WO2017004560A1

WO2017004560A1 - Methods and compositions for amyloid aggregates

Info

Publication number: WO2017004560A1
Application number: PCT/US2016/040772
Authority: WO
Inventors: Jagesh SHAH; Dominic Walsh; Jerry Yang
Original assignee: University of California Berkeley; University of California San Diego UCSD
Current assignee: University of California Berkeley; University of California San Diego UCSD
Priority date: 2015-07-02
Filing date: 2016-07-01
Publication date: 2017-01-05
Anticipated expiration: 2018-01-02

Abstract

There are provided inter alia compounds and method for fluorescence correlation spectroscopy, useful for the detection of amyloid aggregates in a biological sample.

Description

METHODS AND COMPOSITIONS FOR AMYLOID AGGREGATES

CROSS-REFERENCES TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Patent Application Publication No.

62/188,198, filed July 2, 2015, the disclosure of which is incorporated by reference herein in its entirety.

REFERENCE TO A "SEQUENCE LISTING," A TABLE, OR A COMPUTER

PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII FILE

[0002] NOT APPLICABLE

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0003] This invention was made with Government support under grant number AG046275 awarded by the National Institutes of Health. The Government has certain rights in this invention.

BACKGROUND

[0004] Aggregation and deposition of certain proteins is a common facet of many neurological disorders. Specifically, a defining feature of Alzheimer's disease pathology is the presence of abundant amyloid plaques, the principle component of which is the amyloid β peptide (Αβ). Without wishing to be bound by theory, it is believed that intermediates in Αβ aggregation, referred to as oligomers, are the initiators of a complex molecular cascade that, over a course of decades, leads to dementia. To date the real time study of Αβ aggregation in solution has been limited by methods that best detect abundant assemblies of protofibrils and mature fibrils[3].

[0005] In response to this need, there is provided novel amyloid (Αβ) aggregate-binding probe, aryl cyano amide (ARCAM), which exhibits a large enhancement in fluorescence properties (~5X) upon binding to aggregates (compared to free probe in solution) when assayed by Probe- Enabled Fluorescence Correlation Spectroscopy (PE-FCS). This compound and embodiments thereof are useful in detecting potentially pathological aggregation intermediates from real patient samples (e.g., cerebral spinal fluid) for diagnosis of disease (e.g., Alzheimer's disease), Without wishing to be bound by any theory, it is believed that by virtue of its design and stability in solution, the disclosed methods are significantly more sensitive than alternative methods currently available.

SUMMARY

[0006] Provided herein, inter alia, are compositions and methods for the detection of amyloid aggregates for use in the detection, diagnosis, prognosis, and treatment of neurological diseases propagated by aggregated amyloid proteins. Accordingly, in some aspects, provided herein is a method for detecting an amyloid aggregate in a biological sample. The method includes contacting a biological sample containing an amyloid aggregate with an amyloid-binding fluorophore thereby forming a fluorescent amyloid aggregate complex comprising said amyloid aggregate anon-covalently bound to said amyloid-binding fluorophore, and detecting said fluorescent amyloid aggregate complex using fluorescence correlation spectroscopy. In some aspects, the method also includes determining the size of said amyloid aggregate. In some aspects, determining the size of said amyloid aggregate comprises measuring a number of fluorescent burst events for a resident time in a confocal volume. In some aspects, the size of the amyloid aggregate is less than 1000 nm.

[0007] In one aspect, the amyloid-binding fluorophore includes the structure of Formula (I),

EDG TTCE

NC WSG

(I).

In Formula (I), EDG is an electron donor group; 7lCE is a pi-conjugation element; and WSG is a water soluble group.

[0008] In embodiments, EDG is R^-substituted or unsubstituted alkyl, R^substituted or unsubstituted cycloalkyl, R¹- substituted or unsubstituted heteroalkyl, R¹ -substituted or unsubstituted heterocycloalkyl, R^substituted or unsubstituted aryl, R^substituted or unsubstituted heteroaryl, -OR², -NR⁴C(0)R³, -NR⁴R⁵, -SR⁶, or -PRV.R¹ is halogen, -OR⁹, -NR¹⁰Rⁿ, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R 12 -substituted or unsubstituted heteroaryl. R⁴ and R⁵ are optionally joined together to form an R¹²-substituted or unsubstituted heterocycloalkyl, or R¹²-substituted or unsubstituted heteroaryl; R⁹, R¹⁰ and R¹¹ are

independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl, or R 12 -substituted or unsubstituted heteroaryl. R¹⁰ and R¹¹ are optionally joined together to form an R¹²-substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl; R 12 is halogen, -OR 13 , -NR 14 R 15 , R¹⁶-substituted or unsubstituted alkyl, R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶- substituted or unsubstituted cycloalkyl, R¹⁶-substituted or unsubstituted heterocycloalkyl, R¹⁶- substituted or unsubstituted aryl, or R¹⁶-substituted or unsubstituted heteroaryl. R¹³, R¹⁴ and R¹⁵ are independently hydrogen or unsubstituted alkyl. R¹⁶ is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0009] In embodiments, the pi-conjugation element has the formula:

or -L*-(A L⁴-A³-L²-(A²)r-L³-. q and r are independently 0 or 1; L¹, L², L and L are independently a bond or a linking group having the formula: V / χ , x is an integer from 1 to 50. A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted arylene, or

R 17 -substituted or unsubstituted heteroarylene. R 17 is halogen, -OR 18 , -NR19 R 20 , R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl. R 18 , R 19 and R 20 are

independently hydrogen, R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl. R 21 is halogen, -OR 22 , -NR 23 R 24 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. R 22 , R 23 and R 24 are independently hydrogen or unsubstituted alkyl.

[0010] In embodiments, A 1 , A 2 and A 3 are independently R 21 -substituted or unsubstituted naphthylene, or R 21 -substituted or unsubstituted phenylene. In embodiments, x is an integer from 1 to 10. In embodiments, the water soluble group is R -substituted or unsubstituted alkyl, R - substituted or unsubstituted heteroalkyl, R 25 -substituted or unsubstituted cycloalkyl, R 25 - substituted or unsubstituted heterocycloalkyl, R 25 -substituted or unsubstituted aryl, R 25 - substituted or unsubstituted heteroaryl. R 25 is halogen, -OR 26 , -NR 27 R 28 , R 29 -substituted or

9Q 9Q

unsubstituted alkyl, R -substituted or unsubstituted heteroalkyl, R -substituted or unsubstituted

9Q 9Q

cycloalkyl, R -substituted or unsubstituted heterocycloalkyl, R -substituted or unsubstituted aryl, or R 29 -substituted or unsubstituted heteroaryl. R 26 , R 27 and R 28 are independently hydrogen,

9Q 9Q 9Q

R -substituted or unsubstituted alkyl, R -substituted or unsubstituted heteroalkyl, R -

9Q 9Q substituted or unsubstituted cycloalkyl, R -substituted or unsubstituted heterocycloalkyl, R - substituted or unsubstituted aryl, or R 29 -substituted or unsubstituted heteroaryl, wherein R 27 and

R 28 are optionally joined together to form an R 29 -substituted or unsubstituted heterocycloalkyl, or

R 29 -substituted or unsubstituted heteroaryl. R 29 is halogen, -OR 30 , -NR 31 R 32 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. R 30 , R 31 and R 32 are independently hydrogen or unsubstituted , the water soluble group is an ethylene glycol moiety having the formula:

y is an integer from 1 to 50. In embodiments, R is -OH.

[0012] In embodiments, the amyloid-binding fluorophore comprises the structure:



[0013] In embodiments, the biological sample is a biofluid. In some aspects, the biofluid is blood, urine, saliva, or cerebrospinal fluid. In some aspects, the biological sample further comprises a buffer.

[0014] In some aspects, the invention includes a fluorescent amyloid aggregate complex including an amyloid aggregate non-covalently bound to said amyloid-binding fluorophore. In embodiments, the complex is within a vessel. In embodiments, the vessel further comprises a buffer. In embodiments, the buffer has a pH of between 4.5 and 7.5. In embodiments, the buffer has a pH of between 5.0 and 7.0.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] FIGS. 1A-1C. Structure and spectroscopic properties of fluorescent probe 1. FIG. 1A) Structures of fluorescent, aggregate-binding compound ARCAM 1. Excitation (FIG. IB) and emission (FIG. 1C) properties of 1 in the presence or absence of aggregated Αβ(1-42).

[0016] FIGS. 2A-2B. Comparison of aggregate size measured using ThT, ARCAM 1 and fluorescently labeled Αβ peptide. Dyes (ThT or ARCAM 1) or TAMRA-labeled Αβ(1-42) peptides were added to pre-aggregated Αβ samples. FCS measurements, after a thirty minute incubation, revealed larger species in the ThT labeled sample (FIG. 2A, p <0.05 for 0.1, Is delay time). ARCAM 1 revealed larger species in the pre-aggregated samples when compared to TAMRA-Αβ (1-42) peptides (FIG. 2B, p<0.05 for 10s delay time). Note that pre-aggregated samples were matched for measurements in (FIG. 2A) and (FIG. 2B) independently, resulting in the different ARCAM 1 FCS spectra.

[0017] FIGS. 3A-3B. Monitoring the kinetics of aggregation of Αβ (1-42) peptides by bulk fluorescence and FCS burst analysis. FIG. 3A) Increase in total fluorescence (blue squares) or fluorescent burst number (squares) in solutions containing Αβ and fluorescent ARCAM 1. FIG. 3B) Detection of aggregates interacting with ARCAM 1 by monitoring the intensity and number of fluorescent bursts within a 120 second acquisition time window as a function of the concentration of total peptide.

[0018] FIG. 4. Representative raw FCS auto-correlation curves with fitting curves at three time points (t=0,120 and 600 minutes) are plotted together for comparison. Time points of t=0 min and t=120 min share the same left y-axis and the t=600 min time point uses the right y-axis with a different scale. The amplitude of autocorrelation curve G(0) increases along the reaction time point 0, 120 and 600 min, indicating a decrease in particle number for the dominant fluorescent species. The autocorrelation functions show increasing contribution from long delay times indicating the increasing size of the particles.

[0019] FIG. 5. A plot of fluorescence intensity versus concentration of ARCAM 1 in the presence of aggregated A ?(l-42) peptide at pH 7.4. The K_d was determined by fitting data to a one-site specific binding algorithm: Y = B_mx-X/(K_d + X), where X is the concentration of the probe, Y is the specific binding fluorescence intensity, and B_max corresponds to the apparent maximal observable fluorescence upon binding of probes to aggregated A ?(l-42) peptide [26].

[0020] FIG. 6. Hydrolytic stability studies of ARCAM 1 in pH 7.4 IX PBS over 24 hours.

[0021] FIGS. 7A-7D: pH dependence of ARCAM 1 fluorescence free in solution (FIG. 7A and 7C) and bound to Αβ(1-42) aggregates (FIG. 7B and 7D). Kd dependence on pH shown for pH 5.6, and 9.1 (and 7.4 in FIG. 5). RFI = relative fluorescence intensity.

[0022] FIG. 8. Unbound dye 2PE emission and fluorescence brightness.

[0023] FIG. 9. Αβ(1-42) aggregation kinetics are not perturbed by the addition of ARCAM 1 or ThT.

[0024] FIG. 10. Αβ monomer was incubated in the absence or presence of ThT and bulk fluorescence monitored at intervals. Maximal fluorescence was attained after 140 min and remained constant thereafter. Samples lacking ThT were collected after 240 min and used as the aggregate standard.

[0025] FIG. 11. The intensity trace of Αβ peptide (5 μΜ) alone by FCS measurement. [0026] FIG. 12A-12C. Burst selection method. (FIG. 12A) the histogram of intensity trace with 1 ms resolution at 240 min of Αβ reaction. The solid line indicates the modal value of the histogram. This value is treated as the average of intensity background due to the majority of intensity events. The dashed line is the position at four times of the standard deviation of this intensity trace distribution above the average background. FIG. 12B: solid and dash lines are the same as FIG. 12A, showing the relative positions of cutoff. FIG. 12C: Selected bursts clearly shown with all other intensities set as zero.

[0027] FIG. 13. The diffusion coefficients of large species by two-component analysis at different time points.

[0028] FIGS. 14A-14H. NMR spectra. FIG. 14A: 1H NMR spectrum of Cmpd A at 500 MHz in CDC1₃; FIG. 14B: ¹³C NMR spectrum of Cmpd A at 125 MHz in CDCI3; FIG. 14C: 1H NMR spectrum of Cmpd B at 500 MHz in CDCI₃; FIG. 14D: ¹³C NMR spectrum of Cmpd B at 125 MHz in CDCI₃; FIG. 14E: 1H NMR spectrum of Cmpd C at 500 MHz in CDC1₃; FIG. 14F: ¹³C NMR spectrum of Cmpd C at 125 MHz in CDC1₃; FIG. 14G: 1H NMR spectrum of Cmpd 1 at 500 MHz in CDCI₃; FIG. 14H: ¹³C NMR spectrum of Cmpd 1 at 125 MHz in CDCI₃.

DETAILED DESCRIPTION

I. Definitions

[0029] The abbreviations used herein have their conventional meaning within the chemical and biological arts. The chemical structures and formulae set forth herein are constructed according to the standard rules of chemical valency known in the chemical arts.

