyadenosine (1) (197 mg, 0.3 mmol) was dissolved in 1.6 ml of tert-BuOH and acrylonitrile (0.42 ml, 0.6 mmol) and Cs2CO3 (112 mg, 0.3 mmol) were added. The mixture was vigorously stirred at room temperature for 2 h. After filtration and concentration, the crude material was purified by flash column chromatography (MeOH in CH2C12 0 - 1%) to give the title compound (177 mg, 83%). 3 '-O-Cyanoethyl-6-N-benzoyl-2 '-deoxy-adenosine (3). 3 '-O-Cyanoethyl-5 '-O- (dimethoxyltrityl)-6-N-benzoyl-2'-deoxyadenosine (2) (142 mg, 0.2 mmol) was dissolved in 20 ml of CH2C12 / EtOH (1 : 1) and PTSA (114 mg, 0.6 mmol) was added at O⁰C. The resulting mixture was stirred at room temperature for 30 min. The reaction was quenched with saturated NaHCO3 and the residue was extracted with CH2C12. Combined organic layers were washed with brine and dried with Na2SO4. After removal of solvent, the residue was purified by flash column chromatography (MeOH in CH2C12 0 - 3%) to give 3'-O-cyanoethyl-6-N-benzoyl-2'- deoxy-adenosine (73 mg, 89%).

Synthesis of 3 '-O-cyanoethyl-2'-deoxyadenosine-5 '-triphosphate (4). 3'-O-Cyanoethyl-6-N- benzoyl-2 '-deoxy-adenosine (3) (41 mg, 0.1 mmol) was dissolved in anhydrous pyridine (1 ml) and evaporated to dryness. Anhydrous pyridine (100 μl) was injected through the septum followed by anhydrous dioxane (300 μl). A solution of 2-chloro-4H-l,2,3-dioxaphosphorin-4- one (30 mg, 0.15 mmol) in anhydrous dioxane (300 μl) was then injected into the well-stirred solution of the nucleoside. After 10 min a well-vortexed mixture of a 0.5 M solution of bis(tri-n- butylammonium) pyrophosphate in anhydrous DMF (400 μl) and tri-n-butylamine (100 μl) was quickly injected and the reaction mixture was stirred for 10 min. A solution of 1% iodine in pyridine / water (98 / 2, v / v) (2 ml) was then added. After 15 min excess iodine was destroyed by adding a few drops of a 5% aqueous solution of NaHSO3, and the reaction solution evaporated to dryness. The residue was dissolved in water (10 ml). After standing at room temperature for 30 min, concentrated ammonia (20 ml) was added. The resulting mixture was stirred at room temperature overnight and then evaporated to dryness, the residue was dissolved in water, and the solution applied to a DEAE Sephadex column, which was eluted with a linear gradient of 800 ml each of 0.05 M and 0.5 M TEAB. The product was eluted between 0.28 and 0.33 M buffer. The title compound (4) was further purified by HPLC to give an oil (21.8 mg, 23%). Example 4

Synthesis of 3 '-O-cyanoethyl-5-(3-aminoprop-l-ynyl)-2'-deoxyuridine-5 '-triphosphate (9).

CF₃ODOH CHO₃ / MeOH

N-Benzoyl-2',3'-O-trimethylsilanyl-5-iodo-2'-deoxyuridine (2). To a solution of 5-iodo-2'- deoxyuridine (1) (700 mg, 2 mmol), which was rendered anhydrous by repeated coevaporation with dry pyridine, in dry pyridine (20 ml) were added ethyldiisopropylamine (1.74 ml, 10 mmol) and chlorotrimethylsilane (0.63 ml, 5 mmol). After the mixture was stirred at room temperature for 30 min, benzoyl chloride (0.35 ml, 3 mmol) was added. The resulting mixture was stirred for another 1 h at room temperature and then KH2PO4 (1.6 g) and ice-water (10 ml) were added to the mixture with cooling. After the mixture has been stirred for several minutes, a crystalline precipitation appears. The crystals were collected by filtration and washed with water. After dried by oil pump overnight, the desired compound (2) was obtained as pale-yellow crystals (1.2O g, 99%).

