WO2008075955A2

WO2008075955A2 - Process for the preparation of 1,4,5-trisubstituted triazoles and 3,4,5-trisubstituted triazoles

Info

Publication number: WO2008075955A2
Application number: PCT/NL2007/050676
Authority: WO
Inventors: Floris Petrus Johannes Theodorus Rutjes; Jeroen. Johannes Lambertus Maria Cornelissen; Sander Sebastiaan Van Berkel; Antonius Johannes Dirks
Original assignee: Stichting Voor De Technische Wetenschappen; Stichting Katholieke Universiteit Nijmegen
Priority date: 2006-12-21
Filing date: 2007-12-20
Publication date: 2008-06-26
Also published as: WO2008075955A3

Abstract

The present invention relates to a process for the preparation of 1,4,5- trisubstituted triazoles and 3,4,5-trisubstituted triazoles according to Formulas (Ia) and 5 (Ib), and mesomers and tautomers thereof, wherein a compound according to Formula (II) or Formula (III): is reacted with a compound according to Formula (IV). The process is very useful for the selective and site-specific addition of azide compounds to optionally activated alkynes to form 1,4,5-trisubstuted triazoles and mesomers and tautomers thereof and the application of this process to the covalent functionalisation of biomolecules.

Description

Process for the preparation of 1,4,5-trisubstituted triazoles and 3,4,5- trisubstituted triazoles

Field of the invention

The present invention relates to a process for the selective and site-specific addition of azide compounds to optionally activated alkynes to form 1,4,5-trisubstuted triazoles and 3,4,5-trisubtituted triazoles and mesomers and tautomers thereof and the application of this process to the covalent functionalisation of biomolecules.

In this application, reference is made to various scientific publications which are indicated by a reference number. At the end of this description, full references of these scientific publications are included.

Background of the invention

The development of selective and site-specific conjugation methods are upon the most investigated processes in chemical biology. A wide range of effective methods such as, Staudinger ligation¹, native chemical ligation², genetic incorporation³, expressed protein ligation⁴, Huisgen azide-alkyn cycloaddition⁵, and the Diels-Alder ligation⁶ are frequently employed in selective modification of proteins and other biomolecules.

The Cu(I) catalyzed variant of the Huisgen 1,3 -dipolar cycloaddition ' , also referred to as the "click reaction", has been applied in various fields of chemistry as a versatile and mild ligation method⁹. These important features allow for the synthesis of complex materials including bioconjugates¹⁰, glycopeptides¹¹, functionalized polymers¹², virus particles¹³ and therapeutics¹⁴. However, due to the toxicity of the copper catalyst to both bacterial cells and mammalian cells, applications regarding in vivo ligation are limited. In addition, WO 03/101972 and US 2005/0222427, both to Sharpless et al, also disclose this Cu(I) catalysed click reaction between an terminal alkyne and an azide compound, wherein the Cu(I) catalyst is made in situ by contacting Cu(II) with a reducing agent. In order to circumvent the use of copper Bertozzi and coworkers¹⁵ devised a strain promoted [3+2] cycloaddition reaction using an azide and a cyclooctyne. Recent reports also demonstrate copper free 1,3 -dipolar cycloaddition using either elevated temperatures¹⁶ or electron-deficient alkynes¹⁷. Furthermore, WO 2004/055160 to Li et al. discloses a process for covalently linking a biomolecule having an azide group, preferably an azido labeled DNA fragment, to a component having an alkyne group, said component having an alkyne group optionally being immobilised on a surface.

EP A 1.471.059 discloses a process for the preparation of substituted triazoles, wherein an α,β-unsaturated carbonyl compound is reacted with an azide in the presence of a catalytic amount of Cu(I).

EP A 1.602.663 discloses a process for the preparation of triazole-linked glycoamino acids and glycopeptides wherein a saccharide having an azide group is reacted with an amino acid having a terminal alkyne moiety, said process providing 1 ,4- disubstituted triazoles wherein the saccharide moiety occurs at position 1 and the amino acid moiety at position 4 or vice versa.

US 6.664.399 discloses a process for the preparation of triazole linked carbohydrates, wherein a carbohydrate having an azide substituent is reacted with a carbohydrate having an alkyne substituent, said process providing disubstituted triazoles having carbohydrate residues either at positions 1 and 4 or positions 1 and 5.

However, the processes known from the prior art have several drawbacks, in particular because they are unsuitable to be performed under physiological conditions occurring in a vertebrate, in particular a mammal. For example, some processes need to be performed in the presence of toxic catalysts or require elusive alkynes such as cyclooctyne. Other processes need elevated temperatures. The present invention provides a solution to these problems by providing a spontaneous tandem cycloaddition-retro Diels-Alder ligation method that can be performed under essentially physiological conditions in an aqueous environment, i.e. without the presence of toxic catalysts and without the use of elevated temperatures. Summary of the invention

The present invention relates to a process for the preparation of 1,4,5-trisubstituted triazoles and 3,4,5-trisubstituted triazoles according to Formulas (Ia) and (Ib), and mesomers and tautomers thereof:

(Ia)

(Ib)

wherein a compound according to Formula (II) or Formula (III):

is reacted with a 1 ,3 -dipolar compound, preferably a compound according to Formula (IV), R⁸ N₃

(IV)

at a temperature in the range of 15 - 50⁰C, wherein:

R is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 12, preferably 1 - 6 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, - C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched C₂ -C₁₂ alkenyl groups, Ce - Cn aryl groups, C7 - C₁₂ arylalkyl groups, and C7 - C₁₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur;

R is selected from the group consisting of electron-withdrawing groups, said electron- withdrawing group having a Hammett σ_p constant of more than 0;

R¹ and R² may optionally form a four to nine membered ring structure comprising a heteroatom selected from the group consisting of O and N;

R¹ and R² may independently also be selected from the group of functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle, label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer; R³ and R⁴ are independently selected from the group consisting of electron-withdrawing groups, said electron-withdrawing group having a Hammett σ_p constant of more than 0; R⁵ and R are independently selected from the group consisting of hydrogen, linear or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, Ce - Cn aryl groups, C7 - C₁₂ arylalkyl groups, and C7 - C₁₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; or R⁵ and R⁶ form, together with the carbon atoms of the bicycloheptane ring to which they are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S; R⁵ and R⁶ may independently and optionally also form, together with R³ and R⁴, respectively, and the carbon atoms of the bicycloheptane ring to which R , R , R⁵ and R are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S; X is selected from the group consisting of =CR⁷, =NR⁷, =0, =S, =SO, =SO₂, =PR⁷, and =P(O)R⁷;

R⁷ is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, Ce - Cn aryl groups, Cj - C₁₂ arylalkyl groups, and Cj - Cn alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; R⁸ is selected from the group consisting of linear or branched Ci - C₁₂ alkyl groups, linear or branched C₂ - C₁₂ alkenyl groups, Ce - Cn aryl groups, Cj - Cn arylalkyl groups, Cj - Cn alkylaryl groups, the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted, a group having biological or pharmaceutical activity, and a functional group having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

The present invention further relates to compounds according to Formula (I).

The present invention also relates to compounds according to Formula (III) and a process for preparing these compounds.

Finally, the present invention relates to the use of compounds according to Formula (II) and Formula (III) in a cycloaddition reaction. Detailed description of the invention

Definitions

The verb "to comprise" as is used in this description and in the claims and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there is one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

1,3 -Dipolar compounds are well known in the art (cf. for example F. A. Carey and RJ. Sundberg, Advanced Organic Chemistry, 3^rd Ed., page 635 - 637, 1990) and are preferably selected from the group consisting of nitrile oxides according to the formula R⁸-C≡N⁺-O^" (<→ R⁸-C⁺=N-O^~) azides according to the formula R⁸-N^~-N=N⁺ (cf. Formula (IV) described above), diazomethanes according to the formula R -^~CH₂-N=N⁺, nitrones according to the formula (R )₂ ⁺C-N(R ^a) -O^" wherein R ^a is a group as defined for R or hydrogen, or nitrilamines according to the formula R8-⁺C=N-N-R ^a, most preferably from the group of azides according to Formula (IV).

A fluorinated hydrocarbyl group comprising 1 - 12, preferably 1 - 6 carbon atoms may be a saturated or unsaturated hydrocarbyl group and is constituted of carbon atoms, fluorine atoms and hydrogen atoms. However, the fluorinated hydrocarbyl group may also be substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -Ci₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, Ce - Ci₂ aryl groups, Cj - Ci₂ arylalkyl groups, and C7 - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur. According to the invention, it is preferred that the fluorinated hydrocarbyl group is substituted with one of these substituents. As will be understood by a person skilled in the art, a hydrocarbyl group encompasses (cylo)aliphatic groups and aromatic groups and the fluorinated hydrocarbyl group may therefore be pentafluorophenyl.

Obviously, an alkyl group or an alkenyl group can only be a cyclic group when it contains al least three carbon atoms or two carbon atoms and a heteroatom, e.g. an oxygen atom, so that it represents an oxiranyl group (epoxide group) as will be understood by a person skilled in the art.

An electron-withdrawing group is here defined as a group having a Hammett σ_p constant of more than 0 (cf. J. March, Advanced Organic Chemistry, 4* Ed., page 280 (Table 9.4), 1992).

Linear or branched C₁ - C₆ alkyl and linear or branched Ci -C 12 alkenyl groups are hydrocarbyl groups which may optionally be substituted or interrupted with one or more heteroatoms selected from the group consisting of O, S and N. For example, the alkyl group may be methoxy methyl or 2-methoxy butyl as will be apparent to those skilled in the art. If required, such a heteroatom may itself be substituted with a hydrocarbyl group, i.e. an alkyl group, an aryl group, an alkylaryl group or an arylalkyl group, so that the alkyl group is for example ethoxy, phenoxy or p-methylphenoxy.

A C_O - C₁₂ aryl group is an hydrocarbyl group and may consists of only one aromatic Cό-ring, wherein the other carbon atoms are incorporated in substituents, although it may also be an aryl substituted aromatic C_ό-ring, e.g. a 4-phenyl-phenyl group, or a bicyclic aromatic group, e.g. a naphtyl group. The C₆ - C₁₂ aryl group may be substituted with one or more substituents that may comprise one or more heteroatoms selected from the group consisting of O, S and N.

A C7 - C 12 arylalkyl group is also an hydrocarbyl group and is for example 3- phenylpropyl and a C7 - C₁₂ alkylaryl group is for example 3-propylphenyl as is well known to the skilled person in the art.

Functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label are to be understood as substituents which are substituted by a biomolecule, a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

However, it is within the scope of this invention that the biomolecule, the UV- label-, the fluorescence-label, the luminescence-label, the radioactive label, the dye, the chromophore, a magnetic particle label or the affinity label are directly bonded., that is that e.g. substituent R itself may be a biomolecule, a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label moiety, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer, as will be understood by a person skilled in the art. In this document, such functional groups are defined by the term "biologically active groups". In addition, these biologically active groups may be provided with a linking moiety or a spacer, preferably a Ci - C₄ alkylene glycol chain having 1 - 100 alkylene glycol units or a poly(meth)acrylate chain having a Ci - Ce alkyl group and comprising 1 - 100 (meth)acrylate units.

The term "biomolecule", "biologically active group" and "biomolecule moiety" includes "biological" organic compounds occurring in a living system, e.g. a mammal, a plant and the like, such as hormones, proteins, peptides, carbohydrates, amino acids, biomacromolecules, antibodies, DNA fragments, RNA fragments, lipids, vitamins, enzymes and the like.

The terms functional groups, biologically active groups, biomolecules and biological moiety are well known to the person skilled in the art and are for example also disclosed in WO 2004/055160, expressly incorporated by reference herein.

Detailed description of the invention

Compounds according to Formula (I) are suitable for use in immobilization processes on surfaces as is for example also disclosed in WO 2004/055160, in imaging techniques involving UV, fluorescence or luminescence detection, positron electron tomography and the like. According to the invention, it is preferred that the electron-withdrawing group has a Hammett σ_p-constant of more than 0. Examples for suitable electron-withdrawing groups are halogens, in particular fluorine (σ_p = 0.15), chlorine (σ_p = 0.24), -COOH (σ_p = 0.44). The values for σ_p are taken from J. March, Advanced Organic Chemistry, 4^th Ed., page 280 (Table 9.4), 1992. Preferred electron-withdrawing groups include groups having a Hammett σ_p-constant of more than 0 and as high as feasible, although the upper limit will usually be limited to about 2.5, preferably 2. Such preferred electron-withdrawing groups include halogens, in particular fluorine; - C(O)OR* groups, wherein R* is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched d - Ce alkenyl groups, CO - C₁₂ aryl groups, C7 - C₁₂ arylalkyl groups, and C7 - C₁₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, in particular by one or more fluorine atoms, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur; -C(O)N(R*)₂, wherein R* is as defined above; -⁺N(R*)₄, wherein R* is as defined above and wherein two or more R*'s may form a saturated or unsaturated four to nine membered ring structure (e.g. pyridinium) including the N-atom; and -S(O)₂R*, wherein R* is as defined above. If the electron-withdrawing group has more than one substituent, their combined electron-withdrawing capability is preferably equivalent to a Hammett σ_p- constant of more than 0. It is well known to the person skilled in the art which combinations of substituents provide such an electron-withdrawing capability.

According to another embodiment of the present invention, R² is independently selected from the group consisting of the groups enumerated for R¹.

A particular advantage of the present invention is that the process of the tandem cycloaddition-retro Diels-Alder ligation method disclosed herein proceeds under mild conditions, i.e. that it is envisaged that it proceeds under physiological condition. According to the invention the process is therefore preformed in an aqueous medium, preferably water, and preferably in the absence of an added catalyst. Obviously, when occurring in a physiological environment, e.g. within a vertebrate, the reaction may be catalysed by components occurring therein which may have a catalytic effect. According to the invention, it is preferred that a compound according to Formula (III) is reacted with a compound according to Formula (IV).

