ES2786983T3 - Modified nucleotides for polynucleotide sequencing - Google Patents

Abstract

A nucleotide molecule having a ribose or deoxyribose sugar moiety and a base linked to a detectable marker through a cleavable linker, wherein the sugar moiety comprises a blocking group attached through a 3 'oxygen atom , in such a way that the 3 'carbon atom has attached a group of the structure: -OZ where Z is -C (R') 2-N3; where each R 'is H.

Description

DESCRIPTION

Modified nucleotides for polynucleotide sequencing

The invention relates to modified nucleotides. In particular, this invention discloses nucleotides having a removable protecting group, their use in polynucleotide sequencing methods and a method for chemical deprotection of the protecting group.

Advances in the study of molecules have been guided, in part, by improvements in technologies used to characterize molecules or their biological reactions. In particular, the study of nucleic acids DNA and RNA has benefited from developing techniques used for sequence analysis and the study of hybridization events.

An example of technologies that have improved the study of nucleic acids is the development of fabricated arrays of immobilized nucleic acids. These arrangements typically consist of a high density matrix of polynucleotides immobilized on a solid support material. See, for example, Fodor et al., Trends Biotech. 12: 19-26, 1994, which describes ways of assembling nucleic acids using a chemically sensitized glass surface protected by a mask, but exposed in defined areas to allow the binding of appropriately modified nucleotide phosphoramidites. The fabricated arrays can also be manufactured by the technique of "spotting" known polynucleotides on a solid support at predetermined positions (eg, Stimpson et al., Proc. Natl. Acad. Sci. USA 92: 6379-6383, 1995). Sequencing by DNA synthesis ideally requires the controlled incorporation (that is, one at a time) of the correct complementary nucleotide opposite to the oligonucleotide being sequenced. This allows for precise sequencing by adding nucleotides in multiple cycles as each nucleotide residue is sequenced one at a time, thus avoiding the occurrence of an uncontrolled series of incorporations. The incorporated nucleotide is read using an appropriate tag attached to it prior to removal of the tagged building block and the subsequent subsequent round of sequencing. In order to ensure that only single incorporation occurs, a structural modification ("blocking group") of nucleotides in sequencing is required to ensure individual nucleotide incorporation but to avoid any additional nucleotide incorporation into the polynucleotide chain. The blocking group must be removable, under reaction conditions that do not interfere with the integrity of the DNA being sequenced. The sequencing cycle can then continue with the incorporation of the next blocked, labeled nucleotide. In order to be of practical use, the entire process should consist of highly specific, high throughput chemical and enzymatic steps to facilitate multiple cycles of sequencing.

To be useful in DNA sequencing, the nucleotide, and more usually the nucleotide triphosphates, generally require a 3'OH blocking group in such a way as to prevent the polymerase used to incorporate it into a polynucleotide chain from continuing to replicate once the base on the nucleotide is added. There are many limitations on the suitability of a molecule as a blocking group. It should be such that it prevents additional nucleotide molecules from being added to the polynucleotide chain while simultaneously being easy to remove from the sugar moiety without causing damage to the polynucleotide chain. Additionally, the modified nucleotide must be tolerated by the polymerase or other appropriate enzymes used to incorporate it into the polynucleotide chain. The ideal blocking group will therefore exhibit long-term stability, be efficiently incorporated by the polymerase enzyme, produce a total blocking of secondary or additional incorporations, and have the ability to be removed under moderate conditions that do not damage the polynucleotide structure. , preferably under aqueous conditions. These stringent requirements are formidable obstacles to the design and synthesis of the required modified nucleotides.

Reversible blocking groups for this purpose have been previously described but none of them generally satisfy the above criteria for polynucleotide chemistry, eg, that they are compatible with DNA. Metzker et al., (Nucleic Acids Research, 22 (20): 4259-4267, 1994) discloses the synthesis and use of eight 3 'modified 2-deoxyribonucleoside 5'-triphosphates (3' modified dNTPs) and their testing in two DNA template assays for incorporation activity. The 3 'modified dNTPs included 3'allyl deoxyriboadenosine 5'-triphosphate (3'-allyl ATP). However, the 3'alyl-blocked compound was not used to demonstrate a complete cycle of termination, deprotection, and reinitiation of DNA synthesis: the only test results presented were those that showed the ability of this compound to terminate synthesis. of DNA in a single termination assay, and of the eight such assays carried out, each was carried out with a different DNA polymerase.

WO 02/29003 (The Trustees of Columbia University in New York City) describes a sequencing method which may include the use of an allyl protecting group to cap the 3'-OH group in a growing strand of DNA in a reaction with polymerase. The allyl group is introduced according to the Metzker procedure ( infra) and is said to be removed using the methodology reported by Kamal et al (Tet. Let, 40, 371-372, 1999).

Kamal's deprotection methodology employs sodium iodide and chlorotrimethylsilane in such a way that Generate the iodotrimethylsilane in situ , in acetonitrile solvent, stopping it with sodium thiosulfate. After extraction into ethyl acetate and drying (sodium sulfate), subsequent concentration under reduced pressure and column chromatography (ethyl acetate: hexane; 2: 3 as eluent), the free alcohols were obtained in a yield of 90 -98%.

In WO 02/29003, the deprotection of allyls according to Kamal is suggested as directly applicable in the sequence of DNA without modification, the Kamal conditions being moderate and specific.

While Metzker reports the preparation of a 3'alyl blocked nucleotide or nucleoside and WO 02/29003 suggests the use of allyl functionality as a cap for 3'-OH during sequencing, none of these papers actually teach the deprotection of a hydroxyl group with 3'-allyl in the context of a sequencing protocol. While the use of an allyl group as a hydroxyl protecting group is well known - it is easy to introduce and stable throughout the entire pH range and at elevated temperatures - there is to date no concrete realization of a successful cleavage of a 3'-allyl group under conditions compatible with DNA, that is, conditions under which the integrity of the DNA is not totally or partially destroyed. In other words, it has not been possible to date to carry out DNA sequencing using 3'OH allyl blocked nucleotides.

Kamal's methodology is inappropriate to be carried out in an aqueous medium since the TMS chloride will hydrolyze avoiding the in situ generation of TMS iodide. Attempts to carry out Kamal deprotection (in acetonitrile) in sequencing have been unsuccessful in our hands.

The present invention is based on the surprising development of a number of reversible blocking groups and methods for deprotecting them under conditions compatible with DNA. Some of these blocking groups are novel per se; others have been disclosed in the prior art but, as noted above, the possibility of using these blocking groups in DNA sequencing has not been demonstrated. WO91 / 06678 discloses DNA sequencing methods.

Nucleosides, Nucleotides and Nucleic Acids., Vol. 19, no 10-12, pages 1977-1991 discloses the synthesis of derivatives of ribonucleosides 2'- and 3'-O-azidomethyl.

Protecting groups comprising acetal functionality have previously been used as blocking groups. However, the removal of such groups and ethers requires strongly acidic deprotections that are harmful to DNA molecules. However the hydrolysis of an acetal results in the formation of an unstable hemiacetal intermediate which hydrolyzes under aqueous conditions to the natural hydroxyl group. The inventors have used this concept and have subsequently applied it in such a way that this feature of the invention resides in the use of blocking groups that include protecting groups to protect intermediate molecules that would normally hydrolyze under aqueous conditions. These protecting groups comprise a second functional group that stabilizes the structure of the intermediate but can be removed at a later stage after incorporation into the polynucleotide. Protecting groups have been used in organic synthesis reactions to temporarily mask the characteristic chemistry of a functional group since it interferes with another reaction.

The invention is defined by the claims according to a first aspect wherein a modified nucleotide or nucleoside molecule is provided comprising a purine or pyrimidine base and a ribose or deoxyribose sugar structural unit having a removable 3'-OH blocking group attached. covalently to it, such that the 3 'carbon atom has an attached group of the structure

-O-Z

where Z is any of -C (R ') 2-OR ", -C (R') 2-N (R") 2, -C (R ') 2-N (H) R ", -C ( R ') 2-SR "and C (R') 2-F,

wherein each R "is or is part of a removable protecting group;

each R 'is independently a hydrogen atom, an alkyl, substituted alkyl, arylalkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclic, acyl, cyano, alkoxy, aryloxy, heteroaryloxy, or amido group, or a detectable label attached through a linking group; or (R ') 2 represents an alkylidene group of the formula = C (R "') 2 wherein each R" 'may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; Y

wherein said molecule can be reacted to produce an intermediate in which each R "is exchanged for H or, where Z is -C (R ') 2-F, the F is exchanged for OH, SH or NH2, preferably OH, which immediately dissociates under aqueous conditions to generate a molecule with a free 3'OH;

with the proviso that where Z is -C (R ') 2-S-R ", both R' groups are not H.

In a further aspect the disclosure provides a method for controlling the incorporation of a nucleotide molecule complementary to the nucleotide in a target single stranded polynucleotide in a synthesis reaction. or sequencing comprising incorporating into the growing complementary polynucleotide a molecule according to the disclosure, such that the incorporation of said molecule prevents or blocks the introduction of subsequent nucleoside or nucleotide molecules into said growing complementary polynucleotide.

In a further aspect, the disclosure provides a method for determining the sequence of a target single-stranded polynucleotide, which comprises monitoring the sequential incorporation of complementary nucleotides, wherein at least one incorporation, and preferably all incorporations are of one nucleotide according to with the invention as described hereinabove which preferably comprises a detectable label linked to the base of the nucleoside or nucleotide by means of a cleavable linker and wherein the identity of the incorporated nucleotide is determined by detecting the label, said blocking group and said label being removed before of the introduction of the next complementary nucleotide.

From a further aspect, the disclosure provides a method for determining the sequence of a target single-stranded polynucleotide, comprising:

(a) providing a plurality of different nucleotides according to the above-described disclosure, nucleotides that preferably are linked from the base to a detectable marker by a cleavable linker and wherein the detectable marker linked to each type of nucleotide can be distinguished by detection of the detectable marker used for other types of nucleotides;

(b) incorporating the nucleotide into the complement of the target single stranded polynucleotide;

(c) detecting the nucleotide marker of (b), thereby determining the type of nucleotide incorporated;

(d) removing the nucleotide tag from (b) and the blocking group; Y

(e) optionally repeating steps (b) - (d) one or more times;

thereby determining the sequence of a target single stranded polynucleotide.

Additionally, in another aspect, the disclosure provides a kit, comprising:

(a) a plurality of different individual nucleotides of the invention; Y

(b) packaging materials therefor.

The nucleosides or nucleotides according to or used in the methods of the present disclosure comprise a purine or pyrimidine base and a ribose or deoxyribose sugar structural unit which has a blocking group covalently linked thereto, preferably at the 3'O position. , which makes the molecules useful in techniques that require blocking of the 3'-OH group to avoid the incorporation of additional nucleotides, such as for example in sequencing reactions, polynucleotide synthesis, nucleic acid amplification, hybridization assays nucleic acid studies, single nucleotide polymorphism studies, and other such techniques.

When the term "blocking group" is used herein in the context of the invention, it encompasses both the allyl and "Z" blocking groups described herein. However, it will be apparent that, in the methods as described and claimed herein, where mixtures of nucleotides are used, these most preferably each comprise the same type of blocker, that is, "Z" blocked. When "Z" blocked nucleotides are used, each "Z" group will generally be the same group, except in those cases where the detectable marker is part of the "Z" group, that is, it is not bound to the base.

Once the blocking group has been removed, it is possible to add another nucleotide to the free 3'-OH group.

The molecule can be linked through the base to a detectable label by a desirable linker, which linker can be a fluorophore, for example. If desirable, the detectable label may instead be incorporated into the blocking groups of formula "Z". The binder can be acid labile, photolabile, or contain a disulfide bond. Other linkages, in particular azide-containing phosphine cleavable linkers, may be employed in the invention as described in greater detail.

Preferred markers and linkers include those disclosed in WO 03/048387.

Methods where nucleotides are incorporated, for example, where the incorporation of a nucleotide molecule complementary to the nucleotide in a target single-stranded polynucleotide is controlled in a synthesis or sequencing reaction of the invention, incorporation of the molecule can be achieved. through a terminal transferase, a polymerase or a reverse transcriptase.

Preferably, the molecule is incorporated by a polymerase and particularly from Thermococcus sp., Such as a 9 ° N. Even more preferably, the polymerase is a 9 N A485L mutant and even more preferably it is a Y409V and A485L double mutant.

In methods for determining the sequence of a target single-stranded polynucleotide comprising monitoring the sequential incorporation of complementary nucleotides of the invention, it is preferred that the blocking group and the label can be removed in a single chemical treatment step. Thus, in a preferred embodiment of the invention, the blocking group is cleaved simultaneously with the marker. This will of course be an inherent characteristic of those blocking groups of formula Z that incorporate a detectable label.

Additionally, preferably the modified and labeled blocked and modified nucleotide constructs of nucleotide bases A, T, C and G are recognized as substrates by the same polymerase enzyme.

In the methods described here, each of the nucleotides can be contacted with the target sequentially, with removal of unincorporated nucleotides prior to the addition of the next nucleotide, where detection and removal of the marker and blocking group is carried out. either after the addition of each nucleotide, or after the addition of all four nucleotides.

In the methods, all nucleotides can be contacted with the target simultaneously, that is, a composition comprising all the different nucleotides is contacted with the target, and unincorporated nucleotides are removed prior to detection and subsequent to elimination of the marker and the blocking group.

The methods may comprise a first stage and a second stage, wherein in the first stage, a first composition comprising two of the four types of modified nucleotides is brought into contact with the target, and the unincorporated nucleotides are removed prior to removal. detection and subsequent removal of the marker and blocking group, and where in the second step, a second composition comprising the two nucleotides not included in the first composition is brought into contact with the target, and the unincorporated nucleotides are removed before of detection and subsequent removal of the marker and blocking group, and wherein the first steps and the second step may optionally be repeated one or more times.

In the methods described herein they may also comprise a first stage and a second stage, where in the first stage, a composition comprising one of the four nucleotides is brought into contact with the target, and the unincorporated nucleotides are removed before detection. and subsequent to the removal of the marker and the blocking group, and where in the second step, a second composition comprising the three nucleotides not included in the first composition is brought into contact with the target, and the unincorporated nucleotides are removed prior to detecting and subsequently removing the marker and blocking group, and wherein the first steps and the second step can optionally be repeated one or more times.

