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WO2006055644A2 - Synthese et utilisation de reactifs glycodendrimeres - Google Patents

Synthese et utilisation de reactifs glycodendrimeres Download PDF

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Publication number
WO2006055644A2
WO2006055644A2 PCT/US2005/041549 US2005041549W WO2006055644A2 WO 2006055644 A2 WO2006055644 A2 WO 2006055644A2 US 2005041549 W US2005041549 W US 2005041549W WO 2006055644 A2 WO2006055644 A2 WO 2006055644A2
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Prior art keywords
protein
glycosylated
scheme
proteins
tren
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WO2006055644A3 (fr
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Richard R. Bott
Benjamin G. Davis
John Bryan Jones
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University of Toronto
Oxford University Innovation Ltd
Danisco US Inc
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University of Toronto
Oxford University Innovation Ltd
Genencor International Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
    • C07H5/08—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium
    • C07H5/10—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to sulfur, selenium or tellurium to sulfur

Definitions

  • chemically modified mutant proteins having modified glycosylation patterns with respect to a precursor protein from which they are derived.
  • chemically modified mutant proteins including a cysteine residue substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate.
  • methods of producing the chemically modified mutant proteins and glycosylated methanethiosulfonate reagents Further disclosed are methods of modifying the functional characteristics of a protein by reacting the protein with a glycosylated methanethiosulfonate reagent.
  • methods of determining the structure-function relationships of chemically modified mutant proteins are BACKGROUND OF THE INVENTION
  • Enzymes are now widely accepted as useful catalysts in organic synthesis.
  • natural, wild-type, enzymes can never hope to accept all structures of synthetic 0 chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis.
  • This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering.
  • modifying enzyme properties by s protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis.
  • Glycoproteins occur naturally in a number of forms (glycoforms) (Rademacher et al., Annu. Rev. Biochem., 57:785-838 (1988)) that possess the same peptide backbone, but differ in both the nature and site of glycosylation. The differences exhibited (Rademacher et al., Annu. Rev. Biochem.. 57:785-838 (1988); Parekh et al., Biochem..
  • Neoglycoproteins (Krantz et al, Biochem.. 15:3963-3968 (1976)), formed via unnatural linkages between sugars and proteins, provide an invaluable alternative source s of carbohydrate-protein conjugates (For reviews see Stowell et al., Adv. Carbohvdr. Chem. Biochem., 37:225-281 (1980); Neoglvcoconiugates: Preparation and Applications, Lee et al., Eds., Academic Press, London (1994); Abelson et al., Methods Enzymol., 242: (1994); Lee et al., Methods EnzvmoL, 247: (1994); Bovin et al., Chem. Soc.
  • glycosylation pattern of a protein is modified in a predictable and repeatable manner. Generally, the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.
  • a chemically modified mutant (“CMM”) protein differs from a precursor protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate.
  • the glycosylated O thiosulfonate is an alkylthiosulfonate, most preferably a methanethiosulfonate.
  • a method of producing a chemically modified mutant protein comprising the steps of: (a) providing a precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated s thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto.
  • the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a 0 DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.
  • the present invention also relates to novel glycosylated thiosulfonates.
  • the glycosylated thiosulfonate is a methanethiosulfonate.
  • the glycosylated methanethiosulfonate comprises a chemical structure s including:
  • R comprises - ⁇ -Glc, -Et- ⁇ -Gal, -Et- ⁇ -Glc, -Et- ⁇ -Glc,-Et- ⁇ -Man, -Et-Lac, - ⁇ -Glc(Ac) 2 , -P-GIc(Ac) 3 , -P-GIc(Ac) 4 , -Et-(XrGIc(Ac) 2 , -Et-O-GIc(Ac) 3 , -Et-O-GIc(Ac) 4 , -Et- ⁇ -Glc(Ac) 2 , -Et-P-GIc(Ac) 3 , -Et- ⁇ -Glc(Ac) 4 , -Et- ⁇ -Man(Ac) 3 , -Et- ⁇ -Man(Ac) 4 , -Et- ⁇ -Gal( Ac) 3 , o
  • Another aspect of the present invention is a method of modifying the functional characteristics of a protein including reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. Accordingly, the present invention provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.
  • the present invention relates to methods of determining the structure-function relationships of chemically modified mutant proteins.
  • One method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified mutant proteins and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • Another method involves providing first and second chemically modified mutant proteins of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting said cysteine residue with a glycosylated thiosulfonate, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the chemically modified mutant proteins of the present invention provide an alternative to site-directed mutagenesis and chemical modification for introducing unnatural amino acids into proteins.
  • the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure- activity relationships ("SARs")) of non-natural variants of the proteins.
  • SARs structure-activity relationships
  • An advantage of the present invention is that it is possible to introduce predetermined glycosylation patterns into proteins in a simple and repeatable manner. This advantage provides an ability to modify critical protein characteristics such as partitioning, solubility, cell-signaling, catalytic activity, biological activity and pharmacological activity. Additionally, certain methods of the present invention provide for a mechanism of "masking" certain chemically or biologically important protein sites, for example, sites which are critical s for immunological or allergenic responses or sites which are critical to proteolytic degradation of the modified protein.
  • Another advantage of the present invention is the ability to glycosylate a protein which is not generally glycosylated, or to modify the glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is improved synthetic methods for glycosylating a protein which is not generally glycosylated, or for modifying the glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is novel reagents for glycosylating a protein which is not generally glycosylated, or for modifying the s glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is to produce enzymes that have altered catalytic activity.
  • the inventors have shown that it is possible to modify the substrate specificity of a protease to increase its ability to degrade lectins, selectin or integrins or members of other adhesion receptor families. Degradation of 0 these proteins may have many specific effects which include, but are not limited to, degradation of biofilms and especially pathogenic biofilms. As an example, pathogenic biofilms from the organism Pseudomonas may be degraded.
  • production of enzymes with altered catalytic or substrate binding activity may have therapeutic uses related to cancers, as they relate, for example, to metastasis. Similarly, modifications of substrate s specificity would be expected when utilizing the present invention with other enzymes.
  • Figure 1 shows dendrimer methanethiosulfonate (“MTS”) reagents.
  • FIG. 2 shows dendrimer methanethiosulfonate (“MTS”) reagents and hybrid dendrimer methanethiosulfonate (“MTS”) reagents.
  • Figure 3 shows a glycodendrimer protein binding to carbohydrate binding sites on a lectin, selectin, integrin or member of other adhesion receptor family.
  • X, Y and Z represent optional carbohydrate linkers; the lengths of these linkers, if present, need not be equal to each other.
  • Figure 4 is a schematic illustration of a glycodendriprotein showing terminal carbohydrate moieties, optional linkers, disulfide linkages, dendrimer cores, and a model enzyme, SBL.
  • Figure 5 shows two different synthetic approaches for generating glycodendrimers, i.e., normal addition, and inverse addition.
  • Figure 6 shows several synthetic schemes for generating tethered and direct-linked carbohydrate MTS reagents.
  • Figure 7 shows additional synthetic schemes for generating direct-linked carbohydrate MTS reagents.
  • Figure 8 shows the X-ray crystal structure of the MTS reagent 5 ⁇ .
  • Figure 9 shows the X-ray crystal structure of the MTS reagents 10a and lO ⁇ .
  • Figure 10 illustrates the use of a glycodendriprotein to digest a lectin, selectin, integrin or member of other adhesion receptor family.
  • Figure 11 illustrates components of a glycodendriprotein.
  • the Y shaped 0 dendrimer core illustrated is TREN-type, but also may represent Penta-E type, ArGal-type or other dendrimer core structures.
  • Figure. 12 shows a normal addition synthetic scheme for producing two different first-generation glycodendrimer MTS reagents, and glycodendriproteins produced from these reagents.
  • Figure 13 shows a normal addition synthetic scheme for producing a glycodendriprotein.
  • Figure 14 shows a normal addition synthetic scheme for producing a second generation glycodendrimer reagent.
  • Figure 15 shows glyco MTS reagent 12 ⁇ , and diglycosyl disulfides 18 and o 19 resulting from the use of 12 ⁇ or 5 ⁇ in the in situ reduction approach described in Example 2.
  • Figure 15 also illustrates an inverse addition synthesis scheme (Scheme 9) for a first generation glycodendrimer reagent.
  • Figure 16 shows an inverse addition synthesis scheme for a multi- generation glycodendrimer reagent.
  • Figure 17 shows Scheme 11, illustrating synthetic approaches for producing bis-MTS reagents; Scheme 12, for producing thioglycoses; and Scheme 13, illustrating another synthetic method for generating a first generation glycodendrimer reagent.
  • Figure 18 shows another synthetic scheme for producing a glycodendrimer MTS reagent.
  • Figure 19 shows a synthetic scheme (Scheme 15) for producing a first generation glycodendrimer MTS reagent, and a second generation hybrid glycodendrimer MTS reagent; and an improved synthesis scheme (Scheme 16) for producing sodium methanethiosulfonate (“NaMTS").
  • Figure 20 shows synthetic scheme 17 for producing ArGal-based glycodendrimer reagent 42; and synthetic scheme 18 for producing ArGal-based glycodendrimer reagent 44, bearing two deprotected sugars.
  • Figure 21 illustrates synthetic scheme 19 and synthetic scheme 20 for producing glycodendrimer reagents.
  • Figure 22 illustrates synthetic scheme 21 for producing a glycodendriprotein.
  • Figure 23 illustrates a glycosylated variant of Bacillus lentus subtilisin mutant S 156C.
  • a method wherein the glycosylation pattern of a protein is modified in a predictable and repeatable manner.
  • the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.
  • a chemically modified mutant protein differs from a precursor protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate.
  • the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • a method of producing a chemically modified mutant protein comprising the steps of: (a) providing a s precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto.
  • the glycosylated thiosulfonate is an O alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.
  • the amino acid residues to be substituted with cysteine residues according to the present invention may be replaced using site-directed mutagenesis methods s or other methods well known in the art. See, for example, PCT Publication No. WO 95/10615, which is hereby incorporated by reference.
