Process for preparing a peptide

Abstract

The present disclosure is directed to an efficient and scalable process for synthesizing a crystalline form of a compound of Formula I.

Description

Attorney Docket No.: 771417: MKRA-026PCPROCESS FOR PREPARING A PEPTIDECROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application No. 63 / 717,609 filed November 7, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing electronically submitted as an XML Document file entitled “771417_MKRA-026PC_SEQUENCE_LISTING.xml” created on November 4, 2025, and having a size of 136,281 bytes. The information contained in the Sequence Listing is incorporated by reference herein.FIELD OF USE

[0003] The present disclosure relates to a process for preparing a crystalline peptide of FormulaI:wherein A is an anion, a process for preparing intermediates to make a compound of Formula I or a pharmaceutically acceptable salt thereof, as well as crystalline forms of intermediates.BACKGROUND

[0004] Identification of compounds and / or agents effective in the treatment of cardiovascular affliction is highly desirable. Also desirable are compounds and / or agents effective in the antagonism of PCSK9’s role in LDL regulation. However, because PCSK9 circulates in blood- 1 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC and has modest binding affinity to cell surface LDL receptors, previous attempts to utilize this mechanism in treatment of diseases related to high serum LDL levels have been focused on the use of large biomolecules, such as antibodies. The therapeutic potential of small peptides or small molecules as drugs targeting PCSK9 has only just begun to be explored; see for example, Tombling et al., Atherosclerosis 330 (2021) 52-60. Moreover, there is a paucity of compounds that are amenable to being formulated into a dosage form for oral administration, which would be highly desirable for the provision of therapy for conditions in which regulation of the activities of PCSK9 could play a role.

[0005] As therapeutic agents, macrocyclic peptides combine a high degree of specific binding with more favorable pharmacological properties, such as metabolic stability and cell permeability, compared to linear peptides. However, the practicality and economics of the chemical synthesis of macrocyclic peptides on a manufacturing scale presents significant challenges. In particular, chemo- and regioselectivity challenges, arising from competing functional groups in amino acids and peptides, typically require extensive use of protecting groups and complex purification steps, thereby decreasing the efficiency of the synthesis. Furthermore, macrocyclization of peptides is usually achieved using stoichiometric chemical coupling reagents and high dilution to minimize the formation of undesirable oligomers, limiting its application on a manufacturing scale.

[0006] WO 2019 / 246349 discloses cyclic peptide compounds, including a compound of Formula I, useful in the treatment of cardiovascular disease and conditions related to PCSK9 activity. WO 2024 / 040125 discloses crystalline forms of a compound of Formula I. The present disclosure advances the state of the art by providing cost-effective, efficient, and scalable process for manufacturing such compounds.SUMMARY

[0007] The present disclosure is directed to an efficient and scalable process for synthesizing a crystalline form of a compound of Formula I- 2 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCwherein A is an anion. Formula I corresponds to an enlicitide salt. In some embodiments, Ar is caprate, or decanoate. As such, the present disclosure is directed to an efficient and scalable process for synthesizing one or more crystalline forms of Compound 7.

[0008] In some aspects, A is suberate. As such, the present disclosure is directed to an efficient and scalable process for synthesizing one or more crystalline forms of Compound 6.

[0009] The instant process is a significantly more convergent route than prior syntheses, with only 10 linear steps. The process of the present disclosure requires fewer protecting groups than prior methods of making a compound of Formula I, which allows for a more efficient synthesis by decreasing the number of steps and operations. This process utilizes both chemical and enzymatic steps to provide an effective and efficient manufacturing process. By using an enzyme in the present disclosure, the process can use a lower volume of solvents during the macrocyclization step, which significantly increases production of a compound of Formula I. The utilization of enzymes for macrocyclizations also enables the reaction to be carried out under catalytic conditions in a more environmentally friendly solvent mixture and at higher concentration to maximize production efficiency on manufacturing scale.

[0010] Unlike other synthetic processes, the process of the present disclosure synthesizes a compound of Formula I without the isolation of intermediates. Additionally, the present disclosure allows one to control the anion, such that the salt of the compound of Formula I can be switched to increase purity.

[0011] The present disclosure is also directed to crystalline forms of Compound 6, or a solvate thereof. In some aspects, crystalline solvate forms Form I, Form II, Form III, and Form IV of Compound 6 are provided.- 3 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0012] Other embodiments, aspects, and features of the present disclosure are either further described in or will be apparent from the ensuing description, examples, and appended claims.

[0013] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1 is an X-ray powder diffraction pattern of a crystalline Form I of Compound 6. The graph plots the intensity of the peaks as defined by counts per second versus the diffraction angle 2 theta (29) in degrees.

[0015] FIG. 2 is an X-ray powder diffraction pattern of a crystalline Form II of Compound 6. The graph plots the intensity of the peaks as defined by counts per second versus the diffraction angle 2 theta (29) in degrees.

[0016] FIG. 3 is an X-ray powder diffraction pattern of a crystalline Form III of Compound 6. The graph plots the intensity of the peaks as defined by counts per second versus the diffraction angle 2 theta (29) in degrees.

[0017] FIG. 4 is an X-ray powder diffraction pattern of a crystalline Form IV of Compound 6. The graph plots the intensity of the peaks as defined by counts per second versus the diffraction angle 2 theta (29) in degrees.DETAILED DESCRIPTION

[0018] The present disclosure pertains to a scalable synthesis for preparing a crystalline form of a compound of Formula I:- 4 -81358249. v1Atorney Docket No.: 771417: MKRA-026PCI wherein A is an anion. In one embodiment, the present disclosure is directed to a chemo- enzymatic process comprising a two-step sequence using Compound 1 and Compound 3, followed by two consecutive crystallizations to yield a compound of Formula I.

[0019] In a first embodiment of the present disclosure, the process for preparing a crystalline form of a compound of Formula I, comprises the steps of: a) combining Compound 11 with Compound 3to obtain a reaction product comprising Compound 4- 5 -81358249. v1Attomey Docket No.: 771417: MKRA-026PCwherein X is a mixture of counterions thereof; and b) adding a macrocyclase enzyme to the reaction product comprising Compound 4 to obtain a crystalline form of a compound of Formula I.

[0020] In a further embodiment, the process for preparing a crystalline form of a compound ofFormula I, comprises the steps of: a) combining Compound 1 with an aldehyde, a trialkylamine base and acetonitrile to obtain a mixture comprising Compound 22 b) reacting the mixture comprising Compound 2 with a coupling reagent andCompound 3 to obtain a reaction product comprising Compound 4;- 6 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC c) adding a macrocyclase enzyme and a buffer solution to the reaction product comprising Compound 4; and d) adding an acid to obtain a crystalline form of a compound of Formula I.

[0021] In a second embodiment, the present disclosure is directed to a process for preparing Compound 6, or a solvate thereof,comprising the steps of: a) combining Compound 1 with salicylaldehyde, triethylamine (TEA or EtsN) and acetonitrile to obtain a mixture comprising Compound 2; b) reacting the mixture comprising Compound 2 with diphenylphosphinic chloride(DPPC1) and Compound 3 to obtain a reaction product comprising Compound 4wherein X is mixture of chloride, 1,2-ethanedi sulfonate or diphenylphosphinate; c) adding a macrocyclase enzyme and a solution comprising 3-(N- morpholino)propanesulfonic acid (MOPS), water and MeCN to the reaction product comprising- 7 -81358249. v1Atorney Docket No.: 771417: MKRA-026PCCompound 4 to obtain a solution comprising a compound of Formula I wherein A is a mixture of chloride, 3-(N-morpholino)propanesulfonate, or 1,2-ethane disulfonate; d) combining the solution comprising a compound of Formula I from step c with aqueous potassium bicarbonate and acetonitrile to obtain a solution comprising Compound 8e) adding suberic acid and a solution of n-propanol (nPrOH) and Methyl tert-butyl ether (MTBE) to the solution comprising Compound 8 to obtain a crystalline form of Compound 6, or a solvate thereof. In various embodiments, the nPrOH is 1-propanol (1-PrOH).

