WO2013181697A1

WO2013181697A1 - Bicyclo[6.1.0]non-4-yne compounds suitable for use as linkers in biological applications

Info

Publication number: WO2013181697A1
Application number: PCT/AU2013/000591
Authority: WO
Inventors: Spencer John Williams; Paul Stephen Donnelly; Paul Mulvaney; Christine SCHIEBER; Isaac Ojea JIMENEZ
Original assignee: The University Of Melbourne
Priority date: 2012-06-05
Filing date: 2013-06-05
Publication date: 2013-12-12

Abstract

The present invention relates to compounds that can be used link substrates to molecules, typically for biological applications such as imaging and the like. The compounds contain a cyclooctyne which is joined, via a linking group, to a moiety such as a squarate, amine, aminooxy, hydrazide, semicarbazide or carbohydrazide. The cyclooctyne moiety acts as the coupling partner to an azide-functionalised substrate or molecule, whilst the other moiety acts as the coupling partner to an amine-, aldehyde- or ketone-functionalised molecule or substrate. The invention therefore also relates to processes of synthesis of such compounds and further use of the compounds to link a substrate to a molecule.

Description

NANOPARTICLE

FIELD OF THE INVENTION

The invention relates to compounds to link substrates to molecules, typically for biological applications. In particular, the invention relates to compounds that can be used to link nanoparticles to biological molecules to allow for applications such as imaging and the like.

BACKGROUND

There are a number of applications in which there is a need to link molecules to substrates. Examples of such applications include certain detection assays such as microarrays and in the imaging of biological systems. Dye-labelling used in biological systems typically involves the process of attaching a fluorophore or other photodetectable dye to a molecule, such as a protein or nucleic acid. Generally this is achieved by using a reactive derivative of the dye that selectively binds to a functional group contained in the target molecule. For example, commonly used fluorescently- labelled molecules are antibodies, which are then used as highly-specific probes for optical detection of a particular target. Fluorescent dye labelling is typically accomplished using a chemically-reactive derivative of a fluorophore. Common reactive groups include amine-reactive isothiocyanate derivatives such as fluorescein isocyanate (FITC) and tetramethylrhodamine (TRITC); amine-reactive succinimidyl esters such as NHS- fluorescein; and sulfhydryl-reactive maleimide-activated fluorophores such as fluorescein-5-maleimide or iodoacetamide-derivatives such as 5-(iodoacetamido) fluorescein (5-IAF). Reaction of any of these reactive dyes with another molecule results in a covalent bond being formed between the fluorophore and the labelled molecule. Following a fluorescent labelling reaction, it is often necessary to remove any unreacted fluorophore from the labelled target molecule. This is often accomplished by size-exclusion chromatography, taking advantage of the size difference between the fluorophore and labelled protein, nucleic acid, etc. Fluorophores may interact with the separation matrix and reduce the efficiency of separation. For this reason, specialized dye removal columns that account for the hydrophobic properties of fluorescent dyes are sometimes used.

Reactive fluorescent dyes are available from many sources and can be obtained with different reactive groups for attachment to various functional groups within the target molecule. They are also available in labelling kits that contain all of the components necessary to carry out a labelling reaction.

Fluorescent labels are generally used for detection of a protein or other labelled molecule via fluorescence microscopy, flow cytometry (FCM), or some other fluorescence measuring technique. High resolution optical microscopy, western blot assays, and other immunoanalytical methods may also be useful in localization of a target within a cell. More flexibility is typically required of these dyes, and the traditional dyes are often unable to meet expectations. To this end, semiconducting nanoparticles, hereinafter referred to as quantum dots (QDs), typically provide distinct advantages over traditional organic dyes, which include:

(i) superior brightness (owing to their high absorption cross-sections);

(ii) increased stability (owing to reduced photobleaching);

(iii) the ability to acquire many consecutive focal-plane images that can be reconstructed into a high-resolution three-dimensional image (broader absorption, narrower luminescence emission spectra); and

(iv) real-time tracking of single molecules and cells over extended periods of time. For example, in one study quantum dots could be observed in lymph nodes of mice for more than 4 months after administration (Ballou, B. , Lagerholm, B.C., Ernst, L.A., Bruchez, M.P.; Waggoner, A.S., (2004). "Noninvasive imaging of quantum dots in mice." Bioconj. Chem. 15 (1 ): 79-86).

Semiconductor quantum dots have been employed for in vitro imaging of pre-labelled cells. The ability to image single-cell migration in real time is important to several research areas such as fluorescence microscopy, histology, flow cytometry, fluorescence in-situ hybridization, DNA sequencing, immuno-assays, binding assays, separation, etc.

Core-shell CdSe/ZnS quantum dots (QDs) are semiconducting nanocrystals that exhibit unique optical and physical features. The size of the QDs can be adjusted to tune the optical properties to give substances with tunable, narrow emission profiles, high quantum yields and excellent photostability. QDs are therefore an attractive alternative to organic fluorescent dyes and QDs tethered to molecules such as antibodies or peptides have great promise for use in biological imaging applications.

However, in order to take advantage of the fluorescence qualities of QDs it is necessary to find means to tether a QD to a biological molecule, which requires reliable bioconjugation. Current bioconjugation strategies suffer two major drawbacks. Firstly, the reactive groups present in the chemically-reactive fluorescent dye reagents often hydrolyse over time, so that reagents have limited shelf-life and reaction in solution is only possible in limited pH ranges and often suffer from competing hydrolysis side reactions.

Secondly, the linker molecules or conjugation strategy may destabilize the nanocrystals leading to unwanted aggregation or non-specific binding of the nanocrystals to the biological target or surfaces. There is therefore a need for a conjugation strategy using molecules that use stable reactive group chemistry, can be stored for long periods in solution or as pure reagents, will work under a wide range of buffer and pH conditions, and are bio- orthogonal, i.e. cross reactions with other chemical groups present in biological systems is minimal.

The present invention relates to a series of compounds that meets the above requirements and enable bioconjugations to be carried out between a wide range of substrates and a wide range of molecules. It is an aspect of the current invention to disclose how such compounds may be prepared and purified.

It is a further aspect of the current invention to disclose experimental procedures and protocols, which enable such bioconjugation to be carried out in a general way.

It is also an aspect of the current invention to disclose how said bioconjugates may be purified. SUMMARY OF INVENTION

The present invention relates to a compound, which can be used to tether substrates to molecules.

The molecules that can be tethered to the substrates include any molecule suitable for conjugation, including polyatomic ions. Molecules used in the present invention for linking to the substrates include, but are not limited to, biological molecules. Some examples of the biological molecules for use in the present invention include large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and nucleic acids as well as small molecules such as natural products and derivatives thereof.

The substrates may include any surface or material that may be conjugated by the methods disclosed in this invention. Some substrates may be particles. A skilled addressee would appreciate that the particles may have different shapes and sizes. Typical particles for conjugation may include microparticles, mesoparticles, nanoparticles or any other particle suitable for conjugation. Other examples include semiconducting, metallic and magnetic nanocrystals (or mixtures thereof); particles of non-spherical shapes such as nanorods and nanowires; complex particles consisting of a core and shell or alloy structure; silica or silicic acid based particles; or organic semiconducting polymer-based particles that have useful labelling properties may also be conjugated through the methods outlined herein. In some embodiments the substrates may be proteins or peptides. The current invention discloses a method of conjugation, which can be applied to dendrimers, various polymers such as PEG-polymers and dextran polymers, carbon structures such as C60, C70 and carbon nanotubes and their derivatives, and also to particles carrying further functional molecules such as dyes, surface-enhanced Raman-scattering labels or stabilizing molecules.

For the purposes of describing the invention in detail, semiconducting nanocrystals (also termed nanoparticles or quantum dots) are used to highlight the features of the invention. However, the protocols may be suitably varied to enable any particle to be conjugated.

In one aspect the invention relates to a compound, which contains a cyclooctyne moiety and a moiety for conjugation to an amine-, aldehyde- or ketone-functionalised substrate or molecule. The following moieties may form covalent bonds on exposure to amine, aldehyde or ketone groups: squarate, hydrazide, semicarbazide, carbohydrazide, aminooxy and amine groups. The amine, aldehyde and ketone groups on the substrate or molecule of interest may be native to the substrate or molecule or they can be created via oxidation or reduction of suitable reactive groups. The cyclooctyne moiety acts as the coupling partner to an azide-functionalised substrate such as a QD or biological molecule, via a [3+2] cycloaddition, whilst the squarate, hydrazide, semicarbazide, carbohydrazide, aminooxy or amine moiety acts as the coupling partner to an amine-, aldehyde- or ketone-functionalised substrate such as QD or biological molecule. The cyclooctyne and aldehyde moieties are joined via a linking group.

The present invention relates to solution-stable linker molecules that can be used to conjugate particles and other structures to each other or to biological molecules and substrates of interest (e.g. cells.). For conjugation to an amine, aldehyde or ketone group, this invention discloses a variety of novel molecules. In some embodiments, hydrazide, semicarbazide, carbohydrazide, aminooxy and amine derivatives can be used for conjugation to aldehyde or ketone groups. For conjugation to an extant amine group, squarate derivatives can be employed. In some embodiments the substrates for conjugation are particles. In some embodiments the particles are microparticles, mesoparticles and/or nanoparticles. In other embodiments the particles are QDs. In some embodiments the molecules for conjugation to a substrate are biological molecules. In some embodiments the molecules are synthetic molecules or naturally occurring molecules. In some embodiments the molecules are large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and/or nucleic acids as well as small molecules such as natural products

In one aspect the invention relates to a compound which can be used to tether QDs to biological molecules. The compound contains a cyclooctyne moiety and a second moiety for conjugation to QDs and biological molecules. The cyclooctyne moiety acts as the coupling partner to an azide-functionalised QD or biological molecule, via a [3+2] cycloaddition, whilst the second moiety acts as the coupling partner to an amine- aldehyde- or ketone-functionalised QD or biological molecule. Typical moieties which react with aldehyde or ketone functional groups may include N-containing moieties such as an amine, aminooxy, hydrazide, semicarbazide and carbohydrazide. Typical moieties which react with amine functional groups may include a squarate. The cyclooctyne moiety and the second moiety are joined via a linking group.

In one aspect, the present invention provides a compound of the Formula (I):

Formula (I)

wherein each R^a and R^b is independently-selected from the group consisting of: H, OH, halogen, optionally-substituted alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally- substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally- substituted OC_2-i2 alkenyl, optionally-substituted OC_2-i2 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;

each R¹ is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1.12 alkyl, optionally-substituted

haloalkyl, optionally-substituted C_2-12 alkenyl, optionally- substituted 02-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted 03-12 cycloalkenyl, optionally-substituted 02-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted C_6-18 aryl, optionally-substituted CM S heteroaryl, optionally-substituted C^ .^2 alkyloxy, optionally-substituted 02-12 alkenyloxy, optionally-substituted 02-12 alkynyloxy, optionally-substituted 02-12 heteroalkyloxy, optionally-substituted 03-12 cycloalkyloxy, optionally-substituted 03-12 cycloalkenyloxy, optionally-substituted C2-12 heterocycloalkyloxy, optionally-substituted C2-12 heterocycloalkenyloxy, optionally- substituted C6-18 aryloxy, optionally-substituted CM S heteroaryloxy, optionally- substituted C1-12 alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d, or any two R¹ on adjacent carbon atoms form a fused substituent;

L¹ is a bond or linking group;

R² is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C3-12 cycloalkyl, optionally substituted C3-12 cycloalkenyl, NH₂, ONH₂, NHNH₂, NHNR^aR^p, NH(CO)(optionally-substituted Ci_₆ alkyl)ONH₂, NH(CO)(optionally-substituted Ci_₆ alkyl)NH₂, NH(CO)(optionally-substituted Ci_₆ alkyl)NHNH₂, NH(CO)(optionally-substituted Ci_₆ alkyl)NHNR^aR^p, (CO)NH(optionally- substituted Ci_₆ alkyl)ONH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNH₂, (CO)NHNR^aR^p, NH(CO)NHNH_2, NH(CO)NHNR^aR^p, NHNH(CO)NHNH₂ and NHNH(CO)NHNR^aR^p; each FT and R^p is independently selected from the group consisting of: H, OH, optionally-substituted C^₂ alkyl, optionally-substituted C_2-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

wherein each of R^X and R⁵ is independently selected from the group consisting of H , optionally-substituted

alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; and

R^C, R^D and R^E are each independently-selected from the group consisting of: H , OH , halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OC6-18 aryl, and optionally-substituted OCM S heteroaryl;

wherein the stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo.

In another aspect the invention provides a process for the preparation of a compound of Formula (V):

Formula (V)

wherein each R¹ is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1-12 alkyl, optionally-substituted Ci-12 haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted C_2-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted C-M2 alkyloxy, optionally-substituted 02-12 alkenyloxy, optionally-substituted 02-12 alkynyloxy, optionally-substituted C_2-12 heteroalkyloxy, optionally-substituted C_3-i₂ cycloalkyloxy, optionally-substituted C_3-12 cycloalkenyloxy, optionally-substituted 02-12 heterocycloalkyloxy, optionally-substituted 02-12 heterocycloalkenyloxy, optionally- substituted Ce-18 aryloxy, optionally-substituted Ci-ie heteroaryloxy, optionally- substituted C1-12 alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d or any two R¹ on adjacent carbon atoms form a fused substituent;

R^c, R^d and R^e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C^ .^2 alkyl, optionally-substituted C_2-12 heteroalkyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OCM S heteroaryl;

wherein the stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo;

L is of the Formula (III): X³(CR⁸R⁹)_p [X⁴(CR¹⁰R^{1 1})_q]_r (CR¹²R¹³)_S X⁵

Formula (III)

X³ is selected from the group consisting of: O, NH and NR¹⁴;

X⁴ is independently selected from the group consisting of: a bond, O, NH, NR¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: O, NH, NR¹⁴, optionally-substituted

C-i-12 alkyl, HN-(optionally-substituted Ci-12 alkyl)-, R¹⁴N-(optionally-substituted Ci-12 alkyl)-, O-(optionally-substituted Ci-12 alkyl)-, optionally-substituted Cs-12 aryl, HN- (optionally-substituted Cs-12 aryl)-, R¹⁴N-(optionally-substituted Cs-12 aryl)-, O- (optionally-substituted C_5-i₂ aryl)-, HN(CO)-(optionally-substituted C_5-i₂ aryl)-, R¹⁴N(CO)-(optionally-substituted C5-12 aryl)- O(CO)-(optionally-substituted C5-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)- (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted Cs-12 aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C_2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C_2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, HN(CO)-(optionally- substituted C2-12 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C2-12 heteroaryl)-, O(CO)-(optionally-substituted C2-12 heteroaryl)-, (CO)HN-(optionally-substituted C2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C_2-12 heteroaryl)- and (CO)O- (optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C_3-i₂ cycloalkyl, optionally substituted C_3-i₂ cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted C_1-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted C_1-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted C_1-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted C_1-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted C1-12 alkyl, optionally-substituted C_2-i₂ alkenyl, optionally- substituted C_2-i₂ alkynyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted C3-i₂ cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC_3-i₂ cycloalkyl and optionally-substituted OC_3-i₂ cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted

alkyl, optionally-substituted C_2-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

each R⁸ R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C_2-12 alkenyl, optionally-substituted C_2-12 alkynyl, optionally- substituted C3-i₂ cycloalkyl, optionally-substituted C3-i₂ cycloalkenyl, optionally- substituted OCi-i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC3-i₂ cycloalkyl and optionally-substituted OC3- 2 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted Ci-i₂ alkyl and N-protecting group;

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, the process comprising:

(a) coupling a compound of Formula (VI):

Formula (VI) wherein

R¹ and n are as defined above and LG is a leaving group; in a coupling reaction with a compound of Formula (Vll-a): X^3A(CR⁸R⁹)_P [X⁴(CR^{1 0}R^{1 1} )_Q]_R (CR¹²R^{1 3})s X^5A

Formula (Vll-a)

wherein

X^3a is independently selected from the group consisting of: OH, NH₂ and NHR¹⁴;

X^5a is selected from the group consisting of: OH, NH₂, NHR¹⁴, OR¹⁸ CO₂R¹⁹; each R⁸ R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted

alkyl, optionally-substituted C_2-12 alkenyl, optionally-substituted C_2-12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally- substituted OCi-12 alkyl, optionally-substituted OC_2-i2 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3- 12 cycloalkenyl;

R¹⁴ is independently selected from the group consisting of: optionally- substituted C-i-12 alkyl and N-protecting group;

R¹⁸ is selected from the group consisting of: optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl and O-protecting group;

R¹⁹ is selected from the group consisting of: H, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl optionally-substituted C_6-18 aryl, optionally-substituted C_6-18 alkylaryl, optionally- substituted C-i-18 heteroaryl, optionally-substituted CM S alkylheteroaryl, optionally- substituted Ci-12 alkyloxy, optionally-substituted C2-12 alkenyloxy, optionally- substituted C2-12 alkynyloxy, optionally-substituted C2-12 heteroalkyloxy, optionally- substituted C3-12 cycloalkyloxy, optionally-substituted C3-12 cycloalkenyloxy and optionally-substituted C_2-12 heterocycloalkyloxy,

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; to provide a compound of Formula (VIII):

Formula (VIII)

(b) reacting the compound of Formula (VIII) with a compound of Formula (IX):

R²⁰ B R²

Formula (IX)

wherein

B is selected from the group consisting of: a bond, an optionally-substituted d.

12 alkyl group, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally-substituted 05-12 aryl group and optionally-substituted 02-12 heteroaryl group, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl; each R² and R²⁰ is selected independently from the group consisting of: OH, CO2R²², optionally-substituted OC1-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl, optionally-substituted OC3-12 cycloalkenyl, NHR¹⁷, NHNHR¹⁷, NHNR^aR^p, ONHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHNHR¹⁷, NH(CO)(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷, and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or FT and R^p when combined together provide the group of Formula (l-a):

of H, optionally-substituted C-.-₂ alkyl, optionally-substituted C_2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

each R¹⁷ and R²¹ is independently selected from the group consisting of: H, optionally-substituted Ci-i₂ alkyl and N-protecting group;

R²² is selected from the group consisting of: H, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted C_2-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted Ce-18 alkylaryl, optionally-substituted CM S heteroaryl, optionally-substituted C-M S alkylheteroaryl,

wherein R¹ , n, X³, R⁵, R⁹ p, X⁴, R¹⁰, R¹¹ , q, r, R¹², R¹ , s and X^5a are as discussed above, to provide a compound of Formula (V).