  • the present invention also relates to glycosylated thiosulfonate.
  • the glycosylated thiosulfonate comprises methanethiosulfonate. More preferably, the methanethiosulfonate comprises the chemical structure:
  • R comprises - ⁇ -Glc, -Et- ⁇ -Gal, -Et- ⁇ -Glc, -Et- ⁇ -Glc,-Et- ⁇ -Man, -Et-Lac, - ⁇ -Glc(Ac) 2 , -P-GIc(Ac) 3 , -P-GIc(Ac) 4 , -Et-O-GIc(Ac) 2 , -Et-OC-GIc(Ac) 3 , -Et-O-GIc(Ac) 4 , -Et- ⁇ -Glc(Ac) 2 , -Et-P-GIc(Ac) 3 , -Et-P-GIc(Ac) 4 , -Et- ⁇ -Man(Ac) 3 , -Et- ⁇ -Man(Ac) 4 , -Et- ⁇ -Gal(Ac) 3 ,
  • Another aspect of the present invention is a method of modifying the functional characteristics of a protein including providing a protein and reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein.
  • the functional characteristics of a protein which may be altered by the o present invention include, but are not limited to, enzymatic activity, the effect on a human or animal body, the ability to act as a vaccine, the tertiary structure (i.e., how the protein folds), whether it is allergenic, its solubility, its signaling effects, its biological activity and its pharmacological activity (Paulson, "Glycoproteins: What are the sugar chains for?" Trends in Biochem. Sciences, 14:272-276 (1989), which is hereby incorporated by reference).
  • glycosylated thiosulfonates as thiol-specific modifying reagents in a method of the present invention allows virtually unlimited alterations of protein residues, hi addition, this method allows the production of pure glycoproteins with predetermined and unique structures and therefore, unique functional characteristics, with control over both the site and level of glycosylation.
  • the methods of modifying the functional characteristics of a protein allow the preparation of single glycoforms through regio- and glycan-specific protein glycosylation at predetermined sites. Such advantages provide an array of options with respect to modification of protein properties which did not exist in the prior art.
  • the present invention provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.
  • glycosylation pattern means the composition of a carbohydrate moiety.
  • the present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins.
  • the first method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified s mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the second method involves providing a first and second chemically modified mutant protein of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting O said cysteine residue with a glycosylated thiosulfonate evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the chemically modified mutant proteins of the present invention provide s a valuable source of carbohydrate-protein conjugates. Moreover, the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships ("SARs")) of non-natural variants of the proteins. 0 [0063]
  • the protein of the invention may be any protein for which a modification of the glycosylation pattern thereof may be desirable. For example, proteins which are naturally not glycosylated may be glycosylated via the invention.
  • proteins which exist in a naturally glycosylated form may be modified so that the glycosylation pattern confers improved or desirable properties to the protein.
  • proteins useful in the s present invention are those in which glycosylation plays a role in functional characteristics such as, for example, biological activity, chemical activity, pharmacological activity, or immunological activity.
  • Glycosylated proteins as referred to herein means moieties having carbohydrate components which are present on proteins, peptides, or amino acids.
  • the glycosylation is provided, for example, as a result of reaction of the glycosylated thiosulfonate with the thiol hydrogen of a cysteine residue thereby producing an amino acid residue which has bound thereto the carbohydrate component present on the glycosylated thiosulfonate.
  • Glycosylation also may be accomplished, according to the present invention, by attachment of glycodendrimer reagents such as those described in the examples below. Such reagents comprise one or more dendrimer core portions, optionally a linker (or tether), and one or more carbohydrate moieties.
  • the invention provides for synthetic schemes for producing glycodendrimer reagents.
  • Said schemes include normal addition schemes in which a carbohydrate alkylthiosulfonate is reacted with a dendrimer core, said core comprising a free sulfhydryl group.
  • inverse addition synthesis schemes in which a thioglycose is reacted with a dendrimer core alkylthiosulfonate.
  • a preferred synthesis scheme involves reacting a carbohydrate with an alkylthiosulfonate and a phase transfer catalyst under refluxing toluene conditions.
  • the alkylthiosulfonate is a sodium salt of methanethiosulfonate
  • the phase transfer catalyst is tetrabutylammonium s iodide (Bu 4 NI).
  • the protein is an enzyme.
  • the term "enzyme” includes proteins that are capable of catalyzing chemical changes in other substances without being changed themselves.
  • the enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include pullulanases, proteases, cellulases, 0 amylases, isomerases, lipases, oxidases, and reductases.
  • the enzyme is a protease.
  • the enzyme can be a wild-type or mutant protease. Wild-type proteases can be isolated from, for example, Bacillus lentus or Bacillus amyloliquefaciens (also referred to as BPN').
  • Mutant proteases can be made according to the teachings of, for example, PCT Publication Nos. WO 95/10615 and WO 91/06637, which are hereby incorporated by reference.
  • Functional characteristics of enzymes which are suitable for modification according to the present invention include, for example, enzymatic activity, solubility, partitioning, cell-cell signaling, substrate specificity, substrate binding, stability to temperature and reagents, ability to mask an antigenic site, physiological functions, and pharmaceutical functions (Paulson, "Glycoproteins: What are the Sugar Chains For?” Trends in Biochem Sciences. 14:272-276 O (1989), which is hereby incorporated by reference.)
  • the protein is modified so that a non-cysteine residue is substituted with a cysteine residue, preferably by recombinant means.
  • the amino acids replaced in the protein by cysteine are selected from the group consisting of asparagine, leucine or serine. Orthogonal protection schemes that are well known in the art may be used when modification is to be carried out at more than one site within a protein.
  • thiol side chain group thiol containing group
  • thiol s side chain are terms which can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine.
  • the cysteine occurs in the native protein sequence, while in other embodiments, a cysteine replaces one or more amino acids in the protein.
  • the thiol side chain group includes a sulfur through which the thiol side chain groups defined above are attached to the thiol sulfur of the cysteine.
  • glycosylated thiosulfonates of the invention are those which are capable of reacting with a thiol hydrogen of a cysteine to produce a glycosylated amino acid residue.
  • glycosylated is meant that the thiosulfonate has bound thereto a sugar or carbohydrate moiety that can be transferred to a protein or dendrimer (which may be bound to a protein) pursuant to the present invention.
  • die glycosylated thiosulfonates are s glycosylated alkylthiosulfonates, most preferably, glycosylated methanethiosulfonates.
  • Such glycosylated methanethiosulfonates have the general formula:
  • the methanethiosulfonate R group comprises: - ⁇ -Glc,
  • the carbohydrate moiety of the present invention is a dendrimer moiety.
  • Multiple functionalization of chemically modified mutant proteins can be s achieved by dendrimer approaches, whereby multiple-branched linking structures can be employed to create poly-functionalized chemically modified mutant proteins.
  • Dendrimer moieties of the present invention may contain one or more branch points.
  • first generation refers to dendrimer moieties that contain a single branch point.
  • second generation refers to a o dendrimer moiety that contains multiple branch points.
  • the number of branch points in a second generation dendrimer will depend on the number of arms that can be attached to the central core of the dendrimer building block. In general, the number of branch points for a dendrimer comprising central cores having the same number of arms is equal to
  • N equals the number of arms and G is the generation number.
  • the TREN-type dendrimers such as those illustrated in Fig. 1 have three arms attached to the central TREN core.
  • N 4
  • the dendrimer reagent structures would include methanethiosulfonates with simple branching such as:
  • a first generation glycodendrimer reagent is synthesized as shown in Figure 12, Scheme 6. This approach can be extended to cover larger dendrimers. More specifically, by leaving one "arm" of the glycodendrimer free for conversion to a methanethiosulfonate, the remaining arms can be further branched to synthesize highly- functionalized glycodendrimer reagents as shown in Figure 14, Scheme 8.
  • a glycodendrimer comprising the steps of providing at least a first dendrimer core building block, at least one alkylthiosulfonate, and at least one carbohydrate having a first sulfhydryl group; reacting the first dendrimer core building block with the alkylthiosulfonate to produce a modified dendrimer core building block having an alkylthiosulfonate group; reacting the resultant modified dendrimer core building block with the carbohydrate to produce a glycodendrimer.
  • the carbohydrate is preferably a disaccharide, and may optionally be a monosaccharide or a polysaccharide.
  • the alkylthiosulfonate can be methanethiosulfonate, ethanethiosulfonate, propanethiosulfonate, or a C 1 -C 6 alkylthiosulfonate.
  • the methanethiosulfonate can be a salt, such as an alkali metal salt, e.g., lithium, sodium, or potassium salt, or an alkali earth metal salt, e.g., magnesium or calcium salt.
  • the first dendrimer core building block can be tris(2-aminoethyl)amine (TREN).
  • TREN tris(2-aminoethyl)amine
  • the first dendrimer core building block can be pentaerythritol, mesitylene or mesitylene tribromide.
  • the carbohydrate can be directly or indirectly linked to the first sulfhydryl group or, alternatively, tethered to the first sulfhydryl group.
  • the disaccharide can optionally be selected from the group consisting of Gal-Gal, Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and Glu-Man, and acetyl or benzoyl derivatives thereof.
  • the polysaccharide can optionally be selected from the group consisting of Gal-Lac, Glu-Lac, Man-Lac, and Lac-Lac, and acetyl or benzoyl derivatives thereof.
  • Gal refers to the saccharide unit galactose.
  • GIu refers to the saccharide unit glucose.
  • Man refers to the saccharide unit manose.
  • Lac refers to the disaccharide unit lactose.
  • either the first or the second, or both, saccharide units may be linked to the dendrimer core.
  • the terms "Gal,” “GIu,” “Man,” and “Lac” are meant to encompass saccharide units that are mono-, di-, tri-, tetra-, or otherwise polysubstituted. Such substitution is preferably with acetyl or benzoyl groups.
