WO2025022130A1

WO2025022130A1 - Dendrimers comprising fluorescent dyes

Info

Publication number: WO2025022130A1
Application number: PCT/GB2024/051974
Authority: WO
Inventors: Jon Andrew Preece; Mark Shepherd; Alex Philip Graham Robinson; Michael BUTLIN; Parvez IQBAL; Sareena SUND; Sunil CLAIRE; Karolis VIRZBICKAS
Original assignee: Chromatwist Ltd
Current assignee: Chromatwist Ltd
Priority date: 2023-07-27
Filing date: 2024-07-26
Publication date: 2025-01-30
Anticipated expiration: 2026-01-27
Also published as: GB202311550D0

Abstract

A molecular scaffold is provided having n successive generations. The molecular scaffold having a core having plural arms (Al), n-1 sets of branching moieties each having plural arms (A2, A3, A4), a functional group (FG1) capable of forming a bond with a second species and plural identifier molecules (IM). Where present each branching moiety of the first set is bonded to one of the arms (Al) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set. The plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=l the plural identifier molecules are each bonded to an arm (Al) of the core. The one or more or all of the identifier molecules (IM) have the following general structure (1). The core has the following general structure (Formula I). The molecular scaffold is useful for labelling molecules, e.g. biomolecules such as antibodies, that can be excited at the same, or similar wavelengths, but that emit at different wavelengths. In particular, it is useful as a labelling system for use in flow cytometry.

Description

DENDRIMERS COMPRISING FLUORESCENT DYES

This invention relates generally to a molecular scaffold. More specifically, although not exclusively, this invention relates to a molecular scaffold comprising plural identifier molecules, e.g. luminescent moieties or compounds, the molecular scaffold being capable of forming a bond, e.g. a covalent bond, with a second species, e.g. a biomolecule, for use as a chemical sensor or a biosensor.

Dyes, e.g. fluorescent dyes, are frequently used as labels, tags, or probes, e.g. in assays, for cells and/or in tissue samples. Such dyes may be used in a wide range of biological applications, for example, in immunofluorescence assays, flow cytometry, fluorescence microscopy, Western blot, and cellular imaging.

Some of the most common fluorescent dyes include xanthene derivatives, e.g. fluorescein, eosin, rhodamine, Oregan green (RTM), and Texas red (RTM). Many other organic fluorophore families are known.

One of the most widely used classes of fluorescent dye is the Alexa Fluor(RTM) series designed by Molecular Probes and currently marketed by ThermoFisher Scientific (Waltham, Massachusetts, United States). The Alexa Fluor(RTM) series comprises more than twenty different fluorescent dyes that exhibit emission spectra that span the near-UV, visible, and near-IR spectral range.

It is known to conjugate fluorescent dyes to a biomolecule or species of interest by forming a covalent bond between the fluorescent dye and the biomolecule. For example, it is known to conjugate fluorescent dyes to antibodies. This may be used to track the conjugated antibodies to visualise its interaction with specific antigens. Typically, the fluorescent dye forms a covalent bond to the biomolecule via a functional group located on the fluorescent dye, for example, a thiol-reactive or amine-reactive functional group.

It is known to use fluorescent dyes in multiplex assay systems. Multiplex assays combine assays for multiple target analytes in a single reaction volume. This reduces workflow and sample volume problems. It is known to use combinations of different fluorescent dyes to detect different analytes. However, often the different dyes require different excitation wavelengths, which adds complexity to the system. It would be advantageous to provide a molecular scaffold for use in labelling molecules, e.g. biomolecules such as antibodies, that can be excited at the same, or similar wavelengths, but that emit at different wavelengths.

In particular, it would be useful to have a labelling system for use in flow cytometry.

It would be useful to have a labelling system which provides larger signal to noise ratios than prior art systems. It would also be useful to have a system which allows for the provision of unique or tuned signals.

Accordingly, a first aspect of the invention provides a molecular scaffold (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecular scaffold comprising: a core having or comprising plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set; wherein the plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moiety in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core; wherein one or more or preferably all of the identifier molecules (IM) have the following general structure (1 ):

wherein X represents one of NH, O, S, Se;

R represents an aromatic group and/or an aliphatic group which may be further functionalised; q and s are independently integers of 1 , 2, 3, or 4; p is an integer of 1 or 2;

Y¹, Y², Y³ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^x (e.g. -OCH₃, -O(CH₂CH₂O)nCH₂COOH or -O(CH₂CH₂O)_nCH₃ wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH₃), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C₂-6 alkenyl, C1-6 haloalkyl, C₂-e alkynyl; wherein one or more of Y¹, Y², Y³ and/or R comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

Optionally, when the molecular scaffold carries a charge, e.g. a single negative charge, there is a counterion associated therewith, e.g. selected from Na⁺ or K⁺.

Advantageously, it has been found that a molecular scaffold (e.g. a dendrimer or a dendron) having plural identifier molecules is able to provide an amplified signal in comparison to the use of only one identifier molecule as a label, tag, or probe.

In embodiments, the identifier molecules (IM) may all be the same molecule. Alternatively, one or more of the identifier molecules (IM) may be different from the other identifier molecules (IM). Advantageously, functionalising the molecular scaffold with different identifier molecules (IM) may provide the scaffold with a unique emission signature or fingerprint.

In embodiments, X represents an oxygen atom or a sulphur atom, preferably an oxygen atom.

In embodiments one or more of Y¹, Y², Y³ may independently comprise a heterocyclic moiety. In embodiments two or more of Y¹, Y², Y³ may combine together to form a condensed or fused ring (e.g. a condensed or fused aromatic ring), which may be substituted. In embodiments, one or more or all of Y¹, Y², and Y³, may independently represent a polyglycol group, e.g. a PEG (poly(ethylene glycol)) moiety (e.g. -O(CH2CH2O)bCH3) wherein b is an integer selected from 1 , 2, 3, 4, or 5. Advantageously, it has been found that the use of one or more polyglycol groups, e.g. a PEG group, provides increased water solubility, which increases the utility of the molecular scaffold, e.g. in biological systems or assays.

In embodiments, one or more of Y¹, Y², Y³ and/or R may comprise a spacing portion. In embodiments, the spacing portion may comprise a continuous chain of between 3 and 20 atoms, and further comprising said linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

In embodiments, the linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4) is located at a or the terminus of the spacing portion.

In embodiments, the spacing portion may comprise a continuous chain of between 3 and 20 atoms selected from carbon atoms or a combination of carbon atoms and heteroatoms, e.g. oxygen atoms or nitrogen atoms.

In embodiments, the spacing portion may comprise or consist of a polyether chain comprising a continuous chain of between five and twenty atoms, e.g. 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms selected from carbon atoms and oxygen atoms.

In embodiments, the spacing portion and the linker group (L) may consist of an -O- (CH2CH2O)_ZCH2-L moiety, wherein z may be an integer of 1 , 2, 3, 4, or 5. In embodiments, the spacing portion and the linker group (L) may consist of an -O-(CH2CH2O)2CH2-L moiety.

In embodiments, the R group may comprise the linker group (L) and for example a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon atoms or a combination or carbon atoms and heteroatoms, for example oxygen atoms. In embodiments, the linker group (L) may be located at the terminus of the spacing portion.

In embodiments, the R group only may comprise the linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4) and for example a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon atoms or a combination or carbon atoms and heteroatoms, for example oxygen atoms. In embodiments, the linker group (L) may be located at the terminus of the spacing portion .

In embodiments, R may consist of an -O-(CH2CH2O)_ZCH2-L moiety, wherein z is an integer of 1 , 2, 3, 4, or 5.

In embodiments, R may be represented by the following general structure:

wherein Ar represents a substituted or unsubstituted aryl group, e.g. comprising one, two, or three fused rings; e and f are independently integers of 0, 1 , 2, 3;

G is an alkyl group, e.g. -CH2CH2-;

L is the linker group (L).

Advantageously, the G moiety may be used to modify the aqueous buffer solubility of the molecular scaffold.

In embodiments, Ar may represent a phenyl, naphthyl, anthracyl, triphenyl, pyrenyl, pyridyl, thiophenyl, furanyl, indonyl group.

In embodiments, the aryl group (Ar) may comprise substituents selected from H, D, F, Cl, Br, I, CN, NO2, OH, COOH, OR^X, a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C-i- 6 haloalkyl, C2-6 alkynyl, NH2, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-6 alkynyl. In embodiments, R may be represented by one of the following general structures (i), (ii), (iii):

wherein G, e, f, and L are defined as above; and the remainder of Q¹, Q², Q³, Q⁴, Q⁵, Q⁶, Q⁷, Q⁸, Q⁹, Q¹⁰, Q¹¹, Q¹², Q¹³, Q¹⁴, Q¹⁵, Q¹⁶, Q¹⁷, Q¹⁸, Q¹⁹, Q²⁰, Q²¹ independently comprise, consist of, or represent H, D, F, Cl, Br, I, CN, NO2, OH, COOH, OR^X, a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C1-6 haloalkyl, C2-e alkynyl, NH2, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-e alkynyl.

In preferable embodiments, Q¹, Q², Q³, Q⁴, Q⁵, Q⁶, Q⁷, Q⁸, Q⁹, Q¹⁰, Q¹¹, Q¹², Q¹³, Q¹⁴, Q¹⁵, Q¹⁶, Q¹⁷, Q¹⁸, Q¹⁹, Q²⁰, Q²¹ independently comprise, consist of, or represent C3-9 aryl, CH3, NO2, F, NR^yR^z, OR^X, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-ealkynyl.

In embodiments, one or more or each of the identifier molecules (IM) may be luminescent. In embodiments, one or more or each of the identifier molecules (IM) may be a fluorophore. In embodiments, one or more or each of the identifier molecules (IM) may comprise a core comprising a polycyclic aromatic hydrocarbon, e.g. comprising five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty or more fused ring, e.g. 6-membered rings. In embodiments, one or more or each of the identifier molecules (IM) may comprise a core comprising a polycyclic aromatic hydrocarbon comprising plural six fused 6-membered rings. In embodiments, one or more or each of the identifier molecules may be or comprise a triphenylene derivative.

In embodiments, one or more or each of the identifier molecules (IM) may comprise or be a luminescent compound as disclosed in any one of our earlier applications PCT/GB2019/050809, PCT/GB2019/050806, PCT/GB2020/052324, PCT/GB2020/052325 or PCT/GB2020/052323.

In embodiments, each identifier molecule (IM) may be independently selected from Compounds 1 to 6 shown in the Figures.

Advantageously, the provision of plural identifier molecules (IM), e.g. luminescent and/or fluorescent identifier molecules, may amplify the signal, e.g. the luminescent or fluorescent signal, of the identifier, which may thereby improve identification (as signal-to-noise ratio will improve).

In embodiments, the identifier molecules (IM) may have any of the general structures shown below in place of general structure (1 ). In embodiments, one, or each of, or more than one, or all of the identifier molecule (IM) is represented by the following general formula (2):

wherein X represents one of NH, O, S, Se;

R represents an aromatic group and/or an aliphatic group which may be further functionalised;

Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. - O(CH₂CH2O)nCH₂COOH or -O(CH₂CH₂O)_nCH₃wherein n is an integer of 1 , 2, 3, 4, or 5, a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl; two or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ may combine together to form or comprise a condensed or fused ring (e.g. a condensed or fused aromatic ring); wherein one or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ and R comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4); and optionally wherein the molecular scaffold carries a charge, e.g. a single negative charge, there is a counterion associated therewith, e.g. selected from Na⁺ or K⁺.

In embodiments, one or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ may independently comprise a heterocyclic moiety. In embodiments two or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ may combine together to form a condensed or fused ring (e.g. a condensed or fused aromatic ring), which may be substituted.

In embodiments, one or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³, may independently represent a polyglycol group, e.g. a PEG (poly(ethylene glycol)) moiety (e.g. - O(CH2CH2O)bCH3) wherein b is an integer selected from 1 , 2, 3, 4, or 5. Advantageously, it has been found that the use of one or more polyglycol groups, e.g. a PEG group, provides increased water solubility, which increases the utility of the molecular scaffold in biological systems.

In embodiments, one or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ and/or R may comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, and further comprising said linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

In embodiments, the spacing portion comprises a continuous chain of between 3 and 20 atoms selected from carbon atoms ora combination of carbon atoms and heteroatoms, e.g. oxygen atoms.

In embodiments, the spacing portion comprises or consists of a polyether chain comprising a continuous chain of between five and twenty atoms, e.g. 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms selected from carbon atoms and oxygen atoms.

In embodiments, the spacing portion and the linker group (L) consists of an -O- (CH2CH2O)_ZCH2-L moiety, wherein z is an integer of 1 , 2, 3, 4, or 5. In embodiments, the spacing portion and the linker group (L) may consist of an -O-(CH2CH2O)2CH2-L moiety.

In embodiments, one or more or all Y⁴ Y⁷, Y⁸, Y¹¹, Y¹² independently represent an alkoxy group, e.g. an -OR’ group, e.g. a OC5H11 group or a OCH3 group.

In embodiments, one or more ofY⁵, Y⁶, Y⁹, Y¹⁰, Y¹³ (for example each) represent a hydrogen atom. In embodiments, R comprises the spacing portion and the linker group (L). In embodiments, R is defined in general formula (2) in the same way as for general formula (1 ).

In embodiments, the R group may comprise the linker group (L) and for example a spacing portion comprising a continuous chain of between 3 and 20 atoms. In embodiments, the linker group (L) may be located at the terminus of the spacing portion.

In embodiments, the R group only may comprise the linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

In embodiments, one, or each of, or more than one, or all of the identifier molecule (IM) is represented by the following general formula (3):

wherein X represents one of NH, O, S, Se;

J¹, J², J³, J⁴, J⁵ and Z independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. -OCH₃, -O(CH2CH₂O)nCH₂COOH or - O(CH2CH2O)_nCH3wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C1-6 haloalkyl, C2-6 alkynyl, NH2, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-e alkynyl; two or more of J¹, J², J³, J⁴, J⁵ and/or Z may combine together to form a condensed ring (e.g. a condensed aromatic ring);

A¹, A², A³, A⁴ independently represents a hydrogen atom, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group (e.g. CH3, C5H9, - (CH2CH2O)_nCH2COOH or - (Cf^CFfeOJnCHawherein n is an integer of 1 , 2, 3, 4, or 5), ), a polyether group; wherein one or more of A¹, A², A³, A⁴ and Z comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4); and optionally wherein the molecular scaffold carries a charge, e.g. a single negative charge, there is a counterion associated therewith, e.g. selected from Na⁺ or K⁺.

In embodiments, one or more or all of A⁴ and/or R and/or Z comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon and oxygen atoms, and further comprising said linker group (L), e.g. located at the terminus of the spacing portion, forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

In embodiments, the spacing portion and the linker group (L) consists of an -O- (CH₂CH₂O)2CH2-L moiety.

In embodiments, Z represents the spacing portion comprising a linker group. In embodiments, Z consists of an -O-(CH2CH2O)2CH2-L moiety.

In embodiments, one or more or all A¹, A², A³, A⁴ independently represent an alkyl group, e.g. a C5H11 group or a CH3 group, and/or J¹, J², J³, J⁴, J⁵ each represent a hydrogen atom. In embodiments, one, or each of, or more than one, or all of the identifier molecule (IM) is represented by the following general formula (4):

wherein X represents one of NH, O, S, Se;

J¹, J², J³, J⁴, J⁵ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. -OCH₃, -O(CH₂CH₂O)nCH₂COOH or - O(CH₂CH₂O)_nCH3wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl; two or more of J¹, J², J³, J⁴, J⁵ may combine together to form a condensed ring (e.g. a condensed aromatic ring);

A¹, A², A³, A⁴, A⁵ independently represents a hydrogen atom, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group (e.g. CH3, - O(CH₂CH₂O)_nCH₂COOH or -O(CH₂CH₂O)nCH₃ wherein n is an integer of 1 , 2, 3, 4, or 5),), a polyether group; wherein R comprises a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4); and optionally wherein the molecular scaffold carries a charge, e.g. a single negative charge, there is a counterion associated therewith, e.g. selected from Na⁺ or K⁺. In embodiments, R comprises a spacing portion comprising a continuous chain of between

3 and 20 atoms, e.g. wherein the linker group (L) is located at the terminus of the spacing portion.

In embodiments, the R group (for example, the R group only) may comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, and further comprising a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

In embodiments, R, e.g. the spacing portion of R, is defined as for general structure (1 ), (2), (3), or (5).

In embodiments, X represents an oxygen atom or a sulphur atom.

