Aromatic alkyne compounds useful for labeling biomarkers and biomolecules

Abstract

This invention relates to a compound of formula I or a salt or solvate thereof, wherein: A, B, R 1 R 2 L 1 As defined in the specification, m and n are compounds that can be used to form Raman-labeled bioconjugates with biomarkers and / or biomolecules and can be detected by Raman spectroscopy.

Description

Technical Field

[0001] This disclosure relates to Raman-labeled compounds that can be used for biomarkers and / or biomolecules. Associated Raman-labeled bioconjugates, uses, methods, kits, and procedures are also disclosed. Background Technology

[0002] Raman spectroscopy is an analytical technique that identifies chemical functional groups based on the inelastic scattering of light between specific vibrational modes. Therefore, Raman spectroscopy is used to characterize biological samples and other types of samples to achieve label-free imaging of particles, cells, and tissues, which can be distinguished based on their chemical composition.

[0003] Current Raman spectroscopy methods can detect categories of biomolecules (e.g., proteins, DNA, RNA), but cannot detect or distinguish specific biomolecules of interest, such as differentiating two (or more) different proteins. Single-particle detection methods are of significant value because they can be used to measure the inherent heterogeneity of biological samples, which is impossible with whole-sample measurements.

[0004] While label-free techniques offer significant advantages (e.g., by measuring natural samples and avoiding sample degradation), distinguishing compounds of the same class (e.g., different membrane-bound proteins) remains a challenge due to the nature of Raman spectroscopy. To address this challenge, compounds exhibiting strong Raman signals can be bound to appropriate targeting ligands (e.g., antigen-binding epitopes). Ideally, these compounds will exhibit strong Raman signals in the Raman silencing region (1800 cm⁻¹). -1 -2800cm -1 This refers to the presence of Raman signals in specific regions where no Raman signal is found in most biological samples.

[0005] Min and colleagues first reported Raman activity imaging tags based on oligoalkynes (oligo-alkyne) derivatives (Nat Methods 15, 194–200 (2018); WO 2019 / 028430). This was the first report of multiplexed Raman imaging using oligoalkynes. The multiplexing of Raman activity tags has been well-established; however, applying these tags to the imaging of non-lipid-based biomolecules remains a challenge due to the inherent hydrophobicity of the compounds, resulting in poor solubility in water.

[0006] Other general teachings in this field include: Tian, ​​S., Li, H., Li, Z. et al., Nat Commun 11,81 (2020), which describes a polydiacetylene-based ultra-strong bioorthogonal Raman probe for targeted live-cell Raman imaging. WO 2005 / 031301 A2 relates to Raman-characterized probes and their use in the detection and imaging of molecular processes and structures; CN105823770 A relates to optically interference-free Raman-labeled probes, their preparation methods, and applications; WO 2020 / 182174 A1 relates to aggregation-induced emission (AIE) compounds with fluorescence, photoacoustic, and Raman properties; and WO 2011 / 078794 A1 relates to surface-enhanced Raman spectroscopy-based SERS-based analyte detection.

[0007] The inventors have now discovered novel compounds as Raman tags that, when conjugated with biomarkers and biomolecules, enhance the detection and analysis of said biomarkers and biomolecules (specifically, non-lipid-based biomarkers and biomolecules) in biological samples via Raman technology. In this regard, relevant performance metrics that can be used to evaluate at least some examples of this disclosure are Raman signal intensity and water solubility.

[0008] Listing or discussing any background information in previously published documents or this specification should not be construed as an admission that the documents or background information are part of the prior art or common general knowledge. One or more aspects / examples of this disclosure may or may not solve one or more of these background problems. Attached Figure Description

[0009] The following figures are provided to illustrate various aspects of the inventive concept and are not intended to limit the scope of the invention, unless specifically stated herein.

[0010] Figure 1 The spontaneously normalized Raman spectrum of a sulfonated oligoacetylene according to an example is shown.

[0011] Figure 2 A representative example of using Raman spectroscopy to detect extracellular vesicles expressing CD63 is shown.

[0012] Figure 3 The UV-Vis spectrum of a bioconjugate with Raman labeling according to an example is shown.

[0013] Figure 4 A representative molecular weight curve is shown based on an example of a successful Hercepti bioconjugation.

[0014] Figure 5 An overview of an example procedure for detecting biomolecules on a single particle is shown in schematic form.

[0015] Figure 6 A schematic diagram of an automated particle trapping system for particle analysis, particularly suitable for Raman spectroscopy analysis, is shown as an example.

[0016] Figure 7 It shows that according to Figure 6 An example process flow for the automated particle capture and data acquisition of an automated particle capture system.

[0017] Figure 8 A flowchart illustrating a method for analyzing biological samples based on an example is shown.

[0018] Figure 9 A flowchart is shown for a method for detecting the presence or absence of biomarkers and / or biomolecules in a sample, according to an example.

[0019] Figure 10 A flowchart illustrating a method for diagnosing a subject's disease or condition, based on an example, is shown.

[0020] Figure 11 A flowchart of an in vitro or ex vivo method for imaging biological samples, according to an example, is shown. Detailed Implementation

[0021] According to a first aspect of the invention, a compound of formula I is provided.

[0022] I

[0023] in:

[0024] A and B are each an aromatic group independently;

[0025] Each R 1 Independently selected from -S(O)2OH, -S(O)OH, -OS(O)2OH, -S(O)2NH2, -P(O)(OH)2, -OP(O)(OH)2, -P + (R 3 3. -N + (R 3 )3, -OH, -C(O)OH, -NHC(O)OH, 5- or 6-membered heterocyclic alkyl groups, 5- or 6-membered heteroaryl groups, zwitterionic groups and PEG groups;

[0026] L 1 For bonds or suitable linking groups;

[0027] R 2 A group that can conjugate with biomarkers and / or biomolecules;

[0028] Each R 3Independently selected from hydrogen and C 1-4 alkyl groups;

[0029] m is 1, 2, or 3; and

[0030] n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

[0031] Or its salts or solvates.

[0032] These compounds (including their salts and solvates) may be referred to herein as "compounds of the present invention".

[0033] Salts of compounds of formula I can be prepared using techniques well known to those skilled in the art. For example, compounds of formula I can be reacted with suitable organic or inorganic acids. Salt conversion techniques can also be used to convert one salt into another.

[0034] Examples of salts include base addition salts; metal salts formed with bases, such as sodium and potassium salts. Acid addition salts may also be mentioned, such as salts formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid, and salts formed with carboxylic acids or organic sulfonic acids.

[0035] The compounds disclosed herein can exist in both solvated and solvated forms with solvents such as water, and the present invention is intended to cover both solvated and non-solventized forms of the compounds of the present invention.

[0036] The term "solvent" refers to a complex of variable stoichiometry formed by a solute and a solvent. Such solvents used for the purposes of this invention may not interfere with the activity of the solute. Examples of suitable solvents include, but are not limited to, water, methanol, ethanol, and acetic acid. Solvates in which water is a solvent molecule are generally referred to as hydrates. Hydrates include compositions containing a stoichiometric amount of water, as well as compositions containing a variable amount of water.

[0037] Compounds of Formula I may contain double bonds and therefore may exist as E (opposite-side) and Z (same-side) geometric isomers with respect to each individual double bond. All such isomers and mixtures thereof are included within the scope of this invention.

[0038] The compounds of this invention may also exhibit tautomerism. All tautomer forms (or tautomers) and mixtures thereof are included within the scope of this invention. The terms "tautomer" or "tautomer form" refer to structural isomers of different energies that can be interconverted through a low energy barrier. For example, proton tautomers (also known as proton-heterotautomers) involve interconversions via proton migration, such as keto-enol and imine-enamine isomerization. Valence tautomers involve interconversions through some recombination of bound electrons.

[0039] The compounds of the present invention may also contain one or more asymmetric (e.g., carbon or sulfur) atoms and thus may exhibit optical and / or diastereomeric isomerism. Diastereomers can be separated using conventional techniques, such as chromatography or fractional crystallization. Various stereoisomers can be separated by separating racemic or other mixtures of the compounds using conventional techniques, such as fractional crystallization or HPLC. Alternatively, desired optical isomers can be prepared by reacting a suitable optically active starting material with a chiral auxiliary under conditions that do not induce racemization or epimerization (i.e., the “chiral pool” method), which can then be removed at an appropriate stage by derivatization (i.e., resolution, including dynamic resolution), for example with an isochoric acid, followed by separation of the diastereomeric derivative under conditions known to those skilled in the art by conventional methods such as chromatography, or by reaction with a suitable chiral reagent or chiral catalyst.

[0040] All stereoisomers (including, but not limited to, diastereomers, enantiomers, and throttling isomers) and mixtures thereof (e.g., racemic mixtures) are included within the scope of this invention.

[0041] In the structures shown herein, all stereoisomers are considered and included in the compounds of the invention when no particular chiral atom is specified in the stereochemistry. When the stereochemistry is specified by a solid wedge or dashed line indicating a particular configuration, the stereoisomers are thus specified and defined.

[0042] Unless otherwise stated, alkyl groups as defined herein can be straight-chain or branched when a sufficient number (e.g., at least three) of carbon atoms are present. Alkyl groups that may be mentioned include methyl, ethyl, propyl, isopropyl, butyl, and tert-butyl.

[0043] When used herein, "alkylene" (i.e., alkyl diel) refers to a divalent alkyl group. Specific alkylene groups that may be mentioned include, for example, methylene (i.e., -CH2-).

[0044] An alkenyl group is an unsaturated alkyl group (i.e., having at least one carbon-carbon double bond). Unless otherwise stated, an alkenyl group as defined herein may be straight-chain or branched when a sufficient number of carbon atoms are present (i.e., at least two or three, as the case may be). Permissible alkenyl groups include vinyl, propenyl, and butenyl groups.

[0045] When used herein, "alkylene group" (i.e., alkenyl group) refers to a divalent alkenyl group. Specific alkylene groups that may be mentioned include, for example, -CH=CH-.

[0046] Heterocyclic alkyl groups that may be mentioned include non-aromatic monocyclic heterocyclic alkyl groups, wherein at least one (e.g., one and four) of the atoms in the ring system is not carbon (i.e., a heteroatom, such as sulfur, oxygen, or especially nitrogen), and wherein the total number of atoms in the ring system is four to six. The linking point of the heterocyclic alkyl group can be any atom in the ring system containing (where appropriate) a heteroatom (such as a nitrogen atom), or an atom on any fused carbon ring that can be present as part of the ring system. Heterocyclic alkyl groups can also be in N- or S-oxidized form (i.e., those heteroatoms can be substituted by one or two =O substituents, as the case may be). For the avoidance of doubt, optional substituents include substituents as defined herein. Heterocyclic alkyl groups that may be mentioned include, but are not limited to, pyrrolidone, pyrrolyl, pyrrolinyl, pyranyl, pyrazolyl, tetrahydropyranyl, piperazinyl, piperidinyl, tetrahydrothiophene, sulfolane, tetrahydrothiophene, etc.

[0047] Aromatic (i.e., aryl) groups that may be mentioned include C 6-14 aryl groups (e.g., C) 6-10 Aryl groups. These groups can be monocyclic, bicyclic, or tricyclic, and have 6 to 14 ring carbon atoms, at least one of which is aromatic. The bonding point of the aryl group can be located on any atom in the ring system. However, when the aryl group is bicyclic or tricyclic, these aryl groups are connected to the rest of the molecule through an aromatic ring. C 6-14 Aryl groups include, but are not limited to, phenyl, naphthyl, biphenyl, fluorene, etc.

[0048] Unless otherwise stated, when used herein, the term "heteroaryl" refers to an aromatic group containing one or more heteroatoms (e.g., one to four heteroatoms) preferably selected from N, O, and S. Heteroaryl groups that may be mentioned include, but are not limited to, pyrroleyl, imidazolyl, pyrazinyl, pyrazolyl, pyridazinyl, pyridinyl, pyridinyl, etc.

[0049] As used herein, the term zwitterion refers to a portion containing both positively charged (cationic) and negatively charged (anionic) groups within the same part. Typically, the charges on the positively and negatively charged groups are balanced, resulting in a portion with a net charge of zero. However, the charges on the cation and anionic groups need not be balanced. Specific zwitterions that may be mentioned include, but are not limited to, amino acid groups (e.g., ...). ), betaine group and the following groups: , , and .

[0050] As provided in this article, This indicates the connection point with the compound of formula I.

[0051] Unless otherwise stated, the term PEG group refers to a group containing polyethylene glycol (i.e., polyethylene glycol). The portion of residues, where z is, for example, 2 to 10.

[0052] As used herein, the term "linking group" refers to a group suitable for linking a solubilizing group and / or a group capable of conjugating to a biomarker and / or biomolecule to the compounds of this invention. Any group in the part. For example, the linking group will connect group R in the compound of formula I. 1 Attached to group A.

[0053] In specific embodiments, the "linking group" comprises a series of 1 to 100 (e.g., 1 to 50, such as 1 to 20, preferably 1 to 10) linked connector portions. In some embodiments, the connector portion is a straight or branched chain comprising any combination of alkyl or alkenyl chains or aromatic groups and main chain heteroatoms (e.g., O, S, N, P, etc.), which are optionally substituted by one or more (e.g., 1, 2, or 3) independently selected substituents from the following, as appropriate: -OH, =O, -C(O)OH, -C(O)O(C 1-4 Alkyl groups, -NH2, =NH, -C(O)NH, -C(O)N(C 1-4 Alkyl), -SH, -C(S)(C 1-4 Alkyl groups), -S(O)2NH2 and -S(O)2(C 1-4 Alkyl groups). For example, each connector portion can be independently selected from C16. 1-4 Alkylene (e.g., -CH2-), C 2-4 alkenyl groups (e.g., -CH=CH-), -C(OH)H-, -C(NH2)H-, -O-, -C(O)-, -NH-, -S-, -S(O)-, -S(O)2- and Specific linking groups that may be mentioned include -(CH2)-, -(CH2)-O-(CH2)-, -(CH2)3-O-(CH2)-, -O-(CH2)2-O-, and -OC(O)-NH-.

[0054] Those skilled in the art will understand that groups capable of conjugating to biomarkers and / or biomolecules are groups capable of forming covalent bonds between the compound and the biomarker and / or biomolecule. For example, covalent bonds (e.g., amide bonds) can be formed, for example, by a reaction between a carboxylic acid (i.e., -CO2H) moiety present on the compounds of the present invention as defined above and an amine (i.e., -NH2) group present in the biomarker and / or biomolecule.