[0022] In some embodiments, the solution of nPrOH and MTBE in step (e) of the second embodiment comprises MTBE and 1-PrOH in an about 3:1 ratio, 2.5:1 ratio, 2.4:1 ratio, or an about 2: 1 ratio (v / v) of MTBE to 1-PrOH. In some embodiments, the solution of nPrOH and MTBE comprises an about 2.4:1 ratio (v / v). In some embodiments, this solution comprises MTBE and 1-PrOH in a 2.4: 1 ratio (v / v).

[0023] In a third embodiment, the present disclosure is directed to a process for preparing a crystalline form of Compound 781358249. v1Attorney Docket No.: 771417: MKRA-026PC comprising the steps of: a) combining Compound 1 with salicylaldehyde, TEA and acetonitrile to obtain a mixture comprising Compound 2; b) reacting the mixture comprising Compound 2 with DPPC1 and Compound 3 to obtain a reaction product comprising Compound 4, wherein X is a mixture of chloride, 1,2- ethanedi sulfonate or diphenylphosphinate; c) adding a macrocyclase enzyme and a solution comprising MOPS, water and MeCN to the reaction product comprising Compound 4 to obtain a solution comprising a compound of Formula I wherein A is a mixture of chloride, 3-(N-morpholino)propanesulfonate and 1,2-ethanedisulfonate; d) combining the solution comprising the compound of Formula I from step c with aqueous potassium bicarbonate and acetonitrile to obtain a solution comprising Compound 8; e) adding suberic acid and a solution of nPrOH and MTBE to the solution comprising Compound 8 from step d to obtain a crystalline form of Compound 6, or a solvate thereof; f) combining Compound 6, or a solvate thereof, with aqueous potassium bicarbonate and acetonitrile to obtain a solution comprising Compound 8; and g) adding capric acid and a solution of nPrOH and MTBE to the solution comprising Compound 8 of the prior step to obtain a crystalline form of Compound 7. In various embodiments, the nPrOH is 1 -propanol (1-PrOH).

[0024] In some embodiments, the solution of nPrOH and MTBE in step (e) of the third embodiment comprises MTBE and 1-PrOH in an about 3:1 ratio, 2.5:1 ratio, 2.4:1 ratio, or an about 2: 1 ratio (v / v) of MTBE to 1-PrOH. In some embodiments, the solution of nPrOH and MTBE comprises an about 2.4:1 ratio (v / v). In some embodiments, this solution comprises MTBE and 1-PrOH in a 2.4: 1 ratio (v / v).

[0025] In some aspects, the present disclosure is directed to Compound 4:- 9 -81358249. v1Atorney Docket No.: 771417: MKRA-026PCwherein X is a mixture of counterions. In a further embodiment, X is a mixture of chloride, 1,2- ethanedi sulfonate or diphenylphosphinate.

[0026] In an embodiment, the present disclosure is directed to Compound 6, or a solvate thereof:

[0027] In an embodiment, the disclosure is directed to a crystalline form of Compound 6:- 10 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0028] In a further embodiment, the crystalline form is a crystalline solvate form of Compound 6.

[0029] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form I) can include one or more peaks from Table 1.

[0030] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form I) has an X-ray diffraction pattern substantially similar to that set forth in Figure 1.

[0031] In an embodiment, the crystalline form of Compound 6 (Form I) is characterized by an X-ray powder diffraction pattern having peaks, expressed in degrees-2-theta at angles of (±0.2°) measured using Cu Ka radiation, selected from the group consisting of about 7.83 and about 16.23° 29. That is, this diffraction pattern includes peaks at about 7.83 and about 16.23° 29, expressed in degrees-2-theta at angles of (±0.2°), measured using Cu Ka radiation. In a further embodiment, the crystalline form of Compound 6 (Form I) is characterized by an X-ray powder diffraction pattern having peaks, expressed in degrees-2-theta at angles of (±9.2°) measured using Cu Ka radiation, selected from the group consisting of about 7.83, 16.23 and 18.76° 29. In a further embodiment, the crystalline form of Compound 6 (Form I) is characterized by an X-ray powder diffraction pattern having peaks, expressed in degrees-2-theta at angles of (±9.2°) measured using Cu Ka radiation, selected from the group consisting of about 4.53, 7.54, 7.83, 8.98, 19.16, 12.79, 16.23, 17.51 and 18.76° 29. In various embodiments, the X-ray powder diffraction pattern is obtained using a Cu Ka radiation source (1.54 A).

[0032] In another embodiment, the crystalline Form I is characterized by an X-ray powder diffraction pattern having peaks shown in Table 1 (expressed in degrees-2-theta at angles ±0.2°).

[0033] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form II) can include one or more peaks from Table 2.

[0034] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form II) has an X-ray diffraction pattern substantially similar to that set forth in Figure 2.

[0035] In an embodiment, the X-ray powder of the crystalline form of Compound 6 (Form II) is characterized by an X-ray powder diffraction pattern having peaks, expressed in degrees-2- theta at angles of (±0.2°) measured using Cu Ka radiation, selected from the group consisting of about 3.95 and 5.99° 29. In an embodiment, the X-ray powder of the crystalline form of Compound 6 (Form II) is characterized by an X-ray powder diffraction pattern having peaks, expressed in degrees-2-theta at angles of (±9.2°) measured using Cu Ka radiation, selected from the group consisting of about 3.95, 5.99, 9.93 and 9.77° 29. In another embodiment, the- 11 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC crystalline Form II is characterized by an X-ray powder diffraction pattern having peaks shown in Table 2 (expressed in degrees-2-theta at angles ±0.2°).

[0036] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form III) can include one or more peaks from Table 3.

[0037] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form III) has an X-ray diffraction pattern substantially similar to that set forth in Figure 3.

[0038] In an embodiment, the crystalline form of Compound 6 (Form III) is characterized by an X-ray powder diffraction pattern that includes peaks at about 6.24 and about 6.84° 29. In a further embodiment, the crystalline form of Compound 6 (Form III) is characterized by an X-ray powder diffraction pattern that includes peaks at about 4.34, 6.24 and 6.84° 29. In a further embodiment, the crystalline form of Compound 6 (Form III) is characterized by an X-ray powder diffraction pattern that includes peaks at about 4.34, 5.23, 6.24, 6.84, 9.72, 14.45, 18.24, and 29.92° 29.

[0039] In another embodiment, the crystalline Form III is characterized by an X-ray powder diffraction pattern having peaks shown in Table 3 (expressed in degrees-2-theta at angles ±0.2°).

[0040] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form IV) can include one or more peaks from Table 4.

[0041] In an embodiment, the X-ray powder diffraction pattern of the crystalline form of Compound 6 (Form IV) has an X-ray diffraction pattern substantially similar to that set forth in Figure 4.

[0042] In an embodiment, the crystalline form of Compound 6 (Form IV) is characterized by an X-ray powder diffraction pattern that includes peaks, expressed in degrees-2 -theta at angles of (±0.2°) measured using Cu Ka radiation, at about 6.93 and about 8.10° 29. In a further embodiment, the crystalline form of Compound 6 (Form IV) is characterized by an X-ray powder diffraction pattern that includes peaks at about 6.93, 8.10 and 10.24° 29. In a further embodiment, the crystalline form of Compound 6 (Form IV) is characterized by an X-ray powder diffraction pattern that includes peaks at about 6.93, 8.10, 8.77, 10.24, 11.77, 12.70, 14.78, 15.40, 16.39, 17.68, 18.81, and 20.41° 29.

[0043] In another embodiment, the crystalline Form IV is characterized by an X-ray powder diffraction pattern having peaks shown in Table 4 (expressed in degrees-2-theta at angles ±0.2°).