The methods described herein may also comprise a first stage and a second stage, wherein in the first stage, a first composition comprising three of the four nucleotides is brought into contact with the target, and the unincorporated nucleotides are removed prior to removal. detection and subsequent removal of the marker and blocking group and where in the second step, a composition comprising the nucleotide not included in the first composition is brought into contact with the target, and the unincorporated nucleotides are removed prior to detection and subsequent to the removal of the marker and the blocking group, and wherein the first steps and the second step can optionally be repeated one or more times.

The incorporation step in the methods of the invention can be achieved through a terminal transferase, a polymerase or a reverse transcriptase as defined hereinbefore. The detectable label and / or the cleavable linker may be of sufficient size to avoid incorporation of a second nucleotide or nucleoside into the nucleic acid molecule.

In certain methods described herein for determining the sequence of a target single-stranded polynucleotide, each of the four nucleotides, one of which will be complementary to the first unpaired base in the target polynucleotide, can be contacted with the Target sequentially, optionally with removal of unincorporated nucleotides prior to addition of the next nucleotide. The determination of the incorporation success can be carried out either after the provision of each nucleotide, or after the addition of all the added nucleotides. If it is determined after the addition of less than four nucleotides that one of them has been incorporated, it is not necessary to provide additional nucleotides in order to detect nucleotides complementary to the incorporated nucleotide.

Alternatively, all nucleotides can be contacted with the target simultaneously, that is a composition comprising all the different nucleotides (i.e. A, T, C and G or A, U, C and G) is contacted with the target. target, and unincorporated nucleotides are removed prior to marker detection and removal. Methods that involve sequential nucleotide adhesion may comprise a first substep and optionally one or more subsequent substeps. In the first sub-stage a composition is provided comprising one, two or three of the four possible nucleotides, that is, it is brought into contact with the target. Any unincorporated nucleotides can then be removed and a detection step can be carried out to determine if one of the nucleotides has been incorporated. If one of them has been incorporated, cleavage of the linker can be effected. In this way the identity of a nucleotide in the target polynucleotide can be determined. The nascent polynucleotide can then be extended to determine the identity of the next unpaired nucleotide in the target oligonucleotide.

If the previous first substep does not lead to the incorporation of a nucleotide, or if this is not known, since the presence of incorporated nucleotides is not looked for immediately after the first substep, one or more subsequent substeps may be carried out in which some or all of those nucleotides not provided in the first substep are provided either, as appropriate, simultaneously or subsequently. After this any unincorporated nucleotides can be removed and a detection step can be carried out to determine if one of the nucleotide classes has been incorporated. If one of them has been incorporated, cleavage of the linker can be effected. In this way, the identity of a nucleotide in the target polynucleotide can be determined. The nascent polynucleotide can then be extended to determine the identity of the next unpaired nucleotide in the target oligonucleotide. If necessary, a third and optionally a fourth sub-stage can be performed in a similar manner to the second sub-stage. Obviously, once four substeps have been performed, all possible nucleotides will have been provided and one of them will have been incorporated.

It is desirable to determine whether a nucleotide type or class has been incorporated after any particular combination comprising one, two or three nucleotides has been provided. In this way, the unnecessary cost and time spent in providing the other nucleotides is obviated. However, this is not a required feature of the invention.

It is also desirable, when the sequencing method comprises one or more sub-steps, to remove any unincorporated nucleotides before new nucleotides are provided. Again, this is not a required feature of the invention. Obviously, it is necessary that at least some and preferably as many of the unincorporated nucleotides as practicable be removed prior to detection of the incorporated nucleotide. Kits of the disclosure include: (a) individual nucleotides in accordance with the invention described hereinbefore, wherein each nucleotide has a base that is linked to a detectable label through a cleavable linker, or a detectable label linked through a linker cleavable to a blocking group of formula Z, and where the detectable label attached to each nucleotide can be distinguished by detecting the detectable label used for the other three nucleotides; and (b) packaging materials therefor. The kit may additionally include an enzyme to incorporate the nucleotide into the complementary nucleotide chain and appropriate regulators for the action of the enzyme in addition to the appropriate chemical agents for removal of the blocking group and the detectable label, which can preferably be removed by the same stage of chemical treatment.

Nucleotides / nucleosides are suitable for use in many different DNA-based methodologies, including protocols for DNA synthesis and DNA sequencing.

The invention can be understood with reference to the accompanying drawings in which:

Figure 1 shows exemplary nucleotide structures. For each structure, X can be H, phosphate, diphosphate or triphosphate. R ¹ and R ² can be the same or different, and can be selected from H, OH, or any group that can be transformed into an OH, including, but not limited to, a carbonyl. Some suitable functional groups for R ¹ and R ² include the structures shown in Figure 3 and Figure 4.

Figure 2 shows linker structures, including (1) disulfide linkers and acid-labile linkers, (2) dialkoxybenzyl linkers, (3) Sieber linkers, (4) indole linkers, and (5) Sieber t-butyl linkers. . Figure 3 shows some functional molecules, including some cleavable linkers and some suitable hydroxyl protecting groups. In these structures, R ¹ and R ² can be the same or different, and can be H, OH, or any group that can be transformed into an O ^h group, including a carbonyl. R ³ represents one or more substituents independently selected from alkyl, alkoxy, amino or halogen groups. R ⁴ and R ⁵ can be H or alkyl, and R ⁶ can be alkyl, cycloalkyl, alkenyl, cycloalkenyl, or benzyl. X can be H, phosphate, diphosphate or triphosphate.

Figure 4 is a schematic illustration of some of the Z blocking groups that can be used.

Figure 5 shows two cycles of incorporation of labeled and blocked DGTP, DCTP and dATP respectively (compounds 18, 24 and 32).

Figure ⁶ shows six cycles of incorporation of labeled and blocked DTTP (compound ⁶ ).

The present invention relates to nucleotide or nucleoside molecules that are modified by binding reversible covalent of 3'-OH blocking groups thereto, and molecules that can be used in reactions where blocked nucleotide or nucleoside molecules are required, such as in sequencing reactions, polynucleotide synthesis and the like.

As used herein the term "biological molecule" is used to encompass any molecule or class of molecule that performs a biological role. Such molecules include, for example, polynucleotides such as DNA or RNA, oligonucleotides, and individual nucleotides. In addition, peptides and peptide mimics, such as enzymes and hormones, etc. are covered.

Particularly preferred compounds however are polynucleotides (including oligonucleotides) and nucleotides and nucleosides, preferably those that contain a base to which a detectable marker linked through a cleavable linker is attached. Such compounds are useful in determining oligonucleotide sequences as described herein.

Suitable ligands are any phosphine or mixed nitrogen-phosphine ligands known to those skilled in the art, characterized in that these ligands are derived in such a way that they become soluble in water, for example, by introducing one or more sulfonate, amine, hydroxyl residues (preferably a plurality of hydroxyl) or carboxylate). When amine residues are present, the formation of amine salts can aid in the solubilization of the ligand, and thus the metal-allyl complex. Examples of suitable ligands are triaryl phosphines, for example triphenyl phosphine, derived in such a way that it becomes soluble in water. Also preferred are trialkyl phosphines, for example tri-C ^1-6 -alkyl phosphines such as triethyl phosphines; such trialkyl phosphines are derived in the same way such that they become soluble in water. The sulfonate-containing and carboxylate-containing phosphines are particularly preferred; an example of the first 3,3 ', 3 "-phosphinidintris (benzenesulfonic acid) which is commercially available from Aldrich Chemical Company as the trisodium salt; and a preferred example of the latter is tris (2-carboxyethyl) phosphine which is available from Aldrich as the hydrochloride salt.

The derived water-soluble phosphines and nitrogen-containing phosphines described herein can be used as their salts (for example as the hydrochloride or sodium salts) or, for example, in the case of the sulfonic and carboxylic acid containing phosphines described here, in the form of the free acids. Thus 3,3 ', 3' '-phosphinidinetris (benzenesulfonic acid) and tris (2-carxyethyl) phosphines can be introduced either as triacids or trisodium salts. Other suitable salts will be apparent to those skilled in the art. Existence in saline form is not particularly important since phosphines are soluble in aqueous solution.

Other ligands that can be used include the following:

as hydrochloride salt, compound as hydrochloride salt, compound

neutral and trisodium salt neutral and trisodium salt

The intermediates produced advantageously spontaneously dissociate under aqueous conditions to return to the natural 3 'hydroxy structure, allowing for further incorporation of another nucleotide.

An example of groups of structure -OZ where Z is C (R ') ² -N (R ^{") 2} are those in which - N (R ^{") 2} is azido (-N ³ ). One such example is azidomethyl where each R 'is H.

The "Het" groups are of the formula C (R ') ² N ³ in which the R' groups are hydrogen

When the molecules contain Z groups of formula C (R ') ² N3, the azido group can be converted to amino by contacting such molecules with the phosphine or nitrogen-containing phosphine ligands described in detail in connection with the metal complexes. of transition that serve to cleave the allyl groups of the compounds of formula PN-O-allyl, formula RO-allyl, RaN (allyl), RMH (allyl ^{) 2} and RS-allyl. When transforming azido to amino, however, a transition metal is not necessary. Alternatively, the azido group in the Z groups of formula C (R ') ² N ³ can be converted to amino by contacting such molecules with the thiols, in particular water soluble thiols such as dithiothreitol (DTT).

As is known in the art, a "nucleotide" consists of a nitrogen base, a sugar, and one or more phosphate groups. They are monomeric units of a nucleic acid sequence. In RNA, the sugar is a ribose, and in DNA a deoxyribose, that is, a sugar lacking a hydroxyl group that is present on ribose. The nitrogen base is a derivative of purine or pyrimidine. The purines are adenine (A) and guanine (G) and the pyrimidines are cytosine ( C) and thymine (T) (in the context of RNA, uracil (U)). The C-1 atom of deoxyribose is linked to N-1 of a pyrimidine or N-9 of a purine. A nucleotide is also a phosphate ester or nucleoside, with esterification present at the hydroxyl group attached to the sugar C-5. Nucleotides are usually mono, di- or triphosphates.

A "nucleoside" is structurally similar to a nucleotide, but lacks the phosphate building blocks. An example of a nucleoside analog would be one in which the label is attached to the base and there is no phosphate group attached to the sugar molecule.

Although base usually refers to a purine or a pyrimidine, the skilled person will know that derivatives and analogs are available that do not alter the ability of the nucleotide or nucleoside to undergo Watson-Crick base pairing. "Derivatives" or "analogs" means a compound or molecule whose central structure is the same as or closely similar to that of an parent compound, but which has a chemical or physical modification, such as a different or additional side group, or groups 2 'and / or 3' blockers, which allow the nucleotide or nucleoside derivative to bind to another molecule. For example, the base can be a de-glitter. Derivatives should be able to undergo Watson-Crick mating. "Derivative" and "analog" also mean a nucleotide or synthetic nucleoside derivative having modified base structural units and / or modified sugar structural units. Such derivatives and analogs are discussed, for example, in Scheit, Nucleotide Analogs (John Wiley & Son, 1980) and Uhlman et al., Chemical Reviews 90: 543-584, 1990. Nucleotide analogs can also comprise modified phosphodiester linkages, including phosphorothioate, phosphorodithioate, alkyl phosphonate, phospharanilidate, and phosphoramidate linkages. Analogs should be able to undergo Watson-Crick base pairing. "Derivatives", "analogs" and "modified" as used herein can be used interchangeably, and are encompassed by the terms "nucleotides" and "nucleosides" defined herein.

In the context of the present invention, the term "incorporating" means becoming part of a nucleic acid molecule (eg DNA) or oligonucleotide or primer. An oligonucleotide refers to a synthetic or natural molecule that comprises a covalently linked sequence of nucleotides that is formed by a phosphodiester or modified phosphodiester bond between the 3 'position of pentose on a nucleotide from the 5' position of pentose in a nucleotide. adjacent.

The term "alkyl" covers straight chain, branched chain and cycloalkyl groups. Unless the context indicates otherwise, the term "alkyl" refers to groups having 1 to 10 carbon atoms, for example 1 to 8 carbon atoms, and typically 1 to 6 carbon atoms, for example 1 to 4 carbon atoms. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, 2-pentyl, 3-pentyl, 2-methyl butyl, 3-methyl butyl, and n-hexyl and its isomers.

Examples of cycloalkyl groups are those having 3 to 10 ring atoms, including particular examples those derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane and cycloheptane, bicycloheptane and decalin.

When alkyl groups (including cycloalkyl) are substituted, particularly when they form either R 'groups of the molecules of the invention, examples of appropriate substituents include halogen substituents or functional groups such as hydroxyl, amino, cyano, nitro, carboxyl and the like. . Such groups may also be substituents, where appropriate, for other R 'groups on the molecules of the invention.

The term amino refers to groups of the type NR * R ** where R * and R ** are independently selected from hydrogen, an alkyl group C ^1-6 (also referred to as C ^1-6 alkylamino or dialkylamino C ^{1 to 6} ).

The term "halogen" as used herein includes fluorine, chlorine, bromine, and iodine.

The nucleotide molecules of the present invention are suitable for use in many different methods where nucleotide detection is required.

DNA sequencing methods, such as those outlined in U.S. Patent No.

5,302,509 can be carried out using the nucleotides.

The present invention can make use of conventional detectable markers. Detection can be carried out by any suitable method, including fluorescence spectroscopy or by other optical means. The preferred marker is a fluorophore, which, after energy absorption, emits radiation at a defined wavelength. Many suitable fluorescent markers are known. For example, Welch et al. (Chem. Eur. J.

5 (3): 951-960, 1999) describes dansyl functionalized fluorescent building blocks that can be used in the present invention. Zhu et al. (Cytometry 28: 206-211, 1997) describes the use of Cy3 and Cy5 fluorescent markers, which can also be used in the present invention. Suitable markers for use are also disclosed in Prober et al. (Science 238: 336-341, 1987); Connell et al. (BioTechniques 5 (4): 342-384, 1987), Ansorge et al. (Nucl. Acids Res. 15 (11): 4593-4602, 1987) and Smith et al. (Nature 321: 674, 1986). Other commercially available fluorescent markers include, but are not limited to, fluorescein, rhodamine (including TMR, Texas red, and Rox), alexa, bodipy, acridine, coumarin, pyrene, benzanthracene, and the cyanines. Multiple markers can also be used in the invention. For example, FRET biofluorophor cassettes (Tet. Let. 46: 8867-8871, 2000) are well known in the art and can be used in the present invention. Multifluorine dendrimeric systems can also be used (J. Amer. Chem. Soc. 123: 8101-8108, 2001).

