Detection and Functionalisation of S-nitrosylated

Polypeptides

Field of the Invention The present invention relates to the detection and functionalisation of S-nitrosylated polypeptides, and more particularly to methods and kits in which an S-nitrosothiol (SNO) group present in a polypeptide is reacted with an SNO binding reagent via a free radical reaction.

Background of the Invention

Nitric oxide (NO) has an established role as a key- signalling molecule in a plethora of cellular events. Classically, the actions of NO are attributed to activation of soluble guanylate cyclase and the generation of cGMP . In a second distinct pathway, NO reacts covalently with cysteine residues, leading to formation of a S-nitrosothiol (SNO) group, a process termed S-nitrosylation . Post- translational modification by S-nitrosylation can alter protein function in many diverse ways, such as regulating the catalytic site of an enzyme or the activation of ion channels (Takahashi et al . , 2007, Whalen et al . , 2007) . Important roles for S-nitrosylation include inhibition of NMDA receptor activation under hypoxic conditions, regulation of G-protein-coupled receptor kinase 2 activity and activation of TRPC5 channels (Takahashi et al . , 2007: Whalen el al., 2007; Yoshida et al., 2006) .

However, despite the emerging concept that S-nitrosylation is an important signalling pathway akin to phosphorylation, it remains a relatively poorly understood cellular event. In particular, work in this area has been hampered by the current tools used to study protein S-nitrosylation. The most commonly used technique for detecting the presence of

S-nitrosothiol groups in proteins uses a method known as "biotin-switch" . In this method, S-nitrosylated cysteines are transiently biotinylated via a multi-step approach (Jeffrey et al . , 2001; Hao et al . , 2006; WO 02/039110; WO 2005/101019) . This method involves an initial step in which cellular proteins are denatured and free thiol groups are blocked by alkylation. It is assumed that the alkylating reagent will label all free thiol groups and is then completely removed prior to the subsequent steps. SNO bonds are then decomposed by ascorbate with the assumption that disulphide bonds are not affected by this reaction (Huang et al., 2006; Landino et al, 2006; Zhang et al., 2005) . Free thiols formed during this reaction are then labelled with a sulphydryl-specific biotinylation reagent via formation of a disulfide bond and biotinylated proteins can then be detected by Western blot, or extracted for proteomic analysis by cleavage of the disulfide linkage. From the description above, it will be apparent that the biotin switch method is based on a number of assumptions that are often not true and has the disadvantages that it will only detect denatured proteins in cell lysates and relies on the relative stability of the transient SNO group.

Summary of the Invention Broadly, the present invention is based on methods in which a polypeptide comprising one or more S-nitrosothiol (SNO) groups is contacted with an SNO binding reagent (a "SNOB reagent") to react with the SNO groups in a free radical reaction. The reaction may be used to label the polypeptide using the SNO groups where the SNO binding reagent comprises a label or to modify the polypeptide where the SNO binding reagent comprises a transfer moiety. The methods of the present invention generally differ from the prior art biotin switch reaction as the free radical mechanism leads to

direct and specific reaction between the SNO binding reagent and the S-nitrosothiol groups, typically forming a thio- . oxime after the free radical addition to the alkene bond. In preferred embodiments, as the SNO-binding reagents may incorporate many different functional groups, the reaction makes it possible to introduce these functional groups into polypeptides that contain SNO groups for a range of different purposes, including detection and functionalisation of S-nitrosylated endogenous polypeptides to explore physiological roles and signalling pathways of NO, the detection of S-nitrosylation profiles in living systems with molecular imaging techniques and the functionalisation of polypeptides with labels and transfer moieties .

In the present invention, this reaction proceeds via a free radical mechanism in which the SNO group (s) react with the alkene group of the SNO binding reagent to introduce the label or other functionalising group into the polypeptide at the site of the starting SNO group (s) . Accordingly, the method is capable of directly and specifically detecting or labelling the SNO groups, in contrast to the multi-step biotin switch method of the prior art. As the present invention does not involve the initial step of alkylating free thiol groups in the polypeptide, it is not limited to the use of denatured polypeptides in cell lysates and may be used for a range of applications beyond detecting SNO groups in polypeptides. The present methods may further help to avoid the requirement to first block free thiol groups or denitrosylate polypeptides with - ascorbate . This approach- allows more efficient detection of polypeptide nitrosylation profiles using Western blot polypeptide detection or other proteomic approaches, and can be utilized to detect S-

nitrosylation of individual cysteines in biological samples and living cells.

There is one literature report of nitrosylated thiols undergoing thermal and photolytic rearrangement with simple alkenes to yield stable thioethers (Cavero et al., 2002) . This reaction proceeds via a free radical process, making it extremely specific towards nitrosothiols . Rearrangement (tautomerisation) to the final thio-oxime product is quite facile and occurs spontaneously (see Figure 1) . However,

Cavero et al . do not disclose or suggest the application of this reaction for any particular purpose and certainly do not suggest that it might be used to solve the problems with the biotin switch assay for the labelling or detection of SNO groups in biological systems, such as polypeptides or proteins .

Accordingly, in a first aspect, the present invention provides a method which comprises contacting a polypeptide having at least one S-nitrosothiol (SNO) group with a SNO binding reagent comprising an alkene group and a label or a transfer moiety, wherein the SNO group reacts with the alkene group of the SNO binding reagent via a free radical reaction to transfer the label or transfer moiety to the polypeptide. In embodiments of the present invention where the method is for labelling the polypeptide, the SNO binding reagent comprises a label. Alternatively or additionally, where the method is for modifying the polypeptide, the SNO binding reagent comprises a transfer moiety which is transferred to the polypeptide in the SNOB reaction.

Accordingly, the methods disclosed herein can be used for a wide range of different purposes. Therefore, in a further aspect, the present invention provides a method for

detecting S-nitrosothiol (SNO) groups in a polypeptide, the method comprising (a) contacting the polypeptide with a SNO binding reagent comprising an alkene group and a label, wherein the SNO groups react with the alkene groups via a free radical reaction to transfer the label to the polypeptide and (b) detecting the label to determine or observe the presence of the S-nitrosothiol (SNO) groups in the polypeptide. This method may involve determining the number or location of the S-nitrosothiol (SNO) groups in the polypeptide.

