Antimicrobial vaccine compositions

Abstract

To provide antimicrobial vaccine compounds.SOLUTION: Provided is antimicrobial vaccine compounds and compositions comprising oligosaccharide β-(1→6)-glucosamine groups having from 3 to 12 glucosamine units and linked through a linker group to tetanus toxoid.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims priority to U.S. Provisional Application No. 62 / 892,400, filed August 27, 2019, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present invention relates to antibacterial vaccine compounds and compositions comprising oligosaccharide β-(1→6)-glucosamine groups having 3 to 12 glucosamine units attached via a linker group to tetanus toxoid, where the toxoid is predominantly in monomeric form. The present invention also relates to vaccine compositions that confer natural immunity against microorganisms whose cell wall structures contain oligosaccharide N-acetyl-β-(1→6)-glucosamine (PNAG) structures. [Background technology]

[0003] Oligosaccharide antigens bound to toxoid carriers are known to induce weak immune responses, especially in children and the elderly. When oligosaccharides are conjugated to toxoid carriers to form vaccines, it is desirable to conjugate or load as many oligosaccharide groups as possible onto the carrier in order to improve the overall immune response generated. In general, vaccines containing more oligosaccharide antigens loaded onto the carrier will produce higher antibody titers than similar vaccines containing less oligosaccharide antigen.

[0004] Vaccines that use tetanus toxoid with multiple copies of oligosaccharides attached as a carrier are known in the art. Traditionally, the attachment of the oligosaccharide group to the toxoid has been achieved via a linker that binds to a reactive amino group on the toxoid (e.g., -NH2 found on lysine residues). While such chemistry is well established, there are many complexities involved in working with toxoid chemistry.

[0005] First, tetanus toxoid is prepared by treating tetanus toxoid with chemicals such as formaldehyde, which detoxifies the toxin but still retains its antigenicity when administered. Formaldehyde reacts with reactive amino groups on the toxin, thereby reducing the number of remaining reactive amino groups on the toxoid that are available for oligosaccharide coupling. Furthermore, the number of reactive amino groups on the treated toxoid varies depending on the manufacturer. Second, the manufacturing process for tetanus toxoid also introduces low-molecular-weight contaminants into the tetanus toxoid composition. These contaminants include low-molecular-weight reactive amino functionalities that compete with the toxoid for oligosaccharide coupling.

[0006] Previously, the background art has disclosed antibacterial vaccines containing penta-β-(1→6)-glucosamine groups conjugated to tetanus toxoid, with loading factors for the attachment of these penta-β-(1→6)-glucosamine groups being as low as 12 to 20 (Gening, et al., Infect. Immun., 78(2):764-772 (2010)). However, this loading factor is lower than desired, and clearly reflects the fundamental synthetic challenges associated with toxoids and coupling chemistry.

[0007] Therefore, it would be desirable to provide a higher level of loading of tetanus toxoid. Summary of the Invention

[0008] The present invention relates to the discovery that a vaccine compound having a loading level of at least 25, preferably about 31 to 39, oligomeric β-(1→6)-glucosamine linking groups on a tetanus toxoid having at least 25, preferably 31, reactive amino functional groups is possible, provided that the toxoid component in the vaccine compound contains at least 85% of the toxoid in monomeric form. In one embodiment, the toxoid component in the vaccine compound contains at least 90% of the toxoid in monomeric form, or any subvalue or subrange therebetween. In some embodiments, the toxoid contains at least 90% to 99.9% of the toxoid in monomeric form, preferably at least 95% to 99.9% of the toxoid in monomeric form, or any subvalue or subrange therebetween. In one embodiment, the amount of low molecular weight reactive amino compounds does not exceed 3% by weight based on the weight of the toxoid present. In another embodiment, the amount of low molecular weight amino compound in the composition is less than 2% by weight based on the weight of the toxoid present, preferably less than 1% by weight, more preferably less than 0.5% by weight based on the weight of the toxoid present, hi another preferred embodiment, the amount of monomer is greater than 99 area %, for example, based on HPLC.

[0009] Thus, in one embodiment, the present invention provides a vaccine composition comprising at least 25, preferably about 31 to about 39, oligomeric-β-(1→6)-glucosamine group units linked via a linker to a tetanus toxoid carrier, wherein the oligomers contain 3 to 12 repeating β-(1→6)-glucosamine units, with the proviso that less than about 40% of the total number of such units are N-acetylated, and further, the tetanus toxoid contains at least 25, preferably at least 31, reactive amino functional groups, and at least 85% of the toxoid component is in monomeric form, or in some embodiments, at least 90% of the toxoid component is in monomeric form. Such a vaccine composition confers effective immunity to a patient against infection with a microorganism whose cell wall contains oligomeric N-acetyl-β-(1→6)-glucosamine structures.

[0010] In one embodiment, the present invention provides a compound of formula I: (AB) x -CI wherein A is a compound of the formula: [ka] containing 3 to 12 repeating β-(1→6)-glucosamine units or mixtures thereof; B is a compound of the formula: [ka] (where the left side of the formula binds to C and the right side binds to A); C is a tetanus toxoid having at least 31 reactive amino functional groups; x is an integer from about 31 to about 39; y is an integer from 1 to 10; and R is hydrogen or acetyl, provided that no more than 40% of the R groups are acetyl; wherein the tetanus toxoid contains at least 31 reactive amino groups and at least 90% by number of the toxoid is in monomeric form. The present invention provides a compound represented by the formula:

[0011] In one embodiment, the present invention provides a vaccine composition useful against microorganisms that contain oligomeric N-acetyl-β-(1→6)-glucosamine structures in their cell walls, comprising a pharmaceutically acceptable carrier and a compound of formula I: (AB) x -CI wherein A is a compound of the formula: [ka] or mixtures thereof; B is a compound of the formula: [ka] (wherein the left side of the formula binds to C and the right side binds to A), wherein C is a tetanus toxoid having at least 31 reactive amino functional groups; x is an integer from about 31 to about 39; y is an integer from 1 to 10; and R is hydrogen or acetyl, provided that no more than 40% of the R groups are acetyl; wherein the tetanus toxoid contains at least 31 reactive amino groups and at least 90% by number of the toxoid is in monomeric form. The present invention provides a vaccine composition comprising an effective amount of a vaccine represented by the formula: wherein the vaccine composition confers effective immunity to a patient against infection with a microorganism containing oligomeric N-acetyl-β-(1→6)-glucosamine structures in its cell wall.

[0012] In one embodiment of Formula I above, Formula II: (A'-B) x -C II wherein A' is a compound of the formula: [ka] a penta-β-(1→6)-glucosamine (carbohydrate ligand) group of B, C, and x are as defined above, with the proviso that at least 85% by number of the toxoids are in monomeric form, or in some embodiments, at least 90% are in monomeric form. The compound of formula (I) is provided.

[0013] In one embodiment, the present invention provides a vaccine composition against a microorganism that contains oligomeric N-acetyl-β-(1→6)-glucosamine structures in its cell wall, comprising a pharmaceutically acceptable carrier and a compound of formula II: (A'-B) x -C II wherein A' is a compound of the formula [ka] and B, C, and x are as defined above, with the proviso that at least 85% by number of the toxoid components are in monomeric form, or in some embodiments, at least 90% are in monomeric form. and an effective amount of the vaccine represented by formula (I) above.

[0014] In one embodiment, the present invention provides a method for providing a patient with effective immunity from microorganisms that contain oligomeric N-acetyl-β-(1→6)-glucosamine groups in their cell walls, comprising administering a compound of Formula I or II above.

[0015] In one embodiment, the present invention provides a method for providing a patient with effective immunity from microorganisms that contain oligomeric N-acetyl-β-(1→6)-glucosamine groups in their cell walls, comprising administering to the patient a pharmaceutical composition of the present invention as described above.

[0016] In one embodiment, compounds of the present invention include those in which x is 33 to 39. In another embodiment, compounds of the present invention include those in which x is 35 to 38.

[0017] Representative compounds of the present invention are shown in the table below: [ka] [Table 1]

[0018] In one embodiment, the compositions of the present invention contain no more than about 3% by weight of low molecular weight amino groups, based on the total weight of the compounds of Formula I or II.

[0019] In one embodiment, the present invention provides a method of immunizing a patient against a microorganism that contains an oligosaccharide β-(1→6)-glucosamine group in its cell wall, comprising administering to a subject an antibody of Formula I: (AB) x -CI wherein A, B, C and x are as defined above or elsewhere herein.

[0020] In one embodiment, the present invention provides a method of immunizing a patient against microorganisms containing N-acetyl oligosaccharide β-(1→6)-glucosamine groups in their cell walls, comprising administering to the patient an effective amount of a compound of Formula I, or a mixture thereof, as defined above or elsewhere herein, wherein y is 2, 3, or 4.

[0021] In one embodiment, the present invention provides a method of immunizing a patient against a microorganism that contains N-acetyl oligosaccharide β-(1→6)-glucosamine groups in its cell wall, the microorganism comprising a polypeptide of formula II: (A'-B) x -C II wherein A' is a compound of the formula: [ka] a penta-β-(1→6)-glucosamine (carbohydrate ligand) group of B, C and x are as defined above or elsewhere herein. The method comprises administering to said patient an effective amount of a compound of the formula:

[0022] In one embodiment, the present invention provides a method for providing effective immunity to a subject against a microorganism that contains N-acetyl oligosaccharide β-(1→6)-glucosamine groups in its cell wall, comprising administering to a subject a pharmaceutically acceptable diluent and a compound of formula I: (AB) x -CI wherein A, B, C and x are as defined above or elsewhere herein.

[0023] In one embodiment, the present invention provides a method for providing a subject with effective immunity against microorganisms containing N-acetyl oligosaccharide β-(1→6)-glucosamine groups in their cell walls, comprising administering to the patient a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of a compound of Formula I, as defined above or elsewhere herein, where y is 2, 3, or 4.

[0024] In one embodiment, the present invention provides a method for providing effective immunity to a subject against a microorganism that contains N-acetyl oligosaccharide β-(1→6)-glucosamine groups in its cell wall, comprising administering to a subject a pharmaceutically acceptable diluent and a compound of formula II: (A'-B) x -C II wherein A' is a compound of the formula: [ka] a penta-β-(1→6)-glucosamine (carbohydrate ligand) group of wherein B, C and x are as defined above or elsewhere herein].

[0025] In one embodiment, the present invention provides a method for conferring effective immunity in a subject against microorganisms containing β-(1→6)-glucosamine groups in N-acetyl oligosaccharides in their cell walls, comprising administering to the patient an effective amount of a compound described above in a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of the compound, wherein the patient has a white blood cell count of at least 2,000.

[0026] In some embodiments, in one or more of the above methods, the pharmaceutical composition can include, for example, about 3% by weight or less of a low molecular weight amino compound, or in alternative embodiments, less than 1% by weight and any subvalue or subrange between 3% by weight and 0% by weight of a low molecular weight amino compound. [Brief explanation of the drawings]

[0027] [Figure 1] FIG. 1 shows the 1H NMR of compound 17 (described below).

[0028] [Figure 2] FIG. 2 shows the 13C NMR of compound 17.

[0029] [Figure 3] FIG. 3 shows an HPLC spectrum separating tetanus toxoid monomer from oligomers and low molecular weight amino compounds.