In embodiments, A¹, A², A³, A⁴, A⁵ may independently represent a polyglycol group, e.g. a PEG (poly(ethylene glycol)) moiety (e.g. -(CF^CFWJbCHa) wherein b is an integer selected from 1 , 2, 3, 4, or 5. Advantageously, it has been found that the use of one or more polyglycol groups, e.g. a PEG group, provides increased water solubility, which increases the utility of the molecular scaffold in biological systems.

In embodiments, A¹, A², A³, A⁴, A⁵ may independently represent an alkyl group, e.g. CH3, or C5H11. In embodiments, one or more or all of J¹, J², J³, J⁴, J⁵ each represent a hydrogen atom, A¹, A², A³, A⁴, A⁵ each represent an alkyl group, e.g. a CH3 group, or a C₅Hn group.

In embodiments, R represents a -O-(CH2CH2O)_nCH2CH2L moiety wherein L represents the linker group and n is an integer of 1 , 2, 3, 4, or 5.

In embodiments, R consists of an -O-(CH2CH2O)2CH2-L moiety.

In embodiments, the identifier molecule (IM) has the following general structure (5):

wherein X, R, q, s, Y¹, Y², Y³, L are defined as shown above in general structure (1 ); p is an integer selected from a number between 1 to 10, e.g. 2, 3, or 4. Preferably, p is 2.

Throughout this specification, the term “the linker group (L)” is intended to mean the functional group resulting from reaction of a functional group (FG2) on the identifier molecule (IM) which is capable of reacting with a complementary functional group (FG3) located on the arm (A1 , A2, A3, or A4) of the molecular scaffold.

In embodiments, the linker group (L) may be an amide, e.g. formed from reaction of an amine (e.g. FG3 located on an arm A1 , A2, A3, or A4 of the molecular scaffold), and a carboxylic acid (e.g. FG2 located on the identifier molecule (IM)).

In embodiments, the linker group (L) may be a triazole, e.g. formed via click chemistry from reaction of an alkyne (e.g. FG2 or FG3) and an azide (e.g. FG2 or FG3).

In embodiments, FG2 or FG3 may be a carboxylic acid, or a derivative of a carboxylic acid, i.e. an active ester, e.g. -C=O(Y) wherein Y is replaced by a group on the arm of the molecular scaffold in a reaction. For example, Y may be selected from OH, OD, OR^X, NH2, ND2, SH, NHR^X, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from C1-6 alkyl optionally substituted with one or more substituents, for example selected from H, D, F, Cl, Br, I, OH, NH2. In embodiments, FG2 or FG3 may comprise or be a thiol (-SH), a thioacetate (-SAc), an aliphatic alcohol (-OH) or a phenol, an amine (-NH2), an alkyne, an azide (-N3), a maleimide group, an isothiocyanate (-N=C=S), an aldehyde, a ketone.

Advantageously, the identifier molecule (IM) may comprise one or more moieties to improve its solubility in water. In embodiments, the moieties for improving water solubility may be a PEG (poly(ethylene glycol)) moiety.

For example, the identifier molecule (IM) may comprise a PEG (poly(ethylene glycol)) moiety (e.g. -O(CH2CH2O)bCH3) wherein b is an integer selected from 1 , 2, 3, 4, or 5, a PEI (polyethylenimine) moiety, a carboxylic acid moiety.

In embodiments, R may be an alkyl group, for example, a straight or branched alkyl chain. In embodiments, R may be a methyl, ethyl, propyl, butyl group.

In embodiments wherein R is an aromatic group, the aromatic group may be one of, or a combination of, an aromatic hydrocarbon group, and/or an aromatic heterocyclic group.

In embodiments, R may be selected from C1-20 alkyl, C2-20 alkylene, C3-9 cycloalkyl, Cs-garyl, C3-9 heteroaryl, C3-9 heterocyclic.

In embodiments wherein R is an aromatic hydrocarbon group, the aromatic hydrocarbon group may comprise one of, or a combination of, a phenyl ring and/or a substituted phenyl ring. There may be one, two, three, four, or five additional substituents on the phenyl ring. The substituents are bonded directly to the phenyl ring, and may be one of, ora combination of, fluorine, chlorine, bromine, iodine, a hydroxyl group, an amine group, a nitro group, an alkoxy group, a carboxylic acid, an amide, a cyano group, a trifluoromethyl, an ester, an alkene an alkyne, an azide, an azo, an isocyanate, a ketone, an aldehyde, an alkyl group consisting of a hydrocarbon chain, or a hydrocarbon ring, an alkyl group consisting of other heteroatoms such as fluorine, chlorine, bromine, iodine, oxygen, nitrogen, and/or sulphur. The alkyl group may comprise a hydroxyl group, an amine group, a nitro group, an ether group, a carboxylic acid, an amide, a cyano group, trifluoromethyl, an ester, an alkene an alkyne, an azide, an azo, an isocyanate, a ketone, an aldehyde, for example. The substituents may be another aromatic group, for example, R may comprise a phenyl substituted with a further phenyl ring. In embodiments, the R group may be a phenyl ring, substituted with a second phenyl ring, which in turn is substituted with a third phenyl ring. In embodiments, R, R¹, R², or R³ may represent a p-fluorophenyl group, a m-fluorophenyl group, an o-fluorophenyl group, a thiophene group, a cyanophenyl moiety (e.g. a p- cyanophenyl moiety), a trifluoromethylphenyl moiety (e.g. a p-trifluoromethylphenyl moiety), an iodophenyl moiety (e.g. an o-iodophenyl moiety), a chlorophenyl moiety (e.g. an o- chlorophenyl moiety), a bromophenyl moiety (e.g. an o-bromophenyl moiety), an aminophenyl moiety (e.g. a mono-substituted ordi-substituted or trisubstituted aminophenyl moiety), a nitrophenyl moiety (e.g. a p-nitrophenyl moiety), a phenol moiety.

In embodiments wherein R is an aromatic group, the aromatic group may be a polycyclic aromatic hydrocarbon, for example, naphthalene, anthracene, phenanthrene, tetracene, chrysene, triphenylene, pyrene, pentacene, benzo[a]pyrene, corannulene, benzo[ghi]perylene, coronene, ovalene, fullerene, and/or benzo[c]fluorene. The R, R¹, R², and/or R³ group may be bonded to the triphenylene derivative by any isomer of the polycyclic aromatic hydrocarbons described, for example, 1 -napthalene, 2-napthalene, 2- anthracene, 9-anthracene. The polycyclic aromatic hydrocarbon group may be substituted with other moieties such as aryl groups, alkyl groups, heteroatoms, and/or other electron withdrawing or electron donating groups.

In embodiments, R is naphthalene.

In embodiments wherein R is an aromatic heterocyclic group, the heterocyclic group may be a three membered ring, a four membered ring, a five membered ring, a six membered ring, a seven membered ring, an eight membered ring, a nine membered ring, a ten membered ring, or a fused ring. In embodiments, the heterocyclic group may be furan, benzofuran, isobenzofuran, pyrrole, indole, isoindole, thiophene, benzothiophene, benzo[c]thiophene, imidazole, benzimidazole, purine, pyrazole, indazole, oxazole, benzoxazole, isoxazole, benzisoxazole, thiazole, benzothiazole, pyridine, quinoline, isoquinoline, pyrazine, quinoxaline, acridine, pyrimidine, quinozoline, pyridazine, cinnoline, phthalazine, 1 ,2,3-triazine, 1 ,2,4-triazine, 1 , 3, 5-triazine. pyridine or thiophene. In embodiments wherein R is an aliphatic group, the aliphatic group may be one of, or a combination of, an n-alkyl chain, a branched alkyl chain, an alkyl chain comprising unsaturated moieties, an alkyl chain comprising heteroatoms, for example, fluorine, chlorine, bromine, iodine, oxygen, sulphur, nitrogen. The alkyl chain may comprise unsaturated portions, comprising alkenes, or aromatic moieties. The alkyl chain may comprise functional groups for further derivatisation of the polycyclic aromatic hydrocarbon, e.g. triphenylene, derivative. For example, the functional groups may be one or more of an azide, a carbonyl group, an alcohol, a halogen, an alkene, or a thioacetate.

In embodiments, R comprise a crown ether.

The term C_m-n refers to a group with m to n carbon atoms.

The term “C1-20 alkyl” refers to a linear or branched hydrocarbon chain containing 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, for example methyl, ethyl, n-propyl, /so-propyl, n-butyl, /so-butyl, sec-butyl, fert-butyl, n-pentyl and n-hexyl, n- heptyl, n-octyl, n-decyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n- pentadecyl, n-hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, or n-eicosyl, and so on.

The term “C2-20 alkylene” refers to a divalent alkyl group, which is a linear or branched hydrocarbon chain containing 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. Alkylene groups are divalent alkyl groups and may likewise be linear or branched and have two points of attachment to the remainder of the molecule. Furthermore, an alkylene group may, for example, correspond to one of those alkyl groups listed in this paragraph. For example, C1-6 alkylene may be -CH2-, -CH2CH2-,-CH2CH(CH3)-, - CH2CH2CH2- or -CH2CH(CH3)CH2-. The alkyl and alkylene groups may be unsubstituted or substituted by one or more substituents. Possible substituents are described herein. For example, substituents for an alkyl or alkylene group may be halogen, e.g. fluorine, chlorine, bromine and iodine, OH, C1-C4 alkoxy, -NR’R” amino, wherein R’ and R” are independently H or alkyl, e.g. C1-6 alkyl. Substituents may also be ring systems such as an aromatic ring (e.g. a five or six membered aromatic ring), a saturated ring system (e.g. a five or six membered saturated ring system), a bridged ring system, a spiro bicyclic ring system, or a heterocyclic ring system. The ring system may itself be substituted or unsubstituted. Other substituents for the alkyl group may alternatively be used. In embodiments, the molecular scaffold (e.g. a dendrimer or dendron) has one generation (i.e. n is 1 ) and comprises: a core having or comprising plural (e.g. 2, 3, 4, or 5) arms (A1 ); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); wherein the plural identifier molecules are each bonded to an arm (A1 ) of the core, wherein the identifier molecules (IM) have the general structure (1 ), (2), (3), (4), and/or (5).

In embodiments, the molecular scaffold (e.g. a dendrimer or dendron) has two successive generations (i.e. n is 2) and comprises: a core having or comprising plural (e.g. 2, 3, 4, or 5) arms (A1 ); one set of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); wherein each branching moiety is bonded to one of the arms (A1 ) of the core; and the plural identifier molecules are each bonded to an arm (A2) of a branching moieties; wherein the identifier molecules (IM) have the general structure (1 ) , (2), (3), (4), and/or (5).

In embodiments, the molecular scaffold (e.g. a dendrimer or dendron) has three successive generations (i.e. n is 3), the molecular scaffold comprising: a core having or comprising plural (e.g. 2, 3, 4, or 5) arms (A1 ); two sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); wherein each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and each branching moiety of the second set is bonded to an arm (A2) of a branching moiety of the first set; wherein the plural identifier molecules are each bonded to an arm (A3) of a branching moieties in the second set; wherein the identifier molecules (IM) have the general structure (1 ) , (2), (3), (4), and/or (5).

In embodiments, one or more or each of the identifier molecules (IM) may be located at a or the terminus of the arm of the core (A1 ) or the branching moiety (A2, A3, A4) of the final set of branching moieties via the linker group (L). In embodiments, the molecular scaffold may comprise at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more identifier molecules (IM). For example, the molecular scaffold may comprise between 2 and 30 identifier molecules (IM). For example, the molecular scaffold may comprise any one of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 identifier molecules (IM). Preferably, the molecular scaffolds comprise 3, 9, or 27 identifier molecules (IM).

The number of identifier molecules (IM) in successive generations will depend on the number of arms (A2, A3, A4) of the branching moieties.

In embodiments wherein n=1 , the number of identifier molecules (IM) in the molecular scaffold may be 2, 3, 4, or 5. Preferably, when n=1 , there are three arms (A1 ) and three identifier molecules (IM).

In a specific embodiment, n=2, the core has three arms (A1 ), there is one set of three branching moieties each having three arms (A2), and therefore the total number of identifier molecules (IM) in the molecular scaffold is nine (3 branching moieties x 3 arms (A2) = 9 identifier molecules (IM)). In another specific embodiment, n=3, the core has three arms (A1 ), there are two sets of branching moieties, the first set having three branching moieties, and the second set having nine branching moieties, each branching moiety having three arms (A2, A3), and therefore the total number of identifier molecules (IM) in the molecular scaffold is 27 (9 branching moieties in the second set x 3 arms (A3) = 27 identifier molecules (IM)).

In embodiments, the core may comprise a ring, for example a six membered ring, e.g. a six membered aliphatic ring.

In embodiments, the core may comprise an aromatic moiety.

In embodiments, the core may comprise a ring or a ring system. In embodiments, the ring system may be selected from C3-9 cycloalkyl, Ca-g aryl, C3-9 heteroaryl, or C3-9 heterocyclic ring systems.

The term “C3-9 cycloalkyl” includes a saturated hydrocarbon ring system containing 3, 4, 5, 6, 7, 8, or 9 carbon atoms. For example, the “C3-C6 cycloalkyl” may be cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, bicyclo[2.1 ,1]hexane or bicyclo[1 .1 ,1]pentane. Suitably the “C3-C6 cycloalkyl” may be cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl.

The term “aromatic” when applied to a substituent as a whole includes a single ring or polycyclic ring system with 4n + 2 electrons in a conjugated TT system within the ring or ring system where all atoms contributing to the conjugated TT system are in the same plane.

The term “Ca-g aryl” includes an aromatic hydrocarbon ring system containing 3, 4, 5, 6, 7, 8 or 9 carbon atoms. The term “aryl” includes an aromatic hydrocarbon ring system. The ring system has 4n +2 electrons in a conjugated TT system within a ring where all atoms contributing to the conjugated TT system are in the same plane. For example, the “aryl” may be phenyl and naphthyl. The aryl system itself may be substituted with other groups.

The term “heteroaryl” includes an aromatic mono- or bicyclic ring incorporating one or more (for example 1 -4, particularly 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur. The term “C3-9 heteroaryl” includes an aromatic mono- or bicyclic ring incorporating one or more (for example 1 -4, particularly 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur, and comprising 3, 4, 5, 6, 7, 8 or 9 carbon atoms. The ring or ring system has 4n + 2 electrons in a conjugated TT system where all atoms contributing to the conjugated TT system are in the same plane.

The term “heterocyclic” includes a non-aromatic saturated or partially saturated monocyclic or fused, bridged, or spiro bicyclic heterocyclic ring system. The term “C3-9 heterocyclic” includes a non-aromatic saturated or partially saturated monocyclic or fused, bridged, or spiro bicyclic heterocyclic ring system containing 3, 4, 5, 6, 7, 8, 9 carbon atoms. Monocyclic heterocyclic rings may contain from about 3 to 12 (suitably from 3 to 7) ring atoms, with from 1 to 5 (suitably 1 , 2 or 3) heteroatoms selected from nitrogen, oxygen or sulfur in the ring. Bicyclic heterocycles may contain from 7 to 12-member atoms in the ring. Bicyclic heterocyclic(s) rings may be fused, spiro, or bridged ring systems. The heterocyclyl group may be a 3-12, for example, a 3- to 9- (e.g. a 3- to 7-) membered non-aromatic monocyclic or bicyclic saturated or partially saturated group comprising 1 , 2 or 3 heteroatoms independently selected from O, S and N in the ring system (in other words 1 , 2 or 3 of the atoms forming the ring system are selected from O, S and N). By partially saturated it is meant that the ring may comprise one or two double bonds. This applies particularly to monocyclic rings with from 5 to 7 members. The double bond will typically be between two carbon atoms but may be between a carbon atom and a nitrogen atom. Bicyclic systems may be spiro-fused, i.e. where the rings are linked to each other through a single carbon atom; vicinally fused, i.e. where the rings are linked to each other through two adjacent carbon and/or nitrogen atoms; or they may be share a bridgehead, i.e. the rings are linked to each other through two non-adjacent carbon or nitrogen atoms (a bridged ring system). Examples of heterocyclic groups include cyclic ethers such as oxiranyl, oxetanyl, tetrahydrofuranyl, dioxanyl, and substituted cyclic ethers. Heterocycles comprising at least one nitrogen in a ring position include, for example, azetidinyl, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, tetrahydrotriazinyl, tetrahydropyrazolyl, tetrahydropyridinyl, homopiperidinyl, homopiperazinyl, 2,5-diaza-bicyclo[2.2.1]heptanyl and the like. Typical sulfur containing heterocycles include tetrahydrothienyl, dihydro-1 ,3- dithiol, tetrahydro-2H-thiopyran, and hexahydrothiepine. Other heterocycles include dihydro oxathiolyl, tetrahydro oxazolyl, tetrahydro-oxadiazolyl, tetrahydrodioxazolyl, tetrahydrooxathiazolyl, hexahydrotriazinyl, tetrahydro oxazinyl, tetrahydropyrimidinyl, dioxolinyl, octahydrobenzofuranyl, octahydrobenzimidazolyl, and octahydrobenzothiazolyl. For heterocycles containing sulfur, the oxidized sulfur heterocycles containing SO or SO2 groups are also included. Examples include the sulfoxide and sulfone forms of tetrahydrothienyl and thiomorpholinyl such as tetrahydrothiene 1 ,1 -dioxide and thiomorpholinyl 1 ,1 -dioxide. A suitable value for a heterocyclyl group which bears 1 or 2 oxo (=0), for example, 2 oxopyrrolidinyl, 2-oxoimidazolidinyl, 2-oxopiperidinyl, 2,5- dioxopyrrolidinyl, 2,5-dioxoimidazolidinyl or 2,6-dioxopiperidinyl. Particular heterocyclyl groups are saturated monocyclic 3 to 7 membered heterocyclyls containing 1 , 2 or 3 heteroatoms selected from nitrogen, oxygen or sulfur, for example azetidinyl, tetrahydrofuranyl, tetrahydropyranyl, pyrrolidinyl, morpholinyl, tetrahydrothienyl, tetrahydrothienyl 1 ,1 -dioxide, thiomorpholinyl, thiomorpholinyl 1 ,1 -dioxide, piperidinyl, homopiperidinyl, piperazinyl or homopiperazinyl. As the skilled person would appreciate, any heterocycle may be linked to another group via any suitable atom, such as via a carbon or nitrogen atom. For example, the term “piperidino” or “morpholino” refers to a piperidin- 1-yl or morpholin-4-yl ring that is linked via the ring nitrogen.