In a further aspect, the present invention provides a method of labelling a polypeptide comprising at least one SNO group, the method comprising contacting the polypeptide with a SNO binding reagent comprising an alkene group and a label wherein the SNO group reacts with the alkene group via a free radical reaction to transfer the label to the polypeptide .

In a further aspect, the present invention provides a method for imaging S-nitrosothiol (SNO) groups in a polypeptide, the method comprising (a) contacting the polypeptide with a SNO binding reagent comprising an alkene group and a label, wherein the SNO groups react with the alkene groups via a free radical reaction to transfer the label to the polypeptide and (b) detecting the label to image the S- nitrosothiol (SNO) groups in the polypeptide. In this aspect, the method may be for imaging polypeptides in an in vivo system, for example, in a living animal , or in a cell culture or in a cell lysate:

In a further aspect, the present invention provides a method for functionalising a polypeptide comprising at least one S- nitrosothiol (SNO) group, the method comprising contacting

the polypeptide with a SNO binding reagent comprising an alkene group and a transfer moiety, wherein the SNO group reacts with the alkene group via a free radical reaction to functionalise the polypeptide with the transfer moiety at the site of the SNO group.

In some embodiments, the method may include the initial step of introducing at least one SNO group into the polypeptide, for example by reacting one of more thiol groups in the polypeptide (e.g. present in cysteine residues) with a nitrosylating agent, to produce one or more S-nitrosothiol (SNO) groups for subsequent functionalisation by a SNOB reagent. The method may additionally involve the step of introducing a cysteine residue, or another residue that may be reacted to produce a thiol group, in the polypeptide at a site where it is desired to functionalise the polypeptide. This may be useful in situations where a convenient cysteine residue for reaction according to the present invention is not present in a starting or wild-type polypeptide. Conveniently, this may be achieved using site directed mutagenesis of the polypeptide.

In preferred embodiments of the present invention, the alkene group of the SNO binding reagent is a terminal alkene group and may comprise, for example, an allyl amide group. Generally, the SNOB reagents can be represented by the general formulae: label group-optional linker-alkene group or transfer moiety-optional linker-alkene group. When present, the linker may be used to fine tune or modify the properties of the polypeptide that is labelled or functionalised with the SNOB reagent. By way of example, in some embodiments in which the label or transfer moiety is a tagging group to allow the polypeptide to be manipulated, e.g. through its interaction with a specific binding

partner, it may be advantageous to include a linker that is sufficiently long to allow the label or transfer moiety to interact with the specific binding partner. This may help to avoid steric hindrance that may otherwise result if the linker was short or not present, with the result that the label or transfer moiety was present in close proximity to the polypeptide. However, in other embodiments, it may be advantageous to dispense with a linker or else have a short linker, for example where it is important to introduce the label or transfer moiety in close proximity to the SNO group in the polypeptide, for example to help prevent cleavage of the label or transfer group. By way of illustration, this may be useful where the polypeptide is glycosylated or pegylated using the methods disclosed herein or where labelling in the immediate vicinity of SNO group is desired.

Examples of linker groups include:

a substituted or unsubstituted Ci-20 alkyl group; or

a -[(CH ₂ )In-X] _n -CH ₂ - group where: n is 1 to 4, m is 1 to 10, preferably 1 to 5, and is selected independently for each repeat unit [] _n (for example, when n=3, m may take a different value in each of the three -

(CH ₂ U-X- units), and

X is NH or -CO-Y- where Y is NH or 0, preferably NH, and where X is selected independently for each repeat unit [] _n ; or

a - (CH ₂ ) p-X- (CH ₂ ) q-X- (CH ₂ ) r-X-CH ₂ - group where: p, q and r are independently 1 to 10, preferably 1 to 5, and

X is NH or -CO-Y- where Y is NH or 0, preferably NH, and where each x is selected independently; or

a - (CH ₂ ) p-X- (CH ₂ ) C ₁ -X-CH ₂ - group where:

P and q are independently 1 to 10, preferably 1 to 5, and X is NH or -CO-Y- where Y is NH or 0, preferably NH, and where each x is selected independently; or

a - (CH ₂ ) p-X—CH ₂ - group where:

P is 1 to 10, preferably 1 to 5, and

X is NH or -CO-Y- where Y is NH or 0, preferably NH; or

a -[ (CH ₂ ) _In -CO-NH-CH ₂ ] _n - (NH) ₃ -CH ₂ group where n is 1 to 4, m is 1 to 10, preferably 1 to 5, and is selected independently for each repeat unit [] _n , and a is 0 or 1; or

a - (CH ₂ ) p-CO-NH- (CH ₂ Jq-NH-CH ₂ - group where:

P and q are independently 1 to 10, preferably 1 to 5; or

a - (CH ₂ ) _p -CO-NH- (CH ₂ Jq-CO-NH-CH ₂ - group where:

P and q are independently 1 to 10, preferably 1 to 5; or

a -[X- (CH ₂ ) _m ] _n - group, where n is 1 to 4, ra is 1 to 10, preferably 1 to 5, and is selected independently for each repeat unit [] _n , and

X is NH or -CO-Y- where Y is NH or 0, preferably NH, and where X is selected independently for each repeat unit [] _n ; or

a -X-CH ₂ - group, where

X is NH or -CO-Y- where Y is NH or 0, and preferably X is

-CO-NH-.

Preferably, the alkene group may be represented by the formula :

wherein Ri, R ₂ and R ₃ are independently selected from hydrogen, halogen (preferably fluorine, chlorine or bromine) , a substituted or unsubstituted, straight chain or branched alkyl group (e.g. a Ci- ₂₀ alkyl group) or a hetero atom containing group, and more particularly where the hetero atom is oxygen, nitrogen or sulphur; and wherein the linker group is preferably as defined above.

