Methods for coupling dialkylamines and peptides

Abstract

This invention provides an innovative means and method for coupling dialkylamines and polypeptides or peptides capable of forming random coil conformations. This invention also provides innovative compounds prepared by the methods detailed herein, wherein the compounds are characterized by formula (I): A-[B-] b C-D. The compounds comprise polypeptides / peptides capable of forming a random coil conformation, an N-terminal protecting group, a dialkylamino group, and optionally a linker. Furthermore, the present invention provides lipid nanoparticles comprising the compounds characterized in formula (I), and means and methods for preparing the lipid nanoparticles. Formulations comprising the compounds of the present invention and / or lipid nanoparticles are also provided. Furthermore, the present invention relates to the use of the innovative compounds, lipid nanoparticles, and / or formulations disclosed herein.

Description

[0001] This invention provides an innovative means and method for coupling dialkylamines and polypeptides or peptides capable of forming random coil conformations. This invention also provides innovative compounds prepared by the methods detailed herein, wherein the compounds are characterized by formula (I): A-[B-] b CD. The compounds comprise polypeptides / peptides capable of forming a random coil conformation, an N-terminal protecting group, a dialkylamino group, and optionally a linker. Furthermore, the present invention provides lipid nanoparticles comprising the compounds characterized in formula (I), and means and methods for preparing the lipid nanoparticles. Formulations comprising the compounds of the present invention and / or lipid nanoparticles are also provided. Furthermore, the present invention relates to the use of the innovative compounds, lipid nanoparticles, and / or formulations disclosed herein.

[0002] Lipid formulations, such as lipid nanoparticles (LNPs), are commonly used as drug delivery systems for delivering active ingredients and therapeutic agents. Major obstacles to drug delivery via LNPs include plasma half-life, endosoma lentrapment, and LNP stability. Coupling certain polymers, particularly polyethylene glycol (PEG), to LNPs (or lipids contained within such LNPs) has been shown to overcome or at least reduce these obstacles. In particular, the prior art provides methods for coupling polyethylene glycol (PEG) to lipids (“PEGylated lipids”) to improve the plasma half-life of such lipids or LNPs.

[0003] PEGylated lipids have been used, in particular, in mRNA vaccines against SARS-CoV-2 (including, in particular, known and recently used SARS-CoV-2 vaccines from known sources). More than one billion doses of such vaccines have been administered globally (see, for example, Ju (2022) ACS Nano 16, 11769–11780). However, studies have shown that administration of such SARS-CoV-2 mRNA vaccines can induce up to 68.5-fold increases in anti-PEG immunoglobulins (anti-PEG IgM and IgG), which may lead to an immune response against PEG polymers (i.e., PEG immunity; see, for example, Ju; ibid.). Some studies have suggested that hospitalizations and deaths following SARS-CoV-2 mRNA vaccination are due to severe allergic reactions to polyethylene glycol (PEG) (see, for example, Moghimi (2021) Molecular Therapy 29(3), 898–900). Furthermore, PEG immunization may lead to accelerated clearance of PEGylated therapies, thereby reducing therapeutic efficacy (see, for example, Yang (2015) Wiley Interdiscip Rev NanomedNanobiotechnol.7(5), 655-677). Therefore, we need to develop lipids and LNPs that do not induce adverse allergic reactions like PEGylated lipids / LNPs.

[0004] To overcome some of the drawbacks of PEGylation technology, several recombinant peptide mimics have been developed in this field, some of which are based on natural amino acid sequences or synthetic amino acid fragments. Most natural amino acid sequences do not exhibit the ideal random coil structure in physiological solutions, unlike polyethylene glycol (PEG), which is a key characteristic of PEG. Natural amino acid sequences either tend to adopt a folded conformation (secondary structure) or, even when unfolded, are generally insoluble in water and form aggregates.

[0005] Novel conformationally disordered polypeptides containing small residues Pro, Ala, and Ser have been developed and named “PAS” polypeptides. (Schlapschy, M., Binder, U., Börger, C., Theobald, I., Wachinger, K., Kisling, S., Haller D. & Skerra, A. (2013) PASylation: a biological alternative to PEGylation for extending the plasma half-life of pharmaceutically active proteins by retarding renal filtration. Protein Eng.Des. Sei, 26(8), 489-501; WO 2008 / 155134, EP-B1 2 173 890, US 8,563,521 and WO2017 / 109087). This “PASylation®” technology has previously been used to increase the hydrodynamic volume, especially of pharmaceutically active proteins, thereby extending their plasma half-life. Similar methods for modifying therapeutic proteins with peptides composed of Pro and Ala (“P / A” peptides) have also been proposed (e.g., see WO 2011 / 144756, EP-B1 2 571510, US 9,221,882 and / or WO 2018 / 234455). P / A- and PAS- peptides are hydrophilic, uncharged biopolymers with biophysical properties very similar to polyethylene glycol (PEG). Furthermore, they are biodegradable, thus avoiding accumulation in organs, while exhibiting stability in plasma.

[0006] Unlike the specific interactions of structural proteins, the conformationally disordered nature of polypeptides often poses a challenge to the immune system in generating corresponding antibodies, a characteristic that pathogens exploit to evade immune responses (Giri (2016) Front. Cell. Infect. Microbiol. 6-144; Goh (2016) Mol. BioSyst. 12, 1881-1891). Therefore, conformationally disordered P / A- and PAS- polypeptides lack toxicity and immunogenicity in mice, and thus may be an effective alternative to the PEGylation modification of lipids and LNPs.

[0007] Krishnamurthy (2019) Nanomedicine: NBM, Vol. 18; pp. 169-178) constructed PAS-modified lipid-polymer nanoparticles (“nanoghosts”) by expressing PAS peptides on the outer surface of mammalian cell membranes encapsulated with poly(lactic-glycolic acid copolymer) (PLGA). However, some chemical bonds that might be required in certain situations were not achieved in this study.

[0008] Zhang (2023) Nanomedicine: NBM, Vol. 47, pp. 102-622) attempted to chemically couple P / A- and PAS-peptides with lipids and prepared liposomes containing these peptides. However, the resulting liposome formulations exhibited high turbidity, and precipitation of liposomes or their components was visible.

[0009] Therefore, existing technologies do not provide technical guidance on how to successfully couple P / A- and / or PAS- peptides to lipids. Thus, the technical challenge of this invention lies in providing convenient and reliable means and methods for coupling P / A- and / or PAS- peptides to lipids.

[0010] This technical problem is solved by providing the embodiments described below, and the features as described in the appended claims.

[0011] Therefore, this invention provides an innovative means and method for chemically coupling P / A- and PAS- peptides with di(alkyl)amines. Specifically, this invention provides a method for preparing compound (I):

[0012] A-[B-] b CD (I)

[0013] Wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group; and b) purifying compound (I).

[0014] As mentioned above, Krishnamurthy (2019, ibid.) prepared PAS-modified lipid-polymer nanoparticles (“nanoghosts”) by expressing PAS-peptides on the outer surface of mammalian cell membranes encapsulated with poly(lactic-co-glycolic acid) (PLGA). However, this method only achieved recombinant expression of PAS-peptides in lipid particles and did not involve the chemical (direct) coupling of P / A-peptides and / or PAS-peptides with lipids. It is foreseeable that using recombinant expression rather than chemical coupling to bind PAS-peptides to lipids or lipid particles may increase the chance of introducing potential cellular contaminants and / or increase the number of purification steps required. Furthermore, the PAS-modified “nanoghosts” based on PLGA nanoparticles described by Krishnamurthy (2019, ibid.) have a structure and composition significantly different from typical LNPs; therefore, they are likely unsuitable for standard LNP applications (e.g., as components of vaccines or therapeutics for the delivery of mRNA, siRNA, and similar substances).

[0015] Zhang et al. (2023, ibid.) chemically coupled P / A or PAS-peptides to lipids, producing / preparing P / A-lipids containing a single C16 or two C11 hydrocarbon chains, or PAS-lipids containing a single C16 hydrocarbon chain. However, how Zhang (2023, ibid.) performed the aforementioned chemical coupling is unclear, and the method employed by the authors clearly has significant drawbacks. Specifically, PAS-lipids cause high turbidity and visible precipitation in liposome formulations containing them. Furthermore, compared to liposomes without external modification, the double-chain P / A-peptides showed almost no extension of the blood circulation time of the liposomes. This latter phenomenon may be due to a weakened anchoring effect of the double-chain P / A-lipids; therefore, the authors concluded that the double-chain P / A-lipids may not be suitable for stabilizing lipid particles with a diameter of approximately 100 nm or larger. In summary, this demonstrates the difficulty in using P / A-lipids and PAS-lipids in lipid particles and illustrates the need to provide P / A-lipids and PAS-lipids suitable for lipid particles.

[0016] This invention addresses these deficiencies through unique means and methods involving the coupling of di(alkyl)amines with compounds comprising P / A- and / or PAS- polypeptides.

[0017] The means and methods of this invention have successfully overcome these deficiencies: specifically, as shown in the appended examples, this invention provides a convenient and / or reliable method for coupling P / A- and / or PAS- peptides to lipids, i.e., di(alkyl)amines. This invention has broad applicability, particularly evident in the inventors' successful coupling of a wide variety of different P / A- and / or PAS- peptides to di(alkyl)amines, demonstrating excellent and sequence-independent coupling efficiency using the innovative means and methods provided herein. Furthermore, the compounds obtained / obtainable through the means and methods provided herein exhibit significant and excellent purity, as can also be seen from the non-limiting figures provided herein. This contrasts sharply with the non-practical teachings provided by Zhang (2023, ibid.).

[0018] This invention also provides innovative compounds that are obtained or obtainable through the means and methods detailed herein. These innovative compounds are characterized by formula (I):

[0019] A-[B-] b CD (I)

[0020] Where A is a di(alkyl)amino group, B is a linker, b is 1 or 0, which means that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, which contains an amino acid sequence consisting of alanine, proline and serine, or an amino acid sequence consisting of alanine and proline, and D is an N-terminal protecting group.

[0021] The di(alkyl)amine-containing compounds presented herein are likely to be particularly less prone to cleavage than, for example, similar compounds containing phospholipids instead of di(alkyl)amines. Therefore, these compounds may exhibit higher anchoring efficiency, particularly in LNPs, as will be further detailed below. This document provides polypeptides (P / A- or PAS-peptides) capable of forming random coil conformations and being coupled with di(alkyl)amines. In the accompanying examples, these innovative conjugates are compared with PEGylated di(alkyl)amines. As shown in the accompanying non-limiting examples, the innovative compounds / conjugates presented herein, among other advantages, are even superior to the reference PEGylated di(alkyl)amines tested herein. Specifically, as shown in the accompanying examples, the compounds presented herein, among other things, exhibit favorable effects on cell viability parameters compared to the reference PEGylated lipids.

[0022] The compounds comprising di(alkyl)amines and P / A- and / or PAS- peptides described herein have not been mentioned in the prior art. WO 2011 / 144756 indicates that P / A- peptides can be coupled to lipid solvents, but does not provide detailed technical guidance on specific means and methods for coupling P / A- and / or PAS- peptides to lipids or lipid solvents. Similarly, lipid nanoparticles (LNPs) comprising the compounds described herein have not been mentioned in the prior art.

[0023] Therefore, the present invention also provides a method for generating / preparing lipid nanoparticles comprising a compound of formula (I), wherein the method comprises the steps of: a) providing a compound of formula (I) in the form of an ethanol solution; b) mixing the ethanol solution with an aqueous solution to prepare / generate lipid nanoparticles; and c) optionally dialyzing the lipid nanoparticles. Therefore, the present invention also provides lipid nanoparticles and / or lipid nanoparticles comprising the innovative compound of formula (I) that are obtained / obtainable by the means and methods detailed herein. Furthermore, the present invention provides lipid nanoparticle colloidal dispersions comprising the lipid nanoparticles of the present invention. In the context of the present invention, a lipid nanoparticle colloidal dispersion refers to a suspension comprising the lipid nanoparticles of the present invention and / or a suspension comprising lipid nanoparticles containing the innovative compound characterized by formula (I).

[0024] The lipid nanoparticles and dispersions provided herein exhibit high stability, showing no turbidity or visible precipitation compared to the lipid nanoparticles provided by Zhang (2023, ibid.). Furthermore, compared to LNPs containing a reference PEGylated compound, the lipid nanoparticles provided herein demonstrate superior characteristics, particularly in RNA encapsulation efficiency and LNP shielding ability. This is particularly evident in the examples provided herein.

[0025] Furthermore, this invention provides methods, means, and uses for generating / preparing pharmaceutical and non-pharmaceutical formulations comprising innovative compounds of formula (I) and / or lipid nanoparticles or lipid nanoparticle colloidal dispersions of this invention. Additionally, uses of pharmaceutical and non-pharmaceutical formulations comprising the lipid nanoparticles, lipid nanoparticle colloidal dispersions, and / or innovative compounds of formula (I) provided herein are also disclosed herein.

[0026] The innovative compound of formula (I) provided by the present invention, and the method for generating / preparing said compound, will be described in more detail below. This detailed description relates to and applies to all aspects of the invention, including not only the compound or the method for generating / preparing said compound, but also lipid nanoparticles containing said compound, lipid nanoparticle suspensions, pharmaceutical formulations, and non-pharmaceutical formulations. This detailed description also relates to any use of the compound, lipid nanoparticles containing said compound, lipid nanoparticle suspensions, pharmaceutical formulations, and non-pharmaceutical formulations, as well as methods for generating / preparing them and methods for using them.

[0027] This invention provides compounds according to formula (I), and methods for generating / preparing such compounds of formula (I):

[0028] A-[B-] b CD (I)

[0029] in

[0030] A is a di(alkyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, and C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0031] D is the N-terminal protecting group.

[0032] Consistent with the above, when b is 0, the compound of formula (I) can be a compound of formula ACD; when b is 1, the compound of formula (I) can be a compound of formula ABCD.

[0033] In the above formula (I), A is a di(alkyl)amino.

[0034] The compounds of this invention comprise a dialkylamino group (A group). Therefore, the means and methods described in detail herein refer to coupling a di(alkyl)amine with compound LBCD or compound LCD to prepare / generate a di(alkyl)amine coupled to a B group or a C group of compound LBCD or compound LCD, respectively. In other words, this invention provides P / A- and / or PAS-peptides coupled to a di(alkyl)amine.

[0035] Those skilled in the art will understand that the di(alkyl)amino group comprises two alkyl chains linked by nitrogen atoms, wherein the nitrogen atoms form a tertiary amine, and the di(alkyl)amino group is further linked to either the B group or the C group in compounds ABCD or ACD, respectively. Therefore, the A group comprises two alkyl chains, and said alkyl chains are independently straight-chain or branched alkyl chains. For this purpose, the A group in formula (I) can be represented by formula -N(R A1)2 is used to represent the two R atoms connected to the nitrogen atom. A1 The groups are independently straight-chain or branched alkyl chains.

[0036] Preferably, both alkyl chains are straight chains. Furthermore, each alkyl chain may independently contain about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, and more preferably about 14 carbon atoms.

[0037] In a preferred embodiment, group A comprises two straight-chain alkyl chains, each containing 14 carbon atoms. Therefore, as shown in the appended examples and provided in the specific embodiments, group A can in particular be bis(tetradecyl)amino. It should be understood that, for example, when group A is bis(tetradecyl)amino, the bis(alkyl)amine coupled to compound LBCD or compound LCD is a bis(tetradecyl)amine.

[0038] The accompanying examples exemplify that the A group is not particularly limited in the context of the means and methods provided herein. For example, Examples 1 and 2 illustrate the efficient coupling of di(tetradecyl)amine with P / A-peptides and PAS-peptides, and Example 9 illustrates that di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine, and di(octadecyl)amine can be efficiently coupled with, for example, PAS-peptides. Thus, the two alkyl chains in the A group can independently contain about 8 to about 20 carbon atoms, preferably about 10 to about 18 carbon atoms, more preferably about 10, about 12, about 14, about 16, or about 18 carbon atoms.

[0039] In another preferred embodiment, group A comprises two straight-chain alkyl chains, each containing 10 carbon atoms. Thus, as shown in the appended examples and provided in the specific embodiments, group A may in particular be di(decyl)amino.

[0040] In another preferred embodiment, group A comprises two linear alkyl chains, each containing 12 carbon atoms. Thus, as shown in the appended examples and provided in the specific embodiments, group A may in particular be bis(dodecyl)amino.

[0041] In another preferred embodiment, group A comprises two linear alkyl chains, each containing 16 carbon atoms. Thus, as shown in the appended examples and provided in the specific embodiments, group A may in particular be bis(hexadecyl)amino.

[0042] In another preferred embodiment, group A comprises two linear alkyl chains, each containing 18 carbon atoms. Thus, as shown in the appended examples and provided in the specific embodiments, group A may in particular be bis(octadecyl)amino.

[0043] In the context of this invention, besides di(alkyl)amines, di(alkenyl)amines can also be coupled to compounds LBCD or LCD to prepare / generate di(alkenyl)amino groups respectively coupled to the B or C group of compounds A'-BCD or A'-CD. Therefore, this invention II provides a method for generating / preparing compounds of formula (II):

[0044] A'-[B-] b CD (II)

[0045] Wherein A is a di(alkenyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkenyl)amine with compound LBCD or compound LCD to obtain compound (II), wherein L is a leaving group; and b) purifying compound (II).

[0046] Therefore, the present invention also provides innovative compounds characterized by formula (II):

[0047] A'-[B-] b CD (II)

[0048] Where A is a di(enyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence of alanine, proline, and serine, or an amino acid sequence of alanine and proline, and D is an N-terminal protecting group. Therefore, the A group in formula (II) can be represented by the formula -N(R A2 )2 is used to represent the two R atoms connected to the nitrogen atom. A2 The groups are independently straight-chain or branched alkenyl chains. Preferably, the two alkenyl chains of the di(alkenyl)amine and / or the A' group may independently contain about 8 to about 20 carbon atoms. Preferably, the two alkenyl chains of the di(alkenyl)amine and / or the A' group may each contain about 1 or about 2 double bonds.

[0049] In the context of this invention, besides di(alkyl)amines, di(alkynyl)amines can also be coupled to compounds LBCD or LCD to prepare / generate di(alkynyl)amino groups coupled to the B or C groups of compounds A'-BCD or A'-CD, respectively. Therefore, this invention II provides a method for generating / preparing compounds of formula (III):

[0050] A''-[B-] b CD (III)

[0051] Wherein A is a di(alkynyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkynyl)amine with compound LBCD or compound LCD to obtain compound (II), wherein L is a leaving group; and b) purifying compound (II).

[0052] Therefore, the present invention also provides an innovative compound characterized by formula (III):

[0053] A''-[B-] b CD (III)

[0054] Where A'' is a di(alkynyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence composed of alanine, proline, and serine, or an amino acid sequence composed of alanine and proline, and D is an N-terminal protecting group. Therefore, the A'' group in formula (III) can be represented by formula -N(R A3 )2 is used to represent the two R atoms connected to the nitrogen atom. A3 The group is independently a straight-chain or branched alkynyl chain. Preferably, the two alkynyl chains of the di(alkynyl)amine and / or the A'' group may independently contain about 8 to about 20 carbon atoms. Preferably, the two alkenyl chains of the di(alkynyl)amine and / or the A'' group may each contain about 1 or about 2 triple bonds.

[0055] The definition of the A group provided herein, after necessary modifications, applies to all means and methods of this invention, as well as to all innovative compounds provided herein.

[0056] This invention provides compounds according to formula (I), and methods for generating / preparing such compounds of formula (I):

[0057] A-[B-] b CD (I)

[0058] in

[0059] A is a di(alkyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, and C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0060] D is the N-terminal protecting group.

[0061] In the above formula (I), B is an optional connector.

[0062] The innovative compound may contain an optional linker. Therefore, the B group may or may not be present. It should be understood that if the B group is absent, the A group is directly connected to the C group; and if the B group is present, the A group and the C group are connected through the linker (i.e., the B group), yielding compounds ACD or ABCD, respectively. In this invention, "linker" and "linker group" are used interchangeably. It should be understood that when b = 0, B is absent. Therefore, when b = 1, B is present.

[0063] Those skilled in the art will understand suitable connectors / connector groups that can be used in this invention. Exemplary and non-limiting examples of such connectors / connector groups are provided below.

[0064] The B group may contain one or more amino acid residues, which may be naturally occurring or non-natural amino acids. Therefore, the B group may contain a carboxyl terminus and an amino terminus (i.e., a C-terminus and an N-terminus, respectively). Furthermore, the B group contains at least two carbon atoms between the amino and carboxyl groups, providing a distance of at least two carbon atoms between the amino and carboxyl groups of the B group (e.g., when the B group is ω-amino-C). 3-15 This is the case for alkyl acids—such as ε-aminohexanoic acid—or for alkyl acids where only one carbon atom is provided between the amino and carboxyl groups of the B group (e.g., when the B group is alanine). Since the B group can contain one or more amino acid residues, which can be natural or non-natural, the B group can be, for example, -HN-(C 2-12 hydroxyl)-C(O)-, wherein, optionally, is contained in the -HN-(C 2-12 In the hydrocarbon moiety of -C(O)-, one or more -CH2- units are each independently selected from -O-, -S-, -NH-, and -N(C 1-4 The alkyl group is replaced, and further, wherein optionally, it is contained in the -HN-(C 2-12 One or more =CH- units (if present) in the hydrocarbon moiety of -C(O)- are each replaced by =N-. The hydrocarbon moiety contained in -HN-(C 2-12The hydrocarbon moiety in the cyclic hydrocarbon group (C(O)-) can be, for example, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, or any combination thereof (e.g., alkylaryl or aralkyl, such as benzyl, phenethyl, or methylphenyl). Furthermore, the hydrocarbon moiety can have 3 to 10 carbon atoms, preferably 4 to 8 carbon atoms. Additionally, the two connection points on the aforementioned cyclic hydrocarbon group (e.g., the aryl or cycloalkyl group; also including any specific cyclic group mentioned below, such as the phenyl group contained in -HN-(CH2)0-2-phenyl-(CH2)0-2-C(O)- mentioned in subsequent paragraphs) cannot be located on the same ring carbon atom or on adjacent ring carbon atoms; if such a cyclic group has six ring atoms (e.g., phenyl or cyclohexyl), 1,4-connection (para position) or 1,3-connection (meta position) is preferred, especially 1,4-connection. Furthermore, it is preferred that the cyclic hydrocarbon moiety contained in the -HN-(C)- is... 2-12 The -CH2- and =CH- units (if present) of the hydrocarbon moiety in -C(O)- are not replaced by the aforementioned heterogroups (i.e., the -CH2- unit is not replaced by -O-, -S-, -NH-, or -N(C)-). 1-4 Alkyl group is substituted, and the =CH- unit (if present) is not substituted with =N-. Therefore, the B group is preferably -HN-(C 2-12 (Hydrocarbon group)-C(O)-.

[0065] If the B group provides an amino terminus (e.g., -HN-(C) 2-12 If the amino terminus is an NH- group in (e.g., -HN-(C)-), then the amino terminus typically forms a bond with the C group of the polypeptide. If the B group provides a carboxyl terminus (e.g., -HN-(C)-), then the amino terminus typically forms a bond with the C group of the polypeptide. 2-12 If the -C(O)- group is present in the alkyl group (-C(O)-), then the carboxyl terminus typically forms a bond with a di(alkyl)amino group A. Therefore, those skilled in the art will understand that di(alkyl)amino group A and connector B can form an amide.

[0066] Accordingly, the B group can be selected from -HN-(C 2-12 Alkyl)-C(O)-, -HN-(CH2) 0-2 -phenyl-(CH2) 0-2 -C(O)- and -HN-(CH2) 0-2 -(C 3-8 (cycloalkyl)-(CH2) 0-2 -C(O)-. Therefore, the B group can be selected from -HN-CH2-(C 1-11 Alkyl)-C(O)-, -HN-(C 1-11 Alkyl group -CH2-C(O)-, -HN-(CH2) 0-2 -phenyl-(CH2) 0-2 -C(O)- and -HN-(CH2)0-2 -(C 3-8 (cycloalkyl)-(CH2) 0-2 -C(O)-. Accordingly, the B group can be selected from -HN-CH2CH2-C(O)-, -HN-CH2CH2-(C 1-10 Alkyl)-C(O)-, -HN-(C 1-10 Alkyl group -CH2CH2-C(O)-, -HN-(CH2) 0-2 -phenyl-(CH2) 0-2 -C(O)- and -HN-(CH2) 0-2 -(C 3-8 (cycloalkyl)-(CH2) 0-2 -C(O)-. Therefore, group B can be, for example, selected from -HN-(CH2). 2-12 -C(O)-、-HN-(CH2) 0-2 -phenyl-(CH2) 0-2 -C(O)- and -HN-(CH2) 0-2 -

[0067] Cyclohexyl-(CH2) 0-2 -C(O)-. Accordingly, group B can be selected from -HN-(CH2). 3-10 -C(O)-, -HN-phenyl-C(O)- and -HN-cyclohexyl-C(O)-.

[0068] As shown in the appended embodiments, group B can be selected from -HN-(CH2)4-C(O)-, -HN-(CH2)5-C(O)-, -HN-(CH2)6-C(O)-, -HN-(CH2)7-C(O)-, -HN-(CH2)8-C(O)-,

[0069] and Accordingly, the B group can be selected from ε-aminohexanoic acid, δ-aminovaleric acid, 7-aminoheptanoic acid, 8-aminooctanoic acid, 9-aminononanoic acid, p-aminobenzoic acid, and p-aminocyclohexanecarboxylic acid (i.e., 4-aminocyclohexanecarboxylic acid).

[0070] Using natural amino acids (containing at least two carbon atoms between their amino and carboxyl groups) as B-group amino acids (especially standard proteogenic α-amino acids such as alanine or proline) can also be advantageous because these amino acids are considered safe and well-tolerated. Therefore, the B-group can also be a standard proteogenic α-amino acid containing at least two carbon atoms between its amino and carboxyl groups, particularly alanine or proline.

[0071] Therefore, the B group can also be selected from, for example, alanine (e.g., L-alanine or D-alanine), proline (e.g., L-proline), β-alanine, γ-aminobutyric acid (GABA), δ-aminovaleric acid (Ava), ε-aminohexanoic acid (Ahx), 7-aminoheptanoic acid, 8-aminooctanoic acid (Aoa), 9-aminononanoic acid, p-aminobenzoic acid (Abz), p-aminocyclohexanecarboxylic acid (ACHA; e.g., cis-ACHA or trans-ACHA) and p-(aminomethyl)cyclohexanecarboxylic acid (AMCHA; e.g., cis-AMCHA or trans-AMCHA).

