Chemical probes for unfolded, misfolded or intrinsically disordered proteins

Abstract

Disclosed herein are chemical probes for unfolded, misfolded or intrinsically disordered proteins, in particular chemical probes having the structure of Formula I or salts thereof, and to the use of the probes for various applications relating to unfolded, misfolded or intrinsically disordered proteins, for example, for diagnosing or prognosing a disease or disorder associated with an unfolded, misfolded or intrinsically disordered protein.

Description

CHEMICAL PROBES FOR UNFOLDED, MISFOLDED OR INTRINSICALLY DISORDERED PROTEINSCROSS REFERENCE

[0001] The present application claims priority to Australian Provisional Patent Application no. 2024900570, filed 5 March 2024, the entire contents of which is incorporated herewith by cross reference in its entirety.FIELD OF THE INVENTION

[0002] The invention relates to chemical probes for unfolded, misfolded or intrinsically disordered proteins, and to the use of the probes for various applications relating to unfolded, misfolded or intrinsically disordered proteins, for example, for diagnosing or prognosing a disease or disorder associated with an unfolded, misfolded or intrinsically disordered protein. However, it will be appreciated that the invention is not limited to this particular field of use. BACKGROUND

[0003] The following discussion of the prior art is provided to place the invention in an appropriate technical context and enable the advantages of it to be more fully understood. It should be appreciated, however, that any discussion of the prior art throughout the specification should not be considered as an express or implied admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0004] Disorder in protein structure can be essential for some biological functions but can also lead to diseases like neurodegenerative diseases. Characterizing the folding status of proteins on a proteome-wide scale directly in their biological matrices, however, has been challenging.

[0005] The classical structure-function paradigm is in general centered on the intricate folding of the polypeptide chain, creating a three-dimensional structure essential for protein functionality. Cells rely on a proteostasis network to maintain the proper folding of the proteome. Impairment of this quality control system can result in the accumulation of unfolded proteins that are susceptible to misfolding and aggregation. Apart from aberrant disordered proteins from unfolding, it is estimated that more than half of the proteome in eukaryotes are natively unfolded, containing significant intrinsically disordered regions under physiological conditions. Being highly dynamic and flexible, these intrinsically disordered proteins (IDPs) have a higher tendency to interact with other molecules such as proteins and RNAs, and play a central role in regulating cellular signalling pathways. So far, more than 50 human diseases have been linked to the abnormal folding of proteins and IDPs, which include neurological disorders like Alzheimer’s and Parkinson’s disease and non-neurological diseases like type II diabetes, cystic fibrosis, amyloidosis and certain cancers.

[0006] Identifying endogenous disordered protein species in the complex cellular environment, however, has been challenging due to the lack of tools. Common structural biology methods, such as X-ray crystallography and cryogenic electron microscopy, are not suitable for studyingunfolded proteins that are highly heterogenous and dynamic. Nuclear magnetic resonance (NMR)-based methods require the expression / labeling of a particular protein and thus may not be efficient for a proteome-wide study. Mass spectrometry-based proteomics are ideal for a proteome-wide application, but many reported methods require cell lysis prior to the processing / labeling of cellular proteins, and the protein extraction step could alter protein structures before the unfolded species are captured. Accordingly, there is a need to develop further tools for identifying disordered proteins.

[0007] It is an object of the present invention to overcome or ameliorate one or more the disadvantages of the prior art, or at least to provide a useful alternative.SUMMARY OF THE INVENTION

[0008] The present inventors have developed a novel bifunctional class of chemical probes, that may be capable of capturing in situ, enriching and / or quantifying global endogenous protein disorder extracellularly and in cells. In certain embodiments, the probes may display a fluorescence turn-on effect upon selective conjugation with proteins with free cysteines located in a surface exposed and flexible environment, the signature of disorder in proteins. By using an affinity-based proteomic approach, in certain embodiments the probes may be capable of identifying both basal intrinsically disordered proteins and proteins that change their degree of foldedness in cells in response to stress. In certain embodiments, the probes may be used for identifying proteins of low abundance that cannot be detected by conventional abundance profiling methods.

[0009] In a first aspect of the invention there is provided a compound of Formula I, or a salt thereofFormula I wherein A, B, C, and D are independently selected from the group consisting of H, CN, unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: said substituted aryl and said substituted heteroaryl is independently substituted with one or more groups independently selected from the group consisting of Z-Y- C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3- Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5- C2-C6 alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7- aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20- SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; wherein at least two of A, B, C, and D are independently selected from the group consisting of unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-Ce alkynyl, Z-C2-Ce alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E; wherein: each incidence of Z is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z1is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z2is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z3is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z4is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z5is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z6is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z7is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z8is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z9is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z10is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z11is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z12is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH;each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C1-C10 alkenyl, and C1- C heteroalkenyl, each of which may be optionally substituted with =0; each incidence of R is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, SO2-C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-C alkenyl, and Z16- C1-C10 heteroalkenyl; each incidence of R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, SO2-C1-C6 alkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-C alkenyl, and Z16- C1-C10 heteroalkenyl; each incidence of Z13is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z14is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z15is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Z16is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; andE is a group which is capable of reacting with a thiol group.

[0010] The following options may be used in conjunction with the first aspect, either individually or in any combination.[0001 1 ] In certain embodiments, each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C2-C10 alkenyl, and C2-C10 heteroalkenyl; and each incidence of R and R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl.

[0012] In certain embodiments, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2- Ce alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide, also comprises one or more other substituents on the aryl or heteroaryl ring which are independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C3- Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl,Z5-Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8- aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7 cycloalkyl, Z9-C3-C? cycloalkyl, Z10-Y11- C3-C7 cycloheteroalkyl, Z10-C3-C7 cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2. For example, one of A, B, C, and D may be substituted with Z-Y-C2-C6 alkynyl and Z6-C2-Ce heteroalkenyl. In other alternative embodiments, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide, does not have any other substituents on the aryl or heteroaryl ring.

[0013] In certain embodiments, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent which is selected from the group consisting of Z12-Y17- E and Z12-E, also comprises one or more other substituents on the aryl or heteroaryl ring which are independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5- Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7 cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15- COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2. For example, one of A, B, C, and D may be substituted with Z12-E and Z6-Y7-C2-Ce heteroalkenyl. In other alternative embodiments, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E, does not have any other substituents on the aryl or heteroaryl ring.

[0014] In certain embodiments, each incidence of Z is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0015] In certain embodiments, each incidence of Z1is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0016] In certain embodiments, each incidence of Z2is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0017] In certain embodiments, each incidence of Z3is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0018] In certain embodiments, each incidence of Z4is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0019] In certain embodiments, each incidence of Z5is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0020] In certain embodiments, each incidence of Z6is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0021] In certain embodiments, each incidence of Z7is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0022] In certain embodiments, each incidence of Z8is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0023] In certain embodiments, each incidence of Z9is independently selected from the group consisting of a bond, O, NR1, SO2, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0024] In certain embodiments, each incidence of Z10is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0025] In certain embodiments, each incidence of Z11is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0026] In certain embodiments, each incidence of Z12is independently selected from the group consisting of a bond, O, NR1, SO2, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1.

[0027] In certain embodiments, Y is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y is a bond.

[0028] In certain embodiments, Y1is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y1is a bond.

[0029] In certain embodiments, Y2is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y2is a bond.

[0030] In certain embodiments, Y3is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y3is a bond.

[0031] In certain embodiments, Y4is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y4is a bond.

[0032] In certain embodiments, Y5is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y5is a bond.

[0033] In certain embodiments, Y6is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y6is a bond.

[0034] In certain embodiments, Y7is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y7is a bond.

[0035] In certain embodiments, Y8is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y8is a bond.

[0036] In certain embodiments, Y9is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y9is a bond.

[0037] In certain embodiments, Y10is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y10is a bond.

[0038] In certain embodiments, Y11is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y11is a bond.

[0039] In certain embodiments, Y12is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y12is a bond.

[0040] In certain embodiments, Y13is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y13is a bond.

[0041] In certain embodiments, Y14is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y14is a bond.

[0042] In certain embodiments, Y15is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y15is a bond.

[0043] In certain embodiments, Y16is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y16is a bond.

[0044] In certain embodiments, Y17is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y17is a bond.

[0045] In certain embodiments, Y18is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y18is a bond.

[0046] In certain embodiments, Y19is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y19is a bond.

[0047] In certain embodiments, Y20is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y20is a bond.

[0048] In certain embodiments, Y21is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y21is a bond.

[0049] In certain embodiments, Y22is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y22is a bond.

[0050] In certain embodiments, Y23is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y23is a bond.

[0051] In certain embodiments, Y24is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, each of which may be optionally substituted with =0, optionally Y24is a bond.

[0052] In certain embodiments, R is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, CrCe heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci- Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-Cio alkenyl, and Z16-Ci-C heteroalkenyl.

[0053] In certain embodiments, R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci- Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-Cio alkenyl, and Z16-Ci-C heteroalkenyl.

[0054] In certain embodiments, each incidence of Z13is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH.

[0055] In certain embodiments, each incidence of Z14is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH.

[0056] In certain embodiments, each incidence of Z15is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH.

[0057] In certain embodiments, each incidence of Z16is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH.

[0058] In certain embodiments, E is selected from the following: a maleimide, a halo-acetamide, a vinyl acetamide, an aryl acetamide,wherein R’ is independently selected from the group consisting of H, C1-C12 alkyl, and aryl, and X is halo;optionally wherein

[0059] In certain embodiments, the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E are the same group, i.e. they are one of A, B, C, or D. In alternative embodiments, the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E are different groups, i.e. they are two of A, B, C, and D.

[0060] In certain embodiments, A and D are independently selected from the group consisting of unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; and C and B are independently selected from the group consisting of H, CN, unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl.

[0061] In certain embodiments, A and D are independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline; and C and B are independently selected from the group consisting of H, CN, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline.

[0062] In certain embodiments, one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2- C2-C6 alkenylazide; and another of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E.

[0063] In certain embodiments, one of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole, or quinoline, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z- C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and another of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole, or quinoline, comprising a substituent which is selected from the group consisting of Z12- Y17-E and Z12-E.

[0064] In certain embodiments at least two of A, B, C, and D are substituted phenyl; wherein one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following:Z-C2-C6 alkynyl, azide, Z1-Ci-Ce alkylazide, and Z2-C2-Ce alkenylazide; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E.

[0065] In certain embodiments, two of A, B, C, and D are unsubstituted phenyl.

[0066] In certain embodiments, one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR2-C2-C3 alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E; wherein R2is H or C2-C4 alkyl-OH.

[0067] In certain embodiments, one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR3-C2-C3alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is:; wherein R3is H or C2-C4 alkyl-OH.

[0068] In certain embodiments, one of A, B, C, and D is substituted phenyl, which is substituted with O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, or NR4-C2-C3 alkynyl; and another of A,B, C, and D is substituted phenyl, which i; wherein R4is H or C2-C4 alkyl-OH.

[0069] In certain embodiments,• two of A, B, C, and D are unsubstituted phenyl;• one of A, B, C, and D are substituted phenyl, which is substituted with the following group:• the other of A, B, C and D are substituted phenyl, which is substituted with the following group:

[0070] In certain embodiments where A is not the same as B or where C is not the same as D, the compound of Formula I, or a salt thereof may be a mixture of cis and trans isomers. In certain alternative embodiments where A is not the same as B or where C is not the same as D, the compound of Formula I, or a salt thereof is a trans isomer. In certain alternative embodiments where A is not the same as B or where C is not the same as D, the compound of Formula I, or a salt thereof is a cis isomer.

[0071] In certain embodiments, two of A, B, C, and D are unsubstituted phenyl; two of A, B, C, and D are H; or one of A, B, C, and D is H and another of A, B, C, and D is CN.

[0072] In certain embodiments, Z12-Y17-E and Z12-E are selected from the group consisting of:

[0073] In certain embodiments, one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, and Z1-Ci-Ce alkylazide, optionally wherein one of A, B, C and D is substituted phenyl.

[0074] In certain embodiments, one of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole, or quinoline, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z- C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, and Z1-Ci-Ce alkylazide, optionally wherein one of A, B, C and D is substituted phenyl.

[0075] In certain embodiments, Z is O, NR1, or a bond.

[0076] In certain embodiments, Y is a bond.

[0077] In certain embodiments, one of A, B, C, and D is aryl or heteroaryl substituted with C2 alkynyl, O-C2 alkynyl, O-C1-C4 alkyl azide, or NR100-C2 alkynyl, and is further optionally substituted with one or more groups selected from the group consisting of NH2, NHC1-C3 alkyl,N(Ci-Cs alkyl)2, OC1-C3 alkyl,wherein R100is H, C1-C3 alkyl, Ci-C3alkyl-OH, or Ci-C3alkyl-OCO-Ci-C3alkyl.

[0078] In certain embodiments, one of A, B, C, and D is phenyl, naphthyl, furan, pyrrole, or quinoline, substituted with C2alkynyl, O-C2alkynyl, O-C1-C4 alkyl azide, or NR100-C2alkynyl, and is further optionally substituted with one or more groups selected from the group consisting ofNH2, NHC1-C3 alkyl, N(Ci-C3alkyl)2, OC1-C3 alkyl,wherein R100is H, C1-C3 alkyl, Ci-C3alkyl-OH, or Ci-C3alkyl-OCO-Ci-C3alkyl.

[0079] In certain embodiments, one of A, B, C, and D is aryl, optionally phenyl or naphthyl, substituted with C2alkynyl, O-C2alkynyl, O-C1-C4 alkyl azide, or NR100-C2alkynyl, and is further optionally substituted with one or more groups selected from the group consisting of NH2, NHC1- C3alkyl, N(Ci-C3alkyl)2, OC1-C3 alkyl,wherein R100is H, C1-C3 alkyl, Ci-C3alkyl-OH, or Ci-C3alkyl-OCO-Ci-C3alkyl.

[0080] In certain embodiments, A, B, C, and D are independently selected from the group consisting of: H, CN,

[0081] In certain embodiments, A and D are independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline, each of which may be independently substituted with one or more groups independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4- Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10- C3-C7 cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19- NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; andC and B are independently selected from the group consisting of hydrogen, CN, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline, each of which may be independently substituted with one or more groups independently selected from the group consisting of Z-Y- C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-Cs- C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24- halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21- CONH2, Y22-NHSO3H, and Y23-SO2NH2;wherein: one of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole or quinoline, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2- C2-C6 alkenylazide; and one of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole or quinoline, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E.

[0082] In certain embodiments, A and D are independently selected from the group consisting of optionally substituted phenyl, and optionally substituted quinoline, each of which may be independently substituted with one or more groups independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4- Y5-CI-C6heteroalkyl, Z4-CI-C6heteroalkyl, Z5-Y6-C2-C6alkenyl, Z5-C2-C6alkenyl, Z6-Y7-C2-C6heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10- C3-C7 cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19- NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; andC and B are independently selected from the group consisting of hydrogen, CN, optionally substituted phenyl, and optionally substituted quinoline, each of which may be independently substituted with one or more groups independently selected from the group consisting of Z-Y- C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-Cs- C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24- halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21- CONH2, Y22-NHSO3H, and Y23-SO2NH2; wherein: one of A, B, C, and D is substituted phenyl or quinoline, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and one of A, B, C, and D is substituted phenyl or quinoline, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E.

[0083] In certain embodiments, A and D are each optionally substituted phenyl, which may be independently substituted with one or more groups independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-CI-C6heteroalkyl, Z4-CI-C6heteroalkyl, Z5-Y6-C2-C6alkenyl, Z5-C2-C6alkenyl, Z6-Y7-C2-C6heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10- C3-C7 cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19- NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; andC and B are independently selected from the group consisting of hydrogen, CN, and optionally substituted phenyl, which may be substituted with one or more groups independently selected from the group consisting of Z-Y-C2-Ce alkynyl, Z-C2-Ce alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-CI-C6alkyl, Z4-Y5-CI-C6heteroalkyl, Z4-CI-C6heteroalkyl, Z5-Y6-C2-C6alkenyl, Z5-C2-C6alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10- C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17- E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: Z-Y-C2-Ce alkynyl, Z-C2-Ce alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and one of A, B, C, and D is substituted phenyl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E.

[0084] In certain embodiments, the compound or salt thereof is a compound of Formula II, or salt thereof:wherein:B1and B2are independently selected from hydrogen, cyano, and phenyl;A1is phenyl; andA2is an aryl or heteroaryl group selected from the group consisting of phenyl, indole, and quinoline; wherein: one of A1or A2is substituted with a group selected from -C2-C4 alkynyl, -N(RA)-C2-C4 alkynyl, -O-C2-C4 alkylazide, and -O-C2-C4 alkynyl, wherein RAis selected from C1-C4 alkyl-O-CO-Ci-C4 alkyl, C1-C4 alkyl-COO-Ci-C4 alkyl, and -SO2-Ci-C4 alkyl; and one of A1or A2is substituted with a group selected from:wherein X is a halogen, optionally chlorine; andA1and A2are optionally substituted with one or more groups selected from the following: -N(Ci-C4 alkyl)2, -O-C1-C4 alkyl, -O-C2-C4 alkynyl, -COO-C1-C4 alkyl, halogen, and cyano.

[0085] In certain embodiments, the compound or salt thereof is selected from the following:and salts and / or cis / trans isomers thereof.

[0086] In certain embodiments, the compound is selected from the following:and / or cis / trans isomers thereof.

[0087] In certain embodiments, the compound or salt thereof has the following structure:salt and / or cis / trans isomer thereof.

[0088] In a second aspect of the invention there is provided a method of diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• contacting a biological sample of the subject with the compound or salt thereof according to the first aspect; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to determine whether the subject has, or is likely to develop, the disease or disorder.

[0089] The following options may be used in conjunction with the second aspect, either individually or in any combination.

[0090] In certain embodiments, the sample may include cells from the subject, and the method may include a further step of lysing the cells. In certain embodiments, the lysing may be performed after contacting the biological sample with the compound or salt thereof. In certain embodiments, the lysing may be performed by, for example, a sonication step. In certain alternative embodiments, the sample may be a blood, e.g. plasma, sample.

[0091] In certain embodiments, the method further comprises the following steps: o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a biotin binding agent, optionally wherein the biotin binding agent is a streptavidin-functionalised agent, optionally wherein the streptavidin-functionalised agent is a streptavidin-functionalised bead, optionally a streptavidin-functionalised agarose bead; or o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally azide or alkyne functionalised bead, and o collecting the protein-agent conjugate.

[0092] In certain embodiments, reacting the bound compound or salt thereof with an azide functionalized biotin or alkyne functionalized biotin may include a copper-catalyzed azide-alkyne cycloaddition. In such embodiments, a skilled person will understand that in the case where thecompound or salt thereof includes an azide functional group, then the bound compound or salt thereof may be reacted with an alkyne functionalized biotin, and alternatively, in the case where the compound or salt thereof includes an alkyne functional group, then the bound compound or salt thereof may be reacted with an azide functionalized biotin. In certain embodiments, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized biotin or alkyne functionalized biotin with a copper solution, optionally copper sulfate. In certain specific embodiments, the reaction may be performed by a step of mixing the bound compound or salt thereof in the form of a cell lysate optionally having a concentration of proteins of from about 0.1 mg / mL to about 10 mg / mL, or from about 1 mg / mL to about 5 mg / mL, or about 0.1 , 0.2, 0.5, 1 , 2, 5, or 10 mg / mL, and an azide or alkyne functionalized biotin, such as / V-(3-azidopropyl)biotinamide, at a concentration of from about 0.05 mM to about 1 mM, or from about 0.1 mM to about 1 mM, or about 0.05, 0.1 , 0.2, 0.25, 0.3, 0.4, 0.5, or 1 mM.

[0093] In certain embodiments, reacting the bound compound or salt thereof with an azide functionalized agent or alkyne functionalized agent may include a copper-catalyzed azidealkyne cycloaddition. In such embodiments, a skilled person will understand that in the case where the compound or salt thereof includes an azide functional group, then the bound compound or salt thereof may be reacted with an alkyne functionalized agent, and alternatively, in the case where the compound or salt thereof includes an alkyne functional group, then the bound compound or salt thereof may be reacted with an azide functionalized agent. In certain embodiments, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized agent or alkyne functionalized agent with a copper solution, optionally copper sulfate.

[0094] In certain embodiments, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers. In such embodiments, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected. The method may further include a cleavage step, for example, an acid cleavage step, to cleave the bound protein from the solid phase or resin thereby enabling separation and isolation of the bound protein from the solid phase or resin.

[0095] In certain embodiments, the method may include a protein precipitation step, which may, for example, include a pH modification, optionally an acidification, optionally using trichloroacetic acid. The protein precipitation step may be performed prior to the step of collecting the protein-biotin conjugate using a biotin binding agent.

[0096] In certain embodiments, the biotin binding agent includes streptavidin, for example a streptavidin functionalized solid phase, such as a streptavidin functionalized bead. The methodmay further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate.

[0097] In certain embodiments, detecting the compound or salt thereof in the form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence or an absorbance, optionally a UV absorbance.

[0098] In certain embodiments, detecting the compound or salt thereof in the form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises performing mass spectrometry, optionally LC-MS / MS; or electrophoresis, optionally SDS-PAGE or Western blot.

