HK1172036B - Cyclic amino acid molecules and methods of preparing the same - Google Patents

Description

Cyclic amino acid molecules and methods of making the same

Technical Field

The present invention relates to cyclic amino acid molecules and a preparation method thereof, particularly to macrocyclization after an amino acid or a linear peptide reacts with amphoteric amino aldehyde and isonitrile.

Background

Peptides play a key role by mediating a variety of biological processes, acting as hormones, antibiotics and signal transduction molecules. Peptides have been widely used in the medical field due to their highly specific interaction with their biological targets. However, due to their low bioavailability, the enormous therapeutic potential of peptides is often difficult to achieve. This disadvantage is caused by the low in vivo stability of peptides due to degradation of the peptides by exo-and endopeptidases. Cyclic peptides are more resistant to degradation than their linear counterparts. There are two main reasons for this stability. First, exopeptidases cannot cleave cyclic peptides at their termini (absent). Second, cyclic peptides, particularly cyclic peptides having small to medium ring sizes, can prevent degradation of endopeptidases because the constrained cyclic peptide backbone can prevent conversion to the extended conformation required during protein cleavage. In addition, the reduction of charge and intramolecular hydrogen bonding within cyclic peptides contributes to passive membrane permeability, which contributes to their bioavailability. Most importantly, the conformational constraints imposed by the cyclic topology on the amino acid sequence maximize the enthalpy of interaction (enthalpic interaction) between the cyclic peptides and their biochemical targets, while ensuring favorable entropy of binding.

Naturally occurring and synthetic cyclic peptides have shown great interest as scaffolds with amino acid sequences predisposed to rigid configurations¹. Of the large number of known cyclic peptides, small to medium sized rigid loops are of particular interest. Various cyclic amidation and non-peptide cyclization processes have been developed²A method.

Macrocyclization of linear precursors is perturbed by several thermodynamic and kinetic problems arising from the conformational preference of linear peptides. For the synthesis of cyclic molecules from non-cyclic precursors, the main difficulty is the chain/ring configuration balance. Short chain peptides readily form a cyclic configuration, driven by ion pairing between the N-and C-termini (scheme 1, A)³. Although entropy is a disadvantage, the thermodynamics of these cyclic configurations is favorable due to the enthalpy accumulated through electrostatic and other polar interactions. As shown in scheme 1, conventional activation reagents tend to remove the zwitterionic character of the peptide, rendering it incapable of forming ion pairs. Thus, the activated peptides form a random linear configuration without enthalpic contributions from electrostatic and other polar interactions (scheme 1, B). In order for macrocyclization to occur, the activated peptide must form a pre-cyclization conformation (C) prior to formation of the desired cyclic molecule (D). 10^-4Or higher order high dilution for restricted Loop dimerization⁴Cyclotrimerization and multimerization⁵The formation of the resulting by-products is necessary. Unfortunately, dilution results in extended reaction times, which in turn cause background processes such as epimerization. Try to makeCyclization of linear peptides containing less than seven residues is the most challenging cyclization^6，7。

Scheme 1 general peptide macrocyclization strategy

Another common challenge in developing the chemical space of macrocycles is associated with late-stage modifications. This is a historical problem in the biotechnology field and in the construction of libraries of macrocyclic compounds by pharmaceutical companies. Their classical cyclization techniques (ring-closing metathesis, Huisgen cycloaddition) do not lend themselves naturally to further modification (halogenation). A functional group handle must be constructed prior to cyclization to achieve this goal.

Disclosure of Invention

In one aspect, provided herein is a method of making a cyclic amino acid molecule comprising reacting an amino acid molecule having an amino terminus and a carboxy terminus with an isonitrile and a compound having formula (Ia) and/or (Ib):

wherein:

n is 0 or 1, R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an alkenyl group; a heterocycle; a cycloalkyl group; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lowLower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lower alkylaryl; OR lower alkyl-OR_dWherein R is_dIs a suitable protecting group or OH group; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents; and

its aldehyde component optionally in the form of its bisulphite adduct;

and the amino acid molecule is an amino acid, a linear peptide, or a salt thereof, provided that if the amino acid molecule is a linear peptide, then the compound comprises an aziridine (aziridine) chiral center adjacent the aldehyde, which has matched stereochemistry at the carbon atom adjacent the amino terminus of the peptide.

In another aspect, provided herein are cyclic amino acid molecules made using the methods described herein.

In another aspect, provided herein are cyclic amino acid molecules of formula (II):

wherein n is 0 or 1, R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an aryl group; a heteroaryl group; an alkenyl group; a heterocycle; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lower alkylaryl; OR lower alkyl-OR_dWherein R is_dIs a suitable protecting group or OHA group; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents;

the bonds [ a ] and [ b ] are on the same side (syn) as one another;

r' is the amino acid side chain of the amino-terminal amino acid;

r' is an optionally substituted amide;

and the amino acid molecule is an amino acid or a linear peptide, wherein N 'is the nitrogen of the amino terminus of the amino acid molecule and C' is the carbon of the carboxy terminus of the amino acid molecule, and with the proviso that if the amino acid molecule is a linear peptide, the bonds [ a ] and [ C ] are located heterolaterally (anti) to each other.

In another aspect, provided herein is the use of a compound for cyclizing an amino acid molecule, wherein the compound has the formula (Ia)/(Ib):

comprises an alpha-stereocenter (alpha-stereoenter) at a carbon atom adjacent to the aldehyde group, having matched stereochemistry to a carbon atom adjacent to the amino terminus of the amino acid molecule; and

n is 0 or 1, R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an aryl group; a heteroaryl group; an alkenyl group; a heterocycle; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lower alkylaryl; OR lower alkyl-OR_dWhich isIn R_dAre suitable protecting groups; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents;

and the amino acid molecule is an amino acid, a linear peptide, or a salt thereof, with the proviso that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center adjacent to the aldehyde, which has matched stereochemistry with a carbon atom adjacent to the amino terminus of the peptide.

Drawings

Embodiments of the invention may best be understood by referring to the following description and accompanying drawings. In the description and drawings, like numbers refer to like structures or methods. In the drawings:

FIG. 1 is a schematic representation of a crude reaction mixture using leucine isomers¹H NMR comparison.

FIG. 2 is a scheme depicting a non-matching reaction to give an aminal product (aminal product).

FIG. 3 is a matching reaction to obtain a cyclic product.

Fig. 4 shows the chemical structures of cyclic products synthesized by methods 1-3.

FIG. 5 is a drawing of the cyclic product 1¹H and¹³c NMR spectrum.

FIG. 6 is a drawing of cyclic product 2¹H and¹³c NMR spectrum.

FIG. 7 shows the cyclic product 3¹H and¹³c NMR spectrum.

FIG. 8 is a drawing of cyclic product 4¹H and¹³c NMR spectrum.

FIG. 9 is a drawing of the cyclic product 5¹H and¹³c NMR spectrum.

FIG. 10 is a drawing of cyclic product 6¹H and¹³c NMR spectrum.

FIG. 11 is a drawing of cyclic product 7¹H and¹³c NMR spectrum.

FIG. 12 is a drawing of the cyclic product 8¹H and¹³c NMR spectrum.

FIG. 13 is a drawing of the cyclic product 9¹H NMR spectrum.

FIG. 14 shows a cyclic product 10¹H and¹³c NMR spectrum.

FIG. 15 shows the cyclic product 11¹H and¹³c NMR spectrum.

FIG. 16 is a drawing of the cyclic product 12¹H and¹³c NMR spectrum.

FIG. 17 is a drawing of the cyclic product 13¹H and¹³c NMR spectrum.

FIG. 18 shows the cyclic product 14¹H and¹³c NMR spectrum.

FIG. 19 is a drawing of the cyclic product 15¹H and¹³c NMR spectrum.

FIG. 20 is a drawing of the cyclic product 16¹H. COSY and¹³c NMR spectrum.

FIG. 21 shows the cyclic product 17¹H and¹³c NMR spectrum.

FIG. 22 shows the cyclic product 18¹H and¹³c NMR spectrum.

FIG. 23 is a drawing of the cyclic product 19¹H and¹³c NMR spectrum.

FIG. 24 is a drawing of the cyclic product 20¹H and¹³c NMR spectrum.

FIG. 25 is a drawing of cyclic product 21¹H and¹³c NMR spectrum.

FIG. 26 shows the cyclic product 22¹H and COSY NMR spectra.

FIG. 27 is a drawing of the cyclic product 23¹H NMR spectrum。

FIG. 28 depicts crude LC-MS (ESI) analysis of HATU-mediated Pro-Gly-Gly-Gly cyclization at 0.2M.

FIG. 29 depicts crude LC-MS (ESI) analysis of isonitrile/aminoaldehyde-mediated Pro-Gly-Gly-Gly cyclization at 0.2M.

FIG. 30 shows nucleophilic opening of aziridine-containing cyclic peptides.

FIG. 31 depicts pK of thiol and ammonium ions_a' and Δ pK of ion pair_a。

FIG. 32 depicts crude LC-MS (ESI) analysis of a reaction mixture of thiol ring-opening products.