The term “fused ring system” takes the IUPAC definition and includes aliphatic and aromatic systems.

The term “bridged ring systems" includes ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages 131 -133, 1992. Suitably the bridge is formed between two non- adjacent carbon or nitrogen atoms in the ring system. The bridge connecting the bridgehead atoms may be a bond or comprise one or more atoms. Examples of bridged heterocyclyl ring systems include, aza-bicyclo[2.2.1]heptane, 2-oxa-5- azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza-bicyclo[3.2.1]octane, and quinuclidine.

The term “spiro bi-cyclic ring systems” includes ring systems in which two ring systems share one common spiro carbon atom, i.e. the heterocyclic ring is linked to a further carbocyclic or heterocyclic ring through a single common spiro carbon atom. Examples of spiro ring systems include 3,8-diaza-bicyclo[3.2.1]octane, 2,5-diaza-bicyclo[2.2.1]heptane, 6-azaspiro[3.4]octane, 2-oxa-6-azaspiro[3.4]octane, 2-azaspiro[3.3]heptane, 2-oxa-6- azaspiro[3.3]heptane, 6-oxa-2-azaspiro[3.4]octane, 2,7-diaza-spiro[4.4]nonane, 2- azaspiro[3.5]nonane, 2-oxa-7-azaspiro[3.5]nonane and 2-oxa-6-azaspiro[3.5]nonane.

In embodiments, the core may comprise an aliphatic moiety. In embodiments, the core may comprise a C1-20 alkyl or a C2-20 alkylene chain.

In embodiments, the core may comprise a ring, for example a six membered ring, e.g. a six membered aromatic ring.

In embodiments, the core may comprise a number of arms (A1 ) selected from 2, 3, 4, or 5.

In embodiments, the functional group FG1 may be located on or bonded to the core.

In embodiments, the core has the following general structure:

wherein A1 represents an arm, and FG1 represents the functional group capable of forming a bond with a second species. In embodiments, the arms (A1 ) may all be the same. In embodiments, one or more or all of the arms (A1 ) may be different to one or more or all of the other arms (A1 ) within the molecular scaffold.

In embodiments, one or more or all arms (A1 ) may be aliphatic or comprise an aliphatic moiety. In embodiments, one or more or all arms (A1 ) may be aromatic or comprise an aromatic moiety.

In embodiments, one or more or all of the arms (A1 ) may comprise a continuous chain of from 3 to 20 atoms, e.g. from 4 to 18 atoms, or from 5 to 16 atoms, or from 6 to 14 atoms, or from 7 to 12 atoms, or from 8 to 10 atoms, e.g. 9 atoms. In embodiments, one or more of all of the arms (A1 ) may comprise a continuous chain of atoms comprising one of 3, 4,

5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. In embodiments, the atoms may be selected from carbon atoms, e.g. an alkyl chain, or a combination of carbon atoms and heteroatoms, e.g. oxygen atoms and/or nitrogen atoms. The continuous chain may be formed of carbon atoms, or a combination of carbon atoms and heteroatoms, covalently bonded in an unbroken linear chain of between 3 to 20 atoms to form a backbone. The carbon atoms and/or heteroatoms (which form the backbone of one or more of the arms) may have other atoms, e.g. hydrogen atoms, branching alkyl or aryl groups, attached or bonded thereto, which are not included as part of the definition of the continuous chain of between 3 to 20 atoms.

Advantageously, by separating the core from the branching moieties of the molecular scaffold with one or more of the arms (A1 ), inter identifier molecule (IM) quenching may be reduced and/or avoided.

In embodiments, the arms (A1 ) may comprise one or more amino acids, e.g. 1 , 2, 3, 4, 5,

6, 7, 8, 9, 10 amino acids. In embodiments, one or more of the amino acids may be lysine, glycine, alanine, serine, threonine, cysteine, valine, leucin, proline, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine and/or arginine.

In embodiments, one or more or each of the arms (A1 ) may comprise one or more moieties to improve water solubility. In embodiments, the moieties for improving water solubility may be selected from one or more of a PEG (poly(ethylene glycol)) moiety, a PEI (polyethylenimine) moiety, a carboxylic acid moiety.

In embodiments, one or more or each of the arms (A1 ) may comprise one or more PEG moieties. In embodiments, one or more or each of the arms (A1 ) may comprise from 1 to 20 polyethylene glycol repeating units, e.g. between 2 and 15, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 repeating units. Preferably, one or more or each of the arms (A1 ) comprise two poly(ethylene) glycol repeating units.

In embodiments, one or more, or each, arm (A1 ) of the core may comprise the following structure:

wherein x is selected from 1 , 2, 3, 4, or 5. Preferably, x is 2.

The molecular scaffold comprises n-1 sets of branching moieties each having plural arms. In embodiments, n=1 and the molecular scaffold does not have any sets of branching moieties. In embodiments, n=2 and the molecular scaffold has one set of branching moieties. In embodiments, n=3 and the molecular scaffold has two sets of branching moieties. In embodiments, n=4 and the molecular scaffold has three sets of branching moieties. In embodiments, n=5 and the molecular scaffold has four sets of branching moieties.

In embodiments, one or more or each of the branching moieties may have the same structure as the core.

In embodiments, one or more or all of the branching moieties may comprise an aliphatic moiety. In embodiments, one or more or all of the branching moieties may comprise a ring, for example a six membered ring, e.g. a six membered aliphatic ring.

In embodiments, one or more or all of the branching moieties may comprise an aromatic moiety. In embodiments, one or more or all of the branching moieties may comprise a ring, for example a six membered ring, e.g. a six membered aromatic ring. In embodiments, one or more or all of the branching moieties may comprise a number of arms (A2, A3, A4) selected from 2, 3, 4, or 5.

In embodiments, one or more or all of the branching moieties in the first set has the following general structure:

wherein A2 represents an arm, and CG represents a connecting group forming a bond with an arm A1 .

The branching moieties in subsequent sets may have the same structure as that shown for the first set.

Throughout this specification, the connecting group “CG" is intended to mean the functional group resulting from reaction of a functional group on an arm (e.g. A1 , A2, A3, A4) which is capable of reacting with a complementary functional group located on the branching moiety of the molecular scaffold.

In embodiments, the connecting group CG may be an amide, e.g. formed from reaction of an amine (e.g. located on an arm) and a carboxylic acid (e.g. located on a branching moiety).

In embodiments, the connecting group CG may be a triazole, e.g. formed via click chemistry from reaction of an alkyne and an azide.

In embodiments, the connecting group CG may be formed from a carboxylic acid, or a derivative of a carboxylic acid, i.e. an active ester, e.g. -C=O(Y) wherein Y is replaced by a group on the arm of the molecular scaffold in a reaction. For example, Y may be selected from OH, OD, OR^X, NH2, ND2, SH, NHR^X, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from C1-6 alkyl optionally substituted with one or more substituents, for example selected from H, D, F, Cl, Br, I, OH, NH2. In embodiments, the connecting group CG may be formed from reaction of a thiol (-SH), a thioacetate (-SAc), an aliphatic alcohol (-OH) or a phenol, an amine (-NH2), an alkyne, an azide (-N3), a maleimide group, an isothiocyanate (-N=C=S), an aldehyde, a ketone, with a complementary functional group.

It is understood by the skilled person that the connecting group CG should be different to that used to conjugate the identifier molecule (IM) to the arms of the molecular scaffold.

In embodiments, the arms (A2) may all be the same. In embodiments, one or more or all of the arms (A1 ) may be different to one or more or all of the other arms (A2) within the molecular scaffold.

In embodiments, one or more or all arms (A2) may be aliphatic or comprise an aliphatic moiety. In embodiments, one or more or all arms (A2) may be aromatic or comprise an aromatic moiety.

In embodiments, one or more or all of the arms (A2) may comprise a continuous chain of from 3 to 20 atoms, e.g. from 4 to 18 atoms, or from 5 to 16 atoms, or from 6 to 14 atoms, or from 7 to 12 atoms, or from 8 to 10 atoms, e.g. 9 atoms. In embodiments, one or more of all of the arms (A1 ) may comprise a continuous chain of atoms comprising one of 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. In embodiments, the atoms may be selected from carbon atoms, e.g. an alkyl chain, or a combination of carbon atoms and heteroatoms, e.g. oxygen atoms and/or nitrogen atoms. The continuous chain may be formed of carbon atoms, or a combination of carbon atoms and heteroatoms, covalently bonded in an unbroken linear chain of between 3 to 20 atoms to form a backbone. The carbon atoms and/or heteroatoms (which form the backbone of one or more of the arms) may have other atoms, e.g. hydrogen atoms, branching alkyl or aryl groups, attached or bonded thereto, which are not included as part of the definition of the continuous chain of between 3 to 20 atoms.

Advantageously, by separating the sets of branching moieties of the molecular scaffold with one or more of the arms (A2, A3, A4), inter identifier molecule (IM) quenching may be reduced and/or avoided.

In embodiments, the arms (A2, A3, A4) may comprise one or more amino acids, e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids. In embodiments, one or more of the amino acids may be lysine, glycine, alanine, serine, threonine, cysteine, valine, leucin, proline, isoleucine, methionine, proline, phenylalanine, tyrosine, tryptophan, aspartic acid, glutamic acid, asparagine, glutamine, histidine and/or arginine.

In embodiments, one or more or each of the arms (A2, A3, A4) may comprise one or more moieties to improve water solubility. In embodiments, the moieties for improving water solubility may be selected from one or more of a PEG (poly(ethylene glycol)) moiety, a PEI (polyethylenimine) moiety, a carboxylic acid moiety.

In embodiments, one or more or each of the arms (A2, A3, A4) may comprise one or more PEG moieties. In embodiments, one or more or each of the arms (A2, A3, A4) may comprise from 1 to 20 polyethylene glycol repeating units, e.g. between 2 and 15, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 repeating units. Preferably, one or more or each of the arms (A2, A3, A4) comprise two poly(ethylene) glycol repeating units.

In embodiments, one or more, or each, arm (A2, A3, A4) of the core may comprise the following structure:

wherein x is selected from 1 , 2, 3, 4, or 5. Preferably, x is 2.

In embodiments, the functional group (FG1 ) capable of forming a bond with a second species may be capable of forming a covalent bond with a second species.

In embodiments, the functional group (FG1 ) capable of forming a bond with a second species may be a derivative of a carboxylic acid, i.e. -C=O(Y) wherein Y is replaced by the second species in a reaction. In embodiments, Y may be selected from OH, OD, OR^x (e.g. OCH3), NH2, ND2, SH, NHR^X, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from C1-6 alkyl optionally substituted with one or more substituents, for example selected from H, D, F, Cl, Br, I, OH, NH₂.

In embodiments, the functional group (FG1 ) capable of forming a bond with a second species may be a thiol (-SH), a thioacetate (-SAc), an aliphatic alcohol (-OH) or a phenol, an amine (-NH2), an alkyne, an azide (-N3), a maleimide group, an isothiocyanate (-N=C=S), an aldehyde, a ketone. The molecular scaffold may be described as being oligomeric or polymeric. In embodiments, the molecular scaffold may be described as being a dendrimer or a dendron.

In embodiments, the core of the molecular scaffold comprises the functional group capable of forming a bond with a second species. In embodiments, the molecular scaffold may be termed a “dendron” for this reason.

A further aspect of the invention provides a dendrimer or dendron comprising plural identifier molecules (IM) selected from one or more of the general structures (1 ), (2), (3), (4), and/or (5).

In embodiments, the second species may be a small molecule and/or a biomolecule. For example, the biomolecule may be an amino acid, a peptide, a protein, a nucleic acid, a polynucleotide, or an antibody. In embodiments, the second species is a therapeutic species or a pharmaceutically active molecule, e.g. a drug molecule.

Advantageously, the molecular scaffold of the invention is capable of being covalently bonded to a second species which may specifically bind to a molecule of interest, and hence may be used as chemical sensors or biosensors.

We define a biosensor as a sensor comprising or consisting of the identifier molecules which are usable for the detection of, or to determine the concentration of, a chemical or biological substance.

Additionally or alternatively, the second species may be a solid support, e.g. for use in solid phase synthesis. Additionally or alternatively, the second species may be a nanoparticle, e.g. a nanoparticle comprising or formed from a metal or a metal alloy, carbon, clay, a polymer, and/or a ceramic material.

A further aspect of the invention provides a molecular scaffold according to the invention bonded, e.g. covalently bonded, to a second species.

In embodiments, the second species may be a small molecule and/or a biomolecule. For example, the second species may be an amino acid, a peptide, a protein, a nucleic acid, a polynucleotide. The second species need not be a molecule. For example, the second species may be an antibody. In embodiments, the second species is a therapeutic species or a pharmaceutically active molecule, e.g. a drug molecule.

Advantageously, the molecular scaffolds according to the invention are suitable for being covalently bonded to a second species, e.g. a biomolecule or a small molecule or a drug molecule. Therefore, the molecular scaffolds are usable as biosensors, as the molecular scaffolds according to the invention comprise identifier molecules.

The biomolecule may be an antibody, e.g. monoclonal antibodies or polyclonal antibodies. The biomolecule may be an avidin, e.g. streptavidin. The biomolecule may be biotin.

In embodiments, the second species may be a molecule for recognition of a species in a biological system. For example, the biomolecule may be capable of binding to a protein or receptor on the surface of a cell.

A yet further aspect of the invention provides a biomolecule, e.g. an antibody, covalently bonded to a molecular scaffold of the invention.

A yet further aspect of the invention provides a kit of parts suitable for use in covalently bonding the molecular scaffold of the invention to a second species, e.g. a biomolecule, the kit of parts comprising a molecular scaffold of the invention and a solvent suitable for dissolving or suspending the molecular scaffold.

The solvent may comprise water, for example, the solvent may be an aqueous media and/or a buffer solution. In embodiments, the solvent may comprise an organic solvent, for example, THF or DMSO or combinations thereof. In embodiments, the solvent may comprise a first solvent comprising an aqueous media and a second solvent comprising an organic solvent, e.g. THF or DMSO or combinations thereof.

In some embodiments, the kit of parts may further comprise the second species.

The kit of parts may further comprise a set of instructions that explain how to covalently bond the molecular scaffold of the invention to a second species. The kit of parts may further comprise a catalyst and/or a coupling agent for use in covalently bonding the molecular scaffold of the invention to a second species.

The kit of parts may further comprise a measuring means, e.g. a pipette or pipette tips. The kit of part may further comprise a vessel suitable for carrying out the reaction of covalently bonding the molecular scaffold to a second species. The kit of parts may further comprise a means to purify the final conjugate comprising the molecular scaffold and the second molecule. The means to purify the final conjugate may be a filter.