[0099] In a third aspect of the invention there is provided use of the compound or salt thereof according to the first aspect, for diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.[000100] In a fourth aspect of the invention there is provided the compound or salt thereof of according to the first aspect, for use in diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.[000101] In a fifth aspect of the invention there is provided a method of treating or preventing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• determining whether the subject has, or is likely to develop, the disease or disorder using the method according to the second aspect; and• if the subject has the disease or disorder, administering a therapeutically effective amount of a medicament capable of treating or preventing said disease or disorder to the subject.[000102] In a sixth aspect of the invention there is provided a method for enriching an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps: o contacting the compound or salt thereof according to the first aspect with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein; or o contacting the compound or salt thereof of according to the first aspect with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein,o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, and o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein.[000103] The following options may be used in conjunction with the sixth aspect, either individually or in any combination.[000104] In certain embodiments, the streptavidin-functionalized agent may be a streptavidin functionalized solid phase, such as a streptavidin functionalized bead. The method may further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein.[000105] In certain embodiments, reacting the bound compound or salt thereof with an azide functionalized agent or alkyne functionalized agent may include a copper-catalyzed azidealkyne cycloaddition. In such embodiments, a skilled person will understand that in the case where the compound or salt thereof includes an azide functional group, then the bound compound or salt thereof may be reacted with an alkyne functionalized agent, and alternatively, in the case where the compound or salt thereof includes an alkyne functional group, then the bound compound or salt thereof may be reacted with an azide functionalized agent. In certain embodiments, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized agent or alkyne functionalized agent with a copper solution, optionally copper sulfate.[000106] In certain embodiments, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers. In such embodiments, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected. The method may further include a cleavage step, for example, an acid cleavage step, to cleave the bound protein from the solid phase or resin thereby enable enrichment of the unfolded, misfolded or intrinsically disordered protein by separation and isolation of the bound protein from the solid phase or resin.[000107] In a seventh aspect of the invention there is provided a method for identifying an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps: o contacting the compound or salt thereof according to the first aspect with the sample containing the unfolded, misfolded or intrinsically disordered proteinto thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein; or o contacting the compound or salt thereof according to the first aspect with the sample containing the unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein.[000108] The following options may be used in conjunction with the seventh aspect, either individually or in any combination.[000109] In certain embodiments, the streptavidin-functionalized agent may be a streptavidin functionalized solid phase, such as a streptavidin functionalized bead. The method may further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein.[0001 10] In certain embodiments, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers. In such embodiments, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected. The method may further include a cleavage step, for example, an acid cleavage step, to cleave the bound protein from the solid phase or resin thereby enableenrichment of the unfolded, misfolded or intrinsically disordered protein by separation and isolation of the bound protein from the solid phase or resin.[0001 1 1] In an eighth aspect of the invention there is provided a method of detecting an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps:• contacting the sample with the compound or salt thereof according to the first aspect;• optionally enriching the unfolded, misfolded or intrinsically disordered protein, according to the method according to the sixth aspect; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to thereby detect the unfolded, misfolded or intrinsically disordered protein, optionally wherein detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence; absorbance, optionally UV absorbance; and / or performing liquid chromatography and / or mass spectrometry.[0001 12] In a ninth aspect of the invention there is provided a method for screening a drug candidate for treating a disease or disorder associated with an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps: contacting said drug candidate with a sample containing the unfolded, misfolded or intrinsically disordered protein; and detecting the unfolded, misfolded or intrinsically disordered protein in the sample according to the method of the eighth aspect to determine whether the drug candidate is capable of treating the disease or disorder.[0001 13] The following options may be used in conjunction with the ninth aspect, either individually or in any combination.[0001 14] In certain embodiments of the second, third, fourth, fifth, sixth and ninth aspects, the disease or disorder is selected from the group consisting of Huntington’s disease; Alzheimer's disease; Parkinson’s disease; thrombosis; stroke; autism; obesity and metabolic diseases; cardiovascular diseases; chronic liver disease and cirrhosis; nephritis; nephrotic syndrome; nephrosis; Creutzfeldt-Jakob diseases; cystic fibrosis; Gaucher’s disease; hereditary cerebral haemorrhage; dementia; Niemann-Pick disease; multiple system atrophy; Creutz-Jakob disease; fatal insomnia; Gerstmann-Straussler-Scheinker disease; Spongiform encephalopathy; Creutzfeldt-Jakob disease; Kuru; hereditary sensory and autonomic neuropathy; Pick disease; progressive supranuclear palsy; argyrophilic grain disease; Guam Parkinson dementia complex; frontotemporal lobar degeneration; chronic traumatic encephalopathy; ganglioglioma; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy, tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; amyloidosis; familial amyloidotic polyneuropathy; familial amyloid cardiomyopathy; amyloidosis; Type II diabetes; insulinoma; medullary carcinoma of the thyroid; motor neuron diseases; amyotrophic lateral sclerosis; atrial amyloidosis; pituitary prolactinoma; Gelatinous drop-like corneal dystrophy; calcifying epithelialodontogenic tumors; pulmonary alveolar proteinosis; hypotrichosis simplex; lattice corneal dystrophy; cancer, e.g. prostate cancer and / or breast cancer; myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS); and long COVID. In certain specific embodiments of the second, third, fourth, fifth, sixth and ninth aspects, the disease or disorder is breast cancer, myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), prostate cancer, Huntington’s disease or Parkinson’s disease, optionally Huntington’s disease or Parkinson’s disease.BRIEF DESCRIPTION OF THE DRAWINGS[0001 15] Figure 1 Chemical structure of an example biomarker: TME and the RUBICON workflow. a) Structure of TME and purposes of each functional group. b) Distinct TME reactivity and fluorescence towards folded proteins and unfolded proteins. TME reacts with unfolded proteins via access to buried cysteine (Cys) thiols in a surface exposed and flexible environment. c) Schematic of the RUBICON workflow and its potential applications for biomarker discovery and disease stratification. d) TME fluorescence differentiates cell subpopulations during pre-inclusion formation for disease mechanism study.[0001 16] Figure 2: TME specifically labels protein thiols located in a surface-exposed and flexible environment. a) In vitro fluorescence kinetic traces of TME with folded / unfolded BLG, GSH and unfolded BLG pre-treated with NMM. “U” and “F” denote “unfolded” and “folded” respectively, n = 4 biological replicates; mean ± s.d. Figure 8a shows the emission spectra measured at the end of the kinetics measurement. b) Comparison of the nearby environment of surface-exposed cysteines shown as yellow sphere in DsbA (PDB ID: 1 A2L) and orange sphere C33A (PDB ID: 1TI1) using B-factor analysis. Black dotted boxes denote the region of interest where C33A mutation renders confirmational flexibility of the nearby loop. High conformational flexibility is denoted by the orange to red color and wider tube, corresponding to a high B factor, where green to blue color and narrower tube, corresponding to a lower B factor, denote low conformational flexibility. c) In vitro fluorescence kinetic traces of TME with folded / unfolded DsbA-wildtype (hereinafter, DsbA) and DsbA-C33A (hereinafter, C33A). n = 4 biological replicates; mean ± s.d. d-e) In-gel fluorescence of Figure 2a and 2c showing the comparison of the conjugation rates of TME with folded / unfolded DsbA and C33A. Coomassie channel shows the total protein amount. Figure 8c-d shows the kinetics curves using the same aliquot of in-gel fluorescence samples.[0001 17] Figure 3: TME binders contain significantly more unfolded regions.a-c) TME binders in control (a) and tunicamycin treated (b) conditions. Proteins that became more unfolded upon tunicamycin treatment were labeled with gene names in b and c. Color represents protein abundance. Red points denote proteins undetectable by the lysate profiling proteomics. d) Venn diagram of TME binders in control and tunicamycin treated conditions. e) TME binders in Figure 3a and 3b were not significantly enriched in functional cysteines, indicating TME displayed a completely distinct reactivity towards cellular cysteines compared to the iodoacetamide (IA) alkyne probe. On top of each bar shows the fold enrichment, followed by the significance level. f) Enrichment analysis of TME binders in Figure 2a. “Control binders” refers to TME binders in control condition. g-i) IUPred2, ANCHOR2, degree and betweenness value of TME binders in Figure 3a and 3b were significantly higher than the proteome average.[0001 18] Figure 4: RUBICON identifies disordered proteins to effectively discriminate disease from healthy cohorts. a-c) TME binders in lymphoblasts from healthy control (a) and idiopathic PD (b) groups. Proteins that became more unfolded in healthy (left) and PD samples (right) were labeled with gene names (c). n = 4 participants in each group. Color represents protein abundance. Red points denote proteins undetectable by the lysate profiling proteomics. d) Venn diagram of TME binders in healthy control and PD groups. e) Parallel comparison of principal component analysis results of protein abundance profiling using cell lysate (left) and protein unfoldedness profiling using RUBICON (right).[0001 19] Figure 5: mHttexI distribution determines cellular proteostasis. a) Principle of PulSA. b) Non-inclusion-forming 25Q displayed one stage and inclusion-forming 46Q and 97Q displayed a three-stage transformation with the increase of Httexl expression levels. Cytograms showing the height (H) versus the width (W) of the mCherry signals in Neuro- 2a cells expressing mCherry-fused 25 / 46 / 97Q. Color denotes the expression level, which was estimated from area (A). c) Httexl aggregation is polyQ-length and concentration-dependent. Percentages of inclusion-forming cells at different expression levels of the proteins were obtained based on the gating strategy shown in (b). d) TME signals identify two subpopulations in stage n . Cytograms showing mCherry.H versus mCherry.W of 25 / 46 / 97Q. Color denotes TME signals. e) Time-course pulse shape monitoring of cells expressing 46Q. f) Impact of protein expression on cellular proteostasis determined by TME signals. The mCherry A value was binned into 25 intervals. The shaded red, blue and pink areas denote stage I , n , and HI, respectively, as defined in Figure 5b.g-h) Impact of protein distribution on cellular proteostasis determined by TME signals. Large / small H or W is defined in Figure 14c-d.[000120] Figure 6: Huntington inclusion clears cellular unfolded protein load by sequestering potential TME binders. a) Cells transfected with 46Q were sorted into four populations based on Figure 5d. Total cell lysate (T) was further separated into supernatant (S) and pellet (P) by ultracentrifuge. Cluster-D and cluster-A were defined as proteins with descending and ascending abundance respectively, from Stage I to Stage HI. b) Enrichment analysis of P-cluster-A. c) Comparison of P- n a and P- n n. Red dots denote proteins with higher abundance in P- H a. d) Venn diagram showing relations of enriched functional terms of TME binders in Figure 3f and T / S / P-cluster-A / D. e-g), IUPred2, ANCHOR2, degree and betweenness values of P-cluster-A / D. h-i) Protein distribution models for non-inclusion-forming (h) and inclusion-forming (i) Httexl .[000121] Figure 7: Crystal structure and photophysical characterization of TME. a) Crystal structures of thiol reactive TME and its isomer 4-E. b) DPT calculations for TME and its thiol adduct TME-S showing a loss of the LUMO energy level from the maleimide and the photoinduced electron transfer effect upon reaction with thiol. c-d) Changes of TME-S fluorescence intensity in solvent mixture of ethylene glycol: glycerol with increase of glycerol fraction (vol%) demonstrating restriction of intramolecular motion (RIM) effect arising from viscosity increase (c) and in water: DMSO with increase of water fraction (vol%) demonstrating the RIM effect arising from aggregation (d). Concentration of TME: 10 pM; excitation wavelength: 322 nm; emission wavelength: 475 nm.[000122] Figure 8: TME fluorescence reveals protein unfolding in vitro. a) End-point emission spectra of samples in Figure 2a. b) Fluorescence of TME with unfolded BLG in the presence of 0.025 - 8 mM GSH.Arrow indicates intracellular protein to glutathione thiol ratio, i.e., 7:3. n = 4 biological replicates; mean ± s.d. c-d) TME fluorescence measured by the plate reader in Figure 2d and 2e. Sample solutions were aliquoted from the in-gel fluorescence samples, n = 4 technical replicates; mean ± s.d. Results were consistent with kinetic curves (Figure 2a and 2c).[000123] Figure 9: TME fluorescence is more sensitive and consistent for protein stability quantification compared to intrinsic tryptophan fluorescence.a-b) Comparison of protein denaturation curves measured by intrinsic tryptophan (Trp) (a) and TME (b) fluorescence. The transition region was set from 3.5 - 7 M urea, n = 3 biological replicates; mean ± s.d. c-d) Plots showing AGApparent versus [Urea] using Trp (c) and TME (d) fluorescence measurement. Points were linearly fitted, and the absolute value of Pearson's correlation coefficient was shown. e) GH2O was calculated as 7.55 ± 0.91 kcal / mol for intrinsic tryptophan fluorescence measurement and 8.49 ± 0.13 kcal / mol for TME fluorescence measurement respectively, which were consistent with a previous value (7.8 ± 1 .4 kcal / mol) using circular dichroism, differential UV absorption measurements. The standard deviation of TME measurement was much smaller than Trp measurement, n = 3 biological replicates; mean ± s.d. f) TME measurement exhibited a higher signal-to-noise ratio, compared to Trp fluorescence measurement. Plots show the ratio of maximum to minimum signals detected in one run. n = 3 biological replicates; mean ± s.d.[000124] Figure 10: TME biocompatibility and its functionality to enrich unfolded proteins in vitro and cellular proteins from cell lysate via RUBICON workflow. a) Cell viability assays for cells stained with 25 - 75 pM TME for 30 and 60 min. Cells were also incubated with the same amount of DMSO as vehicle controls, n = 4 biological replicates; mean ± s.d. b) Confocal microscopy images of cells stained with TME and co-stained with ER- Tracker and DRAQ5 for endoplasmic reticulum (ER) and nucleus respectively. Scale bar: 20 pm. c-d) Only TME labeled model proteins (c) and proteins from cell lysates (d) were enriched by the RUBICON workflow, which displayed evident fluorescence in TME, SYPRO Ruby and Coomassie Blue channels. The input loading amount for BLG and cell lysate was 7.5 and 50 pg respectively. SYPRO Ruby were used as a sensitive stain to visualize all proteins while Coomassie Blue were used to quantify total protein amount. “TME- / BLG-” and “TME- / Lysate-” denote samples that contained beads only. e) Flow cytometry results showing that proteostatic stressor (i.e., tunicamycin and MG132) treated cells displayed significantly higher TME fluorescence compared to control. Gating strategies are shown in Figure 17. n = 3 biological replicates; mean ± s.d.[000125] Figure 11 : Characterization of TME binders in the tunicamycin dataset. a) Strategies to interpret proteomics results. In each dataset, there were four experimental conditions: TME- / Drug-, TME+ / Drug-, TME- / Drug+, TME+ / Drug+. Each experimental condition was processed parallelly with the RUBICON workflow. Note that unlabeled proteins can be potentially enriched by Dynabeads, i.e., non-specific binders.Comparison of TME- / Drug- and TME+ / Drug- identified TME binders in control condition, e.g., Figure 3a. Comparison of TME- / Drug+ and TME+ / Drug+ identified TME binders in drug treated conditions, e.g., Figure 3b. Comparison of TME+ / Drug- and TME+ / Drug+ identified proteins with higher abundance upon drug treatment using RUBICON, e.g., Figure 3c. Among these proteins, the TME labeled ones were proteins that became more unfolded in stressed conditions. They were identified as the intersections of significant hits in Figure 3b and 3c, for example, and labeled with their gene name. b) Western Blot results for validation of proteins enriched from untreated or tunicamycin treated cells with or without TME using the RUBICON workflow. c) The number of proteins (protein counts) detected in TME labeled / unlabeled, and tunicamycin treated / untreated cells, shown on the top of each bar plot. Protein counts in each condition were calculated as the number of proteins detected in all triplicates. For this calculation, mass spectra were searched using MaxQuant without ‘Match between runs’. d) TME binders in Figure 3a and 3b were enriched in intracellular proteins (e.g., nucleus, cytoplasm, ER). On top of each bar shows the fold enrichment, followed by the significance level. e) Enrichment analysis of TME binders in Figure 3b. “Tunicamycin binders” refer to TME binders in tunicamycin treated condition. f) Protein-protein interaction network of the six proteins that became more unfolded upon tunicamycin treatment. Green nodes denote the first neighbor of these proteins.[000126] Figure 12: Characterization of TME binders in the MG132 dataset. a-c) TME binders in control (a) and MG132 treated (b) conditions. Proteins that became more unfolded upon MG132 treatment were labeled with gene names (b and c). Color represents protein abundance. Red points denote proteins undetectable by the lysate profiling proteomics. d) Venn diagram of TME binders in control and MG132 treated conditions. e) TME binders in Figure 12a and 12b were not heavily enriched in functional cysteines, indicating TME displayed a completely distinct reactivity towards cellular cysteines compared to the IA alkyne probe. On top of each bar shows the fold enrichment, followed by the significance level. f) TME binders in Figure 12a and 12b were enriched in intracellular proteins (e.g., nucleus, cytoplasm, ER) but depleted in membrane and secreted proteins. On top of each bar shows the fold enrichment, followed by the significance level. g-h) Enrichment analysis of TME binders in Figure 12a (g) and 12b (h). ‘MG132 binders’ refer to TME binders in MG 132 treated condition. i-k) IUPred2, ANCHOR2, degree and betweenness value of TME binders in Figure 12a and 12b were significantly higher than the proteome average.[000127] Figure 13: Characterization of TME binders in the PD and healthy control lymphoblast datasets. a) TME binders in Figure 4a and 4b were not heavily enriched in functional cysteines, indicating TME displayed a completely distinct reactivity towards cellular cysteines compared to the IA alkyne probe. On top of each bar shows the fold enrichment, followed by the significance level. b) TME binders in Figure 4a and 4b were enriched in intracellular proteins (e.g., nucleus, cytoplasm, ER). On top of each bar shows the fold enrichment, followed by the significance level. c-d) Enrichment analysis of TME binders in Figure 4a (c) and 4b (d). ‘Healthy binders’ refer to TME binders in the healthy control group and ‘PD binders’ refer to TME binders in the PD group. e-g) IUPred2, ANCHOR2, degree and betweenness value of TME binders in Figure 4a and 4b were significantly higher than the proteome average. h) Parallel comparison of scree plots of protein abundance profiling using cell lysate (left) and protein unfoldedness profiling using RUBICON (right).[000128] Figure 14: Supplementary data for the pulse shape analysis of Httexl . a) Data of vehicle control, i.e. cells expressing mCherry only. Legends are the same as in Figure 5b-d. b) Flow cytometry results confirming negligible crosstalk between mCherry and TME fluorescence. Cytograms show mCherry. A versus TME fluorescence of monochromic cells, i.e., cells either only stained with TME or only transfected with mCherry or mHttexI . The shaded red, blue and pink areas denote stage I , n , and HI, as defined in Figure 5b, respectively. The rectangular gate denotes high TME signals, inside which shows the percentages of TME+ cells. c-d) Examples of separation of 97Q by H (d) and W (e) in the expression bin ranked 7, 17 and 25. Large and small values are referred to as top 25% and bottom 25% of H and W, respectively. In each expression bin, two-sample t-test was performed between TME signals from large and small H, and from large and small W. Representative confocal images of cells are shown in Figure 18.[000129] Figure 15: 46Q overexpression accelerates translation and triggers stress response and quality control. a) Protein abundance of 46Q from Stage I to HI in T, S and P fractions. b-c) Comparison of T- H a and T- H n, and S- H a and S- H n. Red dots denote proteins with higher abundance in H a. d-f) Heatmap of cluster-A and cluster-D in T, S and P fraction (top); enrichment analysis of P / T / S-cluster-A (bottom).[000130] Figure 16: Supplementary data of 46Q T / S-cluster-A / D. a-f) IUPred2, ANCHOR2, degree and betweenness value of T-cluster-A / D (a-c) and of S-cluster-A / D (d-f).[000131] Figure 17 shows gating strategies for flow cytometry. Briefly, the main cell population was selected by SSC.A versus FSC.A, and single cells were selected by FSC.H versus FSC.W, and SSC.H versus SSC.W. Live cells were selected by TO-PRO-3 negative (APC.A channel) cells.[000132] Figure 18 shows representative confocal images of cells expressing mCherry only, Httex1-25Q-mCherry, Httex1 -46Q-mCherry and Httex1-97Q-mCherry stained by TME. Results confirm mCherry and 25Q did not form inclusions while 46Q and 97Q could form inclusions (non-inclusion-forming cells denoted by white dashed circles; cellular inclusions marked by white arrows). Scale bars: 30 pm.[000133] Figure 19 shows NMR spectra for example probes and their synthesis intermediates: a)1H NMR for 1 in CDCI3. b)13C NMR for 1 in CDCI3. c)1H NMR for 2-Ein CDCI3. d)13C NMR for 2-Ein CDCI3. e)1H NMR spectra for 2-Zin CDCI3. f)13C NMR spectra for 2-Zin CDCI3. g)1H NMR spectra for 3-E in CDCI3. h)13C NMR spectra for 3-E in CDCI3. i)1H NMR spectra for 3-Z in CDCI3. j)13C NMR spectra for 3-Z in CDCI3. k)1H NMR spectra for 4-E in CDCI3. l)13C NMR spectra for 4-E in CDCI3. m)1H NMR spectra for TME (4-Z) in CDCI3. n)13C NMR spectra for TME (4-Z) in CDCI3. o)1H NMR spectra for TME-S in CDCI3. p)13C NMR spectra for TME-S in CDCI3. q)1H NMR spectra for PT_N012 in D6-DMSO. r)13C NMR spectra for PT_N012 in D6-DMSO. s)1H NMR spectra for PT_N022 in D6-DMSO. t)13C NMR spectra for PT_N022 in D6-DMSO. u)1H NMR spectra for PT_N024 in D6-DMSO. v)13C NMR spectra for PT_N024 in D6-DMSO. w)1H NMR spectra for PT_N038 in D6-DMSO. x)13C NMR spectra for PT_N038 in D6-DMSO. y)1H NMR spectra for PT_N048 in D6-DMSO.z)13C NMR spectra for PT_N048 in D6-DMSO. a’)1H NMR spectra for PT_N049 in D6-DMSO. b’)13C NMR spectra for PT_N049 in D6-DMSO. c’)1H NMR spectra for PT_N077 in D6-DMSO. d’)13C NMR spectra for PT_N077 in D6-DMSO. e’)1H NMR spectra for PT_N057 in D6-DMSO. f’)13C NMR spectra for PT_N057 in D6-DMSO. g’)1H NMR spectra for PT_N056 in CDCI3. h’)13C NMR spectra for PT_N056 in CDCI3. i')19F NMR spectra for PT_N056 in CDCI3. j’)1H NMR spectra for PT_N067 in D6-DMSO. k’)13C NMR spectra for PT_N067 in D6-DMSO.I’)1H NMR spectra for PT_N086 in D6-DMSO. m’)13C NMR spectra for PT_N086 in D6-DMSO. n’)1H NMR spectra for PT_N095 in D6-DMSO. o’)13C NMR spectra for PT_N095 in D6-DMSO. p’)1H NMR spectra for PT_N098 in D6-DMSO. q’)13C NMR spectra for PT_N098 in D6-DMSO. r’)1H NMR spectra for PT_N109 in D6-DMSO. s’)13C NMR spectra for PT_N109 in D6-DMSO. t’)1H NMR spectra for PT_N110 in D6-DMSO. u’)13C NMR spectra for PT_N110 in D6-DMSO.[000134] Figure 20 shows UV and photoluminescence data for example probes: a) Absorbance spectra for compound MI-SLDPAN. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPC>4. b) Absorbance spectra for compound CI-SLDPAN. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPC>4. c) Absorbance spectra for compound MI-SLDPAN. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPC>4. d) Absorbance spectra for compound 11 (HPC-MI). 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPC>4. e) Fluorescence spectra for compound MI-DPAN. 8 mM of GSH (G), 250 pM - Lactoglobulin (P), 8 M Urea (U) and 1 mM N-methylmaleimide (N) were used for the respective conditions. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPO4. For denaturation conditions with urea, GSH and p-Lactoglobulin were incubated with urea for 30 min, before treatment with N-methylmaleimide for 15 min, subsequent treatment with compound 7 for 15 min followed by fluorescence intensity measurement.f) Fluorescence spectra for compound CI-DPAN. 8 mM of GSH (G), 250 pM p- Lactoglobulin (P), 8 M Urea (U) and 1 mM N-methylmaleimide (N) were used for the respective conditions. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPO4. For denaturation conditions with urea, GSH and p-Lactoglobulin were incubated with urea for 30 min, before treatment with N-methylmaleimide for 15 min, subsequent treatment with compound 7 for 15 min followed by fluorescence intensity measurement. g) Fluorescence spectra for compound MI-SLDPAN. 8 mM of GSH (G), 250 pM - Lactoglobulin (P), 8 M Urea (U) and 1 mM N-methylmaleimide (N) were used for the respective conditions. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPO4. For denaturation conditions with urea, GSH and p-Lactoglobulin were incubated with urea for 30 min, before treatment with N-methylmaleimide for 15 min, subsequent treatment with compound 7 for 15 min followed by fluorescence intensity measurement. h) Fluorescence spectra for compound 11 (HPC-MI). 8 mM of GSH (G), 250 pM p- Lactoglobulin (P), 8 M Urea (U) and 1 mM N-methylmaleimide (N) were used for the respective conditions. 10 pM of dye was used for all conditions. PBS was 20 mM Na2HPO4. For denaturation conditions with urea, GSH and p-Lactoglobulin were incubated with urea for 30 min, before treatment with N-methylmaleimide for 15 min, subsequent treatment with compound 7 for 15 min followed by fluorescence intensity measurement.[000135] Figure 21 shows U2OS cells treated with environmental stress heat shock for 45min or 2h and followed up with 4% PFA fixation. Cells were stained with example probe compound 11 (HPC-MI) and DRAQ5™ (nucleic acid stain) to indicate changes in cell nuclei. Scale bar:20 uM.[000136] Figure 22: a-c) Violin (left two panels) and bar (right two panels) plots for “Lysate,” “Binder,” and “Undetectable” in the MG132 dataset, using Neuro-2a cell line proteomic dataset (a), the PaxDb Brain (Integrated) dataset (b), and the PaxDb Whole Organism (Integrated) dataset (c) as standard references. Violin plots show the distribution of protein abundance. Bar plots illustrate the percentages of proteins within each percentile, with percentiles determined by the respective standard references. ‘U’ in the last column of the bar plot denotes ‘Unmapped,’ referring to proteins not found in the corresponding standard references. “Lysate,” “Binder,” and “Undetectable” denote: proteins detected by lysate profiling methods, TME binders identified by RUBICON exclusive of undetectable proteins, and TME binders only detected by RUBICON but not by lysate profiling methods (red dots in Figure 12 a-b), respectively. Notably, referencing the Cell Line dataset resulted in the most unmapped proteins, followed by the Brain dataset, which left some proteins unmapped. Using the Whole Organism dataset, nearly all protein abundances could then be mapped.[000137] Figure 23: Classification of identified proteins by protein disorder in the proteome, tunicamycin and MG132 datasets, a) Schematic diagram showing the classification of proteins into 5 types. Type 1 : Structured proteins with less than 5% of residues having an IUPred2 score greater than 0.5. Type 2a-c: Proteins with IDRs, where the percentage of residues with an IUPred2 score greater than 0.5 is between 5% and 80%. Type 2a: Proteins with cysteines only located in the structured regions. Type 2b: Proteins with cysteines only located in the disordered regions. Type 2c: Proteins with cysteines located in both structured and disordered regions. Type 3: IDPs, where the percentage of residues with an IUPred2 score greater than 0.5 is greater than 80%. Non-cysteine containing proteins, if any, are discarded from analysis. The red circle denotes the cysteine, b-c) Classification of proteins in the tunicamycin dataset with threshold length of 15 (b) and 30 (c) amino acids in the continuous disordered sequences, d-e) Classification of proteins in the MG132 dataset with threshold length of 15 (d) and 30 (e) amino acids in the continuous disordered sequences. On top of each bar the fold enrichment is shown, followed by the significance level.[000138] Figure 24: Violin plots and bar plots of control and tunicamycin binders using Neuro- 2a cell line proteomic dataset (a), PaxDb Brain (Integrated) dataset (b), PaxDb Whole organism (Integrated) dataset (c) as standard reference, respectively. Legends and notations are the same as Figure 22.[000139] Figure 25: Violin plots and bar plots of healthy and PD binders using PaxDb Lympho Node (Integrated) dataset (a) and PaxDb Whole organism (Integrated) dataset (b) as standard reference, respectively. Legends and notations are the same as Figure 22.[000140] Figure 26: a-d) Correlation between biological replicates in the (a) tunicamycin, (b) MG132, (c) PD, and (d) mHttex1 -46Q datasets. Graphs shows scatter plot in the bottom left corner, density plot on the diagonal, and the Pearson correlation coefficient heatmap in the top right corner.[000141 ] Figure 27: Original gel figures of in vitro fluorescence kinetics for (a) Figure 2d and (b) Figure 2e.[000142] Figure 28: Original gel figures for (a) Figure 10c and (b) Figure 10d.[000143] Figure 29: Original western blot results for Figure 1 1 b.[000144] Figure 30: Reactions with model proteins, p-lactoglobulin (P-Lac) and bovine serum albumin (BSA) show differences in reactivity of chemical probes as well as sensitivity toward structure of proteins. Urea (6M) was used to unfold proteins, (a) Fluorescence measurements showing an increase in reactivity for both probes and BSA, for p-Lac when unfolded. 38 (PT_N038), unlike TME, has increased fluorescence suggesting increased reactivity for BSA upon unfolding conditions (b) Coomassie blue staining for total protein, confirming that the changes in fluorescence is due to reacted chemical probes and not differences in protein amount. 50 pM of chemical probe was used.[000145] Figure 31 : Reactions with model proteins, p-lactoglobulin (P-Lac) and bovine serum albumin (BSA) show differences in reactivity of chemical probes as well as sensitivity toward structure of proteins. Urea (6M) was used to unfold proteins, (a) Fluorescence measurements showing an increase in reactivity for both chemical probes for p-Lac when unfolded, but not for BSA. (b) Coomassie blue staining for total protein, confirming that the changes in fluorescence is due to reacted chemical probes and not differences in protein amount. 50 pM of chemical probe was used.[000146] Figure 32: Human Plasma experiments with 38 (PT_N038), 56 (PT_N056) and TME showing differences in labelling between chemical probes and enriched proteins, (a) Fluorescence measurements showing reacted chemical probes and different protein reactivities, (b) Coomassie blue stained gel, for total protein, showing enrichment of specific proteins unique to each chemical probe. 50 pM of chemical probe was used.[000147] Figure 33: Human plasma proteomics experiments with 56 (PT_N056) and TME demonstrating the ability to identify plasma proteins and showing enrichment of specific proteins unique to each chemical probe using the RUBICON workflow.[000148] Figure 34: Human fibroblast proteomics experiments with TME demonstrating the ability to identify unique proteins and showing enrichment of specific proteins unique to healthy control and myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) using the RUBICON workflow.[000149] Figure 35: Human fibroblast experiments with TME showing differences in labelling of cell lysate between healthy control (HC) and myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) patient. Coomassie blue stained gel, for total protein, showing enrichment of specific proteins unique to each condition. 50 pM of chemical probe was used.[000150] Figure 36: Human plasma experiments differences in labelling between healthy control (HC) and breast cancer (BC) patient as an example of both probes 38 (PT_N038) and 56 (PT_N056) able to chemically react and enrich proteins, (a) Fluorescence measurements for probe 38 showing reacted chemical probes and different protein reactivities between BC and HC. (b) Coomassie blue stained gel, for total protein, showing successful enrichment of proteins with chemical probe 38. 50 pM of chemical probe was used, (c) Coomassie blue stained gel, for total protein, showing successful enrichment of proteins with chemical probe 56. 50 pM of chemical probe was used.[000151 ] Figure 37: Reactions with model proteins, p-lactoglobulin (P-Lac) show differences in reactivity of chemical probes as well as sensitivity toward structure of proteins. Urea (6M) was used to unfold proteins, (a) Fluorescence measurements showing differences in reactivities when comparing all probes for p-Lac when unfolded. Probes 86 (PT_N086) and 98 (PT_N098) increased fluorescence suggesting increased reactivity for p-Lac upon unfolding conditions (b) Coomassie blue staining for total protein, confirming that the changes in fluorescence is due toreacted chemical probes and not differences in protein amount. 50 pM of chemical probe was used.DEFINITIONS[000152] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions set out below. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.[000153] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one having ordinary skill in the art to which the invention pertains.[000154] Unless the context clearly requires otherwise, throughout the description and the claims, the terms “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”. For example, a composition, mixture, process or method that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, mixture, process or method.[000155] The transitional phrase “consisting of” excludes any element, step, or ingredient not specified. If in the claim, such would close the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith. When the phrase “consisting of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.[000156] The transitional phrase “consisting essentially of” is used to define a composition, process or method that includes materials, steps, features, components, or elements, in addition to those literally disclosed, provided that these additional materials, steps, features, components, or elements do not materially affect the basic and novel characteristic(s) of the claimed invention. The term “consisting essentially of” occupies a middle ground between “comprising” and “consisting of”.