[0030] Where substituent groups are specified by their conventional chemical formulae, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, e.g., -CH₂0- is equivalent to -OCH₂-.

[0031] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight (i.e., unbranched) or branched carbon chain (or carbon), or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include mono-, di- and multivalent radicals, having the number of carbon atoms designated (i.e., C₁-C₁₀ means one to ten carbons). Alkyl is an uncyclized chain. Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs and isomers of, for example, n-pentyl, n- hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. An alkoxy is an alkyl attached to the remainder of the molecule via an oxygen linker (-0-).

[0032] The term "alkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkyl, as exemplified, but not limited by, - CH₂CH₂CH₂CH₂-. Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being preferred herein. A "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term "alkenylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from an alkene.

[0033] The term "heteroalkyl," by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or combinations thereof, including at least one carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S), and wherein the nitrogen and sulfur atoms may optionally be oxidized, and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) (e.g., N, S, Si, or P) may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Heteroalkyl is an uncyclized chain. Examples include, but are not limited to: -C¾- CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -CH₂-CH₂, -S(O)- CH₃, -CH₂-CH₂-S(0)₂-CH₃, -CH=CH-0-CH₃, -Si(CH₃)₃, -CH₂-CH=N-OCH₃, -CH=CH-N(CH₃)- CH₃, -0-CH₃, -0-CH₂-CH₃, and -CN. Up to two or three heteroatoms may be consecutive, such as, for example, -CH₂-NH-OCH₃ and -CH₂-0-Si(CH₃)₃. A heteroalkyl moiety may include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include two optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include three optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include four optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include five optionally different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl moiety may include up to 8 optionally different heteroatoms (e.g., O, N, S, Si, or P). [0034] Similarly, the term "heteroalkylene," by itself or as part of another substituent, means, unless otherwise stated, a divalent radical derived from heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy,

alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. For example, the formula -C(0)₂R'- represents both -C(0)₂R'- and -R'C(0)₂-. As described above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as - C(0)R', -C(0)NR', -NR'R", -OR', -SR', and/or -S0₂R'. Where "heteroalkyl" is recited, followed by recitations of specific heteroalkyl groups, such as -NR'R" or the like, it will be understood that the terms heteroalkyl and -NR'R" are not redundant or mutually exclusive. Rather, the specific heteroalkyl groups are recited to add clarity. Thus, the term "heteroalkyl" should not be interpreted herein as excluding specific heteroalkyl groups, such as -NR'R" or the like.

[0035] The terms "cycloalkyl" and "heterocycloalkyl," by themselves or in combination with other terms, mean, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl," respectively. Cycloalkyl and heterocycloalkyl are not aromatic. Additionally, for

heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6- tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1- piperazinyl, 2-piperazinyl, and the like. A "cycloalkylene" and a "heterocycloalkylene," alone or as part of another substituent, means a divalent radical derived from a cycloalkyl and

heterocycloalkyl, respectively.

[0036] The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl" are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(Ci-C₄)alkyl" includes, but is not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. [0037] The term "acyl" means, unless otherwise stated, -C(0)R where R is a substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

[0038] The term "aryl" means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (preferably from 1 to 3 rings) that are fused together (i.e., a fused ring aryl) or linked covalently. A fused ring aryl refers to multiple rings fused together wherein at least one of the fused rings is an aryl ring. The term "heteroaryl" refers to aryl groups (or rings) that contain at least one heteroatom such as N, O, or S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. Thus, the term "heteroaryl" includes fused ring heteroaryl groups (i.e., multiple rings fused together wherein at least one of the fused rings is a heteroaromatic ring). A 5,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 5 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. Likewise, a 6,6-fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 6 members, and wherein at least one ring is a heteroaryl ring. And a 6,5- fused ring heteroarylene refers to two rings fused together, wherein one ring has 6 members and the other ring has 5 members, and wherein at least one ring is a heteroaryl ring. A heteroaryl group can be attached to the remainder of the molecule through a carbon or heteroatom. Non- limiting examples of aryl and heteroaryl groups include phenyl, naphthyl, pyrrolyl, pyrazolyl, pyridazinyl, triazinyl, pyrimidinyl, imidazolyl, pyrazinyl, purinyl, oxazolyl, isoxazolyl, thiazolyl, furyl, thienyl, pyridyl, pyrimidyl, benzothiazolyl, benzoxazoyl benzimidazolyl, benzofuran, isobenzofuranyl, indolyl, isoindolyl, benzothiophenyl, isoquinolyl, quinoxalinyl, quinolyl, 1- naphthyl, 2-naphthyl, 4-biphenyl, 1 -pyrrolyl, 2-pyrrolyl, 3 -pyrrolyl, 3 -pyrazolyl, 2-imidazolyl, 4- imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4- isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3 -pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5 -benzothiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, 1 -isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5 -quinoxalinyl, 3- quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below. An "arylene" and a "heteroarylene," alone or as part of another substituent, mean a divalent radical derived from an aryl and heteroaryl, respectively. A heteroaryl group substituent may be -O- bonded to a ring heteroatom nitrogen.

[0039] Spirocyclic rings are two or more rings wherein adjacent rings are attached through a single atom. The individual rings within spirocyclic rings may be identical or different.

Individual rings in spirocyclic rings may be substituted or unsubstituted and may have different substituents from other individual rings within a set of spirocyclic rings. Possible substituents for individual rings within spirocyclic rings are the possible substituents for the same ring when not part of spirocyclic rings (e.g. substituents for cycloalkyl or heterocycloalkyl rings). Spirocylic rings may be substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkyl or substituted or unsubstituted heterocycloalkylene and individual rings within a spirocyclic ring group may be any of the immediately previous list, including having all rings of one type (e.g. all rings being substituted heterocycloalkylene wherein each ring may be the same or different substituted heterocycloalkylene). When referring to a spirocyclic ring system, heterocyclic spirocyclic rings means a spirocyclic rings wherein at least one ring is a heterocyclic ring and wherein each ring may be a different ring. When referring to a spirocyclic ring system, substituted spirocyclic rings means that at least one ring is substituted and each substituent may optionally be different.

[0040] The symbol "— ~" denotes the point of attachment of a chemical moiety to the remainder of a molecule or chemical formula.

[0041] The term "oxo," as used herein, means an oxygen that is double bonded to a carbon atom.

[0042] The term "alkylarylene" as an arylene moiety covalently bonded to an alkylene moiety (also referred to herein as an alkylene linker). In embodiments, the alkylarylene group has the formula:

[0043] An alkylarylene moiety may be substituted (e.g. with a substituent group) on the alkylene moiety or the arylene linker (e.g. at carbons 2, 3, 4, or 6) with halogen, oxo, -N₃, -CF₃, - CC1₃, -CBr₃, -CI₃, -CN, -CHO, -OH, -NH₂, -COOH, -CONH₂, -N0₂, -SH, -S0₂CH₃ -S0₃H, , - OSO₃H, -SO₂NH₂, -NHNH₂, -ONH₂, -NHC(0)NHNH₂, substituted or unsubstituted C₁-C5 alkyl or substituted or unsubstituted 2 to 5 membered heteroalkyl). In embodiments, the alkylarylene is unsubstituted.

[0044] Each of the above terms (e.g., "alkyl," "heteroalkyl," "cyclalkyl," "heterocycloalkyl," "aryl," and "heteroaryl") includes both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below.

[0045] Substituents for the alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to, -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(0)R', - C(0)R', -CO₂R', -CONR'R", -OC(0)NR'R", -NR"C(0)R', -NR'-C(0)NR"R"', -NR"C(0)₂R', -NR- C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(0)R', -S(0)₂R', -S(0)₂NR'R", -NRS0₂R',

-NR'NR"R"', -ONR'R", -NR'C(0)NR"NR"'R"", -CN, -N0₂, -NR'S0₂R", -NR'C(0)R", - NR'C(0)-OR", -NR'OR", in a number ranging from zero to (2m'+l), where m' is the total number of carbon atoms in such radical. R, R', R", R'", and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl (e.g., aryl substituted with 1-3 halogens), substituted or unsubstituted heteroaryl, substituted or

unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl groups. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" group when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 4-, 5-, 6-, or 7-membered ring. For example, -NR'R" includes, but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of

substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF₃ and -CH₂CF₃) and acyl (e.g., -C(0)CH₃, -C(0)CF₃, -C(0)CH₂OCH₃, and the like).

[0046] Similar to the substituents described for the alkyl radical, substituents for the aryl and heteroaryl groups are varied and are selected from, for example: -OR', -NR'R", -SR', -halogen, - SiR'R"R"', -OC(0)R', -C(0)R', -C0₂R', -CONR'R", -OC(0)NR'R", -NR"C(0)R', -NR'- C(0)NR"R"', -NR"C(0)₂R', -NR-C(NR'R"R"')=NR"", -NR-C(NR'R")=NR"', -S(0)R', -S(0)₂R', - S(0)₂NR'R", -NRS0₂R', -NR'NR"R"', -ONR'R", -NR'C(0)NR"NR"'R"", -CN, -N0₂, -R', -N₃, - CH(Ph)₂, fluoro(Ci-C₄)alkoxy, and fluoro(Ci-C₄)alkyl, -NR'S0₂R", -NR'C(0)R", -NR'C(O)- OR", -NR'OR", in a number ranging from zero to the total number of open valences on the aromatic ring system; and where R', R", R'", and R"" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl. When a compound described herein includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R'", and R"" groups when more than one of these groups is present.

[0047] Substituents for rings (e.g. cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkylene, heterocycloalkylene, arylene, or heteroarylene) may be depicted as substituents on the ring rather than on a specific atom of a ring (commonly referred to as a floating substituent). In such a case, the substituent may be attached to any of the ring atoms (obeying the rules of chemical valency) and in the case of fused rings or spirocyclic rings, a substituent depicted as associated with one member of the fused rings or spirocyclic rings (a floating substituent on a single ring), may be a substituent on any of the fused rings or spirocyclic rings (a floating substituent on multiple rings). When a substituent is attached to a ring, but not a specific atom (a floating substituent), and a subscript for the substituent is an integer greater than one, the multiple substituents may be on the same atom, same ring, different atoms, different fused rings, different spirocyclic rings, and each substituent may optionally be different. Where a point of attachment of a ring to the remainder of a molecule is not limited to a single atom (a floating substituent), the attachment point may be any atom of the ring and in the case of a fused ring or spirocyclic ring, any atom of any of the fused rings or spirocyclic rings while obeying the rules of chemical valency. Where a ring, fused rings, or spirocyclic rings contain one or more ring heteroatoms and the ring, fused rings, or spirocyclic rings are shown with one more floating substituents (including, but not limited to, points of attachment to the remainder of the molecule), the floating substituents may be bonded to the heteroatoms. Where the ring heteroatoms are shown bound to one or more hydrogens (e.g. a ring nitrogen with two bonds to ring atoms and a third bond to a hydrogen) in the structure or formula with the floating substituent, when the heteroatom is bonded to the floating substituent, the substituent will be understood to replace the hydrogen, while obeying the rules of chemical valency. [0048] Two or more substituents may optionally be joined to form aryl, heteroaryl, cycloalkyl, or heterocycloalkyl groups. Such so-called ring-forming substituents are typically, though not necessarily, found attached to a cyclic base structure. In one embodiment, the ring-forming substituents are attached to adjacent members of the base structure. For example, two ring- forming substituents attached to adjacent members of a cyclic base structure create a fused ring structure. In another embodiment, the ring-forming substituents are attached to a single member of the base structure. For example, two ring-forming substituents attached to a single member of a cyclic base structure create a spirocyclic structure. In yet another embodiment, the ring- forming substituents are attached to non-adjacent members of the base structure.

[0049] Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally form a ring of the formula -T-C(0)-(CRR')_q-U-, wherein T and U are independently -NR-, -0-, - CRR'-, or a single bond, and q is an integer of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH₂)_r-B-, wherein A and B are independently -CRR'-, -0-, -NR-, -S-, -S(O) -, - S(0)₂-, -S(0)₂NR'-, or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X'- (C"R"R"')d-, where s and d are independently integers of from 0 to 3, and X' is -0-, -NR'-, -S-, -S(O)-, -S(0)₂-, or -S(0)₂NR'-. The substituents R, R', R", and R'" are preferably independently selected from hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, and substituted or unsubstituted heteroaryl.

[0050] As used herein, the terms "heteroatom" or "ring heteroatom" are meant to include oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si).

[0051] A "substituent group," as used herein, means a group selected from the following moieties:

(A) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -N0₂, -SH, -S0₃H, -S0₄H, - S0₂NH₂, -NHNH₂, -ONH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, -NHS0₂H, -NHC= (O)H, - NHC(0)-OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(B) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(i) oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -N0₂, -SH, -S0₃H, -S0₄H, - S0₂NH₂, -NHNH₂, -ONH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, -NHS0₂H, -NHC= (O)H, - NHC(0)-OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl, and

(ii) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from:

(b) alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, substituted with at least one substituent selected from: oxo, halogen, -CF₃, -CN, -OH, -NH₂, -COOH, -CONH₂, -N0₂, -SH, - S0₃H, -S0₄H, -S0₂NH₂, -NHNH₂, -ONH₂, -NHC=(0)NHNH₂, -NHC=(0) NH₂, -NHS0₂H, - NHC= (O)H, -NHC(0)-OH, -NHOH, -OCF₃, -OCHF₂, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, unsubstituted heteroaryl.