Although fluorescent labels are preferred, other forms of detectable labels will be apparent as useful to those of ordinary skill. For example, microparticles, including quantum dots (Empodocles et al., Nature 399: 126-130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72: 6025-6029, 2000) and microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97 (17): 9461-9466, 2000) can all be used. Multi-component markers can also be used in the invention. A multicomponent marker is one that is dependent on the interaction with an additional compound for its detection. The most common multicomponent marker used in biology is the biotin-streptavidin system. Biotin is used as the marker attached to the nucleotide base. The streptavidin is then added separately to allow detection to occur. Other multi-component systems are available. For example, dinitrophenol has a commercially available fluorescent antibody that can be used for detection.

The invention has been and will be described with reference to nucleotides. However, unless otherwise indicated, reference to nucleotides is also intended to apply to nucleosides. The invention will also be further described with reference to DNA, although the description will also apply to RNA, PNA, and other nucleic acids, unless otherwise indicated.

The modified nucleotides of the invention can use a cleavable linker to attach the tag to the nucleotide. The use of a cleavable linker ensures that the label, if required, can be removed after detection, avoiding any interference signal with any subsequently incorporated labeled nucleotide.

Those skilled in the art will know the utility of dideoxynucleoside triphosphates in so-called Sanger sequencing methods and related protocols (Sanger type), which are based on the randomized termination of strands in a particular type of nucleotide. An example of a Sanger-like sequencing protocol is the BASS method described by Metzker ( infra). Other Sanger-like sequencing methods will be well known to those of skill in the art.

The Sanger and Sanger-type methods generally operate by running an experiment in which eight types of nucleotides are provided, four of which contain a 3'OH group; and four of which omit the OH group and which are marked differently from each other. Nucleotides used that omit the 3'OH group - dideoxynucleotide - are conventionally abbreviated as ddNTPs. It is also known to the skilled person, since the ddNTPs are labeled differently, by determining the positions of the incorporated terminal nucleotides, and by combining this information, the sequence of the target oligonucleotide can be determined.

The nucleotides of the present invention, as will be recognized, may be useful in Sanger methods and related protocols since the same effect achieved using ddNTPs can be achieved using the novel 3'-OH blocking groups described here: both prevent the incorporation of subsequent nucleotides.

The use of the nucleotides according to the present invention in the Sanger and Sanger-type sequencing methods, wherein the linker connecting the detectable marker to the nucleotide may or may not be cleavable, forms a still further aspect of this invention. Viewed from this aspect, the invention provides the use of such nucleotides in a Sanger or Sanger-type sequencing method.

When the 3'-OH Z-blocked nucleotides are used in accordance with the present invention, it will be apparent that the detectable labels attached to the nucleotides need not be connected via cleavable linkers. Since in each case when a labeled nucleotide of the invention is incorporated, it is not necessary to subsequently incorporate nucleotides and thus the label does not need to be removed from the nucleotide.

Furthermore, it will be apparent that the monitoring of the incorporation of the 3'OH blocked nucleotides can be determined by using radioactive 32P at the attached phosphate groups. These can be present either in the ddNTPs themselves or in the primers used for extension. When the blocking groups are of formula "Z", this represents a further aspect of the invention.

Viewed from this aspect, the invention provides the use of a nucleotide having a 3'OH group blocked with a "Z" group in a Sanger or Sanger-type sequencing method. In this embodiment, a detectable 32P marker may be present in either any of the ddNTPs used in the primer used for extension.

Cleavable linkers are known in the art, and conventional chemistry can be applied to attach a linker to a nucleotide base and a label. The linker can be cleaved by any suitable method, including exposure to acids, bases, nucleophiles, electrophiles, radicals, metals, reducing or oxidizing agents, light, temperature, enzymes, etc. The linker as discussed herein can also be cleaved with the same catalyst used to cleave the 3'O blocking group bond. Suitable linkers can be adapted from standard chemical blocking groups, as disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, John Wiley & Sons. Additional suitable cleavable linkers used in solid phase synthesis are disclosed in Guillier et al. (Chem. Rev. 100: 2092-2157, 2000).

Use of the term "cleavable linker" is not intended to imply that all linker is required to be removed from, for example, the nucleotide base. When the detectable label is attached to the base, the nucleoside cleavage site can be located at a position on the linker that ensures that part of the linker remains attached to the nucleotide base after cleavage.

When the detectable label is attached to the base, the linker can be attached at any position on the nucleotide basis provided that Watson-Crick base pairing can still be performed. In the context of purine bases, it is preferable if the linker is attached via the 7-position of the purine or preferred dezapurine analog, via an 8-modified purine, via an N-6 modified adenosine, or into an N-2 modified guanine. For pyrimidines, binding is preferably through the 5-position on cytosine, thymidine, or uracil and the N-4 position on cytosine. Suitable nucleotide structures are shown in Figure 1. For each structure in Figure 1 X can be H, phosphate, diphosphate or triphosphate. R ¹ and R ² can be the same or different, and are selected from H, OH, or the formula Z as described herein or any other group that can be transformed into an OH, including, but not limited to, a carbonyl , provided that at least one of R ¹ and R ² is O-allyl or formula Z as described herein. Some suitable functional groups for R ¹ and R ² include the structures shown in Figures 3 and 4.

Suitable linkers are shown in Figure 3 and include, but are not limited to, disulfide linkers (1), acid-labile linkers (2, 3, 4 and 5; including dialkoxybenzyl linkers (e.g. 2), Sieber linkers ( e.g. 3) indole linkers (e.g. 4), Sieber t-butyl linkers (e.g. 5)), electrophilically cleavable linkers, nucleophilically cleavable linkers, photoscissile linkers, cleavage under reducing conditions, oxidative conditions, cleavage through use of security binders, and cleavage by elimination mechanisms.

A. Electrophilically cleaved linkers

Electrophilic cleaved linkers are typically proton cleaved and include acid sensitive cleavages. Suitable binders include modified benzyl systems such as trityl, p-alkoxybenzyl esters, and p-alkoxybenzyl amides. Other suitable linkers include tert-butyloxycarbonyl (Boc) groups and the acetal system.

The use of thiophilic metals, such as nickel, silver or mercury, in the cleavage of thioacetal or other sulfide-containing protecting groups can also be considered for the preparation of suitable binding molecules. B. Nucleophilically cleaved linkers.

Nucleophilic cleavage is also a well recognized method in the preparation of linker molecules. Groups such as esters that are water-labile (that is, they can simply be cleaved at basic pH) and groups that are non-aqueous nucleophilic labile can be used. Fluoride ions can be used to cleave silicon-oxygen bonds into groups such as triisopropylsilane (TIPS) or t-butyldimethylsilane (TBDMS).

C. Photo-cleavable linkers.

Photo-cleavable binders have been used extensively in carbohydrate chemistry. It is preferable that the light required to activate the cleavage does not affect the other components of the modified nucleotides. For example, if a fluorophore is used as a marker, it is preferable that it absorbs light of a different wavelength than that required to cleave the linker molecule. Suitable binders include those based on O-nitrobenzole compounds and nitroveratryl compounds. Binders based on benzoin chemistry can also be used (Lee et al., J. Org. Chem. 64: 3454-3460, 1999).

D. Cleavage under reducing conditions.

There are many known binders that are susceptible to reductive cleavage. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in the art.

E. Cleavage under oxidative conditions.

Methodologies based on oxidation are well known in the art. They include oxidation of p-alkoxybenzyl groups and oxidation of sulfur and selenium linkers. The use of aqueous iodine to cleave disulfides and other sulfur- or selenium-based binders also falls within the scope of the invention.

F. Secure hold bindings.

Secure hold binders are those that are cleaved in two stages. In a preferred system the first step is the generation of a reactive nucleophilic center followed by a second step that involves intramolecular cyclization that results in cleavage. For example, levulinic ester linkers can be treated with hydrazine or photochemistry to release an active amine, which can then be cyclized to cleave an ester anywhere in the molecule (Burgess et al., J. Org. Chem. 62 : 5165-5168, 1997).

G. Excision by elimination mechanisms.

Elimination reactions can also be used. For example, base-catalyzed removal of groups such as Fmoc and cyanoethyl, and palladium-catalyzed reductive removal of allyl systems can be used. As well as the cleavage site, the linker can comprise a spacer unit. Spacer distances, eg, nucleotide base of cleavage site or marker. The length of the linker is not important since the marker is kept at a sufficient distance from the nucleotide such that it does not interfere with any interaction between the nucleotide and an enzyme.

In a preferred embodiment the linker may consist of the same functionality as the block. This will make the deprotection and unblocking process more efficient, since only a simple treatment will be required to remove both the marker and the block.

Particularly preferred binders are binders containing phosphine cleavable azide.

A method of determining the sequence of a target polynucleotide can be carried out by contacting the target polynucleotide separately with the different nucleotides to complement that of the target polynucleotide, and detecting incorporation of the nucleotides. Such a method makes use of polymerization, whereby a polymerase enzyme extends the complementary strand by incorporating the correct nucleotide complementary to that of the target. The polymerization reaction also requires a specific primer to initiate polymerization.

For each cycle, the incorporation of the modified nucleotide is carried out by the polymerase enzyme, and the incorporation event is then determined. There are many different polymerase enzymes, and it will be apparent to the person of ordinary skill which one is the most appropriate for use. Preferred enzymes include DNA polymerase I, the Klenow fragment, DNA polymerase III, T4 or T7 DNA polymerase. Taq polymerase or Vent polymerase. Polymerases engineered to have specific properties can also be used. As noted above, the molecule is preferably incorporated by a polymerase and particularly from Thermococcus sp., Such as 9 ° N. Even more preferably, the polymerase is a 9 ° N A485L mutant and even more preferably is a Y409V and A485L double mutant. An example of such a preferred enzyme is Thermococcus sp. 9 ° N exo-Y409V A485L available from New England Biolabs. Examples of such suitable polymerases are disclosed in Proc. Natl. Acad. Sci. USA, 1996 (93), pp 5281-5285, Nucleic Acids Research, 1999 (27), pp 2454 2553 and Acids Research, 2002 (30), pp 605-613.

Sequencing methods are preferably carried out with the target polynucleotide arranged on a solid support. Multiple target polynucleotides can be immobilized on the solid support via binding molecules, or they can be attached to particles, for example microspheres, which can also be attached to a solid support material. Polynucleotides can be attached to the solid support by a number of means, including the use of biotin-avidin interactions. Methods for immobilizing polynucleotides on a solid support are well known in the art, and include lithographic techniques and "staining" with individual polynucleotides at defined positions on a solid support. Suitable solid supports are known in the art, and include glass sheets and beads, ceramic and silicon surfaces, and plastic materials. The support is usually a flat surface although microscopic beads (microspheres) can also be used and in turn can be attached to other solid supports by known means. The microspheres can be of any suitable size, typically in the range of 10 nm to 100 nm in diameter. In a preferred embodiment, the polynucleotides are attached directly to a flat surface, preferably a flat glass surface. The attachment will preferably be by means of a covalent bond. Preferably, the arrangements that are used are single molecule arrangements comprising polynucleotides in areas with different optical resolution, for example, as disclosed in International Application No. WO 00/06770.

The sequencing method can be carried out in both single polynucleotide and multipolinucleotide molecule arrays, that is, arrays of different individual polynucleotide molecules and arrays of different regions comprising multiple copies of a single polynucleotide molecule. The arrangements of individual molecules allow each individual polynucleotide to be resolved separately. The use of single molecule arrangement is preferred. The sequencing in arrays of individual molecules allows in a non-destructive way the formation of a spatially addressable array.

The method makes use of the polymerization reaction to generate the complementary sequence of the target. Conditions compatible with polymerization reactions will be apparent to the skilled person. To carry out the polymerase reaction it will usually be necessary to first fuse a primer sequence to the target polynucleotide, the primer sequence being recognized by the polymerase enzyme and acting as an initiation site for subsequent extension of the complementary strand. The primer sequence can be added as a separate component from the target polynucleotide. Alternatively, the primer and target polynucleotide may each be part of a single stranded molecule, the primer portion forming an intramolecular duplex with a part of the target, that is, a hairpin loop structure. This structure can be immobilized to the solid support at any point in the molecule. Other necessary conditions for carrying out the reaction with polymerase, including temperature, pH, compositions of the buffers etc., will be apparent to those of skill in the art.

The modified nucleotides of the invention are then brought into contact with the target polynucleotide to allow polymerization to occur. Nucleotides can be added sequentially, that is, separate addition of each type of nucleotide (A, T, G, or C), or added together. If added together, it is preferable that each type of nucleotide is labeled with a different marker.

The polymerization step is allowed to proceed long enough to allow incorporation of a nucleotide.

Nucleotides that are not incorporated are then removed, for example, by subjecting the array to a washing step, and detection of the incorporated labels can then be carried out.

The detection can be carried out by conventional means, for example if the marker is a fluorescent structural unit, the detection of a built-in base can be carried out using a confocal scanning microscope to scan the surface of the array with a laser, to form an image of a fluorophore directly bound to the incorporated base. Alternatively, a sensitive 2-D detector, such as a charge-coupled detector (CCD), can be used to visualize the individual signals generated. However, other techniques such as scanning near-field optical microscopy (SNOM) are available and can be used when imaging dense arrays. For example, using SNOM, individual polynucleotides can be distinguished when they are separated by a distance of less than ^{10 0} nm, for example 10 nm at 10 pm. For a definition of scanning near-field light microscopy see Moyer et al., Laser Focus World 29:10, 1993. Suitable apparatus used for imaging arrays of polynucleotides are known and technical structuring will be apparent to the experienced person.

After detection, the label can be removed using suitable conditions that cleave the linker and the 3'OH block to allow for the incorporation of additional modified nucleotides of the invention. Appropriate conditions may be those described herein for "Z" group deprotections. These conditions can serve to deprotect both the linker (if cleavable) and the blocking group.

This invention may be further understood with reference to the following examples which serve to illustrate the invention.