[0072] Alternatively, the B group can be an L-lysine residue with a side chain providing an ε-amino group, which is connected via CO-(C 1-8 The linker group is attached to the N atom of a di(alkyl)amino group (A group). Such linker groups can be obtained, for example, by reacting the lysine side chain with an N-hydroxysuccinimide ester of iodoacetate, followed by coupling with the A group. Other activated iodocarboxylic acids, such as iodopropionic acid, iodobutyric acid, iodovallic acid, or iodohexanoic acid, can be used in addition to iodoacetate or other activated iodocarboxylic acids; these acids are always used in their activated form, such as as an N-hydroxysuccinimide (NHS) ester. In addition to iodoacetate or other activated iodocarboxylic acids, activated bromocarboxylic acids or activated chlorocarboxylic acids (e.g., bromoacetate-NHS ester and chloroacetate-NHS ester) can also be used.

[0073] Alternatively, the B group may contain a cysteine ​​residue that provides a thiol group on its side chain, which can be linked to a di(alkyl)amine. The inventors have unexpectedly discovered a method for coupling PA- / PAS-peptides, each containing a cysteine ​​residue, to a di(alkyl)amine. Example 10 illustrates this by way of example. Figure 14 The potential reaction mechanism was shown. Figure 15 The purity of the resulting compound is shown. The inventors unexpectedly coupled the thiol group of the cysteine ​​residue to a di(alkyl)amine using a bispecific crosslinking agent (see Example 10). In other words, as described herein, the inventors unexpectedly coupled a compound of formula LBCD to compound A using an active ester. Accordingly, in the context of this invention, the bispecific crosslinking agent is an active ester.

[0074] In summary, Example 10 illustrates that an organic solvent containing dichloromethane and methanol, and a bispecific crosslinking agent (e.g., succinimide-trans-4-(N-maleimide-methyl)cyclohexane-1-carboxylic acid ester (SMCC)) can be used to couple a compound LBCD (e.g., an N-terminal protected PAS-Cys polypeptide) containing Cys residues in B with a di(alkyl)amino group (e.g., di(tetradecyl)amine) to form a compound ABCD (e.g., PAS(20)C-linker-di(tetradecyl)amine).

[0075] Therefore, in one embodiment of the invention, it is also contemplated that the means and methods provided herein may further include coupling a compound of formula (I) to compound A using a bispecific crosslinking agent. In other words, the methods provided herein may further include a bispecific crosslinking agent for preparing the compound of formula (I). The bispecific crosslinking agent may be selected from: succinimidyl-trans-4-(N-maleimidemethyl)cyclohexane-1-carboxylic acid ester (SMCC), m-maleimidebenzoyl-N-hydroxysuccinimidyl ester (MBS), N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), succinimidyl-4-(p-maleimidephenyl)butyrate ester (SMBP), N-(γ-maleimidebutyryloxy)succinimidyl ester (GMB). S), succinimide-6-((iodoacetyl)amino)hexanoate (SIAX), succinimide-4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (SIAC), succinimide-6-((((4-(iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (SIACX), p-nitrophenyl iodoacetate (NPIA), and N-hydroxysuccinimide iodoacetate (or one of their analogues), preferably SMCC.

[0076] When a Cys residue is coupled to another molecule using a dual-specific crosslinking agent such as SMCC, the agent specifically reacts with the thiol group of the Cys residue (e.g., via Michael addition). Those skilled in the art will understand that after the coupling reaction, the hydrogen (i.e., H+) contained in the thiol group... - It will be included in the bispecific crosslinking agent or a portion thereof.

[0077] Therefore, without being bound by theory, the leaving group L contained in the compound to be coupled, LBCD (where B contains Cys residues), can be the H- contained in the thiol group of the Cys residues, which can then be included in the bispecific crosslinking agent or a portion thereof after the coupling reaction.

[0078] The bispecific crosslinking agent may be contained in an organic solvent. As described above, the organic solvent may include dichloromethane and methanol. If the organic solvent contains the bispecific crosslinking agent, it contains about 0.5 to about 10 molar equivalents of the bispecific crosslinking agent relative to compound A, preferably about 0.5 to about 5 molar equivalents, preferably about 1 to about 5 molar equivalents, preferably about 1 to about 4 molar equivalents, preferably about 1 to about 3 molar equivalents, preferably about 1 to about 2 molar equivalents, more preferably about 1 molar equivalent.

[0079] Those skilled in the art will understand that when two molecules are coupled using a bispecific crosslinker, the bispecific crosslinker or a portion thereof may be included in the resulting compound. Therefore, when compound LBCD is coupled with compound A, the bispecific crosslinker or a portion thereof may be included in the resulting compound ABCD. Such a portion of the bispecific crosslinker can be considered as forming part of the resulting (and then modified) connector B. Those skilled in the art can readily determine which portions of the bispecific crosslinker may be included in the resulting compound and which portions may be removed during the coupling reaction. The portions of the aforementioned crosslinker (e.g., SMCC) removed during the coupling reaction may be, for example, N-hydroxysuccinimide (NHS) or nitrophenol. This is also described, for example, in Bioconjugate Techniques, G. Hermanson, 3rd edition (2013), the entire text of which is incorporated herein by reference.

[0080] like Figure 15 As shown, a bispecific crosslinking agent (e.g., SMCC) can react with compound LBCD (e.g., PAS(20)C peptide) in the first reaction step; see [link to previous section]. Figure 15 (left-hand reaction scheme) or with compound A (e.g., di(tetradecyl)amine; see also) Figure 15 The reaction scheme on the right reacts to generate different intermediate reaction products. In the second reaction step, the intermediate reaction products can react with compound A or compound LBCD, respectively. Therefore, in the context of this invention, it is also contemplated that the intermediate reaction products can be isolated and subsequently used in the means and methods provided herein.

[0081] Therefore, in one embodiment, the present invention provides a method for preparing compound (I):

[0082] ABCD (I)

[0083] in

[0084] A is a di(alkyl)amino group.

[0085] B is the connector.

[0086] Preferably, B contains Cys residues.

[0087] Wherein B has previously reacted with a bispecific crosslinking agent, thereby coupling the bispecific crosslinking agent or a portion thereof to B.

[0088] C is a polypeptide capable of forming a random coil conformation and containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0089] D is the N-terminal protecting group.

[0090] The method includes the following steps:

[0091] a) Coupling a di(alkyl)amine with a compound LBCD (or a derivative thereof) yields a compound of formula (I), wherein L is a leaving group (e.g., N-hydroxysuccinimide); and

[0092] b) Purification of the compound of formula (I), wherein the compound of formula (I) comprises the bispecific crosslinking agent or a portion thereof.

[0093] In another embodiment, the present invention provides a method for preparing compound (I):

[0094] ABCD (I)

[0095] in

[0096] A is a di(alkyl)amino group.

[0097] Wherein A has previously reacted with a bispecific crosslinking agent, thereby coupling the bispecific crosslinking agent or a portion thereof with A.

[0098] B is the connector.

[0099] Preferably, B contains Cys residues.

[0100] C is a polypeptide capable of forming a random coil conformation and containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0101] D is the N-terminal protecting group.

[0102] The method includes the following steps:

[0103] a) Coupling of a di(alkyl)amine (or a derivative thereof) with compound LBCD yields compound (I), wherein L is a leaving group (e.g., H- contained in the thiol group of the Cys residue); and

[0104] b) Purification of the compound of formula (I), wherein the compound of formula (I) comprises the bispecific crosslinking agent or a portion thereof.

[0105] For compounds LBCD or A that have previously reacted with a bispecific crosslinking agent, the term "or its derivatives" refers to the aforementioned intermediate reaction products. Such intermediate reaction products can also be considered as activated compounds LBCD or activated compounds A.

[0106] Illustrative (non-limiting) examples of the B group are included in SEQ IDs NO: 1, 2 and 6, and are illustrated in the appended examples.

[0107] As mentioned above, if b is 0, the B group may not exist. Therefore, in one aspect of the invention, b is 0 (and thus, B does not exist).

[0108] The definition of the B group provided herein, with necessary modifications, applies to all means and methods of this invention, as well as to all innovative compounds provided herein.

[0109] This invention provides compounds according to formula (I), and methods for generating / preparing such compounds of formula (I):

[0110] A-[B-] b CD (I)

[0111] in

[0112] A is a di(alkyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, and C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0113] D is the N-terminal protecting group.

[0114] In formula (I) above, C is a polypeptide capable of forming a random coil conformation and contains an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0115] The C group contained in the compound of formula (I) is a polypeptide or peptide capable of forming a random coil conformation and contains an amino acid sequence consisting of alanine, proline and serine, or an amino acid sequence consisting of alanine and proline.

[0116] As used herein, the term "random coil" generally refers to any conformation of a polymer molecule, including amino acid polymers / amino acid sequences / peptides, wherein the individual monomeric elements constituting the polymer structure are substantially randomly oriented toward adjacent monomeric elements, but are still chemically bonded to said adjacent monomeric elements. Specifically, peptides, amino acid sequences, or amino acid polymers that employ / have / form / are capable of forming a "random coil conformation" substantially lack well-defined secondary and tertiary structures. Therefore, in the context of this invention, peptides capable of forming random coils are conformationally disordered peptides, and are interchangeably referred to herein as "random coil peptides."

[0117] Those skilled in the art are familiar with the properties of peptides that form random coil conformations and their experimental identification methods, as described in scientific literature (Cantor (1980) Biophysical Chemistry, 2nd ed., WH Freeman and Company, New York; Creighton (1993) Proteins - Structures and Molecular Properties, 2nd ed., WH Freeman and Company, New York; Smith (1996) Fold Des TR95-R106). Such peptides are particularly prone to forming random coil conformations in aqueous environments (e.g., aqueous solutions or aqueous buffers). The presence of random coil conformations can be determined using methods known in the art, particularly spectroscopic techniques such as circular dichroism (CD) spectroscopy. CD spectroscopy is an optical absorption spectroscopy method that determines the presence of a substance by measuring the difference in absorbance of dextrorotatory and levorotatory circularly polarized light. The secondary structure of proteins can be determined by CD spectroscopy using far-ultraviolet spectroscopy with wavelengths between approximately 190 and 250 nm. Within these wavelength ranges, various secondary structures commonly found in peptides can be analyzed, as α-helices, parallel and antiparallel β-sheets, and random coil conformations each produce characteristic CD spectral shapes and intensities. Therefore, using CD spectroscopy, those skilled in the art can readily determine whether a peptide (or a segment thereof) forms / adopts a random coil conformation in aqueous solution or under physiological conditions. Other established biophysical methods include nuclear magnetic resonance (NMR) spectroscopy, absorption spectroscopy, infrared and Raman spectroscopy, measurement of hydrodynamic volume by size exclusion chromatography, analysis of ultracentrifugation or dynamic / static light scattering, and measurement of friction coefficients or intrinsic viscosity (Cantor (1980) ibid.; Creighton (1993) ibid.; Smith (1996) ibid.).

[0118] In addition to the experimental methods described above, there are many theoretical methods for predicting protein secondary structures. One example of such theoretical methods is the Chou-Fasman method (Chou and Fasman, ibid.), which is based on the analysis of the relative frequencies of amino acids in known protein structures (e.g., structures obtained through X-ray crystallography) within α-helical, β-sheet, and turn regions. However, theoretical predictions of protein secondary structures are unreliable. As shown below, amino acid sequences expected to adopt an α-helical secondary structure according to the Chou-Fasman method may, experimentally, form random coils. Therefore, theoretical methods such as the Chou-Fasman algorithm may have limited value in predicting whether a given polypeptide will adopt a random coil conformation, as illustrated in the attached examples and figures. Nevertheless, the theoretical predictions described above are often the preferred method for assessing the assumed secondary structure of a given polypeptide / amino acid sequence. Theoretical predictions of random coil structures also generally suggest that it may be worthwhile to verify, through the aforementioned experimental methods, whether a given polypeptide / amino acid sequence indeed has a random coil conformation.

[0119] Those skilled in the art will understand that when the amino acid sequence / peptide contains residues other than proline, alanine, and optionally serine as minor components, the amino acid sequence / peptide may also form a random coil conformation. The term "minor component" as used herein means that in the random coil peptide encoded by this invention, the amino acid residues different from proline, alanine, or serine account for at most 5 mol% or at most 10 mol%. This means that, out of 100 amino acids, there can be a maximum of 10 different from proline, alanine, and optionally serine, preferably a maximum of 8 mol%, that is, a maximum of 8 different from proline, alanine, and optionally serine, more preferably a maximum of 6 mol%, that is, a maximum of 6 different from proline, alanine, and optionally serine, more preferably a maximum of 5 mol%, that is, a maximum of 5 different from proline, alanine, and optionally serine, particularly preferably a maximum of 4 mol%, that is, a maximum of 4 different from proline, alanine, and optionally serine, more preferably a maximum of 3 mol%, that is, a maximum of 3 different from proline, alanine, and optionally serine, even more specifically preferably a maximum of 2 mol%, that is, a maximum of 2 different from proline, alanine, and optionally serine; and most preferably a maximum of 1 mol%, that is, a maximum of 1 different from proline, alanine, and optionally serine. The amino acid, which is different from proline, alanine, and optionally serine, may be selected from Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr, Trp, Tyr, and Val, including post-translational modified amino acids or non-natural amino acids (see, for example: Budisa (2004) Angew Chem Int Ed Engl 43:6426-6463; Young (2010) J Biol Chem 285:11039-11044; Liu (2010) Annu Rev Biochem 79:413-444; Wagner (1983) AngewChem Int Ed Engl 22:816-828; Walsh (2010) Drug DiscovToday 15: 773-780). In some cases, the P / A-peptide may also contain serine as a small amount. For example, if the encoded random coiled polypeptide is composed of proline and alanine, then serine can also be considered a minor component.

[0120] Typically, preferably, these “few” amino acids (excluding proline, alanine, and optionally serine) are not present in the encoded random coil polypeptide described herein or in the encoded random coil polypeptide as part / fragment of a fusion protein. According to the invention, the encoded random coil polypeptide / amino acid sequence can in particular consist entirely of proline, alanine, and optionally serine residues (i.e., no other amino acid residues are present in the encoded random coil polypeptide or amino acid sequence).

[0121] In one embodiment, the C group comprises an amino acid sequence consisting of alanine and proline residues (P and A). Therefore, in the context of this invention, a polypeptide comprising an amino acid sequence consisting of alanine and proline residues (e.g., the C group in this specific embodiment) may be referred to as a "P / A-polypeptide" or simply "P / A".

[0122] Preferably, P / A (or the C group in this specific embodiment) is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from proline and alanine residues. Preferably, P / A comprises at least one proline residue and at least one alanine residue.

[0123] More preferably, in P / A (or the C group in this specific embodiment), the proportion of proline residues in P / A to the total number of amino acid residues in P / A is preferably ≥10 mol% and ≤70 mol%, more preferably ≥20 mol% and ≤50 mol%, and even more preferably ≥25 mol% and ≤40 mol%. Therefore, it is preferred that 10 mol% to 70 mol% of the total number of amino acid residues in P / A be proline residues; more preferably 20 mol% to 50 mol% of the total number of amino acid residues in P / A be proline residues; and even more preferably 25 mol% to 40 mol% (e.g., 25 mol%, 30 mol%, 35 mol%, or 40 mol%) of the total number of amino acid residues in P / A be proline residues. Furthermore, it is preferred that P / A does not contain any continuous proline residues (i.e., does not contain any partial PP sequence or multiples thereof). Furthermore, preferably, P / A (or C group in this specific embodiment) contains no more than 6 identical consecutive amino acid residues (i.e., it does not contain any partial AAAAAA sequence or multiples thereof, where “A” in the context refers to an alanine residue and “AAAAAA” refers to 6 consecutive alanine residues).

[0124] As described above, the preferred amino acid residue percentage of P / A is at least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, even more preferably at least 99 mol%, and most preferably 100 mol% (or the C group in this specific embodiment) independently selected from proline and alanine. The remaining amino acid residues in P / A are preferably selected from 20 standard proteogenous α-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine. Therefore, it is preferred that P / A is composed of proline, alanine, glycine, and serine residues (wherein less than 10 mol% of the amino acid residues in P / A, preferably less than 5 mol%, are glycine or serine residues), and most preferably P / A is composed of proline and alanine residues, i.e., only proline and alanine residues. It should be understood that, as stated above, P / A contains at least one proline residue and at least one alanine residue.

[0125] The number of amino acid residues constituting P / A (or the C group in this specific embodiment) is preferably about 10 to about 300 amino acid residues, more preferably about 10 to about 250 amino acid residues, even more preferably about 10 to about 200 amino acid residues, even more preferably about 15 to about 150 amino acid residues, even more preferably about 10 to about 140 amino acid residues, even more preferably about 10 to about 130 amino acid residues, even more preferably about 15 to about 120 amino acid residues, even more preferably about 15 to about 110 amino acid residues, and even more preferably about 20 to about 100 amino acid residues. The P / A sequence may more preferably comprise about 20, about 40, or about 100 amino acid residues.

[0126] Examples of preferred P / A amino acid sequences particularly include such amino acid sequences that contain (or more preferably, consist of) the following: (i) the sequence AAPAPAPAAPAAPAPAPAAPA (SEQ ID NO: 9; also known as “P / A#1”); or (ii) the sequence AAPAAAPAPAAPAAPAPAAP (SEQ ID NO: 10; also known as “P / A#2”); or (iii) the sequence APAAAPAPAAAPAPAAAPAPAAAP (SEQ ID NO: 9). 13; also referred to as “P / A#5”); or (iv) a fragment of any one of these sequences; or (v) a combination of two or more of these sequences (these sequences may be the same or different, i.e., combinations of any two or more (e.g., two, three, four, five, six, seven, eight, nine, ten or more) of sequences P / A#1, P / A#2 and / or P / A#5); a corresponding example is a dimer of P / A#1 (“P / A#1-P / A#1”); further examples include P / A#1-P / A#2, P / A#1- P / A#5, P / A#2-P / A#1, P / A#2-P / A#2, P / A#2-P / A#5, P / A#5-P / A#1, P / A#5-P / A#2, P / A#5-P / A#5, P / A#1-P / A#1-P / A# 1. P / A#1-P / A#1-P / A#2, P / A#1-P / A#1-P / A#5, P / A#1-P / A#2-P / A#1, P / A#1-P / A#2-P / A#2, P / A#1-P / A#2-P / A#5, P / A#1-P / A#5-P / A#1, P / A#1-P / A#5-P / A#2, P / A#1-P / A#5-P / A#5, P / A#2-P / A#1-P / A#1, P / A#2-P / A#1-P / A#2, P / A#2- P / A#1-P / A#5, P / A#2-P / A#2-P / A#1, P / A#2-P / A#2-P / A#2, P / A#2-P / A#2-P / A#5, P / A#2-P / A#5-P / A#1, P / A#2-P / A# 5-P / A#2, P / A#2-P / A#5-P / A#5, P / A#5-P / A#1-P / A#1, P / A#5-P / A#1-P / A#2, P / A#5-P / A#1-P / A#5, P / A#5-P / A#2-P / A#1, P / A#5-P / A#2-P / A#2, P / A#5-P / A#2-P / A#5, P / A#5-P / A#5-P / A#1, P / A#5-P / A#5-P / A#2 or P / A#5-P / A#5-P / A#).

[0127] In another embodiment, the C group comprises an amino acid sequence consisting of alanine, proline, and serine residues (P, A, and S). Therefore, in the context of this invention, a polypeptide comprising an amino acid sequence consisting of alanine, proline, and serine residues (e.g., the C group in this specific embodiment) may be referred to as a "PAS-polypeptide" or simply "PAS".

[0128] Preferably, PAS (or the C group in this specific embodiment) is a polypeptide comprising an amino acid sequence consisting of amino acid residues independently selected from alanine, proline, and serine residues. Preferably, PAS comprises at least one proline residue and at least one alanine residue.

[0129] More preferably, the encoded amino acid sequence in PAS (or the C group in this specific embodiment) comprises more than about 4 mol%, preferably more than about 6 mol%, more preferably more than about 10 mol%, more preferably more than about 15 mol%, more preferably more than about 20 mol%, more preferably more than about 22 mol%, 23 mol% or 24 mol%, more preferably more than about 26 mol%, 29 mol% or 30 mol%, more preferably more than about 31 mol%, 32 mol%, 33 mol%, 34 mol% or 35 mol%, and most preferably more than about 25 mol%. The encoded amino acid sequence preferably comprises less than about 40 mol%, more preferably less than 38 mol%, 35 mol%, 30 mol%, or 26 mol% of proline residues, with lower values ​​being preferred. Furthermore, it is preferred that PAS does not contain any consecutive proline residues (i.e., does not contain any partial PP sequence or multiples thereof). Furthermore, the preferred PAS (or the C group in this specific embodiment) contains no more than 6 identical consecutive amino acid residues (i.e., it does not contain any partial AAAAAA sequence or multiples thereof, where “A” in the context refers to an alanine residue and “AAAAAA” refers to 6 consecutive alanine residues).

[0130] As described above, the preferred amino acid residue percentage of PAS is at least 90 mol%, preferably at least 92 mol%, more preferably at least 93 mol%, more preferably at least 94 mol%, more preferably at least 95 mol%, more preferably at least 96 mol%, more preferably at least 97 mol%, even more preferably at least 98 mol%, even more preferably at least 99 mol%, and most preferably 100 mol% (or the C group in this specific embodiment) independently selected from proline, alanine, and serine. The remaining amino acid residues in PAS are preferably selected from 20 standard proteogen α-amino acids, more preferably from proline, alanine, serine, glycine, valine, asparagine, and glutamine, and even more preferably from proline, alanine, glycine, and serine. Therefore, PAS is preferably composed of proline, alanine, glycine, and serine residues (where less than 10 mol% of the amino acid residues in PAS are glycine or serine residues), and most preferably PAS is composed of proline and alanine residues, i.e., only proline, alanine, and serine residues. It should be understood that, as described above, PAS contains at least one proline residue, at least one alanine residue, and at least one serine residue.

[0131] The number of amino acid residues constituting PAS (or the C group in this specific embodiment) is preferably from about 10 to about 300 amino acid residues, more preferably from about 10 to about 250 amino acid residues, even more preferably from about 10 to about 200 amino acid residues, even more preferably from about 15 to about 150 amino acid residues, even more preferably from about 10 to about 140 amino acid residues, even more preferably from about 10 to about 130 amino acid residues, even more preferably from about 15 to about 120 amino acid residues, even more preferably from about 15 to about 110 amino acid residues, and even more preferably from about 20 to about 100 amino acid residues. It is more preferable to include a PAS sequence of about 20, about 40, or about 100 amino acid residues.

[0132] Non-limiting examples of preferred PAS amino acid sequences particularly include such amino acid sequences that contain (or more preferably, consist of) the following: (i) the sequence ASPAPAPASPAAPAPSAPA (SEQ ID NO: 16; also known as “PAS#1”), or (ii) the sequence AAPASPAPAAPSAPAPAAPS (SEQ ID NO: 17; also known as “PAS#2”), or (iii) the sequence SSPSAPSPSSPASPSPSSPA (SEQ ID NO: 16; also known as “PAS#1”). NO:20; also known as “PAS#5”), or (iv) a fragment of any one of these sequences; or (v) a combination of two or more of these sequences (these sequences may be the same or different, i.e., combinations of any two or more (e.g., two, three, four, five, six, seven, eight, nine, ten or more) of sequences PAS#1, PAS#2 and / or PAS#5); a corresponding example is a dimer of PAS#1 (“PAS#1-PAS#1”); further examples include PAS#1-PAS#2, PAS# 1-PAS#5, PAS#2-PAS#1, PAS#2-PAS#2, PAS#2-PAS#5, PAS#5-PAS#1, PAS#5-PAS#2, PAS#5-PAS#5, PAS#1-PAS#1-PA S#1, PAS#1-PAS#1-PAS#2, PAS#1-PAS#1-PAS#5, PAS#1-PAS#2-PAS#1, PAS#1-PAS#2-PAS#2, PAS#1-PAS#2-PAS#5, P AS#1-PAS#5-PAS#1, PAS#1-PAS#5-PAS#2, PAS#1-PAS#5-PAS#5, PAS#2-PAS#1-PAS#1, PAS#2-PAS#1-PAS#2, PAS#2 -PAS#1-PAS#5, PAS#2-PAS#2-PAS#1, PAS#2-PAS#2-PAS#2, PAS#2-PAS#2-PAS#5, PAS#2-PAS#5-PAS#1, PAS#2-PAS# 5-PAS#2, PAS#2-PAS#5-PAS#5, PAS#5-PAS#1-PAS#1, PAS#5-PAS#1-PAS#2, PAS#5-PAS#1-PAS#5, PAS#5-PAS#2-PA S#1, PAS#5-PAS#2-PAS#2, PAS#5-PAS#2-PAS#5, PAS#5-PAS#5-PAS#1, PAS#5-PAS#5-PAS#2 or PAS#5-PAS#5-PAS#5).

[0133] As described above, the C group (i.e., the P / A and / or PAS polypeptide) may comprise a combination of two or more of the exemplary and non-limiting sequences detailed in any of SEQ ID NO: 9 to 22. Combinations of the same P / A or PAS sequences (i.e., multiples of the same P / A or PAS sequence) are most preferred herein. Therefore, in the context of this invention, "combination of two or more sequences" also includes multiples thereof. The term multiple may in particular refer to 2, 3, 4, 5, 6, 7, 9, or 10 polypeptides (e.g., but not limited to those comprising the amino acid sequence SEQ ID NO: 16; PAS#1) that combine to form a single polypeptide. For illustrative purposes only, SEQ ID NO: 4 comprises the amino acid sequence shown in SEQ ID NO: 16 (PAS#1) twice, while SEQ ID NO: 5 and 6 each comprise the amino acid sequence shown in SEQ ID NO: 16 (PAS#1) five times. It will be apparent to those skilled in the art that any other combination of such P / A sequences with PAS sequences is also contemplated herein. Therefore, in the context of this invention, the C group may comprise an amino acid sequence selected from any one of SEQ ID NO: 9 to 22, or multiples thereof.

[0134] Such examples are listed as further illustrative and non-limiting examples in SEQ ID NO: 1 to 8.

[0135] Further examples of the P / A and / or PAS amino acid sequences (or P / A and / or PAS polypeptides containing such amino acid sequences) described in detail above (and below) are provided, in particular WO 2008 / 155134, WO 2011 / 144756, WO 2017 / 109087 and WO 2018 / 234455, the full text of which are incorporated herein by reference.

