Conjugate for selective responsiveness to adjacent diols

Abstract

The present invention provides a boronized sensor that can simultaneously possess desirable selectivity or suitable affinity for a specific adjacent diol while exhibiting reduced affinity for other diols. [Solution] A boronated adjacent diol-responsive sensor compound can sense the level of a specific adjacent diol and respond to these molecules in the body. In certain embodiments, the adjacent diol is a cis diol, such as a hexose such as glucose. In certain embodiments, the sensor is conjugated to a drug substance (API), and the sensor can alter the biophysical properties, pharmacokinetics, and / or activity of the API in response to the adjacent diol. The API may be or contain a polypeptide, such as insulin, human endocrine or incretin peptides, or analogs thereof, and may contain one or more modified amino acids containing the adjacent diol-responsive sensor.

Description

[Technical Field]

[0001] Cross-reference of related applications This application was filed on 31 March 2020 and claims priority and benefits of U.S. Provisional Patent Application No. 63 / 002,662, entitled “INSULINS CONTAINING MODIFIED AMINO ACIDS,” the entirety of which is incorporated herein by reference.

[0002] Sequence List This application is filed together with an electronic sequence listing. The sequence listing is provided as a file titled "203819_ST25.txt" created on March 30, 2021, and is 8795 bytes in size. The electronic information of the sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0003] Boronic acids are generally considered Lewis acids because they tend to bind to hydroxyls, as they can form complexes with Lewis bases, such as hydroxide anions. Therefore, molecules containing boronic acid salts have a general tendency to bind to hydroxyl groups. This binding tendency can be used to detect hydroxyl-containing groups with boron-oxidized labeling reagents, where the boronic acid group binds to the hydroxyl group, and depending on the solvent and buffer conditions, the boronic acid salt can form a hydrolyzable boronic acid ester bond to the hydroxyl group of the hydroxyl-containing molecule. The strength and reversibility of the boronic acid ester bond are generally influenced by various factors, including the type of boronic acid salt, the buffer conditions, and the composition of the hydroxyl-containing molecule to which they bind. [Overview of the project]

[0004] One or more embodiments of the present disclosure relate to a boronized sensor that can simultaneously have desirable selectivity or suitable affinity to a specific adjacent diol while having reduced affinity to other diols. In certain embodiments, the boronized sensor can be used to modulate the pharmacokinetics and pharmacodynamics of a drug substance in the body in response to a specific level of a specific adjacent diol.

[0005] One or more embodiments of this disclosure include the following embodiments 1 to 15: 1. A compound represented by formula I, [ka] In equation I, R is selected from formulas FF1 to FF24. Z a) NH2 or OH, b) Covalent bonding to the active pharmaceutical ingredient, either directly or via any linker. c) Covalent bonding of one or more amino acids in the polypeptide drug substance to the N-terminal amine or epsilon-amino group, either directly or via any linker, and d) [ka] Selected from one of the bases represented by, [ka] The index k is an integer in the range of 3 to 14. J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula I'. [ka] In equation I', [ka] However, it indicates a binding site to the rest of the polypeptide active pharmaceutical ingredient, * indicates the connection point to the rest of Z, The index n is an integer in the range of 1 to 8. Regarding formulas FF1-FF24, [ka] [ka] X represents a covalent bond to Z in equation I, either directly or via any linker. The index i is an integer in the range of 1 to 20. B1 and B2 are either the same or different, and each independently represents a base selected from formulas F1 to F9. B3 is a base represented by one selected from formulas F1 to F11, [ka] For each of the formulas F1 to F9, One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- represents, and --- represents the covalent bond to the remainder of R in equation I, R1, which is 0, 1, or 2, each independently produces F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, The index m is an integer in the range of 1 to 14. In F5, one R1 represents B(OH)2, All remaining R1s represent H, Compounds in formula F10 where index j is an integer in the range of 1 to 13.

[0006] 2. A compound represented by formula II,

Chemical

Chemical

Chemical

Chemical Formula

Chemical Formula

[0007] 3. A compound containing an active pharmaceutical ingredient, wherein the active pharmaceutical ingredient contains insulin, and the insulin contains one or more modified amino acids represented by formula III. [ka] In Equation III, R is selected from formulas FF1 to FF24. Z is any linker, [ka] Selected from, [ka] The index k is an integer in the range of 3 to 14. J is expressed by equation III', [ka] In equation III', [ka] However, it shows the binding site to the rest of insulin, * indicates the connection point to the rest of Z, The index n is an integer in the range of 1 to 8. Regarding formulas FF1-FF24, [ka] [ka] X represents a covalent bond to Z in Equation III, either directly or via any linker. The index i is an integer in the range of 1 to 20. B1 and B2 are either the same or different, and each independently represents a base selected from formulas F1 to F9. B3 represents the base selected from formulas F1 to F11. [ka] For each of the formulas F1 to F9, One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- represents, and --- represents the covalent bond to the remainder of R. R1, which is 0, 1, or 2, each independently produces F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, The index m is an integer in the range of 1 to 14. In F5, one R1 represents B(OH)2, All remaining R1s represent H, Compounds in formula F10 where index j is an integer in the range of 1 to 13.

[0008] 4. Any linker is an L- or D-amino acid having at least one functional group directly conjugated to R, or any linker is selected from formulas FL1 to FL9. [ka] In formulas FL1 to FL9, Z'' represents a covalent bond with respect to Z, R'' represents a covalent bond with respect to R, p is an integer in the range of 1 to 5. q is an integer in the range of 1 to 5. A compound from any one of embodiments 1 to 3, wherein r is an integer in the range of 1 to 5.

[0009] 5. Any one of the compounds from Embodiments 1 to 3, wherein the compound is a drug substance further modified as described in Embodiments 1 to 3, and / or one or more amines are independently acetylated or alkylated.

[0010] 6. A compound from any one of Embodiments 1 to 3, wherein the active pharmaceutical ingredient is insulin containing human insulin or an analogue thereof, and the insulin comprises A chain and B chain.

[0011] 7. The compound of Embodiment 1 or 2, wherein the active pharmaceutical ingredient comprises a polypeptide active pharmaceutical ingredient or a human peptide hormone.

[0012] 8. The compound of Embodiment 6, wherein insulin comprises one or two peptide sequences each independently attached to the A and / or B chains of insulin, and each peptide sequence independently comprises 1 to 20 consecutive residues.

[0013] 9. The compound of Embodiment 6, comprising 2 to 10 amino acids, each independently represented by formula I, II, or III, wherein insulin is modified.

[0014] 10. Insulin contains one or more modifications, each independently represented by formula I, II, or III, and each of the one or more modifications is (i) on the side chains of up to 20 amino acids attached to the N-terminus and / or C-terminus of the A-chain and / or B-chain of insulin, and / or at the N-terminus of the polypeptide, and / or (ii) Within four residues of B1, B21, B22, B29, A1, A22, or A3 residues in the insulin A chain or B chain, and / or (iii) on the side chains of amino acids attached to or incorporated into the A and / or B chains of insulin, and / or located at the N-terminus of the polypeptide, wherein the polypeptide is in sequence (X2) n X1(X2) m The compound of Embodiment 6, comprising the formula, wherein X1 is a lysine residue, the side chain of the lysine residue is modified to be represented by formula I, II, or III, each X2 is independently selected from the group of amino acids K, P, E, G, N, M, A, R, L, W, S, F, V, C, H, D, I, Y, Q, T, or X1, the index m is an integer in the range of 0 to 20, and the index n is an integer in the range of 0 to 18.

[0015] 11. A conjugate comprising the compound according to Embodiment 1 or 2, either directly or via a covalent linker, wherein the conjugation is not via Z, when Z is NH2 in formula II.

[0016] 12. One of the compounds from Embodiments 1 to 3, wherein one of the compounds from Embodiments 1 to 3 is used as an intermediate compound for the production of any of the compounds in Embodiments 1 to 11.

[0017] 13. The compound contains one or more modifications represented by formula IV, V, or VI. Regarding equation IV, [ka] [ka] However, it indicates a binding site to the rest of the active pharmaceutical ingredient, The index n is an integer in the range of 1 to 8. R is selected from the group consisting of formulas F111, F222, F333, F444, and F555. [ka] In formulas F111, F222, F333, F444, and F555, The index n is an integer in the range of 1 to 8. R 1 Each carbon atom bonded to it independently has either (R) or (S) stereochemistry. Each R1 independently has -H, -OR 3 , -N(R 3 )2, -SR 3 -OH, -OCH3, -OR 5 NHC(O)CH3, -CH2R 3 , -C(O)NHOH, -NHC(O)CH3, -CH2OH, -CH2OR5 -NH2, -CH2R 4 , -OR 8 , -R 6 , -R 8 , and -R 7 Selected from, Each R 3 However, independently, -H, acetyl, phosphate, and -R 2 , -SO2R 2 ,-S(O)R 2 , -P(O)(OR 2 )2, -C(O)R 2 , -CO2R 2 , and -C(O)N(R 2 ) Selected from 2, Each R 2 However, independently, -H, and C as arbitrarily substituted. 1-6 Selected from an aliphatic ring, an optionally substituted phenyl ring, an optionally substituted 5-6 member monocyclic heteroaryl ring having 1-4 heteroatoms selected from nitrogen, oxygen, and sulfur, a 4-7 member heterocyclic ring having 1-2 heteroatoms selected from nitrogen, oxygen, and sulfur, and an alkyl or amide covalent bond to R in formula IV, Each R 4 However, independently, -H, -OH, -OR 3 , -N(R 3 )2, -OR 5 , and -SR 3 Selected from, Each R 5 However, they are independently selected from monosaccharides, disaccharides, trisaccharides, pentoses, and hexoses. Each R 6 However, independently, -NCOCH2-, -(OCH2CH2) n -, -OC 1-9 Alkylene group and substituted C 1-9 Selected from alkylene groups, one or more methylene groups are optionally -O-, -(CH2) n -, -OCH2-, -N(R 2 )C(O)-, -N(R 2 )C(O)N(R 2 )-, -SO2-, -SO2N(R 2 )-,-N(R 2 )SO2-, -S-, -N(R2 )-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R 2 )-, or -N(R 2 )SO2N(R 2 ) is replaced by -, where index n is an integer in the range of 1 to 8, Each R 7 However, independently, -N(R 2 )2, -F, -Cl, -Br, -I, -SH, -OR 2 , -SR 2 -NH2, -N3, -C≡CR 2 , -CH2C≡CH, -C≡CH, -CO2R 2 , -C(O)R 2 , -OSO2R 2 -N(R 2 )2, -OR 2 , -SR 2 Selected from -CH3, -CH2NH2, and direct bonding to R in formula IV, R 8 However, (i) it is one side chain of L-serine, D-serine, L-threonine, D-threonine, L-alrosreonine, or D-alrosreonine, corresponding to R in formula IV, with index n=1 in formula IV, (ii) it is an amide bond to the C-terminus of lysine, cysteine, or 2,3-diaminopropionic acid, or (iii) it is -CH2C(CH2OH)2CH2NH2, Structures F111, F222, F333, F444, and / or F555 optionally contain one or more acetyl, acetylene, acetonide, and / or pinacol protecting groups. Regarding equation V, [ka] [ka] However, it indicates a binding site to the rest of the active pharmaceutical ingredient, The index n is an integer in the range of 1 to 8. R represents X and Y, X is a covalent bond selected from the group consisting of a triazole, an amide bond, an imine bond, or a thioether bond, Y is selected from the group consisting of structures represented by formulas F200 to F203,

Chemical Structure

Chemical Structure

Chemical Structure

Chemical Structure

[0018] 14. A method for producing any one of the compounds in Embodiments 1 to 13, wherein B1 and B2 are optionally first conjugated to one of the structures represented by FF1 to FF33, and the resulting conjugation is then covalently bonded to the active pharmaceutical ingredient, or optionally, the structures represented by FF1 to FF33 are first conjugated to the active pharmaceutical ingredient, and then B1 and B2 are covalently bonded to the corresponding structures in FF1 to FF33.

[0019] 15. A method for administering one of the compounds from Embodiments 1 to 13 to a human subject as a therapeutic or prophylactic agent. [Brief explanation of the drawing]

[0020] The features and advantages of the embodiments of this disclosure will be better understood by referring to the following detailed description, in conjunction with the accompanying drawings.

[0021] [Figure 1] This is a mass spectrum plot confirming the synthesis of Example 1. [Figure 2] This is a mass spectrum plot confirming the synthesis in Example 2. [Figure 3] This is a mass spectrum plot confirming the synthesis in Example 3. [Figure 4] This is a mass spectrum plot confirming the synthesis of Example 4. [Figure 5] This is a mass spectrum plot confirming the synthesis of Example 5. [Figure 6] This is a mass spectrum plot confirming the synthesis of Example 6. [Figure 7] This is a mass spectrum plot confirming the synthesis in Example 7. [Figure 8] This is a mass spectrum plot confirming the synthesis of Example 8. [Figure 9] This is a mass spectrum plot confirming the synthesis of Example 9. [Figure 10] This is a mass spectrum plot confirming the synthesis of Example 10. [Figure 11] This is a mass spectrum plot confirming the synthesis of Example 11. [Figure 12] This is a mass spectrum plot confirming the synthesis of Example 12. [Figure 13] This is a mass spectrum plot confirming the synthesis of Example 13. [Figure 14] This is a mass spectrum plot confirming the synthesis of Example 14. [Figure 15] This is a mass spectrum plot confirming the synthesis of Example 15. [Figure 16] This is a mass spectrum plot confirming the synthesis of Example 16. [Figure 17] This is a mass spectrum plot confirming the synthesis of Example 17. [Figure 18] This is a mass spectrum plot confirming the synthesis of Example 18. [Figure 19] This is a mass spectrum plot confirming the synthesis of Example 19. [Figure 20] This is a mass spectrum plot confirming the synthesis of Example 20. [Figure 21] This is a mass spectrum plot confirming the synthesis of Example 21. [Figure 22] This is a mass spectrum plot confirming the synthesis of Example 22. [Figure 23] This is a mass spectrum plot confirming the synthesis of Example 23. [Figure 24] This is a mass spectrum plot confirming the synthesis of Example 24. [Figure 25] This is a mass spectral plot confirming the synthesis of modified insulin 1. [Figure 26A] This is a mass spectral plot confirming the synthesis of the modifier conjugated to modified insulin 2. [Figure 26B] This is a mass spectral plot confirming the synthesis of modified insulin 2. [Figure 27] This is a mass spectral plot confirming the synthesis of modified insulin 3. [Figure 28] This is a mass spectral plot confirming the synthesis of modified insulin 4. [Modes for carrying out the invention]

[0022] The ability of a sensor (e.g., a molecular sensor) to selectively bind and respond to a specific adjacent diol in the body is facilitated by binding to the adjacent diol of interest while reducing binding to other diols or other adjacent diols. While most boronic acid salts bind to diols containing molecules, achieving selectivity using boronic acid salts (e.g., boronic acid-based sensors) is not always straightforward, due to their general tendency to bind to most diols, including cis-diols, to varying degrees. Improved binding affinity of such sensors to a specific adjacent diol of interest is often achieved at the expense of selectivity or affinity, and the development of selectivity for a particular adjacent diol within the range of physiological levels is facilitated by the identification of specific molecular skeletons that can distinguish between the hydroxyl orientations of different adjacent diols. The development of skeletons that position boronic acid salts in a specific or particular shape to increase selectivity for a specific adjacent diol while simultaneously maintaining affinity to the diol of interest is facilitated by understanding or identifying which of the different pendant groups on the boronic acid salt influences hydroxyl binding along with which specific skeleton shape.

[0023] In certain embodiments of this disclosure, specific skeletal molecules have been identified that orient the boronic acid so spatially (e.g., in three-dimensional space) that the hydroxyl group of the boronic acid is oriented to engage with a hexose containing an adjacent diol, and matching the orientation of the hydroxyl on the boron group and the hydroxyl in the adjacent diol molecule provides enhanced selectivity. To further provide selectivity, the boronic acid is modified with specific functional groups on the benzene ring of the phenylboronic acid, together with a suitable or preferred skeletal, which can provide higher selectivity for binding toward the adjacent diol of interest and away from other diols in the body. In certain embodiments, the adjacent diol sensor is conjugated to the drug substance, and the adjacent diol sensor provides intramolecular and intermolecular interactions with the drug substance and / or proteins in the body, e.g., circulating proteins in blood and / or plasma, including albumin and / or globulin. In certain embodiments, selective binding of the sensor to specific adjacent diols alters the degree of their intramolecular and intermolecular binding, thereby modulating the pharmacokinetics and overall activity of the drug substance in the body, and this effect can be controlled by the level of adjacent diols present. In certain embodiments, the active pharmaceutical ingredient (API) is a peptide hormone, and in certain embodiments, the peptide hormone is a human peptide hormone such as insulin, glucagon, or another incretin hormone. In certain embodiments, the sensor is selective for adjacent diols in glucose, and this selectivity is enhanced while maintaining affinity for glucose and simultaneously reducing affinity for other sugars in the blood. In certain embodiments, pendant groups on the skeleton and boronic acid (e.g., in combination) allow for control of the overall activity and / or pharmacokinetics of the conjugated API based on the levels of glucose and / or other adjacent diols in the blood.

[0024] One or more embodiments of the present disclosure provide a sensor containing a specific skeleton molecule having a conjugated boronic acid, the skeleton being used to orient the boronic acid in a three-dimensional shape such that the hydroxyl groups of the boronic acid are oriented close to each other and within a distance that helps engage with the specific hydroxyl orientation of a selected hexose, such as glucose. The sensor molecules presented in the present disclosure enhance selectivity through three mechanisms: (1) the skeleton facilitates matching the orientation of hydroxyls on the boron groups in the phenylboronic acid and hydroxyls in adjacent diol molecules to enhance selectivity; (2) further selectivity is achieved by identifying specific functional groups bonded to or near the benzene ring of the phenylboronic acid that affect the electronic structure of the phenylboronic acid, thereby favoring reversible bonding to adjacent diols at physiological pH; and (3) the functional groups bonded to the phenylboronic acid or the sensor skeleton help provide steric hindrance that reduces bonding to unwanted hexoses while maintaining bonding to a desired sugar, such as glucose. These effects, in embodiments of this disclosure, are combined to provide desired or preferred selectivity for binding toward the adjacent diol-containing molecule of interest and away from other diols in the body. In certain embodiments, the adjacent diol sensor is conjugated to the drug substance, and the adjacent diol sensor provides intramolecular and / or intermolecular interactions with proteins in the body. Such proteins may include circulating proteins in blood and / or human plasma, such as albumin, glycosylated proteins, and / or immunoglobulins. In certain embodiments, the selective binding of the sensor to a specific adjacent diol in the molecule of interest alters the degree of intramolecular and intermolecular binding, thereby modulating the pharmacokinetics and overall activity of the drug substance in the body. In certain embodiments, the drug substance is a peptide hormone, and in those particular embodiments, the peptide hormone is an incretin hormone such as insulin, and the adjacent diol-containing molecule is glucose, but this disclosure is not limited thereto.

[0025] definition The following description illustrates and illustrates selected exemplary embodiments of the subject matter of this disclosure. As those skilled in the art will recognize, the subject matter of this disclosure may be embodied in many different forms and should not be construed as being limited to the embodiments described herein.

[0026] The following detailed description includes numerous specific details to provide a more complete understanding of some of the embodiments of the Disclosure. However, those skilled in the art will understand that embodiments of the Disclosure may be carried out in a variety of preferred forms and are not necessarily limited to these specific details. All disclosed features may be replaced by similar features serving the same, equivalent, or similar purpose unless otherwise expressly stated. Thus, unless otherwise expressly stated, each disclosed feature is merely an example of a set of equivalent or similar features. Similarly, unless otherwise indicated, features of one embodiment may be incorporated into other embodiments without departing from the spirit and scope of the Disclosure.

[0027] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those generally understood by those skilled in the art to the extent of this disclosure. Terms such as those defined in commonly used dictionaries should be construed to have meanings consistent with their meanings in the relevant art and / or context of this specification, and should not be construed in an idealized or overly formal sense unless expressly defined herein.

[0028] For example, unless otherwise defined, all chemical terms and functional group names used throughout this specification are as defined in Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thIdentified according to the ed. and inside cover. Specific functional groups are identified in *Organic Chemistry*, Thomas Sorrell, University Science Books, Sausalito, 1999; *Larock*, *Comprehensive Organic Transformations*, VCH Publishers, Inc., New York, 1989; and *Carruthers*, *Some Modern Methods of Organic Synthesis*, 3. rd Edition, Cambridge University Press, Cambridge, 1987, and Smith and March, March's Advanced Organic Chemistry, 5 th As described in Edition, John Wiley & Sons, Inc., New York, 2001, general principles of organic chemistry are given, as well as their meanings expressed by specific functional groups and reactivity.

[0029] The terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit this disclosure. Where used herein, singular nouns such as “a,” “an,” and “the” are intended to include plural nouns unless the context explicitly indicates otherwise, and vice versa. Where used herein, the terms “comprises,” “comprising,” “includes,” and “including,” and their variations thereof, identify the presence of the described additives, materials, features, integers, actions, operations, elements, groups, components, and / or parts, but do not exclude the presence or addition of one or more other additives, materials, features, integers, actions, operations, elements, groups, components, or parts. Where used herein, the term “and / or” includes any and all combinations of one or more of the enumerated items relating to the disclosure. Where preceding a list of elements, expressions such as “at least one of the elements” modify the entire list of elements, not the individual elements of the list.

[0030] Terms such as “First,” “Second,” and “Third” may be used herein to describe various elements, components, regions, layers, and / or sections, but it will be understood that these elements, components, regions, layers, and / or sections should not be limited by these terms. These order-indicating terms are used to distinguish one element, component, region, layer, or section from another element, component, region, layer, or section. Thus, the first element, component, region, layer, or section described below may be referred to as the second element, component, region, layer, or section without departing from the spirit and scope of this disclosure.