[0030] [Figure 4] FIG. 4 provides an HPLC trace of the conversion of the disulfide, compound 16, to two equivalents of the monosulfide, compound 17.

[0031] Detailed Description of the Invention The present invention provides antibacterial vaccine compounds and compositions comprising at least 25, preferably 31-39, oligosaccharide β-(1→6)-glucosamine groups, each having 3-12 glucosamine units, each attached via a linker to a tetanus toxoid protein, wherein no more than 40% of the individual glucosamine units contain an N-acetyl group, and wherein the tetanus toxoid contains at least 25, preferably at least 31, reactive amino groups, and wherein at least 85%, 90%, 95%, 99%, or any subvalue or subrange within the range of 85%-99%, by number, of the toxoid moieties is in monomeric form.

[0032] The vaccine compositions described herein provide effective immunity to patients against microbial infections in which the microorganisms contain oligomeric N-acetyl-β-(1→6)-glucosamine structures in their cell walls.

[0033] Before describing the present invention in more detail, we will first define the following terms: If a term used herein is not defined, it has its generally accepted scientific or medical meaning.

[0034] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0035] "May" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0036] The term "about" when used before a numerical designation, e.g., temperature, time, amount, concentration, etc., indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween, inclusive of ranges. Preferably, the term "about" when used in reference to dosage means that the dose may vary by + / - 10%.

[0037] "Comprising" or "comprises" is intended to mean that compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of," when used to define compositions and methods, shall mean excluding other elements that are essential to the combination for the stated purpose. Thus, a composition consisting essentially of the elements defined herein will not exclude other materials or steps that do not materially affect the basic and novel characteristic(s) of the claimed invention. "Consisting of" shall mean excluding more than trace elements and substantial method steps of other components. Embodiments defined by each of these transition terms are within the scope of the present invention.

[0038] The term "β-(1→6)-glucosamine unit" or "glucosamine unit" means [ka] [wherein the 6-hydroxyl group is condensed with the 1-hydroxyl group of the preceding glucosamine unit, and the dashed lines indicate the bond sites to the preceding and succeeding glucosamine units]. When combined with another "β-(1→6)-glucosamine," the resulting disaccharide has the following structure: [ka] It has.

[0039] The term "β-(1→6)-glucosamine unit" having an N-acetyl group refers to a unit having the structure: [ka] where the 6-hydroxyl group of the second unit condenses with the 1-hydroxyl group of the previous glucosamine unit.

[0040] The term "oligosaccharides containing a β-(1→6)-glucosamine group" refers to that group on a compound that mimics a portion of the cell wall of a pathogenic bacterium, defined as an "oligosaccharide β-(1→6)-glucosamine structure" (defined below). Again, such groups are limited to 3 to 12 β-(1→6)-glucosamine units, where up to 40% of the units can have an N-acetyl group. In one embodiment, fewer than 30% of the β-(1→6)-glucosamine units are N-acetylated. In another embodiment, fewer than 20% of the β-(1→6)-glucosamine units are N-acetylated. In yet another embodiment, fewer than 10% of the β-(1→6)-glucosamine units are N-acetylated. In yet another embodiment, none of the β-(1→6)-glucosamine units are N-acetylated.

[0041] The terms "oligosaccharides containing N-acetyl β-(1→6)-glucosamine structures" or "polysaccharides containing N-acetyl β-(1→6)-glucosamine structures" refer to those structures found in the cell walls of microorganisms. Microbial walls contain many of these structures, which are conserved across many microbial lineages. These structures are primarily N-acetyl β-(1→6)-glucosamine, but also contain regions of deacetylated sugars resulting from the action of enzymes such as poly-β-1,6-D-glucosamine-N-deacetylase. Thus, the vaccines of the present invention generate antibodies, including those that target such deacetylated oligosaccharide regions. Without being limited to any theory, antibodies against such deacetylated sugars are cytotoxic in vivo against such microorganisms.

[0042] As used herein, the term "vaccine composition" refers to a composition comprising a compound of Formula I or II above, including an adjuvant and a pharmaceutical carrier. These compositions may also contain limited amounts of low-molecular-weight amino compounds, including those in which the amount of such amino compounds is 3% by weight or less, preferably 2% by weight or less, and more preferably 1% by weight or less, based on the weight of the toxoid present. These compositions confer effective immunity against microorganisms containing oligosaccharides / polysaccharides with N-acetyl-β-(1→6)-glucosamine structures in their cell walls. Therefore, unlike traditional vaccines that vaccinate against a single bacterium, the vaccine compositions described herein can confer effective immunity against any microorganism containing the oligosaccharide structures described herein. Such microorganisms include, but are not limited to, Gram-positive bacteria, Gram-negative bacteria, antibiotic-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus), fungi, and the like.

[0043] As used herein, the term "effective immunity" refers to the ability of a defined amount of a vaccine composition to generate an antibody response in vivo sufficient to treat, prevent, or ameliorate a microbial infection in which the microorganism contains oligosaccharides / polysaccharides containing N-acetyl-β-(1→6)-glucosamine in its cell wall.

[0044] The term "vaccine compound" refers to compounds of formulas I and II. These compounds may exist as solvates, particularly hydrates. Hydrates may form during the preparation of the compounds or compositions containing the compounds, or hydrates may form over time due to the hygroscopic nature of the compounds. The compounds of the present invention may also exist as organic solvates, including, inter alia, DMF, ether, and alcohol solvates. The identification and preparation of specific solvates is within the skill of a person skilled in the art of synthetic organic chemistry or medicinal chemistry.

[0045] The term "toxoid" refers to both monomeric and oligomeric forms of tetanus toxoid. The presence of oligomeric tetanus toxoid components reduces the average number of exposed reactive amino groups because the surface area of ​​each monomeric toxoid in the oligomer is reduced by oligomerization, resulting in a lower factor of oligosaccharide binding to the toxoid.

[0046] By "subject" is meant a mammal. The mammal can be a human or a non-human mammal, but is preferably a human.

[0047] "Treating" or "treatment" of a disease or disorder in a subject means 1) preventing the disease or disorder from occurring in a subject who is susceptible to or who does not yet exhibit symptoms of the disease or disorder; 2) inhibiting or arresting the development of the disease or disorder; or 3) ameliorating or causing regression of the disease or disorder.

[0048] By "effective amount" is meant an amount of a vaccine composition of the present invention sufficient to treat a disease or disorder afflicting a subject or to prevent said subject or patient from developing such a disease or disorder.

[0049] By "reactive amino functionality" is meant the primary amino groups (-NH2) found in the side chains of lysines and guanidines of tetanus toxoid, and does not include the amide (-NHC(O)-) groups found in peptide bonds or amide side chains of tetanus toxoid, such as glutamine.

[0050] "Low molecular weight amino compounds" refers to amino-containing compounds present as contaminants in tetanus toxoid compositions, including toxoid fragments, amino-containing buffers, reaction quenchers such as lysine and ammonium sulfate, toxin detoxifiers such as formalin, and other amino-containing reagents that come into contact with tetanus toxoid. Typically, such low molecular weight reactive amino compounds have a molecular weight of less than about 10,000, preferably less than 1,000. In one embodiment, such low molecular weight amino compounds are identified by the elution peaks in Figure 3.

[0051] General synthesis method The compounds of the present invention can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (i.e., reaction temperatures, times, molar ratios of reactants, solvents, pressures, etc.) are given, it will be understood that other process conditions can also be used unless otherwise specified. Optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.

[0052] Furthermore, as will be apparent to those skilled in the art, conventional protecting groups may be necessary to prevent certain functional groups from undergoing undesired reactions. Suitable protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting particular functional groups, are well known in the art. For example, numerous protecting groups are described in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and references cited therein.

[0053] The starting materials for the following reactions are generally known compounds or can be prepared by known procedures or obvious modifications thereof. For example, many of the starting materials are available from commercial suppliers such as Sigma-Aldrich (St. Louis, Missouri, USA), Bachem (Torrance, California, USA), and Emka-Chemce (St. Louis, Missouri, USA). Others are available from Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-15 (John Wiley and Sons, 1991), Rodd's Chemistry of Carbon Compounds, Volumes 1-5, and Supplementals (Elsevier Science Publishers, 1989), Organic Reactions, Volumes 1-40 (John Wiley and Sons, 1991), and March's Advanced Organic Chemistry, (John Wiley and Sons, 5 th These compounds can be prepared by procedures described in standard textbooks such as The Organic Chemistry of America (VCH Publishers Inc., 1989), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof.

[0054] Synthesis of Representative Vaccine Compounds of the Invention The general synthesis of the vaccine compounds of the present invention is known in the art and is disclosed in U.S. Patent Application Serial No. 10 / 713,790, and U.S. Patent Nos. 7,786,255 and 8,492,364, each of which is incorporated herein by reference in its entirety.

[0055] In one embodiment of the vaccine compounds described herein, the β-(1→6)-glucosamine groups in Formulae I-III are limited to 4-6 units, preferably 5 units, eg, y=2-4.

[0056] In some embodiments, the compounds are homogeneous in that y is a single integer selected from 1 to 10, inclusive. Thus, the compounds disclosed herein can be designed to be homogeneous with y=1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the compounds of Formula I can be heterogeneously designed with two or more values ​​of y, such as mixtures of y=1 and 2, or y=2 and y=3, or y=3 and y=4, or y=4 and y=5, or y=5 and y=6, or y=6 and y=7, or y=7 and y=8, or y=8 and y=9, or y=9 and y=10. Such pairings of y need not be consecutive. Thus, the compounds may include mixtures of any combination of two or more different values ​​of y, such as y=1 and y=3, or y=1 and y=4, or y=2 and y=4, or y=2 and y=5. In some embodiments, the compounds may be heterogeneous with values ​​of y of 3 or more, or values ​​of y of 4 or more, or values ​​of y of 5 or more, up to a maximum of all different values ​​of y being 10. In some embodiments, each occurrence of y in a compound of Formula I is independent.

[0057] In some embodiments, two or more compounds of Formula I can be used in pharmaceutical compositions in which each individual compound of Formula I is homogeneous in y, and the other compound(s) of Formula I have different y values. In such embodiments, the homogeneous compounds used are simply mixed together at a defined weight percentage. For example, a pharmaceutical composition can include a compound of Formula I (where y=1) in a mixture with a compound of Formula I (where y=2). When a pharmaceutical composition or method includes a heterogeneous mixture of compounds of Formula I, the mixture can be defined in terms of the relative weight percentage of each compound of Formula I. For example, a mixture can include 50% by weight of a compound of Formula I (where y=1) and 50% by weight of a compound of Formula I (where y=2). Any combination of compounds totaling 100% is contemplated, for example, 1, 2, 3, 4, 5, or more compounds with different y values ​​can each be mixed at a known relative weight percentage of the total 100%. Thus, any combination of weight percentages of compounds of Formula I can be used in the pharmaceutical compositions and methods disclosed herein. Thus, in the case of a combination of two compounds of Formula I, the percentage can be expressed as the ratio of the two compounds and can range anywhere from 0.1:99.9 to 99.9:0.1, inclusive, and any value therebetween, for example, 1:99, 5:95, 10:90, 15:85, 20:80, etc., up to 99:1 (including fractional values). Similarly, when three, four, five, or more compounds of Formula I are used in a pharmaceutical composition, the relative weight percentage of each compound can vary from 0.1% up to 99% by weight, provided that the total amounts of the different compounds of Formula I add up to 100%.

