Methods for providing sustained treatment against microorganisms comprising pnag

Abstract

To provide a method for providing sustained immune protection against PNAG microorganisms.SOLUTION: A combination of an antibacterial vaccine containing an oligosaccharide β - (1 → 6) - glucosamine group and a monoclonal antibody is utilized, the monoclonal antibody targets PNAG, the PNAG vaccine generates an endogenous immune response while performing immediate treatment to PNAG microorganisms, and the immune response generated by the vaccine complements the action of the monoclonal antibody. These combinations provide methods that allow for sustained therapy against PNAG microorganisms from the start of treatment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 939,331, filed November 22, 2019, and U.S. Provisional Patent Application No. 62 / 994,130, filed March 24, 2020, which are incorporated herein by reference in their entireties.

[0002] The present invention relates to methods for providing sustained therapy against microorganisms, including PNAG. In particular, these methods utilize a combination of a PNAG vaccine and a monoclonal antibody. The monoclonal antibody targets PNAG and provides immediate therapy against such microorganisms, while the PNAG vaccine generates an endogenous immune response, and once effective, the immune response generated by the vaccine complements the monoclonal antibody to the extent that it provides an additional means of therapy beyond that provided by the antibody. This combination allows for sustained therapy from the start of treatment.

[0003] The art has previously disclosed antibacterial vaccines comprising oligosaccharide β-(1→6)-glucosamine groups, where the number of repeating glucosamine units ranges from 1 up to 300. One such example is provided in U.S. Provisional Patent Application No. 62 / 892,400, which is pending conversion to a standard patent application and is incorporated herein by reference in its entirety.

[0004] Data generated to date indicate that these vaccines confer protective immunity against microorganisms containing such oligosaccharide β-(1→6)-glucosamine structures, including their N-acetylated versions, in their cell walls. However, effective immunity in treated patients begins approximately four weeks or more after vaccination. During this incubation period, patients are at risk for microbial infection. This is particularly troublesome for patients who have already experienced or are at significant risk of developing a microbial infection during this incubation period.

[0005] To treat patients requiring immediate protection against microbial infections, monoclonal antibodies have been developed that target microorganisms whose cell walls contain oligosaccharide N-acetyl-β-(1→6)-glucosamine structures. These monoclonal antibodies have demonstrated efficacy against such microorganisms and provide immediate antibacterial protection after injection. One such monoclonal antibody is F-598, disclosed in U.S. Patent No. 7,786,255, which is incorporated herein by reference in its entirety. The antibody is recognized to bind to several N-acetylglucosamine groups of PNAG. The efficacy provided by a single administration of this monoclonal antibody typically lasts for up to about four weeks after injection.

[0006] However, there are problems in treating patients who require immediate and long-term immune protection, especially those experiencing or at risk of microbial infection. These include elderly patients, burn patients, premature infants, patients undergoing chemotherapy or radiation therapy, and other related conditions. However, there is concern that if a doctor administers a vaccine during the period of active protection with a monoclonal antibody, there is a risk that at least a portion of the monoclonal antibody will cross-react with the oligosaccharide structure of the vaccine, reducing or negating the effectiveness of both the vaccine and the monoclonal antibody.

[0007] Therefore, to avoid this problem, it is necessary to ensure that patients no longer have active immunity due to the presence of monoclonal antibodies before administering the vaccine. Furthermore, due to the inherent delay in achieving effective immunity after vaccination, a significant incubation period is required to switch patients from monoclonal antibody therapy to the immune protection provided by vaccination, which then places them at risk of infection or subjects them to alternative, potentially less effective, treatment methods. Because the natural immunity resulting from vaccination is more durable than the immunity provided by monoclonal antibodies, the benefits of such natural immunity greatly favor vaccination.

[0008] Thus, there is a continuing need to provide patients with sustained immune protection using both monoclonal antibodies and vaccination. Summary of the Invention

[0009] The present invention is based on the discovery that monoclonal antibody F-598 can function as a complementary therapy to the vaccines disclosed herein for the treatment of PNAG-type microorganisms. Accordingly, the present invention relates to a method for providing sustained immune protection against PNAG-type microorganisms by co-administering an oligosaccharide β-(1→6)-glucosamine vaccine and the F-598 monoclonal antibody. In one aspect, the vaccine relates to a specific class of tetra-, penta-, and hexa-β-(1→6)-glucosamine-linked tetanus toxoid vaccines, which provide effective immunity to patients against microbial infections, where the microorganisms contain PNAG structures in their cell walls.

[0010] Surprisingly, these vaccines generate an endogenous immune response, but the oligosaccharide β-(1→6)-glucosamine groups of the vaccines do not appreciably cross-react with the F-598 antibody. This surprising result allows for the simultaneous administration of both the vaccine and the antibody. Such simultaneous administration further enables clinicians to provide patients with continuous complementary immune protection. In some embodiments, the complementary immune protection is synergistic.

[0011] Thus, in one embodiment, the present invention provides a method for providing sustained immune protection against PNAG microorganisms by using a vaccine comprising a β-(1→6)-glucosamine oligosaccharide-linked tetanus toxoid vaccine that provides effective immunity to a patient against microbial infection, wherein the microorganism contains β-(1→6)-glucosamine structures in its cell wall. In one embodiment, antibodies to the vaccine bind to the β-(1→6)-glucosamine structures. In some embodiments, the vaccine does not cross-react with F-598 monoclonal antibody, and further, the oligosaccharide contains 3 to 12 β-(1→6)-glucosamine units. In some embodiments, the vaccine generates antibodies complementary to F-598. Here, the vaccine disclosed herein selectively binds to β-(1→6)-glucosamine structures, and F-598 selectively binds to acetylated β-(1→6)-glucosamine structures, i.e., N-acetylglucosamine.

[0012] In one embodiment, the present invention provides a vaccine against a microorganism that comprises an oligosaccharide β-(1→6)-glucosamine structure in its cell wall, wherein said vaccine comprises a polypeptide of formula I: (AB) x -CI wherein A contains 3 to 12 β-(1→6)-glucosamine (carbohydrate ligand) groups or mixtures thereof, and the oligosaccharide portion of the vaccine has the following formula:

[0013] [ka] and B is a compound of the formula:

[0014] [ka] and wherein A is as defined above and C is tetanus toxoid; x is an integer from about 30 to about 39; y is an integer between 1 and 10) It is expressed as:

[0015] In one embodiment, the present invention provides a vaccine against a microorganism that contains an oligosaccharide β-(1→6)-glucosamine structure in its cell wall, wherein said vaccine comprises a polypeptide of formula II: (A'-B) x -C II wherein A′ is a penta-β-(1→6)-glucosamine (carbohydrate ligand) group of the formula:

[0016] [ka] B, C, and x are as defined above) It is expressed as:

[0017] In one embodiment, the present invention provides a pharmaceutical composition comprising a pharmaceutically acceptable diluent and an effective amount of a vaccine of Formula I and / or Formula II.

[0018] In one embodiment, the present invention provides a method for providing immunity to a patient from a microorganism that contains oligosaccharide β-(1→6)-glucosamine groups in its cell wall, comprising administering to the patient the vaccine of Formula I and / or Formula II.

[0019] In one embodiment, the present invention provides a method for providing effective immunity to a patient from microorganisms that contain oligosaccharide β-(1→6)-glucosamine groups in their cell walls, comprising administering to said patient a pharmaceutical composition of the present invention.

[0020] Representative vaccines of the present invention are shown in the table below.

[0021] [ka]

[0022] [Table 1]

[0023] In embodiments, the present invention provides a method for providing immunity to a patient from a microorganism that contains oligosaccharide β-(1→6)-glucosamine groups in its cell wall, comprising administering to said patient the vaccine of Formula I and / or Formula II simultaneously with monoclonal antibody F-598.

[0024] As used herein, "concurrently" can include before or during administration of the vaccine. In some embodiments, simultaneous can include administration of the Formula I and / or Formula II vaccine within about ±6 hours, ±4 hours, or ±2 hours of administration of F-598. In embodiments, the two can be administered as part of the same bolus injection. Administration is "concurrent" as long as the patient is able to mount an immune response based on the individual components. The order in which F-598 and the Formula I or II vaccine are administered is not important. Simultaneous administration can correspond to any period other than 2 or 6 hours, but can be simultaneous as long as both sets of antibodies (those from F-598 and those generated from the vaccine) are effectively providing antibody coverage against their respective targets for the overlapping period.