In any of the aspects of the present invention, it is possible to employ a SNOB reagent that comprises a plurality of labels and/or transfer moieties (e.g. 2, 3, 4 or more), where the labels or transfer moieties may be the same or different, for example where it is desired to introduce more than one functionality into a polypeptide at the site of a given SNO group. Alternatively or additionally, a similar end result may be reached by reacting a plurality of SNO groups in a polypeptide with a plurality of different SNOB reagents (e.g. 2, 3, 4 or more), each reagent comprising a different label or transfer group. In this latter case, preferably the reactions are carried out sequentially to maintain control over the site of reaction of the different SNOB reagent with the polypeptide, by the use of appropriate protecting groups and/or by sequentially creating and reacting the SNO groups .

More specifically, the SNOB reagent may be represented by the represented by the formula:

wherein:

X is the transfer moiety or label;

Ri, R ₂ and R ₃ are independently selected from hydrogen, halogen (preferably fluorine, chlorine or bromine) , a substituted or unsubstituted, straight chain or branched alkyl group (e.g. a Ci- ₂₀ alkyl group) or a hetero atom containing group, and more particularly where the hetero atom is oxygen, nitrogen or sulphur; Y is an optional linker group that, when present, preferably comprises a substituted or unsubstituted alkyl group (e.g. a substituted or unsubstituted Ci- ₂₀ alkyl group) or a substituted or unsubstituted polyethylene glycol group.

By way of example, in some embodiments of the present invention, the SNOB reagent comprises a biotin group for use in labelling or manipulating polypeptides when SNO groups of the polypeptide are reacted in accordance with the methods disclosed herein. A first group of SNOB reagents may be represented by the formula:

wherein n≥l, and is preferably between 1 and 10.

A further group of biotin containing SNOB reagents may be represented by the formula:

wherein n is between 0 and 4 and preferably is 1 or 2.

The methods disclosed herein are free radical reactions and are typically may be initiated by heat or light. Using heat typically comprises heating a reaction mixture containing the polypeptide and the SNO-binding reagent from ambient

(room) temperature up to about 60 ⁰ C. The use of temperatures between about 35°C and about 40 ⁰ C, for example as commonly employed in cell culture or in vivo is preferred

(e.g. a temperature of about 37 ⁰ C. Initiating the reaction using light preferably employs white light and may also include the use of free-radical initiating chemicals.

In a further aspect, the present invention provides kits for carrying out one or more of the above methods, the kits comprising:

(a) a SNO binding reagent binding reagent comprising an alkene group and a label or a transfer moiety; (b) optionally instructions for carrying out the method; and

(c) optionally one or more of an NO donor for polypeptide functionalisation, an antibody conjugate for detection/visualisation and/or affinity media for purification of functionalised polypeptides or peptides.

Typically, nitrosylation using an NO donor would be carried out immediately prior to SNOB functionalisation, and

performed according to standard conditions for achieving S- nitrosylation with the respective NO donor. Examples of nitrosylating agents that may be used include: DEA NONOate (2- (N, N-Diethylamino) -diazenolate-2-oxide) , SNAP (S-nitroso-N-acetylpenicillamine) , GSNO (S- nitrosoglutathione) , sulfo NONOate (hydroxydiazenesulfonic acid 1-oxide) , and DETA/NO (diethylenetriamine/NO) , and salts and derivatives thereof.

Embodiments of the present invention will now be described by way of example and not limitation with reference to the accompanying figures.

Brief Description of the Figures Figure 1. Chemical reaction of terminal alkenes of SNOB reagents with S-nitrosothiol groups of polypeptides

Figure 2. Western blot detection of S-nitrosylated proteins with SNOB-I. Upper panel represents proteins resolved using SDS-PAGE, transferred to nitrocellulose membrane and detected using horseradish peroxidase conjugated to streptavidin. Lower panel represents biotinylated proteins resolved by SDS-PAGE and detected by Commasie Blue staining. Lane 1 contains pre-biotinylated markers. Protein identities indicated in the upper panel with a ratio of number of cysteines present to protein molecular weight, indicated in parenthesis.

Figure 3. Structures of SNOB reagents 1-3.

Figure 4. General synthesis of SNOB reagents 1-3.

Figure 5. Reaction of SNOB 1 with S-No glutathione.

Figure 6 . Reaction of SNOB 1 with S-nitroso pencillanime .

Figure 7. Structures of SNOB reagents 4-6.

Figure 8. Comparison of different SNOB reagents under reducing or nonreducing conditions. BSA reacted with 1 mM DEA NONOate, incubated- with 450 μM SNOB at pH 7 temp 60 "C for 15 min. M: Biotinylated BSA within markers supplied by Biorad.

Figure 9. Temperature and time dependence of SNOB- 6 reaction with nitrosylated bovine serum albumin (BSA) . BSA reacted with 1 mM DEA NONOate, incubated with 450 μM SNOB-6 at a range of temperatures (4 "C to 37 "C) from 0 to 60 min at pH 6.0. SNOB-6 biotinylation of BSA quantified as a percentage of signal from a pre-biotinylated BSA marker (M) protein from Biorad.

Figure 10. pH dependence of SNOB-6 reaction with nitrosylated bovine serum albumin (BSA) . (A) BSA reacted with 1 mM DEA NONOate as indicated in figure, incubated with 450 μM SNOB-6 at 37 "C for 30 min at the indicated pH. Prebiotinylated BSA indicated as M. (B-C) SNOB-6 biotinylation of BSA quantified as a percentage of signal from a pre-biotinylated BSA marker (M) protein from Biorad. Quantification of SNOB-6 reaction with BSA in the presence of the NO donor (B) and the absence of the NO donor (C) .

Figure 11. Detection of endogenous SNO groups on the surface of live cells, human platelets. 450 μM SNOB-6 reacted with acutely isolated human platelets at 37 °C or ambient room temperature (20-22 'C) for 5 minutes. To disrupt SNO groups, platelets were then incubated with 20 mM DTT as indicated. Several bands detected in wild type proteins

that are no longer detected following incubation in DTT. Biotinylation proteins detected by SDS PAGE and Western blot using neutravidin HRP to detect biotin groups.