[0055] Specific groups that can conjugate with biomarkers and / or biomolecules include, but are not limited to:

[0056] Carboxylic acids and their derivatives (e.g., -C(O)OR) 5 and -C(O)Cl, where R 5 (as defined in this article)

[0057] Isocyanates (i.e., -NCO);

[0058] Isothiocyanates (i.e., -NCS);

[0059] Thiols (i.e., -SH);

[0060] Thioesters (i.e., -C(O)SR) 6 , where R 6 As defined in this article)

[0061] Maleimide group (e.g.) );as well as

[0062] Iodoacetamide group (e.g.) ).

[0063] "Biomarkers and / or biomolecules" include the meaning of naturally occurring biomolecules or their components or fragments, the measurement of which can provide information useful for the prognosis and / or diagnosis of a disease or condition. For example, a biomarker can be a naturally occurring protein or carbohydrate fraction, or its antigenic component or fragment.

[0064] For example, in a specific implementation, the biomarker and / or biomolecule is selected from antigens, antibodies, peptides, proteins, nucleic acids, vesicles, or carbohydrates. Specific biomarkers and biomolecules that may be mentioned are those containing lysine residues.

[0065] To avoid any doubt, in compounds of Formula I, where two or more substituents may have the same identity, the actual identities of the corresponding substituents are not related to each other in any way.

[0066] When a group is referred to herein as optionally substituted, it is specifically expected that such optional substituents may be absent (i.e., the reference to such optional substituents can be removed), in which case the optionally substituted group may be referred to as unsubstituted in some embodiments.

[0067] In this specification, the structure may or may not be represented by a chemical name. In case of any issues regarding nomenclature, the structure shall prevail. Where the compound may exist as a tautomer (e.g., in an alternative resonance form), the depicted structure represents one of the possible tautomer forms, wherein the actual tautomer form observed may vary depending on environmental factors such as solvent, temperature, or pH. All tautomer (and resonance) forms and mixtures thereof are included within the scope of this invention.

[0068] Unless otherwise indicated, all technical and scientific terms used herein shall have the common meaning as understood by one of ordinary skill in the art to which this invention relates.

[0069] For the avoidance of doubt, those skilled in the art will understand that references to specific aspects of the invention (such as the first aspect of the invention) herein will include references to all embodiments and their specific features, which may be combined to form further embodiments and features of the invention.

[0070] Preferably, A and B are each a phenyl group. Therefore, specific compounds of the present invention that may be mentioned include compounds of formula IA.

[0071] IA

[0072] Where R 1 R 2 m and n are as defined in this paper.

[0073] The compounds of the present invention contain one or more "solubilizing groups", that is, groups that promote the dissolution of the compounds of the present invention in a suitable (e.g., aqueous) medium.

[0074] The water solubility of the compounds of the present invention enables the synthesis of water-soluble Raman-labeled bioconjugates (e.g., antigen-targeting molecules with different Raman tags), thereby enabling the easy identification of biomolecules (e.g., peptides, proteins, and / or antibodies) in complex biological samples (e.g., cells, tissues, patient samples).

[0075] More specifically, the improved water solubility of the compounds of the present invention makes it possible to label significantly large quantities of said compounds (i.e. Raman tags) onto biomarkers / biomolecules (e.g., peptides, proteins, antibodies, etc.) to form Raman-active bioconjugates, enabling the design and detection of biomarkers / biomolecules using multiplexable oligoacetylene derivatives of the compounds of Formula I.

[0076] Specific "solubilizing groups" that may be mentioned include -S(O)2OH, -S(O)OH, -OS(O)2OH, -S(O)2NH2, -P(O)(OH)2, and -OP(O)(OH)2. Salts (e.g., -P) + (R 3 3) Quaternary ammonium salts (e.g., -N) + (R 3 3) -OH, -C(O)OH, -NHC(O)OH, 5- or 6-membered heterocyclic alkyl groups, 5- or 6-membered heteroaryl groups, zwitterionic groups and PEG groups, as defined herein.

[0077] Therefore, in the specific implementation plan, R 1 Selected from -S(O)2OH, -P(O)(OH)2 and -N + (R 3 3. Preferably, R 1 It is -S(O)2OH.

[0078] As this article points out, L 1 For bonds or suitable linking groups. In this context, suitable linking groups include 1 to 50 (e.g., 1 to 20, such as 1 to 10) connected linking portions, each independently selected from C 1-4 Alkylene (e.g., -CH2-), C 2-4 alkenyl groups (e.g., -CH=CH-), -O-, -C(O)-, -NH-, -S-, -S(O)-, -S(O)2- and In the specific implementation plan, L 1 It is -(CH2)-. Preferably, L 1 For key.

[0079] As noted herein, the compounds of the present invention have at least one (e.g., 1, 2, or 3) solubilizing (i.e., R) 1 In a specific embodiment, the compound of the present invention has an R group. 1 Group (i.e., m is 1).

[0080] Therefore, the specific compounds of the present invention that may be mentioned include compounds of formula IB.

[0081] IB

[0082] Where R 1 R 2 And n is as defined in this article.

[0083] In the specific implementation plan, R 1 and R 2 Different. Therefore, the specific compounds of the present invention that may be mentioned are compounds of formula I (including all embodiments and specific features, and combinations thereof), provided that R 1 and R 2 Different (i.e. not the same).

[0084] The compounds of the present invention comprise a group capable of conjugating to biomarkers and / or biomolecules. This group can be directly bonded to group B. Alternatively, the group can be connected to group B via a suitable linker. Therefore, in a specific embodiment, R... 2 for

[0085] Groups, in which

[0086] L 2 For bonds or suitable linking groups;

[0087] R 4 Selected from -C(O)OR 5 , -C(O)Cl, -NCO, -NCS, -SH, -C(O)SR 6 , and ;

[0088] R 5 Selected from H, , and C 1-4 alkyl group, the C 1-4 The alkyl group is optionally replaced by one or more phenyl groups; and

[0089] R 6 Selected from hydrogen and C 1-4 Alkyl groups.

[0090] In the specific implementation plan, R 4 Selected from -C(O)OR 5 ,

[0091] -NCO, -NCS, and ,in

[0092] R 5 Selected from H, and .

[0093] Preferably, R 4 -C(O)OR as defined in this paper 5 Groups, such as -C(O)OH.

[0094] As this article points out, L 2 For bonds or suitable linking groups. In this context, suitable linking groups include 1 to 30 (e.g., 1 to 10, such as 1 to 5, preferably 1) linked joint portions, each independently selected from C 1-4 Alkylene (e.g., -CH2-), C 2-4 alkenyl groups (e.g., -CH=CH-), -O-, -C(O)-, -NH-, -S-, -S(O)-, -S(O)2- and Specific linking groups that may be mentioned include -(CH2)- and -(CH2)-O-(CH2)- (i.e., Preferably, L 2 For key or Group.

[0095] Therefore, in the specific implementation plan,

[0096] R 4 -C(O)OR 5 ;and

[0097] L 2 For key or Group.

[0098] Specific embodiments of the invention that may be mentioned are those in which R 4 It is -C(O)OH and L 2 The preferred embodiment of the invention is where R is a bond. Therefore, the preferred compound of the invention is one in which R is a bond. 2 Compounds that are -C(O)OH.

[0099] Therefore, in other embodiments, specific compounds of the present invention may be mentioned, including compounds of formula IC.

[0100] IC

[0101] Where n is as defined in this article.

[0102] The compounds of the present invention contain 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 alkyne moieties, and therefore can be referred to as polyalkynes. Preferably, the compounds of the present invention contain 2, 3, or 4 alkyne moieties; for example, 2 or 3 alkyne moieties (i.e., n is 2 or 3).

[0103] Particularly preferred compounds (or their salts or solvates) of the present invention are described in Table 1 below.

[0104] Example number Compound Name Compound Structure 1 4-((2-sulfophenyl)but-1,3-diyne-1-yl)benzoic acid 2 4-((3-sulfophenyl)but-1,3-diyn-1-yl)benzoic acid 3 4-((4-sulfophenyl)but-1,3-diyn-1-yl)benzoic acid 4 4-((3-sulfophenyl)hexa-1,3,5-triyne-1-yl)benzoic acid

[0105] Table 1.

[0106] preparation

[0107] The compounds of the present invention as described herein can be prepared according to techniques well known to those skilled in the art, such as those described in the examples provided below.

[0108] Compounds of Formula I can be obtained from available starting materials by methods similar to those known in the literature or by conventional synthetic procedures, using appropriate reagents and reaction conditions according to standard techniques. In this regard, those skilled in the art may in particular refer to BM Trost and I. Fleming, “Comprehensive Organic Synthesis,” Pergamon Press, 1991.

[0109] According to a second aspect of the invention, a method is provided for preparing a compound as defined in the first aspect of the invention (i.e., a compound of formula I), the method comprising reacting a protected derivative of a compound of formula I in the presence of a suitable deprotecting agent.

[0110] For example, where R 1 It is -S(O)2OH and R 2 Compounds of formula I with -C(O)OH (i.e., compounds of formula IC as defined herein) can be prepared according to procedures known to those skilled in the art by reacting a compound of formula II with a suitable deprotecting agent (e.g., trifluoroacetic acid) in the presence of a suitable solvent (e.g., dichloromethane):

[0111] II

[0112] Where n is as defined in this paper, and P 1 For a suitable sulfonic acid protecting group (such as 2,2,2-trifluoro-1-(p-tolyl)ethyl group), and P 2 For a suitable carboxylic acid protecting group (such as a tert-butyl group).

[0113] Compounds of Formula II can be prepared according to procedures known to those skilled in the art by reacting the following compounds in the presence of a copper(I) salt (e.g., CuCl), a suitable reducing agent (hydroxylamine hydrochloride), and a suitable solvent (e.g., diethyl ether): Compounds of Formula III

[0114] III

[0115] With compounds of formula IV,

[0116] IV

[0117] Where P 1 and P 2 As defined above, X is a suitable halogen atom (such as a bromine atom), and q is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11.

[0118] Compounds of Formula III are commercially available, known in the literature, or can be obtained from available starting materials using conventional synthetic procedures, according to standard techniques, and with appropriate reagents and reaction conditions. For example, compounds of Formula III can be prepared according to procedures known to those skilled in the art by reacting compounds of Formula V with a suitable halogenating agent (e.g., N-bromosuccinimide) and a suitable deprotecting / activating agent (e.g., silver fluoride (I)) in the presence of a suitable solvent (e.g., acetonitrile).

[0119] V

[0120] Where P 2 And q as defined above.

[0121] Compounds of formula V are commercially available, known in the literature, or can be obtained from available starting materials using standard synthetic procedures, appropriate reagents, and reaction conditions, according to standard techniques.

[0122] Compounds of Formula IV are commercially available, known in the literature, or can be obtained from available starting materials using conventional synthetic procedures, according to standard techniques, and with appropriate reagents and reaction conditions. For example, compounds of Formula IV can be prepared according to procedures known to those skilled in the art by reacting compounds of Formula VI with a suitable deprotecting agent (e.g., TBAF) in the presence of a suitable solvent (e.g., THF).

[0123] VI

[0124] Where P 1 As defined above.

[0125] Compounds of Formula VI are commercially available, known in the literature, or can be obtained from available starting materials using conventional synthetic procedures, according to standard techniques, and with appropriate reagents and reaction conditions. For example, compounds of Formula VI can be prepared according to procedures known to those skilled in the art by reacting the following compounds in the presence of a suitable coupling agent (e.g., bis(triphenylphosphine)palladium(II) chloride), a suitable copper(I) salt (e.g., CuCl), a suitable ligand (e.g., triphenylphosphine), a suitable base (e.g., N,N-diisopropylamine), and a suitable solvent (e.g., toluene): compounds of Formula VII

[0126] VII

[0127] Compounds of formula VIII,

[0128] VIII

[0129] Where P 1 As defined above, and X is a suitable halogen atom (such as a bromine atom).

[0130] Compounds of Formula VII are commercially available, known in the literature, or can be obtained from available starting materials using conventional synthetic procedures, according to standard techniques, and with appropriate reagents and reaction conditions. For example, compounds of Formula VII can be prepared according to procedures known to those skilled in the art by reacting compounds of Formula IX with a suitable protecting agent (e.g., 2,2,2-trifluoro-1-(p-tolyl)ethanol) in the presence of a suitable base (e.g., 1,4-diazabicyclo(2.2.2)octane) and a suitable solvent (e.g., CH₂Cl₂).

[0131] IX

[0132] Where X is as defined above and Z is a suitable leaving group (e.g., a chlorine atom).

[0133] The compounds of formulas VIII and IX are commercially available, known in the literature, or can be obtained from available starting materials using standard synthetic procedures, appropriate reagents, and reaction conditions, according to standard techniques.

[0134] use

[0135] It has been found that the compounds of this invention exhibit Raman quiescent region (1800 cm⁻¹). -1 -2800cm -1 The compounds exhibit a strong Raman signal that is unaffected by biological samples, thus enabling the identification of biomolecules of interest within the sample. Therefore, the compounds of this invention can be considered "Raman tags" or "Raman labels".

[0136] Furthermore, it has been found that the compounds of the present invention exhibit higher Raman signals compared to other Raman-active molecules (e.g., alkynes, azides, fully deuterated compounds, or nitriles), which, although exhibiting signals in the Raman silent region, have much lower relative intensities compared to the compounds of the present invention.

[0137] Therefore, one use of the compounds of the present invention is their ability to form Raman-labeled bioconjugates with biomarkers and / or biomolecules. More specifically, the compounds of the present invention are useful because their water solubility enables the formation of water-soluble Raman-labeled bioconjugates that exhibit a high signal-to-noise ratio in the Raman silent region. Thus, such bioconjugates can be used as highly sensitive tag molecules in the Raman spectra of biological systems, thereby enabling the easy identification of biomolecules (e.g., peptides, proteins, and / or antibodies) in complex biological samples (e.g., cells, tissues, patient samples).

[0138] Previously reported compounds based on oligohydryne structures exhibit reduced solubility in aqueous environments due to their carbon backbone, which limits their application as vibrational tags for biomolecular detection, as the conjugation of these compounds leads to instability of biomolecules in aqueous media.