[0025] Without being bound by theory, the methods disclosed herein are complementary and synergistic due to the respective selectivities of the F-598 antibody and the antibodies generated from the vaccines of Formulas I and II. F-598 has been found to specifically bind to the N-acetyl-rich region of the PNAG structure in microbial cell walls, as described in "Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetyl glucosamine," J. Biol. Chem. 293(14) 5079-5089 (2018), incorporated herein by reference in its entirety. The vaccines of Formulas I and II provide selectivity for the non-N-acetylated region of the PNAG cell wall structure. In some embodiments, the presence of both antibody populations can minimize cross-reactivity and provide complete protection against microorganisms with cell wall structures containing PNAG.

[0026] In some embodiments, F-598 is co-administered during the entire treatment period.

[0027] In some embodiments, F-598 is co-administered only until the time that the vaccine of Formula I and / or Formula II produces sufficient antibody titers to effectively treat the patient, after which time administration of F-598 may be terminated.

[0028] In some embodiments, F-598 may be terminated immediately after a sufficient titer of antibodies produced by the vaccine is measured. In embodiments, F-598 may be terminated one week after a sufficient titer of antibodies produced by the vaccine is measured. In embodiments, F-598 may be terminated two weeks after a sufficient titer of antibodies produced by the vaccine is measured. In embodiments, F-598 may be terminated one month after a sufficient titer of antibodies produced by the vaccine is measured. Those skilled in the art will understand the exact time period, which may be determined by the patient's particular condition / condition.

[0029] In some embodiments, administration of the vaccine of Formula I and / or Formula II may include a regimen of one to three doses. For example, in some patients, one dose may be sufficient. In some patients, two doses may be required. In some patients, three doses may be required. Factors that may contribute to the number of doses include the age and condition of the patient. Very young patients with newly formed immune systems may require multiple doses. Similarly, older patients with weakened immune systems may require multiple doses.

[0030] In some embodiments, the treatment regimen includes monitoring the patient for depletion of F-598 and / or the need for additional doses of vaccine based on antibody titers. For example, a burn victim may require additional doses of F-598 due to secretion of antibodies at the wound site. Thus, in some embodiments, serum antibody concentrations are assessed periodically throughout the treatment regimen to maintain adequate titers. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 shows the 1H NMR of compound 17 (described below). [Figure 2] FIG. 2 shows the 13C NMR of compound 17. DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention provides an antibacterial vaccine comprising an oligosaccharide β-(1→6)-glucosamine group having 3 to 12 glucosamine units linked to an immunogenic protein.

[0033] Before describing the present invention in more detail, the following terms will first be defined. 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] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes instances in which the event or circumstance occurs and instances in which it does not occur.

[0036] When the term "about" is used before a numerical designation, for example, temperature, time, amount, concentration, and others including ranges, it indicates an approximation that may vary by (+) or (-) 10%, 5%, 1%, or any subrange or subvalue therebetween. Preferably, the term "about" when used in reference to a dose means that the dose may vary by + / - 10%.

[0037] "Comprising" or "comprises" is intended to mean that the compositions and methods include the recited elements, but do not exclude others. When used to define compositions and methods, "consisting essentially of" 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 characteristics of the claimed invention. "Consisting of" shall mean excluding other components beyond trace elements and substantial method steps. Embodiments defined by each of these transitional phrases are within the scope of the present invention.

[0038] The term "β-(1→6)-glucosamine unit" or "glucosamine unit" refers to an individual glucosamine structure such as:

[0039] [ka] (where the 6-hydroxyl group is condensed with the 1-hydroxyl group of the preceding glucosamine unit, and the dashed lines represent the attachment sites to the preceding and following glucosamine units.) When combined with another "β-(1→6)-glucosamine unit," the resulting disaccharide structure is as follows:

[0040] [ka]

[0041] The term "β-(1→6)-glucosamine unit with an N-acetyl group" refers to the following structure:

[0042] [ka] (where the 6-hydroxyl group of the second unit is condensed with the 1-hydroxyl group of the preceding glucosamine unit, as shown above, despite the absence of an N-acetyl group).

[0043] As used herein, the term "linker" refers to any organic fragment that serves as a means for covalently linking tetanus toxoid to the oligosaccharide domains disclosed herein. While any suitable linker known to those skilled in the art can be used, such a linker is generally selected so that it is not readily cleavable and separates the oligosaccharide from its attachment to the toxoid structure. For example, the linker can be one of the linkers disclosed in U.S. Patent Nos. 4,671,958; 4,867,973; 5,691,154; 5,846,728; 6,472,506; 6,541,669; 7,141,676; 7,176,185; or 7,232,805, each of which is incorporated herein by reference. Linkers generally comprise a C2-C6 heteroatom with any number of intervening heteroatoms, particularly nitrogen, sulfur, and oxygen. 20The linker may include an alkylene fragment. Carbon atoms may be substituted with alkyl, oxo, etc. At the reducing end of the oligosaccharide, the linker can be attached via an N-, O-, or S-linkage at the anomeric center, although a C-linkage is also possible. At the toxoid terminus, the linker can be linked to a heteroatom on the toxoid. In some embodiments, the linkage is via an amine functionality on the toxoid. In some such embodiments, the linker is attached by forming an amide bond to an amino group on the toxoid. The intervening atoms between the terminal oligosaccharide attachment point and the terminal toxoid attachment point may be beneficial to have a structure that does not interfere with the antigenicity of the oligosaccharide, but are generally of little importance. In some embodiments, the linker may also be branched, allowing for the attachment of two or more oligosaccharides per unit amino group on the toxoid via the linker.

[0044] The term "oligosaccharides containing a β-(1→6)-glucosamine group" refers to that group on the vaccine that mimics a portion of a cell wall that contains an oligosaccharide containing a "β-(1→6)-glucosamine structure" (as defined below).

[0045] The term "oligosaccharides containing β-(1→6)-glucosamine structures" refers to structures found in microbial cell walls. Microbial walls contain many of these structures that are conserved across many microbial lineages. These structures are found in microbial cell walls and contain oligosaccharides in which the majority of their units are β-(1→6)-glucosamine.

[0046] As used herein, the term "vaccine" refers to the ability of the compounds of the present invention (Formulas I and II) to provide effective immunity against any microorganism containing oligosaccharides with β-(1→6)-glucosamine structures in their cell walls. Therefore, unlike classical vaccines that vaccinate against a single bacterium, the vaccines described herein can provide 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, provided that such microorganisms contain such oligosaccharides containing β-(1→6)-glucosamine structures.

[0047] As used herein, the term "effective immunity" refers to the ability of an effective amount of a vaccine to generate an in vivo antibody response sufficient to treat, prevent, or ameliorate a microbial infection, wherein the microorganism contains oligosaccharides containing β-(1→6)-glucosamine in its cell wall. Assays for assessing antibody responses are conventional in the art and include assays to assess the titer of antibodies responding to the microorganism.

[0048] The vaccines and intermediates ("compounds") of the present invention 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 DMF, ether, and alcohol solvates, among others. The identification and preparation of specific solvates is within the skill of one of ordinary skill in the art of synthetic organic or medicinal chemistry.

[0049] "Subject" refers to a mammal. The mammal may be a human or a non-human animal mammal.

[0050] "Treating" or "treatment" of a disease or disorder in a subject refers to 1) preventing the disease or disorder from occurring in a subject who is predisposed 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.

[0051] An "effective amount" refers to an amount of a vaccine of the present invention sufficient to treat a disease or disorder afflicting a subject or to prevent such disease or disorder from occurring in said subject or patient.

[0052] The term "sustained immune protection" means that the patient has therapeutic titers of antibodies in their serum, whether those titers include F-598 antibodies, vaccine-generated polyclonal antibodies alone, or a combination of both.

[0053] 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 temperature, time, molar ratio of reactants, solvent, pressure, 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, although such conditions can be determined by one skilled in the art by routine optimization procedures.

[0054] 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.Proper protecting groups for various functional groups, as well as suitable conditions for protecting and deprotecting specific functional groups, are well known in the art.For example, many protecting groups are described in TW Greene and PGM Wuts, Protecting Groups in Organic Synthesis, Third Edition, Wiley, New York, 1999, and the references cited therein.

