Illumina Cambridge Limited

Silke Ruediger

Xiaohai Liu

John Milton

Xiaolin Wu

Colin Lloyd Barnes

Mark Smith

Joseph Brennan

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a Csubstituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a Csubstituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). 

Labelled nucleotides

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached thereto, such that the 3&#x2032; carbon atom has attached a group of the structure &#x2014;O&#x2014;Z wherein Z is any of &#x2014;C(R&#x2032;)2-O&#x2014;R&#x2033;, &#x2014;C(R&#x2032;)2-N(R&#x2033;)2, &#x2014;C(R&#x2032;)2-N(H)R&#x2033;, &#x2014;C(R&#x2032;)2-S&#x2014;R&#x2033; and &#x2014;C(R&#x2032;)2-F, wherein each R&#x2033; is or is part of a removable protecting group; each R&#x2032; 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&#x2032;)2 represents an alkylidene group of formula &#x2550;C(R&#x2032;&#x2033;)2 wherein each R&#x2032;&#x2033; may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R&#x2033; is exchanged for H or, where Z is &#x2014;C(R&#x2032;)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3&#x2032;OH; with the proviso that where Z is &#x2014;C(R&#x2032;)2-S&#x2014;R&#x2033;, both R&#x2032; groups are not H.

Modified nucleotides

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterized in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a Csubstituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl), T is hydrogen or a Csubstituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). 

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterized in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH, and NQ wherein Q is a Csubstituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH, and N(allyl), T is hydrogen or a Csubstituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). 

Provided herein is a method for replacing the allyl group in a compound of formula R&#x2014;O-allyl, RN(allyl), RNH(allyl), RN(allyl)or R&#x2014;S-allyl with a hydrogen, using a transition metal and one or more phosphine ligands.

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterised in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a Csubstituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl). T is hydrogen or a Csubstituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside).

The invention provides a nucleotide or nucleoside having a base attached to a detectable label via a cleavable linker, characterized in that the cleavable linker contains a moiety selected from the group comprising: Formula (I) (wherein X is selected from the group comprising O, S, NH and NQ wherein Q is a Csubstituted or unsubstituted alkyl group, Y is selected from the group comprising O, S, NH and N(allyl). T is hydrogen or a Csubstituted or unsubstituted alkyl group and * indicates where the moiety is connected to the remainder of the nucleotide or nucleoside). 

The invention provides modified nucleotide or nucleoside molecule comprising a purine or pyrimidine base and a ribose or deoxyribose sugar moiety having a removable 3'-OH blocking group covalently attached thereto, such that the 3&#x2032; carbon atom has attached a group of the structure &#x2014;O&#x2014;Z wherein Z is any of &#x2014;C(R&#x2032;)2-O&#x2014;R&#x2033;, &#x2014;C(R&#x2032;)2-N(R&#x2033;)2, &#x2014;C(R&#x2032;)2-N(H)R&#x2033;, &#x2014;C(R&#x2032;)2-S&#x2014;R&#x2033; and &#x2014;C(R&#x2033;)2-F, wherein each R&#x2033; is or is part of a removable protecting group; each R&#x2032; 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&#x2032;)2 represents an alkylidene group of formula &#x2550;C(R&#x2032;&#x2033;)2 wherein each R&#x2032;&#x2033; may be the same or different and is selected from the group comprising hydrogen and halogen atoms and alkyl groups; and wherein said molecule may be reacted to yield an intermediate in which each R&#x2033; is exchanged for H or, where Z is &#x2014;C(R&#x2032;)2-F, the F is exchanged for OH, SH or NH2, preferably OH, which intermediate dissociates under aqueous conditions to afford a molecule with a free 3&#x2032;OH; with the proviso that where Z is &#x2014;C(R&#x2032;)2-S&#x2014;R&#x2033;, both R&#x2032; groups are not H.

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Nr Saffron Walden, United Kingdom

Silke Ruediger