  • the glycodendrimer produced by the above method can optionally be a first-generation glycodendrimer. Further, the first-generation glycodendrimer can optionally comprise an alkylthiosulfonate group, which can be methanethiosulfonate.
  • the above disclosed methods for producing a glycodendrimer can further comprise a step of reacting the modified dendrimer core building block having an alkylthiosulfonate group with a second dendrimer core building block having a second sulfhydryl group to produce a multi-generation glycodendrimer.
  • the multi-generation glycodendrimer is a second-generation glycodendrimer.
  • the method for producing a glycodendrimer can comprise the steps of providing a first dendrimer core building block and a carbohydrate having a first s sulfhydryl group; and reacting said dendrimer core building block with said carbohydrate to produce a glycodendrimer; where the carbohydrate is a disaccharide.
  • the disaccharide can be any disaccharide, and is preferably selected from the group consisting of 4-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-2,3,6-tri-O- benzoyl-1-thio- ⁇ -D-galactopyranoside, Gal-Gal, Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and
  • the dendrimer core building block can be selected from the group consisting of TREN, TREN-Boc, TREN-Boc-Ac(Cl) 2 ,
  • the glycodendrimer is selected from the group consisting of TREN-BoC-(SGaI) 2 , TREN-(SGaI) 2 ,
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are each independently and optionally selected from the group consisting of hydrogen, alkyl, alkylacyl (alkyl-C(O)-), arylacyl (aryl-C(O)-) and arylalkylacyl (arylalkyl-C(O)-); and
  • R 8 is selected from the group consisting of hydrogen, alkali metal, alkali earth metal, aluminum, organic cation, and inorganic cation. 2s [0083] In another aspect, disclosed herein is a compound selected from the group consisting of the following structures:
  • the present invention relates to a glycodendrimer reagent composition having the generic structure:
  • a, b, c, and d are each independently selected from the group consisting of all integers from O to 10, inclusive, such as, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10,
  • X may be either SR or R, wherein R is a disaccharide, substituted disaccharide, polysaccharide, or substituted polysaccharide selected from the group consisting of Gal-Gal, Glu-Glu, Man-Man, Lac-Lac, Gal-Glu, Gal-Man, Gal-Lac, Glu-Man, and Glu-Lac.
  • X may be
  • subtilisins Alkaline serine proteases
  • subtilisins are increasingly used in biocatalysis, particularly in chiral resolution, regioselective acylation of polyfunctional compounds, peptide coupling, and glycopeptide synthesis.
  • subtilisins can be used to catalyze peptide bond formation. From an ester substrate, subtilisins can form an acyl enzyme intermediate which then reacts with a primary amine to form a peptide product.
  • subtilisins do not meet these requirements. Nonetheless, improved esterase to amidase selectivities are desired in the art. By using the methods described s herein, it is now possible to produce subtilisins that have advantageous properties.
  • Bacillus lentus subtilisin is selected O for illustrated purposes only, as it does not contain a natural cysteine and is not naturally glycosylated.
  • the substrate binding site of an enzyme consists of a series of subsites across the surface of the enzyme.
  • the portion of substrate that corresponds to the subsites are labeled P and the subsites are labeled S.
  • the subsites are labeled S 1 , S 2 , S 3 , s S 1 ', S 2 ', etc.
  • a discussion of subsites can be found in Berger et al., Phil Trans. Ro yl Soc. Lond.
  • any of the S 1 , S 1 ', or S 2 subsites is selected as a 0 suitable target for modification.
  • the amino acid residues corresponding to N62, L217, S 156, and S 166 in naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as Bacillus lentus subtilisin were selected for modification to cysteine.
  • An amino acid residue of an enzyme is equivalent to a residue of a s referenced enzyme (e.g., B. amyloliquefaciens subtilisin) if it is either homologous (corresponds in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (has the same or similar functional capacity to combine, react, or interact chemically).
  • a s referenced enzyme e.g., B. amyloliquefaciens subtilisin
  • the amino acid sequence of 0 the subject enzyme for example, a serine hydrolase, cysteine protease, aspartyl protease, metalloprotease, etc.
  • a reference enzyme for example, B. amyloliquefaciens subtilisin in the case of a subtilisin type serine protease
  • a set of residues known to be invariant in all enzymes of that family e.g. subtilisins
  • the residues equivalent to particular amino acids in the primary sequence of the reference enzyme e.g., B. amyloliquefaciens subtilisin
  • Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of at least approximately 75% or at least approximately 50% of conserved residues are also adequate to define equivalent residues.
  • Conservation of a catalytic triad, (for example, Asp32/His64/Ser221) is preferably maintained for serine hydrolases.
  • the conserved residues may be used to define the corresponding amino acid residues in other related enzymes.
  • the two sequences one a "reference” and the other a "target" are aligned in order to produce the maximum homology of conserved residues.
  • a number of deletions are seen in the thermitase sequence as compared to B. amyloliquefaciens subtilisin ⁇ see, e.g. U.S. Patent 5,972,682).
  • amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217 in Figure 5B-2 of the 5,972,682 patent.
  • the particular "corresponding" residues may be substituted by a different amino acid to produce a mutant carbonyl hydrolase because they the correspond in primary structures.
  • Corresponding residues may also be determined as homologous or corresponding at the level of tertiary structure for a particular enzyme. Tertiary structure may be determined by for example x-ray crystallography or other known techniques. Corresponding residues may be defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the reference sequence (e.g., B. amyloliquefaciens subtilisin) and the sequence in question (target sequence) (N on N, CA on CA, C on C, and O on O) are within 0.2, 0.15, or 0.13 nm and preferably approximately 0.1 nm after alignment.
  • the reference sequence e.g., B. amyloliquefaciens subtilisin
  • target sequence target sequence
  • Alignment may be achieved after the model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the enzyme in question to the reference sequence.
  • the best model is the crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.
  • Corresponding residues may also be determined as functionally analogous s to a specific residue of a reference sequence.
  • corresponding residues are defined as those amino acid residues, which may adopt a conformation such that they will alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the reference sequence as described herein.
  • corresponding residues are those residues of the sequence (typically for which a O tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the main chain atoms of a given residue.
  • the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 nm of the corresponding side chain atoms of the reference sequence residue(s).
  • the three dimensional structures are to be aligned as described above. For an illustration of such an alignment procedure see U.S. s Patent 5,972,682, which is incorporated by reference herein in its entirety.
  • mutated subtilisins were produced through standard site directed mutagenesis techniques and the obtained mutant subtilisin was reacted with certain glycosylated alkylthiosulfonates, particularly glycosylated methanethiosulfonates, as provided in the examples appended hereto.
  • glycosylated alkylthiosulfonates particularly glycosylated methanethiosulfonates, as provided in the examples appended hereto.
  • Proteins or other compositions obtained using the methods described herein may be used in any application in which it is desired to use such proteins, especially those in which having modified functional capabilities is advantageous. Proteins modified as provided herein may be used in the medical field for pharmaceutical compositions and in diagnostic preparations.
  • proteins such as enzymes, that are modified as s described herein may be used in applications which generally are known for such enzymes including industrial applications such as cleaning products, textile processing, feed modification, food modification, brewing of grain beverages, starch processing, as antimicrobials, health care formulations and in personal care formulations.
  • industrial applications such as cleaning products, textile processing, feed modification, food modification, brewing of grain beverages, starch processing, as antimicrobials, health care formulations and in personal care formulations.
  • the unique functionalities made possible by the methods described herein may result in new and o unforeseen uses for proteins which have not heretofore been recognized.
  • Example 1 Synthesis and Characterization of Carbohydrate Methanethiosulfonate Reagents
  • Cationic carbohydrate-methanethiosulfonates have been prepared previously, as described in United States Patent Application Serial No. 09/347,029 "Chemically Modified Proteins with a Carbohydrate Moiety," the entire disclosure of which is hereby incorporated by reference in its entirety.
  • the following examples are those that have been prepared as novel compounds for use in glycodendriproteins.
  • the sugar-MTS reagents required for attachment to the tips of the dendrimer were prepared.
  • MTS reagent 3 could also be prepared from 2 using this tetrabutylammonium method, but the yields and rate were no improvement on that described in Scheme 1. This method was also used to prepare the directly linked glucofuranosyl MTS reagent 8 (Fig. 6, Scheme 3). The product was an inseparable anomeric mixture. No further effort has yet been made to obtain pure 8 ⁇ or 8 ⁇ .
  • Glucofuranose 6 is the first readily available crystalline peracylated glucofuranose. Furneaux, R.H.; Rendle, P.M.; Sims, LM. /.
  • Acetobromogalactose 1 (1.1 g, 2.68 mmol) and sodium methanethiosulfonate (0.45 g, 3.35 mmol) in toluene (50 mL) were concentrated in vacuo to approximately 30 mL to remove any water as the azeotrope. The mixture was made up to 50 mL with more toluene and again concentrated to 30 mL. A catalytic amount of tetrabutylammonium iodide was added and the mixture heated at reflux for 75 minutes. After cooling, Celite (to stop the formation of a salt cake on top of the column) was added and the whole mixture loaded directly on to a flash silica column.
  • TLC Thin Layer Chromatography
  • Lectins, selectins, integrins and members of other adhesion receptor families are sugar binding proteins. The sugar binding sites are relatively shallow and hence binding is comparatively weak. Lectins, selectins, integrins and members of other adhesion o receptor families, however often bind many saccharides of an oligosaccharide to give a strong, selective affinity between the lectin, selectin, integrin or members of another adhesion receptor family and a particular combination of saccharides. This is illustrated in Figure 3. Briefly, the aim is to attach many sugars to the surface of a dendrimeric structure that is in turn attached to a protein to mimic the natural system.
  • the model protein we used is subtilisin Bacillus lentus (SBL), a serine protease enzyme. If the glycodendrimer system synthesized has a strong affinity to the lectin, selectin, integrin or members of another adhesion receptor family being targeted, then the attached SBL (being a protease) should start 'cutting' up the lectin, selectin, integrin or members of another adhesion receptor family. This is shown schematically in Figure 10. The specific model glycodendrimer that is the initial synthetic target of this project is shown in Figure 11. SBL has no natural cysteines (and hence no thiols present). One can be introduced by way of site-directed mutagenesis.