[0055] 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 SigmaAldrich (St. Louis, Missouri, USA), Bachem (Torrance, California, USA), and Emka-Chemce (St. Louis, Missouri, USA). Others are described in 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 Organic synthesis compounds may also be prepared by procedures described in standard reference texts such as The American Journal of Biological Chemistry (Eds. Edition, 2001), and Larock's Comprehensive Organic Transformations (VCH Publishers Inc., 1989), or obvious modifications thereof.

[0056] Synthesis of Representative Compounds of the Invention The general synthesis of the vaccines of the present invention is known in the art and is disclosed in U.S. Patent Application 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. As disclosed in co-pending U.S. patent application Ser. No. 62 / 934,925, entitled "Low-Contamination Antibacterial Vaccines," which is incorporated herein by reference in its entirety, the toxoid itself can be purified to contain low levels of contaminants by stepwise filtration before conjugating oligosaccharides to the toxoid. Briefly, the toxoid is first purified by stepwise filtration to remove toxoids that are higher in oligomer order than the dimer toxoid. The monomers and dimers pass through the filtrate. The low molecular weight impurities are then separated by a small filter that isolates the monomers and dimer toxoids, allowing the small molecular weight impurities to pass through with the filtrate. In this manner, good yields of conjugate vaccines containing primarily monomers and dimer toxoids can be prepared.

[0057] For certain vaccines described herein, the β-(1→6)-glucosamine group is limited to 4-6 units, preferably 5 units. Formation of the linker group is accomplished by art-recognized synthetic techniques, exemplified, but not limited to, by those found in U.S. Pat. No. 8,492,364 and the Examples below. In one embodiment, the first portion of the aglycone is attached to a reducing β-(1→6)-glucosamine unit and has the following formula III:

[0058] [ka] (wherein y is an integer from 2 to 4) As shown in Figure 1, it carries a thiol (-SH) group.

[0059] The second portion of the linker, as shown in Formula IV, is attached to the tetanus toxoid in the following manner:

[0060] [ka]

[0061] In this formula, the separate moieties of tetanus toxoid are shown with wavy lines and are merely illustrative in nature and are not intended to provide the complete structure of the toxoid. The disulfide bridge is represented by a single line connecting the moieties. For clarity, only a single second moiety of the linker is shown, although there are multiple such second moieties covalently attached to amino groups found on the toxoid.

[0062] 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 oligosaccharide β-(1→6)-glucosamine group of the tetanus toxoid via the combined linker, as shown below for a vaccine structure where y is defined herein:

[0063] [ka]

[0064] It is understood that the number of β-(1→6)-glucosamine group-linker-moieties attached to tetanus toxoid is stoichiometrically controlled so that the desired amount of such moieties is attached to the toxoid, thereby providing the vaccine of the present invention.

[0065] Methods, Uses and Pharmaceutical Compositions The vaccines used in the combinations of the present invention can mount an effective immune response against microorganisms that have PNAG oligosaccharide β-(1→6)-glucosamine structures in their cell walls, with up to about 20% of the oligosaccharides being N-deacetylated. An effective immune response occurs approximately four weeks after inoculation into a patient. This creates a latency period during which the vaccine becomes ineffective prophylactically or therapeutically. When the vaccine is administered prophylactically and the latency period is acceptable, the vaccines of the present invention are useful for preventing subsequent microbial infections in which the microorganism in question has a cell wall that contains PNAG.

[0066] When used in this manner, the vaccine of the present invention is administered to patients at risk of microbial infection caused by such microorganisms. Such patients include, by way of example only, elderly people, burn patients, particularly those with burns covering 20% ​​or more of the body, patients undergoing elective surgery, and patients traveling to destinations where microbial infections are prevalent. The vaccine is typically administered intramuscularly to immunocompetent patients with a suitable adjuvant to enhance the immune response. After an incubation period, patients acquire natural immunity against such microorganisms.

[0067] In another embodiment, the vaccine of the present invention can be used therapeutically, especially when the microbial infection is localized and / or not life-threatening.In such cases, the vaccine of the present invention is administered to patients suffering from microbial infection caused by such microorganisms.The vaccine is usually administered intramuscularly to immunocompetent patients together with a suitable adjuvant to enhance immune response.Once administered, effective immunity is generated within about 4 weeks.If the patient is still infected, the natural immunity generated by the vaccine promotes recovery.

[0068] Clearly, it would be beneficial to combine antibacterial therapy with a vaccine, particularly for antibiotic-resistant infections. This would allow immediate therapeutic treatment of the patient's infection, rather than waiting for a latent period. Monoclonal antibodies generated against PNAG are known to be therapeutically effective. One such example is the monoclonal antibody designated F-598 and disclosed in U.S. Patent No. 7,786,255, which is incorporated herein by reference in its entirety.

[0069] The use of such monoclonal antibodies with the vaccines described herein presents a problem in that the monoclonal antibodies are designed to bind to PNAG, and therefore, when administered with the vaccine, the antibody will bind to the polyglucosamine portion of the vaccine, rendering both ineffective.

[0070] Surprisingly, the vaccine described herein induces an endogenous immune response in patients without cross-reacting with the F-598 monoclonal antibody. This combination allows for simultaneous administration of the vaccine and the F-598 antibody, allowing for immediate antibody-only treatment during the incubation period followed by endogenous antibody production after the incubation period. This allows for treatment of patients with the F-598 monoclonal antibody during the incubation period between administration of the vaccine and the development of effective immunity. In this embodiment, therapeutic treatment of patients suffering from infections mediated by microorganisms expressing PNAG in their cell walls can be immediately initiated with the antibody, while simultaneously administering the vaccine to the patient to develop innate immunity against the microorganism. For completeness, innate immunity refers to the immune response to an antigen, which generates antibodies that, alone or in combination with other components of the immune system, kill the microorganism in question.

[0071] When so used, the vaccines of the present invention are administered in therapeutically effective amounts by any of the accepted modes of administration for agents that serve similar utilities. The actual amount of the vaccine of the present invention, i.e., the active ingredient, will depend on many factors, including the severity of the disease being treated, the age and relative health of the subject, the potency of the vaccine used, the route and form of administration, and other factors well known to those skilled in the art.

[0072] An effective or therapeutically effective amount of a vaccine of the present invention refers to that amount of vaccine that produces antibodies of sufficient titer to ameliorate symptoms or prolong survival in a subject. The toxicity and therapeutic efficacy of such vaccines can be determined by standard pharmaceutical procedures in cell cultures or experimental animals.

[0073] The vaccines 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.

[0074] Similarly, the F-598 monoclonal antibody will be administered in a therapeutically effective amount by any of the accepted modes of administration for agents that serve similar utilities. The actual amount of antibody will depend on many factors, including the severity of the disease being treated, the age and relative health of the subject, the route and form of administration, and other factors well known to those skilled in the art.

[0075] An effective or therapeutically effective amount of a vaccine of the present invention refers to that amount of antibody that results in a sufficient titer of antibody to ameliorate symptoms or prolong survival of a subject. The antibody is preferably administered intravenously as an injectable sterile aqueous composition containing one or more conventional ingredients well known in the art, including, by way of example only, preservatives, etc.

[0076] In some embodiments, the patient being treated is a burn patient. Such patients are known to exude fluids from their burns, which contain antibodies. Therefore, over time, the titer of antibodies, particularly F-598, decreases, resulting in a suboptimal antibody concentration in the patient. In such cases, it is preferable to monitor the patient's antibody titer against F-598 and adjust it as needed by administering F-598 periodically or continuously.

[0077] In embodiments, a method for treating a patient at risk of developing a biofilm is provided, the method comprising administering to the patient a combination of a vaccine disclosed herein together with an F-598 antibody. In embodiments, the method can include identifying a patient at risk of developing a biofilm. Such patient populations include, but are not limited to, patients who have undergone certain surgical implants, such as knee or hip replacements, stents, or catheters.

[0078] In embodiments, a method for treating a patient at risk of developing a biofilm may include administering an F-598 antibody before any surgery. In some such embodiments, administration may occur at least 24 hours before surgery, or at least 72 hours before surgery, or at least one week before surgery, or at least two weeks before surgery. If the patient is undergoing emergency surgery, the F-598 antibody can be administered immediately before or during surgery. In treating a patient at risk of developing a biofilm, a PNAG vaccine can be administered simultaneously with or sequentially to the F-598 antibody. If administered sequentially, the PNAG vaccine is preferably administered within 24 hours of the administration of the F-598 antibody.