[0058] Formation of the linker group is accomplished by art-recognized synthetic techniques, exemplified but not limited to those found in U.S. Patent 8,492,364 and the Examples below. In one embodiment, a compound of formula III: [ka] As shown in the formula: [wherein y is an integer from 1 to 10, and optionally up to 40% of the amino groups are N-acetyl groups], the first portion of the aglycone is attached to a reducing β-(1→6)-glucosamine unit and bears a thiol (—SH) group.

[0059] The second portion of the linker has formula IV: [ka] The antibody binds to tetanus toxoid in the following manner as shown in Figure 1.

[0060] In this formula, individual moieties of tetanus toxoid are represented by serpentine lines, which are merely illustrative in nature and are not intended to provide the complete structure of the toxoid. Any disulfide bridges are shown with single lines connecting the moieties. For clarity, only one second moiety of the linker is illustrated, although there are multiple such second moieties covalently bonded to amino groups found on the toxoid.

[0061] When the first and second portions of the linker are combined under coupling conditions, a thioether bond is formed. The reaction is carried out in an inert diluent, optionally in the presence of a base, to remove the acid generated. The thioether bond connects the first and second portions of the linker, thereby providing a covalent bond to the β-(1→6)-glucosamine group of the tetanus toxoid oligosaccharide via the combined linker, where y is as defined herein, in a vaccine compound, as exemplified below. [ka] wherein up to 40% of the amino groups may optionally be N-acetyl groups.

[0062] It is understood that the number of β-(1→6)-glucosamine linker groups attached to tetanus toxoid is stoichiometrically controlled so that from about 31 to about 39 such groups are attached to the toxoid to provide a vaccine compound of the invention.

[0063] Methods, Uses and Pharmaceutical Compositions The vaccine compositions of the present invention can elicit an effective immune response against microorganisms that have the β-(1→6)-glucosamine structure of the PNAG oligosaccharide in their cell walls. After inoculation into a patient, an effective immune response is generated approximately four weeks later. After an effective immune response is generated, the patient is provided with protection against subsequent microbial infections in which the offending microorganisms have cell walls that contain PNAG.

[0064] When used in this manner, the vaccine composition of the present invention is administered to patients at risk for microbial infections resulting from such microorganisms. Such patients include, by way of example only, the elderly, patients scheduled for elective surgery, and patients traveling to destinations where microbial infections are prevalent. The vaccine is typically administered intramuscularly to immunocompetent patients along with an appropriate adjuvant to enhance the immune response. After a latent period, the patient will have acquired natural immunity to such microorganisms. Such immunocompetent patients have an effective immune system capable of mounting an immune response to the antigen. Preferably, such patients have an activated white blood cell (WBC) count of at least about 1,000 WBC / microliter, preferably at least about 1,500 WBC / microliter, more preferably at least about 2,000 WBC / microliter, even more preferably about 3,000 WBC / microliter, and most preferably about 4,000 WBC / microliter.

[0065] In another embodiment, the vaccine composition of the present invention can be used therapeutically, especially when the microbial infection is localized and / or non-life-threatening. In such cases, the vaccine composition of the present invention is administered to patients suffering from a microbial infection caused by such a microorganism. The vaccine is typically administered intramuscularly to immunocompetent patients with an appropriate adjuvant to enhance the immune response. Administration results in effective immunity within about four weeks. If the patient is still suffering from the infection, the natural immunity generated by the vaccine promotes recovery.

[0066] When so used, the vaccine compositions of the present invention will be administered in therapeutically effective amounts by any of the accepted modes of administration for drugs that serve similar functions. The actual amount of the vaccine compound, i.e., active ingredient, of the present invention will depend on many factors, such as the severity of the disease being treated, the age and relative health of the subject, the potency of the vaccine compound used, the route and form of administration, and other factors well known to those skilled in the art.

[0067] An effective or therapeutically effective amount of a vaccine compound of the invention refers to that amount of vaccine compound that produces a sufficient antibody titer to ameliorate symptoms or prolong survival in a subject. The toxicity and therapeutic efficacy of such vaccine compounds and vaccine compositions can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0068] The vaccine compositions described herein are typically administered as injectable sterile aqueous compositions containing one or more conventional ingredients well known in the art, including, by way of example only, adjuvants, stabilizers, preservatives, etc.

[0069] combination The vaccine compounds and compositions of the present invention can be used in conjunction with other therapeutic compounds or other suitable agents as deemed appropriate by the attending clinician. Where selected, the vaccine compounds of the present invention can be administered simultaneously with antibiotics to treat bacterial infections and agents to enhance the immune response induced by the vaccine compounds and / or compositions. In the case of antibiotics, the selection of the appropriate antibiotic or antibiotic cocktail and the amount to be administered to a patient is within the skill of the attending clinician, based on the type of causative organism, the severity of the bacterial infection, and the patient's age, weight, and other relative health status. If appropriate, immune-boosting agents or adjuvants may also be co-administered by the attending clinician in combination with the vaccines described herein.

[0070] The vaccine compositions of the present invention may be administered with an adjuvant to enhance the patient's immune response to the antigen. Adjuvants include, but are not limited to, aluminum compounds such as gel, aluminum hydroxide, and aluminum phosphate, and Freund's complete or incomplete adjuvant (e.g., an antigen incorporated into the aqueous phase of a stabilized water-in-paraffin oil emulsion). Obviously, paraffin oil can be replaced with other types of oil, such as squalene or peanut oil. Other materials with adjuvant properties include BCG (attenuated Mycobacterium tuberculosis) calcium phosphate, levamisole, isoprinosine, polyanions (e.g., polyA:U), lentinan, pertussis toxin, lipid A, saponin, QS-21, and peptides, such as muramyl dipeptide, and immunostimulatory oligonucleotides, such as CpG oligonucleotides. Rare earth salts, such as lanthanum and cerium, can also be used as adjuvants. The amount of adjuvant used will depend on the subject being treated and the particular antigen being used, and can be readily determined by one skilled in the art. [Example]

[0071] The present invention will be further understood by reference to the following examples, which are intended to be purely exemplary of the invention. The present invention is not limited in scope by the exemplified embodiments, which are intended only as illustrations of single aspects of the invention. Any methods that are functionally equivalent are within the scope of the invention. Various modifications of the present invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are encompassed within the scope of the appended claims.

[0072] As used herein, the following terms are used and have the following meanings: If not defined, an abbreviation has its commonly recognized definition. [Table 2]

[0073] Example 1 - Preparation of Tetanus Toxoid A crude tetanus toxoid sample containing monomeric toxoid containing at least 25, and preferably at least 31, free amino groups was concentrated and chromatographed on a Superdex® 200 size-exclusion column (commercially available from Sigma-Aldrich, St. Louis, Missouri, USA) using two different loads—0.6% and 1.2% column volume. The elution profile was monitored by absorbance at A280. As shown in Figure 3, six distinct peaks (pools 1–5 and the monomeric pool) were observed, with the monomeric fraction exhibiting the largest peak area. Pools were generated based on analytical SEC HPLC analysis of individual fractions. Crude tetanus toxoid and each individual pool were analyzed by SEC HPLC, and the results are summarized in Table 1 and Figure 1. [Table 3]

[0074] The monomer pool showed a single symmetrical peak with an elution volume consistent with monomeric TT (99.9 area%), with no additional peaks detected. The column load contained 58.8 area% monomer, confirming the effectiveness of the preparative Superdex purification protocol under these conditions. The remaining fractions from the Superdex 200 column, as monitored by SEC HPLC, contained primarily high molecular weight material (pools 1 and 2) or low molecular weight species (pools 3–5) compared to the TT monomer. Mass balance throughout the run was assessed by protein recovery (BCA), and the results are summarized in Table 2. [Table 4] It is understood that other size exclusion chromatography methods can be used to achieve the same results.

[0075] Protein recovery from the spin concentration step was 83%, with losses primarily due to removal of low molecular weight protein / peptide contaminants via the filtrate (data not shown). After purification by preparative Superdex 200 chromatography, the yield of TT monomer was 51%, with the remaining protein recovered in the high molecular weight / aggregate and low molecular weight fractions. Finally, TT monomer was recovered in 87% yield after buffer exchange into reaction buffer. In this example, the overall run recovery from crude tetanus toxoid to purified / formulated TT monomer was 35% based on protein recovery.

[0076] The stability of purified TT monomer was evaluated after storage at pH 9.0 (4°C or -70°C) or pH 7.5 (-70°C) for up to 4 weeks. Specifically, monomer content (SEC HPLC) and protein concentration were monitored at weekly intervals. TT monomer showed no significant changes in SEC fingerprint or protein concentration over 4 weeks at 4°C (pH 9.0) or frozen at -70°C (pH 7.5 or 9.0). In this study, due to limited methods for demonstrating stability, we decided to purify TT monomer prior to each production campaign and store purified TT in reaction buffer (50 mM HEPES, pH 8.0) at 4°C, using it within 7 days of production.

[0077] Example 2 - Binding of SBAP to TT monomers Step 1: Preparation of N-BABA: [ka] Commercially available β-alanine, compound 1, is converted to N-BABA (bromoacetyl-β-alanine), compound 2, by reaction with at least a stoichiometric amount of commercially available bromoacetyl bromide. In a first vessel, β-alanine is combined with sodium bicarbonate or other suitable base in water to remove the acid generated during the reaction. The aqueous solution is stirred at about 20±5°C until a solution is obtained. The solution is then maintained at about 5±5°C. In a separate vessel, the required amount of bromoacetyl bromide is added, followed by dichloromethane. The contents of both vessels are combined. After the reaction is complete, 6N HCl is added and mixed until the pH is about 2. The resulting N-BABA is extracted from the solution with a suitable solvent, such as ethyl acetate. The organic phase is concentrated under conventional conditions, e.g., under vacuum at an elevated temperature, such as 60°C. Heptane is then added to precipitate the N-BABA, which is then collected on a filter and dried in a vacuum oven at 40°C. This product is used directly in the next step.

[0078] Step 2: Preparation of SBAP: [ka] N-BABA, compound 2, is reacted with N-hydroxysuccinimide (NHS) under conventional conditions well known in the art to produce SBAP, compound 3. Specifically, N-BABA is combined with at least a stoichiometric amount of NHS in a suitable inert solvent, such as methanol, ethanol, or isopropanol. The resulting solution is stirred at approximately 20±5°C until a clear solution is obtained. N-diisopropylcarbodiimide is then added to the reaction mixture and mixed with the solid product. The system is then cooled to 0±5°C, and the resulting SBAP is obtained by filtration. Further purification requires pre-cooling a mixture of isopropanol and heptane, washing the filter cake, and then drying the wet cake in a vacuum oven at approximately 30°C. The resulting SBAP is then used directly in the coupling reaction with the TT monomer.