  • SBL subtilisin Bacillus lentus
  • Methane thiosulfonate (MTS) reagents react specifically and quantitatively with thiols (Wynn, R.; Richards, F.M. Methods Enzymol 1995, 201, 351) giving an excellent method for the attachment of the glycodendrimer to the protein.
  • glycodendrimer reagents were prepared according to the methods described in Davis, B. G., "The controlled glycosylation of a protein with a bivalent glycan: towards a new class of glycoconjugates, glycodendriproteins," Chem.
  • the initial dendrimeric core building block was prepared from tris(2- aminoethyl)amine (TREN) 1 using literature methodology (Tecilla, P.; Tonellato, U.;
  • the next step is the attachment of the sugars to the dendrimeric core. Because of the stability problems of the dithiol 3, many attempts were made to generate it in j situ. This type of coupling is one of the most important reactions in building of multi- generation glycodendriproteins (Scheme 8) and hence an elegant, high yielding reaction would be very useful. These attempts to generate the dithiol 3 in situ took two forms, either (a) reducing the disulfide 4 or (b) deprotecting the diacetate 2.
  • chemically modified mutant proteins having modified glycosylation patterns with respect to a precursor protein from which they are derived.
  • chemically modified mutant proteins including a cysteine residue s substituted for a residue other than cysteine in a precursor protein, the substituted cysteine residue being subsequently modified by reacting the cysteine residue with a glycosylated thiosulfonate.
  • methods of producing the chemically modified mutant proteins and glycosylated methanethiosulfonate reagents Further disclosed are methods of modifying the functional characteristics of a protein by reacting the protein with a o glycosylated methanethiosulfonate reagent.
  • methods of determining the structure-function relationships of chemically modified mutant proteins - 2 -
  • Enzymes are now widely accepted as useful catalysts in organic synthesis.
  • natural, wild-type, enzymes can never hope to accept all structures of synthetic 0 chemical interest, nor always be transformed stereospecifically into the desired enantiomerically pure materials needed for synthesis.
  • This potential limitation on the synthetic applicabilities of enzymes has been recognized, and some progress has been made in altering their specificities in a controlled manner using the site-directed and random mutagenesis techniques of protein engineering.
  • modifying enzyme properties by s protein engineering is limited to making natural amino acid replacements, and molecular biological methods devised to overcome this restriction are not readily amenable to routine application or large scale synthesis.
  • Glycoproteins occur naturally in a number of forms (glycoforms) (Rademacher et al., Annu. Rev. Biochem.. 57:785-838 (1988)) that possess the same peptide backbone, but differ in both the nature and site of glycosylation. The differences exhibited (Rademacher et al., Annu. Rev. Biochem.. 57:785-838 (1988); Parekh et al., Biochem.. 28:7670 7679 ( 1989); Knight, Biotechnol..
  • Neoglycoproteins (Krantz et al, Biochem.. 15:3963-3968 (1976)), formed via unnatural linkages between sugars and proteins, provide an invaluable alternative source of carbohydrate-protein conjugates (For reviews see Stowell et al., Adv. Carbohydr. Chem. Biochem.. 37:225-281 (1980); Neoglycoconiugates: Preparation and Applications. Lee et al., Eds., Academic Press, London (1994); Abelson et al., Methods Enzymol., 242: (1994); Lee et al., Methods EnzvmoL. 247: (1994); Bovin et al., Chem. Soc. Rev..
  • glycosylated proteins which have well defined properties, for example, by having predetermined glycosylation patterns.
  • a method wherein the glycosylation pattern of a protein is modified in a predictable and repeatable manner. Generally, the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.
  • a chemically modified mutant (“CMM") protein is provided, wherein said mutant protein differs from a precursor protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by reacting said cysteine residue with a glycosylated thiosulfonate.
  • the glycosylated O thiosulfonate is an alkylthiosulfonate, most preferably a methanethiosulfonate.
  • a method of producing a chemically modified mutant protein comprising the steps of: (a) providing a precursor protein; (b) substituting an amino acid residue other than cysteine in said precursor protein with a cysteine; (c) reacting said substituted cysteine with a glycosylated S thiosulfonate, said glycosylated thiosulfonate comprising a carbohydrate moiety; and (d) obtaining a modified glycosylated protein wherein said substituted cysteine comprises a carbohydrate moiety attached thereto.
  • the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a 0 DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.
  • the present invention also relates to novel glycosylated thiosulfonates.
  • the glycosylated thiosulfonate is a methanethiosulfonate.
  • the glycosylated methanethiosulfonate comprises a chemical structure s including:
  • R comprises - ⁇ -Glc, -Et- ⁇ -Gal, -Et- ⁇ -Glc, -Et- ⁇ -Glc,-Et- ⁇ -Man, -Et-Lac, - ⁇ -Glc(Ac) 2 , -P-GIc(Ac) 3 , - ⁇ -Glc(Ac) 4 , -Et- ⁇ -Glc(Ac) 2> -Et- ⁇ -Glc(Ac) 3 , -Et-OrGIc(Ac) 4 , -Et- ⁇ -Glc(Ac) 2 , -Et-P-GIc(Ac) 3 , -Et-P-GIc(Ac) 4 , -Et- ⁇ -Man(Ac) 3 , -Et- ⁇ -Man(Ac) 4 , -Et-p-Gal(Ac) 3 , o -Et-P-GaI
  • Another aspect of the present invention is a method of modifying the functional characteristics of a protein including reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein. Accordingly, the present invention s provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.
  • the present invention relates to methods of determining O the structure-function relationships of chemically modified mutant proteins.
  • One method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified mutant s proteins and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • Another method involves providing first and second chemically modified mutant proteins of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting said cysteine residue with 0 a glycosylated thiosulfonate, evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the chemically modified mutant proteins of the present invention provide s an alternative to site-directed mutagenesis and chemical modification for introducing unnatural amino acids into proteins.
  • the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships ("SARs")) of non-natural variants of the 0 proteins.
  • SARs structure-activity relationships
  • An advantage of the present invention is that it is possible to introduce predetermined glycosylation patterns into proteins in a simple and repeatable manner.
  • This - 8 - advantage provides an ability to modify critical protein characteristics such as partitioning, solubility, cell-signaling, catalytic activity, biological activity and pharmacological activity.
  • certain methods of the present invention provide for a mechanism of "masking" certain chemically or biologically important protein sites, for example, sites which are critical for immunological or allergenic responses or sites which are critical to proteolytic degradation of the modified protein.
  • Another advantage of the present invention is the ability to glycosylate a protein which is not generally glycosylated, or to modify the glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is improved synthetic methods for glycosylating a protein which is not generally glycosylated, or for modifying the glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is novel reagents for glycosylating a protein which is not generally glycosylated, or for modifying the glycosylation pattern of a protein which is generally glycosylated.
  • Another advantage of the present invention is to produce enzymes that have altered catalytic activity.
  • the inventors have shown that it is possible to modify the substrate specificity of a protease to increase its ability to degrade lectins, selectin or integrins or members of other adhesion receptor families. Degradation of these proteins may have many specific effects which include, but are not limited to, degradation of biofilms and especially pathogenic biofilms. As an example, pathogenic biofilms from the organism Pseudomonas may be degraded.
  • production of enzymes with altered catalytic or substrate binding activity may have therapeutic uses related to cancers, as they relate, for example, to metastasis. Similarly, modifications of substrate specificity would be expected when utilizing the present invention with other enzymes.
  • Figure 1 shows dendrimer methanethiosulfonate (“MTS”) reagents.
  • FIG. 2 shows dendrimer methanethiosulfonate (“MTS”) reagents and hybrid dendrimer methanethiosulfonate (“MTS”) reagents. - 9 -
  • Figure 3 shows a glycodendrimer protein binding to carbohydrate binding sites on a lectin, selectin, integrin or member of other adhesion receptor family.
  • X, Y and Z represent optional carbohydrate linkers; the lengths of these linkers, if present, need not be equal to each other.
  • Figure 4 is a schematic illustration of a glycodendriprotein showing terminal carbohydrate moieties, optional linkers, disulfide linkages, dendrimer cores, and a model enzyme, SBL.
  • Figure 5 shows two different synthetic approaches for generating glycodendrimers, i.e., normal addition, and inverse addition.
  • Figure 6 shows several synthetic schemes for generating tethered and direct-linked carbohydrate MTS reagents.
  • Figure 7 shows additional synthetic schemes for generating direct-linked carbohydrate MTS reagents.
  • Figure 8 shows the X-ray crystal structure of the MTS reagent 5 ⁇ .
  • Figure 9 shows the X-ray crystal structure of the MTS reagents 10a and lO ⁇ .
  • Figure 10 illustrates the use of a glycodendriprotein to digest a lectin, selectin, integrin or member of other adhesion receptor family.
  • Figure 11 illustrates components of a glycodendriprotein.
  • the Y shaped 0 dendrimer core illustrated is TREN-type, but also may represent Penta-E type, ArGal-type or other dendrimer core structures.
  • Figure 12 shows a normal addition synthetic scheme for producing two different first-generation glycodendrimer MTS reagents, and glycodendriproteins produced from these reagents.
  • Figure 13 shows a normal addition synthetic scheme for producing a glycodendriprotein.
  • Figure 14 shows a normal addition synthetic scheme for producing a second generation glycodendrimer reagent.
  • Figure 15 shows glyco MTS reagent 12 ⁇ , and diglycosyl disulfides 18 and 0 19 resulting from the use of 12 ⁇ or 5 ⁇ in the in situ reduction approach described in Example 2.
  • Figure 15 also illustrates an inverse addition synthesis scheme (Scheme 9) for a first generation glycodendrimer reagent. - 10 -
  • Figure 16 shows an inverse addition synthesis scheme for a multi- generation glycodendrimer reagent.