[0031] Where used herein, the terms “substantially,” “about,” and similar terms are used as approximations, not as terms of degree, and are intended to describe inherent deviations in measured or calculated values ​​that would be recognized by those skilled in the art. Furthermore, where “may be” is used to describe embodiments of the Disclosure, it refers to “one or more embodiments of the Disclosure.” Where used herein, the terms “use,” “to use,” and “to be used” may be considered synonymous with the terms “to utilize,” “to utilize,” and “to be utilized,” respectively. Also, the term “exemplary” is intended to refer to an example or description.

[0032] Any numerical range enumerated herein is intended to include all subranges of the same numerical precision that are contained within the enumerated range. For example, the range "1.0 to 10.0" is intended to include all subranges between (and including) the enumerated minimum value of 1.0 and the enumerated maximum value of 10.0, i.e., all subranges having a minimum value of 1.0 or greater and a maximum value of 10.0 or less, such as 2.4 to 7.6. Any maximum numerical limit enumerated herein is intended to include all lower numerical limits contained within it, and any minimum numerical limit enumerated herein is intended to include all upper numerical limits contained within it. Accordingly, the applicant reserves the right to modify this specification, including the claims, to explicitly enumerate any subranges contained within the ranges explicitly enumerated herein.

[0033] As used herein, the term "CAS#," which is interchangeable with the terms "CASRN" or "CAS number," refers to a unique numerical identifier assigned by the Chemical Abstracts Service (CAS) to every chemical substance listed in publicly available scientific literature.

[0034] In certain embodiments, the terms “covalently connected,” “covalently conjugated,” or “via covalent bond” may be used interchangeably to indicate that two or more atoms, groups, or chemical moieties are bonded or connected via a chemical bond. In certain embodiments, a chemical bond (which may be called a covalent bond in certain embodiments) may be one or more shared electron pairs directly between two atoms, groups, or chemical moieties (e.g., in a single, double, or triple bond), as indicated by the term “directly bonded.” In certain embodiments, a chemical (covalent) bond may further include one or more atoms or functional groups, which may be referred to using the corresponding names of those functional groups in the art. For example, a covalent bond containing an -SS- group may be called a disulfide bond, a covalent bond containing an -(C=O)- group may be called a carbonyl bond, and a covalent bond containing an -(CF2)- group may be called a difluoromethylene bond, and so on. The type of bond or functional group within a covalent bond is not limited unless explicitly stated, for example, if it is stated that it includes or is selected from a particular group. The preferred type or kind of covalent bond will be understood from the description and / or context.

[0035] In certain embodiments, the side chains of amino acids may be covalently bonded (e.g., bonded or crosslinked) via any number of chemical bonds (e.g., bonding sites) as commonly described in Bioconjugate Techniques (Third edition), edited by Greg T. Hermanson, Academic Press, Boston, 2013. For example, the side chains may be covalently bonded via amides, esters, ethers, thioethers, isoureas, imines, triazoles, or any suitable covalent chemistry available in the art for covalently bonding one peptide, protein, or synthetic polymer to a second peptide, protein, or synthetic polymer. The term polymer includes polypeptides. The term "covalent chemistry" may refer to one or more functional groups contained in the bonding site and / or the chemical reactions used to form the bonding site.

[0036] The term "adjacent diol" refers to a group of molecules in which two hydroxyl groups occupy adjacent positions, that is, are bonded to adjacent atoms. Such molecules may include, but are not limited to, sugars such as hexoses, glucose, mannose, and fructose.

[0037] In certain embodiments, peptides, proteins, or synthetic polymers may be bound to insulin modified using click chemistry reactions understood and defined in the art. Non-limiting examples of suitable click chemistry reactions may include cycloaddition reactions, e.g., 3+2 cycloaddition, strain-enhanced alkyne-nitrone cycloaddition, strain alkenes, alkenes, and tetrazine reverse-demand Diels-Alder reactions, copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC), strain-enhanced azide-alkyne cycloaddition, Staudinger ligation, nucleophilic ring-opening reactions, and addition to carbon-carbon multiple bonds. Some of these reactions are described, for example, in H. Kolb, M. Finn and K. Sharpless (2001); Click Chemistry: Diverse Chemical Function from a Few Good Reactions, Angewandte Chemie International Edition 40(11):2004-2021, Kolb and Sharpless, Drug Discovery Today 8:1128-1137, 2003, Huisgen, R. Angew. Chem. Int. Ed. Engl. 1963, 2,565, and Agard, NJ; Baskin, JM; Prescher, JA; Lo, A.; Bertozzi, CRACS Chem. Biol. 2006, 1,644. Those skilled in the art can select buffers, pH, and reaction conditions suitable for such click reactions. For example, the use of chelating agents such as EDTA should be avoided in CuAAC reactions. In certain embodiments, covalent bonding may be the result of a "biorthogonal reaction" as known in the art.Such reactions are described, for example, by Sletten, Ellen M.; Bertozzi, Carolyn R. (2009). Bioorthogonal Chemistry: Fishing for Selectivity in a Sea of ​​Functionality, Angewandte Chemie International Edition 48(38):6974-98. and by Presser, Jennifer A; Bertozzi, Carolyn R (2005). Chemistry in living systems, Nature Chemical Biology 1(1):13-21. In certain embodiments, units may be linked using natural chemical ligation, for example, by Dawson, PE; Muir, TW; Clark-Lewis, I.; Kent, SB (1994). Synthesis of proteins by native chemical ligation, Science 266(5186):776-778.

[0038] The term "substituted" indicates that at least one hydrogen atom of the designated group is replaced by a non-hydrogen atom, a functional group, a peptide, a linker, etc. Substitutive structures (sometimes referred to herein as "substituents") are not particularly limited unless expressly stated otherwise and may include any suitable functional groups, amino acids, polypeptides, etc., available in the art. In certain embodiments, substituents may be further substituted themselves.

[0039] The term “insulin” encompasses both wild-type and modified forms of insulin that can bind to and activate insulin receptors, or, when administered in vivo, cause a measurable reduction in blood glucose. In certain embodiments, insulin includes insulin of any species, whether in purified, synthetic, or recombinant form, and may include, for example, human insulin, porcine insulin, bovine insulin, sheep insulin, and rabbit insulin.

[0040] In certain embodiments, the insulin may be or may contain proinsulin known in the art (e.g., an insulin precursor), which can be further processed into mature insulin.

[0041] If insulin is a modified form of insulin, it may be modified using any preferred technique in the art. For example, insulin may be chemically modified (e.g., by the addition of chemical moieties such as PEG groups or fatty acyl chains) and / or mutated (e.g., including the addition, deletion, or substitution of amino acids). If insulin contains one or more mutations, those mutations may be indicated using standard terminology in the art, but it is understood that insulin analogs may contain one or more mutations known in the art, and some of these mutations may alter (enhance) various aspects of the molecule, including biophysical properties or stability and degradation resistance. In certain embodiments, for example, the term "desB30" refers to insulin lacking the B30 amino acid residue.

[0042] In certain embodiments, the term “homology percentage” refers to the percentage of sequence identity between two sequences after optimal alignment, where identical sequences have a homology percentage of 100%. Optimal alignment may be performed using any preferred homology alignment algorithm described by the Similarity Search Method in Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444 (1988), or by general methods described by Neddleman and Wunsch, J. Mol. Biol. 48:443 (1970) for similarity searching, including implementations of these algorithms or visual comparisons. “Insulin A chain” is the insulin chain having the highest percentage homology to the A chain of wild-type human insulin. “Insulin B chain” is the insulin chain having the highest percentage homology to the B chain of wild-type human insulin. In certain embodiments, the A and B chains of insulin may be linked together via one or more peptides, such as the c-peptide known in the art, or a shortened version thereof.

[0043] In certain embodiments, the term “albumin” refers to human serum albumin, or a protein having at least 60% homology to human serum albumin protein. In certain embodiments, it should be understood that albumin may be further chemically modified for the purpose of conjugation. Such modifications may include one or more covalently linked linkers.

[0044] In certain embodiments, “therapeutic composition” as used herein refers to a substance or mixture of substances intended to have a therapeutic effect, such as a pharmaceutical composition, genetic material, biologics, and other substances. Pharmaceutical compositions may be configured to function within the body in terms of therapeutic quality, concentration that reduces the frequency of supplementation, etc. In certain embodiments, “therapeutic effective dose” and “preventive effective dose” refer to a quantity that provides therapeutic benefit in the treatment, prevention, or management of a disease or its apparent symptoms. A therapeutic effective dose may treat a disease or condition, symptoms of a disease, or predisposition to a disease for the purpose of treating, curing, reducing, alleviating, modifying, correcting, improving, or influencing the disease, symptoms of a disease, or predisposition to a disease. A therapeutically effective set or specific quantity can be readily determined by a typical healthcare professional and may vary depending on factors known in the art, such as the type of disease, the patient’s medical history and age, the stage of the disease, and the administration of other therapeutic agents.

[0045] Adjacent Diol Sensor The sensor skeletons and specific boronic acid functional groups presented herein provide a molecular (sensor) framework that can distinguish between adjacent diol-containing molecules and other diol-containing molecules, for example, by preferentially binding one adjacent diol-containing molecule to another. For example, a sensor skeleton having a suitable or preferred boronic acid can be synthesized using the methods presented herein to provide a sensor molecule that can bind to a specific hexose while rejecting or ignoring other sugars having a similar structure that lacks an adjacent diol. For example, a sensor can be developed that binds to glucose but actively rejects or ignores (e.g., does not bind to) lactate and / or fructose. Without being bound by the correctness of any theory in the explanation, the sensor molecules presented in this disclosure may enhance selectivity through any combination of three mechanisms: (1) the skeleton may position the boron hydroxyl group in the phenylboronic acid and the hydroxyl group in the adjacent diol molecule in a complementary orientation; (2) specific functional groups bonded to or near the benzene ring of the phenylboronic acid may alter the electronic structure of the phenylboronic acid to preferentially enable reversible bonding to the adjacent diol at physiological pH; and (3) functional groups bonded to the phenylboronic acid and / or the sensor skeleton may increase steric hindrance and reduce bonding to unwanted (e.g., untargeted) hexoses (diols) while maintaining bonding to the target molecule such as glucose. These effects, individually or in combination in embodiments of this disclosure, provide selective bonding toward the target adjacent diol and away from other diols in the body. In certain embodiments, the adjacent diol sensor is conjugated to the drug substance, and the adjacent diol sensor may provide and / or enhance intramolecular and / or intermolecular interactions between the drug substance and one or more proteins in the body.

[0046] The effect of the above mechanism on sensor selectivity can be partially explained by the data provided in Table 1. Selectivity can first be achieved or enhanced through the appropriate or preferred use of the skeletal molecule (e.g., fragment). For example, the compounds of Examples 9, 10, 11, 12, and 13 all utilize similar phenylboronates but exhibit significantly different affinities for glucose. As shown in Table 1, Example 9 provides the lowest Kd value (e.g., highest affinity) for glucose within the group, while Example 11 provides the highest Kd (e.g., lowest affinity) for fructose. Since all of these examples utilize similar nitro-substituted phenylboronates, this counterintuitive selectivity response is primarily driven by the skeletal molecule. A comparison of Examples 9 and 10 shows that an additional CH2-CH2 group in the skeleton (e.g., as in Example 10) can substantially disrupt the glucose bond with little effect on the fructose bond. Conversely, the addition of a CH2-CH2 group in the skeleton increases the affinity for lactate (e.g., reduces the Kd value for lactate). Therefore, glucose affinity is reduced by the extra distance between boronic acid salts, while lactate affinity is increased. This example demonstrates that the skeleton presented in this disclosure can significantly influence the ability to selectively bind (e.g., with higher affinity to a series of competing hexoses) to a specific hexose of an adjacent diol sensor.

[0047] As an example, another comparison can be made between two sensors that utilize the same boronic acid but have different skeletal molecules (e.g., fragments). A comparison of the diol affinity of Examples 2 and 14 from Table 1 shows that the skeletal structure of Example 14 provides higher selectivity for glucose than lactate, while the skeletal structure of Example 2 provides higher selectivity for fructose than lactate. This unexpected result was discovered through experimental identification of the skeletal structures of Examples 2 and 14 and subsequent analysis of their binding specificity.

[0048] A second factor influencing bond selectivity is the position and nature (e.g., composition) of the functional groups on the benzene ring of the phenylboronate. Both the bond site of the phenylboronate on the adjacent diol sensor (e.g., the bond site on the benzene ring to the boron bond (substituent) on the benzene ring), and the position and identity (e.g., composition) of other functional groups on the benzene ring (e.g., ortho, meta, or para to the boron group on the phenylboronate ring) affect selectivity. Electron-withdrawing groups on the phenylboronate generally provide lower pKa values ​​(e.g., because they aid in ionization), and generally, ring distortion of five-membered oxabolol ring borons (e.g., formulas F2, F13, or F29) distorts the shape and also results in lower pKa values. Fluorine and / or CF3 groups can be used as electron-withdrawing groups, but the introduction of nitro groups into the benzene ring can have a dramatic effect on lowering the pKa. These effects are most easily observed when the skeletal molecule is kept constant while the boronate is modified. For example, the compounds of Examples 4-8 utilize the same skeletal molecule but have phenylboronic acid salts containing different functional groups and exhibit different binding selectivity for glucose, fructose, and lactate. This example demonstrates, for instance, that the presence of an NO2 group on the phenylboronic acid ring can enhance affinity for glucose, and that heterobifunctional sensors containing two different boronic acid salts or phenylboronic acid salts exhibit different sugar selectivity than adjacent diol sensors containing two similar (or identical) boronic acid salts. Furthermore, aspects of this disclosure include nitro-substituted boronic acid salts combined with boroxol boronic acid on the same skeletal structure, as shown, for example, by the affinity comparison of Examples 5 and 7 in Table 1. The homobifunctional boronic acid group of Example 5 provides a worse affinity for glucose compared to the heterobifunctional boronic acid salt of Example 7. The use of ring-strained boroxol provides an approximately seven-fold increase in affinity for glucose, fructose, and lactate, even though the rest of the compound structure is similar between Examples 5 and 7.

[0049] Similarly, for example, a comparison of the compounds in Examples 9 and 15 demonstrates that the introduction of a nitro group in the boronate enhances glucose affinity in specific skeletons, and that this affinity enhancement is not simply due to the electron-withdrawing properties of the boronate's functional group (since fluorine is also electron-withdrawing). Thus, contrary to the long-held assumption that stronger electron-withdrawing groups on the phenylboronate ring always enhance sugar bonding at physiological pH by regulating the boronate's pKa, this is not always the case (for example, other aspects of the functional group may be at play). Examples 9 and 15 demonstrate that for some skeletons of the present disclosure, a nitro group enhances affinity more than an equivalent fluoro group on the phenylboronate ring. The importance of functional groups on phenylboronates is further highlighted by comparisons of exemplary compounds having similar skeleton structures. For example, the importance of functional groups to sensor selectivity can be seen in comparisons of Example 14 versus Example 18, Example 11 versus Example 20, Example 12 versus Examples 21 and 23, as well as comparisons within Examples 1-3 or within Examples 4-8, and the corresponding affinities of these molecules listed in Table 1. These examples demonstrate that the effect of functional group configuration on the phenylboronate ring of a given skeletal molecule can enhance sensor binding and selectivity to a target sugar, e.g., glucose, away from other hydroxyl-containing molecules, including fructose or lactate. Accordingly, in some embodiments, the identified skeletal molecules and specific boronates conjugated to these skeletons described herein include adjacent diol sensors that have preferential binding selectivity to a target adjacent diol (e.g., glucose) and away from other adjacent diols (e.g., fructose) or hydroxyl-containing molecules (e.g., lactate).

[0050] A third structural factor influencing selectivity is steric hindrance or charge effect that prioritizes binding to one sugar molecule over another. For example, the effect of amine (amide) groups on acid groups on the skeleton can be seen by comparing Examples 1-3 in Table 1. Substituting acid or amide groups on the skeleton can contribute to the difference in binding affinity of these sensors to glucose versus lactate or fructose. A comparison of examples with skeletons containing acid or amide groups with Examples 1-3 and 4-8 in Table 1 shows the overall effect of acid versus amide groups on the skeleton, and such an effect can be further extended to substituents on boronic acid, which does not directly affect the electronic structure of the boronic acid but can sterically hinder the engagement of the sensor between one pair of adjacent diols and another, thereby affecting selectivity. In summary, the combined effects of the skeleton molecule, the functional groups on the phenylboronic acid ring, and any functional groups immediately adjacent to or on the phenylboronic acid ring, as included in the specific embodiments of this disclosure, represent some of the approaches by which the disclosed sensors achieve binding to specific adjacent diols. These examples and the relevant binding affinities in Table 1 demonstrate at least some of the effects identified in selective enhancement.

[0051] In certain embodiments, the adjacent diol sensor is conjugated to an incretin peptide to control pharmacokinetics in the body in response to a specific adjacent diol, such as glucose. In certain embodiments, the incretin peptide is a polypeptide, which may be, for example, insulin. Insulin is an important regulator of blood glucose levels. In healthy individuals, insulin, when present and released by the pancreas, acts to lower blood glucose levels. Diabetes mellitus (DM), commonly known as diabetes, is a group of metabolic diseases characterized by persistently high blood glucose levels.

[0052] In certain embodiments, the adjacent diol sensor may contain a single boronic acid molecule (or group) or multiple boronic acid molecules (or groups), and the sensor skeleton and / or boronic acid salt may be directly conjugated to or include a naphthalene, anthracene, biphenyl, anthraquinone, phenanthrene, chrysene, pyrene, coronene, corannarene, tetracene, pentacene, or triphenylene skeleton. These skeletons may, but are not limited to, include additional substituents such as nitro, fluoro, alcohol, thiol, trifluoromethyl, and / or methoxy functional groups. Two or more skeletons may be conjugated together either directly or via one or more amino acids. The skeletons may be further conjugated to a drug or active pharmaceutical ingredient to confer the ability to identify a desired diol-containing molecule or protein. Certain embodiments may include multiple copies of these skeletons that may provide further selectivity and functionality.

[0053] In certain uses, the modified insulin described herein may be delivered to the body by injection or by other routes and may reversibly bind to soluble glucose in a non-depot form. In certain uses, the modified insulin described herein may be delivered to the body by injection or by other routes and may reversibly bind to soluble glucose in a depot and / or soluble form. In certain embodiments, the modified insulin described herein may also be released over a long period from a local depot in the body. In certain embodiments, the modified insulin may bind to proteins in the blood and / or plasma, such as serum albumin, and the release of the modified insulin may depend on the level of glucose in the blood, such that elevated blood glucose levels release more modified insulin from serum albumin. Such release rates may depend on blood glucose levels or the levels of other small molecules in the blood, including diol-containing molecules. In certain embodiments, the release, bioavailability, and / or solubility of the modified insulin described herein may be controlled as a function of blood and / or serum glucose concentrations and / or concentrations of other small molecules in the body. Certain embodiments include intermediate compounds of any of the compounds described herein, which may optionally contain one or more protecting groups (e.g., Boc, Fmoc, etc.), and in certain embodiments, one or more protecting groups are independently located on any of the compounds or subsets of intermediates of the Disclosure.

[0054] Modified insulin refers to insulin that has been chemically altered compared to wild-type insulin, such as by the addition of a chemical moiety, including but not limited to a PEG group or a fatty acyl chain. Modified insulin can be mutated, including by the addition, deletion, or substitution of amino acids. Different protomers of insulin may arise from these changes and be incorporated into specific embodiments. Generally, the active form of insulin has fewer than 11 such modifications (e.g., 1-4, 1-3, 1-9, 1-8, 1-7, 1-6, 2-6, 2-5, 2-4, 1-5, 1-2, 2-9, 2-8, 2-7, 2-3, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-9, 4-8, 4-7, 4-6, 4-5, 5-9, 5-8, 5-7, 5-6, 6-9, 6-8, 6-7, 7-9, 7-8, 8-9, 9, 8, 7, 6, 5, 4, 3, 2, or 1). The wild-type sequence of human insulin (chain A and chain B) consists of chain A having the amino acid sequence GIVEQCCTSICSLYQLENYCN (SEQ ID NO: 1) and chain B having the amino acid sequence FVNQHLCGSHLVEALYLVCGERGFFYTPKT (SEQ ID NO: 2).

[0055] Human insulin differs from rabbit, pig, bovine, and sheep insulin at amino acids A8, A9, A10, and B30, which are Thr, Ser, Ile, Thr for humans; Thr, Ser, Ile, Ser for rabbits; Thr, Ser, Ile, Ser for pigs; Thr, Ser, Ile, Ala for sheep; Ala, Gly, Val, Ala for sheep; and Ala, Ser, Val, Ala for bovines. Modifications to insulin may, in certain embodiments, include insulin mutated at positions B1, B2, B28, or B29 of the B chain, or at positions B28 and B29. Modifications to insulin may, in certain embodiments, include insulin mutated at positions A1, A2, A21, or other positions of the A chain. For example, insulin lispro is a rapid-acting modified insulin in which the lysine and proline residues at the C-terminus of the B chain are reversed. Insulin aspart is a rapid-acting modified insulin in which proline is substituted with aspartic acid at position B28. In certain embodiments of this disclosure, the mutations at B28 and B29 are intended to be accompanied by additional mutations. Insulin glulysine is a rapid-acting modified insulin in which aspartic acid is substituted at position B3 by a lysine residue and lysine is substituted at position B29 by a glutamic acid residue.