Detailed Description

Described herein is a one-step process for providing cyclic peptides in high yield and selectivity while avoiding the difficulties typically encountered in conventional cyclization reactions. Later diversification of large circular molecules can now be achieved in a seamless manner (seamlesswashion). The macrocyclization product is equipped with specific modification sites that enable later structural modification of the cyclic peptide. Post-cyclization diversity is a historical challenge in the biotechnology field and the pharmaceutical industry in constructing macrocyclic libraries. The macrocyclization process of the present invention solves this problem in particular.

The inclusion of unprotected aziridine and aldehyde groups useful for synthesis is demonstrated herein⁸The use of a stable class of amphoteric aminoaldehydes. Leading amphoteric amino aldehyde to react reversibly with amino and carboxyl terminals of peptide. The resulting electrophilic intermediate reacts rapidly with the nucleophilic aziridine moiety due to the high molar concentration. As a result, the unfavorable chain/ring equilibrium is altered using Le Chatelier principle, ensuring efficient conversion to cyclic products. In another aspect, cyclization is tolerant to aliphatic, acidic, and basic amino acid side chain residues. In particular peptides comprising acidic and basic residues are within the scope of the method, in each caseA single diastereoisomeric product may be obtained. It is known to stabilize the secondary structure of peptides and to promote polar interactions⁹Trifluoroethanol ("TFE") is a preferred reaction medium. Ugi four-component condensation is a well-known reaction involving carboxylic acids, amines, aldehydes and isonitriles¹⁰Is a preferred method of forming zwitterionic macrocyclized precursors. From the reaction mechanism, it is known that the Ugi reaction proceeds along a series of reversible transformations that contribute to the thermodynamic driving force for amide bond formation. When alpha-amino acids, aldehydes and isonitriles are used as starting materials, the reaction proceeds through a zwitterionic imine (iminium) ionic intermediate, which upon attack by isonitriles produces an electrophilic mixed anhydride. Subsequent reaction with methanol gives linear peptide esters as a mixture of diastereomers¹¹. This reaction was attempted to be used for the synthesis of cyclic peptides from linear peptides and common (monofunctional) aldehydes. However, the lack of diastereoselectivity and the preponderance of cyclodimerization products are accompanied by low yields.

In one aspect, provided herein is a method of making a cyclic amino acid molecule comprising reacting an amino acid molecule having an amino terminus and a carboxy terminus with an isonitrile and a compound having formula (Ia) and/or (Ib).

Wherein: n is 0 or 1, and R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an alkenyl group; a heterocycle; a cycloalkyl group; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lowerAn alkylaryl group; OR lower alkyl-OR_dWherein R is_dIs a suitable protecting group or OH group; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents; and

its aldehyde component optionally in the form of its bisulfite adduct;

and the amino acid molecule is an amino acid, a linear peptide, or a salt thereof, with the proviso that if the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center adjacent to the aldehyde, which has matched stereochemistry with a carbon atom adjacent to the amino terminus of the peptide.

Preferably, if the amino acid molecule is an amino acid, the amino terminus is a primary or secondary amino group, but when the amino acid molecule is a peptide, the amino terminus is a secondary amino group.

In one embodiment, R₁-R₅Any of which is H. Preferably, n is 0 and R₂And R₃Is H or R₁-R₃Is H.

In a specific embodiment, R₁Is CH₂OTBDMS or CH₂ ⁱPr。

In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino-terminal amino acid of the linear peptide is selected from the group consisting of proline and amino-substituted NHBn, NHCH₂CH₂SO₂Ph or NHCH₂CH₂CN substituted amino acids.

In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine (selenocysteine), serine, tyrosine, threonine, tryptophan, and valine.

The amino acid molecule may be an alpha-amino acid, a beta-amino acid, or a gamma-amino acid.

In one embodiment, the isonitrile is selected from the group consisting of: (S) - (-) - α -methylbenzylisocyanitrile; 1, 1, 3, 3, -tetramethylbutylisonitrile; 1-pentylisonitrile; 2, 6-dimethylphenyliisonitrile; 2-morpholinylethylisocyanitrile; 2-naphthyl isonitrile; 2-pentylisonitrile; 4-methoxyphenyl isonitrile; a benzylisonitrile; butyl isonitrile (Cutyl isocyanide); cyclohexyl isonitrile; isopropyl isonitrile; p-toluenesulfonylmethyl isonitrile; dichlorophenyl isonitrile; tert-butyl isonitrile; (trimethylsilyl) methylisonitrile; 1H-benzotriazol-1-ylmethyl-isonitrile; 2-chloro-6-methylphenyl isonitrile; di-tert-butyl 2-isocyanosuccinate; tert-butyl 2-isocyano-3-methylbutyrate; tert-butyl 2-isocyano-3-phenylpropionate; 2-Isocyanopropionic acid tert-butyl ester; and tert-butyl 3-isocyanoacetate, preferably tert-butylisonitrile.

In some embodiments, the process is carried out in a non-nucleophilic reaction medium, preferably trifluoroethanol or HFIP (mixed with water).

In one embodiment, if the amino acid molecule is an amino acid, the method is performed in water.

In some embodiments, the method further comprises binding (conjugate) a fluorescent label to the cyclic amino acid molecule by nucleophilic opening of the aziridine moiety.

In some embodiments, the peptide is 2-30 amino acids in length.

In another aspect, provided herein are cyclic amino acid molecules made using the methods described herein.

It will be appreciated that in some cases, cyclization requires and should include protection of certain peptide or amino acid side chains in a manner known to those skilled in the art.

In another aspect, provided herein are cyclic amino acid molecules of formula (II):

wherein the content of the first and second substances,

n is 0 or 1, R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an aryl group; a heteroaryl group; an alkenyl group; a heterocycle; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lower alkylaryl; OR lower alkyl-OR_dWherein R is_dIs a suitable protecting group or OH group; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents;

the bonds [ a ] and [ b ] are on the same side of each other;

r' is the amino acid side chain of the amino-terminal amino acid;

r' is an optionally substituted amide;

and the amino acid molecule is an amino acid or a linear peptide, wherein N 'is the nitrogen at the amino terminus of the amino acid molecule and C' is the carbon at the carboxy terminus of the amino acid molecule, and with the proviso that if the amino acid molecule is a linear peptide, then the bonds [ a ] and [ C ] are located heterolaterally to each other.

Preferably, if the amino acid molecule is an amino acid, the amino terminus is a primary or secondary amino group, but when the amino acid molecule is a linear peptide, the amino terminus is a secondary amino group.

In one embodiment, R₁-R₅Any of which is H. Preferably, n is 0 and R₂And R₃Is H or R₁-R₃Is H.

In a specific embodiment, R₁Is CH₂OTBDMS or CH₂ ⁱPr。

In one embodiment, the amino acid molecule is a linear peptide. Preferably, the amino-terminal amino acid of the linear peptide is selected from the group consisting of proline and amino-substituted NHBn, NHCH₂CH₂SO₂Ph or NHCH₂CH₂CN substituted amino acids.

In another embodiment, the amino acid molecule is a D or L amino acid selected from the group consisting of: alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, selenocysteine, serine, tyrosine, threonine, tryptophan, and valine.

The amino acid molecule may be an alpha-amino acid, a beta-amino acid, or a gamma-amino acid.

In one embodiment, R' is t-butylamide.

In another aspect, provided herein is the use of a compound for cyclizing an amino acid molecule, wherein the compound has the formula (Ia)/(Ib):

comprises an alpha-stereocenter on a carbon atom adjacent to the aldehyde group having matched stereochemistry to a carbon atom adjacent to the amino terminus of the amino acid molecule; and

n is 0 or 1, R₁、R₂、R₃、R₄And R₅Independently selected from H; a lower alkyl group; an aryl group; a heteroaryl group; an alkenyl group;a heterocycle; of the formula-C (O) OR^*In which R is^*Selected from alkyl and aryl; formula-C (O) NR^**R^***In which R is^**And R^***Independently selected from alkyl and aryl; -CH₂C (O) R, wherein R is selected from-OH, lower alkyl, aryl, lower alkylaryl or-NR_aR_bWherein R is_aAnd R_bIndependently selected from H, lower alkyl, aryl or lower alkylaryl; -C (O) R_cWherein R is_cSelected from lower alkyl, aryl or lower alkylaryl; OR lower alkyl-OR_dWherein R is_dAre suitable protecting groups; all groups are optionally substituted at one or more substitutable positions with one or more suitable substituents;

the amino acid molecule is an amino acid, a linear peptide, or a salt thereof, provided that the amino acid molecule is a linear peptide, the compound comprises an aziridine chiral center adjacent to the aldehyde, which has matched stereochemistry with a carbon atom adjacent to the amino terminus of the peptide.

The term "amino acid molecule" as used herein is intended to include single amino acids and also peptides.

The term "amino acid" as used herein refers to a molecule comprising an amino group, a carboxylic acid group, and variable side chains. Amino acids are intended to include not only the twenty amino acids typically found in proteins, but also non-standard amino acids and non-natural amino acid derivatives known to those skilled in the art, and thus include, but are not limited to, alpha-, beta-, and gamma-amino acids. Peptides are polymers of at least two amino acids and may comprise standard, non-standard and unnatural amino acids.