  • Figure 17 shows Scheme 11, illustrating synthetic approaches for producing bis-MTS reagents; Scheme 12, for producing thioglycoses; and Scheme 13, s illustrating another synthetic method for generating a first generation glycodendrimer reagent.
  • Figure 18 shows another synthetic scheme for producing a glycodendrimer MTS reagent.
  • Figure 19 shows a synthetic scheme (Scheme 15) for producing a first O generation glycodendrimer MTS reagent, and a second generation hybrid glycodendrimer MTS reagent; and an improved synthesis scheme (Scheme 16) for producing sodium methanethiosulfonate (“NaMTS").
  • Figure 20 shows synthetic scheme 17 for producing ArGal-based glycodendrimer reagent 42; and synthetic scheme 18 for producing ArGal-based s glycodendrimer reagent 44, bearing two deprotected sugars.
  • Figure 21 illustrates synthetic scheme 19 and synthetic scheme 20 for producing glycodendrimer reagents.
  • Figure 22 illustrates synthetic scheme 21 for producing a glycodendriprotein.
  • Figure 23 illustrates a glycosylated variant of Bacillus lentus subtilisin mutant S 156C.
  • a method wherein the glycosylation pattern of a s protein is modified in a predictable and repeatable manner.
  • the modification of the protein occurs via reaction of a cysteine residue in the protein with a glycosylated thiosulfonate.
  • a chemically modified mutant protein differs from a precursor 0 protein by virtue of having a cysteine residue substituted for a residue other than cysteine in said precursor protein, the substituted cysteine residue being subsequently modified by - 11 - reacting said cysteine residue with a glycosylated thiosulfonate.
  • the glycosylated thiosulfonate is an alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • a method of producing a chemically modified mutant protein comprising the steps of: (a) providing a
  • the glycosylated thiosulfonate is an O alkylthiosulfonate, most preferably, a methanethiosulfonate.
  • the substitution in said precursor protein is obtained by using recombinant DNA techniques by modifying a DNA encoding said precursor protein to comprise DNA encoding a cysteine at a desired location within the protein.
  • the amino acid residues to be substituted with cysteine residues according to the present invention may be replaced using site-directed mutagenesis methods s or other methods well known in the art. See, for example, PCT Publication No. WO 95/10615, which is hereby incorporated by reference.
  • the present invention also relates to glycosylated thiosulfonate.
  • the glycosylated thiosulfonate comprises methanethiosulfonate. More preferably, the methanethiosulfonate comprises the chemical structure:
  • R comprises - ⁇ -Glc, -Et- ⁇ -Gal, -Et- ⁇ -Glc, -Et- ⁇ -Glc,-Et- ⁇ -Man, -Et-Lac, - ⁇ -Glc(Ac) 2 , - ⁇ -Glc(Ac) 3 , - ⁇ -Glc(Ac) 4 , -Et-Ct-GIc(Ac) 2 , -Et-Ct-GIc(Ac) 3 , -Et-CC-GIc(Ac) 4 , -Et- ⁇ -Glc(Ac) 2 , -Et- ⁇ -Glc(Ac) 3 , -Et- ⁇ -Glc(Ac) 4 , -Et- ⁇ -Man(Ac) 3 , -Et- ⁇ -Man(Ac) 4 , -Et- ⁇ -Gal( Ac) 3 , -Et- ⁇ -
  • Another aspect of the present invention is a method of modifying the functional characteristics of a protein including providing a protein and reacting the protein with a glycosylated thiosulfonate reagent under conditions effective to produce a glycoprotein with altered functional characteristics as compared to the protein.
  • the functional characteristics of a protein which may be altered by the 0 present invention include, but are not limited to, enzymatic activity, the effect on a human or - 12 - animal body, the ability to act as a vaccine, the tertiary structure (i.e., how the protein folds), whether it is allergenic, its solubility, its signaling effects, its biological activity and its pharmacological activity (Paulson, "Glycoproteins: What are the sugar chains for?" Trends in Biochem. Sciences, 14:272-276 (1989), which is hereby incorporated by reference).
  • glycosylated thiosulfonates as thiol-specific modifying reagents in a method of the present invention allows virtually unlimited alterations of protein residues.
  • this method allows the production of pure glycoproteins with predetermined and unique structures and therefore, unique functional characteristics, with control over both the site and level of glycosylation.
  • the methods of modifying the functional characteristics of a protein allow the preparation of single glycoforms through regio- and glycan-specific protein glycosylation at predetermined sites.
  • the present invention provides for modified protein, wherein the protein comprises a wholly or partially predetermined glycosylation pattern which differs from the glycosylation pattern of the protein in its precursor, natural, or wild type state and a method for producing such a modified protein.
  • glycosylation pattern means the composition of a carbohydrate moiety.
  • the present invention also relates to methods of determining the structure-function relationships of chemically modified mutant proteins.
  • the - 13 - first method includes providing first and second chemically modified mutant proteins of the present invention, wherein the glycosylation pattern of the second chemically modified mutant protein differs from the glycosylation pattern of the first chemically modified mutant protein, evaluating a functional characteristic of the first and second chemically modified s mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the second method involves providing a first and second chemically modified mutant protein of the present invention, wherein at least one different cysteine residue in the second chemically modified mutant protein is modified by reacting O said cysteine residue with a glycosylated thiosulfonate evaluating a functional characteristic of the first and second chemically modified mutant proteins, and correlating the functional characteristic of the first and second chemically modified mutant proteins with the structures of the first and second chemically modified mutant proteins.
  • the chemically modified mutant proteins of the present invention provide s a valuable source of carbohydrate-protein conjugates. Moreover, the methods of the present invention allow the preparation of pure glycoproteins (i.e., not mixtures) with predetermined and unique structures. These glycoproteins can then be used to determine structure-function relationships (e.g., structure-activity relationships ("SARs")) of non-natural variants of the proteins. 0 [0063]
  • the protein of the invention may be any protein for which a modification of the glycosylation pattern thereof may be desirable. For example, proteins which are naturally not glycosylated may be glycosylated via the invention.
  • proteins which exist in a naturally glycosylated form may be modified so that the glycosylation pattern confers improved or desirable properties to the protein.
  • proteins useful in the s present invention are those in which glycosylation plays a role in functional characteristics such as, for example, biological activity, chemical activity, pharmacological activity, or immunological activity.
  • Glycosylated proteins as referred to herein means moieties having carbohydrate components which are present on proteins, peptides, or amino acids.
  • the glycosylation is provided, for example, as a result of reaction of the glycosylated thiosulfonate with the thiol hydrogen of a cysteine residue thereby producing an amino acid residue which has bound thereto the carbohydrate component present on the - 14 - glycosylated thiosulfonate.
  • Glycosylation also may be accomplished, according to the present invention, by attachment of glycodendrimer reagents such as those described in the examples below.
  • Such reagents comprise one or more dendrimer core portions, optionally a linker (or tether), and one or more carbohydrate moieties.
  • the invention provides for synthetic schemes for producing glycodendrimer reagents. Said schemes include normal addition schemes in which a carbohydrate alkylthiosulfonate is reacted with a dendrimer core, said core comprising a free sulfhydryl group. Also included are inverse addition synthesis schemes in which a thioglycose is reacted with a dendrimer core alkylthiosulfonate.
  • a preferred synthesis scheme involves reacting a carbohydrate with an alkylthiosulfonate and a phase transfer catalyst under refluxing toluene conditions, hi a particularly preferred scheme, the alkylthiosulfonate is a sodium salt of methanethiosulfonate, and the phase transfer catalyst is tetrabutylammonium s iodide (Bu 4 NI).
  • the protein is an enzyme.
  • the term "enzyme” includes proteins that are capable of catalyzing chemical changes in other substances, without being changed themselves.
  • the enzymes can be wild-type enzymes or variant enzymes. Enzymes within the scope of the present invention include pullulanases, proteases, cellulases, 0 amylases, isomerases, lipases, oxidases, and reductases.
  • the enzyme is a protease.
  • the enzyme can be a wild-type or mutant protease. Wild-type proteases can be isolated from, for example, Bacillus lentus or Bacillus amyloliquefaciens (also referred to as BPN').
  • Mutant proteases can be made according to the teachings of, for example, PCT Publication Nos. WO 95/10615 and WO 91/06637, which are hereby incorporated by reference.
  • Functional characteristics of enzymes which are suitable for modification according to the present invention include, for example, enzymatic activity, solubility, partitioning, cell-cell signaling, substrate specificity, substrate binding, stability to temperature and reagents, ability to mask an antigenic site, physiological functions, and pharmaceutical functions (Paulson, "Glycoproteins: What are the Sugar Chains For?” Trends in Biochem Sciences, 14:272-276 o (1989), which is hereby incorporated by reference.)
  • the protein is modified so that a non-cysteine residue is substituted with a cysteine residue, preferably by recombinant means.
  • the amino - 15 - acids replaced in the protein by cysteine are selected from the group consisting of asparagine, leucine or serine. Orthogonal protection schemes that are well known in the art may be used when modification is to be carried out at more than one site within a protein.
  • thiol side chain group “thiol ' containing group,” and “thiol side chain” are terms which can be used interchangeably and include groups that are used to replace the thiol hydrogen of a cysteine.
  • the cysteine occurs in the native protein sequence, while in other embodiments, a cysteine replaces one or more amino acids in the protein.
  • the thiol side chain group includes a sulfur through which the thiol side chain groups defined above are attached to the thiol sulfur of the cysteine.
  • glycosylated thiosulfonates of the invention are those which are capable of reacting with a thiol hydrogen of a cysteine to produce a glycosylated amino acid residue.
  • glycosylated is meant that the thiosulfonate has bound thereto a sugar or carbohydrate moiety that can be transferred to a protein or dendrimer (which may be bound to a protein) pursuant to the present invention.
  • the glycosylated thiosulfonates are glycosylated alkylthiosulfonates, most preferably, glycosylated methanethiosulfonates.
  • Such glycosylated methanethiosulfonates have the general formula:
  • the methanethiosulfonate R group comprises: - ⁇ -Glc,
  • the carbohydrate moiety of the present invention is a dendrimer moiety.