[0139] The Raman signals of Raman-labeled bioconjugates can be synthesized using multiplexed signals, enabling the simultaneous identification of multiple bioconjugates by adjusting the number of alkyne units within the structure. Unlike previously reported alkyne derivatives, the water solubility of the compounds of this invention makes it possible to label a significantly large number of compounds onto biomarkers and / or biomolecules of interest (e.g., peptides, proteins, antibodies, etc.) to provide water-soluble Raman-active bioconjugates, thus improving the multiplexing capabilities of all oligoalkyne derivatives.

[0140] The data provided in the examples indicate that bioconjugation experiments with the water-insoluble derivatives showed 0.31 tags per antibody. However, using the compounds of the present invention, it is possible to achieve at least more than 6 tags per antibody. This enhanced labeling efficiency makes the signals on the biomolecules more readily detectable, thereby lowering their detection limits.

[0141] According to a third aspect of the invention, a Raman-labeled bioconjugate is provided, consisting of a compound of formula I as defined herein and a biomarker and / or biomolecule. These bioconjugate compounds (including their salts and solvates) may be referred to herein as “bioconjugates of the invention”.

[0142] The bioconjugates of the present invention are characterized by the presence of a carboxylic acid (i.e., -CO2H) moiety on a compound of formula I as defined above (e.g., in R...). 2 At least one covalent bond (e.g., amide bond) is formed by the reaction between the substance and an amine (i.e., -NH2) group present in biomarkers and / or biomolecules.

[0143] Specific biomarkers and / or biomolecules that may be mentioned are selected from antigens, antibodies, peptides, proteins, nucleic acids (e.g., RNA, DNA, etc.), vesicles, and carbohydrates. For example, specific extracellular markers such as CD63, or cancer-specific markers such as HER2, LIV-1 EGFR, TROP2, and EpCAM.

[0144] Those skilled in the art will recognize that the compounds of the present invention can be used to prepare bioconjugates of the present invention as defined herein (i.e., Raman-labeled bioconjugates of the third aspect of the present invention). Therefore, in a fourth aspect of the present invention, the use of compounds of formula I as defined herein in the preparation of Raman-labeled bioconjugates as defined herein is provided.

[0145] As noted herein, the bioconjugates of the present invention comprise biomarkers and / or biomolecules. These biomarkers and / or biomolecules may have pharmacological activity and / or may be metabolized in vivo after oral or parenteral administration to form pharmacologically active compounds. Therefore, the bioconjugates of the present invention may also possess the aforementioned pharmacological activity. Accordingly, according to a fifth aspect of the present invention, a Raman-labeled bioconjugate for medical use is provided as defined above (i.e., a Raman-labeled bioconjugate as defined in the third aspect of the present invention).

[0146] The bioconjugates of the present invention are particularly useful for detecting the presence or absence of biomarkers and / or biomolecules in samples using Raman spectroscopy.

[0147] According to a sixth aspect of the present invention, a method for detecting the presence or absence of biomarkers and / or biomolecules in a sample is provided, wherein the method comprises the following steps:

[0148] a) Provide samples;

[0149] b) The sample is incubated with a compound of formula I as defined herein under conditions that allow the formation of Raman-labeled bioconjugates as defined herein, wherein Raman-labeled bioconjugates are formed if biomarkers and / or biomolecules are present in the sample.

[0150] c) Remove any non-conjugated compounds of formula I; and,

[0151] d) Use Raman spectroscopy to analyze the sample to detect the presence or absence of Raman-labeled bioconjugates.

[0152] Preferably, the method is an in vitro or ex vivo method.

[0153] It should be understood that a non-conjugated compound of formula I refers to any compound of formula I that has not formed a conjugate after being incubated with the sample. It should also be understood that 'removing any non-conjugated compound of formula I' in step (c) means removing at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% of the non-conjugated compound of formula I.

[0154] As used herein, when referring to measurable values ​​such as the amount of a compound, dosage, time, temperature, etc., the term "about" refers to a change of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount. It is anticipated that in each case, such terms could be replaced with the symbol "±10%" or similar (or by indicating a change in a specific amount calculated based on the relevant value). It is also anticipated that in each case, such terms could be deleted.

[0155] In principle, any Raman spectroscopy technique can be used to determine the presence and / or absence of Raman-labeled bioconjugates in a sample. Examples include spontaneous Raman spectroscopy, enhanced Raman spectroscopy (e.g., surface-enhanced Raman spectroscopy, tip-enhanced Raman spectroscopy, surface plasmon polariton-enhanced Raman scattering, surface-enhanced resonance Raman spectroscopy), nonlinear Raman spectroscopy, non-resonant Raman spectroscopy, resonance Raman spectroscopy, confocal Raman spectroscopy, coherent anti-Stokes Raman spectroscopy, spatially shifted Raman spectroscopy, depth Raman spectroscopy, and morphologically oriented Raman spectroscopy. Where applicable, any of these techniques can be combined with techniques for sample trapping, such as spontaneous Raman spectroscopy coupled with optical tweezers.

[0156] Preferably, the presence and / or absence of the biomarker in step (d) can be detected by detecting the Raman silent region (1800 cm⁻¹). -1 -2800cm -1 The signal in the compound is used to determine the compound of the present invention, which is associated with and indicates the compound of the present invention.

[0157] Therefore, in a specific implementation, the presence of a biomarker is indicated by a Raman signal in a Raman silent region that is greater than the Raman signal of a negative control containing only the biomarker to be measured, as defined by the user.

[0158] In other implementations, the absence of a biomarker is indicated by the indistinguishability of the Raman signal in the Raman silent region compared to the Raman signal of a blank buffer sample.

[0159] As defined herein, a sample is an article to be analyzed (e.g., by Raman spectroscopy). In the context of this invention, the term sample encompasses articles containing, including, containing, and characterized at the single-particle level (or similar) biomolecules, bioparticles, and biomaterials or systems (wherein the expression "particle" in the context of this disclosure encompasses micron and nanoparticles as well as other articles capable of exhibiting a Raman response, such as polymeric particles and vesicles for drug delivery systems, such as liposomes and polymeric vesicles, and extracellular vesicles or exosomes).

[0160] Suitable samples that may be mentioned include biological samples, chemical samples (e.g., non-aqueous solutions, suspensions, colloids, emulsions, and foams), water samples (e.g., aqueous solutions, suspensions, colloids, emulsions, and foams), food samples, environmental samples (e.g., water, soil, biological materials, and waste (liquid, solid, or sludge) collected from the environment), and process samples (including bioprocesses).

[0161] Preferably, the sample is a biological sample. Preferably, the biological sample is provided by a mammal. The mammal can be any domesticated or farm animal (e.g., rat, mouse, guinea pig, cat, dog, horse, or primate). Preferably, the mammal is a human.

[0162] Biological samples as used herein include any relevant biological samples that can be used for molecular profiling analysis, such as tissue sections such as biopsies or tissue sections removed during surgery or other procedures, body fluids (e.g., liquid biopsies), autopsy samples and frozen sections for histological testing, and samples containing cells. Such biological samples include blood or blood fractions or products (e.g., serum, buccal membrane, plasma, platelets, erythrocytes, etc.), sputum, malignant effusions, buccal cell tissue, cultured cells (e.g., primary cultures, explants, and transformed cells), feces, urine, other biological or body fluids (e.g., prostatic fluid, gastric juice, intestinal fluid, renal fluid, pulmonary fluid, cerebrospinal fluid, etc.). Biological samples may contain biological material as fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissue blocks or within RNA preservatives and formalin fixatives. Preferably, the biological sample is a cell or tissue sample (or a derivative thereof), such as a sample containing cancer cells or composed of cancer cells.

[0163] Biological samples can be processed using techniques understood by those skilled in the art. Samples can be, but are not limited to, fresh, frozen, or fixed cells or tissues. Biological samples can contain cultured cells, including primary or immortalized cell lines derived from subject samples. Biological samples can also refer to extracts from subject samples. For example, biological samples can contain DNA, RNA, or proteins extracted from tissues or body fluids. Many techniques and commercial kits are available for such purposes. Fresh biological samples from subjects can be treated with reagents to preserve RNA before further processing, such as cell lysis and extraction. Biological samples can include frozen samples collected for other purposes. Biological samples can be associated with relevant information such as the subject's age, sex, and presenting clinical symptoms; the source of the sample; and the methods used for sample collection and storage.

[0164] The detection of the presence or absence of biomarkers and / or biomolecules in samples can be widely applied, but not limited to, the monitoring of water, food and environmental contaminants, bioprocess reactions, drug adulteration, drug overdose, therapeutic drugs in patients, and exposure to environmental toxins.

[0165] The detection of the presence or absence of biomarkers and / or biomolecules in biological samples can be particularly useful for diagnosing diseases or conditions in subjects.

[0166] According to a seventh aspect of the present invention, a method for diagnosing a disease or symptom in a subject is provided, wherein the method comprises the following steps:

[0167] a) Provide biological samples previously obtained from the subject;

[0168] b) The biological sample is incubated with a compound of formula I as defined herein under conditions that allow the formation of Raman-labeled bioconjugates as defined herein, wherein a Raman-labeled bioconjugate is formed if a biomarker and / or biomolecule is present in the biological sample.

[0169] c) Remove any non-conjugated compounds of formula I; and,

[0170] d) Use Raman spectroscopy to analyze biological samples to detect the presence or absence of Raman-labeled bioconjugates.

[0171] The presence of Raman-labeled bioconjugates indicates that the subject has a disease or condition.

[0172] In the specific implementation plan, the disease or condition is selected from cancer, cardiovascular disease (such as heart failure), diabetic nephropathy, diabetes (such as type 2 diabetes), insulin resistance, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, pain, obesity, inflammation (including chronic inflammatory diseases), autoimmune diseases, osteoporosis, enteropathy and hyperinsulinemia associated with obesity or cardiovascular disease, hyperinsulinemia and related conditions, fibrotic conditions, and neurodegenerative diseases.

[0173] Those skilled in the art will understand that the term "cancer" includes one or more diseases belonging to a classification of conditions characterized by uncontrolled cell division and the ability of these cells to invade other tissues by invading, proliferating directly into adjacent tissues, or by metastasizing and implanting at distant sites. "Proliferation" includes an increase in the number and / or size of cancer cells. "Metastasis" refers to the movement or migration (e.g., invasive) of cancer cells from the primary tumor site in the subject to one or more other areas of the subject's body (where the cells can then form a secondary tumor).

[0174] Therefore, the method of the seventh aspect of the present invention is suitable for diagnosing any type of cancer, including all tumors (non-solid and preferably solid tumors, such as carcinoma, adenoma, adenocarcinoma, leukemia, regardless of the organ). For example, cancer cells can be selected from cancer cells of the breast, bile duct, brain, colon, stomach, reproductive organs, thyroid, hematopoietic system, lungs and airways, skin, gallbladder, liver, nasopharynx, nerve cells, kidneys, prostate, lymph nodes, and gastrointestinal tract. Preferably, the cancer is selected from colon cancer (including colorectal adenoma), breast cancer (e.g., postmenopausal breast cancer), endometrial cancer, hematopoietic system cancers (e.g., leukemia, lymphoma, etc.), thyroid cancer, kidney cancer, esophageal adenocarcinoma, ovarian cancer, prostate cancer, pancreatic cancer, gallbladder cancer, liver cancer, and cervical cancer. More preferably, the cancer is selected from colon cancer, prostate cancer, and especially breast cancer. In cases where the cancer is a non-solid tumor, it is preferably a hematopoietic tumor, such as leukemia (e.g., acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL)). Preferably, the cancer cells are breast cancer cells.

[0175] Those skilled in the art will understand that the term "diabetes" (i.e., diabetes mellitus) refers to both type 1 (insulin-dependent) diabetes and type 2 (insulin-independent) diabetes, both of which involve dysfunction of glucose homeostasis. The method of the seventh aspect of the invention is suitable for diagnosing type 1 and / or type 2 diabetes.

[0176] In addition to its use in diagnosing diabetes, the method of the seventh aspect of this invention is also suitable for diagnosing diabetic nephropathy. Diabetic nephropathy refers to kidney damage caused by diabetes and is a serious complication of both type 1 and type 2 diabetes. Diabetic nephropathy affects the kidneys' ability to remove waste products from the blood for excretion as urine and can lead to kidney failure.

[0177] Furthermore, the method of the seventh aspect of the present invention is suitable for diagnosing chronic kidney disease, including chronic kidney disease without type 2 diabetes. "Chronic kidney disease" is a condition characterized by the gradual loss of kidney function over time. Chronic kidney disease is usually caused by one or more other diseases or conditions affecting the kidneys, such as hypertension, diabetes, high cholesterol, kidney infection, glomerulonephritis, polycystic kidney disease, urinary tract obstruction (i.e., impaired urine flow), and long-term medication use.

[0178] Those skilled in the art will understand that the term "hyperinsulinemia or related conditions" includes hyperinsulinemia, type 2 diabetes, impaired glucose tolerance, insulin resistance, metabolic syndrome, dyslipidemia, childhood hyperinsulinemia, hypercholesterolemia, hypertension, obesity, fatty liver disease, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular diseases such as stroke, systemic lupus erythematosus, neurodegenerative diseases such as Alzheimer's disease, and polycystic ovary syndrome. Other disease states include progressive kidney disease, such as chronic renal failure.

[0179] Specifically, the method of the seventh aspect of the present invention may be suitable for diagnosing obesity associated with hyperinsulinemia and / or cardiovascular disease associated with hyperinsulinemia.

[0180] The method of the seventh aspect of the present invention is also suitable for diagnosing cardiovascular diseases, such as heart failure, wherein the cardiovascular disease is not related to hyperinsulinemia.

[0181] Conditions / symptoms in which fibrosis plays a role include (but are not limited to) scar healing, keloids, scleroderma, pulmonary fibrosis (including idiopathic pulmonary fibrosis), renal systemic fibrosis and cardiovascular fibrosis (including endocardial myocardial fibrosis), systemic sclerosis, cirrhosis, macular degeneration, retinal and vitreoretinal diseases, Crohn's disease / inflammatory bowel disease, postoperative scar tissue formation, radiation and chemotherapy-induced fibrosis, and cardiovascular fibrosis.

[0182] Neurodegenerative diseases that may be mentioned include Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis (ALS), polyglutamine diseases such as spinal and bulbar muscular atrophy (SBMA), dentary and pallidoid muscular atrophy (DRPLA), and various spinocerebellar ataxias (SCA).

