US20120190579A1

US20120190579A1 - Functionalisation of Solid Substrates

Info

Publication number: US20120190579A1
Application number: US13/389,613
Authority: US
Inventors: Mark Smith; Stephen Caddick; James Baker
Original assignee: UCL Business Ltd
Current assignee: UCL Business Ltd
Priority date: 2009-08-10
Filing date: 2010-08-09
Publication date: 2012-07-26
Also published as: US20160176942A1; WO2011018612A3; IL218010A0; EP2464654B1; EP2464654A2; EP2464972A1; BR112012003089A2; IL218010A; US20120190814A1; CN102597773B; IL245506A; ES2537623T3; US20120190124A1; JP2013501766A; US10174094B2; JP5954789B2; EP2889624A2; WO2011018612A2; AU2010283632B2; AU2010283632A1

Abstract

The present invention relates to a product comprising a solid substrate and a moiety of formula (I) linked thereto: wherein X, X′ and R are as defined herein. The product is useful for immobilising target molecules such as molecules of biochemical interest to solid substrates for numerous applications, such as affinity chromatography, ELISA, biotechnological assay techniques and solid phase peptide synthesis.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

Pursuant to 35 U.S.C. §371, this application is a National Stage Application of PCT/GB2010/001504, filed Aug. 9, 2010, and claims priority to GB 0913967.6, filed Aug. 10, 2009, GB 0913965.0, filed Aug. 10, 2009 and GB 0914321.5, filed Aug. 14, 2009. The entire contents of each of the aforementioned applications are incorporated herein by reference as if set forth in their entirety.

INTRODUCTION

It is well known that it can be desirable to link a wide variety of molecules to a solid substrate. For example, immobilisation of biomolecules such as antibodies, antigens and proteins on a surface provides the basis for numerous sensitive and selective detection and purification techniques, such as enzyme-linked immunosorbent assays, pull-down assays and affinity chromatographic methods. Microarray techniques, in which a plurality of biomolecules or synthetic molecules linked to discrete points on a substrate are simultaneously screened for their ability to bind to a test substance, are revolutionising screening techniques in the biotechnological and pharmaceutical fields. Meanwhile, solid phase peptide synthesis (SPPS), a technique pioneered by Nobel laureate R. B. Merrifield in which a peptide chain is grown in a step-wise manner while attached to a solid phase, is by far the most convenient and high-yield method for producing synthetic peptides on a laboratory scale.
An abundance of basic solid substrate materials have been used to carry out such immobilisation techniques. These include glass, silica, mica and both natural and synthetic polymers. Solid substrates are also routinely used in various different forms tailored to a specific application, being present for example as unitary chips or membranes, as beaded materials or in the hollow tubular form characteristic of carbon nanotubes.
Important considerations when designing conjugates in which a solid substrate is linked to a particular target molecule include the characteristics of the bond between the substrate and the target molecule and the ease and specificity with which this bond is formed.
In order to preserve the integrity of the conjugate formed between a solid substrate and a molecule of interest, it is often desirable that the components be linked via a covalent bond (rather than a weaker bond such as an electrostatic interaction). Clearly, in order to achieve that, a solid substrate needs to carry at least one functional group that is capable of reacting with a corresponding functional group on the target molecule. Fortunately, functionalisation of solid substrate materials to provide surface-accessible functional groups is a well known technique. Accordingly, appropriately functionalised solid substrates are routinely and commercially available. Functional groups attached to solid substrate materials, typically via an inert linker moiety, include groups capable of attaching to amine, carboxyl, thiol, aldehyde and active hydrogen functional groups on the target molecule.
Coupling of a solid substrate to a target molecule through a reactive thiol group can be of particular interest due to the unique nucleophilic characteristics of the thiol moiety as well as the fact that these functional groups often have low abundance in the biomolecules frequently targeted in immobilisation techniques. These features open up the possibility of highly selective immobilisation procedures based on thiol reactions.
Conventionally, iodoacetyl-activated solid substrates have been used as a reagent for immobilising thiol-containing molecules such as proteins. For example, iodoacetyl-activated agarose or acrylamide-based resins are commercially available from Thermo Fisher. However, it is known that the reaction of haloacetyl functional groups is not always specific to thiol compounds. For example, haloacetyl groups can react with various functional groups present within a protein: the imidazolyl side chain nitrogens of histidine, the thioether of methionine and the primary ε-amine group of lysine residues and N-terminal α-amines. The reaction between an iodoacetyl group on a solid substrate and a thiol (e.g., a cysteine residue in a protein) is also irreversible. This can place severe limitations on its practical utility, for example when carrying out procedures involving proteins that are difficult to express, such as many GPCRs (G-protein coupled receptors). Clearly, it also precludes the exploitation of thiol immobilisation methodology in areas where lability of the bond between the solid substrate and the target molecule is important (for example, where the solid substrate is being used in the context of solid state protein synthesis).
Other compounds that have been used as reagents to link a thiol-containing biomolecule to a secondary molecule (typically another biomolecule) include 1,2-dicarbonyl ethene derivatives and thiosulfonates. 1,2-dicarbonyl ethene derivatives, such as maleimides, are generally recognised to be the most selective reagents for reaction with a thiol and in particular with a cysteine moiety. Unfortunately, though, this reaction, like that of a thiol with an iodoacetyl compound, is again chemically irreversible.
The present invention is based on the surprising finding that it is advantageous to incorporate an electrophilic leaving group onto the C═C double bond of a 1,2-dicarbonyl ethene cross-linking reagent. That chemical modification enables a thiol-containing target molecule to be readily attached via the cross-linking reagent to a solid substrate, while retaining the C═C double bond. Similarly, it enables a thiol-containing solid substrate to be attached via the cross-linking reagent to a target molecule, again while retaining the C═C double bond. This has the following advantages:

- The reaction between the cross-linker and the thiol can often be carried out rapidly and with high yield using a substantially stoichiometric amount of cross-linker.
- The thioether bond between the cross-linker and the thiol is readily reversible, and in particular can be cleaved in a controlled manner at a time chosen by the skilled worker.
- The retention of the double bond in the product obtained after linking the thiol to the cross-linker constitutes a reaction site for linking to further functional compounds. It is therefore easy to add extra functionally active molecules to the solid substrate-target molecule conjugate.

The new cross-linking methodology is readily applicable across the full spectrum of known methods involving conjugation of target molecules to solid substrate materials.

SUMMARY OF THE INVENTION

The present invention provides a product, which comprises (a) a solid substrate and (b) a moiety of formula (I) linked thereto
wherein X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl and either

(i) R represents an electrophilic leaving group Y, the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I); or
(ii) the solid substrate carries a thiol moiety, R represents a bond to the sulfur atom of said thiol moiety and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I); or
(iii) the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and R represents a group of formula —S—F₁or —S-L-F₂, wherein L represents a linker group and —S—F₁and —F₂represent a functional moiety;
wherein said functional moiety is selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.

The present invention also provides use of a compound containing a moiety of formula (III) as a reagent for linking a solid substrate to a functional moiety
wherein:

- X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl;
- Y is an electrophilic leaving group; and
- the functional moiety is a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA or an RNA.

The present invention further provides a process, which comprises:

- providing a product of the present invention, in which either:
- (i) a solid substrate carrying a thiol moiety is attached to the 2-position of the formula (I) via the sulfur atom of said thiol moiety; or
- (ii) a group —S—F₁or —S-L-F₂is attached to the 2-position of the formula (I); and
- cleaving the thiol bond at the 2-position of the formula (I).

The present invention also provides a product of formula (Va) or (Vb)
wherein

- R₁represents:
- (i) a solid substrate carrying a thiol moiety, which solid substrate is attached to the 2-position of the formula (Va) or (Vb) via the sulfur atom of said thiol moiety; or
- (ii) —S—F₁or —S-L-F₂;
- X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl;
- R₂represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG;
- either:
  - R₃and R₃′ are the same or different and each represents a hydrogen atom, Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG; or
  - R₃and R₃′ together form a group of formula —O— or —N(R_33′), wherein R_33′ represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG; or
  - R₃and R₃′ together form a group of formula —N(R_33′)—N(R_33′)—, wherein each R_33′ is the same or different and represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG;
- R₄is a halogen atom, a hydroxyl, C_1-6alkoxy, thiol, C_1-6alkylthio or C_1-6alkylcarbonyloxy group, or a group of formula F₃;
- Sol represents a solid substrate;
- each —S—F₁, F₂and F₃is the same or different and represents a functional moiety selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;
- each E and Y is the same or different and represents an electrophilic leaving group;
- each Nu is the same or different and represents a nucleophile selected from —OH, —SH, —NH₂and —NH(C_1-6alkyl);
- each L is the same or different and represents a linker group;
- each Z is the same or different and represents a reactive group attached to a moiety L;
- each n is the same or different and is 1, 2 or 3;
- each m is the same or different and is an integer having a value of from zero to n;
- each IG is the same or different and represents a moiety which is a C_1-20alkyl group, a C_2-20alkenyl group or a C_2-20alkynyl group, which is unsubstituted or substituted by one or more halogen atom substituents, and in which (a) 0, 1 or 2 carbon atoms are replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, wherein:
- (i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl) and nitro groups; and
- (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by —C(O)— groups; and
- at least one of the groups R₂and R₄comprises a group of formula F₃;
  with the proviso that the product contains one solid substrate.

The present invention further provides a plurality of products of the present invention, arranged in an array.
Still further, the present invention provides an assay process, which comprises:

- providing a plurality of products of the present invention, wherein each product comprises a functional moiety selected from an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a peptide, a protein, a cell, a DNA and an RNA;
- incubating said plurality of products with a sample comprising a test substance; and
- detecting whether any of said test substance is bound to any of said plurality of products.

The present invention provides a detection process, which comprises:

(i) providing a product of the present invention, wherein said product comprises an antibody or an antigen;
(ii) incubating said product with a sample;
(iii) removing any material which is not bound to said antibody or antigen; and
(iv) detecting any substance that is bound to the antibody or antigen.

The present invention also provides a process for purifying a specific substance from a sample, which comprises:

(i) providing a product of the present invention, wherein said product comprises a functional moiety that is capable of selectively binding to said substance;
(ii) incubating said product with said sample;
(iii) removing any material which is not bound to said functional moiety; and
(iv) separating said substance from said product.

The present invention further provides a process for producing a peptide or protein, which comprises:

(i) providing a product of the present invention;
(ii) attaching an amino acid to said product; and
(iii) attaching one or more further amino acids to said amino acid, thus producing a peptide or protein moiety that is linked to a solid substrate; and
(iv) cleaving said peptide or protein moiety from said solid substrate.

The present invention also provides a product containing a moiety of formula (VI) having: (a) a functional moiety; and (b) a solid substrate; linked thereto
wherein:

- X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl; and
- said functional moiety is selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the results of the protocol described in Example 1 wherein protein/biotin-PEG-bromomaleimide adduct and unmodified model protein solutions (In) were added to neutravidin-coated agarose beads, centrifuged, the flow-through (FT) collected, the beads washed with PBS and both wash fractions collected (W1 and W2), protein released from the beads by incubation in PBS containing β-mercaptoethanol, the sample centrifuged and the eluant (El) containing cleaved protein collected.

FIG. 2 shows the generation of somatostatin-maleimide adducts from halomaleimides according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) Top left: Generation of somatostatin adduct from dichloromaleimide (circle), dibromomaleimide (square) and diiodomaleimide (triangle). Top right: Generation of somatostatin adduct from monobromomaleimide (circle), N-methylmonobromomaleimide (square) and N-methyldibromomaleimide (triangle). Bottom left: Generation of somatostatin adduct from N-fluorescein-dibromomaleimide (circle), N-biotin-dibromomaleimide (square), N-PEG 5000-dibromomaleimide (triangle), N-PEG-5000-dithiophenolmaleimide (diamond) and N-PEG 300-dibromomaleimide (oval).

FIG. 3 shows the generation of somatostatin-maleimide adducts from dithiomaleimides according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) Top left: Generation of somatostatin adduct from di-2-mercaptoethanolmaleimide at 1 eq. (circle), 5 eq. (square) and 10 eq. (triangle). Top right: Generation of somatostatin adduct from dicysteinemaleimide at 1 eq. (circle), 5 eq. (square) and 10 eq. (triangle). Bottom left: Generation of somatostatin adduct from dithiophenolmaleimide at 1 eq. (circle), 5 eq. (square) and 10 eq. (triangle). Bottom right: Generation of somatostatin adduct from di-2-mercaptopyridinemaleimide at 1 eq. (circle), 5 eq. (square) and 10 eq. (triangle).

FIG. 4 shows cleavage of maleimide bridged somatostatin with various reducing agents according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time in minutes (min), hours (h) and days (d)). Top left: Total modified somatostatin-maleimide with DTT (hollow circle) and total amount of side products (filled circle). Top middle: Total modified somatostatin-maleimide with 2-mercaptoethanol (hollow circle) and total amount of side products (filled circle). Top right: Total modified somatostatin-maleimide with GSH (hollow circle) and total amount of side products (filled circle). Bottom left: Total modified somatostatin-maleimide with TCEP (hollow circle) and total amount of side products (filled circle). Bottom right: Total modified somatostatin-maleimide with 1,2-ethanedithiol (hollow circle) and total amount of side products (filled circle).

FIG. 5 shows cleavage of maleimide bridged somatostatin with various amounts of DTT and 2-mercaptoethanol according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) Left Regeneration of somatostatin by DTT at 50 eq. (hollow circle), 20 eq. (hollow triangle) and 10 eq. (hollow square). Right: Regeneration of somatostatin by 2-mercaptoethanol at 50 eq. (hollow circle), 20 eq. (hollow triangle) and 10 eq. (hollow square) and total amount of side products at 50 eq. (filled circle), 20 eq. (filled triangle) and 10 eq. (filled square).

FIG. 6 shows catalysed cleavage of bridged somatostatin according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) Shown on the Figure are regeneration of somatostatin by 2-mercaptoethanol (hollow circle), 2-mercaptoethanol with NaI (hollow square) and 2-mercaptoethanol with benzeneselenol (hollow triangle), as well as total side products when using 2-mercaptoethanol (filled circle), 2-mercaptoethanol with NaI (filled square) and 2-mercaptoethanol with benzeneselenol (filled triangle).

FIG. 7 shows cleavage of N-functionalised maleimide bridged somatostatin by 2-mercaptoethanol according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time in minutes (min), hours (h) and days (d)). Top left: cleavage of N-methylmaleimide somatostatin adduct to give somatostatin (hollow circle) and total side products (filled circle). Top middle: cleavage of N-biotin maleimide somatostatin adduct to give somatostatin (hollow circle) and total side products (filled circle). Top right: cleavage of N-fluorescein maleimide somatostatin adduct to give somatostatin (hollow circle) and total side products (filled circle). Bottom left: cleavage of N-PEG 5000 maleimide somatostatin adduct to give somatostatin (hollow circle) and total side products (filled circle). Bottom middle: cleavage of N-PEG 300 maleimide somatostatin adduct to give somatostatin (hollow circle) and total side products (filled circle).

FIG. 8 shows cleavage of the diaddition product of monobromomaleimide with somatostatin according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/hours). Top left: Somatostatin-maleimide (hollow circle), somatostatin-bis-maleimide (filled circle) and total side products (triangle) using 2-mercaptoethanol. Top right: Somatostatin-maleimide (hollow circle), somatostatin-bis-maleimide (filled circle) and total side products (triangle) using DTT. Bottom left: Somatostatin-maleimide (hollow circle), somatostatin-bis-maleimide (filled circle) and total side products (triangle) using TCEP.

FIG. 9 shows comparable in situ bridging of somatostatin with various amounts of dithiomaleimides according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) The Figure shows generation of bridged somatostatin using TCEP initiator and thiophenol in a ratio of 3:5 (circle), selenol initiator with thiophenol in a ratio of 5:10 (square) and selenol initiator with 2-mercaptoethanol in a ratio of 10:20 (triangle).

FIG. 10 shows in situ PEGylation of somatostatin according to the protocol described in Reference Example 116 as measured by LC-MS (y-axis=signal %; x-axis=time/min) The Figure shows generation of PEGylated somatostatin using 5 eq. N-PEG5000-dithiophenolmaleimide and 3 eq. TCEP (circle) and using 10 eq. N-PEG5000-dithiophenolmaleimide and 5 eq. benzeneselenol (square).

FIG. 11 shows whole cell patch-clamp current recordings obtained in the patch clamp assay described in Reference Example 116. The Figure shows representative traces recorded from the GIRK 1/2A cell line expressing SSTR2. The cells were clamped at −60 mV and 20 μM of somatostatin or its derivatives were applied for 20 s. Top left: somatostatin (in the axes shown the vertical line represents 1000 pA and the horizontal line represents 20 ms). Top right: dibromomaleimide-bridged somatostatin (in the axes shown the vertical line represents 1000 pA and the horizontal line represents 20 ms). Bottom left: fluorescein dibromomaleimide-bridged somatostatin (in the axes shown the vertical line represents 1000 pA and the horizontal line represents 20 ms). Bottom right: PEGylated dibromomaleimide-bridged somatostatin (in the axes shown the vertical line represents 1000 pA and the horizontal line represents 20 ms).

FIG. 12 shows the amplitudes of the currents activated by somatostatin and its analogues in the patch clamp assay described in Reference Example 116. The x-axis represents current amplitude in pA/pF. Top two bars are from fluorescein dibromomaleimide-bridged somatostatin (black bar is after pre-treatment of cell with Pertussis toxin for 24 hr; grey bar is with no pre-treatment), next two bars are from PEGylated dibromomaleimide-bridged somatostatin (black bar is after pre-treatment of cell with Pertussis toxin for 24 hr; grey bar is with no pre-treatment), next three bars are from dibromomaleimide-bridged somatostatin (white bar is after preincubation with the GIRK inhibitor TertiapinQ, 100 nM for 5 minutes; black bar is after pre-treatment of cell with Pertussis toxin for 24 hr; grey bar is with no pre-treatment) and bottom two bars are from somatostatin (black bar is after pre-treatment of cell with Pertussis toxin for 24 hr; grey bar is with no pre-treatment).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “solid substrate” means an object which is a solid under standard conditions (temperature of about 20° C. and pressure of about 100 kPa) and which is capable of interacting with a functional moiety, optionally via a linker, to form a product comprising both the solid substrate and the functional moiety. The solid substrates used in the present invention may be microscopic or macroscopic in dimension, but typically have at least one dimension that is greater than or equal to 0.001 μm, preferably 0.1 μm and most preferably 1 μm. The solid substrates of the present invention can have any shape, including substrates having at least one substantially flat surface (for example, “slide”-, “membrane”- or “chip”-shaped substrates) and substrates having a curved surface (for example, bead-shaped substrates and tube-shaped substrates).
Those of skill in the art will be familiar with the variety of materials, shapes and sizes of solid substrates that are used routinely in the art. Typically, the solid substrates used in the present invention are solid substrates that are suitable for immobilising biomolecules or other molecules of biological interest and thus they include any solid substrate that is known in the art to be suitable for such purposes. Commercial suppliers of such materials include Pierce, Invitrogen and Sigma Aldrich.
Solid substrates of the present invention include nanotubes, metallic substrates, metal oxide substrates, glass substrates, silicon substrates, silica substrates, mica substrates and polymeric substrates. Preferred metallic substrates include gold, silver, copper, platinum, iron and/or nickel substrates, with gold substrates being particularly preferred.
Polymeric substrates include natural polymers and synthetic polymers. Clearly, a “polymeric substrate” is a substrate comprising a plurality of polymer molecules. Preferred polymeric substrates include polystyrene substrates, polypropylene substrates, polycarbonate substrates, cyclo-olefin polymer substrates, cross-linked polyethylene glycol substrates, polysaccharide substrates, such as agarose substrates, and acrylamide-based resin substrates, such as polyacrylamide substrates and polyacrylamine/azlactone copolymeric substrates. Preferred substrates include gold substrates, glass substrates, silicon substrates, silica substrates and polymeric substrates, particularly those polymeric substrates specified herein. Particularly preferred substrates are glass substrates, silicon substrates, silica substrates, polystyrene substrates, cross-linked polyethylene glycol substrates, polysaccharide substrates (for example, agarose substrates) and acrylamide-based resin substrates. In another preferred embodiment, the solid substrate is a nanotube, particularly a carbon nanotube.
As used herein, the term “nanotube” means a tube-shaped structure, the width of which tube is of the order of nanometres (typically up to a maximum of ten nanometres). Nanotubes can be carbon nanotubes or inorganic nanotubes. Carbon nanotubes can be single-walled nanotubes (SWNTs) or multi-walled nanotubes (MWNTs). Inorganic nanotubes are nanotubes made of elements other than carbon, such as silicon, copper, bismuth, metal oxides (for example, titanium dioxide, vanadium dioxide and manganese dioxide), sulfides (for example, tungsten disulphide and molybdenum disulphide), nitrides (for example, boron nitride and gallium nitride) and selenides (for example, tungsten selenide and molybdenum selenide). Preferably, the nanotube is a carbon nanotube.
In one preferred embodiment of the present invention, the solid substrate is an implantable device (i.e., a device that is suitable for implantation into the human or animal, preferably human, body). Preferred implantable devices include prostheses, implantable drug delivery devices and stents, with stents being particularly preferred.
In the product of the present invention the substrate is linked to the moiety of formula (I) and clearly therefore it is necessary for the solid substrate to be capable of forming a strong (for example covalent) bond to a substance immobilised thereon. Accordingly, a solid substrate typically contains a reactive group. Certain solid substrates may comprise suitable reactive groups without the need to carry out any activation of their surface (for example, the end groups of individual polymer chains within a polymeric substrate may constitute reactive groups). Alternatively, the reactive groups may be provided by chemical activation of the solid substrate.
Chemical activation of solid substrates to lend them surface activity is well known in the art. For example, silane compounds carrying at least one alkoxysilicon bond are commonly used to activate metal oxide, glass, mica, silicon and silica substrates. These compounds are thought to react with free hydroxyl groups on the solid surface, leading to formation of a strong silicon-oxygen bond. The silane compound can of course also carry a linker group which itself carries one or more further functional moieties, tailored for reacting with a particular target molecule of interest. Thiol compounds or thioether compounds carrying such linker groups are often used to activate gold surfaces through formation of the strong gold-sulfur bond. Clearly, polymeric substrates can either be specifically designed to carry reactive groups (for example, through judicious choice of monomers) or alternatively can be activated after polymerization is complete. All of these techniques are entirely routine and activated solid substrates carrying reactive groups capable of reacting with practically any common functional group on a target molecule (e.g., amine, carboxyl, thiol, aldehyde and active hydrogen functional groups) are now commercially available.
For the avoidance of doubt, the term “solid substrate” as used herein includes solid substrates which inherently contain reactive groups and also solid substrates which have been chemically activated (i.e., solid substrates carrying reactive groups such as those provided by the surface activation techniques described above). The solid substrate may, for example, be a substrate that bears an affinity tag (or partner) such as a biotin or (stept)avidin tag.
As those of skill in the art would appreciate, where chemical activation of the solid substrate is required to lend it the ability to bond covalently to a substance to be immobilised thereon, this opens the possibility of at least two strategies for attaching the solid substrate to the 1,2-dicarbonyl ethene crosslinking reagent. In a first strategy, a chemically activated solid substrate bearing a particular reactive group (for example, a commercially available chemically activated substrate) may be provided and reacted with a corresponding reactive group on the crosslinking reagent. In an alternative possibility, the crosslinking reagent could be attached directly to a solid substrate, without any prior activation of the substrate. Clearly, this could be achieved by designing the crosslinking reagent so that it carries a reactive group that is capable of activating the target solid substrate. For example, where the solid substrate is a glass substrate, the crosslinking reagent could carry an alkoxysilane group. Under these circumstances, activation of the solid substrate and addition of the crosslinking group take place simultaneously.
Another useful technique for linking the solid substrate to the moiety of formula (I) is via complementary affinity tag partners, i.e. where one of the substrate and the moiety of formula (I) comprises an affinity tag and the other comprises the relevant affinity partner, where “affinity tag” and “affinity partner” are as herein defined. This strategy provides an alternative, typically non-covalent, means for linking the solid substrate to the moiety of formula (I). For example, the biotin/(strept)avidin pair could be used as the basis for attaching the two species: one of the solid substrate and the moiety of formula (I) comprises a biotin group and the other of the solid substrate and the moiety of formula (I) comprises a (strept)avidin group.
As used herein, the term “functional moiety” means a moiety which forms part of a product of the invention and which is one of a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.
As will be readily understood by those of skill in the art, a functional moiety comprised within a product (for example, within a product comprising a solid substrate and a linker group attached thereto) is obtainable by attaching a corresponding “functional compound” thereto. When a functional compound is attached to a reactive group on a solid substrate or to a cross-linker reagent, it is necessary for a bond somewhere in that functional compound to be broken so that a new bond can form. Examples of such processes include the loss of a leaving group from the functional compound when it becomes a functional moiety bound to a solid substrate or to a cross-linker, the loss of a proton when the functional compound reacts via a hydrogen-atom containing nucleophilic group such as an —OH or —SH group, or the conversion of a double bond in the functional compound to a single bond. Those skilled in the art would thus understand that a functional moiety that is, for example, a “protein” means a moiety that is formed by attaching a protein compound to a reactive group on a solid substrate or to a cross-linker reagent, with concomitant loss of a internal bond compared to the corresponding protein compound (for example, loss of a proton from an —OH, —SH or —NH₂moiety).
A functional moiety is typically a moiety that has a discrete biological significance in its native form (i.e., when it is not part of a bioconjugate). Preferably any functional moiety used in the present invention has a relative molecular weight of at least 200, more preferably at least 500, most preferably at least 1000. Preferably a functional moiety as described herein is a biomolecule.
As used herein, the term “detectable moiety” means a moiety which is capable of generating detectable signals in a test sample. Clearly, the detectable moiety can be understood to be a moiety which is derived from a corresponding “detectable compound” and which retains its ability to generate a detectable signal when it is linked to another substance via a cross-linker in a product of the present invention. Detectable moieties are also commonly known in the art as “tags”, “probes” and “labels”. Examples of detectable moieties include chromogenic moieties, fluorescent moieties, radioactive moieties and electrochemically active moieties. In the present invention, preferred detectable moieties are chromogenic moieties and fluorescent moieties. Fluorescent moieties are most preferred.
A chromogenic moiety is a moiety which is coloured, which becomes coloured when it is incorporated into a product of the invention, or which becomes coloured when it is incorporated into a product of the invention and the product subsequently interacts with a secondary target species (for example, where the product of the invention comprises a protein which then interacts with another target molecule).
Typically, the term “chromogenic moiety” refers to a group of associated atoms which can exist in at least two states of energy, a ground state of relatively low energy and an excited state to which it may be raised by the absorption of light energy from a specified region of the radiation spectrum. Often, the group of associated atoms contains delocalised electrons. Chromogenic moieties suitable for use in the present invention include conjugated moieties containing Π systems and metal complexes. Examples include porphyrins, polyenes, polyynes and polyaryls. Preferred chromogenic moieties are
A fluorescent moiety is a moiety which comprises a fluorophore, which is a fluorescent chemical moiety. Examples of fluorescent compounds which are commonly incorporated as fluorescent moieties into the products of the present invention include:

- the Alexa Fluor® dye family available from Invitrogen;
- cyanine and merocyanine;
- the BODIPY (boron-dipyrromethene) dye family, available from Invitrogen;
- the ATTO dye family manufactured by ATTO-TEC GmbH;
- fluorescein and its derivatives;
- rhodamine and its derivatives;
- naphthalene derivatives such as its dansyl and prodan derivatives;
- pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole derivatives;
- coumarin and its derivatives;
- pyrene derivatives; and
- Oregon green, eosin, Texas red, Cascade blue and Nile red, available from Invitrogen.

