Cyclic cell penetrating peptides

Abstract

The present invention relates to cell penetrating cyclic peptides obtained using the CuAAC reaction. The invention further relates to conjugates of such peptides for intracellular transport of a molecular cargo.

Description

[0001] P7529PC00

[0002] Cyclic cell penetrating peptides

[0003] Technical field

[0004] The invention pertains to compounds useful for delivery of cargo across cellular membranes. In particular, the invention relates to cyclic peptides useful for delivery of cargo across cellular membranes. The cyclic peptides are thus useful for intracellular delivery of molecules, cellular uptake and / or transport across the blood-brain barrier for example for therapeutic applications.

[0005] Background

[0006] In recent years, research in the fields of chemical biology and bio-pharmaceutics has focused on molecules capable of delivering exogenous cargo into cells. Such molecules, particularly cyclic peptides, represent a promising modality for addressing intracellular protein-protein interactions (PPIs), which are often deemed “undruggable” using conventional small molecules or biologics. The unique structural properties of cyclic peptides, including their ability to bind flat PPI interfaces with high specificity and affinity, make them highly suitable for therapeutic and diagnostic applications.

[0007] However, their clinical potential has been severely hampered by poor cell permeability, metabolic instability, and susceptibility to proteolytic degradation. Despite significant advancements in combinatorial library technologies for identifying potent cyclic peptides, their application remains limited by these inherent challenges.

[0008] One of the primary barriers to effective intracellular delivery of cyclic peptides is their inability to cross the cell membrane. This limitation arises from the hydrophilic nature of their peptide bonds, high molecular weight, and conformational flexibility. While strategies such as Na-methylation and the introduction of intramolecular hydrogen bonds have improved the cell permeability of certain cyclic peptides, most remain impermeable. Further complicating their development, many promising peptides are unable to escape endosomal entrapment following uptake, thus failing to reach their intracellular targets. Alternative approaches, such as conjugating peptides with cellpenetrating motifs or using permeable nanoparticles, have been explored, but these methods introduce additional complexities and are often insufficient to fully overcome these limitations.

[0009] A growing area of research has focused on cyclic cell-penetrating peptides (cCPPs) as a means to improve intracellular delivery. Some of these peptides, characterized byP7529PC00

[0010] their cyclic structure, show enhanced proteolytic stability and some also show a moderate membrane permeability compared to linear counterparts.

[0011] Summary

[0012] It is therefore an objective of the present disclosure to provide cyclic peptides with good cell permeability and / or intracellular delivery capabilities. Specifically, the aim is to develop cyclic peptides that overcome at least some of the challenges of membrane impermeability, metabolic instability, and endosomal entrapment, thereby enabling the effective targeting of intracellular proteins and protein complexes.

[0013] The present disclosure addresses the above-mentioned problem by providing circular peptides containing a triazole moiety, designed to overcome the challenges of cell membrane impermeability. These circular peptides exhibit cell permeability and can be incorporated into conjugates to transport various types of cargo across cell membranes. The circular structure typically confer enhanced stability and resistance to enzymatic degradation, making the peptides effective in diverse biological environments. A further benefit of the present disclosure is the wide cell line compatibility demonstrated by these circular peptides, enabling their use across multiple cellular systems and conditions. The disclosure also encompasses conjugates comprising the circular peptides and one or more cargo molecules, as well as methods of facilitating intracellular transport, and kits of parts for use in delivering cargo into cells. These aspects provide a versatile platform for enabling effective delivery of peptides and cargo(s) in order to reach intracellular targets.

[0014] Thus, the cyclic peptides of the invention may have one or more of the following properties:

[0015] • Good cell permeability, which may even be improved compared to known cyclic peptides

[0016] • Capability of transporting cargo into cells, which may even be improved compared to known cyclic peptides

[0017] • Good transport across the blood-brain barrier, which may even be improved compared to known cyclic peptidesP7529PC00

[0018] • Capability of transporting cargo across the blood-brain barrier, which may even be improved compared to the capability of known cyclic peptides to transport cargo

[0019] • Capability to locate to desirable sub-cellular compartment(s)

[0020] • Good stability and low susceptibility to proteolytic degradation, which may even be improved compared to known cyclic peptides

[0021] Typically, the cyclic peptides of the invention comprise at least one amino acid with a hydrophobic side chain, which may enhance interaction with lipid bilayers.

[0022] Furthermore, the peptides preferably comprise a proline or a substituted proline, which may induce β-turns. Finally, the peptides also comprise positively charged amino acids, which may facilitate endosomal escape and / or targeting to specific subcellular compartments.

[0023] In one aspect, the present disclosure concerns a compound of formula (I):

[0024] P1 - A1 - P2 - A2 - P3

[0025]

[0026] -, formula (I),

[0027] wherein:

[0028] A1 is any amino acid or derivative thereof;

[0029] A2 is any amino acid or derivative thereof;

[0030] P1 is an amino acid sequence of 1 to 20 amino acids;

[0031] P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:

[0032] i) at least one amino acid is AP; or

[0033] ii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;

[0034] P3 is an amino acid sequence of 1 to 20;

[0035] the connection between A1 and A2 contains a 1,2,3-triazole moiety;

[0036] ^NH onr1 r2arg* is defined as:

[0037]

[0038] ' 'n NR3R4or

[0039] X

[0040]

[0041] , wherein R1, R2, R3, R4and R6independentlyP7529PC00

[0042] are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0043] AP is defined as:

[0044] , wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0045]

[0046] , wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0047]

[0048] , wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group;

[0049] and

[0050] AN is defined as:

[0051]

[0052] , wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings;

[0053] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0054] In one major aspect, the present disclosure concerns a compound of formula (I):

[0055] P1 - A1 P2 A2 - P3

[0056]

[0057] , formula (I),

[0058] wherein:

[0059] A1 is any amino acid or derivative thereof;

[0060] A2 is any amino acid or derivative thereof;P7529PC00

[0061] P1 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0062] P2 is an amino acid sequence of 3 to 5 amino acids, wherein at least one amino acid is AP and at least one amino acid is arg*;

[0063] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0064] the connection (depicted herein as a full line) between A1 and A2 contains a 1,2,3-triazole moiety;

[0065] i N NR1R2arg* is defined as:

[0066]

[0067] n NR'4orn NR3R4or

[0068] ^NH R6

[0069] I,. I u.

[0070]

[0071] n NR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl such as aminopropyl, X' is a negative counter ion; and n is 1, 2, 3, 4, or 5, with a preferred substitution with methyl and butyl for R1'6

[0072] and

[0073] AP is defined as:

[0074]

[0075] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0076] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0077] In a second aspect, the present disclosure concerns a conjugate comprising:

[0078] i) the compound according to formula (I);

[0079] and

[0080] ii) a cargo bound to the compound, optionally by a linker connecting the cargo and the compound.

[0081] In a third aspect, the present disclosure concerns a method of facilitating intracellular transport, comprising:P7529PC00

[0082] i) providing a compound according to formula (I) or a conjugate as defined herein;

[0083] and

[0084] ii) bringing said compound or conjugate in contact with one or more cells.

[0085] In a fourth aspect, the present disclosure concerns a kit of parts for intracellular transport, comprising:

[0086] i) a compound according to formula (I) or a conjugate as defined herein; ii) optionally a cargo to be transported;

[0087] and

[0088] iii) a cell sample.

[0089] In a fifth aspect, the present disclosure concerns a compound according to formula (I) or a conjugate as defined herein for use in reaching intracellular targets for the treatment of a disease, such as a disease of the brain.

[0090] Description of Figures

[0091] Figure 1. Fluorescence quantification analysis of live cells uptake by flow cytometry. Screening of cCPP15 (15), TAT, and other cyclic and linear peptides 1-14 and 16 by quantifying mean fluorescence intensity (MFI) through flow cytometry, upon treatment of MCF-7 cells with peptides at 1 pM and 37°C for 1 h, and blank group without treating any peptide under same conditions, comparisons between blank and peptide uptake were statistically significant by one-way ANOVA with Dunnett's test (****p < 0.0001).

[0092] Figure 2. cCPP15 uptake in different cell lines, a, Imaging of cCPP15 uptake by SDCM, the cell membrane was stained in purple color with CellMask™ Deep Red while the imported fluorophore ATOTA presents a green color, scale bars, 20 pm; b, quantification of fluorescence intensity by flow cytometry. These results represent the mean SD of three independent experiments. Comparisons between MCF-7 and other cell lines uptake were statistically significant by one-way ANOVA with Dunnett's test (**p < 0.01).

[0093] Figure 3. Endocytosis inhibition on MCF-7 and HeLa cells. Influence of different cell import inhibitors for cCPP15 import into MCF-7 cells (a) and HeLa (b) were investigated by established flow cytometry method. These results represent the meanP7529PC00

[0094] and SD of three independent experiments. Comparisons between control and inhibited uptake were statistically significant by one-way ANOVA with Dunnett's test (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

[0095] Figure 4. MCF-7 and HeLa two different cell lines were used to investigate the penetration of EGFP-cCPP17. There are three groups: EGFP, EGFP and cCPP17 mixture group and EGFP-cCPP17 conjugate group, which were treated with EGFP at 100 pM, mixture of EGFP and cCPP17, both at 100 pM, and EGFP-cCPP17 conjugate at 30 pM and 37°C for 0.5 h, respectively. All samples were imaged by SpinSR 10-Spin Disk Confocal Microscope, scale bars, 20 pm. Each experiment was performed in triplicates and in three independent repeats.

[0096] Figure 5. 3D live cell imaging of peptide uptake visualized using Lattice Light-Sheet Microscopy. HeLa cells were incubated at 37°C, 5% CO2 for 1h with a concentration of 1 pM of each sample. The peptides CPP1, CPP2 and CPP3 attached to the fluorophore ATOTA were imaged and peptides with ATOTA were excited using a 488 nm laser, while the membrane was stained with CellMask™ Deep Red membrane stain and excited at 640 nm. Images show fluorescence of CPP1, CPP2, and CPP3 resulting from fluorophore excitation, with brighter (whiter) regions indicating higher fluorescence intensity.

[0097] Figure 6. 3D live cell imaging of peptide uptake visualized using Lattice Light-Sheet Microscopy. HeLa cells were incubated at 37°C, 5% CO2 for 1h with a concentration of 1 pM of each sample. The peptide CPP1-AlexaFluor attached to the fluorophore Alexa Fluor 488 was imaged and the peptide with Alexa Fluor 488 were excited using a 488 nm laser, while the membrane was stained with CellMask™ Deep Red membrane stain and excited at 640 nm. Images show fluorescence of CPP1-AlexaFluor resulting from fluorophore excitation, with brighter (whiter) regions indicating higher fluorescence intensity.

[0098] Figure 7. 3D live cell imaging of peptide uptake visualized using Lattice Light-Sheet Microscopy. MCF-7 and HeLa cells were incubated at 37°C, 5% CO2 for 1h with a concentration of 1-2 pM of each sample. The peptides CPP3 attached to the fluorophore ATOTA were imaged and peptides with ATOTA were excited using a 488 nm laser, while the membrane was stained with CellMask™ Deep Red membrane stainP7529PC00

[0099] and excited at 640 nm. (A) Image show fluorescence of CPP3 resulting from fluorophore excitation in MCF-7 cells; (B) Image show fluorescence of CPP3 resulting from fluorophore excitation in HeLA cells. Brighter (whiter) regions indicate higher fluorescence. Scale bar 20 pm.

[0100] Figure 8. 3D live cell imaging of peptide uptake visualized using Lattice Light-Sheet Microscopy. MCF-7 and HeLa cells were incubated at 37°C, 5% CO2 for 1h with a concentration of 1-2 pM of each sample. The peptides ATOTA-CW-1 and ATOTA-CW-2 attached to the fluorophore ATOTA were imaged and peptides with ATOTA were excited using a 488 nm laser, while the membrane was stained with CellMask™ Deep Red membrane stain and excited at 640 nm. (A-B) Images show fluorescence of ATOTA-CW-1 (A) and ATOTA-CW-2 (B) in MCF-7 cells; (C-D) Images show fluorescence of ATOTA-CW-1 (C) and ATOTA-CW-2 (D) in HeLa cells. Brighter (whiter) regions indicate higher fluorescence. Scale bar 20 pm.

[0101] Detailed description

[0102] Definitions

[0103] A wavy line drawn over a chemical bond in a formula, as used herein, indicates a variable bond or connection to another unspecified chemical entity. For example, X— I

[0104] ?—

[0105] indicates a variable bond from the chemical entity X to another unspecified chemical entity. Such notation is commonly used in chemistry.

[0106] This connection may for example represent a single bond, a double bond, or another type of bond, depending on the context, or it may denote flexibility in the chemical structure where the exact bonding configuration or attached group is not predetermined.

[0107] The term “amino acid” as used herein refers to any amino acid, such as any canonical and non-canonical amino acid. Free amino acids have the structure H2

[0108] N-C(H)(R)-COOH. The term amino acid as used herein is also used to cover amino acids being part of a peptide, in which case the amino acid will have the structure -NH-C(H)(R)-CO-, H2N-C(H)(R)-CO- or -NH-C(H)(R)-COOH. The R group of an amino acid is also referred to as the “side chain”.P7529PC00

[0109] Amino acids in particular amino acids wherein the R-group comprises an amino group, such as arginine residues, may exist in protonated or non-protonated forms depending on the pH of the surrounding environment. Throughout the present application, amino acids and peptides may be represented in either a protonated (charged) or a nonprotonated (neutral) form. Unless explicitly stated otherwise, any representation of an amino acid or peptide in a protonated form shall be understood to also encompass the corresponding non-protonated form, and any representation in a non-protonated form shall likewise be understood to encompass the corresponding protonated form, as well as mixtures thereof, as may occur under physiological or other relevant conditions.

[0110] The terms ‘Orn’ and ‘orn’ as used herein refers to the amino acid: ornithine, such as L-or D-ornithine, respectively. The term ‘Pra’ and ‘pra’ as used herein refers to the amino acid: propargylglycine, such as L- or D-propargylglycine, respectively. The term ‘P8’ as used herein refers to the amino acid: ‘(2S,4R)-1-(((9H-fluoren-9-yl )methoxy)carbonyl)-4-((tert-butoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-2-carboxylic acid. The term ‘1 Nal’ as used herein refers to the amino acid: 3-(1-naphthyl)-D-alanine. The term ‘2Nal’ as used herein refers to the amino acid: 3-(2-naphthyl)-D-alanine.

[0111] The term ‘Y comprises one and no more than one’ as used herein, refers to the inclusion of exactly one instance of the variable Y, explicitly excluding the presence of more than one instance of Y.

[0112] The term ‘beta-structure inducing amino acid’ as used herein, refers to an amino acid that promotes the formation of beta-sheet or beta-turn secondary structures in peptides or proteins. This property is typically influenced by the amino acid's side chain characteristics and its ability to stabilize specific conformations. Amino acids commonly associated with beta-structure induction include valine (Vai), isoleucine (lie), threonine (Thr) and other beta-branched amino acids, such as glucosaminic acid or cysteine (Cys). These amino acids, through their hydrophobic or specific steric properties, contribute to the stabilization and formation of beta-sheet or beta-turn regions within peptides and proteins.

[0113] The term “canonical amino acid” as used herein refers to a proteinogenic amino acid. Preferably, the proteinogenic amino acid is one of the 20 amino acids encoded by theP7529PC00

[0114] standard genetic code. The IUPAC one and three letter codes are used to name amino acids.

[0115] The term ‘CuAAC’ as used herein, refers to copper(l)-catalyzed azide-alkyne cycloaddition, a click chemistry reaction that forms a stable 1,2,3-triazole linkage between an azide and an alkyne.

[0116] The term ‘conjugate’ as used herein, refers to a compound formed by the covalent or non-covalent attachment of two or more chemical entities. For example, as described herein, a cell-penetrating peptide attached a cargo via a linker may be understood as a conjugate.

[0117] The term ‘cargo’ as used herein, refers to a chemical entity, such as any molecule, compound, or structure intended to be delivered into a cell, including but not limited to proteins, peptides, nucleic acids, small molecules, or nanoparticles.

[0118] The term ‘linker’ as used herein, refers to a chemical entity, which may be a chemical bond or a more complex structure that joins two or more components together within a compound or conjugate.

[0119] The term “peptide” as used herein refers to a molecule composed of two or more amino acids linked by peptide bonds. A peptide bond is a covalent bond formed between the carboxyl group (-COOH) of one amino acid and the amino group (-NH2) of another through a condensation reaction, resulting in the release of a molecule of water.

[0120] The term ‘treatment’ as used herein, refers to the combating of a disease or disorder. ‘Treatment’ or ‘treating’, as used herein may be ameliorating, preventive, curative and / or slowing down disease progression. Preferably, treatment is ameliorating treatment, curative treatment and / or treatment slowing down disease progression, such as slowing down the progression of cancer. Thus, treatment may be any improvement in one or more measurable markers of the disease or condition being treated.

[0121] ‘Treatment’ or ‘treating’ does not necessarily indicate complete eradication or cure of the disease or condition, or associated symptoms thereof. In some embodiments, the term ‘treatment’ encompasses amelioration and prevention..P7529PC00

[0122] Compounds

[0123] In one aspect, the present disclosure concerns a compound of formula (I):

[0124] P1 - A1 - P2 - A2 - P3

[0125]

[0126] -, formula (I),

[0127] wherein:

[0128] A1 is any amino acid or derivative thereof;

[0129] A2 is any amino acid or derivative thereof;

[0130] P1 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid may be arg*;

[0131] P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:

[0132] i) at least one amino acid is AP; or

[0133] ii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;

[0134] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid may be arg*;

[0135] the connection between A1 and A2 contains a 1,2,3-triazole moiety;

[0136] ^NH R1^NHNR2R3°NR1 R2arg* is defined as:

[0137]

[0138] nNR4 nNR3R4or

[0139] ^NH R6

[0140] O^^ ^NR^R2X-

[0141]

[0142] nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0143] AP is defined as:

[0144]

[0145] , wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; orP7529PC00

[0146] , wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0147]

[0148] , wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group;

[0149] and

[0150] ^NH

[0151] AN is defined as:

[0152]

[0153] , wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings;

[0154] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0155] In one aspect, the present disclosure concerns a compound of formula (I):

[0156] ....

[0157]

[0158] -, formula (I),

[0159] wherein:

[0160] A1 is any amino acid or derivative thereof;

[0161] A2 is any amino acid or derivative thereof;

[0162] P1 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid is arg*;

[0163] P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:

[0164] i) at least one amino acid is AP; or

[0165] ii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;

[0166] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0167] the connection between A1 and A2 contains a 1,2,3-triazole moiety;P7529PC00

[0168] ^NH R1^NHOYN*Y arg* is defined as:

[0169]

[0170] nNR4,J'Tr nNRN3RR41R2or

[0171] ^NH R6

[0172] X“

[0173]

[0174] nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0175] AP is defined as:

[0176] ^N-\ ^RS

[0177] I )— NH

[0178] i)

[0179]

[0180] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or

[0181] Vf _ __p8

[0182] zryrTR

[0183] \_R9

[0184] ii)

[0185]

[0186] , wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; orZN-AN / -R

[0187] iii)

[0188]

[0189] ', wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;

[0190] and

[0191] AN is defined as:

[0192]

[0193] , wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0194] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0195] In one aspect, the present disclosure provides a compound according to formula (I),P7529PC00

[0196] P1 - A1 - P2 - A2 - P3

[0197]

[0198] -, formula (I),

[0199] wherein:

[0200] A1 is any amino acid or derivative thereof;

[0201] A2 is any amino acid or derivative thereof;

[0202] P1 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0203] P2 is an amino acid sequence of 3 to 5 amino acids, wherein at least one amino acid is AP and at least one amino acid is arg*;

[0204] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0205] the connection between A1 and A2 contains a 1,2,3-triazole moiety;

[0206]

[0207]

[0208] , wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0209] and

[0210] AP is defined as:

[0211]

[0212] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0213] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0214] The connection between A1 and A2 is depicted by a full line above. The connection is typically a chemical moiety which is covalently linked to the side chain of A1 at one end and covalently linked to the side chain of A2 at the other end and comprises a 1,2,3-tnazole moiety.P7529PC00

[0215] The compound of formula (I) is preferably a cyclic peptide. Preferably, the cyclic peptide comprises a peptide sequence, wherein two amino acids of said sequence are covalently connected to each other. Said two amino acids are also referred to as A1 and A2 herein.

[0216] Thus, the compound of formula (I) may comprise three peptide sequences P1, P2, and P3 and two intervening amino acids A1 and A2. In particular, said compound may be a cyclic peptidenmade up of three peptide sequences P1, P2, and P3 and two intervening amino acids A1 and A2. Peptide sequences P1, P2, and P3 may each comprise one or more amino acids connected by peptide bonds. The amino acids A1 and A2 may be positioned between P1 and P2 and between P2 and P3 and may be connected to said peptide sequences by peptide bonds. The peptide sequences P1, P2, and P3 and amino acids A1 and A2 may be connected to form a linear peptide sequence comprising or consisting of P1-A1-P2-A2-P3.

[0217] The amino acids A1 and A2 may be configured in such a way that a connection is established between the sidechain of A1 and the sidechain of A2 by means of a 1,2,3-triazole moiety, whereby a fraction of the linear peptide sequence comprising P1, P2, P3, A1, and A2 is closed into a cycle. Accordingly, the compound of formula (I) may be construed as a circular peptide. P1, P2, P3, A1, and A2 may comprise a wide combination of amino acids used to achieve the desired properties of the compound of formula (I), including cell permeability. The compound of formula (I) may comprise one or more amino acids with a hydrophobic side chain, or a sidechain that can be charged under physiological conditions, or a sidechain that allows for the connection of one or more sidechains via a 1,2,3-triazole moiety. The compound of formula (I) may comprise a range of amino acids or derivatives thereof within the peptide sequences P1, P2, and P3.

[0218] Thus, in some embodiments, the amino acids of P1, P2 and / or P3 are linked via peptide bonds.

[0219] In some embodiments, P1, P2 and P3 are linked to A1 and / or A2 via peptide bonds.P7529PC00

[0220] In some embodiments, P1 is an amino acid sequence of 1 to 20 amino acids, and preferably 1 to 5 amino acids.

[0221] In some embodiments, P2 is an amino acid sequence of 3 to 5 amino acids, and preferably of 4 amino acids.

[0222] In some embodiments, P3 is an amino acid sequence of 1 to 20 amino acids, and preferably 1 to 5 amino acids.

[0223] In some embodiments, P1 consists of 2 amino acids.

[0224] In some embodiments, P2 consists of 4 amino acids.

[0225] In some embodiments, P3 consists of 2 amino acids.

[0226] Said amino acids may be any amino acids, for example they may be canonical amino acids, or canonical amino acids containing one or more additional substituents.

[0227] The compound of formula (I) may comprise one or more amino acids with a sidechain amenable to becoming positively charged under physiological conditions, such as an arg* amino acid as defined herein. While not intending to be limited by theory, it is believed that amino acids with polar sidechain groups amenable to becoming positively charged, e.g. by protonation, under physiological conditions contribute favourably to cell permeability. It is further believed that the location and number of such polar sidechain groups can contribute to cell permeability. The compound of formula (I) may comprise three or more arg* amino acids located in P1, P2, and P3. In some preferred embodiments of the disclosure, P1, P2, and P3 each contain one arg* amino acid.

[0228] Thus, in some preferred embodiments, P1 comprises one and no more than one arg* amino acid.

[0229] In some preferred embodiments, P2 comprises two and no more than two arg* amino acids.P7529PC00

[0230] In some preferred embodiments, P3 comprises one and no more than one arg* amino acid.

[0231] In some embodiments, P1 and P3 are configured to include one arg* amino acid and one beta-structure-inducing amino acid. In some embodiments, P1 and P3 are configured to include one arg* amino acid and an amino acid which is Vai, lie, Leu, Phe, Tyr, Trp, Thr or Cys, such as valine, leucine, or phenylalanine. Similarly, P2 preferably comprises two arg* residues and one AP residue. The combination of these features may allow the compound to adopt secondary structures favourable for cellular penetration.

[0232] Thus, in some embodiments, P1 contains an arg* amino acid and an amino acid known to induce beta-structure, for example an amino acid selected from the group consisting of val, ile, thr and other beta-branched amino acids, such as an amino acid selected from the group consisting of val, ile, thr, glucosaminic acid and cys.. In some embodiments, P1 contains an arg* amino acid and an amino acid selected from the group consisting of val, ile, leu, phe, tyr, trp, thr or cys. Said amino acid may for example be of D or L form.

[0233] In some embodiments, P3 contains an arg* amino acid and an amino acid known to induce beta-structure, for example an amino acid selected from the group consisting of val, ile, thr and other beta-branched amino acids, such as an amino acid selected from the group consisting of val, ile, thr, glucosaminic acid and cys. In some embodiments, P3 contains an arg* amino acid and an amino acid selected from the group consisting of Val, Ile, Leu, Phe, Tyr, Trp, Thr or Cys.

[0234] In some embodiments, P1 comprises or consists of the sequence thr-arg* or arg*-thr.

[0235] In some embodiments, P2 comprises or consists of arg*-arg*-AP-A3, arg*-arg*-A3-AP, arg*-AP-arg*-A3, arg*-AP-A3-arg*, arg*-A3-arg*-AP, arg*-A3-AP-arg*, AP-arg*-arg*-A3, AP-arg*-A3-arg*, AP-A3-arg*-arg*, A3-arg*-arg*-AP, A3-arg*-AP-arg*, or A3-AP-arg*-arg*, wherein A3 may be any amino acid.

[0236] In some embodiments, P3 comprises or consists of arg*-leu or leu-arg*.P7529PC00

[0237] In some embodiments, the present disclosure provides a compound according to formula (I) wherein:

[0238] P1 is thr-arg*;

[0239] P2 is arg*-AP-A3-arg*;

[0240] P3 is arg*-leu;

[0241] and

[0242] A3 is any amino acid.