A yet further aspect of the invention provides a method of conjugating the molecular scaffold of the invention to a second species, the method comprising providing the molecular scaffold according to the invention, providing a second species, and forming a covalent bond between the molecular scaffold and the second species.

The method may comprise adding the second species, e.g. a solution or suspension of the second species, to a solution of the molecular scaffold. The method may comprise adding the molecular scaffold, e.g. a solution of the molecular scaffold, to a solution or suspension of the second species.

Advantageously, the molecular scaffolds of the invention when conjugated (i.e. covalently bonded) to a second species are usable to locate and/or track the second species, e.g. in vivo, as the molecular scaffold according to the invention comprise plural identifier molecules, e.g. luminescent moieties or compounds. The conjugated molecular scaffold and second species may also be usable to locate and/or track a target species of the second species.

A yet further aspect of the invention provides use of one or more of the molecular scaffolds according to the invention or conjugates thereof (e.g. to a second species), in a composition for cell or tissue imaging.

By conjugates of the molecular scaffold, we mean the molecular scaffold when covalently bonded to a second species.

A yet further aspect of the invention provides use of the molecular scaffold or conjugates thereof in an immunofluorescence technique. A yet further aspect of the invention provides use of the molecular scaffold or conjugates thereof in flow cytometry.

A yet further aspect of the invention provides a method of fabricating a molecular scaffold or conjugate thereof, the method comprising:

(i) providing a molecule (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecule comprising: a core having or comprising plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set;

(ii) providing plural identifier molecule having the general structure selected from (1 ), (2), (3), (4), or (5);

(iii) forming a bond between each identifier molecules with an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core.

In embodiments, the plural identifier molecules may be the same as one another.

In embodiments, the plural identifier molecules are different from one another. In this case, the method may comprise providing a mixture of two or more, e.g. three, four or five, different identifier molecules (IM), e.g. in a 1 :1 ratio.

In embodiments, the method may further comprise converting the functional group (FG1 ) of the molecule to a different functional group (FG1 ’) subsequent to the step (iii) of forming a bond between each identifier molecule and an arm (A1 , A2, A3, A4) of the branching moieties. For example, when the identifier molecules are bonded to an arm (A1 , A2, A3, A4), the functional group (FG1 ) may be a derivative of a carboxylic acid, i.e. -C=O(Y) wherein Y is selected from OH, OD, OR^x (e.g. OCH3), NH2, ND2, SH, NHR^X, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from C1-6 alkyl optionally substituted with one or more substituents, for example selected from H, D, F, Cl, Br, I, OH, NH2. However, in some embodiments it has been appreciated that a Y group that provides good yields and/or chemoselectivity when bonding the identifier molecules could be less suitable for the subsequent method step of reacting the functional group (FG1 ) with the second species. In other words, although functional group (FG1 ) is capable of forming a bond with the second species, the reactivity and/or selectivity and/or yield could be improved. As such, in these examples, in may be beneficial following step (iii) to modify the functional group (FG1 ) of the molecule to a different functional group (FG1 ’) showing modified (e.g. improved) reactivity to the second species.

For example, the different functional group (FG1 ’) may be a different derivative of a carboxylic acid, i.e. -C=O(Y’) where Y’ is selected from OH, OD, OR^x (e.g. OCH3), NH2, ND2, SH, NHR^X, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from C1-6 alkyl optionally substituted with one or more substituents, for example selected from H, D, F, Cl, Br, I, OH, NH2, and wherein Y’ is selected to be different to Y. For example, Y may be OR^X such that the functional group (FG1 ) of the molecule when the identifier molecules are bonded is an ester group. Then, subsequently, the functional group (FG1 ) may be modified by hydrolysis (e.g. to a carboxylic acid group where Y’=OH or OD), aminolysis (e.g. to an amide where Y’ = NH2, ND2, NHR^X, NR^yR^z) or converted to a thioester (e.g. Y’=SH).

Alternatively, the different functional group (FG1 ’) may be a different functional group to that of the functional group (FG1 ) of the molecule when the identifier molecules are bound. For example, the initial functional group (FG1 ) and the different functional group (FGT) may be selected to be different groups from the following: a carboxylic acid derivative (-C=OY), a thiol (-SH), a thioacetate (-SAc), an aliphatic alcohol (-OH) or a phenol, an amine (-NH2), an alkyne, an azide (-N3), a maleimide group, an isothiocyanate (-N=C=S), an aldehyde, a ketone.

The molecular scaffold and conjugates thereof may be used to quantify and/or detect the presence of, or the target of, the second species, e.g. in an assay. The molecular scaffold and conjugates thereof may be used to quantify and/or detect the presence of a biomolecule, e.g. DNA, RNA, a protein, a hormone, an antibody, or a cell. In this way, the molecular scaffold and conjugates thereof may be used as biosensors. The molecular scaffold according to the invention may be used to form covalent bonds with a second species present in vivo or in vitro. For example, the covalently bond between the molecular scaffold and the second species may be formed in a tissue culture, or a cell culture, e.g. a plant, animal, or microbial cell culture.

The conjugates of the molecular scaffold of the invention to a second species may be usable in vivo, for example, to track or locate the second species in vivo, or to track or locate a target of the second species in vivo. The conjugates of the molecular scaffold may be usable to produce an image, e.g. of an organ in which the conjugates have accumulated. The image, e.g. produced using a fluorescence imaging technique, may be usable in the diagnosis of a disease.

More advantageously, the molecular scaffolds according to the invention are tunable. For example, the molecular scaffold according to the invention are tunable according to the selection of the identifier molecules, e.g. luminescent moieties or compounds. Further, modification of the molecular scaffold or one or more of the identifier molecules, e.g. the R groups of one or more of the identifier molecules, provides a series of molecular scaffolds comprising identifier molecules that require the same excitation wavelength but exhibit different emission spectra. Even more advantageously, the molecular scaffolds of the invention, e.g. the identifier molecules of the molecular scaffold, may be designed to emit wavelengths across the entire visible spectrum by varying the structure of the R group of one or more of the identifier molecules.

Additionally, the molecular scaffolds of the invention may be used in a multiplexed system. For example, two or more different identifier molecules (IM), e.g. luminescent moieties or compounds, may be conjugated to a respective second species via the molecular scaffold of the invention. The identifier molecules (IM), e.g. luminescent moieties or compounds, of the molecular scaffold(s) are able to be excited at the same wavelengths but may emit at different wavelengths. This enables two different second species, e.g. biomolecules, to be studied, e.g. in a biological system, whilst using a single light source. For example, two or more different identifier molecules, e.g. luminescent moieties or compounds, may be used to observe or track two different second species, e.g. biomolecules, in a system, e.g. in vivo or ex vivo. It has also been surprisingly found that the molecular scaffolds of the invention may be usable with multi-photon excitation microscopy. As is known in the art, in multiphoton microscopy (also known as two-photon microscopy) two or more photons of light are absorbed for each excitation. This technique differs from traditional fluorescence microscopy in which the excitation wavelength is shorter than the emission wavelength. Two-photon excitation microscopy typically uses near-infrared excitation light. In some embodiments, multiphoton microscopy is carried out by irradiating the luminescent compound, e.g. the conjugated luminescent compound, using a light source which emits a wavelength in the range of from 500 to 1000 nm, or from 800 to 900 nm. The use of multiphoton microscopy is advantageous since it uses lower energy light and is thus less damaging to biological samples. Advantageously, this prevents or mitigates phototoxicity when the luminescent molecules are used in living systems. More advantageously, the light penetrates deeper through tissues and is less likely to photobleach the luminescent compound.

The molecular scaffold(s) may emit light in the visible spectrum, i.e. between 380 nm and 750 nm and/or may exhibit a Stokes shift of between 8000 cm ¹ to 25,000cm’¹, for example, between 15,000 cm’¹ to 25,000 cm’¹. In embodiments, the molecular scaffold(s) may exhibit a conductivity value of 5.0 x 10’¹³ S cm’¹ and 1 .5 x 10’¹¹ S cm’¹, for example, between 6 x 10’¹² S cm^-1 and 1.5 x 10’¹¹ S cm’¹. The molecular scaffold(s) may exhibit a photoconductivity when irradiated at 350 nm of between 1.5 x 10’¹⁰ S cm’¹ and 1 x 10’³ S cm’¹, for example, between 1 x 10’⁸ S cm’¹ and 1 x 10’³ cm’¹.

Within the scope of this application it is expressly intended that the various aspects, embodiments, examples and alternatives set out in the preceding paragraphs, in the claims and/or in the following description and drawings, and in particular the individual features thereof, may be taken independently or in any combination. That is, all embodiments and/or features of any embodiment can be combined in any way and/or combination, unless such features are incompatible. For the avoidance of doubt, the terms “may”, “and/or”, “e.g.", “for example” and any similar term as used herein should be interpreted as non-limiting such that any feature so-described need not be present. Indeed, any combination of optional features is expressly envisaged without departing from the scope of the invention, whether or not these are expressly claimed. The applicant reserves the right to change any originally filed claim or file any new claim accordingly, including the right to amend any originally filed claim to depend from and/or incorporate any feature of any other claim although not originally claimed in that manner.

Embodiments of the invention will now be described by way of example only with reference to the accompanying drawings in which:

Referring to Figure 1 there is shown examples (Compounds 1 to 6) of identifier molecules (IM) that may be used to synthesise Examples of the invention;

Figure 2 is a method of synthesising part of a molecular scaffold according to an Example of the invention;

Figure 3 is a schematic synthetic route for a first generation molecular scaffold according to the invention;

Figure 4 is a schematic synthetic route for a first generation molecular scaffold according to the invention;

Figure 5 shows the photophysical properties of the first generation molecular scaffold shown in Figure 4;

Figure 6 is a schematic synthetic route for a first generation molecular scaffold according to the invention;

Figure 7 is a schematic synthetic route for a first generation molecular scaffold according to the invention;

Figures 8A, 8B, 8C are schematics to show first, second, and third generation (n = 1 , 2, 3 respectively) molecular scaffolds according to the invention;

Figures 9A and 9B are Examples of third generation (n=3) molecular scaffold of the invention;

Figures 10A to 10F show synthetic routes to Compounds 1 to 6 according to Examples of the invention;

Figure 11 shows a photograph of a successful Conjugation Check Kit result;

Figure 12 shows flow cytometry scatterplots for beads stained in accordance with embodiments of the present invention; and

Figure 13 shows fluorescence profiles of beads stained in accordance with an embodiment of the present invention.

Referring now to Figure 1 there is shown Examples (Compounds 1 to 6) of identifier molecules for use in the invention. Other suitable identifier molecule (IM) structures may be used as part of the invention, which are described along with the methods for their synthesis in our earlier applications PCT/GB2019/050809, PCT/GB2019/050806, PCT/GB2020/052324, PCT/GB2020/052325 and PCT/GB2020/052323.

Referring now to Figure 2, there is shown a synthesis of a molecular scaffold according to the invention. The synthesis of these compounds is described in Sousa-Herves, A., Novoa- Carballal, R., Riguera, R. et al. GATG Dendrimers and PEGylated Block Copolymers: from Synthesis to Bioapplications. AAPS J 16, 948-961 (2014).

In Step A, 2-[2-(2-chloroethoxy)ethoxy]ethanol (CAS: 5197-62-6) 1 is reacted with sodium azide in dimethyl sulfoxide (DMSO) at 80°C for three hours under nitrogen to form azido triethylene glycol 2.

In Step B, the alcohol group of the azido triethylene glycol 2 is functionalised with tosylchloride in 5 M sodium hydroxide and tetrahydrofuran (THF) at room temperature for 2.5 hours to form the tosylate ester 3.

In Step C, gallic acid 4 is reacted with sulphuric acid and methanol under reflux for 20 hours to form methyl gallate 5.

In Step D, methyl gallate 5 and tosylate ester 3 are then reacted together with potassium carbonate and potassium iodide in dimethylformamide (DMF) at 80°C for 20 hours to form GAGT dendrimer 6. Note that DMF may be replaced by another polar aprotic solvent, for example acetonitrile.

In Step E, the terminal azide groups on the PEG arms are reduced to amino groups in the presence of zinc, ammonium chloride, water/ethanol under reflux for 2 hours. The resulting precursor 7 comprises a gallic acid based core and hydrophilic polyethylene glycol (PEG) arms with terminal amino groups.

Referring now to Figure 3 there is shown the functionalisation of the precursor 7 according to an Example of the invention. In this embodiment, the identifier molecule (IM) comprises n CH2CH2O groups, wherein n is an integer of 1 , 2, 3, 4, or 5. It is understood that different identifier molecules (IM) may be used to form the molecular scaffold of the invention , e.g. Compounds 1 to 6 shown in Figure 1 . In Step F, each of the terminal amino groups precursor ? are functionalised with an identifier molecule (IM) to form molecular scaffold 8 using dicyclohexylcarbodiimide (DCC), 4- dimethylaminopyridine (DMPA) and DCM (DCM) under nitrogen at room temperature for 20 hours. In this Example, the linker group (L) is an amide bond formed from a carboxylic acid located on the identifier molecule (IM) and an amine group located on the arms of the molecular scaffold.

In other embodiments of the invention, the linker group (L) may be a triazole formed via click chemistry from reaction of an alkyne and azide.

Molecular scaffold 8 is a first generation dendron wherein n=1. In this Example, FG1 is a methyl ester.

In Step G, the methyl ester is hydrolysed in the presence of sulphuric acid and THF under reflux for 20 hours, resulting in molecular scaffold 9 wherein FG1 ’ for attachment to a second molecule is a carboxylic acid moiety.

The combination of high water solubility from the PEG groups and the hydrophobic core is well suited for the covalent attachment or physical encapsulation of large payloads of therapeutic molecules. This is advantageous because plural fluorophores can be attached to a single molecule of interest, increasing the signal to noise ratio.

Referring now to Figure 4, there is shown functionalisation of precursor 10 according to an Example of the invention to provide a first generation molecular scaffold (i.e. dendron 1 ) 12. Precursor 10 has a gallic acid based core with hydrophilic polyethylene glycol (PEG) arms with terminal azide groups.

In step H, precursor 6 was hydrolysed in basic conditions in the presence of sodium hydroxide and MeOH at room temperature for 20 hours.

In step I each of the terminal azide groups of precursor 10 are functionalised with an identifier molecule (IM) (compound 1 , Figure 1 and Figure 9A) to form molecular scaffold 12 (i.e. dendron 1 ) by mixing the precursors 10, compound 1 with copper sulfate and sodium ascorbate at room temperature for 4 days. In this example, the linker group (L) is a triazole group formed by an azide-alkyne cycloaddition wherein the azide group is located on precursor 10 and the alkyne group is located on the compound 1 identifier molecule (IM). The full synthesis of molecular scaffold 12 (i.e. dendron 1 ) is provided in the below Experimental section.

Referring now to Figure 5, the photophysical properties of dendron 1 (i.e. molecular scaffold 12 in Figure 4) are compared to the photophysical properties of TpMeOPhpPEGCOOH. Absorption spectra (A, a) were recording using a Shimadzu UV3600i UV-vis spectrometer. Emission spectra (B, b) and excitation spectra (C, c) were recording using a Horiba Fluorolog-3 (L-configuration) fluorescence spectrometer equipped with a 450 W Xenon light source, R928P photomultiplier tube, double monochromators and a 345 nm longpass filter fitted in the emission channel.

Table 1 shows a comparison of the photophysical properties of dendron 1 and TpMeOPhpPEGCOOH in DMF. The molar absorpitivity was calculated using the Beer- Lambert law from the absorption spectra shown in Figure 5 at five concentrations (0.1 -1 .4 pM). Quantum yield (QY) calculations were performed using a relativistic method (Horiba Scientific Limited, “A Guide to Recording Fluorescence Quantum Yields”, https://static.horiba.com/fileadmin/Horiba/Application/Materials/Material_Research/Quantu m_Dots/quantumyieldstrad.pdf accessed 25^th July 2024), using the same solutions prepared for molar absorptivity measurements, the reference compound was TpOx-n-Bu in ethyl acetate solvent (Of = 0.18). Data was compared with TpMeOx-PhpPEGCOOH (Figure 1 and Table 1 ) in the same solvent and concentration range.

Table 1

Referring now to Figure 6, there is shown functionalisation of precursor 10 according to an

Example of the invention to provide a first generation molecular scaffold 16 (i.e. dendron 2) and an N-hydroxysuccinimide ester derivative thereof (i.e. dendron 2-NHS). Precursor 10 has a gallic acid based core with hydrophilic polyethylene glycol (PEG) arms with terminal azide groups.