According to a preferred embodiment of the present invention, R has the formula - CpFqHr, wherein p is in the range of 1 - 6 and q and r are in the range of 3 - 13, provided that q + r = 2p + 1. Most preferably, Ri is -CF₃.

According to an other preferred embodiment of the present invention, R has the formula C_pF_qH_r, wherein p is in the range of 6 - 18 and q and r are in the range of 7 - 17, provided that q + r = p - 1.

According to another preferred embodiment of the present invention, R is a group according to Formula (V):

-(CF₂VZ

wherein n is in the range of 1 - 25, preferably n = 1, and Z is selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, - PO₃H and -SO3H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, Ce - Ci₂ aryl groups, C7 - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted as is described above.

According to yet another preferred embodiment of the present invention, R is selected from the group of functional groups or "biologically active groups" consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label.

It is furthermore preferred that R² is selected from the group consisting of - COOR , wherein R is selected from the group consisting of hydrogen, linear or branched Ci - Ci₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, , Ce - Ci₂ aryl groups, C7 - Ci₂ arylalkyl groups, and C7 - Ci₂ alkylaryl groups, the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted; -N⁺(R¹⁰)4; -CN; -COR¹⁰; -C_pF_qH_r, wherein p is in the range of 1 - 6 and q and r are in the range of 3 - 13, provided that q + r = 2p + 1 , and C_pF_qH_r, wherein p is in the range of 6 - 18 and q and r are in the range of 7 - 17, provided that q + r = p - 1.

However, according to another preferred embodiment of the present invention, R² is selected from the group of functional groups or "biologically active groups" consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer.

Preferably, R and R are selected from the group consisting of preferably hydrogen, optionally substituted, linear, cyclic or branched C₁ - C₆ alkyl and linear, cyclic or branched d - Ce alkenyl.

R⁵ and R may be substituted with a substituent that allows for a polymer supported reaction.

According to the present invention, X is preferably =0.

The process according to the present invention wherein a compound of Formula (II) or Formula (III), preferably a compound according to Formula (III), is reacted with a compound according to Formula (IV), can be conducted with a wide range of molar ratios of the reactants. However, it is preferred that the molar ratio between the compound according to Formula (II) or Formula (III) and the compound according to Formula (IV) is between 1 : 5 to 5 : 1 , more preferably between 1 : 2 and 2 : 1 and in particular between 1 : 1.5 and 1.5 : 1.

The present invention also relates to compounds according to Formula (I) and mesomers and tautomers thereof:

(Ib)

wherein:

R¹ is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 6 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; - C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -Ci₂ alkyl groups, linear, cyclic or branched C₂ - Ce alkenyl groups, Ce - Cn aryl groups, C7 - Ci₂ arylalkyl groups, and C7 - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur;

R and R may optionally form a four to nine membered ring structure comprising a heteroatom selected from the group consisting of O and N;

R¹ and R² may independently also be selected from the group of functional groups or "biologically active groups" consisting of synthetic polymers such as polyethylene glycols, biomolecules, pharmaceuticals, functional groups having as a substituent a UV- label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer; and

R⁸ is selected from the group consisting of linear or branched Ci - C₁₂ alkyl groups, linear or branched C2 - C₁₂ alkenyl groups, Ce - Ci₂ aryl groups, C7 - C₁₂ arylalkyl groups, C7 - C₁₂ alkylaryl groups, the alkyl groups, alkenyl aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted, a group having biological or pharmaceutical activity, and a functional group having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer..

The present invention also relates to the valuable intermediates, in particular compounds according to Formula (III):

wherein R is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 6 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, - CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched C₂ - C_O alkenyl groups, Ce - Ci₂ aryl groups, C7 - Ci₂ arylalkyl groups, and C7 - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur;

R and R may independently also be selected from the group of functional groups or "biologically active groups" consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label, or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

R and R are independently selected from hydrogen and optionally substituted, linear or branched C₁ - C₆ alkyl and linear, cyclic or branched C2 - C₆ alkenyl; R and R are independently selected from hydrogen and linear or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched C2 - C₆ alkenyl groups, C₆ - C₁₂ aryl groups, C7 - C₁₂ arylalkyl groups, and C7 - C₁₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; or R and R form, together with the carbon atoms of the bicycloheptane ring to which they are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S;

R⁵ and R⁶ may independently and optionally also form, together with R³ and R⁴, respectively, and the carbon atoms of the bicycloheptane ring to which R³, R⁴, R⁵ and R⁶ are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S; X is selected from the group consisting of =CR⁷, =NR⁷, =0, =S, =SO, =Sθ2, =PR⁷, and =P(O)R⁷; and

R is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C2 - C₆ alkenyl groups, C₆ - C₁₂ aryl groups, Cj - C₁₂ arylalkyl groups, and C7 - C₁₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted;

The present invention also relates to a process for the preparation of a compound according to Formula (III), wherein a compound according to Formula (V):

(V)

is reacted with a compound according to Formula (II), wherein the substituents X, R , R , R³, R⁴, R⁵ and R⁶ are as defined above.

The present invention also relates to a process for covalently linking a biomolecule, a functional group or a biologically active group as defined in this document to a second molecule comprising contacting the biomolecule, the functional group or the biologically active group, said biomolecule, functional group or biologically active group having an azide group, with the second molecule, wherein the second molecule is selected from the group of molecules according to formulas (II) and (III).

The process is very useful for the selective and site-specific addition of azide compounds to optionally activated alkynes to form 1,4,5-trisubstuted triazoles and 3,4,5- trisubstuted triazoles and mesomers and tautomers thereof and the application of this process to the covalent functionalisation of biomolecules.

Examples

1. Synthesis

The Diels-Alder oxanorbomadiene adducts used in the following examples were prepared according to literature procedures¹⁸'¹⁹.

Dimethyl 7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (1)

Furan (0.64 mL, 10 mmol) and dimethyl acetylenedicarboxylate (DMAD, 1.22 mL, 10 mmol) were dissolved in 4 mL ether. The mixture was stirred for 7 days at room temperature. Water (10 mL) was added and the layers were separated. The water layer was extracted with ether (15 mL). The combined ether layers were washed with brine (20 mL). The organic layer was dried over Na2SO₄ and concentrated in vacuo. The obtained liquid was purified by column chromatography (EtOAc/n-heptane, 1/1) resulting in the desired product (1.48 g (70%), light yellow liquid). R_f = 0.6 (EtOAc/n-heptane 1/1). ¹H- NMR (300 MHz, CDCl₃) δ (ppm): 7.22 (s, 2H), 5.68 (s, 2H), 3.83 (s, 6H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 162.7, 152.5, 142.8, 84.6, 51.9. LCQ MS(ESI+) m/z (%) 210.9 (100) [M+H]⁺.

3-(methoxycarbonyl)-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxylic acid (2)

Oxanorbomadiene 1 (0.13 g, 0.62 mmol) was dissolved in 4 mL THF. The mixture was cooled to 0 ⁰C and 4 mL NaOH (aq) (0.25 M) was added drop wise. The conversion of the reaction was monitored with TLC (100% EtOAc) and after full conversion the reaction was quenched with 1 mL HCl (aq) (IM) and subsequently extracted into EtOAc (10 mL). The combined organic layers were dried over Na₂SO₄ and concentrated in vacuo resulting in the desired product as a dark yellow oil (97 mg (80 %)). H-NMR (300 MHz, CDCl₃) δ (ppm): 7.27 (dd, J= 1.8, 3.3 Hz, IH), 7.19 (dd, J= 1.8, 3.3 Hz, IH), 5.83 (t, J = 1.8 Hz, IH), 5.78 (t, J = 1.8 Hz, IH), 3.98 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 166.3, 161.8, 161.3, 151.8, 143.1, 142.8, 85.2, 84.2, 54.1. LCQ MS(ESI-) m/z (%) 194.93 (100) [M-H]^".

Dimethyl 7-N-Boc-bicyclo[2.2.1 ]hepta-2,5-diene-2,3-dicarboxylate (3)

N-Boc-pyrrole (1.67 mL, 10 mmol) and dimethylacetylenedicarboxylate (DMAD, 1.23 mL, 10 mmol) were dissolved in EtOAc (4.5 mL). The solution was placed in a 7.5 mL teflon vial. The vial was sealed and placed in high-pressure reactor at 1.5 GPa at 50 ⁰C for 18 hours. The crude reaction mixture was purified by column chromatography (EtOAc/n-heptane, 1/5) and was obtained as a slightly yellow solid (1.2 g (40%)). Rf = 0.5 (EtOAc/n-heptane, 1/5). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 7,13 (s, 2H), 5.46 (s, 2H), 3.82 (s, 6H), 1.41 (s, 9H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 163.1, 153.8, 152.2, 143.1, 142.4, 81.3, 69.0, 52.2, 27.9. LCQ MS(ESI-) m/z (%) 308.12 (100) [M-H]^"

3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid ethyl ester (4)

Ethyl 2-fluorobut-2-ynoate (1.00 g, 0.86 mL, 6.02 mmol) was placed in a Schlenk tube which was fitted with a stopper, evacuated and back-filled with argon. Furan (498 mg, 468 μL, 7.32 mmol) was added and the reaction mixture was heated to 40 ⁰C. The reaction was stirred at 40 ⁰C under an argon atmosphere for 4 days. The resulting mixture was washed out with ether and concentrated in vacuo. The crude mixture was purified by column chromatography (EtOAc/n-heptane, 1/4) resulting in compound 4 as a slightly yellow oil (1.00 g (71%)). R_f = 0.43 (EtOAc/n-heptane, 1/4). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.30 (dd, J = 5.3, 1.9 Hz, IH), 7.20 (dd, J = 5.3, 1.9 Hz, IH), 5.70 (m, IH), 5.66 (t, J= 1.7 Hz, IH), 4.29 (m, 2H), 1.30 (t, J = 7.1 Hz, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 161.39, [151.14, 151.08, 150.99, 150.48] (q), 143.4, 142.2, 137.4, [126.5, 122.9, 119.4, 115.8] (q, CF₃), 84.7, 83.5, 61.4, 13.4. LCQ MS(ESI+) m/z (%) 235.07 (100) [M+H]⁺. Example 5

3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxylic acid (5)

Oxanorbornadiene 4 (500 mg, 2.13 mmol) was dissolved in THF (30 mL) and cooled to 0 ⁰C. 4.85 mL NaOH (aq) (IM) was added drop wise. The mixture was stirred for 30 min. at 0 ⁰C and 1-2 hours at room temperature. After complete conversion the volume of the mixture was reduced to 50% of the original volume and H2O (20 mL) and EtOAc (15 mL) were added. The layers were separated and the aqueous layer was acidified to pH 4-5 with HCl (aq) (2M). The water layer was extracted with EtOAc (2 x 20 mL). The combined organic layers were dried over MgSO₄ and evaporated to dryness. The solid was washed with CH2CI2 (2 x 10 mL) to removed traces of EtOAc and THF. Compound 5 was obtained as an off- white solid (363 mg (83%)). Rf = 0.1 (n- heptane/EtOAc, 2/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.80-7.57 (brs, IH), 7.30 (dd, J= 5.3, 1.9 Hz, IH), 7.22 (dd, J= 5.3, 1.9 Hz, IH), 5.74 (s, IH), 5.70 (d, J= 1.3 Hz, IH). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 166.2, [155.1, 154.6, 154.1, 153.6] (q), [150.7, 150.6] (d), 143.9, 142.6, [126.7, 123.1, 119.5, 115.9] (q), 85.0, 84.2. LCQ MS(ESI-) m/z (%) 205.00 (100) [M-H]^".

Example 6

3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxyl-Gly-OMe (6)

Oxanorbornadiene carboxylic acid 5 (20.6 mg, 0.1 mmol), H-GIy-OMe-HCl (13.8 mg, 0.1 1 mmol) and 4-(dimethylamino)-pyridine (DMAP, 24.2 mg, 0.2 mmol) were dissolved in 2 mL CH₂CI₂ and cooled to 0 ⁰C. l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 21 mg, 0.11 mmol) was added and the reaction mixture was stirred at 0 ⁰C for 30 min. The mixture was allowed to warm to room temperature and was stirred for an additional 16 hours. The reaction was quenched with 2 mL HCl (aq) (2M) and extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na₂SO₄ and concentrated in vacuo. The crude mixture was purified by preparative TLC (CHiCVMeOH, 9/1) resulting in compound 6 as a slightly yellow solid (15.5 mg (56%)). R_f= 0.55 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.33 (dd, J = 5.3, 2.0 Hz, IH), 7.16 (dd, J = 5.3, 2.0 Hz, IH), 6.41 (brs, IH, NH), 5.68 (m, 2H), 4.15 (dq, J = 18.5, 18.5, 18.5, 5.2 Hz, 2H) 3.80 (s, 3H). ¹³C-NMR (50 MHz, CDCl₃) δ (ppm): 166.6, 154.9, 154.2, 150.7, 144.0, 142.7, [124.8, 1 18.6] CF₃, 85.1, 84.3. LCQ MS(ESI-) m/z (%) 276.1 (100) [M-H]^".