[0052] A "size-limited substituent" or " size-limited substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C₂o alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-Cio aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl.

[0053] A "lower substituent" or " lower substituent group," as used herein, means a group selected from all of the substituents described above for a "substituent group," wherein each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C7 cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-Cio aryl, and each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl.

[0054] In some embodiments, each substituted group described in the compounds herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted cycloalkylene, substituted heterocycloalkylene, substituted arylene, and/or substituted heteroarylene described in the compounds herein are substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. In other embodiments, at least one or all of these groups are substituted with at least one lower substituent group.

[0055] In other embodiments of the compounds herein, each substituted or unsubstituted alkyl may be a substituted or unsubstituted C₁-C₂₀ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 8 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-Cio aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 10 membered heteroaryl. In some embodiments of the compounds herein, each substituted or unsubstituted alkylene is a substituted or unsubstituted C₁-C₂₀ alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₈ cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 8 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C₆-Cio arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 10 membered heteroarylene.

[0056] In some embodiments, each substituted or unsubstituted alkyl is a substituted or unsubstituted Ci-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₃-C₇ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 3 to 7 membered heterocycloalkyl, each substituted or unsubstituted aryl is a substituted or unsubstituted C₆-Cio aryl, and/or each substituted or unsubstituted heteroaryl is a substituted or unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each substituted or unsubstituted alkylene is a substituted or unsubstituted Q-Cg alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene is a substituted or unsubstituted C₃-C₇ cycloalkylene, each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 3 to 7 membered heterocycloalkylene, each substituted or unsubstituted arylene is a substituted or unsubstituted C₆-Cio arylene, and/or each substituted or unsubstituted heteroarylene is a substituted or unsubstituted 5 to 9 membered heteroarylene. In some embodiments, the compound is a chemical species set forth in the Examples section, figures, or tables below.

[0057] Certain compounds of the present invention possess asymmetric carbon atoms (optical or chiral centers) or double bonds; the enantiomers, racemates, diastereomers, tautomers, geometric isomers, stereoisometric forms that may be defined, in terms of absolute

stereochemistry, as (R)-or (S)- or, as (D)- or (L)- for amino acids, and individual isomers are encompassed within the scope of the present invention. The compounds of the present invention do not include those that are known in art to be too unstable to synthesize and/or isolate. The present invention is meant to include compounds in racemic and optically pure forms. Optically active (R)- and (S)-, or (D)- and (L)-isomers may be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. When the compounds described herein contain olefinic bonds or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers. [0058] As used herein, the term "isomers" refers to compounds having the same number and kind of atoms, and hence the same molecular weight, but differing in respect to the structural arrangement or configuration of the atoms.

[0059] The term "tautomer," as used herein, refers to one of two or more structural isomers which exist in equilibrium and which are readily converted from one isomeric form to another.

[0060] It will be apparent to one skilled in the art that certain compounds of this invention may exist in tautomeric forms, all such tautomeric forms of the compounds being within the scope of the invention.

[0061] Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention.

[0062] Unless otherwise stated, structures depicted herein are also meant to include

compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention.

[0063] Unless otherwise stated, structures depicted herein are also meant to include

[0064] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium ( H), iodine-125 (¹²⁵I), or carbon-14 (¹⁴C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are encompassed within the scope of the present invention.

[0065] It should be noted that throughout the application that alternatives are written in Markush groups, for example, each amino acid position that contains more than one possible amino acid. It is specifically contemplated that each member of the Markush group should be considered separately, thereby comprising another embodiment, and the Markush group is not to be read as a single unit.

[0066] "Analog," or "analogue" is used in accordance with its plain ordinary meaning within Chemistry and Biology and refers to a chemical compound that is structurally similar to another compound (i.e., a so-called "reference" compound) but differs in composition, e.g., in the replacement of one atom by an atom of a different element, or in the presence of a particular functional group, or the replacement of one functional group by another functional group, or the absolute stereochemistry of one or more chiral centers of the reference compound. Accordingly, an analog is a compound that is similar or comparable in function and appearance but not in structure or origin to a reference compound.

[0067] The terms "a" or "an," as used in herein means one or more. In addition, the phrase "substituted with a[n]," as used herein, means the specified group may be substituted with one or more of any or all of the named substituents. For example, where a group, such as an alkyl or heteroaryl group, is "substituted with an unsubstituted C₁-C₂₀ alkyl, or unsubstituted 2 to 20 membered heteroalkyl," the group may contain one or more unsubstituted C₁-C₂₀ alkyls, and/or one or more unsubstituted 2 to 20 membered heteroalkyls.

[0068] Moreover, where a moiety is substituted with an R substituent, the group may be referred to as "R-substituted." Where a moiety is R-substituted, the moiety is substituted with at least one R substituent and each R substituent is optionally different. Where a particular R group is present in the description of a chemical genus (such as Formula (I)), a Roman alphabetic symbol may be used to distinguish each appearance of that particular R group. For example, where multiple R¹³ substituents are present, each R¹³ substituent may be distinguished as R^13A, R^13B, R^13C, R^13D, etc., wherein each of R^13A, R^13B, R^13C, R^13D, etc. is defined within the scope of the definition of R 13 and optionally differently.

[0069] A "covalent cysteine modifier moiety" as used herein refers to a subtituent that is capable of reacting with the sulfhydryl functional group of a cysteine amino acid (e.g. cysteine YYY of the XXX (e.g., human XXX)) to form a covalent bond. Thus, the covalent cysteine modifier moiety is typically electrophilic. [0070] A "detectable moiety" as used herein refers to a moiety that can be covalently or noncovalently attached to a compound or biomolecule that can be detected for instance, using techniques known in the art. In embodiments, the detectable moiety is covalently attached. The detectable moiety may provide for imaging of the attached compound or biomolecule. The detectable moiety may indicate the contacting between two compounds. Exemplary detectable moieties are fluorophores, antibodies, reactive dies, radio-labeled moieties, magnetic contrast agents, and quantum dots. Exemplary fluorophores include fluorescein, rhodamine, GFP, coumarin, FITC, Alexa fluor, Cy3, Cy5, BODIPY, and cyanine dyes. Exemplary radionuclides include Fluorine- 18, Gallium-68, and Copper-64. Exemplary magnetic contrast agents include gadolinium, iron oxide and iron platinum, and manganese.

[0071] Descriptions of compounds of the present invention are limited by principles of chemical bonding known to those skilled in the art. Accordingly, where a group may be substituted by one or more of a number of substituents, such substitutions are selected so as to comply with principles of chemical bonding and to give compounds which are not inherently unstable and/or would be known to one of ordinary skill in the art as likely to be unstable under ambient conditions, such as aqueous, neutral, and several known physiological conditions. For example, a heterocycloalkyl or heteroaryl is attached to the remainder of the molecule via a ring heteroatom in compliance with principles of chemical bonding known to those skilled in the art thereby avoiding inherently unstable compounds.

[0072] Fluorescence correlation spectroscopy (FCS), as known in the art, is useful for high- resolution spatial and temporal analysis of extremely low concentrations of biomolecules. In contrast to other fluorescence techniques, the parameter of primary interest is not the emission intensity itself, but rather spontaneous intensity fluctuations caused by the minute deviations of the small system from thermal equilibrium. In general, all physical parameters that give rise to fluctuations in the fluorescence signal are accessible by FCS. It is, for example, rather straightforward to determine local concentrations, mobility coefficients or characteristic rate constants of inter- or intramolecular reactions of fluorescently labeled biomolecules in nanomolar concentrations.

[0073] The extreme sensitivity of PE-FCS can enable significantly improved sensitivity for monitoring the aggregation of amyloidogenic proteins vs. current methods which measure the fluorescence of an amyloid-binding dye, e.g., Thioflavin T (ThT) or Thioflavin S (ThS), using bulk fluorescence measurements. A significant limitation of the conventional method is that neither ThT nor ThS fluoresce appreciably over background fluorescence until the size and number of amyloid or amyloid-like species is quite large. Hence, one cannot detect early formed aggregate intermediates that are implicated as the most active species involved in the pathology of amyloid- associated diseases.

[0074] The term "amyloid" is used herein according to its customary meaning in the art.

Amyloids contain a plurality of associated amyloid peptides, such as aggregates of amyloid peptides. Thus, in some embodiments, amyloids include an amyloid peptide aggregated with one or more amyloid peptides. In some embodiments, amyloids include "amyloid plaques," amyloid deposits," "amyloid aggregates" or "aggregates of amyloid peptides." The compounds described herein can associate with (e.g., bind) an amyloid peptide and/or an amyloid. In certain embodiments, the compounds described herein can associate with an amyloid by hydrophobic interactions.

[0075] The term "fluorescent amyloid aggregate complex" is used herein to indicate an amyloid binding fluorophore in complex with an amyloid aggregate. In embodiments, the amyloid aggregate can vary in size. In embodiments, the amyloid aggregate can be a native amyloid aggregate. In embodiments, the amyloid aggregate can be a non-native amyloid aggregate. In embodiments, the amyloid binding fluorophore is non-covalently bound to the amyloid aggregate.

[0076] The term "biofluid" is used herein to describe a biological or bodily fluid that can be obtained from a subject, such as a mammalian subject (e.g. a human). In embodiments, a biofluid is uring, blood, semen, saliva or cerebrospinal fluid. In embodiments, a biofluid may be contained within a suitable buffer.

[0077] The term "fluorescent burst event" or "fluorescence burst event" is used herein to describe fluorescence measured by FCS that is detectably above background levels. Fluorescence can be measured via FCS across a set period of time. Fluorescence events can be measured for time course and intensity. Fluorescence burst events can be recorded in both their numerical quantity for a given time or by calculating a normalized bulk fluorescence correlating the number of burst events with the intensity of each event. II. Amyloid Binding Fluorophores

[0078] Amyloid binding fluorophores useful in the methods and compositions provided herein are compounds that are capable of binding an amyloid, amyloid peptide or amyloid aggregate and are fluorescent. In embodiments, the amyloid binding fluorophore is not a fluorescently labeled peptide. In embodiments, the amyloid binding fluorophore does not include an amino acid sequence. In embodiments, the amyloid binding fluorophore is not a biomolecule (i.e. a molecule found in nature). In embodiments, the amyloid binding fluorophore does not include a biomolecule or biomolecule moiety. In embodiments, the amyloid binding fluorophore is less than 900 daltons. In embodiments, the amyloid binding fluorophore is less than 800 daltons. In embodiments, the amyloid binding fluorophore is less than 700 daltons. In embodiments, the amyloid binding fluorophore is less than 600 daltons. In embodiments, the amyloid binding fluorophore is less than 500 daltons.

[0079] In embodiments, the amyloid binding fluorophore has the structure of Formula (I),

EDG nCE

NC WSG

(I).

[0080] In Formula I, "EDG" is an electron donor group. "7rCE" is a π-conjugation element. "WSG" is a water soluble group.

[0081] In some embodiments, EDG is substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OR ,

-NR⁴C(0)R³, -CONR⁴R⁵, -NR⁴R⁵, -SR⁶, or -PR⁷R⁸. In some embodiments, EDG is substituted alkyl, substituted cycloalkyl, substituted heteroalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, -OR², -NR⁴C(0)R³, -CONR⁴R⁵, -NR⁴R⁵, -SR⁶, or -PR⁷R⁸. In some embodiments, EDG is R^substituted or unsubstituted alkyl, R^substituted or unsubstituted cycloalkyl, R^substituted or unsubstituted heteroalkyl, R^substituted or unsubstituted heterocycloalkyl, R^substituted or unsubstituted aryl, R^substituted or unsubstituted heteroaryl,

-OR², -NR⁴C(0)R³, -CONR⁴R⁵, -NR⁴R⁵, -SR⁶, or -PR⁷R⁸. In some embodiments, EDG is R¹- substituted alkyl, R^substituted cycloalkyl, R^substituted heteroalkyl, R^substituted heterocycloalkyl, R^substituted aryl, R^substituted heteroaryl, -OR², -NR⁴C(0)R³, -CONR⁴R⁵, -NR⁴R⁵, -SR⁶, or -PR⁷R⁸.

[0082] In some embodiments, R¹ is halogen, -CN, -OR⁹, -CONR¹⁰Rⁿ, -NR¹⁰Rⁿ, -SR⁹, -SOR⁹, -S0₂R⁹,-COR⁹, -COOR⁹, -NR¹⁰COR⁹, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R¹ is halogen, -CN, -OR⁹, -CONR¹⁰Rⁿ, -NR¹⁰Rⁿ, -SR⁹, -SOR⁹,

-S0₂R⁹,-COR⁹, -COOR⁹, -NR¹⁰COR⁹, R^12a-substituted or unsubstituted alkyl, R^12a-substituted or unsubstituted heteroalkyl, R^12a-substituted or unsubstituted cycloalkyl, R^12a-substituted or unsubstituted heterocycloalkyl, R^12a-substituted or unsubstituted aryl, or R^12a-substituted or unsubstituted heteroaryl. In some embodiments, R¹ is halogen, -OR⁹, -NR¹⁰Rⁿ, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In some embodiments, R¹ is -OR⁹, -NR¹⁰Rⁿ, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted

heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In some embodiments, when R¹ is attached to alkyl, cycloalkyl, or aryl, R¹ includes at least one heteroatom. In some embodiments, R¹ includes at least one heteroatom. In some embodiments, R¹ is -OR⁹ or -NR¹⁰Rⁿ. In some embodiments, R¹ is -NR¹⁰Rⁿ.