[0136] The amino acid residues constituting the C group (i.e., P / A or PAS) can have any configuration. Specifically, each α-amino acid residue contained in P / A can be in either an L- or D-configuration. Therefore, any proline residue in P / A can be in either L-proline or D-proline, and any alanine residue in P / A can be in either L-alanine or D-alanine. It should be understood that not all amino acids have definite L- or D-configurations; in particular, glycine residues have only one configuration. Of the α-amino acid residues contained in P / A that can have either an L- or D-configuration, the number of said α-amino acid residues is preferably at least 75 mol%, more preferably at least 80 mol%, even more preferably at least 90 mol%, further preferably at least 95 mol%, even more preferably at least 98 mol%, and most preferably 100 mol% are in the L-configuration.

[0137] The definition of the C group provided herein, with necessary modifications, applies to all means and methods of this invention, as well as to all innovative compounds provided herein.

[0138] This invention provides compounds according to formula (I), and methods for generating / preparing such compounds of formula (I):

[0139] A-[B-] b CD (I)

[0140] in

[0141] A is a di(alkyl)amino group, B is a linker, b is 1 or 0, allowing B to be present or absent, and C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline.

[0142] D is the N-terminal protecting group.

[0143] In formula (I) above, D is an N-terminal protecting group.

[0144] In the compound of formula (I), the D group is a protecting group attached to the N-terminal amino group, particularly the N-terminal α-amino group, of a polypeptide (group B) capable of forming a random coil conformation. In the context of this invention, the terms "protecting group" and "protecting group" are used interchangeably.

[0145] Those skilled in the art will understand suitable protective bases that can be used in this invention. Exemplary and non-limiting examples of such protective bases are provided below.

[0146] In the context of this invention, the D group may be selected from formyl (i.e., -CHO), -CO (C1-6 alkyl), pyroglutamyl (i.e., 5-oxopyrrolidone-2-yl-carbonyl), and high-pyroglutamyl (i.e., 6-oxopyridine-2-yl-carbonyl); wherein the alkyl portion contained in -CO (C1-6 alkyl) may optionally be replaced by one or more groups (e.g., one, two, or three groups) independently selected from -OH, -O (C1-4 alkyl), -NH (C1-4 alkyl), -N (C1-4 alkyl) (C1-4 alkyl), and -COOH. Furthermore, the D group may be selected from formyl, -CO (C1-4 alkyl), pyroglutamyl, and high-pyroglutamyl; wherein the alkyl portion contained in -CO (C1-4 alkyl) may optionally be replaced by one or two groups, which are independently selected from -OH, -O (C1-4 alkyl), -NH (C1-4 alkyl), -N (C1-4 alkyl)(C1-4 alkyl), and -COOH.

[0147] Therefore, the D group can be selected from formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl (i.e., -CO-CH2-COOH), propionyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 2-methoxypropionyl, 3-methoxypropionyl, 2-ethoxypropionyl, 3-ethoxypropionyl, succinyl (i.e., -CO-CH2CH2-COOH; or cyclosuccinyl, i.e., -CO-CH2CH2-CO-), butyryl, 2-hydroxybutyryl, 3-hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3-methoxybutyryl, 4-methoxybutyryl, glycine betaine (i.e., -CO-CH2-N) + (-CH3)3), glutaryl (i.e., -CO-CH2CH2CH2-COOH), pyroglutamyl, and high-pyroglutamyl. Preferably, the D group is selected from acetyl and pyroglutamyl, and especially preferably pyroglutamyl.

[0148] Illustrative (non-limiting) examples of the D group are included in SEQ IDs NO 1 to 8 and are illustrated in the appended examples.

[0149] The definition of the D group provided herein, after necessary modifications, applies to all means and methods of this invention, as well as to all innovative compounds provided herein.

[0150] This invention provides a method for generating / preparing a compound of formula (I):

[0151] A-[B-] b CD (I)

[0152] in

[0153] A is a di(alkyl)amino group.

[0154] B is the connector.

[0155] b is 1 or 0, allowing B to exist or not. C is a polypeptide capable of forming a random coil conformation, containing an amino acid sequence of alanine, proline, and serine, or an amino acid sequence of alanine and proline.

[0156] D is the N-terminal protecting group.

[0157] The method includes the following steps:

[0158] a) Coupling of a di(alkyl)amine with compound LBCD or compound LCD yields compound (I), wherein L is a leaving group; and

[0159] b) Purification of compound (I).

[0160] In the above method, L is a leaving group.

[0161] In the context of this invention, during the coupling of a di(alkyl)amine with compound LBCD and / or with compound LCD, the leaving group L is replaced by an A group. This means that, in the context of this invention, when a di(alkyl)amine is coupled with compound LBCD or compound LCD, the leaving group L is replaced by a di(alkyl)amino group, resulting in the production or preparation of compound ABCD or compound ACD. As those skilled in the art will understand, the di(alkyl)amino group A is provided by the di(alkyl)amine used in the coupling reaction via the replacement of one hydrogen atom of the di(alkyl)amine with a bond formed between the di(alkyl)amino group and a linking group B (if present) or polypeptide C, respectively. Those skilled in the art will understand suitable leaving groups that can be used in this invention. Exemplary and non-limiting examples of such leaving groups are provided below. In the context of this invention, compound LBCD and / or compound LCD generally contain the group -C(O)-L, where L represents a leaving group and is preferably selected from -OH, -I, -Br, -Cl, -H and -OR. 3 , where R 3 The leaving group is a C1-C6 alkyl group, more preferably -OH. In another preferred embodiment, the leaving group may also be an alcohol corresponding to an active ester (e.g., N-hydroxysuccinimide, NHS, or 1-hydroxybenzotriazole, HOBT), such that the -C(O)-L group represents an active ester group (e.g., an NHS ester group or a HOBT ester group). Examples 1 and 2 provide non-limiting examples of such leaving groups.

[0162] It will be apparent to those skilled in the art that if b is 0, then B is absent, and the di(alkyl)amino group of compound (I) is coupled / linked to the C group (i.e., a polypeptide capable of forming a random coil conformation). In other words, if B is absent, the di(alkyl)amino group is coupled to compound LCD, thereby preparing / generating compound ACD.

[0163] In the context of this invention, and as described above and below, compound LCD comprises a polypeptide (especially P / A- and / or PAS- polypeptides) capable of forming a random coil conformation. As described above and illustratively shown in the appended non-limiting examples, for compound ACD, a di(alkyl)amino group is typically linked via a nitrogen atom (N) to the carbon atom (C) of the carboxyl group at the carboxyl terminus of a polypeptide (especially the P / A- and / or PAS- polypeptides) capable of forming a random coil conformation, thereby forming an amide. When a di(alkyl)amine is coupled to the C-terminal carboxyl group of a polypeptide (especially the P / A- and / or PAS- polypeptides) capable of forming a random coil conformation, the leaving group L may be -OH, which is part of the C-terminal carboxyl group.

[0164] Therefore, it will be apparent to those skilled in the art that when a di(alkyl)amine is coupled to a polypeptide capable of forming a random coil conformation (especially the P / A- and / or PAS- polypeptides), the C-terminal carboxyl group may already contain a leaving group L (i.e., -OH). In the context of this invention, when B is absent, the leaving group L can be -OH, which forms part of the C-terminal carboxyl group of a polypeptide capable of forming a random coil conformation (especially the P / A- and / or PAS- polypeptides). Thus, in the compound LCD, L can be -OH, thereby being included in the C-terminal carboxyl group of the C group. As detailed above, L is preferably selected from -OH, -I, -Br, -Cl, and -OR. 3 , where R 3 It is a C1-C6 alkyl group; most preferably -OH. Therefore, when B is absent, L can also be selected from -OH, -I, -Br, -Cl, -H and -OR. 3 , where R 3 It is a C1-C6 alkyl group. In the context of this invention, when B is absent, -I, -Br, -Cl, and -OR... 3 (where R) 3 The -OH group of the C-terminal carboxyl group of a polypeptide (especially the P / A- and / or PAS- polypeptide) that is capable of forming a random coil conformation can be replaced by a C1-C6 alkyl group. In another preferred embodiment, the leaving group may also be an alcohol corresponding to an active ester (e.g., N-hydroxysuccinimide, NHS, or 1-hydroxybenzotriazole, HOBT), such that the -C(O)-L group represents an active ester group (e.g., an NHS ester group or a HOBT ester group).

[0165] Figure 1 An exemplary and non-limiting coupling is illustrated, wherein L is the C-terminal -OH of a polypeptide (especially the P / A- and / or PAS- polypeptide) capable of forming a random coil conformation.

[0166] As stated above, the prior art does not provide any means or methods for coupling peptides capable of forming random coil conformations with di(alkyl)amines. According to the present invention, an innovative method for targeted and / or directional coupling of such peptides with di(alkyl)amines has been discovered.

[0167] In the context of this invention, it is desirable to obtain compounds comprising a single polypeptide capable of forming a random coil conformation and a single di(alkyl)amino group via chemical coupling. Therefore, it is desirable to couple a polypeptide containing only a single reactive group to a single di(alkyl)amine. As detailed above and below, polypeptides capable of forming a random coil conformation comprise an amino acid sequence consisting of alanine and proline, or alanine, proline, and serine; therefore, in the context of this invention, the polypeptide preferably does not contain any reactive amino acid side chains (i.e., preferably does not contain any carboxyl, amino, or thiol groups). Furthermore, in the context of this invention, the N-terminus of the polypeptide is connected to and protected by an N-terminal protecting group. The structural features of the N-terminal protecting group have been disclosed above. Therefore, coupling of polypeptides capable of forming a random coil structure is preferably carried out via its C-terminal carboxyl group or via a linker attached to the C-terminal carboxyl group. Accordingly, the present invention provides a method for generating / preparing a compound of formula (I), wherein the di(alkyl)amine and the compound LBCD or the compound LCD each contain only a single reactive group, and wherein the di(alkyl)amine and the compound LBCD or the compound LCD are coupled via said reactive group. According to the present invention, the terms "link," "couple," "attach," and "connect" are used interchangeably in the context.

[0168] Therefore, the present invention provides a method and means for targeting and / or directionally coupling a single P / A- and / or PAS-peptide, or a compound containing a P / A- and / or PAS-peptide, to a nitrogen atom of a single di(alkyl)amine via an unprotected C-terminal carboxyl group or a linker attached to the C-terminal carboxyl group of the P / A- and / or PAS-peptide. It is through extensive experimental exploration that the inventors have discovered reaction / coupling reagents and conditions that allow for the convenient and reliable generation / preparation of compounds of formula (I) disclosed herein, as particularly illustrated in the appended examples.

[0169] As described above, P / A- and PAS-peptides are capable of forming random coil conformations in aqueous environments, and are therefore highly polar / hydrophilic / oleophobic. Only a few polar protic solvents (e.g., water and methanol) and polar aprotic solvents (e.g., dimethyl sulfoxide (DMSO) and dimethylformamide) are suitable for dissolving P / A- or PAS-peptides. Conversely, di(alkyl)amines (e.g., but not limited to di(tetradecyl)amine) exhibit particularly strong nonpolar / hydrophobic / oleophilic properties due to their two alkyl chains. Diethyl ether and dichloromethane can be used to dissolve di(alkyl)amines (e.g., but not limited to di(tetradecyl)amine). Therefore, the inventors needed to identify a solvent or combination of solvents that allowed for the simultaneous dissolution of P / A- or PAS-peptides and di(alkyl)amines.

[0170] Those skilled in the art will be familiar with methods and means for assessing whether a solvent or solvent mixture can solubilize a compound (i.e., a solute) or a combination of compounds. In particular, the presence of precipitation and / or turbidity may indicate to those skilled in the art that a particular solvent-solute combination has failed to effectively dissolve and / or solubilize the solute. The presence of precipitation and / or turbidity can be easily assessed by those skilled in the art by spectrophotometry or simply by visual inspection.

[0171] The results showed that none of the above solvents could simultaneously dissolve P / A- or PAS- peptides (or compounds containing them) and di(alkyl)amines (e.g., but not limited to, di(tetradecyl)amine). Tetrahydrofuran, dioxane, and 1-methoxy-2-propanol are commonly used to dissolve reactants with very different polarities, but they also could not simultaneously dissolve di(tetradecyl)amines and P / A- or PAS- peptides or compounds containing them. Of all the tested combinations of the above solvents, only a mixture of the polar protic solvent methanol and an excess of the aprotic solvent dichloromethane was able to dissolve both reactants simultaneously. Therefore, the present invention provides a method for generating / preparing a compound of formula (I), wherein, during the coupling process, the di(alkyl)amine and compound LBCD or compound LCD are contained in one or more organic solvents, preferably said organic solvents containing methanol and dichloromethane.

[0172] Therefore, the present invention provides a method for generating / preparing a compound of formula (I):

[0173] A-[B-] b CD (I)

[0174] Wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the di(alkyl)amine and compound LBCD or compound LCD are contained in one or more organic solvents, preferably the organic solvents comprising methanol and dichloromethane; and b) purifying compound (I).

[0175] In the context of this invention, at least one di(alkyl)amine, or compound LBCD or compound LCD, can be provided in an organic solvent comprising dichloromethane and methanol. This means, in particular, that compound LBCD or compound LCD can be provided in an organic solvent comprising dichloromethane and methanol, and that the di(alkyl)amine can be added to / dissolved in the solution comprising the organic solvent and compound LBCD or compound LCD. Alternatively, in particular, it is also apparent from non-limiting embodiments that compound LBCD or compound LCD can be dissolved in methanol, and di(alkyl)amine can be dissolved in dichloromethane. Obviously, these two solutions can be mixed to simultaneously provide compound LBCD or compound LCD and di(alkyl)amine in an organic solvent comprising dichloromethane and methanol.

[0176] The inventors have unexpectedly discovered that when coupling di(alkyl)amines with compounds LBCD or LCD in an organic solvent containing dichloromethane and methanol, the coupling is particularly effective if the solvent contains a dichloromethane to methanol volume ratio of at least about 3 parts by volume of dichloromethane and about 1 part by volume of methanol (volume ratio approximately 3:1) to about 8 parts by volume of dichloromethane and 1 part by volume of methanol (volume ratio approximately 8:1). Studies have found that when the dichloromethane to methanol volume ratio in the solvent is high (>8:1; >8:1; greater than about 8 parts by volume of dichloromethane and about 1 part by volume of methanol), the solubility of the P / A- and / or PAS- peptides provided herein may be impaired, thereby negatively impacting the coupling efficiency of the P / A- and / or PAS- peptides with the di(alkyl)amine. Furthermore, it was found that volume ratios less than approximately 3:1 (<3:1; <3:1; less than approximately 3 parts by volume of dichloromethane and approximately 1 part by volume of methanol) also negatively impacted the coupling efficiency of the P / A- and / or PAS- peptides with di(alkyl)amines, because under these conditions, the generation / preparation of P / A- and / or PAS- peptide methyl esters occurred more frequently.

[0177] Therefore, the present invention provides a method for generating / preparing a compound of formula (I), wherein, during the coupling process, the di(alkyl)amine and the compound LBCD or the compound LCD are contained in an organic solvent comprising dichloromethane and methanol, wherein the organic solvent comprises about 3 parts by volume, about 3.5 parts by volume, about 4 parts by volume, about 4.5 parts by volume, about 5 parts by volume, about 5.5 parts by volume, about 6 parts by volume, about 6.5 parts by volume, about 7 parts by volume, about 7.5 parts by volume, or about 8 parts by volume of dichloromethane and about 1 part by volume of methanol.

[0178] Therefore, the present invention provides a method for generating / preparing a compound of formula (I):

[0179] A-[B-] b CD (I)

[0180] Wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the di(alkyl)amine and compound LBCD or compound LCD are contained in an organic solvent comprising about 3 parts by volume, about 3.5 parts by volume, about 4 parts by volume, about 4.5 parts by volume, about 5 parts by volume, about 5.5 parts by volume, about 6 parts by volume, about 6.5 parts by volume, about 7 parts by volume, about 7.5 parts by volume, or about 8 parts by volume of dichloromethane and about 1 part by volume of methanol; and b) purifying compound (I).

[0181] In the context of this invention, for example, a 3:1 dichloromethane:methanol volume ratio (3:1; 3 parts by volume of dichloromethane and 1 part by volume of methanol) refers to the volume ratio of two solvents (e.g., dichloromethane and methanol), wherein the content of the first solvent (e.g., dichloromethane) is 3 times that of the second solvent (e.g., methanol). For example, a 3:1 volume ratio (3:1; 3 parts by volume of dichloromethane and 1 part by volume of methanol) corresponds to 75 ml of dichloromethane and 25 ml of methanol in a total volume of 100 ml of the solvent. For example, a 4:1 volume ratio (4:1; 4 parts by volume of dichloromethane and 1 part by volume of methanol) corresponds to 80 ml of dichloromethane and 20 ml of methanol in a total volume of 100 ml of the solvent. For example, a 5:1 volume ratio (5:1; 5 parts by volume of dichloromethane and 1 part by volume of methanol) corresponds to approximately 83.33 ml of dichloromethane and approximately 16.67 ml of methanol in a total volume of 100 ml of the solvent. Those skilled in the art can calculate the individual mixing amounts / volumes of dichloromethane and methanol to obtain a solvent with a specific volume ratio.

[0182] Surprisingly, a volume ratio of approximately 5:1 (approximately 5 to 1; approximately 5 parts by volume of dichloromethane and approximately 1 part by volume of methanol) is most favorable for solubilizing P / A- and / or PAS-peptides and di(alkyl)amines, and is most favorable for reducing the production / preparation of P / A- and / or PAS-peptide methyl esters.

[0183] Therefore, the present invention provides a method for preparing a compound of formula (I), wherein, during the coupling process, the di(alkyl)amine and the compound LBCD or the compound LCD are contained in an organic solvent comprising dichloromethane and methanol, wherein the organic solvent comprises about 5 parts by volume of dichloromethane and about 1 part by volume of methanol.

[0184] Therefore, the present invention provides a method for generating / preparing a compound of formula (I):

[0185] A-[B-] b CD (I)

[0186] Wherein A is a di(alkyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a di(alkyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the di(alkyl)amine and compound LBCD or compound LCD are contained in an organic solvent comprising about 3 parts by volume, about 3.5 parts by volume, about 4 parts by volume, about 4.5 parts by volume, about 5 parts by volume, about 5.5 parts by volume, about 6 parts by volume, about 6.5 parts by volume, about 7 parts by volume, about 7.5 parts by volume, or about 8 parts by volume of dichloromethane and about 1 part by volume of methanol, preferably about 5 parts by volume of dichloromethane and 1 part by volume of methanol; and b) purifying compound (I).

[0187] In a preferred aspect, the present invention provides a method for generating / preparing a compound of formula (I):

[0188] A-[B-] b CD (I)

[0189] Wherein A is a bis(tetradecyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a bis(tetradecyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the bis(tetradecyl)amine and compound LBCD or compound LCD are contained in an organic solvent comprising dichloromethane and methanol; and b) purifying compound (I).

[0190] The present invention also provides a method for generating / preparing a compound of formula (I):

[0191] A-[B-] b CD (I)

[0192] Wherein A is a bis(tetradecyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a bis(tetradecyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the bis(tetradecyl)amine and compound LBCD or compound LCD are contained in an organic solvent comprising about 3 parts by volume, about 3.5 parts by volume, about 4 parts by volume, about 4.5 parts by volume, about 5 parts by volume, about 5.5 parts by volume, about 6 parts by volume, about 6.5 parts by volume, about 7 parts by volume, about 7.5 parts by volume, or about 8 parts by volume of dichloromethane and about 1 part by volume of methanol, preferably about 5 parts by volume of dichloromethane and about 1 part by volume of methanol; and b) purifying compound (I).

[0193] The present invention also provides a method for generating / preparing a compound of formula (I):

[0194] A-[B-] b CD (I)

[0195] Wherein A is a bis(tetradecyl)amino group, B is a linker, b is 1 or 0 such that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline, D is an N-terminal protecting group, and the method comprises the following steps: a) coupling a bis(tetradecyl)amine with compound LBCD or compound LCD to obtain compound (I), wherein L is a leaving group, and wherein during the coupling process, the bis(tetradecyl)amine and compound LBCD or compound LCD are contained in an organic solvent comprising about 5 parts by volume of dichloromethane and about 1 part by volume of methanol; and b) purifying compound (I).

[0196] In the context of this invention, the organic solvents described above, particularly suitable for coupling di(alkyl)amines with P / A- or PAS-peptides, may further comprise other / additional / supporting reagents that are advantageous for the coupling reaction. Such other / additional / supporting reagents may (in particular) be coupling agents.

[0197] Therefore, the inventors have identified coupling agents that allow for efficient and convenient coupling of P / A- or PAS-peptides, or compounds comprising them, to di(alkyl)amines (e.g., but not limited to di(tetradecyl)amine). Consequently, the present invention also provides 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU) as a highly efficient coupling agent, exhibiting a coupling rate of approximately 70% with the P / A-peptides disclosed herein and approximately 40% with the PAS-peptides disclosed herein. The inventors tested various coupling agents (including propylphosphonic anhydride (T3P), pentafluorophenyl trifluoroacetate, and ethyl 2-cyano-2-(hydroxyimino)ethyl acetate (OxymaPure®)) and unexpectedly found that TBTU resulted in superior coupling rates (2-fold to 50-fold increases in coupling rate) compared to all other tested coupling agents. Therefore, the present invention further provides a method for generating / preparing a compound of formula (I), wherein a di(alkyl)amine is coupled with compound LBCD or compound LCD using a coupling agent, and wherein said coupling agent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU). Studies have found that adding approximately 1 to 3 molar equivalents of TBTU to the coupling reaction is beneficial / advantageous in increasing the coupling rate of P / A- or PAS- peptides with di(alkyl)amines (e.g., but not limited to, di(tetradecyl)amine). Compared to using, for example, 2 molar equivalents of TBTU, adding more than 5 molar equivalents of TBTU to the coupling reaction results in a significantly reduced coupling rate of P / A- or PAS- peptides with di(alkyl)amines (e.g., but not limited to, di(tetradecyl)amine). The inventors unexpectedly discovered that the addition of approximately 2 molar equivalents of TBTU was particularly beneficial / advantageous in increasing the coupling rate between P / A- or PAS- peptides and dialkylamines (e.g., but not limited to, di(tetradecyl)amine). Therefore, the present invention further provides a method for generating / preparing a compound of formula (I), wherein a di(alkyl)amine is coupled to compound LBCD or compound LCD using a coupling agent, and wherein said coupling agent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU), and wherein approximately 2 molar equivalents of TBTU are added to the reaction.

[0198] The present invention further relates to a nonnucleophilic base for use in accordance with the method detailed herein. Therefore, the present invention relates to a method for generating / preparing a compound of formula (I), wherein a di(alkyl)amine is further coupled with compound LBCD or compound LCD using a nonnucleophilic base, and wherein said nonnucleophilic base is selected from N,N-diisopropylethylamine (DIPEA), 1,8-diazabicycloundec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), triethylamine (TEA), preferably N,N-diisopropylethylamine (DIPEA). The inventors have unexpectedly discovered that the addition of about 1.5 molar equivalents of DIPEA is particularly beneficial / advantageous in increasing the coupling rate of P / A- or PAS- peptides with dialkylamines (e.g., but not limited to di(tetradecyl)amine). However, in the context of the present invention, the addition of 1 to 10 molar equivalents of DIPEA in the coupling reaction is also conceivable. Therefore, the present invention also relates to a method for generating / preparing a compound of formula (I), wherein a di(alkyl)amine is further coupled to a compound LBCD or a compound LCD using a nonnucleophilic base, wherein the nonnucleophilic base is N,N-diisopropylethylamine (DIPEA), and wherein about 1.5 molar equivalents are added to the reaction.

[0199] The inventors unexpectedly discovered that the coupling of P / A- or PAS- peptides with di(alkyl)amines (e.g., but not limited to di(tetradecyl)amine) can be completed in a single reaction. Therefore, the present invention also relates to a method for generating / preparing a compound of formula (I), wherein the di(alkyl)amine is coupled with compound LBCD or compound LCD in a single reaction.

[0200] According to the present invention, the single reaction may be incubated under an argon atmosphere for at least about 1 min, at least about 5 min, at least about 10 min, at least about 20 min, at least about 30 min, at least about 40 min, at least about 50 min, at least about 60 min, at least about 10 min, preferably about 60 min. Therefore, the present invention relates to a method for generating / preparing a compound of formula (I), wherein a di(alkyl)amine reacts with compound LBCD or compound LCD for at least about 1 min, at least about 5 min, at least about 10 min, at least about 20 min, at least about 30 min, at least about 40 min, at least about 50 min, at least about 60 min, at least about 10 min, preferably about 60 min.

[0201] The conditions and reagents detailed above, as well as the innovative solvent combinations, are further illustrated in the following non-limiting examples and figures. Specifically, Figure 1An exemplary reaction scheme, detailed herein but not limiting, is illustrated schematically for the coupling of PAS40-peptide with di(tetradecyl)amine.

[0202] Illustrative and non-limiting examples of the compound LBCD are found in SEQ IDs NO: 1, 2, and 6, while illustrative and non-limiting examples of the compound LCD are found in SEQ IDs NO: 3 to 5, 7, and 8. SEQ IDs NO: 7 and 8 contain a lysine residue (K) at position 12. In the context of this invention, the free amino group on the side chain of the lysine residue can be used to couple the compound LCD with the di(alkyl)amine provided herein to generate / prepare the compound ACD. The means and methods provided herein (especially, non-limitingly, illustratively shown in Example 2) allow those skilled in the art to perform such coupling reactions. Those skilled in the art will recognize that such lysine residues can be readily introduced at any position in any of the P / A and / or PAS sequences disclosed herein. Preferably, the lysine residue can be introduced at the central position of a given amino acid sequence encoding a P / A- and / or PAS-peptide provided herein. In other words, in the amino acid sequence encoding, for example, a random coiled polypeptide of 40 amino acid residues, preferably, such lysine residues can be introduced at position 21 to obtain a polypeptide containing 41 amino acid residues. The advantage of coupling compound LCD with the di(alkyl)amine provided herein is that compound ACD can be generated / prepared, which comprises a branched random coiled polypeptide. Non-limiting examples of such branched P / A- and / or PAS- polypeptides are shown in SEQ ID NO: 7 and 8.

[0203] Those skilled in the art will understand that the methods and means for coupling di(alkyl)amines with compounds LBCD or LCD as described above and / or below can be used interchangeably for coupling di(alkenyl)amines or di(alkynyl)amines with compounds LBCD or LCD as described above.

[0204] The present invention also relates to methods and means for purifying compounds of formula (I).

[0205] After coupling the P / A- or PAS-peptide with a di(alkyl)amine (e.g., but not limited to di(tetradecyl)amine), about 8 volume equivalents of a methanol / H2O mixture (about 10 parts by volume of methanol and about 1 part by volume of water) can be added to precipitate the unreacted di(alkyl)amine. Furthermore, the reaction system can be incubated on ice or at about 0°C for about 20 min. Subsequently, the reaction system can be filtered, for example (but not limited to) using a glass fiber filter with a pore size of about 1 µm, to remove the precipitate. Therefore, the present invention also relates to the purification of compounds of formula (I), wherein at least a portion of the reagent is precipitated from the reaction by the addition of a hydrophilic solvent.