  • Multiple functionalization of chemically modified mutant proteins can be achieved by dendrimer approaches, whereby multiple-branched linking structures can be employed to create poly-functionalized chemically modified mutant proteins.
  • Dendrimer moieties of the present invention may contain one or more branch points.
  • first generation refers to dendrimer moieties that contain a single branch point.
  • second generation refers to a dendrimer moiety that contains multiple branch points.
  • the number of branch points in a - 16 - second generation dendrimer will depend on the number of arms that can be attached to the central core of the dendrimer building block. In general, the number of branch points for a dendrimer comprising central cores having the same number of arms is equal to
  • the TRE ⁇ -type dendrimers such as those illustrated in Fig. 1 have three arms attached to the central TRE ⁇ core.
  • ⁇ 3
  • the dendrimer reagent structures would include methanethiosulfonates with simple branching such as:
  • a first generation glycodendrimer reagent is synthesized as shown in Figure 12, Scheme 6. This approach can be extended to cover larger dendrimers. More specifically, by leaving one "arm" of the glycodendrimer free for conversion to a methanethiosulfonate, the remaining arms can be further branched to synthesize highly- functionalized glycodendrimer reagents as shown in Figure 14, Scheme 8. - 17 -
  • a glycodendrimer comprising the steps of providing at least a first dendrimer core building block, at least one alkylthiosulfonate, and at least one carbohydrate having a first sulfhydryl group; reacting the first dendrimer core building block with the alkylthiosulfonate to produce a modified dendrimer core building block having an alkylthiosulfonate group; reacting the resultant modified dendrimer core building block with the carbohydrate to produce a glycodendrimer.
  • the carbohydrate is preferably a disaccharide, and may optionally be a monosaccharide or a polysaccharide.
  • the alkylthiosulfonate can be methanethiosulfonate, ethanethiosulfonate, propanethiosulfonate, or a C 1 -C 6 alkylthiosulfonate.
  • the methanethiosulfonate can be a salt, such as an alkali metal salt, e.g., lithium, sodium, or potassium salt, or an alkali earth metal salt, e.g., magnesium or calcium salt.
  • the first dendrimer core building block can be tris(2-aminoethyl)amine (TREN).
  • TREN tris(2-aminoethyl)amine
  • the first dendrimer core building block can be pentaerythritol, mesitylene or mesitylene tribromide.
  • the carbohydrate can be directly or indirectly linked to the first sulfhydryl group or, alternatively, tethered to the first sulfhydryl group.
  • the disaccharide can optionally be selected from the group consisting of
  • Gal-Gal Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and Glu-Man, and acetyl or benzoyl derivatives thereof.
  • the polysaccharide can optionally be selected from the group consisting of Gal-Lac, Glu-Lac, Man-Lac, and Lac-Lac, and acetyl or benzoyl derivatives thereof.
  • Gal refers to the saccharide unit galactose.
  • GIu refers to the saccharide unit glucose.
  • Man refers to the saccharide unit manose.
  • Lac refers to the disaccharide unit lactose. In the above disaccharides, either the first or the second, or both, saccharide units may be linked to the dendrimer core.
  • Gal “GIu,” “Man,” and “Lac” are meant to encompass saccharide units that are mono-, di-, tri-, tetra-, or otherwise polysubstituted. Such substitution is preferably with acetyl or benzoyl groups.
  • the glycodendrimer produced by the above method can optionally be a first-generation glycodendrimer. Further, the first-generation glycodendrimer can optionally comprise an alkylthiosulfonate group, which can be methanethiosulfonate.
  • the above disclosed methods for producing a glycodendrimer can further comprise a step of reacting the modified dendrimer core building block having an alkylthiosulfonate group with a second dendrimer core building block having a second - 18 - sulfhydryl group to produce a multi-generation glycodendrimer.
  • the multi-generation glycodendrimer is a second-generation glycodendrimer.
  • the method for producing a glycodendrimer can comprise the steps of providing a first dendrimer core building block and a carbohydrate having a first sulfhydryl group; and reacting said dendrimer core building block with said carbohydrate to produce a glycodendrimer; where the carbohydrate is a disaccharide.
  • the disaccharide can be any disaccharide, and is preferably selected from the group consisting of 4-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-2,3,6-tri-O- benzoyl-1-thio- ⁇ -D-galactopyranoside, Gal-Gal, Glu-Glu, Man-Man, Gal-Glu, Gal-Man, and Glu-Man, and acetyl or benzoyl derivatives thereof.
  • the dendrimer core building block can be selected from the group consisting of TREN, TREN-Boc, TREN-B OC-Ac(Cl) 2 , CbZ 2 -TREN, Cbz 2 -TREN-Boc, and Cbz 2 -TREN-CO-(CH 2 ) 4 -SH.
  • the glycodendrimer is selected from the group consisting of TREN-BoC-(SGaI) 2 , TREN-(SGaI) 2 , MTS-(CEb) 4 -CO-TREN-(SGaI) 2 , (2,3, 4,6-tetra-O-acetyl-Gal ⁇ ( 1,4)2,3, 6-tri-0-benzoyl- Gal ⁇ S) 2 TREN-Boc, (Gal ⁇ (l,4)Gal ⁇ S) 2 TREN-Boc, (Gal ⁇ (l,4)Gal ⁇ S) 2 TREN, and (Gal ⁇ (l,4)Gal ⁇ S) 2 TREN-CO(CH 2 ) 4 MTS.
  • R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , and R 7 are each independently and optionally selected from the group consisting of hydrogen, alkyl, alkylacyl (alkyl-C(O)-), arylacyl (aryl-C(O)-) and arylalkylacyl (arylalkyl-C(O)-); and
  • R 8 is selected from the group consisting of hydrogen, alkali metal, alkali earth metal, aluminum, organic cation, and inorganic cation. [0083] In another aspect, disclosed herein is a compound selected from the group consisting of the following structures: -19-
  • the present invention relates to a glycodendrimer reagent composition having the generic structure:
  • a, b, c, and d are each independently selected from the group consisting of all integers from 0 to 10, inclusive, such as, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10, - 21 -
  • X may be either SR or R, wherein R is a disaccharide, substituted disaccharide, polysaccharide, or substituted polysaccharide selected from the group consisting of Gal-Gal, Glu-Glu, Man-Man, Lac-Lac, Gal-Glu, Gal-Man, Gal-Lac, Glu-Man, and Glu-Lac.
  • X may be
  • subtilisins Alkaline serine proteases
  • subtilisins are increasingly used in biocatalysis, particularly in chiral resolution, regioselective acylation of polyfunctional compounds, peptide coupling, and glycopeptide synthesis.
  • subtilisins can be used to catalyze peptide bond formation. From an ester substrate, subtilisins can form an acyl enzyme intermediate which then reacts with a primary amine to - 22 - form a peptide product.
  • subtilisins do not meet these requirements. Nonetheless, improved esterase to amidase selectivities are desired in the art. By using the methods described s herein, it is now possible to produce subtilisins that have advantageous properties.
  • Bacillus lentus subtilisin is selected O for illustrated purposes only, as it does not contain a natural cysteine and is not naturally glycosylated.
  • the substrate binding site of an enzyme consists of a series of subsites across the surface of the enzyme.
  • the portion of substrate that corresponds to the subsites are labeled P and the subsites are labeled S.
  • the subsites are labeled S 1 , S 2 , S 3 , s S 1 ', S 2 ', etc.
  • a discussion of subsites can be found in Berger et al., Phil Trans. Royl Soc. Lond.
  • any of the S 1 , S 1 ', or S 2 subsites is selected as a 0 suitable target for modification.
  • the amino acid residues corresponding to N62, L217, S 156, and S 166 in naturally-occurring subtilisin from Bacillus amyloliquefaciens or to equivalent amino acid residues in other subtilisins, such as Bacillus lentus subtilisin were selected for modification to cysteine.
  • An amino acid residue of an enzyme is equivalent to a residue of a s referenced enzyme (e.g., B. amyloliquefaciens subtilisin) if it is either homologous (corresponds in position in either primary or tertiary structure) or analogous to a specific residue or portion of that residue in B. amyloliquefaciens subtilisin (has the same or similar functional capacity to combine, react, or interact chemically).
  • a s referenced enzyme e.g., B. amyloliquefaciens subtilisin
  • the amino acid sequence of 0 the subject enzyme for example, a serine hydrolase, cysteine protease, aspartyl protease, metalloprotease, etc.
  • a reference enzyme for example, B. amyloliquefaciens subtilisin in the case of a subtilisin type serine protease
  • primary sequence - 23 - and particularly to a set of residues known to be invariant in all enzymes of that family (e.g. subtilisins) for which sequence is known.
  • the residues s equivalent to particular amino acids in the primary sequence of the reference enzyme e.g., B. amyloliquefaciens subtilisin
  • Alignment of conserved residues preferably should conserve 100% of such residues. However, alignment of at least approximately 75% or at least approximately 50% of conserved residues are also adequate to define equivalent residues.
  • Conservation of a catalytic triad, (for example, Asp32/His64/Ser221) is preferably O maintained for serine hydrolases.
  • the conserved residues may be used to define the corresponding amino acid residues in other related enzymes.
  • the two sequences one a "reference” and the other a "target" are aligned in order to produce the maximum homology of conserved residues.
  • a number of deletions are seen in the thermitase sequence as compared to B. amyloliquefaciens subtilisin ⁇ see, e.g. U.S. Patent 5,972,682).
  • amyloliquefaciens subtilisin in thermitase is the particular lysine shown beneath Tyr217 in Figure 5B-2 of the 5,972,682 patent.
  • the particular "corresponding" residues may be substituted by a different amino acid to produce a mutant carbonyl hydrolase because they the correspond in primary structures.
  • Corresponding residues may also be determined as homologous or corresponding at the level of tertiary structure for a particular enzyme.
  • Tertiary structure may s be determined by for example x-ray crystallography or other known techniques.