[0079] Alternatively, SBAP can be prepared by the method described in U.S. Patent 5,286,846, which is incorporated herein by reference in its entirety. Specifically, the method described therein is provided by the following synthetic scheme: [ka]

[0080] Step 3 - Conjugation Purified TT monomer contains 43 lysine residues / mol, as quantified by the free amine assay, as described above. Reaction with increasing concentrations of SBAP, from 0 to 170 molar equivalents of TT monomer, results in a corresponding decrease in free amine content over the range of 15 to 110 molar equivalents of SBAP. Steady-state conversion was achieved at SBAP charge >110 equivalents. Assuming that the loss of free amines is directly proportional to the SBAP linker loading, the linker density at saturation was estimated to be 43 moles of SBAP / TT monomer. We also assessed the monomer / aggregate content and protein concentration of the linker-TT / monomer intermediate at each titration point. The monomer content before linker addition was 99.7%, and there was no significant change in monomer levels (no aggregates detected) with increasing amounts of SBAP linker added. Protein recovery was also similar across the titration steps. Based on this collective data, a value of 110 molar equivalents of SBAP for 1 h at ambient temperature was selected as the appropriate reaction conditions for all subsequent syntheses.

[0081] Example 3 - Synthesis of Oligosaccharides Composition of Components The following reaction schemes illustrate the synthetic steps used to prepare compounds 3, 5 and 8, detailed below. [ka]

[0082] Synthesis of Compound D Compound C (120.6 g, 252.6 mmol), commercially available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside, and toluene (200 mL) were added to a 1 L Buchi flask and swirled at 40 °C until dissolved (<5 min). The solvent was evaporated to provide a foam. Toluene (200 mL) was added to the flask and swirled at 40 °C until dissolved (<5 min). The solvent was evaporated again to dryness. A crystalline solid formed and adhered to the walls. Dichloromethane (800 mL) was added to the flask and swirled at ambient temperature until dissolved; the resulting dark brown solution was added to a 5 L jacketed reactor, and the flask was rinsed with additional dichloromethane (200 mL). The heating / cooling jacket was set to 20 °C, and the contents of the reactor were mechanically stirred. Ethanethiol (40 mL, 540 mmol) was dissolved in 50 mL of dichloromethane and added to the vessel, and the flask was rinsed with 50 mL of dichloromethane. Boron trifluoride diethyl etherate (50 mL, 390.1 mmol) was dissolved in 50 mL of dichloromethane and added to the reactor, rinsing with 50 mL of dichloromethane and adding to the vessel. The mixture was stirred at 20 °C for 2 h. The reaction was monitored by TLC for residual C. The mobile phase was toluene:ethyl acetate (3:1, v / v), product Rf ~0.45, C Rf ~0.3, UV visualization. If a significant amount of C was present, the reaction time needed to be extended.

[0083] The stirring was set to high speed and 4 M aq. sodium acetate (1.25 L, 5100 mmol) was added. This phase was mixed thoroughly for 30 minutes. The pH of the aqueous phase was checked with a dipstick and found to be approximately pH = 7. The stirring was stopped and the reaction mixture was allowed to settle for 70 minutes.

[0084] The phases were separated and collected. The reactor was charged with the organic phase (lower layer, 1.2 L) and ethanol (840 mL, 14,400 mmol). The jacket was set to 60°C and the solvent was distilled at atmospheric pressure (dichloromethane at 40°C and ethanethiol at 35°C, receiver flask in ice bath). When the distillation slowed, the jacket temperature was increased to 70°C. After 1,300 mL of distillate had been collected, the contents of the vessel were sampled and 1 The dichloromethane to ethanol ratio was measured by H-NMR and confirmed to be less than 10 mol%. If more dichloromethane was present, further distillation was required. Additional ethanol (400 mL) was added, followed by seed crystals of D. The jacket was cooled to 5°C over 30 minutes. The crystal slurry was stirred at 5°C for 3 days. The solid was collected in a sinter funnel and washed with petroleum ether (60-80°C): 1 x 500 mL slurry, 1 x 300 mL plug. The solid was transferred to a 500 mL RBF and dried to constant weight (approximately 4+ hours) on a rotary evaporator (bath temperature 45°C) to yield an off-white solid. Expected yield: approximately 86 g (71% from C).

[0085] Synthesis of Compound 1 A 50 mL round-bottom flask was charged with anhydrous methanol (33 mL). Sodium methoxide in methanol (30% solution, 25 μL, 0.135 mmol) was added, and the resulting solution was stirred at ambient temperature for 5 minutes. Ethyl 3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-thio-D-glucopyranoside (Compound D) (3.09 g, 6.44 mmol) was added in portions (approximately 200 mg) over 10 minutes at a rate that dissolved the solid during the addition. The reaction was stirred at ambient temperature for 2.5 hours. TLC (EtOAc) showed complete consumption of Compound D (Rf = 0.9) and the formation of one more polar spot (Rf = 0.5). A sample was taken and the reaction was determined to be complete by IPC by HPLC (2.5 μL of the reaction mixture in 0.8 mL of acetonitrile and 0.2 mL of water), passing the NMT 1.00 area % for Compound D. Acetic acid (8 μL, 0.1397 mmol) was added. The pH was checked with a dipstick and confirmed to be approximately pH 5-6. The mixture was concentrated to near dryness on a rotary evaporator (50 °C). EtOAc (15 mL) was added and most of it evaporated. The residue was dissolved / slurried in 15 mL of EtOAc and removed from the rotary evaporator. 2 mL of petroleum ether was added and the mixture was stirred at ambient temperature. The crystal slurry was stirred overnight. The solid was collected on a sinter funnel, washed with petrol (2 × 10 mL), dried on a rotary evaporator (45 °C bath temperature) and brought to constant weight. Expected yield: 1.94 g (85% from compound D).

[0086] Synthesis of compound 2 Compound 1 (2.040 g) was dissolved in pyridine (28 mL), and the solution was concentrated to approximately half its volume (approximately 14 mL) on a rotary evaporator at a bath temperature of 40 °C to yield a yellow solution. Additional pyridine (14 mL) was added, and the solution was again concentrated to approximately 14 mL using the same method. This solution was placed under argon, and trityl chloride (2.299 g, 1.36 eq) was added. An air-cooled condenser was then attached, and the solution was heated to 50 °C with stirring. After 4 h, an IPC (HPLC; 5 μL in 800 μL MeCN, residual compound 1 NMT 3.00 area%) was performed. Once the IPC was filled, the reaction was cooled to 10–15 °C. Benzoyl chloride (1.60 mL, 2.34 eq) was added dropwise over a 20 min period, maintaining the reaction temperature below 20 °C. After the addition was complete, the reaction was allowed to warm to ambient temperature and stirred for at least 3 h. At this point, IPC (HPLC; 5 μL in 1500 μL of MeCN, 3.00 area% total residual mono-Bz derivative NMT of compound 1) was performed. Once the IPC was full, the reaction was cooled to 0° C. and quenched by the slow addition of methanol (0.8 mL), ensuring the reaction temperature remained below 20° C. The quenched reaction was then warmed to ambient temperature.

[0087] The product mixture was diluted with toluene (20 mL) and stirred at ambient temperature for 1 hour, after which the precipitate was removed by filtration through a sintered funnel. The toluene solution was then washed with citric acid (20% w / w, 4 x 20 mL), followed by saturated NaHCO3 (9% w / v, 20 mL), which resulted in a side reaction with any residual citric acid present. The toluene (top) phase was then washed with brine (20 mL) before being evaporated on a rotary evaporator at a bath temperature of 40°C to give a yellow / orange syrup (6.833 g). This syrup was purified by IPC (H 1 NMR, passing condition NMT (30 wt% residual toluene). Expected yield: about 6.833 g (147%).

[0088] Synthesis of compound 3 Glacial acetic acid (648 mL) and ultrapure water (72 mL) were mixed together to obtain a 90% aqueous acetic acid solution. A portion of the acetic acid solution (710 mL) was added to crude compound 2 (111 g) along with a stir bar. An air-cooled condenser was attached to the flask, and the mixture was heated to 70 °C. Due to the viscous nature of 2, the mixture did not completely dissolve until 1 hour and 20 minutes later, at which point stirring was initiated. After 2 hours, an IPC (HPLC; 5 μL in 800 μL MeCN, residual compound 2NMT 3.00 area %) was run. Once the IPC met specifications, the reaction was cooled to ambient temperature. The mixture was transferred to a sinter funnel, and the precipitated trityl alcohol (31.09 g) was filtered using house vacuum. The flask was rinsed with an additional portion of 90% acetic acid (40 mL), and the entire wash was transferred to a mixing vessel. Toluene (700 mL) and water (700 mL) were added and mixed thoroughly. The aqueous (lower) phase was a cloudy white solution and the pH was tested (expected to be less than 2). The wash was repeated two more times with water (2x700 mL; pH approx. 2.4 and approx. 3, respectively, clear, colorless solution). Saturated NaHCO3 (9% w / v, 700 mL) was added to the mixing vessel, causing a side reaction (gassing). The toluene (upper) phase was then washed with brine (700 mL) and then evaporated on a rotary evaporator at a bath temperature of 40°C to give a yellow / orange solid / liquid mixture (86 g). This mixture was dissolved in 400 mL of toluene (300 mL + 100 mL wash) and loaded onto a silica column (450 g silica) equilibrated with 3 column volumes (CV) of petroleum ether:toluene (1:1, v:v). The column was eluted using a step gradient, collecting 1 CV (790 mL) fractions. The gradient used was: Petroleum ether: 4 vol% ethyl acetate in toluene (1:1 v:v, 4 CV) Petroleum ether: 8 vol% ethyl acetate in toluene (1:1 v:v, 12 CV) Petroleum ether: 15 vol% ethyl acetate in toluene (1:1 v:v, 4 CV) Petroleum ether: 20 vol% ethyl acetate in toluene (1:1 v:v, (4 CV) Petroleum ether: 30 vol% ethyl acetate in toluene (1:1 v:v, 1 CV).

[0089] The product eluted over 14 fractions. TLC was used to identify fractions containing the product. All fractions were subjected to IPC (HPLC, NMT 1.50 area % for the peak at 10.14 min and NMT 1.50 area % for the peak at 10.94 min). Fractions that did not satisfy the IPC were set aside for further processing to compound 4. The fractions were combined and evaporated on a rotary evaporator at a bath temperature of 45° C. to give a colorless syrup. Expected yield: approximately 60 g (78%).

[0090] Synthesis of compound 4 Crude compound 3 (39.54 g, containing approximately 21 g of compound 3, approximately 37 mmol, taken immediately before chromatography) was dissolved in toluene (7.2 mL) and dry pyridine (14.2 mL, 176 mmol, approximately 4.8 eq) was added to obtain a homogeneous solution. 7.2 mL of acetic anhydride (76 mmol, approximately 2.1 eq) was added, and the mixture was stirred at 25 °C for 18 h. A solid precipitated during the reaction; some of this precipitate may have been compound 4. Samples were taken from the reaction and analyzed by IPC. If the amount of compound 3 detected was greater than 1.00 area%, additional dry pyridine (1.4 mL, 17 equiv) was added, and the reaction was continued until the remaining compound 3 in the liquid phase was ≤ 1.00 area%.