[0183] The method of the seventh aspect of the present invention is suitable for the diagnosis of non-alcoholic fatty liver disease (NAFLD).

[0184] Nonalcoholic fatty liver disease (NAFLD) is defined as excessive accumulation of fat in the form of triglycerides in the liver (steatodegeneration) (histologically termed accumulation of more than 5% of hepatocytes). NAFLD is the most common liver disease in developed countries (for example, affecting approximately 30% of adults in the United States), and most patients are asymptomatic. If left untreated, the condition can gradually worsen and eventually lead to cirrhosis. NAFLD is particularly prevalent in obese individuals, with approximately 80% of obese patients having the disease.

[0185] A diagnosis of NAFLD can be made if the patient's alcohol consumption is not considered a major contributing factor. The typical threshold for diagnosing fatty liver disease as "alcohol-independent" is: less than 20g of alcohol per day for female subjects and less than 30g of alcohol per day for male subjects.

[0186] Specific diseases or conditions associated with NAFLD include metabolic conditions such as diabetes, hypertension, obesity, dyslipidemia, abeta-lipoproteinemia, glycogen storage disease, Weber-Christian disease, acute fatty liver of pregnancy, and lipodystrophy. Other non-alcoholic factors associated with fatty liver disease include malnutrition, total parenteral nutrition, severe weight loss, refeeding syndrome, jejunoileal bypass, gastric bypass, polycystic ovary syndrome, and diverticulosis.

[0187] Nonalcoholic steatohepatitis (NASH) is the most severe form of NAFLD and is a condition characterized by excessive fat accumulation (steatohepatitis) accompanied by liver inflammation. In advanced stages, NASH can lead to the development of scar tissue in the liver (fibrosis) and eventually cirrhosis.

[0188] Specific autoimmune diseases known to those skilled in the art include Crohn's disease / inflammatory bowel disease, systemic lupus erythematosus, and type 1 diabetes.

[0189] Specific intestinal diseases that should be mentioned include Crohn's disease / inflammatory bowel disease and gastrointestinal cancers.

[0190] Those skilled in the art will understand that references to a “diagnosis” (or similarly, “diagnosing” the stated condition) for a particular symptom will have their normal meaning in the medical field. Specifically, these terms may refer to determining the presence or absence of a disease state in an individual (e.g., determining whether an individual has cancer).

[0191] Those skilled in the art will understand that such a diagnosis will be performed on subjects in need of it. Those skilled in the art can assess a subject's need for such a diagnosis using conventional techniques. In the context of this invention, "subjects in need of a diagnosis" includes subjects suspected of having a condition or a condition as defined herein. As used herein, the terms "disease" and "symptom" (and similarly, the terms symptom, ailment, medical problem, etc.) are used interchangeably.

[0192] Technicians will recognize that Raman spectroscopy is a useful technique for imaging biological samples. For example, vibrational microscopy based on Raman scattering has been recognized in recent years as one of the most promising and powerful cell imaging tools, capable of directly visualizing detailed molecular structure information, quantitative relationships between signal intensity and substance concentration, and narrowband multicolor imaging, among other things.

[0193] As indicated herein, the compounds of the present invention can be used to prepare Raman-labeled bioconjugates (e.g., bioconjugates of the third aspect of the present invention). These Raman-labeled bioconjugates can be detected using Raman spectroscopy, thereby facilitating the imaging of biological samples.

[0194] According to an eighth aspect of the present invention, an in vitro or ex vivo method for imaging biological samples is provided, wherein the method includes the following steps:

[0195] a) Provide biological samples;

[0196] b) Incubate a compound of formula I as defined herein with a biological sample to form a Raman-labeled bioconjugate as defined herein, wherein the Raman-labeled bioconjugate is formed if a biomarker and / or biomolecule is present in the biological sample.

[0197] c) Remove any non-conjugated compounds of formula I; and

[0198] d) Image biological samples using Raman spectroscopy.

[0199] The methods disclosed in this specification are applicable to the detection / imaging of any Raman-labeled bioconjugates as defined herein (e.g., according to a third aspect of the invention).

[0200] As provided herein, the compounds of the present invention (such as compounds of formula I) may be provided in the form of a kit along with instructions for using compounds of formula I in, for example, the methods described herein.

[0201] Therefore, according to a ninth aspect of the present invention, a reagent kit is provided, the reagent kit comprising:

[0202] (a) Compounds of formula I as defined herein; and

[0203] (b) A specification for the use of a compound of formula I as defined herein in in vitro or ex vivo methods for detecting the presence or absence of biomarkers and / or biomolecules in a sample, methods for diagnosing a disease or condition in a subject, and / or methods for imaging biological samples.

[0204] In one specific implementation, the specification relates to in vitro or ex vivo methods for detecting the presence or absence of biomarkers and / or biomolecules in biological samples, methods for diagnosing diseases or conditions in subjects, and / or methods for imaging biological samples.

[0205] Unbound by theory, the compounds of this invention are considered to exhibit enhanced solubility, enabling efficient labeling of biomarkers and / or biomolecules, thereby providing water-soluble bioconjugates. The water solubility of the bioconjugates lowers their detection limits and enables enhanced detection of biomarkers and / or biomolecules in biological samples via Raman spectroscopy. This enhanced detection of biomarkers and / or biomolecules is particularly useful for methods involving Raman spectroscopy in detection, imaging, and diagnostics.

[0206] According to another aspect, the present invention provides a computer program that can be distributed via electronic data transmission or via a computer-readable medium, the computer program including computer program code means adapted to cause the automated particle capture system to execute any of the programs described herein when the program is loaded onto the automated particle capture system.

[0207] Illustrative embodiments

[0208] The present invention is illustrated in more detail in the following non-limiting embodiments.

[0209] Figure 1 Spontaneously normalized Raman spectra 100 of sulfonated oligoyne according to one example are shown. Different oligoyne (2-yne to 4-yne) derivatives (Examples 1, 4, and 5) exhibit baseline-separated Raman spectra based on their symmetrical C≡C stretching frequencies. With the addition of additional alkyne units, the symmetrical C≡C shifts to lower wavenumbers due to the increased conjugation length of the alkyne bond and the lower energy of the conjugated π bond. In the case of 4-yne (precursor of Example 5), the Raman spectrum of the protected derivative is shown.

[0210] Figure 2 Representative examples of using Raman spectroscopy to detect HEK293F extracellular vesicles (EVs) and HEK293F EVs overexpressing CD63 are shown. SPARTA, mentioned and described below, is used. ® The system collected Raman spectra at 200 nm and showed that HEK293FEV and HEK293F EVs overexpressing CD63 were in the Raman silent region (1800 cm⁻¹). -1 -2800cm -1 No Raman signal is displayed. The antibody recognizing CD63 is conjugated with sulfonated oligoacetylene (…). Example 2) This enables dose-dependent detection of CD63 protein on EVs (as seen in Raman spectra 200a, particularly in the illustration), where the Raman-labeled antibody (RAb) is in the amount of 100 or 1000 molar equivalents relative to the EV concentration.

[0211] Figure 3The UV-Vis spectrum 300 of a Raman-tagged bioconjugate according to an example is shown. The UV-Vis spectrum 300 confirms successful bioconjugation, as can be seen from the increase in relative absorbance at 345 nm due to the presence of the Raman tag (compared to the absorbance of the bioconjugate (CD63 / IgG) at 280 nm). By varying the degree of Raman tag functionalization on the bioconjugate, the UV-Vis data allows for the calculation of the extinction coefficient of the Raman tag; in this embodiment, the extinction coefficient was calculated to be 16,747 cm⁻¹ using the peak at 345 nm. -1 M -1 Using the peak at 280 nm, the extinction coefficient of the IgG antibody was 210,000 cm⁻¹. -1 M -1 The ratio of the extinction coefficient at 345 nm to that at 280 nm is 0.834, which is used as a correction factor in these concentration calculations to account for the tag's contribution at 280 nm.

[0212] Figure 4 A representative molecular weight curve 400 of a successful Herceptin bioconjugation, measured using an Agilent 2100 Bioanalyzer, is shown according to an example. In curve 400, the relative molecular weight of the IgG antibody (with 7 tags bioconjugated to the surface) is highlighted by a black arrow 401. The IgG antibody has a relative molecular weight of 150 kDa, which can be observed on the molecular weight curve obtained from this technique (note that the position of the 150 kDa peak in the representative molecular weight curve is due to the calibration of the sample molecular weight). This peak is observed in curve 400, and there are no other large peaks near 150 kDa. Figure 3 The exemplary UV-Vis curve of the Raman tag shown confirms that a successful biological conjugation has occurred.

[0213] SPARTA (Single Particle Automated Capture and Analysis) ® )

[0214] As described above, the compounds disclosed herein are similar to those described in International Patent Application No. PCT / EP2019 / 066106 (published as WO 2019 / 243375 A1) and SPARTA (Single Particle Automated Raman Trapping Analysis) by Penders, J., Pence, IJ, Horgan, C. et al., in Nat Commun 9, 4256 (2018). ® (The system is used in conjunction with other systems.)

[0215] Typical methods for detecting specific biomolecules on single particles face two major challenges: 1) ensuring that, for a given sample, the information obtained from a single particle is representative of the sample as a whole; and 2) being able to detect specific biomolecules on a single particle. SPARTA ® It can overcome the first challenge; however, it cannot detect specific biomolecules. Other imaging techniques, such as immunostaining combined with super-resolution microscopy, can address the second challenge; however, these techniques are limited by their ability to measure enough particles to demonstrate that the measurements performed are representative of the whole sample.

[0216] The second challenge, as mentioned above, can be overcome by using SPARTA ® This is overcome by binding to the synthesized water-soluble Raman-active labeling molecule (Raman tag) described herein. This is achieved through the bioconjugation of the Raman tag with the labeling antibody of interest, as discussed above. The resulting Raman-active antibody (Raman body) is used to label particles, which can then be processed using SPARTA. ® These particles were detected. Compared to polyacetylenic derivatives that have not been modified with solubilizing groups or other Raman-active compounds, the improved water solubility of these tags favorably promotes Raman signal enhancement, thus enabling detection via SPARTA. ® Conduct testing.

[0217] Figure 5 An overview of an example procedure 500 for the detection of biomolecules on a single particle is illustrated in schematic form. According to this procedure, nanoparticles such as extracellular vesicles (EVs) are exemplarily present in a patient's blood sample. In an alternative example, the EVs may be derived from other bodily fluids. A Raman-active biomarker 502 is synthesized to detect a specific biomolecule of interest. Furthermore, multiple biomolecule biomarkers can be detected simultaneously. When the Raman-active biomarker is mixed with the extracellular vesicle, a specific interaction occurs between the biomolecule of interest and the Raman-active biomarker, thereby labeling the particle with a unique Raman signal. The labeled EVs are analyzed (e.g., using SPARTA). ® )503 is used to detect the presence of biomolecules of interest based on single particles.

[0218] The following discussion covers SPARTA related to the compounds and Raman-labeled bioconjugates described herein. ® The relevant aspects and exemplary uses of the system are described. Nevertheless, for a comprehensive discussion, readers are advised to refer to SPARTA. ® The system is described in its original text. Furthermore, these exemplary uses are not intended to more generally limit the detection and / or analysis of the compounds and Raman-labeled bioconjugates described herein by means of Raman techniques (both spectroscopy and imaging).

[0219] Generally speaking, SPARTA ®The system is configured to provide high throughput, conventional size, and / or compositional analysis based on single particles, unaffected by substrate interference. More specifically, Raman spectroscopy is combined with optical trapping, a process in which particles are suspended or trapped by the radiation pressure generated by a focused laser. Nanoparticles in the Rayleigh limit (r ≪ λ) are trapped due to their polarizability difference compared to the solution, resulting in a dipole gradient force. This force varies in magnitude with laser intensity and decreases with increasing distance from the focusing volume, guiding the particles to optical trapping at the laser's focal point.

[0220] Figure 6 A schematic diagram of an automated particle trapping system 600 for particle analysis, particularly suitable for Raman spectroscopy analysis, is shown according to an example. System 600 includes an electromagnetic radiation source 604, which in this embodiment is a light source and more preferably a laser. A dichroic mirror 605 directs laser radiation 606 toward a sample 607 via a focusing element 608 for analysis. Electromagnetic radiation 609 from the illuminated sample 607 is directed to a detector 610 via the dichroic mirror 605. Elastically scattered radiation from the sample 607 at the wavelength of the laser beam is filtered out at the detector 610 by a suitable filter (not shown), and Raman scattered radiation is transmitted to the detection device of the detector 610.

[0221] Substrate 611 is configured to receive sample 607 for analysis. This substrate may be covered with a suitable cap 612, for example, to limit sample evaporation. Cap 612 (if used) is transparent to electromagnetic radiation 606, 609. In another example, the cap may include a focusing element 608, which may be the objective of the system, allowing the sample to come into direct contact with the object (e.g., water immersion). Sample 607 includes any fluid medium 613 capable of transporting particles 614 therein for analysis. The expression "capable of transporting" is intended to cover any fluid medium 613 capable of providing suspension of particles 614 within the medium while allowing movement or transport of particles within or through the fluid medium itself. For example, movement / transportation of particles 614 may be by diffusion within the medium 613 or by flow of the medium, or a combination of both. In the illustrated embodiment, where the volume of sample 607 is generally static on substrate 611, diffusion may be the primary mechanism for moving particles 614 relative to the impact beam 606. In other arrangements, substrate 611 may be configured for microfluidic control of the movement of fluid medium 613 and particles 614 suspended therein, for example, using microfluidic channels to move a sample to the appropriate location. Preferably, fluid medium 613 is a liquid. The liquid may be a "non-transparent" or "non-redispersible" liquid, such as water or an aqueous buffer (e.g., phosphate-buffered saline or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid) with a small amount of solvent (e.g., ethanol or dimethyl sulfoxide) added.

[0222] The control system 615 is coupled to an electromagnetic radiation source (e.g., a laser) 604 and a detector 610 to perform the functions described below.

[0223] return Figure 5 In the analysis step 503 shown, a focused beam of electromagnetic radiation (e.g., from electromagnetic radiation source 604) passes through a medium in which particles are suspended. The beam has a waist portion 516 at its focal point, which defines a particle trapping region in which particles 514a can be retained using optical trapping effects or single-beam gradient force trapping effects. The highly focused beam provides attractive or repulsive forces based on, for example, refractive index mismatch, to physically hold and move microscopic dielectric objects, such as particles. For nanoparticles with diameters smaller than the wavelength of the trapping beam, the mechanism can be understood as based on dipole absorption and re-radiation of light.