[000157] Where applicants have defined an invention or a portion thereof with an open-ended term such as “comprising”, it should be readily understood that (unless otherwise stated) the description should be interpreted to also describe such an invention using the terms “consisting essentially of” or “consisting of”. In other words, with respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter may include the use of either of the other two terms. Thus, in some embodiments not otherwise explicitly recited, any instance of “comprising” may be replaced by “consisting of” or, alternatively, by “consisting essentially of”. [000158] Other than in the claims or operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to beunderstood as modified in all instances by the term “about”. The examples are not intended to limit the scope of the invention.[000159] In what follows, or where otherwise indicated, “%” will mean “weight %”, “ratio” will mean “weight ratio” and “parts” will mean “weight parts”.[000160] The terms “predominantly”, “predominant”, and “substantially” as used herein shall mean comprising more than 50% by weight, unless otherwise indicated.[000161 ] As used herein, with reference to numbers in a range of numerals, the terms “about,” “approximately” and “substantially” are understood to refer to the range of -10% to +10% of the referenced number, preferably -5% to +5% of the referenced number, more preferably -1 % to + 1 % of the referenced number, most preferably -0.1 % to +0.1 % of the referenced number. Moreover, with reference to numerical ranges, these terms should be construed as providing support for a claim directed to any number or subset of numbers in that range. For example, a disclosure of from 1 to 10 should be construed as supporting a range of from 1 to 8, from 3 to 7, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, from 8 to 10, and so forth.[000162] The terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention.[000163] Reference throughout this specification to ‘one embodiment’ or ‘an embodiment’ or ‘certain embodiments’ means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearance of the phrases ‘in one embodiment’ or ‘in an embodiment’ or ‘certain embodiments’ in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more combinations.[000164] The term "alkyl" refers to a straight-chain or branched (including cyclic branched) alkyl substituent containing from, for example, 1 to about 36 carbon atoms. Examples of suitable alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, secbutyl, isobutyl, tert-butyl, pentyl, isoamyl, 2-methylbutyl, 3-methylbutyl, hexyl, heptyl, 2- methylpentyl, 3-methylpentyl, 4-methylpentyl, 2-ethylbutyl, 3-ethylbutyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain. Non-limiting examples of C10 alkyl groups include the following structures:[000165] The term "alkenyl" refers to a straight-chain or branched (including cyclic branched) alkenyl substituent containing from, for example, 2 to about 36 carbon atoms. Examples of suitable alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropenyl, butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, hexadienyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.[000166] The term "alkynyl" refers to a straight-chain or branched alkynyl substituent containing from, for example, 2 to about 36 carbon atoms. Examples of suitable alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, butadienyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, undecynyl, dodecynyl and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.[000167] The term "cycloalkyl" refers to a saturated non-aromatic cyclic hydrocarbon. The cycloalkyl ring may include a specified number of carbon atoms. For example, a 3 to 8 membered cycloalkyl group includes 3, 4, 5, 6, 7 or 8 carbon atoms. The cycloalkyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Nonlimiting examples may include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and the like.[000168] The term "cycloalkenyl" or “cycloalkene” refers to a cyclic hydrocarbon having at least one double bond, which is not aromatic. The cycloalkenyl ring may include a specified number of carbon atoms. The cycloalkenyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). For example, a 5 membered cycloalkenyl group includes 5 carbon atoms. Non-limiting examples may include cyclopentenyl and cyclopenta-1 ,3-dienyl.[000169] The term "cycloalkynyl" or “cycloalkyne” refers to a cyclic hydrocarbon having at least one triple bond, which is not aromatic. The cycloalkynyl ring may include a specified number of carbon atoms. The cycloalkynyl group may be monocyclic, bicyclic or tricyclic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). For example, a 5 membered cycloalkynyl group includes 5 carbon atoms. Non-limiting examples may include cyclopentynyl.[000170] The term “aryl” refers to an aromatic carbocyclic substituent, as commonly understood in the art. It is understood that the term aryl applies to cyclic substituents in which at least one ring is planar and comprises 4n+2 electrons, according to Huckel’s Rule. Aryl groups may be monocyclic, bicyclic or tricyclic. Examples of aryl groups include, but are not limited to, phenyl, naphthyl and 1 ,2,3,4-tetrahydronaphthyL An aryl group may be monocyclic,bicyclic or tricyclic, provided that at least one ring is aromatic. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings).[000171] The term "heteroalkyl" refers to a straight-chain or branched (including cyclic branched) alkyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 1 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. Examples of suitable heteroalkyl groups include, but are not limited to, methoxy, ethoxy, propyloxy, isopropyloxy, and the like. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain. In the case where a terminal carbon is replaced by a heteroatom, such as N, O, the heteroalkyl group can be an alkyl amine, or alkyl alcohol, respectively. Non-limiting examples of Ce heteroalkyl groups include the following[000172] The term "heteroalkenyl" refers to a straight-chain or branched (including cyclic branched) alkenyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 2 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.[000173] The term "heteroalkynyl" refers to a straight-chain or branched alkynyl substituent in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. It may contain from, for example, 2 to about 36 carbon atoms. For example, between 1 and 4 carbon atoms may be replaced by heteroatoms independently selected from N, S and O. The number of carbons referred to relates to the carbon backbone and carbon branching but does not include carbon atoms belonging to any substituents, for example the carbon atoms of an alkoxy substituent branching off the main carbon chain.[000174] The term “heterocyclic” or “heterocyclyl” as used herein, refers to a cycloalkyl or cycloalkenyl group in which one or more carbon atoms have been replaced by heteroatoms independently selected from N, S and O. For example, between 1 and 4 carbon atoms in each ring may be replaced by heteroatoms independently selected from N, S and O. The heterocyclyl group may be monocyclic, bicyclic or tricyclic in which at least one ring includes a heteroatom.When more than one ring is present the rings may be fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Each of the rings of a heterocyclyl group may include, for example, between 5 and 7 atoms. Examples of heterocyclyl groups include tetrahydrofuranyl, tetrahydrothiophenyl, pyrrolidinyl, pyrrolinyl, dithiolyl, 1 ,3-dioxanyl, dioxinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, pyranyl, 1 ,4-dithiane, and decahydroisoquinoline. In one embodiment, heterocyclyl may be optionally substituted by =0. [000175] The term “heteroaryl”, as used herein, refers to a monocyclic, bicyclic or tricyclic ring of up to 7 atoms in each ring, wherein at least one ring is aromatic and said at least one ring contains from 1 to 4 heteroatoms selected from the group consisting of O, N and S. When more than one ring is present the rings are fused together (for example, a bicyclic ring is fused if two atoms are common to both rings). Consideration must be provided to tautomers of heteroatom containing ring systems containing carbonyl groups, for example, when determining if a ring is a heterocyclyl or heteroaryl ring. Heteroaryl includes, but is not limited to, 5-membered heteroaryls having one hetero atom (e.g., thiophenes, pyrroles, furans); 5 membered heteroaryls having two heteroatoms in 1 ,2 or 1 ,3 positions (e.g., oxazoles, pyrazoles, imidazoles, thiazoles, purines); 5-membered heteroaryls having three heteroatoms (e.g., triazoles, thiadiazoles); 5-membered heteroaryls having four heteroatoms (e.g., tetrazoles); 6- membered heteroaryls with one heteroatom (e.g., pyridine, quinoline, isoquinoline); 6- membered heteroaryls with two heteroatoms (e.g., pyridazines, cinnolines, phthalazines, pyrazines, pyrimidines, quinazolines, quinoxalinone, quinazolinone); 6-membered heteroaryls with three heteroatoms (e.g., 1 ,3,5- triazine); and 6-membered heteroaryls with four heteroatoms. Examples of heteroaryl include thiophene, benzothiophene, benzofuran, benzimidazole, benzoxazole, benzothiazole, benzisothiazole, furan, pyrrole, imidazole, pyrazole, pyridine, pyrazine, pyrimidine, pyridazine, indole, isoindole, 1 H-indazole, purine, quinoline, isoquinoline, phthalazine, naphthyridine, quinoxaline, cinnoline, carbazole, phenanthridine, acridine, phenazine, thiazole, isothiazole, phenothiazine, oxazole, isooxazole, furazane, and phenoxazine. Further exemplary heteroaryl groups may include, for example, indoline or 2,3-dihydrobenzofuran. In one embodiment, heteroaryl may be optionally substituted by =0.[000176] Whenever a range of the number of atoms in a structure is indicated (e.g., a C1 C12, Ci-Ce alkyl, etc.), it is specifically contemplated that any sub-range or individual number of carbon atoms falling within the indicated range also can be used. Thus, for instance, the recitation of a range of 1 -12 carbon atoms (e.g., C1-C12), 1 -6 carbon atoms (e.g., Ci-Ce) as used with respect to any chemical group (e.g., alkyl, etc.) referenced herein encompasses and specifically describes 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , and / or 12 carbon atoms, as appropriate, as well as any sub-range thereof (e.g., 1 -2 carbon atoms, 1 -3 carbon atoms, 1 -4 carbon atoms, 1 -5 carbon atoms, 1 -6 carbon atoms, 1 -7 carbon atoms, 1 -8 carbon atoms, 1 -9 carbon atoms, 1 -10 carbon atoms, 1 -11 carbon atoms, 1 -12 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5carbon atoms, 2-6 carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-1 1 carbon atoms, 3-12 carbon atoms, 4-5 carbon atoms, 4-6 carbon atoms, 4-7 carbon atoms, 4-8 carbon atoms, 4-9 carbon atoms, 4-10 carbon atoms, 4-11 carbon atoms, and / or 4-12 carbon atoms, etc., as appropriate).[000177] As used herein, “halo” refers to a halogen atom, especially I, F, Cl or Br; more especially F or Cl; most especially F.[000178] As used herein, the term “optionally substituted” means that any number of hydrogen atoms on the optionally substituted group are replaced with another moiety. Unless defined otherwise, said moiety is independently selected from the group consisting of C1-C12 alkyl (or Ci-Ce alkyl), C2-C6 alkenyl, C2-C6 alkynyl, aryl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, heteroaryl, R120-CONH-R130, R120-NHCO-R130, R120-CSNH-R130, R120-NHCS-R130, R120-CO-R130, =0, =S, cyano, CF3, nitro, and halogen; wherein:R120is selected from the group consisting of Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, heteroaryl, and a bond; andR130is selected from the group consisting of Ci-Ce alkyl, C2-C6 alkenyl, C2-C6 alkynyl, aryl, Ci-Ce heteroalkyl, C2-C6 heteroalkenyl, C2-C6 heteroalkynyl, heteroaryl, and hydrogen.[000179] As used herein, the phrase “a group which is capable of reacting with a thiol group”, means a group which is capable of reacting with a thiol group so as to form a bond with the thiol group. For example, such a group may react with the thiol group to form a disulfide group, or it may react with the thiol group and form a thioamide group, or it may react with the thiol group and form a thioether group, or it may react with the thiol group and form a thioacetal group. In certain embodiments, it may mean a group selected from the following: a maleimide, a halo-acetamide, a vinyl acetamide, an aryl acetamide,, wherein R’ is independently selected from the group consisting of H, C1-C12 alkyl, and aryl, and X is halo. In certain embodiments, it may mean a group which is capable of reacting with the thiol of a cysteine group within a polypeptide or protein.[000180] It will be appreciated by a person of skill in the art that the compounds of the first aspect may, due to the core double bond, present as geometric isomers, such as cis / trans and E / Z isomers. While drawn in one configuration herein for the sake of convenience, it should be appreciated that all compounds of the first aspect may be in the E or Z form, and every structure drawn herein is explicitly considered to be represented in both the E and Z isomeric forms. Synthesis of the compounds of the first aspect may result in substantially pure forms of E or Z isomer, or a mixture of E and Z forms, which forms may be used in any of the methods and applications described herein in that particular form.ABBREVIATIONS[000181] AGC: automatic gain control; BLG: p-lactoglobulin; CuAAC: Cu-catalyzed Azide- Alkyne Cycloaddition; Cys: cysteine; DTT: dithiothreitol; EBV: Epsteine-Barr virus; ER: endoplasmic reticulum; FACS: fluorescence-activated cell sorting; FBS: fetal bovine serum; GSH: glutathione; HD: Huntington’s disease; Httexl : Huntingtin exon 1 ; IA: iodoacetamide; IDP: intrinsically disordered protein; LFQ: label-free quantification; NMM: / V-methylmaleimide; Ml: maleimide; ND: normal distribution; NMR: nuclear magnetic resonance; PBMC: peripheral blood mononuclear cell; PCA: Principal component analysis; PD: Parkinson’s disease; PPI: proteinprotein interaction; PulSA: pulse-shape analysis; RFU: relative fluorescence units; RIM: restriction of intramolecular motion; RUBICON: Reactive Unfolded protein Based Identification of Cysteine On eNrichment; TBAF: tetrabutylammonium fluoride; TCEP: tris(2- carboxyethyl)phosphine; TEAB: tetraethylammonium bromide; THPTA: Tris(3- hydroxypropyltriazolylmethyl)amine; TME: Z-1 -(4-(2-(4-ethynylphenyl)-1 ,2-diphenylvinyl)phenyl)- 7 A7-pyrrole-2, 5-dione; TMS: trimethylsilane; TPE: tetraphenylethene; Trp: tryptophan.[000182] Preferred features, embodiments and variations of the invention may be discerned from the following Examples which provides sufficient information for those skilled in the art to perform the invention. The following Examples are not to be regarded as limiting the scope of the preceding Summary of the Invention in any way.EXAMPLES[000183] Disclosed herein are methods that enable the capture of unfolded protein species including IDPs in situ in living cells, which can subsequently be detected by a fluorescence readout or enriched and analyzed by mass spectrometry-based proteomics, for the global analysis of a protein disorder (Figure 1 ). Core to this method are novel chemical probes, for example TME. The chemical probe (e.g. TME) selectively reacts with unfolded proteins with free cysteines in an exposed and flexible environment, with little interference from folded proteins with buried cysteines or surface exposed cysteines in a rigid environment. The reaction with unfolded proteins generates a fluorescence turn-on effect, allowing the study of the impact of disease-related protein aggregation on the global proteome foldedness in a complex cellular environment. Coupled with mass spectrometry-based proteomics, disclosed herein is a workflow named RUBICON (Reactive Unfolded protein Based Identification of Cysteine OneNrichment), to enrich and quantify probe (e.g. TME)-labeled unfolded proteins. RUBICON allows the mapping of the basal and stress-specific unfolded proteome, for the first time, with coverage to proteins even of low abundance. Using blood cells, the potential of using the RUBICON pipeline for biomarker discovery is demonstrated, and for improving the discrimination of disease and healthy cohorts using Parkinson’s disease as an example. Harnessing the high-throughput TME fluorescence and proteomics, further demonstrated is a generic cellular quality control mechanism to adapt to proteostatic stress by aggregation-prone distributions and sequestering I DPs in a Huntington’s disease model.[000184] In lymphoblastoid cells from Parkinson’s disease (PD) patients and healthy controls, the inventive probe (e.g. TME) can differentiate PD from the healthy population more effectively compared to protein abundance profiling methods. By using high-throughput probe (e.g. TME) fluorescence and proteomics, a generic cellular quality control mechanism is revealed in which cells adapt to proteostatic stress by aggregation-prone distributions and sequestration of disordered proteins, using cell models of Huntington’s disease.Overall design strategy and workflow[000185] The major characteristics of protein disorder is the increased accessibility / exposure and structural flexibility and dynamics. Disclosed herein is a method to detect proteins that are inherently unfolded, as well as those resulting from active protein unfolding. To address the current technological gap, a new method would preferably 1 ) be compatible with a live-cell setup to capture unfolded protein species in situ to minimize artifactual structural changes; 2) have high sensitivity to detect the disordered regions of not only high-to-medium abundant proteins but low abundant proteins that are often overlooked by existing methods; and 3) be robust to study protein disorder in a wide range of cell types and in a high-throughput manner. [000186] To address these criteria, new chemical probes, e.g. TME, which consists of three components - a tetraphenylethene (TPE) fluorophore, a maleimide (Ml) group and an alkyne handle (Figure 1 a), were synthesised. TPE is an aggregation-induced emission fluorophore meaning its fluorescence is activated by restriction of intramolecular motion (RIM). Ml is a thiol (e.g. cysteine) reactive group which can conjugate selectively with free cysteines in physiological conditions. On one hand, the bulkiness of TPE can prevent TME from reaching and reacting with buried cysteines in folded proteins, thus enhancing the selectivity towards cysteines situated in a conformationally flexible environment (Figure 1b). On the other hand, this design enables a two-step fluorescence activation process - the reaction with a thiol to remove the photoinduced electron transfer quenching effect of the Ml group (Figure 7b) and the reaction with proteins but not small biothiols that provide physical constraints to enable the RIM effect (Figure 7c-d). The unique alkyne handle of TME can be readily clicked with biotin-azide via Cu- catalyzed Azide-Alkyne Cycloaddition (CuAAC), allowing the subsequent affinity purification and mass spectrometry-based identification and quantification of the TME-labeled unfolded protein species. Hence, the RUBICON workflow was developed as follows (Figure 1c). Live cellsamples to be investigated (e.g., cell lines treated with stressors, patient samples, etc.) were first treated with cell permeable TME to label endogenous unfolded proteins in situ. In a second step, the proteome was extracted and subjected to in vitro CuAAC. Labeled unfolded proteins were then selectively enriched by streptavidin beads and analyzed using a proteomic approach. In vitro validation of TME specificity towards protein disordered regions[000187] TME’s reactivity towards several model proteins with at least one buried free cysteine thiol in the core of their folded state was investigated. Small biothiol glutathione (GSH), which comprises the major pool of cellular non-protein thiols, was included as a control. Being non- fluorescent itself, TME fluorescence exhibited a dramatic increase upon reaction with urea- unfolded p-lactoglobulin (BLG) (Figure 2a). This fluorescence increase was completely counteracted if unfolded BLG was pre-treated with the th iol-blocking reagent, N- methylmaleimide (NMM), indicating TME’s specificity towards the cysteine thiol. Conversely, TME showed only small fluorescence enhancement in the presence of native BLG and almost no fluorescence with GSH even above its physiological concentration in cells (i.e., ~ 5 mM) (Figure 2a). Strikingly, TME displayed superior resistance to the damping effect of GSH, suggesting a higher reactivity with unfolded proteins than GSH (Figure 8b).[000188] Next, how surface exposure and flexibility jointly affect TME reactivity towards free cysteines was explored. DsbA is a thiol oxidase from Escherichia coli that encompasses a Cys30-Phe-His-Cys33motif in which one cysteine is surface-exposed (C30) and the other is buried (C33) (Figure 2b). Consistent with BLG, TME displayed a substantial fluorescence increase when conjugated to unfolded DsbA but not folded DsbA (Figure 2c). TME displayed low reactivity with folded DsbA even though there is a surface accessible Cys. Mutation of the buried C33 in DsbA to an alanine (hereinafter “C33A”) rendered higher flexibility and dynamics around the nearby loop containing C30 (Figure 2b). The increase of the flexibility resulted in an increase of TME reactivity to folded C33A as exemplified by 4-fold fluorescence increase compared to folded DsbA (Figure 2c). Without being bound by theory, it is possible that the bulkiness of TME may have hampered its access to the structurally constrained surface exposed Cys on folded DsbA, while unstructured cysteine residues, like those on unfolded proteins, may be better recognized by TME.[000189] It was further confirmed that the observed TME fluorescence strongly correlated to its bioconjugation with the proteins by using in-gel fluorescence of the above samples (Figure 2d- e). Linearization, denaturation, and immobilization of proteins in the gel matrix homogenize the local environment that can modulate the RIM process to affect TME fluorescence. Consistently, in-gel fluorescence displayed similar outcomes, showing higher TME labeling on unfolded BLG and DsbA over folded ones, as well as folded C33A over folded DsbA (Figure 2d-e and Figure 8c-d). Collectively, TME displayed selective reactivity towards cysteines located in an exposed, flexible and dynamic environment. These cysteines are a signature of both inherent and acquired protein disorder. It was further demonstrated that TME fluorescence can be used tostudy protein denaturation and stability in vitro, showing higher selectivity and consistency compared to intrinsic tryptophan fluorescence measurement, using the model protein BLG (Figure 9).Probe binders contain significantly more disordered regions[000190] The binding targets of TME (hereinafter “TME binders”) were determined in a murine cell line (Neuro-2a cells) and the RUBICON workflow was evaluated for capturing and analyzing endogenous proteome disorder in cells. The biocompatibility of TME was confirmed, including low cytotoxicity (Figure 10a) and good cell permeability (Figure 10b), followed by its functionality to enrich unfolded model proteins (Figure 10c) and cellular proteins from cell lysate (Figure 10d). Two classical proteostatic stressors were then applied, i.e., tunicamycin (an inhibitor of N- glycosylation) and MG132 (a proteasome inhibitor), to the cells to promote accumulation of unfolded proteins. Flow cytometry using TME fluorescence confirmed the elevated level of unfolded protein load in live cells treated with tunicamycin or MG132 (Figure 10e).[000191] To evaluate protein disorder in individual proteins of the proteome, mass spectrometry proteomics was employed. By using the RUBICON workflow (strategy shown in Figure 11a), TME binders were identified and quantified in control (Figure 3a) and in tunicamycin treated conditions (Figure 3b), as well as proteins that became more unfolded upon tunicamycin treatment (Figure 3c). Western blotting was used to further validate this result (Figure 11 b). Approx. 60% more proteins were identified in the labeled samples compared to the unlabeled ones (Figure 11c), which further confirmed the validity of the RUBICON workflow to enrich probe (e.g. TME) labeled proteins over background proteins.[000192] The abundance of the RUBICON workflow enriched proteins was then quantified. The intensity-Based Absolute Quantification (iBAQ) values of the TME binders ranged from high to low and remarkably, even proteins which were undetectable by using lysate profiling methods (red dots in Figure 3a-b and Figure 12a-b). To further quantify the abundance of these undetectable proteins, Protein Abundance Database (PaxDb) and proteomic datasets were used as standard references (Figure 22 for the MG132 dataset and Figure 24 for the tunicamycin dataset). Using the MG132 dataset as an example, it was evident that in ‘lysate’, higher protein abundance increases the likelihood of detection when using lysate profiling methods (grey bar). ‘Binder’, exclusive of the 'Undetectable', covers proteins spanning from low to high abundance, with proteins of higher abundance more likely to be labeled by TME. Interestingly, ‘Undetectable1proteins showed a relatively even distribution across all protein abundance levels, without a preference for proteins of high abundance, in all three standard references. The analysis also showed that RUBICON was capable of detecting proteins down to the 5th percentile of low-abundance proteins.[000193] Probe (e.g. TME) labeling does not depend on protein abundance. The RUBICON workflow enriched proteins with abundance levels ranging from high to low and remarkably, even proteins of very low abundance (red dots in Figure 3a-b) which were not detectable byusing abundance-based profiling methods. These results were compared with results utilizing a small-sized iodoacetamide (IA) alkyne probe to profile intrinsic cysteine reactivity. IA alkyne enriched hyper-active cysteines mainly in functional residues involving in catalytic function, post- translational modification and redox reaction. A similar analysis was performed on TME binders on the protein level, focusing on the following cysteine functional annotations: nucleophile, active-site and redox-active disulfide. TME binders were slightly enriched in ‘active-site’ and ‘redox-active disulfide’ where fold enrichment ranged from 0 to 2.5, while IA alkyne binders were remarkably enriched in those functional classes with fold enrichment up to 143 and 225 respectively (Figure 3e). This is consistent with TME being much bulkier than the IA alkyne probe, and displaying a disparate reactivity towards cellular cysteines, and not preferentially targeting hyper-active and functional cysteines that are usually first reacted with small thiol probes. TME binders were anchored in all cellular organelles, and enriched in intracellular proteins (e.g., nucleus, cytoplasm, ER) (Figure 11d). This further confirmed the membrane permeability of TME and the broad-spectrum functionality of RUBICON to target proteins of any localization in cells.[000194] The TME binders were further characterized in control and stress. Up to 60-75% TME binders remained the same upon tunicamycin (Figure 3d) and MG132 (Figure 12d) treatment, compared to their corresponding controls. Similar enriched functional terms were identified among controls (Figure 3f for tunicamycin and Figure 12g for MG132), tunicamycin (Figure 11 e) and MG132 binders (Figure 12h). These function terms were grouped into seven functional clusters highlighted in different colors. Among these seven clusters, protein quality control (e.g. protein folding (G0:0006457), positive regulation of macroautophagy (G0:0016239), proteasome complex (G0:0000502), chaperone binding (G0:0051087)), biomolecular condensates (e.g., P-body assembly (G0:0033962) and cytoplasmic ribonucleoprotein granule (G0:0036464)) are related to unfolded protein. Most of the enriched functional clusters, such as mRNA processing, nucleotide binding, translation initiation and regulation, cell cycle, and biomolecular condensates, were also reported to be classified as intrinsically disordered proteins (IDPs).[000195] Hence, the protein disorder of TME binders was next assessed using IUPred2A which allows energy estimation-based predictions to identify disordered protein regions and disordered binding regions. TME binders contained significantly more unfolded regions compared to the proteome average (Figure 3g). It was reported that structural flexibility promotes binding diversity and thus IDPs are frequently involved in key cellular, regulatory, and metabolic processes. The role of TME binders was then investigated in the protein-protein interaction (PPI) network. Consistently, degrees (Figure 3h) and betweenness centrality (Figure 3i) of TME binders were significantly higher than the proteome average, suggesting that these TME binders may function as hubs and bottlenecks in the PPI network (Figure 11f). Following this, cysteine-containing proteins were classified into structured proteins, proteins withintrinsically disordered regions (IDRs) and IDPs (Type 1 , Type 2a-c, and Type 3 in Figure 23a, respectively). Notably, TME binders in both tunicamycin and MG132 datasets included a substantial portion of proteins with IDRs, and were highly enriched in IDPs and depleted in structured proteins (Figure 23), supporting the specificity of TME towards protein disorder (Figure 2, Figure 3g and Figure 12i). Proteins that become more disordered upon tunicamycin (Figure 3c) and MG132 treatment (Figure 12c) were either structured proteins or proteins with IDRs (Table 1). In summary, TME binders identified by RUBICON contained significantly more unfolded regions and played a key role in proteome integrity through extensive and bridging interactions within cells.Tabie 1. Classification of proteins that become more disordered after tunicamycin and MG 132 treatment.Disordered region threshold length: 15 i Disordered region threshold length: 30RUBICON, as a diagnostic tool, effectively discriminates disease from healthy cohorts [000196] The above findings confirmed the capability and accuracy of TME to probe cellular protein disorder. It was further considered whether the disordered proteome, as a novel type of molecular signature, could be used as a potential diagnostic tool. Using Parkinson’s disease (PD) as an example, the feasibility of using TME and RUBICON in clinically relevant, biologically heterogenous samples was explored. Lymphoblastoid cell lines (hereinafter, lymphoblasts) were generated from PBMCs of individuals with idiopathic PD and their age / gender-matched healthy controls. Like other cell lines, lymphoblasts can also be stained by TME and processed using RUBICON. The disordered proteomes, i.e., TME binders, associated with the healthy and the PD groups were identified, as well as proteins that are more unfolded in either the healthy or the PD group (Figure 4a-c).[000197] The TME binders enriched from the human lymphoblasts were further characterized (Figure 13a-g). They were enriched in similar functional clusters as the dataset generated from the mouse cell line (Figure 13c-d). Consistently, the TME binders contained significantly more unfolded regions (Figure 13e), as well as more hubs and bottlenecks in the PPI network (Figure 13f-g), compared to the proteome average. Since RUBICON (1) captures low abundant proteins that are not detectable by abundance-based lysate profiling methods and (2) focuses on protein disorder which may provide an additional dimension of information beyond protein abundance (Figure 4a-c), the effectiveness of establishing predictive models to discriminate the PD and the healthy cohorts were compared by using traditional protein abundance profiling (‘cell lysate’) and using protein disorder profiling (‘RUBICON’). Principal component analysis (PCA) showedRUBICON clearly distinguished between the healthy and RD samples, while protein abundance levels of these two groups were extremely similar (Figure 4e). The first two principal components of the PCA results of RUBICON constituted a significantly larger percentage of the total variations compared to those of the protein abundance profiling (Figure 13h). These results demonstrate a proof of concept of using RUBICON for characterizing heterogeneous clinical samples and for discriminating disease from healthy cohorts for disease diagnosis and classification.Probe fluorescence reveals the correlation between protein expression levels, distribution, and cellular proteostasis[000198] The above results using RUBICON confirm the possibility of using the inventive probes (e.g. TME) to capture endogenous unfolded proteins for protein disorder analysis in cultured cells and patient-derived materials. Next, TME was applied in a Huntington’s disease (HD) model to investigate how the expression of HD-related proteins affects the cellular proteostasis. Huntingtin exon 1 (Httexl ), mutated to contain variable polyglutamine (polyQ) lengths, has recapitulated characteristic HD features in transgenic mice. A flow cytometry-based pulse-shape analysis (PulSA) was used to examine Neuro-2a cells transiently transfected with Httexl fused to C-terminal mCherry and their impact on TME fluorescence. Note that Httexl - mCherry proteins do not contain any cysteine and thus were not labeled by TME. Three parameters recorded in the PulSA workflow, i.e., area (A), width (W) and height (H), inferred the mCherry-fused Httexl expression level (A) and distributions (W and H) in cells respectively. Suppose A is the same, diffuse protein distribution displays large W and small H, while small W and large H correspond to protein aggregation which can further evolve to inclusion (Figure 5a). PulSA revealed that inclusion-forming mutants (46Q and 97Q) displayed three stages as expression levels increased, while there was only one stage observed for non-inclusion-forming proteins, 25Q (Figure 5b; vehicle control shown in Figure 14a). With the increase of A, 25Q exhibited larger H and slightly larger W, indicating proteins accumulate successively with its pulse shape becoming wider and higher. In the case of 46Q and 97Q, increasing A first manifested larger H and larger W (Stage I ), then larger H but smaller W (Stage n ), and eventually larger H and slightly larger W (Stage HI). Cells in stage n displayed a sharp W decrease, suggesting Httexl reached the concentration threshold for aggregation and inclusions started to shape. These three stages corresponded to protein accumulation, preinclusion formation and inclusion maturation, respectively. Figure 5c clearly depicts how expression levels of Httexl affected inclusion formation, where the threshold of expression levels of 97Q to form inclusions was much lower than that of 46Q. This is consistent with previous reports concluding that Httexl aggregation is both polyQ-length- and concentrationdependent.[000199] When TME fluorescence was taken into account in PulSA, stage n could be further divided into two subpopulations, na(‘aggregation-prone’) and nn(‘non-aggregation-prone’)(Figure 5d). nadisplayed low TME signals and small W, while nnshowed high TME signals and large W. These two subpopulations were characterized and are shown and discussed in Figure 6. Time-course pulse shape monitoring further confirmed the progressive aggregation of 46Q through these three stages along with the increase of expression levels (Figure 5e). To better understand the impact of protein expression levels on the cell’s unfolded protein load, the A value in mCherry channel was binned into 25 intervals ranging from low to high. 25Q displayed significantly higher TME fluorescence than 46Q and 97Q at almost any expression bin, and TME signals decreased when 46 / 97Q reached stage n and HI (Figure 5f).[000200] To understand the impact of protein distribution on the cell’s unfolded protein load, in each expression bin cells were divided into subsets with large and small H (top and bottom 25% respectively), and subsets with large and small W (top and bottom 25% respectively), and compared the TME signals among them (Figure 14c-d). Cells with large H or small W, exhibited significantly lower TME signals than those with small H or large W respectively, in almost all expression bins (Figure 5g-h), suggesting the aggregation-prone distribution may reduce cellular protein disorder. Previous research has reported the protective role of inclusion formation in reducing intracellular level of diffuse huntingtin, sequestrating potentially toxic protein aggregated species that have escaped degradation by the ubiquitin-proteasome pathway and facilitating aggregate clearance and degradation by the autophagy-lysosomal system. This PulSA analysis based on TME signals spanning across all expression levels further generalized the above conclusions. Not only inclusion formation but the aggregation- prone distribution, i.e. large H, small W, at low-to-medium expression levels, was protective against unfolded protein load. It may be a generic strategy for cellular quality control to sequester not only protein aggregations but also overexpressed protein species into inclusions or locally aggresome-like structures when the folding capacity is compromised, to reduce cellular protein disorder (further discussed in Figure 6).Huntingtin inclusion clears cellular unfolded protein load by sequestering potential probe binders[000201] Using PulSA and TME fluorescence, a unique three-stage, four-subpopulation pattern was revealed during the process of inclusion formation by the disease-causing forms of huntingtin (Figure 5). Using 46Q as an example, cells were first sorted by fluorescence- activated cell sorting (FACS) into four subpopulations based on Figure 5d, aliquoted and lysed (total lysate T), and then further fractionated into supernatant (S) and pellet (P) by ultracentrifugation to separate the soluble and aggregated proteins (Figure 6a). Subsequently, the samples were analyzed by mass spectrometry-based proteomics. 