[0206] The present invention also relates to the liquid chromatography analysis of compounds of formula (I), wherein the liquid chromatography preferably refers to high performance liquid chromatography (HPLC) of compounds of formula (I). According to the present invention, compounds of formula (I) can be subjected to liquid chromatography analysis (e.g., HPLC) after precipitation of at least some of the reagents as detailed above.

[0207] The purification method for compounds of formula (I) detailed herein is further illustrated in non-limiting examples. Specifically, Figure 2 An exemplary high-performance liquid chromatogram of PAS40-lipids is illustrated, clearly demonstrating the efficient purification of this compound.

[0208] As described above and below, this invention provides innovative compounds of formula (I), or such compounds that are obtained / available by the means and methods described herein. In any embodiment described below or above, when referring to a compound of formula (I), this invention also always relates to salts and solutions of or containing salts and solutions of compounds of formula (I).

[0209] Therefore, the present invention provides an innovative compound having the characteristics of formula (I):

[0210] A-[B-] b CD (I)

[0211] Where A is a di(alkyl)amino group, B is a linker, b is 1 or 0, which means that B may or may not be present, C is a polypeptide capable of forming a random coil conformation, which contains an amino acid sequence consisting of alanine, proline and serine, or contains an amino acid sequence consisting of alanine and proline, and D is an N-terminal protecting group.

[0212] Appendix Figures 3 to 8Illustrative and non-limiting examples of compounds according to formula (I) of the present invention are provided. Exemplary and non-limiting structures of the said compounds are provided herein, along with illustrative HPLC chromatograms and mass spectrometry analysis results. Those skilled in the art will appreciate that the innovative compounds may also be referred to herein as, for example, Pga-PAS20-di(tetradecyl)amine (a non-limiting example of compound ACD), Pga-P / A20-Ahx-di(tetradecyl)amine (a non-limiting example of compound ABCD), Pga-PAS40-di(tetradecyl)amine (a non-limiting example of compound ACD), or Pga-P / A40-Ahx-di(tetradecyl)amine (a non-limiting example of compound ABCD), or Pga-PAS100-K-di(tetradecyl)amine (a non-limiting example of compound ACD). Those skilled in the art will also appreciate that the reference compound PEG2k (2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide) may also be simply referred to as PEG2k-N,N-di(tetradecyl)acetamide.

[0213] As described above, the innovative compound represented by formula (I) provided herein does not, in one respect, contain an optional linker B group (i.e., therefore, its general formula is ACD). Those skilled in the art will appreciate that, in the context of this invention, such a compound can also be represented by the following formula:

[0214] D-PP-C(O)-NR 1 R 2

[0215] Where D is the N-terminal protecting group (D group) detailed above, -PP-C(O)- corresponds to polypeptide C (C group) as defined above, providing a functional group -C(O)- at its C-terminus for attaching the A group (di(alkyl)amino) detailed above, -NR 1 R 2 Corresponding to group A (the di(alkyl)amino), wherein R1 and R2 are two independent straight-chain or branched alkyl chains, preferably the two alkyl chains independently contain about 8 to about 20 carbon atoms, more preferably about 12 to about 15 carbon atoms, and more preferably about 14 carbon atoms.

[0216] As described above, the innovative compound represented by formula (I) provided herein contains, in one aspect, an optional linker B group (i.e., therefore, its general formula is AB:-CD). Those skilled in the art will appreciate that, in the context of this invention, such a compound can also be represented by the following formula:

[0217] D-PP-C(O)-HN-(C 2-12 (Hydrocarbon group)-C(O)-NR 1 R2

[0218] Where D is the N-terminal protecting group (D group) detailed above, -PP-C(O)- corresponds to the polypeptide C (C group) defined above, providing a functional group -C(O)- at its C-terminus for attaching the B group (the linker group detailed above), -HN-(C 2-12 The hydrocarbon group (-C(O)-) corresponds to the linker group B, and -NR 1 R 2 Corresponding to group A (the di(alkyl)amino), wherein R1 and R2 are two independent straight-chain or branched alkyl chains, preferably the two alkyl chains independently contain about 8 to about 20 carbon atoms, more preferably about 12 to about 15 carbon atoms, and more preferably about 14 carbon atoms.

[0219] As detailed above, in compounds of formula (I), where B is present (i.e., compounds ABCD), the amide bond is preferably formed between the carboxyl group -C(O)- provided by the B group and the di(alkyl)amino group A. As detailed above, in compounds of formula (I), where B is absent (i.e., compounds ACD), the amide bond is preferably formed between the C-terminal carboxyl group -C(O)- of polypeptide C and the di(alkyl)amino group A.

[0220] The compound of formula (I) provided in this paper, after transfection of HeLa cells at a concentration of 10 µg / ml, resulted in a cell viability exceeding 70% as determined by the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole) method. Furthermore, compared to the reference PEG2k compound (2-[(polyethylene glycol)-2000]-N,N-di(tetradecyl)acetamide), the compound of formula (I) provided in this paper, after transfection of HeLa cells at a concentration of 10 µg / ml, showed improved cell viability as determined by the MTS method.

[0221] Therefore, the compound of formula (I) provided herein is clearly beneficial / advantageous in its effect on cell viability compared to compounds in the prior art. This means, in particular, that the compound provided herein has lower cytotoxicity compared to the reference PEG2k compound. This means, in particular, that the compound provided herein has lower or reduced cytotoxicity / lower or reduced toxicity / lower or reduced negative impact on cell viability compared to the reference PEG2k compound. In the context of this invention, an ideal criterion / feature is that the compound of formula (I) provided herein has the lower or reduced cytotoxicity / lower or reduced toxicity / lower or reduced negative impact on cell viability as detailed herein compared to the reference PEG2k compound.

[0222] The surprising yet beneficial / advantageous effects of the compounds of formula (I) provided herein will be further elaborated in the accompanying examples. In particular, Example 5 and Table 3 further illustrate these beneficial / advantageous effects of the compounds of formula (I) provided herein.

[0223] The present invention also provides a method for generating / preparing lipid nanoparticles comprising one or more compounds of formula (I). Furthermore, the present invention relates to the use of compounds of formula (I) in the generation / preparation of lipid nanoparticles.

[0224] Therefore, the present invention provides a method for generating / preparing lipid nanoparticles comprising a compound of formula (I), wherein the method comprises the following steps: a) providing a compound of formula (I) in the form of an ethanol solution; b) mixing the ethanol solution with an aqueous solution to prepare / generate lipid nanoparticles; and c) optionally dialyzing the lipid nanoparticles.

[0225] In the context of this invention, providing an ethanol solution of a compound of formula (I) may also refer to obtaining and / or preparing an ethanol solution comprising a compound of formula (I). Means and methods for generating / preparing lipid nanoparticles are known to those skilled in the art. Such means and methods are detailed below; further illustrative examples can be found in the various embodiments (specifically, Example 3), but these examples are not limiting.

[0226] In the context of this invention, the term "ethanol solution" may be used interchangeably with the term "alcohol solution." As used herein, ethanol solution / alcohol solution refers to a non-aqueous solution composed of ethanol. In the context of this invention, an aqueous solution refers to a non-ethanol solution composed of water. In the context of this invention, the ethanol solution may also contain cationic lipids, non-cationic lipids, and sterols. The ethanol solution may contain about 2 mM to about 60 mM of total lipids, preferably about 7.5 mM to about 30 mM, more preferably 9.43 mM of total lipids. In the context of this invention, "total lipids" includes any lipids contained in the ethanol solution (e.g., compounds of formula (I), cationic lipids, non-cationic lipids, and sterols). Thus, in the context of this invention, a compound of formula (I) is a lipid. With respect to the ethanol solution, the detailed molar percentage (mol%) below refers only to the molar percentage (mol%) of a specific compound (e.g., compound of formula (I), cationic lipid, non-cationic lipid, or sterol) relative to the total amount of lipids (total lipids) in the ethanol solution, without regard to ethanol molecules in the ethanol solution. The ethanol solution may contain about 1 mol% to about 5 mol%, preferably about 1.5 mol%, of the compound of formula (I). Therefore, the ethanol solution contains about 99 mol% to 95 mol%, preferably about 98.5 mol%, of the other lipids (i.e., cationic lipids, non-cationic lipids, and sterols) mentioned above.

[0227] According to the present invention, the ethanol solution may contain about 45 mol% to about 55 mol%, preferably about 50 mol%, of cationic lipids. Generally, cationic lipids are amphiphilic molecules containing a positively charged hydrophilic head group, two (or more) lipophilic tails, or a steroidal moiety, and a linker between these two domains. Preferably, the cationic lipid carries a net positive charge at near physiological pH. In the context of the present invention, cationic lipids may refer to lipids having a constitutively positively charged head group or to ionizable cationic lipids with an apparent pKa value below about 7, the latter potentially containing a neutral or positively charged head group. In the context of the present invention, ionizable cationic lipids are preferred. Therefore, in the context of the present invention, the cationic lipid may be DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butyrate).

[0228] The present invention also relates to a non-cationic lipid, wherein the non-cationic lipid may be anionic lipid, preferably wherein the anionic lipid is a phospholipid, more preferably distearate phosphatidylcholine (DSPC). According to the present invention, the ethanol solution may contain about 7 mol% to about 13 mol%, preferably about 10 mol% of non-cationic lipid.

[0229] Furthermore, the ethanol solution may contain about 35 mol% to about 42 mol%, preferably about 38.5 mol% of sterols. In the context of this invention, the sterols may be cholesterol.

[0230] In the context of this invention, the method disclosed herein for generating / preparing lipid nanoparticles containing a compound of formula (I) may include introducing / including one or more active ingredients into the lipid nanoparticles. The one or more active ingredients may be contained in the aqueous solution. Therefore, mixing an aqueous solution containing the one or more active ingredients with an ethanolic solution containing a compound of formula (I) as detailed in the context may result in the generation / preparation of lipid nanoparticles containing the one or more ingredients and the compound of formula (I). According to the invention, the one or more active ingredients are one or more nucleic acids, one or more polypeptides, one or more proteins, or combinations thereof, preferably one or more nucleic acids. The one or more nucleic acids may be selected from: mRNA, small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA of gene editing systems, DNA, plasmids, antisense oligonucleotides, and combinations thereof, preferably mRNA.

[0231] An aqueous solution containing one or more active ingredients may also contain one or more acids and / or one or more buffer solutions / buffer systems, preferably one or more acids. Therefore, the aqueous solution may contain one or more acids or one or more buffer solutions / buffer systems at concentrations between about 5 mM and about 50 mM, preferably between about 10 mM and about 30 mM, more preferably about 11 mM, preferably the one or more acids. Exemplary buffer solutions / buffer systems suitable for methods of generating / preparing lipid nanoparticles are known to those skilled in the art. Therefore, the one or more acids may be selected from acetic acid, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and citric acid, preferably acetic acid.

[0232] In the context of this invention, one or more active ingredients and one or more buffer solutions / buffer systems or acids, preferably one or more acids, may be contained in the aqueous solution in a volume ratio of about 1:3 to about 1:4, preferably about 1:3. Those skilled in the art will understand that, for example, a volume ratio of one or more active ingredients to one or more acids (or one or more buffer solutions / buffer systems) of about 1:3 may refer to about one volume part of one or more active ingredients or a solution containing one or more active ingredients, and about three volume parts of one or more acids, one or more buffer solutions / buffer systems, or a solution containing them.

[0233] As detailed in the context, methods for producing / preparing lipid nanoparticles may include an optional step of dialysis of the lipid nanoparticles. In one aspect of the invention, the lipid nanoparticles may not be dialyzed. In another aspect, the lipid nanoparticles may be dialyzed using a buffer solution, preferably selected from phosphate buffer, phosphate-buffered saline, and phosphate-buffered saline containing sucrose, more preferably phosphate-buffered saline. In the context of this invention, the term "buffer solution" may be used interchangeably with the term "buffer system" and may also refer to a solution containing it. The preparation of such buffer solutions / buffer systems / solutions containing them is a standard method in the art.

[0234] The method for generating / preparing lipid nanoparticles described above is illustrated in more detail in the appended non-limiting examples, namely Example 3.

[0235] This invention also relates to lipid nanoparticles comprising a compound of formula (I) and / or lipid nanoparticles obtained / obtainable by the methods described above for generating / preparing lipid nanoparticles comprising formula (I). Therefore, this invention provides innovative lipid nanoparticles comprising an innovative compound of formula (I) and / or innovative lipid nanoparticles obtained / obtainable by the innovative methods described above.

[0236] As will be described in detail below, the lipid nanoparticles containing the compound of formula (I) contain features that may be highly beneficial / advantageous in the context of this invention, but which were unexpectedly discovered by the inventors.

[0237] Therefore, the innovative lipid nanoparticles detailed herein, after dialyzing with phosphate-buffered saline for approximately 3 hours, exhibit a zeta potential of less than 10 mV as determined by diffraction light scattering (DLS). This means, in particular, that the lipid nanoparticles, after dialyzing with phosphate-buffered saline for approximately 3 hours, have a lower / reduced zeta potential as determined by DLS compared to reference lipid nanoparticles containing an equal amount of a reference PEG2k compound but not the compound of formula (I). Those skilled in the art will appreciate that, for example, the zeta potential of lipid nanoparticles can provide insights into the surface charge of the lipid nanoparticles. For example, the surface charge of lipid nanoparticles may be influenced by molecules (e.g., PEG polymers, P / A-peptides, or PAS-peptides) on the surface of the lipid nanoparticles that can shield it. Therefore, for example, the zeta potential of lipid nanoparticles can provide relevant insights into the ability of molecules (e.g., PEG polymers, P / A-peptides, or PAS-peptides) located on the surface of the lipid nanoparticles to shield the surface of the lipid nanoparticles. Therefore, in the context of this invention, a low Zeta potential of lipid nanoparticles comprising the compound of formula (I) is desirable and indicates that the lipid nanoparticles and / or the surface of the lipid nanoparticles are successfully shielded / protected. This is further demonstrated in illustrative but non-limiting Example 4. In the context of this invention, the terms "shielded" and "protected" are used interchangeably.

[0238] Furthermore, the lipid nanoparticles comprising formula (I) provided herein exhibit an RNA encapsulation efficiency of at least 91% when measured using the RiboGreen assay. This means, in particular, that the lipid nanoparticles have an improved / increased RNA encapsulation efficiency when measured using the RiboGreen assay, compared to reference lipid nanoparticles comprising an equal amount of a reference PEG2k compound but not formula (I). As shown in the accompanying illustrative but non-limiting example, Example 7, the compound of formula (I) provided herein, when incorporated into lipid nanoparticles, is superior to a reference PEG2k compound in shielding / protecting the lipid nanoparticles and / or the membranes containing the lipid nanoparticles. In the context of this invention, this may be a desirable characteristic of the compound of formula (I) and lipid nanoparticles comprising the compound of formula (I).

[0239] Furthermore, compared to reference lipid nanoparticles containing an equal amount of a reference PEG2k compound but not the compound of any one of claims 35 to 38, the lipid nanoparticles comprising formula (I) provided herein exhibit improved / increased transfection rates, as measured by luminescent readings, when transfecting Jurkat, A549, HepG2, and / or C2C12 cells. This is further illustrated in the appended non-limiting example 8. In the context of this invention, high / increased / improved transfection rates are desirable.

[0240] The present invention also provides lipid nanoparticle suspensions and / or lipid nanoparticle colloidal dispersions comprising the lipid nanoparticles detailed above, wherein the lipid nanoparticles comprise a compound of formula (I). Therefore, the present invention also provides lipid nanoparticle suspensions and / or lipid nanoparticle colloidal dispersions comprising a compound of formula (I). According to the present invention, the term "lipid nanoparticle" as used herein may also refer to the "lipid nanoparticle suspension" and / or the "lipid nanoparticle colloidal dispersion" comprising the lipid nanoparticles and / or a compound of formula (I). The lipid nanoparticle suspensions and / or lipid nanoparticle colloidal dispersions can be stored at about 4°C for at least about 4 days without turbidity and / or visible precipitation of the lipid nanoparticle suspension or its components.

[0241] As described above, the lipid nanoparticles / lipid nanoparticle suspensions / lipid nanoparticle colloidal dispersions containing formula (I) exhibit unexpected and / or desirable beneficial / advantageous effects compared to reference lipid nanoparticles / reference lipid nanoparticle suspensions / reference lipid nanoparticle colloidal dispersions that do not contain the compound of formula (I). The only difference between the lipid nanoparticles / lipid nanoparticle suspensions / lipid nanoparticle colloidal dispersions provided herein and the reference lipid nanoparticles / reference lipid nanoparticle suspensions / reference lipid nanoparticle colloidal dispersions is that the former contains the compound of formula (I), while the latter contains the reference PEG2k compound (2-[(polyethylene glycol)-2000]-N,N-bis(tetradecyl)acetamide). Therefore, any technical advantages of the lipid nanoparticles / lipid nanoparticle suspensions / lipid nanoparticle colloidal dispersions provided herein, as detailed in the context, can be attributed to the compound of formula (I) provided herein contained in the lipid nanoparticles / the lipid nanoparticle suspensions / the lipid nanoparticle colloidal dispersions. Therefore, any technical advantages of the lipid nanoparticles / liquid nanoparticle suspensions / liquid nanoparticle colloidal dispersions provided herein further contribute to the inventiveness of the innovative compounds of formula (I) provided herein. This is further confirmed in the appended non-limiting examples.

[0242] Those skilled in the art will understand that the innovative compounds provided herein can also be used to generate / prepare lipid (or lipid-like) particles and / or lipid (or lipid-like) formulations different from those exemplarily provided herein. Therefore, lipid (or lipid-like) particles (including, but not limited to, lipid nanoparticles) and / or lipid (or lipid-like) formulations produced by any of the following (or any combination thereof) and / or according to the means and methods described herein are also contemplated; thereby, the entirety of the foregoing is incorporated herein by reference, for example, WO 2022 / 180213, WO 2010 / 053572, WO 2012 / 000104, WO 2010 / 053572, WO 2014 / 028487, WO 2015 / 095351, WO 2018 / 089540, WO 2017 / 218704.

[0243] The present invention also relates to means, methods and uses for generating / preparing compounds comprising formula (I), lipid nanoparticles comprising said compound and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound.

[0244] Therefore, the present invention relates to the use of compounds of formula (I), lipid nanoparticles comprising said compound, and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound in the production / preparation of pharmaceutical compositions. The present invention also provides a method for producing / preparing a pharmaceutical composition, wherein the method comprises formulating a compound of formula (I), lipid nanoparticles comprising said compound, and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound into a pharmaceutical composition.

[0245] The method of generating / preparing the pharmaceutical composition may include formulating the compound, the lipid nanoparticles, and / or the lipid nanoparticle suspension using a pharmaceutically acceptable carrier. In one aspect, the pharmaceutical composition may be formulated for intramuscular or intravenous administration. Therefore, the pharmaceutical composition may be formulated for use as a drug or as a vaccine.

[0246] This invention also relates to pharmaceutical compositions comprising a compound of formula (I), lipid nanoparticles comprising said compound, and / or a lipid nanoparticle suspension comprising said lipid nanoparticles and / or said compound. Furthermore, this invention relates to pharmaceutical compositions obtained / available through the means, methods, and uses detailed in the context.

[0247] The present invention also relates to the use of the pharmaceutical composition in treating diseases and / or medical conditions, or in methods of treating said diseases and / or medical conditions.

[0248] The present invention also relates to means, methods and uses for generating / preparing compounds comprising formula (I), lipid nanoparticles comprising said compound and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound.

[0249] Therefore, the present invention relates to the use of compounds of formula (I), lipid nanoparticles comprising said compound, and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound in the generation / preparation of non-pharmaceutical compositions. The present invention also provides a method for generating / preparing a non-pharmaceutical composition, wherein the method comprises formulating a compound of formula (I), lipid nanoparticles comprising said compound, and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound into a non-pharmaceutical composition.

[0250] This invention also relates to non-pharmaceutical compositions comprising a compound of formula (I), lipid nanoparticles comprising said compound, and / or lipid nanoparticle suspensions comprising said lipid nanoparticles and / or said compound. Furthermore, this invention relates to non-pharmaceutical compositions obtained / available through the means, methods, and uses detailed in the context.

[0251] The present invention also relates to the use of the non-pharmaceutical composition.

[0252] In the context of this invention, a non-pharmaceutical composition may be a cosmetic composition.

[0253] In this document, the terms “polypeptide” and “peptide” are used interchangeably and refer to a polymer consisting of two or more amino acids linked by an amide bond, wherein the amide bond is formed between the amino group of one amino acid and the carboxyl group of another amino acid. The amino acids (also known as amino acid residues) contained in the peptide or protein may be selected from 20 standard proteogenic α-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), but may also be selected from non-proteogenic and / or non-standard α-amino acids (e.g., ornithine, citrulline, homolysine, pyrrolyllysine, 4-hydroxyproline, α-methylalanine (i.e., 2-aminoisobutyric acid), valine, leucine, tert-leucine, labonion, or alanine or glycine whose side chain is substituted with a cyclic group (e.g., cycloalkyl, heterocycloalkyl, aryl, or heteroaryl), such as cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, etc.). The peptide or protein contains acids, thienylalanine, cyclohexylglycine, or phenylalanine, as well as β-amino acids (e.g., β-alanine), γ-amino acids (e.g., γ-aminobutyric acid, isoglutamine, or statins), and δ-amino acids. Preferably, the amino acid residues contained in the peptide or protein are selected from α-amino acids, more preferably from 20 standard proteogenic α-amino acids (which may be present in L-isomers or D-isomers, preferably all in L-isomers). The peptide or protein may be unmodified or modified, for example, at functional groups at its N-terminus, its C-terminus, and / or in the side chains of any of its amino acid residues (particularly at the side chain functional groups of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and / or Arg residues). Such modifications may include, for example, the attachment of any protecting group, see Wuts for details. PGM, Greene's Protective Groups in Organic Synthesis, 5th Edition, John Wiley & Sons, 2014, describes the protecting groups for the corresponding functional groups. Such modifications may also include, for example, glycosylation and / or acylation with one or more fatty acids (e.g., one or more C44-C ... 8-30 Alkyl or enoic acids; forming fatty acid acylated peptides or proteins. The amino acid residues contained in a peptide or protein can exist, for example, as a straight-chain molecular chain (forming a straight-chain peptide or protein), or can form one or more rings (corresponding to cyclic peptides or proteins) or branched structures. Peptides or proteins can also form oligomers composed of two or more identical or different molecules.

[0254] As used herein, the term "amino acid" specifically refers to any one of the 20 standard proteogenic α-amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro (also known as imino acids), Ser, Thr, Trp, Tyr, or Val), but also refers to non-proteogenic and / or non-standard α-amino acids (e.g., ornithine, citrulline, homolysine, pyrrolidone, 4-hydroxyproline, α-methylalanine (i.e., 2-aminoisobutyric acid), valine, leucine, tert-leucine, labonion, or those with cyclic groups on their side chains (e.g., cyclic cyclophosphamides). Alanine or glycine substituted with alkyl, heterocyclic alkyl, aryl, or heteroaryl groups, such as cyclopentylalanine, cyclohexylalanine, phenylalanine, naphthylalanine, pyridylalanine, thienoylalanine, cyclohexylglycine, or phenylalanine, or β-amino acids (e.g., β-alanine), γ-amino acids (e.g., γ-aminobutyric acid, isoglutamine, or statins), or δ-amino acids, or any other compound containing at least one carboxylic acid group and at least one amino group. Unless otherwise defined, the term "amino acid" preferably refers to α-amino acids, more preferably any one of the 20 standard proteogenic α-amino acids (which may be in the form of L-isomers or D-isomers, but preferably in the form of L-isomers).

[0255] The term "hydrocarbon chain" refers to a hydrocarbon group consisting of carbon and hydrogen atoms.

[0256] As used herein, the term "hydrocarbon group" refers to a monovalent hydrocarbon group, which can be acyclic (i.e., non-cyclic) or cyclic, and can be composed of acyclic and cyclic groups / subunits. Acyclic hydrocarbon groups or acyclic subunits within hydrocarbon groups can be straight-chain or branched, and can be saturated or unsaturated. Cyclic hydrocarbon groups or cyclic subunits within hydrocarbon groups can be saturated, partially unsaturated (i.e., unsaturated but non-aromatic), or aromatic. "C2- 12 "Hydrocarbon group" refers to a hydrocarbon group having 2 to 12 carbon atoms. Exemplary hydrocarbon groups include, in particular, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, or complex groups consisting of two or more of the above groups (e.g., alkylcycloalkyl, alkylcycloalkenyl, alkylarylalkenyl, arylalkyl, or alkynylaryl). However, it should be understood that if the hydrocarbon group is attached to the parent moiety and further substituted, for example, as residue H2N-(C 2-12 In the case of (hydrocarbon group)-COOH, the corresponding hydrocarbon group in the residue can also be considered as divalent.

[0257] As used herein, the term "alkyl" refers to a monovalent, saturated, acyclic (i.e., non-cyclic) hydrocarbon group, which can be straight-chain or branched. Therefore, "alkyl" does not contain any carbon-carbon double or triple bonds. "C" 1-4"alkyl" refers to an alkyl group having 1 to 4 carbon atoms. Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl). Unless otherwise defined, the term "alkyl" preferably refers to C14. 1-4 alkyl.

[0258] As used herein, the term "alkenyl" refers to a monovalent unsaturated acyclic hydrocarbon group, which may be straight-chain or branched and contains one or more (e.g., one or two) carbon-carbon double bonds, but does not contain any carbon-carbon triple bonds. The term "C..." 2-4 "Alkenyl" refers to an alkenyl group having 2 to 4 carbon atoms. Preferred exemplary alkenyl groups are vinyl, propenyl (e.g., propyl-1-en-1-yl, propyl-1-en-2-yl, or propyl-2-en-1-yl), butenyl, or butadienyl (e.g., buten-1,3-dien-1-yl or buten-1,3-dien-2-yl). Unless otherwise defined, the term "alkenyl" preferably refers to C 2-4 Alkenyl group.

[0259] As used herein, the term "alkynyl" refers to a monovalent unsaturated acyclic hydrocarbon group, which may be straight-chain or branched, and contains one or more (e.g., one or two) carbon-carbon triple bonds and optionally one or more (e.g., one or two) carbon-carbon double bonds. The term "C..." 2-4 "Alynyl" refers to an alkynyl group having 2 to 4 carbon atoms. Preferred exemplary alkynyl groups are ethynyl, propynyl (e.g., propynyl), or butynyl. Unless otherwise defined, the term "alkynyl" preferably refers to C... 2-4 Alkyne group.