Formula (V)

wherein each R¹ is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1-12 alkyl, optionally-substituted C-i-₁₂ haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted C_2-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted C-M2 alkyloxy, optionally-substituted 02-12 alkenyloxy, optionally-substituted 02-12 alkynyloxy, optionally-substituted C_2-12 heteroalkyloxy, optionally-substituted C_3-i₂ cycloalkyloxy, optionally-substituted C_3-12 cycloalkenyloxy, optionally-substituted 02-12 heterocycloalkyloxy, optionally-substituted 02-12 heterocycloalkenyloxy, optionally- substituted Ce-18 aryloxy, optionally-substituted Ci-ie heteroaryloxy, optionally- substituted C₁-₁₂ alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d or any two R¹ on adjacent carbon atoms form a fused substituent;

the stereochemistry of the cyclopropane-fused cyclooctyne may be either endo or exo;

L is of the Formula (III): X³(C R⁸R⁹)_P [X⁴(C R^{1 0}R^{1 1} )_Q]_R (C R^{1 2}R^{1 3})_S X⁵

Formula (III)

X³ is selected from the group consisting of: OH, NH and NR¹⁴;

X⁵ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted C-i-₁₂ alkyl, HN-(optionally-substituted C-i-₁₂ alkyl)-, R¹⁴N- (optionally-substituted

alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted 05-12 aryl)-, R¹⁴N-(optionally- substituted 05-12 aryl)-, O-(optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)- R¹⁴N(CO)-(optionally-substituted C5-12 aryl)- O(CO)- (optionally-substituted Cs-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)- (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C_2-12 heteroaryl)-, O-(optionally-substituted C_2-12 heteroaryl)-, HN(CO)-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N(CO)-(optionally- substituted C2-12 heteroaryl)-, O(CO)-(optionally-substituted C2-12 heteroaryl)-, (CO)HN-(optionally-substituted C2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C_2- 12 heteroaryl)- and (CO)O-(optionally-substituted C_2-i₂ heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted Ci_i₂ alkyl, optionally-substituted C_2-i₂ alkenyl, optionally- substituted C_2-i₂ alkynyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted C3-i₂ cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC3-i₂ cycloalkyl and optionally-substituted OC3-i₂ cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted Ci_i₂ alkyl, optionally-substituted C_2-i₂ alkenyl, optionally- substituted C_2-i₂ alkynyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R³, R⁴ R⁶ R⁷, R⁸ R⁹, R¹⁰ R^{1 1} , R¹² R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C1-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C₂-12 alkynyl, optionally-substituted 03-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC₂-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted C^.^ 2 alkyl and N-protecting group; and

(a) coupling a compound of Formula (Vll-b):

X^3a(CR⁸R⁹)_p [X⁴(CR^{1 0}R^{1 1} )_q]_r (CR¹²R^{1 3})_S X⁵ R²

Formula (Vll-b)

X^3a is selected from the group consisting of: OH, NH₂ and NHR¹⁴;

X⁴ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶; X⁵ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted alkyl, HN-(optionally-substituted alkyl)-, R¹⁴N- (optionally-substituted

alkyl)-, O-(optionally-substituted Ci-12 alkyl)-, optionally- substituted C_5-i₂ aryl, HN-(optionally-substituted C_5-i₂ aryl)-, R¹⁴N-(optionally- substituted 05-12 aryl)-, O-(optionally-substituted 05-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R¹⁴N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C2-12 heteroaryl, HN(CO)-(optionally-substituted C2- 12 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C2-12 heteroaryl)-, O(CO)-(optionally- substituted C_2-12 heteroaryl)-, (CO)HN-(optionally-substituted C_2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted 02-12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C-M2 alkyl, optionally substituted C_2-12 alkenyl, optionally substituted C_2-12 alkynyl, optionally substituted C3-12 cycloalkyl, optionally substituted C3-12 cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO) (optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH (optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH (optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH (optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted C₂-12 alkenyl, optionally- substituted C₂-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i2 alkenyl, optionally-substituted OC₂-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC₃-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted Ci-₁₂ alkyl, optionally-substituted C₂-₁₂ alkenyl, optionally- substituted C₂-₁₂ alkynyl, optionally-substituted C3-₁₂ cycloalkyl, optionally-substituted C_3-12 cycloalkenyl;

each R⁸ R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci-₁₂ alkyl, optionally-substituted C₂-₁₂ alkenyl, optionally-substituted C₂-₁₂ alkynyl, optionally- substituted C_3-i₂ cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally- substituted OC-i-₁₂ alkyl, optionally-substituted OC₂-₁₂ alkenyl, optionally-substituted 0C_2-12 alkynyl, optionally-substituted OC_3-i₂ cycloalkyl and optionally-substituted OC_3- ₁₂ cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted

alkyl and N-protecting group; and

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; coupling reaction with a compound of Formula (VI):

Formula (VI) wherein

R¹ and n are as defined above and LG is a leaving group; to provide the compound of Formula (V).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a schematic of a linker of the invention showing its reactive ends available to form bonds with an azide functionalised material (a Quantum dot in this case) and an amino functionalised biological molecule (transferrin in this case).

Figure 2 shows the idealised structure of the linker shown in figure 1 after binding.

Figures 3 shows the mass spectral analysis of the reaction of the linker of example 3 with transferrin at a ratio of linker to transferrin of 2:1 .

Figure 4 shows the mass spectral analysis of the reaction of the linker of example 3 with transferrin at a ratio of linker to transferrin of 4:1 .

Figure 5 shows the reaction product of the linker bound to transferrin with a small molecule azide, azidoacetanilide.

Figure 6 shows the mass spectral analysis of the reaction product of the linker with transferrin. Figure 7 shows the product of reaction of the mixture analysed in figure 6 with an azide showing reactivity of the transferrin-bound cyclo-octyne moiety with an azide. Figure 8 shows the stylised product of the linker of example 3 bound to an amino- Alexa dye and an azide-modified Quantum dot.

Figure 9 shows the normalised absorbance of separate quantum dot and the dye. Figure 10 shows the fluorescent intensity with differing amounts of dye bound through the linker to the quantum dot.

Figure 1 1 shows an agarose gel electrophoresis of QD's and QD plus transferrin under UV lamp.

Figure 12 shows the agarose gel electrophoresis of QD's and QD plus transferrin after staining with Coomassie blue.

Figure 13 shows Fe₂-transferrin uptake into HeLa cells using A568-Fe₂Tf (A1 -A3), QD100-Fe₂Tf (B1 -B3) and QD100 (C1 -C3).

Figure 14 shows time dependent QD uptake into cells and co-staining with early and late endosomal markers, a) QD100-Fe₂Tf uptake into HeLa cells were performed for 15 min at 37°C. Cells were fixed in 4% paraformaldehyde and stained with rabbit anti-EEA1 (early endosome antigen 1 ) followed by A568-conjugated anti-rabbit IgG, and mouse anti-CD63 followed by A647-conjugated mouse IgG. (A, D) EEA1 has been pseudo-colored green; (B, E) QD100-Fe2Tf has been pseudo-colored red. D-H show 2x magnification of the boxed region. G is overlay of D and E; H is overlay of E and Fb) QD100-Fe2Tf uptake into HeLa cells was performed for 2 h at 37°C. Cells were fixed in 4% paraformaldehyde and stained and pseudo-colored as for a). D-H show 2x magnification of the boxed region. G is overlay of D and E; H is overlay of E and F. Representative regions of overlap are indicated by arrows.

Figure 15 Mass spectral analysis of herceptin.

Figure 16 and figure 17 show mass spectral analysis of Herceptin conjugated to the linker of example 3 using a ratio of antibody to linker of 1 : 10 (figure 16) and a ration of 1 :20 (figure 17).

Figure 18 is an absorption spectrum of Herceptin-linker-azido-Fluor 585 dye conjugates. After oxidation of glycan residues on an antibody with Nal0₄, a linker of example 8 was reacted in a ratio of 4: 1 , 10:1 and 100: 1 of linker to antibody in an aniline buffer, and then finally with azide-AlexaFluor585. DETAILED DESCRIPTION OF THE INVENTION

In this specification a number of terms are used which are well known to a skilled addressee. Nevertheless for the purposes of clarity a number of terms will be defined.

The term "substrate" may include any surface or material for conjugation by the methods disclosed in this invention. Some substrates include deposited or adhered particles, which may include coatings or layers. In some embodiments of the invention, the term substrate refers to particles. A skilled addressee would appreciate that the term "particle" encompasses particles of different shapes and sizes. Typical particles for conjugation may include microparticles, mesoparticles, nanoparticles or any other particle suitable for conjugation. Other examples include semiconducting, metallic and magnetic nanocrystals (or mixtures thereof); particles of non-spherical shapes such as nanorods and nanowires; complex particles consisting of a core and shell or alloy structure; silica or silicic acid based particles; or organic semiconducting polymer-based particles.

Quantum dots are semiconductors whose electronic characteristics are closely related to the size and shape of the individual crystal. The term "quantum dot" is readily understood by the skilled person in the art.

An "azide-functionalised" substrate or an "azide-functionalised" molecule refers to a substrate or a molecule bearing one or more azide (-N₃) groups. The azide groups are present at the surface of the quantum dot or biological molecule such that the azide group can react with other molecules or particles, which possess other reactive functional groups. For example, the azide group can react with other molecules or particles, which possess an alkyne group or cycloalkyne group.

Similarly, an "amine-functionalised" substrate or an "amine-functionalised" molecule refers to a substrate or molecule, which has one or more reactive amine groups. The amine groups are present at the surface of the substrate or molecule such that the amine group can react with molecules or particles, which possess other reactive functional groups. For example, the amine group can react with other molecules or particles, which possess a squarate group, a carboxylic acid group, or an activated equivalent such as an NHS ester.

Similarly, an "aldehyde-functionalised" substrate or an "aldehyde-functionalised" molecule refers to a substrate or molecule, which has one or more reactive aldehyde groups. The aldehyde moiety may be present in free or hydrated form. The aldehyde groups are present at the surface of the substrate or molecule such that the aldehyde group can react with molecules or particles, which possess other reactive functional groups. For example, the aldehyde group can react with other molecules or particles, which possess an amine, aminooxy, hydrazide, semicarbazide or carbohydrazide group.

Similarly, a "ketone-functionalised" substrate or a "ketone -functionalised" molecule refers to a substrate or molecule, which has one or more reactive ketone groups. The ketone moiety may be present in free or hydrated form. The ketone groups are present at the surface of the substrate or molecule such that the ketone group can react with molecules or particles, which possess other reactive functional groups. For example, the ketone group can react with other molecules or particles, which possess an amine, aminooxy, hydrazide, semicarbazide or carbohydrazide group.

The term "molecule" is readily understood by a skilled addressee to include polyatomic compounds held together by covalent bonds. The molecules of the present invention may also include polyatomic ions. The term "biological molecule" refers to a synthetic or naturally occurring molecule produced or used by an organism, cell or cellular fraction. This includes, but is not limited to, large polymeric molecules such as proteins, peptides, polysaccharides, lipids, hormones and nucleic acids as well as small molecules such as natural products and derivatives thereof.

The term "[3+2] cycloaddition" reactions is readily understood by the skilled addressee. The [3+2] cycloaddition reactions refer to the reaction of a dipolarophile with a 1 ,3-dipolar compound that leads to 5-membered (hetero)cycles. Examples of dipolarophiles are alkenes and alkynes and molecules that possess related heteroatom functional groups (such as carbonyls and nitriles). 1 ,3-Dipolar compounds typically contain one or more heteroatoms and can be described as having at least one mesomeric structure that represents a charged dipole. Examples of 1 ,3-dipolar compounds are azides, nitrile oxides, nitrones and diazoalkanes.

A "leaving group" is a chemical group that is readily displaced by a nucleophilic incoming chemical moiety. Accordingly in any situation the choice of leaving group will depend upon the ability of the particular group to be displaced by the incoming chemical moiety. Suitable leaving groups are well known in the art, see for example "Advanced Organic Chemistry" Jerry March 4^th Edn. pp 351 -357, Oak Wick and Sons NY (1997). Examples of suitable leaving groups include, but are not limited to, halogen, alkoxy (such as ethoxy, methoxy), sulfonyloxy, optionally-substituted arylsulfonyl. Specific examples include chloro, iodo, bromo, fluoro, ethoxy, methoxy, methansulphonyl, triflate and the like.

The term "normal chain" refers to the direct chain joining the two ends of a linking moiety.

The term "optionally-substituted" as used throughout the specification denotes that the group may or may not be further substituted or fused (so as to form a condensed polycyclic system), with one or more non-hydrogen substituent groups. In certain embodiments the substituent groups are one or more groups independently selected from the group consisting of halogen, =O, =S, -CN, -NO2, -CF₃, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, cycloalkylalkyl, heterocycloalkylalkyl, heteroarylalkyl, arylalkyl, cycloalkylalkenyl, heterocycloalkylalkenyl, arylalkenyl, heteroarylalkenyl, cycloalkylheteroalkyl, heterocycloalkylheteroalkyl, arylheteroalkyl, heteroarylheteroalkyl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxycycloalkyl, alkyloxyheterocycloalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkyloxycarbonyl, alkylaminocarbonyl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, phenoxy, benzyloxy, heteroaryloxy, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, alkylsulfinyl, arylsulfinyl, aminosulfinylaminoalkyl, -C(=0)OH, -C(=0)R^f, -C(=0)OR^f, C(=0)NR^fR^g, C(=NOH)R^f, C(=NR^f)NR^gR^h, NR^fR^g, NR^fC(=0)R^g, NR^fC(=0)OR^g, NR^fC(=0)NR^gR^h, NR^fC(=NR^g)NR^hR', NR^fS0₂R^g, -SR^f, S0₂NR^fR^g, -OR^f OC(=0)NR^fR^g, OC(=0)R^f and acyl, wherein R^f, R^g, R^h and R' are each independently selected from the group consisting of H, C-i-C-12 alkyl, C1-C12 haloalkyl, C2-C12 alkenyl, C2-C12 alkynyl, C1-C10 heteroalkyl, C3-C12 cycloalkyl, C3-C12 cycloalkenyl, C1-C12 heterocycloalkyl, C1-C12 heterocycloalkenyl, C₆-Ci₈ aryl, Ci-Ci₈ heteroaryl, and acyl, or any two or more of R^e, R^f, R^g and R^h, when taken together with the atoms to which they are attached form a heterocyclic ring system with 3 to 12 ring atoms.

In some embodiments each optional substituent is independently selected from the group consisting of: halogen, =0, =S, -CN, -NO2, -CF₃, -OCF3, alkyl, alkenyl, alkynyl, haloalkyl, haloalkenyl, haloalkynyl, heteroalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, heterocycloalkenyl, aryl, heteroaryl, hydroxy, hydroxyalkyl, alkyloxy, alkyloxyalkyl, alkyloxyaryl, alkyloxyheteroaryl, alkenyloxy, alkynyloxy, cycloalkyloxy, cycloalkenyloxy, heterocycloalkyloxy, heterocycloalkenyloxy, aryloxy, heteroaryloxy, arylalkyl, heteroarylalkyl, arylalkyloxy, amino, alkylamino, acylamino, aminoalkyl, arylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, aminoalkyl, -COOH, -SH, and acyl.

Examples of particularly suitable optional substituents include =0, F, CI, Br, I, CH₃, CH2CH3, OH, OCH3, OCH2CH3, CF₃, OCF3, NO₂, NH₂, and CN.

In the definitions of a number of substituents below it is stated that "the group may be a terminal group or a bridging group". This is intended to signify that the use of the term is intended to encompass the situation where the group is a linker between two other portions of the molecule as well as where it is a terminal moiety. Using the term alkyl as an example, some publications would use the term "alkylene" for a bridging group and hence in these other publications there is a distinction between the terms "alkyl" (terminal group) and "alkylene" (bridging group). In the present application no such distinction is made and most groups may be either a bridging group or a terminal group. "Acyl" means an R-C(=0)- group in which the R group may be an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group as defined herein. Examples of acyl include acetyl and benzoyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.

"Acylamino" means an R-C(=0)-NH- group in which the R group may be an alkyl, cycloalkyl, heterocycloalkyl, aryl or heteroaryl group as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

"Alkenyl" as a group or part of a group denotes an aliphatic hydrocarbon group containing at least one carbon-carbon double bond and which may be straight or branched preferably having 2-12 carbon atoms, more preferably 2-10 carbon atoms, most preferably 2-6 carbon atoms, in the normal chain. The group may contain a plurality of double bonds in the normal chain and the orientation about each is independently E or Z. The alkenyl group is preferably a 1 -alkenyl group. Exemplary alkenyl groups include, but are not limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl and nonenyl. The group may be a terminal group or a bridging group.

"Alkenyloxy" refers to an alkenyl-O- group in which alkenyl is as defined herein. Preferred alkenyloxy groups are C1-C6 alkenyloxy groups. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Alkyl" as a group or part of a group refers to a straight or branched aliphatic hydrocarbon group, preferably a C1-C12 alkyl, more preferably a C1-C10 alkyl, most preferably CrC₆ unless otherwise noted. Examples of suitable straight and branched C1-C6 alkyl substituents include methyl, ethyl, n-propyl, 2-propyl, n-butyl, sec-butyl, t- butyl, hexyl, and the like. The group may be a terminal group or a bridging group. "Alkylamino" includes both mono-alkylamino and dialkylamino, unless specified. "Mono-alkylamino" means an Alkyl-NH- group, in which alkyl is as defined herein. "Dialkylamino" means a (alkyl)₂N- group, in which each alkyl may be the same or different and are each as defined herein for alkyl. The alkyl group is preferably a Ci- C₆ alkyl group. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

"Alkylaminocarbonyl" refers to a group of the formula (alkyl)_x(H)_yNC(=0)- in which alkyl is as defined herein, x is 1 or 2, and the sum of X+Y =2. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon.

"Alkyloxy" refers to an alkyl-O- group in which alkyl is as defined herein. Preferably the alkyloxy is a C1-C6 alkyloxy. Examples include, but are not limited to, methoxy and ethoxy. The group may be a terminal group or a bridging group.

"Alkyloxyalkyl" refers to an alkyloxy-alkyl- group in which the alkyloxy and alkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.

"Alkyloxyaryl" refers to an alkyloxy-aryl- group in which the alkyloxy and aryl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the aryl group.

"Alkyloxycarbonyl" refers to an alkyl-0-C(=0)- group in which alkyl is as defined herein. The alkyl group is preferably a C1-C6 alkyl group. Examples include, but are not limited to, methoxycarbonyl and ethoxycarbonyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the carbonyl carbon. "Alkyloxycycloalkyl" refers to an alkyloxy-cycloalkyl- group in which the alkyloxy and cycloalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the cycloalkyl group.

"Alkyloxyheteroaryl" refers to an alkyloxy-heteroaryl- group in which the alkyloxy and heteroaryl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroaryl group.

"Alkyloxyheterocycloalkyl" refers to an alkyloxy-heterocycloalkyi- group in which the alkyloxy and heterocycloalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heterocycloalkyl group.

"Alkylsulfinyl" means an alkyl-S-(=0)- group in which alkyl is as defined herein. The alkyl group is preferably a CrC₆ alkyl group. Exemplary alkylsulfinyl groups include, but not limited to, methylsulfinyl and ethylsulfinyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.

"Alkylsulfonyl" refers to an alkyl-S(=0)₂- group in which alkyl is as defined above. The alkyl group is preferably a C1-C6 alkyl group. Examples include, but not limited to methylsulfonyl and ethylsulfonyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.

"Alkynyl" as a group or part of a group means an aliphatic hydrocarbon group containing a carbon-carbon triple bond and which may be straight or branched preferably having from 2-12 carbon atoms, more preferably 2-10 carbon atoms, more preferably 2-6 carbon atoms in the normal chain. Exemplary structures include, but are not limited to, ethynyl and propynyl. The group may be a terminal group or a bridging group. "Alkynyloxy" refers to an alkynyl-O- group in which alkynyl is as defined herein. Preferred alkynyloxy groups are C1-C6 alkynyloxy groups. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Aminoalkyl" means an NH₂-alkyl- group in which the alkyl group is as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group. "Aminosulfonyl" means an NH₂-S(=0)2- group. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.

"Aryl" as a group or part of a group denotes (i) an optionally-substituted monocyclic, or fused polycyclic, aromatic carbocycle (ring structure having ring atoms that are all carbon) preferably having from 5 to 12 atoms per ring. Examples of aryl groups include phenyl, naphthyl, and the like; (ii) an optionally-substituted partially saturated bicyclic aromatic carbocyclic moiety in which a phenyl and a Cs-7 cycloalkyl or Cs-7 cycloalkenyl group are fused together to form a cyclic structure, such as tetrahydronaphthyl, indenyl or indanyl. The group may be a terminal group or a bridging group. Typically an aryl group is a C6-C18 aryl group.

