WO2025085506A1 - Deprotection of fmoc in peptide synthesis - Google Patents

Abstract

Compositions and methods of compound preparation, such as peptide preparation, comprising, providing a compound, such as a peptide, having a Fmoc-protected primary amine, and deprotecting the primary amine under acidic hydrogenolysis conditions. The method suppresses nucleophilic reactivity of the resulting primary amine on the peptide. The method results in a peptide tolerant of N-Boc protected amino groups within the peptide. In embodiments, the invention provides pharmaceutical compositions and methods for treating a condition or disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a peptide that has been deprotected under acidic hydrogenolysis conditions.

Description

DEPROTECTION OF FMOC IN PEPTIDE SYNTHESIS

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63/590,697 filed October 16, 2023, which application is incorporated herein by reference.

GOVERNMENT SPONSORSHP

[0002] This invention was made with government support under grant NS 109075 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

FIELD OF THE INVENTION

[0003] The present disclosure relates to processes for peptide synthesis.

BACKGROUND OF THE INVENTION

[0004] The 9-fluorenylmethoxy carbonyl (Fmoc) group represents one of the most widely used groups for the protection of amines in organic synthesis.^1,2 This protecting group has found extensive application due to its easy removal under mildly basic conditions such as with piperidine, diethylamine or morpholine. Fmoc also has the advantage of being tolerant to acidic conditions. The Fmoc protecting group is resistant to many other reaction conditions such as oxidation and reduction in multi-step total synthesis of natural products.³ The highly electrophilic by-product of Fmoc deprotection, dibenzofulvene (Dbf), can induce a variety of side reactions by capture of incipient nucleophiles;³ conditions that limit Fmoc use in peptide synthesis where an excess of the nitrogenous base is needed to achieve both complete removal of the Fmoc group and quenching of the resulting Dbf ³ An additional burden is the need to remove excess nitrogenous base before proceeding to the next peptide coupling step.

[0005] Many peptide syntheses have faced the problems described above; in essence, unmasking of reactive groups while maintaining orthogonality of protection groups for NH2, COOH and side-chain functional groups. In particular, exposure of a nucleophilic primary amine by Fmoc deprotection in the presence of a reactive electrophile can lead to unwanted side reactions.^4,5 Similarly, the 2° amine employed in the deprotection step (e.g. diethylamine or piperidine) can readily engage reactive electrophiles to produce undesired side-products including polymeric materials. Paradoxically, the reagents used for Fmoc deprotection and the exposed 1° amine product are liabilities when the deprotected peptide has electrophilic functionalities.

[0006] The response of the base labile Fmoc group to acidic deprotection has not been widely studied. Lewis acid catalyzed Fmoc deprotection utilized A1C13 with toluene as a Friedel-Crafts scavenger,⁶ however, this method is limited by the requirement for deactivation of the unmasked 1° amine by acidification in a separate step. This poses a risk for side reactions between the free amine and electrophilic functional groups in the product molecule. Exposure to a strong Lewis acid may result in undesired side reactions in sensitive molecules, or premature deprotection of acid-sensitive protecting groups used in peptide synthesis (e.g. Boc, i.e., tert-butyloxycarbonyl protecting group). It was reported that Pd/C-catalyzed hydrogenolysis of the Fmoc group was employed in the presence of acetonitrile to produce ethylamine (the reduction product of acetonitrile), which subsequently aids in scavenging the Dbf side-product after Fmoc removal.⁷ Despite the claim of neutrality for this method, in-situ generation of nucleophilic EtNH2 would promote capricious side reactions with electrophiles. In evermore complex peptide syntheses that require maximum flexibility in their ‘end-game strategies’, the following unmet need arises: alternative methods for Fmoc removal orthogonal to conventional treatment with 2° amines that mitigates both the liabilities of nucleophilic 2° -amine reagents and the revealed exposure of 1° -amines within peptides containing electrophilic functionalities.

SUMMARY OF THE INVENTION

[0007] This invention provides for the removal of an Fmoc protecting group from a molecule containing an amine, such as a peptide, and converting the resulting free amine to a protonated amine. The protonated amine is much less reactive than the free amine, and thus does not lead to side reactions, making the Fmoc group much more useful in peptide chemistry.

[0008] The present invention provides compositions and methods of peptide preparation comprising, providing a peptide having a Fmoc-protected primary amine, and deprotecting the primary amine under acidic hydrogenolysis conditions to prepare the peptide. The method suppresses nucleophilic reactivity of the resulting primary amine on the peptide. The method results in a peptide tolerant of 7V-Boc protected amino groups within the peptide. The methods provide Fmoc-removal reaction conditions, including the structure of the Bronsted acid, acid concentration and the molar equivalents of Pd. The methods provide that acidic hydrogenolysis conditions are produced by 2-3 equivalents of HC1 in MeOH.

[0009] The invention provides methods for removal of a Fmoc protecting group in synthetic organic chemistry, especially with peptides, with applications for laboratory, industnal and clinical purposes. In embodiments, the invention provides pharmaceutical compositions and methods for treating a condition or disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a peptide that has been deprotected under acidic hydrogenolysis conditions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1. Irreproducible literature route for Z-R-K-AOMK synthesis.⁵

[0011] Figure 2A. Failed synthesis of Z-R-K-AOMK (1) due to the basic Fmoc removal step or due to the reactivity of the deprotected amine from compound 4. Figure 2B. Successful synthesis of Z-R-K-AOMK (1) using the novel Fmoc deprotection strategy described in this manuscript.