The term "suitable substituents" as used in the context of the present invention is intended to independently include H; a hydroxyl group; a cyano group; alkyl groups such as lower alkyl groups, e.g., methyl, ethyl, propyl, n-butyl, t-butyl, hexyl, and the like; alkoxy groups such as lower alkoxy groups, e.g., methoxy, ethoxy, and the like; aryloxy groups such as phenoxy and the like; a vinyl group; alkenyl groups such as hexenyl and the like; an alkynyl group; a formyl group;haloalkyl radicals, e.g. including CF₃、CCl₃Lower haloalkyl, etc.; a halide; aryl groups such as phenyl and naphthyl; heteroaryl groups such as thienyl and furyl, and the like; amides such as C (O) NR_aR_bWherein R is_aAnd R_bIndependently selected from lower alkyl, aryl or benzyl, etc.; acyl radicals, e.g. C (O) -C₆H₅Etc.; esters such as-C (O) OCH₃Etc.; ethers and thioethers such as O-Bn and the like; a thioalkoxy group; a phosphine group; and-NR_aR_bWherein R is_aAnd R_bIndependently selected from lower alkyl, aryl or benzyl, etc. It is to be understood that suitable substituents used in the context of the present invention are intended to represent substituents that do not interfere with the formation of the desired product by the process of the present invention.

As used in the context of the present invention, the term "lower alkyl" as used herein alone or in combination with another substituent denotes a cyclic, straight or branched alkyl substituent comprising one to six carbon atoms and includes, for example, methyl, ethyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl and the like. With respect to the number of carbon atoms, it is understood that "lower alkoxy", "lower thioalkyl", "lower alkenyl", and the like have similar terminology usage. For example, "lower alkoxy" as used herein includes methoxy, ethoxy, t-butoxy.

The term "alkyl" includes lower alkyl groups and also includes alkyl groups having more than six carbon atoms, for example, acyclic, straight chain or branched alkyl substituents having from seven to ten carbon atoms.

The term "aryl" as used herein alone or in combination with another substituent group, denotes an aromatic monocyclic ring system or an aromatic polycyclic ring system. For example, the term "aryl" includes a benzene or naphthalene ring, and may also include larger aromatic polycyclic ring systems, such as fluorescent (e.g., anthracene) or radioactive labels and derivatives thereof.

The term "heteroaryl" as used herein alone or in combination with another substituent group, denotes a 5-, 6-or 7-membered unsaturated heterocyclic ring containing 1-4 heteroatoms selected from nitrogen, oxygen and sulfur and forming an aromatic (aromatic system). The term "heteroaryl" also includes polycyclic aromatic systems comprising 5, 6, or 7-membered unsaturated heterocycles containing 1-4 heteroatoms selected from nitrogen, oxygen, and sulfur.

The term "cycloalkyl" as used herein alone or in combination with another substituent denotes a cycloalkyl substituent including, but not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl.

The term "cycloalkylalkyl" as used herein denotes an alkyl group to which a cycloalkyl group is directly attached, including, but not limited to, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, 1-cyclopentylethyl, 2-cyclopentylethyl, cyclohexylmethyl, 1-cyclohexylethyl, and 2-cyclohexylethyl. It is to be understood that as used herein, in arylalkyl, aryl lower alkyl (e.g., benzyl), lower alkyl alkenyl (e.g., allyl), heteroarylalkyl, and the like, "alkyl" or "lower alkyl" terms have similar applications. For example, the term "arylalkyl" denotes an alkyl group to which an aryl group is bonded. Examples of arylalkyl groups include, but are not limited to, benzyl (benzyl), 1-phenylethyl, 2-phenylethyl, and phenylpropyl.

The term "heterocycle" as used herein alone or in combination with another group means a monovalent group derived by removing one hydrogen from a three-to seven-membered saturated or unsaturated (including aromatic) heterocycle containing one to four heteroatoms selected from nitrogen, oxygen, and sulfur. Examples of such heterocycles include, but are not limited to, aziridine, epoxide, azetidine, pyrrolidine, tetrahydrofuran, thiazolidine, pyrrole, thiophene, hydantoin, diazepine, imidazole, isoxazole, thiazole, tetrazole, piperidine, piperazine, homopiperidine, homopiperazine, 1, 4-dioxane, 4-morpholine, 4-thiomorpholine, pyridine-N-oxide, pyrimidine, and the like.

The term "alkenyl" as used herein, alone or in combination with another group, is intended to denote an unsaturated, non-cyclic, straight-chain group containing two or more carbon atoms and in which at least two are bonded to each other by a double bond. Examples of such groups include, but are not limited to, ethenyl (vinyl), 1-propenyl, 2-propenyl, and 1-butenyl.

The term "alkynyl" as used herein is intended to mean an unsaturated, non-cyclic, straight-chain group containing two or more carbon atoms and in which at least two are bonded to each other by a triple bond. Examples of such groups include, but are not limited to, ethynyl, 1-propynyl, 2-propynyl, and 1-butynyl.

The term "alkoxy" as used herein alone or in combination with another group denotes the group-O- (C)_1-n) Alkyl, wherein alkyl is as defined above, which contains 1 or more carbon atoms and includes, for example, methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy and 1, 1-dimethylethoxy. Where n is 1-6, the term "lower alkoxy" as used herein above applies to the term "lower alkoxy" and the term "alkoxy" includes "lower alkoxy" and alkoxy groups wherein n is greater than 6 (e.g. n-7-10). The term "aryloxy" as used herein, alone or in combination with another group, denotes-O-aryl, wherein aryl is as defined above.

A peptide is a polymer of two or more amino acids.

The following examples illustrate various aspects of the invention and do not limit the broad aspects of the invention as disclosed herein.

Examples

Example 1

Materials and methods

Anhydrous toluene and Dimethylformamide (DMF) were purchased and used as received. Tetrahydrofuran (THF) was distilled under argon with sodium benzophenone carbonyl (sodium benzophenone ketyl). All other solvents, including TFE (2, 2, 2, -trifluoroethanol) and HFIP (1, 1, 1, 3, 3, 3-hexafluoro-2-isopropanol), have reagent grade quality. Melting points were obtained on a MelTemp melting point apparatus and were not corrected.

The method comprises the following steps: the amino acid/peptide cyclization reaction was carried out using the following method. To a screw-capped glass vial fitted with a magnetic stir bar was added the peptide (0.2mmol) and 1ml of TFE and stirring was carried out until a homogeneous solution was obtained. Aziridine aldehyde dimer (0.1mmol) and isonitrile (0.2mmol) were then added in succession and the resulting mixture was stirred for the time indicated in Table 1. The reaction was monitored by electrospray ionization mass spectrometry ("ESI-MS") and/or thin layer chromatography ("TLC") analysis at 60 eV. After the reaction was completed, 1ml of water and 1ml of Et were added₂O and the mixture was shaken vigorously and then cooled on ice. The resulting precipitate was filtered and washed with hexanes and cold Et₂O (1ml) was washed to obtain a cyclic peptide. For water-soluble or non-precipitating products, the reaction mixture is concentrated under reduced pressure and then treated with Et₂O and hexane (0.2ml) were triturated to obtain the cyclic peptide product.

The method 2 comprises the following steps: the amino acid/peptide cyclization reaction was performed by replacing TFE with HFIP (method 2). To a screw-capped glass vial fitted with a magnetic stir bar was added the peptide (0.2mmol) and 1ml HFIP and stirred until a homogeneous solution was obtained. Aziridine aldehyde dimer (0.1mmol) and isonitrile (0.2mmol) were then added in succession and the resulting mixture was stirred for the time indicated in Table 1. The reaction was monitored by ESI-MS and/or TLC analysis at 60 eV. After completion of the reaction, the mixture was concentrated under reduced pressure and then treated with Et₂O and hexane (0.2ml) were triturated to obtain the cyclic peptide product.

The method 3 comprises the following steps: the amino acid/peptide cyclization reaction was performed using HFIP and water. To a screw-capped glass vial fitted with a magnetic stir bar was added peptide (0.2mmol) and 1ml HFIP and 47 ml H₂O and stirring uniformly. Aziridine aldehyde dimer (0.1mmol) and isonitrile (0.2mmol) were then added in succession and the resulting mixture was stirred for the time indicated in Table 1. The reaction was monitored by ESI-MS and/or TLC analysis at 60 eV. After completion of the reaction, the mixture was concentrated under reduced pressure and then treated with Et₂O and hexane were triturated to obtain the cyclic peptide product.

Note that: in reactions involving cysteine or peptides containing thiol residues, the solvent was degassed with argon for two hours prior to the reaction. The reaction was then carried out under an argon atmosphere.

Some of the cyclic products that can be synthesized by each of methods 1-3 are summarized in table 3 and depicted in fig. 4.

Table 3 methods for amino acid/peptide cyclization reactions.

Method of producing a composite material	Cyclic product
		1	1，2，3，4，7，9，11，12，15，16，17，18，19，20，21，22
2	5，8，10，13，14
		3	6

By mixing each one¹The correlation of the methine (methine) region of the H NMR spectrum with the piperazinone 1 region establishes the relative stereochemistry of each cyclic product listed in table 3.

Chromatography: flash column chromatography was performed using silica 230-. Thin Layer Chromatography (TLC) was performed on Macherey Nagel pre-coated glass TLC plates (SIL G/UV254, 0.25mm) and visualized using UV lamps (254nm) and iodine staining.