46Q displayed a gradual increase of expression levels from Stage I to HI in their T and P fractions, while in the S fraction, the expression level was relatively similar in Stage H and HI (Figure 15a). This indicated that most 46Q aggregated when overexpressed.[000202] In the abundance analysis, two clusters of proteins were focused on: one exhibited descending abundance (cluster-D) and the other ascending abundance (cluster-A) going through Stage I to HI (Figure 6a). When 46Q was overexpressed and aggregation occurred from Stage I to HI, cells sped up translation and triggered a series of stress response and quality control machinery, as indicated from the enrichment analysis of T-cluster-A (Figure 15e) and S-cluster-A (Figure 15f). P-cluster-A were enriched in the same functional clusters as TME binders (Figure 6b vs. Figure 3f, except for “cytoskeleton organization”). The relation of the enriched functional annotations between TME binders, cluster-A and cluster-D was next explored. The Venn diagram shows that the enriched terms of P-cluster-A and T-cluster-A, but not S-cluster-A, were highly similar to those of TME binders, with more than 50% overlap (Figure 6d). In contrast, cluster-D were mutually exclusive to both TME binders and cluster-A in T, S and P (only max. 3 terms in common). This indicated that 46Q overexpression induced upregulation and co-aggregation of proteins that belong to similar functional categories of TME binders. Consistently, P-cluster-A exhibited higher IUPred2 score, ANCHOR2 score, degrees and betweenness than the proteome average, while proteins belonging to P-cluster-D were more folded (Figure 6e-g; data for T / S-cluster-D shown in Figure 16). This suggests that the global protein disorder is reduced resulting from co-aggregation of proteins with significantly more disordered regions and re-solubilization of those with less disordered regions.Furthermore, proteins co-aggregated with 46Q in aggregation-prone distribution were enriched in translation regulation and ribonucleoprotein (Figure 6c). This was consistent with the enrichment analysis result of TME binders (Figure 3f).[000203] Collectively, non-inclusion forming Httexl exhibits only one stage (Figure 6h) while inclusion forming Httexl displays three stages when protein expression levels increase (Figure 6i). Upon Httexl -46Q aggregation, cells activate stress response and protein quality control pathways by expressing more proteins with disordered regions like I DPs that are potential TME binders and play essential roles as hubs and bottlenecks in the PPI network. Httexl -46Q proteins co-aggregate with these TME binders to clear cellular unfolded protein load as an adaptive response, as indicated by the TME fluorescence (Figure 6i).Discussion[000204] Bi-functional chemical probes, such as TME, were used for global analysis of endogenous protein disorder in cells. The probes specifically react with free cysteines situated in an exposed and flexible environment, which represents a signature of protein disordered regions. TME can penetrate living cells to capture endogenous unfolded proteins with disordered regions in their native biological matrix. This is advantageous as extensive studies have shown that the cellular environment greatly impacts protein structure and interactions. Adapting the RUBICON workflow allows the enrichment of TME labeled proteins for subsequent large-scale analysis of protein disorder through mass spectrometry proteomics.[000205] TME labeled unfolded proteins (i.e. TME binders) were characterized, and it was discovered that they contain significantly more disordered regions than the proteome average. TME binders include a substantial portion of proteins with intrinsically disordered regions, and are highly enriched in intrinsically disordered proteins (IDPs). IDPs make up a key component of biological dark matter, which exhibit inherently flexible, heterogenous, dynamic and peculiar features and possess no well-defined 3-D structure under physiological conditions. These proteins are engaged in essential biological processes and regulatory pathways such as translation regulation, the cell cycle and protein quality control through extensive and bridging interactions with other proteins. Upon drug treatment that induces stress, constitutions of the unfolded proteome change but still belong to the same functional categories. This indicates the ubiquitous existence of IDPs in the physiological condition, which constitute a large part or even the vast majority of the unfolded proteome, and the robustness of cellular quality control to conserve proteome integrity to cope with proteostatic stress.[000206] Notably, this work showed that the TME binders cover proteins from high to low abundance, and RUBICON could identify proteins of very low abundance that cannot be detected by lysate-based protein profiling methods. Using biologically heterogenous, complex samples like cells derived from human blood samples, it was demonstrated that the disordered proteome, measured by RUBICON, better discriminated between healthy controls and individuals with Parkinson’s disease than abundance information. RUBICON identifies IDPs and low abundant proteins, which are often overlooked by conventional methods, thereby providing hidden information of non-canonical biomarkers and potential drug targets.[000207] Applied in a Huntington’s disease model, TME fluorescence revealed a characteristic pattern correlated to huntingtin inclusion formation. Cells oppose proteostatic stress by changing huntingtin protein distribution and co-aggregating potential TME binders. The aggregation-prone protein distribution, which displays a large H and small W in the flow cytometric pulse shape, ameliorate proteostatic stress induced by overexpression of exogenous protein, huntingtin in this case, even at low to medium expression levels. The co-aggregated proteins are enriched in similar functional categories of TME binders, indicating the essential regulatory roles of unfolded proteins including IDPs to change protein distribution through extensive protein-protein interactions as an adaptive response.General Probe synthetic methods and characterisation information[000208] Anhydrous solvents were dried by molecular sieves (4A). Hexanes refer to the fraction of boiling point range 40-60°C. Flash chromatography was carried out on silica gel (Merck Kieselgel 60 (230 - 400 mesh)) under pressure of nitrogen. 4-Aminobenzophenone was obtained from Alfa Aesar. Acetic anhydride, ethylene glycol, glycerol, toluene and triethylamine were obtained from Ajax Finechem. (Trimethylsilyl)acetylene was obtained from Matrix Scientific. Maleic Anhydride was obtained from BDH. Chloroform, K2CO3, Na2SC>4 and NaOAc were obtained from Chem Supply. Hexanes were obtained from Fisher Scientific. Acetonitrileand ethanol, were obtained from Merck. 4-Bromobenzophenone, CDCI3, copper (I) iodide, DMSO, methyl thioglycolate, titanium (IV) tetrachloride, tetrabutylammonium fluoride (1 M solution in THF), triphenylphosphine and zinc powder were obtained from Sigma Aldrich. DCM and THF were obtained from RCI Labscan Limited.[000209] NMR spectra were acquired on an Agilent MR400. The chemical shift data for each signal are given as 5. High-resolution mass spectra were acquired using a Thermo Scientific Q Exactive Plus Orbitrap LC-MS / MS instrument.[000210] Absorbance spectra were obtained on a Cary UV-Vis Compact Peltier instrument. Fluorescence emission spectra were obtained on a Cary Eclipse Fluorescence Spectrophotometer.[00021 1] DFT calculations were carried out using B3LYP / 6-31g+(d,p) on Gaussian16 for all calculations. Visualisation of structures were done using Avogadro 1 .20 and GaussView 5.0. No negative frequencies for optimised structures were observed.[000212] X-ray diffraction intensity data for compounds TME and 4-E were collected on the MX1 and MX2 beamlines at the Australian Synchrotron respectively. The structures were solved by direct methods and difference Fourier synthesis. Thermal ellipsoid plots were generated using the program Mercury integrated within the WINGX suite of programs.Synthetic procedures and characterization data[000213] The synthesis of TME is shown in Scheme 1 below. In short, a palladium catalyzed Sonogashira coupling installed the protected alkyne, ethynyltrimethylsilane, on 4- bromobenzophenone. A McMurry coupling reaction formed the tetraphenylethylene (TPE) core, with EZZ isomers isolated as pure isomeric intermediates. Deprotection of trimethylsilane (TMS) group on alkyne by tetrabutylammonium fluoride (TBAF) occurred in excellent yields before installation of the cysteine specific maleimide group, giving our final product, TME in respectable yields. Model reaction to validate maleimide reactivity toward thiol was then carried out with methyl thioglycolate to give TME-S in good yield. Synthetic products were characterized by1H and13C NMR and high-resolution mass spectrometry. The pure isomers (TME and 4-E) were further confirmed by single-crystal X-ray crystallography. Characterization data are shown herein.Scheme 1. Synthesis scheme for TME and its isomer.Synthesis of 4-(ethynyltrimethylsilane)benzophenone (1):[000214] In a round bottom flask under constant N2flow was added Cui (0.31 g, 1 .62 mmol), Pd(PPh3)Cl2 (0.56 g, 0.80 mmol), PPh3, (0.63 mg, 2.40 mmol), 4-bromobenzophenone (5.20 g, 20.0 mmol) and dry THF: NEt3(200 mL, 1 :1 ) was added. The reaction vessel was purged with N2three times before being warmed to room temperature and ethynyltrimethylsilane (3.54 mL, 25.0 mmol) was added. The reaction mixture was then heated to 60 °C and stirred for 24 h. Upon cooling to room temperature, the reaction mixture was filtered, extracted with DCM (x3) and washed with water. The organic layer was collected and concentrated then dried over Na2SC>4. The crude product was purified by silica-gel chromatography (100% hexanes) to give a pale-brown solid in 87% yield (4.86 g).1H NMR (400 MHz, CDCI3) 5: 7.80 - 7.71 (m, 2H), 7.61 - 7.55 (m, 3H), 7.50 - 7.46 (m, 2H), 0.27 (s, 9H).13C NMR (CDCI3, 100MHz), 5: 195.89, 137.36, 136.95, 132.52, 131.76, 129.93, 129.87, 128.33, 127.31 , 104.05, 97.81 , -0.16. HRMS (ESI+): mlz 279.12006 [C HigOSi (M+H)+, calcd 279.1 1997], NMR of compound 1 are shown at Figure 19 (a-b).Synthesis of 4-(1 ,2-diphenyl-2-(4-((trimethylsilyl)ethynyl)phenyl)vinyl) aniline (2)[000215] In a two-necked flask under constant N2flow was added zinc powder (3.76 g, 57.4 mmol) and 80 mL of THF. After the mixture was cooled to -5 °C, TiCL (5.46 g, 28.7 mmol) was slowly added by a syringe. The mixture was then warmed to room temperature and stirred for 10 minutes before being heated to reflux. A solution of 4-aminobenzophenone (1.42 g, 7.18 mmol) and 1 (2.00 g, 7.18 mmol) in dry THF (70 mL) was then added slowly. After completeaddition, the reaction mixture was heated at reflux for 24 hr. After cooling to room temperature, the reaction mixture was quenched with 10% K2CO3 aqueous solution and then extracted with DCM and washed with water. The organic layer was collected and concentrated then dried over Na2SC>4. The crude mixture of geometric isomers was purified by (1 :1 hexanes : DCM) silica-gel chromatography to give the isomerically pure products, 2-E and 2-Z.[000216] Compound 2-E was obtained as a yellow solid in 12% yield (360 mg).1H NMR (400 MHz, CDCI3) 6: 7.23 (d, J = 8.2 Hz, 2H), 7.14 - 6.91 (m, 12H), 6.78 (d, J = 8.4 Hz, 2H), 6.42 (d, J = 8.4 Hz, 2H), 3.60 (s, 2H), 0.23 (s, 9H).13C NMR (100 MHz, CDCI3) 6: 145.05, 144.93, 143.88, 143.74, 141.72, 138.54, 133.59, 132.50, 131.42, 131.35, 131.26, 127.60, 127.51 , 126.39, 126.23, 120.39, 1 14.35, 105.46, 94.05, -0.02. HRMS (ESI+): m / z 444.21448 [C3iH30NSi (M+H)+, calcd 444.21420]. NMR of compound 2-E are shown at Figure 19 (c-d).[000217] Compound 2-Z was obtained as a yellow solid in 10% yield (330 mg).1H NMR (400 MHz, CDCI3) 6: 7.19 (d, J = 8.2 Hz, 2H), 7.15 - 7.00 (m, 10H), 6.94 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.41 (d, J = 8.3 Hz, 2H), 3.58 (s, 2H), 0.23 (s, 9H).13C NMR (100 MHz, CDCI3) 5: 144.93, 144.77, 143.92, 143.82, 141.69, 138.49, 133.68, 132.47, 131.43, 131.37, 131.29, 131.23, 127.72, 127.66, 126.48, 126.22, 120.37, 114.24, 105.39, 94.04, -0.04. HRMS (ESI+): m / z 444.21609 [CsiHsoNSi (M+H)+, calcd 444.21420]. NMR of compound 2-Z are shown at Figure 19 (e-f).Synthesis of (E)-4-(2-(4-ethynylphenyl)-1 ,2-diphenylvinyl)aniline (3-E):[000218] 3-E (300mg, 6.76 mmol) was added to 1 M TBAF in THF (10 mL) and stirred at room temperature for 24 h. The reaction mixture was then extracted with DCM (x3) and washed with water (x3). The organic layer was collected and concentrated then dried over Na2SC>4. The crude product was purified by silica-gel chromatography (20:1 hexanes : ethyl acetate) to give the pure products as a yellow solid in 80% yield (200 mg).1H NMR (400 MHz, CDCI3) 6: 7.20 (d, J = 8.1 Hz, 2H), 7.16 - 7.00 (m, 10H), 6.94 (d, J = 8.2 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H), 6.41 (d, J = 8.4 Hz, 2H), 3.58 (s, 2H), 3.02 (s, 1 H).13C NMR (100 MHz, CDCI3) 6 145.05, 144.96, 143.90, 143.77, 141.82, 138.40, 133.67, 132.48, 131.43, 131.39, 131.36, 131.34, 127.77, 127.68, 126.53, 126.26, 1 19.40, 114.26, 83.91. HRMS (ESI+): m / z 372.17475 [C28H22N (M+H)+, calcd 372.17468]. NMR of compound 3-E are shown at Figure 19 (g-h).Synthesis of (Z)-4-(2-(4-ethynylphenyl)-1 ,2-diphenylvinyl)aniline (3-2):[000219] 3-Zwas prepared following the same procedure of 3-E. the pure product was obtained as a yellow solid in 95% yield (240 mg).1H NMR (400 MHz, CDCI3) 6: 7.25 (d, J = 8.3 Hz, 2H), 7.14 - 6.91 (m, 12H), 6.79 (d, J = 8.4 Hz, 2H), 6.43 (d, J = 8.4 Hz, 2H), 3.61 (s, 2H), 3.04 (s, 1 H).13C NMR (100 MHz, CDCI3) 6: 145.19, 145.08, 143.84, 143.72, 141.85, 138.43, 133.53, 132.49, 131.51 , 131.41 , 131.31 , 127.64, 127.52, 126.42, 126.26, 1 19.41 , 1 14.38, 83.97. HRMS (ESI+): m / z 372.17469 [C28H22N (M+H)+, calcd 372.17468]. NMR of compound 3- Z are shown at Figure 19 (i-j).Synthesis of 1-(4-(2-(4-ethynylphenyl)-1 ,2-diphenylvinyl)phenyl)-1 H-pyrrole-2, 5-dione (4): [000220] Into a round bottom flask was added 3 (160 mg, 0.431 mmol), maleic anhydride (84.5 mg, 0.862 mmol) and THF (5 mL) then stirred at room temperature for 24 h. After solvent evaporation, anhydrous sodium acetate (1 10 mg, 1 .34 mmol) and acetic anhydride (10 mL) were added to the reaction mixture and stirred at 75 °C for 6 h. After cooling to room temperature, the reaction mixture was quenched with 20% Na2COs, extracted with DCM (x3) and washed with brine (x3). The organic layer was collected, concentrated, and dried over Na2SC>4. The crude product was purified by silica-gel chromatography (1 :1 hexanes : ethyl acetate) to give the pure product.[000221 ] 4-Ewas obtained as a yellow solid in 41 % yield (80 mg).1H NMR (400 MHz, CDCI3) 5: 7.22 (d, J = 8.2 Hz, 2H), 7.16 - 7.06 (m, 10H), 7.06 - 7.00 (m, 4H), 6.97 (d, J = 8.2 Hz, 2H), 6.81 (s, 2H), 3.03 (s, 1 H).13C NMR (100 MHz, CDCI3) 6: 169.36, 144.26, 142.93, 142.82, 140.87, 140.68, 134.14, 131.85, 131.47, 131.31 , 131.28, 131.22, 129.54, 127.94, 127.91 , 126.91 , 126.86, 124.82, 1 19.98, 83.73. HRMS (ESI+): m / z 452.16491 [C32H22NO2 (M+H)+, calcd 452.16451]. NMR of compound 4-E are shown at Figure 19 (k-l).[000222] 4-Z (TME) was obtained as a yellow solid in 51% yield (1 10 mg).1H NMR (400 MHz, CDCI3) 6: 7.25 (d, J = 6.4 Hz, 2H), 7.16 - 7.08 (m, 10H), 7.03 - 6.99 (m, 6H), 6.82 (s, 2H), 3.04 (s, 1 H).13C NMR (100 MHz, CDCI3) 6: 169.36, 144.05, 143.05, 142.72, 140.87, 140.69, 134.17, 131.86, 131.65, 131.31 , 131.27, 129.69, 127.76, 126.75, 124.97, 120.17, 83.71 , 77.40. HRMS (ESI+): m / z 452.16323 [C32H22NO2 (M+H)+, calcd 452.16451]. NMR of compound 4-Z are shown at Figure 19 (m-n).[000223] The reaction of TME with a model thiol is shown in Scheme 2 below.TME TME-S(95%)Scheme 2. Reaction of TME with model thiol.Synthesis of the thiol conjugate of TME (TME-S):[000224] TME (20.0 mg, 0.04 mmol), methyl thioglycolate (9.14 mg, 0.09 mmol) and DCM (1 mL) were added together and the reaction mixture was stirred for 24 h at room temperature. The residual solvent was evaporated before purification by silica-gel chromatography (1 :1 Hexanes : CHCI3) to afford the product as a pale-yellow solid in 95% (23.0 mg).1H NMR (400 MHz, CDCI3) 6: 7.25 (d, J = 7.8 Hz, 2H), 7.16 - 7.05 (m, 10H), 7.02 - 6.98 (m, 6H), 4.17 - 4.14 (m, 1 H), 3.95 (d, = 15.9 Hz, 1 H), 3.76 (s, 3H), 3.42 (d, = 15.9 Hz, 1 H), 3.32 - 3.25 (m, 1 H), 3.04 (s, 1 H), 2.69 - 2.63 (m, 1 H).13C NMR (100 MHz, CDCI3) 6: 175.03, 173.14, 170.02,143.94, 143.71 , 142.99, 142.96, 141.10, 140.55, 131.88, 131.67, 131.49, 131.32, 131.26,129.82, 127.96, 127.77, 126.79, 125.53, 120.25, 83.69, 77.45, 52.72, 38.35, 35.37, 32.87. MS(ESI+): m / z 558.17436 [C35H28NO4S (M+H)+, calcd 558.17336], NMR of compound TME-S are shown at Figure 19 (o-p).Crystal data and structure refinement[000225] Crystal data and structure refinement for TME.Identification code TMEEmpirical formula C32 H21 N 02Formula weight 451.50Temperature 100(2) KWavelength 0.71073 ACrystal system MonoclinicSpace group P 21 / cUnit cell dimensions a = 22.856(5) A a = 90°. b = 9.879(2) A P = 95.61(3)°. c = 20.731 (4) A y = 90°.Volume 4658.5(16) A3Z 8Density (calculated) 1 .287 Mg / m3Absorption coefficient 0.080 mm-1F(000) 1888Crystal size 0.10 x 0.05 x 0.05 mm3Theta range for data collection 0.895 to 31.568°.Index ranges -30<=h<=30, -13<=k<=13, -25<=l<=25Reflections collected 83249Independent reflections 12550 [R(int) = 0.0901]Completeness to theta = 25.242' 97.1 %Refinement method Full-matrix least-squares on F2Data / restraints / parameters 12550 / 0 / 632Goodness-of-fit on F21.039Final R indices [l>2sigma(l)] R1 = 0.0556, wR2 = 0.1219R indices (all data) R1 = 0.0994, wR2 = 0.1444Extinction coefficient 0.0200(8)Largest diff. peak and hole 0.340 and -0.269 e.A’3[000226] Crystal data and structure refinement for 4-E.Identification code 4-EEmpirical formula C32 H21 N 02Formula weight 451.50Temperature 100(2) KWavelength 0.82566 ACrystal system OrthorhombicSpace group P 21 21 2Unit cell dimensions a = 16.697(3) A a = 90°. b = 26.199(5) A = 90°. c = 5.5980(11) A y = 90°.Volume 2448.8(8) A3Z 4Density (calculated) 1.225 Mg / m2Absorption coefficient 0.105 mm’1F(000) 944Crystal size 0.1 x 0.01 x 0.01 mm2Theta range for data collection 1.680 to 32.063°.Index ranges -19<=h<=19, -33<=k<=33, -6<=l<=6Reflections collected 27094Independent reflections 4872 [R(int) = 0.1162]Completeness to theta = 29.696' 98.5 %Absorption correction NONERefinement method Full-matrix least-squares on F2Data / restraints / parameters 4872 / 0 / 317Goodness-of-fit on F21.105Final R indices [l>2sigma(l)] R1 = 0.0634, wR2 = 0.1784 R indices (all data) R1 = 0.0684, wR2 = 0.1872Absolute structure parameter 0.6(6)Extinction coefficient 0.066(9)Largest diff. peak and hole 0.464 and -0.293 e.A’3[000227] The synthesis of example probe compound 11 is shown in Scheme 3 below.Scheme 3: Synthesis of probe compound 112-(Phenylamino)ethan-1-ol (5)[000228] To a solution of aniline (908 pL, 10.0 mmol, 1.00 eq), 2-chloroethanol (2.00 mL, 30.0 mmol, 3.00 eq) and potassium iodide (336 mg, 2.00 mmol 0.20 eq) in a mixture of MeCN (30.0 mL) and H2O (20.0 mL) was added sodium carbonate (2.00 g, 20.0 mmol, 4.00 eq) and the solution refluxed for 16 h. Upon completion, the reaction was cooled to room temperature and extracted into ethyl acetate (2 x 50.0 mL). The combined organic phases were dried over anhydrous Na2SO4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (10-60% EtOAc in petrol) to give the title compound (5) as a yellow oil (516 mg, 38%).2-(Phenyl(prop-2-yn-1-yl)amino)ethan-1-ol (6)[000229] To a solution of 2-(phenylamino)ethan-1 -ol (5) (574 mg, 4.19 mmol, 1.00 eq) and proparyl bromide (796 mL, 8.38 mmol, 2.00 eq) in DMSO (10.0 mL) was added sodium hydrogen carbonate (704 mg, 8.38 mmol, 2.00 eq) and the solution stirred at room temperature for 16 h. Upon completion, the reaction was diluted with toluene (20.0 mL) and washed sequentially with brine (2 x 20.0 mL) and H2O (2 x 20.0 mL). The organic phase was dried over anhydrous Na2SC>4, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography (20-50% EtOAc in petrol) to give the title compound (6) as a brown oil (522 mg, 71%).1H NMR (400 MHz, CDCI3) 6 7.31 - 7.25 (m, 2H), 6.95 (d, = 8.1 Hz, 2H), 6.85 (t, J = 7.3 Hz, 1 H), 4.08 (d, J = 2.4 Hz, 2H), 3.84 (t, J = 5.4 Hz, 2H), 3.56 (t, J = 5.4 Hz, 2H), 2.25 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, CDCI3) 6 148.1 , 129.4, 119.2, 114.9, 79.9, 72.7, 60.2, 54.1 , 41.2. HRMS (ESI+): calculated for CuHuNO [M+H+]: m / z = 176.1070, m / z found 176.1075.2-(Phenyl(prop-2-yn-1-yl)amino)ethyl acetate (7)[000230] To a solution of 2-(phenyl(prop-2-yn-1 -yl)amino)ethan-1 -ol (6) (522 mg, 2.98 mmol, 1 .00 eq) and triethylamine (622 mL, 4.49 mmol, 1 .50 eq) in CH2CI2(9.00 mL) was cooled to 0 °C and acetyl chloride (317 pL, 4.49 mmol, 1.50 eq) added dropwise. The resultant solution was warmed to room temperature, stirred for 16 h, diluted with CH2CI2(20.0 mL) and washed with H2O (2 x 20 mL). The organic phase was dried over anhydrous Na2SC>4, filtered and concentrated under pressure to give the title compound (7) as a yellow oil (51 1 mg, 79%).1H NMR (400 MHz, CDCI3) 6 7.31 - 7.24 (m, 2H), 6.89 (d, J = 8.0 Hz, 2H), 6.82 (t, J = 7.3 Hz, 1 H), 4.30 (t, J = 6.1 Hz, 2H), 4.07 (d, J = 2.4 Hz, 2H), 3.65 (t, J = 6.1 Hz, 2H), 2.23 (t, J = 2.4 Hz, 1 H), 2.05 (s, 3H).13C NMR (101 MHz, CDCI3) 6 171.1 , 147.8, 129.4, 118.6, 114.0, 79.8, 72.4, 61.9, 50.0, 40.6, 21.0. HRMS (ESI+): calculated for Ci3Hi5NO2Na [M+H+]: m / z =240.0995, m / z found 240.0997.4-((2-Hydroxyethyl)(prop-2-yn-1-yl)amino)benzaldehyde (8)[000231 ] A solution of 2-(phenyl(prop-2-yn-1 -yl)amino)ethan-1 -ol (7) (550 mg, 2.53 mmol, 1.00 eq) in anhydrous DMF (1.00 mL) was cooled to 0 °C and POCI3 (355 pL, 3.80 mmol, 1.50 eq) added dropwise. The resultant solution was heated to 40 °C and stirred for 4 h. Upon consumption of the starting material (monitored by TLC), the reaction was cooled to room temperature, and diluted with sat. NaOAC(aq) (5.00 mL) and stirred at room temperature for 16 h. The resultant solution was diluted with EtOAc (15.0 mL) and washed sequentially with brine (2 x 15.0 mL) and H2O (15.0 mL). The organic phase was dried over anhydrous Na2SC>4, concentrated under reduced pressure to give title compound (8) as a yellow oil (213 mg, 34%). HRMS (ESI+): calculated for C11H14NO [M+H+]: m / z = 204.1020, m / z found 204.1024.(Z)-3-(4-((2-Hydroxyethyl)(prop-2-yn-1-yl)amino)phenyl)-2-(4-nitrophenyl)acrylonitrile (9)[000232] To a solution of 4-((2-hydroxyethyl)(prop-2-yn-1-yl)amino)benzaldehyde (8) (404 mg, 2.00 mmol, 1.00 eq) and 2-(4-nitrophenyl)acetonitrile (324 mg, 2.00 mmol, 1.00 eq) in ethanol (5.00 mL) was added piperidine (394 mL, 4.00 mmol, 2.00 eq) and the solution refluxed for 24 h. The reaction was cooled to 0 °C, the resultant precipitate collected by filtration and dried under vacuum to give the title compound (9) as a red solid (575 mg, 83%).1H NMR (400 MHz, CDCI3) 5 8.30 - 8.25 (m, 2H), 7.97 - 7.93 (m, 2H), 7.81 - 7.77 (m, 2H), 7.56 (s, 1 H), 6.98 - 6.92 (m, 2H), 4.22 (d, J = 2.4 Hz, 2H), 3.93 (t, J = 5.4 Hz, 2H), 3.70 (t, J = 5.4 Hz, 2H), 2.33 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, CDCI3) 6 150.3, 147.3, 145.2, 141.8, 132.3, 126.1 , 124.5, 122.9, 118.5, 1 13.4, 103.7, 79.0, 73.2, 60.4, 53.8, 41 .3. HRMS (ESI+): calculated for C2oHi7N303Na [M+H+]: m / z = 370.1 163, m / z found 370.1 156.(Z)-2-(4-Aminophenyl)-3-(4-((2-hydroxyethyl)(prop-2-yn-1-yl)amino)phenyl)acrylonitrile(10)[000233] To a solution of (Z)-3-(4-((2-hydroxyethyl)(prop-2-yn-1 -yl)amino)phenyl)-2-(4- nitrophenyl)acrylonitrile (9) (500 mg, 1 .44 mmol, 1 .00 eq) was added SnCl2 (684 mg, 3.60 mmol, 2.50 eq) and the solution refluxed for 16 h. The reaction mixture was cooled to room temperature, filtered through a pad of Celite® and concentrated under reduced pressure. The crude residue was redissolved in CH2CI2 (20.0 mL) and washed with sat. NaHCO3(aq> (20.0 mL). The organic phase was dried over anhydrous Na2SC>4 and concentrated under reduced pressure to give title compound (10) as an orange solid (394 mg, 86%).1H NMR (400 MHz, DMSO-G ) 5 7.79 - 7.73 (m, 2H), 7.49 (s, 1 H), 7.39 - 7.32 (m, 2H), 6.91 - 6.85 (m, 2H), 6.67 - 6.58 (m, 2H), 5.45 (s, 1 H), 4.76 (s, 1 H), 4.21 (d, J = 2.1 Hz, 1 H), 3.67 - 3.56 (m, 1 H), 3.48 (t, J = 6.2 Hz, 1 H), 3.16 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO-cfe) 5 149.2, 148.8, 137.3, 130.1 , 126.1 , 122.6, 121.8, 119.4, 1 14.0, 112.4, 104.7, 80.5, 74.2, 58.3, 52.7. HRMS (ESI+): calculated for C20H20N3O [M+H+]: m / z = 318.1601 , m / z found 318.1596.(Z)-2-((4-(2-Cyano-2-(4-(2,5-dioxo-2,5-dihydro-1 / +pyrrol-1-yl)phenyl)vinyl)phenyl)(prop-2- yn-1-yl)amino)ethyl acetate (11)[000234] To a solution of (Z)-2-(4-aminophenyl)-3-(4-((2-hydroxyethyl)(prop-2-yn-1 - yl)amino)phenyl)acrylonitrile (10) (317 mg, 1.00 mmol, 1.00 eq) in THF was added maleic anhydride (196 mg, 2.00 mmol, 2.00 eq) and the solution stirred at room temperature for 16 h then concentrated under reduced pressure. The residue was redissolved in AC2O (5.00 mL) followed by the addition of NaOAc (205 mg, 2.50 mmol, 2.50 eq). The reaction mixture was then stirred at 75 °C for 7.5 h, neutralised with sat. NaHCO3(aq.) and extracted into CH2CI2 (3 x 15.0 mL). The organic phases were combined, dried over Na2SO4, filtered and concentrated under reduced pressure. The crude residue was purified using flash column chromatography (20-60% EtOAc in petrol) to give the title compound (11 ) as an orange solid (200 mg, 46%).1H NMR (400 MHz, CDCI3) 6 7.93 - 7.86 (m, 2H), 7.76 - 7.71 (m, 2H), 7.48 - 7.40 (m, 3H), 6.92 - 6.89 (m, 2H), 6.87 (s, 2H), 4.33 (t, J = 6.1 Hz, 2H), 4.15 (d, J = 2.4 Hz, 2H), 3.74 (t, J = 6.1 Hz, 2H), 2.27 (t, J = 2.4 Hz, 1 H), 2.06 (s, 3H).13C NMR (101 MHz, CDCI3) 6 171.0, 169.4, 149.5, 142.9, 135.0, 134.5, 131.6, 131.3, 126.4, 126.3, 123.4, 1 18.9, 113.0, 105.3, 79.0, 72.8, 68.6, 61.6, 49.8, 40.5, 27.9, 22.3, 21.0. HRMS (ESI+): calculated for C2oHi7N303Na [M+H+]: m / z = 462.1425, m / z found 462.1426. UV and photoluminescence data for compound 11 is shown at Figure 20 (a, h).[000235] The synthesis of example probe compound MI-SLQUI is shown in Scheme 4 below.Scheme 4: Synthesis of probe compound MI-SLQUINO2-SLQUI[000236] 8-Nitroquinaldine (848 mg, 4.48 mmol), 4-(Diethylamino)-2-(2-propyn-1 - yloxy)benzaldehyde (1.24 g, 5.38 mmol), Fe(OAc)2 (39.0 mg, 0.22 mmol) and trifluoroacetic acid (34.3 pL, 0.45 mmol) was suspended in toluene (7.65 mL). The reaction mixture was then stirred at 105 °C. Upon completion of the reaction, the reaction mixture was cooled to room temperature, and poured into saturated aqueous NaHCOs (30 mL), extracted with ethyl acetate (3 x, 30 mL). The organic phase was then washed with saturated aqueous NaHCOs (3 x, 50 mL) and brine (1 x, 50 mL). The organic phase was then collected and dried over MgSC , filtered through a short pad of silica, with an ethyl acetate elution. The filtered material was then concentrated, subjected to column chromatography (n-hexane: ethyl acetate: triethylamine, 99:0:1 to 79:20:1) to obtain the pure product as a red solid in 18% yield (320 mg).1H NMR (400 MHz, DMSO) 5 8.40 (d, J =8.7 Hz, 1 H), 8.15 (d, J = 7.8 Hz, 2H), 7.94 (d, J = 16.2 Hz, 1 H), 7.85 (d, J = 8.8 Hz, 1 H), 7.61 - 7.56 (m, 2H), 7.15 (d, J = 16.2 Hz, 1 H), 6.37 - 6.36 (m, 2H), 4.95 (d, J = 2.1 Hz, 2H), 3.62 (t, J = 2.2 Hz, 1 H), 3.40 (dd, J = 13.9, 6.9 Hz, 4H), 1.14 (t, J = 6.9 Hz, 6H).13C NMR (101 MHz, DMSO) 5 158.97, 157.14, 149.63, 147.38, 138.71 , 136.40, 131.81 , 131.57, 129.19, 127.35, 124.14, 123.38, 122.17, 121.50, 1 11.85, 105.04, 95.89, 79.39, 78.41 , 55.72, 43.97, 12.64.NH2-SLQUI & MI-SLQUI[000237] NO2-SLQUI (120 mg, 0.30 mmol), ammonium chloride (160 mg, 2.99 mmol) and iron powder (167 mg, 2.99 mmol) was suspended in EtOH (4.24 mL) and H2O (1 .25 mL). The reaction mixture was then stirred and heated at 85 °C for 5 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before the pH was adjusted by addition of 1 M NaOH (30 mL). The reaction mixture was then extracted with diethyl ether (3 x, 30 mL). The organic phase was then washed with 1 M NaOH (3 x, 50 mL) and brine (1 x, 50 mL). The organic phase was then collected and dried over MgSO4, filtered through a short pad of silica, with a diethyl ether elution wash. The filtered material was then concentrated to obtain the product (NH2-SLQUI) as a yellow solid that was used without further purification.[000238] NH2-SLQUI from the previous step was then added with and maleic anhydride (59.0 mg, 0.60 mmol) was suspended in THE (5 mL). The reaction mixture was then stirred at room temperature overnight. Upon completion of the reaction, the reaction mixture was concentrated. Sodium acetate (159 mg, 1.94 mmol) and acetic anhydride (1.59 mL) was then added to the reaction mixture and heated at 75 °C for 6 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before being poured into saturated aqueous Na2COs (50 mL), extracted with diethyl ether (3 x, 15 mL). The organic phase was then washed with saturated aqueous Na2COs (3 x, 15 mL) and brine (1 x, 15 mL). The organic phase was then collected and dried over MgSC , filtered through a short pad of celite, with diethyl ether elution. The filtered material was then concentrated, to obtain the pure product (MI-SLQUI) as an orange solid in 16% yield (21 mg).1H NMR (400 MHz, DMSO) 5 8.33 (d, J = 8.6 Hz, 1 H), 8.01 (dd, J = 8.2, 1.1 Hz, 1 H), 7.88 (d, J = 16.1 Hz, 1 H), 7.72 (dd, J = 7.3, 1 .2 Hz, 1 H), 7.64 (d, J = 8.6 Hz, 1 H), 7.59 - 7.51 (m, 2H), 7.35 (s, 2H), 7.05 (d, J = 16.1 Hz, 1 H), 6.37 - 6.34 (m, 2H), 4.92 (d, J = 2.2 Hz, 2H), 3.65 (t, J = 2.2 Hz, 1 H), 3.42 - 3.39 (m, 4H), 1.14 (t, J = 7.0 Hz, 6H).13C NMR (101 MHz, DMSO) 5 171.00, 156.96, 149.38, 143.73, 136.53, 135.19, 130.78, 130.09, 129.05, 128.99, 128.61 , 127.30, 124.76, 122.00, 121 .38, 1 11 .98, 105.03, 95.94, 79.51 , 78.31 , 55.78, 43.94, 12.64.[000239] The synthesis of example probe compounds MI-DPAN and CI-DPAN is shown in Scheme 5 below.Scheme 5: Synthesis of probe compounds MI-DPAN and CI-DPANNO2-DPAN[000240] 4-Nitrophenylacetonitrile (1.00 g, 6.17 mmol) and 4-(propargyloxy)benzaldehyde (1.09 g, 6.79 mmol) was suspended in EtOH (20 mL). Piperidine (4 drops) were then added to the reaction mixture and then stirred at reflux for 4 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before precipitation in 1 M HCI (approx. 100 mL). The solid precipitate was then washed with 1 M HCI (3 x, 10 mL) followed by water (3 x, 20 mL) and dried to obtain the pure product as an orange solid in 90% yield (1.68 g).1H NMR (400 MHz, DMSO) 5 8.34 (d, J = 9.0 Hz, 2H), 8.23 (s, 1 H), 8.02 (t, J = 9.0 Hz, 4H), 7.19 (d, J = 8.9 Hz, 2H), 4.92 (d, J = 2.3 Hz, 2H), 3.63 (t, J = 2.3 Hz, 1 H). 13C NMR (101 MHz, DMSO) 5 159.76, 147.07, 145.91 , 140.51 , 131.72, 126.64, 126.45, 124.33, 1 17.76, 115.49, 105.52, 78.74, 78.70, 55.77.NH2-DPAN[000241 ] NO2-DPAN (980 mg, 3.22 mmol) and iron powder (967 mg, 16.1 1 mmol) was suspended in EtOH (6.44 mL), THF (6.44 mL) and H2O (644 pL). Acetic acid (967 pL, 16.1 1 mmol) was then added to the reaction mixture. The reaction mixture was then stirred and heated at 85eC for 5 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before the pH was adjusted by addition of saturated aqueous Na2COs (100 mL). The reaction mixture was then extracted with ethyl acetate (3 x, 25 mL). The organic phase was then washed with Na2COs (2 x, 25 mL) and brine (1 x, 25 mL). The organic phase was then collected and dried over MgSC , filtered through a short pad of silica, with an ethyl acetate elution wash (3 x, 10 mL). The filtered material was then concentrated to obtain the product as a yellow solid in 92% yield (814 mg) that was used without further purification.MI-DPAN[000242] NH2-DPAN (200 mg, 0.729 mmol) and maleic anhydride (143 mg, 1.46 mmol) was suspended in THF (4 mL). The reaction mixture was then stirred at room temperature overnight. Upon completion of the reaction, the reaction mixture was concentrated. Sodium acetate (365 mg, 4.45 mmol) and acetic anhydride (3.65 mL) was then added to the reaction mixture and heated at 80eC for 6 h. Upon completion of the reaction, the reaction mixture was cooled to roomtemperature before being poured into saturated aqueous Na2COs (50 mL), extracted with ethyl acetate (3 x, 15 mL). The organic phase was then washed with saturated aqueous Na2COs (3 x, 15 mL) and brine (1 x, 15 mL). The organic phase was then collected and dried over MgSC>4, filtered through a short pad of silica, with an ethyl acetate elution was (3 x, 10 mL). The filtered material was then concentrated to obtain the pure product as a yellow solid in 39% yield (101 mg).1H NMR (400 MHz, DMSO) 5 8.01 (s, 1 H), 7.97 (d, J = 8.9 Hz, 2H), 7.84 (d, J = 8.7 Hz, 2H), 7.49 (d, J = 8.7 Hz, 2H), 7.21 (s, 2H), 7.17 (d, J = 8.9 Hz, 2H), 4.91 (d, J = 2.3 Hz, 2H), 3.62 (t, J = 2.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.74, 159.12, 143.01 , 134.81 , 133.32, 131.94, 131.11 , 127.09, 126.84, 126.05, 118.18, 115.37, 106.81 , 78.80, 78.65, 55.69.CI-DPAN[000243] NH2-DPAN (200 mg, 0.729 mmol) and triethylamine (1.19 mL, 0.856 mmol) was suspended in DCM (20 mL). Chloroacetyl chloride (624 pL, 0.785 mmol) in DCM (10 mL) was then added dropwise at 0eC and the reaction mixture was then stirred at room temperature overnight. Upon completion of the reaction, the reaction mixture was concentrated and resuspended in ethyl acetate (50 mL) and washed with 1 M aqueous HCI (50 mL x 3) followed by brine (1 x, 50 mL). The organic phase was then collected and dried over MgSC , filtered through a short pad of silica, with an ethyl acetate elution was (3 x, 20 mL). The filtered material was then concentrated to obtain the pure product as a yellow solid in 41% yield (105 mg).1H NMR (400 MHz, DMSO) 5 10.50 (s, 1 H), 7.94 - 7.90 (m, 3H), 7.71 (s, 4H), 7.15 (d, J = 8.8 Hz, 2H), 4.90 (d, J = 2.2 Hz, 2H), 4.28 (s, 2H), 3.62 (t, J = 2.1 Hz, 1 H). 13C NMR (101 MHz, DMSO) 5 164.86, 158.85, 141.11 , 139.05, 130.86, 129.37, 127.05, 126.19, 119.67, 118.28, 1 15.31 , 107.25, 78.84, 78.62, 55.66, 43.57.[000244] The synthesis of example probe compound MI-SLDPAN is shown in Scheme 6 below.Scheme 6: Synthesis of probe compound MI-SLDPANNO2-SLDPAN[000245] 4-Nitrophenylacetonitrile (770 mg, 4.75 mmol) and 4-(diethylamino)-2-(2-propyn-1 - yloxy)benzaldehyde (1.00 g, 4.32 mmol) was suspended in EtOH (20 mL). Piperidine (4 drops) were then added to the reaction mixture and then stirred at reflux overnight. Upon completion of the reaction, the reaction mixture was cooled to room temperature before precipitation in 1 M NaOH (approx. 200 mL). The solid precipitate was then washed with 1 M NaOH (3 x, 50 mL) followed by water (3 x, 50 mL) and dried to obtain the pure product as a dark solid in 90% yield (1.46 g).1H NMR (400 MHz, DMSO) 5 8.27 (d, J = 8.9 Hz, 2H), 8.15 (d, J = 9.1 Hz, 1 H), 8.06 (s, 1 H), 7.80 (d, J = 8.9 Hz, 2H), 6.51 (dd, J = 9.1 , 1 .9 Hz, 1 H), 6.37 (s, 1 H), 4.98 (d, J = 2.0 Hz, 2H),3.66 (t, J = 2.0 Hz, 1 H), 3.47 (dd, J = 13.7, 6.7 Hz, 4H), 1.16 (t, J = 6.9 Hz, 6H).13C NMR (101 MHz, DMSO) 5 158.56, 151.86, 145.98, 142.08, 139.17, 129.08, 125.46, 124.42, 119.05, 109.49, 105.21 , 98.82, 95.25, 78.98, 78.82, 56.14, 44.20, 12.60.NH2-SLDPAN[000246] NO2-SLDPAN (400 mg, 1.06 mmol) and iron powder (595 mg, 10.66 mmol) was suspended in EtOH (3.22 mL), THE (3.22 mL) and H2O (644 pL). Acetic acid (575 pL, 10.66 mmol) was then added to the reaction mixture. The reaction mixture was then stirred and heated at 85eC for 5 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before the pH was adjusted by addition of 1 M NaOH (30 mL). The reaction mixture was then extracted with diethyl ether (3 x, 30 mL). The organic phase was then washed with 1 M NaOH (3 x, 50 mL) and brine (1 x, 50 mL). The organic phase was then collected and dried over MgSO4, filtered through a short pad of silica, with a diethyl ether elution wash. The filtered material was then concentrated to obtain the product as an orange solid in 72% yield (263 mg) that was used without further purification.MI-SLDPAN[000247] NH2-SLDPAN (100 mg, 0.292 mmol) and maleic anhydride (57.3 mg, 0.584 mmol) was suspended in THE (4 mL). The reaction mixture was then stirred at room temperature overnight. Upon completion of the reaction, the reaction mixture was concentrated. Sodium acetate (146 mg, 1.78 mmol) and acetic anhydride (1.46 mL) was then added to the reaction mixture and heated at 90eC for 6 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before being poured into saturated aqueous Na2COs (50 mL), extracted with ethyl acetate (3 x, 15 mL). The organic phase was then washed with saturated aqueous Na2COs (3 x, 15 mL) and brine (1 x, 15 mL). The organic phase was then collected and dried over MgSC>4, filtered through a short pad of silica, with an ethyl acetate elution. The filtered material was then concentrated, subjected to column chromatography (n-hexane: ethyl acetate, 100:0 to 7:3) to obtain the pure product as an orange solid in 41% yield (51 mg).1H NMR (400 MHz, DMSO) 5 8.08 (d, J = 9.0 Hz, 1 H), 7.89 (s, 1 H), 7.68 (d, J = 8.6 Hz, 2H), 7.43 (d, J = 8.7 Hz, 2H), 7.20 (s, 2H), 6.49 (dd, J = 9.1 , 2.0 Hz, 1 H), 6.38 (d, J = 2.0 Hz, 1 H), 4.95 (d, J = 2.1 Hz, 2H), 3.63 (t, J = 2.2 Hz, 1 H), 3.45 (dd, J = 13.8, 6.8 Hz, 4H), 1.16 (t, J = 6.9 Hz, 6H).13C NMR (101 MHz, DMSO) 5 169.79, 157.94, 151.06, 137.13, 134.77, 134.67, 130.99, 128.67, 127.18, 125.35, 119.35, 109.72, 104.85, 101 .07, 95.49, 79.12, 78.68, 56.11 , 44.09, 12.60.