[0044] In the embodiments of the instant disclosure, the processes of the disclosure may be conducted as a through process, or the steps may be conducted sequentially. For clarity, it should be noted that steps and reactions of the present disclosure may occur simultaneously, or- 12 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC sequentially, unless otherwise specifically designated. In embodiments, the intermediate products may optionally be isolated.

[0045] In an embodiment, the aldehyde is selected from the group consisting of salicylaldehyde, benzaldehyde, 2 -hydroxy-3 -methoxybenzaldehyde, 2-hydroxy-3- methylbenzaldehyde, 2-hydroxy-5-methoxybenzaldehyde, 3,5-dibromo-2-hydroxybenzaldehyde, 5-bromo-2-hydroxybenzaldehyde, picolinaldehyde and the like. In a further embodiment, the aldehyde is salicylaldehyde.

[0046] In an embodiment, the trialkylamine base is selected from the group consisting of TEA, DIPEA, DEMA and the like. In a further embodiment, the trialkylamine base is TEA.

[0047] In an embodiment, the coupling reagent is selected from the group consisting of DPPC1, T3P, HATU, EDC, HC1 and the like. In a further embodiment, the coupling reagent is DPPC1.

[0048] In an embodiment, the buffer solution is selected from the group consisting of MOPS, Tris, Borate, Bicine, Bis-Tris, Bis-Tris propane, EPPS, HEPES, Triethanolamine, and mixtures thereof. In a further embodiment, the buffer solution is a mixture of MOPS.

[0049] In an embodiment, the acid is selected from the group consisting of suberic acid, capric acid, sebacic acid, adipic acid and the like. In a further embodiment, the acid is suberic acid. In a further embodiment, the acid is capric acid.

[0050] In an embodiment, A is defined as an anion, which is independently selected from the group consisting of caprate, suberate, bicarbonate, chloride, 3-(N-morpholino)propanesulfonate or 1,2-ethanedi sulfonate or mixtures thereof. In a particular embodiment, Ar is caprate. In an embodiment, Ar is suberate. In an embodiment, Ar is bicarbonate. In an embodiment, Ar is chloride, 3-(N-morpholino)propanesulfonate or 1,2-ethanedisulfonate or mixtures thereof.

[0051] In an embodiment, a macrocyclase enzyme as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73. In a further embodiment, the macrocyclase enzyme is selected from SEQ ID NO: 73. In a further embodiment, the macrocyclase enzyme is selected from SEQ ID NO: 70. The disclosure of U.S. Ser. No.63 / 717,514, filed on November 7, 2024, which is directed to the macrocyclase enzymes is herein incorporated by reference in its entirety.

[0052] It should also be noted that when a term is used more than once, such as anion, aldehyde, or acid, the definition at each instance is independent of a prior selection. For example, the same, or a different, anion may be chosen for each step of the process independently of a previous selection.- 13 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCDefinitions

[0053] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0055] As used herein, the articles “a” and “an” refer to one or more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. As used herein, including the appended claims, the singular forms of words such as “a,” “an,” and “the,” include their corresponding plural references unless the context clearly dictates otherwise.

[0056] As used herein, the terms “at least one” item or “one or more” item each include a single item selected from the list as well as mixtures of two or more items selected from the list.

[0057] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values. As an example, temperature ranges, percentages, ranges of equivalents, and the like described herein include the upper and lower limits of the range and any value in the continuum there between. “About” when used to modify a numerically defined parameter (e.g., the temperature, the length of time for a reaction, or the peaks in an X-ray powder diffraction pattern, as described herein) means that the parameter may vary by as much as 10% below or above the stated numerical value for that parameter; where appropriate, the stated parameter may be rounded to the nearest whole number. For example, a temperature of about 30°C may vary between 25°C and 35°C. In addition, the term “or,” as used herein, denotes alternatives that may, where appropriate, be combined; that is, the term “or” includes each listed alternative separately.

[0058] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the- 14 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC presence of other ingredients / steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0059] The term “substantially similar” used in reference to X-ray powder diffraction patterns means that the X-ray powder diffraction pattern of a polymorph may display “batch-to-batch” variations due to differences in the types of equipment used for the measurements, and fluctuations in both experimental conditions (e.g. purity and grain size of the sample) and instrumental settings (e.g. X-ray wavelengths; accuracy and sensitivity of the diffractometer; and “instrumental drift”) normally associated with the X-ray diffraction measurement. Due to these variations, the same polymorph may not contain X-ray powder diffraction peaks at exactly the same positions or intensities shown in the figures disclosed herein. Accordingly, the term “about” used in reference to the peaks in an X-ray powder diffraction pattern takes into account these variations and a skilled artisan would readily appreciate the scope.

[0060] The compounds of the present disclosure may contain one or more asymmetric centers and can thus occur as “stereoisomers” including racemates and racemic mixtures, enantiomeric mixtures, single enantiomers, diastereomeric mixtures and individual diastereomers. Additional asymmetric centers may be present depending upon the nature of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and it is intended that all of the possible optical isomers and diastereomers in mixtures and as pure or partially purified compounds are included within the scope of this disclosure. The present disclosure is meant to comprehend all such isomeric forms of these compounds. When bonds to the chiral carbon are depicted as straight lines in the formulae of the disclosure, it is understood that both the (R) and (S) configurations of the chiral carbon, and hence both enantiomers and mixtures thereof, are embraced within a given formula. For example, Formula I shows the structure of the compound with the designation of specific stereochemistry. When the compounds of the present disclosure contain one chiral center, the term “stereoisomer” includes both enantiomers and mixtures of enantiomers, such as the specific 50:50 mixture referred to as a racemic mixture.

[0061] The independent syntheses of these diastereomers or their chromatographic separations may be achieved as known in the art by appropriate modification of the methodology disclosed herein. Their absolute stereochemistry may be determined by the X-ray crystallography of crystalline products or crystalline intermediates which are derivatized, if necessary, with a- 15 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC reagent containing an asymmetric center of known absolute configuration. If desired, racemic mixtures of the compounds may be separated so that the individual enantiomers are isolated. The separation can be carried out by methods well known in the art, such as the coupling of a racemic mixture of compounds to an enantiomerically pure compound to form a diastereomeric mixture, followed by separation of the individual diastereomers by standard methods, such as fractional crystallization or chromatography. The coupling reaction is often the formation of salts using an enantiomerically pure acid or base. The diastereomeric derivatives may then be converted to the pure enantiomers by cleavage of the added chiral residue. The racemic mixture of the compounds can also be separated directly by chromatographic methods utilizing chiral stationary phases, which methods are well known in the art. Alternatively, any enantiomer of a compound may be obtained by stereoselective synthesis using optically pure starting materials or reagents of known configuration by methods well known in the art.

[0062] Some of the compounds described herein may exist as tautomers which have different points of attachment of hydrogen accompanied by one or more double bond shifts. For example, a ketone and its enol form are keto-enol tautomers. The individual tautomers as well as mixtures thereof are encompassed by the compounds of the present disclosure.

[0063] The term “salts” refers to salts prepared from acceptable bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particular embodiments include the ammonium, calcium, magnesium, potassium, and sodium salts. Salts in the solid form may exist in more than one crystal structure and may also be in the form of hydrates. Salts derived from organic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, A,7V'-dibenzylethylene-diamine, diethylamine, 2- diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine, ethylenediamine, N- ethylmorpholine, 7V-ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

[0064] When the compound of the present disclosure is basic, salts may be prepared from acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic,- 16 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Additional examples of such acids include aryl sulfonic acids, such as but not limited to / ?-toluenesulfonic acid, 3- methyl-toluenesulfonic acid, 2-methyl-toluenesulfonic acid, benzenesulfonic acid, 2-naphthalene sulfonic acid, 2,6-naphtalene sulfonic acid, as well as hydrochloric acid, hydrobromic acid, sulfuric acid, acetic acid, phenyl acetic acid, trimethylacetic acid, tetrafluoroboric acid, tetraphenylboric acid, maleic acid, fumaric acid, oxalic acid, or camphorsulfonic acid. Particular embodiments include the citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, fumaric, and tartaric acids. If a compound of the present disclosure simultaneously contains acidic and basic groups in the molecule, the disclosure also includes, in addition to the salt forms mentioned, inner salts or betaines (zwitterions). Salts can be obtained from the compounds of the present disclosure by customary methods which are known to the person skilled in the art, for example, by combination with an organic or inorganic acid or base in a solvent or dispersant, or by anion exchange or cation exchange from other salts. The present disclosure also includes all salts of the compounds which, owing to low physiological compatibility, are not directly suitable for use in pharmaceuticals but which can be used, for example, as intermediates for chemical reactions or for the preparation of pharmaceutically acceptable salts.