[0243] In another main aspect, the present disclosure provides a compound according to formula (I),

[0244] P1 - A1 - P2 - A2 - P3

[0245]

[0246] -, formula (I),

[0247] wherein:

[0248] A1 is any amino acid or derivative thereof;

[0249] A2 is any amino acid or derivative thereof;

[0250] P1 is thr-arg* or pra-thr-arg*;

[0251] P2 is arg*-AP-A3-arg* or arg*-pro-AN-arg*;

[0252] P3 is arg*-leu;

[0253] ^NH R1^NH o N NR2R3 nr1 r2arg* is defined as:

[0254]

[0255] nNR4,nNR3R4or

[0256] ^NH R6

[0257] 0^Y^^N^NRIR2X~

[0258]

[0259] nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0260] AP is defined as:

[0261]

[0262] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;P7529PC00

[0263] \_R9

[0264] ii)

[0265]

[0266] , wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or y*, _ pio

[0267] N^R

[0268] )^R11

[0269] iii)

[0270]

[0271] °, wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;

[0272] and

[0273] ^NH

[0274] Oy^R’

[0275] AN is defined as:

[0276]

[0277] , wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0278] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0279] In another main aspect, the present disclosure provides a compound according to formula (I),

[0280]

[0281] -, formula (I),

[0282] wherein:

[0283] A1 is any amino acid or derivative thereof;

[0284] A2 is any amino acid or derivative thereof;

[0285] P1 is thr-arg*;

[0286] P2 is arg*-AP-A3-arg*;

[0287] P3 is arg*-leu;

[0288] the connection between A1 and A2 contains a 1,2,3-triazole moeity;P7529PC00

[0289] arg* is defined as:

[0290]

[0291] X

[0292]

[0293] , wherein R1, R2, R3, R4, and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0294] AP is defined as:

[0295]

[0296] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0297] and

[0298] A3 is any amino acid;

[0299] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0300] In some embodiments, the sidechain of A1 and the sidechain of A2 are linked to the 1,2,3-triazole moiety.

[0301] In some embodiments, the 1,2,3-triazole moiety was formed in a CuAAC click reaction between two complementary units, one unit being contained in the sidechain of A1 and the other unit being contained in the sidechain of A2.

[0302] In some embodiments, the 1,2,3-triazole moiety was formed from an azide unit reacting with an alkyne unit, said azide unit being contained within the sidechain of A1 or A2 and said alkyne unit being contained within the sidechain of A1 or A2. The precursors of A1 and A2, i.e. the amino acid reacting with each other to form A1 and A2 covalently connected to each other may also be referred to herein as A1* and A2*.

[0303] In some embodiments, an azide unit or an alkyne unit contained in the sidechain of A1 has reacted with an azide or an alkyne unit contained in the sidechain of A2 to form theP7529PC00

[0304] 1,2,3-triazole moiety. Thus, A1* may comprise an azide unit and A2* may comprise an alkyne unit or vice versa.

[0305] In some embodiments, an azide unit contained in the sidechain of A1 has reacted with an alkyne unit contained in the sidechain of A2 to form the 1,2,3-triazole moiety.

[0306] In some embodiments, an alkyne unit contained in the sidechain of A1 has reacted with an azide unit contained in the sidechain of A2 to form the 1,2,3-triazole moiety.

[0307] In some embodiments, either A1 or A2 is a derivative of an azido amino acid preferably a derivative obtained after reaction of said azido moiety with an alkyne moiety..

[0308] In some embodiments, either A1 or A2 is a derivative of an alkyne-containing amino acid, preferably a derivative obtained after reaction of said alkyne moiety with an azido moiety.

[0309] In some embodiments, either A1 or A2 is a D- or L-form of azido homolysine, azidolysine, azido-ornithine, 4-azido-2-amino butyric acid, 3-azido-2-amino propanoic acid, 4-azidophenylalanine or a derivative thereof, preferably a derivative obtained after reaction of said azido moiety with an alkyne moiety.

[0310] In some embodiments, either A1 or A2 is a D- or L-form 2-aminotetra-3-ynoic acid, 2-aminopent-4-ynoic acid, 2-aminohex-5-ynoic acid, 2-, 3-, or 4-acetylenephenylalanine or a derivative thereof, preferably a derivative obtained after reaction of the alkyne moiety with an azido moiety.

[0311] In some embodiments, A1 is azido-ornithine or a derivative thereof and A2 is propargylglycine or a derivative thereof. Preferably A1 is a derivative of azido-ornithine and A2 is a derivative of propargylglycine, wherein said derivatives are obtained by reaction of said azido-ornithine with said propargylglycine.

[0312] In some embodiments A1* is D-Orn(N3) and A2* is D-Pra, and A1-A2 may thus be formed by reacting D-Orn(N3) with D-Pra.P7529PC00

[0313] A3 may be any amino acid, preferably any canonical amino acid, even more preferably a non-polar canonical amino acid. In some embodiments, A3 is glycine.

[0314] In some embodiments, the present disclosure provides a compound of formula (I) wherein:

[0315] A1 is ornithine or a derivative thereof;

[0316] A2 is propargylglycine or a derivative thereof;

[0317] P1 is thr-arg* or pra-thr-arg*;

[0318] P2 is arg*-AP-gly-arg* or arg*-pro-AN-arg*;

[0319] and

[0320] P3 is arg*-leu.

[0321] In some embodiments, the present disclosure provides a compound of formula (I) wherein:

[0322] A1 is ornithine or a derivative thereof;

[0323] A2 is propargylglycine or a derivative thereof;

[0324] P1 is thr-arg*;

[0325] P2 is arg*-AP-gly-arg*;

[0326] and

[0327] P3 is arg*-leu.

[0328] 2 3

[0329] N=N

[0330] In some embodiments, the triazole moiety is defined by:

[0331]

[0332] , wherein said triazole moiety connects A1 and A2 by a connection at the nitrogen in position 1 and by a connection at the carbon in position 4 or 5.

[0333] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 4 or 5 to A2.

[0334] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 4 or 5 to A1.

[0335] In some embodiments, said triazole moiety connecting A1 and A2 is 1,4-disubstituted.P7529PC00

[0336] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 4 to A2.

[0337] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 4 to A1.

[0338] In some embodiments, said triazole moiety connecting A1 and A2 is 1,5-disubstituted.

[0339] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 5 to A2.

[0340] In some embodiments, said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 5 to A1.

[0341] ^NH R1

[0342] ° N NR2R3

[0343] Each arg* may independently b

[0344]

[0345] enNR4

[0346] ^NH ^NH R6O^ / i^N^NR1R2O^^N^NR1R2x-

[0347]

[0348] " T n NR3R4or n NR3R4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5. Thus, each arg* of the compound of formula (I) may be different or one or more arg* may be the same. In some embodiments, all arg* of compounds of formula (I) are the same.

[0349] Preferably, at least one arg* and more preferably each arg* may independently be ^NH R1

[0350] NR2R3

[0351]

[0352] J''Tr nNR4, wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.P7529PC00

[0353] In another embodiment, preferably, at least one arg* and more preferably each arg*

[0354] ^NH

[0355] Onr1R2

[0356] may independently b

[0357]

[0358] enNR3R4wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

[0359] In some embodiments, R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, benzyl, isopropyl or aminoalkyl.

[0360] In some embodiments, one or more of R1, R2, R3, R4and R6is C1-20 alkyl.

[0361] In some embodiments, one or more of R1, R2, R3, R4and R6are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eiosyl or branched or cyclic derivatives thereof.

[0362] In some embodiments, one or more of R1, R2, R3, R4and R6are H.

[0363] In some embodiments, all of R1, R2, R3, and R4are H. In some embodiments. Arg* is ^NH R1

[0364] N YNR2R3

[0365]

[0366] nNR4, and all of R1, R2, R3, and R4are H; and n is 3.

[0367] In some embodiments, all of R1, R2, R3, and R4are methyl.

[0368] In some embodiments, all of R1, R2, R3, and R4are butyl.

[0369] In some embodiments, n is 2, 3, 4, or 5, and preferably n is 3.

[0370] In some embodiments, n is 3.

[0371] In some embodiments, R1, R2, R3, R4and R6of one or all arg* are H.P7529PC00

[0372] In some embodiments, R1, R2, R3, and R4of one or all arg* are C1-C12 alkyl.

[0373] In some embodiments, R1, R2, R3, and R4of one or all arg* are methyl.

[0374] In some embodiments, R1, R2, R3, and R4of one or all arg* are butyl.

[0375] ^NH

[0376] onr1 r2

[0377] In some embodiments, arg* is defined as:

[0378]

[0379] nNR3R4, wherein R1, R2, R3, R4are methyl; and n is 3.

[0380] ^NH

[0381] nr1R2

[0382] In some embodiments, arg* is defined as:

[0383]

[0384] nNR3R4wherein R1, R2, R3, R4are buthyl; and n is 3.

[0385] In some embodiments, one or all arg* are orn(tmG), wherein tmG is tetramethylguanidinylation.

[0386] In some embodiments, one or all arg* are orn(tbG), wherein tbG is tetrabuthylguanidinylation.

[0387] In some embodiments, AP is:

[0388] , wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0389]

[0390] , wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; orP7529PC00

[0391]

[0392] , wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group.

[0393] AP is preferably

[0394]

[0395] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl or another planar, aromatic compound.

[0396] In some embodiments, R5is phenyl, 1-napthyl, 2-naphthyl, 1-anthracenyl, 2-anthracenyl, 9-anthracenyl, 1 -phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, 9-phenanthryl, 2-indolyl, 3-indolyl, 2-benzothiophenyl, or 3-benzothiophenyl.

[0397] In some embodiments, R5is phenyl.

[0398] In some embodiments, R5is 1-napthyl or 2-naphthyl.

[0399] In some embodiments, R5is 2-naphthyl.

[0400] In some embodiments, R5is 1-anthracenyl, 2-anthracenyl, or 9-anthracenyl.

[0401] In some embodiments, R5is 1 -phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, or 9-phenanthryl.

[0402] In some embodiments, R5is 2-indolyl or 3-indolyl.

[0403] In some embodiments, R5is 2-benzothiophenyl or 3-benzothiophenyl.P7529PC00

[0404] In another embodiment, AP is preferably

[0405]

[0406] wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.

[0407] In some embodiments, R8and R9independently are phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.

[0408] In some embodiments, R8and R9is 1-naphthyl or 2-naphthyl.

[0409] In yet another embodiment, AP is preferably

[0410]

[0411] wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group.

[0412] In some embodiments, R10and R11independently are phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, 2-naphthyl, or a terminal C2-C12 alkynyl group.

[0413] In some embodiments, R10is 1-naphthyl or 2-naphthyl and R11is a terminal C2-C12 alkynyl group.

[0414] In some embodiments, R10is 1-naphthyl or 2-naphthyl and R11is a terminal C4 alkynyl group.

[0415] In some embodiments, AN is:

[0416] NH

[0417]

[0418] , wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings.P7529PC00

[0419] Preferably R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.

[0420] In some embodiments, R7is phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl. In some embodiments, R7is 1-naphthyl or 2-naphthyl.

[0421] In some embodiments, the compound of formula (I) is given wherein:

[0422] A1 is ornithine or a derivative thereof;

[0423] A2 is propargylglycine or a derivative thereof;

[0424] R1, R2, R3, and R4are H;

[0425] n is 3; and

[0426] R5is 2-naphthyl.

[0427] In some embodiments, the compound is of formula (1a):

[0428]

[0429] preferably formula 1a is as follows:P7529PC00

[0430]

[0431] In some embodiments, the compound as described herein is or comprises:P7529PC00

[0432]

[0433] Me

[0434]

[0435]

[0436] In some embodiments, the amino acids are of D-form, L-form, or a mixture of D- and L-form.

[0437] In some embodiments, the amino acids are primarily of the D-form.

[0438] In some embodiments, A1 is of the D-form.

[0439] In some embodiments, A2 is of the D-form.

[0440] In some embodiments, A3 is of the D-form.

[0441] In some embodiments, arg* is of the D-form, L-Form, or a combination of the D-form and L-form.

[0442] In some embodiments, arg* is of the D-form.

[0443] In some embodiments, AP is of the L-form.P7529PC00

[0444] In some embodiments, the amino acids are primarily of the D-form and AP is of the L-form. In some embodiment all amino acids except AP are of the D-form.

[0445] In some embodiments, A1 is of the D-form;

[0446] A2 is of the D-form;

[0447] A3 is glycine or an amino acid of the D-form;

[0448] Arg* is of the D-form; and

[0449] AP is of the L-form.

[0450] In some embodiments, the compound is of formula (1b):

[0451]

[0452] , formula (1b).

[0453] In some embodiments, the compound as described herein is or comprises:P7529PC00

[0454]

[0455] P7529PC00

[0456]

[0457] P7529PC00

[0458]

[0459] In some embodiments, the compound of formula (I) is an enantiomer of any of the compounds described herein.

[0460] The compound for formula (I) may also comprise or consist of the sequence Thr-Arg-Xi-Arg-X2-X3-Arg-X4-Arg-Leu is set forth in SEQ. ID. NO.: 1, wherein X1to X4are glycine or any amino acid.

[0461] In some embodiments, Xi is Ornithine, X2 is AP as defined herein, preferably wherein R5is 2-naphthyl, X3 is Glycine and X4 is propargylglycine.

[0462] In embodiments wherein the compound is cCPP15, the compound is as set forth in in SEQ. ID. NO.: 1 (Thr-Arg-X1-Arg-X2-X3-Arg-X4-Arg-Leu), wherein X1is Ornithine, X2is AP as defined herein, wherein R5is 2-naphthyl, X3is Glycine and X4is propargylglycine.

[0463] Synthesis

[0464] The present disclosure comprises compounds of formula (I) that may be synthesized using any suitable methodology known to the skilled practitioner. T

[0465] The methods frequently comprises synthesizing a linear peptide, followed by cyclization. Said linear peptide may in particular be a compound of the formula P1-A1*-P7529PC00

[0466] P2-A2*-P3, wherein P1, P2 and P3 are as described above, and A1* and A2* may connected with each other to form -A1-A2-.

[0467] Many methods for synthesis of linear peptides are known to the skilled person.

[0468] One possible approach involves the use of an Fmoc-based solid-phase peptide synthesis strategy, where linear peptides are assembled on a solid support, such as amino-functionalized poly(ethylene glycol) acrylate (PEGA800) resin. In such methods, amino acid building blocks are sequentially coupled under standard activation conditions, followed by deprotection steps to remove Fmoc groups. Side-chain protecting groups can be cleaved using standard protocols, such as treatment with trifluoroacetic acid solutions. The resulting linear peptides may then be purified using techniques like high-performance liquid chromatography (HPLC).

[0469] For obtaining cyclic peptides, cyclization can be achieved through various methods, including Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry. This process may involve introducing two non-natural amino acids, A1* and A2* into the linear peptide, wherein one contains an azide group and the other contain an alkyne group to facilitate the formation of stable 1,2,3-triazole linkages. Thus, A1* may comprise an azide group and A2* may comprise an alkyne or vice versa. Said A1* and A2* may for example be D-Pra and D-Orn(N3), respectively, or vice versa.

[0470] Alternative cyclization methods known in the field, such as lactamization or disulfide bond formation, may also be employed for obtaining cyclic peptides. Cyclization reactions can be monitored using analytical techniques such as liquid chromatographymass spectrometry (LC-MS), and the products can be purified and lyophilized for subsequent use.

[0471] For obtaining cyclic peptides with alkylated arginine residues, various methods are known to the skilled person, including methods for introducing monoalkylated, bisalkylated, trisalkylated, tetraalkylated, and / or pentaalkylated arginine residues, wherein the tetraalkylated and pentaalkylated arginine residues comprise positively charged guanidinium side chains. Alkylated arginine residues may be prepared by chemical modification of ornithine residues present in a linear or cyclic peptide, optionally in combination with one or more alkylation steps.P7529PC00

[0472] In such approaches, the amino-containing side chain of an ornithine residue is converted into an arginine-type side chain by a guanidinylation reaction, wherein the peptide is treated with a suitable guanidinylating reagent capable of transferring a guanidine or guanidinium group to the side-chain amine. Guanidinylation may be carried out using activated amidinium or guanidinium reagents, including reagents bearing one or more alkyl substituents, thereby introducing a substituted guanidinium functionality onto the former ornithine side chain.

[0473] The degree of alkylation of the resulting arginine residue may be controlled by the nature of the guanidinylating reagent and, where applicable, by subsequent alkylation reactions, allowing the formation of monoalkylated, bisalkylated, trisalkylated, tetraalkylated, or pentaalkylated guanidinium side chains. In the case of tetraalkylated and pentaalkylated arginine residues, the guanidinium functionality typically carries a permanent positive charge that is delocalised over the guanidinium moiety. Suitable guanidinylating reagents for obtaining tetra-alkylated arginine residues include, but are not limited to, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylammonium tetrafluoroborate (TBTU), N-butyl-N-(chloro(dibutylamino)methylene)butan-1-aminium (14), and 1, 1,3,3-tetrabutyl-2-methylisothiouronium halides activated by reaction with 1,1,2-trimethyldisulfan-1-ium reagents.

[0474] Guanidinylation reactions may be performed in polar solvents and in the presence of a base to maintain the nucleophilicity of the ornithine side-chain amine and to neutralise any acid generated during the reaction. Where peptides contain multiple ornithine residues, guanidinylation may lead to conversion of some or all of the ornithine side chains into alkylated arginine residues, resulting in peptides comprising multiple alkylated guanidinium motifs.

[0475] Following introduction of the alkylated arginine residues, the peptide may be cyclised, if not already cyclic, and purified using methods known to the skilled person.

[0476] Arginine residues or Arg* residues, including, mono-alkylated, bis-alkylated, trisalkylated, and tetra-alkylated arginine residues typically comprise a positively charged guanidinium moiety due to protonation, while penta-alkylated arginine residues typically comprise a permanently charged guanidinium moiety due to the 5thnitrogen alkylation.P7529PC00

[0477] The nature of the counterion of any of these positively charged arginine residues may include, but are not limited to inorganic and organic anions commonly encountered in peptide synthesis and purification, such as halide ions (e.g. chloride, bromide, or iodide), carboxylate ions (e.g. acetate, trifluoroacetate), sulfonate ions (e.g. methanesulfonate, p-toluenesulfonate), tetrafluoroborate, hexafluorophosphate, or other pharmaceutically or synthetically acceptable anions. In some cases, the counterion may be derived from the guanidinylation reagent itself, from an acid used during deprotection or cleavage, or from ion exchange occurring during purification. The skilled person will appreciate that the counterion may also be deliberately exchanged or selected using methods known in the art. Accordingly, the alkylated arginine residues and peptides comprising such residues may be present as salts with a single counterion or as mixtures of salts with different counterions.

[0478] The identity of the counterion is not believed to be critical for the function of the peptides and may vary depending on parameters such as the biological environment, pH, synthetic route, work-up, and isolation conditions.

[0479] These methods are illustrative and not limiting, as the skilled practitioner would recognize that other suitable peptide synthesis strategies, both solid-phase and solution-phase, may also be applied to prepare the compounds of the present disclosure. The choice of method may depend on factors such as the desired structural features, functional modifications, the specific application of the resulting compounds, or merely on the materials and instrumentation available to the skilled practitioner.

[0480] Conjugates

[0481] The present disclosure also provides a conjugate comprising:

[0482] iii) the compound of formula (I) as described herein;

[0483] and

[0484] iv) a cargo bound to the compound of formula (I), optionally by a linker connecting the cargo and the compound.

[0485] The conjugates of the present disclosure may comprise the compound of formula (I), a cargo bound to said compound and optionally a linker connecting the cargo and the compound. The compound of formula (I) may provide the conjugates of the presentP7529PC00

[0486] disclosure with the advantages and properties of the those compounds, such as cell permeability. Examples 2-5 demonstrate that cargo structures may be transported into cells, such as the transport of a synthetic fluorophore (examples 2-4) and a modified protein (example 5) by connecting said cargo to the compound of the present disclosure using covalent linkers. The exemplified cargo exhibit no to very limited cell permeability on its own, but is however successfully being transported due to the cell permeability of the conjugate provided for by the compounds of the present disclosure.

[0487] The linker is usually a convenient mean by which the compound of the present disclosure and a cargo is connected. The linker is optional, it may only be one chemical bond, and it will usually not contribute to cell permeability. The cargo and the compound of formula (I) may be bound to each covalently, such as by an optional linker, or by association without being covalently connected, for example by one or more of the following: electrostatic interactions, hydrogen bonds, hydrophobic interactions, van der Waals forces, pi-pi stacking, cation-pi interactions, salt bridges, cooperative binding processes, metal ion coordination, or a combination thereof. It will be apparent to the skilled practitioner that the conjugates of the present disclosure are not limited to any specific type of cargo. It is preferred that the cargo is compatible with the physiological conditions within a cell and it is also preferred that the cargo does not interfere with the cell permeability provided for by the compound of formula (I).

[0488] Thus, in some embodiments, the cargo is a peptide, a protein, a nucleic acid sequence, a carbohydrate, or a nanoparticle. The peptide or the protein may be an antibody, enzyme, or any peptide based drug. The protein may be an antibody or an antigen binding protein. The antigen binding protein for example monoclonal antibodies, polyclonal antibodies, recombinant antibodies, nanobodies, single domain antibodies, antibody fragments, single chain antibodies, bi-or multispecific antibodies.

[0489] The nucleic acid sequence may be DNA, RNA, mRNA, siRNA, CRISPR-Cas9 complexes, or any nucleic acid based drug. The nucleic acid may also be aptamers.

[0490] In some embodiments, the cargo is a detection agent or a therapeutic agent. The detection agent may be a contrast agent, a luminescent agent, or a radioactive agent. The luminescent agent may be a fluorescent agent or a phosphorescent agent. The radioactive agent may be a radiolabelled biomolecule or a radiolabelled complex.P7529PC00

[0491] The therapeutic agent may be a chemotherapeutic agent or a biopharmaceutical agent.

[0492] In some embodiments, the cargo comprises a ligand in which a metal ion is bound.

[0493] In some embodiments, the cargo is bound to the conjugate by non-covalent interactions.

[0494] In some embodiments, the cargo is ATOTA. In some embodiments, the cargo is a fluorescent protein, for example EFGP.

[0495] The skilled practitioner will realise that the conjugates of the present disclosure are also not limited to any specific type of linker. The linker is preferablt compatible with the physiological conditions within a cell and the linker does preferably also not interfere with the cell permeability provided for by the compound of formula (I). The skilled practitioner will further be aware of the general use of linkers in bioconjugation, where linkers are a well established tool for facilitating the attachment of two or more molecules, such as a cell-penetrating peptide and its cargo, without compromising the biological activity or stability of the components. These linkers are selected based on their chemical properties, stability under physiological conditions, and cleavage mechanisms to achieve controlled release when needed. Common linkers used in chemical biology and bioconjugation, which may be useful with the conjugates of the invention include peptide linkers, bifunctional lipid linkers, polyethylene glycol (PEG) linkers, disulfide linkers, acid-labile linkers, photocleavable linkers, click chemistry linkers, heterobifunctional linkers, maleimide-thiol linkers, NHS ester linkers, enzyme-cleavable linkers, hydrazone linkers, azide-alkyne cycloaddition linkers, or thioether linkers. The aforementioned linkers may also be used in combination.

[0496] Thus, in some embodiments, the linker is a peptide chain or a bifunctional lipid chain, which may be cleaved with a protease or a lipase, respectively.

[0497] In some embodiments, the linker is a chain comprising one or more units selected from: –CH2–, –C(O)–, –C(O)NH–, –(CH2CH2O)–, and –(CH2CH2)–. In some embodiments, the linker is a chain comprising one or more units selected from -CH(R)-C(O)-NH-, wherein R of each unit is individually any side chain of an amino acid,P7529PC00

[0498] preferably R of each unit is individually a side chain of a canonical amino acid. The linker may also be a chain comprising a mixture of aforementioned units.

[0499] In some embodiments, the linker comprises an amino acid with a triazole unit and one O

[0500] * N O

[0501] or more units

[0502]

[0503] of H

[0504] In some embodiments, the linker is

[0505]

[0506]

[0507] In some embodiments, the linker is the one or more chemical bond(s) between the conjugate and the cargo.

[0508] In some embodiments, the conjugate is according to formula (2):

[0509]

[0510] preferably formula (2) is:P7529PC00

[0511]

[0512] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof. In some embodiments, the conjugate as described herein is:P7529PC00

[0513]

[0514] P7529PC00

[0515]

[0516] P7529PC00

[0517]

[0518] stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof. In some embodiments, the conjugate is according to formula (3):

[0519]

[0520] or a tautomer, solvate, or pharmaceutically acceptable salt thereof.P7529PC00

[0521] In some embodiments, the conjugate as described herein is:

[0522]

[0523] P7529PC00

[0524] Me

[0525]

[0526] Bu, orP7529PC00

[0527]

[0528] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0529] Facilitation of intracellular transport

[0530] The compounds or conjugates of the present disclosure are preferably capable of transporting different cargo structures, such as synthetic molecules or proteins, across cell membranes (see e.g. examples 2-5). The compounds or conjugates of the present disclosure further preferably have the advantage of being compatible with a wide range of cell lines (see e.g. example 3). Thus, the conjugates of the invention are useful for intracellular transport, because they preferably facilitate transport for a wide range of cargos.

[0531] The present disclosure also provides methods of facilitating intracellular transport, comprising:

[0532] i) providing a compound of formula (I) or a conjugate of the present disclosure;

[0533] and

[0534] ii) bringing said compound or conjugate in contact with one or more cells.

[0535] The present disclosure also provides a kit of parts for intracellular transport, comprising:

[0536] i) a compound of formula (I); and

[0537] ii) a cargo to be transported.P7529PC00

[0538] In some embodiments, the compound and the cargo are associated with each other such that the compound is capable of transporting the cargo across a membrane.

[0539] In some embodiments, the compound and the cargo are associated with each other by means of:

[0540] i) covalent linkage; or

[0541] ii) non-covalent interaction.