N-Benzoyl-5-iodo-2'-deoxyuridine (3). N-Benzoyl-2',3'-O-trimethylsilanyl-5-iodo-2'- deoxyuridine (2) (1.20 g, 2 mmol) was dissolved in 20 ml of CHC13 / MeOH (1 : 1) and CF3COOH was added. The mixture was stirred for 30 min at room temperature. The solvent was removed in vacuum and the residue was crystallized from MeOH to give N-benzoyl-5-iodo-2'- deoxyuridine as a white solid. The mother liquid was repeated to crystallize for two times to give the combined solid (0.78 g, 86%).

N-Benzoyl-5'-O-MMTr-5-iodo-2'-deoxyuridine (4). N-Benzoyl-5-iodo-2'-deoxyuridine (3) (4.57 g, 10 mmol) was dissolved in 20 ml of dry pyridine and 40 ml of DMF, and then Et3N (2.3 ml, 15 mmol) and MMTrCl (4.0 g, 13 mmol) were added. The resulting mixture was stirred for 19 h. Another part of MMTrCl (0.3 g, 1 mmol) was added and the mixture was stirred for 3 h. The reaction was quenched by addition of 20 ml of EtOH. The solvent was removed in vacuum, and traces of pyridine were evaporated by repeated coevaporation with toluene. The residue was dissolved in 100 ml of dichloromethane and the resulting solution was washed with 5% NaHCO3, brine and concentrated. Crude material was purified by flash column chromatography (MeOH in CH2C12 0 - 2%) to give the title compound (4.73 g, 64%).

N-Benzoyl-3 '-O-cyanoethyl-5 '-O-MMTr-5-iodo-2'-deoxyuridine (5). N-Benzoyl-5 '-0-MMTr- 5-iodo-2'-deoxyuridine (4) (4.5 g, 6.2 mmol) was dissolved in 31 ml of t-BuOH, and then acrylonitrile (8.25 ml, 126 mmol) and Cs2CO3 (2.21 g, 6.3 mmol) were added. The mixture was stirred vigorously at room temperature for 3 h. After filtration and concentration, the crude material was purified by flash column chromatography (MeOH in CH2C12 0 - 1%) to give N- benzoyl-3 '-O-cyanoethyl-5 '-O-MMTr-5-iodo-2'-deoxyuridine (3.88 g, 80%). 3 '-O-Cyanoethyl-5 '-O-MMTr-5-iodo-2'-deoxyuridine (6). N-Benzoyl-3 '-O-cyanoethyl-5 '-O- MMTr-5-iodo-2'-deoxyuridine (5) (120 mg, 0.13 mmol) was dissolved in 6 ml of MeOH and aqueous ammonia (2 ml) was added at O⁰C. The resulting mixture was stirred at room temperature for 2 h. After removal of the solvent, the residue was purified by flash column chromatography (MeOH in CH2C12 0 - 1%) to give 3'-O-cyanoethyl-5'-O-MMTr-5-iodo-2'- deoxyuridine (66 mg, 75%). 3 '-O-Cyanoethyl-5-iodo-2'-deoxyuridine (7). 3 '-O-Canoethyl-5 '-O-MMTr-5-iodo-2'- deoxyuridine (6) (680 mg, 1 mmol) was dissolved 10 ml of CHC13 and 80% AcOH (10 ml) was added at O⁰C. The resulting mixture was stirred at room temperature for 30 min. After removal of the solvent, the residue was purified by flash column chromatography (MeOH in CH2C12 0 - 5%) to give the title compound (370 mg, 91%). 3'-O-Cyanoethyl-5-[3-(trifluoroacetamido)-prop-l-ynyl]-2'-deoxyuridine (8). Amberlite resin IRA-67 (1.5 g) was added to a solution of 3'-O-cyanoethyl-5-iodo-2'-deoxyuridine (7) (300 mg, 0.74 mmol) and CuI (29 mg, 0.15 mmol) in 10 ml of dry DMF. After stirring for 5 min, N- propargytrifluoroacetamide (223 mg, 1.48 mmol) and Pd(PPh3)2C12 (50 mg, 0.07 mmol) were added and the reaction mixture was stirred in the dark for 24 h. The crude mixture was concentrated and then purified by flash column chromatography (MeOH in CH2C12 0 - 5%) to give 3'-O-cyanoethyl-5-[3-(trifluoroacetamido)-prop-l-ynyl]-2'-deoxy-uridine (226 mg, 71%). 3 '-O-Cyanoethyl-5-(3-aminoprop- 1 -ynyl)-2'-deoxyuridine-5 '-triphosphate (9). 3 '-O- Cyanoethyl-5-[3-(trifluoroacetamido)-prop-l-ynyl]-2'-deoxyuridine (8) (44 mg, 0.1 mmol) was dissolved in anhydrous pyridine (1 ml) and evaporated to dryness. Anhydrous pyridine (100 μl) was injected through the septum followed by anhydrous dioxane (300 μl). A solution of 2- chloro-4H-l,2,3-dioxaphosphorin-4-one (30 mg, 0.15 mmol) in anhydrous dioxane (300 μl) was then injected into the well-stirred solution of the nucleoside. After 10 min a well-vortexed mixture of a 0.5 M solution of bis(tri-n-butylammonium) pyrophosphate in anhydrous DMF (400 μl) and tri-n-butylamine (100 μl) was quickly injected and the reaction mixture was stirred for 10 min. A solution of 1% iodine in pyridine / water (98 / 2, v / v) (2 ml) was then added. After 15 min excess iodine was destroyed by adding a few drops of a 5% aqueous solution of NaHSO3, and the reaction solution evaporated to dryness. The residue was dissolved in water (10 ml). After standing at room temperature for 30 min, concentrated ammonia (20 ml) was added. After the resulting mixture was stirred at room temperature overnight and then evaporated to dryness, the residue was dissolved in water, and the solution applied to a DEAE Sephadex column, which was eluted with a linear gradient of 800 ml each of 0.05 M and 0.5 M TEAB. The product was eluted between 0.30 and 0.35 M buffer. The title compound was further purified by HPLC to give a pale-yellow oil (10.8 mg, 11%). Example 5