[0224] Figure 7 It shows that according to Figure 6An example process 700 flow of an automated particle trapping system for automated particle trapping and data acquisition. In a first step, an electromagnetic radiation beam is switched on and focused onto the sample to confine the particle trapping zone to the beam waist (step 721). In a first data acquisition procedure, a Raman-scattered radiation signal is detected at a detector (step 722). At least a portion of this Raman signal (e.g., the spectral portion) is tested against a threshold to determine whether a particle has been trapped or not (step 723). Further details can be found in WO 2019 / 243375 A1. If no particle is detected from the Raman signal, and if the number of particle trapping failures does not exceed a predetermined number (step 724), the process is repeated to test the Raman signal against a threshold (step 722). Short trapping wait periods may exist between the successive steps of the first data acquisition procedure (step 721) (step 725) to allow time for the particle to move into the particle trapping zone via the primary transport mechanism (e.g., particle diffusion within the medium).

[0225] If a particle is detected with a Raman signal at step 723, a second data acquisition procedure (step 726) is executed. The second data acquisition procedure may also include detecting the radiation signal from the Raman scattering. Preferably, the second data acquisition procedure (step 726) includes a higher quality data signal acquisition procedure than the first data acquisition procedure (step 722). Higher quality data acquisition may include, for example, any one or more of the following: (i) extending the data acquisition period to obtain a higher signal-to-noise ratio than the first data acquisition procedure; (ii) acquiring spectral extension data, i.e., data over a larger frequency bandwidth or frequency selection; (iii) collecting data using different modes (e.g., modes other than Raman spectral signals); (iv) multiple data acquisition periods for averaging; (v) changing the laser power to increase the signal-to-noise ratio; (vi) collecting data and adding / combining this data with the data from the first data acquisition procedure to improve the signal-to-noise ratio of the data from the first data acquisition procedure. Such other modes may include, for example, fluorescence signals / spectroscopy, absorption signals / spectroscopy, and other spectroscopic techniques. Data acquired during the second data acquisition procedure 726 can be stored for later analysis together with subsequently captured datasets, or can be processed / analyzed in real time or pseudo-real time.

[0226] After the second data acquisition procedure (step 726), the bundle is disabled (step 727) to allow the captured particles to escape from the particle capture area during the escape waiting period or escape delay period (step 728).

[0227] In one arrangement, a first data acquisition procedure (step 722) includes acquiring Raman spectral data over a time period of 1 to 2 seconds, and a second data acquisition procedure (step 726) includes acquiring Raman spectral data over a time period of 5 to 10 seconds. These times can be adjusted to optimize for various factors, such as the intensity of Raman scattering by the particles due to their composition or size, the desired signal-to-noise ratio of the generated data, or any other external constraints or requirements on the generated data.

[0228] More typically, the first data acquisition procedure (step 722) may include acquiring a Raman response signal to a degree sufficient to enable the detection of the presence of particles (e.g., over an integration time period) by comparing the spectral profile obtained by sensing a spectral profile obtained in the absence of particles. The spectral profile may include the amplitude and / or shape of one or more peaks, the area under a peak or one or more peaks, the area under one or more portions of a Raman spectrum, the shape of at least a portion of the spectrum, or some other feature.

[0229] The process loop (steps 721-725) can be repeated a predetermined number of times i (e.g., over multiple sampling time periods) as tested in step 724. The number of allowed iterations can be determined based on several factors, and detecting particle failure within the predetermined number of iterations (step 729) can trigger a restart of the entire process or a recalibration process, as described later.

[0230] The disabling of the beam in step 727 can be achieved by directly controlling the laser, such as powering on / off the laser, or by shielding or otherwise attenuating the beam. In this regard, particularly in step 727, it should be noted that it may not be necessary to completely cut off the beam; it may only be necessary to reduce or attenuate the beam to a sub-trapping level, i.e., an intensity level insufficient to retain particles in the particle trapping region by the beam gradient force trapping effect. This can be achieved by a modulation device (not shown), for example, inserted into the beam path that emerges from the laser and precedes the mirror.

[0231] In some cases, the data captured during the first data acquisition procedure may have a sufficient signal-to-noise ratio to also serve the requirements of the second data acquisition procedure. In this case, the system can be configured to use / acquire the data from the first data acquisition procedure as the required high signal-to-noise ratio data in place of the second data acquisition procedure (step 726), and save this data for subsequent analysis or real-time processing.

[0232] The above discussion illustrates the following methods.

[0233] Figure 8 A flowchart of a method 800 for analyzing a biological sample according to an example is shown. Method 800 includes:

[0234] -841 provides biological samples incubated with compounds of Formula I under conditions that allow for the formation of Raman-labeled biological conjugates.

[0235] -842 focuses an electromagnetic radiation beam onto a biological sample to define a particle trapping region for capturing candidate particles within the beam.

[0236] -843 executes the first data acquisition procedure to test particle capture.

[0237] -844 If no particle trapping is detected, repeat step 843.

[0238] -845 If particle trapping is detected, acquire particle data of the trapped particles to determine the presence or absence of Raman-labeled bioconjugates.

[0239] -846 ​​reduces the beam intensity to a sub-capture level to release particles from the particle trapping zone, and

[0240] -847 Repeat steps 842-846 for continuous particles in the particle conveying medium.

[0241] Biological samples can be incubated together with any compound of Formula I as described herein. Steps 845 and optional step 843 include a Raman data acquisition procedure, preferably a Raman spectroscopy data acquisition procedure. One or more steps of method 800 may involve procedures generally as described in the references. Figure 6 The automated particle capture system described herein (i.e., the SPARTA system as generally described and mentioned herein) ® The use of the system. Steps 842-846 are repeated. Step 847 may include references. Figure 7 One or more steps of the example process described.

[0242] Automated particle capture systems as described above (e.g., SPARTA) ® Any program described herein in the data acquisition and analysis procedure may be executed when the corresponding computer program code or instructions are executed. The computer program code or instructions may be distributed via electronic data transmission or through computer-readable media / products.

[0243] Other example methods

[0244] Figure 9 A flowchart of an in vitro or ex vivo method 900 for detecting the presence or absence of biomarkers and / or biomolecules in a sample, according to an example, is shown. Method 900 includes:

[0245] -951 provides samples.

[0246] -952 The sample is incubated with a compound of Formula I under conditions that allow for the formation of Raman-labeled bioconjugates, wherein Raman-labeled bioconjugates are formed if biomarkers and / or biomolecules are present in the sample.

[0247] -953 removes any non-conjugated compounds of formula I.

[0248] -954 uses Raman spectroscopy to analyze samples to detect the presence or absence of Raman-labeled bioconjugates.

[0249] Figure 10 A flowchart of a method 1000 for diagnosing a subject's disease or condition according to an example is shown. Method 1000 includes:

[0250] -1061 provides biological samples previously obtained from the subject.

[0251] -1062 The biological sample is incubated with a compound of Formula I under conditions that allow for the formation of Raman-labeled bioconjugates, wherein Raman-labeled bioconjugates are formed if biomarkers and / or biomolecules are present in the biological sample.

[0252] -1063 removes any non-conjugated compounds of formula I.

[0253] -1064 uses Raman spectroscopy to analyze biological samples to detect the presence or absence of Raman-labeled biological conjugates, where the detection of the presence of Raman-labeled biological conjugates indicates that the subject has a disease or condition.

[0254] Figure 11 A flowchart of an in vitro or ex vivo method 1100 for imaging a biological sample according to an example is shown. Method 1100 includes:

[0255] -1171 provides biological samples;

[0256] -1172 The biological sample is incubated with a compound of Formula I under conditions that allow for the formation of Raman-labeled bioconjugates, wherein a Raman-labeled bioconjugate is formed if a biomarker and / or biomolecule is present in the biological sample.

[0257] -1173 removes any unconjugated compounds of formula I.

[0258] -1174 uses Raman spectroscopy to image biological samples.

[0259] refer to Figures 8-11 The method, wherein the compound of formula I can be any compound of formula I as described herein. Steps 954 and 1064 may involve performing the reference. Figure 8 The methods described (such methods optionally refer to references) Figure 7 (One or more steps of the described example process). Advantageously, this enables high-throughput Raman measurements of biological samples that have been difficult to characterize until now. This is achieved by using a region that was originally Raman silent (1800 cm⁻¹). -1 -2800cm -1 Biological samples are labeled with water-soluble Raman-tagged compounds, such as those disclosed herein, that exhibit relatively strong Raman signals, and then preferably with SPARTA. ® The platform performs automated Raman analysis.

[0260] Exemplary reaction schemes and methods

[0261] The reaction schemes described below are intended to provide a general description of the methods used in the preparation of the compounds of the present invention. The examples provided herein are for illustrative purposes, but do not limit, the compounds of the present invention, and the preparation of such compounds and intermediates.

[0262] abbreviation

[0263] CDCl3: Deuterated chloroform

[0264] d: double peak

[0265] d of d: double peak of double peak

[0266] d of t: the second peak of a triplet

[0267] DI: Deionized

[0268] ESI: Electrospray Ionization

[0269] eq.: equivalent

[0270] EtOAc: Ethyl acetate

[0271] HPLC: High Performance Liquid Chromatography

[0272] J: Coupling constant

[0273] m: multiple peaks

[0274] min: minutes

[0275] MS: Mass spectrometry

[0276] m / z: Mass-to-charge ratio

[0277] NMR: Nuclear Magnetic Resonance

[0278] NEt3: Triethylamine

[0279] sat: saturated

[0280] s: singlet

[0281] t: triple peak

[0282] TBAF: Tetra-n-Butylammonium Fluoride

[0283] t of d: triplet of a doublet

[0284] TFA: Trifluoroacetic acid

[0285] THF: Tetrahydrofuran

[0286] TIPS: Triisopropylsilyl

[0287] Analytical methods

[0288] GC-MS parameters: Column characteristics: 0.22 mm ID × 0.25 μm film thickness × 12 m length, 5% phenyl-95% methylpolysiloxane; Carrier gas: Helium at a constant flow rate of 1 ml / min; Temperature program: Start at 50 °C, hold for 2 min, then ramp to 240 °C at 20 °C / min, ramp to 300 °C at 35 °C / min, hold for 3 min; Injector temperature: 230 °C; Injection method: 1 μL split injection (1:50); Detector: Quadrupole mass spectrometer (MS), mass range used m / z 25-450; Analysis date: 10 / 05 / 2022; GC-MS analysis number: AC-95198-1-301A-1.

[0289] Reference C6D6: 7.16 ppm, 128.0 ppm

[0290] Reference CDCl3: 7.26 ppm and 77.0 ppm

[0291] Trajan (formerly SGE) capillary GC column BPX5, 12m × 0.22mm inner diameter, 0.25μm film thickness, per column.

[0292] General Procedure

[0293] Option 1: Protection with sulfonic acid to form acid-instantaneous sulfonates.

[0294] A sulfonyl chloride derivative (1.0 eq., scale 1.96 mmol) was dissolved in anhydrous CH2Cl2 to a concentration of 0.49 mmol / mL, and 2,2,2-trifluoro-1-(p-tolyl)ethanol (1.1 eq.) was added. The reaction was placed on ice, and 1,4-diazabicyclo(2.2.2)octane (1.3 eq.) was dissolved in anhydrous CH2Cl2 to a concentration of 2 mmol / mL. The diazabicyclo(2.2.2)octane solution was added dropwise over 10 minutes, and then the mixture was warmed to room temperature and stirred overnight. The reaction mixture was diluted in CH2Cl2 (50 mL) and washed twice with water (30 mL) and brine (30 mL). The resulting organic layer was dried (via MgSO4) and then dried under vacuum. The reaction mixture was dissolved in CH2Cl2 (0.1% NEt3) and dried before being loaded onto silica. The title compound was obtained by purification by rapid column chromatography (hexane (0.1% NEt3) to 50% CH2Cl2).

[0295] Option 2: Sonogashira reaction:

[0296] Add the protected halobenzene derivative (1.0 eq., up to 1.57 mmol), bis(triphenylphosphine)palladium(II) dichloride (0.02 eq), cuprous(I) iodide (0.02 eq), triphenylphosphine (0.04 eq.), and a magnetic stir bar to the vial. Seal the vial with a diaphragm, and then add anhydrous degassed toluene (5 mL) and degassed N,N-diisopropylamine (0.63 mL). Bubble the reaction mixture with argon for 10 min, and then add argon-purged (triisopropylsilyl)acetylene (0.422 mL, 1.88 mmol, 1.2 eq). Heat the reaction mixture at 70 °C for 12 h. Cool the reaction mixture to room temperature, dilute in hexane, and filter through diatomaceous earth. The filtrate was then dried under vacuum and the crude reaction mixture was purified by rapid column chromatography (hexane (0.1% Net3) to 10% ethyl acetate / hexane (0.1% Net3). The reaction mixture was then dissolved in toluene (30 mL) and 150 mL of diethyl ether was added. The mixture was filtered, the filtrate was collected and dried under vacuum to give the title compound.

[0297] Option 3: Use TBAF to remove TIPS protection:

[0298] Add 1.0 eq. of TIPS-protected alkyne (up to 0.59 mmol) and a stir bar to a vial. Purge with nitrogen and add anhydrous THF (0.3 mL / mmol). Degas the solvent for 10 min and add 1 M TBAF in THF (1.4 eq.). Stir for 2 h. Dry the reaction mixture under vacuum and add CH2Cl2 (50 mL). Wash with brine (50 mL) and extract the organic layer. Purify by rapid column chromatography (1:1 hexane (0.1% NEt3):CH2Cl2) to give the title compound.

[0299] Option 4: One-pot TIPS deprotection and bromination:

[0300] TIPS-protected alkynes (1.0 eq., up to 7.89 mmol) were added to a round-bottom flask and dissolved in HPLC-grade acetonitrile (10 mL / mmol). The reaction mixture was covered with aluminum foil and placed on a hot plate stirrer. N-bromosuccinimide (1.21 eq.) was added to the reaction mixture at room temperature, followed by silver(I) fluoride (1.22 eq.). After stirring for 2 hours, the reaction mixture was filtered through diatomaceous earth, washed with acetonitrile (2 × 25 mL), and then washed with dichloromethane (3 × 30 mL) into a separatory funnel. The organic layer was then washed with water (3 × 100 mL) and brine (1 × 100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated by rotary evaporation. The crude solids were dried onto silica using dichloromethane and purified by rapid chromatography using 5% ethyl acetate / hexane to give the title compound.