Preferred fluorescent moieties for use in the present invention include fluorescein, rhodamine, coumarin, sulforhodamine 101 acid chloride (Texas Red) and dansyl. Fluorescein and dansyl are especially preferred.
A radioactive moiety is a moiety that comprises a radionuclide. Examples of radionuclides include iodine-131, iodine-125, bismuth-212, yttrium-90, yttrium-88, technetium-99m, copper-67, rhenium-188, rhenium-186, gallium-66, gallium-67, indium-111, indium-114m, indium-114, boron-10, tritium (hydrogen-3), carbon-11, carbon-14, sulfur-35 and fluorine-18.
In one embodiment, the radioactive moiety may consist of the radionuclide alone. In another embodiment, the radionuclide may be incorporated into a larger radioactive moiety, for example by direct covalent bonding to a linker group (such as a linker containing a thiol group) or by forming a co-ordination complex with a chelating agent. Suitable chelating agents known in the art include DTPA (diethylenetriamine-pentaacetic anhydride), NOTA (1,4,7-triazacyclononane-N,N′,N″-triacetic acid), DOTA (1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid), TETA (1,4,8,11-tetraazacyclotetra-decane-N,N′,N″,N′″-tetraacetic acid), DTTA (N¹-(p-isothiocyanatobenzyl)-diethylene-triamine-N¹,N²,N³-tetraacetic acid) and DFA (N′-[5-[[5-[[5-acetylhydroxyamino)pentyl]amino]-1,4-dioxobutyl]hydroxyamino]pentyl]-N-(5-aminopentyl)-N-hydroxybutanediamide).
An electrochemically active moiety is a moiety that comprises a group that is capable of generating an electrochemical signal in an electrochemical method such as an amperometric or voltammetric method. Typically, an electrochemically active moiety is capable of existing in at least two distinct redox states.
A person of skill in the art would of course easily be able to select a detectable compound that would be suitable for use in accordance with the present invention from the vast array of detectable compounds that are routinely available. The methodology of the present invention can thus be used to produce a product comprising a detectable moiety, which can then be used in any routine biochemical technique that involves detection of such species. For example, in one such technique the detectable moiety may be one member of a donor chromophore/acceptor chromophore pair suitable for use in a FRET analytical method.
As used herein, the term “enzymatically active moiety” means an enzyme, a substrate for an enzyme or a cofactor for an enzyme. Preferably, the enzymatically active moiety is an enzyme.
As used herein, the term “affinity tag” means a chemical moiety which is capable of interacting with an “affinity partner”, which is a second chemical moiety, when both the affinity tag and the affinity partner are present in a single sample. Typically, the affinity tag is capable of forming a specific binding interaction with the affinity partner. A specific binding interaction is a binding interaction which is stronger than any binding interaction that may occur between the affinity partner and any other chemical substance present in a sample. A specific binding interaction may occur, for example, between an enzyme and its substrate.
Affinity tags can be useful in applications such as detection or purification of biomolecules such as proteins. In such applications, a product comprising a solid substrate and an affinity tag can be used to detect or purify a bioconjugate comprising a biomolecule and a corresponding affinity partner, by exploiting the specific binding interaction between the affinity tag and its affinity partner.
One affinity tag/affinity partner pair that is particularly widely used in biochemistry is the biotin/(strept)avidin pair. Avidin and streptavidin are proteins which can be used as affinity partners for binding with high affinity and specificity to an affinity tag derived from biotin (5-[(3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl]pentanoic acid). Other affinity tag/affinity partner pairs commonly used in the art include amylase/maltose binding protein, glutathione/glutathione-S-transferase and metal (for example, nickel or cobalt)/poly(His). As one of skill in the art would appreciate, either member of each pair could function as the “affinity tag”, with the other member of the pair functioning as the “affinity partner”. The terms “affinity tag” and “affinity partner” are thus interchangeable.
A person of skill in the art would be aware of the routine use of affinity tag/affinity partner interactions in biochemistry and in particular in the context of bioconjugate technology. A person of skill in the art would thus have no difficulty in selected an affinity tag for use in accordance with the present invention. The methodology of the present invention can therefore be used to produce products adapted for use in routine biochemical techniques that make use of affinity tag/affinity partner interactions. Particularly preferred affinity tags for use in the products of the present invention are avidin, streptavidin, amylase and glutathione.
As used herein, the term, the term “hapten” means a moiety which comprises an epitope, which is not capable of stimulating an in vivo immune response in its native form, but which is capable of stimulating an in vivo immune response when linked to an immunogenic carrier molecule. Typically, a hapten is a non-proteinaceous moiety of relatively low molecular weight (for example, a molecular weight of less than 1000). An epitope is the part of a molecule or moiety which is recognized by the immune system and stimulates an immune response.
As used herein, the term “immunogenic carrier” means an antigen that is able to facilitate an immune response when administered in vivo and which is capable of being coupled to a hapten. Examples of immunogenic carriers include proteins, liposomes, synthetic or natural polymeric moieties (such as dextran, agarose, polylysine and polyglutamic acid moieties) and synthetically designed organic moieties. Commonly used protein immunogenic carriers have included keyhole limpet hemocyanin, bovine serum albumin, aminoethylated or cationised bovine serum albumin, thyroglobulin, ovalbumin and various toxoid proteins such as tetanus toxoid and diphtheria toxoid. Well known synthetically designed organic molecule carriers include the multiple antigentic peptide (MAP).
As a person of skill in the biochemical art would be aware, hapten-immunogenic carrier conjugates are widely used in, for example, immunology and proteomics. A person of skill in the art would recognise that the methodology of the present invention could readily be applied to produce conjugates comprising a hapten and an immunogenic carrier, which conjugates are immobilised to a solid substrate.
As used herein, the term “antibody or antibody fragment” means a protein that is capable of binding to a specific antigen via an epitope on the antigen, or a fragment of such a protein. Antibodies include monoclonal antibodies and polyclonal antibodies. Monoclonal antibodies are preferred.
As used herein, the term “antigen” means a substance that is capable of instigating an immune response when administered in vivo and which is capable of binding to an antibody produced during said immune response.
As used herein, the term “ligand” means a moiety that is able to interact with a biomolecule (for example, a protein) in such a way as to modify the functional properties of the biomolecule. Typically, the ligand is a moiety that binds to a site on a target protein. The interaction between the ligand and the biomolecule is typically non-covalent. For example, the interaction may be through ionic bonding, hydrogen bonding or van der Waals' interactions. However, it is also possible for some ligands to form covalent bonds to biomolecules. Often, a ligand is capable of altering the chemical conformation of the biomolecule when it interacts with it.
Examples of ligands capable of interacting with a protein include substrates (which are acted upon by the enzyme upon binding, for example by taking part in a chemical reaction catalysed by the enzyme), inhibitors (which inhibit protein activity on binding), activators (which increase protein activity on binding) and neurotransmitters.
As used herein, a ligand candidate is a moiety which is suspected of being a ligand for a particular biomolecule. Ligand candidates are routinely used in biochemistry to carry out screening techniques. Typically, in such techniques a plurality of ligand candidates are provided and then assayed to establish whether they are able to interact with a biomolecule of interest (for example, a protein). As those of skill in the art would appreciate, a particular ligand candidate may constitute a ligand (where it does interact with the biomolecule) or it may not constitute a ligand (where it is found not to interact with the biomolecule). Typically, a plurality of ligand candidates to be assayed against a specific biomolecule share certain common structural features, but are deliberately chosen to differ from one another in at least one aspect of their structure.
Typically, ligand candidates are small molecules (for example, molecules having a molecular weight of less than 1000, or less than 500). In a preferred embodiment, the plurality of ligand candidates are presented on a plurality of products of the present invention arranged in an array (i.e., in a single array structure) so that they can be screened simultaneously. Such array systems are commonly known in the art as “chemical compound microarrays” or “small molecule arrays”. In such systems, the plurality of ligand candidates may be selected, for example, using so-called “diversity-oriented synthesis” (DOS) approaches. A comprehensive review of the application of immobilised ligand candidates in this field of assay technology can be found in Uttamchandani et al. (Current Opinion in Chemical Biology 2005, 9:4-13), the content of which is herein incorporated by reference in its entirety.
As used herein, the term “biologically active moiety” means a moiety that is capable of inducing a biochemical response when administered in vivo.
The biologically active moiety can be a drug. Drugs include cytotoxic agents such as doxorubicin, methotrexate and derivatives thereof, cytotoxin precursors which are capable of metabolising in vivo to produce a cytotoxic agent, anti-neoplastic agents, anti-hypertensives, cardioprotective agents, anti-arrhythmics, ACE inhibitors, anti-inflammatories, diuretics, muscle relaxants, local anaesthetics, hormones, cholesterol lowering drugs, anti-coagulants, anti-depressants, tranquilizers, neuroleptics, analgesics such as a narcotic or anti-pyretic analgesics, anti-virals, anti-bacterials, anti-fungals, bacteriostats, CNS active agents, anti-convulsants, anxiolytics, antacids, narcotics, antibiotics, respiratory agents, anti-histamines, immunosuppressants, immunoactivating agents, nutritional additives, anti-tussives, diagnostic agents, emetics and anti-emetics, carbohydrates, glycosoaminoglycans, glycoproteins and polysaccharides, lipids, for example phosphatidyl-ethanolamine, phosphtidylserine and derivatives thereof, sphingosine, steroids, vitamins, antibiotics, including lantibiotics, bacteristatic and bactericidal agents, antifungal, anthelminthic and other agents effective against infective agents including unicellular pathogens, small effector molecules such as noradrenalin, alpha adrenergic receptor ligands, dopamine receptor ligands, histamine receptor ligands, GABA/benzodiazepine receptor ligands, serotonin receptor ligands, leukotrienes and triodothyronine, and derivatives thereof.
In a preferred embodiment of the present invention, the biologically active moiety is a drug. In this embodiment, the solid substrate is typically an implantable device, for example a stent.
As used herein, the term “liposome” means a structure composed of phospholipid bilayers which have amphiphilic properties. Liposomes suitable for use in accordance with the present invention include unilamellar vesicles and multilamellar vesicles.
As used herein, the term “polymeric moiety” means a single polymeric chain (branched or unbranched), which is derived from a corresponding single polymeric molecule. Polymeric moieties may be natural polymers or synthetic polymers. Typically, though, the polymeric molecules are not polynucleotides. As is well known in the art, polymeric moieties can be used to modify the surface properties of solid substrates. For example, where the solid substrate is administered in vivo (e.g., as an implantable device such as a stent), polymeric moieties can modify the resulting biological interactions such as biostability, blood compatibility and the immune response engendered by the immune system against the foreign body.
A person of skill in the art would therefore recognise that the methodology of the present invention can be used to prepare a product comprising a solid substrate and a polymeric moiety. A person of skill in the art would easily be able to select suitable polymeric moieties for use in accordance with the present invention, on the basis of those polymeric moieties used routinely in the art. Clearly, the nature of the polymeric moiety will depend upon the intended use of the product of the present invention. Exemplary polymeric moieties for use in accordance with the present invention include polysaccharides, polyethers, polyamino acids (such as polylysine), polyvinyl alcohols, polyvinylpyrrolidinones, poly(meth)acrylic acid and derivatives thereof, polyurethanes and polyphosphazenes. Typically such polymers contain at least ten monomeric units. Thus, for example, a polysaccharide typically comprises at least ten monosaccharide units.
Two particularly preferred polymeric molecules are dextran and polyethylene glycol (“PEG”), as well as derivatives of these molecules (such as monomethoxypolyethylene glycol, “mPEG”). Preferably, the PEG or derivative thereof has a molecular weight of less than 20,000. Preferably, the dextran or derivative thereof has a molecular weight of 10,000 to 500,000.
As used herein, the term “amino acid” means a moiety containing both an amine functional group and a carboxyl functional group. However, preferably the amino acid is an α-amino acid. Preferably, the amino acid is a proteinogenic amino acid, i.e. an amino acid selected from alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, phenylalanine, pyrrolysine, selenocysteine, serine, threonine, tryptophan, tyrosine and valine. However, the amino acid can also be a non-proteinogenic amino acid. Examples of non-proteinogenic amino acids include lanthionine, 2-aminoisobutyric acid, dehydroalanine, gamma-aminobutyric acid, ornithine, citrulline, canavanine and mimosine. A particularly preferred amino acid according to the present invention is cysteine.
As used herein, the terms “peptide” and “protein” mean a polymeric moiety made up of amino acid residues. As a person of skill in the art will be aware, the term “peptide” is typically used in the art to denote a polymer of relatively short length and the term “protein” is typically used in the art to denote a polymer of relatively long length. As used herein, the convention is that a peptide comprises up to 50 amino acid residues whereas a protein comprises more than 50 amino acids. However, it will be appreciated that this distinction is not critical since the functional moieties identified in the present application can typically represent either a peptide or a protein.
As used herein, the term “polypeptide” is used interchangeable with “protein”.
As used herein, a peptide or a protein can comprise any natural or non-natural amino acids. For example, a peptide or a protein may contain only α-amino acid residues, for example corresponding to natural α-amino acids. Alternatively the peptide or protein may additionally comprise one or more chemical modifications. For example, the chemical modification may correspond to a post-translation modification, which is a modification that occurs to a protein in vivo following its translation, such as an acylation (for example, an acetylation), an alkylation (for example, a methylation), an amidation, a biotinylation, a formylation, glycosylation, a glycation, a hydroxylation, an iodination, an oxidation, a sulfation or a phosphorylation. A person of skill in the art would of course recognise that such post-translationally modified peptides or proteins still constitute a “peptide” or a “protein” within the meaning of the present invention. For example, it is well established in the art that a glycoprotein (a protein that carries one or more oligosaccharide side chains) is a type of protein.
As used herein, the term “cell” means a single cell of a living organism.
As used herein, the term “carbohydrate” includes monosaccharides and oligosaccharides. Typically an oligosaccharide contains from two to nine monosaccharide units. Thus, as used herein, a polysaccharide is classified as a “polymeric moiety” rather than as a carbohydrate. However, a person of skill in the art will appreciate that this distinction is not important, since the functional moieties used in accordance with the invention can typically constitute either of a “carbohydrate” and a “polysaccharide”.
As used herein, the term “DNA” means a deoxyribonucleic acid made up of one or more nucleotides. The DNA may be single stranded or double stranded. Preferably, the DNA comprises more than one nucleotide.
As used herein, the term “RNA” means a ribonucleic acid comprising one or more nucleotides. Preferably, the RNA comprises more than one nucleotide.
Preferred functional moieties are detectable moieties, enzymatically active moieties (preferably enzymes), affinity tags, antibodies or antibody fragments, antigens, ligands or ligand candidates, amino acids, peptides, proteins, cells, DNAs and RNAs. Particularly preferred functional moieties are enzymatically active moieties (preferably enzymes), affinity tags, antibodies or antibody fragments, antigens, amino acids, peptides, proteins, DNAs and RNAs.
As used herein, “conjugate” means a molecule which comprises a functional moiety as defined herein and at least one of a solid substrate and a further functional moiety.
Accordingly, when a product of the present invention (which necessarily contains a solid substrate) carries a functional moiety it constitutes a conjugate. The functional moiety and the solid substrate are then covalently linked to one another via a cross-linker moiety, as described herein.
As used herein, the terms “group” and “moiety” are used interchangeably.
As used herein, the term “electrophilic leaving group” means a substituent attached to a saturated or unsaturated carbon atom which can be replaced by a nucleophile following a nucleophilic attack at that carbon atom. Those of skill in the art are routinely able to select electrophilic leaving groups that would be suitable for locating on a particular compound and for reacting with a particular nucleophile.
As used herein, the term “nucleophile” means a functional group or compound which is capable of forming a chemical bond by donating an electron pair.
As used herein, the term “linker group” means a group which is capable of linking one chemical moiety to another. The nature of the linker groups used in accordance with the present invention is not important. A person of skill in the art would recognise that linker groups are routinely used in the construction of conjugate molecules. Typically, a linker group for use in the present invention is an organic group. Typically, such a linker group has a molecular weight of 50 to 1000, preferably 100 to 500. Examples of linker groups appropriate for use in accordance with the present invention are common general knowledge in the art and described in standard reference text books such as “Bioconjugate Techniques” (Greg T. Hermanson, Academic Press Inc., 1996), the content of which is herein incorporated by reference in its entirety.
As used herein, a “reactive group” means a functional group on a first substance that is capable of taking part in a chemical reaction with a functional group on a second substance such that a covalent bond forms between the first substance and the second substance. Reactive groups include leaving groups, nucleophilic groups, and other reactive groups as described herein.
As used herein, the term “alkyl” includes both saturated straight chain and branched alkyl groups. Preferably, an alkyl group is a C_1-20alkyl group, more preferably a C_1-15, more preferably still a C_1-12alkyl group, more preferably still, a C_1-6alkyl group, and most preferably a C_1-4alkyl group. Particularly preferred alkyl groups include, for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl and hexyl. The term “alkylene” should be construed accordingly.
As used herein, the term “alkenyl” refers to a group containing one or more carbon-carbon double bonds, which may be branched or unbranched. Preferably the alkenyl group is a C_2-20alkenyl group, more preferably a C_2-15alkenyl group, more preferably still a C_2-12alkenyl group, or preferably a C_2-6alkenyl group, and most preferably a C_2-4alkenyl group. The term “alkenylene” should be construed accordingly.
As used herein, the term “alkynyl” refers to a carbon chain containing one or more triple bonds, which may be branched or unbranched. Preferably the alkynyl group is a C_2-20alkynyl group, more preferably a C_2-15alkynyl group, more preferably still a C_2-12alkynyl group, or preferably a C_2-6alkynyl group and most preferably a C_2-4alkynyl group. The term “alkynylene” should be construed accordingly.
Unless otherwise specified, an alkyl, alkenyl or alkynyl group is typically unsubstituted. However, where such a group is indicated to be unsubstituted or substituted, one or more hydrogen atoms are optionally replaced by halogen atom substituents. Preferably, a substituted alkyl, alkenyl or alkynyl group has from 1 to 10 substituents, more preferably 1 to 5 substituents, more preferably still 1, 2 or 3 substituents and most preferably 1 or 2 substituents, for example 1 substituent. Preferably, though, an alkyl, alkenyl or alkynyl group is unsubstituted.
In the moiety that is an alkyl, alkenyl or alkynyl group or an alkylene, alkenylene or alkynylene group, in which (a) 0, 1 or 2 carbon atoms may be replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups may be replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, a total of 0, 1 or 2 of said carbon atoms and —CH₂— groups are preferably replaced, more preferably a total of 0 or 1. Most preferably, none of the carbon atoms or —CH₂— groups is replaced.
Preferred groups for replacing a —CH₂— group are —O—, —S— and —C(O)— groups. Preferred groups for replacing a carbon atom are phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups. As used herein, the reference to “0, 1 or 2 carbon atoms” means any terminal or non-terminal carbon atom in the alkyl, alkenyl or alkynyl chain, including any hydrogen atoms attached to that carbon atom. As used herein, the reference to “0, 1 or 2 —CH₂— groups” refers to a group which does not correspond to a terminal carbon atom in the alkyl, alkenyl or alkynyl chain.
As used herein, a C_6-10aryl group is a monocyclic or polycyclic 6- to 10-membered aromatic hydrocarbon ring system having from 6 to 10 carbon atoms. Phenyl is preferred. The term “arylene” should be construed accordingly.
As used herein, a 5- to 10-membered heteroaryl group is a monocyclic or polycyclic 5- to 10-membered aromatic ring system, such as a 5- or 6-membered ring, containing at least one heteroatom, for example 1, 2, 3 or 4 heteroatoms, selected from O, S and N. When the ring contains 4 heteroatoms these are preferably all nitrogen atoms. The term “heteroarylene” should be construed accordingly.
Examples of monocyclic heteroaryl groups include thienyl, furyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl and tetrazolyl groups.
Examples of polycyclic heteroaryl groups include benzothienyl, benzofuryl, benzimidazolyl, benzothiazolyl, benzisothiazolyl, benzoxazolyl, benzisoxazolyl, benztriazolyl, indolyl, isoindolyl and indazolyl groups. Preferred polycyclic groups include indolyl, isoindolyl, benzimidazolyl, indazolyl, benzofuryl, benzothienyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl and benzisothiazolyl groups, more preferably benzimidazolyl, benzoxazolyl and benzothiazolyl, most preferably benzothiazolyl. However, monocyclic heteroaryl groups are preferred.
Preferably the heteroaryl group is a 5- to 6-membered heteroaryl group. Particularly preferred heteroaryl groups are thienyl, pyrrolyl, imidazolyl, thiazolyl, isothiazolyl, pyrazolyl, oxazolyl, isoxazolyl, triazolyl, pyridinyl, pyridazinyl, pyrimidinyl and pyrazinyl groups. More preferred groups are thienyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, pyrrolyl and triazinyl, most preferably pyridinyl.
As used herein, a 5- to 10-membered heterocyclyl group is a non-aromatic, saturated or unsaturated, monocyclic or polycyclic C_5-10carbocyclic ring system in which one or more, for example 1, 2, 3 or 4, of the carbon atoms are replaced with a moiety selected from N, O, S, S(O) and S(O)₂. Preferably, the 5- to 10-membered heterocyclyl group is a 5- to 6-membered ring. The term “heterocyclyene” should be construed accordingly.
Examples of heterocyclyl groups include azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, methylenedioxyphenyl, ethylenedioxyphenyl, thiomorpholinyl, S-oxo-thiomorpholinyl, S,S-dioxo-thiomorpholinyl, morpholinyl, 1,3-dioxolanyl, 1,4-dioxolanyl, trioxolanyl, trithianyl, imidazolinyl, pyranyl, pyrazolinyl, thioxolanyl, thioxothiazolidinyl, 1H-pyrazol-5-(4H)-onyl, 1,3,4-thiadiazol-2(3H)-thionyl, oxopyrrolidinyl, oxothiazolidinyl, oxopyrazolidinyl, succinimido and maleimido groups and moieties. Preferred heterocyclyl groups are pyrrolidinyl, imidazolidinyl, oxazolidinyl, isoxazolidinyl, thiazolidinyl, isothiazolidinyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, dithiolanyl, dioxolanyl, pyrazolidinyl, piperidinyl, piperazinyl, hexahydropyrimidinyl, thiomorpholinyl and morpholinyl groups and moieties. More preferred heterocyclyl groups are tetrahydropyranyl, tetrahydrothiopyranyl, thiomorpholinyl, tetrahydrofuranyl, tetrahydrothienyl, piperidinyl, morpholinyl and pyrrolidinyl groups.
For the avoidance of doubt, although the above definitions of heteroaryl and heterocyclyl groups refer to an “N” moiety which can be present in the ring, as will be evident to a skilled chemist the N atom will be protonated (or will carry a substituent as defined below) if it is attached to each of the adjacent ring atoms via a single bond.
As used herein, a C_3-7carbocyclyl group is a non-aromatic saturated or unsaturated hydrocarbon ring having from 3 to 7 carbon atoms. Preferably it is a saturated or mono-unsaturated hydrocarbon ring (i.e. a cycloalkyl moiety or a cycloalkenyl moiety) having from 3 to 7 carbon atoms, more preferably having from 5 to 6 carbon atoms. Examples include cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl and their mono-unsaturated variants. Particularly preferred carbocyclic groups are cyclopentyl and cyclohexyl. The term “carbocyclylene” should be construed accordingly.
Where specified, 0, 1 or 2 carbon atoms in a carbocyclyl or heterocyclyl group may be replaced by —C(O)— groups. As used herein, the “carbon atoms” being replaced are understood to include the hydrogen atoms to which they are attached. When 1 or 2 carbon atoms are replaced, preferably two such carbon atoms are replaced. Preferred such carbocyclyl groups include a benzoquinone group and preferred such heterocyclyl groups include succinimido and maleimido groups.
Unless otherwise specified, an aryl, heteroaryl, carbocyclyl or heterocyclyl group is typically unsubstituted. However, where such a group is indicated to be unsubstituted or substituted, one or more hydrogen atoms are optionally replaced by halogen atoms or C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl) or nitro groups. Preferably, a substituted aryl, heteroaryl, carbocyclyl or heterocyclyl group has from 1 to 4 substituents, more preferably 1 to 2 substituents and most preferably 1 substituent. Preferably a substituted aryl, heteroaryl, carbocyclyl or heterocyclyl group carries not more than 2 nitro substituents. Preferred substituents are halogen atoms and C_1-4alkyl and C_1-4alkoxy groups. Particularly preferred substituents are halogen atoms. Preferably, though, an aryl, heteroaryl, carbocyclyl or heterocyclyl group is unsubstituted.
As used herein, halogen atoms are typically F, Cl, Br or I atoms, preferably Br or Cl atoms, more preferably Br atoms.
As used herein, a C_1-6alkoxy group is a C_1-6alkyl (e.g. a C_1-4alkyl) group which is attached to an oxygen atom.
As used herein, a C_1-6alkylthiol group is a C_1-6alkyl (e.g. a C_1-4alkyl) group which is attached to a sulfur atom.
As used herein, a 5- to 10-membered heterocyclylthiol is a 5- to 10-membered (e.g., a 5- to 6-membered) heterocyclyl group which is attached to a sulfur atom.
As used herein, a C_6-10arylthiol is a C_6-10aryl (e.g., a phenyl) group which is attached to a sulfur atom.
As used herein, a C_3-7carbocyclylthiol is a C_3-7carbocyclyl (e.g., a C_5-6carbocyclyl) group which is attached to a sulfur atom.
The product of the present invention necessarily comprises a solid substrate and the moiety of formula (I) together with at least one functional moiety. This moiety of formula (I) constitutes a cross-linking reactive moiety which is capable of linking the solid substrate to a further functional moiety.
Preferably X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NH. More preferably, X and X′ are the same or different and each represents oxygen or sulfur. Preferably at least one of X and X′ represents oxygen. Most preferably, X and X′ are both oxygen.
Y is preferably a halogen atom or a triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, C_1-6alkylthiol, 5- to 10-membered heterocyclylthiol, C_6-10arylthiol, C_3-7carbocyclylthiol, —OC(O)CH₃, —OC(O)CF₃, phenyloxy, —NR_xR_yR_z ⁺ or —PR_xR_yR_z ⁺ group. More preferably, Y is a halogen atom or a triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, C_1-6alkylthiol, 5- to 10-membered heterocyclylthiol, C_6-10arylthiol or C_3-7carbocyclylthiol. More preferably still Y is a halogen atom or a C_1-6alkylthiol, 5- to 10-membered heterocyclylthiol, C_6-10arylthiol or C_3-7carbocyclylthiol. Most preferably Y is a halogen atom, particularly a bromine atom.
R_x, R_yand R_zare each preferably selected from hydrogen atoms and C_1-6alkyl groups. More preferably R_x, R_yand R_zare each preferably selected from hydrogen atoms and methyl and ethyl groups. Preferably, in a particular —NR_xR_yR_z ⁺ or —PR_xR_yR_z ⁺ group, R_x, R_yand R_zare the same.
In a first embodiment (i), R represents an electrophilic leaving group and the solid substrate is linked to the 1-, 3- or 4-position of the formula (I). The product of embodiment (i) is capable of reacting with a functional moiety carrying a thiol group, such that the functional moiety attaches to the 2-position of the formula (I) via the sulfur atom of its thiol group, i.e. by displacing the electrophilic leaving group Y. The product of embodiment (i) already carries one or more further functional moieties linked to the 1-, 3- and/or 4-positions of the formula (I), i.e. it constitutes a conjugate.
In a second embodiment (ii), the solid substrate carries a thiol moiety and R represents a bond to the sulfur atom of said thiol moiety. The product of embodiment (ii) comprises one or more functional moieties linked to the 1-, 3- and/or 4-positions of the formula (I) (i.e., it constitutes a conjugate comprising both a solid substrate and one or more functional molecules). The product also constitutes a solid substrate reagent that is “primed” to react with a functional moiety. As those of skill in the art would appreciate, such a reaction could occur through any reactive functional group present on the moiety of formula (I), including addition across the carbon-carbon double bond between the 2- and 3-positions of formula (I).
In a third embodiment (iii), the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and R represents a group of formula —S—F₁or —S-L-F₂, wherein L represents a linker group and —S—F₁and —F₂represent a functional moiety. Thus, in this embodiment the product also constitutes a conjugate.
For the avoidance of doubt, the moiety —S—F₁constitutes a functional moiety which is attached to a second moiety via a sulfur atom S. Similarly, —S-L- represents a linker group of the present invention attached to a sulfur atom, which sulfur atom is then attached to a second moiety.
It will be clear that the moiety of formula (I) represents the key reactive moiety which according to the present invention allows a thiol-containing functional moiety to be conjugated to a solid substrate, or a thiol-containing solid substrate to be conjugated to a functional moiety. In the moiety of formula (I) (and the moiety of formula (III), as described in detail elsewhere), the symbol
means a point of attachment to another group. It will be appreciated that the identity of the groups attached via these points of attachment is unimportant to the present invention. Those of skill in the art would readily understand that it would be possible to choose the groups attached via these points of attachment to suit a particular purpose, for example based on the specific identity of the functional moieties and solid substrates to be linked together. As would be well known to those of skill in the art, cross-linking reagents are routinely designed which carry functional groups adapted to react with functional moieties and solid substrates having particular reactive groups and which are spaced by linker groups (which typically do not play a significant role in the reactions). A person of skill in the art would immediately understand that the moiety of formula (I) could readily be incorporated into routine cross-linker reagents, for example, by replacing conventional moieties designed to react with thiol groups (for example, maleimide groups or iodoacetyl groups). Detailed information on the design of cross-linker reagents suitable for adaptation in this manner can be found, for example, in “Bioconjugate Techniques” (Greg T. Hermanson, Academic Press Inc., 1996), the content of which is herein incorporated by reference in its entirety.
Preferably, the product of the present invention has the formula (II)
wherein:

- X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl;
- R₁represents:
- (i) an electrophilic leaving group Y; or
- (ii) a solid substrate carrying a thiol moiety, which solid substrate is attached to the 2-position of the formula (II) via the sulfur atom of said thiol moiety; or
- (iii)-S—F₁or —S-L-F₂;
- R₂represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG;
- either:
  - R₃and R₃′ are the same or different and each represents a hydrogen atom, Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG; or
  - R₃and R₃′ together form a group of formula —O— or —N(R_33′), wherein R_33′ represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG; or
  - R₃and R₃′ together form a group of formula —N(R_33′)—N(R_33′)—, wherein each R_33′ is the same or different and represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG;
- Sol represents a solid substrate;
- each —S—F₁, F₂and F₃is the same or different and represents a functional moiety selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;
- each E and Y is the same or different and represents an electrophilic leaving group;
- each Nu is the same or different and represents a nucleophile selected from —OH, —SH, —NH₂and —NH(C_1-6alkyl);
- each L is the same or different and represents a linker group;
- each Z is the same or different and represents a reactive group attached to a moiety L;
- each n is the same or different and is 1, 2 or 3;
- each m is the same or different and is an integer having a value of from zero to n; and
- each IG is the same or different and represents a moiety which is a C_1-20alkyl group, a C_2-20alkenyl group or a C_2-20alkynyl group, which is unsubstituted or substituted by one or more halogen atom substituents, and in which (a) 0, 1 or 2 carbon atoms are replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, wherein:
- (i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl) and nitro groups; and
- (ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by —C(O)— groups;
  with the proviso that the product contains one solid substrate and at least one functional moiety.

Preferably, R₁represents (a) an electrophilic leaving group Y or (b) —S—F₁or —S-L-F₂.
R₂is preferably a hydrogen atom, Sol, -L-Sol, F₃, Y, -L(F₃)_m(Z)_n-mor IG. More preferably R₂is a hydrogen atom, Sol, F₃, Y, -L(Z)_nor IG and more preferably still a hydrogen or halogen atom, Sol, F₃or a C_1-6alkyl group. Most preferably, R₂is a hydrogen or halogen atom or a C_1-6alkyl group.
When R₃and R₃′ are the same or different and each represents a hydrogen atom, Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG, preferably R₃and R₃′ are the same or different and each represents Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG. More preferably in this embodiment at least one of R₃and R₃′ represents Sol or -L-Sol, most preferably Sol.
Preferably R₃and R₃′ are the same or different and each represents a hydrogen atom, Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG; or R₃and R₃′ together form a group of formula —N(R_33′), wherein R_33′ represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG. More preferably R₃and R₃′ together form a group of formula —N(R_33′).
R_33′ preferably represents a hydrogen atom, Sol, -L-Sol, F₃, -L(F₃)_m(Z)_n-mor IG. More preferably, R_33′ represents a hydrogen atom, Sol, F₃, or IG. More preferably still, R_33′ represents a hydrogen atom, Sol, F₃, or a C_1-6alkyl group. Most preferably R_33′ represents Sol.
E is preferably a halogen atom or a C_1-6alkoxy, thiol, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl), triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl, phenyloxy or nitrophenyloxy group. More preferred groups of formula E are halogen atoms and triflate, tosylate and mesylate groups.
Preferred groups X, X′ and Y in the formula (II) are as defined above.
Nu is preferably a group of formula —OH or —SH. In another embodiment, Nu is preferably a group of formula —OH, —NH₂or —SH, more preferably —NH₂or —SH.
The linker moiety L links together two other moieties in the products of the present invention (i.e., it is at least a divalent moiety). However, in some embodiments certain linker moieties L may link together more than two other moieties (for example, where R₂, R₃, R₃′ or R_33′ represents -L(F₃)_m(Z)_n-mwherein n is 2 or 3), in which case it is to be understood that the third other moiety and any further other moiety each replace a hydrogen atom on the corresponding divalent linker moiety L.
L preferably represents a moiety which is a C_1-20alkylene group, a C_2-20alkenylene group or a C_2-20alkynylene group, which is unsubstituted or substituted by one or more halogen atom substituents, and in which (a) 0, 1 or 2 carbon atoms are replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, wherein:

(i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl) and nitro groups; and
(ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by —C(O)— groups.

More preferably, L represents a moiety which is an unsubstituted C_1-6alkylene group, C_2-6alkenylene group or C_2-6alkynylene group, in which (a) 0 or 1 carbon atom is replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups, wherein said phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or two substituents selected from halogen atoms and C_1-4alkyl and C_1-4alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S— and —C(O)— groups.
Most preferably, L is a moiety which is an unsubstituted C_1-4alkylene group, in which 0 or 1 carbon atom is replaced by an unsubstituted phenylene group.
Z represents a reactive group attached to a group of formula L. The reactive groups Z are selected so as to connect a functional moiety to the cross-linker (i.e., to link the functional moiety to the solid substrate). As those of skill in the art would understand, the nature of the reactive group itself is not important. A very wide range of reactive groups are routinely used in the art to connect functional moieties to cross-linker reagents. Such reactive groups may be capable, for example, of attaching an amine compound, a thiol compound, a carboxyl compound, a hydroxyl compound, a carbonyl compound or a compound containing a reactive hydrogen, to a cross-linker. Those of skill in the art would of course immediately recognise that any such reactive group would be suitable for use in accordance with the present invention. Those of skill in the art would therefore be able to select an appropriate reactive group from common general knowledge, with reference to standard text books such as “Bioconjugate Techniques” (Greg T. Hermanson, Academic Press Inc., 1996), the content of which is herein incorporated by reference in its entirety.
Z is preferably:

(a) a group of formula -LG, —C(O)-LG, —C(S)-LG or —C(NH)-LG wherein LG is an electrophilic leaving group;
(b) a nucleophile Nu′ selected from —OH, —SH, —NH₂, —NH(C_1-6alkyl) and —C(O)NHNH₂groups;
(c) a cyclic moiety Cyc, which is capable of a ring-opening electrophilic reaction with a nucleophile;
(d) a group of formula —S(O₂)(Hal), wherein Hal is a halogen atom;
(e) a group of formula —N═C═O or —N═C═S;
(f) a group of formula —S—S(IG′) wherein IG′ represents a group of formula IG as defined herein;
(g) a group AH, which is a C_6-10aryl group that is substituted by one or more halogen atoms;
(h) a photoreactive group capable of being activated by exposure to ultraviolet light;
(i) a group of formula —C(O)H or —C(O)(C_1-6alkyl);
(j) a maleimido group;
(k) a group of formula —C(O)CHCH₂;
(l) a group of formula —C(O)C(N₂)H or -PhN₂ ⁺, where Ph represents a phenyl group; or
(m) an epoxide group.

Most preferably, Z is selected from:

(a) groups of formula -LG, —C(O)-LG and —C(S)-LG, wherein LG is selected from halogen atoms and —O(C_1-6alkyl), —SH, —S(C_1-6alkyl), triflate, tosylate, mesylate, N-hydroxysuccinimidyl and N-hydroxysulfosuccinimidyl groups;
(b) groups of formula —OH, —SH and —NH₂;
(c) a group of formula

and
(d) a maleimido group.

LG is preferably selected from halogen atoms and —O(IG′), —SH, —S(IG′), —NH₂, NH(IG′), —N(IG′)(IG″), —N₃, triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl and azide groups, wherein IG′ and IG″ are the same or different and each represents a group of formula IG.
Nu′ is preferably selected from —OH, —SH and —NH₂groups.
Cyc is preferably selected from the groups
Hal is preferably a chlorine atom.
AH is preferably a phenyl group that is substituted by at least one fluorine atom.
The photoreactive group is preferably selected from:

(a) a C_6-10aryl group which is substituted by at least one group of formula —N₃and which is optionally further substituted by one or more halogen atoms;
(b) a benzophenone group;
(c) a group of formula —C(O)C(N₂)CF₃; and
(d) a group of formula -PhC(N₂)CF₃, wherein Ph represents a phenyl group.

n is preferably 1 or 2, and most preferably 1.
IG preferably represents a moiety which is an unsubstituted C_1-6alkyl group, C_2-6alkenyl group or C_2-6alkynyl group, in which (a) 0 or 1 carbon atom is replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups, wherein said phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or two substituents selected from halogen atoms and C_1-4alkyl and C_1-4alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S— and —C(O)— groups.
More preferably, IG represents a moiety which is an unsubstituted C_1-6alkyl group, in which (a) 0 or 1 carbon atom is replaced by a group selected from unsubstituted phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups.
Most preferably, IG represents an unsubstituted C_1-6alkyl group.
Preferably the product of formula (II) is a product of formula (IIa)
wherein X, X′, R₁, R₂and R_33′ are all as herein defined.
In the product of formula (II) it is particularly preferred that

- —X and X′ each represent oxygen;
- R₁represents:
- (i) a halogen atom; or
- (ii) a solid substrate carrying a thiol moiety, which solid substrate is attached to the 2-position of the formula (II) via the sulfur atom of said thiol moiety; or
- (iii) —S—F₁;
- R₂represents a hydrogen or halogen atom, a solid substrate, F₃or a C_1-6alkyl group;
- R₃and R₃′ together form a group of formula —N(R_33′), wherein R_33′ represents a hydrogen atom, a solid substrate, F₃or a C_1-6alkyl group; and
- —S₁—F₁and F₃are the same or different and represent a functional moiety selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;
  with the proviso that the product contains one solid substrate and at least one functional moiety.

In a preferred embodiment, the product of formula (II) comprises one functional moiety —S—F₁, F₂or F₃, with that functional moiety preferably being the functional moiety —S—F₁.
In a particularly preferred embodiment, in the product of formula (II) R₃and R₃′ together form a group of formula —N(R_33′) and R_33′ represents a solid substrate.
Preferably either the solid substrate or the at least one functional moiety is attached at the 2-position of the formula (I) or the formula (II).
It will be appreciated that in some embodiments the product of the present invention will contain a maleimide ring. Specifically, this occurs when in the moiety of formula (I) the carbon atoms at positions 1 and 4 are linked together via a group —N(R_33′)—. In this case the present invention further provides a process which comprises effecting ring opening of the maleimide ring in the product of the invention. Ring opening of maleimide rings can be effected by hydrolysis reactions that are known in the art.
Effecting ring opening of the maleimide may be advantageous in certain applications since it can render the functional moieties and/or solid substrate irreversibly bound to the conjugate.
The products of the present invention can be obtained using a reagent which is a compound containing a moiety of formula (III)
wherein:

- X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl; and
- Y is an electrophilic leaving group.

Clearly, the precise details of the process for producing a product of a present invention depend on the nature of the desired product.
Where the moiety of formula (III) already comprises a functional moiety the reagent containing a moiety of formula (III) can be reacted directly with the solid substrate to obtain the product. Accordingly, this process comprises reacting a solid substrate with a compound containing a moiety of formula (III), thus obtaining a product which comprises (a) a solid substrate and (b) a moiety of formula (I) linked thereto, wherein either

(i) R represents an electrophilic leaving group Y, the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I); or
(ii) the solid substrate carries a thiol moiety, R represents a bond to the sulfur atom of said thiol moiety and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I).

Where the desired product has the solid substrate linked to the 1-, 3- or 4-position of the formula (I), the reaction is carried out by reacting a reactive group on the solid substrate with a reactive group linked to the 1-, 3- or 4-position of the formula (III). The solid substrate may be a chemically activated solid substrate such as a solid substrate carrying a linker moiety which itself carries a reactive group. That reactive group can then be attached to the 1-, 3- or 4- position of the formula (I), either directly or via a further linker group, for example a linker group as defined herein. For example, an activated glass substrate, such a glass substrate treated with an alkoxysilane-bearing linker compound which itself carries another reactive group, could be used as the solid substrate. Alternatively, the solid substrate may simply carry surface groups that can react directly with an appropriate reactive group linked to the 1-, 3- or 4-position of the formula (III). For example, a glass substrate could be reacted directly with the reagent containing a moiety of formula (III) where an alkoxysilane-bearing reactive group is linked to the 1-, 3- or 4-position of the formula (III). In a still further alternative, the solid substrate and the moiety of formula (III) may each carry one of an affinity tag and its respective affinity partner, with the reaction then comprising attachment of the affinity tag to its affinity partner.
Where the desired product comprises a solid substrate that carries a thiol moiety and the sulfur atom of said thiol moiety is attached at the 2-position in the formula (I), the reaction carried out involves allowing the thiol group on the solid substrate to attack at the 2-position of the formula (III) and hence displace the electrophilic leaving group Y. The thiol moiety on the solid substrate may inherently be present on the surface in certain embodiments (for example, where the solid substrate is a polymeric substrate comprising polymers having terminal thiol groups). Alternatively, the thiol groups can easily be supplied by activating a solid substrate with a suitable cross-linker reagent. For example, a glass substrate could be activated using a cross-linker comprising both an alkoxysilane end (to react with the glass surface) and a thiol end. In that specific example, the solid substrate would therefore be a glass surface attached to a thiol-containing linker.
Where the moiety of formula (III) does not already carry a functional moiety it can be reacted with the solid substrate first and the functional moiety second, with the functional moiety first and the solid substrate second, or with both the solid substrate and the functional moiety at the same time. Clearly, the specific choice of solid substrate, functional moiety and reagent may determine how best to carry out the reaction. Such considerations are a matter of routine for those skilled in the art.
One exemplary production process comprises:

a) reacting a solid substrate with a compound containing a moiety of formula (III), thus obtaining a product which comprises (a) a solid substrate and (b) a moiety of formula (I) linked thereto, wherein either
- (i) R represents an electrophilic leaving group Y and the solid substrate is linked to the 1-, 3- or 4-position of the formula (I); or
- (ii) the solid substrate carries a thiol moiety and R represents a bond to the sulfur atom of said thiol moiety; and
b) reacting said product with a functional moiety.

A second exemplary production process comprises:

a) reacting a functional moiety with a compound containing a moiety of formula (III); and
b) reacting the compound obtained from step a) with a solid substrate, thus obtaining a product of the present invention.

Clearly, in both the first and second exemplary production processes above the compound containing a moiety of formula (III) must carry at least one reactive group linked to the 1-, 3- or 4-position. This ensures that both the solid substrate and the functional moiety can become accommodated into the conjugate product.
Preferably the compound containing a moiety of formula (III) is a compound of formula (IV)
wherein:

- R_2arepresents a hydrogen atom, Y, Nu, -L(Z′)_nor IG;
- either:
  - R_3aand R_3a′ are the same or different and each represents a hydrogen atom, E, Nu, -L(Z′)_nor IG; or
  - R_3aand R_3a′ together form a group of formula —O— or —N(R_33a′)—, wherein R_33a′ represents a hydrogen atom, Y, Nu, -L(Z′)_nor IG; or
  - R_3aand R_3a′ together form a group of formula —N(R_33a′)—N(R_33a′)—, wherein each R_33a′ is the same or different and represents a hydrogen atom, Y, Nu, -L(Z′)_nor IG;
- Z′ represents Z or a group of formula —Si(O(C_1-6alkyl)(G₁)(G₂), wherein G₁and G₂are the same or different and represent H, C_1-6alkyl or O(C_1-6alkyl); and
- X, X′, Y, Nu, L, Z, n, IG and E are all as herein defined.