[0065] Some of the compounds of the present disclosure may form solvates with water ( / .< ., a hydrate) or common solvents such as but not limited to, n-propanol and MTBE. Such solvates and hydrates, particularly the pharmaceutically acceptable solvates and hydrates, of the present compounds are likewise encompassed within the scope of this disclosure, along with unsolvated and anhydrous forms.

[0066] The preparation of the pharmaceutically acceptable salts and other typical pharmaceutically acceptable salts is more fully described by Berg et al., “Pharmaceutical Salts,” J. Pharm. Sci., 1977:66: 1-19.

[0067] The terms “engineered,” “recombinant,” “variant,” and “non-naturally occurring,” when used with reference to, e.g., a polynucleotide, polypeptide, or cell, refers to a material, or a material corresponding to the natural or native form of the material, or that has been modified in a manner that would not otherwise exist in nature. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0068] As used herein, “polynucleotide” and “nucleic acid’ refer interchangeably to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., - 17 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCDNA), or comprised of mixtures of ribo- and 2' deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded regions and doublestranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0069] The terms “macrocyclase enzyme” or “macrocyclase polypeptide” refer to a polypeptide having a capability of catalyzing an intramolecular amide or ester bond formation in the generation of macrocyclic peptides. The present disclosure describes the use of nonribosomal peptide synthetase (NRPS) macrocyclase enzymes capable of catalyzing amide bond formation in macrocycles or 11-mer peptides in a protecting group-free manner from activated aminoacylthioesters, aminoacyl-esters, and cyclic peptides. In embodiments, the NRPS macrocyclase enzymes described herein can couple two peptides that can be further used in the synthesis of multimer peptides or can macrocyclize linear peptides. The terms “macrocyclase enzyme,” “NRPS macrocyclase enzyme,” or “macrocyclase polypeptide” are used interchangeably herein.

[0070] Macrocyclase polypeptide as used herein includes naturally occurring (wild-type) macrocyclase polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. An exemplary wild-type macrocyclase enzyme known as Ulml6 is set forth herein as SEQ ID NO: 1 (nucleic acid sequence), SEQ ID NO: 2 (amino acid sequence), Genbank Accession ID ATU31793.1, and Uniprot ID A0A2D3E317. In some embodiments, a macrocyclase enzyme as disclosed and used herein may have an amino acid sequence selected from any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.Engineered Macrocyclase Polypeptides

[0071] This present disclosure utilizes macrocyclase enzymes (or macrocyclase polypeptides) capable of catalyzing amide bond formation in the generation of macrocyclic peptides.

[0072] In certain embodiments, a polypeptide (e.g., a macrocyclase polypeptide) described herein has an amino acid sequence comprising one or more amino acid differences as compared to a reference amino acid sequence of a wild-type polypeptide that result in an improved enzyme property.- 18 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCIn some embodiments, the NRPS macrocyclase enzymes described herein are the product of directed evolution from a wild-type NRPS macrocyclase from Streptomyces sp. KCB13F003 described in literature by Son et al. (J. Nat. Prod. 2017, 80, 3025-3031) and Matsuda, Zhai, et al. (Nat. Catal. 2020, 3, 507-515). This protein, Ulml6, is encoded by a polynucleotide of SEQ ID NO: 1 and has a polypeptide sequence as set forth below in SEQ ID NO: 2: MHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGTLTTWEFGTEEHA GGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPEAEAEAEIEVEAGS GTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGFLALPVGPWPAPG SFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDPGSLPAGGLAGSAA DLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADGWGPGLGRFGPAG NRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVDALRDADIDVGVH RPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGARELAQPLAHRTFSSRG AGFLGRFVTDARSDAVHALQYSGRTLLREAGESRRARSH (SEQ ID NO: 2).

[0073] In some examples, the polynucleotide of the wild-type NRPS macrocyclase enzyme described herein and used to discover the desired reactions has been codon optimized for production in specific hosts such as E. coli and a short poly-His polypeptide has been added at the N-terminus. Such construct is encoded by polynucleotide of SEQ ID NO: 3 and a polypeptide sequence as set forth below in SEQ ID NO: 4: MGSSHHHHHHSSGHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGT LTTWEFGTEEHAGGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPE AEAEAEIEVEAGSGTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGF LALPVGPWPAPGSFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDP GSLPAGGLAGSAADLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADG WGPGLGRFGPAGNRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVD ALRDADIDVGVHRPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGAREL AQPLAHRTFSSRGAGFLGRFVTDARSDAVHALQYSGRTLLREAGESRRARSH (SEQ ID NO: 4).

[0074] In other examples, the polynucleotide of the wild-type NRPS macrocyclase enzyme described herein and subsequently used for directed evolution has been codon optimized for production in specific hosts such as E. coli and a short poly-His polypeptide added at the C- terminus. Such construct is encoded by polynucleotide of SEQ ID NO: 5 and a polypeptide sequence as set forth below in SEQ ID NO: 6: MHGDYADPADCGAGDGAPVGLDLDRLARDCDVVGGQLALHHQGTLTTWEFGTEEHA GGRPVHVGSAFPYGSVTKAFTATAVLQLAGDGDLDLDRPVRELLPEAEAEAEIEVEAGS- 19 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCGTGARADGGHPALAATLRQLLSHTAGLPSDHDDERAPSLRRWLTGFLALPVGPWPAPG SFSYSNVGYGIAGRVVEAVTGLTWSEAVRDFLLHPLGTAITVLPTDPGSLPAGGLAGSAA DLVRLGRLHLDEPGDPDLARLADPDALREMARPTAGADPFGLADGWGPGLGRFGPAG NRWLGHDGTLDGATCHLRIHPGRGTVVALTTNSPTGQALWDAVVDALRDADIDVGVH RPAPPPAIAAAAFADCTGTYRNGDLAVTVGIDGPYLVLELPGGARELAQPLAHRTFSSRG AGFLGRFVTDARSDAVHALQYAGRTLLREAGESRRARSHGSHHHHHH (SEQ ID NO: 6)

[0075] In embodiments, the NRPS macrocyclase enzymes described herein are the result of directed evolution from an NRPS macrocyclase having the amino acid sequence of SEQ ID NO: 6.

[0076] In embodiments, NRPS macrocyclase enzymes of the disclosure may demonstrate improvements relative to the NRPS macrocyclase enzyme of SEQ ID NO: 6, such as increases in enzyme activity, regioselectivity, thermostability, organic cosolvent tolerance, and reduction in competing side reactions.

[0077] The present disclosure provides numerous exemplary NRPS macrocyclases capable of generating cyclic peptides. Those exemplary polypeptides were evolved from SEQ ID NO: 6 and exhibit improved properties, particularly improved activity in the conversion of various peptidyl- thioesters and peptidyl-esters. These exemplary engineered NRPS macrocyclase enzymes having macrocyclizing or intermolecular coupling activity have amino acid sequences that include two or more residue differences as compared to SEQ ID NO: 6 as depicted in the accompanying sequence listing with sequence identifiers SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.