Synthesis of the trifunctional linker unit.

_Q NaN₃, NaI

EtOH, 6d reflux

NaH, dry THF

2-(2-(2-Azidoethoxy)ethoxy)ethanol (2). 2-(2-(2-Chloroethoxy)ethoxy)ethanol (1). (10.0 g, 56.9 mmol, 1.0 eq.) were dissolved in 50 ml ethanol. To this solution sodium iodide (1.7 g, 11.9 mmol, 0.2 eq.) and sodium azide (11.1 g, 170.8 mmol, 3.0 eq.) were added and the resulting mixture refluxed for 6 days. The reaction mixture was filtered and concentrated under reduced pressure. The residue was dissolved in methylene chloride and stored overnight at 4°C. After filtration and concentration the slightly yellow oil was purified by distillation. 9.2 g (92%) of the desired compound were obtained as clear oil. 2-((2-(2-(2-Azidoethoxy)ethoxy)ethoxy)-methyl)oxirane (3). To a suspension of sodium hydride (151 mg, 6.28 mmol, 1.1 eq.) in dry THF (6 ml) 2-(2-(2-azidoethoxy)ethoxy)ethanol (2) (1 g, 5.7 mmol, 1.0 eq.) was added dropwise and the resulting mixture stirred for 2 h at room temperature. Epichlorohydrin (2.3 ml, 28.54 mmol, 5.0 eq.) were added dropwise and the mixture was stirred for 16 h at room temperature. The reaction mixture was then neutralised with 30% methanolic H2SO4 and poured into 40 ml brine. The aqueous layer was extracted two times with ethyl acetate (30 ml), and two times with methylene chloride (30 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The resulting oily residue was purified by flash column chromatography using a gradient from hexane / ether = 1 : 2 + 1% NH3 to hexane / ether = 1 : 5 + 1% NH3. 884 mg (67%) of the title compound were obtained as colourless oil.

4-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-3-hydroxybutanenitrile (4). To a solution of 2-((2-(2-(2- azidoethoxy)ethoxy)ethoxy)-methyl)oxirane (3) (500 mg, 2.16 mmol, 1.0 eq.) in ethanol (2 ml) water (10 ml) was added. After stirring for 5 min, sodium cyanide (153 mg, 3.03 mmol, 1.4 eq.) were added and stirring was continued for 16 h at room temperature. The reaction mixture was then concentrated to about half the volume under reduced pressure, extracted two times with ethyl acetate (20 ml), and one time with methylene chloride (20 ml), dried over Na2SO4 and concentrated. The oily residue was purified by flash column chromatography using DCM / MeOH = 97 : 3 as eluent. The desired compound were obtained as clear oil (498 mg, 89%).