Example 7

N- {Boc} -N'- {3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxyl} -3,6- dioxaoctane-l ,8-diamine (7)

To a solution of oxanorbornadiene 5 (34.7 mg, 0.17 mmol) and Boc-l-amino-3,6- dioxa-8-octanediamine (41.8 mg, 0.17 mmol) in CH₂CI₂ (1.5 mL) was added 4-(dimethyl amino)-pyridine (DMAP, 41.1 mg, 0.34 mmol). The mixture was cooled to 0 ⁰C and 1- ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 21 mg, 0.1 1 mmol) was added slowly. The mixture was stirred for 30 min. at 0 ⁰C and 16 hours at room temperature. The reaction mixture was acidified with HCl (2M) to a pH of 1-2 and extracted with CH2CI2 (2 x 5 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by preparative TLC (CH₂Cl₂/Me0H, 9/1) resulting in compound 7 as a slightly yellow oil (47.8 mg (64%)). R_f= 0.50 (CH₂Cl₂/Me0H, 9/1). ¹H- NMR (400 MHz, CD₃OD) δ (ppm): 7.30 (dd, J = 5.3, 1.9 Hz, IH), 7.21 (dd, J = 5.3, 1.9 Hz, IH), 5.65 (t, J = 1.6, 1.6 Hz, IH), 5.55 (m, IH), 3.59 (s, 4H) 3.56 (dd, J = 10.5, 5.0 Hz, 2H), 3.49 (t, J = 5.7, 5.7 Hz, 2H), 3.45 (dd, J = 11.3, 5.5 Hz, 2H), 3.20 (t, J= 5.7 Hz, 2H), 1.41 (s, 9H). ¹³C-NMR (50 MHz, CD₃OD) δ (ppm): 165.2, 158.5, 144.7, 143.6, [126.6, 121.2] CF₃, 87.1, 84.4, 80.1, 71.3, 70.3, 41.2, 40.4, 28.8.

N,N'-{3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2-carboxyl}-3,6- dioxaoctane-l,8-diamine-TFA (8)

To a cooled solution (0 ⁰C) of 7 (47.8 mg, 0.11 mmol) in dry CH₂Cl₂ (2 mL) was added drop wise trifluoroacetic acid (TFA, 0.5 mL). The reaction was stirred at 0 ⁰C for 1 hour after which the reaction was complete. The solvent was evaporated and the crude mixture was dissolved in H₂O (5 mL) and dioxane (5 mL) and freeze-dried to afford compound 8 as a light yellow solid (49.0 mg (+99%)). R_f = 0.09 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CD₃OD) δ (ppm): 7.30 (ddd, J = 5.3, 1.9 Hz, 0.7 Hz, IH), 7.22 (ddd, J = 5.3, 1.9, 0.7 Hz, IH), 5.66 (s, IH), 5.55 (s, IH), 3.76 (t, J = 4.9, 2H) 3.64, (s, 4H), 3.57 (t, J = 5.9 Hz, 2H), 3.38-3.55 (m, 4H), 3.09 (t, J = 5.7 Hz, 2H). ¹³C-NMR (50 MHz, CD₃OD) δ (ppm) 165.3, 156.2, 144.6, 143.7, 127.8, [126.5, 121.2] CF₃, 87.1, 84.5, 71.3, 70.3, 67.9, 40.7, 40.3.

Example 9

N-[{thioureido-benzyl}-DTPA]-N'-{3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5- diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine (9)

To solution of 8 (29.7 mg, 0.066 mmol) in 0.6 mL dry CH₂Cl₂ was added 2-(4- isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-Bn-DTPA, 40 mg, 0.06 mmol) in H₂O (0.9 mL). NaHCO₃ (aq) (0.1 mL, IM) was added and the reaction mixture was stirred vigorously. The conversion was monitored by LCQ analysis and was found to be complete after 4 hours. The CH₂Cl₂ was removed under reduced pressure and the obtained residue was diluted with H₂O (2 mL) and dioxane (2 mL) and freeze-dried. LCQ MS(ESI+) m/z (%) 876.0 (100) [M+H]⁺

3,6,9-Trioxadodecan-12-oic acid, l-[[3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5- diene-2-carboxyl]oxy]-l,l-dimethylethyl ester (10)

A mixture of 5 (103 mg, 0.50 mmol), tert-butyl-12-hydroxy-4,7,10-trioxadodecanoate (139 mg, 0.50 mmol) and 4-(dimethylamino)-pyridine (DMAP, 121 mg, 1.00 mmol) in CH₂Cl₂ (6 mL) was cooled to 0 ⁰C before adding l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 105 mg, 0.55 mmol). The mixture was stirred for 5 min. at 0 ⁰C and 18 hours at room temperature. The reaction mixture was acidified with HCl (2M) to a pH of 1-2 and extracted with CH₂Cl₂ (2 x 10 mL). The combined organic layers were dried over MgSO₄ and concentrated in vacuo. The crude mixture was purified by preparative TLC (CH₂Cl₂/Me0H, 9/1) resulting in compound 10 as a slightly yellow oil (65 mg (27%)). R_f= 0.81 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.19 (dd, J = 5.3, 1.9 Hz, IH), 7.29 (dd, J = 5.3, 1.9 Hz, IH), 5.71 (dd, J = 2.9, 1.6 Hz, IH), 5.66 (t, J = 1.7 Hz, IH), 4.36 (ddt, J = 11.9, 11.9, 7.1, 4.9 Hz, 2H), 3.73 (t, J = 4.9, 2H), 3.70 (t, J = 6.6 Hz, 2H), 3.64 (s, 6H), 3.60 (m, 2H), 2.49 (t, J= 6.6 Hz, 2H), 1.44 (s, 9H). LCQ MS(ESI+) m/z (%) 489.0 (50) [M+Na]⁺, 410.9 (50) [M-(CH₃)₂C=C]⁺

3,6,9-Trioxadodecan-12-oic acid, l-[[3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5- diene-2-carboxyl]oxy]-12-carboxylic acid (11)

A solution of compound 10 (65 mg, 0.13 mmol) in CH2CI2 (2 mL) was cooled to 0 ⁰C before trifluoroacetic acid (TFA, 55 μL) was added drop wise. The mixture was stirred for 30 min. at 0 ⁰C and 16 hours at room temperature. The solvent was removed and the residue was dissolved in H2O (5 mL) and dioxane (3 mL) and subsequently freeze-dried. The product was purified by preparative TLC (CH₂Cl₂ZMeOH, 9/1), resulting in compound 11 as a colorless oil (44.6 mg (84%)). R_f = 0.25 (CH₂Cl₂/Me0H, 9/1). ¹H- NMR (400 MHz, CDCl₃) δ (ppm): 7.60 (brs, IH), 7.30 (dd, J = 5.3, 1.9 Hz, IH), 7.20 (dd, J = 5.3, 1.9 Hz, IH), 5.72 (m, IH), 5.66 (t, J = 1.72 Hz, IH), 4.44-4.31 (m, 2H), 3.79-3.72 (m, 4H), 3.65 (s, 4H), 3.64 (s, 4H), 2.63 (t, J = 6.28 Hz, 2H). ¹³C-NMR (50 MHz, CDCl₃) δ (ppm): 176.2, 161.7, 151.3, 144.0, 142.7, [124.3, 119.1] (CF₃), 109.7, 85.2, 84.1 , 70.6, 70.4, 68.7, 66.4, 64.8, 34.9. LCQ MS(ESI+) m/z (%) 410.9 (40) [M+H]⁺, 433.0 (100) [M+Na]⁺

3,6,9-Trioxadodecan-12-oic acid, l-[[3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5- diene-2-carboxyl]oxy]- 12-N-hydroxysuccinimide (12) A mixture of 11 (29.6 mg, 0.07 mmol) and N-hydroxysuccinimide (9.7 mg, 0.084 mmol) in anhydrous DMF (1.5 mL) was cooled to 0 ⁰C before adding l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 19.1 mg, 0.10 mmol). The mixture was stirred 30 min. at 0 ⁰C and 20 hours at room temperature. After the addition Of H₂O (1 mL) and acetone (1 mL), the mixture was extracted with ethyl acetate and ether (2 x 5 mL) (1/1 v/v). The combined organic layers were dried over MgSO₄ and concentrated in vacuo. The crude product was purified by preparative TLC (100% EtOAc) resulting in compound 12 as a slightly yellow oil (16.4 mg (46%)). Rf = 0.8 (100% EtOAc). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.29 (d, J = 5.3 Hz, IH), 7.20 (d, J = 5.3 Hz, IH), 5.72 (s, IH), 5.66 (s, IH), 4.44-4.30 (m, 2H), 3.85 (t, J = 6.4 Hz, 2H), 3.75 (t, J = 4.8 Hz, 2H), 3.65 (s, 8H), 2.90 (t, J = 6.4 Hz, 2H) 2.83, (brs, 4H). ¹³C-NMR (50 MHz, CDCl₃) δ (ppm): 168.6, 166.4, 143.6, 142.3, 84.8, 83.7 (d), 70.2 (t), 68.3, 65.4, 64.5, 42.6, 31.8, 25.2. LCQ MS(ESI+) m/z (%) 530.0 (100) [M+Na]⁺

Example 13

Dimethyl 5-methyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (13a) and dimethyl 6-methyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2,3-dicarboxylate (13b)

3-methylfuran (0.52 g (0.56 ml), 6.3 mmol) and dimethyl but-2-ynedioate (0.89 g (0.76 ml), 6.3 mmol) were dissolved in ether (3 mL). The mixture was allowed to stir at room temperature for 7 days. TLC analysis showed that not all the starting material was consumed so the mixture was allowed to stir for an additional 6 days. No further progress was observed so the mixture was purified by column chromatography (EtOAc/n-heptane 1/4), resulting in desired product as a white solid (1.05 g (81%)). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.59 (m, IH), 5.59 (dt, J= 1.5 Hz, J= 0.6 Hz, IH), 5.35 (d, J= 1.5 Hz, IH), 3.82 (s, 3H), 3.81 (s, 3H), 2.01 (d, J = 2.1 Hz, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 155.3, 153.7, 152.4, 134.0, 88.4, 85.7, 52.3, 14.3. LCQ MS(ESI+) m/z (%) 224.93 (98) [M+H]⁺, 246.87 (100) [M+Na]⁺. Example 14

5-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxylic acid ethyl ester (14a) and 6-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2- carboxylic acid ethyl ester (14b)

A mixture of 4,4,4-trifluorobutynoate (0.61 g, 3.65 mmol) and 3-methylfuran (0.30 g, 3.65 mmol) was stirred under an Ar-atmosphere for 4 days. The crude mixture was washed out with ether, concentrated, and purified by column chromatography (EtO Ac/n- heptane 1/5), resulting in a mixture of two regio-isomers (ratio 1:1.4 for 14b and 14a respectively) as a slightly yellow oil (0.65 g (72%)).

Data of compound 14a: ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.65 (m, IH), 5.38 (s, IH), 5.30 (d, J= 1.5 Hz, IH), 4.28 (m, 2H), 1.99 (d, J= 2.0 Hz, 3H), 1.31 (t, J= 6.9 Hz, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 162.1, 154.8, 152.0 (q), 150.8, 134.5, 122.2 (q, CF₃), 85.9, 84.8, 61.7, 14.2, 14.0. LCQ MS(ESI+) m/z (%) 249.00 (100) [M+H]⁺. Data of compound 14b: ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.58 (m, IH), 5.61 (s, IH), 5.56 (d, J = 1.5 Hz, IH), 4.27 (m, 2H), 2.05 (d, J = 2.0 Hz, 3H), 1.32 (t, J = 6.9 Hz, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 162.3, 156.2, 151.2 (q), 150.3, 133.7, 121.5 (q, CF₃), 88.6, 87.4 (d), 61.7, 14.3, 14.0. LCQ MS(ESI+) m/z (%) 249.00 (100) [M+H]⁺.

Example 15

5-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene-2-carboxylic acid (15a) and 6-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2. l]hepta-2,5-diene-2-carboxylic acid (15b) A mixture of 14a and 14b (0.30 g, 1.22 mmol) was dissolved in THF (16 ml) and cooled to 0 ⁰C. 1.22 mL NaOH (aq) (1 M) was added drop wise and the mixture was stirred overnight at room temperature. TLC analysis showed still some starting material after reacting overnight and another 1.22 ml NaOH (aq) (1 M) was added. After 30 min. the reaction was completed and the volume of THF was reduced to 50 % of the original volume by evaporation using a N2 airflow. The mixture was diluted with HCl (aq) (5 ml, IM) and extracted with EtOAc (2 x 75 ml). The organic layer was dried over Na2SO4 and evaporated under reduced pressure to obtain a yellowish solid (0.26 g (95%)). A mixture of two regio-isomers was obtained in a ratio of 1 : 1.4 for 15b and 15a respectively. Data of compound 15b:

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.67 (t, J = 2.0 Hz, IH), 5.56 (s, IH), 5.34 (d, J = 1.5 Hz, IH), 2.01 (d, J= 2.0, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 166.4, 150.9 (q), 134.4, 121.0 (q, CF₃), 84.9, 84.8, 14.1. LCQ MS(ESI-) m/z (%) 219 (100) [M-H]^" Data of compound 15b:

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.59 (t, J = 2.0 Hz, IH), 5.60 (s, IH), 5.42 (d, J = 1.5 Hz, IH), 2.05 (d, J= 2.0 Hz, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 166.7, 149.9 (q), 133.2, 121.2 (q, CF₃), 88.3, 87.5, 14.0. LCQ MS(ESI-) m/z (%) 219 (100) [M-H]^"

Example 16

5-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1 ]hepta-2,5-diene -2 -carboxyl glycine methyl ester (16a) and 6-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene- 2-carboxyl glycine methyl ester (16b)

Oxanorbornadiene carboxylic acids 15a and 15b (40 mg, 0.18 mmol), glycine methyl ester.HCl (25 mg, 0.20 mmol) and 4-(dimethylamino)-pyridine (DMAP, 44 mg, 0.36 mmol) were dissolved in 2 mL CH2CI2 and cooled to 0 ⁰C. l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 38 mg, 0.20 mmol) was added and the reaction mixture was stirred at 0 ⁰C for 30 min. The mixture was allowed to warm to room temperature and was stirred for an additional 16 hours. The reaction was quenched with 2 mL HCl (aq) (2M) and extracted with EtOAc (2 x 5 mL). The combined organic layers were washed with brine (5 mL), dried over Na₂SO₄ and concentrated in vacuo. The crude mixture was purified by preparative TLC (CH2Cl2/MeOH, 9/1) resulting in a mixture of compounds 16a and 16b (ratio of 1.25:1) as a slightly yellow oil

(15 mg (27 %)).