3'-OH protected with an azidomethyl group as a protected form of a heniaminal:

Nucleotides that carry this blocking group in the 3 'position have been synthesized, proving to be successfully incorporated by DNA polymerases, to block efficiently and can be subsequently eliminated under neutral aqueous conditions using phosphines or water-soluble thiols that allow a subsequent extension:

To a solution of 5-iodo-2'-deoxyuridine (1.05 g, 2.96 mmol) and Cul (114 mg, 0.60 mmol) in dry DMF (21 ml) was added triethylamine (0.9 ml). After stirring for 5 minutes, trifluoro-N-prop-2-ynyl-acetamide (1.35 g, 9.0 mmol) and Pd (PPh ^{3) 4} (330 mg, 0.29 mmol) were added to the mixture and the reaction was stirred at temperature environment in the dark for 16 hours. Methanol (MeOH) (40 ml) and Dowex bicarbonate were added to the reaction mixture and stirred for 45 minutes. The mixture was filtered and the filtrate was washed with MeOH and the solvent was removed under vacuum. The crude mixture was purified by chromatography on silica (ethyl acetate and (EtOAc) to EtOAc: MeOH 95: 5) to give slightly yellow crystals (794 mg, 71%). 1H NMR (d ⁶ dimethylsulfoxide (DMSO)) ⁶ 2.13-2.17 (m, 2H, H-2 '), 3.57-3.65 (m, 2H, H-5'), 3.81-3.84 (m, 1H, H-4 '), 4.23-4.27 (m, 3H, H-3', CH ² N), 5.13 (t, J = 5.0 Hz, 1H, OH), 5.20 (d, J = 4.3 Hz, 1H, OH), 6.13 (t, J = 6.7 Hz, 1H, H-1 '), 8.23 (s, 1H, H- ⁶ ), 10.11 (t, J = 5.6 Hz, 1H, NH), 11.70 (br s, 1H, NH) . Mass (-ve electrospray) calculated for C ¹⁴ H ¹⁴ F ³ N ³ O ⁶ 377.08, found 376.

5'-O- (tert-butidimethylsilyl) -5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyuridine (2).

To a solution of (1) (656 mg, 1.74 mmol) in dry DMF (15 ml) was added t-butyldimethylsilyl chloride (288 mg, 1.91 mmol) in small portions, followed by imidazole (130 mg, 1.91 mmol). The reaction was followed by TLC and was completed after stirring for 8 hours at room temperature. The reaction was quenched with saturated aqueous NaCl solution. EtOAc (25 ml) was added to the reaction mixture and the aqueous layer was extracted with EtOAc three times. After drying the combined organic phases (MgSO ⁴ ), the solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: petroleum ether 8: 2) gave (2) as slightly yellow crystals (676 mg, 83%). 1H NMR (da DMSO) 50.00 (s, 6H, CH ³ ), 0.79 (s, 9H, tBu), 1.93-2.00 (m, 1H, H-2 '), 2.06 2.11 (m, 1H, H-2' ), 3.63-3.75 (m, 2H, H-5 '), 3.79-3.80 (m, 1H, H-4'), 4.12-4.14 (m, 3H, H-3 ', CH ² N), 5.22 ( d, J = 4.1 Hz, 1H, OH), 6.03 (t, J = 6.9 Hz, 1H, H-1 '), 7.86 (s, 1H, H-6), 9.95 (t, J = 5.4 Hz, 1H , NH), 11.61 (br s, 1H, NH). Mass (-ve electrospray) 'calculated for C ²⁰ ^ ⁸ F ³ ^ O ⁶ Si 491.17, found 490.

5'-O- (tert-Butidimethylsilyl) -3'-O-methylthiomethyl-5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyuridine (3).

Acetic acid (3.2 ml) and acetic anhydride (10.2 ml) were added to a solution of (2) (1.84 g, 3.7 mmol) in dry DMSO (7 ml). The mixture was stirred for 2 days at room temperature, before being quenched with saturated aqueous NaHCO ³ . EtOAc (50 ml) was added and the aqueous layer was extracted three times with ethyl acetate. The combined organic layers were washed with saturated aqueous NaHCO3 solution and dried (MgSO4). After removing the solvent under reduced pressure, the product (3) was purified by chromatography on silica (EtOAc: petroleum ether 8: 2) yielding a clear sticky oil (1.83 g, 89%), 1H NMR (d6 DMSO): 50.00 (s, 6H, CH3), 0.79 (s, 9H, tBu), 1.96-2.06 (m, 1H, H-2 '), 1.99 (s, 3H, SCH ³ ), 2.20-2.26 (m, 1H, H-2 '), 3.63-3.74 (m, 2H, H-5'), 3.92-3.95 (m, 1H, H-4 '), 4.11-4.13 (m, 2H, CH ² ), 4.28-4.30 ( m, 1H, H-3 '), 4.59 (br s, 2H, CH ² ), 5.97 (t, J = 6.9 Hz, 1H, H-1'), 7.85 (s, 1H, H-6), 9.95 (t, J = 5.3 Hz, 1H, NH), 11.64 (s, 1H, NH). Mass (-ve electrospray) calculated for C ²² H32F3N3O6SSi 551.17, found 550.

3'-O-Azidomethyl-5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyuridine (4).

To a solution of (3) (348 mg, 0.63 mmol) and cyclohexene (0.32 ml, 3.2 mmol) in dry CH ² CI ² (5 ml) at 4 ° C, sulfuryl chloride (1M in CH ² Cl ² , 0.76 ml, 0.76 mmol) dropwise under N ² . After 10 minutes TLC indicated complete consumption of nucleoside (3). The solvent was evaporated and the residue was placed under high vacuum for 20 minutes. It was then redissolved in dry DMF (3 ml) and treated with NaN ³ (205 mg, 3.15 mmol). The resulting suspension was stirred under room temperature for 2 hours. The reaction was quenched with CH ² Cl ² and the organic layers were washed with saturated aqueous NaCl solution. After removing the solvent, the resulting yellow gum was redissolved in THF (2 ml) and treated with TBAF (1 M in THF, 0.5 ml) at room temperature for 30 minutes. The solvent was removed and the reaction was manipulated with CH ² O ² and saturated aqueous NaHCO ³ solution. The aqueous layer was extracted three times with CH ² Cl ² . Purification by chromatography on silica (EtOAc: petroleum ether 1: 1 to EtOAc) gave (4) (100 mg, 37%) as a pale yellow foam. 1H NMR (da DMSO) 82.15-2.26 (m, 2H, H-2 '), 3.47-3.57 (m, 2H, H-5'), 3.88-3.90 (m, 1H, H-4 '), 4.14 ( d, J = 4.7 Hz, 2H, CH ² NH), 4.24-4.27 (m, 1H, H-3 '), 4.75 (s, 2H, CH ² N ³ ), 5.14 (t, J = 5.2 Hz, 1H , OH), 5.96-6.00 (m, 1H, H-1 '), 8.10 (s, 1H, H-6), 10.00 (s, 1H, NHCOCF ³ )), 11.26 (s, 1H, NH).

Preparation of bis (tri-n-butylammonium) pyrophosphate (0.5 M solution in DMF)

Tetrasodium diphosphate decahydrate (1.5 g, 3.4 mmol) was dissolved in water (34 ml) and the solution was applied to a Dowex column in the H + form. The column was eluted with water. The eluent was dropped directly into a cooled (ice bath) and stirring solution of tri-n-butylamine (1.6 ml, 6.8 mmol) in EtOH (14 ml). The column was washed until the pH of the eluent increased to 6. The aqueous ethanol solution was evaporated to dryness and then coevaporated twice with ethanol and twice with anhydrous DMF. The residue was dissolved in DMF (6.7 ml). The pale yellow solution was stored on 4A molecular sieves.

3'-O-Azidomethyl-5- (3-amino-prop-1-ynyl) -2'-deoxyuridine 5'-O-nucleoside triphosphate (5).

The nucleoside (4) and the proton sponge were dried over P ² O ⁵ under vacuum overnight. A solution of (4) (92 mg, 0.21 mmol) and the proton sponge (90 mg, 0.42 mmol) in trimethylphosphate (0.5 ml) with 4A molecular sieves were added for 1 hour. Freshly distilled POCl ³ (24 µl, 0.26 mmol) was added and the solution was stirred at 4 ° C for 2 hours. The mixture was slowly warmed to room temperature and bis (tri-n-butyl ammonium) pyrophosphate (1.7 ml, 0.85 mmol) and anhydrous tri-n-butyl amine (0.4 ml, 1.7 mmol) were added. After 3 minutes, the reaction was quenched with 0.1 M TEAB (triethylammonium bicarbonate) buffer (15 mL) and stirred for 3 hours. The water was removed under reduced pressure and the resulting residue was dissolved in concentrated ammonia (p 0.88, 15 ml) and stirred at room temperature for 16 hours. The reaction mixture was then evaporated to dryness. The residue was dissolved in water and the solution was applied to a DEAE-Sephadex A-25 column. MPLC was run with a linear TEAB gradient. The triphosphate was eluted in buffer between 0.7 M and 0.8 M. The fractions containing the product were combined and evaporated to dryness. The residue was dissolved in water and subsequently purified by HPLC. HPLC: rt (5): 18.8 min (Zorbax C18 preparative column, gradient: 5% to 35% B in 30 min, 0.1M TEAB buffer A, MeCN buffer B). The product was isolated as a white foam (76 OD, 7.6 pmol, 3.8%, £ ²⁸⁰ = 10,000). 1H NMR (D ² O) 81.79 (s, CH ² ), 2.23-2.30; 2.44-2.50 (2 xm, 2H, H-2 '), 3.85 (m, CH ² NH), 4.10-4.18 (m, 2H, H-5'), 4.27 (br s, H-4 '), 4.48 -4.50 (m, H-3 '), 4.70-4.77 (m, CH ² N ³ ), 6.21 (t, J = 6.6 Hz, H-1'), 8.32 (s, 1H, H-6). 31P NMR (D ² O) 8 -6.6 (m, 1P, Pv), -10.3 (d, J = 18.4 Hz, 1P, Pa), -21.1 (m, 1P, Pp). Mass (-ve electrospray) calculated for C ¹³ H ¹⁹ N ⁶ O ¹⁴ P ³ 576.02, found 575.

The starting disulfide (4.0 mg, 13.1 Limol) was dissolved in DMF (300 ^ L) and diisopropylethylamine (4 ^ L) was added slowly. The mixture was stirred at room temperature and a solution of Cy-3 dye (5 mg, 6.53 µmol) in DMF (300 µL) was added over 10 minutes. After 3.5 hours, at the end of the reaction, the volatiles were evaporated under reduced pressure and the crude residue was purified by HPLC on a Zorbax SB-C18 analytical column with a flow rate of Iml / minute in 0.1M triethylammonium bicarbonate buffer. (regulator A) and CH ³ CN (regulator B) using the following gradient: 5 min 2% B; 31 min 55% B; 33 min 95% B; 37 min 95%; 39 min 2% B; 44 min.

2% B. The expected Cy3-disulfide binder was eluted at rt: 21.8 min. in 70% yield (based on a UV measurement; £ ⁵⁵⁰ 150,000 cm-1 M-1 in H ² O) as a hygroscopic solid. 1H NMR (D ² O) 51.31-1.20 (mt, J = 7.2 Hz, 5H, CH ² + CH ³ ), 1.56-1.47 (m, 2H, CH ² ), 1.67 (s, 12H, 4 CH ³ ), 1.79-1.74 (m, 2H, CH ² ), 2.11 (t, J = 6.9 Hz, 2H, CH ² ), 2.37 (t, J = 6.9 Hz, 2H, CH ² ), 2.60 (t, J = 6.3 Hz , 2H, CH ² ), 2.67 (t, J = 6.9 Hz, 2H, CH ² ), 3.27 (t, J = 6.1 Hz, 2H, CH ² ), 4.10-4.00 (m, 4H, ² CH ² ), 6.29 (dd, J = 13.1, 8.1 Hz, 2H, 2 = CH), 7.29 (dd, 2H, J = 8.4, 6.1 Hz, 2 = CH), 7.75-7.71 (m, 2H, 2 = CH), 7.78 (s, 2H, = CH), 8.42 (t, J = 12.8 Hz, 1H, = CH). Mass (-ve electrospray) calculated for C ³⁶ H ⁴⁷ K ³ O ⁹ S ⁴ 793.22, found 792 (MH), 396 [M / 2].

A mixture of Cy3 disulfide binder (2.5 ^ mol), disuccinimidyl carbonate (0.96 mg, 3.75 ^ mol), and DMAP (0.46 mg, 3.75 ^ mol) was dissolved in dry DMF (0.5 ml) and stirred at room temperature for 10 minutes. The reaction was monitored by TLC (MeOH: CH2Cl23: 7) until the dye binder was consumed. Then a solution of (5) (7.5 µmol) and n-Bu3N (30 µl, 125 µmol) in DMF (0.2 ml) were added to the reaction mixture and stirred at room temperature for 1 hour. TLC (MeOH: CH2Cl24: 6) showed complete consumption of the activated ester and a dark red spot appeared on the baseline. The reaction was stopped with TEAB buffer (0.1M, 10 ml) and loaded onto a DEAE Sephadex column (2 x 5 cm). The column was eluted first with 0.1 M TEAB buffer (100 ml) to wash off organic residues and then 1 M TEAB buffer (100 ml). The desired triphosphate analog (6) was eluted with 1M TEAB buffer. The fractions containing the product were combined, evaporated and purified by HPLC. Conditions for HPLC: rt (6): 16.1 min (Zorbax C18 preparative column, gradient: 2% to 55% B in 30 min, 0.1M TEAB buffer A, MeCN buffer B). The product was isolated as a dark red solid (1.35 ^ mol, 54%, e55o = 150000). 1H NMR (D2O) S 1.17-1.28 (m, 6H 3 x CH ² ), 1.41-1.48 (m, 3 H, CH ³ ), 1.64 (s, 12H, 4 x CH ³ ), 1.68-1.71 (m, 2H, CH ² ), 2.07-2.10 (m, 3H, H-2 ', CH ² ), 2.31-2.35 (m, 1H, H-2'), 2.50-2.54 (m, 2H, CH ² ), 2.65 (t, J = 5.9 Hz, 2H, CH ² ), 2.76 (t, J = 7.0 Hz, 2H, CH ² ), 3.26-3.31 (m, 2H, CH ² ), 3.88-3.91 (m, 2H CH ² ), 3.94-4.06 (m, 3H, CH ² N, H-5 '), 4.16 (br s, 1H, H-4'), 4.42-4.43 (m, 1H, H-3 '), 4.72-4.78 (m, 2H, CH ² N ³ ), 6.24 (dd, J = 5.8, 8.2 Hz, H-1 '), 6.25 (dd, J = 3.5, 8.5 Hz, 2H, HAr), 7.24, 7.25 (2d, J = 14.8 Hz, 2 x = CH), 7.69-7.86 (m, 4H, HAr, H-6), 8.42 (t, J = 13.4 Hz, = CH). 31P NMR (D ² O) S -4.85 (m, 1P, PY), -9.86 (m, 1P, Pa), -20.40 (m, 1P, Pp). Mass (-ve electrospray) calculated for C ⁴⁹ H ⁶⁴ N ⁹ O ²² P ³ S ⁴ 1351.23, found 1372 (M-2H + Na), 1270 [M-80], 1190 [M-160].