Nuclear magnetic resonance spectroscopy: recording on a Varian Mercury 400 or 500MHz spectrometer¹H and¹³c NMR spectrum.¹The H NMR spectrum was referenced to TMS (0ppm),¹³c NMR spectra reference CDCl₃(77.23 ppm). Peak multiplicities are designated with the following abbreviations: s, singlet; bs, broad singlet; d, doublet; t, triplet; q, quartet; m multiplets; ds, double singlet; dd, doublet of doublets; ddd, doublet; bt, broad triplet; td, triplet peak; tdd, three sets of doublets.

Mass spectrometry analysis: high resolution mass spectra were obtained at 70eV on a VG 70-250S (double focusing) mass spectrometer or on an ABI/Sciex Qstar mass spectrometer with ESI source, MS/MS and accurate mass analysis capability. Low resolution mass spectra (ESI) were obtained at 60eV, 70eV and 100 eV.

Synthetic route for aziridine aldehyde dimer: all of the dimeric aminoaldehydes used in this study were prepared according to the synthetic routes outlined in supplementary FIGS. 1 and 2.

Results

Various piperazinones were prepared in a one-step process (table 1).

TABLE 1 Range of piperazinone syntheses (products derived from L-amino acids are shown)

^aUnless otherwise indicated, the reaction was carried out at room temperature using 0.2mmol of isonitrile and amino acid and 0.1mmol of aminoacid dimer in TFE solution (0.2M).^b% isolated yield.^cWill HFIP/H₂O (20: 1) was used as solvent (0.2M).^dHFIP was used as solvent (0.2M).^eCyclization occurs at the more acidic alpha-carboxylic acid.^fTFE was degassed before use.^gThe lysine side chain is protonated.

The step of altering the ring/chain equilibrium is the trans-ring collapse of the nucleophilic NH aziridine onto the electrophilic mixed anhydride, which is generated by subjecting the amphoteric amino aldehyde to Ugi reaction conditions (scheme 2). This reaction occurs under stoichiometric conditions and yields a cyclic peptide product. The harsh conditions of high dilution typically required to achieve high yields of cyclic peptide synthesis are not necessary.

Scheme 2.a. macrocyclization with monofunctional aldehydes; B. macrocyclization mediated by amphoteric aminoaldehydes.

Medium-sized rings, which are generally difficult to prepare, can be prepared in good yield in a few hours using large equipment (Table 2).

TABLE 2 representative ranges for macrocyclization of linear peptides

^aUnless otherwise indicated, the reaction was carried out at room temperature using 0.2mmol of isonitrile and amino acid and 0.1mmol of aminoacid dimer in TFE solution (0.2M).^bThe isolation yield.^cBy passing each crude reaction mixture¹H NMR analysis confirmed a diastereoselectivity of > 20: 1. By combining each¹The methine region of the H NMR spectrum correlates with the region of piperazinone 1 to establish the relative stereochemistry (see x-ray crystal structure of 1).^dHFIP was used as solvent (0.2M).

Of the cyclic products of Table 3¹H and¹³the C NMR spectrum is shown in FIGS. 5 to 27.

A one-step process is provided for the preparation of a variety of cyclic peptides with high stereoselectivity and chemoselectivity to linear peptides, isonitriles and amphoteric amino aldehydes. The Ugi reaction is carried out with the replacement of the monofunctional aldehyde by the amphoteric aminoaldehyde. When L-phenylalanine was reacted with tert-butylisonitrile and aziridinal, cyclic piperazinones were obtained in 92% yield in about one hour (table 1, entry 1). The relative stereochemistry of 1 was determined by x-ray analysis. The reaction was performed with a series of amino acids and the corresponding piperazinone product was obtained as a single diastereomer under all conditions and no linear peptide was formed (figure 1).

By analysing the crude reaction mixture¹H NMR(DMSO-d₆Or CDCl₃) The presence of epimer product was investigated. In all reactions studied, there was no detectable signal corresponding to the epimer. Thus, the resulting crude cyclic peptide was obtained with a selectivity of > 20: 1.

The effect of medium-sized peptide chain length on the reaction results was investigated (table 2). Challenging, medium-sized rings can be easily prepared; the reaction time is less than 10 hours, and the product has high yield and diastereoselectivity. A preferred work-up operation involves precipitation of the product from diethyl ether and hexane. Further purification of the cyclic peptide by HPLC is not required. In addition, no racemization was detected during the entire reaction or in the product isolation. The absence of epimerization is further demonstrated by high stereoselectivity; aziridine aldehydes having an S stereocenter adjacent to the carbonyl group undergo macrocyclization with peptides containing L-amino acid residues at the N-terminus. A "mismatch" reaction with a peptide having a D-amino acid at the terminus results in no harvest, resulting in the formation of a stable aminal.

In the case of peptides, it has been determined that the α -stereocenter of the N-terminal amino acid (here proline) should match the α -stereocenter of aziridine aldehyde to allow cyclization. In the non-matched reaction, the only product formed is the corresponding aminal (FIG. 2). Even if the reaction time is prolonged and more equivalents of isonitrile are added, no cyclic peptide formation is observed. This limitation does not exist in the cyclization of individual amino acids.

When the opposite enantiomer of the aminoaldehyde is used in the same reaction, both proline and the aminoaldehyde have the S-configuration at their α -stereocenters. This matching situation allows easy peptide cyclisation to obtain the corresponding cyclic product in high yield (figure 3).

This stereoselective process is of great importance in cases where epimerization at the N-terminus is possible. Because cyclization occurs requiring matched stereochemistry, the resulting cyclic peptide must contain matched stereocenters. Furthermore, the fact that no cyclic peptide formation was observed in the non-matched reaction indicates that no epimerization to form the matched substrate occurred under the bulk conditions of the present invention.

Due to the mechanism controlling the cyclization, it is likely that consistent yields will be obtained in this chemistry. The difference from the Ugi reaction using monofunctional aldehydes becomes evident at this time. When using monofunctional aldehydes in combination with isonitriles and peptides, low diastereoselectivity is observed. More importantly, unfavorable ring dimerization occurs in the cyclization of linear peptides containing less than six residues; cyclization of tripeptides only yields cyclic dimers¹³. This low selectivity is due to the slow trans-ring attack of the amino group on the mixed anhydride (scheme 2, a), thus making the intermolecular process kinetically competitive. By adding an amphoteric aminoaldehyde to the cyclization reaction mixture containing the secondary amino-terminal peptide, the slow ring-spanning attack is replaced by a fast attack of the nucleophilic aziridine located outside the mixed anhydride ring (scheme 2, B). This provides an unimpeded line of attack. Since TFE is a non-nucleophilic solvent, no premature solvolysis (pre-catalyst solvolysis) of mixed anhydrides was observed. This reaction mechanism ensures that the C-terminus is activated only after formation of the intermediate cyclic mixed anhydride, which is then attacked by the exocyclic aziridine. This not only ensures selectivity for intramolecular macrocyclization, but also avoids prolonged C-terminal activation and potential epimerization. Thus, the harsh conditions of high dilution typically required to achieve high yields in conventional cyclic peptide synthesis are not necessary⁶. In addition, no oligomeric or polymeric by-products were detected in the subject assay. The subject reaction was compared in parallel with the traditional lactamization of linear tetrapeptides widely used for cyclic peptide synthesis. Aminoaldehyde-mediated macrocyclization withFacilitates rapid, selective and efficient cyclic peptide formation. In contrast, only trace amounts of cyclic peptides were detected in many structures formed during lactamization. Unfavorable cyclodimerization⁵Predominate in this process, which demonstrates slow kinetics in conventional intramolecular processes. Peptide macrocyclization reactions are usually carried out at extreme dilution. A typical scenario must use 10^-4Molar concentration of M. This high dilution increases the selectivity of intramolecular cyclization, thereby limiting unfavorable cyclodimerization and trimerization.

The characterization of the relative stereochemistry of each cyclic product listed in table 3 is set forth below.

(3S, 5R, 6R, 7S) -3-benzyl-N-tert-butyl-7- ((tert-butyldimethylsilyloxy) methyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (1)

¹H NMR(CDCl₃，400MHZ)：7.36-7.18(m，5H)，6.75(bs，1H)，3.86(dd，J＝11.6Hz，4.8Hz，1H)，3.74(dd，J＝11.2Hz，4.8Hz，1H)，3.54(bs，1H)，3.47(dd，J＝3.6Hz，1.2Hz，1H)，3.25(dd，J＝14.4Hz，3.2Hz，1H)，3.10(m，1H)，2.52(dd，J＝14.4Hz，10.4Hz，1H)，2.40(m，1H)，1.80(bs，1H)，1.02(s，9H)，0.90(s，9H)，0.10(s，3H)，0.08(s，3H)ppm.¹³C NMR(CDCl₃，100MHz)：184.8，169.0，138.5，129.7，128.9，127.0，63.9，57.5，53.6，50.7，44.6，39.0，35.5，28.5，26.1，18.6，-5.0ppm.HRMS(ESI)[MH]⁺Calculated value 446.2833, found value 446.2831

(3R, 5R, 6R, 7S) -3-benzyl-N-tert-butyl-7- ((tert-butyldimethylsilyloxy) methyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (2)

¹H NMR(CDCl₃，400MHz)：7.35-7.20(m，5H)，6.32(bs，1H)，3.86(m，IH)，3.73(dd，J＝11.5Hz，4.2Hz，1H)，3.86(bs，1H)，3.73(dd，，J＝11.5Hz，4.2Hz，1H)，3.25(dd，J＝14.4Hz，3.2Hz，1H)，3.10(m，1H)，2.52(dd，J＝14.4Hz，10.4Hz，1H)，2.40(m，1H)，1.80(bs，1H)，1.31(s，9H)，0.90(s，9H)，0.10(s，3H)，0.08(s，3H)ppm.13CNMR(CDCl₃，75MHz)：184.0，169.1，136.2，129.2，129.1，127.3，63.0，59.5，51.4，49.6，42.2，38.7，36.0，28.8，26.1，18.7，-5.1ppm。MS(ESI)[MH]⁺Calculated 446.3, found 446.3.