[0079] combination The combination of the present invention can be used in combination with other therapeutic compounds or other appropriate drugs as deemed appropriate by the attending physician. In selected cases, the combination of the present invention can be administered simultaneously with antibiotics, antifungal agents, etc. to treat bacterial infections. In the case of antibiotics, the selection of the appropriate antibiotic or antibiotic cocktail and the amount to administer to the patient are well within the skill of the attending physician, based on the specifics of the bacteria in question, the severity of the bacterial infection, the age, weight, and other relative health conditions of the patient. In the case of antifungal therapy, an effective amount of an antifungal drug can be administered simultaneously to the patient.

[0080] The vaccines of the present invention can be administered with an antigen to enhance the patient's immune response to the antigen. Adjuvants include, but are not limited to, gels, aluminum compounds such as aluminum hydroxide and aluminum phosphate, and Freund's complete or incomplete adjuvants (e.g., antigens incorporated into the stabilized water phase of a 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]

[0081] The present invention will be further understood by reference to the following examples of the invention, which are intended to be purely illustrative. 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 present invention. Various modifications of the present invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications fall within the scope of the appended claims.

[0082] The following terms are used herein and have the following meanings: If not defined, abbreviations have their conventionally recognized definitions. Å = angstrom aq.=aqueous solution Biotage=Biotage, Div. Dyax Corp., Charlottesville, Virginia, USA bp=boiling point CAD = Charged Aerosol Detector DCM = dichloromethane deg = degrees DMSO = dimethyl sulfoxide eq.=equivalent EtOAc = ethyl acetate FEP = Fluorinated Ethylene Propylene g = grams H 1 -NMR = proton nuclear magnetic resonance h=time HDPE = High Density Polyethylene HPLC = High-Performance Liquid Chromatography MeCN = acetonitrile kg = kilograms mbar = millibar MeOH = methanol mg = milligram mL = milliliters mM = millimolar concentration mmol = millimolar N=normality NBS = N-bromosuccinimide NIS = N-iodosuccinimide NMT = N-methyltryptamine PP = Polypropylene qHNMR = quantitative proton nuclear magnetic resonance RBF = round bottom flask RO = Reverse Osmosis SEC HPLC = Size Exclusion Chromatography HPLC SIM = Secondary Ion Mass TCEP = Tris(2-carboxyethyl)phosphine TLC = thin layer chromatography TMSOTf = methanesulfonic acid, 1,1,1-trifluoro-, trimethylsilyl ester TT = tetanus toxoid μL = microliter μm = microns w / w = weight to weight w / v = weight to volume [Example 1]

[0083] Tetanus Toxoid Stepwise Filtration A sample of crude tetanus toxoid preparation containing monomeric and dimeric toxoids is first passed through a 3- to 5-micron filter to remove higher-order oligomers. This can be done by gradually decreasing the filter pore size. Thus, the toxoid preparation can be passed through a 5-micron filter, followed by a 3-micron filter. Alternatively, the toxoid preparation can be passed through a 5-micron filter, followed by a 4-micron filter, followed by a 3-micron filter. The effectiveness of the 5-micron filtration is assessed by light scattering techniques, which can be used to detect the presence of higher-order oligomers. If necessary, additional stepwise filtrations can be performed to further remove higher-order oligomers. The resulting filtrate contains monomeric and dimeric toxoids. After the oligosaccharide attachment chemistry is fully purified, the filtrate is passed through a 2.5-micron filter, separating the monomeric and dimeric toxoids as a filter cake, while low-molecular-weight impurities pass through with the filtrate. After each filtration step (high and low molecular weight), a filter cake rinse can be performed.

[0084] In one embodiment, prior to attaching the oligosaccharide β-(1→6)-glucosamine structures to the toxoid, the toxoid can be prepared to contain primarily monomers and dimers and less than 3% small molecular weight impurities. See U.S. Provisional Patent Application No. 62 / 934,925, which is incorporated herein by reference in its entirety. [Example 2]

[0085] Attachment of SBAP to TT monomers Step 1: Preparation of N-BABA:

[0086] [ka] [1] Commercially available beta-alanine, i.e., compound 1, is converted to N-BABA (bromoacetyl-β-alanine), i.e., compound 2, by reacting it with at least a stoichiometric amount of commercially available bromoacetyl bromide. In a first vessel, β-alanine is combined with water along with sodium bicarbonate or other suitable base to remove the acid generated during the reaction. The aqueous solution is mixed 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 approximately 2. The resulting N-BABA is extracted from the solution with a suitable solvent, such as ethyl acetate. The organic layer is concentrated under conventional conditions, such as under reduced pressure at an elevated temperature, e.g., 60°C. Heptane is then added to precipitate the N-BABA, which is collected on a filter and dried in a vacuum oven at 40°C. This product is used directly in the next step.

[0087] Step 2: Preparation of SBAP:

[0088] [ka] N-BABA, i.e., compound 2, is reacted with N-hydroxysuccinimide (NHS) under conventional conditions known in the art to produce SBAP, i.e., 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 to form a solid. 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 TT monomer.

[0089] Alternatively, SBAP can be prepared in the manner described in U.S. Patent No. 5,286,846, which is incorporated herein by reference in its entirety. Specifically, the method described therein is provided by the following synthetic scheme:

[0090] [ka]

[0091] Step 3: Conjugation As described above, purified TT monomer contains 43 lysine residues / mol, as quantified by free amine assay. Reaction of TT monomer with increasing concentrations of SBAP, from 0 to 170 molar equivalents, resulted in a corresponding decrease in free amine content over the range of 15–110 molar equivalents of SBAP. Steady-state conversion was achieved by loading >110 equivalents of SBAP. Assuming that the loss of free amines is directly proportional to the loading of the SBAP linker, the linker density at saturation was estimated to be 43 moles of SBAP / TT monomer. The monomer / aggregate content of the linker-TT / monomer intermediate and the protein concentration at each titration point were also assessed. The monomer content before linker addition was 99.7%, and increasing the amount of SBAP linker did not significantly change the monomer level (no aggregates were detected). Protein recovery throughout the titration process was also similar. 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 condition for all subsequent syntheses. [Example 3]

[0092] Oligosaccharide synthesis Building Block Synthesis The following reaction schemes illustrate the synthetic steps used to prepare compounds 3, 5 and 8, which are detailed below.

[0093] [ka]

[0094] Synthesis of compound D. Commercially available 1,3,4,6-tetra-O-acetyl-2-deoxy-2-N-phthalimido-β-D-glucopyranoside, Compound C (120.6 g, 252.6 mmol), and toluene (200 mL) were placed in a 1 L Buchi flask and rotated at 40 °C until dissolved (<5 min). The solvent was evaporated to give a foam. Toluene (200 mL) was charged to the flask and rotated 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 rotated around until dissolved; the resulting dark brown solution was charged to a 5 L jacketed reactor, and the flask was rinsed with additional dichloromethane (200 mL) to initiate the reaction. 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 diethyl etherate (50 mL, 390.1 mmol) was dissolved in dichloromethane (50 mL) and added to the reactor, rinsed with dichloromethane (50 mL) and added to the vessel. The mixture was stirred at 20 °C for 2 hours. The reaction was checked for residual C by TLC. 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.

[0095] Stirring was set to high speed and 4 M aqueous sodium acetate (1.25 L, 5100 mmol) was added. The phases were mixed well for 30 minutes. The pH of the aqueous layer was checked with a dipstick and found to be approximately pH=7. Stirring was stopped and the reaction mixture was allowed to stand for 70 minutes.

[0096] The layers were separated and collected. The organic layer (bottom, 1.2 L) and ethanol (840 mL, 14,400 mmol) were charged to the reactor. The jacket was set to 60°C and the solvent was distilled at atmospheric pressure (dichloromethane bp 40°C and ethanethiol bp 35°C, receiving 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, a sample of the vessel contents was taken to determine the ratio of dichloromethane to ethanol. 1 H-NMR confirmed less than 10 mol% dichloromethane. If more dichloromethane was present, further distillation was necessary. 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 give an off-white solid. Expected yield: approximately 86 g (71% from C).