In step J, the carboxylic functional group of precursor 10 is functionalised to provide an amide group by reacting the amine group of Compound 3 (Figure 1 , Figure 9C) with the carboxylic acid group of compound 10 and provide precursor 14.

In step K, the terminal butyl ester group (originating from Compound 3) is hydrolysed to provide precursor 15 having a terminal carboxylic acid group.

In step L, each of the terminal azide groups of precursor 15 (originating from precursor 10) are functionalised with an identifier molecule (IM) (compound 1 , Figure 1 and Figure 9A) to form molecular scaffold 16 (i.e. dendron 2) by mixing the precursor 15 and compound 1 with copper sulfate and sodium ascorbate at room temperature for 20 hours. In this example, the linker group (L) is a triazole group formed by an azide-alkyne cycloaddition wherein the azide group is located on precursor 10 and the alkyne group is located on the compound 1 identifier molecule (IM). The full synthesis of molecular scaffold 16 (i.e. dendron 2) is provided in the below Experimental section.

In step M, the terminal carboxylic acid group is esterified using N-hydroxysuccinimide (NHS) at room temperature for 21 hours to provide dendron 2-NHS. Dendron 2-NHS may then be used directly for antibody conjugation.

Referring now to Figure 7, there is shown functionalisation of precursor 15 (the synthesis of which is shown in Figure 6) according to an Example of the invention to provide a first generation molecular scaffold 21 (i.e. dendron 3) and an N-hydroxysuccinimide ester derivative 22 thereof (i.e. dendron 3-NHS).

In step N, the terminal azide groups on the PEG arms are reduced to amino groups in the presence of zinc, aqueous ammonium chloride and ethanol under reflux for 3 hours. The resulting precursor 17 has hydrophilic polyethylene glycol (PEG) arms with terminal amino groups. In step Q, the precursor 17 is reacted with an N-hydroxysuccinimide ester precursor 20, synthesised via steps O and P.

In step O, the precursor compound 6 (see Figure 1 with synthesis shown in Figure 10F and described below) is mixed with N-hydroxysuccinimide (NHS) in an argon environment in the presence of N,N’-dicyclohexylcarbodiimide (DCC) and N.N’dimethylformamide (DMF) for 24 hours. Following centrifugation, the supernatant containing the NHS-ester is mixed with a solution of 3-amino-2-sulfopropanoic acid in a sodium bicarbonate buffer at room temperature for 1 hour to provide precursor 18 which is a black solid.

In step P, precursor 18 is mixed with N-hydroxysuccinimide (NHS) in an argon environment in the presence of N,N’-dicyclohexylcarbodiimide (DCC) and N.N’dimethylformamide (DMF) for 24 hours to provide the NHS ester 19 of precursor 18.

Returning back to step Q, the NHS ester 19 is mixed with precursor 17 in a sodium bicarbonate buffer at room temperature for 19 hours. Following removal of the solvent, the resulting residue is dissolved in methanol and sodium hydroxide and stirred for 30 minutes at room temperature, providing the first generation molecular scaffold 20 (i.e. dendron 3).

In step R, the terminal carboxylic acid group of molecular scaffold 20 is esterified using N- hydroxysuccinimide (NHS) at room temperature for 21 hours in the presence of N,N’- dicyclohexylcarbodiimide (DCC) and N.N’dimethylformamide (DMF) to provide molecular scaffold 21 (i.e. dendron 3-NHS). Molecular scaffold 21 (i.e. dendron 3-NHS) may then be used directly for antibody conjugation.

Referring now to Figures 8A, 8B, and 8C there are shown general formulae of first (n=1 ), second (n=2), and third (n=3) generation molecular scaffolds according to the invention.

The molecular scaffold shown in Figure 8A is a first generation dendron wherein n=1 comprising a core C, a functional group FG1 for attachment to a second species, three arms A1 and three identifier molecules IM. There are no branching moieties.

The identifier molecules are preferably luminescent moieties or compounds. Advantageously, the provision of multiple identifier molecules (IM) allows for amplification of the identifier signal. The identifier molecules IM may be the same as each other or they may be different to each other. Advantageously, when one or more of the identifier molecules IM are different to the other identifier molecules IM, a unique luminescent signal can be provided.

The identifier molecules IM may comprise any of the Examples (Compounds 1 to 6) shown in Figure 1.

The arms of the molecular scaffold A1 may be the same or they may be different. For ease of synthesis, preferably the arms A1 are the same as one another. The arms A1 may be polyethylene chains or polyethylene glycol. The polyethylene chains may be the same or different lengths, e.g. may be in the range 1 to 20, e.g. between 2 and 15, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15.

The functional group FG1 allows attachment, e.g. via covalent bonding, of the molecular scaffold to a second molecule. Preferably, the functional group FG1 is or comprises a terminal carboxylic acid. The functional group FG1 may comprise a chain of atoms with a terminal carboxylic acid. The chain of atoms may be in the range 1 to 20 atoms, e.g. carbon atoms, e.g. between 2 and 15, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 atoms, e.g. carbon atoms. Advantageously, being able to extend the chain length of the functional group FG1 may assist in reducing steric hinderance.

The molecular scaffold shown in Figure 8B is a second generation dendron wherein n=2 comprising a core C, a functional group FG1 for attachment to a second species, three arms A1 , three branching moieties BM1 , and nine identifier molecules IM. There is one set of branching moieties in this embodiment.

BM1 are branching moieties which each comprise an aryl group. BM1 may be the same or they may be different.

The molecular scaffold shown in Figure 8C is a third generation dendron wherein n=3 comprising a core C, a functional group FG1 for attachment to a second species, the core C having three arms A1 each bonded to one of a first set of three branching moieties BM1 . Each branching moiety BM1 has three arms (A2) such that there are nine arms (A2) each bonded to one of a second set of nine branching moieties BM2. Each branching moiety BM2 has three arms (A3), which are each bonded to an identifier molecules IM such that there are 27 identifier molecules IM in this embodiment. There is two sets of branching moieties in this embodiment.

The identifier molecules (IM) may be the same as each other or they may be different to each other.

Referring now to Figure 9A there is shown a second generation (n=2) molecular scaffold 23 synthesised from the precursor 7 shown in Figure 2 and the first generation (n=1 ) molecular scaffold 9 shown in Figure 3. The second generation molecular scaffold was formed by reacting precursor 7 with molecular scaffold 9 in the presence of dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMPA) and dichloromethane (DCM) under nitrogen at room temperature for 20 hours.

Figure 9B shows the molecular scaffold 23, wherein the gallic acid core is reformed from the methyl ester in the presence of sulphuric acid and THF under reflux for 20 hours to form molecular scaffold 24.

Advantageously, the molecular scaffolds according to the invention have applications in drug and gene delivery, as antiviral agents, for the treatment of neurodegenerative diseases, in diagnosis and as tools to study multivalent carbohydrate recognition and dendrimer dynamics.

Further, the differences in the solubility properties of the components of the dendrimers can be exploited in the preparation of micelles and other nano structures of biomedical interest.

The invention is exemplified with the following Examples, which are shown in Figure 1 .

Experimental Syntheses

Identifier Molecules (IM) before reaction to form the linker (L) with the remaining part of the molecular scaffold, were synthesised and characterised as follows:

Compound 1 :

The synthesis of Compound 1 shown in Figure 1 is shown in Figure 10A.

Synthesis of Compound 601 : A solution of NaOH (3.90 g, 97.49 mmol) in tetraethylene glycol (42 ml, 243.74 mmol) was heated at 70 °C for 0.5 h attached with a CaCl2 drying tube, followed by the addition of chloropentyne (5.08 g, 49.53 mmol). The resultant solution was further heated at 70 °C for 20 h with a CaCl2 drying tube. The solution was allowed to cool to room temperature and H2O (50 ml) was added. The organic layer was extracted with EtOAc (3 x 100 ml) and the combined organic layers were dried (MgSO4), filtered and the solvent removed in vacuo. The crude material was purified by column chromatography (silica, wet loaded with DCM, gradient elution: 0-80 % EtOAc in hexane, increase polarity by 20 % every 200 ml of elute). The solvent was removed in vacuo to yield a colorless oil (8.11 g, 63 %). The colorless oil (8.00 g, 30.77 mmol) was dissolved in THF (30 ml) and 5 M NaOH(aq) (37 ml, 184.62 mmol) was added, followed by the dropwise addition of a solution of TsCI (11 .54 g, 61 .54 mmol) in THF (30 ml) at room temperature. The resultant solution was further stirred at room temperature for 3 h followed by addition of H2O (50 ml) and the organic layer was extracted with EtOAc (3 x 50 ml). The combined organic layers were washed with brine (50 ml), dried (MgSO4), filtered and solvent removed in vacuo to yield a colorless oil (12.27 g, 96 %). d_H (¹H NMR, 300 MHz) 7.82 (d, 2H), 7.37 (d, 2H), 4.65-4.60 (m, 2H), 4.28-4.19 (s, 6H), 4.18-4.13 (m, 9H), 3.90-3.83 (m, 2H), 3.62-3.82 (m, 12H), 2.23- 2.28 (m, 2H), 1.91 (s, 1 H), 1.83-1.74 (m, 2H); ESMS (m/z): 414 ([M]⁺, 100 %).

Synthesis of TpueOx-PhpPEG-alkyne: A slurry of TpueOx-PhpOH (600) (106 mg, 0.21 mmol), K2CO3 (57 mg, 0.41 mmol) and TsO-PEG-alkyne (601 ) (121 mg, 0.40 mmol) in MeCN (10 mL) was heated at reflux with a CaCl2 drying tube for 20 h. The reaction was allowed to cool to room temperature and the solid was filtered off via suction filtration. The solid was washed thoroughly with DCM (20 mL) and the solvent was removed from the filtrate in vacuo. The crude solid was purified via flash column chromatography (silica, wet loaded with DCM, gradient elution from 0 to 10 % EtOAc in DCM). The solvent was removed in vacuo and further dried under high vacuum for 1 h to afford an orange solid (75 mg, 48 %). d_H (¹H NMR, 300 MHz) 10.02 (s, 1 H), 8.22-8.20 (m, 2H), 7.90-7.75 (m, 4H), 7.05-7.01 (m, 2H), 4.66-4.60 (m, 2H), 4.29-4.20 (s, 6H), 4.18-4.12 (m, 9H), 3.90-3.82 (m, 2H), 3.60- 3.80 (m, 12H), 2.25-2.29 (m, 2H), 1.92 (s, 1 H), 1.83-1.75 (m, 2H); MALDI (m/z): 753 ([M]⁺, 100 %).

Compound 2:

The synthesis of Compound 2 shown in Figure 1 is shown in Figure 10B.

TpMe(PEG-COOMe)Ox-PhpMe: A solution of TpMeOx-Ph Me (200 mg, 0.39 mmol) in anhydrous DCM (50 mL) was stirred at -10° C under N2 for 10 min and 1 M boron tribromide in DCM (0.59 mL, 0.59 mmol) was added dropwise. The resultant black solution was stirred under N2 for 2 h at room temperature followed by quenching of the reaction via pouring the reaction mixture over crushed ice and stirring until the ice melted. The organic layer was extracted with DCM (20 mL) followed by washing of the organic layer with H2O (3 x 20 mL). The solvent was removed in vacuo yielding a brown solid. The crude TpMe(OH)Ox-PhpMe (190 mg, 0.38 mmol), K2CO3 (212 mg, 1.54 mmol), CI-PEGCOOMe (151 mg, 0.77 mmol) and KI (63 mg, 0.39 mmol) in MeCN (20 mL) was heated at reflux with a CaCl2 drying tube for 20 h. The reaction was allowed to cool to room temperature and the solid was filtered off via suction filtration. The solid was washed thoroughly with DCM (20 mL) and the solvent was removed from the filtrate in vacuo. The crude solid was adsorbed onto silica and purified via flash column chromatography (silica, gradient elution from 0 to 30 % EtOAc in DCM. The solvent was removed in vacuo and further dried under high vacuum for 1 h to afford Tpue(PEG-COOMe)Ox-PhpMe as a yellow solid (50 mg, 19 %). C/H (¹H NMR, 300 MHz) 10.09 (s, 1 H), 8.22-8.21 (m, 2H), 7.91-7.82 (m, 4H), 7.30-7.23 (m, 2H), 4.65-4.61 (m, 2H), 4.29 (s, 3H), 4.21-4.04 (m, 14H), 3.86-3.83 (m, 2H), 3.76-3.72 (m, 2H), 3.34-3.32 (m, 2H), 2.48 (s, 3H); MALDI (m/z): 655 ([M]⁺, 100 %).

TpMe(PEG-COOH)Ox-PhpMe: To a solution of TpMe(PEG-COOMe)Ox-PhpMe (50 mg, 0.98 mmol) in THF:MeOH (1 :1 , 6mL), an aqueous solution of NaOH (16 mg, 0.40 mmol, 1 mL) was added. The resultant solution was heated at 65 °C for 2 h. The reaction was allowed to cool to room temperature and 1 M HCI(_aq) (10 mL) was added followed by extraction of the organic layer with DCM (3 x 10 ml). The combined organic layers were dried (MgSOi), filtered and the solvent removed in vacuo. The crude solid was adsorbed onto silica and purified via flash column chromatography (silica, gradient elution from 0 to 10 % MeOH in DCM. The solvent was removed in vacuo to yield a light brown solid (20 mg, 41 %) :d_H (¹H NMR, 300 MHz) 10.09 (s, 1 H), 8.22-8.21 (m, 2H), 7.91 -7.79 (m, 4H), 7.38- 7.25 (m, 2H), 4.66-4.62 (m, 2H), 4.28 (s, 3H), 4.21-4.05 (m, 11 H), 3.86-3.83 (m, 2H), 3.76- 3.72 (m, 2H), 3.36-3.35 (m, 2H), 2.48 (s, 3H); MALDI (m/z): 641 ([M]⁺, 100 %).

Compound 3:

The synthesis of Compound 3 shown in Figure 1 is shown in Figure 10C.

8-(naphthalen-2-yl)triphenyleno[1,2-d]oxazole-2,3,6,11,12-pentaol (Tp(OH)₅Ox-2-Np): AlCh (484 mg, 3.631 mmol, 10 eq.) was charged to a dried, N2 purged flask and anhydrous toluene (60 mL) was added. The resulting slurry was cooled to 0 °C. A solution of TpOx-2- Np (300 mg, 0.363 mmol, 1 eq.) in anhydrous toluene (30 mL) was added dropwise over 15 mins ensuring the temperature of the slurry did not exceed 1 °C. The reaction was stired at 0 °C and continuously degassed with N2 for 20 mins before being heated under reflux for 1 h. The solution was cooled to r.t. and poured over acidified crushed ice (150 mL ice containing 1 M HCI(_aqj (6 mL)) and stired until all the ice had melted. The toluene layer was seperated and the aqueous layer was extracted with ethyl acetate (3 x 20 mL). The combined organic phases were washed with water (3 x 20 mL), dried over MgSO4, and the organic solvent was removed yielding a cream solid which was purified by prepHPLC. Fractions eluting between 11 -19 mins were collected and the solvent was removed by freeze drying to yield Tp(OH)₅Ox-2-Np as a yellow solid (66 mg, 38 %). ¹H NMR (300 MHz, MeOD) 6_H: 9.92 (s, 1 H), 8.84 (s, 1 H), 8.48 (dd, J = 8.5, 1.7 Hz, 1 H), 7.98 (m, 2H), 7.85 (m, 5H), 7.51 (dd, J = 6.2, 3.2 Hz, 2H) ppm. ¹³C NMR{¹H} (100 MHz, MeOD) 5_C: 162.7, 147.1 , 146.4, 146.4, 146.0, 141.7, 141.6, 140.7, 136.0, 134.5, 129.8, 129.7, 128.9, 128.7, 128.7, 128.6, 127.9, 125.9, 125.4, 125.1 , 124.8, 124.1 , 124.0, 116.5, 114.2, 109.7, 108.8, 108.3, 106.6 ppm. MALDP m/z 475.2 ([M]⁺ 100 %), 476.2 ([M+H]⁺ 90 %), 477.2 ([M+H+1]⁺ 40 %). IR v (neat): 3264s, br (O-H), 1627m (N=C), 1605m (aromatic C=C), 1523m (aromatic C=C) cm’¹.