[0260] As used herein, the term "aryl" refers to an aromatic hydrocarbon cyclic group, including monocyclic aromatic rings and bridged and / or fused ring systems containing at least one aromatic ring (e.g., a ring system consisting of two or three fused rings, wherein at least one fused ring is an aromatic ring; or a bridged ring system consisting of two or three rings, wherein at least one bridged ring is an aromatic ring). "Aryl" may, for example, refer to phenyl, naphthyl, dinaphthyl (i.e., 1,2-dihydronaphthyl), tetranaphthyl (i.e., 1,2,3,4-tetrahydronaphthyl), indenyl, indenyl (e.g., 1H-indenyl), anthracene, phenanthryl, 9H-fluorenyl, or azulel. Unless otherwise defined, the term "aryl" preferably has 6 to 14 ring atoms, more preferably 6 to 10 ring atoms, even more preferably phenyl or naphthyl, and most preferably phenyl.

[0261] As used herein, the term "heteroaryl" refers to an aromatic ring group, including monocyclic aromatic rings and bridged and / or fused ring systems containing at least one aromatic ring (e.g., a ring system consisting of two or three fused rings, wherein at least one fused ring is an aromatic ring; or a bridged ring system consisting of two or three rings, wherein at least one bridged ring is an aromatic ring), wherein the aromatic ring group comprises one or more (e.g., one, two, three or four) cyclic heteroatoms independently selected from O, S and N, and the remaining ring atoms are carbon atoms, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) may optionally be oxidized, and wherein one or more carbon ring atoms may optionally be oxidized (i.e., forming an oxo group). For example, each heteroatom-containing ring contained in the aromatic ring group may contain one or two O atoms and / or one or two S atoms (which may optionally be oxidized) and / or one, two, three or four N atoms (which may optionally be oxidized), provided that the total number of heteroatoms in the corresponding heteroatom-containing ring is 1 to 4, and that there is at least one carbocyclic atom in the corresponding heteroatom-containing ring (which may optionally be oxidized)."Heteroaryl" can refer to, for example, thienyl (i.e., thiophenyl), benzo[b]thienyl, naphtho[2,3-b]thienyl, thianyl, furanyl (i.e., furanyl), benzofuranyl, isobenzofuranyl, chromanyl, chromenyl (e.g., 2H-1-benzopyranyl or 4H-1-benzopyranyl), isochryneyl (e.g., 1H-2-benzopyranyl), chromone, xanthonyl, phenoxathiinyl, pyrroleyl (e.g., 1H-pyrroleyl), imidazoyl, pyrazolyl, and pyridinyl (i.e., pyridinyl). ; for example, 2-pyridyl, 3-pyridyl or 4-pyridyl), pyrazinyl, pyrimidinyl, pyridazinyl, indoleyl (e.g., 3H-indoleyl), isoindoleyl, indazoleyl, indoleazinyl, purineyl, quinolinyl, isoquinolinyl, phthalazinyl, naphridinyl, quinoxalinyl, cyclolinyl, pteridinyl, carbazoyl, β-carbazoyl, phenanthridineyl, acridineyl, perimidinyl, phenanthroxolinyl (e.g., [1,10]phenanthroxolinyl, [1,7]phenanthroxolinyl or [4,7]phenanthroxolinyl), phenazinyl, thiazoyl, isothiazinyl, phenthiazoyl, oxazinyl, isoxazinyl, oxadiazinyl (e.g., 1,2,4-oxadiazinyl, 1,2,5-oxadiazolyl (i.e., furazolidyl or 1,3,4-oxadiazolyl), thiadiazolyl (e.g., 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl or 1,3,4-thiadiazolyl), phenoxazinyl, pyrazolo[1,5-a]pyrimidinyl (e.g., pyrazolo[1,5-a]pyrimidin-3-yl), 1,2-benzisoxazol-3-yl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, benzoisoxazolyl, benzimidazolyl, benzo[b]thiophene (i.e., benzothiophene), triazolyl (e.g., 1H-1,2,3-triazolyl, 2H-1,2,3-triazolyl, 1H-1,2,4-triazolyl, or 4H-1,2,4-triazolyl), benzotriazolyl, 1H-tetrazolyl, 2H-tetrazolyl, triazine (e.g., 1,2,3-triazine, 1,2,4-triazine, or 1,3,5-triazine), furano[2,3-c]pyridyl, dihydrofuranopyridyl (e.g., 2,3-dihydrofurano[2,3-c]pyridyl or 1,3-dihydrofurano[3,4-c) Pyridyl), imidazopyridyl (e.g., imidazo[1,2-a]pyridyl or imidazo[3,2-a]pyridyl), quinazolinyl, thienopyridyl, tetrahydrothienopyridyl (e.g., 4,5,6,7-tetrahydrothieno[3,2-c]pyridyl), dibenzofuranyl, 1,3-benzodioxanepentenyl, benzodioxane (e.g., 1,3-benzodioxane or 1,4-benzodioxane), or coumarinyl.Unless otherwise defined, the term "heteroaryl" preferably refers to a 5- to 14-membered (more preferably 5- to 10-membered) monocyclic or fused ring system comprising one or more (e.g., one, two, three, or four) cyclic heteroatoms independently selected from O, S, and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbide ring atoms are optionally oxidized; more preferably, "heteroaryl" refers to a 5- or 6-membered monocyclic ring comprising one or more (e.g., one, two, or three) cyclic heteroatoms independently selected from O, S, and N, wherein one or more S ring atoms (if present) and / or one or more N ring atoms (if present) are optionally oxidized, and wherein one or more carbide ring atoms are optionally oxidized. In addition, unless otherwise defined, particularly preferred examples of “heteroaryl” include pyridyl (e.g., 2-pyridyl, 3-pyridyl or 4-pyridyl), imidazole, thiazolyl, 1H-tetrazole, 2H-tetrazole, thienyl (i.e., thiophenyl) or pyrimidinyl.

[0262] As used herein, the term "cycloalkyl" refers to a saturated hydrocarbon cycloalloy, including monocyclic rings and bridged, spirocyclic, and / or fused ring systems (which may consist of, for example, two or three rings; for example, fused ring systems consisting of two or three fused rings). "Cycloalkyl" may, for example, refer to cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, decalinyl (i.e., decahydronaphthyl) or adamantyl. Unless otherwise defined, "cycloalkyl" preferably refers to C14. 3-11 Cycloalkyl, and more preferably C 3-7 Cycloalkyl. Particularly preferred “cycloalkyl” is a monocyclic saturated hydrocarbon ring having 3 to 7 ring members. Furthermore, unless otherwise defined, a particularly preferred example of “cycloalkyl” is cyclohexyl.

[0263] As used herein, the term "cycloalkenyl" refers to an unsaturated alicyclic (non-aromatic) hydrocarbon cyclic group, including monocyclic and bridged, spirocyclic, and / or fused cyclic systems (which may consist of, for example, two or three rings; for example, fused cyclic systems consisting of two or three fused rings), wherein the hydrocarbon cyclic group contains one or more (for example, one or two) carbon-carbon double bonds and does not contain any carbon-carbon triple bonds. "Cycloalkenyl" may, for example, refer to cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl, or cycloheptadienyl. Unless otherwise defined, "cycloalkenyl" preferably refers to C 3-11 Cycloalkenyl, more preferably C 3-7 Cycloalkenyl. Particularly preferred “cycloalkenyl” is a monocyclic unsaturated alicyclic hydrocarbon ring having 3 to 7 ring members and containing one or more (e.g., one or two; preferably one) carbon-carbon double bonds.

[0264] As used herein, the term "nucleic acid" refers to a compound containing at least two deoxyribonucleotides or ribonucleotides (which may be single-stranded, double-stranded, or triple-stranded, including DNA, RNA, and their hybrids). DNA may exist as antisense molecules, plasmid DNA (pDNA), linear or circular DNA, PCR products, or vectors. RNA may be in the following forms: self-amplifying RNA (saRNA) or small hairpin RNA (shRNA), small interfering RNA (siRNA), chemically modified or unmodified messenger RNA (mRNA), antisense RNA, circular RNA (circRNA) containing at least one coding sequence, microRNA (miRNA), micRNA, multivalent RNA, transfer RNA (tRNA), single-stranded guide RNA (sgRNA), replicating RNA (repRNA), Dicer substrate RNA or viral RNA (vRNA), antisense oligonucleotides (ASO), double-stranded RNA (dsRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, or non-natural, and have binding properties similar to reference nucleic acids. Examples of such analogues include, but are not limited to, phosphate thioesters, phosphoramide esters, methylphosphonates, chiral methylphosphonates, 2'-O-methylribonucleotides, and peptide nucleic acids (PNAs). Unless otherwise specified, the term covers nucleic acids containing known natural nucleotide analogues that have similar binding properties to a reference nucleic acid. Unless otherwise stated, a specific nucleic acid sequence also implicitly covers variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, orthologs, single nucleotide polymorphisms, complementary sequences, and explicitly stated sequences.

[0265] The term “lipid” refers to a class of organic compounds, including but not limited to fatty acid esters, and is typically characterized by being poorly soluble in water but readily soluble in a variety of organic solvents. They are generally classified into at least three categories: (1) “simple lipids”, which include fats, oils and waxes; (2) “complex lipids”, which include phospholipids and glycolipids; and (3) “derived lipids”, such as steroids.

[0266] "Catonic lipids" refer to lipids capable of carrying a positive charge. Exemplary cationic lipids contain one or more positively charged amino groups. Preferred cationic lipids are ionizable, and therefore they can exist in a positively charged or neutral form depending on the pH value. The ionization of cationic lipids affects the surface charge of lipid nanoparticles under different pH conditions.

[0267] The term "neutral lipid" refers to any of a variety of lipids that exist as uncharged or neutral zwitterions at a selected pH value.

[0268] The term "ionizable lipid" refers to any of a variety of lipids that can exist in a positively or negatively charged form within a useful physiological range (e.g., pH ~3 to pH ~9), regardless of pH. Ionizable lipids can be synthetic or of natural origin.

[0269] The “effective amount” or “therapeutic effective amount” of an active agent (e.g., nucleic acid) refers to an amount sufficient to produce the desired effect, such as an increase or inhibition of target sequence expression compared to the normal expression level detected in the absence of nucleic acid. An increase in target sequence expression is achieved when any measurable level of the expression product, which is absent in the absence of nucleic acid, is detected. If the expression product is already at a certain level before contact with nucleic acid, an increase in expression is considered achieved when the fold increase relative to the control obtained using nucleic acid (e.g., mRNA) is approximately 1.05, 1.1, 1.2, 1.3, 1.4, 1.5, 1.75, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, 250, 500, 750, 1000, 5000, 10000, or greater. Inhibition of target gene or sequence expression is achieved when the value obtained relative to the control using nucleic acids (such as antisense oligonucleotides) is approximately 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, or 0%. Assays suitable for measuring target gene or sequence expression include, for example, detection of protein or RNA levels using techniques known to those skilled in the art (such as dot blotting, Northern blotting, in situ hybridization, ELISA, immunoprecipitation, enzyme function, fluorescence, or luminescence of a suitable reporter protein), and phenotypic assays known to those skilled in the art.

[0270] The present invention also relates to compositions comprising at least one compound according to formula (I) and at least one active agent. Preferably, the at least one active agent is contained in an effective amount.

[0271] The active ingredients used in this article include any molecule or compound capable of producing a desired effect on cells, tissues, organs, or subjects. Such effects can be biological, physiological, or cosmetic. Active agents can be any type of molecule or compound, including, for example, nucleic acids, nucleic acid analogs, peptides and polypeptides, including, for example, antibodies (such as polyclonal antibodies, monoclonal antibodies, antibody fragments), humanized antibodies, recombinant antibodies, recombinant human antibodies and Primatized™ antibodies, cytokines, growth factors, apoptosis factors, differentiation-inducing factors, cell surface receptors and their ligands, hormones, and small molecules (including small organic molecules or compounds).

[0272] In one embodiment, the active agent is a therapeutic agent, or a salt or derivative thereof. The therapeutic agent derivative may itself have therapeutic activity or may be a prodrug that becomes active after further modification.

[0273] In one implementation, the therapeutic agent includes any therapeutically effective substance or drug, such as anti-inflammatory compounds, antidepressants, stimulants, analgesics, antibiotics, contraceptives, antipyretics, vasodilators, anti-angiogenic agents, cellular vascular drugs, signal transduction inhibitors, cardiovascular drugs (e.g., antiarrhythmic drugs), vasoconstrictors, hormones, and steroids.

[0274] In one implementation, the therapeutic agent is an oncology drug, which may also be referred to as an antitumor drug, anticancer drug, tumor drug, antitumor agent, etc. Examples of oncology drugs that can be used according to the present invention include, but are not limited to: doxorubicin, alkeran, allopurinol, hexamethylmelamine, amifostine, anastrozole, arsenic trioxide, azathioprine, bexarotine, biCNU, bleomycin, intravenous busulfan, oral busulfan, capecitabine (Xeloda), carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin, cladribine, cyclosporine A, cytarabine, cytosine alabinoside, daunorubicin, cyclophosphamide, dexamethasone, dexazosone, docetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estradiol, etoposide phosphate, etoposide and VP-16, exemestane, FK506, fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar), and gemcitabine. Toxascar - Ozomicin, Goserelin Acetate, Hydraea, Hydroxyurea, Idarubicin, Ifosfamide, Imatinib Mesylate, Interferon, Irinotecan (Camptostar, CPT-111), Letrozole, Calcium Leufosinate, Leustatin, Leuprorelin, Levamisole, Aliretinoin, Medroxyprogesterone Acetate, Melphalan, L-PAM, Mesna, Methotrexate, Methoxam, Scrubicin, Mitomycin, Mitomycin Toxantrone, nitrogen mustard, paclitaxel, pamidronate disodium, pegademase, pentostatin, porphyrin sodium, prednisone, rituximab, streptozotocin, STI-571, tamoxifen, docetaxel, temozolomide, teniposide, VM-26, topotecan (Hycamtin), toremifene, retinoic acid, all-trans retinoic acid, pentorubicin, velban, vincristine, VP16, and vinorelbine. Other examples of oncology drugs that can be used according to the invention include elliptic roseine and elliptic roseine analogs or derivatives, epothilones, intracellular kinase inhibitors, and camptothecins.

[0275] In a preferred embodiment, at least one active agent is selected from proteins, peptides, carbohydrates, nucleic acids and nucleic acid analogs, organic molecules with a molecular weight up to 1000 g / mol, and combinations thereof.

[0276] In general, any known protein is applicable. Exemplary proteins include glycoproteins and apolipoproteins. As used herein, the term "apolipoprotein" or "lipoprotein" refers to apolipoproteins and their variants and fragments known to those skilled in the art, as well as apolipoprotein agonists, analogs, or fragments thereof, and chimeric structures of apolipoproteins. The apolipoproteins used in this invention also include recombinant, synthetic, semi-synthetic, or purified apolipoproteins.

[0277] Generally, any known peptide is applicable. The term peptide according to the invention includes peptide-like peptides. The length of a peptide or peptide-like peptide can be from about 5 to 50 amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids. A “cell-permeating peptide” is capable of permeating cells, such as microbial cells (e.g., bacterial or fungal cells) or mammalian cells (e.g., human cells). Microbial cell-permeating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinPI), disulfide-containing peptides (e.g., α-defensins, β-defensins, or bacteriocins), or peptides containing only one or two major amino acids (e.g., PR-39 or indole-containing peptides). Cell-permeation peptides can also contain nuclear localization signals (NLS). For example, a cell-permeating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the NLS of the fusion peptide domain of HIV-1 gp41 and the SV40 large T antigen.

[0278] In one implementation, the targeting peptide tethered by the iRNA reagent and / or carrier oligomer can be an amphiphilic α-helical peptide.

[0279] Peptides and peptide-like ligands include those that have naturally occurring or modified peptides, such as D- or L-peptides; α-, β-, or γ-peptides; N-methyl peptides; azapeptides; peptides having one or more amide bonds (i.e., peptide bonds) replaced by one or more urea bonds, thiourea bonds, carbamate bonds, or sulfonylurea bonds; or cyclic peptides.

[0280] Generally speaking, any known carbohydrate will be applicable. For example, carbohydrates include dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid.

[0281] As described herein, the compositions of the present invention are particularly suitable for delivering nucleic acids or nucleic acid analogs, including, for example, siRNA molecules, mRNA molecules, plasmids, microRNAs, antagomir, aptamers, and ribozymes. Therefore, the compositions of the present invention can be used to regulate the expression of target genes and proteins in vitro and in vivo by contacting cells with a composition of the present invention associated with a nucleic acid (e.g., siRNA or microRNA) capable of reducing the expression of a target gene, or a nucleic acid (e.g., mRNA or plasmid encoding the desired protein) capable of increasing the expression of a desired protein.

[0282] Any known nucleic acid, nucleic acid analogue, or plasmid is generally applicable. Their preparation methods include, but are not limited to, chemical synthesis, enzymatic cleavage of longer precursors, chemical cleavage, or in vitro transcription. Methods for the synthesis of DNA and RNA nucleotides are widely used and well-known in this field.

[0283] Nucleic acids and nucleic acid analogs include polymers containing at least two single-stranded, double-stranded, or triple-stranded deoxyribonucleotides or ribonucleotides, and include DNA, RNA, and their hybrids. DNA can be linear DNA, circular DNA, plasmid DNA (pDNA), antisense molecules, PCR products, or vectors. RNA can be in the following forms: chemically modified or unmodified messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA) containing at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), micro RNA (miRNA), dicer substrate RNA, antisense oligonucleotides (ASO), transfer RNA (tRNA), single-guide RNA (sgRNA), or viral RNA (vRNA), and combinations thereof. Nucleic acids may include one or more oligonucleotide modifications.

[0284] The length of the nucleic acids in this invention varies and generally depends on the specific form of the nucleic acid. For example, in some embodiments, the length of the plasmid or gene may be from about 1,000 to 100,000 nucleotide residues. In some embodiments, the length of the oligonucleotide may be from about 10 to 100 nucleotides. In various related embodiments, the length of the oligonucleotide (including single-stranded, double-stranded, and triple-stranded forms) may be from about 10 to about 50 nucleotides, from about 20 to about 50 nucleotides, from about 15 to about 30 nucleotides, and from about 20 to about 30 nucleotides.

[0285] The term "circular DNA" includes any DNA that forms a closed circular shape without endpoints. Examples of circular DNA include plasmid DNA, microcircular DNA, and dog bone DNA (dbDNA).

[0286] For plasmid DNA, the preparation for embodiments of the present invention typically involves (but is not limited to) in vitro amplification and isolation of the plasmid DNA in a bacterial liquid culture containing the target plasmid. If the target plasmid contains a gene encoding resistance to a specific antibiotic (such as penicillin, kanamycin, etc.), bacteria containing the target plasmid can selectively grow in a culture medium containing the antibiotic. Methods for isolating plasmid DNA are widely used and well-known in the art. Plasmid isolation can be performed using a variety of commercially available kits, including but not limited to Plasmid Plus (Qiagen), GenJET plasmidMaxiPrep (Thermo), and Pure Yield MaxiPrep (Promega) kits, as well as commercially available reagents.

[0287] In a preferred embodiment, the present invention specifically relates to compositions for delivering mRNA or siRNA molecules.

[0288] For mRNA, the main preparation methods include, but are not limited to, enzymatic synthesis (also known as in vitro transcription), which represents the most efficient method currently available for producing long, sequence-specific mRNAs. In vitro transcription describes template-guided RNA molecule synthesis starting from an engineered DNA template, which consists of an upstream phage promoter sequence (e.g., sequences from, but not limited to, those from, T7, T3, and SP6 E. coli phages) linked to a downstream sequence encoding a target gene. The template DNA required for in vitro transcription can be derived from a variety of sources and prepared using appropriate techniques known in the art, including but not limited to plasmid DNA and polymerase chain reaction amplification.

[0289] RNA transcription is performed in vitro using a linearized DNA template, in the presence of appropriate RNA polymerases and rNTPs (adenosine, guanosine, uridine, and cytidine ribonucleoside triphosphates), under conditions that support polymerase activity and minimize potential degradation of the resulting mRNA transcripts. In vitro transcription can be performed using a variety of commercially available kits, including but not limited to the RiboMax Large Scale RNA Production System (Promega), MegaScript Transcription kits (Life Technologies), and commercially available reagents including RNA polymerases and rNTPs. Methods for in vitro mRNA transcription are well known in the art.

[0290] The desired in vitro transcribed mRNA is then purified from unwanted components of the transcription or related reactions, including unincorporated rNTPs, proteases, salts, short RNA oligomers, etc. Techniques for isolating mRNA transcripts are well known in the art. Common methods include phenol / chloroform extraction or precipitation with alcohols (ethanol, isopropanol) in the presence of monovalent cations or lithium chloride.

[0291] Other non-limiting examples of available purification methods include size exclusion chromatography, silica affinity chromatography, and polyacrylamide gel electrophoresis. Purification can be performed using a variety of commercially available kits, including but not limited to the SV Total Isolation System (Promega) and the In Vitro Transcription Cleanup and Concentration Kit (Norgen Biotek).

[0292] Furthermore, while reverse transcription can produce large amounts of mRNA, its products may contain numerous aberrant RNA impurities associated with undesirable polymerase activity, which need to be removed from the full-length mRNA preparation. These impurities include short RNAs resulting from transcription initiation failure, and double-stranded RNAs (dsRNAs) resulting from RNA-dependent RNA polymerase activity, RNA primer transcription of the RNA template, and self-complementary 3' extension. Studies have shown that these contaminants with dsRNA structures can lead to undesirable immunostimulatory activity by interacting with various innate immune sensors in eukaryotic cells, which are capable of recognizing specific nucleic acid structures and inducing strong immune responses. In turn, this significantly reduces mRNA translation due to decreased protein synthesis during innate cellular immune responses. Therefore, various techniques for removing these dsRNA contaminants have been developed, which are well known in the art, including but not limited to scalable HPLC purification. HPLC-purified mRNA has been reported to yield significantly higher levels of translation, especially in primary cells and in vivo.

[0293] Various modification methods have been described in this art for altering specific properties of in vitro transcribed mRNA and enhancing its application value. These methods include, but are not limited to, modifications to the 5' and 3' ends of mRNA. Endogenous eukaryotic mRNA typically contains a cap-like structure at the 5' end of mature molecules, which plays a crucial role in mediating the binding of mRNA cap-binding proteins (CBPs), which in turn enhance mRNA stability within the cell and improve translation efficiency. Therefore, capped mRNA transcripts achieve the highest protein expression levels. The 5' cap contains a 5'-5'-triphosphate bond between the 5' terminal nucleotide and the guanine nucleotide. The conjugated guanine nucleotide is methylated at the N7 position. Other modifications include methylation of the 2'-hydroxyl groups of the last and penultimate 5' nucleotides.

[0294] A variety of cap structures can be used to generate the 5' cap for in vitro transcribed mRNA. Capping the 5' end of synthetic mRNA can be done using chemical cap analogs, simultaneously with the transcription process (i.e., capping during in vitro transcription). For example, the anti-reverse cap analog (ARC A) contains a 5'-5'-guanine triphosphate-guanine bond, where one guanine contains an N7 methyl group and a 3'-O-methyl group. However, up to 20% of transcripts remain uncapped during this transcription process, and the synthetic cap analogs are not entirely identical to the 5' cap structure of real cellular mRNA, which may reduce its translatability and cellular stability. Alternatively, synthetic mRNA molecules can also be enzymatically capped post-transcriptionally. These methods generate more realistic 5' cap structures that more closely mimic the endogenous 5' cap, both structurally and functionally. Such structures exhibit enhanced binding to cap-binding proteins, a prolonged half-life, and reduced sensitivity to 5' endonucleases and / or reduced 5' capping. Several synthetic 5' cap analogues have been developed, which are known in the art to enhance the stability and translatability of mRNA.

[0295] During RNA processing, a long chain of adenine nucleotides (poly-A tail) is typically added to the 3' end of the mRNA molecule. After transcription, the 3' end of the transcription product is cleaved, releasing a 3' hydroxyl group. Poly-A polymerase then adds a string of adenine nucleotides to the RNA through a process called polyadenylation. Numerous studies have shown that the poly-A tail can improve the translation efficiency and stability of mRNA.

[0296] Poly(A) tails can be added to in vitro transcribed mRNA using various methods, including but not limited to cloning poly(T) sequences into a DNA template or post-transcriptional addition using poly(A) polymerase. The first method imparts a poly(A) tail of a specific length to the in vitro transcribed mRNA (the length depends on the size of the poly(T) sequence), but requires additional template processing. The latter method involves enzymatically adding poly(A) tails to the in vitro transcribed mRNA using poly(A) polymerase, which catalyzes the incorporation of adenine residues into the 3' end of the RNA. No additional DNA template processing is required, but the resulting mRNA has poly(A) tails of uneven length. Both 5' capping and 3' poly(A) tailing can be performed using various commercially available kits, including but not limited to the Poly(A) Polymerase Tailing kit (Epicenter), mMESSAGE mMACHINE T7 Ultrakit, and Poly(A) Tailing kit (Life Technologies), as well as commercially available reagents, various ARCA caps, and poly(A) polymerases.

[0297] In addition to 5' cap and 3' polyadenylation, other modifications to in vitro transcripts have been reported to provide benefits for translation efficiency and stability. Pathogenic DNA and RNA are known to be recognized by a variety of sensors in eukaryotes, triggering a strong innate immune response. The ability to distinguish pathogenic DNA and RNA from self-generated DNA and RNA has been shown to depend at least in part on their structure and nucleoside modifications, as most naturally derived nucleic acids contain modified nucleosides. In contrast, in vitro synthesized RNA lacks these modifications and is therefore immunostimulatory, which in turn inhibits efficient mRNA translation, as described above. Introducing modified nucleosides into in vitro transcribed mRNA can prevent recognition and activation by RNA sensors, thereby mitigating this undesirable immunostimulatory activity and improving translational capacity. Modified nucleosides and nucleotides used to synthesize modified RNA can be prepared, monitored, and utilized using methods and procedures known in the art. Various nucleoside modification methods are available, which can be used alone or in combination with other modified nucleosides to incorporate to some extent into in vitro transcribed mRNA, for example, as disclosed in US 2012 / 0251618. According to reports, in vitro synthesized nucleoside-modified mRNAs have reduced activation of immune sensors, while simultaneously enhancing translational capabilities.

[0298] Other components of mRNA can also be modified to provide benefits for translatability and stability, including the 5' and 3' untranslated regions (UTRs). Optimization of the UTR (where favorable 5' and 3' UTRs can be obtained from cellular or viral RNA), whether by optimizing both the 5' and 3' UTRs simultaneously or by optimizing one of them individually, has been shown to improve the stability and translation efficiency of in vitro transcribed mRNA.