5- [3- (2,2,2-Trifluoroacetamido) -prop-1-ynyl] -2'-deoxycytidine ( 7).

To a solution of 5-iodo-2'-deoxycytidine (10 g, 28.32 mmol) in DMF (200 ml) in a round bottom flask protected from light under argon atmosphere, CuI (1.08 g, 5.67 mmol) was added , triethylamine (7.80 ml, 55.60 mmol), 2,2,2-trifluoroN-prop-2-ynyl-acetamide (12.8 g, 84.76 mmol) and finally Pd (PPh) ³ ) ⁴ (3.27 g, 2.83 mmol). After 18 hours at room temperature, Dowex bicarbonate (20 mg) was added and the mixture was stirred for an additional 1 hour. Filtration and evaporation of the volatiles under reduced pressure gave a residue that was purified by flash chromatography on silica gel (CH ² Cl ² , CH ² Ch: EtOAc 1: 1, EtOAc: MeOH 9: 1). The expected product (7 ) was obtained as a beige solid in quantitative yield. 1H NMR (D ² O) 82.24-2.17 (m, 1H, H-2 '), 2.41-2.37 (m, 1H, H-2'), 3.68 (dd, J = 12.5, 5.0 Hz, 1H, H- 5 '), 3.77 (dd, J = 12.5, 3.2 Hz, 1H, H-5'), 3.99 (m, 1H, H-4 '), 4.27 (s, 2H, CH ² N), 4.34 (m, 1H, H-3 '), 6.11 (t, J = 6.3 Hz, 1H, H-1'), 8.1 (br s, 1H, NH); MS (ES): m / z (%) (MH) 375 (100).

5'-O- (tert-Butyldimethylsilyl) -5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxycytidine (8).

To a solution of the starting material (7) (1.0 g, 2.66 mmol) and imidazole (200 mg, 2.93 mmol) in DMF (3.0 ml) at 0 ° C, TBDMSC ¹ (442 mg, 2.93 mmol) was slowly added in four servings for 1 hour. After 2 hours, the volatiles were evaporated under reduced pressure and the residue was adsorbed on silica gel and purified by flash chromatography (EtOAc, EtOAc: MeOH 9.5: 0.5). The expected product (8) was isolated in the form of a crystalline solid (826 mg, 64%). 1H NMR (d6 DMSO) 80.00 (s, 1H, CH ³ ); 0.01 (s, 1H, CH3), 0.79 (s, 9H, tBu), 1.87 1.80 (m, 1H, H-2 '), 2.12 (ddd, J = 13.0, 5.8 and 3.0 Hz, 1H, H-2 '), 3.65 (dd, J = 11.5, 2.9 Hz, 1H, H-5'), 3.74 (dd, J = 11.5, 2.5 Hz, 1H, H-5 '), 3.81-3.80 (m, 1H, H4 '), 4.10-4.09 (m, 1H, H-3'), 4.17 (d, 2H, J = 5.1 Hz, NCH ² ), 5.19 (d, 1H, J = 4.0 Hz, 3'-OH), 6.04 (t, J = 6.6 Hz, 1H, H1 '), 6.83 (br s, 1H, NHH), 7.78 (br s, 1H, NHH), 7.90 (s, 1H, H-6), 9.86 (t, J = 5.1 Hz, 1H, -H ² CNH); MS (ES): m / z (%) (MH) + 491 (40%).

4-N-Acetyl-5'-O- (tert-butyldimethylsilyl) -3'-O- (methylthiolmethyl) -5- [3- (2,2,2-trifluoroacetamide) -prop-1-inM] 2'- deoxycytidine (9).

To a solution of the starting material (8) (825 mg, 1.68 mmol) in DMSO (6.3 ml) and N ² atmosphere, acetic acid (AcOH) (1.3 ml, 23.60 mmol) was slowly added followed by acetic anhydride (Ac ² O) (4.8 ml, 50.50 mmol). The solution was stirred at room temperature for 18 hours and stopped at 0 ° C by adding saturated NaHCO ³ (20 ml). The product was extracted with EtOAc (3 x 30 ml), the organic extracts were combined, dried (MgSO ⁴ ), filtered, and the volatiles were evaporated. The crude residue was purified by flash chromatography on silica gel (EtOAc: petroleum ether 1: 1) to give the expected product in the form of a colorless oil (9) (573 mg, 62%). 1H NMR (da DMSO) 50.00 (s, 6H, 2 x CH ³ ), 0.78 (s, 9H, tBu), 2.01 (s, 3H, SCH ³ ), 2.19-1.97 (m, 2H, 2 x H2 ') , 2.25 (s, 3H, COCH ³ ), 3.67 (dd, 1H, J = 11.5 Hz, H-5 '), 3.78 (dd, 1H, J = 11.5, 3.3 Hz, H-5'), 4.06-4.05 (m, 1H, H-4 '), 4.17 (d, 2H, J = 5.1 Hz, N-CH ² ), 4.30-4.28 (m, 1H, H-3'), 4.63 (s, 2H; CH ² -S), 5.94 (t, 1H, J = 6.5 Hz, H-1 '), 8.17 (s, 1H, H-6), 9.32 (s, 1H, NHCO), 9.91 (t, 1H, J = 5.4 Hz, NHCH ² ); MS (ES): m / z (%) (MH) + 593.

4-N-Acet¡l-3'-O- (az¡dome¡l) -5'-O- (tert-but¡ld¡met¡ls¡l¡l) -5- [3- (2, 2,2-trifluoroacetamide) -prop-1-¡n¡l] -2'-deoxyc¡t¡d¡na (10).

To a solution of starting material (9) (470 mg, 0.85 mmol) in dichloromethane (DCM) (8 ml) under N ² atmosphere and cooled to 0 ° C, cyclohexene (430 | jl, 4.27 mmol) was added followed by SO ² Cl ² (1 M in DCM, 1.0 ml, 1.02 mmol). The solution was stirred for 30 minutes at 0 ° C, and the volatiles were evaporated. The residue was immediately dissolved in DMF (8 ml) stirred under N ² and sodium azide (275 mg, 4.27 mmol) was added slowly. After 18 hours, the crude product was evaporated to dryness, dissolved in EtOAc (30 ml) and washed with Na ² CO ³ (3 x 5 ml). The combined organic layers were kept separately. A second extraction of the product from the aqueous layer was carried out with DCM (3 x 10 ml). All the combined organic layers were dried (MgSO ⁴ ), filtered and the volatiles evaporated under reduced pressure to give oil identified as the expected product (10) (471 mg, 94% yield). This was used without any further purification. 1H NMR (from DMSO) 50.11 (s, 3H, CH ³ ), 0.11 (s, 3H, CH ³ ), 0.88 (s, 9H, tBu), 2.16-2.25 (m, 1H, H-2 '), 2.35 (s, 3H, COCH ³ ), 2.47-2.58 (m, 1H, H-2 '), 3.79 (dd, J = 11.6, 3.2 Hz, 1H, H-5'), 3.90 (dd, J = 11.6, 3.0 Hz, 1H, H-5 '), 4.17 4.19 (m, 1H, H-4'), 4.28 (s, 2H, NCH ² ), 4.32-4.35 (m, 1H, H-3 '), 4.89 ( dd, J = 14.4, 6.0 Hz, 2H, CH ² -N ³ ), 6.05 (t, J = 6.4 Hz, 1H, H-1 '), 8.25 (s, 1H, H-6), 9.46 (br s , 1H, NHH), 10.01 (br s, 1H, NHH).

4-N-Acetyl-3'-O- (azidomethyl) -5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxycytidine and 3'-O- (Azidomethyl) -5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxycytidine (11).

To a solution of the starting material (11) (440 mg, 0.75 mmol) in THF (20 ml) at 0 ° C and N ² atmosphere, TBAF in 1.0 M THF (0.82 ml, 0.82 mmol) was added. After 1.5 hours, the volatiles were evaporated under reduced pressure and the residue was purified by flash chromatography on silica gel (EtOAc: petroleum ether 8: 2 to EtOAc 100% to EtOAc: MeOH 8: 2). Two compounds were isolated and identified as described above. The first eluted 4-N-acetyl (11), (53 mg, 15%) and the second ⁴ -NH ² (12) (271 mg, 84%). 4-N-acetyl compound (11): 1H NMR (da DMSO) 51.98 (s, 3H, CH ³ CO), 2.14-2.20 (m, 2H, HH-2 '), 3.48-3.55 (m, 1H, H-5'), 3.57-3.63 (m , 1H, H-5 '), 3.96-4.00 (m, 1H, H-4'), 4.19 (d, J = 5.3 Hz, 2H, CH ² -NH), 4.23-4.28 (m, 1H, H3 ' ), 4.77 (s, 2H, CH ² -N ³ ), 5.2 (t, 1H, J = 5.1 Hz, 5'-OH), 5.95 (t, J = 6.2 Hz, 1H, H-1 '), 8.43 (s, 1H, H-6), 9.34 (s, 1H, CONH), 9.95 (t, J = 5.3 Hz, 1H, NHCH ² ).

Compound ⁴ -NH ² (12): 1H NMR (d6 DMSO) 51.98-2.07 (2H, CHH-2 '), 3.50-3.63 (m, 2H, CHH-5'), 3.96-4.00 (m, 1H, H -4 '), 4.09 (d, J = 5.3 Hz, 2H, CH ² -NH), 4.24-4.28 (m, 1H, H-3'), 4.76 (s, 2H, CH ² -N ³ ) 5.13 ( t, J = 5.3 Hz, 1H, 5'-OH), 5.91 (br s, 1H, NHH), 6.11 (t, J = 6.4 Hz, 1H, H-1 '), 8.20 (t, J = 5.3 Hz , 1H, NCH ² ), 8.45 (s, 1H, H-6), 11.04 (br s, 1H, NHH).

The starting material (8) (10 g, 20.43 mmol) was subjected to azeotroping in dry pyridine (2 x 100 ml) and then dissolved in dry pyridine (160 ml) under N ² atmosphere. Chlorotrimethylsilane (10 ml, 79.07 mmol) was added dropwise to the solution and it was stirred for 2 hours at room temperature. Benzoyl chloride (2.6 ml, 22.40 mmol) was then added to the solution and it was stirred for an additional 1 hour. The reaction mixture was cooled to 0 ° C, distilled water (50 ml) was slowly added to the solution and stirred for 30 minutes. Pyridine and water were evaporated from the mixture under high vacuum to produce a brown gel that was portioned between 100 ml of saturated aqueous NaHCO3 solution (100 ml) and DCM. The organic phase was separated and the aqueous phase was extracted with additional (2 x 100 ml) of DCM. The organic layers were combined, dried (MgSO ⁴ ), filtered and the volatiles evaporated under reduced pressure. The resulting brown oil was purified by flash chromatography on silica gel (DCM: MeOH 99: 1 to 95: 5) to yield a light yellow crystalline solid (13) (8.92 g, 74%). 1H NMR (d6 DMSO): 50.00 (s, 6H, CH ³ ), 0.78 (s, 9H, tBu), 1.94 (m, 1H, H-2 '), 2.27 (m, 1H, H-2'), 3.64 (d, 1H, J = 11.6 Hz, H-5 '), 3.75 (d, 1H, J = 11.6 Hz, H-5'), 3.91 (m, 1H, H-4 '), 4.09 (br m , 3H, CH ² NH, H-3 '), 5.24 (s, 1H, 3'-OH), 6.00 (m, 1H, H-1'), 7.39 (m, 2H, Ph), 7.52 (m, 2H, Ph), 7.86 (m, 1H, Ph), 8.0 (s, 1H, H-6), 9.79 (t, 1H, J = 5.4 Hz, NHCH2), 12.67 (br s, 1H, N ^h ). Mass (+ ve electrospray) calculated for C27H33F3N4O6Si 594.67, found 595.

4-N-Benzoyl-5'-O- (tert-butyldimethylsilyl) -3'-O-methylthiomethyl-5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] 2'-deoxycytidine

(14).

The starting material (13) (2.85 g, 4.79 mmol) was dissolved in dry DMSO (40 ml) under a N ² atmosphere. Acetic acid (2.7 ml, 47.9 mmol) and acetic anhydride (14.4 ml, 143.7 mmol) were added sequentially and slowly to the starting material, which was then stirred for 18 hours at room temperature. Saturated NaHCO ³ solution (150 ml) was carefully added to the reaction mixture. The aqueous layer was extracted with EtOAc (3 x 150 ml). The organic layers were combined, dried (MgSO ⁴ ), filtered and evaporated to produce an orange colored liquid which was subsequently azeotroped with toluene (4 x 150 ml) until the material solidified. The crude residue was purified on silica gel (petroleum ether: EtOAc 3: 1 to 2: 1) to yield a yellow crystalline solid (14) (1.58 g, 50%). 1H NMR (d6 DMSO): 50.00 (s, 6H, CH ³ ), 0.78 (s, 9H, tBu), 1.99 (s, 3H, CH ³ ), 2.09 (m, 1H, H-2 '), 2.28 ( m, 1H, H-2 '), 3.66 (d, 1H, J = 11.5, 2.9 Hz, H-5'), 3.74 (dd, 1H, J = 11.3, 2.9 Hz, H-5 '), 3.99 ( m, 1H, H-4 '), 4.09 (m, 1H, CH ² NH), 4.29 (m, 1H, H-3'), 4.61 (s, 2H, CH ² S), 6.00 (m, 1H, H-1 '), 7.37 (m, 2H, Ph), 7.50 (m, 2H, Ph), 7.80 (d, 1H, J = 7.55 Hz, HAr), 7.97 (s, 1H, H-6), 9.79 (br t, 1H, NHCH ² ), 12.64 (br s, 1H, NH). Mass (-ve electrospray) calculated for C ²⁹ H ³⁷ F ³ N ⁴ O ⁶ SSÍ 654.79, found 653.2.

4-N-Benzoyl-5'-O- (tert-butyldimethylsilyl) -3'-O-azidomethyl-5- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxycytidine (fifteen).