[0542] In some embodiments, the covalent linkage is selected from an amide or peptide bond, a urea or carbamate linkage, a triazole linkage, a thioether or disulfide linkage, an oxime or hydrazone linkage, an ester or ether linkage, or an imine or secondary amine linkage. In some embodiments, the covalent linkage is preferably selected from an amide or peptide bond, or a triazole linkage. In some embodiments, the one or more non-covalent interactions are selected from electrostatic interaction, hydrogen bonding, hydrophobic interaction, π–π stacking, and host-guest complexation.

[0543] Medical use

[0544] As shown herein the compounds and conjugates of the invention are useful e.g. for intracellular transport of cargo. This property highlights their potential to overcome common challenges associated with delivering cargo to intracellular targets, such as limited membrane permeability. Consequently, these compounds or conjugates may be particularly useful in the treatment of diseases with intracellular drug targets, including conditions requiring precise modulation of protein-protein interactions, enzyme activity, or nucleic acid functions. It has further been demonstrated (see e.g. example 4) that the compounds or conjugates may enter cells by dynamin-promoted endocytosis, which is also a useful transport mechanism for crossing the blood-brain barrier (BBB).

[0545] Thus, the present disclosure also provides a compound according to formula (I) or a conjugate as defined herein for use in methods of treatment of a disease.

[0546] In some embodiments, the present disclosure is a compound according to formula (I) or a conjugate as defined herein for use in transport across a membrane in living cells.P7529PC00

[0547] In some embodiments, the present disclosure is a compound according to formula (I) or a conjugate as defined herein for use in a method of treatment of a disease of the brain or the central nervous system.

[0548] In some embodiments, the present disclosure is a compound according to formula (I) or a conjugate as defined herein for use in a method of treatment of brain diseases or disorders, comprising neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and prion diseases; cancers of the brain, including brain cancer, astrocytoma, and meningioma; inflammatory and autoimmune brain disorders, including multiple sclerosis, brain disorders resulting from autoimmune responses, encephalitis, and meningitis; neurological disorders, including epilepsy and stroke; psychiatric disorders, including schizophrenia and bipolar disorder; and other brain-related conditions such as dementia, wherein the method involves therapeutic interventions targeting the underlying pathophysiology of these conditions.P7529PC00

[0549] Items

[0550] The invention may further be defined by the following items:

[0551] 1. A compound according to formula (I),

[0552] P1 - A1 - P2 - A2 - P3

[0553]

[0554] -, formula (I),

[0555] wherein:

[0556] A1 is any amino acid or derivative thereof;

[0557] A2 is any amino acid or derivative thereof;

[0558] P1 is an amino acid sequence of 1 to 20 amino acids;

[0559] P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:

[0560] i) at least one amino acid is AP; or

[0561] ii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;

[0562] P3 is an amino acid sequence of 1 to 20;

[0563] the connection between A1 and A2 contains a 1,2,3-triazole moiety;

[0564] ^NH R1^NHNR2R3°NR1 R2arg* is defined as:

[0565]

[0566] nNR4 nNR3R4or

[0567] ^NH R6

[0568] O^^ ^NR^R2X-

[0569]

[0570] nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0571] AP is defined as:

[0572]

[0573] , wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; orP7529PC00

[0574] , wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0575]

[0576] , wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group;

[0577] and

[0578] AN is defined as:

[0579]

[0580] , wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings;

[0581] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0582] 2. The compound according to item 1, wherein the aryl or heteroaryl is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.

[0583] 3. The compound according to any one of the preceding items, wherein P1 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid is Arg*.

[0584] 4. The compound according to any one of the preceding items, wherein P3 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid is Arg*.

[0585] 5. The compound according to any one of the preceding items, wherein the compound is according to formula (I),

[0586] P1 - A1 - P2 - A2 - P3

[0587]

[0588] , formula (I),P7529PC00

[0589] wherein:

[0590] A1 is any amino acid or derivative thereof;

[0591] A2 is any amino acid or derivative thereof;

[0592] P1 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid is arg*;

[0593] P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:

[0594] i) at least one amino acid is AP; or

[0595] ii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;

[0596] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0597] the connection between A1 and A2 contains a 1,2,3-triazole moiety;

[0598] ^NH R1^NH NNR2R3 NR1 R2arg* is defined as:

[0599]

[0600] nNR4,nNR3R4or

[0601] ^NH R6

[0602] O^z^ N^NR1R2x-

[0603]

[0604] n NR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0605] AP is defined as:

[0606] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or

[0607]

[0608] , wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; orP7529PC00

[0609]

[0610] , wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;

[0611] and

[0612] AN is defined as:

[0613]

[0614] , wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0615] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0616] 6. The compound according to any one of the preceding items, wherein the compound is according to formula (I),

[0617] P1 - A1 - P2 - A2 - P3

[0618]

[0619] -, formula (I),

[0620] wherein:

[0621] A1 is any amino acid or derivative thereof;

[0622] A2 is any amino acid or derivative thereof;

[0623] P1 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0624] P2 is an amino acid sequence of 3 to 5 amino acids, wherein at least one amino acid is AP and at least one amino acid is arg*;

[0625] P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;

[0626] the connection between A1 and A2 contains a 1,2,3-triazole moiety;P7529PC00

[0627] ^NH R1^NHOYN*Y arg* is defined as:NR1R

[0628]

[0629] nNR4,J'Tr nNR3R4 2

[0630] or

[0631] ^NH R6

[0632] X“

[0633]

[0634] nNR3R4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0635] and

[0636] AP is defined as:

[0637]

[0638] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0639] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0640] 7. The compound according to any one of the preceding items, wherein:

[0641] P1 is an amino acid sequence of 1 to 20 amino acids, and preferably 1 to 5 amino acids.

[0642] 8. The compound according to any one of the preceding items, wherein:

[0643] P2 is an amino acid sequence of 3 to 5 amino acids, and preferably 4 amino acid.

[0644] 9. The compound according to any one of the preceding items, wherein:

[0645] P3 is an amino acid sequence of 1 to 20 amino acids, and preferably 1 to 5 amino acids.

[0646] 10. The compound according to any one of the preceding items, wherein:

[0647] P1 comprises one and no more than one arg* amino acid.

[0648] 11. The compound according to any one of the preceding items, wherein:

[0649] P2 comprises two and no more than two arg* amino acids.P7529PC00

[0650] 12. The compound according to any one of the preceding items, wherein:

[0651] P3 comprises one and no more than one arg* amino acid.

[0652] 13. The compound according to any one of the preceding items, wherein:

[0653] P1 consists of 2 amino acids.

[0654] 14. The compound according to any one of the preceding items, wherein:

[0655] P2 consists of 4 amino acids.

[0656] 15. The compound according to any one of the preceding items, wherein:

[0657] P3 consists of 2 amino acids.

[0658] 16. The compound according to any one of the preceding items, wherein the amino acids of P1, P2 and / or P3 are linked via peptide bonds.

[0659] 17. The compound according to any one of the preceding items, wherein P1, P2 and P3 are linked to A1 and / or A2 via peptide bonds.

[0660] 18. The compound according to any one of the preceding items, wherein:

[0661] P1 contains an arg* amino acid and an amino acid known to induce betastructure.

[0662] 19. The compound according to any one of the preceding items, wherein:

[0663] P1 contains an arg* amino acid and an amino acid selected from the group consisting of val, ile, thr and other beta-branched amino acids, such as an amino acid selected from the group consisting of val, ile, thr, glucosaminic acid and cys.

[0664] 20. The compound according to any one of the preceding items, wherein:

[0665] P1 contains an arg* amino acid and an amino acid selected from the group consisting of val, ile, leu, phe, tyr, trp, thr or cys.P7529PC00

[0666] 21. The compound according to any one of the preceding items, wherein:

[0667] P3 contains an arg* amino acid and an amino acid known to induce betastructure.

[0668] 22. The compound according to any one of the preceding items, wherein:

[0669] P3 contains an arg* amino acid and an amino acid selected from the group consisting of val, ile, thr and other beta-branched amino acids, such as an amino acid selected from the group consisting of val, ile, thr, glucosaminic acid and cys.

[0670] 23. The compound according to any one of the preceding items, wherein:

[0671] P3 contains an arg* amino acid and an amino acid selected from the group consisting of Val, Ile, Leu, Phe, Tyr, Trp, Thr or Cys.

[0672] 24. The compound according to any one of the preceding items, wherein:

[0673] P1 is thr-arg* or arg*-thr.

[0674] 25. The compound according to any one of the preceding items, wherein:

[0675] P2 is arg*-arg*-AP-A3, arg*-arg*-A3-AP, arg*-AP-arg*-A3, arg*-AP-A3- arg*, arg*-A3-arg*-AP, arg*-A3-AP-arg*, AP-arg*-arg*-A3, AP-arg*-A3- arg*, AP-A3-arg*-arg*, A3-arg*-arg*-AP, A3-arg*-AP-arg*, or A3-AP- arg*-arg*,

[0676] wherein A3 may be any amino acid.

[0677] 26. The compound according to any one of the preceding items, wherein:

[0678] P3 is arg*-leu or leu-arg*.

[0679] 27. The compound according to any one of the preceding items, wherein:

[0680] P1 is thr-arg*;

[0681] P2 is arg*-AP-A3-arg*;

[0682] P3 is arg*-leu;

[0683] and

[0684] A3 is any amino acid.P7529PC00

[0685] 28. The compound according to any one of the preceding items, wherein the compound comprises or consist of the sequence Thr-Arg*-A1-Arg*-AP-A3-Arg*- A2-Arg-Leu as set forth in SEQ. ID. NO.: 1, wherein.

[0686] 29. The compound according to any one of the preceding items, wherein the compound is a compound according to formula (I),

[0687] P1 - A1 - P2 - A2 - P3

[0688]

[0689] -, formula (I),

[0690] wherein:

[0691] A1 is any amino acid or derivative thereof;

[0692] A2 is any amino acid or derivative thereof;

[0693] P1 is thr-arg* or pra-thr-arg*;

[0694] P2 is arg*-AP-A3-arg* or arg*-pro-AN-arg*;

[0695] P3 is arg*-leu;

[0696] ^NH R1^NH O<YZ^^^^NR2R3arg* is defined as:

[0697]

[0698] nNR4 J'r\r nNR3R4or

[0699] X

[0700]

[0701] , wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0702] AP is defined as:

[0703] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0704]

[0705] , wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; orP7529PC00

[0706]

[0707] , wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;

[0708] and

[0709] ^NH

[0710] AN is defined as:

[0711]

[0712] , wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0713] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0714] 30. A compound according to formula (I),

[0715] P1 - A1 - P2 - A2 - P3

[0716]

[0717] -, formula (I),

[0718] wherein:

[0719] A1 is any amino acid or derivative thereof;

[0720] A2 is any amino acid or derivative thereof;

[0721] P1 is thr-arg*;

[0722] P2 is arg*-AP-A3-arg*;

[0723] P3 is arg*-leu;

[0724] the connection between A1 and A2 contains a 1,2,3-triazole moeity;

[0725] ^NH R1^NH N NR2R3 NR1 R2arg* is defined as:

[0726]

[0727] nNR4 nNR3R4or

[0728] ^NH R6

[0729] O^ / ^N^NR^2X“

[0730]

[0731] nNR3R4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;P7529PC00

[0732] AP is defined as:

[0733]

[0734] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;

[0735] and

[0736] A3 is any amino acid;

[0737] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0738] 31. The compound according to any one of the preceding items, wherein:

[0739] the sidechain of A1 and the sidechain of A2 are linked to the 1,2,3-triazole moiety.

[0740] 32. The compound according to any one of the preceding items, wherein: the 1,2,3- triazole moiety was formed in a CuAAC click reaction between two complementary units, one unit being contained in the sidechain of A1 and the other unit being contained in the sidechain of A2.

[0741] 33. The compound according to any one of the preceding items, wherein:

[0742] the 1,2,3-triazole moiety was formed from an azide unit reacting with an alkyne unit, said azide unit being contained within the sidechain of A1 or A2 and said alkyne unit being contained within the sidechain of A1 or A2.

[0743] 34. The compound according to any one of the preceding items, wherein an azide unit or an alkyne unit contained in the sidechain of A1 has reacted with an azide or an alkyne unit contained in the sidechain of A2 to form the 1,2,3-triazole moiety.

[0744] 35. The compound according to any one of the preceding items, wherein an azide unit contained in the sidechain of A1 has reacted with an alkyne unit contained in the sidechain of A2 to form the 1,2,3-triazole moiety.

[0745] 36. The compound according to any one of the preceding items, wherein an alkyne unit contained in the sidechain of A1 has reacted with an azide unit contained in the sidechain of A2 to form the 1,2,3-triazole moiety.P7529PC00

[0746] 37. The compound according to any one of the preceding items, wherein either A1 or A2 is a derivative of an azido amino acid, preferably a derivative obtained after reaction of said azido moiety with an alkyne moiety.

[0747] 38. The compound according to any one of the preceding items, wherein either A1 or A2 is a derivative of an alkyne-containing amino acid, preferably a derivative obtained after reaction of said alkyne moiety with an azido moiety.

[0748] 39. The compound according to any one of the preceding items, wherein either A1 or A2 is a derivative of azido-lysine or azido-ornithine, preferably a derivative obtained after reaction of said azido moiety with an alkyne moiety.

[0749] 40. The compound according to any one of the preceding items, wherein A1 or A2 is a derivative of propargylglycine, preferably a derivative obtained after reaction of the alkyne moiety with an azido moiety.

[0750] The compound according to any one of the preceding items, wherein A1 is azido-ornithine or a derivative thereof and A2 is propargylglycine or a derivative thereof, preferably A1 is a derivative of azido-ornithine and A2 is a derivative of propargylglycine, wherein said derivatives are obtained by reaction of said azido-ornithine with said propargylglycine.

[0751] 41. The compound according to any one of the preceding items, wherein A3 is glycine.

[0752] 42. The compound according to any one of the preceding items, wherein:

[0753] A1 is ornithine or a derivative thereof;

[0754] A2 is propargylglycine or a derivative thereof;

[0755] P1 is thr-arg* or pra-thr-arg*;

[0756] P2 is arg*-AP-gly-arg* or arg*-pro-AN-arg*;

[0757] and

[0758] P3 is arg*-leu.

[0759] 43. The compound according to any one of the preceding items, wherein:P7529PC00

[0760] A1 is ornithine or a derivative thereof;

[0761] A2 is propargylglycine or a derivative thereof;

[0762] P1 is thr-arg*;

[0763] P2 is arg*-AP-gly-arg*;

[0764] and

[0765] P3 is arg*-leu.

[0766] 44. The compound according to any one of the preceding items, wherein the compound comprises or consists of the sequence Thr-Arg*-A1-Arg*-AP-Gly- Arg*-A2-Arg-Leu as set forth in SEQ. ID. NO.: 1, wherein A1 is X1is ornithine and A2 is propargylglycine.

[0767] 45. The compound according to any one of the preceding items, wherein the

[0768] 2 3

[0769] N=N

[0770] triazole moiety is defined by:

[0771]

[0772] 5 x, wherein said triazole moiety connects A1 and A2 by a connection at the nitrogen in position 1 and by a connection at the carbon in position 4 or 5.

[0773] 46. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 4 or 5 to A2.

[0774] 47. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 4 or 5 to A1.

[0775] 48. The compound according to any one of the preceding items, wherein said triazole moiety connecting A1 and A2 is 1,4-disubstituted.

[0776] 49. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 4 to A2.P7529PC00

[0777] 50. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 4 to A1.

[0778] 51. The compound according to any one of the preceding items, wherein said triazole moiety connecting A1 and A2 is 1,5-disubstituted.

[0779] 52. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A1 and at the carbon position in position 5 to A2.

[0780] 53. The compound according to any one of the preceding items, wherein said triazole moiety is connected at the nitrogen in position 1 to the sidechain of A2 and at the carbon position in position 5 to A1.

[0781] 54. The compound according to any one of the preceding items, wherein at least

[0782] NRR3 one Arg* and preferably all Arg* are defined a

[0783]

[0784] s wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

[0785] 55. The compound according to any one of the preceding items, wherein all Arg* are the same.

[0786] 56. The compound according to any one of the preceding items, wherein:

[0787] one or more of R1, R2, R3, R4and R6is C1-20 alkyl.

[0788] 57. The compound according to any one of the preceding items, wherein:

[0789] one or more of R1, R2, R3, R4and R6are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, eiosyl or branched or cyclic derivatives thereof.P7529PC00

[0790] 58. The compound according to any one of the preceding items, wherein one or more of

[0791] R1, R2, R3, R4and R6are H.

[0792] 59. The compound according to any one of the preceding items, wherein arg* is ^NH R1

[0793] NR2R3

[0794] defined as:

[0795]

[0796] nNR4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

[0797] 60. The compound according to any one of the preceding items, wherein arg* is ^NH R1

[0798] N YNR2R3

[0799] defined as:

[0800]

[0801] nNR4, wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

[0802] 61. The compound according to any one of the preceding items, wherein ^NH

[0803] onr1 r2

[0804] arg* is defined as:

[0805]

[0806] nNR3R4wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;

[0807] 62. The compound according to any one of the preceding items, wherein arg* is ^NH R6

[0808] O^ / I^^N^NR1R2X~

[0809] defined as:

[0810]

[0811] nNR3R4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.P7529PC00

[0812] 63. The compound according to any one of the preceding items, wherein

[0813] R1, R2, R3, R4and R6of one or all arg* are H.

[0814] 64. The compound according to any one of the preceding items, wherein one or ^NH R1

[0815] NR2R3

[0816] more arg* is defined as:

[0817]

[0818] nNR4, wherein R1, R2, R3and R4are H; and n is 3.

[0819] 65. The compound according to any one of the preceding items, wherein

[0820] R1, R2, R3, and R4of one or all arg* are C1-C12 alkyl.

[0821] 66. The compound according to any one of the preceding items, wherein

[0822] R1, R2, R3, and R4of one or all arg* are methyl.

[0823] 67. The compound according to any one of the preceding items, wherein

[0824] R1, R2, R3, and R4of one or all arg* are butyl.

[0825] 68. The compound according to any one of the preceding items, wherein ^NH

[0826] onr1 r2

[0827] arg* is defined as:

[0828]

[0829] nNR3R4wherein R1, R2, R3, R4are methyl; and n is 3.

[0830] 69. The compound according to any one of the preceding items, wherein ^NH

[0831] N NR1R2

[0832] arg* is defined as:

[0833]

[0834] nNR3R4wherein R1, R2, R3, R4are buthyl; and n is 3.

[0835] 70. The compound according to any one of the preceding items, wherein one or all arg* are orn(tmG), wherein tmG is tetramethylguanidinylation.P7529PC00

[0836] 71. The compound according to any one of the preceding items, wherein one or all arg* are orn(tbG), wherein tbG is tetrabuthylguanidinylation.

[0837] 72. The compound according to any one of the preceding items, wherein AP is:

[0838] , wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0839]

[0840] , wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or

[0841]

[0842] , wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group.

[0843] 73. The compound according to any one of the preceding items, wherein AP is

[0844]

[0845] , wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.

[0846] 74. The compound according to any one of the preceding items, wherein R5is phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.

[0847] 75. The compound according to any one of the preceding items, wherein AP is

[0848]

[0849] , wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.P7529PC00

[0850] 76. The compound according to any one of the preceding items, wherein R8and R9independently are phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.

[0851] 77. The compound according to any one of the preceding items, wherein AP is

[0852] OyA /

[0853]

[0854] °, wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group.

[0855] 78. The compound according to any one of the preceding items, wherein R10and R11independently are phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, 2-naphthyl, or a terminal C2-C12 alkynyl group.

[0856] 79. The compound according to any one of the preceding items, wherein AN is:

[0857] ^NH

[0858]

[0859] , wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings.

[0860] 80. The compound according to any one of the preceding items, wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl.

[0861] 81. The compound according to any one of the preceding items, wherein R7is phenyl, 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.

[0862] 82. The compound according to any one of the preceding items, wherein: R5, R8, R9, R10, R11independently are indolyl, naphthyl, anthracenyl, or a terminal C2- C12 alkynyl group.

[0863] 83. The compound according to any one of the preceding items, wherein:

[0864] R5is 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.P7529PC00

[0865] 84. The compound according to any one of the preceding items, wherein:

[0866] n is 2, 3, 4, or 5, and preferably n is 3.

[0867] 85. The compound according to any one of the preceding items, wherein:

[0868] n is 3.

[0869] 86. The compound according to any one of the preceding items, wherein:

[0870] R5is phenyl, 1-napthyl, 2-naphthyl, 1-anthracenyl, 2-anthracenyl, 9-anthracenyl, 1 -phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, 9-phenanthryl, 2- indolyl, 3-indolyl, 2-benzothiophenyl, or 3-benzothiophenyl.

[0871] 87. The compound according to any one of the preceding items, wherein:

[0872] R5is phenyl.

[0873] 88. The compound according to any one of the preceding items, wherein:

[0874] R5is 1-napthyl or 2-naphthyl.

[0875] 89. The compound according to any one of the preceding items, wherein:

[0876] R5is 2-naphthyl.

[0877] 90. The compound according to any one of the preceding items, wherein:

[0878] R5is 1-anthracenyl, 2-anthracenyl, or 9-anthracenyl.

[0879] 91. The compound according to any one of the preceding items, wherein:

[0880] R5is 1 -phenanthryl, 2-phenanthryl, 3-phenanthryl, 4-phenanthryl, or 9- phenanthryl.

[0881] 92. The compound according to any one of the preceding items, wherein:

[0882] R5is 2-indolyl or 3-indolyl.

[0883] 93. The compound according to any one of the preceding items, wherein:

[0884] R5is 2-benzothiophenyl or 3-benzothiophenyl.

[0885] 94. The compound according to any one of the preceding items, wherein R8and R9are naphthyl, such as 1-napthyl and / or 2-naphthyl.P7529PC00

[0886] 95. The compound according to any one of the preceding items, wherein R10and R11independently are naphthyl, such as 1 -naphthyl or 2-naphthyl, or a terminal C2-C12 alkynyl group, such as a terminal C2 alkynyl, such as a terminal C3 alkynyl, such as a terminal C4 alkynyl, such as a terminal C5 alkynyl, or such as a C6terminal alkynyl.

[0887] 96. The compound according to any one of the preceding items, wherein:

[0888] A1 is ornithine or a derivative thereof;

[0889] A2 is propargylglycine or a derivative thereof;

[0890] R1, R2, R3, and R4are H;

[0891] n is 3; and

[0892] R5is 2-naphthyl.

[0893] 97. The compound according to any one of the preceding items, wherein:

[0894] A1 is ornithine or a derivative thereof;

[0895] A2 is propargylglycine or a derivative thereof;

[0896] R1, R2, R3, and R4are methyl, and R6is H;

[0897] n is 3; and

[0898] P2 is arg*-AP-gly-arg* or arg*-pro-AN-arg*; wherein:

[0899] , wherein R5is 2-naphthyl;

[0900]

[0901] , wherein R7is 1-naphthyl or 2-naphthyl.

[0902] 98. The compound according to any one of the preceding items, wherein:

[0903] A1 is ornithine or a derivative thereof;

[0904] A2 is propargylglycine or a derivative thereof;

[0905] R1, R2, R3, and R4are C4 alkyl, and R6is H;

[0906] n is 3; and

[0907] P2 is arg*-AP-gly-arg*; wherein

[0908] AP is:P7529PC00

[0909] 5

[0910] , wherein R5is 2-naphthyl;

[0911] , wherein R8and R9are each 2-naphthyl; or

[0912]

[0913] , wherein R10is 2-naphthyl and R11is a terminal C4 alkynyl group.

[0914] 99. The compound according to any one of the preceding items, wherein the compound is or comprises:P7529PC00

[0915]

[0916] MeP7529PC00

[0917] Me

[0918] MeN Me Me Bu

[0919] BuN Bu

[0920]

[0921] Bu100. The compound according to any one of the preceding items, wherein the compound is of formula (1a):

[0922]

[0923] P7529PC00

[0924] 101. The compound according to any one of the preceding items, wherein the amino acids are of D-form, L-form, or a mixture of D- and L-form.

[0925] 102. The compound according to any one of the preceding items, wherein the amino acids are primarily of the D-form.

[0926] 103. The compound according to any one of the preceding items, wherein A1 is of the D-form.

[0927] 104. The compound according to any one of the preceding items, wherein A2 is of the D-form.

[0928] 105. The compound according to any one of the preceding items, wherein A3 is of the D-form.

[0929] 106. The compound according to any one of the preceding items, wherein all arg* is of the D-form, L-Form, or some arg* are of D-form and some of L-form.

[0930] 107. The compound according to any one of the preceding items, wherein arg* is of the D-form.

[0931] 108. The compound according to any one of the preceding items, wherein AP is of the L-form.

[0932] 109. The compound according to any one of the preceding items, wherein the amino acid amino acids are primarily of the D-form and AP is of the L-form.

[0933] 110. The compound according to any one of the preceding items, wherein all amino acids are of D-form except AP, wherein AP is of the L-form.

[0934] 111. The compound according to any one of the preceding items, wherein:P7529PC00

[0935] A1 is of the D-form;

[0936] A2 is of the D-form;

[0937] A3 is glycine or an amino acid of the D-form;

[0938] Arg* is of the D-form; and

[0939] AP is of the L-form.

[0940] 112. The compound according to any one of the preceding items, wherein the compound is or comprises:P7529PC00

[0941]

[0942] P7529PC00

[0943]

[0944] P7529PC00

[0945]

[0946] Bu

[0947] 113. The compound according to any one of the preceding items, wherein the compound is of formula (1b):

[0948]

[0949] P7529PC00

[0950] 114. The compound according to any of the preceding items, wherein said compound is an enantiomer of any of the compounds defined above.

[0951] 115. The compound according to any one of the preceding items, wherein the compound is a cyclic peptide.