[0012] Figure 3. pH-dependent inhibition of cathepsin B cleavage activity in the presence of serial diluted inhibitor comparing newly synthesized Z-R-K-AOMK (1) versus an authentic sample of Z-R-K-AOMK from previous studies.⁵ Inhibitory potencies were evaluated by IC50 (n=4) without preincubation of inhibitor and enzyme, as described in the methods. [0013] Figure 4A. Example of successful Fmoc removal from a molecule containing a Boc group and an a-chloroketone moiety. Figure 4B. Michael addition after Fmoc removal using acidic hydrogenolysis Figure 4C. Mannich reaction after successful Fmoc removal using acidic hydrogenolysis.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present disclosure provides compositions and methods of peptide preparation comprising, providing a peptide having a Fmoc-protected primary amine, and deprotecting the primary amine under acidic hydrogenolysis conditions to prepare the peptide. The method suppresses nucleophilic reactivity of the resulting primary amine on the peptide. The method results in a peptide tolerant of N-Boc protected amino groups within the peptide. The methods provide Fmoc-removal reaction conditions, including the structure of the Bronsted acid, acid concentration and the molar equivalents of Pd. The methods provide that acidic hydrogenolysis conditions are produced by 2-3 equivalents of HC1 in MeOH.

[0015] In embodiments, the invention provides pharmaceutical compositions and methods for treating a condition or disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a peptide that has been deprotected under acidic hydrogenolysis conditions.

[0016] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the present disclosure belongs. Any materials and methods similar or equivalent to those described herein can be used to practice the present invention. The practice of the present invention may employ conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al, 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ. Gait, ed., 1984); Methods in Molecular Biology⁷, Humana Press; Cell Biology: A Laboratory Notebook (J .E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R.I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J.P. Mather and P.E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J.B. Griffiths, and D.G. Newell, eds., 1993- 1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D .M. Weir and CC. Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (J.M. Miller and M.P. Calos, eds., 1987); Current Protocols in Molecular Biology (F .M. Ausubel et al , eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al, eds., 1994); Current Protocols in Immunology (J.E. Coligan et al, eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J.D. Capra, eds., Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (V. T. DeVita et al, eds., J.B. Lippincott Company, 1993). Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein. For the purposes of the present disclosure, the following terms are defined below. Additional definitions are set forth throughout this disclosure.

[0017] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0018] As used herein, the terms “comprises,” “comprising,” “includes,” ‘including,” “has,” “having,” “contains”, “containing,” “characterized by,” or any other variation thereof, are intended to encompass a non-exclusive inclusion, subject to any limitation explicitly indicated otherwise, of the recited components. For example, a pharmaceutical composition, and/or a method that “comprises” a list of elements (e.g., components, features, or steps) is not necessarily limited to only those elements (or components or steps), but may include other elements (or components or steps) not expressly listed or inherent to the pharmaceutical composition and/or method. Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0019] Various further aspects and embodiments of the disclosure are provided by the following description. Before further describing various embodiments of the presently disclosed inventive concepts in more detail by way of exemplary description, examples, and results, it is to be understood that the presently disclosed inventive concepts are not limited in application to the details of methods and compositions as set forth in the following description. The presently disclosed inventive concepts are capable of other embodiments or of being practiced or carried out in various ways. As such, the language used herein is intended to be given the broadest possible scope and meaning; and the embodiments are meant to be exemplary, not exhaustive. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting unless otherwise indicated as so. Moreover, in the following detailed description, numerous specific details are set forth in order to provide a more thorough understanding of the disclosure. However, it will be apparent to a person having ordinary skill in the art that the presently disclosed inventive concepts may be practiced without these specific details. In other instances, features which are well known to persons of ordinary skill in the art have not been described in detail to avoid unnecessary complication of the description. All of the compositions and methods of production and application and use thereof disclosed herein can be made and executed without undue experimentation in light of the present disclosure.

[0020] The term “and/or” when used in a list of two or more items, means that any one of the listed items can be employed by itself or in combination with any one or more of the listed items. For example, the expression “A and/or B” is intended to mean either or both of A and B, i.e. A alone, B alone or A and B in combination. The expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination or A, B, and C in combination.

[0021] It should be understood that the terms “a” and “an” as used herein refer to “one or more” of the enumerated components unless otherwise indicated. The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.

[0022] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Values or ranges may be also be expressed herein as “about,” from “about” one particular value, and/or to “about” another particular value. When such values or ranges are expressed, other embodiments disclosed include the specific value recited, from the one particular value, and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another embodiment.

[0023] It will be further understood that there are a number of values disclosed therein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. In embodiments, “about” can be used to mean, for example, a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length that varies by as much as 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length. In various embodiments, the term “about” or “approximately” refers a range of quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%, ± 2%, or ± 1% about a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight or length.

[0024] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0025] In embodiment, the invention provides methods of deprotection, the method comprising: providing a precursor compound comprising a Fmoc-protected amine; and deprotecting the Fmoc-protected amine under acidic hydrogenolysis conditions to prepare a compound.