[0097] Synthesis of Compound 1 Anhydrous methanol (33 mL) was charged to a 50 mL round-bottom flask. 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 over 10 minutes (approximately 200 mg) at a rate sufficient to dissolve the solid during the addition. The reaction was stirred at ambient temperature for 2.5 hours. TLC (EtOAc) indicated complete consumption of Compound D (Rf = 0.9) and the formation of one more polar spot: Rf = 0.5. A sample was taken and submitted for HPLC completion IPC (2.5 μL of the reaction mixture in 0.8 mL of acetonitrile and 0.2 mL of water), and the pass condition was NMT 1.00 area % Compound D. Acetic acid was added (8 μL, 0.1397 mmol). 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 the majority of the residue 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), and dried to constant weight on a rotary evaporator (bath temperature 45 °C). Expected yield: 1.94 g (85% from compound D).

[0098] 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, yielding a yellow solution. Additional pyridine (14 mL) was added, and the solution was concentrated to approximately 14 mL in the same manner. The solution was placed under argon, and trityl chloride (2.299 g, 1.36 equiv.) was added. After this, an air-cooled condenser was attached, and the solution was heated to 50 °C with stirring. After 4 h, an IPC was performed (HPLC; 5 μL in 800 μL MeCN, residual compound 1 NMT 3.00 area %). Once the IPC was full, the reaction was cooled to 10–15 °C. Benzoyl chloride (1.60 mL, 2.34 equiv.) was added dropwise over 20 min, 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, an IPC was performed (HPLC; 5 μL in 1500 μL MeCN, total NMT 3.00% by area of ​​residual mono-Bz derivative of compound 1). 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.

[0099] The product mixture was diluted with toluene (20 mL) and stirred at ambient temperature for 1 hour before filtering through a sintered funnel to remove the precipitate. 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 reacted only slightly with any residual citric acid present. The toluene (top) layer 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). The syrup was subjected to IPC (H 1 NMR, acceptable condition NMT 30 wt% residual toluene). Expected yield: about 6.833 g (147%).

[0100] Synthesis of compound 3 Glacial acetic acid (648 mL) and ultrapure water (72 mL) were mixed together to obtain a 90% 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 then heated to 70 °C. Because 2 was viscous, the mixture only fully dissolved after 1 hour and 20 minutes, at which point stirring was initiated. After 2 hours, an IPC was performed (HPLC; 5 μL in 800 μL MeCN, residual compound 2 NMT 3.00 area %). 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 off using house vacuum. The flask was rinsed with additional 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 (bottom) layer was a cloudy white solution and was tested for pH (expected to be <2). Two more water washes were repeated (2 × 700 mL; pH ≈2.4 and ≈3, respectively, clear, colorless solutions). Saturated NaHCO3 (9% w / v, 700 mL) was added to the mixing vessel, resulting in a slight reaction (gas evolution). The toluene (top) layer 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 of 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 stepwise gradient, collecting 1 CV (790 mL) fractions. The gradient used was as follows: 4 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 4 CV) 8 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 12 CV) 15 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 4 CV) 20 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, (4 CV) 30 vol% ethyl acetate:toluene in petroleum ether (1:1 v:v, 1 CV)

[0101] The product eluted over 14 fractions. TLC was used to identify the location of product-containing fractions. All fractions were subjected to IPC (HPLC, NMT 1.50 area % peak at 10.14 min and NMT 1.50 area % peak at 10.94 min). Fractions not satisfying IPC were saved for processing to compound 4. The combined fractions were evaporated on a rotary evaporator at a bath temperature of 45° C. to give a colorless syrup. Expected yield: approximately 60 g (78%).

[0102] Synthesis of compound 4 Crude compound 3 (39.54 g, containing approximately 21 g of compound 3, approximately 37 mmol, collected immediately prior to chromatography) was dissolved in toluene (7.2 mL) and dry pyridine (14.2 mL, 176 mmol, approximately 4.8 equiv.) was added to obtain a homogeneous solution. 7.2 mL of acetic anhydride (76 mmol, approximately 2.1 equiv.) was added, and the mixture was stirred at 25 °C for 18 h. During precipitation of the reaction solid, some of this precipitate was likely compound 4. The reaction was sampled for IPC, and if the amount of compound 3 detected was >1.00 area %, additional dry pyridine (1.4 mL, 17 equiv.) was added and the reaction was continued until the remaining compound 3 was ≤1.00 area % in the liquid phase.

[0103] The reaction was diluted with dichloromethane (112 mL), followed by the addition of water (2.8 mL) and methanol (2.8 mL). The mixture was stirred at 25°C for 3 hours. This stirring period proved 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 process was repeated until the pH of the aqueous phase was ≤2 (typically two additional washes). The combined citric acid wash was 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 collected 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.

[0104] 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 ​​with stirring over 3 hours 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 vacuum gave compound 4 (16.04 g, 77% from 2). Expected yield: 16.0 g (77% from compound 2).

[0105] 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 × 90 mL), water (90 mL), and brine (90 mL). The DCM (bottom) layer was then rotary evaporated at a bath temperature of 40 °C to yield 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 cooled to room temperature. Petroleum ether (4 mL) was slowly added to the stirred solution along with a seed crystal, at which point the product slowly began to crystallize. Once most of the product had precipitated, the final portion of petroleum ether (17 mL) was slowly added (total solvent added: ethyl acetate:petroleum ether 1:3, 21 mL). The product was then filtered under reduced pressure 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%).

[0106] 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 equiv.) at ambient temperature. The solution was then cooled to −10°C. NBS (0.639 g, 3.59 mmol, 2.08 equiv.) was added in one portion. An exotherm of approximately +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, pass criteria: less than 2.00 area % of compound 4 remaining). If the reaction was not complete, 1.00 equiv. of NBS (0.307 g, 1.73 mmol, 1.00 equiv.) was added in one portion, and the reaction was held at −10°C for an additional 15 min, followed by another IPC run. The reaction was quenched by the addition of aqueous NaHCO3 (5% w / v, 5 mL). Cooling was discontinued, and the mixture was allowed to warm to 10–20 °C during the following addition. After stirring for 3–5 min, additional 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 for 20 min at 10–20 °C, and then the solid was collected by filtration. The vessel was rinsed on the filter pad with NaHCO3 (5% w / v, 25 mL), and this rinse was filtered off. The filter cake was then rinsed successively with NaHCO3 (5% w / v, 25 mL), then water (25 mL). The (still wet) filter cake was dissolved in DCM (20 mL) and washed with two portions of NaHCO (5% w / v, 20 mL), then once with water (20 mL). The dichloromethane layer was dried by rotary evaporation and then dissolved in ethyl acetate (36 mL) at 65 °C. Petroleum ether 60-80 (10 mL) was then added slowly with stirring, and the mixture was cooled to 45 °C and stirred at 45 °C for 30 min. Additional petroleum ether 60-80 (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 reduced pressure to give compound 5 (0.805 g, 83% yield; combined purity of α and β anomers by HPLC was 98%).

[0107] Synthesis of compound 7 Compound 4 (500 mg) and Intermediate 3.1 (211 mg, 1.2 equiv.) 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 reduced pressure 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 to dry. After 10 min, dry NIS (400 mg, 2.0 equiv.) was added, and the solution was stirred at room temperature for 30 min. TMSOTf (8 μL, 5 mol%) was then added quickly, causing the solution to change color from red / orange to dark red / brown. The reaction temperature also increased from 22 °C to 27 °C. Immediately after the addition of TMSOTf, an IPC was performed for reference (HPLC; 10 μL in 1 mL MeCN-HO (8:2)). The reaction was then quenched by the addition of pyridine (20 μL, 0.245 mmol) and stirred at ambient temperature for 5 min. The DCM solution was filtered to remove the molecular sieves, washed with 10% NaSO (3 × 5 mL), brine (5 mL), and concentrated on a rotary evaporator (40 °C bath temperature) to give crude compound 7 as a foamy yellow oil (616 mg). Expected yield: approximately 616 mg (99%).

[0108] Synthesis of compound 8 Crude compound 7 (16.6 g) was dried by evaporation from toluene (2 × 30 mL) and then anhydrous DCM (30 mL) to produce a yellow foam / oil. The flask was then placed under an argon atmosphere, and 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 equiv.) 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 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 adding N-methylmorpholine (a total of 7.0 mL 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 combined DCM layers were then washed with brine (8 mL) and then rotary evaporated 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) and loaded onto a silica column (85 g silica) equilibrated with 3 column volumes (CV) of 30 vol% ethyl acetate in petroleum ether. The column was eluted using a stepwise gradient, collecting 1 CV (140 mL) fractions. The gradient used was as follows: 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, impurity peak with NMT 1.50 area % at 230 nm). The combined fractions were evaporated on a rotary evaporator at a bath temperature of 40° C. to give an off-white foam, which solidified to give 8 as a crisp solid (10.45 g). Expected yield: 10.45 g (66%). [Example 4]

[0109] Synthesis of disulfide (compound 17)

[0110] [ka]

[0111] compound 17 The overall synthetic procedure for synthesizing compound 17 is described in the synthetic scheme below.