[0078] Table A below provides exemplary engineered NRPS macrocyclase polypeptides with the ability to form macrocyclic peptides. Each row lists two SEQ ID NOs, with the odd number referring to the nucleotide sequence encoding the amino acid sequence provided by the even number. The residue differences are based on comparison to a reference sequence such as of SEQ ID NO: 6, an NRPS macrocyclase derived from Streptomyces sp. KCB13F003 that differs from the naturally occurring enzyme (SEQ ID NO: 2) in having a codon optimized sequence for E. coli expression and a short hexahistidine tag at the C-terminus. The column listing the number of mutations ( / .< ., residue changes) refers to the number of amino acid substitutions as compared to the NRPS thioesterase enzyme of SEQ ID NO: 6. Polypeptide SEQ ID NO: 48 contains a two- residue insertion and all subsequent polypeptides derived from this lineage are listed in Table B relative to SEQ ID NO: 48.- 20 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCTable A: Activity of NRPS Macrocyclase for Cyclic Peptide Formation Relative to SEQ ID NO: 6- 21 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 22 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCTable B: Activity of NRPS Macrocyclase for Cyclic Peptide Formation Relative to SEQ IDNO: 48- 23 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC

[0079] In some embodiments, a polypeptide (e.g., macrocyclase polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2, 4, or 6.

[0080] These differences between these variants and SEQ ID NO:2, 4, or 6 can be amino acid insertions, deletions, substitutions, or any combinations of such changes. In some embodiments, the amino acid sequence differences can comprise non-conservative, conservative, as well as a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the amino acid sequence differences are conservative amino acid substitutions. In other embodiments, the amino acid sequence differences are non-conservative amino acid substitutions.- 24 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0081] In some embodiments, the amino acid sequence comprises or consists of any amino acid sequence set forth in Table A or Table B.

[0082] In some embodiments, the amino acid sequence comprises or consists of any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73. In some embodiments, the amino acid sequence comprises SEQ ID NO: 73. In some embodiments, the amino acid sequence consists of SEQ ID NO: 73. In some embodiments, the amino acid sequence comprises SEQ ID NO: 70. In some embodiments, the amino acid sequence consists of SEQ ID NO: 70.

[0083] For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 8. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 8. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 8.

[0084] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 10. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 10. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 10.

[0085] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 12. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 12. Namely, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 12. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the- 25 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC amino acid sequence of SEQ ID NO: 12. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 12.

[0086] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 14. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 14. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 14. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 14. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 14.

[0087] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 16. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 16. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 16. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 16.

[0088] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 18. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 18. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 18. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 18. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 18.

[0089] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 20. For example, provided herein is a - 26 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 20. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 20. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 20. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 20.

[0090] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 22. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 22. Namely, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 22. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 22. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 22.

[0091] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 24. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 24. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 24. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 24. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 24.

[0092] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 26. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 26. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 26. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the- 27 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC amino acid sequence of SEQ ID NO: 26. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 26.

[0093] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 28. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 28. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 28. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 28. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 28.

[0094] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 30. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 30. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 30. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30.

[0095] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 32. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 32. Notably, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 32. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 32. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 32.

[0096] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 34. For instance, provided herein is a - 28 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 34. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 34.Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 34. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 34.

[0097] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 36. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 36. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 36. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 36. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 36.

[0098] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 38. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 38. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 38. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 38. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 38.

[0099] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 41. For instance, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 41. Moreover, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 41. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting - 29 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC essentially of the amino acid sequence of SEQ ID NO: 41. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 41.

[0100] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 44. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 44. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 44. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 44. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 44.

[0101] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 46. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 46. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 46. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 46. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 46.

[0102] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 48. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 48. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 48. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 48. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 48.- 30 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0103] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 51. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 51. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 51. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 51. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 51.

[0104] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 54. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 54. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 54. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 54. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 54.

[0105] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 57. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 57. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 57. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 57. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 57.

[0106] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 61. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at - 31 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC least 99% sequence identity to SEQ ID NO: 61. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 61. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 61. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 61.

[0107] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 64. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 64. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 64. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 64. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 64.

[0108] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 67. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 67. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 67. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 67. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 67.

[0109] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 70. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 70. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 70. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 70. Additionally, provided herein is a - 32 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 70.

[0110] Provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 73. For example, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 73. Specifically, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) comprising the amino acid sequence of SEQ ID NO: 73. Furthermore, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 73. Additionally, provided herein is a polypeptide (e.g., a macrocyclase polypeptide) consisting of the amino acid sequence of SEQ ID NO: 73.[oni] In some embodiments, the polypeptide further comprises a tag (e.g., an epitope tag). Any suitable tag may be used, e.g., a His tag, a FLAG tag, a fluorescent protein tag (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)), a hemagglutinin (HA) tag, an ALFA-tag, a V5-tag, a Myc-tag, a SPOT-tag, a T7-tag, or an NE-tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the His tag comprises the amino acid sequence of GSHHHHHH (SEQ ID NO: 74). In some embodiments, the polypeptide comprises an N-terminal methionine residue, and the epitope tag is inserted immediately following the N-terminal methionine residue, e.g., relative to a reference sequence.

[0112] In some embodiments, the polypeptide comprises one or more substitutions set forth in any one of Tables A or B.

[0113] In some embodiments, the polypeptide does not comprise the sequence of SEQ ID NO: 2, 4, or 6.

[0114] In another embodiment, provided herein is an engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 2, 4, or 6.

[0115] In some embodiments, the polypeptide is isolated.

[0116] In some embodiments, the polypeptide is a macrocyclase (e.g., a NRPS macrocyclase).

[0117] Also provided herein are functional fragments of any of the polypeptides disclosed herein. Also provided herein are analogues of any of the polypeptides disclosed herein.- 33 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0118] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the polypeptide. The composition may include one or more carriers or diluents.Polynucleotides Encoding Macrocyclase Polypeptides

[0119] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., macrocyclase polypeptides, NRPS macrocyclase enzymes) disclosed herein. The polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression vectors containing a heterologous polynucleotide encoding the polypeptide (e.g., macrocyclase polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0120] Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acids to be made, all of which encode the polypeptides (e.g., macrocyclase polypeptides) disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates using each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein.

[0121] In various embodiments, the codons are preferably selected to be suitable for the host cell in which the polypeptide is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. By way of example, the polynucleotide of SEQ ID NO: 1 has been codon optimized for expression in E. coli to yield SEQ ID NO: 3 and SEQ ID NO: 5.

[0122] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., macrocyclase polypeptide) since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the polypeptides (e.g., - 34 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC macrocyclase polypeptides) may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0123] Provided herein is a polynucleotide that encodes a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 8, 10, 12, 14, 16, 18. 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 41, 44, 46, 48, 51, 54, 57, 61, 64, 67, 70, or 73.

[0124] For example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 40, 42, 43, 45, 47, 49, 50, 52, 53, 55, 56, 58, 59, 60, 62, 63, 65, 66, 68, 69, 71, or 72. In some examples, the polynucleotide sequence is modified to no longer code for an N-terminal His tag or C-terminal His tag.

[0125] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2'- deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2'- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5' to 3' direction in accordance with common convention, and the phosphates are not indicated.