[0083] In certain embodiments, R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or

unsubstituted aryl, or substituted or unsubstituted heteroaryl. In certain embodiments, R 2 , R 3 , R 4 ,

R 5^J, R 6, R 7' and R 8° are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 - substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl, R 12 - substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R 12 - substituted or unsubstituted heteroaryl. In some embodiments, R⁴ and R⁵ are optionally joined together to form a substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl. In some embodiments, R⁴ and R⁵ are optionally joined together to form R¹²- substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl.

[0084] In some embodiments, R⁹, R¹⁰ and R¹¹ are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R⁹, R¹⁰ and R¹¹ are independently hydrogen, R 12 -substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl,

R 12 -substituted or unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl,

R 12 -substituted or unsubstituted aryl, or R 12 -substituted or unsubstituted heteroaryl. In certain embodiments, R¹⁰ and R¹¹ are optionally joined together to form an substituted or unsubstituted heterocycloalkyl, or substituted or unsubstituted heteroaryl. In certain embodiments, R¹⁰ and R¹¹ are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or

R 12 -substituted or unsubstituted heteroaryl.

[0085] R¹² and R^12a are independently halogen, -CN, -SR¹³, -SOR¹³, -S0₂R¹³,-OR¹³, -NR¹⁴R¹⁵, -COR¹⁵, -COOR¹⁵, CONR¹⁴R¹⁵, -NR¹⁴COR¹⁵, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R 12 and R 12έΐ are independently halogen, -CN, -SR 13 , -SOR 13 ,

-S0₂R¹³,-OR¹³, -NR¹⁴R¹⁵, -COR¹⁵, -COOR¹⁵, CONR¹⁴R¹⁵, -NR¹⁴COR¹⁵, R¹⁶-substituted or unsubstituted alkyl, R¹⁶- substituted or unsubstituted heteroalkyl, R¹⁶-substituted or unsubstituted cycloalkyl, R¹⁶-substituted or unsubstituted heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, or R¹⁶- substituted or unsubstituted heteroaryl. In some embodiments, R¹² is -OR¹³, -NR¹⁴R¹⁵, R¹⁶-substituted or unsubstituted alkyl, R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted or unsubstituted cycloalkyl, R¹⁶- substituted or unsubstituted heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, or R¹⁶-substituted or unsubstituted heteroaryl.

[0086] R¹³, R¹⁴ and R¹⁵ are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R¹³, R¹⁴ and R¹⁵ are independently hydrogen R¹⁶-substituted or unsubstituted alkyl, R¹⁶-substituted or unsubstituted heteroalkyl, R¹⁶-substituted or unsubstituted cycloalkyl, R¹⁶-substituted or unsubstituted heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, or R¹⁶- substituted or unsubstituted heteroaryl. In some embodiments, R¹³, R¹⁴ and R¹⁵ are independently hydrogen, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R¹³, R¹⁴ and R¹⁵ are independently hydrogen or unsubstituted alkyl. [0087] R¹⁶ is halogen, -NH₂, -OH, -SH, -COOH, -COH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

[0088] In some embodiments, R¹² is -OR¹³ or -NR¹⁴R¹⁵. In some embodiments, R^12a is -OR¹³ or -NR¹⁴R¹⁵. In some embodiments, where R^12a forms part of an R¹ substituent (e.g. where R¹ is an alkyl, cycloalkyl or aryl), R^12a includes a heteroatom. In some embodiments, where R^12a forms part of an R¹ substituent (e.g. where R¹ is an alkyl, cycloalkyl or aryl), R^12a is -OR¹³ or -NR¹⁴R¹⁵.

[0089] In some embodiments, R⁴ and R⁵ are independently hydrogen or R¹²-substituted or unsubstituted alkyl. In some embodiments, R⁴ and R⁵ are independently hydrogen, R¹²-

12

substituted or unsubstituted C1-C20 (e-g-, Ci-Cio) alkyl, or R -substituted or unsubstituted heteroalkyl. In some embodiments, R⁴ and R⁵ are optionally joined together to form an R¹²- substituted or unsubstituted heterocycloalkyl. The R 12 -substituted or unsubstituted

heterocycloalkyl can be R 12 -substituted or unsubstituted piperidinyl, R 12 -substituted or unsubstituted morpholinyl, R 12 -substituted or unsubstituted tetrahydrofuranyl, R 12 -substituted or unsubstituted tetrahydrothienyl, or R 12 - substituted or unsubstituted piperazinyl. In some embodiments, R¹² is R¹⁶-substituted or unsubstituted Ci-C₂o (e-g-, Ci-Cio) alkyl or R¹⁶- substituted or unsubstituted heteroalkyl. R¹⁶ can be unsubstituted C₄-C₈ heterocycloalkyl.

[0090] In some embodiments, R⁴ and R⁵ are joined together to form R¹²-substituted or unsubstituted heteroaryl. The R 12 -substituted or unsubstituted heteroaryl can be R 12 -substituted or unsubstituted purinyl, R 12 -substituted or unsubstituted pyrimidinyl, R 12 -substituted or unsubstituted imidazolyl, R 12 -substituted or unsubstituted pyrrolopyridinyl (e.g., lH-pyrrolo[2,3- b]pyridinyl), R 12 -substituted or unsubstituted pyrimidinyl, R 12 -substituted or unsubstituted indazolyl (e.g., lH-indazolyl), or R 12 -substituted or unsubstituted pyrrolopyrimidinyl (e.g., 7H- pyrrolo[2,3-d]pyrimidinyl). In some embodiments, R⁴ and R⁵ are joined together to form R¹²- substituted or unsubstituted pyrrolopyrimidinyl, R 12 -substituted or unsubstituted indolyl, R 12 - substituted or unsubstituted pyrazolyl, R 12 -substituted or unsubstituted indazolyl, R 12 -substituted or unsubstituted imidazolyl, R 12 -substituted or unsubstituted thiazolyl, R 12 -substituted or unsubstituted benzothiazolyl, R 12 -substituted or unsubstituted oxazolyl, R 12 -substituted or unsubstituted benzimidazolyl, R 12 -substituted or unsubstituted benzoxazolyl, R 12 -substituted or unsubstituted isoxazolyl, R 12 -substituted or unsubstituted benzisoxazolyl, R 12 -substituted or unsubstituted triazolyl, R -substituted or unsubstituted benzotriazolyl, R -substituted or unsubstituted quinolinyl, R 12 -substituted or unsubstituted isoquinolinyl, R 12 -substituted or unsubstituted quinazolinyl, R 12 -substituted or unsubstituted pyrimidinyl, R 12 -substituted or unsubstituted pyridinyl N-oxide, R 12 -substituted or unsubstituted furanyl, R 12 -substituted or unsubstituted thiophenyl, R 12 -substituted or unsubstituted benzofuranyl, R 12 -substituted or unsubstituted benzothiophenyl, R 12 -substituted or unsubstituted imidazopyridazinyl (e.g., imidazo[l,2b]pyridazinyl). In some embodiments, R⁴ and R⁵ are joined together to form R¹²- substituted or unsubstituted 6,5 fused ring heteroaryl, R 12 -substituted or unsubstituted 5,6 fused ring heteroaryl, R 12 -substituted or unsubstituted 5,5 fused ring heteroaryl, or R 12 -substituted or unsubstituted 6,6 fused ring heteroaryl. In other embodiments, R⁴ and R⁵ are joined together to form a R 12 -substituted or unsubstituted 5 or 6 membered heteroaryl having at least 2 (e.g. 2 to 4) ring nitrogens.

[0091] In some embodiments, the pi-conjugation element has the formula: -L 1 -(A1 )_q-L 2 -(A 2 )_r- L³- or -L¹-(A¹)_q-L⁴-A³-L²-(A²)_r-L³-. L¹, L², L³ and L⁴ are independently a bond or a linking group having the formula:

In the formula above, the symbol x is an integer from 1 to 50. In some embodiments, x is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some embodiments, x is an integer from 1 to 3. In some embodiments, x is an integer of 1. R^17a and R^17b are independently hydrogen, halogen, -CN, -OR¹⁸, -CONR¹⁹R²⁰, -NR¹⁹R²⁰, -SR¹⁸, -SOR¹⁸, -S0₂R¹⁸,-COR¹⁸,

-COOR 18 , -NR 1 COR 20 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted

heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R^17a and R^17b are independently hydrogen, halogen, -CN, -OR¹⁸,

-CONR¹⁹R²⁰, -NR¹⁹R²⁰, -SR¹⁸, -SOR¹⁸, -S0₂R¹⁸,-COR¹⁸, -COOR¹⁸, -NR¹⁹COR²⁰, R²¹- substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl. [0092] A 1 , A2 and A 3 are independently substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 - substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene. The symbols q and r are independently 0 or 1.

[0093] In some embodiments, the pi-conjugation element has the formula: -L 1 -(A1 )_q-L 2 -(A 2 )_r-

L 3 -. In certain embodiments, L 1 and L3 are bonds, L 2 is a linking group (as defined above or below), A 1 and A 2 are substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, and q and r are 1. In certain embodiments, L 1 and L3 are bonds, L 2 is a linking group (as defined above or below), A 1 and A 2 are R 17 -substituted or unsubstituted arylene, or

R 17 -substituted or unsubstituted heteroarylene, and q and r are 1. In some embodiments, L 1 , L2 and L 3 are bonds, A 1 and A 2 are R 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene, and q is 1 and r is 0.

[0094] In some embodiments, the pi-conjugation element has the formula: -L 1 -(A 1 )_q-L 4 -A 3 -L 2 -

(A 2")_r-L 3 -. In some embodiments, L 1 and L 3 are bonds, L 2 and L 4 are linking groups (as defined above or below), A 1 , A2 and A 3 are substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene, and q and r are 1. In some embodiments, L 1 and L3 are bonds, L 2 and L 4 are linking groups (as defined above or below), A 1 , A2 and A 3 are R 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene, and q and r are 1. In some embodiments, the pi-conjugation element is substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene. In some embodiments, the pi-conjugation element is

R 17 -substituted or unsubstituted arylene or R 17 -substituted or unsubstituted heteroarylene. In certain embodiments, the pi-conjugation element is substituted or unsubstituted phenylene or substituted or unsubstituted naphthylene. In certain embodiments, the pi-conjugation element is

R 17 -substituted or unsubstituted phenylene or R 17 -substituted or unsubstituted naphthylene.

[0095] In certain embodiments, compounds disclosed herein can exhibit increased

fluorescence when bound to amyloids. In certain embodiments, the pi-conjugation element is in a planar or substantially planar orientation when bound to an amyloid. In some embodiments, negative charge donated from EDG can enhance the fluorescent properties of the compounds herein and improve detection of amyloids (e.g., a amyloid plaque). [0096] In some embodiments, a linking group (L . , L2 , L3 and L 4 ) has the formula:

The symbol x is an integer from 1 to 50. In some embodiments, x is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some embodiments, x is an integer from 1 to 5, 1 to 3, 2 or 1. In some embodiments, x is an integer from 1 to 3. In some embodiments, x is an integer of 1. In some embodiments, L 1 , L2 , L 3 and L 4 are independently a bond.

[0097] In some embodiments, A 1 , A2 and A 3 are independently substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted arylene, or R 17 -substituted or unsubstituted heteroarylene. In certain embodiments, q and r are independently 0 or 1. In some embodiments, q is 1 and r is 0. In some embodiments, q is 0 and r is 1. In some embodiments, A 1 , A2 and A 3 are independently substituted or unsubstituted phenylene, or substituted or unsubstituted naphthylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted phenylene, or R 17 -substituted or unsubstituted naphthylene. In some embodiments,

A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted phenylene. In some

embodiments, A 1 , A2 and A 3 are independently substituted or unsubstituted phenylene. In some embodiments, A 1 , A2 and A 3 are independently substituted or unsubstituted naphthylene. In some embodiments, A 1 , A2 and A 3 are independently R 17 -substituted or unsubstituted

naphthylene.

[0098] In some embodiments, R¹⁷ is independently halogen, -CN, -OR¹⁸, -CONR¹⁹R²⁰, -NR¹⁹R²⁰, -SR¹⁸, -SOR¹⁸, -S0₂R¹⁸,-COR¹⁸, -COOR¹⁸, -NR¹⁹COR²⁰, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R 17 is independently halogen, -CN, -OR 18 , -CONR¹⁹R²⁰, -NR¹⁹R²⁰, -SR¹⁸, -SOR¹⁸, -S0₂R¹⁸,-COR¹⁸, -COOR¹⁸, -NR¹⁹COR²⁰, R²¹- substituted or unsubstituted alkyl, R- 21 substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted heteroaryl. In some embodiments, R 17 is

-OR 18 , -NR 19 R 20 , R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 -substituted or unsubstituted 17 21

heteroaryl. In some embodiments, R is R -substituted or unsubstituted C1-C20 (e-g-, Ci-Cio) alkyl, or R 21 -substituted or unsubstituted heteroalkyl.

