Method for fluorescence labeling of biological materials, thermally removable fluorescent labels for this method, and methods of their preparation and use

A method for fluorescence labeling of nucleosides, nucleotides and oligonucleotides with fluorescent dyes wherein a moiety of general formula 1 or 2 or 3 or 4 with the double bond configuration E is attached to a nucleoside, nucleotide or oligonucleotide

• wherein:
• n is 1 or 2
• —marks the bonding site with the nucleoside or nucleotide or oligonucleotide
• R1 means:
• hydrogen, methyl, amino group, carbonyl, benzyl, naphthyl, naphthylmethyl, phenyl, benzyl, quinolinemethyl,
• benzyl substituted with one or more the same or different substituents:
• chlorine, fluorine, methyl,
• saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, saturated alkyl containing from 2 to 8 carbon atoms substituted with a phenyl, amino group, hydroxyl group or simultaneously phenyl and hydroxyl group or phenyl and amino group,
• R2 means:
• hydrogen, methyl, benzyl, phenyl, naphthyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond,
• R3 are the same or different and represent hydrogen, methyl, saturated or unsaturated alkyl containing from 2 to 8 carbon atoms and one double bond, amino group, nitro group, azido group, a group of general formula 5
• R4 means carbon or nitrogen
• R5 and R6 are the same or different and represent hydrogen, methyl, benzyl, phenyl, naphthyl, halogen (F, Cl, Br, I), hydroxyl, amino group, nitro group, carboxyl, saturated or unsaturated alkyl containing 2 to 8 carbon atoms and one double bond, saturated or phenyl substituted unsaturated alkyl containing 2 to 8 carbon atoms and one double bond,
• in a chemical reaction of a free primary hydroxyl group of the nucleoside, nucleotide or oligonucleotide with a compound of general formula 6 or 7 or 8 or 9
• wherein A represents a group of general formula 10 or 11
• wherein n and substituents R1, R2, R3, R4, R5, and R6 have the same meanings as given above, with the compounds of the formulas 6 and 7 being obtained directly in the reaction medium as reaction products between an alcohol of general formula 12 or 13
• wherein n and substituents R1, R2, R3, EC, R5, and R6 have the same meanings as above, with a carbonyldimidazole of general formula 14 or a carbonyldi(1,2, 4-triazole) of general formula 15
• forming an intermediate of general formula 6 or 7, with the compound of formula 8 being added into the reaction medium or being obtained directly in the reaction medium by reacting an alcohol of general formula 12 or 13 wherein n and substituents R1, R2, R3, R4, R5, and R6 have the same meaning as above, with N,N,N′,N′-bis(diisopropylamino)chlorophosphine of formula 16,
• whereat the reaction proceeds in two steps, with first step in which an alcohol of general formula 12 or 13 reacts with N,N,N′,N′-bis(diisopropylamino)chlorophosphine and the second step in which 1-H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole is added in order to enable further metathesis resulting in a product that reveals rests A introduced in reaction with the alcohol of formula 12 or 13, and whereat the compound of formula 9 is injected into the reaction medium or is obtained directly in the reaction medium by reacting an alcohol of formula 12 or 13, wherein n and substituents R1, R2, R3, R4, R5, and R6 have the same meaning as above, with 2-cyanoethyl-N,N,N′,N′-bis(diisopropylamino)phosphine of formula 17
• in the presence of 1H-tetrazole or 2-ethylthiotetrazole or 4,5-dicyanoimidazole or 5-benzyl-mercaptotetrazole, wherein after the reaction between a nucleoside, nucleotide or oligonucleotide and the intermediate of formula 8 or 9 the reaction product is subjected to standard agents used in the oxidation of phosphorus (III) compounds, whereat in the case when CSO is used, methylamine or 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) or concentrated aqueous ammonia solution is added after the oxidation step is completed.