Figure 12. The action of a NO donor on SNOB- 6 binding to acutely isolated human platelets. 450 μM SNOB-6 reacted with platelets at 37 °C for 30 minutes. Some samples were pre-incubated with 5 mM DEA NONOate for 20 min at 37 "C, prior to labelling with SNOB-6. The exogenous NO donor increases biotinylation above SNOB-6 binding for untreated platelets. Biotinylation proteins detected by SDS PAGE and Western blot using neutravidin HRP to detect biotin groups.

Detailed Description The following applications of the present invention are provided by way of example and not limitation. The methods disclosed herein are generally applicable to a range of uses based on the reaction being capable of specifically and directly introducing functional groups into reaction targets such as peptides, polypeptides and proteins at the site of SNO groups. These applications can be broadly divided into (a) methods involving detecting or imaging the SNO groups, for example for studying the nitrosylation of proteins, (b) methods for introducing labels into proteins, for example where a SNO group is used as a site for labelling a protein, and (c) methods for modifying or functionalising a protein using SNO binding reagents comprising a transfer moiety, for example where the transfer moiety is a label or a molecule for introducing further physical and/or chemical properties to the protein. The methods disclosed herein are applicable for use in both in vivo and in vitro systems.

Reaction targets

Any of the methods disclosed herein may be used to label or

functionalise a wide range of polypeptides, proteins or other reaction targets that include one or more SNO groups. The methods described are applicable to any size of polypeptide and have been successfully employed for labelling or transferring functional groups to reaction targets from single amino acids and peptides to polypeptides and proteins having molecular weights over 10OkDa. In contrast to the prior art biotin switch methods, the methods of the present invention are also capable of employing proteins, i.e. retaining the secondary and/or tertiary structure of the protein in the reaction. For convenience, the methods herein are generally described by reference to polypeptides and this should be taken to include shorter sequences of amino acids (e.g. from 3, 5 or 10 amino acids in length to 40 or 50 amino acids in length) , sometimes referred to in the art as peptides.

By way of example, the polypeptides include molecules of therapeutic, biological, diagnostic or functional interest, or that may be used for structural research and quantification purposes. Polypeptides that might be used in accordance with the present invention include hormones, growth factors, signalling molecules, chemokines, cytokines, ion-channel polypeptides, enzymes, antibodies, ligands and receptors.

Methods of detecting and/or labelling SNO groups As set out above, the presently used biotin switch method of detecting nitrosylation in proteins suffers from a number of significant drawbacks. The methods disclosed herein using

SNO binding (SNOB) reagents that are capable of directly and specifically labelling S-nitrosothiol groups on proteins of interest may help to overcome these problems by bypassing the requirement to block free thiol groups with alklyating

reagents or denitrosylate proteins prior to labelling of previously nitrosylated thiol groups. Finally, biotin switch methods require the purification of proteins and/or protein digests prior to visualisation by Western blot analysis.

In contrast, the proteomic approach described here allows direct detection and functionalisation of S-nitrosylated proteins, and in particular provides a method of detecting nitrosylation in endogenous systems, thereby providing a tool for exploring the physiological roles of NO. Moreover, a single step labelling of SNO groups will facilitate the detection of S-nitrosylation profiles in living systems with molecular imaging techniques.

SNOB reagents could be used to detect in vitro cell surface or intracellular S-nitrosylation. For cell surface detection, the SNOB transfer moiety could include (a) biotin (in direct detection with steptavidin-conjugated to a fluorophore) or (b) a membrane impermeable fluorophore for direct detection using fluorescent imaging technologies . Alternatively the SNOB transfer moiety could be a membrane permeable fluorescent reagent (e.g. BODIPY) that will be trapped within the cell upon binding to a S- nitrosylated protein. Secondary antibodies attached to gold particles for biotin detection using transmission electron microscopy. Suitable fluorescent groups for use as labels or transfer moieties are well known in the art and may be chosen by the skilled person according to the application for which they are intended.

The SNOB reagents may be used for transferring labels to proteins for a range of different purposes, for example for

detecting the protein itself, detecting other species or binding partners with which the protein interacts with or for determining whether a protein of interest is nitrosylated.

The use of labelled SNOB reagents, where the reagent comprises a detectable label which can be transferred to the SNO group of polypeptide of interest, such as a fluorophore, a chromophore, a magnetic probe, a radioactively labelled molecule or any other spectroscopic probe, allows the present invention to be used to specifically and covalently attach the detectable label to the polypeptide at the site of the SNO groups to enable then to be detected or quantitated. The proteins may be present in a cell, on the surface of a cell, in a cell lysate or sample. This allows the detection and characterization of the SNO groups in vivo or in vitro, for example facilitating the study of the nitrosylation of proteins.

Methods of modifying or functionalising proteins

The present invention provides a convenient method for modifying or functionalising a protein of interest using SNO groups that are present in, or have been previously introduced into, the protein. These reactions can be used to introduce a desired functional group, referred to herein as a "transfer moiety", to the protein by using a SNOB reagent which comprises the transfer moiety. By way of example, the transfer moiety can be conjugated to the alkene group that reacts with the SNO groups of the protein, optionally by the use of a linker.

In preferred embodiments, the methods disclosed herein employ reagents and conditions that are well adapted for modifying proteins and other biological materials . In

particular, the mild reaction conditions that are used in the present methods helps to avoid the problem that proteins are susceptible to denaturation and side reactions when harsher reagents and conditions are employed. In this connection, it is also an advantage of the present invention that the reactions may be carried out in vitro or in vivo. Additionally or alternatively, as the methods are generally selective, the methods are amenable to the production of particular functionalised proteins, as compared with non- specific chemical derivatisation reactions that have a tendency to produce a mixture of different products.

As mentioned above, the protein of interest may be modified using existing SNO groups or by introducing SNO groups in an initial step of the method, for example by converting one or more thiol groups in a protein (e.g. cysteine residues) to a SNO group, or by otherwise introducing a SNO group or a precursor thereof into the protein. Preferably, this initial reaction is selective to retain the selectivity of the methods of the invention disclosed herein. The method may additionally involve the step of introducing a cysteine residue, or another residue that may be reacted to produce a thiol group, in the polypeptide at a site where it is desired to functionalise the polypeptide. This may be useful in situations where a convenient cysteine residue for reaction according to the present invention is not present in a starting or wild-type polypeptide. Conveniently, this may be achieved using site directed mutagenesis of the polypeptide, the use of which is well established in the art.