[0091] The reaction was diluted with dichloromethane (112 mL), and water (2.8 mL) and methanol (2.8 mL) were added. The mixture was stirred at 25°C for 3 hours. This stirring period was sufficient to quench excess acetic anhydride. The mixture was washed with citric acid monohydrate / water 20 / 80 w / w (112 mL). The aqueous phase was back-extracted with dichloromethane (50 mL). The dichloromethane used for back-extraction was set aside and used to back-extract the aqueous phase from the remaining citric acid wash. The main dichloromethane extract was returned to the vessel, and the citric acid wash procedure was repeated until the pH of the aqueous phase was ≤ 2 (typically two additional washes). The citric acid washes were combined and back-extracted. The back-extract and main dichloromethane extract were then combined. The resulting dichloromethane solution was washed with 5% w / v NaHCO3 (100 mL), and the dichloromethane phase was removed and washed with water (100 mL). The dichloromethane phase was transferred to an evaporation vessel, ethyl acetate (50 mL) was added, and the solution was concentrated to a syrup.

[0092] Ethyl acetate (150 mL) was added and heated to 55°C with stirring to dissolve the product. Petroleum ether 60-80 (200 mL) was added, and the solution was reheated to 55°C and held for 5 minutes. The solution was cooled to 45°C, seed crystals (30 mg) were added, and then cooled to 18°C ​​over 3 hours with stirring and held at 18°C ​​for at least 1 hour. The crystals were collected by filtration and washed with ethyl acetate / petroleum ether (1 / 2 v / v, 60 mL). Drying under reduced pressure gave compound 4 (16.04 g, 77% from 2). Expected yield: 16.0 g (77% from compound 2).

[0093] Synthesis of compound 3.1 3-Aminopropan-1-ol (7.01 g, 93 mmol) was dissolved in DCM (70 mL) and cooled to 0 °C. Benzyl chloroformate (5.40 mL, 32 mmol) was dissolved in DCM (20 mL) and added dropwise, maintaining the internal reaction temperature below 10 °C. Upon completion, the flask was stirred at room temperature for 2 h. A sample removed for NMR analysis (IPC: 20 μL + 0.6 mL d6-DMSO) indicated that the benzyl chloroformate reagent had been consumed. The product mixture was then washed with citric acid (10% w / w, 2 x 90 mL), water (90 mL), and brine (90 mL). The DCM (lower) phase was then evaporated on a rotary evaporator at 0 °C bath temperature to give a slightly cloudy oil / liquid (6.455 g). This oil was dissolved in ethyl acetate (7 mL), warmed to 40 °C as needed to dissolve precipitated solids, and then allowed to cool to room temperature. Petroleum ether (4 mL) was slowly added to the stirred solution along with the seed crystals, at which point the product slowly began to crystallize. Once most of the product had precipitated, the final portion of petroleum ether (17 mL) (total solvent added: ethyl acetate:petroleum ether 1:3, 21 mL) was slowly added. The product was then filtered under vacuum and washed with petroleum ether (5 mL) to give the product as a fine white powder (4.72 g). Expected yield: approximately 4.7 g (61%).

[0094] Synthesis of compound 5 Compound 4 (1.05 g, 1.73 mmol) was dissolved in dry acetone (12 mL, 0.06% w / w water) and water (39 μL, 2.15 mmol, 1.3 eq.) at ambient temperature. The solution was then cooled to −10° C. NBS (0.639 g, 3.59 mmol, 2.08 eq.) was added in one portion. An exotherm on the order of +7° C. was expected, and the solution was then immediately recooled to −10° C. 15 min after the NBS addition, the reaction mixture was subjected to IPC (HPLC, less than 2.00 area % of compound 4 remaining, acceptable). If the reaction was incomplete, 1.00 eq. of NBS (0.307 g, 1.73 mmol, 1.00 eq.) was added in one portion, and the reaction was then held at −10° C. for an additional 15 min, followed by another IPC. The reaction was quenched by the addition of aqueous NaHCO3 (5% w / v, 5 mL). Cooling was removed, and the mixture was allowed to warm to 10–20 °C between additions. After stirring for 3–5 min, an additional portion of aqueous NaHCO3 (5% w / v, 5 mL) was added, and stirring was continued for 5 min. A final aliquot of aqueous NaHCO3 (5% w / v, 10 mL) was added with stirring, followed by sodium thiosulfate (20% w / v, 5 mL). The mixture was stirred at 10–20 °C for 20 min, and then the solid was collected by filtration. The vessel was rinsed into a filter pad with NaHCO3 (5% w / v, 25 mL), and this rinse was removed by filtration. The filter cake was then rinsed sequentially with NaHCO3 (5% w / v, 25 mL) and then water (25 mL). The (still wet) filter cake was dissolved in DCM (20 mL) and washed with two portions of NaHCO3 (5% w / v, 20 mL), then once with water (20 mL). The dichloromethane phase was dried by rotary evaporation and then dissolved in ethyl acetate (36 mL) at 65 °C. Next, petroleum ether (10 mL) was slowly added with stirring, and the mixture was cooled to 45 °C and stirred at 45 °C for 30 min. Additional petroleum ether (22 mL) was added with stirring, and the stirred mixture was cooled to 15 °C over 2 h. The product was collected by filtration, washed with petroleum ether / ethyl acetate 2 / 1 v / v (20 mL), and then dried under vacuum to give compound 5 (0.805 g, 83% yield; α and β anomeric purity by HPLC was 98%).

[0095] Synthesis of compound 7 Compound 4 (500 mg) and intermediate 3.1 (211 mg, 1.2 eq) were weighed into a dry flask, toluene (5 mL) was added, and the solution was concentrated on a rotary evaporator (bath temperature 45 °C). This was repeated once more, after which the starting material was concentrated from anhydrous DCM (5 mL). Once all solvent was removed, the remaining solid was dried under vacuum for 10 min. After drying, the starting material was placed under argon, dissolved in anhydrous DCM (5.0 mL), and activated 4 Å molecular sieves (450 mg, pellets) were added. At this point, the NIS reagent was placed under high vacuum and dried. After 10 min, dried NIS (400 mg, 2.0 eq) was added, and the solution was stirred at room temperature for 30 min. Next, TMSOTf (8 μL, 5 mol%) was quickly added, resulting in a solution color change from red / orange to dark red / brown. The reaction temperature also increased from 22 °C to 27 °C. Immediately after the addition of TMSOTf, IPC (HPLC; 10 μL in 1 mL of MeCN-HO (8:2)) was performed for informational purposes only. The reaction was then quenched by adding pyridine (20 μL, 0.245 mmol) and stirred at ambient temperature for 5 min. The DCM solution was filtered to remove the molecular sieves, then washed with 10% NaSO (3 × 5 mL), brine (5 mL), and subsequently concentrated on a rotary evaporator (bath temperature 40 °C) to give crude compound 7 as a foamy yellow oil (616 mg). Expected yield: approximately 616 mg (99%).

[0096] Synthesis of compound 8 Crude compound 7 (16.6 g) was dried by evaporation from toluene (2 × 30 mL) followed by anhydrous DCM (30 mL) to produce a yellow foam / oil. Next, the flask was placed under an argon atmosphere, and then anhydrous DCM (100 mL) and dry MeOH (260 mL) were added, and the mixture was stirred. The flask was then cooled to 0 °C. Acetyl chloride (3.30 mL, 2.0 eq.) was added dropwise, maintaining the internal temperature below 10 °C. Upon completion of the addition, the mixture was stirred at ambient temperature for 16 h. At this point, an IPC was performed (HPLC; 20 μL in 1 mL of MeCN, residual compound 7 was less than 3 area %). The flask was then cooled to 0 °C, and the pH of the product solution was adjusted to pH 6.5-7.5 by the addition of N-methylmorpholine (7.0 mL total required). The product mixture was diluted with DCM (50 mL) and washed with HO (2 × 200 mL). The second HO wash was cloudy and contained the target material by TLC, so it was back-extracted with DCM (50 mL). The DCM phases were combined and then washed with brine (8 mL) before being evaporated on a rotary evaporator at a bath temperature of 40°C to give an off-white foam / oil (approximately 16.8 g). This mixture was dissolved in 140 mL of toluene (100 mL + 40 mL wash), loaded onto a silica column (85 g silica) and equilibrated with 3 column volumes (CV) of 30 vol% ethyl acetate in petroleum ether. The column was eluted using a step gradient, collecting fractions of 1 CV (140 mL). The gradient used was: 30 vol% ethyl acetate in petroleum ether (3 CV) 35 vol% ethyl acetate in petroleum ether (4 CV) 40 vol% ethyl acetate in petroleum ether (9 CV) 50 vol% ethyl acetate in petroleum ether (4 CV) 60 vol% ethyl acetate in petroleum ether (3 CV). The product eluted over 12 fractions. All fractions were subjected to IPC (HPLC, NMT 1.50 area % of any impurity peak at 230 nm). The fractions were combined and evaporated on a rotary evaporator at a bath temperature of 40° C. to give an off-white foam, which solidified to give compound 8 (10.45 g) as a crisp solid. Expected yield: 10.45 g (66%).

[0097] Example 4 - Synthesis of Disulfide (Compound 17) [ka] The overall synthetic procedure for the synthesis of compound 17 is described in the synthetic scheme below. [ka]

[0098] Synthesis of compound 9 Compound 5 (1620 g, 1.18 eq.) and toluene (18 kg) were charged in that order to a 50 L Buchi bowl. The bowl was warmed in a water bath set at 50 ± 10 °C for 30 minutes. Evaporation was carried out under vacuum using a water bath temperature of 50 ± 10 °C until no more solvent was distilled. The water bath was cooled to 20 ± 10 °C. Trichloroacetonitrile (7.1 kg, 21 equiv.) and dry DCM (6.5 kg) were charged to the bowl under a nitrogen atmosphere. A suspension of sodium hydride (5.6 g, 0.060 equiv.) in dry DCM (250 g) was charged to the bowl under a nitrogen atmosphere. The contents of the bowl were mixed by rotation at a water bath temperature of 20 ± 10 °C for 1-2 hours. Compound 5 dissolved during the reaction. A sample was taken from the contents of the bowl and subjected to reaction completion IPC (H 1NMR analysis integrated the triplet peak at 6.42 ppm (product) relative to the triplet peak at 6.35 ppm (starting material); acceptance criteria: ≤5% residual starting material. Compound 3 (1360 g, 2.35 mol), dry DCM (12.3 kg), and powdered 4Å molecular sieves (136 g) were charged, in that order, to a 50 L reactor. The reactor contents were mixed for 24 h. The reactor contents were sampled via syringe filter and analyzed by Karl Fischer (AM-GEN-011, acceptance criteria: ≤0.03% w / w). After reaching the moisture threshold (approximately 24 h), the reactor contents were adjusted to 0 ± 5 °C. The contents of the Buchi bowl were transferred to the reactor header as volume permitted. A solution of trimethylsilyl trifluoromethanesulfonate (100 g, 0.18 eq.) in dry DCM (1250 g) was charged to the reactor under a nitrogen atmosphere. The reactor contents were maintained at 0 ± 10 °C throughout the addition, while the header contents were discharged into the reactor. The addition took 15-20 min. Dry DCM (1250 g) was charged to a Buchi bowl and then transferred to the reactor header. The reactor contents were maintained at 0 ± 10 °C throughout the addition, while the header contents were discharged into the reactor. The reactor contents were stirred at 0 ± 5 °C for 60 min. The reactor contents were sampled and the reaction was determined to be complete using IPC (HPLC, acceptance criteria ≤ 5% starting material). The reaction was quenched by charging N-methylmorpholine (85 g, 0.36 eq.) to the reactor. The reactor contents were sampled and quenched using IPC (wet pH paper, pass criterion ≥ pH 7). Silica gel (4.9 kg) was charged to a Buchi bowl. The reactor contents were transferred to a Buchi bowl. Evaporation was carried out under vacuum using a water bath temperature of 40 ± 10 °C until no more solvent distilled. Silica gel (1.4 kg) was charged to the Buchi bowl, followed by rinsing the reactor with dichloromethane (7.0 kg). The bowl contents were swirled to ensure no solids adhered to the bowl surface. Evaporation was carried out under vacuum using a water bath temperature of 40 ± 10 °C until no more solvent distilled. The bowl contents were divided into three portions and subjected to silica gel chromatography. A 150 L KP-SIL cartridge was attached to the Biotage system.Ethyl acetate (7.8 kg) and petroleum ether (22 kg) were charged to a 50 L reactor along with one-third of the reaction mixture adsorbed onto silica gel, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The contents of the solvent reservoir were eluted through the column to condition the column. The eluate was collected in a 20 L jerry can and discarded. The column was run in three batches, each eluted with ethyl acetate / petroleum ether as described below: a. Ethyl acetate (1.6 kg) and petroleum ether (4.4 kg) were charged to a Biotage solvent reservoir, mixed thoroughly, and then eluted through the column. The column effluent was collected in a 20 L jerry can. b. Ethyl acetate (25 kg) and petroleum ether (26 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. c. Ethyl acetate (31 kg) and petroleum ether (22 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 5 L glass lab bottle. d. Ethyl acetate (16 kg) was charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 20 L jerry can. e. Repeat the column as above to prepare the remaining two portions of dry loaded silica.