The starting material (14) (1.65 g, 2.99 mmol) was dissolved in DCM (18 ml) and cooled to 0 ° C. Cyclohexene (1.5 ml, 14.95 mmol) and SO ² O ² (0.72 ml, 8.97 mmol) were added and stirred for 1 hour in an ice bath. TLC indicated that the starting material was still present whereupon an additional aliquot of SO ² O ² (0.24 ml) was added and the mixture was stirred for 1 hour at 0 ° C. The volatiles were removed by evaporation to produce a light brown solid which was redissolved in 18 ml of dry DMF (18 ml) under N ² . Sodium azide (0.97 g, 14.95 mmol) was then added to the solution and it was stirred for 2.5 hours at room temperature. The reaction mixture was passed through a bed of silica and eluted with EtOAc and the volatiles were removed by evaporation under high vacuum. The resulting brown gel was purified by flash chromatography (petroleum ether: EtOAc 4: 1 to 2: 1) to yield the desired product as a white crystalline solid (15) (0.9 g, 55%). 1H NMR (d6 DMSO); ⁶ 0.00 (s, ^6H, CH ^3), 0.78 (s, 9H, tBu), 2.16 (m, 1H, H-2 '), 2.22 (m, 1H, H-2'), 3.70 (d, 1H , J = 11.5 Hz, H-5 '), 3.75 (d, 1H, J = 11.3 Hz, H-5'), 4.01 (m, 1H, H-4 '), 4.10 (m, 1H, CH ² NH ), 4.23 (m, 1H, H-3 '), 4.76 (s, 2H, CH ² S), 5.99 (m, 1H, H-1'), 7.37 (m, 2H, Ph), 7.50 (m, 2H, Ph), 7.81 (d, 1H, J = 7.4 Hz, Ph), 7.95 (s, 1H, H- ⁶ ), 9.78 (br s, 1H, NHCH ² ), 12.64 (br s, 1H, NH) . Mass (-ve electrospray) calculated for C ²⁸ H ³⁴ FaN ⁷ O ⁶ Si 649.71, found 648.2

The starting material (15) (140 mg, 0.22 mmol) was dissolved in THF (7.5 ml). TBAF (1 Molar solution in THF, 0.25 ml) was added slowly and stirred for 2 hours at room temperature. Volatile material was removed under reduced pressure to produce a brown gel and was purified by flash chromatography (EtOAc: DCM 7: 3) to give the desired product (16) as a light colored crystalline solid (0.9 g, 76% ). 1H ^r M ⁿ (d6 DMSO): ⁶ 2.16 (m, 1H, H-2 '), 2.22 (m, 1H, H-2'), 3.70 (d, 1H, J = 11.5 Hz, H-5 ') , 3.75 (d, 1H, J = 11.3 Hz, H-5 '), 4.01 (m, 1H, H-4'), 4.10 (m, 1H, CH ² NH), 4.23 (m, 1H, H-3 '), 4.76 (s, 2H, CH ² S), 5.32 (s, 1H, 5'OH), 5.99 (m, 1H, H-1'), 7.37 (m, 2H, Ph), 7.50 (m, 2H, Ph), 7.81 (d, 1H, J = 7.35 Hz, Ph), 7.95 (s, 1H, H- ⁶ ), 9.78 (br s, 1H, NHCH ² ), 12.64 (br s, 1H, NH) . Mass (-ve electrospray) calculated for C ²² H ²⁰ F ³ N ⁷ O ⁶ 535.44, found 534.

To a solution of (11) and (12) (290 mg, 0.67 mmol) and proton sponge (175 mg, 0.82 mmol) (both previously dried under P2O5 for at least 24 hours) in PO (OMe) 3 (600 pl ), at 0 ° C under argon atmosphere, POCl3 (freshly distilled) (82 µl, 0.88 mmol) was added slowly. The solution was vigorously stirred for 3 hours at 0 ° C and then quenched by adding tetra-tributylammonium diphosphate (0.5 M) in DMF (5.2 ml, 2.60 mmol), followed by nBu3N (1.23 ml, 5.20 mmol) and bicarbonate 0.1 M triethylammonium (TEAB) (20 ml). After 1 hour at room temperature aqueous ammonia solution (p 0.88, 20 ml) was added to the mixture. The solution was stirred at room temperature for 15 hours, the volatiles were evaporated under reduced pressure and the residue was purified by MPLC with a TEAB gradient from 0.05 M to 0.7 M. The expected triphosphate was eluted from the column at approximately 0.60 M TEAB . A second HPLC purification was performed on a Zorbax SB-C18 column (21.2 mm id x 25 cm) eluted with 0.1 M TEAB (pump A) and 30% CH3CN in 0.1M TEAB (pump B) using a gradient as follows: 0-5 min 5% B, 0.2 ml; 5-25 min 80% B, 0.8 ml; 25-27 min 95% B, 0.8 ml; 27-30 min 95% B, 0.8 ml; 30-32 min 5% B, 0.8 ml; 32-35 min 95% B, 0.2 ml, generating the product described above with an rt (17): 20.8 (14.5 pmol, 2.5% yield); 31P NMR (D ² O, 162 MHz) 5 -5.59 (d, J = 20.1 Hz, Px), -10.25 (d, J = 19.3 Hz, 1P, Pa), -20.96 (t, J = 19.5 Hz, 1P , Pp); 1H NMR (D ² O) 52.47-2.54 (m, 1H, H-2 '), 2.20-2.27 (m, 1H, H-2'), 3.88 (s, 2H, CH ² N), 4.04-4.12 ( m, 1H, HH-5 '), 4.16-4.22 (m, 1H, HH-5'), 4.24-4.30 (m, 1H, H-4 '), 4.44-4.48 (m, 1H, H-3' ), 6.13 (t, J = 6.3 Hz, 1H, H1 '), 8.35 (s, 1H, H-6); MS (ES): m / z (%) (MH) 574 (73%), 494 (100%).

Disulfide binder Alexa488.

Commercially available Alexa fluorine 488-NHS (35 mg, 54 pmol) was dissolved in DMF (700 pL) and, to ensure complete activation, 4-DMAP (7 mg, 59 pmol) and N, N carbonate were added sequentially '-disuccinimidyl (15 mg, 59 pmol). After 15 minutes in full activation, a solution of the starting disulfide (32.0 mg, 108 pmol) in DMF (300 pL) containing diisopropylethylamine (4 pL) was added to the dye solution activated. Diisopropylethylamine (20 pL) was subsequently added to the final mixture, sonicated for 5 minutes, and reacted for 18 hours at room temperature in the dark. The volatiles were evaporated under reduced pressure and the crude residue was purified first by passing it through a short column of ion exchange resin Sephadex -DEAE A-25 (40-120 p), eluting first with 0.1 M TEAB (25 ml) then 1.0 M TEAB (75 ml). The latter contained the two final compounds and was concentrated and the residue was purified by HPLC on a Zorbax SB-C18 column (21.2 mm id x 25 cm) eluted with 0.1M TEAB (pump A) and CH ³ CN (pump B) using a gradient as follows: 0-2 min 2% B, $ .2 ml; 2-4 min 2% B, $ .8 ml; 4-15 min 23% B, $ .8 ml; 15-24 min 23% B, $ .8 ml; 24-26 min 95% B, $ .8 ml; 26-28 min 95% B, $ .8 ml, 28-30 min 2% B, $ .8 ml, 30-33 min 2% B, $ .2 ml generating both compounds detailed above with tr: 19.0 (regioisomer left) and tr: 19.5 (right regioisomer). Both regioisomers were passed respectively through a column with Dowex ion exchange resin, generating respectively 16.2 pmol and 10.0 pmol, 62% total yield (based on commercially available Alexa fluorine 488-NHS of 76% purity); £ ⁴⁹³ = 71,000 cm-1 M-1 in H ² O. 1H NMR (D ² O) (left regioisomer) 62.51 (t, J = 6.8 Hz, 2H, CH ² ), 2.66 (t, J = 6.8 Hz, 2H, CH ² ), 2.71 (t, J = 5.8 Hz, 2H, CH ² ), 3.43 (t, J = 5.8 Hz, 2H, CH ² ), 6.64 (d, J = 9.2 Hz, 2H, HAr), 6.77 (d, J = 9.2 Hz, 2H, HAr), 7.46 (s, 1H, HAr), 7.90 (dd, J = 8.1 and 1.5 Hz, 1H, HAr), 8.20 (d, J = 8.1 Hz, 1H, HAr). 1H NMR (D ² O) (right regioisomer) 62.67 (t, J = 6.8 Hz, 2H, CH ² ), 2.82 (t, J = 6.8 Hz, 2H, CH ² ), 2.93 (t, J = 6.1 Hz, 2H, CH ² ), 3.68 (t, J = 6.1 Hz, 2H, CH ² ), 6.72 (d, J = 9.3 Hz, 2H, HAr), 6.90 (d, J = 9.3 Hz, 2H, HAr) , 7.32 (d, J = 7.9 Hz, 1H, HAr), 8.03 (dd, J = 7.9, 1.7 Hz, 1H, HAr), 8.50 (d, J = 1.8 Hz, 1H, HAr) Mass (-ve electrospray) Calcd for C ²⁶ H ²³ N ³ O ¹² S ⁴ 697.02, found 692 (MH), 347 [M / 2].

To a solution of the disulfide binder Alexa fluorine 488 (3.4 pmol, 2.37mg) in DMF (200 pL) was added 4-DMAP (0.75 mg, 5.1 pmol) and N, N-disuccinimidyl carbonate (1.70 mg, 5.1 pmol) . The mixture was stirred for 15 until complete activation of the acid, then it was added to the solution of nucleotide (17) (3.45 mg, 6.0 pmol) in DMF (0.3 ml) containing nBu ³ N (40 pL) at 0 ° C. The mixture was sonicated for 3 minutes and then continuously stirred for 16 hours in the absence of light. The volatiles were evaporated under reduced pressure and the residue was first purified by filtration through a short column of Sephadex -DEAE A-25 ion exchange resin, first eluted with 0.1 M TEAB (50 ml) removing the unreacted dye-binder , then 1.0 M TEAB (100 ml) to collect the expected product (18). After concentration the residue was purified by HPLC on a Zorbax SB-C18 column (21.2 mm id x 25 cm) eluted with 0.1M TEAB (pump A) and CH ³ CN (pump B) using a gradient as follows: 0-2 min 2% B, $ .2 ml; 2-4 min 2% B, $ .8 ml; 4-15 min 23% B, $ .8 ml; 15-24 min 23% B, $ .8 ml; 24-26 min 95% B, $ .8 ml; 26-28 min 95% B, $ .8 ml, 28-30 min 2% B, $ .8 ml, 30 33 min 2% B, $ .2 ml generating the product detailed above with an rt (18): 19.8 (0.26 pmols, 12% yield based on UV measurements); Amax = 493 nm, e 71,000 cm-1 M-1 in H ² O); 31P NMR (D ² O, 162 MHz) 6 -5.06 (d, J = 20.6 Hz, 1P, Px), -10.25 (d, J = 19.3 Hz, 1P, Pa), -21.21 (t, J = 19.5 Hz , 1P, Pp); 1H NMR (D ² O) 62.09 2.17 (m, 1H, HH-2 '), 2.43-2.50 (m, 1H, HH-2'), 2.61 (t, J = 6.8 Hz, 2H, H ² CS), 2.83 (2H, S-CH ² ), 3.68 (t, J = 6.0 Hz, 2H, ArCONCH ² ), 4.06 (s, 2H, CH ² N), 4.08-4.17 (m, 4H, HH-5 '), 4.25-4.29 (m, 1H, H-4'), 4.46-4.50 (m , 1H, H-3 '), 6.09 (t, J = 6.4 Hz, 1H, H-1'), 6.88 (d, J = 9.1 Hz, 1H, HAr), 6.89 (d, J = 9.3 Hz, 1H , HAr), 7.15 (d, J = 9.3 Hz, 1H, HAr), 7.17 (d, J = 9.1 Hz, 1H, HAr), 7.64 (br s, 1H, HAr), 8.00-7.94 (m, 2H, HAr), 8.04 (s, 1H, H-6); MS (ES): m / z (%) (MH) 1253 (46%), (M-H + Na) '1275 (100%)

7-Desaza- [3- (2,2,2-trifluoroacetamido) -prop-1 -inyl] -2'-deoxyguanosine (1.9).

Low N ² , a suspension of 7-desaza-7-iodo-guanosine (2 g, 2.75 mmol), Pd (PPh ³ ) ⁴ (582 mg, 0.55 mmol), CuI (210 mg, 1.1 mmol), Et ³ N (1.52 ml, 11 mmol) and the propagylamine (2.5 g, 16.5 mmol) in DMF (40 ml) was stirred at room temperature for 15 hours and under N ² . The reaction was protected from light with an aluminum foil. After TLC indicated complete consumption of the starting material, the reaction mixture was concentrated. The residue was diluted with MeOH (20 ml) and treated with Dowex-HCO ³ -. The mixture was stirred for 30 minutes and filtered. The solution was concentrated and purified by silica gel chromatography (petroleum ether: EtOAc 50:50 to petroleum ether: EtOAc: MeOH 40:40: 20), giving (19) as a yellow powder (2.1 g, 92 %). 1H NMR (d6 DMSO) 2.07-2.11 (m, 1H, H-2 '), 2.31-2.33 (m, 1H, H-2'), 3.49-3.53 (m, 2H, H-5 '), 3.77 (br s, 1H, H-4 '), 4.25 (d, J = 4.3 Hz, 2H, ECCH ² ), 4.30 (br s, 1H, H-3'), 4.95 (t, J = 5.2 Hz, 1H , 5'OH), 5.25 (d, J = 3.4 Hz, 1H, 3'-OH), 6.27-6.31 (m, 1H, H-1 '), 6.37 (s, 2H, NH ² ), 7.31 (s , 1H, H-8), 10.10 (br s, 1H, NHCOCF ³ ), 10.55 (s, 1H, NH). Mass (-ve electrospray) calculated for C ¹⁶ H ¹⁶ F ³ N ⁵ O ⁵ 415, found 414.20 * 53

5'-O- (tert-Butyldiphenyl) -7-desaza-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyguanosine (20).

A solution of (19) (2.4 g, 5.8 mmol) in pyridine (50 ml) was treated with tert-butyldiphenylsilyl chloride (TBDPSCl) (1.65 ml, 6.3 mmol) dropwise at 0 ° C. The reaction mixture was then warmed to room temperature. After 4 hours, another portion of TBDPSCl (260 ^ L, 1 mmol) was added. The reaction was monitored by TLC, until complete consumption of the starting material. The reaction was quenched with MeOH (-5 ml) and evaporated to dryness. The residue was dissolved in DCM and saturated aqueous NaHCO3 was added. The aqueous layer was extracted three times with DCM. The combined organic extracts were dried (MgSO ⁴ ) and concentrated under vacuum. Purification by chromatography on silica (EtOAc to EtOAc: MeOH 85:15) gave (20) a yellow foam (3.1 g, 82%).