[0126] As used herein, the terms “amino acid” or “residue” as used in context of the polypeptides disclosed herein refers to the specific monomer at a sequence position. Amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by International Union of Pure and Applied Chemistry (TUPAC) - International Union of Biochemistry (TUB) Biochemical Nomenclature Commission.Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0127] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).EXAMPLES

[0128] Exemplary methods and materials are described herein, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the- 35 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC present disclosure. The materials, methods, and examples are illustrative only and not intended to be limiting.GENERIC SCHEME- 36 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 37 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 38 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0129] In step I of the route of the present disclosure, Compound 1 is selectively coupled to Compound 3 to form the corresponding intermediate 4, utilizing DPPC1 as a coupling reagent and salicylaldehyde as a transient protecting group. In step II, a macrocyclase enzyme catalyzes the macrocyclization of intermediate 4 to form Compound 5 under buffered aqueous conditions. In step III, following enzyme denaturation, a solution of Compound 8 is prepared by salt metathesis of Compound 5. In step IV, Compound 8 is converted to Compound 6, which is then isolated as crystalline salt in nPrOH / MTBE solvent mixture. In step V, a second salt metathesis to switch from the suberate (Compound 6) to the bicarbonate (Compound 8), then finally to the caprate salt allows to generate Compound 7: enlicitide caprate (or enlicitide decanoate). This process for turning a quaternary ammonium suberate salt into a quaternary ammonium caprate salt in high yields, reproducibility and control was robust and unique. This caprate salt is then crystallized through a co-feed in 2.4: 1 MTBE / nPrOH solvent composition.ABBREVIATIONSMEASUREMENTS: eq. / equiv. Equivalent mg Milligram min minutes h hours ml, mL MilliliterL LiterM MolarMHz Megahertz w / w% weight percent mmHg millimeters of Mercury psi pounds per square inchNMR nuclear magnetic resonanceCHEMICALS:ACN acetonitrileBis-Tris 2-bis(2 -hydroxy ethyl)amino-2(hydroxymethyl)-l,3-propanediolBis-Tris propane l,3-bis(tris(hydroxymethyl)methylamino)propaneDIPEA Di sopropy 1 ethyl amineDEMA DiethylmethylamineDPPC1 diphenylphosphinic chloride- 39 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCEDC l-Ethyl-3-(3-dimethylaminopropyl)carbodiimideEDSA Ethanedisulfonic acidEPPS 3-[4-(2-Hydroxyethyl)piperazin-l-yl]propane-l -sulfonic acidHATU Hexafluorophosphate Azabenzotri azole Tetramethyl UroniumHEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acidMe methylMeCN AcetonitrileMOPS 3-(N-morpholino)propanesulfonic acidMTAC Methyltrioctylammonium chlorideMTBE Methyl tert-butyl ether nPrOH n-propanol1-PrOH 1 -propanol iPr isopropylT3P Propanephosphonic acid anhydrideTEA triethylamineTris tris(hydroxymethyl)aminomethane

[0130] Additional abbreviations may be defined throughout this disclosure.

[0131] The following examples are meant to be illustrative and should not be construed as further limiting. The process for preparing Compound 1 is described in U.S Provisional Application No. 63 / 717,514, filed on November 7, 2024, the contents of which are expressly incorporated herein by reference. The process for preparing Compound 3 is described in International Patent Application No. PCT / US2025 / 036006, filed on July 1, 2025, the contents of such are expressly incorporated herein by reference. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.- 40 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 41 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC

[0132] In a 50-L cylindrical vessel under nitrogen, Compound 1 (2.25 kg, 1.73 mol, 1 eq) was added to a stirring mixture of salicylaldehyde (254 g, 2.08 mol, 1.2 eq) and triethylamine (919 g, 9.09 mol, 5.25 eq) at 0 °C. The reaction mixture was agitated at 0 °C for 1 h to form Compound2. Compound 3 (1.10 kg, 1.90 mol, 1.1 eq) was charged, and the resulting mixture was stirred at 0 °C for 0.5 h. Diphenylphosphinic chloride (50 wt% in acetonitrile, 1.16 kg, 2.34 mol, 1.35 eq) was slowly added over 0.75 h. The resulting mixture was agitated at 0 °C for 1 h. The reaction was quenched by adding a solution of aqueous HC1 (0.25 M, 14 L, 7 vol) then warmed up to 20 °C. The pH was adjusted to 2-2.5 by addition of 5 M HC1. In another embodiment, the reaction was quenched using 5 M HC1 (0.7 L, 0.35 vol), followed by the addition of water (13.3 L, 6.65 vol) and adjustment of the pH to 2-2.5 using additional 5 M HC1. The quenched reaction mixture was washed twice with a solution of MTAC in toluene (15 wt%, 14 L, 7 vol), then washed once- 42 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC with heptane (14 L, 7 vol). The resulting aqueous layer (19.0 kg) was pH-adjusted back to 5-7 with 5 M NaOH, providing Compound 4.EXAMPLE 2Macrocyclase enzyme, MOPS, water / MeCNCompound 4 pH 7.5, 25 °C

[0133] To an aqueous mixture containing 2.63 kg of Compound 4 in a 50 L cylindrical jacketed vessel equipped with pH meter and thermometer was added water (14.6 L) at 22 °C, which was followed by the addition of MOPS (0.787 kg) and additional water (0.37 L). NaOH (5 N, 0.87 L) was slowly added to the vessel to adjust the pH from 4.7 to 7.7, which was followed by the addition of water (0.79 L). The macrocyclase enzyme (SEQ ID NO. 73) (2 wt%, 52.6 g), was dissolved in 0.60 L of 0.1 M MOPS buffer (at pH 7.6) and added to the vessel at 22 °C, which was followed by the addition of 1.37 L water. The mixture was agitated at 22-26 °C for 17.5 h to yield Compound 5. In particular, the mixture may be agitated at about 25 °C.

[0134] In another embodiment, the macrocyclase enzyme (SEQ ID NO. 73) (2 wt%, 52.6 g) was added directly to the vessel, which was followed by the addition of water (1.44 L). In various alternate procedures, 1.0, 1.5, or 2.0 wt% of the macrocyclase enzyme was used. In a particular procedure, 1.0 wt% was used. In another alternate procedure, about 0.60 L of 0.1 M MOPS buffer at pH 7.5 or pH 8.0 was added to the vessel.- 43 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCEXAMPLE 3Compound 5 1. MeCN, 40 °C2. NaH2PO4(25 wt%), water, 35 °C3. KHCO3(3 M in water), 35 °C4. Suberic acid, 25 °C5. n-PrOH / MTBE (2.5 / 1 ), 25 °C

[0135] Acetonitrile (24.8 L, 10 vol.) was then charged in the mixture containing Compound 5 and the resulting solution was warmed up at 40 °C before the addition of NaEhPCL (9.57 kg). The quenched reaction mixture was then stirred for 1 h while maintaining the temperature at 40 °C. The stream was filtered through a bed of celite to remove the denatured enzyme and the waste cake was washed with acetonitrile (4.96 L, 2 vol.). The biphasic filtrate was transferred into a 100 L cylindrical vessel and warmed up at 35 °C. The layers were separated, and the organic layer was successively washed a 4 M aqueous solution of NaEhPCL (24.8 L, 10 vol.), and a 3 M aqueous solution KHCO3 (24.8 L, 10 vol.) at 35 °C. A 4 / 1 mixture of acetonitrile / water (v / v, 2.48 L, 1 vol.) was added to the organic layer following by a 3 M aqueous solution KHCO3 (24.8 L, 10 vol.). The biphasic mixture was agitated at 35 °C and the phases were separated. The organic layer was again combined and a 4 / 1 mixture of acetonitrile / water (v / v, 2.48 L, 1 vol.) was charged following by a 3 M aqueous solution KHCO3 (24.8 L, 10 vol.) while maintaining the temperature at 35 °C. The biphasic mixture was agitated at 35 °C and the phases were separated. In another embodiment, the biphasic filtrate was transferred into a 100 L cylindrical vessel and warmed up at 35 °C. The layers were separated, and the organic layer was washed with a 3 M- 44 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC aqueous solution KHCO3 (24.8 L, 10 vol.) at 35 °C. A 4: 1 mixture of acetonitrile / water (v / v, 2.48 L, 1 vol.) was added to the organic layer following by a 3 M aqueous solution KHCO3 (24.8 L, 10 vol.). The biphasic mixture was agitated at 35 °C and the phases were separated. The organic layer was again combined and a 4: 1 mixture of acetonitrile / water (v / v, 2.48 L, 1 vol.) was charged following by a 3 M aqueous solution KHCO3 (24.8 L, 10 vol.) while maintaining the temperature at 35 °C. The biphasic mixture was agitated at 35 °C and the phases were separated.