[0099] R 18 , R 1 and R 20 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. R 18 , R 19 and R 20 are independently hydrogen, R 21 -substituted or unsubstituted alkyl,

R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 - substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 - substituted or unsubstituted heteroaryl. In some embodiments, R 21 is halogen, -OR 22 , -NR 23 R 24 , halogen, -CN, -OR²², -CONR²³R²⁴, -NR²³R²⁴, -SR²², -SOR²², -S0₂R²²,-COR²², -COOR²²,

-NR 23 COR 24 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. R 21 can be halogen, -OR 22 , -NR²³R²⁴, halogen, -CN, -OR²², -CONR²³R²⁴, -NR²³R²⁴, -SR²², -SOR²², -S0₂R²²,-COR²², -COOR²², -NR²³COR²⁴, R^21a- substituted or unsubstituted alkyl, R^21a-substituted or unsubstituted heteroalkyl, R^21a- substituted or unsubstituted cycloalkyl, R^21a-substituted or unsubstituted heterocycloalkyl, R^21a- substituted or unsubstituted aryl, or R^21a-substituted or unsubstituted heteroaryl. In some embodiments, R^21a is halogen, -NH₂, -OH, -SH, -COOH, -COH, unsubstituted alkyl, unsubstituted, heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. R 22 , R 23 and R 24 are

independently hydrogen or unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R 22 , R 23 and R 24 are independently hydrogen or unsubstituted alkyl.

[0100] In some embodiments, the water soluble group is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, the water soluble group is substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, or substituted heteroaryl. In some embodiments, the water soluble group is R 25 -substituted or unsubstituted alkyl, R 25 -substituted or unsubstituted heteroalkyl, R 25 -substituted or unsubstituted cycloalkyl,

R 25 -substituted or unsubstituted heterocycloalkyl, R 25 -substituted or unsubstituted aryl, R 25 - substituted or unsubstituted heteroaryl. In some embodiments, the water soluble group is R 25 - substituted alkyl, R 25 -substituted heteroalkyl, R 25 -substituted cycloalkyl, R 25 -substituted heterocycloalkyl, R 25 -substituted aryl, R 25 -substituted heteroaryl.

[0101] R²⁵ is halogen, -CN, -OR²⁶, -CONR²⁷R²⁸, -NR²⁷R²⁸, -SR²⁶, -SOR²⁶, -S0₂R²⁶,-COR²⁶,

-COOR 26 , -NR 27 COR 28 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted

heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R²⁵ is halogen, -CN, -OR²⁶, -CONR²⁷R²⁸, -NR²⁷R²⁸, -SR²⁶, -SOR²⁶, -S0₂R²⁶,-COR²⁶, -COOR²⁶, -NR²⁷COR²⁸, R²⁹- substituted or unsubstituted alkyl, R²⁹-substituted

9Q 9Q

or unsubstituted heteroalkyl, R -substituted or unsubstituted cycloalkyl, R -substituted or

9Q 9Q

unsubstituted heterocycloalkyl, R -substituted or unsubstituted aryl, or R -substituted or unsubstituted heteroaryl. In some embodiments, R 26 , R 27 and R 28 are independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R²⁶, R²⁷ and

R 28 are independently hydrogen, R 29 -substituted or unsubstituted alkyl, R 29 -substituted or

9Q 9Q

unsubstituted heteroalkyl, R -substituted or unsubstituted cycloalkyl, R -substituted or

9Q 9Q

unsubstituted heterocycloalkyl, R -substituted or unsubstituted aryl, or R -substituted or unsubstituted heteroaryl. In certain embodiments, R 27 and R 28 are optionally joined together to form a substituted or unsubstituted heterocycloalkyl, or a substituted or unsubstituted heteroaryl.

In certain embodiments, R 27 and R 28 are optionally joined together to form a R 29 -substituted or

9Q

unsubstituted heterocycloalkyl, or a R -substituted or unsubstituted heteroaryl.

[0102] R²⁹ is halogen, -CN, -OR³⁰, -CONR³¹R³², -NR³¹R³², -SR³⁰, -SOR³⁰, -SO₂R³⁰,-COR³⁰,

-COOR 30 , -NR 31 COR 32 , substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted

heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In

9Q

some embodiments, R is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl. In some embodiments, R 30 , R 31 and R 32 are independently hydrogen or substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl. In some embodiments, R 30 , R 31 and R 32 are independently hydrogen or unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl or unsubstituted heteroaryl. In some embodiments, R 30 , R 31 and R 32 are independently hydrogen or unsubstituted alkyl.

[0103] In some embodiments, the water soluble group can include a moiety that increases the water solubility of a molecule. In some embodiments, the water soluble group can include a moiety containing a heteroatom (e.g., oxygen). In some embodiments, the heteroatom can be oxygen or nitrogen.

[0104] In some embodiments, the water soluble group is an ethylene glycol moiety having the

some embodiments, y is an integer from 1 to 50. In some embodiments, y is an integer from 1 to 10, from 1 to 20, from 1 to 30, or from 1 to 40. In some

9Q

embodiments, R is -OMe.

9Q

[0105] In some embodiments, the water soluble group is R -substituted or unsubstituted C_\-

9Q

C₂o (e-g-, C₁-C₁₀) alkyl or R -substituted or unsubstituted heteroalkyl. In some embodiments,

9Q

R^{^} is -OH. In some embodiments, the water soluble group can be -(CH₂)_b-(CH₂0H)-CH₂0H, and b is an integer from 0 to 20, or from 0-10.

[0106] In some embodiments, the compound has the structure:

(Ila).

In Formula Ila, q and r are independently 0 or 1, and y is an integer from 1 to 10. L 1 , L2 , L 3 , A 1 ,

A 2", R 4", R 5^J and R 29 are as defined above.

[0107] In some embodiments, the compound has the structure:

R⁵ (lib).

In Formula lib, q and r are independently 0 or 1, and y is an integer from 1 to 10. L 1 , L2 , L 3 , L 4 , A¹, A², A³, R⁴, R⁵ and R²⁹ are as defined above.

[0108] In some embodiments, the compound has the structure:

In Formula Ilia, m is an integer from 0 to 4, z is an integer from 0 to 4, and y is an integer from 1 to 10. L¹, L², L³, R⁴, R⁵, R¹⁷ and R²⁹ are as defined above.

[0109] In some embodiments, the compound has the structure:

(Illb).

In Formula Illb, m is an integer from 0 to 6, z is an integer from 0 to 6, and y is an integer from 1 to 10. L 1 , V 2, If 3, R 4", R 5^J, R 17 and R 29 are as defined above. In some embodiments, m is 0. In some embodiments, z is 0. In some embodiments, m is 1. In some embodiments, z is 1.

[0110] In some embodiments, the compound has the structure:

In Formula IVa, y is an integer from 1 to 10, and z is an integer from 0 to 4. R , R , R and R

9Q

are as defined above. In some embodiments, R is -OMe.

[0111] In some embodiments, the compound has the structure:

In Formula IVb, y is an integer from 1 to 10, and z is an integer from 0 to 6. R , R , R and R

9Q

are as defined above. In some embodiments, R is -OMe. In some embodiments, m is 0. In some embodiments, z is 0. In some embodiments, m is 1. In some embodiments, z is 1.

[0112] In some embodiments, the compound has the structure:

In Formula IVc, m is an integer from 0 to 4, x is an integer from 1 to 10, y is an integer from 1 to 10, and z is an integer from 0 to 4. In some embodiments, x is 1, and m and z are 0. R⁴, R⁵, R¹⁷

9Q 9Q

and R are as defined above. In some embodiments, R is -OMe. [0113] In some embodiments, each substituted group described above in the compounds of the Formulae provided herein is substituted with at least one substituent group. More specifically, in some embodiments, each substituted alkyl, substituted heteroalkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted alkylene, substituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or

unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene described above in the compounds of the Formulae provided herein is substituted with at least one substituent group. In other embodiments, at least one or all of these groups are substituted with at least one size-limited substituent group. Alternatively, at least one or all of these groups are substituted with at least one lower substituent group.

[0114] In other embodiments of the compounds of the Formulae provided herein, each substituted or unsubstituted alkyl is a substituted or unsubstituted C^C^ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 20 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₄-C₈ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 4 to 8 membered heterocycloalkyl, each substituted or unsubstituted alkylene is a substituted or unsubstituted C_j-

C₂₀ alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 20 membered heteroalkylene, each substituted or unsubstituted cycloalkylene substituted or unsubstituted C₄-C₈ cycloalkylene, and each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 4 to 8 membered heterocycloalkylene.

[0115] Alternatively, each substituted or unsubstituted alkyl is a substituted or unsubstituted C_j-C₈ alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8 membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C₅-C₆ cycloalkyl, each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5 to 7 membered heterocycloalkyl, each substituted or unsubstituted alkylene is a substituted or unsubstituted C_j-C₈ alkylene, each substituted or unsubstituted heteroalkylene is a substituted or unsubstituted 2 to 8 membered heteroalkylene, each substituted or unsubstituted cycloalkylene substituted or unsubstituted C₅-C₆ cycloalkylene, and each substituted or unsubstituted heterocycloalkylene is a substituted or unsubstituted 5 to 7 membered heterocycloalkylene. [0116] In some embodiments, the compounds of the Formulae provided herein are one or more of the compounds set forth in Table 1 below:

Table 1. Compounds.

III. Pharmaceutical Compositions

[0117] In another aspect, pharmaceutical compositions disclosed herein (i.e., formulations) can include a compound described herein in combination with a pharmaceutically acceptable excipient (e.g., carrier). The pharmaceutical compositions include optical isomers,

diastereomers, or pharmaceutically acceptable salts of the inhibitors disclosed herein. For example, in some embodiments, the pharmaceutical compositions include a compound disclosed herein and citrate as a pharmaceutically acceptable salt. The compound included in the pharmaceutical composition may be covalently attached to a carrier moiety, as described above. Alternatively, the compound included in the pharmaceutical composition is not covalently linked to a carrier moiety. [0118] A "pharmaceutically acceptable carrier," as used herein refers to pharmaceutical excipients, for example, pharmaceutically, physiologically, acceptable organic or inorganic carrier substances suitable for enteral or parenteral application that do not deleteriously react with the active agent. Suitable pharmaceutically acceptable carriers include water, salt solutions (such as Ringer's solution), alcohols, oils, gelatins, and carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethycellulose, and polyvinyl pyrrolidine. Such preparations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring, and/or aromatic substances and the like that do not deleteriously react with the compounds disclosed herein.

[0119] The compounds disclosed herein can be administered alone or can be coadministered to the subject or biological sample from a subject. Coadministration is meant to include simultaneous or sequential administration of the compounds individually or in combination (more than one compound). The preparations can also be combined, when desired, with other active substances (e.g. to reduce metabolic degradation).

IV. Fluorescence Correlation Spectroscopy (FCS)

[0120] Fluorescence correlation spectroscopy (FCS) is a time-resolved spectroscopic technique that can measure the concentration and size of fluorescently labeled particles[6]. This method may be used to study aggregation phenomenon such as protein oligomerization (e.g. p53 [7,8]) or formation of large aggregates such as prions [9,10]. FCS has also been used to study Αβ aggregation using fluorescently labeled peptides [11-13]. However, the effect of the fluorescent label covalently attached to the peptide on assembly dynamics in these prior studies remains unclear. Moreover, the requirement of incorporation of exogenously added fluorescent Αβ peptides complicates translation to detect amyloid species in human biofluids such as cerebrospinal fluid [15]. In order to circumvent these limitations, a novel fluorescent probe that undergoes strong fluorescence emission when bound to aggregates of amyloidogenic proteins and has a low unbound fluorescence was used. Fluorescence enhancement in the bound state can dramatically increase the signal to noise and permit fluorescent intensity fluctuations (required for FCS measurements) that are derived primarily from amyloid-bound probe rather than unbound probe in solution. In embodiments, this form of Probe Enhancement-FCS (PE-FCS), using the ARCAM 1 probe, can detect Αβ aggregates at earlier time points along the aggregation pathway in comparison to standard bulk fluorescence measurements.

[0121] Fluorescence correlation spectroscopy (FCS) is a technique that directly measures the spontaneous fluorescence fluctuation of systems in thermodynamic equilibrium. It is an ultrasensitive technique operating at the level of single fluorescent molecules diffusing in and out of the confocal volume created by a focused laser beam. That is, in FCS, a sharply focused laser beam illuminates a femtoliter volume. This volume is so small that it typically hosts only one particle out of the many under analysis at a given moment in time. The single molecules diffusing through the illuminated volume give rise to bursts of fluorescence light quanta. Each individual burst, resulting from a single molecule, can be registered. In a typical FCS instrument, the photons are recorded in a time resolved manner by a highly sensitive single-photon detection device. All signals resulting from the diffusion of a series of molecules through the confocal volume are recorded. The quanta belonging to particular fluorescing molecules are identified using autocorrelation software. The number of molecules in the illuminated volume, as well as their characteristic translational diffusion times, can be determined.

[0122] The spontaneous fluorescence fluctuating quantity is the number of observed molecules in a defined unit volume, and the diffusion coefficient and the kinetic coefficients of the system are two quantities that are generally measured. In particular, FCS detects the time-dependent spontaneous intensity fluctuations in the fluorescence signal which may derive from Brownian motion, flow, and chemical reactions, such as binding.