[0026] In embodiments, the invention provides methods of peptide preparation comprising, providing a peptide having a Fmoc-protected primary amine, and deprotecting the primary amine under acidic hydrogenolysis conditions to prepare the peptide. The method suppresses nucleophilic reactivity of the resulting primary amine on the peptide. The method results in a peptide tolerant of A-Boc protected amino groups within the peptide. The methods provide Fmoc-removal reaction conditions, including the structure of the Bronsted acid, acid concentration and the molar equivalents of Pd.

[0027] In embodiments, the invention provides pharmaceutical compositions and methods for treating a condition or disease comprising administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a peptide that has been deprotected under acidic hydrogenolysis conditions.

[0028] In an aspect, the disclosure provides a method of treating or preventing a disease or disorder in a subject in need thereof, comprising administering to the subject an effective amount of a peptide produced by the methods described herein.

[0029] The following is a non-limiting listing of embodiments of the disclosure.

[0030] Embodiment 1. A method of deprotection, the method comprising, consisting essentially of, or consisting of:

[0031] providing a precursor compound comprising a Fmoc-protected amine, and

[0032] deprotecting the Fmoc-protected amine under acidic hydrogenolysis conditions to prepare a compound.

[0033] Embodiment 2. The method of Embodiment 1, wherein the acidic hydrogenolysis conditions are produced by a reaction medium that comprises, consists essentially of, or consists of-

[0034] an acid,

[0035] a metal, and

[0036] a liquid.

[0037] Embodiment 3. The method of Embodiment 2, wherein the acid comprises, consists essentially of, or consists of a Bronsted acid.

[0038] Embodiment 4. The method of Embodiment 3, wherein the Bronsted acid comprises, consists essentially of, or consists of a hydrogen halide, such as HC1.

[0039] Embodiment 5. The method of any of Embodiments 2 to 4, wherein about 2 to about 3 equivalents of the acid are present (see Table 1, which explains that the “equivalents” of acid are relative to the starting material, e.g., the precursor compound). [0040] Embodiment 6. The method of any of Embodiments 2 to 5, wherein the metal comprises, consists essentially of, or consists of a platinum group metal.

[0041] Embodiment 7. The method of Embodiment 6, wherein the platinum group metal comprises, consists essentially of, or consists of Pd.

[0042] Embodiment 8. The method of any of Embodiments 2 to 7, wherein the metal is present at a relative amount of about 1 % to about 30 %, or about 10 % to about 20 % molar metal/C equivalents to starting material (i.e., the precursor compound) see Table 1).

[0043] Embodiment 9. The method of any of Embodiments 2 to 8, wherein the liquid comprises, consists essentially of, or consists of a solvent in which the compound and/or the precursor compound is/are soluble.

[0044] Embodiment 10. The method of Embodiment 9, wherein the liquid comprises, consists essentially of, or consists of an organic liquid, such as a polar organic liquid, which may be a C1-C5 alcohol, such as methanol.

[0045] Embodiment 11. A composition comprising, consisting essentially of, or consisting of a compound prepared according to any of the preceding Embodiments.

[0046] Embodiment 12. A composition comprising, consisting essentially of, or consisting of a compound that has been deprotected under acidic hydrogenolysis conditions.

[0047] Embodiment 13. A pharmaceutical composition comprising, consisting essentially of, or consisting of -

[0048] the compound prepared according to any of the preceding Embodiments; and

[0049] a pharmaceutically acceptable carrier.

[0050] Embodiment 14. A method of preventing or treating a condition or disease, the method comprising, consisting essentially of, or consisting of - [0051] administering to a subject in need thereof an effective amount of a pharmaceutical composition of Embodiment 13.

[0052] Embodiment 15. A method of preventing or treating a condition or disease, the method comprising, consisting essentially of, or consisting of -

[0053] administering to a subject in need thereof an effective amount of a pharmaceutical composition comprising a compound that has been deprotected under acidic hydrogenolysis conditions.

[0054] Embodiment 16. The method or composition of any of the preceding Embodiments, wherein the Fmoc-protected amine is a Fmoc-protected primary amine or a Fmoc-protected secondary amine.

[0055] Embodiment 17. The method or composition of any of the preceding Embodiments, wherein the precursor compound comprises, consists essentially of, or consists of a precursor peptide.

[0056] Embodiment 18. The method or composition of any of the preceding Embodiments, wherein the compound comprises, consists essentially of, or consists of a peptide, such as a peptide that includes one or more protecting groups other than Fmoc, such as Boc.

[0057] Embodiment 19. The method or composition of any of the preceding Embodiments, wherein a deprotected amine (i.e., the amine from which the Fmoc group was removed) of the compound has a suppressed nucleophilic reactivity.

[0058] Embodiment 20. The method or composition of any of the preceding Embodiments, wherein the precursor compound and the compound comprises an amine protected by an acid-sensitive protecting group, such as a Boc-protected amine.