Preferably R_3aand R_3a′ are the same or different and each represents a hydrogen atom, E, Nu, -L(Z′)_nor IG; or R_3aand R_3a′ together form a group of formula —N(R_33a′)—, wherein R_33a′ represents a hydrogen atom, Y, Nu, -L(Z′)_nor IG
Groups of formula —Si(O(C_1-6alkyl)(G₁)(G₂) are alkoxysilane groups and are known to be excellent reactive groups for activating substrates such as glass substrates, mica substrates, silica substrates and silicon substrates. Typically, therefore, a group of formula —Si(O(C_1-6alkyl)(G₁)(G₂) is introduced into the compound of formula (IV) when it is intended for the compound of formula (IV) to react with, and attach to, a glass, mica, silica or silicon substrate. Typically, not more than one such group is present on the compound of formula (IV). Preferably, the group of formula —Si(O(C_1-6alkyl)(G₁)(G₂) is a group of formula —Si(O(C_1-4alkyl)(G₁)(G₂) where G₁and G₂are the same or different and represent C_1-4alkyl or O(C_1-4alkyl), more preferably O(C_1-4alkyl). Exemplary groups of formula —Si(O(C_1-6alkyl)(G₁)(G₂) are —Si(OEt)₃, —Si(OEt)₂(OMe), —Si(OEt)(OMe)₂and —Si(OMe)₃, with —Si(OEt)₃and —Si(OMe)₃being particularly preferred.
In a further embodiment, the present invention relates to the use of a compound containing a moiety of formula (III) as a reagent for linking a solid substrate to a functional moiety. This use allows one of skill in the art to obtain a product of the present invention. The use typically involves a process in which the compound containing a moiety of formula (III) is reacted with (a) a solid substrate and (b) a functional moiety, thus obtaining a product of the present invention which comprises a functional moiety. For example, the use may involve carrying out a production process as defined herein for obtaining a product of the present invention. In the use of the present invention, the compound containing a moiety of formula (III) is preferably a compound of formula (IV).
In an alternative embodiment, the use may instead comprise a step in which either a functional moiety or a solid substrate containing an alkene moiety is attached to the moiety of formula (III) by engaging in a photocatalytic [2+2] cycloaddition with the carbon-carbon double bond between the 2- and 3-positions of the moiety of formula (III). This procedure results in a cyclobutane ring moiety containing the 2- and 3-carbon atoms from the moiety of formula (III) and in which the carbon-carbon double bond has been saturated.
In a still further embodiment, when R_3aand R_3a′ together form a group of formula —O— and the solid substrate or the functional moiety carries a nucleophilic group, such as a primary or secondary amine group, this nucleophilic group can link to the moiety of formula (IV) by engaging in a nucleophilic ring-opening and then nucleophilic ring closing reaction. For example, when X and/or X′ are O and R_3aand R_3a′ together form a group of formula —O—, the moiety of formula (IV) is a cyclic acid anhydride. Thus, it can be seen that the nucleophilic group, for example an amine group, can engage in nucleophilic ring-opening and then nucleophilic ring closing with the overall effect that the group —O— is replaced by the nucleophilic group.
It will be appreciated that in some embodiments the use of the present invention will produce a product that contains a maleimide ring. In this case the use of the invention may further comprise effecting ring opening of the maleimide ring. Ring opening of maleimide rings can be effected by hydrolysis reactions that are known in the art. Effecting ring opening of the maleimide may be advantageous in certain applications since it can render the functional moieties and/or solid substrate irreversibly bound to the conjugate.
As would be understood by those of skill in the art, where a reagent (for example, a compound carrying a functional moiety or a cross-linker reagent) carries more than one reactive group, it may be desirable to effect chemical protection of reactive groups that are not intended to take part in the reaction. For example, it may be necessary to protect groups such as hydroxyl, amino and carboxy groups, where these are desired in the final product, to avoid their unwanted participation in the reactions (see, for example, Greene, T. W., “Protecting Groups in Organic Synthesis”, John Wiley and Sons, 1999). Conventional protecting groups may be used in conjunction with standard practice. In some instances deprotection may be used in an intermediate or final step in the production processes and use defined herein.
When the product of the present invention has either the solid substrate or a functional moiety attached at the 2-position of the formula (I) (or the formula (II)), the thiol bond at that position can readily be cleaved to release the solid substrate or functional moiety, respectively. The present invention therefore also provides a process which involves cleaving this thiol bond.
The cleavage can be effected using routine methods for cleaving a thiol bond at an unsaturated carbon centre, specifically using routine methods for cleaving a thiol attached to an electron deficient alkene. Thus, preferably the cleavage is effected by incubating the product with a reagent that is capable of acting as a nucleophile in a Michael reaction. Examples of reagents that are well known to be capable of acting as a nucleophile in a Michael reaction include phosphine compounds, phosphite compounds, thiols, selenols, amines and soft carbon nucleophilic compounds. Phosphine compounds and phosphite compounds both contain a trivalent phosphorous atom. In a phosphine, the phosphorous atom is attached to hydrogen or carbon atoms, while in a phosphite the phosphorous atom is attached to oxygen atoms (it being understood that the carbon atoms and oxygen atoms are themselves further attached to other groups in the respective compounds). Thiols are organic compounds containing a thiol group —SH. Selenols are organic compounds containing an —SeH group. Amines are compounds containing an amine functional group. Soft carbon nucleophiles are compounds which contain a soft nucleophilic carbon centre. Exemplary soft carbon nucleophiles are disclosed in U.S. Pat. No. 5,414,074, the content of which is herein incorporated by reference in its entirety. Those of skill in the art would of course be able to select appropriate reagents that are capable of acting as a nucleophile in a Michael reaction as a matter of routine, for example by routinely selecting a suitable reagent from amongst the exemplified list of classes of reagent herein described.
For the avoidance of doubt, as used herein, the term “reagent that is capable of acting as a nucleophile in a Michael reaction” means a reagent that is capable of reacting with an α,β-unsaturated moiety in a compound, and in particular a moiety of formula (IV)
wherein X is as herein defined. Such reagents are sometimes known as “reagents that are capable of acting as a nucleophile in a conjugate addition reaction”. Clearly, the reagents are not limited to reagents which react through a nucleophilic carbon centre (e.g., soft carbon nucleophiles), but also include reagents which react through a nucleophilic non-carbon centre, such as the exemplary reagents that are described herein.
Presently preferred reagents are phosphine compounds and thiols. A particularly preferred phosphine is tris(2-carboxyethyl)phosphine, which is also known as “TCEP” and is commonly used in the art to reduce disulfide bonds in compounds, for example, in proteins. Tris(2-carboxyethyl)phosphine can also be supplied in the form of a salt, such as its hydrochloride salt. A particularly preferred thiol is glutathione. Further preferred thiols are 2-mercaptoethanol and dithiothreitol (i.e., HSCH₂CH(OH)CH(OH)CH₂SH, commonly known as DTT). A preferred group of reagents is 1,2-ethanedithiol, 2-mercaptoethanol, dithiothreitol, glutathione and tris(2-carboxyethyl)phosphine.
Typically when the product is a product in which a solid substrate carrying a thiol moiety is attached to the 2-position of the formula (I) via the sulfur atom of said thiol moiety, the step of cleaving the thiol bond generates a solid substrate. Typically, when the product is a product in which a group —S—F₁or —S-L-F₂is attached to the 2-position of the formula (I), the step of cleaving the thiol bond generates a functional compound of formula HS—F₁or HS-L-F₂. In a preferred embodiment the product is a product in which a group —S—F₁or —S-L-F₂is attached to the 2-position of the formula (I).
In a further embodiment, the present invention also provides a product of formula (Va) or (Vb). It will be appreciated that these products constitute a conjugate molecule since they comprise a solid substrate and at least one functional moiety. Furthermore, these products comprise a single, rather than a double, carbon-carbon bond between the 2-position and the 3-position. However, unlike products which could be prepared, for example, using conventional maleimide reagents, the compounds of formula (Va) and (Vb) contain at least either a solid substrate and a functional moiety at the 2- and 3-positions or at least two functional moieties at the 2- and 3-positions (as a skilled worker would be aware, a conventional maleimide reagent is capable only of reacting with a single thiol-containing solid substrate or functional moiety, which adds across the double bond between the 2- and 3-positions).
The products of formula (Va) and (Vb) can be prepared using straightforward methods. In a first method, the product of formula (Va) and (Vb) is prepared from a product of the present invention which comprises a moiety of formula (I) and either a solid substrate or a functional moiety attached to the 2-position of the formula (I), but no functional moiety at the 3-position. In that case, the product of formula (Va) and (Vb) can be prepared by an electrophilic addition reaction of a functional moiety F₃or a solid substrate, respectively, across the carbon-carbon double bond between the 2-position and the 3-position of the formula (I). In a second method, the product of formula (Va) and (Vb) is prepared from a product of the present invention which comprises a moiety of formula (I) and either a solid substrate or a functional moiety attached to both the 2- and 3-positions of the formula (I). In that case, the product of formula (Va) and (Vb) can be prepared by carrying out an electrophilic addition reaction to saturate the double bond. This electrophilic addition reaction may involve the addition of a further functional moiety or a solid substrate (for example, a thiol-containing further functional moiety or a thiol-containing solid substrate). Alternatively, it may involve any other reagent routinely used to carry out electrophilic addition reactions at unsaturated carbon-carbon centres. For example, the reagent may be a hydrogen halide, a dihalogen, sulfuric acid, water, an alcohol, H₂S, a mercaptan or a carboxylic acid.
It will be appreciated that the positions at which the groups belonging to a particular reagent add across the double bond between the 2- and 3-positions will depend on the precise structure of the respective reagents and the reaction conditions under which the reaction is carried out. For example, the location of addition might normally be expected to follow from where the most stable cationic intermediate can form, in accordance with Markovnikov's rule. A person skilled in the art would appreciate that if a specific location is desired for addition of a particular group across the double bond, routine selection of reaction conditions and the identity of the various groups on the respective reagents may be capable of achieving such regioselectivity.
As will be clear to those of skill in the art, the methodology of the present invention is broadly applicable to known products, processes and methods which involve conjugation of functional moieties to a solid substrate. Typically, conventional products, processes and methods of this type can straightforwardly be modified by replacing a conventionally known thiol-reactive group on a cross-linking molecule which links together a solid substrate and a functional moiety (such as a maleimide group or an iodoacetyl group) by the moiety of formula (III) of the present invention.
Examples of such routine products, process and methods include array products and associated multiplex assays, detection methods such as ELISA methods, affinity purification techniques including pull-down assays and solid phase peptide or oligonucleotide syntheses.
The present invention thus also provides a plurality of products of the present invention, arranged in an array. As used herein, a “plurality of products arranged in an array” typically means a single, continuous array which has a plurality of areas each of which can be identified as a product of the invention. Thus, typically each of the plurality of products comprises a solid substrate which forms part of a single array solid substrate. However, each of the plurality of products comprises a separate moiety of formula (I).
Preferably, in the plurality of products of the present invention each product comprises a functional moiety selected from an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a peptide, a protein, a cell, a DNA and an RNA.
More preferably in the plurality of products:

(a) each product comprises a different antibody or antibody fragment; or
(b) each product comprises a different antigen; or
(c) each product comprises a different ligand or ligand candidate; or
(d) each product comprises a different peptide; or
(e) each product comprises a different protein; or
(f) each product comprises a different cell; or
(g) each product comprises a different DNA; or
(h) each product comprises a different RNA.

The array system of the present invention can be usefully applied to methods in which a test substance is assayed for its ability to interact with a particular functional moiety (and in particular functional moieties such as those listed under (a) to (h) above). The present invention therefore also provides an assay process wherein a sample comprising a test substance is incubated with an array which is a said plurality of products of the invention. The process then involves detecting whether any of said test substance is bound to any of the plurality of products. Common detection strategies employ labelling of the target substance with, for example, a fluorophore. Other detection strategies include calorimetric detection, chemiluminescent detection and surface plasmon resonance detection.
Assays of this type are often known as “multiplex assays” since they combine a plurality of distinct assays (i.e., an assay run to detect whether a test substance interacts with a single product of the present invention) into a single experimental test. Such assays are also commonly referred to as microassays, although it is emphasised that there is no limitation on the physical scale of the array and assay techniques of the present invention. Numerous array systems involving, for example, the immobilisation of a plurality of proteins, DNAs or RNAs on a single array substrate are well known and commercially available. Arrays involving smaller molecules, such as ligand candidate species of both fundamental research interest or applied therapeutic interest, are also now commonly known in the art, as reviewed for example in Uttamchandani et al. (Current Opinion in Chemical Biology 2005, 9:4-13), the content of which is herein incorporated by reference in its entirety. Those of skill in the art would immediately appreciate that the technology of the present invention could be routinely applied to modify these known systems.
A significant advantage of applying the technology of the present invention to array assays such as those described herein is that the thiol bond formed to the cross-linker can be readily cleaved using the methods described herein. Where the thiol bond to the cross-linker consists of a sulfur atom attached to the solid substrate, this reversibility allows the solid substrate to be completely regenerated after an assay has been carried out. Where the thiol bond to the cross-linker consists of a sulfur atom attached to the functional moiety (e.g., a sulfur atom in a cysteine residue of a protein) the reversibility allows the functional moiety itself to be readily cleaved from the array. This could be advantageous for example in certain detection methodologies or for recovery of valuable functional moieties after an assay is complete.
The present invention also provides a detection process which makes use of a product of the present invention that comprises an antibody or an antigen. The product is incubated with a sample and any material which is not bound to the antibody or antigen is removed. Removal of unbound material can be carried out, for example, using washing techniques routinely employed in the art. Finally, any substance that remains bound to the antibody or antigen is detected, for example, using a chromogenic or fluorogenic label.
In a preferred embodiment of the invention, the detection process is an ELISA assay. Examples of ELISA assays are indirect ELISA, sandwich (or “capture”) ELISA, competitive ELISA and reverse ELISA. All of these assay processes are routine immunoassay processes and would be familiar to those of skill in the art.
Typically, in an ELISA process the detecting step is carried out by adding a conjugate compound comprising an enzyme and an antibody which is capable of specifically binding to the substance bound to the antibody or antigen, and also adding a substrate for the enzyme which generates a detectable signal when it is turned over by the enzyme (such as generating a coloured or fluorescent product). Preferred enzymes include horseradish peroxidase, alkaline phosphatase, β-galactrosidase and glucose oxidase.
The present invention also provides a process for purifying a specific substance from a sample. This process involves incubating a sample with a product of the present invention which comprises a functional moiety that is capable of selectively binding to said substance.
As used herein a “functional moiety that is capable of selectively binding to a substance” means that the functional moiety binds to that substance with a strength that is sufficiently greater than the binding strength to any other substances (in particular any other substances typically found in the relevant substances) that it enables the bound functional moiety-substance entity to be isolated from a sample substantially without also isolating any functional moiety bound to other substances.
Functional moieties suitable for use in the present invention include affinity tags or affinity partners where the specific substance comprises a corresponding affinity partner or affinity tag, enzymes where the specific substance is a substrate for an enzyme and antibodies where the substance is a corresponding antigen.
The process for purifying a specific substance further involves removing any material which is not bound to said functional moiety. Removal of unbound material can be carried out, for example, using washing techniques routinely employed in the art. Finally, the substance is separated from the product using routine elution techniques.
In a preferred embodiment, the specific substance constitutes a bait substance S_band the sample possibly further comprises one or more prey substances S_pcapable of binding to S_b. As those of skill in the art would appreciate, this specific embodiment constitutes a process which is commonly known in the art as a “pull-down assay”. Pull-down assays are routinely used to indirectly purify substances (the “prey”) which by purifying another substance (the “bait”) to which they (the prey) are bound. Preferably, the bait substance is a protein and the prey substance is also a protein.
In another embodiment, the present invention relates to a process for producing a peptide or protein. This process involves attaching an amino acid to a product of the present invention, then attaching one or more further amino acids to said amino acid to produce a peptide or protein moiety linked to a solid substrate. Finally, the peptide or protein moiety is cleaved from the solid substrate.
Techniques for solid phase synthesis or peptides and proteins are very well known in the art. A large number of solid supports activated with a range of reactive groups are commercially available, for example from Sigma Aldrich. Those of skill in the art would thus have no difficulty in applying the methodology of the present invention to carry out a process for producing a peptide or protein. One common reference textbook which discusses the various routine techniques now used in the art to synthesise peptides is “Amino Acid and Peptide Synthesis” (John Jones, Oxford Scientific Publications, 1992), the content of which is herein incorporated by reference in its entirety.
In a particularly preferred embodiment, the product of the present invention used in this process is (a) a product carrying an electrophilic leaving group Y at the 2-position of the formula (I) or (b) a product carrying the amino acid cysteine at the 2-position of the formula (I), wherein the cysteine is attached to the 2-position via its sulfur atom. When the product carries an electrophilic leaving group Y at the 2-position, the amino acid to be attached to the product is typically a cysteine atom, which attaches to the 2-position by displacing the electrophilic leaving group Y. The one or more further amino acids are then attached to the immobilised cysteine residue using routine solid phase peptide synthesis reaction methodology.
As those of skill in the art will immediately appreciate, a significant advantage associated with growing the peptide or protein chain from an immobilised cysteine residue in this manner is that the final peptide or protein can be very easily cleaved from the solid support using the cleavage process described herein.
The present invention further provides a product containing a moiety of formula (VI) having: (a) a functional moiety; and (b) a solid substrate; linked thereto
wherein:

This product typically contains one solid substrate.
Preferably either:

- said functional moiety carries a thiol moiety and is linked to the 2-position of the moiety of formula (VI) via the sulfur atom of said thiol moiety and said solid substrate is attached to either the 5- or 6-position of the moiety of formula (VI); or
- said solid substrate carries a thiol moiety and is linked to the 2-position of the moiety of formula (VI) via the sulfur atom of said thiol moiety and said functional moiety is attached to either the 5- or 6-position of the moiety of formula (VI).

Preferably the solid substrate is attached to either the 5- or 6-position of the moiety of formula (VI).
This product may contain more than one functional moiety, in which case each functional moiety is independently selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.
One process for producing this product involves:

- reacting a compound containing a moiety of formula (III) with a thiol-carrying solid substrate or functional moiety, to obtain an intermediate product in which the electrophilic leaving group Y in the moiety of formula (III) has been replaced by the solid substrate or functional moiety (the linkage being via the said thiol group); and
- reacting this intermediate product with a functional moiety or solid substrate containing an alkene moiety to effect a photocatalytic [2+2] cycloaddition reaction between said alkene moiety and the carbon-carbon double bond between the 2- and 3-positions of the intermediate product.

Preferably the product containing a moiety of formula (VI) having (a) a functional moiety and (b) a solid substrate linked thereto has the formula (VIa)
wherein:

- X, X′, R₁, R₂, R₃and R₃′ are each as defined in relation to the product of formula (II);
- Each of R_alk1, R_alk2, R_alk3and R_alk4is the same or different and is a group of formula R₂as defined in relation to the product of formula (II);
- each functional moiety is the same or different and is selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;
  with the proviso that the product contains one solid substrate and at least one functional moiety.

Preferably one of R_alk1, R_alk2, R_alk3and R_alk4represents Sol or -L-Sol and preferably R₁represents —S—F₁or —S-L-F_2..

EXAMPLES

The following Examples illustrate the scientific principles underlying the present invention. The majority of the Examples are Reference Examples since they do not involve linkage of a functional moiety to a solid substrate. However, linkage of a functional moiety to a secondary moiety, including numerous large functional moieties such as proteins, for a very wide range of functional moieties and linking groups, has been demonstrated, evidencing the broad applicability of the present invention.

A) Preliminary Examples

¹H and ¹³C NMR spectra were recorded at room temperature on a Bruker Avance 500 instrument operating at a frequency of 500 MHz for ¹H and 125 MHz for ¹³C. ¹H NMR spectra were referenced to the CDCl₃(7.26 ppm) signal. ¹³C NMR spectra were referenced to the CDCl₃(77.67 ppm) signal.
Infra-red spectra were run on a PerkinElmer Spectrum 100 FT-IR spectrometer operating in ATR mode with frequencies given in reciprocal centimetres (cm⁻¹). Mass spectra and high resolution mass data were recorded on a VG70-SE mass spectrometer (EI mode and CI mode).
Melting points (m.p.) were taken on a Gallenkamp heating block and are uncorrected. Optical rotation measurements were carried out using a PerkinElmer 343 polarimeter with a cell length of 10 cm.

Abbreviations

Boc Tert-butyloxycarbonyl group.
Cys Cysteine
Mal Maleimide
DMF Dimethylformamide
TCEP (tris(2-carboxyethyl)phosphine)
LC-ESI-MS Liquid chromatography electron spray ionisation mass spectroscopy

Reference Example 1

Preparation of Bromomaleimide (Compound 1)

To maleimide (2.00 g, 0.02 mol) in chloroform (15 mL) was added bromine (1.16 mL, 0.02 mol) in chloroform (15 mL) dropwise. The reaction mixture was refluxed for 2 hours and left to cool to room temperature over 1 hour. Solid yellow precipitate was filtered off and washed with cold chloroform (2×50 mL) to afford white crystals of crude 2,3-dibromosuccinimide (4.09 g, 0.016 mol). The crude succinimide was dissolved in tetrahydrofuran (50 mL) and triethylamine (2.4 mL, 0.017 mol) in tetrahydrofuran (10 mL) was added over 5 minutes at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 48 hours. The solid was filtered off and washed with tetrahydrofuran (50 mL) to afford a pale yellow powder (2.14 g, 0.012 mol) in 59% yield.
¹H NMR (500 MHz, CDCl₃): δ=7.67 (br s, 1H, NH), 6.89 (s, 1H, C═CH); ¹³C NMR (125 MHz, CDCl₃): δ=173.8 (C═O), 170.5 (C═O), 136.9 (—(Br)C═C—), 135.4 (—C═CH—); IR (solid, cm⁻¹): 3235 (s), 1709 (s); MS (CI+) m/z, (%): 178 (⁸¹M+, 32), 176 (⁷⁹M+, 32), 125 (25), 86 (100); Mass calculated for C₄H₃O₂N⁷⁹Br: 175.93472. Found: 175.93493; m.p. 148-151° C.

Reference Example 2

Preparation of N-Methylbromomaleimide (Compound 2)

To N-methyl maleimide (0.5 g, 4.5 mmol) in methanol (22.5 mL) was added bromine (0.52 mL, 10 mmol) dropwise. The reaction mixture was stirred at room temperature for 24 hours. Solvent was removed in vacuo and the reaction mass was dissolved in tetrahydrofuran (20 mL) and triethylamine (0.8 mL, 5.85 mmol) added, then stirred for 24 hours at room temperature. The material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, 7:3) to afford a pale white powder (0.761 g, 4.0 mmol) in 89% yield.
¹H NMR (500 MHz, CDCl₃): δ=6.90 (s, 1H, C═CH), 3.09 (s, 3H, N—CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=168.6 (C═O), 165.4 (C═O), 131.9 (—C═CH—), 131.4 ((Br)C═C—), 24.7 (—N—CH₃); IR (solid, cm⁻¹): 3106 (s), 1708 (s); MS (CI) m/z, (%): 192 (⁸¹M+, 99), 190 (⁷⁹M+, 100); Mass calculated for C₅H₅O₂N⁷⁹Br: 189.95037. Found: 189.95052; m.p: 77-79° C.

Reference Example 3

Preparation of N-Boc-Cys(Mal)-OMe (Compound 4)

To a stirring solution of N-Boc-Cys-OMe (compound 3) (36 mg, 0.153 mmol) and sodium acetate (13 mg, 0.153 mmol) in methanol (3 mL) was added bromomaleimide (30 mg, 0.169 mmol) in methanol (3 mL). After 1 minute solvent was removed in vacuo. The material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford a pale yellow powder (51 mg, 0.153 mmol) in 100% yield.
¹H NMR (500 MHz, CDCl₃): δ=7.63 (s, 1H, NH), 6.27 (s, 1H, C═CH), 5.40 (d, 1H, J=6.8, NH), 4.67 (ddd, 1H, J=6.8, 5.4 and 5.1, —HN—CH—C(O)—), 3.80 (s, 3H, O—CH₃), 3.48 (dd, 1H, J=13.8 and 5.1, —S—CHH—), 3.62 (dd, 1H, J=14.1 and 5.4, —S—CHH—) 1.45 (s, 9H, 3×CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=170.2 (C═O), 168.9 (C═O), 167.6 (C═O), 155.2 (C═O), 155.9 (—C═CH—), 119.7 (—C═CH—), 81.1 ((CH₃)CO—), 53.3 (O—CH₃), 52.7 (CH), 34.0 (CH₂), 28.3 (3×CH₃); IR (solid, cm⁻¹) 3236 (w), 1715 (s); MS (CI+) m/z, (%): 331 (M+H, 5), 275 (20), 231 (100); Mass calculated for [C₁₃H₁₈O₆N₂S]+H, 331.09638. Found: 331.09684; ²⁰α_D: −41.9° (c=1.0, Methanol); m.p. 145-147° C.

Reference Example 4

Preparation of N-Boc-Cys(N-Me-Mal)-OMe (Compound 5)

To a stirring solution of N-Boc-Cys-OMe (32 mg, 0.136 mmol) in methanol (4 mL) was added sodium acetate (82 mg, 0.408 mmol). To this was added N-methyl bromomaleimide (25.8 mg, 0.136 mmol) in methanol (4 mL) over 10 minutes. The reaction turned light yellow. The solvent was removed in vacuo and the residue was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford a pale white powder (39.3 mg, 0.114 mmol) in 84% yield.
¹H NMR (500 MHz, CDCl₃): δ=6.26 (s, 1H, C═CH), 5.36 (d, 1H, J=6.3, NH), 4.66 (m, 1H, —HN—CH—), 3.79 (s, 3H, O—CH₃), 3.46 (dd, 1H, J=5.2 and 5.0, —S—CHH—), 3.35 (dd, 1H, J=13.7 and 5.1, —S—CHH—), 3.00 (s, 3H, —N—CH₃), 1.44 (s, 9H, 3×CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=170.2 (C═O), 169.5 (C═O), 167.9 (C═O), 155.0 (C═O), 149.9 (—C═CH—), 118.7 (—C═CH—), 80.9 ((CH₃)₃CO—), 53.1 (O—CH₃), 52.7 (CH), 33.8 (CH₂), 28.3 (3×CH₃), 24.1 (—N—CH₃); IR (solid, cm⁻¹) 3367.8, 2977.1, 1694.7; MS (ES+) m/z, (%): 367(46), 311 (M, 100); Mass calculated for C₁₄H₂₀N₂O₆NaS: 367.0940. Found: 367.0931; ²⁰α_D: −18.55° (c=1.0, Methanol); m.p. 101-103° C.

Reference Example 5

Preparation of N-Boc-Cys(Succ)-OMe (Compound 6)

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) in methanol (3 mL) was added maleimide (17 mg, 0.169 mmol) in methanol (3 mL). After 1 minute solvent was removed in vacuo. The material was purified by flash chromatography on silica gel (dichloromethane:methanol, gradient elution from 99:1 to 7:3) to afford a colourless oil (51 mg, 0.153 mmol) in 100% yield that was a 1:1 mixture of diastereomers.
¹H NMR (500 MHz, CDCl₃): δ=9.00 (s, 1H, NH), 8.95 (s, 1H, NH), 5.59 (1H, d, J=7.6, NH), 5.41 (d, 1H, J=7.6, NH), 4.65-4.56 (m, 2H, 2×—HN—HC—C(O)—) C═CHH), 3.93 (dd, 1H, J=9.3 and 3.9, CH), 3.86 (dd, 1H, J=9.2 and 4.2, CH), 3.76 (s, 3H, OCH₃), 3.76 (s, 3H, OCH₃), 3.51 (dd, 1H, J=13.8 and 4.6, —CHH—), 3.36 (dd, 1H, J=14.1 and 6.0, —CHH—), 3.19-3.11 (m, 3H, 3×—CHH—), 2.96 (dd, 1H, J=13.1 and 7.1, —CHH—), 2.54-2.02 (m, 2H, —CHH—) 1.43 (s, 18H, 9×CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=177.2 (C═O), 177.1 (C═O), 175.1 (C═O), 175.0 (C═O), 172.0 (C═O), 171.5 (C═O), 155.5 (C═O), 155.3 (C═O), 80.6 (2×—OCCH₃), 53.6 (CH), 52.91 (OCH₃), 52.85 (OCH₃), 50.8 (CH), 40.6 (CH), 40.0 (CH), 37.3 (CH₂), 37.0 (CH₂), 34.6 (CH₂), 34.1 (CH₂), 28.3 (6×CH₃); IR (oil, cm⁻¹) 3233 (w), 2980 (w), 1783 (w), 1709 (s); MS (CI+) m/z, (%): 333 (M+H, 15), 277 (50), 233 (100); Mass calculated for C₁₃H₂₀O₆N₂S: 332.10420. Found: 332.10475;

Reference Example 6

Demonstration that Maleimide does not Displace Thiol from N-Boc-Cys(Mal)-OMe and that Bromomaleimide does not Displace Thiol from N-Boc-Cys(Succ)-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodium acetate (13 mg, 0.153 mmol) in methanol (3 m L) was added bromomaleimide (30 mg, 0.169 mmol) in methanol (3 mL). After 10 minutes maleimide (17 mg, 0.169 mmol) was added. Solvent was removed in vacuo and ¹H NMR analysis showed only compound 4 and unreacted maleimide.
To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodium acetate (13 mg, 0.153 mmol) in methanol (3 m L) was added maleimide (17 mg, 0.169 mmol) in methanol (3 mL). After 10 minutes bromomaleimide (30 mg, 0.169 mmol) was added. Solvent was removed in vacuo and ¹H NMR analysis showed only compound 6 and unreacted bromomaleimide.

Reference Example 7

Competition Reaction Between Bromomaleimide and Maleimide for N-Boc-Cys-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and sodium acetate (13 mg, 0.153 mmol) in methanol (3 m L) was added a mixture of bromomaleimide (30 mg, 0.169 mmol) and maleimide (17 mg, 0.169 mg) in methanol (3 mL). After 1 minute solvent was removed in vacuo. The material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford a pale yellow powder 4 (36 mg, 0.108 mmol) in 70% yield and a colourless oil 6 (15 mg, 0.045 mmol) in 30% yield.
Reference Example 5 demonstrated that, once attached to a succinimide or maleimide moiety, the cysteine moiety is not capable of detaching in the presence of these reagents. Reference Example 6 therefore demonstrates that the cysteine reagent reacts more rapidly with bromomaleimide than with maleimide (i.e., the reaction kinetics are more favourable for formation of compound 4).

Reference Example 8

Demonstration of Selectivity of the Bromomaleimide Reagent for N-Boc-Cys-OMe Compared to Propylamine

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.153 mmol) and propylamine (10 μL, 0.153 mmol) in methanol (3 m L) was added bromomaleimide (30 mg, 0.169 mmol) in methanol (3 mL). After 1 minute solvent was removed in vacuo. The material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford a pale yellow powder (51 mg, 0.153 mmol) in 100%. Data matched those obtained above for N-Boc-Cys(Mal)-OMe 4.

Reference Example 9

Cleavage of N-Boc-Cys(Mal)-OMe to Regenerate N-Boc-Cys-OMe

To a stirring solution of 4 (50 mg, 0.151 mmol) in dimethylformamide (2 mL) was added 20 mL of an aqueous buffer (150 mM NaCl, 100 mM NaH₂PO₄, pH 8.0). Tris(2-carboxyethyl)phosphine (430 mg 1.51 mmol) in 20 mL of an aqueous buffer (150 mM NaCl, 100 mM NaH₂PO₄, pH 8.0) was added to the solution. After 5 minutes the aqueous solution was extracted with ethyl acetate (3×25 mL), washed with saturated lithium chloride solution (5×25 mL), water (25 mL) and brine (25 mL) and dried over MgSO₄. Solvent was removed in vacuo to afford a colourless oil (34.5 mg, 0.148 mmol) in 98% yield. ¹H and ¹³C NMR of this oil showed it to be the commercially available N-Boc-cysteine methyl ester 3.

Reference Example 10

Reaction of 2,3-Dibromomaleimide with Somatostatin

Somatostatin is peptide hormone which is known to exist in a form in which two cysteine residues within the molecule are attached via a disulfide bridge.
1 mg of lyophilised somatostatin (Sigma-Aldrich) was resolubilised in 2 ml of 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN, 2.5 DMF. 500 μl were transferred to a Eppendorf reaction tube and diluted in the same buffer to a final concentration of 0.25 mg/ml (152.6 μM). 2.0 equivalents of TCEP (100× stock solution in 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN) were added and the reaction incubated for 1 hour at ambient temperature. After reduction of the disulfide bond 1.4 equivalents of 2,3-dibromomaleimide (Sigma-Aldrich, 100× stock solution in 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN, 2.5% DMF) were added, the solution gently mixed and incubated for a further 12 h at 4° C.
Maleimide-bridged somatostatin was detected by LC-ESI-MS (ES⁺/ES⁻). Controls included untreated peptide and somatostatin treated with 2,3-dibromomaleimide or TCEP only. Complete reduction was detected by the reaction of TCEP-treated peptide with maleimide (Sigma-Aldrich, 100× stock solution in 50 mM NaHPO₄ ⁻, pH 6.2, 40% MeCN, 2.5% DMF).

Experimental Data:

Untreated somatostatin: [ES+]1638.04 (m/z 1), 819.82 (m/z 2), 546.95 (m/z 3).
Maleimide-bridged somatostatin: [ES+]1734.14 Da (m/z 1), 867.40 Da (m/z 2), 578.73 (m/z 3).

Reference Example 11

Expression of GrB2-SH2 Domain L111C

The protein GrB2-SH2 domain L111C was used as a model protein. This model protein contains a single cysteine residue.
LC-MS was performed on a Waters Acquity uPLC connected to Waters Acquity Single Quad Detector (SQD). Column: Acquity uPLC BEH C18 1.7 μm 2.1×50 mm Wavelength: 254 nm Mobile Phase: 95:5 Water (0.1% Formic Acid):MeCN (0.1% Formic Acid) Gradient over 4 min (to 5:95 Water (0.1% Formic Acid):MeCN (0.1% Formic Acid). Flow Rate: 0.6 mL/min MS Mode: ES+. Scan Range: m/z=85-2000. Scan time: 0.25 sec. Data obtained in continuum mode. The electrospray source of the MS was operated with a capillary voltage of 3.5 kV and a cone voltage of 50 V. Nitrogen was used as the nebulizer and desolvation gas at a total flow of 600 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt 1 algorithm preinstalled on MassLynx software.
The model protein was over-expressed in E. coli, and the hexa-His-tagged protein purified using both Ni-affinity chromatography and size-exclusion chromatography via standard techniques. Analysis using LC-MS showed a single protein species of mass 14169 which corresponds to the desired protein.
To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 10 mins, after which the mixture was analysed by LC-MS. Analysis showed that a single reaction had occurred yielding a single product with a mass of 14366 showing that C111 was available for functionalisation.

Reference Example 12

Reaction of GrB2-SH2 Domain L111C with Bromomaleimide

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponds to the desired protein.
The mixture was treated with Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 10 mins after which the mixture was analysed by LC-MS. Analysis showed that no reaction with Ellman's reagent was evident, highlighting that bromomaleimide functionalisation had occurred at C111.

Reference Example 13

Reaction of GrB2-SH2 Domain L111C with N-methylbromomaleimide

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponds to the desired protein.
The mixture was treated with Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 00° C. for 10 mins after which the mixture was analysed by LC-MS. Analysis showed that no reaction with Ellman's reagent was evident, highlighting that N-methylbromomaleimide functionalisation had occurred at C111.

Reference Example 14

Phosphine-Mediated Reductive Cleavage of GrB2-SH2 Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponds to protein/bromomaleimide adduct.
The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding GrB2-SH2 domain L111C (mass=14168) in 85% yield. The remaining material was unreacted protein/bromomaleimide adduct.

Reference Example 15

Phosphine-Mediated Reductive Cleavage of GrB2-SH2

Domain L111C/N-methylbromomaleimide Adduct
To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponds to protein/N-methylbromomaleimide adduct.
The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-methylbromomaleimide adduct had been cleanly cleaved yielding GrB2-SH2 domain L111C (mass=14168) in 85% yield. The remaining material was unreacted protein/N-methylbromomaleimide adduct.

Reference Example 16

Synthesis of GrB2-SH2 Domain L111C/bromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponds to protein/bromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide/2-mercaptoethanol adduct had been formed (mass=14339) in 55% yield. The remaining material was GrB2-SH2 domain L111C.

Reference Example 17

Synthesis of GrB2-SH2 Domain L111C/N-Methylbromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponds to protein/N-methylbromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-methylbromomaleimide/2-mercaptoethanol adduct had been formed (mass=14356) in 61% yield. The remaining material was GrB2-SH2 domain L111C.

Reference Example 18

Synthesis of GrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponds to protein/dibromomaleimide adduct.

Reference Example 19

Synthesis of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponds to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14572).

Reference Example 20

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct at Physiological Relevant Glutathione Concentration (5 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponds to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14572).
The mixture was treated with glutathione (5 μL, 100 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that GrB2-SH2 domain L111C was the only protein species present (mass=14173).

B) Further Examples

General Procedures

¹H and ¹³C NMR spectra were recorded at room temperature on a Bruker Avance 500 instrument operating at a frequency of 500 MHz for ¹H and 125 MHz for ¹³C. ¹H NMR spectra were referenced to the CDCl₃(7.26 ppm) signal. ¹³C NMR spectra were referenced to the CDCl₃(77.67 ppm) signal. Infra-red spectra were run on a PerkinElmer Spectrum 100 FT-IR spectrometer operating in ATR mode with frequencies given in reciprocal centimeters (cm⁻¹). Mass spectra and high resolution mass data for small molecules were recorded on a VG70-SE mass spectrometer (EI mode and CI mode). Melting points were taken on a Gallenkamp heating block and are uncorrected. 3,4-Dibromomaleimide, lyophilized somatostatin, PEG5000, TCEP and benzeneselenol were purchased from Sigma-Aldrich and used without further purification.

Protein and Peptide Mass Spectrometry

LC-MS was performed on protein samples using a Waters Acquity uPLC connected to Waters Acquity Single Quad Detector (SQD). Column: Acquity uPLC BEH C18 1.7 μm 2.1×50 mm Wavelength: 254 nm Mobile Phase: 95:5 Water (0.1% Formic Acid):MeCN (0.1% Formic Acid) Gradient over 4 min (to 5:95 Water (0.1% Formic Acid):MeCN (0.1% Formic Acid). Flow Rate: 0.6 mL/min MS Mode: ES+. Scan Range: m/z=85-2000. Scan time: 0.25 sec. Data obtained in continuum mode. The electrospray source of the MS was operated with a capillary voltage of 3.5 kV and a cone voltage of 50 V. Nitrogen was used as the nebulizer and desolvation gas at a total flow of 600 L/h. Total mass spectra were reconstructed from the ion series using the MaxEnt 1 algorithm pre-installed on MassLynx software. MALDI-TOF analysis was performed on a MALDI micro MX (Micromass). Data was obtained in reflectron positive ion mode with a source voltage of 12 kV and a reflectron voltage of 5 kV at a laser wavelength of 337 nm. Samples were prepared as outlined below and those containing peptide were dialysed for 24 h in deionised H₂O. The peptide and its derivates (0.1-0.3 mg/ml) were spotted onto a MALDI plate in 2 μl sinapinic acid (10 mg/ml) after pre-spotting of trifluoroacetic acid (10 mg/ml). ACTH (10 ng/ml) was used for mass calibration.