[0056] In certain embodiments, the isoelectric point of insulin as described herein may be shifted relative to wild-type human insulin by the addition or substitution of amino acids or by other means, and in certain embodiments, the isoelectric point of modified insulin may be modulated by glucose. For example, insulin glargine is basal insulin in which two arginine residues are added to the C-terminus of the B peptide and A21 is substituted with glycine. Insulin may not have one or more of the residues B1, B2, B3, B26, B27, B28, B29, and B30. In certain embodiments, the insulin molecule contains additional amino acid residues on the N-terminus or C-terminus of the A-chain or B-chain. In certain embodiments, one or more amino acid residues are located at or missing from positions A0, A21, B0, and / or B31. In certain embodiments, the insulin molecule of this disclosure is mutated so that one or more amino acids are substituted in an acidic form. For example, asparagine may be replaced with aspartic acid or glutamic acid, and similarly glutamine may be replaced with aspartic acid or glutamic acid. In certain embodiments, A21 may be aspartic acid, B3 may be aspartic acid, or both positions may contain aspartic acid (e.g., simultaneously). Those skilled in the art will recognize that any previously reported or widely accepted mutations or modifications can be made to insulin while retaining its biological activity, and that modified insulin can be used in embodiments of this disclosure. In certain embodiments, insulin may be bound to a fatty acid at any position, or may be acylated with a fatty acid at any amino group, including one on the side chain of lysine or the alpha-amino group at the N-terminus of insulin, and the fatty acid may include C8, C9, C10, C11, C12, C14, C15, C16, C17, and C18. In certain embodiments, a combination of fatty acids or fatty diacitates and PEG linker linking to modified insulin is used to increase the serum half-life of modified insulin or to confer sustained-release properties to modified insulin, such sustained release of 12 hours to 7 days. In certain embodiments, the fatty acid chain is 8 to 20 carbon atoms long.By way of example, such a modification may be similar to the modification in insulin detemir, where myristic acid is covalently bound to lysine at B29, and B30 is deleted or absent. In certain embodiments, position B28 of the insulin molecule is lysine, and the epsilon (ε)-amino group of this lysine is conjugated to a fatty acid, a modified fatty acid or a diacid. In certain embodiments, the lysine at or near the C-terminus of the B chain of insulin is substituted with an amino acid represented by Formulas I to III. In certain embodiments, the activity, bioavailability, solubility, isoelectric point, charge, and / or hydrophobicity of modified insulin can be controlled by chemical modification, or as a result of the interaction of a small molecule such as a sugar with the modified insulin described herein, which is either covalently bound to or mixed with insulin.

[0057] In certain embodiments, the modified insulin molecules of the present disclosure include, but are not limited to, the following insulin molecules: N εB29 -octanoyl-Arg B0 Gly A21 Asp B3 Arg B31 Arg B32 -HI, N εB29 -octanoyl-Arg B31 Arg B32 -HI, N εB29 -octanoyl-Arg A0 Arg B31 Arg B32 -HI, N εB28 -myristoyl-Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI, N εB28 -myristoyl-Gly A21 Gln B3 Lys B28 Pro B30 Arg B31 Arg B32 -HI, N εB28 -myristoyl-Arg A0 Gly A21 LysB28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Milistil-Arg A0 Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Milistil-Arg A0 Gly A21 Asp B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Milistil-Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Milistil-Arg A0 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoil-Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoil-Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoyl-Arg A0 Gly A21 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB29 -Palmitoyl-HI, N εB29 -Milistil-HI, N εB28 -Palmitoil-Lys B28 Pro B29 -HI,NεB28 -Milistil-Lys B28 Pro B29 -HI,N εB29 -Palmitoyl-des(B30)-HI, N εB30 -Milistil-Thr B29 Lys B30 -HI,N εB30 -Palmitoil-Thr B29 Lys B30 -HI,N εB29 -(N-palmitoyl-γ-glutamyl)-des(B30)-HI, N εB29 -(N-litocoryl-γ-glutamyl)-des(B30)-HI, N εB29 -(ω-carboxyheptadecanoyl)-des(B30)-HI,N εB29 -(ω-carboxyheptadecanoyl)-HI,N εB29 -Octanoyl-HI, N εB29 -Milistil-Gly A21 Arg B31 Arg B31 -HI,N εB29 -Milistil-Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Milistil-Arg A0 Gly A21 Arg B31 Arg B32 -HI,N εB29 -Arg A0 Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Milistil-Arg A0 Gly A21 Asp B3 Arg B31 Arg B32 -HI,N εB29 -Milistil-Arg B31 Arg B32 -HI,N εB29 -Milistil-Arg A0 Arg B31 Arg B32 -HI,NεB29 -Octanoil-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Octanoil-Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB29 -Octanoyl-Arg A0 Gly A21 Arg B31 Arg B32 -HI,N εB29 -Octanoyl-Arg A0 Gly A21 Gln B3 Arg B31 Arg B32 -HI,N εB28 -Octanoyl-Arg A0 Gly A21 Gln B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoyl-Arg A0 Gly A21 Asp B3 Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoil-Lys B28 Pro B29 Arg B31 Arg B32 -HI,N εB28 -Octanoyl-Arg A0 Lys B28 Pro B29 Arg B31 Arg B32 -HI.N εB29 -Pentanoyl-Gly A21 Arg B31 Arg B32 -HI,N αB1 -Hexanoyl-Gly A21 Arg B31 Arg B32 -HI,N αA1-Heptanoyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Octanoil-N αB1 -Octanoil-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Propionyl-N αA1 -Propionyl-Gly A21 Arg B31 Arg B32 -HI,N αA1 -acetyl-N αB1 -Acetyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -Formyl-N αA1 -Formyl-N αB1 -Formyl-Gly A21 Arg B31 Arg B32 -HI,N εB29 -formyl-des(B26)-HI,N αB1 -acetyl-Asp B28 -HI,N εB29 -Propionyl-N αA1 -Propionyl-N αB1 -Propionyl-Asp B1 Asp B3 Asp B21 -HI,N εB29 -Pentanoyl-Gly A21 -HI,N αB1 -Hexanoyl-Gly A21 -HI,N αA1 -Heptanoyl-Gly A21 -HI,N εB29 -Octanoil-N αB1 -Octanoil-Gly A21 -HI,N εB29 -Propionyl-N αA1 -Propionyl-Gly A21 -HI,N αA1 -acetyl-N αB1 -Acetyl-Gly A21 -HI,N εB29 -Formyl-N αA1 -Formyl-NαB1 -Formyl-Gly A21 -HI,N εB29 -Butyryl-des(B30)-HI, N αB31 -Butyryl-des(B30)-HI, N αA1 -Butyryl-des(B30)-HI, N εB29 -Butchiril-N αB31 -Butyryl-des(B30)-HI, N εB29 -Butchiril-N αA1 -Butyryl-des(B30)-HI, N αA1 -Butchiril-N αB31 -Butyryl-des(B30)-HI, N εB29 -Butchiril-N αA1 -Butchiril-N αB31 -Butyryl-des(B30)-HI, Lys B28 Pro B29 -HI (insulin lispro), Asp B28 -HI (insulin aspart), Lys B3 Glu B29 -HI (insulin glulysin), Arg B31 Arg B32 -HI (insulin glargine), N εB29 -Myristoyl-des(B30)-HI (insulin detemir), Ala B26 -HI, Asp B1 -HI, Arg A0 -HI, Asp B1 Glu B13 -HI EntryGly A21 -HI EntryGly A21 Arg B31 Arg B32 -HI, Arg A0 Arg B31 Arg B32 -HI, Arg A0 Gly A21 Arg B31 Arg B32 -HI, des(B30)-HI, des(B27)-HI, des(B28-B30)-HI, des(B1)-HI, des(B1-B3)-HIN εB29 -Tridecanoyl-des(B30)-HI,N εB29-Tetradecanoyl-des(B30)-HI,N εB29 -Decanoyl-des(B30)-HI, N εB29 -Dodecanoyl-des(B30)-HI,N εB29 -Tridecanoil-Gly A21 -des(B30)-HI, N εB29 -Tetradecanoil-Gly A21 -des(B30)-HI, N εB29 -Decanoil-Gly A21 -des(B30)-HI, N εB29 - Dodecanoil - Gly A21 -des(B30)-HI, N εB29 -Tridecanoil-Gly A21 Gln B3 -des(B30 )-HI, N εB29 -Tetradecanoil-Gly A21 Gln B3 -des(B30)-HI, N εB29 -Decanoil-Gly A21 -Gln B3 -des(B30)-HI, N εB29 - Dodecanoil - Gly A21 -Gln B3 -des(B30)-HI, N εB29 -Tridecanoil-Ala A21 -des(B30)-HI, N εB29 -Tetradecanoil-Ala A21 -des(B30)-HI, N εB29 -Decanoil-Ala A21 -des(B30)-HI, N εB29 -Dodecanoil-Ala A21 -des(B30)-HI, N εB29 -Tridecanoil-Ala A21 -Gln B3 -des(B30)-HI, N εB29 -Tetradecanoil-Ala A21 Gln B3 -des(B30)-HI, N εB29 -Decanoil-Ala A21 Gln B3 -des(B30)-HI, NεB29 -Dodecanoil-Ala A21 Gln B3 -des(B30)-HI, N εB29 -Tridecanoil-Gln B3 -des(B30)-HI, N εB29 -Tetradecanoyl-Gln B3 -des(B30)-HI, N εB29 -Decanoil-Gln B3 -des(B30)-HI, N εB29 - Dodecanoil - Gln B3 -des(B30)-HI, N εB29 -Z1-Gly A21 -HI,N εB29 -Z2-Gly A21 -HI,N εB29 -Z4-Gly A21 -HI,N εB29 -Z3-Gly A21 -HI,N εB29 -Z1-Ala A21 -HI,N εB29 -Z2-Ala A21 -HI,N εB29 -Z4-Ala A21 -HI,N εB29 -Z3-Ala A21 -HI,N εB29 -Z1-Gly A21 Gln B3 -HI,N εB29 -Z2-Gly A21 Gln B3 -HI,N εB29 -Z4-Gly A21 Gln B3 -HI,N εB29 -Z3-Gly A21 Gln B3 -HI,N εB29 -Z1-Ala A21 Gln B3 -HI,N εB29 -Z2-Ala A21 Gln B3 -HI,N εB29 -Z4-Ala A21 Gln B3 -HI,N εB29 -Z3-Ala A21 Gln B3-HI、N εB29 -Z1-Gln B3 -HI、N εB29 -Z2-Gln B3 -HI、N εB29 -Z4-Gln B3 -HI、N εB29 -Z3-Gln B3 -HI、N εB29 -Z1-Glu B30 -HI、N εB29 -Z2-Glu B30 -HI、N εB29 -Z4-Glu B30 -HI、N εB29 -Z3-Glu B30 -HI、N εB29 -Z1-Gly A21 Glu B30 -HI、N εB29 -Z2-Gly A21 Glu B30 -HI、N εB29 -Z4-Gly A21 Glu B30 -HI、N εB29 -Z3-Gly A21 Glu B30 -HI、N εB29 -Z1-Gly A21 Gln B3 Glu B30 -HI、N εB29 -Z2-Gly A21 Gln B3 Glu B30 -HI、N εB29 -Z4-Gly A21 Gln B3 Glu B30 -HI、N εB29 -Z3-Gly A21 Gln B3 Glu B30 -HI、N εB29 -Z1-Ala A21 Glu B30 -HI、N εB29 -Z2-Ala A21 Glu B30 -HI、N εB29 -Z4-Ala A21 Gln B30 -HI、N εB29 -Z3-Ala A21Glu B30 -HI,N εB29 -Z1-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z2-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z4-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z3-Ala A21 Gln B3 Glu B30 -HI,N εB29 -Z1-Gln B3 Glu B30 -HI,N εB29 -Z2-Gln B3 Glu B30 -HI,N εB29 -Z4-Gln B3 Glu B30 -HI,N εB29 -Z3-Gln B3 Glu B30 -Includes one of HI, including mutations and / or chemical modifications, where Z1 is tridecanoyl, Z2 is tetradecanoyl, Z3 is dodecanoyl, Z4 is decanoyl, and HI is human insulin.

[0058] In certain embodiments, the insulin molecule undergoes the following mutations and / or chemical modifications: N εB28 -XXXXX-Lys B28 Pro B29 -HI,N αB1 -XXXXX-Lys B28 Pro B29 -HI,N αA1 -XXXXX-Lys B28 Pro B29 -HI,N εB28 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI,N εB28 -XXXXX-N αA1 -XXXXX-Lys B28 ProB29 -HI、N αA1 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI、N εB28 -XXXXX-N αA1 -XXXXX-N αB1 -XXXXX-Lys B28 Pro B29 -HI、N εB29 -XXXXX-HI、N αB1 -XXXXX-HI、N αA1 -XXXXX-HI、N εB29 -XXXXX-N αB1 -XXXXX-HI、N εB29 -XXXXX-N αA1 -XXXXX-HI、N αA1 -XXXXX-N αB1 -XXXXX-HI、N εB29 -XXXXX-N αA1 -XXXXX-N αB1 -XXXXX-HI、N εB29 -YYYYY-HI、N αB1 -YYYYY-HI、N αA1 -YYYYY-HI、N εB29 -YYYYY-N αB1 -YYYYY-HI、N εB29 -YYYYY-N αA1 -YYYYY-HI、N αA1 -YYYYY-N αB1 -YYYYY-HI、N εB29 -YYYYY-N αA1 -YYYYY-N αB1 -YYYYY-HI、N εB28 -YYYYY-Lys B28 Pro B29 -HI、N εB21 -YYYYY-Lys B28 Pro B29 -HI、N αA1 -YYYYY-Lys B28 Pro B29 -HI、N εB28 -YYYYY-N αB1 -YYYYY-Lys B28 Pro B29 -HI、N εB28-YYYYY-N αA1 -YYYYY-Lys B28 Pro B29 -HI,N αA1 -YYYYY-N αB1 -YYYYY-Lys B28 Pro B29 -HI,N εB28 -YYYYY-N αA1 -YYYYY-N αB1 -YYYYY-Lys B28 Pro B29 It has -HI, where YYYYY is one of acetyl or formyl, XXXXX is one of propionyl, butyryl, pentanoyl, hexanoyl, heptanol, octanoyl, nonanoyl, or decanoyl, and HI is human insulin.

[0059] As discussed herein, insulin molecules can be conjugated via reactive moieties that are naturally present within the insulin structure and / or added before conjugation, including, for example, carboxyl or reactive esters, amines, hydroxyls, aldehydes, sulfhydryls, maleimidyls, alkynyls, and azides. Insulin naturally contains reactive alpha-terminal amines and epsilon-aminelysine groups that can covalently bond NHS-esters, isocyanates, and / or isothiocyanates. In certain embodiments, modified insulin may be used in which suitable amino acids (e.g., lysine and / or non-natural amino acids) are added to or substituted in the amino acid sequence to provide alternative (e.g., additional) binding sites in addition to the modified amino acids of the embodiments described herein. Furthermore, it will be understood that the conjugation process can be controlled by selectively blocking or protecting specific reactive moieties before conjugation. In particular embodiments, insulin may include any combination of these modifications, and it should be understood that this disclosure also includes modified forms of non-human insulin (e.g., porcine insulin, bovine insulin, rabbit insulin, sheep insulin, etc.) that include any one of the aforementioned modifications.Certain embodiments refer to these and certain other previously described modified insulins, for example, U.S. Patents No. 5,474,978, No. 5,461,031, No. 4,421,685, No. 7,387,996, No. 6,869,930, No. 6,174,856, No. 6,011,007, No. 5,866,538, No. 5,750,4976, No. 906,028, No. 6,551,992, No. 6,465,426, No. 6,444,641, No. 6,335,316, and No. 6,268. It is understood that this may include those described in U.S. Patent Application No. 335, No. 6,051,551, No. 6,034,054, No. 5,952,297, No. 5,922,675, No. 5,747,642, No. 5,693,609, No. 5,650,486, No. 5,547,929, and No. 5,504,188, as well as those described in U.S. Patent Application No. 2015 / 0353619, and includes non-natural amino acids described or referenced herein, and includes such modifications to non-human insulin described herein. It should also be understood that in certain embodiments, insulin may be covalently bonded to a polyethylene glycol polymer with Mn 218,000 or less, or to albumin.

[0060] In certain embodiments, modified insulin is further conjugated to a non-boronized polypeptide by the use of an enzyme. In certain embodiments, the N-terminal or C-terminal residue of the peptide fragment can function as a recognition sequence for a peptide ligase to enable the conjugate of the peptide to insulin, and in certain other embodiments, insulin can be expressed using one or more additional amino acids such that one of the ends of the A or B chain of insulin is recognized by an enzyme, and then the desired non-boronized polypeptide is added to the insulin. In certain embodiments, the polypeptide is added to the C-terminus of the insulin A and / or B chain using a protein ligase. In certain embodiments, the polypeptide is added to the N-terminus of the insulin A and / or B chain using a protein ligase. In certain embodiments, the polypeptide is conjugated to modified insulin using a protein ligase selected from the group consisting of saltase, buterase, tripsyligase, subtilisin, peptiligases, or enzymes having at least 75% homology to these ligases. In certain embodiments, this is achieved by expressed protein ligation as described in Muir TW, Sondhi D, Cole PA. Expressed protein ligation: a general method for protein engineering. Proc Natl Acad Sci US A. 1998;95(12):6705-6710. In certain other embodiments, the polypeptide is bound to modified insulin using Staudinger ligation, for example, Nilsson, BL; Kiessling, LL; Raines, RT (2000). "Staudinger ligation: A peptide from a thioester and azide". Org. Lett. 2(13):1939-1941.In certain other embodiments, the polypeptide is conjugated to modified insulin using Ser / Thr ligation, for example, as described in Zhang Y, Xu C, Kam HY, Lee CL, Li X. 2013, "Protein chemical synthesis by serine / threonine ligation." Proc. Natl. Acad. Sci. USA. 17:6657-6662. In certain embodiments, the B chain itself has fewer than 32 amino acids or 34 amino acids, and in certain embodiments, the insulin has 4 disulfide bonds instead of 3.

[0061] The covalent binding of modified insulin to peptides, proteins, synthetic polymers, or modified insulin itself, and its molecular properties, can be tested by LC-MS or SDS-polyacrylamide gel shift assays to verify conjugation and modify stoichiometry. Different linker chemistry and terminal functionalizations can be tested. Some of these linkers may contain orthogonal chemistry to the protein, and in certain embodiments, the linker covalently bonds the adjacent diol sensor to the drug substance, and any molecule further interacting with the adjacent diol sensor can be achieved by what is known as click chemistry, or by various similar biorthogonal chemical reactions, for example, by a copper-catalyzed 3+2 cycloaddition reaction (click reaction) using a suitable or preferred copper coordination ligand, as described, for example, in Rostovtsev, VV, Green, LG, Fokin, VV & Sharpless, KBA stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective “ligation” of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596-2599 (2002). In addition, copper-free conjugate of terminal azides to alkynes or alkynyl probes can be used as described by Liang, Y., Mackey, JL, Lopez, SA, Liu, F. & Houk, KN: Control and design of mutual orthogonality in bioorthogonal cycloadditions. J. Am. Chem. Soc. 134, 17904-17907 (2012) and Beatty, KE et al.: Live-cell imaging of cellular proteins by a strain-promoted azide-alkyne cycloaddition. Chembiochem 11, 2092-2095 (2010).

[0062] In certain embodiments, further modifications to the compounds of the Disclosure may include the attachment of one or more hydroxyl-containing chemicals that interact with the adjacent diol sensor. In certain embodiments, the groups that interact with the adjacent diol sensor include groups such as carbohydrates, one or more cis-diol-containing molecules, one or more phosphate groups, one or more catechol groups, one or more farnesyl groups, isofarnesyl groups, fatty acid or diacid groups, and / or other diol-containing molecules.

[0063] In certain embodiments, the active pharmaceutical ingredient is insulin to which additional groups are added that interact with adjacent diol sensors to modulate the sensor's response profile to glucose levels in the body. In those particular embodiments, the amino acid side chains in the modified insulin contain one or more chemical structures, or proteins and / or polypeptides to which the modified insulin is conjugated, and in those particular embodiments, one or more chemical structures are represented by formulas F111, F222, F333. [ka] During the ceremony, ·Each R 1 It can independently have (R) or (S) stereochemistry, and independently have H, OR 3 , N(R 3 )2, SR 3 OH, OCH3, OR 5 , R 6 -R 7 NHC(O)CH3,CH2R 3 NHC(O)CH3,CH2OH,CH2OR 5 NH2, R 2 , or CH2R 4 Selected from, ·Each R 2 These are, independently, H, or C 1-6 Selected from optionally substituted groups, which are either a 5-6 member monocyclic heteroaryl ring having 1-4 heteroatoms selected from aliphatic, phenyl, nitrogen, oxygen, or sulfur, or a 4-7 member heterocyclic ring having 1-2 heteroatoms selected from nitrogen, oxygen, or sulfur. ·Each R 3 These are independently H, acetyl, phosphate, and R 2 SO2R 2 S(O)R 2 , P(O)(OR 2 )2, C(O)R 2 CO2R 2 , or C(O)N(R 2 ) Selected from 2, ·Each R 4 These are independently H, OH, OR 3 , N(R 3 )2, OR 5 , or SR 3 Selected from, ·Each R 5 These are independently selected from monosaccharides, disaccharides, trisaccharides, pentoses, or hexoses. ·Each R 6 These are independently linker, NCOCH2, OCH2CH2, and OC. 1-9 Alkylene, substitution C 1-9 Selected from alkylenes, one or more methylene groups are optionally -O-, -CH2-, -OCH2-, -N(R) 2 )C(O)-, -N(R 2 )C(O)N(R 2 )-, -SO2-, -SO2N(R 2 )-,-N(R 2 )SO2-, -S-, -N(R 2 )-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R 2 )-, or -N(R 2 )SO2N(R 2 )- is replaced by, ·Each R 7 Independently, N(R) 2 )2, F, Cl, Br, I, SH, OR 2 , SR 2 NH2, N3, C≡CR 2 CH2C≡CH, C≡CH, CO2R 2 , C(O)R 2 , or OSO2R 2 , N(R 2 )2, OR 2 , SR 2, or selected from CH2NH2, In certain embodiments, structures F111, F222, and F333 can be covalently bound to modified insulin or a drug or protein to which modified insulin is covalently bound via various linkers.