6,6,11 ,11 -tetramethyl-2-(naphthalen-2-yl)bis([1 ,3]dioxolo)[4',5':6,7;4",5":10,11 ] triphenyleno[1,2-d]oxazol-15-ol (Tp(ACET)₂(OH)Ox-2-Np)

2,2-dimethoxypropane (1.18 mL, 9.66 mmol, 17.6 eq.) was added dropwise to a solution of Tp(OH)₅Ox-2-Np (260.9 mg, 0.55, 1 eq.) and p-toluenesulfonic acid (10.5 mg, 0.06 mmol, 0.1 eq.) in anhydrous and degassed acetone:toluene (1 :15) (16 mL). The reaction was heated under reflux under for 26 h before being cooled to r. t. and the solvent removed in vaccuo. The crude product was purified by flash column chromatrography (Alumina (neutral), 0-8 % methanol:DCM) yielding Tp(ACET)2(OH)Ox-2-Np as a green solid (40.5 mg, 13 %). ¹H NMR (300 MHz, CDCI₃) 5_H: 9.93 (s, 1 H), 8.75-8.69 (m, 1 H), 8.39 (dd, J =

8.5, 1.7 Hz, 1 H), 7.96-7.81 (m, 4H), 7.74 (s, 1 H), 7.71 (s, 1 H), 7.70 (s, 1 H), 7.59-7.47 (m, 2H), 1 .87 (s, 6H), 1 .79 (s, 6H) ppm. ¹³C NMR{¹H} (100 MHz, CDCI3) 5_C: 161 .6, 148.1 , 147.6,

147.5, 147.3, 140.5, 139.0, 134.7, 133.1 , 129.1 , 128.7, 128.3, 128.0, 127.9, 127.7, 127.6, 126.9, 125.7, 124.4, 124.4, 124.3, 124.2, 124.2, 118.6, 118.3, 116.8, 106.5, 106.2, 102.0, 101.3, 100.8, 26.3, 26.2 ppm. MALDP m/z 557 ([M+H+1]⁺ 20 %), 556 ([M+H]⁺ 75 %), 555 ([M]⁺ 100 %). IR v (neat): 3401w (O-H), 2987m (C-H), 2963m (C-H), 2930m (C-H), 1636w (aromatic C=C) cm ¹. methyl 2-(2-(2-((6,6,11,11 -tetramethyl-2-(naphthalen-2-yl)bis([1,3]dioxolo)

[4',5':6,7;4",5":10,11]triphenyleno[1,2-d]oxazol-15-yl)oxy)ethoxy)ethoxy)acetate (Tp(ACET)₂(PEGCOOMe)Ox-2-Np) methyl 2-(2-(2-chloroethoxy)ethoxy)acetate (18.3 pL, 0.107 mmol, 1 .5 eq.) was added to a slurry of Tp(ACET)₂(OH)Ox-2-Np (39.7 mg, 0.072 mmol, 1 eq.), potassium carbonate (24.7 mg, 0.179 mmol, 2.5 eq.), potassium iodide (12.0 mg, 0.072 mmol, 1 eq.) and acetonitrile (23.5 mL). The slurry was heated under reflux for 48 h. The slurry was cooled to r.t. and filtered under vacuum, the filtrate was collected and the solvent removed in vaccuo yielding Tp(ACET)₂(PEGCOOMe)Ox-2-Np as a crude brown solid (68.9 mg, quant, yield (crude)) which was used without further purification. MALD m/z: 717 ([M+H+1]⁺ 15 %), 716 ([M+H]⁺ 50 %), 715 ([M]⁺ 100 %). methyl 2-(2-(2-((2,3,11,12-tetrahydroxy-8-(naphthalen-2-yl)triphenyleno[1,2-d]oxazol- 6-yl)oxy)ethoxy)ethoxy)acetate (T p(OH)₄(PEGCOOMe)Ox-2-Np)

Trifluoroacetic acid (534 pL, 6.936 mmol, 72 eq.) was added dropwise over 1 min to a stiring solution of Tp(ACET)2(PEGCOOMe)Ox-2-Np (68.9 mg, 0.096 mmol, 1 eq.) in anhydrous, degassed DCM (15.7 mL), the reaction was stirred at r.t. for 19 h. The solvent and trifluoroacetic acid were removed in vaccuo and the resulting solid was purified by prep HPLC (method 1 ) yielding Tp(OH)4(PEGCOOMe)Ox-2-Np as a crude brown solid (49 mg, 79 % (crude)) which was used without further purification. MALD m/z: 637 ([M+H+1]⁺ 100 %), 636 ([M+H]⁺ 90 %), 635 ([M]⁺ 75 %). methyl 2-(2-(2-((2,3,11,12-tetrakis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-8-

(naphthalen-2-yl)triphenyleno[1,2-d]oxazol-6-yl)oxy)ethoxy)ethoxy)acetate

(Tp(PEG)₄(PEGCOOMe)Ox-2-Np)

1-(2-bromoethoxy)-2-(2-methoxy-ethoxy)ethane (225 pL, 0.913 mmol, 12 eq.) was added dropwise over 5 mins to a slurry of Tp(OH)4(PEGCOOMe)Ox-2-Np (48 mg, 0.076 mmol, 1 eq.), potassium carbonate (84 mg, 0.609 mmol, 8 eq.), potassium iodide (10 mg, 0.061 mmol, 0.8 eq.) and anhydrous acetonitrile (25 mL). The reaction was heated under reflux with N₂ bubbling through the slurry for 5.5h before being cooled to r.t. and filtered under vacume. The filtrate was collected, solvent removed in vaccuo and purified by flash column chromatography (silica; 0-6 % MeOH in DCM) yielding Tp(PEG)4(PEGCOOMe)Ox-2-Np as a yellow solid (4 mg, 4 %). ¹H NMR (300 MHz, CDCI₃) 6_H: 10.22 (s, 1 H), 8.90 (d, J = 1.7 Hz, 1 H), 8.46 (dd, J = 8.6, 1.7 Hz, 1 H), 8.06 (s, 1 H), 8.04 (s, 1 H), 8.02 (s, 1 H), 7.95-7.94 (m, 2H), 7.92 (s, 1 H), 7.63-7.57 (m, 2H), 4.71 (t, J = 5.2, 2H), 4.50-4.41 (m, 6H), 4.32 (s, 2H), 4.17 (t, J = 5.2 Hz, 2H), 4.07-3.97 (m, 8H), 3.93-3.86 (m, 2H), 3.87-3.80 (m, 9H), 3.77- 3.69 (m, 12H), 3.70-3.62 (m, 10H), 3.58-3.51 (m, 10H), 3.36 (s, 3H), 3.36 (s, 3H), 3.36 (s, 3H), 3.33 (s, 3H) ppm. MALDI⁺ m/z 1260 ([M+H+K]⁺ 75 %), 1259 ([M+K]⁺ 100 %), 1244 ([M+H+Na]⁺ 25 %), 1243 ([M+Na]⁺ 30 %), 1222 ([M+H+1]⁺ 25 %), 1221 ([M+H]⁺ 75 %), 1220 ([M]⁺ 100 %). IR v (neat): 2926m (C-H), 2873m (C-H), 1693m (C=O), 1616w (aromatic C=C), 1511 m (aromatic C=C) cm’¹. 2-(2-(2-((2,3,11,12-tetrakis(2-(2-(2-methoxyethoxy)ethoxy)ethoxy)-8-(naphthalen-2- yl)triphenyleno[1 ,2-d]oxazol-6-yl)oxy)ethoxy)ethoxy)acetic acid

(Tp(PEG)₄(PEGCOOH)Ox-2-Np)

Aqueous sodium hydroxide (1 M solution, 43 pL, 0.043 mmol, 12 eq.) was added to a solution of Tp(PEG)4(PEGCOOMe)Ox-2-Np (4 mg, 0.004 mmol, 1 eq.) in methanol (15 mL) and the reaction was heated under reflux for 1.5 h. The reaction was cooled to r.t. and acidified from pH8 to pH5 by dropwise addition of aqueous hydrochloric acid (1 M solution, ~6 drops). Water (20 mL) was added and the aqueous solution was extracted with DCM (6 x 10 mL). The organic extracts were combined and evaporated to dryness. The resulting brown solid was purified by prepHPLC yielding Tp(PEG)4(PEGCOOH)Ox-2-Np as a brown oil (2.3 mg, 58 %). ¹H NMR (300 MHz, CDCI₃) 5_H: 10.24 (s, 1 H), 8.91 (s, 1 H), 8.47 (dd, J = 8.7, 1.7 Hz, 1 H), 8.09-8.00 (m, 3H), 7.99-7.89 (m, 4H), 7.64-7.57 (m, 2H), 4.72 (t, J = 5.2 Hz, 2H), 4.51-4.42 (m, 4H), 4.33 (s, 2H), 4.20-4.15 (m, 2H), 4.08-3.99 (m, 6H), 3.93-3.84 (m, 2H), 3.88-3.80 (m, 6H), 3.77-3.70 (m, 5H), 3.70-3.61 (m, 20H), 3.58-3.52 (m, 5H), 3.37 (s, 3H), 3.37 (s, 3H), 3.36 (s, 3H), 3.33 (s, 3H) ppm. MALDF m/z. 1245 ([M+K]⁺ 20 %), 1230 (M+H+Na]⁺ 80 %), 1229 ([M+Na]⁺ 100 %), 1208 ([M+H+1]⁺ 25 %), 1207 ([M+H]⁺ 75 %, 1206 ([M]⁺ 85 %). IR v (neat): 3375br (O-H), 2923m (C-H), 2854m (C-H), 1721s (C=O), 1618w (aromatic C=C), 1514w (aromatic C=C) cm’¹.

Compound 4:

The synthesis of Compound 4 is shown in Figure 10D.

TpMe(PEG-N3)Ox-PhpMe: Synthesis procedure as described for the preparation of TpMe(PEG-COOMe)Ox-PhpMe using TsO-PEGNa (189 mg, 0.57 mmol) and KI was not added. Isolated as an orange solid (32 mg, 12 %); OIH (¹H NMR, 300 MHz) 10.12 (s, 1 H),

8.22-8.21 (m, 2H), 7.92-7.81 (m, 4H), 7.39-7.26 (m, 2H), 4.67-4.64 (m, 2H), 4.29 (s, 3H),

4.22-4.06 (m, 13H), 3.86-3.84 (m, 2H), 3.76-3.73 (m, 2H), 3.37-3.36 (m, 2H), 2.48 (s, 3H); MALDI (m/z): 652 ([M]⁺, 100 %).

The synthesis of Compound 5 is shown in Figure 10E.

TpMe(PEG-alkyne)Ox-PhpMe: Synthesis procedure as described for the preparation of Tpue(PEG-COOMe)Ox-PhpMe using TsO-PEG-alkyne (236 mg, 0.57 mmol) and KI was not added. Isolated orange solid (42 mg, 15 %) ; d_H (¹H NMR, 300 MHz) 10.11 (s, 1 H), 8.22- 8.21 (m, 2H), 7.91 -7.80 (m, 4H), 7.38-7.25 (m, 2H), 4.66-4.62 (m, 2H), 4.28 (s, 3H), 4.22- 4.06 (m, 23H), 2.48 (s, 3H), 2.25-2.29 (m, 2H), 1.92 (s, 1 H), 1.83-1.75 (m, 2H); MALDI (m/z): 737 ([M]⁺, 100 %).

Compound 6:

The synthesis of Compound 6 is shown in Figure 10F.

TpMeOx-PhpPEG-COOMe: Synthetic procedure as described for the preparation of TpMe(PEG-alkyne)Ox-PhpMe using TpMeOx-PhpOH (200 mg, 0.36 mmol), K2CO3 (197 mg, 1.47 mmol), KI (59 mg, 0.36 mmol), CI-PEGCOOMe (140 mg, 0.71 mmol) and MeCN (20 mL). Orange solid isolated (130 mg, 54 %) ,d_H (¹H NMR, 300 MHz) 10.06 (s, 1 H), 8.23-8.21 (m, 2H), 7.89-7.74 (m, 4H), 7.05-7.02 (m, 2H), 4.67-4.61 (m, 2H), 4.26 (s, 6H), 4.20-4.04 (m, 14H), 3.87-3.81 (m, 2H), 3.79-3.71 (m, 2H), 3.37-3.35 (m, 2H) MALDI (m/z): 671 ([M]+, 100 %). TpMeOx-PhpPEG-COOH: Synthesis procedure as described for the preparation of Tp_Me(PEG-COOH)Ox-PhpMe using Tp_MeOx-PhpPEG-COOMe (130 mg, 97 %), NaOH (31 mg, 0.78 mmol), THF (5 mL), MeOH (5 mL) and H2O (1 mL). Aorangish brown solid isolated (124 mg, 97 %). d_H (¹H NMR, 300 MHz) 10.05 (s, 1 H), 8.22-8.20 (m, 2H), 7.90-7.74 (m, 4H), 7.04-7.01 (m, 2H), 4.66-4.60 (m, 2H), 4.25 (s, 6H), 4.20-4.05 (m, 11 H), 3.86-3.82 (m, 2H), 3.76-3.73 (m, 2H), 3.37-3.36 (m, 2H) MALDI (m/z): 657 ([M]⁺, 100 %).

Precursor 10:

Synthesis of precursor 10: to a solution of precursor 6 (5.63 g, 8.64 mmol) in MeOH (20 mL), was added 13 M NaOH(_aq) (16 mL). The resultant solution was stirred at room temperature for 20 h. The solvent was removed in vacuo and EtOAc (50 mL) was added. The EtOAc solution was washed with 1 M HCI_aq (100 mL), and the aqueous layer was separated. The aqueous layer was extracted with EtOAc (3 x 50 mL). The combined organic layers were dried (MgSO4), filtered and the solvent was removed in vacuo. The crude pale yellow oil was purified by column chromatography (silica, elution degraded 0 to 100 % EtOAc in hexane) and the solvent was removed to afford a colourless oil (4.04 g, 73 %). 1 H NMR (300 MHz, CDCI₃, Me₄Si, 25 °C) 6H ppm 10.40 (br s, 1 H), 7.34 (s, 2H), 4.29-4.21 (m, 6H), 3.72-3.66 (m, 24H), 3.38 (t, J = 5 Hz, 6H); 13H NMR (300 MHz, CDCI3, Me₄Si, 25 °C) 5c ppm 169.5, 151.8, 142.6, 124.2, 109.1 , 72.2, 70.4, 70.2, 70.1 , 69.7, 69.5, 68.4, 50.3; ESMS (m/z): 641 ([M]+). Precursor 17:

R ^s Rj

The synthesis of precursor 15 is shown in Figure 6.

Synthesis of precursor 15: To a solution of 4 (0.70 g, 0.78 mmol) in MeOH (10 mL) was added 5 M NaOH(_aq)(3.8 mL, 19.00 mmol) and the resultant solution was stirred at room temperature for 20 h. The solvent was removed in vacuo and EtOAc (10 mL) was added. The EtOAc solution was washed with 1 M HCI_aq (10 mL), and the aqueous layer was separated and extracted with EtOAc (3 x 10 mL). The combined organic layers were dried (MgSO4), filtered and the solvent was removed in vacuo. The crude pale yellow oil was purified by column chromatography (silica, elution degraded 0 to 5 % MeOH in DCM) and the solvent was removed to afford a colourless oil (0.50 g, 73 %)(Figure 6).1 H NMR (300 MHz, CDCI3, Me₄Si, 25 oC) 5H ppm 7.39 (t, 1 H, J = 5.3 Hz), 7.12 (s, 2H), 4.23-4.18 (m, 6H), 3.89-3.86 (m, 4H), 3.82-3.79 (m, 2H), 3.76-3.65 (m, 32H), 3.38 (t, 6H, J = 5.1 Hz), 2.53 (t, 2H, J = 6.0 Hz); 13C NMR (300 MHz, CDCI3, Me₄Si, 25 °C) 5c ppm 173.4, 167.3, 152.3, 145.0, 141.1 , 129.8, 107.9, 107.1 , 72.3, 70.8, 70.7, 70.6, 70.5, 70.3, 70.2, 70.0, 69.8, 68.9, 66.5, 50.7, 40.1 , 34.8; MALDI (m/z): 867 ([M+Na]+).