[000248] The synthesis of example probe compound PT_NO12 is shown in Scheme 7 below.Scheme 7: Synthesis of probe compound PT_NO12PT_N003[000249] 4-Nitrophenylacetonitrile (1.00 g, 6.17 mmol) and vanillin (0.938 g, 6.79 mmol) were suspended in EtOH (20 mL). Piperidine (5 drops) were then added to the reaction mixture and then stirred at 80 °C for 16 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before precipitation in 1 M HCI(aq). The solid precipitate was then washed with 1 M HCI and water to obtain the product as an orange solid in 96% yield (1 .76 g).1H NMR (400 MHz, DMSO) 5 10.13 (s, 1 H), 8.34 - 8.24 (m, 2H), 8.1 1 (s, 1 H), 7.98 - 7.88 (m, 2H), 7.71 (d, J = 2.1 Hz, 1 H), 7.52 (dd, J = 8.4, 2.1 Hz, 1 H), 6.94 (d, J = 8.3 Hz, 1 H), 3.83 (s, 3H).13C NMR (101 MHz, DMSO) 5 150.69, 147.63, 146.70, 146.43, 140.80, 126.20, 125.14, 124.60, 124.24, 1 18.11 , 1 15.85, 1 12.98, 103.46, 55.51.PT_N006[000250] PT_N003 (1.50 g, 5.06 mmol) and K2CO3 (0.770 g, 5.57 mmol) were suspended in acetone (20 mL). A solution of 80 wt% propargyl bromide in toluene (0.60 mL, 5.57 mmol) was added to the reaction mixture and then stirred at 65 °C for 16 h. The solvent was then removed by rotary evaporation, and the crude solid resuspended in water and petroleum spirits. The suspension was filtered, washed with 1 M Na2COs(aq), water and petroleum spirits, and the product collected from the filter as a microcrystalline yellow solid in 99% yield (1 .68 g).1H NMR (400 MHz, DMSO) 5 8.33 (d, J = 8.5 Hz, 2H), 8.21 (s, 1 H), 7.99 (d, J = 8.6 Hz, 2H), 7.75 (d, J = 2.1 Hz, 1 H), 7.63 (dd, J = 8.5, 2.1 Hz, 1 H), 7.22 (d, J = 8.5 Hz, 1 H), 4.92 (d, J = 2.4 Hz, 2H), 3.84 (s, 3H), 3.63 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.42, 148.88, 147.00, 146.12, 140.45, 126.66, 126.52, 124.30, 124.04, 1 17.82, 1 13.49, 112.51 , 105.43, 78.78, 78.69, 56.06, 55.50.PT_N009[000251 ] PT_N006 (1 .40 g, 4.19 mmol) and iron powder (1 .17 g, 20.9 mmol) were suspended in EtOH (10 mL), THE (10 mL) and H2O (1 mL). Glacial acetic acid (1.20 mL, 20.9 mmol) was thenadded to the reaction mixture. The reaction mixture was then stirred and heated at 80 °C for 24 h. After cooling to room temperature, the reaction mixture was filtered and washed with EtOH and THF. The filtrate was collected and the solvent evaporated. The resulting oil was redissolved in 30 mL MeOH and poured into an aqueous solution of trisodium citrate dihydrate (12.3 g, 41.9 mmol), causing a yellow solid to precipitate. The mixture was filtered and washed with water, and the product collected from the filter as a yellow solid in 94% yield (1.20 g).1H NMR (400 MHz, DMSO) 5 7.61 (s, 1 H), 7.59 (d, J= 2.1 Hz, 1 H), 7.44 (dd, J= 8.7, 2.1 Hz, 1 H), 7.41 (d, J= 8.3 Hz, 2H), 7.13 (d, J = 8.5 Hz, 1 H), 6.65 (d, J = 8.3 Hz, 2H), 5.55 (s, 2H), 4.86 (d, J = 2.4 Hz, 2H), 3.82 (s, 3H), 3.59 (t, J = 2.0 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.80, 148.86, 147.66, 136.68, 128.06, 126.50, 122.02, 121.05, 118.75, 1 13.89, 1 13.68, 11 1.93, 108.54, 78.97, 78.54, 56.00, 55.46.PT N012[000252] PT_N009 (300 mg, 0.99 mmol) and maleic anhydride (97 mg, 0.99 mmol) were dissolved in THF (5 mL). The reaction mixture was then stirred at room temperature for 2 h. Then, 5 mL of petroleum spirits was added to precipitate the solid amic acid intermediate, and the mixture was filtered and washed with petroleum spirits. The solid on the filter was collected, mixed with sodium acetate (485 mg, 5.91 mmol) and acetic anhydride (5 mL), and the mixture was heated at 80 °C for 6 h. The reaction mixture was then cooled to room temperature before adding 15 mL of Et20. The resulting suspension was filtered, washed with Et20 and water, and the product collected from the filter as a yellow solid in 59% yield (222 mg).1H NMR (400 MHz, DMSO) 5 8.01 (s, 1 H), 7.84 (d, J= 8.6 Hz, 2H), 7.71 (d, J= 2.1 Hz, 1 H), 7.57 (dd, J= 8.5, 2.1 Hz, 1 H), 7.49 (d, J = 8.6 Hz, 2H), 7.27 - 7.15 (m, 3H), 4.91 (d, J = 2.5 Hz, 2H), 3.84 (s, 3H), 3.62 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.70, 148.88, 148.72, 143.26, 134.77, 133.27, 131.92, 127.1 1 , 127.05, 125.97, 123.20, 1 18.25, 1 13.56, 112.27, 106.79, 78.81 , 78.68, 56.03,55.50. NMR of compound PT_N012 are shown at Figure 19 (q-r).[000253] The synthesis of example probe compound PT_NO22 is shown in Scheme 8 below.Scheme 8: Synthesis of probe compound PT_NO22PT_N004[000254] 4-Nitrophenylacetonitrile (1.00 g, 6.17 mmol) and 3,4-dihydroxybenzaldehyde (0.938 g, 6.79 mmol) were suspended in EtOH (20 mL). Piperidine (5 drops) were then added to the reaction mixture and then stirred at 80 °C for 16 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature before precipitation in 1 M HCI(aq). The solid precipitate was then washed with 1 M HCI(aq) and water to obtain the product as an orange solid in 85% yield (1 .48 g).1H NMR (400 MHz, DMSO) 5 9.76 (br s, 2H), 8.35 - 8.23 (m, 2H), 8.02 (s, 1 H), 7.94 (d, J = 8.9 Hz, 2H), 7.61 (d, J = 2.2 Hz, 1 H), 7.36 (dd, J = 8.4, 2.2 Hz, 1 H), 6.89 (d, J = 8.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.91 , 146.66, 146.57, 145.59, 141.02, 126.26, 124.74, 124.35, 124.22, 1 18.02, 1 15.97, 115.86, 103.10.PT_N007[000255] PT_N004 (1.20 g, 4.25 mmol) and K2CO3 (1.29 g, 9.35 mmol) were suspended in acetone (20 mL). A solution of 80 wt% propargyl bromide in toluene (1.00 mL, 9.35 mmol) was added to the reaction mixture and then stirred at 65 °C for 16 h. The solvent was then removed by rotary evaporation, and the crude solid resuspended in water and petroleum spirits. The suspension was filtered, washed with Na2COs(aq) 1 M, water and petroleum spirits, and the product collected from the filter as a dark green powder in 98% yield (1 .50 g).1H NMR (400 MHz, DMSO) 5 8.38 - 8.28 (m, 2H), 8.20 (s, 1 H), 8.00 (d, J = 8.9 Hz, 2H), 7.79 (d, J = 2.1 Hz, 1 H), 7.71 (dd, J = 8.6, 2.1 Hz, 1 H), 7.26 (d, J = 8.6 Hz, 1 H), 4.94 (d, J = 2.4 Hz, 2H), 4.85 (d, J = 2.4 Hz, 2H), 3.65 (t, J = 2.2 Hz, 1 H), 3.61 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.87, 147.04, 146.60, 145.82, 140.39, 126.58, 126.47, 124.72, 124.29, 1 17.61 , 1 15.15, 113.85, 105.75, 78.89, 78.86, 78.60, 56.21 , 56.13.PT_N019[000256] PT_N007 (1.30 g, 3.63 mmol) and anhydrous tin(ll) chloride (4.13 g, 21.8 mmol) were suspended in MeOH (30 mL) and 32% HCI(aq) (3.6 mL). The reaction mixture was then stirred and heated at 70 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (21.3 g, 72.5 mmol), causing an orange solid to precipitate. The mixture was filtered and washed with water, and the product collected from the filter as an orange solid in 83% yield (0.99 g).1H NMR (400 MHz, DMSO) 5 7.62 (d, J = 2.1 Hz, 1 H), 7.59 (s, 1 H), 7.53 (dd, J = 8.6, 2.0 Hz, 1 H), 7.41 (d, J = 8.6 Hz, 2H), 7.18 (d, J = 8.6 Hz, 1 H), 6.65 (d, J = 8.6 Hz, 2H), 5.56 (s, 2H), 4.89 (d, J = 2.4 Hz, 2H), 4.82 (d, J = 2.4 Hz, 2H), 3.61 (t, J = 2.2 Hz, 1 H), 3.58 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.85, 148.13, 146.58, 136.31 , 127.87, 126.53, 122.79, 120.98, 118.54, 114.57, 113.98, 1 13.88, 108.85, 78.88, 78.80, 78.67, 56.19, 56.04.PT_N022[000257] PT_N019 (300 mg, 0.91 mmol) and maleic anhydride (90 mg, 0.91 mmol) were dissolved in THE (5 mL). The reaction mixture was then stirred at room temperature for 2 h. Then, 15 mL of petroleum spirits was added to precipitate the solid amic acid intermediate, and themixture was filtered and washed with petroleum spirits. The solid on the filter was collected, mixed with sodium acetate (450 mg, 5.48 mmol) and acetic anhydride (5 mL), and the mixture was heated at 80 °C for 5 h. The solvent was then removed in a rotary evaporator, redissolved in 5 mL THF, precipitated with 1 mL of petroleum spirits and then diluted with 50 mL Et20. The resulting suspension was filtered, washed with Et20 and water, and the product collected from the filter as a tan-orange solid in 53% yield (197 mg).1H NMR (400 MHz, DMSO) 5 8.01 (s, 1 H), 7.86 (d, J = 8.6 Hz, 2H), 7.76 (d, J = 2.1 Hz, 1 H), 7.67 (dd, J = 8.7, 2.1 Hz, 1 H), 7.51 (d, J = 8.6 Hz, 2H), 7.26 (d, J = 8.6 Hz, 1 H), 7.22 (s, 2H), 4.94 (d, J = 2.4 Hz, 2H), 4.86 (d, J = 2.4 Hz, 2H), 3.65 (t, J = 2.4 Hz, 1 H), 3.62 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.70, 149.21 , 146.61 , 142.96, 134.78, 133.22, 131.98, 127.06, 126.93, 126.03, 123.97, 1 18.04, 114.91 , 113.91 ,107.11 , 78.80, 78.72, 78.69, 56.23, 56.09. NMR of compound PT_N022 are shown at Figure 19 (s-t).[000258] The synthesis of example probe compound PT_NO24 is shown in Scheme 9 below.Scheme 9: Synthesis of probe compound PT_NO24PT N010[000259] 2,4-Dihydroxybenzaldehyde (1.00 g, 7.24 mmol) and K2CO3 (2.20 g, 15.9 mmol) were suspended in acetone (40 mL). A solution of 80 wt% propargyl bromide in toluene (1 .70 mL, 15.9 mmol) was added to the reaction mixture and then stirred at 60 °C for 24 h. The solvent was then removed by rotary evaporation, and the crude solid resuspended in water and petroleum spirits. The suspension was filtered, washed with Na2COs(aq) 1 M, water and petroleum spirits, and the product collected from the filter as a tan powder in 90% yield (1 .40 g).1H NMR (400 MHz, DMSO) 5 10.18 (s, 1 H), 7.71 (d, J = 8.7 Hz, 1 H), 6.85 (d, J = 2.3 Hz, 1 H), 6.77 (dd, J = 8.7, 2.2 Hz, 1 H), 4.99 (d, J = 2.4 Hz, 2H), 4.93 (d, J = 2.4 Hz, 2H), 3.64 (app. dt, J = 5.0, 2.4 Hz, 2H).13C NMR (101 MHz, DMSO) 5 187.17, 163.51 , 161.08, 129.85, 1 19.20, 107.85, 101.05, 79.14, 78.94, 78.40, 78.38, 56.57, 56.06.PT_N013[000260] PT_N010 (1.19 g, 5.55 mmol) and 4-nitrophenylacetonitrile (0.900 g, 5.55 mmol) were suspended in EtOH (30 mL). Piperidine (5 drops) were then added to the reaction mixture andthen stirred at 80 °C for 16 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and the solid rinsed with MeOH and petroleum spirits to obtain the product as a yellow powder in 80% yield (1.59 g).1H NMR (400 MHz, DMSO) 5 8.32 (d, J = 8.4 Hz, 2H), 8.12 (s, 1 H), 8.07 (d, J = 8.7 Hz, 1 H), 7.93 (d, J = 8.4 Hz, 2H), 6.87 (s, 1 H), 6.84 (d, J = 8.8 Hz, 1 H), 4.97 (d, J = 1 .6 Hz, 2H), 4.93 (d, J = 1 .6 Hz, 2H), 3.64 (br app s, 2H).13C NMR (101 MHz, DMSO) 5 161.23, 157.54, 147.08, 140.44, 140.40, 129.44, 126.63, 124.39, 117.59, 1 15.99, 107.33, 106.85, 101.08, 79.10, 78.82, 78.56, 78.50, 56.54, 55.99.PT_N021[000261 ] PT_N013 (1.40 g, 3.91 mmol) and anhydrous tin(ll) chloride (4.44 g, 23.4 mmol) were suspended in MeOH (30 mL) and 32% HCI(aq) (3.9 mL). The reaction mixture was then stirred and heated at 70 °C for 20 h. After cooling to room temperature, the reaction mixture was poured into a separatory funnel with EtOAc. The organic phase was washed multiple times with 1 M aqueous trisodium citrate, water and brine, then collected, dried with MgSO4, filtered and the solvent removed by rotary evaporation. To the resulting yellow oil was added Et20. The solvent was once again removed by rotary evaporation, and the product was obtained as orange solid in 96% yield (1 .24 g).1H NMR (400 MHz, DMSO) 5 7.89 (d, J = 8.6 Hz, 1 H), 7.57 (s, 1 H), 7.45 - 7.31 (m, 2H), 6.81 (d, J = 2.5 Hz, 1 H), 6.77 (dd, J = 8.7, 2.3 Hz, 1 H), 6.64 (d, J = 8.6 Hz, 1 H), 5.56 (s, 2H), 4.92 (d, J = 2.5 Hz, 2H), 4.88 (d, J = 2.5 Hz, 2H), 3.61 (br app s, 2H).13C NMR (101 MHz, DMSO) 5 159.52, 156.51 , 149.87, 131.21 , 128.70, 126.53, 121.12, 118.49, 117.20, 1 13.94, 1 10.01 , 106.85, 101.01 , 78.82, 78.76, 78.60, 56.32, 55.81 .PT_NO24[000262] PT_N021 (300 mg, 0.91 mmol) and maleic anhydride (90 mg, 0.91 mmol) were dissolved in THE (1 mL) and Et20 (5 mL). The reaction mixture was then stirred at room temperature for 2 h. The resulting suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (450 mg, 5.48 mmol) and acetic anhydride (5 mL), and the mixture was heated at 80 °C for 6 h. After cooling to room temperature, the reaction mixture was diluted with 20 mL of Et20 and 20 mL of petroleum spirits, then filtered and washed with water, Et20, and petroleum spirits, and the product collected from the filter as a yellow powder in 41% yield (151 mg).1H NMR (400 MHz, DMSO) 5 8.02 (d, J = 8.6 Hz, 1 H), 7.94 (s, 1 H), 7.79 (d, J = 8.6 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 7.21 (s, 2H), 6.86 (d, J = 2.4 Hz, 1 H), 6.83 (dd, J = 9.0, 2.2 Hz, 1 H), 4.95 (d, J = 2.5 Hz, 2H), 4.92 (d, J = 2.4 Hz, 2H), 3.63 (app q, J = 2.3 Hz, 2H).13C NMR (101 MHz, DMSO) 5 169.67, 160.56, 157.12, 137.73, 134.77, 133.31 , 132.01 , 129.13, 127.10, 126.03, 1 17.99, 1 16.38, 108.25, 107.13, 101.10, 79.00, 78.74, 78.67, 78.60, 56.47, 55.91. NMR of compound PT_N024 are shown at Figure 19 (u-v).[000263] The synthesis of example probe compound PT_NO38 is shown in Scheme 10 below.Scheme 10: Synthesis of probe compound PT_N038PT_N016[000264] Methyl 5-formyl-2-hydroxybenzoate (2.00 g, 1 1 .1 mmol) and K2CO3 (1 .69 g, 12.2 mmol) were suspended in DMSO (10 mL). A solution of 80 wt% propargyl bromide in toluene (1 .20 mL, 1 1 .2 mmol) was added to the reaction mixture and then stirred at 80 °C for 90 min. The reaction mixture was cooled to room temperature, then diluted with water causing evolution of CO2. The resulting suspension was filtered, washed with Na2COs(aq) 1 M, water and petroleum spirits, and the product collected from the filter as a tan powder in 93% yield (2.26 g).1H NMR (400 MHz, DMSO) 5 9.93 (s, 1 H), 8.21 (d, J= 2.2 Hz, 1 H), 8.10 (dd, J= 8.7, 2.2 Hz, 1 H), 7.42 (d, J= 8.7 Hz, 1 H), 5.04 (d, J = 2.4 Hz, 2H), 3.84 (s, 3H), 3.68 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 191.02, 164.98, 160.34, 134.35, 132.55, 129.33, 121.00, 114.39, 79.27, 78.15, 56.61 , 52.25.PT_N026[000265] PT_N016 (2.21 g, 10.1 mmol) and 4-nitrophenylacetonitrile (1.64 g, 10.1 mmol) were suspended in MeOH (30 mL). Piperidine (10 drops) was added to the reaction mixture and then stirred at 70 °C for 64 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, filtered, and the solid rinsed with MeOH to obtain the product as a yellow powder in 94% yield (3.30 g).1H NMR (400 MHz, DMSO) 5 8.36 (d, J = 2.4 Hz, 1 H), 8.33 (d, J = 8.9 Hz, 2H), 8.28 (s, 1 H), 8.24 (dd, J = 8.9, 2.5 Hz, 1 H), 8.00 (d, J= 8.9 Hz, 2H), 7.43 (d, J= 9.0 Hz, 1 H), 5.02 (d, J = 2.4 Hz, 2H), 3.84 (s, 3H), 3.68 (t, J = 2.1 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 165.14, 158.1 1 , 147.18, 144.76, 140.09, 134.14, 133.07, 126.72, 125.96, 124.28, 120.92, 1 17.35, 1 14.64, 106.82, 79.15, 78.32, 56.51 , 52.26.PT_N032[000266] PT_N026 (1.10 g, 3.04 mmol) and tin(ll) chloride dihydrate (4.11 g, 18.2 mmol) were suspended in MeOH (50 mL) and glacial acetic acid (1.7 mL). The reaction mixture was then stirred and heated at 70 °C for 16 h. After cooling to room temperature, the reaction mixture was filtered to remove undissolved solids. The filtrate was collected and poured into an aqueous solution of trisodium citrate dihydrate (17.9 g, 60.7 mmol). The suspension was then filtered,washed with water, and the product collected from the filter as a yellow powder in 97% yield (0.981 g).1H NMR (400 MHz, DMSO) 5 8.19 (d, J = 2.4 Hz, 1 H), 8.07 (dd, J = 8.9, 2.4 Hz, 1 H), 7.68 (s, 1 H), 7.43 (d, J = 8.7 Hz, 2H), 7.35 (d, J = 8.9 Hz, 1 H), 6.65 (d, J = 8.7 Hz, 2H), 5.59 (s, 2H), 4.97 (d, J = 2.4 Hz, 2H), 3.83 (s, 3H), 3.64 (t, J = 2.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 165.48, 156.52, 150.02, 135.02, 132.98, 131.43, 127.35, 126.67, 120.88, 120.72, 118.28, 1 14.49, 1 13.85, 109.81 , 78.94, 78.56, 56.36, 52.16.PT N038[000267] PT_N032 (300 mg, 0.90 mmol) and maleic anhydride (89 mg, 0.90 mmol) were dissolved in THF (5 mL). The reaction mixture was then stirred at room temperature for 3 h, and then 10 mL Et20 was added. The resulting suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (444 mg, 5.42 mmol) and acetic anhydride (5 mL), and the mixture was heated at 80 °C for 6 h. After cooling to room temperature, the reaction mixture was diluted with 10 mL of Et20 and 5 mL of petroleum spirits, then filtered and washed with dilute aqueous Na2COs, water, Et20, and petroleum spirits, and the product collected from the filter as a yellow powder in 82% yield (305 mg).1H NMR (400 MHz, DMSO) 5 8.32 (d, J = 2.4 Hz, 1 H), 8.20 (dd, J = 8.9, 2.5 Hz, 1 H), 8.09 (s, 1 H), 7.86 (d, J= 8.6 Hz, 2H), 7.50 (d, J = 8.7 Hz, 2H), 7.41 (d, J = 8.8 Hz, 1 H), 7.21 (s, 2H), 5.01 (d, J = 2.4 Hz, 2H), 3.84 (s, 3H), 3.66 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.67, 165.28, 157.51 , 141.85, 134.78, 133.67, 132.96, 132.43, 132.14, 127.03, 126.42, 126.13, 120.92, 117.79, 1 14.60, 108.16, 79.08,78.42, 56.45, 52.24. NMR of compound PT_N038 are shown at Figure 19 (w-x).[000268] The synthesis of example probe compound PT_NO48 is shown in Scheme 1 1 below.Scheme 11 : Synthesis of probe compound PT_N048PT_N031[000269] 3-Hydroxybenzaldehyde (0.800 g, 6.55 mmol) and K2CO3 (0.996 g, 7.21 mmol) were suspended in EtOH (30 mL). A solution of 80 wt% propargyl bromide in toluene (0.77 mL, 7.21 mmol) was added to the reaction mixture and then stirred at 80 °C for 18 h. To the reaction mixturewas then added piperidine (5 drops) followed by 4-nitrophenylacetonitrile (1.062 g, 6.55 mmol), and the mixture was stirred at 80 °C for a further 4 h. The reaction mixture was then poured into 50 mL of 2 M methanolic NaOH, then diluted with water. The resulting suspension was then filtered and washed with NaOH(aq) 1 M, water and petroleum spirits. The green-blue solid was then recrystallized from hot MeCN, and the product collected as a yellow powder in 44% yield (0.877 g) over two steps.1H NMR (400 MHz, DMSO) 5 8.34 (d, J= 8.9 Hz, 1 H), 8.25 (s, 1 H), 8.02 (d, J = 8.9 Hz, 1 H), 7.63 (d, J = 7.9 Hz, 1 H), 7.60 (t, J = 2.1 Hz, 1 H), 7.51 (t, J = 8.0 Hz, 1 H), 7.20 (dd, J = 8.1 , 2.6 Hz, 1 H), 4.86 (d, J = 2.4 Hz, 1 H), 3.61 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 157.35, 147.39, 146.06, 139.91 , 134.46, 130.19, 127.01 , 124.28, 122.54, 1 17.82, 1 17.16, 1 15.99, 108.79, 78.81 , 78.58, 55.62.PT_N048[000270] PT_N031 (0.800 g, 2.63 mmol) and tin(ll) chloride dihydrate (3.56 g, 15.8 mmol) were suspended in EtOH (30 mL) and 32% HCI(aq) (2.6 mL). The reaction mixture was then stirred and heated at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into a separatory funnel with EtOAc. The organic phase was washed with an aqueous solution of trisodium citrate dihydrate (15.5 g, 52.6 mmol), water and brine, then collected, dried with MgSC , filtered and the solvent removed by rotary evaporation. 450 mg of the crude product was then directly used in the next reaction by stirring with maleic anhydride (161 mg, 1 .64 mmol) in THE (5 mL) at room temperature for 6 h, and then 10 mL Et20 was added. The resulting suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (807 mg, 9.84 mmol) and acetic anhydride (5 mL), and the mixture was heated at 90 °C for 6 h. After cooling to room temperature, the reaction mixture was diluted with 10 mL of Et20 and 5 mL of petroleum spirits, then filtered and washed with water, Et20, and petroleum spirits, and the product collected from the filter as a tan powder in 30% yield over three steps (176 mg).1H NMR (400 MHz, DMSO) 5 8.06 (s, 1 H), 7.88 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.1 Hz, 2H), 7.57 (d, J = 3.4 Hz, 2H), 7.53 - 7.42 (m, 3H), 7.21 (s, 2H), 7.17 (dd, J = 8.3, 2.6 Hz, 1 H), 4.87 (d, J = 2.5 Hz, 2H), 3.60 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.66, 157.35, 143.14, 134.89, 134.79, 132.84, 132.36, 130.10, 127.03, 126.34, 122.19, 117.62, 117.1 1 , 1 15.65, 109.93, 78.88, 78.54, 55.60. NMR of compound PT_N048 are shown at Figure 19 (y-z).[000271 ] The synthesis of example probe compound PT_NO49 is shown in Scheme 12 below.PTJW49Scheme 12: Synthesis of probe compound PT_N049PT N044[000272] 3,5-Dibromo-4-hydroxybenzaldehyde (1.00 g, 3.57 mmol) and NaOH (0.157 g, 3.93 mmol) were suspended in EtOH (30 mL). A solution of 80 wt% propargyl bromide in toluene (0.42 mL, 3.93 mmol) was added to the reaction mixture and then stirred at 80 °C for 16 h. To the reaction mixture was then added piperidine (5 drops) followed by 4-nitrophenylacetonitrile (0.579 g, 3.57 mmol), and the mixture was stirred at 80 °C for a further 6 h. Then, 32% HCI(aq) (3.6 mL) and tin(ll) chloride dihydrate (4.84 g, 21 .4 mmol) were added to the mixture, which was stirred at 80 °C for an additional 6 h. After cooling to room temperature, the reaction mixture was poured into a separatory funnel with EtOAc. The organic phase was washed with an aqueous solution of trisodium citrate dihydrate (15.5 g, 52.6 mmol), NaOH(aq) 1 M, water and brine, then collected, dried with MgSC , filtered and the solvent removed by rotary evaporation. The resulting oil was redissolved in minimal acetone and then an excess of petroleum spirits was added to trigger precipitation. The suspension was filtered, washed with petroleum spirits, and the product collected from the filter as an orange powder in 33% yield (514 mg) over three steps.1H NMR (400 MHz, DMSO) 5 8.13 (s, 2H), 7.63 (s, 1 H), 7.42 (d, J = 8.6 Hz, 1 H), 6.65 (d, J = 8.7 Hz, 1 H), 5.72 (s, 2H), 4.82 (d, J = 2.6 Hz, 2H), 3.68 (t, J = 2.5 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 151.51 , 150.61 , 134.05, 132.39, 132.08, 127.03, 120.05, 118.29, 117.64, 1 13.79, 1 12.69, 79.59, 77.97, 60.59.PT_N049[000273] PT_N044 (450 mg, 1.04 mmol) and maleic anhydride (102 mg, 1.04 mmol) were dissolved in THE (5 mL). The reaction mixture was then stirred at room temperature for 16 h, and then 10 mL Et20 was added. The resulting suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (513 mg, 6.25 mmol) and acetic anhydride (5 mL), and the mixture was heated at 90 °C for 6 h. After cooling to room temperature,the reaction mixture was diluted with 10 mL of Et20, then filtered and washed with dilute aqueous K2CO3, water and Et20, and the product collected from the filter as a beige powder in 51% yield (274 mg).1H NMR (400 MHz, DMSO) 5 8.25 (s, 2H), 8.04 (s, 1 H), 7.85 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3 Hz, 2H), 7.22 (s, 2H), 4.87 (d, J = 2.5 Hz, 2H), 3.70 (t, J = 2.5 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.61 , 152.59, 139.48, 134.81 , 133.14, 133.06, 132.68, 132.31 , 127.04, 126.42, 1 18.49, 1 17.11 , 11 1.45, 79.74, 77.86, 60.66. NMR of compound PT_N049 are shown at Figure 19 (a’-b’).[000274] The synthesis of example probe compound PT_NO77 is shown in Scheme 13 below.Scheme 13: Synthesis of probe compound PT_N077PT N036[000275] 4- Hydroxybenzyl cyanide (3.00 g, 22.5 mmol) and K2CO3 (3.74 g, 27.0 mmol) were suspended in EtOH (50 mL). A solution of 80 wt% propargyl bromide in toluene (2.80 mL, 26.4 mmol) was added to the reaction mixture and then stirred at 80 °C for 48 h. The reaction solvent was removed by rotary evaporation, and the resulting oily slurry was resuspended in Et20 and filtered. The filtrate was collected and the solvent removed by rotary evaporation, providing the product in 99% yield (3.85 g) as a dark orange oil which crystallizes on standing.1H NMR (400 MHz, CDCI3) 6 7.28 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.7 Hz, 2H), 4.72 (d, J = 2.4 Hz, 3H), 3.71 (s, 2H), 2.55 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, CDCI3) 6 157.38, 129.23, 122.92, 1 18.17, 1 15.69, 78.34, 75.90, 56.00, 22.96.PT_N040[000276] KOtBu (328 mg, 2.92 mmol) was fully dissolved in EtOH (20 mL), and to the solution was added PT_N036 (500 mg, 2.92 mmol), followed by 4-nitrobenzaldehyde (441 mg, 2.92 mmol). The mixture was stirred at room temperature for 24 h. Upon completion of the reaction, the reaction mixture was poured into water, filtered, and the solid rinsed with water and Et20 to obtain product as a yellow powder in 74% yield (654 mg).1H NMR (400 MHz, DMSO) 5 8.35 (d, J = 8.8 Hz, 2H), 8.10 (d, J = 9.0 Hz, 2H), 8.08 (s, 2H), 7.76 (d, J = 8.8 Hz, 2H), 7.15 (d, J = 8.9 Hz, 2H), 4.89 (d, J = 2.4 Hz, 2H), 3.62 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 158.53,147.53, 140.22, 138.27, 129.93, 127.62, 126.21 , 123.99, 117.22, 115.57, 113.55, 78.79, 78.58, 55.67.PT_N076[000277] PT_N040 (1.47 g, 4.83 mmol) and tin(ll) chloride dihydrate (6.54 g, 29.0 mmol) were suspended in EtOH (40 mL) and 32% HCI(aq) (4.8 mL). The reaction mixture was then stirred and heated at 80 °C for 3 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (28.4 g, 96.6 mmol). The suspension was then filtered, washed with water, and the product collected as a yellow solid in 95% yield (1 .26 g).1H NMR (400 MHz, DMSO) 5 7.70 (d, J = 8.7 Hz, 2H), 7.59 (d, J = 8.2 Hz, 2H), 7.07 (d, J = 8.9 Hz, 1 H), 6.64 (d, J = 8.7 Hz, 1 H), 5.94 (s, 2H), 4.84 (d, J = 2.4 Hz, 2H), 3.58 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 156.96, 151.51 , 141.78, 131.10, 128.1 1 , 126.27, 121.05, 119.40, 1 15.39, 1 13.38, 101.50, 79.04, 78.38, 55.55.PT_N077[000278] PT_N076 (300 mg, 1.09 mmol) and maleic anhydride (107 mg, 1.09 mmol) were dissolved in THE (5 mL). The reaction mixture was then stirred at room temperature for 3 h. Then, 10 mL of petroleum spirits was added to precipitate the solid amic acid intermediate, and the mixture was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (538 mg, 6.56 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 3 h. The reaction mixture was then cooled to room temperature before adding 10 mL of Et20 and 5 mL of petroleum spirits. The resulting suspension was filtered, washed with water and Et20, and the product collected from the filter as a yellow solid in 73% yield (284 mg).1H NMR (400 MHz, DMSO) 5 8.05 - 7.93 (m, 3H), 7.74 (d, J = 8.8 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.22 (s, 2H), 7.14 (d, J = 8.8 Hz, 2H), 4.89 (d, J = 2.4 Hz, 2H), 3.61 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.62, 158.05, 140.06, 134.81 , 133.01 , 132.88, 129.34, 127.23, 126.69, 126.64, 1 17.81 , 1 15.52, 110.51 , 78.88, 78.52, 55.63. NMR of compound PT_N077 are shown at Figure 19 (c’-d’).[000279] The synthesis of example probe compound PT_NO57 is shown in Scheme 14 below.Scheme 14: Synthesis of probe compound PT_N057PT N002[000280] 4-Nitrophenylacetic acid (1.00 g, 5.52 mmol) and 4-hydroxybenzaldehyde (0.672 g, 5.52 mmol) were transferred into a Schlenk tube, to which was added piperidine (0.55 mL, 5.56 mmol). The mixture was carefully evacuated, and the Schlenk flask was then closed with a static vacuum inside. The slurry was then carefully heated to 100 °C for 1 h, during which the flask was briefly evacuated multiple times to remove evolved CO2 and avoid over-pressuring the flask. After CO2 evolution mostly subsided, the mixture was heated further to 150 °C for 2 h under a static vacuum. The reaction mixture was then allowed to cool and the solid mass scraped out, crushed into a powder and dissolved in boiling MeOH, to which 10 mL glacial AcOH was added. The solution was then poured into an excess of water, and the resulting suspension filtered, washed with water, and the product collected from the filter as an orange solid in 82% yield (1.09 g).1H NMR (400 MHz, DMSO) 5 9.79 (s, 1 H), 8.19 (d, J = 8.8 Hz, 2H), 7.78 (d, J = 8.6 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 16.4 Hz, 1 H), 7.16 (d, J = 16.4 Hz, 1 H), 6.81 (d, J = 8.2 Hz, 2H).13C NMR (101 MHz, DMSO) 5 158.32, 145.59, 144.70, 133.46, 128.75, 127.33, 126.67, 123.98, 122.94, 1 15.70.PT_N029[000281 ] PT_N002 (1.00 g, 4.15 mmol) and K2CO3 (0.630 g, 4.56 mmol) were suspended in DMSO (10 mL). A solution of 80 wt% propargyl bromide in toluene (0.49 mL, 4.56 mmol) was added to the reaction mixture and then stirred at 80 °C for 90 min. The reaction mixture was then cooled to room temperature and poured into a mixture of dilute aqueous Na2COs and petroleum spirits. The resulting suspension was filtered, rinsed with dilute aqueous Na2COs, water and a small amount of MeOH, and the product collected from the filter as an orange powder in 67% yield (775 mg).1H NMR (400 MHz, DMSO) 5 8.19 (d, J = 8.5 Hz, 2H), 7.80 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 8.3 Hz, 2H), 7.46 (d, J = 16.3 Hz, 1 H), 7.26 (d, J = 16.5 Hz, 1 H), 7.03 (d, J = 8.7 Hz, 2H), 4.83 (d, J = 2.4 Hz, 2H), 3.58 (t, J = 2.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 157.67, 145.83, 144.37, 132.82, 129.60, 128.50, 126.90, 124.40, 123.99, 1 15.19, 79.06, 78.36, 55.50.PT N033[000282] PT_N029 (650 mg, 2.33 mmol) and tin(ll) chloride dihydrate (3.15 g, 14.0 mmol) were suspended in EtOH (30 mL) and 32% HCI(aq) (2.3 mL). The reaction mixture was then stirred and heated at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (13.7 g, 46.6 mmol). The suspension was then filtered, washed with water, and the product collected as a yellow solid in 81 % yield (467 mg).1H NMR (400 MHz, DMSO) 5 7.44 (d, J= 8.5 Hz, 2H), 7.24 (d, J= 8.2 Hz, 2H), 6.99 - 6.89 (m, 3H), 6.84 (d, J = 16.4 Hz, 1 H), 6.56 (d, J = 8.1 Hz, 2H), 5.24 (s, 2H), 4.79 (d, J = 2.4 Hz, 2H), 3.56 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 156.04, 148.39, 131.36, 127.32, 127.29, 126.81 , 125.00, 122.33, 1 15.03, 113.90, 79.31 , 78.15, 55.43.PT N057[000283] PT_N033 (300 mg, 1.20 mmol) and maleic anhydride (1 18 mg, 1.20 mmol) were dissolved in THE (5 mL). The reaction mixture was then stirred at room temperature for 36 h. Then, 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (592 mg, 7.22 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 6 h. The reaction mixture was then cooled to room temperature before adding 10 mL of Et20 and 10 mL of petroleum spirits. The resulting suspension was filtered, washed with water and Et20, and the product collected from the filter as an orange solid in 73% yield (284 mg).1H NMR (400 MHz, DMSO) 5 7.67 (d, J = 8.2 Hz, 2H), 7.58 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.1 Hz, 2H), 7.26 (d, J = 16.5 Hz, 1 H), 7.21 - 7.11 (m, 2H), 7.01 (d, J = 8.3 Hz, 2H), 4.83 (d, J = 2.4 Hz, 2H), 3.58 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.88, 157.00, 136.74, 134.68, 130.31 , 130.19, 128.78, 127.82, 126.82, 126.44, 125.60, 1 15.1 1 , 79.17, 78.28, 55.47. NMR of compound PT_N057 are shown at Figure 19 (e’-f’).[000284] The synthesis of example probe compound PT_NO56 is shown in Scheme 15 below.Scheme 15: Synthesis of probe compound PT_N056PT_N047[000285] KOtBu (3.14 g, 28.0 mmol) was fully dissolved in THF (100 mL), and to the solution was added 4-hydroxy-3-nitrobenzaldehyde (4.45 g, 26.6 mmol). To the resulting slurry was added a solution of 80 wt% propargyl bromide in toluene (3.0 mL, 28.0 mmol) and 1 .72 g (5.33 mmol) of tetrabutylammonium bromide. The mixture was heated to 70 °C for 72 h, after which the reaction solvent was removed by rotary evaporation. The crude oil obtained was then redispersed in EtOAc and transferred to a separatory funnel. The organic layer was washed with NaOH(aq) 1 M, water and brine, then collected, dried with MgSC , filtered and the solvent removed by rotary evaporation, providing the product in 94% yield (5.15 g) as a brown oil which crystallizes on prolonged standing.1H NMR (400 MHz, DMSO) 59.95 (s, 1 H), 8.43 (d, J = 2.1 Hz, 1 H), 8.21 (dd, J = 8.7, 2.1 Hz, 1 H), 7.61 (d, J = 8.7 Hz, 1 H), 5.16 (d, J = 2.4 Hz, 2H), 3.76 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 190.35, 153.88, 139.83, 134.59, 129.41 , 126.39, 1 15.99, 79.99, 77.46, 57.62.PT_N052[000286] KOtBu (0.574 g, 5.12 mmol) was fully dissolved in EtOH (100 mL), and to the solution was added 4-fluorophenylacetonitrile (0.69 mL, 5.12 mmol), followed by PT_N047 (1.00 g, 4.87 mmol). The mixture was stirred at room temperature for 24 h. Upon completion of the reaction, the reaction mixture was filtered, and the solid rinsed with water and EtOH to obtain product as a light orange solid in 77% yield (1.21 g).1H NMR (400 MHz, DMSO) 5 8.46 (d, J = 2.3 Hz, 1 H), 8.24 (dd, J = 8.9, 2.4 Hz, 1 H), 8.04 (s, 1 H), 7.79 (dd, J = 8.8, 5.3 Hz, 2H), 7.60 (d, J = 9.0 Hz, 1 H), 7.37 (t, J = 8.8 Hz, 2H), 5.12 (d, J = 2.4 Hz, 2H), 3.75 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 162.59 (d, J = 247.9 Hz), 151.04, 140.07 (d, J = 2.1 Hz), 139.45, 134.25, 129.92 (d, J = 3.1 Hz), 128.04 (d, J = 8.6 Hz), 126.88, 125.99, 117.48, 116.22 (d, J = 22.1 Hz), 116.16, 109.61 , 79.80, 77.72, 57.37.19F NMR (376 MHz, DMSO) 5 -1 11 .64 - -1 11 .88 (m).PT_N053[000287] PT_N052 (1.10 g, 3.41 mmol) and tin(ll) chloride dihydrate (4.62 g, 20.5 mmol) were suspended in EtOH (30 mL) and 32% HCI(aq) (3.4 mL). The reaction mixture was then stirred and heated at 80 °C for 3 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (20.1 g, 68.3 mmol). The suspension was then filtered, washed with water, and the product collected as a yellow solid in 96% yield (0.960 g).1H NMR (400 MHz, DMSO) 5 7.81 - 7.67 (m, 3H), 7.32 (dd, J = 10.1 , 7.6 Hz, 2H), 7.29 (d, J = 2.3 Hz, 1 H), 7.16 (dd, J = 8.5, 2.2 Hz, 1 H), 7.01 (d, J = 8.4 Hz, 1 H), 5.03 (s, 2H), 4.88 (d, J = 2.5 Hz, 2H), 3.61 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 162.12 (d, J = 246.4 Hz), 146.30 , 143.50 (d, J = 1.7 Hz), 138.26 , 130.89 (d, J = 3.2 Hz), 127.66 (d, J = 8.5 Hz), 127.17 , 1 18.77 , 118.28 , 1 16.01 (d, J = 22.0 Hz), 1 13.62 , 1 12.10 , 105.64 , 79.14 , 78.46 , 55.77.19F NMR (376 MHz, DMSO) 5 -1 13.01 - -113.15 (m).PT N056[000288] PT_N053 (943 mg, 3.23 mmol) and maleic anhydride (316 mg, 3.23 mmol) were dissolved in THF (8 mL). The reaction mixture was then stirred at room temperature for 16 h. Then, 10 mL of petroleum spirits was added, and the suspension was filtered and washed with Et20. The solid on the filter was then recrystallized in 20 mL hot MeCN to provide 49% yield (621 mg) of purified amic acid intermediate. 606 mg (1.55 mmol) of intermediate was then mixed with sodium acetate (764 mg, 9.31 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 3 h. The reaction mixture was then cooled to room temperature before adding MeOH (20 mL) and water (50 mL). The resulting suspension was filtered, washed with dilute aqueous K2CO3 and water. The solid retained on the filter was then recrystallized from a boiling mixture of EtOH (15 mL) and MeCN (3 mL) followed by cooling to -20 °C for 16 h. The crystals were filtered and washed with a small amount of MeOH. Subsequently, the material was purified by column chromatography using gradient elution from petroleum spirits:DCM 1 :3 to neat DCM, yielding the product as a pale yellow solid in 25% overall yield (287 mg) from the aniline.1H NMR (400 MHz, CDCI3) 6 8.04 (dd, J = 8.8, 2.4 Hz, 1 H), 7.71 (d, J = 2.3 Hz, 1 H), 7.65 - 7.56 (m, 2H), 7.37 (s, 1 H), 7.25 (d, J = 8.7 Hz, 1 H), 7.17 - 7.08 (m, 2H), 6.88 (s, 2H), 4.74 (d, J = 2.4 Hz, 2H), 2.55 (t, J = 2.4 Hz, 1 H).13C NMR (101 MHz, CDCI3) 5 169.21 , 163.31 (d, J = 250.1 Hz), 155.08, 140.24 (d, J = 1 .9 Hz), 134.75, 131 .56, 131 .51 , 130.71 (d, J = 3.3 Hz), 127.92 (d, J = 8.3 Hz), 127.70, 121 .04, 1 17.92, 1 16.30 (d, J = 22.1 Hz), 1 14.08, 109.82, 77.30, 76.94, 56.87.19F NMR (376 MHz, CDCI3) 5 -1 11 .52 - -11 1 .73 (m). NMR of compound PT_N056 are shown at Figure 19 (g’-i').