[0059] Embodiment 21. The method or composition of any of the preceding Embodiments, wherein the acidic hydrogenolysis conditions prevent, or reduce the likelihood of, the removal of acid-sensitive protecting groups, such as Boc, from the precursor compound and/or the compound. [0060] As used herein, “acidic hydrogenolysis conditions” means an acidic reaction medium that includes a metal, such as a mildly acidic reaction medium that includes a platinum group metal, such as Pd. A “mildly acidic reaction medium” is one that prevents, or reduces the likelihood of, the removal of acid-sensitive protecting groups, such as Boc. The “acidic hydrogenolysis conditions”, as described herein, are effective to remove an Fmoc protecting group from an Fmoc-protected amine.

[0061] As used herein, a subject in need refers to an animal, a non-human mammal or a human. As used herein, “animals” include a pet, a farm animal, an economic animal, a sport animal and an experimental animal, such as a cat, a dog, a horse, a cow, an ox, a pig, a donkey, a sheep, a lamb, a goat, a mouse, a rabbit, a chicken, a duck, a goose, a primate, including a monkey and a chimpanzee.

[0062] As used herein, the term “amount” refers to “an amount effective” or “an effective amount” of a treatment to achieve a beneficial or desired prophylactic or therapeutic result, including clinical results. As used herein, “therapeutically effective amount” refers to an amount of a pharmaceutically active compound(s) that is sufficient to treat or ameliorate, or in some manner reduce the symptoms associated with diseases and medical conditions. When used with reference to a method, the method is sufficiently effective to treat or ameliorate, or in some manner reduce the symptoms associated with diseases or conditions. For example, an effective amount in reference to diseases is that amount which is sufficient to block or prevent onset; or if disease pathology has begun, to palliate, ameliorate, stabilize, reverse or slow progression of the disease, or otherwise reduce pathological consequences of the disease. In any case, an effective amount may be given in single or divided doses.

[0063] As used herein, the terms “treat,” “treatment,” or “treating” embraces at least an amelioration of the symptoms associated with diseases in the subject, where amelioration is used in a broad sense to refer to at least a reduction in the magnitude of a parameter, e.g. a symptom associated with the disease or condition being treated. As such, “treatment” also includes situations where the disease, disorder, or pathological condition, or at least symptoms associated therewith, are completely inhibited (e.g. prevented from happening) or stopped (e.g. terminated) such that the subject no longer suffers from the condition, or at least the symptoms that characterize the condition. [0064] As used herein, and unless otherwise specified, the terms "prevent," "preventing" and "prevention" refer to the prevention of the onset, recurrence or spread of a disease or disorder, or of one or more symptoms thereof. In certain embodiments, the terms refer to the treatment with or administration of a compound or dosage form provided herein, with or without one or more other additional active agent(s), prior to the onset of symptoms, particularly to subjects at risk of disease or disorders provided herein. The terms encompass the inhibition or reduction of a symptom of the particular disease. In certain embodiments, subjects with familial history of a disease are potential candidates for preventive regimens. In certain embodiments, subjects who have a history of recurring symptoms are also potential candidates for prevention. In this regard, the term "prevention" may be interchangeably used with the term "prophylactic treatment."

[0065] As used herein, and unless otherwise specified, a "prophylactically effective amount" of a compound is an amount sufficient to prevent a disease or disorder, or prevent its recurrence. A prophylactically effective amount of a compound means an amount of therapeutic agent, alone or in combination with one or more other agent(s), which provides a prophylactic benefit in the prevention of the disease. The term "prophylactically effective amount" can encompass an amount that improves overall prophylaxis or enhances the prophylactic efficacy of another prophylactic agent.

[0066] Pharmaceutically active: The term “pharmaceutically active” as used herein refers to the beneficial biological activity of a substance on living matter and, in particular, on cells and tissues of the human body. A “pharmaceutically active agent” or “drug” is a substance that is pharmaceutically active and a “pharmaceutically active ingredient” (API) is the pharmaceutically active substance in a drug. As used herein, pharmaceutically active agents include synthetic or naturally occurring small molecule drugs and more complex biological molecules.

[0067] Pharmaceutically acceptable: The term “pharmaceutically acceptable” as used herein means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopoeia, other generally recognized pharmacopoeia in addition to other formulations that are safe for use in animals, and more particularly in humans and/or non-human mammals. [0068] Pharmaceutically acceptable salt: The term “pharmaceutically acceptable salt” as used herein refers to acid addition salts or base addition salts of compounds, such as peptides in the present disclosure. A pharmaceutically acceptable salt is any salt which retains the activity of the parent compound and does not impart any deleterious or undesirable effect on a subject to whom it is administered and in the context in which it is administered. Pharmaceutically acceptable salts may be derived from amino acids including, but not limited to, cysteine. Methods for producing compounds as salts are known to those of skill in the art (see, for example, Stahl et al., Handbook of Pharmaceutical Salts: Properties, Selection, and Use, Wiley-VCH; Verlag Helvetica Chimica Acta, Zurich, 2002; Berge et al., J Pharm. Sci. 66: 1, 1977). In some embodiments, a “pharmaceutically acceptable salt” is intended to mean a salt of a free acid or base of a compound represented herein that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, Berge, et al., J. Pharm. Sci., 1977, 66, 1-19. Preferred pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of subjects without undue toxicity, irritation, or allergic response. A compound described herein may possess a sufficiently acidic group, a sufficiently basic group, both types of functional groups, or more than one of each type, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.