1H NMR (da DMSO) 1.07 (s, 9H, CH ³ ), 2.19-2.23 (m, 1H, H-2 '), 2.38-2.43 (m, 1H, H-2'), 3.73-3.93 (m , 2H, H-5 '), 4.29 (d, J = 5.0 Hz, 2H, CH ² N), 4.42-4.43 (m, 1H, H-3'), 5.41 (br s, 1H, OH), 6.37 (t, J = 6.5 Hz, H-1 '), 6.45 (br s, 2H, NH ² ), 7.24-7.71 (m, 11H, H-8, HAr), 10.12 (t, J = 3.6 Hz, 1H , NH), 10.62 (s, 1H, H-3). Mass (+ ve electrospray) calculated for C ³² H ³⁴ F ³ N ⁵ O ⁵ Si 653, found 654.

5'-O- (tert-Butyldiphenyl) -7-desaza-3'-O-methylthiolmethyl-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyguanosine (21 ).

A solution of (20) (1.97 g, 3.0 mmol) in DMSO (15 ml) was treated with AC ² O (8.5 ml, 90 mmol), and AcOH (2.4 ml, 42 mmol) and stirred at room temperature for 15 hours , then 2 hours at 40 ° C. The reaction mixture was diluted with EtOAc (200 ml) and stirred with saturated aqueous NaHCO3 (200 ml) for 1 hour. The aqueous layer was washed with EtOAc twice. The organic layer was combined, dried (MgSO ⁴ ), and concentrated in vacuo. Purification by chromatography on silica (EtOAc: Hexane 1: 1 to EtOAc: Hexane: MeOH 10: 10: 1) gave (21) as a yellow foam (1.3 g, 60%). 1H NMR (CDCb) 51.04 (s, 9H, CH ³ ), 2.08 (s, 3H, SCH ³ ), 2.19-2.35 (m, 2H, H-2), 3.67-3.71 (m, 2H, H-5 ' ), 3.97-3.99 (m, 2H, H-4 ', H-3'), 4.23 (br s, 2H, CH ² N), 4.58 (s, 2H, CH ² S), 6.31 (dd, J = 5.7, 7.9 Hz, H-1 ') 7.19-7.62 (m, 11H, H8, HAr). Mass (+ ve electrospray) calculated for C ³⁴ H ³⁸ F ³ N ⁵ O ⁵ SSi 713, found: 714.

To a solution of (21) (1.3 mg, 1.8 mmol), cyclohexene (0.91 ml, 9 mmol) in CH ² Cl ² (10 ml) at 4 ° C, sulfuryl chloride (1M in C ^ C h) was added (1.1 ml, 1.1 mmol) dropwise under N ² . After 30 minutes, TLC indicated complete consumption of nucleoside (22). After evaporating to remove the solvent, the residue was then placed under high vacuum for 20 minutes, and then treated with NaN ³ (585 mmol, 9 mmol) and DMF (10 ml). The resulting suspension was stirred under room temperature for 2 hours. Extraction with C ^ C ^ / NaCl (10%) gave a yellow gum, which was treated with TBAF in THF (1 M, 3 ml) and THF (3 ml) at room temperature for 20 minutes. Evaporation to remove solvents, extraction with EtOAc / saturated aqueous NaHCO ³ , followed by purification by chromatography on silica (EtOAc to EtOAc: MeOH 9: 1) gave (22) as a yellow foam (420 mg, 50%) . 1H NMR (d6 DMSO): 52.36-2.42 (m, 1H, H-2 '), 2.49-2.55 (m, 1H, H-2'), 3.57-3.59 (m, 2H, H-5 '), 3.97 -4.00 (m, 1H, H-4 '), 4.29 (m, 2H, CH ² N), 4.46-4.48 (m, 1H, H-3'), 4.92-4.96 (m, 2H, CH ² N ³ ), 5.14 (t, J = 5.4 Hz, 1H, 5'-OH), 5.96-6.00 (dd, J = 5.7, 8.7 Hz, 1H, H-1 '), 6.46 (br s, 2H, NH ² ) , 7.39 (s, 1H, H-6), 10.14 (s, 1H, NH), 10.63 (s, 1H, H-3).

5'-O-nucleoside triphosphate

(2. 3).

Tetrasodium diphosphate decahydrate (1.5 g, 3.4 mmol) was dissolved in water (34 ml) and the solution was applied to a column of Dowex 50 in the H + form. The column was washed with water. The eluent was dripped directly onto a chilled (ice bath) and stirred solution of tri-n-butyl amine (1.6 ml, 6.8 mmol) in EtOH (14 ml). The column was washed until the pH of the eluent increased to 6. The aqueous ethanol solution was evaporated to dryness and then coevaporated twice with ethanol and twice with anhydrous DMF. The residue was dissolved in DMF (6.7 ml). The pale yellow solution was stored on 4A molecular sieves. The nucleoside (22) and the proton sponge were dried over P ² O ⁵ under vacuum overnight. A solution of (22) (104 mg, 0.22 mmol) and proton sponge (71 mg, 0.33 mmol) in trimethylphosphate (0.4 ml) was stirred with 4A molecular sieves for 1 hour. Freshly distilled POCh (25 µl, 0.26 mmol) was added and the solution was stirred at 4 ° C for 2 hours. The mixture was slowly warmed to room temperature and bis (tri-n-butyl ammonium) pyrophosphate (1.76 ml, 0.88 mmol) and anhydrous tri-n-butyl amine (0.42 ml, 1.76 mmol) were added. After 5 minutes, the reaction was quenched with 0.1M TEAB (triethylammonium bicarbonate) as a buffer (15 ml) and stirred for 3 hours. The water was removed under reduced pressure and the resulting residue was dissolved in concentrated ammonia (p 0.88, 10 ml) and stirred at room temperature for 16 hours. The reaction mixture was then evaporated to dryness. The residue was dissolved in water and the solution applied to a DEAE-Sephadex A-25 column. MPLC was carried out with a linear gradient of 2 L each of 0.05 M and 1 M TEAB. The triphosphate was eluted between 0.7 M and 0.8 M buffer. The fractions containing the product were combined and evaporated to dryness. The residue was dissolved in water and subsequently purified by HPLC. tr (23) = 20.5 min (Zorbax C18 preparative column, gradient: 5% to 35% B in 30 min, 0.1M TEAB buffer A, MeCN buffer B). The product was isolated as a white foam (225 OD, 29.6 mmol, 13.4%, £ ²⁶⁰ = 7,600). 1H NMR (D ² O) S 2.43-2.5 (m, 2H, H2 '), 3.85 (m, 2H, CH ² N), 3.97 4.07 (m, 2H, H-5'), 4.25 (br s, 1H , H-4 '), 4.57 (br s, 1H, H-3'), 4.74-4.78 (m, 2H, CH ² N ³ ), 6.26-6.29 (m, 1H, H-1 '), 7.41 ( s, 1H, H-8). 31P-NMR (D ² O) S -8.6 (m, 1P, PY), -10.1 (d, J = 19.4 Hz, 1P, Pa), -21.8 (t, J = 19.4 Hz, 1P, Pp). Mass (-ve electrospray) calculated for C15H21N8O13P3614, found 613.

A mixture of disulfide-linked Cy3 (2.5 ^ mol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC) (0.95 mg, 5 ^ mol), 1-hydroxybenzotriazole (HOBt) (0.68 mg, 5 ^ mol ) and N-methyl-morpholine (0.55 ^ L, 5 ^ mol) in DMF (0.9 ml) was stirred at room temperature for 1 hour. A solution of (23) (44 OD, 3.75 µmol) in 0.1 ml of water was added to the reaction mixture at 4 ° C, and left at room temperature for 3 hours. The reaction was stopped with TEAB buffer (0.1M, 10 ml) and loaded onto a DEAE Sephadex column (2 x 5 cm). The column was eluted first with a 0.1 M TEAB buffer (100 ml) and then 1 M TEAB buffer (100 ml). The desired triphosphate product was eluted with 1M TEAB buffer. The fraction containing the product was concentrated and HPLC was applied. tr (24) = 23.8 min (Zorbax C18 preparative column, gradient: 5% to 55% B in 30 min, 0.1M TEAB buffer A, MeCN buffer B). The product was isolated as a red foam (0.5 ^ mol, 20%, £ max = 150,000). 1H NMR (D ² O) 51.17-1.71 (m, 20H, 4 x CH ² , 4 x CH ³ ), 2.07-2.15 (m, 1H, H-2 '), 2.21-2.30 (m, 1H, H- 2 '), 2.52-2.58 (m, 2H, CH ² ), 2.66-2.68 (m, 2H, CH ² ), 2.72-2.76 (m, 2H, CH ² ), 3.08-3.19 (m, 2H, CH ² ), 3.81-3.93 (m, 6H, CH ² , H-5 '), 4.08-4.16 (m, 1H, H-4'), 4.45-4.47 (m, 1H, H-3 '), 4.70-4.79 (m, 2H, CH ² N ³ ), 6.05-6.08 (m, 2H, HAr), 6.15-6.18 (m, 1H, H-1 '), 7.11 (s, 1H, H-8), 7.09-7.18 (m, 2H, CH), 7.63-7.72 (m, 4H, HAr), 8.27-8.29 (m, 1H, CH). 31P NMR (D ² O) 5 -4.7 (m, 1P, P ^y ), -9.8 (m, 1P, Pa), -19.7 (m, 1P, Pp). Mass (-ve electrospray) calculated for C ⁵¹ H ⁶⁶ N ¹¹ O ²¹ P ³ S ^{4 1 3 89} . ^{2 5} , found 1388 (MH), 694 [M-2H], 462 [M-3H].

7-Desaza-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyadenosine (25).

To a suspension of 7-desaza-7-iodo-2'-deoxyadenosine (1 g, 2.65 mmol) and CuI (100 mg, 0.53 mmol) in dry DMF (20 ml) was added triethylamine (740 µl, 5.3 mmol). After stirring for 5 minutes, trifluoro-N-prop-2-ynyl-acetamide (1.2 g, 7.95 mmol) and Pd (PPh ^{3) 4} (308 mg, 0.26 mmol) were added to the mixture and the reaction was stirred at temperature environment in the dark for 16 hours. MeOH (40 ml) and Dowex bicarbonate were added to the reaction mixture and it was stirred for 45 minutes. The mixture was filtered. The filtrate was washed with MeOH and the solvent was removed under vacuum. The crude mixture was purified by chromatography on silica (EtOAc to EtOAc: MeOH 95:20) to give a slightly yellow powder (25) (1.0 g, 95%). 1H NMR (da DMSO) 82.11-2.19 (m, 1H, H-2 '), 2.40 2.46 (m, 1H, H-2'), 3.44-3.58 (m, 2H, H-5 '), 3.80 (m , 1H, H-4 '), 4.29 (m, 3H, H-3', CH ² N), 5.07 (t, J = 5.5 Hz, 1H, OH), 5.26 (d, J = 4.0 Hz, 1H, OH), 6.45 (dd, J = 6.1, 8.1 Hz, 1H, H-1 '), 7.74 (s, 1H, H-8), 8.09 (s, 1H, H-2), 10.09 (t, J = 5.3 Hz, 1H, NH).

5'-O- (tert-Butyldiphenylsilyl) -7-desaza-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyadenosine (26).

The nucleoside (25) (1.13 g, 2.82 mmol) was coevaporated twice in dry pyridine (2 x 10 ml) and dissolved in dry pyridine (18 ml). To this solution, t-butyldiphenylsilyl chloride (748 µl, 2.87 mmol) was added in small portions at 0 ° C. The reaction mixture was then allowed to warm to room temperature and stirred overnight. The reaction was quenched with saturated aqueous NaCl solution. EtOAc (25 ml) was added to the reaction mixture and the aqueous layer was extracted with EtOAc three times. After drying the combined organic extracts (MgSO ⁴ ) the solvent was removed under vacuum. Purification by chromatography on silica (DCM then EtOAc to EtOAc: MeOH 85:15) gave (26) as a slightly yellow powder (1.76 g, 97%). 1H Rm N (d6 DMSO) 81.03 (s, 9H, tBu), 2.25-2.32 (m, 1H, H-2 '), 2.06-2.47 (m, 1H, H-2'), 3.71-3.90 (m, 2H, H-5 '), 3.90-3.96 (m, 1H, H-4'), 4.32 (m, 2H, CH ² N), 4.46 (m, 1H, H-3 '), 5.42 (br s, 1H, OH), 6.53 (t, J = 6.7 Hz, 1H, H-1 '), 7.38-7.64 (m, 11H, H-8 and HAr), 8.16 (s, 1H, H-2), 10.12 ( t, J = 5.3 Hz, 1H, NH).

5'-O- (tert-Butyldiphenylsilyl) -7-desaza-4-N, N-dimethylformadin-7- [3- (2,2,2-trifluoroacetamido) -prop-1inyl] -2'-deoxyadenosine (27) .

A solution of nucleoside (26) (831 mg, 1.30 mmol) was dissolved in a mixture of MeOH: N, N-dimethylacetal (30 ml: 3 ml) and stirred at 40 ° C. The reaction, monitored by TLC, was complete after 1 hour. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: MeOH 95: 5) gave (27) as a slightly brown powder (777 mg, 86%). 1H NMR (da DMSO) 50.99 (s, 9H, tBu), 2.22-2.29 (m, 1H, H-2 '), 2.50-2.59 (m, 1H, H-2'), 3.13 (s. 3H, CH ³ ), 3.18 (s. 3H, CH ³ ), 3.68-3.87 (m, 2H, H-5 '), 3.88-3.92 (m, 1H, H-4'), 4.25 (m, 2H, CH ² N ), 4.43 (m, 1H, H-3 '), 6.56 (t, J = 6.6 Hz, 1H, H-1'), 7.36-7.65 (m, 10H, HAr), 7.71 (s, 1H, H- 8), 8.33 (s, 1H, CH), 8.8 (s, 1H, H-2), 10.12 (t, J = 5.3 Hz, 1H, NH).

5'-O- (tert-Butyldiphenylsilyl) -7-desaza-4-N, N-dimethylformadin-3'-O-methylthiomethoxy-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl ] -2'-deoxyadenosine (28).