Data for compound 16a:

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.69 (m, IH), 5.56 (br s, IH), 5.31 (d, J = 1.5 Hz,

IH), 4.13 (m, 2H), 3.78 (s, 3H), 1.98 (d, J= 1.5 Hz, 3H)

Data for compound 16b:

¹H-NMR (300 MHz, CDCl₃) δ (ppm): 6.56 (m, IH), 5.56 (m, IH), 5.37 (br s, IH), 4.13

(m, 2H), 3.78 (s, 3H), 2.09 (d, J= 1.5 Hz, 3H).

Example 17

α-methoxy ω-(trifluoromethyl-7-oxa-bicyclo[2.2.1.]hepta-2,5-diene-2-carbonyl) poly(ethylene glycol) (17)

A mixture of oxanorbornadiene carboxylic acid 5 (80 mg, 0.39 mmol), α-methoxy poly(ethylene glycol) (mPEG, 152 mg, 0.076 mmol) and 4-(dimethylamino)-pyridine (DMAP, 22 mg, 0.18 mmol) in anhydrous CH₂Cl₂ (4 mL) was cooled to 0 ⁰C. l-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 94.5 mg, 0.49 mmol) was added and the mixture was stirred for 1.5 hours at 0 ⁰C. The mixture was allowed to warm to room temperature and stirred for another 36 hours. After dilution with CH₂Cl₂ (50 mL) the reaction mixture was washed with a saturated aqueous NaHCO₃ solution (2 x 50 mL) and a saturated aqueous NH₄Cl solution (2 x 50 mL). Subsequently, the organic phase was dried over Na₂SO₄ and concentrated in vacuo, affording a yellowish solid (142 mg (86%)). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.27 (dd, J = 1.9, 5.3 Hz, IH), 7.17 (dd, J= 1.9, 5.3 Hz, IH, oxanorbornadiene), 5.72 (m, IH, oxanorbornadiene), 5.63 (t, J = 1.7, IH, oxanorbornadiene), 4.34 (m, 2H, 0-CH₂-CH₂-CO₂), 3.62 (br s, 180Η, O-

(CH₂CH₂) -O), 3.35 (s, 3Η, CH₃-O).

ESI-ToF (ESI+) analysis shows a clear shift of the molecular weight distribution towards higher molecular weight. The [M+Na] peaks for n = 38 were assigned for both compounds; ToF (ESI+) [M+Na] hydroxyl functionalized mPEG m/z = 1727.94 (calc m/z = 1728.02), [M+Na] oxanorbornadiene functionalized mPEG (17) m/z = 1915.86 (calc m/z = 1916.03). By subtracting the two peaks, the expected difference of m/z = 187.92 (calc m/z = 188.01) is found. Note: No fresh calibration sample was acquired while measuring the samples.

Example 18

α-methoxy-ω-propyl-(trifmoromethyl-7-oxa-bicyclo[2.2.1.]hepta-2,5-diene-2- carboxamide) poly(ethylene glycol) (18)

Compound 18 can be synthesized according to the method described for compound 17, using α-methoxy poly(ethylene glycol)propyl-l -amine instead of α-methoxy poly(ethylene glycol).

Example 19

5-(dimethylamino)-N-(3-hydroxypropyl)naphthalene-l -sulfonamide (19) To a flame dried 50 mL round bottom flask was added, dansyl chloride (283 mg, 1.05 mmol), anhydrous CH₂Cl₂ (20 mL) and 2,4,6-collidine (280 μL, 2.11 mmol). Subsequently, 3-aminopropanol (160 μL, 2.11 mmol) was added and the mixture was allowed to stir under an Ar atmosphere for 1.5 hours. The reaction mixture was diluted with EtOAc (100 mL) and washed with an aqueous 10% (w/w) solution of citric acid (3 x 75 mL) and a saturated aqueous NaHCO₃ solution (2 x 75 mL). After drying over Na₂SO₄, the mixture was concentrated in vacuo, yielding a greenish solid (65 mg (20%)). Rf = 0.1 (EtOAc/n-heptane, 1/1). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 8.53 (dt, J= 1.0, 1.0, 8.6 Hz, IH), 8.28 (m, IH), 8.23 (dd, J= 1.3, 7.3 Hz), 7.53 (m, 2H), 7.17 (dd, J= 0.7, 7.6 Hz, IH), 5.46 (t, J = 5.9 Hz, IH, NH), 3.64 (t, J = 5.7 Hz, 2H), 3.04 (q, J = 6.1 Hz, 2H), 2.88 (s, 6H), 1.63 (m, 2H).

Example 20

3-(5-(dimethylamino)naphthalene-l-sulfonamido)propyl 3-(trifluoromethyl)-7-oxa- bicyclo[2.2.1]hepta-2,5-diene-2-carboxylate (20)

A mixture of oxanorbornadiene carboxylic acid 5 (46.0 mg, 0.22 mmol), 5- (dimethylamino)-N-(3-hydroxypropyl)naphthalene-l -sulfonamide (19) (60.0 mg, 0.19 mmol) and 4-(dimethylamino)-pyridine (DMAP, 55.2 mg, 0.45 mmol) in anhydrous CH₂Cl₂ (4 mL) was cooled to 0 ⁰C. l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 64.5, 0.34 mmol) was added and the mixture was stirred at 0 ⁰C for 1 hour. The mixture was allowed to warm to room temperature and stirred for another 18 hours. The mixture was concentrated in vacuo before purification by preparative TLC (CH₂Cl₂ZMeOH, 9/1). The product was obtained as a greenish solid (24 mg (25%)). Rf = 0.8 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (300 MHz, CDCl₃) δ (ppm): 8.55 (m, IH), 8.25 (m, IH), 7.54 (m, 2H) 7.21 (m, 3H), 5.63 (m, 2H), 4.89 (t, J = 6.3 Hz, IH, NH), 4.16 (m, 2Η), 2.98 (q, J = 6.6 Hz, 2H), 2.89 (s, 6H), 1.79 (m, 2H).

Example 21

Hen Egg white Lysozyme (HEL) was functionalized with an oxanorbornadiene moiety by employing an EDC peptide coupling between oxanorbornadiene 11 and one of the primary amines on the surface of HEL:

Typical procedure for the functionalization of Hen Egg white Lysozyme (HEL) via an EDCI coupling at pH 5.5.

Hen Egg white Lysozyme (5.7 mg, 4.0 ^• 10^~4 mmol) was dissolved in 1 mL of sodium acetate buffer (100 mM, pH 5.5). After the addition of an azide or oxanorbornadiene functionalized carboxylic acid (14 ^• 10^" mmol, as a solution in 100 μL THF), EDCI (3.8 mg, 19 ^• 10^"3 mmol) was added as a solution in sodium acetate buffer (100 mM, pH 5.5, 200 μL). The reaction mixture was shaken at room temperature for 14 hours. Subsequently, the protein was separated from low molecular weight compounds by a sephadex G50 column using a sodium acetate solution (20 mM, pH 5.5) as the eluent.

Example 22

Azidoacetic acid (22)

2-bromoacetic acid (2.00 g, 14.4 mmol) was dissolved in water (15 mL) and cooled using an ice-bath. After the addition of NaN₃ (4.00 g, 61.5 mmol) the mixture was allowed to warm to room temperature during a period of 16 hours. The reaction mixture was acidified to pH 1 by the addition of concentrated HCl, and subsequently extracted with EtOAc (3 x 75 mL). The combined organic phases were dried over Na₂SO₄ and concentrated in vacuo. After co-evaporation with CH2CI2 (4 x 50 mL) the product was obtained as a slightly yellow liquid (1.36 g (93%)). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.25 (s, IH), 3.98 (s, 2H). ¹³C-NMR (CDCl₃, 75 MHz) δ (ppm): 174.4, 50.1. FT-IR (ATR): 3451 (OH), 2110 (N₃), 1724 (C=O), 1215.

Example 23

Azidoacetyl-Gly-Gly-Arg-Gly-Asp-Gly-OH (23)

Azide functionalized GGRGDG (23) was synthesized by standard solid-phase methods using a 'Wang' resin. ' ⁵ A suspension of Wang resin (30 g) in DMF (300 mL) was cooled in an ice bath, after which Fmoc-Gly-OH (13.5 g, 45 mmol), 1- hydroxybenzotriazole hydrate (HOBt, 9.2 g, 60 mmol) and N.AP-diisopropylcarbodiimide (DIPCDI, 4.3 g, 34 mmol) were added. This mixture was shaken for 6 hours. The functionalized resin was filtered and washed repeatedly with CH₂CI₂, DMF, and isopropyl alcohol. Unfunctionalized groups on the resin were capped by adding benzoyl chloride (10.2 mL) and pyridine (8.4 mL) to a suspension of the resin in CH₂CL₂ (300 mL) at 0⁰C. The mixture was shaken for 30 min, filtered and washed repeatedly with CH₂CI₂, DMF, and isopropyl alcohol. Then the Fmoc-Gly funtionalized Wang resin (I g, loading; 0.67 mmol/g) was swollen in DMF (20 mL) and filtered three times. Subsequently, the mixture was shaken in a 20% (v/v) solution of piperidine in DMF (20 mL) for 30 min to remove the Fmoc protecting group. A positive Kaiser test ' ⁷ indicated the completeness of this reaction. After filtering and washing with DMF (3 x 20 mL), the next amino acid was coupled by adding a mixture of Fmoc-Asp(OtBu)-OH (500 mg, 1.22 mmol), HOBt (405 mg, 3.00 mmol) and DIPCDI (340 mg, 2.70 mmol) in DMF (20 mL). The mixture was shaken for 45 min., after which it was filtered and washed with DMF (3 x 20 mL). A negative Kaiser test indicated the completeness of the reaction. The deprotection-coupling sequence was repeated with the following amino acids: Fmoc-Gly- OH (210 mg, 0.706 mmol), Fmoc-Arg(PMC)-OH (700 mg, 1.77 mmol), and Fmoc-Gly- OH (210 mg, 0.706 mmol) twice. After deprotection of the terminal Fmoc group, 2- azidoacetic acid (158 mg, 1.56 mmol) was coupled to the peptide by shaking the mixture with HOBt (405 mg, 3.00 mmol) and DIPCDI (340 mg, 2.70 mmol) in DMF (20 mL) for 45 min. The mixture was washed repeatedly with DMF and MeOH. Subsequently, the resin was stirred in a mixture of TFA/triisopropyl silane/water (95/2.5/2.5, 3.5 mL) to cleave the peptide from the resin. The peptide was precipitated in Et2θ and stirred in TF A/water (95/5, 3 mL) for 4 hours to achieve a complete deprotection of the amino acid residues. Upon precipitation in Et2θ and drying in vacuo the peptide was obtained as an off-white solid (350 mg (87%)) ¹H-NMR (400 MHz, D₂O) δ (ppm): 4.82 (m, IH), 4.35 (dd, J= 5.7, 8.7 Hz, IH), 4.11 (s, 2H), 4.07 - 3.89 (m, 8H), 3.21 (t, J = 6.7 Hz, 2H), 2.92 (ddd, J = 6.6, 17.2, 25.0 Hz, 2H), 1.97 - 1.85 (m, IH), 1.85 - 1.73 (m, IH), 1.73 - 1.56 (m, 2H). FT-IR (ATR): 3285, 3092, 2928, 2114, 1651, 1540, 1186 cm^"1. ESI-ToF (ESI-): calc. m/z = 599.229 [M-H]^", found m/z = 599.233 [M-H]^". Example 24

Azido-7-hydroxycoumarin (24)

This compound was prepared according to a literature procedure.⁸⁸ 1H-NMR (DMSO, 400 MHz): δ = 10.53 (s, IH), 7.60 (s, IH), 7.48 (d, J = 8.5 Hz, IH), 6.81 (dd, J= 8.5, 2.3 Hz, 1 H), 6.76 (d, J= 2.3 Hz, IH). FT-IR (ATR): 3291 (OH), 2115 (N₃), 1679 (C=O), 1616, 1303.

3. Cycloaddition reactions with oxanorbornadiene derivatives.

Example 25

Cycloaddition of oxanorbornadiene 1 with benzyl azide.

Dimethyl 1 -benzyl- IH- 1,2, 3 -triazole-4,5-dicarboxy late (25)

Compound 1 (210 mg, 1.0 mmol) and benzyl azide (666 mg, 5 mmol) were dissolved in 5 mL TΗF and stirred for 18 hours. The solvent was evaporated and the crude mixture was purified by column chromatography (EtOAc/n-heptane, 3/1) resulting in compound 25 as a white powder (160 mg (60%)). Rf= 0.3 (EtOAc/n-heptane, 3/1). Η- NMR (CD₃OD, 400 MHz) δ (ppm): 7.34 (m, 3H), 7.27 (d, J = 7.5, 2.0 Hz, 2H), 5.81 (s, 2H), 3.91 (s, 3H), 3.87 (s, 3H). ¹³C-NMR (75 MHz, CDCl₃) δ (ppm): 159.9, 158.3, 139.7, 133.4, 129.3, 128.5, 128.4, 127.5, 53.5, 52.8, 52.2. LC/MS (ESI+) m/z (%) 276.10 (100) [M+H]⁺.

Example 26

Cycloaddition of azanorbornadiene 3 with benzyl azide.

A solution of azanorbornadiene 3 (15.4 mg, 0.05 mmol) in CD₃OD (0.5 mL) was added to a test tube containing benzyl azide (31.3 μL, 0.25 mmol). The mixture was briefly stirred using a vortex and then added to an NMR tube. Directly after the addition, the tube was placed in a Varion inova 400 NMR apparatus at 25 ⁰C, and reaction was monitored following a preset measurement schedule. The conversion to triazole 25, determined by ¹H-NMR, was found to be 90% after 14 hours. ¹H-NMR (CD₃OD, 400 MHz) δ (ppm): 7.34 (m, 3H), 7.25 (m, 2H), 5.76 (s, 2H), 3.88 (s, 3H), 3.83 (s, 3H).