[0299] In one embodiment, the RNA is a self-amplifying RNA. A self-amplifying RNA molecule (replicon), even without any protein, can produce multiple daughter RNAs through self-transcription (via its own generated antisense copies) after delivery to a vertebrate cell. Therefore, in some embodiments, the self-amplifying RNA molecule is: a (+)-chain molecule that can be directly translated upon delivery to the cell, a translation process that provides an RNA-dependent RNA polymerase, which subsequently produces antisense and sense transcripts from the delivered RNA. Thus, the delivered RNA results in the production of multiple daughter RNAs. These daughter RNAs, along with collinear subgenomic transcripts, can be self-translated to express the encoded protein in situ; or they can be transcribed to produce additional transcripts with the same sense as the delivered RNA, which, after translation, can express the protein in situ. The final result of this series of transcriptions is an amplification of the number of introduced self-amplifying RNAs, thus making the encoded protein the major polypeptide product of the host cell.

[0300] In one embodiment, the RNA is circular RNA (circRNA), a single-stranded RNA that, unlike linear RNA, forms a covalently closed continuous loop by linking the 3' and 5' ends, which are typically present in RNA molecules. Similar to mRNA, circRNA can be programmed to encode and express proteins. In a specific embodiment, the oligonucleotide (or one strand thereof) of the present invention specifically hybridizes to or is complementary to the target polynucleotide.

[0301] In one embodiment, the RNA is a hairpin-shaped siRNA with a double-stranded region of length equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The length of this double-stranded region can be equal to or less than 200, 100, or 50 nucleotide pairs. In some embodiments, the length of the double-stranded region ranges from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs. The hairpin can have a single-stranded overhang or an unpaired terminal region. In some embodiments, the overhang is 2 to 3 nucleotides long. In some embodiments, the overhang is located on the sense strand side of the hairpin, while in other embodiments, the overhang is located on the antisense strand side of the hairpin.

[0302] In one implementation, the RNA is siRNA. siRNA is a double-stranded RNA, typically 16 to 30 nucleotides in length, that can be associated with a cytoplasmic polyprotein complex called the RNAi-induced silencing complex (RISC). The RISC loaded with siRNA mediates the degradation of homologous mRNA transcripts, thus allowing siRNA to be programmed to knock down protein expression with high specificity. Unlike other antisense technologies, siRNA functions through a natural mechanism that evolved to control gene expression via non-coding RNA.

[0303] The term "single-stranded siRNA compound" as used in this article refers to an siRNA compound consisting of a single molecule. It can contain double-stranded regions formed by intrastrand pairing; for example, it can be or contain hairpin or pan-handle structures. Single-stranded siRNA compounds can be antisense relative to the target molecule.

[0304] The single-stranded siRNA compound can be long enough to enter RISC and participate in RISC-mediated target mRNA cleavage. The single-stranded siRNA compound is at least 14 nucleotides long, and in other embodiments, it is at least 15, 20, 25, 29, 35, 40, or 50 nucleotides long. In some embodiments, it is less than 200, 100, or 60 nucleotides long.

[0305] The term "double-stranded siRNA compound" as used in this article refers to an siRNA compound containing more than one, and in some cases two, strands, where interstrand hybridization can form a double-stranded structural region.

[0306] The antisense strand of a double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. Its length can also be equal to or less than 200, 100, or 50 nucleotides. The length range can be 17 to 25 nucleotides, 19 to 23 nucleotides, and 19 to 21 nucleotides. In this document, the term "antisense strand" refers to the strand of the siRNA compound that is fully complementary to the target molecule (e.g., the target RNA).

[0307] The positive strand length of a double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 25, 29, 40, or 60 nucleotides. Its length can also be equal to or less than 200, 100, or 50 nucleotides. The length range can be 17 to 25 nucleotides, 19 to 23 nucleotides, and 19 to 21 nucleotides.

[0308] The length of the double-stranded portion of a double-stranded siRNA compound can be equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs. Its length can also be equal to or less than 200, 100, or 50 nucleotide pairs.

[0309] The length can range from 15 to 30, 17 to 23, 19 to 23, and 19 to 21 nucleotide pairs.

[0310] In many implementations, the siRNA compound is large enough to be cleaved by endogenous molecules (such as Dicer) to produce smaller siRNA compounds (such as siRNA agents).

[0311] The sense and antisense strands can be selected so that the double-stranded siRNA compound contains single-stranded or unpaired regions at one or both ends of its molecule. Therefore, the double-stranded siRNA compound may contain a sense strand and an antisense strand, which pair to form overhangs, such as one or two 5' or 3' overhangs, or 3' overhangs of 1 to 3 nucleotides. Overhangs can be due to one strand being longer than the other, or due to the misalignment of two strands of equal length. Some embodiments will have at least one 3' overhang. In one embodiment, the siRNA molecule has 3' overhangs at both ends. In some embodiments, the overhangs are 2 nucleotides.

[0312] In some embodiments, the length of the double-stranded region is 15 to 30 nucleotides, or 18, 19, 20, 21, 22, and 23 nucleotides, for example, within the range of the siRNA compounds described above. The length and structure of the siRNA compound can be similar to the product of natural long dsiRNA processed by Dicer. The invention also includes embodiments in which the two strands of the siRNA compound are joined (e.g., covalently joined). Hairpin structures, or other single-stranded structures providing the desired double-stranded region and 3' overhangs, are also included within the scope of the invention.

[0313] The siRNA compounds described herein, including double-stranded and single-stranded siRNA compounds, are capable of mediating the silencing of target RNAs (e.g., mRNAs, such as transcripts of proteins-encoding genes). For convenience, these mRNAs are also referred to as mRNAs to be silenced. These genes are also referred to as target genes. Typically, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, other RNAs besides mRNA, such as tRNA and viral RNA, can also serve as targets.

[0314] In this article, the phrase “mediated RNAi” refers to the ability to silence target RNA in a sequence-specific manner. While it is desirable to be free from theoretical constraints, it is generally believed that such silencing requires an RNAi mechanism or process and a guide RNA, such as a 21- to 23-nucleotide siRNA compound.

[0315] In one embodiment, the siRNA compound is “fully complementary” to the target RNA (e.g., target mRNA), enabling the siRNA compound to silence the production of the protein encoded by the target mRNA. In another embodiment, the siRNA compound is “exactly complementary” to the target RNA; for example, the target RNA and the siRNA compound anneal to form a hybrid consisting entirely of Watson-Crick base pairs in the perfectly complementary region. The “fully complementary” target RNA may contain an inner region (e.g., at least 10 nucleotides) that is perfectly complementary to the target RNA. Furthermore, in some embodiments, the siRNA compound is capable of specifically distinguishing differences in individual nucleotides. In this case, the siRNA compound mediates RNAi only when a perfectly complementary sequence is found in a region of individual nucleotide difference (e.g., within 7 nucleotides of that region).

[0316] In addition to traditional siRNA, dicer substrate siRNA can also serve as a less immunogenic alternative. DsiRNA is 25 to 30 nucleotides in length. After being taken up by cells, it is further cleaved and processed by the Dicer enzyme to convert it into its active form, and then associated with RISC.

[0317] Antisense RNA targeting a target polynucleotide. The term "antisense RNA" or simply "antisense" is intended to include RNA complementary to a target polynucleotide sequence. Antisense RNA is a single-stranded RNA complementary to a specially selected sequence, such as target gene mRNA. Antisense RNA is thought to repress gene expression by binding to complementary mRNA. Binding to target mRNA can prevent translation of the complementary mRNA strand or cause gene expression repression by leading to the degradation of the target mRNA. In a particular embodiment, the antisense RNA contains about 10 to about 50 nucleotides, more preferably about 15 to about 30 nucleotides. The term also covers antisense RNA that is not perfectly complementary to the desired target gene.

[0318] MicroRNAs (miRNAs) are a class of highly conserved small RNA molecules transcribed from DNA in the genomes of plants and animals, but not translated into proteins. Processed miRNAs are single-stranded RNA molecules of 17 to 25 nucleotides (nt) that are integrated into the RNA-induced silencing complex (RISC) and have been identified as key regulators of development, cell proliferation, apoptosis, and differentiation.

[0319] In one implementation, the RNA is transfer RNA (tRNA). A transfer RNA is a linker molecule composed of RNA, typically 76 to 90 nucleotides in length, that acts as a physical link between mRNA and the amino acid sequence of a protein. Transfer RNA performs this function by carrying amino acids to the cell's protein synthesis mechanism, called the ribosome. The trinucleotide codon in messenger RNA (mRNA) is complementary to the trinucleotide anticodon in tRNA, thus enabling the synthesis of mRNA-encoded proteins. Therefore, tRNA is an essential component of the translation process (i.e., the biological process of synthesizing new proteins according to the genetic code).

[0320] In one implementation, the nucleic acid is a single-guide RNA used to guide CRISPR / Cas9-mediated gene editing. This single-guide RNA hybridizes to a target sequence in the cell genome and forms a complex with the Cas9 protein at the target site, thereby initiating single-strand or double-strand breaks.

[0321] In one embodiment, at least one active agent is selected from antisense oligonucleotides, aptamers, ribozymes, immunostimulatory oligonucleotides, decoy oligonucleotides, supermir, miRNA mimics, antimir or miRNA inhibitors, and UI adaptors.

[0322] Antagomir are RNA-like oligonucleotides that carry a variety of modifications for the protection and pharmacological properties of RNAse, such as enhancing tissue and cellular uptake. They differ from normal RNA in, for example, complete 2'-O-methylation of sugars, a phosphate-thiolated backbone, and, for example, a cholesterol moiety at the 3' end.

[0323] Aptamers are nucleic acid or peptide molecules capable of binding to specific target molecules with high affinity and specificity. Successfully prepared DNA or RNA aptamers can bind to a wide variety of entities, from large proteins to small organic molecules. Aptamers can be RNA- or DNA-based and may contain riboswitches. Riboswitches are part of the mRNA molecule that can directly bind to small target molecules, and their binding to the target molecule affects gene activity. Aptamers can be prepared by any known method, including synthetic, recombinant, and purified methods, and can be used alone or in combination with other aptamers specific to the same target. Furthermore, as described in more detail herein, the term "aptamer" specifically includes "secondary aptamers," which contain a shared sequence derived by comparing two or more known aptamers with a given target.

[0324] Ribozymes are RNA molecular complexes with specific catalytic domains that possess endonuclease activity. For example, many ribozymes can accelerate phosphoester transfer reactions with high specificity, typically cleaving only one of several phosphate groups in an oligonucleotide substrate. This specificity is attributed to the fact that the substrate must bind to the ribozyme's internal guide sequence (IGS) via specific base-pairing interactions before the chemical reaction can occur.

[0325] The nucleic acids associated with the lipid particles of the present invention may be immunostimulatory, including immunostimulatory oligonucleotides (ISS; single-stranded or double-stranded) that can induce an immune response when administered to a subject (which may be a mammal or other patient).

[0326] Because transcription factors can recognize their relatively short binding sequences even in the absence of surrounding genomic DNA, short oligonucleotides carrying binding sequences common to specific transcription factors can be used as tools to regulate gene expression in living cells. This strategy involves the intracellular delivery of such "bait oligonucleotides," which are then recognized and bound by target factors. Once the bait oligonucleotides occupy the DNA binding sites of the transcription factors, they prevent the transcription factors from binding to the promoter regions of the target genes.

[0327] Supermir refers to a single-stranded, double-stranded, or partially double-stranded oligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), or a modified form thereof, whose nucleotide sequence is substantially identical to that of miRNA and is antisense relative to its target. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars, and covalently linked nucleotides (backbone), as well as non-natural moieties containing at least one functionally similar portion. Such modified or substituted oligonucleotides are superior to their natural forms because they possess desired properties, such as enhanced cellular uptake, increased affinity for nucleic acid targets, and increased stability in the presence of nucleases.

[0328] miRNA mimics are a class of molecules that can mimic the gene silencing ability of one or more miRNAs. Therefore, the term "microRNA mimic" refers to synthetic non-coding RNAs that can enter the RNAi pathway and regulate gene expression (i.e., the miRNA is not purified from an endogenous miRNA source).

[0329] The terms “antimir,” “microRNA inhibitor,” “miR inhibitor,” or “inhibitor” are synonymous and refer to oligonucleotides or modified oligonucleotides that interfere with the function of a specific miRNA. Typically, such inhibitors are nucleic acids or modified nucleic acids, including oligonucleotides, comprising RNA, modified RNA, DNA, modified DNA, locked nucleic acids (LNA), or any combination thereof. Modifications include 2' modifications and internucleotide modifications (e.g., phosphate thioester modifications), which affect delivery, stability, specificity, intracellular compartmentalization, or potency. Furthermore, miRNA inhibitors may contain conjugates that can affect delivery, intracellular compartmentalization, stability, and / or potency. Inhibitors can take various conformations, including single-stranded, double-stranded (RNA / RNA or RNA / DNA duplexes), and hairpin structures. Typically, microRNA inhibitors contain one or more sequences or portions of sequences that are complementary or partially complementary to the mature strand (one or more) of the target miRNA. Additionally, miRNA inhibitors may contain additional sequences located at the 5' and 3' ends of the inverse complementary sequence of the mature miRNA. These additional sequences can be the reverse complementary sequences of the pri-miRNA from which the mature miRNA is derived, or the additional sequences can be any sequences (containing a mixture of A, G, C, or U).

[0330] The Ul adaptor, which represses the poly A site, is a bifunctional oligonucleotide possessing a "target domain" complementary to the site of the terminal exon of the target gene, and a "Ul domain" capable of binding the Ul small nuclear RNA component in the Ul snRNP. The Ul snRNP is a ribonucleoprotein complex whose primary function is to guide the early steps of spliceosome formation by binding to the exon-intron boundary of the precursor mRNA. Nucleotides 2-11 at the 5' end of the Ul snRNA base-pair with the 5'ss of the precursor mRNA. In one embodiment, the oligonucleotide of the present invention is the Ul adaptor.

[0331] In a preferred embodiment, the at least one active agent is selected from: linear or circular DNA, plasmid DNA (pDNA), self-amplifying RNA (saRNA), chemically modified or unmodified messenger RNA (mRNA), circular RNA (circRNA) containing at least one coding sequence, small hairpin RNA (shRNA), small interfering RNA (siRNA), microRNA (miRNA), dicer substrate RNA, antisense oligonucleotide (ASO), transfer RNA (tRNA), single-guide RNA (sgRNA), or viral RNA (vRNA); and combinations thereof.

[0332] In one embodiment, the at least one active agent is an organic molecule with a molecular weight up to 1000 g / mol, also known as a small molecule in the pharmaceutical field. Preferably, the organic molecule is selected from paclitaxel, doxorubicin, irinotecan, vincristine, and oxaliplatin.

[0333] The compositions according to the invention may further comprise a compound selected from the following: a lipid other than a compound of formula (I), such as an ionizable lipid, cationic lipid, neutral lipid or structural lipid, sterol or sterol derivative; a buffer; a pharmaceutically acceptable salt; a cryoprotectant; or any combination thereof.

[0334] According to the invention, suitable lipids that may further exist and are different from compounds of formula (I) are, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides, or mixtures thereof. Lipids with various acyl chains of different chain lengths and saturations are available or can be isolated or synthesized by known techniques. In one embodiment, lipids containing saturated fatty acids with carbon chain lengths of C10 to C20 are preferred. In one embodiment, lipids containing monounsaturated or diunsaturated fatty acids with carbon chain lengths of C10 to C20 are used. Furthermore, lipids having a mixture of saturated and unsaturated fatty acid chains may also be used. The preferred lipids are 1,2-dioleoyl-sn-glycerol-3-phosphate ethanolamine (DOPE), 1,2-distearate-sn-glycerol-3-phosphate choline (DSPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), dipalmitoylphosphatidylcholine (DPPC), or any related phosphatidylcholine.

[0335] Other suitable lipids consist of sphingomyelin, dihydrosphingomyelin, or phospholipids with other head groups (such as serine and inositol), as well as sterols, especially cholesterol and phytosterols.

[0336] In one embodiment, the other lipid is an ionizable lipid, preferably selected from 1,2-distearyl-3-dimethylammonium-propane, 1,2-dipalmitoyl-3-dimethylammonium-propane, 1,2-dimyristoyl-3-dimethylammonium-propane, 1,2-dioleoyl-3-dimethylammonium-propane, 1,2-dioleoyloxy-3-dimethylaminopropane, (6Z,9Z,28Z,31Z)-heptadecane-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butyrate, and 9-heptadecyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate. N,N-Dimethyl-2,2-di-(9Z,12Z)-9,12-octadecadien-1-yl-1,3-dioxolane-4-ethylamine, [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate).

[0337] In one embodiment, the other lipids are cationic lipids, preferably salts selected from the following: 1,2-di-O-octadecenyl-3-trimethylammonium propane, 1,2-dioleoyl-3-trimethylammonium-propane, N1-[2-((1S)-1-[(3-aminopropyl)amino]-4-[di(3-aminopropyl)amino]butylcarbamoylamino)ethyl]-3,4-di[oleoyloxy]benzamide, N4-cholesterolyl-spermine, 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol, O,O'-di(tetradecanoyl)-N-(α-trimethylammoniumacetyl)diethanolamine, 1,2-dilauroyl-sn-glycerol-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, 1,2-Dipalmitoyl-sn-glycerol-3-ethylphosphocholine, 1,2-distearyl-sn-glycerol-3-ethylphosphocholine, 1,2-dioleoyl-sn-glycerol-3-ethylphosphocholine, 1-palmitoyl-2-oleoyl-sn-glycerol-3-ethylphosphocholine, 1,2-dimyristoyl-sn-glycerol-3-ethylphosphocholine, dimethyl dioctadecylammonium, 1,2-dimyristoyl-3-trimethylammonium-propane, 1,2-dipalmitoyl-3-trimethylammonium-propane, 1,2-stearoyl-3-trimethylammonium-propane, N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)prop-1-amine and 3β-[N-(N',N'-dimethylaminoethane))-carbamoyl]cholesterol. The salt can be any pharmaceutically acceptable salt, preferably a fluoride or chloride salt.

[0338] Other lipids suitable for use in the compositions of the present invention include, but are not limited to, phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysine phosphatidylglycerol, and other anionic modifying groups linked to neutral lipids.

[0339] In one embodiment, the other lipids are selected from phospholipids, aminolipids, and sphingolipids. Representative phospholipids include sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoyl phosphatidylcholine, distearate phosphatidylcholine, or dilinoleoyl phosphatidylcholine. Other phosphorus-deficient compounds, such as sphingolipids, glycosphingolipids, diacylglycerols, and β-acyloxy acids, may also be used. Furthermore, this amphiphilic lipid can be readily mixed with other lipids, such as triglycerides and sterols.

[0340] In one embodiment, the other lipids are selected from polysorbate 80 (also known as Tween 80, IUPAC name 2-[2-[3,4-bis(2-hydroxyethoxy)oxacyclopentan-2-yl]-2-(2-hydroxyethoxy)ethoxy]ethyl octadec-9-enoate), Myrj52 (polyoxyethylene (40) stearate), and Brij S10 (polyoxyethylene (10) stearate), or combinations thereof. These lipids are known stabilizers in the art and may also be present in the compositions of the invention, in addition to the compound (I) of the present invention and any other lipids described herein.

[0341] A cryoprotectant is a reagent that can protect a composition from the adverse effects of freeze-thaw cycles. For example, in this invention, cryoprotectants such as polyols and / or carbohydrates can be added to prevent significant particle aggregation.

[0342] Buffers can also be added. Suitable buffers include phosphates, acetates, citrates, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid, amino acids and other organic compounds; antioxidants, including ascorbic acid and methionine.

[0343] In addition, the composition may contain at least one of the following additives: preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride, benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids. Examples of carboxylic acids include glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates, including monosaccharides, disaccharides, and other sugar compounds such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., zinc-protein complexes), carriers, binders, disintegrants, and immunoadjuvants (e.g., cell-penetrating peptides such as human lactoferrin or fragments thereof, Tat, Ant, Rev, FHV, HSV-1). Proteins VP22, C6, C6M1, PF20, NAP, POD, polyarginine, polylysine, PTD-5, Transportan, MAP, TP10, Pep-7, Azurin p18, Azurin p28, hCT18-32, Bac7, CTP, K5-FGF, HAP-1, 293P-1, KALA, GALA, LAH4-L1, melioflavone peptide, penetratin, EB1, MPG, CATY, Pep4, preferably human lactoferrin or its fragments; fillers (diluents), lubricants, flow aids, tableting aids, colorants, sweeteners, suspending / dispersing agents, film-forming / coating agents, flavoring agents, and printing inks.

[0344] In one embodiment, the composition (preferably lipid nanoparticles) comprises at least one activator in a weight ratio of 1:0.01 to 1:100 with respect to the compound of formula (I).

[0345] In one embodiment, the composition (preferably lipid nanoparticles) comprises at least one other compound selected from: one or more lipids different from the compound of formula (I); a buffer; a pharmaceutically acceptable salt different from the buffer; a cryoprotectant; or any combination thereof. In a preferred embodiment, the composition (preferably lipid nanoparticles) further comprises one or more lipids different from the compound of formula (I), more preferably additional lipids of one, two, or three compounds different from formula (I). In a preferred embodiment, the composition (preferably lipid nanoparticles) consists of a compound of formula (I), at least one activator, and one or more lipids different from the compound of formula (I), more preferably additional lipids of one, two, or three compounds different from formula (I).

[0346] In one embodiment of the composition (preferably lipid nanoparticles), the compound of formula (I) is present at a concentration of about 0.1 to 10 mol% (based on total lipid content). In one embodiment, the compound of formula (I) is present at a concentration greater than 10 mol% (based on total lipid content). In one embodiment, the compound of formula (I) is present at a concentration of 0.5 mol% to 5 mol% (based on total lipid content). In some embodiments, the compound of formula (I) is present at a concentration of 1.5 mol%.

[0347] In one embodiment of the composition (preferably lipid nanoparticles), an additional lipid, different from the compound of formula (I), is contained, and this lipid is a cationic lipid, preferably present in a proportion of about 10 to about 80 mol% (based on total lipid content). In one embodiment, the cationic lipid is present in a proportion of about 50 mol% (based on total lipid content).

[0348] In one embodiment of the composition (preferably lipid nanoparticles), an additional lipid, different from the compound of formula (I), is contained, and this lipid is an ionizable lipid, preferably present in a proportion of about 10 to about 80 mol% (based on total lipid content). In one embodiment, the ionizable lipid is present in a proportion of about 50 mol% (based on total lipid content).

[0349] In one embodiment of the composition (preferably lipid nanoparticles), an additional lipid, different from the compound of formula (I), is contained, and this lipid is a structural lipid, also known as an "auxiliary lipid," carrying a neutral or negative net charge. This structural lipid is preferably present in a proportion of about 10 to about 40 mol% (based on total lipid content). In one embodiment, the structural lipid is present in a proportion of about 10 mol% (based on total lipid content).

[0350] In one embodiment of the composition (preferably lipid nanoparticles), an additional lipid, different from the compound of formula (I), is contained, and this lipid is a sterol, such as cholesterol or phytosterol or a derivative thereof, preferably present in a proportion of about 10 to about 60 mol% (based on total lipid content). In one embodiment, the sterol is present in a proportion of about 35 to about 41 mol% (based on total lipid content). In one embodiment, the sterol is present in a proportion of about 38.5 mol% (based on total lipid content).

[0351] In one embodiment of the composition (preferably lipid nanoparticles), an additional lipid different from the compound of formula (I) is contained, and the lipid is a stabilizer, which is preferably present in a proportion of about 0 to about 10 mol% (based on total lipid content).

[0352] In one embodiment of the composition (preferably lipid nanoparticles), at least one buffer is present at a molar concentration of 0.1 mM to 1000 mM relative to the total volume of the solution in which the composition is dispersed.

[0353] In one embodiment of the composition (preferably lipid nanoparticles), at least one cryoprotectant is present at a mass concentration of 0.1 wt% to 50 wt% relative to the total volume of the solution in which the composition is dispersed.

[0354] The compositions of the present invention can be formulated into solid, semi-solid, liquid, or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols. Typical routes of administration for such pharmaceutical compositions include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal administration. The term "parenteral" as used herein includes subcutaneous injection, intravenous, intramuscular, intradermal, intracisional, or intravenous infusion.

[0355] The compositions of the present invention (preferably pharmaceutical compositions) are formulated to allow that the active ingredient contained therein is bioavailable when administered to a patient. In some embodiments, the composition to be administered to a subject or patient is in the form of one or more dose units; for example, a tablet may be a single dose unit, while a container of the compound of formula (I) of the present invention in aerosol form may contain multiple dose units. Practical methods for preparing such dose forms are known or obvious to those skilled in the art. In some embodiments, the composition to be administered will in any case contain a therapeutically effective amount of the compound of formula (I) of the present invention or a pharmaceutically acceptable salt thereof for treating a target disease or condition in accordance with the teachings of this disclosure.

[0356] The compositions of the present invention (preferably pharmaceutical compositions) may be in solid or liquid form. In one aspect, the carrier is particulate, so the composition may be in tablet or powder form, for example. The carrier may be liquid, and the composition is, for example, an oral syrup, an injection, or an aerosol, which may be used for, for example, inhalation administration.

[0357] When the compositions of the present invention (preferably pharmaceutical compositions) are intended for oral administration, they are preferably in solid or liquid form, wherein semi-solid, semi-liquid, suspension and gel forms are all included in the forms considered herein as solid or liquid.

[0358] As a solid composition for oral administration, the composition (preferably a pharmaceutical composition) can be formulated as a powder, granules, compressed tablets, pills, capsules, chewing gum, or wafer. Such solid compositions typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders, such as carboxymethyl cellulose, ethyl cellulose, microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch, lactose, or dextrin; disintegrants, such as alginate, sodium alginate, Primogel, corn starch, etc.; lubricants, such as magnesium stearate or Sterotex; flow aids, such as colloidal silica; sweeteners, such as sucrose or saccharin; flavoring agents, such as peppermint, methyl salicylate, or orange flavoring; and coloring agents. When the pharmaceutical composition of some embodiments is in capsule form, such as gelatin capsules, in addition to the materials of the types described above, it may also contain a liquid carrier, such as polyethylene glycol or oil.

[0359] The compositions of the present invention (preferably pharmaceutical compositions) may be in liquid form, such as elixirs, syrups, solutions, emulsions, or suspensions. For example, the liquid may be used for oral administration or delivery by injection. When used for oral administration, the preferred compositions, in addition to the compound of formula (I), contain one or more sweeteners, preservatives, pigments / coloring agents, and flavor enhancers. In compositions intended for injection, one or more surfactants, preservatives, wetting agents, dispersants, suspending agents, buffers, stabilizers, and isotonic agents may be included.