[0069] Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne- 1,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, methylsulfonates, propylsulfonates, besylates, xylenesulfonates, naphthalene- 1 -sulfonates, naphthal ene-2-sulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y-hydroxybutyrates, glycolates, tartrates, and mandelates. [0070] The terms “pharmaceutically acceptable carrier” as used herein refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and/or vehicle with which a compound, such as a peptide, is administered. Such earners may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a earner. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. See, e.g., Remington, The Science and Practice of Pharmacy, 20th ed., (Lippincott, Williams & Wilkins 2003). Except insofar as any conventional media or agent is incompatible with the active compound, such use in the compositions is contemplated.

[0071] The peptides of the present invention can be prepared for delivery or storage in a lyophilized form such as well known to those skilled in the art.

[0072] The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound and a combination partner (e.g., another drug as explained below, also referred to as “therapeutic agent” or “co-agenf ’) may be administered independently at the same time or separately within time intervals, especially where these time intervals allow that the combination partners show a cooperative, e g., synergistic effect. The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one active ingredient and includes both fixed and nonfixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.

[0073] The pharmaceutical compositions, alone or in combination with other active ingredient(s), described herein may be formulated as solutions, emulsions, suspensions, or dispersions in suitable pharmaceutical solvents or carriers, or as pills, tablets, lozenges, suppositories, sachets, dragees, granules, powders, powders for reconstitution, or capsules along with solid earners according to conventional methods known in the art for preparation of various dosage forms. A peptide, alone or in combination with other active ingredient(s), described herein, and preferably in the form of a pharmaceutical composition, may be administered by a suitable route of delivery, such as oral, parenteral, rectal, nasal, topical, or ocular routes, or by inhalation. In some embodiments, the compositions are formulated for intravenous or oral administration.

[0074] For oral administration, a peptide, alone or in combination with another active ingredient, may be provided in a solid form, such as a tablet or capsule, or as a solution, emulsion, or suspension. To prepare the oral compositions, a peptide, alone or in combination with other active ingredient(s), may be formulated to yield a dosage of, e.g., from about 0.01 to about 50 mg/kg daily, or from about 0.05 to about 20 mg/kg daily, or from about 0.1 to about 10 mg/kg daily. Oral tablets may include the active ingredient(s) mixed with compatible pharmaceutically acceptable excipients such as diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservative agents. Suitable inert fillers include sodium and calcium carbonate, sodium and calcium phosphate, lactose, starch, sugar, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol, and the like. Exemplary liquid oral excipients include ethanol, glycerol, water, and the like. Starch, polyvinyl-pyrrolidone (PVP), sodium starch glycolate, microcrystalline cellulose, and alginic acid are exemplary disintegrating agents. Binding agents may include starch and gelatin. The lubricating agent, if present, may be magnesium stearate, stearic acid, or talc. If desired, the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract, or may be coated with an enteric coating.

[0075] Capsules for oral administration include hard and soft gelatin capsules. To prepare hard gelatin capsules, active ingredient(s) may be mixed with a solid, semi-solid, or liquid diluent. Soft gelatin capsules may be prepared by mixing the active ingredient with water, an oil, such as peanut oil or olive oil, liquid paraffin, a mixture of mono and di-glycerides of short chain fatty acids, polyethylene glycol 400, or propylene glycol.

[0076] Liquids for oral administration may be in the form of suspensions, solutions, emulsions, or syrups, or may be lyophilized or presented as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid compositions may optionally contain: pharmaceutically-acceptable excipients such as suspending agents (for example, sorbitol, methyl cellulose, sodium alginate, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel and the like); non-aqueous vehicles, e.g., oil (for example, almond oil or fractionated coconut oil), propylene glycol, ethyl alcohol, or water; preservatives (for example, methyl or propyl p-hydroxybenzoate or sorbic acid); wetting agents such as lecithin; and, if desired, flavoring or coloring agents.

[0077] The compositions may be formulated for parenteral use, including intravenous, intramuscular, intraperitoneal, intranasal, or subcutaneous routes, a peptide, alone or in combination with other active ingredient(s), may be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity or in parenterally acceptable oil. Suitable aqueous vehicles can include Ringer's solution and isotonic sodium chloride. Such forms may be presented in unit-dose form such as ampoules or disposable injection devices, in multi-dose forms such as vials from which the appropriate dose may be withdrawn, or in a solid form or pre-concentrate that can be used to prepare an injectable formulation. Illustrative infusion doses range from about 1 to 1000 pg/kg/minute of agent admixed with a pharmaceutical carrier over a period ranging from several minutes to several days. [0078] For nasal, inhaled, or oral administration, a peptide, alone or in combination with other active ingredient(s), may be administered using, for example, a spray formulation also containing a suitable carrier.

[0079] For topical applications, a peptide, alone or in combination with other active ingredient(s), are preferably formulated as creams or ointments or a similar vehicle suitable for topical administration. For topical administration, a peptide, alone or in combination with other active ingredient(s), may be mixed with a pharmaceutical carrier at a concentration of about 0.1% to about 10% of drug to vehicle. Another mode of administering a peptide, alone or in combination with other active ingredient(s), may utilize a patch formulation to effect transdermal delivery.