[0952] 116. A conjugate comprising:

[0953] i) the compound according to any one of the preceding items;

[0954] and

[0955] ii) a cargo bound to the compound, optionally by a linker connecting the cargo and the compound.

[0956] 117. The conjugate according to any one of the preceding items, wherein the cargo is a peptide, a protein, a nucleic acid sequence, a carbohydrate, or a nanoparticle.

[0957] 118. The conjugate according to any one of the preceding items, wherein the cargo is a detection agent or a therapeutic agent.

[0958] 119. The conjugate according to any one of the preceding items, wherein the detection agent is a contrast agent, a luminescent agent, or a radioactive agent.

[0959] 120. The conjugate according to any one of the preceding items, wherein the detection agent is a luminescent agent, such as a fluorescent agent or a phosphorescent agent.

[0960] 121. The conjugate according to any one of the preceding items, wherein the detection agent is a radioactive agent, such as a radiolabelled biomolecule or a radiolabelled complex.

[0961] 122. The conjugate according to any one of the preceding items, wherein the therapeutic agent is a chemotherapeutic agent or a biopharmaceutical agent.

[0962] 123. The conjugate according to any one of the preceding items, wherein the cargo comprises a ligand in which a metal ion is bound.P7529PC00

[0963] 124. The conjugate according to any one of the preceding items, wherein the cargo is bound to the conjugate by non-covalent interactions.

[0964] 125. The conjugate according to any one of the preceding items, wherein the cargo is ATOTA.

[0965] 126. The conjugate according to any one of the preceding items, wherein the cargo is EFGP.

[0966] 127. The conjugate according to any one of the preceding items, wherein the linker is a peptide chain or a bifunctional lipid chain, which may be cleaved with a protease or a lipase, respectively.

[0967] 128. The conjugate according to any one of the preceding items, wherein the linker is a chain comprising one or more units selected from: –CH₂–, –C(O)–, –C(O)NH–, –(CH₂CH₂O)–, and –(CH₂CH₂)–.

[0968] 129. The conjugate according to any one of the preceding items, wherein the linker is a chain comprising one or more units selected from -CH(R)-C(O)-NH-, wherein R of each unit is individually any side chain of an amino acid, preferably R of each unit is individually a side chain of a canonical amino acid..

[0969] 130. The conjugate according to any one of the preceding items, wherein the linker comprises an amino acid with a triazole unit and one or more units of

[0970]

[0971] P7529PC00

[0972] 131. The conjugate according to any one of the preceding items, wherein the

[0973]

[0974] 132. The conjugate according to any one of the preceding items, wherein the linker is the one or more chemical bond(s) between the conjugate and the cargo.

[0975] 133. The conjugate according to item 10, wherein the conjugate is:P7529PC00

[0976]

[0977] P7529PC00

[0978]

[0979] P7529PC00

[0980]

[0981] 134. The conjugate according to any one of the preceding items, wherein the conjugate is according to formula (2a):

[0982]

[0983] or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.P7529PC00

[0984] 135. The conjugate according to item 10, wherein the conjugate is:

[0985]

[0986] P7529PC00

[0987] Me

[0988]

[0989] Bu, orP7529PC00

[0990]

[0991] Bu

[0992] 136. The conjugate according to any one of the preceding items, wherein the conjugate is according to formula (2b):

[0993]

[0994] or a tautomer, solvate, or pharmaceutically acceptable salt thereof.

[0995] 137. A method of facilitating intracellular transport, comprising:

[0996] i) providing a compound according to any one of the preceding items or a conjugate according to any one of the preceding itemsP7529PC00

[0997] and

[0998] ii) bringing said compound or conjugate in contact with one or more cells.

[0999] 138. A kit of parts for intracellular transport, comprising:

[1000] i) a compound according to any one of the preceding items; and ii) a cargo to be transported.

[1001] 139. The kit of parts for intracellular transport according to any one of the preceding items, wherein the compound and the cargo are associated with each other such that the compound is capable of transporting the cargo across a membrane.

[1002] 140. The kit of parts for intracellular transport according to any one of the preceding items, wherein the compound and the cargo are associated with each other by means of:

[1003] i) covalent linkage; or

[1004] ii) non-covalent interaction.

[1005] 141. The kit of parts for kit of parts for intracellular transport according to any one of the preceding items, wherein the covalent linkage is selected from an amide or peptide bond, a urea or carbamate linkage, a triazole linkage, a thioether or disulfide linkage, an oxime or hydrazone linkage, an ester or ether linkage, or an imine or secondary amine linkage.

[1006] 142. The kit of parts for kit of parts for intracellular transport according to any one of the preceding items, wherein the covalent linkage is selected from an amide or peptide bond, or a triazole linkage.

[1007] 143. The kit of parts for kit of parts for intracellular transport according to any one of the preceding items, wherein the one or more non-covalent interactions are selected from electrostatic interaction, hydrogen bonding, hydrophobic interaction, π–π stacking, and host-guest complexation.

[1008] 144. The compound, the conjugate or the kit-of-parts according to any one of the preceding items for use in a method of treatment of a disease of the brain.P7529PC00

[1009] 145. The compound, the conjugate or the kit-of-parts according to any one of the preceding items for use in a method of treatment of brain diseases or disorders, comprising neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS), and prion diseases; cancers of the brain, including brain cancer, astrocytoma, and meningioma; inflammatory and autoimmune brain disorders, including multiple sclerosis, brain disorders resulting from autoimmune responses, encephalitis, and meningitis; neurological disorders, including epilepsy and stroke; psychiatric disorders, including schizophrenia and bipolar disorder; and other brain-related conditions such as dementia, wherein the method involves therapeutic interventions targeting the underlying pathophysiology of these conditions.

[1010] 146. The compound, the conjugate or the kit-of-parts according to any one of the preceding items for use treatment of a disease.

[1011] 147. The compound, the conjugate or the kit-of-parts according to any one of the preceding items for use in transport across a membrane in living cells.

[1012] Examples

[1013] General methods

[1014] Reagents and cells

[1015] All chemicals were purchased from Chem-lmpex, Sigma-Aldrich, Biosynth Carbosynth, Iris Biotech GmbH, MedChemExpress, Tokyo Chemical Industry (TCI) and Carl Roth GmbH and used as received. All solvents were of HPLC quality and were purchased from VWR Chemicals. PEGA800 resin was purchased from Polymer Laboratories Ltd. All cell lines (MCF-7, HeLa, U2OS, HEK293T, SH-SY5Y, HT-29, RPE-1) were bought from American Type Culture Collection (ATCC). All bio-Consumables were purchased from Sigma-Aldrich, Bio-Rad, Thermo Fisher Scientific, BD Biosciences, Avantor®.

[1016] Instruments

[1017] TC20 Cell Counter, NanoDrop™ 2000 / 2000c Spectrophotometers, VWR® pHenomenal® pH 1100L, Ultra Clear® RO DI Series system, BenchTop CO₂ Incubators The OASIS™ 6400 Series, SpectraMax® i3x Multi-Mode MicroplateP7529PC00

[1018] Reader, Eppendorf® ThermoMixer® C, Thermo Scientific™ Fresco™ 17 Microcentrifuge, Eppendorf® Centrifuge 5702, Kojair Biosafety Cabinet, BD FACSJazz™ Cell Sorter, SpinSR 10-Spinning Disk Confocal Microscope (SDCM) (Olympus, Tokyo, Japan).

[1019] 1H and13C NMR spectra were recorded on a Bruker instrument at 500 MHz and 126 MHz, respectively. Chemical shift values are quoted in ppm and coupling constants (J) in Hz. The residual solvent peak from CDCl3(1H: 7.26,13C: 77.16) or DMSO-d6(1H: 2.50 ppm,13C: 39.5 ppm) was used as reference.

[1020] LC-MS spectra were recorded on a Bruker micrOTOF-Q III instrument with an Acclaim™ RSLC 120 C18 column (2.2 pm, 120 A, 2.1x100 mm) and a Dionex UltiMate 3000 autosampler from Thermo Scientific, using a linear gradient of acetonitrile in water with 0.1% formic acid running from 0% to 90% acetonitrile over 10 min at a flow rate of 0.5 mL / min, with UV absorption peaks measured at 214, 225, 250, and 275 nm. High resolution mass spectrometry (HRMS) spectra were recorded on a Bruker Autoflex™ speed MALDI-TOF instrument by using positive electrospray ionization with α-Cyano-4-hydroxycinnamic acid (α-CHCA) as matrix.

[1021] Peptide purification was performed on a custom-built preparative Waters HPLC (Gardient Controller, 2487 Dual I Absorbance Detector, two 515 HPLC Pumps, an Inline Degasser and an Injection Module - 2 mL loop) equipped with a Vydac-218TP1022 C18 RP-column. For the mobile phase, two buffers were used: buffer A (0.1% trifluoroacetic acid in water) and buffer B (0.1% trifluoroacetic acid in 9:1 MeCN / water). Fibrillization assays were conducted using a SpectraMax® i3x plate reader.

[1022] Cell culture method

[1023] Adherent cell lines were propagated in standard culture medium, composed of DMEM, supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin.

[1024] Cultivation occurred within an incubator set to 37 °C, under an atmosphere containing 5% CO2.

[1025] For subculturing, a 0.05% Trypsin-EDTA solution (1-2 mL) was employed once cellular confluence approached 80-90% within T75 flasks. Prior to enzymatic detachment, the culture medium was aspirated, and flasks were rinsed with sterile PBS (2 mL x 2 times) free of calcium and magnesium and shook mildly every time. Subsequently, cells were exposed to the Trypsin-EDTA solution and relocated to the incubator chamber for no more than 3 minutes to facilitate detachment.P7529PC00

[1026] The enzymatic digesting action of trypsin was quenched by addition of culture medium (2 mL), and the flask bottom was washed mildly. Following collection, the cell suspension was centrifuged to form a pellet (5 min at 6000 rpm), after which the supernatant was discarded. The resultant cell pellet was then resuspended in fresh culture medium. Cell density was ascertained utilizing an automated cell counter (TC20 Cell Counter), and seeding was performed accordingly. To prevent over confluence, cells were routinely passaged every 3-4 days, contingent upon visual confluence assessments by microscope.

[1027] Live-Cell Imaging by SpinSR 10-Spinninq Disk Confocal Microscope (SDCM) All images of cells uptake, stained or treated with a peptide sample, were captured by using SpinSR 10-Spinning Disk Confocal Microscope (SDCM), with an oil-immersion 60x magnification objective (Olympus) and a numerical aperture of 1.4, connected to a CMOS camera (Photometries PRIME 95B) with an effective pixel size of 183 nmx183 nm. The CellMask™ Deep Red Plasma Membrane Stains (excitation max / emission max: 649 / 666 nm), used for labelling plasma membrane, were excited with a 5% intensity of 640 nm laser; and the fluorophore ATOTA (excitation max / emission max: 488 / 503 nm) was excited with a 10% intensity of 488 nm laser. Images were captured every 30 seconds, with an exposure time of 30.04 ms. Pre-imaging, peptide samples were dissolved in DMSO (1 mg / mL) as stock solution, and furtherly, was diluted into desired concentration by DMEM as testing solution in following experiments.

[1028] MCF-7 or other cell lines, were seeded and inoculated onto ibidi p-slides 8 wells, and subsequently cultivated under standard culture conditions for an overnight. Notably, a variation in seeding density was employed across the different cell lines: the MCF-7, HeLa, U2OS, HEK293T, and RPE-1 cell lines were each seeded at a density of 2×104cells / well, whereas the HT-29 and SH-SY5Y cell lines were seeded at a higher density of 1×105cells / well.

[1029] Prior to imaging, cells were washed gently with PBS (pH 7.4, 37°C, 2x200 pL), then incubated for 10 min with 1 pL of CellMask™ Deep Red Plasma Membrane Stains (1:100 diluted with 10 mM HEPES in HBSS) in 200 pL DMEM, followed by additional gently washing with PBS (pH 7.4, 37°C, 3x200 pL). After washing, cell membrane imaging was conducted for 3 min by using SDCM with the 640 nm laser at 5% intensity and exposure time of 30.04 ms. Images were captured every 30 seconds, for a total of 6 frames. Subsequently, 200 pL peptide testing solution at certain concentration was added to each cell chamber of p-slides. Cells were transferred to SDCM equipped withP7529PC00

[1030] incubator immediately, followed closely by that images were captured for 30 min by using SDCM system with the 488 nm laser at 5% intensity and exposure time of 30.04 ms. Images were captured every 30 seconds, for a total of 60 frames.

[1031] To mitigate potential phototoxic effects during the 1 h peptide treatment, a similar protocol was followed. After the 1 h exposure to the testing solution, cells were stained with CellMask™ Deep Red Plasma Membrane Stain, and imaging was performed using a 640 nm laser, a 488 nm laser, and bright-field microscopy.

[1032] Fluorescence quantification by Flow Cytometry (FC)

[1033] To quantify cell penetrating performance of peptide samples, flow cytometry (FC) was used to measure fluorescence intensity in different cell lines after being treated with peptide solution under various conditions. Quantification of fluorescence intensity was determined and analyzed by using BD FACSJazz™ Cell Sorter and FlowJo software (v10.8.1, Tree Star, Inc.) respectively.

[1034] Cells were seeded onto 24 well plate with certain density, supplemented by standard cell culture medium (400 pL, DMEM containing 10% FBS and 1% penicillin / streptomycin) and grown at 37 °C under an atmosphere containing 5% CO2. Particularly, a difference in seeding density was employed across the different cell lines: the MCF-7, HeLa, U2OS, HEK293T, and hTERT RPE-1 cell lines were each seeded at a density of 2.5×105cells / well, whereas the HT-29 and SH-SY5Y cell lines were seeded at a higher density of 5.0x105cells / well.

[1035] After being cultivating for 24 h, the culture medium was discarded, cells were washed gently with PBS (37°C, 1x300 pL) and incubated with peptide / DMEM solutions at certain concentration (37°C, 300 pL) for desired period at 37°C under 5% CO2 atmosphere. Afterwards, peptide / DMEM solution was discarded and cells were washed gently with PBS (37°C, 2x300 pL) to remove unbound peptide, and incubated for 10 min at 37 °C with trypsin-EDTA (200 pL) to detach from plate and remove noninternalized peptide bounding on the surface of the cell.

[1036] Subsequently, fresh culture medium (800 pL, 25°C, DMEM containing 10% FBS and 1% penicillin / streptomycin) was added to terminate trypsinization process, cells were pelleted at 3000 rpm at 4°C for 5 min. After removing supernatant, the pellet was resuspended in PBS (4°C, 1000 pL) and centrifuged at 3000 rpm at 4°C for 5 min, which was repeated for 3 times to wash cells.

[1037] A blue fluorescent dye used for DNA staining, propidium iodide (PI, excitation max / emission mas: 535 / 615 nm), was included in all the flow cytometry experiments,P7529PC00

[1038] serving as a cell live / dead indicator. The supernatant was carefully removed and the pellet was mildly resuspended in a solution of 3.75 pM (2.5 p / mL) PI / PBS (1 mL) at4°C to counterstain. The Cells were transferred to a filter-topped FASC tubes through 50 pm sterile cup-type filcon, and kept on ice prior to analysis. Subsequently, all processed cell samples were determined fluorescence intensity by flow cytometer. ATOTA fluorophore and PI (excitation max / emission max: 493 / 636 nm) were excited at 488 nm and monitored with 488 / 15 band-pass and 586 / 42 long-pass filters, respectively, on FACSJazz™ Cell Sorter. Events corresponding to cellular debris were removed by gating on forward- and side- scatter and then by PI fluorescence.

[1039] Each sample was tested in triplicate (3x10000 events) at the same day of measurement and the entire experiment was repeated three times on different days within one week. The standard error was calculated from at least three independent trials on different days. Control groups (not incubated with peptide, but DMEM) were analyzed to determine background and auto-fluorescence levels or cutoff point and thus differentiates them from the ATOTA labelled cells. The geometric mean of the fluorescence values, produced by PI and ATOTA, were determined from the histograms using FlowJo v10.8.1 software, while dead cells were removed according to PI staining.

[1040] Example 1: Synthetic procedures

[1041] Synthesis of amino acid P8

[1042] The amino acid P8 is required for the synthesis of cCPP15 and EGFP-cCPP17. The synthesis of P8 is outlined below in Scheme 1.

[1043] Teoc-OSu NaHCO3(sat. aq.) Mel, K2CO3CBr4, PPh3n NaN3, DMF - ► - ► HO" - ►Bri^ / 'Y' oMe - - Dioxane DMF, r.t. THF \-N. 70°C Teoc Teoc M11 M12 R" HO BOC2O PPh3, H2O, THF NaBHCN, AcOH NaHCO3(sat. aq.) 60°C DCM THF, r.t.

[1044] Teoc M13 M14 M15 Boc TBAF LIOH Boc,.. N. R THF Methanol, H2O NaHCO3(aq. sat.) Teoc Fmoc

[1045]

[1046] M16 M17 P8 Scheme 1. Synthesis of P8, R”= 2-2-naphthalenemethyl.P7529PC00

[1047] 2-Naphthaldehyde

[1048]

[1049] To a 100 mL round bottom flask, charged with 2-(bromomethyl)naphthalene (442 mg, 2 mmol, 1 equiv.) and sodium periodate (NaIO4) (428 mg, 2 mmol, 1 equiv.) was added 30 ml dried N, N-dimethyl formamide (DMF). The reaction mixture was heated at 150°C to reflux for 1 h. After the reaction was completed, monitored by thin layer chromatography, the reaction solution was cooled and treated with 20 ml of water, and then was extracted with ethyl acetate (3x30 ml). The combined ethyl acetate layer was dried over anhydrous sodium sulfate (Na2SO4), filtered off and concentrated in vacuo. Purification by flash chromatography on silica gel (v / v, ethyl acetate / heptane =1 / 20, 1 / 15) gave 2-naphthaldehyde (1662 mg, 95.7% yield) as white powder.

[1050] 1H NMR (500 MHz, CDCh-d): 5 10.19 (s, 1H), 8.37 (d, J= 1.3 Hz, 1H), 8.04 (dd, J = 8.2, 1.3 Hz, 1 H), 8.01 - 7.96 (m, 2H), 7.96 - 7.92 (m, 1 H), 7.65 (dddd, J = 28.0, 8.2, 6.9, 1.3 Hz, 2H).13C NMR (126 MHz, CDCI3): 5 192.27, 136.48, 134.56, 134.14, 132.67, 129.55, 129.13, 129.12, 128.10, 127.11, 122.80. HRMS(ESI+) m / z calculated for C11H8O [M+H]+: 157.061, found 157.1131.

[1051] (2S,4R)-4-Hydroxy-1-((2-(trimethylsilyl)ethoxy)carbonyl)pyrrolidine-2-carboxylic acid

[1052]

[1053] To a solution of (2S,4R)-4-hydroxypyrrolidine-2-carboxylic acid (656 mg, 5 mmol) dissolved in 12 mL saturated aqueous NaHCO3, TeocOSu (1430 mg, 5.5 mmol, 1.1 equiv.) in dioxane was added and the resulting mixture was stirred for overnight at room temperature. The reaction solution was diluted with water, and was acidified to pH 4 with 0.5 M hydrochloric acid. Then it was extracted three times with dichloromethane. The combined organic layers were washed twice with water, then dried over anhydrous Na2SO4. Removal of the drying agent by filtration and evaporation provided 1300 mg (94.4%) (2S,4R)-4-hydroxy-1-((2-(trimethylsilyl)ethoxy)carbonyl)pyrrolidine-2-carboxylic acid as a yellow oil.P7529PC00

[1054] 1H NMR (500 MHz, CDCI3-d): 54.51 - 4.39 (m, 2H), 4.18 (td, J= 8.0, 3.1 Hz, 1H), 4.12 (q, J= 7.6 Hz, 1H), 3.57 (d, J= 17.7 Hz, 1H), 3.53 (d, J= 3.9 Hz, 1H), 2.39 -2.09 (m, 2H), 1.02 - 0.88 (m, 2H).13C NMR (126 MHz, CDCI3): 6 175.79, 173.71, 158.66, 71.34, 70.89, 66.47, 65.90, 59.45, 58.84, 56.39, 56.00, 40.47, 39.03, 26.85, 19.19. HRMS(ESI+) m / z calculated for C11H21NO5Si [M+H]+: 276.122, found 276.2143.

[1055] 2-Methyl 1 -(2-(trimethylsilyl)ethyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (M11):

[1056] o

[1057]

[1058] Nx

[1059] Teoc

[1060] A mixture of (2S,4R)-4-hydroxy-1-((2-(trimethylsilyl)ethoxy) carbonyl)pyrrolidine-2-carboxylic acid (1655 mg, 6 mmol) and potassium carbonate (1250 mg, 9 mmol, 1.5 equiv.) was stirred in DMF (30 mL) for 5 min. Then, methyl iodide (1200 mg, 72 mmol, 1.5 equiv.) was added to the mixture and the leading solution was stirred for extra 4 hours. After the complete consumption of starting material, water (30 mL) was poured into reaction solution to quench it, and extracted with dichloromethane (3x30 mL). The combine organic layers was washed with water (2x30 mL), dried over anhydrous sodium sulfate, filtered and concentrated, purified though flash chromatography over silica gel (v / v, ethyl acetate / heptane =1 / 1) to provide 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (1662 mg, 95.7% yield) as light yellow oil.

[1061] 1H NMR (500 MHz, CDCI3-d): 54.53 - 4.36 (m, 2H), 4.21 - 4.05 (m, 2H), 3.70 (d, J = 11.5 Hz, 3H), 3.63 (dd, J= 11.5, 4.4 Hz, 1H), 3.58 (d, J= 12.1 Hz, 1H), 3.51 - 3.44 (m, 1H), 2.35 -2.19 (m, 1H), 2.06 (ddd, J= 13.1, 7.8, 4.8 Hz, 1H), 0.95 (ddd, J= 36.7, 13.2, 7.5 Hz, 2H).13C NMR (126 MHz, CDCI3): 571.77, 70.98, 65.33, 59.19, 59.02, 56.52, 56.01, 53.80, 53.68, 40.70, 39.93, 19.23. HRMS(ESI+) m / z calculated for C12H23NO5Si [M+H]+: 290.138, found 290.0648.

[1062] 2-Methyl 1 -(2-(trimethylsilyl)ethyl) (2S,4S)-4-bromopyrrolidine-1,2-dicarboxylate (M12):

[1063] o

[1064]

[1065] TeocP7529PC00

[1066] To a flask charged with 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-hydroxypyrrolidine-1,2-dicarboxylate (1655 mg, 5.7 mmol) and CBr4 (2585 mg, 7.8 mmol, 1.5 equiv) was added CH2CI2 (30 mL). The mixture was cooled to 0 °C. PPh3(2112 mg, 8.1 mmol, 1.55 equiv) was added in portions over 10 min. The reaction mixture was warmed to room temperature and stirred for 14 h. It was concentrated to about 3 mL in vacuo to give the crude, which was purified by flash chromatography (SiO2, v / v, ethyl acetate / heptane =1 / 3, 1 / 2) to give 1-benzyl 2-methyl (2S,4S)-4-bromopyrrolidine-1,2-dicarboxylate as light yellow oil (1950 mg, 97.1% yield) (Ding et al and Lin et al).

[1067] 1H NMR (500 MHz, CDCI3-d): 54.38 (ddd, J = 29.7, 8.6, 5.2 Hz, 1 H), 4.27 (p, J = 6.1 Hz, 1H), 4.20- 4.13 (m, 2H), 4.13 -4.07 (m, 1H), 4.02 (dd, J= 11.9, 6.4 Hz, 1H), 3.73 (d, J = 5.6 Hz, 3H), 2.86- 2.71 (m, 1H), 2.43 (ddt, J= 19.4, 13.8, 5.6 Hz, 1H), 0.94 (ddd, J= 37.3, 10.7, 6.5 Hz, 2H), -0.07 (s, 9H).13C NMR (126 MHz, CDCI3): 6 173.39, 173.22, 156.15, 155.74, 65.62, 65.55, 59.64, 59.46, 57.20, 56.89, 54.01, 53.90, 43.57, 42.95, 42.51, 41.46, 19.25. HRMS(ESI+) m / z calculated for C12H22BrNO4Si [M+H]+: 352.054, found 352.2820.

[1068] 2-Methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate (M13):

[1069] N3- OMe

[1070]

[1071] Teoc

[1072] Sodium azide (2160 mg, 33.2 mmol, 6 equiv.) was added to a solution of 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4S)-4-bromopyrrolidine-1,2-dicarboxylate (1950 mg, 5.5 mmol) in DMF (30 mL). The mixture was heated at 70 °C and stirred for 15 h. The reaction mixture was diluted with 40 mL water, and the resulting solution was extracted with dichloromethane. The extract was washed with brine and then dried over anhydrous Na2SO4. Concentration of the extraction in vacuo gave an oily residue, which was purified by column chromatography (SiO2, v / v, ethyl acetate / heptane =1 / 3, 1 / 2) to give 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate as a colorless oil (1170 mg, 67.7% yield) (Lin et al and Makoto et al).

[1073] 1H NMR (500 MHz, CDCI3-d): δ 4.47 – 4.32 (m, 1H), 4.17 (ddt, J = 14.4, 7.6, 4.3 Hz, 2H), 4.11 (td, J= 7.5, 4.7 Hz, 1H), 3.71 (d, J= 10.1 Hz, 3H), 3.68 (t, J= 5.7 Hz, 1H), 3.64 - 3.44 (m, 1H), 2.31 (dddd, J = 21.2, 13.1, 8.1, 4.6 Hz, 1H), 2.16 (dq, J= 13.1, 6.5 Hz, 1H), 1.03-0.85 (m, 2H).13C NMR (126 MHz, CDCI3): 5 174.14, 174.09, 156.42, 155.96, 65.62, 65.53,P7529PC00

[1074] 65.47, 60.81, 60.68, 60.13, 59.79, 59.12, 58.92, 54.00, 53.96, 53.86, 53.15, 53.07, 52.77, 52.69, 52.49, 37.84, 37.67, 36.79, 36.57, 19.27, 19.25, 19.23. HRMS(ESI+) m / z calculated for C12H22N4O4Si [M+H]+: 315.144, found 315.1740.