The method is also amenable to use with a range of different transfer moieties. One preferred class of transfer moiety are carbohydrate groups for use in

glycosylating a protein of interest with one or more carbohydrate groups . The carbohydrate group may be a naturally occurring or synthetic monosaccharide, oligosaccharide or polysaccharide. This approach can be used to add glycosylation to a protein of interest, or to introduce glycosylation where glycosylation is desired but is not introduced in the course of the production of the protein, e.g. by virtue of the fact that the protein has been produced in bacterial cells or, particularly for peptides, where they have been produced by chemical synthesis .

The ability to control glycosylation at defined sites using the present invention represents a useful tool for engineering recombinant proteins, for example therapeutic proteins and antibodies, and fragments thereof, in their manufacture and in controlling their immunogenicity and pharmacological properties such as half life. At present, the manufacture of recombinant protein therapeutics is expensive and slow as mammalian cell lines are often used for manufacture to ensure that the proteins are glycosylated. The methods disclosed herein may be used to add glycosylation to a polypeptide after production in bacterial cell lines, in which expression is generally more efficient, thereby helping to improve the speed and/or economy of protein production, while retaining the glycosylation. Alternatively, for polypeptides expressed in cell lines that glycosylate expression products, the present invention may be used as modify or add glycosylation.

In preferred embodiments, the carbohydrates employed may comprise chemically modified derivatives of naturally occurring branched oligosaccharides commonly displayed on

N- or 0- linked glycoproteins, or degradation products thereof. Chemical modifications include the attachment of a terminal alkene for carrying out SNOB reactions. Carbohydrate groups that may be used in the present invention are well known in the art and include carbohydrate groups found in the N- and O-linked glycosylation of eukaryotic proteins and man made carbohydrate groups, e.g. see the carbohydrate groups and methods of producing and identifying them disclosed in WO 03/025133 and WO2004/083807.

The methods disclosed herein can also be employed to engineer proteins containing other transfer moieties, in particular moieties useful for modifying the pharmacological properties of therapeutic proteins. One well known example is the pegylation, the conjugation of proteins with polyethylene glycol (PEG) that is used to enhance the half life of the proteins. The present methods provide the opportunity to pegylate proteins of interest in a selective way depending on where the SNO groups are present in the protein. Pegylation is a known strategy for modifying the properties of therapeutic polypeptides, such as peptides, proteins and antibodies. In general, the attachment of PEG molecules to polypeptides is used to alter their conformation, electrostatic or hydrophobic properties, and lead to improvements in their biological and pharmacological properties, such as increasing drug solubility, reducing dosage frequency, modulating (especially increasing) circulating half-life, increasing drug stability and increasing resistance to proteolytic degradation Pegylation works by increasing the molecular weight of the therapeutic polypeptide by conjugating the polypeptide to one or more PEG polymer molecules. The methods of the present invention have the advantage that the site of introduction of the PEG

molecules into a polypeptide is defined by the presence of SNO groups .

A further class of transfer moiety are spectroscopic probes, which have already been discussed above.

A further class of transfer moiety are affinity tags that can be introduced into a protein to enable it to be manipulated in one or more subsequent steps. A wide range of affinity tags are known in the art suitable affinity tags include members of specific binding pairs, antibodies and antigens, biotin which binds to streptavidin and avidin, polyhistidine (e.g. hexa-His tags) or amino di- or tri- carboxylates which bind to metal ions such as Ni ²⁺ or Co ²⁺ , Flag or GIu epitopes which bind to anti-Flag antibodies, S- tags which bind to streptavidin, calmodulin binding peptide which binds to calmodulin in the presence of Ca ²⁺ , and ribonuclease S which binds to aporibonuclease S. Examples of other affinity tags that can be used in accordance with the present invention will be apparent to those skilled in the art. Protein including these affinity tags can be easily purified and manipulated.

A further class of transfer moiety are selenium containing molecules that can be introduced into a protein that is in either solution or crystalline form, to allow phase determination for protein X-ray crystallographic studies .

Substituents In embodiments of the present invention where a SNOB reagent is substituted, preferably the substituent (s) are independently selected from one of more of halo; hydroxy; ether (e.g., Ci- ₇ alkoxy) ; formyl; acyl (e.g., Ci_ ₇ alkylacyl , C ₅ - ₂ oarylacyl) ; carboxy; ester; acyloxy; amido; acylamido;

thioamido; tetrazolyl; amino; nitro; nitroso; azido; cyano; isocyano; cyanato; isocyanato; thiocyano; isothiocyano; sulfhydryl; thioether (e.g., Ci_ ₇ alkylthio) ; sulfonic acid; sulfonate; sulfone; sulfonyloxy; sulfinyloxy; sulfamino; sulfonamino; sulfinamino; sulfamyl; sulfonamido; Ci_ ₇ alkyl (including, e.g., unsubstituted Ci_ ₇ alkyl, Ci_ ₇ haloalkyl, Ci_ ₇ hydroxyalkyl, Ci- ₇ carboxyalkyl, Ci- ₇ aminoalkyl) .

Examples The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to practice the invention, and are not intended to limit the scope of the invention. In this study we have developed a novel compound to directly detect S- nitrosylated cysteines in cell lysates or recombinant proteins .

Methods

General procedure for synthesis of SNOB reagents (1-3) Pentfluorophenyl TFA (0.20 ml, 1.16 mmol) was added to a stirred solution of biotin (200 mg, 0.82 mmol) in pyridine (3.0 ml) . The reaction was stirred for 1 hour, then the amine (3 mol equiv.) was added and stirred for a further 30 minutes. The mixture was concentrated in vacuo and purified by flash chromatography (10% MeOH/CHCl ₃ ) to give the corresponding amide as a white amorphous powder.