[0080] One of ordinary skill in the art may modify the formulations within the teachings of the specification to provide numerous formulations for a particular route of administration. In particular, a peptide, alone or in combination with other active ingredient(s), may be modified to render them more soluble in water or other vehicle. It is also well within the ordinary skill of the art to modify the route of administration and dosage regimen of a particular peptide, alone or in combination with other active ingredient(s), in order to manage the pharmacokinetics of the present compounds for maximum beneficial effect in a patient.

[0081] The disclosure is now described with reference to the following Examples. These Examples are provided for the purpose of illustration only and the disclosure should in no way be construed as being limited to these Examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0082] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the methods of the present disclosure and practice the claimed methods. The following working examples therefore, specifically point out embodiments of the present disclosure, and are not to be construed as limiting in any way the remainder of the disclosure. Examples

[0083] The peptide derivative Z-Arg-Lys-AOMK (1) was developed as a neutral pH-selective inhibitor of cathepsin B.⁵ Cathepsin B is believed to be released upon cellular injury from acidic lysosomes into the neutral pH cytoplasm and there initiate and mediate inappropriate proteolytic degradation. Such sequelae are thought to be relevant to the pathogenesis observed with traumatic brain injury and neuropathies such as Alzheimer’s Disease. Ongoing in-vivo evaluations of Z-Arg-Lys-AOMK for its potential neurotherapeutic potential necessitated the scaled-up synthesis of this inhibitor.⁵ Upon embarking on this endeavor, several problems were encountered with the methodology employed in the previously published synthesis (Figure 1), which utilized solid phase peptide synthesis (SPPS) and produced only a low yield Z-Arg-Lys-AOMK (1, Z-R-K- AOMK).⁵ These problems included irreproducibility of some steps and very low yields for others. The step involving resin-loading upon a poly-(aminomethyl-styrene semicarbazide) posed significant challenges due to sensitivity of the semicarbazide linker to moisture, resulting in a very low yield at best.^5,8 The biggest challenge in this synthetic route was the required removal of the Fmoc protecting group during the resin loading step, a deprotection that resulted in the formation of multiple undesired products from reaction of the newly liberated free amine with the highly reactive electrophilic AOMK group (Figure 2A). Additionally, the published synthetic procedure required a final cumbersome preparative HPLC purification.⁹ In this example, multiple attempts to re-synthesize the desired compound Z-R-K-AOMK using these published procedures were unsuccessful. Therefore, this example reverted to a solution phase synthesis as described below.

[0084] In the revamped synthesis (Figure 2), commercially purchased Fmoc- Lys(Boc)-COCH2Cl was acylated with 2,6-dimethylbenzoic acid to produce Fmoc- Lys(Boc)-AOMK (4). Multiple attempts to remove the Fmoc group from 4 using different bases (diethylamine, triethylamine, and piperidine), under different organic solvent regimes (e.g. DMF or DCM), were unsuccessful (Figure 2A). Similarly, the reported ‘neutral’ H2/Pd deprotection of Fmoc was unproductive, presumably due to unwanted reactions of the revealed nucleophilic L-amine (Figure 2A).⁷ A careful analysis of the byproducts resulting from H2/Pd removal of the Fmoc group revealed that the deprotected a- amino group of Lys engaged in a nucleophilic attack on the electron-deficient carbon between the carbonyl ketone and the AOMK moiety, leading to the formation of complex polymeric side products (Figure 2A).

[0085] To address this issue, Fmoc-removal by Pd-promoted catalytic hydrogenolysis under mildly acidic conditions (Figure 2B) was explored, to produce a non- nucleophilic ammonium salt immediately upon Fmoc deprotection, thereby suppressing undesired side products. Indeed, the inclusion of acid within this hydrogenolysis process not only led to the creation of a masked salt derivative of the deprotected amine, but also effectively deterred the unwanted reduction of the ketone into a secondary alcohol. This improved synthetic procedure for Z-R-K-AOMK employs a solution phase synthetic strategy (Figure 2B) in which the resulting deprotected ammonium salt (10) was coupled with Z-Arg(Boc2)-OH to give compound 11 in high yield. Finally, the target compound Z-R-K-AOMK (1) was obtained as the TFA salt by simultaneous deprotection of all three Boc protecting groups using TFA/CH2CI2 in near quantitative yield, without the need for further purification (Figure 2B). The overall yield for the synthesis of Z-R-K-AOMK using the newly developed route is 40.5% while in the previous synthetic route to Z-R-K-AOMK (Figure 1) yields were not reported for the final steps.⁵

[0086] The cathepsin B inhibitory activity of the newly synthesized Z-R-K-AOMK (1) was assessed by IC50 and compared to that of authentic samples. Similar levels of cathepsin B inhibitory activity were observed at pH 4.6 and 7.2 for both newly synthesized compound 1 from this study and an authentic sample from the previous work (Figure 3).⁵

[0087] The success of this new route to 1 inspired exploration of the scope and limitations of these new Fmoc-removal reaction conditions, including the structure of the Bronsted acid, acid concentration and the molar equivalents of Pd (Table 1). One molecule used in this study, compound 4, contained the acid-sensitive Boc protecting group that we desired to not remove in this reaction sequence, as well as the potentially reactive electrophilic a-chloroketone functionality (Figure 2A). Hence, we avoided the use of strong acid conditions that might cause this Boc deprotection. Optimal conditions included 2-3 equivalents of HC1 in MeOH, which was sufficient to achieve the efficient in-situ Fmoc removal and conversion of the liberated amine to its deactivated ammonium salt. The number of Pd/C equivalents did not significantly impact the yield, however a 20% molar equivalent to the starting material was slightly more efficient. Summarizing, Fmoc- deprotection in the presence of a Bronsted acid yielded the desired amine salts in high yield while preserving the Boc protecting group and a-chloroketone functionality (Table 1, Entry 8).