[1075] 2-Methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate (M14):

[1076] o

[1077] OMe

[1078]

[1079] \^Nx

[1080] Teoc

[1081] To a solution of 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-azidopyrrolidine-1,2-dicarboxylate (1162 mg, 3.7 mmol) in THF (25 mL) under nitrogen was added PPha (1460 mg, 5.6 mmol, 1.5 equiv.) and water (0.27 mL, 14.8 mmol, 4 equiv.). The reaction mixture was refluxed with stirring for 16 h. Then the solution was allowed to return to room temperature and added methanol 10 mL. After being concentrated under reduced pressure, the residue was purified by column chromatography (SiO2, v / v, methanol / dichloromethane=1 / 25, 1 / 20) to afford the 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate as yellow oil (715 mg, 97.7% yield) (Lin et al and Makoto et al).

[1082] 1H NMR (500 MHz, CDCh-d): 64.41 (ddd, J= 18.9, 8.6, 5.1 Hz, 1H), 4.20 -4.04 (m, 2H), 3.76 - 3.63 (m, 5H), 3.20 (ddd, J= 59.8, 10.5, 4.8 Hz, 1H), 2.13 (dq, J= 11.2, 5.6 Hz, 1H), 2.02 (ddt, J = 37.0, 13.7, 7.3 Hz, 1H), 0.94 (dt, J= 37.8, 8.6 Hz, 2H), -0.05 (s, 9H).13C NMR (126 MHz, CDCI3): 6 174.66, 174.57, 156.79, 156.31, 65.25, 65.21, 59.56, 59.37, 55.98, 55.77, 53.76, 53.67, 51.82, 50.96, 40.80, 40.16, 19.25, 19.21. HRMS(ESI+) m / z calculated for C12H24N2O4Si [M+H]+: 289.154, found 289.1775.

[1083] 2-Methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-((naphthalen-2-ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (M15):

[1084]

[1085] V

[1086] Teoc

[1087] To a stirred solution of 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate (750 mg, 2.60 mmol), dodecanal (487 mg, 3.12 mmol, 1.2 equiv.) and acetic acid (0.15 mL, 2.60 mmol, 1.0 equiv.) in DCM (25 mL) at O °C under N2, was added sodium cyanoborohydride (1105 mg, 5.20 mmol, 2 equiv.) portionwise over 5P7529PC00

[1088] minutes. Alternatively, 2-naphthaldehyde (487 mg, 3.12 mmol, 1.2 equiv.) was added instead of dodecanal. The reaction mixture was allowed to warm to room temperature over 17 hours. After this time, the reaction mixture was quenched with water and was allowed to stir at room temperature for an additional 5 minutes. The mixture was then extracted with dichloromethane (3x30 mL) and the organic layers were combined, washed with brine (2 x 30 mL), and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give the crude amine. Further purification by column chromatography (SiO2, v / v, ethyl acetate / heptane=1 / 4, 1 / 2, 1 / 1) afforded the 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-((naphthalen-2-ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (681 mg, 61.1%) as colorless oil (Cabrera-Pardo et al).

[1089] 1H NMR (500 MHz, CDCI3-d): 57.83 - 7.69 (m, 4H), 7.47 - 7.38 (m, 3H), 4.49 - 4.36 (m, 1H), 4.13 (dtd, J= 15.5, 7.7, 5.1 Hz, 2H), 3.96- 3.85 (m, 2H), 3.80 - 3.72 (m, 1H), 3.69 (d, J= 11.6 Hz, 3H), 3.58- 3.46 (m, 1H), 3.35 (ddd, J= 59.9, 10.6, 5.2 Hz, 1H), 2.21 - 2.03 (m, 2H), 1.00 - 0.88 (m, 2H).13C NMR (126 MHz, CDCI3):13C NMR (126 MHz, CDCI3) 6 174.70, 156.79, 134.85, 134.21, 129.73, 129.15, 129.12, 128.02, 127.93, 127.86, 127.81, 127.60, 127.21, 65.22, 65.16, 59.58, 59.41, 57.66, 56.65, 53.81, 53.76, 53.65, 53.55, 38.56, 37.61, 19.26, 19.21. HRMS(ESI+) m / z calculated for C23H32N2O4Si [M+H]+: 429.216, found 429.2177.

[1090] 2-Methyl 1 -(2-(trimethylsilyl)ethyl) (2S,4R)-4-((tert-butoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (M16):

[1091] Boc

[1092] 1

[1093] N,

[1094]

[1095] Teoc

[1096] To the stirring solution of 2-methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-((naphthalen-2 ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (653 mg, 1.52 mmol) in THF (7 mL) and NaHCO3saturated aqueous (7 mL), was added with di-tert-butyl dicarbonate (400 mg, 1.83 mmol, 1.2 equiv.). Then the leading mixture was stirred for overnight. The reaction solution was quenched by 20 mL water and extracted with dichloromethane (3x30 mL). The combined organic phases were dried with anhydrous sodium sulfate and concentrated under reduced pressure to give rude products, which was purified by by flash chromatography over silica gel (v / v, ethyl acetate / heptane=1 / 4, 1 / 2) to offer 2-methyl 1 - (2- (tri m ethy I s i ly l)ethy I) (2S,4R)-4-((tert-butoxycarbonyl)(naphthalen-2-P7529PC00

[1097] ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (492 mg, 61.3%) as colorless oil (Nguyen et al).

[1098] 1H NMR (500 MHz, CDCI3-d): 67.82 (dt, J = 18.5, 7.2 Hz, 3H), 7.59 (s, 1 H), 7.49 (dq, J = 7.2, 3.5 Hz, 2H), 7.36 - 7.29 (m, 1 H), 4.71 - 4.48 (m, 3H), 4.36 (dd, J = 62.1, 9.3 Hz, 1 H), 4.11 (dq, J= 18.1, 9.0 Hz, 2H), 3.78 (t, J= 9.5 Hz, 1H), 3.69 (d, J= 9.5 Hz, 4H), 3.55 -3.38 (m, 1H), 2.43 (dq, J= 13.2, 9.0 Hz, 1H), 2.06 (s, 1H), 1.45 (s, 9H), 0.92 - 0.89 (m, 2H).13C NMR (126 MHz, CDCI3):13C NMR (126 MHz, CDCI3) 6 174.25, 137.95, 134.92, 134.23, 130.03, 129.34, 129.25, 129.23, 129.17, 127.81, 127.74, 127.32, 126.04, 82.34, 78.76, 78.50, 78.25, 65.36, 65.27, 59.32, 59.20, 56.18, 55.26, 53.88, 53.81, 50.16, 29.88, 19.33, 19.23. HRMS(ESI+) m / z calculated for C28H40N2O6Si [M+H]+: 529.269, found 529.2888.

[1099] (2S,4R)-4-((tert-Butoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-2-carboxylic acid (M17):

[1100]

[1101] A mixture of 2-Methyl 1-(2-(trimethylsilyl)ethyl) (2S,4R)-4-((tert-butoxycarbonyl)(naphthalen-2-ylmethyl) amino)pyrrolidine-1,2-dicarboxylate (479 mg, 0.91 mmol) and tetrabutylammonium fluoride (5 mL, 1 M in THF) was stirred under nitrogen at room temperature for 15 h. After being concentrated, the leading residue was not conducted further purification (Davies et al). The crude products (330 mg) in methanol (4 mL) and water (4 mL) was added lithium hydroxide (103.02 mg, 4.29 mmol, 5 equiv.). After stirring at room temperature for 15 h, methanol was removed under reduced pressure. The remaining solution was diluted in water and acidified with 1 N HCI to pH 5. The mixture was then extracted with ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous Na2SO4. After being filtered, and concentrated in vacuo, the residue was purified through flash chromatography over silica gel (v / v, methanol / dichloromethane =1 / 3, 1 / 1) to provide the (2S,4R)-4-((tert-butoxycarbonyl) (dodecyl)amino)pyrrolidine-2-carboxylic acid (273.02 mg, 73.7 yield) as white solid (Nguyen et al).

[1102] 1H NMR (500 MHz, MeOD-d): 67.91 - 7.82 (m, 3H), 7.72 (s, 1 H), 7.50 (tt, J = 6.9, 5.1 Hz, 2H), 7.41 (dd, J= 8.5, 1.8 Hz, 1H), 4.67 (s, 2H), 4.28 (d, J = 55.4 Hz, 2H), 3.51 -3.35 (m, 2H), 2.46 (ddd, J= 13.6, 9.4, 6.8 Hz, 1H), 2.39-2.28 (m, 1H), 1.52 (s, 9H).P7529PC00

[1103] 13C NMR (126 MHz, MeOD-d): 6156.79, 137.20, 134.89, 134.34, 129.71, 128.75, 127.47, 127.08, 127.03, 126.18, 82.49, 28.67, 23.71. HRMS(ESI+) m / z calculated for C21H26N2O4[M+H]+: 371.193, found 371.2179.

[1104] (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((tert- butoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-2-carboxylic acid (P8):

[1105]

[1106] Fmoc

[1107] To a stirred solution of (2S,4R)-4-((tert-butoxycarbonyl) (naphthalen-2- ylmethyl)amino)pyrrolidine-2-carboxylic acid acid (260 mg, 0.70 mmol) in NaHCO3saturated aqueous (4 mL) was added Fmoc-OSu (284.14 mg, 0.84mmol, 1.2 equiv.) dissolved in THF. The leading mixture was stirred under room temperature for overnight. The reaction solution was added 10 water and then extracted with dichloromethane (3x15 mL). The organic layers were combined, washed with brine (2 x 20 mL), and dried over anhydrous sodium sulfate (Na2SO4). The solvent was removed in vacuo to give the crude amine. Further purification by column chromatography (SiO2, v / v, MeOH / CH2CI2=1 / 15, 1 / 10, 1 / 5) (2S,4R)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-4-((tert-butoxycarbonyl)(dodecyl)amino) pyrrolidine-2-carboxylic acid (316.64 mg, 63.6%) as white solid.

[1108] 1H NMR (500 MHz, CDCh-d): 57.88 - 7.78 (m, 3H), 7.71 (dd, J = 7.6, 2.8 Hz, 2H), 7.61 (d, J= 18.2 Hz, 1H), 7.50 (qd, J= 8.5, 4.2 Hz, 4H), 7.39-7.31 (m, 3H), 7.21 (ddd, J = 23.6, 17.4, 7.4 Hz, 2H), 4.67 (d, J= 16.5 Hz, 1H), 4.58 (d, J= 16.4 Hz, 1H), 4.53- 4.39 (m, 2H), 4.33 - 4.24 (m, 1 H), 4.15 (dt, J = 28.9, 6.8 Hz, 1 H), 3.83 - 3.70 (m, 1 H), 3.54- 3.43 (m, 1H), 2.50-2.39 (m, 1H), 2.38-2.28 (m, 1H), 2.20 (s, 1H), 1.42 (s, 9H).13C NMR (126 MHz, CDCI3): 6174.40, 155.72, 155.46, 154.14, 143.93, 143.71, 143.57, 141.32, 141.23, 136.16, 133.41, 133.36, 132.74, 132.66, 128.60, 127.78, 127.75, 127.71, 127.63, 127.61, 127.54, 127.06, 127.03, 126.40, 126.29, 125.91, 125.79, 125.06, 124.98, 124.86, 119.94, 119.89, 81.21, 81.16, 68.02, 67.55, 57.95, 57.42, 54.62, 48.72, 47.13, 47.01, 31.77, 28.37, 28.29. HRMS(ESI+) m / z calculated for C36H36N2O6[M+H]+: 593.261, found 593.2788.P7529PC00

[1109]

[1110] cCPP15 was synthesized manually at room temperature according to Fmoc-based solid-phase peptide synthesis strategy. By introducing unnatural amino acids D-Pra and D-Orn(N3), cyclic peptides were cyclized by Cu(l)-catalysed azidealkyne cycloaddition (CuAAC) click reaction.

[1111] Step 1. Synthesis ofATOTA-thr-arg-orn(N3)-arg-P8-gly-arg-pra-arg-leu-OH:

[1112] Adiabatic washes were conducted on 1 mL of amino-functionalized poly(ethylene glycol)acrylate 800 (PEGA800) resin (0.4 mmol / g) with 3 vol DMF, and then resin was immersed in 1 vol DMF for 1 h. Followingly, the resin underwent additional washes with 3 vol DMF, and the 4-hydroxymethylbenzoic acid (HMBA linker) was conjugated with PEGA800 resin. The base labile HMBA linker (4.0 equiv.), 1-[(1-(Cyano-2-ethoxy-2-oxoethylideneaminooxy)-dimethylamino-morpholino)] uronium hexafluorophosphate (COMU, 4.0 equiv.), N-ethylmorpholine (NEM, 0.5 equiv.) and ethyl cyano(hydroxyimino)acetate (Oxyma, 1.0 equiv.) were premixed in DMF (1 vol) and allowed to react for 5 min before addition. The reaction mixture was added to the preswollen PEGA800 resin and allowed to react for 1 h, followed by washing with DMF (5 vol) and CH2CI2 (5 vol).

[1113] First building block was esterified on to HMBA by mixing with solution of Fmoc-leu-OH (3.0 equiv.) preactivated (3.0 min) with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole (MSNT, 4.0 equiv.) and 1 -methylimidazole (Melm, 3.5 equiv.) in CH2CI2 (1 vol) and reacting for 1 h. Then the resin was washed with CH2CI2 (5 vol) and DMF (5 vol). Fmoc group was removed by repetitive treating with 20% (v / v) piperidine / DMF first for 5 min and then 3 min. The resin was washed with DMF (5 vol).

[1114] Removing DMF, then the pre-activated (5min) second Fmoc-arg-OH (4.0 equiv.) with COMU (4.0 equiv.), NEM (4.4 equiv.) and Oxyma (1.0 equiv.) in DMF (1 vol) was added to resin and reacted for 1 h. The resin was washed with DMF (5 vol).

[1115] Subsequently, Fmoc group was removed by repetitive treating with 20% (v / v)P7529PC00

[1116] piperidine / DMF first for 3 min and then 3 min. Then the resin was washed with DMF (5 vol). The following other Fmoc-protected amino acids (Fmoc-pra-OH, Fmoc-arg-OH, Fmoc-gly-OH, Fmoc-P8-OH, Fmoc-arg-OH, Fmoc-orn(N3)-OH, Fmoc-arg-OH, and Fmoc-thr-OH) were coupled by using standard COMU and Oxyma couplings conditions stated above. Besides, to economize P8 consumption, the relevant coupling conditions were changed as follows: P8 (2 equiv.) mixing with COMU (2.0 equiv.), NEM (2.2 equiv.) and Oxyma (1.0 equiv.) in 1 vol DMF; reacting for overnight. After the last coupling and Fmoc cleaving, the resin was washed with DMF (5 vol), DOM (5 vol) and dried for 1 h under stable nitrogen flow.

[1117] After synthesis of the linear peptide on the resin, it was coupled to ATOTA.

[1118] The side-chain protecting groups were removed using a solution of TFA / water (95 / 5) for 1 h or 3 h. After washing resin with DOM (5 vol) and with 2% (v / v) piperidine / DMF (5 vol), then the resin was washed with DMF (6 vol) and water (20 vol) respectively. Subsequently, 0.1 M NaOH / Water (1 vol) was added to resin and soaked for 1 h. After that, the PH of the resin was changed with 0.1 M HCI reaching 7-8. Collected the filtrates and washed resin with water (5 vol) and solution of acetonitrile / water (1:1) till that target peptide cannot be detected in the washing solution. Filtrate was sampled and were characterized by LC-MS. All solution was frozen by dry ice for 0.5 hour, and dried by lyophilizer.

[1119] Step 2. CuACC cyclization to obtain cCPP15:

[1120] After drying, the peptide powder was dissolved in water leading to 20 mg / mL concentration. 30 mg catalyst tetrakisacetonitrile copper(l) triflate (Cu(NCCH3)4·CF3SO3) was dissolved in 1 mL DMSO under N2 flow, followed by 10 mg ligand tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) and 25 pL N, N-diisopropylethylamine (DI PEA) addition, the resulting solution was bubbled under nitrogen for around 10 min. The peptide solution was added to pre-activated catalyst system dropwise with a syringe over 1 h and reacted for overnight. Reaction solution was analyzed by LC-MS. If there was no precipitate, mixture was purified by Pre-HPLC directly or filtered before purifying. All fractions containing pure peptide was collected and frozen by dry ice, and then remove solvent by lyophilizer.P7529PC00

[1121] ESI-TOF MS: Calculated for C90H126N29O16+[M+]: 1868.9932; Found [M++2H+] 935.5015, [M++3H+] 623.6725.

[1122] Synthesis of EGFP-cCPP17

[1123] The synthesis of EGFP-cCPP17 is outlined below in Scheme 2.

[1124]

[1125] EGFP-cCPP17 Scheme 2. Synthesis of EGFP-cCPP17.

[1126] Step a. Synthesis of EG FP-N3:

[1127] Azido-PEG4-NHS ester (4 mg) was dissolved in 1000 pL HEPES buffer (20 mM) and led to 4 mg / mL (10.3 pM) solution. 100 µL (15.5 mg / mL) was diluted with 300 pL HEPES (20 mM), then mixed with 100 pL Azido-PEG4-NHS ester / HEPES solution (4 mg / mL, 10.3 pM). The led solution was shaken under room temperature for 30 min to form EGFP-N3 (EGFP(H6G)-PEG4-N3). Reaction solution was transferred to dialysis membrane (10 kDa) and mildly stirred in 1 L PBS (0.2 M NaH2PO4 and 0.2 M Na2HPO4, pH 8.5) for overnight to purify EGFP-N3. The purified solution was analyzed by MALDI.

[1128] Step b. Synthesis of cCPP17 and CuAAC reaction to obtain EFGP-cCPP17:

[1129] cCPP17 was synthesized by following general peptide synthesis method above described and same scaffold. In other words, the cyclic peptide (cCPP17 contains the same amino acids and modified amino acids as cCPP15. But ATOTA fluorophore was replaced by Fmoc-AEEP and D-propargylglycine after cyclization. Besides, cCPP17 powder was dissolved in DMSO for following experiment. 1 vol Cu(CH3CN)4O-SO2-CF3(Tetrakisacetonitrile copper(l) triflate) / PBS (2 mM) was premixed with 1 vol 5-fold excess of the chelating ligand THPTA (10 mM), catalyst solution was degassed with nitrogen for 10 min. Then, cCPP17 (420 pL, 5 equiv., 5 mg / mL, 2.76 mM) was diluted with 4.8 mL PBS and injected into 5 mL degassed catalyst solution under nitrogen. Subsequently, EGFP-N3 / PBS solution (10.0 mg / mL) was dropwise injected into mixture of cCPP17 and catalyst solution under nitrogen. The produced solution was mildlyP7529PC00

[1130] stirred under nitrogen environment for overnight. After reaction, solution was purified with dialysis membrane (10 kDa) mildly stirred in 1 L PBS for overnight and analyzed by MALDI.

[1131] EGFP-cCPP17 was subsequently characterised by MALDI.

[1132] MALDI: calculated for 32847.53 Da; found 32847.906 Da.

[1133] Synthesis of tetra-methylated cCPPs (CPP1, CPP2, and CPP3)

[1134] The linear sequence precursors of the three different peptides, CPP1, CPP2, and CPP3 and are shown in Table A.

[1135] Table A. Sequences of the linear precursor peptides containing D-amino acids except L-proline derivate P8.

[1136] Peptide: Sequence of linear precursor peptides:

[1137] CPP1 ATOTA-pra-thr-orn-orn(N3)-orn-pro-1Nal-orn-pra-orn- leu-OH

[1138] CPP2 ATOTA-pra-thr-orn-orn(N3)-orn-pro-2Nal-orn-pra-orn- leu-OH

[1139] CPP3 ATOTA-pra-thr-orn-orn(N3)-orn-P8-G-orn-pra-orn-leu- OH

[1140]

[1141] Abbreviated amino acid residues are in bold: pra: D-propargylglycine, orn(N3): D-δ-azido ornithine, 1 Nal: 3-(l-naphthyl)-D-alanine, 2Nal: 3-(2-naphthyl)-D-alanine, P8: (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-( (tert-butoxycarbonyl) (naphthalen-2-ylmethyl)amino)pyrrolidine-2-carboxylic, ATOTA: 2-((3-carbonylpropyl(methyl)amino)-6,10-bis(diemethylamino) trioxatriangulenium hexafluorophosphate.

[1142] CPP1 contains a 1-napthylmethyl group, while CPP2 features a 2-napthylmehtyl group. Both modified amino acids, N-Fmoc-3-(1-naphthyl)-D-alanine (Fmoc-1Nal-OH) and N-Fmoc-3-(2-naphthyl)-D-alanine (Fmoc-2Nal-OH), were commercially available and directly incorporated during SPPS. CPP3, in contrast, exhibits a distinct structural variation. In this peptide, the napthylmethyl group is introduced via a modified trans-4-amino-L-proline, where the aromatic group is attached to a secondary amine on position 4. This building block P8 was synthesized through a multi-step synthesis before being incorporated into the peptide sequence. The naphthyl amino acids are shown in Scheme A.P7529PC00

[1143] HO

[1144] Fmoc-1Nal-OH

[1145]

[1146] Scheme A. Naphthyl amino acids: Fmoc-1Nal-OH, Fmoc-2Nal-OH, and P8.

[1147] The synthesis of CPP1 and CPP2 was carried out using SPPS on PEGA900 resin. Firstly, the peptide chain was assembled by classical SPPS. The HMBA linker was coupled followed by the esterification of the first amino acid. The coupling of the first amino acid was repeated a second time to achieve a sufficient resin loading. Subsequent amino acids were coupled under standard procedure. For CPP1, Fmoc-1Nal-OH was used, whereas CPP2 incorporated Fmoc-2Nal-OH.

[1148] After assembling the first ten amino acids, the terminal Na-Fmoc was removed and the CuAAC reaction was performed on-resin. Cyclization occurred between the azide-functionalized ornithine (Orn(N3)) and propargylglycine.

[1149] To confirm successful cyclization, a small resin sample (100 pL) was cleaved from the resin. The solution was degassed and treated with tris(2-carboxyethyl)phosphine (TCEP), a phosphine reagent that reduces unreacted azide groups to primary amines (Fawcett et al. 2025).

[1150] Mass spectrometry was used to evaluate the outcome. Successful CuAAC reaction would result in no change in mass, whereas incomplete reaction would be indicated by a loss of mass of 26 Da. As a control, a sample of the linear peptide prior to CuAAC was treated under the same conditions, confirming the expected mass shift.

[1151] Following successful CuAAC, the synthesis was continued with the coupling of the final amino acid. Here the Fmoc-protecting group from the N-terminal a-amine was not removed.

[1152] After completion of the peptide chain, the side chain groups were removed using a cleavage mixture of 95% TFA in water and 2% TIPS for 2 h, yielding free primary amines on the ornithine side chains.P7529PC00

[1153] The tetramethylated guanidinium groups were introduced by treating the peptide with 20% NEM in DMF, followed by an excess of 2-(1H-Benzotriazole-1-yl)-1, 1,3,3-tetramethylammonium tetrafluoroborate (TBTU) (Scheme B).

[1154]

[1155] Scheme B. Tetramethylguanidinylation of the four side chains of the cyclic tetra ornithine peptide attached to PEGA-solid support via the base labile HMBA linker using TBTU and NEM as a base.

[1156] A small quantity of peptide was cleaved from the resin and analyzed by LC-MS and MALDI and the predominant mass corresponded to full conversion of all four ornithine residues to tetramethylated arginines.

[1157] The synthesis was continued, and a fluorophore was coupled on, with the peptide still bound to the resin. Finally, CPP1 and CPP2 was obtained after cleavage from the resin.

[1158] In contrast to CPP1 and CPP2, where the napthylmethyl group was introduced via modified alanine residues, CPP3 features a structurally distinct approach. Here the naphthylmethyl moiety was incorporated into a synthesized L-proline derivate.

[1159] The desired amino proline derivate P8 was synthesized starting from trans-4-amino-L-proline. The synthesis involved a seven-step route to get to the final compound (Scheme C). To ensure compatibility with SPPS, appropriate protecting groups were introduced to selectively mask the secondary amines. The nitrogen within the pyrrolidine ring was protected with an Fmoc group, and the exocyclic amine at the 4-position was protected with a Boc group. This protecting strategy ensured the stability of both functional groups during SPPS and allowed for controlled deprotection in later stages.P7529PC00

[1160] SOCl2, MeOH reflux, 2.5h

[1161] 2-naphthaldehyde, OMe OMe NaBH3CN, PPh3, H2O, THF AcOH, DCM reflux, ovn rt, ovn 67% 52%

[1162] 5

[1163] Boc2O, THF,

[1164] NaOH (s), 0.8M CaCl2, sat. NaHCO3iPrOH / H2O 50°C, ovn rt, ovn

[1165] 92%

[1166]

[1167] P8 Scheme C. The synthesis of building block P8 starting of with trans-4-amino-L-proline (1) involving seven steps. Ovn: overnight.

[1168] In a first step the primary amine of 1 was converted into an azide via diazo transfer reaction using imidazole-1 -sulfonyl azide hydrochloride in the presence of CuSO4, yielding compound 2. This azide served as a temporary protecting group for the exocyclic amine, allowing selective protection of the secondary amine in the pyrrolidine ring. The secondary amine was protected with an Fmoc group, affording compound 3 in 54% yield. Subsequently, the carboxylic acid was converted into a methyl ester using thionyl chloride and methanol under reflux.