Example 6

Synthesis of thymidine 5 '-triphosphate, protected at the 3'-0 position by a cleavable unit carrying additionally a detectable moiety.

It is well established that nucleoside containing a methylthiomethyl substituents can react upon activation with appropriate electrophile like sulfuryl chloride, N-bromosuccinimide or CuBr2 with a spectrum of nucleophilic reagents. Here, the secondary alcohol (2) was used as the nucleophile and was found to react smoothly with the activated compound (1) to form derivative (3), conforming the results of Sawada and Ito (Tet. Lett. 2001, 42, 2501-2504) who found that primary, secondary and even tertiary alcohols react efficiently to form appropriate formacetal derivatives. The following removal of MMTr group and introduction of the triphosphate residue was described in the earlier Examples. The reduction of the azido group upon treatment with triphenyl- or tributylphosphine in pyridine to form an amino function is a standard procedure of organic chemistry. The resulted amine can be reacted with a spectrum of labeling moieties. The conveniently detectable labels - fluorophores and the efficient methods for their activation to other chemical functions can be found in the catalogue from Molecular Probes. For instance application of fluorescein isothiocyanate as the derivatizing reagent results with the formation of title compound (4).

Example 7

Synthesis of uridine 5 '-triphosphate reversibly protected at the 3 '-position and conjugated on the base with a cleavable moiety carrying an additional detectable function.

NaHCO₃ or Et₃N

The alcohol (1) can react with carbonyl diimidazole (CDI), disuccinamidyl carbonate (DSC) or phosgene to form a reactive derivative (2). Such derivatives can react with primary and secondary amines like the amine group present in the compound (3) to form the carbamido derivative (4). All reactions represent standard procedures, known from the textbooks of organic chemistry. A following process consisting of four consecutive reactions is introducing the triphosphate moiety on the 5 '-position, and a labeling function on the base. These steps were listed in the Example above. Example 8 Cleavage of the 3'-O- CEM from a primer containing this modification.

Synthesis of the 24-mer oligonucleotide 5' TGC AGGC ATGC AAGCTTGGCGT AT -CEM 3 'containing the cyanoethoxymethyl group at the 3 '-end was performed in 5' to 3' direction using commercial inverted amidites and inverted support. The final coupling was made using thymidine 3'-0-CEM -5'-phosphoramidite made in our laboratory. The deprotected and HPLC purified material was treated with TBAF under different conditions, desalted and analyzed.

Experiments were performed according to the specific conditions presented for every RP-HPLC chromatogram.

The analysis was made on Gemini C 18, Phenomenex HPLC column using buffer A: 5% ACN in

0.1M TEAAc and buffer B: 80% ACN in 0.1M TEAAc, gradient: 0-40% B in 12 min.

Example 9

Cleavage of the 3'-O- CE from a primer containing this modification. TBAF solution (IM in dry THF, 50OmL) was added to -400 pmol 3 '-O-CE labelled primer and the sample was incubated at 37 ⁰C for 5min. Water (50OmL) was added to quench the reaction and the sample was desalted (NAPlO) followed by RP-HPLC analysis. The analysis was made on Gemini C18, Phenomenex, Buffer A: 5% ACN in 0.1M TEAAc, Buffer B: 80% ACN in 0.1 M TEAAc, gradient: 0-40% B in 12 min

1

prim e r 2 (without CNE)

{ 3 7 8 9

Example 10

Template-based incorporation of TTP-CEM by RevertAid, and stability of the terminating function during the reaction.

Full extension f" products

5 15 I 60 I 90

Incubation time, πύn

Experimental scheme:

2,7μM RevertAid M-MuLV RT was incubated for the indicated time at 37°C (buffer Tango) with 50μM TTP-CNE and 10OnM of labeled DNA duplex.

After 90 min. of incubation, lOOμM TTP was added and reaction was allowed to proceed for an additional 15 minutes at 37°C. Example 11

Template-based incorporation of TTP-CEM into DNA primer catalyzed by HIV-I polymerase.