[0064] In certain embodiments, the anomeric carbon is bonded to the OR 5 The resulting glycosidic bond may have an α:DOWN or β:UP configuration. In certain embodiments, the modified insulin may be aminoethyl glucose, aminoethyl bimannose, aminoethyltrimannose, D-glucose, D-galactose, D-allose, D-mannose, D-glucose, D-idose, D-talose, N-azidomannasamine (ManNAz) or N-azidogalactoseamine (GalNAz), or N-azidoglucosamine (GlcNAz), 2'-fluororibose, 2'-deoxyribose, glucose, sucrose, maltose, mannose, or their derivatives (e.g., glucosamine, mannosamine, methylglucose, methyl The active pharmaceutical ingredient is mixed with or covalently bonded to one or more covalent bonds from the group consisting of, for example, L-DOPA or L-3,4-dihydroxyphenylalanine, including molecules containing cis-diols (such as lumannose, ethyl glucose, ethylmannose), sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, and / or higher-order combinations thereof (such as linear and / or branched bimannose, linear and / or branched trimannose), molecules containing cis-diols, catechol, tris, DOPA molecules, for example, L-DOPA or L-3,4-dihydroxyphenylalanine, or a modified drug substance from its original form to contain one or more covalent bonds. In certain embodiments, modified insulin is conjugated to catechol.

[0065] In certain embodiments, the structures represented by F111, F222, and F333 may be covalently bonded to modified insulin or the active pharmaceutical ingredient via various linkers, such as via amide bonds, one or more alkyl groups, triazole bonds, any covalent linkers, or combinations thereof.

[0066] In certain embodiments, modified insulin containing one or more adjacent diol sensors may include aminoethyl glucose, aminoethyl bimannose, aminoethyl trimannose, D-glucose, D-galactose, D-allose, D-mannose, D-growth, D-idose, D-talose, N-azidomannasamine (ManNAz) or N-azidogalactoseamine (GalNAz), or N-azidoglucosamine (GlcNAz), 2'-fluororibose, 2'-deoxyribose, glucose, sucrose, maltose, mannose, or derivatives thereof (e.g., glucosamine, The modified insulin is mixed with or covalently bonded to one or more covalently bonded substances, partially containing or selected from the group consisting of mannosamine, methyl glucose, methyl mannose, ethyl glucose, ethyl mannose, etc., sorbitol, inositol, galactitol, dulcitol, xylitol, arabitol, and / or higher-order combinations thereof (linear and / or branched bimannose, linear and / or branched trimannose, etc.), molecules containing cis-diols, catechol, tris, DOPA molecules, for example, L-DOPA or L-3,4-dihydroxyphenylalanine. In certain embodiments, the modified insulin contains amino acids including: [ka]

[0067] In certain embodiments, modified insulin containing one or more adjacent diol sensors is conjugated to a modified glucose such as azidoglucose. For example, in the case of M-azido-M-deoxy-D-glucose, M is one of 1, 2, 3, 4, 5, or 6. In certain embodiments, the azido-containing sugar can be conjugated to a terminal alkyne, for example, via click chemistry (such terminal alkynes may exist as side chains of amino acids, such as L-homopropagylglycine or other amino acids described herein having alkyne side chains). The azido group on the sugar can be conjugated to the alkyne group, for example, by a copper-catalyzed click reaction resulting in a triazole bond, or, in certain embodiments, to cyclooctin which is conjugated to the side chain of an amino acid by itself. In certain embodiments, the modified insulin can interact with albumin in the blood, either by itself or via covalent modifications such as covalent bonding to a fatty acyl or fatty diacitic acid, and in certain embodiments, the affinity of this interaction can be regulated based on glucose. In certain embodiments, insulin is mixed as part of a pharmaceutically acceptable carrier comprising a sugar polymer, a diol-containing polymer, and / or a polysaccharide.

[0068] In certain embodiments, one or more artificial amino acids may be included in a linker bound to the structure of modified insulin or an adjacent diol sensor. There are 20 different natural (standard) amino acids, which are the building blocks of all natural proteins. Non-standard or artificial amino acids have different side chains from standard amino acids and are generally not present in proteins. The incorporation of artificial amino acids into recombinant proteins and / or synthetic peptides enables selective functionalization and the introduction of modifiable chemical groups. This is particularly useful in the development of modified insulin because it allows for the selective chemical modification of insulin at specific positions in the protein sequence. In certain embodiments, artificial amino acids can be used in modified insulin to modulate pKa, local hydrophobicity of the protein domain, and aggregation and folding properties, or to introduce new chemical and / or chemical and / or physical properties, including thermal stability, aggregation behavior, solution stability, reduced aggregation, conformational changes, and / or movement of insulin A and B chains relative to each other. In certain embodiments, one or more of the following artificial amino acids represented by formulas FX15-28 may be used in modified insulin: [ka] During the ceremony, Each R1 is independently selected from H, NH2, NO2, Cl, CF3, I, COCH3, CN, C≡CH, N3, or Br. Each R2 is independently selected from NH2, CF3, H, or CH3. a. Each R3 is independently selected from C≡CH, H, N3, or a vinyl group. b. Each R4 is independently selected from NH2, R2, or R3. c. Each R5 is independently selected from S or NH. d. The index n is an integer in the range of 1 to 4. j is an integer in the range of 1 to 14. k is an integer in the range of 1 to 14.

[0069] Furthermore, in certain embodiments, one or more previously disclosed protein-constitutive or non-protein-constitutive artificial amino acids may be used either as part of a structure that binds an adjacent diol sensor to the drug substance, and / or as part of the drug substance, and if the drug substance is insulin or another peptide, the artificial amino acids may be present in the insulin or peptide. For example, in certain embodiments, one or more of the following artificial amino acids may be used based on the method described and referenced therethrough in Liu, CC; Schultz, PG (2010) “Adding new chemistries to the genetic code”. Annual Review of Biochemistry 79:413-44, and the list of amino acids provided therein. In certain embodiments, the artificial amino acids may be incorporated by peptide synthesis, which includes the amino acids referenced herein and previously reported non-protein-constitutive amino acids. For example, a portfolio of such non-protein-constitutive amino acids, including β-amino acids, is commercially available from Sigma Aldrich and includes amino acids such as 2,3-diaminopropionic acid, 2,4-diaminopropionic acid, ornithine, and any beta or alpha amino acids.As an example, the protein-constituting artificial amino acids described in F26-F41 are covered by U.S. Patents and Patent Applications US2008 / 0044854, US8518666, US8980581, US2008 / 0044854, US2014 / 0045261, US2004 / 0053390, US7229634, US8236344, US2005 / 0196427, US2010 / 0247433, US7198915, US7723070, US2002 / 0042097, US2004 / 0058415, and US2008 / 0026. The proteins can be incorporated by recombinant protein expression using the methods and approaches described in US No. 422, US2008 / 0160609, US2010 / 0184193, US2012 / 0077228, US2014 / 025599, US7198915, US7632492, and US7723070, and other protein-constituting artificial amino acids can be recombinantly introduced using the methods and approaches described in US7736872, US7816320, US7829310, US7829659, US7883866, US8097702, and US8946148.

[0070] In certain embodiments, cyclic amino acids, such as 3-hydroxyproline, 4-hydroxyproline, aziridine-2-carboxylic acid, azetidine-2-carboxylic acid, piperidine-2-carboxylic acid, 3-carboxymorpholine, 3-carboxythiamorpholine, 4-oxaproline, pyroglutamic acid, 1,3-oxazolidine-4-carboxylic acid, 1,3-thiazolidine-4-carboxylic acid, 3-thiaproline, 4-thiaproline, 3-selenoproline, 4-selenoproline, 4- Ketoproline, 3,4-dehydroproline, 4-aminoproline, 4-fluoroproline, 4,4-difluoroproline, 4-chloroproline, 4,4-dichloroproline, 4-bromoproline, 4,4-dibromoproline, 4-methylproline, 4-ethylproline, 4-cyclohexylproline, 3-phenylproline, 4-phenylproline, 3,4-phenylproline, 4-azidoproline, 4-carboxyproline, α-methylproline, α-ethylproline, α-proline Ropyruproline, α-allylproline, α-benzylproline, α-(4-fluorobenzyl)proline, α-(2-chlorobenzyl)proline, α-(3-chlorobenzyl)proline, α-(2-bromobenzyl)proline, α-(4-bromobenzyl)proline, α-(4-methylbenzyl)proline, α-(diphenylmethyl)proline, α-(naphthylmethyl)-proline, D-proline, or L-homoproline, (2S,4S)-4-fluoro-L-proline, (2S (4R)-4-fluoro-L-proline, (2S)-3,4-dihydro-L-proline, (2S,4S)-4-hydroxy-L-proline, (2S,4R)-4-hydroxy-L-proline, (2S,4S)-4-azido-L-proline, (2S)-4,4-difluoro-L-proline, (2S)-azetidine-2-carboxylic acid, (2S)-piperidine-2-carboxylic acid, or (4R)-1,3-thiazolidine-4-carboxylic acid can be used in modified insulin.

[0071] In certain embodiments, it should be understood that the amino acid set or specific orientation is achieved, for example, by the synthesis of modified insulin using the method described in Albericio, F. (2000). Solid-Phase Synthesis: A Practical Guide (1st ed.). Boca Raton: CRC Press. P.848.

[0072] In certain embodiments, modified insulin can be bound to polymers of diols, catechols, hexose sugars, glucose, xylose, fucose, galactosamine, glucosamine, mannosamine, galactose, mannose, fructose, galacturonic acid, glucuronic acid, iduronic acid, mannuronic acid, acetylgalactosamine, acetylglucosamine, acetylmannosamine, acetylmuramic acid, 2-keto-3-deoxy-glycero-galactonononic acid, acetylneuraminic acid, glycolylneuraminic acid, neurotransmitters, dopamine, and / or disaccharides, and / or sugars and / or diols.

[0073] In certain embodiments, a set or specific modified insulin having the desired biophysical properties, including binding to a target protein (or target molecule) or binding to and responsiveness to a target small molecule, can be obtained by screening a library of modified insulins that are recombinantly expressed and either chemically modified and / or chemically synthesized using standard FMOC or BOC-protected amino acid synthesis on a solid support.

[0074] In certain embodiments, the modified insulin is further conjugated to a chemical structure represented by the following structure: [ka] During the ceremony, ·Each R 1 These are independently H, F, Cl, CH3, B(OH)2, C≡N, NO2, R 4 Selected from, or two adjacent R 1The groups are CH2-O--- and B(OH)---, where --- represents two adjacent R groups. 1 It is a bond between groups, ·Each R 2 These are independently H, C≡N, (SO2)NH(R 4 ), or R 4 Selected from, ·Each R 3 These are independently C≡N, CONH(R 4 ), NH(R 4 ), (SO2)NH(R 4 ), or R 4 Selected from, ·Each R 4 These are independently H, N3, C≡CH, -CH2N(R 5 ), or selected from the linker, ·Each R 5 The linker is independently selected from H, or a linker that covalently bonds the structure to an amino acid side chain, such as the lysine side chain, via an amide bond to the epsilonamine of lysine.

[0075] In certain embodiments, modified insulin or drug substance conjugated with adjacent diol sensors may be further covalently bonded using amide bonds to structures represented by the following formulas F500-F520: [ka]

[0076] In certain embodiments, such modifications involve the use of an N-methyliminodiacetic acid (MIDA) group, which can be used to produce MIDA conjugate boronic acid or MIDA boronic acid, and such modifications can be used during the preparation of the boronic acid for the final structure of use. In certain embodiments, pinacol boronic acid ester is used for the final structure, and the pinacol group can be readily removed by those skilled in the art. MIDA-protected boronic acid esters are readily handled, stable in air, chromatographically compatible, nonreactive under standard anhydrous cross-coupling conditions, and readily deprotected at room temperature under mild aqueous basic conditions such as 1 M NaOH, or even NaHCO3, or as described by Lee, S.Jet al. J.Am.Chem.Soc.2008, 130, 466.

[0077] As previously reported in Nature 493, 241-245 (2013); Menting, J Get al. Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl. Acad. Sci. USA 111, E3395-3404 (2014), the biological mechanism by which wild-type insulin binds to the insulin receptor. In certain embodiments, the binding of glucose to an adjacent diol on modified insulin can be used to modulate the bioavailability, solubility, and / or ability of insulin to engage with the insulin receptor. The activity of such insulin may, for example, be influenced by, but is not limited to, TyrA14- 125I. Measurement can be performed by using an in vitro insulin receptor that binds to human insulin as a tracer and utilizing antibody-conjugated beads together with an insulin receptor monoclonal antibody. In one or more embodiments, animal models can be used for in vivo evaluation of insulin activity, including during a glucose challenge, using methods readily apparent to those skilled in the art. In certain embodiments, modified insulin is further modified or manipulated to bind to glucose transporters so that changes in the concentration of soluble glucose can modulate the affinity with which modified insulin binds to glucose transporters. In certain embodiments, modified insulin can bind to small molecules orally administered in the body, and in certain embodiments, such binding can be used to modulate the activity of modified insulin. In certain embodiments, modified insulin can bind to other proteins and / or drugs that directly or indirectly affect blood glucose levels and / or metabolism in the body. In addition to insulin, in certain embodiments, the conjugate diol sensor is conjugated to peptides and / or incretin hormones selected from the group consisting of glucagon, GLP-1, GLP-1 analogs, GLP-1 receptor agonists, IGF1, amylin, and relaxin. In certain embodiments, insulin and / or these incretins contain at least one structure represented by formula I, II, or III. In certain embodiments, insulin contains at least two structures, each independently represented by formula I, II, or III. In certain embodiments, at least one peptide sequence comprising 2 to 20 amino acids can be independently added to or removed from the A and / or B chains of insulin.

[0078] In certain embodiments, modified insulin is expressed partially or completely as a recombinant protein, and the side chains corresponding to formulas I-VI are introduced via chemical modification into the side chains of existing amino acids such as lysine. The process for insulin expression in E. coli is known and can be readily carried out by those skilled in the art using the procedures outlined in Jonasson, Eur. J. Biochem. 236:656-661 (1996), Cowley, FEBS Lett. 402:124-130 (1997), Cho, Biotechnol. Bioprocess Eng. 6:144-149 (2001), Tikhonov, Protein Exp. Pur. 21:176-182 (2001), Malik, Protein Exp. Pur 55:100-111 (2007), Min, J. Biotech. 151:350-356 (2011). In the most common process, the protein is expressed as a splitting protein or a single-chain proinsulin construct with affinity tags. Modified insulin can be expressed as part of proinsulin and then chemically modified to bind to the desired boronic acid via an amide bond. This approach offers good yields, reduces experimental complexity by decreasing the number of processing steps, and allows for refolding in a natural-like manner; see, for example, Jonasson, Eur. J. Biochem. 236:656-661 (1996), Cho, Biotechnol Bioprocess Eng. 6:144-149 (2001), Tikhonov, Protein Exp. Pur. 21:176-182 (2001), Min, J. Biotech. 151:350-356 (2001). When expressed in E. coli, proinsulin is usually found in inclusion bodies and can be readily purified by those skilled in the art.

[0079] In certain embodiments, modified insulin containing one or more adjacent diol sensors may be formulated for injection. For example, it may be formulated for injection into subjects such as humans, and the composition may be a pharmaceutical composition such as a sterile injectable pharmaceutical composition. The composition may be formulated for subcutaneous injection. In certain embodiments, the composition may be formulated for transdermal, intradermal, transmucosal, transnasal, inhalation, or intramuscular administration. The composition may be formulated in oral or pulmonary administration form. Pharmaceutical compositions suitable for injection may include, for example, a sterile aqueous solution containing sugars, polyhydric alcohols such as mannitol and sorbitol, phenol, metacresol, and sodium chloride; the dispersion may be prepared in glycerol, liquid polyethylene glycol, and mixtures thereof, and in oil; and the carrier may be, for example, a solvent or dispersion medium containing water, sugars, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and / or suitable mixtures thereof. Those skilled in the art will recognize that sets or specific formulations may be developed to best suit the application and use of the modified insulin described herein. General considerations regarding the formulation and manufacture of pharmaceutical compositions, routes of administration, and suitable pharmaceutically acceptable carriers can be found, for example, in Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Co., Easton, Pa., 1995. In certain embodiments, the pharmaceutical composition may contain zinc, i.e., Zn 2+and / or polysaccharides may be included. Certain zinc preparations are described, for example, in U.S. Patent No. 9,034,818. For example, a pharmaceutical composition may contain zinc in a molar ratio of about M:N to modified insulin, where M is 1 to 11 and N is 6 to 1. In certain embodiments, such modified insulin may be stored in a pump, which is either outside or inside the body, and releases the modified insulin. In some cases, the pump may be used to release a fixed amount of modified insulin, and the insulin is glucose-responsive based on an adjacent diol sensor on the insulin, and its activity can be automatically adjusted based on the level of glucose in the blood or the rate of release from the injection site. In some cases, the composition may be formulated in dose unit form for ease of administration and uniformity of dosage. In some cases, the pharmaceutical composition may further include a second insulin type that provides rapid-acting or basal insulin in addition to the effect provided by the modified insulin.

[0080] In another embodiment, the disclosure includes a kit comprising modified insulin containing an adjacent diol sensor and a pharmaceutically acceptable carrier, and may include a syringe or pen for injection. In various embodiments, the kit may include a syringe or pen pre-filled with a pharmaceutical composition comprising modified insulin together with a liquid carrier. In certain embodiments, the kit may include a separate container, such as a vial, containing a pharmaceutical composition comprising modified insulin together with a dry carrier and an empty syringe or pen. In certain embodiments, such a kit may include a separate container having a liquid carrier that can be used to reconstitute a given composition that can then be taken up by the syringe or pen. In certain embodiments, the kit may include instructions. In certain embodiments, the kit may include a blood glucose measuring device for calculating an appropriate or preferred dose of modified insulin to be injected at a given time or at regular intervals, either locally or remotely. Such a dosing regimen is patient-specific and may be provided, for example, as instructions for programming a pump, either by a human or by a computer. The kit may include an electronic device that transfers blood glucose measurements to a second computer, either locally or elsewhere (e.g., in the cloud), which then calculates the correct amount of modified insulin that the patient needs to use at a specific time.

[0081] In some embodiments, embodiments of the present disclosure relate to a method for treating a disease or condition in a subject, comprising administering a composition comprising modified insulin as described herein, wherein the insulin contains a glucose-responsive adjacent diol sensor. In some cases, the disease or condition may be hyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1 diabetes, obesity, metabolic syndrome X, or dyslipidemia, diabetes during pregnancy, prediabetes, Alzheimer's disease, MODY 1, MODY 2, or MODY 3 diabetes, mood disorders, and / or psychiatric disorders. It will be understood that this combination approach may also be used in insulin-resistant patients receiving insulin sensitizers or secondary drugs for diabetes (e.g., biguanides such as metformin, glitazone, etc.) and / or insulin secretagogues (e.g., sulfonylureas, GLP-1, exendin-4, etc.) and / or amylin.

[0082] The modified insulin of this disclosure may be administered to a patient receiving at least one additional therapy or taking at least one additional drug or therapeutic protein. At least one additional therapy is intended to treat the same disease or disorder as the administered modified insulin. In certain embodiments, at least one additional therapy is intended to treat a side effect of the modified insulin. The timeframes of the two therapies may be different or the same, and they may be administered on the same or different schedules, as long as there is a period during which the patient benefits from both therapies. Two or more therapies may be administered in the same or different formulations, as long as there is a period during which the patient benefits from both therapies. Any of these approaches may be used to target two or more antidiabetic drugs.

[0083] In one or more embodiments, a therapeutically effective amount of modified insulin is used that is suitable or sufficient to treat a disease or condition (e.g., improving its symptoms, slowing its progression, preventing or delaying its recurrence, or delaying its onset) with a reasonable benefit-to-risk ratio. This may involve balancing efficacy with additional safety and toxicity. Additional safety means that even when the patient is not actively monitoring the level of the molecule, such as blood glucose levels, during a given time frame, for example, while sleeping, the modified insulin may be responsive to changes in blood glucose levels or levels of other molecules to which the peptide responds. Generally, therapeutic efficacy and toxicity are measured using standard pharmacological procedures with cell cultures or in vivo experimental animals, e.g., the ED of the therapeutic index of the drug. 50 and LD 50 This can be determined by measuring [the relevant parameters]. In various embodiments, the average daily dose of insulin, including modified insulin, is in the range of 5 to 400 U (for example, 30 to 150 U, if one unit of insulin is approximately 0.04 mg). In certain embodiments, a certain amount of modified insulin is administered daily, every other day, every three days, or every four days. In certain embodiments, the criteria are determined by an algorithm that can be calculated by computer. In certain embodiments, amounts of modified insulin having 5 to 10 times these doses are administered weekly or at regular intervals. In certain embodiments, amounts of modified insulin having 10 to 20 times these doses are administered every other week or at regular intervals. In certain embodiments, amounts of modified insulin having 20 to 40 times these doses are administered monthly.