"Arylalkenyl" means an aryl-alkenyl- group in which the aryl and alkenyl are as defined herein. Exemplary arylalkenyl groups include phenylallyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.

"Arylalkyl" means an aryl-alkyl- group in which the aryl and alkyl moieties are as defined herein. Preferred arylalkyl groups contain a d-salkyl moiety. Exemplary arylalkyl groups include benzyl, phenethyl, 1 -naphthalenemethyl and 2- naphthalenemethyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group. "Arylalkyloxy" refers to an aryl-alkyl-O- group in which the alkyl and aryl are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Arylamino" includes both mono-arylamino and di-arylamino unless specified. Mono-arylamino means a group of formula arylNH-, in which aryl is as defined herein. Di-arylamino means a group of formula (aryl)₂N- where each aryl may be the same or different and are each as defined herein for aryl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

"Arylheteroalkyl" means an aryl-heteroalkyl- group in which the aryl and heteroalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.

"Aryloxy" refers to an aryl-O- group in which the aryl is as defined herein. Preferably the aryloxy is a Ce-Cisaryloxy, more preferably a C6-Cioaryloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Arylsulfonyl" means an aryl-S(=0)2- group in which the aryl group is as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.

A "bond" is a linkage between atoms in a compound or molecule. The bond may be a single bond, a double bond, or a triple bond. "Cycloalkenyl" means a non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and preferably having from 4-10 carbon atoms per ring. Exemplary monocyclic cycloalkenyl rings include cyclobutenyl, cyclopentenyl, cyclohexenyl or cycloheptenyl. The cycloalkenyl group may be substituted by one or more substituent groups. Exemplary monocyclic cycloalkenyl rings are substituted with one or more =0 group, and/or one or more OMe group and/or one or more OEt. A cycloalkenyl group typically is a C3-C12 alkenyl group. The group may be a terminal group or a bridging group. "Cycloalkyi" refers to a saturated monocyclic or fused or spiro polycyclic, carbocycle preferably containing from 3 to 9 carbons per ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like, unless otherwise specified. It includes monocyclic systems such as cyclopropyl and cyclohexyl, bicyclic systems such as decalin, and polycyclic systems such as adamantane. A cycloalkyi group typically is a C3-C12 alkyl group. The group may be a terminal group or a bridging group.

"Cycloalkylalkyl" means a cycloalkyl-alkyl- group in which the cycloalkyi and alkyl moieties are as defined herein. Exemplary monocycloalkylalkyl groups include cyclopropylmethyl, cyclopentylmethyl, cyclohexylmethyl and cycloheptylmethyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.

"Cycloalkylalkenyl" means a cycloalkyl-alkenyl- group in which the cycloalkyi and alkenyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.

"Cycloalkylheteroalkyl" means a cycloalkyl-heteroalkyl- group in which the cycloalkyi and heteroalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.

"Cycloalkyloxy" refers to a cycloalkyl-O- group in which cycloalkyi is as defined herein. Preferably the cycloalkyloxy is a Ci-C6cycloalkyloxy. Examples include, but are not limited to, cyclopropanoxy and cyclobutanoxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom. "Cycloalkenyloxy" refers to a cycloalkenyl-O- group in which the cycloalkenyl is as defined herein. Preferably the cycloalkenyloxy is a Ci-C₆cycloalkenyloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Haloalkyl" refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom selected from the group consisting of fluorine, chlorine, bromine and iodine. A haloalkyl group typically has the formula C_nH(2n+i _-m) m wherein each X is independently selected from the group consisting of F, CI, Br and I. In groups of this type n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3. m is typically 1 to 6, more preferably 1 to 3. Examples of haloalkyl include fluoromethyl, difluoromethyl and trifluoromethyl.

"Haloalkenyl" refers to an alkenyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.

"Haloalkynyl" refers to an alkynyl group as defined herein in which one or more of the hydrogen atoms has been replaced with a halogen atom independently selected from the group consisting of F, CI, Br and I.

"Halogen" represents chlorine, fluorine, bromine or iodine.

"Heteroalkyl" refers to a straight- or branched-chain alkyl group preferably having from 2 to 12 carbons, more preferably 2 to 6 carbons in the chain, in which one or more of the carbon atoms (and any associated hydrogen atoms) are each independently replaced by a heteroatomic group selected from S, 0, P and NR' where R' is selected from the group consisting of H, optionally-substituted C1 -C12 alkyl, optionally- substituted C3-C12 cycloalkyl, optionally-substituted C6-C18 aryl, and optionally- substituted Ci-Ci₈ heteroaryl. Exemplary heteroalkyls include alkyl ethers, secondary and tertiary alkyl amines, amides, alkyl sulfides, and the like. Examples of heteroalkyl also include hydroxyCi-C6 alkyl, Ci-C6-alkyloxyCi-C6 alkyl, amino-Ci-C6 alkyl, C1 -C6- alkylamino C1 -C6 alkyl, and di(Ci-C6-alkyl)amino C1 -C6 alkyl. The group may be a terminal group or a bridging group. "Heteroalkyloxy" refers to a heteroalkyl-O- group in which heteroalkyl is as defined herein. Preferably the heteroalkyloxy is a C2-C6 heteroalkyloxy. The group may be a terminal group or a bridging group.

"Heteroaryl" either alone or part of a group refers to groups containing an aromatic ring (preferably a 5 or 6 membered aromatic ring) having one or more heteroatoms as ring atoms in the aromatic ring with the remainder of the ring atoms being carbon atoms. Suitable heteroatoms include nitrogen, oxygen and sulphur. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, naphtho[2,3-b]thiophene, furan, isoindolizine, xantholene, phenoxatine, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, tetrazole, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, phenoxazine, 2-, 3- or 4- pyridyl, 2-, 3-, 4-, 5-, or 8- quinolyl, 1 -, 3-, 4-, or 5- isoquinolinyl 1 -, 2-, or 3- indolyl, and 2-, or 3-thienyl. A heteroaryl group is typically a C1-C18 heteroaryl group. The group may be a terminal group or a bridging group.

"Heteroarylalkyl" means a heteroaryl-alkyl group in which the heteroaryl and alkyl moieties are as defined herein. Preferred heteroarylalkyl groups contain a lower alkyl moiety. Exemplary heteroarylalkyl groups include pyridylmethyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group.

"Heteroarylalkenyl" means a heteroaryl-alkenyl- group in which the heteroaryl and alkenyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.

"Heteroarylheteroalkyl" means a heteroaryl-heteroalkyl- group in which the heteroaryl and heteroalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.

"Heteroaryloxy" refers to a heteroaryl-O- group in which the heteroaryl is as defined herein. Preferably the heteroaryloxy is a Ci -Ci ₈ heteroaryloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Heterocyclic" refers to saturated, partially unsaturated or fully unsaturated monocyclic, bicyclic or polycyclic ring system containing at least one heteroatom selected from the group consisting of nitrogen, sulfur and oxygen as a ring atom. Examples of heterocyclic moieties include heterocycloalkyi, heterocycloalkenyl and heteroaryl. "Heterocycloalkenyl" refers to a heterocycloalkyi group as defined herein but containing at least one double bond. A heterocycloalkenyl group typically is a C2-C12 heterocycloalkenyl group. The group may be a terminal group or a bridging group.

"Heterocycloalkyi" refers to a saturated monocyclic, bicyclic, or polycyclic ring containing at least one heteroatom selected from nitrogen, sulfur, oxygen, preferably from 1 to 3 heteroatoms in at least one ring. Each ring is preferably from 3 to 10 membered, more preferably 4 to 7 membered. Examples of suitable heterocycloalkyi substituents include pyrrolidyl, tetrahydrofuryl, tetrahydrothiofuranyl, piperidyl, piperazyl, tetrahydropyranyl, morphilino, 1 ,3-diazapane, 1 ,4-diazapane, 1 ,4- oxazepane, and 1 ,4-oxathiapane. A heterocycloalkyi group typically is a C2-C12 heterocycloalkyi group. The group may be a terminal group or a bridging group.

"Heterocycloalkylalkyl" refers to a heterocycloalkyl-alkyl- group in which the heterocycloalkyi and alkyl moieties are as defined herein. Exemplary heterocycloalkylalkyl groups include (2-tetrahydrofuryl)methyl,

(2-tetrahydrothiofuranyl) methyl. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkyl group. "Heterocycloalkylalkenyl" refers to a heterocycloalkyl-alkenyl- group in which the heterocycloalkyl and alkenyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the alkenyl group.

"Heterocycloalkylheteroalkyl" means a heterocycloalkyl-heteroalkyl- group in which the heterocycloalkyl and heteroalkyl moieties are as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the heteroalkyl group.

"Heterocycloalkyloxy" refers to a heterocycloalkyl-O- group in which the heterocycloalkyl is as defined herein. Preferably the heterocycloalkyloxy is a C1-C6 heterocycloalkyloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Heterocycloalkenyloxy" refers to a heterocycloalkenyl-O- group in which heterocycloalkenyl is as defined herein. Preferably the heterocycloalkenyloxy is a Ci- C6 heterocycloalkenyloxy. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the oxygen atom.

"Hydroxyalkyi" refers to an alkyl group as defined herein in which one or more of the hydrogen atoms has been replaced with an OH group. A hydroxyalkyi group typically has the formula C_nH(2n+i-x₎(OH)_x. In groups of this type n is typically from 1 to 10, more preferably from 1 to 6, most preferably 1 to 3. x is typically 1 to 6, more preferably 1 to 3.

"Sulfinyl" means an R-S(=O)- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or heteroaryl group as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom. "Sulfinylamino" means an R-S(=0)-NH- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or heteroaryl group as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

"Sulfonyl" means an R-S(=O)2- group in which the R group may be OH, alkyl, cycloalkyl, heterocycloalkyl; aryl or heteroaryl group as defined herein. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the sulfur atom.

"Sulfonylamino" means an R-S(=O)2-NH- group. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom. "N-protecting group" means a group that can prevent the nitrogen atom reacting during further derivatisation of the protected compound and which can be readily removed when desired. Examples of N-protecting groups include alkyl amines, benzyl amines, t-Boc, Alloc, CBz and Fmoc. Further examples of these groups are found in: Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, Second edition; Wiley-lnterscience: 1991 ; Chapter 7; McOmie, J. F. W. (ed.), Protective Groups in Organic Chemistry, Plenum Press, 1973; and Kocienski, P. J., Protecting Groups, Second Edition, Theime Medical Pub., 2000.

"O-protecting group" means a group that can prevent the oxygen atom reacting during further derivatisation of the protected compound and which can be readily removed when desired. Examples of O-protecting groups include silyl ethers (e.g. trimethylsilyl ether, tert-butyldimethylsilyl ether), acetyl, benzoyl, benzyl trityl. Further examples of these groups are found in: Greene, T. W. and Wuts, P. G. M., Protective Groups in Organic Synthesis, Second edition; Wiley-lnterscience: 1991 ; Chapter 7; McOmie, J. F. W. (ed.), Protective Groups in Organic Chemistry, Plenum Press, 1973; and Kocienski, P. J., Protecting Groups, Second Edition, Theime Medical Pub., 2000.

The compounds of the invention include a linking moiety (L¹) which links the moiety containing the cyclooctyne to the second moiety (R² or X⁵-R²). In some embodiments L¹ is a linking group that serves to act as a spacer between the cyclooctyne moiety and the second moiety. The linking group separates the cyclooctyne moiety and second moiety, which can individually react with a functionalised substrate and a functionalised molecule, tethering the substrate and molecule. As such whilst it is desirable that there be a certain degree of separation between the two in order to ensure that the two moieties do not interfere with each other it is also important that the two are not so far removed such that the substrate tethered to the molecule loses application and/or activity.

In some embodiments L¹ is a linking moiety having from 1 to 25 atoms in the normal chain. In some embodiments L¹ is a linking moiety having from 1 to 20 atoms in the normal chain. In some embodiments L¹ is a linking moiety having from 1 to 15 atoms in the normal chain. In some embodiments L¹ is a linking moiety having from 1 to 12 atoms in the normal chain. In some embodiments L¹ is a linking moiety having from 1 to 10 atoms in the normal chain. In some embodiments L¹ is a linking moiety having from 1 to 8 atoms in the normal chain. In some embodiments L¹ has 8 atoms in the normal chain. In some embodiments L¹ has 7 atoms in the normal chain. In some embodiments L¹ has 6 atoms in the normal chain. In some embodiments L¹ has 5 atoms in the normal chain. In some embodiments L¹ has 4 atoms in the normal chain. In some embodiments L¹ has 3 atoms in the normal chain. In some embodiments L¹ has 2 atoms in the normal chain. In some embodiments L¹ has 1 atom in the normal chain. A wide range of possible moieties may be used to create a linking moiety of this type. Examples of suitable moieties that may be used in the creation of L¹ include optionally-substituted C1-C12 alkyl, optionally-substituted C1 -C12 heteroalkyl, optionally-substituted C3-C12 cycloalkyl, optionally-substituted C6-C18 aryl, and optionally-substituted C1-C18 heteroaryl.

In some embodiments, L¹ is of the Formula (II):

Formula (II)

wherein

A is bonded to the cyclopropane group of Formula (I) and A is selected from the group consisting of: a bond and (CR³R⁴)_m;

X¹ , is selected from the group consisting of: a bond, 0, NH, NR⁵, S and CR⁶R⁷; X² is selected from the group consisting of: 0 and S;

L² is bonded to the R² group and is of the Formula X³(CR⁸R⁹)_p [X⁴(CR¹⁰R^{1 1} )_q]_r (CR¹²R¹³)_S X⁵

Formula (III)

X³ is selected from the group consisting of: a bond, 0, NH, NR¹⁴, S and

_{C R}15_R16.

X⁴ is selected from the group consisting of: a bond, 0, NH, NR¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, 0, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted alkyl, HN-(optionally-substitute alkyl)-, R¹⁴N- (optionally-substituted

alkyl)-, 0-(optionally-substituted kyl)-, optionally- substituted C_5-12 aryl, HN-(optionally-substituted C_5-i₂ aryl)-, R¹⁴N-(optionally- substituted C_5-i₂ aryl)-, 0-(optionally-substituted C_5-i₂ aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R¹⁴N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted 05-12 aryl)-, (CO)NH-(optionally-substituted Cs-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C_2-12 heteroaryl, HN-(optionally-substituted C_2-12 heteroaryl)-, R¹⁴N-(optionally-substituted 02-12 heteroaryl)-, 0-(optionally-substituted 02-12 heteroaryl)-, optionally-substituted 02-12 heteroaryl, HN(CO)-(optionally-substituted C2- 12 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted 02-12 heteroaryl)-, 0(CO)-(optionally- substituted C_2-12 heteroaryl)-, (CO)HN-(optionally-substituted C_2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted 02-12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-; each R⁵ and R¹⁴ is independently selected from the group consisting of: optionally-substituted

alkyl and N-protecting group;

each R³, R⁴, R⁶, R⁷, R⁸ R⁹ R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-12 alkyl, optionally-substituted C_2-12 alkenyl, optionally-substituted C_2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally- substituted OC_3-i2 cycloalkenyl;

n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; m is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; and each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. This provides compounds of Formula (ll-a):

Formula (ll-a)

wherein

each R is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1-12 alkyl, optionally-substituted

haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_2-12 heteroalkyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl, optionally-substituted 02-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-M S heteroaryl, optionally-substituted Ci-12 alkyloxy, optionally-substituted C_2-12 alkenyloxy, optionally-substituted C_2-12 alkynyloxy, optionally-substituted C_2-12 heteroalkyloxy, optionally-substituted C_3-i₂ cycloalkyloxy, optionally-substituted C3-12 cycloalkenyloxy, optionally-substituted 02-12 heterocycloalkyloxy, optionally-substituted 02-12 heterocycloalkenyloxy, optionally- substituted Ce-18 aryloxy, optionally-substituted Ci-ie heteroaryloxy, optionally- substituted C_{1 -12} alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d, or any two R¹ on adjacent carbon atoms form a fused substituent;

A is selected from the group consisting of: a bond and (CR³R⁴)_m;

X¹ , is selected from the group consisting of: a bond, O, N H, NR⁵, S and CR⁶R⁷; X² is selected from the group consisting of: O and S;

L² is of the Formula (III): X³(CR⁸R⁹)_p [X⁴(CR^{1 0}R^{1 1} )_q]_r (CR¹²R^{1 3})_S X⁵

Formula (III)

R² is selected from the group consisting of: optionally substituted C-M2 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C_3-i₂ cycloalkyl, optionally substituted C_3-i₂ cycloalkenyl, NH₂, ONH₂, NHNH₂, NHNR^aR^p, NH(CO) (optionally-substituted C_{1 -6} alkyl)ONH₂, NH(CO) (optionally-substituted Ci_-6 alkyl)NH₂, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNH₂, NH(CO) (optionally-substituted C_{1 -6} alkyl)NHNR^aR^p, (CO)NH (optionally-substituted Ci_-6 alkyl)ONH₂, (CO)NH (optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH (optionally-substituted Ci_-6 alkyl)NHNH₂, (CO)NH (optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNH₂, (CO)N HNR^aR^p, N H(CO)NHN H_2, NH(CO)NHNR^aR^p, NHNH(CO)NHNH₂ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC_2-i2 alkynyl, optionally-substituted OC_3-i₂ cycloalkyl and optionally-substituted OC_3-i₂ cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

X³ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S and

_{C R}15_R16.

X⁴ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted

alkyl, HN-(optionally-substituted Ci-12 alkyl)-, R¹⁴N- (optionally-substituted Ci-i₂ alkyl)-, O-(optionally-substituted Ci-i₂ alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted Cs-12 aryl)-, R¹⁴N-(optionally- substituted Cs-12 aryl)-, O-(optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally- substituted C_5-i₂ aryl)-, R¹⁴N(CO)-(optionally-substituted C_5-i₂ aryl)-, O(CO)- (optionally-substituted C_5-i₂ aryl)-, (CO)NH-(optionally-substituted C_5-i₂ aryl)-, (CO)R¹⁴N-(optionally-substituted C_5-i₂ aryl)-, (CO)O-(optionally-substituted C_5-i₂ aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C_2-12 heteroaryl, HN(CO)-(optionally-substituted C_2- 12 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C2-12 heteroaryl)-, O(CO)-(optionally- substituted C2-12 heteroaryl)-, (CO)HN-(optionally-substituted C2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C2-12 heteroaryl)- and (CO)O-(optionally-substituted C_2-12 heteroaryl)-;

each R⁵ and R¹⁴ is independently selected from the group consisting of: optionally-substituted Ci-12 alkyl and N-protecting group;

each R³, R⁴, R⁶, R⁷, R⁸ R⁹ R¹⁰, R^{1 1} , R¹², R^{1 3}, R¹⁵ and R¹⁶ is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-i₂ alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted 0C_2-12 alkynyl, optionally-substituted OC_3-i ₂ cycloalkyl and optionally- substituted OC3-12 cycloalkenyl;

n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; m is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; and each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.

A skilled addressee will recognise that due to the difficulty of preparation of pure samples having precisely one value of p, q, r and n, it is often necessary to use as a reagent mixture which provides molecules with various values of p, q, r and n. In some embodiments the present invention includes mixtures of compounds wherein the value of p may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of q may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of r may vary within the mixture of compounds. In some embodiments the present invention includes mixtures of compounds wherein the value of s may vary within the mixture of compounds.