Reference Example 21

Preparation of Bromomaleimide

To maleimide (2.00 g, 0.02 mol) in chloroform (15 mL) was added bromine (1.16 mL, 0.02 mol) dropwise in chloroform (15 mL). The reaction mixture was refluxed for 2 hours and left to cool to room temperature over 1 hour. Solid yellow precipitate was filtered off and washed with cold chloroform (2×50 mL) to afford off white crystals of crude 2,3-dibromosuccinimide (4.09 g, 0.016 mol). The crude succinimide was dissolved in tetrahydrofuran (50 mL) and triethylamine (2.4 mL, 0.017 mol) in tetrahydrofuran (10 mL) was added over 5 minutes at 0° C. The reaction mixture was allowed to warm to room temperature and stirred for 48 hours. The solid was filtered off and washed with tetrahydrofuran (50 mL). Purification by flash chromatography (5% ethyl acetate in petroleum ether) afforded the desired compound as a pale yellow powder (2.14 g, 0.012 mol) in 59% yield. δ_H(500 MHz, CDCl₃) 7.67 (br s, 1H, NH), 6.89 (s, 1H, H-3); δ_C(125 MHz, CDCl₃) 173.8 (C═O), 170.5 (C═O), 136.9 (C2), 135.4 (C3); IR (solid, cm⁻¹) 3235 (s), 1709 (s); MS (CI+) m/z, (relative intensity): 178 ([⁸¹M+H], 32), 176 ([⁷⁹M+H], 32), 125 (25), 86 (100); Mass calcd for [C₄H₂O₂N⁷⁹Br]+H, 175.9347 Found 175.9349 (CI+); m.p. 148-151° C.; UV (Acetonitrile) ε₂₄₂=13800 and ε₂₇₆=1700 cm⁻¹M⁻¹d³.

Reference Example 22

Preparation of N-Methylbromomaleimide

To N-methylmaleimide (0.5 g, 4.5 mmol) in methanol (10 mL) was added bromine (232 μL, 4.5 mmol) dropwise in methanol (5 mL). The reaction mixture was stirred at room temperature for 12 hours. The solvent was removed in vacuo and dissolved in tetrahydrofuran (20 mL). Triethylamine (815 μL, 5.9 mmol) in tetrahydrofuran (5 mL) was added over 5 minutes, whereupon a precipitate formed. The reaction mixture was stirred for 24 hours. The solid was filtered off and washed with tetrahydrofuran (50 mL). Purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded the desired compound as a pale yellow powder (563 mg, 2.96 mmol) in 66% yield. δ_H(500 MHz, CDCl₃) 6.90 (s, 1H, H-3), 3.09 (s, 3H, H₃-6); δ_C(125 MHz, CDCl₃) 168.6 (C═O), 165.4 (C═O), 131.9 (C3), 131.4 (C2), 24.7 (C6); IR (solid, cm⁻¹) 3106 (s), 1708 (s); MS (CI+) m/z, (relative intensity): 192 ([⁸¹M+H], 99), 190 ([⁷⁹M+H], 100); Exact mass calcd for [C₅H₄O₂N⁷⁹Br]+H requires 189.9504 Found 189.9505 (CI+); m.p: 77-79° C.; UV (Acetonitrile) ε₂₀₉=17100, ε₂₃₈=13200, ε₂₉₉=290 cm⁻¹M⁻¹d³.

Reference Example 23

Preparation of N-Phenylbromomaleimide

To N-phenylmaleimide (2 g, 11.50 mmol) in chloroform (15 mL) was added bromine (0.65 mL, 12.70 mmol) dropwise in chloroform (5 mL). The reaction mixture was refluxed for 1 hour, and then allowed to cool to room temperature. The precipitate was filtered off and washed with chloroform (50 mL). This solid (2.70 g, 8.10 mmol) was dissolved in tetrahydrofuran (50 mL) and to this was added dropwise dropwise a solution of triethylamine (1.2 mL, 8.9 mmol) in tetrahydrofuran (10 mL) at 0° C. and the mixture was stirred for 2 hours. The mixture was allowed to warm to room temperature and solvent removed in vacuo. The residue was dissolved in ethyl acetate and washed with H₂O (50 mL) brine (50 mL) and dried (Na₂SO₄). Solvent was removed in vacuo to afford the desired compound as a pale yellow solid (1.80 g, 7.14 mmol) in 62% yield. Data matched literature: Sahoo et al., Synthesis, 2003, 346

Reference Example 24

Preparation of N-Phenyldibromomaleimide

Aniline (72 μL, 0.788 mmol) was added to a solution of dibromomaleic anhydride (200 mg, 0.788 mmol) in AcOH (10 mL). The mixture was stirred for 3 h at RT and at 130° C. for 90 mins. After cooling, the mixture was concentrated to dryness and traces of AcOH removed by azeotrope with toluene. The tan residue was purified using silica flash chromatography (5% EtOAc/95% petroleum ether) to yield the desired compound as a pale yellow solid (166 mg, 60%). δ_H(600 MHz, CDCl₃) 7.48 (m, 2H, ArH), 7.41 (tt, 1H, J=7.4 and 1.1 Hz, ArH), 7.33 (m, 2H, ArH); δ_C(150 MHz, CDCl₃) 163.0, 131.0, 130.0, 129.5, 128.8, 126.2.

Reference Example 25

Preparation of 3,4-Diiodo-pyrrole-2,5-dione

To dibromomaleimide (500.0 mg, 2.0 mmol) in acetic acid (50 ml) was added sodium iodide (886.5 mg, 5.9 mmol). The reaction mixture was heated to 120° C. and refluxed for 2 h. The reaction was allowed to cool down to RT, H₂O (50 ml) was added and kept at 4° C. for 15 h. The yellow precipitate was filtered off and air dried to afford the desired compound as an orange crystalline powder (415 mg, 60%). ¹H NMR (500 MHz, MeOD): no signals; ¹³C NMR (125 MHz, MeOD): δ=169.3 (C), 119.5 (C); IR (solid, cm⁻¹): 3244 (s), 2944 (m), 2833 (m); MS (EI) m/z, (%): 349 (M, 83), 179 (100); Mass calc. for C₄H₁₂O₂N, 348.80912. Found: 348.81026. m.p. 238-241° C. (Literature: 254-255° C.).

Reference Example 26

Preparation of 3,4-Bis-(2-hydroxy-ethylsulfanyl)-pyrrole-2,5-dione

To 2-mercaptoethanol (683.8 μl, 9.8 mmol) in buffer (100 ml, 150 mM NaCl, 100 mM sodium phosphate, pH 8.0, 5.0 DMF) was added di-bromomaleimide (1 g, 3.9 mmol) in DMF (2.5 ml, final concentration DMF 7.5%). The reaction was stirred for 30 min at RT and lithium chloride (20 g) was added. The aqueous reaction mixture was extracted with ethyl acetate (7×150 ml). The organic layers were combined, the solvent removed in vacuo and the residual material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 1:1 to 1:9). Fractions containing the product were collected and the solvent were removed in vacuo. The still impure product was purified by flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-10.0% methanol) to afford the desired compound as a yellow solid (518 mg, 53%). λ_max(50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/318 nm (ε/dm³mol⁻¹cm⁻¹1855); ¹H NMR (500 MHz, MeOD): δ=3.74 (t, 4H, J=6.4, 2×HO—CH₂), 3.41 (t, 4H, J=6.3, 2×S—CH₂) ¹³C NMR (125 MHz, MeOD): δ=168.5 (C), 137.2 (C), 62.3 (CH₂), 34.4 (CH₂); IR (solid, cm⁻¹): 3344 (s), 2500 (m), 2078 (w); MS (EI) m/z, (%): 250 (M, 43), 232 (100), 161 (37); Mass calc. for C₈H₁₁O₄NS₂: 250.02077. Found: 250.02126; m.p. 46-50° C.

Reference Example 27

Preparation of 3,4-Bis-phenylsulfanyl-pyrrole-2,5-dione

To dibromomaleimide (80.0 mg, 0.3 mmol) and sodium hydrogencarbonate (130.2 mg, 1.6 mmol) in methanol (6 ml) was slowly added benzenethiol (66.6 μl, 0.7 mmol) in methanol (1 ml). The reaction was stirred for 15 min at RT. The solvent was removed in vacuo and the residual material was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 9:1 to 7:3) to afford the desired product as bright yellow crystals (73 mg, 75%). λ_max(50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/412 nm (ε/dm³mol⁻¹cm⁻¹2245); ¹H NMR (500 MHz, MeOD): δ=7.27-7.22 (m, 6H, Ar—H), 7.16-7.14 (m, 4H, Ar—H); ¹³C NMR (125 MHz, MeOD): δ=169.3 (C), 137.6 (C), 135.4 (C), 132.4 (CH), 130.1 (CH), 129.1 (CH); IR (solid, cm⁻¹): 3285 (m), 3059 (w), 2924 (w), 1774 (m), 1715 (s); MS (CI) m/z, (%): 314 (M+H, 100), 206 (13), 111 (12); Mass calc. for C₁₆H₁₁O₂NS₂[+H]: 314.0231. Found: 314.0309; m.p. 102-104° C. (Literature: 123-126° C.).

Reference Example 28

Preparation of 3,4-Bis-(pyridine-2-ylsulfanyl)-pyrrole-2,5-dione

To dibromomaleimide (300.0 mg, 1.2 mmol) and sodium acetate (480.0 mg, 5.9 mmol) in methanol (15 ml) was slowly added 1H-pyridine-2-thione (275.8 mg, 2.5 mmol) in methanol (4 ml). The reaction was stirred for 15 min at RT. The solvent was removed in vacuo and the residual material was purified by flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-3.0%) to afford the desired product as a dark yellow powder (190 mg, 51%). λ_max(50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)/395 nm (ε/dm³mol⁻¹cm⁻¹3508); ¹H NMR (500 MHz, MeOD): δ=8.37 (d, 2H, J=3.8, 2×N—CH), 7.70 (t, 2H, J=6.9, 2×C—CH—CH), 7.38 (d, 2H, J=7.9, 2×C—CH), 7.26 (t, 2H, J=6.5, 2×N—CH—CH); ¹³C NMR (125 MHz, MeOD): δ=168.5 (C), 154.7 (C), 150.9 (CH), 140.0 (C), 139.0 (CH), 126.8 (CH), 123.7 (CH); IR (solid, cm⁻¹): 2926 (m), 2734 (w), 1771 (w), 1726 (s), 1619 (m); MS (CI) m/z, (%): 316 (M+H, 5), 152 (10), 126 (34), 112 (100); Mass calc. for C₁₄H₉O₂N₃S₂[+H]: 316.0214. Found: 316.0223; m.p. 70-72° C.

Reference Example 29

Preparation of N-PEG300 dibromomaleimide

The reaction was carried out under strictly dry conditions. To triphenylphosphine (193.9 mg, 0.7 mmol) in THF (5 mL) was added drop-wise diisopropyl azodicarboxylate (145.6 μl, 0.7 mmol) at −78° C. The reaction was stirred for 5 min and PEG300 (200.0 mg, 0.6 mmol) in THF (4 mL) was added drop-wise. The reaction was stirred for 5 mM and neopentyl alcohol (45.8 mg, 0.5 mmol) in THF (1 ml) was added. The reaction was stirred for 5 min and 3,4-dibromomaleimide (189.4 mg, 0.7 mmol) in THF (2 ml) was added. The reaction was stirred for 10 mM, the cold bath removed and stirred for 20 h at ambient temperature. The solvent was removed in vacuo and the residual material was purified by flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-5.0% methanol). Fractions containing the product were collected and the solvent was removed in vacuo. The still impure product was purified by flash chromatography on silica gel (petroleum ether:ethyl acetate, gradient elution from 7:3 to 2:8) to afford the desired compound as a yellow oil (137 mg, 40%) with 97.5% purity. ¹H NMR (500 MHz, CDCl₃): δ=3.76 (t, 2H, J=5.6, N—CH₂), 3.64-3.52 (m, 24H, 12×CH₂—O), 3.49 (t, 2H, J=4.4, N—CH₂—CH₂), 3.32 (s, 3H, O—CH₃); ¹³C NMR (125 MHz, CDCl₃): δ=163.8 (2×C), 129.5 (2×C), 72.0 (CH₂), 70.7-70.5 (9×CH₂), 70.1 (2×CH₂), 67.5 (CH₂), 59.1 (CH₃), 39.0 (CH₂); IR (solid, cm⁻¹): 3496 (w), 2869 (m), 1786 (m), 1720 (s), 1594 (m); MS (CI) m/z, (%): 580 (⁸¹M+H, 12), 578 (^81,79M+H, 23), 576 (⁷⁹M+H, 12), 279 (100), 84 (61); Mass calc. for C₁₉H₃₁ ⁷⁹Br₂O₉N[+H]: 576.0444. Found: 576.0437.

Reference Example 30

Preparation of N-PEG5000 dibromomaleimide

The reaction was carried out under strictly dry conditions. To triphenylphosphine (154.6 mg, 0.6 mmol) in a mixture of THF (8 mL) and DCM (3 mL) was added drop-wise diisopropyl azodicarboxylate (116.0 μl, 0.6 mmol) at −78° C. The reaction was stirred for 5 min and PEG5000 (2950.0 mg, 0.6 mmol) in dichloromethane (7 mL) was added drop-wise. The reaction was stirred for 5 min and neopentyl alcohol (26.5 mg, 0.3 mmol) in a mixture of THF (1 ml) and DCM (1 ml) was added. The reaction was stirred for 5 min and 3,4-dibromomaleimide (150.0 mg, 0.6 mmol) in THF (2 ml) was added. The reaction was stirred for 5 min, the cold bath removed and stirred for 20 h at ambient temperature. The solvent was removed in vacuo and the residual material was purified by flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-5.0% methanol). Fractions containing the product were collected and the solvent was removed in vacuo. The still impure product was purified by very slow flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-6.0% methanol) to afford desired compound as a pale green crystalline powder (417 mg, 13%). ¹H NMR (500 MHz, CDCl₃): δ=3.58 (s, 4×n H, CH₂); ¹³C NMR (125 MHz, CDCl₃): δ=163.8 (C), 129.5 (C), 70.6 (CH₂); IR (solid, cm⁻¹): 3517 (w), 2872 (s), 1977 (w), 1727 (m), 1641 (w); m.p. 51-55° C.

Reference Example 31

Preparation of N-PEG5000 dithiophenolmaleimide

The reaction was carried out under strictly dry conditions. To triphenylphosphine (167.7 mg, 0.6 mmol) in a mixture of THF (8 ml) and DCM (3 ml) was added drop-wise diisopropyl azodicarboxylate (125.9 μl, 0.6 mmol) at −78° C. The reaction was stirred for 5 min and PEG5000 (1600.0 mg, 0.3 mmol) in DCM (7 ml) was added drop-wise. The reaction was stirred for 5 min and neopentyl alcohol (56.3 mg, 0.6 mmol) in a mixture of THF (1 ml) and DCM (1 ml) was added. The reaction was stirred for 5 min and 3,4-dithiophenolmaleimide (200.0 mg, 0.6 mmol) in THF (3 ml) was added. The reaction was stirred for 5 min, the cold bath removed and stirred for 20 h at ambient temperature. The solvent was removed in vacuo and the residual material was purified by flash chromatography on silica gel (methanol:dichloromethane, gradient elution from 0.5-10.0% methanol). Fractions containing the product were collected and the solvent was removed in vacuo. The still impure product was purified by flash chromatography on TLC grade silica gel (methanol:dichloromethane, gradient elution from 0.0-10.0% methanol) to afford the desired compound as a bright yellow crystalline powder (1.24 g, 73%). ¹H NMR (500 MHz, CDCl₃): δ=7.26 (dd, H, J=7.7, J=4.5, CH), 7.23 (dd, 2H, J=8.4, J=6.6, CH), 7.19 (dd, 2H, J=8.4, J=6.8, CH), 3.63 (s, 4×n H, CH₂); ¹³C NMR (125 MHz, CDCl₃): δ=166.7 (C), 135.7 (C), 131.9 (CH), 129.1 (C), 129.0 (CH), 128.4 (CH), 70.6 (CH₂); IR (solid, cm⁻¹): 3498 (w), 2881 (s), 1959 (w), 1711 (m); m.p. 57-59° C.

Reference Example 32

Preparation of 2,3-dibromo-maleic anhydride

Under an inert atmosphere, a solution of maleic anhydride (1.50 g, 15.3 mmol, 1 eq), aluminium trichloride (300 mg, 0.21 mmol, cat.) and bromine (4.95 g, 30.6 mmol, 2 eq) was heated at 160° C. in a sealed ampule (note—blast shield) for 16 h. Upon cooling to 21° C. the reaction mixture was stirred for a further 24 h and carefully opened to air. EtOAc was added and the solid filtered off and repeatedly washed with further EtOAc. The filtrate was finally concentrated in vacuo to give the title compound was a yellow solid which was used without further purification (3.05 g, 11.9 mmol, 78% yield). m.p 107-110° C.; ¹³C NMR (150 MHz, CD₃OD) δ 163.33 (s), 125.28 (s); IR (MeOH) 1769, 1706, 1590 cm⁻¹; HRMS (CI) calcd for C₄O₃Br₂[M]⁺253.82087, 253.82082 observed.

Reference Example 33

Preparation of tert-Butyl N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate

A solution of di-tert-butyl-dicarbonate (1.10 g, 5.00 mmol, 1 eq) in CH₂Cl₂(5 mL) was added dropwise to a solution of 2-[2-(2-aminoethoxy)ethoxy]ethanamine (7.32 mL, 50.0 mmol, 10 eq) in CH₂Cl₂(15 mL). The resulting reaction mixture was stirred at 21° C. for 24 h. The CH₂Cl₂was then removed in vacuo to leave a colourless residue. Addition of EtOAc (125 mL) caused formation of a white precipitate, which was washed with a saturated solution of Na₂CO₃(3×50 mL), dried over MgSO₄, and concentrated in vacuo. Further purification by column chromatography (8:2 CH₂Cl₂/MeOH) furnished the desired monoprotected amine as a colourless oil (0.69 g, 2.80 mmol, 56% yield). ¹H NMR (500 MHz, CDCl₃) δ 5.27 (bs, 1H, NH), 3.54-3.52 (m, 4H, OCH₂), 3.47-3.42 (m, 4H, OCH₂), 3.23-3.22 (m, 2H, NCH₂), 2.80 (t, J=5.0, 2H, NCH₂), 2.05 (bs, 2H, NH), 1.35 (s, 9H, CH₃); ¹³C NMR (125 MHz, CDCl₃) δ 156.08 (s), 79.09 (s), 73.19 (t), 70.21 (t), 70.16 (t), 41.59 (t), 40.32 (t), 28.40 (q), * 1 t missing; IR (neat) 3344, 2869, 1692 cm⁻¹; HRMS (CI) calcd for C₁₁H₂₅N₂O₄[M+H]⁺ 249.18143, observed 249.18251.

Reference Example 34

Preparation of tert-Butyl-N-(2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethyl)carbamate

A solution of biotin (0.59 g, 2.42 mmol, 1.5 eq), HBTU (0.79 g, 2.10 mmol, 1.3 eq) and DIEA (0.45 mL, 2.60 mmol, 1.6 eq) in DMF (15 mL) was stirred for 20 min at 21° C. before being added dropwise to a solution of tert-butyl-N-(2-(2-(2-aminoethoxy)ethoxy)ethyl)carbamate (400 mg, 1.61 mmol, 1 eq) in DMF (10 mL). The reaction mixture was stirred for 2 h at 21° C., after which the DMF was removed in vacuo to give a yellow residue. The crude product was purified by column chromatography (gradient 2-10% MeOH/CH₂Cl₂) to yield the desired compound as a white solid (0.61 g, 1.29 mmol, 80% yield). m.p. 106-108° C.; [α]_D ^20.0+23.0 (c 0.6, CH₂Cl₂); ¹H NMR (500 MHz, CDCl₃) δ 4.55 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 4.36 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 3.62 (bs, 6H, OCH₂), 3.59-3.55 (m, 2H, OCH₂), 3.46 (m, 2H, NCH₂), 3.31 (m, 2H, NCH₂), 3.17 (dt, 3.0, 5.0 Hz, 1H, SCH), 2.92 (dd, J=5.0, 13.0 Hz, 1H, SCHH), 2.79 (d, J=13.0 Hz, 1H, SCHH), 2.27 (t, J=7.0 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.71 (m, 4H, NHC(O)CH₂CH₂CH₂CH₂), 1.47 (br, 11H, C(CH₃)₃& NHC(O)CH₂CH₂CH₂CH₂); ¹³C NMR (125 MHz, CDCl₃) δ 173.69 (s), 163.92 (s), 155.99 (s), 79.14 (s), 70.03 (t), 69.69 (br t), 61.58 (d), 60.06 (d), 55.19 (d), 40.16 (t), 39.96 (t), 38.91 (t), 35.44 (t), 28.09 (q), 27.80 (t), 27.67 (t), 25.23 (t), * 2 t absent; IR (neat) 3307, 2933, 1691 cm⁻¹; HRMS (ES) calcd for C₂₁H₃₈N₄O₆NaS [M+Na]⁺497.2410, observed 497.2423.

Reference Example 35

Preparation of 2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium; 2,2,2-trifluoroacetate

A solution of tert-butyl N-(2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethyl)carbamate (0.61 g, 1.29 mmol) in CH₂Cl₂(5 mL) and TFA (5 mL) was stirred at 21° C. for 24 h. Toluene was then added (×2) and the solvent removed in vacuo to yield the desired compound as an oil (0.63 g, 1.29 mmol, 100% yield). [α]_D ^20.0+41.0 (c 0.49, MeOH); ¹H NMR (400 MHz, CD₃OD) δ 4.53 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 4.33 (dd, J=5.0, 7.5 Hz, 1H, NHC(O)NHCH), 3.71 (t, J=5.0 Hz, 2H, OCH₂CH₂NH₃), 3.65 (br, 4H, OCH₂), 3.57 (t, J=5.0 Hz, 2H, OCH₂), 3.38 (t, J=5.0 Hz, 2H, OCH₂), 3.22 (dt, J=5.0, 8.5 Hz, 1H, SCH), 3.13 (t, J=5.0 Hz, 2H, C(O)NHCH₂CH₂O), 2.94 (dd, J=5.0, 13.0 Hz, 1H, SCHH), 2.74 (d, J=13.0 Hz, 1H, SCHH), 2.24 (t, J=7.5 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.76-1.43 (m, 6H, NHC(O)CH₂CH₂CH₂CH₂); ¹³C NMR (100 MHz, CD₃OD) δ 174.98 (s), 164.76 (s), 69.92 (t), 69.83 (t), 69.22 (t), 66.46 (t), 62.08 (d), 60.36 (d), 55.59 (d), 39.65 (t), 39.24 (t), 38.77 (t), 35.29 (t), 28.29 (t), 28.06 (t), 25.44 (t); IR (MeOH) 3300, 2941, 1686 cm⁻¹; HRMS (ES) calcd for C₁₆H₃₁N₄O₄S [M+H]⁺375.2066, observed 375.2060.

Reference Example 36

Preparation of N-(2-(2-(2-(3-bromo-2,5-dioxo-pyrrol-1-yl)ethoxy)ethoxy)ethyl)-5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanamide

Monobromomaleic anhydride (45.0 mg, 0.25 mmol, 1 eq) was added in one portion to a solution of 2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium 2,2,2-trifluoroacetate (124 mg, 0.25 mmol, 1 eq) in AcOH (10 mL) and the reaction mixture heated to 170° C. for 3 h. Upon cooling to 21° C. toluene was added and the AcOH azeotropically removed in vacuo (×2) to give crude product. Column chromatography (gradient 2-10% MeOH/CH₂Cl₂) yielded the desired compound as a white solid (70.0 mg, 0.13 mmol, 52% yield). m.p. 95-98° C.; [α]_D ^20.0+65.1 (c 0.15, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 7.17 (s, 1H, CHCBr), 4.51 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 4.33 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 3.77 (t, J=5.5 Hz, 2H, OCH₂), 3.68 (t, J=5.5 Hz, 2H, OCH₂), 3.63 (m, 2H, OCH₂), 3.58 (m, 2H, OCH₂), 3.53 (t, J=5.5 Hz, 2H, NCH₂), 3.37 (t, J=5.5 Hz, 2H, NCH₂), 3.24 (td, J=5.0, 8.0 Hz, 1H, SCH), 2.95 (dd, J=5.0, 12.5 Hz, 1H, SCHH), 2.73 (d, J=12.5 Hz, 1H, SCHH), 2.26 (t, J=7.0 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.69 (m, 4H, CH₂CH₂CH₂), 1.47 (quintet, J=7.0 Hz, 2H, CH₂CH₂CH₂); ¹³C NMR (150 MHz, CD₃OD) δ 176.12 (s), 170.13 (s), 166.97 (s), 166.08 (s), 133.63 (s), 132.05 (d), 71.22 (t), 71.11 (t), 70.61 (t), 68.69 (t), 63.35 (d), 61.61 (d), 57.03 (d), 41.09 (t), 40.31 (t), 39.09 (t), 36.75 (t), 29.78 (t), 29.50 (t), 26.87 (t); IR (MeOH) 3355, 2970, 1737 cm⁻¹; HRMS (ES) calcd for C₂₀H₂₉N₄O₆NaSBr [M+Na]⁺555.0889, observed 555.0905.

Reference Example 37

Preparation of N-(2-(2-(2-(3,4-dibromo-2,5-dioxo-pyrrol-1-yl)ethoxy)ethoxy)ethyl)-5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanamide

Dibromomaleic anhydride (108 mg, 0.42 mmol, 1 eq) was added in one portion to a solution of 2-(2-(2-(5-(2-oxo-1,3,3a,4,6,6a-hexahydrothieno(3,4-d)imidazol-6-yl)pentanoylamino)ethoxy)ethoxy)ethylammonium 2,2,2-trifluoroacetate (205 mg, 0.42 mmol, 1 eq) in AcOH (10 mL) and the reaction mixture heated to 170° C. for 2 h. Upon cooling to 21° C. toluene was added and the AcOH azeotropically removed in vacuo (×2) to give crude product. Column chromatography (gradient 2-7% MeOH/CH₂Cl₂) yielded the desired compound as a white solid (123 mg, 0.20 mmol, 48% yield). m.p. 100-102° C.; [α]_D ^20.0+71.0 (c 0.15, MeOH); ¹H NMR (600 MHz, CD₃OD) δ 4.53 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 4.34 (dd, J=5.0, 8.0 Hz, 1H, NHC(O)NHCH), 3.82 (t, J=5.5 Hz, 2H, OCH₂), 3.70 (t, J=5.5 Hz, 2H, OCH₂), 3.63 (m, 2H, OCH₂), 3.59 (m, 2H, OCH₂), 3.53 (t, J=5.5 Hz, 2H, NCH₂), 3.37 (t, J=5.5 Hz, 2H, NCH₂), 3.24 (dt, J=5.0, 8.0 Hz, 1H, SCH), 2.96 (dd, J=5.0, 13.0 Hz, 1H, SCHH), 2.73 (d, J=13.0 Hz, 1H, SCHH), 2.26 (t, J=7.5 Hz, 2H, NHC(O)CH₂CH₂CH₂), 1.74 (m, 4H, CH₂CH₂CH₂), 1.49 (quintet, J=7.5 Hz, 2H, CH₂CH₂CH₂); ¹³C NMR (150 MHz, CD₃OD) δ 174.83 (s), 164.71 (s), 164.06 (s), 129.00 (s), 69.80 (t), 69.72 (t), 69.24 (t), 67.19 (t), 61.97 (d), 60.22 (d), 55.64 (d), 39.67 (t), 39.03 (t), 38.56 (t), 35.42 (t), 28.39 (t), 28.11 (t), 25.47 (t); IR (MeOH) 2970, 1724, 1365, 1217 cm⁻¹; HRMS (ES) calcd for C₂₀H₂₈N₄O₆NaSBr₂[M+Na]⁺631.9916, observed 631.9937.

Reference Example 38

Preparation of N-Fluorescein bromomaleimide

Dibromomaleic anhydride (346 mg, 1.95 mmol) was added in one portion to a solution of fluoresceinamine isomer 1 (678 mg, 1.95 mmol) in acetic acid (65 mL) and the reaction mixture was stirred for 12 hours at room temperature in a sealed tube. The reaction mixture was then heated to 150° C. for 3 h. Upon cooling to room temperature the solid was filtered and dried (toluene azeotrope) to afford the desired compound as an orange solid (722 mg, 1.43 mmol, 73% yield). ¹H NMR (600 MHz, DMSO) δ 7.99 (d, 1H, J=1.7, 1H, H-11), 7.77 (dd, 1H, J=1.9 and 8.2, 1H, H-7), 7.73 (s, 1H, H-3), 7.43 (d, J=8.2, 1H, H-8), 6.69 (m, 6H, 2×H-16, 2×H-17, 2×H-18); ¹³C NMR (175 MHz, DMSO) δ 167.93 (C═O), 167.63 (C═O), 164.48 (C═O), 159.62 (2×C18), 151.79 (2×C20), 151.52 (C6), 133.68 (C7), 133.02 (Ar), 132.90 (C3), 131.23 (C), 129.15 (2×Ar—H), 126.73 (C), 124.82 (C11), 122.29 (C8), 112.77 (2×Ar—H), 109.08 (2×Ar), 102.30 (2×Ar—H), 83.36 (C14); IR (solid, cm⁻¹) 3064 (w), 1726 (s); MS (ES+) m/z, (relative intensity): 508 ([⁸¹M], 95), 506 ([⁷⁹M], 100); Exact mass calcd for [C₂₄H₁₃O₇N⁷⁹Br] requires 505.9875 Found 505.9833 (ES+).

Reference Example 39

Preparation of N-Fluorescein dibromomaleimide

Dibromomaleic anhydride (77.0 mg, 0.30 mmol) was added in one portion to a solution of fluoresceinamine isomer 1 (105 mg, 0.30 mmol) in acetic acid (10 mL) and the reaction mixture was stirred for 6 h at room temperature. The solid was then filtered off, washed with ethyl acetate, and redissolved in acetic acid (10 mL). The reaction mixture was then heated to reflux for 3 h. Upon cooling to room temperature toluene (10 ml) was added and the solvent removed in vacuo, affording the desired compound as an orange solid (148 mg, 0.25 mmol, 84% yield). δ ¹H NMR (400 MHz, CD₃OD) δ 8.07 (d, 1H, J=1.5, H-11), 7.81 (dd, 1H, J=1.5 and 8.0, H-7), 7.34 (d, 1H, J=8.5, H-8), 6.71-6.58 (m, 6H, 6×Ar—H); ¹³C NMR (100 MHz, CD₃OD) δ 170.23 (C═O), 164.34 (2×C═O), 161.63 (2×C), 154.18 (2×C), 152.93 (C), 134.59 (C), 134.19 (Ar—H), 131.01 (C), 130.35 (Ar—H), 129.25 (2×C), 126.25 (2×Ar—H), 123.63 (Ar—H), 113.84 (2×Ar—H), 111.02 (2×C), 103.55 (2×Ar—H); IR (solid, cm') 3064 (w), 1732 (s); MS (ES+) m/z, (relative intensity): 586 ([⁸¹⁺⁸¹M], 30), 584 ([⁷⁹⁺⁸¹M], 100), 582 ([⁷⁹⁺⁷⁹M], 100); Exact mass calcd for [C₂₄H₁₀O₇N⁷⁹Br₂] requires 581.8824 Found 581.8824 (ES+).

Reference Example 40

Preparation of Tert-butyl 2-aminoethylcarbamate

Di-tertbutyldicarbonate (3.26 g, 15 mmol, 1 eq) in DCM (30 mL) was added, dropwise, to a solution of ethylenediamine (10 ml, 150 mmol, 10 eq) in DCM (30 mL) under an argon atmosphere over two hours using an autoinjecter. Based on TLC analysis (eluent: 90% EtOAc:10% MeOH R_f(8)=0.23) the reaction reached completion 30 minutes after the end of the addition. The DCM was removed under reduced pressure using a Büchi. The resultant residue was taken up in EtOAc (40 mL) and washed with saturated Na₂CO₃(3×20 mL), dried over MgSO₄, and concentrated in vacuo to obtain the desired product (2.08 g; 12.98 mmol, 87%) as a white foam. mp (104-106° C.), δ_H ¹H NMR (300 MHz CDCl3): 4.95 (broad singlet, 1H, NH), 3.12 (q, J=6.4 Hz, 2H, CH₂), 2.78 (t, J=5.9 Hz, 2H, CH₂), 1.42 (s, 9H, 3CH₃). 13C NMR (CDCl3): 28.06, 41.51, 43.02, 78.82, 155.9 IR: 3354.9 cm⁻¹, [M+H]⁺: 161.00

Reference Example 41

Preparation of tert-butyl 2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethylcarbamate

A round bottom flask was flame dried and equipped with a stirrer bar and a solution of amine (0.57 g, 3.6 mmol, 1 eq) in dry DCM (150 mL) under an argon atmosphere. Dansyl chloride (1.05 g, 3.92 mmol, 1.1 eq) in dry DCM (150 mL) and triethylamine (1.3 ml, 9.29 mmol, 2.5 eq) were added through a septum in one portion. Reaction was monitored by TLC (eluent: 35% EtOAc:65% Petroleum ether R_f(9)=0.27, fluorescent green under long UV), the reaction was complete after 4 hours. Following purification by column chromatography (eluent: 35% EtOAc:65% Petroleum ether), the desired compound was formed (1.24 g, 3.15 mmol, 88%) as a sticky, clear green oil. δ_H ¹H NMR (CDCl3): 8.55 (d, J=8.55 Hz, 1H, CH), 8.46 (d, J=8.51 Hz, 1H, CH), 8.33 (d, J=8.67 Hz, 1H, CH), 7.57 (m, 2H, 2×CH), 7.26 (d, J=7.08 Hz, 1H, CH), 3.07 (quartet, J=6.58 Hz, 2H, CH₂), 2.89 (m, 2H, CH₂), 2.85 (s, 6H, 2×CH₃), 1.35 (s, 9H, 3×CH₃). 13C NMR (CDCl₃): 158.01, 153.05, 136.5, 131.49, 131.09, 130.02, 129.54, 124.48, 120.59, 116.41, 80.41, 45.9, 43.81, 41.5, 28.5. MS: [M+H]+: 393.16.

Reference Example 42

Preparation of 5-(3-aminopropylsulfonyl)-N,N-dimethylnaphthalen-1-amine 2,2,2-trifluoroacetate

To a flask containing BOC-carbamate (1.24 g, 3.15 mmol), TFA (40 ml) was added in one portion. The resulting grey solution was stirred for 2 hours at room temperature (ca. 25° C.). Upon completion the solution was concentrated in vacuo and azeotroped with toluene (5×10 ml). The resultant crude product was then purified by column chromatography (eluent: EtOAc 1:2 Petroleum ether R_f(10)=0.20). After concentrating the relevant fractions in vacuo, to the yellow oil that resulted, DCM (100 ml) was added and the solution placed in an ice bath for 2 hours, this solution was fluorescent under long-wave UV. The desired compound (1.25 g, 3.10 mmol, 97%) crashed out of solution as a white solid and it was filtered off and washed with diethyl ether under gravity. Mp (114-116° C.); δ_H ¹H NMR (500 MHz MeOD): 8.64 (d, J=8.3 Hz, 1H, CH), 8.35 (d, J=8.45 Hz, 1H, CH), 8.32 (d, J=8.65 Hz, 1H, CH), 7.67 (m, 2H, 2×CH), 7.34 (d, J=7.3 Hz, 1H, CH), 3.03 (quartet, 4H, 2×CH₂), 2.84 (s, 6H, 2×CH₃). δ_c ¹³C NMR (500 MHz MeOD): 153.41, 135.78, 131.69, 131.32, 130.81, 130.56, 129.48, 124.29, 120.07, 116.635, 66.91, 45.79, 41.27, 40.78, 15.45. ¹⁹F NMR (300 MHz CDCl₃); −76.89; IR: 3092 cm⁻¹, 2901.5 cm⁻¹, MS: [M+H]+: 294

Reference Example 43

Preparation of (E)-2-bromo-4-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethylamino)-4-oxobut-2-enoic acid

An oven-dried 500 ml round bottomed flask was equipped with a stirring bar Amine salt (1.09 g) was dissolved in 25 ml acetic acid and added to the flask. To the resulting light yellow solution, Bromomaleic anhydride was added and reaction was monitored by TLC (eluent; 10% methanol: 90% EtOAc, R_f(11)=0.7). After 1.5 hours of stirring at room temperature (25° C.) the acetic acid was removed in vacuo. The desired compound was used without further purification. 1H NMR (500 Mz CDCl3 (Crude)): δ_H8.6 (d, J=8.56 Hz, 1H, CH), 8.35 (d, 1H, J=8.27 Hz, CH), 8.22 (d, 1H, J=8.57 Hz, CH), 7.64 (m, 2H, 2×CH), 7.30 (d, J=7.60 Hz, 1H, CH), 5.48 (s, 1H, CH)/5.03 (s, 1H, CH), 3.00 (m, 4H, 2×CH₂), 2.88 (s, 9H, 2×CH₃)

Reference Example 44

Preparation of N-(2-(3-bromo-2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-5-(dimethylamino)naphthalene-1-sulfonamide

The acid was dissolved in acetic acid (25 mL) and loaded into an oven dried 500 ml round bottom flask. A condenser was fitted and the reaction was placed under reflux (170° C.) for 2 hours. The acetic acid was then removed from the crude mixture in vacuo and the resultant oil was aziotroped with toluene (5×10 ml). The resultant oil was purified by column chromatography (eluent: 30% ethylacetate:70% petroleum ether, R_f(12)=0.2 in the aforementioned eluent system). Once the very slow column was completed, the more mobile fraction was collected and the solvent removed. The resultant brown oil was left to stand in neat ethyl acetate (50 ml) for 1 hour in an ice bath. The desired product (0.961 g, 80%) crashed out of solution as a brown solid (powder like texture), this was filtered under gravity and washed with diethyl ether (20 ml). mp (166-170° C.); ¹H NMR (600 MHz DMSO): δ_H8.53 (d, J=8.46 Hz, 1H, CH), 8.21 (d, 1H, J=8.40 Hz, CH), 8.17 (d, 1H, J=8.58 Hz, CH), 7.56 (m, 2H, 2×CH), 7.18 (d, J=7.50 Hz, 1H, CH), 6.46 (s, 1H, maleimide olefin C—H), 5.11 (t, J=6.24, 1H, NH), 3.56 (m, 2H, CH₂), 3.2 (m, 2H, CH₂), 3.91 (s, 6H, 2×CH₃). δ_C ¹³C NMR (600 MHz DMSO): 168.62, 165.33, 151.38, 151.38, 135.62, 132.33, 130.13, 129.60, 129.09, 128.85, 128.30, 127.96, 123.61, 118.99, 115.22, 45.11, 40.05, 39.37, 38.47.