Example 27

Cycloaddition of oxanorbornadiene 4 with benzyl azide.

Ethyl l-benzyl-5-(trifluoromethyl)-lH-l,2,3-triazole-4-carboxylate (26a) and ethyl 1- benzyl-4-(trifluoromethyl)-lH-l ,2,3-triazole-5-carboxylate (26b)

Benzyl azide (39.9 mg, 0.3 mmol) was added to a solution of oxanorbornadiene 4 (70.2 mg, 0.3 mmol) in CD3OD (3 mL) and the reaction mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure and the crude mixture was purified by preparative TLC (EtOAc/n-heptane, 3/1) resulting in compounds 26a (53 mg (60%)), R_f = 0.70 (EtOAc/n-heptane, 3/1), ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.35 (m, 3H), 7.22 (dd, J = 6.6, 2.8 Hz, 2H), 5.76 (s, IH), 4.45 (q, J = 7.1 , 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H) and 26b (24 mg (27%)), R_f = 0.75 (EtOAc/n-heptane, 3/1), ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.34 (s, 5H), 5.94 (s, 2H), 4.39 (q, J = 7.2, 7.2 Hz, 2H), 1.35 (t, J = 7.2 Hz, 3H) as slightly yellow oils. NMR techniques such as gHSQC and NOESY were employed to elucidate the configuration of the regio-isomers. Unfortunately, both techniques were inconclusive and we therefore set out to crystallize one of the isomers as described in the section below.

Example 28

Benzyl-5-(trifluoromethyl)-lH-l ,2,3-triazole-4-carboxylic acid (27)

NaOH (aq) (1.25 mL, 2M) was added to a solution of 26a (53 mg, 0.18 mmol) in TΗF (4 mL) and the mixture was stirred at room temperature for 16 hours. Η2O (2 mL) and EtOAc (4 mL) were added and the layers were separated. The aqueous layer was acidified with HCl (2M) to a pH of 1-2 and extracted with CH₂Cl₂ (2 x 10 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo resulting in an off-white semi-solid (43.7 mg (91%)). R_f= 0.15 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.37 (m, 3H), 7.24 (dd, J = 6.6, 2.8 Hz, IH), 5.78 (s, 2H), 4.08 (brs, IH). Example 29

1 -Benzyl-4-(trifluoromethyl)-lH-l ,2,3-triazole-5-carboxylic acid (28)

NaOH (aq) (0.75 mL, 2M) was added to a solution of 26b (24 mg, 0.08 mmol) in TΗF (2 mL) and the mixture was stirred at room temperature for 16 hours. Η2O (1 mL) and EtOAc (2 mL) were added and the layers were separated. The aqueous layer was acidified with HCl (2M) to a pH of 1 -2 and extracted with CH₂Cl₂ (2 x 5 mL). The organic layer was dried over MgSO₄ and concentrated in vacuo, resulting in an off- white semi-solid (16.7 mg (77%)). R_f= 0.12 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 7.33 (s, 5H), 5.95 (s, 2H), 3.54 (brs, IH).

4-nitrophenyl l-benzyl-5-(trifluoromethyl)-lH-l,2,3-triazole-5-carboxylate (29)

Compound 28 (43.7 mg, 0.16 mmol), /?-NO₂-phenol (22.4 mg, 0.16 mmol) and 4- (dimethylamino)-pyridine (DMAP, 38.0 mg, 0.32 mmol) were dissolved in CH₂Cl₂ (4 mL). The mixture is cooled to 0 ⁰C and l -ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 32.8 mg, 0.17 mmol) was added. The mixture was stirred 15 min. at 0 ⁰C and 16 hours at room temperature. After completion of the reaction 2 mL HCl (IM) was added and the aqueous layer was extracted with CH₂Cl₂ (2 x 4 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by preparative TLC (CH₂Cl₂/Me0H, 9/1) resulting in compound 29 as an off-white solid. After crystallization from n-heptane / EtOAc (1 :1 v/v) colorless crystals were obtained (25 mg (40%)). R_f = 0.5 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.32 (d, J = 9.0 Hz, 2H), 7.46 (d, J = 9.0 Hz, 2H), 7.41-7.35 (m, 3H), 7.30-7.24 (m, 2H), 5.82 (s, IH).

Jb

Table 2. Results of test reaction between oxanorbornadiene derivatives and azido compounds (at 100 niM, scheme S3) obtained by monitoring the reactions with H-NMR spectroscopy (400 MHz).

5 1

a 50 mM instead of 100 mM, ^b Determined after 11 5 h, ^c Exclusively one regio-isomer was observed nd = not determined

Table 2. Results of test reaction between activated alkynes and azido compounds (at 100 mM, Scheme S4) obtained by monitoring the reactions with H-NMR spectroscopy (400 MHz)

M -J

Example 31

Cycloaddition of oxanorbornadiene 5 with 5'-azido-5'-deoxy-2'-0-methyl-(7V-Bz)- adenosine.

5 '-(4-(trifluoromethyl)-lH-l ,2,3-triazole-5-carboxylic acid)-5'-deoxy-2'-0-methyl-(7V- Bz)-adenosine (30a) and 5'-(5-(trifluoromethyl)-lH-l,2,3-triazole-4-carboxylic acid)- 5'-deoxy-2'-0-methyl-(7V-Bz)-adenosine (30b)

To a solution of 5'-azido-5'-deoxy-2'-0-methyl-(7V-Bz)-adenosine^S9 (50 mg, 0.12 mmol) in THF (2 mL) was added oxanorbornadiene 5 (50 mg, 0.24 mmol). The reaction was stirred at room temperature for 48 hours. The reaction mixture was concentrated under reduced pressure and purified by gradient column chromatography going from 2% MeOH in CH₂Cl₂ to 5% MeOH in CH₂Cl₂ to MeCN/H₂O 20:1. Compound 30a (35.7 mg (54%)) Rf = 0.10 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (300 MHz, DMSO-d6) δ (ppm): 11.22 (brs, IH), 8.73 (s, IH), 8.67 (s, IH), 8.04 (d, J = 7.5 Hz, 2H), 7.58 (m, 3H), 6.15 (s, IH), 5.70 (s, IH), 4.85 (m, 2H), 4.60 (brs, IH), 4.31 (brs, IH), 3.37 (s, 3H). Compound 30b (14.3 mg, (22%)) Rf = 0.15 (CH₂Cl₂/Me0H, 9/1). ¹H-NMR (300 MHz, DMSO-d6) δ (ppm): 11.20 (brs, IH), 8.74 (s, IH), 8.71 (s, IH), 8.03 (d, J = 7.5 Hz, 2H), 7.56 (m, 3H), 6.14 (d, J = 6.0 Hz, IH), 5.71 (d, J = 4.2 Hz, IH), 5.03 (m, 2H), 4.54 (m, IH), 4.42 (d, J= 2.4 Hz, 2H), 3.37 (s, 3H).

Example 32

Cycloaddition of oxanorbornadiene functionalized PEG (17) with 3-azido-7-hydroxy coumarin (24).

α-methoxy-ω-(3 -(5 -(trifluoromethyl)- IH- 1 ,2,3 -triazol-4-carbonyl)-7-hydroxy- coumarin) poly(ethylene glycol) (31) A mixture of oxanorbornadiene functionalized PEG (17) (7.7 mg, 3.5 ^■ 10^~3 mmol) and 3-azido-7-hydroxy coumarin (24) (2.6 mg, 0.013 mmol) in CH₂Cl₂ (3 mL) was stirred for 15 hours at room temperature. The presence of fluorescence (when irradiated by UV light of λ = 366 nm) indicated that the reaction took place. The mixture was concentrated in vacuo and characterized without further purification. From

¹H-NMR analysis the level of functionalization was determined to be 88%. ¹H-NMR

(400 MHz, CDCl₃,) δ (ppm): 8.12 (s, IH, coumarin), 7.52 (d, J = 8.5 Hz, IH, coumarin), 7.00 (d, J = 1.9 Hz, IH, coumarin), 6.96 (dd, J = 2.2, 8.4 Hz, IH, coumarin), 4.46 - 4.40 (m, 2H, 0-CH₂-CH₂-CO₂), 3.63 (br s, 180H, 0-(CH₂CH₂) -O), 3.37 (s, 3Η, CH₃-O).

After the cycloaddition reaction, SEC analysis using UV detection at λ = 340 nm showed a clear signal at the elution time of PEG. As the oxanorbornadiene functionalized PEG shows almost no UV response, this confirms that the coumarin is covalently attached to the PEG. As expected, in the RI traces no significant differences could be observed before and after the cycloaddition reaction.

Example 33

Cycloaddition of oxanorbornadiene functionalized PEG (17) with 2-azidoactyl-Gly-

Gly-Arg-Gly-Asp-Gly-OΗ (23).

α-methoxy-ω-(3 -(5 -(trifluoromethyl)- IH- 1 ,2,3 -triazol-4-carbonyl)acetyl-Gly-Gly-Arg- Gly-Asp-OΗ) polyethylene glycol) (32)

A mixture of oxanorbornadiene functionalized PEG (17) (14.7 mg, 6.7 ^■ 10^"3 mmol) and 2-azidoactyl-Gly-Gly-Arg-Gly-Asp-Gly-OΗ (23) (10.7 mg, 0.018 mmol) in H₂O (2 mL) was stirred for 36 hours at 37 ⁰C. The mixture was concentrated in vacuo and characterized without further purification. By comparing the ¹H-NMR integral of an oxanorbornadiene bridgehead signal (δ = 5.83) with the -CH₂-triazole signals (δ = 5.68 - 5.62) of the product, the conversion was determined to be 80%. MALDI-ToF analysis (using indoleacrylic acid (IAA) as a matrix) of the mixture clearly showed a shift of the molecular weight distribution towards higher mass.

In principle the mass of a single polymer chain can be defined as the sum of masses of the end groups and the number (N) of repeating units. Therefore, the mass of the end groups can be derived from a molecular weight distribution by plotting the molecular mass against N. Consequently, the intercept (N = 0) affords the mass of the end groups. Prior knowledge to the likely nature of these end groups gives an appropriate choice of N, which in case of the obtained PEG-GIyGIy ArgGlyAspGly-OH was defined as 34 for the peak assigned with m/z = 2293.696. Using this peak as a reference also N for the other peaks was determined and a plot of mass versus N was constructed. Linear regression (using Origin 6.1 software) of this dataset resulted in the following equation:

Mass = 44.08 x N + 794.67

Herein, 44.08 is in line with the mass of one repeating unit of PEG (calc 44.03) and the intercept of 794.67 corresponds to the α-methoxy (calc 31.02) and ω-Gly-Gly-

Arg-Gly-Asp-Gly-OH (calc 721.23) end groups, plus an additional proton, sodium and water.

Example 34

Typical procedure for the cycloaddition reaction with functionalized Hen Egg white

Lysozyme.

Functionalized HEL (typically, 300 μL of a 1.5 mg/mL solution, 3.3 ^■ 10^"5 mmol) and an azido compound or oxanorbornadiene derivative (depending on the functionality on HEL) (1.6 ^■ 10^~3 mmol) were shaken at room temperature for 36 hours. The mixtures were analyzed without further purification.

Example 35 Employing the oxanorbornadiene-azide ligation in bioconjugation to proteins.

The oxanorbornadiene functionalized HEL (21) was mixed with 3-azido-7- hydroxy-coumarin and shaken for 36 hours (Scheme S5). As a control experiment, unfunctionalized HEL was also incubated with 3-azido-7-hydroxy-coumarin under the same conditions. After 36 hours the crude mixtures were analyzed by SD-PAGE (15%), and for the reaction a clear fluorescent band was observed at the position of HEL. As expected the control experiment showed no fluorescent band (Figure S8A). Since 3-azido-coumarin derivatives are known to become strongly fluorescent upon undergoing a cycloaddition^S8 the observed fluorescent band furthermore proved that the coumarin is covalently attached to the HEL. Upon staining with coomassie blue, as expected, no mass differences were observed between the reaction mixture and the control experiment .

Scheme S5

Example 36

N-ll-O-fert-butyl-DTPA-acetamideJ^V'-IS-trifluoromethyl-T-oxa-bicycloIl.l.l]- hepta-2,5-diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (33)

Compound 8 (49 mg, 0.11 mmol) was dissolved in dry CH₂Cl₂ (2 mL) and 4-(dimethyl amino)-pyridine (DMAP, 26.6 mg, 0.22 mmol) and diethylenetriamine-7V,7V,7V"7V"- tetra-tert-butyl acetate -N -acetic acid ((DTPA-tert-butyl ester) 67.8 mg, 0.11 mmol) were added. The mixture was cooled to 0⁰C followed by addition of l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 23.1 mg, 0.12 mmol). After 1 hour stirring at 0⁰C the mixture was allowed to warm to room temperature and was stirred for an additional 4 hours. The reaction mixture was quenched with 1 M HCl (2 mL) and the water layer was extracted with CH₂Cl₂ (2 x 2 mL). The combined organic layers was dried over Na₂SO₄ and subsequently evaporated. The crude product was purified by preparative TLC (MeOH/CH₂Cl₂ 1 :9) to obtain pure product as a colorless oil (38 mg, (81%)). ¹H-NMR (400 MHz, CDCl₃) δ (ppm): 8.23 (br s, NH IH), 7.32 (dd, J = 5.3, 2.1 Hz, IH), 7.13 (dd, J = 5.3, 2.1 Hz, IH) 6.81 (br s, NH IH), 5.62 (dd, J = 1.4, 0.5 Hz, 2H), 3.62-3.50 (m, 8H), 3.49-3.42 (m, 4H), 3.39 (s, 8H), 3.12 (s, 2H), 2.77 (t, J = 6.6 Hz, 4H), 2.61 (t, J = 6.5 Hz, 4H), 1.43 (s, 36H). ¹³C-NMR (50 MHz, CDCl₃) δ (ppm):172.2, 170.5 (4C), 162.2, 143.7, 141.9, 86.0, 83.5, 81.0 (4C), 70.5, 70.0, 69.7, 69.4, 58.5, 55.8 (4C), 53.7, 53.4, 52.1 (2C), 39.4, 38.6, 28.1. LRMS(ESI+) m/z calc. for C₄₄H₇₄F₃N₅Ol₃ [M+H]⁺ 936.52, found 936.40.