  • Corresponding residues may be defined as those for which the atomic coordinates of two or more of the main chain atoms of a particular amino acid residue of the reference sequence (e.g., B. amyloliquefaciens subtilisin) and the sequence in question (target sequence) (N on N, CA on CA, C on C, and O on O) are within 0.2, 0.15, or 0.13 nm and preferably 0 approximately 0.1 nm after alignment.
  • the reference sequence e.g., B. amyloliquefaciens subtilisin
  • target sequence target sequence
  • Alignment may be achieved after the model has been oriented and positioned to give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the enzyme in question to the reference sequence.
  • the best model is the - 24 - crystallographic model giving the lowest R factor for experimental diffraction data at the highest resolution available.
  • Corresponding residues may also be determined as functionally analogous s to a specific residue of a reference sequence.
  • corresponding residues are defined as those amino acid residues, which may adopt a conformation such that they will alter, modify or contribute to protein structure, substrate binding or catalysis in a manner defined and attributed to a specific residue of the reference sequence as described herein.
  • corresponding residues are those residues of the sequence (typically for which a O tertiary structure has been obtained by x-ray crystallography) which occupy an analogous position to the main chain atoms of a given residue.
  • the atomic coordinates of at least two of the side chain atoms of the residue lie with 0.13 run of the corresponding side chain atoms of the reference sequence residue(s).
  • the three dimensional structures are to be aligned as described above. For an illustration of such an alignment procedure see U.S. s Patent 5,972,682, which is incorporated by reference herein in its entirety.
  • mutated subtilisins were produced through standard site directed mutagenesis techniques and the obtained mutant subtilisin was reacted with certain glycosylated alkylthiosulfonates, particularly glycosylated methanethiosulfonates, as provided in the examples appended hereto.
  • glycosylated alkylthiosulfonates particularly glycosylated methanethiosulfonates, as provided in the examples appended hereto.
  • Proteins or other compositions obtained using the methods described herein may be used in any application in which it is desired to use such proteins, especially those in which having modified functional capabilities is advantageous. Proteins modified as provided herein may be used in the medical field for pharmaceutical compositions and in diagnostic preparations.
  • proteins such as enzymes, that are modified as s described herein may be used in applications which generally are known for such enzymes including industrial applications such as cleaning products, textile processing, feed modification, food modification, brewing of grain beverages, starch processing, as antimicrobials, health care formulations and in personal care formulations.
  • industrial applications such as cleaning products, textile processing, feed modification, food modification, brewing of grain beverages, starch processing, as antimicrobials, health care formulations and in personal care formulations.
  • the unique functionalities made possible by the methods described herein may result in new and 0 unforeseen uses for proteins which have not heretofore been recognized. - 25 -
  • Example 1 Synthesis and Characterization of Carbohydrate Methanethiosulfonate Reagents
  • Cationic carbohydrate-methanethiosulfonates have been prepared previously, as described in United States Patent Application Serial No. 09/347,029 "Chemically Modified Proteins with a Carbohydrate Moiety," the entire disclosure of which is hereby incorporated by reference in its entirety.
  • the following examples are those that have been prepared as novel compounds for use in glycodendriproteins.
  • the sugar-MTS reagents required for attachment to the tips of the dendrimer were prepared.
  • MTS reagent 3 could also be prepared from 2 using this tetrabutylammonium method, but the yields and rate were no improvement on that described s in Scheme 1. This method was also used to prepare the directly linked glucofuranosyl MTS reagent 8 (Fig. 6, Scheme 3). The product was an inseparable anomeric mixture. No further effort has yet been made to obtain pure 8 ⁇ or 8 ⁇ .
  • Glucofuranose 6 is the first readily available crystalline peracylated glucofuranose. Furneaux, R.H.; Rendle, P.M.; Sims, I.M. J.
  • This method has also allowed the preparation of novel directly-linked s mannose MTS reagents 10a and lO ⁇ (Fig. 7, Scheme 4), whose identity was again confirmed by X-ray crystallography (Fig. 9). In addition it ⁇ allowed the more efficient preparation of ⁇ - gluco MTS reagent 12 ⁇ (Fig. 7, Scheme 5).
  • Acetobromogalactose 1 (1.1 g, 2.68 mmol) and sodium methanethiosulfonate (0.45 g, 3.35 mmol) in toluene (50 mL) were concentrated in vacuo to approximately 30 mL to remove any water as the azeotrope. The mixture was made up to 50 mL with more toluene and again concentrated to 30 mL. A catalytic amount of j tetrabutylammonium iodide was added and the mixture heated at reflux for 75 minutes. After cooling, Celite (to stop the formation of a salt cake on top of the column) was added and the whole mixture loaded directly on to a flash silica column.
  • Lectins, selectins, integrins and members of other adhesion receptor families are sugar binding proteins. The sugar binding sites are relatively shallow and hence binding is comparatively weak. Lectins, selectins, integrins and members of other adhesion
  • the aim is to attach many sugars to the surface of a dendrimeric structure that is in turn attached to a protein to mimic the natural system.
  • the model protein we used is subtilisin Bacillus lentus (SBL), a serine protease enzyme. If the glycodendrimer system synthesized has a strong affinity to the lectin, selectin, integrin or members of another s adhesion receptor family being targeted, then the attached SBL (being a protease) should start 'cutting' up the lectin, selectin, integrin or members of another adhesion receptor family. This is shown schematically in Figure 10. The specific model glycodendrimer that is the initial synthetic target of this project is shown in Figure 11.
  • SBL has no natural cysteines (and hence no thiols present).
  • O Methane thiosulfonate (MTS) reagents react specifically and quantitatively with thiols (Wynn, R.; Richards, F.M. Methods Enzymol. 1995, 201, 351) giving an excellent method for the attachment of the glycodendrimer to the protein.
  • the initial dendrimeric core building block was prepared from tris(2- aminoethyl)amine (TREN) 1 using literature methodology (Tecilla, P.; Tonellato, U.; s Veronese, A. /. Org. Chem. 1997, 62, 7621) and that described in Scheme 7 (Fig. 13). Excess 1 is reacted with BoC 2 O to selectively give the mono-Boc protected TREN. After chromatographic purification, the remaining amines were protected with chloroacetates and the chlorines displaced with thioacetates to give 2. The acetates were deprotected under mild basic conditions to give the dithiol 3.
  • the next step is the attachment of the sugars to the dendrimeric core. Because of the stability problems of the dithiol 3, many attempts were made to generate it in s situ. This type of coupling is one of the most important reactions in building of multi- generation glycodendriproteins (Scheme 8) and hence an elegant, high yielding reaction would be very useful. These attempts to generate the dithiol 3 in situ took two forms, either (a) reducing the disulfide 4 or (b) deprotecting the diacetate 2.
  • This acylating reagent ⁇ i.e., 3- bromopropionoyl chloride
  • TREN 1 Scheme 14, Fig. 8
  • Amine 30 (Scheme 15, Fig. 19) was treated in the same way to give the expected thiol 34 in moderate yield. This is a useful product for the preparation of second-generation glycodendrimers (by reaction with bis-MTS reagents of type 21). 34 was then used in the synthesis of the di-Gal-TREN-MTS 39 via a nitrosylation reaction and s reaction with methanesulfinate (Scheme 15, Fig. 19). 34 was also used to synthesize the tetra-Gal-TREN/araGal hybrid-MTS 41 (Scheme 15, Fig. 19).
  • Second- generation galactodendrimer MTS reagent tetra-Gal-TREN/AraGalhybrid-MTS 41 s [0134] Tris(methanethiosulfonatomethyl)mesitylene 40 (26 mg, 0.05 mmol) and triethylamine (15 ⁇ L, 0.10 mmol) were dissolved in DMF (20 mL) in an ice/salt bath.
  • NaMTS was synthesized in high yield by refluxing sodium sulfinate with sulphur in methanol O (Scheme 16, Fig. 19), described in further detail below). Although formation of small amounts of an unknown by-product was observed, it could be easily separated from NaMTS.
  • NaMTS Sodium methanethiosulfonate
  • a fourth isolated compound (white viscous foam, 0.088 g) is believed to be 48; analytical o data of 45: 1 H NMR (300 MHz, CDCl 3 ) ⁇ 2.29 (s, 3H, CH 3 ), 2.36 (s, 6H, COCH 3 ), 2.39 (s, 6H, CH 3 ), 3.36 (s, 3H, SO 2 CH 3 ), 4.20 (s, 4H, CH 2 SAc), 4.48 (s, 2H, CH 2 S); 13 C NMR (75 MHz, CDCl 3 ) ⁇ 16.3 (CH 3 ), 16.4 (CH 3 ), 29.6 (CH 2 SAc), 30.4 (CH 3 CO), 36.7 (CH 2 SSO 2 ), -42 -
  • glycoconjugates Glycosylated macromolecules, also known as glycoconjugates, are present on the surfaces of all mammalian cells. Interactions of glycoconjugates with carbohydrate- binding structures on other cells, viruses, bacteria or toxins are playing a crucial role in biological processes such as cell-cell recognition, inflammation, invasion by pathogens or the s onset of cancer metastasis. Sugar-binding proteins, also known as lectins, show a highly specific affinity to complex carbohydrate structures. Sharon et al., Essays Biochem. 1995, 30, 59.
  • a new class of structures is produced by ligation by a dendrimeric supported carbohydrates to proteins: glycodendriproteins. The glycodendriproteins may be useful tools in the study of protein-carbohydrate interactions as o well as in targeting lectins specifically with enzymes.
  • glycodendriproteins To synthesize glycodendriproteins, chemical synthesis of the glycodendrimer and site-directed mutagenesis of the enzyme were used, although other - 44 - methods may also be used.
  • MTS reagents may be used. MTS reagents may be reacting in a specific and high yielding reaction with SH-functions introduced by site-directed mutagenesis. Matsumoto et al, Chem. Commun. 2001, 903-904; Davis et al, Bioorg. Med. Chem. 2000, 8, 1527-1535.
  • One of the steps in the assembly of the protein modifying reagents is the attachment of the sugars via nucleophilic substitution.