Reference Example 45

Preparation of 4-Bromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione (BrDDPD)

A mixture of monobromomaleic anhydride (177 mg, 1.0 mmol) and N,N′-diethylhydrazine (88 mg, 1.0 mmol) in glacial AcOH (3 mL) was heated at 130° C. for 16 h. The solvent was removed in vacuo and the crude residue purified by column chromatography (neat CH₂Cl₂-5% MeOH/CH₂Cl₂) to give 4-bromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione as a yellow solid (159 mg, 0.64 mmol, 64%): ¹H NMR (600 MHz, CDCl₃) δ 7.31 (s, 1H), 4.14 (q, J=7.0 Hz, 2H), 4.07 (q, J=7.0 Hz, 2H), 1.26 (t, J=7.0 Hz, 3H), 1.22 (t, J=7.0 Hz, 3H); ¹³C NMR (150 MHz, CDCl₃) δ 156.2 (s), 154.3 (s), 136.0 (d), 133.7 (s), 41.9 (t), 40.7 (t), 13.3 (q), 13.3 (q); IR (solid) 3058, 2979, 2938, 1631, 1595 cm⁻¹; LRMS (CI) 249 (100, [M⁸¹Br+H]⁺), 247 (100, [M⁷⁹Br+H]⁺); HRMS (CI) calcd for C₈H₁₂BrN₂O₂[M+H]⁺249.0082, observed 249.0086.

Reference Example 46

Preparation of 4,5-Dibromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dione (DiBrDDPD)

A mixture of dibromomaleic anhydride (256 mg, 1.0 mmol) and N,N′-diethylhydrazine (88 mg, 1.0 mmol) in glacial AcOH (3 mL) was heated at 130° C. for 16 h. The solvent was removed in vacuo and the crude residue purified by column chromatography (neat CH₂Cl₂-5% MeOH/CH₂Cl₂) to give 4,5-dibromo-1,2-diethyl-1,2-dihydro-pyridazine-3,6-dioneas a yellow solid (202 mg, 0.62 mmol, 62%): ¹H NMR (600 MHz, CDCl₃) δ 4.17 (q, J=7.0 Hz, 4H), 1.28 (t, J=7.0 Hz, 6H); ¹³C NMR (150 MHz, CDCl₃) δ 153.3 (s), 136.1 (s), 42.4 (t), 13.2 (q); IR (solid) 2979, 2937, 1630, 1574 cm⁻¹; LRMS (EI) 328 (50, [M⁸¹Br⁸¹Br]^+•), 326 (100, [M⁸¹Br⁷⁹Br]^+•), 324 (50, [M⁷⁹Br⁷⁹Br]^+•); HRMS (EI) calcd for C₈H₁₀Br₂N₂O₂[M⁷⁹Br⁷⁹Br]^+• 323.9104, observed 323.9097.

Reference Example 47

Preparation of N-Boc-Cys(Mal)-OMe

To a stirring solution of N-Boc-Cys-OMe (36 mg, 0.15 mmol) and sodium acetate (13 mg, 0.15 mmol) in methanol (3 mL) was added bromomaleimide (30 mg, 0.17 mmol) in methanol (3 mL). After 1 minute solvent was removed in vacuo. Purification by flash chromatography (gradient elution in 50% ethyl acetate in petroleum ether to ethyl acetate) afforded a pale yellow powder N-Boc-Cys(Mal)-OMe (51 mg, 0.15 mmol) in 100%. δ_H(500 MHz, CDCl₃) 7.63 (s, 1H, mal-NH), 6.27 (s, 1H, 9-H), 5.40 (d, 1H, J=6.8, NH), 4.67 (ddd, 1H, J=5.1, 5.4 and 6.8, H-4), 3.80 (s, 3H, H₃-6), 3.48 (dd, 1H, J=5.1 and 13.8, HH-7), 3.62 (dd, 1H, J=5.4 and 14.1, HH-7) 1.45 (s, 9H, 3×H₃-1); δ_C(125 MHz, CDCl₃) 170.2 (C═O), 168.9 (C═O), 167.6 (C═O), 155.2 (C═O), 155.9 (C8), 119.7 (C9), 81.1 (C2), 53.3 (C6), 52.7 (C4), 34.0 (C7), 28.3 (3×C1); IR (solid, cm⁻¹) 3236 (w), 1715 (s); MS (CI+) m/z, (relative intensity): 331 ([M+H], 5), 275 (20), 231 (100); Mass calcd for [C₁₃H₁₈O₆N₂S]+H requires 331.0964 Found 331.0968 (CI+); ²⁰α_D: −41.9° (c=1.0, Methanol); m.p. 145-147° C.; UV (Acetonitrile) ε₂₄₅=14200 and ε₃₃₉=8600 cm⁻¹M⁻¹d³.

Reference Example 48

Preparation of N-Boc-Cys(N′-Me-Mal)-OMe

To a stirring solution of N-Boc-Cys-OMe (32 mg, 0.136 mmol) in methanol (4 mL) was added sodium acetate (82 mg, 0.408 mmol). To this was added N-methyl bromomaleimide (25.8 mg, 0.136 mmol) in methanol (4 mL) over 10 minutes. The solvent was removed in vacuo purification by flash chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate in petroleum ether) to afford the desired compound as a pale white powder (39.3 mg, 0.114 mmol) in 84% yield. δ_H(500 MHz, CDCl₃) 6.26 (s, 1H, H-9), 5.36 (d, 1H, J=6.3, ‘Boc’ NH), 4.66 (m, 1H, H-4), 3.79 (s, 3H, H₃-6), 3.46 (dd, 1H, J=5.0 and 5.2, HH-7), 3.35 (dd, 1H, J=5.1 and 13.7, HH-7), 3.00 (s, 3H, H₃-13), 1.44 (s, 9H, 3×H₃-1); δ_C(125 MHz, CDCl3) 170.2 (C═O), 169.5 (C═O), 167.9 (C═O), 155.0 (C═O), 149.9 (C8), 118.7 (C9), 80.9 (C2), 53.1 (C6), 52.7 (C4), 33.8 (C7), 28.3 (3×C1), 24.1 (C13); IR (solid, cm⁻¹) 3368 (m), 2977 (m), 1695 (s); MS (ES+) m/z, (relative intensity): 311 (M+, 100); Mass calcd for C₁₄H₂₀N₂O₆NaS requires 367.0940. Found: 367.0931; ²⁰α_D: −18.55 o (c=1.0, Methanol); m.p. 101-103° C.

Reference Example 49

Preparation of 2,3-Di(N-Boc-Cys-OMe)succinimide (mix of diastereomers)

To a stirred solution of bromomaleimide (50 mg, 0.28 mmol) in aqueous buffer (100 mM sodium phosphate, 150 mM NaCl, pH 8.0):DMF, 95:5 (9.25 mL) was added N-Boc-Cys-OMe (660 mg, 2.81 mmol) in DMF (0.25 mL). After 5 minutes the aqueous reaction mixture was extracted with ethyl acetate (3×25 mL) and the combined organic layers washed with saturated lithium chloride solution (aq) (5×25 mL), water (25 mL) and brine (25 mL), dried (MgSO₄), filtered and the solvent removed in vacuo. Purification by column chromatography (10-40% ethyl acetate in petroleum ether) afforded 2,3-Di(N-Boc-Cys-OMe)succinimide (mix of diastereomers) as a yellow waxy oil (150 mg, 0.27 mmol, 94% yield), an inseparable 1:1 mix of two symmetrical diastereomers; δ_H(400 MHz, CDCl₃) 8.62 (s, 1H, maleimide NH from one symmetrical diastereomer), 8.66 (s, 1H, maleimide NH from one symmetrical diastereomer), 5.62 (d, 2H, J=8.4, 2×‘Boc’ NH from one symmetrical diastereomer), 5.51 (d, 2H, J=8.0, 2×‘Boc’ NH from one symmetrical diastereomer), 4.72-4.58 (m, 4×H-4 from both diastereomers), 3.80 (s, 6H, 2×H₃-6 from one symmetrical diastereomer), 3.79 (s, 6H, 2×H₃-6 from one symmetrical diastereomer), 3.68 (s, 2H, 2×H8 from one symmetrical diastereomer), 3.64 (s, 2H, 2×H-8 from one symmetrical diastereomer), 3.46 (dd, 2H, J=4.8 and 12.0 Hz, 2×HH-7* from one symmetrical diastereomer), 3.37 (dd, 2H, J=6.0 and 14.4, 2×HH-7† from one symmetrical diastereomer), 3.21 (dd, 2H, J=4.8 and 14.0 Hz, 2×HH-7† from one symmetrical diastereomer), 3.11 (dd, 2H, J=6.4 and 14.0 Hz, 2×HH-7* from one symmetrical diastereomer), 1.463 (s, 18H, 6×H₃-1 from one symmetrical diastereomer), 14.460 (s, 18H, 6×H₃-1 from one symmetrical diastereomer);
*—signals shown as part of the same AB system by HMQC data
†—signals shown as part of the same AB system by HMQC data
δ_C(125 MHz, CDCl₃) 174.32 (2×C═O), 171.25 (2×C═O), 155.33 (2×C═O), 80.61 (2×C2), 80.58 (2×C2), 53.51 (2×C4), 53.18 (2×C4), 52.91 (2×C6), 52.90 (2×C6), 48.45 (2×C8), 47.89 (2×C8), 34.66 (2×C7), 34.59 (2×C7), 28.37 (6×C1), 28.36 (6×C1) Several carbon signals are missing due to overlap of the diastereomers; IR (thin film, neat) 3348, 2978, 1719 cm⁻¹; MS (EI) m/z (relative intensity): 566 ([M+H], 20), 564 ([M−H], 100); Exact mass calcd for [C₂₂H₃₅N₃O₁₀S₂]—H requires 564.1669 Found 564.1686.

Reference Example 50

Preparation of N—Ac-Cys(Mal)-Benzylamine

To N—Ac-Cys-Benzylamine (1.00 g, 4.00 mmol) above) in methanol (42 mL), was added bromomaleimide (777 mg, 4.37 mmol) in methanol (42 mL) dropwise over 5 minutes. After 10 minutes, solvent removed in vacuo and residue subjected to flash chromatography using 10% ethyl acetate in petroleum ether afford the desired compound as an off-white solid (429 mg, 1.2 mmol) in 100% yield, based on 69% recovery of the bromomaleimide. δ_H(500 MHz, MeOD) 7.32-7.20 (m, 5H, 5×Ar—H), 6.45 (s, 1H, H-12), 4.71 (t, 1H, J=7.3, H-3), 4.38 (d, 2H, J=2.7, H₂-5), 3.40 (dd, 1H, J=7.0 and 13.6, HH-10), 3.25 (dd, 1H, J=7.2 and 13.6, HH-10), 1.99 (s, 3H, H₃-1); δ_C(125 MHz, MeOD) 173.51 (C═O), 172.22 (C═O), 171.44 (C═O), 170.51 (C═O), 151.58 (C11), 139.48 (C6), 129.54 (2×Ar—H), 128.51 (2×Ar—H), 128.26 (C9), 121.01 (C12) 53.04 (C3), 44.25 (C5), 33.72 (C10), 22.42 (C1); IR (film, cm⁻¹) 3187 (w), 1717 (s), 1646 (s); MS (ES+) m/z (relative intensity): 370 ([M+Na], 20), 337 (50), 325 (90), 309 (100); Exact Mass Calcd for [C₁₆H₁₇N₃O₄SN]+Na requires m/z 370.0873 Found 370.0852 (ES+); UV (Acetonitrile) ε₂₁₃=19400, ε₂₄₇=4800 and ε₃₃₇=2700 cm⁻¹M⁻¹d³; White solid decomposes at 180° C.

Reference Example 51

Preparation of N-Methyl hexylsulfanylmaleimide

To N-methyl bromomaleimide (100 mg, 0.53 mmol) and sodium acetate trihydrate (70 mg, 0.53 mmol) in methanol (15 mL) was added hexanethiol (74 μL, 0.58 mmol) in methanol (100 mL) dropwise over 1 hour with vigorous stirring. After 5 minutes solvent was removed in vacuo. Purification by column chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a bright yellow solid (99 mg, 0.44 mmol) in 83% yield. δ_H(600 MHz, CDCl₃) 6.03 (s, 1H, H-2), 3.01 (s, 3H, H₃-5), 2.89 (t, 2H, J=7.6, 2H, H₂-11), 1.76-1.71 (m, 2H, H₂-10), 1.46-1.41 (m, 2H, H₂-9), 1.33-1.27 (m, 4H, H₂-7 and CH₂-8), 0.89 (t, 3H, J=6.5, H₃-6); δ_C(125 MHz, CDCl₃) 171.47 (C═O), 169.94 (C═O), 151.84 (C3), 117.27 (C2), 31.92 (C11), 31.31 (CH₂), 28.64 (CH₂), 27.75 (CH₂), 24.10 (C5), 24.10 (C7), 14.09 (C6); IR (oil, cm⁻¹) 2727 (w), 1708 (s); MS (FAB+) m/z (relative intensity): 250 ([M+Na], 40), 228 (35), 199 (30), 176 (100); Exact Mass Calcd for [C₁₁H₁₇NO₂S]+Na requires m/z 250.0878 Found 250.0880 (FAB+)

Reference Example 52

Preparation of 2,3Dihexylsulfanylsuccinimide and Hexylsulfanylmaleimide

Method A

To bromomaleimide (300 mg, 1.69 mmol) and sodium acetate (138 mg, 1.69 mmol) in methanol (60 mL) was added hexanethiol (356 μL, 2.50 mmol). After 5 minutes solvent removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded 2,3 dihexanethiosuccinimide as a bright yellow paste (13 mg, 0.04 mmol) in 2% and hexylsulfanylmaleimide as a cream powder (alkene310 mg, 1.46 mmol) in 86% yield.

2,3Dihexylsulfanylsuccinimide

δ_H(500 MHz, CDCl₃) 8.21 (s, 1H, NH), 3.49 (s, 2H, 2×H-7), 2.89-2.83 (m, 2H, 2×HH-6), 2.79-2.83 (m, 2H, 2×HH-6), 1.71-1.57 (m, 4H, 2×CH₂), 1.44-1.37 (m, 4H, 2×CH₂), 1.34-1.26 (m, 8H, 4×CH₂), 0.89 (t, 6H, J=6.8, 2×H₃-1); δ_C(125 MHz, CDCl₃) 174.60 (2×C═O), 48.23 (2×C7), 32.34 (2×CH₂), 31.26 (2×CH₂), 28.99 (2×CH₂) 28.46 (2×CH₂), 22.56 (2×CH₂), 14.27 (2×C1); IR (solid, cm⁻¹) 3198 (m), 2928 (m), 1703 (s); No mass ion found.

Hexylsulfanylmaleimide

δ_H(500 MHz, CDCl₃) 7.35 (s, 1H, NH), 6.04 (s, 1H, H-8), 2.91 (t, 2H, H₂-6), 1.78-1.72 (m, 2H, H₂-5), 1.48-1.42 (m, 2H, CH₂), 1.33-1.30 (m, 4H, 2×CH₂), 0.90 (t, 3H, J=6.9, H₃-1); δ_C(125 MHz, CDCl₃) 169.06 (C═O), 167.69 (C═O), 152.74 (C7), 118.24 (C8), 32.06 (C6), 31.26 (CH₂), 28.58 (CH₂), 27.70 (CH₂), 22.52 (CH₂), 14.03 (C1); IR (solid, cm⁻¹) 3200 (m), 2918 (m), 1703 (s); MS (ES−) m/z (relative intensity): 212 ([M−H], 100); Exact Mass Calcd for [C₁₀H₁₅NO₂S]—H requires m/z 212.0745 Found 212.0753 (ES−); m.p. 99-101° C.; UV (Acetonitrile) ε₂₄₇=12000 and ε₃₄₇=9500 cm⁻¹M⁻¹d³.

Method B

To bromomaleimide (300 mg, 1.69 mmol) and sodium acetate (138 mg, 1.69 mmol) in methanol (100 mL) was added hexanethiol (237 μL, 1.69 mmol). After 5 minutes solvent removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded hexylsulfanylmaleimide as a cream powder (362 mg, 1.69 mmol) in 100% yield.

Reference Example 53

Preparation of N-Methylenecyclohexane hexylsulfanylmaleimide

To N-methylenecyclohexane bromomaleimide (50 mg, 0.19 mmol) in methanol (50 mL), was added hexanethiol (52 μL, 0.37 mmol) and sodium acetate (50 mg, 0.37 mmol) in methanol (50 mL) dropwise over 5 minutes. After 10 minutes, solvent removed in vacuo and residue subjected to flash chromatography (petroleum ether) to afford the desired compound as an off-white solid (29 mg, 0.09 mmol) in 84% yield. δ_H(600 MHz, CDCl₃) 6.01 (s, 1H, H-3), 6.27 (s, 1H, 9-H), 3.42 (d, 1H, J=6.8, NH), 4.67 (ddd, 1H, J=5.1, 5.4 and 6.8, H-4), 3.80 (s, 3H, H₃-6), 3.48 (dd, 1H, J=5.1 and 13.8, HH-7), 3.62 (dd, 1H, J=5.4 and 14.1, HH-7) 1.45 (s, 9H, 3×H₃-1); δ_C(125 MHz, CDCl₃) 170.23 (C═O), 16844 (C═O), 151.49 (C2), 117.08 (C3), 44.36 (C16), 37.00 (C15), 31.91 (2×CH₂), 31.32 (2×CH₂), 30.73 (CH₂), 28.66 (CH₂), 27.78 (CH₂), 26.33 (CH₂), 25.73 (2×CH₂), 22.58 (CH₂), 14.10 (C6); IR (solid, cm⁻¹) 2927 (m), 1700 (s); MS (ES+) m/z, (relative intensity): 310 ([M+H], 100), 180 (40); Mass calcd for [C₁₇H₂₇O₂NS]+H requires 310.1841 Found 310.1828 (ES+).

Reference Example 54

Preparation of 3-Mercaptopropylthiomaleimide and 1,5-Dithio-8-aza-bicyclo[5,3,0]decan-7,9-dione

To bromomaleimide (30 mg, 0.17 mmol) and sodium acetate (14 mg, 0.17 mmol) in methanol (6 mL) was added 1,3-propanedithiol (17 μl, 0.17 mmol). After five minutes solvent was removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded 3-mercaptopropylthiomaleimide and 1,5-dithio, 8-aza-bicyclo[5,3,0]decan-7,9-dione as a pale yellow powder that was a mix of two inseparable isomers 3-mercaptopropylthiomaleimide (7 mg, 0.03 mmol) in 21% yield, 1,5-dithio, 8-aza-bicyclo[5,3,0]decan-7,9-dione (12 mg, 0.06 mmol) in 34% yield. δ_H(500 MHz, MeOD) 6.28 (s, 1H, H-5), 4.41 (s, 3.2H, 2×H-10), 3.15 (t, 2H, J=7.3, H₂-3), 2.82-2.77 (m, 3.2H, CH₂), 2.35 (t, 3.2H, J=13.1, CH₂), 2.30-2.25 (m, 2H, CH₂), 2.20-2.13 (m, 2H, CH₂), 1.91-1.83 (m, 3.2H, CH₂); δ_C(125 MHz, MeOD) 177.79 (2×C11), 172.33 (C═O), 170.56 (C═O), 152.37 (C4), 120.30 (C5), 54.52 (2×C10), 34.94 (2×C9), 32.16 (CH₂), 31.10 (CH₂) 30.96 (CH₂), 27.49 (CH₂); IR (solid, cm⁻¹) 3246 (m), 1703 (s); MS (ES−) m/z (relative intensity): 202 ([M−H], 100); Exact Mass Calcd for [C₇H₉NO₂S₂]—H requires m/z 201.9996 Found 201.9996 (ES−).

Reference Example 55

Preparation of N-Phenyl hexylsulfanylmaleimide

To hexanethiol (111 μL, 0.79 mmol) and sodium acetate trihydrate (108 mg, 0.79 mmol) in methanol (60 mL) was added in N-phenyl monobromomaleimide (200 mg, 0.79 mmol) in methanol (60 mL) dropwise over 1 hour with vigorous stirring. After 5 minutes solvent was removed in vacuo. Purification by column chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a pale yellow solid (109 mg, 0.38 mmol) in 48% yield. δ_H(600 MHz, CDCl₃) 7.45 (dd, 2H, J=7.1 and 8.0, 2×H-12), 7.36 (d, 2H, J=6.0, H-11), 7.35 (d, 2H, J=8.1, 2×H-13), 6.19 (s, 1H, H-2), 2.96 (t, 2H, J=7.9, H₂-10), 1.81-1.76 (m, 2H, H₂-9), 1.50-1.45 (m, 2H, H₂-8), 1.34-1.32 (m, 4H, H₂-6 and H₂-7), 0.91 (t, 3H, J=6.9, H₃-5); δ_C(125 MHz, CDCl₃) 168.59 (C═O), 166.96 (C═O), 152.20 (C3), 131.53 (C14), 129.21 (2×Ar—H), 127.93 (C11), 126.09 (2×Ar—H), 117.24 (C2), 32.03 (C10), 31.33 (CH₂), 28.68 (CH₂), 27.78 (CH₂), 22.59 (CH₂), 14.11 (C5); IR (oil, cm⁻¹) 2931 (w), 1703 (s); MS (CI+) m/z (relative intensity): 290 ([M+H], 100); Exact Mass Calcd for [C₁₆H₂₀NO₂S]+H requires m/z 290.1215 Found 290.1224 (CI+);

Reference Example 56

Preparation of Phenylthiomaleimide

To thiophenol (57 μL, 0.56 mmol) and sodium acetate trihydrate (136 mg, 0.56 mmol) in methanol (30 mL) was added in monobromomaleimide (100 mg, 0.56 mmol) in methanol (30 mL) dropwise over 1 hour with vigorous stirring. After 5 minutes solvent was removed in vacuo. Purification by column chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a pale yellow solid (22 mg, 0.11 mmol) in 19% yield. δ_H(600 MHz, CDCl₃) 7.56 (dd, 2H, J=1.6 and 7.8, 2×H-7), 7.50-7.48 (m, 3H, 3×Ar), 5.63 (s, 1H, H-2); δ_C(125 MHz, CDCl₃) 169.42 (C═O), 167.98 (C═O), 153.60 (C3), 134.45 (2×Ar—H), 130.68 (C5), 130.42 (2×Ar—H), 127.27 (C8), 119.91 (C2); IR (oil, cm⁻¹) 3265 (m), 1770 (m), 1701 (s); MS (CI+) m/z (relative intensity): 206 ([M+H], 100), 111 (40); Exact Mass Calcd for [C₁₀H₇NO₂S]+H requires m/z 206.0276 Found 206.0273 (CI+);

Reference Example 57

Preparation of 1,4-Dithia-7-aza-spiro[4.4]nonane-6,8-dione

To bromomaleimide (30 mg, 0.17 mmol) and sodium acetate (14 mg, 0.17 mmol) in methanol (6 mL) was added 1,2-ethanedithiol (17 μl, 0.17 mmol). After five minutes solvent removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded the desired compound as a pale yellow powder (13 mg, 0.07 mmol) in 41% yield. δ_H(500 MHz, CDCl₃) 8.39 (s, 1H, NH), 3.75-3.69 (m, 2H, HH-2 and HH-3), 3.60-3.53 (m, 2H, HH-2 and HH-3), 3.30 (s, 2H, H₂-9); δ_C(125 MHz, CDCl₃) 177.93 (C═O), 172.76 (C═O), 61.23 (C5), 43.12 (C9), 41.05 (C2 and C3); IR (solid, cm⁻¹) 3290 (m), 1703 (m), 1629 (s); MS (ES−) m/z (relative intensity): 188 ([M−H], 100); Exact Mass Calcd for [C₆H₇NO₂S₂]—H requires m/z 187.9840 Found 187.9839 (ES−); m.p. 112-115° C.

Reference Example 58

Preparation of (S)-methyl 2-(tert-butoxycarbonylamino)-3-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-ylthio)propanoate

Dansyl-bromomaleimide (100 mg) was dissolved in methanol (200 ml) by briefly heating the stirring solution using a heat gun. To the resulting pale yellow solution, N-Boc-Cys-OMe (22 μl, 0.1 mmol, 0.5 eq) and sodium acetate (14.5 mg, 0.1 mmol, 0.5 eq)) were added over 3 hours. The reaction was monitored by TLC (eluent: 40% EtOAc:60% Petroleum ether) Once the addition was complete the methanol was removed in vacuo to yield a yellow oil. Purification by column chromatography yielded the desired product (48.29 mg, 0.08 mmol, 79.7%). ¹H NMR (600 MHz CDCl₃): δ_H8.54 (d, J=8.56 Hz, 1H, CH), 8.21 (d, 1H, J=8.27 Hz, CH), 8.13 (d, 1H, J=8.57 Hz, CH), 7.55 (m, 2H, 2×CH), 7.25 (d, J=7.60 Hz, 1H, CH), 5.92 (s, 1H, CH), 4.44 (m, 1H, HN—CH—CO), 3.77 (s, 3H, OMe), 3.48 (m, 2H, CH₂), 3.44 (m, 2H, CH₂), 3.38 (s, 6H, 2×CH₃), 3.13 (t, J=5.79, 2H, S—CH₂) 2.88 (s, 9H, 3×CH₃). ¹³C NMR (600 MHz CDCl₃): 173.08, 170.52, 169.69, 168.96 (4×C═O), 157.73 (—C═CH), 153.16, 150.71, 136.48, 131.39, 131.25, 131.18, 130.74, 130.65, 129.31, 124.35, 120.61, 119.22, 81.12, 53.58, 52.95, 45.89, 41.53, 33.98, 28.66. IR: 3324.7 cm⁻¹, 1775 cm⁻¹. [M+H]+: 605.1756, calculated; 605.1740

Reference Example 59

Preparation of (2R,2′R)-dimethyl-3,3′-(1-(2-(5-(dimethylamino)naphthalene-1-sulfonamido)ethyl)-2,5-dioxopyrrolidine-3,4-diyl)bis(sulfanediyl)bis(2-(^tbutoxycarbonylamino)propanoate)

Dansyl/maleimide/cysteine adduct (15 mg, 0.0247 mmol, 1 eq) was dissolved in methanol (100 ml). To the resulting clear solution, N-Boc-Cys-OMe (3.1 μl, 0.0247 mmol, 1 eq) was added over 1 hour. The reaction was monitored by TLC (eluent: 40% EtOAc:60% Petroleum ether) Once the addition was complete the methanol was removed in vacuo to yield the desired product (12.51 mg, 0.08 mmol, 60%). ¹H NMR (600 MHz CDCl₃): δ_H8.44 (d, J=8.56 Hz, 1H, CH), 8.13 (m, 2H, 2×CH), 7.49 (m, 2H, 2×CH), 7.17 (m, 2H, 2×CH), 4.49 (bs, 1H, HN—CH—CO), 3.77 (s, 3H, OMe), 3.48 (m, 2H, CH₂), 3.44 (m, 2H, CH₂), 3.38 (s, 6H, 2×CH₃), 3.13 (t, J=5.79, 2H, S—CH₂) 2.88 (s, 9H, 3×CH₃).

Reference Example 60

Preparation of Di-dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (100 mg, 0.16 mmol), TCEP (46 mg, 1 eq) and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. Dibromomaleimide (36 mg, 0.9 eq), in MeOH (5 ml) was then added to the reaction mixture. After 30 mins NaOAc (56 mg, 4 eq), was added to the reaction mixture and the solvent evaporated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄), filtered and concentrated in vacuo. Purification by flash chromatography (silica gel, 0-20% EtOAc-DCM) afforded the desired compound as a yellow gum (40 mg, 40%). ¹HNMR (CDCl₃, 600 MHz), δ8.5 (2H, d J 8.5 Hz aromatic H's), δ8.2 (4H, m aromatic H's), δ7.53 (1H, s CONH), δ7.46 (4H, m, aromatic H's), δ7.1 (2H, d, J=7.4 Hz aromatic H's), δ5.65 (2H, t, J 6.27 SO₂NH), δ3.3 (4H, t, J 6.0 SCH₂), δ3.17 (4H, q, J 6.0 NHCH₂), δ2.8 (12H, s NCH₃); ¹³CNMR (CDCl₃, 150 MHz), δ165.9, 152.0, 136.5, 134.7, 130.7, 129.94, 129.85, 129.62, 129.57, 128.63, 123.3, 118.8, 115.4, 45.5, 43.6, 31.8; IR (cm⁻¹) 3288 (br) 1720 (s) MS (Na+) m/z relative intensity: 736 (M, 100); Exact mass calculated for [C₃₂H₃₅N₅O₆NaS₄] requires m/z 736.1368. Found 736.1390 (Na+).

Reference Example 61

Preparation of Bromo-dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (48 mg, 0.08 mmol), TCEP (23 mg, 1 eq), and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. Dibromomaleimide (41 mg, 2 eq) in MeOH (10 ml), was added to the reaction mixture. After 16 hrs, the reaction mixture was concentrated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄) and purified by flash chromatography (silica gel, 0-15% EtOAC-DCM) to yield the desired compound (17 mg, 22%). ¹HNMR (CDCl₃, 600 MHz), δ8.5 (1H, d J 8.5 Hz aromatic H's), δ8.2 (2H, m aromatic H's), δ7.6 (1H, s CONH), δ7.53 (2H, m, aromatic H's), δ7.15 (1H, d, J=7.4 Hz aromatic H's), δ5.30 (1H, t, J 5.6 SO₂NH), δ3.38 (2H, t, J 6.3 SCH₂), δ3.26 (2H, q, J 6.3 NHCH₂), δ2.88 (6H, s NCH₃); ¹³CNMR (CDCl₃, 150 MHz), δ165.5, 162.9, 152.2, 142.5, 134.5, 130.95, 129.94, 129.92, 129.5, 128.7, 123.3, 119.0, 118.5, 115.4, 45.5, 43.7, 30.5; IR (cm⁻¹) 3295 (br) 1726 (s) MS (ES+) m/z relative intensity: 485 (M, 100); Exact mass calculated for [C₁₈H₁₉N₃O₄S₂Br] requires m/z 484.0000. Found 783.9982.

Reference Example 62

Preparation of Dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (100 mg, 0.16 mmol), TCEP (46 mg, 1 eq), and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. Bromomaleimide (56 mg, 2 eq) in MeOH (5 ml), was added to the reaction mixture. After 16 hrs, the reaction mixture was concentrated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄) and purified by flash chromatography (silica gel, 0-30% EtOAC-CHCl₃) to yield the desired compound (73 mg, 55%). ¹HNMR (CDCl₃, 600 MHz), δ8.5 (1H, d J 8.5 Hz aromatic H's), δ8.2 (2H, m aromatic H's), δ8.1 (1H, s CONH), δ7.5 (2H, m, aromatic H's), δ7.15 (1H, d, J=7.5 Hz aromatic H's), δ6.0 (1H, s, CO₂CH) δ5.89 (1H, t, J 6.4 SO₂NH), δ3.20 (2H, q, J 6.7 NHCH₂), δ3.99 (2H, t, J 6.9 SCH₂), δ2.86 (6H, s NCH₃); ¹³CNMR (CDCl₃, 150 MHz), 6169.6, 168.0, 152.0, 150.7, 134.4, 131.0, 129.9, 129.7, 129.5, 128.8, 123.4, 119.3, 118.7, 115.6, 45.5, 41.0, 31.8; IR (cm⁻¹) 3277 (br) 1720 (s) MS (ES−) m/z relative intensity: 404 (M, 100); Exact mass calculated for [C₁₈H₁₈N₃O₄S₂] requires m/z 404.0739. Found 404.0733.

Reference Example 63

Preparation of N-propionic-acid-methyl-ester-di-dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (132 mg, 0.214 mmol), TCEP (61 mg, 1 eq) and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. The dibromomaleimide (70 mg, 1 eq), in MeOH (5 ml) was then added to the reaction mixture. After 30 mins NaOAc (88 mg, 5 eq), was added to the reaction mixture and the solvent evaporated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄), filtered and concentrated in vacuo. Purification by flash chromatography (silica gel, 0-20% EtOAc-DCM) afforded the desired compound as a yellow gum (32 mg, 20%). ¹HNMR (CDCl₃, 300 MHz), δ8.5 (2H, d J 8.5 Hz aromatic H's), δ8.2 (4H, m aromatic H's), δ7.53 (1H, s CONH), δ7.45 (4H, m, aromatic H's), δ7.1 (2H, d, J 7.5 Hz aromatic H's), δ5.7 (2H, t, J 6.1 SO₂NH), δ3.75 (2H, t, J 7.0 CONCH₂), δ3.6 (3H, s, OCH₃), δ3.2 (4H, m, SCH₂), δ3.18 (4H, m, NHCH₂), δ2.9 (12H, s, NCH₃), δ2.6 (2H, t, J 7.1 NHCH₂); ¹³CNMR (CDCl₃, 75 MHz), δ171.3, 165.9, 135.8, 134.8, 130.5, 129.8, 129.5, 129.4, 128.4, 123.3, 119.0, 115.3, 51.9, 45.5, 43.5, 34.3, 32.6 31.9; IR (cm⁻¹) 3295 (br) 2948 (br) 1702 (s) MS (ES−) m/z relative intensity: 798 (M, 100); Exact mass calculated for [C₃₆H₄₀N₅O₈S₄] requires m/z 798.1760. Found 798.1715.

Reference Example 64

Preparation of N-propionic-acid-methyl-ester-bromo-dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (66 mg, 0.107 mmol), TCEP (31 mg, 1 eq) and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. The dibromomaleimide (70 mg, 0.5 eq), in MeOH (5 ml) was then added to the reaction mixture. After 16 hrs NaOAc (88 mg, 5 eq), was added to the reaction mixture and the solvent evaporated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄), filtered and concentrated in vacuo. Purification by flash chromatography (silica gel, 0-2% MeOH—CHCl₃) afforded the desired compound as a yellow gum (20 mg, 16%). ¹HNMR (CDCl₃, 300 MHz), δ8.5 (1H, m, aromatic H), δ8.2 (2H, m aromatic H's), δ7.5 (2H, m, aromatic H's), δ7.2 (1H, d, J=7.5 Hz aromatic H), δ5.2 (1H, t, J 6.1 SO₂NH), δ3.8 (2H, t, J 7.0 CONCH₂), 63.7 (3H, s, OCH₃), δ3.4 (2H, m, SCH₂), δ3.3 (2H, m, NHCH₂), δ2.9 (6H, s, NCH₃), δ2.6 (2H, t, J 7.1 NHCH₂); ¹³CNMR (CDCl₃, 75 MHz), δ170.95, 165.5, 163.3, 141.6, 134.5, 130.8, 129.8, 129.5, 128.5, 123.2, 118.6, 115.3, 52.0, 45.4, 43.6, 34.8, 32.5 30.6; IR (cm⁻¹) 3296 (br) 2948 (br) 1713 (s)

Reference Example 65

Preparation of N-diethylene-glycol-monomethyl-ether-di-dansyl-cystamine-maleimide

A round bottomed flask was charged with di-dansyl cystamine (155 mg, 0.25 mmol), TCEP (72 mg, 1 eq) and MeOH (10 ml). The reaction mixture was stirred at ambient temperature under argon for 3 hrs. PEG-dibromomaleimide (100 mg, 1 eq), in MeOH (5 ml) was then added to the reaction mixture. After 16 hrs NaOAc (102 mg, 5 eq), was added to the reaction mixture and the solvent evaporated in vacuo. The residue was worked up with DCM and brine. The organic layers were combined, dried (MgSO₄), filtered and concentrated in vacuo. Purification by flash chromatography (silica gel, 0-10% THF-DCM) afforded the desired compound as a yellow gum (13 mg, 6%). ¹HNMR (MeOH, 300 MHz), δ8.5 (2H, m, aromatic H's), δ8.3 (2H, m aromatic H's), δ8.13 (2H, m, aromatic H's), δ7.5 (4H, m, aromatic H's), δ7.2 (2H, m, aromatic H's), δ3.5 (12H, m, CONCH₂, OCH₂), δ3.3 (3H, s, OCH₃), δ3.1 (8H, m, SCH₂, NHCH₂), δ2.8 (12H, s, NCH₃); ¹³CNMR (CDCl₃, 150 MHz), δ167.4, 153.2, 136.9, 136.2, 131.3, 131.2, 130.9, 130.2, 129.6, 124.3, 120.5, 116.4, 72.9, 71.4, 71.3, 71.1, 68.7, 59.1, 45.8, 44.5, 38.98, 36.97; IR (cm⁻¹) 3323 (br) 2946 (br) 2946 (s) 1017 (s) MS (Na+) m/z relative intensity: 882 (M, 100); Exact mass calculated for [C₃₉H₄₉N₅O₉NaS₄] requires m/z 882.2311. Found 882.2294 (Na+).