Example 37

N-^-DTPA-acetamide^'-IS-trifluoromethyl-T-oxa-bicycloβ^Λlhepta^S-diene- 2-carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (34)

To a solution of (33) (49 mg, 0.11 mmol) in DCM (2 mL), 200 μL TFAA was added. This mixture was stirred for 6 days (until MS analysis did not show starting material). The product was obtained a white solid after lyophilized from H2θ/dioxane (28 mg, (+99%)). ¹H-NMR (400 MHz, CD₃OD) δ (ppm): 7.32 (dd, J = 5.3, 1.9 Hz, IH), 7.23 (dd, J = 5.3, 1.9 Hz, IH), 5.67 (s, IH), 5.58 (s, IH), 4.24 (br s, NH, IH), 3.66 (s, 2H) 3.62-3.57 (m, 16H) ppm 3.50-3.37 (m, 8H), 3.20 (br s, 4H). ¹³C-NMR (50 MHz, CD₃OD) δ (ppm): 174.31, 174.28, 174.15,174.14, 167.2, 164.9, 155.9, 144.3, 143.4, 127.6 (q, CF₃), 86.9, 84.2, 71.1, 71.0, 70.04, 70.00, 67.9, 55.8 (4C), 53.8 (2C), 50.9, 50.8, 40.2, 40.1. LRMS(ESI-) m/z calc. for C₂₈H₃₉F₃N₅Ol₃ [M-H]^" 710.25, found 710.30.

Example 38

l,4-dimethyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2-carboxylic acid ethyl ester (35)

To a solution of ethyl 4,4,4-trifiuoro-2-butynoate (436 mg, 2.62 mmol) in dioxane (0.5 mL), 2,5-dimethylfuran (279 μL, 2.62 mmol) was added. The mixture was heated to 103⁰C under a nitrogen atmosphere and stirred overnight. The reaction mixture was cooled and diluted with CH₂Cl₂ after which all the solvents were evaporated. The crude product was purified by column chromatography (MeOHZCH₂Cl₂ 1 :9) to obtain the pure product as a white solid (238 mg, (91%)). ¹H NMR (300 MHz, CDCl₃) δ (ppm): 7.01 (d, J = 5.1 Hz, IH), 6.91 (d, J = 5.1 Hz, IH), 4.30 (m, 2H), 1.81 (s, 3H), 1.74 (s, 3H), 1.31 (t, J= 7.1 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ (ppm): 163.7, 154.6, 149.0 (q, J = 35 Hz), 147.5, 146.7, 121.9 (q, CF₃, J = 270 Hz), 92.6, 91.4, 61.6, 15.1, 14.8, 13.9. Both HRMS and LRMS techniques were employed to acquire the mass of the described compound. Unfortunately, none of the techniques used gave a comprehensible mass spectrum.

Example 39

^-{BocJ-N'-iS-methyl-S-trifluoromethyl-T-oxa-bicycloIl.l.llhepta-l^-diene-l- carboxyl}-3,6-dioxaoctane-l,8-diamine (36a) and iV-{Boc}-./V'-{6-methyl-3- trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2-carboxyl}-3,6-dioxaoctane- 1,8-diamine (36b)

To a solution of a mixture of compounds 15a and 15b (49 mg, 0.21 mmol) and Boc-1- amino-3,6-dioxa-8-octanediamine (50.9 mg, 0.21 mmol) in dry CH₂Cl₂ (1.5 mL) was added 4-(dimethyl amino)-pyridine (DMAP, (50.6 mg, 0.41 mmol)). The mixture was cooled to 0 ⁰C and l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 44.1 mg, 0.23 mmol) was added slowly. The mixture was stirred for 30 minutes at 0 ⁰C and 16 hours at room temperature. The reaction mixture was acidified with HCl (2 M) to a pH of 1-2 and extracted with CH₂Cl₂ (2 x 5 mL). The combined organic layers were dried over MgSO₄, concentrated in vacuo, and purified by preparative TLC (CH₂Cl₂ZMeOH, 9/1) resulting in compounds xa and xb as a slightly yellow oil (44.4 mg, (47%)) A mixture of two regio-isomers was obtained in a ratio of 1 :1.4 for 36b and 36a respectively. 1H-NMR (300 MHz, CDCl₃) peaks assigned to compound 36a δ (ppm): 6.70 (d, J= 6.7 Hz, IH), 6.44 (br s, NH, IH), 5.54 (s, IH), 5.28 (s, IH), 4.96 (br s, NH IH), 3.60-3.52 (m, 10H), 3.30 (q, J = 5.2 Hz, 2H), 1.97 (d, J = 1.6 Hz, 3H), 1.44 (s, 9H). Peaks assigned to compound 36b δ (ppm): 6.55 (d, J= 6.6 Hz, IH), 6.44 (br s, NH, IH), 5.54 (s, IH), 5.34 (s, IH), 4.96 (br s, NH, IH), 3.30 (q, J= 5.2 Hz, 2H), 3.60-3.52 (m, 10H), 3.30 (q, J = 5.2 Hz, 2H), 2.10 (d, J = 1.9 Hz, 3H), 1.44 (s, 9H). ¹³C NMR (75 MHz, CDCl₃) δ (ppm): 162.4, 155.9, 154.5, 153.8, 134.8, 133.1, 122.8 (q, CF₃), 89.4, 86.7, 84.1, 70.4-69.4 (4 signals, spacer), 40.3, 39.4, 28.4, 14.0. HRMS(ESI+): m/z calc.for C₂₀H₃₀F₃N₂O₆ [M+H]⁺ 451.2056, found 451.2078.

Example 40

N^/V'-IS-methyl-S-trifluoromethyl-T-oxa-bicycloIl.l.llhepta-ljS-diene-l-carboxyl}- 3,6-dioxaoctane-l,8-diamine (37a) and N_r/V'-{6-methyl-3-trifluoromethyl-7-oxa- bicyclo[2.2.1]hepta-2,5-diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine (37b)

To a cooled solution (0 ⁰C) of a mixture of 36a and 36b (42 mg, 0.093 mmol) in dry CH₂Cl₂ (2 mL) was added drop wise trifiuoroacetic acid (TFA, 0.5 mL, 2.85 mmol). The reaction was stirred at 0 ⁰C for 1 hour after which the reaction was complete. The solvent was evaporated and the crude mixture was dissolved in H₂O (5 mL) and dioxane (5 mL) and freeze-dried to afford compound x as a light yellow solid (43.1 mg (+99%)). A mixture of two regio-isomers was obtained in a ratio of 1:1.4 for 37b and 37a respectively. 1H-NMR (400 MHz, CDCl₃) peaks assigned to compound 37a δ (ppm): 7.84 (br s, NH₃ 3H), 7.92 (br s, NH, IH), 6.66 (s, IH), 5.52 (s, IH), 5.27 (s, IH), 3.70-3.46 (m, 10H), 3.15 (m, 2H), 1.96 (s. 3H). Peaks assigned to compound 37b δ (ppm): 7.84 (br s, NH₃, 3H), 7.92 (br s, NH, IH), 6.55 (s, IH), 5.52 (s, IH), 5.31 (s, IH), 3.70-3.46 (m, 10H), 3.15 (s, 2H), 2.06 (s, 3H). ¹³C NMR (50 MHz, CDCl₃) δ (ppm): 159.1, 154.0, 151.1, 134.8, 133.1, 105-100 (4 signals, spacer), 86.8, 86.5, 40.3, 14.0. HRMS(ESI+): m/z calc.for Ci₅H₂₂F₃N₂O₄ [M+H]⁺ 351.1532, found 351.1541

Example 41

N-{2-0-fert-butyl-DTPA-acetamide}^V'-{5-methyl-3-trifluoromethyl-7-oxa- bicyclo[2.2.1]hepta-2,5-diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (38a) and N-{2-0-fert-butyl-DTPA-acetamide}^V'-{6-methyl-3-trifluoromethyl-7-oxa- bicyclo[2.2.1]hepta-2,5-diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (38b) Compounds 37a and 37b (39 mg, 0.084 mmol) were dissolved in dry CH₂Cl₂ (2 mL) and 4-(dimethyl amino)-pyridine (DMAP, 20.4 mg, 0.17 mmol) and diethylenetriamine-7V_>7V_>7V"7V"-tetra-tert-butyl acetate -N'-acetic acid ((DTPA-tert-butyl ester) 53 mg, 0.084 mmol) were added. The mixture was cooled to 0⁰C followed by addition of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCHCl, 17.5 mg, 0.09 mmol). After 1 hour stirring at 0⁰C the mixture was allowed to warm to room temperature and was stirred for an additional 4 hours. The reaction mixture was quenched with 1 M HCl (2 mL) and the water layer was extracted with CH₂Cl₂ (2 x 2 mL). The combined organic layers was dried over Na₂SO₄ and subsequently evaporated. The crude product was purified by preparative TLC (CH₂Cl₂/Me0H, 9:1) to obtain pure product as a light brown oil (35 mg, (44%)). A mixture of two regio- isomers was obtained in a ratio of 1 :1.4 for 38b and 38a respectively. 1H-NMR (400 MHz, CDCl₃) peaks assigned to compound 38a δ (ppm): 8.22 (br s, NH, IH), 6.75 (br s, NH, IH), 6.69 (t, J = 1.9 Hz, IH), 5.53 (s, IH), 5.27 (s, IH), 3.60-3.39 (m, 18H), 3.11 (s, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.61 (t, J = 6.5 Hz, 2H), 1.96 (d, J = 1.3 Hz, 3H), 1.43 (s, 36H). Peaks assigned to compound 38b δ (ppm): 8.22 (br s, NH, IH), 6.75 (br s, NH, IH), 6.54 (t, J = 1.9 Hz, IH), 5.51 (s, IH), 5.33 (s, IH), 3.60-3.39 (m, 18H), 3.11 (s, 2H), 2.77 (t, J = 6.5 Hz, 2H), 2.61 (t, J = 6.5 Hz, 2H), 2.09 (d, J = 1.7 Hz, 3H), 1.43 (s, 36H). ¹³C NMR (75 MHz, CDCl₃) δ (ppm): 172.2, 170.5, 162.5, 155.2 (q), 153.7, 142.5, 134.4, 122.8 (q, CF₃), 89.3, 86.7, 84.0, 81.0, 70.5, 70.0, 69.8, 69.4, 58.6, 55.8, 53.8, 52.1, 39.4, 38.6, 28.1, 14.0. HRMS(ESI+): m/z calc.for C₄₅H₇₅F₃N₅Oi₃ [M+H]⁺ 950.5313, found 950.5361.

Example 42

N-{2-DTPA-acetamide}^V'-{5-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta- 2,5-diene-2-carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (39a) and iV-{2 -DTPA- acetamide}_r/V'-{6-methyl-3-trifluoromethyl-7-oxa-bicyclo[2.2.1]hepta-2,5-diene-2- carboxyl}-3,6-dioxaoctane-l,8-diamine*TFA (39b)

To a solution of compounds 38a and 38b (25 mg, 0.026 mmol) in 2 ml Of CH₂Cl₂, 200 μL TFAA was added. This mixture was stirred for 6 days (until MS analysis did not show starting material). The product was obtained as a white solid in quantitative yield (+99%) after lyophilized from H₂O/dioxane. A mixture of two regio-isomers was obtained in a ratio of 1 :1.4 for 39b and 39a respectively.

¹H-NMR (400 MHz, CD₃OD) peaks assigned to compound 39a δ (ppm): 6.96 (t, J = 1.7 Hz, IH), 5.50 (s, IH), 5.36 (s, IH), 4.31 (br s, 2H), 3.66-3.58 (m, 18H), 3.49-3.42

(m, 2H), 3.30-3.15 (m, 8H), 1.99 (d, J= 1.5 Hz, 3H). Peaks assigned to compound 39b δ (ppm): 6.63 (m, IH), 5.56 (s, IH), 5.30 (s, IH), 4.31 (br s, 2H), 3.66-3.58 (m, 18H),

3.48-3.42 (m, 2H,), 3.28-3.14 (m, 8H), 2.07 (d, J = 1.7 Hz, 3H). ¹³C NMR (75 MHz,

CD₃OD) δ (ppm): 174.6 (4C), 167.2, 165.6, 155.8, 135.4, 90.5, 87.7, 84.1, 71.4, 71.3, 70.3, 68.2, 55.9, 55.6, 54.3, 50.9, 40.5, 40.4, 14.0. HRMS(ESI+): m/z calc.for

C₂₉H₄₃F₃N₅O_n [M+H]⁺ 726.2809, found 726.2837.