V. Detection of Amyloid Aggregation

[0123] In embodiments, fluorescence correlation spectroscopy (FCS) and a novel amyloid- binding fluorescent probe, ARCAM 1, can be used to monitor the aggregation of the Alzheimer's disease-associated amyloid-β peptide (Αβ). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Αβ assemblies, making it an excellent candidate for probe

enhancement FCS (PE-FCS). ARCAM 1 binding does not change Αβ aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Αβ, when compared to thioflavin T (ThT) or an Αβ peptide modified with a fluorophore. In embodiments, using fluorescent burst analysis (via PE-FCS) to follow aggregation of Αβ, soluble aggregates can be detected at significantly earlier time points compared to typical bulk fluorescence measurements. In embodiments, an amyloid aggregate is a non-native molecule (e.g. a biological sample containing amyloid aggregates has been subjected to physical or chemical denaturing forces.) In embodiments, an amyloid aggregate is native.

[0124] In embodiments, PE-FCS/ARCAM 1 based assays can detect and provide some characterization of small Αβ aggregation intermediates during the assembly process, which enables monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases. In embodiments, biofluids include urine, blood, semen, saliva, cerebrospinal fluids, or others. In embodiments, the biofluid is mixed with a biological buffer (e.g. phosphate buffered saline, HEPES, tris-HCL, etc...) In embodiments, a buffer has a pH of about 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, or more. In embodiment, a buffer has a pH of about 2.5 to about 8.5, about 3.0 to about 8.0, about 3.5 to about 7.5, about 4.0 to about 7.5, about 4.5 to about 6.5, about 5.0 to about 7.0, about 5.5 to about 6.5, about 5.5 to about 7.5, about 6.0 to about 7.0, or about 5.6 to about 6.5.

[0125] In embodiments, detection of an aggregate includes contacting a biological sample (e.g. a biofluid) containing an amyloid aggregate with an amyloid-binding fluorophore thereby forming a fluorescent amyloid aggregate complex comprising said amyloid aggregate anon- covalently bound to said amyloid-binding fluorophore. In some embodiments, detecting said fluorescent amyloid aggregate complex using fluorescence correlation spectroscopy.

[0126] In embodiments, detection and/or characterization of an amyloid aggregate can be used in the diagnosis, prognosis or treatment determination of diseases. In embodiments, the disease can include Alzheimer's disease, bovine spongiform encephalopathy (BSE), Parkinson's disease, Huntington's disease, Down's Syndrome, Dementia with Lewy Body, or Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the amyloid peptide is Αβ peptide and the disease is Alzheimer's disease. In some embodiments, the methods of treating or diagnosing described herein include a method of treating Alzheimer's disease. In some embodiments, the methods of treating or diagnosing described herein include a method of treating Parkinson's disease.

[0127] In embodiments, characteristics of the amyloid aggregate are indicative of disease progression (e.g. smaller aggregates correlate to a less advanced disease, larger aggregates correlate to a more advanced disease state.) In embodiments, the size of an amyloid aggregate is less than about 5,000nm, about 4,000nm, about 3,000nm, about 2,000nm, about 1000 nm, about

500nm, or about lOOnm. In embodiments, the size of an amyloid aggregate is about lOOnm to about 5,000nm, about 500nm, to about 4,000nm, about 500nm to about 3,000nm, about 500nm to about 2,000nm, about 500nm to about Ι,ΟΟΟηηι, or about Ι,ΟΟΟηηι to about 2,000.

[0128] Measuring a size of a fluorescent compound is possible using FCS as fluorescence is monitored in a defined confocal volume. Molecular resident time in the confocal volume is proportional to rates of diffusion and molecular size. In embodiments, a size of an amyloid aggregate can be determined by measuring a number of fluorescent burst events for a given molecule while it resides within the confocal volume. In embodiments, autocorrelation analysis is used in determining the size of the amyloid aggregates.

EXAMPLES

Example 1; Materials and Methods [0129] Synthesis of ARCAM 1.

Scheme 1. Synthesis of ARCAM 1

i. p-TsCl, pyridine, DCM. ii. NaN₃, DMF. iii. PPh₃, Et₂0, H₂0. iv.

Cyanoacetic acid, EDC, HOBt, DCM. v. C, piperidine (cat), THF

[0130] (A) 2-(2-(2-methoxyethoxy)ethoxy)ethyl 4-methyl-benzene-sulfonate.

Methoxyethoxy)ethoxy ethanol (20.0 g, 0.122 mol) was added to a solution of dry pyridine (49.0 mL) and CH₂C1₂ (152 mL). The solution was cooled to 0 °C and p-toluenesulfonyl chloride (27.9 g, 0.146 mol) was added in one portion with stirring. The reaction was allowed to come to room temperature and stir for 24 hours. The reaction was then concentrated in vacuo and the solution was filtered to remove solids. The filtrate was purified by flash silica column chromatography (0-3% MeOH/EtOAc) to give A (20.5 g, 53%) as a clear pale yellow oil. (A): R_f = 0.85 (9% MeOH/EtOAc); 1H NMR (500 MHz, CDC1₃) δ 7.79 (d, J = 8.0 Hz, 2H), 7.33 (d, / = 8.0 Hz, 2H), 4.15 (m, 2H), 3.52-3.69 (m, 10H), 3.37 (s, 3H), 2.44 (s, 3H); ¹³C (125 MHz, CDC1₃) δ 144.9, 133.0, 129.9, 128.1, 72.0, 72.0, 71.5, 70.9, 7038, 70.7, 70.7, 70.7, 69.4, 68.8, 59.2, 42.9, 21.8; HRMS calcd for Ci₄H₂₂0₆SNa [M+Na]⁺ 341.1029, found 341.1030 by ESI.

[0131] (B) l-azido-2-(2-(2-methoxyethoxy)ethoxy)ethane. To a solution of A (1.0 g, 3.14 mmol) in DMF (125 mL) in a flask equipped with a condenser was added sodium azide (0.51 g, 7.85 mmol) and the reaction was heated to 67 °C for 15 hours. The reaction was then cooled to room temperature, diluted with water (125 mL), and stirred for 30 minutes. The reaction mixture was poured into ice (150 mL) and extracted with diethyl ether (3 x 50 mL). The combined organic extracts were washed with water (2 x 30 mL) and dried with anhydrous MgS0₄. The solvent was then removed in vacuo and the residue was purified via silica gel flash

chromatography (10-70% EtOAc/hexanes) to give B (355 mg, 60%) as a clear colorless oil. (B): R/= 0.6 (50 % EtOAc/hexanes); 1H NMR (500 MHz, CDC1₃) δ 3.65 (m 8H), 3.55-3.56 (m, 2H), 3.39-3.40 (m, 2H), 3.38 (s, 3H); ¹³C (125 MHz, CDC1₃) δ 72.0, 70.8, 70.8, 70.2, 59.2, 50.8; HRMS calcd for C₇Hi₅N₃0₃Na [M+Na]⁺ 212.1006, found 212.1006 by ESI.

[0132] (C) 2-cyano-N-(2-(2-(2-methoxyethoxy)ethoxy)ethyl)acetamide. B (3.0 g, 15.7 mmol) was dissolved in diethyl ether (628 mL) and cooled to 0 °C. Triphenylphosphine (5.0 g, 18.8 mmol) was added in one portion and the mixture was allowed to stir at 0 °C for 1 hour and then at room temperature for 6 hours. Water (200 mL) was then added and the reaction was allowed to stir for 12 hours. Toluene (150 mL) was then added and the reaction was allowed to stir for an additional 12 hours. The water layer was then isolated and washed with toluene (1 x 200 mL) and then removed in vacuum to give the corresponding amine as a clear yellow oil which, without further purification, was subjected to the next reaction. A solution of amine (1.79 g, 10.97 mmol), prepared as described above, and hydroxybenzotriazole (HOBt) (1.48 g, 10.97 mmol) in CH₂C1₂ (10 mL) was added dropwise via syringe to a cold (0 °C) solution of cyanoacetic acid (0.621 g, 7.3 mmol) in CH₂C1₂ (15 mL) under argon. The reaction mixture was then allowed to stir for 10 minutes at 0 °C. l-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) (2.1g, 10.97 mmol) was then added in one portion and the reaction was allowed to stir overnight at 0 °C. The reaction was then concentrated in vacuo and purified via silica gel flash chromatography (0-2% MeOH/CH₂Cl₂) to give C (1.4 g, 83%) as a clear yellow oil. (C): R = 0.36 (10 % MeOH/EtOAc); 1H NMR (500 MHz, CDC1₃) δ 7.04 (bs, 1H), 3.63-3.65 (m, 6H), 3.55-3.59 (m, 4H), 3.45-3.48 (m, 2H), 3.39 (s, 2H), 3.37 (s, 3H); ¹³C (125 MHz, CDC1₃) δ 161.5, 114.8, 71.9, 70.6, 70.4, 70.2, 69.3, 59.0, 40.1, 25.9; HRMS calcd for Ci₀H₁₈N₂O₄Na [M+Na]⁺ 253.1159, found 253.1161 by ESI.

[0133] (ARCAM 1) (E)-2-cyano-N-(2-(2-(2-methoxyethoxy)ethoxy)-ethyl)-3-(6-(piperidin-l- yl)naphthalen-2-yl)acrylamide. To a solution of 6-(piperidin-l-yl)naphthalene-2-carbaldehyde (0.15 g, 0.627 mmol) and C (0.115 g, 0.501 mmol) in THF (2.5 mL) was added piperidine (0.01254 mmol) and the reaction was heated to 50 °C for 16 hours. The reaction was then concentrated in vacuo, adsorbed on to silica, and purified via silica gel flash chromatography (0- 20% acetone/toluene) to give 1 (138 mg, 61%) as an orange solid. (ARCAM 1): R ₌ 0.25 (5 % acetone/toluene); 1H NMR (500 MHz, CDCI3) δ 8.33 (s, 1H), 8.11 (s, 1H), 8.00-8.02 (dd, = 8.5 Hz, 1.5 Hz, 1H), 7.69-7.71 (d, = 9.5 Hz, 1H), 7.60-7.62 (d, = 8.5 Hz, 1H), 7.24-7.26 (m, 1H), 7.01 (bs, 1H), 6.84 (m, 1H), 3.64-3.66 (m, 6H), 3.62-3.63 (m, 4H), 3.54-3.55 (m, 2H), 3.35 (s, 3H), 3.12-3.34 (m, 4H), 1.69 (m, 4H), 1.61-1.62 (m, 2H); ¹³C (125 MHz, CDC1₃) δ 161.2, 152.9, 151.6, 137.2, 133.8, 130.3, 127.2, 126.6, 126.1, 125.7, 119.4, 117.8, 108.6, 100.5, 71.9, 70.6, 70.6, 70.5, 69.4, 59.0, 49.5, 40.2, 25.5, 24.3; HRMS calcd for C₂₆H₃₂N₂0₅Na [M+Na]⁺ 474.2363, found 474.2363 by ESI.

[0134] Preparation of aggregated Αβ(1-42).

[0135] Aggregation of Αβ(1-42) was monitored using a continuous ThT assay and material which exhibited maximal fluorescence (t_max) was used as our aggregate standard [24,25].

Briefly, solutions of Αβ(1-42) monomer were isolated as described above, but in 10.9 mM

HEPES pH 7.8, diluted to 10 μΜ in the same buffer. A portion was held on ice and ThT added to the remainder to achieve a final concentration of 20 μΜ. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 700 rpm in a VorTemp 56 shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). ThT

fluorescence was measured every 20 minutes using a SpectraMax M2 microplate reader

(Molecular Devices, Sunnyvale, CA) with excitation and emission of 435 nm and 485 nm, respectively. Aggregation was allowed to proceed until the maximal fluorescence reached a plateau (see FIG. 9), then a portion of the same SEC-isolated monomer sample that had been held on ice was used for a repeat experiment exactly as described above, but adding an equal volume of MQ water in place of ThT.

[0136] FCS measurement of pre-aggregated Αβ with ThT, ARCAM 1 and TAMRA- Αβ.

[0137] ThT (20μΜ) and ARCAM 1 (2.5μΜ) were added into pre-aggregated Αβ (5μΜ) for comparison or TAMRA- Αβ (2.5μΜ, AnaSpec, Fremont, CA) and ARCAM 1 (2.5μΜ) were added into pre-aggregated Αβ for comparison in FCS measurement.

[0138] Monitoring the aggregation of Αβ(1-42) using ARCAM 1 and bulk fluorescence measurements.

[0139] Solutions of SEC-isolated Αβ(1-42) monomer were diluted to 10 μΜ with 20 mM ammonium bicarbonate pH 8.2 and ARCAM 1 was added to a final concentration of 2.5 μΜ. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96- well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 500 rpm in a VorTemp 56 shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). The fluorescence of ARCAM 1 was measured every 20 minutes using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 410 nm and 570 nm, respectively. Data are presented as normalized bulk fluorescence plotted vs. time.

[0140] Monitoring the aggregation of Αβ(1-42) using ARCAM 1 and FCS.

[0141] Aggregation of Αβ(1-42) by FCS was monitored using a modified version of method used for bulk fluorescence measurements. Briefly, Αβ(1-42) monomers were diluted to 10 μΜ with 20 mM ammonium bicarbonate pH 8.2 and incubated in the presence of fluorescence probe at a final concentration of 2.5 μΜ. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 500 rpm in a VorTemp 56

shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). Aliquots were removed every 20 minutes and these aliquots were loaded onto microscope slides (treated with 0.1% BSA solution to reduce the non-specific adsorption) and used for FCS. [0142] FCS setup and data analysis.