Reference Example 66

Preparation of Glu-Cys(Mal)-Gly

To glutathione (47 mg, 0.15 mmol) in methanol (3 mL) was added bromomaleimide (30 mg, 0.15 mmol) in methanol (3 mL). After five minutes solvent removed in vacuo to afford the desired compound as a thick colourless oil (62 mg, 0.15 mmol) in 100% yield. δ_H(500 MHz, MeOD) 6.47 (s, 1H, H-12), 4.79 (dd, 1H, J=5.7 and 8.2, H-6), 4.06 (t, 1H, J=6.5, H-2), 3.95 (s, 2H, H₂-8), 3.49 (dd, 1H, J=5.8 and 13.9, HH-10), 3.29 (dd, 1H, J=8.3 and 13.6, HH-10), 2.61 (t, 2H, J=7.1, H₂-4), 2.29-2.15 (m, 2H, H₂-3); δ_C(125 MHz, MeOD) 174.68 (C═O), 172.81 (C═O), 172.39 (C═O), 171.89 (C═O), 171.62 (C═O), 170.59 (C═O), 151.75 (C11), 120.91 (C12), 53.79 (C6), 52.76 (C2), 42.01 (C8), 33.92 (C10) 32.42 (C4), 27.03 (C3); IR (oil, cm⁻¹) 3259 (m), 2928 (m), 1717 (s); MS (ES−) m/z (relative intensity): 401 ([M−H], 100), 272 (30); Exact Mass Calcd for [C₁₄H₁₈N₄O₈S]—H requires m/z 401.0767 Found 401.0773 (ES−); UV (Acetonitrile) ε₂₀₄=8100, ε₂₅₃=5600 and ε₃₄₂=1900 cm⁻¹M⁻¹d³.

Reference Example 67

Preparation of Preparation of Boc-Cys(MeMal)-Phe-ⁱPr

O-(Benzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (344 mg, 0.82 mmol) was added to a stirred solution of (2R)-2-[(tert-butoxycarbonyl)amino]-3-[(1-methyl-2,5-dioxo-2,5-dihydro-1H-pyrrol-3-yl)sulfanyl]propanoic acid (313 mg, 0.95 mmol) and 1-hydroxybenzotriazole hydrate (139 mg) in DMF (2 mL) and the reaction was stirred at 21° C. for 3 mins A solution of (2S)-1-oxo-3-phenyl-1-(propan-2-ylamino)propan-2-ammonium trifluoroacetate (262 mg, 0.82 mmol) in DMF (1.5 mL) was added to the reaction mixture followed by N,N-diisopropylethylamine (294 μL, 1.64 mmol) and the reaction stirred at 21° C. for 4 h. The solvent was removed in vacuo and the residue dissolved in EtOAc (60 mL) and washed with 1 M HCl (×3), H₂O (×1), sat NaHCO₃(×3), 10% LiCl (×1) and sat. NaCl (×1), dried (MgSO₄), filtered and the solvent removed in vacuo. Purification by precipitation (CHCl₃/petroleum ether 40-60) gave the desired compound as a pale brown solid (359 mg, 0.69 mmol, 84% yield): ¹H NMR (600 MHz, CD₃CN, 25° C.) δ 7.32-7.28 (m, 2H), 7.25-7.21 (m, 3H), 7.11 (d, J=7.7 Hz, 1H), 6.46 (d, J=6.3 Hz, 1H), 6.42 (s, 1H), 4.46 (td, J=7.6, 6.5 Hz, 1H), 4.31 (td, J=7.3, 6.4 Hz), 3.88 (septets of doublet, J=6.6, 6.3 Hz, 1H), 3.30 (dd, J=13.7, 5.8 Hz, 1H), 3.16 (dd, J=13.7, 7.4 Hz, 1H), 2.95 (dd, J=13.8, 7.5 Hz, 1H), 2.93 (s, 3H), 1.43 (s, 9H), 1.07 (d, J=6.6 Hz, 3H), 1.02 (d, J=6.6 Hz, 3H); ¹³C NMR (151 MHz, CD₃CN, 25° C.) δ 169.39, 168.84, 168.77, 167.85, 155.17, 149.33, 136.77, 129.10, 127.98, 126.30, 118.57, 79.49, 54.10, 52.62, 40.91, 37.44, 32.43, 27.15, 22.95, 21.24, 21.17; IR (thin film) 3301, 2973, 1770, 1701, 1674, 1641, 1525 cm⁻¹; LRMS (EI) 518 (24%, [M]^+•), 432 (23), 219 (33), 149 (21) 110 (27), 86 (37), 84 (100); HRMS (EI) calcd for C₂₅H₃₄N₄O₆S [M]^+• 518.2194, observed 518.2199.

Reference Example 68

Deprotection of Boc-Cys(MeMal)-Phe-ⁱPr

Tris(2-carboxyethyl)phosphine hydrochloride (138 mg, 0.48 mmol) in 150 mM phosphate buffer (pH 8, 25 mL) was added to a stirred solution of Boc-Cys(MeMal)-Phe-ⁱPr (50 mg, 97 mmol) in MeCN (25 mL) and the reaction stirred at 21° C. for 10 min. Synthesis of Boc-Cys-Phe-ⁱPr was confirmed by LCMS (ES⁻) 408.7 (100%).

Reference Example 69

Cloning and Expression of Grb2-SH2 L111C Mutant

Sequence of Grb2-SH2 L111C (residues 53-163): M G I E M K P H P W F F G K IP R A K A E E M L S K Q R H D G A F L I R E S E S A P G D F S L S V K F G N D V Q H F K V C R D G A G K Y F L W V V K F N S L N E L V D Y H R S T S V S R N Q Q I F L R D I E Q V P Q Q P T Y V Q A G S R S H H H H H H Stop.
Calculated mass=14171
The DNA construct for the Grb2 SH2 domain contained the primary amino acid sequence 53-163 and was cloned on plasmid QE-60 (Qiagen). The Grb2 SH2 L111C mutant was constructed by site-directed mutagenesis (Stratagene Kit) using oligonucleotides coding for the mutated residue. Both constructs were expressed in Escherichia coli (M15[pREP4], Qiagen) using a T5 promoter and a C-terminal 6-His Tag was incorporated for the purification. Cultures (1 L) were grown at 37° C. in T.B. from a single colony, and expression was induced with 1.0 mM IPTG when an O.D._λ600of 0.9 was reached. Cultures were allowed to express protein for roughly 3 h before the cells were pelletised. Pellets were lysed in 0.1M sodium phosphate, 300 mM NaCl, 50 mM imidazole, pH 7.2 containing a protease inhibitor cocktail (Roche). The lysate was centrifuged, and the supernatant was applied to a Ni-NTA column (Qiagen). Grb2-SH2 L111C was eluted from the Ni-NTA column with 0.1M sodium phosphate, 300 mM NaCl, 200 mM imidazole at pH 7.2. The collected Grb2 SH2 L111C was ˜95% pure as visualized by Coomassie-stained SDS-PAGE. Dimerization of Grb2 SH2 domain through domain-swapping has been previously observed. Dimeric and monomeric Grb2-SH2 were separated on a Sephacryl S-100 column (320 mL) that had been pre-equilibrated with 0.1 M sodium phosphate and 150 mM NaCl at pH 8.0. Two peaks eluted, corresponding to the molecular weights of monomer (˜14 kDa) and dimer (˜28 kDa) Grb2-SH2. Almost, 60% of the Grb2-SH2 L111C domain eluted from the column as monomer. Separated monomer and dimer were found to be surprisingly kinetically stable, as very little interconversion was seen over a course of months at 4° C. The monomer was concentrated using Amicon® Ultra-4 centrifugal filter units (Millipore) and the final concentration of the protein was determined by absorbance at 280 nm using the extinction coefficient obtained by McNemar and coworkers (15,600M⁻¹). The protein was frozen at 2 mg/mL concentration in 100 mL aliquots which were thawed as required for experiments. The mass of the monomeric protein (mass 14170) was obtained using ESI-MS.
To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 10 mins after which the mixture was analysed by LC-MS. Analysis showed that a single reaction had occurred yielding a single product with a mass of 14370 showing that C111 was available for functionalisation.

Reference Example 70

Preparation of GrB2-SH2 Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative conversion (mass 14266).
The mixture was treated with Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 10 mins after which the mixture was analysed by LC-MS. Analysis showed that no reaction with Ellman's reagent was evident highlighting that bromomaleimide functionalisation had occurred at C111.

Reference Example 71

Preparation of GrB2-SH2 Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative conversion (mass 14280).
The mixture was treated with Ellman's reagent (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 10 mins after which the mixture was analysed by LC-MS. Analysis showed that no reaction with Ellman's reagent was evident highlighting that N-methylbromomaleimide functionalisation had occurred at C111.

Reference Example 72

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponded to protein/bromomaleimide adduct.
The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14169) in 80% conversion

Reference Example 73

β-Mercaptoethanol-Mediated Reductive Cleavage of GrB2-SH2 Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponded to protein/bromomaleimide adduct.
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14173) in quantitative conversion.

Reference Example 74

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponded to protein/bromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14173) in quantitative conversion.

Reference Example 75

Phosphine-Mediated Reductive Cleavage of GrB2-SH2 Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponded to protein/N-methylbromomaleimide adduct.
The mixture was treated with TCEP.HCl (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-methylbromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14168) in 85% conversion.

Reference Example 76

β-Mercaptoethanol-Mediated Reductive Cleavage of GrB2-SH2 Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14280 which corresponded to protein/N-methylbromomaleimide adduct.
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed that the protein/N-methylbromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14173) in quantitative conversion.

Reference Example 77

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/N-Methylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14280 which corresponded to protein/N-methylbromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed that the protein/N-methylbromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14173) in quantitative conversion.

Reference Example 78

Ethanedithiol-Mediated Cleavage of GrB2-SH2 Domain L111C/bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponded to protein/bromomaleimide adduct.
The mixture was treated with ethanedithiol (5 μL, 282 mM solution in H₂O), vortexed for 1 s and maintained at 37° C. for 4 h. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14173) in quantitative conversion.

Reference Example 79

Preparation of GrB2-SH2 Domain L111C/Bromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponded to protein/bromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide/2-mercaptoethanol adduct had been formed (mass=14345) in 54% yield. The remaining material was GrB2-SH2 domain L111C.

Reference Example 80

Preparation of GrB2-SH2 Domain L111C/N-Methylbromomaleimide/2-Mercaptoethanol Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponded to protein/N-methylbromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product (mass=14359) had been formed in 90% yield. The remaining material was GrB2-SH2 domain L111C.

Reference Example 81

Preparation of GrB2-SH2 Domain L111C/Bromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14265 which corresponds to protein/bromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide/glutathione adduct had been formed (mass=14574) in 44% conversion. The remaining material was GrB2-SH2 domain L111C.

Reference Example 82

Preparation of GrB2-SH2 Domain L111C/N-Methylbromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-methylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed a single protein species of mass 14278 which corresponded to protein/N-methylbromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 3 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-methylbromomaleimide/glutathione adduct had been formed (mass=14588) in 95% conversion. The remaining material was GrB2-SH2 domain L111C.

Reference Example 83

Preparation of GrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14345).

Reference Example 84

2-Mercaptoethanol-Mediated Reductive Cleavage of the GrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with 2-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14171) in quantitative yield yield.

Reference Example 85

Glutathione-Mediated Reductive Cleavage of the GrB2-SH2 Domain L111C/Dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/bromomaleimide adduct had been cleanly cleaved yielding the desired product (mass=14170) in quantitative yield.

Reference Example 86

Preparation of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product had been formed (mass=14573) in quantitative conversion.

Reference Example 87

Preparation of GrB2-SH2 Domain L111C/Dibromomaleimide/β-1-Thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with β-1-thioglucose, sodium salt (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product (mass=14461) was formed in near quantitative yield.

Reference Example 88

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14573).
The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product (mass=14173) was formed in quantitative yield.

Reference Example 89

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct at Physiologically Relevant Glutathione Concentration (5 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14573).
The mixture was treated with glutathione (5 μL, 100 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product (mass=14173) was formed in quantitative yield.

Reference Example 90

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct at Physiologically Relevant Glutathione Concentration (1 mM)

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14573).
The solution of protein/dibromomaleimide/glutathione adduct was subjected to a buffer swap (Micro Bio-Spin 6 Chromatography Column, Bio-Rad) yielding the adduct (95 μL, [adduct] 0.2 mg/mL, 20 mM HEPES, 100 mM KCl, 1 mM MgCl₂, 1 mM EDTA, pH 7.4). To this was added glutathione (5 μL, 20 mM solution in 20 mM HEPES, 100 mM KCl, 1 mM MgCl2, 1 mM EDTA, pH 7.4). The mixture was vortexed for 1 s then maintained at 37° C. for 4 h. Analysis showed that Grb2-SH2 (L111C) was formed (mass)) 14170) in quantitative conversion.

Reference Example 91

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/Glutathione Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with glutathione (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/glutathione adduct was the only protein species present (mass=14573).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that the desired product (mass=14172) was formed in quantitative conversion.

Reference Example 92

Glutathione-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/β-1-thioglucose adduct was the only protein species present (mass=14461).
The mixture was treated with glutathione (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14173) in quantitative conversion.

Reference Example 93

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/Dibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 4 h. Analysis using LC-MS showed a single protein species of mass 14346 which corresponded to protein/dibromomaleimide adduct.
The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/dibromomaleimide/β-1-thioglucose adduct was the only protein species present (mass=14461).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14172) in quantitative conversion.

Reference Example 94

Preparation of GrB2-SH2 Domain L111C/N-Phenylbromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14351).

Reference Example 95

Preparation of GrB2-SH2 Domain L111C/N-Phenyldibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES, 150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14431).

Reference Example 96

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/N-Phenyldibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES, 150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that protein/N-phenyldibromomaleimide adduct had been formed in quantitative yield (mass 14431).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14179) in quantitative conversion.

Reference Example 97

Preparation of GrB2-SH2 Domain L111C/N-Phenyldibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES, 150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/N-phenyldibromomaleimide adduct had been formed in quantitative yield (mass 14431).
The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-phenyldibromomaleimide/β-1-thioglucose adduct was the only protein species present (mass=14547).

Reference Example 98

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/N-Phenyldibromomaleimide/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 20 mM MES, 150 mM NaCl, pH 6) at 0° C. was added N-phenylbromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/N-phenyldibromomaleimide adduct had been formed in quantitative yield (mass 14431).
The mixture was treated with β-1-thioglucose (5 μL, 2.82 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/N-phenyldibromomaleimide/β-1-thioglucose adduct was the only protein species present (mass=14547).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14178) in quantitative conversion.

Reference Example 99

Preparation of GrB2-SH2 Domain L111C/Biotin-PEG-bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added biotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14634).

Reference Example 100

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/Biotin-PEG-bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added biotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/biotin-PEG-bromomaleimide adduct had been formed in quantitative yield (mass 14634).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14180) in quantitative conversion.

Reference Example 101

Preparation of GrB2-SH2 Domain L111C/Biotin-PEG-dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added biotin-PEG-dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 2 h. Analysis using LC-MS showed that the desired product had been formed in >80% yield (mass 14701).

Reference Example 102

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/Biotin-PEG-dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added biotin-PEG-dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/biotin-PEG-dibromomaleimide adduct had been formed in >80% conversion (mass 14701).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 0° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14171) in >80% conversion.

Example 1

Pull-Down and Release of GrB2-SH2 Domain L111C/Biotin-PEG-Bromomaleimide Adduct onto Neutravidin Coated Agarose Beads

To a solution of model protein (200 μL, [Protein] 1.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added biotin-PEG-bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14634).
Protein/biotin-PEG-bromomaleimide adduct (200 μL) and unmodified model protein (200 μL) were washed independently with PBS buffer (3×500 μL) in a concentrator (Vivaspin, cut off 10 k) yielding protein solutions (300 μL) (In). For each of the protein solutions obtained, neutravidin-coated agarose beads (750 μL of 50% aqueous slurry) were washed with PBS (2×500 μL). Protein solution (300 μL) was then added to the beads and the mixture incubated at 4° C. for 30 mins. The mixture was centrifuged and the flow through (FT) collected. The beads were washed with PBS (2×500 μL) and both wash fractions collected (W1 and W2). Protein was released from the beads by incubation in PBS (300 μL) containing β-mercaptoethanol (25 mM) for 2 h at 37° C. The sample was centrifuged and the eluant (El) containing cleaved GrB2-SH2 domain L111C collected. The results are shown in FIG. 1.
The amount of protein recovered was determined as 44% by comparison with a protein series dilution via densitometry. However, correcting for irreversibly physisorbed protein (determined using the unmodified protein control) the corrected recovery was 71%.

Reference Example 103

Preparation of GrB2-SH2 Domain L111C/N-Fluorescein bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluorescein bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in 90% conversion (mass 14597).

Reference Example 104

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/N-Fluorescein bromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluorescein bromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/fluorescein bromomaleimide adduct had been formed in 90% conversion (mass 14597).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14171) in 87% conversion.

Reference Example 105

Preparation of GrB2-SH2 Domain L111C/N-Fluorescein dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added N-fluorescein dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in 61% conversion (mass 14675).

Reference Example 106

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/N-Fluorescein dibromomaleimide Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 7.0) at 0° C. was added N-fluorescein dibromomaleimide (5 μL, 2.82 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 0° C. for 1 h. Analysis using LC-MS showed that the protein/fluorescein dibromomaleimide adduct had been formed in 61% conversion (mass 14597).
The mixture was treated with β-mercaptoethanol (5 μL, 282 mM solution in H₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C. for 4 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14171) in 85% conversion.

Reference Example 107

Preparation of GrB2-SH2 Domain L111C/BrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14348).

Reference Example 108

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/BrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the protein/BrDDPD adduct had been formed in quantitative yield (mass 14348).
The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150 mM NaCl, pH 8.0) and treated with β-mercaptoethanol (5 μL, 2.82 M solution in H₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14180) in quantitative conversion.

Reference Example 109

Preparation of GrB2-SH2 Domain L111C/DiBrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14427).

Reference Example 110

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/DiBrDDPD Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the protein/DiBrDDPD adduct had been formed in quantitative yield (mass 14427).
The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150 mM NaCl, pH 8.0) then treated with β-mercaptoethanol (5 μL, 2.82 M solution in H₂O) at 37° C.
The mixture was vortexed for 1 s and maintained at 37° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14180) in quantitative conversion.

Reference Example 111

Preparation of GrB2-SH2 Domain L111C/DiBrDDPD/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the protein/DiBrDDPD adduct had been formed in quantitative yield (mass 14427).
The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150 mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at RT for 1 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/DiBrDDPD/β-1-thioglucose adduct was the only protein species present (mass=14543).

Reference Example 112

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2

Domain L111C/DiBrDDPD/β-1-thioglucose Adduct
To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added DiBrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the protein/DiBrDDPD adduct had been formed in quantitative yield (mass 14427).
The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150 mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at RT for 1 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/DiBrDDPD/β-1-thioglucose adduct was the only protein species present (mass=14543).
The mixture was treated with β-mercaptoethanol (5 μL, 2.82 M solution in H₂O) at RT. The mixture was vortexed for 1 s and maintained at RT for 30 mins after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14180) in quantitative conversion.

Reference Example 113

Preparation of GrB2-SH2 Domain L111C/BrDDPD/β-1-thioglucose Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added BrDDPD (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the protein/BrDDPD adduct had been formed in quantitative yield (mass 14348).
The mixture was dialysed for 40 h at 4° C. (100 mM sodium phosphate, 150 mM NaCl, pH 8.0) then treated with β-1-thioglucose (5 μL, 28.2 mM solution in H₂O) at 0° C. The mixture was vortexed for 1 s and maintained at 37° C. for 1 h after which the mixture was analysed by LC-MS. Analysis showed that the protein/BrDDPD/β-1-thioglucose adduct was formed in 17% conversion (mass=14543).

Reference Example 114

Preparation of GrB2-SH2 Domain L111C/Z-2,3-Dibromo-but-2-enedioic acid dimethyl ester Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Z-2,3-dibromo-but-2-enedioic acid dimethyl ester (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed (mass 14440).

Reference Example 115

β-Mercaptoethanol-Mediated Cleavage of GrB2-SH2 Domain L111C/Z-2,3-Dibromo-but-2-enedioic acid dimethyl ester Adduct

To a solution of model protein (100 μL, [Protein] 2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0° C. was added Z-2,3-dibromo-but-2-enedioic acid dimethyl ester (5 μL, 282 mM solution in DMF). The mixture was vortexed for 1 s then maintained at 37° C. for 1 h. Analysis using LC-MS showed that the desired product had been formed in quantitative yield (mass 14370).
The mixture was treated with β-mercaptoethanol (5 μL, 2.82 M solution in H₂O) at 37° C. The mixture was vortexed for 1 s and maintained at 37° C. for 2 h after which the mixture was analysed by LC-MS. Analysis showed that desired product was formed (mass=14180) in quantitative conversion.

Reference Example 116

Modification and Regeneration of Somatostatin

Preparation of Reduced Somatostatin

Lyophilised somatostatin (mass=1638) was solubilised in buffer (50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) to yield a concentration of 152.6 μM (0.25 mg/ml) and reduced with 1.1 equiv of TCEP for 1 h at ambient temperature. Completeness of the reduction was confirmed by addition of 4 equiv of dibromomaleimide to an aliquot of the sample and analysis by LC-MS.
Bridging of Somatostatin with Halomaleimides and Derivatives
Reduced somatostatin was generated as described. 1.1 equiv of the halomaleimides or dibromomaleimide derivates (100× stocks in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-15.0% DMF) were added at ambient temperature and the generation of product monitored over 1 h by LC-MS. The results are shown in FIG. 2.
Bridging of Somatostatin with Dithiomaleimides
Reduced somatostatin was generated as described. Various amounts of dithiomaleimide (100× stocks in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) were added at ambient temperature and the generation of product monitored over 1 h by LC-MS. The results are shown in FIG. 3.
Modification of Somatostatin with Bromomaleimide
Reduced somatostatin was generated as described. 2.1 equiv of bromomaleimide (100× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 7.5% DMF) were added at ambient temperature and complete conversion to the di-addition product observed by LC-MS within 1 h.
Modification of Somatostatin with Dibromomaleic Anhydride
Reduced somatostatin was generated as described. 5 equiv of dibromomaleic anhydride (in DMF) were added and the generation of products monitored by LC-MS. 17.3% bridged somatostatin were generated within 90 min
Cleavage of Bridged Somatostatin with Various Reducing Agents
Maleimide bridged somatostatin was prepared as described. 100 equiv of various reducing agents (1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and the generation of unmodified peptide and side products (mixed disulfides of the reducing agents with the free peptide-cysteines) monitored at 4° C. over 2 d by LC-MS. Mixed disulfides of somatostatin with GSH could only be detected by MALDI-TOF MS. The results are shown in FIG. 4.
Cleavage of Bridged Somatostatin with Various Amounts of DTT and 2-mercaptoethanol
Maleimide bridged somatostatin was prepared as described. Various amounts of DTT or 2-mercaptoethanol (1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and the generation of unmodified peptide and side products (mixed disulfides of the reducing agents with the free peptide-cysteines) monitored at 4° C. over 6 h by LC-MS. The results are shown in FIG. 5.

Catalysed Cleavage of Bridged Somatostatin

Maleimide bridged somatostatin was prepared as described. 20 equiv of 2-mercaptoethanol (1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added followed by either buffer or 5 equiv of sodium iodide or benzeneselenol (100× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 7.5% DMF) and the generation of unmodified peptide and side products (mixed disulfides of 2-mercaptoethanol or benzeneselenol with the free peptide-cysteines) monitored at ambient temperature over 20 min by LC-MS. The results are shown in FIG. 6.

Cleavage of N-Functionalised Maleimide Bridged Somatostatin

Somatostatin was reduced and bridged with N-functionalised maleimide derivates as described. 100 equiv of 2-mercaptoethanol (1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and the generation of unmodified peptide and side products (mixed disulfides of 2-mercaptoethanol with the free peptide-cysteines) monitored at 4° C. over 2 d by LC-MS. The results are shown in FIG. 7.

Cleavage of Di-Addition Product of Monobromomaleimide to Somatostatin

Reduced somatostatin was reacted with 2.1 equiv of monobromomaleimide to generate the di-addition product. Next 100 equiv of 2-mercaptoethanol (1000× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) were added and the generation of mono-addition product, unmodified peptide and side products (mixed disulfides of 2-mercaptoethanol with the free peptide-cysteines) monitored at ambient temperature over 2.5 h by LC-MS. The results are shown in FIG. 8.

Comparable In Situ Bridging of Somatostatin

To somatostatin were added various amounts of dithiomaleimides (100× stock in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) and the reaction was incubated at ambient temperature for 10 min. Next various amounts of TCEP or benzeneselenol (100× stocks, freshly prepared in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) were added and the generation of bridged somatostatin was monitored over 1 h at ambient temperature by LC-MS. The results are shown in FIG. 9.

In Situ Pegylation of Somatostatin

To somatostatin were added either 5 equiv of N-PEG5000-dithiophenolmaleimide or 10 equiv of N-PEG5000-dithiophenolmaleimide (100× stocks in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) and the reaction was incubated at ambient temperature for 10 min. Next 3 equiv of TCEP respectively 5 equiv of benzeneselenol (100× stocks, freshly prepared in 50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5-7.5% DMF) were added and the generation of PEGylated somatostatin was monitored over 2 h at ambient temperature by LC-MS. The results are shown in FIG. 10.
Modification of Somatostatin with DiBrDDPD
Lyophilized somatostatin (mass=1638) was solubilized in buffer (50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) to yield a concentration of 152.6 μM (0.25 mg/mL) and reduced with 1.1 equiv of TCEP for 1 h at 21° C. Completeness of the reduction was confirmed by LCMS (mass=1640). DiBrDDPD (100 mol eq) was added and the reaction maintained at 21° C. for 10 mins Somatostatin/DiBrDDPD adduct was observed to form quantitative conversion (mass=1803).

Demonstration the Retained Biological Activity of Bridged Somatostatins Using Patch-Clamping

To examine whether the bridging modification had a deleterious effect on the activity of the resultant somatostatin analogues we tested the dibromomaleimide bridged analogue, the PEGylated-dibromomaleimide bridged analogue, and the fluorescein dibromomaleimide-bridged analogue via a patch clamp assay. HEK 293 cells expressing HKIR3.1/3.2 channel and human somatostatin receptor 2 were treated with these compounds, and whole cell patch-clamp current recordings taken. All three analogues induced a robust activation of GIRK currents in an amplitude comparable to somatostatin iteslf. As a control when cells were treated with Pertussis toxin, or by the GIRK inhibitor Tertiapin Q, currents were largely inhibited. This data confirms that the bridged somatostatin analogues retain the biological activity of somatostatin for agonism of the somatostatin receptor 2.

Cell Culture

Cell-culture methods and the generation of stable cell lines were carried out as described in J Biol Chem 275, 921-9 (2000). HEK293 cells (human embryonic kidney cell line) stably expressing Kir3.1 and Kir3.2A channels were maintained in minimum essential medium supplemented with 10% foetal calf serum and 727 μg of G418 (Invitrogen), at 37° C. in humidified atmosphere (95% O₂, 5% CO₂). Cells were transiently transfected with SSTR2DNA (Missouri S&T cDNA Resource Center) along with pEGFP-N1 (Clontech) for visualization of transfected cells using epifluorescence. Transfections were performed with 5 μl of Fugene HD (Roche) and 800 ng SSTR2-DNA and 40 ng EGFP-DNA per 97 μl of cell culture medium (containing no serum or antibiotics).

Preparation of Somatostatin and Analogues for Patch-Clamp Experiments

Bridged somatostatins were prepared as described above. Somatostatin and its analogues were dialysed for 24 h at 4° C. in buffer (50 mM sodium phosphate, pH 6.2) to remove the organic solvents. After dialysis the concentration was determined and the peptides stored at 4° C. A final concentration of 20 μM somatostatin and analogues were used (dilution was done in the extracellular patch-clamp buffer).

Electrophysiology

Whole cell patch-clamp current recordings were performed with an Axopatch 200B amplifier (Axon Instruments) using fire-polished pipettes with a resistance of 3-4 MΩ pulled from filamented borosilicated glass capillaries (Harvard Apparatus, 1.5 mm OD×1.17 mm ID). Data was acquired and analysed via a Digidata 1322A interface (Axon Instruments) and pCLAMP software (version 8.1, Axon Instruments). A fast perfusion system was used to apply somatostatin and analogues (Rapid Solution Changer, RSC-160, Bio-Logic France). Cells were clamped at −60 mV. The extracellular solution was (mM): NaCl 80, KCl 60, CaCl₂2, MgCl ₂1, HEPES 10, NaH₂PO₄0.33, glucose 10, pH 7.4; while the intracellular solution was (mM): K gluconate 110, KCl 20, NaCl 10, MgCl ₂1, MgATP 2, EGTA 2 GTP 0.3, pH 7.4. After agonist application, current activated with a delay “lag” followed by a rapid rise to peak amplitude “time to peak”. After removal of the agonist, the current decays back to baseline. For each cell it was assessed if flow artifacts resulting from the pressure of drug application were present. This was done by applying bath solution from one of the sewer pipes at the beginning of the recordings. Tertiapin, an inhibitor of GIRK current (Alomone), was used at a final concentration of 100 nM. Cells were incubated overnight with pertussis toxin (Sigma, 100 ng/ml), an inhibitor of Gi/o proteins. Drugs were prepared as concentrated stocks solutions and kept at −20° C.
The results are shown in FIGS. 11 and 12.

Reference Example 117

Preparation of Propylaminomaleimide

To propylamine (75 μL, 1.09 mmol) and sodium acetate (92 mg, 1.12 mmol) in methanol (15 mL) was added bromomaleimide (200 mg, 1.12 mmol) dropwise in methanol (15 mL). After 10 minutes, solvent was removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded the desired compound as a bright yellow waxy solid (82 mg, 0.53 mmol) in 49% yield. δ_H(500 MHz, CDCl₃) 7.36 (s, 1H, NH), 5.45 (s, 1H, NH), 4.80 (d, 1H, J=1.3, H-5), 3.14 (dt, 2H, J=6.2 and 7.2, H₂-3), 1.71-1.63 (m, 2H, H₂-2), 0.99 (t, 3H, J=7.4, H₃-1); δ_C(125 MHz, CDCl₃) 172.31 (C═O), 167.73 (C═O), 149.83 (C4), 85.29 (C5), 46.16 (C3), 21.91 (C2), 11.42 (C1); IR (solid, cm⁻¹) 3190 (m), 2962 (m), 1693 (m), 1627 (s); MS (EI) m/z (relative intensity): 154 (M+, 60), 125 (98), 84 (100); Exact Mass Calcd for [C₇H₁₀N₂O₂]+ requires m/z 154.0737 Found 154.0734 (EI); UV (Acetonitrile) ε₂₄₀=7400 and ε₃₄₈=5700 cm⁻¹M⁻¹d³.

Reference Example 118

Preparation of But-3-enylaminomaleimide

To 3-butenylamine hydrochloride (200 mg, 1.12 mmol) and sodium acetate (184 mg, 2.24 mmol) in methanol (15 mL) was added bromomaleimide (200 mg, 1.12 mmol) dropwise in methanol (15 mL). After 10 minutes, solvent was removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded the desired compound as a bright yellow waxy solid (142 mg, 0.85 mmol) in 76% yield. δ_H(500 MHz, CDCl₃) 7.10 (s, 1H, NH), 5.77 (tdd, 1H, J=6.9, 10.7 and 17.4, H-2), 5.38 (s, 1H, NH), 5.18-5.15 (m, 2H, H₂-1), 4.83 (d, 1H, J=1.3, H-6), 3.24 (t, 2H, J=6.7, H₂-4), 2.40 (dtd, 2H, J=1.2, 6.8 and 6.9, H₂-3); δ_C(125 MHz, CDCl₃) 171.94 (C═O), 167.45 (C═O), 149.53 (C5), 133.89 (C2), 118.51 (C1), 85.80 (C6), 43.30 (C4), 32.68 (C3); IR (solid, cm⁻¹) 3290 (m), 1703 (m), 1629 (s); MS (ES−) m/z (relative intensity): 165 ([M−H], 100); Exact Mass Calcd for [C₈H₁₀N₂O₂]—H requires m/z 165.0659 Found 165.0664 (ES−); m.p. 68-76° C.; UV (Acetonitrile) ε₂₄₁=8300 and ε₃₄₈=6100 cm⁻¹M⁻¹d³.

Reference Example 119

Preparation of N-Methyl propylaminomaleimide

To propylamine (52 μL, 0.78 mmol) and sodium acetate (64 mg, 0.78 mmol) in methanol (30 mL) was added N-methylmonobromomaleimide (150 mg, 0.78 mmol) dropwise in methanol (30 mL). After 10 minutes, solvent was removed in vacuo and purification by flash chromatography (10% ethyl acetate in petroleum ether) afforded the desired compound as a bright yellow waxy solid (41 mg, 0.24 mmol) in 31% yield. δ_H(500 MHz, CDCl₃) 5.43 (s, 1H, NH), 4.80 (s, 1H, H-2), 3.16-3.13 (m, 2H, H₂-9), 2.98 (s, 3H, H₃-6), 1.71-1.64 (m, 2H, H₂-8), 0.99 (t, J=7.5, H₃-7); δ_C(125 MHz, CDCl₃) 172.71 (C═O), 167.66 (C═O), 149.51 (C3), 83.84 (C2), 46.01 (C9), 23.44 (C6), 21.87 (C8), 11.38 (C7); IR (film, cm⁻¹) 3317 (m), 2944 (w), 1698 (s), 1651 (s); MS (EI) m/z (relative intensity): 168 (M+, 70), 139 (100), 111 (40); Exact Mass Calcd for [C₈H₁₂N₂O₂]+ requires m/z 168.0893 Found 168.0887 (EI); UV (Acetonitrile) ε₂₁₀=15900, ε₂₄₀=2800, ε₂₈₃=500 and ε₃₆₈=500 cm⁻¹M⁻¹d³.

Reference Example 120

Preparation of 2,9-azatricyclo[5,3,0,0^10-4]decan-1,3-dione

But-3-enylaminomaleimide (42 mg, 0.25 mmol) was dissolved in acetonitrile (25 mL), to provide a 0.01M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 4 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in ethyl acetate to 5% methanol in ethyl acetate) afforded the desired compound as an off-white solid (39 mg, 0.23 mmol) in 93% yield. δ_H(500 MHz, CDCl₃) 3.50 (ddd, 1H, J=2.6, 4.8 and 11.8, HH-8), 3.18-3.12 (m, 2H, HH-8 and H-6), 2.98 (dd, 1H, J=3.9 and 10.7, H-4), 2.21 (ddd, 1H, J=4.0, 8.6 and 13.2, HH-5), 2.01 (ddd, 1H, 5.8, 10.5 and 13.4, HH-5), 1.79 (m, 2H, H₂-7); δ_C(125 MHz, CDCl₃) 179.04 (C═O), 178.95 (C═O), 70.85 (C10), 48.43 (C8), 44.25 (C4), 43.82 (C6), 32.93 (C7) 24.96 (C5); IR (solid, cm⁻¹) 3198 (m), 2944 (m), 1701 (s); MS (EI) m/z (relative intensity): 166 (M+, 45), 125 (100); Exact Mass Calcd for [C₈H₁₀N₂O₂]+ requires m/z 166.07387 Found 166.07386 (EI); m.p. 110-113° C.

Reference Example 121

Preparation of (4SR,6RS,7SR)2-Aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (22.5 mL) and acrylonitrile (2.5 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution 10% ethyl acetate in petroleum ether to 50% ethyl acetate in petroleum ether) afforded (4SR,6RS,7SR)2-Aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione as a thick colourless oil (9 mg, 0.034 mmol) in 29% yield and (4RS,7RS,5RS)2-aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione as a thick colourless oil (12 mg, 0.045 mmol) in 39% yield.
(4SR,6RS,7SR)2-Aza-4-hexylsulfanyl-6-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione δ_H(500 MHz, CDCl₃) 3.53 (dt, 1H, J=1.4 and 8.1, H-6), 3.16-3.10 (m, 2H, HH-5 and H-7), 2.89-2.80 (m, 2H, H₂-13), 2.56-2.50 (m, 1H, HH-5), 1.67-1.55 (m, 4H, H₂-12 and H₂-11), 1.42-1.37 (m, 2H, H₂-10), 1.33-1.27 (m, 2H, H₂-9), 0.89 (t, 3H, J=6.9, H₃-8); δ_C(125 MHz, CDCl₃) 174.49 (C═O), 172.91 (C═O), 116.82 (C4), 52.38 (C14), 44.16 (C6), 31.33 (C13), 30.87 (C7), 30.29 (CH₂), 29.26 (CH₂), 28.64 (CH₂), 25.92 (CH₂), 22.82 (C5), 14.11 (C8); IR (oil, cm⁻¹) 3223 (w), 2926 (w), 1778 (w), 1714 (s); MS (CI+) m/z (relative intensity): 267 ([M+H], 40), 213 (70), 180 (100); Exact Mass Calcd for [C₁₃H₁₈N₂O₂S]+H requires m/z 267.1167 Found 267.1175 (CI+).
(4RS,7RS,5RS)2-Aza-4-hexylsulfanyl-5-carbonitrile-bicyclo[3.2.0]heptan-1,3-dione δ_H(500 MHz, CDCl₃) 3.66 (dd, 1H, J=6.0 and 9.5, H-5), 3.23 (dd, 1H, J=5.2 and 10.9, H-7), 3.01-2.82 (m, 3H, HH-6 and H₂-13), 2.67 (ddd, 1H, J=5.3, 9.6 and 14.7, HH-6), 1.65-1.60 (m, 2H, H₂-12), 1.42-1.36 (m, 2H, H₂-11), 1.32-1.27 (m, 4H, H₂-9 and H₂-10), 0.88 (t, 3H, J=6.8, H₃-8); δ_C(125 MHz, CDCl₃) 175.08 (C═O), 174.82 (C═O), 117.13 (C4), 51.24 (C14), 44.26 (C5), 31.36 (C13), 30.96 (C7), 29.82 (CH₂), 29.19 (CH₂), 28.62 (CH₂), 25.72 (C6), 22.58 (CH₂), 14.26 (C8); IR (oil, cm⁻¹) 3247 (w), 2927 (w), 1717 (s); MS (CI) m/z (relative intensity): 267 ([M+H], 75), 214 (100), 180 (70); Exact Mass Calcd for [C₁₃H₁₈N₂O₂S]+H requires m/z 267.1167 Found 267.1158 (CI).