[0136] 1-Propanol (29.76 L, 12 vol.) was added to the resulting organic phase and cooled at 5 °C. The heterogenous solution was cooled at 5 °C and aged for 16 h. The mixture was filtered, and the filter was rinsed with 1-propanol (4.96 L, 2 vol) to produce Compound 8. To the combined filtrate was added suberic acid (169 g, 0.65 eq) and the mixture was stirred at rt to achieve dissolution (around 15 min). Acetonitrile solvent was replaced with 1-propanol by continuous distillation under vacuum (65 °C, 50 mbar). 1 -Propanol was added to the concentrated residue until reaching < 0.5 wt% water in the distillate and a total volume of 24.8 L (10 vol.) (solution A). The mixture was cooled to 35 °C then aged overnight at the same temperature. The resulting slurry was aged at 35 °C for 1 h then cool to rt and aged another 1 h. MTBE (9.24 L, 4 vol.) was then added dropwise over 8-10 h. The slurry was filtered, and the wet cake was washed through a first displacement wash mixture of MTBE / 1-propanol (1 :2.4, v / v, 4.62 L, 2 vol.), then another displacement wash with a solution of MTBE / 1-propanol (4.3: 1, v / v, 4.62 L, 2 vol.). The resulting wet cake was dried under vacuum to yield Compound 6.Alternate process:

[0137] Alternatively, the solution A can be seeded with crystalline Compound 6 (1 wt%). The resulting slurry was aged at 35 °C for 1 h then cool to rt and aged another 1 h. MTBE (9.24 L, 4 vol.) was then added dropwise over 8-10 h. The slurry was filtered, and the wet cake was washed through a first displacement wash mixture of MTBE / 1-propanol (1 :2.4, v / v, 4.62 L, 2 vol.), then another displacement wash with a solution of MTBE / 1-propanol (4.3: 1, v / v, 4.62 L, 2 vol.). The resulting wet cake was dried under vacuum to yield Compound 6.

[0138] In another embodiment, the wet cake was washed through a first displacement wash mixture of MTBE / 1 -propanol (1 :2.4 ratio, v / v, 4.62 L, 2 vol.), then another displacement wash with a solution of MTBE / 1 -propanol (3.9: 1 ratio, v / v, 4.62 L, 2 vol.). The resulting wet cake was dried under vacuum to yield Compound 6.- 45 -81358249. v1Attorney Docket No.: 771417: MKRA-026PCEXAMPLE 41 . MeCN / water (4 / 1 , v,v), 35 °C2. KHCO3(3 M in water), 35 °CCompound 6 3. Capric acid, 25 °C4. MTBE / n-PrOH (2.4 / 1 , v,v), 25 °C

[0139] Compound 6 (the suberate salt, 1.1 kg as suberate salt) was dissolved in a 4 / 1 mixture of acetonitrile-water (11 L) at 35 °C and stirred for 30 min until achieving dissolution. The solution was mixed with 3.0 M aqueous KHCO3 (11.0 L; 1 the solution of KHCO3 was prepared and kept at 35 °C to prevent precipitation), stirred for 10 min, and the layers were separated while maintaining the temperature at 35 °C. The organic phase was combined with a 4 / 1 mixture of acetonitrile-water (1.1 L) and 3.0 M aqueous KHCO3 (11.0 L), stirred for 10 min, and the layers were separated while maintaining the temperature at 35 °C. The organic phase was again combined with a 4 / 1 mixture of acetonitrile-water (0.7 L) and 3.0 M aqueous KHCO3 (11.0 L), stirred for 10 min, and the layers were separated while maintaining the temperature at 35 °C. 1- propanol (11.0 L) was added to the resulting organic layer. The heterogenous solution was cooled at 4 °C and aged for 16 h. The mixture was filtered, and the filter was rinsed with 1 -propanol (2.2 L, 2 vol.) to form Compound 8. To the combined filtrate was added a solution of decanoic acid (142 g) in 1.1 L of 1-propanol and the mixture was stirred for 15 min at rt. Acetonitrile solvent was replaced with 1-propanol by continuous distillation under vacuum. 1 -Propanol was added to the concentrated residue until reaching a total volume of 3.7 vol. (including 3 vol. of nPrOH total). Water (67.5 mL) was added to reach 2.1 wt% of water. The mixture was stirred at room- 46 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC temperature and MTBE (1.5 L; 1.5 vol.) was slowly added (Solution #1). A separate flask was charged MTBE (4.92 L, 4.5 vol.) (Solution #2).

[0140] In a separate vessel, 3.7 L (3.0 vol.) solution of 2.0 / 1 MTBE / l-PrOH containing 0.7 wt% water was heated at 30 °C (Solution #3).

[0141] Solutions #1 and #2 were then simultaneously added to the solution #3 over the course of 14 h while maintaining temperature at 30 °C and aged during 48h. At the end of the cofeed, an additional 1.87 L (1.7 vol.) of MTBE was added to the slurry over the course of 4 h at 30 °C. The resulting slurry was cooled at rt and aged for 1 h. The slurry containing Compound 7 was filtered under nitrogen atmosphere. The wet cake was washed through a first displacement wash with a 3.3 L of a solution of 2.4: 1 (v:v) MTBE / l-PrOH containing 0.7 wt% water, then a slurry wash with a solution of 3.3 L of 8 / 2 (m:m) MTBE / l-PrOH. The cake was dried under a nitrogen flow in order to remove MTBE and part of the 1-PrOH solvent. Humid drying with humidified nitrogen (50% RH) was applied to remove residual 1-PrOH affording Compound 7.

[0142] Alternatively, in a separate vessel, the seed bed was prepared by addition 33 g of Compound 7 seeds suspended in a 3.7 L (3.5 vol.) solution of 2.0: 1 MTBE / l-PrOH containing 0.7 wt% water. The resulting slurry was aged at 30 °C for 1 h.

[0143] Solutions #1 and #2 were simultaneously added to a stirred Compound 7 seed slurry over the course of 14 h while maintaining temperature at 30 °C. At the end of the cofeed, an additional 1.87 L (1.7 vol.) of MTBE was added over the course of 4 h at 30 °C. The resulting slurry was cooled at rt and aged for 1 h. Compound 7 slurry was filtered under nitrogen atmosphere. The wet cake was washed through a first displacement wash with a 3.3 L of a solution of 2.4: 1 (v:v) MTBE / l-PrOH containing 0.7 wt% water, then a slurry wash with a solution of 3.3 L of 8 / 2 (m:m) MTBE / l-PrOH. In another embodiment, the wet cake was washed through a first displacement wash with a 3.3 L of a solution of 2.4: 1 (v:v) MTBE / l-PrOH containing 0.7 wt% water, a slurry wash with a solution of a 3.3 L of 2.4: 1 (v:v) MTBE / l-PrOH containing 0.7 wt% water MTBE / l-PrOH, then a displacement wash with a 3.3 L of a solution of 2.4: 1 (v:v) MTBE / l-PrOH containing 0.7 wt% water. The cake was dried under a nitrogen flow in order to remove MTBE and part of the 1-PrOH solvent. Humid drying with humidified nitrogen (50% RH) was applied to remove residual 1-PrOH affording Compound 7.- 47 -81358249. v1Atorney Docket No.: 771417: MKRA-026PCEXAMPLE 5Method for Obtaining X-Ray Powder Diffraction Patterns

[0144] X-ray powder diffraction was done using an X’Pert Pro instrument (PANalytical B.V., Almelo, The Netherlands). Samples were prepared on Si zero-return wafers. A typical scan is from 29 of 2° to 40°, with an Empyrean Cu-LFF source of wavelength (1.5406A) operating at 45 kV and 40 mA.