(3S, 5R, 6R, 7S) -N-tert-butyl-7- ((tert-butyldimethylsilyloxy) methyl) -3-methyl-2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (3)

¹H NMR(CDCl₃，400MHz)：7.42(s，NH)，3.88(dd，J＝11.2Hz，4.8Hz，1H)，3.70(dd，J＝11.2Hz，5.2Hz，1H)，3.55(d，J＝0.8Hz，1H)，3.46(dd，J＝3.6Hz，1.6Hz，1H)，3.07(m，1H)，2.36(td，J＝4.8Hz，3.6Hz，1H)，1.8(bs，NH)，1.39(s，9H)，1.23(d，J＝6.8Hz，3H)，0.90(s，9H)，0.09(s，3H)，0.08(s，H)ppm.13C NMR(CDCl₃，100MHz)：185.5，169.3，63.9，53.8，51.2，51.0，44.5，39.3，28.9，26.1，16.6，15.0，-5.0ppm.HRMS(ESI)[MH]⁺Calculated value 370.2520, found value 370.2520

(1S, 3aS, 8R, 8aS) -N-tert-butyl-1- ((tert-butyldimethylsilyloxy) methyl) -3-oxooctahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] pyrazine-8-carboxamide (4)

¹H NMR(CDCl₃，400MHz)：6.17(s，1H)，3.76(dd，J＝11.6Hz，4.4Hz，1H)，3.67(dd，J＝11.6Hz，4.8Hz，1H)，3.70(d，J＝6.2Hz，1H)，3.12(dt，J＝4.8Hz，9.2Hz，1H)，2.97(dd，J＝6.4Hz，3.6Hz，1H)，2.93-2.89(m，1H)，2.60(q，J＝4.4Hz，1H)，2.25-2.05(m，2H)，1.90-1.75(m，3H)，1.70-1.51(m，1H)，1.48(s，9H)，0.85(s，9H)，0.09(s，3H)，0.08(s，3H)ppm.¹³C NMR(CDCl₃，100MHz)：183.0，168.6，65.0，63.4，63.2，54.6，51.3，43.4，41.0，29.0，26.1，22.2，22.0，18.9，-5.1ppm.HRMS(ESI)[MH]⁺Calculated value 396.2676, found value 396.2656

(3R, 5S, 6S, 7S) -N-tert-butyl-3- (3-guanidinopropyl) -7-isobutyl-2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (5)

¹H NMR(D₂O，400MHz)：3.59(t，J＝5.6Hz，1H)，3.46(m，1H)，3.13(m，1H)，2.94(m，1H)，3.37(m，1H)，1.78-1.44(m，8H)，1.22(s，9H)，0.83(d，J＝2.8Hz，3H)，0.81(d，J＝2.8Hz，3H)ppm.¹³H NMR(D₂O，400MHz)：186.7，172.0，156.9，54.9，54.5，54.0，44.5，42.8，28.7，27.9，26.5，25.7，25.0，24.1，22.2，21.5ppm.MS(ESI)[MH]⁺Calculated value 367.3, found 367.2, [ M +2H]²⁺Measured value of/2 was 184.1.

(3S, 5S, 6S, 7S) -3- (4-Aminobutyl) -N-tert-butyl-7-isobutyl-2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (6)

NMR (containing 10% v/v CD)₃D of COOD₂O，400MHz)：5.14(ddd，J＝9.6Hz，4.4Hz，2.4Hz，1H)，4.12(d，J＝2.4Hz，1H)，3.07(dd，J＝7.6Hz，4.4Hz，1H)，2.85(t，J＝7.6Hz，2H)，1.82-1.46(m，10H)，1.32(s，9H)，0.83(d，J＝6.8Hz，3H)，0.81(d，J＝6.8Hz，3H)ppm.¹³C NMR (containing 10% v/v CD)₃D of COOD₂O，100MHz)：178.0，173.8，97.2，58.6，54.5，53.0，40.6，39.3，29.3，27.8，27.6，27.2，26.8，24.6，22.2，21.9，20.8ppm.MS(ESI)[MH]⁺Calculated value 339.3, found 339.2, [ M +2H]²⁺Measured value of/2 was 170.1.

(5R, 6R, 7S) -N-tert-butyl-7- ((tert-butyldimethylsilyloxy) methyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (7)

¹H NMR(400MHz，CDCl₃)：6.98(s，1H)，3.87(dd，J＝11.5，4.5Hz，1H)，3.78(dd，J＝11.5，4.5Hz，1H)，3.34-3.24(m，2H)，3.13(d，J＝4.6Hz，1H)，2.40(td，J＝4.5，3.4Hz，1H)，2.17-1.96(m，1H)，1.43-1.36(m，9H)，0.94-0.86(m，9H)，0.13-0.05(m，6H).¹³C NMR(101MHz，CDCl₃)：183.93，168.37，77.48，77.16，76.84，63.02，55.81，51.25，48.72，45.27，39.64，28.87，26.06，18.55，0.15，-5.09，-5.12.

2- ((3S, 5R, 6R, 7R) -5- (tert-butylcarbamoyl) -7-isobutyl-2-oxo-1, 4-diazabicyclo [4.1.0] heptan-3-yl) acetic acid (8)

¹H NMR(400MHz，CD₃OD)：4.69-4.58(m，1H)，3.92(d，J＝2.0Hz，1H)，3.76(dd，J＝9.8，3.6Hz，1H)，3.66(dd，J＝7.8，4.0Hz，1H)，2.90(dd，J＝17.5，3.6Hz，1H)，2.81(dd，J＝16.7，4.0Hz，1H)，2.69-2.53(m，2H)，1.36-1.32(m，9H)，0.98(dt，J＝8.5，4.2Hz，6H).¹³C NMR(101MHz，CD₃OD)188.21，172.40，158.66，158.62，56.05，55.25，55.23，51.95，44.17，43.55，43.35，42.55，42.39，42.16，41.98，29.85，28.90，28.85，28.10，27.37，27.16，26.85，26.51，23.19，22.63，21.05.

(3R, 5R, 6R, 7S) -N- (tert-butyl) -7- (((tert-butyldimethylsilyl) oxy) methyl) -3- (mercaptomethyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (9)

Note: since the spectra show extremely broad peaks, it is difficult to perform proper spectral characterization of the compound. This is due to the inherent instability of 9 due to the presence of the nucleophilic thiol and its susceptibility to oxidation and aziridine ring opening.

¹H NMR(CDCl3，400MHz)：3.47(s，1H)，3.41(d，J＝6.5Hz，1H)，4.35-2.42(m，7H)，1.70-1.42(bs，10H)，0.90(s，9H)，0.10(s，3H)，0.08(s，3H)ppm。MS(ESI)[MH]⁺Calculated 402.6, found 402.2.

(3S, 5R, 6R, 7S) -3- ((1H-imidazol-4-yl) methyl) -N- (tert-butyl) -7- (((tert-butyldimethylsilyl) oxy) methyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (10)

¹H NMR(CDCl₃，400MHz)：7.56(s，1H)，7.25(s，1H)，6.87(s，1H)，6.22(s，1H)，4.46(m，1H)，3.72(dd，J＝11.2Hz，4.8Hz，1H)，3.60(m，1H)，3.44(dd，J＝3.6Hz，1.5Hz，1H)，3.27(dd，J＝14.1Hz，3.1Hz，1H)，2.99(dd，J＝14.7Hz，4.2Hz，1H)，2.80(dd，J＝15.2Hz，8.1Hz，1H)，2.40(m，1H)，2.05(bs，1H)，1.36(s，9H)，0.87(s，9H)，0.07(s，3H)，0.05(s，3H)ppm.¹³C NMR(CDCl₃，100MHz)：185.4，171.4，169.5，168.6，135.1，64.3，62.7，60.6，54.7，51.2，44.5，39.3，28.9，26.0，18.4，-5.3ppm。MS(ESI)[MH]⁺Calculated 436.6, found 436.2.