[000289] The synthesis of example probe compound PT_NO67 is shown in Scheme 16 below.PT_NO64 PT_NO66 PT_ 067Scheme 16: Synthesis of probe compound PT_N067PT N064[000290] PT_N047 (1 .00 g, 4.87 mmol) and 2-cyanophenylacetonitrile (0.693 g, 4.87 mmol) were suspended in EtOH (30 mL). Piperidine (5 drops) were then added to the reaction mixture and then stirred at 80 °C for 64 h. Upon completion of the reaction, the reaction mixture was cooledto room temperature, poured into water, filtered, and the solid rinsed with MeOH and Et2O to obtain the product as a pale orange powder in 72% yield (1.16 g).1H NMR (400 MHz, DMSO) 5 8.51 (d, J = 2.3 Hz, 1 H), 8.29 (dd, J = 9.0, 2.3 Hz, 1 H), 8.03 (dd, J = 7.8, 1.3 Hz, 1 H), 7.92 (s, 1 H), 7.87 (td, J = 7.7, 1.4 Hz, 1 H), 7.80 (d, J = 8.3 Hz, 1 H), 7.74 - 7.58 (m, 2H), 5.15 (d, J = 2.4 Hz, 2H), 3.76 (d, J = 2.4 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 151 .72, 146.85, 139.52, 137.63, 134.53, 134.27, 133.99, 130.08, 129.79, 126.23, 126.03, 117.02, 116.77, 1 16.40, 1 10.23, 107.30, 79.90, 77.62, 57.49.PT_N066[000291 ] PT_N064 (1.10 g, 3.34 mmol) and tin(ll) chloride dihydrate (4.52 g, 20.0 mmol) were suspended in EtOH (30 mL) and 32% HCI(aq) (3.4 mL). The reaction mixture was then stirred and heated at 80 °C for 16 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (19.6 g, 66.8 mmol). The suspension was then filtered, washed with water, and the product collected as a yellow solid in 92% yield (0.921 g).1H NMR (400 MHz, DMSO) 5 8.02 - 7.94 (m, 1 H), 7.82 (td, J = 7.7, 1.4 Hz, 1 H), 7.74 (d, J = 8.4 Hz, 1 H), 7.64 (dd, J = 7.7, 1.4 Hz, 1 H), 7.61 (s, 2H), 7.32 (d, J = 2.2 Hz, 1 H), 7.18 (dd, J = 8.4, 2.2 Hz, 1 H), 7.05 (d, J = 8.4 Hz, 1 H), 5.11 (s, 2H), 4.90 (s, 1 H), 3.62 (s, 1 H).13C NMR (101 MHz, DMSO) 5 149.96, 146.96, 138.79, 138.48, 134.17, 133.83, 129.62, 129.41 , 126.47, 1 19.20, 1 17.64, 1 17.30, 1 13.53, 1 12.15, 110.20, 103.04, 79.02, 78.56, 55.82.PT_N067[000292] PT_N066 (300 mg, 1.09 mmol) and maleic anhydride (107 mg, 1.09 mmol) were dissolved in THE (5 mL). The reaction mixture was then stirred at room temperature for 36 h. Then, 20 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (536 mg, 6.54 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 2 h. The solvent was then evaporated under reduced pressure, and the resulting solid was suspended in water, sonicated, then filtered, washed with dilute aqueous K2CO3 and water, and the product collected from the filter as a tan solid in 44% yield (180 mg).1H NMR (400 MHz, DMSO) 5 8.13 (dd, J = 8.9, 2.3 Hz, 1 H), 8.01 (d, J = 6.8 Hz, 1 H), 7.90 - 7.82 (m, 3H), 7.79 (d, J = 7.7 Hz, 1 H), 7.67 (t, J = 7.6 Hz, 1 H), 7.47 (d, J = 8.9 Hz, 1 H), 7.26 (s, 2H), 4.95 (d, J = 2.4 Hz, 2H), 3.65 (t, J = 2.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.48, 155.56, 147.88, 138.09, 135.15, 134.22, 133.94, 131.72, 131.65, 129.83, 129.75, 126.32, 120.83, 117.14, 114.25, 1 10.25, 105.52, 79.20, 78.06, 56.58. NMR of compound PT_N067 are shown at Figure 19 G’-k’).[000293] The synthesis of example probe compound PT_NO86 is shown in Scheme 17 below.Scheme 17: Synthesis of probe compound PT_086PT_N079[000294] KOtBu (430 mg, 3.83 mmol) was fully dissolved in MeOH (30 mL), and to the solution was added PT_N047 (748 mg, 3.65 mmol), followed by methyl 4-(Cyanomethyl)benzoate (639 mg, 3.65 mmol). The mixture was stirred at room temperature for 16 h. Upon completion of the reaction, the reaction mixture was diluted with water (70 mL), filtered, and the solid rinsed with water to obtain product as a tan solid in 89% yield (1 .18 g).1H NMR (400 MHz, DMSO) 5 8.51 (d, J = 2.3 Hz, 1 H), 8.29 (dd, J = 9.0, 2.4 Hz, 1 H), 8.22 (s, 1 H), 8.06 (d, J = 8.5 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.9 Hz, 1 H), 5.13 (d, J = 2.5 Hz, 2H), 3.87 (s, 3H), 3.76 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 165.54, 151.42, 142.03, 139.40, 137.72, 134.61 , 129.96, 129.93, 126.58, 126.40, 125.97, 117.22, 116.18, 109.50, 79.84, 77.67, 57.42, 52.28.PT_N083[000295] PT_N079 (1.10 g, 3.04 mmol) and tin(ll) chloride dihydrate (4.11 g, 18.2 mmol) were suspended in MeOH (40 mL) and glacial acetic acid (1.7 mL). The reaction mixture was then stirred and heated at 70 °C for 20 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (17.9 g, 60.7 mmol). The suspension was then filtered, washed with water, and the product collected from the filter as a yellow powder in 88% yield (0.884 g).1H NMR (400 MHz, DMSO) 5 8.04 (d, J = 8.5 Hz, 2H), 7.95 (s, 1 H), 7.86 (d, J = 8.3 Hz, 2H), 7.36 (d, J = 2.3 Hz, 1 H), 7.22 (dd, J = 8.4, 2.2 Hz, 1 H), 7.03 (d, J = 8.4 Hz, 1 H), 5.07 (s, 2H), 4.89 (d, J = 2.4 Hz, 2H), 3.87 (s, 3H), 3.62 (t, J = 2.3 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 165.67, 146.78, 145.41 , 138.85, 138.34, 129.86, 129.20, 126.95, 125.63, 1 19.54, 1 18.01 , 1 13.68, 112.08, 105.46, 79.08, 78.53, 55.79, 52.23.PT_N086[000296] PT_N083 (400 mg, 1.20 mmol) and maleic anhydride (1 18 mg, 1.20 mmol) were dissolved in THF (15 mL). The reaction mixture was then stirred at room temperature for 16 h. Then, 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. Thesolid on the filter was collected, mixed with sodium acetate (592 mg, 7.22 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 3 h. The reaction mixture was then cooled to room temperature before adding 15 mL of Et2O. The resulting suspension was filtered, washed with water, Et2O and a small amount of MeOH. The tan solid on the filter was collected, redissolved in hot MeCN (5 mL), and precipitated with MeOH (30 mL), then cooled to -20 °C for 16 h. The mixture was then filtered cold and rinsed with a small amount of MeOH, and the product collected from the filter as a light brown solid in 34% yield (169 mg).1H NMR (400 MHz, DMSO) 5 8.19 - 8.12 (m, 2H), 8.07 (d, J = 8.5 Hz, 2H), 7.95 - 7.82 (m, 3H), 7.45 (d, J = 8.8 Hz, 1 H), 7.26 (s, 2H), 4.93 (d, J = 2.4 Hz, 2H), 3.88 (s, 3H), 3.64 (t, J = 2.1 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.50, 165.61 , 155.23, 143.26, 138.21 , 135.15, 131.79, 129.93, 129.70, 126.86, 125.91 ,120.71 , 1 17.54, 114.12, 107.92, 79.15, 78.12, 56.53, 52.27. NMR of compound PT_N086 are shown at Figure 19 (I’-nT).[000297] The synthesis of example probe compound PT_NO95 is shown in Scheme 18 below.Scheme 18: Synthesis of probe compound PT_N095PT N088[000298] 4-Aminobenzyl cyanide (2.00 g, 15.1 mmol) was dissolved in pyridine (20 mL) and the solution cooled to 0 °C, followed by dropwise addition of mesyl chloride (1.30 mL, 16.8 mmol). After full addition, the mixture was warmed to room temperature and allowed to stir for 16 h. The solvent was then removed by rotary evaporation, and the crude solid redissolved in DCM and transferred to a separatory funnel. The organic phase was washed 1 M HCI(aq), 20% K2CO3(aq), water and brine, then collected, dried with MgSC , filtered and the solvent removed by rotary evaporation, providing the product in 35% yield (1.1 1 g) as brown crystals.1H NMR (400 MHz, DMSO) 5 9.81 (s, 1 H), 7.31 (d, J = 8.4 Hz, 2H), 7.23 (d, J = 8.5 Hz, 2H), 3.97 (s, 2H), 2.99 (s, 3H).13C NMR (101 MHz, DMSO) 5 137.84, 129.08, 126.56, 120.16, 119.25, 39.27, 21.76.PT_N089[000299] PT_N088 (1.1 1 g, 5.28 mmol) and K2CO3 (0.803 g, 5.81 mmol) were suspended in acetone (40 mL). A solution of 80 wt% propargyl bromide in toluene (0.59 mL, 5.54 mmol) was added to the reaction mixture and then stirred at 65 °C for 64 h. The mixture was then filtered, the filtrate collected and the solvent removed by rotary evaporation, providing the product in 99% yield (1 .30 g) as a brown oil which crystallizes slowly on standing.1H NMR (500 MHz, CDCI3) 6 7.59 (d, J = 8.5 Hz, 2H), 7.37 (d, J = 8.6 Hz, 2H), 4.43 (d, J = 2.5 Hz, 2H), 3.76 (s, 2H), 3.02 (s, 3H), 2.50 (t, J = 2.5 Hz, 1 H).13C NMR (126 MHz, CDCI3) 6 139.99, 130.08, 129.18, 127.97, 1 17.49, 78.74, 74.77, 41.04, 39.07, 23.29.PT_N090[000300] KOtBu (452 mg, 4.03 mmol) was fully dissolved in EtOH (30 mL), and to the solution was added PT_N089 (1 .00 g, 4.03 mmol), followed by 4-nitrobenzaldehyde (609 mg, 4.03 mmol). The mixture was stirred at room temperature for 16 h. Upon completion of the reaction, the reaction mixture was diluted with water (150 mL), filtered, and the solid rinsed with water and a small amount of MeOH and Et20 to obtain product as a yellow powder in 49% yield (747 mg).1H NMR (500 MHz, DMSO) 5 8.39 (d, J = 8.8 Hz, 2H), 8.24 (s, 1 H), 8.15 (d, J = 8.8 Hz, 2H), 7.88 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.6 Hz, 2H), 4.58 (d, J = 2.5 Hz, 2H), 3.40 (t, J = 2.3 Hz, 1 H), 3.12 (s, 3H).13C NMR (126 MHz, DMSO) 5 147.90, 141.02, 140.83, 139.91 , 132.13, 130.24, 127.59, 127.07, 124.09, 1 17.03, 1 13.24, 78.67, 76.66, 39.94, 38.40.PT_N092[000301 ] PT_N090 (700 mg, 1.84 mmol) and tin(ll) chloride dihydrate (2.48 g, 11.0 mmol) were suspended in EtOH (30 mL) and glacial acetic acid (1.0 mL). The reaction mixture was then stirred and heated at 80 °C for 1 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (10.8 g, 36.7 mmol). The suspension was then filtered, washed with water, and the product collected from the filter as a yellow powder in 98% yield (632 mg).1H NMR (400 MHz, DMSO) 5 7.75 (s, 1 H), 7.75 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 8.6 Hz, 2H), 7.54 (d, J = 8.7 Hz, 2H), 6.66 (d, J = 8.7 Hz, 2H), 6.07 (s, 2H), 4.52 (d, J = 2.5 Hz, 2H), 3.39 (t, J = 2.4 Hz, 1 H), 3.09 (s, 3H).13C NMR (101 MHz, DMSO) 5 152.08, 143.95, 138.79, 134.37, 131.61 , 127.87, 125.70, 120.73, 119.20, 1 13.39, 100.56, 78.81 , 76.54, 38.21.PT_N095[000302] PT_N092 (450 mg, 1.28 mmol) and maleic anhydride (126 mg, 1.28 mmol) were dissolved in THE (7 mL). The reaction mixture was then stirred at 60 °C for 3 h. The reaction mixture was allowed to cool, then 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (630 mg, 7.68 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 3 h. The reaction mixture was then cooled to room temperature before adding 15 mL of Et20. The resulting suspension was filtered, washed with water and Et20, and the product collected from the filter as a yellow powder in 74% yield (408 mg).1H NMR (400 MHz, DMSO) 5 8.13 (s, 1 H), 8.03 (d, J =8.7 Hz, 2H), 7.85 (d, J = 8.8 Hz, 2H), 7.63 (d, J = 8.7 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H), 7.23 (s, 2H), 4.57 (d, J = 2.5 Hz, 2H), 3.40 (t, J = 2.3 Hz, 1 H), 3.1 1 (s, 3H).13C NMR (101 MHz, DMSO) 5 169.60, 142.60, 140.25, 134.84, 133.33, 132.69, 129.62, 127.68, 126.70, 126.67, 117.58,1 10.06, 78.70, 76.63, 39.99, 38.31 . NMR of compound PT_N095 are shown at Figure 19 (n’-o’).[000303] The synthesis of example probe compound PT_NO98 is shown in Scheme 19 below.Scheme 19: Synthesis of probe compound PT_N098PT_N093[000304] 4-Ethynylbenzaldehyde (562 mg, 4.32 mmol) and 4-nitrophenylacetonitrile (700 mg, 4.32 mmol) were suspended in EtOH (30 mL). Piperidine (5 drops) was then added to the reaction mixture and then stirred at 80 °C for 5 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, poured into water, filtered, and the solid rinsed with water, MeOH, MeCN and Et20 to obtain the product as a yellow powder in 81% yield (960 mg).1H NMR (400 MHz, DMSO) 5 8.34 (d, J = 9.1 Hz, 2H), 8.29 (s, 1 H), 8.03 (d, J = 9.0 Hz, 2H), 7.99 (d, J = 8.6 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 4.45 (s, 1 H).13C NMR (101 MHz, DMSO) 5 147.45, 145.26, 139.81 , 133.47, 132.25, 129.71 , 127.08, 124.44, 124.29, 1 17.09, 109.20, 83.66, 82.95 PT_N097[000305] PT_N093 (900 mg, 3.28 mmol) and tin(ll) chloride dihydrate (4.44 g, 19.7 mmol) were suspended in EtOH (30 mL) and glacial acetic acid (1.9 mL). The reaction mixture was then stirred and heated at 80 °C for 3 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (19.3 g, 65.6 mmol). The suspension was then filtered, washed with water, and the product collected from the filter as a yellow powder in 95% yield (759 mg).1H NMR (400 MHz, DMSO) 5 7.85 (d, J = 8.1 Hz, 2H), 7.69 (s, 1 H), 7.58 (d, J = 8.2 Hz, 2H), 7.45 (d, J = 8.6 Hz, 2H), 6.65 (d, J = 8.6 Hz, 2H), 5.67 (s, 2H), 4.34 (s, 1 H).13C NMR (101 MHz, DMSO) 5 150.37, 135.18, 134.86, 132.04, 128.67, 126.95, 122.37, 120.52, 1 18.07, 1 13.83, 1 11.74, 83.25, 82.60.PT_N098[000306] PT_N097 (300 mg, 1.23 mmol) and maleic anhydride (120 mg, 1.23 mmol) were dissolved in THF (5 mL). The reaction mixture was stirred at room temperature for 16 h, then 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (604 mg, 7.37 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 4 h. The reaction mixture was then cooled to room temperature before adding 15 mL of Et20. The resulting suspension was filtered, washed with water and Et20, and the product collected from the filter as a yellow powder in 81% yield (322 mg).1H NMR (400 MHz, DMSO) 5 8.10 (s, 1 H), 7.96 (d, J = 8.4 Hz, 2H), 7.88 (d, J = 8.7 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.7 Hz, 2H), 7.21 (s, 2H), 4.41 (s, 1 H).13C NMR (101 MHz, DMSO) 5 169.65, 142.35, 134.80, 133.93, 132.74, 132.47, 132.20, 129.34, 127.02, 126.40, 123.73, 1 17.54, 1 10.37, 83.25, 83.05. NMR of compound PT_N098 are shown at Figure 19 (p’- q’)-[000307] The synthesis of example probe compound PT_N109 is shown in Scheme 20 below._Scheme 20: Synthesis of probe compound PT_N109PT_N101[000308] lndole-3-carboxaldehyde (1.00 g, 6.89 mmol) and 4-nitrophenylacetonitrile (1.12 g, 6.89 mmol) were suspended in EtOH (30 mL). Piperidine (5 drops) was then added to the reaction mixture and then stirred at 80 °C for 6 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, glacial acetic acid (0.50 mL) was added, and the mixture poured into water, filtered, and the solid rinsed with water to obtain the product as orange crystals in 55% yield (1.09 g).1H NMR (400 MHz, DMSO) 5 12.23 (s, 1 H), 8.51 (s, 1 H), 8.49 (s, 1 H), 8.27 (d, J = 8.9 Hz, 2H), 8.13 (d, J = 7.7 Hz, 1 H), 8.01 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 7.7 Hz, 1 H), 7.33 - 7.09 (m, 2H).13C NMR (101 MHz, DMSO) 5 145.99, 141.11 , 138.04, 135.85, 128.83, 127.29, 125.52, 124.20, 123.13, 121.10, 119.36, 118.94, 112.48, 1 10.96, 99.81.PT_N102[000309] PT_N101 (1.00 g, 3.46 mmol) and K2CO3 (526 mg, 3.80 mmol) were suspended in acetone (40 mL). A solution of 80 wt% propargyl bromide in toluene (0.41 mL, 3.80 mmol) was added to the reaction mixture and then stirred at 65 °C for 24 h. The mixture was cooled to room temperature, glacial acetic acid (1.0 mL) was added, and the solvent was removed by rotary evaporation. The crude solid was resuspended in MeOH (50 mL) and filtered, then rinsed with water and MeOH to provide the product as an orange powder in 90% yield (1 .02 g).1H NMR (400 MHz, DMSO) 5 8.59 (s, 1 H), 8.49 (s, 1 H), 8.28 (d, J = 9.0 Hz, 2H), 8.18 (d, J = 7.9 Hz, 1 H), 8.04 (d, J = 9.0 Hz, 2H), 7.65 (d, J = 8.2 Hz, 1 H), 7.36 (t, J = 7.6 Hz, 1 H), 7.30 (t, J = 7.4 Hz, 1 H), 5.33 (d, J = 2.7 Hz, 2H), 3.58 (t, J = 2.5 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 146.14, 140.85, 137.19, 135.43, 130.53, 127.93, 125.71 , 124.19, 123.44, 121 .68, 1 19.34, 1 19.00, 1 11 .06, 110.71 , 100.75, 78.00, 76.85, 36.09.PT_N107[000310] PT_N102 (950 mg, 2.90 mmol) and tin(ll) chloride dihydrate (3.93 g, 17.4 mmol) were suspended in EtOH (30 mL), THE (15 mL) and glacial acetic acid (1.6 mL). The reaction mixture was then stirred and heated at 80 °C for 6.5 h. After completion of the reaction, the mixture was concentrated by rotary evaporation to a viscous oil, then redissolved in MeOH (30 mL) and poured into an aqueous solution of trisodium citrate dihydrate (17.1 g, 58.0 mmol). The suspension was then filtered, washed with water, and the product collected from the filter as an orange powder in 83% yield (714 mg).1H NMR (400 MHz, DMSO) 5 8.33 (s, 1 H), 8.04 (d, J = 7.9 Hz, 1 H), 7.88 (s, 1 H), 7.61 (d, J = 8.1 Hz, 1 H), 7.47 (d, J = 8.6 Hz, 2H), 7.31 (t, J = 7.6 Hz, 1 H), 7.23 (t, J = 7.4 Hz, 1 H), 6.66 (d, J = 8.7 Hz, 2H), 5.46 (s, 2H), 5.27 (d, J = 2.6 Hz, 2H), 3.51 (t, J = 2.2 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 149.12, 135.27, 128.06, 127.74, 127.63, 126.08, 122.85, 121.35, 120.77, 1 19.85, 1 19.04, 1 13.99, 110.96, 110.63, 104.75, 78.48, 76.33, 35.69.PT_N109[00031 1 ] PT_N107 (450 mg, 1.51 mmol) and maleic anhydride (148 mg, 1.51 mmol) were dissolved in THE (5 mL). The reaction mixture was stirred at room temperature for 16 h, then 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, mixed with sodium acetate (745 mg, 9.08 mmol) and acetic anhydride (5 mL), and the mixture was heated at 85 °C for 3 h. The reaction mixture was then cooled to room temperature before adding 15 mL of Et20. The resulting suspension was filtered, washed with water and Et20, and the product collected from the filter as a red powder in 77% yield (439 mg).1H NMR (400 MHz, DMSO) 5 8.51 (s, 1 H), 8.29 (s, 1 H), 8.14 (d, J = 7.9 Hz, 1 H), 7.90 (d, J = 8.6 Hz, 2H), 7.65 (d, J = 8.1 Hz, 1 H), 7.47 (d, J = 8.6 Hz, 2H), 7.35 (t, J = 7.4 Hz, 1 H), 7.28 (t, J = 7.4 Hz, 1 H), 7.21 (s, 2H), 5.32 (d, J = 2.6 Hz, 2H), 3.55 (t, J = 2.5 Hz, 1 H).13C NMR (101 MHz, DMSO) 5 169.77, 135.37, 134.74, 134.33, 133.50, 131.11 , 129.29, 127.85, 127.07, 125.47, 123.19, 121.29, 119.41 , 119.21 , 110.90, 1 10.63, 102.43, 78.25, 76.60, 35.91 . NMR of compound PT_N109 are shown at Figure 19 (r’-s’).[000312] The synthesis of example probe compound PT_N110 is shown in Scheme 21 below.Scheme 21 : Synthesis of probe compound PT_N110PT N018[000313] 4- Hydroxybenzaldehyde (1.51 g, 12.3 mmol) and 4-nitrophenylacetonitrile (2.00 g, 12.3 mmol) were suspended in EtOH (40 mL). Piperidine (10 drops) was then added to the reaction mixture and then stirred at 80 °C for 4 h. Upon completion of the reaction, the reaction mixture was cooled to room temperature, glacial acetic acid (1 .0 mL) was added, and the mixture poured into water, filtered, and the solid rinsed with water to obtain the product as an orange powder in 93% yield (3.07 g).1H NMR (400 MHz, DMSO) 5 10.46 (br s, 1 H), 8.29 (d, J = 8.9 Hz, 2H), 8.12 (s, 1 H), 7.95 (d, J = 9.0 Hz, 2H), 7.92 (d, J = 8.9 Hz, 2H), 6.93 (d, J = 8.6 Hz, 2H).13C NMR (101 MHz, DMSO) 5 161.00, 146.74, 146.26, 140.83, 132.20, 126.29, 124.27, 124.24, 118.00, 116.05, 103.52.PT_N027[000314] PT_N018 (1.50 g, 5.63 mmol) and K2CO3 (856 mg, 6.20 mmol) were suspended in acetone (50 mL). 1 -Bromo-3-chloropropane (0.72 mL, 7.32 mmol) was added to the reaction mixture and then stirred at 65 °C for 64 h. After completion of the reaction, the mixture was concentrated by rotary evaporation to a slurry, then dispersed in MeOH (30 mL) and poured into dilute K2CC>3(aq). The resulting suspension was filtered, rinsed with water and a small amount of MeOH and MeCN to provide the product as a yellow powder in 76% yield (1 .47 g).1H NMR (400 MHz, DMSO) 5 8.32 (d, J = 8.9 Hz, 2H), 8.20 (s, 1 H), 8.07 - 7.93 (m, 4H), 7.16 (d, J = 9.0 Hz, 2H), 4.21 (t, J = 6.1 Hz, 2H), 3.82 (t, J = 6.4 Hz, 2H), 2.21 (p, J = 6.2 Hz, 2H).13C NMR (101 MHz,DMSO) 6 161.01 , 146.92, 145.88, 140.53, 131.87, 126.48, 125.88, 124.24, 117.77, 115.08, 104.96, 64.71 , 41.81 , 31.52.PT_N039[000315] PT_N027 (1 .40 mg, 4.08 mmol) and tin(ll) chloride dihydrate (5.53 g, 24.5 mmol) were suspended in EtOH (30 mL) and glacial acetic acid (2.3 mL). The reaction mixture was then stirred and heated at 80 °C for 3 h. After cooling to room temperature, the reaction mixture was poured into an aqueous solution of trisodium citrate dihydrate (19.3 g, 65.6 mmol). The suspension was then filtered, washed with water, and the product collected from the filter as a yellow powder in 98% yield (1.25 g).1H NMR (400 MHz, DMSO) 5 7.84 (d, J = 8.8 Hz, 2H), 7.60 (s, 1 H), 7.40 (d, J = 8.6 Hz, 2H), 7.07 (d, J = 8.8 Hz, 2H), 6.64 (d, J = 8.6 Hz, 2H), 5.54 (s, 2H), 4.16 (t, J = 6.0 Hz, 2H), 3.80 (t, J = 6.4 Hz, 2H), 2.19 (p, J = 6.3 Hz, 2H).13C NMR (101 MHz, DMSO) 5 159.32, 149.71 , 136.52, 130.34, 127.13, 126.46, 121.12, 1 18.70, 114.79, 113.86, 108.12, 64.47, 41.87, 31.59.PT_N105[000316] PT_N039 (1.51 g, 12.3 mmol) and sodium azide (2.00 g, 12.3 mmol) were suspended in DMF (10 mL), and the reaction mixture was stirred at 70 °C for 48 h. Upon completion, the reaction mixture transferred to a separatory funnel with EtOAc. The organic phase was washed multiple times with water and brine, then collected, dried with MgSO4, filtered, and solvent removed by rotary evaporation to obtain the product as a red-yellow oil in 86% yield (1 .05 g).1H NMR (400 MHz, DMSO) 5 7.84 (d, J = 8.5 Hz, 2H), 7.60 (s, 1 H), 7.40 (d, J = 8.3 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H), 6.64 (d, J = 8.4 Hz, 2H), 5.53 (s, 2H), 4.11 (t, J = 6.1 Hz, 2H), 3.52 (t, J = 6.7 Hz, 2H), 2.00 (p, J = 6.2 Hz, 2H).13C NMR (101 MHz, DMSO) 5 159.36, 149.70, 136.53, 130.33, 127.09, 126.46, 121.13, 1 18.71 , 114.78, 113.87, 108.08, 64.83, 47.65, 28.04.PT_N110[000317] PT_N105 (250 mg, 0.78 mmol) and maleic anhydride (77 mg, 0.78 mmol) were dissolved in THE (5 mL). The reaction mixture was stirred at room temperature for 16 h, then 10 mL of Et20 was added, and the suspension was filtered and washed with Et20. The solid on the filter was collected, suspended in DCM (5 mL) plus one drop of DMF, and the mixture was cooled to 0 °C under nitrogen before dropwise addition of oxalyl chloride (0.10 mL, 1.17 mmol). The mixture was allowed to warm to room temperature and stirred under nitrogen for 16 h. Next, the solvent was evaporated under vacuum, the solid was redissolved in another aliquot of DCM (5 mL) under nitrogen, and lastly triethylamine (0.33 mL, 2.35 mmol) was added dropwise. The mixture was stirred at room temperature for 2 h, then petroleum spirits (15 mL) was added. The precipitate was filtered, washed with petroleum spirits, water and Et20, and the product collected from the filter as an orange powder in 74% yield (233 mg).1H NMR (500 MHz, DMSO) 5 8.00 (s, 1 H), 7.96 (d, J = 8.4 Hz, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.48 (d, J = 8.7 Hz, 2H), 7.21 (s, 2H), 7.13 (d, J = 9.0 Hz, 2H), 4.14 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 6.7 Hz, 2H), 2.01 (p, J = 6.4 Hz, 2H).13C NMR (126 MHz, DMSO) 5 169.72, 160.41 , 143.10, 134.79, 133.39, 131.85, 131.26, 127.05,126.26, 125.98, 118.24, 114.99, 106.30, 64.99, 47.64, 28.01. NMR of compound PT_N110 are shown at Figure 19 (t’-u’).Protein expression and purification.[000318] Recombinant DsbA, DsbA(C33A), and DsbL from Escherichia coliwere overexpressed using the autoinduction method and then purified by Nickel affinity chromatography. TEV protease was used to cleave the / V-terminal Hise-tag and the cleaved proteins were further purified by size exclusion chromatography. Proteins were then reduced by incubating at 4 °C for 1 h with 50 mM reduced (GSH) glutathione (Sigma Aldrich, Australia). Excess redox agents in the reaction mixtures were removed by size exclusion chromatography using a Superdex 75 10 / 300 GL (GE Healthcare, USA) equilibrated in 25 mM HEPES, pH 7.0, 150 mM NaCI and 1 mM EDTA. The final redox state of the proteins was confirmed by the Ellman assay.In-vitro protein fluorescence measurement.[000319] TME was dissolved in DMSO as 1 mM stocks and stored at -20 °C in the dark. TME, proteins (i.e., BLG, DsbA, C33A, and DsbL), and GSH, were freshly prepared at high concentrations as stock solutions using proper buffers that maintained the same solution components across different experimental conditions when measured. 30 pL in total of proteins, GSH, PBS or urea was first mixed in each well of a 384-well plate. The fluorescence was then measured using a plate reader (CLARIOstar, BMG Labtech). The settings were as follows: 10 pL of TME or the same amount of DMSO was injected into each well using the automatic injection mode, followed by the kinetics measurement mode. Excitation / Emission settings were 350±20 / 430±30 nm. At the end of the kinetics measurement, emission spectra were measured using the fluorometer (Cary Eclipse, Agilent, CA, USA). Excitation was set as 350 ± 5 nm and emission spectra were collected from 370 to 680 nm. The final concentrations for BLG experiments were: [BLG] = 250 pM, [TME] = 50 pM, [urea] = 6 M, [GSH] = 8 mM, [NMM] = 2.5 mM. The final concentrations for Dsb protein experiments were: [DsbA] = [C33A] = [DsbL] = 25 pM, [TME] = 25 pM, [urea] = 6 M.Semi-quantification of TME conjugation using in-gel fluorescence.[000320] Protein samples were prepared as above. TME stock solutions were added. The final concentrations for BLG experiments were: [BLG] =50 pM, [TME] = 50 pM, [urea] = 6 M. The final concentrations for Dsb protein experiments were: [DsbA] = [C33A] = [DsbL] = 25 pM, [TME] = 25 pM, [urea] = 6 M. At each time point, an aliquot of protein samples was quickly mixed with NMM (final concentrations of 2.5 mM) to fully block unreacted free cysteines. Proteins samples were then mixed with SDS loading buffer, boiled at 95 °C for 5 min, and analyzed by the SDS-polyacrylamide gel electrophoresis (SDS-PAGE). For BLG, 7.5 pg of BLG was loaded into each lane. For Dsb proteins, 15 pL of 25 pM DsbA, C33A or DsbL was loaded into each lane. Note the molecular weight of DsbA (~ 21.13 kD), C33A (~ 21.10 kD) and DsbL (~ 21 .66 kD). An aliquoted of the same batch of protein samples was also measured using aplate reader. Excitation / Emission settings were 350±20 / 430±30 nm. Original gel figures are shown in Figure 27.Protein denaturation curve analysis and protein stability quantification using TME fluorescence.[000321 ] BLG was unfolded in 0 - 9 M urea in phosphate buffer, pH 2.3, without or with TME labeling for 2 h, prior to measurement using a plate reader. The final concentrations of each component are: [BLG] = 50 pM, [TME] = 100 pM, [phosphate] = 20 mM. Excitation / Emission settings for the intrinsic tryptophan fluorescence (without TME labeling) measurement were 295±10 / 360±20 nm. Excitation / Emission settings for TME fluorescence measurement were 350±20 / 430±30 nm. Deduction of the thermostability calculation is shown below.Thermostability Calculation[000322] Consider the simplest form of an unfolding reaction:where only folded (F) and unfolded ((7) states but not any intermediate states are significantly populated in proteins, i.e., two-state folding model. Upon unfolding equilibrium, the ratio of unfolded and folded fractions reaches a constant value, i.e., equilibrium constant:where fr and fu denote the fraction of the protein present in the folded and unfolded states, respectively, and fF+ fu = [3]Assume that values of / characteristics (i.e., intrinsic tryptophan fluorescence and TME fluorescence) of the folded and unfolded states are / F and yu, respectively, / characteristics of any point in the transition region of the protein denaturation curve is simply the linear combination of / and yu, that is, y = fFx yF+ fu x yu [4]Combining [2], [3] and [4],where the equilibrium constant / <can be expressed by measurement of / characteristics.Further, / <can be linked to the Gibbs free energy change of denaturation by Nernst equation:AG = —RT InK [6] where R is the universal gas constant, Tis the absolute temperature in kelvin. Of note is that [F], [U], fF, fu, K, / and AG in [2] - [6] are functions of denaturant concentrations D which can be explicitly expressed (e.g., [F](D), [U](D)) and termed “apparent” when observed and measured in a given denaturant concentration. yFand yu are constants and can be obtained by extrapolation from the linear portions of the denaturation curve at low and high denaturant concentrations to the transition region, respectively.The free energy change in the absence of denaturant, AGH2°, is a useful quantitative measure of protein structural stability, which reflects the intrinsic thermodynamic property of proteins. The simplest method to estimate AGH2° is to assume the linear dependence of AG(£>) on denaturant concentration at least in the transition region:AG(D) = AGH2° + m[D] [7] where m value is the slope of the linear dependence of AG(£>) on denaturant concentration. By linear extrapolation, AGH2° can be estimated as the y-intercept of least-squares fitting of AG(£>) ~ [D] in [7], Cell culture[000323] Neuro-2a cells were obtained from ATCC and maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% atmospheric CO2. Lymphoblastoid cell lines from Parkinson’s patients and healthy donors were created by Epsteine-Barr virus (EBV)-mediated transformation of cells from the peripheral blood mononuclear cell (PBMC) layer at the interface of Ficoll-paque Plus (Sigma-Aldrich) gradients. In each case, an aliquot of 5x106PBMCs was transformed with EBV from the cell line B95.8 using cyclosporin A (0.5 pg / mL, Sigma-Aldrich) to reduce the innate immune response and rejection of the virus. Upon successful establishment, cell lines were expanded into larger cultures. Human lymphoblasts were maintained in Gibco Roswell Park Memorial Institute (RPMI) 1640 Medium supplemented with 10% fetal bovine serum (FBS) at 37 °C in a humidified incubator with 5% atmospheric CO2. Access to clinical samples used in this study was approved by the Ethics, Integrity and Biosafety Team of La Trobe University under human ethics HEC20445. Clinical information is shown in Table 2.Table 2. Information of clinical samples shown in Figure 4.Drug treatment and transfection[000324] Neuro-2a cells were seeded overnight in a 12-well plate for flow cytometry measurement, in an 8-well p-slide for confocal microscopy imaging, in a 55-mm Petri dish for proteomic profiling, or in a 145-mm Petri dish for fluorescence-activated cell sorting (FACS), to reach around 80 % confluency. For proteostatic stressor treatment, Neuro-2a cells were treated with 5 pg / mL tunicamycin for 18 h, or 50 pM MG132 for 4 h as experimental groups, as well as the same amount of DMSO as control. For Httexl overexpression, Neuro-2a cells weretransiently transfected with the vectors using Lipofectamine 3000 according to manufacturer’s instructions for 48 h. Representative confocal images of cells expressing mCherry only, Httexl - 25Q-mCherry, Httex1-46Q-mCherry and Httex1 -97Q-mCherry are shown in Figure 18.Probe cell staining[000325] TME was dissolved in DMSO as 1 mM stocks and stored at - 20 °C in the dark. Neuro-2a cells or lymphoblasts were first rinsed with PBS and then incubated with freshly diluted 50 pM TME in cysteine-free DMEM for 30 min at 37 °C. Cells were rinsed with PBS again prior to downstream analysis or treatment. Cells were optionally stained with TO-PRO-3 for gating live / dead in flow cytometry, or DRAQ5 and ER-Tracker Red for staining nucleus and ER respectively for confocal imaging.Cell viability assay[000326] Neuro-2a cells were stained with 25, 50 or 75 pM TME for 30 or 60 min as above. Cells were also incubated with the same amount of DMSO as vehicle controls. After staining, cells were incubated with 10% AlamarBlue reagent (Thermo Fisher) in complete DMEM at 37 °C for 4 h in darkness. Fluorescence was measured using a plate reader. Excitation / Emission was set to 560±15 / 590±20 nm. Positive controls (by killing cells with 70% ethanol) and negative controls (cultured in complete DMEM without any treatment) were set. Cell viability was calculated by the following formula: Normalized cell viability = (sample RFU - positive control RFU) / (negative control RFU - positive control RFU), where RFU represents “relative fluorescence units”.Confocal microscopy[000327] Probe (e.g. TME) stained cells and vehicle controls were prepared as above, fixed with 4% Paraformaldehyde and imaged on a Zeiss LSM800 confocal microscope. Excitation / Emission settings were 405 / 450-520 nm for TME, 561 / 560-620 nm for ER-Tracker Red, 561 / 565-620 nm for mCherry, and 640 / 650-700 nm for DRAQ5.Flow cytometry and cell sorting[000328] For flow cytometric measurement, cells were harvested in PBS supplemented with 1 mM EDTA and 1% FBS and analyzed by a BD FACS Canto II flow cytometer. For cell sorting, cells were resuspended in PBS supplemented with 10 U / mL DNase I, filtered by 100-pm cell strainers, and sorted by a BD FACS Aria III cell sorter. Excitation / Emission settings were 405 / 450±40 nm for TME fluorescence, 561 / 610±20 nm for mCherry fluorescence, and 635 / 660±20 nm for TO-PRO-3 fluorescence. Pulse height, width and area parameters for each channel were collected. The gating strategies are shown in Figure 17. Flow cytometry data were analyzed with in-house R scripts.RUBICON workflow[000329] Probe (e.g. TME) labeled cells and vehicle controls (cells treated with the same amount of DMSO) were prepared in triplicates as above. Cells were washed with PBS, resuspended in PBS supplemented with 1 mM PMSF and protease inhibitor, and lysed bysonication. 100 g of cell lysate was aliquoted for protein abundance profiling. Cu-catalyzed Azide-Alkyne Cycloaddition (CuAAC) was initiated by mixing the following components to their final concentrations: 1 mM CuSO THPTA (1 :5), 5 mM aminoguanidine hydrochloride, 0.25 mM biotin-azide, 2 mg / mL cell lysate, and 5 mM sodium ascorbate. The reaction was carried out at room temperature for 2 h. Proteins were then precipitated by trichloroacetic acid (TCA) precipitation and resolubilized in 8 M urea. Proteins were diluted to a final concentration of 1 M urea with wash buffer (2 M NaCI, 1% Tween 20 in PBS) and incubated with Dynabeads Streptavidin (Thermo Fisher) to enrich TME labeled proteins. Beads were gently washed by wash buffer three times to remove non-specific bindings, followed by 50 mM TEAB wash for three times.Probe labeled proteins on SDS-PAGE[000330] For BLG samples, 0.6 mM BLG was labeled with 0.12 mM TME and unfolded by heating at 75 °C for 1 h. For cell lysate, cells were stained and lysed as above. Both protein samples were processed by the RUBICON workflow. Washed Dynabeads were resuspended in 0.1% SDS and boiled for 5 min to release immobilized biotinylated proteins. Eluents were visualized by SDS-PAGE. Original gel figures are shown in Figure 28.Western blotting[000331] Proteins enriched from the RUBICON workflow were loaded on a 4-12% Bis-Tris gel (Invitrogen) for western blot analysis. The Mini Gel Tank system (ThermoFisher) with MES SDS (ThermoFisher) as running buffer was used. Electrophoresis was carried out at 120 V for 60 mins. Proteins were transferred to a nitrocellulose membrane (Cytiva) during a wet transfer (1 x Tris-Glycine buffer with 20% EtOH) for 2.5 h at a constant 200 mA. Membranes were blocked with 5% non-fat dry milk in TBS-T. Antibodies were diluted in 1% milk in TBS-T and incubated overnight at 4 °C while rotating. The following antibodies were used: P4hb (1 :1000, Proteintech, 11245-1 -AP), Emc8 (1 :1000, Proteintech, 19889-1 -AP), Bcatl (1 :2000, Proteintech, 13640-1- AP). Membranes were washed with TBS-T. HRP-coupled secondary antibodies (1 :10.000, Dako, mouse P0161 , rabbit P0448) were incubated for 1 h at room temperature. Membranes were developed with ECL Detection Reagent (Cytiva). Chemiluminescence was detected on a ChemiDoc MP (Bio-Rad) and analyzed using the ImageLab 6.1 software (Bio-Rad). Membranes were re-probed repeatedly following antibody stripping for 2x 5 mins in mild stripping buffer (0.2 M Glycine, 0.1% SDS, 1% Tween20, pH 2.2), followed by PBS and TBS-T washes and reblocking in 5% milk. Original western blot results are shown in Figure 29.Cell fractionation by ultracentrifugation.[000332] Sorted Neuro-2a cells were resuspended in 50 pL ice-cold Buffer 1 (50 mM Tris-HCI pH 7.4, 150 mM NaCI, 1% (v / v) IGEPAL CA-630, 10 U / mL DNase I, 1 mM PMSF, protease inhibitor) and extruded through a 27 G needle for 25 times, followed by a 31 G needle for 10 times. 0.5 M EDTA was added a final concentration of 2 mM. A 10 pL of aliquot was designated as the total (T) sample. The rest of 40 pL of aliquot was then centrifuged at 100,000 g for 20 minat 4 °C. The resultant supernatant was designated as the supernatant (S) sample. The pellet was carefully washed in Buffer 1 (without DNase I) three times, each followed by centrifugation at 100,000 g for 20 min at 4 °C. The resultant pellet was designated as the pellet (P) fraction and resuspended by Buffer 2 (Buffer 1 , 2% SDS, 2 mM DTT). 2% SDS and 2 mM DTT were also added to T and S. T, S and P were then heated at 95 °C for 20 min. P was cooled down at room temperature and urea was added to a final concentration of 8 M to fully dissolve the pellet. T, S and P were finally absorbed and immobilized into the gel matrix for mass spectrometry analysis.Sample preparation for proteomics.[000333] For RUBICON, Dynabeads were reduced with 10 mM TCEP, alkylated with 55 mM iodoacetamide in the dark, and digested with trypsin overnight with shaking at 37 °C.Dynabeads were discarded by centrifugation and the supernatant was transferred to a new Eppendorf low-binding tube. For lysate profiling, 100 pg of cell lysate was precipitated with acetone at -20 °C overnight and resolubilized with 8 M urea in 50 mM TEAB pH 8.0. Protein solutions were reduced with 10 mM TCEP, alkylated with 55 mM iodoacetamide in the dark, diluted to final 1 M urea, and digested with trypsin overnight with shaking at 37 °C. For Httexl samples, gels were cut into small pieces, washed with LC-MS grade water three times, and fixed with fixation buffer (50% methanol, 10% acetic acid). The gels were then washed with destaining buffer (50% acetonitrile, 25 mM ammonium bicarbonate) three times and 100% acetonitrile three times, before being reduced with 10 mM DTT, alkylated with 55 mM iodoacetamide in the dark, and digested with trypsin overnight with shaking at 37 °C. Gel pieces were discarded by centrifugation and the supernatant was transferred to a new Eppendorf low- binding tube. The resultant peptides from the above on-bead, in-solution and in-gel digestions were desalted using StageTips and freeze dried. Peptides were resuspended in 2% acetonitrile and 0.05% trifluoroacetic acid, quantified using a Micro BCA assay (Thermo Fisher), and finally subjected to mass spectrometer for label-free quantification.NanoESI-LC-MS / MS analysis.