[1169] In a next step, the azide was reduced to a primary amine via a Staudinger reduction using Triphenylphosphine in aqueous THF, yielding compound 5 in 67%. The primary amine was now accessible for further reaction. The next step was the key step involving the reductive amination of the primary amine 5 with 2-naphtaldehyde. The reaction was carried out in the presence of sodium cyanoborohydride under mildly acidic conditions (acetic acid in DCM), forming the secondary amine 6 in 52% yield. To protect the newly introduced secondary amine and make this compound suitable for SPPS, a Boc group was installed. Finally the methyl ester was hydrolyzed to the free acid P8 using method adapted from Che et al. 2017 and Pascal and Sola 1998.P7529PC00

[1170] Using solid NaOH in a mixture of 0.8M CaCl2in i-PrOH and water allowed selective ester cleavage, without affecting the Fmoc functionality. After overnight stirring at room temperature, the free carboxylic acid was obtained in 92%. The final compound P8 was then incorporated in the sequence of CPP3.

[1171] Assembly was carried out following a similar strategy as for CPP1 and CPP2. After attaching the HMBA linker to the resin, the first amino acid was coupled twice to ensure sufficient resin loading. Subsequent amino acids were coupled stepwise and instead of the napthyl-alanines used in CPP1 and CPP2, glycine was incorporated in the turnsequence and then the synthesized building block P8 was incorporated in the subsequent position. After the tenth amino acid, the Click reaction was performed before coupling the last amino acid. Upon treatment with 95% TFA and 2% TIPS for 2 h to remove ornithine side chain protection, the guanidinylation to install tetramethylated arginines was carried out using TBTU and 20% NEM in DMF. After successful reaction, the Fmoc group on the a-amine was now cleaved and the peptide was ready for attachment of a fluorophore, before cleaving it off the resin.

[1172] Materials and methods:

[1173] All chemicals were purchased from Biosynth Carbosynth Chem Impex, Combi-Blocks, Iris GmbHand Roth and Sigma-Aldrich. All solvents were of HPLC quality and purchased from VWR Chemicals and Sigma Aldrich. Cell culture reagents and consumables were purchased from Sigma-Aldrich, Thermo Fisher Scientific Inc. Filcon, VWR Chemicals, ibidi Gmbh. Bio-Rad Laboratories, Inc. HeLa cell line was bought from American Type Culture Collection (ATCC).

[1174] A spectrophotometer from Thermo Scientific NanoDrop 2000 was used for UV measurement at 295 nm and 300 nm to determine resin loading.

[1175] Peptide purification was carried out using a custom-built preparative RP- HPLC systems (Waters Inc.) equipped with a gradient controller, two 515 HPLC pumps, an inline degasser, a 2 mL injection loop and a 2487 dual-wavelength UV absorbance detector. A Vydac 218TP1022 C18 reverse-phase preparative column (25 cm x 22 mm). The mobile phase consisted of solvent A: 0.1 % TFA in MQ-water, and solvent B: 0.1 % TFA in 9:1 ACN / MQ- water. The instrument ran linear gradient, running from 100 % solvent A to 100 % solvent B with a flow rate of 8 ml / min.P7529PC00

[1176] LC-MS measurements were performed using a Waters Acquity™ UPLC system coupled to a Xevo G2-S QTOF mass spectrometer. A BEH C-18 column (1.7 µm, 2.1×50 mm) was used with a linear gradient of ACN in MQ-water containing 0.1% formic acid. The flow rate was set to of 0.6 mL / min with a run time of 7 min.

[1177] Matrix-assisted laser desorption / ionization time-of-f light mass spectrometry (MALDI-TOF- MS) was performed on a Bruker Autoflex™ speed MALDI-TOF instrument with a-cyano-4- hydroxycinnamic acid as the matrix.

[1178] Proton (1H), carbon (13C) and 2D (COSY) NMR spectra were recorded on a Bruker 500 MHz and 126 MHz instrument. Chemical shifts (5) are reported in ppm and coupling constants (J) in Hz.

[1179] Molecular modelling of peptide structures was conducted using the software Molecular Operating Environment (MOE) from Chemical Computing Group.

[1180] General Procedure for SPPS:

[1181] The PEGA900 resin was washed with DMF (3 vol) and DCM (3vol) and was swollen in DMF for 20 min. To attach the base labile HMBA linker, HMBA (5 eq), the coupling reagent COMU (5 eq) and catalyst OXYMA (1.27 eq) were dissolved in DMF in a glass vial. The base NEM (5 eq) was added and preactivated for 5 min before adding to the resin. It was allowed to react for 2 h and the resin was washed with DMF (5 vol), DCM (5 vol) and dry DCM (1 vol). For the first amino acid coupling- an esterification- the amino acid (5 eq) and MSNT (5 eq) were dissolved in dry DCM in a vial and Melm (5 eq) and it was added to the resin to react for 1.5 h. With UV-Vis measurement the resin loading (see Section 6.8) was determined and the step was repeated until the desired loading was reached. The resin was washed with DCM (5 vol) and DMF (5 vol) and treated with a 20% (v / v) piperidine in DMF solution for Fmoc removal. It was reacted for 20min before washing the resin with DMF (5 vol). The other amino acids were coupled in the following way: In a vial the amino acid (5 eq), COMU (5 eq) and OXYMA (1.27 eq) were dissolved in DMF and NEM (5 eq) was added and preactivated for 5min before adding to the resin. It reacted for 1.5 h followed by washing the resin with DMF (5 vol), DCM (2 vol) and DMF (5 vol). Fmoc was removed by treating the resin with 20%P7529PC00

[1182] (v / v) piperidine in DMF for 5 min. Then the resin was washed with DMF (5 vol) and the next amino acid was coupled.

[1183] Because of high costs of Orn(N₃), these coupling conditions were also carried out by using only 2 eq of the amino acid, COMU, OXYMA and NEM and it was coupled overnight.

[1184] General Procedure for Side Chain Deprotection:

[1185] The resin was washed with DMF (5 vol) and DCM (5 vol) and dried under a nitrogen flow for 30 min. The side chain protecting groups were removed by treating the resin with a TFA / water (95 / 5)+2% TIPS solution for 2 h. The resin was washed with DMF (5 vol), DCM (5 vol), 2% (v / v) piperidine in DMF (5 vol) and DMF (5 vol).

[1186] General Procedure for Tetramethylguanidinylation:

[1187] 20% (v / v) NEM in DMF was added to the resin and an excess of TBTU (20 eq) was added and it was allowed to react overnight. The resin was washed with DMF (10 vol) and DCM (5vol) and DMF (5vol).

[1188] General Procedure for Peptide-Cleavage from Solid Support:

[1189] To cleave off the peptide the resin was treated with 0.1M NaOH (1 vol) for 1 h. The resin was washed with water and the filtrate was collected. The pH was adjusted by treating the resin with 0.1M HCI (1 vol) and the resin was washed with 70% (v / v) acetonitrile in water. The filtrate was purified by preparative RP- HPLC and the fractions containing the pure peptide were frozen on dry ice and lyophilized.

[1190] General Procedure for CuAAC reaction on resin:

[1191] The resin was washed with DMF (5 vol), DCM (5 vol) and MQ-water (10 vol). The resin was then transferred to a 20 mL glass vial and the solvent was degassed for 30 min. Then 30 mg catalyst tetrakis(acetonitrile)copper(l) triflate and 5 mg ligand tris(3-hydroxypropyltriazolylmethyl) amine (THTPA) were added. The mixture was stirred overnight at room temperature. The resin was transferred back to a syringe and was washed with water (10 vol), 0.1M HCI (1 vol), water (5 vol) and DMF (5 vol).

[1192] General Procedure for Staudinger Reaction:

[1193] To verify the completion of the CuAAC reaction, a Staudinger reaction was performed on two peptide samples: one prior to the click reaction (unclicked peptide) and oneP7529PC00

[1194] following the CuAAC reaction. Both samples were dissolved in MQ- water and degassed for 10 min. Tris(2-carboxyethyl)phosphine (TCEP) was added and the vials were placed on the shaker for 1h before analyzing by LCMS.

[1195] Resin loading:

[1196] To determine the loading of the resin after the MSNT coupling, 100 pL of the resin were taking into a second syringe and washed with DCM and DMF. The resin was treated with 20% (v / v) piperidine in DMF for 5 min and then washed with 5 mL DMF. The filtrate was collected in a vial and then the UV absorption was measured with the Nanodrop machine 2000. First a blank with pure DMF was measured and then from the sample at 295 and 300 nm. The MSNT coupling was repeated until the absorbance was between 0.2 and 0.3.

[1197] General Procedure for Fluorophore Coupling:

[1198] The fluorophore (ATOTA or Alexa Fluor 488) (1.1 eq) was coupled under standard peptide coupling. Because of its high costs only 1.1 eq were used. Therefore COMU, OXYMA and NEM were adjusted to 1.1 eq accordingly and it was coupled overnight. The resin was washed with DMF (10 vol), DCM (10 vol) and DMF (10 vol) and water (10 eq).

[1199] Synthesis of 1H-imidazole-1 -sulfonyl azide hydrochloride:

[1200] O

[1201]

[1202] •HCl

[1203] Sulfuryl chloride (2.99 mL, 37.05mmol) was added dropwise to an ice-cooled suspension of Sodium azide (2.41 g, 37.05 mmol, 1 eq) in acetonitrile (50 mL) and the mixture was stirred overnight at room temperature. The mixture was cooled to 0°C and imidazole (4.79 g, 70.39 mmol, 1.9 eq) was added portion-wise and it was stirred for 3h at room temperature. The mixture was diluted with EtOAc (100mL) and washed with water (2 x 100mL) followed by saturated NaHCO₃ (2 x 100mL). The organic phase was dried over Na₂SO₄ and filtered. A solution of HCI in EtOH (obtained by drop-wise addition of AcCI (3.97 mL, 55.57 mmol, 1.5 eq) to ice-cooled dry EtOH (30 mL)) was added dropwise to the ice-cooled filtrate. It was filtered and the filter cake was washedP7529PC00

[1204] with EtOAc (3 x 50 mL) to give the final compound as a colourless powder (7.10 g, 33.9 mmol, 91%) (Goddard-Borger and Stick 2007).

[1205] 1H NMR (500 MHz, D2O) 59.16 (s, 1 H), 7.97 (d, J = 6.0, 3.2 Hz, 1 H), 7.57 – 7.50 (d, 1H).

[1206] 13C NMR (126 MHz, D2O) δ 137.73, 124.00, 119.98.

[1207] LC-MS m / z: calculated exact mass: 173.0007, [M+H]+: 174.0008; found 174.0069.

[1208] Synthesis of (2S,4R)-4-azidopyrrolidine-2-carboxylic acid (2):

[1209] OH

[1210]

[1211] Trans-4-amino-L-proline (1, 1.04 g, 8 mmol) and Copper (II) sulfate pentahydrate (39.95 mg, 0.16 mmol, 0.02 eq) were dissolved in water (50 mL). The pH was adjusted to 9 with K2CO3. 1H-imidazole-1 -sulfonyl azide hydrochloride (2.01 g, 9.6 mmol, 1.2 eq) was added followed by a readjustment of the pH to 9 by using K2CO3. The reaction was stirred overnight at room temperature. It was concentrated and the crude (2) used without further purification.

[1212] Synthesis of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4- azidopyrrolidine-2-carboxylic acid (3):

[1213]

[1214] The crude 2 (1.25 g, 8 mmol) was dissolved in saturated NaHCO₃ (30 mL) and Fmoc-Osu (5.49 g, 16 mmol, 2 eq) was dissolved in THF (20 mL) and added to the mixture. The mixture was stirred overnight at room temperature, THF was removed in vacuo and the remaining aqueous phase was acidified to pH 2 using 1M HCI. It was extracted with EtOAc (3 x 50 mL), dried over Na2SO4, filtered and concentrated. The residue was purified by column chromatography (SiO2, v / v, MeOH / DCM, 1 / 20 + 0.1% AcOH) to provide (2S,4R)-1-(((9H-fluoren-9- yl)methoxy)carbonyl)-4-azidopyrrolidine-2-carboxylic acid, 3 (1.63 g, 4.31 mmol, 54%) as a colourless powder.P7529PC00

[1215] 1H NMR (500 MHz, Methanol-d₄) δ 7.86 - 7.78 (m, 2H), 7.71 - 7.60 (m, 2H), 7.41 (ddd, J= 8.0, 6.3, 4.5 Hz, 2H), 7.33 (tddd, J = 9.0, 4.2, 3.4, 1.4 Hz, 2H), 4.49 - 4.38 (m, 2H), 4.36 (dq, J = 6.9, 3.6 Hz, 1 H), 4.33 - 4.28 (m, 1 H), 4.22 (t, J = 6.9 Hz, 1 H), 3.73 -3.59 (m, 2H), 2.52 -2.37 (m, 1H), 2.25 (dddd, J = 35.2, 13.4, 7.4, 5.4 Hz, 1H).

[1216] 13C NMR (126 MHz, Methanol-d₄) δ 174.89, 142.61, 128.87, 128.23, 126.22, 126.12, 126.08, 120.97, 68.85, 61.09, 60.32, 53.05, 52.71, 48.37, 37.46, 36.42, 26.31.

[1217] LC-MS m / z: calculated exact mass: 378.1328, [M+Na]+: 401.1226; found 401.1190.

[1218] Synthesis of 1-((9H-fluoren-9-yl) methyl) 2-methyl (2S,4R)-4- azidopyrrolidine-1,2-dicarboxylate (4):

[1219]

[1220] The compound 3 (800 mg, 2.11 mmol) was dissolved in MeOH (20 mL) and cooled to 0°C. Thionyl chloride (800 mg, 4.23 mmol, 2 eq) was added drop-wise and the mixture was stirred under reflux for 2.5h. The crude was concentrated under vacuo

[1221] and the brown oil was used without further purification.

[1222] Synthesis of 1-((9H-fluoren-9-yl) methyl) 2-methyl (2S,4R)-4-aminopyrrolidine-1,2-dicarboxylate (5):

[1223] OMe

[1224]

[1225] Triphenylphosphine (449 mg, 1.71 mmol, 1.5 eq) was added to a solution of the previous crude azide 4 (448 mg, 1.14 mmol) in THF (30 mL) under inert atmosphere and water (82 pL, 4.57 mmol, 4 eq) was added. The mixture was stirred at reflux for 8h. The mixture was concentrated and dissolved in Et20 (25 mL) and 0.1M HCI (25 mL). The aqueous phase was extracted 3 times with Et20 (3x25 mL). The aqueous phase was neutralized with saturated Na₂CO₃ and extracted with DCM (3x 30mL). It was dried over Na₂SO₄ and concentrated. The residue was purified by columnP7529PC00

[1226] chromatography (SiO₂, MeOH / DCM, 1 / 20, 1 / 10) to afford the desired compound, 5 (280 mg, 0.76 mmol, 67%) as a colourless powder.

[1227] 1H NMR (500 MHz, Methanol-d₄) δ 7.83 (dd, J= 7.8, 3.3 Hz, 2H), 7.62 (dq, J= 20.3, 7.6, 7.0 Hz, 2H), 7.42 (t, J = 7.7 Hz, 2H), 7.34 (tt, J = 7.8, 5.4 Hz, 2H), 4.58 - 4.44 (m, 2H), 4.43 - 4.37 (m, 1 H), 4.24 (dt, J = 47.8, 6.3 Hz, 1 H), 4.01 - 3.88 (m, 1 H), 3.82 (ddd, J= 11.9, 6.5, 2.4 Hz, 1H), 3.64 (s, 3H), 2.47-2.34 (m, 2H).

[1228] 13C NMR (126 MHz, MeOD) δ 173.33, 142.65, 128.89, 128.19, 126.01, 125.92, 125.83, 125.76, 121.04, 68.94, 68.86, 58.88, 58.35, 53.33, 53.15, 51.16, 48.83, 48.66, 48.49, 35.38, 34.36.

[1229] LC-MS m / z: calculated exact mass: 366.1580, [M+H]+: 367.1653; found: 367.1667.

[1230] Synthesis of 1-((9H-fluoren-9-yl)methyl) 2-methyl (2S,4R)-4-((naphthalen-2-yl methyl) amino)pyrrolidine- 1, 2-dicarboxylate (6):

[1231]

[1232] To a solution of 1-((9H-fluoren-9-yl)methyl) 2-methyl (2S,4R)-4- aminopyrrolidine-1,2-dicarboxylate (5) (300 mg, 0.82 mmol) and 2-naphthaldehyde (153 mg, 0.98 mmol, 1.2 eq) in dry DCM (15 mL) was added acetic acid (46.8 pL, 0.82 mmol, 1 eq) under inert atmosphere. The mixture was cooled to 0°C and sodium cyanoborohydride (102.9 mg, 1.64 mmol, 2 eq) was added. It was allowed to warm up to room temperature and the reaction was stirred overnight. The mixture was quenched with water (20 mL), extracted with DCM (3x20 mL), dried over Na₂SO₄ and concentrated. Further purification through column chromatography (SiO₂, v / v, heptane / EtOAc, 2 / 1, 1 / 1) provided 1-((9H-fluoren-9-yl)methyl) 2-methyl (2S,4R)-4- ((naphthalen-2-ylmethyl)amino)pyrrolidine-1, 2-dicarboxylate, 6 (214 mg, 0.42 mmol, 52%) as a colourless powder.

[1233] 1H NMR (500 MHz, Chloroform-d) δ 7.82 (ddd, J = 14.3, 7.3, 3.9 Hz, 4H), 7.75 (dd, J = 7.6, 3.0 Hz, 2H), 7.59 - 7.53 (m, 2H), 7.50 - 7.45 (m, 3H), 7.38 (td, J = 7.1, 2.8 Hz, 2H), 7.30 (td, J = 7.6, 4.7 Hz, 2H), 4.49 (t, J = 6.5 Hz, 1 H), 4.47 - 4.33 (m, 3H), 4.29 -P7529PC00

[1234] 4.19 (m, 1 H), 4.20-4.08 (m, 1 H), 4.01 -3.93 (m, 2H), 3.84 (dd, J= 10.6, 6.3 Hz, 1H), 3.72 (s, 2H), 3.69- 3.57 (m, 3H), 3.57-3.45 (m, 1H), 2.29-2.16 (m, 2H).

[1235] 13C NMR (126 MHz, Chloroform-d) δ 141.32, 141.29, 133.35, 128.57, 127.74, 127.71, 127.67, 127.10, 126.51, 126.44, 126.36, 126.16, 125.14, 125.11, 125.01, 124.95, 119.97, 67.62, 58.14, 57.75, 52.44, 47.27, 47.14, 29.71.

[1236] LC-MS m / z: calculated exact mass: 506.2206, [M+H]+: 507.2279; found 507.2265.

[1237] Synthesis of 1-((9H-fluoren-9-yl)methyl) 2-methyl (2S,4R)-4-((tert-butoxycarbonyl) (naphthalen-2-ylmethyl) amino)pyrrolidine- 1, 2-dicarboxylate (7):

[1238]

[1239] To a solution of the amine (6) (91 mg, 0.18 mmol) in THF (3 mL) and saturated NaHCO₃ (3mL) was added di-tert-butyl decarbonate (47 mg, 0.22 mmol, 1.2 eq) and stirred overnight at 50°C. The mixture was quenched with water (3 mL) and extracted with DCM (3 x5 mL). It was dried over Na₂SO₄ and concentrated under reduced pressure. The crude yellow-white powder, 7 was used without further purification.

[1240] Synthesis of (2S,4R)-1-(((9H-fluoren-9-yl)methoxy)carbonyl)-4-((tert-butoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-2-carboxylic acid (P8):

[1241]

[1242] To a solution of 1-((9H-fluoren-9-yl)methyl) 2-methyl (2S,4R)-4-P7529PC00

[1243] ((tertbutoxycarbonyl)(naphthalen-2-ylmethyl)amino)pyrrolidine-1,2-dicarboxylate (7) (80 mg, 0.13 mmol) in 0.8M CaCl2in i-PrOH / H₂O (v / v, 7 / 3) was added solid Sodium hydroxide (7.9 mg, 0.19 mmol, 1.5 eq). The mixture was stirred overnight at room temperature. Then the pH of the mixture was adjusted to 5 with 1M citric acid. / -PrOH was removed under reduced pressure and water (5 mL) and DCM (5 mL) was added. It was extracted three times, dried over Na₂SO₄ and concentrated under vacuo (Che et al. 2017; and Pascal and Sola 1998).

[1244] The crude yellow oil was purified by column chromatography (SiO₂, v / v, MeOH / DCM, 1 / 20, 1 / 10, 1 / 5) and a colourless solid (72 mg, 0.12 mmol, 92%).

[1245] 1H NMR (500 MHz, Chloroform-d) δ 7.88 – 7.74 (m, 3H), 7.73 - 7.64 (m, 2H), 7.59 (d, J= 15.8 Hz, 1H), 7.55 - 7.41 (m, 4H), 7.39 - 7.28 (m, 3H), 7.17 (dt, J = 14.1, 7.4 Hz, 1H), 4.66 (d, J = 16.1 Hz, 1H), 4.60 - 4.41 (m, 2H), 4.43 - 4.34 (m, 1H), 4.34 - 4.18 (m, 2H), 4.13 (dt, J= 14.7, 7.0 Hz, 1H), 3.81 - 3.57 (m, 1H), 3.59- 3.40 (m, 1H), 2.46 (dd, J= 17.6, 7.6 Hz, 1H), 2.32 (d, J = 12.7 Hz, 1H), 2.19 (q, J = 5.9, 4.2 Hz, 1H), 1.44 (d, J = 17.3 Hz, 9H).

[1246] 13C NMR (126 MHz, CDCl₃) δ 162.79, 155.91, 141.25, 136.21, 128.58, 127.72, 127.05, 126.37, 125.89, 119.94, 77.28, 77.03, 76.78, 68.03, 58.06, 48.98, 47.19, 47.01, 36.58, 31.53, 29.71, 28.38.

[1247] LC-MS m / z: calculated exact mass: 592.2573, [M+Na]+: 615.2471; found 615.2417.

[1248]

[1249] (CPP1)

[1250] 1-(3-(4-((2,10-bis(dimethylamino)-4,8,12-trioxadibenzo[cd,mn]pyren-3a2(4H)-ylium- 3a2-ylium-6-yl)(methyl)amino)butanamido)-thr-orn(tmG)-orn(N₃)-orn(tmG)-pro-1Nal-P7529PC00

[1251] orn(tmG)-pra-orn(tmG)-leu-OH where tmG is tetramethylguanidinylation.

[1252]

[1253] Alexa-fluor-488-pra-thr-orn(tmG)-orn(N₃)-orn(tmG)-pro-1Nal-orn(tmG)-pra-orn(tmG)-leu-OH where tmG is tetramethylguanidinylation.

[1254] To 3 mL of resin the HMBA linker was attached and the first amino acid, D-leucine, was coupled twice for sufficient resin loading (Table B). The ten amino acids were attached and Fmoc was removed. CuACC reaction was carried out followed by the coupling of the last amino acid. Fmoc was not cleaved off and the side chains were deprotected. The tetramethylguanidinylation was performed before removing the Fmoc and attaching the fluorophore ATOTA or Alexa Fluor 488.

[1255] CPP1:

[1256] The peptide was cleaved off the resin and purified by preparative RP-HPLC, yielding 10.4 mg, corresponding to 9% of the theoretical yield. LC-MS m / z: calculated exact mass: 2177.2989, M5+: 435.4598, found: 435.4657.

[1257] CPP1-AlexaFluor:

[1258] The coupling product of MCCPP1 and Alexa-Fluor was cleaved off the resin and purified by preparative RP- HPLC, yielding 0.21 mg, corresponding to 8% of the theoretical yield. LC-MS m / z: calculated exact mass: 2224.0932, M3+: 741.3644, found: 741.3682.P7529PC00

[1259] Table B. Reagents used for CPP1 and CPP1-AlexaFluor.

[1260] AA eq MW M COMU NEM Oxy ma (mg) (mg) (uL) (mg) AA- Fmoc-leu- MSNTester 5 353,16 211,9 174,05 48,22 0 1 OH

[1261] AA- Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 2 orn(Boc)- OH AA- Fmoc- Standard 5 335,12 201,07 251,66 82,5 21,6 3 pra- OH AA- Fmoc- Standard 5 454,21 9272,5 251,66 82,5 21,6 4 orn(Boc)- 3

[1262] OH

[1263] Aa Fmoc- Standard 5 437,16 262,3 251,66 82,5 21,6 - 5 1Nal- OH

[1264] Aa Fmoc-p- Standard 5 337,13 202,28 251,66 82,5 21,6 - 6 OH

[1265] Aa Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 - 7 orn(Boc)- OH

[1266] Aa Fmoc- Standard 2 380,15 228,09 251,66 82,5 21,6 - 8 orn(N3)- OH

[1267] Aa Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 - 9 orn(Boc)- OH

[1268] Aa Fmoc- Standard 5 397,19 238,31 251,66 82,5 21,6 t(tBu)- OH

[1269] 10

[1270] Aa Fmoc- Standard 5 335,12 201,07 251,66 82,5 21,6 pra- OH

[1271] 11

[1272] Aa- ATOTA Standard 1,5 631,5 113,67 75,33 24,75 6,48

[1273]

[1274] 12P7529PC00

[1275] Synthesis of CPP2:

[1276]

[1277] Me

[1278] 1-(3-(4-((2,10-bis(dimethylamino)-4,8,12-trioxadibenzo[cd,mn]pyren-3a2(4H)-ylium- 3a2-ylium-6-yl)(methyl)amino)butanamido)-pra-thr-orn(tmG)-orn(N₃)-orn(tmG)-pro-2Nal-orn(tmG)-pra-orn(tmG)-leu-OH where tmG is tetramethylguanidinylation.

[1279] To 3 mL of resin the HMBA linker was attached and the first amino acid, D-leucine, was coupled twice for sufficient resin loading. The ten amino acids were attached and Fmoc was removed (Table C). CuACC reaction was carried out followed by the coupling of the last amino acid. Fmoc was not cleaved off and the side chains were deprotected. The tetramethylguanidinylation was performed before removing the Fmoc and attaching the fluorophore ATOTA. The peptide was cleaved off the resin and purified by preparative RP- HPLC, yielding 10 mg, corresponding to 10% of the theoretical yield. LC-MS m / z: calculated exact mass: 2177.2989, M5+: 435.4598, found: 435.4657.