[0112] [ka]

[0113] Synthesis of compound 9 Compound 5 (1620 g, 1.18 equiv.) and toluene (18 kg) were charged in this 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 reduced pressure 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 to 2 hours. Compound 5 dissolved during the reaction. The contents of the bowl were sampled and the reaction completion IPC (H 1NMR was performed, integrating the triplet peak at 6.42 ppm (product) compared to 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 with a syringe filter and analyzed by Karl Fischer (AM-GEN-011, acceptance criteria: ≤0.03% w / w). After the moisture threshold was reached (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 equiv.) in dry DCM (1250 g) was charged to the reactor under a nitrogen atmosphere. The contents of the header were discharged into the reactor, and the reactor contents were maintained at 0±10°C during the addition. The addition took 15-20 minutes. Dry DCM (1250 g) was charged to the buchi bowl and transferred to the reactor header. The contents of the header were discharged into the reactor, and the reactor contents were maintained at 0±10°C during the addition. The reactor contents were stirred at 0±5°C for 60 minutes. The reactor contents were sampled for reaction completion using IPC (HPLC, pass criterion ≤5% starting material). The reaction was quenched by charging N-methylmorpholine (85 g, 0.36 equiv.) to the reactor. The reactor contents were sampled for quench completion using IPC (wet pH paper, pass criterion ≥ pH 7). Silica gel (4.9 kg) was charged to the buchi bowl. The reactor contents were transferred to the buchi bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40±10°C until no more solvent was distilled. Silica gel (1.4 kg) was loaded into the Buchi bowl, followed by rinsing the reactor with dichloromethane (7.0 kg). The contents of the bowl were swirled to prevent solids from adhering to the surface of the bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40±10°C until no more solvent was distilled. The contents of the bowl were divided into three portions for silica gel chromatography. A 150LKP-SIL cartridge was installed in 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 silica gel-adsorbed reaction mixture, mixed thoroughly, and then transferred to the Biotage solvent reservoir. The contents of the solvent reservoir were eluted through the column to condition it. 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.

[0114] Ethyl acetate (1.6 kg) and petroleum ether (4.4 kg) were charged to a Biotage solvent reservoir and mixed thoroughly before eluting from the column. The column effluent was collected in a 20 L jerry can.

[0115] 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.

[0116] 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.

[0117] Ethyl acetate (16 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can.

[0118] The remaining two portions of dry loaded silica were prepared and the columns were repeated as above.

[0119] Column fractions were sampled for product purity (TLC [10% acetone in toluene, Rf 0.5]) to identify fractions containing product. The accepted column fractions were combined and placed in a 100 L buchi bowl. Toluene was used to rinse the crystalline material from the accepted fraction container into the bowl. Evaporation was carried out under reduced pressure using a water bath temperature of 40 ± 10 °C 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 evaporated to 6 L. The mixture was transferred to a Nutsche filter and the solvent removed by vacuum filtration. t-Butyl methyl ether (620 g) was charged to a bowl, transferred to the Nutsche filter, and filtered through the filter cake. The filter cake was air-dried in the filter and then transferred to a vacuum oven and dried under reduced pressure at a setting 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 or below -15 °C. Expected yield: 1.68-1.94 kg of compound 9 (65-75%).

[0120] Synthesis of compound 10 The reagents were prepared as follows: N-iodosuccinimide (241 g, 2.20 equiv.) was dried under reduced pressure 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.

[0121] Compound 8 (355 g, 0.486 mol) and compound 9 (634 g, 1.10 equiv.) were charged to a 20 L buchi bowl, followed by toluene (1500 g) and heated at 40 ± 5°C until dissolved. Evaporation was carried out under reduced pressure 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 reduced pressure 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 ± 5°C. Dry dichloromethane (710 g) was charged to the buchi bowl. The bowl was swirled to rinse the bowl surface, and the solution was transferred to the 5 L reactor. The contents of the reactor were mixed in a 200 mL flask with a 200 mL ethanol solution at a reagent ratio of IPC (H 1 The contents of the reactor were sampled for reaction completion (HPLC). Dry N-iodosuccinimide was charged to the reactor under a nitrogen atmosphere, and the reactor was stirred for 5-15 minutes. The contents of the reactor were adjusted to 20°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (5.94 g, 0.055 eq) in dry DCM (60 g) was charged to the reactor over 5-15 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. The contents of the reactor were sampled for reaction completion (HPLC). N-methylmorpholine (98 g, 2 eq) was charged to the reactor and mixed thoroughly. One portion of the sodium thiosulfate solution prepared above was charged to a 50 L reactor. 5 L of the reactor contents were transferred to the 50 L reactor containing the sodium thiosulfate solution and mixed thoroughly. The bottom layer was drained into an HDPE jerry can.

[0122] DCM (570 g) was charged to a 5 L reactor along with the top layer from the 50 L reactor and mixed thoroughly. The bottom layer was combined with the bottom layer from the previous 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 a 50 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into an HDPE jerry can. The top layer was held in the HDPE jerry can until yield was confirmed. The sodium chloride solution was charged to a 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 of the reactor was charged to the Buchi bowl. The contents of the bowl were swirled to prevent adsorption to the bowl, and evaporation was carried out under reduced pressure using a water bath temperature of 40 ± 5 °C until no solids distilled over. The contents of the bowl were divided into two equal portions. Silica gel (200 g) was charged into a Buchi bowl, followed by dichloromethane (700 g). The contents of the bowl were swirled to prevent solids from adhering to the surface of the bowl. The bowl was evaporated under reduced pressure at 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 one portion was added to each of the previous silica gel samples.

[0123] Each portion was independently purified on silica gel using the following procedure (samples were stored below ≦15°C while awaiting purification): A 150L KP-SIL cartridge was installed in 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 the columns. The eluate was collected in a 20 L jerry can and discarded. A portion of the dry packed silica from above was loaded into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can until fraction 11, then in a 5 L HDPE jerry can.

[0129] Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0130] Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0131] Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0132] Column fractions were sampled for product purity (TLC to identify fractions containing product). Fractions representing 75-95% area of ​​compound 10 from the first two columns were combined in a Buchi bowl packed with silica gel (400 g) and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. The contents of the bowl were purified as follows: 150L KP-SIL cartridges were installed in 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 jerry can and discarded. The contents of the bowl were loaded into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0133] 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] Ethyl acetate (29.7 kg) and petroleum ether (22.9 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 20 L jerry can until fraction 11, then in a 5 L HDPE jerry can.

[0138] Ethyl acetate (15.5 kg) and petroleum ether (11.0 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0139] Ethyl acetate (29.7 kg) and petroleum ether (13.2 kg) were loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0140] Ethyl acetate (15.5 kg) was charged to the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L HDPE jerry can.

[0141] The accepted column fractions from all three columns were combined in a Buchi bowl and evaporated under reduced pressure using a water bath temperature of 40±10°C until no more solvent distilled. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤-15°C. Expected yield: 440-540 kg (52-64% yield).

[0142] Synthesis of compound 11 Dichloromethane was charged to a buchi bowl containing compound 10 (635 g, 0.345 mol) (PN0699) and heated at 30 ± 10 °C until dissolved. Methanol (3.2 kg) was charged 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 charged 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 for reaction completion IPC (HPLC, passed). The contents of the bowl were adjusted to 0 ± 3 °C. N-methylmorpholine (139 g, 4 equiv.) was charged to the bowl and mixed thoroughly. The contents of the bowl were sampled for quench completion IPC (pH paper, passed ≤ pH 7). The contents of the bowl were concentrated under reduced pressure in a 35 ± 10 °C water bath. Ethyl acetate (4.8 kg) and water (5.5 kg) were charged 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 equipped with a rotary evaporator, and the contents were concentrated under reduced pressure in a 35±10°C water bath. The bottom layer of the HDPE jerry can was charged to a 50 L reactor containing 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 equipped with a rotary evaporator, and the contents were concentrated under reduced pressure in a 35±10°C water bath. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤-15°C. Expected yield: 518-633 kg (90-110% yield).