Example 43

The DTPA linked oxanorbornadiene systems (34 and 39a/b) were ¹¹¹In labeled by dissolving oxanor-DTPA 34 or Me-oxanor-DTPA 39a/b (5 μg, 7.0 nmol) in 5 μL H₂O. Subsequently metal free NH₄Ac buffer (90 μL, 0.25 M, pH 5.5) and 5 μL (-100 μCi) ¹¹¹InCl₃ were added to each of the reaction mixtures. The mixtures were allowed to incubate for 1 hour at room temperature after which they are checked for radiochemical purity by high-performance liquid-chromatography (HPLC) (HPI lOO series, LC system, Agilent Technologies, Palo Alto, CA, USA) using a RP-C 18 column (5 μm, 4.6 mm x 250 mm, Alltech, Deerfield, IL, USA) eluated with a gradient mobile phase (0- 100% B over 20 min, solvent A = 0.1% TFA in water, solvent B = 0.1% TFA in acetonitrile) at 1 mL/min. The radioactivity of the eluates was monitored using an in- line NaI radiodetector (Raytest GmbH, Straubenhardt, Germany.)

Example 44

Dimethyl l^-dimethyl-T-oxabicycloβ.l.lJhepta-ZjS-diene-ZjS-dicarboxylate (40) In 0.5 ml of dioxane, dimethyl acetylene carboxylate was dissolved. To this, 2,5- dimethylfuran was added. The mixture was heated to 103⁰C under a N₂ atmosphere and allowed to stir overnight. Then the solvent was evaporated and the product was further purified on silica column (1:9 MeOfLCH₂Cl₂). The product was obtained in a yield of 100%.

¹H NMR (CDCl₃, 300 MHz, δ=ppm): 6.94 (s, 2H, vinylic), 3.79 (s, 6H, methoxy), 1.79 (s, 6H, methyl) ¹³C NMR (CDCl₃, 75 MHz, δ=ppm): 163.9 (carbonyls), 154.3 (vinylic near carbonyls), 146.8 (vinylic), 91.6 (bridgeheads), 51.7 (methoxy), 14.9 (methyl) MS: calc. [M+H] 239.09195 [M+Na] 261.07389 found [M+H] 239.09077 [M+Na] 261.07219

Example 45

2-(4-bromo-3-methoxyphenoxy)ethanol (41) (method 1)

A solution of 4-bromophenol (1.00 g, 5.38 mmol) and NaOH (323 mg, 8.07 mmol) in a stoichiometric mixture of water and THF (10 mL) was stirred for 15 min followed by the addition of 2-chloroethanol (1.08 mL, 16.1 mmol) dissolved in water/THF (20 mL). After 3 days, extra 2-chloroethanol was added (323 μL, 4.01 mmol) and the mixture was stirred for an additional 2 days. For workup, 1 mL HCl (2M) and a saturated aqueous NaCl solution were added three times extracted with ethyl acetate. The organic layers were combined, dried over anhydrous Na₂SO₄ and the solvent was removed in vacuo. Column chromatography (n-Heptane/EtOAc, 2/1) afforded 41 (991 mg, 80% yield) as an off-white semisolid. R_f = 0.48 (n-Heptane/EtOAc, 1/1); ¹H NMR (CDCl₃, 400 MHz): δ = 7.40 (d, J= 8.7 Hz, IH), 6.81 (d, J= 3.0, IH), 6.63 (dd, J= 8.7, 3.0 Hz, IH), 4.05 (m, 2H), 3.95 (m, 2H), 2.36 (s, 3H) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 157.7, 138.9, 132.8, 117.1, 115.8, 113.5, 69.3, 61.3, 23.1 ppm; IR v_max (film): 3382, 2911, 2358, 2336, 1476, 1238, 1027 cm^"1; HRMS (EI+) m/z calcd for C₉HnO₂Br 229.9942, found 229.9945 [M]^+*. Example 46

2-(4-bromo-3-methoxyphenoxy)ethanol (41) (method 2)

Under Ar atmosphere, potassium carbonate (207 mg, 1.50 mmol) was added to a solution of 3-methyl-4-bromophenol (134 mg, 0.75 mmol) and ethylene carbonate (264 mg, 3.00 mmol) in destilled toluene (5 mL). The mixture was stirred and heated for 24h at 115 ⁰C. After completion, water was added to extract two times with EtOAc. The organic layers were combined, dried over anhydrous Na₂SO₄ and the solvent was evaporated in vacuo. Further purification was accomplished by column chromatography (n-Heptane/EtOAc, 2/1) to afford 2-(4-bromo-3- methoxyphenoxy)ethanol as a off-white semisolid (169 mg, 98%).

Example 47

(2-(4-bromo-3-methylphenoxy)ethoxy)(tert-butyl)dimethylsilane (42)

To a stirred solution of 2-(4-bromo-3-methoxyphenoxy)ethanol (1,000 g, 4.35 mmo), 41, in DMF (30 mL) were added TBDMSCl (978 mg, 6.52 mmol) and imidazole (888 mg, 13.04 mmol). The reaction was finished after 1.5h of stirring at room temperature. The DMF was evaporated in vacuo and column chromatography (n-Heptane/EtOAc, 2/1) afforded (2-(4-bromo-3-methylphenoxy)ethoxy)(tert-butyl) dimethylsilane as a colorless oil (1.477 g, 99%). R_f = 0.86 (n-Heptane/EtOAc, 1/1); ¹H NMR (CDCl₃, 400 MHz): δ = 7.38 (d, J = 8.7 Hz, IH), 6.80 (d, J = 3.0, IH), 6.62 (dd, J = 8.7, 3.0 Hz, IH), 4.00 (m, 2H), 3.95 (m, 2H), 2.36 (s, 3H), 0.91 (s, 9H), 0.10 (s, 6H) ppm; ¹³C NMR (CDCl₃, 75 MHz): δ = 158.2, 138.7, 132.7, 117.2, 115.4, 113.6, 69.5, 62.0, 25.9, 23.1, 18.4, -5.2 ppm; IR v_max (film): 2923, 1473, 1241, 1128, 828, 776 cm^"1; HRMS (ESI+) m/z calcd for Ci₅H₂₆O₂BrSi 345.08854, found 345.09071 [M+H].

Example 48

3,6-bis(tert-butyldimethylsilyloxy)-9H-xanthen-9-one (43)

Dissolved in 45 mL dry DMF was 3,6-dihydroxy-9H-xanthen-9-one (0.500 g, 2.20 mmol) where after TBDMSCl (1.993 g, 13.2 mmol) and imidazole (1.496 g, 22.0 mmol) were added. After stirring at room temperature for 2h, the reaction mixture was diluted with toluene, washed extensively three times with water and dried over NaSO₄. Evaporation in vacuo left a light brown solid, which was recrystallized from ethanol to give 43 as off white needle crystals (0.841 g, 84% yield). R_f = 0.90 (n-Heptane/EtOAc, 1/2); ¹H NMR (CDCl₃, 400 MHz): δ = 8.20 (td, J = 9.14, 1.18, 1.18 Hz, 2H), 6.85 (dd, J = 9.16, 2.23 Hz, 2H), 6.84 (s, 2H), 1.01 (s, 18H), 0.29 (s, 12H) ppm; ¹³C NMR (CDCl₃, 50 MHz): δ = 161.3, 159.1, 157.7, 128.2, 117.6, 116.4, 107.3, 25.5, 18.3, -4.4 ppm; IR v_max: (cm^"1) 2924, 16.15, 1279,1270, 850, 840; HRMS (CI+) m/z calcd for C₂₅H₃₇O₄Si₂ 457.223, found 457.2230 [M+H]⁺.

Example 49

6-hydroxy-9-(4-(2-hydroxyethoxy)-2-methylphenyl)-3H-xanthen-3-one (44)

In a flame-dried Schlenk-fmger under Ar atmosphere, Mg (36.5 mg, 1.50 mmol) was suspended in less dry Et₂O while stirring. After activation of the Mg with a drop of dibromoethane, 42 (329 mg, 0.96 mmol) dissolved in Et₂O (1.5 mL) was slowly added to the mixture while gas formation was maintained by intervallic short warming with a heat gun. The solution became grey unclear. When no gas formation was obtained without appending a lot of heat, the mixture was stirred for another half hour at room temperature and then cooled to 0 ⁰C. Following, 43 (325 mg, 0.71 mmol) dissolved in 4 mL THF was slowly dripped into the reaction mixture. When warmed to room temperature, the color went from yellow to brownish to deep purple in 1.5h. The mixture was quenched with MeOH and the remaining Mg was filtered off. The deprotection with 8 mL 2M HCl took 10 minutes where after the solution was washed three times with EtOAc. The organic layers where combined, dried over NaSO4, the solvent was removed in vacuo and further purification using column chromatography (MeOH/DCM, 1/19). The resulting orange sticky oil was lyophilized that led to 44 as a red solid (211 mg, 82% yield). R_f = 0.30 (MeOH/DCM, 1/9); ¹H NMR (MeOH, 400 MHz) δ = 7.24 (d, J = 9.15 Hz, IH), 7.17 (d, J = 8.41 Hz, IH), 7.09 (d, J = 2.33 Hz, IH), 7.05 (dd, J = 8.36, 2.48 Hz, IH), 6.86 (d, J = 2.16 Hz, IH), 6.84 (dd, J = 9.15, 2.19 Hz, IH), 4.17 (m, 2H), 3.94 (m, 2H), 2.04 (s, 3H) ppm; ¹³C NMR (MeOH, 75 MHz): δ = 161.8, 159.7, 158.6, 158.5, 139.1, 132.9, 131.6, 125.6, 122.5, 122.5, 117.8, 117.2, 113.5, 104.2, 70.8, 61.7, 20.1 ppm; IR v_max: (cm^"1) 3377, 2915, 1592, 1461, 1382, 1244, 1207, 1109, 621, 609; HRMS (ESI+) m/z calcd for C₂₂Hi₈NaIO₅ 385.10519, found 385.10237 [M+Na]⁺.

Example 50

2-(4-(3-hydroxy-6-oxo-6H-xanthen-9-yl)-3-methylphenoxy)ethyl 3- (trifluoromethylJ-T-oxa-bicycloβ.l.ljhepta-ljS-diene-l-carboxylate (45)

For the preparation of the oxanorbornadiene propionic anhydride, 5 (52 mg, 0.25 mmol) was dissolved in destilled DCM (3 mL) in a flame-dried Schlenk-finger under Ar atmosphere and cooled to -18 ⁰C. Triethylamine (42 μL, 0.30 mmol) and ethyl chloro formate (23 μL, 0.24 mmol) were added and the reaction mixture was warmed to 0 ⁰C and stirred for Ih. After adding 44 (100 mg, 0.27 mmol) suspended in MeCN (3 mL), the mixture was allowed to warm further to r.t. and became completely clear after 1.5h; an indication that the fluoresceine has been converted into the desired product. For workup, DCM was added and the solution was extracted once with water. The latter layer was washed with DCM where after the combined organic layers where dried over Na₂SO₄ and concentrated in vacuo. Purification using gradient column chromatography (n- Heptane/EtOAc, 1/4 to 1/7) left 45 after lyophylization as a orange fluffy solid (30 mg, 23% yield). R_f = 0.28 (n-Heptane/EtOAc, 1/4); ¹R NMR (CDCl₃, 400 MHz) δ = 7.38 (dd, J = 5.30, 1.94 Hz, IH), 7.34 (d, J = 2.23 Hz, IH), 7.28 (dd, J = 5.29, 1.98 Hz, IH), 7.13 (d, J = 8.76 Hz, IH), 7.09 (d, J = 8.33 Hz, IH), 7.02 (ddd, J = 8.76, 2.25, 0.75 Hz, IH), 7.01 (d, J = 9.76, IH), 6.97 (d, J = 2.48, IH), 6.94 (dd, J = 8.35, 2.46, IH), 6.65 (dd, J = 9.76, 1.89, IH), 6.45 (d, J = 1.89, IH), 5.85 (m, IH), 5.77 (t, J = 1.74, IH), 4.18 (m, 2H), 4.04 (m, 2H), 2.06 (s, 3H) ppm; ¹³C NMR (MeOH, 75 MHz): δ = 185.6, 159.2, 158.0, 152.6, 152.6, 147.3, 143.5, 142.2, 137.6, 130.6, 130.2, 130.1, 128.9, 124.0, 120.7, 118.8, 117.2, 116.3, 111.8, 109.5, 105.8, 84.7, 84.0, 68.9, 60.9, 292, 19.6 ppm; IR v_max: (cm^"1) 3378, 2924, 1639, 1598, 1242, 1182, 611; HRMS (ESI+) m/z calcd for C₃₀H₂₂F₃O₇ 551.13176, found 551.13476 [M+H]⁺.

References

[1] a) E. Saxon, J.I. Armstrong, CR. Bertozzi, Org. Lett. 2000, 2, 2141-2143, b) A.Watzke, M. Kohn, M. Gutierrez-Rodriguez, R. Wacker, H. Schroder, R. Breinbauer, J. Kuhlmann, K. Alexandrov, CM. Niemeyer, R.S. Goody, H. Waldmann, Angew. Chem. Int. Ed. 2006, 45, 1408-1412 c) M.-L. Tsao, F. Tian, P.G. Schultz, ChemBioChem, 2005, 6, 2147-2149

[2] P.E. Dawson, S.B.H. Kent, Annu. Rev. Biochem. 2000, 69, 923-960

[3] A. Deiters, T.A. Cropp, M.Mukherji, J.W. Chin, J.C Anderson, P.G. Schultz, J.

Am. Chem. Soc. 2003, 125, 11782-11783 [4] a) D. Schwarzer, P.A. Cole, Curr. Opin. Chem. Biol. 2005, 9, 561-569, b) N.

Budisa, ChemBioChem 2004, 5, 1176-1179

[5] R. Huisgen, G. Szeimies, L. Mobius, Chem Ber. 1967, 100, 2494-2507

[6] a) A. Dantes de Araύjo, J. M. Palomo, J. Cramer, M. Kohn, H. Schroder, R. Wacker, C. Niemeyer, K. Alexandrov, H. Waldmann, Angew. Chem. Int. Ed. 2006, 45,

296-301 b) A. Dantes de Araύjo, J. M. Palomo, J. Cramer, O. Seitz, K. Alexandrov, H.