[1280] Table C. Reagents used for CPP2.

[1281] AA eq MW M COMU NEM Oxyma (mg) (mg) (uL) (mg) Aa - Fmoc- MSNTester 5 353,16 211,9 174,05 48,22 0 1 leu- OH

[1282] Aa - Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 2 orn(Boc)-

[1283]

[1284] OHP7529PC00

[1285] Aa - Fmoc- Standard 5 335,12 201,07 251,66 82,5 21,6 3 pra-OH

[1286] Aa - Fmoc- Standard 5 454,21 9272,53 251,66 82,5 21,6 4 orn(Boc)- OH

[1287] Aa - Fmoc- Standard 5 437,16 262,3 251,66 82,5 21,6 5 1Nal- OH

[1288] Aa - Fmoc-p- Standard 5 337,13 202,28 251,66 82,5 21,6 6 OH

[1289] Aa - Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 7 orn(Boc)- OH

[1290] Aa - Fmoc- Standard 2 380,15 228,09 251,66 82,5 21,6 8 orn(N3)- OH

[1291] Aa - Fmoc- Standard 5 454,21 272,53 251,66 82,5 21,6 9 orn(Boc)- OH

[1292] Aa - Fmoc- Standard 5 397,19 238,31 251,66 82,5 21,6 10 t(tBu)- OH

[1293] Aa - Fmoc- Standard 5 335,12 201,07 251,66 82,5 21,6 11 pra- OH

[1294] Aa- ATOTA Standard 1,5 631,5 113,67 75,33 24,75 6,48

[1295]

[1296] 12P7529PC00

[1297] Synthesis of CPP3:

[1298]

[1299] Me

[1300] Click cyclized 1-(3-(4-((2,10-bis(dimethylamino)-4,8,12-trioxadibenzo[cd,mn]pyren- 3a2(4H)-ylium-3a2-ylium-6-yl)(methyl)amino)butanamido)-pra-thr-orn(tmG)-orn(N3)-orn(tmG)-P8-Gly-orn(tmG)-pra-orn(tmG)-leu-OH.

[1301] To 0.5 mL of resin the HMBA linker was attached and the first amino acid, D-leucine, was coupled twice for sufficient resin loading. The ten amino acids were attached and Fmoc was removed (Table D). CuACC reaction was carried out followed by the coupling of the last amino acid. Fmoc was not cleaved off and the side chains were deprotected. The tetramethylguanidinylation was performed before removing the Fmoc and attaching the fluorophore ATOTA. The peptide was cleaved off the resin and purified by preparative RP-HPLC, yielding 1 mg, corresponding to 6% of the theoretical yield.

[1302] LC-MS m / z: calculated exact mass: 2192.3098, M5+:438.4620; found 438.4077.

[1303] Table D. Reagents used for CPP3.

[1304] AA eq MW M COMU NEM Oxyma (mg) (mg) (uL) (mg) Aa - Fmoc-leu- MSNTester 5 353,16 70,63 58,02 16,07 0 1 OH

[1305] Aa - Fmoc- Standard 5 454,21 90,84 83,89 27,5 7,2 2 orn(Boc)- OH AA- Fmoc-pra- Standard 5 335,12 67,02 83,89 27,5 7,2

[1306]

[1307] 3 OHP7529PC00

[1308] Aa - Fmoc- Standard 5 454,21 90,84 83,89 27,5 7,2 4 orn(Boc)- OH

[1309] Aa - Fmoc-G-OH Standard 5 297,1 59,42 83,89 27,5 7,2 5

[1310] Aa - Fmoc-L- Standard 2 592,69 47,42 34 11 3 6 Pro(NH-2- naphtylmeth

[1311] yi)

[1312] Aa - Fmoc- Standard 5 454,21 90,84 83,89 27,5 7,2 7 orn(Boc)- OH

[1313] Aa - Fmoc- Standard 2 380,15 76,03 83,89 27,5 7,2 8 orn(N3)-OH

[1314] Aa - Fmoc- Standard 5 454,21 90,84 83,89 27,5 7,2 9 orn(Boc)- OH

[1315] Aa - Fmoc- Standard 5 397,19 79,44 83,89 27,5 7,2 10 t(tBu)-OH

[1316] Aa - Fmoc-pra- Standard 5 335,12 67,02 83,89 27,5 7,2 11 OH

[1317] Aa- ATOTA Standard 1,5 631,5 37,89 25,11 8,25 2,16

[1318]

[1319] 12P7529PC00

[1320] Synthesis of tetra-butylated cCPPs (CW-1, CW-2, and CW-3)

[1321] Peptide couplings were performed on a 10 mL scale, 6 mL scale, and 3 mL scale as indicated in Table E, F, and G, respectively.

[1322] Table E. The first amino acid couplings were performed on a 10 mL bead scale.

[1323] AA Method eq mw AA MSNT / COMU NEM / MEIM OXYMA (mg) (mg) (µL) (mg) 0 HMBA Std 5 304.1 838.88 275 72 1 Fmoc-I- MSNT 5 706.3 580.16 160.7 0 OH

[1324] 2 Fmoc- Std 5 908.4 838.88 275 72 O(Boc)- OH

[1325] 3 Fmoc- Std 5 670.2 838.88 275 72 Pra-OH

[1326] 4 Fmoc- Std 2.5 454.2 419.44 137.5 36 O(Boc)- OH

[1327] 5 Fmoc- Std 5 594.2 838.88 275 72

[1328]

[1329] G-OH

[1330] Table F. The later amino acid couplings were performed on a 6 mL bead scale.

[1331] AA Method eq mw AA MSNT / COMU NEM / MEIM OXYMA (mg) (mg) (µL) (mg) 6 Fmoc- Std 2 209.5 201.3 66 17.3 P(NH- Alloc)

[1332] 7 Fmoc- Std 5 545 503.33 165 43.2 O(Boc)- OH

[1333] 8 Fmoc- Std 1.65 150 165.56 54.3 14.2 O(N3)- OH

[1334] 9 Fmoc- Std 5 545 503.33 165 43.2 O(Boc)- OH

[1335] 10 Fmoc- Std 5 476.6 503.33 165 43.2 T(tBu)-

[1336]

[1337] OH

[1338] Table G. The coupling with 4-pentynoic acid was performed on a 3 mL bead scale.P7529PC00

[1339] AA Method eq mw AA MSNT / COMU NEM / MEIM OXYMA (mg) (mg) (µL) (mg) 11 4- Std 5 58.8 251.66 82.5 21.6 pentynoic

[1340]

[1341] acid

[1342] 10 ml beads swelled in 3:7 EtOH / H2O were added to a 100 ml filter column with polyethylene frit. The beads were washed 3 times with DMF and swelled in DMF overnight. HMBA (304.1 mg), COMU (838.88 mg) and OXYMA (72 mg) were dissolved in a vial with DMF to a total volume of 10 ml and 275 pl NEM was added to the solution and it turned yellow. The solution was then added to the beads on the column and left for 1.5 h.

[1343] The column was drained until the beads became a more slushy suspension and the suspension was washed with 5 vol of DMF and then 3 vol DCM.

[1344] 2 vials of Fmoc-d-leucine-OH (706.3 mg, 5 eq) and MSNT (580.16 mg) were prepared. The content of the first vial was suspended in 10 ml of dry DCM and then 160 ul MEIM was quickly added. The suspension turned dark yellow and it was immediately added to the beads which was then quickly stirred using a plastic pipette and a septum was added. A clear layer formed immediately on the bottom and the reaction was left for 1 h before it was washed with DCM (x3) and then DMF (x3). The process was then repeated once.

[1345] 100 pL beads were transferred to a smaller filter column and suspended in 500 pL 20% piperidine in DMF for 20 min. The filter column was drained into a smaller vial and washed with DMF until a total volume of 5 ml was collected. UV-VIS measurements were then performed on 1 drop of solution repeated 3 times for determining the loading on the beads.

[1346] Amino acid couplings started with Fmoc deprotection by suspending the beads in 20% piperidine in DMF for 20 min, then washed with DMF (x3). Amino acid, COMU and OXYMA were suspended in DMF in a vial before NEM was added and the mixture was mixed with a pipette for 3 min and then added to the bead mixture. The filter column was plugged with a septum and left for 1 h before washing with DMF (x3).P7529PC00

[1347] The third amino acid coupling was deprotected for 3 min with a quick stir using a cut-up plastic pipette, when the piperidine mixture was added and when the activated coupling reagent was added, it was quickly stirred again.

[1348] After coupling the first 5 amino acids, the bead solution was split into smaller portions, and the following couplings were performed on 6 ml beads.

[1349] After 10 amino acid couplings the beads were cleaved to confirm the amino acid sequence.

[1350] Cleaving peptide from solid support:

[1351] The beads were cleaved by first deprotection in 20% piperidine in DMF for 20 min, then washing DMF (x3) and DCM (x3) and then suspended in 95% TFA in water for 1h. Then the beads were drained completely dry, washed in DCM (x3) then DMF (x3) 2% DIPEA in DMF (x3) DMF (x3) and then MILIQ water (x10). The beads were then suspended in freshly prepared 0.1 M NaOH and covered with parafilm for 1 h. The filter column was then drained and collected in a small vial and the filter column was washed with the same volume of 0.1 M HCI as the 0.1 M NaOH added.

[1352] Click cyclization reaction:

[1353] After coupling the 10 first amino acids, a click reaction was performed by first taking out a small sample of -100 pL beads, which were saved for later analysis / comparison. The rest of the beads were washed with MILIQ water (x10) and transferred to a vial with MILIQ water resulting in a total volume of 10 ml. The bead solution was then degassed using N2. Meanwhile 60 mg tetrakis acetonitrile Cu(l)triflate, 35 mg THPTA in 5 ml DMSO were degassed in another vial and then added to the degassed vial with the beads. The vial was then tightly sealed with N2 atm and placed on a shaker overnight. The now yellow solution was then transferred to a filter column and washed with MILIQ water (x10) DMF (x5) and then DCM (x10). The beads were then washed quickly with 0.1 M HCI to remove excess Cu.

[1354] To test if the click reaction was complete the beads from the click reaction and the -100 pL sample were cleaved and a small spatula of TCEP was added to reduce any leftover azide to the corresponding amine and left over night.P7529PC00

[1355] Alloc deprotection:

[1356] The bead solution was washed with DCM 10 times, then swelled in dry DCM and then degassed on sonicator with N2 bubbling for 10 min and then sealed under N2 atm. Meanwhile a solution of 68 mg Pd(PPh3)4in dry DCM and 0.48 mL PhSiH3in dry DCM were degassed for 10 min. First the PhSiH3solution was added to the beads and thenthe Pd(PPh3)4solution and the reaction was swirled manually and left for 1 h under N2 atm. The beads were then drained clean, washed with 5 x DCM, 5 x DMF and then quickly washed with 0.1 M HCI to remove leftover Pd. The procedure was repeated 3 times.

[1357] Reductive amination:

[1358] 3 ml beads were washed 3 times in 4:1 DMF MeOH. 383 mg 2-naphthaldehyde was dissolved in 2 mL 4:1 DMF MeOH with 1% AcOH and then added to the beads for 10 min at 45 °C and placed on the shaker. After 10 min 25 mg NaCNBH3was added every 10 minutes a total of 3 times. The reaction was the left with a cap on over night at room temperature and then washed 3 times in 4:1 DMF MeOH, 5 x DCM and then 5x DMF.

[1359] 4-pentynoic acid coupling:

[1360] A peptide coupling with 4-pentynoic acid is carried out as described earlier starting with Fmoc deprotection by suspending the beads in 20% piperidine in DMF for 20 min, then washed with DMF (x3). 4-pentynoic acid, COMU and OXYMA was suspended in DMF in a vial before NEM was added and the mixture was mixed with a pipette for 3 min and then added to the bead mixture. The filter column was sealed with a septum and left for 1 h before washing with DMF (x3).

[1361] Synthesis of N-butyl-N-(chloro(dibutylamino)methylene)butan-1-aminium (14):

[1362] Cl

[1363]

[1364] To a solution of 1,1,3,3-tetrabutylurea (3.24 mL, 10 mmol) in dry DCM was added oxalyl chloride (4.29 mL, 50 mmol, 5 eq) under inert atmosphere. The mixture was stirred at reflux overnight. The solvent was removed in vacuo and ACN (40 mL) and heptane (40 mL) was added and extracted. The ACN layer was then concentrated underP7529PC00

[1365] reduced pressure and N-butyl-N-(chloro(dibutylamino)methylene)butan-1-aminium (2.84 mg, 9.34 mmol, 93%) was obtained as a brown oil.

[1366] 1H NMR (500 MHz, Chloroform-d) δ 3.93 - 3.54 (m, 6H), 3.33 (t, J = 8.2 Hz, 2H), 1.71 - 1.51 (m, 8H), 1.31 (ddt, J= 14.9, 12.5, 7.4 Hz, 8H), 0.96-0.81 (m, 12H).

[1367] 13C NMR (126 MHz, CDCl3) δ 159.15, 77.41 77.16, 76.90, 55.53, 29.93, 19.91- 19.87, 13.62.

[1368] LC-MS m / z: calculated exact mass: 303.2562; found 303.2284.

[1369] Guanidinylation:

[1370]

[1371] (CW-1: Click cyclized Pent-4-ynamido-thr-orn(tbG)-orn(N₃)-orn(tbG)-P8-Gly-orn(tbG)-pra-orn(tbG)-leu-OH where tbG is tetrabutylguanidinylation).

[1372] (CW-2: Click cyclized Pent-4-ynamido-thr-orn(tbG)-orn(N3)-orn(tbG)-pro(4-(di-naphth-2-ylmethyl) amino)-Gly-orn(tbG)-pra-orn(tbG)-leu-OH where tbG is

[1373] tetrabutylguanidinylation).P7529PC00

[1374] (CW-3: Click cyclized Pent-4-ynamido-thr-orn(tbG)-orn(N3)-orn(tbG)-pro(4-((N-(naphth-2-ylmethyl))-pent-4-ynamido))-Gly-orn(tbG)-pra-orn(tbG)-leu-OH where tbG is tetrabutylguanidinylation).

[1375] 3 mL beads were suspended in 95% TFA in water for 1 h, then washed with 3 x DCM, 3x DMF, 3 x 2% DI PEA in DMF and then 3x DMF. The beads were then suspended in 20% NEM in DMF. 166.5 mg (10 eq) of guanidinylation reagent 14 was dissolved in 1-2 mL 20% NEM in DMF and then added to the bead solution. The solution was stirred quickly and was left with a cap on for 1 h. The bead solution was then washed with with 3 x DMF, 3 x water, 3 x DCM and then 3 x DMF again. The procedure was repeated 3 and an extra 4thtime with 240 mg guanidinylation reagent 14, to yield mixture of compounds CW-1, CW-2, and CW-3.

[1376] ESI MS spectrum at 4.50 min TIC peak showing mass corresponding to cyclic peptide CW-1 Calc (M) (C131H236N26O134+) 595.4645 Found 595.3209.

[1377] ESI MS spectrum at 4.88 min TIC peak showing mass corresponding to cyclic peptide CW-2 Calc (M) (C142H244N26O134+) 630.4802 Found 630.3322.

[1378] ESI MS spectrum at 4.25 min TIC peak showing mass corresponding to cyclic peptide CW-3 Calc (M) (C136H240N26O144+) 615.4711 Found 615.3274.

[1379] Synthesis of ATOTA-N3 (6-((4-((3-azidopropyl)amino)-4-oxobutyl)(methyl)amino)-2, 10-bis(dimethylamino)-3a2H-4,8, 12-trioxadibenzo[cd,mn]pyren-3a2(4H)-ylium-3a2-ylium hexafluorophosphate(V)):

[1380]

[1381] In a 20 ml glass vial, ATOTA-COOH (126 mg, 199.75 pmol) and COMU (1 eq, 85.5 mg) was dissolved in 2 mL DMF. NEM (1.1 eq, 28 pL) was then added to the solution followed by 3-Azidopropylamine (1 eq, 19.6 pL). After 2.5 h additional COMU was added (0.5 eq, 42.75 mg) and the reaction was left over night. The deep red crude mixture of ATOTA-N3 was evaporated and then used in the next reactions without further purification.

[1382] MALDI-MS of crude mixture containing ATOTA-N3. Calc (M+4) (C31H34N7O4+) 568.2666 Found 568.8.P7529PC00

[1383] Synthesis ofATOTA-CW-1 and ATOTA-CW-2:

[1384] BUN

[1385]

[1386] BUBU(ATOTA-CW-1) Click cyclized 1-(3-(4-((2,10-bis(dimethylamino)-4,8,12-trioxadibenzo[cd,mn]pyren- 3a2(4H)-ylium-3a2-ylium-6-yl)(methyl)amino)butanamido)propyl)-4-1H-1,2,3-triazoyl-3-propamido-thr-orn(tbG)-orn(N3)-orn(tbG)-P8-Gly-orn(tbG)-pra-orn(tbG)-leu-OH where tbG is tetrabutylguanidinylation.

[1387]

[1388] (ATOTA-CW-2) Click cyclized 1-(3-(4-((2,10-bis(dimethylamino)-4,8,12-trioxadibenzo[cd,mn]pyren- 3a2(4H)-ylium-3a2-ylium-6-yl)(methyl)amino)butanamido)propyl)-4-1H-1,2,3-triazoyl-3-propamido-thr-orn(tbG)-orn(N3)-orn(tbG)-pro(4-di-(naphth-2-ylmethyl) amino)-Gly-orn(tbG)-pra-orn(tbG)-leu-OH where tbG is tetrabutylguanidinylation.

[1389] Procedure: 1 mL resin containing peptide mixture CW-1, CW-2, and CW-3 were washed in water (10x) and then transferred to a 20 mL glass vial. The crude ATOTA-N3 mixture was then added to the vial and a white / light red precipitate formed. DMSO wasP7529PC00

[1390] then added to the reaction mixture until the precipitate was dissolved resulting in a total volume of ~10 mL. The reaction mixture was then degassed by first sonicating for 2 min, then bubbling with N₂ for 10 min. The degassing was then repeated one more time. In another 20 mL glass vial, tetrakis acetonitrile Cu(l)triflate (27 mg), THPTA (16 mg) in 3 ml DMSO were degassed for 20 min by bubbling with N₂. After degassing both vials, the tetrakis acetonitrile Cu(l)triflate solution was then added to the resin mixture under N₂. The reaction vial was then degassed by N₂ bubbling for 10 min before it was sealed and placed on a shaker overnight. The reaction mixture was transferred to a filter column and the filtrate was collected. The resin was then washed with water (10x), 0.1 M HCI (1x) DMF (10x), DCM (10x), DMF (3x), 2% DIPEA in DMF (1x) and then DMF (3x). The resin was then washed in water (10x) and transferred to a 20 mL glass vial with the collected filtrate and degassed for 20 min. Meanwhile tetrakis acetonitrile Cu(l)triflate (27 mg), THPTA (16 mg) in 1 ml DMSO was degassed for 20 min in another vial and the Cu(l) solution was then added to the resin mixture. The reaction mixture was then degassed for 10 min before it was sealed and left on a shaker overnight. The resin was then washed with water (10x), 0.1 M HCI (1x) DMF (10x), DCM (10x), DMF (3x), 2% DIPEA in DMF (1x) and then DMF (3x).

[1391] Purification: 1 mL resin containing the mixture of ATOTA-peptides (AT0TA-CW1 and AT0TA-CW2) was washed in MILIQ water (10x) and then 4 mL freshly prepared 0.1 M NaOH was added before the filter column was sealed and left for 1.5 h. The colorless filtrate was then collected in a vial and the resin was washed with water and the resulting colorless filtrate was then collected in another vial. The resin was now washed with acetonitrile and the collected filtrate was deep orange. The acetonitrile wash was repeated 7 times total until the filtrate was no longer strongly colored. The resin was then washed with water (2x) and then 0.1 M HCI, all resulting in colorless filtrates. Then the resin was again washed with acetonitrile resulting in another deep orange filtrate and then repeated until the collected filtrates were nearly colorless. The washes with water, HCI and MeCN were all tried again but none resulting in colored filtrates. All the colored filtrates in acetonitrile were combined and evaporated in vacuo.

[1392] The deep red / orange crude was then purified on a custom HPLC with a C18 column using solvents MeCN / H₂O 9:1 + 0.1% FA and H₂O + 0.1% FA. ATOTA-CW-1 elutes at 36.28-38.03 min and ATOTA-CW-2 elutes at ~40min+ (the timer stopped).P7529PC00

[1393] ATOTA-CW-1:

[1394] ESI MS spectrum at UV peak 4.77 min TIC peak showing mass corresponding to ATOTA-1 Calc (M) (C162H270N33O175+) 590.0250 Found 590.0169.

[1395] ATOTA-CW-2:

[1396] ESI MS spectrum at UV peak 4.87 min TIC peak showing mass corresponding to ATOTA-2 Calc (M) (C162H270N33O175+) 618.0375 Found 618.0199.

[1397] Synthesis of eGFP-CW-1:

[1398]

[1399] Where the sequence is: CW1-1-triazoyl-glycyl- MAHHHHHHGHHHQLVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRD HMVLLEFVTAAGITLGMDELYK-OH (CW1-1-triazoyl-glycyl-SEQ ID NO. 6)

[1400] and CW1 is linked to the 1,2,3-triazol-4-yl as the click-cyclized prop-3-ylamido-thr-orn(tbG)-orn(N₃)-orn(tbG)-P8-Gly-orn(tbG)-pra-orn(tbG)-leu-OH, and tbG is tetrabutylguanidinylation.

[1401] The crude reaction mixture of click coupling of CW-1 and azidoglycinyl-hexa-histidyl-eGFP (~0.3 mg) in 1 mL reaction buffer from the reaction above was separated using a 2 x 40 co G15 Sephadex column with a flow of water (5 mL / min) and fractions of 3 mL were collected. According to fluorescence microscopy a drop of each fraction on a microscope slide the GFP was mainly in fraction 17. Fraction 15-18 were concentratedP7529PC00

[1402] by lyophilization and the product was dissolved in 0.1 mL of water and applied to MCF7 cells.

[1403] Example 2: Uptake of cCPP15 in MCF-7 cells

[1404] Aim:

[1405] To investigate the cell penetration capability of cCPP15 in MCF-7 cells in comparison with TAT and other linear and cyclic peptides 1-14 and 16 (Table 1).

[1406] Table 1. Structures of TAT and peptides 1-14 and 16 (Table 1).

[1407]

[1408]

[1409] P7529PC00

[1410]

[1411] P7529PC00

[1412]

[1413] Method:

[1414] Stock solutions (1 mg / mL in DMSO) of peptides TAT, 1-14 and 16 were diluted into 1 pM by DMEM, and MCF-7 cell line was set as model cell line in the following experiments. MCF-7 cells, seeded on p-Slide 8 Well slide with density of 2.0×104cells / well, were incubated for 24 h by utilizing standard culture conditions and general cell culture method as described under general methods. All cells were recorded images and quantified by general SDCM and FC approaches as described under general methods. Each sample was investigated in triplicate at the same day ofP7529PC00

[1415] measurement and the entire experiment was repeated three times on different days within one week.

[1416] Results:

[1417] The results in Figure 1 show that cCPP15 exhibit a superior cell permeability over TAT and the other linear and cyclic peptides 1-14 and 16.

[1418] Example 3: cCPP15 uptake in different cell lines

[1419] Aim:

[1420] To investigate the penetrating ability of cCPP15 in different cell lines, including: HeLa, U2OS, HEK293T, RPE-1, HT-29 and SH-SY5Y.

[1421] Method:

[1422] Cells were seeded and cultured with different density under standard culture conditions, and then they were treated with cCPP15 for 1 h at 1 pM under standard culture conditions as described under general methods. Subsequently, images were captured using SDCM and fluorescence quantification conducted by FC as described under general methods.

[1423] Results:

[1424] The results in Figure 2 show that all the cell lines have taken up cCPP15. These results demonstrate that cCPP15 permeability is not driven by a cell type specific mechanism and can be applied across a wide range of plasma membranes.

[1425] Example 4: Cellular uptake mechanism

[1426] Aim:

[1427] To explore the mechanism of cellular uptake for cCPP15 in MCF-7 and HeLa cells using various inhibitors of endocytosis.

[1428] Method:

[1429] MCF-7 or HeLa cells (2.5×105cells / well) were seeded and incubated onto 24 well plate, according to standard culture conditions as described under general methods. After 24 h incubation (37°C, 5% CO2), cells were washed with PBS (37°C, 1x300 pL)P7529PC00

[1430] and treated with different endocytosis inhibitors dissolved in DMEM (50 pM EIPA, 50 pM Dynasore, 5 pM Cytochalasin D, 100 pM Chloroquine, 50 pM Nystatin and 3 mM Methyl-β-Cychlodextrin) for 0.5 h.

[1431] Then, the inhibitors solutions were discarded and cells were washed with PBS (37°C, 2x300 pL). The PBS was replaced with fresh DMEM containing 1 pM cCPP15. After incubating for 1 h and following washing with PBS (2x100 pL, 37°C), a Trypsin-EDTA solution was added to detach the cells. Subsequently, all cells were processed with the general fluorescence quantification determined by FC. The cells were washed, collected, centrifugated and resuspended in PI / PBS solution. Once resuspended, cells were filtered into glass tubes and stored in ice for flow cytometry analysis as described under general methods.

[1432] Results:

[1433] The results in Figure 3 show that when the dynamin dependent route was inhibited with Dynasore, the penetration of cCPP15 was significantly inhibited, leading to the conclusion that cCPP15 generally enters cells by dynamin-promoted endocytosis.

[1434] Example 5: EGFP-cCPP uptake

[1435] Aim:

[1436] To evaluate cell penetration of EGFP-cCPP17 in MCF-7 and HeLa cell lines.