SNOB 1

Yield was 225 mg, 97%. 1H NMR (400 MHz), DMSO: δ 7.97 (IH, appt, J 5.47, NH), 6.48 (IH, s, NH), 6.40{lH, s, NH), 5.77 (IH, appdquintet, J117.22, 10.17, 5.09, 2-H) , 5.08 (IH, ddd, J 17.22, 3.52, 1.96, l'-H), 4.76 (IH, ddd, .J 10.27, 3.52, 1.56, 1"H), 4.30 (IH, dd, J 7.43, 5.09, 10-H) , 4.13 (IH, m, 9-H) ,

3 67 (2H, m, 3'-H and 3"-H) , 3.09 (IH, m, 8-H) , 2.81 (IH, dd, J 12.52, 5.09, ll'-H) . 2.57 (IH, d, J 12.52, 11"H) , 2.09 (2H, t, J7.43, 4-H) , 1.27-1.27 (6H, m, 5-7-H) .

¹³ C NMR (100 MHz) , DMSO: δ 172.0 (C) , 162.9 (C) , 135.6 (CH) , 11 5 0 (CH ₂ ) . 61.1 (CH) , 59.3 (CH) , 55.5 (CH) , 40.8 (CH ₂ ) , 40.1 (CH ₂ ) , 35.2 (CH ₂ ) , 28.3 (CH ₂ ) , 28.1 (CH ₂ ) , 25.4 (CH ₂ ) .

SNOB 3 Yield was 234 mg, 98%.

¹ H NMR (400 MHz); δ 7.77 (IH, appt, J 5.48, NH), 6.45 (IH, s, NH), 6.38 (IH, s, NH), 4.30 (IH, dd, J 7.43, 5.09, 10-H) , 4.12 (IH, dd, J l 39, 4.30, 9-H) , 3.09 (IH, dd, J 10.17, 4.30, 8-H), 2.97 (IH, J 7.04, 5.87, 3-H) , 2.81 (IH, dd, J 12.52, 5.09, ll'-H), 2.57 (IH, d, J 12.52, 11"-H), 2.04 (2H, t, J 7.43, 4-H), 1.42-1.61 (4H, m, 5-7-H), 1.33-1.40 (2H, m, 2-H), 1.26-1.32 (2H, m, 5-7-H), 0.82 (3H, t, J 7.43).

¹³ C NMR (100 MHz) δ 171.9 (C) , 162.8 (C) , 61.1 (CH) , 59.3 (CH) , 55.5 (CH) , 40.3 (CH ₂ ) , 40.1 (CH ₂ ) , 35.3 (CH ₂ ) , 28.3 (CH ₂ ) , 28.1 (CH ₂ ) , 25 4 (CH ₂ ) , 22.5 (CH2) , 11.5 (Me) .

SNOB 4 1H NMR (400 MHz) , CDCl ₃ : δ 7.79-7.82 (2H, m, o-H) , 7.52 (IH, bs, NH) , 7.37-7.41 (IH, m, p-H) , 7.23-7.31 (2H, m, m-H) , 5.82 (IH, dquintet, J 117.22, 10.96, 5.48, 2-H) , 5.13 (IH, ddd, J 17.22, 2.74, 1.32, l'-H) , 5.04 (IH, ddd, J 10.17, 2.74, 1.32, 1"-H) , 5.96 (2H, tt, J 5.48, 1.57, 3'-H and 3"- H) .

¹³ C NMR (100 MHz) , CDCl ₃ : δ 167.5 (C) , 134.0 (C) , 133.9 (CH) , 131.0 (CH) , 128.0 (CH) , 127.9 (CH) , 115.7 (CH ₂ ) , 42.1 (CH ₂ ) .

Alternative synthesis of SNOB4

Benzoyl chloride (5.0 ml, 43.07 mmmol) was added to a solution of pyridine (50 ml) at 0°C, allyl amine (2.93 ml, 39.15 mmol) was added dropwise and the reaction was stirred at 0 ⁰ C for 40 minutes. The liquor was reduced in vacuo, removing residual water with toluene, the resulting oil was partitioned between CHC13 (50 ml) and water (50 ml) . The aqueous layer was further extracted CHCl ₃ (50 ml) and the combined organics were washed with brine (40 ml), dried (MgSO ₄ ) , filtered and reduced in vacuo. The oil was purified by flash chromatography eluting with petroleum ether and ethyl acetate to give the desired product as an oil 5.72 g (91% yield) . (Found: MNa ⁺ , 184.0729. CioHnNONa requires MNa ⁺ , 184.0733) .

¹ H NMR (400 MHz) CDCl ₃ δ 7.79-7.82 (2H, m, o-H) , 7.52 (IH, bs, NH), 7.37-7.41 (IH, m, p-H) , 7.23-7.31 (2H, m, zn-H) , 5.82 (IH, dquintet, J 17.22, 10.96, 5.48, 2-H) , 5.13 (IH, ddd, J 17.22, 2.74, 1.32, l'-H), 5.04 (IH, ddd, J 10.17, 2.74, 1.32, 1"-H), 5.96 (2H, tt, J 5.48, 1.57, 3'-H and 3"-H) .

¹³ C NMR (100 MHz) CDCl ₃ ; δ 167.5 (C) , 134.0 (C) , 133.9 (CH) , 131.0 (CH) , 128.0 (CH) , 127.9 (CH) , 115.7 (CH ₂ ) , 42.1 (CH ₂ ) .

SNOB5

Triethylamine (0.03 ml, 0.20 mmol) and allyl amine (0.04 ml, 0.46 mmol) were added to a stirred solution of succinimidyl- biotinamide hexonate (46 mg, 0.1 mmol) in DCM (1.5 ml) . The reaction was stirred for 1.5 hours and reduced in vacuo, purification by flash chromatography eluting with 15% MeOHiCHCl ₃ to give a white amorphous powder 41 mg, 100% yield. (Found: MH ⁺ , 397.2275. Ci ₉ H ₃₃ N ₄ O ₃ S requires Mlf, 397.2273) .