Synthesis of precursor 17: To a solution of precursor 15 (100 mg, 0.11 mmol, 1 e.q.) in ethanol (3 mL), was added a solution of NH4CI (41 mg, 0.77 mmol, 7 e.q.) in water (1 mL) followed by Zn powder (42 mg, 0.65 mmol, 6 e.q.). The resultant slurry was heated at 78 °C for 3 h. The reaction was allowed to cool to room temperature and tetrahydrofuran (THF)(15 mL) was added. The resulting slurry was filtered and the filtrate was reduced in vacuo to yield Compound 7 a colourless viscous oil (85 mg, 93 %). 1 H NMR (300 MHz, D₂O) 5H: 7.16 (s, 2H), 4.37-4.22 (m, 6H), 4.03-3.90 (m, 4H), 3.88-3.82 (m, 2H), 3.80-3.51 (m, 36H), 3.21-3.14 (m, 4H) ppm. MALDI-TOF+ m/z: 767.6 ([M+H]+ 100%), 789.6 ([M+Na]+ 75%).

Precursor 18

R’ = H

The synthesis of precursor 15 is shown in Figure 7.

Compound 6 (170 mg, 0.26 mmol, 1 e.q.) and N-Hydroxysuccinimide (NHS) (30 mg, 0.26 mmol, 1 e.q.) are charged to an oven dried flask and the solids are purged with argon for 5 mins. A solution of N.N'-Dicyclohexylcarbodiimide (DCC) (53 mg, 0.26 mmol, 1 e.q.) in N,N- Dimethylformamide (DMF) (5.6 mL) is added to the flask via needle through a rubber septa forming a straw coloured solution. The reaction is stirred at room temperature for 24 h. The reaction is divided between several Eppendorf tubes and centrifuged to remove the solid dicyclohexylurea byproduct. The supernatant containing the NHS ester is collected via pipette and added dropwise to a cooled (4 °C) solution of 3-Amino-2-sulfopropanoic acid (53 mg, 0.31 mmol, 1 .2 e.q.) in sodium bicarbonate buffer (6.8 % w/v, 12 mL). The reaction is allowed to warm to room temperature and stirred for 1 h. The reaction is acidified to pH 2 by addition of HCI_aq (1 M) and the solvent is then removed under reduced pressure. The resulting residue is purified by flash column chromatography (Silica-C18 stationary phase; 10-100 % acetonitrile in water (0.1 % trifluoroacetic acid) mobile phase yielding Compound 9 as a black solid (14.5 mg, 7 %). 1 H NMR (300 MHz, DMSO-D6) 6H: 12.19 (s br, 1 H), 10.04 (s, 1 H), 8.27 (d, J = 8.9 Hz, 2H), 8.16 (s, 1 H), 8.14 (s, 1 H), 8.07 (s, 1 H), 8.07 (s, 1 H), 7.95 (s, 1 H), 7.83-7.73 (m, 1 H), 7.27 (d, J = 8.9 Hz, 2H), 4.31 -4.25 (m, 5H), 4.14 (s, 3H), 4.12-4.06 (m, 11 H), 3.86-3.80 (m, 2H), 3.68-3.63 (m, 2H), 3.63-6.59 (m, 2H) ppm. MALDI- TOF+ m/z: 809.4 ([M]+ 100%). HPLC: 92.5 %. Precursor 19 hteO

H SC ^Q^O^O^K^S-bR* MeO O e 0 0

R* = MHS

The synthesis of precursor 18 is shown in Figure 7.

Precursor 18 (14.5 mg, 0.02 mmol, 6 e.q.) and N-Hydroxysuccinimide (NHS) (8.5 mg, 0.08 mmol, 4 e.q.) are charged to an oven dried flask and the solids are purged with argon for 5 mins. A solution of N,N'-Dicyclohexylcarbodiimide (DCC) (5.7 mg, 0.03 mmol, 1.5 e.q.) in

N,N-Dimethylformamide (DMF) (2 mL) is added to the flask via needle through a rubber septa forming a straw coloured solution. The reaction is stirred at room temperature for 24 h. The reaction is divided between several Eppendorf tubes and centrifuged (5000 r.p.m., 10 mins) to remove the solid dicyclohexylurea byproduct. The supernatant containing precursor 19 is collected via pipette.

Dendron 1 :

The synthesis of Dendron 1 (i.e. molecular scaffold 12) is shown in Figure 4.

Synthesis of Compound 1 : shown in Figure 9A and described above.

Synthesis of Dendron 1 : To a solution of precursor 10 (22 mg, 0.03 mmol) and compund 1 (150 mg, 0.20 mmol) in THF (5 mL), aqueous solutions of 0.05 M CuSO4 (0.42 mL) and

O.50 M sodium ascorbate (1 .20 mL) were added and the resultant slurry was stirred at room temperature for 20 h. The precipitate was filtered off and the residue was washed with copious amounts of acetone and MeOH until the residue no longer glowed when exposed to UV light. The filtrate was removed in vacuo followed by addition of H2O (5 mL) and the organic layer was extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried (MgSO4), filtered and the filtrate was removed in vacuo. The crude solid was adsorbed onto silica and purified via flash column chromatography (silica, gradient elution from 0 to 20 % MeOH in DCM and the solvent was removed in vacuo to yield an orange solid (47 mg, 47 %) (Figure 4). 1 H NMR (300 MHz, CDCI3, Me4Si, 25 oC) 5H ppm 9.62-9.65 (m, 3H), 8.01 -8.05 (m, 6H), 7.59-7.41 (m, 15H), 7.33 (s, 2H), 6.99- 6.93 (m, 6H), 4.49-4.45 (m, 6H), 4.29-4.01 (m, 58H), 3.93-7.87 (m, 6H), 3.81-3.42(m, 66H), 2.78-2.63 (m, 6H), 1.93-1.88 (m, 6H); 13H NMR (300 MHz, CDCI₃, Me₄Si, 25 °C) 6c ppm 161.1 , 148.6, 147.8, 142.7, 140.0, 139.1 , 128.9, 126.2, 123.1 , 122.9, 122.7, 120.0, 115.7, 114.7, 109.1 , 104.5, 103.7, 102.6, 72.4, 70.8, 70.5, 70.0, 69.7, 69.6, 68.8, 68.7, 67.6, 55.9, 55.8, 55.6, 50.7, 50.1 , 29.3, 22.2; MALDI (m/z): 2925 ([M+Na]+).

Dendron 1 (i.e. molecular scaffold 12) was dissolved in anhydrous DMF to form a 43 pM stock solution which was then titrated in 10 pL portions into a 1 cm square quartz cuvette containing DMF (3 mL) for photophysical measurements shown in Figure 5.

Dendron 2:

The synthesis of Dendron 2 (i.e. molecular scaffold 16) is shown in Figure 6.

Synthesis of precursor 14: To a N? purged mixture of precursor 10 (1 .55 g, 2.43 mmol), tert-butyl 12-amino-4,7,10-trioxadodecanoate (3) (1.01 g, 3.64 mmol), DCC (0.75 g, 3.64 mmol) and DMAP 0.04 g, 0.36 mmol) was added anhydrous DCM (20 mL). The resultant solution was stirred at room temperature under N2 atmosphere for 4 days. A precipitate formed overtime, which was filtered off after the reaction was complete and was washed with copious amounts of DCM. The filtrate was reduced in vacuo, and the resulting crude yellow oil was purified by flash column chromatography (silica, elution degraded 0 to 5 % MeOH in DCM) and the solvent was removed in vacuo to afford a colourless oil (1 .70 g, 76 %) (Figure 6).1 H NMR (300 MHz, CDCI₃, Me₄Si, 25 oC) 6H ppm; 7.08 (s, 2H), 6.90-6.87 (br s, 1 H), 4.22-4.18 (m, 6H), 3.88-3.72 (m, 6H), 3.69-3.58 (m, 32H), 3.39-3.37 (m, 6H), 2.47 (t, 2H, J = 6.0 Hz), 1.43 (s, 9H); 13H NMR (300 MHz, CDCI3, Me4Si, 25 °C) 6c ppm 170.9, 167.0, 152.5, 141.3, 129.9, 107.0, 80.6, 72.4, 70.8, 70.7, 70.6, 70.5, 70.4, 70.3, 70.1 , 70.0, 69.8, 69.0, 66.9, 50.7, 40.0, 36.2, 28.1 ; ESMS (m/z): 923 ([M+Na]+, 100 %); m/z (HRMS): found 923.4463. Calc, mass for C3sH6₄NioOi5Na: 923.4450.

Synthesis of precursor 15: described above.

Synthesis of Compound 1 : shown in Figure 9A and described above. Synthesis of Dendron 2: To a solution of precursor 15 (33 mg, 0.04 mmol) and Compound 1 (180 mg, 0.24 mmol) in DMF (1.50 mL), was added aqueous solutions of 0.05 M CuSO4 (0.20 mL) and 0.10 M sodium ascorbate (1 mL) and the resultant slurry was stirred at room temperature for 20 h. H2O (5 mL) was added and the organic layer was extracted with EtOAc (3 x 10 mL). The combined organic layers were washed with brine (10 mL), dried (MgSO4) and the solvent removed in vacuo. The crude solid was adsorbed onto silica and purified via flash column chromatography (silica, gradient elution from 0 to 20 % MeOH in DCM). The solvent was removed in vacuo to afford a vicious orange oil (104 mg, 85 %)(Figure 6). 1 H NMR (300 MHz, CDCI3, Me₄Si, 25 oC) 6H ppm 9.71 (s, 2H), 9.68 (s, 1 H), 8.10-8.02 (m, 6H), 7.55 (s, 2H), 7.53 (3, 1 H), 7.52 (s, 2H), 7.50 (s, 3H), 7.48 (s, 1 H), 7.44 (s, 3H), 7.40 (s, 2H), 7.31 (s, 1 H), 7.14 (s, 2H), 7.02-6.96 (m, 6H), 4.47-4.38 (m, 6H), 4.20 (t, J = 4.8 Hz, 10H), 4.13 (s, 6H), 4.12 (s, 3H), 4.09 (s, 6H), 4.08 (s, 3H), 4.07 (s, 12H), 4.03 (s, 6H), 3.92-3.88 (m, 8H), 3.83-3.51 (m, 76H), 3.50-3.42 (m, 6H), 2.72 (t, J = 7.7 Hz, 6H), 2.53 (s br, 2H), 1 .94-1 .82 (m, 6H). 13C NMR (300 MHz, CDCI3, Me₄Si, 25 °C) 5c ppm 167.2, 161.2, 161.1 , 152,2m 148.7, 148.2, 148.0, 147.5, 142.9, 140.2, 139.2, 129.8, 129.0, 126.4, 123.8, 123.2, 123.0, 122.9, 122.1 , 120.0, 115.8, 114.8, 109.2, 107.2, 104.6,

103.8, 102.8, 100.9, 70.9, 70.6, 70.5, 70.4, 70.1 , 69.7, 69.6, 69.5, 68.7, 67.6, 56.0, 55.9,

55.8, 55.7, 50.1 , 29.7, 29.6, 21.8; MALDI (m/z): 3128 ([M+Na]+). Purity by HPLC: 92.22 %.

Dendron 2-NHS

Synthesis of Dendron 2-NHS: A solution of N.N'-dicyclohexylcarbodiimide (DCC) (0.047 mg, 0.225 mmol, 1 eq.) in dimethylformamide (DMF) (50 pL) was added to a solution of Dendron 2 (0.7 mg, 0.225 mmol, 1 eq.) and N-hydroxysuccinimide (NHS)(0.026 mg, 0.225 mmol, 1 eq.) in dimethylformamide (DMF)(100 pL). The reaction was vortexed to mix, sealed under argon and left at room temperature for 21 h in light free conditions (Figure 6). MALDI (m/z): 3225 ([M+Na]+), 3203 ([M]+).

Dendron 3

The supernatant containing precursor 19 (as described above) is added dropwise to a cooled (4 °C) solution of Compound 7 (2.4 mg, 0.003 mmol, 1 e.q.) in sodium bicarbonate buffer (6.8 % w/v, 870 pL). The reaction is allowed to warm to room temperature and stirred for 19 h. The reaction is acidified to pH 2 by addition of HCI_aq (1 M) and the solvent is then removed under reduced pressure. The resulting residue is purified by flash column chromatography (Silica-C18 stationary phase; 10-100 % acetonitrile in water (0.1 % trifluoroacetic acid) mobile phase. The purified residue was then dissolved in MeOH (1 mL) and NaOHaq (1 M, 4 e.q.) was added with stirring for 30 mins at room temperature. The solvent was then removed under reduced pressure to yield Dendron 3 as a yellow solid (4 mg, 38 %). 1 H NMR (300 MHz, CDCI3) 6H: 10.14 (s, 2H), 10.09 (s, 1 H), 8.31 (d, J = 8.8 Hz, 4H), 8.28 (d, J = 8.9 Hz, 2H), 7.90 (s, 2H), 7.87 (s, 3H), 7.86 (s, 3H), 7.84 (s, 4H), 7.81 (s, 2H), 7.08 (d, J = 8.6 Hz, 6H), 4.36-4.23 (m, 31 H), 4.22-4.02 (m, 52H), 4.01 -3.90 (m, 10H), 3.90-3.79 (m, 9H), 3.79-3.58 (m, 28H), 3.39 (t, J = 5.2 Hz, 2H) ppm. MALDI-TOF+ m/z: 2708.1 ([M-TpMeOx-Ph]+ 10%), 2087.5 ([M-(TpMeOx-PhpOCH2CH2OCH2)2]+ 5%), 670.4 ([TpMeOx-PhpPEGCONHCH2]+ 50 %), 656.4 ([TpMeOx-PhpPEGCONH]+ 100 %), 641 .8 ([TpMeOx-PhpPEGCO]+ 60 %).

Dendron 3-NHS

A solution of N.N'-dicyclohexylcarbodiimide (DCC) (0.131 mg, 0.001 mmol, 1 eq.) in dimethylformamide (DMF) (136 pL) was added to a solution of Dendron 3 (1 mg, 0.001 mmol, 1 eq.) and N-hydroxysuccinimide (NHS)(0.073 mg, 0.001 mmol, 1 eq.) in dimethylformamide (DMF)(273 pL). The reaction was vortexed to mix, sealed under argon and left at room temperature for 21 h in light free conditions.

Second Generation Dendron G1 :

G1 -Gen 2 (TpueOx-PhpPEG-alkyne) ester: A slurry of Compound 1 (TpMeOx-PhpPEG- alkyne) (40 mg, 0.05 mmol), 1^st generation dendron-ester (9 mg, 0.03 mmol), CUSO4.5H2O (40 mg, 16 mmol) and sodium ascorbate (50 mg, 0.25 mmol) in THF (5 mL) was stirred for 20 h at room temperature. The solute was filtered off and the residue was washed with copious amounts of acetone and MeOH until the residue no longer glowed when exposed with UV light. The solvent was removed in vacuo and the crude solid was adsorbed onto silica and purified via flash column chromatography (silica, gradient elution from 0 to 20 % MeOH in DCM. The solvent was removed in vacuo (17 mg, 45 %). MALDI (m/z): 2939 ([M+Na]⁺, 100 %). Antibody Conjugation

Reagents

• BupH™ MES Buffered Saline Packs - ThermoFischer Scientific Catalog Number: 28390

• Anhydrous (Extra Dry) DMF - ThermoFisher Scientific Catalog number: 348431000

• Sodium Bicarbonate Buffer (100 mM) - ThermoFisher Scientific Catalogue number: 44710001

• Pierce™ EDC, No-Weigh™ Format - ThermoFischer Scientific Catalog number: A35391

• Sulfo-NHS (N-hydroxysulfosuccinimide), No-Weigh™ Format - ThermoFisher Scientific Catalogue number: A39269

• CD4 (SK3) Unconjugated Antibody - Purified Mouse Anti-Human CD4 (0.5 mg/mL) - Biolegend Catalogue number: 344602

• Ultrapure HyClone™ Water, Molecular Biology Grade, Cytiva - Fisher Catalogue number: 10787944

• UltraComp eBeads™ Compensation Beads - ThermoFisher Scientific Catalogue number: 01-2222-42 (100 tests)

• Cell Staining Buffer - Biolegend Catalogue number: 420201 (500 mL)

1 . Active Ester Dendron Formation

In a first step, Pierce™ EDC, No-Weigh™ Format (1 mg) is reconstituted in dry DMF (100 mM, 52.2 pL, 22 °C). The solution is then vortexed for 2 mins, sonicated for 10 mins (30 °C), vortexed for a further 2 mins and sonicated for 10 mins (25 °C). The EDC is then diluted from 100 mM to 20 mM by taking 100 mM solution (2.0 pL) and add anhydrous DMF (8.0 ML).