Waldmann, Chem. Eur. J. 2006, 12, 6095-6109

[7] V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless, Angew. Chem. 2002,

114, 2708-2721; Angew. Chem. Int. Ed. 2002, 41, 2596-2599 [8] CW. Tornoe, C. Christensen, M. Medal, J. Org Chem. 2002, 67, 3057-3064

[9] For a review, see; V.D. Bock, H. Hiemstra, J.H. van Maarseveen, Eur. J. Org.

Chem. 2006, 7, 51-68

[10] a) AJ. Dirks, S. S. van Berkel, N.S. Hatzakis, J.A. Opsteen, F.L. van Delft,

J.J.L.M. Cornelissen, A.E. Rowan, J.C.M. van Hest, F.P.J.T. Rutjes, R.J.M. Nolte, Chem Commun. 2005, 4172-4174, b) AJ. Link, D.A. Tirell, J. Am. Chem. Soc. 2003,

125, 11164-11165

[11] a) B.H.M. Kuijpers, S. Groothuys, A.R. Keereweer, PJ.L.M. Quaedflieg, R.H.

Blauw, F.L. van Delft, F.P.J.T. Rutjes, Org. Lett. 2004, 6, 3123-3126, b) A. Dondoni,

P.P. Giovannini, A. Massi, Org. Lett. 2004, 6, 2929-2931 [12] a) J.A. Opsteen, J.C.M. van Hest, Chem. Commun. 2005, 57-59 b) V. Ladmiral,

G. Mantovani, GJ. Clarkson, S.Cauet, J.L. Irwin, D. M. Haddleton, J. Am. Chem. Soc.

2006, 128, 4823-4830

[13] a) S. S. Gupta, J. Kuzelka, P. Singh, W.G. Lewis, M. Manchester, M.G. Finn

Bioconj. Chem. 2005, 16, 1572-1579, b) Q. Wang, T.R. Chan, R. Hilgraf, V.V. Fokin, K.B. Sharpless, M.G. Finn, J. Am. Chem. Soc. 2003, 125, 3192-3193

[14] a) L.V. Lee, M.L. Mitchell, S.-J. Huang, V.V. Fokin, K.B. Sharpless, C-H. Wong,

J. Am. Chem. Soc. 2003, 125, 9588-9589, b) R. Manetsch, A. Krasinski, Z. Radic, J.

Raushel, P. Taylor, K.B. Sharpless, H.C. KoIb, J. Am. Chem. Soc. 2004, 126, 12809-

12818, c) G.G. Kochendoerfer, Curr. Opin. Chem. Biol. 2005, 9, 555-560 [15] NJ. Agard, J.A. Prescher, CR. Bertozzi, J. Am. Chem. Soc. 2004, 126, 15046-

15047

[16] a) SJ. Coats, J.S. Link, D. Gauthier, DJ. Hlasta, Org. Lett. 2005, 7, 1469-1472, b)

T.S. Seo, Z. Li, H. Ruparel, J. Ju, J. Org. Chem. 2003, 68, 609-612 [17] Z. Li, T.S. Seo, J. Ju, Tet. Lett. 2004, 45, 3143-3146

[18] a) A. Nezes, J. Fayn, A. Cambon, J. Fluor. Chem. 1991, 53, 285-295, b) M.G. Barlow, N.N.E. Suliman, A.E. Tipping, J. Fluor. Chem. 1995, 70, 59-69 [19] D. Cristina, M. De Amici, C. De Micheli, Tetrahedron 1981, 37, 1349-1357 Sl L. Fisera, D. Pavlovic, Coll. Czech. Chem Commun. 1984, 49, 1990-2000.

52 S. Niwayama, J. Org. Chem. 2000, 65, 5834-5836.

53 a) A. Nezes, J. Fayn, A. Cambon, J. Fluor. Chem. 1991, 53, 285-295, b) M.G. Barlow, N.N.E. Suliman, A.E. Tipping, J. Fluor. Chem. 1995, 70, 59-69.

54 E. Atherton, R.C. Sheppard, Solid-Phase Peptide Synthesis, IRL Press: Oxford England, 1989.

55 G.B. Fields, R.L. Noble, Int. J. Pept. Protein Res. 1990, 35, 161-214.

56 V.K. Sarin, S.B.H. Kent, J.P. Tarn, R.B. Merrifield, Anal. Biochem. 1981, 147.

57 E. Kaiser, R.L. Colescott, CD. Bossinger, P.I. Cook, Anal. Biochem. 1970, 34, 595.

58 K. Sivakumar, F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q. Wang, Org. Lett, 2004, 6, 4603-4606.

59 Unpublished results by A. Jawalekar according to literature procedure: MJ. Robins, B. Doboszewski, B. L. Nilsson, M.A. Peterson, Nucleosides, Nucleotides & Nucleic Acids 2000, 19(1 & 2), 69-86.

Claims

1. A process for the preparation of 1,4,5-trisubstituted triazoles and 3,4,5- trisubstituted triazoles according to Formulas (Ia) and (Ib), and mesomers and tautomers thereof:

(Ia)

wherein a compound according to Formula (III):

is reacted with a 1,3 -dipolar compound at a temperature in the range of 15 - 50⁰C, wherein: the 1,3 -dipolar compound is selected from the group consisting of nitrile oxides according to the formula R⁸-C≡N⁺-O^" (<→ R⁸-C⁺=N-O^"), azides according to the formula R⁸-N^"-N=N⁺, diazomethanes according to the formula R⁸-^"CH2-N=N⁺, nitrones according to the formula (R⁸)2⁺C-N(R^8a) -O^" and nitrilamines according to the formula R⁸-⁺C=N-N-R^8a, wherein R^8a is a group as defined for R⁸ or hydrogen;

R¹ is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 12 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, Ce - Ci₂ aryl groups, C₇ - C i2 arylalkyl groups, and C7 - C12 alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur; R² is selected from the group consisting of electron-withdrawing groups, said electron-withdrawing group having a Hammett σ_p constant of more than O; R¹ and R² may optionally form a four to nine membered ring structure comprising a heteroatom selected from the group consisting of O and N; R¹ and R² may independently also be selected from the group of functional groups or biologically active groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer; R³ and R⁴ are independently selected from electron-withdrawing groups, said electron-withdrawing group having a Hammett σ_p constant of more than 0; R⁵ and R⁶ are independently selected from hydrogen and linear or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, C₆ - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; or R⁵ and R⁶ form, together with the carbon atoms of the bicycloheptane ring to which they are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S;

R⁵ and R⁶ may independently and optionally also form, together with R³ and R⁴, respectively, and the carbon atoms of the bicycloheptane ring to which R³, R⁴, R⁵ and R⁶ are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing

3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S; X is selected from the group consisting of =CR⁷, =NR⁷, =0, =S, =SO, =SC>2, =PR⁷, and =P(O)R⁷;

R⁷ is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -C₁₂ alkyl groups, linear, cyclic or branched C₂ -C12 alkenyl groups, Ce - C₁₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted;

R⁸ is selected from the group consisting of linear or branched Ci - Ci₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, linear or branched Ci - C i2 alkenyl groups, C₆ - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, C₇ - Ci₂ alkylaryl groups, the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted, a group having biological or pharmaceutical activity, and a functional group having as a substituent a UV- label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

2. Process according to Claim 1, wherein the 1,3 -dipolar compound is a compound according to Formula (IV):

R⁸ N₃

(IV)

wherein R⁸ is selected from the group consisting of linear or branched Ci - Ci₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, linear or branched Ci - C₁₂ alkenyl groups, Ce - Ci₂ aryl groups, C₇ - C₁₂ arylalkyl groups, C₇ - C₁₂ alkylaryl groups, the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted, a group having biological or pharmaceutical activity, and a functional group having as a substituent a UV- label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

3. Process according to Claim 1 or Claim 2, wherein R¹ has the formula -C_pF_qH_r, wherein p is in the range of 1 - 6 and q and r are in te range of 3 - 13, provided that q + r = 2p + 1.

4. Process according to Claim 1 or Claim 2, wherein R¹ has the formula C_pF_qH_r, wherein p is in the range of 6 - 18 and q and r are in the range of 7 - 17, provided that q + r = p - 1.

5. Process according to Claim 3, wherein R¹ is -CF₃.

6. Process according to any one of Claims 1 - 5, wherein R¹ is a group according to Formula (V):

-(CF₂VZ

wherein n is 1 - 25 and Z is selected from the group consisting of halogen, -YH, - C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched C1 -C12 alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, C₆ - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted.

7. Process according to any one of Claims 1 - 6, wherein R¹ is selected from the group of functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer.

8. Process according to any one of the preceding claims, wherein R² is selected from the group consisting of:

-COOR¹⁰, wherein R¹⁰ is selected from the group consisting of hydrogen, linear or branched Ci - C 12 alkyl groups, linear or branched C₂ - C 12 alkenyl groups, Ce - C₁₂ aryl groups, C₇ - C₁₂ arylalkyl groups, and C₇ - C₁₂ alkylaryl groups, the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted;

-N⁺(R¹⁰)₄;

-CN; -COR¹⁰;

-CpFqHr, wherein p is in the range of 1 - 6 and q and r are in te range of 3 - 13, provided that q + r = 2p + 1 ; and

CpFqHr, wherein p is in the range of 6 - 18 and q and r are in the range of 7 - 17, provided that q + r = p - 1.

9. Process according to any one of Claims 1 - 8, wherein R² is selected from the group of functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label; said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer.

10. Process according to any one of the preceding claims, wherein X is =0.

11. Process according to any one of the preceding claims, wherein the process is conducted in the absence of a catalyst.

12. Process according to any one of the preceding claims, wherein the molar ratio between the compound according to Formula (III) and the compound according to Formula (IV) is between 1 : 5 to 5 : 1.

13. Process according to any one of the preceding claims, wherein the process is conducted in an aqueous medium.

14. Process according to Claim 13, wherein the process is conducted in water.

15. A compound according to Formula (I) and mesomers and tautomers thereof:

(Ib)

wherein:

R¹ is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 6 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and

-SO₃H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, C₆ - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur; R² is selected from the group consisting of electron-withdrawing groups, said electron-withdrawing group having a Hammett σ_p constant of more than 0; R¹ and R² may optionally form a four to nine membered ring structure comprising a heteroatom selected from the group consisting of O and N; R¹ and R² may independently also be selected from the group of functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;; and R⁸ is selected from the group consisting of linear or branched Ci - C 12 alkyl groups, linear or branched C₂ - C 12 alkenyl groups, C₆ - C 12 aryl groups, C₇ - C 12 arylalkyl groups, C₇ - C 12 alkylaryl groups, the alkyl groups, aryl groups, arylalkyl groups and alkylaryl groups optionally being substituted, a group having biological or pharmaceutical activity, and a functional group having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

16. A compound according to Formula (III):

wherein R¹ is selected from the group consisting of fluorinated hydrocarbyl groups comprising 1 - 6 carbon atoms, said hydrocarbyl groups optionally being substituted with one or more substituents selected from the group consisting of halogen, -YH, -C(Y)R^a, -CYR^a; -C(Y)OR^a, -C(Y)N(R^a)₂, -CN, -NO₂, -PO₃H and -SO3H, wherein halogen is chlorine, bromine or iodine, Y is O or S, and R^a is selected from the group consisting of hydrogen and linear, cyclic or branched Ci - C 12 alkyl groups, C₆ - C 12 aryl groups, linear, cyclic or branched C2 -C 12 alkenyl groups, C₇ - C 12 arylalkyl groups, and C₇ - C 12 alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted, or wherein the hydrocarbyl groups are interrupted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulphur; R² is selected from the group consisting of electron-withdrawing groups, said electron-withdrawing group having a Hammett σ_p constant of more than 0; R¹ and R² may optionally form a four to nine membered ring structure comprising a heteroatom selected from the group consisting of O and N; R¹ and R² may independently also be selected from the group of functional groups consisting of biomolecules, pharmaceuticals, functional groups having as a substituent a UV-label-, a fluorescence-label, a luminescence-label, a radioactive label, a dye, a chromophore, a magnetic particle label or an affinity label, said functional groups or biologically active groups optionally being provided with a linking moiety or a spacer;

R³ and R⁴ are independently selected from hydrogen and optionally substituted, linear or branched C₁ - C₆ alkyl and linear, cyclic or branched C₂ -C12 alkenyl; R⁵ and R⁶ are independently selected from hydrogen and linear or branched Ci - C₁₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, C₆ - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; or R⁵ and R⁶ form, together with the carbon atoms of the bicycloheptane ring to which they are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S;

R⁵ and R⁶ may independently and optionally also form, together with R³ and R⁴, respectively, and the carbon atoms of the bicycloheptane ring to which R³, R⁴, R⁵ and R⁶ are bonded, a cyclic, optionally unsaturated hydrocarbyl group containing 3 - 20 carbon atoms, wherein the hydrocarbyl group is optionally substituted and optionally comprises one or more heteroatoms selected from the group consisting of O, N and S;

X is selected from the group consisting of =CR⁷, =NR⁷, =0, =S, =SO, =SO₂, =PR⁷, and =P(O)R⁷; and R⁷ is selected from the group consisting of hydrogen and linear, cyclic or branched Ci -Ci₂ alkyl groups, linear, cyclic or branched C₂ -Ci₂ alkenyl groups, Ce - Ci₂ aryl groups, C₇ - Ci₂ arylalkyl groups, and C₇ - Ci₂ alkylaryl groups, wherein the alkyl groups, alkenyl groups, aryl groups, arylalkyl groups and alkylaryl groups may be substituted; 17. A process for the preparation of a compound according to Formula (III), wherein a compound according to Formula (V):

is reacted with a compound according to Formula (II), wherein X, R¹, R², R³, R⁴, R⁵ and R⁶ are as defined in claim 1. 18. Use of a compound according to Formula (III) in a cycloaddition reaction.