[0143] Synthesis of compound 12 The reagents were prepared as follows: Two portions of N-iodosuccinimide (143 g, 3.90 equivalents) were dried under reduced pressure 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.

[0144] Compound 9 (504 g, 1.30 equiv.) was charged to a 50 L buchi bowl containing compound 11 (607 g, 0.327 mol), followed by toluene (1500 g) and heated at 40 ± 5 °C until dissolved. Evaporation was carried out under reduced pressure 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 reduced pressure 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 remaining 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 bowl surface, 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 a reactor under a nitrogen atmosphere. The reactor contents were adjusted to -40°C ± 3°C. Trimethylsilyl trifluoromethanesulfonate (9.09 g, 0.25 effective equivalents) 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. The reactor contents were sampled for reaction completion. N-methylmorpholine (33.1 g, 2 effective equivalents) was charged to the reactor and mixed thoroughly. One 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 from the 5 L lab bottle. The combined organic phase was charged to a 5 L reactor, followed by another portion of sodium thiosulfate and mixed thoroughly. The bottom layer was drained into a 5 L lab bottle. The upper portion of the sodium chloride solution was charged to the reactor, followed by the contents of the previous lab bottle. The bottom layer from the reactor was charged to a buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent distilled. The reactor was washed and dried.

[0145] The second portions of compounds 9 and 11 were charged to the reactor and treated identically to the first batch. Following the organic extraction of the second batch, the reaction mixtures were combined in the reactor. The sodium chloride solution portion was charged to the reactor and thoroughly mixed. Silica gel (1700 g) was charged to a buchi bowl and a rotavapor was attached. The bottom layer of the reactor was charged to the buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent was distilled. The contents of the bowl were divided into two equal portions and purified independently with silica gel. A 150L KP-SIL cartridge was installed in a Biotage system (commercially available from Biotage, a division of Dyax Corporation, Charlottesville, Virginia, USA). Ethyl acetate (7.7 kg) and petroleum ether (22.0 kg) were charged to a 50 L reactor, thoroughly mixed, and then transferred to two Biotage solvent reservoirs. The contents of the solvent reservoir 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 packed silica from above was packed into a Biotage Sample-Injection Module (SIM) and eluted with ethyl acetate / petroleum ether as follows:

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Ethyl acetate (15.5 kg) was loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L glass lab bottle.

[0151] Column fractions were sampled for product purity (TLC to identify fractions containing product). Fractions representing 75-95% area of ​​compound 12 from the first two columns were combined in a buchi bowl packed with silica gel (400 g) and evaporated under reduced pressure 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 to condition them. The eluate was collected in a 20 L jerry can and discarded. The dry packed silica containing the impure product was loaded into a Biotage Sample-Injection Module (SIM) and eluted as detailed below:

[0152] 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.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] Ethyl acetate (15.5 kg) was loaded into the Biotage solvent reservoir and then eluted from the column. The column effluent was collected in a 5 L glass lab bottle.

[0157] Column fractions were sampled for product purity (TLC to identify fractions containing product, HPLC acceptance criteria ≥ 95% compound 12, and > 2.5% no single impurity). Accepted column fractions from all three columns were combined in a Buchi bowl and evaporated under reduced pressure using a water bath temperature of 40 ± 10 °C until no more solvent distilled. The contents of the bowl were sampled for analysis and retention. The bowl was sealed and transferred to storage at ≤ -15 °C. Expected yield: 494-584 kg (52-64% yield).

[0158] 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 number 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. The solution was purged of oxygen by pressurizing with nitrogen to 10 bar and then released. This was repeated two more times. The contents of the reactor were pressurized under hydrogen to 10 bar and then released. 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 and then released. 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 × 5.5 kg). The combined filtrate was evaporated under reduced pressure (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 approximately 316 g. A Biotage system was fitted with a 150MKP-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 a 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 it. 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 a SIM and passed through a column. The reaction mixture was chromatographed as follows:

[0159] 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.

[0160] The contents of the solvent reservoir were eluted through the column via the SIM and the eluate was collected in a 20 L jerry can.

[0161] 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 transferred to the Biotage solvent reservoir.

[0162] The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can.

[0163] 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.

[0164] The contents of the solvent reservoir were eluted through the column and the eluate was collected in approximately 2.5 L fractions into a 5 L jerry can.

[0165] Ethyl acetate (31.4 kg), glacial acetic acid (4.1 kg) and methanol (3.40 kg) were charged to a 50 L reactor, mixed thoroughly and then transferred to the Biotage solvent reservoir.

[0166] The contents of the solvent reservoir were eluted through the column and the eluate was collected in a 5 L jerry can.

[0167] The fractions containing compound 13 were combined and evaporated under reduced pressure (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) until the pH of the aqueous medium was ≥ 8. The ethyl acetate phase was evaporated under reduced pressure (bath temperature 40±5°C). The contents of the bowl were sampled for analysis and retention. Expected yield: 182-207 g (71-81%).

[0168] Synthesis of compound 16 Dry dichloromethane (2.5 kg) was charged to a buchi bowl containing compound 13 (211 g, 76.5 mmol, 1.00 equiv.) 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 reduced pressure at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the bowl and removed under reduced pressure twice at a water bath temperature of 40 ± 5 °C. Toluene (0.8 kg) was added to the bowl and dissolved in the residue. Silica gel (557 g) was placed in the reaction vessel, and the solvent was removed under reduced pressure at a water bath temperature of 40 ± 5 °C. A Biotage system was fitted with a 150M KP-SIL cartridge equipped 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:

[0169] The column was conditioned by passing solvent A through it and eluting it. The eluate was discarded.

[0170] The dried packed silica gel was transferred to the SIM.

[0171] 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).

[0172] Solvent B was eluted through the column and the eluate was collected in a 5 L jerry can.

[0173] 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).

[0174] Solvent C is eluted through the column and the eluate is collected in a 5 L jerry can.

[0175] 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).

[0176] Solvent D was eluted through the column and the eluate was collected in a 5 L jerry can.

[0177] 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).

[0178] Solvent E was eluted through the column and the eluate was collected in a 5 L jerry can.

[0179] Fractions containing compound 16 (acceptance criteria: ≥ 90% compound 16 and > 2.5% absence of a single impurity) were combined and evaporated under reduced pressure (bath temperature 40 ± 5 °C). The residue was dissolved in tetrahydrofuran (4.4 kg) and concentrated under reduced pressure at a water bath temperature of 40 ± 5 °C. The contents of the bowl were sampled for analysis and retention. Expected yield: 169-192 g (76-86%).

[0180] Synthesis of compound 17 Prior to start-up, the reactor was marked at the 2.5 L, 3.5 L, and 3.9 L levels and attached to a vacuum controller. Dichloromethane was charged to a buchi bowl containing 140 g of compound 16 and transferred to the Reactor Ready vessel. The contents of the buchi bowl were transferred to the Reactor Ready vessel using two rinses of DCM (333 g). Ethanol (2.50 kg) was added to the Reactor Ready. The reaction mixture was concentrated to the 2.5 L mark (target vacuum 250 mbar). Ethanol (1.58 kg) was added to the Reactor Ready 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 the nitrogen. A slow flow of nitrogen was maintained throughout the reaction. Hydrazine monohydrate (1.13 kg, 1.11 L) was charged into a 5 L Reactor Ready vessel under a nitrogen atmosphere. The temperature gradient was set as follows: initial temperature 20°C, final temperature 60°C, linear temperature gradient over 50 minutes (0.8 degrees / min), and active control of the reactor contents. The vessel temperature was held at 60°C for 45 minutes. The cooling gradient temperature was set at -2 degrees / min, with a final temperature of 20°C. The contents were drained into suitable HDPE jugs and weighed. Equal amounts were transferred to eight polypropylene centrifuge vessels with FEP encapsulation seals. Each centrifuge vessel was filled with ethanol (750 g) and stirred for 30 minutes at ambient temperature. The vessel was centrifuged (5300 RCF, 15°C, 30 minutes). Residual hydrazine on the outside of the vessel was removed by rinsing the outside of the bottle with acetone and then water before removing from the fume hood. The supernatant from 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, while aerating the solution using a sparger. The reaction was then stirred overnight at 20°C in a sealed vessel. The reaction was considered complete when IPC indicated a free pentamer composition of less than 3% (reported total 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 after 15 minutes of stirring. 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 LE water (140 g) was added to each container. Each pellet was dissolved in water by agitating the mixture at ambient temperature using an orbital shaker until the pellet was dissolved. Acetone (630 g) was added to each container and stirring continued 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 additional LE water (66 g each), with the rinse transferred to the same tray. The product was lyophilized at a shelf temperature of -0.5°C for 16-20 hours, then at 20°C until dry. The lyophilized product was sampled for analysis and storage. The Lyoguard trays were double-wrapped, labeled, and stored in a freezer (≤-15°C). The potency of the lyophilized product was determined using qH NMR. This procedure yielded crude penta dimer 17. Expected yield: 26.1-35.5 g (61-83%).