[0145] Tables 1-4 provide the major 29 peaks and d-spacings for each of the isolated crystalline forms of Compound 6.Table 1: Diffraction peaks and corresponding d-spacings for Form 1Pos. Height[°29] [cts]4.53 129.61 19.47 5.787.54 1986.13 11.71 88.567.83 2242.79 11.29 199.998.98 187.75 9.84 8.3719.16 149.64 8.79 6.6712.79 254.71 6.96 11.3616.23 596.11 5.46 22.5717.51 263.83 5.96 11.7618.76 765.74 4.73 34.14Table 2: Diffraction peaks and corresponding d-spacings for Form 2TT • u+d- Rel.Pos. Height .TF [° 22991] l [ecttss!]sP[aAci]n§[I%nt]-3.95 1775.31 22.37 97.785.99 1815.69 14.75 199.999.93 149.26 9.78 8.229.77 74.65 9.95 4.11Table 3: Diffraction peaks and corresponding d-spacings for Form 3Pos. Height[°29] [cts]4.34 1401.94 20.34 51.625.23 379.41 16.87 13.976.24 2451.64 14.15 90.266.84 1435.47 12.92 52.859.72 472.41 9.10 17.3914.45 647.70 6.12 23.85- 48 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC6.93 140.31 12.75 19.188.10 731.65 10.90 100.008.77 84.50 10.07 11.5510.24 133.14 8.63 18.2011.77 73.17 7.51 10.0012.70 162.16 6.97 22.1614.78 109.88 5.99 15.0215.40 88.51 5.75 12.1016.39 220.24 5.40 30.1017.68 225.52 5.01 30.8218.81 458.38 4.71 62.6520.41 256.23 4.35 35.02

[0146] It will be appreciated that various of the above-discussed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. It will also be appreciated that various presently unforeseen or unanticipated alternatives, modifications, variations, or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

[0147] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0148] All figures and references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.- 49 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-50-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 51 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-52-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 53 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-54-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 55 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-56-81358249. v1Attomey Docket No.: 771417: MKRA-026PC-57-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 58 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-59-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 60 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 61 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 62 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 63 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 64 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 65 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 66 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 67 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 68 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 69 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 70 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 71 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 72 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 73 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 74 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 75 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 76 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 77 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 78 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 79 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-80-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 81 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-82-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 83 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC-84-81358249. v1Attorney Docket No.: 771417: MKRA-026PC- 85 -81358249. v1

Claims

Atorney Docket No.: 771417: MKRA-026PCWhat is claimed is:

1. A process for preparing a crystalline form of a compound of Formula I,wherein A is an anion, comprising the steps of: a) combining Compound 1with Compound 3- 86 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC to obtain a reaction product comprising Compound 4wherein X is a mixture of counterions thereof; and b) adding a macrocyclase enzyme to the reaction product comprising Compound 4 to obtain a crystalline form of a compound of Formula I.

2. The process according to Claim 1, comprising the steps of: a) combining Compound 1with an aldehyde, a trialkylamine base and acetonitrile to obtain a mixture comprisingCompound 2- 87 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC2 b) reacting the mixture comprising Compound 2 with a coupling reagent and Compound 3to obtain a reaction product comprising Compound 4- 88 -81358249. v1Attomey Docket No.: 771417: MKRA-026PC c) adding a macrocyclase enzyme and a buffer solution to the reaction product comprising Compound 4; and d) adding an acid to obtain a crystalline form of a compound of Formula I.

3. A process for preparing Compound 6, or a solvate thereof,comprising the steps of: a) combining Compound 1with salicylaldehyde, TEA and acetonitrile to obtain a mixture comprising Compound 2- 89 -81358249. v1Atorney Docket No.: 771417: MKRA-026PCb) reacting the mixture comprising Compound 2 with DPPC1 and Compound 3to obtain a reaction product comprising Compound 4wherein X is mixture of chloride, 1,2-ethanedi sulfonate or diphenylphosphinate;- 90 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC c) adding a macrocyclase enzyme and a solution comprising 3-(N- morpholino)propanesulfonic acid (MOPS), water and MeCN to the reaction product comprising Compound 4 to obtain a solution comprising a compound of Formula Iwherein A is a mixture of chloride, 3-(N-morpholino)propanesulfonate, or 1,2- ethanedi sulfonate; d) combining the solution comprising a compound of Formula I from step c with aqueous potassium bicarbonate and acetonitrile to obtain a solution comprising Compound 8e) adding suberic acid and a solution of n-propanol (nPrOH) and Methyl tert-butyl ether (MTBE) to the solution comprising Compound 8 to obtain a crystalline form of Compound 6, or a solvate thereof.

4. The process of Claim 3 further comprising the steps of a) combining Compound 6, or a solvate thereof, with aqueous potassium bicarbonate and acetonitrile to obtain a solution comprising Compound 8; and- 91 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC b) adding capric acid and a solution of nPrOH and MTBE to the solution comprising Compound 8 of the prior step to obtain a crystalline form of Compound 75. The process of claim 4, wherein the solution of nPrOH and MTBE comprises MTBE and1-propanol (1-PrOH) in an about 2.4:1 ratio (v / v).

6. A compound having the structurewherein X is mixture of counterions.- 92 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC7. A compound having the structureor a solvate thereof.

8. A crystalline form of the compound of Claim 7:

9. The crystalline form of Claim 8, wherein the crystalline form is a crystalline solvate form.

10. The crystalline form (Form I) of Claim 8 or 9, characterized by an X-ray powder diffraction pattern including peaks at about 7.83 and about 16.23° 29, expressed in degrees-2- theta at angles of (±0.2°), measured using Cu Ka radiation.

11. The crystalline form of any one of claims 8-10, characterized by an X-ray powder diffraction pattern including peaks at about 7.83, 16.23 and 18.76° 29, expressed in degrees-2- theta at angles of (±0.2°), measured using Cu Ka radiation.- 93 -81358249. v1Attorney Docket No.: 771417: MKRA-026PC12. The crystalline form of any one of claims 8-11, characterized by an X-ray powder diffraction pattern including peaks at about 4.53, 7.54, 7.83, 8.98, 10.16, 12.70, 16.23, 17.51 and 18.76° 20 (±0.2°).

13. The crystalline form of any one of claims 8-12, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 1.

14. The crystalline form (Form II) of Claim 8 or 9, characterized by an X-ray powder diffraction pattern including peaks at about 3.95 and 5.99° 20, expressed in degrees-2 -theta at angles of (±0.2°), measured using Cu Ka radiation.

15. The crystalline form of Claim 14, characterized by an X-ray powder diffraction pattern including peaks at about 3.95, 5.99, 9.03 and 9.77° 20 (±0.2°).

16. The crystalline form of Claim 14 or 15, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 2.

17. The crystalline form (Form III) of Claim 8 or 9, characterized by an X-ray powder diffraction pattern including peaks at about 6.24 and 6.84° 20, expressed in degrees-2 -theta at angles of (±0.2°), measured using Cu Ka radiation.

18. The crystalline form of Claim 17, characterized by an X-ray powder diffraction pattern including peaks at about 4.34, 6.24 and 6.84° 20 (±0.2°).

19. The crystalline form of Claim 17 or 18, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 3.

20. The crystalline form (Form IV) of Claim 8 or 9, characterized by an X-ray powder diffraction pattern including peaks at about 6.93 and 8.10° 20, expressed in degrees-2 -theta at angles of (±0.2°), measured using Cu Ka radiation.

21. The crystalline form of Claim 20, characterized by an X-ray powder diffraction pattern including peaks at about 6.93, 8.10 and 10.24° 20 (±0.2°).- 94 -81358249. v1Atorney Docket No.: 771417: MKRA-026PC22. The crystalline form of Claim 20 or 21, having an X-ray diffraction pattern substantially similar to that set forth in FIG. 4.- 95 -81358249. v1

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