1 - DNA after the incubation with the HIV-I polymerase in the presence of TTP-CEM;

2 - The same as in lane 1, but after the addition of TTP and incubation for an additional 15 minutes Experimental scheme:

Polymerases was incubated for a 30 min at 37°C (buffer Tango) with 50 μM TTP-CEM and 1OnM of labeled DNA duplex.

For 2, reaction samples were taken, enriched with lOOμM of TTP and further incubated for an additional 15 minutes in order to test if reaction products contain the blocking 3' group (and thus are resistant to the extension).

Example 12

Template-based incorporation of TTP-CE into DNA by HIV polymerases

K 1 2 1 2

HiV-1 HIV-2 1 - DNA after the incubation with the indicated polymerase in the presence of dTTP-CE;

2 - the same as in lane 1, but after the addition of dTTP and incubation for an additional 15 minutes.

Experimental scheme:

Polymerases were incubated for a 60 min at 37°C (buffer Tango) with 50 μM TTP-CE and 10 nM of labeled DNA duplex.

For Probe 2, reaction samples were taken, enriched with lOOμM of TTP and further incubated for an additional 15 minutes in order to test if reaction products contain the blocking 3' group (and thus are resistant to the extension).

Claims

1. A compound of the general structure I:

or a salt thereof, wherein:

B is a nucleobase,

X and Y independently are oxygen or sulphur,

Z is a chemical group characterized by strong electron withdrawing properties,

Rl is hydrogen, hydroxyl or a protected hydroxyl, R2 and R3 are together or separately hydrogen or a hydrocarbyl,

R4 is hydrogen or a chemical moiety consisting of a linker molecule L, linking a detectable group to the rest of the structure (I), R5 is hydrogen or an additional electron withdrawing group, R5 can be identical with Z or different, R6 is hydrogen or a chemical moiety consisting of a linker unit L, a cleavable group and a detectable group joined together in the following order R6 = -L-cleavable group-detectable group, while the detectable group is placed either on R4, or R6 or is absent, thus either R4 is hydrogen the detectable group is placed on R6, or R6 is hydrogen and the detectable group is placed on R4, or both R4 and R6 are hydrogens, m and n are independently 0 or 1.

2. A compound according to claim 1 wherein Z is selected from the group containing a cyano (CN), trifluoromethyl (CF3), trichloromethyl (CC13), tribromomethyl (CBr3), sulfone, sulfoxide or nitro (NO2) group.

3. A compound according to claim 1 and 2 wherein the detectable label is located on the nucleobases via the substituent R6.

4. A compound according to claims 1 to 3 wherein the detectable label is a fluorescent label.

5. A compound according to any of the preceding claims wherein the fluorescent label is selected from the group containing fluorescein, substituted fluorescein, rhodamin, substituted rhodamin, Texas Red, bodipy, Cy 3, Cy 4 and Cy 5.

6. A compound according to any of the preceding claims wherein Z = CN and R2 = R3 = R4 = R5 = hydrogen and the whole 3'-O- substituent thus becoming cyanoethoxy methyl group -CH2-O-CH2-CH2-CN.

7. A compound according to any of the preceding claims wherein n= 0, Z = CN, R4 = R5 = hydrogen and the whole 3'-O- substituent thus becoming cyanoethyl group -CH2-CH2-CN.

8. A compound according to any of the preceding claims wherein n = 0, m = 0, Z = CN, R5 = hydrogen and the whole 3'-O- substituent thus becoming cyanomethyl group -CH2-CN.

9. A compound according to any of the preceding claims being capable to regenerate free 3'- hydroxyl upon the cleavage of the 3'-O-substituent.

10. A compound according to any of the preceding claims being capable to regenerate free 3'- hydroxyl upon cleavage of the 3'-O-substituent by means of fluoride anions.

11. A compound according to claims 1 to 9 being capable to regenerate free 3'- hydroxyl upon the cleavage of the 3'-O-substituent by means of a nucleophile ions.

12. A compound according to claims 11 wherein the nucleophilic ions capable to cleave the 3'- O- substituent are selected from the group of thiols, alkoxy ions, and hydroxyl.

13. A compound according to claims 1 to 9 being capable to regenerate free 3'- hydroxyl upon the cleavage of the 3'-O-substituent by means of strong bases.

14. A compound according to claims 11 wherein the strong bases capable to cleave the 3'-O- substituent are selected from the group of metal hydroxides, and amines.