[0143] FCS setup and data analysis were described as before 7 ' 10. For the burst analysis, the raw intensity traces were analyzed in the following four steps. First, the histogram of intensity trace (l ms binning time windows) was plotted and the maximum peak (mode of the intensity trace) was used to designate the background signal (see Fig. 12A). The standard deviation (or width) of this histogram was also determined. Second, any signals from the trace with intensities greater than four times the width of the histogram above the background baseline (mode) were selected as burst candidates. Third, due to the diffusion properties, there may be bursts in rapid succession without any intervening time. These bursts are merged as single burst event. Finally, based on the previous three steps, a final burst trace was generated that was used to calculate the burst number.

[0144] Determination of binding constant.

[0145] Aggregated A ?(l-42) (t_max) at a final concentration of 10 μΜ (based on the molecular weight of monomer) was mixed with increasing concentration of ARC AM 1 in 5% DMSO in IX PBS at pH 7.4, 5.6, or 9.1. K_d's were determined as previously described [26]. Binding curves at pH 7.4 are shown in Figure 5. Binding curves at pH 5.6 or 9.1 are shown in FIG. 7A-7D.

[0146] Hydrolytic stability studies of ARCAM 1 in PBS solution.

[0147] A solution of ARCAM 1 was prepared at 100 μΜ in pH 7.4 IX PBS with 5% DMSO (by volume) and incubated quiescently at room temperature. Aliquots from this solution were removed at selected time points and flash frozen at -78°C. Aliquots were then warmed to room temperature and probe stability monitored by LC-UV-MS equipped with a CapCell MGIII C18 column with a 3μιη particle size. The absorption was measured at 254 nm, 280 nm, and 480 nm using a solvent gradient of 2.5% to 100% MeCN in deionized H₂0 with 0.1% formic acid at a flow rate of 0.3 mL/min. Relative probe stability was determined by integrating the peak area of the probe at different time intervals relative to the peak area at time zero.

[0148] The half-life of ARCAM 1 for hydrolysis was determined by linear regression of the relative concentration of 1, [l]/[l]o, as a function of time, t. The half-life was obtained at [l]/[l]o = 0.5

[0149] Measurements of pH dependence of ARCAM 1 fluorescence. [0150] ARC AM 1 was prepared to a final concentration of 4 μΜ in 5% DMSO in IX PBS at various pH values both as free probe in solution and in the presence of 5 μΜ aggregated Αβ(1- 42) (t_max). Sample preparations were incubated at room temperature for 10 minutes prior to measurement.

[0151] Emission spectra were collected with 450 nm excitation wavelength on a PTI

QuantaMaster 40 spectrofluorometer using FelixGX software.

[0152] 2PE excitation comparison of ThT and ARC AM 1.

[0153] The intensities of fluorescence from 20μΜ ThT in HEPES or 2.5 μΜ ARCAM 1 in HEPES were compared using FCS setup (see below). The two-photon excitation wavelengths were set at 780nm, 800nm, 820nm, 840nm, 860nm and 880nm for comparison.

[0154] Αβ(1-42) monomer Preparation.

[0155] Αβ(1-42) was synthesized, purified, and characterized. The peptide mass and purity (98%) were determined by electro spray/ion trap mass spectrometry and purified by reverse phase HPLC, respectively.

[0156] Production of aggregate-free solutions of Αβ(1-42) monomers involved 2 steps. First, Αβ(1-42) was dissolved at 1 mg/mL in disaggregation buffer (50 mM Tris-HCl, pH 8.5 containing 7 M guanidium hydrochloride (GuHCl) and 200 μΜ EDTA) and incubated overnight at room temperature. Then monomer was isolated from the resulting solution by size exclusion chromatography (SEC) using a Superdex 75 10/300 GL column eluted at 0.5 mL/min in 20 mM Ammonium Bicarbonate pH 8.2. The concentration of the monomer peak fraction was determined using 8257 = 1361 M^"1 cm^"1 [27].

[0157] Comparing the aggregation kinetics of Αβ(1-42) with ARCAM 1 and ThT using bulk fluorescence measurements.

[0158] Solutions of SEC-isolated Αβ(1-42) monomer were diluted to 10 μΜ with 20 mM ammonium bicarbonate pH 8.2 and ARCAM 1 was added to a final concentration of 2.5 uM. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96- well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 500 rpm in a VorTemp 56 shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). The fluorescence of ARC AM 1 was measured at 20 minute intervals using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 410 nm and 570 nm, respectively. Data are presented as normalized bulk fluorescence plotted vs. time.

[0159] Preparation of aggregated Αβ(1-42) to determine limit of detection by FCS.

[0160] Aggregation of Αβ(1-42) was monitored using a continuous ThT assay and material which exhibited maximal fluorescence (t_max) was used as the aggregate standard [28,29]. Briefly, solutions of Αβ(1-42) monomer were isolated as described above, but in 10.9 mM HEPES pH 7.8, diluted to 10 μΜ in the same buffer. A portion was held on ice and ThT added to the remainder to achieve a final concentration of 20 μΜ. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were sealed with an adhesive plastic cover (WVR, Radnor, PA) and incubated at room temperature with shaking at 700 rpm in a VorTemp 56 shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). ThT fluorescence was measured at 20 minute intervals using a SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA) with excitation and emission of 435 nm and 485 nm, respectively. Aggregation was allowed to proceed until the maximal fluorescence reached a plateau, then a portion of the same SEC-isolated monomer sample that had been held on ice was used for a repeat experiment exactly as described above, but adding an equal volume of MQ water in place of ThT.

[0161] Monitoring the aggregation of Αβ(1-42) using ARC AM 1 and FCS

[0162] Aggregation of Αβ(1-42) by FCS was monitored using a modified version of method used for bulk fluorescence measurements. Briefly, Αβ(1-42) monomers were diluted to 10 μΜ with 20 mM ammonium bicarbonate pH 8.2 and incubated in the presence of fluorescence probe at a final concentration of 2.5 μΜ. Aliquots (120 μί) of the peptide solutions were then dispensed into the wells of an ice-cold 96-well black microtiter plate (Nunc, Roskilde, Denmark) and read immediately. Plates were then sealed with an adhesive plastic cover (WVR, Radnor,

PA) and incubated at room temperature with shaking at 500 rpm in a VorTemp 56

shaker/incubator with an orbit of 3 mm (Labnet International, Windsor, UK). Aliquots were removed every 20 minutes and these aliquots were loaded onto microscope slides (treated with

0.1% BSA solution to reduce the non-specific adsorption) and used for FCS. [0163] FCS setup

[0164] Two-photon FCS was performed on a customized setup based on an inverted Nikon TE2000 microscope. A collimated 820nm IR laser (Mai Tai, Ti:Sapphire laser with 80MHz and 100 fs pulse width, Spectra-Physics, CA, USA) was aligned through an inverted Nikon microscope (TE-2000 U) into the back aperture of a Nikon 40X Plan Apochromat oil immersion objective (N.A.=1.0). The back aperture of the objective was slightly overfilled, creating a diffraction-limited focal spot. The laser power was set to lOmW (at entrance to microscope) to reduce photobleaching of the fluorescent probe. The emission fluorescence (collected via epifluorescence) was passed through an emission filter (HQ525/50m-2p for ARCAM1,

HQ485/70m-2p for ThT, HQ605/90m-2p for TAMRA-Αβ, Chroma Tech) and focused onto a photomultiplier tube (H7421, Hamamatsu, Japan) configured for single photon counting. Each autocorrelation curve measured was collected for 120 seconds using Flex02-01D/C correlator (correlator.com) and transferred to a personal computer through a high speed USB port. The raw intensity traces with 1 μ8 resolution were also record corresponding to each autocorrelation curve.

[0165] FCS burst analysis

[0166] The raw intensity trace has 1 μ8 time resolution. By binning the time windows new time traces can be generated with 1 ms time resolution, which was analyzed by the burst analysis method.

[0167] To find a fluorescent burst, the following four steps were analyzed. First, the histogram of new intensity trace was plotted and the maximum peak (mode of the intensity trace) was used to designate the background signal (Figure S8). The standard deviation (or width) of this histogram was also determined. Second, any signals from the trace with intensities greater than four times the width of the histogram above the background baseline (mode) were selected as burst candidates. Third, due to the diffusion properties, there may be bursts in rapid succession without any intervening time. These bursts were merged as single burst event. Finally, based on the previous three steps, a final burst trace was generated that was used to calculate the burst number.

[0168] FCS correlation curve analysis

[0169] All FCS curves were analyzed by custom-written Matlab code (Mathworks Inc, Waltham, MA) using a nonlinear least-squares fitting algorithm. The fitting formula of auto- correlation analysis for single-component diffusion was adapted from Krichevsky and Bonnet [30]:

where N is the average particle number of species in the sampling volume, T_D is the residence time of species within the sampling volume, T_D

D is the diffusion coefficient of the species, and ω = co_z/co_Xy is the aspect ratio of the sampling volume. co_z is the axial size of the excitation volume and co_Xy is its radius. Brightness (Q) was calculated by dividing the average fluorescence intensity by average particle number (TV).

[0170] For two-component analysis, the following formula was used for fitting:

G(T) = — (i + _L_r ^> (i + -J—)-¹-² + i— (i + _L)-' (i +

N₂ CO T_D2

where N and N₂ are the average particle number of small species and large species in the sampling volume, individually.

and ¾ are the particle brightness for small species and large species. T_{D I} and T_D2 are the residence time of small species and large species, individually. From single component FCS curve analysis at time=0 minute, small species brightness

and residence time T_DI can be obtained. These values were fixed in two-component analysis for small species. The average particle number N for small species and N₂ for large species, the brightness ¾ and residence time T_D2 for large species can be obtained through the above two- component analysis.

[0171] For the FCS curve fitting analysis at time 0 minutes, a single-component diffusion formula was used and obtained the initial diffusion coefficient Dl=148±25 μιη /s for small species. The Dl value was fixed for the following time points with two-component analysis (Figure S9).

[0172] Diffusion coefficient calculation for rod shaped species

[0173] According to Stokes' diffusion equation [31], the diffusion coefficient (D) and radius (r) of sphere-shape species have the following relationship: βττητ

wherein k is Boltzmann constant (k=l.38x10 -^"23 m 2 kgs -^"2 K -^"1 ), T is temperature (T=298K here) and η is viscosity of the solution (=8.9xl0^~4 m^kgs^"1). A rod-shaped species has a Stokes relationship that takes into account the long axis (a) and short axis (b) radii through the following relationship: = Z__ln(¾

6 H]r b

[0174] FCS fitting results gave the D=3.42±0.43 μι ²/8, this corresponds to a 64-82 nm radius for spherical species and 306-420 nm for rod shape species (~8 nm diameter).

[0175] NMR spectra.

[0176] NMR spectra for compounds disclosed herein are disclosed in FIGS. 10A-10H.

Example 2. Real-time monitoring of Alzheimer's-related amyloid aggregation via Probe Enhancement - Fluorescence Correlation Spectroscopy (PE-FCS).

[0177] Aggregation and deposition of certain proteins is a common facet of many neurological disorders. Specifically, a defining feature of Alzheimer's disease pathology is the presence of abundant amyloid plaques, the principle component of which is the amyloid β peptide (Αβ) [1]. How the intrinsically disordered Αβ monomer converts to the fibrillar aggregates found in amyloid plaques and the relationship between Αβ aggregation and disease remain poorly understood [2] . However, it is widely believed that intermediates in Αβ aggregation, referred to as oligomers, are the initiators of a complex molecular cascade that, over a course of decades, leads to dementia [3]. To date the real time study of Αβ aggregation in solution has been limited by methods that best detect abundant assemblies of protofibrils and mature fibrils[3].

[0178] Real time detection of aggregates typically relies upon the use of fluorophores, such as Thioflavin T (ThT), that are applied in bulk fluorescence measurement assays[4]. When ThT is introduced to a solution of amyloidogenic proteins or peptides, its emission intensity increases with increasing population of aggregates. A major limitation of amyloid aggregation assays that use ThT, however, is that the bulk fluorescence intensity increases above background only once protofibril and fibril structures have become abundant in solution, precluding the capability to detect small, transient intermediates [4,5]. Moreover, ThT has a significant fluorescence as an unbound dye, decreasing the signal to noise ratio for sensitive measurements of small assemblies.

[0179] A family of fluorescent probes that bind Αβ assemblies in solution and in tissue are described herein and previously [16-18]. These probes exhibit a large enhancement in fluorescence properties upon binding to aggregates compared to the weaker fluorescence of the free compounds in solution. For the experiments in this study, a novel Αβ aggregate-binding probe, aryl cyano amide (ARCAM) 1 (FIG. 1A) was designed and synthesized. This probe displayed an ~8-fold increase (at a _em(bound) of -540 nm) in fluorescence emission upon binding to aggregated Αβ in solution versus probe without Αβ (FIG. 1C). The affinity of ARCAM 1 for aggregated Αβ (K_d = 870 + 280 nM at pH 7.4) was comparable to the binding of similar fluorescent probes [18] (see FIG. 5). An important advantage of ARCAM 1 for aggregation studies is its stability in aqueous solutions (FIG. 6) and broad insensitivity of fluorescence as a function of pH (FIG. 7A-7D). For instance, the half-life of ARCAM 1 in phosphate buffered saline (PBS) at room temperature was -150 hours. Importantly, there is negligible change in the effective concentration of ARCAM 1 over the aggregation time-courses. In addition, ARCAM 1 shows similar multi-photon excitation (used in our FCS setup) to that of ThT (FIG. 8).