[0181] 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 reference solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the reference solution was added to the sample. The composition of the final test sample 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 and13 The C NMR spectra are shown in Figures 1 and 2, respectively. [Example 5]

[0182] Conversion of crude pentadimer to the free base form. Amberlite FPA91 (1.46 kg; 40 g / g crude pentadimer—corrected for potency) was loaded into a large column. 8 L of 1.0 M NaOH solution was prepared by adding NaOH (32 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 the flow-through pH was <8.0. 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 an additional LE water (200 g), and these washes were added to the contents of the Schott bottle. The crude pentadimer solution was carefully poured onto the top of the resin. A 1 L Schott bottle was rinsed with LE water (200 g) and added to the resin. The Amberlite tap was opened, allowing the crude pentadimer solution 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 using a 0.2 μm nylon filter membrane. The solution was divided evenly into 5-6 Lyoguard trays. The filter vessel was rinsed with LE water (100 g) and divided between the trays. The material was lyophilized within the trays. The shelf temperature was set to -10°C for 16-20 hours, then to +10°C until the material was dry. LE water (150g) was filled into all but one of the Lyoguard trays and transferred to the remaining tray containing the dried material. Each empty tray was rinsed with more LE water (100g) and this rinse was added to the final Lyoguard tray. The final Lyoguard tray was freeze-dried. The shelf temperature was set to -10°C for 16-20 hours, then +10°C until the material was dry.The product was sampled for analysis and retention. The dried material was transferred to HDPE or PP containers and stored at ≦−15°C. Expected yield: 31-34 g (86-94%).

[0183] 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 1 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 pentaglucosamine monomer (peak at approximately 10 min) was nearly complete (pentaglucamine dimer peak at approximately 11.5 min)—see Figure 4. The remaining unannotated peaks were obtained from the sample matrix. Based on the equilibrium chemical equation, the added TCEP was primarily converted to TCEP oxide, and the residual TCEP was then inhibited by air oxidation back to the dimer before being added to the conjugation reaction. For simplicity, the glucosamine pentasaccharide can be added based on the input dimer, assuming >95% conversion to monomer under these conditions.

[0184] The identity of the pentadimer was confirmed using a 500 MHz instrument. 1 H and 13 The NMR spectra were determined by C NMR. A reference solution of t-butanol was prepared at 25 mg / mL in DO. Samples were prepared at 13 mg / mL in DO, and the reference solution was added to the sample. The composition of the final test sample 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. [Example 5]

[0185] Conversion of the TT-linker of Example 2 to the pentasaccharide monomer of Example 4 to provide 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 indicated saturation of payload density at ≥50 pentameric glucosamine equivalents. Based on SEC-HPLC analysis, aggregate content increased with increasing pentasaccharide monomer charge, appearing to reach a steady-state level of approximately 4% increase starting at 30 pentameric glucosamine equivalents. Based on these results, the pentasaccharide dimer charge selected for subsequent conjugation reactions was 25 molar equivalents, corresponding to 50 molar equivalents of the theoretical charge of pentameric glucosamine.

[0186] A series of three test syntheses followed by a GMP synthesis of compound 18 were prepared as described above. The potency (by ELISA assay) and payload density (pentameric glucosamine to tetanus toxoid molar ratio) of each resulting product were evaluated. The results are shown in the table below.

[0187] [Table 2]

[0188] These results demonstrate the extremely high loading capacity of the compounds of the present invention. The foregoing description is provided merely to illustrate the present invention and is not intended 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. [Example 6]

[0189] Monoclonal antibody F-598 The monoclonal antibody designated F-598 is disclosed in U.S. Patent No. 7,786,255, which is incorporated herein by reference in its entirety. This monoclonal antibody is also commercially available from Creative Biolabs, Shirley, New York, USA, as TAB-799CL and AFC-765CL. The amino acid sequence of F-598 is set forth in SEQ ID NOs: 1-5. [Example 7]

[0190] A 45-year-old, 175-pound firefighter with burns over 45% of his body. Patients are immediately identified as being at high risk for developing sepsis. To minimize this risk, the patient is first administered a therapeutic dose of monoclonal antibody (mAb) F-598, which provides immediate immunotherapy to the patient. Approximately two hours later, the patient is administered the vaccine of Formula I disclosed herein.

[0191] Patients are monitored to ensure that therapeutic levels of monoclonal and polyclonal antibodies remain in the patient's serum. If necessary, additional treatments of monoclonal antibodies are administered to maintain therapeutic serum concentrations. Similarly, the titer of polyclonal antibodies produced by the compounds described herein is measured. If necessary, additional vaccines can be administered to patients to maintain therapeutic serum concentrations. Treatment is continued until the patient is no longer at risk.

Claims

1. 1. A method for providing sustained protection against microbial infection, comprising administering a therapeutically effective amount of monoclonal antibody F-598 and a compound of formula I: (A-B) ｘ -C I wherein A contains 3 to 12 β-(1→6)-glucosamine (carbohydrate ligand) groups or mixtures thereof, and the oligosaccharide portion of the vaccine has the following formula A: 【Chemistry 1】 is expressed as B is a linker; wherein A is as defined above and C is tetanus toxoid; x is an integer from about 30 to about 39; y is an integer from 1 to 10. administering to said patient a vaccine of the formula:

2. 10. The method of claim 1, wherein the vaccine of Formula I is administered simultaneously with F-598.

3. 10. The method of claim 1, wherein the vaccine of Formula I is administered within about 6 hours of administering F-598.

4. 10. The method of claim 1, wherein the vaccine of Formula I is administered within about 4 hours of administering F-598.

5. 10. The method of claim 1, wherein the vaccine of Formula I is administered within about 2 hours of administering F-598.

6. 10. The method of claim 1, wherein F-598 is co-administered during the entire treatment period.

7. The linker has the following formula: 【Chemistry 2】 (wherein A and C are not included in the linker) The method of claim 1 , wherein

8. 10. The method of claim 1, wherein F-598 is co-administered to the point where the vaccine of Formula I produces sufficient antibody titers to effectively treat the patient.

9. 10. A method for providing effective immunity to a patient from microorganisms containing oligosaccharide β-(1→6)-glucosamine groups in their cell walls, comprising administering to said patient the vaccine of claim 7.

10. 1. A method for inhibiting biofilm formation comprising administering a therapeutically effective amount of monoclonal antibody F-598 and a compound of Formula I: (A-B) ｘ -C I wherein A contains 3 to 12 β-(1→6)-glucosamine (carbohydrate ligand) groups or mixtures thereof, and the oligosaccharide portion of the vaccine has the following formula A: 【Transformation 3】 is expressed as B is a linker; wherein A is as defined above and C is tetanus toxoid; x is an integer from about 30 to about 39; y is an integer from 1 to 10. administering to a patient a vaccine of the formula:

11. 11. The method of claim 10, wherein the vaccine of Formula I is administered simultaneously with F-598.

12. 11. The method of claim 10, wherein the vaccine of Formula I is administered within about 6 hours of administering F-598.

13. 11. The method of claim 10, wherein the vaccine of Formula I is administered within about 4 hours of administering F-598.

14. 11. The method of claim 10, wherein the vaccine of Formula I is administered within about 2 hours of administering F-598.

15. 11. The method of claim 10, wherein F-598 is co-administered during the entire treatment period.

16. The linker has the following formula: 【Chemistry 4】 (wherein A and C are not included in the linker) The method of claim 10, wherein

17. 11. The method of claim 10, wherein F-598 is co-administered to the point where the vaccine of Formula I produces sufficient antibody titers to effectively treat the patient.

18. 17. A method for providing effective protection against biofilms formed by microorganisms containing oligosaccharide β-(1→6)-glucosamine groups in their cell walls, comprising administering to said patient a vaccine according to claim 16.

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