[0084] The following examples and experimental data are provided for illustrative purposes only and are not intended to limit the scope of the embodiments of this disclosure. [Examples]

[0085] Preparation of small molecule diol sensors and modified insulin. Example 1 (3-((2R,4R)-4-(5-Borono-2-(methylsulfonyl)benzamide)-2-Carbamoylpyrrolidine-1-carbonyl)-4-(methylsulfonyl)phenyl)boronic acid [ka] Synthesis of Example 1: Link-amide resin (1.2 mmol / eq, 150 mg) was swollen in DMF (5 mL) for 20 minutes. The solution was removed under a nitrogen stream, and a 20% piperidine solution in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL). A solution containing (2R,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid (280 mg, 0.5 mmol) in DMF (5 mL), hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (HATU, 190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 20 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 5-Borono-2-(methylsulfonyl)benzoic acid (244 mg, 1 mmol) in DMF (5 mL), HATU (380 mg, 1 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (2 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes. The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA. The pure fractions were isolated, combined, frozen, and freeze-dried to obtain Example 1 as a white powder (20 mg). Expected mass [M+H]: 582.11, Measured mass [M+H]: 582.07

[0086] Figure 1 is a mass spectrum plot confirming the synthesis of Example 1.

[0087] Example 2 ((2S,4S)-1-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carbonyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)pyrrolidine-2-carbonyl)glycine [ka] Synthesis of Example 2: Chlorotrityl resin (1.5 mmol / eq, 300 mg) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under a stream of nitrogen, and a solution of Fmoc-glycine (0.5 M) in DIPEA (1 M) in DCM was immediately added and gently mixed for 1 hour. The mixture was washed with DCM, and any unreacted areas were capped with a solution of 20% MeOH in a solution of DCM and DIEA (1 M), and mixed for 1 hour. The resin was washed with DCM (2 × 5 mL) and then with DMF (2 × 5 mL). The solution was removed under a stream of nitrogen, and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL). A solution containing (2R,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid (280 mg, 0.5 mmol) in DMF (5 mL), hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (HATU, 190 mg, 0.5 mmol), and DIPEA (200 μl) was added to the resin and mixed at 50°C for 20 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (177 mg, 1 mmol) in DMF (5 mL), HATU (380 mg, 1 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (3 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes. The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA. The pure fractions were isolated, combined, frozen, and freeze-dried to obtain Example 2 as a white powder (27 mg).Expected mass [M+H]: 508.16, measured mass [M+H]: 508.13.

[0088] Figure 2 is a mass spectrum plot confirming the synthesis of Example 2.

[0089] Example 3 ((2S,4S)-1-(5-Borono-2-nitrobenzoyl)-4-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)pyrrolidine-2-carbonyl)glycine [ka] Synthesis of Example 3. Chlorotrityl resin (1.5 mmol / eq, 300 mg) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under a stream of nitrogen, and a solution of Fmoc-glycine (0.5 M) in DIPEA (1 M) in DCM was immediately added and gently mixed for 1 hour. The mixture was washed with DCM, and any unreacted areas were capped with a solution of 20% MeOH in a solution of DCM and DIEA (1 M), and mixed for 1 hour. The resin was washed with DCM (2 × 5 mL) and then with DMF (2 × 5 mL). The solution was removed under a stream of nitrogen, and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL). A solution containing (2R,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl)amino)pyrrolidine-2-carboxylic acid (258 mg, 0.5 mmol) in DMF (5 mL), hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (HATU, 190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 20 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 5-borono-2-nitrobenzoic acid (105 mg, 0.5 mmol) in DMF (5 mL), HATU (190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 4% hydrazine in DMF was added to the resin (3 × 5 mL) and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (89 mg, 0.5 mmol) in DMF (5 mL), HATU (190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (3 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes.The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in 0.1% TFA water to 60% ACN in 0.1% TFA water. The pure fractions were isolated, combined, frozen, and lyophilized to obtain Example 3 as a white powder (15 mg). Predicted mass [M+H]: 541.15, measured mass [M+H]: 541.13.

[0090] Figure 3 is a mass spectrum plot confirming the synthesis of Example 3.

[0091] Example 4 (S)-(3-((1-amino-3-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)-1-oxopropan-2-yl)carbamoyl)-5-nitrophenyl)boronic acid [ka] Synthesis of Example 4: Link-amide resin (1.2 mmol / eq, 150 mg) was swollen in DMF (5 mL) for 20 minutes. The solution was removed under a nitrogen stream, and a 20% piperidine solution in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL). Fmoc-N in DMF (5 mL) βA solution containing -(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-methylbutyl-L-2,3-diaminopropionic acid (266 mg, 0.5 mmol), hexafluorophosphate 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide (HATU, 190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 20 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 20% piperidine in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 3-borono-5-nitrobenzoic acid (105 mg, 0.5 mmol) in DMF (5 mL), HATU (190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL), and a solution of 4% hydrazine in DMF was added to the resin (3 × 5 mL) and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (89 mg, 0.5 mmol) in DMF (5 mL), HATU (190 mg, 0.5 mmol), and DIPEA (200 μL) was added to the resin and mixed at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) followed by DCM (3 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes. The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA. The pure fractions were isolated, combined, frozen, and lyophilized to obtain Example 4 as a white powder (11 mg). Predicted mass [M+H]: 457.13, Measured [M+H]: 457.00 [M+H-H2O]: 440.13

[0092] Figure 4 is a mass spectrum plot confirming the synthesis of Example 4.

[0093] Example 5 (S)-(3-((1-amino-3-(5-borono-2-nitrobenzamide)-1-oxopropan-2-yl)carbamoyl)-4-nitrophenyl)boronic acid [ka] Example 5 was synthesized in the same manner as Example 4 and contains F27 and F1. Expected mass [M+H]: 490.11, Measured mass [M+H]: 490.00

[0094] Figure 5 is a mass spectrum plot confirming the synthesis of Example 5.

[0095] Example 6 (S)-(3-((1-amino-3-(1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)-1-oxopropan-2-yl)carbamoyl)-5-nitrophenyl)boronic acid [ka] Example 6 was synthesized in the same manner as Example 4 and contains F27, F1, and F2. Expected mass [M+H]: 525.11, Measured mass [M+H]: 525.00 [M+H-H2O]: 508.07

[0096] Figure 6 is a mass spectrum plot confirming the synthesis of Example 6.

[0097] Example 7 (S)-(3-((1-amino-3-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)-1-oxopropan-2-yl)carbamoyl)-4-nitrophenyl)boronic acid [ka] Example 7 was synthesized in the same manner as Example 4 and contains F27, F1, and F2. Expected mass [M+H]: 457.13, Measured mass [M+H]: 457.07 [M+H-H2O]: 439.07

[0098] Figure 7 is a mass spectrum plot confirming the synthesis of Example 7.

[0099] Example 8 (S)-(2-((1-amino-3-(3-boronothiophene-2-carboxamide)-1-oxopropan-2-yl)carbamoyl)thiophene-3-yl)boronic acid [ka] Example 8 was synthesized in the same manner as Example 4 and contains F27, F1, and F2. Expected mass [M+H]: 411.05, [M+H-2xH2O]: 376.00

[0100] Figure 8 is a mass spectrum plot confirming the synthesis of Example 8.

[0101] Example 9 N-(3-(3-Borono-5-nitrobenzamide)propyl)-N-(3-Borono-5-nitrobenzoyl)glycine [ka] Synthesis of Example 9: Chlorotrityl resin (1.5 mmol / eq, 300 mg) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under a stream of nitrogen, and a solution of bromoacetic acid (1 M) containing DIPEA (1 M) in DCM was immediately added and gently mixed for 1 hour. The mixture was washed with DCM, and any unreacted areas were capped with a solution of 20% MeOH in a solution of DCM and DIEA (1 M), and mixed for 1 hour. The resin was washed with DCM (2 × 5 mL) and then with DMF (2 × 5 mL). A solution of 1,3-diaminopropane (1 M) in DMF (5 mL) was added to the resin and heated at 50°C for 10 minutes. The resin was washed with DMF (3 × 5 mL) and a solution containing 3-borono-5-nitrobenzoic acid (0.2 M, 5 mL) in DMF, along with 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL) and oxima (0.5 M, 2 mL) in DMF, and heated at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (3 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes. The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA. The pure fractions were isolated, combined, frozen, and freeze-dried to obtain Example 9 as a white powder (15 mg). Expected mass [M+H]: 519.13, Measured mass [M+H]: 519.20, [M+H-H2O]: 501.33

[0102] Figure 9 is a mass spectrum plot confirming the synthesis of Example 9.

[0103] Example 10 N-(4-(3-Borono-5-nitrobenzamide)butyl)-N-(3-Borono-5-nitrobenzoyl)glycine [ka] Example 10 was synthesized in the same manner as in Example 9 and derived from FF2 and F1. Expected mass [M+H]: 533.14, Measured mass [M+H]: 533.27, [M+H-H2O]: 515.2

[0104] Figure 10 is a mass spectrum plot confirming the synthesis of Example 10.

[0105] Example 11 N-(5-(3-Borono-5-nitrobenzamide)pentyl)-N-(3-Borono-5-nitrobenzoyl)glycine [ka] Example 11 was synthesized in the same manner as in Example 9 and derived from FF2 and F1. Expected mass [M+H]: 547.16, Measured mass [M+H]: 547.18, [M+H-H2O]: 529.17 Figure 11 is a mass spectrum plot confirming the synthesis of Example 11.

[0106] Example 12 N-(4-((3-Borono-5-nitrobenzamide)methyl)benzyl)-N-(3-Borono-5-nitrobenzoyl)glycine [ka] Example 12 was synthesized in the same manner as in Example 9 and derived from FF8 and F1. Expected mass [M+H]: 581.14, Measured mass [M+H-H2O]: 563.16

[0107] Figure 12 is a mass spectrum plot confirming the synthesis of Example 12.

[0108] Example 13 N-(3-((3-Borono-5-nitrobenzamide)methyl)benzyl)-N-(3-Borono-5-nitrobenzoyl)glycine [ka] Example 13 was synthesized in the same manner as in Example 9 and derived from FF4 and F1. Expected mass [M+H]: 581.14, Measured mass [M+H]: 581.18, [M+H-H2O]: 563.16

[0109] Figure 13 is a mass spectrum plot confirming the synthesis of Example 13.

[0110] Example 14 N-(2-amino-2-oxoethyl)-1-hydroxy-N-((1R,2R)-2-(1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide)cyclohexyl)-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxamide [ka] Synthesis of Example 14: Link-amide resin (1.2 mmol / eq, 150 mg) was swollen in DMF (5 mL) for 20 minutes. The solution was removed under a stream of nitrogen, and a 20% piperidine solution in DMF (5 mL) was added to the resin and mixed for 5 minutes. The resin was washed with DMF (3 × 5 mL). Bromoacetic acid (1 M, 5 mL) in DMF containing 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL) in DMF was added to the resin and heated at 50°C for 10 minutes. The reaction mixture was washed with DMF (2 × 5 mL). A solution of (1R,2S)-cyclohexane-1,2-diamine (2 M, 5 mL) in DMF was added to the reaction mixture and heated at 50°C for 10 minutes. The resin was washed with DMF (3 × 5 mL), and a solution containing 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (0.2 M, 5 mL) in DMF, 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL), and oxima (0.5 M, 2 mL) in DMF was added and heated at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (3 × 5 mL). A solution of trifluoroacetic acid containing triisopropylsilane and water (95:2.5:2.5, 5 mL) was added to the resin and mixed for 90 minutes. The solution was collected, dried under vacuum, dissolved in DMSO (100 μL), and fractionated over 10 minutes by reverse-phase (RP) flash chromatography on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 60% ACN in water containing 0.1% TFA. The pure fractions were isolated, combined, frozen, and freeze-dried to obtain Example 14 as a white powder (5 mg). Expected mass [M+H]: 492.19, Measured mass [M+H]: 492.14, [M+H-H2O]: 475.27, [M+Na]: 513.07

[0111] Figure 14 is a mass spectrum plot confirming the synthesis of Example 14.

[0112] Example 15 N-(3-(3-Borono-4-Fluorobenzamide)propyl)-N-(3-Borono-4-Fluorobenzoyl)glycine [ka] Example 15 was synthesized in the same manner as in Example 9 and derived from FF2 and F1. Expected mass [M+H]: 465.14, Measured mass [M+H]: 465.2, [M+H-H2O]: 447.1

[0113] Figure 15 is a mass spectrum plot confirming the synthesis of Example 15.

[0114] Example 16 N-(5-(3-Borono-4-Fluorobenzamide)pentyl)-N-(3-Borono-4-Fluorobenzoyl)glycine [ka] Example 16 was synthesized in the same manner as in Example 9 and derived from FF2 and F1. Expected mass [M+H]: 493.17, Measured mass [M+H]: 493.1

[0115] Figure 16 is a mass spectrum plot confirming the synthesis of Example 16.

[0116] Example 17 N-(3-((3-Borono-4-fluorobenzamide)methyl)benzyl)-N-(3-Borono-4-fluorobenzoyl)glycine [ka] Example 17 was synthesized in the same manner as in Example 9 and derived from FF4 and F1. Expected mass [M+H]: 527.15, Measured mass [M+H]: 527.1, [M+H-H2O]: 509.1

[0117] Figure 17 is a mass spectrum plot confirming the synthesis of Example 17.

[0118] Example 18 N-((1S,2R)-2-(3-Borono-4-Fluorobenzamide)cyclohexyl)-N-(3-Borono-4-Fluorobenzoyl)glycine [ka] Example 18 was synthesized in the same manner as in Example 9 and derived from FF5 and F1. Expected mass [M+H]: 505.17, Measured mass [M+H]: 505.1

[0119] Figure 18 is a mass spectrum plot confirming the synthesis of Example 18.

[0120] Example 19 N-(3-(4-Borono-3-fluorobenzamide)propyl)-N-(4-Borono-3-fluorobenzoyl)glycine [ka] Example 19 was synthesized in the same manner as in Example 9 and contains FF2 and F1. Expected mass [M+H]: 465.14, Measured mass [M+H]: 465.1, [M+H-H2O]: 447.1 Figure 19 is a mass spectrum plot confirming the synthesis of Example 19.

[0121] Example 20 N-(5-(4-Borono-3-fluorobenzamide)pentyl)-N-(4-Borono-3-fluorobenzoyl)glycine [ka] Example 20 was synthesized in the same manner as in Example 9 and contains FF2 and F1. Expected mass [M+H]: 493.17, Measured mass [M+H]: 493.1, [M+H-H2O]: 475.1

[0122] Figure 20 is a mass spectrum plot confirming the synthesis of Example 20.

[0123] Example 21 N-(4-((4-Borono-3-fluorobenzamide)methyl)benzyl)-N-(4-Borono-3-fluorobenzoyl)glycine [ka] Example 21 was synthesized in the same manner as in Example 9 and contains FF8 and F1. Expected mass [M+H]: 527.15, Measured mass [M+H]: 527.0

[0124] Figure 21 is a mass spectrum plot confirming the synthesis of Example 21.

[0125] Example 22 N-(3-((4-Borono-3-fluorobenzamide)methyl)benzyl)-N-(4-Borono-3-fluorobenzoyl)glycine [ka] Example 22 was synthesized in the same manner as in Example 9 and contains FF4 and F1. Expected mass [M+H]: 527.15, Measured mass [M+H]: 527.05

[0126] Figure 22 is a mass spectral plot confirming the synthesis of Example 22.

[0127] Example 23 (3-((4-((N-(2-amino-2-oxoethyl)-3-borono-5-bromobenzamide)methyl)benzyl)carbamoyl)-5-bromophenyl)boronic acid [ka] Example 23 was synthesized in the same manner as in Example 9 and contains FF8 and F1. Expected mass [M+H]: 648.87, Measured mass [M+H]: 648.9

[0128] Figure 23 is a mass spectrum plot confirming the synthesis of Example 23.

[0129] Example 24 N-(3-((3-Borono-5-bromobenzamide)methyl)benzyl)-N-(3-Borono-5-bromobenzoyl)glycine [ka] Example 24 was synthesized in the same manner as in Example 9 and contains FF4 and F1. Expected mass [M+H]: 648.87, Measured mass [M+H]: 648.9

[0130] Figure 24 is a mass spectrum plot confirming the synthesis of Example 24.

[0131] Examples of compounds containing insulin as the active pharmaceutical ingredient: Modified insulin 1 [ka] Synthesis of modified insulin 1 Synthesis of modified insulin containing two modified amino acids from formulas I-VI: The following describes exemplary methods for producing insulin having modified amino acids. These methods are merely examples of methods for synthesizing insulin having modified amino acids. It should be understood that other methods may be suitably used to produce similar insulin having similar desirable properties. Furthermore, while the described methods may relate to the synthesis of modified insulin in specific examples, those skilled in the art can use the described methods to synthesize other insulin analogs and / or their associated sequences. In addition, those skilled in the art can similarly use the described methods to select suitable A-chain, B-chain, and / or complete insulin and combine them with the various sensor molecules described herein.

[0132] The following insulin chain sequences are described or referenced below: GIVEQCCTSICSLYQLENYCN(Sequence ID 1) FVNQHLCGSHLVEALYLVCGERGFFYTPKT(Sequence ID 2) GKFVNQHLCGSHLVEALYLVCGKRGFFYTPKT(Sequence ID 4) KPFVNQHLCGSHLVEALYLVCGERGFFYTPKT(Sequence ID 5) KPGSEHESAFVNQHLCGSHLVEALYLVCGERGFFYTPK (Sequence ID 6) FVNQHLCGSHLVEALYLVCGKRGFFYTPKT(Sequence ID 7) KGPEGESAGSEGESVNQHLCGSHLVEALYLVCGKRGFFYTPRT(Sequence ID 8) GIVEQCCTSICSLYQLENYCNASEKPSEA (Sequence ID 9) KPGSEVGESAIKPGSEGESVNQHLCGSHLVEALYLVCGERGFFYTPKT(Sequence ID 10) KPGSSAEEGESAKPGSEGESVNQHLCGSHLVEALYLVCGKRGFFYTPKT(Sequence ID 11) GIVEQCCTSICSLYQLENYCNKLSESG (Sequence ID 12) KGREDEAYGNIKPGWEGESKPFVNQHLCGSHLVEALYLVCGKRGFFYTPKT(Sequence ID 13) KPSGERSEGAIKPGSEGSEKFVNQHLCGSHLVEALYLVCGKRGFFYTPKT(Sequence ID 14) KPGSEHESAFVNQHLCGSHLVEALYLVCGKEGFFYTPKT(Sequence ID 15) GIVEQCCTSICSLYQLENYCNAEGSK (Sequence ID 16) KPGSEHESAFVNQHLCGSHLVEALYLVCGERGFFYTPRT(Sequence ID 17) KPGIVEQCCTSICSLYQLENYCN (Sequence ID 18) KPGSEHESAFVNQHLCGSHLVEALYLVCGERGFFYTPK (Sequence ID 19) GIVKPCCTSICSLYQLENYCN (Sequence ID 20)

[0133] Complete insulin synthesis can occur through a combination of two chains: chain A and chain B (e.g., separate synthesis and then conjugation). In an exemplary synthesis of modified insulin 1, chain B is modified with a sensor before conjugation of chains A and B. The following protocol describes the general synthesis of the first chain of insulin, chain A.

[0134] Synthesis of chain A: Array: GIVEQC(Acm)C(Acm)TSIC(Acm)SLYQLENYCN The synthesis of A chains and modified A chains (e.g., A chains conjugated to sensors) was achieved using conventional solid-phase peptide synthesis (SPPS).

[0135] Tentagel S RAM low-load (LL) resin (0.26 mmol / eq) was swollen with a mixture of DMF:DCM (50:50, v:v) for 5 minutes. The Fmoc protecting groups on the resin were removed with 20% piperidine in DMF (4 mL) at 90°C for 2 minutes. The deprotected resin was washed with DMF (4 × 5 mL). A solution of 0.5 M N,N'-diisopropylcarbodiimide (DIC, 1 mL), 0.5 M oxima (0.5 mL), and 0.2 M Fmoc-Asp(α-tBu)-OH (0.2 M) in DMF was bonded to the resin at 90°C. Each amino acid binding step included i) deprotection with 20% piperidine in DMF at 90°C, ii) washing with DMF, iii) activation and binding of the Fmoc-protected amino acid with 0.5 M N,N'-diisopropylcarbodiimide (DIC, 1 mL), 0.5 M oxima, and 0.2 M Fmoc-amino acid in DMF at 90°C, and iv) washing with DMF.

[0136] Total deprotection and isolation of the A chain. The crude peptide was deprotected with TFA:TIPS:H2O (95:2.5:2.5) and gently stirred for 2 hours. The crude solution was filtered, the peptide was precipitated in cold ether, centrifuged, and washed with additional cold ether. The supernatant was decanted, and the crude peptide was dried under a gentle stream of nitrogen gas. The crude peptide was dissolved in 20% ACN in water and fractionated by RP-HPLC on a C18 column.

[0137] The following protocol describes the general synthesis of insulin's second chain, chain B.

[0138] B chain synthesis: Synthesis of B chains and modified (e.g., sensor-conjugated) B chains using solid-phase peptide synthesis (SPPS).

[0139] MPA resin (0.22 mmol / eq) was swollen with a mixture of DMF:DCM (50:50, v:v). A solution of potassium iodide (125 mM) in DIPEA (1 M) in DMF was added to the reaction vessel along with Fmoc-Thr(tBu)-OH (0.2 M). The reaction vessel was heated to 90°C. Each amino acid bonding step included i) deprotection with 20% piperidine in DMF at 90°C, ii) washing with DMF, iii) activation and bonding of the Fmoc-protected amino acid with 0.5 M N,N'-diisopropylcarbodiimide (DIC), 0.5 M oxima, and 0.2 M Fmoc-amino acid (2.5 mL) in DMF at 90°C, and iv) washing with DMF. Fmoc-Arg(Pbf)-OH was bonded twice using the above method. The last residue in the sequence was conjugated as Boc-Gly-OH using the method described above, yielding a crude peptide containing the resin-bound sequence Boc-GK(Dde)FVNQHLC(Acm)GSHLVEALYLVCGK(Dde)RGFFYTPKT.