¹ H NMR (400 MHz) CD ₃ OD; δ 5.83 (IH, m, 2-H) , 5.17 (IH, -ddd, J 17.22, 3.13, 1.96, l' -H) , 5.10 (IH, ddd, J 10.17, 3.13, 1.57, 1" -H) , 4.49 (IH, m, 15-H) , 4.30 (IH, m, 14-H) , 3.76- 3.81 (2H, 3-H) , 3.22 (IH, m, 13-H) , 3.16 (2H, m, CH ₂ ) , 2.93 (IH, dd, J 12.52, 5.09, 16' -H) , 2.70 (IH, d, J 12.52, 16"- H) , 2.15-2.27 (4H, m, CH ₂ ) , 1.29-1.76 (12H, CH ₂ ) .

¹³ C NMR (100 MHz) CD ₃ OD δ 176.0 (C) , 175.9 (C) , 166.1 (C) , 135.6 (CH) , 116.1 (CH ₂ ) , 63.4 (CH) , 61.6 (CH) , 57.0 (CH) ,

42.7 (CH ₂ ) , 41.0 (CH ₂ ) , 40.2 (CH ₂ ) , 36.9 (CH ₂ ) , 36.8 (CH ₂ ) ,

30.1 (CH ₂ ) , 29.8 (CH ₂ ) , 29.5 (CH ₂ ) , 27.6 (CH ₂ ) , 26.9 (CH ₂ ) , 26.7 (CH ₂ ) .

SNOB 6

Allyl amine (0.50 ml, 6.68 mmol) ws added to a stirred solution of biotinamidohexanoyl-6-amino-hexanoic acid N- hydroxysuccinimide ester (50 mg, 0.088 mmol) in DMF (5 ml) . The reaction was stirred for 2.5 hours and reduced invacuo, acetonitrile (10 ml) was added and the suspension filtered under suction. The filtrand was washed with acetonitrile (2 X 5 ml) and the solid collected, reduced in vacuo to give a white amorphous powder 41 mg, 91% yield. (Found: MNa ⁺ , 532.2925. C ₂₅ H ₄₃ N ₅ O ₄ SNa requires MNa ⁺ , 532.2933) .

¹ H NMR (400 MHz) CD ₃ OD; δ 5.83 (IH, m, 2-H), 5.17 (IH, ddd, J 17.22, 3.50, 1.57, l'-H), 5.10 (IH, dd, J 10.17, 1.57, 1"- H), 4.50 (IH, dd, J 7.83, 4.70, 20-H) , 4.31 (IH, dd, J 7.83, 4.26, 19-H) , 3.76-3.81 (2H, 3-H), 3.20 (IH, m, 18-H) , 3.16 (2H, m, CH ₂ ), 2.93 (IH, dd, J 12.91, 5.09, 21'-H), 2.71 (IH, d, J 12.91, 21"-H), 2.14-2.26 (6H, m, CH ₂ ), 1.30-1.80 (2OH, CH ₂ ) .

¹³ C NMR (100 MHz) CD ₃ OD δ 176.0 (C), 176.0 (C), 175.0 (C), 166.1 (C), 135.6 (CH), 116.1 (CH ₂ ), 63.4 (CH), 61.6 (CH), 57.0 (CH), 42.7 (CH ₂ ), 42.7 (CH ₂ ), 41.1 (CH ₂ ), 40.2 (CH ₂ ), 40.2 (CH ₂ ), 37.0 (CH ₂ ), 36.9 (CH ₂ ), 36.8 (CH ₂ ), 30.1 (CH ₂ ), 29.8 (CH ₂ ), 29.5 (CH ₂ ), 27.6 (CH ₂ ), 26.9 (CH ₂ ), 26.7 (CH ₂ ), 26.7 (CH ₂ ) , 26.3 (CH ₂ ) .

SNOB detection of SNO containing proteins

All reactions were performed in physiological salt solutions containing (in mM) 130 NaCl, 5 KCl, 1.5 CaCl ₂ , 1 MgCl ₂ . 5

NaHCO ₃ , 1.5 KH ₂ PO ₄ , 25 Hepes, 10 glucose (pH 7.3 with NaOH).

Recombinant proteins (Broad range prestained SDS-PAGE standards, Biorad, Herfordshire, UK) were subjected to S- nitrosylation by addition of an NO donor, 100 μM DEA NONOate (Sigma Aldrich, UK) , for 10 minutes at ambient room temperature. Control samples were left untreated without addition of NONOate. Control and nitrosylated protein samples were then incubated with 300 μM SNOB-I or SNOB-3 reagent for 15 mins at 60 ⁰ C or 4 ⁰ C for control reactions.

Bovine serum albumin (Sigma Aldrich, UK) was subjected to S- nitrosylation by addition of a NO donor, 1-5 mM DEA NONOate

(Sigma Aldrich, UK), for 5-60 minutes from 4 °C - 60 °C. Control samples were left untreated without addition of DEA NONOate. Control and nitrosylated protein samples were then incubated with 300 - 450 μM SNOB reagent for 0-60 mins at 4

"C to 60 ⁰ C.

In control reactions, SNOB reagents were added immediately before running samples on SDS-PAGE. In control experiments, reaction components were substituted for equal volumes of physiological salt solution. Control and labelled proteins were run with pre-biotinylated SDS-PAGE standard markers

(Biorad, Hertfordshire, UK) as a positive control. Proteins were resolved using SDS-PAGE and were transferred onto nitrocellulose membrane. Membranes were blocked with 5% bovine serum albumin and biotinylated proteins were detected using horseradish peroxidase conjugated to streptavidin or neutravidin. After additional wash steps, bound streptavidin was detected by the ECL detection system

(Amershan, GE Healthcare) .