Acetic acid (775 µl, 13.35 mmol) and acetic anhydride (2.54 ml, 26.7 mmol) were added to a solution of (27) (623 mg, 0.89 mmol) in dry DMSO (8 ml). The mixture was stirred overnight at room temperature. The reaction was then poured into EtOAc and saturated aqueous NaHCO ³ (1: 1) and stirred vigorously. The organic layer was washed once more with saturated aqueous NaHCO ³ and dried over MgSO ⁴ . After removing the solvent under reduced pressure, the product (28) was purified by chromatography on silica (EtOAc: petroleum ether 1: 2, then EtOAc) yielding (28) (350 mg, 52%). 1H NMR (d6 DMSO): 5.10 (s, 9H, tBu), 2.09 (s, 3H, SCH ³ ), 2.41-2.48 (m, 1H, H-2 '), 2.64-2.72 (m, 1H, H-2 '), 3.12 (s, 3H, CH ³ ), 3.17 (s, 3H, CH ³ ), 3.66-3.89 (m, 2H, H-5'), 4.04 (m, 1H, H-4 ' ), 4.26 (m, J = 5.6 Hz, 2H, CH ² ), 4.67 (m, 1H, H-3 '), 4.74 (br s, 2H, CH ² ), 6.49 (t, J = 6.1, 8.1 Hz , 1H, H-1 '), 7.37-7.48 (m, 5H, HAr), 7.58-7.67 (m, 5H, HAr), 7. 76 (s, 1H, H8), 8.30 (s, 1H, CH) , 8.79 (s, 1H, H-2), 10.05 (t, J = 5.6 Hz, 1H, NH).

3'-O-Azidomethyl-5'-O- (tert-butyldiphenylsilyl) -7-desaza-4-N, N-dimethylformadin-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl ] -2'-deoxyadenosine (29).

To a solution of (28) (200 mg, 0.26 mmol) and cyclohexene (0.135 ml, 1.3 mmol) in dry CH ² O ² (5 ml) at 0 ° C, sulfuryl chloride (32 µl, 0.39 mmol) was added low N ² . After 10 minutes, TLC indicated complete consumption nucleoside (28). The solvent was evaporated and the residue was subjected to high vacuum for 20 minutes. It was then redissolved in dry DMF (3 ml), cooled to 0 ° C and treated with NaN ^{3 (} 86 mg, 1.3 mmol). The resulting suspension was stirred under room temperature for 3 hours. The reaction was partitioned between EtOAc and water. The aqueous phases were extracted with EtOAc. The combined organic extracts were combined and dried over MgSO ⁴ . After removing the solvent under reduced pressure, the mixture was purified by chromatography on silica (EtOAc) yielding an oil (29) (155 mg, 80%), 1H NMR (from DMSO): ⁶ 0.99 (s, 9H, tBu) , 2.45-2.50 (m, 1H, H-2 '), 2.69-2.78 (m, 1H, H-2'), 3.12 (s, 3H, CH ³ ), 3.17 (s, 3H, CH ³ ), 3.67 -3.88 (m, 2H, H-5 '), 4.06 (m, 1H, H-4'), 4.25 (m, 2H, CH ² ), 4.61 (m, 1H, H-3 '), 4.84-4.97 (m, 2H, CH ^2), 6.58 (t, J = ^{6 6} Hz, 1H, H-1 '), 7.35-7.47 (m, 5H, haf), 7.58-7.65 (m, 5H, HAF) 7.77 (s, 1H, H- ⁸ ), 8.30 (s, 1H, CH), 8.79 (s, 1H, H-2), 10.05 (brs, 1H, NH).

3'-O-Azidomethyl-7-deaza-4-N, N-dimethylformadin-7- [3- (2,2,2-trifluoroacetamido) -prop-1-ynyl] -2'-deoxyadenosine (30).

A solution of (29) (155 mg, 0.207 mmol) in solution in tetrahydrofuran (THF) (3 ml) was treated with TBAF (1 M in THF, 228 µl) at 0 ° C. The ice bath was then removed and the reaction mixture was stirred at room temperature. After 2 hours TLC indicated complete consumption of the nucleoside. The solvent was removed. Purification by chromatography on silica (EtOAc: MeOH 95: 5) gave (30) ⁽ 86 mg, 82%) as a pale brown oil. 1H NMR (d6 DMSO) ⁶ 2.40-2.48 (dd, J = 8.1, 13.6 Hz, 1H, H-2 '), 2.59-2.68 (dd, J = 8.3, 14 Hz, 1H, H-2'), 3.12 (s, 3H, CH ³ ), 3.17 (s, 3H, CH ³ ), 3.52-3.62 (m, 2H, H5 '), 4.02 (m, 1H, H-4'), 4.28 (d, J = 5.6 Hz, 2H, CH ² NH), 4.47 (m, 1H, H-3 '), 4.89 (s, 2H, CH ² N ³ ), 5.19 (t, J = 5.6 Hz, 1H, OH), 6.49 (dd , J = 8.1, 8.7 Hz, 1H, H-1 '), 7.88 (s, 1H, H- ⁸ ), 8.34 (s, 1H, CH), 8.80 (s, 1H, H-2), 10.08 (s , 1H, NH) .25 * 30

7- (3-Aminoprop-1-ynyl) -3'-O-azidomethyl-7-desaza-2'-deoxyadenosine 5'-O-nucleoside triphosphate (31).

The nucleoside (30) and the proton sponge were dried over P ² O ⁵ under vacuum overnight. A solution of (30) (150 mg, 0.294 mmol) and the proton sponge (126 mg, 0.588 mmol) in trimethyl phosphate (980 µl) was stirred with 4A molecular sieves for 1 hour. Freshly distilled POCU (36 µl, 0.388 mmol) was added and the solution was stirred at 4 ° C for 2 hours. The mixture was allowed to warm slowly to room temperature and 0.5 M bis (tri-n-butyl ammonium) pyrophosphate in solution in DMF (2.35 ml, 1.17 mmol) and anhydrous tri-n-butyl amine (560 µl, 2.35 mmol) were added. ). After 5 minutes, the reaction was quenched with 0.1M TEAB (triethylammonium bicarbonate) as a buffer (15 ml) and stirred for 3 hours. The water was removed under reduced pressure and the resulting residue was dissolved in concentrated ammonia (p 0.88, 15 ml) and stirred at room temperature for 16 hours. The reaction mixture was then evaporated to dryness. The residue was dissolved in water and the solution was applied to a DEAE-Sephadex A-25 column. MPLC was carried out with a linear gradient from 0.05 M to 1 M TEAB. The fractions containing the product were combined and evaporated to dryness. The residue was dissolved in water and subsequently purified by HPLC. HPLC: rt (31): 19.94 min (Zorbax C18 preparative column, gradient: 5% to 35% B in 20 min, 0.1M TEAB buffer A, MeCN buffer B). Product (31) was isolated as a white foam (17.5 pmol, 5.9%, £ ²³⁰ = 15000). 1H NMR (D ² O) 52.67-2.84 (2m, 2H, H-2 '), 4.14 (m, 2H, CH ² NH), 4.17-4.36 (m, 2H, H-5'), 4.52 (br s , H-4 '), 6.73 (t, J = 6.6 Hz, H-1'), 3.06 (s, 1H, H-8), 3.19 (s, 1H, H-2). 31P NMR (D ² O) 5 -5.07 (d, J = 21.3 Hz, 1P, PY), -10.19 (d, J = 19.3 Hz, 1P, Pa), -21.32 (t, J = 19.3 Hz, 1P, Pp). Mass (-ve electrospray) calculated for C ¹⁵ H ²¹ N ³ O ¹² P ³ 593.05, found 596.

To the disulfide binder Cy3 (1.3 ^ mol) in solution in DMF (450 ^ l) are added at 0 ° C 50 ^ l of a mixture of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, 1-hydroxybenzotrizole hydrate and N -methylmorpholine (26 ^ M each) in DMF. The reaction mixture was stirred at room temperature for 1 hour. The reaction was monitored by TLC (MeOH: CH ² Cl ² 3: 7) until the dye binder was consumed. Then DMF (400 ^ l) was added at 0 ° C, followed by nucleotide (31) (1.2 i ^ mol) in solution in water (100 i ^ l) and the reaction mixture was stirred at room temperature overnight . TLC (MeOH: CH2Cl24: 6) showed complete consumption of the activated ester and a dark red spot appeared on the baseline. The reaction was stopped with TEAB buffer (0.1M, 10 ml) and loaded onto a DEAE Sephadex column (2 x 5 cm). The column was eluted first with 0.1 M TEAB buffer (100 ml) to wash the organic residues and then 1 M TEAB buffer (100 ml). The desired triphosphate (32) was eluted with 1M TEAB buffer. The fractions containing the product were combined, evaporated and purified by HPLC. Conditions for HPLC: rt (32): 22.44 min (Zorbax C13 preparative column, gradient: 5% to 35% B in 20 min, 0.1M TEAB buffer A, MeCN buffer B). The product was isolated as a dark pink solid (0.15 μmol, 12.5%, £ ⁵⁵⁰ = 150,000). 1H NMR (D ² O) 52.03 (t, 2H, CH ² ), 2.25 (m, 1H, H-2 '), 2.43 (m, 1H, H-2'), 2.50 (m, 2H, CH ² ) , 2.66 (m, 2H, CH ² ), 3.79 (m, 2H CH ² ), 3.99 (m, 4H, CH ² N, H-5 '), 4.13 (br s, 1H, H-4'), 6.02 , 6.17 (2d, J = 13.64 Hz, 2H, HAr), 6.30 (dd, J = 6.06, 3.53 Hz, H-1 '), 7.03, 7.22 (2d, 2H, 2 x = CH), 7.53-7.32 ( m, 5H, HAr, H-2, H-3), 3.29 (m, = CH). 31P NMR (D ² O) 5 -4.33 (m, 1P, Py), -10.06 (m, 1P, Pa), -20.72 (m, 1P, Pp).

Enzyme incorporation of 3'-azidomethyl dNTPs

To a 100 nM DNA / template primer (previously labeled with 32 P and T4 polynucleotide kinase) in Tris HCl pH ^{8 0.8} 50 mM, Tween-20 to 0.01%, and MgSO ⁴ , 4 mM, was added compound ^{6, j} 2 M, and 100 nM polymerase (Thermococcus sp. 9 ° N exo -Y409V A485L supplied by New England Biolabs) . The template consisted of a 10-base adenine run to show the effect of the block. The reaction was heated to 65 ° C for 10 minutes. To show complete blockage, a test is performed with the four natural, unblocked nucleoside triphosphates. The quantitative incorporation of an individual azidomethyl blocked dTTP could be observed and thus it can be seen that the azidomethyl group acts as an effective block for further incorporation.

By linking a hairpin DNA (covalently linked autocomplementary primer / template) to a streptavidin bead, the reaction can be carried out with multiple cycles as shown in Figures 5 and ⁶ .

Preparation of streptavidin beads

The storage buffer is removed and the beads are washed 3 times with TE buffer (Tris-HCl pH ⁸ , 10 mM and EDTA, 1 mM). Resuspend in B&W buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA and 2.0 M NaCl), add biotinylated 32P-labeled hairpin DNA with an appropriate pendant template sequence. Let it stand at room temperature for 15 minutes. The buffer is removed and the beads are washed 3 times with TE buffer. Incorporation of fully functional nucleoside triphosphate (FFN)

To a solution of 50 mM Tris-HCl pH ^{8 .8} , 0.01% Tween-20, 4 mM MgSO ⁴ , 0.4 mM MnCh (except cycle 1, 0.2 mM), 2 jM FFN and 100 nM polymerase are added. This solution is then added to the beads and mixed thoroughly and incubated at 65 ° C for 10-15 minutes. The reaction mixture is removed and the beads are washed 3 times with TE buffer.

Unlocking stage

Tris- (2-carboxyethyl) phosphine (TCEP) trisodium salt (0.1M) is added to the beads and mixed thoroughly. The mixture was then incubated at 65 ° C for 15 minutes. The deblocking solution is removed and the beads are washed 3 times with TE buffer.

Coverage stage

Iodoacetamide (431 mM) in 0.1 mM phosphate pH 6.5 is added to the beads and mixed thoroughly, then left at room temperature for 5 minutes. The covering solution is removed and the beads are washed 3 times with TE buffer.

Repeats as required

The reaction products can be analyzed by placing the bead solution in the well of a standard 12% polyacrylamide DNA sequencing gel in 40% formamide loading regulator. The gel is run under denaturing conditions causing the DNA to be released from the beads onto the gel. DNA band shifts are affected both by the presence of dye and by the addition of extra nucleotides and thus cleavage of the dye (and the block) with phosphine produces a shift in mobility on the gel.

Figures 5 and ^{6 show} two cycles of incorporation with compounds 18 (C), 24 (G) and 32 (A) and six cycles with compound ⁶ .

Claims (14)

1. A nucleotide molecule having a ribose or deoxyribose sugar moiety and a base linked to a detectable marker through a cleavable linker, wherein the sugar moiety comprises a blocking group attached through an oxygen atom 3 ', in such a way that the 3' carbon atom has joined a group of the structure: -O-Z where Z is -C (R ') ² -N ³ ; where each R 'is H. 2. The nucleotide according to claim 1, wherein the base is a purine or a pyrimidine. 3. The nucleotide according to claim 1, wherein the base is a deazapurine. 4. The nucleotide according to any preceding claim which is a deoxyribonucleotide triphosphate. The nucleotide according to any of the preceding claims, wherein the detectable marker is a fluorophore. ⁶ . A kit comprising a plurality of nucleotides according to any one of claims 1 to 5. 7. The kit according to claim ⁶ containing four nucleotides according to any one of claims 1 to 5. ⁸ . The kit according to claim ⁶ or claim 7 further containing a polymerase. 9. The kit according to claim ⁸ , wherein the polymerase is a Thermococcus sp. 10. A method for determining the sequence of a target single-stranded polynucleotide comprising monitoring the sequential incorporation of complementary nucleotides, wherein at least one incorporation is of a nucleotide according to any one of claims 1 to 5, wherein it is determined nucleotide identity by detection of the base bound label and the blocking group and label are removed prior to introduction of the next complementary nucleotide. The method of claim 10, wherein the blocking group and the cleavable structural unit are removed in a single chemical treatment step. 12. The method of claims 10 or 11, the method comprising: (a) providing a plurality of four different nucleotides according to any one of claims ¹ to ⁶ , wherein the fluorophore bound to each type of nucleotide can be distinguished upon detection; (b) incorporating the nucleotide into the complement of the target single stranded polynucleotide; (c) detecting the fluorophore of the nucleotide of (b), thereby determining the type of nucleotide incorporated; (d) removing the nucleotide tag from (b) and the blocking group; Y (e) repeating steps (b) - (d) one or more times; thereby determining the sequence of a target single stranded polynucleotide. The method of claim 12, wherein step (d) is achieved using a water soluble phosphine. The method of claim 13, wherein the phosphine is a derivatized trialkylphosphine with one or more functionalities selected from the group consisting of amino, hydroxyl, carboxyl, and sulfonate groups.