(3S, 5R, 6R, 7S) -N- (tert-butyl) -7- (((tert-butyldimethylsilyl) oxy) methyl) -3- (hydroxymethyl) -2-oxo-1, 4-diazabicyclo [4.1.0] heptane-5-carboxamide (11)

¹H NMR(CDCl₃，400MHz)：6.80(bs，1H)，6.15(bs，1H)，4.02(d，J＝11.6Hz，1H)，3.85(dd，J＝13.3Hz，6.7Hz，1H)，3.76(dd，J＝14.2Hz，3.0Hz，1H)，3.68(bs，1H)，3.53(d，J＝5.0Hz，1H)，3.45(dd，J＝3.5Hz，2.4Hz，1H)，3.13(m，1H)，2.55(m，1H)，2.42(m，1H)，1.39(s，9H)，0.90(s，9H)，0.10(s，3H)，0.08(s，3H)ppm.¹³C NMR(CDCl₃，100MHz)：185.3，168.9，63.5，61.4，55.8，53.9，51.2，44.9，39.6，28.9，26.1，18.6，-5.1ppm。MS(ESI)[MH]⁺Calculated 386.6, found 386.2.

(1S, 4S, 6aS, 11R, 11aS) -N-tert-butyl-1- ((tert-butyldimethylsilyloxy) methyl) -4-isobutyl-3, 6-dioxodecahydro-1H-aziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7] triazacyclononane-11-carboxamide (12)

¹H NMR(CD₃OD，400MHz)：4.47(m，1H)，4.14(dd，J＝8.4Hz，6Hz，1H)，4.07-3.99(m，2H)，3.84(dd，J＝12Hz，3.2Hz，1H)，3.32(d，J＝5.6Hz，1H)，3.26-3.15(m，2H)，2.42-2.30(m，2H)，1.92-1.78(m，3H)，1.74(q，J＝6.8Hz，2H)，1.59(sept，J＝6.8Hz，IH)，1.22(s，9H)，0.90(d，J＝6.8Hz，3H)，0.87(d，J＝6.4Hz，3H)，0.84(s，9H)，0.06(s，3H)，0.03(s，3H)ppm.¹³C NMR(CD₃OD，100MHz)：174.1，164.1，157.4，86.5，62.4，60.7，60.6，57.7，54.2，52.1，50.2，39.6，29.0，25.8，25.2，25.1，22.1，21.0，20.5，17.9，-6.5ppm。MS(ESI)[MH]⁺Calculated 509.3, found 509.3.

(1S, 4S, 6aR, 11S, 11aR) -N-tert-butyl-4- (3-guanidinopropyl) -1-isobutyl-3, 6-dioxodecahydro-1H-aziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7] triazacyclononane-11-carboxamide (13)

¹H NMR(CD₃OD，400MHz)：4.26(t，J＝5.6Hz，1H)，4.18(dd，J＝8.8Hz，2.4Hz，1H)，3.58(dd，J＝8.8Hz，5.2Hz，1H)，3.28-3.24(m，2H)，3.00(d，J＝5.6Hz，1H)，2.70(dd，J＝10.4Hz，6.4Hz，1H)，2.32-2.18(m，2H)，1.98-1.52(m，HH)，1.28(s，9H)，1.01(d，J＝6.4Hz，3H)，0.96(d，J＝6.4Hz，3H)ppm.¹³C NMR(CD₃OD，100MHz)：178.0，160.8，159.1，157.4，86.9，64.3，63.5，60.2，54.5，53.7，51.0，42.6，41.4，29.4，28.8，26.1，24.9，24.5，22.7，21.3，20.4ppm。MS(ESI)[MH]⁺Calcd for 464.3, found 464.3, [ M +2H]²⁺Measured value of/2 232.6.

2- ((1S, 4S, 6aR, 11S, 11aR) -11- (tert-butylcarbamoyl) -1-isobutyl-3, 6-dioxodecahydro-1H-aziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7] triazacyclononane 4-yl) acetic acid (14)

¹H NMR(CD₃OD，400MHz)：4.46(t，J＝9.2Hz，1H)，4.34(dd，J＝8.4Hz，2.0Hz，1H)，3.81(dd，J＝8.4Hz，5.6Hz，1H)，3.38-3.30(m，1H)，3.02(dd，J＝17.2Hz，2.4Hz，1H)，2.85-2.73(m，1H)，2.57(dd，J＝17.2Hz，8.4Hz，1H)，2.45-2.32(m，2H)，2.05-1.62(m，7H)，1.58-1.48(m，2H)，1.27(s，9H)，1.01(d，J＝6.4Hz，3H)，0.98(d，J＝6.4Hz，3H)ppm。MS(ESI)[MH]⁺Calculated value 423.3, foundValue 423.2.

(1S, 4S, 9aS, 14R, 14aS) -N-tert-butyl-1- ((tert-butyldimethylsilyloxy) methyl) -4-isobutyl-3, 6, 9-trioxadetrahydrocyclopentaaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10] tetraazacyclododecane-14-carboxamide (15)

¹H NMR(CD₃OD，400MHz)：4.55(ddd，J＝8Hz，3.2Hz，1.6Hz，1H)，4.32(dd，J＝10.4Hz，4Hz，1H)，4.21(dd，J＝14Hz，6Hz，1H)，4.15-4.05(m，3H)，3.91(dd，J＝12Hz，3.2Hz，1H)，3.41Hz(dd，J＝6Hz，1H)，3.36-3.28(m，IH)，2.50-2.30(m，2H)，2.00-1.50(m，7H)，1.31(s，9H)，0.98-0.92(m，6H)，0.92(s，9H)，0.13(s，3H)，0.11(s，3H)ppm.¹³C NMR(CD₃OD，100MHz)：178.3，166.4，165.1，157.6，86.7，62.7，61.0，60.1，54.2，53.7，52.6，50.5，44.0，41.6，28.9，25.6，25.2，25.1，25.1，22.6，20.7，20.5，17.9，-6.5，-6.5ppm。MS(ESI)[MH]⁺Calculated 566.4, found 566.4.

(1R, 1aS, 2R, 6aS, 12S, 18S) -18-benzyl-N-tert-butyl-1, 12-diisobutyl-7, 10, 13, 16, 19-pentaoxoeicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecane-2-carboxamide (16)

¹H NMR(CDCl₃，500MHz)：7.82(bs，NH)，7.40-7.20(m，5H)，7.18(bs，NH)，7.05(bs，NH)，6.81(bs，2NH)，4.64(q，J＝6.8Hz，1H)，4.13(dd，J＝16Hz，7.6Hz，1H)，4.10-3.90(m，2H)，3.57(dd，J＝16Hz，4.8Hz，1H)，3.50-3.39(m，2H)，3.26(dd，J＝14Hz，5.6Hz，1H)，3.12(dd，J＝14Hz，7.2Hz，1H)，3.12-3.08(m，1H)，3.00-2.92(m，2H)，2.63(t，J＝3.6Hz，1H)，2.28-2.18(m，IH)，1.90-1.70(m，2H)，1.37-1.25(m，9H)，1.10-0.88(m，12H)ppm.13CNMR(CDCl₃，125MHz)：181.4，176.1，175.7，172.1，170.1，169.2，136.4，130.1，128.7，126.8，56.2，63.0，43.2，42.5，40.2，38.6，37.9，31.2，30.0，29.8，29.5，28.8，28.7，27.2，25.1，24.9，24.3，23.3，23.1，22.8，22.7，22.6，22.4，22.3，21.9，MS(ESI)[MH]⁺Calculated 682.4, found 682.4.

(1S, 12aS, 17R, 17aR) -N-tert-butyl-1-isobutyl-3, 6, 9, 12-tetraoxohexadecahexahydro-1H-aziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13] pentaazacyclopentadecane-17-carboxamide (17)

¹H NMR(400MHz，CDCl₃)：4.57-4.44(m，1H)，4.32-3.70(m，10H)，3.38(d，J＝5.4Hz，1H)，3.28(s，1H)，3.14(dd，J＝11.2，5.6Hz，1H)，2.52-2.41(m，1H)，2.37(d，J＝6.9Hz，1H)，2.15-1.65(m，8H)，1.64-1.48(m，2H)，1.42-1.23(m，8H)，1.00(dt，J＝21.4，7.4Hz，6H)。

¹³C NMR(100MHz，CD₃OD)：176.18，171.23，168.97，165.90，159.24，86.37，65.77，63.49，61.48，59.76，55.41，51.43，47.38，45.65，45.23，44.59，44.43，44.19，43.41，42.46，29.85，29.43，28.97，28.58，28.46，27.72，27.13，26.74，25.68，23.84，23.68，23.14，23.06，22.16，21.62。

Tert-butyl 2- ((1S, 4S, 7S, 13S, 15aS, 20R, 20aS) -20- (tert-butylcarbamoyl) -1- (((tert-butyldimethylsilyl) oxy) methyl) -4-methyl-3, 6, 9, 12, 15-pentaoxo-13- (3- (3- ((2, 2, 4, 6, 7-pentamethyl-2, 3-dihydrobenzofuran-5-yl) sulfonyl) guanidino) propyl) eicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecan-7-yl) acetate (18)

¹H NMR(400MHz，CDCl₃)：7.84(d，J＝9.7Hz，2H)，7.58(dd，J＝18.1，9.1Hz，2H)，7.07(s，IH)，6.41(s，2H)，5.68(s，1H)，4.85(dq，J＝13.7，6.8Hz，1H)，4.70-4.58(m，1H)，4.35(q，J＝8.1Hz，1H)，4.17-4.07(m，1H)，3.97(dd，J＝14.2，7.5Hz，2H)，3.74(s，1H)，3.69-3.57(m，1H)，3.56-3.42(m，2H)，3.42-3.13(m，4H)，2.96(s，2H)，2.93(d，J＝2.3Hz，1H)，2.89-2.76(m，4H)，2.66-2.53(m，7H)，2.42-2.15(m，2H)，2.10(s，3H)，2.07-1.98(m，1H)，1.87-1.55(m，8H)，1.46(s，6H)，1.44(s，9H)，1.34(s，9H)，0.87(s，9H)，0.09(s，3H)，0.05(s，3H).¹³C NMR(101MHz，CDCl₃)：187.07，176.14，174.46，171.46，171.04，169.11，166.93，158.77，156.39，138.63，133.66，132.55，124.65，117.55，105.24，86.49，82.64，63.92，63.84，60.81，57.07，51.69，51.31，50.37，49.56，45.47，43.51，40.43，37.55，35.99，30.28，29.17，28.83，28.22，26.11，24.61，19.49，18.71，18.19，17.31，12.69，-5.11，-5.44。MS(ESI)[MH]⁺Calculated 1104.6, found 1104.6.