[000334] Samples were analyzed by LC-MS / MS using Orbitrap Lumos / Eclipse mass spectrometer (Thermo Scientific) fitted with nanoflow reversed-phase-HPLC (Ultimate 3000 RSLC, Dionex). The nano-LC system was equipped with an Acclaim Pepmap nano-trap column (Dionex - C18, 100 A, 75 pm x 2 cm) and an Acclaim Pepmap RSLC analytical column (Dionex - C18, 100 A, 75 pm x 50 cm). Typically for each LC-MS / MS experiment, 2 pL of the peptide mix was loaded onto the enrichment (trap) column at an isocratic flow of 5 pL / min of 3% Acetonitrile containing 0.1% formic acid for 6 min before the enrichment column is switched inline with the analytical column. The eluents used for the LC were 0.1% v / v formic acid in water (solvent A) and 100% Acetonitrile / 0.1 % formic acid v / v (Solvent B). For RUBICON samples, the gradient used was 3% B to 23% B for 29 min, 23% B to 40% B in 10 min, 40% B to 80% B in 5 min and maintained at 80% B for the final 5 min before equilibration for 10 min at 3% B prior tothe next analysis. For lysate samples, the gradient used was 3% B to 23% B for 89 min, 23% B to 40% B in 10 min, 40% B to 80% B in 5 min and maintained at 80% B for the final 5 min before equilibration for 10 min at 3% B prior to the next analysis. All spectra were acquired in positive mode with full scan MS spectra scanning from m / z 375-1400 at 70000 resolution with AGC target of 3e6 with a maximum accumulation time of 50 ms. The 15 most intense peptide ions with charge state2-5 were isolated with an isolation window of 1 .2 m / z and fragmented with a normalized collision energy of 30 at 17500 resolution with AGC target of 5e4 with a maximum accumulation time of 50ms. Underfill threshold was set to 2% for triggering of precursor for MS2. Dynamic exclusion was activated for 30s.Proteomic data analysis.[000335] Mass spectra were searched against the UniProtKB / Swiss-Prot Homo sapiens (Organism ID: 9606) and Mus musculus (Organism ID: 10090) database (with additional protein sequences of mCherry and Httex1 -46Q-mCherry for Httexl samples) using MaxQuant (version 2.0.3.0.). Default parameters were used unless otherwise specified. ‘Label-free quantification’, ‘iBAQ’ and ‘Match between runs’ were checked. Oxidation (M) and acetyl (Protein N-term) were selected as variable modifications for all samples. For cell lysate and Httexl samples, carbamidomethyl (C) was selected as the fixed modification. For RUBICON samples, it should be noted that TME labeled peptides remained on Dynabeads and were not eluted, therefore only non-cysteine containing peptides were used to quantify the protein abundance. To discard all cysteine-containing peptides for analysis of RUBICON samples, a pseudo-cysteine modification (i.e., C(1000) H(1000)) was manually created and set as the fixed modification instead of carbamidomethyl (C). Normalized label-free quantification (LFQ) intensity was used for quantification and analysis. Proteins marked as ‘Potential contaminants’, ‘only identified by site’ or ‘reverse identification’ were discarded. Proteins with valid values in at least three biological replicates in at least one experimental condition were selected for analysis. Missing values were imputed using a left-censored normal distribution (ND). Briefly, missing values were replaced by random drew values from a normal distribution on the left tail of the abundance distribution of the complete dataset. The mean and standard deviation of this left-censored normal distribution was determined as: p, == 0.3om(p = mean, o = standard deviation, i = values for imputation, m = measured values). Resulting P-values were adjusted by Benjamini-Hochberg procedure as the multi-test adjustment method for controlling the false discovery rate. Differential proteins were identified with fold change ratios > 1 .5 (or < -1 .5) and adjusted P < 0.05. For RUBICON samples, the non-cysteine-containing proteins were removed for downstream analysis (see Table 3). Characterization of the quantitative performance and reproducibility of all datasets is shown in Figure 26.Table 3. Summary of the total number of TME binders and non-cysteine-containing proteins in different datasets.Protein abundance quantification[000336] Intensity-Based Absolute Quantification (iBAQ) is an approximation of protein copy numbers based on the sum of peptide-feature intensities of all peptides matching to a protein divided by the number of theoretically observable peptides. The protein abundance of TME binders in the tunicamycin, MG132 (Neuro-2a cells) and PD (lymphoblastoid cells) datasets was estimated as the iBAQ value in their corresponding lysate profiling datasets using MaxQuant as described above. TME binders that were only detected by RUBICON but not the lysate profiling methods were denoted as “undetectable”. To further examine the protein abundance of these undetectable proteins, Protein Abundance Database (PaxDb) and previously reported proteomic datasets were used as standard reference for protein abundance. Considering the experimental conditions, protein coverage, data availability and data relevance, protein abundance quantification in a reported deep proteomic analysis of Neuro-2a cells, in M.musculus - Whole organism (Integrated) (Coverage: 90%, URL: https: / / pax-db.org / dataset / 10090 / 3224231993 / ) and M.musculus - Brain (Integrated) (Coverage: 51%, URL: https: / / pax- db.org / dataset / 10090 / 961549256 / ) was mapped to the tunicamycin and MG132 datasets (Neuro- 2a cell line). Protein abundance quantification in H. sapiens - Whole organism (Integrated) (Coverage: 99%, URL: https: / / pax-db.org / dataset / 9606 / 3902734007 / ) and H. sapiens - Lymph node (Integrated) (Coverage: 53%, URL: https: / / pax-db.org / dataset / 9606 / 952965544 / ) was mapped to the PD (lymphoblastoid cells) dataset. P value was calculated using the unpaired two- samples two-sided Wilcoxon test.Analysis of cysteine functional annotation and cellular localization.[000337] Custom R-scripts were developed to query the UniProtKB / Swiss-Prot Protein Knowledgebase release 2022 03 downloaded on 01 / 09 / 2022. Cysteine functional annotation and subcellular location of the relevant UniProt entry was mined. “Active-site” denotes proteins that contain at least one cysteine annotated as “ACT_SITE”; “Nucleophile” denotes proteins that contain at least one cysteine annotated as “Nucleophile”; “Redox-active Disulfide” denotes proteins that contain at least one pair of disulfide bond annotated as “Redox-active”. P value was calculated by Fisher's Exact Test.Functional enrichment analysis.[000338] The list of protein of interest (POI) was analyzed by the R package “clusterProfiler” and “ReactomePA”. Functional terms under investigation included Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and REACTOME pathway database. Filtering criteria for enriched terms were fold enrichment3 and FDR-adjusted P < 0.05. Each enriched functional term generated a subgraph showing the protein-protein interaction (PPI) network of all genes in the list of POI that belong to this functional term. The PPI information was gleaned from the R package “STRINGdb”, version = “11”, score_threshold = 200. For each PPI subgraph, node size, node color and edge transparency denote number of interactors (degrees), class of functional clusters and combined_score, respectively. Representative terms were visualized as non-transparent with labeled description text while other terms were shown as 10% transparency. X and Y coordinates denote minus logarithmic P value and fold enrichment of the term.Prediction and analysis of protein disorder[000339] Protein amino acid sequence was analyzed by IUPred2A. Predictions were made on a scale of 0-1 , where any residue with a score over 0.5 was predicted to be in a disordered region, and any residue scoring below 0.5 was predicted to be ordered. For each protein, mean IUPred2 and ANCHOR2 score were calculated as the mean of IUPred2 and ANCHOR2 scores of all residues of a protein. P value was calculated using the unpaired two-samples two-sided Wilcoxon test. Disordered regions are defined as continuous sequences exceeding a threshold length, wherein all IUPred2 scores were greater than 0.5. The threshold length was set at 15 and 30. Proteins were then classified into six types, defined as follows. Type 0: Non-cysteine containing proteins, which were discarded from subsequent analysis. Type 1 -3 were all cysteine containing proteins (Figure 23a). Specifically, Type 1 were structured proteins with less than 5% of residues having an IUPred2 score greater than 0.5. Type 2 were proteins with IDRs, where the percentage of residues with an IUPred2 score greater than 0.5 was between 5% and 80%. Within the proteins with IDRs, Type 2a were proteins with cysteines only located in the structured regions; Type 2b were proteins with cysteines only located in the disordered regions; and Type 2c were proteins with cysteines located in both structured and disordered regions. Type 3 were IDPs, where the percentage of residues with an IUPred2 score greater than 0.5 was greater than 80%. P value was calculated by Fisher's Exact Test.Protein-protein interaction (PPI) network analysis[000340] For protein-protein interaction (PPI) network analysis, the PPI information was gleaned from the R package “STRINGdb”, version = “11”, score_threshold = 600. Degrees and betweenness centrality were calculated using the R package “igraph”. P value was calculated using the unpaired two-samples Wilcoxon test.Clustering of proteins upon 46Q overexpression and aggregation.[000341 ] Protein abundance across populations of Stage I , nn, naand HI was analyzed by label-free quantification, for T, S and P respectively. Filtering criteria for cluster-D were (1 ) P1> P2n + Iog2(1 .5) or P1 > P2a + Iog2(1 .5); (2) P1 > P3 + Iog2(1 .5); (3) P2n > P3 or P2a > P3 where P1 , P2n, P2a and P3 denotes mean intensity of triplicates of Stage I , nn, naand HI respectively. Filtering criteria for cluster-A were (1) P3 > P2n + Iog2(1 .5) or P3 > P2a + Iog2(1 .5); (2) P3 > P1 + Iog2( 1 .5); (3) P2n > P1 or P2a > P1 . Dendrograms were visualized by the R package “ComplexHeatmap”.Data visualization.[000342] Plots were visualized using R packages “ggplot2”, “ComplexHeatmap”, “ggprism”, “cowplot”, “grid” and “ggraph”. Statistical significance level was visualized as: ‘ns’, no significance; *, P < 0.05; “, P < 0.01 ; P < 0.001 ; P < 0.0001 .Chemical probe screening[000343] The final loading concentration for protein lactoglobulin (P-Lac) and bovine serum albumin (BSA) was set as 100 pM for all lanes. Upon 0.5 h of labelling by 50 pM of respective chemical probe, proteins were then mixed with loading buffer, containing 5% 2-mercaptoethanol and analyzed by SDS-PAGE.Plasma sample preparation, chemical probe screening and SDS-PAGE[000344] For plasma samples, 500 pg of protein was labelled by 50 pM of respective chemical probe for 0.5 h and subsequently treated with 1 mM PMSF and protease inhibitor for 1 h. Then copper-catalyzed azide-alkyne cycloaddition (CuAAC) was initialized by mixing the following components to their final concentrations: 1 mM CuSC / THPTA (1:5 dilution), 5 mM aminoguanidine hydrochloride, 0.25 mM biotin azide, 2 mg ml-1cell lysate and 5 mM sodium ascorbate. The reaction was carried out at room temperature for 2 h. Proteins were then precipitated by trichloroacetic acid precipitation and resolubilized in 8 M urea. Proteins were diluted to a final concentration of 1 M urea with wash buffer (2 M NaCI, 1% Tween 20 in PBS) and incubated with Dynabeads Streptavidin (Thermo Fisher) to enrich probe-labeled proteins. Beads were gently washed by wash buffer three times to remove nonspecific bindings, followed by 50 mM TEAB washing buffer for three times. Washed Dynabeads were resuspended in 0.1% SDS and boiled for 10 min to release immobilized biotinylated proteins, mixed with loading buffer, containing 5% 2-mercaptoethanol and visualized by SDS-PAGE.[000345] Chemical probes (TME, 38 (PT_N038), 56 (PT_N056), 67 (PT_N067)) were tested through reaction with model proteins, p-lactoglobulin (P-Lac) and bovine serum albumin (BSA) and show differences in reactivity of chemical probes as well as sensitivity toward structure of proteins (Figure 30 - 31). When comparing fluorescence measurements for TME and 38 (PT_N038), an increase in reactivity was observed for both probes and BSA, for p-Lac when unfolded. 38, unlike TME, had increased fluorescence suggesting increased reactivity for BSA upon unfolding conditions (Figure 30a). Comparing fluorescence intensities for changes in structure of p-Lac, it was observed that probes 38 (PT_N038) and 56 (PT_N056) had excellent responses toward unfolded p-Lac (Figure 30, 31). Since p-Lac could easily demonstrate probe sensitivity toward protein structural change, this was relied upon for further screening,demonstrating that probes 86 (PT_N086), 95 (PT_N095), 98 (PT_N098) and 109 (PT_N109) were also capable of detecting structural changes, with 86 (PT_N086) and 98 (PT_N098) giving a higher fluorescence, suggesting an increased reactivity with unfolded p-Lac (Figure 37). [000346] Following screening experiments, validation of the probes was performed to see if they could be used in different sample matrix and the RUBICON workflow. This was tested with whole human plasma, and differences in labelling were observed for chemical probes toward plasma including enriched proteins (Figure 32). Probe 56 (PT_N056) and TME were selected for plasma proteomic experiments and identified plasma proteins, further demonstrating enrichment of specific proteins unique to each chemical probe using the RUBICON workflow (Figure 33). Following the successful plasma experiments and seeing that SDS-PAGE gel could be used as a screen for further successful proteomics experiments, probes 38 and 56 were tested on whole plasma from healthy control and breast cancer patients. There were differences in labelling observed between healthy control (HC) and breast cancer (BC) patient (Figure 36a). Successful enrichment of proteins was also observed using 38 (PT_N038) and 56 (PT_N056), showing that the probes were compatible with the RUBICON workflow enabling downstream applications (Figure 36b-c).[000347] Looking at human fibroblasts, from health control and myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS) patients, preliminary experiments were performed using TME which showed differences in labelling of cell lysates between health control and ME / CFS patient as well as in the enrichment workflow (Figure 35). With this promising result, proteomics experiments were performed, demonstrating with TME the ability to identify unique proteins and showing enrichment of specific proteins unique to healthy control and ME / CFS using the RUBICON workflow (Figure 34).[000348] Other embodiments of the invention as described herein are defined in the following paragraphs:1 . A compound of Formula I, or a salt thereofFormula I wherein A, B, C, and D are independently selected from the group consisting of H, CN, unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: said substituted aryl and said substituted heteroaryl is independently substituted with one or more groups independently selected from the group consisting of Z-Y- C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3- Ci-C6alkyl, Z4-Y5-CI-C6heteroalkyl, Z4-CI-C6heteroalkyl, Z5-Y6-C2-C6alkenyl, Z5-C2-C6 alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7- aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24- halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20- SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; wherein at least two of A, B, C, and D are independently selected from the group consisting of unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E; wherein: each incidence of Z, Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, and Z12is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C1-C10 alkenyl, and C1- C heteroalkenyl, each of which may be optionally substituted with =0; each incidence of R and R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, SO2-C1-C6 alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-C alkenyl, and Z16-Ci-C heteroalkenyl; each incidence of Z13, Z14, Z15, and Z16is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; andE is a group which is capable of reacting with a thiol group; optionally, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide, also comprises one or more other substituents on the aryl or heteroaryl ring which are independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8- aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7 cycloalkyl, Z9-C3-C? cycloalkyl, Z10-Y11- C3-C7 cycloheteroalkyl, Z10-C3-C7 cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2. For example, one of A, B, C, and D may be substituted with Z-Y-C2-C6 alkynyl and Z6-C2-Ce heteroalkenyl; optionally, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide, does not have any other substituents on the aryl or heteroaryl ring; optionally the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent which is selected from the group consisting of Z12-Y17- E and Z12-E, also comprises one or more other substituents on the aryl or heteroaryl ring which are independently selected from the group consisting of Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3-Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5- Y6-C2-Ce alkenyl, Z5-C2-Ce alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8- aryl, Z7-aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7 cycloalkyl, Z9-Cs-C7 cycloalkyl, Z10-Y11- C3-C7 cycloheteroalkyl, Z10-C3-C7 cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24-halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20-SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SC>2NH2. For example, one of A, B, C, and D may be substituted with Z12-E and Z6-Y7- C2-C6 heteroalkenyl; optionally, the one of A, B, C, and D that is substituted aryl or substituted heteroaryl, which comprises a substituent which is selected from the group consisting of Z12-Y17- E and Z12-E, does not have any other substituents on the aryl or heteroaryl ring; optionally, each incidence of Z is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z1is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z2is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z3is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z4is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z5is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z6is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z7is independently selected from the group consisting of a bond, O, NR1, OCO,and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z8is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z9is independently selected from the group consisting of a bond, O, NR1, SO2, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z10is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z11is independently selected from the group consisting of a bond, O, NR1, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, each incidence of Z12is independently selected from the group consisting of a bond, O, NR1, SO2, OCO, and COO, optionally selected from the group consisting of a bond, O and NR1; optionally, Y is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y1is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y2is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y3is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y4is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y5is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y6is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y7is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y8is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y9is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y10is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y11is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y12is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y13is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y14is a bond or a linker selected from C1- C alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y15is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y16is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y17is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y18is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y19is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y20is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y21is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y22is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y23is a bond or a linker selected from C1-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, Y24is a bond or a linker selected from Ci-C10 alkyl, and C1-C10 heteroalkyl, optionally a bond; optionally, R is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-Cio heteroalkyl, Z15-Ci-C alkenyl, and Z16-Ci-C heteroalkenyl; optionally, R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each of Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13- C1-C10 alkyl, Z14-Ci-Cio heteroalkyl, Z15-Ci-C alkenyl, and Z16-Ci-C heteroalkenyl; optionally, each incidence of Z13is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; optionally, each incidence of Z14is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; optionally, each incidence of Z15is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; optionally, each incidence of Z16is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; optionally one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E are the same group, i.e. they are one of A, B, C, or D; optionally the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and the one of A, B, C, and D which is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E are different groups, i.e. they are two of A, B, C, and D; optionally, A and D are independently selected from the group consisting of unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; and C and B are independently selected from the group consisting of H, CN, unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; optionally A and D are independently selected from the group consisting of optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline; and C and B are independently selected from the group consisting of H, CN, optionally substituted phenyl, optionally substituted naphthyl, optionally substituted furan, optionally substituted pyrrole, and optionally substituted quinoline; optionally, one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazideand Z2-C2-Ce alkenylazide; and another of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E; optionally one of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole, or quinoline, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2- C2-C6 alkenylazide; and another of A, B, C, and D is substituted phenyl, naphthyl, furan, pyrrole, or quinoline, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E; optionally Z12-Y17-E and Z12-E are selected from the group consisting of:optionally A, B, C, and D are independently selected from the group consisting of: H, CN,2. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein: each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C2-C10 alkenyl, and C2- C heteroalkenyl; and each incidence of R and R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl.3. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein E is selected from the following: a maleimide, a halo-acetamide, a vinyl acetamide, an aryl acetamide,wherein R’ is independently selected from the group consisting of H, C1-C12 alkyl, and aryl, and X is halo; optionally wherein4. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein at least two of A, B, C, and D are substituted phenyl; wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: Z-C2-C6 alkynyl, azide, Z1-Ci-Ce alkylazide, and Z2-C2-Ce alkenylazide; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E.5. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein two of A, B, C, and D are unsubstituted phenyl.6. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR2-C2-C3 alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E; wherein R2is H or C2-C4 alkyl-OH.7. The compound or salt thereof of according to any one or more of the preceding paragraphs, wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR3-C2-Cs alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is:wherein R3is H or C2-C4 alkyl-OH.8. The compound or salt thereof according to any one or more of the preceding paragraphs, wherein one of A, B, C, and D is substituted phenyl, which is substituted with O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, ynyl; and another of A, B, C, and D is substituted phenyl, which is substituted wit; wherein R4is H or C2-C4 alkyl-OH.9. The compound or salt thereof of according to any one or more of the preceding paragraphs, wherein:• two of A, B, C, and D are unsubstituted phenyl;• one of A, B, C, and D are substituted phenyl, which is substituted with the following group:• the other of A, B, C and D are substituted phenyl, which is substituted with the following group:10. The compound or salt thereof according to any one or more of the preceding paragraphs, which is a compound of Formula II, or salt thereof:Formula II wherein:B1and B2are independently selected from hydrogen, cyano, and phenyl;A1is phenyl; andA2is an aryl or heteroaryl group selected from the group consisting of phenyl, indole, and quinoline; wherein: one of A1or A2is substituted with a group selected from -C2-C4 alkynyl, -N(RA)-C2-C4 alkynyl, -O-C2-C4 alkylazide, and -O-C2-C4 alkynyl, wherein RAis selected from C1-C4 alkyl-O-CO-Ci-C4 alkyl, C1-C4 alkyl-COO-Ci-C4 alkyl, and -SO2-C1-C4 alkyl; and one of A1or A2is substituted with a group selected from:, wherein X is a halogen, optionally chlorine; andA1and A2are optionally substituted with one or more groups selected from the following: -N(Cr C4alkyl)2, -O-C1-C4 alkyl, -O-C2-C4 alkynyl, -COO-C1-C4 alkyl, halogen, and cyano.11 . The compound or salt thereof according to any one or more of the preceding paragraphs, which is selected from the following:and salts and / or cis / trans isomers thereof.12. The compound or salt thereof according to any one or more of the preceding paragraphs, which has the following structure:salt and / or cis / trans isomer thereof.13. A method of diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• contacting a biological sample of the subject with the compound or salt thereof according to any one or more of the preceding paragraphs; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to determine whether the subject has, or is likely to develop, the disease or disorder;optionally, the sample may include cells from the subject, and the method may include a further step of lysing the cells; optionally, the lysing may be performed after contacting the biological sample with the compound or salt thereof; optionally, the lysing may be performed by, for example, a sonication step; optionally, the sample may be a blood, e.g. plasma, sample. 14. The method according to any one or more of the preceding paragraphs, further comprising the following steps: o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a biotin binding agent, optionally wherein the biotin binding agent is a streptavidin-functionalised agent, optionally wherein the streptavidin-functionalised agent is a streptavidin-functionalised bead, optionally a streptavidin-functionalised agarose bead; or o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally azide or alkyne functionalised bead, and o collecting the protein-agent conjugate; optionally, reacting the bound compound or salt thereof with an azide functionalized biotin or alkyne functionalized biotin may include a copper-catalyzed azide-alkyne cycloaddition; optionally, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized biotin or alkyne functionalized biotin with a copper solution, optionally copper sulfate; optionally, the reaction may be performed by a step of mixing the bound compound or salt thereof in the form of a cell lysate optionally having a concentration of proteins of from about 0.1 mg / mL to about 10 mg / mL, or from about 1 mg / mL to about 5 mg / mL, or about 0.1 , 0.2, 0.5, 1 , 2, 5, or 10 mg / mL, and an azide or alkyne functionalized biotin, such as / V-(3-azidopropyl)biotinamide, at a concentration of from about 0.05 mM to about 1 mM, or from about 0.1 mM to about 1 mM, or about 0.05, 0.1 , 0.2, 0.25, 0.3, 0.4, 0.5, or 1 mM; optionally, reacting the bound compound or salt thereof with an azide functionalized agent or alkyne functionalized agent may include a copper-catalyzed azide-alkyne cycloaddition; optionally, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized agent or alkyne functionalized agent with a copper solution, optionally copper sulfate; optionally, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers; optionally, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected; optionally the method may further include a cleavage step, for example, an acid cleavage step,to cleave the bound protein from the solid phase or resin thereby enabling separation and isolation of the bound protein from the solid phase or resin; optionally the method may include a protein precipitation step, which may, for example, include a pH modification, optionally an acidification, optionally using trichloroacetic acid; optionally the protein precipitation step may be performed prior to the step of collecting the protein-biotin conjugate using a biotin binding agent; optionally the biotin binding agent includes streptavidin, for example a streptavidin functionalized solid phase, such as a streptavidin functionalized bead; optionally the method may further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate.15. The method according to any one or more of the preceding paragraphs, wherein detecting the compound or salt thereof in the form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence or an absorbance, optionally a UV absorbance; performing mass spectrometry, optionally LC-MS / MS; or electrophoresis, optionally SDS-PAGE or Western blot.16. Use of the compound or salt thereof according to any one or more of the preceding paragraphs, for diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.17. The compound or salt thereof according to any one or more of the preceding paragraphs, for use in diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.18. A method of treating or preventing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• determining whether the subject has, or is likely to develop, the disease or disorder using the method according to any one or more of the preceding paragraphs; and• if the subject has the disease or disorder, administering a therapeutically effective amount of a medicament capable of treating or preventing said disease or disorder to the subject.19. A method for enriching an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps: o contacting the compound or salt thereof according to any one or more of the preceding paragraphs with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein;or o contacting the compound or salt thereof according to any one or more of the preceding paragraphs with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, and o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein; optionally, the streptavidin-functionalized agent may be a streptavidin functionalized solid phase, such as a streptavidin functionalized bead; optionally, the method may further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein; optionally, reacting the bound compound or salt thereof with an azide functionalized agent or alkyne functionalized agent may include a copper-catalyzed azide-alkyne cycloaddition; optionally, the reaction may be performed by a step of mixing the bound compound or salt thereof and an azide functionalized agent or alkyne functionalized agent with a copper solution, optionally copper sulfate; optionally, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers; optionally, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected; optionally, the method may further include a cleavage step, for example, an acid cleavage step, to cleave the bound protein from the solid phase or resin thereby enable enrichment of the unfolded, misfolded or intrinsically disordered protein by separation and isolation of the bound protein from the solid phase or resin.20. A method for identifying an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps: o contacting the compound or salt thereof according to any one or more of the preceding paragraphs with the sample containing the unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate,o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein; or o contacting the compound or salt thereof according to any one or more of the preceding paragraphs with the sample containing the unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein; optionally, the streptavidin-functionalized agent may be a streptavidin functionalized solid phase, such as a streptavidin functionalized bead; optionally the method may further include a step of washing the streptavidin functionalized bead, and subsequently eluting the protein-biotin conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein; optionally, the azide functionalised agent or alkyne functionalised agent is an azide-functionalised solid phase or alkyne-functionalised solid phase, such as, for example, an azide-functionalised resin or alkyne-functionalised resin, optionally an agarose resin that is functionalized with azide or alkyne groups and acid-cleavable linkers; optionally, the solid phase or resin may be washed to remove any non-bound species, and subsequently collected; optionally, the method may further include a cleavage step, for example, an acid cleavage step, to cleave the bound protein from the solid phase or resin thereby enable enrichment of the unfolded, misfolded or intrinsically disordered protein by separation and isolation of the bound protein from the solid phase or resin.21 . A method of detecting an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps:• contacting the sample with the compound or salt thereof according to any one or more of the preceding paragraphs;• optionally enriching the unfolded, misfolded or intrinsically disordered protein, according to the method according to any one or more of the preceding paragraphs; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to thereby detect the unfolded, misfolded or intrinsically disordered protein, optionally wherein detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence; absorbance, optionally UV absorbance; and / or liquid chromatography and / or performing mass spectrometry.22. A method for screening a drug candidate for treating a disease or disorder associated with an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps:• contacting said drug candidate with a sample containing the unfolded, misfolded or intrinsically disordered protein; and• detecting the unfolded, misfolded or intrinsically disordered protein in the sample according to the method according to any one or more of the preceding paragraphs to determine whether the drug candidate is capable of treating the disease or disorder.23. The method, use, or compound or salt thereof for use according to any one or more of the preceding paragraphs, wherein the disease or disorder is selected from the group consisting of Huntington’s disease; Alzheimer's disease; Parkinson’s disease; thrombosis; stroke; autism; obesity and metabolic diseases; cardiovascular diseases; chronic liver disease and cirrhosis; nephritis; nephrotic syndrome; nephrosis; Creutzfeldt-Jakob diseases; cystic fibrosis; Gaucher’s disease; hereditary cerebral haemorrhage; dementia; Niemann-Pick disease; multiple system atrophy; Creutz-Jakob disease; fatal insomnia; Gerstmann-Straussler-Scheinker disease; Spongiform encephalopathy; Creutzfeldt-Jakob disease; Kuru; hereditary sensory and autonomic neuropathy; Pick disease; progressive supranuclear palsy; argyrophilic grain disease; Guam Parkinson dementia complex; frontotemporal lobar degeneration; chronic traumatic encephalopathy; ganglioglioma; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy; tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; amyloidosis; familial amyloidotic polyneuropathy; familial amyloid cardiomyopathy; amyloidosis; Type II diabetes; insulinoma; medullary carcinoma of the thyroid; motor neuron diseases; amyotrophic lateral sclerosis; atrial amyloidosis; pituitary prolactinoma; Gelatinous drop-like corneal dystrophy; calcifying epithelial odontogenic tumors; pulmonary alveolar proteinosis; hypotrichosis simplex; lattice corneal dystrophy; cancer, e.g. prostate cancer and breast cancer; myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS); and long COVID.24. The method, use, or compound or salt thereof for use according to any one or more of the preceding paragraphs, wherein the disease or disorder is prostate cancer, breast cancer,Huntington’s disease or Parkinson’s disease, optionally Huntington’s disease or Parkinson’s disease.[000349] Although the invention has been described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. In particular, features of any one of the various described examples may be provided in any combination in any of the other described examples. Various modifications and alterations to this invention will become apparent to those skilled in the art without departing from the scope and spirit of this invention. It should be understood that this invention is not intended to be unduly limited by the illustrative embodiments and examples set forth herein and that such examples and embodiments are presented by way of example only.