[0360] The liquid compositions (preferably liquid pharmaceutical compositions) of the present invention, whether in solution, suspension, or other similar form, may include one or more of the following adjuvants: sterile diluents, such as water for injection, physiological saline (preferably physiological saline), Ringer's solution, isotonic sodium chloride solution, fixative oils (e.g., synthetic monoglycerides or diglycerides that can be used as solvents or suspension media), polyethylene glycol, glycerol, propylene glycol, or other solvents; antibacterial agents, such as benzyl alcohol or methylparaben; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid; buffers, such as acetate, citrate, or phosphate; and agents for adjusting tension, such as sodium chloride or glucose; and agents used as cryoprotectants, such as sucrose or trehalose. The injectable composition may be packaged in ampoules made of glass or plastic, disposable syringes, or multi-dose vials. Physiological saline is a preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

[0361] The compositions of the present invention (preferably pharmaceutical compositions) can be used for topical application, wherein the carrier may suitably comprise a solution, emulsion, ointment, or gel matrix. For example, the matrix may comprise one or more of the following: petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents (such as water and alcohol), and emulsifiers and stabilizers. Thickeners may be present in pharmaceutical compositions for topical application. If intended for transdermal application, the composition may comprise a transdermal patch or iontophoresis device.

[0362] The compositions of the present invention (preferably pharmaceutical compositions) can be used for rectal administration, for example, in the form of suppositories that dissolve and release the drug within the rectum. Compositions for rectal administration may contain an oily matrix as a suitable, non-irritating excipient. Such matrices include, but are not limited to, lanolin, cocoa butter, and polyethylene glycol.

[0363] The compositions of the present invention (preferably pharmaceutical compositions) may contain a variety of materials for altering the physical form of solid or liquid dosage units. For example, the composition may contain a material that forms a coating around the active ingredient. The material forming the coating is typically inert, and may be selected from sugars, shellac, and other enteric coating agents.

[0364] The compositions of the present invention (preferably pharmaceutical compositions) may consist of dosage units that can be administered as an aerosol. The term "aerosol" is used to refer to a variety of systems, ranging from colloidal systems to systems consisting of pressurized packaging. Delivery may be made by liquefied or compressed gas or by a suitable pump system for dispensing the active ingredient. Aerosols of compounds of formula (I) of the present invention may be delivered in single-phase, two-phase, or three-phase systems to deliver the active ingredient. The delivery of the aerosol includes necessary containers, activators, valves, sub-containers, etc., which together constitute an assembly.

[0365] In a preferred embodiment, the composition is a lipid nanoparticle. In a specific embodiment, the active agent is encapsulated within the aqueous interior of the lipid nanoparticle. In other embodiments, the active agent is present within one or more lipid layers of the lipid nanoparticle. In other embodiments, the active agent binds to the external or internal lipid surface of the lipid nanoparticle. Lipid nanoparticles include, but are not limited to, liposomes. As used herein, a liposome is a structure having a lipid-containing membrane encapsulating an aqueous interior. Liposomes may have one or more lipid membranes. Liposomes may be monolayered (referred to as a unilamellar) or multilayered (referred to as a multilamellar). When complexed with nucleic acids, lipid particles may also be liposome complexes consisting of a cationic lipid bilayer sandwiched between DNA layers.

[0366] The lipid nanoparticles of the present invention can be formulated into pharmaceutical compositions, which may, for example, also contain pharmaceutically acceptable diluents, excipients or carriers, such as physiological saline or phosphate buffer, selected according to the route of administration and standard pharmaceutical practice.

[0367] In some embodiments, the lipid nanoparticles of the present invention are prepared according to standard techniques and further comprise a pharmaceutically acceptable carrier. Typically, physiological saline is used as a pharmaceutically acceptable carrier. Other suitable carriers include, for example, water, buffered water, 0.9% physiological saline, 0.3% glycine, etc.; and glycoproteins, such as albumin, lipoproteins, globulins, etc., for enhancing stability. In compositions comprising physiological saline or other salt-containing carriers, the carrier is preferably added after lipid nanoparticle formation. Therefore, after lipid nanoparticle formation, the composition can be diluted with a pharmaceutically acceptable carrier, such as physiological saline.

[0368] The resulting pharmaceutical formulation can be sterilized using conventional, well-known sterilization techniques. The aqueous solution can then be packaged for use, or filtered and lyophilized under aseptic conditions. The lyophilized formulation is then combined with the sterile aqueous solution prior to administration. As needed, the composition may contain pharmaceutically acceptable excipients to approximate physiological conditions; such as pH adjusters and buffers, osmotic pressure regulators, etc., for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. Furthermore, the lipid suspension may contain lipid protectants to protect lipids from damage caused by free radicals and lipid peroxidation during storage. Lipophilic free radical scavengers, such as α-tocopherol, and water-soluble iron-specific chelating agents, such as ferrioxamine, are suitable.

[0369] The term "lipid nanoparticle" refers to a particle with at least one dimension in the nanometer range (e.g., 1-1,000 nm) and comprising one or more compounds of formula (I). In some embodiments, lipid nanoparticles comprising at least one compound of formula (I) are included in formulations that can be used to deliver therapeutic agents (e.g., nucleic acids (such as mRNA)) to target sites (e.g., cells, tissues, organs, tumors, etc.). In some embodiments, lipid nanoparticles comprise both a compound of formula (I) and a nucleic acid. In some embodiments, the therapeutic agent (e.g., nucleic acid) may be encapsulated in the lipid portion of the lipid nanoparticle or in an aqueous space encapsulated by some or all of the lipid portion of the lipid nanoparticle, thereby protecting it from enzymatic degradation or other adverse effects caused by mechanisms of the host organism or cells (e.g., adverse immune responses).

[0370] In various embodiments, the average diameter of the lipid nanoparticles is about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 nm to about 90 nm, about 80 nm to about 90 nm, about 70 nm to about 80 nm, or about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm, which is determined by dynamic light scattering, preferably according to ISO. The procedure was performed according to standard 22412:2017, in which the sample was diluted 1:10 in RNase-free water, corresponding to an RNA concentration of 5 ng / µL. The Malvern Zetasizer NanoZS was preferably used for the measurements.

[0371] Certain administration techniques can achieve systemic delivery of some active ingredients, but not all. Systemic delivery refers to the exposure of an effective amount (preferably a therapeutic amount) of the active ingredient to a large portion of the body. Systemic delivery of lipid nanoparticles can be achieved by any method known in the art, including, for example, intravenous, intra-arterial, subcutaneous, and intraperitoneal delivery. In some embodiments, systemic delivery of lipid nanoparticles is via intravenous delivery.

[0372] As used in this article, "local delivery" refers to the direct delivery of an active ingredient to a target site within a living organism. For example, drugs can be delivered locally to disease sites (such as tumors), other target sites (such as inflammatory sites), or target organs (such as the liver, heart, pancreas, and kidneys) via direct injection. Local delivery can also include topical or local injection techniques, such as intramuscular, subcutaneous, or intradermal injection. Local delivery does not preclude systemic pharmacological effects.

[0373] The compositions of the present invention may also be administered simultaneously, before, or after one or more other active agents. Such combination therapies include: administration of a single pharmaceutical dosage form of the composition of the present invention and one or more additional active agents; and administration of the composition of the present invention and each active agent in their own pharmaceutical dosage form. For example, the compositions of the present invention and other active agents may be administered to a patient as a single oral dosage composition (e.g., tablets or capsules), or each agent may be administered in an independent oral dosage formulation. If an independent dosage formulation is used, the compound of formula (I) of the present invention and one or more additional active agents may be administered substantially simultaneously (i.e., concurrently) or independently at staggered times (i.e., sequentially); combination therapies should be understood to include all of the above-described options.

[0374] The compositions of the present invention (preferably pharmaceutical compositions) can be prepared using methods known in the pharmaceutical industry. For example, pharmaceutical compositions intended for injection can be prepared by mixing the lipid nanoparticles of the present invention with sterile distilled water or other carriers to form a dispersion. Surfactants may be added to promote the formation of homogeneous solutions or suspensions. Surfactants are compounds that interact non-covalently with the compounds disclosed herein, thereby promoting the dissolution of the compounds of the present invention in aqueous delivery systems or the formation of homogeneous suspensions.

[0375] The compositions of the present invention are administered in a therapeutically effective amount, which will vary depending on a variety of factors, including the activity of the particular therapeutic agent used; the metabolic stability and duration of action of the therapeutic agent; the patient's age, weight, general health condition, sex, and diet; the method and timing of administration; the rate of excretion; the combination of drugs; the severity of the particular condition or symptom; and the subject receiving treatment.

[0376] In one preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating illness in humans. In another preferred embodiment, the composition of the present invention is a pharmaceutical composition for treating illness in mammals.

[0377] As used herein, the terms “optional,” “optionally,” and “may” indicate that the described feature may or may not be present. Whenever the terms “optional,” “optionally,” and “may” are used, the invention specifically refers to both possibilities, namely, the presence or absence of the corresponding feature. For example, in the context of this invention, the term “polypeptide comprising proline, alanine, and optionally serine” may refer to a polypeptide composed of proline and alanine, or may refer to a polypeptide composed of proline, alanine, and serine.

[0378] As used herein, unless otherwise expressly stated or the context otherwise requires, the terms “a,” “an,” and “the” are used interchangeably with “one or more” and “at least one.” Thus, for example, a composition comprising “a” conjugate of the present invention can be interpreted as a composition comprising “one or more” conjugates of the present invention.

[0379] As used herein, the term "about" preferably refers to ±10% of the indicated value, more preferably ±5% of the indicated value, and especially to the precise indicated value. If the term "about" is used to refer to the endpoints of a range, it preferably refers to the range from -10% of the lower endpoint to +10% of the upper endpoint of the indicated value, more preferably from -5% of the lower endpoint to +5% of the upper endpoint, and even more preferably the range defined by the precise values ​​of the lower and upper endpoints. If the term "about" is used to refer to the endpoints of an open range, it preferably refers to the corresponding range starting from -10% of the lower endpoint or +10% of the upper endpoint, more preferably from -5% of the lower endpoint or +5% of the upper endpoint, and even more preferably the open range defined by the precise values ​​of the corresponding endpoints. If the term "about" is used to refer to a parameter quantified in integer form (e.g., the number of amino acid residues in a protein), the value corresponding to ±10% or ±5% of the indicated value should be rounded to the nearest integer (using the "round up" rounding rule).

[0380] As used herein, the terms “comprising” and “containing”, unless otherwise expressly stated or the context otherwise requires, mean “especially containing,” that is, “containing, among other optional elements…”. Furthermore, the term also includes the narrower meanings of “consistently composed of…” and “composed of…”. For example, the term “A contains B and C” means “A especially contains B and C,” where A may contain other optional elements (e.g., “A contains B, C, and D” is also included), but the term also includes the meanings of “A is essentially composed of B and C” and “A is composed of B and C” (i.e., A does not contain any other components besides B and C).

[0381] Unless otherwise expressly stated, the term “produce” as used herein may be used interchangeably with the term “prepare”.

[0382] The term “method” refers to the manner, means, techniques and procedures for accomplishing a given task, including but not limited to those known to practitioners in the fields of chemistry, biology and biophysics, or those that can be easily developed from known manner, means, techniques and procedures.

[0383] For example, “treatment” for a condition or disease can cause the progression of the condition or disease (e.g., worsening of symptoms) to stop or the progression of the condition or disease to slow down (if the cessation of progression is only temporary). “Treatment” for a condition or disease can also cause partial remission (e.g., symptom relief) or complete remission (e.g., symptom disappearance) in a patient / subject suffering from said condition or disease. Therefore, “treatment” for a condition or disease can also refer to a reduction in the condition or disease, for example, this can cause the progression of the condition or disease to stop or slow down. Relapse may occur after such partial or complete remission. It should be understood that the response of a subject / patient to treatment can be diverse (e.g., the exemplary responses described above). Treatment for a condition or disease can particularly include curative treatment (preferably resulting in complete remission and ultimately curing the condition or disease) and palliative treatment (including symptom relief).

[0384] As used herein, the term "prevention" refers to a condition or disease, which is well known in the art. For example, patients / subjects suspected of being susceptible to a certain condition or disease can particularly benefit from prevention of that condition or disease. The subject / patient may have a susceptibility or predisposition to a certain condition or disease, including but not limited to a genetic predisposition. This predisposition can be determined by standard methods or assays, such as using genetic markers, phenotypic indicators, or biomarkers. It should be understood that the condition or disease to be prevented according to the present invention has not yet been diagnosed in the patient / subject, or cannot be diagnosed (e.g., the patient / subject does not exhibit any clinical or pathological symptoms). Therefore, the term "prevention" includes using the conjugates of the present invention before an attending physician diagnoses or determines any clinical and / or pathological symptoms, or before being able to diagnose or determine any clinical and / or pathological symptoms.

[0385] In the context of this invention, the groups (A, B, C, and D) of the compound of formula (I) may be referred to as "A", "B", "C", and "D" respectively, both above and below, and may also be referred to as "group A", "group B", "group C", and "group D" respectively. Furthermore, in the context of the compound of formula (I), the terms "group" and "each group" may be used interchangeably with the terms "part" and "each part". Accordingly, the groups (A, B, C, and D) of the compound of formula (I) may also be referred to as "part A", "part B", "part C", and "part D" respectively, both above and below. Similarly, compounds ABCD, ACD, LBCD, and LCD may be referred to as "ABCD", "ACD", "LBCD", and "CD" respectively, and may also be referred to as "compound ABCD", "compound ACD", "compound LBCD", and "compound LCD" respectively. Furthermore, compounds ABCD and ACD may be referred to individually or collectively as "compound of formula (I)".

[0386] It should be understood that the present invention specifically relates to combinations of each feature and embodiment described herein, including any combination of general features / exemplifications and / or preferred features / exemplifications. In particular, the present invention specifically refers to combinations (including general and / or preferred meanings) of the various groups and variables contained in the P / A peptides and conjugates according to the present invention.

[0387] This specification references several documents, including patents, patent applications, and scientific literature. While the disclosures in these documents are not considered to be relevant to the patentability of this invention, their entire contents are incorporated herein by reference. More specifically, all cited documents are incorporated herein by reference as if each individual document were specifically and individually indicated to be incorporated herein by reference.

[0388] References to any existing publications (or information derived from such publications) in this specification shall not be construed as, nor constitute an acknowledgment, endorsement, or any form of implication that such existing publications (or information derived from such publications) are of general common knowledge in the technical field covered by this specification.

[0389] The following table (Table 1) also lists the non-limiting amino acid sequences disclosed herein. However, these sequences are also included in the sequence list in the appendix, which is a specific part of this disclosure and the specification of this invention. In the following table, all amino acid sequences are represented by single-letter codes. Furthermore, the letter "X" at position 1 indicates an N-terminal pyroglutamic acid ("Pga" or "pyrrolidone carboxylic acid"; used synonymously herein) protecting group, and the letter "X" at positions 22 or 42 indicates a C-terminal ε-aminocaproic acid ("Ahx" or "acp"; used synonymously herein) residue (linker).

[0390] Table 1: Non-restrictive amino acid sequences disclosed in this paper

[0391]

[0392]

[0393] The present invention will be further described with reference to the following non-limiting drawings. The drawings show:

[0394] Figure 1: Reaction scheme for coupling PAS-peptide with di(tetradecyl)amine. (In the case of a nonnucleophilic base...) N,N In the presence of diisopropylethylamine (DIPEA, Hünig base), and using a mixture of dichloromethane (DCM) and methanol as a solvent, N-terminal protected PAS-peptides (e.g., Pga-PAS40) are activated at their C-terminus with 2-(1H-benzotriazole-1-yl)-N,N,N′,N′-tetramethylurea tetrafluoroborate (TBTU). The resulting peptide-based hydroxybenzotriazole active ester then undergoes a selective reaction with a single amino group of di(tetradecyl)amine, releasing free 1-hydroxybenzotriazole (HOBt).

[0395] Figure 2: Exemplary chromatogram of Pga-PAS40-di(tetradecyl)amine purified by RP-HPLC. The eluent contains unreacted starting material and byproducts, while Pga-PAS40-di(tetradecyl)amine exhibits a broad single peak during DCM gradient elution.

[0396] Figure 3: Characterization of Pga-PAS20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0397] Figure 4: Characterization of Pga-PAS40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0398] Figure 5: Characterization of Pga-PAS100-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0399] Figure 6: Characterization of Pga-P / A20-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0400] Figure 7: Characterization of Pga-P / A40-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0401] Figure 8: Characterization of Pga-PAS100-K-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0402] Figure 9: Characterization of RNA encapsulation efficiency of PEG2k-LNP, P / A-LNP, and PAS-LNP by agarose gel electrophoresis. LNPs were prepared as described in Example 3, loaded into wells of a 1% agarose gel, and electrophoresed using a Thermo Fisher Scientific E-Gel Power Snap system as described in Example 6. Lane M shows the Ribo Ruler High Range RNAladder, lane 1 shows 20 µl of Flux mRNA as a positive control, and lanes 3 to 7 show entries g to l from Example 3, respectively. Only one clear band was observed in lane 1, corresponding to Flux mRNA. Therefore, all tested LNPs (entries g to l from Example 3) effectively encapsulated the encapsulated mRNA.

[0403] Figure 10: Transfection efficiency of PEG2k-LNP, P / A-LNP, and PAS-LNP characterized by luciferase assay. LNPs were prepared as described in Example 3 and transfected into HeLa, Jurkat, A549, HepG2, and C2C12 cell lines according to the method in Example 8. jetMessenger and jetPEI were used as positive controls, and untreated cells were used as negative controls. Transfection efficiency was determined one day after transfection using a Luciferase Assay System (Promega GmbH, Walldorf, Germany). The luminescent signal was quantified using an Infinite 200 PRO multi-mode microplate reader. Although transfection efficiency varied among cell lines, all tested LNPs showed high transfection efficiency, with some LNPs even exceeding the transformation efficiency of the positive controls (jetMessenger and jetPEI).

[0404] Figure 11: Characterization of Pga-PAS40-di(decyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0405] Figure 12: Characterization of Pga-PAS40-di(dodecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0406] Figure 13: Characterization of Pga-PAS40-di(hexadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0407] Figure 14: Characterization of Pga-PAS40-di(octadecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0408] Figure 15: Reaction scheme for coupling C-terminal cysteine-containing PAS-peptides with di(acyl)amines. Using a mixture of dichloromethane (DCM) and methanol as solvent, the N-terminally protected PAS-Cys peptides (e.g., Pga-PAS20C) are activated via thiol groups using the bispecific cross-linking agent succinimide-trans-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC). In the presence of non-nucleophilic bases... N,N In the presence of diisopropylethylamine (DIPEA, Hünig base), the SMCC active ester of the generated peptide selectively reacts with a single secondary amino group of di(tetradecyl)amine, releasing free N-hydroxysuccinimide (NHS) (left-hand reaction scheme). Alternatively, the bispecific crosslinking agent may first react with a single secondary amino group of di(tetradecyl)amine, followed by a reaction of the resulting intermediate with the thiol group of the PAS-Cys polypeptide (right-hand reaction scheme). The resulting compound comprises a PAS-polypeptide linked to a di(acyl)amine via a linker (i.e., trans-4-(N-maleimide methyl)cyclohexane-1-carboxylate (MCC)).

[0409] Figure 16: Characterization of Pga-PAS20C-MCC-di(tetradecyl)amine. Chemical formula and calculated molecular weight (top), analytical RP-HPLC (middle) and deconvolution ESI-MS mass spectra (bottom).

[0410] Example

[0411] Some embodiments of the invention will now be described with reference to the following examples, which are for illustrative purposes only and are not intended to limit the general scope of the above description.

[0412] Example 1: Di(tetradecyl)amine is coupled to P / A- and PAS-peptides via its single carboxyl group.

[0413] Prepare 30 mM solutions of the N-terminally blocked peptides Pga-P / A20-Ahx (P / A20; SEQ ID No.: 1, Almac Group, London, UK), Pga-P / A40-Ahx (P / A40; SEQ ID No.: 2, Almac), Pga-PAS20 (PAS20; SEQ ID No.: 3, XL-protein, Freising, Germany), Pga-PAS40 (PAS40; SEQ ID No.: 4, XL-protein), or Pga-PAS100 (PAS100; SEQ ID No.: 5, XL-protein) in a dichloromethane (DCM) / methanol mixture (5:1). 1.5 mol equivalents of di(tetradecyl)amine (Ambeed, Arlington Hts, IL, USA) were dissolved in solid form in a P / A- or PAS-peptide solution, followed by the addition of 2 mol equivalents of DIPEA base (N,N-diisopropylethylamine; Merck) liquid and 1.5 mol equivalents of TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate; 400 mM dimethylformamide (DMF; Carl Roth, Karlsruhe, Germany) stock solution, and the mixture was incubated at 25°C for 1 hour under an argon atmosphere. Figure 1 Alternatively, the P / A- or PAS- peptide can be dissolved separately in methanol, followed by the addition of a DCM solution of di(tetradecyl)amine. To precipitate unreacted di(tetradecyl)amine, 8 volumes of a 10:1 methanol / water mixture are added, and the mixture is incubated on ice for 20 min. The precipitate is removed by filtration (Acrodisc 1 µm glass fiber syringe filter, PALL, Port Washington, NY, USA), and the coupling product is purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a VP 250 / 10 Nucleodur C18 Gravity column (Macherey-Nagel, Düren, Germany). For 20-mer and 40-mer P / A- or PAS-peptide-conjugated lipids, the mobile phase was 85% (v / v) MeOH, 10% (v / v) H2O, 5% (v / v) DCM, and 0.1% (v / v) formic acid. The conjugated products were eluted using a 0-100% gradient of MeOH / DCM (1:1). Figure 2 For Pga-PAS100-di(tetradecyl)amine, the mobile phase consisted of 70% MeOH, 30% H2O and 0.1% formic acid. The elution process employed gradient elution with a gradient range of 0% to 100%, wherein the eluent composition was 90% (v / v) MeOH, 10% (v / v) DCM and 0.1% (v / v) formic acid.

[0414] The elution fractions were combined, concentrated using a SpeedVac concentrator, diluted 1:10 with H2O, and then lyophilized. Analytical reversed-phase HPLC was performed using a ResourceRPC polystyrene / divinylbenzene column (1 ml, Cytiva) and the same mobile phase used in the preparative RP-HPLC for P / A- or PAS-peptide-di(tetradecyl)amine described above. Additionally, 10–50 µg of P / A- or PAS-di(tetradecyl)amine was dissolved in 50% acetonitrile aqueous solution containing 0.1% formic acid and analyzed by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode using a maXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany).

[0415] Based on RP-HPLC and ESI-MS data, the P / A- and PAS- compounds clearly exhibit homogeneity; this is evident from... Figures 3 to 7 This phenomenon is clearly confirmed by the presence of a single peak in all the analytical RP-HPLC and ESI-MS analyses shown.

[0416] Example 2: Di(tetradecyl)amine is coupled to the Pga-PAS100-K peptide via the ε-amino group of its C-terminal lysine residue.

[0417] The N-terminally protected Pga-PAS100-K peptide (PAS100-K; SEQ ID No.: 6) was dissolved in 100 mM sodium bicarbonate buffer (pH 8.4) and incubated with iodoacetic acid N-hydroxysuccinimide ester (CAS 39028-27-8, Apollo Scientific, Bredbury, UK) at 25°C for 1 h to iodoacetylate the ε-amino group of its C-terminal lysine side chain. After dialyzing with ultrapure water and lyophilization, the activated PAS100-K peptide was dissolved in MeOH / DCM (1:7). For alkylation, 10 molar amounts of di(tetradecyl)amine and 2 molar amounts of DIPEA were added, and the coupling reaction was carried out at 25°C for 24 h. Eight volume equivalents of a H₂O / MeOH (1:10) mixture were added, and the mixture was incubated on ice for 20 min. Unreacted di(alkyl)amine precipitate was removed by filtration (Acrodisc glass fiber 1 µm syringe filter, PALL). The coupling product was purified by RP-HPLC using a VP250 / 10 Nucleodur C18 Gravity column (Macherey-Nagel, Düren, Germany). The mobile phase was a mixture of 70% (v / v) MeOH, 30% (v / v) H₂O, and 0.1% (v / v) formic acid. The alkylated PAS-peptide was eluted from the C18 column using a 0-100% gradient of 90% MeOH (v / v), 10% (v / v) DCM, and 0.1% formic acid. After evaporating the DCM under vacuum, the eluent was diluted with H₂O and lyophilized. Based on RP-HPLC and ESI-MS analysis, the Pga-PAS100-K compound exhibits significant homogeneity, such as... Figure 8 As shown, only a single peak appeared in both RP-HPLC and ESI-MS analyses.

[0418] Example 3: Preparation of LNP

[0419] CleanCap® Fluc mRNA is sourced from TriLink BioTechnologies (San Diego, CA, USA).

[0420] D-Lin-MC3-DMA was obtained from MedCHemExpress (Monmouth Junction, NJ, USA), cholesterol from Merck KGaA, and DSPC from NOF (White Plains, NY, USA).

[0421] All cell lines were provided by the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ, Braunschweig, Germany).

[0422] An aqueous solution (or aqueous phase) containing 0.133 g / L FLuc mRNA and 11 mM acetic acid was mixed with an ethanolic solution (or ethanolic phase) containing 9.43 mM total lipids (50 mol% DLin-MC3-DMA, 38.5 mol% cholesterol, 10 mol% DSPC, 1.5 mol% PEG2k-compound, P / A-compound, or PAS-compound) at a volume ratio of 3:1 by pipetting. The crude colloidal LNP dispersion was dialyzed in phosphate-buffered saline (PBS) for 3 h (3x buffer exchange). The purified colloidal LNP dispersion (i.e., LNP or purified colloidal dispersion) was stored at 4°C until subsequent use.

[0423] The purified LNP remained stable at 4°C for at least 4 days. No turbidity or precipitation was observed in any of the tested LNPs.

[0424] Example 4: Determination of particle size (Z-mean), polydispersity index (PDI), and zeta potential of PEG2k-LNP, P / A-LNP, and PAS-LNP

[0425] The results are shown in Table 2. Measurements were performed using a Zetasizer NanoZS instrument from Malvern Instruments GmbH (Herrenberg, Germany). A DTS1070 transparent disposable folded capillary cell from Malvern Panalytical GmbH (Kassel, Germany) was used. For particle size measurements, samples were diluted 1:10 with RNase-free water, corresponding to an RNA concentration of 5 ng / µl. For Zeta potential measurements, purified (after dialysis) and crude (before dialysis) colloidal LNP dispersions from Example 3 were diluted 1:30 with RNase-free water, corresponding to an RNA concentration of 1.67 ng / µl. Z-mean (i.e., average particle size), polydispersity index (PDI; i.e., the width of the fitted Gaussian distribution), and average Zeta potential were measured before and after dialysis and calculated based on data from at least 10 runs.