[0099] Column fractions were sampled for product purity (TLC [10% acetone in toluene, Rf 0.5]) and fractions identified with product. The identified column fractions were combined and placed in a 100 L Buchi bowl. Toluene was used to rinse the crystalline material from the identified fraction container into the bowl. Evaporation was carried out under vacuum using a 40±10°C water bath temperature until no more solvent was distilled. Toluene (1.7 kg) was charged to the bowl and the contents were swirled until the solids dissolved. t-Butyl methyl ether (4.4 kg) was charged to the bowl over 20-40 minutes. The contents of the bowl were swirled at a temperature of 20±5°C for 12-24 hours. The contents of the bowl were then poured into 6 L of a 100 L Buchi bowl. The mixture was transferred to a Nutsche filter and the solvent removed by vacuum filtration. t-Butyl methyl ether (620 g) was placed in a bowl, transferred to the Nutsche filter, and passed through the filter cake. The filter cake was allowed to air dry in the filter and then transferred to a vacuum oven and dried under vacuum at a set temperature of 30°C to remove residual solvent. The solid was sampled for analysis and retention. The solid was transferred to a screw-cap Nalgene container and stored at ≤ -15°C. Expected yield: 1.68-1.94 kg of compound 9 (65-75%).

[0100] Synthesis of compound 10 The reagents were prepared as follows: N-iodosuccinimide (241 g, 2.20 eq) was dried under vacuum in a vacuum oven set at 30°C for 24 hours. A solution of sodium chloride (300 g) in water (3000 g) was prepared in a 5 L lab bottle. A solution of sodium thiosulfate (1100 g) in water (6000 g) was prepared in a 50 L reactor and divided into two portions.

[0101] Compound 8 (355 g, 0.486 mol) and compound 9 (634 g, 1.10 eq.) were charged to a 20 L Buchi bowl, followed by toluene (1500 g) and heated to 40±5° C. until dissolved. Evaporation was carried out under vacuum using a water bath temperature of 35±10° C. until no more solvent was distilled. Toluene (1500 g) was charged to the Buchi bowl. Evaporation was carried out under vacuum using a water bath temperature of 35±10° C. until no more solvent was distilled. Dry dichloromethane (4000 g) was charged to the Buchi bowl. The bowl was swirled until the solids dissolved, and the solution was transferred to a 5 L reactor with a jacket temperature of 20° C.±5° C. Dry dichloromethane (710 g) was charged to the Buchi bowl. The bowl was swirled to rinse the surface of the bowl, and the solution was transferred to the 5 L reactor. A sample was taken from the reactor contents and the reagent ratio IPC(H 1 The reaction mixture was analyzed by NMR. Dry N-iodosuccinimide was charged to the reactor under a nitrogen atmosphere and the reactor was stirred for 5 to 15 minutes. The reactor contents were adjusted to 20°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (5.94 g, 0.055 eq.) in dry DCM (60 g) was dosed to the reactor over 5 to 15 minutes, maintaining the temperature of the contents at 20°C ± 3°C. The reaction mixture was stirred at 20°C ± 3°C for 20 ± 3 minutes. A sample was taken from the reactor contents to determine reaction completion (HPLC). N-methylmorpholine (98 g, 2 equiv.) was charged to the reactor and thoroughly mixed. A portion of the sodium thiosulfate solution prepared above was charged to a 50-L reactor. The contents of the 5-L reactor were transferred to the 50-L reactor containing the sodium thiosulfate solution and thoroughly mixed. The bottom layer was drained into an HDPE jerry can.

[0102] DCM (570 g) was charged to the 5 L reactor along with the top layer from the 50 L reactor and mixed thoroughly. The bottom layer was combined with the previous bottom layer in the HDPE jerry can. The top layer was transferred to another HDPE jerry can and held until yield was confirmed. The combined organic phase (bottom layer) was charged to the 50 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into the HDPE jerry can. The top layer was held in the HDPE jerry can until yield was confirmed. The sodium chloride solution was charged to the 50 L reactor along with the organic phase (bottom layer) and mixed thoroughly. Silica gel (1300 g) was charged to a Buchi bowl and attached to a rotary evaporator. The bottom layer from the reactor was charged to the Buchi bowl. The contents of the bowl were swirled to prevent adsorption to the bowl and evaporated under vacuum using a water bath temperature of 40 ± 5 °C until no solids distilled over. The contents of the bowl were divided into two equal parts. Silica gel (200 g) was added to a Buchi bowl, followed by dichloromethane (700 g). The contents of the bowl were swirled to ensure that no solids remained on the surface of the bowl. The bowl was evaporated under vacuum at a water bath temperature of 40°C ± 10°C until no more solvent was distilled. The contents of the bowl were divided into two portions, and one portion was added to each of the previous silica gel samples.

[0103] Each portion was independently purified on silica gel using the following procedure (samples were stored at ≦15°C while awaiting purification): A 150 L KP-SIL cartridge was attached to a Biotage system. Ethyl acetate (15.5 kg) and petroleum ether (16.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns, conditioning the columns. The eluate was collected in a 20 L jerry can and discarded. A portion of the above dry loaded silica was loaded into a Biotage Sample Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows: a. Ethyl acetate (6.2 kg) and petroleum ether (6.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can. b. Ethyl acetate (19.5 kg) and petroleum ether (19.2 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. c. Ethyl acetate (13.6 kg) and petroleum ether (12.3 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. d. Ethyl acetate (14.2 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. e. Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were charged to the Biotage solvent tank and then eluted through the column. The column effluent was collected in a 20 L jerry can until fraction 11, then in a 5 L HDPE jerry can. f. Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L HDPE jerry can. g. Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L HDPE jerry can. h. Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and eluted through the column. The column effluent was collected in a 5 L HDPE jerry can.

[0104] Column fractions were sampled for product purity (TLC to identify fractions containing product). Fractions from the first two columns that were 75-95% area 10 were combined in a Buchi bowl charged with silica gel (400 g) and evaporated under vacuum using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. The contents of the bowl were purified as follows: A 150 L KP-SIL cartridge was attached to a Biotage system. Ethyl acetate (15.5 kg) and petroleum ether (16.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns to condition them. The eluate was collected in a 20 L jerrican and discarded. The contents of the bowl were loaded into a Biotage sample injection module (SIM) and then eluted with ethyl acetate / petroleum ether as follows: a. Ethyl acetate (6.2 kg) and petroleum ether (6.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can. b. Ethyl acetate (19.5 kg) and petroleum ether (19.2 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. c. Ethyl acetate (13.6 kg) and petroleum ether (12.3 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. d. Ethyl acetate (14.2 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. e. Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 20 L jerry can up to fraction 11 and then in a 5 L HDPE jerry can. f. Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L HDPE jerry can. g. Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L HDPE jerry can. h. Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L HDPE jerry can.

[0105] The observed column fractions from all three columns were combined in a Buchi bowl and evaporated under vacuum using a water bath at 40°C ± 10°C until no more solvent distilled. A sample was taken from the contents of the bowl for analysis and retention. The bowl was sealed and transferred to storage at ≤ -15°C. Expected yield: 440-540 kg (52-64% yield).

[0106] Synthesis of compound 11 Compound 10 (635 g, 0.345 mol) (PN0699) was added to a Buchi bowl in dichloromethane and heated to 30 ± 10 °C until dissolved. Methanol (3.2 kg) was added to the bowl. The contents of the bowl were adjusted to 0 ± 3 °C. Acetyl chloride (54.1 g, 2 equiv.) in dichloromethane (660 g) was added to the bowl while maintaining the temperature of the contents at 0 ± 10 °C. The contents of the bowl were adjusted to 20 ± 3 °C, and the mixture was stirred for 40-48 h. The contents of the bowl were sampled and the reaction was complete by IPC (HPLC, passing). The contents of the bowl were adjusted to 0 ± 3 °C. N-methylmorpholine (139 g, 4 equiv.) was added to the bowl and mixed thoroughly. The contents of the bowl were sampled and the reaction was quenched by IPC (pH paper, passing ≤ pH 7). The contents of the bowl were concentrated under vacuum in a 35±10°C water bath. Ethyl acetate (4.8 kg) and water (5.5 kg) were added to a Buchi bowl and swirled to dissolve the contents of the bowl. The contents of the bowl were transferred to a 50 L reactor and mixed thoroughly. The bottom layer was drained into an HDPE jerry can. The top layer was transferred to a Buchi bowl fitted with a rotary evaporator, and the contents were concentrated under vacuum in a 35±10°C water bath. The bottom layer from the HDPE jerry can was added to a 50 L reactor along with ethyl acetate (1.5 kg) and mixed thoroughly. The bottom layer was drained into an HDPE jerry can and held until yield was confirmed. The top layer was transferred to a Buchi bowl fitted with a rotary evaporator, and the contents were concentrated under vacuum in a 35±10°C water bath. A sample was taken from the contents of the bowl, analyzed, and held. The bowl was sealed and transferred to storage at ≤-15°C. Expected yield: 518-633 kg (90-110% yield).