Table 1. Optimization of removal conditions for the Fmoc protecting group using acidic hydrogenolysis.

* Commercial 10% w/w Pd on C and 1 atm of Hz

* * Isolated yields when greater than 10%.

*** For HCl/MeOH, a 3M stock solution was used

⁺ In all entries the reactions were run for 12 hours, except for entry #9 for which the reaction time was 4 hours.

NP: No product was observed

[0088] The scope and limitations of the method in the presence of other electrophiles were explored. Michael acceptors are defined by carbon double bonds conjugated to powerful electron- withdrawing substituents. ^{10 12} However, in many cases an undesired by-product results from the reaction of the amines used for the deprotection of Fmoc (or the resulting deprotected amine) and the enone in another molecule. Deprotection of Fmoc using acidic hydrogenolysis offers a non-basic option for the deprotection and in-situ protection of the resulting deprotected amine. Because no secondary amines have been used in the deprotection sequence, after removal of the Fmoc group, the reaction can be done in a single pot by addition of the enone to achieve the Michael addition product (Figure 4B). Various intramolecular Michael addition reactions can be employed using this method. Similarly, the Mannich reaction involving a three- component acid-catalyzed reaction of aldehydes or ketones with 1° or 2° amines to produce 0-amino-carbonyl compounds, is conveniently performed following Fmoc deprotection.¹³ The Fmoc group of compound 17 was removed under acid catalyzed hydrogenolysis conditions to produce the amine salt. This amine was neutralized in-situ and reacted with formaldehyde and furan to produce product 19 in quantitative yield (Figure 4C). To date, amine protection for this type of reaction has been limited to the Boc group due to the traditional incompatibility of the deprotection conditions for Fmoc which require the use of a secondary amine.

[0089] Fmoc removal using Fb/Pd in acidic media directly delivers the ammonium salt, preventing engagement in adverse nucleophilic reactions with electrophiles. In the case of Z-R-K-AOMK (1), this reaction was utilized to achieve a clean removal of the Fmoc group, providing the deactivated HC1 salt form of the resulting amine. This approach effectively avoided the undesired nucleophilic side reaction with the AOMK moiety. Furthermore, the application of these conditions was expanded to the deprotection of amines in the presence of a-chloroketone moieties; this reaction yielded the desired deprotected amines in quantitative yield. The use of Fh/Pd in acidic media for Fmoc- protected amine deprotection, along with the concurrent in-situ deactivation of the resulting free amine, represents a promising and versatile method that can find broad applicability in amine deprotection strategies to mitigate undesired side reactions in the presence of highly reactive electrophiles.

REFERENCES

(1) Li, W.; O’Brien-Simpson, N. M.; Hossain, M. A.; Wade, J. D.; Li, W.; O’Brien- Simpson, N. M.; Hossain, M. A.; Wade, J. D. The 9-Fluorenylmethoxy carbonyl (Fmoc) Group in Chemical Peptide Synthesis - Its Past, Present, and Future. Aust. J. Chem. 2019, 73 (4), 271-276. https://doi.org/10.1071/CH19427.

(2) Behrendt, R.; White, P.; Offer, J. Advances in Fmoc Solid-Phase Peptide Synthesis. J. Pept. Set. 2016, 22 (1), 4-27. https://doi.org/10.1002/PSC.2836.

(3) Ralhan, K.; Krishnakumar, V. G.; Gupta, S. Piperazine and DBU: A Safer Alternative for Rapid and Efficient Fmoc Deprotection in Solid Phase Peptide Synthesis. https://doi.org/10.1039/x0xx00000x.

(4) Harris, P. W. R.; Brimble, M. A. A Comparison of Boc and Fmoc SPPS Strategies for the Preparation of C-Terminal Peptide a-Thiolesters: NY-ESO-1 39Cys-68Ala-COSR. Pept. Sci. 2013, 700 (4), 356-365. https://doi.org/10.1002/BIP.22223. (5) Yoon, M. C.; Solania, A.; Jiang, Z.; Christy, M. P.; Podvin, S.; Mosier, C.; Lietz, C. B.; Ito, G.; Gerwick, W. H.; Wolan, D. W.; Hook, G.; O’Donoghue, A. J.; Hook, V. Selective Neutral PH Inhibitor of Cathepsin B Designed Based on Cleavage Preferences at Cytosolic and Lysosomal PH Conditions. ACS Chem. Biol. 2021, 16 (9), 1628. https ://doi. org/10. 1021/AC SCHEMBIO. 1 COO 138.