Claims

Claims:1 . A compound of Formula I, or a salt thereofFormula I wherein A, B, C, and D are independently selected from the group consisting of H, CN, unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: said substituted aryl and said substituted heteroaryl is independently substituted with one or more groups independently selected from the group consisting of Z-Y- C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1-azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide, Z2-C2-Ce alkenylazide, Z3-Y4-Ci-Ce alkyl, Z3- Ci-Ce alkyl, Z4-Y5-Ci-Ce heteroalkyl, Z4-Ci-Ce heteroalkyl, Z5-Y6-C2-Ce alkenyl, Z5- C2-C6 alkenyl, Z6-Y7-C2-Ce heteroalkenyl, Z6-C2-Ce heteroalkenyl, Z7-Y8-aryl, Z7- aryl, Z8-Y9-heteroaryl, Z8-heteroaryl, Z9-Y10-C3-C7cycloalkyl, Z9-C3-C7cycloalkyl, Z10-Y11-C3-C7cycloheteroalkyl, Z10-C3-C7cycloheteroalkyl, Z11-Y12-R, Z11-R, Y24- halo, Y13-CN, Y14-OH, Y15-COOH, Y16-CF3, Z12-Y17-E, Z12-E, Y18-NH2, Y19-NO2, Y20- SO3H, Y21-CONH2, Y22-NHSO3H, and Y23-SO2NH2; wherein at least two of A, B, C, and D are independently selected from the group consisting of unsubstituted aryl, substituted aryl, unsubstituted heteroaryl, and substituted heteroaryl; wherein: one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent selected from the following: Z-Y-C2-C6 alkynyl, Z-C2-C6 alkynyl, Y1- azide, Z1-Y2-Ci-Ce alkylazide, Z1-Ci-Ce alkylazide, Z2-Y3-C2-Ce alkenylazide and Z2-C2-Ce alkenylazide; and one of A, B, C, and D is substituted aryl or substituted heteroaryl, comprising a substituent which is selected from the group consisting of Z12-Y17-E and Z12-E; wherein: each incidence of Z, Z1, Z2, Z3, Z4, Z5, Z6, Z7, Z8, Z9, Z10, Z11, and Z12is independently selected from the group consisting of a bond, O, S, NR1, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C1-C10 alkenyl, and C1- C heteroalkenyl, each of which may be optionally substituted with =0; each incidence of R and R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, SO2-C1-C6 alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl, wherein each ofCi-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl are optionally substituted with one or more groups selected from Z13-Ci-C alkyl, Z14-Ci-C heteroalkyl, Z15-Ci-C alkenyl, and Z16-Ci-C heteroalkenyl; each incidence of Z13, Z14, Z15, and Z16is independently selected from the group consisting of a bond, O, S, SO2, NHSO2, SO2NH, CO, OCO, COO, NHCO, and CONH; andE is a group which is capable of reacting with a thiol group.

2. The compound or salt thereof of claim 1 , wherein: each incidence of Y, Y1, Y2, Y3, Y4, Y5, Y6, Y7, Y8, Y9, Y10, Y11, Y12, Y13, Y14, Y15, Y16, Y17Y18, Y19, Y20, Y21, Y22, Y23, and Y24is independently selected from the group consisting of a bond and L, wherein L is a linker group selected from C1-C10 alkyl, C1-C10 heteroalkyl, C2-C10 alkenyl, and C2- C heteroalkenyl; and each incidence of R and R1is independently selected from the group consisting of H, CF3, Ci-Ce alkyl, Ci-Ce heteroalkyl, C2-C6 alkenyl, and C2-C6 heteroalkenyl.

3. The compound or salt thereof of claim 1 or 2, wherein E is selected from the following: a maleimide, a halo-acetamide, a vinyl acetamide, an aryl acetamide,wherein R’ is independently selected from the group consisting of H, C1-C12 alkyl, and aryl, and X is halo; optionally wherein4. The compound or salt thereof of any one of claims 1 to 3, wherein at least two of A, B, C, and D are substituted phenyl; wherein:one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: Z-C2-C6 alkynyl, azide, Z1-Ci-Ce alkylazide, and Z2-C2-Ce alkenylazide; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E.

5. The compound or salt thereof of any one of claims 1 to 4, wherein two of A, B, C, and D are unsubstituted phenyl.

6. The compound or salt thereof of any one of claims 1 to 5, wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR2-C2-C3 alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is Z12-E; wherein R2is H or C2-C4 alkyl-OH.

7. The compound or salt thereof of any one of claims 1 to 6, wherein: one of A, B, C, and D is substituted phenyl, comprising a substituent selected from the following: O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, and NR3-C2-Cs alkynyl; and another of A, B, C, and D is substituted phenyl, comprising a substituent which is:wherein R3is H or C2-C4 alkyl-OH.

8. The compound or salt thereof of any one of claims 1 to 7, wherein one of A, B, C, and D is substituted phenyl, which is substituted with O-Ci-Ce alkylazide, C2-C3 alkynyl, O-C2-C3 alkynyl, or NR4-C2-C3alkynyl; and another of A, B, C, and D is substituted phenyl, which is substitutedwherein R4is H or C2-C4 alkyl-OH.

9. The compound or salt thereof of any one of claims 1 to 8, wherein:• two of A, B, C, and D are unsubstituted phenyl;• one of A, B, C, and D are substituted phenyl, which is substituted with the following group:• the other of A, B, C and D are substituted phenyl, which is substituted with the following group:

10. The compound or salt thereof of claim 1 , which is a compound of Formula II, or salt thereof:Formula II wherein:B1and B2are independently selected from hydrogen, cyano, and phenyl;A1is phenyl; andA2is an aryl or heteroaryl group selected from the group consisting of phenyl, indole, and quinoline; wherein: one of A1or A2is substituted with a group selected from -C2-C4 alkynyl, -N(RA)-C2-C4 alkynyl, -O-C2-C4 alkylazide, and -O-C2-C4 alkynyl, wherein RAis selected from C1-C4 alkyl-O-CO-Ci-C4 alkyl, C1-C4 alkyl-COO-Ci-C4 alkyl, and -SO2-C1-C4 alkyl; and one of A1or A2is substituted with a group selected from:, wherein X is a halogen, optionally chlorine; andA1and A2are optionally substituted with one or more groups selected from the following: -N(Cr C4 alkyl)2, -O-C1-C4 alkyl, -O-C2-C4 alkynyl, -COO-C1-C4 alkyl, halogen, and cyano.1 1 . The compound or salt thereof of claim 1 , which is selected from the following:optionally wherein the compound is selected from the following:and salts and / or cis / trans isomers thereof.

12. The compound or salt thereof of claim 1 , which has the following structure:salt and / or cis / trans isomer thereof.

13. A method of diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• contacting a biological sample of the subject with the compound or salt thereof of any one of claims 1 to 12; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to determine whether the subject has, or is likely to develop, the disease or disorder.

14. The method of claim 13, further comprising the following steps: o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a biotin binding agent, optionally wherein the biotin binding agent is a streptavidin-functionalised agent, optionally wherein the streptavidin-functionalised agent is a streptavidin-functionalised bead, optionally a streptavidin-functionalised agarose bead; or o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally azide or alkyne functionalised bead, and o collecting the protein-agent conjugate.

15. The method of claim 13 or 14, wherein detecting the compound or salt thereof in the form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence or an absorbance, optionally a UV absorbance; performing mass spectrometry, optionally LC-MS / MS; or electrophoresis, optionally SDS-PAGE or Western blot.

16. Use of the compound or salt thereof of any one of claims 1 to 12, for diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.

17. The compound or salt thereof of any one of claims 1 to 12, for use in diagnosing or prognosing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject.

18. A method of treating or preventing a disease or disorder which is associated with an unfolded, misfolded or intrinsically disordered protein in a subject, said method comprising the following steps:• determining whether the subject has, or is likely to develop, the disease or disorder using the method according to any one of claims 13 to 15; and• if the subject has the disease or disorder, administering a therapeutically effective amount of a medicament capable of treating or preventing said disease or disorder to the subject.

19. A method for enriching an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps: o contacting the compound or salt thereof of any one of claims 1 to 12 with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate, and o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein; or o contacting the compound or salt thereof of any one of claims 1 to 12 with a sample containing an unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, and o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein.

20. A method for identifying an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps: o contacting the compound or salt thereof of any one of claims 1 to 12 with the sample containing the unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised biotin or alkyne functionalised biotin to form a protein-biotin conjugate,o collecting the protein-biotin conjugate using a streptavidin-functionalised agent to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein; or o contacting the compound or salt thereof of any one of claims 1 to 12 with the sample containing the unfolded, misfolded or intrinsically disordered protein to thereby bind said compound or salt thereof to the unfolded, misfolded or intrinsically disordered protein, o reacting the bound compound or salt thereof with an azide functionalised agent or alkyne functionalised agent to form a protein-agent conjugate, optionally wherein the azide or alkyne functionalised agent is an azide or alkyne functionalised solid phase, optionally an azide or alkyne functionalised bead, o collecting the protein-agent conjugate to thereby enrich the unfolded, misfolded or intrinsically disordered protein, and o analysing the enriched unfolded, misfolded or intrinsically disordered protein to thereby identify the unfolded, misfolded or intrinsically disordered protein.21 . A method of detecting an unfolded, misfolded or intrinsically disordered protein in a sample, said method comprising the following steps:• contacting the sample with the compound or salt thereof of any one of claims 1 to 12;• optionally enriching the unfolded, misfolded or intrinsically disordered protein, according to the method of claim 18; and• detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein to thereby detect the unfolded, misfolded or intrinsically disordered protein, optionally wherein detecting the compound or salt thereof in a form which is bound to the unfolded, misfolded or intrinsically disordered protein comprises measuring a fluorescence; absorbance, optionally UV absorbance; and / or liquid chromatography and / or performing mass spectrometry.

22. A method for screening a drug candidate for treating a disease or disorder associated with an unfolded, misfolded or intrinsically disordered protein, said method comprising the following steps:• contacting said drug candidate with a sample containing the unfolded, misfolded or intrinsically disordered protein; and• detecting the unfolded, misfolded or intrinsically disordered protein in the sample according to the method of claim 21 to determine whether the drug candidate is capable of treating the disease or disorder.

23. The method of any one of claims 13 to 15, 18 and 22, use of claim 16, or compound or salt thereof for use according to claim 17, wherein the disease or disorder is selected from the group consisting of Huntington’s disease; Alzheimer's disease; Parkinson’s disease; thrombosis; stroke; autism; obesity and metabolic diseases; cardiovascular diseases; chronic liver disease and cirrhosis; nephritis; nephrotic syndrome; nephrosis; Creutzfeldt-Jakob diseases; cystic fibrosis; Gaucher’s disease; hereditary cerebral haemorrhage; dementia; Niemann-Pick disease; multiple system atrophy; Creutz-Jakob disease; fatal insomnia; Gerstmann-Straussler-Scheinker disease; Spongiform encephalopathy; Creutzfeldt-Jakob disease; Kuru; hereditary sensory and autonomic neuropathy; Pick disease; progressive supranuclear palsy; argyrophilic grain disease; Guam Parkinson dementia complex; frontotemporal lobar degeneration; chronic traumatic encephalopathy; ganglioglioma; meningioangiomatosis; subacute sclerosing panencephalitis; lead encephalopathy; tuberous sclerosis; Hallervorden-Spatz disease; lipofuscinosis; amyloidosis; familial amyloidotic polyneuropathy; familial amyloid cardiomyopathy; amyloidosis; Type II diabetes; insulinoma; medullary carcinoma of the thyroid; motor neuron diseases; amyotrophic lateral sclerosis; atrial amyloidosis; pituitary prolactinoma; Gelatinous drop-like corneal dystrophy; calcifying epithelial odontogenic tumors; pulmonary alveolar proteinosis; hypotrichosis simplex; lattice corneal dystrophy; cancer, e.g. prostate cancer and breast cancer; myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS); and long COVID.

24. The method, use, or compound or salt thereof for use according to claim 23, wherein the disease or disorder is prostate cancer, breast cancer, myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), Huntington’s disease or Parkinson’s disease, optionally Huntington’s disease or Parkinson’s disease.