[1437] Method:

[1438] Cells were seeded on ibidi p-slides 8 and cultured under general method and conditions. Subsequently, culture medium was discarded and cells were treated with 100 pM EGFP-cCPP17 / PBS conjugate solution for 1 h. Particularly, to demonstrate EGFP-cCPP17 permeability, EGFP / PBS (100 pM) group and mixture of EGFP-N3 / PBS and cCPP17 / PBS group also were set and investigate. After washing with PBS (3x200 pL), images were taken by SDCM system as described in detail under general methods. MCF-7 and HeLa cells were selected as model lines to evaluate the cellular penetration of EGFP-cCPP17 conjugate. Cells were seeded onto ibidi p-slides 8-well and cultured under standard conditions. Following incubation, the culture medium was removed, and the cells were treated with a 100 pM solution of EGFP-cCPP17 in PBS (200 pL / well) for 0.5 h. To assess the permeability of the EGFP-cCPP17 conjugate, two control groups were included: one treated with EGFP in PBS (100 pM, 200 pL / well)P7529PC00

[1439] and another with a mixture (200 pL / well) of EGFP-N3 (100 pM) and cCPP17 (100 pM) in PBS. After thorough washing with PBS (3x200 pL), images were acquired using a spinning disk confocal microscopy (SDCM) system under standard imaging conditions as described in detail under general methods.

[1440] Results:

[1441] As shown in Figure 4, only the group treated with the EGFP-cCPP17 conjugate demonstrated significant cellular penetration. In contrast, both the EGFP-alone group and the mixture of EGFP and cCPP17 showed no observable uptake, indicated by the lack of fluorescence and the presence of a dark background, confirming

[1442] impermeability.P7529PC00

[1443] Example 6: Uptake of tetra-methylated cCPPs in cells

[1444] Aim:

[1445] To investigate the permeability and cellular uptake of the synthesized peptides CPP1, CPP1-AlexaFluor, CPP2 and CPP3 (prepared as described in Example 1), a series of cellular assays were conducted. Experiments were primarily performed on HeLa cells, a widely used cervical cancer cell line.

[1446] In a separate uptake experiment, CPP3 was imaged both in MCF-7 and HeLa cells.

[1447] Materials and methods:

[1448] All chemicals were purchased from Biosynth Carbosynth Chem Impex, Combi-Blocks, Iris GmbHand Roth and Sigma-Aldrich. All solvents were of HPLC quality and purchased from VWR Chemicals and Sigma Aldrich. Cell culture reagents and consumables were purchased from Sigma-Aldrich, Thermo Fisher Scientific Inc. Filcon, VWR Chemicals, ibidi Gmbh. Bio-Rad Laboratories, Inc. HeLa cell line was bought from American Type Culture Collection (ATCC).

[1449] Cell counting was performed using a TC20™ Automated Cell Counter (Bio-Rad Laboratories).

[1450] Cells were imaged using the ZEISS Lattice light-sheet 7 microscope. It was equipped with diode lasers operating at 488 nm, 561 nm, and 640 nm wavelengths. For illumination, a 13.3x magnification excitation objective with a 0.44 numerical aperture (NA) was employed, projecting a 30 pm x 1 pm light-sheet. Image collection was performed using a 44.83x magnification detection objective with a 1 NA. During image acquisition, the system captured data along the Y-axis at 0.2 pm intervals. Laser power for the 488 nm line was adjusted between 5–10% and exposure time between 5-10 ms, depending on the brightness of the fluorophores. For the 640 nm laser the power was adjusted between 2-5% and exposure time between 5-10 ms. To maintain optimal image quality, aberrations were corrected using a value of 180, and auto-immersion was performed every 20 minutes. For live cell imaging, a humidity chamber (70% humidity) was used to control temperature and provide 5% CO2. Finally, images were acquired using two Hamamatsu ORCA-Fusion cameras and were deskewed using ZEN (Zeiss) software.P7529PC00

[1451] All cell culture procedures were conducted under sterile conditions within a Class II biosafety cabinet.

[1452] General Cell Culture Methods:

[1453] All cell culture procedures were conducted under sterile conditions within a Class II biosafety cabinet.

[1454] Resuscitation of frozen cells

[1455] The growth medium, consisting of Dulbecco’s Eagle Medium (DMEM) supplemented with 20% feal bovine serum (FBS) and 1% penicillin-streptomycin (PS), was prewarmed to 37°C. Cryopreserved cell vials were taken from the liquid nitrogen storage and immediately transferred to a 37°C water bath. It is important to slowly freeze and rapidly thaw cells. The exterior of the vial was disinfected with a tissue moistened with 70% ethanol and was placed in the sterile cabinet. The vial cap was loosened under a tissue dampened with 70% ethanol and growth medium (3 mL) was pipetted into the cell vial. The suspension was gently pipetted up and down three rimes and transferred to the T75 flask containing 10mL of growth medium. The flask was incubated at 37°C with 5% CO2. The next day the cells were examined under the inverted microscope and the growth medium was changed every 2-3 days.

[1456] Freezing cells

[1457] The day prior to freezing the culture medium was replaced with fresh pre-warmed growth medium. The next day the cells were examined under the inverted microscope to ensure the absence of bacterial or fungal contamination. The confluency should be between 50 and 70%. Cells were harvested using Trypsin-EDTA, following the protocol for subculturing cells (see Section 6.14.3). The suspension was transferred to a 15 mL flask followed by cell counting using the Automatic Cell Counter. Cells were pelleted by centrifugation at 400 g for 5 min. The supernatant was discarded and the cell pellet resuspended in fresh growth medium to achieve a final cell density of approximately1 3·103cells / mL. DMSO was then added to the suspension to a final concentration of 10% (v / v). The mixture was kept on ice and aliquots of 1 mL were dispensed into cryoprotective vials, which had been pre-labeled with the cell line name, passage number, date and operator initials. The vials were stored at -80°C for 3 days before transferring to the liquid nitrogen storage tank.P7529PC00

[1458] Subculturinq cells

[1459] Prior to passaging, cultures were inspected under the inverted microscope. Based on the degree of confluency, an appropriate split ratio was determined. The medium in the flask was aspirated and the cells were washed with PBS. PBS was removed and cells were incubated with a 0.25% solution of Trypsin-EDTA to detach the cells. The flask was gently rotated to ensure even distribution of the trypsin solution. After 3-5 min growth medium was added to quench the trypsine. The cell suspension was gently pipetted up and down five times to ensure complete resuspension. Cells were centrifuged at 400 g for 5 min and the supernatant was aspirated. The cell pellet was resuspended in an appropriate volume of fresh growth medium and the cell density was measured with the Automatic cell counter (TC20 Cell Counter) and seeding was performed accordingly. Culture was returned to the incubator for continued growth. To prevent confluence cells were passages every 3-4 days.

[1460] Imaging of cells:

[1461] For all peptides the same procedure was applied. HeLa cells were seeded in Ibidi 8-well plates with a seeding density of 2·104cells / mL and incubated overnight. Prior to imaging, cells were washed with PBS and then treated with 200 pL of the different peptide solution in DMEM, at a concentration of 1 pM. Following a 1 h incubation at 37°C and 5% CO2, cells were washed and stained with CellMask™ Deep Red plasma membrane stain for 10 min. After a final washing step, imaging was performed. The membrane stain was excited at 640 nm while the attached fluorophore ATOTA was excited at 488 nm.

[1462] Microscopy analysis was performed using a ZEISS Lattice light-sheet 7 microscope for 3D single-cell imaging of CPP1-CPP3.

[1463] The CellMask™ Deep Red membrane stain was excited at 640nm, while the ATOTA fluorophore attached to the peptides was excited with a laser at 488nm.

[1464] Results:

[1465] All three peptides CPP1, CPP2, and CPP3 demonstrated evident cellular uptake (Figure 5).P7529PC00

[1466] CPP1 and CPP2 exhibited similar permeability. In both cases, peptide-associated signal was detected by fluorescence beyond the membrane boundary and within the central region of the cell, consistent with efficient crossing of the plasma membrane. Additionally, nuclear accumulation was observed, with CPP1 and CPP2 localizing not only to the cytosol but also into the nucleus. Their uptake patterns were very similar, suggesting that the positional variation of the naphthyl substituent on the alanine residue does not significantly affect cellular uptake. Some punctate signals were observed outside the cells, which may reflect peptide aggregation.

[1467] CPP3 also exhibited efficient cellular uptake with a slightly distinct intracellular localization pattern. The peptide crossed the plasma membrane and appeared in “worm-like”, filamentous structures in the cytosol. Similar to CPP1 and CPP2, CPP3 also demonstrated nuclear accumulation. The experiment with CPP3 was independently repeated on a different day under identical conditions, yielding in similar results.

[1468] Overall, all peptides CPP1, CPP2 and CPP3 demonstrated cellular uptake and similar intracellular behaviour. A small difference that can be noted is the intracellular distribution of the peptides. CPP1 and CPP2 exhibited a more clustered and “fragmented” localization within the cytosol, whereas CPP3 showed a more “worm like”, filamentous pattern.

[1469] The peptide conjugated to Alexa Fluor 488 (CPP1-AlexaFluor) similarly demonstrated evident cellular uptake (Figure 6), as peptide-associated signal was detected by fluorescence beyond the membrane boundary and within the central region of the cell, consistent with efficient crossing of the plasma membrane.

[1470] The peptide CPP3, when imaged in a separate uptake experiment, similarly demonstrated evident cellular uptake in MCF-7 and HeLa cells (Figure 7), as peptide-associated signal was detected by fluorescence beyond the membrane boundary and within the central region of the cell, consistent with efficient crossing of the plasma membrane in both cell types.P7529PC00

[1471] Example 7 Uptake of tetra-buthylated cCPPs in cells

[1472] Aim:

[1473] To investigate the permeability and cellular uptake of the synthesized peptides ATOTA-CW-1 and ATOTA-CW-2 (prepared as described in Example 1), a series of cellular assays were conducted.

[1474] The aim of the butylation of arginines is to change the profile of the import into cells, preferably by release.

[1475] Method:

[1476] The peptide sequence was assembled with ornithine protected with Boc. The sequence was cyclized using CuAAC reaction to form the triazole The product was alloc deprotected and reacted with naphthylaldehyde under reductive amination conditions to provide a product. The aminoproline was fmoc deprotected and reacted with 4-pentynoic acid on the N-terminal. Boc-groups were removed with TFA and the perbutylation of arginines were achieved by tetrabytylguanidinylation of all 4 ornitines. This was achieved as described in the experimental part. The product was ready for clicking fluorophores using CuAAC reaction conditions with tetrakis-acetonitrile Cu(1) triflate and an azide derivative of ATOTA made by cornu promoted coupling to 3-azidopropylamine. The product was separated by preparative HPLC and characterized.

[1477] Results:

[1478] The products with 1 and 2 naphthyl groups on the aminoproline were obtained and separated as ATOTA-CW-1 and ATOTA-CW-2. The ATOTA-labelled CW1 and CW2 were dissolved and used at 1 micromolar for 1.5 h using MCF7 cells in cell growth medium to study cell import. The images are shown in Figure 8. The result is that the fluorophore was imported into the cells with an efficiency similar to that of the nonalkylated peptides.

[1479] It is evident from Figure 8 that cellular uptake takes place for ATOTA-CW-1 and ATOTA-CW-2 in both MCF-7 and HeLa cells, since peptide-associated signal was detected by fluorescence beyond the membrane boundary and within the central region of the cell, consistent with efficient crossing of the plasma membrane in both cell types.P7529PC00

[1480] The images show that there is endosomal release with ATOTA-CW-1 and that the release is best with this CPP.

[1481] Sequence overview

[1482] SEQ ID NO.: 1 (Consensus sequence)

[1483] TRX1RX2X3RX4L

[1484] SEQ ID NO.: 2 (TAT -ver. 1)

[1485] GRRRGRKKRRQ

[1486] SEQ ID NO.: 3 (Consensus sequence)

[1487] TRX1RX2X3RX4RL

[1488] SEQ ID NO.: 4 (Consensus sequence)

[1489] X5TRX1RX2X3RX4RL

[1490] SEQ ID NO.: 5 (TAT -ver. 2)

[1491] GRKKRRQRRRA

[1492] SEQ ID NO.: 6 MAHHHHHHGHHHQLVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKL TLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFF KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQK NGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRD HMVLLEFVTAAGITLGMDELYK

[1493] References

[1494] Dougherty, P. G.; Sahni, A.; Pei, D. Understanding Cell Penetration of Cyclic Peptides.

[1495] Chem Rev 2019, 779 (17), 10241-10287.

[1496] Ding, H.; Roberts, A. G.; Chiang, R.; Harran, P. G. Cascading Auto-oxidative Biproline Guanylations Form Optically Active Dispacamide Dimers and Permit an Eight-Step Synthesis of (-)-Ageliferin. J Org Chem 2018, 83 (13), 7231-7238.

[1497] Lin, T. H.; Lin, C. H.; Liu, Y. J.; Huang, C. Y.; Lin, Y. C.; Wang, S. K. Controlling Ligand Spacing on Surface: Polyproline-Based Fluorous Microarray as a Tool in SpatialP7529PC00

[1498] Specificity Analysis and Inhibitor Development for Carbohydrate-Protein Interactions. ACS Appl Mater Interfaces 2017, 9 (48), 41691-41699.

[1499] Makoto Tamaki, G. H., Victor J. Hruby Practical and Efficient Synthesis of Orthogonally Protected Constrained 4-Guanidinoprolines. J. Org. Chem. 2001, 66 (3), 1038-1042.

[1500] Cabrera-Pardo, J. R.; Trowbridge, A.; Nappi, M.; Ozaki, K.; Gaunt, M. J. Selective Palladium(ll)-Catalyzed Carbonylation of Methylene beta-C-H Bonds in Aliphatic Amines. Angew Chem Int Ed Engl 2017, 56 (39), 11958-11962.

[1501] Nguyen, T.; Decker, A. M.; Langston, T. L.; Mathews, K. M.; Siemian, J. N.; Li, J. X.; Harris, D. L.; Runyon, S. P.; Zhang, Y. Discovery of Novel Proline-Based Neuropeptide FF Receptor Antagonists. ACS Chem Neurosci 2017, 8 (10), 2290-2308.

[1502] Davies, H. M. L.; Saikali, E.; Young, W. B. Synthesis of (,+-.)-ferruginine and (.+-.)-anhydroecgonine methyl-ester by a tandem cyclopropanation / Cope rearrangement. J. Org. Chem. 2002, 56 (19), 5696-5700.

[1503] Fawcett, C., et al., Comparative Study of Click Handle Stability in Common Ligation Conditions. Bioconjugate Chemistry, 2025, 36, 5, 1054-1065.

[1504] Che, Y. F., Yiqing; Hayward, Matthew Merrill; Hepworth, David; Jones, Peter; Kaila, Neelu; Papaioannou, Nikolaos, Cyclic Peptides as C5 A Receptor Antagonists. U. S Patent WO2018020358A1, 2017.

[1505] Pascal, R. and Sola, R., Preservation of the Fmoc protective group under alkaline conditions by using CaCl2. Applications in peptide synthesis. Tetrahedron Letters, 39 (1998) 5031-5034.

[1506] Goddard-Borger, E. D. and R. V. Stick, An Efficient, Inexpensive, and Shelf-Stable Diazotransfer Reagent: Imidazole-1-sulfonyl Azide Hydrochloride. Organic Letters, 2007. 9(19): p. 3797-3800.

Claims

P7529PC00Claims1. A compound according to formula (I),P1 - A1 - P2 - A2 - P3-, formula (I),wherein:A1 is any amino acid or derivative thereof;A2 is any amino acid or derivative thereof;P1 is an amino acid sequence of 1 to 20 amino acids;P2 is an amino acid sequence of 3 to 6 amino acids, wherein either:i) at least one amino acid is AP; orii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;P3 is an amino acid sequence of 1 to 20;the connection between A1 and A2 contains a 1,2,3-triazole moiety;, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;AP is defined as:, wherein R5is an aryl or heteroaryl, each comprising 1, 2, or 3 rings; orP7529PC00, wherein R8and R9independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings; or, wherein R10and R11independently are an aryl or heteroaryl, each comprising 1, 2, or 3 rings, or a terminal C2-C12 alkynyl group;andAN is defined as:, wherein R7is an aryl or heteroaryl, each comprising 1, 2, or 3 rings;or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein the compound is according to formula (I),-, formula (I),wherein:A1 is any amino acid or derivative thereof;A2 is any amino acid or derivative thereof;P1 is an amino acid sequence of 1 to 20 amino acids, wherein at least one amino acid is arg*;P2 is an amino acid sequence of 3 to 5 amino acids, wherein either:i) at least one amino acid is AP; orii) P2 comprises two adjacent amino acids that are proline and AN; and at least one amino acid is arg*;P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;P7529PC00the connection between A1 and A2 contains a 1,2,3-triazole moiety;^NH R1^NH N NR2R3O^Z^O'NR1 R2arg* is defined as:nNR4, ■zTr nNR3R4or^NH R6O^^^^4^NR1R2 XJ'Tr nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;AP is defined as:, wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or, wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or, wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;and^NHAN is defined as:, wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.P7529PC003. The compound according to claim 1, wherein the compound is according to formula (I),P1 - A1 - P2 - A2 - P3-, formula (I),wherein:A1 is any amino acid or derivative thereof;A2 is any amino acid or derivative thereof;P1 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;P2 is an amino acid sequence of 3 to 5 amino acids, wherein at least one amino acid is AP and at least one amino acid is arg*;P3 is an amino acid sequence of 1 to 20, wherein at least one amino acid is arg*;the connection between A1 and A2 contains a 1,2,3-triazole moiety;, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;andAP is defined as:, wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

4. The compound according to any one of the preceding claims, wherein the compound is a compound according to formula (I),P7529PC00P1 - A1 - P2 - A2 - P3-, formula (I),wherein:A1 is any amino acid or derivative thereof;A2 is any amino acid or derivative thereof;P1 is thr-arg* or pra-thr-arg*;P2 is arg*-AP-A3-arg* or arg*-pro-AN-arg*;P3 is arg*-leu;^NH R1^NHO<5^^N^NR2R3O<^<>N^XNR1 R2arg* is defined as:NNR4,nNR3R4or^NH R6O^yJ^-N^NR'R2x-nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;AP is defined as:, wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;, wherein R8and R9independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl; or / OFR11iii)1, wherein R10and R11independently are phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, benzothiophenyl, or a terminal C2-C12 alkynyl group;andP7529PC00AN is defined as:, wherein R7is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

5. The compound according to claim 1, wherein the compound is a compound according to formula (I),-, formula (I),wherein:A1 is any amino acid or derivative thereof;A2 is any amino acid or derivative thereof;P1 is thr-arg*;P2 is arg*-AP-A3-arg*;P3 is arg*-leu;the connection between A1 and A2 contains a 1,2,3-triazole moeity;^NH R1^NH °^YZ^(^^^NR2R3°^^(^N^NR1R2arg* is defined as:nNR4,nNR3R4or^NH R6OiJ^>'^N^NR'R2x-nNR3R4, wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;AP is defined as:, wherein R5is phenyl, naphthyl, anthracenyl, phenanthryl, indolyl, or benzothiophenyl;andP7529PC00A3 is any amino acid;or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

6. The compound according to any one of the preceding claims, wherein:A1 is ornithine or a derivative thereof;A2 is propargylglycine or a derivative thereof;P1 is thr-arg* or pra-thr-arg*;P2 is arg*-AP-gly-arg* or arg*-pro-AN-arg*;andP3 is arg*-leu.

7. The compound according to any one of the preceding claims, wherein:A1 is ornithine or a derivative thereof;A2 is propargylglycine or a derivative thereof;P1 is thr-arg*;P2 is arg*-AP-gly-arg*;andP3 is arg*-leu.

8. The compound according to any one of the preceding claims, wherein arg* is ^NH R1N NR2R3defined as:nNR4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

9. The compound according to any one of the preceding claims, wherein ^NHNR1 R2arg* is defined as:nNR3R4wherein R1, R2, R3, and R4independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5;P7529PC0010. The compound according to any one of the preceding claims, wherein arg* is ^NH R60<^^N^NR1R2 Xdefined as:nNR3R4wherein R1, R2, R3, R4and R6independently are H or C1-12 alkyl, C5-6 cycloalkyl, substituted or unsubstituted benzyl, isopropyl or aminoalkyl; and n is 1, 2, 3, 4, or 5.

11. The compound according to any one of the preceding claims, whereinR1, R2, R3, and R4of one or all arg* are H.

12. The compound according to any one of the preceding claims, whereinR1, R2, R3, and R4of one or all arg* are C1-C12 alkyl.

13. The compound according to any one of the preceding claims, whereinR1, R2, R3, and R4of one or all arg* are methyl.

14. The compound according to any one of the preceding claims, whereinR1, R2, R3, and R4of one or all arg* are butyl.

15. The compound according to any one of the preceding claims, wherein one or all arg* are orn(tmG), wherein tmG is tetramethylguanidinylation.

16. The compound according to any one of the preceding claims, wherein one or all arg* are orn(tbG), wherein tbG is tetrabuthylguanidinylation.

17. The compound according to any one of the preceding claims, wherein: R5, R8, R9, R10, R11independently are indolyl, naphthyl, anthracenyl, or a terminal C2- C12 alkynyl group.

18. The compound according to any one of the preceding claims, wherein:R5is 2-indolyl, 3-indolyl, 1-naphthyl, or 2-naphthyl.

19. The compound according to any one of the preceding claims, wherein:R5is 2-indolyl or 3-indolyl.P7529PC0020. compound according to any one of the preceding claims, wherein:R5is 1-naphthyl or 2-naphthyl.

21. The compound according to any one of the preceding claims, wherein R8and R9are naphthyl, such as 1-napthyl and / or 2-naphthyl.

22. The compound according to any one of the preceding claims, wherein R10and R11independently are naphthyl, such as 1-naphthyl or 2-naphthyl, or a terminal C2-C12 alkynyl group, such as a terminal C2 alkynyl, such as a terminal C3 alkynyl, such as a terminal C4 alkynyl, such as a terminal C5 alkynyl, or such as a C6terminal alkynyl.

23. The compound according to any one of the preceding claims, wherein:A1 is ornithine or a derivative thereof;A2 is propargylglycine or a derivative thereof;R1, R2, R3, and R4are H;n is 3; andR5is 2-naphthyl.

24. The compound according to any one of the preceding claims, wherein:A1 is ornithine or a derivative thereof;A2 is propargylglycine or a derivative thereof;R1, R2, R3, and R4are methyl, and R6is H;n is 3; andP2 is arg*-AP-gly-arg* or arg*-pro-AN-arg*; wherein:NH, wherein R5is 2-naphthyl;, wherein R7is 1-naphthyl or 2-naphthyl.

25. The compound according to any one of the preceding claims, wherein:P7529PC00A1 is ornithine or a derivative thereof;A2 is propargylglycine or a derivative thereof;R1, R2, R3, and R4are C4 alkyl, and R6is H;n is 3; andP2 is arg*-AP-gly-arg*; whereinAP is:, wherein R5is 2-naphthyl;, wherein R8and R9are each 2-naphthyl; or, wherein R10is 2-naphthyl and R11is a terminal C4 alkynyl group.

26. The compound according to any one of the preceding claims, wherein the compound is or comprises:P7529PC00MeP7529PC00MeMeN Me Me BuBuN BuBuP7529PC00Bu27. The compound according to any one of the preceding claims, wherein the compound is of formula (1a):P7529PC0028. The compound according to any one of the preceding claims, wherein all amino acids are of D-form except AP, wherein AP is of the L-form.

29. The compound according to any one of the preceding claims, wherein the compound is or comprises:161P7529PC00P7529PC00Bu30. The compound according to any one of the preceding claims, wherein the compound is of formula (1b):, formula (1b).

31. A conjugate comprising:i) the compound according to any one of the preceding claims;andP7529PC00ii) a cargo bound to the compound, optionally by a linker connecting the cargo and the compound.

32. The conjugate according to claim 10, wherein the conjugate is:164P7529PC00P7529PC00The conjugate according to claim 10, wherein the conjugate is according to formula (2a):or a stereoisomer, tautomer, solvate, or pharmaceutically acceptable salt thereof.

33. A method of facilitating intracellular transport, comprising:P7529PC00i) providing a compound according to any one of claims 1 to 9 or a conjugate according to any one of claims 10 to 11andii) bringing said compound or conjugate in contact with one or more cells.

34. A kit of parts for intracellular transport, comprising:i) a compound according to any one of claims 1 to 9; andii) a cargo to be transported.

35. The kit of parts for intracellular transport according to claim 34, wherein the compound and the cargo are associated with each other such that the compound is capable of transporting the cargo across a membrane.

36. The kit of parts for intracellular transport according to claims 34-35, wherein the compound and the cargo are associated with each other by means of:i) covalent linkage; orii) non-covalent interaction.

37. The kit of parts for kit of parts for intracellular transport according to claims 34- 36, wherein the covalent linkage is selected from an amide or peptide bond, a urea or carbamate linkage, a triazole linkage, a thioether or disulfide linkage, an oxime or hydrazone linkage, an ester or ether linkage, or an imine or secondary amine linkage.

38. The kit of parts for kit of parts for intracellular transport according to claims 34- 37, wherein the covalent linkage is selected from an amide or peptide bond, or a triazole linkage.

39. The kit of parts for kit of parts for intracellular transport according to claims 34- 38, wherein the one or more non-covalent interactions are selected from electrostatic interaction, hydrogen bonding, hydrophobic interaction, π–π stacking, and host-guest complexation.

40. The conjugate according to any one of claims 31-32 or the kit of parts according to any one of claims 34-38, wherein the cargo is a peptide, a protein, a nucleicP7529PC00acid sequence, a carbohydrate, a nanoparticle, a detection agent or a therapeutic agent.

41. The compound, the conjugate or the kit-of-parts according to any one of the preceding claims for use in a method of treatment of a disease of the brain.

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