15. A compound according to claim 3 wherein the detectable label is connected to the nucleobase via a cleavable group.

16. A compound according to claim 15 wherein the cleavable group is cleaved under the same conditions as it is used to cleave the 3'- O protecting group.

17. A compound according to claim 16 wherein the cleavable group is cleavable upon treatment with fluoride anions.

18. A compound according to claim 16 and 17 wherein the cleavable group is selected from any of existing beta eliminating functions, or siloxyl or disyloxyl group

19. A compound according to claim 15 wherein the cleavable group is cleaved under different conditions then what is needed to cleave the 3'- O protecting group.

20. A compound according to claim 19 wherein the cleavable group is selected from a group consisting of functions cleavable under reducing conditions, functions cleavable under oxidative conditions, functions cleavable under photo illumination conditions, functions cleavable upon treatment with transient metal ions, functions cleavable under acidic conditions.

21. A compound according to claim 1 and 2 wherein the detectable label is located on the on the function protecting the 3 '-OH via the substituent R6.

22. A compound according to claim 21 wherein the fluorescent label is selected from the group containing fluorescein, substituted fluorescein, rhodamin, substituted rhodamin, Texas Red, bodipy, Cy 3, Cy 4 and Cy 5.

23. A method for determining the sequence of a nucleic acid characterized by: a) providing a single-stranded template comprising the nucleic acid to be determined, b) hybridizing a primer to said template c) at least partially synthesizing a complementary nucleic acid molecule in a stepwise serial manner by the addition of nucleotides in which the identity of each nucleotide incorporated into the complementary nucleic acid molecule is determined subsequent to its incorporation, wherein said nucleotides are compounds of Formula I as defined in any one of claims 1 to 22, and wherein the 3 '-blocking group is removed from the nucleotide after its incorporation to permit further extension of the nucleic acid molecule.

24. The method according to claim 23, wherein the 3'- blocking group is removed by treatment with fluoride anions.

25. The method according to claim 23, wherein the 3'- blocking group is removed by treatment with nucleophilic reagents.

26. The method according to claim 23 wherein the detectable group is present on the nucleobase and the cleavable group is cleaved under the same conditions as it is used to cleave the 3'- O protecting group.

27. The method according to claim 23 to 26 wherein the detectable group is present on the nucleobase, and the cleavable group located on the base is cleaved upon treatment with fluoride anions.

28. The method according to claim 23, 24, 25, 26 and 27 wherein the cleavable group located on the base is selected from any of existing beta eliminating functions, or siloxyl or disyloxyl group

29. The method according to claim 23 wherein the cleavable group located on the base is cleaved under different conditions then what is needed to cleave the 3'- O protecting group.

30. The method according to claim 29 wherein the cleavable group is selected from a group consisting of functions cleavable under reducing conditions, functions cleavable under oxidative conditions, functions cleavable under photo illumination conditions, functions cleavable upon treatment with transient metal ions, functions cleavable under acidic conditions.

31. A method according to claim 23, wherein the extension is done by a polymerase.

32. A method according to claim 31, where the polymerase is modified to better accept the modified nucleoside 5 '-triphosphate of Claim 1.

33. A method according to claim 23, where the primer is attached to a solid support.

34. A method according to claim 23, where at least two primers with different sequences are attached to the same solid support.

35. A method according to claim 23, where a large set of primers with different sequences are attached to a solid support forming an array.

36. A method according to claim 23 to claim 35, where the solid support is made of material that do not interact with the reagent used for the cleavage of the 3'-O-protecting group and is not reacting with the reagent used for the cleavage of the cleavable group connected to R6.

37. A method according to claim 23 to claim 35, where the solid support that otherwise reacts with the reagents used for the cleavage of 3'- O-protecting group and for the cleavage of the cleavable group connected to R6, is covered by another material, that do not interact with the reagent used for the cleavage of the 3 '-O-protecting group and is not reacting with the reagent used for the cleavage of the cleavable group connected to R6.

38. Method of preparing a compound of claim 1-22.

39. Kit comprising, in separate containers, the compound of claim 1-22, and necessary enzymes and/or nucleotides and/or reagents for preparing oligo- or polynucleotides.

40. Kit comprising, in separate containers, the compound of claim 1-22, and necessary enzymes and/or nucleotides and/or reagents, for determining the sequence of a nucleic acid.