[0107] Synthesis of compound 12 The reagents were prepared as follows: Two portions of N-iodosuccinimide (143 g, 3.90 eq.) were dried under vacuum in a vacuum oven set at 30°C for 24 hours. A solution of sodium chloride (450 g) in water (1850 g) was prepared in a 5 L lab bottle and divided into two approximately equal portions. A solution of sodium thiosulfate (230 g) in water (2080 g) was prepared in a 5 L lab bottle and divided into four approximately equal portions. A 50 L Buchi bowl containing compound 11 (607 g, 0.327 mol) was charged with compound 9 (504 g, 1.30 eq.), followed by toluene (1500 g) and heated to 40 ± 5 °C until dissolved. Evaporation was carried out under vacuum using a water bath temperature of 35 ± °C until no more solvent was distilled. Toluene (1500 g) was charged to the Buchi bowl. Evaporation was carried out under vacuum using a water bath temperature of 35 ± 10 °C until no more solvent was distilled. Dry DCM (2400 g) was charged to the Buchi bowl. The bowl was swirled until the solids dissolved, and half of the solution was transferred to a 5 L reactor with a jacket temperature of 20 ± 5 °C. The other half of the solution was transferred to a 5 L lab bottle. Dry DCM (710 g) was charged to the Buchi bowl. The bowl was swirled to rinse the surface of the bowl, and half of the solution was transferred to the 5 L reactor. The remaining half was charged to the 5 L lab bottle described above and stored under nitrogen for use in the second batch. A portion of the dried N-iodosuccinimide was charged to the reactor under a nitrogen atmosphere. The reactor contents were adjusted to -40°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (9.09 g, 0.25 effective equiv.) in dry dichloromethane (90 g) was charged to the reactor over 15 minutes while maintaining the temperature of the contents at -40°C ± 5°C. The reaction mixture was stirred at -40°C ± 3°C for 30 ± 5 minutes, then adjusted to -30°C ± 3°C and stirred for 150 minutes. A sample was taken from the reactor contents to complete the reaction. N-methylmorpholine (33.1 g, 2 effective equiv.) was charged to the reactor and mixed thoroughly. A portion of the sodium thiosulfate solution prepared above was charged to the 5 L reactor and mixed thoroughly. The bottom layer was drained into a 5 L lab bottle. DCM (400 g) was charged to a 5 L reactor and mixed thoroughly. The bottom layer was combined with the previous bottom layer in a 5 L lab bottle. The organic phases were combined and charged to the 5 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into the 5 L lab bottle. A portion of the sodium chloride solution from above was charged to the reactor, followed by the contents of the previous lab bottle. The bottom layer in the reactor was charged to a Buchi and evaporated under vacuum using a water bath temperature of 40±10°C until no more solvent was distilled. The reactor was washed and dried.

[0108] The second portion of compound 9 and compound 11 were charged to the reactor and treated similarly to the first batch. After the organic extraction of the second batch, the reaction mixtures were combined in the reactor. A portion of the sodium chloride solution was charged to the reactor and mixed thoroughly. Silica gel (1700 g) was charged to a Buchi bowl and attached to a rotavapor. The bottom layer in the reactor was charged to the Buchi and evaporated under vacuum using a water bath temperature of 40±10°C until no more solvent was distilled. The contents of the bowl were divided into two portions and purified independently with silica gel. A 150 L KP-SIL cartridge was used. A Biotage system (Biotage, a division of Dyax Corporation, Charlottesville, Virginia, USA) was attached. Ethyl acetate (7.7 kg) and petroleum ether (22.0 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the column to condition it. The eluate was collected in a 20 L jerry can and discarded. A portion of the dry loaded silica from above was loaded into a Biotage sample injection module (SIM) and eluted with ethyl acetate / petroleum ether as follows: Ethyl acetate (1.5 kg) and petroleum ether (4.4 kg) were charged into an HDPE jerry can, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can. b. Ethyl acetate (18.6 kg) and petroleum ether (8.8 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. c. Ethyl acetate (19.2 kg) and petroleum ether (8.4 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. d. Ethyl acetate (29.7 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. e. Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L glass lab bottle.

[0109] Column fractions were sampled for product purification (TLC to identify fractions containing product). Fractions from the first two columns representing 75-95% area of ​​compound 12 were combined in a Buchi bowl charged with silica gel (400 g) and evaporated under vacuum using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. Ethyl acetate (7.7 kg) and petroleum ether (22.0 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoirs were eluted through the columns, conditioning them. The eluate was collected in a 20 L jerrican and discarded. The dry loaded silica containing the impure product was loaded into a Biotage sample injection module (SIM) and eluted as detailed below: a. Ethyl acetate (1.5 kg) and petroleum ether (4.4 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The column effluent was collected in a 20 L jerry can. b. Ethyl acetate (19.2 kg) and petroleum ether (8.4 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. c. Ethyl acetate (18.6 kg) and petroleum ether (8.8 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. d. Ethyl acetate (29.7 kg) and petroleum ether (11.9 kg) were charged to a 50 L reactor, mixed thoroughly, transferred to two Biotage solvent reservoirs, and then eluted through the column. The column effluent was collected in a 20 L jerry can. e. Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted through the column. The column effluent was collected in a 5 L glass lab bottle.

[0110] Column fractions were sampled for product purification (TLC to identify fractions containing product, HPLC acceptance criteria: ≥ 95% compound 12, no single impurity greater than 2.5%). The recognized column fractions from all three columns were combined in a Buchi bowl and evaporated under vacuum using a water bath temperature of 40 ± 10°C until no more solvent was distilled. The contents of the bowl were sampled for analysis and retained. The bowl was sealed and transferred to storage at ≤ -15°C. Expected yield: 494-584 kg (52-64% yield).

[0111] Synthesis of compound 13 Glacial acetic acid (7.5 kg) and ethyl acetate (6.5 kg) were combined in a suitable container and labeled "GAA / EA Solution." Sodium bicarbonate (0.5 kg) was dissolved in RO water (10 kg) and labeled "5% w / w sodium bicarbonate solution." Palladium on activated carbon (100 g, specifically Johnson Matthey, Aliso Viejo, California, USA, Product No. A402028-10) and GAA / EA solution (335 g) were charged, in that order, to a reaction vessel. Compound 12 (270 g) was dissolved in GAA / EA solution (1840 g) and transferred to a 50 L reaction vessel. Oxygen was purged from the solution by pressurizing with nitrogen to 10 bar, followed by venting. This was repeated two more times. The contents of the reactor were pressurized under hydrogen to 10 bar, followed by venting. The reaction mixture was hydrogenated at 20 bar H2 for 1.5 days. The pressure was then released and the solution was purged of hydrogen by pressurizing with nitrogen to 10 bar, followed by venting. This was repeated once. The reaction mixture was filtered through a pad of Celite (300 g). The Celite cake was washed with the GAA / EA solution (2 x 5.5 kg). The combined filtrates were evaporated under vacuum (bath temperature 40 ± 5 °C). The residue was co-evaporated with ethyl acetate (2.3 kg) in two portions. The expected weight of the crude product was 316 g. A Biotage system was fitted with a 150 M KP-SIL cartridge equipped with a 5 L sample injection module (SIM). Ethyl acetate (10.6 kg) and glacial acetic acid (1.4 kg) were charged to the 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The contents of the solvent reservoir were eluted through the column to condition the column. The eluate was discarded. The crude product was dissolved in ethyl acetate (422 g) and glacial acetic acid (55 g). The resulting solution was loaded onto the SIM and passed through the column. The reaction mixture was chromatographed as follows: a. Ethyl acetate (13.8 kg) and glacial acetic acid (1.8 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. b. The contents of the solvent reservoir were eluted through the SIM on the column and the eluate was collected in a 20 L jerry can. c. Ethyl acetate (10.3 kg), glacial acetic acid (1.3 kg), and methanol (206 g) were charged to a 50 L reactor, mixed thoroughly, and then collected in the Biotage solvent reservoir. d. The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can. e. Ethyl acetate (6.6 kg), glacial acetic acid (0.9 kg) and methanol (340 g) were charged to a 50 L reactor, mixed thoroughly and then transferred to the Biotage solvent reservoir. f. The contents of the solvent reservoir were eluted through the column and the eluate was collected in approximately 2.5 L fractions in a 5 L jerry can. g. Ethyl acetate (31.4 kg), glacial acetic acid (4.1 kg), and methanol (3.4 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir. h. The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can.

[0112] Fractions containing compound 13 were combined and evaporated under vacuum (bath temperature 40±5°C). The residue was dissolved in ethyl acetate (3.1 kg) and washed with 5% w / w sodium bicarbonate solution (9.3 kg), ensuring the pH of the aqueous medium was ≥ 8. The ethyl acetate phase was evaporated under vacuum (bath temperature 40±5°C). A sample was taken from the contents of the bowl, analyzed, and retained. Expected yield: 182-207 g (71-81%).

[0113] Synthesis of compound 16 Dry dichloromethane (2.5 kg) was added to a Buchi bowl containing compound 13 (211 g, 76.5 mmol, 1.00 eq.) and swirled without heating until dissolved. A solution of (2,5-dioxopyrrolidin-1-yl) 4-acetylsulfanylbutanoate (25.8 g, 99.4 mmol, 1.30 equiv.) in dry dichloromethane (200 g) was added to the Buchi bowl. The bowl was swirled at ambient temperature for 1 hour and then concentrated under vacuum at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the bowl and removed twice under vacuum at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the residue to dissolve it. Silica gel (557 g) was placed in a reaction vessel, and the solvent was removed under vacuum at a water bath temperature of 40 ± 5 °C. The Biotage system was equipped with a 150M KP-SIL cartridge with a 5 L sample injection module (SIM). Toluene (10.1 kg) and acetone (1.0 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent A). The reaction mixture was purified as follows: a. Solvent A was eluted through the column to condition the column. The eluate was discarded. b. The dried loaded silica gel was transferred to the SIM. c. Toluene (9.6 kg) and acetone (1.5 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent B). d. Solvent B was eluted through the column and the eluate was collected in a 5 L jerry can. e. Toluene (53.6 kg) and acetone (12.2 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent C). f. Solvent C was eluted through the column and the eluate was collected in a 5 L jerry can. g. Toluene (8.4 kg) and acetone (2.6 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (solvent D). h. Solvent D was eluted through the column and the eluate was collected in a 5 L jerry can. i. Toluene (23.4 kg) and acetone (9.2 kg) were charged to a 50 L reactor, mixed thoroughly, and then transferred to the Biotage solvent reservoir (Solvent E). j. Solvent E was eluted through the column and the eluate was collected in a 5 L jerry can. Fractions containing compound 16 (acceptance criteria ≥ 90% compound 16, no single impurity > 2.5%) were combined and evaporated under vacuum (bath temperature 40 ± 5 °C). The residue was dissolved in tetrahydrofuran (4.4 kg) and concentrated under vacuum at a water bath temperature of 40 ± 5 °C. A sample was taken from the contents of the bowl, analyzed, and retained. Expected yield: 169-192 g (76-86%).