(6) Leggio, A.; Liguori, A.; Napoli, A.; Siciliano, C.; Sindona, G. New Strategies for an Efficient Removal of the 9-Fluorenylmethoxycarbonyl (Fmoc) Protecting Group in the Peptide Synthesis. European J. Org. Chem. 2000, 2000 (4), 573-575. https://doi.Org/https://doi.org/10. 1002/(SICI)1099-0690(200002)2000:4<573::AID- EJOC573>3,O.CO;2-I.

(7) Maegawa, T.; Fujiwara, Y.; Ikawa, T.; Hisashi, H.; Monguchi, Y.; Sajiki, H. Novel Deprotection Method of Fmoc Group under Neutral Hydrogenation Conditions. Amino Acids 2009, 36 (3), 493-499. https://doi.org/10.1007/S00726-008-0109-7/FIGURESZ2.

(8) Vazquez, J.; Albericio, F. A Convenient Semicarbazide Resin for the Solid-Phase Synthesis of Peptide Ketones and Aldehydes. Tetrahedron Lett. 2006, 47 (10), 1657-1661. https://doi.Org/10.1016/J.TETLET.2005.12.101.

(9) NMR Data Was Not Reported for the Final Products in the Previous Report (Reference 5).

(10) Zhang, Y.; Wang, W. Recent Advances in Organocatalytic Asymmetric Michael Reactions. Catal. Sci. Technol. 2011, 2 (1), 42-53. https://doi.org/10.1039/ClCY00334H.

(11) Malkar, R. S.; Jadhav, A. L.; Yadav, G. D. Innovative Catalysis in Michael Addition Reactions for C-X Bond Formation. Mol. Catal. 2020, 485, 110814. https://doi.Org/10.1016/J.MCAT.2020.110814.

(12) Poon, T.; Mundy, B. P.; Shattuck, T. W. The Michael Reaction. J. Chem. Educ.

2002, 79 (2), 264. https://doi.org/10.1021/ED079P264/SUPPL_FILE/JCE2002P0264W.ZIP. (13) Shi, Y.; Wang, Q.; Gao, S. Recent Advances in the Intramolecular Mannich Reaction in Natural Products Total Synthesis. Org. Chem. Front. 2018, 5 (6), 1049-1066. https://doi.org/10.1039/C7Q001079F.

(14) Wood, W. J. L.; Huang, L.; Ellman, J. A. Synthesis of a Diverse Library of Mechanism-Based Cysteine Protease Inhibitors. J. Comb. Chem. 2003, 5 (6), 869-880. https://doi.Org/10.1021/CC034008R/SUPPL_FILE/CC034008RSI20030518_111248.PDF.

Claims

CLAIMS What is claimed is:

1. A method of deprotection, the method comprising: providing a precursor compound comprising a Fmoc-protected amine; and deprotecting the Fmoc-protected amine under acidic hydrogenolysis conditions to prepare a compound.

2. The method of claim 1, wherein the acidic hydrogenolysis conditions are produced by a reaction medium comprising an acid, a metal, and a liquid.

3. The method of claim 2, wherein the acid comprises a Bronsted acid.

4. The method of claim 2, wherein about 2 to about 3 equivalents of the acid, relative to the precursor compound, are present in the reaction medium.

5. The method of claim 2, wherein the metal comprises a platinum group metal.

6. The method of claim 5, wherein the metal comprises Pd.

7. The method of claim 2, wherein the metal is present in the reaction medium at a relative amount of about 10 % to about 20 % molar metal/C equivalents.

8. The method of claim 2, wherein the liquid comprises a solvent in which (i) the precursor compound is soluble, (ii) the compound is soluble, or (iii) both the precursor compound and the compound are soluble.

9. The method of claim 2, wherein the liquid comprises a polar organic liquid.

10. The method of claim 9, wherein the polar organic liquid comprises methanol.

11. The method of claim 1, wherein the acidic hydrogenolysis conditions are produced by a reaction medium comprising Pd, a hydrogen halide, and a C1-C5 alcohol.

12. The method of claim 1, wherein the Fmoc-protected amine is a Fmoc-protected primary amine or an Fmoc-protected secondary amine.

13. The method of claim 1, wherein the compound comprises a deprotected amine from which the Fmoc protecting group is removed, and the deprotected amine has a suppressed nucleophilic reactivity.

14. The method of claim 1, wherein the precursor compound comprises a precursor peptide, and the compound comprises a peptide.

15. The method of claim 14, wherein the precursor peptide and the peptide comprise a protecting group other than Fmoc.

16. The method of claim 15, wherein the protecting group other than Fmoc comprises Boc.

17. The method of claim 1, wherein the precursor compound and the compound comprise an amine protected by an acid-sensitive protecting group, and the acidic hydrogenolysis conditions do not remove the acid-sensitive protecting group from the precursor compound and the compound.

18. A composition comprising a peptide that has been deprotected under acidic hydrogenolysis conditions.

19. A pharmaceutical composition comprising: the compound prepared according to the method of any one of claims 1 to 17; and a pharmaceutically acceptable carrier.

20. A method of preventing or treating a condition or disease, the method comprising: administering to a subject in need thereof an effective amount of a pharmaceutical composition of a peptide that has been deprotected under acidic hydrogenolysis conditions.