[0140] Deprotection of Lys-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)ethyl (Dde) on the Lys residue in the B chain and addition of ((1S,2R)-2-aminocyclohexyl)glycine. The Dde protecting group on the lysine residue was removed with 4% hydrazine in DMF (3 × 5 mL, mixed for 3 minutes), and then washed with DMF (5 × 5 mL). The side chain of the lysine residue was bonded to (3-(aminomethyl)benzyl)glycine via submonomer synthesis. Bromoacetic acid (1 M, 5 mL) in DMF containing 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL) in DMF was added to the crude B-chain peptide and heated at 50°C for 10 minutes. The reaction mixture was washed with DMF (2 × 5 mL). A solution of 1,3-phenylenedimethanamine (2 M, 5 mL) in DMF was added to the reaction mixture and heated at 50°C for 10 minutes to provide Boc-GK((3-(aminomethyl)benzyl)glycine)FVNQHLC(Acm)GSHLVEALYLVCGK((3-(aminomethyl)benzyl)glycine)RGFFYTPKT.

[0141] Addition of 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid to (3-(aminomethyl)benzyl)glycine on the crudely modified B chain. The free amine of (3-(aminomethyl)benzyl)glycine was bonded to 1-hydroxy-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (0.2M, 5mL) in DMF containing 1M N,N'-diisopropylcarbodiimide (DIC, 1M, 1mL) and oxima (0.5M, 2mL), and the mixture was heated at 50°C for 30 minutes. The resin was washed with DMF (3×5mL) to obtain a functionalized sequence.

[0142] Overall deprotection, resin cleavage, and addition of DTDP to the crude B chain. The crude functionalized B-chain sequence from the previous step was deprotected whole with 2,2-dithiopyridine (DTDP, 100 mg) in TFA:TIPS:H2O (95:2.5:2.5, 5 mL) and gently stirred at room temperature for 2 hours. The crude peptide was precipitated in cold ether (50 mL), centrifuged, decanted, washed with additional cold ether (50 mL), and centrifuged again. The supernatant was decanted, and the crude peptide was dried under a gentle stream of nitrogen gas. The crude peptide was dissolved in 20% CAN in water and fractionated over 30 minutes by RP-HPLC on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 50% ACN in water containing 0.1% TFA. The fractions were collected, frozen, and lyophilized.

[0143] Insulin and modified insulin A and B chain combinations. Two synthetic chains (e.g., chain A and chain B) were combined in a 1:1 molar ratio in 0.2 M NH4HCO3 containing 6 M urea at pH 8. The mixture was gently stirred for 1 hour, diluted with water, and fractionated over 45 minutes by RP-HPLC on a C18 column with a gradient from 20% ACN in water containing 0.1% TFA to 50% ACN in water containing 0.1% TFA.

[0144] Deprotection of the Cys-Acm protecting group, oxidation of free thiols, and final folding of modified insulin. The combined intermediate from the previous step was dissolved in glacial acetic acid and water and vigorously vortexed. A solution of iodine in glacial acetic acid (20 equivalents) was added to the reaction mixture and gently stirred for 10 minutes. A solution of ascorbic acid (5 mM) was added directly to the reaction mixture. The mixture was fractionated by RP-HPLC on a Higgins C18 column over 45 minutes with a gradient from 20% ACN in water containing 0.1% TFA to 50% ACN in water containing 0.1% TFA. The fractions were isolated, combined, frozen, and lyophilized to obtain Example 25 as a white powder (1.1 mg). Expected mass: 6940. Measured mass: [M+5-4H2O] +5 :1383.6, [M+4-4H2O] +4 :1729.05

[0145] Figure 25 is a mass spectrum plot confirming the synthesis of Example 25.

[0146] Modified insulin 2 [ka] Synthesis of modified insulin 2: In an exemplary synthesis of modified insulin 2, the modifier (e.g., a sensor precursor) is bound to complete insulin (with A and B chains already bound), thereby producing modified insulin. For example, the following exemplary method illustrates the synthesis of the modifier and its binding to wild-type insulin. [ka]

[0147] Synthesis of modifying agents Chlorotrityl resin (1.5 mmol / eq, 300 mg) was swollen in dry DCM (5 mL) for 30 minutes. The solvent was removed under a stream of nitrogen, and a solution of Fmoc-beta-Ala-OH (0.5 M) in DIPEA (1 M) was immediately added to the DCM and gently mixed for 1 hour. The mixture was washed with DCM, and any unreacted areas were capped with a solution of 20% MeOH in a solution of DCM and DIEA (1 M), and mixed for 1 hour. The resin was washed with DCM (2 × 5 mL) and then with DMF (2 × 5 mL). A solution of 20% piperidine in DMF (3 × 5 mL) was added to the resin and washed with DMF (3 × 5 mL). A solution of bromoacetic acid (1 M) in DMF containing 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL) was heated at 50°C for 30 minutes. A solution of 1,3-diaminopropane (1M) in DMF (5 mL) was added to the resin and heated at 50°C for 10 minutes. The resin was washed with a solution of DMF (3 × 5 mL) and 3-borono-5-nitrobenzoic acid (0.2 M, 5 mL) in DMF, containing 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL) and oxima (0.5 M, 2 mL), and heated at 50°C for 30 minutes. The resin was washed with DMF (3 × 5 mL) and then with DCM (3 × 5 mL). A cleavage solution of 20% 1,1,1,3,3,3-hexafluoropropane-2-ol (HFIP) in DCM (5 mL) was added to the resin and stirred for 90 minutes. The solution was collected and the resin was washed with an additional solution of HFIP in DCM (5 mL). The solutions were combined and dried under vacuum to obtain the crude product. The crude product was dissolved in dry DMF, and 3-(ethyliminomethyleneamino)-N,N-dimethylpropan-1-amine (EDC, 60 mg, 2 equivalents assuming 100% yield from the previous step) and N-hydroxysuccinimide (NHS, 30 mg, 2 equivalents assuming 100% yield from the previous step) were added to the crude product, and the mixture was stirred for 90 minutes. Dilute acid (100 mM HCl in water, 20 mL) was added to the mixture, and the product was extracted with ethyl acetate (2 × 50 mL).The ethyl acetate layer was combined, dried over magnesium sulfate, filtered, and then dried under vacuum to obtain crude crystals of (3-((3-((3-borono-5-nitrophenyl)(2-((2,5-dioxopyrrolidine-1-yl)oxy)-2-oxoethyl)amino)propyl)amino)-5-nitrophenyl)boronic acid. The crude product was dissolved in DMSO (100 μL) and fractionated by flash chromatography on a C18 column. The pure fractions were combined, frozen, and lyophilized to obtain a pure NHS activating modifier. Predicted mass [M+H]. +1 :671.18, measured [M+H] +1 :671.33.

[0148] Figure 26A is a mass spectrum plot confirming the synthesis of the modifier.

[0149] Addition of modifying agents to WT insulin Wild-type (WT) insulin (10 mg) was dissolved in 1 mL of 100 mM potassium phosphate at pH 11.5. NHS-activated modifiers were dissolved in DMSO (10 mg / mL), and 50 μL was added to the WT insulin solution. The mixture was gently stirred for 1 hour, diluted with 3 mL of 20% ACN in water, and fractionated by RP-HPLC on a C18 column. The pure fractions were combined, frozen, and lyophilized to obtain pure modified insulin. Expected mass [M+4H] +4 1595.75, measured [M+4H-4H2O] +4 :1577.8.

[0150] Figure 26B is a mass spectral plot confirming the synthesis of modified insulin.

[0151] Modified insulin 3 [ka] Synthesis of modified insulin 3. The A chain of modified insulin 3 was synthesized using the method described in relation to modified insulin 1. Furthermore, a crude peptide having the sequence Boc-GK(Dde)FVNQHLC(Acm)GSHLVEALYLVCGK(Dde)RGFFYTPK(Dde)T, bound to a resin, was synthesized using the method described for the B chain of modified insulin 1.

[0152] Continuation of B chain synthesis: Deprotection of Lys-N-ε-1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)ethyl (Dde) on the Lys residue in the B chain and addition of 4-aminopyrrolidine-2-carboxylic acid (4-Pro). The Dde protecting group on the lysine residue was removed with 4% hydrazine in DMF (3 × 5 mL, mixed for 3 minutes), and then washed with DMF (5 × 5 mL). The side chain of the lysine residue was bonded to 1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)pyrrolidine-2-carboxylic acid (Fmoc-4-amino-Fmoc-Pro-OH) (0.2 M, 5 mL) in DMF containing 1 M N,N'-diisopropylcarbodiimide (DIC, 1 M, 1 mL), and to oxima (0.5 M, 2 mL) in DMF, and heated at 50°C for 30 minutes. The Fmoc protecting group on 4-amino-Pro was removed with 20% piperidine (2 x 3 mL) in DMF at 50°C, and the molecule was washed with DMF (3 x 5 mL) to obtain the sequence: Boc-GK(4-Pro)FVNQHLC(Acm)GSHLVEALYLVCGK(4-Pro)RGFFYTPK(4-Pro)T.

[0153] Addition of 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid to 4-Pro of the modified B chain. The free amine of 4-aminoproline (4-Pro) was bonded to 1-hydroxy-4-(trifluoromethyl)-1,3-dihydrobenzo[c][1,2]oxabolol-6-carboxylic acid (0.2M, 5mL) and oxima (0.5M, 2mL) in DMF containing 1M N,N'-diisopropylcarbodiimide (DIC, 1M, 1mL), and the mixture was heated at 50°C for 30 minutes. The resin was washed with DMF (3×5mL) to obtain a functionalized sequence.

[0154] Overall deprotection, resin cleavage, and addition of DTDP to the crude B chain. The crude functionalized B-chain sequence from the previous step was deprotected and combined with the A-chain using a method similar to that described in relation to modified insulin 1. The resulting complete insulin was further deprotected as described in relation to modified insulin 1 to provide modified insulin 3. Expected mass [M+4]+4: 1924.5 Measured mass [M+4-6H2O] +4 July 1897.

[0155] Figure 27 is a mass spectral plot confirming insulin synthesis.

[0156] Example 28: Modified insulin 4 [ka] Modified insulin 4 was synthesized in the same way as modified insulin 3. Expected mass [M+5] +5 :1359.5 Measured value [M+5-6H2O] +5 1338.1.

[0157] Figure 28 is a mass spectral plot confirming insulin synthesis.

[0158] Modified insulin 5 [ka] Modified insulin 5 can be prepared in the same way as modified insulin 2.

[0159] Modified insulin 6 [ka] Modified insulin 6 can be prepared in the same way as modified insulin 1.

[0160] Modified insulin 7 [ka] Modified insulin 7 can be prepared in the same way as modified insulin 1.

[0161] Modified insulin 8 [ka] Modified insulin 8 can be prepared in the same way as modified insulin 1.

[0162] Modified insulin 9 [ka] Modified insulin 9 can be prepared in the same way as modified insulin 1.

[0163] Modified insulin 10 [ka] Modified insulin 10 can be prepared in the same way as modified insulin 1.

[0164] Modified insulin 11 [ka] Modified insulin 11 can be prepared in the same way as modified insulin 1.

[0165] Modified insulin 12 [ka] Modified insulin 12 can be prepared in the same way as modified insulin 3.

[0166] Modified insulin 13 [ka] Modified insulin 13 can be prepared in the same way as modified insulin 1.

[0167] Modified insulin 14 [ka] Modified insulin 14 can be prepared in the same way as modified insulin 1.

[0168] Modified insulin 15 [ka] Modified insulin 15 can be prepared in the same way as modified insulin 1.

[0169] Modified insulin 16 [ka] Modified insulin 16 can be prepared in the same way as modified insulin 1.

[0170] Modified insulin 17 [ka] Modified insulin 17 can be prepared in the same way as modified insulin 1.

[0171] Modified insulin 18 [ka] Modified insulin 18 can be prepared in the same way as modified insulin 1.

[0172] Modified insulin 19 [ka] Modified insulin 19 can be prepared in the same way as modified insulin 1.

[0173] Modified insulin 20 [ka] Modified insulin 20 can be prepared in the same way as modified insulin 1.

[0174] Modified insulin 21 [ka] Modified insulin 21 can be prepared in the same way as modified insulin 1.

[0175] Modified insulin 22 [ka] Modified insulin 22 can be prepared in the same way as modified insulin 1.

[0176] Modified insulin 23 [ka] Modified insulin 23 can be prepared in the same way as modified insulin 1.

[0177] Modified insulin 24 [ka] Modified insulin 24 can be prepared in the same way as modified insulin 1.

[0178] Modified insulin 25 [ka] Modified insulin 25 can be prepared in the same way as modified insulin 1.

[0179] Modified insulin 26 [ka] Modified insulin 26 can be prepared in the same way as insulin 1.

[0180] Modified insulin 27 [ka] Modified insulin 27 can be prepared in the same way as insulin 1.

[0181] Modified insulin 28 [ka] Modified insulin 28 can be prepared in the same way as insulin 1.

[0182] Modified insulin 29 [ka] Modified insulin 29 can be prepared in the same way as insulin 1.

[0183] Modified insulin 30 [ka] Modified insulin 30 can be prepared in the same way as insulin 1.

[0184] Modified insulin 31 [ka] Modified insulin 31 can be prepared in the same way as insulin 1.

[0185] Modified insulin 32 [ka] Modified insulin 32 can be prepared in the same way as insulin 1.

[0186] Modified insulin 33 [ka] Modified insulin 33 can be prepared in the same way as modified insulin 1.

[0187] Modified insulin 34 [ka] Modified insulin 34 can be prepared in the same way as modified insulin 2.

[0188] Determination of glucose binding (Kd) using the Alizarin Red S (ARS) substitution assay. The association constants of the binding events between Alizarin Red S (ARS) and each of the compounds in Examples 1-24 were determined using standard methods in the art. 10 in 0.1 M phosphate buffer, pH 7.4 -5 Three titrations of M ARS were performed in 96-well plates at 25°C for serial dilutions of the example compound in the concentration range of 0 to 0.1 M. The example compound-ARS solutions were incubated at 25°C for 5 to 45 minutes, and the absorbance was measured using an excitation wavelength of 468 nm and an emission wavelength of 585 nm. The change in intensity was plotted against the concentration of the example compound, and the association constant of the ARS bond was obtained by fitting the intensity data.

[0189] The association constant of the binding between the target sugar compound (e.g., glucose) and the boronic acid compound was determined by substitution of ARS bound to the example compound. 10 in 0.1 M phosphate buffer, pH 7.4 -5 Three wells containing M ARS and 0.1M example compounds were titrated in a 96-well plate at 25°C against serial dilutions of the target glycoside in the concentration range of 0–2.0M. The boronic acid-ARS-carbohydrate solution was incubated at 25°C for 30–60 minutes, and the intensity of each well was measured using a plate reader at excitation wavelength 468 nm and emission wavelength 585 nm. The change in intensity was plotted against the concentration of the target glycoside, and the data were fitted to a one-site competition equation. y = minimum(y) + (maximum(y) - minimum(y)) / (1 + 10 x-logEC50 ) The association constants for the boronic acid compound-target sugar compound binding event were obtained.

[0190] Table 1 shows the binding constants of Examples 1-24 to glucose, fructose, and lactate. [Table 1]

[0191] One or more embodiments of this disclosure include the following embodiments 1 to 43: 1. A compound represented by formula I, [ka] In equation I, R is selected from formulas FF1 to FF24. Z a) NH2 or OH, b) Covalent bonding to the active pharmaceutical ingredient, either directly or via any linker. c) Covalent bonding of one or more amino acids in the polypeptide drug substance to the N-terminal amine or epsilon-amino group, either directly or via any linker, and d) [ka] Selected from one of the bases represented by, [ka] If index k is an integer in the range of 3 to 14, for example, 4 to 12, 5 to 10, or 6 to 8, J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula I'. [ka] In equation I', [ka] However, it indicates a binding site to the rest of the polypeptide active pharmaceutical ingredient, * indicates the connection point to the rest of Z, If index n is an integer in the range of 1 to 8, for example, 1, 2, 3, 4, 5, 6, 7, or 8, Regarding formulas FF1-FF24, [ka] [ka] X represents a covalent bond to Z in equation I, either directly or via any linker. If index i is an integer in the range of 1 to 20, for example, 2 to 18, 3 to 16, 4 to 14, 6 to 12, or 8 to 10, B1 and B2 are either identical or different, and each is independently represented by one selected from formulas F1 to F9. B3 is a base represented by one selected from formulas F1 to F11, [ka] For each of the formulas F1 to F9, One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- represents, and --- represents the covalent bond to the remainder of R in equation I, R1, which is 0, 1, or 2, each independently produces F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, If the index m is an integer in the range of 1 to 14, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7, In F5, one R1 represents B(OH)2, All remaining R1s represent H, Compounds in formula F10 where index j is an integer in the range of 1 to 13, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7.

[0192] 2. A compound represented by formula II, [ka] In Equation II, (i) R is selected from formulas FF25 to FF31, In FF25-FF31, B1 and B2 are either the same or different, and each is independently selected from formulas F12-F19. Is Z NH2 and not conjugated with any active pharmaceutical ingredient? or (ii) R is selected from formulas FF25 to FF31, B1 and B2 are each independently selected from formulas F20 to F27. Z a) OH b) Covalent bonding to the active pharmaceutical ingredient, either directly or via any linker. c) Covalent bonding of one or more amino acids in the polypeptide drug substance to the N-terminal amine or epsilon-amino group, either directly or via any linker, and d) [ka] Selected from one of the bases represented by, [ka] If index k is an integer in the range of 3 to 14, for example, 4 to 12, 5 to 10, or 6 to 8, J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula II', or (iii) R is selected from formulas FF32 to FF33, In FF32, B1 and B2 are each independently selected from formulas F28 to F35. In FF33, B1 and B2 are each independently selected from formulas F36 to F43. Z a) Active pharmaceutical ingredient, b) Covalent bonding of amino acids in the polypeptide drug material to the N-terminal amine or epsilon-amino group, either directly or via any linker, and c) [ka] Selected from one of the bases represented by, [ka] If index k is an integer in the range of 3 to 14, for example, 4 to 12, 5 to 10, or 6 to 8, J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula II'. Regarding equation II', [ka] [ka] However, it indicates a binding site to the rest of the polypeptide active pharmaceutical ingredient, * indicates the connection point to the rest of Z, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, Regarding formulas FF25~FF33, [ka] X represents a covalent bond to Z in equation II, either directly or via any linker. If index i is an integer in the range of 1 to 20, for example, 1, 2, 3, 4, 2 to 18, 3 to 16, 4 to 14, 6 to 12, or 8 to 10, For each of the formulas F12 to F19, [ka] One R1 from either B1 or B2 represents a covalent bond to the active pharmaceutical ingredient, either directly or via any linker. In both B1 and B2, one R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- and --- represent the covalent bond of R to the remainder in equation II, In B1 and B2, 0, 1, or 2 R1 independently produce COOH, F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, If the index m is an integer in the range of 1 to 14, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7, All remaining R1s represent H, For each of the formulas F20 to F25, [ka] One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- and --- represent the covalent bond of R to the remainder in equation II, (a) One or two R1s on the same B1 and / or B2 represent a COOH, and at least one COOH is not conjugated to the active pharmaceutical ingredient, and / or (b) One or two R1s independently produce NO2, CH3, OCH3, and O(CH2) m CH3, -(SO2)NH CH3, -(SO2)NH(CH2) m CH3 is represented, and the index m is an integer in the range of 1 to 14, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7. 0, 1, or 2 R1s each independently represent NO2, F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CH3, CF3, or OCF3. All remaining R1s represent H, For each of equations F26 to F27, [ka] One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m --- and --- represent the covalent bond of R to the remainder in equation II, R1, which is 0, 1, or 2, can each independently produce COOH, F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, If the index m is an integer in the range of 1 to 14, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7, All remaining R1s represent H, For each of the formulas F28 to F35, [ka] One R1 in B1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m - represents a covalent bond to Z in equation II, either directly or via any linker. For each of B1 and B2, one R1 is a covalent bond between B1 and B2, and the covalent bonds are -(S=O)-, -(S(=O)(=O)-, -(CF2)-, -(C=O)-, -(CH2) m SCH2CO(CH2) k -,-(CH2) m S(CH2)2CO(CH2) k -, and -(CH2) m (CO)NH(CH2) k - Selected from, (i) The two R1 groups in B2 are COOH and these two R1 groups are not conjugated in the active pharmaceutical ingredient, or (ii) One or two R1 groups in either B1 and / or B2 are independently NO2, CH=O, CH3, OCH3, O(CH2) m CH3, -(SO2)NH CH3, or -(SO2)NH(CH2) m It represents either CH3 or something else. In either B1 or B2, 0, 1, or 2 R1s each independently represent CH=O, F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CH3, CF3, CHF2, or OCF3. The remaining R1 represents H, If index k is an integer in the range of 1 to 7, for example, 2 to 6 or 3 to 5, If the index m is an integer in the range of 1 to 7, for example, 2 to 6 or 3 to 5, For each of the formulas F36 to F43, [ka] For each of B1 and B2, one R1 is a covalent bond to a sulfoximine group such that B1 and B2 are bonded together by the sulfoximine group, and the amino group of the sulfoximine is covalently bonded to Z in formula II, either directly or via any linker through an acid-containing linker. (i) Two R1 groups in B1 and / or B2 are COOH and these two R1 groups are not conjugated in the drug substance, or (ii) one or two R1 groups in either B1 and / or B2 are independently NO2, CH=O, CH3, OCH3, O(CH2) m CH3, -(SO2)NH CH3, or -(SO2)NH(CH2) m It represents either CH3 or something else. In either B1 or B2, 0, 1, or 2 R1s each independently represent CH=O, F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CH3, CF3, CHF2, or OCF3. The remaining R1 represents H, If index k is an integer in the range of 1 to 7, for example, 2 to 6 or 3 to 5, A compound in which the index m is an integer in the range of 1 to 7, for example, 2 to 6 or 3 to 5.