[0180] To determine whether ARCAM 1 could be used to monitor Αβ (1-42) aggregation kinetics, a 10 μΜ solution of peptide monomers completely free of aggregates was prepared [19,20], added to ARCAM 1 and monitored total fluorescence at intervals until a stable maximal fluorescence was achieved. In parallel, an identical time course was monitored by ThT.

Experiments were conducted at room temperature and were shaken in between sample readings (see below). ARCAM 1 and ThT time courses looked identical when the probe was present in solution throughout the aggregation process (see FIG. 9).

[0181] Probe-Enhancement FCS relies on the increase in fluorescence that occurs as a result of the probe binding to its target. To compare fluorescent probes for detecting Αβ aggregates, FCS curves were measured for solutions containing diluted, pre-aggregated Αβ and the probes. Either ThT or ARCAM 1 was added to pre-aggregated Αβ samples (FIG. 10). Separately, TAMRA- labeled Αβ (1-42) peptides or ARCAM 1 was added to another set of matched samples. FCS measurements of the probe-Αβ solutions were taken after a 30 minutes incubation at room temperature to permit probe binding (for ThT and ARCAM 1) or monomer incorporation (for TAMRA-Αβ). Autocorrelation spectra of ThT-Αβ solutions showed the presence of primarily large aggregates, whereas ARCAM-Αβ solutions of the match sample showed a range of small and large aggregates (FIG. 2A). Conversely, TAMRA-Αβ solutions showed small aggregates whereas measurements of the matched ARCAM-Αβ solution showed a range of small and large aggregates (FIG. 2B). Together these point to ARCAM 1 detecting a larger dynamic range of aggregate sizes compared to ThT or TAMRA-labeled peptide, making it a useful choice for further studies by PE-FCS methods.

[0182] Bulk fluorescent measurement of an Αβ (1-42) aggregation time course (FIG. 3A), in the presence of ARCAM 1 were also carried out in parallel with FCS measurements. Aliquots were collected at 20 minute intervals and the samples were analyzed by multi-photon FCS [21]. The presence of bright fluorescent bursts in the intensity traces are indicative of aggregated amyloid species that were bound by ARCAM 1 diffusing through the multi-photon excitation volume (FIG. 3B and FIG. 1). Burst analysis of these intensity traces counts the statistically significant events (intensity values in top 0.01%) in a fixed time window (see below and FIG. 12B for details) to determine if ARCAM 1-bound material is present in significant quantities. This analysis revealed the presence of ARCAM 1-bound aggregated species at significantly earlier time points compared to aggregates that could be observed using bulk fluorescence measurements (FIG. 3 A, circles compared to squares). Moreover, analysis of solutions of preformed aggregates (see FIG. 10) revealed that the probe could detect aggregated species at total peptide concentrations as low as -100 nM (FIG. 3B). Notably, solutions containing fluorescent ARCAM 1 alone (FIG. 3B, bottom trace) or Αβ peptide alone (FIG. 11) showed no fluorescence bursts by PE-FCS measurement.

[0183] In order to gain additional insight into the properties of the aggregates detected by PE- FCS, the size of the species at the onset of increased burst activity was also estimated (i.e., during the first 120 min of the aggregation of Αβ monomers, FIG. 3A). To determine the size of the diffusing species, intensity time traces (e.g. FIG. 3B) were subjected to autocorrelation analysis [10,21]. The result is a correlation function G(x) that is proportional to the number of burst events while a molecule is resident in the excitation volume for the delay time, τ (FIG. 4, and below). The residence time is directly related to the translational diffusion constant and, therefore, its size. For mixed species solutions, individual species sizes can be distinguished when they exhibit sufficiently different diffusion constants (~5-fold difference in size). However, for more complex mixtures, FCS can only provide an average diffusion constant that is biased towards the more fluorescent species. In addition, the G(0) point is inversely proportional to the number of particles in the solution. For ARC AM 1-Αβ (1-42) solutions, there were two diffusing species: unbound ARCAM 1 and bound to Αβ . The FCS curve at time = 0 min was analyzed by a one component model (FIG. 13), producing a diffusion coefficient (Dl=148 ± 25 μιη /s) consistent with the free diffusion of ARCAM 1 [22]. A two-component fitting model, to distinguish free dye from bound, was used for the analysis of the aggregation time series. Three representative FCS curves with fitting are shown in FIG. 4 for different time points.

[0184] Time autocorrelation analysis of intensity fluctuations revealed diffusing assemblies with a mean diffusion constant of 3.42 μι ²/8 (range: 2.99-3.85 μι ²/8) at 120 min (FIG. 13). This mean diffusion constant corresponds to a hydrodynamic radius between 64-82 nm in size for a spherical particle and 300-420 nm for an 8 nm diameter rod (see below). This

hydrodynamic radius and proposed rod length are consistent with protofibrils, an early assembly intermediate in Αβ aggregation [4] .

[0185] The data presented herein demonstrated that the combination of a novel amyloid- binding fluorophore and FCS enabled the sensitive direct detection of amyloid assemblies at time points at earlier stages than conventional bulk fluorescence measurements. Here, in real-time, amyloid aggregates were detected that were only a few hundred nanometers in length, which is consistent in size with protofibrillar forms of Αβ intermediates [3,4]. Moreover, ARCAM 1 performed well in the detection of a large dynamic range of aggregate sizes in FCS

measurements when compared to ThT or TAMRA-Αβ peptides. This PE-FCS method can detect low concentrations of early amyloid assembly intermediates that were consistent in size with a previous FCS study that used covalently labeled Αβ peptides [11]. A major advantage of the method reported in this work is that using an exogenously added fluorescence reporter (as opposed to fluorescently labeled peptides) may make it possible to analyze patient samples containing a large mixture of native aggregated species. The combination of a fluorescent reporter and PE-FCS -based detection of aggregated Αβ represents a potentially important step towards establishing a reliable method for studying aggregate intermediates that may be present in human CSF [23]. REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A method for detecting an amyloid aggregate in a biological sample, said method comprising:

contacting a biological sample comprising an amyloid aggregate with an amyloid- binding fluorophore thereby forming a fluorescent amyloid aggregate complex comprising said amyloid aggregate non-covalently bound to said amyloid-binding fluorophore; and

detecting said fluorescent amyloid aggregate complex using fluorescence correlation spectroscopy.

2. The method of claim 1, further comprising determining the size of said amyloid aggregate.

3. The method of claim 2, wherein determining the size of said amyloid aggregate comprises measuring a plurality of fluorescent burst events for a resident time in a confocal volume.

4. The method of any of claims 1-3, wherein the amyloid-binding fluorophore has the structure of Formula (I),

EDG nCE

NC WSG

(I)

wherein

EDG is an electron donor group;

7lCE is a pi-conjugation element; and

WSG is a water soluble group.

5. The method of claim 4, wherein said EDG is R^-substituted or unsubstituted alkyl, R^substituted or unsubstituted cycloalkyl, R^substituted or unsubstituted heteroalkyl, R^substituted or unsubstituted heterocycloalkyl, R^substituted or unsubstituted aryl, R^substituted or unsubstituted heteroaryl, -OR², -NR⁴C(0)R³, -NR⁴R⁵, -SR⁶, or -PR⁷R⁸, wherein R¹ is halogen, -OR⁹, -NR¹⁰Rⁿ, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl;

R², R³, R⁴, R⁵, R⁶, R⁷ and R⁸ are independently hydrogen, R¹²-substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or

unsubstituted cycloalkyl, R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl or R¹²- substituted or unsubstituted heteroaryl, wherein R⁴ and R⁵ are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 - substituted or unsubstituted heteroaryl;

R⁹, R¹⁰ and R¹¹ are independently hydrogen, R¹²-substituted or unsubstituted alkyl, R 12 -substituted or unsubstituted heteroalkyl, R 12 -substituted or unsubstituted cycloalkyl,

R 12 -substituted or unsubstituted heterocycloalkyl, R 12 -substituted or unsubstituted aryl, or R 12 - substituted or unsubstituted heteroaryl, wherein R¹⁰ and R¹¹ are optionally joined together to form an R 12 -substituted or unsubstituted heterocycloalkyl, or R 12 -substituted or unsubstituted heteroaryl;

R¹² is halogen, -OR¹³, -NR¹⁴R¹⁵, R¹⁶-substituted or unsubstituted alkyl, R¹⁶- substituted or unsubstituted heteroalkyl, R¹⁶- substituted or unsubstituted cycloalkyl, R¹⁶- substituted or unsubstituted heterocycloalkyl, R¹⁶-substituted or unsubstituted aryl, or R¹⁶- substituted or unsubstituted heteroaryl;

R¹³, R¹⁴ and R¹⁵ are independently hydrogen or unsubstituted alkyl; and

R¹⁶ is unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl.

6. The method of claim 5, wherein said pi-conjugation element has the formula:

or -L¹-(A¹)_Q-L⁴-A³-L²-(A²)_R-L³- wherein

q and r are independently 0 or 1 ;

1 , L2 , L 3 and L 4 are independently a bond or a linking group having

wherein x is an integer from 1 to 50;

AA 11 ,, AA22 aanndd AA 33 aarree iinnddeeppeennddently R 17 -substituted or unsubstituted arylene, or R 17 - substituted or unsubstituted heteroarylene; R is halogen, -OR , -NR R , R -substituted or unsubstituted alkyl, R - substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl, R 21 - substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 - substituted or unsubstituted heteroaryl;

R 18 , R 1"9 and R 2^{^}0 are independently hydrogen, R 21 -substituted or unsubstituted alkyl, R 21 -substituted or unsubstituted heteroalkyl, R 21 -substituted or unsubstituted cycloalkyl,

R 21 -substituted or unsubstituted heterocycloalkyl, R 21 -substituted or unsubstituted aryl, or R 21 - substituted or unsubstituted heteroaryl;

R 21 is halogen, -OR 22 , -NR 23 R 24 , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; and

R 22 , R 2"3^J and R 2^{^}4 are independently hydrogen or unsubstituted alkyl.

7. The method of claim 6, wherein A 1 , A 2 and A 3 are independently R 21 - substituted or unsubstituted naphthylene, or R 21 -substituted or unsubstituted phenylene.

8. The method of claim 6, wherein x is an integer from 1 to 10.

9. The method of any of claims 4-8, wherein said water soluble group is

R 25 -substituted or unsubstituted alkyl, R 25 -substituted or unsubstituted heteroalkyl, R 25 - substituted or unsubstituted cycloalkyl, R 25 -substituted or unsubstituted heterocycloalkyl, R 25 - substituted or unsubstituted aryl, R 25 -substituted or unsubstituted heteroaryl;

wherein

R²⁵ is halogen, -OR²⁶, -NR²⁷R²⁸, R²⁹-substituted or unsubstituted alkyl, R²⁹-

9Q 9Q substituted or unsubstituted heteroalkyl, R -substituted or unsubstituted cycloalkyl, R -

9Q 9Q substituted or unsubstituted heterocycloalkyl, R -substituted or unsubstituted aryl, or R - substituted or unsubstituted heteroaryl;

R 26 , R 2"7 and R 2"8° are independently hydrogen, R 29 -substituted or unsubstituted

9Q 9Q

alkyl, R -substituted or unsubstituted heteroalkyl, R -substituted or unsubstituted cycloalkyl,

9Q 9Q 9Q

R -substituted or unsubstituted heterocycloalkyl, R -substituted or unsubstituted aryl, or R - substituted or unsubstituted heteroaryl, wherein R 27 and R 28 are optionally joined together to

9Q 9Q

form an R -substituted or unsubstituted heterocycloalkyl, or R -substituted or unsubstituted heteroaryl; R is halogen, -OR , -NR R , unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, or unsubstituted heteroaryl; and

R 30 , R 3^J1¹ and R 3^J2" are independently hydrogen or unsubstituted alkyl.

10. The method of claim 9, wherein said water soluble group is an ethylene glycol moiety having the formula: VR²⁹ wherein y is an integer from 1 to 50.

11. The method of claim 9, wherein R is -OH.

12. The method of claim 1, wherein the amyloid-binding fluorophore comprises the structure:

PCT/US2016/040772

13. The method of claim 2, wherein the size of said amyloid aggregate is less than 1000 nm.

14. The method of claim 1, wherein said biological sample is a biofluid.

15. The method of claim 14, wherein said biofluid is blood, urine, saliva, or cerebrospinal fluid.

16. The method of claim 14 or 15, wherein the biological sample further comprises a buffer.

17. A fluorescent amyloid aggregate complex comprising said amyloid aggregate non-covalently bound to said amyloid-binding fluorophore.

18. The fluorescent amyloid aggregate complex of claim 17, wherein the complex is within a vessel.

19. The fluorescent amyloid aggregate complex of claim 18, wherein the vessel further comprises a buffer.

20. The fluorescent amyloid aggregate complex of claim 19, wherein the buffer has a pH of between 4.5 and 7.5.

21. The fluorescent amyloid aggregate complex of claim 20, wherein the buffer has a pH of between 5.0 and 7.0.