Reference Example 122

Preparation of (5RS,9SR)2-Aza-4-hexylsulfanyl-2-aza-tricylo[3.5.0.0^5.9]di-1,3-one

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (22.5 mL) and cyclopentene (3 mL, 36 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution 10% ethyl acetate in petroleum ether to 50% ethyl acetate in petroleum ether) afforded the desired compound as a thick colourless oil (12 mg, 0.045 mmol) in 77% yield, a 1:1 mix of two inseparable diastereomers. COSY analysis shows that certain signal arise from the same compound, denoted by subscripts ‘a’ and ‘b’, but the specific identity of each diastereomer is unknown. Overlap of signals prevents NOe analysis. δ_H(500 MHz, CDCl₃) 3.15-3.07 (m, 2H, H-5_aand H-10_a), 3.00 (t, 1H, J=6.8, H-5_b), 2.94 (td, 1H, J=3.9 and 6.6, H-9_b), 2.87-2.82 (m, 2H, H-9_aand HH-16_a), 2.64-2.59 (m, 1H, HH-16_b), 2.52-2.47 (m, 3H, H-10_b, HH-16_aand HH-16_b), 2.07 (dd, 2H, J=6.3 and 6.9, HH-15_aand HH-15_b), 1.96-1.89 (m, 2H, HH-15_aand HH-15_b) 1.88-1.82 (m, 4H, H₂-7_aand H₂-7_b) 1.64-1.50 (m, 8H, H₂-6_a, H₂-6_b, H₂-8_aand H₂-8_b), 1.38-1.25 (m, 12H, H₂-12_a, H₂-12_b, H₂-13_a, H₂-13_b, H₂-14_aand H₂-14_b), 0.89-0.86 (m, 6H, H₃-11_aand H₃-11_b); δ_C(125 MHz, CDCl₃) 179.09 (C═O), 177.12 (C═O), 176.93 (C═O), 171.83 (C═O), 51.31 (C4), 51.33 (C4), 50.68 (C10), 45.32 (C10), 43.26 (C9), 41.70 (C5), 32.38 (CH₂), 30.98 (CH₂), 30.95 (CH₂), 30.78 (CH₂), 28.92 (CH₂), 28.50 (CH₂), 28.30 (CH₂), 28.22 (CH₂), 28.10 (CH₂), 28.13 (CH₂), 25.09 (CH₂), 22.14 (CH₂), 22.11 (CH₂), 13.66 (2×C11) Several carbon signals are missing due to overlap of the diastereomers; IR (oil, cm⁻¹) 3120 (w), 2927 (m), 1711 (s), 1627 (s); MS (ES−) m/z (relative intensity): 280 ([M−H], 50), 212 (100); Exact Mass Calcd for [C₁₅H₂₃NO₂S]-H requires m/z 280.1371 Found 280.1382 (ES−).

Reference Example 123

Preparation of 4-Hexylsulfanyl-1-phenyl-1,7-dihydro-2H-azepine-3,6-dione and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (133 μL, 1.2 mmol) added and the solution irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded 4-hexylsulfanyl-1-phenyl-1,7-dihydro-2,1-azepine-3,6-dione as a thick colourless oil (11 mg, 0.034 mmol) in 30% yield and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a thick colourless oil (26 mg, 0.082 mmol) in 70% yield

4-Hexylsulfanyl-1-phenyl-1,7-dihydro-2,1-azepine-3,6-dione

δ_H(600 MHz, CDCl₃) 7.34-7.24 (m, 5H, 5×Ar—H), 6.15 (d, 1H, J=1.5, H-5), 4.11 (t, 1H, J=7.7, H-1), 3.01 (ddd, 1H, J=1.5, 7.8 and 15.8, HH-7), 2.96 (dd, 1H, J=7.8 and 15.6, HH-7), 2.36-2.26 (m, 2H, H₂-13), 1.50-1.41 (m, 2H, H₂-12), 1.35-1.33 (m, 6H, H₂-9, H₂-10 and H₂-11), 0.85 (t, 3H, J=7.0, H₃-8); δ_C(150 MHz, CDCl₃) 170.96 (C═O), 169.95 (C═O), 147.26 (C4), 141.10 (C14), 129.41 (C5), 128.89 (2×Ar—H), 127.86 (C17), 127.71 (2×Ar—H), 47.24 (C1), 32.47 (C13), 31.43 (CH₂), 29.19 (CH₂), 28.61 (CH₂), 22.60 (CH₂), 14.10 (C8); IR (oil, cm⁻¹) 3288 (w), 2928 (w), 1775 (w), 1717 (s); MS (FAB+) m/z (relative intensity): 340 ([M+Na], 20), 329 (35), 207 (20), 176 (100); Exact Mass Calcd for [C₁₆H₂₃NO₂S]+Na requires m/z 340.1347 Found 340.1351 (FAB+).

(4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.77 (s, 1H, NH), 7.39-7.31 (m, 5H, 5×Ar—H), 4.05 (t, 1H, J=8.8, H-5), 3.17 (dd, 1H, J=3.4 and 10.9, H-7), 3.04 (ddd, 1H, J=8.4, 11.1 and 12.7, HH-6), 2.63 (ddd, 1H, J=3.6, 9.0 and 12.8, HH-6), 2.43 (ddd, 1H, J=6.7, 7.9 and 11.3, HH-17), 2.13 (ddd, 1H, J=6.6, 8.0 and 11.3, HH-17), 1.30-1.08 (m, 8H, H₂-13, H₂-14 and H₂-15 and H₂-16), 0.83 (t, 3H, J=7.3, H₃-12); δ_C(150 MHz, CDCl₃) 178.76 (C═O), 177.62 (C═O), 136.51 (C11), 128.77 (2×Ar—H), 128.70 (2×Ar—H), 128.03 (C8), 57.17 (C4), 45.70 (C5), 43.87 (C7), 31.26 (C17), 28.70 (CH₂), 28.65 (CH₂), 28.52 (CH₂), 26.22 (C6), 22.46 (CH₂), 14.10 (C12); IR (oil, cm⁻¹) 3218 (w), 2926 (w) 1771 (m), 1703 (s); MS (FAB+) m/z (relative intensity): 340 ([M+Na], 20), 199 (25), 176 (100); Exact Mass Calcd for [C₁₆H₂₃NO₂S]+Na requires m/z 340.1347 Found 340.1357 (FAB+).

Reference Example 124

Preparation of (4RS,7SR,5RS)2-Aza-4-hexylsulfanyl-5-carboxylic acid methyl ester-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (21.9 mL) and methyl acrylate (3.1 mL, 36 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 50% ethyl acetate in petroleum ether) afforded the desired compound as a thick colourless oil (17 mg, 0.056 mmol) in 48% yield. δ_H(600 MHz, CDCl₃) 8.50 (s, 1H, NH), 3.81 (s, 3H, H₃-8), 3.57 (dd, 1H, J=5.8 and 8.5, H-5), 3.18 (dd, 1H, J=5.0 and 10.7, H-7), 3.11 (ddd, 1H, J=5.5, 11.0 and 12.9, HH-6), 2.73 (dt, 1H, J=7.5 and 11.5, HH-15), 2.64 (dt, 1H, J=7.5 and 11.5, HH-15), 2.29 (ddd, 1H, J=5.2, 8.5 and 13.2, HH-6), 1.52-1.47 (m, 2H, H₂-14), 1.35-1.30 (m, 2H, H₂-13), 1.29-1.21 (m, 4H, H₂-11 and H₂-12), 0.87 (t, 3H, J=6.7, H₃-10); δ_C(150 MHz, CDCl₃) 176.65 (C═O), 171.13 (C═O), 170.48 (C═O), 52.59 (C8), 52.39 (C4), 44.56 (C7), 44.06 (C5), 31.41 (C15), 29.73 (CH₂), 29.16 (CH₂), 28.68 (CH₂), 23.57 (C12), 22.57 (C11), 14.11 (C10); IR (oil, cm⁻¹) 3244 (w), 2928 (w) 1778 (w), 1714 (s); MS (FAB+) m/z (relative intensity): 322 ([M+Na], 100), 300 (30), 214 (25); Exact Mass Calcd for [C₁₄H₂₁NO₄SN]+Na requires m/z 322.1089 Found 322.1082 (FAB+).

Reference Example 125

Preparation of (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-carboxylic acid phenyl ester-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, phenyl acrylate (160 μL, 1.20 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a thick colourless oil (21 mg, 0.058 mmol) in 48% yield and hexylsulfanylmaleimide dimer (12 mg, 0.028 mmol) in 47% yield. δ_H(600 MHz, CDCl₃) 8.41 (s, 1H, NH), 7.41-7.39 (m, 2H, 2×Ar—H), 7.27-7.25 (m, 1H, H-8), 7.20 (d, 1H, J=7.8, 2×Ar—H), 3.80 (dd, 1H, J=5.1 and 8.5, H-5), 3.29 (dd, 1H, J=5.1 and 10.7, H-7), 3.20 (ddd, 1H, J=5.5, 10.9 and 13.0, HH-6), 2.62 (dt, 1H, J=7.5 and 11.5, HH-18), 2.73 (dt, 1H, J=7.5 and 11.5, HH-18), 2.40 (ddd, 1H, J=5.6, 8.8 and 13.5, HH-6), 1.54-1.48 (m, 2H, H₂-17), 1.33-1.16 (m, 6H, H₂-14, H₂-15 and H₂-16), 0.84 (t, 3H, J=6.9, H₃-13); δ_C(150 MHz, CDCl₃) 176.35 (C═O), 176.19 (C═O), 168.85 (C═O), 150.66 (C11), 129.64 (2×Ar—H), 126.34 (C8), 121.54 (2×Ar—H), 52.59 (C4), 44.78 (C7), 44.17 (C5), 31.36 (CH₂), 29.94 (C18), 29.09 (CH₂), 28.68 (CH₂), 23.83 (C6), 22.56 (CH₂), 14.08 (C13); IR (oil, cm⁻¹) 3213 (w), 2927 (w) 1757 (m), 1715 (s); MS (CI+) m/z (relative intensity): 362 ([M+H], 35), 268 (100), 149 (25); Exact Mass Calcd for [C₁₉H₂₃NO₄S]+H requires m/z 362.1426 Found 362.1431 (CI+).

Reference Example 126

Preparation of (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(p-amino)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, 4-vinyl aniline (136 μL, 1.2 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a thick colourless oil (7 mg, 0.021 mmol) in 17% yield. δ_H(600 MHz, CDCl₃) 8.17 (s, 1H, NH), 7.10 (d, 2H, J=8.5, 2×Ar—H), 6.67 (d, 2H, J=8.5, 2×Ar—H), 3.94 (t, 1H, J=9.0, H-5), 3.13 (dd, 1H, J=3.7 and 11.1, H-7), 2.98 (ddd, 1H, J=8.8, 11.2 and 12.9, HH-6), 2.58 (ddd, 1H, J=3.5, 9.1 and 12.8, HH-6), 2.42 (dt, 1H, J=7.5 and 11.5, HH-17), 2.17 (dt, 1H, J=7.5 and 11.5, HH-17), 1.34-1.29 (m, 2H, H₂-16), 1.25-1.11 (m, 6H, H₂-13, H₂-14 and H₂-15), 0.80 (t, 3H, J=7.4, H₃-12); δ_C(150 MHz, CDCl₃) 178.64 (C═O), 177.48 (C═O), 146.28 (C8), 129.81 (2×Ar—H), 126.20 (C11), 114.81 (2×Ar—H), 57.97 (C4), 45.48 (C5), 43.79 (C7), 31.35 (CH₂), 29.07 (CH₂), 28.65 (C17), 26.41 (C6), 22.54 (CH₂), 14.12 (C12); IR (oil, cm⁻¹) 3214 (w), 2928 (w) 1769 (m), 1715 (s); MS (CI+) m/z (relative intensity): 333 ([M+H], 55), 119 (100); Exact Mass Calcd for [C-₁₈H₂₄N₂O₂S]+H requires m/z 333.1637 Found 333.1642 (CI+).

Reference Example 127

Preparation of 4-Hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2H-azepine-3,6-dione, (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, 3-nitrostyrene (136 μL, 1.2 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded 4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2,1-azepine-3,6-dione as a thick colourless oil (23 mg, 0.063 mmol) in 55% yield, (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione as a thick colourless oil (0.5 mg, 0.001 mmol) in 1% yield (alongside 4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2,1-azepine-3,6-dione), and (4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione as a thick colourless oil (12 mg, 0.33 mmol) in 21% yield (alongside dimer).

2-Aza-4-hexylsulfanyl-1-(m-nitro)phenyl-1,7-dihydro-2,1-azepine-3,6-dione

δ_H(600 MHz, CDCl₃) 8.22 (s, 1H, H-16), 8.14 (d, 1H, J=8.5, Ar—H), 7.68 (t, 1H, J=7.6, Ar—H), 7.53 (t, 1H, J=7.8, H-19), 7.20 (s, 1H, NH), 6.29 (s, 1H, H-5), 4.25 (t, 1H, J=7.9, H-1), 3.03 (dd, 1H, J=8.4 and 15.4, HH-7), 2.98 (dd, 1H, J=7.4 and 15.2, HH-7), 2.37-2.26 (m, 2H, H₂-13), 1.30-1.24 (m, 2H, H₂-12), 1.21-1.08 (m, 6H, 3×CH₂), 0.81 (t, 3H, J=7.1, H₃-8); δ_C(150 MHz, CDCl₃) 170.70 (C═O), 169.54 (C═O), 148.60 (C16), 146.35 (C4), 143.94 (C14), 133.83 (Ar—H), 129.96 (Ar—H), 129.86 (C5), 122.96 (Ar—H), 122.59 (Ar—H), 46.78 (C1), 32.52 (C7), 31.56 (CH₂), 31.37 (C13), 29.03 (CH₂), 28.52 (CH₂), 22.57 (CH₂), 14.11 (C8); IR (oil, cm⁻¹) 3282 (w), 2928 (m) 1775 (w), 1717 (s); Mass ion not found.

(4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione signals are bold

δ_H(600 MHz, CDCl₃) 8.59 (s, 0.2H, NH), 8.22-8.16 (m, 0.4H, 2×Ar—H), 8.15 (d, 1H, J=8.4, H-10), 8.07 (s, 1H, H-8), 7.84 (s, 1H, NH), 7.66 (d, 0.2H, J=7.5, Ar—H), 7.62 (d, 1H, J=7.6, H-12), 7.56 (t, 0.2H, J=8.0, H-11), 7.53 (t, 1H, J=8.0, H-11), 4.14 (t, 0.2H, J=8.6, H-5), 4.10 (t, 1H, J=9.4, H-5), 3.30 (dd, 1H, J=6.1 and 10.4, H-7), 3.23 (dd, 0.2H, J=3.0 and 11.6, H-7), 3.17 (dt, 1H, J=10.3 and 13.3, HH-6), 3.05 (ddd, 0.2H, J=8.5, 11.1 and 13.1, HH-6), 2.73 (ddd, 0.2H, J=3.6, 9.0 and 12.9, HH-6), 2.68-2.56 (m, 311, HH-6 and H₂-19), 2.45 (ddd, 0.2H, J=6.7, 8.1 and 11.4, HH-19), 2.13 (ddd, 0.2H, J=6.8, 8.0 and 11.3, HH-19), 1.40-1.35 (m, 2.4H, H₂-18 and H₂-18), 1.31-1.23 (m, 6H, H₂-15, H₂-16 and H₂-17), 1.21-1.08 (m, 1.2H, H₂-15, H₂-16 and H₂-17), 0.87 (t, 3H, J=6.9, H₃-14), 0.81 (t, 0.6H, J=7.1, H₃-14); δ_C(150 MHz, CDCl₃) 176.25 (C═O), 174.16 (C═O), 148.44 (C9), 139.02 (C13), 133.88 (Ar—H), 129.69 (Ar—H), 123.07 (Ar—H), 121.92 (Ar—H), 57.34 (C4), 46.76 (C5), 44.18 (C7), 31.36 (CH₂), 30.21 (CH₂), 29.33 (C19), 28.68 (CH₂), 26.07 (C6), 22.57 (CH₂) 14.11 (C14); IR (oil, cm⁻¹) 2934 (w), 1719 (s); MS (CI+) m/z (relative intensity): 363 ([M+H], 65), 214 (90), 180 (100); Exact Mass Calcd for [C₁₈H₂₂N₂O₄S]+H requires m/z 363.1379 Found 363.1397 (CI+).

(4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-(m-nitro)phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.59 (s, 1H, NH), 8.22-8.16 (m, 2H, 2×Ar—H), 7.66 (d, 1H, J=7.5, Ar—H), 7.56 (t, 1H, J=8.0, H-11), 4.14 (t, 1H, J=8.6, H-5), 3.23 (dd, 1H, J=3.0 and 11.6, H-7), 3.05 (ddd, 1H, J=8.5, 11.1 and 13.1, HH-6), 2.73 (ddd, 1H, J=3.6, 9.0 and 12.9, HH-6), 2.45 (ddd, 1H, J=6.7, 8.1 and 11.4, HH-19), 2.13 (ddd, 1H, J=6.8, 8.0 and 11.3, HH-19), 1.30-1.24 (m, 2H, H₂-18), 1.21-1.08 (m, 6H, H₂-15, H₂-16 and H₂-17), 0.81 (t, 3H, J=7.1, H₃-14); δ_C(150 MHz, CDCl₃) 177.94 (C═O), 176.84 (C═O), 148.14 (C9), 138.76 (C13), 135.29 (Ar—H), 129.24 (Ar—H), 123.39 (Ar—H), 123.16 (Ar—H), 56.71 (C4), 45.09 (C5), 43.69 (C7), 31.26 (CH₂), 28.88 (CH₂), 28.84 (C19), 28.51 (C6), 26.33 (CH₂), 22.57 (CH₂), 14.06 (C14); IR (oil, cm⁻¹) 3214 (w), 2928 (w) 1773 (m), 1709 (s); MS (CI+) m/z (relative intensity): 363 ([M+H], 10), 214 (15), 84 (100); Exact Mass Calcd for [C₁₈H₂₂N₂O₄S]+H requires m/z 363.1379 Found 363.1394 (CI+).

Reference Example 128

Preparation of (4RS,5SR,7RS)2-Aza-4-(N-Boc-L-Cys-OMe)-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Boc-Cys(Mal)-OMe (50 mg, 0.15 mmol) was dissolved in acetonitrile (30 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (136 μL, 1.2 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a thick colourless oil (16 mg, 0.037 mmol) in 24% yield as a mixture of two major diastereomers (small signals suggest two other diastereomers, possibly regioisomers regarding the addition of the styrene). Reanalysis of the crude suggests that the reaction was successful in at least 80%. δ_H(600 MHz, CDCl₃) 8.08 (s, 2H, 2×H-2), 7.40-7.31 (m, 10H, 10×Ar—H), 5.0 (d, 1H, J=8.2, H-15), 4.9 (d, 1H, J=7.5, H-15), 4.26-4.23 (m, 1H, H-16), 4.18-4.12 (m, 1H, H-16), 4.06 (t, 2H, J=8.5, 2×H-5), 3.669 (s, 3H, H₃-18), 3.674 (s, 3H, H₃-18), 3.19 (ddd, 1H, J=2.4 and 11.0, H-7), 3.11 (dd, 1H, J=3.2 and 11.0, H-7) 3.04-2.93 (m, 3H, 2×HH-6 and HH-19), 2.91 (dd, 1H, J=6.6 and 12.8, HH-19), 2.64-2.60 (m, 2H, 2×HH-6), 2.51 (dd, 1H, J=4.6 and 12.8, HH-19), 2.43 (dd, 1H, J=7.3 and 13.0, HH-19), 1.45 (s, 9H, 3×H₃-12), 1.43 (s, 9H, 3×H₃-12); δ_C(150 MHz, CDCl₃) 178.41 (C═O), 177.25 (C═O), 177.20 (C═O), 171.40 (C═O), 171.10 (C═O), 170.98 (C═O), 155.28 (C═O), 155.18 (C═O), 136.28 (C11), 136.25 (C11), 128.94 (2×Ar—H), 128.93 (2×Ar—H), 128.49 (2×Ar—H), 128.46 (2×Ar—H), 128.38 (C8), 128.33 (C8), 80.44 (2×C13), 56.71 (C4), 56.48 (C4), 53.03 (C16), 52.87 (C16), 52.78 (C18), 52.75 (C18), 45.92 (C5), 45.82 (C5), 43.76 (C7), 43.61 (C7), 31.28 (C6), 31.09 (C6), 28.38 (6×C12), 26.33 (C19), 26.21 (C19); IR (oil, cm⁻¹) 3215 (w), 2971 (w) 1738 (s), 1715 (s); MS (CI+) m/z (relative intensity): 435 ([m+H], 10), 379 (30), 335 (100); Exact Mass Calcd for [C₂₁H₂₇N₂O₆S]+H requires m/z 435.1590 Found 435.1576 (CI+).

Reference Example 129

Preparation of 1-(p-Methoxy)phenyl-4-Hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione, (4RS,5RS,7RS)2-Aza-4-Hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-4-Hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, 4-methoxy styrene (154 μL, 1.20 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded 1-(p-methoxy)phenyl-4-Hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione as a colourless oil (10 mg, 0.037 mmol) in 25% yield and (4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione (major) and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione (minor) as a colourless oil (27 mg, 0.77 mmol) in 67% yield as a mixture of diastereomers (10:1).
4-Hexylsulfanyl-1-(p-methoxy)phenyl-1,7-dihydro-2H-azepine-3,6-dione δ_H(600 MHz, CDCl₃) 7.21 (d, 2H, J=8.5, 2×Ar—H), 7.18 (s, 1H, NH), 6.84 (d, 2H, J=9.0, 2×Ar—H), 6.13 (s, 1H, H-5), 4.08 (t, 1H, J=7.9, H-1), 3.80 (s, 3H, H₃-18), 2.99 (dd, 1H, J=7.2 and 15.7, HH-7), 2.91 (dd, 1H, J=8.7 and 15.7, HH-7), 2.35-2.26 (m, 2H, H₂-13), 1.51-1.43 (m, 2H, H₂-12), 1.33-1.16 (m, 6H, H₂-9 H₂-10 and H₂-11), 0.85 (t, 3H, J=7.0, H₃-8); δ_C(150 MHz, CDCl₃) 171.03 (C═O), 170.05 (C═O), 159.06 (C17), 147.37 (C4), 132.92 (C14), 129.37 (C5), 128.79 (2×Ar—H), 114.18 (2×Ar—H), 55.38 (C18), 46.61 (C1), 32.59 (C7), 31.45 (C13), 31.38 (CH₂), 29.22 (CH₂), 28.64 (CH₂), 22.61 (CH₂), 14.14 (C8); IR (oil, cm⁻¹) 3275 (w), 2927 (m) 1774 (w), 1717 (s); MS (CI+) m/z (relative intensity): 347 ([M+], 15), 237 (70), 230 (100), 202 (60); Exact Mass Calcd for [C₁₉H₂₅NO₃S]+ requires m/z 347.1550 Found 363.1553 (CI+).
(4RS,5RS,7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione (in bold) and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-(p-methoxy)phenyl-bicyclo[3.2.0]heptan-1,3-dione δ_H(600 MHz, CDCl₃) 8.71 (s, 1H, NH), 8.45 (s, 0.1H, NH), 7.24 (d, 2H, J=7.86 2×Ar—H), 7.15 (d, 0.2H, J=8.7, 2×Ar—H), 6.90 (d, 1H, J=8.6, 2×Ar—H), 6.86 (d, 0.2H, J=8.6, 2×Ar—H), 3.99 (t, 1H, J=8.8, H-5), 3.94 (dd, 0.1H, J=8.4 and 10.1, H-5), 3.81 (s, 3H, H₃-8), 3.77 (s, 0.3H, H₃-8), 3.20 (dd, 0.1H, J=4.5 and 11.9, H-7), 3.13 (dd, 1H, J=3.4 and 11.0, H-7), 3.09 (dt, 0.1H, J=10.6 and 13.2, HH-6), 2.98 (ddd, 1H, J=8.7, 11.2 and 12.9, HH-6), 2.67 (dt, 0.1H, J=7.3 and 11.5, HH-18), 2.63-2.53 (m, 1.1H, HH-6 and HH-18), 2.54 (ddd, 1H, J=4.5, 8.4 and 11.9, HH-6), 2.43 (ddd, 1H, J=6.7, 8.2 and 11.3, HH-18), 2.15 (ddd, 1H, J=6.7, 8.4 and 11.4, HH-18), 1.56-1.52 (m, 0.2H, H₂-17), 1.39-1.33 (m, 0.2H, CH₂), 1.31-1.09 (m, 8.4H, H₂-14, H₂-15, H₂-16, H₂-17 and 2×CH₂) 0.87 (t, 0.3H, J=7.1, H₃-13), 0.83 (t, 3H, J=7.1, H₃-13); Only signals of major diastereoisomer shown δ_C(150 MHz, CDCl₃) 177.81 (C═O), 175.48 (C═O), 159.39 (C9), 129.94 (2×Ar—H), 128.56 (C12), 113.65 (2×Ar—H), 57.63 (C4), 55.38 (C8), 45.25 (C5), 43.78 (C7), 31.33 (CH₂), 29.23 (CH₂), 29.01 (CH₂), 28.62 (CH₂), 26.53 (C18), 22.52 (C6), 14.10 (C13); IR (oil, cm⁻¹) 3216 (w), 2928 (m) 1771 (m), 1707 (s); MS (CI+) m/z (relative intensity): 348 ([M+H], 20), 135 (20), 134 (100); Exact Mass Calcd for [C₁₉H₂₅NO₃S]+H requires m/z 348.1633 Found 363.1642 (CI+).

Reference Example 130

Preparation of (4RS,5SR,7RS)2-Aza-4-(N—Ac-L-Cys-Benzylamine)-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N—Ac-Cys(Mal)-Benzylamine (29 mg, 0.084 mmol) was dissolved in acetonitrile (50 mL) to provide a 0.002M solution. The resulting solution was degassed for 30 minutes, styrene (10 μL, 0.084 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in 30% ethyl acetate in petroleum ether to 10% methanol in ethyl acetate) afforded the desired compound as a colourless oil (33 mg, 0.073 mmol) in 87% as a mixture of diastereomers of the [2+2] reaction. Reanalysis of the crude suggests the reaction was successful in around 70%.
N—Ac-Cys(Mal)-Benzylamine (58 mg, 0.17 mmol) was dissolved in acetonitrile (80 mL) to provide a 0.002M solution. The resulting solution was degassed for 30 minutes, styrene (191 μL, 1.70 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in 30% ethyl acetate in petroleum ether to 10% methanol in ethyl acetate) afforded the desired compound as a colourless oil (14 mg, 0.031 mmol) in 19% yield. Reanalysis of the crude suggests the reaction was successful in at least 75%.
δ_H(600 MHz, CDCl₃) 8.69 (s, 1H, H-2), 7.36-7.26 (m, 8H, 8×Ar—H), 7.18 (d, 2H, J=7.0, 2×Ar—H), 6.65 (t, 1H, J=5.6, H-17), 6.51 (d, 1H, J=7.4, H-14), 4.33 (d, 2H, J=6.0, H₂-18), 4.30 (td, 1H, J=1.2 and 5.4, H-15), 4.05 (t, 1H, J=8.9, H-5), 3.16 (dd, 1H, J=3.1 and 11.1, H-7), 3.04 (ddd, 1H, J=8.9, 11.1 and 12.8, HH-23), 2.94 (dd, 1H, J=6.6 and 13.3, HH-6), 2.59 (ddd, 1H, J=3.4, 8.9 and 12.5, HH-23), 2.31 (dd, 1H, J=5.3 and 13.6, HH-6), 1.98 (s, 3H, H₃-12); δ_C(150 MHz, CDCl₃) 179.07 (C═O), 176.97 (C═O), 171.10 (C═O), 169.67 (C═O), 137.67 (Ar), 136.16 (Ar), 129.02 (2×Ar—H), 128.80 (2×Ar—H), 128.55 (2×Ar—H), 128.35 (Ar—H), 128.78 (2×Ar—H), 127.66 (Ar—H), 57.44 (C4), 52.61 (C15), 46.09 (C5), 43.68 (C7), 43.66 (C18), 30.58 (C6), 26.15 (C23), 23.17 (C12); IR (oil, cm⁻¹) 3437 (w), 1726 (s); MS (FAB+) m/z (relative intensity): 695 ([M+H], 10), 439 (10), 286 (100); Exact Mass Calcd for [C₃₂H₃₄N₆O₈S₂]+H requires m/z 695.1958 Found 695.1964 (FAB+).

Reference Example 131

Preparation of (4RS,5RS,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Methyl hexylsulfanylmaleimide (27 mg, 0.119 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (136 μL, 1.19 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded (4RS,5RS,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (5 mg, 0.015 mmol) in 13% and (4RS,5SR,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (23 mg, 0.069 mmol) in 58% yield.

(4RS,5RS,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 7.32-7.30 (m, 3H, 3×Ar—H), 7.13 (d, 2H, J=7.9, 2×H-10), 3.99 (dd, 1H, J=7.8 and 10.2, H-5), 3.20 (dd, 1H, J=4.8 and 10.3, H-7), 3.13-3.07 (m, 1H, HH-6), 2.93 (s, 3H, H₃-18), 2.65 (td, 1H, J=7.5 and 11.4, HH-17), 2.58 (td, 1H, J=7.5 and 11.8, HH-17), 2.46 (ddd, 1H, J=4.9, 7.7 and 12.0, HH-6), 1.55-1.25 (m, 8H, H₂-13, H₂-14, H₂-15 and H₂-16), 0.87 (t, 3H, J=7.0, H₃-12); δ_C(150 MHz, CDCl₃) 177.78 (C═O), 175.17 (C═O), 137.21 (C11), 128.71 (2×Ar—H), 127.93 (C8), 127.24 (2×Ar—H), 48.23 (C5), 42.71 (C7), 31.42 (CH₂), 30.07 (C17), 29.33 (CH₂), 28.69 (CH₂), 25.71 (CH₂), 25.25 (C18), 22.58 (CH₂), 14.12 (C12); IR (oil, cm⁻¹) 2927 (w) 1715 (s); MS (CI+) m/z (relative intensity): 332 ([M+H], 40), 228 (40), 86 (70), 84 (100); Exact Mass Calcd for [C₁₉H₂₅NO₂S]+H requires m/z 332.1684 Found 333.1697 (CI+).

(4RS,5SR,7RS)2-Aza-2-methyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 7.39-7.30 (m, 5H, 5×Ar—H), 3.90 (t, 1H, J=8.7, H-5) 3.13-3.11 (m, 4H, H₃-18 and H-7), 3.00 (ddd, 1H, J=8.5, 11.0 and 12.8, HH-6), 2.53 (ddd, 1H, J=3.7, 9.1 and 12.8, HH-6), 2.40 (ddd, 1H, J=6.4, 8.1 and 11.3, HH-17), 2.06 (ddd, 1H, J=6.5, 8.3 and 11.3, HH-17), 1.25-1.08 (m, 8H, H₂-13, H₂-14, H₂-15 and H₂-16), 0.82 (t, 3H, J=7.4, H₃-12); δ_C(150 MHz, CDCl₃) 178.73 (C═O), 177.70 (C═O), 136.77 (C11), 128.89 (2×Ar—H), 128.27 (2×Ar—H), 127.99 (C8), 55.69 (C4), 45.62 (C5), 42.61 (C7), 31.29 (CH₂), 28.94 (CH₂), 28.68 (CH₂), 28.55 (C17), 26.26
(C6), 25.70 (C18), 22.58 (CH₂), 14.09 (C12); IR (oil, cm⁻¹) 2927 (w) 1703 (s); MS (CI+) m/z (relative intensity): 332 ([M+H], 100); Exact Mass Calcd for [C₁₉H₂₅NO₂S]+H requires m/z 332.1684 Found 332.1680 (CI+).

Reference Example 132

Preparation of (4RS,5RS,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione, (4RS,5SR,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and 1-Phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione

N-Phenyl hexylsulfanylmaleimide (34 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (135 μL, 1.18 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded (4RS,5RS,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (37 mg, 0.94 mmol) in 80% yield as a mixture of diastereoisomers (11:2) and 1-phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione as a colourless oil (0.5 mg, 0.001 mmol) in 1% yield (4RS,5RS,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

(4RS,5RS,7RS)2-Aza-2-phenyl-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione in bold

δ_H(600 MHz, CDCl₃) 7.54-7.52 (m, 11H, 2×Ar—H), 7.46-7.42 (m, 4H, 4×Ar—H), 7.41-7.37 (m, 46H, 8×Ar—H and 2×Ar—H), 7.27-7.26 (m, 2H, 2×Ar—H), 7.06-7.05 (m, 2H, 2×Ar—H), 4.09 (t, 1H, J=8.5, H-5), 4.08 (t, 5.5H, J=8.5, H-5), 3.35 (dd, 1H, J=4.7 and 10.5, H-7), 3.29 (dd, 5.5H, J=4.0 and 10.9, H-7), 3.20 (td, 1H, J=10.3 and 13.3, HH-6), 3.09 (ddd, 5.5H, J=8.2, 11.0 and 13.0, HH-6), 2.78 (td, 1H, J=7.4 and 11.7, HH-17), 2.72-2.68 (m, 6.511, HH-6 and HH-6), 2.66 (dd, 1H, J=4.8 and 7.4, HH-17), 2.52 (ddd, 5.511, J=6.5, 8.2 and 11.4, HH-17), 2.16 (ddd, 5.511, J=6.6, 8.4 and 11.4, HH-17), 1.63-1.58 (m, 2H, H₂-16), 1.42-1.37 (m, 2H, H₂-15), 1.36-1.09 (m, 24H, 4×CH₂and 2×CH₂), 0.88 (t, 3H, J=6.7, H₃-12), 0.83 (t, 16.5H, J=7.3, H₃-12); δ_C(150 MHz, CDCl₃) 177.65 (C═O), 176.82 (C═O), 176.67 (C═O), 174.08 (C═O), 136.96 (C11), 136.82 (C11), 132.06 (C21), 131.86 (C21), 129.40 (2×Ar—H), 129.22 (Ar—H), 128.96 (2×Ar—H), 128.84 (Ar—H), 128.79 (Ar—H), 128.34 (2×Ar—H), 128.08 (Ar—H), 127.51 (Ar—H), 126.56 (2×Ar—H), 126.34 (Ar—H), 55.57 (C4), 55.47 (C4), 48.78 (C5), 45.87 (C5), 44.88 (C7), 42.79 (C7), 31.45 (C17), 31.31 (C17), 30.27 (CH₂), 29.46 (CH₂), 29.08 (CH₂), 28.75 (CH₂), 28.56 (CH₂), 26.95 (CH₂), 22.60 (CH₂), 22.52 (CH₂), 14.14 (C12), 14.10 (C12) Several carbon signals are missing due to overlap of the diastereomers; IR (oil, cm⁻¹) 2926 (w) 1709 (s); MS (CI+) m/z (relative intensity): 394 ([M+H], 70), 290 (100), 105 (100); Exact Mass Calcd for [C₂₄H₂₇NO₂S]+H requires m/z 394.1841 Found 394.1834 (CI+).

1-Phenyl-3-phenyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione

δ_H(600 MHz, CDCl₃) 7.54-7.27 (m, 10H, 10×Ar—H), 6.32 (s, 1H, H-6), 4.19 (t, 1H, J=88.0, H-1), 3.14-3.03 (m, 2H, H₂-2), 2.39-2.29 (m, 2H, H₂-13), 1.61-1.10 (m, 8H, 4×CH₂), 0.89-0.81 (m, 3H, H₃-8); δ_C(150 MHz, CDCl₃) 146.31 (C═O), 141.19 (C═O), 130.19 (Ar), 129.41 (Ar), 129.20 (Ar—H), 128.93 (2×Ar—H), 128.49 (2×Ar—H), 127.90 (Ar—H), 127.76 (2×Ar—H), 126.03 (2×Ar—H), 47.19 (C1), 32.70 (CH₂), 31.43 (CH₂), 29.83 (CH₂), 29.20 (CH₂), 28.63 (CH₂), 22.61 (CH₂), 14.13 (C8); IR (oil, cm⁻¹) 2926 (m) 1715 (s); MS (CI+) m/z (relative intensity): 394 ([M+H], 40), 278 (100); Exact Mass Calcd for [C₂₄H₂₇NO₂S]+H requires m/z 394.1841 Found 394.1829 (CI+).

Reference Example 133

Preparation of (4RS,5SR,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5RS,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

Phenylthiomaleimide (17 mg, 0.082 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.003M solution. The resulting solution was degassed for 30 minutes, styrene (111 μL, 0.82 mmol) added and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded (4RS,5SR,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (1.5 mg, 0.005 mmol) in 6% yield and (4RS,5RS,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (17.5 mg, 0.056 mmol) in 69% yield.