For human platelets, venous blood was taken from healthy volunteers who had taken no medication in the previous two weeks. Acid-citrate-dextrose (ACD) (85 mM trisodium citrate) 78 mM citric acid and 111 mM glucose) was added to isolated blood at a blood: ACD ratio of 8.5:1.5. Blood was centrifuged at 700 g for 5 min and platelet rich plasma was isolated. Platelet rich plasma was centrifuged for 20 min at 350 g and pelleted platelets were resuspended in platelet saline containing (in mM) 145 NaCl, 5 KCl, 1 MgCl ₂ , 10 HEPES, 10 glucose, pH 7.3 with NaOH. The platelet suspension was recentrifuged for 20 min at 350 g and resuspended in platelet saline. Throughout the protocol, platelets were maintained in the dark at 37 °C. Where indicated, DEA NONOate (5 mM) was added to the platelet suspension and incubated for 20 min. Also, where indicated, 20 mM DTT was added for 20 min to the platelet suspension. Finally, SNOB-6 was added to the washed platelets following the prior treatments and incubated for 5 min at 37 °C. After the 5 min incubation, cells were centrifuged for 20 min at 350 g and the platelet resuspended in lysis buffer containing (in mM) 150 NaCl, 20 TRIS (HCl pH 7.4 ) , 1 MgCl ₂ , 1 CaCl ₂ , 1 % Triton X-100 and 2 % protease inhibitor cocktail. Control and labelled proteins were run with pre- biotinylated markers as a positive control for detection of biotin as well as the molecular weight standards. Proteins

were resolved by SDS PAGE and transferred to nitrocellulose prior to blocking with 5 % bovine serum albumin and detected using horseradish peroxidase conjugated to neutravidin. After additional wash steps, bound neutravidin was detected by the ECL detection system (Amershan, GE Healthcare) .

Results

Synthesis of SNOB reagents

We have investigated the chemical reaction of S-nitroso cysteine derivatives with several molecules containing a terminal alkene, with the exception of SNOB 3, which served as a negative control to establish the requirement for an alkene group for the reaction to proceed. See Figure 2.

The S-NO biotinylating (SNOB) reagents 1-3 were synthesised according to a general procedure (Figure 4) . Briefly, the activated biotin derivative was generated in situ from D- biotin 5 and penta-fluorophenyl trifluoroacetate in dry pyridine. The terminal amine was then added directly to the solution of activated biotin to yield the desired amides 1 - 3. SNOB reagents 4-6 were synthesized successfully as detailed above.

Reaction of SNOB reagents with S-nitrosoglutathione Each of the SNOB reagents 1-3 were treated with commercially available S-nitrosoglutathione (6) in water at 50 ⁰ C and the products analysed by high resolution MS. SNOB reagent 1 was found to form the oxime 7. (Figure 5) . The SNOB reagents 3 did not undergo reaction with S-nitrosoglutathione. In the case of SNOB 3, this result shows that the alkene is essential for a reaction to occur with the nitrosothiol .

Reaction of SNOB reagents with S-nitroso penicillamine The SNOB 1 reagent was treated with commercially available

S-nitroso penicillamine (9) in water at 50°C and the products analysed by high resolution MS (Figure 6) . SNOB-I was found to react with (9) to give the thio-linked oxime (10) . This reaction shows that the SNOB reagent is also reactive towards very sterically hindered S-nitrosyl groups.

Detection of SNO groups in known proteins

To investigate the ability of SNOB reagents to detected S- nitrosylated proteins, known recombinant proteins were treated with a NO donor followed by detection of SNOB reagents (SNOB-I, SNOB-3, SNOB-5 and SNOB-6) with avidin containing reagents. Using this approach, SNOB-I, SNOB-5 and SNOB-6 binding were all detected as protein biotinylation in the presence of a NO donor heating the sample to 60 °C while SNOB-3 binding (an inactive analogue of SNOB-I) was not detected in the presence or absence of a NO donor (Figure 8) . Using this approach, SNOB-6 binding occurred efficiently in the presence of the NO donor heating the sample to 37 "C - 60 "C (Figures 8-10) . At 10 ⁰ C to 37 °C, SNOB-6 binding could be detected within 5 min and increased with time (up to 60 min) at pH 6 (Figure 9) . At 4 "C at pH 6, SNOB-6 binding was detected following 15 min incubation that increased with time up to 60 min (Figure 9) . SNOB-6 binding was optimal at pH 7 - 9 following incubation with nitrosylated protein at 37 "C for 30 min; low background labelling in the absence of the NO donor was evident at pH 11 at 37 "C (Figure 10) . At 10 "C and 22 ⁰ C, background labelling was undetectable at pH 5-11 where un- nitrosylated protein was incubated with SNOB-6 for 30 min (Figure 10) . Under non-denaturing conditions where the predicted number of nitrosylated cysteine groups in the presence of an NO donor would be reduced, avidin-containing reagents most efficiently detected SNOB-6 binding compared

to SNOB-I and SNOB-5; under these conditions SNOB-3 did not bind to the nitrosylated protein (Figure 8) .

We have developed a single step technique to selectively functionalize S-nitrosylated cysteines in the presence of free thiols and disulphide groups. This technique represents a significant advance over current methodologies.

Detection of endogenous S-nitrosylated proteins Endogenous S-nitrosylated proteins have been detected in cell lysates from endotoxin primed murine macrophages. Using this approach, protein biotinylation was detected when cell lysates were reacted with SNOB at 37°C. To increase to the detection of endogenous S-nitrosylated proteins, SDS was added to the sample buffer to denature protein and increase access to SNO groups.

Endogenous S-nitrosylated proteins have been detected on the cell surface of acutely isolated human platelets. Using this approach, isolated platelets were incubated with SNOB-6 either in the presence or absence of a NO donor or dithiothreitol (DTT) at 37 "C. Following incubation with SNOB-6, platelets were isolated by centrifugation that will also remove excess SNOB reagents and protein biotinylation detected by SDS-PAGE. Protein biotinylation was detected in platelets (in the absence of a NO donor) and reduced by incubation with DTT expected to remove SNO groups from proteins (Figure 11) . Incubation of platelets with a NO donor increased protein biotinylation above that detected in untreated platelets (Figure 12). This technique represents a novel approach to detect cell surface S-nitrosylation in live cells at 37 ⁰ C.

References :

All publications, patent and patent applications cited herein or filed with this application, including references filed as part of an Information Disclosure Statement are incorporated by reference in their entirety. WO 02/039110 and WO 2005/101019.

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