[0301] Option 5: Cadiot-Chodkiewicz coupling:

[0302] Add a magnetic stir bar to a 7 mL vial fitted with a rubber septum. Evacuate the flask and refill it with nitrogen five times. Then add nitrogen-purged ether (1 mL / 0.2 mmol). Next, add a solution containing CuCl (0.05 eq.) dissolved in n-butylamine (6 mL / 0.2 mmol) to the reaction vial. Add a solution of hydroxylamine hydrochloride dissolved in ether (excess but always <0.1 g) to the reaction mixture to give a colorless solution. Then add alkyne (1.2 eq., 0.24 mmol to 0.024 mmol) dissolved in purged ether (5 mL / 0.2 mmol) to the reaction to give a yellow precipitate. Dropwise add bromoalkyne (1.0 eq., 0.20 mmol to 0.02 mmol) dissolved in nitrogen-purged ether (5 mL / 0.2 mmol) and stir the reaction mixture overnight at room temperature. The reaction mixture was diluted with diethyl ether (50 mL), washed with saturated NH4Cl solution, water (3×), and brine, dried over Na2SO4, filtered, and loaded onto silica by rotary evaporation. The crude reaction mixture was purified using a gradient from 0% EtOAc / hexane to 20% EtOAc / hexane by rapid column chromatography.

[0303] Option 7: Deprotection conditions for acid-labile protecting groups:

[0304] The oligoalkyne fraction (up to 0.05 mmol) was added to a vial containing a stir bar and dissolved in anhydrous DCM (10 mL / mmol). TFA (10 mL / mmol) was added, and the reaction was stirred at room temperature for 2 hours. The reaction mixture was precipitated, filtered, and the precipitate was washed with diethyl ether (2 × 10 mL) and then dried under vacuum to give the title compound.

[0305] Compound Examples

[0306] Intermediate S1-OC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 2-bromobenzenesulfonic acid

[0307]

[0308] The reaction was carried out according to Scheme 1, using 2-bromobenzenesulfonyl chloride as the starting material. Column chromatography (using a gradient of hexane containing 0.1% triethylamine to 20% ethyl acetate) yielded intermediate S1-OC, a colorless oil that solidified into a white solid (86%) upon standing.

[0309] 1H NMR (400MHz, CDCl3): d 7.92 (1H, m, Hd), 7.66 (1H, m, Ha), 7.36(2H, m, Hb & Hc), 7.26 (2H, d, Hf), 7.09 (2H, d, J = 9.78Hz, Hg), 5.69 (1H,q, J = 6.59Hz, He), 2.32 (3H, s, Hh).

[0310] 13 C NMR (150MHz, CDCl3): d 140.85, 136.54, 135.70, 134.77, 131.75, 129.41, 128.41, 127.50, 126.18, quartet (125.15, 123.28, 121.43, 119.56, J = 281.86Hz), 121.22, quartet (75.90, 79.56, 79.21, 78.87, J = 34.01Hz), 21.39.

[0311] 19 F NMR (400MHz, CDCl3): d -75.61.

[0312] HPLC: Elution time 6.6 min.

[0313] Intermediate S2-OC:

[0314] 2-((triisopropylsilyl)ethynyl)benzenesulfonic acid-2,2,2-trifluoro-1-(p-tolyl)ethyl ester

[0315]

[0316] The reaction was carried out according to Scheme 2, using intermediate S1-OC as the starting material. Intermediate S2-OC was obtained by column chromatography (using a gradient of hexane containing 0.1% triethylamine to ethyl acetate of 5%), as a pale yellow oil (81%).

[0317] 1¹H NMR (400MHz, CDCl₃): d 7.74 (¹H, d of d, J = 7.74 & 1.53Hz, Hd), 7.53 (¹H, d of d, J = 7.74 & 1.20Hz, Ha), 7.42 (¹H, t of d, J = 7.64 & 1.18Hz, Hb), 7.26 (¹H, Hc), 7.22 (2H, d, J = 7.97Hz, Hf), 7.02 (2H, d, J = 7.72Hz, Hg), 5.64 (¹H, quartet, J = 6.41Hz, He), 2.28 (3H, s, Hh), 1.20 (2¹H, m, Hi & Hj).

[0318] 13 C NMR (101MHz, CDCl3): d 140.46, 137.50, 135.72, 134.78, 133.11, 129.58, 129.42, 129.24, quartet (128.27, 126.42, 123.29, 119.27, J = 279.37Hz) 127.91, 102.00, 101.71, quartet (79.71, 79.37, 79.03, 78.69, J = 34.30Hz), 21.35, 18.71, 11.52.

[0319] 19 F NMR (400MHz, CDCl3): d -75.78,

[0320] HPLC: Elution time 7.6 min.

[0321] MS (ESI-MS, m / z): [C26H33F3O3SSi +Na] + The calculated value is 533.18, and the actual measured value of the mass is 533.2.

[0322] Intermediate S3-OC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 2-ethynylbenzenesulfonic acid

[0323]

[0324] The reaction was carried out according to Scheme 2, using intermediate S2-OC as the starting material. Intermediate S3-OC was obtained by column chromatography (a gradient of hexane containing 0.1% triethylamine to 10% ethyl acetate) as a pale yellow oil (100%).

[0325] 1H NMR (600MHz, CDCl3): d 7.84 (1H, d, J = 8.18Hz, Hd), 7.57 (1H, d, J= 7.87Hz, Ha), 7.48 (1H, t, J = 7.46Hz, Hb), 7.35 (1H, t, J = 7.46Hz, Hc), 7.24 (2H, d, J = 7.94Hz, Hf), 7.07 (2H, d, J = 7.94Hz, Hg), 5.74 (1H, quartet, J = 6.26Hz, He), 3.35 (1H, s, Hi), 2.29 (3H, s, Hh).

[0326] 13 C NMR (150MHz, CDCl3): d 140.64, 137.98, 135.53, 133.45, 129.69, 129.33, 128.72, 128.31, 126.42, quartet (125.25, 123.33 121.42, 119.59, J = 280.16Hz), 121.77, 86.29, quartet (79.33, 79.09, 78.86, 78.68, J = 34.0Hz), 79.16, 21.32.

[0327] MS (ESI-MS, m / z): [C17H13F3O3S +Na] + The calculated value is 377.03, and the actual measured mass is 377.0.

[0328] Intermediate S1-MC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-bromobenzenesulfonic acid

[0329]

[0330] The reaction was carried out according to Scheme 1, using 3-bromobenzenesulfonyl chloride as the starting material. The product was separated by column chromatography (a gradient of hexane containing 0.1% triethylamine to 20% ethyl acetate) to obtain intermediate S1-MC, which was an oily substance that solidified upon standing (76%).

[0331] 1¹H NMR (400MHz, CDCl₃): d 7.72 (¹H, t, J = 1.75Hz, Hd), 7.70 (¹H, m, Ha), 7.66 (¹H, m, Hc), 7.29 (¹H, t, J = 8.25Hz, Hb), 7.19 (2H, d, J = 8.11Hz, Hf), 7.11 (2H, d, J = 8.04Hz, Hg), 5.67 (¹H, quartet, J = 6.10Hz, He), 2.34 (3H, s, Hh).

[0332] 13 C NMR (150MHz, CDCl3): d 141.10, 138.21, 137.05, 130.91, 130.60, 129.59, 128.37, 126.48, 126.11, 123.01, quartet (125.09, 123.22, 121.36, 119.50, J = 280.88Hz), quartet (79.49, 79.26, 79.03, 78.90, J = 33.28Hz), 21.45.

[0333] 19 F NMR (400MHz, CDCl3): d -75.86,

[0334] HPLC: Elution time 6.6 min.

[0335] Intermediate S2-MC:

[0336] 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-(triisopropylsilyl)ethynyl)benzenesulfonic acid

[0337]

[0338] The reaction was carried out according to Scheme 2, using intermediate S1-MC as the starting material. Intermediate S2-MC was obtained by column chromatography (with a gradient of hexane containing 0.1% triethylamine to 5% ethyl acetate), which was an oily substance that solidified (75%) upon standing.

[0339] 1H NMR (400MHz, CDCl3): d 7.73 (1H, t, J = 1.64Hz, Hd), 7.65 (1H, m,Ha), 7.59 (1H, m, Hb), 7.33 (1H, t, J = 7.95Hz, Hb), 7.17 (2H, d, J = 8.35Hz,Hf), 7.07 (2H, d, J = 7.91Hz, Hg), 5.65 (1H, q, J = 6.10Hz, He), 2.31 (3H, s,Hh), 1.13 (m, 21H, Hi & Hj).

[0340] 13 C NMR (101MHz, CDCl3): d 140.69, 137.04, 136.57, 131.09, 129.35, 128.95, 128.12, 127.06, 126.17, quartet (124.98, 123.12, 121.26, 119.40, J = 280.34Hz), 124.94, 94.14, quartet (79.03, 78.81, 78.58, 78.35, J = 36.49Hz), 21.25, 18.63, 11.23. 19 F NMR (400MHz, CDCl3): d -75.93,

[0341] HPLC: Elution time 7.7 min.

[0342] MS (ESI-MS, m / z): [C26H33F3O3SSi+Na] + The calculated value is 533.19, and the actual measured mass is 533.2.

[0343] Intermediate S3-MC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-ethynylbenzenesulfonic acid

[0344]

[0345] The reaction was carried out according to Scheme 3, using intermediate S2-MC as the starting material. Intermediate S3-MC was obtained by column chromatography (a gradient of hexane containing 0.1% triethylamine to 10% ethyl acetate) as a colorless oil (78%).

[0346] 1H NMR (400MHz, CDCl3): d 7.75 (1H, t, J = 1.53Hz, Hd), 7.71 (1H, m,Ha), 7.63 (1H, m, Hc), 7.37 (1H, t, J = 7.95Hz, Hb), 7.19 (2H, d, J = 8.28Hz,Hf), 7.09 (2H, d, J = 8.28Hz, Hg), 5.67 (1H, quartet, J = 6.60Hz, He), 3.17(1H, s, Hi), 2.33 (S, 3H, Hh).

[0347] 13 C NMR (101MHz, CDCl3): d 140.97, 137.24, 136.95, 131.48, 129.57, 129.23, 128.34, 127.84, 126.28, quartet (125.67, 124.69, 123.26, 121.4, J =280.10Hz), 123.76, 81.23, 79.90, quartet (79.30, 79.07, 78.84, 78.61, J =32.34Hz), 21.42.

[0348] 19 F NMR (400MHz, CDCl3): d -75.89,

[0349] MS (ESI-MS, m / z): [C17H13F3O3S+H] + The calculated value is 355.05, and the actual measured mass is 355.1.

[0350] Intermediate S1-PC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 4-iodobenzenesulfonic acid

[0351]

[0352] The reaction was carried out according to Scheme 1, using 4-iodobenzenesulfonyl chloride as the starting material. The product was separated by column chromatography (a gradient of hexane containing 0.1% triethylamine to 20% ethyl acetate) to give intermediate S1-PC, which was a white crystalline solid (78%).

[0353] 1H NMR (600MHz, CDCl3): d 7.75 (2H, d, J = 8.33Hz, Hb), 7.41 (2H, d, J= 8.73Hz, Ha), 7.16 (2H, d, J = 8.25Hz, Hd), 7.09 (2H, d, J = 7.94Hz, He), 5.63 (1H, h, J = 6.33Hz, Hc), 2.33 (3H, s, Hf).

[0354] 13 C NMR (101MHz, CDCl3): d 140.99, 138.47, 136.10, 129.57, 129.18, 128.29, 126.32, quartet (125.49, 123.70, 120.90, 119.11, J = 278.89Hz), 102.04, quartet (79.35, 79.01, 78.66, 78.32, J = 34.78Hz), 21.46.

[0355] 19 F NMR (400MHz, CDCl3): d -75.91,

[0356] HPLC: Elution time 6.7 min.

[0357] MS (ESI-MS, m / z): [2 C15H12F3IO3S+Na] + The calculated value is 934.9, and the actual measured mass value is 934.9.

[0358] Intermediate S2-PC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 3-ethynylbenzenesulfonic acid

[0359]

[0360] The reaction was carried out according to Scheme 2, using intermediate S1-PC as the starting material. The product was separated by column chromatography (a gradient of hexane containing 0.1% triethylamine to 20% ethyl acetate) to obtain intermediate S2-PC, which was a colorless oil (80%).

[0361] 1H NMR (600MHz, CDCl3): d 7.65 (2H, d, J = 8.71, Hb), 7.45 (2H, d, J =8.56, Ha), 7.18 (2H, d, J = 8.01Hz, Hd), 7.10 (2H, d, J = 7.94Hz, He), 5.64(1H, h, J = 6.46Hz, Hc), 2.33 (3H, s, Hf), 1.14 (21H, m, Hg & Hh).

[0362] 13 C NMR (101MHz, CDCl3): d 140.84, 135.43, 132.47, 129.53, 128.26, 127.87, 126.49, quartet (125.54, 123.75, 120.95, 118.16, J = 284.6Hz), 104.89, 96.66, quartet (79.22, 78.87, 78.53, 78.19, J = 31.87Hz), 21.36, 18.75, 11.36. 1

[0363] 9 F NMR (400MHz, CDCl3): d -75.95,

[0364] HPLC: Elution time 7.8 min.

[0365] Intermediate S3-PC: 2,2,2-trifluoro-1-(p-tolyl)ethyl 4-ethynylbenzenesulfonic acid

[0366]

[0367] The reaction was carried out according to Scheme 3, using intermediate S2-PC as the starting material. The product was separated by column chromatography (a gradient of hexane containing 0.1% triethylamine to 10% ethyl acetate) to obtain intermediate S3-PC, which was a white crystalline solid (80%).

[0368] 1H NMR (600MHz, CDCl3): d 7.69 (2H, d, J = 8.70, Hb), 7.49 (2H, d, J =8.60, Ha), 7.19 (2H, d, J = 7.35Hz, Hd), 7.11 (2H, d, J = 7.35Hz, He), 5.65 (1H, q, J = 6.19Hz, Hc), 3.28 (1H, s, Hg), 2.34 (3H, s, Hf).

[0369] 13 C NMR (101MHz, CDCl3): d 140.79, 136.08, 132.53, 129.41, 128.22, 128.10, 127.79, 126.28, quartet (124.98, 123.12, 121.26, 119.39, J = 273.97Hz), 81.56, quartet (78.96, 78.74, 78.51, 78.28, J = 31.99Hz), 21.24.

[0370] 19 F NMR (400MHz, CDCl3): d -75.95.

[0371] MS (ESI-MS, m / z): [C17H13F3O3S+H] + The calculated value is 355.05, and the actual measured mass is 355.1.

[0372] Intermediate S4B: 4-((triisopropylsilyl)ethynyl)tert-butyl benzoate

[0373]

[0374] The product was synthesized according to the conditions of Scheme 2, using tert-butyl 4-bromobenzoate as the starting material. Intermediate S4B was isolated as a clear, viscous liquid (69%).

[0375] 1 H NMR (400MHz, CDCl3): δ 7.91 (2H, d, J = 8.4Hz, Hb), 7.49 (2H, d, J= 8.5Hz, Ha), 1.59 (9H, s, Hc), 1.14 (21H, s, He & Hd).

[0376] 13C NMR (101MHz, CDCl3): δ 165.37, 131.91, 131.60, 129.34, 127.76, 106.48, 81.42, 28.33, 18.79, 11.43.

[0377] MS (ESI-MS, m / z): [C22H34O2Si+Na] + The calculated value is 381.59, and the measured value is 381.3.

[0378] Intermediate S5B: tert-butyl 4-(bromoethynyl)benzoate

[0379]

[0380] According to scheme 4, intermediate S4B was used as the starting material for synthesis. Intermediate S5B was obtained, which was a colorless oily substance that crystallized into a white solid (91%) when stored in a freezer.

[0381] 1 H NMR (400MHz, CDCl3): δ 7.97 – 7.87 (2H, d, J = 8.6Hz, Hb), 7.51 –7.45 (2H, d, J = 8.6Hz, Ha), 1.59 (9H, s, Hc).

[0382] 13 C NMR (101MHz, CDCl3): δ 165.2, 132.0, 131.9, 129.5, 126.9, 81.6,79.7, 53.0, 28.3.

[0383] Intermediate S6B: 4-((triisopropylsilyl)but-1,3-diyn-1-yl)tert-butyl benzoate

[0384]

[0385] The product was synthesized according to the conditions of Scheme 2, using intermediate S5B as the starting material. Intermediate S6B was produced, which was a white solid (70%).

[0386] 1 H NMR (400MHz, CDCl3): δ 7.91 (2H, d, J = 8.69, Hb), 7.52 (2H, d, J =8.69Hz, Ha), 1.57 (9H, s, Hc), 1.11 (s, 21H, He & Hd).

[0387] 13C NMR (101MHz, CDCl3): δ.

[0388] MS (ESI-MS, m / z): [2C24H34O2Si+Na] + The calculated value is 787.46, and the actual measured value of the mass is 787.5.

[0389] Intermediate S7B: tert-butyl 4-(bromobut-1,3-diyn-1-yl)benzoate

[0390]

[0391] According to scheme 4, intermediate S6B was used as the starting material for synthesis. The intermediate S7B was purified by aqueous posttreatment with CH2Cl2 (50 mL) and washed twice with DI water (25 mL) to separate it as a white solid (91%).

[0392] 1 H NMR (400MHz, CDCl3): δ 7.92 (2H, d, J = 8.73Hz. Hb), 7.52 (2H, , J= 8.67Hz, Ha), 1.58 (9H, s, Hc).

[0393] 13 C NMR (101MHz, CDCl3): δ 164.94, 132.71, 131.65, 129.47, 125.25, 81.65, 76.76, 73.55, 65.32, 46.17, 28.27.

[0394] MS (ESI-MS, m / z): [C15H13BrO2+H] + The calculated value is 304.00, and the actual measured mass is 305 + 307.

[0395] Intermediate S8B: 4-((triisopropylsilyl)hexa-1,3,5-triynyl-1-yl)tert-butyl benzoate

[0396]

[0397] The product was synthesized according to the conditions of Scheme 2, using intermediate S7B as the starting material. Intermediate S8B was produced, which was a white solid (70%).

[0398] 1H NMR (400MHz, CDCl3): δ 7.92 (2H, d, J = 8.26Hz, Hb), 7.52 (2H, d, J= 8.32Hz, Ha), 1.57 (9H, s, Hc), 1.08 (21H, s, He & Hd).

[0399] 13 C NMR (101MHz, CDCl3): δ 206.97, 164.89, 132.90, 129.49, 125.06, 89.66, 87.82, 81.71, 75.71, 68.55, 60.29, 31.01, 28.25, 18.63, 11.39.

[0400] MS (ESI-MS, m / z): [2C26H34O2Si +Na] + The calculated value is 835.47, and the actual measured mass is 835.4.

[0401] Intermediate BD-o-tolyl CF3-protecting group:

[0402] 4-((2-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)phenyl)but-1,3-diyne-1-yl)benzene tert-butyl formate

[0403]

[0404] The synthesis was performed using intermediates S5B and S3-OC as starting materials according to scheme 5. The title compound was isolated as a white solid (98%).

[0405] 1 H NMR (600MHz, DMSO): δ 7.99 (2H, d of t, J = 8.78 & 1.81Hz, Hb), 7.90 (1H, d of d, J = 8.00 & 1.14Hz, Hg), 7.61 (3H, m, Hd, & Ha), 7.52 (1H, tof d, J = 7.36 & 1.15Hz, He), 7.40 (1H, t of d, J = 7.65 & 1.47Hz, Hf), 7.29(2H, d, J = 7.99Hz, Hi), 7.10 (2H, d, J = 7.96Hz, Hj), 5.76 (1H, q, 6.45Hz,Hh), 2.29 (3H, s, Hk), 1.61 (9H, s, Hc). 13C NMR (150MHz, CDCl3): δ 164.92,140.60, 138.21, 135.76, 133.42, 132.79, 132.49, 129.88, 129.52, 129.33,129.07, 128.99, 128.88, 128.25, 126.37, 125.38, 121.31, 116.64, 115.72,81.90, 81.68, 78.77, 75.83, 70.64, 64.49, 28.23, 21.33, 19.16, 13.78. 19 F NMR (400MHz, CDCl3): δ -75.78,

[0406] HPLC: Elution time 7.2 min.

[0407] UV-Vis wavelength peaks (nm): 304, 326, 348.

[0408] MS (ESI-MS, m / z): [C30H25F3O5S+H] + The calculated value is 555.14, and the actual measured value of the mass is 555.2.

[0409] Intermediate BD-m-tolyl CF3-protecting group:

[0410] 4-((3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)phenyl)but-1,3-diyne-1-yl)benzene tert-butyl formate

[0411]

[0412] Synthesized using Scheme 5 with intermediates S5B and S3-MC as starting materials. The title product was isolated as a white solid (51%).

[0413] 1H NMR (600MHz, DMSO): δ 7.97 (2H, d of t, J = 8.69 & 1.86Hz, Hb), 7.74 (2H, m, Hg & Hd), 7.65 (1H, d of t, J = 8.00 & 1.39Hz, He), 7.58 (2H, dof t, J = 8.66 & 1.83Hz, Ha), 7.40 (1H, t, J = 8.12Hz, Hf), 7.19 (2H, d, J =8.20Hz, Hi), 7.11 (2H, d, J = 7.83Hz, Hj), 5.67 (1H, q, 6.34Hz, Hh), 2.35(3H, s, Hk), 1.60 (9H, s, Hc).

[0414] 13 C NMR (150MHz, CDCl3): δ 164.97, 142.67, 141.12, 138.30, 137.40,133.60, 132.86, 137.40, 137.20, 133.60, 132.86, 132.53, 131.78, 129.60,129.41, 128.39, 128.19, 126.18, 125.37, 124.73, 123.29, 120.91, 81.79, 79.85,79.25, 19.94, 76.14, 75.63, 29.85, 28.31, 21.44, 14.28.

[0415] 19 F NMR (400MHz, CDCl3): δ -75.87,

[0416] HPLC: Elution time 7.3 min.

[0417] UV-Vis wavelength peaks (nm): 300, 316, 338.

[0418] Intermediate BD-p-TolylCF3-Prot:

[0419] 4-((4-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)phenyl)but-1,3-diyne-1-yl)benzene tert-butyl formate

[0420]

[0421] Scheme 5 was used to synthesize intermediates S5B and S3-PC as starting materials. Intermediate 17 was isolated as a white solid (38%).

[0422] 1 H NMR (600MHz, CDCl3): δ 7.97 (2H, d of t, J = 8.71 & 1.86Hz, Hb), 7.70 (2H, d of t, J = 8.64 & 1.83Hz, He), 7.58 (2H, d of t, J = 8.60 &1.83Hz, Ha), 7.52 (2H, d of t, J = 8.77 & 1.88Hz, Hd), 7.19 (2H, d, J =8.07Hz, Hg), 7.12 (2H, d, J = 7.86Hz, Hh), 5.67 (1H, q, J = 6.33Hz, Hf), 2.35(3H, s, Hi), 1.60 (9H, s, Hc).

[0423] 13 C NMR (150MHz, CDCl3): δ 174.67, 144.16, 140.92, 138.89, 136.41,132.88, 132.47, 129.54, 129.51, 128.20, 127.96, 127.81, 125.17, 81.74, 80.17,78.93, 78.59, 75.50, 28.23, 21.37, 19.62, 15.52.

[0424] 19 F NMR (400MHz, CDCl3): δ -75.92,

[0425] HPLC: Elution time 7.3 min.

[0426] UV-Vis wavelength peaks (nm): 302, 322, 344.

[0427] intermediate triyne-m-tolyl CF3-protecting group:

[0428] 4-((3-((2,2,2-trifluoro-1-(p-tolyl)ethoxy)sulfonyl)phenyl)hexa-1,3,5-triyne-1-yl) tert-butyl benzoate

[0429]

[0430] Scheme 5 was used to synthesize the product using intermediates S7B and S3-MC as starting materials. The isolated product was a white solid (34%).

[0431] 1H NMR (600MHz, CDCl3): δ 7.96 (2H, d of t, J = 8.57 & 1.37Hz, Hb), 7.77-7.73 (1H, m, He), 7.71 (1H, t, J = 1.59Hz, Hg), 7.65 (1H, d of t, J =7.85 & 1.43Hz, Hf), 7.59 (2H, d of t, J = 8.57 & 1.88Hz, Ha), 7.39 (2H, t ofd, J = 7.85 & 0.37Hz, Hd), 7.18 (2H, d, J = 8.12Hz, Hi), 7.10 (2H, d, J =7.93Hz, Hj), 5.67 (1H, q, J = 6.44Hz, Hh), 2.35 (3H, s, Hk).

[0432] 13 C NMR (150MHz, CDCl3): δ 164.82, 141.11, 137.76, 137.20, 129.65, 129.53, 129.42, 128.46, 128.38, quartet (128.25, 126.02, 124.69, 122.14), 122.53, 81.71, quartet (79.16, 78.92, 78, 63, 78.25), 76.37, 76.13, 67.57, 66.71, 58.72, 28.18, 15.25, 14.15.

[0433] Example 1: 4-((2-sulfophenyl)but-1,3-diyne-1-yl)benzoic acid

[0434]

[0435] Scheme 7 was used to synthesize the product using the intermediate BD-o-tolylCF3-protecting group as the starting material. The product from Example 1 was isolated as a white solid (100%).

[0436] 1 H NMR (600MHz, d6-DMSO): δ 7.97 (2H, m, Hb), 7.81 (1H, d, J =6.24Hz, Hg), 7.74 (2H, m, Ha), 7.59 (1H, d, J = 6.98Hz, Hd), 7.42 (1H, t, J =6.98Hz, He), 7.37 (1H, t, 6.83Hz, Hf).

[0437] 13 C NMR (150MHz, d6-DMSO): δ 166.49, 150.50, 134.63, 132.56, 132.41,131.34, 129.55, 129.12, 128.65, 127.00, 125.19, 117.57, 82.70, 81.11, 77.22,76.77.

[0438] HPLC: Elution time 5.9 min.

[0439] UV-Vis wavelength peaks (nm): 302, 322, 344.

[0440] HR-MS (ESI-MS, m / z): [C17H10O5S-H] - The calculated value is 325.02, and the actual measured mass is 325.0187.

[0441] Example 2: 4-((3-sulfophenyl)but-1,3-diyne-1-yl)benzoic acid

[0442]

[0443] The synthesis was performed using scheme 7 with the intermediate BD-m-tolylCF3-protecting group as the starting material. The isolated product was a white solid (100%).

[0444] 1 H NMR (600MHz, d6-DMSO): δ 7.97 (2H, d, Hb, J = 8.29Hz), 7.77-7.73(3H, m, Ha & Hg), 7.71 (1H, d, J = 7.6Hz, Hf), 7.54 (1H, d, J = 7.6Hz, Hd),7.43 (1H, t, J = 7.74Hz, He).

[0445] 13 C NMR (150MHz, d6-DMSO): δ 166.94, 149.38, 133.11, 133.00, 132.06,130.02, 129.73, 129.12, 127.79, 125.15, 120.05, 83.23, 81.39, 76.26, 73.59.

[0446] HPLC: Elution time 6.0 min.

[0447] UV-Vis wavelength peaks (nm): 300, 320, 342.

[0448] HR-MS (ESI-MS, m / z): [C17H10O5S-H] - The calculated value is 325.02, and the actual measured mass is 325.0187.

[0449] Example 3: 4-((4-sulfophenyl)but-1,3-diyne-1-yl)benzoic acid

[0450]

[0451] The synthesis was performed using scheme 7 with the intermediate BD-p-tolylCF3-protecting group as the starting material. The isolated product was a white solid (100%).

[0452] 1 H NMR (600MHz, d6-DMSO): δ 7.97 (2H, d, J = 8.4Hz, Hb), 7.73 (2H, d,J = 8.3Hz, Ha), 7.64 (2H, d, J = 8.2Hz, He), 7.59 (2H, d, J = 8.2Hz, Hd).

[0453] 13 C NMR (150MHz, d6-DMSO): δ 166.96, 150.20, 133.10, 132.58, 132.04, 126.49, 125.16, 120.47, 83.38, 81.55, 76.26, 74.06.