[0426] Z-average measurements confirmed that the particle size of LNPs was influenced by the molecular weights of the corresponding P / A- and PAS- compounds. The P / A20- and PAS20- compounds had similar molecular weights to the PEG2k- compound. However, surprisingly, the corresponding LNP particle sizes were approximately 10% smaller. Similarly, the P / A40- and PAS40- compounds had molecular weights approximately 55% higher than the PEG2k- compound, but the corresponding LNP particle sizes were comparable. In summary, these results indicate that the P / A- and PAS- compounds unexpectedly formed more compact LNPs.

[0427] The polydispersity indices of PEG2k-LNP, P / A-LNP and PAS-LNP are comparable, therefore, it is clear that the homogeneity of P / A-LNP and PAS-LNP is similar to that of PEG2k-LNP.

[0428] Before dialysis, the average zeta potential of all LNPs tested was comparable, but after dialysis, the zeta potential of PEG2k-LNP was significantly higher than that of all P / A-LNP and PAS-LNPs tested. This indicates that P / A-LNP and PAS-LNP have a stronger ability to shield the charged film of LNPs compared to PEG2k-LNP. This result is particularly surprising considering the smaller particle size of P / A-LNP and PAS-LNP.

[0429] Table 2: Particle size, PDI, and Zeta potential of PEG2k-LNP, P / A-LNP, and PAS-LNP

[0430]

[0431] Example 5: MTS cell viability assay using PEG2k-compound, P / A-compound, and PAS-compound, as well as PEG2k-LNP, P / A-LNP, and PAS-LNP.

[0432] HeLa cells were detected using the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazole (MTS) assay. One day prior to transfection, 10,000 cells were seeded into each well of a 96-well plate with 100 µl of the appropriate culture medium (containing 10% fetal bovine serum (FBS) and 30 µg / ml gentamicin) per well, and cultured at 37°C and 5% CO2 for 24 h. The next day, the old culture medium was removed, and 90 µl of fresh culture medium was added to the cells. For the P / A-compound and PAS-compound assays, the compounds from Examples 1 and 2 were dissolved in sterile water to achieve the final concentrations shown in Table 3, and the sample volume was adjusted to 10 µl. For the LNP assay, the purified colloidal LNP dispersion from Example 3 was diluted with RNase-free water to adjust the mRNA concentration to 5 to 20 ng / µl. 10 µl of the correspondingly diluted sample was added to the cells, so that the amount of mRNA in each well was equal to 50 to 200 ng, with a total volume of 100 µl. The results are shown in Table 4. The cells were cultured at 37°C and 5% CO2 for another 24 h. On day 3, cell viability was measured using the CellTiter 96 AQueous non-radioactive cell proliferation assay kit (MTS) according to the manufacturer's instructions (Promega GmbH). The absorbance signal at 490 nm was quantitatively analyzed using a multi-well microplate reader (Infinite 200PRO, Tecan, Männedorf, Switzerland).

[0433] In MTS assays, only a slight decrease in cell viability was observed when using PEG2k-, P / A-, or PAS- compounds at concentrations of 0.01 µg / ml, 0.1 µg / ml, or 1 µg / ml. However, when using PEG2k compounds at concentrations of 10 µg / ml or 100 µg / ml, cell viability decreased to (or below) 0%. In contrast, at a concentration of 10 µg / ml, P / A- and PAS- compounds showed significantly higher cell viability than PEG2k compounds, with all tested P / A- and PAS- compounds resulting in cell viability above 69%. Therefore, surprisingly, when applied to HeLa cells at a concentration of 10 µg / ml in MTS assays, P / A- and PAS- compounds exhibited significantly lower toxicity to human cells than PEG2k- compounds. Thus, it is evident that the inventors have unexpectedly discovered that P / A- and PAS- compounds are more suitable as pharmaceutical components compared to PEG2k compounds.

[0434] At any test dose, all tests

[0435] PEG2k-LNP, P / A-LNP, or PAS-LNP had almost no effect on the viability of HeLa cells.

[0436] Therefore, P / A-LNP and PAS-LNP are clearly non-toxic to human cells.

[0437] Table 3: MTS cell viability assay results for PEG2k-compound, P / A-compound, and PAS-compound

[0438]

[0439] Table 4: MTS cell viability assay results for PEG2k-LNP, P / A-LNP, and PAS-LNP

[0440]

[0441] Example 6: PEG2k-LNP, P / A-LNP, and PAS-LNP were characterized by agarose gel electrophoresis (AGE).

[0442] RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3 was characterized by agarose gel electrophoresis. This assay was performed using a Thermo Fisher Scientific E-Gel Power Snap electrophoresis system. Samples were evaluated using 20 µL of 1% agarose gel per well. Results are summarized in... Figure 9 The swimmers' pool consisted of several lanes: lane "M" contained the RiboRuler High Range RNA Ladder from ThermoFisher Scientific; lane 1 contained mFluc mRNA (negative control); lane 2 contained mRFLuc-MC3 LNP-PEG2k-N,N-di(tetradecyl)acetamide (mRFluc-MC3 LNP1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000); lane 3 contained mRFLuc-MC3 LNP-PEG2k-N,N-di(tetradecyl)acetamide; lane 4 contained mRFLuc-MC3 LNP-Pga-P / A20-Ahx-di(tetradecyl)amine; lane 5 contained mRFLuc-MC3 LNP-Pga-P / A40-Ahx-di(tetradecyl)amine; lane 6 contained mRFLuc-MC3 LNP-Pga-PAS20-di(tetradecyl)amine; and lane 7 contained mRFLuc-MC3 LNP-Pga-PAS20-di(tetradecyl)amine. LNP-Pga-PAS40-Di(tetradecyl)amine, lane 8 contains mRFLuc-MC3 LNP-Pga-PAS100-K-Di(tetradecyl)amine.

[0443] Agarose gel electrophoresis results showed that all tested LNP compositions completely encapsulated mRNA (within the SYBRSafe staining detection range). Therefore, based on these results, LNPs prepared with P / A-compounds or PAS-compounds had similar mRNA encapsulation rates to the reference sample prepared with conventional PEG2k-compounds.

[0444] Example 7: PEG2k-LNP, P / A-LNP, and PAS-LNP were characterized by RiboGreen assay.

[0445] The RiboGreen assay further characterized the RNA encapsulation efficiency of the purified colloidal LNP dispersions from Example 3. The Thermo Fischer Quant-iT RiboGreen RNA Assay Kit was used. Procedure was performed according to the manufacturer's instructions with minor adjustments. Samples were diluted to a theoretical RNA concentration of 0.4 µg / ml using Tris-EDTA (TE) buffer or Triton buffer and added to 96-well plates in 100 µl volumes. To dissolve the LNPs in Triton buffer, the plates were incubated at 37°C with 5% CO2 for 10 minutes. 100 µl of dye solution was added to each well and thoroughly mixed by pipetting. Fluorescence signals were measured at excitation and emission wavelengths of 480 nm and 520 nm, respectively, using an Infinite 200 PRO microplate reader. All samples and standards were measured repeatedly.

[0446] As shown in Table 5, all tested P / A-LNP and PAS-LNP showed higher RNA encapsulation rates compared to PEG2k-LNP. Surprisingly, the P / A-compound and PAS-compound were more effective at shielding LNP membranes than PEG2k-LNP.

[0447] Table 5: RiboGreen assay results for PEG2k-LNP, P / A-LNP, and PAS-LNP

[0448]

[0449] Example 8: The transfection efficiency of PEG2k-LNP, P / A-LNP, and PAS-LNP was determined by luciferase assay.

[0450] Luciferase assays were performed using various immortalized cell lines (HeLa, Jurkat, C2C12, HepG2, A549). The cell lines were cultured under standard cell culture conditions.

[0451] Transfection and luciferase assay using adherent cells

[0452] HeLa, C2C12, HepG2, and A549 cells were used as adherent cells.

[0453] On day 1, 10,000 cells were seeded into 96-well plates, each well containing 100 µl of the appropriate culture medium (containing 10% FBS and 30 µg / ml gentamicin), and cultured at 37°C and 5% CO2 for 24 h. The culture medium could be Dulbecco's Modified Eagle Medium (DMEM), DMEM / F12, or RPMI 1640. On day 2, the old culture medium was removed, and 90 µl of fresh culture medium (free of FBS and antibiotics) was added to the cells. The purified colloidal LNP dispersion prepared according to Example 3 was diluted with RNase-free water to adjust the mRNA concentration to 10 ng / µl. 10 µl of the correspondingly diluted sample was added to the cells, so that the amount of mRNA in each well was equal to 100 ng, for a total volume of 100 µl. After 4 h, the old culture medium containing residual sample was removed and replaced with 100 µl of fresh culture medium (containing 10% FBS and 30 µg / ml gentamicin). The cells were then cultured at 37°C and 5% CO2 for another 20 h. On the third day, transfection efficiency was determined using a luciferase assay system (Promega GmbH, Walldorf, Germany). The luminescence signal was quantitatively analyzed using an Infinite 200 PRO multi-functional microplate reader.

[0454] Transfection and luciferase assay using suspension cells

[0455] Jurkat cells were used as suspension cells.

[0456] On day 1, 50,000 cells were seeded into 96-well plates, each containing 90 µl of RPMI 1640 medium (containing 10% FBS and 30 µg / ml gentamicin). The mRNA concentration was adjusted to 10 ng / µl by diluting the sample with RNase-free water. 10 µl of the correspondingly diluted sample was added to the cells, bringing the mRNA concentration in each well to 100 ng, for a total volume of 100 µl. Cells were incubated at 37°C and 5% CO2 for 24 h. On day 2, transfection efficiency was determined using a luciferase assay system (Promega GmbH). The luminescence signal was quantitatively analyzed using an Infinite 200 PRO multi-plate reader.

[0457] All transfection experiments used jetMessenger and jetPEI as positive controls. Reagents were prepared according to the manufacturer's instructions and added to each well with an equal dose of RNA as the test sample. Results were summarized in […]. Figure 10 .

[0458] Although transformation efficiencies varied across different cell lines, all tested P / A-LNP and PAS-LNP strains exhibited high transformation efficiencies, some even exceeding those of the positive controls (i.e., jetMessenger and jetPEI). Notably, among all P / A-LNP and PAS-LNP strains, P / A20-LNP and PAS20-LNP showed the highest transformation efficiencies, and their transformation efficiencies on A549, Jurkat, HepG2, and C2C12 cells were even higher than those of PEG2k-LNP. In conclusion, P / A-LNP and PAS-LNP can efficiently transfect a variety of human cell lines.

[0459] Example 9: Di(decyl)amine, di(dodecyl)amine, di(hexadecyl)amine, and di(octadecyl)amine are coupled to the Pga-PAS40 peptide via their single carboxylic acid groups.

[0460] Prepare a 30 mM solution of N-terminally blocked Pga-PAS40 peptide (PAS40; SEQ ID No.:4, XL-protein) in a dichloromethane (DCM) / methanol mixture (5:1). Dissolve 1.5 mol equivalents of di(decyl)amine (TCI Deutschland, Eschborn, Germany), di(dodecyl)amine (TCI Deutschland), di(hexadecyl)amine (Ambeed, Arlington Hts, IL, USA), or di(octadecyl)amine (Ambeed, Arlington Hts, IL, USA) in PAS40 solution; then add 2 mol equivalents of DIPEA base (N,N-diisopropylethylamine; Merck) and 1.5 mol equivalents of TBTU (22-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate; 400 mM stock solution dissolved in dimethylformamide DMF; Carl Roth, Karlsruhe, Germany); and incubate the mixture at 25°C for 1 h under argon protection. To precipitate unreacted di(alkyl)amine, 8 volumes of a methanol / water mixture (10:1) were added to the mixture, and the mixture was incubated on ice for 20 min. The precipitate was removed by filtration (using an Acrodisc 1 µm glass fiber syringe filter, PALL); subsequently, the coupling product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a VP 250 / 10 Nucleodur C18 Gravity column (Macherey-Nagel) with a mobile phase of 85% (v / v) MeOH, 10% (v / v) H2O, 5% (v / v) DCM, and 0.1% (v / v) formic acid. The coupling product was eluted with a 0-100% gradient of MeOH / DCM (1:1).

[0461] The elution fractions were combined, concentrated using a SpeedVac concentrator, diluted 1:10 with H2O, and then lyophilized. Analytical reversed-phase HPLC was performed using the same mobile phase as the preparative RP-HPLC of the corresponding PAS-di(alkyl)amines described above, on a Gemini 3 µm C18 110 Å column (150 x 4.6 mm, Phenomenex, Torrance, CA, USA). Additionally, 10–50 µg of PAS-di(alkyl)amines were dissolved in a 50% (v / v) aqueous solution of acetonitrile containing 0.1% (v / v) formic acid and analyzed by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode using a maXis Q-TOF instrument (Bruker Daltonics, Bremen, Germany). All PAS-di(alkyl)amines were obtained with homogeneous compositions, as evidenced by the single peaks observed in all analytical RP-HPLC and ESI-MS analyses (e.g., Figures 11 to 14 (As shown).

[0462] Example 10: Using a bispecific crosslinking agent, di(tetradecyl)amine is coupled to the Pga-PAS20-C peptide via the thiol group of the C-terminal cysteine ​​residue.

[0463] A 20 mM solution of N-terminally blocked Pga-PAS20-C peptide (PAS20C; SEQ ID NO: 23, XL-protein) was prepared in a dichloromethane (DCM) / methanol mixture (5:1). One molar equivalent of di(tetradecyl)amine (Ambeed) and one molar equivalent of the bispecific cross-linking agent succinimide-trans-4-(N-maleimidemethyl)cyclohexane-1-carboxylate (SMCC, TCI Deutschland) were dissolved in the PAS-peptide solution, followed by the addition of one molar equivalent of DIPEA base (N,N-diisopropylethylamine; Merck). Figure 15 The mixture was incubated at 25°C for 1 h under argon protection. To precipitate unreacted di(alkyl)amine, 8 volumes of a 10:1 methanol / water mixture were added, and the mixture was incubated on ice for 20 min. The precipitate was removed by filtration (Acrodisc 1 µm glass fiber syringe filter, PALL), and the coupling product was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) on a VP 250 / 10 Nucleodur C18 Gravity column (Macherey-Nagel) with a mobile phase of 85% (v / v) MeOH, 10% (v / v) H2O, 5% (v / v) DCM, and 0.1% (v / v) formic acid. The coupling product was eluted with a 0-100% gradient of MeOH / DCM (1:1).

[0464] The elution fractions were combined, concentrated using a SpeedVac concentrator, diluted 1:10 with H2O, and then lyophilized. Analytical reversed-phase HPLC was performed using the same mobile phase as the preparative RP-HPLC on a Gemini 3 µm C18 110 Å column (150 x 4.6 mm, Phenomenex). Additionally, 10–50 µg of PAS-di(alkyl)amine was dissolved in a 50% (v / v) aqueous solution of acetonitrile containing 0.1% (v / v) formic acid and analyzed by electrospray ionization mass spectrometry (ESI-MS) in positive ion mode using a maXis Q-TOF instrument (BrukerDaltonics). All PAS-di(alkyl)amines were obtained with a homogeneous composition, as evidenced by the single peaks observed in both analytical RP-HPLC and ESI-MS analyses (e.g., Figure 16 (As shown).

Claims

1. A method for preparing a compound of formula (I): A-[B-] b C-D (I) in A is a di(alkyl)amino group. B is the connector. b can be 1 or 0, allowing B to exist or not exist. C represents a polypeptide capable of forming a random coil conformation, comprising an amino acid sequence consisting of alanine, proline, and serine, or an amino acid sequence consisting of alanine and proline. D is an N-terminal protecting group. in, The method includes the following steps: a) Coupling of a di(alkyl)amine with compound LBCD or compound LCD yields compound (I), wherein L is a leaving group; and b) Purification of compound (I).

2. The method of claim 1, wherein A comprises two alkyl chains, and wherein the alkyl chains are independently straight-chain or branched alkyl chains.

3. The method of claim 1 or 2, wherein the two alkyl chains independently comprise about 8 to about 20 carbon atoms, preferably about 12 to about 15 carbon atoms, more preferably about 14 carbon atoms.

4. The method according to any one of claims 1 to 3, wherein A is bis(tetradecyl)amino.

5. The method of any one of claims 1 to 4, wherein C comprises an amino acid sequence consisting of alanine and proline, and wherein C comprises at least one alanine and one proline residue.

6. The method of claim 5, wherein the proline residue comprises more than 10 mol% and less than 70 mol% of C.

7. The method of claim 5 or 6, wherein C comprises at least 95 mol% proline and alanine residues.

8. The method of any one of claims 1 to 4, wherein C comprises an amino acid sequence consisting of alanine, proline and serine, and wherein C comprises at least one alanine, one proline residue and one serine residue.

9. The method of claim 8, wherein the proline residues comprise more than 4 mol% and less than 40 mol% of C.

10. The method of claim 8 or 9, wherein C comprises at least 95 mol% of proline, alanine, and serine residues.

11. The method according to any one of claims 5 to 10, wherein C comprises an amino acid sequence consisting of about 10 to about 200 amino acids, preferably about 20, about 40 or about 100 amino acids.

12. The method of any one of claims 5 to 11, wherein C comprises no more than 6 identical consecutive amino acid residues.

13. The method of any one of claims 5 to 12, wherein C comprises an amino acid sequence selected from any one of SEQ ID NO: 9 to SEQ ID NO: 22, or a multiple thereof.

14. The method according to any one of claims 1 to 13, wherein D is selected from pyroglutamyl, formyl, CO(C) 1-4 alkyl) and high pyroglutamyl, wherein the -CO(C) group is... 1-4 The alkyl moiety contained in the alkyl group may optionally be replaced by one or two groups, which are independently selected from -OH, -O(C 1-4 alkyl), -NH(C) 1-4 alkyl), -N(C) 1-4 Alkyl)(C 1-4 Alkyl groups and -COOH.

15. The method of claim 14, wherein D is selected from pyroglutamyl, formyl, acetyl, hydroxyacetyl, methoxyacetyl, ethoxyacetyl, propoxyacetyl, malonyl, propionyl, 2-hydroxypropionyl, 3-hydroxypropionyl, 2-methoxypropionyl, 3-methoxypropionyl, 2-ethoxypropionyl, 3-ethoxypropionyl, succinyl, butyryl, 2-hydroxybutyryl, 3-hydroxybutyryl, 4-hydroxybutyryl, 2-methoxybutyryl, 3-methoxybutyryl, 4-methoxybutyryl, glycine betaine, glutaryl, and high pyroglutamyl, preferably pyroglutamyl.

16. The method of any one of claims 1 to 15, wherein B comprises natural or non-natural amino acids.

17. The method of claim 16, wherein B is -HN-(C 2-12 (Hydrocarbon group)-C(O)-, wherein B is preferably selected from -HN-(CH2). 3-10 -C(O)-, -HN-phenyl-C(O)-, and -HN-cyclohexyl-C(O)-, wherein more preferably B is selected from -HN-(CH2)4-C(O)-, -HN-(CH2)5-C(O)-, -HN-(CH2)6-C(O)-, -HN-(CH2)7-C(O)-, -HN-(CH2)8-C(O)-, and And the bond shown at the N atom links B to polypeptide C.

18. The method of any one of claims 1 to 17, wherein L is replaced by A during the coupling of the di(alkyl)amine with compound LBCD or during the coupling of the di(alkyl)amine with compound LCD.

19. The method of claim 18, wherein the compound LBCD or compound LCD comprises the group -C(O)-L, wherein L represents a leaving group, and is preferably selected from -OH and -OR. 3 , where R 3 It is a C1-C6 alkyl group.

20. The method of any one of claims 1 to 15, wherein b is 0.

21. The method of any one of claims 1 to 20, wherein the di(alkyl)amine and the compound LBCD or the compound LCD are coupled in a single reaction.

22. The method of any one of claims 1 to 21, wherein the di(alkyl)amine and the compound LBCD or the compound LCD each contain only a single reactive group, and wherein the di(alkyl)amine and the compound LBCD or the compound LCD are coupled via said reactive group.

23. The method of any one of claims 1 to 22, wherein during the coupling process, the di(alkyl)amine and compound LBCD or compound LCD are contained in one or more organic solvents, preferably, said organic solvents comprising dichloromethane and methanol.

24. The method of claim 23, wherein the organic solvent comprises about 3 parts by volume, about 3.5 parts by volume, about 4 parts by volume, about 4.5 parts by volume, about 5 parts by volume, about 5.5 parts by volume, about 6 parts by volume, about 6.5 parts by volume, about 7 parts by volume, about 7.5 parts by volume, or about 8 parts by volume of dichloromethane and about 1 part by volume of methanol, preferably 5 parts by volume of dichloromethane and about 1 part by volume of methanol.

25. The method of any one of claims 1 to 24, wherein the di(alkyl)amine and compound LBCD or compound LCD are coupled using a coupling agent, and wherein the coupling agent is 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylurea tetrafluoroborate (TBTU).

26. The method of any one of claims 1 to 25, wherein the di(alkyl)amine is further coupled to the compound LBCD or the compound LCD using a nonnucleophilic base, and wherein the nonnucleophilic base is selected from N,N-diisopropylethylamine (DIPEA), 1,8-diazabicycloundec-7-ene (DBU), 1,5-diazabicyclo[4.3.0]non-5-ene (DBN), triethylamine (TEA), preferably DIPEA.

27. The method of any one of claims 1 to 26, wherein the purification of the compound comprises liquid chromatography of the compound, preferably high-performance liquid chromatography.

28. A compound as defined in any one of claims 1 to 27, or a compound of formula (I) obtained / available by the method of any one of claims 1 to 27.

29. The use of the compound of claim 28 for the preparation of lipid nanoparticles.

30. A method for preparing lipid nanoparticles, the lipid nanoparticles comprising the compound of claim 28, wherein the method comprises the following steps: a) Provide an ethanol solution of the compound of claim 28; b) The ethanol solution is mixed with an aqueous solution to prepare lipid nanoparticles; and c) Optionally, the lipid nanoparticles are dialyzed.

31. The method of claim 30, wherein the ethanol solution further comprises cationic lipids, non-cationic lipids, and sterols.

32. The method of claim 31, wherein the ethanol solution contains about 2 mM to about 60 mM of total lipids, preferably about 7.5 mM to about 30 mM, more preferably 9.43 mM of total lipids.

33. The method of any one of claims 30 to 32, wherein the ethanol solution comprises about 1 mol% to about 5 mol%, preferably about 1.5 mol% of the compound of claim 28.

34. The method of any one of claims 31 to 33, wherein the ethanol solution comprises about 45 mol% to about 55 mol%, preferably about 50 mol% of cationic lipids.

35. The method of any one of claims 31 to 34, wherein the ethanol solution comprises about 7 mol% to about 13 mol%, preferably about 10 mol% of non-cationic lipids.

36. The method of any one of claims 31 to 35, wherein the ethanol solution comprises about 35 mol% to about 42 mol%, preferably about 38.5 mol% of sterols.

37. The method of any one of claims 31 to 36, wherein the sterol is cholesterol.

38. The method of any one of claims 31 to 37, wherein the cationic lipid is DLin-MC3-DMA ([(6Z,9Z,28Z,31Z)-heptadec-6,9,28,31-tetraen-19-yl]4-(dimethylamino)butyrate).

39. The method of any one of claims 31 to 38, wherein the non-cationic lipid is anionic lipid, preferably wherein the anionic lipid is a phospholipid, more preferably distearate phosphatidylcholine (DSPC).

40. The method of any one of claims 30 to 39, wherein the aqueous solution comprises one or more active ingredients.

41. The method of claim 40, wherein the one or more active ingredients are one or more nucleic acids, one or more polypeptides, one or more proteins, or a combination thereof, preferably one or more nucleic acids.

42. The method of claim 41, wherein the one or more nucleic acids are selected from: mRNA, small interfering RNA (siRNA), Dicer substrate dsRNA, small hairpin RNA (shRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), tRNA, rRNA, tRNA, viral RNA (vRNA), self-amplifying RNA, guide RNA of gene editing systems, DNA, plasmids, antisense oligonucleotides, and combinations thereof, preferably mRNA.

43. The method of any one of claims 40 to 42, wherein the aqueous solution further comprises one or more acids.

44. The method of claim 43, wherein the one or more acids are selected from acetic acid, HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and citric acid, preferably acetic acid.

45. The method of claim 44, wherein the aqueous solution comprises one or more acids in a concentration of about 5 mM to about 50 mM, preferably about 10 mM to about 30 mM, more preferably about 11 mM.

46. ​​The method of any one of claims 30 to 45, wherein step c) comprises dialyzing the lipid nanoparticles with a buffer solution, preferably wherein the buffer solution is selected from phosphate buffer, phosphate-buffered saline, and phosphate-buffered saline containing sucrose, more preferably phosphate-buffered saline.

47. A lipid nanoparticle, which is defined by the method of any one of claims 30 to 46, or is obtained / available by the method of any one of claims 30 to 46.

48. A lipid nanoparticle suspension comprising the lipid nanoparticles of claim 47.

49. A method for preparing a pharmaceutical composition, wherein the method comprises formulating the compound of claim 28, the lipid nanoparticles of claim 47, and / or the lipid nanoparticle suspension of claim 48 into a pharmaceutical composition.

50. The method of claim 49, wherein the method comprises formulating the compound of claim 28, the lipid nanoparticles of claim 47, and / or the lipid nanoparticle suspension of claim 48 using a pharmaceutically acceptable carrier.

51. The method of claim 49 or 50, wherein the pharmaceutical composition is formulated for intramuscular or intravenous injection.

52. The method of any one of claims 49 to 51, wherein the pharmaceutical composition is formulated for use as a drug or as a vaccine.

53. A pharmaceutical composition that is as defined by the method of any one of claims 49 to 52, or that is obtained / available by the method of any one of claims 49 to 52.

54. Use of the compound of claim 28, the lipid nanoparticles of claim 47, and / or the lipid nanoparticle suspension of claim 48, for the preparation of pharmaceutical compositions.

55. Use of the compound of claim 28, the lipid nanoparticle of claim 47, and / or the lipid nanoparticle suspension of claim 48 in the preparation of a non-pharmaceutical composition.

56. A method for preparing a non-pharmaceutical composition, wherein the method comprises formulating the compound of claim 28, the lipid nanoparticles of claim 47, and / or the lipid nanoparticle suspension of claim 48 into a non-pharmaceutical composition.

57. The non-pharmaceutical composition as defined in claim 56, or a non-pharmaceutical composition obtained / available by the method of claim 56.

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