(4RS,5SR,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.05 (s, 1H, NH), 7.42-7.41 (m, 2H, 2×Ar—H), 7.35-7.17 (m, 8H, 8×Ar—H), 4.05 (t, 1H, J=10.1, H-5), 3.29 (dd, 1H, J=5.5 and 13.0, H-7), 3.01 (dt, 1H, J=10.3 and 13.0, HH-6), 2.56 (ddd, 1H, J=5.6, 10.1 and 13.4, HH-6); δ_C(150 MHz, CDCl₃) 176.69 (C═O), 174.10 (C═O), 136.56 (C11), 136.01 (2×Ar—H), 130.13 (Ar—H), 129.66 (2×Ar—H), 129.28 (C15), 128.72 (2×Ar—H), 128.01 (Ar—H), 127.32 (2×Ar—H), 60.59 (C4), 46.32 (C5), 43.70 (C7), 25.51 (C6); IR (oil, cm⁻¹) 3226 (w), 2925 (w) 1715 (s); MS (CI+) m/z (relative intensity): 310 ([M+H], 10), 206 (30), 111 (100); Exact Mass Calcd for [C₁₈H₁₅NO₂S]+H requires m/z 310.0902 Found 310.0901 (CI+).

(4RS,5RS,7RS)2-Aza-4-phenylthio-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.24 (s, 1H, NH), 7.43-7.30 (m, 6H, 8×Ar—H), 7.27-7.24 (m, 2H, 2×Ar—H), 4.13 (t, 1H, J=9.0, H-5), 3.20-3.13 (m, 2H, HH-6 and H-7), 2.55 (ddd, 1H, J=5.6, 8.3 and 13.4, HH-6); δ_C(150 MHz, CDCl₃) 177.99 (C═O), 177.17 (C═O), 135.80 (C11), 135.80 (2×Ar—H), 129.64 (Ar—H), 129.54 (2×Ar—H), 128.95 (C15), 128.51 (2×Ar—H), 128.45 (2×Ar—H), 128.22 (Ar—H), 60.72 (C4), 45.80 (C5), 43.39 (C7), 25.20 (C6); IR (oil, cm⁻¹) 3211 (w), 1772 (w) 1707 (s); MS (CI+) m/z (relative intensity): 310 ([M+H], 50), 206 (100), 104 (40); Exact Mass Calcd for [C₁₈H₁₅NO₂S]+H requires m/z 310.0902 Found 310.0905 (CI+).

Reference Example 134

Preparation of (4RS,7RS)2-Aza-4-hexylsulfanyl-bicyclo[3.2.0]hept-5-ene-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.12 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, phenyl acetylene (128 μL, 1.16 mmol) added and irradiated in pyrex glassware for 30 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a colourless oil (4.5 mg, 0.014 mmol) in 18% yield (based on recovered SM) alongside SM (1.5 mg, 0.007 mmol) in 6% yield. δ_H(600 MHz, CDCl₃) 7.86 (s, 1H, NH), 7.68 (d, 2H, J=7.86, 2×Ar—H), 7.40-7.34 (m, 3H, 3×Ar—H), 6.58 (s, 1H, H-5), 3.73 (s, 1H, H-7), 2.57 (dt, 1H, J=2.3 and 7.4, H₂-13), 1.77-1.19 (m, 8H, 4×CH₂), 0.85 (t, 3H, J=7.3, H₃-8); δ_C(150 MHz, CDCl₃) 173.46 (C═O), 173.28 (C═O), 149.72 (C17), 130.25 (C6), 130.02 (C14), 128.85 (2×Ar—H), 126.32 (2×Ar—H), 125.76 (C5), 53.78 (C7), 31.33 (CH₂), 29.70 (CH₂), 29.32 (CH₂), 28.61 (CH₂), 22.55 (CH₂), 14.11 (C8); IR (oil, cm⁻¹) 3228 (w), 2925 (m), 1770 (w) 1709 (s); MS (CI+) m/z (relative intensity): 316 ([M+H], 100), 214 (30); Exact Mass Calcd for [C₁₈H₂₂NO₂S]+H requires m/z 316.0371 Found 316.1365 (CI+).

Reference Example 135

Preparation of 1-Butyl-4-hexylsulfanyl-1,7-dihydro-2H-azepine-3,6-dione and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile (20.7 mL) and hex-1-ene (4.3 mL, 11.6 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded 1-butyl-4-hexylsulfanyl-1,7-dihydro-2,1-azepine-3,6-dione as a colourless oil (4 mg, 0.013 mmol) in 12% yield (alongside (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione) and (4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (16 mg, 0.054 mmol) in 47% yield.

1-Butyl-4-hexylsulfanyl-1,7-dihydro-2,1-azepine-3,6-dione

δ_H(600 MHz, CDCl₃) 7.29 (s, 1H, NH), 6.43 (s, 1H, H-5), 2.91-2.76 (m, 3H, H-1 and H₂-17), 2.70 (dd, 1H, J=1.4 and 5.8, HH-7), 2.64 (dd, 1H, J=1.4 and 7.9, HH-7), 2.49 (t, 1H, J=7.4, H₂-13), 1.81-1.25 (m, 12H, 6×CH₂), 0.93-0.85 (m, 6H, H₃-8 and H₃-14); δ_C(150 MHz, CDCl₃) 174.39 (C═O), 171.40 (C═O), 148.19 (C4), 129.28 (C5), 43.90 (C1), 34.97 (CH₂), 31.53 (CH₂), 30.60 (CH₂), 29.70 (CH₂), 29.07 (CH₂), 28.76 (CH₂), 28.51 (CH₂), 22.66 (CH₂), 22.65 (CH₂), 14.16 (CH₃), 14.13 (CH₃); IR (oil, cm⁻¹) 3226 (w), 2927 (m) 1715 (s); MS (CI+) m/z (relative intensity): 298 ([M+H], 80), 187 (100); Exact Mass Calcd for [C₁₆H₂₇NO₂S]+H requires m/z 298.1841 Found 298.1841 (CI+).

(4RS,5SR,7RS)2-Aza-4-hexylsulfanyl-5-butyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.19 (s, 1H, NH), 3.09 (dd, 1H, J=4.5 and 10.2, H-7), 2.70-2.64 (m, 1H, H-5), 2.53-2.45 (m, 2H, H₂-17), 2.37-2.28 (m, 2H, H₂-6), 1.80-1.73 (m, 1H, HH-11), 1.59-1.50 (m, 2H, HH-11 and HH-10), 1.38-1.20 (m, 11H, HH-10 and 5×CH₂), 0.90 (t, 3H, J=7.1, CH₃), 0.87 (t, 3H, J=7.3, CH₃); δ_C(150 MHz, CDCl₃) 178.73 (C═O), 177.81 (C═O), 56.10 (C4), 44.29 (C7), 40.99 (C5), 31.42 (C17), 29.24 (CH₂), 28.99 (CH₂), 28.90 (CH₂), 28.74 (CH₂), 28.73 (CH₂), 27.92 (CH₂), 22.59 (CH₂), 22.58 (CH₂), 14.12 (CH₃), 14.09 (CH₃); IR (oil, cm⁻¹) 3209 (w), 2927 (m) 1774 (w), 1711 (s); MS (CI+) m/z (relative intensity): 298 ([M+H], 100); Exact Mass Calcd for [C₁₆H₂₇NO₂S]+H requires m/z 298.1841 Found 298.1845 (CI+).

Reference Example 136

Preparation of (4RS,7RS)2-Aza-4-hexylsulfanyl-5-ethyl-6-ethyl-bicyclo[3.2.0]hept-5-ene-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile (21.1 mL) and hex-3-yne (3.9 mL, 11.6 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a colourless oil (17 mg, 0.057 mmol) in 49% yield. δ_H(600 MHz, CDCl₃) 8.26 (s, 1H, NH), 3.88 (s, 1H, H-7), 2.99 (ddd, 1H, J=7.5, 9.1 and 12.8, HH-18), 2.87 (ddd, 1H, J=5.1, 8.7 and 12.9, HH-18), 2.39-2.20 (m, 2H, HH-9 and HH-11), 1.95-1.75 (m, 2H, HH-9 and HH-11), 1.56-1.10 (m, 8H, 4×CH₂), 0.90-0.86 (m, 9H, H₃-8, H₃-10 and H₃-12); δ_C(150 MHz, CDCl₃) 172.66 (C═O), 171.34 (C═O), 148.84 (C═C), 142.53 (C═C), 70.45 (C4), 48.86 (C18), 48.09 (C7), 31.45 (CH₂), 28.51 (CH₂), 23.53 (CH₂), 22.50 (CH₂), 21.89 (CH₂), 21.62 (CH₂) 14.09 (C12), 12.03 (CH₃), 11.84 (CH₃); IR (oil, cm⁻¹) 2931 (m) 1717 (s); MS (CI+) m/z (relative intensity): 312 ([M+OH], 100), 178 (100); Exact Mass Calcd for [C₁₆H₂₅NO₂S]+OH requires m/z 312.1633 Found 312.1648 (CI+).

Reference Example 137

Preparation of 1-Ethyl-2-ethyl-6-hexylsulfanyl-1,2-dihydro-3H-azepine-4,7-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile (21.1 mL) and trans-hex-3-ene (3.9 mL, 11.6 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a colourless oil (2 mg, 0.007 mmol) in 6% yield. δ_H(600 MHz, CDCl₃) 7.32 (s, 1H, NH), 6.49 (s, 1H, H-6), 2.92 (ddd, 1H, J=4.6, 6.3 and 10.8, H-1), 2.83 (ddd, 1H, J=5.2, 9.4 and 12.8, HH-13), 2.75 (dd, 1H, J=5.9 and 10.9, H-2), 2.62 (ddd, 1H, J=6.7, 9.6 and 12.7 HH-13), 2.07-2.00 (m, 1H, HH-17), 1.89-1.83 (m, 1H, HH-15), 1.79-1.40 (m, 6H, HH-15, HH-17 and 2×CH₂), 1.33-1.30 (m, 4H, 2×CH₂), 1.10 (t, 3H, J=7.5, H₃-16), 0.92 (t, 3H, J=7.4, CH₃), 0.89 (t, 3H, J=7.0, CH₃); δ_C(150 MHz, CDCl₃) 171.00 (C═O), 169.53 (C═O), 149.76 (C5), 129.27 (C6), 62.54 (C2), 51.16 (C13), 40.42 (C1), 31.48 (CH₂), 28.65 (CH₂), 23.65 (CH₂), 23.32 (CH₂), 22.52 (CH₂), 17.23 (C17) 14.11 (CH₃), 13.96 (CH₃), 12.33 (CH₃); IR (oil, cm⁻¹) 2962 (m) 1717 (s); MS (CI+) m/z (relative intensity): 314 ([M+OH], 75), 180 (100); Exact Mass Calcd for [C₁₆H₂₆NO₂S]+OH requires m/z 314.1790 Found 314.1799 (CI+).

Reference Example 138

Preparation of (4RS,7RS)2-Aza-4-hexylsulfanyl-5,5-diphenyl-bicyclo[3.2.0]heptan-1,3-dione

Hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile (21 mL) and 1,1-diphenylethyene (203 μL, 1.16 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded the desired compound as a colourless oil (30 mg, 0.075 mmol) in 64% yield. δ_H(600 MHz, CDCl₃) 8.15 (s, 1H, NH), 7.42 (m, 2H, 2×H-8), 7.36-7.21 (m, 8H, 8×Ar—H), 3.54 (dd, 1H, J=10.3 and 12.9, HH-6), 3.34 (dd, 1H, J=5.7 and 10.3, H-7), 3.18 (dd, 1H, J=5.8 and 12.9, HH-6), 2.43 (dt, 1H, J=7.3 and 11.0, HH-17), 2.34 (dt, 1H, J=7.4 and 11.0, HH-17), 1.40-1.34 (m, 2H, H₂-16), 1.26-1.20 (m, 4H, H₂-14 and H₂-15), 1.18-1.13 (m, 2H, H₂-13), 0.84 (t, 3H, J=7.5, H₃-12); δ_C(150 MHz, CDCl₃) 177.16 (C═O), 175.87 (C═O), 142.09 (2×Ar), 141.80 (2×Ar), 128.17 (2×Ar—H), 128.13 (2×Ar—H), 128.10 (2×Ar—H), 128.06 (2×Ar—H), 127.44 (Ar—H), 127.32 (Ar—H), 63.03 (C5), 57.20 (C4), 44.38 (C7), 35.17 (C6), 31.35 (CH₂), 30.07 (C17), 28.77 (CH₂), 28.71 (CH₂) 22.53 (CH₂), 14.11 (C12); IR (oil, cm⁻¹) 2927 (m) 1772 (w), 1709 (s); MS (ES−) m/z (relative intensity): 392 ([M], 10), 212 (100); Exact Mass Calcd for [C₂₄H₂₆NO₂S] requires m/z 392.1684 Found 392.1674 (ES−)

Reference Example 139

Preparation of (4RS,5RS,7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione

N-Methylene hexylsulfanylmaleimide (25 mg, 0.116 mmol) was dissolved in acetonitrile (21 mL) and 1,1-diphenylethyene (203 μL, 1.16 mmol) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes and irradiated in pyrex glassware for 5 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded (4RS,5RS,7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5SR,7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (30 mg, 0.075 mmol) as a mix of diastereoisomers (10:1) in 64% yield.

(4RS,5RS,7RS)2-Aza-2-methylenecyclohexane-4-hexylsulfanyl-5-phenyl-bicyclo[3.2.0]heptan-1,3-dione in bold

δ_H(600 MHz, CDCl₃) 7.39-7.29 (m, 5.2H, 5×Ar—H and 0.2×Ar—H), 7.24 (d, 0.1H, J=7.4, H-8), 7.16 (d, 2H, J=7.4, 0.2×H-10), 4.00 (dd, 0.1H, J=8.2 and 9.9, H-5), 3.89 (t, 1H, J=8.7, H-5), 3.46 (d, 2.2H, J=7.5, H₂-22 and H₂-22), 3.19 (dd, 0.1H, J=5.1 and 10.5, H-7), 3.12 (dd, 1H, J=3.5 and 10.9, H-7), 3.07 (td, 0.1H, J=10.5 and 13.1, HH-6), 3.00 (ddd, 1H, J=8.5, 11.5 and 12.6, HH-6), 2.64 (ddd, 0.1H, J=6.9, 11.4 and 14.8, HH-17), 2.56 (td, 0.1H, J=6.9 and 11.6, HH-6), 2.53 (ddd, 1H, J=3.3, 9.1 and 12.9, HH-6), 2.48 (ddd, 0.1H, J=5.4, 8.0 and 13.2, HH-17), 2.39 (td, 1H, J=7.4 and 11.4, HH-17), 2.08 (td, 1H, J=7.7 and 11.4, HH-17), 1.83-0.99 (m, 2H, 20.9H), 0.87 (t, 3H, J=7.0, H₃-12), 0.82 (t, 311, J=7.5, H₃-12); δ_C(150 MHz, CDCl₃) 179.12 (C═O), 178.35 (C═O), 178.15 (C═O), 175.41 (C═O), 137.23 (C11), 137.12 (C11), 129.08 (2×Ar—H), 128.76 (2×Ar—H), 128.47 (2×Ar—H), 128.16 (C8), 127.99 (C8), 127.51 (2×Ar—H), 55.84 (C4), 55.69 (C4), 48.06 (C5), 45.97 (C5), 45.63 (C22), 45.48 (C22), 42.94 (C7), 42.83 (C7), 36.67 (C21), 36.58 (C21), 31.64 (CH₂), 31.49 (CH₂), 31.08 (CH₂), 31.01 (CH₂), 30.96 (CH₂), 30.34 (CH₂), 30.04 (CH₂), 29.67 (CH₂), 29.28 (CH₂), 28.98 (CH₂), 28.82 (CH₂), 26.76 (CH₂), 26.63 (CH₂), 26.52 (CH₂), 26.45 (CH₂), 25.96 (CH₂), 25.94 (CH₂), 25.82 (CH₂), 14.34 (C12), 14.31 (C12) Several carbon signals are missing due to overlap of the diastereomers; IR (oil, cm⁻¹) 2925 (m) 1703 (s); MS (CI+) m/z (relative intensity): 414 ([M+H], 100), 309 (20); Exact Mass Calcd for [C₂₅H₃₅NO₂S]+H requires m/z 414.2461 Found 414.2452 (CI+)

Reference Example 140

Preparation of (4RS,5SR,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione and (4RS,5RS,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

2,3Dihexylsulfanylmaleimide (38 mg, 0.115 mmol) was dissolved in acetonitrile (25 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (133 μL, 1.2 mmol) added and irradiated in pyrex glassware for 20 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in petroleum ether to 30% ethyl acetate in petroleum ether) afforded (4RS,5SR,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (3 mg, 0.007 mmol) in 6% yield and (4RS,5RS,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione as a colourless oil (3 mg, 0.007 mmol) in 6% yield.

(4RS,5SR,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 7.96 (s, 1H, NH), 7.33 (d, 2H, J=7.0, 2×Ar—H), 7.28 (t, 1H, J=7.0, H-8), 7.21 (d, 2H, J=7.5, 2×Ar—H), 4.02 (t, 1H, J=10.0, H-5), 2.98-2.92 (m, 2H, HH-6 and —S—CHH—), 2.87 (dd, 1H, J=10.8 and 13.6, HH-6), 2.83-2.72 (m, 2H, —S—CHH— and —S—CHH—), 2.69 (dt, 1H, J=7.4 and 10.8, —S—CHH—) 1.68-1.57 (m, 4H, H₂-16 and H₂-22), 1.45-1.31 (m, 4H, H₂-15 and H₂-21), 1.31-1.25 (m, 8H, H₂-13, H₂-14, H₂-19 and H₂-20), 0.86 (t, 6H, J=7.0, H₃-12 and H₃-18); δ_C(150 MHz, CDCl₃) 176.91 (C═O), 172.96 (C═O), 136.09 (C11), 128.83 (2×Ar—H), 128.13 (C8), 127.41 (2×Ar—H), 62.86 (C4), 54.36 (C7), 46.49 (C5), 33.28 (C6), 31.51 (CH₂), 31.47 (CH₂), 30.65 (SCH₂), 30.09 (SCH₂), 29.22 (CH₂), 28.96 (CH₂), 28.80 (CH₂), 28.74 (CH₂), 22.63 (CH₂), 22.60 (CH₂), 14.17 (CH₃), 14.15 (CH₃); IR (oil, cm⁻¹) 3194 (w), 2928 (m) 1774 (w), 1722 (s); MS (CI+) m/z (relative intensity): 432 ([M−H], 5), 332 (50), 316 (95), 207 (100); Exact Mass Calcd for [C₂₄H₃₅NO₂S₂]—H requires m/z 432.2026 Found 432.2029 (CI+).

(4RS,5RS,7SR)2-Aza-4-hexylsulfanyl-5-phenyl-7-hexylsulfanyl-bicyclo[3.2.0]heptan-1,3-dione

δ_H(600 MHz, CDCl₃) 8.10 (s, 1H, NH), 7.41 (d, 2H, J=6.9, 2×Ar—H), 7.37 (t, 1H, J=6.9, H-8), 7.33 (d, 2H, J=6.9, 2×Ar—H), 3.92 (t, 1H, J=8.9, H-5), 2.95 (dd, 1H, J=8.9 and 12.9, HH-6), 2.86 (dt, 1H, J=6.9 and 14.2, —S—CHH—), 2.78-2.66 (m, 2H, HH-6 and —S—CHH—), 2.60 (ddd, 1H, J=6.3, 8.3 and 10.9, —S—CHH—) 2.00 (ddd, 1H, J=5.6, 8.6 and 10.7, —S—CHH—), 1.65-1.60 (m, 2H, HH-16 and HH-22), 1.43-1.06 (m, 14H, HH-16, HH-22, H₂-13, H₂-14, H₂-15, H₂-19, H₂-20 and H₂-21), 0.88 (t, 3H, J=6.7, CH₃), 0.82 (t, 3H, J=7.1, CH₃); δ_C(150 MHz, CDCl₃) 176.59 (C═O), 176.44 (C═O), 136.03 (C11), 129.50 (2×Ar—H), 128.83 (C8), 128.29 (2×Ar—H), 62.32 (C4), 54.58 (C7), 45.33 (C5), 34.85 (C6), 31.48 (CH₂), 31.33 (CH₂), 30.51 (CH₂), 29.21 (CH₂), 29.06 (CH₂), 28.90 (CH₂), 28.76 (CH₂), 28.53 (CH₂), 22.62 (CH₂), 22.50 (CH₂), 14.16 (CH₃), 14.11 (CH₃); IR (oil, cm⁻¹) 3215 (w), 2926 (m) 1774 (w), 1715 (s); MS (CI+) m/z (relative intensity): 432 ([M−H], 5), 329 (60), 207 (100), 161 (60); Exact Mass Calcd for [C₂₄H₃₅NO₂S₂]—H requires m/z 432.2026 Found 432.2034 (CI+).

Reference Example 141

Preparation of (4RS,5RS,7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-dione

2,3-Di-(N-Boc-Cys-OMe)-maleimide (76 mg, 0.135 mmol) was dissolved in acetonitrile (29 mL) to provide a 0.005M solution. The resulting solution was degassed for 30 minutes, styrene (148 μL, 1.35 mmol) added and irradiated in pyrex glassware for 30 minutes with stirring. Solvent was removed in vacuo and purification by flash chromatography (gradient elution in 10% ethyl acetate in petroleum ether to 30% ethyl acetate in petroleum ether) afforded a mixture of (4RS,5RS,7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-diones and (4RS,5SR,7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-diones as a colourless oil (28 mg, 0.042 mmol) in 36% yield. The spectra from this mixture was very complex but MS confirmed the identity of the compounds as all having the same mass. 4RS,5RS,7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-diones and (4RS,5SR,7SR)2-Aza-4-(N-Boc-Cys-OMe)-5-phenyl-7-(N-Boc-Cys-OMe)-bicyclo[3.2.0]heptan-1,3-diones was also isolated alongside a [5+2] product as a colourless oil (46 mg) ¹H NMR and MS data suggest 40% of this (by mass) is the desired conjugation products (18 mg, 0.028 mmol) in 20% yield. δ_H(600 MHz, CDCl₃) 8.32 (d, 4.7H, J=8.7, N—H), 8.05 (s, 1.2H, N—H), 7.79 (s, 1H, N—H), 7.40-7.20 (m, 60H, Ar—H), 5.65 (d, 1H, J=7.2, H—N), 5.57 (d, 5.2H, J=8.2, H—N), 5.45 (d, 4.9H, J=7.3, H—N), 5.40 (d, 1.9H, J=7.8, H—N), 4.92 (d, 2.5H, J=7.3, H-14), 4.74 (d, 3.7H, J=7.8, H-14), 4.62-4.54 (m, 8H, H-14), 4.16-4.11 (m, 2.7H), 4.09-4.06 (m, 3.9H), 3.97-3.9 (m, 7.8H), 3.80-3.75 (m, 50.5H, H₃-12), 3.66 (s, 22.6H, H₃-12) 3.47-3.04 (m, 41.4, H₂-18), 2.99-2.94 (m, 9.5H, H₂-18), 2.82-2.73 (m, 9.5H, H₂-18), 1.46-1.42 (m, 240H, H₃-17); δ_C(150 MHz, CDCl₃) 175.94 (C═O), 175.84 (C═O), 175.82 (C═O), 171.10 (C═O), 171.04 (C═O), 170.95 (C═O), 155.28 (Ar), 129.63 (Ar—H), 129.50 (Ar—H), 128.96 (Ar—H), 128.88 (Ar—H), 128.80 (Ar—H), 128.75 (Ar—H), 128.59 (Ar—H), 128.53 (Ar—H), 128.41 (Ar—H), 80.51 (C16), 80.22 (C16), 52.97 (C12), 52.93 (C12), 52.71 (C14), 52.61 (C14), 45.25 (C5), 32.92 (C18), 32.86 (C18), 31.19 (C18), 31.08 (C18), 29.83 (C6), 28.42 (C17) Several carbon signals are missing due to overlap of the diastereomers; IR (oil, cm⁻¹) 2924 (m), 1712 (s); MS (CI+) m/z (relative intensity): 666 ([M−H], 100); Exact Mass Calcd for [C₃₀H₄₁N₃O₁₀S₂]—H requires m/z 666.2155 Found 666.2188 (CI+).

Claims

1. A product, which comprises (a) a solid substrate and (b) a moiety of formula (I) linked thereto

wherein X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl and either

(i) R represents an electrophilic leaving group Y, the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I); or

(ii) the solid substrate carries a thiol moiety, R represents a bond to the sulfur atom of said thiol moiety and a functional moiety is linked to the 1-, 3- or 4-position of the formula (I); or

(iii) the solid substrate is linked to the 1-, 3- or 4-position of the formula (I) and R represents a group of formula —S—F₁or —S-L-F₂, wherein L represents a linker group and —S—F₁and —F₂represent a functional moiety;

wherein said functional moiety is selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA.

2. A product according to claim 1, which has the formula (II)

wherein:

X and X′ are the same or different and each represents oxygen, sulfur or a group of formula ═NQ, in which Q is hydrogen, hydroxyl, C_1-6alkyl or phenyl;

R₁represents:

(i) an electrophilic leaving group Y; or

(ii) a solid substrate carrying a thiol moiety, which solid substrate is attached to the 2-position of the formula (II) via the sulfur atom of said thiol moiety; or

(iii) —S—F₁or —S-L-F₂;

R₂represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG;

either:

R₃and R₃′ are the same or different and each represents a hydrogen atom, Sol, -L-Sol, F₃, E, Nu, -L(F₃)_m(Z)_n-mor IG; or

R₃and R₃′ together form a group of formula —O— or —N(R_33′)—, wherein R_33′ represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG; or

R₃and R₃′ together form a group of formula —N(R_33′)—N(R_33′)—, wherein each R_33′ is the same or different and represents a hydrogen atom, Sol, -L-Sol, F₃, Y, Nu, -L(F₃)_m(Z)_n-mor IG; -Sol represents a solid substrate;

each —S—F₁, F₂and F₃is the same or different and represents a functional moiety selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;

each E and Y is the same or different and represents an electrophilic leaving group;

each Nu is the same or different and represents a nucleophile selected from —OH, —SH, —NH₂and —NH(C_1-6alkyl);

each L is the same or different and represents a linker group;

each Z is the same or different and represents a reactive group attached to a moiety L;

each n is the same or different and is 1, 2 or 3;

each m is the same or different and is an integer having a value of from zero to n; and

each IG is the same or different and represents a moiety which is a C_1-20alkyl group, a C_2-20alkenyl group or a C_2-20alkynyl group, which is unsubstituted or substituted by one or more halogen atom substituents, and in which (a) 0, 1 or 2 carbon atoms are replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, wherein:

(i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol,

—N(C_1-6alkyl)(C_1-6alkyl) and nitro groups; and

(ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by —C(O)— groups;

with the proviso that the product contains one solid substrate and at least one functional moiety.

3. A product according to claim 2, wherein Y is a halogen atom or a triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, C_1-6alkylthiol, 5- to 10-membered heterocyclylthiol, C_6-10arylthiol, C_3-7carbocyclylthiol, —OC(O)CH₃, —OC(O)CF₃, phenyloxy, —NR_xR_yR_z ⁺ or —PR_xR_yR_z ⁺ group, in which R_x, R_yand R_zare the same or different and are selected from hydrogen atoms and C_1-6alkyl and phenyl groups.

4. A product according to claim 2, wherein E is a halogen atom or a C_1-6alkoxy, thiol, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl), triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl, phenyloxy or nitrophenyloxy group.

5. A product according to claim 2, wherein L represents a moiety which is a C_1-20alkylene group, a C_2-20alkenylene group or a C_2-20alkynylene group, which is unsubstituted or substituted by one or more halogen atom substituents, and in which (a) 0, 1 or 2 carbon atoms are replaced by groups selected from C_6-10arylene, 5- to 10-membered heteroarylene, C_3-7carbocyclylene and 5- to 10-membered heterocyclylene groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S—, —S—S—, —C(O)— and —N(C_1-6alkyl)- groups, wherein:

(i) said arylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or more substituents selected from halogen atoms and C_1-6alkyl, C_1-6alkoxy, C_1-6alkylthiol, —N(C_1-6alkyl)(C_1-6alkyl) and nitro groups; and

(ii) 0, 1 or 2 carbon atoms in said carbocyclylene and heterocyclylene groups are replaced by —C(O)— groups.

6. A product according to claim 2, wherein L represents a moiety which is an unsubstituted C_1-6alkylene group, C_2-6alkenylene group or C_2-6alkynylene group, in which (a) 0 or 1 carbon atom is replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups, wherein said phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or two substituents selected from halogen atoms and C_1-4alkyl and C_1-4alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S— and —C(O)— groups.

7. A product according to claim 2, wherein Z represents:

(a) a group of formula -LG, —C(O)-LG, —C(S)-LG or —C(NH)-LG wherein LG is an electrophilic leaving group;

(b) a nucleophile Nu′ selected from —OH, —SH, —NH₂, —NH(C_1-6alkyl) and —C(O)NHNH₂groups;

(c) a cyclic moiety Cyc, which is capable of a ring-opening electrophilic reaction with a nucleophile;

(d) a group of formula —S(O₂)(Hal), wherein Hal is a halogen atom;

(e) a group of formula —N═C═O or —N═C═S;

(f) a group of formula —S—S(IG′) wherein IG′ represents a group of formula IG as defined in claim 2;

(g) a group AH, which is a C_6-10aryl group that is substituted by one or more halogen atoms;

(h) a photoreactive group capable of being activated by exposure to ultraviolet light;

(i) a group of formula —C(O)H or —C(O)(C_1-6alkyl);

(j) a maleimido group;

(k) a group of formula —C(O)CHCH₂;

(l) a group of formula —C(O)C(N₂)H or -PhN₂ ⁺, where Ph represents a phenyl group; or

(m) an epoxide group.

8. A product according to claim 7, wherein:

LG is selected from halogen atoms and —O(IG′), —SH, —S(IG′), —NH₂, —NH(IG′),

—N(IG′)(IG″), —N₃, triflate, tosylate, mesylate, N-hydroxysuccinimidyl, N-hydroxysulfosuccinimidyl, imidazolyl and azide groups, wherein IG′ and IG″ are the same or different and each represents a group of formula IG as defined in claim 2; and/or

Nu′ is selected from —OH, —SH and —NH₂groups; and/or

Cyc is selected from the groups

and/or

Hal is a chlorine atom; and/or

AH is a phenyl group that is substituted by at least one fluorine atom; and/or

the photoreactive group is selected from:

(a) a C_6-10aryl group which is substituted by at least one group of formula —N₃and which is optionally further substituted by one or more halogen atoms;

(b) a benzophenone group;

(c) a group of formula —C(O)C(N₂)CF₃; and

(d) a group of formula -PhC(N₂)CF₃, wherein Ph represents a phenyl group.

9. A product according to claim 2, wherein Z is selected from:

(a) groups of formula -LG, —C(O)-LG and —C(S)-LG, wherein LG is selected from halogen atoms and —O(C_1-6alkyl), —SH, —S(C_1-6alkyl), triflate, tosylate, mesylate, N-hydroxysuccinimidyl and N-hydroxysulfosuccinimidyl groups;

(b) groups of formula —OH, —SH and —NH₂;

(c) a group of formula

and

(d) a maleimido group.

10. A product according to claim 2, wherein IG represents a moiety which is an unsubstituted C_1-6alkyl group, C_2-6alkenyl group or C_2-6alkynyl group, in which (a) 0 or 1 carbon atom is replaced by a group selected from phenylene, 5- to 6-membered heteroarylene, C_5-6carbocyclylene and 5- to 6-membered heterocyclylene groups, wherein said phenylene, heteroarylene, carbocyclylene and heterocyclylene groups are unsubstituted or substituted by one or two substituents selected from halogen atoms and C_1-4alkyl and C_1-4alkoxy groups, and (b) 0, 1 or 2 —CH₂— groups are replaced by groups selected from —O—, —S— and —C(O)— groups.

11. A product according to claim 2, wherein n is 1.

12. A product according to claim 2, wherein the product of formula (II) is a product of formula (IIa)

wherein X, X′, R₁, R₂and R_33′ are all as defined in claim 2.

13. A product according to claim 2, wherein:

X and X′ each represent oxygen;

R₁represents:

(i) a halogen atom; or

(iii) —S—F₁;

R₂represents a hydrogen or halogen atom, a solid substrate, F₃or a C_1-6alkyl group;

R₃and R₃′ together form a group of formula —N(R_33′), wherein R_33′ represents a hydrogen atom, a solid substrate, F₃or a C_1-6alkyl group; and

—S₁—F₁and F₃are the same or different and represent a functional moiety selected from a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA and an RNA;

14. A product according to claim 2, which comprises one functional moiety —S—F₁, F₂or F₃.

15. A product according to claim 14, wherein said functional moiety is the functional moiety —S—F₁.

16. A product according to claim 2, wherein R₃and R₃′ together form a group of formula —N(R_33′) and R_33′ represents a solid substrate.

17. A method for linking a solid substrate to a functional moiety, which method comprises reacting a compound containing a moiety of formula (III) with (a) a solid substrate and (b) a functional moiety

wherein:

Y is an electrophilic leaving group; and

the functional moiety is a detectable moiety, an enzymatically active moiety, an affinity tag, a hapten, an immunogenic carrier, an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a biologically active moiety, a liposome, a polymeric moiety, an amino acid, a peptide, a protein, a cell, a carbohydrate, a DNA or an RNA.

18. A method according to claim 17, where the compound containing a moiety of formula (III) is a compound of formula (IV)

wherein:

R_2arepresents a hydrogen atom, Y, Nu, -L(Z′)_nor IG;

either:

R_3aand R_3a′ are the same or different and each represents a hydrogen atom, E, Nu, -L(Z′)_aor IG; or

R_3aand R_3a′ together form a group of formula —O— or —N(R_33a′), wherein R_33a′, represents a hydrogen atom, Y, Nu, -L(Z′)_nor IG; or

R_3aand R_3a′ together form a group of formula —N(R_33a′)—N(R_33a′)—, wherein each R_33a′ is the same or different and represents a hydrogen atom, Y, Nu, -L(Z′)_nor IG;

Z′ represents Z or a group of formula —Si(O(C_1-6alkyl)(G₁)(G₂), wherein G₁and G₂are the same or different and represent H, C_1-6alkyl or O(C_1-6alkyl); and

X, X′, Y, Nu, L, Z, n, IG and E are all as defined in claim 2.

19. A process, which comprises:

providing a product as defined in claim 1, in which either:

(i) a solid substrate carrying a thiol moiety is attached to the 2-position of the formula (I) via the sulfur atom of said thiol moiety; or

(ii) a group —S—F₁or —S-L-F₂is attached to the 2-position of the formula (I); and

cleaving the thiol bond at the 2-position of the formula (I).

20. A product of formula (Va) or (Vb)

wherein

—R₁represents:

(i) a solid substrate carrying a thiol moiety, which solid substrate is attached to the 2-position of the formula (Va) or (Vb) via the sulfur atom of said thiol moiety; or

(ii) —S—F₁or —S-L-F₂;

X, X′, —S—F₁, F₂, R₂, R₃and R₃′ are as defined in any one of claims 2 to 13;

R₄is a halogen atom, a hydroxyl, C_1-6alkoxy, thiol, C_1-6alkylthio or C_1-6alkylcarbonyloxy group, or a group of formula F₃as defined in claim 2; and

at least one of the groups R₂and R₄comprises a group of formula F₃;

with the proviso that the product contains one solid substrate.

21. A plurality of products as defined in claim 1, arranged in an array.

22. A plurality of products according to claim 21, wherein each product comprises a functional moiety selected from an antibody or antibody fragment, an antigen, a ligand or ligand candidate, a peptide, a protein, a cell, a DNA and an RNA.

23. A plurality of products according to claim 22, wherein:

(a) each product comprises a different antibody or antibody fragment; or

(b) each product comprises a different antigen; or

(c) each product comprises a different ligand or ligand candidate; or

(d) each product comprises a different peptide; or

(e) each product comprises a different protein; or

(f) each product comprises a different cell; or

(g) each product comprises a different DNA; or

(h) each product comprises a different RNA.

24. An assay process, which comprises:

providing a plurality of products as defined in claim 22;

incubating said plurality of products with a sample comprising a test substance; and

detecting whether any of said test substance is bound to any of said plurality of products.

25. A detection process, which comprises:

(i) providing a product according to claim 1, wherein said product comprises an antibody or an antigen;

(ii) incubating said product with a sample;

(iii) removing any material which is not bound to said antibody or antigen; and

(iv) detecting any substance that is bound to the antibody or antigen.

26. A process for purifying a specific substance from a sample, which comprises:

(i) providing a product according to claim 1, wherein said product comprises a functional moiety that is capable of selectively binding to said substance;

(ii) incubating said product with said sample;

(iii) removing any material which is not bound to said functional moiety; and

(iv) separating said substance from said product.

27. A process according to claim 26, wherein said specific substance is a bait substance S_band said sample possibly further comprises one or more prey substances S_pcapable of binding to S_b.

28. A process for producing a peptide or protein, which comprises:

(i) providing a product according to any one of claim 1;

(ii) attaching an amino acid to said product; and

(iii) attaching one or more further amino acids to said amino acid, thus producing a peptide or protein moiety that is linked to a solid substrate; and

(iv) cleaving said peptide or protein moiety from said solid substrate.

29. A process according to claim 28, wherein step (i) comprises providing a product which comprises a cysteine residue attached to the 2-position of the formula (I) via its sulfur atom.

30. A method according to claim 17, wherein the product obtained by linking said solid substrate to said functional moiety using said compound containing a moiety of formula (III) comprises a maleimide ring and wherein said method further comprises effecting ring opening of said maleimide ring.

31. (canceled)

32. (canceled)