Tert-butyl (4- ((1S, 4S, 7S, 10S, 13S, 15aS, 20R, 20aS) -13- ((R) -1- (tert-butoxy) ethyl) -20- (tert-butylcarbamoyl) -1- (((tert-butyldimethylsilyl) oxy) methyl) -4-methyl-10- (2- (methylthio) ethyl) -3, 6, 9, 12, 15-pentaoxoeicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecane-7-yl) butyl) carbamate (19)

¹H NMR(400MHz，CDCl₃)：7.83(d，J＝8.1Hz，1H)，7.77(s，1H)，7.50(d，J＝21.3Hz，1H)，7.35(m，2H)，7.08(dd，J＝15.7，7.5Hz，1H)，6.90(s，1H)，6.82(s，1H)，4.98(d，J＝27.6Hz，1H)，4.84-4.67(m，1H)，4.60(s，1H)，4.40-4.30(m，1H)，4.16-3.93(m，4H)，3.82(s，2H)，3.77-3.69(m，3H)，3.67(d，J＝6.3Hz，1H)，3.45-3.28(m，2H)，3.27-2.99(m，6H)，3.00-2.86(m，2H)，2.84(s，IH)，2.78-2.55(m，3H)，2.45-2.19(m，5H)，2.12(s，4H)，2.06-1.97(m，3H)，1.89(d，J＝4.7Hz，7H)，1.71-1.55(m，3H)，1.54-1.39(m，21H)，1.36(s，13H)，1.31-1.26(m，12H)，1.06(t，J＝10.5Hz，4H)，0.93-0.82(m，14H)，0.06(s，3H)，0.05(s，3H).13C NMR(101MHz，CDCl₃)：182.99，175.20，171.94，170.75，170.37，168.93，156.44，77.43，75.69，66.32，64.35，63.93，61.77，57.52，56.58，54.28，42.68，40.37，31.21，30.95，30.08，29.88，29.36，28.85，28.66，26.01，24.92，24.16，18.91，18.48，17.89，15.48，-5.25，-5.29。MS(ESI)[MH]⁺Calculated 983.6, found 983.6.

(1S, 7S, 10S, 15aS, 20R, 20aS) -10- ((1H-imidazol-5-yl) methyl) -7- (4- (tert-butoxy) benzyl) -N- (tert-butyl) -1- (((tert-butyldimethylsilyl) oxy) methyl) -3, 6, 9, 12, 15-pentaoxoeicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecane-20-carboxamide (20)

¹H NMR(400MHz，CDCl₃)：9.92(s，1H)，7.49(d，J＝4.8Hz，2H)，7.39-7.27(m，HH)，7.17-7.02(m，HH)，6.78(d，J＝8.4Hz，2H)，6.69(s，1H)，4.48(p，J＝6.9Hz，1H)，4.42-4.18(m，2H)，4.06-3.99(m，1H)，3.91(dd，J＝11.4，3.8Hz，1H)，3.78(dd，J＝11.3，4.4Hz，1H)，3.61(dd，J＝9.8，4.8Hz，1H)，3.48-3.37(m，1H)，3.32-2.99(m，8H)，2.83(dd，J＝6.8，3.4Hz，1H)，2.41-1.95(m，2H)，1.91-1.72(m，2H)，1.61(s，12H)，1.41(s，10H)，1.35-1.24(m，20H)，0.72(s，1H)，0.07(s，3H)，0.05(s，3H).13C NMR(101MHz，CDCl₃)：184.31，175.20，172.61，170.67，169.47，168.12，154.53，142.31，138.19，136.81，131.91，129.97，129.85，128.43，128.37，120.09，77.43，63.49，62.17，61.55，57.84，54.97，52.19，51.15，48.67，43.36，42.53，41.75，36.36，31.84，30.99，29.92，29.88，29.44，29.05，28.67，26.07，24.49，19.80，18.56，-5.19。MS(ESI)[MH]⁺Calculated 866.5, found 866.5.

(1S, 4S, 7S, 13S, 15aS, 20R, 20aS) -13-benzyl-N- (tert-butyl) -1- (((tert-butyldimethylsilyl) oxy) methyl) -4-methyl-3, 6, 9, 12, 15-pentaoxo-7- (2-oxo-2- (tritylamino) ethyl) eicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecane-20-carboxamide (21)

¹H NMR(400MHz，CDCl₃)：7.94(t，J＝9.8Hz，2H)，7.73(d，J＝8.7Hz，1H)，7.51(t，J＝6.7Hz，1H)，7.34-7.11(m，23H)，6.79(s，1H)，4.92-4.72(m，2H)，4.59(dd，J＝16.3，8.6Hz，1H)，4.25(d，J＝11.5Hz，1H)，4.01(d，J＝10.7Hz，1H)，3.82(dd，J＝14.2，7.6Hz，1H)，3.73(s，1H)，3.59-3.51(m，2H)，3.48-3.24(m，3H)，3.09(dd，J＝13.8，8.9Hz，1H)，2.96-2.68(m，6H)，2.59(dd，J＝15.8，3.6Hz，1H)，2.45-2.08(m，2H)，2.07-1.96(m，1H)，1.62(s，5H)，1.52-1.17(m，18H)，0.94(s，9H)，0.15(s，3H)，0.13(s，3H).¹³C NMR(101MHz，CDCl₃)：184.06，175.64，173.93，171.01，170.49，169.15，167.20，144.28，136.98，129.61，128.81，128.49，128.30，127.48，126.75，77.55，77.23，76.91，71.17，63.63，60.72，57.12，52.96，51.54，50.58，49.71，49.27，45.35，44.53，37.27，36.80，33.93，30.22，29.24，26.24，24.32，18.78，17.89，0.22，-5.11，-5.34。MS(ESI)[MH]⁺Calculated 1027.5, found 1027.6.

(4S, 10S, 15aS, 20R, 20aS) -4-benzyl-N- (tert-butyl) -10-isobutyl-3, 6, 9, 12, 15-pentaoxoeicosahydroaziridino [1, 2-a ] pyrrolo [1, 2-d ] [1, 4, 7, 10, 13, 16] hexaazacyclooctadecane-20-carboxamide (22)

¹H NMR(CDCl₃，400MHz)：8.33(bs，IH)，8.17(d，J＝9.1Hz，1H)，8.03(d，J＝7.4Hz，1H)，7.54(s，1H)，7.21(m，5H)，6.04(bs，1H)，5.08(bs，1H)，4.58(m，1H)，3.87(dd，J＝8.1Hz，5.0Hz，1H)，3.80(bs，1H)，2.48(dd，J＝3.6Hz，1.8Hz，2H)，2.16(m，2H)，1.62-1.57(m，7H)，1.48(m，2H)，1.42(m，2H)，1.35(m，2H)，1.20(m，9H)，1.07(m，2H)，0.81(m，6H)ppm。MS(ESI)[MH]⁺Calcd for 626.8, found 626.3, [ M +2H]²⁺Measured value of/2 313.7.

7-mmc combined 22(23)

The cyclic peptide 22 as a crude product is reacted to give the bound peptide 23. (see, page 39)¹H NMR(300MHz，DMSO)8.95(s，1H)，8.68(d，J＝8.2Hz，1H)，8.32(t，J＝6.3Hz，1H)，7.68(d，J＝8.4Hz，1H)，7.51(d，J＝9.4Hz，1H)，7.42-7.08(m，5H)，6.46(s，1H)，6.31(s，1H)，4.47-4.15(m，3H)，3.73(dd，J＝20.5，14.1Hz，3H)，3.45-3.33(m，2H)，3.17(dd，J＝20.9，13.0Hz，2H)，2.98-2.64(m，4H)，2.40(s，2H)，2.22-2.10(m，1H)，1.91(ddd，J＝22.9，13.4，6.6Hz，2H)，1.79-1.40(m，5H)，1.30-1.15(m，9H)，0.93-0.74(m，6H)。MS(ESI)[MH]⁺Calculated 818.4, found 818.3.

HATU-mediated cyclization

Advantageously, the cyclization reaction can be carried out at a starting amino acid/peptide concentration of at least 0.002M. Preferably at a concentration of between 0.002M and 0.2M.

In another embodiment, the cyclization reaction is performed at a starting amino acid/peptide concentration of at least 0.1M. In another embodiment, the method is performed at a starting amino acid/peptide concentration of about 0.2M.