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-a):

Formula (X-a) In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-b):

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-c):

Formula (X-c)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-d):

Formula (X-d)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-e):

Formula (X-e)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-f):

Formula (X-f) In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-g):

Formula (X-g)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-h):

Formula (X-h)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-i):

Formula (X-i) In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-k):

Formula (X-k)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-l):

Formula (X-l) In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-m):

Formula (X-m)

In some embodiments, A, X¹ , X² and L² are chosen and combined such that L¹ is of Formula (X-n):

Formula (X-n)

In some embodiments n is 0. In some embodiments R^a and R^b are independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted

alkyl, and optionally-substituted OCi-12 alkyl. In some embodiments R^a and R^b are independently selected from the group consisting of: hydrogen, OH,

alkyl, and OC-i-12 alkyl. In some embodiments R^a and R^b are hydrogen.

In some embodiments A is a bond. In some embodiments A is (CR³R⁴)_m. In some embodiments m is 1 , in some embodiments m is 2, in some embodiments m is 3, in some embodiments m is 4, in some embodiments m is 5, in some embodiments m is 6. In some embodiments R³ and R⁴ are each H.

In some embodiments A is CH₂. This provides compounds of Formula (ll-b):

Formula (ll-b)

wherein X¹, X², X³, X⁴, X⁵, R¹ , R^a, R^b, R² R⁸ R⁹ R¹⁰, R¹¹, R¹², R¹³, n, p, q, r and s are as discussed above.

In some embodiments A is CH₂ and X¹ is O. This provides a compound of Formula (ll-c):

Formula (ll-c) wherein X², X³, X⁴ X⁵ R¹ , R^a, R^b R² R⁸, R⁹ R¹⁰, R¹¹ , R¹², R¹³, n, p, q, r and s are as discussed above.

In some embodiments A is CH₂ and X¹ is O and X² is O. This provides a compound of Formula (ll-d):

Formula (ll-d) wherein X³, X⁴ X⁵, R¹, R^a, R^b R² R⁸, R⁹, R¹⁰, R¹¹ , R¹², R¹³, n, p, q, r and s are as discussed above. In some embodiments X³ is O or NH. In some embodiments X¹, X² and X³ are selected to form a carbonate group such as -O(C=O)O-. In some embodiments X¹ , X² and X³ are selected to form a carbamate group such as -NH(C=O)O- or -O(C=O)NH-. In some embodiments X¹, X² and X³ are selected to form an amide link such as -(C=O)NH- or -NH(C=O)-. In some embodiments A is CH₂ and X¹ is 0 and X² is 0 and X³ is NH such that L² has the Formula (lll-b): NH(CR⁸R⁹)_p [X⁴(CR¹⁰R¹¹)_q]_r (CR¹²R ³)_S X⁵

Formula (lll-b) wherein a compound of Formula (ll-e) is provided:

Formula (ll-e): wherein X⁴, X⁵, R¹, R^a, R^b R² R⁸, R⁹ R¹⁰ R¹¹, R¹², R¹³, n, p, q, r and s are as discussed above.

In some embodiments R⁸, R⁹, R¹², and R¹³ are H. In some embodiments R¹⁰ and R¹¹ are H.

In some embodiments p is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments p is 0, in some embodiments p is 1 , in some embodiments p is 2, in some embodiments p is 3, in some embodiments p is 4, in some embodiments p is 5, in some embodiments p is 6.

In some embodiments q is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments q is 0, in some embodiments q is 1 , in some embodiments q is 2, in some embodiments q is 3, in some embodiments q is 4, in some embodiments q is 5, in some embodiments q is 6. In some embodiments r is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments r is 0, in some embodiments r is 1 , in some embodiments r is 2, in some embodiments r is 3, in some embodiments r is 4, in some embodiments r is 5, in some embodiments r is 6.

In some embodiments s is an integer independently selected group the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. In some embodiments s is 0, in some embodiments s is 1 , in some embodiments s is 2, in some embodiments s is 3, in some embodiments s is 4, in some embodiments s is 5, in some embodiments s is 6.

In some embodiments X⁴ is O. In some embodiments X⁴ is NH.

In some embodiments A is CH₂, X¹ is O, X² is 0, and R⁸, R⁹, R¹⁰ and R¹¹ are each H, X³ is NH, p = 2, X⁴ is 0, q = 2, r = 2 and s = 0 such that L² has the Formula (lll-c):

Formula (lll-c)

wherein X⁵, R¹, R^a, R^b, R² and n are as discussed above. In some embodiments A is CH₂, X¹ is 0, X² is 0, and R⁸, R⁹, R¹⁰ and R¹¹ are each H, X³ is NH, p = 2, X⁴ is 0, q = 2, r = 3 and s = 0 such that L² has the Formula (lll-d):

Formula (lll-d) wherein the compound of Formula (ll-g) is provided:

Formula (ll-g) wherein X⁵, R¹, R^a, R^b R² and n are as discussed above.

In some embodiments A is CH₂, X¹ is 0, X² is 0, and R⁸ R⁹ R¹⁰ R¹¹ , R¹² and R¹³ are each H, X³ is NH, p = 3, X⁴ is 0, q = 2, r = 3, s = 1 such that L² has the Formula (III- e):

Formula (lll-e) wherein the compound of Formula (ll-h) is provided:

Formula (ll-h) wherein X⁵, R¹, R^a, R^b, R² and n are as discussed above.

In some embodiments A is CH₂, X¹ is 0, X² is 0, and R⁸, R⁹, R¹⁰ and R¹¹ are each H, X³ is NH, p = 3, X⁴ is 0, q = 2, r = 2, s = 0 such that L² has the Formula (lll-i):

Formula (lll-i) wherein the compound of Formula (ll-j) is provided:

Formula (ll-j) wherein X⁵, R¹, R^a, R^b, R² and n are as discussed above. In some embodiments X⁵ is 0. In some embodiments X⁵ is NH, In some embodiments X⁵ is optionally-substituted Ci--i₂ alkyl. In some embodiments X⁵ is HN- (optionally-substituted Ci--i₂ alkyl)-. In some embodiments X⁵ is R¹⁴N-(optionally- substituted Ci-12 alkyl)-. In some embodiments X⁵ is 0-(optionally-substituted Ci-12 alkyl)-. In some embodiments X⁵ is HN(CO)-(optionally-substituted C_5-i₂ aryl). In some embodiments X⁵ is HN(CO)-(optionally-substituted C2-12 heteroaryl)-.

In some embodiments each R¹ is independently selected from the group consisting of H, OH, C_1-6 alkyl and OC_1-6 Alkyl.

In some embodiments R² is an amine (-NH₂). In some embodiments R² is an aminooxy (-ONH₂). In some embodiments R² is a hydrazine (-NHNH₂). In some embodiments R² is a hydrazide (-(CO)NHNH₂). In some embodiments R² is a semicarbazide (-NH(CO)NHNH₂). In some embodiments R² is a carbohydrazide (-NHNH(CO)NHNH₂). In some embodiments R² is an optionally-substituted C₃-i₂ cycloalkenyl. In some embodiments R² is an optionally-substituted C₃-i₂ cycloalkenyl, wherein the optionally-substituted C_3-i₂ cycloalkenyl is substituted with 1 or 2 =0

groups. In some embodiments R² is

In some embodiments the compound is of the Formula (IV):

Formula (IV)

wherein

each R^a and R^b is independently selected from the group consisting of: H, OH, halogen, optionally-substituted

alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally- substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally- substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;

each R¹ is independently selected from the group consisting of: H, halogen,

OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1.12 alkyl, optionally-substituted

haloalkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted C_3-12 cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted C_2-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-M S heteroaryl, optionally-substituted Ci-12 alkyloxy, optionally-substituted 02-12 alkenyloxy, optionally-substituted 02-12 alkynyloxy, optionally-substituted C_2-12 heteroalkyloxy, optionally-substituted C_3-i₂ cycloalkyloxy, optionally-substituted C_3-12 cycloalkenyloxy, optionally-substituted 02-12 heterocycloalkyloxy, optionally-substituted 02-12 heterocycloalkenyloxy, optionally- substituted Ce-18 aryloxy, optionally-substituted CM S heteroaryloxy, optionally- substituted C1-12 alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d; or any two R¹ on adjacent carbon atoms form a fused substituent; L¹ is as defined above;

each R^C, R^D and R^E is independently selected from the group consisting of: H, OH, halogen, optionally-substituted Ci-i₂ alkyl, optionally-substituted C_2-12 heteroalkyl, optionally-substituted C3-₁₂ cycloalkyl, optionally-substituted OC6-18 aryl, and optionally-substituted OCM S heteroaryl.

R^2A is selected from the group consisting of: OH, optionally-substituted OCi-₁₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC3-₁₂ cycloalkyl and optionally-substituted OC3-₁₂ cycloalkenyl.

In some embodiments, L¹ is of the Formula (II):

Formula (II)

wherein

X¹ , is selected from the group consisting of: a bond, O, NH, NR⁵, S and CR⁶R⁷;

X² is selected from the group consisting of: O and S;

L² is bonded to the squarate group and is of the Formula (III): X³(CR⁸R⁹)_p [X⁴(CR¹⁰R^{1 1})_q]_r (CR¹²R¹³)_S X⁵

Formula (III) to provide a compound of Formula (IV-a):

Formula (IV-a) wherein X³, X⁴, X⁵, R¹, R^a, R^b R^2a, R⁸ R⁹, R¹⁰ R¹¹, R¹² R¹³, n, p, q, r and s are as discussed above.

In some embodiments the compounds of the present invention include:

It is understood that included in the family of compounds of Formula (I) and Formula (II) are isomeric forms including diastereoisomers, enantiomers, tautomers, and geometrical isomers. It is also understood that some isomeric forms such as diastereomers, enantiomers, and geometrical isomers can be separated by physical and/or chemical methods by those skilled in the art. For those compounds where there is the possibility of geometric isomerism the applicant has drawn the isomer that the compound is thought to exist as although it will be appreciated that the other isomer may be the correct structural assignment. Some of the compounds of the disclosed embodiments may exist as single stereoisomers, racemates, and/or mixtures of enantiomers and/or diastereomers. All such single stereoisomers, racemates and mixtures thereof, are intended to be within the scope of the subject matter described and claimed.

Additionally, the Formulae illustrated herein are intended to cover, where applicable, solvated as well as unsolvated forms of the compounds. Thus, each formula includes compounds having the indicated structure, including the hydrated as well as the non- hydrated forms.

In other embodiments of the invention, the compound may find a multiple number of applications in which the compound can be used to link nanoparticles to biological molecules. For example, in one embodiment a quantum dot (QD) is linked to a biological molecule, wherein a functionalised QD and a functionalised biological molecule are linked to each end of the previously described compound. The compounds of the present invention possess a cyclooctyne functional group at one end and another functional group at the other end, which is capable of reacting with an amine, aldehyde or ketone group. Examples of functional groups that are capable of reacting with an aldehyde or ketone groups may include amine, aminooxy, hydrazide, semicarbazide and carbohydrazide functional groups. Examples of functional groups that are capable of reacting with an amine may include a squarate. These functional groups provide means to link the functionalised QD and the functionalised biological molecule by reacting the functional groups of the compound with the functionalised QD and/or functionalised biological molecule.

In some embodiments the invention relates to a process of linking a quantum dot (QD) to a biological molecule, the process comprising the step: i) reacting an end of the compound as previously described, with either a functionalised QD or a functionalised biological molecule, linking either the functionalised QD or the functionalised biological molecule to a first end of the compound; and step ii) reacting the remaining end group from the product of step i), with either one of the functionalised QD or the functionalised biological molecule which was not used in step i) to provide a QD linked to a biological molecule. In some embodiments step i) comprises reacting the end of the compound which contains the alkyne group, with a functionalised QD, linking the QD to a first end of the compound; and step ii) comprises reacting the end of the product from step i) which does not contain an alkyne group with a functionalised biological molecule, linking the biological molecule to a second end.

In some embodiments step i) comprises reacting the end of the compound which does not contain the alkyne functional group, with a functionalised biological molecule, linking the biological molecule to a first end of the compound; and step ii) comprises reacting the end of the product from step i) which contains the alkyne group with a functionalised QD, linking the QD to a second end.

In some embodiments the functionalised QD is an azide-functionalised QD. In some embodiments the functionalised biological molecule is an amine-, aldehyde- or ketone-functionalised biological molecule.

In one embodiment a QD can be linked to the biological molecule by reacting a functionalised QD with one end of the compound. This results in a QD which is linked to the compound at one end. The product can be reacted with a functionalised biological molecule, linking the biological molecule to the opposite end of the compound.

In another embodiment the QD can be linked to the biological molecule by reacting a functionalised biological molecule with one end of the compound. This results in a biological molecule which is linked to the compound at one end. The product can be reacted with a functionalised QD, linking the QD to the opposite end of the compound.

For example, the cyclooctyne moiety can react with an either an azide-functionalised QD or an azide-functionalised biological molecule. Typically the cyclooctyne moiety will react with the azide-functionalised QD or azide-functionalised biological molecule via a [3+2] cycloaddition reaction. The amine, aminooxy, hydrazide, semicarbazide or carbohydrazide moiety of the compound can react with an aldehyde- or ketone- functionalised QD or an aldehyde- or ketone-functionalised biological molecule. Similarly, a squarate moiety of the compound can react with an amine-functionalised QD or an amine-functionalised biological molecule. The compound, therefore, acts as a tether, covalently linking the QD to the biological molecule. In some embodiments the cyclooctyne moiety reacts with an azide-functionalised QD and in some embodiments the squarate, amine, aminooxy, hydrazide, semicarbazide or carbohydrazide moiety reacts with an amine-, aldehyde- or ketone-functionalised biological molecule.

An example is illustrated below in Scheme 1. The embodiment shown in Scheme 1 the cyclooctyne moiety reacts with an azide-functionalised QD and the aminooxy moiety reacts with an aldehyde-functionalised biological molecule. As will be appreciated the same approach could also be carried out using a ketone rather than an aldehyde.

Scheme 1

Another example is illustrated below in Scheme 2. The embodiment shown in Scheme 2 the cyclooctyne moiety reacts with an azide-functionalised QD and the hydrazine moiety reacts with an aldehyde-functionalised biological molecule. As will be appreciated the same approach could also be carried out using a ketone rather than an aldehyde. Also, as shown in the results in Figure 18, the same result can be achieved using the hydrazone-protected version of the hydrazine group, for example the linker of example 8. Strain -promoted

Scheme 2

Another example is illustrated below in Scheme 3. The embodiment shown in Scheme 3 the cyclooctyne moiety reacts with an azide-functionalised QD and the squarate moiety reacts with an amine-functionalised biological molecule.

Strain-promoted

Scheme 3

Another example is illustrated below in Scheme 4. The embodiment shown in Scheme 4 the cyclooctyne moiety reacts with an azide-functionalised QD and the hydrazine moiety reacts with an aldehyde-functionalised biological molecule. As will be appreciated the same approach could also be carried out using a ketone rather than an aldehyde.

Scheme 4

Another example is illustrated below in Scheme 5. The embodiment shown in Scheme 5 the cyclooctyne moiety reacts with an azide-functionalised biological molecule and the squarate moiety reacts with an amino-functionalised quantum dot.

linkage

QDs

Scheme 5 Process for providing compounds of Formula (V)

The compounds of Formula (V) as used in the process of the present invention may be synthesized using a number of synthetic routes.

In one embodiment the invention provides a process for the preparation of a compound of Formula (V). The process comprises coupling the compound of Formula (VI):

Formula (VI) wherein

LG is a leaving group, R¹ and n are as defined above, in a coupling reaction with a compound of Formula (Vll-a):

X^3a(CR⁸R⁹)_p [X⁴(CR^{1 0}R^{1 1})_q]_r (CR¹²R^{1 3})_S X^5a

Formula (Vll-a)

wherein

X^3a is selected from the group consisting of: OH, NH₂ and NHR¹⁴;

X^5a is selected from the group consisting of: OH, NH₂, NHR¹⁴, OR¹⁸ CO₂R¹⁹; each R⁸ R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted Ci--i₂ alkyl, optionally-substituted C_2-i₂ alkenyl, optionally-substituted C_2-i₂ alkynyl, optionally- substituted C_3-i₂ cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally- substituted OCi-i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC3-i₂ cycloalkyl and optionally-substituted Ο0_3- i₂ cycloalkenyl;

R¹⁴ is independently selected from the group consisting of: optionally- substituted C1-12 alkyl and N-protecting group;

R¹⁸ is selected from the group consisting of: optionally-substituted C1-12 alkyl, optionally-substituted C_2-i₂ alkenyl, optionally-substituted C_2-i₂ alkynyl, optionally- substituted C_3-i₂ cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl and O-protecting group; R¹⁹ is selected from the group consisting of: H, optionally-substituted C^ .^2 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl and COOH-protecting group; and

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6;

to provide a compound of Formula (VIII):

Formula (VIII)

The compound of Formula (VI) is typically reacted, in the presence of a base, with the compound of Formula (Vll-a).

The solvent is chosen so as not to be reactive with the base. Examples of suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents. Specific examples of suitable solvents include dichloromethane (CH2CI2), chloroform (CCI3), ethanol, methanol, DMSO and DMF. In general a base is used. In some instances the amine of Formula (Vll-a) may also be used as a base. Examples of suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides. Specific examples of suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine. In some instances the base may also act as a solvent.

The amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents

The reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.

The reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR.

The product of Formula (VIII) can then be reacted with a compound of Formula (IX):

R²⁰ B R²

Formula (IX)

wherein

B is selected from the group consisting of: a bond, an optionally-substituted d. 12 alkyl group, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally-substituted 05-12 aryl group and optionally-substituted 02-12 heteroaryl group, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl; each R² and R²⁰ is selected independently from the group consisting of: OH, CO2R²², optionally-substituted OC1-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl, optionally-substituted OC3-12 cycloalkenyl, NHR¹⁷, NHNHR¹⁷, NHNR^aR^p, ONHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHNHR¹⁷, NH(CO)(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷, and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted

alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i2 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted Ci-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C_3-12 cycloalkenyl;

each R¹⁷ and R²¹ is independently selected from the group consisting of: H, optionally-substituted Ci-12 alkyl and N-protecting group;

R is selected from the group consisting of: H, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted C_2-12 alkenyl, optionally-substituted C_2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted C2-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted Ce-18 alkylaryl, optionally-substituted

heteroaryl, optionally-substituted Ci-i₈ alkylheteroaryl to provide a compound of Formula (V):

Formula (V)

wherein

each R¹ is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted C1-12 alkyl, optionally-substituted Ci--i₂ haloalkyl, optionally-substituted C_2-i₂ alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_2-12 heteroalkyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted C3-i₂ cycloalkenyl, optionally-substituted C_2-i₂ heterocycloalkyl, optionally-substituted C_2-i₂ heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted C-MS heteroaryl, optionally-substituted C1-12 alkyloxy, optionally-substituted C_2-i₂ alkenyloxy, optionally-substituted C_2-i₂ alkynyloxy, optionally-substituted C_2-i₂ heteroalkyloxy, optionally-substituted C3-i₂ cycloalkyloxy, optionally-substituted C3-i₂ cycloalkenyloxy, optionally-substituted C_2-i₂ heterocycloalkyloxy, optionally-substituted C_2-i₂ heterocycloalkenyloxy, optionally- substituted C_6-18 aryloxy, optionally-substituted Ci-i₈ heteroaryloxy, optionally- substituted C1-12 alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d or any two R¹ on adjacent carbon atoms form a fused substituent;

R^c, R^d and R^e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted Ci_i₂ alkyl, optionally-substituted C_2-i₂ heteroalkyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OCM S heteroaryl;

L is of the Formula (III):

— X³(CR⁸R⁹)_p [X⁴(CR¹⁰R^{1 1})_q]_r (CR¹²R¹³)_S—

Formula (III)

selected from the group consisting of: NH, O and NR¹⁴; X⁴ is independently selected from the group consisting of: a bond, O, NH, NR¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: O, NH, NR¹⁴, optionally-substituted C-i-12 alkyl, HN-(optionally-substituted C-i-12 alkyl)-, R¹⁴N-(optionally-substituted C-i-12 alkyl)- O-(optionally-substituted Ci-i₂ alkyl)-, optionally-substituted C_5-i₂ aryl, HN- (optionally-substituted Cs-12 aryl)-, R¹⁴N-(optionally-substituted Cs-12 aryl)-, O- (optionally-substituted Cs-12 aryl)-, HN(CO)-(optionally-substituted Cs-12 aryl)-, R¹⁴N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)-(optionally-substituted C5-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted Cs-12 aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C2-12 heteroaryl, HN(CO)-(optionally-substituted C_2-12 heteroaryl)-, R¹⁴N(CO)-(optionally- substituted C2-12 heteroaryl)-, O(CO)-(optionally-substituted C2-12 heteroaryl)-, (CO)HN-(optionally-substituted C2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C_2- 12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_₆ alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_₆ alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R⁸, R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted OC2-12 alkynyl, optionally-substituted OC_3-i2 cycloalkyl and optionally- substituted OC_3-i2 cycloalkenyl;

alkyl and N-protecting group;

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6. The compound of Formula (VIII) is reacted with a compound of Formula (IX) in the presence of a base. The solvent is chosen so as not to be reactive with the base. Examples of suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents. Specific examples of suitable solvents include dichloromethane (CH2CI2), chloroform (CCI3), ethanol, methanol, DMSO and DMF.

In general a base is used. Examples of suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides. Specific examples of suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine. In some instances the base may also act as a solvent. The amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents

The reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR. In some embodiments of the invention the compound of Formula (IX) is of Formula

(IX-a):

Formula (IX-a)

wherein

R¹⁷ is selected from the group consisting of: optionally-substituted Ci-i₂ alkyl and N-protecting group.

In some embodiments of the invention the compound of Formula (IX) is of Formula (IX-b):

Formula (IX-b)

wherein

R¹⁷ is selected from the group consisting of: optionally-substituted

alkyl and N-protecting group.

In some embodiments of the invention the compound of Formula (IX) is of Formula (IX-c):

Formula (IX-c)

wherein

In some embodiments of the invention the compound of Formula (IX) is of Formi. (IX-d):

Formula (IX-d)

wherein

R^2a is selected from the group consisting of: OH, optionally-substituted OCi-₁₂ alkyl, optionally-substituted OC_2-i2 alkenyl, optionally-substituted OC_2-i 2 alkynyl, optionally-substituted OC3-₁₂ cycloalkyl and optionally-substituted OC3-₁₂ cycloalkenyl.

In some embodiments of the invention the compound is of Formula (IX-d), wherein R^2a is OMe. In some embodiments of the invention the compound is of Formula (IX- d), wherein R^2a is OMEt. In some embodiments of the invention the compound of Formula (IX) is of Formula (IX-e):

Formula (IX-e)

In some embodiments the compound produced in step b) is further N-deprotected or N-dealkylated.

In some embodiments the compound of Formula (IX) is hydrazine (NH₂NH₂).

In one embodiment of the invention the compound of Formula (Vll-a) is 4,7,10-trioxa- 1 , 13-tridecanediamine of Formula (Vll-c):

H_?N ^'

Formula (Vll-c)

In one embodiment of the invention the compound of Formula (Vll-a) is the compound of Formula (Vll-d):

Formula (Vll-d)

In one embodiment of the invention the compound of Formula (Vll-a) is the compound of Formula (Vll-e):

Formula (Vll-e)

In some embodiments the compound of Formula (VI) is selected from:

wherein LG is a leaving group, selected independently from the group consisting of:

halogen,

In some embodiments the compound of Formula (VI) is selected from:

The above compounds can be obtained by reacting either:

with a 4-nitrophenyl chloroformate.

The solvent is chosen so as not to be reactive with the base. Examples of suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents. Specific examples of suitable solvents include dichloromethane (CH₂CI₂), chloroform (CCI₃), ethanol, methanol, DMSO and DMF.

The reaction of 4-nitrophenyl chloroformate can be conducted in the presence of a base. Examples of suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides. Specific examples of suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine. In some instances the base may also act as a solvent.

The amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents The reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However, the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C. The reaction is typically conducted until analysis of the mixture shows consumption of starting material. This typically takes from 5 minutes to 24 hours, more typically from 15 minutes to 8 hours, more typically from 30 minutes to 1 hour. As will be appreciated, however, it is typically quite routine for a skilled addressee to monitor the reaction. Some methods of monitoring a reaction might include TLC, HPLC, mass spectrometry and NMR.

Syntheses of linker compound

A process for the preparation of a compound of the present invention, endo-5, may include the process illustrated in Scheme 6:

endo-5

Scheme 6 Synthesis of the compounds, exo-1 and endo-1 are known in the literature. As such the exo-1 and endo-1 compounds can be synthesised as described by Dommerholt J. et al. (see Scheme 7 which depicts synthesis of the exo-isomer). Alternative methodologies to provide either exo-1 or endo-1 can also be employed.

Another process for the preparation of a compound of the present invention, endo-7, ma include the process illustrated in Scheme 8:

Scheme 8

Yet another process for the preparation of a compound of the present invention, endo-9, may include the process illustrated in Scheme 9:

Scheme 9

Another process for providing compounds of Formula (V)

In another embodiment the invention provides another process for the preparation of a compound of Formula (V). The process comprises coupling a compound of Formula (Vll-b):

X^3a(CR⁸R⁹)p [X⁴(CR^{1 0}R^{1 1} )_q]_r (CR¹²R^{1 3})_S X⁵ R²

Formula (Vll-b)

wherein

X^3a is selected from the group consisting of: OH, NH₂ and NHR¹⁴;

X⁴ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted alkyl, HN-(optionally-substituted Ci-12 alkyl)-, R¹⁴N- (optionally-substituted Ci-i₂ alkyl)-, O-(optionally-substituted Ci-i₂ alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted Cs-12 aryl)-, R¹⁴N-(optionally- substituted C_5-i₂ aryl)-, O-(optionally-substituted C_5-i₂ aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)- R¹⁴N(CO)-(optionally-substituted C5-12 aryl)- O(CO)- (optionally-substituted Cs-12 aryl)-, (CO)NH-(optionally-substituted Cs-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)- (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C_2-12 heteroaryl, HN-(optionally-substituted C_2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C2-12 heteroaryl, HN(CO)-(optionally-substituted C_2- 12 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C2-12 heteroaryl)-, O(CO)-(optionally- substituted C_2-i₂ heteroaryl)-, (CO)HN-(optionally-substituted C_2-i₂ heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C2-12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C1-12 alkyl, optionally substituted C_2-i₂ alkenyl, optionally substituted C_2-i₂ alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted C1- 2 alkyl, optionally-substituted C_2-i₂ alkenyl, optionally- substituted C_2-i₂ alkynyl, optionally-substituted C3-i₂ cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted OCi_i₂ alkyl, optionally-substituted OC_2-i₂ alkenyl, optionally-substituted OC_2-i₂ alkynyl, optionally-substituted OC3-i₂ cycloalkyl and optionally-substituted OC3-i₂ cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted Ci-i₂ alkyl and N-protecting group; and

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, and in a coupling reaction with a compound of Formula of Formula (VI):

Formula (VI)

wherein

R¹ and n are as defined above and LG is a leaving group;; to provide the compound of Formula V):

Formula (V)

wherein R¹ is selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF3, optionally-substituted C1-12 alkyl, optionally-substituted Ci-12 haloalkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted 02-12 heteroalkyl, optionally-substituted 03-12 cycloalkyl, optionally-substituted C_3-i₂ cycloalkenyl, optionally-substituted C_2-12 heterocycloalkyl, optionally-substituted 02-12 heterocycloalkenyl, optionally-substituted Ce-18 aryl, optionally-substituted CM S heteroaryl, optionally-substituted Ci-12 alkyloxy, optionally- substituted C2-12 alkenyloxy, optionally-substituted 02-12 alkynyloxy, optionally- substituted C_2-12 heteroalkyloxy, optionally-substituted C_3-i₂ cycloalkyloxy, optionally- substituted 03-12 cycloalkenyloxy, optionally-substituted 02-12 heterocycloalkyloxy, optionally-substituted C2-12 heterocycloalkenyloxy, optionally-substituted Ce-18 aryloxy, optionally-substituted C1-18 heteroaryloxy, optionally-substituted C1-12 alkylamino, SR^C, SO3H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d or any two R¹ on adjacent carbon atoms form a fused substituent ;

R^c, R^d and R^e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C1-12 alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OCe-18 aryl, and optionally-substituted OC1-18 heteroaryl;

L is of the Formula (III): X³(CR⁸R⁹)p [X⁴(CR¹⁰R^{1 1} )_q]_r (CR¹²R¹³)s X⁵

Formula (III)

X³ is selected from the group consisting of: O, NH and NR¹⁴;

X⁴ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S and

_{C R}15_R16.

X⁵ is selected from the group consisting of: a bond, O, NH, NR¹⁴, S, CR¹⁵R¹⁶, optionally-substituted C1-12 alkyl, HN-(optionally-substituted C1-12 alkyl)-, R¹⁴N- (optionally-substituted C1-12 alkyl)-, O-(optionally-substituted C1-12 alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted C5-12 aryl)-, R¹⁴N-(optionally- substituted C5-12 aryl)-, O-(optionally-substituted C5-12 aryl)-, HN(CO)-(optionally- substituted C5-12 aryl)-, R¹⁴N(CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)- (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C2-12 heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C2-12 heteroaryl, HN(CO)-(optionally-substituted C2- 2 heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C_2-12 heteroaryl)-, O(CO)-(optionally- substituted C2-12 heteroaryl)-, (CO)HN-(optionally-substituted C2-12 heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C2-12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

selected from the group consisting of: optionally substituted Ci optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted 03-12 cycloalkyl, optionally substituted 03-12 cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted C-i-i2 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted C-i-i2 alkyl, optionally-substituted 02-12 alkenyl, optionally- substituted C_2-12 alkynyl, optionally-substituted C_3-i₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl; each R³, R⁴, R⁶, R⁷, R⁸ R⁹ R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted C-i-12 alkyl, optionally-substituted 02-12 alkenyl, optionally-substituted 02-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCi-i₂ alkyl, optionally-substituted OC_2-i 2 alkenyl, optionally- substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally- substituted OC3-12 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted Ci-i ₂ alkyl and N-protecting group; and

n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6.

The compound of Formula (VI) is typically reacted with the compound of Formula (VII- a) or Formula (Vll-b) in the presence of a base. The solvent is chosen so as not to be reactive with the base. Examples of suitable bases include chlorinated solvents, alcohols and dipolar aprotic solvents. Specific examples of suitable solvents include dichloromethane (CH₂CI₂), chloroform (CCI₃), ethanol, methanol, DMSO and DMF. In general a base is used. In some instances the compound of Formula (Vll-a) or Formula (Vll-b) may also be used as a base. Examples of suitable bases include amine bases, alkali earth metal carbonates, alkali earth metal acetates and alkali earth metal hydroxides. Specific examples of suitable bases include sodium carbonate, potassium carbonate, caesium carbonate, sodium hydrogen carbonate, potassium hydrogen carbonate, sodium hydroxide, potassium hydroxide, sodium acetate, potassium acetate, triethylamine, diisopropylamine, diisopropylethylamine, pyridine and piperidine. In some instances the base may also act as a solvent.

The amount of base chosen will depend on the desired speed of reaction but is chosen to ensure consumption of starting material is achieved. In general therefore an excess of base on a molar equivalent is used. Typically the amount of base used is from 1 to 5 molar equivalents, more typically 1 to 4 molar equivalents, more typically 1 to 3 molar equivalents, more typically 1 to 2 molar equivalents, more typically 1 to 1 .5 molar equivalents, more typically 1 to 1 .2 molar equivalents The reaction may be carried out at any suitable temperature although it is typically conducted at room temperature. However the reaction may also be conducted at from 0°C to 150°C, more typically from 15°C to 80°C, more typically from 20°C to 35°C.

The product can then be optionally deprotected or dealkylated to provide the product of Formula (V).

Synthesis of linker compound

A process for the preparation of a compound of the present invention, endo-11 , may include the process illustrated in Scheme 10:

endo-11

Scheme 10

EXAMPLES

The agents of the various embodiments may be prepared using the reaction routes and synthesis schemes as described below, employing the techniques available in the art using starting materials that are readily available. The preparation of particular compounds of the embodiments is described in detail in the following examples, but the artisan will recognize that the chemical reactions described may be readily adapted to prepare a number of other agents of the various embodiments. For example, the synthesis of non-exemplified compounds may be successfully performed by modifications apparent to those skilled in the art, e.g. by appropriately protecting interfering groups, by changing to other suitable reagents known in the art, or by making routine modifications of reaction conditions. A list of suitable protecting groups in organic synthesis can be found in T.W. Greene's Protective Groups in Organic Synthesis, 3^rd Edition, John Wiley & Sons, 1991 . Alternatively, other reactions disclosed herein or known in the art will be recognized as having applicability for preparing other compounds of the various embodiments. Reagents useful for synthesizing compounds may be obtained or prepared according to techniques known in the art.

The symbols, abbreviations and conventions in the processes, schemes, and examples are consistent with those used in the contemporary scientific literature. Specifically but not meant as limiting, the following abbreviations may be used in the examples and throughout the specification.

• g (grams)

• L (liters)

• Hz (Hertz)

• rt (room temperature)

• HPLC (high performance liquid chromatography)

• min (minutes)

• CH2CI2 (dichloromethane)

• CHCI3 (chloroform)

• d₆-DMSO (deuterated dimethylsulfoxide)

• EtOAc (ethyl acetate)

• mg (milligrams)

• ml_ (milliliters)

• mmol (millimol)

• μΙ_ (microliters)

• nm (nanometers)

• MHz (megahertz)

• h (hours)

• MeOH (methanol)

• CDCI3 (deuterated chloroform)

• DMF (N,N-dimethylformamide)

• DMSO (dimethylsulfoxide)

• MgS0₄ (magnesium sulfate)

• NaOH (sodium hydroxide)

• l₂ (iodine)

• NH₄CI (ammonium chloride)

• Et₃N (triethylamine) • t-Boc (tert-butoxycarbnyl)

• Fmoc (9-fluorenylmethyloxycarbonyl)

• Cbz (Benzyloxycarbonyl)

• Alloc (allyloxycarbonyl)

Unless otherwise indicated, all temperatures are expressed in °C (degree centigrade). All reactions conducted at room temperature unless otherwise mentioned.

All the solvents and reagents used are commercially available and purchased from Sigma Aldrich, Fluka, Acros, Spectrochem, Alfa Aesar, Avra, Qualigens, Merck, Rankem and Leonid Chemicals.

¹ H NMR and ¹³C NMR spectra were recorded on a Varian Unity 400 or 500 spectrometer (¹H at 500 MHz and ¹³C at 126 MHz; or ¹H at 400 MHz and ¹³C at 100 MHz). All NMR spectra were recorded at room temperature. The reported chemical shifts in parts per million (ppm, δ units) are referenced relative to residual solvent protons. Coupling constants are in units of hertz (Hz). Splitting patterns describe apparent multiplicities and are designated as s (singlet), d (doublet), t (triplet), q (quartet), m (multiplet), or br (broad).

Mass spectra were recorded using the electrospray technique (positive and negative ion) with a Micromass QUATTRO II triple-quadropole electrospray mass spectrometer. HPLC was performed with an HP 1 100 ChemStation system using a Supelco Discovery Cis column (150 nm x 4.6 mm, 5 pm). The HPLC solvents were 0.1 % trifluoroacetic acid in water (solvent A) and 0.1 % trifluoroacetic acid in acetonitrile (solvent B). The gradient was 0-100% solvent B in 0-25 min, the flow rate was 1 .00 mL/min, and the detection was at 280 nm.

UV-vis spectra were recorded in water with a Shimadzu UV-1650PC UV-vis spectrophotometer using the UVPC c3.9 software program. Rabbit polyclonal antibodies to human EEA1 were purchased from Cell Signalling Technology (USA). Mouse monoclonal anti-human CD63 was from Santa Cruz Biotechnology (USA). Alexa Fluor 568-conjugated human transferrin, goat anti-rabbit IgG Alexa Fluor 568 and goat anti-mouse IgG Alexa Fluor 647 were purchased from Life Technologies (USA).

Synthesis of linker compound

The process for the preparation of (1 fl,8S,9r)-bicyclo[6.1 .0]non-4-yn-9-ylmethyl- 4,7, 10-trioxa-13-(2-ethoxy-3,4-dioxocyclobut-1 -enylamino) decylcarbamate (exo-15) is illustrated in Scheme 1 1

exo-15

Scheme 11

Synthesis of the compounds, exo-12 and endo-12 are known in the literature. As such the exo-12 and endo-12 compounds can be synthesised as described by Dommerholt J. et al. (see Scheme 12 which depicts synthesis of the exo-isomer). Alternative methodologies to provide either exo-12 or endo-12 can also be employed.

Example 1 :

(1 ff,8S,9ff)-Bicyclo[6.1.0]non- - n-9-ylmethyl (4-nitrophenyl) carbonate (exo-13)

exo-13

To a solution of exo-12 (400 mg, 2.66 mmol) in CH₂CI₂ (20 mL) was added pyridine (540 μΐ, 6.70 mmol) and 4-nitrophenyl chloroformate (590 mg, 2.93 mmol). After stirring at rt for 30 min the reaction mixture was quenched with saturated NH₄CI solution and extracted with CH2CI2 (3 x 20 mL). The combined organic layers were dried with MgS0₄ and concentrated in vacuo. The crude product was purified by column chromatography (hexanes: EtOAC, 4: 1 ) to afford exo-13 as a colourless oil, that solidified overnight (746 mg, 2.66 mmol, 89%). R_f 0.8 (hexanes: EtOAC, 2: 1 ); ¹H NMR (500 MHz; CDCI₃) δ 8.28 (2 H, d, J 9.2), 7.39 (2 H, d, J 9.2), 4.22 (2 H, d, J 6.9), 2.47-2.44 (2 H, m), 2.34-2.28 (2 H, m), 2.21 -2.17 (2 H, m), 1 .44-1 .40 (2 H, m), 0.88- 0.82 (3 H, m). Example 2:

(1 ff,8S,9ff)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4,7,10-trioxa-13-azatridecyl) carbamate (exo-14)

exo-14

To a solution of exo-13 (400 mg, 1 .27 mmol) in DMF (20 mL) was added 4,7, 10- trioxa-1 , 13-tridecanediamine (1.67 mL, 7.61 mmol) followed by Et₃N (531 μΐ, 3.81 mmol). The reaction mixture was stirred at rt for 1 h. The solvent was removed under reduced pressure, the residue was dissolved in CH2CI2 (40 mL) and extracted with 1 N NaOH (2 x 10 mL), followed by water. The combined aqueous layers were extracted with EtOAc (3 x 10 mL) and washed with brine. The combined organic layers were dried over MgS0₄ and concentrated in vacuo to give a yellow oil. Purification by column chromatography (hexanes: EtOAC, 5: 1 - MeOH + 1 % Et₃N, visualised with l₂) yielded exo-14 as a slightly yellow oil (0.435 mg, 1 .10 mmol, 86%). ¹ H NMR (500 MHz; CDCI₃) δ 5.39 (1 H, brs), 3.96 (2 H, d, J 6.6), 3.65-3.63 (4 H, m), 3.61 -3.54 (8 H, m), 3.28 (2 H, d, J 6.0), 2.81 (2 H, t, J 6.6), 2.41 -2.38 (2 H, m), 2.31 - 2.25 (2 H, m), 2.17-2.12 (2 H, m), 1.79-1.74 (4 H, m), 1 .40-1 .33 (2 H, m), 0.72-0.66 (3 H, m).

Example 3:

(1 /?,8S,9/?)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4,7,10-trioxa-13-(2-ethoxy-3,4- dioxocyclobut-1-enylamino)tridecyl)carbamate (exo-15)

exo-15

To a solution of exo-14 (0.47 g, 1.19 mmol) in ethanol (20 mL) was added diisopropylethylamine (103 μί, 0.59 mmol) followed by 3,4-diethoxy-3-cyclobuten-1 ,2- dione (263 μί, 1 .78 mmol) and the resulting yellow solution was stirred at rt for 30 min. The reaction mixture was evaporated and the residue purified by column chromatography (CH2CI2 - CH₂Cl2:MeOH 9: 1 ) to yield exo-15 as a light yellow oil (0.58 g, 1 .1 1 mmol, 93%). ¹H NMR (500 MHz; d₆-DMSO) δ 5.03-7.01 (1 H, brs), 4.65 (2 H, t, J 5.8), 3.84 (2 H, d, J 6.5), 3.54-3.34 (16 H, m), 3.01 (2 H, q, J 6.4), 2.31 -2.28 (2 H, m), 2.24-2.19 (2 H, m), 2.08-2.05 (2 H, m), 1 .74 (2 H, q, J 6.4), 1 .61 (2 H, t, J 6.4), 1 .36 (3 H, t, J 7.1 ), 1 .34-1 .27 (1 H, m), 0.67-0.69 (2 H, m); ¹³C NMR (125 MHz; de-DMSO) δ 189.2, 182.1 , 176.5, 172.6, 156.3, 98.8, 69.7, 69.5, 69.5, 68.7, 67.9, 67.6, 67.4, 67.3, 54.9, 41 .2, 41 .0, 37.5, 32.8, 30.5, 30.0, 29.7, 23.4, 22.8, 20.8, 15.6; HRMS (ESI⁺) m/z (M+H)⁺ 521 .2973 (521 .2857 calculated for C27H₄i N₂0₈).

Synthesis of linker compound

The process for the preparation of (1 fl,8S,9S)-bicyclo[6.1 .0]non-4-yn-9-ylmethyl (4,7, 10-trioxa-13-(2-ethoxy-3,4-dioxocyclobut-1 -enylamino)tridecyl)carbamate (endo- 15) is illustrated in Scheme 13.

Example 4:

(1 fl,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4-nitrophenyl) carbonate (endo- 13)

endo-13

To a solution of endo-12 (170 mg, 1 .13 mmol) in CH₂CI₂ (10 mL) was added pyridine (228 μΐ, 2.83 mmol) and 4-nitrophenyl chloroformate (251 mg, 1.24 mmol). After stirring at rt for 30 min the reaction mixture was quenched with saturated NH₄CI solution and extracted with CH₂CI₂ (3 x 20 mL). The combined organic layers were dried with MgS0₄ and concentrated in vacuo. The crude product was purified by column chromatography (hexanes: EtOAC, 4: 1 ) to afford endo-13 as a colourless oil, that solidified overnight (310 mg, 0.98 mmol, 87%). R_f O.75 (hexanes: EtOAC, 2: 1 ); ¹H NMR (500 MHz; CDCI₃) δ 8.28 (2 H, d, J 9.2), 7.41 (2 H, d, J 9.2), 4.41 (2 H, d, J 8.3), 2.34-2.23 (6 H, m), 1.65-1.57 (2 H, m), 1 .55-1.49 (2 H, m), 1 .09-1 .04 (2 H, m).

Example 5:

(1 ff,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl 4,7,10-trioxa-13-4.7.10-trioxa-13- aminotridecylcarbamate (endo-14)

endo-14

To a solution of endo-13 (150 mg, 0.48 mmol) in DMF (10 mL) was added 4,7, 10- trioxa-1 , 13-tridecanediamine (0.625 mL, 2.85 mmol) followed by Et₃N (200 μΐ, 1 .43 mmol). The reaction mixture was stirred at rt for 1 h. The solvent was removed under reduced pressure, the residue was dissolved in CH₂CI₂ (20 mL) and extracted with 1 N NaOH (2 x 10 mL), followed by water. The combined aqueous layers were extracted with EtOAc (3 x 10 mL) and washed with brine. The combined organic layers were dried over MgSO₄ and concentrated in vacuo to give a yellow oil. Purification by column chromatography (hexanes: EtOAC, 5: 1 - MeOH + 1 % Et₃N, visualised with l₂) yielded endo-14 as a slightly yellow oil (0.172 mg, 0.43 mmol, 90%). ¹ H NMR (500 MHz; CDCI₃) ¹H NMR (500 MHz; CDCI₃) δ 5.39 (1 H, brs), 4.13 (2 H, d, J 8.2), 3.64-3.59 (12 H, m), 3.28 (2 H, d, J 5.9), 2.88 (2 H, t, J 6.3), 2.36-2.19 (6 H, m), 1 .80-1.75 (4 H, m), 1.62-1 .56 (2 H, m), 1 .37-1 .33 (1 H, m), 0.95-0.91 (2 H, m).

Example 6:

(1 ff,8S,9S)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl (4,7,10-trioxa-13-(2-ethoxy-3,4- dioxocyclobut-1 -enylamino)tridecyl)carbamate (endo-15)

endo-15

To a solution of endo-14 (60 mg, 0.151 mmol) in ethanol (2 ml_) was added diisopropylethylamine (13 μΙ_, 0.076 mmol) followed by 3,4-diethoxy-3-cyclobuten-1 ,2- dione (34μΙ_, 0.227 mmol) and the resulting yellow solution was stirred at rt for 30 min. The reaction mixture was evaporated and the residue purified by column chromatography (CH2CI2 - CH₂Cl2:MeOH 9:1 ) to yield endo-15 as a light yellow oil (63 mg, 0.12 mmol, 80%).¹ H NMR (500 MHz; CDCI₃) ¹ H NMR (500 MHz; CDCI₃) δ 5.19-5.14 (1 H, m), 4.77-4.72 (2 H, m), 4.13 (2 H, d, J 7.8), 3.64-3.61 (12 H, m), 3.54 (2 H, t, J 5.9), 3.27 (2 H, d, J 6.2), 2.69-2.68 (1 H, brs), 2.31 -2.18 (6 H, m), 1 .89-1 .85 (2 H, m), 1.85-1.74 (2 H, m), 1 .61 -1 .54 (2 H, m), 1 .45 (3 H, t, J 6.9), 1.35-1 .32 (1 H, m), 0.95-0.91 (2 H, m); ¹³C NMR (125 MHz; CDCI₃) δ 188.8, 183.2, 176.8, 172.6, 156.8, 98.8, 70.5, 70.4, 70.3, 70.2, 69.6, 69.4, 62.5, 43.5, 39.0, 29.6, 29.5, 29.0, 21 .4, 20.1 , 17.8, 15.8.; HRMS (ESI⁺) m/z (M+H)⁺ 521 .3204 (521 .2857 calculated for

Synthesis of linker compound

The process for the preparation of (1 fl,8S,9fl)-bicyclo[6.1 .0]non-4-yn-9-ylmethyl-2-(2- (2-(6-(2-(propan-2-ylidene)hydrazinyl)nicatinamido)ethoxy)ethoxy)ethylcarbamate (exo-15) is illustrated in Scheme 14.

exo-19

Scheme 14

Example 7:

6-Hydrazinonicotinic Acid Acetone Hydrazone 16 was synthesised following published procedures and activated as the NHS-ester 17 (Bioconjugate Chem, 20, 10, 2009, 1950).

Example 8:

(1 R,8S,9r)-bicyclo[6.1.0]non-4-yn-9-ylmethyl-2-(2-(2-(6-(2-(propan-2- ylidene)hydrazinyl) nicotinamido) ethoxy)ethoxy) ethylcarbamate (exo-19)

To a solution of 17 (25.6 mg, 88.16 μίτιοΐ) in anhydrous DMF (2 mL) was added 18 Synaffix SX-A1004 (26 mg, 80.14μηποΙ) followed by triethylamine (1 1 μΙ_, 0.080 mmol) and the resulting light yellow solution was stirred at rt overnight. The DMF was removed in vacuo and the residue purified by column chromatography (CH2CI2 - CH₂CI₂:MeOH 9: 1 + triethylamine) to yield exo-19 as a light yellow solid (38 mg, 0.076 mmol, 94%). ¹ H NMR (500 MHz; CDCI₃) δ 8.52 (1 H, s), 8.00 (1 H, d, J 7.9), 7.22 (1 H, d, J 8.6), 6.76 (1 H, s), 5.36 (1 H, brs), 4.1 1 (2 H, d, J 7.8), 3.63-3.61 (8 H, m), 3.58 (2 H, t, J 5.9), 3.27 (2 H, d, J 6.2), 2.69 (1 H, brs), 2.28-2.16 (6 H, m), 2.00 (3H, s), 1 .94 (3H, s), 1 .94-1 .90 (2 H, m), 1 .69-1.65 (2 H, m), 1 .53-1.51 (2 H, m), 1.33-1.23 (2H, m), 1 .1 1 -1 .07 (1 H, m), 0.91 -0.88 (2 H, m); ¹³C NMR (125 MHz; CDCI₃) δ 172.9, 165.7, 158.6, 156.8, 148.0, 146.7, 137.5, 121 .3, 106.6, 98.8, 70.2, 70.1 , 62.7, 40.7, 33.9, 33.8, 28.9, 25.3, 21 .3, 20.0, 17.7, 16.1 ; HRMS (EST) m/z (M+H)⁺ 500.2868 (500.2828 calculated for C26H37N5O5).

Example 9:

Quantum Dot Synthesis and Characterisation

CdSe/ZnS core/shell quantum dots were synthesized using high-temperature reaction of organometallic precursors according to previously reported methods (J Van Embden, P Mulvaney, Langmuir 2005, 21 , 10226). The quality of the nanocrystals was assessed by optical analysis: absorption spectra were recorded on a Cary 5 UV- Vis-NIR spectrophotometer. Steady-state photoluminescence spectra were measured on a Horiba Jobin Yvon Fluorolog-3 spectrofluorometer, with slit widths of 1 nm and integration times of 0.1 -0.5 s. The Gaussian full-width-at-half maximum (FWHM) was found to range between 25 and 33 nm, depending on the emission peak position of the QDs.

QD Polymer Encapsulation

Water-soluble QDs were generated following the polymer encapsulation technique described in Lees et al (E lees, T Nguyen, A Clayton, P Mulvaney, ACS Nano 2009, 3, 1 121 ) Briefly, 25-50 nmol of freshly synthesized QDs in ODE were placed in trioctyl phosphine/trioctyl phosphine oxide (TOP/TOPO 20 times excess with respect to the total number of surface atoms) overnight at 60°C to assure uniform coating. The TOP/TOPO-coated QDs were then washed free of excess ligand using a mixture of chloroform and methanol and precipitated with acetone. After re-dispersion in 1 ml of chloroform, polystyrene-co-maleic anhydride polymer (PSMA, Mn 1600, 200 mM in CHCI3) was added to give a 100 times molar excess of PSMA. The reaction mixture was then stirred at rt for 3 h to allow the formation of the PSMA shell. After that, a 5- fold molar excess of Jeffamine M1000 or amino-PEG-azide (H2N-PEG-N3) (200 mM in CHCI3) was added and the sample was left to stir at room temperature overnight. QDs functionalized with different amounts of azide groups on their surface were obtained by varying the ratio of Jeffamine M1000 and H2N-PEG-N3 used. For example, QD10 were obtained using 10% H2N-PEG-N3 mixed with 90% M1000 (v/v). Following surface modification, water (2-3 ml_) containing ethanolamine (20 μΙ_) was added to ring-open remaining maleic anhydride groups. The CHCI3 was removed using a rotary evaporator. Additional CHCI3 (2-3 ml_) was added to the resulting water solution of QDs to extract any hydrophobic ligands. The sample was then centrifuged for 5 min at 6000 rpm and the QD water solution was separated and passed through a 0.22 μηι filter. The filtrate was concentrated using a 50 kDa MWCO Vivaspin filter unit (Sartorius Stedim Biotech). Quantum Yield Measurements

The quantum yield (QY) for the QDs was measured relative to the dye Rhodamine 6G (QY 95% in EtOH) with excitation at 400 nm. Fluorescence spectra of QDs and dye were measured under identical conditions and care was taken that the optical density of each sample did not exceed 0.1 at the excitation wavelength. The QY of QDs in chloroform was typically approximately 40% whereas for the samples in water after polymer encapsulation the QY was measured to be 16-26%.

Example 10

Reaction of a sample Linker with Transferrin

The reaction between the linker (L) of example 3 and iron-loaded transferrin (Fe₂Tf) was performed in 0.5 M borate buffer at pH 9. Stock solutions of linker in DMSO (4 mM) and Fe₂Tf in MilliQ deionized water (0.6 mM) were prepared and both were added to the borate buffer at a final concentration of 0.2-0.8 mM, depending on the ratio of linker to Fe₂Tf required. The resulting mixture was allowed to react for 6 h at RT and analyzed by ESI-TOF mass spectroscopy (Agilent 6220 Accurate-Mass TOF LC/MS fitted with a C4 desalting column). The experiment was conducted using a ratio of linker to transferrin of 2: 1 and 4:1 . The result for a 2: 1 ratio is provided in figure 3. The result with a 4: 1 ratio is provided in figure 4. As can be readily seen from these figures demonstrate that it is possible to conjugate multiple linkers to a molecule with sufficient surface amine groups. Example 11

Reaction of the Octyne moiety of Transferrin bound linker with an Azide

To demonstrate that the bound moiety could react with azide groups the reactivity of the cyclooctyne functional group was tested by reaction of L-Fe₂Tf (produced in example 10) with 2-azido-N-phenylacetamide (MW=176 Da). A mass increase corresponding to addition of one or more molecules of the azide confirmed fast and efficient triazole formation as shown in figures 5, 6 and 7. Figure 5 shows the structure of the desired product of triazole formation. Figure 6 shows the mass spectrum of the solution of various numbers of linkers bound to the transferrin and figure 7 shows the same mixture after reaction. As can be seen the azide formation proceeds smoothly notwithstanding that the cyclooctyne moiety is already bound to the transferrin.

Example 12

Reaction of Azide moiety on azide modified quantum dot with solution phase cyclooctyne group

The reaction between dye (ALEXA594) and linker (L) of example 3 was carried out as described in example 10 at a 2: 1 final concentration of linker (0.8 mM) and dye Alexa Fluor 594 cadaverine (Invitrogen) (0.4 mM). The resulting L-A594 conjugate was reacted with QDs functionalized with different percentages of azide groups (QD0, QD1 , QD10, QD100). The reaction mixture containing the L-A594 conjugate in a 50- fold molar excess was added to QDs in water, except for the sample QD100 for which the excess was 100-fold. The mixture was allowed to react for 6 h at rt. Excess dye was removed by spin filtration (50 kDa MWCO, Vivaspin filter unit) until the filtrate no longer showed a fluorescent signal for the dye (typically 10 washes were required). The structure of the desired product is shown in figure 8. Figure 9 shows the normalised absorbance of the two starting materials. Fluorescence spectra of QDs- linker-dye samples upon excitation at 400 nm show FRET (Forster resonance energy transfer) between QDs and dye with FRET signal increasing with increasing azide loading on the QD (Figure 10). This demonstrates a successful SPAAC reaction between QDs and linker and shows that the number of biomolecules attached to the QDs can be tuned by controlling the percentage of azide groups on the nanoparticle surface. A control reaction of QD0 and L-A594 showed the conjugate is completely removed, confirming the specificity of the SPAAC reaction. Example 13

Conjugation of Quantum dots with transferrin

Conjugation of QDs with Linker-Transferrin

The L-Fe₂Tf conjugate was prepared as outlined in example 10 at a ratio 2:1 linker to protein and final concentration of 0.2 mM of Fe2Tf. The mixture was then added to QDs functionalized with different percentages of azide groups in a 10-fold molar excess and allowed to react for 6 h at RT. Gel Electrophoresis and Size Exclusion Chromatography

Gel electrophoresis was performed using a 0.75% agarose gel in Tris/Borate/EDTA (TBE) buffer 0.5*, pH 9.5, at 100 V. Size exclusion chromatography was performed on an Agilent 1200 Series HPLC apparatus equipped with a BioSep-SEC-s4000 column from Phenomenex. Phosphate buffer (20 mM, pH 7.4) was used as mobile phase, with a flow rate of 1 ml/min and injection volumes ranging from 30 to 100 μΙ_. Detection was at 220, 275 and 400 nm.

The results of gel electrophoresis are shown in figures 1 1 (UV illumination) and 12 (Coomassie Blue staining. As the QD surface is negatively charged, all samples migrate from the negative to the positive pole and their speed is determined by their charge and size. For increasing density of azide groups on the QD surface, the difference in mobility between unconjugated and conjugated samples increases. As Fe₂Tf has a pi of 5.2-6.2 and is negatively charged at the basic pH used for gel electrophoresis, the reduced mobility of the QDx-Fe₂Tf samples can thus be ascribed to an increase in hydrodynamic volume, which indicates the success of the conjugation between QDs and L-Fe₂Tf. As well, only QD10-Fe₂Tf and QD100-Fe₂Tf are reactive to Coomassie blue, signalling the presence of conjugated protein, whereas for QD0+Fe2Tf, the Coomassie stain appears only at the position of the free protein.

Example 14

The uptake of QD100-Fe₂Tf conjugates into HeLa cells

HeLa cells were seeded onto glass coverslips at 0.6 χ 10⁵ cells/ml and grown for 48 hr. Cells were then serum starved for 3 h followed by 10 min incubation on ice to stop endocytosis. Chilled cells were then incubated with pre-chilled 100 μς/Γη Ι (A) Alexa Fluor 568-conjugated human transferrin (Life Technologies, USA), (C) QDs, or (B) QD100-Fe2Tf (diluted in serum-free DMEM) on ice for 30 min. Unbound transferrin was removed using cold PBS washes and internalisation of bound conjugated transferrin performed for the indicated duration at 37 °C in serum-free DMEM. As a control, cells were also incubated on ice. Following incubation, cells were washed in PBS, fixed with cold 4% (w/v) paraformaldehyde and processed for immunofluorescence. Indirect Immunofluorescence Microscopy

Cells were fixed in 4% (w/v) paraformaldehyde for 15 min and then incubated with 50 mM NH₄CI/PBS for 10 min to quench free aldehydes. Cells were washed in PBS, permeabilized in 0.1 % Triton X-100/PBS for 4 min and again washed in PBS. Blocking was achieved by incubating cells in 5% (v/v) FCS/PBS at room temperature for 30 min. Cells were incubated in primary antibodies diluted in 5% (v/v) FCS/PBS for 30 min at room temperature. Excess antibodies were removed by PBS washes. Fluorochrome-conjugated secondary antibodies were added and incubated with cells for 30 min at room temperature followed by further PBS washes. Nuclei of cells were stained with 4,6-diamino-2-phenylindole (DAPI; Sigma-Aldrich, USA) where indicated and cells were washed as before. Cells were mounted in Mowiol (10% (w/v) Hopval 5- 88 (Hoechst, Australia), 25% (w/v) glycerol, 0.1 M Tris). Images of cells were captured using a Leica TCS SP2 laser confocal unit and Leica Confocal Software version 2.61 . For multi-colour labelling, images were collected independently. The results are shown in figure 13 which shows Fe₂-transferrin uptake into HeLa cells performed using (A1 -A3) A568-Fe₂Tf, (B1 -B3) QD100-Fe₂Tf and (C1 -C3) QD100 for the indicated times at 37°C.

The results demonstrate that both A568-Fe₂Tf and QD100-Fe2Tf conjugates were internalized into HeLa cells within 15-30 min (Figure 13). Control experiments with unconjugated QD100 demonstrated that the binding was Fe2Tf dependent (Figure 13, C1 -3). Together these results demonstrate that QD100-Fe2Tf binds to TfRs and are internalized by receptor-mediated endocytosis. After incubation at 37°C for 30 min, both the amount of uptake and the intracellular distribution of QD100-Fe₂Tf and A568-Fe₂Tf were similar. However, 2 h after treatment cells incubated with A568-Fe₂Tf have significantly reduced intracellular fluorescence when compared to at 30 min indicating that the conjugate is trafficked out of the cell by known Fe₂Tf cellular efflux pathways (Figure 13, A2 and A3). In contrast, at 2h in cells treated with QD100-Fe₂Tf significant punctate staining is still evident demonstrating that the two materials have different efflux times.

Example 15

Localisation of QD100-Fe₂Tf conjugates into He La cells

To further investigate the localization of QD100-Fe₂Tf after internalization, cells were stained for markers of endocytic compartments. Early endosome antigen 1 (EEA1 ) and CD63 are markers for early endosomes and late endosomes/lysosomes, respectively. After 15 min, QD100-Fe₂Tf shows extensive co-localization with EEA1 but minimal co-localization with CD63 (Figure 14a). Conversely, after 2 h, QD100-Fe₂Tf shows significant overlap with the lysosomal marker CD63 (Figure 14b). These results demonstrate successful binding of QD100-Fe₂Tf to TfRs and internalization into cells, although the presence of QDs appears to perturb the intracellular trafficking pathway of compared to Alexa Fluor-labelled Fe₂Tf, resulting in retarded efflux. This last observation is consistent with reports by others using Fe₂Tf-QD conjugates with HeLa, HEp-2 and SW480 cells respectively, despite using entirely different conjugation strategies to generate the protein-QD conjugates.

Example 16

Connecting Linker to trastuzumab (Herceptin)

HERCEPTIN (trastuzumab) is a recombinant DNA-derived humanized monoclonal antibody that selectively targets the extracellular domain of the human epidermal growth factor receptor 2 protein (HER2). The antibody is an lgG1 kappa that contains human framework regions with the complementarity-determining regions of a murine anti-p185 HER2 antibody that binds to HER2. Trastuzumab is composed of 1 ,328 amino acids and has a molecular weight of ~148 kDa. Figure 15 shows the MS of Herceptin showing variations in the number of bound hexoses.

To demonstrate the ability of the linker of example 3 to bind to Herceptin two binding studies were carried out using the procedure of example 10 with a ratio of antibody to linker of 1 : 10 and 1 :20. The results for the 1 : 10 study are shown in figure 16 and the results of the 1 :20 reaction are shown in figure 17. As can be seen both studies indicate reaction of linker with the antibody to form a linker-antibody conjugate. Example 17

Fluorescent Confirmation of Bioconjugation to trastuzumab

General

The conjugation buffer was Na₃P0₄ (100 mM), NaCI (150 mM) at pH6. The catalyst buffer was a solution of aniline (10 mM). Azide Fluor 585 dye was purchased from Jena Bioscience.

Oxidation of Trastuzumab

To Trastuzumab (300 μΙ_ of 1 mg/mL solution in PBS) was added Nal0₄ (30 μΙ_ of 100 mM stock solution) and the mixture was left at room temperature for 30 min excluded from light. Excess Nal0₄ was removed via buffer exchange (with Vivaspin 500 μΙ_ 5 kDa spin filter) with the conjugation buffer (see above). The final volume (ca. 345 μΙ_) was divided into three equal portions and is referred to as oxidised trastuzumab (Trastuzumab-ox). Conjugation 1: 4:1 linker to antibody

To trastuzumab-ox (1 15 μΙ_) was added to aniline buffer (10 μΙ_) followed by the linker of example 8 (5.4 μΙ_ of 0.5 mM solution in DMF) and the mixture was incubated at room temperature for 1 hour. To this mixture was added Azide Fluor 585 dye (1 .5 μΙ_ of 3.4 mM solution) and the mixture was left at room temperature overnight. The mixture was purified by spin filtration (Amicon Ultra 10 kDa MWCO) until no more dye was visible in the filtrate. The conjugate was resuspended in buffer and the amount of dye conjugated to the trastuzumab-ox was measured by electronic spectroscopy

The conjugation was repeated with 10:1 and 100:1 ratios of linker to antibody.

10:1 trastuzumab-ox (1 15 μΙ_); aniline buffer (10 μΙ_), the linker of example 8 (2.6 μΙ_ of 5 mM solution in DMF), Azide Fluor 585 dye (8.4 μΙ_ of 3.4 mM solution). 100:1 trastuzumab-ox (1 15 μΙ_); aniline buffer (10 μΙ_), the linker of example 8 (13.6 μΙ_ of 5 mM solution in DMF), 34 mM Azide Fluor 585 dye (3.9 μΙ_ of 34 mM solution). The results are shown in figure 18. As can be seen there is evidence of binding of the fluorescent dye to the Herceptin.

Claims

Claims:

1 . A compound of the Formula (I):

Formula (I)

wherein

each R^a and R^b is independently selected from the group consisting of: H, OH, halogen, optionally-substituted C-i- ₂ alkyl, optionally-substituted C₂-₁₂ alkenyl, optionally-substituted C₂-₁₂ alkynyl, optionally-substituted C3-₁₂ cycloalkyl, optionally- substituted C3-₁₂ cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally- substituted OC₂-i₂ alkenyl, optionally-substituted OC₂-i₂ alkynyl, optionally-substituted OC₃- ₂ cycloalkyl and optionally-substituted OC₃- ₂ cycloalkenyl;

each R is independently selected from the group consisting of: H, halogen, OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted CM₂ alkyl, optionally-substituted CM₂ haloalkyl, optionally-substituted C₂-₁₂ alkenyl, optionally- substituted C₂-₁₂ alkynyl, optionally-substituted C₂. ₂ heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C₂-₁₂ heterocycloalkenyl, optionally-substituted C₆-18 aryl, optionally-substituted C1-18 heteroaryl, optionally-substituted C1-12 alkyloxy, optionally-substituted C₂. ₂ alkenyloxy, optionally-substituted C₂. ₂ alkynyloxy, optionally-substituted C2-12 heteroalkyloxy, optionally-substituted C3-12 cycloalkyloxy, optionally-substituted C3-12 cycloalkenyloxy, optionally-substituted C2-12 heterocycloalkyloxy, optionally-substituted C₂-₁₂ heterocycloalkenyloxy, optionally- substituted C₆-18 aryloxy, optionally-substituted CMS heteroaryloxy, optionally- substituted Ci-₁₂ alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR , NR^cCOR , NR^cCOOR^d, NR^cSO₂R , NR^cCONR R^e and NR^cR^d, or any two R¹ on adjacent carbon atoms form a fused substituent;

L¹ is a bond or linking group;

R² is selected from the group consisting of: optionally substituted C₁-₁₂ alkyl, optionally substituted C₂-₁₂ alkenyl, optionally substituted C₂-₁₂ alkynyl, optionally substituted C3-12 cycloalkyl, optionally substituted C3-12 cycloalkenyl, NH₂, ONH₂, NHNH₂, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONH₂, NH(CO)(optionally-substituted Ci_-6 alkyl)NH₂, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNH₂, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally- substituted Ci_-6 alkyl)ONH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNH₂, (CO)NHNR^aR^p, NH(CO)NHNH_2, NH(CO)NHNR^aR^p, NHNH(CO)NHNH₂ and NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted CM 2 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted CM₂ alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

each R^c, R and R^e is independently selected from the group consisting of: H, OH, halogen, optionally-substituted CM₂ alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C₃- ₂ cycloalkyl, optionally-substituted OC₆-is aryl, and optionally-substituted OCM S heteroaryl.

2. The compound according to claim 1 , wherein L¹ is of the Formula (II):

Formula (II)

wherein

X¹ , is selected from the group consisting of: a bond, O, NH, NR⁵, S and CR⁶R⁷; X² is selected from the group consisting of: O and S;

L² is bonded to the R² group and is of the Formula (III): X³(C R⁸ R⁹)_P [X⁴(C R ⁰ R )_Q]_R (C R ² R ³)_S X⁵

Formula (III)

X³ is selected from the group consisting of: a bond, O, N H , N R¹⁴, S and CR¹⁵R¹⁶;

X⁴ is selected from the group consisting of: a bond, O, N H , N R¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, O, N H , N R¹⁴, S, C R¹⁵R¹⁶, optionally-substituted CM 2 alkyl, H N-(optionally-substituted CM₂ alkyl)-, R¹⁴N- (optionally-substituted CM ₂ alkyl)-, O-(optionally-substituted CM₂ alkyl)-, optionally- substituted C5-12 aryl, H N-(optionally-substituted C₅. ₂ aryl)-, R¹⁴N-(optionally- substituted C5-12 aryl)-, O-(optionally-substituted C5-12 aryl)-, H N (CO)-(optionally- substituted C5-12 aryl)-, R¹⁴N (CO)-(optionally-substituted C5-12 aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-, (CO)N H-(optionally-substituted C5-12 aryl)-, (CO) R¹⁴N-(optionally-substituted C₅.₁₂ aryl)-, (CO)O-(optionally-substituted C₅.₁₂ aryl)-, optionally-substituted C2-12 heteroaryl, H N-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C2-12 heteroaryl)-, optionally-substituted C2-12 heteroaryl, H N (CO)-(optionally-substituted C₂. 12 heteroaryl)-, R¹⁴N (CO)-(optionally-substituted C₂-i₂ heteroaryl)-, O(CO)-(optionally- substituted C2-12 heteroaryl)-, (CO)H N-(optionally-substituted C2-12 heteroaryl)-, (CO) R¹⁴N-(optionally-substituted C2-12 heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

each R⁵ and R¹⁴ is independently selected from the group consisting of: optionally-substituted CM 2 alkyl and N-protecting group;

each R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R^{1 1} , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: H , OH , halogen, optionally-substituted CM ₂ alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCM ₂ alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally- substituted OC₃- ₂ cycloalkenyl;

3. The compound according to claim 2, wherein n = 0.

4. The compound according to claim 2 or 3, wherein R^A and R^B are hydrogen.

5. The compound according to any one of claims 2 to 4, wherein A is (CR³R⁴)_m. 6. The compound according to claim 5, wherein m = 1 and R³ and R⁴ are each H .

7. The compound according to any one of claims 2 to 6, wherein and X¹ is O or N H . 8. The compound according to claim 7, wherein X¹ is O.

9. The compound according to any one of claims 2 to 8 wherein X² is O.

10. The compound according to any one of claims 2 to 9, wherein X³ is independently selected from O or NH.

1 1 . The compound according to claim 10, wherein X³ is NH.

12. The compound according to any one of claims 2 to 1 1 , wherein R⁸, R⁹, R¹², and R¹³ are H.

13. The compound according to any one of claims 2 to 12, wherein p = 2 or 3.

14. The compound according to any one of claims 2 to 13, wherein q = 2 or 3.

15. The compound according to any one of claims 2 to 14, wherein r = 2 or 3. 16. The compound according to any one of claims 2 to 15, wherein s = 0 or 1 .

17. The compound according to any one of claims 2 to 16, wherein X⁴ is O.

18. The compound according to any one of claims 2 to 17, wherein R¹⁰ and R¹¹ are H.

19. The compound according to any one of claims 2 to 4, wherein R⁸, R⁹, R¹⁰ and R¹¹ are each H, A = CH₂, X¹ = O, X² = O, X³ = NH, p = 2, X⁴ = O, q = 2, r = 2 and s = 0 such that L¹ has the Formula (ll-g):

Formula (ll-g)

20. The compound according to any one of claims 2 to 4, wherein R⁸, R⁹, R¹⁰, R¹¹ , R¹² and R¹³ are each H, A = CH₂, X¹ = O, X² = O, X³ = NH, p = 3, X⁴ = O, q = 2, r = 3 and s = 1 such that L¹ has the Formula (ll-h):

Formula (ll-h)

21 . The compound according to any one of claims 2 to 19, wherein X⁵ is HN- (optionally-substituted C1 -12 alkyl)-.

22. The compound according to any one of claims 2 to 19, wherein X⁵ is O- (optionally-substituted C-1 -12 alkyl)-. 23. The compound according to any one of claims 2 to 19, wherein X⁵ is HN(CO)- (optionally-substituted C5-12 aryl)-.

24. The compound according to any one of claims 2 to 19, wherein X⁵ is HN(CO)- (optionally-substituted C2-12 heteroaryl)-.

The compound according to any one of claims 2 to 4, wherein A, X¹, X² and are selected such that L¹ is of the Formula (X-a):

Formula (X-a)

26. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-b):

Formula (X-b)

The compound according to any one of claims 2 to 4, wherein A, X¹, X² and are selected such that L¹ is of the Formula (X-c):

Formula (X-c)

28. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-d):

Formula (X-d)

The compound according to any one of claims 2 to 4, wherein A, X¹, X² and are selected such that L¹ is of the Formula (X-e):

Formula (X-e)

The compound according to any one of claims 2 to 4, wherein A, X¹, X² and are selected such that L¹ is of the Formula (X-f):

Formula (X-f)

31 . The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-g):

Formula (X-g)

32. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-h):

Formula (X-h)

33. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-i):

Formula (X-i)

34. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-k):

Formula (X-k)

35. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-I):

Formula (X-l)

36. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-m):

Formula (X-m)

37. The compound according to any one of claims 2 to 4, wherein A, X¹, X² and L² are selected such that L¹ is of the Formula (X-n):

Formula (X-n)

38. The compound according to any one of claims 2 to 37, wherein R² is NH₂.

39. The compound according to any one of claims 2 to 37, wherein R² is ONH₂.

40. The compound according to any one of claims 2 to 37, wherein R² is NHNH₂. 41 . The compound according to any one of claims 2 to 37, wherein R² is (CO)NHNH₂.

42. The compound according to any one of claims 2 to 37, wherein R² is NH(CO)NHNH₂.

43. The compound according to any one of claims 2 to 37, wherein R² is NHNH(CO)NHNH₂.

44. The compound according to any one of claims 2 to 37, wherein R² is NH(CO)CH₂NH₂ or NH(CO)CH₂ONH₂.

45. The compound according to any one of claims 2 to 37, wherein R² is NHNR¹^¹ and wherein R^a and R^p combine together to provide the group of Formula (l-a):

Formula (l-a)

wherein R^x and R⁵ are each methyl.

46. The compound according to any one of claims 2 to 37, wherein R² is

The compound according to any one of claims 2 to 37, wherein R²

48. The compound according to any one of claims 2 to 37, wherein the compound is of the Formula (IV):

Formula (IV)

wherein

each R^a and R^b is independently selected from the group consisting of: H, OH, halogen, optionally-substituted CM 2 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C₃- ₂ cycloalkyl, optionally- substituted C3-12 cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally- substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl;

R¹ is selected from the group consisting of: H, halogen, OH, NO₂, CN, SH,

NH₂, CF₃, OCH F2, OCF3, optionally-substituted CM₂ alkyl, optionally-substituted CM₂ haloalkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C₃- ₂ cycloalkenyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C2-12 heterocycloalkenyl, optionally-substituted C₆-is aryl, optionally-substituted CMS heteroaryl, optionally-substituted CM₂ alkyloxy, optionally- substituted C2-12 alkenyloxy, optionally-substituted C2-12 alkynyloxy, optionally- substituted C2-12 heteroalkyloxy, optionally-substituted C₃- ₂ cycloalkyloxy, optionally- substituted C3-12 cycloalkenyloxy, optionally-substituted C2-12 heterocycloalkyloxy, optionally-substituted C2-12 heterocycloalkenyloxy, optionally-substituted C₆-is aryloxy, optionally-substituted CMS heteroaryloxy, optionally-substituted CM₂ alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d; or any two R¹ on adjacent carbon atoms form a fused substituent; L¹ is as defined in claim 2;

each R^C, R^D and R^E is independently selected from the group consisting of: H , OH , halogen, optionally-substituted C-i- ₂ alkyl, optionally-substituted C2-₁₂ heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OC₆-is aryl, and optionally-substituted OCM S heteroaryl.

R^2A is selected from the group consisting of: OH , optionally-substituted OC₁-₁₂ alkyl, optionally-substituted OC₂-₁₂ alkenyl, optionally-substituted OC₂-₁₂ alkynyl, optionally-substituted OC3-₁₂ cycloalkyl and optionally-substituted OC3-₁₂ cycloalkenyl.

49. The compound according to claim 1 , wherein the compound is selected from the group consisting of:

50. A process of linking a substrate to a molecule, the process comprising the step:

i) reacting an end group of a compound according to any one of claims 1 to 49, with either a functionalised substrate or a functionalised molecule, linking either the functionalised substrate or the functionalised molecule to a first end of the compound; and ii) reacting the other end group from the product of step i), with either one of the functionalised substrate or the functionalised molecule which was not used in step i) to provide the substrate linked to the molecule. 51 . The process according to claim 50, wherein

step i) comprises reacting the end of the compound according to any one of claims 1 to 49 which contains the alkyne group, with a functionalised substrate, linking the substrate to a first end of the compound; and

step ii) comprises reacting the end of the product from step i) which does not contain an alkyne group, with a functionalised molecule, linking the molecule to a second end.

52. The process according to claim 50, wherein

step i) comprises reacting the end of the compound according to any one of claims 1 to 49 which does not contain an alkyne group, with a functionalised molecule, linking the molecule to a first end of the compound; and

step ii) comprises reacting the end of the product from step i) which contains the alkyne group with a functionalised substrate, linking the substrate to a second end.

53. The process according to any one of claims 50 to 52, wherein the functionalised substrate is an azide-functionalised substrate.

54. The process according to any one of claims 51 to 53, wherein the functionalised molecule is an aldehyde-functionalised, a ketone-functionalised, or an amine-functionalised molecule.

55. A process according to any one of claims 50 to 54 wherein the substrate is a bead.

56. The process according to any one of claims 50 to 54, wherein the substrate is a particle.

The process according to claim 56, wherein the particle is a nanoparticle.

58. The process according to claim 57, wherein the nanoparticle is a quantum dot.

59. The process according to any one of claims 50 to 58, wherein the molecule is a biological molecule.

60. The process according to claim 59, wherein the biological molecule is a protein. 61 . A process for the preparation of a compound of Formula (V):

Formula (V)

wherein

each R¹ is independently selected from the group consisting of: H, halogen,

OH, NO₂, CN, SH, NH₂, CF₃, OCHF₂, OCF₃, optionally-substituted d-₁₂ alkyl, optionally-substituted CM₂ haloalkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C₂. ₂ heterocycloalkenyl, optionally-substituted C₆-18 aryl, optionally-substituted CMS heteroaryl, optionally-substituted C1 -12 alkyloxy, optionally-substituted C2-12 alkenyloxy, optionally-substituted C2-12 alkynyloxy, optionally-substituted C2-12 heteroalkyloxy, optionally-substituted C3-12 cycloalkyloxy, optionally-substituted C₃. ₂ cycloalkenyloxy, optionally-substituted C₂. ₂ heterocycloalkyloxy, optionally-substituted C2-12 heterocycloalkenyloxy, optionally- substituted C₆-18 aryloxy, optionally-substituted CMS heteroaryloxy, optionally- substituted Ci-12 alkylamino, SR^C, SO₃H, SO₂NR^cR^d, SO₂R, SONR^cR^d, SOR^c, COOH, COOR^c, COR^c, CONR^cR^d, NR^cCOR^d, NR^cCOOR^d, NR^cSO₂R^d, NR^cCONR^dR^e and NR^cR^d; or any two R¹ on adjacent carbon atoms form a fused substituent; R^c, R^d and R^e are each independently-selected from the group consisting of: H, OH, halogen, optionally-substituted C₁-₁₂ alkyl, optionally-substituted C₂-₁₂ heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted OC₆-is aryl, and optionally-substituted OCi-i₈ heteroaryl;

L is of the Formula (III): X³(CR⁸R⁹)_p [X⁴(CR ⁰R )_q]_r (CR ²R ³)_S X⁵

Formula (III)

X³ is selected from the group consisting of: NH, O and NR¹⁴;

X⁴ is independently selected from the group consisting of: a bond, O, NH,

NR¹⁴, S and CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: O, NH, NR¹⁴, optionally-substituted C-₁-₁₂ alkyl, HN-(optionally-substituted CM ₂ alkyl)-, R¹⁴N-(optionally-substituted CM ₂ alkyl)-, O-(optionally-substituted C-₁-₁₂ alkyl)-, optionally-substituted C5-₁₂ aryl, HN- (optionally-substituted C5-12 aryl)-, R¹⁴N-(optionally-substituted C5-12 aryl)-, O- (optionally-substituted C5-12 aryl)-, HN(CO)-(optionally-substituted C5-12 aryl)-, R¹⁴N(CO)-(optionally-substituted C_5-i₂ aryl)-, O(CO)-(optionally-substituted C_5-i₂ aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted C5-₁₂ aryl)-, optionally-substituted C₂-₁₂ heteroaryl, HN-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C2-12 heteroaryl)-, O-(optionally-substituted C₂-₁₂ heteroaryl)-, optionally-substituted C₂-₁₂ heteroaryl, HN(CO)-(optionally-substituted C_2-i₂ heteroaryl)-, R¹⁴N(CO)-(optionally- substituted C₂-₁₂ heteroaryl)-, O(CO)-(optionally-substituted C₂-₁₂ heteroaryl)-, (CO)HN-(optionally-substituted C₂-₁₂ heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C₂- ₁₂ heteroaryl)- and (CO)O-(optionally-substituted C₂-₁₂ heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C₁-₁₂ alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C3-₁₂ cycloalkyl, optionally substituted C3-₁₂ cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^AR^P, (CO)NHNHR¹⁷, (CO)NHNR^AR^P, NH(CO)NHNHR¹⁷, NH(CO)NHNR^AR^P, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^AR^P;

each R^A and R^P is independently selected from the group consisting of: H, OH, optionally-substituted CM 2 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C₃- ₂ cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^A and R^P when combined together provide the group of Formula (l-a):

each R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted CM₂ alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C₃- ₂ cycloalkenyl, optionally- substituted OCM₂ alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC₃- 12 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted CM₂ alkyl and N-protecting group;

coupling the compound of Formula (VI)

Formula (VI) wherein

R¹ and n are as defined above and LG is a leaving group;

in a coupling reaction with a compound of Formula (Vll-a):

X^3A(CR⁸R⁹)_P [X⁴(CR¹⁰R¹¹)_Q]_R (CR¹²R¹³)_S X^5A

Formula (Vll-a)

wherein

X^3A is selected from the group consisting of: OH, NH₂ and NHR¹⁴;

X^5A is selected from the group consisting of: OH, NH₂, NHR¹⁴, OR¹⁸, CO₂R¹⁹; each R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: H, OH, halogen, optionally-substituted CM₂ alkyl, optionally- substituted C₂-i₂ alkenyl, optionally-substituted C₂-i₂ alkynyl, optionally-substituted C₃- ^2 cycloalkyl, optionally-substituted C₃-i₂ cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally-substituted OC₂. ₂ alkenyl, optionally-substituted OC₂. ₂ alkynyl, optionally-substituted OC₃-i₂ cycloalkyl and optionally-substituted OC₃-i₂ cycloalkenyl;

R¹⁴ is selected from the group consisting of: optionally-substituted Ci-i₂ alkyl and N-protecting group;

R¹⁸ is selected from the group consisting of: optionally-substituted C-i- ₂ alkyl, optionally-substituted C₂-i₂ alkenyl, optionally-substituted C₂-i₂ alkynyl, optionally- substituted C₃-i₂ cycloalkyl, optionally-substituted C₃-i₂ cycloalkenyl and O-protecting group;

R¹⁹ is selected from the group consisting of: H, optionally-substituted C-i- ₂ alkyl, optionally-substituted C₂-i₂ alkenyl, optionally-substituted C₂-i₂ alkynyl, optionally-substituted C₃-i₂ cycloalkyl, optionally-substituted C₃-i₂ cycloalkenyl optionally-substituted C₆-is aryl, optionally-substituted C₆-is alkylaryl, optionally- substituted C₁-18 heteroaryl, optionally-substituted CMS alkylheteroaryl, optionally- substituted C1-12 alkyloxy, optionally-substituted C2-12 alkenyloxy, optionally- substituted C2-12 alkynyloxy, optionally-substituted C2-12 heteroalkyloxy, optionally- substituted C₃- ₂ cycloalkyloxy, optionally-substituted C₃- ₂ cycloalkenyloxy and optionally-substituted C2-12 heterocycloalkyloxy, each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6; to provide a compound of Formula (VIII):

Formula (VIII)

(b) reacting the compound of Formula (VIII) with a compound of Formula (IX):

R²⁰ B R²

Formula (IX)

wherein

B is selected from the group consisting of: a bond, an optionally-substituted Ci- ₁₂ alkyl group, optionally substituted C₂-₁₂ alkenyl, optionally substituted C₂-₁₂ alkynyl, optionally-substituted C5-₁₂ aryl group and optionally-substituted C₂-₁₂ heteroaryl group, optionally substituted C₃- ₂ cycloalkyl, optionally substituted C₃- ₂ cycloalkenyl; each R² and R²⁰ is selected independently from the group consisting of: OH, CO₂R²², optionally-substituted OC1-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC₂-₁₂ alkynyl, optionally-substituted OC₃-i₂ cycloalkyl, optionally-substituted OC₃-i₂ cycloalkenyl, NHR¹⁷, NHNH R¹⁷, NHN R^aR^p, ONHR¹⁷, NH(CO)(optionally-substituted d-₆ alkyl)ONHR¹⁷, NH(CO)(optionally-substituted d-₆ alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally- substituted Ci_-6 alkyl)NHN R^AR^P, (CO)N H(optionally-substituted Ci_-6 alkyl)ONH R¹⁷, (CO)NH(optionally-substituted d-₆ alkyl)NH₂, (CO)NH(optionally-substituted d-₆ alkyl)NHNH R¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH N R^AR^P, (CO)NHNH R¹⁷, (CO)NHN R^AR^P, NH (CO)NHNH R^{1 7}, NH(CO)N HN R^AR^P, NHNH(CO)NHN H R¹⁷, and NHNH(CO)NHN R^AR^P;

of H, optionally-substituted CM₂ alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

each R¹⁷ and R²¹ is independently selected from the group consisting of: H, optionally-substituted CM₂ alkyl and N-protecting group;

R²² is selected from the group consisting of: H, halogen, optionally-substituted CM2 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted C2-12 heterocycloalkenyl, optionally-substituted C₆-18 aryl, optionally-substituted C₆-18 alkylaryl, optionally-substituted CM₈ heteroaryl, optionally-substituted CMS alkylheteroaryl; to provide a compound of Formula (V).

62. The process according to claim 61 , wherein the compound of Formula (IX) is of Formula (IX-a):

O

HO^^ NR¹⁷

Formula (IX-a)

wherein

R¹⁷ is selected from the group consisting of: optionally-substituted C1 -12 alkyl and N-protecting group.

63. The process according to claim 61 , wherein the compound of Formula (IX) is of Formula (IX-b):

Formula (IX-b)

wherein

R¹⁷ is selected from the group consisting of: optionally-substituted C-1 -12 alkyl and N-protecting group.

64. The process according to claim 61 , wherein the compound of Formula (IX) is of Formula (IX-c):

Formula (IX-c)

wherein

65. The process according to claim 61 , wherein the compound of Formula (IX) is of Formula (IX-d):

Formula (IX-d)

wherein

R is selected from the group consisting of: OH , optionally-substituted OCM ₂ alkyl, optionally-substituted OC₂-₁₂ alkenyl, optionally-substituted OC₂-₁₂ alkynyl, optionally-substituted OC₃- ₂ cycloalkyl and optionally-substituted OC₃- ₂ cycloalkenyl.

66. The process according to claim 61 , wherein the compound of Formula (IX) is of Formula (IX-e):

Formula (IX-e)

67. The process according to any one of claims 61 to 66, wherein the compound produced in step b) is further N-deprotected or N-dealkylated.

68. A process for the preparation of a compound of Formula (V):

Formula (V)

wherein

R¹ is selected from the group consisting of: H , halogen, OH , NO₂, CN , SH , N H₂, CF₃, OCH F₂, OCF3, optionally-substituted CM₂ alkyl, optionally-substituted C1 -12 haloalkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C₃- ₂ cycloalkenyl, optionally-substituted C2-12 heterocycloalkyl, optionally-substituted C2-12 heterocycloalkenyl, optionally-substituted C₆-is aryl, optionally-substituted CM S heteroaryl, optionally-substituted CM 2 alkyloxy, optionally- substituted C2-12 alkenyloxy, optionally-substituted C2-12 alkynyloxy, optionally- substituted C2-12 heteroalkyloxy, optionally-substituted C3-12 cycloalkyloxy, optionally- substituted C₃- ₂ cycloalkenyloxy, optionally-substituted C2-12 heterocycloalkyloxy, optionally-substituted C2-12 heterocycloalkenyloxy, optionally-substituted C₆-18 aryloxy, optionally-substituted CMS heteroaryloxy, optionally-substituted CM₂ alkylamino, SR^C, SO3H, SO₂N R^CR^D, SO₂R, SON R^CR^D, SO R^C, COOH , COOR^C, COR^C, CON R^CR^D, N R^CCOR^D, N R^CCOOR^D, N R^CSO₂R^D, N R^CCON R^D R^E and N R^CR^D ; or any two R¹ on adjacent carbon atoms form a fused substituent;

R^C, R^D and R^E are each independently-selected from the group consisting of: H , OH , halogen, optionally-substituted CM₂ alkyl, optionally-substituted C2-12 heteroalkyl, optionally-substituted C₃. ₂ cycloalkyl, optionally-substituted OC₆-is aryl, and optionally-substituted OCM ₈ heteroaryl;

L is of the Formula (III): X³(C R⁸R⁹)_P [X⁴(C R^{1 0}R^{1 1} )_Q]_R (C R^{1 2}R¹³)s X⁵

Formula (III)

X³ is selected from the group consisting of: O, N H and N R¹⁴;

X⁴ is selected from the group consisting of: a bond, O, N H , N R¹⁴, S and

CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, O, N H , N R¹⁴, S, C R¹⁵R¹⁶, optionally-substituted CM ₂ alkyl, H N-(optionally-substituted CM₂ alkyl)-, R¹⁴N- (optionally-substituted CM ₂ alkyl)-, O-(optionally-substituted CM₂ alkyl)-, optionally- substituted C5-12 aryl, H N-(optionally-substituted C5-12 aryl)-, R¹⁴N-(optionally- substituted C5-12 aryl)-, O-(optionally-substituted C5-12 aryl)-, H N (CO)-(optionally- substituted C₅.₁₂ aryl)-, R¹⁴N (CO)-(optionally-substituted C₅.₁₂ aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-, (CO)N H-(optionally-substituted C5-12 aryl)-, (CO) R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C2-12 heteroaryl, H N-(optionally-substituted C2-12 heteroaryl)-, R¹⁴N-(optionally-substituted C₂. ₂ heteroaryl)-, O-(optionally-substituted C₂. ₂ heteroaryl)-, optionally-substituted C2-12 heteroaryl, H N (CO)-(optionally-substituted C₂. 12 heteroaryl)-, R¹⁴N (CO)-(optionally-substituted C2-12 heteroaryl)-, O(CO)-(optionally- substituted C₂-₁₂ heteroaryl)-, (CO)HN-(optionally-substituted C₂-₁₂ heteroaryl)-, (CO)R¹⁴N-(optionally-substituted C₂-₁₂ heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted C₁-₁₂ alkyl, optionally substituted C₂-₁₂ alkenyl, optionally substituted C₂-₁₂ alkynyl, optionally substituted C3-₁₂ cycloalkyl, optionally substituted C3-₁₂ cycloalkenyl, NHR¹⁷, ONHR¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, (CO)NH(optionally-substituted Ci_-6 alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNR^aR^p, NHNH(CO)NHNHR¹⁷ and NHNH(CO)NHNR^aR^p;

each R³, R⁴, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted CM2 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally-substituted C₃- ₂ cycloalkyl, optionally-substituted C₃- ₂ cycloalkenyl, optionally-substituted OCM₂ alkyl, optionally-substituted OC2-12 alkenyl, optionally- substituted OC₂-₁₂ alkynyl, optionally-substituted OC₃- ₂ cycloalkyl and optionally- substituted OC3-12 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted CM ₂ alkyl and N-protecting group; and

n is an integer selected from the group consisting of: 0, 1 , 2, 3, 4, 5, 6, 7 and 8; each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, the process comprising:

(a) coupling a compound of Formula (Vll-b):

X^3A(CR⁸R⁹)_P [X⁴(CR^{1 0}R^{1 1} )_Q]_R (CR^{1 2}R^{1 3})_S X⁵ R²

Formula (Vll-b)

X^3A is selected from the group consisting of: OH, NH₂ and N H R¹⁴;

X⁴ is selected from the group consisting of: a bond, O, NH, N R¹⁴, S, CR¹⁵R¹⁶;

X⁵ is selected from the group consisting of: a bond, O, NH, N R¹⁴, S, CR¹⁵R¹⁶, optionally-substituted CM₂ alkyl, HN-(optionally-substituted CM₂ alkyl)-, R¹⁴N- (optionally-substituted CM₂ alkyl)-, O-(optionally-substituted CM₂ alkyl)-, optionally- substituted C5-12 aryl, HN-(optionally-substituted C5-12 aryl)-, R¹⁴N-(optionally- substituted C5-₁₂ aryl)-, O-(optionally-substituted C5-₁₂ aryl)-, HN(CO)-(optionally- substituted C_5-i₂ aryl)-, R¹⁴N(CO)-(optionally-substituted C_5-i ₂ aryl)-, O(CO)- (optionally-substituted C5-12 aryl)-, (CO)NH-(optionally-substituted C5-12 aryl)-, (CO)R¹⁴N-(optionally-substituted C5-12 aryl)-, (CO)O-(optionally-substituted C5-12 aryl)-, optionally-substituted C₂-₁₂ heteroaryl, HN-(optionally-substituted C₂-₁₂ heteroaryl)-, R¹⁴N-(optionally-substituted C₂-₁₂ heteroaryl)-, O-(optionally-substituted C₂-₁₂ heteroaryl)-, optionally-substituted C₂-₁₂ heteroaryl, HN(CO)-(optionally-substituted C₂- ₁₂ heteroaryl)-, R¹⁴N(CO)-(optionally-substituted C₂-₁₂ heteroaryl)-, O(CO)-(optionally- substituted C₂-₁₂ heteroaryl)-, (CO)HN-(optionally-substituted C₂-₁₂ heteroaryl)-, (CO) R¹⁴N-(optionally-substituted C₂-₁₂ heteroaryl)- and (CO)O-(optionally-substituted C2-12 heteroaryl)-;

R² is selected from the group consisting of: optionally substituted CM₂ alkyl, optionally substituted C2-12 alkenyl, optionally substituted C2-12 alkynyl, optionally substituted C₃- ₂ cycloalkyl, optionally substituted C₃- ₂ cycloalkenyl, NH R¹⁷, ONH R¹⁷, NHNHR¹⁷, NHNR^aR^p, NH(CO)(optionally-substituted Ci_-6 alkyl)ONHR¹⁷, NH(CO)(optionally-substituted d-e alkyl)NHR¹⁷, NH(CO)(optionally-substituted d-e alkyl)NHNHR¹⁷, NH(CO)(optionally-substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NH(optionally-substituted d-e alkyl)ONHR¹⁷, (CO)NH(optionally-substituted d-e alkyl)NH₂, (CO)NH(optionally-substituted Ci_-6 alkyl)NHNHR¹⁷, (CO)NH(optionally- substituted Ci_-6 alkyl)NHNR^aR^p, (CO)NHNHR¹⁷, (CO)NHNR^aR^p, NH(CO)NHNHR¹⁷, NH(CO)NHNHR¹⁷, NHNH(CO)NHNHR¹⁷, NHNH(CO)NHNR^aR^p;

each R^a and R^p is independently selected from the group consisting of: H, OH, optionally-substituted d-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally-substituted Od-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC3-12 cycloalkyl and optionally-substituted OC3-12 cycloalkenyl; or R^a and R^p when combined together provide the group of Formula (l-a):

Formula (l-a)

wherein each of R^x and R⁵ is independently selected from the group consisting of H, optionally-substituted d-12 alkyl, optionally-substituted C2-12 alkenyl, optionally- substituted C2-12 alkynyl, optionally-substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl;

each R⁸, R⁹, R¹⁰, R¹¹ , R¹², R¹³, R¹⁵ and R¹⁶ is independently selected from the group consisting of: hydrogen, OH, halogen, optionally-substituted d-12 alkyl, optionally-substituted C2-12 alkenyl, optionally-substituted C2-12 alkynyl, optionally- substituted C3-12 cycloalkyl, optionally-substituted C3-12 cycloalkenyl, optionally- substituted Od-12 alkyl, optionally-substituted OC2-12 alkenyl, optionally-substituted OC2-12 alkynyl, optionally-substituted OC₃- ₂ cycloalkyl and optionally-substituted OC₃. 12 cycloalkenyl;

each R¹⁴ and R¹⁷ is independently selected from the group consisting of: H, optionally-substituted d-12 alkyl and N-protecting group; and

each p, q, r and s is an integer independently selected from the group consisting of: 0, 1 , 2, 3, 4, 5 and 6, coupling reaction with a compound of Formula (VI)

Formula (VI) wherein

69. The process according to claim 68, wherein the compound of Formula (V) is further N-deprotected or N-dealkylated.

70. The process according to any one of claims 61 to 69, wherein LG is selected independently from the group consisting of:

halogen, , and

71 . The process according to any one of claims 61 to 70, wherein the compound of Formula (VI) is selected from:

or