[0193] A compound containing an active pharmaceutical ingredient, wherein the active pharmaceutical ingredient contains insulin, and the insulin contains one or more modified amino acids represented by formula III, [ka] In Equation III, R is selected from formulas FF1 to FF24. Z is any linker, [ka] Selected from, [ka] If index k is an integer in the range of 3 to 14, for example, 4 to 12, 5 to 10, or 6 to 8, J is expressed by equation III', [ka] In equation III', [ka] However, it shows the binding site to the rest of insulin, * indicates the connection point to the rest of Z, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, Regarding formulas FF1-FF24, [ka] [ka] X represents a covalent bond to Z in Equation III, either directly or via any linker. If index i is an integer in the range of 1 to 20, for example, 2 to 18, 3 to 16, 4 to 14, 6 to 12, or 8 to 10, B1 and B2 are either identical or different, and each is independently represented by one selected from formulas F1 to F9. B3 is a base represented by one selected from formulas F1 to F11, [ka] For each of the formulas F1 to F9, One R1 is (C=O)---, S(=O)(=O)---, (CH2) m (C=O)---, or (CH2) m--- represents, and --- represents the covalent bond to the remainder of R. R1, which is 0, 1, or 2, each independently produces F, Cl, Br, OH, CH2-NH2, NH2, (C=O)-NH2, SO2CH3, CF3, NO2, CH3, OCH3, O(CH2) m CH3, -(SO2)NHCH3, -(SO2)NH(CH2) m Represents CH3 or OCF3, If the index m is an integer in the range of 1 to 14, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7, In F5, one R1 represents B(OH)2, All remaining R1s represent H, Compounds in formula F10 where index j is an integer in the range of 1 to 13, for example, 2 to 12, 3 to 10, 4 to 8, or 5 to 7.

[0194] 4. Any linker is an L- or D-amino acid having at least one functional group directly conjugated to R, or any linker is selected from formulas FL1 to FL9. [ka] In formulas FL1 to FL9, Z'' represents a covalent bond with respect to Z, R'' represents a covalent bond with respect to R, p is an integer in the range of 1 to 5. q is an integer in the range of 1 to 5. A compound from any one of embodiments 1 to 3, wherein r is an integer in the range of 1 to 5.

[0195] 5. Any one of the compounds from Embodiments 1 to 3, wherein the compound is a drug substance further modified as described in Embodiments 1 to 3, and / or one or more amines are independently acetylated or alkylated.

[0196] 6. A compound from any one of Embodiments 1 to 3, wherein the active pharmaceutical ingredient is insulin containing human insulin or an analogue thereof, and the insulin comprises A chain and B chain.

[0197] 7. The compound of Embodiment 1 or 2, wherein the active pharmaceutical ingredient comprises a polypeptide active pharmaceutical ingredient or a human peptide hormone.

[0198] 8. The compound of Embodiment 6, wherein insulin each independently comprises one or two peptide sequences attached to the A and / or B chains of insulin, and each peptide sequence independently comprises 1 to 20 consecutive residues, for example, 2 to 18, 3 to 16, 4 to 14, 6 to 12, or 8 to 10 consecutive residues.

[0199] 9. The compound of Embodiment 6, comprising 2 to 10 amino acids, each independently represented by formula I, II, or III, wherein insulin is modified.

[0200] 10. Insulin contains one or more modifications, each independently represented by formula I, II, or III, and each of the one or more modifications is (i) on the side chains of up to 20 amino acids attached to the N-terminus and / or C-terminus of the A-chain and / or B-chain of insulin, and / or at the N-terminus of the polypeptide, and / or (ii) Within four residues of B1, B21, B22, B29, A1, A22, or A3 residues in the insulin A chain or B chain, and / or (iii) on the side chains of amino acids attached to or incorporated into the A and / or B chains of insulin, and / or located at the N-terminus of the polypeptide, wherein the polypeptide is in sequence (X2) n X1(X2) mA compound of Embodiment 6, comprising (SEQ ID NO: 3), wherein X1 is a lysine residue, the side chain of the lysine residue is modified to be represented by formula I, II, or III, and each X2 is independently selected from the group of amino acids K, P, E, G, N, M, A, R, L, W, S, F, V, C, H, D, I, Y, Q, T, or X1, the index m is an integer in the range of 0 to 20 (e.g., 1 to 18, 2 to 16, 3 to 14, 4 to 12, 5 to 10, or 6 to 8), and the index n is an integer in the range of 0 to 18 (e.g., 1 to 16, 2 to 14, 3 to 12, 4 to 10, 5 to 9, or 6 to 8). SEQ ID NO: 3 represents the longest variant of the polypeptide sequence and encompasses its shorter subsequences.

[0201] 11. A conjugate comprising the compound according to Embodiment 1 or 2, either directly or via a covalent linker, wherein the conjugation is not via Z, when Z is NH2 in formula II.

[0202] 12. One of the compounds from Embodiments 1 to 3, wherein one of the compounds from Embodiments 1 to 3 is used as an intermediate compound for the production of any of the compounds in Embodiments 1 to 11.

[0203] 13. The compound contains one or more modifications represented by formula IV, V, or VI. Regarding equation IV, [ka] [ka] However, it indicates a binding site to the rest of the active pharmaceutical ingredient, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, R is selected from the group consisting of formulas F111, F222, F333, F444, and F555. [ka] In formulas F111, F222, F333, F444, and F555, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, R 1 Each carbon atom bonded to it independently has either (R) or (S) stereochemistry. Each R1 independently has -H, -OR 3 , -N(R 3 )2, -SR 3 -OH, -OCH3, -OR 5 NHC(O)CH3, -CH2R 3 , -C(O)NHOH, -NHC(O)CH3, -CH2OH, -CH2OR 5 -NH2, -CH2R 4 , -OR 8 , -R 6 , -R 8 , and -R 7 Selected from, Each R 3 However, independently, -H, acetyl, phosphate, and -R 2 , -SO2R 2 ,-S(O)R 2 , -P(O)(OR 2 )2, -C(O)R 2 , -CO2R 2 , and -C(O)N(R 2 ) Selected from 2, Each R 2 However, independently, -H, and C as arbitrarily substituted. 1-6 Selected from an aliphatic ring, an optionally substituted phenyl ring, an optionally substituted 5-6 member monocyclic heteroaryl ring having 1-4 heteroatoms selected from nitrogen, oxygen, and sulfur, a 4-7 member heterocyclic ring having 1-2 heteroatoms selected from nitrogen, oxygen, and sulfur, and an alkyl or amide covalent bond to R in formula IV, Each R 4 However, independently, -H, -OH, -OR 3 , -N(R 3 )2, -OR 5 , and -SR 3 Selected from, Each R 5However, they are independently selected from monosaccharides, disaccharides, trisaccharides, pentoses, and hexoses. Each R 6 However, independently, -NCOCH2-, -(OCH2CH2) n -, -OC 1-9 Alkylene group and substituted C 1-9 Selected from alkylene groups, one or more methylene groups are optionally -O-, -(CH2) n -, -OCH2-, -N(R 2 )C(O)-, -N(R 2 )C(O)N(R 2 )-, -SO2-, -SO2N(R 2 )-,-N(R 2 )SO2-, -S-, -N(R 2 )-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R 2 )-, or -N(R 2 )SO2N(R 2 ) is replaced by -, where index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5. Each R 7 However, independently, -N(R 2 )2, -F, -Cl, -Br, -I, -SH, -OR 2 , -SR 2 -NH2, -N3, -C≡CR 2 , -CH2C≡CH, -C≡CH, -CO2R 2 , -C(O)R 2 , -OSO2R 2 -N(R 2 )2, -OR 2 , -SR 2 Selected from -CH3, -CH2NH2, and direct bonding to R in formula IV, R 8 However, (i) it is one side chain of L-serine, D-serine, L-threonine, D-threonine, L-alrosreonine, or D-alrosreonine, corresponding to R in formula IV, with index n=1 in formula IV, (ii) it is an amide bond to the C-terminus of lysine, cysteine, or 2,3-diaminopropionic acid, or (iii) it is -CH2C(CH2OH)2CH2NH2, Structures F111, F222, F333, F444, and / or F555 optionally contain one or more acetyl, acetylene, acetonide, and / or pinacol protecting groups. Regarding equation V, [ka] [ka] However, it indicates a binding site to the rest of the active pharmaceutical ingredient, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, R represents X and Y, X is a covalent bond selected from the group consisting of a triazole bond, an amide bond, an imine bond, or a thioether bond. Y is selected from the group consisting of structures represented by equations F200 to F203. [ka] X1 represents a covalent bond with respect to X, X2 represents SH, OH, or NH2. If index m is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, Regarding Equation VI, [ka] [ka] However, it indicates a binding site to the rest of the active pharmaceutical ingredient, If index n is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, Z is an amino acid, -(CH2) p -, -CH2(OCH2CH2) p -, -SCH2-, -S(CH2)2-, -NH-, -NH(CO)-, -(CO)NH-, -S(CH2) kNH-,-triazole-(CH2) k Selected from the group consisting of -NH-, triazole, amide bond, imine bond, and thioether bond, The index k is an integer in the range of 3 to 5. If index p is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, R is selected from the group consisting of structures represented by equations F203 to F205. [ka] In the equation, X3 represents a covalent bond with Z, X4 represents SH, OH, or NH2. If index q is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5, The compound of Embodiment 5 or 6, wherein the index m is an integer in the range of 1 to 8, for example, 2 to 7, 3 to 6, or 4 to 5.

[0204] 14. A method for producing any one of the compounds in Embodiments 1 to 13, wherein B1 and B2 are optionally first conjugated to one of the structures represented by FF1 to FF33, and the resulting conjugation is then covalently bonded to the active pharmaceutical ingredient, or optionally, the structures represented by FF1 to FF33 are first conjugated to the active pharmaceutical ingredient, and then B1 and B2 are covalently bonded to the corresponding structures in FF1 to FF33.

[0205] 15. A method for administering one of the compounds from Embodiments 1 to 13 to a human subject as a therapeutic or prophylactic agent.

[0206] 16. A compound according to any of Embodiments 1 to 13, wherein one or more amine groups are independently acetylated or alkylated.

[0207] 17. The compound of Embodiment 6, wherein insulin comprises two, three, or four modifications, each independently represented by formula I, II, or III.

[0208] 18. Compounds of Embodiments 1-3, wherein the active pharmaceutical ingredient is a human polypeptide hormone, or a peptide having at least 10% homology with one, two, three, or four different human peptide hormones, and comprises a dual or triple agonist, or a hybrid synthetic peptide based on one or more human polypeptide hormones or their analogues.

[0209] 19. Compounds of Embodiments 1 to 3, wherein the active pharmaceutical ingredient is insulin, and the amino acid at residue 21 of the B chain is a modified amino acid represented by formula I, II, or III.

[0210] 20. Compounds of Embodiments 1 to 3, wherein the active pharmaceutical ingredient is insulin, and one or more residues within four residues of residue 22 of the B chain of insulin are each independently represented by formula I, II, or III, and one or more additional residues in the polypeptide attached to the C-terminus and / or N-terminus of the B chain and / or A chain are each independently represented by formula I, II, or III.

[0211] 21. The compounds of Embodiments 1 to 3, wherein the active pharmaceutical ingredient is insulin, and the modified amino acid either substitutes an amino acid at a given residue in the peptide sequence of the A chain and / or the B chain, or the modified amino acid is added to the peptide sequence of the A chain and / or the B chain either at the terminal and / or within the peptide sequence of the A chain and / or the B chain.

[0212] 22. The compounds of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, the amino acid at residue 21 of the B chain is a modified amino acid represented by formula IV, V, or VI, and the residue at the C-terminus of the A chain is represented by formula I, II, or III.

[0213] 23. The compounds of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more residues located within four residues of the C-terminus of chain A, or attached to the C-terminus of chain A, are each independently represented by formula I, II, or III, and one or more residues located within four residues of residue 22 of chain B, are each independently represented by formula IV, V, or VI.

[0214] 24. The compounds of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more residues located within four residues of the C-terminus of the A chain, or attached to the C-terminus of the A chain, are each independently represented by formula IV, V, or VI, and one or more residues located within four residues of residue 22 of the B chain are each independently represented by formula I, II, or III.

[0215] 25. Any one of embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and two modified amino acids are introduced into the B chain of insulin at any position between the C-terminal cysteine ​​of the B chain and the C-terminus of the B chain, and two additional modified amino acids are introduced at any position in the A chain of insulin, including being added to one or both ends of the A chain.

[0216] 26. A compound from any one of Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and (i) within 4 residues of residue 21 of the B chain, and / or (ii) within 6 residues of the N-terminus or C-terminus of the A chain and / or the B chain, and / or (iii) within 4 residues of residue 13 of the A chain, and / or (iv) each independently one or more residues represented by formula I, II, III, IV, V, or VI, and each of the four or more residues within the C-terminus of the A chain, each independently represented by formula I, II, III, IV, V, or VI.

[0217] 27. Modified insulin according to any one of Embodiments 1, 2, or 3, wherein two or more amino acids in the B chain in the range of B1 to B29 are substituted with natural, non-standard, or synthetic amino acids.

[0218] 28. Any one of the compounds from Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more amino acids in the A chain or B chain are substituted with natural or non-standard amino acids.

[0219] 29. Any one of the compounds from Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is further conjugated either directly or via an optional linker to a polypeptide containing up to 31 amino acids.

[0220] 30. Any one of the compounds from Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is conjugated to a polypeptide containing up to 31 amino acids at the N-terminus or C-terminus of the A-chain or B-chain.

[0221] 31. Any one of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is conjugated to a polypeptide containing up to 31 amino acids at the N-terminus or C-terminus of the A-chain or B-chain, and the polypeptide is bound to insulin via peptide bonds.

[0222] 32. Any one of the compounds of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, which is further conjugated to a polypeptide containing up to 31 amino acids, either directly or via any linker, and one or more pairs of the side chains of the polypeptide are covalently bonded, and in a particular embodiment, the covalent bond between the side chains is a bond selected from the group consisting of a triazole bond, a bond resulting from azido-alkyne cyclization, a disulfide bond, a thioester bond, an oxime bond, an amide bond, a lactam bond, an ester bond, an olefin bond, an imine bond, an ester bond, and a thioether bond.

[0223] 33. Any one of the compounds from Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and at least one primary amine group or one secondary amine group in R of formula I is covalently bonded to the side chains of L- and D-gamma-gamma-glutamic acid via an amide bond, and the N-terminus of glutamic acid is covalently bonded via an amide bond to an unsubstituted or monosubstituted alkyl diacitate chain containing 3 to 16 carbon atoms, e.g., 4 to 14, 5 to 12, 6 to 11, or 7 to 9 carbon atoms.

[0224] 34. Modified insulin of Embodiment 2, in which, for formula FF25, index i is 0.

[0225] 35. A compound according to any one of embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and 1 to 10 amino acids are added to the polypeptide sequence of insulin, these are N-terminated to residue 1 of the B chain of insulin, and the N-terminus of the inserted residue is a modified amino acid represented by formula I, II, or III.

[0226] 36. Any one of the compounds from Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and 1 to 10 amino acids are added to the C-terminus of the B chain of insulin, and the residue at position B29 of insulin is a modified amino acid represented by formula I.

[0227] 37. A compound from any one of Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and up to six residues are added to the polypeptide sequence of insulin, at least two of which are modified amino acids represented by formulas I-VI.

[0228] 38. Any one of the compounds from Embodiments 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is modified to have four or five intramolecular disulfide bonds.

[0229] 39. Any one of the compounds from Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a polypeptide using an enzyme.

[0230] 40. Any one of the compounds from Embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a non-boronized polypeptide containing up to 31 amino acids using an enzyme.

[0231] 41. Any one of embodiments 1-3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a polypeptide containing up to 31 amino acids, and the side chains of at least two amino acids in the polypeptide sequence are covalently linked together or via any linker.

[0232] 42. The active pharmaceutical ingredient is insulin, and the insulin is covalently bonded using amide bonds to a structure represented by formulas F411-F416, or a structure in which F411 is further covalently bonded using amide bonds to a structure represented by formulas F412-F416. [ka] In the formula, R represents either a primary or secondary amine at the N-terminus of modified insulin, or a primary or secondary amine in the side chain of a subset of amino acids in modified insulin, the binding to R is the binding site to modified insulin, the index n represents an integer in the range of 1 to 14 (e.g., 2 to 12, 3 to 10, 4 to 8, or 5 to 7), the index m represents an integer in the range of 1 to 12 (e.g., 3 to 10, 4 to 8, or 5 to 7), the index o represents an integer in the range of 1 to 6 (e.g., 2 to 5 or 3 to 4), and the index p represents an integer in the range of 1 to 12 (e.g., 3 A compound from any one of Embodiments 1-3 and 13, wherein Z represents (~10, 4-8, or 5-7), and Z represents one of -(C=O)-OH, -NH2, cholesterol, 7-OH cholesterol, 7,25-dihydroxycholesterol, cholic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid, glycocholic acid, glycodecoxycholic acid, glycocolic acid, glycochenodeoxycholic acid, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol.

[0233] 43. The active pharmaceutical ingredient contains one or more structures represented by formulas FX15 to FX28. [ka] During the ceremony, Each R1 is independently selected from H, NH2, NO2, Cl, CF3, I, COCH3, CN, C≡CH, N3, or Br. Each R2 is independently selected from CF3, H, or CH3. Each R3 is independently selected from C≡CH, H, N3, or a vinyl group. Each R4 is independently selected from NH2, R2, or R3. Each R5 is independently selected from S or NH. One of the compounds from embodiments 1 to 3 and 13, wherein the index n is an integer in the range of 1 to 4, for example, 2 to 3.

[0234] While this disclosure has been described in relation to specific exemplary embodiments, it should be understood that this disclosure is not limited to the disclosed embodiments, but rather is intended to cover a variety of modifications and equivalent configurations that fall within the spirit and scope of the following claims and their equivalents.

Claims

1. A compound represented by formula I, 【Chemistry 1】 In equation I, R is selected from formulas FF1 to FF24. Z is a) NH ２ Or OH, b) Covalent bonding to the active pharmaceutical ingredient, either directly or via any linker. c) Covalent bonding of one or more amino acids in the polypeptide drug substance to the N-terminal amine or epsilon-amino group, either directly or via any of the linkers, and d) 【Chemistry 2】 Selected from one of the bases represented by, 【Transformation 3】 The index k is an integer in the range of 3 to 14. J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula I'. 【Chemistry 4】 In equation I', 【Transformation 5】 However, it indicates a binding site to the remaining portion of the polypeptide active pharmaceutical ingredient, * indicates the connection point to the rest of Z. The index n is an integer in the range of 1 to 8. Regarding formulas FF1 to FF24, 【Chemistry 6-1】 【Chemistry 6-2】 X represents a covalent bond to Z in formula I, either directly or via any of the linkers. The index i is an integer in the range of 1 to 20. B １ and B ２ However, they may be identical or different, and each independently represents a base selected from formulas F1 to F9. B ３ However, this represents a base selected from formulas F1 to F11, 【Transformation 7】 For each of the formulas F1 to F9, one R １ is (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ --- represents, --- represents the covalent bond to the remainder of R in formula I, 0, 1, or 2 R １ However, each is independent of F, Cl, Br, OH, and CH. ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ CF ３ NO ２ ,CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NHCH ３ ,-(SO ２ )NH(CH ２ ) ｍ CH ３ , or OCF ３ This represents, The index m is an integer in the range of 1 to 14. One R in F5 １ However, B(OH) ２ This represents, All remaining R １ However, it represents H, A compound in formula F10 in which the index j is an integer in the range of 1 to 13.

2. A compound represented by formula II, 【Transformation 8】 In Equation II, (i) R is selected from formulas FF25 to FF31, B in FF25-FF31 １ and B ２ However, they may be the same or different, and each is independently selected from formulas F12 to F19. Z is NH ２ Therefore, it is not conjugated with any active pharmaceutical ingredient. or (ii) R is selected from formulas FF25 to FF31, B １ and B ２ However, each is independently selected from formulas F20 to F27. Z is a) OH b) Covalent bonding to the active pharmaceutical ingredient, either directly or via any linker. c) Covalent bonding of one or more amino acids in the polypeptide active pharmaceutical ingredient to the N-terminal amine or epsilon-amino group, either directly or via any of the linkers, and d) 【Chemistry 9】 Selected from one of the bases represented by, 【Chemistry 10】 The index k is an integer in the range of 3 to 14. J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula II', or (iii) R is selected from formulas FF32 to FF33, B in FF32 １ and B ２ However, each is independently selected from formulas F28 to F35. B in FF33 １ and B ２ However, each is independently selected from formulas F36 to F43. Z is a) Active pharmaceutical ingredient, b) Covalent bonding of one or more amino acids in the polypeptide drug material to the N-terminal amine or epsilon-amino group, either directly or via any linker, and c) 【Chemistry 11】 Selected from one of the bases represented by, 【Chemistry 12】 The index k is an integer in the range of 3 to 14. J is an amino acid in the polypeptide drug substance or one or more amino acids, and each of the one or more amino acids in the polypeptide drug substance is represented by formula II'. Regarding equation II', 【Chemistry 13】 【Chemistry 14】 However, it indicates a binding site to the remaining portion of the polypeptide active pharmaceutical ingredient, * indicates the connection point to the rest of Z. The index n is an integer in the range of 1 to 8. Regarding formulas FF25 to FF33, 【Chemistry 15】 X represents a covalent bond to Z in Equation II, either directly or via any of the linkers. The index i is an integer in the range of 1 to 20. For each of the formulas F12 to F19, 【Chemistry 16】 B １ or B ２ One R from any of the following １ However, this represents a covalent bond to the active pharmaceutical ingredient, either directly or via any linker. B １ and B ２ One R in each of １ are (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ --- and --- represents the covalent bond of R to the remainder in equation II, B １ and B ２ 0, 1, or 2 R in each of １ However, independently, COOH, F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ CF ３ NO ２ ,CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NHCH ３ ,-(SO ２ )NH(CH ２ ) ｍ CH ３ , or OCF ３ This represents, The index m is an integer in the range of 1 to 14. All remaining R １ However, it represents H, For each of the formulas F20 to F25, 【Chemistry 17】 One R １ are (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ --- and --- represents the covalent bond of R to the remainder in equation II, (a) Same B １ and / or B ２ The one or two Rs above １ However, it represents COOH, and at least one COOH is not conjugated to the active pharmaceutical ingredient, and / or (b) one or two R １ are each independently NO ２ , CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ , -(SO ２ )NH CH ３ , -(SO ２ )NH(CH ２ ) ｍ CH ３ , and the index m is an integer in the range of 1 to 14, 0, 1, or 2 R １ However, each is independent, NO ２ , F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ ,CH ３ CF ３ , or OCF ３ This represents, All remaining R １ However, it represents H, For each of the formulas F26 to F27, [Chemistry 18] One R １ are (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ --- and --- represents the covalent bond of R to the remainder in equation II, 0, 1, or 2 R １ However, each is independent of COOH, F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ CF ３ NO ２ ,CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NHCH ３ ,-(SO ２ )NH(CH ２ ) ｍ CH ３ , or OCF ３ This represents, The index m is an integer in the range of 1 to 14. All remaining R １ However, it represents H, For each of the formulas F28 to F35, 【Chemistry 19】 B １ One R in １ are (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ - represents a covalent bond to Z in equation II, either directly or via any linker. B １ and B ２ One R for each of １ However, B １ and B ２ It is a covalent bond between and the, and the covalent bond is -(S=O)-, -(S(=O)(=O)-, -(CF ２ )-,-(C=O)-,-(CH ２ ) ｍ SCH ２ CO(CH ２ ) ｋ -, - (CH ２ ) ｍ S (CH ２ ) ２ CO(CH ２ ) ｋ -, and - (CH ２ ) ｍ (CO)NH(CH ２ ) ｋ - Selected from, (i) B ２ Two R's in １ The group is COOH, and these two R １ The base is not conjugated to the active pharmaceutical ingredient, or (ii) B １ and / or B ２ One or two R in any of the following １ However, each is independent, NO ２ CH=O, CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NH CH ３ , or - (SO ２ )NH(CH ２ ) ｍ CH ３ It represents either one of the following: B １ and / or B ２ 0, 1, or 2 R in any of the following １ However, each is independent of CH=O, F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ ,CH ３ CF ３ CHF ２ , or OCF ３ This represents, Remaining R １ However, it represents H, The index k is an integer in the range of 1 to 7. The index m is an integer in the range of 1 to 7. For each of the formulas F36 to F43, 【Chemistry 20】 B １ and B ２ One R for each of １ However, B １ and B ２ The sulfoximine group is covalently bonded to the sulfoximine group such that the two groups are bonded together by the sulfoximine group, and the amino group of the sulfoximine is covalently bonded to Z in formula II either directly or via any linker through an acid-containing linker. (i) B １ and / or B ２ Two R's in １ The group is COOH, and these two R １ The base is not conjugated to the active pharmaceutical ingredient, or (ii) B １ and / or B ２ One or two R in any of the following １ However, each is independent, NO ２ CH=O, CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NH CH ３ , or - (SO ２ )NH(CH ２ ) ｍ CH ３ It represents either one of the following: B １ and / or B ２ 0, 1, or 2 R in any of the following １ However, each is independent of CH=O, F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ ,CH ３ CF ３ CHF ２ , or OCF ３ This represents, Remaining R １ However, it represents H, The index k is an integer in the range of 1 to 7. A compound in which the index m is an integer in the range of 1 to 7.

3. A compound comprising an active pharmaceutical ingredient, wherein the active pharmaceutical ingredient comprises insulin, and the insulin contains one or more modified amino acids represented by formula III, 【Chemistry 21】 In equation III, R is selected from formulas FF1 to FF24. Z is any linker, 【Chemistry 22】 Selected from, 【Chemistry 23】 The index k is an integer in the range of 3 to 14. J is expressed by formula III', 【Chemistry 24】 In equation III', 【Chemistry 25】 However, it indicates the binding site to the remaining portion of the insulin, * indicates the connection point to the rest of Z. The index n is an integer in the range of 1 to 8. Regarding formulas FF1 to FF24, 【Chemistry 26-1】 【Chemistry 26-2】 X represents a covalent bond to Z in Equation III, either directly or via any of the linkers. The index i is an integer in the range of 1 to 20. B １ and B ２ However, they may be identical or different, and each independently represents a base selected from formulas F1 to F9. B ３ However, this represents a base selected from formulas F1 to F11, 【Chemistry 27】 For each of the formulas F1 to F9, One R １ are (C=O)---, S(=O)(=O)---, (CH ２ ) ｍ (C=O)---, or (CH ２ ) ｍ --- represents, and --- represents the covalent bond to the remainder of R, 0, 1, or 2 R １ However, each is independent of F, Cl, Br, OH, CH ２ -NH ２ NH ２ (C=O)-NH ２ SO ２ CH ３ CF ３ NO ２ ,CH ３ , OCH ３ , O(CH ２ ) ｍ CH ３ ,-(SO ２ ) NHCH ３ ,-(SO ２ )NH(CH ２ ) ｍ CH ３ , or OCF ３ This represents, The index m is an integer in the range of 1 to 14. One R in F5 １ However, B(OH) ２ This represents, All remaining R １ However, it represents H, A compound in formula F10 in which the index j is an integer in the range of 1 to 13.

4. The arbitrary linker is an L- or D-amino acid having at least one functional group directly conjugated to R, or the arbitrary linker is selected from formulas FL1 to FL9. 【Chemistry 28】 In formulas FL1 to FL9, Z'' represents a covalent bond with respect to Z, R'' represents a covalent bond with respect to R, p is an integer in the range of 1 to 5. q is an integer in the range of 1 to 5. The compound according to any one of claims 1 to 3, wherein r is an integer in the range of 1 to 5.

5. The compound according to any one of claims 1 to 3, wherein the compound is a drug substance further modified as described in claims 1 to 3, and / or one or more amines are independently acetylated or alkylated.

6. The compound according to any one of claims 1 to 3, wherein the active pharmaceutical ingredient is insulin comprising human insulin or an analog thereof, and the insulin comprises an A chain and a B chain.

7. The compound according to claim 1 or 2, wherein the active pharmaceutical ingredient comprises a polypeptide active pharmaceutical ingredient or a human peptide hormone.

8. The compound according to claim 6, wherein the insulin each independently comprises one or two peptide sequences attached to the A chain and / or B chain of insulin, and each peptide sequence independently comprises 1 to 20 consecutive residues.

9. The compound according to claim 6, wherein the insulin comprises 2 to 10 amino acids, each independently modified by formula I, II, or III.

10. The insulin described above includes one or more modifications, each independently represented by formula I, II, or III, and each of the one or more modifications is (i) on the side chains of up to 20 amino acids attached to the N-terminus and / or C-terminus of the A-chain and / or B-chain of insulin, and / or at the N-terminus of the polypeptide, and / or (ii) Within four residues of B1, B21, B22, B29, A1, A22, or A3 residues in the insulin A chain or B chain, and / or (iii) Located on the side chains of amino acids attached to or incorporated into the A and / or B chains of insulin, and / or located at the N-terminus of a polypeptide, wherein the polypeptide has sequence (X ２ ) ｎ X １ (X ２ ) ｍ Includes, in the formula, X １ However, each X is a lysine residue, and the side chain of the lysine residue is modified as represented by formula I, II, or III. ２ However, independently, amino acids K, P, E, G, N, M, A, R, L, W, S, F, V, C, H, D, I, Y, Q, T, or X １ The compound according to claim 6, selected from the group, wherein index m is an integer in the range of 0 to 20, and index n is an integer in the range of 0 to 18.

11. The compound according to claim 1 or 2 is conjugated to the active pharmaceutical ingredient either directly or via a covalent linker, wherein the conjugation is such that Z is NH in formula II. ２ In this case, a conjugate comprising the compound according to claim 1 or 2, not via Z.

12. A compound according to any one of claims 1 to 3, wherein the compound according to any one of claims 1 to 3 is used as an intermediate compound for the production of any compound according to claims 1 to 11.

13. The compound contains one or more modifications represented by formula IV, V, or VI, Regarding formula IV, 【Chemistry 29】 【Transformation 30】 However, it indicates a binding point to the remaining portion of the active pharmaceutical ingredient, The index n is an integer in the range of 1 to 8. R is selected from the group consisting of formulas F111, F222, F333, F444, and F555. 【Chemistry 31】 In formulas F111, F222, F333, F444, and F555, The index n is an integer in the range of 1 to 8. R １ Each carbon atom bonded to it independently has (R) or (S) stereochemistry, Each R １ However, independently, -H, -OR ３ , -N(R ３ ) ２ , -SR ３ -OH, -OCH ３ , -OR ５ NHC(O)CH ３ ien-CH ２ R ３ , -C(O)NHOH, -NHC(O)CH ３ ien-CH ２ OH, -CH ２ OR ５ , -NH ２ ien-CH ２ R ４ , -OR ８ , -R ６ , -R ８ , and -R ７ Selected from, Each R ３ However, independently, -H, acetyl, phosphate, and -R ２ , -SO ２ R ２ , -S(O)R ２ , -P(O)(OR ２ ) ２ , -C(O)R ２ , -CO ２ R ２ , and -C(O)N(R ２ ) ２ Selected from, Each R ２ However, independently, -H, and C as arbitrarily substituted. １－６ Selected from an aliphatic ring, an optionally substituted phenyl ring, an optionally substituted 5-6 member monocyclic heteroaryl ring having 1-4 heteroatoms selected from nitrogen, oxygen, and sulfur, a 4-7 member heterocyclic ring having 1-2 heteroatoms selected from nitrogen, oxygen, and sulfur, and an alkyl or amide covalent bond to R in formula IV, Each R ４ However, independently, -H, -OH, -OR ３ , -N(R ３ ) ２ , -OR ５ , and -SR ３ Selected from, Each R ５ However, they are independently selected from monosaccharides, disaccharides, trisaccharides, pentoses, and hexoses. Each R ６ However, independently, -NCOCH ２ -, - (OCH ２ CH ２ ) ｎ -, -O-C １－９ Alkylene group and substituted C １－９ Selected from alkylene groups, one or more methylene groups are optionally -O-, -(CH ２ ) ｎ -, -OCH ２ -, -N(R ２ )C(O)-,-N(R ２ ) C(O)N(R ２ ) -, -SO ２ -, -SO ２ N(R) ２ )-,-N(R ２ ) SO ２ -, -S-, -N(R ２ )-, -C(O)-, -OC(O)-, -C(O)O-, -C(O)N(R ２ )-, or -N(R ２ ) SO ２ N(R) ２ ) is replaced by -, and index n is an integer in the range of 1 to 8, Each R ７ However, independently, -N(R ２ ) ２ , -F, -Cl, -Br, -I, -SH, -OR ２ , -SR ２ , -NH ２ , -N ３ -C≡CR ２ ien-CH ２ C≡CH, -C≡CH, -CO ２ R ２ , -C(O)R ２ , -OSO ２ R ２ -N(R) ２ ) ２ , -OR ２ , -SR ２ ien-CH ３ ien-CH ２ NH ２ , and selected from direct bonding to R in formula IV, R ８ However, (i) is one side chain of L-serine, D-serine, L-threonine, D-threonine, L-alrosreonine, or D-alrosreonine, corresponding to R in formula IV, with index n=1 in formula IV, (ii) is an amide bond to the C-terminus of lysine, cysteine, or 2,3-diaminopropionic acid, or (iii)-CH ２ C (CH ２ OH) ２ CH ２ NH ２ And, Structures F111, F222, F333, F444, and / or F555 optionally contain one or more acetyl, acetylene, acetonide, and / or pinacol protecting groups. Regarding equation V, 【Chemistry 32】 【Transformation 33】 However, it indicates a binding point to the remaining portion of the active pharmaceutical ingredient, The index n is an integer in the range of 1 to 8. R represents X - Y, X is a covalent bond selected from the group consisting of a triazole bond, an amide bond, an imine bond, or a thioether bond. Y is selected from the group consisting of structures represented by formulas F200 to F203, 【Transformation 34】 X １ However, this represents a covalent bond with respect to X, X ２ However, SH, OH, or NH ２ This represents, The index m is an integer in the range of 1 to 8. The index n is an integer in the range of 1 to 8. Regarding formula VI, 【Chemistry 35】 【Transformation 36】 However, it indicates a binding point to the remaining portion of the active pharmaceutical ingredient, The index n is an integer in the range of 1 to 8. Z is an amino acid, -(CH ２ ) ｐ -ien-CH ２ (OCH ２ CH ２ ) ｐ -, -SCH ２ -, -S (CH ２ ) ２ -, -NH-, -NH(CO)-, -(CO)NH-, -S(CH ２ ) ｋ NH-,-triazole-(CH ２ ) ｋ Selected from the group consisting of -NH-, triazole, amide bond, imine bond, and thioether bond, The index k is an integer in the range of 3 to 5. The index p is an integer in the range of 1 to 8. R is selected from the group consisting of structures represented by formulas F203 to F205, 【Chemistry 37】 In the formula, X ３ However, this represents a covalent bond with respect to Z, X ４ However, SH, OH, or NH ２ This represents, The index q is an integer in the range of 1 to 8. The compound according to claim 5 or 6, wherein the index m is an integer in the range of 1 to 8.

14. Optionally, B １ and B ２ However, first it is conjugated to one of the structures represented by FF1 to FF33, and the resulting conjugation is then covalently bonded to the active pharmaceutical ingredient, or, optionally, one of the structures represented by FF1 to FF33 is first conjugated to the active pharmaceutical ingredient, and then B １ and B ２ A method for producing the compound according to any one of claims 1 to 13, wherein the compound is covalently bonded to the corresponding structure in FF1 to FF33.

15. A method for administering a compound according to any one of claims 1 to 13 to a human subject as a therapeutic or prophylactic agent.

16. The compound according to any one of claims 1 to 13, wherein one or more amine groups are independently acetylated or alkylated.

17. The compound according to claim 6, wherein the insulin comprises two, three, or four modifications, each independently represented by formula I, II, or III.

18. The compound according to any one of claims 1 to 3, wherein the active pharmaceutical ingredient is a human polypeptide hormone or a peptide having at least 10% homology with one, two, three, or four different human peptide hormones, and comprises a dual or triple agonist, a hybrid synthetic peptide based on one or more human polypeptide hormones or their analogues.

19. The compound according to any one of claims 1 to 3, wherein the active pharmaceutical ingredient is insulin, and the amino acid of residue 21 of the B chain is a modified amino acid represented by formula I, II, or III.

20. The compound according to any one of claims 1 to 3, wherein the active pharmaceutical ingredient is insulin, and one or more residues within four residues of residue 22 of the B chain of insulin are each independently represented by formula I, II, or III, and one or more additional residues in the polypeptide attached to the C-terminus and / or N-terminus of the B chain and / or A chain are each independently represented by formula I, II, or III.

21. The compound according to any one of claims 1 to 3, wherein the active pharmaceutical ingredient is insulin, and the modified amino acid either substitutes an amino acid at a given residue in the peptide sequence of the A chain and / or the B chain, or the modified amino acid is added to the peptide sequence of the A chain and / or the B chain either at the terminal or / or within the peptide sequence of the A chain and / or the B chain.

22. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, the amino acid at residue 21 of the B chain is a modified amino acid represented by formula IV, V, or VI, and the residue at the C-terminus of the A chain is represented by formula I, II, or III.

23. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more residues located within four residues of the C-terminus of chain A, or attached to the C-terminus of chain A, are each independently represented by formula I, II, or III, and one or more residues located within four residues of residue 22 of chain B, are each independently represented by formula IV, V, or VI.

24. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more residues located within four residues of the C-terminus of chain A, or attached to the C-terminus of chain A, are each independently represented by formula IV, V, or VI, and one or more residues located within four residues of residue 22 of chain B, are each independently represented by formula I, II, or III.

25. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and two modified amino acids are introduced into the B chain of insulin at any position between the C-terminal cysteine ​​of the B chain and the C-terminus of the B chain, and two additional modified amino acids are introduced at any position in the A chain of insulin, including being added to one or both ends of the A chain.

26. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and (i) within four residues of residue 21 of the B chain, and / or (ii) within six residues of the N-terminus or C-terminus of the A chain and / or the B chain, and / or (iii) within four residues of residue 13 of the A chain, and / or (iv) each independently one or more residues represented by formula I, II, III, IV, V, or VI, and one or more residues within four residues of the C-terminus of the A chain, each independently represented by formula I, II, III, IV, V, or VI.

27. Modified insulin according to any one of claims 1, 2, or 3, wherein two or more amino acids in the B chain in the range of B1 to B29 are substituted with natural, non-standard, or artificial amino acids.

28. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and one or more amino acids of the A chain or B chain are substituted with natural or non-standard amino acids.

29. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is further conjugated directly or via an optional linker to a polypeptide containing up to 31 amino acids.

30. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is conjugated at the N-terminus or C-terminus of the A-chain or B-chain to a polypeptide containing up to 31 amino acids.

31. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a polypeptide containing up to 31 amino acids at the N-terminus or C-terminus of the A-chain or B-chain, and the polypeptide is conjugated to the insulin via a peptide bond.

32. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, the insulin is further conjugated to a polypeptide comprising up to 31 amino acids, either directly or via any linker, and one or more pairs of side chains of the polypeptide are covalently bonded, and in a particular embodiment, the covalent bond between the side chains is a bond selected from the group consisting of a triazole bond, a bond resulting from azido-alkyne cyclization, a disulfide bond, a thioester bond, an oxime bond, an amide bond, a lactam bond, an ester bond, an olefin bond, an imine bond, an ester bond, and a thioether bond.

33. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and at least one primary amine group or one secondary amine group in R of formula I is covalently bonded to the side chains of L- and D-gamma-gamma-glutamic acid via an amide bond, and the N-terminus of the glutamic acid is covalently bonded to an unsubstituted or monosubstituted alkyl diacid chain containing 3 to 16 carbon atoms via an amide bond.

34. The modified insulin according to claim 2, wherein, with respect to formula FF25, the index i is 0.

35. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and 1 to 10 amino acids are added to the polypeptide sequence of insulin, and these are added at the N-terminus to residue 1 of the B chain of insulin, and the residue inserted at the N-terminus of residue 1 is a modified amino acid represented by formula I, II, or III.

36. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and 1 to 10 amino acids are added to the C-terminus of the B chain of insulin, and the residue at position B29 of insulin is a modified amino acid represented by formula I.

37. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and up to six residues are added to the polypeptide sequence of insulin, and at least two of these residues are modified amino acids represented by formulas I to VI.

38. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is modified to have four or five intramolecular disulfide bonds.

39. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a polypeptide using an enzyme.

40. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, and the insulin is bound to a non-boronized polypeptide containing up to 31 amino acids using an enzyme.

41. The compound according to any one of claims 1 to 3 and 13, wherein the active pharmaceutical ingredient is insulin, the insulin is bound to a polypeptide comprising up to 31 amino acids, and the side chains of at least two amino acids in the polypeptide sequence are covalently bonded together or via any linker.

42. The active pharmaceutical ingredient is insulin, and the insulin is covalently bonded using an amide bond to a structure represented by formulas F411 to F416, or a structure in which F411 is further covalently bonded using an amide bond to a structure represented by formulas F412 to F416. 【Chemistry 38】 In the formula, R represents either a primary or secondary amine at the N-terminus of the modified insulin, or a primary or secondary amine in the side chain of a subset of amino acids in the modified insulin, the binding to R is the binding site to the modified insulin, index n represents an integer in the range of 1 to 14, index m represents an integer in the range of 1 to 12, index o represents an integer in the range of 1 to 6, index p represents an integer in the range of 1 to 12, and Z represents -(C=O)-OH, -NH ２ A compound according to any one of claims 1 to 3 and 13, representing one of the following: cholesterol, 7-OH cholesterol, 7,25-dihydroxycholesterol, cholic acid, chenodeoxycholic acid, lithocholic acid, deoxycholic acid, glycocholic acid, glycodeoxycholic acid, glycocolic acid, glycochenodeoxycholic acid, α-tocopherol, β-tocopherol, γ-tocopherol, δ-tocopherol, α-tocotrienol, β-tocotrienol, γ-tocotrienol, or δ-tocotrienol.

43. The aforementioned active pharmaceutical ingredient comprises one or more structures represented by formulas FX15 to FX28. 【Chemistry 39】 During the ceremony, Each R １ However, independently, H, NH ２ NO ２ Cl, CF ３ I, COCH ３ ,CN,C≡CH,N ３ , or selected from Br, Each R ２ However, independently, CF ３ , H, or CH ３ Selected from, Each R ３ However, independently, C≡CH, H, N ３ Selected from, or vinyl group, Each R ４ However, it became independent, NH ２ , R ２ , or R ３ Selected from, Each R ５ However, independently selected from S or NH, The compound according to any one of claims 1 to 3 and 13, wherein the index n is an integer in the range of 1 to 4.

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