[0454] HPLC: Elution time 6.0 min.

[0455] UV-Vis wavelength peaks (nm): 300, 320, 342.

[0456] HR-MS (ESI-MS, m / z): [C17H10O5S-H] - The calculated value is 325.02, and the actual measured mass is 325.0186.

[0457] Example 4: 4-((3-sulfophenyl)hexa-1,3,5-triyne-1-yl)benzoic acid

[0458]

[0459] Using scheme 7, the intermediate triyne-m-tolyl CF3 was used as the starting material to synthesize the title compound, which was a white solid (100%).

[0460] 1H NMR (600MHz, d6-DMSO): δ 7.97 (2H, d, J = 7.62Hz, Hb), 7.79-7.76(3H, m, Ha & Hf), 7.72 (1H, d, J = 7.62Hz, He), 7.61 (1H, d, J = 7.60Hz, Hc),7.43 (1H, t, J = 7.59Hz, Hd).

[0461] 13 C10 NMR (150MHz, d6-DMSO): δ 166.84, 133.71, 133.42, 130.36, 130.03, 129.17, 128.92, 124.15, 119.13, 112.97, 111.07, 78.78, 76.22, 69.99, 59.65, 50.80, 42.01. HPLC: elution time 6.5 min.

[0462] UV-Vis wavelength peaks (nm): 316, 340, 366.

[0463] HR-MS (ESI-MS, m / z): [C19H10O5S-H] - The calculated value is 349.02, and the actual measured mass is 349.0189.

[0464] Example 5: 4-((2-sulfophenyl)octa-1,3,5,7-tetrayne-1-yl)benzoic acid

[0465]

[0466] The title compound was prepared from methyl 4-((2-((2,2,2-trifluoroethoxy)sulfonyl)phenyl)octa-1,3,5,7-tetrayne-1-yl)benzoate (as follows) according to procedures known to those skilled in the art.

[0467] 4-((2-((2,2,2-trifluoroethoxy)sulfonyl)phenyl)octa-1,3,5,7-tetrayne-1-yl)methyl benzoate Formation of (Precursor of Example 5)

[0468]

[0469] Using scheme 2, methyl 4-(bromohexa-1,3,5-triyne-1-yl)benzoate and 2,2,2-trifluoroethyl 2-ethynylbenzenesulfonic acid were used as starting materials to synthesize the title compound, which was a yellow solid (17%).

[0470] 1H NMR (400MHz, CDCl3): δ 8.06 (1H, dd, J = 8, 1.4Hz), 8.02 (2H, d, J= 8.4Hz), 7.79 (1H, dd, J = 7.6, 1.4Hz), 7.67 (1H, td, J = 7.7, 1.4Hz), 7.63– 7.56 (3H, m), 4.53 (2H, q, J = 7.9Hz), 3.93 (3H, s).

[0471] UV-Vis wavelength peaks (nm): 322, 348, 374, 404.

[0472] MS (ESI-MS, m / z): [C 24 H 13 F3O5SNa] + The calculated value is 493.03, and the actual measured mass value is 493.0.

[0473] Raman spectra of the compound examples

[0474] The compounds were measured in dry powder form: the compounds were initially dissolved in methanol, aliquoted onto calcium fluoride glass slides, and allowed to dry at room temperature. Data were acquired using a 532 nm excitation laser and single-point spectroscopy. All data were processed in WITec's FIVE software and preprocessed by 1) shape-fitting background subtraction and 2) normalization to symmetric C≡C peak intensities. The data showed 2050 cm⁻¹ values ​​obtained from compounds in Examples 1 and 4, and the precursors in Example 5 (2-yne, 3-yne, and 4-yne, respectively). -1 -2300cm -1 The Raman spectra between them.

[0475] The results are shown graphically. Figure 1 middle.

[0476] Antibody conjugation example 1

[0477] Extracellular vesicles (EVs) were isolated from the HEK293F cell line overexpressing CD63 using the method previously described in Penders et al., ACS Nano 15, 18192–18205. EV isolates were measured using nanoparticle tracking analysis (NTA, Nanosight NS300) at a resolution of 1 × 10⁻⁶. 11 The concentration of particles per mL should be matched, and diluted with PBS if necessary.

[0478] The EV was incubated at room temperature for 2 hours with Raman-labeled antibody added at a concentration of 100 or 1000 equivalents (Ab:EV). The Raman-labeled antibody was generated according to the preparation method described in Antibody Conjugation Example 2 (below). SPARTA was then performed. ® Measurements were performed (as previously described in Penders et al., ACS Nano 15, 18192–18205). Measurement runs without Rab were performed as a control. The obtained SPARTA was then processed. ® Raw spectra (see spectral processing as previously described in Penders et al., ACS Nano 15, 18192–18205).

[0479] The results are shown graphically. Figure 2 middle.

[0480] Antibody conjugation example 2

[0481] Take Raman labels ( Example 2 The antibody was dissolved in DMSO to prepare a stock solution. The solution was vortexed, and a series of dilutions of the tag were prepared in anhydrous DMSO. 2.5 μL of the resulting concentration series was added to 50 μL of CD63 antibody in BBS solution (1.0 eq.), and the solution was incubated at room temperature for 3 hours. The resulting solution was diluted in DPBS (0.5 mL, pH 7.4) and filtered using a 10 kDa MWCO 0.5 mL Viva spin filter by centrifugation at 16500 rcf for 6.5 min. This was repeated 6 times, and the resulting supernatant was then brought to a final volume of 30 μL. 5 μL of this solution was diluted with 95 μL of DPBS and subjected to UV-Vis spectroscopy.

[0482] The results are shown graphically. Figure 3 middle.

[0483] Antibody conjugation example 3

[0484] The molecular weight of the conjugated antibody was measured using an Agilent 2100 bioanalyzer. Samples were run according to the Protein 230 assay protocol as per the manufacturer's instructions. 6 μL of ladder aliquots were thoroughly mixed with 4 μL of 10 μM Raman-labeled antibody aliquots and then loaded into wells.

[0485] The results are shown graphically. Figure 4 The results indicate that the Raman-labeled antibodies did not aggregate.

[0486] Antibody conjugation example 4

[0487] An experiment was conducted to modify the anti-CD63 antibody with a Raman tag derivative lacking solubilizing groups. To prepare the activated Raman tag solution, a 40 mM Raman tag stock solution was prepared in DMSO (25 μL). EDC in 1× PBS (1.5 eq, 12.5 μL, 120 mM) was added to this solution, and the mixture was incubated at 37 °C for 10 min. Then, sulfonyl-NHS in 1× PBS (2 eq, 12.5 μL, 160 mM) was added, followed by another 50 μL of DMSO, and the reaction was incubated at 37 °C for 30 min. The activated Raman tag solution (30 eq, 3 μL, 10 mM) was mixed with the anti-CD63 antibody (50 μL, 20 μM BBS solution, pH = 8.0) and incubated at room temperature for 4 h. The reaction mixture was then transferred to a 10 kDa MWCO 0.5 mL VivaSpin filter and washed with 1× PBS solution (6 × 0.5 mL). The resulting solution was analyzed by UV-Vis spectroscopy, and the antibody concentration (14.6 μM) was determined by absorbance at 280 nm. The absorbance at 326 nm and 26499 M were measured using a Raman tag. -1 cm -1 The concentration of the Raman tag conjugated with the anti-CD63 antibody was determined by the extinction coefficient. A correction factor of 0.502 was applied to account for the absorbance of the Raman tag at 280 nm (13309 M). -1 cm -1 / 26499M -1 cm -1 The Raman-labeled antibody stock solution was diluted 1:10 (v / v) in 1× PBS, where the measured antibody concentration was 3.74 μM and the Raman tag concentration was 1.17 μM. This yielded an average modification degree of 0.31 Raman tags per anti-CD63 antibody.

[0488] Label concentration calculation

[0489] Example work on calculating tag bioconjugation efficiency and tags for each antibody using UV-Vis spectroscopy.

[0490] The extinction coefficients of the Raman-tagged molecules (i.e., the compounds of this invention) were calculated using a dilution series of Raman tags of known concentrations. Similarly, the extinction coefficients of the functionalized (i.e., Raman-labeled) antibody moieties were calculated. Peak correction factors were used to account for the contribution of the Raman tag at 280 nm (the absorbance of the antibody) to allow for accurate characterization of the Raman tag-to-protein ratio.

[0491] An example of using an antibody labeled with anti-human serum albumin (anti-HSA) is described. The Raman-labeled antibody was generated according to the preparation method described in antibody conjugation example 2 (as above).

[0492] The extinction coefficient of the Raman tag, calculated using the peak at 345 nm, is 16,747 cm⁻¹. -1 M -1 The extinction coefficient of the IgG antibody was calculated to be 210,000 cm⁻¹. -1 M -1 The ratio of the peak at 345 nm to that at 280 nm is 0.834, which is used as a correction factor in these calculations to account for the contribution of the Raman tag at 280 nm. An exemplary calculation using data from UV-Vis spectroscopy of the Raman-labeled antibody solution is shown below:

[0493]

[0494]

[0495]

[0496] Working examples of HSA-resistant functionalization:

[0497]

[0498] Other embodiments are intended to fall within the scope of the appended claims.

Claims

1. A compound of formula I, I in: A and B are each an aromatic group independently; Each R 1 Independently selected from -S(O)2OH, -S(O)OH, -OS(O)2OH, -S(O)2NH2, -P(O)(OH)2, -OP(O)(OH)2, -P + (R 3 3. -N + (R 3 )3, -OH, -C(O)OH, -NHC(O)OH, 5- or 6-membered heterocyclic alkyl groups, 5- or 6-membered heteroaryl groups, zwitterionic groups and PEG groups; L 1 For bonds or suitable linking groups; R 2 A group that can conjugate with biomarkers and / or biomolecules; Each R 3 Independently selected from hydrogen and C 1-4 alkyl groups; m is 1, 2, or 3; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. Or its salts or solvates.

2. The compound according to claim 1, wherein the compound of formula I is a compound of formula IA. IA Where R 1 R 2 m and n are as defined in claim 1.

3. The compound according to claim 1 or claim 2, wherein R 1 Selected from -S(O)2OH, -P(O)(OH)2 and -N + (R 3 3.

4. The compound according to any one of claims 1 to 3, wherein R 1 It is -S(O)2OH.

5. The compound according to any one of claims 1 to 4, wherein L 1 For key.

6. The compound according to any one of claims 1 to 5, wherein R 2 for Groups, in which L 2 For bonds or suitable linking groups; R 4 Selected from -C(O)OR 5 , -C(O)Cl, -NCO, -NCS, -SH, -C(O)SR 6 , and ; R 5 Selected from H, , and C 1-4 alkyl group, the C 1-4 The alkyl group is optionally replaced by one or more phenyl groups; and R 6 Selected from hydrogen and C 1-4 Alkyl groups.

7. The compound according to claim 6, wherein: R 4 -C(O)OR 5 ;and L 2 For key or Group.

8. The compound according to any one of claims 1 to 7, wherein R 2 It is -C(O)OH.

9. The compound according to any one of claims 1 to 8, wherein n is 2 or 3.

10. The compound according to any one of claims 1 to 9, wherein m is 1.

11. The compound according to any one of claims 1 to 10, wherein the biomarker and / or biomolecule is selected from antigens, antibodies, peptides, proteins, nucleic acids, vesicles, or carbohydrates.

12. The compound according to any one of claims 1 to 11, wherein the compound is selected from: , , and Or their salts or solvates.

13. A Raman-labeled bioconjugate, said Raman-labeled bioconjugate being formed from a compound of formula I according to any one of claims 1 to 12 and a biomarker and / or biomolecule.

14. The Raman-labeled bioconjugate according to claim 13, wherein the biomarker and / or biomolecule is selected from antigens, antibodies, peptides, proteins, nucleic acids, lipids, vesicles, and carbohydrates.

15. Use of the compound of formula I according to any one of claims 1 to 12 in the preparation of the Raman-labeled bioconjugate according to claim 13 or claim 14.

16. The Raman-labeled bioconjugate according to claim 13 or claim 14, for use in medicine.

17. A method for detecting the presence or absence of biomarkers and / or biomolecules in a sample, wherein the method comprises the following steps: a) Provide samples; b) The sample is incubated with a compound of formula I according to any one of claims 1 to 12 under conditions that allow for the formation of the Raman-labeled bioconjugate according to claim 13 or claim 14, wherein the Raman-labeled bioconjugate is formed if the biomarker and / or biomolecule is present in the sample. c) Remove any non-conjugated compounds of formula I; as well as, d) Use Raman spectroscopy to analyze the sample to detect the presence or absence of the Raman-labeled bioconjugate.

18. A method for diagnosing a disease or condition in a subject, wherein the method comprises the following steps: a) Provide biological samples previously obtained from the subject; b) The biological sample is incubated with a compound of formula I according to any one of claims 1 to 12 under conditions that allow for the formation of the Raman-labeled bioconjugate according to claim 13 or claim 14, wherein the Raman-labeled bioconjugate is formed if the biomarker and / or biomolecule is present in the biological sample. c) Remove any non-conjugated compounds of formula I; as well as, d) Use Raman spectroscopy to analyze the biological sample to detect the presence or absence of the Raman-labeled bioconjugate. The presence of the Raman-labeled bioconjugate indicates that the subject suffers from the disease or condition.

19. An in vitro or ex vivo method for imaging biological samples, wherein the method comprises the following steps: a) Provide biological samples; b) Incubate the compound of Formula I according to any one of claims 1 to 12 with the biological sample to form the Raman-labeled bioconjugate according to claim 13 or claim 14, wherein the Raman-labeled bioconjugate is formed if the biomarker and / or biomolecule is present in the biological sample. c) Remove any non-conjugated compounds of formula I; and d) Image the biological sample using Raman spectroscopy.

20. A reagent kit, the reagent kit comprising: a) A compound of formula I according to any one of claims 1 to 12; as well as b) A specification for the use of a compound of formula I according to any one of claims 1 to 12 in in vitro or ex vivo methods for detecting the presence or absence of biomarkers and / or biomolecules in a sample, methods for diagnosing a disease or condition in a subject, and / or methods for imaging biological samples.

21. A method for preparing a compound of formula I according to any one of claims 1 to 12, the method comprising reacting a protected derivative of the compound of formula I in the presence of a suitable deprotecting agent.

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