In a second step, Sulfo-NHS (N-hydroxysulfosuccinimide), No-Weigh™ Format (2 mg) is reconstituted in MES buffer (150 mM, 61.4 pL, 22 °C). The MES buffer (150 mM) is prepared by adding a pouch of BupH™ MES Buffered Saline to 500 mL of Milli-Q H2O to provide an initial MES buffer solution (0.1 M). NaOH_aq (~1 mL, 1 M) is then added to the initial MES buffer solution (0.1 M, 50 mL) to adjust the pH to between 5.75 and 6.0. The reconstituted Sulfo-NHS solution is vortexed for 2 mins and sonicated for 10 mins (30 °C) before being diluted from 150 mM to 18 mM by taking 150 mM solution (1.2 pL) and add MES (8.8 pL). The diluted solution is again vortexed for 2 mins and sonicated for 10 min (25 °C).

In a third step, Dendron 2 (synthesis described above) is reconstituted in anhydrous DMF to form a 1 .5 mM solution (e.g. dissolve 1 mg of dye in 150 uL of anhydrous DMF) before being vortexed for 3 mins and sonicated for 10 mins (25 °C).

In a fourth step, CT-Blue dye (30.0 pL) is added to a clear Eppendorf (1 .5 mL).

In a final step, add EDC (2.4 pL, 20 mM), followed by Sulfo-NHS (2.8 pL, 18 mM). Vortex (15 sec), incubate (in the dark, 24 hr) to afford the active ester Dendron 2 solution (1 .28 mM).

2. Antibody Solution Preparation

T ransfer sodium bicarbonate buffer (100 mM, 20.8 pL, 22 °C) to a dark eppendorf (1 .5 mL). Then add CD4 antibody (6.3 pL, 0.5 mg per mL, purified Mouse anti-human CD4 SK3, Biolegend), vortex (15 s) and cool on ice (25 min) to afford the antibody solution.

3. Dye-Antibody Conjugation

Combined anhydrous DMF (24 pL) with active ester Dendron 2 solution (3.5 pL, 1.28 mM), and add to the antibody solution. Vortex the solution for 15 seconds and incubate in the dark, on ice (2.5 hr) to afford the Dendron 2«CD4 Conjugate. Store the conjugate in the dark, at 4 °C overnight (~ 18 hr). Conjugate is then ready for use in flow cytometry.

4. Conjugation Check Kit

To confirm conjugation, a confirmation check kit from abeam (Catalogue number ab236554) was used along with the following method:

In a first step, 1 .1 % in ultrapure H2O (0.55g of BSA per 50 mL dH2O) is provided and then vortexed (60 s). In an eppendorf (1 .5 mL), 1.1 % BSA solution (909.1 pL) and 10x Running Buffer (90.1 pL supplied in Conjugation Check Kit, abeam) is added to afford 1x Running Buffer/BSA solution.

Then, in another eppendorf (0.5 mL), 1x Running Buffer/BSA solution (35 pL), followed by Dendron 2«CD4 Conjugate (5 pL) is added and vortexed (10 s) before transfering the solution to a well in a 96-well plate.

A Protein A/G strip (supplied in Conjugation Check Kit, abeam) is added to the well (10 min) before visualising the strip under UV illumination (302 nm, 8W, UV lamp). Successful Dendron 2 CD4 antibody conjugation is indicated by a fluorescent line on the strip. Figure 11 shows a photograph of a strip with the fluorescent line indicated by the arrow.

Bead Staining Protocol

1 . Vortex (20 s) the compensation beads (Invitrogen 01 -2222-42) and transfer (25 pL per sample) to a 1 .5 mL Eppendorf.

2. Add Dendron 2«CD4 Conjugate (2.5, 5.0 and 15.0 pL) to the beads.

3. Incubate the samples in the dark (30 min, 22 °C).

4. Add cell staining buffer (1400 pL) and vortex (3 sec).

5. Centrifuge (600 g, 5 min).

6. Remove supernatant, leaving behind the bead pellet.

7. Resuspend the bead pellet in staining buffer (1400 pL), vortex (3 sec) and centrifuge (600 g, 5 min)

8. Repeat steps 6) and 7) for a total of three washes.

9. Resuspend the bead pellet in 500 pL staining buffer, vortex (3 sec) and transfer the sample to a FACS tube.

10. Samples are analysed on a BD LSR Fortessa X-20 flow cytometer.

Bead Acquisition and Analysis

Samples were acquired on a BD LSR Fortessa X-20 flow cytometer using a UV laser configuration (Aex = 355 nm) with the following filter:

1 . lndo-1 (Violet) H 450/50 for Dendron 2«CD4 Conjugate;

2. Gate for the main bead population (FSC-A vs. SSC-A); 3. Gate for the single bead population (FSC-A vs. FSC-H); and

4. Display data in the lndo-1 (Violet) fluorescence channels

Figure 12 shows the flow cytometry scatterplots for the main bead population (Figure 12A) and the single bead population (Figure 12B).

Figure 13 shows the fluorescence profiles 1300, 1351 , 1352 of unstained beads compared to the fluorescence profiles 1350, 1351 , 1352 of the beads when stained with 2.5 pL (Figure 13A), 5.0 pL (Figure 13B) and 15 pL (Figure 13C) of Dendron 2*CD4 Conjugate.

It will be appreciated by those skilled in the art that any number of combinations of the aforementioned features and/or those shown in the appended drawings provide clear advantages over the prior art and are therefore within the scope of the invention described herein.

Claims

1 . A molecular scaffold (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecular scaffold comprising: a core having plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set; wherein the plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core; wherein one or more or all of the identifier molecules (IM) have the following general structure (1 ):

wherein X represents one of NH, O, S, Se;

Y¹, Y², Y³ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^x (e.g. -OCH₃, -O(CH₂CH₂O)nCH₂COOH or -O(CH₂CH₂O)_nCH₃ wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH₃), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C1-6 haloalkyl, C2-6 alkynyl, NH2, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-6 alkynyl; two or more of Y¹, Y², Y³ may combine together to form a condensed ring (e.g. a condensed aromatic ring), and/or one or more of Y¹, Y², Y³ may independently comprise a heterocyclic moiety; wherein one or more of Y¹, Y², Y³ and/or R comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

2. A molecular scaffold according to Claim 1 , wherein one or more of Y¹, Y², Y³ and/or R comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon and oxygen atoms, and further comprising said linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

3. A molecular scaffold according to Claim 2, wherein the linker group (L) is located at a or the terminus of the spacing portion.

4. A molecular scaffold according to any preceding Claim, wherein the R group comprises the linker group (L) and optionally a spacing portion, e.g. R may consist of an -O-(CH2CH2O)_ZCH2-L moiety, wherein z is an integer of 1 , 2, 3, 4, or 5.

5. A molecular scaffold (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecular scaffold comprising: a core having plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set; wherein the plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core; wherein one or more or all of the identifier molecules (IM) have the following general structure (2):

wherein X represents one of NH, O, S, Se;

Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. - O(CH2CH2O)_nCH₂COOH or -O(CH₂CH₂O)_nCH3wherein n is an integer of 1 , 2, 3, 4, or 5, a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl; two or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ may combine together to form or comprise a condensed or fused ring (e.g. a condensed or fused aromatic ring); wherein one or more of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³ and R comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

6. A molecular scaffold according to Claim 5, wherein one or more or all of Y⁴, Y⁵, Y⁶, Y⁷, Y⁸, Y⁹, Y¹⁰, Y¹¹, Y¹², Y¹³, R, for example one or more or all of Y⁴, Y¹² and/or R, comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon and oxygen atoms, and further comprising said linker group (L), e.g. located at the terminus of the spacing portion, forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

7. A molecular scaffold according to Claim 5 or 6, wherein one or more or all Y⁴, Y⁷, Y⁸, Y¹¹, Y¹² independently represent or comprise an alkoxy group, e.g. an -OR’ group, e.g. a OC5H11 group or a OCH3 group, and/or Y⁵, Y⁶, Y⁹, Y¹⁰, Y¹³ each represent a hydrogen atom, and preferably R comprises a or the spacing portion and linker group.

8. A molecular scaffold (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecular scaffold comprising: a core having plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set; wherein the plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core; wherein one or more or all of the identifier molecules (IM) have the following general structure (3):

wherein X represents one of NH, O, S, Se;

J¹, J², J³, J⁴, J⁵ and Z independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. -OCH₃, -O(CH2CH₂O)nCH₂COOH or - O(CH₂CH₂O)_nCH3wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C2-6 alkenyl, C1-6 haloalkyl, C2-e alkynyl, NH2, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C2-6 alkenyl, C1-6 haloalkyl, C2-e alkynyl; two or more of J¹, J², J³, J⁴, J⁵ and/or Z may combine together to form a condensed ring (e.g. a condensed aromatic ring);

A¹, A², A³, A⁴ independently represents a hydrogen atom, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group (e.g. CH3, C5H9, -(CH2CH2O)_nCH2COOH or -(CF^CFWJnCHswherein n is an integer of 1 , 2, 3, 4, or 5), a polyether group; wherein one or more of A¹, A², A³, A⁴ and Z comprise a linker group (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

9. A molecular scaffold according to Claim 8, wherein one or more or all of A⁴ and/or R and/or Z comprise a spacing portion comprising a continuous chain of between 3 and 20 atoms, e.g. selected from carbon and oxygen atoms, and further comprising said linker group (L), e.g. located at the terminus of the spacing portion, forming a covalent bond with the arm (A1 ) or (A2, A3, A4).

10. A molecular scaffold according to Claim 8 or 9, wherein one or more or all A¹, A², A³, A⁴ independently represent an alkyl group, e.g. a C5H11 group or a CH3 group, and/or J¹, J², J³, J⁴, J⁵ each represent a hydrogen atom, and preferably R comprises the spacing portion and linker group.

11 . A molecular scaffold (e.g. a dendrimer or dendron) having n successive generations (e.g. n is selected from 1 , 2, 3, 4, or 5), the molecular scaffold comprising: a core having plural (e.g. 2, 3, 4, or 5) arms (A1 ); n-1 sets of branching moieties each having plural (e.g. 2, 3, 4, or 5) arms (A2, A3, A4); a functional group (FG1 ) capable of forming a bond with a second species; plural identifier molecules (IM); where present each branching moiety of the first set is bonded to one of the arms (A1 ) of the core, and where present each branching moiety in each subsequent set is bonded to an arm (A2, A3, A4) of a branching moiety of the previous set; wherein the plural identifier molecules are each bonded to an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core; wherein one or more or all of the identifier molecules (IM) have the following general structure (4):

wherein X represents one of NH, O, S, Se;

J¹, J², J³, J⁴, J⁵ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^X (e.g. -OCH₃, -O(CH₂CH₂O)nCH₂COOH or - O(CH₂CH₂O)_nCH3wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH3), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C₂-6 alkenyl, C1-6 haloalkyl, C₂-e alkynyl; two or more of J¹, J², J³, J⁴, J⁵ may combine together to form a condensed ring (e.g. a condensed aromatic ring);

A¹, A², A³, A⁴, A⁵ independently represents a hydrogen atom, a substituted or unsubstituted aryl group, a substituted or unsubstituted alkyl group (e.g. CH3, - O(CH₂CH₂O)_nCH₂COOH or -O(CH₂CH₂O)_nCH₃ wherein n is an integer of 1 , 2, 3, 4, or 5),), a polyether group; wherein R comprises e.g. a spacing portion comprising a continuous chain of between 3 and 20 atoms, and further comprises a linkergroup (L) forming a covalent bond with the arm (A1 ) or (A2, A3, A4), e.g. located at the terminus of a or the spacing portion.

12. A molecular scaffold according to Claim 11 , wherein one or more or all of J¹, J², J³, J⁴, J⁵ each represent a hydrogen atom, A¹, A², A³, A⁴, A⁵ each represent an alkyl group, e.g. a CH3 group or C5H11 group.

13. A molecular scaffold according to any preceding Claim, wherein one of more of the identifier molecules (IM) is derived from one or more of Compounds 1 to 6.

14. A molecular scaffold according to any preceding Claim, wherein at least one of the plural identifier molecules (IM) has a different structure to at least one other of said plural identifier molecules.

15. A molecular scaffold according to any preceding Claim, wherein the linker group (L) is or comprises an amide bond.

16. A molecular scaffold according to any preceding Claim, wherein n=2, the core has three arms (A1 ), there is one set of three branching moieties each having three arms (A2), and the total number of identifier molecules (IM) in the molecular scaffold is nine.

17. A molecular scaffold according to any preceding Claim, wherein the core comprises a six membered aromatic ring and/or three arms (A1 ), for example the core has the following general structure:

wherein A1 represents an arm, and FG1 represents the functional group capable of forming a bond with a second species.

18. A molecular scaffold according to any preceding Claim, wherein the arms (A1 ) comprise a continuous chain of from 3 to 20 atoms selected from carbon atoms, e.g. an alkyl chain, or a combination of carbon atoms and heteroatoms, e.g. oxygen atoms and/or nitrogen atoms, e.g. comprising one or more polyethylene glycol moieties.

19. A molecular scaffold according to any preceding Claim, wherein the branching moieties, where present, comprise a six membered aromatic ring and/or three arms (A2, A3, A4), for example the branching moieties in the first set have the following general structure:

wherein A2 represents an arm, and CG represents a connecting group forming a bond with arm A1 .

20. A molecular scaffold according to any preceding Claim, wherein the arms (A2, A3, A4) comprise a continuous chain of from 3 to 20 atoms selected from carbon atoms, e.g. an alkyl chain, or a combination of carbon atoms and heteroatoms, e.g. oxygen atoms and/or nitrogen atoms, e.g. comprising one or more polyethylene glycol moieties.

21. A molecular scaffold according to any of the preceding Claims, bonded, e.g. covalently bonded, to a second species, wherein the second species is a small molecule and/or a biomolecule e.g. an amino acid, a peptide, a protein, a nucleic acid, a polynucleotide, or an antibody, and/or wherein the second species is a therapeutic species or a pharmaceutically active molecule, e.g. a drug molecule or prodrug molecule, and/or wherein the second species is a solid support, e.g. for use in solid phase synthesis, and/or wherein the second species is a nanoparticle, e.g. a nanoparticle comprising or formed from a metal or a metal alloy, carbon, clay, a polymer, and/or a ceramic material.

22. A method of fabricating a molecular scaffold according to any one of Claims 1 to 21 , the method comprising:

(ii) providing plural identifier molecule having the general structure selected from one or more of (1 ), (2), (3), (4), or (5);

(iii) forming a bond between each of said plural identifier molecules with an arm (A2, A3, A4) of a branching moieties in the n-1 set, or wherein n=1 the plural identifier molecules are each bonded to an arm (A1 ) of the core.

23. A kit of parts suitable for use in covalently bonding the molecular scaffold according to any of Claims 1 to 20 to a second species, e.g. a biomolecule, the kit of parts comprising a molecular scaffold of the invention and a solvent suitable for dissolving or suspending the molecular scaffold.

24. A method of conjugating the molecular scaffold according to Claims 1 to 20 to a second species, the method comprising providing the molecular scaffold, providing a second species, and forming a covalent bond between the molecular scaffold and the second species.

25. A fluorescent dendrimer or dendron comprising plural arms and at least one fluorophore bonded to at least one of said plural arms, wherein said at least one (e.g. or plural or all) fluorophore(s) has the following general structure (1 ):

wherein X represents one of NH, O, S, Se;

Y¹, Y², Y³ independently comprise, consist of, or represent a H, D, F, Cl, Br, I, CN, NO₂, OH, COOH, OR^x (e.g. -OCH₃, -O(CH₂CH₂O)nCH₂COOH or -O(CH₂CH₂O)_nCH₃ wherein n is an integer of 1 , 2, 3, 4, or 5), a substituted or unsubstituted alkyl group (e.g. CH₃), a substituted or unsubstituted aryl group, a polyether chain, a polyglycol group (e.g. a substituted or unsubstituted group comprising a polyglycol moiety, for example one or more polyethylene glycol groups), a PEI (polyethylenimine) moiety, C1-C4 alkoxy, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl, NH₂, NHR^y, NR^yR^z, wherein R^x, R^y, and R^z are independently selected from H, C1-6 alkyl, C₂-6 alkenyl, C1-6 haloalkyl, C₂-6 alkynyl; two or more of Y¹, Y², Y³ may combine together to form a condensed ring (e.g. a condensed aromatic ring), and/or one or more of Y¹, Y², Y³ may independently comprise a heterocyclic moiety; wherein one or more of Y¹, Y², Y³ and/or R comprise a linker group (L) forming a covalent bond with the one of said plural arms; and further comprising a functional group (FG1 ) capable of forming a bond with a second species.