  • This step may be performed by using thio derivatives of sugars and halides of the branched core.
  • Suitable sugar thiols include anomeric thiols of N-acetylglucosamine, galactose and galabiose (galactose- ⁇ (l,4)-galactose) and a 4-thio derivative of mannose and were synthesized to achieve this end.
  • Acetobromo galactose 53 was converted to acetylated thiogalactose 55 via thiouronium salt 54 using thiourea in refluxing acetone, followed by treatment with sodium metabisulfite in a 62% yield over the two steps.
  • Acetylated sugar 55 was deprotected under Zemplen conditions to yield the unprotected sodium salt 56 in very good yield (Scheme 22).
  • the thiol of galabiose was synthesized from a mixture of acetyl 4-0- (2,3,4,6-tetra-O-acetyl- ⁇ -D-galactopyranosyl)-2,3,6,-O-tri-benzoyl- ⁇ -D-galactopyranoside 61 and the corresponding methyl glycoside methyl 4-O-(2,3,4,6-tetra-O-acetyl- ⁇ -D- galactopyranosyl)-2,3,6,-O-tri-benzoyl- ⁇ -D-galactopyranoside 62 (Scheme 24).
  • a method of synthesizing the disaccharides includes starting from monosaccharides or simple sugars.
  • D-Galactose 66 was converted to the corresponding ⁇ - methyl galactoside 67 using HCl in methanol.
  • Benzoylation with benzoyl chloride in pyridine at O °C and subsequent warming to room temperature yielded the selectively 2,3,6 benzoate protected derivative 68, the glycosyl acceptor (Scheme 25).
  • Scheme 25 the glycosyl acceptor
  • D-galactose 66 was acetylated, using acetic anhydride and pyridine (— > 69).
  • the resulting peracetylated galactose 69 was treated with thiophenol and BF 3 -etherate to give thiophenyl galactoside 70.
  • Benzyl and benzoyl protected disaccharide 73 was debenzylated by catalytic hydrogenation with hydrogen (balloon) and palladium on carbon in methanol to liberate four OH functions on the first sugar 74.
  • An initial reaction time of three days was utilized, but could be reduced by increasing the reaction temperature to more than 20 °C (for example, 25 °C or 30 °C) and/or by switching the solvent from methanol to THF (a four hour reaction), yielding >95%.
  • Mannose 75 was converted to l,6-anhydro-2,3-O-isopropylidene-mannose 77 via 1,6-anhydro mannose 76 following a known procedure (Fraser-Reid, J Org. Chern. 1989, 54, 6125-6127) (Scheme 28).
  • Mesylation at 04 ( ⁇ 78), cleavage of the acetal protective group with acetic acid/water (— » 79) and base catalyzed (NaOMe/MeOH) intramolecular nucleophilic substitution yielded 1,6-3,4-anhydro- ⁇ -D-talose 80.
  • a thiobenzoate group was introduced at the 4-position to yield a 4-thio JO mannose derivative 81 by reaction with 80.
  • 80 was treated with thioacetic acid/ pyridine (— > 82) (Scheme 29) to yield a thioacetate.
  • a trivalent bifunctional core was chosen for the synthesis of the sugar displaying dendrons. This core was prepared following procedures described herein.
  • TREN Tris(2-aminoethyl)amine
  • TREN-Boc 84 was reacted with bromo acetyl bromide to synthesize dibromide 86.
  • the product was characterized by electrospray mass spectrometry.
  • TREN-Boc 84 was reacted with Cbz chloride to give Cbz 2 TREN-Boc 87.
  • Base catalyzed addition of ⁇ -butyrolactone under ring-opening gave thiol 89 and the corresponding disulfide 90 in 43% and 27% yield respectively (Scheme 30).
  • MTS reagent 96 was successfully used to modify SBL- mutant S156C to the bivalent glycodendriprotein [Gal] 2 TREN-SI56C 92.
  • Mass spectrometry showed the expected mass of 27770 Da for glycodendriprotein 102.
  • Peanut agglutinin is a galactose binding lectin.
  • PNA protein agglutinin
  • GaI bivalent galactose modified glycodendriprotein
  • Titration with the glycodendriprotein showed the formation of a complex between the two proteins and a micromolar KD was extracted from this data.
  • N-acetylglucosamine (20 g, 90 mmol) and acetylchloride were stirred vigorously over night.
  • Dichloromethane (50 mL) was added to the resulting amber syrup and - 55 - the solution was poured on ice water (100 mL). Layers were separated and the organic layer was poured on saturated sodium bicarbonate solution/ice (100 mL).
  • the neutralization was complete, the org. layer was dried over magnesium sulfate (10 g), concentrated in vacuo and crystallized from dry ether (140 mL). After 20 h at room temperature, solids were collected, washed with ether and dried under high vacuum (25.3 g, 77%).
  • NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.
  • NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.
  • NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. /. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.
  • Methyl glycoside 62 (2.7 g) was dissolved in 25 niL acetic anhydride and cooled to 0 0 C (ice bath). Acetic acid (15 mL) was added and subsequently 0.4 niL concentrated sulfuric acid were added dropwise.
  • TREN tris(2-aminoethyl)amine
  • TREN-Boc 84 (2.2 g, 8.9 mmol) in anhydrous dichloromethane (30 niL) and anhydrous pyridine (3.5 mL) was cooled to 0 0 C (ice bath) and chloroacetic anhydride (3.7 g, 21.4 mmol) was added in portions. The mixture was allowed to warm up to room temperature (90 min) and concentrated in vacuo. Flash chromatography (EtOAc/MeOH 19:1 ⁇ 4:1 v/v) yielded bis(chloroacetamido)-TREN-Boc 85 as a pale yellow oil (1.47 g, 69%).
  • NMR data is reported in, for example, Garegg, et al. Carbohydrate Research 1985, 137, 270-5; Hodosi, et al. J. Am. Chem. Soc. 1997, 119(9), 2335-2336; Hodosi, et al. Carbohydrate Research 1998, 308(1-2), 63-75.
  • TREN-Boc (1.14 g, 4.6 mmol) was dissolved in 10 mL water/dioxane 1:1.
  • a solution of Cbz-chloride (1.43 mL, 10 mmol) in 5 mL dioxane was added drop wise alternating with drop wise addition of 3.5 M K 2 CO 3 to maintain a pH of 7. After addition was complete the mixture was stirred for 1 h at pH 7-8.
  • Deacylated dendrimer 98 was taken up in 1 mL of a mixture of trifluoroacetic acid and water (1:1) and stirred at room temperature for 45 min until TLC indicated complete consumption of the starting material. Solvents were evaporated and the residue was dissolved in water (3 mL) and loaded on Dowex 50WX8-100 (H + ), washed with 0 water, methanol and water (20 mL each) and eluted with 100 mL aqueous ammonia (15%). The filtrate was concentrated and lyophilized from water to yield 99 as 24 mg of a colorless foam (86%).
  • the mixture was purified over a Pharmacia PD-10 column (elution buffer: 5mM MES, 2mM CaCl 2 ), dialyzed against water, 3 x (1 h, 2 L) at 4 s °C, then flash frozen and stored at -18 °C.
  • a cysteine-containing mutant of subtilisin Bacillus lentus, S156C was modified with the glycoMTS reagent 2-( ⁇ -D-galactopyranosyl)ethyl methanethiosulfonate to give the glycoprotein S156C-SS-ethyl 2-( ⁇ -D-galactopyranose) following procedures outlined in Example 3. This resulted in a CMM enzyme ("gal-protease") illustrated in Fig. 18.
  • the S. sanguis receptor for the actinomyces lectin comprises repeating hexasaccharide units with Galactose, N-acetylgalactosamine (GaINAc) termini.
  • the o agglutination of oral streptococci strains by the actinomyces lectin activity blocks attachment of actinomyces to epithelial cells, and this is thought to inhibit the killing of actinomyces by polymorphonuclear leukocytes.
  • a coaggregation experiment was carried out according to methods similar to those described in Kolenbrander PE, Williams BL., "Lactose-reversible coaggregation between oral actinomycetes and Streptococcussanguis," Infect Immun. 1981 Jul;33(l):95- 102.
  • A. naeslundii was pre-treated with subtilisin Bacillu s lentus protease or S156C-SS-ethyl 2-( ⁇ -D-galactopyranose) ("gal-protease") (enzyme concentration 50 ug/ml) in the presence or absence of lactose (60-300 ug/ml), and the ability of the treated A. naeslundi to co-aggregate w/ S. sanguis was determined by microscopic evaluation. The amount of coaggregation (i.e., lectin activity), from highest to lowest is listed in Table 1, below.
  • Attachment was assayed by labeling the bacteria with a fluorescein tag that is internalized by the bacteria (thereby not disturbing the bacteria's adhesive structures (i.e., the surface fimbrae).
  • the number of bacteria adhering to the buccal cells was analyzed by running the reaction mix through a flow cytometer. The counts shown below in Table 2, are average - 67 - counts per buccal cell, and so roughly correspond to the number of bacteria attaching to each cell.

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Abstract

La présente invention concerne une protéine mutante chimiquement modifiée, incluant un reste cystéine substitué en un résidu autre qu'une protéine précurseur de cystéine n, le reste cystéine étant sensiblement modifié par réaction du reste cystéine avec un thiosulfonate glycosylé. L'invention concerne également un méthane-thiosulfonate glycosylé. Un autre aspect de l'invention est un procédé de modification des caractéristiques fonctionnelles d'une protéine par réaction de ladite protéine avec un méthane-thiosulfonate glycosylé dans des conditions propres à produire une glycoprotéine aux caractéristiques fonctionnelles modifiées par rapport à celle de la protéine. L'invention concerne en outre des procédés permettant de déterminer les relations entre structures et fonctions de protéines mutantes chimiquement modifiées. L'invention concerne enfin des techniques de synthèse pour la production de thio-glucose, les thio-glucoses ainsi obtenus, et des procédés de production de réactifs glycodendrimères.
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