[0114] Synthesis of compound 17 The reactor was marked at the 2.5 L, 3.5 L, and 3.9 L levels prior to starting and equipped with a vacuum controller. Dichloromethane was charged to a Buchi bowl containing 140 g of compound 16 and transferred to the ReactorReady vessel. The contents of the Buchi bowl were transferred to the ReactorReady vessel using two rinses of DCM (333 g). Ethanol (2.50 kg) was added to the ReactorReady vessel. The reaction mixture was concentrated to the 2.5 L mark (target vacuum 250 mbar). Ethanol (1.58 kg) was added to the ReactorReady vessel and concentrated to the 3.5 L mark. The reaction was diluted with ethanol to the 3.9 L mark. The contents of the reactor were placed under inert gas by applying a partial vacuum and venting with nitrogen. A gentle reflux of nitrogen was maintained throughout the reaction. Hydrazine monohydrate (1.13 kg, 1.11 L) was charged to a 5 L ReactorReady vessel under a nitrogen atmosphere. The temperature ramp was set to an initial temperature of 20°C, a final temperature of 60°C, a linear temperature ramp (0.8 deg / min) over 50 minutes, and active control of the reactor contents. The vessel temperature was held at 60°C for 45 minutes. The cooling ramp temperature was set to -2 deg / min with a final temperature of 20°C. The contents were drained into appropriate HDPE jugs and weighed. Equal amounts were transferred to eight polypropylene centrifuge vessels with FEP seals. Each centrifuge vessel was charged with ethanol (750 g) and stirred at ambient temperature for 30 minutes. The vessels were centrifuged (5300 RCF, 15°C, 30 minutes). Residual hydrazine on the outside of the vessel was removed by washing the outside of the vessel with acetone followed by water before removing it from the fume hood. The supernatant in the centrifuge vessel was decanted, and the remaining pellet was dissolved in low-endotoxin water (LE water) (1960 g) and transferred to a 5 L Reactor Ready vessel. The contents were stirred at medium speed for approximately 15-20 minutes every 1.5 hours, bubbling air through the solution using a dispersion tube. The mixture was then stirred overnight at 20°C in a sealed container. The reaction was considered complete when IPC showed that the free pentamer composition was less than 3% (reported overall area %). Filtration (using a P3 sintered glass funnel and a 5 L Buchner flask) was required if insoluble material was present in the reaction mixture.The reactor contents were freeze-dried in two Lyoguard trays. The shelf temperature was set at -0.5°C for 16-20 hours, then at 20°C until dry. The freeze-dried product was dissolved in LE water (840 g) and divided equally among six centrifuge bottles. Acetone (630 g) was added to each container, which was stirred for 15 minutes. Isopropanol (630 g per container) was added to each container, and stirring continued for 20 minutes. The contents were centrifuged at 5300 RCF and 15°C for 1 hour. The supernatant was discarded, and each pellet was dissolved in water by adding LE water (140 g) to each container and then stirring the mixture at ambient temperature using an orbital shaker until the pellet was dissolved. Acetone (630 g) was added to each container, which was stirred for 15 minutes. Isopropanol (630 g per container) was added to each container, and stirring continued for 20 minutes. The contents were centrifuged at 5300 RCF and 15°C for 1 hour. The supernatant was discarded, and each pellet was dissolved in water by adding LE water (100 g) and stirring at ambient temperature. The solution was transferred to a Lyoguard tray, and the bottles were rinsed with more LE water (66 g each), and the rinse was transferred to the same tray. The product was lyophilized by setting the shelf temperature at -0.5 °C for 16-20 hours and then at 20 °C until dry. Samples were taken from the lyophilized product for analysis and retention. The Lyoguard trays were double-bagged, labeled, and stored in a freezer (≤ -15 °C). The potency of the lyophilized product was determined using qH NMR. This procedure yielded crude pentadimer 17. Expected yield: 26.1-35.5 g (61-83%).

[0115] The identity of compound 17 was confirmed using a 500 MHz instrument. 1 H and 13 The NMR spectra were determined by C NMR. A standard solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the standard solution was added to the sample. The final test sample composition was 10 mg / mL pentadimer and 5 mg / mL t-butanol. 1 H and 13 C spectra were acquired and integrated, and the resulting chemical shifts were assigned by comparison with theoretical shifts.1 H NMR and 13 The C NMR spectra are shown in Figures 1 and 2, respectively.

[0116] Example 5 - Conversion of crude pentadimer to the free base form A large column was loaded with Amberlite FPA91 (1.46 kg; 40 g / g crude pentadimer—corrected for potency). 8 L of 1.0 M NaOH solution was prepared by adding NaOH (320 g) to LE water (8.00 kg) in a 10 L Schott bottle. This solution was passed through the Amberlite resin over 1 hour. LE water (40.0 kg) was passed through the Amberlite resin. The resin was flushed with additional LE water (approximately 10 kg aliquots) until a pH of <8.0 was achieved in the flow-through. Crude pentadimer (49 g, PN0704), stored in a Lyoguard tray, was warmed to ambient temperature. LE water (400 g) was added to the Lyoguard tray containing the crude pentadimer (49 g) and, after complete dissolution, transferred to a 1 L Schott bottle. The tray was rinsed with additional LE water (200 g), and these rinses were added to the contents of the Schott bottle. The crude pentadimer solution was carefully poured onto the top of the resin. The 1 L Schott bottle was rinsed with LE water (200 g) and loaded onto the resin. The Amberlite tap was opened, and the crude pentadimer solution was allowed to slowly transfer onto the resin over approximately 5 minutes. The tap was stopped, and the material was allowed to sit on the resin for approximately 10 minutes. LE water was poured onto the top of the resin. The tap was opened, and elution was performed with LE water, collecting approximately 16 500 mL fractions. Each fraction was analyzed by TLC charring (10% H2SO4 in EtOH). All carbohydrate-containing fractions were combined and filtered through a Millipore filter with a 0.2 μm nylon filter membrane. The solution was divided equally among 5-6 Lyoguard trays. The filter vessel was rinsed with LE water (100 g) and divided among the trays. The material was lyophilized in the trays. The shelf temperature was set at -10°C for 16-20 hours, then set to +10°C until the material was dry. LE water (150g) was added to all but one Lyoguard tray and transferred to the remaining tray containing the dried material. Each empty tray was rinsed with additional LE water (100g) and this rinse volume was added to the final Lyoguard tray. The final Lyoguard tray was freeze-dried. The shelf temperature was set at -10°C for 16-20 hours, then set to +10°C until the material was dry.The product was sampled, analyzed, and retained. The dried material was transferred to HDPE or PP containers and stored at or below -15°C. Expected yield: 31-34g (86-94%).

[0117] TCEP reduction of the disulfide bond in the dimer is rapid and nearly stoichiometric. Using stoichiometric reduction with TCEP, approximately two equivalents of glucosamine pentasaccharide monomer were obtained. Specifically, the pentasaccharide dimer was dissolved in a reaction buffer (50 mM HEPES buffer, pH 8.0) containing one molar equivalent of TCEP. After 1 h at ambient temperature, the reaction was analyzed by HPLC with CAD detection. Under these conditions, the conversion to the pentaglucosamine monomer (peak at approximately 10 min) was nearly complete (pentaglucosamine dimer peak at approximately 11.5 min)—see Figure 4. The remaining unannotated peaks are due to the sample matrix. Based on the equilibrium equation, the added TCEP was largely converted to TCEP oxide, and the residual TCEP inhibited air oxidation back to the dimer before addition to the conjugation reaction. For simplicity, glucosamine pentasaccharide can be added based on the input dimer, assuming >95% conversion to monomer under these conditions.

[0118] The identity of the pentadimer was confirmed using a 500 MHz instrument. 1 H and 13 The solubility of t-butanol was determined by C NMR. A standard solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the standard solution was added to the samples. The final test sample composition was 10 mg / mL pentadimer and 5 mg / mL t-butanol. 1 H and 13 C spectra were acquired and integrated, and the resulting chemical shifts were assigned by comparison with theoretical shifts. 1 H and 13 The C NMR spectra are shown in Figures 1 and 2, respectively.

[0119] Example 5 - Conversion of Example 4 to the pentasaccharide monomer with the TT linker of Example 2 provided for the vaccine of the present invention (compound 18) The TT monomer linker intermediate from Example 2 was reacted with increasing concentrations of 4 to 70 pentameric glucosamine molar equivalents (2 to 35 pentasaccharide dimer molar equivalents) for 4 hours at ambient temperature. Crude conjugates from each titration point were purified by partitioning through a 30 kDa MWCO membrane. Each purified conjugate sample was analyzed for protein content, payload density by SEC-MALS, and monomer / aggregate content by SEC-HPLC. The data showed saturation of payload density above 50 pentameric glucosamine equivalents. Based on SEC-HPLC analysis, aggregate content increased as the charge of the pentasaccharide monomer increased, appearing to reach a steady-state level of approximately 4% increase starting at 30 pentameric glucosamine equivalents. Based on these results, the charge of the pentasaccharide dimer selected for the next conjugation reaction was 25 molar equivalents, corresponding to 50 molar equivalents of the theoretical charge of pentameric glucosamine.

[0120] A series of three trial syntheses following GMP synthesis of compound 18 were prepared as described above. Each of the resulting products was evaluated for potency (by ELISA assay) and payload density (molar ratio of pentameric glucosamine to tetanus toxoid).

[0121] The table below provides the results. [Table 5]

[0122] The foregoing description is set forth merely to illustrate the present invention and is not meant to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the present invention may occur to those skilled in the art, the present invention should be broadly construed to include all modifications within the scope of the claims and their equivalents.

Claims

1. Formula I: (A-B) x -C I wherein A is a group represented by the formula: 【Chemical 1】 or mixtures thereof; B is a compound of the formula: 【Chemistry 2】 (where the left side of the formula binds to C and the right side binds to A); C is a tetanus toxoid having at least 31 reactive amino functions; x is an integer from about 31 to about 39; y is an integer from 1 to 10; and R is hydrogen or acetyl; provided that not more than 40% of the R groups are acetyl; wherein the tetanus toxoid contains at least 31 reactive amino groups and at least 90% by number of the toxoid is in monomeric form. A compound represented by the formula:

2. A, 【Chemistry 3】 2. The compound of claim 1, wherein:

3. Formula II: (A–B) x -C II wherein A' is 【Chemistry 4】 and B is a group of formula: 【Chemistry 5】 (where the left side of the formula binds to C and the right side binds to A); C is a tetanus toxoid having at least 31 reactive amino functions; x is an integer from about 31 to about 39; y is an integer from 1 to 10; and R is hydrogen or acetyl; provided that not more than 40% of the R groups are acetyl; wherein the tetanus toxoid contains at least 31 reactive amino groups and at least 85% by number of the toxoid is in monomeric form. A compound represented by the formula:

4. 4. The compound of any one of claims 1 to 3, wherein the amount of non-monomeric toxoid is less than about 5% by weight.

5. 4. The compound of any one of claims 1 to 3, wherein the amount of non-monomeric toxoid is less than about 0.5% by weight.

6. 10. A pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of the compound of claim 1.

7. 10. A pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of the compound of claim 2, wherein the composition contains 3% by weight or less of a low molecular weight amino compound.

8. 10. A pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of the compound of claim 3, wherein the composition contains 3% by weight or less of a low molecular weight amino compound.

9. 9. The pharmaceutical composition according to any one of claims 6 to 8, comprising less than 2% by weight of low molecular weight amino compounds.

10. 10. The pharmaceutical composition of claim 9, comprising less than 1% by weight of low molecular weight amino compounds.

11. 5. The pharmaceutical composition of claim 4, wherein the effective amount of the compound of claim 1 is sufficient to kill microorganisms in vivo when the patient has an effective white blood cell (WBC) count of at least about 2,000.

12. The compound is 【Chemistry 6】 【Table 1】 6. The pharmaceutical composition of claim 5, selected from the group consisting of:

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