The presalicyclization cannot be carried out at such high concentrations in the conventional manner. This is demonstrated by direct comparison with the HATU process, which fails because the major products observed derive from oligomerization and polymerization.

O- (7-azabenzotriazol-1-yl) -N, N, N ', N' -tetramethyluronium hexafluorophosphate ("HATU") -mediated Pro-Gly-Gly-Gly cyclization and aminoaldehyde/isonitrile cyclization processes were compared at 0.2M. After the reaction was complete, the crude reaction mixture was analyzed using liquid chromatography electrospray ionization mass spectrometry ("LC-ms (esi)") to evaluate the level of selectivity for the desired cyclic peptide.

Mass Spectrometry ("MS") analysis of the HATU reaction showed that cyclodimerization predominated at 0.2M (FIG. 28). The main peaks at m/z 537.2 and 559.2 correspond to the cyclic dimer product and its sodium chelate, respectively, while only traces of the desired cyclic peptide were detected at m/z 269. Cyclotrimer was also detected (m/z 805). The crude reaction mixture also contains several other unidentified by-products.

From the results from MS peak analysis, 6 components were found.

Component 1: 27.4 scanning peak. The dominant ion (Top ions) is 655566923

And (2) component: 28.4 scanning peak. The primary ion is 982744714

And (3) component: 29.9 scanning peak. The primary ion is 235848538

And (4) component: 30.9 scanning peak. The primary ion is 559827560

And (5) component: 31.9 scanning peak. The primary ion is 752931

And (4) component 6: 32.7 scanning peak. The primary ion is 537295446

Crude LC-ms (esi) analysis of isonitrile/aminoaldehyde mediated cyclization showed high levels of selectivity to the desired cyclic peptide (M/z ═ 479) at 0.2M (fig. 29). No trace peaks of dimer or trimer by-products were detected. Only characteristic fragment peaks were observed and residual Pro-Gly-Gly-Gly remained in the reaction mixture.

From the results from MS peak analysis, 2 components were found.

Component 1: 29.7 scanning peak. The major ion is 287212383 (Pro-Gly-Gly-Gly)

And (2) component: 30.9 scanning peak. The predominant ion is 479396240 (cyclic peptide)

Example 2

Method and material

The method A comprises the following steps: the opening of the aziridine-containing cyclic peptide is carried out with an aromatic thiol as nucleophile (method A). To a screw-capped glass bottle fitted with a magnetic stir bar was added aziridine-containing cyclic peptide (0.06mmol) and aromatic thiol (0.066mmol) to 0.2ml degassed CHCl₃In (1). Then add NEt to the solution₃(0.06mmol) andthe reaction was stirred at room temperature for 3-4 hours. Then using CH₂Cl₂The reaction was diluted and saturated NH₄Aqueous Cl (3ml) was washed twice and then once with brine (3 ml). Then the organic layer was washed with Na₂SO₄Dried, filtered and concentrated under reduced pressure. The crude ring-opened product was of high purity, but only after purification by flash column chromatography (5% MeOH in EtOAc) gave an analytically pure sample.

The method B comprises the following steps: the ring opening of the aziridine-containing cyclic peptide is carried out using an aliphatic thiol or imide as nucleophile (method B). To a screw-capped glass bottle fitted with a magnetic stir bar was added the aziridine-containing cyclic peptide (0.06mmol) and the aliphatic thiol or imide (0.066mmol) was added to 0.2ml degassed CHCl₃In (1). DBU (0.06mmol) was then added to the solution and the reaction was stirred at room temperature for 3-4 hours. Then using CH₂Cl₂The reaction was diluted and saturated NH₄Aqueous Cl (3ml) was washed twice and then once with brine (3 ml). Then the organic layer was washed with Na₂SO₄Dried, filtered and concentrated under reduced pressure. The crude ring-opened product was of high purity, but only after purification by flash column chromatography (5% MeOH in EtOAc) gave an analytically pure sample.

Results

Side chains including, but not limited to, fluorescent substituents are attached to the macrocyclized product (scheme 3).

Scheme 3. fluorescent labels bind site-specifically to cyclic peptides at a later stage.

The incorporation of an activated aziridine ring into the backbone of cyclic peptides provides a useful point for binding to individual side chains by nucleophilic ring opening, a well established method that has been validated using nucleophilic biomolecules from carbohydrates to biotin and farnesyl (farnesyl) derivatives 14. For example, the cyclic peptide binding (conjugation) strategy can be validated by nucleophilic ring-opening of the aziridine moiety using the widely used fluorescent-labeled 7-mercapto-4-methylcoumarin (7-mmc) (scheme 3). On the other hand, late thioester residue synthesis by ring opening of cyclic peptides using commercially available thiobenzoic acid can also be used. This reaction proceeded smoothly, and the ring-opened product was obtained in a yield of 98%. The late introduction of a thioester function avoids the steps normally required to prevent oxidation and nucleophile. Many other natural and unnatural amino acid side chains can be placed through similar ring opening schemes, which provides the possibility of configuration optimization of cyclic peptides at later stages of synthesis. Other nucleophiles, such as aliphatic thiols, acids, secondary amines such as morpholine, and imides, can have equivalent effects in the ring opening process.

On the other hand, a cyclic peptide provided with an aziridine ring is opened (FIG. 30). During this procedure, the side chain of interest can be attached to a cyclic peptide scaffold (scaffold) at a later stage of synthesis. The ring-opening reaction is performed using an appropriate base/nucleophilic system selected based on pKa. A preferred reaction involves the use of a base/nucleophile pair satisfying Δ pKa ═ pKa [ NR3H + ] -Pk [ nucleophile ] ≈ -1 (fig. 31).

These data indicate that strong ion pairing (Δ pKa > 1) between the base and the nucleophile results in reduced reactivity. It was found that optimized ring opening conditions can be carried out smoothly so that a high purity of the modified cyclic peptide can be obtained after post-extraction treatment (fig. 32).

Example 3

X-ray analysis of the cyclic product 1 was performed.

Results

Table 4.k07227 Crystal data and Structure refinement (Structure refinement)

TABLE 5 atomic coordinates (x 10) of k07227⁴) And equivalent isotropic displacement parameterU (eq) is defined as one third of the trace (trace) of the orthogonal Uij tensor.

Bond length of k07227 Table 6Angle of harmony key [ ° ]]

Table 7 isotropic displacement parameters of k07227The isotropic displacement coefficient index is of the form: -2 pi²[h²a^*2U¹¹+...+2h k a^*b^*U¹²]

TABLE 8 hydrogen coordinates (x 10) of k07227⁴) And isotropic displacement parameter

Although preferred embodiments of the present invention have been described herein, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope of the appended claims. All references mentioned herein are incorporated by reference in their entirety.

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Claims (27)

1. A method of making a cyclic amino acid molecule comprising reacting an amino acid molecule having an amino terminus and a carboxy terminus with an aziridine aldehyde and an isonitrile, wherein the aziridine aldehyde has the structure:

2. the process of claim 1, wherein the isonitrile is tert-butyl isonitrile.

3. The process of claim 1, wherein the process is a modified Ugi reaction comprising replacing a monofunctional aldehyde with an amphoteric aminoaldehyde.

4. The method of claim 1, wherein the amino acids of the amino acid molecule have aliphatic, acidic, or basic residues.

5. The method of claim 1, wherein the method is a one-step reaction.

6. The method of claim 1, wherein the reaction is carried out at room temperature.

7. The process of claim 1, wherein the reaction is carried out in TFE.

8. The process of claim 1, wherein the reaction uses HFIP and water as solvents in a ratio of 20: 1.

9. The process of claim 1, wherein the reaction uses HFIP as a solvent.

10. The method of claim 1, further comprising nucleophilic opening of an aziridine ring.

11. The method of any one of claims 1-10, further comprising incorporating an activated aziridine ring into the cyclic amino acid molecule.

12. The method of any one of claims 1-10, wherein the cyclic amino acid molecule is a single diastereoisomeric product.

13. A cyclic amino acid molecule having the structure:

wherein:

the amino acid molecule is a single amino acid or a linear peptide;

the group is an optionally substituted amide;

r is an optional substituent at the amino terminus of the amino acid molecule; and

Rⁿis the side chain at the carboxy terminus of the amino acid molecule.

14. A cyclic amino acid molecule according to claim 13, wherein the amino acid molecule is a peptide.

15. A cyclic amino acid molecule according to claim 13, wherein the amino acid molecule is a single amino acid.

16. A cyclic amino acid molecule according to claim 14, wherein the peptide includes at least one of an acidic and a basic residue.

17. A cyclic amino acid molecule according to claim 13, wherein the aziridine ring is ring opened.

18. The cyclic amino acid molecule of claim 13, wherein a fluorescent label is incorporated into the cyclic amino acid.

19. The cyclic amino acid molecule of claim 13, wherein a thioester residue is incorporated into the cyclic amino acid with thiobenzoic acid.

20. The cyclic amino acid molecule of any one of claims 13-19, wherein an activated aziridine ring is incorporated into the cyclic amino acid molecule.

21. A compound having the structure:

22. a compound having the structure:

23. a compound having the structure:

24. a compound having the structure:

25. a compound having the structure:

26. a compound having the structure:

27. a compound having the structure: